Interstitial flow plays a significant role in vascular system development, mainly including angiogenesis and vasculogenesis. However, compared to angiogenesis, the effect of interstitial flow on vasculogenesis is less explored. Current in vitro models for investigating the effect of interstitial flow on vasculogenesis heavily rely on microfluidic chips, which require microfluidic expertise and facilities, and may not be accessible to biological labs. Here, we proposed a facile approach to building perfusable vascular networks through the self-assembly of endothelial cells in a modified transwell format and investigated the effect of interstitial flow on vasculogenesis. We found that the effect of interstitial flow on vasculogenesis was closely related to the existence of VEGF and fibroblasts in the developed model: (1) In the presence of fibroblasts, interstitial flow (within the range of 0.1–0.6 μm/s) facilitated the perfusability of the engineered vasculatures. Additional VEGF in the culture medium further worked synergically with interstitial flow to develop longer, wider, denser, and more perfusable vasculatures than static counterparts; (2) In the absence of fibroblasts, vasculatures underwent severe regression within 7 days under static conditions. However, interstitial flow greatly inhibited vessel regression and enhanced vascular perfusability and morphogenesis without the need for additional VEGF. These results revealed that the effect of interstitial flow might vary depending on the existence of VEGF and fibroblasts, and would provide some guidelines for constructing in vitro self-assembled vasculatures. The established transwell-based vascularized model provides a simple method to build perfusable vasculatures and could also be utilized for creating functional tissues in regenerative medicine. Blood vessels play a vital role in transporting nutrition and waste, bridging inter-organ communications, and mediating homeostasis . Blood vVascular system development mainly includes angiogenesis and vasculogenesis, which are also orchestrated by multiple biophysical factors . Among tHowever, current in vitro vascularized models for studying the effect of interstitial flow on vasculogenesis mostly rely on microfluidic chips ,13,14. MHere, we proposed a self-assembled vascularized model in a modified transwell format to investigate the effect of interstitial flow on vasculogenesis, mainly focusing on vascular morphology and perfusability. The engineered vascular networks were interrogated by morphological analysis, immunofluorescent assays, finite-element simulation, and particle perfusion tests. Perfusable vascular networks with in vivo-like features were successfully formed through the self-assembly of endothelial cells in this model. Interstitial flow showed different influences on vasculogenesis in the presence or absence of fibroblasts and VEGF, indicating that the effect of interstitial flow on vasculogenesis was context-dependent. These results revealed a partial role of interstitial flow, interplaying with VEGF and fibroblasts, and their combined effects on vasculogenesis. The established vascularized model is simple but effective, and could also be applied for creating vascularized tissues in regenerative medicine.The device was modified from commercially available transwells , with a membrane pore size of 12 μm. Concentric PDMS ring adaptors with an outer diameter of 6 mm, an inner diameter of 2.5 mm, and a height of 1.2 mm, were made by cutting the PDMS membrane using biopsy needles or blunt syringe needles of different sizes. The PDMS ring was then put into the transwell and glued onto the membrane using uncured PDMS. The finished device was heated at 70 °C for 5 h and sterilized under UV radiation. Then, the devices were coated with PDL  to enhance the bond between the fibrin and the tissue reservoir . BrieflyEGFP-HUVECs, RFP-HUVECs, and NHLFs were kindly provided by professor Xin Cheng . HUVECs were cultured with EGM2 medium  on collagen-coated flasks. NHLFs were cultured with DMEM  supplemented with 10% FBS (Gibco), 1% PS (Gibco), 1% Glutamax (Gibco), and 1% NEAA (Gibco). All the cells used for conducting experiments were within the passage of 10.6/mL. NHLFs were suspended in 8 U/mL thrombin  at the concentration of 6 × 106/mL ) at the concentration of 12 × 10ications ,18,19). For the non-NHLFs system, we mixed fresh EGM2 medium with NHLF-conditioned medium at a 1:1 ratio to conduct experiments. Conditioned medium was collected after culturing NHLFs in EGM2 medium for 24 h. 2, more than a third of the total cross-section area of the tissue chamber. The z-stack image acquisition started from the porous membrane, up to 120 μm above the membrane, at the interval of 4 μm. The images from plane 3 (8 μm deep) to plane 20 (80 μm deep) were projected into a single picture. Then, the projected picture was imported to AngioTool to quantify the total length and vessel area percentage, and imported to image J to quantify the vessel diameter and the beads area percentage. The reason that we only chose images taken from 4 μm to 80 μm deep was that the images taken deeper than 80 μm were blurry and hard to quantify due to the optical diffraction. In this stage, 1 μm beads (Sigma) with red fluorescence were diluted in PBS at 1:1000. An amount of 2000 kDa FITC-dextran (Sigma) was diluted in PBS to the concentration of 100 ng/mL. Then, 400 uL PBS with fluorescent molecules was added to the apical chamber of the fixed samples and 400 μL PBS without fluorescent molecules was added to the basolateral chamber, and incubated for 5 h. For the static group, the punctured hole was sealed using molten Vaseline before testing. Then, z-stack images were taken using an Olympus FV1000 confocal microscope. In detail, images were taken in the central region of every sample using a 10× objective, which resulted in an imaging area of 1.6 mmSamples were fixed by 4% PFA, followed by permeabilization using 0.2% Triton. Then, blocking was performed by blocking medium  for 2 h, at room temperature. Then, we incubated the samples with primary antibodies , at 4 °C, for 2 days, followed by rinsing with PBS, and they were further incubated with corresponding secondary antibodies for 2 h, at room temperature. Nuclei were stained with DAPI. Images were taken using an Olympus FV1000 confocal microscope.2O, and no-slip conditions were applied to all the outer surfaces except the inlet and outlet. The simulation was carried out by solving the Brinkman equations, and the resulting velocity magnitude was normalized to the maximum velocity in the fibrin to obtain relative velocity distribution.To verify the differences in velocity field distribution between the fibrin with a planar and the fibrin with a concaved surface, the simulation was conducted using COMSOL Multiphysics. A 2D axisymmetric model was selected to reduce computation demand. The volume of both fibrin blocks was set equally to 50 μL. Additionally, for the concaved surface, the radius was set to 3.6 mm, which was measured through the real photo of the meniscus of 50 μL fibrin in the transwell. The permeability coefficient was determined by previously published methods , and theV) was calculated using the following equations:m0 is the mass of the remaining medium volume in a non-permeable transwell every 24 h, rm is the remaining medium volume in the experimental transwells every 24 h, t is the time between two measures (24 h), and S is the cross-section area of the tissue (4.9 mm2). Due to the low velocity, the pressure head change within a day was less than 50% for most cases. So, the average velocity would be representative of the dynamic interstitial flow that tissues experienced [The culture medium in the apical chamber was collected and measured by weight every 24 h. The average velocity of interstitial flow  for 2 h, followed by rinsing in acetic acid solution. Then, images were taken by the camera.p-Values were calculated using the unpaired student’s t-test. *, p < 0.05; **, p < 0.01, ***, p < 0.001, ****, p < 0.0001, and ns, not significant.Data were analyzed and presented using GraphPad and presented as mean ± standard deviation. In order to investigate the effect of interstitial flow on vasculogenesis, we established a transwell-based vascularized model to build self-assembled perfusable vascular networks. To perform this, the commercially available transwell was integrated with a thin sheet of PDMS ring adaptor using uncured PDMS glue, as shown in 2O. After 7 days, interconnected vascular networks were formed , as shown in In order to investigate the interplay between interstitial flow and VEGF, and their combined effects on vasculogenesis, we added 20 ng/mL VEGF121 into the culture medium for both fluidic and static groups . The hydrostatic pressure difference setting, cell and matrix formulation stayed the same as in the last chapter. Then, similar characterizations were conducted and analyzed, and they are shown in With additional VEGF, the vasculatures under flow developed more junctions (increased 1.5-fold), longer total length (increased 1.4-fold), and better perfusability (increased 2.4-fold) than those without additional VEGF A,B, confVEGF, as a widely explored pro-angiogenic factor, has been proven to modulate endothelial cell proliferation, migration, and homeostasis. ,41. TherWe assume that the synergetic effect between VEGF and interstitial flow could be attributed to multiple factors: ECs might be firstly activated by VEGF, which would make ECs more sensitive to interstitial flow ,6; ThereOther than pro-angiogenic factors, stromal cells also play a vital role in vasculogenesis ,45,46. IWith the addition of the CM, vascular networks were successfully formed under flow. Vasculatures were well interconnected and had high vascular density, although the shape was more irregular than those with NHLFs. Additionally, its diameter was larger and have wider distribution, as shown in It is also worth mentioning that the average flow velocity across the NHLF-free tissue construct was higher than those with NHLFs C. It sugWe have successfully built interconnected vascular networks in the absence of NHLFs. However, most self-assembled vasculatures-on-chips incorporated stromal cells such as fibroblasts, MSCs , or pericytes to promote capillary morphogenesis . StromalDespite that, stromal cells would bring some problems, like overgrowth, nutrition competition , tissue The reason that such large differences existed between the static and the fluidic in the absence of NHLFs would be investigated and discussed below.To determine the reason that such large differences caused by interstitial flow existed in the absence of NHLFs, we further investigate the developed vasculatures on day 4. The results are shown in Blood flow has been extensively proven to regulate vessel regression in vivo. Blood flow activates various signal pathways to prevent regression, including ECs survival, alignment, migration, and vessel dilation et al . SeveralIn summary, we developed a facile self-assembled vascularized model in a modified transwell format and investigated the effect of interstitial flow on vasculogenesis. Perfusable vascular networks with in vivo-like features were successfully developed in this model. We also found that the perfusability of the vasculatures was enhanced under fluidic conditions, but not necessarily with morphological differences. However, interstitial flow could induce vascular morphological differences in the presence of additional VEGF or the absence of fibroblasts. These results will not only facilitate the understanding of the role of interstitial flow during vasculogenesis, but also provide some guidelines for constructing in vitro self-assembled vasculatures. The established vascularized model would provide a facile strategy to construct perfusable vascular networks and could also be applied for creating functional tissues in regenerative medicine.


Furthermore, the slow-transit constipation experiment  was conducted to illustrate the underlying mechanism. The results showed that ROS aided in preventing the occurrence of constipation by improving the gastro-intestinal transit rate and the defecation frequency in mice, and ROS significantly reduced the serum levels of vasoactive intestinal peptide (VIP). In addition, ROS regulated the diversity and structure of intestinal flora. Among them, one specific family and six specific genera were significantly regulated in constipated mice. The targeted screening revealed that 29 targets related to the anti-constipation effects of ROS, indicating ROS may play a role by regulating multiple targets. Furthermore, the network pharmacology analysis showed that Akt1, Stat3, Mapk8, Hsp90aa1, Cat, Alb, Icam1, Sod2, and Gsk3b can be regarded as the core anti-constipation targets. In conclusion, ROS could effectively relieve constipation, possibly by inhibiting the level of neurotransmitters and regulating the gut flora in mice. This study also provides a novel network pharmacology-based targeted screening strategy to reveal the anti-constipation effects of ROS.Raffino-oligosaccharide (ROS), the smallest oligosaccharide of the raffinose family, is a novel food ingredient. However, the anti-constipation effects of ROS remain obscure. This study investigates the anti-constipation effects of ROS based on the loperamide-induced mice model and reveals the underlying mechanism using constipation parameters, neurotransmitter level, 16S rRNA sequencing, and the targeted screening strategy. The prevention effects were firstly investigated by the gastro-intestinal transit rate experiment (50 mice) and defecation status experiment (50 mice), which were divided into five groups ( Constipation is a complex gastrointestinal syndrome, the primary features of which include infrequent bowel movements, prolonged gastrointestinal transit time, and difficulty with defecation, involving the regulation of multiple targets ,2,3,4,5.Raffino-oligosaccharide (ROS) is an α-1,6-linked galactosides trisaccharide  . It is dWith the development of bioinformatics, many target data associated with compounds and diseases have been systematically collected by some databases. Among them, PubChem collects information on more than 100 million compounds, including reports on the biological activity of ROS. DrugBank provides up-to-date information on constipation drugs and related targets. On this basis, the targeted screening becomes a feasible research strategy, which is a new bioinformatics method based on the matching of ROS’s targets and constipation’s targets. The acquisition of intersecting targets helps to further understand molecular mechanisms and provides a systematic perspective. As more data are collected in the database, the more comprehensive the targeted screening can reveal the relevant mechanisms. Based on the above literature, we hypothesized that ROS may play an anti-constipation role by regulating gut microbiota, enteric nervous system, and constipation-related target.In this study, the anti-constipation role of ROS was firstly investigated by evaluating the gastro-intestinal transit rate (GI) and defecation status in mice. Then, the regulation characteristics of ROS were clarified by the level of neurotransmitters and 16S rRNA sequencing. Lastly, the targeted screening strategy was further used to illustrate the gut prebiotic properties of ROS.p < 0.05), indicating that the constipated model was built. The GI was not significantly increased in the low-dose ROS group than in that of the model group, while the GI was increased in the medium-dose and high-dose ROS groups (p < 0.05). Although the ROS groups did not significantly reduce the time to the first black stool defecation (Tfbsd) compared with the model group, low-ROS, medium- ROS, and high-ROS shortened the time to Tfbsd by 86.0 min, 93.7 min, and 79.6 min, respectively (compared to the model group) (p < 0.05). In a word, the results of GI and defecation revealed that ROS could effectively accelerate the intestinal peristalsis and defecation status to prevent the occurrence of constipation at the dose set in the experiment.l group) d. Notablp < 0.05) (p < 0.05) compared with the blank group, while this trend was reversed after the administration of ROS (p < 0.05).The effects of ROS on relieving constipation were further evaluated by measuring serum neurotransmitters (acetylcholine (Ach), substance P (SP), nitric oxide (NO) and vasoactive intestinal peptide (VIP)) in mice. The results showed that no significant difference was observed in the level of SP and NO among all groups c,d. Alth < 0.05) a, it wasp < 0.05). After ROS treatment, the Chao-1, PD whole tree, and observed species indices were remarkably improved , while no statistical differences were observed between the blank group and ROS group, indicating the species richness of intestinal flora recovered after ROS administration. Beta diversity (PCoA) was also evaluated using the unweighted uniFrac matrices. Overall differences were observed in microbial compositions among different groups . At the genus level, ROS administration significantly elevated the levels of Erysipelatoclostridium, Rikenella, Coprobacillus, Ruminococcaceae NK4A214 group, and Ruminococcaceae TCG-009 than in that of the model group, while ROS administration significantly reduced the level of (Eubacterium) coprostanoligenes group in comparison with that of the model group (p < 0.05).At the family level, analysis of variance showed the level of Erysipelotrichaceae was increased in the ROS group compared with that of the model group (Mus musculus) of ROS and 1143 therapeutic targets related to constipation were screened. The intersection of ROS targets and constipation-related targets are shown with a Venn diagram  terms were identified , and theed genes a. A totaed genes . Of themed genes b, included genes , revealied genes . Among tTo verify the results of the targeted screening, the core targets  were selected from the PPI network and R-T-P network for further molecular docking. The crystal structure for Stat3 was available from the Protein Data Bank (PDB entry: 4E68), and the crystal structure for Gsk3b was also obtained from the Protein Data Bank (PDB entry: 6AE3). As the crystal structures of some target proteins were not found, homology modeling was carried out by Swiss-model . Sequences for those proteins were acquired from the UniProt database. The results of homology modeling were shown in Bifidobacterium, and lactobacillus [The supplement of prebiotics and probiotics has been reported to be a practical method to influence intestinal function ,17, for bacillus ,19,20. Tbacillus ,14,15. Ip < 0.05). In addition, the movement of gastrointestinal contents is detected by measuring the status of defecation [Constipation is a complex disease driven by complex interactions between multiple molecules. Lifestyle changes, such as dietary supplement (prebiotics and probiotics) and exercise status (mild to moderate intensity), are the key measures of prevention ,20,21,22The levels of neurotransmitters are closely related to the motility of the gastrointestinal tract ,24. The Erysipelatoclostridium was relatively more abundant in constipated patients [Erysipelatoclostridium in the constipated mice decreased significantly [Erysipelatoclostridium was significantly increased after ROS treatment in constipated mice  belonging to Ruminococcaceae were significantly increased after ROS administration , and ROS included 94% raffinose. The information on the kit and loperamide is consistent with our previous study . LoperamThere are 130 male Kunming mice (20 ± 2 g) that were obtained from the Guangdong Medical Laboratory Animal Center . The mice were maintained under temperature 25 ± 2 °C, humidity 50 ± 5%, 12 h light–dark cycle, and the standard mouse food  and water were provided to feed mice. The Ethics Committee of South China Agricultural University (SYXK2014-0136) approved all the protocols, grant number 2017009.The low-, medium-, and high-dose settings of ROS  for mice were equivalent to 5, 10, and 20 times of the maximum recommended dose for humans (5 g/60 kg·bw) by regulations of China Food and Drug Administration.n = 10); after adaptive feeding for seven days, each of the five mice was placed in a separate cage. The specific experimental workflow for this assay was followed. The blank and model group were given distilled water for seven days ; the ROS groups were given different concentrations of ROS in the same manner, individually. The GI of each mouse was measured using previously described methods when the experiment ended [Fifty mice were randomly separated into five groups  suspended in distilled water was prepared to induce constipation. Each mouse was maintained in a single cage at the beginning of index measurement. At the end of the experiment, Tfbsd, Fn6h, and Fw6h collected during the six hours was measured for each mouse .Thirty mice were randomly divided into three groups after adaptive feeding for seven days. A slightly modified version of the method was adopted to establish the slow-transit constipation model , and theThe paired-end sequenced (2 × 250) was performed on the HiSeq 2500 platform for 16S rRNA sequencing. The detailed information of the sequencing procedures was shown in the previous study .The contents of acetylcholine Ach, SP and VIP in the serum were measured using ELISA kits, and the level of NO in the serum was determined by the nitrate reductase method.For candidate target of ROS, the databases for TCMSP , Swiss TBased on the overlapped targets, GO terms and KEGG pathway enrichment analysis were performed by using STRING bioinformatics resources . CytoscaThe core targets were screened under the condition that the ‘Degree’ of the node in the PPI network and R-T-P network was greater than the mean values. The protein sequences of the mouse  were collected from the UniProt. As no crystal structures were available for them, SWISS-MODEL was used to construct the homology models for them ,55,56. TAutoDock vina software (version 1.1.2) was performed to verify the binding affinity of candidate targets to ROS by molecular docking simulation. The SDF format of ROS was downloaded from Pubchem. ROS was charged and energy-optimized using Chem 3D software (version 15.1), and the structures were saved in MOL 2 format. The pdbqt format of ROS was obtained by AutoDockTools 1.5.6. The binding energy was calculated, and the binding energy ≤ −5 kcal/mol was regarded as an effective combination. The conformation with the lowest binding energy was screened, and PyMOL 2.3 was used for visualization.p < 0.05 is considered as statistical significance.The data are shown as the means ± SD for each group. For analysis of GI and defection, multi-group comparisons of mean values were performed using the one-way ANOVA with LSD, while the Kruskal–Wallis test was used when the variance was not uniform. SPSS 20.0 was used for data analysis. For 16S rDNA sequencing analysis, the Meta-stat 1.5 was used for statistical analysis of the significance of species differences between the two groups based on the Fisher exact test. A The administration of ROS effectively prevents constipation in mice by evaluating the gastro-intestinal transit rate, the number and weight of defecation events in six hours, and time to the first black stool defecation. The underlying mechanism may be through regulating the intestinal flora , reducing the serum levels of inhibitory neurotransmitters (VIP). Based on the network pharmacology, the targeted screening strategy further revealed 29 potential anti-constipation targets of ROS, and the network analysis showed Akt1, Stat3, Mapk8, Hsp90aa1, Cat, Alb, Icam1, Sod2, and Gsk3b can be regarded as the core targets in the ROS regulatory network, which mainly involved the inflammation, immunity, and antioxidation. In a word, ROS is a hopeful candidate for use as an anti-constipation prebiotic, and the combination of gut microbiota, enteric nervous system, and targeted screening strategy provide a novel perspective of ROS and the oligosaccharide to which it belongs.


Chlamydia trachomatis are the most common sexually transmitted bacterial infections in the world. Individuals younger than 25 are the most affected due to the patterns of their sexual activity. In most individuals, these infections do not produce symptoms. Thus, affected individuals usually are not prompted to seek care, and most cases can only be detected through screening. Screenings for chlamydia in United States schools have given an indication of how many adolescents in the population might have chlamydia. In this study, we assessed how quickly chlamydia is acquired within the adolescent population. We determined that 14–19-year-old adolescents are acquiring chlamydia at a pace of 6.6 cases per 100 person-years for boys and 11.9 cases per 100 person-years for girls. Male and female students are acquiring chlamydia within 10 and within 7 months, respectively.After a pathogen that causes disease has been introduced in a human population, an understanding of that disease in the population depends on knowing how many people in the population have the disease , how fast the disease spreads from one person to another within the population (incidence), and how long the disease remains in an individual once acquired (duration). Infections caused by C. trachomatis infection, and provide strong evidence in support of annual chlamydia screening for adolescent boys.Prospective cohort studies of sexually transmitted infections (STIs) are logistically impractical owing to time and expenses. In schools, students are readily available for school-related follow-ups and monitoring. Capitalizing on the logistics that society already commits to ensure regular attendance of adolescents in school, a school-based STI screening in New Orleans made it possible to naturally observe the occurrence of chlamydia and to determine its incidence among 14–19-year-old adolescents. Among participants screened repeatedly, we calculated incidence rates, cumulative incidence, and incidence times. Male (n = 3820) and female (n = 3501) students were observed for 6251 and 5143 person-years, respectively, during which 415 boys and 610 girls acquired chlamydia. Incidence rates per 100 person-years were 6.6 cases for boys and 11.9 cases for girls. In multivariable analysis, the adjusted hazard ratio was 5.34 for boys and 3.68 for girls if the student tested positive for gonorrhea during follow-up, and 2.76 for boys and 1.59 for girls if at first participation the student tested positive for chlamydia, and it increased with age among boys but not among girls. In joinpoint trend analysis, the annual percentage change in the incidence rate was 6.6% for boys  and 0.1% for girls . Annual cumulative incidence was 5.5% among boys and 8.6% among girls. Median incidence time was 9.7 months for boys and 6.9 months for girls. Our findings can be used to refine assumptions in mathematical modeling and in cost analysis studies of  Chlamydia trachomatis is the most common notifiable disease reported in the United States. In 2019, there were 1,808,703 cases reported to the Centers for Disease Control and Prevention (CDC), which, excluding cases with unknown gender, included 644,337 male and 1,160,470 female cases [In the study of the distribution of disease in specified populations, two measures are used to determine the occurrence of disease: prevalence, which counts the number of cases of existing disease in the population at a given time, and incidence, which determines the pace at which a new disease develops within the population ,2,3. InfC. trachomatis can result in urethritis in men and cervicitis in women, with repeat episodes, which are particularly common in adolescents and young adults, increasing the risks of serious complications in women, including pelvic inflammatory disease, chronic pelvic pain, ectopic pregnancy, susceptibility to co-infection with human papillomavirus, and tubal infertility [C. trachomatis that were acquired in 2018 in the United States through sexual contacts by individuals aged 15 to 24 was estimated to be approximately $0.5 billion, 91% of which incurred by women [Infections caused by ertility ,6. The lby women . C. trachomatis and Neisseria gonorrhoeae were implemented in United States public high schools in New Orleans, New York City, and Philadelphia, in addition to smaller-scale screenings in Chicago, Detroit, Pittsburg, San Francisco, and Washington, DC [With the development in the early 1990s of nucleic acid amplification tests (NAATs) for the detection of sexually transmitted infections (STIs) in laboratory specimens that could be obtained without the need to perform clinical examination , programgton, DC . These pC. trachomatis infection in the general adolescent population [The purpose of the present study was to determine the incidence of chlamydia among students tested during 10 school years in the New Orleans STI screening program ; approxipulation ,3,12,13 pulation .Between school years 1995–1996 and 2004–2005, students in a New Orleans public school district were offered chlamydia screening using NAATs with urine specimens ,11. All N. gonorrhoeae [Two opportunities of screening were offered during each of the first two years and one opportunity afterward , for a torrhoeae . A positorrhoeae ,11.During the 10-year period, a cumulative 35,974 students enrolled in at least 3 and as many as 13 participating schools . Of thesAmong the students eligible for analysis, we calculated incidence rates, cumulative incidence, and incidence times ,2,3. Between 1995–1996 and 2004–2005, students were available for testing during different and unequal times because each year a class completed and another class started high school. During the years they enrolled in participating schools, students could take part or not take part in one or the other screening opportunity, students could transfer to or from participating schools, and a school that participated for a year or years may not have participated at other times. To fully account for the unequal times each student contributed to the screening program, person-time denominators were used for calculating incidence rates . For stuCohorts of students tested during 2, 3, and 4 consecutive school years could be identified in the screening program database . For theBecause during follow-up only the first positive test was used to determine incident infection, observation for students who acquired chlamydia was censored at the time they were estimated to become infected. Thus, the numerators for incidence calculations were the numbers of students who acquired chlamydia, which were also identical to the numbers of incident infections in this report . Among students who acquired chlamydia, incidence time was the number of days between the date of the initial negative test or the date of treatment for a previous positive test and the acquisition of the incident infection , i.e., tp-value of 0.05.Analyses were performed separately by gender. For each gender, we computed binomial proportion 95% confidence intervals (CI) of incidence estimates using standard methods. Probability of incident chlamydia was estimated using the time-to-event analysis and summarized in a Kaplan–Meier curve . We used multivariable Cox proportional hazard regression models to assess the independent contributions of risk factors on chlamydia incidence during follow-up   , and 11.Among male students, incidence rates were lower and similar for the 14- and 15-year age cohorts (3.4–4.8 cases/100 person-years) and higher and similar for the 16- through 19-year age cohorts (8.1–9.4 cases/100 person-years) . There wStudents whose chlamydia test results at first participation were positive had significantly higher incidence rates compared to those whose chlamydia test results at first participation were negative  vs. 6.2  cases per 100 person-years, IRR = 3.6, Among male students who had a positive gonorrhea test result during follow-up, the incidence rate was 36.9 cases/100 person-years , and the incidence rate was 6.0 cases/100 person-years  among male students who did not test positive for gonorrhea (IRR = 6.1) . Among fMale students’ incidence rates increased from 5.3 to 7.1 and 8.4 cases/100 person-years when testing was exclusively by the Abbott LCx assay, by both the Abbott and the BD ET assays, and exclusively by the BD ET assay, respectively . Female In both male and female students’ multivariable analyses , the risp = 0.091) for boys and 0.1%  for girls : 12.8 months), decreasing with age; 6 months in cohorts 17 years old and older; and 6.6 months if at first participation the student tested positive for chlamydia . In femaTo determine the incidence of a disease, it is necessary to follow a cohort of individuals initially free from the disease and observe the occurrence of the disease within the cohort. Cohort studies, particularly in their prospective designs, typically involve following large numbers of individuals for many years to observe the occurrence of the disease, which make them time-consuming and expensive ,2,3. TheThe individual longitudinal data gathered from participants in the New Orleans school-based STI screening show a high incidence of chlamydia, especially considering that participants were healthy adolescents who were not seeking care at the time of testing. The risk of acquiring chlamydia was significantly higher among girls than among boys, and increased with age among boys and with follow-up time in both genders. Sexual exposure (gonorrhea infection detected during follow-up and a positive chlamydia test result at first participation) was a stronger risk factor in predicting incident chlamydia than demographic factors. Incidence estimates in this study may be conservative because all students were tested for STIs regardless of sexual activity, which implies that some participants may not have been sexually exposed between screening .C. trachomatis in this screening program was by a commercially available NAAT. The first 444 specimens were tested by the Roche Amplicor chlamydia assay [Testing for ia assay , then byia assay ,20,21. Aia assay , which, ia assay . In the C. trachomatis in the general adolescent population. Findings from this study also contributed evidence that can be assessed for guiding recommendations for screening male adolescents for chlamydia.The observation of 3820 male students developing incident chlamydia over 6253 person-years added data to our understanding of the epidemiology of genital infection with C. trachomatis in the adolescent population [C. trachomatis among adolescent boys and girls are not equal. These findings provide parameter values for mathematical modeling studies that estimate population distributions of genital C. trachomatis infections [The minimal female-to-male ratios of chlamydia incidence in this study are 1.51 for incidence rates and 1.17 for annual cumulative incidence, when the ratios are determined by dividing the girls’ lower limits by the boys’ upper limits of their 95% confidence intervals. These findings have direct implications on how estimates of population distributions of chlamydial infections are calculated. Because data for determining chlamydia incidence are usually unavailable, it became customary to estimate population incidence using available prevalence data and by assuming that infections are of known duration in a steady-state population ,23,24. Upulation ,13. Incifections ,26 or thfections .This longitudinal study also created the opportunity of estimating time to the acquisition of chlamydia. Male and female students aged 17 to 19 acquired chlamydia at a median time of 6 months, with incidence time declining sharply from ages 14 to 17 in both genders. In 2005, 22%, 72%, and 128% more 10th, 11th, and 12th graders in the United States, respectively, reported four or more lifetime sex partners compared to 9th graders . The shaBy estimating the time of infection at the midpoint between the most recent negative test and the following positive test , person-This study assumed a complete tally of infections acquired during follow-up. However, it remains possible that chlamydia could have been diagnosed outside of our program among study subjects that were not counted as incident infections in this analysis. Such incidents were nonetheless unlikely to have occurred in a well-defined group of students to affect trends over time. Additionally, such incidents could only add to an already high incidence in this population.Because consent was required each year, students could not participate during the year(s) for which they did not have consent even if they wanted to, possibly accounting for testing gaps among some study subjects . Longer C. trachomatis. Incidence of chlamydia among adolescent girls is not equal to incidence among adolescent boys [A sufficiently large number of high school adolescents were observed developing chlamydia during a 10-year period in New Orleans, which contributed data that can be used to refine assumptions in mathematical modeling and in cost analysis studies of genital infection with ent boys ,22,24. Tent boys . While ient boys ,29,30,31


Chest pain is a common disease in children. Most cardiac specialists use echocardiography to evaluate the etiology of and screen for children’s cardiac chest pain. We analyzed the etiology and echocardiography results of children with chest pain in pediatric cardiology clinics, clarified the disease spectrum and evaluated the diagnostic value of echocardiography in screening cardiac chest pain in children.P < 0.05). In the preschool group, most of the patients were girls (51.4%), while in the school-age group and the adolescent group, most of the patients were boys (P < 0.05). Among the children  who underwent echocardiography, 108 children had positive results, and 3,097 children had negative results. Among the 108 positive results, 10 cases of cardiac diseases were related to chest pain. The sensitivity, specificity of echocardiography in the diagnosis of cardiac chest pain were 6.7, and 96.9%, while the positive predictive value and negative predictive value was 12.96 and 93.67%, respectively.The clinical data of children with chest pain aged younger than 18 years who admitted to the pediatric cardiology clinic of Beijing Anzhen Hospital between 2005 and 2019 were analyzed. The patients were divided into three groups, including the preschool group, the school-age group and the adolescent group. Total 3,477 children were enrolled in this study. 232 (6.7%) patients were caused by cardiac diseases and chest pain was of non-cardiac origin in 3,245 patients (93.3%). The incidence of non-cardiac chest pain in the adolescent group was significantly lower than the other two groups, respectively (91.4 vs. 94.9 vs. 94.3%, In children with chest pain who are admitted to pediatric cardiology clinics, chest pain is mostly benign and rarely due to cardiac diseases. The use of echocardiography in evaluating cardiac chest pain in children is of little diagnostic value and leads to excess costs for patients and the health care system. Chest pain is common not only in cardiology clinics, but also in other pediatric clinics. Different from the disease spectrum causing adult chest pain, children with chest pain always are benign, and very few are caused by cardiogenic diseases –5. For pTo date, few studies have reported the disease spectrum of chest pain in children who are referred to cardiology clinics , 6. StudFor children with chest pain as a chief complaint, doctors often let them undergo many auxiliary cardiac examinations to exclude cardiac disease when they are admitted to the cardiac clinic, such as electrocardiogram (ECG), echocardiography and Holter monitoring. Echocardiography is a non-invasive auxiliary examination which was used to assessment the structure and function of the heart and is widely used in cardiology clinics. Therefore, is echocardiography of high diagnostic value in assessing the etiology of cardiogenic chest pain in children? To date, no literature has been reported.This study analyzed the disease spectrum of children’s chest pain in pediatric cardiac clinics and evaluated the diagnostic value of echocardiography in screening children with cardiac chest pain.The clinical data of children with chest pain who admitted to pediatric cardiology clinic of Beijing Anzhen Hospital from 2005 to 2019 were analyzed. The disease was coded according to the 10th revision of the International Statistical Classification of Diseases and Related Health Problems (ICD-10). The exclusion criteria included that the clinical data of patients was incomplete. The study was approved by the Institutional Review Board oft Beijing Anzhen Hospital.The demographics, history of the present illness, past medical history, family history, and auxiliary exams of these patients were analyzed. According to the above content and results, we diagnosed the etiology of children with chest pain. And then based on the clinical diagnoses, we determined the disease spectrum of chest pain in children who were admitted to the pediatric cardiology clinic. Based on the cause of chest pain, we divided the patients into the cardiac chest pain group and the non-cardiac chest pain group. According to the age of the patient, we divided these patients into three groups, including the preschool group (3 years ≤ age ≤ 6 years), the school-age group (6 years < age ≤ 12 years), and the adolescent group (12 years < age ≤ 18 years). The sex distribution and the etiology of chest pain in different groups were analyzed. Patients with congenital adrenal insufficiency, systemic lupus erythematous, carnitine deficiency or juvenile rheumatoid arthritis were thought to have positive history of past illnesses. If the family member of the patients which had been diagnosed with pulmonary arterial hypertension, Marfan syndrome, cardiomyopathy, severe familial hypercholesterolemia or had sudden death, we thought the family history was positive.ECG reports were retrieved from the clinical medical records. Abnormal ECG showed pathological Q wave, deviation of the electrical axis, atrial enlargement, pathological ST-segment or T-wave changes, pre-excitation syndrome, high-grade atrioventricular block, frequent premature ventricular contractions, ventricular hypertrophy and supraventricular tachycardia.The results of other auxiliary exams including myocardial enzyme, echocardiography, chest X-ray and Holter monitoring results were analyzed according to the clinical reports.Until now, children with myocarditis, pericarditis, large ventricular septal defect, anomalous origin of the coronary artery, cardiomyopathy, pulmonary arterial hypertension, aortic dissection, moderate or severe left ventricular outflow tract obstruction, pulmonary embolism or arrhythmia were thought to have cardiac chest pain –5, 11–16Children with chest pain without a history of cardiogenic diseases were divided into the echocardiography group and the non-echocardiography group according to whether they underwent echocardiography. The etiology of children in the echocardiography group was analyzed, and the diagnostic value for cardiogenic chest pain was evaluated according to the results of echocardiography.x̄ ± SD). The χ2 test was performed to analyze the sex distribution and causes of chest pain. For the cardiogenic chest pain cohort, we calculated the sensitivity, specificity, positive and negative predictive values, and positive and negative likelihood ratios for echocardiography. The 95% CI for the sensitivity and specificity was computed by the Wilson method. A P < 0.05 was considered statistically significant.For statistical analysis, SPSS 20.0 was used. Count data are expressed as the frequency (percentage), and measurement data are expressed as the mean ± standard deviation (P < 0.05)  reported a chief complaint of chest pain, including 2,083 boys (59.9%) and 1,394 girls (40.1%). Girls (51.4%) were more common in the preschool group, while boys were more common in the school-age group (61.1%) and adolescent group (62.2%), and the sex distribution difference in the three groups was significant ( < 0.05) .P < 0.05) (232 patients (6.7%) were diagnosed as cardiac chest pain, and 3,245 patients (93.3%) were diagnosed as non-cardiac chest pain. In children with non-cardiac chest pain, 1,813 cases (52.1%) were idiopathic (unknown cause), 1,071 (30.8%) cases were skeletomuscular diseases, 348 (10.01%) cases were respiratory diseases, ten cases (0.3%) were gastrointestinal diseases, two cases (0.06%) were mental diseases, and one case (0.03%) was other condition . In the  < 0.05) .n = 166, 71.6%), followed by myocarditis , pulmonary arterial hypertension , Kawasaki disease , frequent premature ventricular contractions , dilated cardiomyopathy , pre-excitation syndrome , anomalous origin of the left coronary artery , and coronary-pulmonary arterial fistula .In 232 patients with cardiac chest pain, suspected myocarditis was the most common cause , followed by slipping rib syndrome , costochondritis , and straight-back syndrome .In 1,071 patients with skeletomuscular diseases, precordial catch syndrome was the most common cause , followed by pneumonia , acute asthmatic bronchitis , asthma , pleuritis , chronic cough , emphysema , and pulmonary tuberculosis .In 348 patients with respiratory diseases, bronchitis was the most common disease , followed by gastritis , esophagitis , and constipation .In ten patients with gastrointestinal diseases, gastroesophageal reflux was the most common cause , followed by musculoskeletal diseases (30.8%),respiratory diseases (10.01%),gastrointestinal diseases (0.3%), psychogenic diseases (0.06%) and other diseases (0.03%). In our study, the proportion of cardiac diseases in children as the cause of chest pain was only 6.7%, extremely low compared with other causes. Our result was consistent with Hanson CL’s, which showed that only one patient (0.7%) had a cardiac cause in children with chest pain admitted to the pediatric cardiology clinic . The proAbout the demographic characteristics of children with chest pain, some literatures have reported that female children with a mean age of about 10 years are often have chest pain , 20. OthEchocardiography is one of the most widely used, safe, non-invasive and cost-effective auxiliary examination in cardiovascular medicine . Most ofIn clinical practice, owing to echocardiography’s availability and non-invasive nature, it was often used by pediatric cardiologists to evaluate chest pain in children. Over the past 20 years, the application of echocardiography examination has grown significantly in both pediatric and adult settings, this trend is in accordance with an increase in the use of all diagnostic imaging in the United States –32. In 2Among the patients we enrolled in the study, 31 patients had a history of cardiac disease. In 3,446 patients with no history of cardiac disease, 3,205 patients underwent echocardiography. Based on the results of echocardiography, we found that the sensitivity, specificity, positive predictive value and negative predictive value of echocardiography for the diagnosis of cardiac chest pain were 6.7, 96.9, 12.96, and 93.67%, respectively. The sensitivity and positive predictive value are low, and the specificity and negative predictive value are high, indicating that the use of echocardiography in evaluating cardiac chest pain in children is of little diagnostic utility and contributes to additional costs for patients and the health care system. This is basically consistent with previous literature reports . TherefoIn this large retrospective study, we found that among preschool children with chest pain, girls were dominant, while for school-age children and adolescents, chest pain is more common in boys. The most common cause of chest pain in Chinese children admitted to pediatric cardiology clinic is idiopathic chest pain, and very few children suffer from chest pain caused by cardiac diseases. Echocardiography has low sensitivity and positive predictive value for the evaluation of cardiac chest pain in children, and it is of little diagnostic utility and contributes to additional costs for patients and the health care system. For the evaluation of children’s cardiac chest pain, a comprehensive analysis should be performed according to a child’s clinical manifestations, past medical history, physical examination, an appropriate auxiliary examination and family history should then be selected to identify and clarify the cause, which can not only save clinical resources but also reduce the economic burden on children and their families.The limitations of this study including: (1) This study was a single center study, which only represents the case composition of a single center. And the relevant data in this study might be affected by the hospital size, patients’ source, first line specialty and other aspects. In the future, to more accurately evaluate the disease composition spectrum of chest pain in Chinese children who are treated in pediatric cardiology clinics, multicenter and large sample research studies should be conducted for analysis. (2) This study was a retrospective study. Some data were missing, which may affect some results. In the future, we will perform further prospective research to clarify the reasonable application time of echocardiography in the diagnosis of cardiac chest pain in children.The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.The studies involving human participants were reviewed and approved by Beijing Anzhen Hospital Ethics Committee. Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin.HD, LC, and GL conceptualized the research hypothesis and analyses. LC researched the data, performed all of the statistical analyses, and wrote the manuscript. XL reviewed and edited the manuscript. GL obtained informed consent and checked 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.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.


ADRB2) gene and has a relationship with obesity; however, this substitution’s effects on food preferences are unclear. Therefore, we determined this relationship among healthy young adults . To evaluate food preferences, four categories of food  along with high-fat foods were evaluated using a self-reporting questionnaire. Male (n = 26) and female subjects (n = 26) were genotyped for the polymorphism and further divided into three groups . Preference for sour foods in the GG group was higher compared with that in the CC group in females (p < 0.05). When sweet foods were classified into low- and high-fat subgroups, preference for high-fat sweet foods in the GG group was higher than that for low-fat sweet foods in all subjects (p < 0.05). The degree of preference for high-fat foods in the GG group was higher than other groups for males (p < 0.05). These results suggest that ADRB2 polymorphism is associated with food preference. Understanding the relationship of ADRB2 substitution to food preference will be valuable for designing individualized anti-obesity strategies.The Gly16Arg polymorphism results in a G to C nucleotide mutation in the human beta 2-adrenergic receptor ( Obesity significantly decreases one’s overall life expectancy and health-related quality of life ,2. It isBecause the consumption of palatable foods is an instinctive behavior that is required for maintaining homeostasis, and may satisfy and relax individuals mentally , it is nThere is limited information regarding the effect of genetics, age, and sex on the sensation of taste and the relationship between taste preference and perception in humans , althougIt is well-known that intake of food is largely regulated by two interacting pathways in the brain. Specifically, one pathway determines food reward and causes the sensation of delight and satisfaction from eating foods or hedonic overeating, whereas the other controls metabolic homeostasis by integrating signals from peripheral tissues with the autonomic nervous system and hormonal system ,22,23. IHigh-fat foods are an indispensable source of energy and include lipid-soluble vitamins and essential fatty acids, such as vitamin A . Due to In addition to its texture-based sensation in the oral cavity, fat has recently been shown to elicit gustatory cues . AccordiIn this context, removing the gustatory lipid receptor, CD36, results in a considerable decrease in fat preference in mice . This imADRB3) and taste preference. The results indicated that a polymorphism in ADRB3 (Trp64Arg) may be associated with fat preference [Obesity is not only associated with environmental factors but also strong genetic factors ,41. Of teference .ADRB2, play a major role in regulating energy expenditure [ADRB3, the beta 2-adrenergic receptor (ADRB2) is a G-protein-coupled receptor family member, which is made up of seven transmembrane domain-containing proteins. ADRB2 is composed of a single 413-amino acid residue-long peptide chain. This receptor is encoded by a single intronless gene, which is found on the distal section of the long arm of chromosome 5 (5q32–q34). Although ADRB2 is expressed in bronchial smooth muscle cells, cardiomyocytes, and vascular smooth muscle cells, it is also expressed in fat cells [ADRB2 regulates the catecholamine-induced adenylate cyclase activation via the action of G proteins [Catecholamines and their corresponding receptors, including enditure . Similarat cells . ADRB2 rproteins ,43. The proteins ,45.ADRB2 gene may be potential genetic factors that explain, in part, the process of human obesity and related traits [ADRB2 gene. Of these, the Gly16Arg polymorphism of ADRB2 is a glycine/arginine change at codon 16. A SNP with a G/C missense mutation in the 46th position of the ADRB2 gene, from GGA to CGA in the ADRB2 protein (reference SNP number: rs1042713) and the 16th amino acid (Gly) is located in the N-terminal extracellular domain of the ADRB2 receptor [ADRB2 is functionally relevant and thus considered important. Interestingly, this polymorphism may alter the cellular trafficking of ADRB2 itself and receptor desensitization [ADRB2 isoform with Arg16 underwent increased downregulation as compared with the ADRB2 isoform with Gly16. This finding was consistent with the observed diminished cyclic adenosine monophosphate response to subsequent isoproterenol treatment after prolonged isoproterenol treatment [Polymorphisms in the d traits ,46. Varireceptor . The gentization ,48,49 intization . In factreatment . Screenireatment ,52; howereatment ,53. In areatment .ADRB2 polymorphism and obesity-related metabolic disorders, studies have shown conflicting results and the underlying mechanisms are still unclear. In some studies, the Gly16Arg polymorphism in ADRB2 was associated with obesity and body mass index (BMI), but the association was not found in others [ADRB2 Gly16Arg (G/C) polymorphism and fatty food preference, which may be associated with the amount of energy intake, because of the essential role of this receptor in fat metabolism. Thus, we examined the effect of the ADRB2 Gly16Arg (G/C) substitution on food preferences using data acquired from self-reporting questionnaires and genotyping of young and healthy Japanese adults (n = 52).With respect to the genetic relationship between this n others ,46. HereInclusion criteria were for young Japanese adult participants (20 to 35 years of age) that were non-smoking and non-medicated, who were healthy when they participated in the testing. Enrollment was also restricted to individuals who achieved a normal saliva flow rate of 1.0 mL/min and 0.1 mL/min during gum-chewing and resting states, respectively . Race anADRB2 Gly16Arg (G/C) polymorphisms, a test for saliva flow rate for the first 5 min during resting and mastication to evaluate salivation ability, and the evaluation of basic characteristics for each subject. The weight, height, body fat percentage , and body mass index (BMI) of each subject were measured with body composition monitors  and a handcrafted stadiometer.Participants attending the laboratory at the Tohoku University Graduate School of Dentistry filled out a self-reporting questionnaire on the condition of their health, lifestyles, and eating habits, including various preferences of taste. The participants took part in tests including genotyping of ADRB2 Gly16Arg (G/C) polymorphism was examined by extracting and analyzing the DNA from each participant enrolled in the study. In detail, to clarify the genotypes  of the ADRB2 Gly16Arg (G/C) polymorphism, a buccal swab from each participant was acquired with a sterile cotton swab . Genotyping was outsourced to EBS  as previously described [The genotype of the escribed . GenomicSalivation in each participant, in terms of an average saliva flow rate, was determined as previously described . The salADRB2 Gly16Arg (G/C) polymorphism and self-recognition of primary taste preference , we used self-reporting questionnaires. In detail, primary taste preferences were collected using a self-reporting questionnaire that was based on questionnaires of previous studies [http://fooddb.mext.go.jp/ (accessed on 13 December 2021)) that shows the fat content contained in each food as follows: (a) low-fat sweet foods (yokan (azuki-bean jelly), manju , and candy with fat contents of 0.2–0.3 g/100 g, 0.2–3.1 g/100 g, and 0 g/100 g, respectively), and (b) high-fat sweet foods ) with fat contents of 2.0–13.6 g/100 g, 34.1–40.4 g/100 g, and 14.7 g/100 g, respectively. The subjects were also asked to rate their degree of greasy (high-fat) food preference on a scale of 1–4, which was previously used in a study by Asano and Watanabe [To evaluate the relationship between the  studies ,36,57. TWatanabe ,36.p-values < 0.05 were defined statistically significant.Statistical analyses were conducted using SigmaPlot 14.5 , SPSS statistics, version 22.0 , or Excel . Differences between group means were identified using appropriate tests as indicated. The relation of age and BMI with each type of food preference was assessed via multiple regression analysis. The allelic and phenotypic frequencies were calculated, and then the Hardy–Weinberg equilibrium was tested for the polymorphism. The chi-square test was employed to compare the frequencies. In each test, n = 26 male; n = 26 female) adults  were analyzed. BMIs ranged from 17.1 to 34.4 kg/m2. No significant difference was evident in the average BMI between male and female subjects. Regarding salivation potential, all subjects enrolled in the present study (n = 52) exhibited a normal saliva flow rate that was greater than 0.1 mL/min and more than 1.0 mL/min during the resting and masticating states, respectively.p > 0.05). In addition, the average degree of preference for each primary tastant was also not significantly different between the two subgroups , we compared the male and female subgroups with data acquired from self-reporting questionnaires. As shown in p > 0.05; ADRB2 GG subgroup, n = 7 in male subjects; n = 6 in female subjects), GC genotype , and CC genotype . To investigate the effect of the ADRB2 Gly16Arg (G/C) substitution on body fat and BMI, we compared the average values among the three subgroups in male and female subjects. As indicated in ADRB2 GG, GC, and CC subgroups (p > 0.05) for either sex.As shown in ADRB2 genotypes, there were significant differences in preference for pickled Japanese plum  and grapefruit  in male subjects , grapefruit , and coffee  in female subjects. However, based on the summarized preference degree for each primary tastant  in each genotype subgroup of the male subjects, no significant difference was observed in preferences for the foods of each taste (p > 0.05) as shown in p < 0.05; GC vs. CC, p < 0.05).Among the subjects . In addiADRB2 GG subgroup only, but not in the other genotype subgroups of ADRB2 , but not in the ADRB2 CC subgroup  polymorphism and the self-recognition of taste preferences for greasy (high-fat) foods among the subjects, we focused on a specific question included in the self-reporting questionnaire , multiple linear regression analysis was performed using the data acquired from the questionnaire in the present study. No significant association was observed between each type of food preference and age or BMI in both male and female subjects .ADRB2 Arg16 allelic frequency (see Introduction for details). Additionally, the observed genotype frequencies of the Gly16Arg polymorphism of the ADRB2 gene in both male and female subjects were in accordance with the Hardy–Weinberg equilibrium, suggesting that the observed genotype frequencies in both males and females represent those of the entire population. Thus, we analyzed the effect of the Gly16Arg (G/C) substitution of ADRB2 on preferences of taste with data from both male and female subjects and found that the preference for sour food was significantly higher in the ADRB2 GG group than that in the ADRB2 CC group, but only for female subjects. When sweet foods were classified into low- and high-fat subgroups, the preference for high-fat sweet foods was higher than that for low-fat sweet foods in the ADRB2 GG group for both male and female subjects. Furthermore, we also found that the degree of preference for high-fat foods was significantly higher in the ADRB2 GG group than that in the ADRB2 CC group, but only for male subjects.In the current study , the frequency in male and female subjects was 44.2% and 51.9%, respectively, which was consistent with the previous findings on the ADRB2 mediates insulin resistance, cardiorespiratory fitness, and obesity [ADRB2, which appears to be of significant importance to ADRB2 function, at least in fat cells, but less so for obesity, may induce a significant decrease in agonist affinity for ADRB2 and/or a significant increase in ADRB2 downregulation [ADRB2 may also be associated with altered glucose metabolism [ obesity . It has  obesity ,59. The gulation ,50. For tabolism . HoweverADRB2 Gly16Alg (G/C) polymorphism and taste preferences in healthy and young male and female subjects. As described above, our results clearly show that there is a specific association of the ADRB2 Gly16Arg (G/C) substitution with average degree of sour food preference in females, but not in males, as well as the average extent of high-fat preference in males, but not in females. Preferences for other basic tastes  were not apparently changed by this alteration. These results suggest that the difference in the degree of affinity of the agonist to ADRB2 and/or in cellular trafficking inducing downregulation of ADRB2 itself (this event might be involved in determining the genotype-dependent lipolytic activity of ADRB2 in fat cells and changing the energy expenditure of the human body) between the GG and CC genotypes for this polymorphism may subsequently induce alterations in taste preferences, rather than that of BMI, at least in a complex and sex-dependent manner, despite an unknown underlying mechanism. In particular, it is unclear why there were no significant differences in fat preference among the three genotype subgroups of the ADRB2 polymorphism in females, but not males.This is the first study to characterize significant associations between the ADRB2 activity and its location in cells (that may subsequently affect the energy expenditure of the human body) on fat preference to some extent in female, but not male subjects.Previous studies indicated that fat preference can be affected by female hormones in mice and rodents ,26. ThusInterestingly, as described above, we also found that in male but not female subgroups, similar to the observed sex-dependent difference in fat preference, the average high-fat sweet food preference was significantly higher than the low-fat sweet preference for the GG genotype, but not the CC genotype only in male subjects. In this context, it should be noted that foods with high-fat sweet tastes can cause a hyperpalatable sensation in the human brain ,61. TherADRB2 receptor activity , compared with those containing the CC genotype [In addition, with respect to the female-specific upregulation of sour-taste food preferences in the subjects with a GG genotype, we should note the following two points: (i) the sour-taste quality can occasionally be a sign of citric acid, which is included in a variety of sour foods, such as grapefruit, oranges, and lemons. In addition, citric acid is a pivotal player in the tricarboxylic acid cycle in mitochondria, which is a sequence in a metabolic pathway that mediates the chemical conversion of carbohydrates, proteins, and fats into water and carbon dioxide to produce energy ; and  substitution affects fat and sour taste preferences was not clarified in the present study. With respect to the altered fat and sour preferences, it should be noted that ADRB2 is an indispensable mediator of fat metabolism [ADRB2 receptor activity and its distribution in cells with this polymorphism, followed by changes in fat metabolism in brown and white adipose tissue, may influence the principal pathways that control intake of food in the brain, such as pathways that regulate (i) metabolic homeostasis and (ii) food reward via an unrevealed mechanism. Therefore, further studies on the effects of gene mutations in ADRB2 on food preferences with respect to brain function on the metabolic regulation of the human body will contribute to reveal the precise mechanism through which the ADRB2 Gly16Arg variant affects preferences of sour and fat.Regardless of the above working hypotheses, the precise mechanism through which the tabolism , which itabolism . Thus, aADRB2 Gly16Arg (G/C) substitution on BMI, our results in addition to several other reports, failed to demonstrate a significant change in BMI associated with the Gly16Arg substitution [ADRB2 Gly16Arg polymorphism) can affect the homeostasis of human body weight [ADRB2. In addition, in this context, it should be noted that long-term moderate exercise training itself can significantly increase the gene expression level of ADRB2 in humans [In relation to the effect of the titution ,42,44,46y weight ,63,64. An humans . Second,ADRB2 Gly16Arg (G/C) polymorphism. Second, the self-reporting style of the questionnaire may have resulted in individual biases that may have affected the results. Our research group prepared the questions presented in Tables in the Japanese language. In particular, the participants received and responded to the Japanese language version of the questionnaire, rather than the translated English language version shown in the tables. Thus, it is possible that some bias may have occurred during translation. A third limitation is the effect of sex differences on taste preferences. Studies have demonstrated the influence of hormones on preferences of taste [ADRB2 Gly16Arg (G/C) polymorphism with taste preferences. Finally, the study subjects were non-smoking, non-medicated, Japanese young adults in good health and may not accurately reflect the entire population.The following study limitations should be considered. First, the small number of subjects may have resulted in low statistical power; however, this population was sufficient to show an association between preference of taste and the of taste ,66. Therof taste . Howeverof taste ,16,67,68ADRB2 in healthy, young, adult Japanese men and women. We concluded that sour and fat preferences were specifically associated with this polymorphism based on self-recognition, and the manner of association was different between males and females. These findings are essential, especially for the advancement of personalized medicine, as genetically altered preferences for both sour and fatty foods (that includes high-fat sweet foods) may be associated with the promotion of inadequate eating habits, such as overeating. This may contribute, in part, to regulating the risk of diabetes, severe obesity, or other lifestyle-related disorders in humans. Taken together, our results suggest that the ADRB2 Gly16Arg (G/C) polymorphism may have a significant correlation with the self-recognition of food preferences in healthy young adults via an unknown mechanism. Further research on the relationship between this polymorphism and fatty and sour food preferences will be highly valuable to identify the control mechanisms underlying the preferences and to develop tools for individualized medicine to analyze personal- and sex-dependent differences in sour and fatty preferences. This will contribute to the assessment of ADRB2-related functional shifts in sour and fatty taste preferences, which may be used to manage or prevent obesity promoted by multiple causative factors, such as this polymorphism.In the current study, we focused on the genetic relationship between taste preferences and the Gly16Arg (G/C) polymorphism of