Seroprevalence surveys provide estimates of the extent of SARS-CoV-2 infections in the population, regardless of disease severity and test availability. In Mexico in 2020, COVID-19 cases reached a maximum in July and December. We aimed to estimate the national and regional seroprevalence of SARS-CoV-2 antibodies across demographic and socioeconomic groups in Mexico after the first wave, from August to November 2020. We used nationally representative survey data including 9,640 blood samples. Seroprevalence was estimated by socioeconomic and demographic characteristics, adjusting by the sensitivity and specificity of the immunoassay test. The national seroprevalence of SARS-CoV-2 antibodies was 24.9% , being lower for adults 60 years and older. We found higher seroprevalence among urban and metropolitan areas, low socioeconomic status, low education and workers. Among seropositive people, 67.3% were asymptomatic. Social distancing, lockdown measures and vaccination programs need to consider that vulnerable groups are more exposed to the virus and unable to comply with lockdown measures. SARS-CoV-2 seroprevalence surveys provide estimates of the extent of prior infection in a population. In this nationally representative survey from Mexico, the authors estimate seroprevalence after the first epidemic wave at ~25%, with variation by region, age, socioeconomic status, and education level. In 2020, SARS-CoV-2 seroprevalence studies were conducted in various countries using representative country-level data. In the first COVID-19 wave (before June 2020), seroprevalence ranged from 1.4% in urban areas in Brazil to 17.1% in Iran .Supplementary Table Table Overall, 67.3% of seropositive participants were asymptomatic, 21.5% symptomatic and 11.2% pauci-symptomatic. The proportion of asymptomatic participants was similar by sex, but varied by age group: 82.4% among children and adolescents, compared to 63.9% among adults 20–39, 56.7% among adults 40–59, and 60.6% among adults 60 and older.Table We aimed to estimate the prevalence of SARS-CoV-2 antibodies in Mexico using a nationally representative sample. The estimated national prevalence of SARS-CoV-2 antibodies in Mexico between August and November 2020 was 24.9%. The highest seroprevalence was observed in the Peninsula region (42.9%), and the lowest in the Central North region (19.1%). Seroprevalence was higher among workers, low socioeconomic groups, low education and urban and metropolitan areas among adults. We also found that 67% of the seropositive cases were asymptomatic.14. Globally, few seroprevalence studies were collected in the second half of 2020. Between August and November 2020, we found seroprevalences that ranged from 4.3% in Florida to 59% in Leticia, Colombia5. The seroprevalence in Mexico City (19.6%) and in the State of Mexico (23.5%) was similar to the one observed in New York (17–23.3%) between August and September4. Other Mexican regions presented similar seroprevalences to Medellin (27%) and Bogotá (30%), but lower than Leticia (59%) and Barranquilla (55%) in Colombia5.The national seroprevalence in Mexico was similar to levels observed in cities in Colombia and some states in the US, collected in similar periods. Most seroprevalence studies were conducted during the first wave  and reported seroprevalences between 5% and 15% , when the surveillance system indicated a cumulative incidence of 586/100,000 inhabitants. In contrast, Mexico City had a 19.6% seroprevalence in August, when the surveillance system estimated a cumulative incidence of 1,067/100,000 inhabitants , disregard for mild and common symptoms, such as fatigue or headache, and bias due to report-by-proxy since symptoms for all household members were reported by the head of the household to reduce collection times.Asymptomatic cases are not regularly tested in the Mexican surveillance system; as a consequence, before this study we had no information about the proportion of the population positive to SARS-CoV-2 that experienced no symptoms. We observed a high proportion of asymptomatic persons, 67%, compared to Austria (20%), UK (20%), Spain (36%), and Iran (36%), but similar to Mexican governmental workers (59%), and people in Lima-Peru (56%) and lower than in China (82%)24, thus, we used our in-house validation with a random sample of all states in Mexico, but we only observed one false positive; thus, cross-reactivity did not seem to be a problem in Mexico.The present study has some limitations to be discussed. Data were collected in different time frames thus, seroprevalence across regions should be interpreted cautiously. As the serologic survey had a low response rate, we conducted a sensitivity analysis to evaluate potential sources of bias. We found that individuals with COVID-19 related symptoms were more likely to participate in the serologic sample, biasing the seroprevalence estimate upwards. After bias quantification, the seroprevalence at the national level was 23.8%, , although the confidence intervals overlapped with the original estimate 24.9% . False positives have been reported in areas where malaria is endemic25. Besides, implementing participatory interventions and designing evidence-based vaccination plans is key to protect the most vulnerable population. One year after the pandemic, we have the chance to address inequity and avoid widening the health gap that already existed in Mexico.In Mexico, the ongoing pandemic affected at least a quarter of the population by the end of November, 2020. Differences in seroprevalence suggest that workers, people with low educational level and living in urban and metropolitan areas were more frequently exposed to the virus. Mexico needs to address structural vulnerabilities and use this opportunity to rethink their public policies grounded on equality. This includes providing healthcare for infected people, food support, preserving jobs, and compensate for salaries’ reduction due to lockdown measuresThe 2020 National Health and Nutrition Survey (Ensanut) focused in understanding the effects of the pandemic on health, food security, dietary quality, and access to healthcare services in Mexico. The survey was conducted from August to November 2020 and used a probabilistic, multistage, stratified, and clustered sampling strategy to be representative of the national, regional, and rural/urban levels. Using this sampling strategy, 10,216 households were selected. At each household, an adult family member was asked to respond to a household questionnaire and a questionnaire on the use of health services and health status of each family member. All research procedures were approved by the ethics, research and biosafety boards from the National Institute of Public Health. The information in the field was collected on tablets through a capture system developed in the CSPro language version 7.5.0. Further details about the sampling strategy and external validity are available in Supplementary methods  and spike (S) proteins. This paper considers the seroprevalence of antibodies to the N protein, since S protein is still under analysis. Blood samples were centrifuged in the field to separate the serum and were frozen to be delivered to the National Institute of Public Health. Samples were then sent to the Institute for Epidemiological Diagnosis and Reference for analysis using the Roche Elecsys Anti-SARS-CoV-2pan-immunoglobulin immunoassay test . According to the manufacturer’s recommendations, samples were considered reactive using a threshold of ≥1.0 AU/ml. We validated the test using pre-COVID-19 serum samples as controls and serum samples from people with confirmed COVID-19 by RT-PCR that were obtained at least 22 days after symptoms onset, when the sensitivity of the antibody tests is the highest. The validation test showed a sensitivity of 92.02% (95% CI 88.57–94.50) and a specificity of 99.52% (95% CI 97.35 to 99.92). Further details about the in-house validation are available in Supplementary methods (section “Between March 2020 and today did you (or any family member) present any of the following symptoms?” Informants were presented with 14 options: cough, fever, headache, sore or burning throat, runny nose, red eyes, muscle or joint pain, difficulty breathing, shortness of air, chest pain, vomiting, diarrhea, loss of smell, or taste, and other symptoms. Supplementary Fig. 26. Participants that did not fulfill the definition but experienced at least one symptom were considered “pauci-symptomatic”14. Participants with no symptoms were considered asymptomatic.We constructed a variable of COVID-19 related symptoms to categorize participants in symptomatic, asymptomatic and pauci-symptomatic. The following question was answered by the head of the household in relation to their experience or that of their family members: Sex, age groups , geographic region and urbanization were used as main demographic characteristics. Participants were classified into nine geographic regions: Mexico City, North Border , Central-Pacific , Central-North , Center , Pacific-North , State of Mexico, Pacific-South , and Peninsula . Urbanization was divided into rural , urban  or metropolitan .Education was categorized according to the maximum level of completed studies into elementary school, middle school, high school, graduate, or postgraduate. Employment status was constructed for individuals 15 years and older based on the question: “During the past week, did you work at least one hour?”. Those who answered “yes”, were considered employees and further divided into formal, if they had social security healthcare access or informal if they did not. Those who answered “no” were then asked about their activities in the week prior to the survey; based on their response they were classified as “students” or “retirees”, while those who answered “looked for a job”, “have a permanent disability that does not allow me to work”, or “unpaid domestic work” were considered unemployed. A socioeconomic status index was constructed using household’ characteristics , own a car, number of household assets  and number of electrical devices . The index was constructed using Principal Component Analysis, with a polychoric correlation matrix. The first component explaining 50.1% of the total variability was selected with an eigenvalue of 4.0 and categorized into tertiles .The observed seroprevalence was calculated as:27Considering that the Elecsys test is imperfect, with 92.02% of sensitivity and 99.52% of specificity, we adjusted the seroprevalence as follows:To calculate confidence intervals, we simulated values of the observed prevalence using a normal distribution using as parameters the confidence intervals of the observed prevalence estimated from the survey and the confidence intervals of sensitivity and specificity estimated in supplementary methods . All analyses considered survey weights using the module “svy” from Stata 14.0 .We used sampling weights to adjust the seroprevalence for the selection probabilities and non-response rates to the serologic subsample. Weights were calculated as the inverse probability of selection, adjusted by non-response by post-stratifying the sample on region, sex, age group , so that the weighted sum of respondents in each stratum matched the total population estimated at the end 2020 by the National Population Council. Additionally, weights were adjusted to resemble the distribution of the reported chronic diseases in the household questionnaire. To estimate adjusted prevalence ratios we used Poisson regression models with robust variance29, to compare results to the adjustment made using our in-house validation.We performed a sensitivity analysis using test performance as reported by the manufacturer n = 36,024) and in the serologic subsample with valid results (n = 9464). We selected variables that could have been associated with seropositivity and with participation in the provision of a blood sample: age, sex, region, education, employment, having reported contact with a suspected case, having experienced a respiratory disease, and having experienced COVID-19 related symptoms. We used raking, a sampling balance method30, to replicate the distribution of key variables from the household questionnaire into the serologic sample. We used “symptoms” by region and age group  as the key variable on raking. After raking the distribution of variables was comparable between the household survey and the serologic sample, with the exception of the “students” category in the education variable (see supplementary Table We performed a sensitivity analysis to assess the potential impact of specific variables that could inform selection bias, considering a low response rate in the serologic subsample. We analyzed differences in socioeconomic characteristics between participants in the household questionnaire (Further information on research design is available in the Supplementary InformationReporting Summary


This paper presents a low-profile ultrawideband antenna for on-body wearable applications. The proposed antenna is based on highly conductive flexible graphite films (FGF) and polyimide (PI) substrate, which exhibits good benefits such as flexibility, light weight and corrosion resistance compared with traditional materials. By introducing flaring ground and an arrow-shaped slot, better impedance matching is achieved. The wearable antenna achieves a bandwidth of 122% from 0.34 GHz to 1.4 GHz, with a reflection coefficient of less than −10 dB, while exhibiting an omnidirectional pattern in the horizontal plane. To validate the proposed design, the wearable antenna with a profile of ~0.1 mm was fabricated and measured. The measured results are in good agreement with simulated ones, which indicates a suitable candidate for on-body wearable devices. Wireless Body Area Networks (WBAN) have been widely applied in many aspects of novel wireless communication systems, e.g., health monitoring, military, and entertainment ,2,3,4,5.Recently, great attention has been paid to the flexible graphite films (FGF), which has been considered as a promising candidate for flexible wearable devices. These advanced carbon material based films exhibit unique properties including high conductivity, flexural endurance, light weight and corrosion resistance, which are studied in previous works ,33.In this paper, we propose a novel flexible ultrawideband antenna for on-body wearable applications. As far as we know, the FGF is firstly adopted to replace traditional metallic materials in wearable antenna design for the sub-GHz region, which shows great superiorities, especially in flexibility, mechanical stability and light weight. The simulated and measured results prove that the proposed antenna achieves an omnidirectional vertical polarized pattern and impedance bandwidth from 0.34 GHz to 1.4 GHz, which operates within the Ultra High Frequency (UHF) band (300–3000 MHz) for military applications . Meanwhi6 S/m, close to the traditional metallic like copper (1.3 × 107 S/m). More importantly, the FGF is much lighter than the copper because the density of the FGF is 1.8 g/cm3, which is around a fifth of the copper density of 8.8 g/cm3 [In the field of wearable antenna design, various conductive materials are adopted to act as radiation structures such as conductive fabrics , copper .8 g/cm3 . The expIn addition, the textile fabric is a porous, anisotropic and compressible material, whose structures and electromagnetic properties can be affected severely by the surroundings , thus flw and g are the widths of the central conductor and the gap between the central conductor and the ground, respectively.Generally speaking, Microstrip antenna has a high quality factor (Q), which leads to a narrow bandwidth . Compare11 of the proposed antenna is shown in 11 < −10 dB), in which two resonances are observed at 0.39 GHz and 0.97 GHz. It is worth mentioning that the original antenna has realized the ultrawideband feature. The distribution plots of surface current at resonant frequencies above are given in All the simulations of the antenna are based on the ANSYS high-frequency structure simulator (HFSS). The simulated reflection coefficient SW1 and R1 (R2) represent the minor-axis and major-axis radii of the elliptical patch, respectively. It can be seen from To achieve a broader operating bandwidth, two rectangle slots are replaced with tapered slots by adopting flaring ground. As depicted in 11 for the fractional bandwidth from 109% to 122%. The variation of S11 with W2 is shown in W2 increases. However, when W2 is too large, the capacitance effect becomes weaker, which provides few contributions to impedance matching. Finally, we select the optimal value of 15 mm. Eventually, we obtain a flexible ultrawideband monopole antenna. 11 obtained from the proposed antenna exhibits that the operating bandwidth of Ant-3 has been effectively improved with the fractional bandwidth of 122%. The optimized geometrical parameters of the proposed antenna are listed in Here a solution is presented by inserting an arrow-shaped slot structure on the radiation patch. Note that the flaring ground and the arrow-shaped slot are adopted for the wideband impedance matching. The simulated surface current distribution of the proposed antenna (Ant-3) is plotted in R. By taking bending states of the body surface into account, we set the bending radii of R = 60 mm, 80 mm and 100 mm. As plotted in y-axis. R, which indicates a slight degradation of S11 around 0.5 GHz and a shift of resonant frequency to lower frequency as well. It proves that the impedance matching of the wearable antenna is well maintained compared with its flat condition.There is no doubt that the bending effects of the wearable antenna degrade the performance compared with its flat condition, and the wearable antenna performance will be also affected by unavoidable deformation even though the antenna is designed for a certain bending radius. In this design, the proposed ultrawideband wearable antenna is conformally integrated over a cylindrical foam with a radius of To study the impact of the loading positions of the proposed antenna, the antenna performance in free space, thigh area and shank area is simulated. As depicted in The simulated realized gain and radiation efficiency are illustrated in As shown in A novel ultrawideband antenna using highly conductive flexible graphite films (FGF) for on-body wearable applications is proposed, fabricated and tested. The ultrawideband characteristic is realized by modifying the planar monopole antenna using the flaring ground and the arrow-shaped slot. In addition, to satisfy some special performance of the wearable antenna, we adopt the FGF as the conductive material, which shows a good candidate for wearable devices. To the best of our knowledge, it is the first time to adopt the FGF to replace traditional metallic materials in wearable antenna design within the sub-GHz region. The fabricated prototype has only a profile of 0.1 mm, making it applicable for conformal and wearable applications. The measured results have demonstrated that the FGF antenna operates from 0.34 GHz to 1.4 GHz with fractional bandwidth of 122% while providing an omnidirectional vertical polarized pattern in the xoz plane although it is bent and working in the proximity of the human body. The proposed antenna has many superiorities such as low profile, favorable flexibility, light weight, ultrawideband, and omnidirectional beam pattern, which may be suitable for an on-body communication system. This work has also demonstrated that the FGF has great potential in wearable antenna design.


Obstructive sleep apnea (OSA) is a complex process that can lead to the dysregulation of the molecular clock, as well as 24 h rhythms of sleep and wake, blood pressure, and other associated biological processes. Previous work has demonstrated crosstalk between the circadian clock and hypoxia-responsive pathways. However, even in the absence of OSA, disrupted clocks can exacerbate OSA-associated outcomes . As we expand our understanding of circadian biology in the setting of OSA, this information could play a significant role in the diagnosis and treatment of OSA. Here, we summarize the pre-existing knowledge of circadian biology in patients with OSA and examine the utility of circadian biomarkers as alternative clinical tools.  The circadian system has been an integral part of evolution for predicting environmental changes and maintaining organisms’ health and fitness . Almost Sleep is a predominant circadian phenotype in humans. Disrupted sleep potentially dysregulates circadian rhythms, impacts multiple biological and physiological processes, and likely contributes to disease initiation or progression . In thisSince ~50% of genes are rhythmic in mammals ,20,21, tIn humans, circadian rhythms are maintained through a natural cycle of 24 h light and dark. Intrinsically photosensitive retinal ganglion cells (IPRGCs) in the human eye receive light and pass this information to the central oscillator (suprachiasmatic nuclei) located in the hypothalamus ,28. ThisAt the cellular level, the circadian clock functions as a transcriptional and translational feedback loop by self-regulating its own activity ,33. The Changes in these molecular and physiological rhythms often serve as diagnostic tools for disease.For decades, sleep–wake patterns, BP, melatonin, cortisol, and core body temperature have been extensively used as biomarkers for measuring circadian physiology in humans ,40,41,42As mentioned, fragmented sleep is a potential hallmark of OSA that occurs due to repeated episodes of arousals . DisruptCortisol is a hormone that exhibits circadian rhythm and is involved in BP regulation, immune responses, and metabolism. Sleep disruption influences hypothalamic–pituitary–adrenal (HPA) activity and increases cortisol secretion . AlthougOnly a limited number of studies have reported on body temperature in patients with OSA ,78,79,80There is an overlap in canonical clock genes and associated biological processes, such as inflammation, metabolism, cell cycle, apoptosis, DNA repair, redox signaling, and oxidative stress, in disease processes of multiple health conditions including cancer, dementia, metabolic, cardiovascular, and hypoxemic disorders ,82,83. OHIF-1α [CRY1 and PER2 [HIF-1α, PER, and CRY genes [A primary regulator of oxygen homeostasis in mammals is hypoxia inducible factor-1 (HIF-1). Under hypoxic conditions, HIF-1α heterodimerizes with HIF-β and binds to the E-box of hypoxia response elements to promote the expression of multiple target genes associated with cellular and biochemical processes . A few sHIF-1α . Interesand PER2 . As a feand PER2 , and CRYand PER2 ,93. One RY genes ,90. A nuRY genes ,96,97,98RY genes . This hyRY genes ,14.PER1 in patients with OSA. However, treatment with CPAP recovered PER1 rhythms similar to those in healthy controls [BMAL1, CLOCK, and CRY2 in patients with OSA. In this study, the authors correlated gene expression profiles with the AHI index, and their observations suggest that the reduction of CRY1 and PER3 levels at specific time points  may serve as predictors for those with severe OSA [BMAL1, PER1, CRY2, and DEC1 in patients with OSA. However, these altered expression patterns were not recovered by short or long-term CPAP treatments [CLOCK gene levels in patients with OSA. Furthermore, no recovery was observed after treatment with CPAP [Several studies have tried to specifically evaluate the responses of canonical clock genes in patients with OSA ,102,103.controls . A time vere OSA . Anothereatments . Additioith CPAP . AdditioThe diagnosis of OSA can be quite challenging and is often entirely dependent on the availability of sleep labs and portable sleep studies. There are no physical assessments that can be used to determine the severity of OSA and an overnight PSG is the only standard tool available for diagnosis. Although OSA has been recognized as a highly prevalent health condition, the field of diagnostics is limited and clinical focus is poorly developed ,44. UndiRodent studies have demonstrated that physiological oxygen fluctuations may alter circadian rhythms and the time-dependent expression of RNA and proteins in cells and tissues ,14,110. The development of biomarkers requires a system-level understanding of gene expression and its role in disease processes. So far, there are no time course studies available that specifically evaluate the circadian transcriptome, circadian proteome, or circadian metabolome in patients with OSA. The available circadian studies are mainly targeted to understanding the expression of canonical clock genes. Therefore, we believe it is worthwhile to further evaluate circadian transcriptome or metabolome expression profiles in patients with OSA and to develop time-dependent biomarkers for use in OSA-associated diagnostics and therapeutics. Here, we provide a strategy to develop circadian biomarkers for these purposes .There are two possible and less invasive methods for developing robust circadian biomarkers in humans, blood collection and skin biopsy. A recent study has shown that the skin clock is robust compared to the circadian clock in blood . HoweverTo develop circadian or time-dependent biomarkers for OSA, we recommend conducting time course studies with at least 3 or 4 h interval sample collections of blood or skin for a period of 24 h from patients with and without OSA. For a comprehensive study design, guidelines for the use of circadian -omics data have already been outlined by experts in the field of circadian biology . SamplesHow would we then use these biomarkers in real-world clinical applications? Once reliable biomarkers are established, we suggest measuring candidate biomarkers from at least two different times of the day based on the peak and trough patterns of their rhythmic expression. This strategy may lead to the identification of circadian biomarkers with significant clinical use for patients with OSA.


Upon activation, maternal platelets provide a source of proinflammatory mediators in the intervillous space of the placenta. Therefore, platelet-derived factors may interfere with different trophoblast subtypes of the developing human placenta and might cause altered hormone secretion and placental dysfunction later on in pregnancy. Increased platelet activation, and the subsequent occurrence of placental fibrinoid deposition, are linked to placenta pathologies such as preeclampsia. The composition and release of platelet-derived factors change over gestation and provide a potential source of predicting biomarkers for the developing fetus and the mother. This review indicates possible mechanisms of platelet-trophoblast interactions and discusses the effect of increased platelet activation on placenta development. Anucleate platelets and their polyploid megakaryocyte progenitors in the bone marrow are only found in mammals. However, when looking at the evolution of mammalian platelets, it becomes apparent that neither live birth nor the presence of a placenta accounts for the evolution of platelets. Although the biological advantage gained from the presence of polyploid megakaryocytes and anucleate platelets has not been identified, a possible role for platelets during pregnancy in mammals has been suggested.9/L (2.5th percentile) and 123 × 109/L , respectively, as thrombocytopenic [In humans, several large population-based studies suggest that the maternal platelet count decreases by approximately 10% in uncomplicated pregnancies at term, and rises again to basal levels postpartum , and is ytopenic . Pregnanytopenic . TherefoFor human pregnancy, both nonhemostatic as well as hemostatic platelet-dependent functions have been discussed to influence the placentation process. Nonhemostatic functions include promotion of trophoblast invasion by activating the chemokine receptor CCR1 in response to granule-stored CCR1 ligands such as CCL5  and MIP-1α (macrophage inflammatory protein-1α or CCL3) . MoreoveOn the other hand, the maternal coagulation system and platelets contribute to the generation of so-called perivillous fibrin-type fibrinoids, which can be detected at the surface of placental villi at sites where fibrinoid focally replaces the villous syncytiotrophoblast (SCT) . Fibrin-Due to the platelets’ ability to become activated in response to biochemical or mechanical stimuli, such as the exposure of the subendothelial extracellular matrix at site of vascular injury, they play an essential role in wound healing . Upon plPlatelet-derived factors are stored in three types of intercellular secretory organelles, including lysosomes, alpha-granules and dense granules  . Alpha-gBeside membrane-bound proteins that become exposed to the platelet surface, such as integrin αIIbβ3 and αVβ3, or the leucine-rich repeat family receptors , alpha-granules also store soluble proteins, such as platelet factor 4 (PF4), von Willebrand Factor (vWF) or vascular endothelial growth factor (VEGF) that are released into the extracellular space . In geneHuman platelets contain about three to eight dense granules per platelet, with a slight acidic lumen . Dense gThe platelet releasate (PR) is defined as a cocktail of soluble and vesicular (exosomes and microparticles) signals, which is released from platelets upon activation into the external milieu . A wide Parsons et al. identified 894 different proteins released from thrombin-induced platelets from 32 healthy adult humans, of which 277 proteins were reproducibly found in every donor and, therefore, defined as core releasate proteins . DependeSince the platelet releasate is not only defined as a mixture of soluble factors, but also of vesicular signals , plateleg and the exosomes to be isolated by centrifugation at 100,000–200,000× g [In order to further distinguish them in size, studies have described and defined different centrifugation protocols that define the microvesicles to be isolated at 10,000–20,000× 0,000× g . The car0,000× g . Althoug0,000× g . Regardi0,000× g  and migh0,000× g ,33.A successful implantation of the embryo into the uterine endometrium begins at day 6–7 post fertilization, with the polar blastocyst attaching to the endometrium. At this time point, the blastocyst is composed of two cell lineages, the outlying trophectoderm (TE) and the inner cell mass, with the latter mass facing towards the maternal side upon attachment . ThereafThe initial syncytium is formed due to the fusion processes of the TE, which then starts to invade into the maternal decidua, resulting in complete embedment of the blastocyst at day 14 post fertilization. This primary syncytium then starts to develop lacunae, which are fluid-filled spaces that later develop into the IVS. The primary syncytium is organized into trabeculae. Throughout the subsequent villous stage of placental development, cytotrophoblasts rapidly proliferate to invade the trabeculae of the primary syncytium. The thereby formed primary villi consist of an inner core of cytotrophoblasts with a surrounding SCT. The following stage of placental development includes the generation of the secondary villi. These secondary villi develop by invasion of fetal mesenchymal cells into the prior formed primary villi. Sequentially, tertiary villi are formed by emergence of fetal vessels within the villi core .Thereafter, cytotrophoblasts penetrate the primary syncytium and generate contact with the maternal decidua. A multilayered structure develops through reorganization of cytotrophoblasts, tailing in the formation of trophoblast cell columns . These mDifferent EVT subtypes have been described. The endovascular EVT (eEVT) directs along the spiral artery, whereas the interstitial EVT (iEVT) moves towards luminal structures such as spiral arteries, uterine veins, and lymphatics, by migration through the decidual interstitium. The iEVT is involved in the remodeling of spiral arteries, and also interacts with decidual stroma cells. This is of great importance for the attachment of the placenta to the uterus ,36. DuriThe IVS is perfused by the maternal spiral arteries. These are tightly coiled vessels, that arise from the uterine arteries of the mother. Due to invasion of EVTs the maternal spiral arteries are remodeled early in gestation, and the EVTs dilate the vessels at the entrance to the IVS into low resistance wide pore vessels . The remBesides remodeling the spiral arteries, EVTs also invade the lumen and plug the spiral arteries during the first trimester of pregnancy. Therefore, maternal blood cells are obstructed to flow into the IVS . MaternaAs mentioned above, EVTs invade the lumen of the spiral arteries from the beginning of gestation onwards, obstructing maternal blood flow into the IVS by plug formation .However, with a diameter of 2–3 µm, platelets are the smallest cells of human blood. Recent immunohistochemical studies of first trimester villous placental tissue led to the assumption that maternal platelets are the first blood cells entering into the IVS a. The plAn electronic microscopic survey showed filopodia formation, as well as fine-grained material in the OCS, in intercellular gaps of distal EVT column parts, indicating considerable platelet activation d. These 2Y1 and P2Y12 (2 (TXA2), is released from adherent platelets and enhances the recruitment and aggregation of additional platelets to the primary plug by binding to TPα and TPβ receptors. It activates platelets during both protective hemostasis and pathologic thrombus formation [2Y12 receptor signaling [There are multiple pathways contributing to platelet activation, granule release and platelet adherence ,46,47,48nd P2Y12 . The agoormation . Plateleormation ,50. Throormation ,52. It bormation ,55,56,57ormation . Thrombiormation . Other cignaling .As platelets are subjected to an unsteady and probably continuously changing shear stress in the IVS, the assumption of platelet activation upon shear stress and turbulence is very important in platelet-trophoblast interaction. Platelet activation regarding shear stress has been the subject of many nonplacenta-related research projects over the past decades.A recent study by Roka-Moiia et al. showed that platelets exposed to continuous shear stress, but not to biochemical agonists, exhibited an increase of phosphatidylserine externalization (PSE) and procoagulant activity. In the study, markers of platelet activation (P-selectin and integrin αIIbβ3 activation) and apoptosis  were examined. Biochemical agonists such as ADP and thrombin are potent inducers of αIIbβ3 activation and/or P-selectin exposure. No integrin αIIbβ3 activation occurred upon shear stress exposure, and P-selectin levels remained nearly unchanged. Shear-mediated platelet activation induced a different pattern of platelet surface activation markers, with enhanced PSE and thrombin generation on the platelet surface .Platelet activation under steady shear stress has been studied intensively over the past decades in regard to shear stress-induced unfolding of vWF and its binding to platelet receptor GPIb, which induced a significant activation of the platelets upon a specific threshold ,61,62,63Recent findings by Pushin et al. also described an analytical approach to platelet activation under unsteady shear stress. The approach was based on the idea that under unsteady flow, the conformation of vWF molecules on the platelet surface are dynamically changing and unfolding. The efficient interaction of vWF and multiple GPIb receptors on the platelet surface should increase and the platelet was assumed to be primed for activation .9/L) [2 observed in normotensive pregnancies is increased above levels reported in normal healthy nonpregnant women [During normotensive healthy pregnancies, a decrease in platelet count occurs, with between 4.4% and 11.6% developing gestational thrombocytopenia (defined as a platelet count below 150 × 109/L) . Platele9/L) ,67. Incr9/L) . Further9/L) ,70,71. Int women . Howevernt women , which ant women .Studies suggest not only different levels of platelet activation during pregnancy but also an altered protein content of the platelet releasate (PR). Szklanna et al. investigated the profile of PRs of 18 women with healthy pregnancies and 13 nonpregnant women. Of 723 identified proteins in the PR, 69 of these proteins were found to be altered in platelet releasate from pregnant women. This includes proteins that are only expressed during pregnancy, such as PSGs and human placental lactogen. Moreover, the population of exosomal vesicles present in the PR is also modified in pregnancy such that the mode size and the particle/mL size are decreased. This demonstrates that platelets and their released cargo are different in physiological stressful situations such as pregnancy. This may represent a promising beginning to understand possible roles of platelet activation in pregnancy complications .Pregnancy is accompanied with remarkable changes in hemostasis towards hypercoagulability  and hypoA study from Sood et al. revealed a differentiation-dependent gene expression program in murine trophoblasts that confers a thromboresistant phenotype onto these cells . In ordeBeside the expression of anti- and procoagulatory mediators, the fibrinolytic system is very important in sustaining a healthy balance in the coagulation system of the placenta. Pregnancy is described as a status of hypofibrinolysis, with remarkable changes in hemostasis, such as an increase of clotting factors and coagulability and a decrease of anticoagulants and fibrinolytic activity . The devThe major key player in the fibrinolytic system are the plasminogen activators urokinase and tissue type plasminogen activator (uPA/tPA), with their corresponding inhibitors the plasminogen activator inhibitor type 1 (PAI-1) and type 2 (PAI-2) . The degOverexpression of PAI-1 has been described to increase fibrin accumulation and insufficient placentation . TowardsPlacental pathologies, such as PE, are associated with an upregulation of PAI-1 in the placenta, as well as with elevated plasma levels. These results suggest that localized elevated levels of PAI-1 may play a role in thrombotic complications. Limited information is available on the factors that regulate the production of PAI-1 within healthy and pathological placentas, but cytokines or growth factors, such as TGF-β, could be key players .Disruption of the SCT, for example as a result of enhanced blood flow velocity and jet-like streams surrounded by turbulence, is replaced by the fibrin-type fibrinoid, which is defined as a product of the coagulation cascade and resulting in the so-called perivillous fibrinoid ,12,83. TThe plasminogen inhibitors are both expressed in the cytoplasm of the CT and the SCT, whereas the SCT also expresses PAI-1 and PAI-2 in its plasma membrane . TrophobOnce exposed to maternal blood, the trophoblastic basal lamina, which contains collagen, fibronectin and laminin, has procoagulant potential, and this suggests that focal degeneration of the SCT results in local blood clotting ,85.Collagen, as a major component of the extracellular matrix, provides structural support , but als2, to tighten the binding of integrin α2β1to collagen, and increases the affinity of integrin αIIβ3 to fibrinogen [Platelets possess several collagen receptors, of which α2β1integrin and the immunoglobulin superfamily member GPVI are the most prominent . The binbrinogen .Trophoblasts release a diverse spectrum of hormones, growth factors and extracellular vesicles (EV) from the first trimester onwards ,90. The Different vesicle fractions transport a diverse repertoire of placenta-derived molecules, which could affect maternal cells in various ways . Hence, For instance, the release of syncytiotrophoblast-derived EVs is elevated in PE compared to healthy pregnancies ,96,98. TSTBEVs interact with platelets and affect their function. Furthermore, they increase the rate and size of thrombus formation in vitro in whole blood under fluidic flow. In general, STBEVs from patients with PE have an increased effect on platelet activation and function, implying that they could contribute to the increased thrombotic risk of PE. The difference in STBEVs composition in PE compared to healthy pregnancies may correlate with the heterogeneity of maternal symptoms .Alterations in the coagulation and fibrinolytic system have been linked to pregnancy pathologies such as PE . With a However, PE provides a broad spectrum of phenotypes with heterogeneous etiology. A study from Than et al. uncovered altered abundance of proteins of the coagulation cascade in maternal proteomics in patients who subsequently developed preterm or term PE. Of note, in preterm cases of PE the extent of changes was larger than in term PE cases . A very 2 (TXA2) production, and hence the consequences of platelet activation, to the onset of PE. Low-dose aspirin (<150 mg daily), as an inhibitor of the cyclooxygenase, is widely used as prophylaxis for PE [In general, changes in platelet function, coagulation and thrombotic factors are strongly associated with the onset of PE . PE has s for PE . A studys for PE . PE is os for PE , which is for PE . TMA is s for PE .For pregnancies with a high-risk of developing PE, the administration of low-dose aspirin from very early stages of gestation onwards is commonly advised. , leadingA study from Roberge et al. described a significant reduction in the prevalence of PE, and a dose-response effect for the prevention of PE, when low dose aspirin (50–150 mg/day) was administered before the 16th week of gestation . However2Y12 similar to clopidogrel, which is the most commonly used thienopyridine, and has been recently introduced into clinical practice. A case report from Tello-Montoliu described a successful pregnancy outcome with prasugrel therapy. However, the use of P2Y12 receptor inhibitors during pregnancy is still under debate [Alternatively, low-molecular weight heparin or prasugrel is used as an anticoagulant substance during pregnancy. Low-molecular-weight heparin (LMWH) does not cross the placenta and has also been described as a promising therapy for especially severe placenta-mediated pregnancy complications . Prasugrr debate . Platelets have been found to carry a complex and dynamic transcriptome, similar to that of many nucleated cells. They are equipped with a complex transcriptome of mRNA, miRNA, long noncoding RNA, pre-mRNA and circular RNA. The majority of transcripts in platelets are already synthesized by megakaryocytes during thrombopoiesis, and only a small number are acquired through cell-cell transfer while circulating in the blood. Since platelets have an active spliceosome, and can process mRNA, proteins are synthesized and might play a role in platelet response, function and in cell-cell interactions  ,118.Studies have shown dynamic changes in the transcriptome of platelets in response to pathogens, stressors or inflammatory signals. A set of the most abundant transcripts is even comparable between species, such as human and murine platelets. A study from Middleton et al. revealed that platelets from septic patients, and platelets from appropriate septic mouse models, undergo similar changes in the transcripts of platelets . Hence, In conclusion, platelets are underestimated contributors to healthy development during pregnancy. Platelets at the fetal-maternal interface are a source of proinflammatory mediators, which might interact with different trophoblast subtypes of the developing placenta. Platelets possess the capability to undergo dynamic changes leading to an altered platelet releasate during pregnancy and a diverse set of transcripts in disease. These findings underline the importance of platelet subtypes (and their cargo) as promising biomarkers in the future.


Dyslipidemia is associated with lifestyle behaviors, while several lifestyle behaviors exist collectively among some populaitons. This study aims to identify lifestyle behavior clusters and their relations to dyslipidemia. This cross-sectional study was conducted in Wuhai City, China. Cluster analysis combined with compositional data analysis was conducted, with 24-h time-use on daily activities and dietary patterns as input variables. Multiple logistic regression was conducted to compare dyslipidemia among clusters. A total of 4306 participants were included. A higher prevalence of newly diagnosed dyslipidemia was found among participants in cluster 1 (long sedentary behavior (SB) and the shortest sleep, high-salt and oil diet) /cluster 5 (the longest SB and short sleep), relative to the other clusters in both age groups (<50 years and ≥50 years). In conclusion, unhealthy lifestyle behaviors may exist together among some of the population, suggesting that these people are potential subjects of health education and behavior interventions. Future research should be conducted to investigate the relative significance of specific lifestyle behaviors in relation to dyslipidemia. Cardiovascular disease (CVD) is one of the most threatening diseases, accounting for 31% of mortality globally in 2016 [Dyslipidemia refers to several lipid disorders that are characterized by at least one of following: raised total cholesterol (TC), raised triglycerides (TG), raised low-density lipoprotein cholesterol (LDL-C), and low high-density lipoprotein cholesterol (HDL-C) . DyslipiIn addition, abnormal biomarkers of dyslipidemia could also lead to other chronic non-communicable diseases among some populations ,10. For In the past decades, with the rapid development of the economy, the living standard of Chinese people has been constantly improved. An increasing proportion of animal fat, oily, and salty foods are consumed in people’s diets, along with less physical activity (PA) and more sedentary behavior (SB) conducted among Chinese people. Regular lifestyle behaviors play significant roles in the prevention of dyslipidemia and other cardio-metabolic disorders . Recent People’s daily activities mainly comprise PA, SB, and sleep ,17. TimeTo address these gaps in the research literature, we aimed to identify lifestyle behaviors among participants in Wuhai City, Inner Mongolia, China, applying compositional data analysis and exploring the associations between these clusters and dyslipidemia.This study was approved by the Peking University Biomedical Ethics Committee . The approval conformed to the provisions of the 1995 Declaration of Helsinki (revised in Edinburgh in 2000). All participants provided their written informed consent prior to the conduct of the survey.This cross-sectional study was conducted based on the project of Adult Chronic Disease and Prevalence Factors Monitoring from June 2014 to October 2014 in Wuhai City. People who aged 18–79 years old and who lived locally for at least six months in the past year were eligible to participate in this study. Multiple stratified cluster random sampling was performed to guarantee the representativeness of sample, comprising two steps. First, 20 streets of Haibowan District, Hainan District, and Wuda District were selected by random sampling. Second, stratified sampling was performed according to participants’ employment status in each street. Employed participants were sampled from 105 work units according to local industry structure for males under 55 years and females under 50 years. Systematic sampling was conducted by employee number to determine the final sample. Unemployed participants were sampled from 100 households in 2 resident committees for males at least 55 years and females at least 50 years, and ultimate participants enrolled were selected by Kish in each household. Participants were excluded if they were pregnant or had neurocognitive difficulties.Participants were required to complete a questionnaire, laboratory measurements, and a physical examination with the assistance of trained surveyors. The questionnaire was well designed and contained: (1) sociodemographic information, such as gender, age, education, and occupation; (2) information about personal lifestyle behaviors, including activities , dietary intake, smoking status, and alcohol intake; and (3) information about history of chronic non-communicable diseases .Activities under investigation comprised PA, SB, and sleep. PA was measured with the long volume of International Physical Activity Questionnaire (IPAQ), an effective and widely used questionnaire for examining PA intensity, frequency, and cumulative time in adults ,20,21. DPhysical examination and laboratory measurements were performed to obtain data on physiological indexes. Fasting venous blood samples were collected with disposal vacuum blood collecting tubes. The concentrations of TC, TG, HDL-C, and LDL-C were measured by local second-class hospitals, where the equipment, instruments, and reagents conformed with the national production and using standard. Weight and height were measured by standard instruments, with participants wearing light clothing and no shoes.Lifestyle behavior clusters in this study were identified as the aggregation of 24-h time-use of activities  and dietary patterns, which were the most important modified behaviors to improve dyslipidemia. PA intensity was classified according to IPAQ criteria , that is2), with a normal range of 18.5–23.9 kg/m2, considering the physiological characteristics of Chinese [Dyslipidemia was defined according to Chinese Guidelines for the Management of Dyslipidemia in Adults (2016 Revision) criteria, as the presence of one or more of the following factors: (1) serum TC concentration of 5.2 mmol/L or greater; (2) TG concentration of 1.7 mmol/L or greater; and (3) HDL-C concentration of under 1.0mmol/L . Specifi Chinese .Flow chart of statistical analysis is presented in Considering potential differences in lifestyle behaviors and risk of dyslipidemia across age groups, participants were divided into a <50 years group and a ≥50 years group ,33,34,35Scree plot was generated to determine the potential cluster number. Subsequently, a k-means partitioning cluster was used. An optimal number of 4 clusters was identified in two age groups based on the results of the scree plot and professional interpretability. To evaluate the stability of the cluster solution, a random subsample of each group (50% of participants in each age group) was clustered via conducting the same procedure. Agreements between solutions were relatively substantial (Cohen’s Kappa= 0.78 in <50 years group and 0.70 in ≥50 years group).Dyslipidemia was compared across lifestyle behavior clusters in two age groups by multinomial logistic analysis with adjustment for covariates, containing age, gender, education, occupation, family annual income, BMI, smoke status, alcohol intake, and history of hypertension and diabetes.χ2 tests . In this study, statistical significance was determined with p < 0.05 (two-tailed). Statistical analyses were performed with R 3.6.2 .Differences among clusters on sociodemographic characteristics and lifestyle behaviors were investigated by ANOVA (continuous variable) or Sociodemographic characteristics of participants are presented in The difference of gender, education, occupation, BMI, smoking status, alcohol use status, and family annual income among clusters were statistically significant in the <50 years group. Age, gender, education, occupation, smoking status, alcohol use status, and family annual income among clusters had statistical differences in the ≥50 years group.2), and a higher family annual income. In ≥50 years group, 309 (21.9%), 539 (38.2%), 196 (13.9%), and 368 (26.1%) belonged to cluster 5 to cluster 8, respectively, with cluster 6 accounting for the highest proportion. Compared with other clusters, cluster 6 was likely to be unemployed or retired, with a lower education level and family annual income.In the <50 years group, 965 (33.3%), 728 (25.2%), 527 (18.2%), and 674 (23.3%) belonged to cluster 1 to cluster 4, respectively, with cluster 1 accounting for the highest proportion. Compared with other clusters, cluster 1 was likely to be female, institution staff, non-smokers and non-drinkers, with a higher education level and a higher BMI  Clusters 1/5 in both age groups were characterized by long cumulative time of SB and short cumulative time of sleep, but high-salt and oil diet existed in the <50 years group, and not in the ≥50 years group. (2) Lifestyle behavior characteristics of cluster 2/6 in both groups were similar, which were both characterized by short cumulative time of SB and long cumulative time of sleep, but cumulative time of sleep in the <50 years group was the longest and cumulative time of SB in the ≥50 years group was the shortest. (3) Lifestyle behavior characteristics in cluster 3 and cluster 7 were different. Cluster 3 was characterized by long cumulative time of MVPA, SB, sleep, and extremely short cumulative time of LPA; however, moderate cumulative time of MVPA and LPA, short cumulative time of SB, the longest cumulative time of sleep, and healthy diet in cluster 7. (4) Long cumulative time of MVPA, short cumulative time of SB, and high-staple diet were observed in clusters 4/8 in two age groups, but cumulative time of MVPA was extremely long and cumulative time of SB was the shortest in the <50 years group; and cumulative time of sleep was short in the ≥50 years group.Blood lipid profiles among lifestyle behavior clusters are shown in p < 0.01) in both age groups. No statistically difference of prevalent cases was found among clusters in both age groups. In particular, among people <50 years, participants in cluster 2 and cluster 4 had a significantly decreased prevalence of newly diagnosed dyslipidemia  relative to cluster 1 after covariate adjustment. Similarly, in the ≥50 years group, participants in cluster 2 and cluster 4 had a significantly decreased prevalence of newly diagnosed dyslipidemia  relative to cluster 1 after adjusting for potential confounders.Among all the participants, 43.80% (1886/4306) were prevalent cases of dyslipidemia, 13.59% (585/4306) were newly diagnosed dyslipidemia cases, with the remaining 42.61% (1835/4306) not suffering from dyslipidemia. The associations between dyslipidemia and cluster memberships by multinomial logistic regression are shown in This study aggregated compositional data of daily activities and dietary patterns into lifestyle behavior clusters and explored their relationships with dyslipidemia. Cluster analyses were conducted among participants aged < 50 years and ≥50 years because age might be a confounding factor influencing the association between lifestyle behavior and dyslipidemia. Cluster analysis is a statistical classification technique for discovering whether the individuals of a population fall into different groups, and the characteristics of the clusters were summarized by the results of cluster analysis. Time length of PA, SB, and sleep of a cluster was classified by comparing their time-use with the mean of PA, SB, and sleep in the total population in each age group.Different lifestyle behavioral characteristics were found among clusters in two age groups. Besides, daily cumulative time of SB and sleep in the <50 years group was almost longer than that in the ≥50 years group. This phenomenon may result from greater need for sleep, prolonged sitting at work, pressure from every aspect, and relatively unhealthy lifestyle behavior among young people . No obviThe average cumulative time of sleep in the <50 years group was higher than that in the ≥50 years group, but both groups met the criteria of the American Sleep Time Duration Recommendation . The resFurthermore, results showed that part of the population focused on healthy lifestyle, but some unhealthy lifestyle behaviors might exist together, such as less PA, more SB, and unhealthy diet. This finding is in line with previous researches ,46,47,48The results of multinomial logistic regression showed that risk of newly diagnosed dyslipidemia might be lower in cluster 2 and cluster 4, but not in cluster 3, compared with cluster 1 in both age groups. This finding indicated that less SB, combined with more MVPA, might decrease risk of newly diagnosed dyslipidemia effectively. However, conducting MVPA may have difficulties in relieving the harm of extremely long cumulative time of SB, so decreasing SB time may be relatively more effective than increasing MVPA time in decreasing risk of newly diagnosed dyslipidemia. However, no significant difference of prevalent dyslipidemia was observed among clusters in both age groups, which might be due to changes in lifestyle behaviors after suffering from dyslipidemia.Age among clusters in ≥50 years varied significantly, with cluster 5 being the oldest. Therefore, age might be an influencing factor of selecting lifestyle behavior, and more SB and less PA might be conducted among participants with older age. The statistical difference on BMI among clusters in the <50 years group was significant, and cluster 5 had the highest BMI, which indicated that BMI might be associated with lifestyle behavior. Cluster 4 in the <50 years group showed different characteristics from the others. Extremely long cumulative time of MVPA, the shortest cumulative time of SB, and more staple foods intake were major characteristics in this cluster. Besides, cluster 4 tended to appear in participants who were male, less educated, consumed cigarettes and alcohol, and had low annual family income. This phenomenon may be explained by the “Health Worker Effect”, which means these young people who were comparatively better in physical fitness often choose manual work without paying much attention to a healthy lifestyle. Therefore, the association between extremely long cumulative time of MVPA and dyslipidemia is unclear. Previous research showed different findings for the health effects of MVPA ,50, so fThe main strength of this study is the application of compositional data analysis to process 24-h time-use on activities, which are recommended for cluster analysis of compositional data. Furthermore, daily cumulative time of activities was considered to develop recommendation on daily PA, SB, and sleep. In addition, participants’ lifestyle behaviors were explored comprehensively, considering cumulative time of all intensities of PA, SB, sleep, as well as dietary patterns, which are the most crucial modified behavior factors to prevent dyslipidemia. To our knowledge, this study is the first to combine compositional data analysis with cluster analysis to identify lifestyle behavior clusters among adults, and their relationship with dyslipidemia. Previous studies have combined compositional data analysis and linear regression to examine the associations between 24-h time-use on activities and physiological indicators related to chronic non-communicable diseases without considering dietary pattern ,53,54,55Several limitations exist in our study. First, the cross-sectional design employed in this study impedes the inference of causation. Further prospective studies should be conducted. Second, information on activities and dietary intake was obtained by IPAQ and FFQ, so slight differences between self-reported data and the actual situation might exist. However, these questionnaires have been widely used among the Chinese population because of their substantial validity and reliability. Finally, the results cannot be generalized to other populations because of exploratory, data-driven nature of cluster analysis and principal component analysis.In summary, this study adds to current evidence that less SB and more MVPA may decrease risk of dyslipidemia, and health education and behavior intervention should focus on the target population. Future studies should also be conducted to investigate the relative significance of specific lifestyle behaviors in relation to dyslipidemia, and make effective interventions.


Research on direct targets of traditional Chinese medicine (TCM) is the key to study the mechanism and material basis of it, but there is still no effective methods at present. We took Compound Danshen dropping pills (CDDP) as a study case to establish a strategy to identify significant direct targets of TCM. As a result, thirty potential active kinase targets of CDDP were identified. Nine of them had potential dose-dependent effects. In addition, the direct inhibitory effect of CDDP on three kinases, AURKB, MET and PIM1 were observed both on biochemical level and cellular level, which could not only shed light on the mechanisms of action involved in CDDP, but also suggesting the potency of drug repositioning of CDDP. Our results indicated that the research strategy including both in silico models and experimental validation that we built, were relatively efficient and reliable for direct targets identification for TCM prescription, which will help elucidating the mechanisms of TCM and promoting the modernization of TCM. It was discovered by Youyou Tu, a Chinese traditional medicine scientist, which is an effective and quick acting antimalarial drug. However, there is still a lack of effective approaches to systematically study its mechanism. The research of reductionism is not capable of answering the essential question of the overall efficacy of TCM. It may lead to deviate from the system theory of TCM, so it needs to be combined with the system theory. In recent years, a variety of "omics" techniques based on system theory have been widely used in TCM research5, for elucidating the pharmacological characteristics of TCM better9, but still cannot fully reveal the nature of it. Comprehensively understanding the mechanism of synergism among the effective components, drug targets and metabolic pathways remains highly demanded. One key to break this dilemma is to carry out the research on the direct target of TCM. It can not only clarify the pharmacological mechanism of TCM from the origin and scientifically interpret its traditional efficacy, but also unveil novel disease-related mechanism10 and provide reasonable estimation for TCM repositioning. At present, technical methods to screen and determine the direct targets for TCM efficiently and accurately are still poorly developed, which hinders elucidating the mechanisms of TCM essentially.Traditional Chinese medicine (TCM) prescriptions are the characteristics of Chinese medicine. They have been practiced for thousands of years and have been proved to be effective in modern clinical practice. These prescriptions embody the dialectical thought of Chinese medicine and the medication holistic view. In recent years, the reductionist research model has accumulated a lot of data, and also provided illuminating research results, such as the discovery of artemisininRadix Salviae (Danshen), Panax Notoginseng (Burk.) F. H. Chen Ex C. Chow (Sanqi), Borneolum Syntheticum (Bingpian). It is widely used in treating coronary artery disease (CAD) and angina pectoris including acute stage and preventive treatment. Although many research articles about CDDP have been published already, the research on its mechanism of action is still not in-depth14. Most studies focused on the genes or proteins regulated by CDDP treatment, most of which can be referred as indirect targets, so far there is no report focusing on the direct targets of CDDP. It is of great significance to obtain the direct targets of TCM.CDDP consists of 15.Kinases is an important class of drug targets. Among them, protein kinases family is the largest group of kinases, which act on specific proteins and modulate their activities. These kinases play a wide range of roles in cell signaling and complex life activities, and their dysfunction plays an important causal role in many human diseases, including cancer, inflammatory diseases, central nervous system diseases, cardiovascular diseases and so onTo obtain the direct targets of the whole prescription, we proposed a hypothesis that the potential direct targets of important components are more likely to be direct targets of the whole prescription. Therefore, the potential targets of components were utilized to speculate the potential direct targets of the whole prescription. More importantly, we adopt a strategy of integrating multi-source data to improve the success rate of validation results. In this study, we took CDDP as a study case to obtain its direct kinase targets. Firstly, the literature database of CDDP was constructed by literature retrieval, and the important components contained in CDDP were extracted. Secondly, the potential targets of important components were obtained through public database querying and Multi-voting chemical similarity ensemble approach (SEA) algorithm predicting. Then, the kinase targets got from KinomeX system was used to filter the potential kinase targets of CDDP. Finally, 30 active targets were obtained and some of them were further validated by a series of experiments Fig. . In sili3719 Chinese-language literatures and 59 English-language literatures were obtained through retrieving the customized terms of the CDDP . Through literature reading manually, the components information of CDDP was extracted. According to the screening criteria of important ingredients, a total of 39 ingredients were collected. In addition, quercetin, a potential important component of CDDP was also included for subsequent analysis. All 40 important components of CDDP are shown in Table Through querying the three public databases, 262 known targets including 55 kinase targets were obtained, and 377 predicted targets including 121 kinase targets were predicted based on the Multi-voting SEA algorithm. By integrating the above two parts of targets, a total of 479 potential direct targets were obtained, including 148 kinase targets Table .Table 2NThrough KinomeX system, we got 288 kinase targets  value and its standard deviation were obtained  than 10 μg/mL derived from the concentration–response curves, and the remainder had an IC50 value better than 35 μg/mL  are the most widely used target identification technologies for active ingredients, such as target fishing technology31. In further study, a variety of state-of-art algorithms based on different principles should be utilized to predict component-target relationship for improving the accuracy of predicting results36, such as in silico models based on network topology parameters33, drug and target structure similarity34, clustering multi-dimensional drug target data35, deep learning and heterogeneous network36 etc. In addition, molecular docking technology can also be used to gain more reliable targets for further experimental verification38.In this study, 106 potential kinase targets of CDDP were tested, and finally 30 active targets were obtained, with an accuracy of 28.3%. As expected, the success rate of the known kinase targets is higher than that of the predicted targets  and CDDP 37 (Catechol) contained in CDDP could act on the common target FYN. However, the binding site for the two components may be different, which may bring the allosteric effect, weakening the inhibition effect under the condition of high concentration. These components may not directly bind to protein active sites, but to the allosteric sites, outside the active sites of the protein, causing the conformational change of proteins and their activity.Among the 14 targets retested at the concentration of 250 µg/mL, the inhibitory activity of four kinases  did not decrease but increased at high concentration. The possible reasons may as follow: firstly, the components with high molecular weight in TCM form great stereo-hindrance effect when the concentration increases, which may hinder the combination between active molecules and targets. The second possibility is potential positive effectors involved in CDDP. The synergy weakens the affinity and internal effectiveness of the ligand on the receptors45. For example, AURKB positively correlates with platelet aggregation and acute myocardial infarction (MI)46. MET shows repair function in cardiomyocytes and blood vessels through pro-angiogenesis, anti-inflammation and preventing fibrosis47. PIM1 plays a role in vascular smooth muscle cells (VSMCs) proliferation, which is closely related to the pathogenesis of atherosclerosis48. Besides, these targets are closely related to some other diseases55, indicating the potential function of CDDP against other indications, especially cancers. Actually, it has been reported the anti-tumor activity of several significant components of CDDP, including Danshensu56, Tanshinone I57, Cryptotanshinone58, Tanshinone IIA59, Rosmarinic acid60, and Ginsenoside Rg161, suggesting the potential anti-tumor effect of CDDP.Three kinase targets  of CDDP, that have been finally validated on cellular level, could provide basis for further elucidating the mechanism of CDDP in treating cardiovascular diseasesComparing with the above three kinases, it is worthy of note that CDDP promoted SYK activity in several cell lines Figs. , 4, whicIn conclusion, 30 direct targets of CDDP were obtained in this study by the strategy we built, which is independent of any specific disease model and can provide a series of potential direct targets of TCM efficiently. Moreover, this strategy takes TCM as a whole research object, which is in line with the holistic view and systematic theory of TCM, conforming to the guiding principles of pharmacology theory of TCM. The direct targets not only provide the theoretical basis for elucidating the mechanism of action and the material basis, but also indicating rationales for the research of drug repositioning, which is of great significance for promoting TCM modernization.https://www.cnki.net/). Similarly, through PubMed (https://pubmed.ncbi.nlm.nih.gov/), "Compound Danshen drilling pills", "Fufang Danshen Diwan", "T89", "dantonic" and "Cardiotonic Pills" were used to get the English-language literatures . Then, the components contained in CDDP were extracted manually and standardized through PubChem database (https://pubchem.ncbi.nlm.nih.gov/)62. Besides the ingredients entering the blood, main metabolites, bioequivalence components compared to the prescription, active components of CDDP reported in literatures, in order to avoid missing critical components included in CDDP, we selected the most extensively studied component in the three single herbs but still unconfirmed in the whole prescription, through retrieving TCM related databases, such as TcmSP63, TCMID64, TCM-ID65, ETCM66, and YaTCM67.In order to review the literatures related to CDDP as comprehensively as possible, we used "Danshen Dropping Pills" as the keyword to obtain the Chinese-language literatures through CNKI  through ingenuity knowledge base in Ingenuity Pathway Analysis (IPA) and subsequently the kinase targets were screened. It is a professional database of functional annotation and biological interaction, which collects millions of detailed annotation information about proteins, genes, compounds, cells, tissues, drugs and diseases, as well as their interaction information. All information was collected from the original literatures and reviewed by hundreds of doctoral experts to ensure its accuracy.The known activity data of 40 important components in CDDP were obtained from three authoritative public databases, namely, ChEMBL70 was utilized to predict potential targets of important components. In this algorithm, prediction models, namely Topological SEA, Morgan SEA, MACCS SEA, Atom Pair SEA and Pharmacophore SEA, were integrated to calculate potential targets of components, which could take advantages of different models and improve the robustness and the success rates of the models. By combining the five models, a flexible forecasting scheme was obtained with precision range from 71 to 90.6%, F0.5-Measure range from 0.663 to 0.684 and F0.25-Measure range from 0.696 to 0.817. Finally, all potential targets of each component were normalized by IPA and the kinase targets were selected.Avoiding missing some important targets, multi-voting SEA algorithmhttps://kinome.dddc.ac.cn/en/)71 is a prediction and analysis platform of single compound regulated kinase spectrum. It enables users to predict its potential kinase targets based on the structure of a given molecule with the average 0.75 area under the receiver operating characteristic curve (auROC), which is significantly higher than other prediction methods78. Therefore, we used the KinomeX to predict the potential protein kinase targets of 40 important components in CDDP.KinomeX system . Firstly, the filter-binding radioactive kinase activity assays were performed at a concentration of 25 µg/mL of CDDP. The kinase activity inhibition rate of the sample was expressed as the percentage of the result of sample compared to the blank group. The kinase activity of the blank was 100%. Generally speaking, if the residual enzyme activity is less than 30%, it is considered to be strongly inhibited. And if the residual enzyme activity is between 30 and 70%, it is considered as moderate inhibition. Considering the weak interaction superposition characteristic and synergistic effect of TCM ingredients50 value and its standard deviation, we chose four targets showing obvious inhibitory action at the concentration of 250 µg/mL to carry out the kinase assay. Pharmaron (Beijing) was commissioned to perform in vitro kinase assays for AURKB, MET, PIM1, SYK. The detailed information about the assays, such as the reagents, instruments, assay procedure, data analysis, and calculation of IC50, for AURKB, MET, PIM1, SYK can be referred in the attachment , CDDP group (0.5 mg/mL), and CDDP group (1.0 mg/mL). Cells at a density of 2 × 105 cells/well in 6-well plates were treated with CDDP according to the groupings. The treatment time was 12 h for MCF7, T47D and 6 h for TPC1, BCPAP.The human breast cancer cell lines MCF7, T47D and thyroid cancer cell lines BCPAP, TPC1 used in this study were purchased from American Type Culture Collection . These four cell lines were maintained in Dulbecco's modified Eagle medium (1640)  supplemented with 10% fetal bovine serum (FBS) . All of them were placed in a 5% COCells were lysed in Triton-X 100 lysis buffer containing 1% (v/v) Triton-X 100, 150 mM NaCl, 50 mM Tris–HCl with protease and phosphatase inhibitors . The cell lysates were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and then transferred to a polyvinyl difluoride membrane . The membranes were then blocked with 5% nonfat milk and incubated overnight with rabbit anti-Aurora B , rabbit anti-pAurora B , mouse anti-MET , rabbit anti-pMET , rabbit anti-Pim1 , rabbit anti-pPim1 , rabbit anti-Syk , and rabbit anti-pSyk , respectively. Peroxidase-conjugated anti-mouse or anti-rabbit IgG  was used as the secondary antibody. Immunoblotting signals were detected using the ECL reagent . All membranes were re-probed with mouse anti-α-tubulin antibody , which served as a loading control. Gray analysis of western blot image was performed using image analysis software . Tanon 4600 SF and GelView 6000 Plus full-automatic digital gelatin/chemiluminescence imaging analysis system software is used for protein gel electrophoresis image acquisition.Statistical significance was determined by a two-tailed, unpaired Student t-test in GraphPad Prism 8. P-value < 0.05 was considered statistically significant.Supplementary Information.


The following indexes were assessed in each subject: BMI, fat-free mass index (FFMI), fat mass index (FMI), tri-ponderal mass index (TMI), waist-to-height ratio (WtHR), and the body mass fat index (BMFI). Thereafter, a threshold value adjusted for age, which could identify MetS, was calculated for each index. A significant correlation was found among all indexes . However, when the area under the curve (AUC) was compared, WtHR performed significantly better in the whole group and in the different age groups, apart from a lack of statistical difference between WtHR and BMFI in the 45–55 years age group. In conclusion, WtHR seems to be a fair index useful for identifying MetS in women with obesity. The use of thresholds appropriate for age can help further improve its accuracy, thus reinforcing the clinical evaluation for MetS screening.The aim of this study was to compare the accuracy of different indexes of adiposity and/or body composition in identifying the metabolic syndrome (MetS) in a group of 1528 Caucasian women with obesity: (age ± standard deviation (SD): 50.8 ± 14 years (range 18–83); body mass index (BMI) 43.3 ± 5.9 kg/m Following the pandemic rise of obesity, a significant increase has been observed in the prevalence of metabolic syndrome (MetS), this finding representing a relevant risk factor for the development of cardiovascular diseases and type 2 diabetes mellitus (T2DM). However, the risk of developing MetS is not limited to the obese population, as it can be present in a significant percentage of normal weighted individuals . The freAs far as the obese population is concerned, one of the main points to be clarified is which level should trigger the start of the investigations aimed to identify the presence of primary or secondary metabolic/endocrine changes. In order to address this crucial problem, on the one hand, it is mandatory to balance high cost and inconvenience to patients undergoing these investigations, while, on the other hand, there is a need to promptly detect the level of obesity triggering the development of MetS. In a recent paper, it was suggested that the body mass index (BMI), which is the most used index in clinical practice, could be considered a useful clinical threshold to help the decision . HoweverBecause of these limits, other indexes have been proposed, such as the fat mass index (FMI) , tri-ponThe objective of this paper was to verify whether these results could also be replicated in a cohort of women with obesity over a large age range. With the background of a worsening metabolic status over the years, women with obesity are particularly at risk of developing MetS and therefore would benefit from an early diagnosis and, consequently, a prompt treatment. Therefore, early recognition of MetS or risk of MetS through easy-to-use indexes would be suitable to enable the implementation of more intensive primary prevention strategies. For this purpose, we evaluated for BMI, BMFI, FFMI, FMI, TMI, and WtHR, the age-adjusted threshold that allows to identify or exclude the presence of MetS, comparing thereafter their sensitivity and specificity, the positive and negative predicted value , and the positive and negative likelihood ratio  in order to establish which index of body composition and/or adiposity is the most reliable in identifying MetS in a large group of women with obesity.2 (30.7–72.9 kg/m2)), consecutively hospitalized at the Division of Metabolic Diseases, Istituto Auxologico Italiano, Piancavallo, Verbania, Italy, were studied. Inclusion criteria were BMI ≥30 kg/m2 and Caucasian origin. None of them suffered from secondary obesity, including genetic , organic , endocrine, ), or iatrogenic forms . At the time of the study, 256 subjects were treated for T2DM (16.7%), while 833 subjects (54.5%) were undergoing therapy for arterial hypertension. A total of 121 patients (7.9%) were treated for dyslipidemia. A total of 54 women were previously diagnosed with T2DM, dyslipidemia, and hypertension (3.5%), 159 with T2DM and hypertension (10.4%), 49 with hypertension and dyslipidemia (3.2%), 7 with T2DM and dyslipidemia (0.4%), 36 with T2DM alone (2.3%), 571 with hypertension alone (37.4%), and 18 with dyslipidemia alone (1.2%).Between January 2015 and December 2018, 1528 women with obesity (aged 50.8 ± 14 years (range 18–83), BMI 43.3 ± 5.9 kg/mn = 466), 45–54 years (n = 390), and 55+ years (n = 672).Subjects were divided according to age in three subgroups: 18–44 years , and all patients gave their written informed consent. The study was performed in accordance with the Declaration of Helsinki and the 2005 Additional Protocol to the European Convention of Human Rights and Medicine concerning Biomedical Research.Weight and height were measured in fasting conditions after voiding. Physical examination was carried out by the same investigators, specifically trained. Standing height was determined by a Harpenden Stadiometer . Body weight was measured to the nearest 0.1 kg, with an electronic scale , in minimal underclothes and no shoes. WC was determined in standing position midway between the lowest rib and the top of the iliac crest after gentle expiration, with a non-elastic flexible tape measure.Diastolic and systolic blood pressure (BP) were measured to the nearest 2 mmHg in the supine position after 5 min rest, using a sphygmomanometer with an appropriately sized cuff. The average of three measurements on different days was used.50), following international guidelines [50) was calculated as the ratio between height (cm)2 and Z50 (Ω). In our laboratory, the within-day CV of Z50, as determined by 10 repeated BIA measurements of 10 obese adults, was 2% [Body composition: fat mass (FM), fat mass percentage (FM%), fat-free mass (FFM), and fat-free mass percentage (FFM%) were assessed by bioelectrical impedance analysis (BIA) . Whole-body impedance was measured at a frequency of 50 kHz , and triglycerides (TG) .2 [Obesity was defined in the presence of a BMI higher than 30 kg/m2 . Accordi2 , MetS wa2 . Hyperte2BMI : weight BMFI : BMI × F2FFMI : fat-fre2FMI : fat mas3TMI : mass inWtHR : WC (cm)The following indexes have been calculated according to the respective formulas:p-value of 0.31 but a graphical analysis and the Cramer–Von–Mises test (p > 0.05) indicated a normal situation.The data were first scrutinized for outliers, using a cutoff of 4.5 standard deviation score. Two patients were excluded on this basis. To explore the data, preliminary analyses were performed. A lognormality test was conducted on the logarithmic transformations of the variables used. The Kolmogorov–Smirnov test was used as a reference, with only one doubtful case for FMI, where the Kolmogorov–Smirnov test showed a t-tests. Pearson correlation coefficients were calculated to assess the relationship between body-composition indexes. ANOVA and the Bonferroni test were used to analyze differences in clinical characteristics among the three age groups. Correlation analyses were used to assess the associations between each body-composition index and each metabolic risk factor component. Fisher’s transformation, changing r to a Z-score, was used to compare correlated correlations.Continuous data were presented as mean ± standard deviation (SD) or with 95% confidence intervals (CIs). Mean values were tested for statistical significance using 2-tailed In order to calculate the growth pattern of body-composition indexes, a quantile regression was used  as an alReceiver operating characteristic (ROC) curves were then generated to obtain the values of area under the curve (AUC), with sensitivity, specificity, and 95% CI, for each age-adjusted standardized body composition index as a predictor of MetS . In addiTo identify the optimal cutoff, the Youden index  was calcp < 0.05. The data were analyzed using SAS Enterprise Guide 4.3 .The significance threshold was set at According to the IDF criteria for MetS, all 1528 women fulfilled the criteria for abdominal obesity. High BP was detected in 1105 subjects (72.3%). Raised triglycerides values were present in 510 patients (33.4%), while 825 had reduced HDL-C levels (54.0%). Hyperglycemia was found in 505 women (33.0%). Overall, the presence of MetS was detectable in 917 patients (60.0%).The clinical characteristics, biochemical data, and values of the different indexes of the whole population, as well as the two subgroups without (MetS−) and with MetS (MetS+), are reported in p < 0.0001 for all but p < 0.01 for FMI), with the exception of FM% and FFM% (p = 0.88 for both). Moreover, MetS+ women were significantly older than the MetS− subjects (p < 0.0001).As expected, all parameters were significantly worse in the MetS+ group than in MetS− , in the MetS+ group , and in the MetS- group , apart from the absent correlation between FMI and FFMI (p = 0.08)  . The valAge-adjusted ROC analysis for the whole group, using clinical cut points for metabolic risks, shows that anthropometric measures are consistent predictors for hypertension and hyperglycemia .In BMI and FMI showed the highest specificity and WtHR the lowest. All indexes showed the same PPV, with only BMI superior to TMI. Considering the negative predictive value, WtHR was significantly higher than the others.The evaluation of the PLR and NLR showed that the post-test probability was not different from the pre-test probability. The (LR+) post-test probability of MetS changed from 7% for TMI to 14% for BMI, and the (LR−) post-test probability of MetS changed from 12% for WtHR to 2% for FMI. Only with BMI and WtHR was there a small improvement in the post-test probabilities.Furthermore, BMI performed better than FMI, FFMI better than BMI and FMI, TMI better than FMI, and, finally, BMFI better than FMI and BMI. Analyzing the single subgroups, it is evident that, in the age range of 18–44 years, WtHR performed better than BMI, FMI, TMI, BMFI, and FFMI and BMFI performed better than FMI and BMI.In the subgroup 45–54 years, WtHR performed better than TMI, BMI, FMI, and FFMI, while BMFI performed better than FMI, TMI, and BMI and BMI performed better than TMI.In the subgroup 55+ years, WtHR performed better than the other indexes and BMFI performed better than FMI.In the present study, the accuracy of different indexes of adiposity and body composition in recognizing MetS was evaluated in a large group of women with obesity aged 18–83 years. Altogether, although a good correlation among indexes was found in the whole group, the WtHR was associated with the best performance. Furthermore, the large age span of the subjects examined allowed to demonstrate that the threshold values of different indexes, beyond which MetS was identified, varied over the years, implying that, for each age group, an appropriate cut-off should be used. This trend should be considered in the clinical praxis in order to identify the patients at risk more precisely. The age-dependent variation of the threshold values may presumably be explained by the physiological changes in body composition that usually occur in women over the years, particularly after the menopause , which iIn all age subgroups of women with obesity , the higher performance in identifying MetS was found with the indexes, which also consider fat distribution, such as WtHR and BMFI. The analysis of correlation between the different indexes and the component of MetS confirmed that WtHR was the better predictor of the metabolic risk factors. In addition, WtHR was the more sensitive index, while BMI and FMI showed the highest specificity.The PPV was similar for all indexes, while WtHR was slightly superior to the other indexes when NPV was considered. Altogether, according to the prevalence of MetS in the study population, the PPV was found to be higher than NPV. PLR and NLR, on the other hand, suggested that these indexes were of little help in predicting the likelihood of a true positive or negative result, with only BMI and WtHR performing slightly better.The accuracy of the indexes was evaluated in the whole group, as well as in the different age subgroups, by comparing the ROC curves. WtHR resulted in the best one, with the exception of a lack of statistical difference between WtHR and BMFI in the 45–54 years age group.These results were consistent with previous studies, demonstrating that WtHR was a valuable screening tool for adult cardiometabolic risk factors  and the In two previous papers by our group, the accuracy of the same indexes evaluated in this study was analyzed in a large group of obese children and adolescents  and in aOn the contrary, in the present study, WtHR performed slightly better than the other indexes. Several possible causes can be taken into consideration in order to explain the different results, such as the large differences in chronological age and therefore the differences in body composition and fat distribution of the present study group. As a matter of fact, associations between anthropometric indexes and regional fat depots varied between children, adolescents, and young and older adults . Further2, >40–50 kg/m2, >50 kg/m2) might represent another critical issue. However, the choice to exclude this analysis was based on the markedly different numerousness of the three BMI-related subgroups . Further additional studies with numerically homogeneous subgroups are mandatory to clarify this relevant aspect. The categorization based on age alone without actually knowing whether women were in their menopause or post-menopause might represent another limitation of the study. Although we are perfectly aware that the measurement of waist circumference may be problematic in severely obese patients, thus representing a possible bias of the present study, the examination of patients was performed by a well-trained and experienced team of a single third level center for severe obesity, thus reducing the risk of significant misinterpretations. Lastly, the present study performed on Caucasian women with obesity cannot allow to extend our conclusions to other ethnic groups.There are some weak points in our study. First of all, the main problem is that our data are derived from a cross-sectional study. It is undoubted that a longitudinal evaluation would have provided more information on the evolution over time of the ability of the different indexes taken into consideration when recognizing MetS. Another limitation is the lack of evaluation of body fat by DEXA, which would actually have been impractical and too expensive to be performed in such a large number of subjects. Moreover, the lack of analysis of our indexes in the different BMI classes  might have influenced the present results. Finally, our results suggest that changes of the different indexes over the years should also be considered in order to ensure effective and age-appropriated rehabilitative approaches, aimed to counteract the complex comorbidities associated with obesity.As a final remark, it must be underlined that all the indexes taken into consideration in the present study should be considered as a support for the clinicians in order to identify the presence of MetS and thus avoid unnecessary investigations. Although both WtHR and BMI represent useful, cheap, practical, and easy-to-use tools, facilitating the work of clinicians and speeding up diagnostics, it must underlined that they cannot replace the clinical judgement at all.