Natural products, including traditional herbal medicine (THM), are known to exert their therapeutic effects by acting on multiple targets, so researchers have employed network pharmacology methods to decipher the potential mechanisms of THM. To conduct THM-network pharmacology (THM-NP) studies, researchers have employed different tools and databases for constructing and analyzing herb–compound–target networks. In this study, we attempted to capture the methodological trends in THM-NP research. We identified the tools and databases employed to conduct THM-NP studies and visualized their combinatorial patterns. We also constructed co-author and affiliation networks to further understand how the methodologies are employed among researchers. The results showed that the number of THM-NP studies and employed databases/tools have been dramatically increased in the last decade, and there are characteristic patterns in combining methods of each analysis step in THM-NP studies. Overall, the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP) was the most frequently employed network pharmacology database in THM-NP studies. Among the processes involved in THM-NP research, the methodology for constructing a compound–target network has shown the greatest change over time. In summary, our analysis describes comprehensive methodological trends and current ideas in research design for network pharmacology researchers. Traditional herbal medicine (THM) has maintained the health of Asian people for thousands of years and built a unique medical system based on empirically accumulated knowledge. Billions of people around the world are taking THM daily, and the drug development field considers THM to be a source of inspiration ,2. The rNetwork pharmacology has emerged as a promising approach to accelerate drug development and elucidate the mechanisms of action of multiple target components . It undeIn the last decade, researchers have employed network pharmacology methods to elucidate the potential targets and toxicity of THMs . THM-netSeveral studies have described THM-NP researches by summarizing network pharmacology databases for THM and illustrating several representative applications ,13,14,15https://www.ncbi.nlm.nih.gov/pubmed/) from January 2000 to December 2018. The search language was restricted to English. The search terms used were NP-related terms  in [title] AND THM-related terms  in [title/abstract]. The search range of THM-related terms was extended to [title/abstract] since the titles of THM studies generally contain only the name of herbs or herbal formulae that are difficult to search.The literature search was performed in PubMed  extract(s) from a single herb; (2) preparation(s) containing multiple herbs; (3) proprietary herbal product(s); and (4) molecule(s) derived from a single herb.Two authors (W.Y. Lee and C.E. Kim) independently examined titles, abstracts, and journals to select eligible THM-NP studies. When articles were duplicated, only the most recent information was included. Then the full text of potentially relevant studies was retrieved. Two authors (W.Y. Lee and C.E. Kim) independently examined the full-text records to determine which studies met the inclusion criteria. Disagreements about the study selection were resolved by rechecking whether the studies met our criteria for inclusion.http://lsp.nwu.edu.cn/tcmsp.php) [Authors extracted the following data from the included THM-NP studies: authors, affiliations, publication years, tools, and databases. Synonyms for tools and databases were merged and counted under a single keyword. DTpre and SysDT  were conmsp.php)  since thTo capture the trends in the methods for constructing compound-target networks, we categorized DTI methods into four groups by their hypothesis and which information was used: the chemogenomic approach, docking simulation approach, ligand-based approach, and others ,19,20. Thttp://www.cytoscape.org/) was used to visualize the networks [The author network and affiliation network were constructed to identify the methodological characteristics of corresponding authors and affiliations. The nodes in each network represent authors or affiliations, and the edges represent co-occurrences of authors or affiliations in THM-NP studies. The frequencies of employed DTI and drug availability methods were counted for each corresponding author or affiliation. These methodologies were mapped to the author network and the affiliation network. Cytoscape 3.7.1  using the Tanimoto coefficient [Because information on the absorption, distribution, metabolism, and excretion (ADME) properties of herbal medicines in humans are lacking, researchers have employed evaluation methods or machine learning tools to predict those properties. We counted the number of THM-NP studies that evaluated the drug availability of herbal ingredients. We found that approximately half of  THM-NP studies evaluated the drug availability of herbal ingredients, and the majority of the studies  employed Obioavail and drug-likeness in combination B. Obioavfficient . They arWe next attempted to determine the frequency of each DTI method for constructing compound–target (C-T) networks . The results showed that TCMSP  and molecular docking  were the most frequently used. In addition, experimental methods, such as microarrays, were also applied A. It is We categorized the methods into four groups: the chemogenomic approach, docking simulation approach, ligand-based approach, and others D. In thehttp://geneontology.org/) [https://www.genome.jp/kegg/) [http://bidd.nus.edu.sg/group/cjttd/) [https://www.omim.org/) [We identified the frequency of biomedical databases employed to analyze the biological processes, pathways, and diseases from the targets of herbal medicines . Most THgy.org/)  for biolp/kegg/)  for path/cjttd/) , Online im.org/) , and Druim.org/) .We identified the combinatorial patterns of each step in THM-NP studies by a Sankey diagram-like representation . The SanThe Sankey diagram-like representation shows the diversity of databases and tools used in THM-NP studies and their combination patterns . We founTo further understand how the methodologies of THM-NP studies are employed among researchers, we constructed a co-author network and an affiliation network that were mapped with drug availability and DTI methods. The nodes in each network denote the author and affiliation, and the edges indicate that two of them appear on the same paper. The methods of DTI and drug-availability used by the corresponding author and affiliation are represented by the pie chart and the outline, respectively.n = 18) and Shao Li (n = 8) appeared most frequently as the corresponding author  and China Academy of Chinese Medical Sciences (n = 17) appeared most frequently. Most affiliations combined various DTI methods (68/143) and employed drug-availability methods (86/143).We also constructed and visualized the affiliation network . NorthweIn this study, we successfully identified the complex methodological trends of THM-NP research fields by analyzing the frequency of the employed methods in THM-NP studies over time and visualizing the combinatorial patterns between them. Our results showed that the number of THM-NP studies and employed databases/tools have been dramatically increased in the last decade. We also found characteristic patterns exist in combining methods of each analysis step in THM-NP studies. Finally, we showed how the diverse methods for THM-NP studies are employed and shared among researchers in the field by analyzing the co-authorship and affiliation networks.http://bionet.ncpsb.org/batman-tcm) and TCM-Mesh (http://mesh.tcm.microbioinformatics.org/), were developed [Among the network pharmacology databases, TCMSP was the most frequently employed database for constructing herb-compound-target networks. This database was developed in 2014 and has been predominantly employed among THM-NP studies . TCMSP peveloped ,45. TheyAmong the processes used in THM-NP research, the methodology for constructing a C-T network has shown the greatest change over time. In the early stages of THM-NP research, DTI methods for identifying targets of herbal ingredients relied on molecular docking simulations, which require high computational resources D. With tTo conduct THM-NP research, various tools and databases are combined in each phase of a study . We founThere are some limitations to our study that should be noted. First, we identified potentially relevant articles using combined keywords consisting of THM-related terms and NP-related terms. Although we carefully selected search terms, we cannot guarantee that our search strategy can fully identify THM-NP studies. Second, we found potentially relevant articles only in PubMed. It is one of the largest electronic database in the world. However, there are other databases which may include other potential THM-NP studies, such as Embase, China Knowledge Resource Integrated Database (CNKI), Research Information Sharing Service (RISS), and Japan Science Technology Information Aggregator (J-stage). Last, we limited the search range of our study to English literature, which might have introduced some bias. In spite of these limitations, our results will help to improve the understanding of the methodological trends of the THM-NP research fields.In conclusion, we investigated the methodological trends in THM-NP studies. Our results provide researchers with the current status of which methodologies are used in THM-NP studies and how they are applied.


Listeria monocytogenes, the causative agent of the food-borne disease listeriosis, which is increasingly prevalent in the European Union. Efficacy of both the fish processing plant self-checking system and official food control conducted by authorities are crucial for L. monocytogenes prevention in the processing of these risky products. However, the impact of official control on L. monocytogenes prevention in the processing of fish products has not been extensively studied. We investigated the occurrence, control measures, and correction of non-compliances predisposing to L. monocytogenes in Finnish fish processing plants. The following features were associated with L. monocytogenes occurrence: (a) frequency of non-compliances concerning processing machinery, (b) recurrence of non-compliances, and (c) frequency of non-compliances for which official control measures were requested by inspecting authorities. Official control of fish processing plants had focused on risky areas, but non-compliances were common and their correction exhibited deficiencies. We conclude that L. monocytogenes prevention in fish processing can be enhanced by strengthening official food control measures and processing plant compliance. In particular, timely correction of all food safety violations must be improved.Vacuum-packaged cold-salted and cold-smoked fish products are considered typical vehicles for  Control measures have thus been insufficient, and foodstuffs contaminated with Listeria monocytogenes are found on the market. Seafood, in particular, are considered typical vehicles, and ready-to-eat vacuum-packaged gravad  and cold-smoked fish products have been implicated in listeriosis outbreaks5. In the most recent investigations by Finnish public food authorities, the prevalence of L. monocytogenes in this type of fish products ranged from 12 to 32%7, and the bacterium was occasionally found from facilities and food contact surfaces of fish processing plants8. Violations in hygiene practices, maintenance, and cleanliness have been reported as drivers of L. monocytogenes contamination10. Stringent interventions targeting such violations have proven successful at reducing L. monocytogenes occurrence in fish processing environments12. Thereby, an efficient self-checking system alongside adequate official control measures are crucial for L. monocytogenes prevention in the production of these risky products.Increased incidence of the severe food-borne disease listeriosis has been reported in Finland and elsewhere in Europe in the 2010s13. The execution of official control must be regular, risk-based, and sufficiently frequent14. Producing vacuum-packaged gravad or cold-smoked fish is considered a highly risky operation requiring 5 to 11 annual inspections15.In Finland, the official food control system is based on European Union food safety legislation complemented by a national act of legislation and decrees notified to the EU. The entire food safety field is overseen by the Finnish Food Safety Authority (Evira) working under the Finnish Ministry of Agriculture and Forestry. Authorities at regional administrative offices supervise and audit the municipal food control units, where official food safety inspectors are employed. Fish-processing plant inspections are carried out by the municipal authorities. If non-compliances of food safety legislation are observed, inspectors can give advice, demand corrections, or use enforcement measures to remove the observed violations16. Food business staff and management may also underestimate food safety risks18. Reportedly, Finnish fish processing plants perceive the food safety risks related to their operations smaller than other food processing plants19, although the processing of vacuum-packaged ready-to-eat fish products evidently contains a risk of L. monocytogenes. In order to elucidate the current state of listeria-related food safety violations and efficacy of official control, we conducted an investigation of ready-to-eat fish processing plants in Finland.Implementation of food safety practices has been shown to necessitate improvement among some fish industry operatorsL. monocytogenes in Finnish food business operators producing gravad and cold-smoked vacuum-packaged fish. Prior to our investigation, no detailed overview was available on the efficacy of official food control in ready-to-eat fish production. With statistical modelling, we identified features of official control and fish-processing plant compliance that associated with L. monocytogenes occurrence, such as deficiencies in processing machinery and correction of violations. Our results highlight ways of improving official food control that support L. monocytogenes prevention in the fish industry.Our aim was to study the efficacy of official control pertaining to occurrence, control measures, and correction of non-compliances predisposing to L. monocytogenes in the facilities and products of each FPP from January 2011 to December 2013. Inspected items, including non-compliances and official control measures, were also extracted from inspection reports .From all over Finland, 21 fish-processing plants (FPPs) producing vacuum-packaged gravad and/or cold-smoked fish participated in this investigation , and their quality varied between inspectors from detailed to cursory. In five FPPs, inspection reports were noticed missing because of discontinued content or reported missing by the inspectors due to either FPP moving from under the jurisdiction of one municipality to another, municipal control unit changing their data storage system, or irregular documentation due to lack of personnel. Thereby, calculation of the annual number of inspections for all FPPs was not possible from the inspection reports obtained. The planned and executed inspections, and inspections carried out during processing, were therefore inquired from the inspectors via an online questionnaire for the year 2014, for which these data could be reliably obtained during conduction of this study in 2015.In addition, we estimated the efficacy of municipal official food control through fulfilment of Finnish Food Safety Authority Evira requirements on number of annual inspectionsThe official inspection reports were read by one researcher (MA-A), and inspected items n = 2803) were extracted into an Excel-file. Each item was assigned the following initial variables: (a) FPP, (b) date, (c) sector of FPP self-checking system (Table 803 were 20 and the grouping (table of contents) used in the guidelines of the Finnish “Oiva” system for official food control21. The link of each non-compliance with L. monocytogenes spread, growth, or contamination was assessed at a 3-level scale  or did not occur (“listeria-negative”), were performed in SPSS version 25. The usage between parametric and non-parametric tests was based on Shapiro-Wilk’s test of normality.Univariate analyses concerning non-compliances and comparison of FPPs, where L. monocytogenes occurred in facilities and products during 2011–2013. GLM was carried out in R version 3.4.032 using ‘glm’  following the data exploration, analysis, and variable selection protocol described by Zuur et al. (2009 and 2010)34. Collinearity was checked using Pearson and Spearman correlation coefficients and Variance Inflation Factor (VIF): only variables with VIF < 3 were included in the model fitting34. The model was initially fitted with eight covariates  proportion (%) of non-compliances given time limit for correction, (e) proportion (%) of non-compliances not corrected, (f) proportion (%) of non-compliances recurred, (g) proportion (%) of non-compliances related to processing equipment, and (h) proportion (%) of non-compliances given an official control measure by inspector. Backward selection based on likelihood ratio tests (‘drop1’) was used to remove variables while the residual deviance remained insignificant on the 0.05-level33. Overdispersion  was observed by dispersion test in ‘AER’ package35 and therefore quasi-Poisson regression was applied for the final model33. No observations exceeded Cook’s distance 1 and hence were not deemed influential36. Explained deviance (or pseudo-R²37) was calculated for the final model, which included intercept and three main effects.The quantitative data describing percentages of non-compliances and control measures for each FPP were used in a generalized linear model (GLM) to explain the number of times L. monocytogenes had occurred in the products or facilities of 9/21 FPPs  FPPs D–I, where contamination was occasional. FPPs A–C suffered from a continuous listeria problem, where L. monocytogenes was repeatedly found in products and on food contact surfaces from several rooms and processing machines. This indicates that the contamination was widespread throughout the processing environment and could have been persistent. In FPPs D–I, L. monocytogenes contamination rarely occurred in products or food contact surfaces and was evidently sporadic. L. monocytogenes did not occur in FPPs J–U  from all studied FPPs, the majority (83%) concerned operations and facilities, whereas 12% concerned plans and 5% documentation. When classified according to sectors of the FPP self-checking system  followed by a demand for correction and less often (11%) by advice without demands. The proportion of non-compliances followed by a demand for correction varied considerably (43–100%) among FPPs. No request for official control measures was given by the inspector for 9% of the non-compliances, while an additional 1.1% did not require intervention from the inspector as the FPP itself reacted. Enforcement measures had been used once in two FPPs during the investigated period. Re-inspection was mentioned in the official inspection reports for 65% of the non-compliances, and out of these, on average 51% were completely and 18% partially corrected during the study period. A third (33%) of the non-compliances recurred: some up to 16 times but generally two to three times.Inspectors gave demands for correction more often for non-compliances posing a listeria risk (either “indirect” or “direct”) than for other non-compliances , but no significant difference was observed in their correction . Furthermore, the higher the deemed listeria-risk was on the scale “no risk–indirect–direct”, the significantly shorter were the time limits given for correction . However, no significant differences were observed in the correction of non-compliances with regard to presence or absence of time limits .L. monocytogenes findings had led to corrective actions initiated by the FPP and required no further intervention from official control, whereas at another 3/9 FPPs, corrective actions for L. monocytogenes were in all cases initiated by a demand for correction from the inspector. In FPPs A, B and C where L. monocytogenes most frequently occurred, 88–100% of L. monocytogenes findings required demands from inspectors to initiate the control measures.In 3/9 of the listeria-positive FPPs, all reported L. monocytogenes occurred in the FPPs during 2011–2013  recurred, (2) concerned processing machinery, and (3) did not result in inspectors requesting official control measures  also reported multiple concurrent non-compliances in fish processing plants, several of which could predispose to outbreaks44. In the current study, prevalence of non-compliances predisposing to L. monocytogenes was somewhat higher in listeria-positive than negative FPPs. Manifestation of such non-compliances in listeria-negative FPPs, however, indicates that not only the presence of non-compliances but also practices of dealing with them can explain the differences in L. monocytogenes occurrence.Finnish FPPs have stated they are able to correct the food safety issues pointed out by inspectorsL. monocytogenes concentrated, were also the sectors inspected more often than others. Thus, inspectors seem to have focused on risky areas, where violations were common. They had evidently also assessed the risks of non-compliances: correction was more often demanded and shorter time-limits given to non-compliances we estimated predisposing to L. monocytogenes to a greater extent. However, correction rates were similar despite estimated listeria risk, and in many FPPs, overall non-compliance recurrence was relatively high and correction rate low. Official control measures consisted mainly of demands for correction, which were generally not strengthened by enforcement measures. The infrequent use of enforcement measures by food control authorities has been discussed by several studies46. Although the enforcement processes can sometimes be lengthy, compliance has been reported for the majority of violations indicative of their utility for prolonged non-compliances45. Recurrence may also reflect more the difficulty FPPs experience with non-compliances than imply inefficacy of time limits or demands, as time limits have previously been associated with successful correction and smaller occurrence of violations46. Long enough time periods may be important for correction40, and thereby, time limits should consider both the FPP resources and the severity of the non-compliance. Ideally, re-inspection should be carried out within the time limit to ensure its efficacy. A protocol for using time limits and re-inspections for all observed non-compliances was recently introduced by the Finnish food control system reform “Oiva”, where follow-up inspections and enforcement measures ensue until non-compliances impairing or jeopardizing food safety have been corrected21. The potential of the reformed system in strengthening the elimination of violations requires further investigation.The sectors of the self-checking system where majority of non-compliances predisposing to L. monocytogenes (FPPs J–U), sporadic findings (FPPs D–I), or a continuous L. monocytogenes problem (FPPs A–C). Since raw fish occasionally contains L. monocytogenes, it is likely that contamination sometimes occurs50. However, persistent contamination often develops in the processing environment and machinery, through which widespread contamination of the end products may ensue53. Stringent cleaning has proven successful in eliminating contamination11, but can only be performed if surfaces remain in good condition. Dirty processing machinery in poor condition undoubtedly played a role in sustaining a continuous, potentially persistent contamination at FPPs A–C. Despite a relatively small sample size for statistical power, we were able to connect L. monocytogenes occurrence at FPPs to the functioning of their self-checking systems and official control. As we exhibited through generalized linear modelling, the larger the proportion of processing-machine-related non-compliances was, the higher was the L. monocytogenes occurrence in the FPPs. Strengthening the self-checking system and official inspection efforts for cleanliness, maintenance, and hygienic handling of the processing machines can help reduce the amount of L. monocytogenes findings particularly in FPPs with potentially persistent contamination.In this study, FPPs had either no occurrence of L. monocytogenes occurrence correlates positively with recurring non-compliances and inversely with those followed by inspector requesting official control measures. This indicates that efficiency in handling all types of non-compliance can reflect upon L. monocytogenes control. If correction is not required for all violations, FPPs may develop a careless attitude even towards the severe ones. We observed that FPPs A–C suffering from continuous listeria problems and widespread contamination demonstrated mistrust against official control, which was not observed in FPPs D–I with occasional contamination nor in listeria-negative FPPs J–U. This finding indicates that lack of mutual understanding may have been at the root of some problems, and FPPs with continuous, potentially persistent contamination may need more encouragement and building of trust from the part of the inspecting authorities. The attitudes of food business operators have been shown to affect processing hygiene and relationship with authorities56. Better understanding of food safety risks and how they reflect upon the business can help appreciate official control57, but knowledge alone may not lead to change of behaviour in food handlers, if management, resources and infrastructure do not support it18. Since listeria control depends on continuous hygienic practices, it needs to be incorporated into everyday routines, and employees must understand its importance58. Professionalism and ability to negotiate from the part of the inspector motivates food business operators to perform the demanded corrections57. Thereby, opportunities for participation in advanced food control training must be ensured for all FPP inspectors: training on building rapport, generating motivation, facilitating behavioural change, and achieving compliance through persuasive communication could be useful for dismantling frustrating situations, which listeria problems and recurring non-compliances often are.Our results also show that L. monocytogenes occurrence is, the more important it is to reduce non-compliances related to processing machinery, demand correction of all non-compliances and prevent their recurrence. Both current EU legislation59 and rules entering into application in December 201960 require verification of efficacy and corrective actions from risk-based official control, and oblige the inspection of hygiene conditions, food processing, machinery, and cleaning and maintenance procedures. Thereby in light of our results, improving L. monocytogenes prevention by means of official control pertains more to enhanced implementation than amendments to the legislative framework itself. History of non-compliances regarding machinery, hygiene, and sanitation should lead to increased inspection frequency by food control authorities. Timely correction of violations should always be demanded, and efficient methods ensuring lasting removal of non-compliances need to be developed and adopted. These could include establishing a systematic re-inspection protocol such as the “Oiva” system21, improving supervision of time limits, enhancing collaborative communication between FPPs and inspectors, and opportune use of enforcement measures.Our main findings indicate that the higher the L. monocytogenes prevention. Non-compliances predisposing to L. monocytogenes were common in many fundamental operations of FPPs and particularly in processing machinery. Official control had focused inspection efforts and control measures towards risky operations, but non-compliances recurred relatively often despite demands for corrections and given time limits, which indicated insufficiency of current methods and communication. L. monocytogenes occurrence can be reduced by always requiring official control measures for non-compliances as well as applying more stringent surveillance of their execution, paying particular attention to non-compliances regarding machinery, hygiene, and sanitation. Recommendations based on our results are applicable to fish processing as well as other food industry areas where L. monocytogenes control is crucial.Our study was the first in-depth investigation on the efficacy of official control in the production of ready-to-eat fish products. We identified control efforts that need improvement and showed that inefficiency of official control can associate with poor Supplementary Table S1Supplementary Table S2


N = 837). Sex-stratified associations between dietary FA and blood lipids were assessed by linear regression in substitution models which represented isocaloric replacements of CHO with saturated FA (SFA), monounsaturated FA (MUFA), n-3 polyunsaturated FA (PUFA) or n-6 PUFA. To assess the interactions with PA, analyses were then performed stratified by tertiles of different PA levels ). Both sexes presented a significant inverse association between MUFA and triglycerides, and females a direct association between n-3 PUFA and high-density lipoprotein. Stratifying by PA tertiles, associations were mainly restricted to participants with the lowest levels of lifestyle PA, or the highest time spent sedentary. The effects of dietary FA on the lipid profile vary in an activity-specific manner, emphasizing possible synergistic roles of diet and PA.The role of consuming different types of fatty acids (FA) at the expense of carbohydrates (CHO), on the blood lipid profile of adolescents is largely unknown, as is the modulating effect of different levels of physical activity (PA). Children from the GINIplus and LISA birth cohorts, with complete data on dietary FA (assessed by food-frequency questionnaires), objectively-measured PA (assessed by accelerometers) and blood lipids (lipoprotein cholesterol and triglycerides) at age 15 years, were included ( Disturbed blood lipid profiles, characterized by low levels of high-density lipoprotein (HDL) cholesterol, and high levels of low-density lipoprotein (LDL) cholesterol and triglycerides (TAG), can be observed as early as childhood and are believed to track into adulthood ,2, repreWith respect to the role of diet in lipoprotein metabolism and CVD risk, fats and carbohydrates (CHO) are frequently discussed in adult populations ,7,8. In We have previously observed that theoretically replacing dietary SFA with the same calories from CHO resulted in increased LDL cholesterol, TAG and total to HDL cholesterol ratio  levels in females . This suTo our knowledge, the integrated role of dietary fatty acid intake and PA with respect to blood lipids during adolescence has not been addressed. Therefore, the present study aimed to assess the association of different types of dietary fatty acids  with blood lipids in a large population of 15-year-olds; as well as the possible modifying role of different levels and duration of objectively-measured PA.N = 5991), and additionally in Leipzig and Bad Honnef between 1997 and 1999 for the LISA study . Both cohorts are population-based, however, GINIplus consists of an observation arm and an intervention arm. For the intervention arm, newborns with a family history of allergy were invited and randomized to receive one of three hydrolyzed formulas or cow’s milk. The aim was to compare the effect of the different formulae on allergy development. Participants who declined to participate in the intervention trial were included in the observation arm, along with those with no family history of allergy. Information on selected exposures and health outcomes was collected by means of questionnaires and medical examinations, carried out at various follow-up assessments. The collection of data relevant to the present analyses is described in detail below. GINIplus and LISA were approved by their local ethics committees  and written consent was obtained from all participants’ families. Since PA was assessed in Munich and Wesel only, this analysis is restricted to subjects from Munich (N = 4413) and Wesel (N = 3390).The present study was based on data obtained from 15-year-old adolescents participating in the GINIplus  and LISA (Influence of Life-style related factors on the development of the Immune System and Allergies in East and West Germany) German birth cohort studies . DetailsBlood samples were collected at the 15-year follow-up physical examination. The concentrations (mmol/L) of total cholesterol, LDL cholesterol, HDL cholesterol, and TAG were measured in serum using homogenous enzymatic colorimetric methods on a Modular Analytics System from Roche Diagnostics GmbH Mannheim according to the manufactures instructions (Roche Diagnostics GmbH Mannheim). External controls were used in accordance with the guidelines of the German Society of Clinical Chemistry and Laboratory Medicine. The ratio of total cholesterol to HDL cholesterol  was calculated by dividing total cholesterol by HDL cholesterol.Dietary intake was assessed by means of a self-completed food frequency questionnaire (FFQ), designed to estimate food and nutrient intake over the past year, and specifically for the estimation of total energy, fatty acid and antioxidant intake in school-aged children. Briefly, the FFQ comprised a list of 80 food items, for which participants were asked to report average consumption frequency and portion sizes. A quality control procedure was applied based on recommendations by Willett, et al. for data cleaning in nutritional epidemiology , and is PA was measured at age 15 years using triaxial accelerometers , worn on the dominant hip for seven consecutive days. Participants for accelerometry were recruited from the study centers Munich and Wesel. This included all of the GINIplus cohort and 64% of the LISA cohort  taking part in the 15-year follow-up. The accelerometry protocol, data management and quality control have been described previously in detail ,38. Brie2-test for categorical variables.Statistical analyses were carried out separately for females and males. Participants were excluded from the analyses if they had a chronic illness affecting diet or if they presented outliers in either an exposure or outcome variable, visually identified using descriptive plots. Subject characteristics at age 15 years were described by means (standard deviation) and medians (25th–75th percentile) for normally-distributed and skewed continuous variables, respectively, or counts (%) for categorical variables. Differences in characteristics between females and males were tested by Student’s t-test and Wilcoxon rank sum test, for continuous variables , and by χRESID and MUFARESID) which were uncorrelated with each other [RESID was included in place of MUFA, along with all other covariates, and vice versa. All models were adjusted for potential confounders, including: Region , study , exact age at blood sampling (years), body mass index , in which case obtained from the 15-year follow-up questionnaire), total daily caloric intake , parental education , pubertal stage   [Linear regression was used to assess the association of different types of dietary fatty acids with blood lipid parameters . In order to assess associations as the theoretical replacement of dietary CHO with different fatty acids, a substitution model approach was applied, in which a model was fit including the different dietary fatty acids , and adjusting for all other energy bearing nutrients  with the exception of CHO (the nutrient being replaced). In this way, the energy intakes of fats, protein, and alcohol are held constant; and by additionally including total energy intake in the model it is possible to interpret the resulting coefficients for each fatty acid as its theoretical substitution for an equal amount of energy (% EI) from CHO, being the only energy-bearing nutrient not accounted for in the model. Given the high inter-correlation typically present amongst dietary components , we calcch other . To avoint scale : Early-;nt scale ,44. The Austria) .N = 9085), blood lipids, dietary intake and PA data were available from 1020 participants at the 15-year follow-up. Complete data for all adjustment variables were provided by 850 participants. Of these, nine participants were excluded due to chronic illness . Finally, four participants were excluded from the analyses as they presented clear outliers, visually identified using descriptive plots . Basic characteristics of the study population are described in The present study analyses were based on data from 837 participants . p-value = 0.049; males: MR = 0.87 , p-value < 0.001). Additionally, in females, n-3 PUFA was directly associated with HDL cholesterol levels , p-value = 0.002). Further adjusting for different levels of PA did not alter the observed associations. However, when adjusting for MVPA in males, a significant direct association between MUFA and HDL cholesterol was observed , p-value = 0.047), which in the main analyses was only borderline significant . Exact values obtained with additional adjustment for different PA levels can be found in Results of analyses assessing the effects on blood lipids of increasing levels of different dietary fatty acids at the cost of CHO are displayed in p-int = 0.045), and when assessing the association of MUFA with TAG in males (p-int = 0.045). Females in the highest tertile of sedentary time presented a significant positive association between n-3 PUFA and HDL cholesterol , p-value = 0.007). Similarly, females in the lowest tertile of lifestyle PA presented a positive association between n-3 PUFA and HDL cholesterol , p-value < 0.001), as well as with LDL cholesterol , p-value = 0.001). Furthermore, in this subgroup of low lifestyle PA, a significant inverse association was observed between SFA and TAG , p-value = 0.013) and between MUFA and TAG , p-value = 0.004). No association between fatty acids and blood lipids was observed in females when stratified by MVPA tertiles. In males, stratifying by tertiles of different levels of PA revealed a significant inverse association between MUFA and TAG only in the subgroups with either the highest sedentary time , p-value = 0.015) or the lowest lifestyle PA time , p-value = 0.015), as well as in the middle tertile of MVPA , p-value = 0.003).Results of the analyses stratified by tertiles of different PA levels are presented for females and males in N = 203, non-fasted N = 269), the effect size of the associations between MUFA and TAG remained almost unaltered regardless of fasting status, although associations were no longer statistically significant, possibly due to reduced power , p-value = 0.082; non-fasted: MR = 0.94 , p-value = 0.132). Males  in the fasted group indicated a stronger inverse association between MUFA and TAG than previously observed , p-value < 0.001), whereas the non-fasted group presented no association , p-value = 0.568). Furthermore, an association was observed in fasted males between SFA and TAG , p-value = 0.008). When considering only fasted individuals, the inverse associations between SFA and TAG and between MUFA and TAG in the lowest tertile of lifestyle PA, were even stronger than previously observed in both sexes , p-value = 0.004; MUFA in females: MR = 0.82 , p-value = 0.001; SFA in males: MR = 0.82 , p-value = 0.003; MUFA in males: MR = 0.77 , p-value < 0.001). The association between MUFA and TAG in males in the highest sedentary tertile was also stronger , p-value = 0.006), whereas no significant associations were present in any of the MVPA tertiles.In females  were evaluated using objectively-measured habitual PA data.We have previously found that replacing dietary SFA with CHO results in a prospective increase in LDL cholesterol, TAG, and total:HDL cholesterol levels in female adolescents . The oppOur stratified analyses indicated that PA modifies the association between dietary fatty acids and blood lipids in adolescents. In females, associations observed between MUFA and TAG were restricted to participants with the lowest levels of lifestyle PA. Females in this subgroup also presented an inverse association between SFA and TAG and positive associations of n-3 PUFA with LDL cholesterol and HDL cholesterol. Most of these associations are in line with existing data. A reduction in TAG levels following the replacement of CHO with SFA, is well documented . EvidencDifferences between females and males in the association between dietary fatty acids and blood lipids have been observed previously in the GINIplus and LISA population . The reaThe present study benefited from the thorough assessment of various crucial behavioral and health-related parameters, including habitual dietary intake, physical activity, and blood lipids, in a large sample of 15-year-old females and males. Our findings add to the limited understanding of the association between dietary fatty acids and blood lipids in adolescents, taking into account the often-neglected context of the nutrients they replace. This was addressed by means of substitution models, which despite being theoretical, allow for a more integrative and disentangled interpretation of the results than single-nutrient models. Generally, in population-based samples, increasing dietary fat tends to be accompanied by a simultaneous reduction in CHO , and henAnother strength of the present study was the use of accelerometers, which allowed for an objective assessment of habitual daily PA. This is a rarely-available method in large epidemiological studies, and permitted a minute-by-minute classification of daily activity into different intensity levels, as well as the definition of tertiles based on the amount of time spent in each PA level. The stratification of our analyses into these subgroups adds novel information to the poorly understood interaction between dietary fatty acids and physical activity  in lipoprotein metabolism.p = 0.032, data not shown). Finally, as often occurring in cohort studies, children of lower social classes were under-represented in our study population. Although we adjusted for parental education, our findings may not be representative of the study area. With this non-random loss-to-follow-up, adolescents in our study might present healthier blood lipid profiles as well as better lifestyle habits than the general population [It must be kept in mind that our findings are based on cross-sectional analyses, and hence the observed associations do not necessarily infer causality. Moreover, the assessment of diet and PA were not performed at exactly the same time, and hence analyses were not adjusted for season. Nevertheless, we observed no association between the season of dietary assessment and fatty acid intakes in bivariate analyses. An association was however observed between the season of PA assessment and lifestyle PA, with greater PA levels occurring in spring and summer compared to winter. We therefore cannot exclude the possibility of residual confounding by seasonal differences related to PA or lack thereof, such as sun exposure and differing levels of vitamin D, which have been suggested to play a beneficial role in glycemic control . Nonethepulation . The facOur findings suggest that replacing dietary CHO with MUFA (both sexes) or n-3 PUFA  may induce beneficial changes in the blood lipid profile of adolescents. Adolescents, and particularly females, who are the least active in their daily lives, seem to be more susceptible to changes in diet involving the substitution of dietary fatty acids for CHO. Our results highlight the complexity of the body as a biological system, where the effects of diet on the lipid profile might vary in an activity-specific manner, emphasizing possible synergistic roles of diet and PA, especially lifestyle PA.


In a logistic regression analysis patients age, LVEF, ST-segment-elevation MI and the presence of MVD were independent predictors of longer hospitals stay (≥8 days). During follow up, there were no significant differences in the rates of clinical events between patients with shorter (<8 days) and longer (≥8 days) hospitalization. In a real-life situation, the LOS, even in low-risk patients is much longer than recommended in the guidelines.According to guidelines, it is safe for low-risk patients with myocardial infarction (MI) to be discharged within 72 h of hospitalization. However, results coming from registries show that the hospital stay is often much longer in a real-life situation. Data on the length of the hospital stay (LOS) of MI patients in Polish centers are lacking. We enrolled 212 consecutive patients with acute MI. Low-risk patients were defined according to PAMI II criteria: age <70 years, left ventricular ejection fraction (LVEF) >45%, no persistent ventricular arrhythmia, and no multi-vessel disease (MVD). The median of the hospitalization length was eight days . In low-risk patients (25%), the median of LOS was six days  ( Risk stratification is crucial in the management of patients with acute myocardial infarction (MI). It allows to implement tailored medical strategy and improve outcome. Previous studies are showing the safety of discharge within 72 h of hospitalization in low-risk patients after an uncomplicated MI ,4,5,6. AThe presented study is a retrospective analysis of consecutive patients diagnosed with acute MI undergoing primary percutaneous coronary intervention (PCI), hospitalized between September 2016 and July 2017 in a single center. Both ST-segment elevation MI (STEMI) and non-ST-segment elevation MI (NSTEMI) patients were included. During the study period, the total number of patients hospitalized with acute MI was 261. Among these patients, eight (3%) did not undergo invasive strategy with coronary angiography, six (2%) patients were referred to coronary artery bypass grafting (CABG), and 35 (14%) were diagnosed with MI with non-obstructive coronary arteries (MINOCA). These patients were excluded from analysis. Finally, 212 patients were enrolled .The study was approved by the Institutional Ethical Board of Jagiellonian University . Patients baseline characteristics and in-hospital course were analyzed according to risk and hospitalization length categories. Low-risk patients were defined according to the criteria described in the second Primary Angioplasty in Myocardial Infarction trial (PAMI II): age <70 years, left ventricular ejection fraction (LVEF) >45%, no persistent ventricular arrhythmia, and no multi-vessel disease (MVD) [U test  or unpaired (two-sample) Student’s t-test  were applied for continuous variables. The χ2 test was used for categorical  variables. A multivariable logistic regression was constructed to identify independent predictors of longer hospital stays . All baseline variables were considered, and forward selection with a probability value for covariates to enter the model was set at the 0.05 level. Results are presented as odds ratios (OR) with an associated 95% confidence interval (CI). The rates of all-cause and cardiovascular death, recurrent MI, and stroke were assessed during the hospital stay, and follow-up and analyzed in relation to the risk and hospitalization length categories. Cardiovascular mortality included death resulting from acute MI, heart failure, stroke, cardiovascular procedures, and sudden cardiac death. Composite endpoint was defined as a combination of cardiovascular mortality, non-fatal recurrent MI, and non-fatal stroke. Non-fatal MI and non-fatal stroke were defined as an episode with survival until hospital discharge. Rates of events assessed during follow-up, included also these which occurred during the index hospitalization. Survival was estimated by using the Kaplan–Meier method, according to the risk and hospitalization length categories with the log-rank test for the full follow-up time available. The level of statistical significance was set at alpha value <0.05. All analyses were performed with SPSS Statistics 24 .Quantitative variables were described using means and standard deviation , or median with interquartile (Q) range (Q1 and Q3) . Quantitative data with normal distribution included: age and glomerular filtration rate (GFR). Non-normal distribution of data presented hospitalization duration and LVEF. Categorical variables were presented as percentages. The Mann–Whitney p < 0.001). Only one patient from the low-risk group was discharged during first 72 h of hospitalization. Hospital discharge within four and five days of hospitalization occurred at rates of five (9%) and 25 (46%) low-risk patients respectively. A histogram of the lengths of hospital stays across the study population is presented in Baseline characteristics are shown in By definition, low-risk patients were younger, with higher LVEF, and did not have MVD and ventricular arrhythmias during hospital stay . Low-risIn In Follow-up was obtained in 200 (94%) patients. The median duration of follow-up was 10  months. Patients from the low-risk group had lower rates of all-cause mortality, cardiovascular mortality, and combined endpoint during an in-hospital course and follow-up, compared to higher-risk patients .There were no significant differences in rates of clinical events occurring in patients with shorter (<8 days) and longer (≥8 days) hospitalization . A KaplaOur most important finding is that in a real-life scenario, the LOS in low-risk patients appeared to be much longer compared to that recommended in the guidelines. In the present study, only one patient from the low-risk group was discharged during the first 72 h of hospitalization.Hospitalization length after MI has been significantly declining over the years. In the 1970s, an average LOS after MI was about 2–3 weeks ,12. LateOn the other hand, data coming from registries are showing that in a real-life situation, the hospital stay is often much longer. There is also a large variance in hospitalization length between different regions in the world ,8,9. A CIn our analysis patients, clinical characteristics explained 23% of the variance in LOS, which is consistent with previous studies. Also, in the above analyses, a large amount of unexplained variance suggests presence of additional factors influencing LOS, which are not included in a typical risk assessment.It is worth mentioning that the PAMI II criteria of low-risk MI patients were established in 1998. The introduction of primary PCI, advancements in pharmacological treatment, and better management of risk factors improved the survival of cardiovascular patients, but also led to a higher age of patients presenting with first or recurrent MI. Nowadays, patients with MI are much older, and with more comorbidities compared to those from 20 years ago, when the PAMI II criteria were designed. Frailty, mental status and comorbidities may increase the risk of in-hospital complications, nosocomial infections, and in the end, affect LOS . PossiblStudy limitations: The presented analysis is a retrospective registry, but it represents real-world data, as consecutive patients were enrolled. Due to its retrospective character, several factors, including biomarkers or additional parameters from echocardiographic examination, were not available. The study is an observational analysis without randomization. The sample size and rates of clinical events in this study are relatively small, which limits other variables that can be incorporated into the regression analysis, and the final conclusions. A follow-up observation was not completed in 100% of patients, and it was relatively short.In real-life situations, the LOS in low-risk patients appeared to be much longer than those recommended in the guidelines. The patients’ characteristics associated with the risk assessment explain the minority of variance in the hospitalization length, thus suggesting an impact of other factors that is not included in typical risk scores.


Helicobacter pylori infection, and gastric epithelial cell proliferation, all of which may be mediators between salt and gastric precancerous/cancerous lesions. These potential mediators were measured using gastric biopsies as: (a) the density of polymorphonuclear and mononuclear cells (gastric inflammation), (b) mucus depletion , and (c) the severity of H. pylori infection. Salt intake was measured with spot urine samples (using urinary sodium/creatinine ratios), self-reported frequency of adding salt to food, and as total added salt. Results: The average sodium/creatinine ratio (at baseline and post-treatment at five months) was associated with increased epithelial damage over the 12-year follow-up period among those with a greater severity of chronic inflammation and among those with continued H. pylori infection after treatment at five months. This association was stronger when both severe gastric inflammation and H. pylori infection were present at five months . Conclusion: In humans, salt was associated with an increase in epithelial damage in stomachs with more severe previous H. pylori-induced chronic inflammation.Background: The process by which salt affects the gastric precancerous process has not been adequately studied in humans. Methods: We investigated the effects of salt on gastric inflammation, epithelial damage, the density of  Gastric cancer is the third leading cause of cancer deaths worldwide ,2. In thThere has been a steady decline in the incidence and mortality of gastric cancer since the first half of the 20th century. This decline correlated with the increased use of refrigeration and decreased use of traditional methods of food preservation, such as salting . This leH. pylori infection and/or gastric epithelial cell proliferation, all of which may be involved in gastric carcinogenesis.Some epidemiological studies have reported an association between salt intake and advanced lesions (dysplasia or gastric cancer) ,24,25,26H. pylori infection density and gastric epithelial cell proliferation) in the gastric precancerous pathway, which may subsequently increase the risk of gastric cancer [H. pylori infection may modify the association between salt intake and gastric precancerous lesions and/or gastric cancer [In animal models, there are reports that salt may affect potential mediators  [Hence, in the current analysis, we explored the associations between salt intake and potential mediators of the gastric precancerous process  . H. pylori infection, all of which are thought to affect the association between salt intake and gastric precancerous progression. In our study, 399 participants provided spot urine samples for the estimation of a sodium/creatinine ratio and 296 participants provided self-reported salt intake measures at baseline. In H. pylori infection (after treatment) at the five-month follow-up   .H. pylori infection and epithelial damage. We observed the sodium/creatinine ratio to be associated with an increase in epithelial damage over the 12-year follow-up period. For every mmol/cg increase in the sodium/creatinine ratio, there was a 0.337 increase in the epithelial damage score in the presence of severe prior gastric inflammation (indicated by the higher density of mononuclear cell infiltrate) at the post-treatment five-month follow-up, particularly among those infected with H. pylori  . When both severe gastric inflammation and H. pylori infection were present at the post-treatment five-month follow-up, the observed effect of the urinary sodium creatinine ratio on epithelial damage at the 11–12-year follow-up was even stronger   gastric inflammation and epithelial damage, and (b) density of , 0.625) . Similar, 2.122) . For eveSalt intake, measured either as the self-reported frequency of adding salt to food or as the amount of salt added to food, showed observed effects on epithelial damage which were less precise compared to the estimates from urinary sodium/creatinine ratios, but the estimates were in the same direction .H. pylori infection at the five-month post-treatment follow-up and the estimated effect tended to be stronger if the density of H. pylori infection was more severe at five months. Furthermore, the presence of prior (five-month) gastric inflammation (mononuclear cells) was also associated with an increase in the estimated effect of salt on epithelial damage at a later stage (11–12 years). The estimated effect of salt intake on epithelial damage at a later stage was even stronger when both severe H. pylori infection (at five months) and gastric inflammation (mononuclear cells at five months) were present. These findings suggest that salt intake in the presence of H. pylori or gastric inflammation may increase epithelial damage at a later stage and that this association is stronger when both severe H. pylori infection and gastric inflammation (mononuclear cells) were previously present. This estimated effect may possibly be due to the potential interaction between salt and H. pylori infection to increase gastric carcinogenesis, as previously suggested in a study with Mongolian gerbils [H. pylori infection. Salt intake may increase epithelial damage at a later stage, particularly in the presence of severe H. pylori-induced gastric inflammation, which may then increase the progression to advanced gastric precancerous lesions (dysplasia) or cancer. We observed that salt intake measured at the five-month follow-up (after treatment) was associated with increased epithelial damage over the 12-year follow-up period, especially among those with more severe  gerbils . This is gerbils , where wH. pylori infection and epithelial damage) were observed in our study with 11–12-year follow-up, which is also consistent with the results of previous animal studies [H. pylori infection [H. pylori infection, and epithelial damage in humans over time, although these associations were not precise. Associations between salt intake during the first five months of follow-up and the outcomes  were done in the original cohort study. Third, we measured the outcomes  using a standardized scoring method (0 (none)–3 (severe)) without objective units. Finally, salt intake was measured as the urinary sodium/creatinine ratio from spot urine samples and self-reported dietary intake. Self-reported measures may not accurately reflect the total intake of salt as the participants may not be aware of the amount of salt added while cooking or naturally present in the food items. Also, sodium/creatinine ratios may not reflect the life-long intake of salt. However, in the Colombian population, individual dietary habits may be more consistent over time than in developed countries. Urinary sodium/creatinine ratios have been reported by multiple studies to be more accurate in measuring salt intake than self-reported measures because participants may not be aware of the amount of salt present in the foods they consume [Our study has some limitations. First, the misclassification of study outcomes may have occurred because the measures of gastric inflammatory cells, epithelial damage and density of  consume ,48,49,50H. pylori infection and gastric epithelial cell proliferation were conducted in animals; here, we estimated the effects in humans. Finally, this was a cohort study with long-term follow-up, which enabled us to study changes over adequate time to estimate the effects of salt intake on histological changes. Despite these limitations, our study has several strengths. First, we used multiple approaches to measure salt intake because each approach may have its own limitations, and included an objective measure which does not rely on self-reports. Second, previous studies estimating the effects of salt intake on gastric inflammation, epithelial damage, the density of H. pylori infection and gastric epithelial cell proliferation), and the outcomes (gastric precancerous lesions/cancer) to evaluate and quantify the potential pathways of gastric carcinogenesis followed over adequate time [The outcomes in this study are potential mediators in the pathway between salt intake and gastric precancerous lesions/gastric cancer. Future studies should conduct formal mediation analyses using methods developed by Van der Weele  in cohorate time . It shouH. pylori infection or who had baseline dysplasia, gastric cancer, or ulcers were excluded from the clinical trial, but not from the long-term follow-up assessment (11 to 12 years post-baseline) [In the current paper, we analyzed data from a prospective cohort study conducted in Pasto, Colombia, that consisted of a 16-week randomized clinical trial in which study participants were randomly assigned to one of the following five treatment regimens: (a) metronidazole, amoxicillin and bismuth subsalicylate for the first two weeks and bismuth subsalicylate alone for the next 14 weeks; (b) calcium carbonate (weeks 1–16); or (c) treatment regimens (a) and (b), (d) tetracycline (weeks 1–16), or (e) placebo (weeks 1–16) . The paraseline) . Salt intake evaluated using dietary recall is problematic as salt intake in food varies and most people do not know the amount of salt contained in their food. Therefore, we focused on a more objective measure using sodium creatinine ratios in addition to other measures of salt intake. We measured salt intake in the following three ways: (1) the ratio of urinary sodium/creatinine, (2) self-reported frequency of adding salt to food, and (3) collecting salt added to food. At baseline, five-month follow-up post-treatment, and 11–12-year follow-up, urine samples from the first void of the day were obtained from all participants during the visit after an overnight fast. Sodium was determined by flame photometry, while creatinine was measured by the Jaffe reaction . We estiAt baseline, research personnel interviewed the participants regarding the frequency with which the study participant, or any individual who cooked in their household, added salt to food; the self-reported frequency was categorized into the following groups: rarely or never adds salt to foods, occasionally adds salt to foods, and always or frequently adds salt to foods. Lastly, we estimated total salt added to food by asking the participants to add the same quantity of salt added to food to a container. At follow-up visits, the amount of salt in the container was measured for analysis.H. pylori infection, gastric inflammation and epithelial damage were measured from biopsies for the entire cohort at each time point [H. pylori infection measured from an average of four biopsies at baseline, four biopsies at the five-month follow-up, and six biopsies at the 11–12-year follow-up. We measured gastric inflammation as the average and maximum (a) density of the polymorphonuclear leukocyte infiltrate, (b) density of the mononuclear leukocyte infiltrate, and (c) combined density of the polymorphonuclear leukocyte infiltrate and the mononuclear leukocyte infiltrate across biopsies. We measured gastric epithelial damage as the average and maximum mucous depletion measured across biopsies. The study pathologists measured the density of H. pylori infection using a modified Steiner stain [H. pylori infection from 0–3, where 0 represented the lowest density of gastric inflammatory cell infiltrate, the lowest density of H. pylori infection and the lowest degree of epithelial damage .me point . The biome point . For eacme point . These her stain . For eacH. pylori density) and not the progression itself. However, since the literature lacks information regarding factors which may confound the association between salt intake and each potential mediator, we adjusted for potential confounders (identified based on the existing literature) for the association between salt intake and gastric precancerous progression. We identified socioeconomic status [We identified potential confounders by creating Directed Acyclic Graphs  based onc status , fruit ac status  and age c status  as potenH. pylori infection density across biopsies; (b) the change in the average and maximum gastric inflammation, epithelial damage and H. pylori density infection across biopsies from baseline to the 11–12 year follow-up; and (c) how epithelial damage would be influenced by prior gastric inflammation and/or density of H. pylori infection across biopsies (two-way and three-way interactions), since both gastric inflammation and epithelial damage may be necessary for progression in the gastric precancerous process. To achieve the above-mentioned objectives, we conducted linear regression analyses, adjusting for the following potential confounders identified by Directed Acyclic Graphs [H. pylori infection as the outcome) among those who did not have H. pylori infection at the five-month post-treatment follow-up, those who continued to be infected with H. pylori at the five-month post-treatment follow-up, and those who were persistently infected with H. pylori at both the five-month post-treatment and the 11–12-year follow-up. This was done to examine if H. pylori infection status affected the associations between salt intake and the outcomes . For each analysis, we estimated beta-coefficients, which in our study, would estimate the effect of a one-unit increase in salt intake on the outcomes. For example, for the estimated effect of salt intake (using sodium/creatinine ratios measured in mmol/cg) on chronic inflammation , the beta-coefficient can be interpreted as an estimate of the change in the chronic inflammation score per mmol/cg of the sodium/creatinine ratio. We used 95% confidence intervals to estimate the precision of these estimates. Consistent with recommendations by the American Statistical Association, our analysis focused on effect estimation rather than statistical significance testing as the appropriate analytic goal [Our objective was to estimate the effects of salt intake  on the following: (a) the average and maximum gastric inflammation, epithelial damage and c Graphs : age, sotic goal ,56.H. pylori infection. Epidemiologic cohort studies with larger sample sizes are needed to further understand the mechanisms by which dietary salt may contribute to the development of gastric cancer. Understanding how salt affects gastric carcinogenesis may be helpful in public health efforts to prevent gastric cancer. Our current results provide preliminary evidence in humans that salt intake may lead to an increase in epithelial damage, especially among those with earlier gastric inflammation and/or


Epicardial fat thickness (EFT) is an emerging cardio-metabolic risk factor and has been shown to be related to atherosclerosis. EFT has not been studied in the context of CAH. This study aimed to evaluate EFT in children with CAH and its relation to carotid artery intima-media thickness (CA-IMT) and left ventricular (LV) functions.Thirty-six children with classical CAH were compared with 36 healthy controls. All patients had confirmed CAH and were receiving steroid substitution therapy. Patients and controls underwent anthropometric evaluation, measurement of fasting lipids, glucose, insulin, homeostasis model assessment for insulin resistance (HOMA-IR). LV functions and EFT were assessed using conventional echocardiography. Duplex ultrasonography was used to measure CA-IMT.Compared to controls, patients had greater EFT (p=0.001), CA-IMT (p=0.01), LV mass index (LVMI) (p=0.001) and prolonged mitral deceleration time (DcT) (p=0.01). CAH patients also had significantly worse HOMA-IR (p=0.001) than controls. Abnormalities were worse in uncontrolled CAH on treatment. Multivariate analysis in CAH subjects showed EFT correlated positively with waist circumference odds ratio (OR) , 17-hydroxyprogesterone , testosterone concentration , LVMI , mitral DcT  and CA-IMT .EFT is elevated in children with classical CAH, particularly in those with poor control, and is correlated with CA-IMT, LV mass and mitral DcT. Measurement of EFT in CAH children may help to identify those at high risk of developing LV dysfunction and subclinical atherosclerosis. What is already known on this topic?There is an increased risk for cardiac abnormalities in children with congenital adrenal hyperplasia. Epicardial fat thickness is an emerging cardio-metabolic risk factor and has been shown to be related to atherosclerosis.What this study adds?To our knowledge, this is the first study assessing epicardial fat thickness (EFT) in children with congenital adrenal hyperplasia (CAH). EFT is higher in children with CAH than in healthy children and correlated with carotid intima media thickness, left ventricular mass and mitral deceleration time. EFT may be used as a possible marker of early atherosclerosis and myocardial function in children with CAH.Congenital adrenal hyperplasia (CAH) is an autosomal recessive condition resulting from mutations in enzymes required for adrenal steroid synthesis . DefectsFew studies have utilized carotid artery intima-media thickness (CA-IMT) to assess vascular structural changes in children with CAH ,5,7. A hThe aim of this study was to evaluate the EFT measurement and its relation to CA-IMT and left ventricular function in a cohort of children with classical CAH.This cross-sectional, controlled study included 36 children  with a confirmed diagnosis of classic CAH . SW patiThe study protocol was approved by the Local Ethics Committee of Assiut University Children Hospital, Assiut, Egypt  and also by the Ethics Committee of the Faculty of Medicine, in accordance to the Declaration of Helsinki. Written informed consent was obtained from the parents of all participants.2. BMI was expressed as standard deviation scores (SDS) using the Egyptian Growth Reference Data , height, weight and body mass index (BMI). Systolic BP (SBP) and diastolic BP (DBP) were measured in all subjects in the right arm with a standard sphygmomanometer (Exacta) by the same operator. Height and weight were measured using a wall-mounted stadiometer (seca 213 l) and a calibrated weight scale , with the child wearing underwear only. BMI was calculated using the following formula: BMI=weight (kg)/height (m)nce Data . Waist cnce Data . A radionce Data . This asnce Data .Blood samples were drawn after an overnight fast for at least 12 hours at 8.00-10.00 a.m. before the first dose of steroids for assessment of serum levels of total cholesterol (TC), triglycerides (TG), high density lipoprotein-cholesterol (HDL-C), low density lipoprotein cholesterol (LDL-C), glucose and insulin. Serum TG and TC were assessed by quantitative enzymatic colorimetric technique . Serum high-density lipoproteins (HDL) were measured by the phosphotungstate precipitation method . LDL cholesterol was calculated by Friedewald’s formula: (TC) - (HDL-C) - 1/5 (TG) . IR was HOMA-IR=Fasting insulin (uU/mL) multiplied by fasting glucose (mmol/L) divided by 22.5. A cut-off level of 2.7 was used for diagnosing IR as previously described . ACTH, pAll echocardiographic examinations were performed according to the recommendations of the American Society of Echocardiography . A PhiliA two-dimensional (2D) echocardiogram, using a standardised procedure, was performed with the patient in the left lateral decubitus position. EFT thickness was measured by an experienced pediatric echocardiologist, who was blinded to the subjects’ clinical and demographic data, using the procedure validated by Iacobellis et al . EFT wasAll participants underwent an ultrasound scan to measure CA-IMT. The studies were performed in the morning between 7:30 and 9:30 a.m. after the children had fasted overnight. All ultrasound scans were performed by an experienced vascular operator who was unaware of the subject’s clinical details. Examination of CA-IMT was manually performed using a color duplex flow imaging system . The examinations were performed while the patients were in a supine position, with their necks slightly extended and their heads turned 450 away from the examination side. From both sides of the head, three images were obtained from the distal common carotid artery, 1-2 cm proximal to the carotid bulb at end diastole. These images were then stored for later offline analyses. All studies were done according to a predetermined, standardized scanning protocol for the right and left carotid arteries . All meaStatistical analysis was performed using the Statistical Package for Social Sciences (SPSS) for Windows, version 16.0 . Data were expressed as means + standard deviation. Comparisons of quantitative variables between the study groups were made using the paired Student t-test. Correlations between EFT and demographic, clinical, and laboratory variables were assessed using Pearson test. Multiple logistic regression analysis was used to determine the factors that were significantly associated with high EFT. The odds ratios, 95% confidence intervals and significances were calculated. For all tests, values of p<0.05 were considered statistically significant.Demographic and anthropometric data of the patients and controls are shown in Laboratory data of the patients and controls are shown in The results of echocardiographic EFT and CA-IMT examinations are shown in EFT thickness showed a statistically significant positive correlation with BMI, waist circumference, SBP, DBP, HOMA-IR, hsCRP, 17-OHP, testosterone, LVMI, mitral DcT and CA-IMT see .Multivariate analysis in children with CAH revealed that EFT was significantly correlated with waist circumference, 17-OHP, HOMA-IR, testosterone, mitral DcT, CA-IMT and LVMI see .This study demonstrates that a) children with classical CAH may have subclinical LV hypertrophy, diastolic dysfunction and subclinical atherosclerosis; b) EFT was higher in patients with CAH than in the healthy controls; c) EFT is correlated to carotid intima media thickness, LV mass and mitral DcT suggesting that EFT may be used as an additional marker of endothelial and myocardial dysfunction in children with CAH. The classical cardiovascular risk factors in children with CAH, namely obesity, hypertension, dyslipidemia, steroid treatment and others have been extensively discussed elsewhere ,5,6.To our knowledge, this study is the first to demonstrate that EFT was significantly increased in children with classical CAH compared with control children (p<0.001). In addition, we showed that EFT was correlated positively with CA-IMT. The multiple linear regression analysis showed that the CA-IMT was the variable that most influenced EFT. EFT was reported to be increased in children with a positive family history of type 2 DM and has been suggested as a risk factor for early atherosclerosis . A meta-Echocardiographic EFT measurements, provide some advantages when used to assess the cardiometabolic risk. They are objective, quantified, non-invasive, low cost, have routine applicability, avoid exposure to radiation and have a potential for monitoring therapeutic effects. They may also be used as a simple marker for identification of CAH patients with higher cardiovascular risk who may need further cardiac evaluation . In the present study, children with CAH had echocardiographic changes indicating the presence of LV hypertrophy, as indicated by increased LVMI. Moreover, our study showed significant positive correlations between EFT and LVMI that remained significant after regression analysis, which suggested a detrimental effect of EFT excess on the myocardium of patients with CAH. This is in agreement with Corradi et al  who repoIn the present study, children with CAH had echocardiographic changes indicating presence of diastolic dysfunction . Regression analysis revealed that EFT in CAH patients correlates with mitral DcT. This is in agreement with the study of Van der Meer  which shBMI and waist circumference are widely accepted measures of generalized adiposity. However they are poor indicators for visceral obesity. It is well known that visceral adipose tissue accumulation is associated with subclinical atherosclerosis and increased cardiovascular mortality and morbidity. In this study, we found a very good correlation between EFT and waist circumference by multiple regression analysis in children with CAH. However we did not find a significant correlation between BMI-SDS and EFT. These findings suggest that waist circumference is a better anthropometric cardiovascular risk predictor and support the evidence that EFT is related to visceral fat, rather than total adiposity . Mavri eTestosterone concentrations were significantly higher in our subjects with CAH compared to controls, particularly in children who were poorly controlled on medical treatment. In addition, testosterone correlated positively and significantly with EFT. Colgecen et al  reportedIn the present study, children with CAH had higher HOMA-IR than controls. EFT correlated positively and significantly with HOMA-IR by multiple regression analysis. This finding is in agreement with Manco et al  who repoWe recognize that this study has some limitations such us; Due to the difficulties in enrolling and studying CAH children, the sample was relatively small in size and included patients with a wide age range. Due to the cross-sectional design, it is difficult to generalize the results to the general population. CA-IMT measurements were performed manually. We were not able to confirm EFT using the standard magnetic resonance imaging methods. However, echocardiographic calculation of epicardial fat has been reported to show good reliability when compared with magnetic resonance epicardial fat measurements . EpicardEFT is increased in children with classic CAH, particularly in those with poor control and is correlated with CA-IMT, LV mass and mitral DcT. Measurement of EFT by echocardiography in CAH children may help to identify those at high risk of developing LV dysfunction and subclinical atherosclerosis. Future prospective and multicenter studies are required to confirm our results.


Background: The physical, cognitive, and learning benefits of physical activity for children have already been established. However, many schools are failing to provide children with sufficient activity at school due to a crowded school curriculum. Physical activity interventions that integrate physical activity with learning is a way to enhance physical and cognitive benefits without loss of academic time. This study evaluated the preliminary efficacy and feasibility of “Thinking While Moving in English”, a primary school program that integrates physical activity into English lessons. Method: Two classes of Grade 4 students  were randomly assigned to the intervention (n = 29) or control (n = 26) conditions. The program components consisted of 3 × 40 min physically active academic lessons per week, delivered over a 4-week period. The following measures were taken at baseline and immediate post-intervention: on-task behavior, cognition (inhibition and working memory), and learning outcomes (spelling and grammar). Results: Results revealed significant improvements in on-task behavior and spelling in the intervention group, compared to the control group. There were no observed improvements in cognitive outcomes or grammar. Conclusions: This study provides preliminary evidence for the efficacy of physically active English lessons to enhance children’s educational outcomes. Emerging evidence from cross-sectional studies suggests that physical activity is positively associated with children’s cognitive functioning and academic performance ,2. NeverSchools are well placed to address the global inactivity pandemic as they possess the necessary facilities, equipment and personnel required to engage children in a range of physical activities during a school day ,9. A mulWith adequate training and support, teachers can successfully increase students’ opportunities for physical activity participation within a school day by using the following: (I) physically active classroom breaks, (II) curriculum-focused physical activity breaks, and (III) physically active academic lessons. The physically active classroom breaks are short bouts of physical activity performed as a break from academic instruction time , whereaThe main difference between curriculum-focused physical activity breaks and physically active academic lessons is duration. Curriculum-focused activity breaks typically involve short bouts of physical activity  performed during a lesson that is usually aligned with the lesson content. For example, during a mathematics lesson, a teacher might provide students with an opportunity to practice multiplication tables while performing aerobic activity . Alternatively, physically active academic lessons require teachers to integrate physical activity specifically within the lesson content to enhance and reinforce learning. Physically active academic lessons are consistent with theories of embodied learning, which advocate the inter-relatedness of action and perception, emphasizing the role of sensorimotor experiences on perception, understanding, and learning ,17. For p < 0.001, ES = 0.45), general mathematics , and spelling , but no benefits were found for reading. Similarly, Riley and colleagues’ EASY Minds program . No significant group-by-time effect was observed for children’s passive engagement . Finally, significant group-by-time effects were observed for off-task behavior, favoring the TWM-E group .Significant group-by-time effects were observed for children’s active engagement in favor of the TWM-E group . The effect sizes were in favor of the TWM-E group. Non-significant group-by-time effects were observed for grammar scores.Significant group-by-time effects were observed for spelling scores [adjusted mean difference = 3.60 , There were no significant group-by-time effects for inhibition and working memory.M = 4.19, SD = 0.86), the teacher was understanding and delivered fun activities , the program timing was appropriate , and the program had an overall positive impact on children’s perceptions about physical activity .Students from the intervention group reported that the overall program was enjoyable  = 111.75, The primary aim of this study was to assess the preliminary efficacy and feasibility of a movement-based English program in primary schools. The Thinking While Moving in English (TWM-E) intervention resulted in significant intervention effects for on-task behavior, and academic achievement in spelling. The TWM-E program was well-received and enjoyed by students and teachers.The significant improvements for children’s on-task behavior and reduced off-task behavior align with previous research showing that school-based physical activity programs can positively influence on-task behavior ,15. The Overall, the current study replicates the results of previous literature on academic performance that integrates physical activity across different learning domains ,26,27,41Concomitantly, existing literature supports the positive impact of physical activity on cognitive and academic performance ,44,45. AFinally, the measure of physical activity demonstrated that on average children in the TWM-E condition performed more steps per English lesson during the study period than the control group for the 4-weeks of program implementation. Our findings support that school-based physical activity interventions are feasible to implement and may contribute to the activity accumulated by young people at school ,55. PrevAlthough the results of this study are promising, some limitations need to be noted. Firstly, the small sample size of the study makes it difficult to generalize the results. Second, the intervention was delivered by a member of the research team within the school context. Training teachers and providing them with professional learning development on how to integrate physical activity within their lessons plans would ensure a higher ecological validity, and would give them more flexibility to adjust the lessons based on their needs. In addition, standardized assessments for grammar and punctuation might be more insightful. Moreover, this study measured physical activity for a very brief time as part of process evaluation only. Possibly, there are potential compensation effects in subsequent activity in the day that cannot be detected by looking at in-class physical activity only. Future studies can include baseline measurements using more accurate tools for physical activity . FinallThe Thinking While Moving in English program was successful in improving on-task behavior and spelling scores in primary school children. The program successfully integrated physical activity into the existing English curriculum, providing a feasible strategy for meeting both academic and physical activity outcomes within the current school context. These findings highlight the potential of school-based physical activity interventions  on improving learning outcomes and increasing physical activity . Based o


Secretory proteins play important roles in the cross-talk of individual functional units, including cells. Since secretory proteins are essential for signal transduction, they are closely related with disease development, including metabolic and neural diseases. In metabolic diseases, adipokines, myokines, and hepatokines are secreted from respective organs under specific environmental conditions, and play roles in glucose homeostasis, angiogenesis, and inflammation. In neural diseases, astrocytes and microglia cells secrete cytokines and chemokines that play roles in neurotoxic and neuroprotective responses. Mass spectrometry-based secretome profiling is a powerful strategy to identify and characterize secretory proteins. This strategy involves stepwise processes such as the collection of conditioned medium (CM) containing secretome proteins and concentration of the CM, peptide preparation, mass analysis, database search, and filtering of secretory proteins; each step requires certain conditions to obtain reliable results. Proteomic analysis of extracellular vesicles has become a new research focus for understanding the additional extracellular functions of intracellular proteins. Here, we provide a review of the insights obtained from secretome analyses with regard to disease mechanisms, and highlight the future prospects of this technology. Continued research in this field is expected to provide valuable information on cell-to-cell communication and uncover new pathological mechanisms. The secretome represents proteins that are secreted from biological units such as organs and cells. Secretory proteins have recently been shown to play important roles in the cross-talk of individual functional units, including facilitating communication between cells ,2,3. SinSecretomics is a sub-field of proteomics that represents a powerful strategy for characterizing and quantifying proteins secreted by a given cell under specific conditions. Secretomics is based on two major proteomics workflows: in-solution digestion coupled with LC-MS/MS, and SDS-gel fractionation/in-gel digestion/LC-MS/MS ,12,13. RThe majority of secretome studies in mammalian cells are performed in vitro by first culturing cells of interest in serum-supplemented medium to obtain a sufficient number of cells for evaluation. In this regard, selection of a suitable cell model is an essential factor for determining the composition of the cell secretome that, along with subsequent steps, will result in a reliable proteomic analysis. After culturing in appropriate media, the cells must then be carefully washed with sterile buffered saline to remove any serum contamination, followed by incubation in serum-free medium for certain times depending on the experimental purpose in consideration of maintaining cellular viability. The conditioned medium (CM) containing secreted proteins is generally collected and concentrated for the next step using centrifugal filters. This step can also eliminate contaminant components from the culture medium as well as serum by the addition of buffered saline or ammonium bicarbonate solution . In addiAfter preparation of the secreted proteins, two major proteomics workflows are typically applied to analyze the secretome profiles: in-solution tryptic digestion coupled with LC-MS/MS and SDS-PAGE/in-gel digestion/LC-MS/MS ,12. In-sFor example, FASP would be selected for a proteome including membrane proteins or cytokine proteins because it uses SDS and urea . ThermalAfter mass analysis, mass raw files are converted to appropriate file formats such as MGF or mzXML, and then compared to a protein database using specially developed search engines, including Mascot, SEQUEST, PEAKS DB, ProteinPilot, pFind, Andromeda, OMSSA, and X!Tandem . Some ofThere are three main methods used to filter secretory proteins from the total identified proteins. First, proteins can be generally identified from Gene Ontology Cellular Components terms of the extracellular region using bioinformatics software such as DAVID bioinformatics resources . Second,Most secretome studies are performed using a cell culture system. The new secretory factors identified from cell culture-based secretomics have been reported that are related with diverse disease including metabolic disease, cancer, and neurodegenerative diseases. Irisin has been identified from CM of skeletal muscle cells using LC-MS/MS analysis . Irisin Some non-classical secretion mechanisms have been reported with regards to the release of intracellular proteins by cells . Severalg centrifugation. The sample is then spiked with protease inhibitors to prevent the degradation of vesicle proteins [g [There are several methods for EVs preparation that are applicable to proteomics, such as ultracentrifugation, immuno-affinity capture, and gel filtration ,68,69. Uproteins ,71. Nextteins [g . During teins [g .w/v) is generated in ultracentrifugation tubes by sequential layering, and the CM is overlaid on this gradient [g for 16 h at 4 °C [g for 2 h at 4 °C, and the resulting pellets are resuspended in PBS [Density-gradient ultracentrifugation has certain advantages in terms of preventing non-vesicular protein contaminants that can be introduced with the conventional ultracentrifugation method . Densitygradient . Ultrace at 4 °C . Each mid in PBS . Some exd in PBS ,75. TablOver 200 adipokines were identified in secretome analyses of adipocytes . Leptin Adiponectin is widely known as a beneficial adipokine because its plasma and protein expression levels are inversely correlated with body weight and abdominal obesity in humans . AdiponeUnder a pathological condition such as severe obesity, the adipose tissue synthesizes and secretes several pro-inflammatory cytokines, which aggravate insulin resistance and the systemic inflammatory status. For example, the tumor necrosis factor-alpha (TNF-α) expression level was shown to be highly increased in adipocytes in a study of obese human subjects . InteresIL-6 is a well-known myokine and popular focus of research into disease mechanisms, particularly in relation to inflammation. IL-6 has been dubbed an “exercise factor” through which skeletal muscles communicate to the peripheral organs. In humans, increased circulating concentrations of IL-6 are known to be affected by both the intensity and duration of skeletal muscle contractions . In vitrAnother important myokine is brain-derived neurotrophic factor (BDNF), which is a member of the neurotrophic factor family. BDNF is also considered to be an exercise factor since its levels are increased as a result of both acute and chronic aerobic exercise . BDNF trIrisin is a fragment of fibronectin type III domain-containing protein 5 (FNDC5) that was recently reported to act as a myokine, although its origin and function are controversial. Irisin plasma levels are increased by diverse types of exercises; thus, it is also considered to be an exercise hormone. Irisin treatment to L6 myotubes resulted in increased glucose uptake in a dose-dependent manner, which was mediated by activation of both AMPK and ACC. Irisin treatment of primary myocytes also upregulated the expression of PGC-1α4, a specific isoform associated with muscle hypertrophy . AnotherFollistatin-like-1 (FSTL-1) is a myokine of the follistatin family that was first identified as a secreted protein from C2C12 mouse myotubes . FSTL-1 Fgf21 knockout mice [Fibroblast growth factor 21 (FGF21) belongs to the FGF superfamily and is a key mediator of fatty acid oxidation and lipid metabolism. In contrast to autocrine/paracrine FGF, FGF21 does not have a heparin sulfate-binding domain, and is thus readily released into the circulation and predominantly acts systemically . A transout mice ; howeverout mice . In summFetuin-A is an important hepatokine regulating systemic metabolism. Fetuin-A is a phosphorylated glycoprotein that is primarily synthesized by hepatocytes, and was originally characterized as a potent inhibitor of the insulin receptor tyrosine kinase in the liver and skeletal muscle . Fetuin-Selenoprotein P (SeP) is mainly synthesized in the liver and appears to show upregulated expression in the liver of patients with T2D, NFALD, and cardiovascular disease . Using sVascular diseases are conditions that affect vasculature, including arteries and/or veins of the circulatory system. pathological changes of vascular endothelial cells by inflammation play an important role in the development of vascular and heart disease, one group analyzed secretory proteins under TNF-α treatment using LC-MS/MS method . In thisVascular smooth muscle cell (VSMC) dysfunction causes major types of vascular disease such as atherosclerosis and hypertension. Recent study identified 349 proteins from VSMC derived microvesicle and exosome . ImportaNeurons are well-recognized as an important cell population for receiving and transmitting information in the nervous system; however, non-neuronal cell types such as astrocytes and glia are also essential for proper regulation of the nervous system . ImportaNumerous studies have now provided a comprehensive profile of astrocyte-derived cytokines and secretory proteins ,130,131.As resident immune cells of the brain, the microglia mediate key functions to support and initiate an immune response to pathogens and damage. Moreover, microglia can regulate neural development and neuronal cell restoration in both healthy and pathological conditions . MicroglNSCs are self-renewing, multipotent cells that generate neurons, astrocytes, and glia of the central nervous system. In addition to generation of new graft-derived neurons and glial cells, accumulating evidence indicates that NSC transplants can improve central nervous system diseases, including animal models of multiple sclerosis, AD, and spinal cord injury, by secreting proteins such as growth factors, cytokines, chemokines, metabolites, and bioactive lipids .Yashura et al.  used an Severino et al.  adopted Extracellular vesicles (EVs) contain proteins and other cellular origin, being used as early pathological condition detection maker in central nervous system (CNS) diseases. Indeed, EVs had been included high level of total Tau, P-T181, P-S396-tau, and Aβ1-42 from fifty-seven Alzheimer’s disease (AD) patients . In partSecretory factors including cytokines such as adipokines, myokines, hepatokines, and EVs are key factors of the secretome, and since their important roles in various diseases are increasingly becoming recognized, the popularity of secretomics analysis is on the rise. Cell-based secretome studies can yield reliable results through diverse and long-term studies, but there are important considerations with regards to secretome preparation. Therefore, optimized and standardized conditions of primary cells are needed for secretome studies, as well as methods for analyzing the secretome at the tissue level, which could provide more direct and reliable results that exclude artifacts. Studies focused on EVs have also recently begun to attract attention. One of the major challenges of secretomics is the inherent sensitivity of the mass spectrometer to cover the wide dynamic range for the low abundance of genuine secreted proteins over the high abundant proteins derived from the serum, which is essential for standard maintenance of the sample . High-re