Waist circumference (WC) and uric acid (UA) are significantly related. Still, their temporal sequence and how the sequence works on future risk of triglyceride glucose (TyG) are unknown, especially in the Chinese population.Cross-lagged panel model was used to analyze the reciprocal, longitudinal relationships among a set of interrelated variables. The mediation model was constructed to test the effect of the relationship between WC and UA on TyG.β = 0.003, P = 0.031) and follow-up TyG ;. Simultaneously, there was no statistical association between the level of baseline UA and the level of follow-up WC . The mediation effects of UA on WC-TyG were estimated to be 18.1% in adults, and 36.2% in women.A total of 5727 subjects were enrolled in our study population, of which 53.5% were women, and the mean age was 59.0  years. After adjusting for traditional confounding factors, the results showed that a higher level of baseline WC was significantly associated with a higher level of follow-up UA (The current study demonstrated that higher baseline level of WC probably preceded UA’ level in general population. In addition, UA mediated the relationship of WC to TyG, especially in females. And the possible mechanism would require further clarification. Insulin resistance (IR) –5 and urRecent researches have reported that visceral fat (determined by waist circumference [WC]) is related to IR –14, and TyG levels, which are associated with IR, play significant roles in predicting the incidence of diabetes and CVD. However, it is unclear whether WC is related to TyG and to what possible extent. To date, there have been few general population studies, particularly prospective cohort studies of Chinese adults who have increased WC and TyG. At the same time, a large American population study showed that the inverse association between serum antioxidant levels and inflammatory markers is moderated by adiposity . On thishttp://charls.pku.edu.cn/en. The study protocol has been approved by ethical committees of Peking University , and all individuals have provided written informed consent.China Health and Retirement Longitudinal Study (CHARLS) was a representative survey research nationwide conducted by the National School of Development of Peking University. Between June 2011 and March 2012 (baseline survey), a multistage process with random cluster sampling was conducted to select a representative sample of individuals older than 45 years in 10,287 households in 450 villages/cities. 17,708 individuals were enrolled in the baseline survey through face-to-face household interviews. After the initial recruitment, all 17,708 individuals have been re-surveyed every two years using the same questionnaire as at the baseline. Blood samples were gathered in the year of 2011 and 2015. The detailed information regarding the CHARLS has been described on the website of CHARLS: This study was a post hoc analysis of CHARLS from 2011 to 2015. Of the 17,708 individuals at baseline, 1099 subjects were younger than 45 years. Participants with detailed follow-up information, major study variables and confounding variables at baseline and follow-up period were included in the study. Participants who had cancer at baseline were excluded. Finally, 5727 subjects were included in our study. The flowchart in Fig. Information on participants demographics , lifestyle factors (smoking status and drinking status), and history of diseases  were collected during the structured household survey. Data on healthy behaviors were obtained from the subjects’ self-reported questionnaire, including smoking status , frequency of alcohol consumption . Data on diabetes, chronic kidney disease, and hypertension were collected by trained health staff members.http://charls.pku.edu.cn/en. The mean of 3 blood pressure values was calculated and used for our analysis. The BMI was calculated using the weight and height indicators; the formula as follows: BMI = weight (Kg) / height2 (m2).Data on collection and measurement of cholesterol indexes, fasting plasma glucose, biochemical blood indexes, and other blood pressure indexes are detailed on the website of CHARLS: The TyG index was calculated according to the following formula: TyG = ln [fasting TG (mmol/L) × FPG (mmol/L) × 0.5 × 159.37]. TG and FPG were measured by enzymatic colorimetric test method.A soft tape was inserted at the navel level to measure WC in a standing pose with a cloth measuring tape. At the same time, all participants needed to do a regular breathing exercise, holding the breath at the end of exhaling and letting the tape out slightly.UA plus method was used to measure UA.P ≤ 0.05 (two-sided) considered statistically significant differences.All analyses were conducted by IBM SPSS version 22.0 and SPSS Amos 22.0 with Percentiles were calculated used to describe the categorical variables, while arithmetic mean with standard deviation (SD) was calculated for the description of continuous variables that met the normal distribution. The median with interquartile range was used to describe the continuous variables that didn’t meet the normal distribution. At the same time, the Student t test, the Mann-Whitney U test or the Pearson’s χ2 test was used for the comparison between male and female.The linear regression model was constructed to explore whether WC (UA) could predict the future variation of TyG. Future TyG variation and baseline WC (UA) were the dependent and independent variables of the model, respectively. Firstly, the multicollinearity problem among independent variables was examined by the variance expansion factor (VIF). VIF greater than 10 was deemed significant multicollinearity. Secondly, three models were constructed to assess the relationship between baseline (examination at 2011) WC (UA) and future (follow-up at 2015) TyG as follows: model 1: adjusted for baseline WC (UA); model 2: model 1 plus sex, ethnicity and age; model 3: model 2 plus drinking, current smoking, education, BMI, LDL-C, HDL-C, HR, DBP, SBP, and creatinine.β1 showed the impact of baseline UA on follow-up WC, and β2 showed the effect of baseline WC on follow-up UA. WC and UA values were adjusted in linear regression analysis by baseline and follow-up variables: sex, age, ethnicity, alcohol consumption, current smoking, education, BMI, LDL-C, HDL-C, HR, DBP, SBP, and creatinine. Residuals were saved and after that, Z-transformation was used to standardize the saved residuals . Root-mean-square residual (RMR) and comparative Fit Index (CFI) and were enrolled for the model fits. RMR < 0.05 and CFI > 90 meant a relatively good model fit in the cross-lagged path model. In addition, this analysis model was constructed in groups of men and women, separately.Longitudinal changes of WC and UA indexes were measured at two follow-up periods. Previous study introduced the theory and application of cross-lagged panel design . OverallWhen the temporal relationship was establised between WC and UA, the mediation model would be fitted to explore the potential influence of the association between UA and WC on TyG. The values of TyG were analyzed by linear regression residual model and standardized by Z-transformation (with mean of 0 and SD of 1). According to the results of the cross-lagged path analysis model, X and M were determined as predictor and mediator, respectively. The detailed mediation model was showed in Fig. The following formula was used to calculate the proportion of the mediation effect, as follows: Mediation effect (%) = In the end, the study enrolled 5727 participants, of which 53.5% was female. The mean age of this population was 59.0 ± 8.62 years. Detailed baseline and follow-up characteristics of the study population are summarized in Table β = 0.003, P = 0.031) and follow-up TyG level .Table β = 0.051, P < 0.001). However, there was no significant association between baseline UA and follow-up WC .Table baseline → UAfollow-up path coefficient is 0.025 (P = 0.021). However, the UAbaseline → WCfollow-up path coefficient is 0.001 (P = 0.921). The study results showed that a wider WC at baseline resulted in increased UA levels at follow-up, but an increased baseline UA level did not lead to a wider WC. The values of the model fitting parameters in the WC with UA model (CFI = 0.991 and RMR = 0.016) showed a relatively good fit to the observed data in our cross-lagged path analysis.Results of the cross-lagged path analysis for WC and UA in the general population with age over 45 are shown in Fig. Figure baseline → UAfollow-up path coefficient is 0.033 (P = 0.014), and the UAbaseline → WCfollow-up path coefficient is 0.019 (P = 0.340). However, for men, we found that the WCbaseline → UAfollow-up path coefficient is 0.015 (P = 0.407), and the UAbaseline → WCfollow-up path coefficient is − 0.013 (P = 0.444). These results indicated that an increase in baseline WC might affect a subsequent UA increase, but only in women, not in men. For women, the percentage of the overall effect mediated by follow-up UA was estimated at 36.2% ; (2) verifying UA mediate effect on the relationship between WC and TyG; (3) estimating the percentages of UA mediation effect on WC - TyG (18.1%); (4) and finding the mediation effect of UA on WC - TyG in women not in men.Results of current research were 4-fold: (1) clarifying the temporal relationship between WC and UA  has been commonly used as measurement index. However, the HOMA-IR is time-consuming and expensive. Some studies have developed and assessed surrogate markers for IR . One of Prevalence of abdominal obesity has increased and has been considered a major health hazard, which is significantly correlated with the risk of mortality, CVD, as well as other diseases. Comparing to generalized obesity, central obesity is more closely linked to IR. WC as a predicted measure of health risk is a convenient and effective index of abdominal obesity. It has a significant, predictable association with intra-abdominal fat area and volume. One cross-sectional study of participants without diabetes demonstrated that the insulin sensitivity index significantly correlated with WC across populations . PreviouThe Finnish Diabetes Prevention Study with impaired glucose tolerance risk population showed that the change in UA was significantly related to the change in fasting insulin . The serAll WC, UA, and TyG measurements were carried out by trained staff using unified and standardized measuring instrument with high repeatability. Most importantly, we conducted the subgroup analysis between males and females about the UA’s mediation effects on WC to IR in a representative sample of the Chinese population with age ≥ 45 years. The study added to the understanding of the relationship between UA and IR in the context of obesity and metabolic syndrome. Additionally, the current study provided information on alcohol consumption, current smoking, education, BMI, LDL-C, HDL-C, HR, DBP, SBP, and creatinine. After excluding the missing data, only 5727 participants were included in our finally analysis.baseline → UAfollow-up, and the mediation effect of UA on WC - TyG was found in women, but not in men. Therefore, early WC detection might be helpful for the primary care physicians to stratify the potential patients that need the further blood screening. Further researches are required to explore the possible underlying mechanism.The results of this research demonstrated that the baseline level of WC and UA was associated with the future risk of IR. Furthermore, the temporal relationship between WC and UA was WC


Different from skeletal muscle, the heart autonomously generates rhythmic contraction independently from neuronal inputs. However, speed and strength of the heartbeats are continuously modulated by environmental, physical or emotional inputs, delivered by cardiac innervating sympathetic neurons, which tune cardiomyocyte (CM) function, through activation of β-adrenoceptors (β-ARs). Given the centrality of such mechanism in heart regulation, β-AR signaling has been subject of intense research, which has reconciled the molecular details of the transduction pathway and the fine architecture of cAMP signaling in subcellular nanodomains, with its final effects on CM function. The importance of mechanisms keeping the elements of β-AR/cAMP signaling in good order emerges in pathology, when the loss of proper organization of the transduction pathway leads to detuned β-AR/cAMP signaling, with detrimental consequences on CM function. Despite the compelling advancements in decoding cardiac β-AR/cAMP signaling, most discoveries on the subject were obtained in isolated cells, somehow neglecting that complexity may encompass the means in which receptors are activated in the intact heart. Here, we outline a set of data indicating that, in the context of the whole myocardium, the heart orchestra (CMs) is directed by a closely interacting and continuously attentive conductor, represented by SNs. After a roundup of literature on CM cAMP regulation, we focus on the unexpected complexity and roles of cardiac sympathetic innervation, and present the recently discovered Neuro-Cardiac Junction, as the election site of “SN-CM” interaction. We further discuss how neuro-cardiac communication is based on the combination of extra- and intra-cellular signaling micro/nano-domains, implicating neuronal neurotransmitter exocytosis, β-ARs and elements of cAMP homeostasis in CMs, and speculate on how their dysregulation may reflect on dysfunctional neurogenic control of the heart in pathology. If we consider cell morphology, myofibers are tapered cells, with varied length depending on the muscle type (from about 7–40 mm), while CMs are smaller in size , with a rod-shaped morphology . Additiospecific . Furtherspecific , the myospecific .It is unquestioned that skeletal muscle contraction, finalized to voluntary movements, initiates upon the neurogenic input provided by the innervating motor neuron (MN), which releases acetylcholine at the Neuro-Muscular Junction (NMJ), a well-characterized neuromuscular synapse . Differevia PKA activation, and subsequent phosphorylation of downstream targets, involved in the mechanism of cell shortening  in both fields, blurring the divide between skeletal and cardiac muscle innervation. While SNs were identified several years ago, in anatomic studies, to account for a significant fraction of the neuronal types in nerves directed to hindlimb muscles , their ivia the β2-AR/Akt/FOXO/ubiquitin ligase pathway, with a mechanism very similar to that active in skeletal muscles . Indeed, we and others have demonstrated that cardiac sympathetic innervation has additional constitutive functions, which include control of heart rate on a beat-to-beat basis , and act muscles .Such evidence supports that, while cardiac and skeletal muscles are unquestionably different for phenotypic and functional aspects, they are not so dissimilar with regards to the relationship with sympathetic nervous components. At this point, it is inevitable to think that a fundamental difference in the way skeletal and cardiac muscle interact with neurons is given by the structures underlaying such interactions. In fact, it has been known for a long time that MNs signal to the innervated fiber in a synaptic way, thanks to the NMJ . The myoStarting from these base notes, this review will focus on the communication occurring between the single SN varicosity and the target CM, operating through intracellular and extracellular signaling microdomains, using the skeletal muscle and the NMJ as a benchmark. Furthermore, starting from the fine characterization of the NMJ structure, we will present a roundup of the literature identifying the molecular bricks which build the NCJ.via receptor-associated Adenylyl Cyclases. Ignition of the β-AR/cAMP pathway is, by no doubts, the mainspring of cardiac “fight-or-flight” reaction, ultimately causing positive chronotropic, lusitropic and inotropic effects, mostly through PKA-dependent phosphorylation of sarcomeric proteins and molecular players regulating Ca2+ dynamics (- vs. β2-AR), if signaling is transduced by the same second messenger  shared with many other receptor types, eliciting different, and sometimes opposed, biological responses?”As stated above, although the process of excitation-contraction coupling occurs in CMs independently from neuronal inputs, heart function is continuously adapted to precisely match the organism requests. Such adaptation results from the integration, by the central nervous system, of signals from peripheral sensors of homeostatic , sensory  and emotional (intrinsic) inputs, and routed to the heart through afferences of the autonomic nervous system . Neurogedynamics . While s2+ currents on the same cell portion, demonstrating the concept of compartmentalization of β-AR/cAMP signaling  and signal regulators  have been shown to impact on the downstream effect of receptor activation . Adding ntration . In detantration . Of notentration .Given the complex and meticulous regulation of the β-AR signaling pathway, which holds a fundamental role in heart physiology, it is not surprising that disruption of the fine space–time organization of the cAMP signal impacts on the adequate CM response to β-AR stimulation. Consistently, results from several studies, in both human and rodents, demonstrate that CMs from failing hearts feature dysregulation in β-AR/cAMP signaling, characterized by desensitization/downregulation of β-ARs, reduced cAMP synthesis and altered phosphorylation of the main PKA targets . In addiAltogether, the examples described above support that the spatial and temporal regulation of signaling of one specific GPCR is restricted to tightly organized compartments within the cell and may occur with singular kinetics. This may represent the receptor “signature,” allowing its stimulation to distinctively activate the proper and specific biological responses in the target cell.While most studies on cAMP signaling, including those related to the fundamental mechanism underlying cardiac cell response to AR stimulation, have been conducted in cultured isolated cells, exposed to natural or artificial AR agonist, very few have aimed to replicate the conditions in which CMs are activated by SNs in the heart. Given that the complexity of cAMP responses, upon activation of β-ARs, depends on the cellular arrangement of the molecular partners of the signaling cascade, it is predictable that localization of the receptors throughout the heterogeneous cell membrane domains, the fraction—or specific subset—of receptors activated by the agonist, and the kinetics and potency of activation, are all critical players. As consequence, since SNs are the endogenous sources of noradrenaline triggering β-ARs in heart, to infer the implications of intracellular β-AR signaling compartmentation in neurocardiac physiology, the microanatomy of interactions between neurotransmitter-releasing neuronal varicosities and target cells, in the intact heart, must be taken into account.Post-ganglionic SNs descend from sympathetic ganglia, located along the cervical and thoracic spine, and reach the epicardium along the great vessels, before entering the heart wall and distributing throughout the myocardial interstitium . We and The idea that SN varicosities could establish specific contacts with CMs, structurally analogous to the well-known NMJ, dates back several years, but the initial studies failed to detect structured neuro-muscular contacts in the heart, thus endorsing the model whereby SN neurotransmitters “diffuse in wide gaps between nerve processes and cellular targets” . In contReappraisal of cardiac synapses started a decade ago, when Based on these results, the authors speculated that specific signaling domain on the myocyte plasma membrane, underscored by accumulation of the scaffolding components AKAP79/150 and SAP97, would be required for physiologic signaling of ARs in cardiac tissue. Interestingly, such post-synaptic organization has been described to be depauperated of β2-ARs, which are internalized upon neuronal noradrenaline discharge, thus supporting that “the subtype-specific distribution of β1- and β2-ARs relative to sympathetic synapses” could contribute to signaling specificity .in vivo, to address neuro-cardiac communication and determine whether the effects of direct synaptic contacts, between neurons and CMs, could be addressed functionally. In line with the previous speculations, by monitoring cAMP, we demonstrated that the tight connection between the neuron and CM membrane enables neuro-cardiac communication to occur via specific extracellular signaling microdomains, with several implications on cardiac β-AR activation. First, the tight assembly of the NCJ minimizes extra-synaptic noradrenaline diffusion, rendering the neuronal varicosity a point source of noradrenaline, suited to activate therefore a subset of β-ARs, as shown by the formation of intracellular cAMP gradients, in the CM, originating from the neuronal contact site  of inputs from an increasing number of simultaneously activated NCJs may allow heart regulation through a wide range of tones, spanning from the acute activation, during the stressful “fight-or-flight” reaction, to long-lasting effects, crucial for the maintenance of heart homeostasis . In otheThe picture emerging is that, in intact hearts, neuro-cardiac communication involves extra-cellular (first-messenger) signaling microdomains and intracellular (second messenger) micro/nanodomains which combine to control, with exquisite precision and flexibility, CM responses to SN activation. The appreciation that neuro-cardiac communication occurs in defined extra-cellular spaces, that it involves the stimulation of defined receptor pools, each with singular and dynamic distribution on the cell membrane, and that it reflects on a tightly organized series of specific intracellular actions, allows new inference based on experimental data which were, thus far, lacking explanation. This relates to the opening question of the current manuscript: “how can CMs maintain specificity of response to—say—β1- and b2-ARs, even if they are activated from the same extra-cellular agonist and transduced, intracellularly, by the same second messenger?”To give an example, as discussed above, we have previously demonstrated that the presence of cSN is required to maintain correct CM mass, through constitutive stimulation of β2-AR signaling. However, it has to be taken into account that β1-ARs have higher affinity for noradrenaline than β2-ARs , and it lowpass filter in β2-AR dependent signaling , one of the main causes of death in Europe.In cardiac physiology, while it is commonly appreciated that catecholamine  incretion occurs by both sympathetic efferences and the adrenal medulla, the effects of such distinct mechanisms of cardiovascular regulation have not been fully agreed upon. It is tempting to speculate that local neurogenic input, and diffuse stimulation by circulating catecholamines, would activate qualitatively different intracellular signaling compartments, with molecular and functionally different effects. This question is still awaiting answer, which we now have the tools to provide.In cardiac pathology, it is well understood that HF features alterations in autonomic control of cardiac function, including decreased responsiveness to β-AR agonists, reduced noradrenaline content in sympathetic endings, increased venous spillover of neuronal noradrenaline and its accumulation in the myocardial interstitium . InteresOur data surmises that the molecular architecture of the cell–cell interaction site may influence the efficiency of neuro-cardiac communication. At the time being, while the components of the NCJ are still partially unresolved, the observation that in culture, the interaction between SNs and CMs is stable in time, as opposed to that SNs establish with, e.g., cardiac fibroblasts , suggestmdx mice, a model characterized by the absence of dystrophin expression  communication, could also impact neurotrophic signaling from CMs to SNs, we set up co-cultures of mixed CMs and SNs from control and  reduced . AltogetAt this point, one can object that what we observed was in a condition far from that of intact heart, and that previous studies failed to detect accumulation of dystrophin in the portion of the CM membrane innervated by the neuron (as occurring at the NMJ). It has to be noted, however, that while each skeletal muscle fiber establishes a single NMJ, with the MN terminal contacting the cell membrane in a well-defined point, the CM membrane is interspersed by repeating contacts with multiple SN varicosities. This implies that, although dystrophin accumulation may occur in the CM portion contacted by the SN varicosity, differences in dystrophin density along the sarcolemma may be less distinct for the overlap of neighboring postsynaptic membrane portions. Furthermore, we cannot exclude that the dystroglycan complex may contribute to stabilize intercellular contacts independent from selective accumulation at the site of the nervously touched CM membrane.The independence of contractions from innervation does not imply that in physiologic conditions the heart beats solo. While neurons, which densely innervate the myocardium, do not ignite contractions, research summarized in this review demonstrates that the finely-built interaction with cardiomyocytes, centered at the NCJ, allows neurons to precisely control multiple cell functions, ranging from contractility, electrophysiology to gene expression. Innervation serves as conductor of the cardiac cellular orchestra, setting the tempo, unifying performers, and shaping the beats of the ensemble. Like a conductor controls interpretation and pace of the music, with gaze and gestures to the performers, neurons do so through direct communication to the cardiomyocytes, guaranteeing the melody and harmony of heartbeats. When diseases break communication between the conductor and the musicians, the orchestra plays out of tune or synchrony, and heartbeats sound turbulent or noisy.MF and LD contributed to manuscript and figure preparation. LV critically discussed data and the review layout. TZ and MM drafted and wrote the manuscript. All authors approved the final version of the manuscript and agree to be accountable for all aspects of the work, in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved, and that all persons designated as authors qualify for authorship and have been listed.This work was supported by STARS-miniheartwork (UNIPD) to MM and STARS-SKoOP (UNIPD) to TZ.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Lactobacillus plantarum, Pediococcus pentosaceus and Enterococcus faecalis inoculated in alfalfa silage alone or in combination on the fermentation quality, dynamics of bacterial community, and their functional shifts using single‐molecule real‐time (SMRT) sequencing technology. Before ensiling, alfalfa was inoculated with L. plantarum (Lp), P. pentosaceus (Pp), E. faecalis (Ef) or their combinations  and sampled at 1, 3, 7, 14 and 60 days. After 60‐days fermentation, the Lp‐, Pp‐ and LpPp‐inoculated silages had lower pH but greater concentrations of lactic acid were observed in Pp, LpEf and LpPpEf‐inoculated silages. The inoculants altered the keystone taxa and the bacterial community dynamics in different manners, where L. plantarum, Weissella cibaria and L. pentosaceus dominated the bacterial communities after 14 days‐fermentation in all treatments. The silages with better fermentation quality had simplified bacterial correlation structures. Moreover, different inoculants dramatically changed the carbohydrate, amino acid, energy, nucleotide and vitamin metabolism of bacterial communities during ensiling. Results of the current study indicate that effect of different inoculants on alfalfa silage fermentation was implemented by modulating the succession of bacterial community, their interactions and metabolic pathways as well during ensiling.The objectives of this study were to investigate the adaptation and competition of  Lactobacillus plantarum, Pediococcus pentosaceus, and Enterococcus faecalis inoculated in alfalfa silage alone or in combination on the fermentation quality, dynamics of bacterial community, and their functional shifts of alfalfa silage. The inoculants altered the keystone taxa, dynamics of bacterial community and their functions in different manners, and the silages with better fermentation quality had simplified bacterial correlation structures.This study were investigated the adaptation and competition of  This agrees well with the report of Cai  during ensiling, which indicated that the Ef strain used in this study was less competitive than the epiphytic bacteria in alfalfa.Silage quality depends on the result of the competition between LAB and spoilage microorganisms, as well as the competition and collaboration between LAB  maintained a high relative abundance during the entire ensiling of alfalfa in the present study . This result suggested that Pp inoculation could accelerate the growth of epiphytic W. cibaria in the silage once fermentation was initiated. As expected, in silages fermented for 1 and 3 days, Pp‐ and Lp‐included inoculants increased the relative abundance of P. pentosaceus or L. plantarum in the treated silages compared with the control. However, the relative abundance of E. faecalis only increased in the silage inoculated with Ef alone. Therefore, it can be concluded that the strains Lp and Pp are more competitive than the strain Ef used in the present study, contrary to the report by Wang et al.  sequencing technology, we figured out the effects of different inoculants on the complicated biological process of the alfalfa ensiling ecosystem in terms of the succession of bacterial community, their interactions and metabolic pathways. The inoculants used in the present study altered the dynamics of bacterial community and the keystone taxa in different manners, and also dramatically changed the carbohydrate, amino acid, energy, nucleotide and vitamin metabolism of bacterial communities during ensiling. Silages with a high fermented quality  had simplified bacterial correlation structures, and the desirable bacterium L. plantarum MTD‐1 , P. pentosaceus  and E. faecalis AH38 (Ef). The strain E. faecalis AH38 was isolated and screened from alfalfa silage after 7 days of ensiling in our laboratory due to its better growth performance and remarkable acid production ability.In this study, the LAB strains used as silage inoculants were ‐1; effective N, P and K in the soil were 82.9, 19.0 and 115.0 mg kg‐1, respectively. Fresh alfalfa was harvested by hand from four randomly selected sites (used as replication for each treatment) within a field of about 0.1 hectare , taken into the laboratory immediately, and wilted to a dry matter (DM) content of 311.9 g kg‐1 of fresh weight (FW). The content of crude protein (CP) in alfalfa before ensiling was 218.7 g kg‐1 DM, neutral detergent fibre (αNDF) was 315.9 g kg‐1 DM, acid detergent fibre (ADF) was 223.4 g kg‐1 DM, water‐soluble carbohydrate (WSC) was 49.9 g kg‐1 DM, and the pH was 6.12. Before ensiling, the wilted forage was manually chopped into lengths of 1–2 cm by paper cutters. For each of the 4 locations, there were 35 piles of forage . The forage piles from each location were then treated separately with distilled water , L. plantarum , P. pentosaceus , E. faecalis , a combination of L. plantarum (5 × 104 CFU g FW‐1) and P. pentosaceus (5 × 104 CFU g FW‐1) (LpPp), a combination of L. plantarum (5 × 104 CFU g FW‐1) and E. faecalis (5 × 104 CFU g FW‐1) (LpEf), and a combination of L. plantarum (3.3 × 104 CFU g FW‐1), P. pentosaceus (3.3 × 104 CFU g FW‐1) and E. faecalis (3.3 × 104 CFU g FW‐1) (LpPpEf). The application rate (1 × 105 CFU g FW‐1) of the inoculants used in each treatment was the least effective dosage for silage inoculation based on the previous study  and harvested in the late bud to early bloom stage in May 2019. The average soil OM in the cropping field was 11.1 g kget al. (3‐N) and water‐soluble carbohydrates (WSC) were tested according to the method described by Licitra et al.  were blended with distilled water (180 ml) at a high speed for 30 s, followed by filtration via four layers of cheesecloth. The pH of the supernatant was measured using a glass electrode pH metre . Concentrations of lactic, acetic and propionic acids were determined according to the methods described by Zhang et al. . The cona et al.  and Ke e ij is the effect of the interaction between the inoculants and ensiling time, and ijkε is the residual error. Means were separated by Tukey’s test. Chemical compositions of 60‐days silage samples were analysed using one‐way ANOVA. Tukey’s test was also used for pair‐wise mean comparisons. Significance was considered at P < 0.05, and the tendency was declared at P < 0.1.The experimental protocol had a 7 × 5 factorial design with seven inoculants and five ensiling times. The data for pH and organic acids were analysed using the general linear model procedure of the Statistical Package for Social Science  according to the model:This work was funded by the National Key R&D Program of China (project no. 2017YFE0104300) and the National Natural Science Foundation of China (project no. 31672487).None declared.


IL-1 family cytokines, HIF1α, SET7 and mTOR, ROS regulators NRF2 and NOX2 partially regulate trained immunity genes; and NRF2 plays roles in downregulating SGs more than that of NOX2 in upregulating SGs. These results provide novel insights on the roles of aorta as immune organ in upregulating secretomes and driving immune and vascular cell differentiations in COVID-19, cardiovascular diseases, inflammations, transplantations, autoimmune diseases and cancers.To determine whether aorta becomes immune organ in pathologies, we performed transcriptomic analyses of six types of secretomic genes (SGs) in aorta and vascular cells and made the following findings: 1) 53.7% out of 21,306 human protein genes are classified into six secretomes, namely, canonical, caspase 1, caspase 4, exosome, Weibel–Palade body, and autophagy; 2) Atherosclerosis (AS), chronic kidney disease (CKD) and abdominal aortic aneurysm (AAA) modulate six secretomes in aortas; and Middle East Respiratory Syndrome Coronavirus  infected endothelial cells (ECs) and angiotensin-II (Ang-II) treated vascular smooth muscle cells (VSMCs) modulate six secretomes; 3) AS aortas upregulate T and B cell immune SGs; CKD aortas upregulate SGs for cardiac hypertrophy, and hepatic fibrosis; and AAA aorta upregulate SGs for neuromuscular signaling and protein catabolism; 4) Ang-II induced AAA, canonical, caspase 4, and exosome SGs have two expression peaks of high (day 7)-low (day 14)-high (day 28) patterns; 5) Elastase induced AAA aortas have more inflammatory/immune pathways than that of Ang-II induced AAA aortas; 6) Most disease-upregulated cytokines in aorta may be secreted  Recentls (Treg) –25 may us (Treg) , facilits (Treg)  and stems (Treg) . Howeverhttps://clinicalinfo.hiv.gov/en/glossary/immune-system). Thus, we compared ECs to prototypic innate immune cells such as macrophages /DAMPs in the blood circulation, the endothelium   could furophages  in more rophages , 3, 32, rophages , 34 in Erophages . ECs exerophages ; and ECsrophages . In addirophages , one typrophages , 40, frorophages , 41, hyprophages , uremia rophages , and hyprophages , which arophages , 20 and rophages , 44. Mitrophages –48. Morerophages  and secrrophages , upregulrophages . Furtherrophages . These frophages –55 and Irophages , 56. In rophages , suggesteceptors , 58. Howeceptors , making First, whether aorta serves as an immune organ by upregulating the expressions of canonical and non-canonical secretomic genes (SGs)  regulators and trained immunity regulators  , 26 in mgulators . Our newhttps://www.ncbi.nlm.nih.gov/gds/) and ArrayExpress (https://www.ebi.ac.uk/arrayexpress/) databases and analyzed with online software GEO2R (https://www.ncbi.nlm.nih.gov/geo/geo2r/), namely, atherosclerotic mouse aorta (GSE10000) and CKD affected rat aorta (GSE146638), abdominal aortic aneurysm in aortas of patients (GSE47472), aorta of Ang-II induced aneurysm model (GSE17901), aorta of elastase induced aneurysm (GSE51229), venous endothelial cell from arteriovenous fistula (GSE46126), MERS-CoV infection in human endothelial cells (GSE79218) and Ang-II treated vascular smooth muscle cells (GSE97470). Thus, a total of 8 datasets were analyzed. In addition, we organized six secretomic pathway gene lists from previous publications, namely, canonical secretome (Atlas website (https://www.proteinatlas.org/)), caspase 1-Gasdermin D (caspase 1) secretome (PMID 18329368), caspase 4-Gasdermin D (caspase 4) secretome (PMID28196878), exosome secretome (http://exocarta.org/download), Weibel-Palade bodies (WPB) (PMID 22468712) and autophagy secretome  –National Center for Biotechnology Information (NCBI)–Gene Expression Omnibus (GEO) (https://metascape.org/gp/index.html#/main/step1) was used for enrichment analysis. This website contains the core of most existing gene annotation portals  to characterize clinical relevance and molecular and cellular functions related to the identified genes in our microarray analysis. Differentially expressed genes were identified and uploaded into IPA for analysis. The core and pathways analysis were used to identify molecular and cellular pathways.We utilized Ingenuity Pathway Analysis   , caspaseownload) , WPB secownload) , suggestSGs) out of 2,640 SGs (68%); caspase 1 dependent secretome had 536 specific SGs out of 964 SGs (56%); caspase 4 dependent secretome had 598 specific SGs out of 1,223 SGs (49%); exosome secretome had 5,443 specific SGs out of 6,560 (83%); WPB secretome had 3 specific SGs out of 37 SGs (8%); and autophagy secretome had 6 specific SGs out of 16 SGs (37.5%). Our results have demonstrated that some secretory proteins can be secreted via different secretomes. Based on their specificities, we ranked six secretomes from high to low in specificities as exosome secretome (83%) > canonical secretome (68%) > caspase 1 secretome (56%) > caspase 4 secretome (49%) > autophagy secretome (37.5%) > WPB secretome (8%).We performed Venn diagram analysis to determine the overlaps between six types of secretomes. As shown in https://metascape.org/gp/index.html#/main/step1). As the top function pathways listed in 2/M progression), mRNA catabolic process, chaperone mediated protein transport, positive regulation of viral process, NABA extracellular matrix (ECM) glycoproteins, cell–cell communications, and interconversion of nucleotide di- and triphosphates. Caspase 4 secretome carried out nine specific pathways, namely, small molecule catabolic process, aromatic compound catabolic process, cofactor metabolic process, oxidation reduction process, alpha amino acid metabolic process, cysteine and methionine metabolism, platelet degranulation, drug catabolic process, and purine ribonucleoside metabolic process. Exosome secretome carried out eight specific pathways, namely, response to virus, cytokine–cytokine receptor interaction, defense system to other organism, G protein-coupled receptor (GPCR) ligand binding, phosphoinositide 3-kinase (PI3K)-protein B kinase  signaling pathway, glycoprotein metabolic process, skeletal system development, and aminoglycan metabolic process. Of note, autophagy had 6 specific SGs, for which no specific pathways were found in this analysis. In addition, canonical and caspase 1 secretomes shared 10 specific pathways, canonical and caspase 4 shared ten specific pathways, canonical and exosome secretomes shared ten specific pathways, canonical and WPB secretomes shared 3 specific pathways, caspase 1 and caspase 4 secretomes shared ten specific pathways, caspase 1 and exosome secretomes shared 5 specific pathways, caspase 4 and exosome secretomes shared 10 specific pathways, and exosome and WPB secretomes shared one specific pathway.We then hypothesized that each secretomic protein carries out specific cellular functions. To examine this hypothesis, we analyzed the SGs with the Metascape signaling database  promotes human aortic EC activation  induced −/−) aorta, CKD accelerated vascular inflammation in rat aorta, MERS-CoV infected human ECs and AAA in patients aortas, deposited in the NIH/NCBI-GeoDatasets database (https://www.ncbi.nlm.nih.gov/gds). As shown in We hypothesized that metabolic CVD such as atherosclerotic mouse aorta and CKD affected rat aorta, MERS-CoV infection in human ECs and AAA in aortas of patients differentially regulate the expression of various types of secretomes. To examine this hypothesis, as shown in We then hypothesized that upregulation and downregulation of secretomes are different in various diseases. As shown in We then hypothesized that the percentages of modulation (upregulation and downregulation) of each type of secretomic genes are different in various vascular cells. As shown in first, atherosclerosis and CKD significantly modulate the expression of six types of secretomes, but AAA modulate the expression of six types of secretomes much less; second, the ratios of upregulation versus downregulation of secretomic genes in atherosclerosis and CKD were much higher than that in AAA. In contrast, the ratios of upregulation versus downregulation of secretomic genes were low in AAA, suggesting that atherosclerosis and CKD-aorta need more general secretomic functions than AAA, but AAA require more focused secretomic functions than atherosclerosis and CKD-aorta; third, in addition to global secretomic genes analysis in aorta, we also examined the expressions of vascular cell secretomic genes. Ang-II treatment of aortic VSMCs and MERS-CoV-infection of ECs significantly modulate the expression of SGs. The ratios of upregulation versus downregulation of caspase 1 SGs and that of caspase 4 SGs are much bigger than that of other SGs in Ang-II treated VSMCs; and the ratios of upregulation versus downregulation of SGs in Ang-II treated VSMC are bigger than that of MERS-infected ECs, and fourth, canonical secretome, exosome secretome, WPB secretome, and autophagy secretome are general pathology secretomes except WPB secretome in atherosclerosis but caspase 1 secretome and caspase 4 secretome are DAMP receptors-focused and inflammatory secretomes such as chronic inflammation, namely, atherosclerosis, CKD, and Ang-II treated VSMC.Taken together, our data have demonstrated that https://maayanlab.cloud/Harmonizome/dataset/PID+Pathways), positive regulation of response to external stimulus, PID AP1 pathway, and regulation of protein kinase activity. These results have demonstrated that atherosclerotic aorta cells modulate different SGs for different functions with cell-type specificities. For example, canonical secretomic genes for metabolic reprogramming, inflammation; caspase 1-secretomic genes for cytoskeletal signaling, leukocyte extravasation signaling; caspase 4 SGs for B cell signaling and NFAT-related cytokine signaling; exosome SGs for T cell signaling and autoimmune signaling; Weibel–Palade body SGs for blood coagulation and platelet activation; and autophagy SGs for positive response to external stimulus and regulation of protein kinase activity.We hypothesized that each type of secretomic genes play different roles in diseases. To examine this hypothesis, we analyzed the functional signaling pathways in differentially modulated secretomic genes in each disease setting. As shown in As shown in As shown in first, aortic cells in three cardiovascular metabolic diseases such as atherosclerosis, CKD, and AAA modulate different SGs for various pathophysiological functions and cell-type specific functions ; second, cell-type specific functions of caspase 4-GSDMD secretome is atherosclerotic aortas-specific, which do not appear in CKD and AAA aortas; third, exosome secretome carry out T cell signaling function not only in atherosclerotic aortas but also in AAA aortas STAT3 pathway    in CKD afirst, atherosclerotic aorta cells differentially modulate the expression of canonical secretomic genes in the three time points; second, at 6 weeks atherosclerotic aortas have more role of pattern recognition receptors, interferon induction and leukocyte extravasation signaling; 32 weeks atherosclerotic aortas have more T cell exhaustion, IL-6 signaling and neuroinflammation signaling; 78 weeks atherosclerotic aortas have more metabolic reprogramming, LPS/IL-1 inhibition of RXR function, dendritic cell maturation, and Toll-like receptor (TLR) signaling.We then hypothesized that atherosclerotic aortas in different time courses differentially modulate the expression of secretomic genes. To test this hypothesis, we collected time courses microarray datasets of atherosclerotic aortas at high fat feeding for 6, 32, and 78 weeks, respectively, in first, at as early as 6 weeks, atherosclerotic aortas have no functional caspase 1-GSDMD secretomic functions; second, starting from 32 to 78 weeks, atherosclerotic aortas have two major functional pathways, namely, integrin signaling and colorectal cancer metastasis signaling; third, estrogen receptor signaling only appears in 32 weeks atherosclerotic aortas whereas four functional pathways appear only in 78 weeks atherosclerotic aortas, namely, actin nucleation by ARP–WASP complex, synaptogenesis signaling, Rac signaling, and ephrin receptor signaling.Caspase 1-GSDMD SGs at 6 weeks atherosclerotic aortas had no functional pathways. However, at 32 weeks had three functional pathways, namely, integrin signaling, estrogen receptor signaling, and colorectal cancer metastasis signaling and at 78 weeks atherosclerotic aortas had six functional pathways, namely actin nucleation by ARP–WASP complex, integrin signaling, synaptogenesis signaling, Rac signaling, ephrin receptor signaling, and colorectal cancer metastasis signaling. These results have demonstrated that first, at as early as 6 weeks, atherosclerotic aortas have no functional caspase 4-GSDMD secretomic functions; second, starting from 32 to 78 weeks, atherosclerotic aortas have six functional pathways such as IL-8 signaling, neuroinflammation, B cell receptor signaling, endothelin-1 signaling, role of PKR in interferon induction and antiviral response and systemic lupus erythematosus in B cell signaling; third, four functional pathways appear only at 32 weeks atherosclerotic aortas, namely, ERK/MAPK, synaptogenesis, Fc epsilon RI, HOTAIR regulatory pathway whereas four other functional pathways appear only at 78 weeks atherosclerotic aortas such as role of NFAT in regulation of immune response, Rac signaling, fMLP signaling in neutrophils, and ephrin receptor signaling.Caspase 4 SGs at 6 weeks atherosclerotic aortas had no functional pathways, however, at 32 weeks had functional pathways, namely, ERK/MAPK signaling, IL-8 signaling, neuroinflammation, synaptogenesis, Fc epsilon RI, role of PKR in interferon induction and antiviral response, B cell receptor signaling, endothelin-1 signaling, HOTAIR (a long non-coding RNA) signaling, and systemic lupus erythematosus in B cell signaling. Caspase 4 SGs at 78 weeks atherosclerotic aortas had functional pathways, namely, role of NFAT in regulation of immune response, B cell receptor signaling, IL-8 signaling, neuroinflammation, role of PKR in interferon induction and antiviral response, Rac  signaling, formyl peptide receptor 1 (fMLP) signaling in neutrophils, ephrin receptor signaling, endothelin-1 signaling, and systemic lupus erythematosus in B cell signaling. These results have demonstrated that Exosome SGs at 6 weeks atherosclerotic aortas had functional pathways, namely, Reelin  signaling, EC nitric oxide synthase (eNOS) signaling, cholecystokinin/gastrin-mediated signaling, cardiac hypertrophy signaling, melatonin signaling, calcium signaling, synaptic long-term depression, apelin cardiomyocyte signaling, cardiac hypertrophy signaling and endothelin-1 signaling. Exosome SGs at 32 weeks atherosclerotic aortas had functional pathways, namely, granulocyte–macrophage colony stimulating factor (GM-CSF) signaling, role of NFAT in regulation of immune response, HLA-F-adjacent transcript 10  cancer signaling, cell cycle control of chromosomal replication, 3-phophoinositide biosynthesis, PDGF signaling, IL-8 signaling, TREM1 signaling, protein kinase Cθ (PKCθ) signaling in T cells, and neuroinflammation signaling. Exosome SGs at 78 weeks atherosclerotic aortas had functional pathways, namely, oncostatin M signaling, PDGF signaling, type-I diabetes signaling, role of NFAT in regulation of immune response, cell cycle control of chromosomal replication, Th1 pathway, systemic lupus erythematosus in B cell signaling, CD28 signaling in T helper cells, neuroinflammation signaling and PKCθ signaling in T cells.first, exosome SGs at 6 weeks atherosclerotic aortas have pathophysiological functional pathways such as eNOS, cardiac hypertrophy, endothelin-1 signaling; second, starting from 32 to 78 weeks, exosome SGs in atherosclerotic aortas have five functional pathways in common, namely, role of NFAT in regulation of immune response, cell cycle control of chromosomal replication, PDGF signaling, neuroinflammation signaling and PKCθ signaling in T cells; third, at 32 weeks exosome SGs in atherosclerotic aortas have several specific functional pathways, namely, GM-CSF, FAT10 cancer signaling, 3-phosphoinositide biosynthesis, IL-8, and TREM1 signaling; whereas at 78 weeks exosome SGs in atherosclerotic aortas have five specific functional pathways, namely, oncostatin M, type-I diabetes, Th1 pathway, systemic lupus erythematosus in B cell signaling, and CD28 signaling in T helper cells.These results have demonstrated that We then hypothesized that abdominal aortic aneurysm aortas in different time courses differentially modulate the expression of secretomic genes. To test this hypothesis, we collected time courses microarray datasets of aneurysm aortas at days 7, 14, and 28. As shown in first, each time point, AAA aortas have their own specific canonical secretomic signaling pathways; second, two pathways are shared in all three time points, namely, cardiac hypertrophy signaling, dendritic cell maturation; one pathway is shared in the day 7 AAA aortas and the day 14 AAA aortas, HMGB1; whereas as many as four pathways are shared by the day 7 AAA aortas and the day 28 AAA aortas, namely, HOTAIR pathway, tumor microenvironment pathway, IL-17 signaling and systemic lupus erythematosus in B cell signaling; and third, these findings suggest that AAA aortas have two-peak signaling processes in the pathogenesis of AAA.We then examined AAA modulated secretomic gene functions by performing Ingenuity Pathway Analysis (IPA). As shown in Caspase 1 SGs at day 7 AAA aortas had functional pathways, namely, epithelial adherens junction signaling, integrin signaling, estrogen receptor signaling, phagosome formation, Fcγ receptor-mediated phagocytosis in macrophages and monocytes, actin cytoskeleton signaling, and insulin secretion signaling. However, caspase 1 SGs at the day 14 AAA aortas and the day 28 AAA aortas have no significant functional pathways.first, once again, caspase 4 SGs at the days 7 and 28 but not day 14 AAA aortas have two-peak signaling processes in the pathogenesis of AAA; second, the caspase 4 SGs in the day 14 AAA aortas have only one functional pathway; and third, the caspase 4 SGs in the day 7 and day 28 AAA aortas share two functional pathways, namely, Fcγ receptor-mediated phagocytosis in macrophages and monocytes and phagosome formation.Caspase 4 SGs at the day 7 AAA aortas had functional pathways, namely, actin nucleation by ARP-WASP complex, phospholipase C signaling, RAC signaling, TEK tyrosine kinase signaling, Fcγ receptor-mediated phagocytosis in macrophages and monocytes, IL-8 signaling, actin cytoskeleton signaling, regulation of actin-based motility by Rho, signaling by Rho family GTPase, and phagosome formation. Caspase 4 SGs at the day 14 AAA aortas had functional pathways of endothelin-1 signaling. Caspase 4 SGs at the day 28 AAA aortas had functional pathways, namely, ephrin receptor signaling, type-1 diabetes signaling, role of NFAT in regulation of immune response, phagosome formation, Fcγ receptor-mediated phagocytosis in macrophages and monocytes, IL-15 signaling, p70S6K signaling, leukocyte extravasation signaling, role of PKR in interferon induction and antiviral response, and endothelin-1 signaling. These results have demonstrated that first, at all three time points one functional pathway, role of hypercytokinemia/hyperchemokinemia in pathogenesis of influenza, is shared; second, four functional pathways are shared by the day 7 AAA aortas and day 28 AAA aortas, namely, phagosome formation, regulation of actin-based motility by Rho, cell cycle control of chromosomal replication, and hepatic fibrosis signaling; third, one functional pathway of neuroprotective role of THOP1 in Alzheimer’s disease is shared by the day 7 and day 14 AAA aortas; and fourth, exosome SGs in AAA aortas have time point-specific functions.Exosome SGs at the day 7 AAA aortas had functional pathways, namely, neuroprotective role of THOP1 in Alzheimer’s disease, regulation of actin-based motility by Rho, osteoarthritis pathway, phagosome formation, TREM1 signaling, HIPPO (an evolutionarily conserved serine/threonine kinase) signaling, hepatic fibrosis signaling, role of hypercytokinemia/hyperchemokinemia in pathogenesis of influenza, cell cycle control of chromosomal replication, and EIF2 signaling. Exosome SGs at the day 14 AAA aortas had functional pathways, namely, superpathway of cholesterol biosynthesis, PD-1, PD-L1 cancer immunotherapy, intrinsic prothrombin activation, endocannabinoid cancer inhibition pathway, PTEN signaling, role of hypercytokinemia/hyperchemokinemia in pathogenesis of influenza, role of PKR interferon induction and antiviral response, role of p14/p19ARF in tumor suppression, neuroprotective role of THOP1 in Alzheimer’s disease, and RHOGDI signaling. Exosome SGs at the day 14 AAA aortas had functional pathways, namely, role of hypercytokinemia/hyperchemokinemia in pathogenesis of influenza, cell cycle control of chromosomal replication, hepatic fibrosis signaling, regulation of actin-based motility by Rho, Th1 pathway, Fcγ (antibody Fc fragment) receptor-mediated phagocytosis in macrophages and monocytes, GP6 signaling, TEC kinase signaling, neuroinflammation signaling and phagosome formation. These results have demonstrated that WPB SGs at the day 7 AAA aortas had one functional pathway, hepatic fibrosis signaling. Autophagy SGs at the day 7 AAA aortas had five functional pathways, namely, neuroinflammation signaling, HMGB1 signaling, IL-17 signaling, systemic lupus erythematosus in B cell signaling, cardiac hypertrophy signaling.first, among six secretomic pathways in Ang-II induced abdominal aortic aneurysm aortas, canonical, caspase 4, and exosome pathways all have two peak (three-phase) of high (day 7)-low (day 14)-high (day 28) expression patterns; second, caspase 1, WPB and autophagy secretomes are only function at the day 7; and third, the early secretomes may function as drivers for trained immunity  designed list of 1,176 cytokines, chemokines and their interactors (https://www.proteinatlas.org/search/cytokine) as we reported  and exosome (14/56 = 25%) SG pathways in atherosclerotic aorta; 2) most IPA designated cytokines, chemokines and interactors are secreted via canonical (14/25 = 56%) and exosome (10/25 = 40%) SG pathways in CKD aorta; 3) most IPA designated cytokines, chemokines and interactors are secreted via canonical (10/13 = 76.9%) and exosome (3/13 = 23%) SG pathways in AAA aorta; 4) no much cytokines, chemokines and interactors are secreted via caspase 1, and caspase 4 secretomes in those three aortic diseases; and 5) most of disease upregulated cytokines, chemokines and interactors in aorta are disease-specific.Taken together, these results have demonstrated that 1) most IPA designated cytokines, chemokines and interactors are secreted Our previous report showed that large numbers of innate immune genes were modulated in human microvascular EC (HMECs) stimulated with wild-type MERS-CoV, a virus homologous to COVID-19 . HoweverThe IPA analysis results of upregulated SGs in MERS-CoV-infected HMECs showed that the ten top pathways for each secretome were different, suggesting that during MERS-CoV infection of ECs, secretomes play their specific roles in secrete functional regulators , immunotIn addition, we hypothesized that each disease uses several types of SGs for different functions. For example, as shown in Taken together, these results have demonstrated that 1) canonical and caspase 1 secretomes play more roles at early MERS-CoV infection of HMECs whereas caspase 4 and exosome secretomes play more roles in the late and chronic phase infection; 2) early secretomes, namely, canonical and caspase 1 secretomes may promote inflammation effectors , 65; 3) Vascular shear stress results from flowing blood generated frictional force, which has major effects on vascular function. Complex blood flow patterns exert low or low oscillatory shear stress at branches and bends of arteries, this mechanical environment promotes vascular dysfunction and atherosclerosis . A signiTaken together, our results have demonstrated that 1) AVF-venous ECs have significant modulations on the gene expressions of five secretomes but not the expression of autophagy SGs; 2) Upregulations of SGs in AVF-venous ECs result in two pathways in canonical SGs and 13 pathways in exosome SGs; 3) AVF-venous EC SGs have one canonical pathways and five unique exosome pathways; and 4) AVF-venous ECs SGs share two pathways with that of atherosclerotic aorta and CKD aorta; five pathways with MERS-CoV infected ECs, but share no any pathways with that of Ang-II induced AAA.http://www.ieom-tm.com/tidb/browse) in five vascular diseases. Of note, the IPA data showed that five pathways promoted by 101 TIGs, namely,TREM1 signaling, role of pattern recognition receptors in recognition of bacteria and viruses, TLR signaling, role of macrophage, fibroblasts and ECs in rheumatoid arthritis, and role of hypercytokinemia/hyperchemokinemia in pathogenesis of influenza. Furthermore, these TIGs further classified into cytokines (21 genes), enzymes (8 genes), kinases (10 genes), transcription factors (13 genes), transmembrane receptors (15 genes), phosphatases (3 genes), and others (31 genes) , trans-differentiation (plasticity) in aorta and pathogenesis of various aortic pathologies , 50, 53.1 genes)  follista1 genes) , in AVF The itaconate pathway and immune-responsive gene 1  play significant role in promoting trained tolerance and inhibiting trained immunity and inflammation , 100 as We then asked whether ROS regulate the expression of trained immunity genes. To answer this question, we used two key ROS regulators, ROS generation enzyme nicotinamide adenine dinucleotide phosphate oxidase 2 (NOX2) deficient  datasets and ROS-suppressive transcription factor (TF) Nuclear factor erythroid 2-related factor 2  KO datasets as model systems as we reported , 35. As IL-1 family cytokines have been reported as drivers and inhibitors of trained immunity . Five IL−/− GEO database (GSE29765), SET7 SiRNA database (GSE53038), and mTOR inhibitor database (GSE138558). As shown in −/− downregulated 4 canonical SGs and 7 exosomes SGs. In MERS-CoV, HIF1α−/− downregulated 1 canonical SG and 11 exosomes SGs. In AAA and AVF, HIF1a−/− did not downregulate any SGs. HIF1a−/− in CKD downregulated one canonical SG, one caspase 4 SG, and one exosome SG. However, SET7 siRNA in atherosclerosis downregulated 57 canonical SGs, 10 caspase 1 SGs, 12 caspase 4 SGs, 81 exosomes SGs, one WBP SG, and two autophagy SGs. SET7 siRNA in MERS-CoV infection downregulated 12 caspase 1 SGs, 8 caspase 4 SGs, 84 exosomes SGs, one WBP SG, and one autophagy SG. In AAA, SET7 siRNA downregulated 5 canonical SGs, one caspase 1 SG, one caspase 4 SG, and two exosomes SGs. In CKD, SET7 siRNA downregulated 20 canonical SGs, 4 caspase 1 SGs, 7 caspase 4 SGs, 39 exosomes SGs, one WBP SG, and one autophagy SGs. In addition, SET7 siRNA in AVF downregulated 9 canonical SGs, 4 caspase 1 SGs, two caspase 4 SGs, 16 exosomes SGs, and two WBP SGs. Furthermore, mTOR inhibitor downregulated 14 canonical SGs and 8 exosomes SGs in atherosclerosis, but in MERS infection, mTOR downregulated one canonical SG, two exosomes SGs, and one autophagy SG. In AAA, mTOR inhibition downregulated only one exosomes SG. However, mTOR inhibition in CKD downregulated 11 canonical SGs, 13 exosomes SGs, and one autophagy SG. Finally, mTOR inhibition in AVF downregulated two canonical SGs and 5 exosomes SGs. These results indicated that SET7 siRNA downregulated more canonical, caspase 1, caspase 4, and exosomes SGs than HIF1a−/− and mTOR inhibition.Recent progress reported that hypoxia inducible factor 1 alpha (HIF1α) , SET domfirst, trained immunity participates in upregulation of secretomes in atherosclerotic aorta, MERS-CoV infection of ECs, Ang-II induced AAA, and CKD aorta but less in AVF venous EC reprogramming; second, trained immunity gene upregulation peak time courses are different: atherosclerosis at 72 weeks, MERS-CoV infection of ECs at 24 h, Ang-II induced AAA at day 7 reach their peak time in upregulating trained immunity genes, suggesting that trained immunity establishment and immune metabolic reprogramming in aorta and aortic ECs are in different pace in various diseases; third, NOX2 upregulates 3 out of 101 trained immunity and NRF2 suppresses 10 out of 101 trained immunity genes, suggesting that ROS partially regulate trained immunity; and fourth, SET7 deficiency play downregulated more canonical, caspase 1, caspase 4, and exosome SGs than HIF1a−/− and mTOR inhibition in atherosclerosis, CKD, AAA, MERS-CoV infection, and AVF.Taken together, these results have demonstrated that −/− dataset; and 47 SGs  were upregulated in NRF2−/− dataset. Among 129 canonical SGs upregulated in CKD aorta, 4 SGs  were downregulated in NOX2−/− dataset; and 13 SGs  were upregulated in NRF2−/− dataset. Among 23 canonical SGs upregulated in AAA aorta, 0 genes  were downregulated in NOX2−/− dataset; and 1 SG  were upregulated in NRF2−/− dataset. Among 120 canonical SGs upregulated in MERS-CoV infected ECs, two SGs  were downregulated in NOX2−/− dataset; and 11 SGs  were upregulated in NRF2−/− dataset. Among 25 canonical SGs upregulated in AVF venous ECs, only one gene  was downregulated in NOX2−/− dataset; and two SGs  were upregulated in NRF2−/− dataset.We hypothesized that ROS regulate the expression of secretomes in aortic pathologies, EC infection by MERS-CoV and AVF venous EC reprogramming. To examine this hypothesis, we first examined the upregulation of 165 ROS regulators in pathologies. Various aortic and vascular cell pathologies resulted in upregulation of ROS regulators: 33 ROS regulators were upregulated in atherosclerosis, 11 ROS regulators were upregulated in CKD-aorta, 15 ROS regulators were upregulated in MERS-CoV infected ECs, but no ROS regulators were upregulated in fistula venous EC reprogramming Table 4.−/− dataset; and 8% (atherosclerotic aorta), 8.33% (CKD aorta), 50% (AAA aorta), 6.98% (MERS-CoV ECs) and 0% genes (AVF venous ECs) (LogFC >1) were upregulated in NRF2−/− dataset, respectively. Among caspase 4 SGs upregulated in atherosclerotic aorta (75 genes), CKD aorta (31 genes), AAA aorta (7 genes), MERS-CoV infected ECs (61 genes) and AVF venous EC reprogramming (11 genes), 4% genes in atherosclerotic aorta were downregulated but no SGs were downregulated in other datasets (LogFC <−1) in NOX2−/− dataset; and 22.67% (atherosclerotic), 19.35% (CKD), 28.57% (AAA), 9.84% (MERS-CoV ECs) and 18.18% SGs (AVF venous ECs) (LogFC >1) were upregulated in NRF2−/− dataset, respectively. Among exosome SG upregulated in atherosclerotic aorta (510 SGs), CKD aorta (198 SGs), AAA aorta (66 SGs), MERS-CoV infected ECs (616 SGs) and AVF venous EC reprogramming (82 SGs), 2.55% SGs in atherosclerotic aorta, 3.54% SGs in CKD aorta, 0% SGs in AAA aorta, 1.79% SGs in MERS-CoV ECs, and 2.44% SGs in AVF venous ECs were downregulated (LogFC <−1) in NOX2−/− dataset; and 16.86% (atherosclerotic), 15.66% (CKD), 10.61% (AAA), 6.01% (MERS-CoV ECs) and 2.44% SGs (AVF venous ECs) (LogFC >1) were upregulated in NRF2−/− dataset, respectively.Among caspase 1 SGs upregulated in atherosclerotic aorta (25 genes), CKD aorta (12 genes), AAA aorta (4 genes), MERS-CoV infected ECs (43 genes) and AVF venous EC reprogramming (8 genes), 0 genes were downregulated (LogFC <−1) in NOX2Taken together, these results have demonstrated that: 1) NRF2 plays more significant roles in downregulating SGs than that of NOX2 in upregulating SGs in aortic and vascular pathologies; 2) NRF2 plays more roles in downregulating SGs in canonical, caspase 4 and exosome secretomes than that in caspase 1 secretome; and 3) NOX2 plays more roles in upregulating SGs in canonical and exosome secretomes than that in caspase 1 and caspase 4 secretomes.via canonical and exosome secretory pathways but not caspase 1 and caspase 4-GSDMD secretory pathways; 8) Canonical and caspase 1 secretomes play roles at early MERS-CoV infection whereas caspase 4 and exosome secretomes play roles in the chronic phase infection; and the early upregulated canonical and caspase 1 secretomes may function as drivers for trained immunity; 9) Venous ECs from AVF upregulates genes in five secretomes, resulting in two pathways in canonical secretome and 13 pathways in exosome secretome; 10) Trained immunity participates in the upregulation of secretomes of atherosclerotic aorta, MERS-CoV infection of ECs, Ang-II induced AAA aorta, and CKD aorta but less in AVF venous EC reprogramming; trained immunity and immune metabolic reprogramming are in different pace in various diseases; NRF2 and NOX2 partially regulate trained immunity; and 11) ROS-suppressing transcription factor NRF2 plays more significant roles in downregulating secretomic genes than that of ROS generating enzyme NOX2 in upregulating secretomic genes in aortic and vascular pathologies; NRF2 plays more roles in downregulating genes in canonical, caspase 4 and exosome secretomes than that in caspase 1 secretome; and NOX2 plays more roles in upregulating genes in canonical and exosome secretomes than that in caspase 1 and caspase 4 secretomes.Our previous work and those of others have demonstrated that ECs are innate immune cells in pathological conditions stimulated by DAMPs/PAMPs. Significant progress has been made to demonstrate that vascular cells and recruited immune cells in aortas in response to DAMPs and PAMPs undergo differentiation and trans-differentiation to promote the pathogenesis of atherosclerosis, CKD-enhanced aortic inflammation, AAA and AVF reprogramming. However, a key question remains to be addressed, by what stimuli, in addition to DAMPs/PAMPs, do those vascular cells and recruited immune cells differentiate and trans-differentiate. To answer the question, we performed extensive transcriptomic analyses of six secretomic gene lists in many microarray datasets deposited in the NIH/NCBI GeoDatasets database and the EMBL-EBI-ArrayExpress repository, and made the following findings: 1) 53.7% out of 21,306 human protein-encoding genes are classified into six secretomes; among which four secretomes, canonical, caspase 1, caspase 4 and exosomes, are the large secretomes with >900 proteins; and six secretomes are ranked from the highest specificity to the lowest as exosome (83%) > canonical (68%) > caspase 1 (56%) > caspase 4 (49%) > autophagy (37.5%) > WPB (8%); 2) Atherosclerosis, CKD, and AAA significantly modulate six secretomes in aortas; and MERS-CoV infection in human ECs and Angiotensin-II treatment in VSMCs also modulate six secretomes; 3) Atherosclerotic aortas upregulate T cell and B cell adaptive immune secretomic genes (SGs); CKD aortas upregulate SGs for cardiac hypertrophy, hepatic fibrosis and senescence; and AAA aortas upregulate SGs for neuromuscular signaling, protein catabolic process and Fcγ receptor-mediated phagocytosis; 4) Canonical secretome in atherosclerotic aortas upregulate pattern recognition receptors, leukocyte extravasation at 6 weeks, T cell exhaustion, fibrosis and neuroinflammation at 32 and 78 weeks; caspase 1, and caspase 4, GSDMD secretomes have no functions at 6 weeks, but have functions of cancer metastasis, NFAT and B cell signaling at 32 and 78 weeks, respectively; and exosome secretome has eNOS, and calcium signaling at 6 weeks but GF-CSF, NFAT, and T cell signaling at 32 and 78 weeks; 5) In Ang-II induced AAA, canonical, caspase 4-GSDMD, and exosome secretomes have two peaks (three-phases) of high (day 7)-low (day 14)-high (day 28) expression patterns; and caspase 1-GSDMD, Weibel–Palade body, and autophagy secretomes are only functional at the day 7; 6) Elastase-induced AAA aortas have more inflammatory and immune pathways than that of Ang-II induced AAA aortas; 7) Most disease-upregulated cytokines and chemokines in aorta are secreted +Foxp3+ Tregs in aorta experience T helper cell (Th) lineage plasticity into Th1-Tregs, Th2-Tregs, Th-17-Tregs, and Tfh-Tregs  become phenotypical switched into macrophages/monocytes-like VSMCs, monocytes become Ly6C+/high proinflammatory ones, macrophages are polarized into M1 and other effector subsets, CD4+ Tregs become plastic into Th1-like, Th17-like and other status in addition to maintain their immunosuppressive functions; second, significantly upregulated six types of secretomes, namely, canonical, caspase 1-GSDMD, caspase 4-GSDMD, exosome, WPB, and authophagy, promote the establishment of niches for immune activation, differentiation (polarization), and trans-differentiation (plastic); and third, trained immunity promoters, IL-1 family cytokine trained immunity promoters, HIF1a, SET7, mTOR, and ROS generator NOX2 (https://singlecell.broadinstitute.org/single_cell?type=study&page=1&terms=aorta) in identifying numerous subtypes of monocytes, endothelial cells, fibroblasts support this new working model. Therefore, aortas can be classified into an immune organ, which express immune regulatomic genes such as receptors for virus infections, DAMPs/PAMPs (PPRs), cytokines (To summarize our findings here, we propose a new working model (tor NOX2  as undertor NOX2  depositeytokines , chemokiytokines , cell deytokines , growth ytokines , immune ytokines , and regytokines -related ytokines , and plaThe original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding author.YL carried out data collections, data analyses and drafted the manuscript. YuS, KX, FS, YiS, CDIV, SW, WH, JY, SPK, JRB, RIVP, JS, XJ, and HW provided material input. XY supervised experimental design and data analyses. XY edited the manuscript. All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.This work was supported by the National Institutes of Health Grants to XY.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Enterococcus genus, including marine isolates, toward which UA-G4K has shown minimum inhibitory concentrations (MICs) very low (0.5–4.3 µM), regardless of their resistance to antibiotics. Time-kill experiments, in addition to confirming the previously reported bactericidal activity of UA against E. faecium, also established it for UA-G4K. Furthermore, cytotoxicity experiments on human keratinocytes revealed that nanomanipulation of UA significantly reduced the cytotoxicity of UA, providing UA-G4K NPs with very high LD50 (96.4 µM) and selectivity indices, which were in the range 22.4–192.8, depending on the enterococcal strain tested. Due to its physicochemical and biological properties, UA-G4K could be seriously evaluated as a novel oral-administrable therapeutic option for tackling difficult-to-treat enterococcal infections.Ursolic acid (UA), a pentacyclic triterpenoid acid found in many medicinal plants and aromas, is known for its antibacterial effects against multi-drug-resistant (MDR) Gram-positive bacteria, which seriously threaten human health. Unfortunately, UA water-insolubility, low bioavailability, and systemic toxicity limit the possibilities of its application in vivo. Consequently, the beneficial activities of UA observed in vitro lose their potential clinical relevance unless water-soluble, not cytotoxic UA formulations are developed. With a nano-technologic approach, we have recently prepared water-soluble UA-loaded dendrimer nanoparticles (UA-G4K NPs) non-cytotoxic on HeLa cells, with promising physicochemical properties for their clinical applications. In this work, with the aim of developing a new antibacterial agent based on UA, UA-G4K has been tested on different strains of the  For many years, antibiotics have made it possible to counteract serious bacterial infections and to perform various medical and surgical procedures in greater safety, thanks to their protection. Unfortunately, over the past 20 years, these drugs have been used unscrupulously, leading to the selection of an increasing number of multidrug-resistant bacteria (MDR), which greatly threaten human health .Today, MDR pathogens of both Gram-positive and Gram-negative species cause infections against which traditional antibiotics are no longer effective and cause an increasing number of deaths in hospitals, long-term care facilities, and community settings. The surveillance of antibiotic resistance is a global public health concern ,5,6,7,8,Acinetobacter baumannii, Pseudomonas aeruginosa, and Stenotrophomonas maltophilia, are now emerging as clinically relevant superbugs, contributing significantly, with their worrying levels of resistance, to numerous therapeutic failures [Streptococcus pneumoniae, methicillin-resistant Staphylococcus aureus (MRSA) and S. epidermidis (MRSE), and enterococcal species, such as Enterococcus faecium and E. faecalis, which express high-level resistance to aminoglycosides and/or resistance to vancomycin (VRE) [Especially Gram-negative pathogens, such as failures . Howeverin (VRE) . Often, in (VRE) . NowadayAromatic plants are the main sources of natural antibacterial products, including monoterpenes, sesquiterpenes, caffeic acid, luteolin, rosmarinic acid, hispidulin, flavonoids, oleanolic acid (OA) and ursolic acid (UA) . The latUA responds to the chemical name 3β-hydroxy-urs-12-en-28-oic-acid  . It is aS. aureus, E. faecalis, S. mutans, S. sobrinus and Mycobacterium tuberculosis [UA is considered a promising compound for cancer prevention and therapy, as it influences cell signalling pathways, inhibiting enzyme activity, inducing apoptosis, and reducing tumor growth. It is abundant in the plant surface extracts ,24,25,26rculosis ,29,30.S. aureus [UA can be used synergistically with antibiotics for enhancing their activity ,32,33 an. aureus  and to i. aureus . Althoug. aureus . Recentl. aureus .Unfortunately, UA also exhibited unwanted features. Reports revealed that UA can trigger unwanted phenomena under certain conditions and can be cytotoxic to human cells ,39. In aIn this regard, considerable progress has been made in the design of the ideal drug carriers, as evidenced by the Food and Drug Administration (FDA) approval obtained by some nanocarriers . MoreoveAmong the nano-systems designed to address these objectives, mainly polymer-based NPs have attracted the interest of scientists due to the versatile characteristics of polymers that allow adjustment of the physicochemical and biological properties of the corresponding nanocarriers. Consequently, several species of polymers have been developed to solubilize and formulate multifunctional drug carriers with properties adapted to the considered application . In thisIn this scenario, with the final aim of making the clinical administration of UA feasible, we have recently increased its poor solubility in water and cancelled its residual toxicity on HeLa cells, trapping it in a cationic fourth-generation, not cytotoxic polyester-based dendrimer (G4K) containing lysine . Highly Moreover, concerning the cytotoxicity experiments performed on HeLa cells, no cytotoxicity (94.3% cell viability) was found, even at the highest concentration tested , thus establishing the capability of the dendrimer reservoir (G4K) to cancel the cytotoxicity of the pristine UA (cell viability of 72.8% at 20 µM concentration).Enterococcus genus. Therefore, we studied in detail the effects of UA-G4K NPs on several isolates of enterococci of different species, including in the study also strains of marine origin isolated from the seawater of the Ligurian coast, obtaining excellent results. This choice was inspired by the idea of comparing the sensitivity to UA-G4K NPs also of enterococcal strains that can be isolated in bathing waters.Encouraged by the new physicochemical characteristics acquired by UA, the UA-G4K NPs obtained were biologically evaluated here, investigating their effects on both bacterial and normal eucaryotic cells. Particularly, a preliminary screening showed a remarkable and selective antibacterial activity against the Enterococcus genus [Finally, to evaluate the feasibility of the clinical application of UA-G4K for the treatment of infections caused by VRE isolates of the us genus , the cytThe biodegradable cationic dendrimer nanoparticles (NPs) loaded with UA (UA-G4K NPs) used in this study were recently synthesized according to the synthetic procedure reported by Alfei and collaborators . ExperimChemical materials and methods, as well as instruments used for the physicochemical and biological characterization of UA-G4K NPs, are consultable in our previous work .® 2  or matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometric technique . Two strains were of Gram-negative species (1 Escherichia coli and 1 MDR Pseudomonas aeruginosa). A total of 29 strains were Gram-positive, including two isolates of the genus Staphylococcus (1 MRSA and 1 MRSE) and 27 clinical isolates of the genus Enterococcus, including 6 vancomycin sensible (VSE) E. faecalis, 2 vancomycin resistant (VRE) E. faecalis, 1 VSE E. faecium, 4 VRE E. faecium, 3 E. casseiflavus, 3 E. gallinarum, 3 E. avium, and 2 E. durans. In addition, three strains of marine isolates of the genus Enterococcus were also included in this study .A total of 31 strains belonging to the Gram-positive and Gram-negative species were used in this study. All were clinical or marine isolates, belonging to a collection obtained from the School of Medicine and Pharmacy of University of Genoa , identified by VITEKThe antimicrobial activity of the substances considered in this study was investigated, determining their MICs. To this end, we followed the microdilution procedures specified by the European Committee on Antimicrobial Susceptibility Testing EUCAST  and descE. faecalis and E. faecium species as previously reported [Killing curve assays for UA and UA-G4K NPs were performed on representative isolates of reported ,51. ExpeIn these experiments, we used human skin keratinocytes cells (HaCaT) obtained and grown as described in a recent study published by us . BrieflyThe viability assay was performed both on the empty dendrimer (G4K), UA and UA-G4K NPs following a procedure previously described . Differep-value ranges: * = p < 0.05, ** = p < 0.01, *** = p < 0.001. Concerning MIC values, experiments were made in triplicate, the concordance degree was 3/3 and ± SD was zero.Concerning cytotoxicity studies, the statistical significance of differences between experimental and control groups was determined via two-way analysis of variance (ANOVA) with the Bonferroni correction. The analyses were performed with Prism 5 software . Asterisks indicate the following The procedure described in Section S1.1 and showed in Scheme S1 (SM) produced the UA-loaded fourth-generation dendrimer NPs (UA-G4K NPs) .p, mV) of UA-G4 NPs are reported in Their characterization by FTIR and NMR analyses, consultable in SM (Section S1.1), confirmed the UA-G4K structure . The resRecently, we encapsulated UA, known for its in vitro antibacterial effects against strains of different Gram-positive genera ,54,55,56E. coli and P. aeruginosa, UA-G4K NPs were also inactive. Furthermore, the screening showed that, unlike free UA, UA-G4K was also poorly active against staphylococci  of UA-G4K NPs. The MIC value of 128 µg/mL was assumed as the cutoff value above which the compound was deemed inactive, regardless of the corresponding µM concentrations, which in all determinations were for UA-G4K 8.1–65.2-fold lower than those of UA. The results showed that, as expected, due to the UA inactivity against Gram-negative species such as ylococci .Enterococcus genus , the UA-G4K NPs displayed very low MICs (0.5–2.1 µM), which were 2.1–35.6-fold lower than those displayed by the untreated UA. This exciting result is due to the high DL% of UA-G4K (which resulted in the presence of 33 UA moles per mole of dendrimer) and to the favorable UA release profile from UA-formulation [As can be seen in mulation . The higmulation ,58 or anmulation ,60,61,62mulation , the onlmulation . TherefoEnterococcus genus, able to inhibit their growth regardless of their multi-resistance to existing antibiotics. Moreover, the physicochemical characteristics of UA-G4K [p-value, polydispersity index, UA release profile and kinetics, solubility in water, and stability in aqueous solution, suggest its suitability for any biomedical applications. As a result, UA-G4K may represent a possible new therapeutic option for addressing severe enterococcal infections caused by MDR species, which are of increasing concern around the world. Enterococci, in fact, are the predominant flora in the gut, which, from this biological district, can extend to the bloodstream or invade as colonizers other tissues, causing endocarditis, urinary tract infections, prostatitis, intra-abdominal infection, cellulitis, wound infection, or infections of the skin and soft tissues [E. faecium possesses an acetylase enzyme (AAC(6ʹ)) that causes higher MICs to aminoglycosides, such as tobramycin and amikacin, which negatively affects their synergistic effect with cell-wall agents [E. faecalis strains have a gene (designated lsa) that encodes an ATP-binding protein that confers intrinsic resistance to quinupristin/dalforistin (Q/D) [E. faecium was found to be the most common enterococcal species isolated from immunocompromised patients [E. faecalis, and vancomycin resistance is much less frequent, E. faecium resistance to antibiotics such as ampicillin  and vancomycin (MIC = 512 µg/mL), which are used to be the cornerstone antibiotics for the treatment of enterococcal infections, is almost “normal” in hospital-associated isolates of E. faecium. Indeed, the infectious Disease Society of America (IDSA) has included E. faecium among the ESKAPE pathogens  for which new therapies are urgently needed [E. faecalis, generally are 0.5 to 4.0 µg/mL (1.4 to 11.4 µM) [E. faecium are 4 to 8 µg/mL (11.4 to 22.8 µM) [E. faecalis and E. faecium  but, according to a study also reporting the MIC of vancomycin against urinary VSE isolates of the Enterococcus genus (MIC = 1.4 µM), were in some cases more potent than vancomycin when effective [In this study, we have identified a new powerful antibacterial agent based on nanotechnologically modified UA (UA-G4K NPs), selectively active against different species of the f UA-G4K , such as tissues . Enterocl agents . Most E.in (Q/D) . In the patients . While ry needed ,68. Agaiy needed ), the us11.4 µM) , and for22.8 µM) , have sh22.8 µM) ,69. SuchTo our knowledge, there is only one study in literature that established the bactericidal activity of both OA and UA on enterococci , while mE. faecium BM4147 strain when added to the culture medium at concentrations above 16 µg/mL (2 times MIC), while it only demonstrated bacteriostatic activity on E. faecalis at concentrations above 8 µg/mL (2 times MIC).In particular, the authors stated that UA showed bactericidal activity on the E. faecium and E. faecalis species at concentrations 5× MICs. In this way, having the same MIC, all strains tested in these experiments were exposed to the same dose of UA , which is like the doses used in the time-kill experiments reported by Horiuchi [E. faecium 21 , steady regrowth began immediately and after 24 h, only a reduction of 2.2 logs was observed   b.P. putida, whose structure should undergo the same degradation process as assumed for our lysine-dendrimers, addressed this issue by administering a mixture of ε-PL/dextrin 50/50, where dextrin, which is a well-known stabilizer, certainly protected ε-PL from self-degradation [Interestingly, these results differed from those observed for the lysine-modified fifth-generation cationic dendrimers G5K  and G5-Pradation . In the 50, which is the dose needed to kill 50% of the cells) on eukaryotic cells. In microbiology, the SI value is given by Equation (1) and provides a measure of the selectivity of the antimicrobial agent for bacteria.The solubility in water and an appropriately high value of the selectivity index (SI) are pivotal requirements to make a molecule worthy of consideration as a new therapeutic agent against bacterial infections. With our previous study , we have50. The obtained LD50 and the MIC values were used to calculate the SI value of UA-G4K and UA against each isolate of the Enterococcus genus considered in this study and when compared, produced very satisfactory results.To determine the SI value for UA-G4K NPs and UA, we performed dose and time-dependent cytotoxicity experiments on human keratinocytes (HaCaT), and the results from the dose-dependent cytotoxicity experiments performed for 24 h were used to compute the LDEnterococcus genus, which have been shown to be the preferred target of the antibacterial activity of UA-G4K NPs, are often the cause of a variety of skin, soft tissue, and wound infections [Dose- and time-dependent cytotoxicity experiments were performed to evaluate the effects of UA-G4K NPs on HaCaT keratinocytes cells. Cytotoxicity experiments under the same conditions were also performed for UA and G4K to evaluate the reciprocal effects on the original cytotoxicity of pristine UA and empty dendrimer. Such experiments were performed on HaCaT keratinocytes cells for several reasons. MDR isolates of the fections . ConsequAs can be seen in Differently, after 12 h of exposure, at concentrations 1–25 µM, G4K showed cytotoxicity comparable or slightly higher than those of pristine UA and UA-G4K NPs, except for the concentration 20 µM, at which (strangely) the more cytotoxic compound was UA-G4K (cells viability of 81.97% vs. 88.8% of G4K and 91.2% of UA). At concentration 50 µM, G4K was significantly more cytotoxic than UA and UA-G4K, while it proved a cytotoxicity significantly lower than that of UA at higher concentrations (cells viability of 56.1% vs. 50.7% at 75 µM and of 53.4% vs. 31.1% at 100 µM), thus establishing that UA was 1.7-fold more cytotoxic than G4K at 100 µM. Moreover, although showed comparable or lower cytotoxicity than that of two ingredients also at lower concentrations (except for 20 µM), evidencing their mutual contribution to reducing the cytotoxicity of the complex, at the concentrations of 75 and 100 µM, UA-G4K NPs were 1.5–1.6-fold less cytotoxic than G4K and 1.6–2.5-fold less cytotoxic than UA, leaving alive the 77% of cells also at the higher concentration tested (100 µM). As expected, the cytotoxic effects of all compounds were more marked after 24 h of cells exposure, but a similar trend to that observed for 12 h of treatment was maintained. Particularly, G4K was the more cytotoxic compound at concentrations 1–50 µM, at 75 µM, its cytotoxicity was higher than that of UA-G4K and comparable with that of UA, while at 100 µM, the more cytotoxic substance was UA (cells viability of 15.0% vs. 23.0% (G4K) and 52.0% (UA-G4K)). Moreover, while after 12 h of exposure to G4K, the viability of cells did not drop below 53.4%, also at 100 µM, after 24 h of exposure, cells viability was remarkably under 50% (36.9%) already at a concentration of 50 µM. At such concentrations, when exposed to untreated UA, cells viability remained higher than 50% (56.6%), but at greater concentrations, it dropped dramatically, reaching 26.8% at 75 µM and 15.0% at 100 µM.Interestingly, when exposed to UA-G4NPs, cells viability remained higher than 50% (52 µM), also at 100 µM, which is 23.2–200-fold higher than the MICs determined on enterococci considered in this study. Finally, while the cytotoxic effects of UA-G4 NPs were comparable to those of UA up to concentration 25 µM, they were significantly lower at concentrations 50, 75 and 100 µM.50/MIC) was determined, getting the LD50 from the data obtained from the cytotoxicity experiments at 24 h of exposure.Collectively, by encapsulating UA in G4K, we realized the reciprocal reduction in the intrinsic cytotoxicity of the two ingredients when alone, achieving water-soluble UA-loaded NPs that showed potent antibacterial activity at not cytotoxic dosage. To have an idea of the selectivity of UA-G4K for bacteria, the SI .The LD50  was reported vs. concentrations (µM), and three dispersion graphs were obtained. The equations of the correspondent linear regression models obtained by the ordinary least squares (OLS) method were used to compute the respective LD50 .2 values, the LD50, and the range of SI for UA and UA-G4K; the SI values computed for each enterococcal isolate considered in this study are observable in 2 , the UA nanotechnologically manipulated resulted 32.2-fold less cytotoxic than untreated UA.In addition, if we consider the amounts of UA that the LD50 of untreated UA (54.9 µM) and the dose of UA-G4K necessary to deliver such amounts of UA (3.0 µM), it can be noted that at the obtained concentration of 3.0 µM, UA-G4K is practically not cytotoxic, leaving alive the 91.2% of cells. This means that the UA-loaded formulation obtained by merging UA and G4K, when will be administered at a dosage able to release a UA amount, which, if untreated, kills the 50% of HaCaT cells, will be lethal for only the 8.8% of exposed cells.Moreover, considering the LD50 of UA-G4K should be 1766.1 µM, the SI of the nanoengineered UA was in the range 410.7–3532.2, that is 66.2–285.6-fold higher than that of untreated UA. Concerning the SI values that can be considered satisfactory, the reported opinions are conflicting. Some authors have hypothesized that SI values ≥ 10 make a molecule worthy of further investigation [Concerning the SI values of UA (6.2–12.5) and UA-G4K (22.5–193.7), they were both much higher than those reported as acceptable to consider the new antibacterial agent suitable for therapeutic uses. Moreover, the SI values of the UA-G4K NPs were 3.6–31.2-fold higher than those of UA, thus establishing its higher suitability for biomedical applications and as a new therapeutic agent. In addition, considering that the UA released by the LDtigation ,76, whiltigation  proposedtigation ,79 reportigation , who destigation . Due to Enterococcus genus, including VRE clinical and marine isolates of E. faecalis and E. faecium. The micromolar MIC values determined for UA-G4K NPs on 31 enterococcal strains were very low (0.5–4.3), regardless of the drug resistance patterns of the tested strains, thus satisfying the urgent need for new antibacterial agents active also against pathogens against which vancomycin is no longer effective. Furthermore, in all cases, the MICs of UA-G4K were lower than those determined for free UA, thus confirming that our innovative strategy, to encapsulate UA in G4K, not only succeeded in obtaining water-soluble UA formulations suitable for possible in vivo applications but also improved the antibacterial potency of the UA.In this study, water-soluble UA-loaded NPs (UA-G4K), previously obtained by UA encapsulation in an auto-biodegradable fourth-generation cationic dendrimer containing lysine (G4K), not possessing antibacterial properties, demonstrated remarkable antibacterial effects against several strains of different species of the E. faecium and E. faecalis species demonstrated that UA possesses bactericidal activity on E. faecium and bacteriostatic effects on E. faecalis.Confirming the observations reported for the first time by Horiuchi and co-workers and never confirmed until now, time-kill experiments performed with UA on representative isolates of E. faecium isolates, while bacteriostatic effects on strains of E. faecalis species.Time-kill curves obtained with water-soluble UA-G4K NPs on the same strains showed that UA-loaded NPs, when administered at the same micromolar concentration of UA (21.8 µM), possessed a behavior identical to that of UA, exerting bactericidal effects on Enterococci, in addition to endocarditis, urinary tract infections, prostatitis, intra-abdominal infections, and concomitant bacteraemia, could also be responsible for serious infections of the skin, soft tissues, and wounds. Consequently, to evaluate the possible future clinical application of UA-G4K as a therapeutic agent against these infections, we evaluated its cytotoxicity on human keratinocyte cells (HaCaT).50 = 96.4 µM . Therefore, the selectivity indices calculated for UA-G4K NPs were very high and in the range 22.5–193.7. As already highlighted, if we compare equal micromolar concentrations of UA, G4K and UA-G4K, without considering the actual amount of UA that the prepared nano-formulation can deliver thanks to the entrapment of UA in G4K dendrimer, the original cytotoxicity of the two ingredients has been reduced by 1.8- and 2-fold, respectively, and the SI of UA was improved by 1.8–31.2 times.The results showed that UA-G4K is practically non-cytotoxic to these cells and well tolerated, being its LDEnterococcus genus and non-cytotoxic toward mammalian cells. Overall, the novelty of our research consists in having successfully tested a new bactericidal agent based on UA potentially administrable in vivo because water-soluble and, consequently endowed with suitable bioavailability, free from cytotoxicity toward eukaryotic cells, and obtained exclusively with a nanotechnological approach which, differently from the nano-emulsion techniques, avoided the use of harmful solvent, co-solvents, and surfactants. To the best of our knowledge, currently, although there is so far no approved dendrimer drug in therapy, six dendrimer derivatives were reported in clinical trials, and seven are available on the market. Particularly, the following dendrimer-derived agents were reported to be in clinical trials: DEP® docetaxel, DEP® cabazitaxel and VivaGel® , a vaccine with the dendrimeric MAG-Tn3 for breast cancer, ImDendrim for inoperable liver cancer, and OP-101 for X-linked adrenoleukodystrophy, while Dendris, 3DNA®, Alert ticketTM, Polyfect®, Stratus CS®, VivaGel® and Superfect® are already present on the market.Differently, if the actual amount of nanoengineered UA delivered by UA-nano-formulation is considered, the cytotoxicity of the untreated UA was decreased by 32.2 times, and the selectivity indices were improved by 32.9–569.7 times. With our study, we have detected a powerful antibacterial compound, highly selective for different species of the In this regard, we believe that the UA-G4K NPs developed here can be considered for future clinical use. Indeed, we are confident that UA-G4K NPs may be suitable for oral administration since previous pharmacokinetic and pharmacodynamic studies on poly(amidoamine) (PAMAM) dendrimer-based drug formulations, administered orally for the treatment of hypercholesterolemia performed in Male albino Sprague-Dawley rats, showed suitable pharmacokinetic performances, even better than those of the suspension of the pure drug. In addition, several formulations of water-soluble drugs, obtained using dendrimers as solubilizing agents, showed better and suitable bioavailability.