Valorisation of the urban plastic waste in high-quality recyclates is an imperative challenge in the new paradigm of the circular economy. In this scenario, a key role in the improvement of the recycling process is exerted by the optimization of waste sorting. In spite of the enormous developments achieved in the field of automated sorting systems, the quest for the reduction of cross-contamination of incompatible polymers as well as a rapid and punctual sorting of the unmatched polymers has not been sufficiently developed. In this paper, we demonstrate that a miniaturized handheld near-infrared (NIR) spectrometer can be used to successfully fingerprint and classify different plastic polymers. The investigated urban plastic waste comprised polyethylene (PE), polypropylene (PP), poly(vinyl chloride) (PVC), poly (PET), and poly(styrene) (PS), collected directly in a recycling plastic waste plant, without any kind of sample washing or treatment. The application of unsupervised and supervised chemometric tools such as principal component analysis (PCA) and partial least squares-discriminant analysis (PLS-DA) on the NIR dataset resulted in a complete classification of the polymer classes. In addition, several kinds of PET  were correctly classified as PET class, and PE samples with different branching degrees were properly separated. The huge amount of urban plastic waste and the continuous growth in human plastic consumption require a high valorisation of the collected waste in the direction of a whole-system economic sustainability. By the numbers, European plastic production reached almost 60 million tonnes in 2016, and 335 million tonnes globally (Source: PlasticsEurope Market Research Group (PEMRG)/Conversio Market & Strategy GmbH). In this framework, over 8.4 million tonnes of plastic waste were collected in order to be recycled inside and outside the EU [Generally, automated sorting processes are widespread in plastic recycling facilities . These sBesides the automated sorting of the plastic stream, manual sorting is still necessary for the full separation of the collected waste. However, manual sorting based only on plastic code recognition is inefficient and time consuming. In some cases, plastic goods can be badly crushed or have missing or unreadable codes. Manufacturers can also use different polymers for the same product, depending on marketing choices. In addition, the high turnover rate can be affected by an improperly trained or unskilled workforce.In this scenario, the possibility of exploiting a well-established technique for polymer identification combined with a miniaturized, portable, low-cost, and real-time spectrometer for local and punctual semi-automated sorting is highly desirable. Urban plastic waste normally contains plastics such as polyethylene (PE), polypropylene (PP), poly(vinyl chloride) (PVC), poly (PET), and poly(styrene) (PS). These polymers have characteristic near-infrared (NIR) spectral fingerprints that can be used for accurate sorting and separation . The proIn the present work, we challenged a compacted and miniaturized NIR spectrometer for a rapid identification of urban plastic waste collected directly from a selection and recycling plant, without any sample treatment. We collected a large amount of plastic samples directly from the plant and we created a robust database of NIR spectra. Different chemometric tools were applied for data evaluation of all the recorded samples. Principal component analysis (PCA) for preliminary data exploration and partial least squares-discriminant analysis (PLS-DA) as supervised pattern recognition were applied to fingerprint and classify the different plastic polymers.Plastic samples were collected at the selection division of the recovery and recycling plant Montello SpA , which receives post-consumer plastic in the form of urban waste for recycling. In total, 250 samples belonging to various classes of polymer were used see . The polTM Pro v3.0 software .Spectra acquisition was realized using MicroNIR On-site  as dispersing element, in contrast to traditional diffraction-based spectrometers . All calculations were performed using MATLAB (R2018a) with chemometric toolboxes ,23.NIR spectroscopy has the key advantage of being a rapid-response analytical tool, recording spectra with no prior manipulation and predicting physical and chemical parameters from a single spectrum . In the The NIR reflectance spectra of the five polymers  in the range 900–1700 nm, obtained by the MicroNIR On-site, are shown in In the absorbance spectra a, the prPrincipal component analysis is a very powerful chemometric tool for analysing data structure . The aimFurthermore, the results of PCA applied to the restricted dataset of PET samples showed no separation among the five kinds of PET samples , confirmThe discrimination between HDPE and LDPE is an important issue in terms of plastic recycling . PE is lAfter the exploratory PCA analysis, a supervised classification tool was applied in order to distinguish the different plastic classes. In PLS-DA, the well-known PLS regression algorithm is modified with a classification goal. The response variable is categorical, reflecting the belonging class of the statistical units. PLS-DA returns the prediction in a vector of size equal to the number of classes in the predictor variables, with values ranging from 0 to 1 .Validation tools are very useful methods in chemometrics, and they are used to verify the capability of the model prediction. Before the model calibration, data were split into training and test sets containing 901 and 386 samples, respectively. The training set was used to calibrate the model both in fitting and in cross-validation, while the test set was employed only at the end of the procedure in order to evaluate the true predictive capability of the calibrated model .n-th sample from the dataset starting from the first one [During the calibration, the selection of the optimal number of latent variables (LVs) is a crucial point, which is performed based on the cross-validation procedure . This shirst one . The selAfter the determination of the optimal number of LVs, the calculation of the PLS-DA model was performed selecting six latent variables and 10 cross-validation groups for internal validation. In The performance of the classification model could be also evaluated by considering the classification parameters derived from the confusion matrix: sensitivity (Sn), specificity (Sp), and non-error rate (NER). These parameters represent the ability of the model to correctly identify the samples of the given class, the ability of a classifier to reject the samples of other classes, and the average of the class sensitivities, respectively . Table 2Furthermore, we analysed the confusion matrix and the classification parameters for the test set, used as an additional trial to validate the model. In the case of the restricted data set composed of HDPE and LDPE spectra, the optimal number of LVs was set to six, assigning 90% of total variability see . With reIn the present study, the use of a handheld NIR spectrometer combined with robust chemometrics tools to fingerprint different urban plastic waste collected directly from a recycling plant was presented. Plastic samples included clear, blue, coloured, opaque, and boxes of PET, PE with different branching degrees (HDPE and LDPE), PP, PVC, and PS. In a first step, PCA was applied to NIR spectra of all polymer classes, and polymers were correctly separated in clusters. Moreover, by limiting the analysis on PE samples, HDPE and LDPE were correctly discriminated. In addition, by comparing samples of different colours, no differences in band positions were found, suggesting that colorants present in the plastic resin matrix do not significantly influence NIR spectra. Moreover, even if samples were not washed or treated prior to the analysis, NIR spectra were not affected by dust contamination or liquid residuals, meaning a successful application directly to the urban plastic waste.After the exploratory PCA analysis, PLS-DA was applied. Before the model calibration, data were split into a training and test set, using the training set to calibrate the model. In both fitting and cross-validation, the performance of the model was very high, proved by the correlation between observed and predicted classes. All the analysed samples were also correctly designated in the case of the restricted data set composed of HDPE and LDPE spectra.The results show promising outcomes in order to improve the reliability and efficiency of the manual sorting of plastic waste, increasing the volume of recycled plastic and the management of multi-component polymer types.


In this review, the mechanisms and implications of this process in the gene regulation mediated by miRNAs will be discussed. This work will also explore techniques for gene silencing in plants that are based on this unique pathway.In plants, proper development and response to abiotic and biotic stimuli requires an orchestrated regulation of gene expression. Small RNAs (sRNAs) are key molecules involved in this process, leading to downregulation of their target genes. Two main classes of sRNAs exist, the small interfering RNAs (siRNAs) and microRNAs (miRNAs). The role of the latter class in plant development and physiology is well known, with many examples of how miRNAs directly impact the expression of genes in cells where they are produced, with dramatic consequences to the life of the plant. However, there is an aspect of miRNA biology that is still poorly understood. In some cases, miRNA targeting can lead to the production of secondary siRNAs from its target. These siRNAs, which display a characteristic phased production pattern, can act in  MicroRNAs (miRNAs) are molecules that play pivotal roles in the control of gene expression, and together with small-interfering RNAs (siRNAs) they form the two major classes of regulatory small RNAs (sRNAs) in plants. Biogenesis of miRNAs relies on the activity of DICER-LIKE 1 (DCL1), an RNAse III enzyme that processes transcripts with imperfectly, self-complementary foldback structures to 21–22 nt long mature miRNA. The miRNA is then loaded into ARGONAUTE (AGO), conferring sequence specificity to the RNA-induced silencing complex (RISC), which promotes cleavage of the target transcript through the slicing activity of AGO. Alternatively, RISC-mediated gene downregulation can also be achieved via translation inhibition, a process still poorly understood in plants TAS transArabidopsis, Nicotiana benthamiana, Medicago, soybean, rice, and petunia [An additional system exploring the transitivity initiated by miRNAs is referred to as miRNA-induced gene silencing (MIGS), which has as its main characteristic the easiness of design ,81. With petunia ,84,85,86CHALCONE SYNTHASE (CHS) was able to cause widespread silencing due to the production of secondary siRNAs by RDR6.A common theme between atasiRNAs and MIGS is the requirement of a trigger miRNA. Therefore, it is important to take into consideration that the spatio-temporal expression pattern of the miRNA could influence the way in which atasiRNAs and MIGS-derived siRNAs are produced. In addition, some species might not code for the miRNA initiating the production of secondary siRNAs from the silencing constructs. To overcome this issue, a collection of plasmids has been created to allow the co-expression of the triggering miRNA and the MIGS/atasiRNA construct from a single vector ,87. AlteThis review has discussed some of the crucial aspects related to the production of secondary siRNA triggered by miRNA and how this process can add valuable features to the control of gene expression mediated by sRNAs. Moreover, different methods to promote gene silencing in plants that are based on this unique ability of certain miRNAs were discussed, showing that they can be important alternatives to well-established systems, such as amiRNA and hpRNAi.In recent years, our understanding of the mechanisms leading to miRNA-triggered secondary siRNA generation, and the importance of these molecules in plant physiology and development, has increased rapidly, yet this pathway is still one of the least understood among different processes involving sRNAs. It is still unknown, for example, how 22 nt long miRNA loaded into AGO1 or the miR390/AGO7 complex can lead to the recruitment of RDR6 to the target transcript. Also, the role and molecular mechanisms behind the activity of many phasiRNAs recently described in different plant species remain elusive. The elucidation of these and other aspects related to miRNA-triggered secondary siRNAs will greatly improve our understanding of how sRNAs impact the proper development of plants and the response to abiotic and biotic stresses. In addition, this new knowledge could be useful for the development of new technologies for biotechnological applications.


Hepatocellular carcinoma (HCC) ranks among the most rapidly evolving cancers in the Western world. The majority of HCCs develop on the basis of a chronic inflammatory liver damage that predisposes liver cancer development and leads to deregulation of multiple cellular signaling pathways. The resulting dysbalance between uncontrolled proliferation and impaired predisposition to cell death with consecutive failure to clear inflammatory damage is a key driver of malignant transformation. Therefore, resistance to death signaling accompanied by metabolic changes as well as failed immunological clearance of damaged pre-neoplastic hepatocytes are considered hallmarks of hepatocarcinogenesis. Hereby, the underlying liver disease, the type of liver damage and individual predisposition to apoptosis determines the natural course of the disease as well as the therapeutic response. Here, we will review common and individual aspects of cell death pathways in hepatocarcinogenesis with a particular emphasis on regulatory networks and key molecular alterations. We will further delineate the potential of targeting cell death-related signaling as a viable therapeutic strategy to improve the outcome of HCC patients. The common hallmark of the vast majority of Hepatocellular carcinomas (HCC) is a chronic inflammatory liver damage induced by a diverse spectrum of etiological risk factors . DependiIn the context of HCC development and progression, special importance can be assigned to the type of liver damage and associated changes to the hepatic micromilieu that create a pro-oncogenic field effect and precede malignant transformation of hepatocytes , 9.Various types of liver injury and associated chronic cell death responses have been identified to trigger inflammatory liver diseases, fibrosis development and, ultimately, hepatocarcinogenesis , 11. AccThe here presented review aims to summarize key cellular and molecular mechanisms involved in liver cell death during hepatocarcinogenesis with a main focus on apoptosis. We will also delineate the importance of predisposition to apoptosis as a key factor for malignant transformation and specify factors that affect differential predisposition to apoptotic stimuli during liver cancer development and therapy. Finally, the impact for personalized medicine and precision oncology will be discussed.Cell death is intrinsically associated with chronic inflammation in various organs including the liver . Herein,c, into the cytoplasm in order to activate the caspase cascade  and PRIMA-1Met are currently evaluated in several clinical trials  has shown the potential of this strategy, as response rates of about 80% can be achieved with single-agent venetoclax even in a relapsed/refractory setting . CurrentIn addition to the therapeutic targeting of critical apoptosis regulators, pan-caspase inhibitors, e.g., Emricasan/IDN-6556, or selective caspase-1,8,9 inhibitors, e.g., GS9450, have been explored in preclinical models as well as clinical trials, mainly in the context of chronic liver diseases . While tAlthough no clinical trials have yet been initiated to test the clinical efficacy of necroptosis inhibition in liver disease, preclinical studies and early phase clinical trials in inflammatory (auto-immune) disease indicate that inhibition of RIPK1 kinase activity might also be a promising therapeutic strategy and prevent apoptosis in chronic liver diseases . HoweverHepatocyte death is a key driver of chronic inflammatory liver diseases and hepatocarcinogenesis. Several lines of evidence suggest that apoptosis and other types of cell death are critically linked to initiation and progression of liver cancer. They participate in shaping the biological trait of the tumor, thus, ultimately determining patient prognosis. Herein, existence and degree of cell death infers several mechanistic and translational implications. While detection of the apoptotic predisposition might be a powerful diagnostic tool, direct targeting of mitochondrial apoptosis might complement the limited therapeutic strategies for HCC. In light of recent advances in immune-oncological approaches, targeting of cell death might also exert synergistic immuno-modulatory properties that could be explored in combination treatment strategies. However, our understanding of the detailed mechanisms and triggers of activation underlying the diverse mechanisms of cell death remains limited. Thus, definition of the actual state of cell death signaling effect in the distinct parenchymal and non-parenchymal cell types within the liver is urgently needed. Furthermore, the relative importance for distinct disease stages, i.e., chronic liver disease, tumor initiation as well as progression should be conclusively dissected to advance the field and before application of specific modulators of cell death in human is warranted. Furthermore, intensive translational research is needed to characterize the molecular hallmarks that operate on the intersection between cell death and inflammation. In this context, individual predisposition to apoptosis of cancer cells or cells within the hepatic microenvironment might be of particular relevance and might require distinct therapeutic strategy, thus, precision oncological approaches. Nevertheless, targeting of apoptosis is a promising avenue of HCC treatment that might yield to novel treatment strategies for this deadly inflammatory-driven cancer.JM and FE conceptualized, wrote, and edited the review.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.


Cell contacts exhibit a considerable influence on tissue physiology and homeostasis by controlling paracellular and intercellular transport processes, as well as by affecting signaling pathways. Since they maintain cell polarity, they play an important role in cell plasticity. The knowledge about the junctional protein families and their interactions has increased considerably during recent years. In contrast to most other tissues, the endometrium undergoes extensive physiological changes and reveals an extraordinary plasticity due to its crucial role in the establishment and maintenance of pregnancy. These complex changes are accompanied by changes in direct cell–cell contacts to meet the various requirements in the respective developmental stage. Impairment of this sophisticated differentiation process may lead to failure of implantation and embryo development and may be involved in the pathogenesis of endometrial diseases. In this article, we focus on the knowledge about the distribution and regulation of the different junctional proteins in the endometrium during cycling and pregnancy, as well as in pathologic conditions such as endometriosis and cancer. Decoding these sophisticated interactions should improve our understanding of endometrial physiology as well as of the mechanisms involved in pathological conditions. Direct cell–cell contacts connect cells to each other to maintain cell polarity, stability and integrity. Moreover, they mediate selective paracellular as well as intercellular transport of molecules . Due to In this review, we focus on the knowledge about the distribution of the different direct cell–cell contacts in the endometrium during cycling and pregnancy, as well as in pathologic conditions such as endometriosis and cancer. Results from human endometrium are correlated with findings from research with human endometrial cell lines and animal models.Tight junctions, adherens junctions, desmosomes and gap junctions were originally identified by their morphological appearance in electron microscopy  and are In recent years, there has been a considerable gain in knowledge about the molecules that contribute to the structure, function and regulation of those cell–cell junctions. Tight junctions are located at the uppermost part of the lateral cell membrane. They build up a selective barrier between the adjacent cells thus regulating and restricting paracellular transport between these cells (gate function). In addition, they maintain the strict organization of the plasma membrane of epithelial cells in an apical and a basolateral compartment (fence function) . MeanwhiAdherens junctions and desmosomes are located just below tight junctions at the lateral plasma membrane. They keep neighboring cells together, thereby maintaining cell and tissue polarity. These adhesions are mediated by the transmembrane protein family of cadherins which include 20 classical members in the human , and whiIn contrast to tight junctions and adherens junctions, gap junctions are intercellular membrane channels connecting directly the cytoplasm of adjacent cells. This intercellular communication allows the exchange of ions, second messengers and small metabolites, and thus influences cell differentiation and proliferation, as well as tissue development, homeostasis and morphogenesis . A gap jSummarizing these aspects, although the different cell–cell junctions comprise different proteins, there are similarities in the roles of specialized transmembrane proteins in forming extracellular adhesive contacts between cells, and intracellular links to the cytoskeleton and signaling pathways which may regulate gene transcription. Thus, all junctions, in addition to their classical function, may also be involved in processes such as proliferation and cell differentiation. Moreover, knowledge about the interaction between the different cell–cell contacts is continuously expanding and may have an impact also on endometrial physiology.In the course of the menstrual cycle, both compartments of the endometrium, the epithelial and the stromal cells, undergo considerable hormonally regulated changes in preparation for embryo implantation. The luminal epithelium is transformed from a non-receptive to a receptive state, which allows adhesion and invasion of the trophoblast ,28, whilAlready in 1982, freeze-fracture electron microscopy had revealed tight junctions in the luminal epithelial cells of the human uterus showing a decrease in junctional complexity from the late proliferative/early luteal phase to the late luteal phase of the menstrual cycle ,31. In pWith increasing knowledge about the composition of cell-to-cell junctions and the discovery of the high variety of proteins involved in these complex structures, knowledge about the changes in cell junction composition became more differentiated and complex in recent years. As members of the tight junction complex, up to now, claudin-1, -2, -3, -4, -5 and -7 have been described in human endometrial epithelial cells. Immunohistochemical staining revealed a localization of the claudins apically in the glandular epithelial cells while weak or no staining has been described in luminal epithelial cells ,35,36,37In addition to claudins, the tight junctional transmembrane protein JAM-1 was found at the basolateral membranes of the luminal and glandular epithelium  and alsoAnalyzing adherens junctions and desmosomes, a change in the localization of the cytoplasmatic plaque proteins desmoplakin 1 and 2, and the transmembrane cadherin desmoglein 2 from the apical pole of the lateral cell membrane in the proliferative phase to an evenly distributed pattern along the lateral cell membranes in the mid- to late luteal phase of the menstrual cycle has been described in the glandular epithelium of human endometrial tissue, while mRNA levels stayed constant. This redistribution was limited to the functionalis layer of the endometrium .Immunostaining for E-cadherin and beta-catenin revealed a subapical localization at the lateral membrane of the glandular epithelial cells during the late proliferative and early luteal phase which disappeared during the mid-to late-luteal phase . Also heGap junctions in the human endometrial epithelial cells were shown to consist of Cx26 and Cx32 ,45,46. ASummarizing these findings, the amount and localization of the different junctional proteins change throughout the cycle, especially in the epithelial compartment, due to the hormonally regulated transformation of endometrial function. These changes are summarized in The findings described above indicate that some of the junctional proteins are regulated throughout the menstrual cycle. To gain more detailed insight into this hormonal regulation, experiments in animals as well as in vitro have been performed.A direct influence of hormonal stimulation on the structure of tight junctions has been described in a freeze fraction study in ovariectomized rats. Application of estrogen resulted in an apical shift of these junctions at the lateral epithelial cell membrane, whereas additional application of progesterone led to their extension down the lateral membrane . LookingUsing primary culture of human endometrial epithelial cells, an upregulation of claudin-1, -3, -4 and -7 protein content by progesterone and an inhibition of this upregulation by estradiol was observed. Furthermore, the barrier function of the tight junction as measured by the transepithelial electrical resistance decreased under the influence of progesterone, but not estradiol, while the treatment did not affect the fence function as determined by BODIPY-sphingomyelin diffusion in the membrane . From thThe hormonal regulation of endometrial gap junction connexins has been studied in a variety of species. Rodents revealed a similar expression pattern like humans showing Cx26 in the uterine epithelium and Cx43 in the stromal cells . Here, iTo sum up, the distribution and function of the junctional protein complexes are partly sensitive to hormonal regulation. These results are summarized in During the luteal phase of the menstrual cycle, the human endometrium is transformed to the receptive state to allow adhesion and invasion of the trophoblast. For successful implantation, however, gene expression in the endometrium in addition is regulated by the implanting blastocyst by precisely synchronized embryo-maternal interactions ,61,62. BIn the rodent, the blastocyst reaches the uterine lumen on 4.5 dpc in the mouse and on 5 dpc in the rat. Degradation of the luminal epithelium and start of trophoblast invasion is observed from 5.5 dpc (mouse) or 6 dpc (rat) onwards ,63. FreeDesmosomes , hemidesSimilar to the situation in humans, gap junction intercellular communication is suppressed in the rodent uterine epithelium during the receptive phase. Prior to implantation, however, Cx26 is induced locally restricted to the luminal epithelium of the implantation chamber ,56. ThisThe crucial function of junctional components in the uterine epithelium might be to control epithelial permeability and thus the uterine milieu as well as regulating trophoblast adhesion to and penetration through the epithelial lining, whereby the various junctional proteins may exhibit different functions. There is evidence that tight junctions are the only junctional complexes that are maintained during the implantation window. The tight connection of the epithelial cells may maintain an optimal uterine micro milieu for the developing blastocyst during the sensitive phase of implantation ,80. In cIn preparation for embryo implantation, not only does the uterine epithelium have to differentiate into a receptive state to allow adhesion and invasion of the trophoblast, but the endometrial stromal cells also undergo a complex differentiation process. They transform to decidual cells, which regulate trophoblast invasion, may be involved in the selection of competent embryos, and, moreover, support angiogenesis to build up an extensive vascular network, which is essential for placental blood supply and successful embryonic development ,81. ThusIn the pre-decidual cells of the human luteal phase endometrium, up to now, only the gap junction protein Cx43 has been described, while there is no knowledge about junctional proteins in the human decidual cells during the early stages of pregnancy. When decidualizing human endometrial stromal cells in vitro, it has been shown that the expression of originally epithelial proteins, including beta-catenin, E-cadherin and ZO-1, is redistributed to the decidualized stromal cells , supportIn rodents, decidualization of endometrial stromal cells is induced with the start of the implantation process. First, the stromal cells surrounding the implantation chamber differentiate to the avascular primary decidual zone, encapsulating the implanting embryo . Here, iTo determine which component of a blastocyst is necessary to induce expression of tight junctional proteins in the decidua, Wang and colleagues examined the expression of various proteins of the tight junction complex in the presence of either a normal blastocyst, trophoblast vesicles or isolated inner cell mass . While bBesides the induction of these junctional proteins, the decidual cells are also extensively connected by gap junctions. In human, baboon and rodent endometrium, Cx43 is the dominantly expressed gap junction subunit in the stromal compartment . In rodeSumming up these findings, there is a considerable induction of various junctional proteins during decidualization which build a selective barrier towards the embryo after breakdown of the epithelial barrier. These proteins play an important role in paracrine signaling within the decidua to sustain differentiation and to support angiogenesis in the maternal compartment as a prerequisite for nutrition of the growing embryo. The clinical significance of these observations is supported by findings that Cx43 levels are reduced in the decidua of women with recurrent early pregnancy loss  and by tIn conclusion, there is a precise temporal and spatial regulation of various junctional proteins in the epithelial, as well as the stromal, compartment of the endometrium during the implantation process  [Since claudin-4 and, with a lesser affinity, also claudin-3 act as epithelial receptors for in (CPE) ,125,126,in (CPE) ,128. Thiin (CPE) . The emein (CPE) .In regard to adhesion contacts, the role of the two adhesion molecules E-cadherin and beta-catenin in the carcinogenesis of endometrial carcinoma has been extensively studied, and the expression of these proteins is discussed as a prognostic marker. Although varying in detail, most studies are consistent that low E-cadherin expression correlates with increasing aggressiveness, poor differentiation, and deep myometrial invasion of the carcinoma ,138,139.CTNNB1) mutations led to decreased cell–cell adhesion and have been reported in about 15% of endometrioid carcinomas [Also for beta-catenin, a decreased level has been demonstrated with increasing grading of endometrial carcinoma . Moreovercinomas ,145. Sinrcinomas .In addition to the roles of tight and adherens junctions described above, there is substantial evidence that an interruption of gap junctional communication or the aberrant expression of connexins constitutes one important step in carcinogenesis . In endoThe junctional proteins regulated in endometrial carcinogenesis are summarized in In conclusion, the different cell–cell contact proteins may exhibit considerable effects on the pathogenesis of endometrial cancer. Besides ensuring cohesion of a healthy cell structure, they may regulate signaling pathways involved in the pathogenesis and progression of endometrial cancer, and thus may represent promising tools for diagnostic and therapeutic approaches in cancer treatment.Direct cell–cell junctions are highly specific and precisely regulated during the physiological changes in the endometrium as well as in pathological conditions. Despite their specific function in cell–cell interaction they also may regulate signaling pathways, thereby influencing gene expression in the different compartments of the endometrial tissue. Moreover, besides close interactions of various proteins within the complex structure of the same junctions and with components of the cytoskeleton, insights are increasing about close relationships between the proteins of different junctional complexes. Thus, a close interaction of the components of the different cell–cell junctions might also play an important role in the different physiological conditions of the endometrium. The number of components building involved in these junctions and their interactions has grown considerably in recent years, and up to now, only some of them have been analyzed in the endometrium. An adequate expression of the different junctional proteins in the endometrium is indispensable, since genetic defects and dysregulation of these interactions can cause different diseases, and may impair the implantation reaction and embryonal or placental development resulting in phenomena like preeclampsia or fetal growth restriction. Still, many questions remain concerning the various functions of junctional proteins and their interactions. Extending our knowledge of these essential functions in endometrial physiology and pathogenesis will provide closer insight in female reproductive health.


Maslow's hierarchy of needs  depicts Environmental behavior varies significantly between ethnic groups. On the one hand, past studies have shown that ethnic minorities in the United States engage in fewer pro-environmental behaviors. This view was shown in a study by Johnson et al. , which tOn the other hand, several recent studies have revealed that non-whites in the U.S. reported greater concerns and engagements about the environment  than Whites , exosystem , and macrosystem . In Bronfenbrenner's view, these systems are interrelated, which means that behaviors occurring at the smallest level of context can be influenced by what occurs in the largest context. In other words, individual pro-environmental behaviors are affected by both individual norms, and the norms and expectations within their social contexts . In the following sections, we discuss the evidence and suggest that both individual and contextual factors play important roles regarding ethnic groups' attitude and behavior on environmental issues. In the end, we offer a mixed-method approach to synthesize both individual and contextual factors in future empirical studies.However, past research on the topic has primarily emphasized individual beliefs toward environmental issues and underestimated the role of social contexts in which members of ethnic minority groups live. Bronfenbrenner's  ecologicConceptually, previous studies investigating ethnic differences in environmental concerns were largely based on value-belief-norm theory (VBN) . Little research is available about the impact of various Eastern cultures on the natural environment, even though past literature has revealed cross-cultural differences on environmental behaviors and perception of environmental concerns across different societies  have been scarce. A recent study by Washinawatok et al.  exploredAnother related, understudied topic of interest to consider is immigrant environmental behavior in the United States. One recent study found that immigrants of New York City, the city with the highest immigrant count in the country, are just as likely and sometimes more likely to engage in environmental behaviors than native-born residents (Pfeffer and Stycos, Future research needs to investigate external factors that influence ethnic minorities' environmental behaviors, such as economic and sociocultural factors (Kollmuss and Agyeman, Sociocultural factors affect environmental action as well. Past research has indicated that age, cohort, political orientations, and educational levels correlate with environmental concern and behaviors (Gifford and Nilsson, It is generally the case that quantitative analysis is used as the predominant framework in the field of Environmental Psychology. However, the quantitative analysis does not provide a comprehensive explanation addressing the complexity of the human experience. A mixed-methods approach is a strong option to consider for future research because it employs qualitative methodologies to enrich the meaning of the quantitative data (Scharf and Mayseless, Triangulation mixed-method design (Creswell and Plano Clark, In contrast to the triangulation design, the explanatory design offers a two-step approach: a primary focus on quantitative analyses of the study and follow-up qualitative data in an effort to provide a comprehensive explanation for the quantitative analyses (Creswell and Plano Clark, In conclusion, globalization and migration have resulted in a growing need to understand ethnic minority members' environmental engagement in a heterogeneous society. However, the literature provides no clear indication of how ethnic minority groups might respond to the environmental issue. This controversy calls for new considerations on theories and methodologies in future research. Bronfenbrenner  ecologicVM wrote an initial draft. AD added a large section of the manuscript. NR provided an initial literature search and summary. DC wrote one section in the initial draft. FJ conceptualized the framework, wrote and revised a significant portion of the manuscript, and advised the writing process.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.


Autophagy, a catabolic degradation system, is utilized for destroying and recycling the damaged or unnecessary cellular components. Brain plasticity refers to the remarkable characteristics of brain neurons that change their structure and function according to previous experience. This review was performed by searching the relevant articles in databases of SCIENCEDIRECT, PUBMED, and Web of Science, from respective inception to January 2019. Here, we review the neuroprotective effect of autophagy in neurological diseases and the mechanism of autophagy in brain plasticity. Moreover, the mechanism of autophagy in the process of brain plasticity can provide the possibility for the development of new treatment methods in the future, thus benefiting patients with neurological diseases. In summary, autophagy and brain plasticity play important roles in neurological diseases. Autophagy is a lysosome-reliant degradation mechanism that regulate many biological courses, such as neuroprotection and cellular stress reactions . There aThe plasticity of the central nervous system(CNS) can be regarded as changes of functional interaction between different types of cells, astrocytes, neurons, and oligodendrocytes . The matAutophagy is involved in the occurrence and treatment for a series of neurological diseases. However, there are only sporadic reports for the relationship between autophagy and some types of the neurological diseases, which have not been accumulated enough to be reviewed. Therefore, in this review, we summarize the relationship between autophagy and brain plasticity in stroke, traumatic brain injury, cerebral tumor, and neurodegenerative diseases.Autophagy plays different roles in various conditions, and both autophagy activation and autophagy inhibition could exert neuroprotective effects in the process of stroke.The co-modulation of autophagy and apoptosis is involved in ischemic stroke (IS)-induced injuries, and apoptosis and mitochondrial autophagy play an important role in this process . RIPer . Liu et There is increasing evidence that autophagy dysfunction leads to the accumulation of damaged organelles and/or abnormal proteins. This accumulation is associated with synaptic functional disorder, neuronal death, and cellular stress . RIPreC In summary, different studies obtained various results for the role of autophagy in the process of stroke, and further studies are required to explore the relationship between autophagy and stroke.Similar to the relationship between autophagy and stroke, the relationship between autophagy and traumatic brain injury is also not simple. Both inhibition and activation of autophagy could exert neuroprotective effects following the occurrence of traumatic brain injury.The promotion of autophagy and neuronal apoptosis are related to the secondary neural injury after Traumatic Brain Injury (TBI). Sevoflurane post-conditioning regulates autophagy through PI3K/AKT signaling, which alleviates the TBI-triggered neuronal apoptosis . Zhang LThe autophagy pathway is associated with the pathophysiological reactions following TBI, and suppression of this pathway might contribute to the alleviation of traumatic injury and functional outcome defects . MelatonAutophagy is the core regulator of central nervous system senescence and neurodegeneration. The delivery of organelles and toxic molecules to lysosomes by autophagy is critical for the health and survival of neurons . Most ofIn summary, autophagy has a close relationship with neurodegenerative disease, and activation of autophagy could improve the neurodegenerative changes, which might be a novel target in clinical treatment for such diseases.The role of autophagy in tumor cell survival and death has attracted much attention in recent years . As for In summary, autophagy provides new therapeutic expectations for cerebral tumor which is a big challenging for human. Therefore, the application of novel therapy for cerebral tumor on the basis of autophagy mechanisms elucidation should be explored in depth.The mTOR-controlled signaling pathways regulate many integrated physiological functions of the nervous system including neuronal development, synaptic plasticity, memory storage, and cognition . MammaliAs a key regulator of autophagy, the mTOR plays an important role in autophagy, translation, cell growth and survival . MammaliIn summary, there is a close relationship among mTOR, brain plasticity and autophagy. The mTOR related pathways play important role in regulating the process of autophagy and brain plasticity.Autophagy acts as a central mediator of cellular disease and health, and this self-balancing process seems to affect synaptic growth and plasticity in the CNS . The inhMoreover, neuronal stimulation led to NMDA receptor (NMDAR)-dependent autophagy via PI3K-Akt-mTOR pathway suppression, which might work in AMPA receptor (AMPAR) degradation, thus showing autophagy as a promoting factor to brain functions and NMDAR-reliant synaptic plasticity . CurcumiThere is increasing evidence that MAPKs can regulate autophagy/macroautophagy . MAPK/ErIn summary, the related signaling pathways of autophagy and brain plasticity has not be well clarified, which is worthy of further exploration.The dysfunction of autophagy pathway is related to a variety of neuropathologic conditions, and numerous studies have demonstrated that autophagy is a potential target for pharmacological regulation of neuroprotection . AutophaBrain plasticity is one of the most fundamental mechanism for neural function recovery following neurological diseases. Autophagy is a crucial lysosome-reliant degradation process that controls various physiological and pathological courses in the brain. The summary for the interaction of autophagy and brain plasticity might provide novel therapy targets for neurological diseases, thus benefiting the patients in clinic.M-MW and FZ conceived the main ideas and wrote the manuscript. Y-SF, JZ, FD, S-DY, and YX searched the references and designed the framework.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.


Inducers of inflammation trigger production of inflammatory mediators, which alter the functionality of tissues and organs and leads to harmful induction of different barrier systems in the body, where the blood-brain barrier, the blood-retinal barrier, blood-nerve barrier, blood-lymph barrier and the blood-cerebrospinal fluid barrier play major roles. The different barriers are unique but structured in a similar way. They are equipped with sophisticated junctional complexes where different connexins, protein subunits of gap junction channels and hemichannels, constitute important partners. The cells involved in the various barriers are coupled in networks, are excitable but do not express action potentials and may be targets for inflammation leading to changes in several biochemical cellular parameters. During any type of inflammation barrier break-down is observed where any form of injury can start with low-grade inflammation and may lead to systemic inflammation.Systemic low-grade inflammation can be initiated  Neuronal signaling in the central nervous system (CNS) requires a balanced and well-controlled composition of the microenvironment around glial cells, neurons, and synapses. Loss of optimal function in the different barriers changes the vascular permeability, resulting in an increased transport of molecules and cells through the tight junctions into immune-privileged sites, resulting in destructive inflammation, as observed in many diseases. The body is composed of different barrier systems, including the blood-brain barrier (BBB), the blood-cerebrospinal fluid barrier (BCSFB), the blood-retinal barrier (BRB), the blood-nerve barrier (BNB), the endothelial-cell barrier (ECB), and the blood-lymph barrier (BLB). They are all responsible for preventing foreign matter penetration and protecting normal cell signaling.The BBB controls the molecular mechanisms and the transport of molecules and cells between the blood and brain , 2. The The present review highlights the different barrier systems and especially substances, molecules and cells that can affect and damage their protective mechanisms. Different types of cellular signaling can be disturbed, which is important in low-grade inflammation that can establish itself and lead to systemic inflammation. The brain and the nervous system play a central role because they can sense all changes that occur in different types of inflammatory processes.Inflammation plays a major role in preserving physiological homeostasis of an organism and is initiated when pathogens, bacteria, viruses, etc. are presented. Inflammation is first initiated on a cellular level, low-grade inflammation, and can from there expand to inflammation involving different inflammatory cascades and organs causing systemic inflammation. Inflammation plays a central role in the body homeostasis and will, protect the body from illness and disease. On the other hand, prolonged or chronic inflammation releases proinflammatory cytokines and may expose the body to unfavorable conditions , 9.The vertebrate inflammatory system is composed of the innate and adaptive inflammatory systems, and both are equally important. The innate immune system consists of macrophages, dendritic cells, mast cells, etc. Its main function is to detect bacteria, viruses, and foreign bodies in the organism as well as to activate the adaptive immune system and complement cascades. The adaptive immune system is highly specific and has developed throughout our lives. It consists of T- and B-lymphocytes, where pattern recognition receptors, including toll-like receptors, along with the induction of cytokines, play a major role in the activation of the adaptive immune system. Cytokines, consisting of small proteins, are produced by a variety of cells, including T- and B-lymphocytes. Cytokines can be proinflammatory or anti-inflammatory and may act as triggers for the release of other cytokines. They are important in chronic inflammation caused by oxidative stress .In recent years, research has begun to focus on the roles of gap junction coupled cells which form networks in different organs in the body. Examples of cells coupled into networks include astrocytes, keratinocytes, chondrocytes, synovial fibroblasts, osteoblasts, connective tissue cells, cardiac and corneal fibroblasts, myofibroblasts, hepatocytes, and different types of glandular cells . They maBarriers at different sites in the body might be affected and can cause spread of inflammatory substances affecting other network-linked gap junction cells in other organs, which can give rise to systemic inflammation .2), carbon dioxide (CO2) and nitric oxide (NO), diffuse through the membranes as well as small lipophilic agents. Transport systems on luminal and abluminal membranes regulate the traffic of small hydrophobic molecules. The combination of intra- and extracellular enzymes provides a metabolic barrier. The different BBB cell types and the molecular constituents have been thoroughly discussed recently  from the blood supply of the choroidal circulation . AlteratThe BCSFB encases the choroid plexus, the CSF-producing structure, and is localized within the brain ventricles. The barrier is formed by the epithelial cells of the choroid plexus as well as the arachnoid epithelium and tanycytes in the other parts that take care of the CSF . BetweenThe endoneurial microenvironment supports the blood-nerve exchange composed of endoneurial vessels and the perineurium. Therefore, the endoneurial microenvironment is protected from substances that can be harmful to Schwann cells and axonal functions .Lymph-to-plasma exchange depends on the pore size of the capillary permeability and the resistance in the endothelial capillary wall .+/K+-ATPase pump and disturbed Ca2+ signaling through gap junction-coupled cells. The different barriers are dynamic structures where nutrients penetrate the barriers by passive diffusion or by transporters that supply the nervous system with necessary nutrients. Barrier disruption causes an increase in the migration of leukocytes through the barriers, and excessive ROS production occurs as well as the activation of matrix metalloproteases (MMPs)  .+/K+-ATPase pump . The main cytoplasmic matrix of proteins, spectrin and actin, are attached to ankyrin B. An intact cytoskeleton is required for the propagation of astrocytic Ca2+ waves (2+ oscillations by changing the balance between the Ca2+-regulating processes (The actin cytoskeleton and microtubules are intimately associated with the plasma membrane . Inflamm2+ waves , and cytrocesses . Actin frocesses , 53. Durrocesses . InvolveSearching for molecular mechanisms to explain how inflammatory processes develop in different cells, tissues and organs and how they can be spread through different systems may be one important task for the treatment of low-grade inflammation that can lead to prolonged pain. The extracellular matrix is an important part of all biological barriers, and it modulates the interchange through these barriers. It controls the migration of leukocytes from the blood, a process that is altered in inflamed tissues in which different extracellular matrix components are involved that influence the inflammatory response. The development of nanotechnology and the application of nanoparticles have led to new research aimed to solve the question of inflammatory processes . EndotheMast cells and glial cells show endogenous homeostatic mechanisms that can be upregulated in response to ongoing inflammation . Mast ceFunctional barriers such as the BBB, BRB, BCSFB, BNB, and BLB enable physiological homeostasis within tissues and organs. The barriers can resist acute inflammation, while prolonged inflammation and oxidative stress may lead to systemic inflammation affecting the nervous system and retina, cartilage, blood vessels, and nerve endings. It is of great importance to understand the different barriers, their functionality and how they act. Although there are still many unsolved questions, understanding why some people are more affected by diseases than others are remains of critical importance.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.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.


There is a rapid increase in the percentage of elderly people in Europe. Consequently, the prevalence of age-related diseases will also significantly increase. Therefore, the main goal of MediHealth, an international research project, is to introduce a novel approach for the discovery of active agents of food plants from the Mediterranean diet and other global sources that promote healthy ageing. To achieve this goal, a series of plants from the Mediterranean diet and food plants from other origins are carefully selected and subjected to in silico, cell-based, in vivo (fly and mouse models), and metabolism analyses. Advanced analytical techniques complement the bio-evaluation process for the efficient isolation and identification of the bioactive plant constituents. Furthermore, pharmacological profiling of bioactive natural products, as well as the identification and synthesis of their metabolites, is carried out. Finally, optimization studies are performed in order to proceed to the development of innovative nutraceuticals, dietary supplements or herbal medicinal products. The project is based on an exchange of researchers between nine universities and four companies from European and non-European countries, exploiting the existing complementary multidisciplinary expertise. Herein, the unique and novel approach of this interdisciplinary project is presented. Current demographic trends suggest a rapid increase in the percentage of elderly people in Europe for the coming decades, due to an increase in life expectancy and consistently low fertility levels . MoreoveMany theories have attempted over the years to explain in pertinent ways the complex set of events leading to ageing and degeneration. The free radical theory of ageing suggests that ageing could be attributed to deleterious effects of gradually accumulating reactive oxygen species (ROS) on various cellular components. The ubiquitous nature of ROS targets  explains the large array of stochastic damage that these molecules bring about in any living organism, causing a progressive functional deterioration of cells, tissues, and organ systems . Two majFor a long period, up to the 1960’s, the structural studies of natural products (NPs) served as the principle driving force for drug discovery and still constitute a unique source for novel leads and pharmacophores for medicinal chemistry. More than 50% of all new chemical entities (NCEs) introduced into pharmacotherapy over the period 1981–2010 derive (directly or indirectly) from NPs . Up to nOne of the geographical areas where nutritional habits have drawn attention as a prototype of nutritional protection against age-related diseases is the Mediterranean region. The traditional Mediterranean diet and its sub-branch, the Cretan diet, is widely considered one of the most important factors for the healthiness and long-life expectancy of populations from Mediterranean regions. It first came to scientific attention with the seven countries study ,16. ThisCichorium spinosum, C. intybus, and Sonchus oleraceus are consumed raw or slightly cooked on a daily basis in some cases, providing a range of polyphenols with strong antioxidant potential [Lotus tetragonolobus, Lathyrus ochrus, Vicia faba, Ceratonia sp.) [Opuntia ficus-indica (Cactaceae) are reported to exhibit positive effects against type-2 diabetes [Olea europaea (Oleaceae) and olive oil, which is an essential component of the Mediterranean diet, have multiple beneficial effects on enzymes involved in diabetes mellitus [The main characteristics of the Mediterranean diet are high consumption of unprocessed plant foods , moderate consumption of fish and red wine, and low consumption of dairy products, eggs, and meat. Wild and semi-cultivated leafy vegetables hold a key position in the daily diet. Many of these plants are cultivated and consumed widely, thus increasing the daily intake of antioxidants and other NPs by the population that adheres to the traditional diet with a potential benefit of protection against ageing and age-related diseases. However, while the “classical” concept of the Mediterranean diet has been a subject of numerous studies , the heaotential . Edible nia sp.)  are reconia sp.) . Exotic diabetes  and dietdiabetes . Olea eumellitus .Phoenix dactilifera, Arecaceae), a well-known Tunisian food plant, has been proven to have a hepatoprotective effect in vivo [Punica granatum, Lythraceae), consumed raw and juiced, contains flavonoids that contribute to the amelioration of early diabetic nephropathy and glucose homeostasis [Sclerocarya birrea (Anacadiaceae), a fruit similar to mango, has great antioxidant and in vitro antidiabetic properties [Carpobrotus edulis (Aizoaceae) has been used as a traditional remedy against diabetes mellitus [Adansonia digitata (Bombacaceae), the known baobab tree, provides fruits extremely rich in antioxidants and reduces glycemic response [Perilla frutescens (Lamiaceae) in stews, a plant that has shown an inhibitory activity against tumor necrosis factor alpha (TNF-α) [Gnetum sp. (Gnetaceae), a promising vascular protective agent, for the preparation of traditional curries [Houttuynia cordata (Saururaceae), a plant with antihyperglycemic properties, is widely used as a raw garnish [Ugni molinae (Myrtaceae) and the “Chilean wineberry” or Aristotelia chilensis (Eleocarpaceae), are used for the preparation of jams or as food supplements due to their antioxidant properties [Lampaya sp. (Verbenaceae), an indigenous Andes shrub, is used for liver and kidney damage [In addition to the Mediterranean diet, a well-known European dietary regime, global ethnic cuisines and dietary habits present similar interest in the consumption of plants with health benefits. Tunisia, belonging to the Mediterranean basin, shares similarities in the dietary habits, such as the high consumption of fruits and vegetables. For example, the date  . Another curries . Houttuy garnish . Moreoveoperties ,45,46,47y damage .However, it is impressive that even if the beneficial effects of healthy eating are greatly recognized and globally acknowledged, the majority of food plants that comprise the ingredients of the most established dietary patterns have not been investigated in depth and consequently the active constituents remain largely unknown. Along these lines, many currently used nutraceuticals or dietary supplements are based on NPs and are used in a broad, and from today’s perspective, rather unspecific way. More importantly, numerous health claims on plant-based products have been issued based on traditional use and eating habits. Nevertheless, those claims are in many cases not clearly associated with distinct bioactive NPs or well chemically characterized extracts and biological activity in vivo.Despite the proven track record of NPs for their health-promoting properties and their noteworthy structural diversity, major drawbacks hinder their exploitation and further development. One of the most important bottlenecks is the complicated and time-consuming process required for tracing the active molecules. Valuable experimental time is wasted in laborious isolation processes with a high risk of “re-discovery” of an already known or inactive NP. For these purposes, some new advanced approaches have been proposed to tackle these obstacles. In particular, a new approach combining the traditional bio-guided fractionation along with modern analytical platforms has been developed. It correlates high-performance liquid chromatography (HPLC)-based (micro)-fractionation of plant extracts and activity determination on-line or at-line and enables an efficient localization of bioactive NPs directly in crude extracts. The obtained data can direct large-scale isolation steps to only metabolites of high interest . In paraIn the search for new biologically active substances, targeted bioassays still play an important role. Nevertheless, despite the technological breakthrough that NPs drug discovery experiences and in spite of enormous efforts and investments, many molecules active in vitro fail when tested in vivo. This is mainly due to limited bioavailability and metabolization processes  before the molecules reach their target. Intestinal absorption, hepatic metabolism, distribution in body tissues, bioconversion by the colonic microbiota with formation of colonic metabolites, and elimination are main efficacy influencing factors. This is a very important drawback which, despite the impressive progress of the last years, does not allow the rationalization and the effectiveness of NPs discovery process. In addition, it is frequently observed that traditional medicinal plant extracts with a high level of ethnopharmacological and even clinical evidence, fail to show cell-based activity. The seeming “inactivity” of many NPs isolated from plants is likely caused because those are often pro-drugs, e.g., glycosides, which must undergo in vivo metabolic conversion (activation) ,54. SomeTherefore, the overall aim of the MediHealth project is to introduce a novel approach for the discovery of active agents in food plants from the Mediterranean diet and other global sources to promote healthy ageing. The MediHealth project incorporates new technologies and methods in NPs discovery and isolation in order to discover natural entities from Mediterranean and global dietary plants, promoting healthy ageing. The focus lies on two key aspects, namely accumulating ROS elicited damages, as well as the development of age-related metabolic diseases. A new approach is introduced by the ab initio parallel in vivo and in vitro testing and metabolization study of extracts and constituents thereof, in combination with sophisticated analytical techniques in a successive and integrated manner. MediHealth aspires to bring, based on scientific evidences, novel nutraceutical products and/or phytotherapeutics to the stage of development. This proceeds through a highly collaborative network between industrial and academic partners from European and non-European countries providing complementary scientific expertise, transfer of knowledge, and training of personnel.Investigation of food plants from the Mediterranean diet as well as from other global origins  based on established health benefits from traditional use, literature, and chemodiversity information.Introduction of a novel approach to assess pharmacological properties of metabolites derived from plant extracts based on newly developed extract metabolization protocols that mimic the processing of plant extracts by the human gut and liver as a prerequisite for meaningful in vitro testing.Use of a sophisticated and integrated concept combining extract metabolization protocols with advanced cell-based assays  and their complementation by innovative tests for ageing and age-associated diseases in flies and mice, respectively, to identify natural entities promoting healthy ageing.Incorporation of state-of–the-art analytical techniques , dereplication, and metabolomics strategies for the characterization of active compounds.Generation of solid data based on experimental scientific findings for the development of a new competitive product in the area of nutraceuticals, dietary supplements, and/or phytotherapeutics.The MediHealth project comprises a unique combination of strategies including the following aspects:The main goal of the MediHealth project is to introduce a new concept for the discovery of small molecules in foods plants from the Mediterranean diet and other global sources with activity on healthy ageing and mainly metabolic disorders. This is achieved by an integrated and multidisciplinary approach and based on a well-organized workflow .The first step of this approach is the collection of food plants with potential anti-ageing activity from diverse geographic regions with special attention to the Mediterranean diet. Interestingly, there is a huge number of food plants that are known to contribute to healthy ageing and to have beneficial effects on metabolic disorders but the available data on their active constituents are scarce. The selection of the plants is carried out mainly based on ethnopharmacological knowledge and literature information. Plant collection is followed by extraction and characterization using metabolic profiling. To lower the complexity of the experimental settings, in the course of this project, neither seasonal variations in the secondary metabolite profiles of plants or the influence of other environmental factors nor possible additive or synergistic effects of the simultaneous intake of two or more plants, which could happen in a complete diet, are considered. The chemodiversity data, including the analysis of the main chemical groups of substances, the complexity, and quantitative and qualitative aspects, facilitates the prioritization and the rational selection of promising plant extracts to be further investigated.These candidate extracts are subjected to comprehensive functional assessment in an integrated in vivo/cell-based/metabolism experimental platform that comprises one of the most innovative aspects of the project. Specifically, in the context of MediHealth, a three-pronged approach to identify novel agents able to reduce ageing associated diseases and to improve healthspan is applied. To this end, the selected plant extracts are tested for potential health benefits in in vivo experiments in mice and flies. In parallel, they are also profiled in advanced cellular assays. In addition to cellular, fly, and mouse models, the same extracts are subjected to metabolization studies since plant extracts do not reach human tissues in their native form in most cases. These studies are crucial for the identification of active principles while offering more complete and meaningful information regarding their mechanism of action and the pharmacological potential of the plants. A human-like in vitro metabolization system, i.e., the simulation of gastrointestinal and microsomal metabolization is incorporated. In order to identify metabolites of a given plant extract that are active in the human body, plant extracts are metabolized in a GIDM including a gastric, an intestinal, and a colonic phase , simulating human gastro-intestinal metabolization. In addition, the dialysates containing the gastro-intestinal metabolization products are treated with hepatic S9 fractions to mimic liver metabolization. Plant extracts metabolized in this way are suitable for testing in cell-based assays. Metabolites generated by the in vitro model are also compared with metabolites actually detected in vivo in mice. In addition, the results obtained in this way are independently verified by the prediction of metabolites from genuine plant constituents using in silico tools. To our knowledge, these methods have so far not been used in NPs research.Drosophila fly from oxidative modifications (a major output of ageing) is examined by measuring the suppression of proteome oxidative damage as well as the activation of proteasome catalytic activity and the Nrf2 pathway. This triple approach including extensive in vivo and metabolic evaluation, combined with advanced cell-based testing in the early stages of plant extract evaluation is unique and offers valuable results that are expected to facilitate and substantiate the development of an agent that is safe, well characterized, and exhibits specific biological activity against age-related oxidative stress and metabolic disorders. Based on the results of these investigations, the derived data are correlated and analyzed, then the most promising plant extracts (hit extracts) are prioritized and forwarded to the next analyses.For the cell-based testing, advanced bioassays for medium-scale screening procedures are used to measure biological effects related to ageing processes. Thus, isolated natural agents from dietary plants are evaluated for their bioactivities against Nox4 and intracellular ROS, the Nrf2/Keap1 signaling pathway, and proteasome activity in living cells. In the context of in vivo evaluation, a second approach is focused specifically on the development and alleviation of age-associated metabolic disorders, and is implemented by direct intervention studies in mice. To this end, mice are fed with extracts and multiple markers for the development of age-associated metabolic disorders are quantified, focusing specifically on dyslipidemia, type 2 diabetes, and CVD markers. In parallel, the capacity of the extracts to protect the proteome of the The hit extracts (prior and after metabolization) are subjected to phytochemical analysis, isolation, and characterization of their chemical constituents. Bioactivity-guided (micro-) fractionation of native and metabolized extracts is incorporated for the isolation and structure elucidation of active compounds. State-of-the-art analytical techniques such as HPLC/ultra-high performance liquid chromatography (UHPLC) coupled with photodiode array detector (PDA), LC-HRMS, and LC-NMR are used for the identification of metabolites. The impact of hyphenated techniques has opened entirely new possibilities for the characterization of NPs in complex matrices such as extracts while instrumentation developments have also occurred in spectroscopic identification . This is complemented by the synthesis of selected bioactive metabolites that might be necessary for verification of the chemical structures, as well as qualitative and quantitative aspects of the observed bioactivity. Finally, the identified active molecules (both precursors and metabolites) are subjected to in silico virtual screening against a panel of relevant pharmacological targets offering better insight into the possible mechanism of action and suggestions for likely additional activities.According to the derived results, metabolomic approaches are applied to different batches of the hit extracts or parent plant materials in order to identify the optimal sources for the procurement of the plants of interest . This defines the lead extracts to proceed with up-scaling and development procedures. To achieve the optimal procedure for metabolite procurement, optimization of extraction focusing on “green” extraction techniques and scaling-up procedures are applied. Based on this, a pilot scale production of the lead extracts and/or enriched extracts are developed while pre-formulation, final product characterization, and stability studies are also performed. To carry the lead agents  to the stage of development, feasibility studies also take place. It is important to underline that the whole research initiative needs to be compliant with local and international guidelines and frameworks related to bioprospecting and the protection of indigenous resources.MediHealth is a project with high scientific goals that aspires to introduce a new approach and pipeline in modern NPs drug discovery process. Along these lines of research, it is obvious that a multidisciplinary consortium is needed to successfully implement such a demanding and ambitious project. Therefore, MediHealth has brought together fifteen different groups of experts from both academia and industry, taking into consideration their competences, complementation, and knowledge but primarily the scientific needs of the project. The MediHealth network is based on three basic levels of expertise, i.e., chemistry , biology , and product development . Specifically, MediHealth has gathered an impressive consortium of experts on an international level that is comprised of seven academic groups from European countries , four European industrial participants , and four academic partners from non-European countries . In addition to the pure experimental and scientific goals of the project, valuable knowledge and know-how is exchanged for the benefit of the researchers and experts involved but also between academia and industry, as well as between European and non-European countries. Furthermore, due to the high degree of expertise and complementarity, meaningful interactions are evolved in the area of NP chemistry, analytical chemistry, biology and pharmacology, metabolization studies, metabolomics, separation and large-scale isolation, synthesis, and product development. Using such an interdisciplinary novel approach increases significantly the chances of successfully identifying NPs that can be used to treat age-associated diseases.The scientific tasks of the MediHealth project can be assigned to five major project parts, as depicted in In the beginning of the project, dietary plants from the Mediterranean diet and other sources with potential anti-ageing profiles are rationally selected. Therefore, based on data from literature and knowledge about traditional use, a series of plant species from the Mediterranean  diet and edible plants used in other areas with potential healthy ageing effects is collected. A small quantity of the plant species  is extracted using an appropriate method  and solvent , respectively. The derived extracts are phytochemically characterized using thin layer chromatography (TLC) and HPLC fingerprint analysis in combination with MS. Finally, they are prioritized based on chemodiversity, ethnopharmacology, traditional use, as well as additional criteria such as plant availability and capability of cultivation or sustainable collection. A selection of plant extracts with diverse chemical profiles and healthy ageing potential is forwarded to the in vitro GIDM as well as to biological evaluation in the in vivo mouse and fly models.Given the fact that most of the compounds present in plant extracts will most likely be metabolized, extracts of the selected plants are subjected to in vitro simulation of gastro-intestinal metabolization, followed by treatment with microsomal S9 fractions to mimic liver metabolization in humans. For this purpose, an in vitro GIDM, including microbial fermentation in the colon, is used, to mimic human metabolization processes and produce metabolites of native plant constituents. The GIDM simulates gastric and intestinal physiological conditions, including digestion pH, digestion time, a pepsin solution for the stomach, gradual pH change from stomach to intestine, a pancreatin-bile extract in the small intestine, body temperature, and an anaerobic environment. The intestinal stage is performed using stirred cells, equipped with a dialysis membrane (MW cut-off 1000 Da) and a dialysis bag. The microbial compartment of the colonic phase is simulated using cultures of fecal slurry (homogenized human stool sample). The continuous removal of the dialyzed components through a dialysis membrane simulates the one-way pathway in the gastro-intestinal tract from lumen to mucosa. This model helps considerably in resolving the mechanism of metabolization and the identification of active principles in complex mixtures such as plant extracts and food materials. For each native plant extract, 6-10 dialysates are collected at different times in the gastric, intestinal, and colonic compartments. While part of each dialysate is forwarded to in vitro pharmacological evaluation, part of it is also studied using a liver metabolization assay, since also in the human body, further metabolization reactions would occur after absorption, mainly in the liver. In specific, the samples are analyzed using microsomal S9 fractions , which consist of cytosol and microsomes. After incubation with S9 fractions, the reaction mixture is treated with sulfatase/glucuronidase enzymes to hydrolyze the formed conjugates. The derived samples are subjected to in vitro pharmacological investigation.After comparing the results of the in vitro analysis of the extracts metabolized in the gastro-intestinal filter, prior to and after liver metabolization, with activities of the genuine extracts observed in the in vivo models, hit plants are selected. Metabolites of these hit plants generated by the in vitro GIDM and the treatment with S9 fractions, are identified by chromatographic and spectroscopic tools. In silico prediction methods are also used to facilitate the identification of derived metabolites. In particular, bioactivity profiler software  is employed to predict likely interactions between established genuine plant constituents and metabolic enzymes. Furthermore, software tools  are used to predict the structure of metabolites formed from the original plant constituents.Moreover, for selected lead extracts that show positive results in the in vivo mouse model, bioavailability studies in mouse models are performed. Blood samples are evaluated by NMR and LC-HRMS using two complementary approaches: (i) targeted analysis for checking the presence of plant metabolites and (ii) untargeted analysis for measuring the changes in endogenous metabolites related to phenotypic effects. The serum metabolites present in mice are also compared with the metabolites generated by the GIDM and the treatment with S9 fractions.Another important part of the MediHealth strategy is the biological evaluation of extracts, fractions, isolated constituents and metabolites thereof, as well as (semi-) synthetic analogues. To identify food plants that have the capacity to induce beneficial metabolic changes, a two-pronged approach is performed, where animal models for ageing and the development of age-related diseases are complemented by cell-based assays.Drosophila flies and in cellular models to harmonize the in vitro and in vivo studies for the analysis of ROS mediated damage to tissues that could influence the progression of age-related diseases.In order to investigate the potential of selected food plant extracts to delay ageing, C57BL/6 mice fed either a control diet or a diabetogenic high fat diet are treated for two weeks with crude plant extracts or vehicle. Metabolic markers related to age-dependent diseases are measured, such as plasma glucose, total triglycerides, free fatty acids, and cholesterol, as well as a detailed plasma lipoprotein profile. In addition, insulin sensitivity is determined as a surrogate marker for the development of type 2 diabetes. Since age-related diseases have been linked to a decrease in basal energy consumption, energy combustion by heat production in response to food plant extract treatment is also determined by analyzing body core temperature profiles and metabolic activity during the course of dietary interventions. On a cellular level, liver, muscle, and adipose tissue as key metabolic organs are studied for changes in the expression of key factors involved in the progression of age-related diseases. These experiments are tightly linked to the analyses in ob/ob mice as well as hyperlipidemic apolipoprotein A5-deficient mice. Taken together, these experiments show whether selected food plants have the power to positively influence body weight and the metabolic profile in extreme models of obesity and hyperlipidemia.Out of the biologically investigated extracts, hit extracts are selected for further analysis, including fractionation and isolation of compounds. Selected fractions generated in this way and their isolated components are tested in the model described above and also for the ability to improve metabolic parameters in leptin-deficient Drosophila flies from oxidative modifications and to activate in vivo the Nrf2/Keap1 pathway and/or the proteasome peptidase activities are evaluated.In order to investigate the potential of selected extracts derived from dietary plants to delay ageing in flies, their capacity to protect the proteome of Drosophila flies are fed with standard food containing the selected NPs under study and the proteome oxidative damage is assayed by measuring proteins’ carbonyl groups.Suppression of proteome oxidative damage. Activation of the Nrf2 signaling pathway. Nrf2 activation is assayed by analyzing the gene expression levels of its transcriptional targets, namely antioxidant and proteasome genes.Activation of the proteasome catalytic activity. The major hydrolyzing enzymatic activities of the proteasome are measured by adding specific fluorogenic peptides (substrates) in lysates of flies’ somatic tissues.Drosophila flies are performed in order to reveal potential in vivo healthspan increasing and/or anti-ageing effects. Extensive statistical analyses in these large cohorts couple these analyses.For the most effective samples, longevity assays in large numbers of The hit extracts  are analyzed further, including fractionation and isolation. Selected fractions and their isolated components are tested in the fly in vivo experimental platform for:Innovative cellular models related to the evaluation of agents with anti-ageing profile are used. The selected extracts, before and after metabolization, are assayed for their effects on Nox4 activity , proteasome activity , for their ability to activate the Nrf2/Keap1 antioxidant signaling pathway , and for anti-diabetic activity such as adipocyte differentiation in 3T3-L1 cells, measuring triglycerides accumulation and differentiation markers. In addition, the same samples are used to determine the concentration range in which no cytotoxicity is observed. This is measured by using the 3--2,5-diphenyltetrazolium bromide (ΜΤΤ) method. These experiments contribute to the prioritization of extracts based on the in vivo assays in mice and flies. Fractions derived from extracts of the hit plants, their isolated constituents, as well as synthesized metabolites are also tested in the cell-based model systems described above. The most active samples are forwarded to more extended biological investigations, including the evaluation of their ability to delay cellular senescence in human umbilical vein endothelial cells (HUVECs) and to prevent UVB-induced senescence of human diploid fibroblasts.The hit plants are also subjected to activity profiling with in silico virtual screening. In particular, the 3D structures of identified active native and metabolized constituents of the hit plants are stored alongside the bioactivity data generated in the cell-based bioassays. These data in combination with a set of in silico target prediction methods, such as pharmacophore modelling and the similarity ensemble approach (SEA), are used to identify potential molecular targets and mechanisms of action for the active (native and metabolized) plant constituents. Experimentally validated pharmacophore models and target-based assay methods for a range of individual targets are used. Through this process, active scaffolds for the selected biological targets are determined and principle structure-activity relationships (SARs) are defined.The bioactive native and metabolized hit extracts are fractionated in a bioactivity guided way, supported by data from the in vitro (mainly metabolized extracts) and in vivo systems (only native extracts), leading to the isolation and purification of the bioactive compounds. Isolation and purification of bioactive plant constituents from native extracts is performed on a preparative scale using state-of-the-art preparative chromatographic separation techniques, e.g., flash or medium pressure liquid chromatography (MPLC) or fast centrifugal partition chromatography (FCPC). The bioactive plant constituents are identified using state-of-the-art spectroscopic techniques including UV-Vis, infrared radiation (IR), circular dichroism (CD), 1D- and 2D-NMR, high resolution NMR equipped with microflow, microprobes, and cryoprobes, as well as HRMS based on time-of-flight or orbital traps technology. The same methods are used to assess the purity of the isolated NPs. An aliquot of the isolated bioactive plant constituents is sent to in vitro metabolization.Active metabolized extracts of the hit plants are submitted to bioactivity guided LC-micro-fractionation in 96-well plates. Bioactive micro-fractions are analyzed by UHPLC coupled with a photodiode array detector (PDA), evaporative light scattering detector (ELSD), and HRMS, in combination with NMR techniques for the identification of the bioactive metabolites. In silico prediction tools are also used to support the identification procedure. Selected bioactive metabolites and structural analogues are (semi-) synthesized using state-of-the-art strategies.Pure bioactive plant constituents and metabolites are subjected to comprehensive pharmacological analysis in vitro (mainly metabolites) and in vivo (only native plant constituents). Based on the results of the detailed phytochemical and pharmacological investigations, lead compounds, extracts, or enriched extracts are identified. Innovative metabolomics strategies are applied for the identification of the optimal plant sources of the leads with respect to industrial needs.2 (SFE-CO2) and subcritical water extraction (SWE) are employed and compared to more conventional techniques. For the development and the optimization of the SFE-CO2 extraction methods, special attention is given to CO2 pressure, extractor and collector temperature, CO2 flow, CO2/starting material ratio, and the percentage of co-solvent . Furthermore, in order to determine the optimal conditions for SWE extraction, the effects of varying parameters  are investigated. Using analytical techniques such as near infrared spectroscopy (NIR) and MS, the obtained extracts can be directly compared and optimal conditions can be identified. For implementation of a fast and cost-effective quality control method for the produced extracts, the data acquired in the analytical screening is used to create an NIR-based model for the determination of extract quality. If a final purification step is needed, this is performed using the centrifugal partition chromatography (CPC) technology. In this case, industrialization processes in CPC seeking optimal conditions for scale-up are considered. Moreover, adsorption resins technology (ART) is used mainly for the production of enriched extracts while spray drying techniques are also incorporated. A series of different resin types  are evaluated for the optimal capacity and selectivity. Furthermore, to provide a smooth transition into industrial implementation and commercialization, all the respective steps of the process as well as the corresponding results are evaluated for their compliance with the demands of industry and regulatory authorities.To carry the lead agents  to the stage of development of nutraceuticals or phytotherapeutic agents, optimization and application of up-scaling procedures are carried out. For the optimization of the extraction and isolation methods, several parameters are taken into consideration . The “green” extraction techniques of supercritical-fluid-extraction-COIn addition, according to their chemical characteristics, the lead agents are assayed using different formulation concepts, with the intention of the development of nutraceuticals and phytotherapeutic agents. For instance, lyophilization and the use of appropriate formulatory agents are encapsulated following the main formulation processes, i.e., extrusion spheronization, solution/suspension layering, powder layering, wet granulation pelletizing, and melt granulation pelletizing.For the formulated lead products, analytical methods for marker components are developed and validated according to the ICH guidelines, including linearity, repeatability (intermediate precision and repeatability on different concentration levels), and accuracy. Based on the respective characteristics and analytical demands of the products, different analytical platforms are incorporated . The microbiological quality of the lead products is also assessed.Moreover, following the development process, stability studies are performed on the lead products under storage conditions in accordance with ICH guidelines using chromatographic techniques. Multiple parameters, such as temperature, humidity, and pH range, are investigated .Finally, to determine the commercialization potential of the lead agents, feasibility studies are conducted. These include a detailed market analysis and the investigation of economic prospects and commercial exploitation of the potential product.The MediHealth project introduces an innovative concept for the investigation and exploitation of dietary plants incorporating an integrated approach combining in silico, cell-based, in vivo, metabolization studies, and state-of-the-art techniques. It aspires to produce valuable and solid data supporting the development of novel nutraceuticals or phytotherapeutics to promote healthy ageing. We hope that sharing the strategy of this novel and unique multidisciplinary approach helps to increase the scientific interest in NPs research with special regard to the exploitation of diet regimes towards healthy ageing.


Nucleic acids are programmable and biocompatible polymers that have beneficial uses in nanotechnology with broad applications in biosensing and therapeutics. In some cases, however, the development of the latter has been impeded by the unknown immunostimulatory properties of nucleic acid-based materials, as well as a lack of functional dynamicity due to stagnant structural design. Recent research advancements have explored these obstacles in tandem via the assembly of three-dimensional, planar, and fibrous cognate nucleic acid-based nanoparticles, called NANPs, for the conditional activation of embedded and otherwise quiescent functions. Furthermore, a library of the most representative NANPs was extensively analyzed in human peripheral blood mononuclear cells (PBMCs), and the links between the programmable architectural and physicochemical parameters of NANPs and their immunomodulatory properties have been established. This overview will cover the recent development of design principles that allow for fine-tuning of both the physicochemical and immunostimulatory properties of dynamic NANPs and discuss the potential impacts of these novel strategies. Food and Drug Administration (FDA) in 2018 . N. N73]. NBesides the activation of multiple functionalities, an important feature of the rational design of the cube/anti-cube NANP system is the ability to fine-tune the physicochemical and immunological properties of NANPs simply by adjusting the ratios of DNA and RNA strands in their composition . With anIt is becoming apparent that interactions between NANPs and the immune system must be defined to permit the successful translation of this technology to the clinic. Foreign nucleic acids can produce a robust and severe response in immune cells. Pro-inflammatory cytokines and type I interferons are characteristic of nucleic acid sensing in immune cells, and in animals, it can produce anywhere from minor inflammation to severe cytokine storms. Therefore, to address fundamental questions regarding the immune recognition of these novel materials in a timely fashion, the relation of features such as the size, shape, composition, and physicochemical properties of various polygonal NANPs  to the aThis very first application of QSAR modeling for NANPS studied measured physical and chemical properties as inputs to predict a given NANP’s ability to generate a pro-inflammatory response B. ImportFollowing these findings, the very first systematic investigation of NANP recognition by immune cells using primary human peripheral blood mononuclear cells (PBMCs) from a cohort of more than 100 healthy human donors was recently designed and executed . DespiteImportantly, the following work has also demonstrated that the functionalization of NANPs with RNAi inducers completely changes their known immunorecognition . HoweverIn conclusion, the responsive behaviors of NANPs can be determined by the specific design principles for individual NANPs, their assembly type, compositions, physicochemical properties, and the presence of any additional functionalities in their structure. The use of NANPs for the controlled design of dynamic stimuli-responsive systems presents multiple advantages: (i) NANPs can be programmed to gather multiple different functionalities for their simultaneous delivery to cells ,37,42,61Predicting and controlling the immune responses triggered by NANPs, especially when the magnitudes of those responses may vary between donors, remains a substantial challenge for the furtherment of nano-TNAs. In addition, due to their negative charges which inhibit free passage across the cell membrane, a carrier or targeting moieties are needed for delivery. While there is a plethora of nanoparticles formulated for nucleic acid delivery, the effects of the carrier on the immunostimulatory properties of NANPs remain unclear. Furthermore, the healthy host immune tolerance to nucleic acids and the generation of antibodies against DNA and RNA after exposure to NANPs is an important safety topic which, so far, has not been addressed. Thus, assessing the immunogenicity of NANPs is a future logical direction in this field.The activation of RNA interference, FRET, RNA aptamers, transcription, and other responses upon the reassociation of cognate NANPs at isothermal conditions exhibits the potential for dynamic constructs to operate only in the presence of a target. Additionally, the degree to which the properties of TNAs contribute to the elicitation of an immune response will allow for their intentional activation or evasion, depending on whether immunotherapy or drug delivery is desired. By optimizing conditional activation and immunostimulation, NANPs can be better designed for future clinical applications.