Cells constantly encounter a wide range of environmental signals and rely on their signaling pathways to initiate reliable responses. Understanding the underlying signaling mechanisms and cellular behaviors requires signal generators capable of providing diverse input signals to deliver to cell systems. Current research efforts are primarily focused on exploring cellular responses to global or local signals, which enable us to understand cellular signaling and behavior in distinct dimensions. This review presents recent advancements in global and local signal generators, highlighting their applications in studying temporal and spatial signaling activity. Global signals can be generated using microfluidic or photochemical approaches. Local signal sources can be created using living or artificial cells in combination with different control methods. We also address the strengths and limitations of each signal generator type, discussing challenges and potential extensions for future research. These approaches are expected to continue to facilitate on-going research to discover novel and intriguing cellular signaling mechanisms. Cells possess the remarkable capability to perceive and respond to a wide array of time-varying signals from their environment. This ability stems from a diverse functional repertoire of genes, proteins, and metabolites that interact in response to various external physical cues, such as matrix stiffness  and fluiThe majority of studies in the field of cell signaling can be broadly categorized into two scenarios: 1) homocellular signaling, which involves signal transduction within identical cell types (monoculture), and 2) heterocellular signaling, which describes signal transmission between two distinct cell types (coculture). In the case of homocellular signaling , a populFurthermore, the microenvironment within the body is subject to rapidly changes influenced by various signaling processes. The presence of transient gradients of signaling molecules facilitates cellular communication and regulates cellular functions. For instance, gonadotropin-releasing hormone (GnRH) is secreted in short pulses, activating the synthesis and release of pituitary gonadotropin hormones, thereby regulating reproductive functions . PulsatiIt is widely recognized that dynamic signal processing are ubiquitous in cellular systems. However, understanding how cells interpret these input signals can be challenging. This challenge arises because population-level measurements often mask the heterogeneous behavior exhibited by individual cells, and conventional methods often lack the ability to generate various types of targeted perturbations other than continuous inputs in signaling pathways, for example, signal molecules simply added in well plates continuously stimulate cells. Moreover, observing cellular events in multiple contexts is essential for multi-dimensional understanding of signaling process, as evident in the distinct cellular responses to global and local inputs . ConsequGlobal signal generators provide uniform inputs that allow for the study of both population-averaged and single-cell responses. Currently, the primary methods employed for generating global inputs include microfluidic molecule delivery and the photodeprotection of caged input molecules. Microfluidic systems can provide a wide range of input modes, such as pulse , continuin vivo biological environments with great accuracy and enable high-content analysis of cells. Microfluidics technology offers precise automation and control of analytical functions, enabling high-resolution manipulation of cells and their microenvironments. With these properties, we can modulate cellular signaling pathways to gain insights into mechanisms underlying cell activation, migration, and intercellular communication. Recent studies investigating temporal signaling dynamics using microfluidics-based global input generators are summarized in In the past decade, microfluidics has made remarkable advancements in exploring temporal cellular behaviors . Microflin vitro, and its flexibility enables easy integration of membrane valves and pumps to create intricate networks of microchannels . Applying a stepwise ramping input of TNFα or interleukine-1β (IL-1β) to fibroblasts revealed that the activity of the NF-κB signaling pathway correlated with the rate of change in cytokine concentrations rather than the absolute cytokine concentrations. In addition, the implementation of sinusoidal inputs was realized using a multiple-layer PDMS device with eight triangular converging valves  by promoting TLR1/2 heterodimerization. In the presence of UV irradiation, trans-P10 is converted to cis-P10, which reduces the activities of APCs by impeding the TLR1/2 heterodimerization  and Resiquimod (R848), which are agonists of Toll-like receptor 7 (TLR7) and TLR7/8, respectively, were conjugated with the photo-protecting group carbamate of 2-(2-nitrophenyl)-propyloxycarbonyl (NPPOC) to suppress their spatial activity. Irradiation with 360 nm UV light deprotected these small-molecule agonists, triggering signal transmission and NF-κB pathway activation in cells  Ryu et . Similarrization . These mPCGlue-NBD, carries multiple guanidinium ion (Gu+) pendants. This functional molecular glue can strongly adhere to the target protein, hepatocyte growth factor (HGF), and cover the region for protein-protein interactions (PPIs) on their surfaces. The PPIs are inactive, suppressing cellular signaling. Upon irradiation with UV light, PCGlue-NBD is photocleaved, reducing the multivalency for the adhesion. Consequently, uncaged HGFs regains its intrinsic PPI affinity toward c-Met, leading to pathway activation and cell migration  or one-dimensional (1D) path  path . The conLiving cell senders serve as natural local signal sources due to their ability to secrete signals within the physiological range. A critical step is the activation of sender cells. This section will discuss recent strategies for the controlled activation of living sender cells, including pre-stimulation, microfluidics-assisted stimulation, photocaged global input, and optogenetic activation. Recent studies on living cell-based local input generators are summarized in To construct a local signaling model, sender cells can be pre-stimulated before cocultured with receiver cells. The activated sender cells become local signal sources, secreting input signals in limited areas. Recently, a diffusion-consumption model has been created using the pre-stimulated T cells as the living senders to produce IL-2, which stimulates surrounding T cell receivers . ImmunofValve-integrated microfluidic devices enable the coculture of a single sender cell  and a population of receiver cells , as well as the control of signal propagation . These din vivo local signaling patterns are typically 2D or 3D. A 2D model of developmental signaling center has been created in microfluidic device . In the presence of 3OC6 HSL, both PFO and BDNF are produced, and BDNF is released through formed PFO pores. In a coculture system, the artificial cells responded to 3OC6 HSL, releasing BDNF that subsequently drives the differentiation of mouse embryonic stem cell-derived neural stem (mNS) cells  and perfringolysin O (PFO) gene expression construct . This syS) cells . CommuniNested (or embedded) architectures involving artificial and living cells provide non-native signaling configurations . In thisGlobal and local signal generators have been utilized in cellular signaling studies to gain insights into the activation of signaling pathways and dynamics of signaling proteins. Each type of generators has its own set of advantages and limitations, which will be discussed in this section .Microfluidic systems have been widely used as global signal generators for investigating temporal signaling dynamics . These sPCGlue-NBD (Another approach of generating global inputs involves light irradiation to induce the photodeprotection of caged input molecules . This meGlue-NBD  or PEG cGlue-NBD  and photLocal signal sources can be established using either living or artificial cell senders. Various strategies have been employed to control the activation of sender cells. A simple 2D signaling model can be constructed by coculturing pre-stimulated living sender cells with receiver cells . This meMicrofluidic cell coculture systems enable controlled local signaling by compartmentalizing sender and receiver cells in closed environments with integrated separation valves. Depending on the sensitivity of receiver cells to global stimuli, sender cells can be either separated from receiver cells  or confiThe photodeprotection of caged input molecules have also been applied in local signal generation . Sender Artificial cells have gained significant attention as substitutes for living cells . These cGlobal and local signal generators have significantly enhanced our understanding of temporal and spatial cellular signaling activities and cellular behaviors. In particular, microfluidic systems have emerged as powerful tools for investigating temporal activity of signaling pathways in single cells  and cellThe development of photocaging and photodeprotection-based global input generators has been a subject of ongoing research for years. This emerging technology has aided our exploration in control of cellular signaling activation. Several small molecule agonists of TLRs conjugated with 2-nitrobenzyl groups have been applied to control the activation of immune signaling pathways . By explLiving cells have been adapted to serve as local signal generators using various approaches, as discussed in this review. These local signal generators can be easily extended to explore other cell types and signaling pathways, offering versatility and flexibility in experimental design. While artificial cells have not been widely applied as local signal generators in observing signaling dynamics in living receiver cells, recent studies have demonstrated their potential in controlled signaling activation in neural and HEK293 cells . The utiIn conclusion, the development of robust platforms for both global and local signal generation holds significant promise in enhancing our understanding of how cells encode and decode diverse input information across spatial and temporal dimensions. The impact of these signal generators is evident in their potential to elucidate the underlying signaling mechanisms governing temporal and spatial signaling dynamics, as well as cellular behaviors. We firmly believe that advancing and expanding upon the techniques discussed in this review will further propel the discovery of novel and intriguing signaling mechanisms.


All cells employ signal transduction pathways to respond to physiologically relevant extracellular cytokines, stressors, nutrient levels, hormones, morphogens, and other stimuli that vary in concentration and rate in healthy and diseased states. A central unsolved fundamental question in cell signaling is whether and how cells sense and integrate information conveyed by changes in the rate of extracellular stimuli concentrations, in addition to the absolute difference in concentration. We propose that different environmental changes over time influence cell behavior in addition to different signaling molecules or different genetic backgrounds. However, most current biomedical research focuses on acute environmental changes and does not consider how cells respond to environments that change slowly over time. As an example of such environmental change, we review cell sensitivity to environmental rate changes, including the novel mechanism of rate threshold. A rate threshold is defined as a threshold in the rate of change in the environment in which a rate value below the threshold does not activate signaling and a rate value above the threshold leads to signal activation. We reviewed p38/Hog1 osmotic stress signaling in yeast, chemotaxis and stress response in bacteria, cyclic adenosine monophosphate signaling in Amoebae, growth factors signaling in mammalian cells, morphogen dynamics during development, temporal dynamics of glucose and insulin signaling, and spatio-temproral stressors in the kidney. These reviewed examples from the literature indicate that rate thresholds are widespread and an underappreciated fundamental property of cell signaling. Finally, by studying cells in non-linear environments, we outline future directions to understand cell physiology better in normal and pathophysiological conditions. All living cells sense and respond to environmental changes . Concent30 = 2.3 * 1032 unique combinations possible. This simple example demonstrates the possible infinite space of modulating the environment of cells in a non-acute manner. This new dimension of dynamic changes of the stimulus molecule may dramatically expand our phenotypic space currently not explored in the laboratory (white star). Including this critical dimension will allow us to observe physiologically relevant phenotypes in different dynamic phenotypes (yellow plane) and under different genetic backgrounds (cyan plane). By designing experiments that consider this new dimension of dynamic changes of the stimulus molecule, we expect to better characterize the accurate and relevant biomedical phenotype (white star) compared to current disease models (grey star) that do not fully consider this new dynamic dimension. As an example of the latest insights gained from dynamic temporal environments, we will focus on the newly discovered concept of a rate threshold. Similar to a concentration threshold, where a concentration above a certain value activates signaling, a rate threshold is a value in the rate of change in the environment that needs to be crossed to activate signaling. We argue that a rate threshold is a novel and phenotypically important mechanism in cell signaling that is relevant in gradual and non-acute changing environments. In this review, we will focus on pathways that respond to gradual environmental changes and describe different examples from the literature to illustrate the importance of non-acute and gradually changing environments from bacteria to humans. We will start with a yeast Mitogen-Activated Protein Kinase (MAPK) pathways rate sensitivity and rate threshold as a paradigm for all the remaining pathways.In contrast, cells of any organism or in any natural environment experience a wide variety of gradual changes in external stimuli. Current treatments and therapies are based on observed phenotypes (purple plane) and current disease models (grey star) in such acute treatments, potentially missing or misinterpreting the response of a cell to the stimulus under physiological conditions. For example, if one uses a dozen concentrations from the previously mentioned dose-response experiment and alters the concentration acutely every 1 min for 30 min, then there are 12Saccharomyces cerevisiae (The high osmolarity glycerol (HOG) MAPK pathway in the budding yeast revisiae  is an idrevisiae  for addrrevisiae . At the revisiae . Activatrevisiae . This durevisiae   decoding these rate thresholds within the same or different cells is elusive and requires further studies.Besides single-cell organisms, rate sensitivity, and rate thresholds were also observed in cells of multicellular organisms. This section will focus on the chemoattractant cyclic adenosine monophosphate (cAMP) sensitivity of single um cells  . These results show that the dynamics of BDNF differentially regulate cell signaling and phenotypes and indicate that BDNF signaling is rate sensitive, and this pathway may encode a rate threshold. Further studies are required to establish this rate sensitivity, the rate threshold, and the proteins regulating these processes.In an independent study, in-vitro, where the cell environment can be precisely controlled  signaling pathway as a significant pathway activated by morphogens. Further, we will focus on the ligand proteins TGF-β, bone morphogenetic proteins (BMPs), and Activin and how their gradual concentration profile that changes over time impact signaling and phenotype . These SMADs can then bind coSMAD SMAD4. R-SMADs  and coSMAD can form complexes in the nucleus and act as transcription factors to regulate target genes. TGF-β signal through SMAD2 and SMAD3, whereas BMPs signal through SMAD1, SMAD5, and SMAD9. TGF-β signaling is induced by the TGF-β ligands binding to one of the five TGF-β type II receptors. TGF-βs bind the TGF-β type II receptor type 2 (TGFBR2), whereas BMPs bind the BMP receptor type 2 (BMPR2). These receptors are serine/threonine receptor kinases that phosphorylate one of the seven TGF-β type I receptors. A specific TGF-β type I receptor phosphorylates one of the five receptor-regulated Mothers against decapentaplegic homolog (MADHs or SMADs) (R-SMAD’s)  encoding the rate threshold. After this protein(s) has been placed, one might investigate the rate threshold mechanism through protein domain deletions, chemical inhibition, or overexpression studies. After identifying a rate threshold protein, a phenotypic assay will establish the biological relevance in different dynamic environments and rate threshold regulator mutants see  Johnson. BesidesIn comparison, the same cells and pathways exposed to a quadratic increasing condition  lead to


The fundamental frequency contour (melody) of cry and non-cry utterances becomes more complex with age. However, there is a lack of longitudinal analyses of melody development during the first year of life.The aim of the study was to longitudinally analyze melody development in typical vocalization types across the first 12 months of life. The aim was twofold: (1) to answer the question whether melody becomes more complex in all vocalization types with age and (2) to characterize complex patterns in more detail.n = 10,988) of 10 healthy infants  over their first year of life were analyzed using frequency spectrograms and fundamental frequency (f0) analyses (PRAAT). Melody complexity analysis was performed using specific in-lab software  in a final subset of 9,237 utterances that contained noise-free, undisturbed contours. Generalized mixed linear models were used to analyze age and vocalization type effects on melody complexity.Repeatedly recorded vocalizations (The vocalization repertoire showed a higher proportion of complex melodies from the second month onward. The age effect was significant, but no difference was found in melody complexity between cry and non-cry vocalizations across the first 6 months. From month 7–12, there was a further significant increase in complex structures only in canonical babbling not in marginal babbling. Melody segmentations by laryngeal constrictions prevailed among complex shapes.The study demonstrated the regularity of melody development in different vocalization types throughout the first year of life. In terms of prosodic features of infant sounds, melody contour is of primary importance, and further studies are required that also include infants at risk for language development. Infants are not yet competent communication partners at birth, but they are already able to perceive auditory information of their surrounding world based on melodic cues , 2, 3, 4Based on many years of research experience in their labs, Wermke and Mende have summarized melody development in pre-speech utterances in a melody development (MD) model , 11, 12.A recent study  corroborThe existence of simple and complex melody structure has also been reported in earlier work on vocal development , 15, 16.For the first time, the present paper aimed to investigate how melody development is accomplished in individual infants longitudinally from birth to 12 months of life. Previous studies have mainly investigated natural crying, while longitudinal studies of melody development in cry and non-cry vocalizations have been lacking in individual infants across the first year of life. The novelty of the present study is its comprehensive objective investigation and description of the age-dependent unfolding of melody patterns within different vocalization types in the same infants. As far as we know, this has not been done yet.Audio files of healthy, term-born  monolingual German infants were analyzed from birth to the age of 373 days of life. Inclusion criteria were an inconspicuous pregnancy, age-appropriate somatic development at birth, an inconspicuous perinatal adjustment (APGAR score of ≥8 at 5 min and ≥9 at 10 min), metabolic indices in the normal range, and passing the newborn hearing screening, i.e., healthy infants. The mean birth weight was 3,201 g ; mean head circumference, 35 cm (range 31–37 cm); and mean birth length, 51 cm (range 47–55 cm). The mean gestational age was 39 weeks (range 37–41 weeks). Based on the archived files, we were able to confirm that none of the children showed any developmental delay or disorder in terms of linguistic or cognitive development during the first 3 years of life.n = 10,988) available in the archive from our participants across the observation age. A vocalization was defined as the total vocal output during a single expiration.For this longitudinal study, the vocalizations of 10  healthy infants were selected. Here, we used all cry and non-cry vocalizations  of the original recording sessions  as well as all the individual sounds, which were previously manually segmented using a commercially available system . Finally, all recordings were archived as anonymized data sets. Each parent had a minimum of high school education, and the monthly family income was reflective of a middle-class standard of living.Cry vocalizations  were recorded under comparable conditions in a hospital (first week) and at home, respectively . As outlined in more detail elsewhere , non-cryn = 1,751; 16%), the finally available database totaled 9,237 vocalizations; comprising of 1,141 spontaneous cry utterances (3–195 days of life) and 4,667 early non-cry vocalizations (4–200 days) recorded across the first 6 months of life. Further data comprised recordings between 7 and 12 months of 3,429 babbling utterances (181–373 days). Note that the term “non-cry vocalizations” comprised vocants, coos, and primitive, mainly nonsyllabic babbling. A differentiation of the babbling utterances in marginal  and canonical (n = 675) babbling was only performed between month 7–12 of life. However, all infants appeared in all  data sets. Table Based on visual inspections of the spectrograms , phonatory noise phenomena  and pheAssignment of vocalizations to a certain vocalization type was based on spectrograms and auditory impression. According to the guidelines for coding pre-speech utterances, force choice approach was applied to differentiate between cry, non-cry, and advanced babbling, respectively . Four voAs elsewhere described in more detail , an autoIn agreement with preceding studies, the analysis of melody pattern was based on a differentiation between simple (singe-arc) and complex melodies Fig. . ComplexBased on the mentioned arc criteria, all vocalization melodies were objectively subdivided into those with only a simple  melody and those with a complex  melody cf. Fig. . SA contMelodies containing at least one complete or incomplete laryngeal constrictions , 31 wereThe analysis focused on two outcome measures: (1) melody complexity: the melody contour of each vocalization was assigned to one of two categories: (1) “simple,” single-arc melody or (2) “complex,” multiple-arc melody. The percentage of these categories was analyzed across the observation age for each vocalization type.(2) Complexity degree: To describe the degree of complexity, melody contours of each vocalization type were divided into double-arc (DA), triple-arc (TA), multiple-arc (MA), or segmented structures (SEG) and their relative occurrence displayed in developmental diagrams.Descriptive information about the different melody structures is presented. Pie charts, column charts, and line charts were used for graphical representation. The evaluated measured variables were examined for their normal distributional properties using Q-Q plots. The line graphs of Figures Because of the hierarchical, two-level data structure and the existence of age-specific vocalization types, generalized linear mixed models for the dichotomous variable “melody complexity” were used for crying (C)/non-crying (NC) across the first 6 months and for marginal babbling (BM)/canonical babbling (BC) between 7 and 12 months of life. The value “0” corresponded to vocalizations with a single-arc melody structure, while “1” was used for those with a complex melody structure. Age in months as a metric predictor and the respective vocalization type as a categorical predictor entered the linear multilevel model as fixed effects. The Akaike information criterion (AIC) was used to assess or compare different models and to consider the best model fit. All statistical analyses were performed using SPSS version 26 .Cry and non-cry vocalizations exhibiting simple, single-arc melodies dominated during the first month. The relative frequency of occurrence of complex melodies increased thereafter. The turning point toward a clear prevailing of complex structures in cry and non-cry vocalizations was observed in the third month of life Fig. . A permaAnalysis of complexity degree in the same repertoire yielded a relative frequency of occurrence of 39% vocalizations with single arcs and 60% complex structures across the whole observation period. Complex contours were found to comprise 21% double-arc melodies, 7% triple-arc melodies, 3% of melodies consisting of four and more arcs, and 30% segmented melodies.p = 0.018), while no significant difference was observed between the two vocalization types (p = 0.594). The AIC value was 25,307.565, the estimator for random effects was 0.276 (p = 0.04).A more detailed analysis of melody analysis adjusted to age and vocalization type was yielded by generalized mixed models to the dichotomous variable melody complexity to all 5,808 cry/non-cry vocalizations of the first 6 months of life. The final model revealed a significant positive age effect across the first 6 months of life (p = 0.293), but a significant higher number of complex structures were found in canonical babbling compared to marginal babbling during this age period (p < 0.001). The AIC value was 14,964.803, the estimator for random effects was 0.006 (p = 0.528).A corresponding generalized mixed model was calculated based on the 3,429 signals of marginal and canonical babbling across the 7th−12th month of life. No age effect was observed , which reflect the degree of melody complexity, there were also marked similarities between the C and NC repertoire Fig. , 6: in bThe second most common category of complex structures were melodies consisting of two combined single arcs to generate a double-arc melody (DA). The DA share was about 15–20% in C and NC over the first 6 months of life. The high share compared to other unsegmented melodies  may have two reasons: (1) a doubling of basic building blocks is the simplest strategy in generating complexity, so one could expect this stage to occur frequently, (2) a high occurrence of double-arc melodies in pre-speech utterances could point to their significance for the acquisition of disyllabic words , 35, 36.From month 7–12, only marginal (BM) and canonical (BC) babbling, the typical non-cry vocalizations at this age, were investigated. For vocal development, the natural crying is hardly or no longer relevant. Besides, it is more difficult to record crying at this age. The most striking finding in the BM development diagram was the high stability in the frequency of occurrence of all types of melody structure. The proportion of complex structures overall was between 55 and 65% and showed a slight cyclical movement in all categories  for producing syllables, words, and sentences later on. Furthermore, the findings indicate the importance of melody to move from expressions of emotion to meaning, to move from emotive sounds to abstract words , 11, 12.The study demonstrated that human infants are able to communicate with melodic patterns long before vocabulary and grammar are established. This provides further evidence of the preparatory function of early melody development for subsequent steps of language development. Increase in melody complexity also reflects increasing vocal control as well as vocal flexibility in all vocalization types, including natural crying, during the first year of life. To produce variable melody structures with tiny vocal folds requires a well-functioning and very responsive laryngeal-respiratory coordination under a steady maturation without marked discontinuity. This is a good prerequisite for approaches to use melody complexity as a risk marker for a reduced vocal control ability.An ethics statement is not applicable because this study is based exclusively on anonymized data. The audio files analyzed here were selected from the anonymized archive of the Centre for Prespeech Development and Developmental Disorders, University Hospital, Würzburg, Germany. Therefore, ethical approval was not required. Original recordings were approved by the respective ethical board and were carried out in accordance with relevant guidelines and regulations to guarantee that the research was conducted ethically in accordance with the World Medical Association Declaration of Helsinki; informed consent signed by parents was given.The authors have no conflicts of interest to declare.There are no funding sources to declare.All authors have made significant contributions to the conception of the paper. K.W. and T.K. designed the research paper. K.W. supervised and coordinated the recordings in several of their earlier projects and supervised the analyses. T.K. and M.W. analyzed the melodies, identified the different vocalization types, and measured the different melody structures. T.K. and M.W. wrote the main text together with K.W. T.K. and M.W. together performed the statistical analysis. All authors jointly interpreted the results and edited the paper.Data are not publicly available on legal grounds. All data generated or analyzed during this study are included in this article. Further inquiries can be directed to the corresponding author.


Steroid hormone secretion is one of the key functions of granulosa cells (GCs). Resveratrol is a natural polyphenol, known for its beneficial health effects, such as improving reproductive health. However, its application is limited due to poor bioavailability. The methoxy derivative of resveratrol (DMU-212) was demonstrated to be more lipophilic, and therefore of greater bioavailability. However, since the addition of methoxy groups to the stilbene scaffold was found to make the molecule insoluble in water, DMU-212 was loaded into liposomes. This study aimed to evaluate how the liposomal formulation of DMU-212 (lipDMU-212) alters estradiol and progesterone secretion of human ovarian GCs in a primary three-dimensional cell culture model.® 3D Cell Viability Assay. The secretion of estradiol and progesterone was determined using commercial ELISA kits. RT-qPCR was conducted to analyze the expression of steroidogenesis-related genes. Finally, the western blot technique was used to analyze the effect of lipDMU-212 and FSH treatments on CYP11A1 and HSD3B1 protein levels.DMU-212-loaded liposomes were prepared by thin film hydration followed by extrusion. Cell viability was measured after exposure of GCs spheroids to the liposomal formulation of DMU-212 using CellTiter-GloCYP11A1, HSD3B1, StAR, CYP17A1, CYP19A1, and HSD17B1 genes. We have also shown that lipDMU-212, used alone and in combination with FSH, significantly increased the expression of the HSD3B1 and CYP11A1 proteins in GCs. Furthermore, our study suggests that lipDMU-212 increases FSH activity.lipDMU-212 was found to significantly increase estradiol and progesterone secretion in a dose-dependent manner by enhancing the expression of StAR and CYP19A1 expression. These findings suggest that lipDMU-212 might have a beneficial effect in the treatment of disorders related to estrogen deficiency and hyperandrogenism, such as PCOS.This is the first study to describe the steroidogenic activity of liposomal formulation of DMU-212, possibly through increasing the  GCs form an important component of the ovary since they play a key role in the production of steroid hormones, mainly estradiol. The synthesis of hormones requires close cooperation between GCs and theca cells, which surround the follicle and produce androgens following luteinizing hormone (LH) stimulation. Once androgens diffuse into GCs, aromatase CYP19A1) converts them to estrogens in response to follicle-stimulating hormone (FSH) 9A1 conve. ResveraSeveral studies have revealed that resveratrol improves ovarian functions, promotes the development of ovarian follicles, protects GCs from degeneration, and enhances their proliferation and differentiation –7. HowevAccording to the structure-biological activity studies, the substitution of the hydroxyl (-OH) groups with methoxy (-OMe) ones in resveratrol may enhance the lipophilicity of the compound, making it more bioavailable. On the other hand, the introduction of methoxy groups to the stilbene scaffold has simultaneously been found to influence the solubility of the molecule .Hence, liposomes that can be loaded with compounds of different lipophilic-hydrophilic nature provide a feasible means to achieve the greater therapeutic potential of methoxy derivatives.Among several methoxy derivatives of resveratrol, 3,4,5,4′-tetramethoxystilbene DMU-212) has been reported to exert potent anticancer activity in several cancer cell lines –21. Furt2 has beeSo far, information regarding the role of resveratrol analogs in the regulation of ovarian steroidogenesis is limited. To the best of our knowledge, there is only one publication concerning the effect of hydroxylated and methylated resveratrol analogs on GCs, however, in a porcine in vitro model . Since tThe study aims to evaluate how liposomal formulation of DMU-212 affects estradiol and progesterone secretion of human ovarian GCs in a primary three-dimensional (3D) cell culture model.sn-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1’-rac-glycerol)  (POPG) were acquired from Avanti Polar Lipids . Chloroform and acetonitrile were purchased from Avantor Performance Materials Inc. . The CellTiter-Glo® 3D Cell Viability Assay Kit was provided by Promega Co. . Estradiol and Progesterone ELISA kits were acquired from DRG Instruments GmbH . Super Signal West Pico PLUS Chemiluminescent Substrate was purchased from Thermo Fisher Scientific . The Trans-Blot Turbo Mini 0.2 µm PVDF Transfer Packs set and the Mini-PROTEAN® TGX™ Precast Gels were provided by Bio-Rad . All other materials were purchased from Sigma-Aldrich Co.  unless otherwise stated.All chemicals used were of analytical grade unless stated otherwise. DMU-212 was purchased from Sigma-Aldrich Co. . 1-palmitoyl-2-oleoyl-Liposomes were prepared by thin film hydration (TLH) followed by extrusion. Chloroform solutions of POPC (50 mg/mL), POPG (50 mg/mL), and DMU-212 (10 mg/mL) were prepared. Volumes of stock solutions were mixed in a glass round-bottom flask. Chloroform was evaporated gradually under reduced pressure at 40 °C in a rotary evaporator . The resulting lipid film was hydrated using mPBS  in a rotary evaporator. The resulting liposome suspension was passed 21 times through polycarbonate membrane equipped with 10 mm drain discs  with pore diameters of 100 nm using a syringe extruder . Unbound material was separated from liposomes by ultrafiltration using Amicon Ultra centrifugal filters with molecular weight cut-off (MWCO) 50 kDa . Liposome samples were stored at 2–8 °C, protected from light.The size and zeta potential of liposomes were determined by dynamic light scattering (DLS) laser Doppler electrophoresis using Malvern Zetasizer Nano ZS , as described elsewhere .Concentration of DMU-212 in liposomal formulation was determined by High-Performance Liquid Chromatography (HPLC) at 40 °C. Liposome samples were diluted 5 times in methanol prior to analysis. The analysis was carried out using Agilent 1260 Infinity II equipped with a DAD detector. The chromatographic separation was achieved on Luna Omega PS C18 column  . A mobile phase gradient of eluent A (water with 5 mM ammonium acetate and 0.1% formic acid) and eluent B (acetonitrile) was applied at 0.8 mL/min. The optimized gradient profile was the following: 60% A was maintained for 3 min, declined to 10% A in 4 min, kept at 10% A for 2 min, then reversed to the original composition of 60% A over 1 min and remained constant for 5 min to re-equilibrate the column . DetectiHuman luteinized GCs were isolated from follicular fluid obtained from 20 women, aged 25-40 years old, undergoing IVF-ICSI procedure, due to tubal or male factor infertility, at the Centre of Diagnosis and Treatment of Infertility in the Division of Infertility and Reproductive Endocrinology at the Poznan University of Medical Sciences. Follicular fluid was obtained by transvaginal ultrasound-guided aspiration, following the controlled ovarian stimulation according to designed protocol, as previously described .After removal of the cumulus oophorus-oocytes-complexes, the freshly collected follicular fluid was centrifuged for 10 min at 250 g at room temperature (rt), and the density gradient centrifugation was performed. Briefly, the pellet of hGCs was suspended in Phosphate Buffered Saline (PBS) and centrifuged on 7 ml of Pancoll human  for 20 min at 400 g. Separated GCs were aspirated by pipetting from the interphase layer, washed in 10 ml of Dulbecco’s Modified Eagle Medium (DMEM), centrifuged at 250 g for 10 min, and resuspended in cell culture medium .2 and 95% air at 37 °C. After 24 h, cells were detached using a trypsin-EDTA solution and seeded in 96-wells U-bottom plates at a density of 10 000 viable cells/well. To enhance spheroids formation, cells were transferred to cell culture medium with methylcellulose  [After isolation, cells were seeded in cell culture flasks and initially maintained for 24 h in DMEM enriched with 10% Fetal Bovine Serum (FBS), supplemented with 10 mg/ml gentamicin, 10,000 μg/mL streptomycin, 10,000 U /ml penicillin, and 4 mM L-glutamine . GCs wern 0,25%) , centrifTo evaluate the effect of the liposomal formulation of DMU-212 (lipDMU-212) in the spheroid model of GCs, the CellTiter-Glo® 3D Cell Viability Assay was performed. After 72 h of incubation of the formed spheroids, the tested compounds (DMU-212 and lipDMU-212) were added in the concentration range of 0-10 μM. DMU-212 was added from the stock solution prepared in DMSO. The final concentration of DMSO in cell treatment solutions was less than 0.1%. The liposomes solutions were added in the volumes, corresponding with the dilution factor as in the lipDMU-212 treatment. The spheroids were incubated with the compounds tested for 72 h. Then the assay was performed according to the manufacturer’s protocol. Briefly, equal volume of reagent was added into treated and untreated hGCs after 72 h, and incubated for 30 min. After that, the lysates were transferred to white 96-well plates for luminescence reading (Tecan Infinite 200 Pro).−6 M androstenedione as a substrate in the presence or absence of lipDMU-212  and FSH (10 ng/ml and 100 ng/ml) for 72 h. The basal secretion of estradiol and progesterone was also analyzed; hGCs spheroids were cultured as described above with the exception of the addition of androstenedione and FSH. After the 72 h incubation time, supernatants were collected and stored at −20 °C until assayed. Quantification of the hormones was assessed by measuring the optical absorbance at 450 nm using an ELx - 800 microplate reader  and KC Junior software (BioTek Instruments). The lower detection limits and the intra-, and inter-assay coefficients of variation for estradiol were 10.6 pg/ml, 8.7–9.2 and 6.9–14.9%, while those of progesterone were 0.14 ng/ml, 5.4–7.0% and 4.3–10.0%, respectively. To meet the assay dynamic range, the samples were diluted 1:100 and 1:600 for estradiol and progesterone measurements, respectively. Subsequently, the dilution factor was taken into account for the calculation of the concentrations. The results obtained from the experiment were expressed in pg/ml for estradiol and ng/ml for progesterone.The secretion of estradiol and progesterone was determined using enzyme-linked immunosorbent assays, according to the manufacturer’s protocols. Briefly, after 24 h of preincubation period, the spheroids were maintained in cell culture medium containing 2% FBS and methylcellulose at a concentration of 0.25%, 10GAPDH , HPRT (hypoxanthine phosphoribosyltransferase) and ACTB (β-actin). Reaction was performed according to the thermocycling protocol: preincubation at 37 °C (30 s); 3 step amplification  for 45 cycles; melting ; cooling at 37 °C (30 s). Target cDNA was quantified using the relative quantification method. For gene expression analysis, the 2 ∆∆Cq method was used [Samples from three independent experiments with three replicates per condition, collected after 72 h of treatment, were used for transcriptomic analysis using real-time quantitative polymerase chain reaction (RT-qPCR). Briefly, total RNA was extracted from human ovarian GCs using Tri Reagent as described elsewhere . RNA conwas used . OligonuAfter treatment, the hGCs spheroids were washed twice with PBS and the protein was extracted, using mix of RIPA lysis buffer and protease inhibitor, according to the manufacturer’s protocol. Subsequently, 10 μg of protein lysates were subjected to western blotting, resuspended in sample buffer, and separated using 4 to 20 % gradient Mini-PROTEAN® TGX™ gel by SDS-PAGE . The protein was then transferred using the Trans-Blot Turbo Mini 0.2 µm PVDF Transfer Pack set, according to the manufacturer’s instructions. Blots were then incubated with primary rabbit antibodies: CYP11A1  and HSD3B1  in Tris-Buffered Saline Tween 0,1% (TBST) with 5% fat-free dry milk powder overnight, at 4 °C. Following several washes in TBST, immunodetection was performed using anti-rabbit secondary IgG, HRP-conjugated goat antibody , utilizing the iBright CL750 Imaging System and iBright Analysis Software . The PVDF membranes were incubated with anti-β-Actin (13E5) rabbit antibody (HRP conjugated) . Finally, Super Signal West Pico PLUS Chemiluminescent was used to reveal the bands. The obtained blots were further analyzed using the ImageJ software .t-test post-hoc, while for the statistical analysis of RT-qPCR results Kruskal-Wallis and Tukey’s tests were used. Western blot bands were quantified using ImageJ software . A p-value < 0.05 was considered statistically significant, although other p-values were also reported.Statistical analysis was conducted using GraphPad Prism software v5.01. Data were collected in triplicate from at least three independent experiments. The results were shown as the means ± standard deviation (SD). Statistical studies for steroid hormones measurements were carried out using one-way analysis of variance (ANOVA) and the Student’s ca. 20% encapsulation efficacy. For further studies lipDMU-212 liposomes were diluted in cell culture medium.The characteristics of lipDMU-212 and non-loaded liposomes is presented in Fig. Cell viability studies showed no cytotoxicity of the liposomal formulation of lipDMU-212 in the concentration range of 0.0–10.0 μM on human ovarian GCs spheroids after 72 h of treatment. No pro- and anti-proliferating activities of non-loaded liposomes in hGCs spheroids were observed. Interestingly, a statistically significant increase in GCs proliferation after 72 h incubation with lipDMU-212 and DMU-212 at the lower concentrations was noted. As shown in Fig. p < 0.001 and p < 0.01), as compared to the controls for all doses tested for estradiol and progesterone assays, respectively. The most pronounced effects were observed after treatments with the highest concentration of lipDMU-212; the estradiol secretion increased to 2719,45 ± 247,94 pg/ml, compared to the control  while the progesterone level increased to 7319,41 ± 1263,04 ng/ml vs. 4024,21 ± 533,028 ng/ml for the control. There were no significant differences after treatment with non-loaded liposomes , as compared to the control.The estradiol and progesterone levels were measured in spent culture media after 72 h of treatments with lipDMU-212 at three different concentrations . As shown in Fig. p < 0.001), while progesterone secretion was not affected (p = 0.1420). lipDMU-212 caused a significant up-regulation in estradiol levels by ~30% (p < 0.001) after treatments with all three concentrations tested combined with FSH stimulation. Regarding progesterone level, only one treatment, lipDMU-212 at the concentration of 0.625 μM, significantly enhanced progesterone secretion . No statistically significant differences after treatment with non-loaded liposomes, compared to controls , were shown.As presented in Fig. p < 0.001; 4973,79 ± 485,27 ng/ml, p < 0.05, respectively) was observed. There were no statistically significant changes after treatment with non-loaded liposomes, compared to controls (p = 0.3908 for estradiol and p = 0.1646 for progesterone assessment).After 72 h of treatment, stimulation with FSH at the concentration of 100 ng/ml significantly increased the estradiol secretion  .To clarify the signaling pathway by which lipDMU-212 enhances steroidogenesis in hGCs, the expression of several genes involved in estradiol and progesterone secretion was studied Fig. . Increas−6 M androstenedione to the culture medium significantly upregulated the transcript levels of all 6 tested genes when compared to the control  and FSH increased the transcript levels of CYP11A1 ~ 9 and ~10 times compared to untreated control, while the addition of FSH was noted to increase the expression 8-fold. Similarly, lipDMU-212 in all concentration tested upregulated the expression of HSD3B1 ~ 10 times when the expression after treatment with FSH alone was increased 7-fold. CYP19A1 expression did not increase significantly after additional lipDMU-212 treatment compared to the 10 ng/ml FSH control.The addition of 10 ng/ml FSH and 10rol Fig. . FurtherStAR and HSD17B1 expression in a dose-dependent manner. CYP11A1 was down-regulated after treatment with the combination of 100 ng/ml FSH and lipDMU-212 at the concentrations of 0.039 μM, compared to the 100 ng/ml FSH control. There was no significant difference after combined treatment with 100 ng/ml FSH and lipDMU-212 at the concentrations of 0.625 μM and 5 μM. Interestingly, lower concentrations of lipDMU-212 when combined with 100 ng/ml FSH, decreased expression level of HSD3B1. Similarly, lipDMU-212 combined with FSH down-regulated the expression of CYP19A1 compared to the control treated with FSH alone. lipDMU-212 was found to enhance FSH activity and increase CYP17A1 expression, but only at concentrations of 0.625 μM and 5 μM.Similarly, in cells stimulated with 100 ng/ml FSH, the expression of the tested genes was significantly elevated compared to the untreated control Fig. . lipDMU-Western blot was employed to analyze the effect of lipDMU-212 and FSH treatments on levels of CYP11A1 and HSD3B1 proteins Fig. . Our stuIn cells treated with 10 ng/ml FSH alone and 10 ng/ml FSH combined with 0.039 μM lipDMU-212, we found an increase in CYP11A1 protein levels by ~30 and ~40%, respectively. Moreover, co-treatment with the highest concentration of lipDMU-212 and 10 ng/ml FSH as well as the treatments with 100 ng/ml FSH alone and combined with lipDMU-212 in all concentrations tested, enhanced the protein expression by ~100%.Due to the fact that DMU-212 is insoluble in water, in the study, DMU-212 was applied in the liposomal formulation. The carrier-mediated administration increases DMU-212 concentration in the water environment and prevents precipitation.2D cell culture models are assumed not to reflect in vivo situation entirely. Concomitantly, 3D is referred to as a bridge between flat in vitro and in vivo experimental parts. Among a plethora of benefits that the 3D culture holds are: appropriate cell polarity, more realistic distribution of nutrients and oxygen, and gene and protein expression levels . Hence, In the first stage of our study, we performed a cell viability assay, which showed no cytotoxicity of the liposomal formulation of DMU-212 at the concentration range of 0.0–10.0 μM on hGCs spheroids after 72 h of treatment. Interestingly, a significant increase in cell viability after treatment with both free DMU-212 and lipDMU-212 was noticed. The most pronounced effect was revealed in cells treated with free DMU-212 and lipDMU-212 at the concentration of 0.625 µm, which might suggest their prominent proliferative effect on hGCs. Hence, 0.625 µm of lipDMU-212 was selected for further studies. Moreover, based on the obtained results, we chose two non-toxic concentrations (0.039 and 5 µM) of the compound tested.GCs play numerous significant roles in ovaries, including the secretion of steroid hormones. In our research, we measured the estradiol and progesterone levels in hGCs treated with lipDMU-212. These analyses revealed that lipDMU-212 significantly enhanced both estradiol and progesterone secretion in a dose-dependent manner. To the best of our knowledge, there is no available data in the literature regarding how DMU-212 influences steroid hormones secretion. However, the activity of several hydroxylated and methylated resveratrol analogs on swine GCs has been studied . In the AhR, ARNT, CYP1A1, and CYP1B1 genes and clinical hyperandrogenism, follicular fluid testosterone levels, and disturbed folliculogenesis was observed [AhR, CYP1A1, and CYP1B1 genes are correlated with impaired folliculogenesis, the inhibition of AhR signaling pathway might be considered as a novel treatment approach for PCOS patients [It is widely known that ERα and ERβ receptors mediate the effects of estrogens and play pivotal roles in various physiological and pathological processes . Accordiobserved . Since hpatients . Interespatients . Hence, To determine the signaling pathways related to the steroidogenic activity of lipDMU-212, we performed RT-qPCR analyses targeting selected genes involved in estradiol and progesterone production.StAR. StAR is known to control cholesterol transfer within the mitochondria, which is the rate-limiting process in the secretion of steroid hormones [We noticed that lipDMU-212 remarkably evoked the up-regulation of the expression of hormones . It is chormones –47.CYP11A1, HSD3B1, CYP17A1, CYP19A1, and HSD17B1 in GCs treated with lipDMU-212. These genes are known to encode the entire steroidogenesis cascade in theca and granulosa cells. Hence, we suggest that the mechanism of steroidogenic actions of lipDMU-212 may be associated with their enhanced expression.In our study, we found a significant increase in the transcript levels of CYP19A1 is then higher in GCs compared to other tissues [via several distinct promoters, which alter its expression by recruiting different transcription factors [CYP19A1 promoter in GCs might be altered by several factors. Solak et al. have revealed that many phytoestrogens enhance the expression of CYP19A1 through the activation of ovarian-specific promoters I.3 and II [CYP19A1 is another enzyme that is crucial for estrogen synthesis as well as ovarian development. In GCs, aromatase catalyzes the demethylation of androstenedione and testosterone, leading to the A ring aromatization, which finally results in production of estrone and estradiol, respectively. Before menopause, estrogen is derived primarily from ovarian GCs and the placenta in healthy women . Moreove tissues . The exp factors . The pro factors , 51. Pre3 and II . Since DCYP19A1 and StAR transcript levels in GCs treated with lipDMU-212 in the lowest concentration tested (0.039 μM). Correspondingly, Morita et al. have demonstrated an increase in the expression of StAR and CYP19A1 in rat GCs, however, after treatment with resveratrol at a higher concentration (100 μM) [In the present study, the most pronounced increase was found in (100 μM) .FSH is a glycoprotein hormone produced by the anterior pituitary in response to gonadotropin-releasing hormone (GnRH) from the hypothalamus . One of It has been revealed that the activity of FSH in GCs depends on the cyclic adenosine monophosphate (cAMP), and the regulation of aromatase activity is associated with the activation of phosphatidylinositol 3-kinase (PI3K) and extracellular signal-regulated kinase (ERK) signaling pathways . In addip < 0.05) increased protein levels of HSD3B1 and CYP11A1 as compared to the control treated only with 100 ng/ml FSH. In this regard, our findings are in line with increased progesterone secretion after combined treatment with 0.039 μM lipDMU-212 and 100 ng/ml FSH.Moreover, the combined treatment with 10 ng/ml FSH and lipDMU-212 in all tested concentrations caused an up-regulation in estradiol levels by ~30% compared to cells treated with 10 ng/ml FSH alone, and a dose-dependent increase in estradiol production after the treatments with a combination of lipDMU-212 and 100 ng/ml FSH. Hence, it can be assumed that lipDMU-212 works synergistically and enhances the activity of FSH. Regarding progesterone levels, a notable increase was found after combined treatment with 10 ng/ml FSH and lipDMU-212 at the concentration of 0.625 μM. The higher concentration of FSH (100 ng/ml) combined with respectively 0.039 μM and 0.625 μM lipDMU-212 has also been shown to upregulate progesterone levels significantly. Surprisingly, we found that the expression of genes involved in the progesterone synthesis, CYP11A1, and HSD3B1, was decreased after combined treatment with 0.039 μM lipDMU-212 and 100 ng/ml FSH as compared to the 100 ng/ml FSH control. To clarify this issue, we investigated the levels of CYP11A1 and HSD3B1 proteins since mRNA expression has been shown to not always reflect in protein level due to the complexity of the regulation of gene expression . ContrarCYP11A1, HSD3B1, StAR, CYP17A1, and HSD17B1 expression after combined treatments with 0.625 μM as well as 5 μM lipDMU-212 and 10 ng/ml FSH. Furthermore, the elevated level of estradiol and progesterone of GCs treated with both 100 ng/ml FSH and higher concentrations of lipDMU-212 might be related to increased expression of StAR, CYP17A, and HSD17B1.To summarize, we suggest that the mentioned increase in the steroid hormones’ secretion might be correlated with the up-regulation of StAR and CYP19A1, which encodes proteins crucial for estradiol secretion. Thus, our results suggest that lipDMU-212 might be potentially beneficial in the treatment of disorders related to estrogen deficiency and hyperandrogenism, such as PCOS.Our study showed for the first time that liposomal formulation of DMU-212 enhanced both estradiol and progesterone secretion in human luteinized ovarian GCs. We reported a remarkable increase in transcript levels of steroidogenesis-related genes, especially


Integrins are transmembrane receptors that possess distinct ligand-binding specificities in the extracellular domain and signaling properties in the cytoplasmic domain. While most integrins have a short cytoplasmic tail, integrin β4 has a long cytoplasmic tail that can indirectly interact with the actin cytoskeleton. Additionally, 'inside-out' signals can induce integrins to adopt a high-affinity extended conformation for their appropriate ligands. These properties enable integrins to transmit bidirectional cellular signals, making it a critical regulator of various biological processes.Integrin expression and function are tightly linked to various aspects of tumor progression, including initiation, angiogenesis, cell motility, invasion, and metastasis. Certain integrins have been shown to drive tumorigenesis or amplify oncogenic signals by interacting with corresponding receptors, while others have marginal or even suppressive effects. Additionally, different α/β subtypes of integrins can exhibit opposite effects. Integrin-mediated signaling pathways including Ras- and Rho-GTPase, TGFβ, Hippo, Wnt, Notch, and sonic hedgehog (Shh) are involved in various stages of tumorigenesis. Therefore, understanding the complex regulatory mechanisms and molecular specificities of integrins are crucial to delaying cancer progression and suppressing tumorigenesis. Furthermore, the development of integrin-based therapeutics for cancer are of great importance.This review provides an overview of integrin-dependent bidirectional signaling mechanisms in cancer that can either support or oppose tumorigenesis by interacting with various signaling pathways. Finally, we focus on the future opportunities for emergent therapeutics based on integrin agonists.Video AbstractThe online version contains supplementary material available at 10.1186/s12964-023-01264-4. The first integrin is identified in 1986, as an integral membrane complex protein that plays a critical role in the association between the extracellular matrix (ECM) and the cytoskeleton . IntegriDuring the development process, integrin exerts an important role in cell rearrangement, migration and differentiation . In myobIn addition, crosstalk between integrins and growth factor receptors (GFRs) is also necessary for the normal development process. For example, the phosphorylation of vascular endothelial growth factor receptor-2 (VEGFR-2) is regulated by integrin αvβ3 to recruit bone marrow cells in angiogenic sites . MeanwhiIntegrins are an overarching regulator of pathophysiological progress, such as wound healing, tissue inflammation, tissue fibrosis, autoimmunity and metabolic disorders, in multicellular contexts of numerous diseases . IntegriDysfunction of normal cells acclimates the initiation and progression of malignancy . IntegriIntegrins control most cell survival, proliferation and differentiation. They act through mediating cytoskeletal linkage between cell adhesion to ECM and nuclear envelope as well as mechanotransduction. Given its fundamental function, integrins play positive or negative roles in numerous diseases . DiffereIncreasing evidence indicates that integrin-mediated RTK signaling pathways are also implicated in tumor migration and invasion through cell–cell adhesion and cell motility. The activation of integrins were initiated through binding interactions involving integrin α/β subunits, extracellular ligands, FAK, Src, and associated activation components like Crk-associated substrate (CAS), paxillin, and talin Fig. . Two impOutside-in signaling: This signaling mediates cellular responses induced by ligand binding to integrins. Integrin-induced adhesion is dependent on the relative activities of RhoA and Rac1. In the area of small nascent adhesions, Rac1 activation is accompanied by RhoA suppression. However, with large focal adhesion formation, RhoA but not Rac1 plays a major role. Activated integrin initiates two major downstream signaling pathways by activating FAK and Src kinases, gathered within the intracellular tails of β integrins. The FAK-Src complex in turn activates adhesion-associated adaptor proteins, such as paxillin and p130Cas subsequently binding and activation downstream adaptors . What’s Inside-out signaling: This signaling activates the ligand binding function of integrins. Rho- and Ras-GTPase encourage the binding of integrins to ligands by converting to a high affinity state. Ras, R-Ras and Rap1-GTPase may cause integrin activation which regulate cytoskeleton remodeling and integrin-dependent adhesion and migration on collagen . CrosstaBesides, the engagement of integrin and ECM protein activates Rho- and Ras-GTPase which in turn controls cell adhesion, migration and invasion by regulating the dynamics of the actin cytoskeleton  . AccordiCancer metastasis is the leading cause of cancer mortality. Metastasis is a cascading process including degrading the basement membrane and escaping from the primary tumor, accessing and surviving in the circulatory system, colonizing and proliferating in the parenchyma of the target organ , 43. TheAs a transmembrane receptor, integrin senses the content and stiffness of the surrounding ECM and biochemical signaling to mediate intracellular signal transduction, which occurs from adhesion sites or endosomes. The binding of ECM ligand  and specific cytoplasmic activators  to integrin lead to integrin-induced non-receptor tyrosine kinases activation. Studies show that the role of integrin in mechanotransduction and malignancy is bound to FAK-Src signaling activation . FAK is botulinum C3 exoenzyme, an inhibitor of Rho GTPase. This exoenzyme can also inhibit integrin αLβ3, αLβ2, α4β1 and α5β1, known to regulate lymphocyte and fibroblast activation [Rho GTPase, a family of small G proteins, plays a crucial role in modulating cytoskeleton dynamics, associated with cell polarity, motility, growth, proliferation and survival. In human, 20 Rho GTPase subfamilies have been verified and Rho (RhoA/B/C), Rac (Rac1/2/3/G) and Cdc42  are well-studied Rho GTPase . Rho GTPtivation . FurtherTransforming growth factor beta (TGFβ) plays crucial homeostatic roles in the pathogenesis of inflammation and fibrosis . There aDuring the wound healing of oral mucosa and skin epidermis, integrin αvβ6-TGFβ crosstalk is specifically increased. It is consistent with basement membrane regeneration, granulation formation and connective tissue remodeling . During As a stiffness-sensor molecule, integrin not only acts as the physical scaffold between the extracellular stiffness and intracellular actin cytoskeleton, but also transduces the extracellular stimulation into cell to mediate mechanotransduction . SeveralThe Hippo signaling is first discovered in Drosophila. Due to the high conservation of the Hippo pathway in mammals, an analogous Hippo core kinase cascade exists which consists of Mst1 and Mst2 (Hpo homolog), WW45 or Sav1 (Sav homolog), Last1 and Lats2 (Wts homolog) and Mob1 (Mats homolog). The kinase cascade phosphorylates YAP (Yki homolog) and leads to YAP/TAZ co-activator inactivation and this inactivation inhibits its nuclear-cytoplasmic translocation . SeveralWnt is an evolutionarily conserved signaling pathway involved in embryo development, tissue homeostasis and a variety of common diseases . Wnt couCellular metabolism under both physiological and malignant condition is extremely associated with extracellular matrix remodeling and integrin traffic, especially in epithelial and endothelial cells with fibroblasts , 101. CaIn clinic, integrin is considered as a double-edged sword. On the one hand, integrin signaling activation promotes the resistance of chemotherapy and radiotherapy. However, since integrin is transmembrane protein sensitive to pharmacological treatment, disease therapeutic strategies targeting integrin may achieve considerable clinical success. Three classes of drug can target integrin: monoclonal antibodies, peptides and small molecule inhibitors . To dateTargeting integrins beyond the ligand binding site , holds the potential to hinder integrin activation by either obstructing the orthosteric site or by maintaining the conformation in a low-affinity state. Within this context, monoclonal antibodies have been categorized into three distinct groups: inhibitory antibodies, activation-specific antibodies, and non-functional antibodies. The primary function of monoclonal antibodies lies in their capacity as competitive inhibitors, with a significant portion exerting allosteric inhibitory effects. These antibodies exhibit specificity for discrete regions within the integrin ectodomain, selectively targeting specific subunits or conformations.Integrin αIIbβ3 is specifically expressed in platelets and megakaryocytes, aggravating cardiovascular and autoimmune diseases. Integrin αIIbβ3 antagonist, Abciximab, is the first drug used in thrombosis-associated disease. Due to the remarkable efficacy of abciximab, eptifibatide and tirofiban are developed immediately afterward , 150. ThIntegrin αVβ3 is mainly expressed in vascular smooth muscle cells and macrophages. Integrin αVβ3 mediates cell-dependent inflammatory angiogenesis, which is essential for the pathology rheumatoid arthritis and related arthropathies. Emerging evidence suggests that as an RGD-binding subfamily, integrin αVβ3 is also associated with ophthalmology and osteoporosis . VitaxinIntegrin α4β1, α4β7 and αLβ2 are leukocyte-specific protein, which contribute to immune response in inflammatory bowel disease (IBD), multiple sclerosis (MS) and dry eye disease (DED). Integrin α4β1 binding to VCAM-1 controls leukocyte diapedesis . In addiIntegrin-ECM interaction triggers cell adhesion-induced drug resistance to chemotherapy, radiotherapy and targeted therapy. This unexpected drug resistance is due to the change of drug targets, substitutability of anti-apoptotic events and invalidation of cell death, especially in the treatment of cancer . In breaAt present, there are emerging clinic trials focusing on integrin-related nanoparticles for DNA/RNA therapeutics, expected to bring detection and therapeutic promise in the biomedical fields. Integrin α4β7 binding to VCAM-1 and MadCAM-1 induces homing to different tissues. MadCAM-1 is crucial for leukocytes adhesion to intestinal endothelium . A recenNatural product compounds targeting integrin and its regulatory components are also proposed as high efficiency, low toxicity and fewer side effects strategy for therapy. Curcumin is a traditional herbal medicine derived from Curcuma longa. Curcumin has been verified as an anti-inflammatory, antiproliferative, antioxidant and antitumor agent used in clinics . In non-Besides, approximately 260 other drugs targeting various integrin subtypes have already been studied preclinically in academic as well as industry clinical trials . FocusinIntegrin, as a crucial transmembrane receptor, with ‘outside-in’ ligand-binding specificities and ‘inside-in’ signaling properties is not yet fully explored. Abnormal activation of the ‘outside-in’ and the ‘inside-out’ bidirectional signaling machinery of integrin is closely associated with common diseases, such as cancer, chronic inflammation and thrombosis. Although studies on integrin are rapidly expanding, given the number of various integrin subtypes and the complexity of integrin-mediating signals, investigation of how integrin traffics and functions in the development and the course of diseases is still rewarding.It is already confirmed that the recruitment of talin and probably kindlin are essential for integrin binding to various ligands and activation, which establishes its mechanical sensitivity. Talin and kindlin induce integrin-ligand complex via binding the tail of integrin β, thereby disrupting the α/β ectodomain. Noteworthy, mechanical forces play a similar indispensable role in integrin regulating adhesion-related mechanotransduction. While at this point, talin and kindlin may not cause integrin heterodimer disruption-induced inside signaling but deliver mechanical forces to the integrin-ligand complex. However, whether kindlin plays a similarly crucial role to talin or exerts an extra potential role in integrin stabilization is still unclear and worth to be explored sequentially. When bound to ligands, integrin aggregates and subsequently participates in the actin skeleton network. The integrin-actin axis regulates several intracellular signalings, such as FAK/Src, Rho GTPase, Ras-ERK and Hippo pathway, as well as cellular behaviors, such as proliferation, migration, invasion, apoptosis, survival and cell stemness.Although numerous studies on integrin have been conducted clinically, however, the approved drugs and therapies are unsatisfactory. Currently, monoclonal antibodies, peptides and small molecules are applied in clinical trials, however, most of the target is on the integrin-ligand binding site or the ligand itself which brings little benefit. Hence, paying more attention to the crosstalk between integrin and other pathways is valuable and will bring unexpected novel opportunities for therapeutics. While the crosstalk between the integrative function of integrins and other pathways, such as TGFβ, Hippo-YAP/TAZ, Wnt-β/catenin and metabolism-related signaling, is initially obscure and complicated as the study progressed, it has become exquisitely clearer. Integrin-dependent regulation of TGFβ is involved in IBD and other immune responses, which could be considered as a therapeutic target. Vedolizumab, as an integrin α4β7 inhibitor, has been used in IBD therapy in clinics. It indicates that integrin-TGFβ signaling will bring increased therapeutic effects. YAP/TAZ nuclear translocation is controlled by integrin β1-Src signaling in skin cancer, basal layer cells as well as other cancer-associated fibroblasts. Dasatinib, as an Src inhibitor, retains YAP/TAZ in the cytoplasm by suppressing integrin β1-Src signaling. Nanoparticles targeting integrin α5 become a novel and effective strategy in metastatic TNBC via modulating Wnt-β/catenin signaling. Besides the synthetic drugs, natural plant compounds as well as nanoparticle-based delivery and RNA-interfered technology have already been used in clinics and enrolled in clinical trials probably due to their high efficiency, low toxicity and fewer side effects.The tumor microenvironment has been the leitmotif during the research on various cancers. Integrin-mediated mechanosensitivity is unignorable for mechanical cues transmission. In the future, studies on integrin should pay attention to the dependency of integrin and the translational biomarkers to ensure clinical efficacy. To be successful, we should take advantage of genetically engineered models to develop effective diagnostic and therapeutic technologies.


Parkinson’s disease (PD) is a chronic and progressive neurodegenerative disorder characterized by a complex pathophysiology and a range of symptoms. The prevalence increases with age, putting the ageing population at risk. Disease management includes the improvement of symptoms, the comfort of the patient’s life, and palliative care. As there is currently no cure, growing evidence points towards the beneficial role of polyphenols on neurodegeneration. Numerous studies indicate the health benefits of the family of urolithins, especially urolithin A (UA). UA is a bacterial metabolite produced by dietary ellagitannins and ellagic acid. An expanding body of literature explores the involvement of the compound in mitochondrial health, and its anti-inflammatory, anti-oxidant, and anti-apoptotic properties. The review organizes the existing knowledge on the role of UA in health and diseases, emphasizing neurodegenerative diseases, especially PD. We gathered data on the potential neuroprotective effect in in vivo and in vitro models. We discussed the possible mechanisms of action of the compound and related health benefits to give a broader perspective of potential applications of UA in neuroprotective strategies. Moreover, we projected the future directions of applying UA in PD management. Neurodegenerative disorders, including primarily Alzheimer’s disease (AD) and Parkinson’s disease (PD), represent a great threat to the ageing society. PD alone affects up to 3% of individuals over 65 years old and its prevalence is expected to increase by 50% by 2030 . PD is aMeanwhile, there is ongoing research and growing evidence of the beneficial role of dietary polyphenols on the neurodegenerative process. The neuroprotective role is related to antioxidant, anti-inflammatory, and anti-apoptotic properties . EllagitIn vivo studies on animal models explore the potential favorable effects of UA on ageing, muscle dysfunctions, cardiovascular disease, inflammatory bowel diseases (IBD), cancer, metabolic disorders, and brain health ,11,12,13Taking into consideration the issues mentioned above, this review aims to organize the existing knowledge and explore the future directions of the role of UA in PD management. The review emphasizes the analysis of the studies focused on the potential neuroprotective effect of this bacteria-derived natural compound, especially in the context of PD. As the data are limited, we also presented possible mechanisms of the action and related other health benefits of UA to support its potential of neuroprotective activity, and to give a broader perspective of potential applications of this compound in PD. Moreover, we discussed a few studies on the antiparkinsonian effects of pomegranate juice treatment which was associated with the presence of UA.Urolithins, a class of organic compounds containing benzo-coumarin scaffolds, are metabolites of ellagitannins and ellagic acid produced by the gut microbiota. These polyphenols are found in plant products, mainly in pomegranates, berries, walnuts, and almonds, but also in some tropical fruits, medicinal plants, and herbal teas ,18. The The catabolic pathway of ellagitannins and ellagic acid to urolithins includes lactone-ring cleavage, decarboxylation, and dehydroxylation reactions. Firstly, ellagitannins are hydrolyzed to ellagic acid by bacterial enzyme tannases. The extensive metabolism results in the creation of pentahydroxy-urolithin (urolithin M5), tetrahydroxy-urolithins , trihydroxy-urolithins (urolithins C and urolithin M7), dihydroxy-urolithins (urolithin A and isourolithin A), and monohydroxy-urolithin (urolithin B) . In receIntestinal conversion of urolithin precursors requires a specific gut microbiome, yet it is not entirely identified. It is believed that only 40% of individuals could naturally convert the polyphenolic precursors to UA . Three mEngagement in mitochondrial homeostasis is observed to be the coherent point in the in vivo model studies based on different types of animals and humans. One of the key modulators in the regulation of mitochondrial health is mitophagy. Mitophagy relates to the elimination of dysfunctional mitochondria in a selective autophagy process . The proUA’s beneficial role is related to the activation of several molecular pathways of mitophagy. In Caenorhabditis elegans, the administration of UA resulted in increased expression of genes responsible for mitophagy and autophagy ,29,35. TA substantial body of research revealed the anti-inflammatory activity of UA. As some diseases, including those related to ageing, are associated with chronic, low-grade inflammation, UA’s ability to modulate inflammatory response is especially investigated . SeveralAnother possible mechanism of action is the beneficial role of the compound against oxidative stress. Studies have explored several processes that may correlate with the reduction of oxidative stress markers ,8,12. PrBeneficial effects of UA, including antioxidant activity, are believed to be mediated through the activation of the Nrf2/Kelch-like ECH-associated protein 1 (Keap1) signaling pathway . Nrf2 isThe available literature supports the use of UA to modulate oxidative stress and alleviate cell damage through different mechanisms . As manyInterest has been generated in the ability of UA to regulate apoptosis. A recent review of the literature on this topic found that UA administration can affect both pro- and anti-apoptotic pathways with related anticancer or cell-loss-protecting effects, respectively . The compound inhibited the pro-apoptotic pathway in both in vivo and in vitro models ,48,49,50UA administration could also promote apoptosis. A growing body of literature suggests that UA is an anti-cancerogenic agent due to the modulation of the expression of genes, proteins, and signaling pathways involved in apoptosis induction ,13,20. IThe induction of cell deaths and expression of relevant proteins is believed to depend on the cell type ,12. ThisThe discussed data on the biological activities of UA has been gathered and presented below .The contribution of these mechanisms  to healtThe impact of UA has been observed in a variety of different health conditions, including in in vivo and in vitro models. The research provides evidence of anti-inflammatory, antiapoptotic, and antioxidant properties, as well as cardioprotective and neuroprotective effects of the metabolite . Many stThe research on the properties attributed to UA exposure or administration, including animal models, increased in the last decade. Experiments show improvement in muscle strength, mobility, and exercise performance both in natural ageing ,54,55 anMeanwhile, the cardioprotective effects of UA include a decrease in risk markers, diastolic cardiac function, and atherosclerotic lesions ,58,59. CUA also presented anticancer properties, including antiproliferation, apoptosis promotion, and anti-angiogenesis. UA-inhibited cell proliferation in both androgen receptor-positive and androgen receptor-negative cells in prostate cancer  due to tUrolithin A was demonstrated to be safe, bioavailable, and well-tolerated in the in vivo animal models but also in human experiments. The variety of potential health benefits tended to be related to the activation of mitophagy, improvement of mitochondrial homeostasis, and reduction of inflammation . HoweverFor several years, increasing evidence of the neuroprotective properties of UA has been observed. In middle-aged and older adults with mild memory complaints, drinking 8 ounces of punicalagin-rich pomegranate juice for 4 weeks caused a significant improvement in verbal and visual memory that correlated well with plasma UA-glucuronide concentration . Urine UThe above-mentioned favorable effects on the brain observed in humans with elevated UA levels in fluids make it a candidate preventive agent for neurodegenerative diseases. This idea is supported by in vivo findings that the administration of the compound improved cognition and reduced amyloid plaques, tau phosphorylation, neuronal loss, and neuroinflammation ,15,16,17C. elegans AD models, supplementation with UA decreased the amyloid-β plaque burden in hippocampus areas and the cerebral cortices, which could be related to improved cognition [The neuroprotective effect of UA administration was reported against neurodegeneration in a wide range of neuronal cell models, whereas the most considerable research was dedicated to AD and hypoxic-ischemic models. In AD model studies, the beneficial properties of UA were observed to be caused by the reduction of inflammation and improvement of mitochondrial health ,55,69,81ognition ,55,69,81ognition ,69. UA aognition . Moreoveognition . Due to ognition ,16,69. ASimilarly, the treatment with UA was shown to have protective properties against brain injuries. UA treatment improved neurological deficit scores and reduced infarct volume or brain oedema in mice subjected to middle cerebral artery occlusion and moderate contusion injury ,70,71. UPD is a chronic and progressive neurodegenerative disorder characterized by a range of motor and non-motor symptoms . The pat+-dependent deacetylase SIRT1 is a pro-survival protein inhibiting apoptosis and oxidative stress by regulating p53 and antioxidant defenses via FOXO family members, respectively. SIRT1 also interacts with PGC-1α to promote mitochondrial biogenesis and maintain mitochondrial homeostasis [Due to the increasing prevalence of the disease in an aging society, decreased life quality, and no available cure or treatment modifying its progress, there has been constant research for substances with neuroprotective properties ,84. In reostasis . In the eostasis . Thus, teostasis . Moreover, given the evidence, which we previously summarized from human and experimental studies, that PD is a prion disorder originating in the gut , the abiAnother aspect of the role of UA in PD management is its inhibitory effects on monoamine oxidase (MAO). As the elevated MAO activity is responsible for the inactivation of monoamine neurotransmitters, such as dopamine, UA can contribute to alleviating the symptoms in PD patients .Furthermore, UA could contribute to the overall beneficial impact reported for pomegranate consumption in PD models ,79,92. TA growing body of evidence suggests a variety of health benefits after UA administration. Numerous in vitro and in vivo experiments investigated the potential advantageous effects of the administration on ageing, muscle dysfunctions, cardiovascular disease, IBD, cancer, metabolic disorders, and brain health . The neuNevertheless, some study limitations should be acknowledged, such as shortages of disease models allowing only for an integrated view of a neuroprotective mechanism in terms of functional improvements with underlying changes in molecular signaling pathways, cell types, and brain networks. PD pathology is, however, diverse with multiple pathomechanisms involved; thus, no single model is currently able to recapitulate human disease . MoreoveFurthermore, to this day, there are numerous gaps in our knowledge of UA mechanisms of action, gut microbial ecologies, and specific impacts on health, including targeted organs and tissues, and primarily the effects on human organisms. Even though the compound can be produced from naturally occurring polyphenols in plant foods – ellagitannins and ellagic acid, the impact of direct UA supplementation on human health and its potential beneficial impact must be assessed. With regard to direct UA intake, the green synthetic access to new urolithin analogues  is a favMoreover, there is a need to identify the specific bacterial genera that can produce the metabolite to establish the specifics of urolithin-producing metabotypes and the related biochemical pathways and enzymes involved. In this context, UA was recently suggested as a potential biomarker of gut dysbiosis and disease stage in PD patients .Taken together, the data gathered herein support the health-promoting activity of UA. Regarding PD, UA-based intervention offers new strategies to improve the prevention, treatment, and even diagnostics of the disease.