The infiltration of naïve tissues by myeloid cells has been long related to their clearance and the physiological cell turnover, however, increasing evidence shows that they can additionally fulfill specific, non-immune functions in different tissues. There is also growing evidence to support that infiltrated granulocytes and monocytes respond to different environments by modulating gene expression and cytokine production, which in turn contribute to the normal function of the host tissue. This review will address the roles of immigrated myeloid cells in different tissues and their crosstalk with the host tissue environments. The generation of an effective immune response typically starts with the infiltration of damaged tissues by neutrophils and monocytes. These cells migrate to the inflamed tissues following cytokine and chemokine gradients that are mainly produced by other sentinel, tissue-resident, myeloid cells . NonetheNeutrophils originate in the bone marrow (BM) from hematopoietic stem cells that give rise to granulocytic progenitors, which in turn proliferate and differentiate to fully mature neutrophils. Around 90% of the neutrophil pool remains in the BM 4–6 days post-differentiation , availabIt has long been acknowledged that in mice the lung functions as a marginal pool for neutrophils under homeostatic conditions. Neutrophils accumulate in pulmonary microvessels –28 durinhighLyC6low, or non-classical, monocytes continuously monitor capillaries, arterioles and venules, crawling on endothelial cells in a LFA/ICAM-dependent mechanism and efficiently scavenging microparticles [A key step for the infiltration of most tissues by myeloid cells is their contact with the vasculature during the extravasation process. In the past decades, significant effort has been put into defining the cascade of events and the molecular cues implicated in leukocyte migration and extravasation during inflammation . Endothearticles , 37. Thearticles  and has articles . Using aarticles . Althoughi CD11clow phenotype in comparison to tissue-resident DCs [During the immune response, monocytes that migrate to damaged or inflamed tissue can also differentiate into DCs, which, upon sensing foreign particles, migrate to draining lymph nodes to initiate an adaptive immune response. Interestingly, however, DC progenitors migrate into tissues also during embryonic and postnatal development. They infiltrate the skin, mucosal surfaces and almost all solid organs in the body, ultimately giving rise to immature conventional DC . In the dent DCs . They fudent DCs –44. Thisdent DCs . Neverthdent DCs . AltogetIn addition to monocytes, it will be important to explore whether the neutrophils contained in the so-called “marginated pool”, within blood vessels, also interact with the endothelial cells to regulate their functions. Interestingly, pro-angiogenic properties of neutrophils have already been shown upon tissue engraftment and have been proposed to involve a distinct subtype of neutrophils . Christo− bacteria. Further, deficiency in eosinophils alters the development of Peyer’s patches, resulting in smaller patches that contain fewer cells, as well as fewer regulatory T cells and CD103+ DC, which are in turn involved in immune tolerance and the generation of IgA+ plasma cells [Over the past decade, the study of immunometabolic pathways in myeloid cells has attracted much attention. Migrating myeloid cells have high rates of metabolic activity to fulfil the energy requirements needed for transmigration into and function within tissues. Paradoxically, the metabolic activity of myeloid cells has been often overlooked in classical metabolic tissues, such as the adipose tissue or the liver. Although neutrophils are present at low levels in steady-state in the adipose tissue , their ima cells . Furtherma cells , and IL-ma cells  upon eosma cells , 59.Csf1op/Csf1op mice, which lack several tissue macrophage populations since early developmental stages, the ductal elongation during the mammary gland development is impaired [In the female reproductive system, neutrophils are recruited to the vaginal lumen and protect against infections, in a process known to be temporally regulated by sex hormones during the estral cycle . At the impaired . Severalimpaired , 69, 70.+ structures in the developing kidney [In the past decade, a profuse number of studies have been published regarding the ontogeny, heterogeneity and plasticity of tissue-resident macrophages , hig kidney . Interesg kidney .Myeloid cells, and in particular granulocytes, are recognized effectors of the immunomodulatory aspects during the steady-state, necessary to maintain tissue homeostasis Fig. . MoreoveThese novel roles of granulocytes in homeostasis raise interesting issues regarding their life cycle and migratory characteristics. The infiltration of naïve tissues by myeloid cells may follow specific patterns that differ from their migratory characteristics during inflammation. In fact, neutrophils show a probing migratory behavior in the steady-state, while their migration towards sites of injury and inflammation follow a directional mode . HoweverAn important outcome of this research focus will be the potential therapeutic approaches of modulating the infiltration of certain tissues by circulating myeloid cells to recover the normal function of damaged tissues. Future studies employing animal models of homeostasis and disease require the consideration of the growing knowledge on the heterogeneity of circulating myeloid cells and that some of these subsets might be predetermined to migrate selectively to different tissues. Single-cell sequencing technologies and mouse models of fate mapping are excellent tools to address these issues, as well as novel in vitro strategies to reprogram myeloid cells as potential cell-based therapeutic approaches.


Background: Diet is one of the leading factors contributing to the prevalence of non-communicable diseases, including neurodegenerative disorders. Dietary polyphenols, antioxidant components and anti-inflammatory agents of plant-based foods rich diets have been shown to modulate neuro-inflammation, adult neurogenesis and brain signaling, all of which are linked to cognitive function. As epidemiological evidence is limited and the results are contradictory, the aim of this study is to explore the association between dietary flavonoid intake and cognitive health among the adult population living in the Mediterranean area. Methods: The demographic and dietary habits of 808 adults living in southern Italy were analyzed. Food frequency questionnaires (FFQs) were used to assess dietary intake. Data on the polyphenol content in foods were estimated using the Phenol-Explorer database. The Short Portable Mental Status Questionnaire was used as a screening tool for cognitive status. Multivariate logistic regression analyses were used to assess the associations. Results: A significant inverse association between higher dietary intake of total flavonoids  and impaired cognitive status was found. Among individual subclasses of flavonoids, flavan-3-ols , catechins , anthocyanins  and flavonols  were associated with cognitive health. Among individual polyphenols, only quercetin was associated with cognitive health . Conclusions: The results of this study suggest that higher dietary intake of flavonoids may be associated with better cognitive health among adult individuals. In recent years, a significant rise in the prevalence of neurodegenerative diseases, including cognitive decline and dementia has been observed . Among mA common characteristic of dietary patterns and individual foods investigated is their richness in polyphenols, a wide range of molecules that naturally occur in plant foods: these compounds have demonstrated antioxidant and anti-inflammatory properties, which have been hypothesized to play a role in a number of chronic non-communicable diseases, including cancer  and cardThe MEAL study is an observational study aiming to investigate the association between nutritional and lifestyle habits characterizing the classical Mediterranean area and non-communicable diseases. The baseline data comprised a sample of 2044 men and women aged 18 or more years old. Details of the study protocol are published elsewhere . BrieflyAn electronic data collection was performed by face-to-face assisted personal interview, using tablet computers. In order to visualize the response options, participants were provided with a paper copy of the questionnaire. However, final answers were registered directly by the interviewer. The demographic data included gender, age at recruitment, highest educational degree achieved, occupation (specifies the character of the most important employment during the year before the investigation) or last occupation before retirement and marital status. Occupational status was categorized as (i) unemployed, (ii) low (unskilled workers), (iii) medium , and (iv) high (skilled workers). Educational status was categorized as (i) low (primary/secondary), (ii) medium (high school) and (iii) high (university). Physical activity was evaluated with the International Physical Activity Questionnaires (IPAQ) , which iThe dietary assessment was performed by the administration of two food frequency questionnaires  that have been previously tested for validity and reliability for the Sicilian population ,17. The The process of the estimation of habitual flavonoid intake has been previously described in detail . BrieflyCognitive health was evaluated using the Short Portable Mental Status Questionnaire (SPMSQ), a 10-item tool administered by the clinician in the office or in a hospital . The preFrequencies were expressed as absolute numbers and percentages, while continuous variables were expressed as means and standard deviations. Individuals were grouped based on variables of exposure (dietary flavonoid intake) and distribution of background characteristics were compared between groups. Differences were tested with the Chi-square test for categorical variables, ANOVA for continuous variables distributed normally and the Kruskall–Wallis test for variables distributed not normally. Energy-adjusted multivariate logistic regression models were used to test the association between dietary flavonoid intake  and cognitive status. A multivariate model adjusted for all other background characteristics  was also performed in order to test whether the observed associations were independent from the aforementioned variables. All reported P-values were based on two-sided tests, and the significance level was 5%. SPSS 17  software was used for all the statistical analyses.The main baseline demographic, socio-economic and health characteristics of the cohort distributed by the quartiles of energy-adjusted total polyphenol intake are presented in Mean intake and standard deviation of total, subclasses and individual flavonoid by cognitive status are presented in Multivariate adjusted analysis revealed a significant association between total dietary flavonoid and cognitive status; however, no clear linear trend was observed . Among iA stratified analysis by sex showed an association between impaired cognitive health and total flavonoids in men and women , flavonols in men , apigenin in women  and catechins in women , with no other significant results (data not shown).A subgroup analysis by age showed an association between impaired cognitive health and total flavonoids , flavanones , hesperetin  and naringenin  in individuals over 65 years old, with no other significant results (data not shown).The results of the present study showed that higher dietary intake of flavonoid and certain subclasses was associated with better cognitive health among Italian adults. In particular, higher dietary intake of flavan-3-ols, catechins, flavonols, anthocyanins and, among individual molecules, quercetin was positively associated with cognitive status. Total flavonoids were associated with better cognitive health in both men and women, with small differences between sexes; concerning elderly individuals, flavanones and the individual compounds investigated hesperetin and naringenin were also associated with cognitive health.Personnes Agées Quid) study, a cohort including 1640 subjects aged 65 years or older free from dementia at the baseline interview, flavonoid intake was associated with better cognitive performance at baseline and with a better evolution of the performance over 10-year follow-up [Dietary flavonoids have been linked to several mental health outcomes, including depression ,24 and sollow-up .Supplementation en Vitamines et Mineraux Antioxydants) study included 2,574 middle-aged adults followed for 13 years and found that among specific groups of flavonoids, both catechins and flavonols resulted in an association with better cognitive function, in line with our results [We also found an association between specific flavonoid subclasses in line with previous reports from the scientific literature. The SU.VI.MAX. ( results . Further results . In cont results .Prevención con Dieta Mediterránea) study reported that urinary polyphenols (measured as markers of dietary polyphenol consumption) were associated with better scores in immediate verbal memory [Among trials investigating dietary interventions to affect cognitive decline and function, the general results are confirming those reported in our study. A study conducted in the frame of the PREDIMED (l memory . Regardil memory . These fl memory .Several mechanisms have been hypothesized to support the effect of dietary flavonoids toward cognitive health, including both direct and indirect pathways of action. Flavonoids may indirectly affect brain health by modulating systemic inflammation and oxidative stress, key risk factors related to aging and playing a crucial role in the pathogenesis of neurodegenerative disorders . In factRecently, the direct mechanisms through which flavonoids could exert neuroprotective effects have gained great attention mainly due to the discovery that certain flavonoid metabolites pass the BBB and have a direct impact on neuronal cells ,41. AmonThe findings of the present study should be considered in light of some limitations. First, this study provided evidence from a cross-sectional analysis, and thus a causal relationship cannot be established. Second, all methods used to assess food consumption and dietary polyphenol intake, even though commonly used and valid as tools applied in nutrition epidemiology, are subjected to recall bias and provide only an estimation, while the real intake and especially exposure to metabolites cannot be estimated. Third, this study explored the association based on information of dietary flavonoid intake and thus the inter-individual variation in response to consumption of flavonoid and actual exposure to the flavonoid metabolites could not be taken into account. Finally, although validated, the SPMSQ may still suffer from limitations in sensitivity and specificity, especially for individuals with limited education.The findings of the present study suggest that higher dietary intake of flavonoids and their subclasses, in particular anthocyanins, flavan-3-ols, catechins and flavonols are related to cognitive health. Further studies are needed to better elucidate the nature of the association between habitual dietary intake of flavonoids and cognition in order to assess whether a protective effect might explain the retrieved findings. Based on the results of this study, future research should focus on selected flavonoids and also emphasize the inter-individual variation in response to consumption of polyphenols, thus investigating the association not only with their dietary intake but also their metabolites. As a future perspective, a focus on the gut microbiota composition should be considered as differences in microbial flora may impact polyphenol metabolite formation and bioactivity.


The indispensible role of mitochondria has been described over a century ago by Otto Warburg which has been serving the fields of cell biology and cancer biology immensely. Mitochondria are the principal site for vital mechanisms which vastly dictate the physiology. The intricacy of mitochondria’s role cancer have been noticed and well addressed in recent times. The underlying mechanisms are surfacing to unveil the nature of mitochondria and its participation in tumor cell motility and metastasis. This addressing may unravel novel therapeutic options. This review summarizes and reweighs the key aspects like underlying and emerging mechanisms which might be useful in designing novel chemotherapy.The Warburg effect has immensely succored the study of cancer biology, especially in highlighting the role of mitochondria in cancer stemness and their benefaction to the malignancy of oxidative and glycolytic cancer cells. Mitochondrial genetics have represented a focal point in cancer therapeutics due to the involvement of mitochondria in programmed cell death. The mitochondrion has been well established as a switch in cell death decisions. The mitochondrion’s instrumental role in central bioenergetics, calcium homeostasis, and translational regulation has earned it its fame in metastatic dissemination in cancer cells. Here, we revisit and review mechanisms through which mitochondria influence oncogenesis and metastasis by underscoring the oncogenic mitochondrion that is capable of transferring malignant capacities to recipient cells.  Tumor cell metabolic reprogramming dictates the difference between normal and tumor cells. Mitochondria play a major role in metabolic reprogramming:It has been shown that tumor mitochondria not only change their structure but also decrease the potential of oxidative phosphorylation (OXPHOS) and apoptosis . ConsideThe balance of mitochondrial fusion and fission is necessary for the regulation of various processes, including the quality of mitochondria, cell metabolism, cell death, proliferation, and cell migration, and is maintained by numerous mitochondrial-shaping proteins. Negative modulations or malfunctions in these processes resulting in changes in mitochondrial dynamics lead to diseases like cancer . MitochoMitochondrial dynamics also massively impact apoptosis. Studies have correlated them to dynamic homeostasis and tumor growth. Strikingly, signaling downstream of mutant KRAS in pancreatic cancers leads to mitochondrial fragmentation and increased activation of Drp1, processes that are required for KRAS-driven tumor growth in vivo. In addition, recent studies also suggest that mitochondrial dynamics are important for regulating metastatic phenotypes such as invasion and migration in breast and thyroid cancers .Examples of high-to-low expressions of Drp1 and Mfn1 have been implicated in metastatic breast cancer . MoreoveMammalian target of rapamycin complex 1 (mTORC1), which is a trigger factor in cancers, stimulates translation of mitochondrial fission process 1 (MTFP1), which is coupled to pro-fission phosphorylation and mitochondrial recruitment of DRP1 in melanoma cells . This shMitochondria have been well-known as crucial factors in various characteristics of cancer biology, including cancer development, metastasis, and drug resistance ,20. The Other aspects to be considered are cross-links with mitochondrial dysfunction and promotion of tumor cells metastasis. Epithelial–mesenchymal transition (EMT) enables cancer cells to obtain the migration abilities to move out of the primary tumor and translocate to new target organs ,35. EMT Deciphering the mechanisms of metastasis involving mitochondria is extremely important in establishing therapeutics. The tumor microenvironment plays a prominent role in the progression of cancer, and it has a similar role in cancer chemoresistance via a mechanism called mitochondrial transfer, which broadly favors further invasion and metastasis. Mitochondrial transfer occurs in cells that fail to perform aerobic respiration due to mtDNA malfunction . On the Ubiquitination of syntaphylin (SNPH)  is regarMitochondria can enormously influence malignant transformation and dictate the tumor plasticity of cancer cells and govern several mechanisms to address tough environmental conditions. Mitochondria are the major source of ROS utilized in OXPHOS. Mitochondrial enzymes, such as pyruvate dehydrogenase (PDH), a-ketoglutarate-dehydrogenase (a-KGDH), acyl-CoA dehydrogenase, and glycerol-3-phosphate dehydrogenase, are involved in ROS generation . The hugSimilar to the above, Sirtuin 3 (SIRT3) is involved in several key processes such as the response to oxidative stress and mitochondrial metabolism regulation. SIRT3, a NAD+-dependent mitochondrial deacetylase that promotes efficient oxidative metabolism, is a key regulator of mitochondrial ROS production and detoxification . LiteratMitochondrial fission is a process commonly implicated in tumor progression, where dynamin-related protein-1 (Drp1) is bonded to one of its receptors, mitochondrial fission factor (MFF), on the mitochondrial outer membrane. Mitochondrial fission has been widely correlated with cell death and mitochondrial integrity . MFF is Mitochondria are responsible for more than just energy production. Recently discovered mechanisms like mitochondrial unfolded protein response (mtUPR) and mitochondrial precursor over accumulation stress (mPOS) are paving new avenues for therapeutics and for understanding diseases better via mitochondria . A genetThe role of mitochondrial chaperones in the cell stress response is quite intriguing. Gamitrinib, for instance, is a mitochondrial targeted HSP90 inhibitor with potential anti-cancer activity. Glioblastoma cells induced with low doses of gamitranib revealed accumulation of unfolded proteins in the mitochondria and a stress response gene characterized by upregulation of chaperones, especially Hsp70. Utilizing this target (mitoUPR) in mitochondria, TRAP-1 or CypD were ablated by genetic or chemical inhibitors. This resulted in the downregulation of NF-kB and related genes. Furthermore, there was an upregulation of pro-apoptotic genes, which aided in mitochondrial-mediated cell death. NF-kB has a wide role in tumor promotion and endorsement in the metastatic environment. Additionally, NF-kB plays a major role in treatment resistance and poor outcomes in cancer. Hence, targeting mitoUPR aids in concomitant loss of NF-kB, which inturn results in exposing the tumors to apoptosis-based therapies . This clThe mitochondrial channels, characterized as either outer or inner membrane channels, are widely targeted in cancer therapies. The outer membrane channels include VDAC and the inner membrane channels include mtKATP, mtBKCa, mtIKCa, mtKv1.3, mtTASK-3, and the nonselective permeability transition pore (MPTP) . MitochoThe basis for mitochondrial ion channels being targeted is due to their role in cancer metastasis. A brief description of the role of potassium channels and their role in cancer progression is that channels like IKCa control OXPHOS. Inhibition of the channel has no or only minor effects on cell proliferation in the presence of glucose, but forcing the cells to generate ATP exclusively via oxidative phosphorylation by culturing them in galactose, allowed researchers to understand that inhibition of the channel decreased proliferation. Kv1.3 is another channel that modulates the cell cycle. Mitochondrial calcium fluxes have also been shown to regulate cancer proliferation. Additionally, calcium channels also drive proliferation. The constitutively active Ca2+transfer from the endoplasmic reticulum (ER) to mitochondria plays a crucial role inensuring viability of tumorigenic cells, and defects in this uptake into mitochondria lead to cancer cell death. The crosstalk between potassium and calcium channels isnot completely clear, but a putative K+/H+transporter, LETM1, has been shown on calcium influx/efflux into/from mitochondria, and silencing ofLETM1 promoted AMPK activation, cell cycle arrest, and autophagy .MPTP can be activated indirectly by different drugs eliciting changes in inner membrane potential, causing ROS production, or leading to calcium overload in the matrix. MPTP opening leads to rupture of the mitochondrial outer membrane (MOM), which contributes to cytochrome-c release, a process required for apoptosome formation and subsequent activation of effector caspases.The energy required for cancer cell migration, invasion, and metastasis is supplied by mitochondria. Suppression of the mitochondrial energy function can reduce the frequency of tumor cell metastasis and invasion . TargetiRecent advances in the field of cancer biology have delineated mitochondrial dysfunctions in cancer. Tumors take advantage of the modulated mitochondrial function to escalate invasiveness. Key mechanisms like respiration are not only essential for tumor growth but also for navigating tumor cells into the circulatory system, facilitating metastasis. Mechanisms connecting mitochondrial dynamics to the development of metastasis remain a puzzle. Moreover, the capability of the mitochondria in allowing cancer cells to adapt to stress should be considered. Consequently, mitochondrial biogenesis might answer these questions and unravel the mechanisms useful for therapeutic strategies for cancer treatment.


Recent developments in organoid technology are revolutionizing our knowledge about the biology, physiology, and function of various organs. Female reproductive biology and medicine also benefit from this technology. Organoids recapitulate features of different reproductive organs including the uterus, fallopian tubes, and ovaries, as well as trophoblasts. The genetic stability of organoids and long-lasting commitment to their tissue of origin during long-term culture makes them attractive substitutes for animal and in vitro models. Despite current limitations, organoids offer a promising platform to address fundamental questions regarding the reproductive system’s physiology and pathology. They provide a human source to harness stem cells for regenerative medicine, heal damaged epithelia in specific diseases, and study biological processes in healthy and pathological conditions. The combination of male and female reproductive organoids with other technologies, such as microfluidics technology, would enable scientists to create a multi-organoid-on-a-chip platform for the next step to human-on-a-chip platforms for clinical applications, drug discovery, and toxicology studies. The present review discusses recent advances in producing organoid models of reproductive organs and highlights their applications, as well as technical challenges and future directions. The female reproductive system is of utmost importance to a woman’s quality of life; it produces sex hormones and oocytes, provides the site for fertilization, and supports the fetal development , 2. DiseConventional in vitro systems that include two-dimensional (2D) and three-dimensional (3D) cell cultures have serious pitfalls that make them unsuitable for studying the female reproductive system. The 2D culture of immortalized cell lines or those derived from tumors in reproductive organs have led to numerous insights about the biology and physiology of the reproductive system; their advantages include reproducibility and ease of access. However, they lack many complex features of in vivo microenvironments, including a cell-cell/cell-extracellular matrix (ECM), the display of a relatively homogenous phenotype, and are not representative of in vivo cellular diversity. Because of genetic alterations in cell lines, they fail to recapitulate key features of native tissues as well as different active cell signaling pathways and function from those in the native cells . MoreoveThe recently developed organoid technology provides new in vitro models to serve as both tissue and organ proxies to bridge the gap between in vitro and in vivo . OrganoiOvarian cancer represents the fifth leading cause of cancer deaths in women in the US . Access Developing organoids from other ovarian disorders such as premature ovarian failure and polycystic ovary would provide a platform to study the mechanisms that underlie these diseases and enable the development of new drugs and treatments.The fallopian tube (oviduct or uterine tube) is a central organ of the female reproductive system that plays an essential role in oocyte maturation and selection, gamete and embryo transportation, sperm reservoir, control of polyspermy, fertilization and early embryonic development . It is bThere are two main methods for fallopian tube organoids, the air-liquid interphase (ALI) system and self-organizing organoids. Chen et al. have applied an ALI system for long-term culture of murine, porcine, and bovine (three common species used in reproductive biology) oviductal epithelial cells (OEC). Isolated OEC have been cultured within an insert submerged in a proliferation-inducing medium for 7 days to form a confluent layer, and then the medium was aspirated from apical compartment to establish an air-liquid interface that supported polarization and differentiation. Subsequently, during the differentiation period, the cells were grown in serum-free or serum-reduced media from the basolateral compartment for two or 3 weeks. During this period, the cells developed from a flat layer to a columnar-shaped layer that consisted of ciliated and secretory cells. The polarized structure was maintained for at least 6 weeks. After the differentiation period, the cells secreted an oviduct fluid surrogate that could support embryonic development up to the blastocyst stage without the addition of embryo culture medium . DespiteScientists reported a human iPSC reprogramming method for generating FTE organoids. In this study, different WNT and BMP signaling were modulated to successful direct differentiation of human pluripotent stem cells into Müllerian cells and subsequent pro-Müllerian growth factors were used to develop FTE precursors. Then, FTE precursors were cultured in Matrigel with phenol red where they formed an organoid structure. However, when cultured in Matrigel without phenol red, they became branched and formed an unorganized matrix . Phenol The described fallopian tube organoid models closely mimic normal physiology and architecture of the human FTE. Therefore, they provide promising models to study the biology and pathology of fallopian tubes with regards to screening technologies, cancer biology, and reproductive medicine . HoweverThe human endometrium is a dynamic tissue that undergoes cyclic changes in response to steroid hormones as well as paracrine and autocrine factors to be prepared for embryo implantation. Embryo implantation is a highly complex process that requires a receptive endometrium, a competent blastocyst, and a synchronized maternal-embryo dialogue . The endFor first time, Bläuer et al. developed and validated a culture condition in which normal human endometrium was cultivated as glandular organoids within Matrigel matrix in co-culture with stromal cells. However, this 3D culture system differed in principle and protocols from the currently adopted organoid concept . SuccessThe placenta is an extraembryonic organ that is essential for survival and development of the mammalian embryo. During implantation in humans, the trophectoderm layer of the blastocyst attaches to the endometrial epithelium and continues to differentiate into trophoblast subtypes: the cytotrophoblast (CTB), extravillous CTB (EVT), and syncytiotrophoblast (STB). Undifferentiated CTB cells grow through the STB to form cell columns and chorionic villi. The CTB cells at the tips of villi differentiate into EVT, which invade the deciduum. EVTs have two cell types: interstitial EVT cells that invade the decidualized endometrium and endovascular EVTs that invade and remodel the spiral arteries , 40. Mul. Turco et al. reported some growth cell clusters of first-trimester placenta were seeded into Matrigel drops and grown in a basal trophoblast organoid medium (TOM) composed of EGF, FGF2, CHIR99021, A83–01, and RSPO1 (Table showed that when villous CTB (vCTBs) were embedded in Matrigel that contained a defined cocktail of growth factors and signaling inhibitors, small cell clusters formed within several days of culture. These clusters rapidly grew and developed into organoids with irregular structures after 2–3 weeks [. Under these two well-defined conditions, organoid structures appeared and homogeneous trophoblast organoids were established within 10–14 days (two passages). To confirm the fetal origin of trophoblast organoids, they used microsatellite analysis and HLA typing. The generated trophoblast organoids were genetically stable after consecutive passages for more than 6 months and had mitochondrial function after cryopreservation. Finally, they provided evidence that the organoids were of clonal origin and that large organoids were formed from single cells within 3–4 weeks [CGB3, GATA3, and PSG6 [+ cells were found in trophoblast organoids, but under an EVT differentiation protocol suggested by Okae et al. [+ cells that migrated out of the organoids appeared and invaded in the 3D culture, and adhered to the plastic dish. Hence, the organoids mimicked the villous placenta in an anatomic, functional, metabolic, and endocrinologic manner [Recently, two independent research groups developed organoid platforms for culturing first-trimester CTBs in vitro–3 weeks . Under t–4 weeks . Identit–4 weeks . Principand PSG6 . The troand PSG6 . The secand PSG6 . HLA-G, and PSG6 . In basie et al.  and by uc manner , 27.FigCumulatively, it is suggested that the trophoblast organoid model is a promising tool to study human placental development and investigate trophoblast dysfunction and insufficiency. Developing an advanced culture system that contains both endometrial and trophoblast organoids would allow researchers to study the mechanisms that underlie maternal-fetal interactions during pregnancy.The discovery and development of a new drug is a complex, time-consuming, and costly process accompanied by a high rate of failure . DespiteAlthough animal models are frequently used for drug screening and toxicity studies , they arCFTR) gene, which results in an accumulation of abnormally viscous, sticky mucus in the gastrointestinal and respiratory tracts [PTEN, PIK3CA, AKT1) [An impressive proof-of-concept has been reported for organoid technology for drug testing in cystic fibrosis (CF) , 69. CF y tracts . Femalesy tracts . Over thy tracts , 71. HenA, AKT1) . These eThere are three types of human reproductive tract infections (RTIs) - STDs such as chlamydia, gonorrhea, syphilis, genital herpes, and human immunodeficiency virus (HIV); endogenous infections, which result from overgrowth of organisms normally present in a healthy women’s genital tract; and iatrogenic infections, which are associated with medical procedures such as induced abortions, poor delivery practices or intrauterine device (IUD) insertions , 73. RTISalmonella enterica [Clostridium difficile [Chlamydia trachomatis (C. trachomatis) serovars D, K, and E for long-term in vitro infection analysis and investigated its effect on epithelial homeostasis [C. trachomatis infection as an epigenetic modulator. C. trachomatis increases hypermethylation of DNA, which is an indicator of accelerated molecular aging [C. trachomatis infections in the development of tubal pathologies, including the initiation of high-grade serous ovarian cancer.Since organoids exhibit enhanced in vivo-like features, including apico-basal polarization, appropriate localization of cell adhesion molecules, cytokine production, responses to antimicrobials and microbial products, support of commensals, and/or susceptibility to infection, they are theoretically well-suited for infectious disease studies. Various pathogens that have been studied using organoids include Helicobacter pylori (stomach organoids) , 96, Salenterica  and Closifficile  in intesifficile . Kesslereostasis . The epiar aging . This orAlong with other infectious models, organoids also have limitations; the site of infection in most pathogens is the apical portion of the epithelium and delivery of the pathogen to the lumen of the cystic organoids is a challenge. To circumvent this limitation, microinjections have been used to deliver pathogens to the luminal side of the organoids.Organoid technology provided a research platform to study reproductive cancers, including ovarian and uterine cancers. Ovarian cancer is the deadliest gynecologic cancer worldwide with approximately a 35% five-year survival rate . It is rUterine malignancies represent the most common diagnosed gynecologic malignancy worldwide and the fourth most common malignancy in women . About 9. Subcutaneously injected cancer organoids generated a cell mass that recapitulated histological and molecular features of the primary tumor. Orthotopic engraftment of high-grade cancer organoids into the uterine horn generated a large, invasive, and highly proliferative mass that had the potential for peritoneal metastasis [Organoids derived from endometrial cancer patients recapitulate the morphology of the primary tumors and show similar immunohistochemistry (IHC) characteristics with the primary tumors. IHC for MUC1 and SOX17 on both tumor and normal organoids has confirmed their glandular origin , 26. Thetastasis . These eOrganoids have been generated from young women with cervical clear cell carcinoma (cCCC), an extremely rare subtype of cervical cancer. The optimized protocol for organoid generation from gynecological tumors was applied to produce cCCC organoids. cCCC organoids were expanded in the laboratory setting by a modified Matrigel bilayer organoid culture. These organoids were successfully cryopreserved and recovered after thawing. A few mutations were identified in cCCC organoids and CCC component following genomic analysis. Two of these mutations were detected both in cCCC organoids and CCC component. Moreover, development of a xenograft was confirmed following cCCC organoid transplantation into nude mice. Spheroids derived from cCCC organoids showed drug sensitivity and proliferative capacity upon exposure to the anti-cancer drugs commonly used for gynecological cancer  .However, additional studies are needed to clearly emphasize genomic heterogeneity and transcriptome patterns between organoids and their tumors of origin before replacing the exciting models with organoids, implement cancer-derived organoids for high-throughput preclinical screenings, and design targeted and personalized therapies.Endometriosis is a gynecological disorder that affects 10–15% of women worldwide and 30–50% of women with infertility. The disease is characterized by the growth of ectopic endometrial tissue outside the uterus . RecentlPatients with Asherman’s syndrome (AS), intrauterine adhesions (IUA), thin dysfunctional endometrium, genital tuberculosis, repeated implantation failure (RIF), and endometrial atrophy (EA) resistant to hormonal treatment are candidates for endometrial reconstruction . AS is a, and genetic and phenotypic stability over a long-term culture [null (NSG) mice [+ endometrial organoids survived in vivo after 6 weeks, expanded, and assembled into organized glandular-type structures in response to E2 and Prog treatment [With the emergence of organoid technology, there is an increasing interest in the use of organoids for regenerative medicine. Advantages of organoids include their ability to be created from a small biopsy sample, massive expansion in vitro culture , 25. Org culture , 4, 25.  culture . Upon tr culture . EngraftTaken together, these studies show that transplanted organoids have the ability to integrate into host tissue, maintain their self-renewal and self-organizing ability, and differentiate to functional tissue-specific cells. Although organoid technology is currently in its infancy, organoids provide a promising platform for harnessing stem cell for regenerative medicine and healing damaged epithelia of specific diseases. The successful engraftment of endometrial organoids and their ability to survive in vivo and differentiate to endometrial specific cells suggests that endometrial organoid transplantation could be a novel approach to treat disorders related to an inadequate endometrium. In addition, autologous transplantation can overcome the traditional hazards associated with allogeneic transplantation.In the past decades, several studies have demonstrated that efficacy and safety of drug treatments are sex-specific and the female sex tends to have a higher risk of developing adverse drug reactions (ADRs) compared with the male sex , 135. ThThe reproductive system-on-a-chip (repro-on-a-chip) is promising model to overcome these gaps and provide a new platform that incorporates reproductive sex hormones in preclinical research to address sexual dimorphism in biological processes, disease development, and improve sex-specific drug development . By deveRecent advances in organoid culture systems, including fallopian tubes, endometrium, testicular, and prostate organoids hold promise for the combination of male and female reproductive organoids with other technologies, such as microfluidics technology Fig. . This coThe current version of reproductive organoids faces some challenges; one limitation in the current organoid culture protocols is the requirement of animal-derived and chemically undefined extracellular matrices such as Matrigel. This could limit high-throughput screens and hinder direct transplantation because of medical legislation. Furthermore, tissues display highly anisotropic, heterogeneous, and nonlinear physical properties that depend on their composition, architecture, and pathology . HoweverDespite current limitations, organoids provide an exciting era as in vitro models of the reproductive system that allow for reproductive biology research, accurate modeling of reproductive organs, clinical decision-making, and individualized medicine for patients with gynecologic cancers and diseases such as endometriosis. In addition, bio-banked organoids can be used for drug screening or in vitro trials to predict individual patient drug responses. Advances on cutting-edge technologies will doubtless improve this novel tool for clinical application.


Sensorineural hearing impairment is the most frequent form of hearing impairment affecting 1–2 in 1000 newborns and another 1 in 1000 adolescents. More than 50% of congenital hearing impairment is of genetic origin and some forms of monogenic deafness are likely targets for future gene therapy. Good progress has been made in clinical phenotyping, genetic diagnostics, and counselling. Disease modelling, e.g. in transgenic mice, has helped elucidate disease mechanisms underlying genetic hearing impairment and informed clinical phenotyping in recent years. Clinical management of paediatric hearing impairment involves hearing aids, cochlear or brainstem implants, signal-to-noise improvement in educational settings, speech therapy, and sign language. Cochlear implants, for example, have much improved the situation of profoundly hearing impaired and deaf children. Nonetheless there remains a major unmet clinical need for improving hearing restoration. Preclinical studies promise that we will witness clinical trials on gene therapy and a next generation of cochlear implants during the coming decade. Moreover, progress in generating sensory hair cells and neurons from stem cells spurs disease modelling, drug screening, and regenerative approaches. This review briefly summarizes the pathophysiology of paediatric hearing impairment and provides an update on the current preclinical development of innovative approaches toward improved hearing restoration. For example, normal hearing is required for acquisition of vocal speech and normal neurocognitive development The electrical CI, put simply, is a medical device that encodes sound by directly stimulating the auditory nerve and thereby bypassing dysfunctional or lost hair cells. CIs consist of an external and an implanted component . The extSLC26A4 gene, children should avoid head- (e.g. due to contact sports) and baro (e.g. due to scuba diving)-trauma and wear head protection for activities with high risk of falling. The hypothesis for the progression of HI due to head trauma is the pressure propagation from the cerebrospinal fluid space into the cochlea via an enlarged vestibular aqueduct that is typically found in Pendred syndrome.Recently, optical stimulation has been demonstrated to fundamentally improve spectral selectivity, as light can be better confined in space  require a careful bioinformatic analysis ideally by a Mutation Mining Team of geneticists and then allows identification of the affected gene in the majority of cases. Although large WES and WGS data sets require the expertise of bio-informaticians, it is an unbiased high throughput method to identify de novo mutations. This holistic approach requires the close collaboration of specialists from different disciplines as medicine, genetics and biology but is an essential step towards personalized medicine.Currently, more than 150 non-syndromic gene loci have been identified in humans In-depth clinical phenotyping starts with otolaryngological and often also neurological examination, physiological and behavioural audiometric measurements of hearing and involves further disciplines as indicated by the results of genetic and clinical analysis. This typically allows to distinguish syndromic and non-syndromic disorders. Moreover, such analysis may already reveal specific pathophysiologies such as auditory synaptopathy or neuropathy with consequences for hearing rehabilitation ,26,27.In vitro analysis and generation of appropriate animal models of human hereditary HI allow the identification of localization, structure, and operation not only of the normal but also the defective protein. Animal models such as knock-out and knock–in mice, which provide access to molecular, cellular, and systems level analysis and preclinical testing of therapeutic strategies (TMC1 gene OTOF gene in DFNB9 Tmc1 and Otof gene function and rescue by virus-mediated expression of the wild-type coding sequence has lent further support to the hypothesized gene function and paved the way for future gene therapy (see below). This way, the requirement of TMC1 for mechanotransducer currents 2+ triggered fusion of synaptic vesicles and their replenishment . Apart from efforts to recover lost hair cells, major efforts have been undertaken to regenerate SGNs and the interested reader is referred to recent reviews on this topic Advancements in human induced pluripotent stem cell (iPSC) technology, genome editing technologies, as well as organoid development offer perspectives for future regenerative therapy. Human neural organoids are self-organized tissues generated by directed differentiation of human embryonic stem cells (ESCs) or iPSCs embedded in a matrix [recent review in ref. ex vivo studies of disease mechanisms. The same models can be used as drug screening platforms or as human preclinical models for gene therapy  normal acquisition of vocal speech. Molecular genetics in close interplay with paediatric otolaryngology and neurology assists counselling of parents and planning of the intervention. The coming decade promises exciting new opportunities for hearing rehabilitation of select patient populations via gene therapy such as for 5Future gene therapy approaches ideally will build upon late preclinical work in non-human primate models of human monogenic HI. Safe and efficient transduction of hair cells is another important development for future gene therapy. Likewise, the optogenetic cochlear implant builds on appropriate AAV administration to the spiral ganglion that avoids spread of virus and neurodegeneration. Converting electrical to optical power, scaling up the number of stimulation channels and their simultaneous activation, as well as higher energy demands per pulse pose engineering challenges for keeping the energy budget compatible with a day-long battery lifetime. For future regenerative approaches, we need to address the nature of HCs generated from human iPSCs. Do HCs in otic organoids resemble HCs in the cochlea or the vestibular organ? And if we attain to generate cochlear HCs do they resemble inner or outer HCs? Moreover, do otic organoids develop other structural features of the inner ear as the tectorial membrane and do the otic cysts that develop contain a solution resembling the endolymph? Another important question concerning OPC implantation studies, is whether the cells injected into the cochlea and give rise to HCs, SGNs and supportive cells. And if so, do they functionally integrate with resident cells?6Data for this Review were identified by searches of Pubmed using search terms such as ear, hearing impairment, deafness, hearing aid, cochlear implant, syndrome, gene, hair cell, synapse, channel, gene therapy, virus, optogenetics, stem cells, organoids, regeneration and combinations thereof. Only articles published in English between 1983 and 2020 were included.All authors performed literature search, writing and figure generation. All authors read and approved the final version of the manuscript.Tobias Moser is co-founder of OptoGenTech company. No conflict of interest for C.W. and M.P.Z.


Extracellular vesicles (EVs) are lipid membranous vesicles that are released from every type of cell. It has become clear that EVs are involved in a variety of biological phenomena, including cancer progression, and play critical roles in intracellular communication through the horizontal transfer of cellular cargoes such as proteins, DNA fragments, RNAs including mRNA and non-coding RNAs  and lipids. The most common cause of death associated with cancer is metastasis. Recent investigations have revealed that EVs are deeply associated with metastasis. Bone is a preferred site of metastasis, and bone metastasis is generally incurable and dramatically affects patient quality of life. Bone metastasis can cause devastating complications, including hypercalcemia, pathological fractures, spinal compression, and bone pain, which result in a poor prognosis. Although the mechanisms underlying bone metastasis have yet to be fully elucidated, increasing evidence suggests that EVs in the bone microenvironment significantly contribute to cancer progression and cancer bone tropism. Emerging evidence on EV functions in bone metastasis will facilitate the discovery of novel treatments. In this review, we will discuss the remarkable effects of EVs, especially on the tumor microenvironment in bone. The existence of membrane-enclosed structures found in the extracellular space has been known for more than 70 years; Chargaff and West first reported platelet-derived procoagulant particles after high-speed centrifugation of human plasma in 1946 ,3. In thEVs are lipid membranous vesicles that are released from every type of cell. They are conventionally classified into three main categories: exosomes (approximately 100 nm), microvesicles (approximately 1 μm), and apoptotic bodies (greater than 1 μm) . As prevIn the 1990s, Raposo et al. demonstrated that exosomes have effects on other cells by analyzing immune cells . ExosomeThe most common cause of death associated with cancer is metastasis. This process involves the growth and invasion of cancer cells, which leads to metastasis. The malignant transformation of cancer has been studied for a long time, and in 1889, Paget proposed the “seed and soil theory” . It has Some organs are common metastatic sites for cancer. Bone is the third most preferred site of metastasis, next to the lung and liver. Approximately 70% of patients with prostate cancer and breast cancer and 30–40% of those with lung cancer eventually develop metastasis in bone. Bone metastasis is generally incurable and dramatically affects patient quality of life. Bone metastasis can cause devastating complications called skeletal-related events (SREs), including hypercalcemia, pathological fractures, spinal compression, and bone pain, which result in a poor prognosis. Moreover, the median survival of bone metastasis from the time of diagnosis is low, including 12–52% for prostate cancer patients, 19–25% for breast cancer patients, and approximately 6–9% for lung cancer patients . TherefoAlthough the mechanisms underlying bone metastasis are yet to be fully elucidated, increasing evidence suggests that EVs in the bone microenvironment significantly contribute to cancer bone tropism. In this review, we will discuss the remarkable effects of EVs, especially on the tumor microenvironment in bone.Cancer-related EVs contribute to the formation and modulation of the microenvironment in the primary tumor site and the metastatic site. In addition to EVs from cancer cells, EVs from noncancerous cells, such as stromal cells, immune cells, inflammatory cells, vascular endothelial cells, and fibroblasts, have also been found to have significant impacts on cancer progression and remission. Many high-quality reports in recent years have revealed the interplay between cancer and the surrounding noncancerous cells and cells of distant metastatic sites through various forms of communication.Cancer cells deliver EVs to surrounding noncancerous cells, altering the properties of surrounding cells to maintain a tumor microenvironment that is favorable for tumor cell survival. Kosaka et al. showed that EVs derived from breast cancer cell lines that have high metastatic abilities contain a large amount of miR-210, which induces angiogenesis, and they affect vascular endothelial cells. EV-miR-210 acts on vascular endothelial cells and inhibits EphrinA3 expression, inducing tumor angiogenesis. Furthermore, the action of these EVs also promotes lung metastasis . ConversMoreover, cancer cells communicate with each other via EVs to gain metastatic capacity. Le et al. demonstrated that EVs from breast cancer cell lines with high metastatic potential could enhance the metastatic potential of breast cancer cell lines with low metastatic potential by providing miR-200 family members .EVs secreted by cancer cells affect not only surrounding cells and fellow cancer cells but also distant cells. It has become clear that cancer cells use EVs to create an environment that facilitates tumor cell survival in the metastatic site before the cells metastasize. The environment in which this occurs is called the premetastatic niche. In a pioneering report, the authors found that EVs secreted by melanoma cells acted on cells in the bone marrow to guide them to the metastatic lung, causing the displaced cells in the bone marrow to form a premetastatic niche . Since tBefore proceeding to the main topic of this review, bone metastasis and EVs, we will describe the normal bone microenvironment and EVs. The bone microenvironment consists of three main cells: osteoclasts, osteoblasts, and osteocytes. Moreover, bone marrow stromal cells (BMSCs), such as mesenchymal stem cells (MSCs), are also deeply associated with the formation of the bone microenvironment. These cells maintain the normal structure and homeostasis of the bone, exchanging various messengers with each other. Osteoclasts are derived from monocytes, differentiate, fuse during maturation and resorb existing bone. Osteoblasts are derived from MSCs in the bone marrow and are responsible for the formation of new bone.Osteoclast differentiation occurs under the influence of macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor kappa-B ligand (RANKL), which are secreted by stromal cells, osteoblasts, osteocytes, and immune cells . The binOther models of coupling mechanisms have been proposed. Zhao et al. focused on the increasing expression of EphrinB2 during osteoclast differentiation and found that osteoclast EphrinB2 affects the receptor EphB4 on osteoblasts to promote osteoblast differentiation and, at the same time, suppress osteoclast differentiation in vitro and in vivo . It has Recently, however, EVs have been shown to play a very important role in maintaining this beneficial cycle as messengers of cell-to-cell exchange, in addition to the cytokines and cell adhesion molecules . If the Protein characterization of EVs isolated from osteoclasts revealed that they express tumor susceptibility gene (TSG) 101, heat shock protein (HSP) 70, β-actin, CD 63, and epithelial cell adhesion molecule (EpCAM) . Sun et Proteomics analysis of osteoblast-derived EVs revealed that proteins from osteoblast EVs are closely involved in the eukaryotic initiation factor (EIF) 2 pathway . EIF2 isEVs from BMSCs can also participate in bone remodeling by directly regulating osteoblast proliferation and activity . MSC-derThere have been a considerable number of reports on EVs in the development and dissemination of cancer. However, the role of EVs in the development and exacerbation of bone metastasis has not been widely reported. Bone metastasis is said to be a complex cascade of processes . The harUnfortunately, the specific EV components that determine bone metastasis have not been identified, but EVs are involved in any of these processes, and EVs associated with cancer cells are key players in carrying cargo that adversely affects bone metabolism and obstructs the vicious cycle in the bone microenvironment. The goal of this review is to consolidate the current findings on bone metastasis-related EVs, which are listed in a table. In the following sections, we summarize the research findings with a focus mainly on prostate cancer , breast Bone is the most frequent metastatic target in men with advanced prostate cancer (PCa). However, the mechanisms that favor to the more frequent development of bone metastasis than soft tissue metastasis in PCa are not well defined. Dai et al. reported that primary PCa cells educate the bone marrow to establish a premetastatic niche through primary PCa cell-derived EV-mediated transfer of pyruvate kinase M2 (PKM2) into BMSCs and the subsequent upregulation of CXCL12, a chemokine involved in cell migration . This reIt is also well known that PCa mainly exhibits osteosclerotic bone metastasis. In fact, bone metastasis in PCa has both osteogenic and osteolytic aspects. EVs involved in PCa may therefore affect osteoclasts and osteoblasts. At the leading edge of tumor invasion in bone metastasis, bone resorption occurs first and is thought to be necessary for subsequent bone formation. First, bone resorption is facilitated by mature osteoclasts. Inder et al. showed that EVs derived from the human PCa cell line PC3 were internalized by murine osteoclast precursor RAW264.7 cells and primary human osteoblasts (hOBs) in vitro, stimulating osteoclastogenesis 37-fold and hOB proliferation 1.5-fold, respectively. This phenomenon was not observed in PC3 cells transfected with a vector containing caveolae associated protein1 (CAVIN1), also known as polymerase I and transcript release factor (PTRF). The authors previously demonstrated that CAVIN1 expression suppressed PC3 tumor growth and metastasis in vivo concomitant with changes in the PC3-EV proteome. They showed that CAVIN1 was not detected in EVs, and then Cavin1-mediated pathways could attenuate metastatic PCa through its associated EV cargo recruitment . On the Subsequent bone formation is performed by osteoblasts. Itoh et al. reported that EVs derived from either the metastatic PCa cell line PC3 or DU145 cell cultures significantly promoted osteoblast differentiation, while EVs from the androgen-sensitive human prostate adenocarcinoma cell line LNCaP did not have this effect. Hormone refractory PCa cell-derived EVs containing ETS1, which is an osteoblast differentiation-associated transcription factor, were transferred into osteoblasts and induced differentiation . Ye et aFurthermore, EVs derived from osteoblasts also have an effect on prostate cancer cells. Morhayim et al. demonstrated that osteoblasts promote PCa cell (PC3) proliferation by the level of mineralization stage-specific proteins in osteoblast-secreted EVs. This group claimed that the stages of differentiation and the mineralization condition were also among the factors that influenced the nature and abundance of EV cargo. Bioinformatics analyses of osteoblast-derived EV proteomes and EV-regulated PCa gene expression profiles showed that these factors converged on pathways involved in cell survival and growth. This finding was verified by in vitro proliferation assays in which osteoblast uptake of EVs led to a 2-fold increase in PC3 cell growth compared to that of the cell-free culture medium-derived vesicle controls .It is well known that breast cancer (BCa) can also easily translocate into the bone and mainly exhibits osteolytic metastasis. Unexpectedly, however, there have been few reports on the exchange of EVs between BCa cells and bone cells, whereas there have been many reports on the involvement of EVs in brain or lung metastasis.Tiedemann et al. showed that BCa cell-derived EVs containing L-plastin mediates osteoclast activation, which in turn created an osteolytic microenvironment that was favorable to breast cancer growth . Liu et There have been some other reports examining EVs derived from BCa cells, which were not limited to those specific to bone metastasis. Wen et al. demonstrated that EVs from highly metastatic BCa cells potentially contributed to establishing a premetastatic niche that was also able to promote metastasis by altering immune cell activity. BCa-derived EVs can suppress CD8 and CD4 T-cell proliferation and reduce NK cytotoxic activity against target tumor cells. This phenomenon also occurs in the bone tumor microenvironment. The acquisition of the ability of tumor cells to escape immune surveillance is also an important factor that promotes metastasis .Most of the reports on the involvement of EVs secreted by BCa cells in bone focus on the dormancy of BCa cells in the bone marrow. Cancer dormancy is generally defined as the arrest of tumor growth in the primary or metastatic site due to cancer cell quiescence. During this period, cancer cells acquire drug resistance until stimulated to awaken, ultimately causing cancer relapse and metastasis. Ono et al. described that coculturing bone metastatic MDA-MB-231 cells with bone marrow-mesenchymal stem cells (BM-MSCs) suppressed tumor cell proliferation and sensitivity to chemotherapy, suggesting the acquisition of a dormant phenotype by tumor cells . InteresLung cancer (LungCa) also has a tendency to induce osteolytic metastasis. Non-small cell lung cancer (NSCLC) cells are said to release factors that alter bone remodeling and increase osteoclast activity through shifting the normal balance of RANKL, RANK and OPG .Taverna et al. showed that NSCLC cells secrete EVs containing epidermal growth factor receptor (EGFR) ligand and amphiregulin (AREG), which stimulate osteoclastogenesis . The relIn contrast, Valencia et al. showed that EVs isolated from the miR-192-overexpressing metastatic human adenocarcinoma A549 cell line could reduce in vivo osteolytic lesions. Altering the cargo of cancer cell-derived EVs via the overexpression of a single anti-angiogenic miRNA (miR-192) repressed tumor-induced angiogenesis, which led to a reduction in the number of bone metastatic lesions .Cancer bone metastasis has been studied in various types of cancers. EV research in primary bone tumors, such as multiple myeloma and osteosarcoma, is also active. These studies could provide crucial hints in the field of bone metastasis. It is important to discover the characteristics that are common to all types of cancer and are specific to bone metastasis. This will lead to future critical treatments that provide a glimmer of hope for the many patients suffering from bone metastasis.Unfortunately, to the best of our knowledge, there are currently no reports of the use of EVs to treat bone metastasis in cancer. However, as the relationship between EVs and bone metastasis becomes clearer, it is possible that more research will be conducted on therapies specific to bone metastasis. In this section, the basic idea of the therapeutic application of EVs and the latest trends will be discussed. There are two main ways in which EVs can be used to reduce the progression of cancer, including bone metastasis: using EVs as DDS carriers or targeting EVs themselves as therapeutic targets . In the In recent years, EVs have been actively investigated as DDS agents . EVs areAmong EVs, especially small EVs, their small size and long half-life in circulation make them ideal drug-delivery vehicles . Thus, TOhno et al. demonstrated that EVs can efficiently deliver miRNAs to breast cancer cells expressing epidermal growth factor receptor (EGFR) . The GE1In addition, in recent years, bovine milk-derived EVs have become a hot topic as drug delivery vehicles ,85. BoviThere are three possible therapeutic strategies for targeting EVs themselves: inhibition of EV production, elimination of circulating EVs, and inhibition of EV absorption .Some reports have described the efficacy of inhibiting EV production to suppress cancer progression. One of the notable targets in this strategy is ceramide, a type of lipid identified in the synthetic pathway that is synthesized by neutral sphingomyelinase (nSMase) 2. Previous attempts have been made to inhibit ceramide production by knocking down nSMase2 or using its inhibitor GW4869 ,86,87. KEliminating circulating EVs could be a relatively new treatment strategy in cancer patients. A method to remove EVs using cancer-specific antigens has been reported . In thisIt has been recently reported that the absorption of EVs is induced by various mechanisms, such as clathrin-mediated endocytosis, phagocytosis, micropinocytosis, and fusion of the plasma and endosomal membranes , and invAlthough therapeutic strategies targeting EVs released from cancer cells are effective, several problems remain before clinical application is feasible. Molecules identified in previous studies play important roles in multiple cellular biological phenomena and can adversely affect normal cell functions when downregulated in normal cells. In other words, the identification of cancer-specific molecules is essential for the future development of EV-targeted anticancer drugs. As we have seen in this review, various factors have complicated mechanisms in the bone microenvironment. The search for specific factors that determine bone metastasis is underway. Some of these factors may have be potential targets for EV therapy.It is well known that the fossil record, which includes skeletal samples of human beings, is used in anthropology. Skeletons are special structures with universality and stationarity that transcend time and space. However, bones are also fluid structures that are constantly metabolized. It is undoubtedly true that bone metastasis is unique and distinct from visceral metastasis. Researchers worldwide have been struggling to understand the mechanisms of bone metabolism and overcome or prevent bone metastasis.Currently, the most prevalent treatment for bone metastasis of PCa, BCa, and LungCa involves the bone modifying agents bisphosphonates and denosumab. Bisphosphonates are antiresorptive agents that block pathological bone resorption by inhibiting osteoclast activation and function . DenosumSimilar to that of cytokines and cell adhesion molecules, the function of EVs in the cell-to-cell communication network is becoming clearer. There is no longer any doubt that EVs are key players in that network. Emerging evidence on EV functions in bone metastasis may allow the discovery of novel treatments. EV research is still in the developmental stage. Many problems remain before clinical application can be achieved; however, we can see the potential of EVs as powerful and promising tools for cancer therapy. It is quite possible that the next emerging therapy for bone metastasis would be a drug targeting EVs or a drug using EVs. In the near future, EV-based therapy will help to dramatically overcome bone metastasis. Above all, we need to understand the messages exchanged between the cells that form the tumor microenvironment in bone. We hope this review will help with that first step.


Studies using whole blood platelet aggregometry as a laboratory research tool, provided important insights into the mechanism and modulators of platelet aggregation. Subsequently, a number of point-of-care (POC) platelet function tests (PFTs) were developed for clinical use, based on the concept that an individual’s thrombotic profile could be assessed in vitro by assessing the response to stimulation of platelet aggregation by specific, usually solo agonists such as adenosine diphosphate (ADP), collagen and thrombin. However, adjusting antiplatelet medication in order to improve the results of such POC PFTs has not translated into a meaningful reduction in cardiovascular events, which may be attributable to important differences between the POC PFT techniques and in vivo conditions, including patient-to-patient variability. Important limitations of most tests include the use of citrate-anticoagulated blood. Citrate directly and irreversibly diminishes platelet function and even after recalcification, it may result in altered platelet aggregation in response to ADP, epinephrine or collagen, and interfere with thrombin generation from activated platelets. Furthermore, most tests do not employ flowing blood and therefore do not assess the effect of high shear forces on platelets that initiate, propagate and stabilize arterial thrombi. Finally, the effect of endogenous thrombolysis, due to fibrinolysis and dislodgement, which ultimately determines the outcome of a thrombotic stimulus, is mostly not assessed. In order to accurately reflect an individual’s predisposition to arterial thrombosis, future tests of thrombotic status which overcome these limitations should be used, to improve cardiovascular risk prediction and to guide pharmacotherapy. Commonly used point-of-care platelet function tests have many limitations and do not accurately reflect the in vivo situation.To assess overall thrombotic status, a global thrombotic stimulus (like high shear) should be used, rather than assessing the response to individual agonist(s) .Native blood is preferable to citrated blood, as the latter may alter platelet aggregation and thrombin generation.Accurate thrombotic status assessment should also assess endogenous thrombolysis.Tests of thrombotic status which overcome these limitations may better reflect physiology, improve cardiovascular risk prediction and help tailor pharmacotherapy., and the development of an instrument to measure aggregation quantitatively, began a flourishing era in thrombosis research [.[The discovery that platelet aggregation is inducible in vitroresearch –3. Plateresearch –6, and mresearch  have beesearch [., 9. Despsearch [.–13. In msearch [., 15. Morsearch [.. Amongstsearch [.. However. One can postulate that this was due to either trial design, assessment of low risk populations, variability in the HTPR definition and possible failure to adequately overcome HTPR. However, it is also possible that POC PFT lacked direct pathological relevance to arterial thrombosis in vivo . Leucocytes and erythrocytes are important mediators of thrombus stability, which impart both structural stability as well as mediate resistance to fibrinolysis. Neutrophils release proteases such as elastase and cathepsin G, which exert a plasmin-independent fibrinolytic effect, including inactivating the major plasmin inhibitor α2-PI . Such pr2-PI .Activation of coagulation is necessary for fibrin formation, which is essential for cross linking platelets within the thrombus, as well as the stimulation of fibrinolytic enzymes such as plasmin and antithrombin. Not only is it important to assess the pro-coagulant tendencies, but also to consider the effectiveness of endogenous thrombolytic/fibrinolytic pathways, which can determine the outcome of a pro-thrombotic stimulus .−1, and may reach 100,000–1 [The characteristics of blood flow, including laminar flow, pulsatility, arterial pressure and local haemodynamic/shear forces, impact the development and stability of the thrombus. Rheological disturbances have an important role in promoting occlusive thrombus formation, as thrombus propagation and vessel occlusion tend to occur on plaques causing marked disturbance of laminar blood flow, rather than on non-stenotic lesions . In seve00,000–1 . High shThe healthy endothelium can exert antithrombotic effects, through release of prostacyclin and nitric oxide, potent inhibitors of platelet and monocyte activation. Endothelial cells can secrete tissue plasminogen activator, which breaks down fibrin, as well as von Willebrand factor (vWF), which can initiate platelet aggregation and contribute to blood coagulation . While eAdvances in research in the past few decades have shed light on the mechanism of occlusive arterial thrombosis, revealing important new determinants that were previously unrecognised, including altered flow conditions at high shear rates (shear gradients), local thrombin generation by shear-activated platelets, the contribution of coagulation factors and NETs, and the importance of fibrin clot stability and fibrinolysis. This new knowledge has relegated to secondary significance the role of specific agonist-induced platelet aggregation in mediating thrombus formation. However, these newer determinants of thrombosis are not reflected in or assessed by many of the POC tests commonly in use in clinical settings.Whilst most tests use whole blood, some such as Platelet Works, the IMPACT: Cone and Plate(Let) Analyzer  (CPA)  and Multiplate Analyzer  although employing whole blood, give results that reflect only platelet aggregation specific to a particular platelet agonist, and do not take account of the contribution of other blood constituents to thrombus formation.Many physiologically-relevant agonists  can cause platelet activation, adhesion and aggregation both in vivo and in vitro. However, in a severely stenosed artery, it is shear-gradient dependent platelet aggregation mechanism which drives thrombus formation –25 while2+), reducing the concentration in the blood sample from ~ 1.2 to ~ 0.1 mmol/L. Citrate anticoagulation is a very convenient way to handle blood samples, allowing storage for a few hours and transfer of samples from the patient to the laboratory for subsequent analysis. However, the “convenience” of anticoagulation comes at a price; namely the rendering of the blood sample highly non-physiological, such that it lacks relevance to the function of whole blood and platelets under thrombotic or haemorrhagic conditions in vivo. The serious drawbacks of citrate anticoagulation have been known for a long time, but there has been no viable alternative proposed and it has not been considered possible to overcome the logistic need for storage of blood prior to analysis. Citrate is added to blood in a fixed concentration and the final plasma iCa concentration after citrate is slightly above the threshold required for activation and aggregation of platelets without triggering blood coagulation. Citrate anticoagulation assumes that plasma iCa concentration in individuals is standard, but this is not the case [.[Most POC PFTs in current clinical use are performed on anticoagulated blood. There are very few exceptions, notably the Global Thrombosis Test and Plateletworks, which employ native non-anticoagulated whole blood. Sodium citrate is the most commonly used anticoagulant for POC PFTs and for platelet aggregometry in platelet-rich plasma or in whole blood. Citrate acts by chelating extracellular ionized calcium  were independently associated with increased levels of fibrinogen and a higher risk of cardiovascular disease . FurtherThe aforementioned adverse effects of citrate highlight the need to consider alternative anticoagulants in POC PFTs. However, heparin enhances primary aggregation and induces aggregation by potentiating platelet release reaction. Akin to that observed in citrated blood, platelets tend to clump in samples collected into unfractionated or low molecular weight heparin, precluding the use of these as anticoagulants in PFTs –49. BecaArterial flow and haemodynamic forces have dramatic impact on thrombus formation, coagulation and lysis of an arterial thrombus , 50. In −1), conformational extension of vWF and its interaction with platelets initiates thrombus formation by causing an explosion of thrombus growth with rapid platelet aggregation, independent of activation [−1 were 12 times more likely to experience cerebrovascular ischemic events than those with lower shear rates [The effect of high shear forces on platelets in initiating, propagating and stabilizing arterial thrombi is two-fold. At pathological shear rates  during recirculation, so called “platelet hammering”, can trigger persistent platelet aggregation. However, in ex vivo devices where platelets are exposed very briefly to high shear while passing through the stenotic region only once, stable aggregate formation requires exposure to much higher shear  [In vivo or in extracorporeal flow chambers with circulating flow loops, the repeated exposure of platelets to lower shear rates  .2 does not generate procoagulant platelets. Some studies report that platelet pretreatment with aspirin or a P2Y12 inhibitor has minimal effect on the generation of procoagulant platelets and their thrombin-generating potential [Blood flow and haemodynamic forces also affect localized thrombin generation, fibrin formation and structure. Upon activation by collagen, thrombin or high shear, a subpopulation of platelets externalize the procoagulant phospholipid phosphatidylserine (PS) and release PS-exposed microvesicles, resulting in localized thrombin generation and fibrin formation , 58. In otential . In the otential –61. Highotential .hypershear-mediated platelet activation that is associated with mechanical circulatory support [The importance of testing blood under pathologically-relevant flow conditions is further reflected by the lack of effect of aspirin at high shear rates. Although the antiplatelet effects of aspirin are well recognized, at shear rates corresponding to an 80% arterial luminal stenosis, aspirin did not affect either the growth rate of thrombus or the thrombotic occlusion time , 63. Thi support . Individ support . This is support .Most POC PFTs do not employ flowing blood under high shear or shear gradient, making these not only nonphysiological but notably, since platelet deposition onto reactive surfaces induced by shear gradients is not prevented by aspirin, clopidogrel or thrombin inhibitors so the effect of these drugs may be overestimated by low-shear tests . Whilst The clinical outcome following an ACS is determined by the interplay between pro-thrombotic and endogenous thrombolytic activities in blood . When thThe intrinsic clot lysis theory was put forward 60 years ago by Alkjaersig, Fletcher, and Sherry in 1959. As such, endogenous thrombolysis is regarded as an intrinsic process, where activators, inhibitors and modulators of fibrinolysis are already localized within the developing thrombus. Major factors distinguishing lysis of an arterial thrombus from plasma or whole blood clot lysis are arterial flow, the presence of platelets, locally-generated thrombin and adherent leukocytes. While it is fibrinolysis which plays the major role in endogenous thrombolysis, the stability of the thrombus in withstanding dislodgement by arterial flow  and clotBlood flow is necessary to accurately assess endogenous thrombolysis. Flow localizes plasminogen during thrombus formation  and promAs the above factors are not present in the static systems commonly used to assess plasma or whole blood fibrinolysis, such tests are not relevant to in vivo endogenous thrombolysis and not suitable to assess endogenous thrombolytic potential in individual patients. Only a test which can form autologous, platelet-rich thrombi from native blood under ex vivo pathological flow conditions and detect the restoration of flow after occlusive thrombus formation can claim to have pathological relevance. Such animal models of thrombolysis have been used for some time in research  but the Based on the importance of the need to reproduce a physiological milieu and the need to avoid the complications of anticoagulation, the ideal POC PFT should employ non-anticoagulated whole blood, which necessitates immediate assessment and avoidance of storage. The test should not only assess platelet activation and aggregation but reflect whole blood thrombus formation. To replicate arterial thrombosis, the test should employ conditions of high shear gradient, during which thrombin is the driving force for thrombus formation and also the main determinant of thrombus stability. Whilst the contribution of the endothelium is difficult to replicate, contact activation due to the artificial surfaces of PFTs contributes toward simulation of coagulation activation by endothelial disruption.Whilst complex ex vivo laboratory models in use and in development, use of animal models of atherosclerosis, flow circuits, platelet-like particles or nanovesicles may better reflect arterial thrombosis than POC tests , 79, sucThe ideal test should employ blood that is flowing under constant arterial pressure, which is subjected to a site of pathological high shear gradient, that leads to occlusive thrombus formation. Such a test should not only assess thrombus growth, but also the susceptibility of the thrombus to endogenous thrombolysis, as well as stability and propensity to embolization.Recognition of the importance of altered flow conditions, hemodynamic forces, procoagulant activity of platelets and endogenous thrombolytic activity is not reflected in most POC PFTs currently in mainstream use. Most tests still rely on specific agonist-induced platelet aggregation using citrate-anticoagulated blood under static conditions, while the significance of high flow, shear and locally-generated thrombin is ignored. To reflect physiological processes, POC tests should assess non-anticoagulated blood at high shear, to reflect and assess the dynamics of thrombus formation, platelet reactivity and thrombus stability, as well as the rate of endogenous thrombolysis of the occlusive thrombus. Future tests should incorporate all these features and physicians employing POC PFTs should chose the most physiological tests to best reflect the thrombotic and thrombolytic status of patients, and to better assess response to pharmacotherapy.


Biosensors are very important for detecting target molecules with high accuracy, selectivity, and signal-to-noise ratio. Biosensors developed using biomolecules such as enzymes or nucleic acids which were used as the probes for detecting the target molecules were studied widely due to their advantages. For example, enzymes can react with certain molecules rapidly and selectively, and nucleic acids can bind to their complementary sequences delicately in nanoscale. In addition, biomolecules can be immobilized and conjugated with other materials by surface modification through the recombination or introduction of chemical linkers. However, these biosensors have some essential limitations because of instability and low signal strength derived from the detector biomolecules. Functional nanomaterials offer a solution to overcome these limitations of biomolecules by hybridization with or replacing the biomolecules. Functional nanomaterials can give advantages for developing biosensors including the increment of electrochemical signals, retention of activity of biomolecules for a long-term period, and extension of investigating tools by using its unique plasmonic and optical properties. Up to now, various nanomaterials were synthesized and reported, from widely used gold nanoparticles to novel nanomaterials that are either carbon-based or transition-metal dichalcogenide (TMD)-based. These nanomaterials were utilized either by themselves or by hybridization with other nanomaterials to develop highly sensitive biosensors. In this review, highly sensitive biosensors developed from excellent novel nanomaterials are discussed through a selective overview of recently reported researches. We also suggest creative breakthroughs for the development of next-generation biosensors using the novel nanomaterials for detecting harmful target molecules with high sensitivity. Early and accurate detection of harmful or interesting molecules is important from the perspective of the biosensor field to prevent their fatal influence on the body or to cure diseases at an early stage ,2,3. To Previously reported biosensors had some essential limitations due to the usage of biomolecules, such as low electrochemical signal strength derived from the biomolecular reaction, instability, and low sensitivity ,16. To sIn biological fields, the importance of nanomaterials in developing biochips for biological applications such as stem cell therapy and drug delivery increased immensely ,26, becaIn this review, highly sensitive biosensors developed by using biomolecules and different types of nanomaterials are discussed in four categories depending on the types of used nanomaterials as NPs, carbon-based nanomaterials, novel nanomaterials, and hybrid nanomaterials. We cover innovative breakthroughs and applications of novel nanomaterials in the development of next-generation biosensors, including practically usable wearable biosensors to detect harmful target molecules with high sensitivity.NPs are the best known and most widely used nanomaterial comprising spherical-shaped structures with a nanometer-sized diameter. Due to their small diameter, NPs possess exceptional properties that do not appear in the bulk product ,37. For An SERS-based biosensor to detect and distinguish two different prostate-specific antigens (PSAs) using Au NPs, magnetic NPs, and SERS nanotags was developed . To overLFA is one of the most widely studied biosensing platforms because of advantages like low cost and ease of use, which are suitable for point-of-care testing (POCT). However, due to the use of the colorimetric band formed on the LFA for target detection, LFA has a sensitivity problem, as it is tens to hundreds of times lower than the sensitivity of other technique-based biosensors. To increase the sensitivity of LFA biosensors, lots of studies were carried out. Among them, using catalytic platinum (Pt) NPs, a sensitive LFA biosensor was reported for detecting the human immunodeficiency virus (HIV) p24 biomarker  . Until n3)63+; RuHex) were introduced as the redox generator since they cannot bind with the positively charged ECP-bound heparin-modified Au-coated magnetic NPs because of the positive charge derived from the ECP captured on the functional NPs. However, they can bind with the negatively charged heparin-modified Au-coated magnetic NPs without ECP, as shown in the schematic image of As mentioned earlier, many electrochemical biosensors based on NPs were reported using the high conductivity and electron transfer facilitating properties of metal NPs. For example, an electrochemical biosensor for detecting microRNA (miRNA) was developed using a uniquely formed bridge DNA aggregated with three Au NPs for amplifying the electrochemical signal through the formation of hugely aggregated Au NP structures and a redox probe for capturing target miRNA . In addiIn addition to these selected NP-based biosensors, various NPs with unique structures and functions were developed and applied widely in the fabrication of highly sensitive biosensors. Also, mentioned researches provided a new scientific approach to develop sensitive biosensors using widely known NPs which were developed a few decades ago. As such, according to new approaches and perspectives, NPs can be applied not only for biosensors, but also for other scientific fields including batteries and energy fields.−14 to 1.0 × 10−8 M). Due to the high conductivity and large surface area of rGO with the conductive polymer, the efficiency for hybridization of target DNA with the single-stranded DNA (ssDNA) probe was increased for effective DNA detection 3+) as the redox probe. In general, the widely used [Fe(CN)6]−3/−4 as a redox probe has a problem of oxidation of Ti3C2–MXene in the measurement range. To prevent this problem, [Ru(NH3)6]3+ was used as the redox probe in this research. As shown in 3C2–MXene in biosensor applications.As another example, a titanium carbide  and thulium (Tm3+) ions were synthesized to achieve an excellent brightness emission signal and sensitivity. Syntheses of two kinds of highly doped UCNPs were confirmed by TEM, power-dependent spectra, and dynamic light scattering. UCNPs were conjugated with anti-PSA and anti-EphA2 antibodies to detect PSA and EphA2. When the target analyte is bound to the antibodies immobilized on the highly doped UCNPs, it can be detected by the increase in emission intensity from the LFA biosensor. As shown in 3+ and 12 times higher for Tm3+ compared to the intensity from the lower-doped UCNPs. The proposed UCNP-based LFA biosensor can detect target analytes with ultra-sensitivity, selectivity (LODs of 89 pg/mL for PSA and 400 pg/mL for EphA2), and good linear correlation (0.01 ng/mL to 100 ng/mL). Also, the target detection was performed with a strip test system based on a single-photon avalanche detector (SPAD) through colorimetric change detection by using the camera of a cellular phone. This UCNP-based LFA biosensing system can be applied to develop portable and wearable devices for the detection of a wide range of biomarkers and analytes.As mentioned before, UCNP-based biosensors that exploit their fluorescent property were reported extensively, particularly in LFA biosensors. In general, large-sized UCNPs up to 400 nm with lots of emitters were used to induce the high brightness of emitted signal from UCNPs. However, large-sized UCNPs hindered the effective flow of UCNPs through the porous region of the LFA chip composed of a nitrocellulose film to develop the biosensors. To solve the size-related problem, small-sized UCNPs (<100 nm) were synthesized and modified with densely doped emitters to synthesize UCNPs capable of efficiently emitting high luminescence with a small size. Using the small-sized UCNPs with densely doped emitters, LFA biosensors were developed to detect PSA and type-A receptor 2 (EphA2). To detect target molecules, the sensing probes were combined with UCNPs which were a prostate cancer biomarker and pancreatic cancer biomarker . To deveAs discussed herein, there were various recently developed novel nanomaterials for application in the biosensor field, and the process is ongoing. In addition, reported novel nanomaterial-based biosensors showed excellent biosensing performances, and they were widely utilized in various biosensing tools by applying various electrochemical and fluorescence methods, thereby showing their versatility. Furthermore, this nanomaterials-based detection of target molecules can be easily achieved using cellular phones.2 hybrid nanomaterial and utilized its exceptional electron transfer property to increase the redox signal derived from metalloprotein to sensitively detect hydrogen peroxide [3O4) NPs and lead sulfide (PbS) NPs were decorated on an ITO photoelectrode to develop a photoelectrochemical hydrogen peroxide biosensor using the photoelectrochemical property of PbS and multienzyme activity derived from Co3O4 [9S5) NPs, UCNPs, and silver sulfide (Ag2S) NPs, which can emit two luminescent signals simultaneously for dual miRNA detection, was reported [In addition to the various nanomaterials mentioned earlier, hybridization of these nanomaterials with other materials was utilized to exploit their exceptional functionality and to develop outstanding hybrid nanomaterials for application in various scientific fields ,79,80. Tperoxide . As anotom Co3O4 . In addireported .Among the various hybrid nanomaterials used in biosensors, an interesting one was developed and utilized in a biosensor for detecting neurotransmitters produced by neurons  Figure a. A sand2 NPs and deposited Au ions by sputter deposition onto a PI substrate to fabricate an Au/MoS2/Au hybrid nanofilm with excellent electron transfer characteristics for enhancing the redox reaction between glucose oxidase and the glucose. High blood glucose is an important bioindicator of diabetes mellitus (a disease that can induce disability and death in humans). Glucose oxidase, the sensing metalloprotein which has a metal ion in its core that reacts with glucose electrochemically, was immobilized on the fabricated hybrid nanofilm through the chemical linker, 6-mercaptohexanoic acid (6-MHA). Fabrication of the Au/MoS2/Au hybrid nanofilm was confirmed by SEM and energy-dispersive X-ray spectroscopy (EDX) mapping. Immobilization of glucose oxidase was verified by CV through the detection of redox signals derived from immobilized glucose oxidase. As shown in 2/Au hybrid nanofilm on the polymer substrate to facilitate the electron transfer. By using the amperometric I–T (current-time) technique, the biosensor could detect glucose excellently (a 10 nM detection limit) and selectively in human serum through the detection of generated electrons by electrochemical reaction between glucose oxidase and added glucose. In addition, it retained its conductive property without the hybrid nanofilm cracking even after numerous bending tests, and its flexibility was verified by using the micro fatigue tester. The most important thing in this research is that the flexible conductive substrate composed of hybrid nanomaterial layers was developed via a simple fabrication process that can be easily applied to the development of other biosensing platforms.Recently, flexible biosensors attracted much attention due to the increased requirement for wearable healthcare devices ,91. Elec−1∙cm−2) was four times higher than the result of the biosensor prepared with only PB   encapsulated by Prussian blue (PB) (AgNCs@PB) was synthesized and used as the template to develop a flexible biosensing platform for detection of hydrogen peroxide . Based o−1∙cm−2) c. IncludIncluding the previously discussed researches, hybrid nanomaterials can be utilized to develop highly sensitive biosensors and flexible electronic devices applied not only in sensors but also in wireless devices. In addition to wide applications in the field of biosensors, novel hybrid nanomaterials can be used widely in biological fields such as cell fate monitoring, spatiotemporal release of drugs, and regenerative medicine. Furthermore, due to its exceptional electrical, electrochemical, and optical properties, developed hybrid nanomaterials can be utilized in various scientific fields such as electrodes for batteries and energy generation ,98.Since the development of nanotechnology began in earnest, various novel nanomaterials were developed and applied in numerous scientific fields. Those nanomaterials provided huge potential in biological fields, particularly in the field of biosensors, because of their superior properties, including electron transfer facilitation, unique optical and plasmonic effects, and biocompatibility, compared with their bulk counterparts. In addition, the exceptionally outstanding properties of nanomaterials helped to solve the intrinsic limitations of biomolecules such as instability and extremely low signals derived from biomolecular reactions, as well as grant high sensitivity to biosensors using their properties such as the high conductivity, efficient fluorescence emission, and reacting properties with harmful molecules. Accordingly, the advantages produced by the combination of biomolecules and nanomaterials are useful for achieving the ultimate goal in the field of biosensors, which is the detection of harmful and interesting molecules with high sensitivity and selectivity.2. Finally, we discussed biosensors constituting biomolecules and hybrid nanomaterials with two or sometimes more different novel nanomaterials, including UCNPs with graphene, and AgNCs encapsulated by PB. Those hybrid nanomaterials showed synergetic effects via the conjugation of two different nanomaterials for developing highly sensitive biosensors. Furthermore, these hybrid nanomaterials provide the possibility for the development of wearable biosensors, which is currently attracting huge attention. Accordingly, numerous superb biosensors were developed using biomolecules and nanomaterials. In this review, we discussed biosensors developed by using biomolecules and various types of nanomaterials. The discussion was conducted sequentially according to four categories of different types of nanomaterials. Firstly, we investigated biosensors based on NPs, the most widely known type of nanomaterial, used in SERS-based, LFA-based, and electrochemical biosensors, and these researches suggested a new approach for developing biosensors using NPs. Next, we discussed biosensors based on carbon nanomaterials, which attracted much attention in recent years. In addition, we considered biosensors developed by using nanomaterials with advantages including luminescence and electrochemical properties, such as UCNPs and MoS