Results indicated that having moderate-to-high scores on commitment attenuated the association between being physically objectified by White men and higher depressive symptoms. However, having high scores on commitment intensified the association between being rejected by Black men and lower feelings of self-worth. Stronger identity commitment may be protective against objectification from White men, though it may also exacerbate negative outcomes related to in-group discrimination. These findings may have important implications for the development of individual and group-level interventions addressing ethnic identity among YSMBM.Racialized Sexual Discrimination (RSD), also known as ‘sexual racism,’ is pervasive within online dating venues. RSD is associated with poor mental health outcomes among young sexual minority Black men (YSMBM), and there is limited research on factors that may mitigate this association. Ethnic identity has been identified as a potential protective factor for racial/ethnic minorities who encounter racialized stressors, though some evidence suggests that ethnic identity may also intensify the negative effects of racial discrimination. Using data from a cross-sectional web-survey of YSMBM ( Online intimate partner-seeking is now widespread among Americans, especially among sexual minority adolescents and young adults. Young sexual minority populations use dating apps and websites at higher rates than their older and heterosexual counterparts, and use of such platforms has been increasing over the past decade ,2. SexuaThe phenomenon of RSD is situated within a broader literature of sexual racism. Sexual racism has been described as the systemic ways in which individuals and society establish a racial hierarchy of desire—wherein White identity/Eurocentric features are considered ideal, interracial intimacy is less socially accepted, and the erotic capital of racial/ethnic minorities are altogether diminished, or wholly afforded through racial fetishization ,17,18. AMuch of our work on RSD has been grounded in Meyer’s Minority Stress Theory (MST), which provides an apt framework for understanding how RSD contributes to adverse health outcomes among sexual minority populations . MST expThere are a number of theories addressing ethnic identity that may complement MST and provide important nuance in investigating RSD among sexual minority men of color. Building upon Erickson’s and Marcia’s seminal work on adolescent identity development ,34,35, PBoth Phinney and other scholars have suggested that higher levels of identity commitment may confer protective benefits and partially mitigate the effects of racial discrimination ,38,39,40There is an extensive body of empirical literature on ethnic identity in general, with most researchers reporting that stronger ethnic identity is associated with positive psychosocial outcomes among Black Americans and other racial/ethnic minority groups ,50,51,52In a study of online discrimination and well-being, Tynes et al. reported that that ethnic identity buffered the association between online discrimination and anxiety symptoms among Black adolescents, but did not significantly alter the association between online discrimination and depressive symptoms . Romero There are two key benefits of examining ethnic identity as a moderator between RSD and health. First, the theoretical and empirical literature has indicated that not all characteristics of ethnic identity are uniformly protective—and in some instances, certain aspects of ethnic identity may actually worsen the negative effects of discrimination ,43,58,59Hypothesis 1; H1) higher scores on RSD would be associated with poorer psychological well-being among the study sample;  higher scores on ethnic identity search would exacerbate the association between RSD and well-being; and  higher scores on ethnic identity commitment would attenuate the association between RSD and well-being.Given the importance of ethnic identity in the context of race-related stress, the current study aims to examine the ways in which ethnic identity might serve to modify the relationship between RSD and psychological well-being among YSMBM. In accordance with prior theoretical and empirical work, we hypothesized that  identify as a man; (2) be assigned male sex at birth; (3) identify primarily as Black, African American, or with any other racial/ethnic identity across the African diaspora ; (4) be between the ages of 18 and 29 inclusive; (5) identify as gay, bisexual, queer, same-gender-loving, or another non-heterosexual identity, or report having had any sexual contact with a man in the last 3 months; (6) report having used a website or mobile app to find male partners for sexual activity in the last 3 months; and (7) reside in the United States.A non-probability convenience sample of YSMBM were recruited using best practices for online survey sampling ,67 betweProspective participants were directed to a survey hosted on Qualtrics upon clicking on the study advertisement. Participants were presented with a set of screening questions to determine their eligibility. Those who met the eligibility criteria were directed to a consent page, which contained detailed study information . Those consenting to participate proceeded to the full survey which lasted 30 to 45 min. Participants were not compensated for taking the survey. While completing the survey, participants were permitted to save their answers and return to the survey at a later time if they were not able to complete it in a single sitting. Study data were kept in an encrypted and firewall-protected server, and the Institutional Review Board at the University of Michigan all study procedures.Outcome Variables. The two dependent variables used in this study include Depressive Symptoms and Feelings of Self-Worth.Depressive Symptoms. We measured depressive symptoms using the Center for Epidemiologic Studies Depression scale ) scored significantly lower on feelings of self-worth. Moderate and low levels of EIC did not significantly modify this association. All four same-race rejection models were significant (p < 0.001) and explained 26–37% of the variance in depressive symptoms and self-worth  and EIC models . Higher scores on White physical objectification were also associated with lower feelings of self-worth in both the EIS  and EIC models . Higher scores on EIC were associated with lower depressive symptoms  and higher feelings of self-worth . A significant interaction effect was observed in the depression and EIC model , with results indicating that EIC attenuated the association between White physical objectification and depressive symptoms. b = 0.003, p < 0.001, 95% CI ) and high  levels of EIC scored significantly lower on depressive symptoms. Low levels of EIC did not significantly modify this association. All four White physical objectification models were significant (p < 0.001) and explained 26–36% of the variance in depressive symptoms and self-worth  and higher feelings of self-worth (p < 0.01); perceived rejection was associated with higher depressive symptoms (p < 0.001) and lower feelings of self-worth (p < 0.001); and self-perceived sexual attractiveness was associated with lower depressive symptoms (p < 0.001) and higher feelings of self-worth (p < 0.001).Age, perceived rejection, and self-perceived sexual attractiveness were significant across all twelve regression models. No other covariates emerged as significant. Across all models, age was associated with lower depressive symptoms , consistent with our prior work, as well as other scholars’ work examining sexual racism among sexual minority men of color [All study covariates performed identically to previous main effects analyses examining the relationship between RSD and psychological well-being. We discuss the implications of these associations at length in prior published work . In addiof color ,27,28,29Participants reported overall moderate to high scores on both ethnic identity subscales. This coincides with extensive research showing that Black sexual minority individuals tend to identify strongly with their racial/ethnic identity ,74. The H2), higher scores on identity search did not significantly exacerbate the association between RSD and psychological well-being in any model. Although our findings do not support the prior theoretical and empirical work in which we grounded our hypothesis [uncertainty is thought to be the underlying component of exploration. Scholars have noted, however, that certain aspects of identity that have a more definitive positive valence , are not only highly correlated with identity search, but may have a reciprocal relationship with identity search [Contrary to our hypothesis . This is consistent with prior research illustrating that ethnic identity may serve as a potential safeguard against racialized stress [In contrast to our null findings for identity search, our results do suggest that identity commitment may significantly modify the relationship between RSD and psychological well-being. Moderate to high levels of commitment buffered the association between White physical objectification and depressive symptoms among the study sample, thus partially supporting one of our central hypothesis  may exacerbate the effects of racial discrimination ,86,87. MThese findings contribute to a growing body of research into the associations between ethnic identity and well-being among sexual minority people of color ,91, and YSMBM with high ethnic identity commitment may require support in grappling with same-race rejection while maintaining the overall benefits of ethnic identity commitment. Critical consciousness interventions, which support participants in understanding and opposing oppressive forces, may help them to understand interpersonal discrimination in terms of broader structures of White supremacy. Various researchers have advocated for critical consciousness interventions in addressing racism and other forms of oppression ,98. In aCritical consciousness interventions are not limited to those who are subject to oppression, but may also be fostered among health professionals and clinical practitioners. A robust cultivation of critical consciousness on the part of clinicians may enable them to more effectively engage with YSMBM, and help them navigate complex intersectional stressors that they encounter in intimate partner-seeking contexts . MoreoveTo our knowledge, this is the first study to examine ethnic identity as a moderator of the association between RSD and psychological well-being among YSMBM. It is also among the first to examine the association between ethnic identity and feelings of self-worth and depressive symptoms in any sexual minority population. Our disaggregation of the search and commitment subscales of the MEIM adds further clarity to the ethnic identity literature, as the majority of studies using the MEIM use the scale as a composite measure . The stuThis study, however, is not without its limitations. The study’s cross-sectional design and lack of representative sample limits generalizability, as well as our capacity to make causal inferences about the relationships between ethnic identity, RSD, and well-being among YSMBM. The study is further limited by its all-Black sample and by its focus on RSD as perpetrated by Black or White users online; thus, the findings cannot generalize to other racial/ethnic groups, nor can they provide insights into RSD as perpetuated by racial/ethnic groups other than Black or White men. The MEIM is also not without critique. The scale has been subject to revisions, including both expanding and shortening the 12-item version used in this study, and researchers have also reported slightly different factor solutions for the scale among different populations ,103. MorAlthough the health and social science literature on RSD has been largely qualitative, there are still many important questions about RSD that call for qualitative or mixed methodological approaches. Most qualitative research on RSD among sexual minority men has focused on the experience of discrimination, but only a handful of studies have focused on strengths for coping with RSD, including identity-based strengths ,13,22. CA closer examination of same-race rejection may be especially important for sexual racism researchers, particularly in light of our unexpected findings. Although commitment to one’s ethnic identity exacerbates the effect of same-race rejection, its overall effect is positive, and YSMBM struggling in the face of same-race rejection are not likely to benefit from lessening their commitment. Given the complexity inherent in navigating this experience, it may be more beneficial to focus on the motivations of YSMBM who are inclined to reject members of their same race. As with other forms of internalized racism, perpetrators of same-race rejection are devalued by the same system of White supremacy in which they participate. However, only a small amount of research on RSD examines same-race discrimination ,105,106.Future research should also examine ethnic identity, RSD, and health among other sexual minority men of color. Patterns of RSD may manifest differentially across different racial/ethnic groups , and some groups may respond differently to RSD than others. For some racial/ethnic minority groups, generational status—or recency of immigration—should also be examined in the context of RSD. Generational status may have some bearing on both self-reported ethnic identification and exposure to RSD over time ,111,112.Using intersectional identity measures  will also add important nuance to work of this nature moving forward. There is a marked deficit of intersectional measures for sexual and gender minority populations, but scholars have long highlighted the importance of examining overlapping identities instead of focusing on singular markers of identity ,118,119.Overall, this study provides valuable insight into the relationship between ethnic identity, RSD, and psychological well-being. Moving forward, researchers should continue to account for factors that may further elucidate these relationships. They should also explore multi-pronged strategies to disrupt harmful behavior perpetrated online. RSD, like any form of racialist discrimination, is neither natural nor inevitable, and combatting racialist ideologies at their root remains critical to advance health equity for marginalized populations.


Nanotechnology carriers have become common in pharmaceutical products because of their benefits to drug delivery, including reduced toxicities and improved efficacy of active pharmaceutical ingredients due to targeted delivery, prolonged circulation time, and controlled payload release. While available examples of reduced drug toxicity through formulation using a nanocarrier are encouraging, current data also demonstrate that nanoparticles may change a drug’s biodistribution and alter its toxicity profile. Moreover, individual components of nanoparticles and excipients commonly used in formulations are often not immunologically inert and contribute to the overall immune responses to nanotechnology-formulated products. Said immune responses may be beneficial or adverse depending on the indication, dose, dose regimen, and route of administration. Therefore, comprehensive toxicology studies are of paramount importance even when previously known drugs, components, and excipients are used in nanoformulations. Recent data also suggest that, despite decades of research directed at hiding nanocarriers from the immune recognition, the immune system’s inherent property of clearing particulate materials can be leveraged to improve the therapeutic efficacy of drugs formulated using nanoparticles. Herein, I review current knowledge about nanoparticles’ interaction with the immune system and how these interactions contribute to nanotechnology-formulated drug products’ safety and efficacy through the lens of over a decade of nanoparticle characterization at the Nanotechnology Characterization Laboratory. Nanotechnology is often used to formulate various drugs to improve their solubility, prolong circulation time, achieve delivery to the target organs and tissues, direct the route of particle uptake into and intracellular distribution within a target cell, and benefit from multifunctional capabilities –6. Many Given the current global emergency use of lipid-nanoparticle (LNP)-based COVID-19 vaccines to combat the COVID-19 pandemic, some experts expect more nanotechnology applications in infectious diseases .Many studies have demonstrated that the reformulation of a drug using a nanocarrier helps to reduce the drug’s toxicity. For example, the anticancer drug doxorubicin (DXR) is known for its accumulation in cardiomyocytes and in relation to its cardiotoxicity; this toxicity is overcome when DXR is delivered using a polyethylene glycol (PEG)-modified liposome (Doxil) . The remWhile reduced drug toxicity through formulation with a nanocarrier is encouraging, available data also suggest that a change in the drug’s biodistribution due to the carrier may also occur and lead to the “relocation” of toxicity from one target organ to another. For example, the liposomal formulation of DXR helped to overcome DXR’s cardiotoxicity ; howeverFor decades, the efforts of the nanotechnology drug delivery community were focused on masking nanoparticles from immune recognition –21. HoweThe mechanisms through which nanocarriers contribute to immunomodulation are incompletely understood. One mechanism commonly discussed in cancer therapy literature includes the induction of so-called immunogenic cell death (ICD) by APIs, which are more precisely delivered to tumors by nanocarriers . The stuThe existing data suggest that nanocarriers may contribute to this phenomenon through other mechanisms than delivering an ICD-inducing drug to tumors. Some of such mechanisms may include nanocarriers inducing chemokines. For example, I reported earlier that liposomes and lipid nanocarriers commonly induce chemokine IL-8 , which iMoreover, nanocarriers can be loaded with immunomodulatory agents that improve the therapeutic outcome of cytotoxic agents. For example, liposomes formulated to co-deliver a PI3K inhibitor with an API  activated anti-tumor T-cell responses . In anotIt is well established now that nanoparticle physicochemical properties such as size, aspect ratio, zeta potential, hydrophobicity, surface area, and functionalization determine interactions between nanoparticles and immune cells. By optimizing these properties, researchers could control undesirable immunotoxicity and achieve desirable immunomodulatory effects. More studies are needed to fully understand the mechanisms by which nanocarriers contribute to API therapeutic efficacy (besides their primary role as drug-delivery vehicles).Regardless of the indication, all new formulations must undergo rigorous safety testing prior to their approval for clinical use. Even after receiving initial approval, drugs undergo post-marketing surveillance and can be removed from the market due to toxicity . One reaInfusion reactions (IRs) are HSRs that occur within minutes to hours of nanoparticle administration . The mecThe complement system plays an essential role in both innate and adaptive immunity . It is cvia three main mechanisms—the lectin pathway, initiated by mannose-binding lectin; the classical pathway, initiated by the antibody; and the alternative pathway initiated by C3b binding. Additionally, autoactivation can occur via the so-called C3-tickover mechanism  before administering drug-loaded nanoparticles  were proposed as effective means of inhibiting complement activation by nanoparticles in vitro and in vivo  assay cascade, PEGylated liposomes, especially those with elongated shapes, had more significant complement activation responses than spherical PEGylated liposomes and other PEGylated nanomaterials, which is consistent with the literature . Other cell types, including platelets, some T cells (mainly regulatory T cells), fibroblasts, endothelial cells, and epithelial cells, can also contribute to the cytokine response during the early phase of inflammation. During this early phase, cytokines act on the nearest cells via the paracrine mechanism and, upon entry into systemic circulation, send the message to cells at other locations via the endocrine mechanism. Cytokines can have similar, overlapping, and unique functions and stimulate the production of other cytokines and secondary messengers, which amplify the response and initiate new responses. Examples of cytokines produced in the early phase of innate immune responses include tumor necrosis factor-alpha (TNFα), interleukins , type I interferons (IFNα and IFNβ), and chemokines . Cytokines coordinate innate and adaptive immune responses; some of them  are also produced by activated T lymphocytes during the adaptive immune response. Understanding cytokine responses helps interpret the results of both safety and efficacy studies. Other aspects of nanoparticle immunocompatibility, such as the immunogenicity topic described later in this review, may provide mechanistic insight into the cytokine responses to nanomaterials due to the known role of antibodies in activating the immune cells and biochemical immune pathways such as the complement pathway.The communication between various immune cells and between the immune cells and other cells in the body can be direct 2 particles with a large aspect ratio were more potent at activating NLRP3 inflammasome and promoting LPS-induced IL-1β induction than their spherical and fibrous low aspect ratio counterparts   and has terparts . In anotterparts . An inteterparts . More exterparts .Iron, silica, chitosan, poly acid (PLGA), liposomes, emulsions, virus-like particles, peptide- and poly(amino acids)-based carriers, synthetic polymers , and DNA origami have been shown in various models to improve the antigen uptake, processing, and presentation, and result in overall better vaccine and immunotherapy performance –86. For Using nanoparticles, researchers were able to direct specific Th1 versus Th2 polarization and major histocompatibility complex (MHC)-restricted cytotoxic T-cell responses that traditional vaccines and therapies could not achieve , 86–88. Other examples of beneficial cytokine response to nanoparticles and nanoparticle-formulated drugs include a recent study of CpG oligonucleotides delivered using particle replication in non-wetting templates (PRINT) nanoparticles. This concept resulted in particle accumulation in the lungs, where local cytokine response to delivered CpG oligonucleotides resulted in a reduction in the tumor size . Unlike In contrast to studies discussed above, an overt production of inflammatory cytokines in response to systemically administered nanoparticles has also been described for certain nanoformulations. For example, adverse immune-mediated reactions to liposomal microRNA formulation MRX34 were so severe that they led to four patient deaths and subsequent discontinuation of the clinical trial . The samvia Scavenger Receptor A-mediated phagocytosis, and this uptake culminates with the production of type I and type III interferons  such as siRNA, anti-sense DNA oligonucleotides, and CpG oligonucleotides, nucleic acid nanoparticles (NANPs) are immunoquiescent in that adding these particles to immune cells does not result in a cytokine response . Howevererferons . Anothererferons .The expression of TLR7 is abundant in airways, and the activation of this innate immune receptor has a bronchodilating effect, decreases allergy-mediating Th2 responses, eosinophilic inflammation, and goblet-cell hyperplasia that make it a therapeutic target in asthma . Since tWhile the initial studies are encouraging, more research is needed to fully evaluate the safety of NANP-mediated TLR7 activation because a recent study provided the first causation link between TLR7 activation and systemic lupus erythematosus (SLE), an autoimmune disorder , which iStructure-activity relationship studies revealed that the magnitude of the interferon response to NANPs could be controlled by the type of nucleic acids used to create these particles, with RNA-based NANPs being more potent interferon inducers; three-dimensional shape, with the globular NANPs being more potent than planar and fibrous NANPs; and size, but not sequence complementarity . More inAn extensive discussion regarding the immunotoxicity of traditional TNAs and NANPs; the role of nanocarriers in mitigating this toxicity; and translational challenges, opportunities, and barriers due to the immunological properties of NANPs are available elsewhere –115.Cytokines are commonly used in preclinical studies as biomarkers of inflammation. Previously, NCL reported an interesting trend showing that lipid-based nanomaterials analyzed in the NCL standardized assay cascade between 2005 and 2015 induced chemokine IL-8 without inducing other pro-inflammatory cytokines such as TNF, IL-1, and IL-6 . The datThe coagulation system’s two main components are platelets and the plasma coagulation system.Platelets, also known as thrombocytes, are the smallest among peripheral blood cells . The maiIt has also been demonstrated that traditional sterilization methods such as gamma irradiation and autoclaving may change nanoparticle surfaces so that the particles become pro-thrombogenic and activate platelets . Howeverin vitro conditions, this cascade can be divided into three pathways: an extrinsic (prothrombin time [PT]) pathway, an intrinsic  pathway, and a common (thrombin time) pathway. Nanoparticle interaction with plasma coagulation depends on particle composition, surface functionalization, and size. For example, amine-terminated polystyrene nanoparticles inhibited plasma coagulation by depleting plasma coagulation factors VII and IX . The ADA can increase or decrease the product’s efficacy, cause alterations in the drug’s pharmacokinetics (PK), accelerate the drug clearance, and mediate systemic and local antibody-mediated toxicities such as anaphylaxis, HSR, kidney toxicity, and neutralization of non-redundant endogenous proteins with overlapping epitopes . The freNanoparticle immunogenicity has been extensively studied using fullerenes, dendrimers, and liposomes. These studies demonstrated that nanoparticles are poor antigens and do not induce antibody responses even in the presence of potent adjuvants. For example, C60 fullerene derivatives in the presence of Freund adjuvant did not induce generation-fullerene-specific antibodies . HoweverMost importantly, unconjugated fullerenes, even in the presence of Freud adjuvants, were not immunogenic . SimilarLike fullerenes and dendrimers, liposomes alone were not immunogenic ; howevervia active immunization with mRNA-PEG-LNPs in a pig model  and other immunological responses to polymers  used in pharmaceutical products and nanomedicines have been reviewed in detail elsewhere . OverallImmunosuppression is a condition in which an individual’s immune response is lowered. It can result from genetic mutations affecting receptors, adaptor proteins, or transcription factors involved in the normal innate and adaptive immunity , 160. FoCytotoxic oncology drugs intend to stop cancer cell proliferation but also affect lymphocytes, thereby decreasing lymphocyte-mediated immune responses . When suBelow, I review some examples of drug-mediated immunosuppression due to bone marrow (BM) and blood lymphocyte inhibition and discuss whether and how nanotechnology platforms influence this toxicity. Whenever available, I will also discuss the immunosuppressive properties of nanocarriers themselves.in vitro exceeded the uptake of iron citrate used as a control. Greater uptake, however, did not influence cell viability and expression of surface markers - and polyisohexyl (PIHCA)- cyanoacrylate nanoparticles, demonstrated differential distribution and toxicity . DXR-PIB markers . Unlike  markers , IONPs w markers . Provideit assay .Accumulation of some nanoparticles in BM resulted in myelosuppressive effects due to particle-mediated apoptosis and hypoplasia. For example, intraperitoneal administration of aluminum oxide nanoparticles to mice decreased total and differential BM counts and altered erythropoiesis . The samSuppression of lymphocyte function may occur due to either immunosuppressive drug payload or nanocarrier per se. Examples of drug-mediated immunosuppression include PLGA-betamethasone and nanoalbumin-paclitaxel (Abraxane), among others –177. Druvia TGFβ produced by alveolar macrophages  may confound the results of such studies  matrix  , 201. Sz) matrix –205.in vitro complement activation assay is used for nanoparticle characterization, it is essential to consider both inter- and intra-species variability in complement activation, which may influence the assay sensitivity and overall conclusions. For example, when mouse plasma from several strains  was used as a matrix to study complement activation by liposomal amphotericin (Ambisome), the highest complement activation was observed in the plasma of Balb/c and CD-1 mice, whereas the lowest activation was seen in plasma of C57BL/6 mice; the activation in plasma of other strains was moderate  is a final product of prothrombin activation that results in platelet activation and fibrinogen-to-fibrin conversion. Thrombin activates transamidase Factor XIIIa, which stabilizes the fibrin network with activated platelets, thereby forming a blood clot. Positive feedback of thrombin activation includes the activation of coagulation factors XI, IX, V, and VIII. The negative feedback controls the thrombin activity: thrombin binding to thrombomodulin expressed on the surface of endothelial cells activates protein C and stops further procoagulant activity. Activated protein C and its cofactor protein S activate proteolytic degradation of activated coagulation factors Va and VIIIa, which function to accelerate the thrombin-generation pathway. Thrombin also activates complement, leukocytes, and other cell types. Activated by thrombin and complement cells contribute to the plasma coagulation by producing cytokines and expressing the phospholipid-protein procoagulant activity complex. This complex initiates plasma coagulation by activating coagulation factor VII.When analyzing the coagulation system in preclinical studies, it is essential to recognize that all components of this system are closely connected in vitro using platelet aggregation, plasma coagulation, and leukocyte procoagulant activity assays . If the cytokine of interest is expressed by cells of low abundance in the whole blood and even in PBMCs , researchers could isolate these cells from the blood and concentrate them prior to analysis o detect Figure 7in vitro assay that is a surrogate of the in vivo T-cell-dependent antibody response (TDAR) for immunosuppression screening is the human lymphocyte activation (HuLa) test that employs PBMCs of healthy donors immunized with the current-year flu vaccine. The HuLa assay was initially developed and validated across immunosuppressive drugs with various mechanisms of action and showed consistent performance (Leukocytes can be activated by mitogens such as plant lectin phytohemagglutinin (PHA) for T cells and lipopolysaccharide for B cells. Antigen-specific lymphocytes can also proliferate in response to their cognate antigens . Proliferating cell expansion can be detected by several commercially available kits and reagents with -2,5-diphenyltetrazolium bromide (MTT), bromodeoxyuridine, (BrdU) and carboxyfluorescein diacetate succinimidyl ester (CFSC), being broadly used , 216. Brformance , 218. Thin vitro and ex vivo. In the in vitro protocol, the BM stem cells are isolated from untreated animals or human-donor volunteers, followed by the in vitro treatment with nanoparticles. In the ex vivo format, the BM cells are obtained from animals exposed to nanoparticles. Although the in vitro method does not account for nanoparticle biodistribution, it allows for rapid identification of potentially toxic formulations and is helpful in cases when amounts of nanoparticles are limited, and the dose information is unavailable, i.e., early in preclinical development. When BM cells are cultured in a methylcellulose-based medium in the presence of SCF, IL-3, and IL-6, it results in the formation of the CFU-GM that can be enumerated. Therefore, the in vitro CFU-GM protocol is used to assess the myelosuppressive properties of cytotoxic oncology drugs or nanoformulations delivering these compounds. The comparison between CFU-GM in the untreated sample (the baseline) and nanoparticle-treated sample (test) allows for the identification of nanomaterials with myelosuppressive properties  in human patients . Depending on the growth factors present in the culture medium, these CFU can be of different cell linage. CFU-GM, for example, assesses the formation of granulocytes and macrophages; CFU-E, erythrocytes; and CFU-GEMM, erythroid and mixed myeloid cells. This method is commonly used to assess the functionality of BM stem cells and the potential effects of test substances on these cells. The method can be conducted operties . When copatients , 221.E. coli) could be done by flow cytometry or confocal microscopy; in this case, the model particulates are conjugated to a fluorescent label. When unconjugated particulates are used as model foreign bodies for monitoring phagocytic function, a luminescence-producing reagent, luminol, is used to detect their uptake by a plate-reader-based assay  cells are staffed with cytoplasmic granules containing cytotoxic proteins, such as perforin and granzymes. These proteins form pores in tumor and virus-infected cells when released, thereby contributing to the innate immune response against abnormal and infected cells. Alterations in the NK cytotoxicity may impair immunity; therefore, NK cell function analysis is an integral part of immunotoxicity studies. Both model cell lines and primary NK cells are used for such studies. For example, NK92 and HepG2 cell lines are frequently used as effector and target cells, respectively; the viability of HepG2 cells in the presence of untreated or nanoparticle-treated NK92 cells can be monitored in real-time using label-free technology . Other evia the route of administration relevant to the intended clinical use of these materials.After the initial immunotoxicity assessment using general toxicity studies, specialized immune function tests can be employed to further interrogate adverse effects on the immune system. Some of these specialized immune function tests are described below. In these methods, test nanomaterials are administered as the dose level, using the dosing regimen and in vitro limulus amoebocyte lysate (LAL) assay , has been validated as a reliable surrogate for LAL and RPT to test not only for endotoxin but for non-endotoxin pyrogens  results in an elevation in body temperature. As such, the rabbit pyrogen test (RPT) was established to detect fever-causing drugs and other medical products to prevent overt responses in patients. The experimental procedure involves the injection of a test material into the ear vein of a rabbit; the animal’s body temperature is monitored before the injection and three hours after the injection with 30-minute intervals. The RPT is standardized for worldwide use in the field of drug development and pharmaceutical analysis for pyrogenicity and is documented in pharmacopoeias of various countries. However, some discrepancies exist between protocols used in various countries with regards of the required number of rabbits, the acceptable initial body temperature, the determination of baseline temperature, and the decision algorithm –229. HisL) assay , 231, thpyrogens , 232–238pyrogens . Current3H-thymidine; five hours after the thymidine injection, the animals are sacrificed, and their draining lymph nodes are analysed by scintillation counting to detect thymidine incorporation into the DNA of proliferating leukocytes. An increase in the thymidine incorporation points to T-cell activation that occurs during allergic sensitization. The LLNA protocol is identical to that of LLNP except for the route of test-material administration. In the LLNA assay, the test material is topically applied to the animal’s skin; this test is applicable to nanomaterials formulated as creams or lotions. In vitro assays myeloid U937 skin sensitization test  and human cell line activation test (h-CLAT) were developed as surrogates for LLNA/LLNP and showed consistent performance in interlaboratory studies  have been developed to test for delayed-type hypersensitivity (DTH) reactions. More recently, the local lymph node proliferation assay (LLNP) was proposed for the prediction of DTH; this method accurately predicted DTH reactions to systemically administered pharmaceuticals . In LLNP studies –243. Hown h-CLAT . Therefoin vivo test for iron oxide formulation Feraheme correlated with the in vitro HuLa assay discussed above; of note, a sex-dependent difference was detected by the TDAR method . Finally, the levels of the antigen-specific IgM and IgG are assessed one and three weeks from the antigen administration . A decreR method . Inhibitr models , 183. HoPigs are infused or injected with nanomedicines, and hemodynamic changes are monitored in real-time, followed by ex vivo blood sample analysis for the presence of complement split products and other inflammatory mediators such as thromboxane; the model reproduces symptoms and molecular markers induced in response to various nanomedicines known to cause IRs in human patients , 246. Clin vitro uptake studies performed using human PBMCs (scid/IL2r common γ chain null (NSG) mice following the transfer of human PBMCs to analyze the functionality of the antigen-specific human regulatory T cells induced by PLGA nanoparticles co-delivering IL-2 and TGF-β to produce the tolerogenic response for lupus therapy , surface moieties , zeta potential , shape , and size  as reviewed in this manuscript and earlier reports from NCL , 261 andvia local routes traditionally used for immunization , in the presence of adjuvants  and intended for use in healthy individuals  warrants studies investigating the risk of autoimmunity. Improving in vitro and in vivo models for assessing nanoparticle immunotoxicity along with harmonization of testing approaches is another important direction in this field. Sharing high quality data generated by “wet” laboratories with bioinformatics researchers is also expected to improve quality of nanotherapeutics, streamline the selection of nanocarriers and aid in developing safer nanomedicines by generating supporting computer-based algorithms and analysis tools.The investigation of nanoparticle immunological properties progresses toward mechanistic studies involving new technological modalities, such as real-time imaging, advanced immunophenotyping, and immunometabolomics. Some examples of mechanistic studies and relevant methods are summarized in The author confirms being the sole contributor of this work and has approved it for publication.This study was funded in whole by federal funds from the National Cancer Institute, National Institutes of Health, under contract 75N91019D00024.I am grateful to Allen Kane and Karolina Wilk for the help with manuscript preparation.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government.


Efficient detection and observation of dynamic RNA changes remain a tremendous challenge. However, the continuous development of fluorescence applications in recent years enhances the efficacy of RNA imaging. Here we summarize some of these developments from different aspects. For example, single-molecule fluorescence in situ hybridization (smFISH) can detect low abundance RNA at the subcellular level. A relatively new aptamer, Mango, is widely applied to label and track RNA activities in living cells. Molecular beacons (MBs) are valid for quantifying both endogenous and exogenous mRNA and microRNA (miRNA). Covalent binding enzyme labeling fluorescent group with RNA of interest (ROI) partially overcomes the RNA length limitation associated with oligonucleotide synthesis. Forced intercalation (FIT) probes are resistant to nuclease degradation upon binding to target RNA and are used to visualize mRNA and messenger ribonucleoprotein (mRNP) activities. We also summarize the importance of some fluorescence spectroscopic techniques in exploring the function and movement of RNA. Single-molecule fluorescence resonance energy transfer (smFRET) has been employed to investigate the dynamic changes of biomolecules by covalently linking biotin to RNA, and a focus on dye selection increases FRET efficiency. Furthermore, the applications of fluorescence assays in drug discovery and drug delivery have been discussed. Fluorescence imaging can also combine with RNA nanotechnology to target tumors. The invention of novel antibacterial drugs targeting non-coding RNAs (ncRNAs) is also possible with steady-state fluorescence-monitored ligand-binding assay and the T-box riboswitch fluorescence anisotropy assay. More recently, COVID-19 tests using fluorescent clustered regularly interspaced short palindromic repeat (CRISPR) technology have been demonstrated to be efficient and clinically useful. In summary, fluorescence assays have significant applications in both fundamental and clinical research and will facilitate the process of RNA-targeted new drug discovery, therefore deserving further development and updating. Discovered in the 1930s, RNA is an essential nucleic acid molecule that participates in genetic information storage, gene expression, and regulation in living cells , microRNFluorescence in situ hybridization (FISH) was first introduced around 1980 . Th. Th77]. Along with the advent of severe acute respiratory syndrome coronavirus 2, also known as SARS-CoV-2, the virus that causes COVID-19, rapid and accurate testing has been vital in slowing its spread. The current gold standard of testing utilizes quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). The basic idea of this method is that viral RNA will be repeatedly copied and duplicated. Fluorescent dyes are added during the copying process, allowing for the amplification and visual detection of viral RNA. The test is extremely accurate, being able to detect just one copy of viral RNA per μL. However, the downside of qRT-PCR is that it requires specialized equipment and, therefore, a centralized lab facility. The run time for the reaction is also several hours. This is why results typically take 1–2 days after collected samples are sent to a lab to be completed.To address these challenges, a clustered regularly interspaced short palindromic repeat (CRISPR)-based technology called Fast Integrated Nuclease Detection In Tandem (FIND-IT) was developed by the Doudna group . As FiguThe tandem use of enzymes is necessary because each enzyme on its own either exhibits reduced efficiency or is unable to produce a detectable signal. For example, when Cas13 is paired to cleave fluorescent dye–quencher pairs, it takes several hours to reach a detectable signal. As for Csm6, recognition of the target sequence normally triggers the synthesis of cyclic tetra- or hexa-adenylates to serve as activators, which are rapidly degraded by the same Csm6. This means that the amount of fluorescent signal Csm6 can generate is limited. Instead, by using both enzymes in sequence, Cas13 is able to cleave a chemically modified activator which is not degraded by Csm6. As a direct result, Csm6 can cleave an indefinite number of dye–quencher pairs, leading to a large, detectable signal from a relatively small amount of substrate. The significance of this assay is that no initial amplification of the viral RNA is necessary, allowing relatively low amounts to be accurately detected. The potential of this technology is immense because, depending on the guide RNA is used, the assay could be programmed to detect a wide array of RNA species associated with different diseases .Overall, this paper summarizes a few developing assays and tools regarding fluorescent probes in combination with RNA, some principles and explanations concerning fluorescence spectroscopy, and the potency and capacities of fluorescence assays in the development of RNA therapeutics in the past few years. However, there are still two significant barriers that need to be overcome. First and foremost, many fluorescence methods are aimed at fixed cells and living cells. The results related to dynamic RNA activities are not as convincing in living organisms. Future efforts could be concentrated on in situ RNA imaging of living animals. Secondly, fluorescence targeting RNA therapeutics are mainly focused on liver cells. In combination with RNA research, an advanced fluorescence assay should be conducted to target cancer or tumor cells in other organs.


Aging is characterized by the accumulation of damage to macromolecules and cell architecture that triggers a proinflammatory state in blood and solid tissues, termed inflammaging. Inflammaging has been implicated in the pathogenesis of many age-associated chronic diseases as well as loss of physical and cognitive function. The search for mechanisms that underlie inflammaging focused initially on the hallmarks of aging, but it is rapidly expanding in multiple directions. Here, we discuss the threads connecting cellular senescence and mitochondrial dysfunction to impaired mitophagy and DNA damage, which may act as a hub for inflammaging. We explore the emerging multi-omics efforts that aspire to define the complexity of inflammaging — and identify molecular signatures and novel targets for interventions aimed at counteracting excessive inflammation and its deleterious consequences while preserving the physiological immune response. Finally, we review the emerging evidence that inflammation is involved in brain aging and neurodegenerative diseases. Our goal is to broaden the research agenda for inflammaging with an eye on new therapeutic opportunities. Aging has been conceptualized as a continuous duel between damage accumulation — due to a combination of environmental and endogenous processes — and resilience mechanisms that cope with such stressors and resolve damage . With agAtop the hierarchy of resilience is the immune system, the aggregate of cells, mediators, and signaling pathways that continuously patrol for pathogens or structural perturbations revealed as “unusual” molecular motifs. The immune system reacts to a variety of threats, such as symbiotic commensal and pathogenic microorganisms, pathogen-associated molecular patterns (PAMPs), and damage-associated molecular patterns (DAMPs) from endogenous and exogenous sources, and orchestrates defense responses aimed at eliminating the specific threat while minimizing damage to the host. While inflammation is important for tissue repair and regeneration, when abnormally intense or persistent, it can drive degeneration and chronic diseases.The immune system undergoes numerous and profound changes with aging, which are extensively reviewed elsewhere –5. HallmConsiderable research in both animal models and humans has examined the causes and consequences of inflammaging . AlthougMechanisms identified as hallmarks of aging biology and immune cell dysfunction have all been hypothesized as causes of inflammation . Aging rGiven the extreme complexity of inflammaging, we focus herein on a few topics that have attracted considerable attention and controversy in the field. First, we discuss cellular senescence as a source of local and systemic inflammation. We highlight evidence that mitochondrial dysfunction is a nexus that binds impaired mitophagy with DNA damage and cellular senescence to ultimately foster a chronic inflammatory state. We then summarize efforts to identify circulating signatures of inflammation through “omics.” Finally, we review emerging data indicating that inflammation is involved in brain aging and dementia. Our intent is to discuss the causes and consequences of inflammaging and to enrich the research agenda toward the development of new therapeutic strategies.Senescent cells and the senescence-associated secretory phenotype (SASP), now widely acknowledged as drivers of aging and age-related diseases, have emerged as key players in inflammaging. Cellular senescence can be initiated by multiple intrinsic and extrinsic nonlethal stresses, including genotoxic, oncogenic, mitochondrial, oxidative, inflammatory, paracrine signaling, and others . HallmarCells of the immune system can undergo cellular senescence or enter a senescent-like state –29. YousINK4a and senescence-associated β-galactosidase  activity accumulate during aging, yet immunomodulatory agents can reverse their expression of these senescence markers , implicating the SASP in the age-related decline of adaptive immunity  and/or modulate the secretory phenotype (senomorphics) as a strategy to mitigate inflammaging and its consequences. Several senolytics reduce or improve inflammatory or age-related phenotypes in preclinical models, specifically atherosclerosis  , 41, ostAn aforementioned complexity of senotherapies is that cellular senescence has beneficial roles in wound healing, tumor suppression, and embryonic development, while also driving inflammaging . RemovalMitochondria provide the energy essential for all cellular activities, including the fueling of resilience mechanisms that counteract macromolecule damage accumulation with aging . Balanci5 modifications per day, many of which are ROS-related single-strand breaks that are detected by the DNA-break sensor poly(ADP-ribose) polymerase 1 (PARP1), and ultimately resolved by defined repair mechanisms   repair, and mitochondrial quality control and biogenesis (SIRT1) pathways, and may also promote senescence  inflammasome, thereby counteracting inflammation . DN. DN66). chanisms . Persist (SIRTs) . Depletinescence . Reducedammation . Indeed,itophagy , 73. Conitophagy .c and precipitates apoptosis , were upregulated well before other gene clusters during the transition from middle to late life. Changes in the expression of these immune gene networks were proposed to originate from the infiltration of T and B cells into a diverse set of organ systems, most prominently in white adipose tissue  a component characterized by simultaneous coexpression of pro- and antiinflammatory proteins that showed a strong positive correlation with age  and (b) a component characterized by an innate immune response that was not correlated with age  . Similare tissue .Several studies have characterized changes in multiple biomarkers following an initial immune challenge. An early study that examined the transcriptional expression response to bacterial endotoxin revealed that different functional networks emerge during the response time course . For exa+ T cells become more abundant, whereas plasmacytoid dendritic cells, naive CD4+ T cells, and CD8+ T cells decline with aging  score that explained a significant portion of the interindividual cytokine response variability . The autE. coli, a large proportion of variation in IL-6 and IL-22 levels after challenge with bacterial stimuli was determined by genetics  and how to specify aspects of inflammation that can be selectively targeted to prevent age-related diseases.Proteomic technologies, including mass spectrometry–based proteomics, aptamer-based arrays (SomaLogic), and proximity extension assay technology (Olink), are now available for the large-scale quantification and validation of thousands of proteins in human biological fluids or tissues –127. AltThe brain is an immune-privileged organ with highly regulated innate and adaptive immune processes designed to maintain homeostasis and quickly clear pathogens while limiting exposure to peripheral immune challenges. However, in the context of inflammaging and dysregulation of the neuro-immune axis, the brain becomes increasingly vulnerable. Immune activation, both within and outside of the CNS, has been observed in many age-related neurological diseases. Mechanisms by which age-related inflammation and neuroimmune activation jointly affect brain health may be targets for disease prevention.Inflammaging can influence homeostatic neurobiological processes and precipitate or perpetuate the development of neuropathology. The blood-brain barrier (BBB) prevents blood molecules from freely entering the CNS and is a key conduit through which inflammatory factors interact with brain targets. The BBB in the brain microvasculature is composed of a continuous endothelial cell layer connected by tight junctions and sheathed by pericytes, basal membranes, and perivascular astrocytes . By shieCirculating TNF-α, IL-1β, and IL-6 bind to receptors in endothelial cells and induce the expression of cellular adhesion proteins  that further promote inflammation by triggering NF-κB signaling  136, 14, 141. ThTREM2, CD33, CR1, GRN, and IL1RAP, and/or are expressed in brain immune cells including microglia  are upregulated to an even greater extent in neurodegenerative conditions such as AD and Parkinson’s disease (PD) , 145–147icroglia . Countericroglia .Neuroinflammation, a CNS-specific process characterized by proinflammatory cellular and molecular changes to microglia, astrocytes, and the BBB, is considered a core feature of aging and age-related neurological conditions, most notably AD, PD, and ALS . Age itsAged glial cells respond less efficiently to antigens, including amyloid-β (Aβ) and α-synuclein, and have reduced phagocytic and antiinflammatory capacity –163. DelThe extent to which neuroinflammation is either protective or pathogenic in the context of age-related changes is unknown and likely dependent on disease etiology and disease stage. For example, in AD and frontotemporal dementia, neuroinflammation and microglia activation potentiate the spreading of pathological tau neurofibrillary tangles , 175. InUnderstanding the causes and consequences of inflammaging is one of the most active areas of research in aging and chronic diseases and one with powerful potential for translation. Work on biomarkers searching for signatures that discriminate between different aspects and triggers of inflammation is currently ongoing. Despite tremendous progress, the robustness and generalizability of developed signatures are not ready for clinical applications . Refinin


Primary bone tumors especially, sarcomas affect adolescents the most because it originates from osteoblasts cells responsible for bone growth. Chemotherapy, surgery, and radiation therapy are the most often used clinical treatments. Regrettably, surgical resection frequently fails to entirely eradicate the tumor, which is the primary cause of metastasis and postoperative recurrence, leading to a high death rate. Additionally, bone tumors frequently penetrate significant regions of bone, rendering them incapable of self-repair, and impairing patients' quality of life. As a result, treating bone tumors and regenerating bone in the clinic is difficult. In recent decades, numerous sorts of alternative therapy approaches have been investigated due to a lack of approved treatments. Among the novel therapeutic approaches, hydrogel-based anticancer therapy has cleared the way for the development of new targeted techniques for treating bone cancer and bone regeneration. They include strategies such as co-delivery of several drug payloads, enhancing their biodistribution and transport capabilities, normalizing accumulation, and optimizing drug release profiles to decrease the limitations of current therapy. This review discusses current advances in functionalized hydrogels to develop a new technique for treating bone tumors by reducing postoperative tumor recurrence and promoting tissue repair. It might be benign or cancerous. The tumor grows and compresses the normal bone tissues in both cases, however benign tumors lack the ability to metastasize and therefore do not spread to other organs of the body. Benign bone tumors can progress to malignancy and pose a risk if remain untreated. Benign bone tumors include osteochondroma, osteoma, osteoblastoma, fibrous dysplasia, and enchondroma  network structure synthesized by crosslinking hydrophilic polymers. Hydrogels have a porous structure similar to that of the extracellular matrix (ECM), are biocompatible, and capable of loading growth factors, which results in effective bone defect repair. As such, they can be utilized as carrier materials for cells proliferation or bone growth to enhance the growth factors in bone tissue. Additionally, because its soft texture is similar to that of many soft biological tissues, it can help to lower the inflammatory reactions of nearby cells and tissues .2.Bone cancers can broadly be divided into primary bone cancers (sarcomas) and secondary bone cancers (metastatic tumors of the bones). The predominant primary bone cancers which constitute nearly two-thirds of cancers include chondrosarcomas, osteosarcomas, and Ewing sarcomas. Primary bone cancers originate from primitive mesenchymal cells in the bones accounting for nearly 0.2% of all malignancies around the world. Secondary bone cancers occur due to various other advanced cancers in the body which spreads to the bones , thus limiting their efficacy , MTX (high dose), or bleomycin, actinomycin-D combinations, and Cytoxan to 27 patients treated without adjuvant chemotherapy - crosslinked hyaluronate (HA) nanoparticle was prepared via a ‘green’ process with responsive behavior toward tumor microenvironment to effectively deliver doxorubicin for the treatment of various stages of osteosarcoma. The developed system exhibited the triggered release at target site with improved survival time, and reduced adverse effects  as a template for enhancing mitochondrial dysfunction caused by Ca2+ overload in cancer therapy while minimizing unwanted off-target consequences  continues to be the main cause of the long-term survival rate of patients associated with osteosarcoma. As a result, new therapeutic options for osteosarcoma are critically needed. The development and applications of nanomaterials in bone ailments have already displayed promising potential. Biomaterials used in bone tumor therapy must accomplish the tasks such as they must target and killing tumor cells as well as stimulating bone repair. Among the various scaffolds used for bone tumors, hydrogels show more benefits over the other counterpart nanosystems and thus are considered the recent research hotspot. These materials show excellent biocompatibility, good drug-loading capacity, biodegradability, and programmable drug release potential and more importantly show less toxicity than other carrier systems , and semi-interpenetrating polymeric network (semi-IPN) hydrogels on the basis of the crosslinking mechanism  polymerization is an adaptable approach for fabricating intelligent hydrogels with improved stimulus-response features, attributed to its ability to effectively build hydrogel precursors with well-defined structures, including block copolymer, graft copolymer, and star copolymer  involved in the cellular defense system against various stressors including heat  was used in the production of a core/shell fiber scaffold, which was 3D printed over alginate-gelatin hydrogel scaffolds, by Liu et al. . Coated 4.5.Injectable biomaterials enable minimally invasive procedures and offer a wide range of uses in bone regeneration. In comparison to conventionally examined scaffolds, injectable hydrogels have several advantages, such as being unique and irreplaceable for filling irregular defects, improving patient compliance with bone injury, and allowing tissue regrowth and regeneration in situ , the blank defect  loaded chitosan (CS) nanoparticles (CCNP) encapsulated silk fibroin (SF)/HA esterified by methacrylate (HAMA) (CCNPs-SF/HAMA) for osteosarcoma therapy and bone regeneration using photocuring and ethanol treatment  is also a type of glycosaminoglycans (GAGs) found in cartilage tissue that interacts with signal transduction, growth factors, and adhesion cells directly or indirectly. In other words, CS is a biocompatible polymer that has been shown to aid bone regeneration /Gel local drug delivery system  Hydrogels are often examined in subcutaneous models that do not accurately depict the complex microenvironment of bone diseases associated with fracture repair or major bone defect repair. (2) While the majority of the test animals are healthy young animals, the practical application of this bone tissue engineering approach is expected to occur in more mature individuals and the elderly, with certain complications. (3) In addition to model challenges, hydrogel storage is a difficult nut to crack, which adds to the complexity of using hydrogels in clinical applications. Hydrogels that swell during storage and transportation are more susceptible to disruption, which can result in medication leakage. To address this problem, dehydration following preparation is recommended; although, this method may alter the structure and properties of reswollen hydrogels. (4) Unlike natural bone tissue, most hydrogels are incapable of self-repair. This disadvantage will result in hydrogels being mechanically damaged during the implantation procedure or bone activity. (5) Numerous functional hydrogels with appropriate mechanical characteristics and high bioactivities have demonstrated beneficial therapeutic effects in vitro and in vivo. However, both the complex preparation techniques and the high cost of hydrogel scaffolds place significant constraints on their further development and use in clinical applications.Based on the foregoing, numerous critical elements should be examined to further develop hydrogel application at the clinical stage. To begin, the rate at which hydrogels degrade should correspond to the rate at which bone defects regenerate. Second, a comprehensive and well-defined clinical criterion for tissue engineering should be developed; concurrently, quality control standards and safety evaluation for these hydrogels should be developed. Additionally, because patients' needs vary, researchers should focus on precision medicine rather than the one-size-fits-all approaches that have gradually become the dominant path of biomaterial design in bone tumor therapy and tissue engineering. Meanwhile, because bone tumor therapy and tissue engineering are complex processes, and each biomaterial has distinct advantages, researchers must select appropriate biomaterials for use as bone repair scaffolds based on the application requirements. As a result, a low-cost, cell-free, and easy-to-manufacture hydrogel system must be designed as a platform for clinical trials, eventually benefiting humans.


Here we present a comprehensive review of the current literature describing mechanisms of damage incurred by cryopreservation, and we highlight several classical and novel strategies to mitigate this damage and increase sperm survival during the cryopreservation process.Cryopreservation is a way to preserve germplasm with applications in agriculture, biotechnology, and conservation of endangered animals. Cryopreservation has been available for over a century, yet, using current methods, only around 50% of spermatozoa retain their viability after cryopreservation. This loss is associated with damage to different sperm components including the plasma membrane, nucleus, mitochondria, proteins, mRNAs, and microRNAs. To mitigate this damage, conventional strategies use chemical additives that include classical cryoprotectants such as glycerol, as well as antioxidants, fatty acids, sugars, amino acids, and membrane stabilizers. However, clearly current protocols do not prevent all damage. This may be due to the imperfect function of antioxidants and the probable conversion of media components to more toxic forms during cryopreservation. Mammalian sperm are among the first cells to be successfully cryopreserved, and over the last seven decades the use of cryopreserved semen for artificial insemination has come to play a crucial role in animal agriculture . For mosIt Is believed that the first successful AI was performed by Italian physiologist Lazzaro Spallanzani in 1780, who inseminated a dog using frozen–thawed semen . SubsequThough references to the preservation of semen date back to the 1600s , it was Cryopreservation is the process by which cells or tissues can survive at low temperatures with reduced or ceased of metabolic activity . During In the presence of extracellular ice during cooling, sperm experience very strong hypertonic conditions because the relative amount of water decreases in the extracellular unfrozen fraction . This rev/v) of permeating cryoprotective agents such as glycerol is almost always included in sperm cryopreservation media in most of the animal species, the equilibration process of sperm with these media can cause large changes in cell volume [The sperm plasma membrane is the major site of damage during cryopreservation . This dal volume . To wit,l volume . 2+ and bicarbonate from the extracellular space [After CPA equilibration, sperm are cooled, which is another source of injury often seen at the plasma membrane . First aar space  that in ar space .The mechanisms responsible for DNA fragmentation after cryopreservation are still not completely understood, though it has been attributed to the increase in oxidative DNA damage . CryoinjHigh DNA fragmentation and low mitochondrial membrane potential are significant contributors to reduced motility after cryopreservation . The mitStudies have demonstrated that cryopreservation can alter the expression level of many proteins related to sperm function . MoreoveSimilar to the results of studies conducted on boar sperm, proteomic analysis of ram sperm found significant changes in the abundance of 51 proteins. The proteins such as T-complex protein CCT subunits, Casein kinase I isoform gamma-2 isoform X2 (CSNK1G2), and TOM1-like protein 1 isoform X2 (TOM1L1) were decreased while the levels of other proteins such as Leukocyte elastase inhibitor (SERPINB1) and Tyrosine-protein kinase Fer isoform X2 (FER) were significantly increased in frozen sperm compared with fresh sperm .These cryopreservation-induced effects on proteins are now being explored in other species. For example, in rainbow trout sperm, cryopreservation can cause the release of proteins related to structure and metabolism from the mitochondria, cytoskeleton, nucleus, and cytosol to the extracellular fluid . A recenEpigenetics encompasses heritable mechanisms that include histone modifications, DNA methylation, and non-coding RNAs . EpigeneFlores et al. found that protein-DNA disulfide bonds and histone H1-DNA binding proteins were altered in boar sperm after cryopreservation . It is sMany methods are available for the cryopreservation of domestic animal sperm. While post-thaw viability varies from species to species and even specimen to specimen, current methods protect only about 50% of sperm against cryoinjury. To improve these results, numerous approaches have been tried to increase sperm resilience during the cryopreservation process. We organize them into two categories. First, we discuss conventional strategies that include cryoprotectants, antioxidants, membrane stabilizers, and other classical media modifications. Second, we introduce some novel recent approaches that have shown some promise. During the last 25 years, the uses of various cryoprotectants and antioxidants have been the conventional strategies against cryoinjury that we will discuss in the following sections.2SO)) [In general, semen extenders must have appropriate osmolarity and buffering capacity along with an adequate pH enabling them to protect sperm cells from cryogenic injury . General2SO)) .Classical penetrating cryoprotective agents (CPAs)  are known to provide a protective effect due to colligative properties . At any Although glycerol has many benefits as a CPA, there are some debates regarding its toxicity when it is applied in high concentrations . Higher On the other hand, a non-permeating cryoprotectant acts extracellularly without crossing the plasma membrane . TherefoEgg yolk is the major component of extenders in most of the animal species and human and low-density lipoprotein is the effective fraction of egg yolk that protects sperm during freeze–thaw . HoweverSperm contains a high amount of polyunsaturated fatty acid (PUFA) that makes it prone to lipid peroxidation due to the massive production of reactive oxygen species (ROS) during cryopreservation . Sperm rThe two main categories of antioxidants are enzymatic and nonenzymatic and both have been well documented as effective strategies for improving cryorecovery and mitigating cryoinjury. The enzyme superoxide dismutase (SOD) in the cytoplasm  and the mitochondria (Mn-SOD) is responsible for combining with two frequent ROS molecules including the superoxide anion  is one of the major compounds with antioxidative properties that is frequently found in the plasma membrane and seminal plasma. This lipophilic antioxidant protects fatty acid contents of membranes against peroxidation  and it h3+ into Fe2+, resulting in a reaction with oxygen or hydrogen peroxide, which then triggers lipid peroxidation [Ascorbic acid (vitamin C) is another water-soluble vitamin attributed with reproduction; however, its exact mechanism is still uncertain . Vitaminxidation .Amino acids are available in seminal plasma and they are counted as non-enzymatic scavengers with antioxidant characteristics. Various types of amino acids such as hypotaurine, glutamine, cysteine, taurine, histidine, proline, and glycine were found to reduce DNA fragmentation and enhance the various post-thaw parameters of ram sperm . SangeetSugars play multifunctional roles not only for regulating osmotic pressure and reducing relative ion concentrations, but also for stabilizing proteins and phospholipid bilayers during the freezing process . They alLactose is another sugar additive that is mainly used in pig semen freezing media . LactoseA brief survey of the types of sugars used in animal semen cryopreservation extender are shown in During cooling, phospholipids in the membrane undergo a phase transition from a liquid phase to the crystalline gel phase. Integral proteins are excluded from the crystalline gel domains and the membrane becomes unstable . AdditioThere are several reports on improved survival rates after the inclusion of cholesterol during cryopreservation of various species’ sperm ,157. ForAs the impact of membrane lipid constituents affects post-thaw viability, it is reasonable to expect that pre-ejaculate diet may impact semen quality, if not cryopreservation success. Towards this, it has been reported that diet components and formulation affect the semen quality in the animal. Zanussi et al. demonstrated that dietary 2% flaxseed oil plus 200 mg/kg vitamin E for 60 days improved aged broiler breeder rooster semen parameters and reproductive performance. This was attributed to possibly increased testosterone, reduced lipid peroxidation, and modification in the content of DPA, DHA, and arachidonic acid of the plasma membrane . In horsThere are significant interactions among cooling rate, warming rate, and survival in cryopreserved semen . From a Classical approaches to improve recovery after cryopreservation have been mostly chemical, with some careful attention to adjusting cooling and warming rates. Here we highlight several novel approaches outside of these paradigms that have been applied to increase sperm cryosurvival. Some of these approaches include mild sublethal stress induced before freezing, induction of a magnetic field before freezing, nanoparticle-enhanced cryopreservation, sperm preservation via freeze-drying, and monolayer centrifugation.Mild sublethal stresses induced before freezing, including hydrostatic , osmoticThe use of “magnetized” extenders treated with a high magnetic field has increased the cryo-survival of sperm in some species. This approach has been employed to interrupt the regular network of water molecules in the cryopreservation medium to reduce the formation of hexagonal ice crystal, which is the most destructive form of ice for sperm. ,189. Via2) nanoparticles have beneficial effects on ram spermatozoa during cooling [2) NPs act as ROS scavengers to protect the integrity of DNA and plasma membranes. As their name suggests, nanoparticles (NPs) are particles at the nanometer scale, with flexible fabrication and a high surface area to volume ratio . In the  cooling . They foIt was reported that selenium nanoparticles (SeNPs) have been used as a scavenger of ROS to protect sperm cells against oxidative stress . Safa etThe freeze-drying of sperm is a simple preservation technology that has been investigated for decades but has sparked much recent enquiry. It is an alternative, low-cost storage option for biodiversity preservation of domestic species . An attrMoreover, other studies reported live offspring in mice , rat 20, rabbit Single-layer centrifugation (SLC) has been shown to separate robust sperm from the rest of the ejaculates; therefore, it can increase the percentage of cells with higher resistance against cryo-damages. This approach, which is based on colloid centrifugation, can select sperm with desirable characteristics and consequently could end up with more viable sperm post-thawing . In thisWe are in the midst of a biodiversity crisis that has been termed the sixth mass extinction event in the history of Earth . Loss ofBiobanking is a rapidly growing industry, covering diverse fields such as human medicine, farm animal production, laboratory animal record-keeping, and wildlife. Different reproductive cell types are now stored in cryobanks: mainly semen and embryos in mammalian species, semen and primordial germ cells in birds, semen and germ cells in fish, and semen and larvae in shellfish. Some cryobanks also include somatic cells with the hope that, in the future, these cells will be reprogrammed to become inefficient reproductive cells . In FranThe cryopreservation of domestic animal sperm is a complex procedure that involves the regulation of many agents to achieve desirable results. To ensure even minimal success, suitable diluents, sperm dilution rates, cooling rates, and thawing rates are needed. Moreover, complete knowledge of the complex sperm physiology for each species is required to maximize the post-thaw recovery of sperm and consequently, fertility.Although the first successful sperm cryopreservation was over 60 years ago, current methods result in post-thaw spermatozoa with about 50% post-thaw viability for most species. Likely due to the wide variation among species’ sperm, a standardized cryopreservation process has not been established. Some measures, including specific cryoprotectants, have been applied to improve the survival and fertility of frozen animal sperm. Factors leading to spermatozoa cryoinjury are complicated and have not been fully explored, and the extensive and severe cryoinjury to the structure and physiological function of domestic animal sperm have been confirmed. Currently, the cryoinjury mechanisms in mammalian and avian spermatozoa are of considerable interest in the field of cryobiology and reproductive science. Because the functional role of spermatozoa is regulated by proteins and nucleic acids, a molecular explanation may be necessary. Research related to cryopreservation of domestic animal semen has progressed greatly in recent years; however, significant room for improvement still exists. The current technological advances, such as vitrification, freeze-drying, and high-throughput sequencing technologies, can provide a new perspective to improve the cryopreservation efficiency of animal sperm.


Malaria remains a global disease of public health concern. Malaria control collaborative initiatives are widely being adopted to reduce malaria burden by various countries. This review sought to describe current and past cross-border malaria control initiatives focusing on key activities, outcomes and challenges. An exhaustive search was conducted in Web of Science, PubMed, Google Scholar and EBSCOhost using the following key words: cross-border malaria control, cross-border malaria elimination, bi-national malaria control and multinational malaria control, in combination with Boolean operators “AND” and “OR”. Eleven studies satisfied the inclusion criteria for this review. The majority of collaborative initiatives have been formed within regional developmental and continental bodies with support from political leadership. The studies revealed that joint vector control, cases management, epidemiological data sharing along border regions as well as resource sharing and capacity building are some of the key collaborative initiatives being implemented globally. Collaborative initiatives have led to significant reduction in malaria burden and mortality. The majority of collaborative initiatives are underfunded and rely on donor support. We concluded that cross-border malaria collaborative initiatives have the capacity to reduce malaria burden and mortality along border regions; however, inadequate internal funding and over-reliance on donor funding remain the biggest threats to the survival of collaborative initiatives.  Malaria remains a major public health concern with at least 240 million cases and over 600,000 deaths recorded globally . Global Key obstacles to the achievement of the malaria elimination agenda include, among others, vector resistance to insecticides, emergence of parasite resistance to current malaria treatment medicines and border malaria . “Cross-One of the significant calls under the GTS is for member states to deepen country-to-country and regional collaboration . In respMalaria control programs exerts a huge financial cost on any economy particularly medium- to low-income countries . The majThreats of malaria re-establishment  and impoWe conducted a scoping review of literature in Web of Science, PubMed, Google Scholar and EBSCOhost (Medline). The search considered articles published in English journals. The search used Boolean operations “AND” and “OR” with a combination of the following key terms: cross-border malaria control, cross-border malaria elimination, bi-national malaria control, multinational malaria control. Additional literature was also obtained through a snowballing technique using bibliographies of previously published articles and reference lists. After the initial search, duplicates were removed. The remaining articles were screened by title and abstract. The process leading to papers selected for full review was informed by the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA)  30]. A . A 30]. Studies were included in the review if they were published in an English peer reviewed journal and reported on a past or existing bi-national, tri-national, regional or continental cross-border malaria control collaborative initiative. Any literature that did not satisfy the inclusion criteria was excluded from the review.A data capturing template shown in A total of eleven (11) articles were included in this study from the initial yield of 1405 papers searched from Web of Science, Google scholar, PubMed and EBSCOhost (Medline). A total of 44 duplicates were removed from those that were screened. The review further removed 1340 papers which were deemed not necessary for this review . Table 1The collaborative organisation and setup were classified into four main categories: (i) Bilateral Initiatives, (ii) Tri-national Initiatives, (iii) Regional Initiatives and (iv) Continental Initiatives.Three papers reported on country-to-country arrangements: the Trans-Kunene, French Guiana–Brazil and Peru–Ecuador  collaborTwo papers reported on the tri-national collaborative partnership of the Lubombo Spatial Development Initiative (LSDI). This partnership was formulated by Mozambique, South Africa and Eswatini (formerly Swaziland) ,34. The Cross-border malaria control’s success is highly dependent on inter-country collaboration as parasites and vectors transcend geographical borders. Asian Pacific regional political leaders formulated the Asia Pacific Malaria Elimination Network Alliance (APMEN) through the Asia Pacific Leaders Malaria Alliance in 2008 . SimilarThe African heads of states formed the African Leaders Malaria Alliance (ALMA) in 2009  to lead A search on the WWW in combination with bibliographical referencing revealed six initiatives outlined in Sharing of health data is critical in assessing performance of interventions as well as assisting in the evaluation of malaria epidemiological trends, vector bionomics and treatment efficacy. The E8, APMEN, French Guiana–Brazil partnership, TKMI, MOSASWA and LSDI initiatives created platforms for the sharing of technical and epidemiological data across member states ,35,36,41Border regions between states often share common climatological, vectoral and community social characteristics. These conditions are critical determinants of an area’s vulnerability and receptivity to malaria transmission. To ensure universal malaria control coverage and standardisation of intervention along border regions the LSDI  and MOSAResource inadequacies significantly affect program performance. In order to close resource gaps in a common border region, health officials in the Peru–Ecuador border region of El Oro and Tumbes shared medicines . SimilarMalaria control interventions require huge monetary resources to finance medicines, equipment and human resources. Countries within the Asia Pacific and Southern African regions have successfully used collaborative initiatives to secure funding from multilateral funders and GF to sustain malaria control and elimination programs. The APMEN , MOSASWAPolitical leadership ownership and support is critical for the sustainability of any health intervention. The French Guiana–Brazil partnership, TKMI, LSDI and MOSASWA were created by countries under the auspices of economic development partnership agreements between states. In addition, APMEN and E8 alliances were created under the auspices of regional development blocks, namely the Asia Pacific Leaders Malaria Alliance (APLMA)  and SADCBorder areas in low resource settings with high malaria burden are often characterised by poor access to health services and low intervention coverages. These regions tend to be highly receptive and vulnerable to malaria transmission. The E8 initiative has embarked on the building of “health posts” along member state border regions . Health It is critical that human resources responsible for the functioning of collaborative partnerships be endowed with the requisite skills to carry out critical tasks related to malaria programming. The APMEN initiative has invested in technical meetings and training programs whilst the China–Africa ,35 initiThe implementation of cross-border collaborative activities has resulted in significant reduction of malaria burden within endemic regions of Africa, Americas and South East Asia. Sharp et al.  and MahaWe reviewed eleven (11) papers which reported on 10 cross-border malaria control collaborative efforts in regions in Africa, the Americas and Asia. These initiatives were formulated based on geographical proximity, common ecological zones and regional integration. The initiatives succeeded in reducing malaria mortality and burden across common border regions. These collaborative initiatives have also facilitated malaria surveillance health information, the conducting of joint malaria control programs and knowledge and technical expertise transfer between member states. Commitment of political leadership to the goals of elimination of cross-border malaria across the globe is evidently growing. Inadequate internal funding capacity and total reliance on donor support are major threats to border malaria control collaborative initiatives.The bi-national and tri-national program outcomes of LSDI, MOSASWA, TKMI and Peru–Ecuador have shown the potential of alliances to reduce malaria mortality and morbidity along border regions. The reduction in malaria burden could be attributed to joint vector control and treatment programs along the border regions. IRS, LLINS and use of anti-malaria medicines have been shown to be effective tools in controlling malaria ,34. JoinThe nexus between malaria and poverty has been established before . CoincidIncreased commitment by political leaders for integrating efforts to combat border malaria and non-border malaria has gained prominence since the mid-1990s. The involvement of regional bodies as well as political leaders ensures long-term sustainability of initiatives . The creData sharing challenges reported by Raman et al. in E8 are a cause of concern . The chaMalaria control interventions such ITNs/LLINs, IRS and case management play an important role in malaria burden reduction. Evidence from TKMI has shown that joint border malaria control interventions could be more effective if they incorporate risk communication and behaviour change activities. Health education and correct risk communication messages improve community ownership, and uptake of community programs. It is critical that all collaborative initiatives incorporate and elevate community engagement initiatives within core interventions such IRS, LLINS distribution and case management. Risk communication and community engagement have been shown to be beneficial as they have the ability to increase case detection, uptake of interventions, access to health and positive behaviour change . Despite the various challenges of implementing cross-border malaria control, it is critical to note that cross-border malaria control and malaria elimination are public goods with positive spill over epidemiological, social and financial benefits to neighbouring countries . The retThe aim of this review was to identify all existing cross-border malaria initiatives and document their key activities, aims and objectives, compositions, outcomes and challenges. However, despite a comprehensive systematic search, only eleven (11) articles were identified and not all the articles documented the required variables. Three reviewed articles did not report both outcomes and challenges ,35,38 whOur review revealed the value of collaborative initiatives by various countries and regional bodies in reducing malaria burden along border regions. Therefore, cross-border malaria control initiatives should continually be adopted across the globe as an effective tool to attain malaria elimination and suppress border malaria. Malaria epidemiological data sharing, joint vector control, surveillance, case management and resource sharing are some of the critical key activities being undertaken by cross-border malaria initiatives globally. Unwillingness by member states to share data and adopt novel methods are key challenges in some regional initiatives. Evidently, there is growing political commitment to a cross-border malaria elimination agenda as evidenced by political leader platforms giving birth to some of the initiatives. The majority of collaborative initiatives in low resource settings are heavily dependent on donor support; therefore, there is a greater need to create sustainable funding mechanisms as well as grow domestic funding of border malaria control programs.


Combining polarization modulation Fourier analysis and spatial information in a joint reconstruction algorithm for polarization-resolved fluorescence imaging provides not only a gain in spatial resolution but also a sensitive readout of anisotropy in cell samples. Polarization-modulated fluorescence microscopy provides molecular orientation mapping in cells. Achieving high polarization sensitivity, however, requires labelling protein domains in a rigid way to limit the orientational fluctuations of the fluorescent label5, which is not always accessible. FPM is also limited to the diffraction scale, which does not permit the study of fine structure organizations, typically occuring in cell organelles made of folded or highly curved membranes.Cells and tissues are made of complex assemblies of proteins and lipids, which nanoscale structure is intrinsically related to their functions. Studying such structures is determining to understand pathologies, however, they cannot be accessible via standard fluorescence microscopy imaging due to their sub-diffraction size. Despite the great progresses made in super-resolution fluorescence imaging to surpass the diffraction scale (~200 nm), an important missing information to reveal such complex architectures is the orientation of proteins and lipids. Orientation is accessible via the readout of fluorescence sensitivity to polarization, typically through the measurement of a signal modulation upon the rotation of a linear excitation polarization. Fluorescence Polarized Microscopy (FPM) has permitted to quantify the degree of anisotropy and orientation of fluorescent labels in lipid membranes7. This method however does not give access to molecular orientation, and raised questions as to whether the nature of the resolution gain is due to polarization modulation. Later a method called super-resolution dipole orientation mapping (SDOM)8 demonstrated that the combined implementation of spatial deconvolution and polarization modulation analysis can provide orientation and intensity information beyond the diffraction limit. SDOM still uses, similarly as SPoD, a sparsity constraint hypothesis of the sample and the need to tune regularization parameters, however, it provides quantitative information of the signal modulation. In parallel with these developments, other demonstrations of super-resolution orientation imaging have been developed, including Polarized Structured Illumination Microscopy (pSIM)9, Single Molecule Orientation and Localization Microscopy (SMOLM)13, or the use of other optical contrasts such as Raman imaging14 or two-photon nonlinear coherent imaging15.Exploiting light polarization to additionally improve spatial resolution in fluorescence imaging is a topic of increasing interest. The fluorescence modulation provided by a rotating linear polarized excitation is indeed an additional parameter that, despite spatial convolution, provides a finer spatial signature of molecular organization, which is sensitive to sub-diffraction scales. To achieve super resolution in polarization microscopy, super-resolution imaging through sparse deconvolution of polarization-modulated fluorescent images (SPoD) has been first implemented, based on a sparsity penalty-enhanced estimation by demodulation (SPEED) algorithmLight: Science & Applications16, M. Guan et al. implement a novel analysis approach, termed optical lock-in detection super-resolution dipole orientation mapping (OLID-SDOM), to push forward the capabilities of dynamic, super-resolved orientation imaging in pure wide field FPM. The algorithm is based on a joint reconstruction of both spatial and polarization modulation information without involving sparsity constraints. The image reconstruction relies on an optimization problem that discriminates the modulated and the unmodulated contributions of the signal. The speed of the algorithm is improved by using Fast Fourier Transform phase extraction of the modulation, providing additionally similar denoising advantages as in a lock-in process and fast data processing as compared to iterative algorithms, such as proposed in a related approach17. Ultimately, both phase and amplitude of the polarized fluorescence modulation are retrieved, providing both molecular orientation and degree of orientational order (named Orientation Uniform Factor) in a super-resolved image. The technique can achieve 100 frames per second, and resolution improvement with orientations distances down to 50 nm scales, with possible extension to 3D imaging.In the article in The striking advantage of the approach is a gain in background rejection, with reduced artifacts under low signal-to-noise ratio (SNR) and therefore ability to measure orientation information under extremely low anisotropy conditions. This appears to be particularly relevant in situations where the polarization modulation is very weak, where the image is not sparse , or if the image is polluted by important background signal, which is often the case in wide-field microscopy. The most remarkable result found is that even in GFP labelled samples that a priori do not exhibit a particular strategy to minimize the wobbling of the label fluorescent protein, a weak fluorescence anisotropy is found. This finding makes orientation mapping a promising and interesting tool to not only understand the fundamental processes at the origin of this anisotropy, but also image biological processes in a large number of subcellular structures with high spatial resolution.The authors imaged different subcellular organelles in yeast encoded with specific GFP-tagged proteins; in lipid particles and vesicles, the Golgi complex, actin, spindle pole and endosome. They also demonstrated high-resolution imaging of mitotic spindles and microtubules, as well as a two-color image of F-actin bundle structures together with weakly anisotropic lysosomes. At last, the ability of OLID-SDOM to produce super-resolution images with a high temporal resolution permitted the imaging of highly dynamic cellular processes such as nanoscale morphological changes of mitochondria, which is an important readout of energy regulation in cells. Millisecond-scale remodeling and growth of sub-mitochondrial regions could be in particular reported, as well as time-lapse orientation dynamics of dendritic spines in live neurons in interconnected networks.5, this makes the potential of this method even stronger due to its sensitivity to small anisotropy changes. Other methods could benefit from the data processing presented in this approach, such as pSIM which intrinsically uses super-resolution imaging9, sectioned spinning disk confocal polarized microscopy18, or fast polarized nonlinear scanning microscopy20. At last, the achievements shown in this work demonstrate the high potential and promising future of polarized microscopy for biological imaging, even though challenges remain open such as the monitoring of 3D orientations in dense samples, a topic so far only addressed at the single molecule scale13.OLID-SDOM is thus a promising tool to study highly resolved spatio-temporal structural information in cells, in particular in neuron where synaptic activity is dynamic. With the recent efforts made in developing strategies to minimize orientational fluctuations of genetically encoded fluorescent protein labelling