T cells recognize antigen fragments from proteolytic products that are presented to them in the form of peptides on major histocompatibility complex (MHC) molecules, which is crucial for the T cell to identify infected or transformed cells. Autophagy, a process that delivers cytoplasmic constituents for lysosomal degradation, has been observed to provide a substantial source of intra- and extracellular antigens for MHC presentation to T cells, which will impact the tumor-specific immune response. Meanwhile, extracellular components are transported to cytoplasm for the degradation/secretion process by the endo-/exosomal pathway and are thus involved in multiple physiological and pathological processes, including immune responses. Autophagy and endo-/exosomal pathways are intertwined in a highly intricate manner and both are closely involved in antigen processing for MHC presentation; thus, we propose that they may coordinate in antigen processing and presentation in anticancer T cell immune responses. In this article, we discuss the molecular and functional crosstalk between autophagy and endo-/exosomal pathways and their contributions to antigen processing for MHC presentation in anticancer T cell immune responses. In eukaryotic cells, MHC presentation monitors two proteolytic routes: the ubiquitin-proteasome and the lysosomal systems. Both of these systems are involved in the degradation of endogenous and exogenous antigens. The lysosomal system degrades and recycles long-lived proteins and defective organelles , 2, in wMacroautophagy (hereafter referred to as autophagy), a cellular self-consumption process, is the main form of autophagy. Basal autophagy enables cells to recycle cytoplasmic constituents and restore metabolic homeostasis, thereby maintaining cellular survival . AberranAutophagy has also been implicated in the exosome secretory pathway . An exosRecent studies suggest that autophagy and endo-/exosomal pathways are closely involved in antigen processing for MHC presentation, which results in the activation of tumor-specific T cells. However, thoroughly understanding the inter-regulations between autophagy and endo-/exosomal pathways in antigen processing is an interesting challenge. In this review, we focus on the crosstalk between autophagy and endo-/exosomal pathways and their contributions to antigen processing for MHC presentation in cancer.More than 30 autophagy-related gene (ATG) proteins are involved in the complex processes of autophagosome formation, encapsulation of target cargoes, and subsequent fusion with the lysosome for degradation , 25. AutPerpendicular to the autophagy process is the endosomal pathway. Numerous studies have shown a close relationship between the autophagy pathway and the biogenesis and secretion of exosomes , 34, 35.Phagocytosis  extracellular vesicles (EVs) formed in vesicular bodies in the endosomal network and can be released by almost all types of cells, including cancer cells. Exosomes play an essential role in cell-to-cell communication, both locally and systemically, by exchanging of their contents, including a subset of proteins, lipids, and functional genetic material derived from the parent cells –61. Emer+ and CD4+ T cells against cancer cells, respectively [Through intercellular communication, exosomes stimulate the immune system to produce antitumor responses, of which the key factor is the APCs, which present MHC-peptide complexes to T cells. Initial studies of the proteome of DEXs revealed a unique molecular composition that endows them with strong immunostimulatory properties in antigen processing and presentation . In 1996ectively . Furtherectively . Additioectively –69. Heatectively .Two mechanisms have been proposed for how DEXs present antigens via their MHC molecules to stimulate T cell responses: direct and indirect pathways Fig. . It was Indirect DEX-to-T cell presentation following interactions of DEXs and DCs is another pathway that stimulates T cell responses and is likely to be the most fundamental pathway in vivo Fig.  63, 71,,71, 75. Different from DEXs, in terms of the immune system, TEXs play dual roles in modulating tumor immunity: immunosuppression and immune activation. TEX properties are distinct from the properties of exosomes secreted by normal cells, except TEXs are rich in various immunosuppressive molecules. TEXs also carry tumor-associated antigens (TAAs), a variety of co-stimulatory proteins and MHC molecules, all of which enable them to stimulate immune responses , 83. The+ T cell activation via the reprocessing or cross-dressing process, which results in tumor rejection in various prophylaxis and therapeutic murine tumor xenograft models [+ T cell-mediated antitumor effect not only on the autologous tumor, but also against other related tumors expressing the same tumor-rejection antigens [+ T cells in vivo and inhibit tumor growth in mice. On the basis of these clues, TEXs have been developed as cancer-specific vaccines for clinical application. In fact, TEX vaccines from patients with metastatic melanoma, advanced colorectal cancer, and non-small cell lung cancer have been tested in phase I and/or phase II clinical trials [Early studies showed that TEXs containing native tumor antigens can be efficiently transferred to DCs and induce antigen-specific CD8t models –87. Moreantigens . Anotherantigens –91. Bothl trials –97.+ T cell recall responses in vivo, leading to a synergistic effect against pre-established tumors [However, the antitumor immune responses induced by TEXs are mild, and thus, many strategies have been adopted to develop modified TEXs to elicit a more efficient antitumor immune response Fig. . One of d tumors .Fig. 3MoThe notion that autophagy and endo-/exosomal pathways are distinct should be reconsidered because they share many components and are intertwined in a highly intricate manner. In essence, autophagy can regulate endosomal secretion to form extracellular vesicles, which can in turn regulate autophagy in a paracrine manner. Recent studies suggest that autophagy plays such a role in the context of anticancer T cell immune responses, while exosomes have been observed to play crucial roles in carrying and presenting functional MHC-peptide complexes to modulate tumor-specific T cell activation. Therefore, we predict that antitumor immune responses could be regulated by modulating the molecular interactions between the autophagy and endo-/exosomal pathways according to the status of cellular metabolism. Despite the major challenges that may be encountered in further investigation of the precise regulation of these two pathways to achieve the expected effective anticancer immune response, the prospect of autophagy- and exosome-associated immunotherapy as a novel cancer treatment remains highly promising.


Brain structural changes in schizophrenia are thought to arise in part from genetic liability, as shown in studies of twins and siblings. Polygenic risk scores (PGRS) derived from large-scale genome-wide association studies (GWAS) have allowed to use measures of genetic liability calculated from large numbers of individual single nucleotide polymorphisms (SNPs). Initial studies on PGRS and structural imaging have, however, failed to provide clear associations. We used three separate measures of brain morphometry  in a sample of healthy subjects to associate them with PGRS for schizophrenia in order to test the hypothesis that gyrification, a putative indicator of early brain development.We analysed high-resolution MRI scans  from n=153 healthy subjects with not current or previous psychiatric condition recruited from the local community. DNA from each subject was analysed using the PsychChip, and polygenic risk scores were calculated for schizophrenia, as well as bipolar disorder and major depression (for assessment of relative specificity of the schizophrenia PGRS). MRI data were pre-processed with the CAT12 toolbox (dbm.neuro.uni-jena.de/cat12) for analysis using a) voxel-based morphometry (VBM), b) cortical thickness, and c) gyrification . We initially used p<0.001 uncorr. on the peak-level and performed correction for multiple-comparisons on the cluster level.We found a negative correlation of the schizophrenia polygenic risk score with gyrification in the left anterior superior cortex , which was significant at the cluster-level for FWE correction (p<0.047). There was not such significant finding for positive correlations, nor for any of the VBM or cortical thickness analyses. Also, there was not significant association (positive or negative) with major depression or bipolar disorder PGRS in any of the three morphometry analyses.Our findings suggest that SNP-based genetic risk for schizophrenia is associated with left temporal gyrification, a putative indicator of early brain development, which again might be affected by multiple schizophrenia risk genes regulating cortical formation and connectivity. Furthermore, our findings are consistent with the notion of specificity for both morphometric marker  as well as diagnosis (with negative findings for major depressive and bipolar disorder risk scores). PGRS might impact on early developmental markers of brain structure (and possibly function), rather than overall liability-related variance.


An organism’s immune response to a vaccine is dependent on a number of factors, including the site of immunization. While muscle is the most common site for vaccine administration, other sites, including the salivary gland, are poised to confer stronger and broader immunoprotection.Studies exploring the salivary gland as an immunization site have involved protein antigens, as well as live pathogens and DNA vaccines. While intraductal instillation of protein antigens into the salivary gland may result in a relatively transient increase in antibody production, DNA or attenuated pathogen vaccination appear to confer a lasting widespread mucosal immune response that includes robust salivary and enteric IgA, as well as high levels of circulating IgG. Furthermore, vaginal and lung antibodies are also seen. For enteric pathogens, a common class of pathogen encountered by travelers, this type of immune response provides for a level of redundant protection against foreign microbes with mucosal targets.The strength of immune response conferred by salivary gland vaccination is generally stronger than that seen in response to the same vaccine at a comparison site. For example, where other routes fail, immunization of the salivary gland has been shown to confer protection in lethal challenge models of infectious pathogens. A host of vaccines currently under development suffer from immunogenicity challenges, adding to the widespread interest and search for novel routes and adjuvants. With its capability to facilitate a strong and broad immune response, the salivary gland warrants consideration as an immunization site, especially for vaccines with immunogenicity challenges, as well as vaccines that would benefit from combined systemic and mucosal immunity. Vaccine delivery leading to systemic antibody-mediated immunity is usually administered through intramuscular injection (IM). Although the resulting humoral immune response forms the basis for protection conferred by nearly all vaccines delivered today, immune responses to the vaccines for bacille Calmette-Guerin (tuberculosis) and zoster trigger a strong cell-mediated immunity component . Most pan = 20) following intranasal delivery, and 380 (n = 20) following sublingual delivery [While most vaccines continue to be administered parenterally, a number of mucosal vaccines are commercially available. The two routes of administration for the currently licensed mucosal vaccines are oral and nasal, and examples of orally-administered vaccines include vaccines for rotavirus, poliovirus, and cholera . Mucosaldelivery . The admdelivery . AnatomiThere are three major bilateral salivary glands, the parotid, submandibular, and sublingual (collectively referred to as the SG). They are encapsulated exocrine glands responsible for the manufacturing of saliva, a complex fluid that includes significant amounts of peptides, proteins, and carbohydrates. The SG is also an immune organ, constantly coming in contact with external pathogens, and may be an optimal immunization site for certain vaccines. In addition to containing dendritic cells, a type of antigen presenting cell, the SG also appears to be a site of B cell activation and isotype switching, along with induction of antigen-specific cytotoxic T lymphocytes , 7.Escherichia coli [Antibodies have long known to be present in saliva, though they have largely been viewed as potential by-product surrogate markers of exposure to various infectious diseases –10. Varyhia coli . This wiWhile immunizations at various sites can result in measurable changes in salivary antibodies, both serum and salivary antibodies have been reported to result from repeated intraductal instillation of either protein or formalin-fixed pathogen into the SG. One early study found a strong IgA response following the injection of bovine serum albumin (BSA) into surgically-exposed SG . HoweverWith an aim toward gaining a better understanding of inflammatory processes in the parotid gland, the group assessed locally-secreted antibodies in response to antigen instillation of the parotid gland. Specifically, the group investigated both salivary and systemic antibodies resulting from intraductal instillation of BSA , 14. TheStreptococcus mutans. Formalin-killed S. mutans was being injected by various routes by different groups in an attempt to vaccinate various animal models from dental caries, including intravenously and subcutaneously in the mouth [S. mutans bilaterally into the parotid glands of non-human primates [S. mutans organisms in a particular plaque were reduced in the immunized group.Around the same time as the Israeli group was exploring correlates of antigen-induced parotiditis, there was broad scientific effort in trying to develop a vaccine for dental caries, and specifically against the main offender, he mouth . One groprimates . While tprimates . Both thmutans three times in a single parotid [A study comparing multiple subcutaneous immunizations in the vicinity of the salivary glands with multiple bilateral immunizations via intraductal instillation found the subcutaneous route induced only a serum response while intraductal instillation resulted in both serum and salivary IgA responses . This fi parotid . While t parotid . ImmunogPorphyromonas gingivalis fimbriae [The first study to target the SG as a potential immunization site using plasmid DNA (pDNA) employed an expression system coding for fimbriae . Strong fimbriae . While tBacillus anthracis  [A more extensive study, also done in rats, involved the bilateral instillation of 88 μg of pDNA coding for either of three antigens, human growth hormone (hGH), HIV envelope protein gp120, or protective antigen (PA) from anthrax) . As companthrax) .Lastly, in an effort to explore the scalability of intraductal instillation of a DNA vaccine to larger animals, the method was extended to beagles in a follow-up study . SolutioThough the SG is a well-established effector site for antibody-secreting B cells, induction of B cells is generally more restricted to specific sites, such as mucosa-associated lymphoid tissue of the ileum . HoweverVaccination using live pathogens has long been demonstrated to elicit broad and lasting immune responses. Indeed, the first vaccines administered were all live . In an eA subsequent comprehensive study by the same group employed the gentler route of intraductal instillation. Here, instilling either a replication-deficient recombinant adenovirus expressing individual MCMV genes or tcMCMV resulted in protection against a lethal systemic challenge with MCMV . Due to Here we have attempted to provide an overview of the SG as an immunization site, and synthesize a number of studies suggesting it be included as a selected immunization site in the development of novel vaccines, especially vaccines that do not elicit strong immune responses. Table As shown in the Table, various groups have demonstrated the SG to be capable of eliciting robust widespread mucosal and systemic protective responses in following antigen presentation, routinely resulting in antibody titers higher than those seen with other immunization targets. It is notable that studies have generally instilled the particular immunogen or vector  in the absence of an adjuvant, yet still observed significant immune responses. Indeed, while complete Freund’s adjuvant has been used in studies employing subcutaneous immunizations delivered in the vicinity of the salivary glands (reviewed in ), very fWhile intraductal instillation into the SG is not an obvious route for vaccination, the potential benefits seen by a robust immune response would warrant the additional research effort. The practicality of mass immunization via the SG may be logistically challenging. However, infusion of the gland is a relatively simple procedure that can be done in an outpatient setting, such as during a routine dental check-up. While intraductal instillation is quick and painless, requiring neither needles nor anesthesia, most health-care workers have only modest familiarity with the procedure; and so training and instrumentation advances that simplify vaccine administration would significantly increase the amenability of the SG as a preferred site.Additionally, saliva contains a number of carbohydrates, peptides, and enzymes, and this complex mixture may require novel formulation approaches to maximize vaccine efficiency and bring an SG-based vaccine into practical application. While the infusate vehicle in most SG immunization studies has been either water or a simple saline solution, the inclusion of mucoadhesives, protease, or nuclease inhibitors could further increase immunization efficiency. For example, adding zinc to the ductal infusate has been reported to result in a 20-fold increase in transgene expression, which would be important for DNA-based vaccines . Neverth


In vivo confocal microscopy (IVCM) shows enlarged and irregular nuclei with hyperreflective cells in OSSN lesions and this has been found to correlate with histopathology findings. Anterior segment optical coherence tomography (AS-OCT) demonstrates thickened hyperreflective epithelium with an abrupt transition between abnormal and normal epithelium in OSSN lesions and this has also been shown to mimic histopathology findings. Although there are limitations to each of these imaging modalities, they can be useful adjunctive tools in the diagnosis of OSSN and could greatly assist the clinician in the management of OSSN patients. Nevertheless, anterior segment imaging has not replaced histopathology's role as the gold standard in confirming diagnosis.Recent advances in anterior segment imaging have transformed the way ocular surface squamous neoplasia (OSSN) is diagnosed and monitored. Ultrasound biomicroscopy (UBM) has been reported to be useful primarily in the assessment of intraocular invasion and metastasis. Invasive squamous cell carcinoma is defined as when the lesion has affected the epithelial basement membrane and substantia propria [Ocular surface squamous neoplasia (OSSN) is the most common tumor affecting the ocular surface in adults . OSSN wa propria , 5. It c propria .OSSN occurs worldwide but has the highest incidence rates in Africa . Risk faThe gold standard for confirming diagnosis of OSSN is excisional biopsy for histopathology. This technique, however, is not without its limitations. Biopsy for histopathology may miss lesions that are not included in the excised tissue as diffuse lesions can be difficult to excise with clear margins. Additionally, since OSSN can recur even after successful treatment, repeated excisional biopsies may cause conjunctival scarring and limbal stem cell deficiency . AdjunctAlthough now rarely done, IC can be useful in diagnosis and has been shown to correlate closely with histopathology , 15. In Another diagnostic test that is inexpensive and helpful in identifying OSSN is dye staining. Diagnostic dyes like lissamine green and rose bengal are routinely used to stain and delineate the extent of OSSN lesions but since these dyes are nonspecific and stain many other ocular surface conditions, it is not possible to diagnose OSSN with the use of these dyes alone. Other vital dyes that have been studied in the diagnosis of OSSN include toluidine blue (ToB) and methylene blue. ToB and methylene blue are acidophilic dyes that stain abnormal tissue dark royal blue. They have an affinity for nucleic acids and, given the increased nuclear material from high rates of mitoses and poor cell-to-cell adhesion in malignancy, these tissues stain more frequently than benign tissues . Several in vivo confocal microscopy (IVCM), and anterior segment optical coherence tomography (AS-OCT) in the diagnosis and monitoring of OSSN. We performed a comprehensive review within the peer reviewed literature using http://pubmed.gov/. The following search terms were used: ocular surface squamous neoplasia, conjunctival intraepithelial neoplasia, corneal intraepithelial neoplasia, carcinoma in situ squamous cell carcinoma, impression cytology, toluidine blue, methylene blue, ultrasound biomicroscopy, in vivo confocal microscopy, and anterior segment optical coherence tomography.Given the limitations of IC and vital dye staining, there is now increased interest in the use of anterior segment imaging techniques to assist in the diagnosis of OSSN. This is becoming especially pertinent since current management options for OSSN include not only surgical excision with cryotherapy but also primary medical therapy with the use of topical chemotherapy such as mitomycin-C, 5-fluorouracil, and interferon alfa-2b. In this review, we will discuss the use of ultrasound biomicroscopy (UBM),Ultrasound biomicroscopy (UBM), which was first developed by Pavlin et al. in 1990, provides cross-sectional visualization of the anterior segment in an intact globe at microscopic resolution . UBM useStudies on the use of UBM in diagnosing OSSN have shown that UBM is most useful in assessing intraocular tumor extension and metastasis , 23. ChaFinger et al. described general ultrasonographic characteristics of conjunctival intraepithelial neoplasia and squamous cell carcinoma in addition to UBM findings in intraocular tumor extension in 11 patients [In vivo confocal microscopy (IVCM) is a noninvasive imaging technique that allows in vivo microscopic examination of all layers of the ocular surface. In brief, it utilizes a point light source that scans the ocular surface and a point detector to increase the resolution [solution . Using csolution . Duchatesolution . in situ with IVCM findings of regular conjunctival epithelium interspersed with complexes of enlarged cells with polymorphic nuclei [Several other reports in the literature have suggested that IVCM may be helpful in establishing the diagnosis of OSSN. Single case reports by Malandrini et al. and Wakuta et al. described IVCM findings of enlarged, irregular cells with bright hyperreflective nuclei in conjunctival and corneal intraepithelial neoplasia , 37. Meac nuclei .Balestrazzi et al. described an atypical case of OSSN in a patient one month after clear corneal phacoemulsification with papillomatous invasion in the area of the side port incision. IVCM demonstrated typical characteristics of the limbal portion of OSSN with very bright intracellular bodies, while the corneal lesion demonstrated large hyperreflective round to oval shaped cells with peripherally displaced nucleus and stromal invasion of neoplasia across an interrupted Bowman layer . The aut in situ keratomileusis (LASIK), and she presented with biopsy proven limbal and conjunctival OSSN. IVCM showed that atypical cells had extended just below the level of basement membrane and Bowman layer along the scars of RK incisions. Because of these findings, the patient underwent surgical excision with a lamellar keratectomy and cryotherapy, followed by topical chemotherapy a few weeks later.Gentile et al. presented a case report of how IVCM was performed to determine the involvement of corneal incisions from previous refractive surgery . The pat ex vivo scraping cytology and histology in 100% of cases [Larger case series by Alomar et al., Parrozzani et al., and Xu et al. also demonstrated correlation between IVCM and histopathology findings , 24, 25.The largest study thus far on the utility of IVCM in differentiating OSSN from benign lesions was conducted by Nguena et al. in Moshi, Tanzania . The stuOther limitations of IVCM include its ability to provide only en face images in contrast to cross-sectional images obtained in tissue histology . AdditioFirst introduced by Izatt et al. in 1994, anterior segment optical coherence tomography (AS-OCT) is a noncontact and noninvasive imaging technique that captures high resolution cross-sectional images of the anterior segment . In AS-Oμm. In a study comparing TD-OCT with UBM, Bianciotto reported that while TD-OCT was useful for the assessment of superficial nonpigmented lesions such as conjunctival tumors, UBM was in general superior for the visualization of all tumor margins and had fewer problems with posterior tumor shadowing [In the original time-domain OCT (TD-OCT), axial resolution was limited at 18 hadowing . UBM prohadowing .μm, while ultra-high resolution OCT (UHR-OCT) can provide axial resolution better than 5 μm [μm allowed the delineation of individual cornea layers [With the development of spectral domain OCT (SD-OCT), higher resolution imaging has become available. High resolution OCT (HR-OCT) is capable of providing axial resolution of 5–10 han 5 μm . Vajzovia layers . The auta layers .Several subsequent studies have further demonstrated that thickened hyperreflective epithelium, abrupt transition from normal to abnormal epithelium, and a sharp plane of cleavage between the lesion and underlying tissue  were allμm, Kieval et al. showed that an epithelial thickness value greater than 140 μm provided 94% sensitivity and 100% specificity for differentiating CCIN from pterygia [μm, Nanji et al. demonstrated that an epithelial thickness cutoff at greater than 120 μm provided 100% sensitivity and specificity for differentiating OSSN from pterygia [Kieval et al. compared UHR-OCT of pterygia with OSSN . Pterygipterygia . In contpterygia . In factpterygia . UHR-OCTpterygia .UHR-OCT can also be used to monitor disease resolution and detect residual subclinical disease. For lesions treated successfully with topical agents, posttreatment UHR-OCT showed normalization of epithelial architecture at the site of the treated lesions. However, in lesions resistant to medical treatment, UHR-OCT will show persistently thickened epithelium with retained abrupt transition between normal and diseased epithelium , 42. ConOther scenarios where UHR-OCT can be useful include ruling out OSSN in the setting of complex ocular pathology and in clinically indeterminate lesions. Thomas et al. described a patient with a past medical history of HIV, vernal keratoconjunctivitis, limbal stem cell deficiency (LSCD), and previously treated OSSN who presented with a change in appearance in the limbal conjunctiva . UHR-OCTAdditional advantages of OCT over other forms of anterior segment imaging include its noncontact method of obtaining images, patients being imaged sitting in an upright and comfortable position, and user friendliness for the operator . HoweverThere are several adjunctive diagnostic modalities available that can assist in the diagnosis and monitoring of OSSN lesions. These include IC, vital dye staining, ultrasound biomicroscopy, IVCM, and AS-OCT. A summary of main findings from major studies on these diagnostic modalities is presented in Given the limitations of IC and vital dye staining, there has been a shift in interest to anterior segment imaging modalities such as UBM, IVCM, and AS-OCT. As discussed in this review, anterior segment imaging can provide the clinician with microscopic lesion detail to make an accurate diagnosis but is equally as important to guide therapeutic decisions. Each device has its limitations, but when combined with clinical examination, anterior segment imaging can greatly aid the clinician. Nevertheless, it is important to note that none of these imaging modalities has replaced histopathology's role as the gold standard for diagnosing OSSN. More research over time and advances in technology will likely provide us with further improved imaging modalities, but to date these devices warrant integration into clinical practice.


In a recent publication in Bioscience Reports “Contaminants in commercial preparations of ‘purified’ small leucine-rich proteoglycans may distort mechanistic studies”, Brown et al. identified by mass spectrometry and immunoblotting that certain commercial preparations of the small leucine-rich proteoglycans (SLRPs) decorin and biglycan, in fact, contained a mix of several proteoglycans that also included fibromodulin and aggrecan. The preparations were thus not suitable to study specific activities of decorin or biglycan. Decorin and biglycan are widely studied SLRPs that are considered to have highly multi-functional effects on cells. Decorin is of interest as a transforming growth factor-β antagonist and is also finding use in tissue engineering materials. This Commentary discusses Brown et al.’s findings and general issues raised for researchers who work with commercially sourced purified proteoglycans. O-linked glycosaminoglycan (GAG) chains on the core protein. Of the secreted proteoglycans of mammals, SLRPs form the largest gene family with the smallest core proteins (approximately 32–45 kDa) that are composed principally of a series of tandem leucine-rich repeats. Decorin and biglycan are closely-related SLRPs: their core proteins have 56% sequence identity in humans and are substituted with one or two chondroitin sulphate/dermatan sulphate-type GAG chains respectively  decorin and biglycan in supporting neurite outgrowth . Proteogectively . NotwithBoth decorin and biglycan are expressed in intervertebral discs, which, in healthy individuals, are innervated only in the outer region. When discs degenerate, as occurs in ageing or after trauma, alterations in the disc ECM provide conditions that can allow for nerve ingrowth into central areas . Althougin vitro), interspersed with stripes of decorin or biglycan. The effects of decorin or biglycan, whether to support or repel neurites extending in from the collagen-coated strips, were then assessed microscopically. The DRG were from chick embryos and the proteoglycans were preparations from bovine articular cartilage, purchased from Sigma [Given the presence of decorin and biglycan in intervertebral discs, the authors’ goal was to examine the activity of these SLRPs as substrata for neurite outgrowth by sensory neurons from dorsal root ganglia (DRG) explants. Their assay involved coating dishes with stripes of collagen I  tissues. Commercial sources for recombinantly expressed decorin and biglycan can provide alternatives. A recombinant preparation was used, for example, to study transforming growth factor-β antagonism by decorin in a spinal cord injury model . Most suosophila . Moving aggrecan .The overall picture is that additional experimental modes, such as side-by-side testing of wild-type and decorin-null cells, gene editing, transcriptional silencing or viral expression of wild-type or mutants, must come to the fore to prove roles of decorin or biglycan in functional processes of interest. The reminder to ‘know your materials’ is timely in view of the current drive from researchers, institutions, funding bodies and publishers to enhance the transparency of research reporting.


Pseudomonas aeruginosa and Escherichia coli, and the morbidity and mortality associated with these infections was striking. Ironically, these pathogens originated from the patient’s own gastrointestinal (GI) tract, based originally on autopsy results and later using molecular studies [3) and fever were found to be at highest risk for developing bacterial and fungal bloodstream infections.In the 1960s, combination chemotherapy (using cytotoxic agents with different mechanisms of action) was adopted to treat cancer patients, resulting in much higher cure rates. During this time, however, a substantial proportion of cancer patients developed bacterial bloodstream infections, predominantly with gram-negative organisms such as  studies . In part3).While treating febrile and neutropenic patients with antibiotics is standard of care, the choice of which antibiotic(s) to use is still largely institution dependent, as a number of antibiotics have been shown to be efficacious in this setting. Thus, cancer and stem cell transplant (SCT) patients often receive multiple antibiotics for long durations of time, and many patients never have a laboratory-documented infection. Cessation of empiric antibiotics before neutrophil recovery, however, is associated with an increased risk of infection [These findings led to sweeping changes in clinical practice whereby initiation of empiric antibiotics for cancer patients with fever and neutropenia became and continues to be standard of care and had a remarkable effect on decreasing the morbidity and mortality associated with these infections . These pnfection . While cnfection . Furthernfection  and the nfection –8. In fanfection , 8–10. TP. aeruginosa or Candida albicans) and cytotoxic chemotherapy  to both deplete neutrophils and damage GI epithelium [The major host immune deficits that promote bacterial and fungal translocation from the GI tract include deficiencies in cellular immunity (particularly neutropenia); impaired intestinal barriers ; and GI microbiota imbalance (often driven by use of broad-spectrum antibiotics). In cancer and SCT patients, as a consequence of cytotoxic chemotherapy, all 3 defense mechanisms are often compromised. In preclinical models emulating the development of gut-derived bacteremia and fungemia, all 3 host defense mechanisms need to be impaired to promote microbial dissemination from the gut: antibiotics to deplete gut commensal microbiota, allowing high levels of pathogenic microbial colonization    or pharalbicans  can be uEnterococcus spp. or Enterobacteriaceae  is associated with a significantly increased risk of developing bloodstream infection with the same bacterial species [Interestingly, in human SCT patients, an expansion of GI  species , 18. The species  or funga species  is suffiE. coli gut microbial burden precedes and significantly increases the risk of E. coli bacteremia in this patient population [E. coli, Klebsiella spp., Enterobacter spp., etc.) in cancer and SCT patients could be used to identify those patients at risk for developing Enterobacteriaceae bacteremia.With the widespread use of advanced gut microbiota profiling tools, such as 16S rRNA sequencing or metagenomic shotgun sequencing (MSS), the question is whether clinicians should be using real-time monitoring of gut microbiota populations in these patients. For instance, given that increased pulation , frequenUnfortunately, both 16S rRNA sequencing and MSS have not been used in the clinical setting because of logistical barriers to implementation—namely, time, cost, and complexity. The turnaround time for 16S rRNA and MSS sequencing data is on the order of weeks to months and thus is far too slow for informing clinical decisions. Another limitation of both 16S rRNA sequencing and MSS is that absolute levels of microbiota cannot be determined, only relative abundance. An increase in gut microbiota relative abundance does not necessarily correlate with an increase in total microbiota levels. In contrast, group or species-specific microbial quantitative polymerase chain reaction (qPCR) performed on patient fecal specimens could theoretically be provided to a clinician within days. qPCR has been applied to 16S rRNA methodology and has been validated for quantification of bacterial groups as well as specific bacterial species within complex bacterial communities , 19. BacE. coli and P. aeruginosa—and relegating broad-spectrum antibiotic use  to when clinical use dictates  bacteremia). More selective pathogen-target intervention strategies are now being investigated: (1) conjugating an antibiotic to a pathogen-specific antibody [Prophylactic and empiric antibiotic therapy in cancer and SCT patients has saved countless lives. However, the use of broad-spectrum and possibly unnecessarily prolonged-duration antibiotic therapy has resulted in increasing microbial resistance and unintended deleterious effects to patients as well. In the short term, perhaps more narrow-spectrum antibiotics should be preferentially used to prophylactically treat cancer patients—focusing on covering the gram-negative organisms such as antibody , (2) genantibody , and (3)antibody . Perhaps


A perspective review of recent strategies involving the use of nano/microvehicles to address the key challenges associated with delivery and (bio)sensing at the cellular level is presented. A perspective review of recent strategies involving the use of nano/microvehicles to address the key challenges associated with delivery and (bio)sensing at the cellular level is presented. The main types and characteristics of the different nano/microvehicles used for these cellular applications are discussed, including fabrication pathways, propulsion  and navigation strategies, and relevant parameters affecting their propulsion performance and sensing and delivery capabilities. Thereafter, selected applications are critically discussed. An emphasis is made on enhancing the extra- and intra-cellular biosensing capabilities, fast cell internalization, rapid inter- or intra-cellular movement, efficient payload delivery and targeted on-demand controlled release in order to greatly improve the monitoring and modulation of cellular processes. A critical discussion of selected breakthrough applications illustrates how these smart multifunctional nano/microdevices operate as nano/microcarriers and sensors at the intra- and extra-cellular levels. These advances allow both the real-time biosensing of relevant targets and processes even at a single cell level, and the delivery of different cargoes  for therapeutics, gene silencing/transfection and assisted fertilization, while overcoming challenges faced by current affinity biosensors and delivery vehicles. Key challenges for the future and the envisioned opportunities and future perspectives of this remarkably exciting field are discussed. H2O2) or biofriendly  fuels, fuel-free nano/micromotors, powered by various external stimuli based on magnetic, electric or ultrasonic fields, exhibit major advantages in directional motion control and have long lifetimes and excellent biocompatibility.The ability to probe cellular activity and target therapeutics inside living cells is of considerable importance as we strive to develop tiny biosensors that monitor complex cellular events and design therapies that can modulate the processes that occur within cells. Such intracellular delivery of drugs can sharply increase the efficiency of various treatment protocols. Nanotechnology researchers have thus been aiming at developing novel nanovehicles capable of entering target cells, probing intracellular processes and ferrying their payload to sub-cellular organelles. Despite the considerable progress made in recent years in biosensing and delivery at the cellular level, current systems are still not able to address some important challenges, such as achieving ultrasensitive detection in a short time in microscale environments, and releasing functional cargoes in a quick and controlled way at specific extra- and intra-cellular locations with limited accessibility. The broad scope of operations and applications, along with the ultrasmall dimensions, accessibility and force offered by synthetic nano/micromotors, open up intriguing possibilities for these purposes, overcoming some of the unfilled gaps and unmet challenges. In this field, apart from chemically powered nano/micromotors propelled by toxic  and enhancing the cooperation of multiple nano/micromotors for complex tasks.Although these unsolved critical issues should be addressed, the high performance demonstrated by nano/micromotors for biosensing and targeted payload delivery at the cellular level has encouraged the scientific community to devote more effort to understanding the fundamental science behind nano/micromotors and expanding their applications. Further developments are expected to provide intelligent, responsive and multifunctional nano/micromachines that mimic the amazing functions of natural systems for a diverse range of biomedical applications. These will allow unprecedented levels of cell manipulation and targeted intervention even at the single cell level, with the possibility of treating diseases more specifically and safely. The immense progress and benefits that nano/micromotors can bring to the fields of biosensing and delivery at the cellular level, along with the potential challenges that lie ahead and the existing gaps and limitations, are discussed in the following sections.2 and active transport systems, holding great potential for a variety of diagnostic and therapeutic applications. The design of miniaturized and versatile nano/microvehicles would allow access throughout the whole human body, leading to new procedures down to the cellular level, and offering localized diagnosis and treatment with greater precision and efficiency.4 Such nano/micromotors with medical potential must be fabricated with non-toxic materials and optimized shapes, and must be propelled by powerful and biocompatible propulsion mechanisms.Nano/microvehicles are expected to become powerful mobile biosensors6 These vehicles can be classified into three different types based on their propulsion mechanism: (1) those with their own built-in propulsion powered by energy-rich molecules, (2) those with external energy sources such as magnetic or acoustic fields, and (3) systems “towed” to the site of interest by living objects with propulsion functions. In the case of autonomous systems, the engine must be equipped with some sort of navigation system to direct the nano/microvehicle to the target, while the direction of externally powered systems must be guided by an operator.6 Another widely accepted classification divides nano/microvehicles into only two major groups: (1) those that are chemically propelled5 and (2) fuel-free nano/micromotors powered by biocompatible external energy sources, such as magnetism and ultrasound (US) .9 Catalytic nano/micromachines include nano/micromotors powered by external15 or natural17 fuels, and micromotors with chemotaxis behavior towards a particular substrate.19 Magnetically actuated flexible/helical swimmers,25 US-powered nanomotors30 and self-motile cell-based micromotors are included in the fuel-free group.32 Many of these nano/micromotors show considerable promise for navigation through complex biological fluids, reaching hard-to-access bodily locations, or releasing their therapeutic payloads at predetermined body destinations.A wide range of nano/microvehicles based on different propulsion mechanisms have been developed in recent years.33 Among the chemically powered nano/micromotors, tubular microengines are particularly attractive for biosensing applications due to their efficient bubble-induced propulsion resulting from the catalytic decomposition of fuel on their internal surface. Fuel-propelled nano/micromotors can perform different types of motion, and all of them are limited by access to fuel in the medium. The inherent lack of a defined trajectory can be solved by including a magnetic component (e.g. nickel) in their structure, thus allowing their guidance in the presence of an external magnetic field.34Fuel-propelled micromotors require a specific fuel and specific composition of their surface. They can drive themselves through aqueous solution using surface reactions to generate local gradients of concentration, electrical potential and gas bubbles.38 acid17 or water,43 is crucial for in vivo applications44 because these systems not only circumvent the need for an external fuel, but also provide selective nano/micromotor activation only in particular environments.The development of biocompatible and biodegradable nano/micromotors, driven by natural fuel sources inherently created in biological systems, such as glucose, urea,in vivo drug delivery applications. Indeed, this group reported the first example of the use of micromotors in vivo, showing the propulsion of Zn motors in the harsh acidic environment of a mouse’s stomach.16 Mg micromotors coated with a pH-responsive enteric polymer were also demonstrated to be useful for site-specific gastrointestinal (GI) delivery.45 These micromotors safely passed through the gastric fluid and were selectively activated in the GI tract of living mice, demonstrating important advantages toward localized tissue delivery. Mg-based micromotors have also been combined with a cargo-loaded pH-responsive film, demonstrating that they can autonomously and temporally neutralize gastric acid, triggering a responsive payload release in the mouse’s stomach.46 This simple but efficient motor configuration opens up future prospects in drug delivery, offering a unique rational on-board release in the presence of specific microenvironments.Wang’s group has reported the use of body fuel-powered micromotors, such as Zn or Mg-based micromotors, for in vivo biomedical applications due to their potential biocompatibility and independence of the chemical composition of the medium in which they operate. The use of magnetic fields to power nano/microswimmers has gained particular interest as magnetic fields can penetrate the human body, allowing for the wireless control of these tiny devices without harming cells and tissues.23 A special type of magnetic helical microswimmer is artificial bacterial flagella (ABF), which can perform 3D navigation in a controllable fashion with micrometer precision in liquid, using low-strength rotating magnetic fields (1000 times lower than the fields used in MRI systems), translating rotational movement into translational motion in a screw-like manner.47 Moreover, Felfoul et al.48 demonstrated that harnessing swarms of microorganisms exhibiting magneto-aerotactic behavior can significantly improve the delivery efficiency of drug nanocarriers to tumor hypoxic regions of limited accessibility.Externally-powered motors, using mainly magnetic and US energies to drive their motion, are extremely interesting for 49 The pioneering work of Mallouk and colleagues at Penn State University demonstrated for the first time that such a gold NW motor, powered by ultrasonic waves, was able to move inside HeLa cells without causing any damage.49 The introduction of US-powered NW motors into living cells has opened a new door for payload delivery and (bio)sensing at the cellular level. In addition, US actuation opens up the possibility of driving and controlling nano/micromotors using deeply penetrative  US to create powerful micromotors with remarkable tissue penetration properties that will be discussed in the following sections.28Nanomotors propelled by acoustic forces also offer great advantages in terms of biocompatibility and cargo delivery. It has been demonstrated that US propulsion facilitates the internalization of functionalized nanowire (NW) motors into living cells and enables them to remain acoustically active, undergoing both directional motion and spinning inside the cells.50 Motile cells have been used as microbiomotors in connection with metal-coated polymer microhelices31 or magnetic microtubes.50The use of self-motile cell-based micromotors provides great flexibility regarding the type of flagellated organism that can be integrated into the microbiomotor. Moreover, the motile cells employed do not need to be cultivated and are readily available, easy to handle, able to swim in highly viscous media and completely harmless.51 Different metal NWs, the simplest 1D structure used as nano- and micromotors, are fabricated by metal electroplating inside a membrane template . These commercially available membranes are sputtered with gold to act as a working electrode in the fabrication process, and are dissolved in the last step using acid or base solutions to release the as-prepared NWs. Janus spherical nano/micromotors, consisting of two hemispheres playing different roles , are commonly fabricated by catalytic metallic  thin film deposition  or polymeric coating onto the surface of nano- or microbeads of different materials  uniformly distributed on glass slides or silicon wafers. Tubular nano/micromotors are different to those using NWs as they have tubular structures with specific inner and outer parts, both of which are suitable to be functionalized.24 Early tubular micromotors  were fabricated by a rolling-up process based on the patterning of a photoresist on a substrate (e.g. Si wafer) bearing a sacrificial layer.33 The subsequent evaporation of metals at a glanced angle created a window that determined the rolling process (occurring after etching the substrate). However, smaller sized microtubes, which are more compatible with certain applications, can be fabricated in a simplified manner by template electroplating using appropriate membranes with conical shaped pores.52 This methodology avoids the need for a cleanroom and significantly reduces the cost and fabrication time. It is also worth mentioning that various chemical compounds, such as polyaniline (PANI), poly (PEDOT), polypyrrole (PPy), poly(3-aminophenylboronic acid) (PAPBA) and molecularly imprinted polymers (MIPs), can be electrodeposited allowing facile further modification. This is therefore an attractive route for fabricating versatile microtubes to be functionalized for different applications. Interestingly, all of these fabrication routes offer many alternative ways to tailor the size, shape, propulsion mechanism and final properties of the synthesized nano/micromotors, as desired for achieving their optimum performance in the pursued applications.These tiny devices can also be classified according to their shape, size and material building block, which determine their propulsion mechanism and the best fabrication route for their preparation.54Affinity biosensors involving analyte–receptor interactions under static conditions have been demonstrated to achieve low detection limits. However, ultrasensitive detection in a short time remains a major challenge due to the small sample volumes and difficulty enhancing analyte–receptor interactions through enhanced mass transport in such microliter samples. The functionalities and capabilities exhibited by advanced nano/micromotors have opened the way to new biosensing applications in previously inaccessible microscale environments. The easy functionalization of the nano/micromotor structure with specific biological or artificial recognition elements, attached onto the motor surface or embedded in the motor material itself, has allowed for their implementation as dynamic biosensing systems, offering specific and fast detection of different target analytes, greatly accelerated by the efficient and fast motion of receptor-functionalized nano/micromotors through the sample, without or with minimal sample treatment.55These novel motile biosensors, in which the continuous movement of receptor-functionalized nano/micromotors around the sample increases the likelihood of target–receptor contacts, have been demonstrated to be highly selective and sensitive, eliminating time-consuming washing steps and largely simplifying and accelerating the overall biosensing procedure. In the particular case of fuel-propelled tubular microrockets, the fast motion and bubble release trigger local vortexes that promote the transport of the target analyte toward the functionalized microjet outer surface, offering the possibility to perform rapid “on-the-fly” recognition events.56 Over the past decade, many nanomaterial-based systems have been developed for drug delivery applications, including liposomes, polymeric nanoparticles, dendrimers, polymersomes and nanoemulsions.57 However, despite the significant progress made to improve the therapeutic efficacy of such drug delivery nanosystems, important challenges, such as issues relating to off-targeting, compatibility with the immune system, low cell and tissue penetration and low therapeutic efficacy, have not yet been overcome. This demands the development of innovative technologies.On the other hand, the development of a new generation of drug delivery vehicles capable of encapsulating, transporting and releasing active substances in a rapid, targeted and controlled manner is of great interest. One of the biggest challenges to face in targeted drug delivery is conquering the innate immune system of the host organism, which readily recognizes and destroys foreign material that enters the body’s circulation.13 It is important to mention at this point (as will be described in more detail later) that the clever delivery approaches proposed by Schmidt’s group, using artificially motorized sperm cells31 and sperm-hybrid micromotors,32 have demonstrated the ability to inhibit the immune response using the specific glycans on the sperm cell membrane. Although still at an early stage, these recent advances will play a major role in the future design of new micro/nanodevices with low immunogenicity that are able to bypass the immune system.In this context, recent delivery strategies involving the use of nano/micromotors have demonstrated the integration of self-driven and navigation capabilities, achieving not only smart drug encapsulation and release but also precise guidance and control.34 In addition, the easy functionalization of the nano/micromotor structure with specific bioreceptors has also enabled efficient modification with a great variety of cargoes and their delivery, both in extra- and intra-cellular spaces. Therefore, drug delivery nano/microvehicles promise to address several key issues for the development of more powerful therapeutics, including the evasion of immune-mediated clearance, improvements to therapeutic efficacy, enabling the combinatorial delivery of multi-cargoes, avoiding negative side effects by delivering the drug precisely at the disease-specific place, and overcoming limited access to certain areas of the body with low drug tissue distribution and cellular barrier penetration.59Nano/micromotors possess a high towing force that represents a major advantage for their implementation as cargo delivery platforms. Furthermore, their directionality can be controlled by the incorporation of magnetic materials in their structure, allowing for the precise motion control required for targeted drug delivery.4 and the compatibility of operation demonstrated in cellular media,51 we journey in this perspective article through recent advances in the use of nano/microvehicles for cellular biosensing and cargo delivery applications. Selected examples of such motor-based operations will be discussed, along with related challenges, envisioned opportunities and future perspectives. As will be discussed below, nano/micromotors have been applied for biosensing and delivery both in extra- and intra-cellular spaces. As depicted in Taking into account the breakthrough biomedical applications of nano/micromotors demonstrated by pioneering groups in this field60 The different biosensing strategies based on receptor-functionalized nano/micromotors are summarized in The ability to dynamically probe intracellular processes and monitor key molecules in individual cells is the next frontier in biomedicine. As introduced above, the nano/micromotor surface can be functionalized with specific bioreceptors used for the detection and/or isolation of different biological targets under dynamic conditions at the extra- and intra-cellular levels. The continuous movement of synthetic nano/micromotors through the samples, and the dramatic modulation of mass transport by the vortex effect resulting from the locomotion of bubble-propelled microengines, significantly enhance the interactions of the nano/micromotor sensing surfaces with the target analytes, thereby improving the recognition efficiency and offering ‘on-the-fly’ recognition of specific biomolecular interactions.2O2 release to extracellular media) and intra- (mature miRNAs) cellular levels. For example, Balasubramanian et al.11 described an immuno-micromachine constructed by anti-carcinoembryonic antigen (CEA) monoclonal antibody modification of Ti/Fe/Au/Pt microrockets prepared by standard photolithography. The modified microvehicle was employed for the in vitro isolation and transport of cancer cells by means of the selective biosensing of CEA overexpressed in pancreatic cancer cells  in just 120 s. This concept, which relied on the asymmetric and continuous release of SDS surfactant and fresh horseradish peroxidase (HRP) from the tubular motors into the sample solution, was applied for the determination of H2O2 in cultures of tumor kidney tubular cells.Moreno-Guzmán in vivo application is still their need of H2O2 fuel for propulsion. With this in mind, motors propelled by external stimulation, such as acoustic fields, appear to be great candidates to work in cell culture environments. The strong energy of the acoustic fields can be utilized to internalize and move nanomotors inside living cells,49 and therefore the efficient movement of US-powered nanomotors can be exploited for intracellular sensing. An illustrative example of this strategy is the construction of a single-step nanomotor for rapid intracellular miRNA biosensing at the single cell level.62 The concept relied on the use of US-propelled dye-labeled single-stranded DNA (ssDNA)/graphene-oxide (GO)-coated gold NWs (AuNWs), with fluorescence quenched by the π–π interaction between GO and the dye-labeled ssDNA. After cell internalization, and in the presence of the target miRNA, the fluorescence signal is recovered due to the displacement of the dye-ssDNA probe from the GO-quenching motor surface, leading to attractive intracellular “OFF–ON” fluorescence switching , both in intra- and extra-cellular spaces. 65 reported for the first time the directed delivery of magnetic polymeric particles loaded with the chemotherapeutic drug doxorubicin (DOX) using magnetically driven flexible nanoswimmers  to perform in vitro single-cell-targeted drug delivery. The results presented demonstrated the feasibility of these microswimmers, steerable with an external magnetic field, to deliver the hydrophilic model drug calcein to C2C12 mouse myoblasts. The uptake of the drug by the target cell was monitored using fluorescence microscopy.Mhanna et al.66 proposed the use of self-propelled multilayer Janus capsule motors for effective drug transportation and controlled release at HeLa cancer cells. These hybrid biocatalytic Janus motors were based on hollow polyelectrolyte multilayer capsules and PtNPs66 or catalase,13 and were propelled by the biocatalytic decomposition of peroxide fuel. The encapsulated drug (DOX) could be rapidly released at predefined sites by US66 or NIR light13 triggering. The same group also reported the first example of self-propelled Janus mesoporous silica nanomotors (MSNs) with sub-100 nm diameters for drug encapsulation and intracellular delivery.14 These Janus nanomotors employed mesoporous silica nanoparticles with chromium/Pt metallic caps and were propelled by decomposing H2O2 to generate oxygen as a driving force with speeds of up to 20.2 mm s–1. The Janus MSNs were applied in vitro for intracellular localization and the slow release of the encapsulated drug (DOX) inside HeLa cells through the catalytic hydrolysis of lipid bilayers by cellular enzymes.Wu 26 Another approach involved the construction of biodegradable, self-propelled bovine serum albumin/poly-l-lysine (PLL/BSA) multilayer microrockets.15 The microrockets’ preparation involved a template-assisted layer-by-layer assembly of PLL/BSA layers followed by the incorporation of a heat-sensitive gelatin hydrogel, which greatly improved the encapsulation capacity, containing AuNPs, DOX and catalase  carrier and as the propulsion force, taking advantage of its swimming capability. A 3D printed four-armed microtube with a nanometric iron layer, called a “tetrapod”, was used to magnetically guide and mechanically release the drug-loaded sperm cell in the desired area , as drug carrier systems. The same group recently reported the use of self-assembled biodegradable PtNP-loaded stomatocyte nanomotors, containing poly(ethylene glycol) (PEG), a semicrystalline poly(ε-caprolactone) (PCL) and a glassy polymer  for anticancer drug delivery.19 The uptake by HeLa cancer cells and fast DOX drug release of these PEG-b-PCL and PEG-b-PS-hybrid based stomatocyte nanomotors was demonstrated during degradation under acidic conditions.Peng et al.24 proposed smart multifunctional tubular nanomachines incorporating a stimuli-responsive building block for targeted drug delivery. The nanomachines consisted of a magnetic Ni nanotube for wireless magnetic propulsion, coated on the outside with Au to allow functionalization with fluorescently tagged thiol-ssDNA. The inner nanotube cavity contained a pH-responsive chitosan hydrogel to carry the drug and selectively release it only in acidic environments. Methylene blue and human epithelial breast cancer cells (MDA-MB-231) were used as model drug and target cells to demonstrate the concept.Hoop 25 introduced for the first time the term medibots, referring to dual-action biogenic hybrid micromotors with functionality both for cell microdrilling and site-directed drug release. These hybrid micromotors are plant-extracted calcified porous microneedles (40–60 μm long), coated via e-beam deposition with a magnetic Fe–Ti layer to facilitate cellular drilling by external magnetic actuation. The loading of the calcified biotubes with the anticancer drug camptothecin enabled specific drug release in the acidic environment of HeLa cancer cells.Schmidt’s groupet al. recently reported a hybrid magnetoelectric nanomotor for on-site magnetically-triggered anticancer drug release in breast cancer cells (MDA-MB-231).64 In this case, the chemotherapeutic drug paclitaxel was adsorbed onto the polydopamine-modified nanomotor surface and was efficiently released upon the application of alternating magnetic fields due to the magnetoelectric effect.Chen 28Wang’s group reported the design of acoustically propelled shell-shaped nanomotors for the “on-the-move” capture and transport of multiple magnetic cargoes (3.3 μm magnetic beads), and their internalization and propulsion inside live MCF-7 cancer cells.in vivo study of artificial PEDOT/Zn micromotors in a living organism, showing efficient propulsion in the harsh acidic environment of mouse stomachs without additional fuel  and acute toxicity profile of these acid-driven propelled motors were evaluated via oral administration. It was demonstrated that the body of the motors gradually dissolved in gastric acid, thus autonomously releasing the carried payloads on the mouse stomach wall while self-destructing non-toxically.The same group also reported the first example of an nal fuel .45 In th67 A similar technology was reported by Soto et al. to develop acoustically-triggered microcannons capable of loading and firing nanobullets  and activated canonical nuclear factor-kappaB (NF-kB) signaling to single HeLa cancer cells.Gene or protein therapy implies the use of DNA or protein therapeutic substances, which are delivered into a patient’s cells to treat diseases such as inherited disorders and cancers.et al.23 for in vitro wirelessly targeted single-cell gene delivery to human embryonic kidney (HEK 293) cells. The successful in vitro transfection of the HEK 293 cells by f-ABFs, steered wirelessly by low-strength rotating magnetic fields, was demonstrated by expressing the encoding Venus protein.ABFs functionalized (f-ABFs) with complexes of cationic lipids and plasmid DNA (lipoplexes) were proposed by Qiu 29 The US-propulsion led to the fast internalization and rapid intracellular movement of the AuNWs, and hence to an accelerated siRNA delivery and silencing response. The strategy allowed 94% silencing of the GFP response in two different cell lines (HEK-293-GFP and MCF-7-GFP) after 5 min treatment with the GFP/RCA-wrapped AuNWs. Interestingly, most of the cells remained alive after the nanomotor treatment, even when using a high nanomotor concentration.The advantages of the rapid internalization and intracellular movement of US-powered nanomotors have also been exploited for intracellular oligonucleotide delivery. Wang’s group described recently an effective intracellular gene silencing strategy, using acoustically-propelled AuNWs wrapped with a Rolling Circle Amplification (RCA) DNA strand, which served to anchor green fluorescent protein (GFP) which was targetedly interfering with the RNA’s (siGFP) payload .29 The U30 The nanomotor allowed 80% apoptosis of cancer cells within only 5 min, suggesting that such nanovehicles may constitute powerful tools for the cytosolic delivery of active therapeutic proteins. Compared to other apoptosis approaches, the nanomotor strategy resulted in the highest apoptosis efficiency while requiring significantly shorter times and smaller amounts of CASP-3.The same group has described recently a method based on the use of US-propelled nanomotors coated with a high pH-responsive polymer, for the rapid internalization and cytosolic delivery of functional caspase-3 (CASP-3), with the aim of causing apoptosis in human gastric adenocarcinoma cells .30 The nin vitro and in vivo settings. Such intracellular delivery can thus sharply increase the efficiency of various treatment protocols.It is worth mentioning that the extraordinary capabilities demonstrated by fuel-free nano/micromotors for targeted gene or protein delivery open the door for a wide portfolio of potential applications in the fields of therapy bioengineering and cell biology in various The discussed examples demonstrate the broad range of applications performed at the cellular level using nano/micromotors. They constitute clear proof of the remarkable progress made in the design and construction of these tiny machines to integrate self-driven, navigation and biosensing capabilities, and perform multitasking, including drug loading, targeted transportation and autonomous or remotely-controlled release in complex environments. These capabilities make them extremely promising vehicles to be employed for biosensing and drug release applications at the cellular level. The reports described so far demonstrate that different types of nano/micromotor can be used as suitable engines for operation in cellular environments. Moreover, at the cellular level, the biosensing applications of nano/micromotors have been much less exploited than those involving cargo delivery (mainly of chemotherapeutic drugs). Such new nanovehicles thus offer considerable promise for the monitoring of complex processes that occur within cells and for modulating such cellular processes and increasing the efficiency of various treatment protocols. While several novel intracellular applications of nanovehicles have been covered in this article, the ability to sense cellular processes and deliver payloads within the cell is still at a very early stage. Future advances in nano/micromotors will be critical to biomedical applications that rely on sensing, delivery and targeting in intracellular space.The studies discussed above show that these smart devices, self-propelled or externally powered by catalytic, magnetic or acoustic sources, offer a myriad of mechanical movements and broad design versatility in terms of building block materials , size  and shape . Since each specific shape and building block material demands a given fabrication technique, determining important functioning properties apart from the vehicle motion mechanism, the design and preparation of motors devoted to a specific application requires the integration of several crucial factors. On the positive side, the large number of available possibilities to tailor the behavior and properties of the constructed engines considerably increases their potential applications.The developed smart vehicles demonstrated outstanding capabilities in terms of speed and force, drug loading and towing capacity , biocompatibility and biodegradability, wireless transport to specific hard-to-reach areas and feasibility to carry a wide variety of cargoes  and release them using biocompatible mechanisms . Importantly, the biosensing and delivery missions undertaken have been performed both at extra- and intra-cellular levels without compromising the viability of the target cells. While a wide variety have been applied in extracellular space, the intracellular applications have been demonstrated primarily using acoustic propelled nano/micromotors.in vitro, and potential difficulties might be encountered for in vivo applications. In addition to the limitation of using fuel-free or biocompatible fuel nanomotors, there are some serious challenges to face to achieve in vivo applications. The smart vehicles must be able to evade the immune system of the host organism and, in the specific case of liposome-based systems, destruction by the reticuloendothelial system. Drug delivery should also overcome the high interstitial pressure in late stage tumors, the endosomal escape challenge and the natural physiological barriers such as the highly acidic gastric environment. Moreover, for in vivo applications, the nano/micromotor must be readily degraded into non-toxic compounds without interference from outside through passive or built-in self-destruction mechanisms activated after finishing their mission.68 Novel smart materials, including biological, responsive or soft materials, are therefore highly desired to provide triggered autonomous actuation and multifunctionality while avoiding irreversible malfunctions in complex physiologically relevant body systems.However, it is important to mention that most of these remarkable capabilities of nano/micromotors have been demonstrated only e.g., Mg, Zn, Al and CaCO3) and the limited power and stability of enzyme-based ones. In this sense, coupling synthetic nanomachines with natural biological materials can minimize these problems, apart from the desired immune evasion and biofouling occurring in complex biological fluids, leading to their enhanced mobility and extended lifetime. The future biomedical operation of nano/micromachines will require individual control of the nano/micromotors wirelessly, for example by the integration of nanoscale control systems and the development and coupling with high-resolution imaging and feedback control systems for the simultaneous localization and mapping of multiple nano/micromotors in the human body.Additional efforts should also be devoted towards developing functional geometries and exploring new synthesis methods for the easy, large scale, high quality, cost-efficient and environmentally friendly fabrication of nano/micromotors. The identification of new energy sources for enhancing tissue penetration and achieving prolonged, biocompatible and fully autonomous operation in complex biological media such as blood (with large drag forces associated with the high viscosity of the blood cells) should also be pursued. Micromotors powered by bio-friendly fuels also require additional improvements for their practical implementation, due to the relatively short lifetimes of those using active material propellants (in vivo environments.45 These pioneering applications have demonstrated the capabilities of nano/micromotors for safely and rapidly yet transiently neutralizing gastric acid, and simultaneously releasing their payload without affecting the stomach function or causing adverse effects in living animals. This type of work represents an important step forward for clinical translation and paves the way for many other breakthrough in vivo applications. The limited tissue penetration has been addressed using US-powered tubular microbullets, in which the ‘bullet-like’ propulsion allows the vehicle to reach different depths of the tissue to perform precise nanosurgery, as well as enabling it to carry large payloads. Indeed, the latest applications in this field have even demonstrated the use of nano/micromotors as efficient vehicles for the direct cytosolic delivery of active therapeutic proteins after internalization in intact target cells. While progress has been made in this direction, effective intracellular delivery to the cytosol remains a challenge. Future efforts could lead to the targeting of specific cytoplasmic organelles, such as the mitochondria, through guided movement within the intracellular space. It is also worth mentioning that, although only cargo delivery applications have been considered in this review, the nearly limitless possibilities demonstrated by nano/microvehicles to deliver a wide variety of payloads with diverse biomedical functions also open up a wide range of other opportunities for therapy, diagnostics and imaging.It is worth noting that despite the long and challenging road ahead, some important steps have been already achieved to address these important issues. The stabilization of the substrates and application of appropriate coatings or preconditioning to the liposomes/cargoes have been employed in the fabrication processes of nano/micromotors to potentially address the endosome escape challenge. Moreover, highly hemo-compatible nano/microvehicles, with a negligible influence from red blood cells on their motion, have already been developed, and more biocompatible nano/micromotors have been proposed involving coatings with different biocompatible materials, such as red blood cell membranes. Most interestingly although still scarcely explored, some authors have already reported the appropriate operation and behavior of these smart systems in in vivo biosensing and cargo delivery. Furthermore, to fully realize the abilities of nano/micromotors in medicine, a closer collaboration between scientists working in the development of nano/micromachines and medical researchers will be required for designing and constructing devices able to meet the demands and needs of the medical community. Overcoming these challenges to further increase the power and functionality of nano/micromotors, together with finding effective paths to take advantage of the emerging novelties in this exciting field toward the most relevant practical and commercial applications demanded by clinical experts, will be compulsory to ensure their utilization in diagnostics and therapy, which is still in its infancy. Therefore, substantial efforts and new innovations are still required to realize the full potential of these tiny motors in a new generation of nano/micromotors. Ideally, they should be fabricated using cutting-edge techniques, be able to mimic the natural intelligence of their biological counterparts, exhibit improved, adaptable and sustainable operation in biological media, have a deformable structure, be able to be precisely controlled, and have the ability to act both individually and collectively with synchronized coordination and self-adaptive and self-replicating capabilities. Nevertheless, the remarkable performance already demonstrated by nano/micromotors to design problem-oriented medical devices for specific sensing or delivery functions undoubtedly represents a tremendous asset in diagnostics and therapeutics. Therefore, the accelerated translation of nano/micromotor research into practical clinical use nowadays seems more reality than science fiction.Advances in this field will likely come from the integration of the tremendous progress made in nano/micromotor research during the last few years with a variety of exciting nano/micromotor-based strategies for the biosensing, transport and release of therapeutic agents at the cellular level, with advances in materials science, nanotechnology and molecular biology. This marriage will be essential to overcome the remaining challenges and promote the wide implementation of these smart, tiny multitasking vehicles for There are no conflicts to declare.


Circadian rhythms orchestrate biochemical and physiological processes in living organisms to respond the day/night cycle. In mammals, nearly all cells hold self-sustained circadian clocks meanwhile couple the intrinsic rhythms to systemic changes in a hierarchical manner. The suprachiasmatic nucleus (SCN) of the hypothalamus functions as the master pacemaker to initiate daily synchronization according to the photoperiod, in turn determines the phase of peripheral cellular clocks through a variety of signaling relays, including endocrine rhythms and metabolic cycles. With aging, circadian desynchrony occurs at the expense of peripheral metabolic pathologies and central neurodegenerative disorders with sleep symptoms, and genetic ablation of circadian genes in model organisms resembled the aging-related features. Notably, a number of studies have linked longevity nutrient sensing pathways in modulating circadian clocks. Therapeutic strategies that bridge the nutrient sensing pathways and circadian clock might be rational designs to defy aging. Saccharomyces cerevisiae, nematode Caenorhabditis elegans, and fruitfly Drosophila melanogaster. These relatively simplified models rendered further analyses of longevity genes and pathways that are activated upon low-energy challenges thus mimicked DR effects  and Cryptochrome (Cry) through the binding of E-box elements. The transcriptional control is also facilitated by recruiting various coactivators including CBP/p300  in the suprachiasmatic nucleus neurons, is critical for the bimodal activity behavior in mice , cataracts, cornea inflammation, osteoporosis, premature hair loss, and failed to form adequate visceral and subcutaneous adipose storage  for systemic synchronization. SCN resides at the anterior hypothalamus and directly contacts optic chiasm for sensing the external photic input. It is composed by paired nuclei lateral to either side of the third ventricle Colwell, . Though Per1 and Per2 are robustly oscillated in the subdivision , and likely also the projections from the raphe nuclei  the change becomes subtle. Further, the peaking time in the early morning in young is reversed to low-amplitude night peaking in the elderly  recordings in the SCN in young (3–5 months) versus aged (13–18 months) mice revealed that the day and night amplitude differences in the older are significantly reduced. Similar decline in neural activity rhythms was also observed in the subparaventricular zone, one of the major SCN output regions  in dopamine neurons thus mimics PD progression particularly for dopamine neuron degeneration. The conditional knockouts have reduced physical activity as early as by 5 months of age, and then dampened for both circadian amplitude and stability. The animals also showed abolished rhythmic locomotion in constant dark or constant light conditions  patients are long recognized for SCN neuronal loss, of that VIP neuron is a prominent case  have sleep symptoms including advanced sleep phase, insomnia and reduced REM sleep  benefits , the rate-limiting enzyme for NAD+ salvage pathway, is a rhythmically expressed protein that under E-box transcriptional control , another longevity mediator that is important for sensing low energy state, contribute to relieve the PER/CRY mediated circadian feedback repression. AMPK phosphorylates and activates casein kinase I epsilon (CKIε) for the subsequent phosphorylation of PER, therefore promote PER degradation , an important sensor of insulin, growth factor, and mitogen inputs, has been revealed in circadian control through many effector proteins. For instance ribosomal S6 protein kinase 1 (S6K1), an important regulator of translation acting downstream of mTOR activation, can rhythmically phosphorylate BMAL1. The particular modification allows BMAL1 to work as a translation factor in a timely manner with response to the mTOR signaling, in addition to the canonical role in circadian transcription (Lipton et al., + precursors (Bonkowski and Sinclair, It is clear that age-related diseases such as cancer, type-2 diabetes, obesity and neurodegenerative disorders are profoundly metabolic-associated (Lopez-Otin et al.,


What made us human? Gene expression changes clearly played a significant part in human evolution, but pinpointing the causal regulatory mutations is hard. Comparative genomics enabled the identification of human accelerated regions (HARs) and other human-specific genome sequences. The major challenge in the past decade has been to link diverged sequences to uniquely human biology. This review discusses approaches to this problem, progress made at the molecular level, and prospects for moving towards genetic causes for uniquely human biology. When the human genome was first sequenced , 2, the The challenge in the post-genomic era has been to determine which of the millions of human-specific non-coding sequence differences are responsible for the unique aspects of our biology. This is a hard problem for many reasons. First, the non-coding genome is vast, requiring methods to prioritize the mutations that matter. The neutral theory of molecular evolution, coupled with redundancy in biological networks, suggests that many human-specific DNA changes had little effect on our biology. Second, we know much less about how sequence determines function of regulatory elements compared to protein or RNA genes. Hence it is difficult to predict the molecular, cellular, and organismal consequences of human-specific regulatory mutations. Furthermore, most uniquely human traits are complex, and there is no doubt that they are encoded by a combination of mutations in different genomic loci. Finally, because gene regulation has diverged significantly between primates and model organisms such as mice, zebrafish or flies, it is hard to test hypotheses about the functional effects of regulatory mutations. In this review, we discuss advances to address these barriers with an emphasis on linking sequence to function, complementing other recent papers that explore genetic and regulatory changes in human evolution –13.Single nucleotide changes can have functional consequences, but currently these are difficult to predict in non-coding regions where small mutations are frequently tolerated and the function of a particular nucleotide is rarely known. Hence, human evolutionary genetics has mostly focused on genome regions with many human-specific differences (reviewed in –17). HumInsertions, deletions, duplications, and rearrangements—collectively known as structural variants (SVs)—contribute many more nucleotides to the genetic difference between humans and chimpanzees than do HARs and other single nucleotide variants. Indeed, the first differences detected between the human and chimp genomes were large SVs discovered prior to genome sequencing . PrimatSequence conservation is a useful tool for predicting which non-coding substitutions will be functional. But many regulatory elements are not conserved , 36, andFunctional genomics data are also very useful for loosening or omitting the requirement that HARs be conserved in other species. Two recent investigations performed evolutionary analyses of putative human regulatory elements and identified many examples of human sequence acceleration missed by studies that relied on deep sequence conservation . Gittelman et al. performed comprehensive evolutionary and population genetics analyses on open chromatin ) from 130 cell types to discover regulatory DNA with evidence of adaptive evolution in humans . They diUsing a similar approach, Dong et al. compared substitution rates of DHSs to nearby ancestral repeats  and identified 3538 accelerated DHSs (ace-DHS) . NotablyAn alternative approach to identify human-specific regulatory elements is to perform functional genomics assays such as open chromatin or protein binding in multiple species and find regions with differential evidence of regulatory activity. Some of the first evidence that human-specific regulatory elements may be common came from the ENCODE project, which found that a high proportion (~50%) of the biochemically active regions identified in the human genome were not conserved across mammals , 40. SevCotney and colleagues performed ChIP-seq for histone H3 lysine 27 acetylation  in human, rhesus macaque, and mouse embryonic limb at four different developmental stages from bud to digit separation, defined as orthologous based on morphology and HOXD gene expression . They foUsing a similar approach, Reilly et al. mapped active promoters and enhancers using di-methylation of histone H2 lysine 27  and H3K27ac, respectively, during three stages of human, rhesus macaque, and mouse cortex development . CompariCADM1 gene and shows steadily increasing H3K27ac signal from macaque to human  of human, chimpanzee and rhesus macaque . The autPrescott et al. compared transcription factor (TF) and co-activator binding, histone modifications, and chromatin accessibility genome-wide in human and chimpanzee cranial neural crest cells (CNCCs) to pinpoint putative enhancers . They detrans or that the catalogs contain many false positives due to the many challenges of cross-species functional genomics, such as antibody affinity, low samples sizes, and matching cell composition and developmental stage. As Prescott et al. note, regions with fewer sequence changes than a typical HAR can show enhancer activity differences [Overall, these studies discovered many genome sequences with human-specific epigenomic signatures, which are exciting catalogs of putative regulatory elements for future studies. Since many of these regions are not deeply conserved across mammals, they complement previously identified HARs. However, most of the elements show little or no evidence of human-specific sequence change, suggesting that the causal mutations are in ferences , though ferences –49. AnotARHGAP11B [SRGAP2C [FOXP2 [One approach to investigating the functional effects of human-specific non-coding sequences is to introduce them into model organisms. Despite differences in anatomy and genetic networks across species , transgeRHGAP11B  and SRGA[SRGAP2C , and humC [FOXP2 , 54. PreC [FOXP2 , a substDespite these caveats, a number of human-specific non-coding sequences have been functionally characterized as enhancers in vivo with reporter assays in transgenic animal models. These include two conserved elements deleted in humans (hCONDELs) , nearly Massively parallel reporter assays (MPRAs) show promise in cell lines and may become practical in whole animals –62. In tAnother way to associate human-specific non-coding regions with traits is to investigate associations between any polymorphisms they harbor and phenotypic variability in humans. The polymorphic sites will be relatively recent mutations that are largely distinct from differences between human and chimpanzee reference genomes. But they may nonetheless shed light on the general function of the accelerated regions in which they occur. For example, gene copy number variants in the human-specific pericentric inversion of chromosome 1 have been associated with human developmental and neurogenetic diseases , 63–65, Cux1 [CUX1 promoter and to GFP show elevated expression of the reporter gene in the developing brain compared to the wild-type allele. But it is important to remember that transgenic enhancer assays cannot capture quantitative expression differences (see above).Leveraging this approach, Doan et al. sequenced HARs in individuals with autism spectrum disorder (ASD) and unaffected controls . ASD wasCux1 –70 is anThe study of Doan et al. demonstrates the potential for human sequencing studies coupled with deep phenotyping to shed light on HAR function. Using associations between specific HARs and ASD, the authors conclude that many HARs are essential for normal development. Even though the ASD-associated mutations are different from the substitutions that happened during human evolution, they nonetheless help to annotate the function of the genomic region containing a HAR. Polymorphic deletions of HARs could similarly be used to annotate their functions. Doan et al. further hypothesize that human-specific mutations in HARs could have altered social and/or cognitive behavior. To test this hypothesis precisely with medical re-sequencing requires that specific human–chimp differences and combinations thereof be polymorphic in living humans or primates. As targeted sequencing  enablestrans . One reason for this is that regulatory elements without sufficient sequence similarity to be aligned across species can direct conserved expression patterns, a phenomenon known as ‘enhancer turnover’ [To measure the effects of non-coding mutations on expression and phenotypes, we must move towards considering the whole regulatory region of a gene, as well as changes in urnover’ –74  . For exaAn emerging approach is to leverage high-throughput genetic screens that can probe combinations of mutations across a locus, for example, via CRISPR genome editing , 82. UsiThe past decade has seen significant progress towards addressing the major hurdles of associating human-specific non-coding elements with traits that make our species unique. Functional genomics has been transformative in terms of annotating and prioritizing HARs, and it has also been used directly to identify novel human-specific regulatory elements that are not divergent enough in sequence to be detectable in genome-wide tests for accelerated sequence evolution. As the mechanisms of gene regulation are increasingly understood, the functional effects of human-specific non-coding mutations are becoming less mysterious. High-throughput functional assays, such as massively parallel reporter assays and CRISPR screens, are increasing the numbers of characterized human-specific regulatory elements by orders of magnitude. Meanwhile, targeted and whole genome sequencing of thousands of people is enabling rare variant association and linkage studies that connect non-coding elements to traits and may even reveal the phenotypes of individuals carrying specific ancestral versus derived haplotypes. Human evolutionary studies will always be challenged by our inability to test specific hypotheses about genetic changes in the correct organismal context . However, we now have a much broader collection of complementary research tools to address this problem, including on one hand genome editing of stem cell-derived cell lines, organoids, human primary cells, and model organisms, and on the other hand deep phenotyping and sequencing of humans and other primates to capture natural variation.Despite these exciting advances towards understanding the role of regulatory changes in human evolution, it is important to ask what hurdles still remain. One major barrier is our very limited ability to assay functions of regulatory elements other than enhancers. Genome editing is helping to address this bias by enabling researchers to introduce individual human mutations or to knock out single human-specific elements , 82. By Another missing piece in the story of human regulatory evolution is the vast sequence differences encoded within SVs, many of which are missing or incorrect in human and non-human primate reference genomes . Even wiThe next decade could be the one in which the regulatory code is cracked, opening the door to reading out the functional effects of non-coding changes that distinguish humans from other primates. To realize this goal, we should not only push to characterize the human–chimp differences for which we already have functional hypotheses. We must also continue to ask ‘what genomic regions are missing from our analyses?’ and ‘where do current models of gene regulation fail to explain divergently expressed genes?’In order to detect acceleration in a particular lineage it is necessary to statistically test for a difference between the substitution rate observed on that lineage and the expected rate given the rest of the tree. The tests to detect acceleration typically use continuous time Markov models of DNA (or protein) evolution to quantify the likelihood of the multiple sequence alignment for a genome region . CompariIs acceleration equal to positive selection? Not necessarily. In order to differentiate positive selection from relaxation of constraint (both mechanisms can produce acceleration) it is important to compare the evolutionary rate of the accelerated element to the local neutral rate. Using this approach, it has been shown that many HARs (~50%) and haDHSs ~90%) are evolving faster than the neutral rate for that part of the genome, which is suggestive of adaptive evolution . These d% are evoHomo sapiens and placed us along with orangutans and chimpanzees in the genus Homo like any other primate, even though he believed that humans were special beings in God’s creation. Since then diverse data  have verified our place on the tree of life, while also revealing numerous differences between humans and other primates, particularly our closest living relatives the chimpanzees. These include distinct behaviors, morphological characteristics, and molecular phenotypes [http://carta.anthropogeny.org/content/about-moca). The uniqueness of the human brain has been investigated in particular detail. Although it is a very typical primate brain overall [When Linnaeus (1758) described our species, he named us enotypes , 92 (see overall , 93–95,  overall , spatial overall , neuropi overall , 99


Epidemiological studies indicate that physical activity and exercise may reduce the risk of developing Parkinson's disease (PD), and clinical observations suggest that physical exercise can reduce the motor symptoms in PD patients. In experimental animals, a profound observation is that exercise of appropriate timing, duration, and intensity can reduce toxin-induced lesion of the nigrostriatal dopamine (DA) system in animal PD models, although negative results have also been reported, potentially due to inappropriate timing and intensity of the exercise regimen. Exercise may also minimize DA denervation-induced medium spiny neuron (MSN) dendritic atrophy and other abnormalities such as enlarged corticostriatal synapse and abnormal MSN excitability and spiking activity. Taken together, epidemiological studies, clinical observations, and animal research indicate that appropriately dosed physical activity and exercise may not only reduce the risk of developing PD in vulnerable populations but also benefit PD patients by potentially protecting the residual DA neurons or directly restoring the dysfunctional cortico-basal ganglia motor control circuit, and these benefits may be mediated by exercise-triggered production of endogenous neuroprotective molecules such as neurotrophic factors. Thus, exercise is a universally available, side effect-free medicine that should be prescribed to vulnerable populations as a preventive measure and to PD patients as a component of treatment. Future research needs to establish standardized exercise protocols that can reliably induce DA neuron protection, enabling the delineation of the underlying cellular and molecular mechanisms that in turn can maximize exercise-induced neuroprotection and neurorestoration in animal PD models and eventually in PD patients. Parkinson's disease (PD) is a common, age-dependent degenerative neurological disorder caused by a severe loss of the nigrostriatal dopaminergic projection , the function of the skeletal musculature and also the function of the broad motor control neural systems including cerebral motor cortices, BG, the cerebellum and the thalamus  are a group of interconnected subcortical nuclei including the striatum, globus pallidus external segment (GPe) and internal segment (GPi), the subthalamic nucleus (STN), the substantia nigra pars compacta (SNc), and pars reticulata (SNr)  protection of the DA neurons or exercise's promotion of DA neuron recovery from MPTP lesion in mice and 6-OHDA lesion in rats , larger presynaptic glutamatergic axon terminals, providing an anatomical foundation for increased glutamatergic synaptic transmission at these remaining synapses , particularly in D2-MSNs; and these exercise-induced cellular changes were accompanied by behavioral improvements . Thus, exercise-induced motoric benefits in PD patients and animal PD models may derive at least partially from exercise's potential effects on MSN activity. There is currently no human study on this topic. To our knowledge, there is only one published study investigating the potential effects of exercise on MSN spiking activity in unilateral MFB 6-OHDA PD rats, performed in our lab  was first observed in cultured peripheral nerve cells in the early 1950s  was purified, sequenced, and cloned   and mesencephalic astrocyte-derived neurotrophic factor (MANF) have also been reported to have trophic effects on nigral DA neurons in normal animals and neuroprotective effects on these neurons in animal PD models -response  relation also needs to be established in both animals and humans. These mundane and incremental studies will build a solid foundation for this clinically important field to move forward.LH, WC, XL and DQ: drafting and editing. F-MZ: conceptualization, drafting, and editing.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.