Moreover, exosomes possess intrinsic properties - a nanosized structure and unique "homing effects" - that make them outstanding drug delivery vehicles. In addition, targeted exosome-based drug delivery systems can be further optimized using active targeting ligands such as nucleic acid aptamers. Indeed, the aptamers themselves can function as therapeutic and/or diagnostic tools based on their attributes of unique target-binding and non-immunogenicity. This review aims to provide readers with a current picture of the research on exosomes and aptamers and their applications in cancer theranostics, highlighting recent advances in their transition from the bench to the clinic.In the past decade, the study of exosomes, nanosized vesicles (50-150 nm) released into the extracellular space via the fusion of multivesicular bodies with the plasma membrane, has burgeoned with impressive achievements in theranostics applications. These nanosized vesicles have emerged as key players in homeostasis and in the pathogenesis of diseases owing to the variety of the cargos they can carry, the nature of the molecules packaged inside the vesicles, and the robust interactions between exosomes and target cells or tissues. Accordingly, the development of exosome-based liquid biopsy techniques for early disease detection and for monitoring disease progression marks a new era of precision medicine in the 21 In addition, increased levels of the neuronal markers neurofilament light (NfL) and synaptophysin were found in the neuron-derived exosomes in plasma from HIV-infected individuals Indeed, exosomes carry information on not only their cells of origin, thus providing readily accessible diagnostic markers, but also the progression and prognosis of a particular disorder. For instance, cancer cell-derived exosomes can carry membrane proteins involved in cancer progression. A recent study on programmed death-ligand 1 (PD-L1) in exosomes from metastatic melanoma cells found that exosomal PD-L1 could inhibit CD8 T cells and facilitate tumor growth The biological features of exosomes endow these vesicles with the ability to efficaciously deliver therapeutic molecules. The “homing effect” of exosomes indicates that exosomal therapeutics delivered are likely to induce more pronounced anticancer effects in cancer cells from which the exosomes are derived ®, a diagnostic test for cancer metastasis that detects circulating cancer cells in the blood, which was approved by U.S. Food and Drug Administration (FDA) in 2004. Liquid biopsy is also indispensable for monitoring therapeutic effectiveness in cancer patients undergoing treatment, as well as for providing real-time feedback and guidance on adjustments to optimize treatment regimens. The presence of EVs released by cancer cells in bodily fluids makes them accessible for simple and repeated sampling. Moreover, in some cases, cancer cells release more exosomes into plasma than do healthy cells, and a number of cancer-related biomarkers have been shown to be overexpressed in these cancer-derived exosomes The survival and quality of life of patients with cancer can be significantly improved when the disease is diagnosed at an early stage. Thus, the early detection of cancer is pivotal for improving treatment outcomes and reducing the cancer burden. The methodologies used for early cancer detection should be sensitive, accurate, minimally invasive and able to be repeatedly performed on demand. To mitigate the limitations of traditional tissue biopsy, a liquid biopsy of biological fluids, such as blood or urine, has been utilized to detect cancer biomarkers with minimal invasiveness, making this technique suitable for obtaining multiple samples over time TM Prostate IntelliScore (EPI) test in June 2019. ExoDxTM, which is an exosomal RNA-based clinical liquid biopsy test based on a simple urine sample that provides a more precise diagnosis of prostate cancer One interesting target protein for liquid biopsy development is EpCAM Fig. 2) that can be used to monitor CD147-CD9 double-positive exosomes secreted from colorectal cancer cells in only 5 μL of patient serum In the past few years, several techniques have been established for the detection of a variety of proteins in exosomes and tumor-associated markers through liquid biopsy. ExoTEST is an enzyme-linked immunosorbent assay (ELISA)-based technique that captures and quantifies exosomes in plasma and other biological fluids by detecting CD63 and Rab5b proteins on exosomes and caveolin-1, a tumor-associated marker The inherent limitations of conventional exosome detection methods include the prolonged time, requirement for large amounts of exosomes and extensive post-labeling processes Table While blood is commonly obtained in liquid biopsy, clinicians may request urinary samples for certain types of cancer, such as bladder cancer in vitro selection strategy known as systematic evolution of ligands by exponential enrichment (SELEX) Fig.  Owing to their advantages over conventional antibodies in terms of low immunogenicity and batch-to-batch variation, high specificity and sensitivity, as well as deeper tumor penetration Aptamers, also known as chemical antibodies, are single-stranded DNA or RNA that fold into 3D structures and specifically bind to their targets with high affinity and specificity Aptamers are emerging as a new class of ligands for tracking tumors at the cellular, subcellular and molecular levels. Aptamers used in super-resolution microscopy offer many advantages over conventional antibody-based immunostaining methods, particularly in cases in which the affinity ligands themselves are larger than the protein of interest. Specifically, aptamer-based probes have improved detection sensitivity due to minimized steric hindrance, increased ligand density and high penetration into cells/tissues in vivo targeting of aptamers to tumor cells can be visualized by labeling with a suitable fluorescent agent or radionuclide. 18F-radiolabeled HER2-targeted DNA aptamers were developed and demonstrated high tumor uptake. Therefore, such aptamers hold promise as specific HER2-positive tumor imaging agents in positron emission tomography (PET) The in silico secondary structure predictions for array-based screening 3O4- Pd@Pt nanozymes are linked by another aptamer and natural enzyme HRP, collectively referred to as nanoprobe 1, to amplify the biosensor signal. To further amplify the signal, nanoprobe 1 is subsequently converted into dendritic DNA nanostructures via links with nanoprobe 2, a structure based on Pd@Pt/HRP/cDNA Simple yet powerful aptamer sensors can be developed on a DNA module platform composed of an aptamer, a joint module for sensing a conformational change in the aptamer, a terminal stem, and a DNAzyme to report target detection in a concentration-dependent manner. Using such a strategy, Tomita and co-workers customized a microarray containing more than 10,000 sequences designed by Since the development of aptamer technology nearly 30 years ago, pegaptanib, an RNA aptamer against vascular endothelial growth factor (VEGF), is the only aptamer that has been approved by the FDA for the treatment of macular degeneration in vivo conditions. After performing studies to mitigate this concern, Tan's group recently reported the causes of this loss of targeting in vivo. Future smart design and engineering are expected to provide effective solutions to this problem.Although aptamers are promising ligands for targeted theranostics, one potential barrier for their translation into the clinic is the possible loss of targeting capacity under The application of aptamer technology in both basic science research and the clinical translation of exosomes is poised to lead to the development of the next generation of diagnostics and therapeutics.To exploit molecular markers on the surface of exosomes for improving exosome enrichment efficiency and facilitating exosome isolation, aptamers can be selected to specifically bind to the markers and thus capture the exosomes. The engineering of aptamer-guided exosome diagnostic tools is outlined in the following sections.By selecting an aptamer against an overexpressed protein on exosomes, an aptamer-guided exosome-capturing nanoplatform system can be developed as a diagnostic tool. Garrido and colleagues developed an aptamer against the A8 peptide that could bind to the extracellular domain of HSP70, which is overexpressed in many cancers Fig. 4).Given their advantages of high sensitivity, rapid response, portability, and low sample volume requirement, aptasensors have become an important diagnostic tool for detecting cancer-derived exosomes 2O2 reduction to influence the production of electrochemical signals as well as a signal amplification step. This is achieved through rolling circle amplification, a reaction that generates high molecular weight products to accomplish signal amplification Wang et al. presented a nanotetrahedron (NTH)-assisted electrochemical aptasensor as a sensitive and rapid tool for the direct capture and detection of exosomes secreted by hepatocellular carcinoma cells 5 particles/ mL. The ability of ExoAPP to identify surface PSMA on target exosomes in blood samples from prostate cancer patients is indicative of its future application in clinical diagnostics in situ electrochemical analysis of exosomes from human serum using DNA aptamer sensors Most aptamer-guided exosome diagnostic tools are based on the principle of selective binding between aptamers and exosome/disease markers. Inorganic materials and polymers are common components used in conjunction with aptamers to form integrated platforms for lab-on-a-chip exosomal diagnostic devices. Zhang and colleagues developed the ExoAPP assay, an aptamer nanoprobe-based exosome profiling system that can be used to phenotype exosome surface proteins and quantify cancer-derived exosomes 5 exosomes in 1 μL within 30 minutes with a detection limit of 500 exosomes per microliter Numerous new platforms have been developed to address the technical challenges in conventional methodologies regarding separation/isolation/c apture of exosomes. Researchers at the Chinese Academy of Sciences have developed an aptamer- based fluorescence polarization assay with a one-step mix-and-read platform for exosome quantification that requires less than 1 µL of plasma and has no purification or amplification step Furthermore, it is often desirable or necessary to release the captured exosomes from aptamers immobilized on a solid matrix. This can be achieved by the use of chelating agents, such as EDTA, which removes magnesium ions and disrupts the 3D structure of the aptamer, resulting in the release of the bound target. Unfortunately, such a chelating agent-based strategy may not work with all aptamers. Ideally, an affinity-based isolation technique should deliver intact exosomes that retain structural integrity and biological function for further downstream applications. To this end, Zhang and colleagues designed a CD63 aptamer- and magnetic bead-based approach that can be used to rapidly isolate exosomes and subsequently release intact captured exosomes within 90 minutes through the use of a DNA structure-switch aptamer 10 aptamer-guided exosomes with a peak size of 105 nm were obtained from ~1 × 107 cells within one hour. Cholesterol-PEG2000 was chosen because of its amphiphilicity and relative rigidity to stabilize exosomes through hydrophobic- hydrophobic interactions at the lipid bilayer. The strategy for synthesizing the cholesterol-PEG2000- aptamers could potentially be utilized for large-scale production of targeted exosomes secreted from immune cells for the treatment of different cancers. This methodology was proposed to be potentially safer than cell-based immunotherapy  because the cells used to prepare the exosomes can be obtained from the patients who need the therapy. Such patient-derived exosomes can be used for drug loading without the need for genetic engineering in vivo.While the technologies for aptamer-based exosome diagnostics have been developed extensively, relatively few studies that have evaluated aptamer-targeted exosome delivery systems have been reported. Wan et al. reported aptamer-targeted exosomes carrying paclitaxel, an anticancer drug commonly included in chemotherapy regimens, as a new platform for clinical implementation in cancer treatment 7 particles/ μL. Fortunately, fluorescence and electrochemical platforms with specifically designed aptamers can be highly sensitive. Compared with antibodies, aptamers have superior performance because they are much smaller. For instance, an EpCAM aptamer was shown to have better tumor penetration, more homogeneous distribution and better retention in vivo than an EpCAM antibody in a mouse xenograft tumor model 7 kDa 2, while a 25-nt aptamer covers an area of ~ 10 nm2. If both the antibody and 25-nt aptamer were conjugated to a single fluorophore or HRP, then the exosome surface area covered by one antibody would be equal to that of multiple aptamers if all other parameters were similar . Therefore, the use of aptamers as binding ligands in lieu of using antibodies will allow the more robust isolation and detection of exosomes. In addition, nucleic acid chemistry enables the large-scale and economical synthesis of aptamers with minimum batch-to-batch variations. Due to these distinct advantages, aptamers are becoming a highly attractive class of targeting ligands for engineering exosomes in theranostic applications (summarized in Table 2).The capture of exosomes using aptamers against exosomal markers such as tetraspanins and other cancer-specific markers, followed by the quantitative assessment of a disease-specific marker, constitutes an invaluable strategy for detecting disease at an early stage, monitoring the treatment response and making accurate prognostic predictions. The fact that there are more circulating exosomes in the peripheral blood of tumor patients than in that of healthy subjects suggests that tumor-derived exosomes are a potentially useful noninvasive diagnostic tool. The commonly used techniques to detect and quantify individual exosomes include nanoparticle tracking analysis (NTA) and flow cytometry. While the results obtained from NTA are subject to interference from contaminating lipoproteins and protein aggregates, which are similar to exosomes in size and density, most flow cytometers are unable to detect vesicles smaller than 100 nm. On the other hand, the specific detection of exosomal surface proteins enables an effective determination of exosomes. However, ELISA requires a relatively large number of exosomes with a detection limit of approximately 10in vivoSince its humble beginnings 30 years ago, aptamer technology is now paving the way for the flourishing development of new and effective diagnostic and therapeutic tools for major diseases A key challenge in exosome research and development is exosome heterogeneity because each exosome is unique in its molecular and biochemical composition


There is a discrepancy about the metastatic rate of internal mammary lymph nodes (IMNs) between clinical and pathologic findings. We aimed to investigate the metastatic rate of IMNs and to provide recommendations on target volume delineation of IMNs for adjuvant radiotherapy in breast cancer patients.We retrospectively analyzed data from 114 breast cancer patients treated with surgery without adjuvant radiotherapy who developed local and/or regional lymph node recurrence/metastasis at our institute from January 2015 to January 2019. Patients with widely lung or pleural metastases were excluded. We first analyzed the recurrence rate with the chest wall, the metastatic rate of internal mammary/anterior mediastinal, ipsilateral axillary and supraclavicular lymph nodes, and then investigated the distribution of the IMNs.Among the 114 included patients, the recurrence rate with the chest wall, metastatic rate of IMNs, IMNs/anterior mediastinal lymph nodes, ipsilateral axillary lymph nodes, and the ipsilateral supraclavicular lymph nodes was 43, 37.7, 59.6, 12.3, and 22.8%, respectively. The metastatic IMNs were mainly located from the first to the second intercostal space. However, metastatic lymph nodes could also be observed above the upper edge of the first rib.The metastatic rate is high in the IMNs and irradiation of the internal mammary lymphatic chain is required. It is suggested that the upper bound of the internal mammary lymphatic chain should be up to the subclavian vein with a 5-mm margin, thus connecting to the caudal border of supraclavicular clinical target volume in breast cancer patients at high risk of recurrence. Breast cancer is the most common malignant tumor in women, and postoperative adjuvant radiotherapy is an important mode of treatment , 2. BaseNevertheless, controversies persist regarding the recommendation of IMNs irradiation in all patients with ALNs metastases , 7. OppoThis is the selection process of patients included in this study Fig. : 126 patEligibility criteria included unilateral histologically confirmed invasive breast carcinoma of stage I, II, or III with any quadrant located primary tumor, irrespective of axillary involvement, eligible patients had undergone mastectomy or breast-conserving surgery and axillary dissection; exclusion criteria included patients that male breast cancer, bilateral breast cancer, and noninvasive breast carcinoma, patients with widely lung or pleural metastases were also excluded; the definition of hormone receptor-positive is estrogen and/or progesterone receptor positive , and the definition of HER2 positive is immunohistochemistry [IHC] 3+ or IHC2+/ in situ hybridization-positive. The definition of TNM stage is according to the 7th edition of AJCC cancer staging. The diagnosis of internal mammary/anterior mediastinal lymph node metastasis was mainly based on clinical symptoms, signs, and imaging modalities (mainly enhanced computed tomography [CT] of the chest). Under normal circumstances, the short diameter of IMNs on the enhanced CT is 2–5 mm. The definition of lymph node metastasis include that the internal mammary lymph nodes whose short diameter is more than 6 mm, or the lymph node is tightly connected to the internal mammary blood vessels regardless of the short diameter, lymph node who meet one of the above conditions can be seen as metastatic lymph node; the anterior mediastinal lymph nodes whose short diameter were more than 10 mm, or the lymph node is tightly connected to the blood vessels, or lymph node with ring-enhancement in contrast-enhanced CT images also can be seen as metastatic lymph node; and the hypermetabolism in PET-CT also be diagnosed as metastases even if the short diameter was less than 6 mm or 10 mm .We first analyzed the recurrence rate with the chest wall, the metastatic rate of internal mammary/anterior mediastinal, ipsilateral axillary and supraclavicular lymph nodes, and then investigated the distribution of the IMNs. In this cross-sectional study, the interval estimation of the population rate was based on approximate normal distribution method when nP and n (1-P) were both greater than five, otherwise look-up table method was used.The patients’ age at onset ranged from 31 to 77 years (median age 45 years old), and none of the included 114 patients had received postoperative adjuvant radiotherapy, patients with widely lung or pleural metastases were excluded. In the present study, most tumors (82.5%) were located at the lateral quadrant; the molecular subtype of Her2 negative (Hormone Receptor (HR)+), Her2 positive (HR+/−) and TNBC (triple negative breast cancer) are 30.7, 34.2, and 35.1% respectively; and the rate of tumor staging that under stage IIIA is 64.9%  cases of chest wall recurrence, 43 (37.7%) cases of IMNs metastases only, and 68 (59.6%) cases of IMNs/anterior mediastinal lymph node metastases. There were also 14 12.3%) cases of ipsilateral ALNs recurrence and 26 (22.8%) cases of ipsilateral supraclavicular lymph node recurrence  . HoweverThe IMNs receive lymphatic drainage from the medial and central parts of the breast and chest wall. The output tube is connected to the supraclavicular lymph nodes, leads to the anterior mediastinal lymph nodes, and enters the vein via the thoracic duct or the right lymphatic duct. Metastasis to the anterior mediastinal lymph node is not as rare as once thought. The anterior mediastinum is a stenotic region between the sternum, pericardium, and mediastinal pleura. Anatomically, the anterior mediastinal lymph nodes include the parasternal and anterior mediastinum group, while the parasternal group corresponds to the IMNs in breast cancer. The anterior mediastinum, which receives lymphatic drainage from the adjacent pleura, is not a region of high-risk lymph node metastasis even in lung cancer, except when there is pleural involvement or reflux of the enlarged mediastinal lymph node is formed. Anatomically, the anterior mediastinal metastatic lymph nodes should be derived from the internal mammary lymphatic chain, excluding widely lung or pleural metastases. Based on these considerations, we propose a concept of the extensive internal mammary lymph node region, which captures the internal mammary lymphatic chain and anterior mediastinal lymph node area (mainly region 3A and 6). While metastatic IMNs might be easily missed at routine imaging examinations, metastatic anterior mediastinal lymph nodes are larger and easier to identify on enhanced CT images therefore reducing the risk metastases being missed. Our results suggest that the metastatic rate to the anterior mediastinal lymph node is 50.9%. When the IMN and anterior mediastinum regions are combined, the recurrence rate increases to 59.6%. As such, we propose that irradiation of the internal mammary lymphatic chain is an indispensable part of adjuvant radiotherapy for breast cancer patients treated with surgery. This proposal is consistent with the NCCN guidelines [Traditionally, the internal mammary lymphatic chain has been defined as the region between the first and third intercostal space ; howeverVeronesi et al.  performeIrradiation of IMNs is problematic because it might increase the cardiac dose, as well as the risk of morbidity and mortality. Recent advances in radiotherapy technology include sophisticated imaging integrated into planning systems with techniques that shield the heart or involve deep inspiration breath holding. Such approaches allow shaping or sculpting radiation doses to suit complex cancer volumes, while reducing treatment time to protect healthy organs. These advances have the potential to further increase the added benefit of radiotherapy beyond surgery and systemic therapy, increasing survival rates.There were several limitations of this study. Firstly, this is a retrospective study, so there may exist selection bias; secondly, most of the patients did not undergo adjuvant radiotherapy owing to that most of them were treated in county hospitals before recurrence, and the others had no indications of radiotherapy according to guidelines at that time; thirdly, our study were mainly based on enhanced-CT, further studies should be done based on more accurate imageological examinations such as PET-CT and so on. In conclusion, the metastatic rate is high in the IMNs, suggesting that irradiation of the internal mammary lymphatic chain is indispensable. We propose that the upper bound of the internal mammary lymphatic chain should be the subclavian vein with a 5-mm margin, thus connecting to the caudal border of supraclavicular CTV in breast cancer patients at high risk of recurrence. Further high-quality prospective randomized trials are needed to validate this conclusion.


Due to their lower systemic toxicity, faster kidney clearance and higher tumor accumulation, ultrasmall gold nanoparticles (less than 10 nm in diameter) have been proved to be promising in biomedical applications. However, their potential applications in cancer imaging and treatment have not been reviewed yet. This review summarizes the efforts to develop systems based on ultrasmall gold nanoparticles for use in cancer diagnosis and therapy. First, we describe the methods for controlling the size and surface functionalization of ultrasmall gold nanoparticles. Second, we review the research on ultrasmall gold nanoparticles in cancer imaging and treatment. Specifically, we focus on the applications of ultrasmall gold nanoparticles in tumor visualization and bioimaging in different fields such as magnetic resonance imaging, photoacoustic imaging, fluorescence imaging, and X-ray scatter imaging. We also highlight the applications of ultrasmall gold nanoparticles in tumor chemotherapy, radiotherapy, photodynamic therapy and gene therapy. Over the last few decades, the use of gold nanoparticles (GNs) in biomedical applications has piqued interest owing to their intrinsic properties, which make them suitable for the diagnosis and treatment of cancer. GNs, like many precious metals, have a unique optical property known as surface plasmon resonance (SPR), which allows them to be used in near-infrared (NIR)-resonant biomedical imaging modalities such as magnetic resonance imaging (MRI), photoacoustic imaging (PAI), fluorescence imaging and X-ray scatter imaging The promising application of GNs in cancer treatment depends largely on their ability to penetrate tumor tissues. The studies of Liang et al. and Hong et al. showed that ultrasmall GNs displayed obvious advantages in penetration of tumor tissue in mice In this review, we focus specifically on ultrasmall GNs, which have diameters of less than 10 nm. We will firstly discuss the methods for synthesizing and surface functionalization of ultrasmall GNs. The use of ultrasmall GNs in imaging techniques for cancer diagnosis will be reviewed briefly. The applications of ultrasmall GNs in cancer therapy, including chemotherapy, gene therapy, radiotherapy and so on, will be discussed in more detail. This information may be significant in the further development of clinical applications for GNs.55(PPh3)12Cl6, which has been widely investigated in models of metal-based catalysis in situ growth in a two-dimensional mixed ligand metal-organic framework nanosheet The wet synthesis method, which is commonly used for preparing larger GNs, has also been used to prepare ultrasmall GNs The surface modification of ultrasmall GNs is necessary to reduce their surfactant-induced toxicity and increase their biocompatibility. The most important method for functionalization of ultrasmall GNs is the thiol gold reaction, which depends on the strong affinity between thiols and Au. GNs with hexadecyl trimethyl ammonium bromide (CTAB) as surfactant can be detoxified and stably dispersed by displacing the CTAB with thiolated species in vivo medical applications. Lee et al. synthesized ultrasmall GNs with a particle size of 1.4 nm using the hepatitis B virus (HBV) core protein as a scaffold. These GNs had significant MRI and magnetic hyperthermia therapy effects on both subcutaneous and deep-tissue tumors in live mice Magnetic resonance imaging (MRI) is an examination technique based on the principle of nuclear spin. MRI is of great value in the diagnosis of degenerative diseases. Superparamagnetic inorganic nanoparticles can be used in MRI and magnetic hyperthermia therapy, but their potential toxicity to the human body limits their clinical application in vivo clearance ability due to their ultrasmall diameter  is a new non-invasive and non-ionizing biomedical imaging method. In PAI, biological tissues are irradiated with non-ionizing laser pulses, and the endogenous molecules (such as hemoglobin or melanin) or exogenous contrast agents absorb light energy. This optical absorption, which is associated with the physiological properties of the tissue, is converted into ultrasonic signals carrying the optical absorption characteristics of the tissue. 2D or 3D optical absorption distribution images can be reconstructed by instrument detection and data analysis r Figure B. These 64Cu-labeled ultrasmall GNs (2.5 nm) as PET imaging contrast agents to assess kidney function in mice for the potential diagnosis of kidney disease Positron emission tomography (PET) is a relatively advanced clinical imaging technique based on positrons released after decay of positron radionuclides, which is especially suitable for early diagnosis of diseases. At present, PET is an important technology for diagnosing tumors. Although a large number of nanoparticles have been reported for the detection and diagnosis of cancer in animal models, their accumulation in tissues means that they cannot be used in clinical applications Fluorescence is a common phenomenon in nature. The principle of fluorescence imaging is based on the linear relationship between the intensity of the fluorescent signal emitted by the excited fluorescent material and the amount of fluorescent substance in a certain range. GNs possess many well-known optical properties and can passively accumulate at tumor sites, which makes them even more promising than small drug molecules in early cancer diagnosis. However, when used in tumor detection, GNs also accumulate extensively in organs of the reticular endothelial system (RES) , which lowers their targeting specificity and hinders their clinical application In recent years, X-ray scatter imaging has become a relatively popular imaging technology. Similar to X-ray imaging technology, the principle of X-ray scatter imaging is based on the difference in X-ray penetration intensity caused by the difference in tissue density and thickness. It is different from typical absorption X-ray imaging and CT imaging in that there is no need to inject a large amount of contrast agent into the patient Chemotherapy is the use of chemical drugs to treat cancer. However, chemotherapeutic drugs are inevitably internalized by normal cells and consequently cause toxic side effects. Furthermore, cancer cells can acquire drug resistance by over-expression of drug efflux pumps, increased drug metabolism, and alteration of drug targets. Therefore, a main challenge of chemotherapy is to increase the accumulation of therapeutic drugs in tumor cells, thereby enhancing the efficacy of the treatment and reducing toxic side effects in vitro proliferation of CT-26 cells was significantly inhibited due to increased apoptosis and cell cycle arrest. Moreover, the experimental results confirmed that the proliferation inhibition effect was dose-dependent. This novel drug delivery system, together with heat treatment, provides a feasible method for killing PD-L1-overexpressing cells. However, DOX is hydrophobic, and to improve its stability and selectivity, DOX-conjugated GNs are generally constructed by covalent attachment of many polyethylene glycol (mPEG) molecules with a thiol group at one end and a drug molecule at the other end Doxorubicin (DOX) is another common chemotherapy drug that effectively induces remission of multiple malignancies. Yook et al. constructed GNs (12 nm) conjugated with DOX and anti-PD-L1 for the targeted chemo- and photothermal therapy of colorectal cancer Many other types of drug molecules have been reported to show improved therapeutic effects when coupled with ultrasmall GNs. Liang et al. synthesized ultrasmall GNs (2 nm) and successfully conjugated a therapeutic peptide (p12) and a targeting peptide (CRGDK) on the surface to study the bioeffects on breast cancer cells in vitro and in vivo radiosensitization studies on 4.8, 12.1, 27.3, and 46.6 nm PEG-coated GNs Radiotherapy is one of the common methods used to treat cancer. It relies on the use of high-energy radiation to kill tumor cells. Radiosensitizers can effectively increase the radiation dose at the cellular level and can therefore enhance the effect of radiotherapy. GNs have the potential to serve as an excellent radiosensitizer in radiotherapy. This is because GNs accelerate the breakage of DNA strands when exposed to gamma or X-rays 25 nanoclusters (1.5 nm) that specifically target a prostate-specific membrane antigen ligand. These clusters provide a high-affinity radiosensitizer for the targeted cancer tissues in mice and therefore significantly enhance the effects of X-ray irradiation While GNs have obvious advantages as radiosensitizers, there are some issues that hamper their use in radiotherapy. The common issue is how to achieve selective targeting and rapid clearance of GNs to reduce the radiation dose and decrease the exposure of healthy tissue in vitro and in vivo studies confirmed that the effect of radiation therapy is dependent on the doses of ultrasmall GNs. Tumor growth was moderately inhibited with 5 mg / kg ultrasmall GNs. Importantly, O2 could be rapidly released from the liquid perfluorooctyl bromide core after ultrasound treatment. This may relieve the tumor hypoxia and prevent DNA repair by fixing the DNA radical intermediates, thus leading to cancer cell death. In conclusion, this dual-DNA-targeting strategy achieved an optimal outcome of radiotherapy.The treatment principle of radiotherapy is to damage intracellular DNA through the direct or indirect effects of external X-ray or γ-ray beams 1O2 production compared to the free photosensitizer Photodynamic therapy (PDT) is a clinically approved cancer therapy which uses non-toxic dyes and harmless visible light in combination with oxygen to produce high level of ROS to kill cells Burda et al. used PEGylated gold nanoparticle 5 nm) to delivery hydrophobic drugs for PDT in a mouse model  nm to deBroome et al. achieved targeted therapy by constructing GNs (5 nm) conjugated with epidermal growth factor (EGF) peptide and the PDT drug Pc 4 1O2 generation, and light illumination triggered drug release from the liposomes. Pérez-García et al. synthesized water-soluble GNs (7~10 nm) functionalized with mixed ligands  and the anionic photosensitizer Na-ZnTCPP 1O2 production compared to the free Na-ZnTCPP. This may be because the gold core enhanced the activity of porphyrin. These results showed that GNs can provide the additional benefit of enhancing the effects of photodynamic therapy effects as well as acting as delivery agents.Deng et al. developed liposomes loaded with GNs (3~5 nm) together with a photosensitizer RB and the antitumor drug DOX Gene therapy, the strategy of using genetic materials to cure diseases, has great potential in cancer treatment There are still many challenges to overcome in terms of enhancing and controlling gene-based therapies. For example, therapeutic nucleic acids must be shielded from enzymatic and physical degradation. Rotello et al. designed a system in which trimethylammonium-modified GNs (2 nm) were combined with DNA by electrostatic interaction to protect the DNA from degradation Mitochondria are a potential target for cancer therapy, because mitochondrial damage leads to altered redox homeostasis, ROS production, and apoptotic cell death. However, intracellular GSH can rapidly neutralize the ROS produced in mitochondria 2O2 and gluconic acid in the presence of dissolved oxygen. This indicated that GNs can be used as a substitute for glucose oxidase (GOx) because their activities are similar 2O2, and the H2O2 was further converted into highly toxic hydroxyl radicals through a Fe3O4-based Fenton reaction which eventually caused cell death. Wu et al. selected a porphyrin metal-organic framework (PCN) with photodynamic therapy and fluorescent imaging abilities. They sandwiched catalase-mimicking Pt nanoparticles between PCN and then embedded ultrasmall GOx-mimicking GNs (2 nm) within the outer shell. The system was coordinated with FA to improve tumor accumulation 2O2 acted as a substrate for the Pt nanoparticles. The studies described here highlight the unique potential of GNs as components of nanozymes for cancer treatment. They also provide ideas for designing catalytic cascade models for antitumor therapy.A nanozyme is a nanomaterial with enzyme-like characteristics. The variable size and higher stability of nanozymes means that they may be more beneficial against solid tumors than natural enzymes, which are restricted by the chemical composition and temperature of the local environment We have reviewed the advantages and biomedical applications of ultrasmall GNs with a spotlight on cancer treatment. Size is one of the key physical parameters of GNs, which directly affects their properties such as toxicity, biocompatibility, etc. Many reports have explored the size effect of GNs, and the results indicated that ultrasmall GNs have more effective renal clearance, higher tumor tissue permeability, better cell uptake and more efficient entry into nuclei. A better understanding of the properties of ultrasmall GNs will help to design ultrasmall gold-based nanoplatforms to further extend their biomedical applications. Surface modifications and functionalization have played key roles in the development of ultrasmall GNs. Currently, ultrasmall GNs with excellent optical and electrical properties have been used as contrast agents in magnetic resonance imaging, photoacoustic imaging, fluorescence imaging, and X-ray scatter imaging. Importantly, ultrasmall GNs can specifically target tumor tissues and deliver agents for photodynamic therapy, chemotherapy, gene therapy and so on, to enhance the efficiency of cancer cell killing and minimize the impact on non-tumor tissues.Although the current results of research on ultrasmall GNs in biomedical applications are indeed encouraging, there are still issues that need more attention. First, based on the physical properties of GNs, more research is needed on integration of diagnosis and treatment. Secondly, as a universal challenge in cancer treatment, the differences between patients complicate the optimization of GNs for cancer therapy, and more efforts are needed to address this obstacle. However, the ease of functionalization of ultrasmall GNs offers more opportunities for personalized medicine. Given the success of ultrasmall GNs for biological imaging and cancer treatment, the development of future strategies to overcome obstacles and realize gold-dependent cancer cell killing is particularly promising. Taking all the evidence together, we believe that ultrasmall GNs offer unique opportunities to translate the insights of basic research into clinical applications.


Clinical and Translational Science Award (CTSA) TL1 trainees and KL2 scholars were surveyed to determine the immediate impact of the COVID-19 pandemic on training and career development. The most negative impact was lack of access to research facilities, clinics, and human subjects, plus for KL2 scholars lack of access to team members and need for homeschooling. TL1 trainees reported having more time to think and write. Common strategies to maintain research productivity involved time management, virtual connections with colleagues, and shifting to research activities not requiring laboratory/clinic settings. Strategies for mitigating the impact of the COVID-19 pandemic on training and career development are described. Clinical and translational research aims to move scientific discoveries to clinical innovations that diagnose, prevent, or treat disease. The Clinical and Translational Science Award (CTSA) program, funded by the National Center for Advancing Translational Sciences (NCATS), supports research training and mentored career awards at CTSA program institutions (hubs). By supporting predoctoral students and postdoctoral fellows through TL1 Awards and early stage investigators through KL2 Awards, NCATS endeavors to increase the number of well-trained clinical and translational scientists who will lead this research to improve human health , “health habits” , “adaptation/home office” , and “virtual connect personal”  more often than did KL2 scholars  and professional skills . The strategies cited most often by both TL1 trainees and KL2 scholars to maintain research productivity, but only by about half of them, were related to time management. Training for more effective time management could be easily implemented by CTSA programs with the assistance of institutional human resource departments, and maintaining good practices can be monitored and encouraged by mentors.Access to mentors was not reported to be a common problem Fig. , but comStrategies mentioned more often by TL1 trainees than KL2 scholars included attention to mental health such as taking breaks, healthy habits such as exercise and nutrition, and virtual connections to family and friends Fig. . KL2 schWhereas many effects of the pandemic were similar between trainees and scholars, a distinct difference emerged for KL2 scholars. Homeschooling and family issues were described by three times as many KL2 scholars as TL1 trainees. This is consistent with KL2 scholars being early stage investigators who are further along in their life journey, with differing non-academic relationships and household responsibilities. However, as early stage investigators, KL2 scholars may also be more driven to maintain research productivity, as research activities were cited the second-most often for maintaining research productivity Fig. .The coordination of work-related activities with family priorities is a particularly challenging issue for KL2 scholars, complicated further for those with patient care responsibilities. As early stage investigators with time-limited KL2 appointments, they expressed concern about the possible adverse impact of decreased productivity on their long-term research goals and perceptions of funding agencies. This anxiety can be alleviated through (1) helping them use their time as productively as possible, on still-available career development activities, on their own research, or on COVID-19-related activities that otherwise align with their career goals; (2) increasing interactions and support from mentors and program directors; (3) extending KL2 appointments and research support using federal or institutional funds when allowable and possible; and (4) facilitating implementation of institutional, private, and federal grant review policies that account for this rare challenge. It should be noted that the NIH has expressed deep concern for the health and safety of people involved in NIH research and the effects of the COVID-19 pandemic on the biomedical research enterprise. In response, the NIH has introduced some flexibilities for the administration of research, training, fellowship, and career development awards , and proWhereas this survey defined some immediate impacts of the COVID-19 pandemic on TL1 and KL2 training and career development activities, it has several limitations. Although developed by stakeholders and survey experts and pre-tested with iterative improvements before launch, this was the first use of this survey instrument; therefore, reliability and validity have not been established. Given the geographic variability in infection rates and institutional responses early in the COVID-19 pandemic, trainees and scholars likely experienced more or less severe impacts depending on their location. Aggregating results for all trainees and scholars prevented detection of pandemic severity-dependent components, and the voluntary nature of the survey allowed potential selection bias; thus, the responses might not be representative of the entire TL1 and KL2 community. The survey did not request gender or other demographic information; thus, gender-specific impacts of the pandemic could not be assessed. The absence of individual-level information, such as training stage, research focus, household composition, and patient care responsibilities, limited granular, explanatory analyses. Although it is likely that the impact on CTSA-sponsored trainees and junior investigators will extend to others at equivalent career stages, CTSA-funded trainees may not be representative of other trainees or junior faculty researchers conducting biomedical research through other funding mechanisms.Future research will be needed to assess how these findings change over time and to capture variations in local institutional and geographic policies influencing the resumption of research and training activities. A subsequent survey after the pandemic will be modified and improved based upon the lessons from this initial survey. For example, themes from qualitative coding could become response choices for new quantitative items, items with little variance could be dropped, new items could be added to address additional domains, and information about trainee characteristics  can allow more detailed analysis. It may be useful to include non-CTSA-funded researchers at similar career stages to assess whether the CTSA programs are protecting the funded trainees against adverse effects from this severe challenge.The rapid institutional responses to the COVID-19 pandemic forced dramatic interruptions in mentored research training. Overall, at this early stage of the pandemic TL1 trainees and KL2 scholars appeared to exhibit good adaptability to the pandemic, but the long-term impact on career development remains unclear. Training leaders at CTSA hubs must be better prepared to communicate with trainees to help them adapt so as to minimize the impact of this crisis on their career development. CTSA training programs must be prepared to implement remote training opportunities and communicate more effectively with TL1 trainees and KL2 scholars about how institutions are adjusting academic programs and about funding decisions that impact training progress and career progression.


Human embryogenesis is hallmarked by two phases of yolk sac development. The primate hypoblast gives rise to a transient primary yolk sac, which is rapidly superseded by a secondary yolk sac during gastrulation. Moreover, primate embryos form extraembryonic mesoderm prior to gastrulation, in contrast to mouse. The function of the primary yolk sac and the origin of extraembryonic mesoderm remain unclear. Here, we hypothesise that the hypoblast-derived primary yolk sac serves as a source for early extraembryonic mesoderm, which is supplemented with mesoderm from the gastrulating embryo. We discuss the intricate relationship between the yolk sac and the primate embryo and highlight the pivotal role of the yolk sac as a multifunctional hub for haematopoiesis, germ cell development and nutritional supply. Why do human embryos need a yolk sac and how does it form? This Perspective by Thorsten Boroviak and Connor Ross explores the development and function of the yolk sac in primate embryogenesis. Despite the absence of yolk in the eggs of placental mammals, the yolk sac has remained an integral part of embryonic development. Human and non-human primates are no exception.The yolk sac is phylogenetically the oldest extraembryonic membrane to support embryogenesis. It evolved in our aquatic ancestors >500 million years agoembryo proper during gastrulation. Interestingly, primate extraembryonic mesoderm is specified prior to gastrulation and the developmental origin of this early mesoderm population has been subject to intensive debate.The mammalian yolk sac originates from the hypoblast , which is specified at the late blastocyst stage prior to embryo implantation. In human and non-human primates, yolk sac formation entails the generation of a transient primary yolk sac—before the establishment of a secondary yolk sac. Primary yolk sac formation coincides with the emergence of extraembryonic mesoderm after implantation. Extraembryonic mesoderm is an integral part of amnion, yolk sac, allantois and chorion Box , with crIn this review, we briefly touch upon the evolutionary origin of the yolk sac and subsequently shift our focus towards the developmental origin of the primate yolk sac. We collate our current knowledge on the emergence of the yolk sac’s founding population, the hypoblast, its progression to the primary yolk sac in the implanting primate embryo and the establishment of the secondary yolk sac. Moreover, we propose a rationale for primary yolk sac formation and early extraembryonic mesoderm specification in primates. We discuss the critical roles of the secondary yolk sac for haematopoiesis, germ cell development and nutritional transport during early primate embryogenesis and conclude by providing an outlook on the exciting prospect of deriving human and non-human primate hypoblast cultures to model yolk sac development in vitro.Box 1 Extraembryonic membranes in human developmentAmnion: The innermost extraembryonic membrane consisting of a fluid-filled sac surrounding the embryo. Amnion provides a protective aqueous environment for the developing foetus.Yolk sac: Human yolk sac development consists of two developmental phases: Initially, the embryo forms a primary yolk sac, which rapidly collapses and is replaced by a secondary yolk sac. The secondary yolk sac is the definitive yolk sac. It gives rise to the first blood cells of the embryo and is highly vascularised. The secondary yolk sac ensures nutritional supply for the early embryo before the chorion is sufficiently developed to perform this function.Allantois: An extraembryonic membrane that extends from the yolk sac into the connecting stalk. Together with the yolk sac, it acts as a source for embryonic blood cells.Chorion: The outermost extraembryonic membrane derived from the trophoblast of the blastocyst. Together with the allantois, the chorion represents the foetal component of the placenta and is highly vascularised for gas exchange, waste management and nutrient transport during foetal growth.3. To accomplish this challenge, amniotes  have developed three additional extraembryonic membranes: amnion, chorion, and allantois3 , where VIT1 is eroded but VIT2 remains functional for an oviparous mode of reproduction. Major milk resource genes, such as caseins, appeared before the loss of VIT genes in the common mammalian ancestor6. This suggests that the emergence of lactation and placentation allowed for the gradual loss of yolk-dependent nourishment during mammalian evolution6.In most cases, viviparity gradually developed by retention of the egg in the mother for prolonged periods of time before hatching until the point at which the egg is kept entirely internal and the newborn baby emerges. Non-mammalian vertebrates and one of the four egg-laying mammals, the platypus, rely on nourishment from yolk rich in vitellogenin during the gestation period. Genomic comparison with chicken vitellogenin showed conserved sequences in human, armadillo and dog; however, the vitellogenin homologues in viviparous species have been degenerated by frame-shift mutations and premature stop codons4. Thus successful adaptation for in utero development required repurposing of extraembryonic membranes . However, recent studies highlight considerable differences in transcriptional regulation and signalling pathway deployment in human and non-human primate hypoblast specification24.The mammalian yolk sac is derived from the hypoblast, an extraembryonic lineage originating from the early inner cell mass (ICM) of the blastocyst. Rodent models have provided invaluable mechanistic insights into hypoblast formation  signalling, as the emerging hypoblast population sustains 34. RSPO3, a potent WNT signalling enhancer48, is one of the predominant primate-specific hypoblast markers50. Conversely, marmoset, cynomolgus monkey and human epiblast cells express WNT ligands, in contrast to the mouse36. NODAL, TGFBR1, TGFBR3 and ACVR1B transcription is earlier and stronger than in the mouse, while FGF4 is delayed in primates36. This divergence in signalling pathway component expression provides a rationale for the fact that FGF signalling inhibition does not entirely block hypoblast formation in human51. Comparative embryo inhibitor experiments in marmoset and mouse showed that blocking of either FGF or WNT signalling slightly reduced hypoblast cell numbers, while combined inhibition ablated hypoblast specification24. This suggests that primate hypoblast specification is regulated by multiple pathways, including FGF and WNT, in contrast to mouse, where FGF signalling is the predominant driver for lineage segregation.Primate early ICM cells exhibit increased expression of WNT, transforming growth factor β (TGFβ)/NODAL and bone morphogenetic protein (BMP) pathway components31. Mouse ICM cells progressively lose developmental plasticity and become irreversibly committed to either lineage53. The hypoblast cells adjacent to the blastocyst cavity polarise and form an epithelium. This process is assisted by atypical protein kinase C , which is required for acquisition of apical–basal polarity and, as such, hypoblast maturation54. Although much less is known about the molecular and functional characteristics of the late primate hypoblast, electron micrographs of rhesus embryos show that hypoblast cells equally consists of a continuous, polarised epithelium55. Therefore, epithelialisation constitutes the final step of hypoblast specification in both rodent and primate blastocysts, which are now ready for implantation.By the late blastocyst stage, the rodent ICM has resolved into mature epiblast and hypoblast lineages as a result of cell migration and apoptosisEmbryo implantation is a developmental milestone, where the embryo undergoes major reorganisation. Extraembryonic tissues grow rapidly to establish a permanent link to the mother and secure a steady supply of metabolites and oxygen.56, embryos undergo interstitial implantation, as the late blastocyst penetrates through the epithelial lining of the endometrium and invades into the underlying connective tissue. Embryos of other primate species, including Old World (rhesus and cynomolgus) and New World monkeys (marmoset), implant superficially, with the blastocyst expanding in the central cavity of the uterus57. Despite this difference in implantation mode, yolk sac formation consists of two developmental phases in probably all primates. In particular, human embryos develop a primary yolk sac between Carnegie stages (CS) 4 and 5, which is then replaced by a secondary yolk sac  and lesser apes (gibbon)sac Fig. . The sec58 and expands beyond the epiblast margin. At this stage, hypoblast cells diversify into visceral and parietal endoderm .Carnegie stages (CS) in human:CS 1 (E1): Fertilisation resulting in the formation of a unicellular embryo, the zygote.CS 2 (E2–4): Cleavage divisions leading to morula formation.CS 3 (E5–6): Blastocyst development and hatching from zona pellucida.CS 4 (E7): Apposition and initial attachment of the blastocyst to the endometrium. Polarisation of the pluripotent epiblast.CS 5A (E8): Epiblast segregates into amnion and the pluripotent embryonic disc. Syncytiotrophoblast formation and invasion of the endometrium.CS 5B (E9–10): Expansion of the amniotic cavity. In the syncytiotrophoblast, small gaps appear, termed lacunae.CS 5C (E11–12): Continuous lacunae formation leads to a nearly complete sphere; extraembryonic mesoderm begins to intrude into prospective primary chorionic villi.CS 6A (E13): Initiation of gastrulation in the embryonic disc and primary chorionic villi formation.CS 6B (E14–15): Primitive streak becomes clearly visible.CS 7 (E16–19): Formation of the notochord and initiation of haematopoiesis in the yolk sac.61). Visceral endoderm plays a critical role in this process, as part of it forms a dynamic signalling centre, the anterior visceral endoderm (AVE). The AVE is specified prior to the formation of the primitive streak and essential for patterning of anterior regions in the mammalian conceptus64.In mammals, the anterior–posterior body axis is established through gradients of conserved signalling pathways (reviewed in refs. 65 and required for the generation of PGCs66. Extraembryonic ectoderm expresses proteases SPC1 (Furin) and SPC4 (Pace4), which activate NODAL in the epiblast67. At the border between epiblast and extraembryonic ectoderm, NODAL re-enforces BMP4 expression, which induces WNT3 in the proximal epiblast68. At the same time, NODAL expression from the epiblast specifies the most distal visceral endoderm cells to secrete NODAL inhibitors CER1 and LEFTY1, as well as WNT inhibitors DKK1, SFRP1 and SFRP569. Distal visceral endoderm cells subsequently migrate towards the prospective anterior side of the mouse egg cylinder, where they form the AVE. Signalling pathway inhibition from the AVE restricts gastrulation towards the opposite side of the AVE, where a combination of WNT, BMP4 and NODAL induces the key mesoderm factor T (Brachyury)70. Primitive streak formation is initiated at the proximal posterior pole of the epiblast, where cells undergo epithelial-to-mesenchymal transition and differentiate into mesoderm and endoderm30 . In cynomolgus, CER1 is initially expressed throughout visceral endoderm and becomes anteriorly restricted within several hours73. The cynomolgus AVE secrets DKK1, presumably to locally inhibit strong WNT3A signalling from the overlying trophoblast and amnion73. Prior to gastrulation, BMP4 is expressed in the amnion and subsequently shifts towards the posterior end of the embryonic disc73. Moreover, there is robust experimental evidence for a conserved function of FGF, BMP and WNT signalling in pluripotent stem cells (PSCs). Human PSCs correspond to the postimplantation epiblast34 and efficiently differentiate into mesoderm and endoderm upon coordinated pathway stimulation mimicking the posterior signalling environment76. In the mouse, definitive endoderm gradually intercalates with visceral endoderm, although a fraction of extraembryonic cells persists at least until the formation of the early gut tube77. The dynamics of visceral endoderm replacement by definitive endoderm in the primate embryo remain unknown and will provide an exciting avenue for further studies.Primate AVE formation is associated with local thickening of the visceral endoderm. This can be observed in human embryos as early as CS 5C and 6A72 Fig. . Equally79 , Collagen  and Laminin111 34. By CS 6, these matrix proteins accumulate around the embryonic stalk region, which connects embryo and placenta79. Notably, single-cell transcriptome analysis of GATA4- and GATA6-positive extraembryonic mesoderm cells showed absence of the mesodermal marker T 34. This is in contrast to rodent extraembryonic mesoderm, which is derived from T-positive epiblast cells in the proximal region of the primitive streak91. It is tempting to speculate that the epithelial-to-mesenchymal transition in the context of primate extraembryonic mesoderm specification from hypoblast is mediated by a distinct transcriptional circuitry, but functional studies and further embryo profiling data will be required to corroborate or refute this hypothesis.Human and non-human primate extraembryonic mesoderm is continuous with, and morphologically indistinguishable from, endodermal cells lining the central cavity of the primary yolk sac74. While this hypothesis has been dismissed in the literature86 on the grounds that extraembryonic mesoderm is already abundantly present prior to primitive streak formation, it is worth taking a closer look at Luckett’s suggestion . One of these CS 5C specimens even exhibits a pronounced curved shape with precocious delamination of mesoderm at the thinner end of the embryonic disc  is already abundantly present at CS 5C and 6 . This leads to the emergence of a small cleft within the visceral endoderm, which sustains specific visceral endoderm marker expression, including SOX17 and FOXA173. In contrast, SOX17- and FOXA1-negative parietal endoderm accumulates around the amnion and stalk regions and appears to differentiate into COL6A1-secreting extraembryonic mesoderm34. Both extraembryonic mesoderm and visceral endoderm express GATA4 and GATA6, consistent with their common endoderm origin34. However, further studies using lineage tracing or spatial transcriptomics in non-human primates will be required to unequivocally clarify the mechanisms of secondary yolk sac formation.The concept of secondary yolk sac formation from visceral endoderm is supported from data in New World and Old World monkeys, where blastocysts grow larger compared to great apes and implant superficially. The extensive mesh-like network of extraembryonic mesoderm observed in the primary yolk sac of great apes is reduced to a layer of elongated and spikey parietal endoderm cells in marmoset and cynomolgus95.Throughout mammalian development, haematopoiesis occurs in three transient waves before haematopoietic stem cells are permanently established in the bone marrow. The first human blood cells are large and nucleated erythrocytes, macrophages and megakaryocytes, which originate in the blood islands of the yolk sac from CS 7 onwards Fig. 93–95.Fig96, which differentiate to form two types of angioblasts: (i) endothelial progenitors, which give rise to constituents of the blood vessels, and (ii) primitive haematopoietic progenitors, encompassing erythroid and myeloid progenitor cells99. Most blood islands are comprised of haematopoietic cells surrounded by endothelial cells and rapidly develop into an extensive vascular plexus, which envelopes the yolk sac in a sophisticated branching-like pattern. In the mouse, the first primitive erythroid progenitors are considered bipotent, giving rise to unipotent megakaryocytes and erythroid progenitors100. The initiation of cardiac contractions marks the onset of the embryo—vitelline circulation, as yolk sac-derived haematopoietic cells are disseminated throughout the developing embryo101. Consequently, primate and rodent primitive erythrocytes can be readily found inside the cardiac cavity103. The first wave of haematopoiesis in the yolk sac is rapidly followed by the second wave, generating erythro-myeloid progenitors and lymphoid progenitors, which transiently seed the foetal liver106. At the sixth week of human gestation, primitive erythroblasts are found in embryonic and extraembryonic blood vessels, followed by an overall decline of haematopoiesis in the yolk sac from the eighth week107. Intraembryonic haematopoietic stem cells arise in the major arteries of the developing embryo through a third and final wave of haematopoiesis108. Towards the end of gestation, blood cell production has translocated to the bone marrow, which becomes the permanent site for haematopoiesis throughout adulthood in mice and humans101.Blood formation is initiated in yolk sac mesoderm of either embryonic or extraembryonic origin, which aggregates into small masses. These condensations of extraembryonic mesoderm have been postulated to be the primordia of blood islands108. Interestingly, mouse yolk sac derived erythro-myeloid progenitors persist into adulthood as a common origin for tissue macrophages109. Evidence from human PSCs suggests that mesoderm destined to a haematopoietic fate is marked by primitive streak genes T (Brachyury), MIXL1 and FOXF1, as well as surface receptors KDR and PDGFRA110. Efficient generation of primitive haematopoietic cells in vitro from nascent mesoderm requires ACTIVIN and BMP signalling111 but does not yield long-term haematopoietic stem cells. Conversely, ACTIVIN inhibition112 or WNT stimulation114 of mesoderm progenitors shifts the balance towards definitive haematopoietic lineages, as defined by the capacity to generate T-lymphocytes113. The fact that primitive and definitive haematopoietic cells can be efficiently derived from human PSCs supports the notion that epiblast-derived mesoderm, at least partially, contributes to yolk sac mesoderm and the first wave of haematopoiesis. Currently, there are no in vitro models for primate hypoblast or hypoblast-derived extraembryonic mesoderm. Further studies and lineage tracing experiments will be required to determine the precise origin of haematopoiesis in primates. While the molecular mechanisms of primary haematopoiesis remain poorly understood, it is clear that the intricate vascular system of the yolk sac plays an indispensable role in transporting nutrients throughout the early stages of gestation. As embryonic development progresses towards organogenesis, the establishment of a complex vascular system is imperative for growth, delivery of nutrients and survival of the conceptus.A central question is whether the first blood cells in the primate yolk sac originate from epiblast- or hypoblast-derived extraembryonic mesoderm. Mouse blood island formation appears to be regulated by conserved signalling cascades, including FGF, BMP, TGFβ and WNT pathways, as well as paracrine crosstalk between yolk sac endoderm and the overlying mesoderm115. PGCs are the founder cells of sperm and egg, which harbour the unique ability to generate a totipotent zygote at fertilisation. The curious fact that PGCs vacate the embryo during the fourth and fifth week of gestation and migrate into the yolk sac shall be the focus of this section.More than 100 years ago, human primordial germ cells (PGCs) were initially discovered in the yolk sac116. Inductive signalling through BMP4 from extraembryonic ectoderm, BMP2 from visceral endoderm and WNT3 from the epiblast are crucial in specifying a cluster of approximately 40 PGCs. The earliest molecular markers include Prdm1 (Blimp1), Prdm14 and Tfap2c (Ap2γ)119. Mouse PGCs traverse the primitive streak and allantois towards the hindgut120 and undergo genome-wide epigenetic reprogramming, which encompasses X-chromosome reactivation, DNA demethylation and erasure of genomic imprints123. Their pilgrimage ends in the genital ridges, where germ cells undergo sex-specific maturation in the developing gonads.In rodents, the epiblast acquires uniform competence for the germ cell fate prior to gastrulation; however, the capacity to form germ cells is swiftly confined to the proximal posterior region73. Primate embryos exhibit a planar, bilaminar structure during the pre-gastrulation period, in contrast to the cup-shaped egg cylinder in rodents. Upon implantation, the primate epiblast segregates amnion and generates the amniotic cavity  and TFAP2C73. However, the proposed amnion model for PGC formation is not observed in bilaminar embryos of other mammalian species. For instance, porcine PGCs are specified in the posterior embryonic disc through autocrine signals from the epiblast at the early primitive streak stage124. Equally, in humans, the epiblast is the likely source of PGCs as suggested by in vitro models of PGC induction from human PSCs125. Conceivable explanations for a difference between human and Old World monkey germ cell specification have been proposed, including the possibility of human amnion segregation prior to PGC formation or a “dual origin” for PGCs from both amnion and posterior epiblast126.PGCs in human and non-human primates were previously considered to originate in the posterior epiblast prior to gastrulation. However, recent evidence in cynomolgus embryos at implantation stages points towards the nascent amnion as a source of germ cellsity Fig. . For cyn73. Importantly, the formation of the primitive gut by lateral folding of the endoderm lining of the yolk sac provides an effective mechanism for PGCs to re-enter the embryo in a central location. Primate PGCs then translocate from within the hindgut into the dorsal mesentery 141 indicates conserved proteolytic activity in the chicken, mouse and human yolk sac. Moreover, the human yolk sac epithelium expresses a wide spectrum of solute carrier (SLC) family transporters to shuttle amino acids, glycose, vitamins, nucleoside sugars and ions from the exocoelomic cavity into the yolk sac cavity12. Elevated levels of the retinol (vitamin A) transporter transthyretin (TTR) supports the notion of active transport of retinol, which is essential for eye development, retinoic acid synthesis and signalling throughout embryogenesis145. In addition, mouse and human yolk sac express abundant levels of metal transporters for zinc, iron, folate and vitamins B12, C and E12.Nutrient uptake in the yolk sac involves tight regulation of receptor-mediated endocytosis and lysosomal degradation Fig. , followe146, propagation of signalling pathways147 and as a precursor for steroid hormones. Importantly, cholesterol is also essential for the activity of sonic hedgehog proteins149, which play a focal role in neural development and organogenesis150. Shortage or complete absence of cholesterol results in severe birth defects, including holoprosencephaly, heart defects and mental retardation148. In the human yolk sac, cholesterol uptake occurs via lipoprotein receptors CUBN (Cubilin), LRP2  and LDLR151. After endocytosis, lipids are shuttled into the vitelline circulation via the cholesterol efflux transporter ABCA1 and lipoprotein complexes containing apolipoproteins12. These hydrophobic vesicles are predominantly found in the blood stream, where they effectively deliver cholesteryl esters, triglycerides and essential fatty acids to all parts of the body. In the adult, apolipoproteins are synthesised and secreted by the liver and intestines. However, prior to the development of the foetal liver, the yolk sac epithelium is the primary site of apolipoprotein synthesis (Fig. APOA1, APOA4, APOB, APOE and APOC3 in chick, mouse and human yolk sacs points towards an evolutionary conserved function for cholesterol transport and lipid metabolism15.Among the most prominent features of the human yolk sac transcriptome are genes associated with cholesterol and lipid metabolism. Cholesterol is required for cell membrane integritysis Fig. . The abuTaken together, the primate yolk sac can be considered a pivotal mediator of histotrophic nutrition during the first weeks of embryonic development. It acts as an upgraded extension of the embryonic gut, evolved to take on endocrine and liver-specific functions during organogenesis. The yolk sac ensures efficient nutrient uptake, provides the molecular framework for metabolism and orchestrates the distribution of metabolites to the developing embryo.81. Moreover, embryonic development must ensue in water, as mammalian cell survival strictly depends on an aqueous environment. Extraembryonic membranes accomplish these challenges by providing nutrient transfer, gas exchange and waste management as well as an “aquarium” for the developing conceptus. In primates, the yolk sac constitutes an essential auxiliary device to process and deliver maternal nutrients to the embryo, long before foetal organs and the placenta are sufficiently matured to take on their later functions (Fig. A famous quotation notes that “the embryo is a machine that needs to function while it is being built”ons Fig. . The visons Fig. . With th50 revealed an imperative role for WNT signalling in primate hypoblast specification24 and the recent generation of naive extraembryonic endoderm from human PSCs provides an exciting avenue152, no embryo-derived hypoblast cell lines have been established so far. The quest for the derivation of authentic human and non-human primate hypoblast cells, corresponding transcriptionally and functionally to the in vivo hypoblast, is ongoing and will provide crucial insights into the signalling cascades regulating yolk sac development. Furthermore, hypoblast cell lines will be an effective tool to functionally interrogate the underlying molecular circuitry and constitute the final missing building block to model primate embryogenesis with synthetic embryos. Recent advances in the field of synthetic embryology157 and protocols to culture human159 and non-human primate embryos89 to postimplantation stages may shine light on the enigmatic primary to secondary yolk sac transition, including extraembryonic mesoderm formation. Nevertheless, non-human primate in vivo studies remain imperative to determine transcriptional and epigenetic signatures for thorough assessment of in vitro models. The exciting prospect of extraembryonic organoid cultures160 might provide a physiological platform to study yolk sac-specific functions in human, including nutrient uptake, vasculogenesis and haematopoiesis. The latter will be particular revealing if combined with embryonic or extraembryonic mesoderm or both. Ultimately, a deeper knowledge of yolk sac development and its complex functions will be imperative to deconvolute the formation of our most ancient extraembryonic organ.Many aspects of primate yolk sac formation remain elusive. While embryo profiling in multiple species


Compared to some other neuropsychiatric disorders, findings of TSPO PET in schizophrenia and related psychotic disorders have been considerably more heterogeneous. Two conflicting meta-analyses have been published on the topic within the last year: one asserting evidence for decreased TSPO uptake, while the other observed increased TSPO uptake in a selection of studies. In this paper, we review and discuss five hypotheses which may explain the observed variability of TSPO PET findings in psychotic illness, namely that (1) an inflammatory phenotype is only present in a subgroup of psychosis patients; (2) heterogeneity is caused by interference of antipsychotic medication; (3) interference of other clinical confounders in the study populations ; or (4) methodological variability between studies ; and (5) the glial responses underlying changes in TSPO expression are themselves heterogeneous and dynamic. Finally, we propose four key recommendations for future research proposals to mitigate these different causes of heterogeneity.Positron emission tomography using ligands targeting translocator protein 18 kDa (TSPO PET) is an innovative method to visualize and quantify glial inflammatory responses in the central nervous system  Heterogeneity seems to come with the territory of psychiatry, and the study of positron emission tomography (PET) imaging of immune alterations in the central nervous system using nuclear ligands targeting translocator protein 18 kDA (TSPO) in psychotic disorders is no different. Ten years after the first studies were published, mixed results have remained an obstinate problem. In 2017, we published the first systematic review on TSPO PET imaging in psychotic illness , bluntlyIt has been proposed that one or more different “immunophenotypes”  may exist within the psychotic population . While tFor obvious ethical and practical reasons, most patients suffering from psychotic illness will be or have previously been exposed to antipsychotic medication. Antipsychotics are known to differentially affect microglial activation  , 25 and A recent multicentric study confirmed significant positive correlations between age and TSPO binding in the frontal and temporal cortex of 140 healthy volunteers, and a significant positive correlation with age in all brain regions in male subjects . It is tNot unimportantly in the predominantly male schizophrenia patient group, the same multicentric study has indicated that females show significantly higher TSPO binding in all regions compared to males. TSPO mediates the rate-limiting step of steroidogenesis. An alteration in TSPO levels could thus affect the production of neurosteroids in the brain, among which female sex hormones, independently of peripheral endocrine sources . FinallyIn a cohort of healthy controls, smokers had a 15.5–17.0% decreased TSPO uptake, both in the satiated state and after a night of abstention, compared to nonsmokers , 33. If Two studies of TSPO PET in alcohol use disorder patients have demonstrated 10–20% decreased TSPO binding in recently detoxified patients , 36. IntT: total amount of tracer in region of interest relative to blood). Marques et al. found a significant increased TSPO level with studies using BP as outcome measure (with five out of six studies using first-generation tracer [11C]PK11195)  , but thePK11195) , 27, 42.in vitro  and a condition’s regional focus, distinct patterns of TSPO upregulation can ensue in different brain pathologies. Secondly, our knowledge on the cellular mechanisms of neuroinflammation is suboptimal . Studies7nAch- and NMDA receptors  or stages throughout the illness course , depending on the differential recruitment from different cellular sources. Both microglia and astrocytes have been implicated in post-mortem research of schizophrenia patients. Kynurenic acid (KA), an astrocyte-derived neuroinhibitory tryptophan neurometabolite, is at the core of the hypothesis linking neuroinflammation to psychotic illness. KA reduces striatal extracellular dopamine through antagonism of αeceptors . Increaseceptors . In our eceptors . Interesin vivo -[11C]PK11195 outcomes, preference should be given to second-generation tracers with arterial input function kinetic modeling. In terms of reporting, we advocate for adherence to the guidelines on the content and format of PET brain data publications which were recently provided in a consensus paper . SpecifiControl cohorts should be matched for age, sex, BMI, smoking status, and prior substance use (up to 3 months before PET scan) as well as genotype and time of scan. If matching for substance use is not possible, a careful history of any substance use in the last 3 months prior to the scan needs to be reported;Longitudinal studies are needed to track the evolution of TSPO expression changes through different phases of the illness, as well as before and after the initiation of antipsychotic medication;Studies should stratify patients according to relevant subgroups such as immunophenotypes, treatment-resistant or ultra-high-risk individuals, and different age groups. Findings should be corrected for (lifetime) cumulative medication exposure.The datasets generated for this study are available on request to the corresponding author.LP devised the main conceptual ideas and proof outline. MM aided in interpreting the results and deciding on the final scope of the manuscript. LP wrote the manuscript in consultation with MM.The authors have received funding from University Hospital Duffel/University of Antwerp to cover open access publication fees.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.


Implementation of standard precautions plus pre-exposure hepatitis B vaccine, postexposure prophylaxis for HIV and HBV, and engineering controls  have dramatically reduced the risks for HIV and HBV acquisition by HCP. Since 1999, only 1 confirmed case of HIV (a laboratory technician who sustained a needle puncture while working with a live HIV culture in 2008) has been reported.2 The rate of HBV infections among HCP decreased ~98% from 1983 to 2010.1 Although some initial pushback occurred with the implementation of standard precautions due to concern that routine wearing of gloves would be poorly received by patients and impair the ability to perform procedures such as placing intravenous catheters, they are routinely practiced and accepted today.In the early 1980s, the human immunodeficiency virus (HIV) epidemic in the United States led to a paradigm shift in infection prevention. Published papers reported that healthcare personnel (HCP) were at risk for acquisition of HIV and hepatitis B virus (HBV) via percutaneous, mucous membrane, or non-intact skin exposure. The number of acute HBV infections among healthcare personnel (HCP) was estimated as 17,000 infections in 1983.Similarly, the threat proffered by the sudden appearance of coronavirus disease 2019 (COVID-19) in the healthcare workplace resulted in the implementation of risk mitigation strategies that may also produce permanent behavioral modification in the healthcare setting. As it also occurred during the 1980s with the HIV/AIDS epidemic, the introduction of a new disease and new risks into the healthcare setting should result in long-lasting changes in patient care that at least offer potential for increased patient and staff safety.3 Increasing evidence has provided a roadmap for protecting HCP from the acquisition of COVID-19. Systematic reviews and meta-analyses have demonstrated the effectiveness of masks5 and eye protection5 to protect HCP. As with HIV and HBV, multiple measures need to be implemented in healthcare facilities to prevent SARS-CoV-2 transmission, including (1) screening of patients, visitors, and HCP for symptoms of COVID-19 prior to entry; (2) routine use of source control masks by patients, visitors, and HCP6; (3) frequent hand hygiene and surface disinfection of shared equipment and devices; (4) enhanced personal protective equipment for HCP performing aerosol-generating procedures and during care of known or suspected patients with COVID-19; and (5) prompt testing of persons with signs or symptoms of COVID-19 and institution of appropriate isolation precautions. Screening of asymptomatic patients at the time of admission may be of benefit in several circumstances, among them: (1) testing prior to admission to a congregate setting ; (2) testing prior to admission to the labor and delivery unit to protect the nursery; and, (3) testing to identify presymptomatic patients because they are clearly infectious. Testing may also be useful for screening HCP who have had COVID-19 exposure as defined by CDC. Testing strategies are changing rapidly, and increased testing of asymptomatic patients and staff may be more practical in the near future, depending on infection prevalence and test availability.7As of June 19, 2020, the COVID-19 pandemic has led to 2,201,399 infections and 118.458 deaths in the United States. The CDC has reported that HCP account for ~11% of COVID-19 infections.Use of a mask and eye protection for all direct patient contacts or at a minimum, use of a mask and eye protection for direct patient contact when the patient is unable  or unwilling to wear a mask. We propose this paradigm shift based on the following: (1) transmission of SARS-CoV-2 has been well described from presymptomatic and likely occurs from asymptomatic COVID-19 patients; (2) A negative COVID-19 test in an asymptomatic patient does not exclude incubating COVID-19, and such patients may become infectious shortly after the test; (3) the use of masks5 and eye protection5 by HCP protects against acquisition of SARS and SARS-CoV-2; and (4) the use of universal pandemic precautions would prevent HCP from an exposure that, per the CDC, would lead to exclusion from work for 14 days.8 We also suggest that the implementation of universal pandemic precautions will likely also offer a potential beneficial effect on the prevention of transmission of other droplet-spread respiratory pathogens in the healthcare setting , especially during seasons in which these and other respiratory viruses are circulating.To protect HCP, patients and visitors from acquisition of COVID-19 and other respiratory pathogens, we propose a paradigm shift; implementation of universal pandemic precautions (UPPs): We understand that implementation of universal pandemic precautions will require careful messaging for our colleagues and patients. We expect that the rationale for aforementioned universal pandemic precautions will persuade our colleagues to accept them. Messaging to patients should focus on describing the rationale for universal pandemic precautions, including noting that their use is one of several precautions that protect patients. In addition, patients should be informed that this routine practice is not specifically focused on them individually. Finally, we realize that institution and discontinuation of universal pandemic precautions should be based on current local case numbers (or rates or burden) and local prevalence of infection in asymptomatic populations (eg, preprocedural test positivity rates because symptom screening cannot detect these potentially infectious patients.9 may not be accepted. Ultimately, however, the use of hand hygiene as advocated by Semmelweis became a keystone of infection prevention. We believe that universal pandemic precautions will be accepted by patients and staff and that it will ultimately result in a safer healthcare environment for all.We realize that paradigm shifts are difficult and, as demonstrated by Semmelweis,


During vertebrate embryonic development, a population of dorsal neural tube-derived stem cells, termed the neural crest (NC), undergo a series of morphogenetic changes and extensive migration to become a diverse array of cell types. Around the developing eye, this multipotent ocular NC cell population, called the periocular mesenchyme (POM), comprises migratory mesenchymal cells that eventually give rise to many of the elements in the anterior of the eye, such as the cornea, sclera, trabecular meshwork, and iris. Molecular cell biology and genetic analyses of congenital eye diseases have provided important information on the regulation of NC contributions to this area of the eye. Nevertheless, a complete understanding of the NC as a contributor to ocular development remains elusive. In addition, positional information during ocular NC migration and the molecular pathways that regulate end tissue differentiation have yet to be fully elucidated. Further, the clinical challenges of ocular diseases, such as Axenfeld-Rieger syndrome (ARS), Peters anomaly (PA) and primary congenital glaucoma (PCG), strongly suggest the need for better treatments. While several aspects of NC evolution have recently been reviewed, this discussion will consolidate the most recent current knowledge on the specification, migration, and contributions of the NC to ocular development, highlighting the anterior segment and the knowledge obtained from the clinical manifestations of its associated diseases. Ultimately, this knowledge can inform translational discoveries with potential for sorely needed regenerative therapies. The neural crest (NC) is an embryonic population of multipotent cells that are extremely important for vertebrate body development. Concomitant with gastrulation, induction at the neural plate border leads to the delamination of cells from the neural ectoderm during primary neurulation . These eAs an extremely dynamic cell population, the NC is of high clinical interest, as disruption of this cell population results in a wide range of congenital abnormalities . Since dCentral to understanding eye development are the concurrent events intertwining cranial NC development and optic cup morphogenesis. During late gastrulation, the developing eye is established as a single eye field located in the medial anterior neural plate at the boundary between the telencephalon and diencephalon  and Rieger anomaly (iris hypoplasia), resulting in corectopia (irregular pupil), and pseudopolycoria (multiple pupils) . Over 50ARS shows genetic heterogeneity, and autosomal dominant mutations in Pitx2 and Foxc1 genes account for up to 70% of cases . Based oAn additional target of Pitx2 is the LIM-homeodomain transcription factor, Lmx1b, which is initially expressed in POM NC cells and then subsequently in the iris, cornea, trabecular meshwork, and hyaloid vasculature. Lmx1b plays a role in NC cell migration and survival, such that homozygous mutant mice have microphthalmia with iris and ciliary body hypoplasia . AutosomFewer studies have focused on Foxc1 and NC cell development. In mice and zebrafish, genetic knockout or morpholino knockdown of Foxc1 are also embryonic lethal, while heterozygous animals show ocular abnormalities consistent with ARS . Foxc1 mTaken together, the regulation and interaction between Pitx2 and Foxc1 in the NC are essential for craniofacial and ocular  development, and mutations in either of their genes manifests as the rare congenital disease, ARS. Nonetheless, additional studies are required to identify further downstream targets of Pitx2 and Foxc1 and determine their roles in anterior segment development.Primary congenital glaucoma (PCG) is due to abnormal formation of the trabecular meshwork and aqueous outflow tracts resulting in increased intraocular pressure. PCG presents between birth and 2 years of age, but most commonly in the first 6 months with the classic triad of photophobia (light sensitivity), epiphora (tearing), and blepharospasm (frequent blinking/eye closure) . These sin vivo targets of Cyp1b1 have yet to be verified. In zebrafish embryos, Cyp1b1 is expressed within the retina in the inferior optic fissure and lesser-known superior optic fissure in correlation with optic fissure patency  is a congenital eye disease characterized by central corneal opacification due to the abnormal separation of the lens from the overlying surface ectoderm. The resulting abnormal migration and differentiation of ocular NC cells leads to iris-corneal adhesions and absence of corneal endothelium and Descemets membrane . In seveGenetic mutations in or chromosomal deletions involving known genes that regulate anterior segment development have also been associated with PA. While Pitx2 and Foxc1 are typically affiliated with ARS and Cyp1b1 is associated with PCG, mutations in these genes have been linked to rare cases of PA. Notably, the underlying molecular pathogenesis of the mutations in these genes that lead to PA versus their more commonly associated congenital diseases is not well understood .Pax6 gene mutations have also been linked to PA. Notably, mutations in Pax6 are more typically associated with aniridia, a panocular congenital disease characterized by foveal hypoplasia, optic nerve hypoplasia, limbal stem cell deficiency, and varying degrees of iris hypoplasia  is the most severe form, and this disease is characterized by neurologic and cognitive disabilities and typical craniofacial abnormalities, such as a thinned vermillion, shortened palpebral fissures, a cleft palate, and/or cleft lip  in the levels of functional Pitx2 and Foxc1 cause disease. Without question, both proteins show dosage sensitivity, as minute deletions of either Foxc1 or Pitx2 lead to ARS by way of haploinsufficiency, and a previous study reported an ARS phenotype that may also result from the genetic duplication of Foxc1 . In addiNotably, in vertebrates, Pax6 function is also hallmarked by gene dosage, and aniridia in humans is linked with mutations that lead to Pax6 haploinsufficiency. Indeed, the high and continuous expression of Pax6 in ectodermal tissues, such as the cornea, corneal epithelium, ciliary epithelium iris, and lens, is indispensable for the expression of structural genes , signaling molecules affecting ocular NC migration (reviewed in Missense mutations in the genes implicated in ocular diseases result in alterations in protein functions. For example, although Pitx2 haploinsufficiency has been demonstrated as a mechanism of ARS, a majority of the reported Pitx2 gene mutations indicate reduced or abolished protein function resulting from defective DNA binding, decreased target gene induction or both . SimilarThe normal formation, migration and differentiation of NC cells is key to the evolution of various tissues and systems. Studying the signaling pathways important for the development of the NC may increase progress in the management of degenerative diseases affecting NC-derived craniofacial and ocular structures. Notably, the extensive migration and differentiation of NC cells into diverse cell types is reminiscent of the migration, invasion and proliferation of metastatic cancer cells and the multipotency of stem cells , suggestAll authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.