We report the case of an 80-year-old female presenting with polymorphic premature ventricular contractions, nonischemic cardiomyopathy, and severe, secondary mitral regurgitation. Despite a low intraprocedural PVC burden, activation mapping and successful ablation of different morphologies were achieved using a novel mapping tool, which facilitates simultaneous mapping of different PVC morphologies. For symptomatic patients with a high burden of polymorphic premature ventricular contractions, catheter ablation is a viable treatment option . So far,We report the case of an 80-year-old female who presented to our outpatient clinic with recurrence of polymorphic PVCs, resulting in palpitations and dyspnea. Preexisting medical conditions included dilated, nonischemic cardiomyopathy with a reduced left ventricular ejection fraction of 40%. Furthermore, a severe, secondary mitral regurgitation was observed in transthoracic echocardiography. Apart from arterial hypertension and grade I obesity (BMI 31), no cardiovascular risk factors were identified .After unsuccessful treatment with beta-blockers , two ablation procedures of PVCs had been performed before: ablation in the right ventricular outflow tract and right coronary cusp  and ablation from the left coronary cusp and distal great cardiac vein (2019) had led to suppression of initially predominant morphologies. However, previous low-frequency PVCs with different morphologies had gained in burden. On presentation to our outpatient clinic, Holter-ECG documented a high burden of polymorphic PVCs (52%).Aside from idiopathic dilated cardiomyopathy, two reasons were identified as potential causes for the aforementioned left ventricular dilation: first, PVCs in a clinically significant number may cause an arrhythmia-induced cardiomyopathy, mimicking dilated cardiomyopathy . Second,Conscious sedation with fentanyl and propofol led to an immediate decrease in the prevalence of PVCs, refractory to provocation with orciprenaline, and after cessation of sedative agents.With most PVCs showing a morphology indicating a left ventricular origin , a retron = 202) registered during a total mapping time of 68 : 23 minutes (2.9 PVCs per minute).Clinical PVCs remained scarce during the procedure, which is evident in the low number of PVCs . Considering their low intraprocedural burden, the different morphologies were mapped simultaneously to record a higher number of PVCs for each respective map.Four different morphologies were mapped, three of which were deemed clinically significant. Even though morphologies 2 and 3  had a veConsecutive RFC-ablation at the corresponding site of the earliest activation led to acute suppression of all targeted morphologies. To consolidate the ablation lesion for PVC 1, complimentary ablation via the distal great cardiac vein was performed. For mapping and ablation, a 3.5 mm tip catheter was used . In total, 20 pulses were applied with a cumulative ablation time of 1375 seconds and titration to a maximum energy of 35 Watts for ablation in the left ventricle and 20 Watts for ablation in the distal great cardiac vein. Considering its very low burden (0.24 PVCs per minute) and remote location of origin, PVC was not addressed with catheter ablation in this intervention.In a follow-up visit three months after ablation, the patient reported a significant improvement of dyspnea and palpitations. A 24-hour Holter-ECG documented a reduction of the PVC-burden to <1%. The occasional episodes of peripheral edema and intermittent dyspnea were still observed. In light of these residual symptoms, transcatheter mitral valve implantation was performed later on, leading to further improvement of the clinical functional status of the patient. Conventional surgery was not a viable option for this frail patient, and valve calcification had ruled out percutaneous mitral valve repair. During a 9-month follow-up, no clinically significant burden of PVCs was detected. Echocardiography showed a good result after mitral valve replacement and a stable left ventricular ejection fraction of 40% despite treatment of mitral regurgitation.In patients with polymorphic PVCs and low intraprocedural burden, successful treatment is especially challenging since both of these features are associated with a lower success rate , 6. A laIf activation mapping is impeded by a low intraprocedural burden of PVCs, pacemapping can be a viable alternative, as studies have shown satisfactory results for mapping and ablation relying solely on pacemaps . HoweverWith sequential activation mapping, PVCs not matching the singular active pattern are often ignored and lost to mapping, which may further impede the construction of a satisfactory map. In contrast, parallel mapping continuously includes all preregistered PVCs into a corresponding map and thereby simplifies the activation mapping of PVCs with low intraprocedural burden. Furthermore, consecutive activation mapping and ablation of different morphologies can be time-consuming. Parallel mapping might reduce the total procedure and fluoroscopy time as well as the dosage of sedative agents, limiting the risk for associated complications .The feasibility of this approach is supported by the here presented case. However, no final conclusions regarding the usefulness of parallel activation mapping of polymorphic PVCs can be drawn from a single case and further studies are warranted.The interplay between polymorphic premature ventricular contractions, nonischemic cardiomyopathy, and severe, secondary mitral regurgitation can challenge clinical decision making : while pGiven the high burden of PVC in this case, it was reasonable to suspect that PVCs also affected the evaluation of the functional mitral valve regurgitation, making it appear more severe in this pathologic hemodynamic state. Secondly, the patient's clinical status correlated well with PVC-burden, making this treatment most likely to improve her symptoms. Still, it seemed likely that both pathologies needed to be addressed. For catheter ablation, a transseptal access to the left ventricle might be necessary. Even though small studies have shown the feasibility of this approach after valve intervention , this isOur case report illustrates that parallel mapping of polymorphic PVCs facilitates simultaneous activation mapping of different morphologies for patients with low intraprocedural PVC-burden, in which the construction of conclusive activation maps might be challenging and time consuming with a sequential mapping approach.


Traumatic spinal cord injury (SCI) is a life-changing event with drastic implications for patients due to sensorimotor impairment and autonomous dysfunction. Current clinical evaluations focus on the assessment of injury level and severity using standardized neurological examinations. However, they fail to predict individual trajectories of recovery, which highlights the need for the development of advanced diagnostics. This narrative review identifies recent advances in the search of clinically relevant biomarkers in the field of SCI.Advanced neuroimaging and molecular biomarkers sensitive to the disease processes initiated by the SCI have been identified. These biomarkers range from advanced neuroimaging techniques, neurophysiological readouts, and molecular biomarkers identifying the concentrations of several proteins in blood and CSF samples. Some of these biomarkers improve current prediction models based on clinical readouts. Validation with larger patient cohorts is warranted.Several biomarkers have been identified—ranging from imaging to molecular markers—that could serve as advanced diagnostic and hence supplement current clinical assessments. Traumatic spinal cord injury (tSCI) is a devastating event and most often leads to serious sensorimotor deficits and autonomous dysfunctions. The worldwide yearly incidence of SCI is estimated at 40 up to 80 cases per million . While tRoutine clinical evaluations for assessing the current clinical impairment include performing the International Standards for the Neurological Classification of Spinal Cord Injury (ISNCSCI) protocol at admission. Based on this assessment, the patient’s overall impairment is classified on the American Spinal Injury Association (ASIA) Impairment scale (AIS) , 5. ThisTrying to predict recovery based on clinical parameters, a prognostic model was formulated including the five variables: age (<65 years vs ≥65 years), L3 and S1 motor, and light touch scores, which were acquired within 15 days post injury. It serves to predict independent walking 1-year post injury . This moA recent approach for classifying a patient population into more homogeneous sub-groups is the unbiased recursive partitioning technique called conditional inference tree (URP-CTREE) that defines simple decision rules for partitioning the population . This moRadiologic examinations are an integral part of the diagnostic evaluation following SCI and play an important role in assessing the level and severity of injury. Computed tomography (CT) provides excellent visualization of osseous anatomy and fractures and allows fast image acquisition . Howeveri) to the a-p diameter at the nearest normal levels (Da and Db) as measured on mid-sagittal T1w images. Similarly, the MSCC is derived as the ratio of the a-p spinal cord diameter at the level of maximum injury (di) to the a-p diameter at the nearest normal levels (da and db), measured on mid-sagittal T2w images [The standard protocol in clinical routine after SCI comprises T1-weighted (T1w) and T2-weighted (T2w) MR images of the lesion level . It servw images . Some stw images , 26. Howw images , 31.FigThe IMLL, measured as the length from the most rostral to the most caudal apices of hyperintensive signal changes within the spinal cord on mid-sagittal T2w images, was found to be a good measure for assessing the severity of SCI and showed greater predictive value when compared to MCC and MSCC , 33••. DAn alternative to these measures is the Brain and Spinal Injury Center (BASIC) score, which was proposed to improve the assessment of injury severity prognostication . This clImportant points to consider when assessing the lesion severity on conventional MR images are the exact anatomical location and local extent of the lesion, as lesions of similar size but different orientations can cause a diverse range and pattern of spared fiber tracts around the lesion . An apprHowever, the T2w signal changes are not specific to the underlying pathophysiology and could represent both transient and irreversible pathologies. Furthermore, there is considerable variation over time and patients, and their quantification relies strongly on the subjective interpretation by the examiner , 31. ThiIn contrast to conventional MRI sequences, which allow macrostructural assessment of neural tissue damage, new imaging protocols aiming at the quantification of specific tissue parameters serve to assess changes at the microstructural level , 44.A prospective quantitative imaging modality that could serve as diagnostic and prognostic measure after SCI is diffusion tensor imaging (DTI). DTI assesses the microstructural integrity of fiber tracts and, thus, has higher sensitivity to early structural changes , 45. MeaQuantitative parameters for the assessment of microstructural changes are magnetization transfer saturation (MTsat) and longitudinal and effective transverse relaxation rates  as implemented in the multi-parameter mapping protocol (MPM) . These qIn summary, recent developments of MRI sequences allow for the assessment of microstructural neuronal integrity and pathophysiological alterations in myelination and iron deposition and might add valuable information for clinical decision-making and outcome prediction. Future studies are necessary for validating and implementing these methods into clinical practice.SCI usually leads to the disruption of the blood spinal cord barrier, resulting in the leakage of several neural tissue components into cerebrospinal fluid (CSF) and blood. Additionally, during the pathophysiological events following SCI, each stage is characterized by up- and downregulations of specific proteins, which renders them an optimal target as biomarker for tracking the stages after SCI . ParticuThese CSF based biomarkers can broadly be categorized into structural and inflammatory factors, as well as markers measured in routine blood analysis , 86. RecStructural biomarkers are mostly cell-specific proteins from neuronal tissue that leak into CSF and blood after trauma. These tissue-specific proteins are produced by different cells such as neurons or glia cells. Changes in the blood and CSF concentrations of several of these proteins following SCI were observed and are described in the following section.Glial fibrillary acidic protein (GFAP) and calcium-binding protein S-100β are mainly derived from glial cells and were repeatedly shown to be elevated in both CSF and blood serum in the early acute phase after SCI in humans and animal models –70. The Neurofilaments are abundant axonal cytoskeletal proteins that can be divided into three subunits, light (NF-L), middle (NF-M), and heavy chain neurofilament (NF-H), of which NF-L and NF-H have been studied most widely as potential biomarkers in SCI . CSF andAnother protein that is expressed at high levels within neurons is Tau. It has an important function as stabilizer of microtubules and has been studied extensively in the context of Alzheimer’s disease due to its involvement in pathogenesis . IncreasNeuron specific enolase (NSE), an enzyme that is mainly located in neuronal cytoplasm, was identified as a biomarker for neuronal damage in different pathologies . NSE expMarkers of inflammation are upregulated and secreted by various cell types due to the neuro-inflammatory processes elicited by the trauma that may further worsen the injury. Therefore, they do not represent specific predictors of neuronal damage but rather reflect the general inflammatory reaction . This seInterleukins (IL) are a large family of cytokines having a diverse range of functions in immune response. Of these, particularly the pro-inflammatory IL-6 and IL-8 have been studied in SCI and seem the most promising biomarkers. IL-6 was reported to be elevated during the acute and subacute phase of SCI in a severity-dependent manner •, 67, 76Monocyte chemoattractant protein-1 (MCP-1) is a chemotactic cytokine (chemokine) produced by various cell types and plays an integral role for immune response . It was Similar to structural CSF and serum biomarkers, some markers of inflammation seem to have the potential of improving the assessment of SCI patients. However, it must be noted that these factors only represent the general inflammatory reaction and are not specific to SCI injury.Investigating the utility of routinely measured blood parameters such as blood albumin levels for the assessment of injury severity and outcome prediction, it could be shown that hypoalbuminemia was associated with poor long-term neurological outcome and thus could serve as a marker for prognostication , 79. AddTwo studies included a plethora of blood parameters measured during the first 2 weeks after SCI into predictive models and tested their contribution to improve the model •, 92. InMicroRNAs, a class of short non-coding RNAs, have drawn a particular interest during recent years as they are involved in several regulatory processes including processes after SCI such as regulation of post-injury inflammation, neuroplasticity, and axon and neuron regeneration , 97. FolNeurophysiologic techniques, such as measuring nerve conduction, motor-evoked potential (MEP), and somatosensory-evoked potentials (SEP), provide objective measures of neuronal integrity and allow the differentiation between demyelination and axonal damage ••, 99. TThis review described the latest progress in identifying reliable biomarkers for traumatic SCI and improving predictive models. Clinical evaluation by standardized neurological examination constitutes the gold standard for assessing injury severity and predicting functional outcome. Nevertheless, these models can be improved by including advanced diagnostics, suggesting that a multimodal approach—including neuroimaging and CSF/blood markers—improves the accuracy of predicting individual trajectories of recovery. Future studies investigating the exact potential of this approach with multivariate models that can accommodate multimodal data are required for demonstrating the utility of combinations of these advanced biomarkers.


BRCA1/2 mutations or HRR deficiency. Moreover, in the recurrent setting, the sequencing of initial tumor does not allow for the detection of reversions or secondary mutations restoring proficient HRR and thus leading to PARPi resistance. Therefore, it becomes crucial to better screen patients who will likely benefit from PARPi treatment, especially those with prior receipt of maintenance PARPi therapy. Recently, patient-derived organoids (PDOs) have been regarded as a reliable preclinical platform with clonal heterogeneity and genetic features of original tumors. PDOs are found feasible for functional testing and interrogation of biomarkers for predicting response to PARPi in EOC. Hence, we review the strengths and limitations of various predictive biomarkers and highlight the role of patient-derived ovarian cancer organoids as functional assays in the study of PARPi response. It was found that a combination of NGS and functional assays using PDOs could enhance the efficient screening of EOC patients suitable for PARPi, thus prolonging their survival time.Epithelial ovarian cancer (EOC) harbors distinct genetic features such as homologous recombination repair (HRR) deficiency, and therefore may respond to poly ADP-ribose polymerase inhibitors (PARPi). Over the past few years, PARPi have been added to the standard of care for EOC patients in both front-line and recurrent settings. Next-generation sequencing (NGS) genomic analysis provides key information, allowing for the prediction of PARPi response in patients who are PARPi naïve. However, there are indeed some limitations in NGS analyses. A subset of patients can benefit from PARPi, despite the failed detection of the predictive biomarkers such as  Epithelial ovarian, tubal, or primary peritoneal cancer (hereinafter referred to as EOC) represents the most devastating gynecologic cancer. Approximately 70% of EOC patients are at advanced stages at diagnosis , 2. DespBRCA1 and BRCA2 genes (BRCA) that encode the breast cancer type 1 and type 2 susceptibility proteins, which are detected in 12–15% and 5–7% of cases respectively [BRCA1/2, only a very small number of other causal mutations in homologous recombination repair (HRR) related genes have been identified, such as RAD51, ATM, ATR and PALB2 [BRCA genes can restore homologous recombination proficiency (HRP), revealing that HRD status is both a dynamic and complex phenotype [With great advances in the last decade in the understanding of the genetics of EOC, the introduction of poly ADP-ribose polymerase (PARP) inhibitors has significantly changed the approaches to EOC management. PARP inhibitors (PARPi) have been approved for the treatment of EOC in both relapsed and front-line settings due to their cytotoxic effects exploiting synthetic lethal, thereby killing tumor cells with homologous recombination deficiency (HRD) –7. Approectively –12. The nd PALB2 . Clear end PALB2 . MoreoveBRCA mutant, are eligible to receive PARPi. There are two main categories of HRD tests: (i) HRR pathway related genes that detect particular causes of HRD, (ii) mutational signatures or genomic ‘scars’ that calibrate the patterns of somatic mutations accumulated in HRD cancers irrespective of the underlying defects. These HRD tests provide key information to predict the PARPi response in patients who are PARPi naïve, however, these tests detect genomic scars that might not correlate well with PARPi sensitivity by current clinical assays [BRCA1/2 mutations or HRD. Moreover, with the common use of PARPi for the management of EOC across the treatment life cycle, de novo and acquired resistance to PARPi have been found in patients [HRD tests driven by next-generation sequencing (NGS) have been developed to better identify which cancers, except l assays –17. A supatients . Mechanipatients , 18. Givpatients –21.In this review, we outline the mechanism of PARPi action, current predictive biomarkers for PARPi response, the available functional assays of HRD to asses PARPi sensitivity, and mechanisms of PARPi resistance. The limitations of genomic profiling in predicting response to PARPi are also addressed, and an important issue is well presented that functional assays dissecting the specific DNA damage repair defects in a tumor substantially improve the selection of candidates for PARPi. The benefits and significant achievement of PDOs in ovarian cancer research are then covered. PDOs are established as a promising tumor model that allows for rapid functional testing and prediction of therapeutic sensitivity to PARPi, where a combination of genomic analysis and functional testing of PDOs allows for the identification of targetable DNA damage repair defects, and also contributes to choosing rational combinatorial approaches for overcoming de novo or acquired resistance to PARPi.DNA damage is a frequent event during cell life, which can be spontaneous or caused by environmental agents. It activates complex cellular process like DNA repair signaling and cell cycle regulation to maintain the integrity of the genome , 23. SinIn 2005, the finding that single-agent PARP inhibition selectively killed BRCA1or BRCA2 deficient cells was a pivotal discovery that ushered in clinical oncology , 35. ThiBRCA1/2 mutations [BRCA mutations or those who are both HRD-positive and platinum-sensitive, providing new treatment options for such patients  or overall survival (OS) in platinum sensitive recurrent ovarian cancer by a series of clinical trials, especially for those with utations –17. Therutations . Hence, BRCA1/2 and other HRR-related genes may be used to infer the presence of HRD. Clinical studies have demonstrated that mutations in other HRR genes have a similar positive impact on overall survival and platinum responsiveness as BRCA1/2 mutations [BRCA mutations had at least one loss-of-function mutation in candidate HRR genes including BRIP1, CDK12, RAD54L, RAD51B, ATM, FANCA, FANCD2, FANCL, RAD51C, RAD52 and XRCC3 [BRCA mutation but carried a mutation in other HRR genes derived a similar benefit to those with a BRCA mutation  and this was of a greater magnitude than that observed in the cohort that lacked mutations in either BRCA or HRR genes (HR 0.71) [BRCA HRR gene mutations, these studies grouped together all HRR genes except BRCA, which makes it difficult to interpret the relevance of any individual HRR gene at present. Though mutations or methylation of RAD51 and ATM alterations were identified associated with PARPi responses in ovarian cancer patients [BRCA HRR genes are not currently part of an FDA-approved test to assess PARPi eligibility in ovarian cancer. Nonetheless, some clinicians identify patients who may benefit from PARPi based on mutations in HRR pathway genes.Testing for germline and somatic mutations in utations , 42. In nd XRCC3 . The cohHR 0.71) . Neverthpatients , 45, theBRCA and wider HRR gene testing is determining the clinical relevance of variants of uncertain significance (VUS) that are typically rarer missense mutations and also include intronic or exonic mutations that may alter RNA splicing [BRCA1 or RAD51C promoter methylation seems not to be sufficiently reliable. Given the difficulty of predicting the functional relevance of an individual point mutation or structural variant within a given gene footprint, corroborating evidence of HRD from a genomic mutation/scar test and/or a functional assay, as discussed below, would ideally be acquired.A major challenge of splicing –48. The splicing . Thus, mBRCA1/2 deficiency and could assess up to three types of genomic scarring patterns: loss of heterozygosity (LOH), telomeric allelic imbalance (TAI), and large-scale transitions (LST) [BRCA mutation testing with a genomic instability score derived from the unweighted sum of TAI, LST and LOH  or with an assessment of fraction of genomic sub-chromosomal LOH  [BRCA1/2 mutations or HRD. ARIEL3, a randomised, double-blind, placebo-controlled, phase 3 trial, assessed rucaparib versus placebo as maintenance treatment after response to second-line or later platinum-based chemotherapy in patients with high-grade, recurrent, platinum sensitive ovarian carcinoma. The NGS assay combines mutation analysis of BRCA1/2 with measurement of the percentage of genome-wide LOH in the cancer tissue (FoundationFocus CDxBRCA) as a biomarker for sensitivity to rucaparib treatment. The result shows that the non-BRCA mutated LOH-low group also benefited significantly from rucaparib compared to placebo  –52. Measedicine) , 54. HRDmutation . FindingIn addition, with the common use of PARPi for the management of EOC across the treatment life cycle, an increasing number of patients have been exposed to prior PARPi with/or without progression on PARPi, hence, the efficacy of PARPi after PARPi treatment need to be explored. Unfortunately, many clinical trials excluded patients who had prior exposure to PARPi, as such, data regarding the efficacy of repeated use of PARPi for recurrent disease of patients who applied PARPi as first-line maintenance treatment remain limited. De novo and acquired resistance to PARPi with various mechanisms have been found in patients, and PARPi resistance mechanisms independent of HRR can result in lack of PARPi response despite HRD (it will be discussed in the next section). Identification of biomarkers for patients who would benefit from PARPi retreatment is currently underway. A prospective randomized controlled trial OReO/ENGOT Ov-38 that sought to assess the efficiency of olaparib retreatment in platinum sensitive recurrent ovarian cancer started in June 2017 (NCT03106987). According to data presented at the 2021 European Society for Medical Oncology Congress, results of OReO/ENGOT Ov-38 trial showed that rechallenge with maintenance olaparib after platinum-based chemotherapy response resulted in longer PFS for patients with relapsed ovarian cancer, regardless of their BRCA status.To date, all of the genomic assays available for HRD have some limitations. Those to detect genomic scars provide snapshots in time measuring past events, but may not reflect the current state. In order to identify real-time HRD, functional assays may produce better evaluation of the dynamic readout of actual, extant, HRR status of the tumor. The complexity of measuring all proteins of interest within a pathway makes it very desirable for a functional assay of HRD to measure a single downstream event integrating multiple upstream components of the HRR pathway. The most common functional assays involve quantification of RAD51 foci. In the process of HRR, RAD51 protein is loaded onto the ends of DSBs, allowing the resected DSBs to invade the sister chromatid. RAD51 forms distinct subnuclear foci after DNA damage, and the inability to form RAD51 foci is a common feature of HRD. This is a functional read-out for HRD without defining the actual cause of HRD. It is the presence of the RAD51 foci that identifies the functional status for homologous recombination up to the stage for RAD51 loading . Since HAt first, RAD51 foci were determined by immunochemistry (IHC) staining on fixed breast cancer biopsies collected before and 24 h after neoadjuvant treatment with DSB-inducing chemotherapy , 57, andIn general, methods for assessing the accumulation of HRR pathway protein in treated biopsies tend to retrospectively evaluate drug response but lack predictive value. To avoid ineffective drug exposure, an improvement of functional analysis is the induction of DSBs before starting treatment of patients. Most commonly, fresh tumor tissue is collected and DSBs are induced ex vivo by ionizing radiation (IR) , 60. TheDespite the potential advantages of RECAP assay, its introduction into routine practice is limited by the need for fresh tumor specimens, ex vivo DNA damage by irradiation, and the need for proliferating cells. To address this, an approach which relies on the presence of endogenous DNA DSB in the tumor at the moment of biopsy collection was studied in two small studies , 64. RADUsing ex vivo, in vivo or in vitro approaches, patient-derived three-dimensional (3D) models are now used in the field of oncology widely in functional assays to evaluate PARPi sensitivity and identify new biomarkers so as to predict patient clinical outcome. 3D tumor models using freshly resected samples, such as solid tumors , spheroids (from fluid paracentesis) or circulating tumor cells (from blood) are used to assess the therapeutic effects of PARPi. It was shown in some studies that short-term cultured PDOs can directly or indirectly predict the PARPi sensitivity of individuals through drug susceptibility tests and DNA damage repair functional assays. Moreover, for PARPi resistant patients, drug screening by PDOs may guide the initial therapy or combination therapy precisely.The incorporation of PARPi as standard first-line maintenance treatment transformed the identification of patients with inherent or acquired PARP resistance into a new urgent need. Numerous mechanisms of PARPi resistance have been described in pre-clinical and clinical studies Fig. . AcquireBRCA1 or BRCA2 that restore the open reading frame of the genes and sufficient HRR function have been fully validated as a mechanism of resistance to PARPi, which also causes platinum-based chemotherapy resistance [BRCA1/2, RAD51C, PALB and other HRR-related genes that restored the open reading frame (ORF) was identified in paired post-progression biopsies [Secondary “revertant” mutations in sistance –69. Secobiopsies , 70. SucDNA end resection is the key of DNA repair choices and often stimulated in the S/G2 phase of cell cycle to promote HRR . In the BRCAmut cells and patients, which challenged the HRR dominance in synthetic lethality [In addition to DNA repair, PARP1 and BRCA1/2 participate in DNA replication. PARP1 plays a key role in mediating the accumulation of regressed forks and avoiding DSB formation . BRCA1/2ethality . As moreethality . MRE11 aethality . In lineethality , 87. Theethality . SLFN11 ethality . Limitinethality . Finallyethality . Downregethality .BRCA1 promoter methylation, which rescues the expression of BRCA1 and confers resistance of PARPi [Epigenetic modification may affect PARPi sensitivity and lead to resistance. Multiple lines of treatment prior PARPi lead to loss of of PARPi . Phosphoof PARPi . Pharmacof PARPi .Further studies are required to develop therapeutic strategies that combat or delay the emergence of acquired resistance. The rationale for the exploit of efficacious PARPi combinations has generally focused on enhancing the antitumor effect of PARPi by creating DNA damage or modulating DNA repair. The strategy of combining PARPi with targeted agents like WEE1, ATR and CHK1/2 inhibitors that impair the ability of tumor cells to stall the cell cycle to process and repair “trapped” PARP-DNA complex has gained some attention. Multiple inhibitors of cell-cycle checkpoint kinases are being developed by pharmaceutical companies and are being tested in clinical trials designed to assess anticancer efficacy in combination with PARPi . In addition, pre-clinical evidences suggest that a series of targeted agents, such as inhibitors of the phosphatidylinositol 3-kinase (PI3K), CDK and histone deacetylases (HDAC) can induce or enhance a BRCAness phenotype in tumor cells, thus causing sensitivity and overcoming resistance to PARPi –97. DataBRCA-mutated cancers can be readily detected by NGS, other mechanisms of resistance to PARPi, such as restoration of HRR or replication fork protection, might be more challenging because these processes are often caused by genomic alterations, such as large deletions or breakpoints in introns, which are less likely to be detected using current sequencing approaches. Thus, there is a critical need for the development of HRD functional assays, which could, as a complementary to genomic studies, predict tumor response in real-time, identify PARPi resistance mechanisms and adopt alternative therapeutic strategies.Resistance to PARPi might be an inevitable consequence of the genomic instability of the HRD tumors. Although targeted approaches designed to overcome resistance to PARPi remain limited, the systematic identification of the vulnerabilities of PARPi resistant tumors will therefore be an important step. An improved understanding of the biology of PARPi resistant tumors will facilitate the development of rational combination treatment strategies to prevent and/or delay the onset of resistance and will ultimately lead to improved long-term outcomes for patients. Although a subset of genetic reversion events leading to resistance to PARPi in patients with One of the major hurdles for the development of novel regimens for cancer treatment is the challenge of translating results from bench to bedside, which is mainly due to that many cancer models poorly recapitulate patient’s tumor, and as a consequence, many drugs that perform well on preclinical cancer models ultimately fail in clinical trials. Although cancer cell lines and patient-derived xenografts (PDXs), as commonly used human cancer models, contributed a lot to cancer research, they have a number of drawbacks. For instance, success rate of establishing cell lines is generally low and involves extensive adaptation and selection to in vitro two-dimensional (2D) culture conditions, thus, the resulting cell lines are the selection of some specific clones . PDXs arOrganoids are 3D in vitro culture systems derived from self-organizing stem cells. The ability to grow organoids with high efficiency from healthy human adult stem cells has paved the way to grow organoids from patient-derived tumor tissue. Briefly, the culture of PDOs begins with mincing up the patient tissue and embedding the cells into Matrigel or Basement Membrane Extract, which are two kinds of solubilized basement membrane extracts derived from Engelbreth-Holm Swarm mouse sarcoma consisting of laminin, collagen IV, entactin, and heparan sulfate proteoglycans. Then, cells are cultured under serum-free conditions and grow into spheroid shapes that are termed “organoids” . PDOs, gIn 2015, Kessler M et al. demonstrated the establishment of long-term, stable 3D organoid cultures from human fallopian tubes which also respond to estradiol and progesterone treatment in a physiological manner . This stBRCA germline mutation carriers undergoing prophylactic bilateral salpingoophorectomy, were subjected to p53 inactivation to model HGSOC. What is noteworthy is that, even after over ten times of passage, PDOs still retain histological and genomic features of the pertinent lesion from original tumors, illustrating intra- and inter-patient heterogeneity, and can be genetically modified. It revealed that PDOs can be used for drug-screening assays and capture different tumor subtype responses to the gold standard platinum-based chemotherapy, including acquisition of chemoresistance in recurrent disease. Furthermore, EOC organoids can be xenografted, enabling in vivo drug-sensitivity assays. This is the first large-scale study presenting a long-term cultured PDO platform that enables in vitro expansion, manipulation and analysis of a wide variety of EOC subtype, indicating a major step in EOC research.Kopper et al. reported for the first time the long-term cultured (over 30 passages) patient-derived ovarian cancer organoids with derivation efficiency of 65% and established a biological sample library of 56 organoid lines from 32 ovarian cancer cases which covers all main histological subtypes . MoreoveRecently, several researches with smaller samples have also demonstrated the successful establishment and application of ovarian cancer PDOs, which maintain good consistency with the original materials, and are feasible for drug sensitivity testing –116. HofCollectively, PDOs provide benefits like high derivation efficiency, 3D spheroid structure, tumor heterogeneity preservation, matched normal controls and are compatible with high throughput drug screening. Thus, the organoid platform might be potentially promising in drug discovery and personalized medicine. Three trials are currently ongoing to evaluate the role of PDOs in predicting the clinical efficacy of anti-cancer drugs (chemotherapy and targeted therapy) in EOC . The NCT04279509 trial is a single-center study aimed at prospectively determining if high-throughput drug screen assays using PDOs can accurately select chemotherapeutic agents that result in objective response in patients with refractory solid tumors . NCT04768270 is a single-center study aimed at verifying whether PDOs can help guide precision treatments for EOC patients. NCT04555473 is a longitudinal observational phase II study of the reliability of HGSOC PDOs as a model for the patients’ response to treatments.. identified personalized responses of four PDOs which were established from three HGSOCs and one ovarian carcinosarcoma to 240 kinds of kinase inhibitors using automated screening platform within one week from harvesting the original tumor [Cancer drug screening in patient-derived cells holds great promise for personalized oncology and drug discovery but lacks standardization. Recent research demonstrated that PDOs could likely be a promising platform for rapid assessment of susceptibility to clinical drugs including PARPi. Using DeathPro, an automated microscopy-based assay assessing cell death and proliferation inhibition, Julia et al. operated a cell sensitivity test in monolayer or organoid culture from ovarian cancer patients with clinically relevant drugs . Interesal tumor . It was al tumor .50 with a proposed cut-off based on in vivo plasma PARPi concentrations, all PDOs were found to be resistant to olaparib, rucaparib and niraparib, in line with HRD classification based on whole-genome sequencing (WGS) data. Moreover, for individual patients, PDOs derived from distinct cancer lesions at a single time point exhibited a differential drug response to at least one drug, which represented intra-patient drug response heterogeneity [BRCA1 variant was found sensitive to DNA damage related agents like cisplatin and olaparib, and the patient’s disease-free period after platinum therapy was longer compared with another HGSOC patient, who showed low response in PDO drug testing [BRCA2 in one organoid, but the result of in vitro drug testing showed resistance towards olaparib [Sequencing studies have shown that EOCs display extensive inter- and intra-tumor heterogeneity on a genetic level, and functional assays in organoids can be used to address the effects of genomic tumor heterogeneity on therapeutic response. de Witte et al. established a living biobank including 36 whole-genome-characterized PDOs from 23 EOC patients with known clinical histories . At firsogeneity . Nanki eogeneity . A HGSOC testing . Howeverolaparib . These dgBRCA1mut, two gBRCA2mut, 1 sRAD51Cmut), which were assumed to be sensitive to PARPi theoretically, they are olaparib resistant and competent for forming RAD51 foci, also consistent with the clinical response. Thus, the magnitude of the HRD signature reflects only the history but not current status of a functional defect in a tumor.The available data also demonstrated that patient-derived organoids are a useful model system for rapid assessment of DNA repair defects in ovarian cancer, and highlight their many advantages , 110. ReBRCA1 mutation carrier with acquired PARPi resistance history, exhibited RAD51 foci indicating HRR restoration, but showed sensitivity to carboplatin, prexasertib (Chk1 inhibitor), VE-822 (ATR inhibitor), and gemcitabine. Interestingly, the patient was later treated with prexasertib and exhibited stable disease [BRCA2 mutation, with de novo pan-resistance to DNA damage agents including olaparib, carboplatin and prexasertib, therapeutic testing may yield useful information even when the drug resistance mechanism is incompletely understood [Organoids can be a useful tool in revealing potential PARPi resistant mechanism and choosing rational mono or combined therapy strategies. For example, through drug testing and functional assays, an organoid from a  disease . For anoderstood . Furtherderstood . Thus, iderstood –127. TheFurthermore, complementary PDO drug screening and genomic analysis allow linkage of genotypes with drug responsiveness patterns to identify candidate biomarkers for drug response. Recently, Kong J et al. presented a machine-learning framework to identify robust drug biomarkers by taking advantage of network-based analyses using pharmacogenomic data derived from 3D organoid culture models . The bioAltogether, a combination of PDO functional or therapeutic assays with genetic features may provide the best predictive accuracy, since genetic features reflect DNA repair ability in the whole cell life cycle while functional assays present this at the time point. The combination could not only predict PARPi sensitivity better, but also further explore the potential resistance mechanism, so as to indirectly or directly guide the choice of individual initial treatment or the combination treatment strategy.The successful development of PARPi over the last decade constitutes one of the prime examples of success in precision medicine to date, which provides an effective therapeutic option for a subset of ovarian cancers with expansion to other biomarker-driven indications expected in the near future. This article has highlighted the limitations of genomic profiling in predicting response to PARPi especially in PARPi reuse setting and emphasized that functional assays dissecting the specific DNA damage repair defects in a tumor substantially improve the selection of candidates for PARPi. Ovarian cancer PDOs have been established as a promising tumor model that allows for both rapid functional testing and prediction of therapeutic sensitivity with such advantages as high success rate of establishing, variety of tissue sources, short time cost and faithful hereditary preservation. Notably, a combination of functional testing of organoids and genomic analysis allows for the identification of targetable DNA damage repair defects, and also encourages rational combinatorial approaches for overcoming de novo or acquired resistance to PARPi Fig. . Howeveret. al showed that 22% of PDOs from pancreatic patient samples were of normal origins [First, it should be noted that there is the potential to grow out normal cells instead of transformed cells. Tirac H  origins , which mAnother important point to consider is the impact of the PDO microenvironment lacking a lasting immune and stroma component on the tumor response to relevant treatment, such as angiogenic and immune checkpoints inhibitors (ICI). For instance, PARPi could increase tumor mutational burden, activate immunosuppressive pathways and reprogram immune microenvironment, offering a novel therapeutic strategy to combine ICI with PARPi . Co-cultFinally, an important factor supporting the clinical applicability of organoids is efficiency and reproducibility. Standardizing guidelines for the procedures of tissue manipulation and culture conditions might promote the widespread usage of this technology in clinical settings. In addition, successful establishment of PDOs requires fragments of sufficient size and concentration of proliferating cancer cells. The possibility of having tissue fragments from laparoscopic biopsies and obtaining tumor specimens in the form of MCSs from malignant effusion fluids or circulating tumor cells would allow for higher efficiency of PDO formation. However, median costs for PDO generation and maintenance are relatively high at the moment, thus representing a potential limitation for their application to routine management of patients.Although longitudinal solid biopsies at diagnosis and relapse can present a significant challenge in ovarian cancers, they remain essential to perform functional assays and to reveal primary or secondary resistance. Future studies with larger patient numbers and longer outcome tracking are required to determine whether organoid testing is a reliable in vitro assay for predicting sensitivity and resistance to PARPi in the clinic.


Diabetes mellitus is characterized by elevated levels of blood glucose and is ultimately caused by insufficient insulin production from pancreatic beta cells. Different research models have been utilized to unravel the molecular mechanisms leading to the onset of diabetes. The generation of pancreatic endocrine cells from human pluripotent stem cells constitutes an approach to study genetic defects leading to impaired beta cell development and function. Here, we review the recent progress in generating and characterizing functional stem cell-derived beta cells. We summarize the diabetes disease modeling possibilities that stem cells offer and the challenges that lie ahead to further improve these models. More than 450 million people worldwide are diagnosed with diabetes, a number unfortunately expected to increase dramatically in the next decades . DiabeteBeta cell failure leading to diabetes occurs in different ways. While in type 1 diabetes, beta cells are destroyed by cytotoxic T lymphocytes , in typeKCNJ11  (in vitro into particular cell types to model the molecular consequences of the genetic variant under study (Differentiated human pluripotent stem cells (hPSCs) represent another source of human beta cells. hPSCs can be derived from human embryos   or from  hiPSCs) . Notablyer study . Importaer study .Here we discuss the possibilities of using hPSCs to model the impact of diabetes-associated genetic variants on the physiology of the beta cell, focusing on the molecular mechanisms impairing insulin secretion.All forms of diabetes have in common the ultimate dysfunction of the pancreatic beta cells and the consequent inadequate circulating insulin levels. Beta cells constitute about 60% of the cells in the human islets. They are highly intermingled with the other endocrine cells, in particular with glucagon producing alpha cells, the second most abundant type, a configuration that is crucial for the optimal function of the beta cells , 44, andIn conjunction with glucagon secreting alpha cells, beta cells keep human fasting blood glucose concentrations around 5 mM, normoglycemia, by adjusting their insulin secretion output . Beta cevia glucose transporters 1 (GLUT1) and 3 (GLUT3). Glucose transporter 2 (GLUT2), the main transporter in rodent beta cells, is expressed at lower levels in human beta cells  harbors genetic variants associated with reduced fasting glycemia (HK1) has been linked to congenital hyperinsulinism (Imported glucose is phosphorylated by glucokinase (GCK), a low affinity hexokinase. Diverse genetic defects in sulinism , 19, 49.glycemia , while tglycemia  hexokinasulinism .via closure of the ATP-sensitive potassium channels (K+ATP), triggering insulin secretion. Thus, oxidative metabolism of pyruvate constitutes a crucial coupling process enabling regulated insulin secretion , a beta cell disallowed gene, results in congenital hyperinsulinism  increase incorporation of glutamate into the TCA cycle leading to congenital hyperinsulinism , another G-protein coupled receptor present in the membrane of beta cells, has also been linked to T2D. A genetic variant increasing the expression of MTNR1B has been shown to lower cAMP levels in beta cells, leading to reduced insulin secretion (ADCY5), which regulates beta cell cAMP levels, has been associated with increased risk of T2D  , present in the membrane of insulin granules, have been associated with T2D susceptibility  , 75–80. tibility , 71. A rc target .While coding and non-coding genetic variants linked to diabetes often impact mechanisms regulating insulin secretion from beta cells, some of them perturb the development of the pancreas, islets, and beta cells themselves , 48. Thein vivo. These protocols have been devised based on knowledge gained from developmental biology, mostly using mouse models, that deciphered the dynamic signaling environment required for pancreas specification, endocrinogenesis, and beta cell formation  in a time-wise manner enhanced the differentiation efficiency of pancreatic progenitors and endocrine cells –132. Howpression , and respression .+ glucagon− beta cells , TGF-beta (ALK5 receptor inhibitors), and EGF (EGF and betacellulin ligands) signaling to trigger NEUROG3 expression. Interestingly, a newly developed 2D planar differentiation protocol generated cells with improved function using latrunculin A to depolymerize the cytoskeleton during endocrine induction, demonstrating that the cytoskeletal state of cells during differentiation can also modulate NEUROG3 expression. These cells were also able to reverse diabetes in STZ treated mice faster than cells generated with a 3D suspension protocol . Appropre+ cells , 156.Other approaches to improve the function of stem cell derived beta cells have relied on enrichment steps at various stages and controlling 3D cluster size. For example, enrichment of GP2+ pancreatic progenitors led to the generation of increased numbers of beta cells , 158, whThe signaling cues required in the later stages of the differentiation protocols to induce maturation are not completely identified. Recent studies have shown that better functioning hPSC-derived beta cells are generated when serum-free media with no added factors is used in the later stages , 163. VeA common problem in the field of hPSC differentiation is the robustness of a given protocol applied to different hPSC lines. In most instances, protocols are optimized specifically for one or few cell lines, and they tend to yield variable differentiation efficiencies when other cell lines are used. In the case of pancreatic differentiation, reports have shown how a particular differentiation protocol results in different percentages of pancreatic progenitors and insulin expressing cell numbers depending on the hPSC line used , 165. Thin vitro differentiation protocols, currently they only partially recapitulate the optimal in vivo signaling environment. Missing signaling cues are probably better tolerated in some cell lines than in others, explaining this apparent genetic background-determined fitness to respond efficiently to a given protocol. A partial solution to the problem of variability in differentiation efficiency across cell lines is the generation of genome edited cell lines. Genome editing technologies have made possible the introduction and correction of point mutations in hPSCs   , 189.+ATP channel genes  the lack of differentiation protocols that robustly generate functional beta cells and are widely applicable to any hPSC line; ii) the high variability of human islet preparations, which makes them a problematic gold-standard control to rely upon; iii) the absence of standardized phenotyping methods for hPSC-derived beta cells and human islets which hinders faithful comparison of results across laboratories.Beta cell differentiation protocols are technically complex: they have multiple stages, last over a month, and utilize combinations of recombinant proteins and small molecules at different dosages. During the course of any differentiation, many aspects can deviate from the optimal parameters, leading to poor reproducibility and consistency across experiments. In order to minimize experimental variation and minimize costs, laboratories differentiating hPSC usually implement standard operating procedures to prepare culture reagents and execute the differentiation experiments. Current approaches to characterize the outcomes of the hPSC differentiations towards beta cells rely on a battery of methods applied at select stages of the differentiation process. These methods commonly include, but are not limited to, flow cytometry, immunohistochemistry, and RT-qPCR. It is not uncommon for differentiation experiments to fail due to poor definitive endoderm induction, limited expression of pancreatic progenitor markers, or reduced number of INS+ cells. Since the differentiation of one cell type into the next is not 100% efficient, it is critical to address the identity of the cells in the population at given time points. The percentage of cells reaching definitive endoderm stage, the abundance of PDX1+NKX6-1+ pancreatic progenitors, and the fraction of INS+NKX6-1+ cells are examples of common flow cytometry quantifications. They are proxies for the efficiency and quality of the differentiation in terms of achieving bona-fide beta cells. The ultrastructure of hPSC-derived beta cells and human islets has been compared using electron microscopy, using insulin granule morphology as another indicator of beta cell maturity , 160.via EGFR-PI3K/AKT-RAC1 signaling resulting in apical polarity inhibition, NOTCH signaling reduction, and induction of NEUROG3 expression  have curiously shown that some important mature beta cell markers associated with functionality are expressed at much lower levels compared to adult beta cells   . It is t3, SIX3) .via a progenitor stage that expresses NKX6-1 before or after NEUROG3 upregulation. Single-cell RNA sequencing approaches enable the transcriptional profiling of thousands of cells simultaneously. Krentz and colleagues used this approach to characterize mouse and hPSC-derived endocrine progenitors , cAMP level modulators , GLP1R ligands , voltage dependent calcium channel agonists (Bay K 8644), non-glucose metabolic substrates , and forced membrane depolarization .The assessment of hPSC-derived beta cell functionality relies on methods established to characterize human islets. The most conventional method is the evaluation of insulin secretion in response to high glucose, either in a static setup or in a dynamic fashion using a perifusion setup. Additionally, different secretagogues can be used to probe the different insulin secretion mechanisms in place: KThe stimulation index (fold increase in insulin secretion from low to high glucose) of hPSC-derived islet cells in static GSIS reported by most studies ranges from 2 to 3, while human islet indexes have a median of about 7 . While sAn important characteristic of human islets is their fine-tuned secretion of insulin in response to glucose. This can be clearly observed in dynamic GSIS assays using perifusion systems, where a robust first insulin secretion phase is followed by sustained second phase of insulin secretion with lower output . The acq2+ influx into the cytoplasm in response to different stimuli. Calcium imaging can be performed on dispersed individual cells or on whole aggregates/islets. It has been used to assess the function of hPSC-derived beta cells in some studies, showing that although calcium dynamics might be similar in a small fraction of cells, they are not as robust as in primary human islet cells (ATP (45% of the cells) and Ca2+ (42% of the cells) channel currents and no Na+ channel currents  that interfere with the glucose-secretion coupling , and the response to glucose can be determined with intraperitoneal, intravenous, or oral glucose tolerance tests. Also, hPSC-derived pancreatic progenitors or endocrine cells can be transplanted within macro- or micro-encapsulation devices (Implantation in the renal subcapsular space is one of the preferred approaches since it is relatively easy to implant and retrieve the cells months later angement , concomiangement , 227. Enangement , 229. Gr devices , 230–232in vivo maturation of pancreatic progenitors since following implant, glucose-stimulated insulin secretion was observed in female mice before males (An interesting phenomenon is the functional maturation of implanted beta cells with time. Rezania and colleagues described the progressive increase in circulating C-peptide levels for several weeks after implanting pancreatic progenitor cells or more matured cells , 131, a re males . The sysre males –238.in vitro (Several studies reporting the generation of hPSC-based diabetes disease models have relied on implanting mice with hPSC-derived beta cells. This constitutes a practical solution to study the function of the beta cells in a systemic environment, especially when the disease phenotype is not apparent in vitro , 192. Itin vitro , 180. OnAn important aspect after implantation of hPSC-derived beta cells, is their capacity to survive the hypoxic environment of the implantation site until they become vascularized. This is a critical stress period that may result in the apoptosis of the most differentiated endocrine cells . Faleo ain vivo maturation process of hPSC-derived beta cells, novel in vivo imaging technologies could be exploited. Radio tracer-based imaging of beta cell mass and function could prove to be particularly useful in this regard (Another concern with stem cell derived islet cell implants, especially when using them on diabetic rodents, is the “pellet” effect, in which the non-regulated basal insulin secretion coming from immature beta cells might be enough to rescue hyperglycemia. This brings up the question of how many cells should be implanted to achieve an optimal working graft, which obviously will depend on the stage of differentiation and quality of the cells in terms of functionality . The coms regard , 242.Although intra islet paracrine signaling between the different endocrine cell types is crucial for fine-tuned insulin secretion , 45, 243Further understanding of the factors playing an important role in the functional maturation of implanted hPSC-derived beta cells will pave the way to the generation of better humanized mouse models to study insulin secretion. Ultimately, optimal control of implantation parameters will reduce the associated variability of these experiments enabling the careful assessment of the impact of genetic variants on insulin secretion.in vitro that closely resemble the native adult counterpart. Arising technologies like CRISPR-Cas9 genome editing and single cell transcriptomics are aiding in the generation of more reliable stem cell models, the refinement of the differentiation protocols and the characterization of the resulting differentiated islet cells. There are still important remaining questions in the quest for more functionally mature beta cells: how can we determine and increase beta cell specification? What are reliable mature beta cell markers and the key triggers of functional maturation? Together with detailed single cell transcriptomic characterization, improved characterization of the metabolism, proteomics and functional genomics of the hPSC-derived islets cells, and their comparison with human islets, will certainly pave the way forward. In this common effort, consensus in standardized characterization of the resulting hPSC progeny, the development of robust and reproducible differentiation protocols, and open dissemination of results, will enable prompt replication and speed up the implementation of successful strategies for beta cell generation and diabetes disease modeling.Derivation of endocrine islet cells from hPSCs has become an attractive possibility to model diabetes disease and screen for new treatments Figure 3Writing—original draft preparation, DB. Writing—review and editing, DB, DI, and TK. Funding acquisition, TK. All authors contributed to the article and approved the submitted version.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.


Despite its healthcare advantages and expanded use during the COVID-19 pandemic, telehealth is not included in many medical school curricula. In this prospective mixed methods study (n=52), we created a novel Telehealth Education Curriculum (TEC) for the third year Obstetrics and Gynecology (Ob/Gyn)rotation at New York Medical College during COVID-19. The TEC included supervised telehealth patient encounters via video conference[and a virtual Objective Structured Clinical Encounter (vOSCE)] designed to simulate a telehealth encounter .We measured student perceptions of the TEC via two 4-point Likert surveys, which included free response questions, administered via SurveyMonkey between April and June 2020.Participation was voluntary and responses were de-identified. We computed means and response distributions across survey questions using SPSS; IBM version 19. The response rate was 92% for both the Telehealth (33/36) and vOSCE (48/52) surveys. Seventy-six percent (25/33) strongly or moderately agreed that telehealth andin-person patient encounters have similar educational value. Eighty-three percent (40/48) strongly or moderately agreed the vOSCE provided a valuable patient interaction.Ninety-seven percent (32/33) strongly or moderately agreed the telehealth encounters should continue during COVID-19 restrictions versus 82% (27/33)agreeing they should be incorporated into the curriculumpost COVID-19. Almost all students responded that the TEC should continue during COVID-19 and most agreed it should be incorporated into the Ob/Gyn clerkship permanently, after COVID-19.We found vOSCEs to be an effective method for teaching telehealth to medical students. Key challenges identified by students included adjusting to a virtual format, lack of body language,and communicating empathy virtually. Positive takeaways included practice with telemedicine and an opportunity for continued clinical education during COVID-19. Telemedicine or “Telehealth” has grown exponentially in the U.S. over the past few decades and current trends in the healthcare environment fuel this expansion into the future  , 2 .TeDespite the multiple innovative applications of digital technology in healthcare, telehealth is not readily adopted by all healthcare providers and many medical schools stilldo not include telehealth in their curricula   , 10 .ADue to the new challenges faced by medical schools during the COVID-19 pandemic, novel curricula must be considered  , 14 .MThe objective of this study was to measure student perceptions of a novel Telehealth Education Curriculum for the virtual obstetrics and gynecology core clerkship during theCOVID-19 pandemic and evaluate best practices for future telehealth curriculum implementation. A secondary objective was to contribute to current medical education literatureon telehealth curricula, and their utility, to continue finding new innovations in medical student education during the COVID-19 pandemic and beyond.This prospective mixed methods study of 52 medical students was conducted at New York Medical College School of Medicine (NYMC SOM), located in Valhalla, New York.It is one of the nation’s oldest private health sciences universities (est. 1860) and provides a comprehensive educational program with the goal of developingwell-rounded medical students who will become resilient, compassionate, and skilled physicians. The SOM is proud of its strong foundational science education,diverse affiliated clinical training sites, and the commitment of its faculty and administration to medical student education.In light of the COVID-19 pandemic, which provided a significant barrier to undergraduate medical education, and specifically clinical education, the third yearObstetrics and Gynecology (Ob/Gyn) medical student core clerkship was converted to a four-week virtual format in April 2020. The students originally scheduled tocomplete the standard Ob/Gyn clerkship in April and May 2020 were automatically enrolled in the virtual Ob/Gyn clerkship. This put the onus on faculty torapidly develop and implement a new virtual curriculum that allowed for clinical learning and interaction in this new digital era. This new curriculum featureddidactic sessions, case-based presentations, and interactive learning workshops via Zoom. Didactic activities included presentation of video-based surgical cases,interactive board question review, and discussion of general obstetrics and gynecology topics. Additionally, a novel Telehealth Education Curriculum (TEC)consisting of virtual Observed Structured Clinical Examinations and actual telehealth patient encounters was implemented as part of the virtual Ob/Gyn third year clerkship.We evaluated the student perceptions of the novel TEC following their successful completion of the virtual Ob/Gyn clerkship.To be considered eligible to participate in the study, the students were required to be in their third year at NYMC SOM, enrolled in the virtual Ob/Gyn clerkship,and participating in the virtual Ob/Gyn clerkship and TEC. Students were provided with explicit information explaining that their participation in surveys was voluntary and theirresponses would be utilized for research purposes only. They were also informed that responses would be de-identified and accessed only by the research team data handlers withno impact on their grading. Two authors, JM and LF, were responsible for all data handling and were not involved in any aspect of medical student grading for the clerkship.NL and JP were able to access student data after it was processed and fully de-identified without the ability to re-identify students given their roles at the School of Medicine.All study procedures were approved by and compliant with the New York Medical College Institutional Review Board (protocol #14323).rd year Ob/Gyn Clerkship. Students then virtually met with their attendings fora telehealth debrief in which verbal and written feedback was provided.The novel Telehealth Education Curriculum (TEC) consisted of two components: the telehealth encounter and the virtual Observed Structured Clinical Examination (vOSCE).For the telehealth encounter, each student completed at least one telehealth visit via a 3-way video conference call with an Ob/Gyn patient and attending physician,before the end of the 4-week virtual clerkship. Following the telehealth visit, students completed a clinical note in the Subjective, Objective, Assessment,and Plan (SOAP) note structure and a survey entitled Telehealth Evaluation for 3The second component of the novel TEC was the virtual OSCE designed to mimic a telehealth patient encounter, conducted via Zoom Video Communications, Inc.The blueprint for the vOSCE included completion of a standardized patient encounter with a physical exam and history (20 minutes), a post-encounter note that included a differential diagnoses, assessment, and plan (15 minutes), followed by two debriefing sessions with faculty focused on communication skills (15 minutes) and clinical reasoning (40 minutes). Students also had the opportunity to collaborate with another student who had independently completed the same case, while individually writing their assessment. The standardized patients presented with a common obstetric and gynecologic complaint . Faculty observed the vOSCEs, reviewed post-encounter assessments, and evaluated students using the following criteria: medical knowledge, interpersonal skills and communication, empathy, and development of a differential diagnoses. Following the vOSCE, students completed the Virtual OSCE Evaluation Form for the 3rd year Ob/Gyn clerkship to provide feedback about their experience with the vOSCE and its value as an educational tool.To assess student perceptions of the novel TEC curriculum, students in the first two cohorts of the virtual Ob/Gyn third year clerkship were asked to complete two4-point Likert surveys consisting of nine student perception questions and four free response questions. Possible answer choices for the Likert survey questions wereas follows:1=Strongly agree, 2=Moderately agree, 3=Moderately disagree, 4=Strongly disagree. Free response questions for both surveys asked the following: 1)What was most successful about the session? 2) What was most challenging about the session? 3) What were my top takeaways from this session? 4) Any Additional comments or suggestions for improvements?The two surveys were distributed to students at the conclusion of their virtual Ob/Gyn clerkship. One survey corresponded to the telehealth encounter and theother to the virtual OSCE. Responses were de-identified upon completion and stored in a secure database.We conducted all analyses using the Statistical Package for the Social Sciences . Likert data were analyzed by computing means,and response distributions, for each question to compare self-reported student agreement with survey statements.For qualitative analysis of free response questions, the research team reviewed all qualitative data individually to identify salient themes.The research team then met as a group to resolve discrepancies in interpretations and reach final theme interpretation. Qualitative data was revieweduntil no new themes emerged from the analysis. Illustrative quotes were collected to represent all salient themes. A total of 52 students participated in the virtual Ob/Gyn clerkship with 25 in the first cohort and 27 in the second. The telehealth encounter componentof the TEC was still being piloted with the first cohort of students, so their participation in the telehealth encounter was optional.Based on faculty availability and student interest, 9 out of the 25 students in the first cohort completed the telehealth encounter.However, all 25 participated in the vOSCE because it was not optional. All 27 students in the second cohort completed both components of the TEC: the telehealth encounter and vOCSE.Of the 52 students who participated in the first two cohorts of the virtual Ob/Gyn clerkship, 36 completed the Telehealth encounter and 52 completed the vOSCE.Of these students, 33 completed the Telehealth survey (92%) and 48 completed the vOSCE survey (92%) Tables  and 2.Student responses to the Telehealth survey revealed that 76% strongly or moderately agreed that the educational value of a telehealth patient interview was similar to anin-person patient interview. Additionally, 97% strongly or moderately agreed that their telehealth encounter provided them with a valuable patient interaction.When asked if the telehealth encounter should continue during the COVID-19 pandemic, 97% strongly or moderately agreed. However, when asked if the telehealth encountershould be incorporated into the Ob/Gyn clerkship curriculum permanently, when the pandemic comes to an end, 82% strongly or moderately agreed.Student answers to free response questions on the Telehealth survey revealed key themes for the first three out of four questions. When asked “What was most successfulabout the session?” students mostly reported: getting virtual clinical exposure to “real patients” during a pandemic, practicing Ob/Gyn history taking,and being taught by knowledgeable attendings who helped develop clinical reasoning skills. When asked “What was most challenging about the session?”most students identified: technical challenges, inability to physically examine the patient, and difficulty communicating nonverbally.When asked “What were my top takeaways from this session?” most students reported: telehealth visits are similar to in person visits,telehealth visits are a valuable tool and save time for patients, communication skills are important in a virtual format, and time management is important whenacquiring patient histories virtually .Medical student responses to the vOSCE survey revealed that 83% strongly or moderately agreed the vOSCE standardized patient experience provided a valuable patient interaction.When asked if the vOSCE session increased their confidence navigating a patient interview using a virtual platform, 90% of students strongly or moderately agreed.Additionally, 83% strongly or moderately agreed that the vOSCE is a valuable tool to train students in telemedicine. Finally, 65% of respondents strongly ormoderately agreed the vOSCEs should be part of the Ob/Gyn clerkship going forward, including after the COVID-19 pandemic.Student answers to free response questions on the vOSCE survey again revealed key themes for the first three out of four questions.When asked “What was most successful about the vOSCE session?” students mostly reported: increased comfort and familiarity with the virtual encounter format,opportunity to experience Ob/Gyn patient encounters during COVID-19, and collaboration with peers. When asked “What was most challenging about the session?”students identified: technical difficulties/adjusting to a virtual format, the virtual physical exam, and difficulty connecting with patients given limitedability to read body language and communicate nonverbally. When asked “What were my top takeaways from this session?”students mostly reported: the vOSCE was helpful for practicing Ob/Gyn history taking and developing a differential diagnosis,ability to experience and practice using telemedicine, telemedicine is a good tool for student learning, and practice with nonverbal communication .When the COVID-19 pandemic hit the Tristate Area and students were dismissed from in person clinical rotations, faculty and student researchers in the Departmentof Obstetrics & Gynecology at New York Medical College drafted and implemented a virtual Ob/Gyn clerkship and novel Telehealth Education Curriculum(TEC) in a matter of weeks. After the first two cohorts of students completed the virtual Ob/Gyn clerkship and novel TEC, we assessed student perceptions of the TEC.In general, most students agreed that the telemedicine encounters provided similar educational opportunities as in-person encounters and were a valuable clinical tool thatshould be incorporated into future Ob/Gyn clerkship curricula, during and after the pandemic. Furthermore, student responses were both positive and thankful for thechance to continue their learning and experience using an emerging healthcare platform in the midst of a rapidly changing learning environment.While students indicated initial difficulty adjusting to a virtual format and communicating with fewer nonverbal cues, most ultimately expressed increasedcomfort using telehealth after completing the TEC.The information presented in this study including the tools used to implement the curriculum and format by which student perceptions were assessed may beutilized by other institutions interested in developing a telehealth curriculum for their students. While some US medical schools have created virtual studentrotations, or exams, and proven them effective for clinical learning, few have incorporated focused teaching on the practice of telehealth itself - 19. ProIn recent years, telehealth has been increasingly employed to deliver quality healthcare to diverse patient groups  , 8 .PrOur data strongly support an urgent need for current and future medical student telehealth education curricula.Still, our study presents with some limitations including a modest sample size. Additionally, we solely evaluated medical students enrolled inthe third-year obstetrics and gynecology rotation during the COVID-19 pandemic at a single institution. Therefore, more studies evaluating medicalstudent perceptions of telehealth education curricula are needed to examine if our findings and conclusions can be extrapolated to other fields ofmedical education. Student free responses indicate areas for continued improvement when implementing telehealth curricula including a demand for moretelehealth encounters, more vOSCE cases and virtual SP feedback, and follow up telehealth visits. Other than these area of improvement,medical students indicated overall great satisfaction with our novel TEC.Given how well received our TEC was at New York Medical College during the COVID-19 pandemic and that students expressed interest in continued telehealth learning,we plan to incorporate the TEC into future clerkship curricula. Currently, the third year Ob/Gyn clerkship is split between virtual and in-person learning.We plan to integrate student feedback from this pilot project by including more telehealth encounters and modify/adapt the curriculum according toemerging student needs and healthcare trends as appropriate. The authors received no specific funding for this work.


Here, we introduce the most recent advances of human embryonic stem cell- or induced pluripotent stem cell-derived organoids to model 3D NMJs.The neuromuscular junction (NMJ), a peripheral synaptic connection between motoneurons and skeletal muscle fibers, controls movement. Dysregulation of NMJs has been implicated in various motor disorders. Because of their large size and easy accessibility, NMJs have been extensively investigated in the neuroscience field and have greatly contributed to our understanding of the fundamental principles of synapses in the central nervous system. Researchers have tried multiple ways to develop models to recreate NMJs. Rapid progress in the research and development of tissue-like organoids has made it possible to produce human NMJ three-dimensional (3D) models  Every breath you take and every move you make…’ is the opening lyric from a popular rock song of the 1980s. Human breathing and movement are delicately controlled by neuromuscular junctions (NMJs) (‘s (NMJs) . Impropes (NMJs) .2). Presynaptic nerve terminals and the postsynaptic membrane align precisely to allow the efficient neuromuscular transmission. Between the presynaptic nerve terminal and the postsynaptic membrane, the synaptic cleft is filled with the synaptic basal lamina that anchors many critical proteins involved in NMJ development and maintenance.In vertebrates, one mature NMJ is in the middle of each muscle fiber and occupies <0.1% of the muscle surface. In response to action potentials, motoneuron terminals release the neurotransmitter acetylcholine (ACh), which activates ACh receptors (AChRs) on the surface of muscle fibers to depolarize myotubes, thereby causing the release of calcium from the sarcoplasmic reticulum to trigger muscle contraction and movement . NMJs coVertebrate NMJ development is regulated by agrin signaling. Motoneuron axon terminals release the ligand agrin to bind to low-density lipoprotein receptor-related protein 4 (LRP4) on myotube surfaces, which leads to the activation of muscle tyrosine kinase (MuSK), a transmembrane receptor. Activated MuSK transfers the extracellular signal into myotubes through the downstream adaptor Dok7, eventually inducing AChRs to cluster with the help of the scaffold protein Rapsyn . Interesin vitro models to recreate NMJs as reviewed recently  cell culture in vitro, organoids display greater cell diversity and remarkably organized tissue architecture. Thus, organoids are promising tools for tissue engineering and disease modeling  . Becausein vivo. Notably, neuromuscular organoids developed the nerve-controlled contractile activity of muscles, which was effectively blocked by curare, a neurotoxin specifically and competitively blocking AChRs, demonstrating that muscle activity was dependent on functional NMJs in the neuromuscular organoids. Note that neuromuscular organoids produced a complex spinal neural network similar to central pattern generator circuits, which produce rhythmic signals critical for walking and breathing.In the neural part of Day 50 neuromuscular organoids, electron microscopy analysis revealed the presence of densely packed longitudinal axons, synaptic clefts, and synaptic vesicles in neurons. Myelinated axons expressing myelin basic protein were also detected. In the neural part close to the skeletal muscle, choline acetyltransferase (ChAT), an ACh-synthesizing enzyme, was clustered. There were parallel-aligned actin filaments, distinct Z-lines, and M-bands in muscle regions, suggesting that mature sarcomeres had formed in the neuromuscular organoids. Strikingly, numerous AChR clusters along the contact sites between neurites and myotubes were labelled by α-bungarotoxin (α-BTX), a peptide that specifically binds to AChRs with a high affinity, indicating the formation of NMJs. Electron microscopy revealed the presence of synaptic vesicles in presynaptic nerve terminals and junctional fold-like structures on the muscle basement membrane. S100β-positive terminal Schwann cells capped the neuronal terminals. Thus, all three cellular components required for the development of functional NMJs were generated and self-organized in the neuromuscular 3D organoids. The number of AChR clusters per muscle fiber declined from Day 50 to Day 100 but was maintained after Day 150, similar to the dynamic remodeling of NMJs during development but stabilizing at the adult stage In contrast to the self-organization of spinal cord and muscle-like tissues from a single progenitor population to generate neuromuscular organoids, the Pasca group recently used a different method to assemble cortico–spinal–muscular triple organoids in one dish .in vitro and was completely blocked by the addition of curare. The ability of hSpS to regulate the activity of human skeletal myoblasts (hSkM) derived from adult muscle biopsies was also tested. 3D hSkM were generated by adding dissociated proliferative hSkM to an extracellular matrix in a silicone well to differentiate into mature multinucleated myotubes, as evidenced by the expression of skeletal muscle markers including Desmin, Titin, and MyHC. To assemble hSpS–hSkM organoids, an hSpS organoid was placed closely near a 3D hSkM organoid on top of a 6-well transwell insert. Approximately 2 weeks later, abundant neurons from hSpS projected to the 3D hSkM. Live imaging with the calcium indicator Cal-590 revealed that the proportion of active hSkM doubled in hSpS–hSkM organoids in comparison to hSkM organoids alone, supporting the idea that motoneuron innervation promotes muscle maturation in vivo. Indeed, retrograde rabies virus tracing verified that most, if not all, of the projections from hSpS to hSkM were from ChAT-positive motoneurons but not GABA-positive interneurons in the hSpS. Using glutamate uncaging, they further showed that these hSpS cholinergic projections were functionally connected to hSkM. Muscle contractions in hSkM were blocked with curare treatment. Immunocytochemistry analysis also showed that the presence of NMJs by labelling α-BTX (the postsynaptic marker of NMJs) and synaptophysin (the presynaptic marker of NMJs) on Desmin-positive myotubes.Using human iPSCs, in vitro over several weeks. This is important because myotubes are easily detached from the dish surface in the current NMJ in vitro models after long culture periods  , which pin vivo. The same population of cells (NMPs) gives rise to neural and muscle populations simultaneously and self-organizes into neuromuscular organoids. Single-cell analysis showed that the organoids reproducibly exhibited posterior identity. In cortico–spinal–muscular assembloids, neurotransmitter identity in the neuronal clusters included glutamatergic, GABAergic, glycinergic, and cholinergic cells.Compared with the monolayer culture of NMJ 2D models  or biomaNMJ development requires intimate interactions among nerve terminals, muscle fibers, and terminal Schwann cells. Their development is influenced commutatively . Glial cCortico–spinal–muscular assembloids generate input from the motor cortex to the skeletal muscles. Neuromuscular organoids produce a spinal neural network that is similar to central pattern generator circuits. These results provide a broader application to study the motor circuits in the future.ex vivo closely resemble that observed in situ (Organotypic slice cultures  in situ . They cain vitro human-specific models is therefore critical to overcome interspecies differences, capture human-specific neurobiological traits, and accelerate the process of drug screening. Thus, human organoid-derived NMJ models provide a useful approach that complements the use of animal models for studying disease.One of the most practical advantages of human organoid-derived NMJ models is disease modeling. At present, most human-related neuromuscular disorders are studied in animal models that may contribute to the low translational success rate of nervous system therapies. The development of Organoid-based NMJ 3D models have exhibited promising potentials for studying mechanisms and disease modeling; however, some challenges remain in the field.in vitro NMJ models, organoid-based NMJ models exhibit scattered α-BTX-positive AChR clusters along each myotube. The junctional folds in NMJ organoids exhibit protrusion-like structure. A better understanding of the mechanisms underlying AChR prepattening in the muscle before innervation would help explain how NMJs form only at the synaptic region. The presence of extracellular matrix at the postsynaptic membrane is also critical for the development and maintenance of the NMJ structure.Remarkable changes occur during NMJ development. In rodents, after birth, neonatal plaque-like AChR clusters transform into multi-perforated elaborate branches and mature NMJs display a pretzel-like morphology at the adult stage . In humaHuman spinal motoneuron somas are restricted in the ventral hood of the spinal cord, while their axons extend a long distance to innervate skeletal muscle fibers. Microfluidics used in previous NMJ models allow spinal motoneurons and muscle cells to grow in different chambers that are connected by axons . This prIn addition to NMJ autoimmune disorders, other motor disorders caused by genetic mutations, such as congenital myasthenia syndrome, amyotrophic lateral sclerosis, Charcot–Marie–Tooth disease, spinal muscular atrophy, and muscular dystrophy, could also be investigated using organoid-derived NMJ models. A complete patient-derived organoids or a combination of healthy and patient-derived organoids could provide new insights into disease mechanisms such as dissecting cell-specific vulnerabilities and cell-autonomous effects of these disorders. Human organoid-derived NMJ models also provide an ideal platform to screen novel drugs for aforementioned diseases on a large scale in the future.in vitro tool, we have to realize that there is some distance from real human tissues in vivo. Using proper controls to ensure functional organoids in each batch of experiments would be important. Also, it relies on further technique development of organoid culture to better mimic tissues in vivo. Organoid-derived human NMJ 3D models could be a complementary tool of 2D cell culture system, animal models, as well as human samples, to study synapses and motor disorders.In general, human organoid system showed great potentials for tissue engineering and disease modeling. However, as an


Diabetic retinopathy (DR), the leading cause of blindness in working-aged adults, remains clinically defined and staged by its vascular manifestations. However, early retinal neurodegeneration may precede vascular pathology, suggesting that this neuronal damage may contribute to disease pathogenesis and represent an independent target for intervention. This review will discuss the evidence and implications for diabetic retinal neurodegeneration.A growing body of literature has identified progressive retinal thinning and visual dysfunction in patients with diabetes even prior to the onset of DR, though advances in retinal vascular imaging suggest that vascular remodeling and choroidal changes occur during these early stages as well. Animal models of diabetes and in vitro studies have also suggested that diabetes may directly affect the retinal neural and glial tissue, providing support to the concept that diabetic retinal neurodegeneration occurs early in the disease and suggesting potentially relevant molecular pathways.Diabetic retinal neurodegeneration may represent a “preclinical” manifestation of diabetic retinal disease and remains an active area of investigation. As the natural history and molecular mechanisms become increasingly understood, it may lead to upcoming developments in not only the treatment options but also the clinical definition of DR. Diabetes mellitus (DM) remains a growing epidemic worldwide with significant morbidity and mortality, and diabetic retinopathy (DR) represents the leading cause of blindness in the working-age population . DR has While the diagnostic criteria for DR were developed at a time when clinical exam, color fundus photographs, and fluorescein angiography were the mainstays of retinal evaluation, the introduction of optical coherence tomography (OCT) imaging to noninvasively and rapidly visualize the layers of the macula in cross-section has revealed alterations in retinal structure prior to the onset of DR. To date, many studies have now used OCT to demonstrate thinning of the inner retina including the retinal nerve fiber layer (RNFL), ganglion cell layer (GCL), and more variably the inner plexiform layer (IPL) in patients with type 1 or type 2 DM without DR , 5. ThesIn addition to inner retinal thinning, functional deficits in contrast sensitivity, perimetry testing, multifocal electroretinogram (mfERG), and dark adaptation have also been described in diabetic patients without DR or with very early DR –13. A coInterestingly, neuronal dysfunction in early diabetic retinal disease may contribute to subsequent DR development and progression. Focal areas of delayed implicit time on mfERG have been associated with an increased risk of DR development at 1 year . ProgresIn summary, changes in inner retinal thickness and visual function deficits occur prior to clinical DR in patients with DM, suggesting a neurodegenerative component of diabetic retinal disease that may be independent from, or at least concurrent with, the vascular manifestations.Akita mouse, which harbors a point mutation in the Insulin 2 gene leading to beta-cell death and subsequent diabetes, develops thinning of the IPL and INL by 22 weeks of hyperglycemia and reduced RGC number in the peripheral retina by 3 months [The early inner retinal neurodegeneration evident in patients with type 1 and type 2 DM has been recapitulated in several animal models, emphasizing the evolutionary conservation of the retinal response to diabetic injury and providing systems within which to study these effects. Thinning of the GCL/IPL on OCT, and in some studies the inner nuclear layer (INL), outer nuclear layer (ONL), and total retina as well, has been described in both mice and rats treated with the pancreatic beta-cell toxin streptozotocin (STZ) to induce diabetes –24. STZ-3 months , 31. RGC3 months .Psammomys obesus, can develop diabetes similar to type 2 DM that has been associated with retinal thinning and vascular changes [Several genetic and induced mouse models of type 2 DM demonstrate inner retinal thinning on OCT and/or retinal function deficits as well. Mice fed a high-fat diet exhibit impairments in a-wave, b-wave, and oscillatory potentials (OPs) on ERG testing, suggesting neuronal injury, though inner retinal thinning was not noted –35. On t changes .Data derived from the various animal models described above, in vitro studies, and, perhaps most relevant, from the ocular fluids of diabetic patients have uncovered various molecular pathways that may mediate retinal neuronal damage in diabetes. Several of these have long been broadly implicated in DR pathogenesis but more recent evidence ties these pathways more specifically to retinal neurodegeneration.−/− db/db mouse [Hyperglycemia itself can trigger cellular damage via protein kinase C (PKC) activation, oxidative stress, increased flux through the polyol pathway in which glucose is converted to sorbitol by aldose reductase, and generation of advanced glycation end-products (AGEs). Wang et al. demonstrated mitochondrial swelling in RGCs and decreased superoxide dismutase (SOD) activity in the retina of STZ-treated rats 4 weeks after induction of hyperglycemia, suggesting that oxidative stress may contribute to RGC loss . Accumuldb mouse . Data frdb mouse . Despitedb mouse . Finallydb mouse .The pro-inflammatory systemic milieu of DM is now well-recognized and increasing evidence suggests a pathologic role for diabetes-related inflammation within the retina. Microglial activation and increased expression of the inflammatory cytokines TNF-alpha and IL-1beta has been demonstrated in the retina of STZ-treated rats, and activated microglia produce factors that can induce retinal neuronal death in cell culture . MicroglReduced thickness of the RNFL, GCL, and GC/IPL complex on OCT in diabetic patients has been associated with diabetic peripheral neuropathy, suggesting that underlying molecular mechanisms of neuronal damage may be similar , 53. EspPharmacologic targeting of the pathophysiologic molecular pathways and mechanisms described above has been able to rescue diabetic RGC death and neuronal dysfunction in rodent models. As described above, subconjunctival insulin administration partially rescued RGC death in the STZ-treated rat model . AdditioAkita mouse [An alternative therapeutic strategy involves enhancement of neuroprotective mechanisms that are impaired in the diabetic retina. Protein and mRNA levels of brain-derived neurotrophic factor (BDNF) are reduced in the retina of STZ-treated rats and aqueous levels of BDNF are decreased in eyes of diabetic patients even without DR compared with nondiabetic controls, suggesting a physiologically relevant role for this neurotrophic factor , 62. Indta mouse . Finallyta mouse . Growth ta mouse . While nta mouse .While considering the early retinal dysfunction that occurs in DM and the potential for neuroprotective strategies in the management of diabetic retinal disease, the question still remains whether retinal neurodegeneration truly precedes vascular pathology. In the HFD mouse model, ERG changes including decreased OP latency indicating bipolar cell dysfunction and pattern ERG deficits indicating that RGC dysfunction are present after 6 months of HFD feeding, preceding the onset of vascular leakage at 12 months , 59. In While most animal models of DR support the argument for diabetic retinal neurodegeneration preceding retinal vascular pathology, the evidence from diabetic patients is less clear in part due to improvements in vascular imaging. Many studies have established that thinning of the inner retina  as assessed by OCT is present in patients with diabetes prior to the diagnosis of clinical DR, consistent with experimental models , 5. ProgHowever, the advent of OCT angiography (OCT-A) technology has allowed greater resolution of the retinal vasculature far beyond what has been detectable by clinical examination or fluorescein angiography, providing clinicians with the ability to visualize the superficial and deep capillary plexi  in a noninvasive manner. OCT-A and other newer imaging modalities, including swept-source OCT imaging which provides penetration into the choroid, have demonstrated preclinical retinal vascular and choroidal changes. Using OCT-A, several abnormal vascular parameters have been described in diabetic patients without DR, including decreased vascular density of the SCP and DCP and increased area of the foveal avascular zone (FAZ) , 72. In Overall, though, findings from OCT-A have raised into question whether vascular or neuronal changes occur first. A concurrent analysis of the retinal microvasculature and retinal thickness in patients with type 1 DM but no DR revealed decreased vessel density in the DCP with no difference in RNFL and GCL thickness compared with controls . On the Studies of patients with DM prior to the onset of DR using newer more sensitive technology to assess retinal architecture, retinal capillary parameters, and visual function continue to reveal the complexity of diabetic retinal disease and expand our understanding of how diabetes affects the retina. Whereas the clinical definition, diagnosis, and staging of DR as well as current medical treatment options reflect the long-held paradigm of DR as a disease of the retinal vasculature, it is very clear now that inner retinal thinning  occurs in the eyes of patients with DM prior to clinical DR, suggesting that diabetic retinal neurodegeneration represents an important, and possibly independent, manifestation of diabetic retinal disease. Animal models and studies of intraocular fluid from diabetic eyes have implicated oxidative stress, hyperglycemia-related production of AGEs, glutamate excitotoxicity, and inflammation as potential underlying molecular mechanisms for retinal neuronal loss and dysfunction in diabetes and targeting these pathways or exogenous administration of neurotrophic growth factors may provide strategies for neuroprotection. OCT-A has allowed detailed analyses of the retinal capillary networks and indicated that preclinical vascular changes also occur in diabetic eyes, and whether these vascular abnormalities precede, follow, or coincide with inner retinal neuronal degeneration remains an open question. Regardless, the current diagnostic criteria for DR do not account for these early “pre-clinical” retinal changes that not only affect visual function but may contribute to subsequent DR progression, and an updated staging system for diabetic retinal disease (DRD) which incorporates retinal neuronal, vascular, and visual function deficits has been proposed ••. RecogThis work was supported by grants to M.M.S. from the National Eye Institute (K08 EY029766) and the Research to Prevent Blindness foundation (CareerDevelopment Award).