Our aim is to use neurophysiological sleep-related consolidation (SRC) phenomena to identify putative pathophysiological mechanisms in CECTS linked to diffuse neurocognitive deficits. We argue that there are numerous studies on the association between seizure aspects and neurocognitive functioning but not as many on interictal variables and neurocognitive deficits. We suggest two additional foci. First, the interictal presentation in CECTS and second, neuronal oscillations involved in SRC processes. Existing data on mechanisms through which interictal epileptiform spikes (IES) impact upon SRC indicate that they have the potential to: (a) perturb cross-regional coupling of neuronal oscillations, (b) mimic consolidation processes, (c) alter the precision of the spatiotemporal coupling of oscillations, and (d) variably impact upon SRC performance. Sleep spindles merit systematic study in CECTS in order to clarify: (a) the state of the slow oscillations (SOs) with which they coordinate, (b) the precision of slow oscillation-spindle coupling, and (c) whether their developmental trajectories differ from those of healthy children. We subsequently review studies on the associations between IES load during NREM sleep and SRC performance in childhood epilepsy. We then use sleep consolidation neurophysiological processes and their interplay with IES to help clarify the diffuse neurocognitive deficits that have been empirically documented in CECTS. We claim that studying SRC in CECTS will help to clarify pathophysiological mechanisms toward diverse neurocognitive deficits. Future developments could include close links between the fields of epilepsy and sleep, as well as new therapeutic neurostimulation targets. At the clinical level, children diagnosed with CECTS could benefit from close monitoring with respect to epilepsy, sleep and neurocognitive functions. Childhood epilepsy with centrotemporal spikes (CECTS) belongs to a common group of idiopathic focal childhood epilepsies (IFEs), often of genetic origin . It is cCentrotemporal spikes are a defining feature of CECTS and are documented through sleep-electroencephalogram (EEG) during NREM sleep . The negThe synaptic homeostasis hypothesis , 2014 puIn the active system consolidation model  new stimSleep-related consolidation, a type of neuroplasticity, correlates with cortical maturation and cognition . It has Studying SRC in CECTS is particularly relevant for a number of reasons. First, there is increasing consensus that CECTS and electrical status epilepticus during slow wave sleep (ESES), an IFE complicated by continuous spike and wave during sleep (CSWS), are within the same continuum, with ESES comprising the severe end of the continuum . It has It has been suggested that IES may interfere with the neurophysiological consolidation processes subserving learning. This hypothesis is strengthened by associations between behavioral and cognitive deficits and IES severity at the acute epileptic phase . It is aAssociations between IES load during NREM sleep and SRC performance in various types of childhood epilepsy have been found in a limited number of studies so far . CollectDuring NREM sleep, neuronal networks from different brain regions “converse” through the temporal coupling of their respective electrophysiological features . Phase aDirect intracranial EEG recordings have provided evidence of the coupling of SOs, spindles and ripples within the human hippocampus during NREM sleep . The afoIES undergo sleep-related reactivation and consolidation processes utilizing physiological mechanisms that were considered to be reserved for meaningful stimuli . To dateBased on adult studies IES couple with slow wave oscillations at different transitional points (up to down states) in comparison to what physiological oscillations do (down to up states) . In healIn epileptogenesis the impact of IES, ranging from proictal to antiictogenic, does not depend only on IES characteristics but also on the complex interaction between IES and specific neuronal networks . In relaSleep spindles merit systematic study in CECTS. Namely, within the hierarchical nesting of one oscillation within the other, spindles play an intermediary role since they are organized by the SOs upstates (in adults) and in their turn they group ripples within their own troughs . In chilOne important line of research would be to concurrently study the characteristics of sleep spindles in CECTS, the state of SOs with which they co-ordinate and NREM sleep-related gains. In fact, sleep spindle abnormalities have already been found in various clinical populations such as in mental disability , autistiPrecise SO-spindle coupling, detected at scalp level, is crucial for consolidation: (a) frontal spindles are thought to be time-locked to SOs “upstates” during childhood and adolescence; (b) the precision of SO-spindle coupling increases in parallel with brain maturation, especially in fronto-parietal areas and (c) subjects whose coupling strength increased from childhood to adolescence also presented with improved SRC performance in adolescence . In adulCECTS is associated with a range of neurocognitive deficits and of varied severity. Deficits have been documented in executive functions , attentiThere are two commonly hypothesized reasons for the diffusely impaired neurocognitive profile in CECTS. The first one is that IES propagate; hence their effect expands beyond the area of initial presentation . The secFrom a neurophysiological perspective, deficits in general intelligence in CECTS could be tentatively explained by the fact that sleep spindles have been identified as an index of intelligence or general aptitude for learning . A gap iIn the latter case scenario, there are interesting implications for SRC processes in CECTS. Even within a neuronal environment burdened by IES, compensatory mechanisms could still enable sleep spindles to adjust their characteristics in order to achieve SRC processes. Hence, this would further support heterogeneous results in terms of SRC alterations in CECTS, possibly leading to heterogeneous long-term neurocognitive profiles.Hippocampus-dependent memory is traditionally studied using declarative memory tasks including verbal and visuospatial tasks . Initial findings suggest that SRC is altered in CECTS  meaning An additional challenge in CECTS is that since there is no clinical need for intracranial recordings, there is a lack of direct recordings of some types of neuronal oscillations. In order to overcome this issue well developed theoretical models are needed with testable hypothesis. There is a also a need to develop measures that can be detected at scalp level, such as the SO-spindle coupling measure based on which data can be inferred related to the quality of the hippocampal-neocortical communication . Future It is crucial that the contribution of each neuronal oscillatory pattern to the SRC process is well understood so that children with CECTS might be able to benefit from the fast developing field of sleep memory manipulation, and, in particular, of oscillatory sleep patterns. In adults various neurostimulatory methods, such as transcranial direct stimulation  and acouWe propose that studying SRC in the context of CECTS will give us further insight into the pathophysiological mechanisms that lead to neurocognitive deficits in this pediatric population in the long term. At the clinical level, children diagnosed with CECTS could benefit from closely monitoring with respect to their epilepsy, sleep and neurocognitive functions.VG conceived the work, searched and studied the literature, and drafted the first version of the manuscript. KS restructured, and discussed the literature and concepts with VG which resulted in the revised version. MK and KG commented on and reviewed the manuscript. 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. The handling editor declared a past co-authorship with one of the authors KG.


Biobanking can promote valuable health research that may lead to significant societal benefits. However, collecting, storing and sharing human samples and data for research purposes present numerous ethical challenges. These challenges are exacerbated when the biobanking efforts aim to facilitate research on public health emergencies and include the sharing of samples and data between low/middle-income countries (LMICs) and high-income countries (HICs). In this article, we explore ethical challenges for COVID-19 biobanking, offering examples from two past infectious disease outbreaks in LMICs where biobanking activities contributed to the perpetuation of global inequities. We focus on how the ethical imperative to promote the common good during public health emergencies can conflict with protecting the interests of biobank participants. We discuss how conducting biobank research under a waiver of informed consent during public health emergencies is ethically permissible, provided guidance is in place to prevent biopiracy and exploitation of vulnerable communities. We also highlight the need for biobank collaborations between LMICs and HICs to promote capacity building and benefit sharing. Finally, we offer guidance to promote the ethical oversight of biobanks and biobank research during the COVID-19 pandemic or other future public health emergencies. Biobanking is a public health imperative during the COVID-19 pandemic.This article provides ethics guidance on the operationalisation and oversight of biobanking activities and research in LMICs during public health emergencies. We first discuss how such emergencies, like COVID-19, exacerbate existing tensions between public health ethics and research ethics in biobanking. We then review the ethical challenges of biobanking in LMICs during previous infectious disease outbreaks. From there, we review how COVID-19 has impacted traditional ethical considerations in biobanking, such as informed consent and sharing of samples, and use South Africa as a case study to illustrate how legislative frameworks affecting biobanking might affect biobanking practice in other LMICs during COVID-19. We conclude with practical steps to guide those involved in the ethical oversight of biobanking activities associated with public health emergencies.Ethical considerations in biobanking are typically aligned with those of research ethics, which focus on protecting the rights and interests of participating individuals and communities. Public health ethics, however, espouses ‘pluralistic values’ including population health and the common good.Solidarity asks that individuals recognise a common purpose and willingness to promote the interests of others,Benefit sharing gives ‘a portion of advantages/profits derived from the use of human (specimen) resources to the resource providers to achieve justice in exchange’.Here, we explore the ethical challenges of biobanking in LMICs during two recent infectious disease outbreaks, H5N1 and Ebola, and their importance for COVID-19 biobanking practices.In 2007, the Indonesian government refused to share samples of H5N1 virus with the WHO unless they were assured access to the vaccines created from the samples. This decision was spurred by historical inequities in vaccine distribution where people in HICs were more likely to be vaccinated than people in LMICs.More than 50 000 samples collected during the recent 2014−16 Ebola epidemic in West Africa were shipped out of the affected countries to unidentified laboratories around the world, primarily in HICs. This shipment was done without state authorisation or proper participant consent,exacerbating pre-COVID-19 global inequities in healthcare.These infectious disease outbreaks serve as important examples for biobanking during the COVID-19 pandemic. In fact, similar dynamics are already at play. For example, despite South Africa participating in AstraZeneca COVID-19 vaccine trials, the country has paid double the price per dose compared with European countries.Given both its similarities to past infectious disease outbreaks and its unique challenges, governance and oversight for COVID-19 biobanking in LMICs should take into account how the pandemic affects a variety of ethical considerations, which we review below: informed consent, community engagement (CE), sample and data sharing processes, and safe handling of collected samples. Those involved in governance and oversight also need to be aware of the biobanking context, which may affect these ethical considerations. COVID-19 biobanking may occur through new biobanks created as a direct response to COVID-19, as well as through existing biobanks expanding their collection efforts to include the collection of COVID-19 samples or repurposing existing samples for COVID-19 research.Informed consent is an ethical cornerstone of research. While volunteerism is important for research participation in general, obtaining informed consent for COVID-19 biobanking research has numerous logistical challenges, some of which may undermine autonomous decision-making. Non-hospitalised patients are instructed to quarantine, making sample collection virtually impossible, and hospitalised patients may be unconscious, heavily sedated, have impaired cognition or otherwise be too sick to engage with the consent process. Similarly, obtaining proxy consent from a patient’s family may be difficult. In South Africa, as in many countries, hospitalised patients with COVID-19 are not permitted visitors, thus limiting opportunities for obtaining proxy consent.Despite logistical difficulties of obtaining informed consent for biobanking, samples are being collected for COVID-19 diagnostic and therapeutic purposes throughout the world. Countries may not have clear regulations that speak to when, if ever, samples collected for these purposes can be repurposed without consent for other research use. For example, in South Africa, health legislation permits collecting samples from hospitalised patients for COVID-19 diagnostic purposes without consent, with provisos, and the Disaster Management Act of 2002 prioritises and makes special allowances for ‘disaster management research.’Collecting and storing samples for research from COVID-19-positive patients under a waiver of consent will have public health benefits and must be weighed against respecting individual patient autonomy. Biobankers should develop a plan to deidentify or anonymise collected samples and data prior to sharing to mitigate any possible risks to individuals.CE contributes to procedural justice to ensure fairness in how significant decisions are made in health research.Coordinated, global sharing of samples and data during a pandemic is critical to expedite research, and biobanks are key in this endeavour. However, prior research indicates that underutilisation of biobank resources is a common problem for biobanks and a source of ethical concern for biobankers.Individuals involved in biobank governance, such as biobankers, RECs/IRBs and researchers, should be cognisant of the ethical complexities surrounding sample and data sharing. The potential for relaxed research regulations during a pandemic, such as waiving informed consent and decreasing community engagement, increases the imperative for biobanks to strengthen the conditions under which samples and data are accessed and shared. It is not uncommon for samples and data to initially be shared with one group, like academic researchers, only for a third party (such as for-profit companies that fund the research) to gain secondary access.At a biobank operational level, COVID-19 biosamples must be handled safely given their potential infectivity.Biobanks should also consider biosecurity measures when dealing with pathogens. ‘Dual use research of concern’ is scientific research that is intended to benefit society but could cause societal harm if misapplied.Given the possible competing tensions of public health ethics and research ethics during public health emergencies, those involved in biobank oversight, such as RECs/IRBs, need to consider innovative, flexible ways to simultaneously protect research participants, promote public health interests and support scientific advancement. Such oversight should include the review of international research collaboration agreements, specifically those that involve partnerships between LMICs and HICs. Such reviews could include questions on whether the research conforms to data sharing, benefit sharing and capacity-building activities between LMICs and HICs.As new information on COVID-19 is constantly emerging, risk–benefit assessments may need to be reviewed on an ongoing basis in both LMICs and HICs. This effort may require additional training, resources and capacity development of RECs/IRBs and others in LMICs to better respond to the changing research landscape during pandemics, or other public health emergencies. There should be clear collaborative efforts and communication networks between those involved in the oversight of biobanks in LMICs and HICs to ensure adequate support and consistency for decision-making.In Box 1Is obtaining informed consent practicable, and would doing so significantly slow research progress?Has a risk–benefit assessment been done to determine the appropriateness of relaxing human research subjects’ protections to facilitate the proposed research? For example, are the risks of not obtaining informed consent outweighed by the societal benefits that are likely to accrue from the proposed research? And would local communities likely see any of these benefits?Is there equitable participant involvement so as to ensure a fair distribution of possible benefits and burdens of the research, including across LMICs and HICs?If using a waiver of consent, are notifications in place so that participants may be made aware of the biobanking research?What community engagement activities are planned?What country-specific legislation permits collecting, storing and sharing samples for research use without informed consent? What, if any, additional mechanisms are in place to protect the privacy of research participants, such as anonymising samples and data?What oversight and monitoring processes are in place for approved research during the public health emergency?Are there clear plans for sample and data sharing to accelerate research?Do research collaborations between LMICs and HICs involve adequate benefit sharing and capacity building?Can data be shared via a secure online data management system to facilitate collaboration rather than proprietary transfer?What mechanisms are in place, such as MTAs and DTAs, to ensure that resources are not shared with unauthorised third parties?What existing legislations, if any, guide sample and data sharing, and do these legislations make allowances for public health emergencies?How will biosamples be safely collected from research participants, especially in contexts where PPE resources may be scarce?What measures are in place to ensure a safe working environment for researchers and laboratory staff?What biosecurity measures are in place?The current COVID-19 pandemic provides an opportunity to reflect on ethical considerations in biobanking during public health emergencies, including respect for research participants, promoting the common good, solidarity, reciprocity and benefit sharing. This requires a review of research ethics guidance and regulatory requirements regarding sample collection, storage and sharing for research so that we are better prepared for the next pandemic or public health emergency. Despite the ethical challenges which we have discussed, there is nevertheless an urgent need to optimise sample sharing to accelerate a research platform that could ultimately benefit all affected by public health emergencies. Similarly, any therapeutic or preventive interventions that result from data and/or sample sharing must be fairly distributed, ensuring equitable access to all. Future research should investigate the ways that existing regulations have been relaxed during the pandemic, such as implementation of waivers of consent, and their resulting effects. For example, to what extent will these changes promote research activities? And to what extent have they affected public trust in biobanking? The latter will be particularly important to consider in ongoing discussions of whether and how research ethics guidance for biobanking during pandemics and public health emergencies ought to be reconceptualised.


The eye is an anatomical extension of the brain where multiple parallels can be drawn between their neurons, vasculature and immune response. Furthermore, both organs modify similarly with disease. As such it stands to reason that multidisciplinary research that investigates both will inform upon each other, especially in the context of neurodegenerative diseases.in-vivo imaging techniques, a plethora of eye tests have been developed to reveal manifestation of systemic pathological changes in the eye. In this special issue we have curated 22 articles  regarding the nexus between the eye and brain.A rapidly emerging area of research is the use of the eye as a window to changes occurring in the brain. These range from reports of symptomatic alterations to vision, functional and structural changes in brain and retina, as well as the presence of key proteinopathy hallmarks of disease. Such findings have implications for both, the effect that neurological diseases have on vision as well as the possibility of using ocular tests as surrogate measures for detection or monitoring of systemic diseases. The eye is the only place in the body where both neurons and blood vessels can be directly visualized. These attributes have led to the common clinical practice for vascular diseases such as diabetes and hypertension, to have ocular assessment as a routine component of their management. With the advancement of Lemmens et al.). They found a significant association with decreased retinal fractal dimension metrics and cognitive impairment/dementia. Additionally, most examined studies exhibited an association between retinal fractal dimension and acute, subacute and chronic stroke settings but more research is needed to examine the potential for prediction .The increasing interest in this field is highlighted by manuscripts which review and provide perspectives on whether different ocular assessments may reflect neurodegenerative and vascular disease and how this may be improved in the future. This includes a systematic review on whether retinal microvasculature metrics  are modified with neurodegenerative disease and stroke . Lee et al. examines post-mortem retinal tissue from Alzheimer's disease patients (n = 15) and control eyes (n = 10). Focusing on the retinal ganglion cell layer they characterize the regional distribution of amyloid-beta and find higher levels in the mid-periphery and the greatest difference between Alzheimer's disease and control retinas occurring in the superior and temporal retinal quadrants. In a complementary study in rodents, they extend previous studies by other groups showing a common herbal supplement can fluorescently highlight retinal amyloid-beta and find that this measure correlates with cortical amyloid beta load, predictive of Alzheimer's disease progression . These works help to direct future in-vivo retinal amyloid-beta studies.Two papers from the same group build upon interesting work in the field investigating the presence of an Alzheimer's disease hallmark, amyloid-beta, in the retina  and electrophysiology measures indicate that the inner retina exhibits thinning of retinal ganglion cell axons and dysfunction of these neurons prior to outer retinal changes in a 5 × FAD transgenic mouse model .Animal models that recapitulate retinal and cortical amyloid-beta deposition are useful to understand the pathology associated with the presence of these protein deposits. Using Octodon degus, a rodent model that naturally accumulates amyloid-beta peptides with advancing age, Romagnoli et al. investigate the function of a particular-type of retinal ganglion cell that is intrinsically photosensitive. This builds on previous work by their group that shows melanopsin containing retinal ganglion cells are lost or abnormal in post-mortem retinas of Alzheimer's disease donors. In the current study, they find that the chromatic pupillary response  exhibited higher variability in mild-moderate Alzheimer's disease patients but were not significantly separated from controls (n = 26/group), whereas the rod mediated transient pupillary light response was altered. They hypothesize that this may reflect abnormalities in melanopsin retinal ganglion cell dendrites in the early stage of the disease before cell body manifestations.Also, focusing on inner retinal ganglion cells Dehghani et al. evaluate corneal dendritic cells in control, mild cognitive impairment and Alzheimer's disease patients (n = 5/group). Using laser scanning in-vivo confocal microscopy they found morphological differences including a larger dendritic cell field area and perimeter in patients with mild cognitive impairment compared to controls. These findings are consistent with an immunologically activated cell state, echoing the retinal microglial changes found in animal models by Chang et al.. Further studies evaluating corneal immunology are warranted in larger populations.In a proof-of-concept study that shifts ocular markers to the anterior eye, Mirzaei et al. who review the current status in the field of retina and Alzheimer's disease. Of note, multiple Alzheimer's disease pathobiological parallels have been found including the presence of pathognomic Alzheimer's proteins  as well as accompanying inflammation, pericyte loss, and neurodegeneration. Brighi et al. then extend our knowledge of the current status in the field by reviewing the literature surrounding the use of induced pluripotent stem cell-derived organoids as a model for brain and retinal tissue in a dish. Such technology may pave the way toward personalized medicines in the future.The consistent themes occurring in eye and brain are eloquently summarized by Cao et al. found in 40 recent small subcortical infarct patients vs. 46 controls that optical coherence tomography angiography was reduced in the macula area  and around the optic nerve head  which correlated with peripapillary retinal nerve fiber layer thickness. In another study, Peng et al. assessed optical coherence tomography angiography on 74 participants with white matter hyper-intensities who were stroke and dementia free at the time of assessment. They found that microvascular density around the optic nerve  and the deep capillary plexus at the macula were associated with disease severity and cognitive function. These two studies lay a foundation for use of optical coherence tomography angiography to evaluate vasculature in patients with cerebral small vessel disease pending longitudinal studies with larger sample sizes. Interestingly, in a pilot functional MRI study that longitudinally examined patients recovering from stroke  Wu et al. found that the sensorimotor network connectivity played a facilitatory role following stroke rehabilitation. They suggested this could be used as a rehabilitation biomarker for future development of interventions and treatments. These studies are in alignment with the aforementioned systematic review which indicated for an association between retinal microvasculature integrity  and stroke .Dementia is thought to be not only a neurological disease but also a disease that has a vascular component. Indeed, cerebral small vessel disease can lead to multiple disorders including dementia cognitive decline and stroke. Neuroimaging features of cerebral small vessel disease include recent small subcortical infarcts and white matter hyper-intensities. Provost et al. extend previous assessment of retinal vasculature in adults to children. They quantify retinal microvasculature indices, including vessel diameter, fractal dimension, lacunarity and tortuosity, from fundus photographs of 190 children. They found that measures reflecting a denser microvascular network, high fractal dimension and low lacunarity, were associated with poorer behavioral and attention outcomes in children assessed via tests from the Neurobehavioral Evaluation System battery. This association of higher retinal fractal dimension has also been shown in adult patients with schizophrenia and bipolar disorder. In contrast, the aforementioned studies indicate for a sparser retinal density with cerebral small vessel disease  associated with a cognitive decline. Whether these differences reflect distinct mechanisms between these diseases or a different time-course of changes  requires further investigation.Huang et al. evaluate diabetic retinopathy patients and conduct dynamic cerebral activity imaging (dynamic amplitude of low frequency fluctuation) as a more sensitive measure than conventional static cerebral activity measures. Here they find that compared with healthy controls (n = 38) diabetic retinopathy patients (n = 34) exhibited significantly increased variability in their dynamic cerebral activity imaging measure in the visual cortex, cerebellum and parahippocampal gyrus.Looking at vascular disease from a flipped perspective (eye disease manifesting in the brain) Following Alzheimer's disease, Parkinson's disease is the second most common neurological disorder.Indrieri et al. provide a comprehensive review of visual changes and parallel molecular mechanisms in Parkinson's disease. Altered visual measures include reduced visual acuity, contrast sensitivity, color vision and more complex visual processing tasks. The retinal manifestations of dopamine, alpha-synuclein and mitochondrial dysfunction are also reviewed. Together these have been shown to manifest as changes in objective assessments such as optical coherence tomography and electroretinography, but the authors suggest that coupling these measures with future imaging of retinal alpha-synuclein will improve specificity.Shen et al. characterize the retinal phenotype of neuromyelitis optica spectrum disorder, an autoimmune inflammatory disease of the central nervous system. More specifically, the BDNF Val66Met genetic polymorphism was evaluated in this study given its known links to neuronal health. In 17 neuromyelitis optica spectrum disorder patients they found the BDNF Val66Met was associated with more severe nerve fiber layer damage using optical coherence tomography imaging in optic neuritis eyes. Met carriers had thinner OCT layers that corresponded to ganglion cells and delayed multifocal VEP in optic neuritis eyes. In contrast no associations were found in non-optic neuritis eyes suggesting that the gene may be associated with optic neuritis and local damage to the optic nerve in these patients.Blades et al. use a Tyro3 knock out mouse model to investigate the role of Tyro3 on structure and function of the retina. They find that Tyro3 deficiency causes a reduction in function across the retina, including from photoreceptor, bipolar, retinal ganglion cells. Of note, the decrease in retinal ganglion cell function was paralleled by in-vivo thinning of ganglion cell axons  and ex-vivo assays of retinal ganglion cell health . Further studies are warranted to examine whether Tyro3 pathways could inform new therapeutic approaches for ganglion cell diseases.Studies in animal models facilitate insight into pathobiological changes that are occurring in disease. Tyro3 is a tyrosine kinase receptor that is expressed in the brain and more recently has also been shown in the retina. Persiconi et al.) shows for the first time that Dp427 is involved in the retinal neurodevelopment process, as reported previously for other central nervous system neurons. Interestingly, in the retina neural alteration during development may be compensated during growth and underlie adult physiological dysfunctions, a role distinct to the retina. This work in conjunction with further functional characterization will inform our understanding of vision changes in Duchenne muscular dystrophy.Duchenne muscular dystrophy is a muscular disease, but patients also suffer from cognitive impairment and visual defects. It is caused by problems with the cytoskeletal protein dystrophin, Dp427 which are expressed on selected neurons including those in the retina. By employing biochemical and immunobiological approached this study  and global  cortical excitation in a manner that modifies with stimulation frequency. These findings may have implications for retinal prostheses to treat retinal degeneration in the future. Together these preclinical studies  form a foundation for our understanding of the potential for novel interventions to treat retinal or cortical disease.In summary, this collection of articles brings together some notable recent advances in the field. It shows that brain changes are reflected in the eye and vice versa. The challenge for the field moving forward may lie in determining whether there are specific changes that occur in the eye for different diseases. Together these articles indicate that neurological diseases may affect the anatomy and function of the eye. Studies like these form the basis for continuing research aiming at understanding the plethora of eye and brain connections.CN, MA, SD, and TS reviewed the submissions, wrote the editorial, and approved it for publication. All authors contributed to the article and approved the submitted version.CN is a Chief Investigator on an Australian Research Council Linkage grant LP160100126 with AstraZeneca Neuroscience and Biogen Inc. TS is the Director and Chief Scientific Officer of Neurexpert Ltd. The remaining 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.


Accurate replication of the entire genome is critical for cell division and propagation. Certain regions in the genome, such as fragile sites , rDNA and telomeres, are intrinsically difficult to replicate, especially in the presence of replication stress caused by, for example, oncogene activation during tumor development. Therefore, these regions are particularly prone to deletions and chromosome rearrangements during tumorigenesis, rendering chromosome fragility. Although, the mechanism underlying their “difficult-to-replicate” nature and genomic instability is still not fully understood, accumulating evidence suggests transcription might be a major source of endogenous replication stress (RS) leading to chromosome fragility. Here, we provide an updated overview of how transcription affects chromosome fragility. Furthermore, we will use the well characterized common fragile sites (CFSs) as a model to discuss pathways involved in offsetting transcription-induced RS at these loci with a focus on the recently discovered atypical DNA synthesis repair pathway Mitotic DNA Synthesis (MiDAS). To proliferate, a cell needs to go through a division cycle, where it duplicates its chromosomes in S phase. The replicated chromosomes are then separated and segregated into daughter cells during mitosis. Due to the large size of human genome, cells start DNA replication from multiple origins and up to thousands of replication forks are established and coordinated to replicate the genome in a very short time period (S phase). The untimely duplication of the whole genome in S phase can lead to cells entering mitosis with under replicated DNA (URD). URD can affect sister chromatids separation and genome stability, which is a hallmark of cancer .There are certain regions in the human genome that are inherently hard-to-replicate, posing a great challenge for the timely duplication of the entire genome. Such regions including fragile sites, rDNA and telomeres have been well documented and are the major cause of chromosome fragility . IntriguDuring transcription, RNA polymerase (RNAP) together with multiple transcription elongation and RNA processing factors form a large complex, which is tightly bound to DNA. In addition, eukaryotic transcription needs to be coupled with other downstream events, like RNA splicing to generate mature mRNA. Thus, transcription machineries together with RNA processing factors could be obstacles for an advancing replication fork. Since DNA replication and transcription compete for the same DNA template, it is inevitable that on some occasions there will be a collision between the two machineries. Indeed, it has long been known that transcription can trigger replication-dependent genome instability that would lead to recombination and mutations .in vitro study suggested that a reconstituted Escherichia coli replisome can remove or bypass a co-directional RNAP and use the newly synthesized mRNA as a primer to carry on DNA synthesis  are only found in less than 5% individuals in the general population, and their fragility is associated with the expansion of dinucleotide or trinucleotide repeats, which can form secondary DNA structures, affecting DNA replication and transcription . FurtherCommon fragile sites (CFSs) are present in all individuals. Some of the characteristics of CFSs might explain their fragility. First, CFSs are late replicating, which might leave insufficient time for cells to clear DNA replication obstacles or to repair damaged DNA following replication perturbations . Second,Early replicating fragile sites (ERFSs) are newly discovered fragile sites that reside in highly transcribed, early replicating genes. Besides, ERFSs are very close to replication origins, which further increases the incidence of transcription and replication collisions . Recent Ribosomal DNA (rDNA) are regions clustered with long tandem repeats, where rRNA are transcribed. rDNA is highly transcribed by RNA polymerase I to meet the demand for the translation activity in cells. Each copy of a repeat harbors a potential replication origin . In yeasTelomeres are regions composed of thousands of TTAGGG repeats that lie at the ends of chromosomes. Usually, telomeres are bound by the shelterin complex. These nucleoprotein structures can prevent telomeres from being recognized as DNA double strand breaks (DSBs), which is critical for genome stability and cell survival . AlthougDespite the high incidence of transcription and replication collisions and the prevalence of R-loops at chromosome fragile loci, cells seem to proliferate with a fairly stable genome. Therefore, it is conceivable that cells have evolved surveillance pathways to avoid or minimize the deleterious effects cause by these transcription and replication collision events. Indeed, multiple strategies or pathways have been identified that can prevent or resolve transcription and replication collisions and R-loops, such as spatial and temporal separation of transcription and replication , the couRecently, an unscheduled mitotic DNA synthesis (MiDAS) pathway functioning in early mitosis has been found to be essential for CFS stability and is recognized as a “salvage pathway” to alleviate RS accumulated at CFSs . In respTumorigenesis is a process involving mutations in tumor suppressor genes and the activation of oncogenes. In the early stage of tumor, oncogene activation induces RS and drives genomic instability. Many oncogenes act as growth factors to support proliferation by upregulating transcription factors which in turn can stimulate RNA synthesis . The inc


Stroke is a leading cause of death and disability worldwide, with limited treatments being available. However, advances in optic methods in neuroscience are providing new insights into the damaged brain and potential avenues for recovery. Direct brain stimulation has revealed close associations between mental states and neuroprotective processes in health and disease, and activity-dependent calcium indicators are being used to decode brain dynamics to understand the mechanisms underlying these associations. Evoked neural oscillations have recently shown the ability to restore and maintain intrinsic homeostatic processes in the brain and could be rapidly deployed during emergency care or shortly after admission into the clinic, making them a promising, non-invasive therapeutic option. We present an overview of the most relevant descriptions of brain injury after stroke, with a focus on disruptions to neural oscillations. We discuss the optical technologies that are currently used and lay out a roadmap for future studies needed to inform the next generation of strategies to promote functional recovery after stroke. Stroke is a debilitating neurological condition that constitutes a major cause of adult disability, affecting 10 million patients annually. Recent advances in treatment have improved the prognosis of stroke survivors, but few treatment options are available for most patients. Tissue plasminogen activator (tPA), the gold standard treatment for ischemic stroke, can break up the clot if administered within a narrow therapeutic window of <4.5 h  sleep, delta oscillations have inhibitory functions that are important for cognitive processing, particularly filtering out unnecessary and distracting stimuli Harmony, . Theta oBeta oscillations are prominent during wakefulness and are involved in maintaining neuronal equilibrium, working memory, sensory information integration, and voluntary movement control  in the motor cortex are present during normal functioning and reaching behaviours in mice. After stroke, reaching behaviour is impaired in correlation with a decrease in power of reaching-associated low-frequency oscillations  signals, can evoke brain oscillations at certain frequencies in rodent models and humans  in order to activate or inhibit specific cell types and circuits with high temporal precision Lin, . The freSeveral studies have already demonstrated the beneficial effects of modulating brain activity with optogenetic tools as a treatment for neurological disorders  as a promising non-invasive method that is able to target deeper tissue with high spatial resolution (Baek et al., Most studies use open-loop paradigms whereby brain stimulation tools are applied with predetermined stimulation parameters for set durations. Although these can effectively induce oscillations, another approach to enhance endogenous oscillations is via a closed-loop paradigm that uses external stimuli to provide feedback to the subject on the levels of desired cortical activity (Kanta et al., Increasing evidence shows an association between the modulation of brain oscillations and their potential for neuroprotection. Environmental and brain stimulation methods are successfully being implemented to measure and modulate brain oscillations at different frequencies. The modulation of gamma oscillations within the brain using these stimulation techniques is a promising approach for recovery after stroke. The next major challenge in stroke recovery research will be to translate preclinical data and methods into clinical trials and practical treatments for patients. However, more research with animal models is first needed to mechanistically understand and optimise different treatment methods, with a particular focus on investigating techniques that modulate natural oscillatory patterns. This research should include measuring changes of both cortical and subcortical neuronal activity, neurogenesis markers, interhemispheric connectivity, and communication following modulation.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Asynchronous health care encounters are becoming an increasingly mainstream form of telehealth. While synchronous phone or video visits have become more widely accepted, US policymakers and other key health care stakeholders have been hesitant to fully embrace asynchronous diagnosis and treatment. This is particularly true in the context of direct-to-consumer (DTC) platforms, where encounters are patient-initiated and there is no preestablished relationship with a provider. This hesitation is compounded by limited research comparing outcomes between asynchronous and synchronous care, especially in the DTC context.The purpose of this study was to explore whether asynchronous care leads to different patient outcomes in the form of medication-related adverse events when compared to synchronous virtual care.Using 10,000 randomly sampled patient records from a prominent US-based DTC platform, we analyzed the rates of patient-reported side effects from commonly prescribed erectile dysfunction medications and compared these rates across modalities of treatment.Asynchronous care resulted in lower but nonsignificant differences in the rates of the reported drug-related side effects compared to synchronous treatment.In some circumstances, such as treatment for erectile dysfunction, asynchronous care can offer the same level of safety in prescribing when compared to synchronous care. More research is needed to evaluate the safety of asynchronous care across a wider set of conditions and measures. Asynchronous technology is becoming an increasingly common component of health care delivery following the explosion of telehealth in the wake of the COVID-19 pandemic ) were treated asynchronously. Overall, patient-reported side effects were rare; less than 2% (n=137) of patients reported experiencing any side effect. In concordance with definitions used by McMurray et al  and MontP=.26), but this difference was not significant.We employed 2 different generalized linear models to determine whether the difference in side effect rates for asynchronously or synchronously treated patients, however small, was statistically significant. The first model was a standard logit model with the dichotomous indicator for whether a patient reported experiencing a side effect as the outcome. We found that asynchronous patients were around 20% less likely to report experiencing a side effect , but this difference was not significant.Because fewer than 2% (n=137) of the patients reported experiencing a side effect, standard logistic regression might bias results toward zero by underestimating the probability of a side effect occurring even with the large sample size . As a seThe recent widespread adoption of telehealth as an acceptable treatment modality and the potential expansion of asynchronous care have prompted deeper exploration of the downstream effects. Research that focuses on telehealth treatment outcomes and the reported side effects can facilitate defining standards, improving quality of care, and identifying opportunities for expanding treatment access to more patients and for more conditions.Our exploration of the rates of reported side effects among people receiving synchronous versus asynchronous care offers a chance to explore any unexpected downstream effects these modalities might have on patient safety and treatment outcomes. Significantly higher rates of side effects for asynchronous treatment might suggest that removing a real time interaction between patient and provider precludes the necessary information gathering in order to make a clinically appropriate assessment of whether a patient should receive medication. Alternatively, significantly lower rates of side effects from asynchronous treatment might indicate that removing the real time patient-provider interaction precludes the necessary rapport building that would encourage patients to contact their provider in case of an adverse event. Overall, we found that though the odds of reporting a side effect were lower for asynchronous patients, they did not significantly differ from the odds among synchronous patients. These results suggest that in this context (DTC treatment for ED), asynchronous care via patient-initiated encounter does not unduly prevent patients from reporting an adverse event, nor does it result in any other notable differences in adverse events when compared to patients who received treatment after a phone or video call. Our findings corroborate those of the handful of other studies in different areas of medicine in which comparisons of patient outcomes across telehealth modalities yielded no disproportionate rates of adverse events ,25.Copious research is required to continue to evaluate the safety of asynchronous care across different conditions and circumstances; however, current evidence suggests that lawmakers and practitioners should continue to consider facilitating its adoption with a prudent approach to implementation that takes into account specific circumstances where asynchronous care is safe and appropriate. These considerations could also factor in circumstances under which certain sites of patients are experiencing access barriers (such as broadband availability) that prevent them from using video technology due to bandwidth limitations , or stigAcknowledging asynchronous care’s departure from the traditional practice of medicine while simultaneously embracing its benefits could entail the design and execution of programs that offer training for providers on best practices for care that relies on store-and-forward technology ,28. AsynThere are several limitations to this study. First, the results are specific to a single DTC platform and might not be generalizable across all DTC platforms and patients. For example, structured online intake forms and requested patient data vary by DTC platform, thus introducing variation in the evidence-based nature, breadth, and quality of information presented to providers for clinical evaluation. Second, the record lookback window in the sample ended in 2019; COVID-19 has likely introduced changes in the DTC patient population. For example, patients who had not previously considered seeking treatment via telehealth might have been prompted by the sudden and unexpected inability to get in-person care. Lastly, the results also might not be generalizable beyond the condition and medications studied. More research needs to be conducted to determine whether the rates of side effects differ across modalities for other conditions that are commonly treated on DTC platforms, especially as these platforms continue to expand their treatment offerings to provide a more comprehensive suite of services.Limitations notwithstanding, this study represents an important step toward a more nuanced approach to evaluating the quality of care delivered via telehealth. As telehealth demonstrated its value during the global pandemic and is becoming an increasingly normalized form of care, research needs to evolve beyond comparisons to in-person care toward identifying the most ideal formats, processes, and approaches for collecting relevant clinical information and safely treating and communicating with patients within the suite of virtual options.


Gap junctions assembled where the tips of the TZPs contact theoocyte plasma membrane, and other contact-dependent signaling may also occur at these sites.Here, I describe early studies of TZPs, which were first identified in the late 19th century,discuss their similarities with classical filopodia, review their structure and function,and compare two models that could account for their origin. Possible priorities and directionsfor future studies close this contribution.Development and differentiation of a functional oocyte that following fertilization isable to give rise to a new individual requires continuous physical contact with the supportingsomatic cells of the ovarian follicle. As the oocyte is surrounded by a thick extracellularcoat, termed the  To overcome this barrier, thin cytoplasmic processesextend from the granulosa cells through the zona pellucida to the oocytesurface. These transzonal projections (TZPs) constitute the only means of contact and contact-dependentcommunication between the oocyte and its somatic environment and thus are indispensable forthe production of an oocyte that is able to give rise to a new individual. Here, I review early studiesof TZPs and cognate structures in non-mammalian species, describe their nature and function,discuss different models that may explain their origin, and conclude with questions for futurestudy.Germ cell differentiation depends on communication with the somatic cells of the gonad, whichconstitute a micro-environment that provides it with essential nutrients and regulatory signalsat each stage of its development . In mamzona pellucida  and bone morphogenetic protein 15 (BMP15), regulatethe differentiation of the granulosa cells (in vivo, they may be presented to the granulosa cells in a membrane-associated context. Such a modelimplies that their receptors would be enriched at the tips of the TZPs, a prediction that couldbe experimentally tested.Oocytes are able to grow partially when detectable gap junctional communication is blockedbut not at all when they are physically separated from the granulosa cells . This ia cells . AlthouRecent studies have identified small vesicles lying between the tips of TZPs and the plasma membraneof the oocyte. In addition, transport of mRNA from the granulosa cell bodies to the tip of TZPs,as well as its transfer to the oocyte, was also observed . Based zona pellucida separating the oocyte and granulosa cells. Afterthe oocytes begin to grow within primary follicles, it is deposited in discrete ‘chunks’that eventually become joined together to establish an intact structure (zona pellucida, intercellular junctionslink the oocyte to its neighboring granulosa cells (zona pellucida becomes establishedaround pre-existing points of contact between the oocyte and granulosa cell and, as the zona pellucida thickens, pushing the granulosa cells away from the oocyte, the cytoplasmstretches to maintain the point of contact (How are TZPs generated? Two mechanisms may be considered, which may be termed stretching andpushing . The structure . Secondcontact .zona pellucida is laid down early during oocyte growth,many granulosa cells adjacent to the oocyte must be born after this event. According to the stretchingmodel, these would not possess TZPs; yet TZPs appear to project from every granulosa cell thatlies adjacent to the zona pellucida. Third, TZPs are destroyed when folliclesare cryopreserved, yet regenerate when they are thawed and placed in culture (Although this model is attractively simple, it is not easily reconciled with several characteristicsof TZPs. First, as reported by several groups based on observation and recently quantified byus, the number of TZPs increases by at least an order of magnitude during oocyte growth (zona pellucida to reachthe oocyte. This model could account both for the continuous increase in the number of TZPs andfor their presence on all zona pellucida-adjacent granulosa cells. To testit, we combined wild-type oocytes that were fully enclosed by a zona pellucidabut were completely stripped of granulosa cells with a mixture of wild-type and fluorescentlytagged granulosa cells and incubated the reaggregates (zona pellucida to reach the oocyte.The pushing model holds that TZPs grow out from granulosa cells, in a manner possibly analogousto filopodial growth, and penetrate through the zona pellucida. In this context, it may be recalled that there exists a small populationof tubulin-rich TZPs. It could be speculated that this sub-population of TZPs is generated throughthe stretching mechanism.Although the both theoretical and experimental evidence suggest that most TZPs are formed denovo, the two models are not entirely incompatible. It is plausible that some TZPs are derivedfrom the points of granulosa cell-oocyte adhesion that existed prior to elaboration of the Gdf9-/- mice, fewer TZPs are found and their structure isdisorganized (What signals trigger TZP formation by the granulosa cells? As described above, GDF9 and BMP15produced by the oocyte regulate differentiation and gene activity in granulosa cells. Strikingly,in ganized .Based on the directionality of TZP growth towards the oocyte, it is tempting to speculate thatthe oocyte provides chemotactic cues that guide the TZP. It is also possible, however, that thereis no preferred direction for TZP growth, which instead occurs wherever it can do so unimpeded.In this context, it is noteworthy that, at the time of formation of primordial follicles, thepre-granulosa cells extend long arms that enclose the oocyte (Ptk2 was deleted from growing oocytes, the number of TZPs as well as gap junctional communicationwere strikingly reduced. This indicates that PTK2 in the oocyte is required to establish or maintainTZP-oocyte contact (Epab, encoding an RNA-binding protein thatpromotes translation, in oocytes caused a decrease in the quantity of E-cadherin in the oocyteand in the number of TZPs (Once the newly elaborated TZP reaches the oocyte surface, the two plasma membranes become associatedby adherens junctions, as discussed above, and gap junctions are assembled to metabolicallycouple the cells. The mechanisms by which these junctions are assembled, as well as whether theTZP-oocyte membrane interaction is stable or dynamic, remains unknown. When contact . AdditiAlthough the basic structure and function of TZPs have long been known, the mechanisms by whichthese indispensable vehicles of communication between the developing oocyte and its follicularenvironment are produced have remained mysterious. Recent results establish that they arecontinuously produced by the granulosa cells surrounding growing oocytes. In view of observationsthat granulosa cell-oocyte communication is impaired in certain disease conditions associatedwith infertility (Are TZPs dynamic or stable structures? If dynamic, what controls the relative rates of elaborationand retraction? Does the stability of a TZP depend on the cell-cycle state of the granulosacell?Are TZPs actively guided towards the oocyte or do they simply grow where there is space?zona pellucida sufficiently porous to permit growing TZPs topenetrate through it or must they digest a path?Is the Do granulosa cells stop generating TZPs once oocytes have reached full size? If so, by whatmechanism?Many studies have reported that TZPs are lost during meiotic maturation . What iThe answers to these and other questions will not only contribute to understanding how the growingoocyte interacts with its environment but will also uncover principles governing the regulationof intercellular contact and communication.


Limited drug diffusion and gradients of oxygen and nutrients in spheroids represent avascular tumor regions containing quiescent and hypoxic tumor cells with high drug resistance. Circulating tumor cells (CTCs) effect metastatic spread and are present in high numbers in malignant diseases such as small-cell lung cancer (SCLC) and in other cancer patients with high tumor load. CTCs are heterogeneous and only a small fraction of these cells survive in the circulation and cause distal lesions. CTCs may circulate as single cells but small CTC clusters or CTC spheroids have been detected in cancer patients and demonstrated to possess increased metastatic potential. At our lab we have obtained 9 permanent SCLC CTC cell lines  of distinct patients exhibiting similar characteristics and spontaneous formation of large spheroids, termed tumorospheres. These aggregates were shown to exhibit high drug resistance compared to the corresponding single cell suspensions. The increased metastatic capability of small circulating tumor clusters/spheroids may be explained by their role as putative precursors of tumorospheres eventually trapped in capillaries. Limited drug penetration and the presence of hypoxic/quiescent cells can readily account for the global drug resistance of advanced SCLC which has resulted in clinical failure of a wide range of chemotherapeutics and low survival. Furthermore, we have detected such tumorospheres in non-small-cell lung cancer (NSCLC) patients progressing under EGFR-directed tyrosine kinase inhibitor therapy which had undergone NSCLC-SCLC transformation.Cancer cell spheroids are used for drug screening as these three-dimensional (3D) assemblies recapitulate tumors more realistic than the widely employed 2D  Since many patients present with metastatic disease or the tumors progress to a disseminated stage despite treatment, cytotoxic or targeted chemotherapy is essential for further care. However, drug resistance limits the efficacy of chemotherapeutics in cancer and, eventually, results in treatment failures and a dismal prognosis. Mechanisms of chemoresistance work at the single cell level but also as a result of the formation of multicellular clusters and at the tumor tissue/tumor microenvironment interphase. For individual cells, reduced drug uptake, increased drug efflux, drug inactivation as well as target modifications and impaired execution of cell death are the most important mechanisms whereas at the tumor level limited drug diffusion, high interstitial pressure, extracellular acidosis, cell contact-mediated mechanisms and irregular vessel supply contribute to refractoriness to treatment. Avascular tumors or metastases often display a gradient in oxygen levels leading to a proliferative zone and a hypoxic core with quiescent cells that are more resistant to chemotherapy, immunotherapy and irradiation.Cancer still remains as one of the diseases with highest mortality affecting the worldwide population. Up to 95% of anticancer drugs in clinical trials failed due to inefficacy and toxicity despite showing efficacy during preclinical testing. To achieve better predictive tests, three-dimensional (3D) cell culture systems were developed because they promised to resemble the in vivo tissue environment more precisely than 2D systems,8. Of the various 3D tumor models under investigation, the most common are the spheroids which can be prepared by a range of techniques relying simply on the prevention of cancer cell attachment to substrates. Spheroids are scaffold-free globular self-assembled aggregates of cancer cells which exhibit an intermediate complexity between 2D in vitro cell cultures and in vivo solid tumors. Advanced 3D models include cocultures of tumor cells with cancer-associated fibroblasts (CAFs) and immune cells, as well as tumor ECM components. Comparison of spheroids with conventional 2D cell monolayers has revealed marked differences in the response to anticancer drugs. In dependence of their size, spheroids display higher chemoresistance due to contact-mediated resistance, exclusion of drugs and their content of quiescent and hypoxic cells as a result of gradients of nutrients and oxygen. In large spheroids, a necrotic core is present when the innermost cells are located beyond the diffusion limit of oxygen approximately 200 μm from the surface of the cellular aggregate-14. This mimics solid tumors because delivery of oxygen to the cells present in the inner regions of the tumor mass is frequently reduced due to the increased oxygen diffusion distances,16. In summary, on the one hand, spheroids represent an important tool for in vitro drug screening and on the other hand, specific multicellular assemblies of cancer cells possibly present in patients may be an important mechanism of resistance to chemotherapy.For successful preclinical drug development, the compounds need to be tested using model systems recapitulating the features of native tumors accurately. Historically, simple two-dimensional (2D) cultures of permanent cancer cell lines and animal models were employed which resulted in a high attrition rate in subsequent clinical trials due to a lack of predictive power. Most cell lines form spheroids under the appropriate non-adherent conditions but properties of these structures vary widely in respect to size, surface density, gradients of nutrients and oxygen as well as homogeneity of the preparations. The transport of oxygen, nutrients and metabolic wastes through the layers of the spheroid is decelerated such creating specific gradients which mimic a physical barrier . The ability to cross these barriers in vivo determines drug absorption, distribution, metabolism, excretion, and toxicity (ADME-Tox).Tumor spheroids seem to provide a better representation of real malignant tissues and are used in drug screening in different forms, depending on the method of preparation. In principle, spheroids can be generated using the hanging drop technique, suspension/spinner flasks, ultra-low attachment plates, micro-patterned plates or microfluidics devices, to mention a few procedures. Accordingly, well-nourished cells in the outer layer exhibit high proliferation whereas the middle layer is characterized by quiescent cells and the depleted core may contain necrotic cells and acellular regions. The presence of diffusive gradients across the spheroids account for the impaired cytotoxicity of various anticancer drugs and of irradiation. Cells in the hypoxic region are resistant to drugs or irradiation which kill cells through reactive oxygen species, while quiescent cells are all less sensitive to drugs requiring active cell cycling. Furthermore, spheroids possess a network of structural and adhesive ECM proteins which regulate response to growth factors, cell proliferation and differentiation. In addition, spheroids closely mimic the physical barriers found in real solid tumors, which obstruct the free penetration of drugs through the whole mass. However, a limiting fact of spheroids is the lack of relevant features of real tumors such as the supporting vasculature, effector cells of the immune system and the fluid dynamics. Furthermore, ECM components expressed in spheroids are of different cellular origin compared to the range of proteins supplied by CAFs in vivo.Large spheroids (~ 400-500 μm diameter) display a layered cell distribution analogous to that observed in solid tumors. However, assays such as formazan or resazurin reduction assays may not be efficient with spheroids because of the incomplete probe penetration.The response of spheroids to cytotoxic drugs can be assessed by variation of morphometric parameters such as their diameter, volume and circularity from bright field microscopy by image analysis. Other methods rely on measurements of membrane integrity , intracellular mitochondrial activity  or ATP content. Alternatively, the effect of drugs on spheroids can be analyzed using immunofluorescence staining, expression of fluorescent proteins and fluorescent probes for checking the state of the constituting cells. The production of large spheroids is difficult since cells tend to adopt irregular shapes and are not any longer suitable for screen drugs in a reproducible manner. Inclusion of CAFs and immune cells in scaffold-embedded structures are technically demanding and significantly increase costs and effort for screening. Additionally, the proof that advanced 3D models show superior predictive power for the selection of promising drugs is largely lacking. The spheroid models are relatively simple with respect to clinical tumors and, therefore, the reliability of their drug-response prediction capacity should not be overestimated.Distinct cancer cell lines and techniques for generating spheroids produce a wide variety of such multicellular aggregates with an extreme range of molecular and physical properties. Exclusion of probes or drugs by spheroids are an important test to confirm the presence of a tight physical surface barrier and to prove the actual representation of an avascular tumor region. Still, the majority of spheroids for drug screening uses permanent cell lines which has been adapted to tissue culture conditions for prolonged times and have lost the lower proliferation rates and various characteristics of primary tumor cells. Most of these spheroids are loose assembly of cancer cells and provide no significant barrier to drug penetration and cytotoxicityin vitro by different methods but such aggregates are found similarly in vivo under special conditions, namely in ascitic fluid and pleural effusions in ovarian and lung cancer among others, respectively. Epithelial ovarian cancer (EOC) is the most lethal gynecological cancer, with a 5-year survival rate below 30% because the current chemotherapeutic regimes for EOC are unable to achieve sustained remission. EOC exhibits local invasion and this peritoneal carcinomatosis is present in a third of patients at diagnosis and in almost all patients at recurrence. For EOC, in vitro cultured spheroids are capable of tumorigenesis in vivo and display a reduced response to chemotherapeutic drugs when compared to monolayers. Therefore, spheroid formation promotes chemoresistance and represents a key component of platinum- and taxane-sensitive recurrence in EOC. Spheroids of EOC arise from collective detachment, rather than aggregation and are more resistant to anoikis than single cells. Subsequently, spheroids can reattach to the mesothelium and invade the ECM during dissemination. In patients, a wide range of sizes of spheroids were observed comprising clusters ranging from less than 20 cells to over 100 cells with approximately 40% of cells positive for the proliferation marker Ki67. The EOC spheroid generates a metabolite gradient that inhibits access of chemotherapy agents to the internal cells and the resulting slowly cycling and/or quiescent cellular states are resistant to therapies involving platinum drugs and taxanes that specifically hit proliferating cells.Spheroids can be easily generated . In advanced stage, malignant pleural effusions (MPEs) are frequently present and constitute an excellent source to culture a wide variety of cancer cells, in particular NSCLC classified as adenocarcinomas,31. In contrast to pleural cells, establishment of cancer cell lines from solid tumor tissue is frequently inefficient, because of loss of growth after a few passages. MPE is caused by a combination of processes such as inflammation, enhanced angiogenesis and vascular leakage. Withdrawal of ascites or pleural effusions is often performed to relieve symptoms and is less invasive than tumor biopsies. These patients have a poor prognosis with median survival times ranging from 2.5 months in lung cancer to approximately 17 months for mesothelioma. Such MPE gave rise to in vitro cultures both in adherent and/or in spheroid conditions in almost all cases. These cultures form large irregular spheroids in non-adherent regular medium or smaller and very compact spheres in serum-free sphere medium and, furthermore, exhibit efficient tumor engraftment in immunocompromised mice.Lung cancer is the most common cause of cancer deaths worldwide. The two broad histological subtypes of lung cancer are small-cell lung cancer (SCLC), which is the cause of 15% of cases, and non-small-cell lung cancer (NSCLC), which accounts for 85% of cases and includes adenocarcinoma, squamous-cell carcinoma, and large-cell carcinoma,33. Generation of cultures from these tumor cells has a high success rate, thereby amenable for sensitivity tests and selection of therapy. For example, single multicellular spheroids from malignant pleural mesothelioma were trapped and tested for chemotherapeutic drug response on a microfluidic platform. Adenocarcinoma MPEs of NSCLC resulted in in vitro cultures both in adherent and/or in spheroid conditions but the ability of such preparations to induce xenografts appeared to correlate with the production of large spheres and not with a particular expression of known surface markers. In conclusion, spheroids are present in malignant peritoneal fluid and pleural effusions but these aggregates tend to exhibit smaller sizes without the hypoxic and necrotic regions observed in larger 3D structures. In these cases, chemoresistance seem to be produced by cell-cell contact, altered gene expression and concurrent resistance to anoikis-induced cell death. Cell-cell interactions in spheroids can control cell signaling, survival, proliferation, and drug sensitivity, in particular under control of E-cadherins.In general, pleural lung cancer spheroids are less than 200 µm in diameter and, in our experience, these aggregates tend to disperse and to grow as monolayers. The number of CTCs has predictive power and their variation in response to therapy indicates the efficacy of treatment. Only a minor fraction of CTCs survives in the circulation, extravasates and is able to establish metastases. Many studies have monitored CTCs in patients’ blood and the investigations showed that, in contrast to single cells, CTC clusters have a high metastastic potential,40. These structures have been described as circulating tumor microemboli, circulating micrometastases or circulating tumor aggregates and are defined as groups of tumor cells that travel together in the bloodstream. Thus, CTCs can exist as single cells or clusters, ranging from two cells to dozens of cells but this number of cells is too low to constitute spheroids with hypoxic cores. Several mechanisms are supposed to account for the higher metastatic potential of CTC clusters and a correspondingly poor prognosis such as an increased lodging in small blood vessels in various organs, increased resistance to cell death after homing in distal organs and acquisition of stem cell signatures within the cell assemblies,43.The major cause of cancer-associated mortality is tumor metastasis which is mediated by circulating tumor cells (CTCs) detached from the primary lesion. CTCs have been detected in variable numbers in almost all tumors with highest numbers in SCLC and inflammatory breast cancer (IBC). Studies showed that the formation of metastases partially depended on the size and concentration of CTC clusters,46. In patients with metastatic breast and cervical carcinomas, tumor cell emboli entrapped in vessels in were proved in the microvasculature of the lungs. The size distribution of vessels was an important determinant of the distribution and survival of CTC clusters. However, a delayed aggregation of CTCs at inflammatory sites or after entrapment in small vessels could not be ruled out. In SCLC, the presence of CTC cluster was reported to be associated with shorter progression-free and overall survival. Although the metastatic process cannot be monitored in patients, CTC clusters may be the precursors of large spheroids in small vessels in advance of extravasation and growth of secondary lesions. Cardiac glycosides were found to disintegrate CTC cluster and were proposed as antimetastatic agents. However, the actual tumor dissemination via CTCs may occur at a time when the initial tumor is of small size and has not been detected clinically thus making a late CTC-directed intervention a futile effort.CTC clusters are expected to have a very short life span in the circulation due to a faster removal by filtration in the circulation compared to single cells. This tumor is disseminated in most cases at first presentation and while exhibiting high initial response rates to first-line platinum-based chemotherapy the disease relapses invariably within approximately 1 year and second-line treatments with either topotecan or epirubicin-based regimens yield low response rates of short duration. Except a minor extension of survival by novel checkpoint immunotherapy, no progress has been made for the last decades with no new drugs approved,52. There is no lack of clinical trials - the NIH clinical trial registry counts over 800 investigations comprising new diagnostic methods, novel drugs, new combinations of drugs and alternating treatment regimens. The therapeutics tested clinically target a host of pathways and effectors ranging from novel platinum compounds, camptothecines, cell cycle inhibitors, PARP inhibitors, modulators of apoptosis and many others. So far, no definite mechanisms of resistance were reported for advanced SCLC which could have been exploited for novel therapeutic strategies. Instead, numerous therapeutics with anticancer activities shown in other malignancies have been administered to SCLC patients and largely demonstrated to be more or less tolerated but to fail to yield significant responses. Genomics has demonstrated inactivation of tumor suppressor proteins p53 and RB1 with broad alterations in the regulation of transcription and putative drugable modifications in small subpopulations of patients.SCLC is a highly aggressive malignancy with a dismal prognosis found mostly associated with heavy tobacco consumptionet al.. In contrast to clusters and spheroids found in pleura and ascites, tumorospheres consist of 104-106 tumor cells. Proliferation marker Ki67 or PCNA as well as carbonic anhydrase IX (CAIX) staining of tumorosphere sections revealed a rim of proliferating cells and a layer of quiescent and hypoxic cells surrounding a necrotic core region, respectively. Accordingly, comparison of the chemosensitivity of CTC single cell suspensions with tumorospheres demonstrated markedly increased resistance of the clusters against chemotherapeutics commonly used for treatment of SCLC. Therefore, global chemoresistance of relapsing SCLC seems to rely on formation of large tumorospheres which reveal limited accessibility for drugs, lower growth fraction and a hypoxic inner microenvironment. The same spheroid-forming ability of SCLC CTCs derived from blood samples of 9 different patients and their similarity of chemosensitivities and molecular markers point to an important role in relapsed SCLC patients-56. Actually, all SCLC CTC lines were established from patients with extended metastatic disease and a high tumor load after 2nd line therapy with poor prognosis, such suggesting an important role of these spheroids in chemoresistance and fatal tumor progression. The CTC tumorospheres show a rather uniform size distribution, depending on the specific CTC line, and may be expanded to large spheroid numbers which are suitable for advanced drug screening. SCLC seems to represent a unique tumor model to study the association of CTCs, metastasis and drug resistance. Of course, tumorospheres cannot exist in the circulation for long but SCLC CTCs or SCLC CTC clusters may be released by primary tumors after first-line chemotherapy or may grow to these large spheroids from smaller aggregates after entrapment in the microvasculature as observed under tissue culture conditions.The rapid dissemination of SCLC is associated with excessive numbers of CTCs in many patients exceeding the numbers of this cell type detectable in colorectal and breast cancers by orders of magnitude. EpCAM-positive SCLC CTCs have been counted using the CellSearch system and other techniques and correlated with prognosis and response to therapy in a range of studies. Furthermore, enriched SCLC CTC fractions were demonstrated to be tumorigenic in immunocompromised mice. In general, CTCs are highly heterogeneous and only a small fraction is competent in inducing metastases. At our institution we were able to initiate 9 permanent SCLC CTC lines  from SCLC patients with a higher tumor load. This panel of SCLC CTC cell lines exhibit the typical SCLC markers, such as chromogranin A, enolase-2, synaptophysin and specific p53 mutations. Upon growth in tissue culture, all CTCs derived from the different patients developed spontaneously large multicellular aggregates up to 1 mm, termed tumorospheres according to a nomenclature proposed by Weiswald . However, SCLC seems to constitute an example of the failure of drug screening that is confined simply to 2D cell line models and loose aggregates of cancer cell lines in suspension. Although SCLC in vivo exhibits aggressive proliferation which results in avascular regions and necrotic areas, drug screening was invariably performed without attempts to use 3D models. Tumorospheres with quiescent cells and hypoxic/necrotic regions are physically resistant to a host of drugs without requiring special molecular mechanisms of drug resistance. In conclusion, CTC tumorospheres represent natural grown and large spheroids which are expected to be involved in tumor dissemination and drug resistance in SCLC and provide a suitable source for 3D testing of drugs. To overcome this type of resistance means to disintegrate spheroids in vivo would be required.SCLC was designated as “graveyard of drug development” since a host of drugs with different targets failed clinically and over 800 studies registered by the US clinical trials site brought little progress in therapeutic modalities with the single exception of a minor prolongation of survival in response to immunotherapy-60. Concurrent development of adenocarcinoma and SCLC was noted in EGFR-mutant tumors before treatment with an EGFR inhibitor. Approximately after one year of EGFR-directed TKI therapy, resistance develops comprising several distinct mechanisms including the most common EGFR T790M mutation in 50%-60% of tumor samples. Resistance mechanisms that bypass EGFR signaling, such as MET and HER2 amplification account for another 15%-20% of resistance to EGFR inhibitors and a less common mechanism is the histological transformation of EGFR-mutant adenocarcinoma to SCLC,64. This process was first described in 2006 in a patient originally diagnosed with adenocarcinoma revealing SCLC with the original EGFR exon 19 deletion mutation in a repeat biopsy. Since then, several other case series have been reported and these SCLCs were identified by morphology and positive immunohistochemical staining for synaptophysin, chromogranin, or NCAM-67.In 3%-15% of patients with NSCLC tumors, transformation to SCLC can occur, preferentially during progression under tyrosine kinase inhibitor (TKI) or rarely under immune checkpoint therapy. This suggests that these SCLCs were not independent de-novo cancers, but a transformed phenotype. SCLC transformation occurs earlier in EGFR-mutant patients but both groups were responsive to platinum/etoposide regimens with approximately 40% response rate. Transformation to SCLC suggests that both adenocarcinoma and SCLC arise from a common cell type,64,69. The initial response to chemotherapy is much greater for patients with advanced SCLC than patients with metastatic NSCLC which suggests inherent differences in tumor biology. Two large case series have investigated the frequency of tumors with combined SCLC and NSCLC histology and reported 2%-10% of such tumors,71. Although EGFR mutations are identified in SCLC, these patients have had mixed responses to EGFR inhibitors.Switch from NSCLC to SCLC is a mechanism of resistance in EGFR-mutant tumors which retain the specific mutation. Thus, atypical SCLC features are visible after transformation and a lower response rate to etoposide/carboplatin EC treatment than classical SCLC is detected. At our institution we have established two cell lines from NSCLC patients progressing after osimertinib TKI treatment which have transformed to SCLC as proved by detection of the typical markers. Single transformed SCLC cells of these lines proved to be highly chemosensitive to cisplatin and topotecan but in tissue culture large spheroids similar to the SCLC CTC tumorospheres were detected and may be held responsible for chemoresistance and a dismal prognosis after a NSCLC-SCLC switch After transformation to SCLC, the median OS was 9-10 months with significant lower OS in the non–EGFR-mutant group. Once the tumor is transformed its survival and response to treatment seems comparable to that of classical SCLC. For example, in 39 patients the median time from diagnosis of NSCLC to the transformation to SCLC was 19 months and the median survival after SCLC diagnosis 6 months. This tumor dormancy is classically defined as the arrest of tumor growth in the primary site or in metastatic dissemination. In cellular dormancy, cancer cells are in a quiescent state characterized by minimum proliferation, minimum death and reversibility. Dormant cancer cells are likely resistant to conventional therapies which target actively proliferating cells. This may be a property common to all cell lines since cancer tissue-originated spheroids could be reversibly held dormant for at least 7 days in hypoxia without growth factor stimulation. Under hypoxic conditions, tumor cells adapt a more aggressive tumor phenotype including the activation of DNA damage repair proteins, altered metabolism, and decreased proliferation.Tumor cells may reside in a protected environment and recur after prolonged times upon reactivation. For tumor like breast cancer this niche is located at less vascularized and partially hypoxic regions within the bone marrow.Tumorospheres seem to fulfil the preconditions for a protected niche for dormant tumor cells: hypoxic conditions, quiescent cells and a protective cover by outer layers which exhibits continuous shedding of tumor cells and fragments (unpublished observation). Thus, in SCLC cells may not seek protection as single cells in the bone marrow but seem to be able to form a hypoxic niche themselves that is not amenable to eradication by chemotherapyin vitro by cells from most cancers. Diverse methods have been used to demonstrate cancer small cell clusters/spheroids consisting of up to 10 cells in the blood of metastatic cancer patients but filtration/image analyses of unprocessed blood showed a size range of up to 30-60 µm obviously assembled by a larger number of tumor cells. SCLC CTC tumorospheres reach sizes exceeding 500-1000 µm and, in vivo, may be the product of the smaller spheroids trapped in the microvasculature. Spheroid-forming potential has been ascribed to cancer stem cells (CSCs) as reported for neurospheres, mammospheres and structures observed in other cancers. The possibility that the spheroids enclose and protect (CSCs) expressing the characteristic SOX2, NANOG and OCT4 markers have bee raised but in the case of the SCLC CTC cell lines no CSC features were found.Spheroids are small multicellular aggregates formed . In this case, the formation of clusters has been linked to a hybrid epithelial/mesenchymal (E/M) phenotype of cells. In IBC, CTC clusters are launched as aggregates of up to 20 cells into the bloodstream which are able to traverse capillary constrictions as single-file chains. Cells in a hybrid E/M phenotype retain at least some levels of E-cadherin and co-express epithelial and mesenchymal markers. However, at least two of the SCLC CTC cell lines exhibit high expression of cadherin and low expression of vimentin constituting a transition epithelial-mesenchymal transition (EMT) phenotype at most that ultimately is not compatible with formation of the tumorospheres. EMT has been found to be dispensable for metastasis in experimental animal models and, so far, clinical attempts to target CSCs were not effective to prolong survival,83.Besides SCLC, the other cancer that is distinguished by extremely high numbers of CTCs is the highly aggressive IBC which can metastasize via clusters. Assembly of SCLC cells into tumorospheres seem to be sufficient for broad-range chemoresistance and, unfortunately, the clinical strategies to attack and eliminate such aggregates in vivo are not available yet. Patient-derived xenograft models employing CTCs should assist in the development of new therapeutic modalities directed to spheroids.SCLC CTC spheroids seem not to rely on features of EMT or CSCs phenotypes for drug resistance. The circulating SCLC clusters/small spheroids may grow to large tumorospheres after entrapment in capillaries and start the process of extravasation with help of proteolytic enzymes. In addition to the overexpression of matrix metalloproteinase-9, SCLC CTC lines release cathepsin S which has been involved in brain metastasis which is frequently observed for this tumor type


Endoscopic retrograde cholangiopancreatography (ERCP) with sphincterotomy and stone extraction is the treatment of choice for choledocholithiasis, reaching a successful clearance of the common bile duct (CBD) in up to 90% of the cases. Endoscopic ultrasound (EUS) has the best diagnostic accuracy for CBD stones, its sensitivity and specificity range being 89–94% and 94–95%, respectively. Traditionally seen as two separate entities, the two worlds of EUS and ERCP have recently come together under the new discipline of bilio-pancreatic endoscopy. Nevertheless, the complexity of both EUS and ERCP led the European Society of Gastrointestinal Endoscopy to identify quality in endoscopy as a top priority in its recent EUS and ERCP curriculum recommendations. The clinical benefits of performing EUS and ERCP in the same session are several, such as benefiting from real-time information from EUS, having one single sedation for both the diagnosis and the treatment of biliary stones, reducing the risk of cholangitis/acute pancreatitis while waiting for ERCP after the EUS diagnosis, and ultimately shortening the hospital stay and costs while preserving patients’ outcomes. Potential candidates for the same session approach include patients at high risk for CBD stones, symptomatic individuals with status post-cholecystectomy, pregnant women, and those unfit for surgery. This narrative review discusses the main technical aspects and evidence from the literature about EUS and ERCP in the management of choledocholithiasis. Endoscopic retrograde cholangiopancreatography (ERCP) with sphincterotomy and stone extraction is the treatment of choice for choledocholithiasis, reaching a successful clearance of the common bile duct (CBD) in up to 90% of cases with relatively low morbidity (about 5%) . The sucEndoscopic ultrasound (EUS) has the best diagnostic accuracy for CBD stones with both radial and linear echoendoscopes, with sensitivity and specificity ranging 89–94% and 94–95%, respectively . MoreoveTraditionally seen as two separate entities, the two worlds of EUS and ERCP have recently come together under the new discipline of bilio-pancreatic endoscopy. Modern endoscopists are very often skilled in both techniques, which they use either in conjunction or alternatively to obtain the best possible outcomes.The purpose of this narrative review is to highlight and discuss the main technical aspects and evidence from the literature about EUS and ERCP in the management of choledocholithiasis.Given the complexity of both EUS and ERCP, the European Society of Gastrointestinal Endoscopy (ESGE) has identified quality in endoscopy as a top priority . UnfortuIn particular, the ESGE maintains that interventional EUS and ERCP are associated with the highest risk of serious complications and mortality among all the endoscopic procedures . For thiThe main training recommendations from the ESGE include structured supervised ERCP/EUS simulator-based learning before hands-on training and participation in formal courses along with self-directed study, for a minimum period of 12 months. At least a further year of dedicated training in a high-volume center is required to reach competence in advanced ERCP and therapeutic EUS. Although the mere number of procedures per se is largely suboptimal to gauge operators’ experience, it is estimated that physicians must have performed at least 250 EUS and 300 ERCP in order to demonstrate competency.Under these premises, as adequate training in both EUS and ERCP is far from easy and swift, to implement a combination of EUS and ERCP in the same session the availability of experienced physicians should be firstly assessed.However, how is competence defined according to the ESGE? It is the ability to independently assess the need for and carry out successful and safe procedures, with good patient satisfaction across a range of case difficulties and clinical contexts. Formal assessments tools should be used regularly during training to assess the acquisition of competence.Finally, a trainee should undergo a formal summative assessment process before commencing independent practice in ERCP and EUS. Once competent in ERCP and EUS, endoscopists should be supported to continue a period of mentored practice with an experienced colleague. Again, we can see how introducing EUS/ERCP combined sessions requires not only an initial good operators experience but also a continuous interaction with other expert centers to maintain competence.Until recently, the guidelines from the American Society for Gastrointestinal Endoscopy (ASGE) suggested that symptomatic patients with gallbladder stones at low risk for CBD stones (<10%) should undergo direct laparoscopic cholecystectomy, while patients at high-risk (>50%) for CBD stones should undergo direct ERCP . In betwAfter reviewing the comprehensive contemporary evidence, the panel of ASGE experts revised the 2010 criterion to decrease the probability of diagnostic ERCP, which has significant risk but minimal benefit . To miniThe ESGE guidelines on the same topic recommended EUS (or magnetic resonance cholangiopancreatography) to diagnose CBD stones in patients with persistent clinical suspicion but insufficient evidence of stones on abdominal ultrasonography . In addiAs we can see, the role of EUS in assessing patients with suspected choledocholithiasis is limited to intermediate-risk groups by both the ASGE and the ESGE guidelines. Moreover, very little is said about same session EUS and ERCP.However, we speculate that EUS is indicated in the majority of the cases before ERCP because it brings important information (beyond the mere diagnosis of CBD stones) that can be beneficial for the technical outcomes of ERCP . AdditioAt intermediate-risk stages for choledocholithiasis, EUS must always be done first to determine whether ERCP is subsequently indicated. Usually, these are the patients with symptomatic biliary disease associated with cholestasis, with or without CBD dilation. In these patients, scheduling same-session EUS/ERCP does not seem justified as the resources might not be well allocated when ERCP is not needed. In fact, a systematic review showed that preliminary EUS avoided ERCP in 67% of the patients by ruling out choledocholithiasis .When the pretest probability of choledocholithiasis is greater, such as with the high-risk cases identified by the ASGE (see above), EUS can still be very informative . AlthougFirst, a significant overall reduction in adverse events in patients with previous EUS versus those who went directly to ERCP was shown, mainly due to a smaller incidence of post-ERCP pancreatitis.Secondly, the excellent diagnostic accuracy of EUS for CBD stones, together with its findings regarding stone number and size, is often useful in planning the best treatment strategy  Figure 1The clinical benefits of performing EUS and ERCP in the same session are multiple, such as benefiting from real-time information from EUS, having one single sedation for both the diagnosis and the treatment of biliary stones, reducing the risk of cholangitis/acute pancreatitis while waiting for ERCP after the EUS diagnosis, and ultimately shortening the hospital stay and costs while preserving patients’ outcomes . Potentin = 33, 28.4%) or within one week  or after more than one week  [Information from EUS tends to get old fast since biliary stones can either migrate into the duodenum or move within the biliary tree. We reported on patients who underwent EUS and ERCP during the same session  . As expe, 35.3%)  Thereforp = 0.001) [Performing EUS and ERCP in a single session does not carry higher complication risks and requires a significantly smaller dose of propofol for sedation compared to performing separate procedures. Vila et al. compared 39 patients who underwent the same session approach to 46 patients who underwent separate procedures. Interestingly, while the overall procedural time did not differ significantly between both groups, the dose of propofol differed significantly being lower with the same session approach  . No diffThe same session approach seems beneficial in reducing the overall risk of adverse events. Benjaminov et al. reporting a 6-year experience at their institution in Israel described 151 patients with EUS-proven CBD stones, with subsequent ERCP . Four , it is sometimes necessary to return to EUS in case of ERCP failures and limitations.EUS rendezvous is an alternative to interventional radiology-guided rendezvous after failed biliary cannulation that can be performed in the same endoscopic session (CP). A recent systematic review and proportion meta-analysis including 342 patients demonstrated an 86% pooled rate of technical success . The pooOther limitations with ERCP can be encountered when cannulation fails due to periampullary diverticulum or post-surgical anatomical changes. Garcia–Alonso et al. reported 170 cases of salvage EUS-guided ductal access and drainage ERCP failures . In apprLastly, EUS-guided gallbladder drainage (GBD) can be coupled to ERCP in non-surgical candidates . We owe EUS and ERCP are highly advanced techniques that have come together to form a new endoscopic art, which is bilio-pancreatic endoscopy. Given the skills required to perform these demanding procedures, accurate planning of training and maintenance of competence are essential. In patients with suspected CBD stones, EUS is always useful to obtain all the diagnostic features that are functional to the appropriate treatment with ERCP. When performed within the same session, EUS and ERCP are mutually beneficial to obtain the best technical and clinical outcomes even in complicated cases, thereby minimizing adverse events and treatment failure.