Current oil spill forensic identification of source oils relies upon hydrocarbon biomarkers resistant to weathering. This international technique was developed by the European Committee for Standardization (CEN), under EN 15522–2 Oil Spill Identification guidelines. The number of biomarkers have expanded at pace with technological advances, while distinguishing new biomarkers becomes more challenging due to interference of isobaric compounds, matrix effects, and high cost of weathering experiments. Application of high-resolution mass spectrometry enabled exploration of potential polycyclic aromatic nitrogen heterocycle (PANH) oil biomarkers. The instrumentation showed reduction in isobaric and matrix interferences, allowing for identification of low-level PANH and alkylated PANHs (APANHs). Weathered oil samples, obtained from a marine microcosm weathering experiment, enabled comparison with source oils to identify new, stable forensic biomarkers. This study highlighted eight new APANH diagnostic ratios that expanded the biomarker suite, increasing the confidence for identifying highly weathered oils back to their source oil.The online version contains supplementary material available at 10.1007/s10661-023-10941-3. Despite intensive research, public awareness, and preventive measures, oil spills are still a common occurrence. Oil finds its way into the environment through both natural and anthropogenic causes, including by leakage from pipelines and shipwrecks  have been studied intensively because of their known toxicity, carcinogenic properties, and high concentration within various petroleum oils , using the EN 15522–2 Oil Spill Identification guidelines, is used to identify source oils from weathered samples CEN, . The CENThe biomarker ratios used in the current CEN method include many well-established oil components such as alkanes, terpanes, steranes, sesquiterpanes, phenanthrenes, dibenzothiophenes, benzonaphthothiophenes, fluorenes, and chrysenes. The inclusion of heterocyclic PAHs as biomarkers is not common, although a recent study by Filewood et al.  demonstrThe goal of the current study was to assess the ability to detect trace levels of PANH and APANH compounds, evaluate their stability during weathering, and finally assess their use in biomarker diagnostic ratio analysis. Microcosm studies were set up using weather conditions experienced in the Pacific Northwest marine environment to analyze the effects on heavy and crude oils. The study aimed to fill a current gap in the classic diagnostic ratio analysis methodology by identifying and screening for low-level PANH. This study took advantage of the high specificity of the gas chromatography quadrupole time-of-flight (GC-QToF) mass spectrometry to asses PANH and APANH levels in six petroleum oils before and after artificial weathering. This novel study aimed to enhance the current oil spill forensic tools and allow for higher confidence when identifying the source oil when a spill occurs in the environment.For details regarding purchased reagents, unweathered extraction and sample preparation, microcosm and weathering conditions, weathered oil extraction and sample preparation, and GC-QToF procedure and data collection, see Supplementary Information (SI).Table Detailed explanation on the data processing was presented in previous work by Kwok et al. and Park et al. . The relative percent difference between the weathered and source oil ratios was compared, with a final acceptance as a match being < 59 out of 105 diagnostic ratios flagged as greater than a 14% relative difference  CEN, .In the current study, the artificially weathered oil samples were used as the “spilled oil” and the corresponding unweathered samples were regarded as the “source oil.” The unweathered oils were analyzed prior to weathering with minimal sample loss or change, and the results were taken as a baseline in this study. One goal of this study was to identify new diagnostic ratios that can withstand Canadian West Coast weathering and reduce the number of flagged ratios present in the current classic CEN method. Flagged ratios were determined if the weathered to unweathered ratios had a percent difference greater than 14% CEN, . The weaThe current result can be observed visually in Fig. In support of the findings of Filewood et al. , the addTo enhance the current CEN plus PASH methodology for application of severe weathering spill source identification, additional PANH and APANH biomarkers were assessed for inclusion SI Table . BiomarkA study completed by Zhang et al.  exploredFor a compound to be employed as a biomarker in oil spill diagnostics, one of the favorable characteristics is to show a relatively small change following weathering. The area and height responses of the PANH and APANH compounds were analyzed for their potential in this regard. The stability of the compounds was determined by examining the chromatographic area and height response following extreme weathering in outdoor microcosms. In the current study, a large number of PANHs and APANHs became undetectable. The exception was a group of APANHs listed in Table Early PANH-related research demonstrated that PANHs are derivatives of PAH, with the assumption that PANH and PAH would be affected similarly by weathering  acceptable diagnostic ratio matches Table . Inclusi8 acceptaThe increase in confidence once the additional APANH diagnostic ratios were included can be visually seen in Fig. Internationally accepted diagnostic ratio analysis is the main tool for determination of the source of a spilled oil by comparison with potential source oils. Recent publications enhanced the diagnostic ratio analysis though the inclusion of sulfur homologues, with the concept that additional non-classical biomarkers add to the fingerprint of an oil. In the current study, this concept was expanded further to screen for trace-level PANH and APANH compounds using the sensitivity and specificity of high-resolution GC-QToF analysis. High consideration was given to determine which of the nitrogen-containing compounds were able to survive extreme weathering conditions corresponding to local exposure in the Pacific Northwest marine waters. Those identified as being the most stable were six APANH compounds, i.e., C1-Benzo[a]carbazole, C2-Benzo[a]carbazole, C3-Benzo[a]carbazole, C4-Benzo[a]carbazole, C2-Benzo[c]acridines, and C3-Benzo[c]acridines. These biomarkers are four membered ring structures, resulting in their high stability during environmental weathering processes, with the inclusion of nitrogen adding variability to their classic APAH counterparts. The selected biomarkers formed the basis of eight ratios that were added to diagnostic ratio forensic analysis, enabling confident identification of oil spill source samples even post-extreme weathering of crude oil. Our study focused on a potential oil spill in a marine environment, while future research of these biomarkers will likely seek to determine their behavior in other matrixes, such as sediment and biota.Supplementary file1 (DOCX 205 KB)Below is the link to the electronic supplementary material.


Digital literacy has gained growing importance in the health agenda. However, the level of digital information use and access among Korean older adults is still low at 54%. It is important to understand digital literacy among older adults to provide healthcare and social connectivity, especially during the COVID-19 pandemic. Objective: This study aims to explore the digital literacy and identify barriers to learning and using digital devices among community-dwelling older adults in urban South Korea.A qualitative study was conducted using a semi-structured interview guide according to the DigComp 2.0 framework which emphasizes the competencies for full digital participation in five categories: information, communication, content creation, safety, and problem solving. Our sample consisted of 14 older adults .Participants reported varying competency of using digital devices for search, communication, and self-management of lifestyle and health. They actively sought help from family and community members to troubleshoot issues related to installation, maintenance, setup software or applications. However, they were passive or evasive in the use of digital devices because of concerns about invasion of privacy or personal information. They struggled to use digital devices owing to physical and cognitive changes associated with aging.Our findings depict current state and barriers of digital literacy in urban older adults in Korea.


Bilateral vocal cord paralysis (BVCP) most commonly occurs secondary to iatrogenic injury and/or malignancy, but can also be a consequence of central nervous system (CNS) pathology. We report a case of BVCP secondary to leptomeningeal consequence in the context of unknown primary malignancy. The aim of this report is to promote awareness for BVCP caused by rare CNS pathology and highlight the importance of complete neoplastic and paraneoplastic workups in new-onset BVCP with unclear etiology. Here, we present a case report and review of the literature. A 68-year-old female presented with new-onset BVCP in the context of progressive dysphagia in addition to rectal and urinary incontinence. She underwent an awake tracheostomy. Her infectious and paraneoplastic workups did not identify a cause for her BVCP. Her brain MRI demonstrated enhancement of multiple cranial nerves, spine MRI demonstrated leptomeningeal enhancement, and cerebrospinal fluid (CSF) cytology was positive for metastatic adenocarcinoma. Her functional status was poor and she was deemed ineligible for chemotherapy and transitioned to palliative care. She died three months following her hospital admission. Leptomeningeal metastasis is a rare cause of new-onset BVCP. Airway management remains a critical component in BVCP. The sudden onset of BVCP in the context of generalized neurologic symptoms or cranial nerve deficits should prompt complete neoplastic and paraneoplastic investigation. Bilateral vocal cord immobility arises from restricted vocal fold movement secondary to neurological or mechanical etiology . In termWe report, to our knowledge, the first case report of sudden-onset BVCP secondary to leptomeningeal metastasis in the context of an unknown primary malignancy. This diagnosis of LM was made in the context of both neuroimaging and gold standard CSF cytology. This report highlights the importance of performing a full neoplastic, paraneoplastic, and neurological work in the presence of new-onset BVCP of unknown etiology.A 68-year-old female presented with a two-week history acute-on-chronic exertional dyspnea and inspiratory stridor. Otherwise, she had a subjective history of dysphagia to both solids and liquids and a 40-pound weight loss. Moreover, she reported a six-month history of nausea and vomiting with reduced oral intake. She did report a number of repeated falls and a gradual loss of independent mobility, multiple episodes of rectal and urinary incontinence, and saddle anesthesia in the months prior to presentation. She had a medical history significant for adenoid cystic carcinoma of her lip that was treated with primary surgical resection. She also had a history of uterine serous adenocarcinoma treated four years prior via total abdominal hysterectomy and bilateral salpingo-oophrectomy, in addition to adjuvant radiation and chemotherapy. She was also noted to have unresectable nodal disease, but was not undergoing any active cancer treatment at time of presentation.Her physical examination demonstrated O2 saturation of 92% on one litre of oxygen, intermittent inspiratory stridor, but no overt work of breathing. Flexible nasopharygolaryngoscopy (NPL) demonstrated bilateral vocal cord immobility (BVCI) and intermittent plica ventricularis Figure . Her vocOn admission, her MRI of her lumbar spine was negative for cauda equina syndrome. Her chest x-ray demonstrated hyperinflation and she was started on management for a presumed chronic obstructive pulmonary disease (COPD) exacerbation. Her CT neck and chest did not demonstrate any lesions underlying her BVCI or inspiratory stridor. Her venous blood gases were suggestive of a compensated respiratory acidosis, but her infectious blood work was unremarkable. She underwent an open tracheostomy and panendoscopy. Intraoperatively, she had mobile cricoarytenoid joints and no mucosal lesions were identified. Her investigations for adrenal insufficiency to explain her ongoing weight loss were negative. She had a negative CT chest/abdomen/pelvis, and an unremarkable colonoscopy and esophagoscopy, helping to rule against a paraneoplastic etiology for her BVCI. Moreover, she underwent a diagnostic bronchoscopy that demonstrated bronchial alveolar lavage and right upper lobe biopsy that were negative for malignancy. Her autoimmune workup was negative and her thyroid function was unremarkable.She was subsequently evaluated by the neurology service for this constellation of symptoms and was found to have hyporeflexia and new-onset right ptosis, but otherwise no focal cranial nerve deficits. She was found to have a non-functional swallow and underwent percutaneous gastrostomy tube. Her additional neurological workup included a lumbar puncture with final cytology demonstrating metastatic adenocarcinoma. A brain MRI with gadolinium demonstrated a heterogeneously enhancing lesion centered within the dorsal fourth ventricle and inferior vermis with involvement of the area postrema, in addition to enhancement of multiple cranial nerves Figure . A subseIn collaboration with neuro-oncology service and family, she was deemed ineligible for chemotherapy due to her worsening functional status. She was transitioned to palliative measures and died peacefully two months following her hospital admission.To the best of our knowledge, this is the first case report demonstrating LM, diagnosed via radiographic and cytologic evidence, as a central etiology for new-onset BVCP. As mentioned previously, BVCP can be secondary to a number of etiologies, including malignancy, iatrogenic, and autoimmune disease Table . One preDespite a complete malignancy workup, we were unable to identify a primary malignancy to explain this development of LM, which is consistent with the literature suggesting that LM with an unknown primary malignancy remains a rare clinical entity . One posIn conclusion, BVCP is a rare consequence of LM, especially in the context an unknown primary malignancy. We propose that patients with BVCP and non-specific neurologic symptoms with a history of solid tumor malignancy warrant prompt diagnostic workup with cytological examination and neuroimaging for CNS pathology.


Intestinal epithelial cells (IECs) are a mucosal immune barrier essential to coordinate host–microbe crosstalk. Sepsis is a systemic inflammatory syndrome with dysfunction in multiple organs including the intestine whose epithelial barrier is deregulated. Thus, IECs are a main contributor to intestinal permeability and inflammation in sepsis. Exosomes emerge as a mediator of intercellular and inter-organic communications. Recently, IEC-derived exosomes and their cargoes, such as microRNAs (miRNAs), in sepsis were shown to regulate the expression of proinflammatory mediators in the inflamed gut tissues. It is a compelling hypothesis that these IEC exosomes exhibit their dynamic activity to deliver their functional miRNA cargoes to immune cells in local and distant organs to regulate proinflammatory responses and alleviate tissue injury. Also, epithelial tight junction (TJ) proteins are downregulated on gut inflammation. Some of the IEC miRNAs were reported to deteriorate the epithelial integrity by diminishing TJ expressions in intestines during sepsis and aging. Thus, it is worth revisiting and discussing the diverse functions of IEC exosomes and miRNAs in reshaping inflammations. This review includes both iterative and hypothetical statements based on current knowledge in this field. Sepsis is a serious illness of multiple organ dysfunction caused by systemic infection and dysregulated immune responses . Almost 2, which corresponds to a half of a badminton court  composed of a monolayer of polarized cells work as an elaborate coordinator of mucosal immune responses to foreign microbes . The suron court . Therefovia transfer of their biological cargoes such as proteins, messenger RNAs (mRNAs), or microRNAs (miRNAs). Consequently, source cell-derived exosomes alter target-cell functions to thereby remodel microenvironmental niches in target tissues. The exosomes secreted from different organs including the lungs, liver, pancreas, or intestine during sepsis are thought to systemically traffic to undergo intertissue crosstalk and induce septic pathogenesis and inflammation in distant tissues. During pancreatitis, pancreas-released exosomes enter into the liver to elicit hepatic cells to secrete functional exosomes, which consequently reach alveolar tissues to spread inflammation by activating lung macrophages , to the 3′-untranslated region (3′-UTR) of target genes and repress their expressions. The miRNAs present within cells and exosomes are capable of affecting the expression of specific genes inside target cells and modifying their functions. Thus, it will be of great importance to examine 1) how cells and exosomes alter their expression and composition of functional miRNAs, depending on the progressions of sepsis and aging and 2) how those modifications of cells and exosomes regulate pathologic progressions of systemic inflammation.The miRNAs, which are single-stranded non-coding RNA molecules, fine-tune cellular function by restricting the target-gene expression within target cells ications . The miRIt is unquestionable that sepsis is a syndrome detrimental to people of all ages. Nonetheless, the elderly patients with sepsis are much more vulnerable to morbidity and complications and show 1.3- to 1.5-times higher mortality than younger patients . Accordivia their miR-223, heart damages in sepsis  signaling, which further mediate epithelial exosomes to shuttle antimicrobial peptides and thus attenuate C. parvum infectivity  through lymphatic vasculature and further reach distant organs such as the lungs in the study using a trauma/hemorrhagic shock model  and in vivo (mice) analyses, disruption of the TJ occludin barrier under the inflammatory condition has proven to be because of the roles played by the IEC miR-122a increased by TNF-α  in remodeling TJ proteins of intestinal epithelia and disrupting the epithelial integrity on intestinal inflammation or aging. Cichon et al. have overviewed the miRNAs engaged in regulating the intercellular permeability in both epithelial and endothelial barriers of different tissues, including the brain , a circRNA transcribed from the HIPK3 gene (Long non-coding RNAs (lncRNAs), which are a class of non-coding RNA molecules and >200 nucleotides in length, regulate the gene expression and a variety of cellular processes . Zou et function . Wang aneability . Specifilaudin-1   . In the via both apical and basolateral sides. IEC exosomes in sepsis have been thought to affect sepsis-induced multiple organ dysfunction. Paradoxically, the IEC exosomes apically released during sepsis may contain the miRNAs to possibly exhibit an anti-inflammatory ability to the inflamed gut. In this regard, it can be considered that basolaterally released IEC exosomes may systemically migrate to distant organs and play a role in alleviating the septic inflamed tissues, presumably through transfer of regulatory miRNAs into inflammatory cells. Aging devastates, or synergizes more with sepsis, pathologic symptoms including intestinal permeability and subsequent inflammation. The TJ proteins in IECs are the main target molecules of intestinal disintegrity during sepsis and aging. A group of miRNAs expressed in IECs engage in post-transcriptionally regulating the mRNAs of those targets during sepsis, inflammation, and aging, while some of lncRNAs are capable of nullifying the miRNAs’ function by binding to them beforehand. Further investigations of IEC exosomes and miRNAs would provide an insight into developing efficacious drug-delivery systems using biomaterials , which would be able to help treat sepsis- and age-associated intestinal disorders in the near future.Due to their polarized morphology and distinct positioning, IECs release the exosomes


Despite strong evidence of the neurodevelopmental origins of psychosis, current pharmacological treatment is not usually initiated until after a clinical diagnosis is made, and is focussed on antagonising striatal dopamine receptors. These drugs are only partially effective, have serious side effects, fail to alleviate the negative and cognitive symptoms of the disorder, and are not useful as a preventive treatment. In recent years, attention has turned to upstream brain regions that regulate striatal dopamine function, such as the hippocampus. This review draws together these recent data to discuss why the hippocampus may be especially vulnerable in the pathophysiology of psychosis. First, we describe the neurodevelopmental trajectory of the hippocampus and its susceptibility to dysfunction, exploring this region’s proneness to structural and functional imbalances, metabolic pressures, and oxidative stress. We then examine mechanisms of hippocampal dysfunction in psychosis and in individuals at high-risk for psychosis and discuss how and when hippocampal abnormalities may be targeted in these groups. We conclude with future directions for prospective studies to unlock the discovery of novel therapeutic strategies targeting hippocampal circuit imbalances to prevent or delay the onset of psychosis. Hippocampal dysfunction is a robust feature in the pathophysiology of psychosis , 2. In pThe hippocampal formation comprises several distinct, densely packed, highly connected areas Fig. . The cirTogether, these hippocampal-cortical circuits form a systematic mental representation of accumulated knowledge and experiences, which can be conceptualised as a ‘cognitive map’ . When a The internal circuitry of the hippocampus develops in a complex and lengthy process. Neural cell proliferation, migration, differentiation, and synaptogenesis all begin prenatally and extend through the first years of postnatal life, with synaptogenesis continuing right through to adolescence . The devDuring early life, sensory information in the environment influences the organisation of the brain through effects on synaptogenesis, pruning, and myelination. Development is rapid through the first year of postnatal life; most structures are already established, and the pace of subsequent synaptogenesis slows . A critiSpecific maturational processes are difficult to characterise in vivo , althougDevelopmental changes within the hippocampus are also not uniform. The posterior/dorsal hippocampus, most often associated with memory and spatial learning, gains in volume as a ratio to its size at age four, over 19 years of subsequent development . ConversThis vulnerability of the hippocampus may particularly impact normal development by its dense connectivity. The hippocampus is part of the so-called “rich club” of hub brain regions , with particularly high network centrality and vulnerability to dysfunction , 44. TheHippocampal volume deficits are evident at the first episode of a psychotic disorder (FEP), when the clinical diagnosis is first made, and are most marked in anterior cornu ammonis (CA) regions . These rAbnormal metabolic activity within the hippocampus may precede and drive hippocampal volume loss . ElevateAlterations in hippocampal structure and function have also been associated with psychotic-like experiences in the general population –63  neural activity is crucial for regulating brain excitability and the synchronisation of signalling between disparate cortical regions involved in cognitive functioning . PsychosIn most cortical regions, ~80% of neurons are excitatory glutamatergic pyramidal cells, whereas in the hippocampus these cells represent ~90% of all neurons . Becausepost-mortem studies [post-mortem and in vivo evidence of synaptic deficits in psychotic disorders [In psychosis, reductions in hippocampal PVI density have been found in  studies , 85 and  studies , 78. Theisorders –89, whicisorders . Experimisorders . These risorders , 86, linPVI dysfunction has also been linked to NMDAR hypofunction in psychosis , 93. NMDSingle-photon emission computed tomography (SPECT) and PET have been used to quantify NMDAR density in vivo in patients with psychosis. While the specificity of these radioligands for the receptor remains controversial, one SPECT study reported reduced NMDAR binding in the left hippocampus relative to the whole cortex in unmedicated patients with psychosis, but this deficit was ameliorated in antipsychotic treated patients . A recen1H-MRS). Meta-analysis of 1H-MRS studies in patients with schizophrenia, FEP, and CHR individuals showed elevations in levels of glutamate + glutamine (Glx) in the basal ganglia of schizophrenia and FEP patients, regardless of whether they were medicated [1H-MRS captures more than hippocampal-striatal projections and includes inputs to the anterior hippocampus. If inhibitory signalling is perturbed, one would predict a Glx decrease.Although currently PVI activity cannot be measured in vivo in humans, levels of glutamate and GABA partially reflect E/I processes  and can edicated . Howeveredicated , althougedicated . One muledicated . This isedicated , though 1H-MRS voxel in this region [1H-MRS studies at both 3T and 7T have not found significant alterations in absolute concentrations of GABA in patients with psychosis compared to healthy controls [1H-MRS may be overcome by using PET imaging. A recent study reported lower GABAA α5 receptor availability (highly expressed in hippocampus) only in the subgroup of patients with schizophrenia that were antipsychotic-free [In terms of GABA levels, relatively few studies have investigated these in the hippocampus in patients with psychosis, partly due to the technical difficulties in reliably positioning a GABA-edited s region . Neverthcontrols , 107. Incontrols . Some oftic-free , suggesttic-free .Environmental stressors  are among the strongest risk factors for psychosis . The hipA possible mechanism by which early life stressors may precipitate metabolic pressures on the hippocampus is through its involvement in stress and emotion processing . The hypOxidative stress results from an impairment in the redox cycle of oxygen metabolism. Redox dysregulation occurs when there is an imbalance between reactive nitrogen and oxygen species (ROS), and the capacity of the cells to detoxify the ROS with antioxidant defences, predominantly through the reduction of glutathione (GSH) . ROS arePVIs display a high rate of firing relative to other neurons, and therefore have relatively high metabolic demands . The genpost-mortem examination [Converging evidence links psychosis with increased oxidative stress –138. Schmination .Oxidative stress is difficult to spatially localise in vivo, so instead researchers look for signs of redox dysregulation, with GSH levels as a widely used marker of an impaired antioxidant system. Animal model evidence for an association of reduced GSH levels with hippocampal abnormalities  implicat1H-MRS studies found a persistent pattern of GSH deficits in psychosis [1H-MRS for measuring hippocampal GABA levels, although one study found higher GSH in the medial temporal lobe of FEP patients versus healthy controls [In humans, recent meta-analyses of sychosis , 143, wicontrols . In thiscontrols  and bloocontrols . One stucontrols , whilst controls . Moreovecontrols . Though Post-mortem analyses have found a 70% reduction of PNN in patients with psychosis in the PFC [The effects of oxidative stress in relation to psychosis may be especially important in preadolescence, when they may affect perineuronal nets (PNN)—an extracellular matrix of molecules that enclose neurons and provide them with structural support . Their p the PFC , however the PFC , 154. Cl the PFC . Further the PFC . Collect the PFC .This section will consider how the aforementioned hippocampal circuit abnormalities may be targeted during neurodevelopment to reduce the cascade to a psychotic disorder Fig. .Fig. 3PrOne method for correcting hippocampal hyperactivity may be through targeting glutamate. For example, in a mouse model of psychosis, an mGluR 2/3 agonist prevented ketamine-induced hyper-metabolism of the hippocampus . FurtherA agonists [A α5 receptor subunit modulators are of great interest, due to their relative specificity for the hippocampus and their effectiveness in animal models [Another strategy to redress E/I imbalance is to target the GABAergic system to restore inhibition within the hippocampus. For instance, in the MAM model for psychosis, the experimental transplantation of GABAergic precursor cells into the ventral hippocampus normalised hippocampal and striatal dopaminergic function . Similaragonists  and theragonists . Levetiragonists , consequagonists  and reguagonists . Severalagonists , 167. Ofagonists , 169. Thl models .Sodium Valproate is another anticonvulsant drug often used to treat mania  which haN-acetyl cysteine (NAC) is an antioxidant and a precursor to GSH. Its administration has been shown to instigate a 23% increase in mPFC GSH, improvement of cognitive symptoms and increase in white matter integrity in the fornix  of earlyA meta-analysis of 7 studies found that in patients with psychosis, NAC improved both positive and negative symptoms after 24 weeks of treatment . HoweverIn addition to NAC, other food supplements may reduce the effects of oxidative stress. Sulforaphane is an extract from broccoli that increases peripheral and hippocampal GSH in healthy volunteers , and is Cannabis use is a robust risk factor for psychosis , and thiPreliminary work using CBD as a treatment of psychosis indicates that it is effective in reducing psychotic symptoms, both alone and when used as an adjunct to antipsychotic medications , 209. InIn theory, stress sensitivity could be targeted through interventions that reduce the risk of exposure to early life stressors, or that provide individuals with strategies to cope with stressors when they are active. Environmental enrichment prevented hippocampal hyperactivity in the MAM model for psychosis , and mayAerobic exercise (AE) is one promising candidate intervention that exerts both antidepressant and anxiolytic effects, and improves resilience to stress . PatientAE is effective at reducing symptoms in schizophrenia patients . HoweverInterestingly, acute exercise induces redox imbalance . HoweverPsychosis is heterogeneous and multiple aetiologies may contribute to a common pathophysiology. Individual differences in hippocampal dysfunction in psychosis likely arise from different genetic risk, the extent of environmental stressors and protective factors, recreational drug or medication use, as well as the neurodevelopmental timing. Moreover, symptoms overlap with other psychiatric disorders. The aforementioned causes of dysfunction—stress sensitivity, oxidative stress and E/I imbalance—are therefore likely to play a role in the aetiology of other disorders , 229, soThe prospect of prophylactic treatments is tantalising but attempting to correct for imbalances in immature brains should be approached cautiously to avoid unintended consequences on neurodevelopment. Preventive interventions could be provided at different stages of neurodevelopment Fig. , with vaLongitudinal studies in CHR and other at-risk populations, as well as large community samples, will be critical to mapping maladaptive hippocampal neurodevelopment leading to adverse clinical outcomes. Moreover, hippocampal circuit abnormalities are often first detectable in anterior hippocampal subregions, before dysfunction spreads to surrounding circuits . ConsequSegmentation and functional imaging of the hippocampus will improve as higher field-strength (7T+) scanners become more available. Higher field-strengths also afford the possibility of cortical layer and column analysis , allowinWe have discussed many neuroimaging techniques used to capture early hippocampal abnormalities; these and several emerging technologies, such as optically-pumped magnetoencephalography , or chemTo detect potential markers, the complexity and heterogeneity of psychosis-risk, and how these patterns are divergent from other mental health disorders, it is critical that this multidimensional information is integrated across scales Fig. . This wiImproving our understanding of the role of the hippocampus as a central hub of abnormality in the pathophysiology of psychosis may unlock the development of novel treatments and much-needed preventive interventions. Preclinical models indicate that hippocampal changes that occur before the onset of frank psychosis can be reversible, suggesting that clinical interventions at this premorbid stage in humans might be able to reduce the risk of illness onset.


We discuss practical considerations and limitations of our approaches as well as new tools on the horizon to facilitate the observation of primary cilia in pancreatic islets.Primary cilia are slender cell-surface organelles that project into the intercellular space. In pancreatic beta cells, primary cilia coordinate a variety of cell responses including GPCR signaling, calcium influx, and insulin secretion, along with likely many underappreciated roles in islet development and differentiation. To study cilia function in islet biology, direct visualization of primary cilia by microscopic methods is often a necessary first step. Ciliary abundance, distribution, and morphology are heterogeneous among islet cells and are best visualized by fluorescence microscopy, the tools for which are readily accessible to most researchers. Here we present a collection of fluorescence imaging methods that we have adopted and optimized for the observation of primary cilia in mouse and human islets. These include conventional confocal microscopy using fixed islets and pancreas sections, live-cell imaging with cilia-targeted biosensors and probes, cilia motion recordings, and quantitative analysis of primary cilia waveform in the  Primary cilia are hair-like protrusions on the surface of most vertebrate cells. As a cellular antenna, primary cilia provide a physical platform for receiving and integrating molecular and mechanical signals to stimulate cellular responses –4. In thThere has been growing interest in understanding the ciliary regulation of beta cell function. Key metabolic receptors are concentrated and uniquely accessible on the surface of primary cilia, providing attractive pharmacological targets. The first step and – often – barrier in studying islet cilia is the visualization of ciliary structures in intact pancreatic and islets, particularly in human specimens. While most of the current knowledge of islet cilia distribution, abundance, and morphology comes from fixed sliced tissues or cell lines using one or two axonemal markers, there is increasing need for visualizing multiple ciliary components in three-dimensional (3D) islet specimens as well as real-time observation of cilia dynamics in live cells. Therefore, imaging methods for primary cilia has become an increasingly hot topic of discussion for islet biologists who are working to understand the structure and function of these small organelles. This methods paper summarizes our current approaches to fluorescence imaging of primary cilia in mouse and human islets. We provide notes on specimen preparation, microscope settings, and practical tips for generating consistent and clean cilia images. Sample images are provided in the Results section along with discussions about antibody selection and imaging parameters, as well as limits and pitfalls of each approach. Our methods described herein are adaptable to various fixation and staining conditions and have been validated by collaborator labs, thus should prove broadly reproducible. Finally, while the images shown here were generated on two Zeiss LSM880 and LSM980 inverted confocal microscopes, our methods are compatible with a variety of imaging systems and can be combined with orthogonal imaging techniques such as intravital microscopy or correlative light and electron microscopy.Mouse pancreatectomy and islet isolations were performed using an established protocol , 37 withPost-isolation or shipping arrival, mouse and human islets are cultured at least overnight in normoglycemia to allow recovery of morphology and function. Cilia length and number may be induced by serum starvation or by other alterations in culture conditions –40, but Pancreas sections allow visualization of larger tissue areas and simultaneous examination of endocrine and non-endocrine structures. In contrast to isolated islets, pancreas tissue sections provide an intact surrounding and are less prone to preparation-induced damages to the islet structure. We typically prepare mouse or human pancreas sections by flash-freezing in optimal cutting temperature (OCT) compound and cut into cryosections, or by fixing in 4% PFA, preferably EM-grade (Electron Microscopy Sciences 15710), then serially dehydrating in 10-30% sucrose gradient solution for three consecutive days, and embedding and cutting as frozen thin sections into 5-10 µm thickness. Tissue staining require different incubation time than isolated islets, which should be optimized before each experiment. Thicker tissue sections (50-100 µm or more) may be used but will require optical clearing. For both islets and pancreas specimens, we recommend considering experimental and biological reproducibility and to use samples from donors of varying age and gender, when possible.Fixed stained islets or pancreas sections on glass slides are first visually examined using brightfield transmitted light or differential interference contrast (DIC). After selecting the target area for imaging, switch to fluorescence and excite with the appropriate lasers, keeping laser power to a minimum (<1%) to prevent photobleaching. Primary cilia are diminutive structures but can be easily recognized with clean staining and by a trained eye. For a whole-islet view, low-magnification objectives such as 20x air on the Zeiss LSM880 confocal system is sufficient and appropriate for capturing all cilia within an intact islet. Single-cilium images are best acquired with 63x or 100x water or oil immersion objectives, which produce superior high-resolution images especially when used in combination with the Airyscan detector . Three-dPost-acquisition, whole-islet or pancreas images can be reconstructed in ImageJ and Imaris software, combining z-stacks or creating 3D rendering or maximal intensity projections. Quantitation of cilia number per high-power field (hpf) can be done by manual counting or by computer-assisted programs like ACDC and CiliaQ, open-source software or ImageJ plugins to automate cilia counting –45. WhilOFF) that expresses GFP-tagged somatostatin receptor-3 (SSTR3) under the control of Ins1-Cre in islet beta cells  can introduce photobleaching and phototoxicity issues, as laser excitation can cause fluorophore decay and produce free radicals that damage cell health. We recommend proactively addressing these issues in experimental design, by minimizing laser power and illumination during image acquisition, limiting the time duration to only what is necessary to record the physiologic process, e.g. glucose-stimulated calcium flux or insulin secretion, and reducing sampling frequency, practices that will also help keep imaging file sizes manageable.The primary cilium, contrary to canonical definition, is not static but constantly remodels and moves. This makes time-lapse imaging in live cells imperative to understand primary cilia dynamics in addition to their fixed distribution and morphology. To visualize live beta cell cilia, we generated a constitutive green fluorescent cilia reporter mouse . Speculatively, the heterogeneity in beta cell cilia movements may be another phenotype in the functional beta cell diversity.While primary cilia may lack vigorous or coordinated motion, they are nonetheless highly dynamic organelles that constantly elongate, shrink, remodel, and traffic molecules in and out, and in certain settings the whole axonemal structure has been observed to move to extracellular or intracellular forces , 76, 77.Live-cell sensors for molecules that traffic in and out of the cilium, such as cAMP, calcium, and tubulin make useful cilia imaging markers. The BacMam vector that carries the cADDis can infect both mouse and human islets, albeit with low efficiency and targeting mostly peripheral cells  , 78. Usi tracing Figure 8Recent advances in superresolution (SR) microscopy techniques herald exciting opportunities for the study of islet cilia structure. In immortalized cell lines and non-islet tissues, the use of expansion microscopy (ExM) using polyelectrolyte gels to physically expand the sample up to ~4-fold in the linear dimension has allowed greater spatial resolution, generating ultrastructural details rivaling conventional electron microscopy –81. ThesIn addition to superresolution imaging modalities, the use of correlative light and electron microscopy (CLEM) is another strategy to obtain ultrastructural information on targeted ciliary regions . For panPrimary cilia are an important sensory organelle and a dynamic structure to study by fluorescence microscopy. Emerging data suggest that primary cilia play pleiotropic roles in pancreatic islets including cell-cell communication, paracrine signaling, glucose-regulated calcium dynamics, and many other cellular processes that can benefit from direct visualization by imaging. The techniques summarized here represent our experiences thus-far and can be used as a field guide for those studying cilia morphology and behavior in the context of islet cell function. For both fixed- and live-cell imaging in both mouse and human islet specimens, the use of 3D imaging is important for visualizing cilia throughout the entire islet volume. Other considerations include imaging depth vs breadth, whether the experiment requires subcellular detail or panoramic whole-organ scale, and for live-cell imaging, the choice to prioritize spatial vs temporal resolution. The choice of imaging modality determines what you see; conventional static imaging using antibody-based staining provides a reliable approach to visualize primary cilia in fixed tissues , while tvia Zenodo at https://doi.org/10.5281/zenodo.6687921.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: The MATLAB program code used for cilia waveform autotrace is deposited Ethical review and approval was not required for the study on human participants in accordance with the local legislation and institutional requirements. Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements. The animal study was reviewed and approved by The Washington University School of Medicine Institutional Animal Care and Use Committee.ZL and JC contributed imaging data. LW wrote the cilia autotrace code. All authors wrote and approved the manuscript.This study was funded by NIH grant DK127748 and DK115795 to JWH. Human islets were acquired from the NIDDK-funded Integrated Islet Distribution Program (IIDP) (RRID : SCR_014387) at City of Hope, NIH grant #2UC4DK098085 and the JDRF-funded IIDP Islet Award Initiative. Light microscopy was performed at the Washington University Center for Cellular Imaging (WUCCI), supported by Washington University School of Medicine, The Children’s Discovery Institute of Washington University and St. Louis Children’s Hospital (CDI-CORE-2015-505 and CDI-CORE-2019-813) and the Foundation for Barnes-Jewish Hospital (3770 and 4642).We are grateful to the donors and families that made human pancreatic islets available to our research. SSTR3-GFP parental mice were a kind gift from Brad Yoder Lab at UAB. We thank members of the Hughes Lab for their discussion of the manuscript and our reviewers for critical feedback. Graphic illustrations were created with BioRender.com.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Esophageal squamous cell carcinoma (ESCC) is one of the most aggressive gastrointestinal cancers with high incidence and mortality. Therefore, it is necessary to identify novel sensitive and specific biomarkers for ESCC detection and treatment. Circular RNAs (circRNAs) are a type of noncoding RNAs featured by their covalently closed circular structure. This special structure makes circRNAs more stable in mammalian cells, coupled with their great abundance and tissue specificity, suggesting circRNAs may present enormous potential to be explored as valuable prognostic and diagnostic biomarkers for tumor. Mounting studies verified the critical roles of circRNAs in regulating ESCC cells malignant behaviors. Here, we summarized the current progresses in a handful of aberrantly expressed circRNAs, and elucidated their biological function and clinical significance in ESCC, and introduced a series of databases for circRNA research. With the improved advancement in high-throughput sequencing and bioinformatics technique, new frontiers of circRNAs will pave the path for the development of precision treatment in ESCC. Esophageal carcinoma is an intractable problem worldwide for human health because of its high morbidity and poor prognosis. According to the epidemiological data in 2020, there will be about 604,000 new cases of esophageal carcinoma and 544,000 deaths globally, ranking seventh in the incidence rate and sixth in mortality of cancer overall . EsophagRecently, circular RNAs (circRNAs) in the field of the tumorigenesis and development of tumors have attracted much attention from researchers. As a novel type of non-coding RNAs (ncRNAs), circRNAs are covalently closed loop structures without 5’ caps and 3’ tails, which makes them more stable and RNA exonuclease resistance. However, when circRNAs were first discovered in the virus in 1976 . Until tMost circRNAs are synthesized from precursor mRNAs (pre-mRNAs) through back-splicing and catalyzing by RNA polymerase II. It has been verified that back-splicing are competed with canonical pre-mRNA splicing , suggestCircRNAs biosynthesis was reported to be associated with a variety of factors. The biogenesis of circRNAs is affected by the transcription rate of circRNA-producing genes. Cells with a high transcription rate normally own a higher expression level of circRNAs in neurons than those with slow transcription elongation speed . RNA witThrough different mechanisms, circRNAs are involved in multiply process in various diseases. There are at least four functions of circRNAs which have been effectively proved by a number of studies Fig. .Fig. 2FuMicroRNAs (miRNAs) could directly bind to miRNA response elements (MREs) of target mRNAs by base-pairing manner, inducing mRNA degradation and translation inhibition. Salmena et al. proposed competitive endogenous RNA (ceRNA) hypothesis that miRNAs could interact with all types of RNA contained with MREs, thus resulting in a competitive combination between miRNAs and other types of RNA and creating a ceRNA interaction network . CircRNAAlthough most circRNAs located in the cellular cytoplasm and predominantly function as miRNA sponges, EIciRNAs and ciRNAs expressed in the nucleus contain few miRNAs binding sites and have been identified to engage in the process of parental genes regulation. For instance, circEIF3J and circPAIP2 can enhance the transcription of their parental genes in cis through forming EIciRNA-U1 snRNP complexes and further interacting with Pol II transcription complex at the promoters of parental genes . SimilarAlthough circRNAs were initially classified as non-coding RNAs, most circRNAs are composed of exons and harbor binding sites of ribosomes, indicating they own the potential to be translated into proteins. Even lacking 5’cap and associated cap-binding protein factors, circRNAs could recruit ribosomal 40S subunits to initiate the translation through a special sequence in 5’ non-coding region which was termed as internal ribosome entry site (IRES) . Such IRWAF1/CIP1) to form a ternary complex, thus extinguishing the effect of CDK2 and blocking cell cycle progression [Several pieces of evidence have shown that circRNAs bind specific proteins to arrest their functions. In some cases, circRNAs can act as “protein sponge” to regulate gene expression at both transcriptional and translational level. For example, circACTN4 binds to far upstream element binding protein 1 (FUBP1) and thus preventing FBP interacting repressor (FIR) from interacting with FUBP1 to lower MYC transcription, resulting in tumorigenesis and metastasis of breast cancer . Likewisgression . Similargression .Recently, the research on circRNAs becomes an emerging hot spot, surging online databases have been developed to meet the increasing requirements of information on circRNAs. Here, we reviewed the circRNAs online database and their usage to provide suggestions for future research Table . CircBasThere are multiply aberrant circRNAs in the oncogenesis and progression of ESCC, a significant percentage of which have been demonstrated to involve in various malignant biological phenotypes, such as cell proliferation, death, metastasis, drug resistance Fig. . Below wEmerging studies have reported the dysregulated expression of circRNAs between ESCC tissues and para-carcinoma tissues, suggesting circRNAs were the initiators in ESCC. This was largely accomplished by the development of high-throughput screening technology. CircRNA microarray analysis and RNA-seq are primarily used in circRNAs high-throughput screening. Unlike linear RNAs, the structural uniqueness and low content of circRNAs put forward new challenges. Circular junction sequence analysis is widely used in circRNAs identification. CircRNA microarray analysis just uses the principle of the specific integration of circRNA junction probe and circRNA junction sites to accurately detect the expression of circRNAs in tissue or plasma samples. However, circular junction reads obtained by RNA-seq are less than 0.1%, making the content of effectively detected circRNA very low. Li et al.  revealedThrough those two high-throughput technologies, accumulating evidence has disclosed that the dysregulated expression of circRNA is widespread in ESCC tissue and plasma. Utilizing circRNA microarray analysis, Song et al. identified 2046 differentially expressed circRNAs between 6 pairs of ESCC tissues and para-carcinoma tissues, of which 1148 were significantly up-regulated and 898 were down-regulated in ESCC tissues . Jiang eCircOGDH expression is higher in ESCC cells than in normal esophageal epithelial cells. Functional analysis indicated that circOGDH inhibits the negative effect of miR-615-5p on PDX1 expression, which leads to cell cycle inhibition . Cell diBesides, some circRNAs also act as tumor suppressors in ESCC progression. ITCH can negatively regulate the canonical Wnt pathway through degradation phosphorylated Dvl2 and arrest the oncogene c-Myc expression. Cir-ITCH, generated from ITCH, could enhance the expression of ITCH via sponging miRNAs such as miR-7, miR-17 and miR-214, thereby increasing c-Myc expression to induce cell proliferation and tumor growth . Tumor sEMT is a critical biological process that influences malignant tumor cells migration and invasion, featured by dysregulation of E-cadherin, N-cadherin, cytokeratin and Vimentin. Some signaling pathways related to circRNAs have been verified to participate in the EMT process, such as TGF-β and PI3K/Akt signaling axis. For instance, circLPAR3 was increased in ESCC and served as ceRNA to rescue c-MET suppression from miR-198. Increased expression of c-MET activated the phosphorylation of Akt and MAPK, thereby infecting the RAS/MAPK and PI3K/Akt pathways to stimulate cell invasion and migration . He et aIn addition, circRNAs also regulate cell migration and invasion via other pathways. CircNTRK2 was increased in ESCC tissue and involved in cell metastasis. This effect of circNTRK2 may be related to its interaction with miR-140-3p to weaken its suppression on E2F3 . AnotherAbnormal regulation of programmed cell death is one of the hallmarks leading to carcinogenesis. A growing body of work showed that circRNAs performed an essential role in mediating cell apoptosis. Circ0120816 functions as a ceRNA of miR-1305 to reduce its inhibition of TXNRD1, which can attenuate the pro-apoptotic proteins including Cleaved PARP, Bax and Cleaved Caspase-3 to dampen ESCC cell apoptosis . In addiChemotherapy and radiotherapy remain reliable strategies for ESCC patients to gain long-term survival. Prevailing radioresistance and drug resistance is one of the major obstacles resulting in poor prognosis of ESCC, but the mechanism of acquiring resistance is still unclear. Recently, many reports have confirmed that circRNAs regulated drug resistance by multiple processes. For instance, circVRK1 overexpression led to a significant activation of ESCC cells radiotherapy sensitivity . Circ100Early detection is the key to harbor successful therapies for tumor patients. It is now well established from various studies that many circRNAs may have the potential to become effective tumor biomarkers. As we mentioned above, circRNAs are associated with diverse pathological processes of ESCC. Besides, the characteristics of circRNAs endow them with evident advantages to be novel biomarkers in tumor diagnosis and prognosis. Firstly, circRNAs present high abundance, evolutionary conservation and own a longer half-life existing in exosomes and blood plasma. Meanwhile, they are expressed in a tissue-specific and developmental-stage-specific manner. With the development of RNA sequencing technology and bioinformatics analysis, circRNAs were easily detected and their roles in ESCC were extensively identified. Area under the curve (AUC) in the receiver operating curve (ROC) analysis is an evaluative criterion commonly used in circRNAs prediction performance. CircRNA-141539 and circRNA-6448–14 perform great diagnostic ability as their AUC in ESCC tissues is up to 0.81 and 0.91 , 107. HoAmple evidence indicates that circRNAs were closely related to the clinicopathological characteristics or prognosis of ESCC, such as TNM stage, lymph node metastasis, and overall survival (OS), which may play a key role in monitoring the prognosis of ESCC. Recent research showed that serum exosomal hsa_circ_0026611 expression was correlated with T stage, N stage, and postoperative radiotherapy and chemotherapy. And hsa_circ_0026611 can predict lymph node metastasis as a potential prognostic biomarker . SimilarThe molecular pathogenesis of circRNAs involved in ESCC has been extensively revealed, circRNAs are proved to own the potential to be developed into promising and useful therapeutic targets. Knockdown of certain tumor-promoting circular RNAs or overexpression of some tumor suppressor circular RNAs can effectively reduce tumor volume, weight and block tumor metastasis in vivo. For instance, Zhou et al. identified that circPDE3B exhibits a tumor-promoting role in ESCC. CircPDE3B knockdown reduces tumor growth and the number of lung micrometastatic nodules . As a tuAs the newly emerging endogenous ncRNAs, circRNAs are being given more attention and have become a hot spot in cancer research. CircRNAs generated from pre-mRNAs back-splicing, they are conserved, abundant, stable, and considered as important regulators of multiple physiological and pathological processes. The rapid advances of high-throughput detection technology have allowed a large number of differentially expressed circRNAs to be detected in ESCC tissues. As described above, many studies on circRNAs related to ESCC have provided convincing evidence that circRNAs participate in ESCC cell proliferation, migration, invasion, apoptosis, and therapy resistance. CircRNAs perform highly specifically roles in tissues and cells, making them have the opportunity to become valuable prognostic and diagnostic biomarkers for ESCC. Furthermore, the unique properties of circRNAs on conformation, stability and immunogenicity will be of interest to explore RNA circle-based technologies, which include regulating innate immune responses, serving as sponges of cellular miRNAs and as aptamers to interfere with intracellular processes . The resA growing body of reports underlines the importance of circRNAs in ESCC and its strong correlation with ESCC pathophysiological feature. And a sea of innovative research on the application of circRNAs is already in full swing. However, not a single circRNA-based medical application has been approved so far. Compared with miRNA and lncRNA research, circRNAs research is still at its sunrise and has many challenges and problems need to be solved. For instance, the mechanism of circRNAs biogenesis, translocation and degradation is still largely unknown. More comprehensive understanding of this mechanism may help the development of novel technologies. Additionally, reports on the functions of circRNAs in ESCC mainly focused on miRNA sponges, but the function of binding protein and coding potential of circRNAs still lags behind. In fact, only a small portion of circRNAs potential capability have been identified, so it is imperative to further elucidate whether there are other unknown effects. How to facilitate the translation of circRNAs research to the clinical setting is the ultimate goal at present. Firstly, novel technologies and detection methods are required to settle the problem of circRNAs low abundance in biological samples and further improve the quality and precision of detection. Besides, their application as noninvasive biomarkers will need a large pool of tumor samples for verification. Studies can focus on building combined detection methods to gain better diagnostic value. At last, more clinical statistics are needed to build the appropriate collection methods and cutoff values of specific circRNAs in tumor diagnosis. Looking forward, despite the limitations mentioned above, with the explosion in RNA circle-based technologies and in-depth understanding of the features and functions of circRNAs, this type of non-coding RNAs will unveil its translational relevance in ESCC prevention, diagnosis and treatment.


Epilepsy affects about 70 million people worldwide and in 60% of these patients the origin of epileptic seizures is due to a localized  alteration of the brain. In about 30% of patients with focal epilepsy, drug treatment is ineffective, a condition defined by the International League Against Epilepsy (ILAE) as drug-resistant epilepsy (DRE) .A significant proportion of patients with medically refractory focal epilepsy can benefit from a surgical treatment, which offers the chance of seizure remission in many cases or decrease in seizure frequency and severity , 3. The Unfortunately, in certain situations traditional epilepsy surgery approaches are not possible, which include bilateral or multifocal seizures, involvement of eloquent cortical areas, or with associated surgical comorbidities , 8. HoweRecently, novel pre-surgical diagnostic methods and new surgical approaches have been developed. Further, a precise definition of the epileptogenic network can provide an opportunity for surgery in complex refractory epilepsies. For patients who are not good candidates for resective treatments, the evolution in neuromodulation devices and other non-resective surgical procedures also offer good chances of seizure control and improved quality of life , 9.During pre-surgical evaluation for conditions such as multifocal or bitemporal epilepsy, periventricular nodular heterotopia (PNH), tuberous sclerosis complex (TSC), Rasmussen encephalitis, seizures arising from eloquent areas, the localization of the ictal onset zone encompasses diagnostic challenges that can be overcome by means of advanced neurophysiological and radiological methods. This can allow for the epileptogenic zone (EZ) identification of difficult-to-localize focal epilepsies , 11.Furthermore, such complex forms of intractable focal epilepsies are challenges for resective epilepsy surgery. Therefore, recent enhancements in surgical techniques, such as laser interstitial thermal therapy (LiTT), magnetic resonance-guided focused ultrasound (MRgFUS), radiofrequency thermocoagulation (RF-THC), radiosurgery  can enable surgical treatment for these patients, targeting the EZ even when this is difficult to approach using classical surgical procedures. Moreover, these new techniques can minimize surgical risks, making surgery possible for patients who would otherwise not be candidates due to their comorbidities , 12–14.®), Deep Brain Stimulation (DBS) and Vagus Nerve Stimulation (VNS)  –18.In this Research Topic of Frontiers in Neurology, we have brought together experts in these new areas of epilepsy research, diagnosis, and treatment. This Research Topic provides a balanced collection of eight original research studies, four case reports, one review, and two perspective articles.Wu et al. , provides a systematic overview on the seizure outcome of stereotactic laser amygdalohippocampectomy in 30 consecutive patients with mesial temporal lobe epilepsy (mTLE). They demonstrate that scalp EEG findings  strongly predicted seizure recurrence after surgery.The first article, from Consales et al. , focus on other important indications for laser ablation. They report their experience with six pediatric cases of hypothalamic hamartomas, one case of TSC, and one case of dysembryoplastic neuroepithelial tumor, treated with magnetic resonance-guided laser interstitial thermal therapy (MR-gLiTT). Taken together, their data show that MR-gLiTT is a highly safe and effective procedure for these epilepsy conditions in children.Welch et al. , provide information on the effectiveness of RNS in a 16-year-old male with drug-resistant primary generalized epilepsy with convulsive and absence seizures. This case report demonstrates that bilateral RNS of the centromedian nuclei brought to a complete resolution of the baseline daily absence seizure activity, and to a significant decrease in convulsive seizures.Ma et al. (China) contribute to the Research Topic describing the use of a new method, named spatio-temporal unifying tomography (STOUT), applied to magnetoencephalography (MEG) to locate a deep source in a patient with insular epilepsy. With this case study they demonstrate that MEG STOUT method can allow the epileptologist to perform a stereo-electroencephalographic (SEEG)-guided RF-THC operation in the event of deep sources, achieving a satisfactory therapeutic effect.Dimova and Minkin  enrich this Research Topic reporting on a patient affected by a drug-resistant epilepsy involving limbic structures on the left side and associated to anti-GAD65 autoantibodies positivity. The authors describe how the patient benefited from a combination of immunotherapies and surgical treatment . This case report sheds light on the possibility to consider epilepsy surgery even in patients with complex etiologies.Iimura et al. (Japan) add another contribution to this Research Topic illustrating how subtotal hemispherotomy dramatically improved epileptic spasms (ES) of a 3-month-old patient with Aicardi syndrome (AS). The authors provide intraoperative neurophysiological evidence as a possible explanation of the successful procedure. They hypothesized that electrocorticography HFOs and phase-amplitude coupling of HFOs and slow wave bands before and after subtotal hemispherotomy could be the biomarkers of efficacy of this surgical procedure in AS with ES.In addition to the contribution given by the aforementioned authors with regard to possible surgical treatments in complex epilepsy cases, other authors have provided important inputs on new diagnostic procedures aimed at improving EZ localization in focal epilepsies.Bacon et al. (China). These authors compare two different tools for quantitative analysis of fluorodeoxyglucose positron emission tomography (FDG-PET) images: statistical parametric mapping (SPM) and its computational anatomy toolbox (SPM-CAT). They demonstrate that SPM-CAT is more efficient than SPM in localizing EZ for DRE. Therefore, this paper underlines the importance of quantitative analysis of FDG-PET images as an objective complementary tool to the visual assessment for EZ localization.The series of contributions to this Research Topic on implemented diagnostic methods in epilepsy opens with an original study by Müller et al.  highlight how quantitative EEG analysis can enhance the accuracy of identification of the brain tissue generating epileptiform events. They showed that non-linear intracranial EEG analysis may provide information relevant for surgery planning in temporal lobe epilepsy.Ganti et al. (India) add to this section of the Research Topic their experience on seizure detection algorithms, especially aimed at improving the treatment of non-localizable epilepsies by targeting the thalamus for neuromodulation. They investigated the thalamograms obtained during SEEG evaluation for epilepsy surgery, using a tool based on temporal Generative Adversarial Networks (TGAN). The authors conclude that this approach can be applied to classify electrographic seizure onset patterns or develop patient-specific seizure detectors from implanted neuromodulation devices.Irannejad et al. (USA) about mapping of the seizure onset zone (SOZ) with direct cortical stimulation (DCS) in TLE. They show that targeted mapping of SOZ in low amplitude fast activity patterns can better distinguish seizure generators (true responders) from hyperexcitable nodes that may be involved in early propagation.The section of the Research Topic dedicated to new diagnostic procedures for epilepsies continues with the original study of Suresh et al. (Canada). In their original research they demonstrate a negative association between intraoperative somatosensory evoked potentials amplitude and seizure reduction after VNS implantation. Therefore, they discuss a method for the identification of patients with DRE who are most likely to benefit from VNS.Finally, this section of the Research Topic ends with the contribution of Iannone et al.  describe efficacy and tolerability of add-on treatment with cannabidiol in drug-resistant patients with Dravet syndrome (DS) and Lennox-Gastaut syndrome (LGS). In their systematic review article, Chin et al. (UK) discuss the need for further high-quality international consensus-based treatment guidelines for LGS, DS, and particularly for CDKL5 Deficiency Disorder. In their original research, Li et al. (China) analyze the treatment outcomes of newly diagnosed epilepsy and the risk factors for refractory epilepsy in a population of 466 adult patients. Finally, the last contribution is a perspective article by Ji et al. (China), who performed a meta-analysis on sodium valproate alone or in combination with topiramate (TPM) for treating DRE. They conclude that sodium valproate combined with TPM is more effective than sodium valproate alone.While most contributions to this Research Topic have focused on surgical treatment or enhanced diagnostic procedures of complex focal epilepsies, three contributions tackle the issue of pharmacological therapy for drug-resistant epilepsies that are not eligible to surgery. In conclusion, this Research Topic embraces various aspects of DRE treatment and provides an up-to-date series of experts' opinions on advanced and new treatment options especially for more complex forms of DRE. Moreover, this article collection offers the possibility to increase the knowledge about unmet needs that might enhance DRE therapy. Finally, it stimulates the identification of new research areas to develop in the field of DRE.GD, VC, EL, RA, and TA edited the Research Topic. All authors contributed and validated the editorial.This work was supported by Italian Ministry of Health Grant NET2013-02355313.TA was a consultant and had received consultant fees from NeuroOne, Monteris Medical, and Zimmer Biomet. 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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The purpose of this revision is to sumarize the most important clinical features of the autoimune retinopathies (AIRs)AIRs are a group of inflammatory conditions affecting the retina characterized by progressive unexplained visual loss, abnormalities and contraction in visual fields, photoreceptor and electroretinographic dysfunction, and the presence of circulating anti-retinal antibodies. The pathogenesis of AIR remains unclear and various antiretinal antibodies have been associated to the disease. The diagnosis of AIR is based on a particular clinical presentation along with the detection of serum antiretinal antibodies. Numerous anti-inflammatory therapeutic alternatives have been described for the treatment of AIR, nevertheless there is no consensus on treatment protocol.Because of its association with different types of malignant tumors, the early diagnosis, multi-disciplinary approach and prompt treatment should be warranted. Autoimmune retinopathy (AIR) is characterized by an inflammatory process affecting the retina that leads to photoreceptor dysfunction, scotoma, visual field defect, and acute or subacute vision loss in association with the presence of antiretinal antibodies (ARA).–4 AIR enThere are certain proteins known to have an antigenic capability. Some of them are specific to the retina, such as recoverin, and others are also found in non-retinal tissues . While recoverin and α-enolase are antigens widely associated with AIRs, others such as carbonic anhydrase, transducin-β, Tubby-like protein 1 (TULP1), neurofilament protein, heat-shock protein-70, photoreceptor-specific nuclear receptor, Müller cell-specific antigen, among others, have been suggested as possible retinal antigenic targets., 5–8Evidence suggests that paraneoplastic AIRs can be triggered by molecular mimicry between tumor antigens and these retinal proteins.–7 PrevioThe non-paraneoplastic presentation of AIR is the most frequent. It usually affects individuals in the fifth or sixth decades of life that tend to be younger on average with female predominance compared to paraneoplastic AIR. –4Personal or family history of rheumatological or autoimmune diseases is common in non-paraneoplastic AIR.–4 CAR anThe disease tends to present in one eye initially, then involving the contralateral eye within 4 days to 2 months. AIR usually presents as bilateral and asymmetric and more rarely, remain unilateral.–4, 7 SymThe diagnosis or AIR starts with detection of visual field constriction, central or paracentral scotomas .1–3) Vi–3 Vi1–3)The electroretinogram (ERG) is one of the main diagnostic tests for AIRs because it presents specific abnormal findings depending on which cell is most affected . The ERG classically shows global retinal dysfunction on multifocal and full field modalities, with reduced a and b waves . The ERGThe OCT may reveal decreased macular thickness associated with outer retinal abnormalities such as loss or disruption of the photoreceptor layer and/or the outer nuclear layer .14)14)When AIR is accompanied by cystoid macular edema, fluorescein angiography (FA) may demonstrate leakage in the macular region. –3 FA is Fundus autofluorescence typically reveals a hyperautofluorescent ring in the parafoveal region, correspondent to the loss of inner segment and outer segment (IS/OS) junction and thinning of the outer nuclear layer on the OCT., 14, 15.Controversy surrounding the diagnosis of AIR continues because the differentiation between what is abnormal ARA (true positive) and a nonpathogenic baseline level of nonspecific circulating antibodies  involves repeat testing to monitor levels of ARA. It is important to consider that there could be variability in the detection and quantification of ARA between laboratories. Despite the uncertainties related to ARA, the presence of these antibodies is still considered essential for the diagnosis of AIR . ARA havARA can be detected using the Western-Blot (WB) method, immuno-histochemistry (IH) or ELISA test (enzyme-linked immunosorbent assay)., 17 EachAlthough most experts believe the detection of ARA is a requirement for the diagnosis of AIR, others believe that they are possibly an epiphenomenon related to the disease, without direct pathogenicity., 11, 18 The differential diagnosis of AIR includes hereditary retinal diseases, various retinal degenerative disorders, toxic retinopathies and diseases of the spectrum of the white dots syndrome, multifocal choroiditis, acute zonal occult outer retinopathy (AZOOR), degenerative retinal disorders and uveitis. Besides the clinical aspect, ARA can also be positive in some inflammatory uveitis, such as Vogt-Koyanagi Harada syndrome, sympathetic ophthalmia, Behcet’s disease, and systemic lupus erythematosus, in infectious uveitis such as toxoplasmosis and syphilis, in degenerative diseases such as age-related macular degeneration, and in genetic conditions such as retinitis pigmentosa; this positivity reaches 90% of cases in some series., 5, 16 ADue to the systemic implications, it is important to differentiate paraneoplastic from non-paraneoplastic AIR with the guidance of an ocular oncologist. In paraneoplastic AIR, the reduction of the primary tumor with surgery, chemo or radiotherapy seems to be the best approach, although additional specific treatment to AIR may be eventually necessary.Ocular treatment includes local and systemic immunosuppression. However, short-term ocular management can be initiated with intravitreal and sub-Tenons triamcinolone. This approach does not address the systemic condition, but help confirm the diagnosis before starting systemic long-term management with immunosuppressive therapy. Systemic or ocular corticosteroid therapy is used as a first-line treatment, followed by conventional immunosuppressants in the antimetabolite group, such as mycophenolate and azathioprine, and T-cell inhibitors, such as cyclosporine., 18, 19 Plasmapheresis seems to be more effective when used before visual loss, decreasing the circulating antibodies, and therefore the damage of the photoreceptors., 3 PlasmUsually systemic treatment is utilized over at least one year, however, these different types of medications still produce variable therapeutic responses, even when compared observation alone., 18, 19 Studies have shown that anti-oxidants vitamins supplements such as beta-carotene (for non-smokers), vitamin C, vitamin E (for non-cardiac patients) and lutein seem to provide extra help against retinal degeneration., 19Patient monitoring and follow up should include visual field and full field ERG every 3–6 months until stabilization is reached., 18, 19 The mechanisms underlying AIR pathogenesis are incompletely understood. Clinical presentation is mild and diverse; temporal correlation to neoplastic diagnosis is not clear. Testing can help determine the presence of retinal autoantibodies, but laboratory methods are imprecise and the pathogenicity of ARA is uncertain. Thus, the diagnosis of AIR can be challenging and requires a thorough workup, and the importance of diligent inquiry and investigation to rule out hereditary retinal dystrophies and malignancy cannot be overstated. Treatment is another challenge since these are uncommon entities to which none of the available therapies showed unquestionable superiority, and most of the patients did not recover vision.Further studies depicting cytokines and immunogenetic profiling of each AIR category are needed in order to establish the pathogenicity of ARA, allowing better standardization of treatment regimens as well more accurate assessment of clinical outcomes. Improving diagnostic tools and achieving consensus on specific management could not only preserve, but restore vision for these patients.


Antimicrobial resistance (AMR) is directly driven by inappropriate use of antibiotics. Although the majority of antibiotics (an estimated 80%) are consumed in primary care settings, antimicrobial stewardship (AMS) activities in primary care remain underdeveloped and factors influencing their implementation are poorly understood. This can result in promising stewardship activities having little-to-no real-world impact. With this narrative review, we aim to identify and summarize peer-reviewed literature reporting on (1) the nature and impact of AMS interventions in primary care and (2) the individual and contextual factors influencing their implementation. Reported activities included AMS at different contextual levels . AMS activities being often combined, it is difficult to evaluate them as stand-alone interventions. While some important individual and contextual factors were reported , this review identified a paucity of information in the literature about the factors that support or hinder implementation of AMS in primary care settings. In conclusion, identifying multilevel barriers and facilitators for AMS uptake is an essential step to explore before implementing primary care AMS interventions. Antimicrobial resistance (AMR) is directly driven by inappropriate use of antibiotics ,2. AntibAntimicrobial stewardship (AMS) programs, defined as “coordinated interventions designed to improve and measure the appropriate use of antibiotics”, are developed to tackle the challenge of over-consumption of antibiotics . AMS remWith this narrative review, we aimed to summarize peer-reviewed literature reporting on (1) the nature and impact of AMS activities targeting physicians in primary care and (2) the specific behavioral and contextual factors that influence the uptake of AMS activities in primary care.We searched for peer-reviewed literature in PubMed and Google scholar databases published prior to October 2022. We considered reviews and primary studies published since 2000 in English. Search terms included “Antimicrobial stewardship”, “AMS”, “primary care”, “qualitative”. Regarding selection criteria, we included clinical trials, reviews and meta-analyses that reported AMS interventions in primary care and nursing homes and/or observational quantitative and qualitative studies, clinical trials, reviews and meta-analyses that evaluated factors influencing the implementation of AMS in the community . We excluded studies which targeted a different setting, such as emergency department or hospital. References of included studies were also screened to identify further relevant articles meeting the selection criteria (having inclusion criteria and not having exclusion criteria) of this narrative review. For included articles, reported AMS activities were identified and categorized according to different contextual levels: (1) Individual level behavior change; (2) Collective  level change, and (3) Structural/policy/legal level change as suggested by the Consolidated Framework for Implementation Research (CFIR) .In this narrative review, we discuss 30 studies reporting on factors influencing the implementation of antimicrobial stewardship in primary care. The characteristics of the studies are shown in A multitude of AMS activities targeting physicians, patients in primary care and/or the public have been reported . A 2014 To guide implementation of the broad range of AMS intervention possibilities, the CDC proposed a framework of four core components targeting different contextual levels in 2016: commitment, action for policy, tracking & reporting and education & expertise .In the following paragraphs, we will briefly review the impact of different AMS interventions and detail the factors influencing their implementation by based on the different contextual levels previously defined.Education for patients can be provided passively by the clinician with the help of leaflets and/or more actively in conjunction with education of prescribers on communication skills, which can enable shared decision-making and explore the patients’ values and preferences . Visual Educational interventions targeting patients are a key component in AMS but there is a lack of evidence demonstrating their impact as a stand-alone intervention .According to a qualitative study conducted in France, primary care prescribers believe that patients’ education is part of their duty . DependiSeveral formats of educational sessions target physicians in primary care: lectures part of continuing medical education, online interventions and interprofessional team discussions . In a siRecently, the nationwide implementation of an online communication skills training illustrated the difficulty of reaching high participation rate in a real-life setting. In this randomized trial, only 3% and 1% of GPs completed the first and second educational trainings, which was the main reason explaining the lack of impact of the intervention on antibiotic prescription .A recent qualitative study identified factors affecting the uptake of educational interventions by GPs: first, facilitators: (1) having flexible and relevant learning strategies; (2) having easy to access information, resources and reminders; and (3) creating a heightened awareness about AMR; and, second, barriers: time pressure (mostly linked to patient volume) .Training physicians in communication skills facilitates shared decision making with patients by clarifying misperceptions and presenting available evidence behind benefits and harms of treatment. A meta-analysis showed moderate quality evidence that interventions aiming to facilitate shared decision making and help physicians communicate with patients reduce antibiotic use for acute RTI in primary care . CommuniDelayed prescribing can be a useful strategy particularly when a patient expects to receive an antibiotic. It is a safe strategy which lead to a reduction in antibiotic use in RTI consultations even in high-risk patients compared to immediate antibiotics ,35. InteComputer-assisted clinical decision support tools (CDSS) provide the prescribers an easy and rapid access to information and assist GPs at the point of prescribing. It can do so by creating alerts, proposing antibiotic order sets or by prompting questions to guide the choice, dosage and duration of treatment in the patient’s electronic health record.Under trial conditions and mostly for RTIs in primary care, CDSS induce a marginal to moderate reduction in antibiotic prescribing . Very feBiomarkers of inflammation are elevated in the acute phase response to tissue injury irrespective of its etiology. They can safely guide clinicians prescribing decision by ruling out severe bacterial infections. A recent Cochrane review including 12 trials concludes that point-of-care C reactive protein (POC-CRP) safely reduces antibiotic prescription among patients with acute RTI in primary care . There wA nationwide prospective web-based survey evaluated GP attitudes towards POC-CRP. It showed that GPs would use lower CRP cut-offs to guide prescribing for more severe RTIs than for uncomplicated RTIs. Faced with intermediate CRP results in non-severe patients, GPs preferred to postpone decision on antibiotic prescription by 3 to 5 days rather than to write a delayed prescription . For botIn a study analyzing factors associated with overruling of POC-PCT guidance in the setting of a clinical trial, some GPs characteristics (GP’s number of years of experience [median of eight year among those who did not overrule PCT guidance versus 10 years among those who overruled PCT guidance] as well as GPs working in an urban setting) were associated with antibiotic prescription in spite of low PCT levels, highlighting the general behavioral problem of overprescription by physicians.  or, less often, publicly funded national institutes. The dissemination of guidelines is classically a top-down intervention and is often part of multifaceted interventions . When usOlder GPs might distrust guidelines in favor of their own clinical impression because of their extensive expertise or might have kept more liberal prescribing practices. Additionally, impact of guidelines dissemination might be lower among older GPs because of lower knowledge of current and updated guidelines as shown in the treatment of cardiovascular diseases ,58.The impact, effectiveness, and safety of implementing the CDC core elements  as a whoProvider feedback is a top-down intervention, which does not give extra work to physicians. Usually, such interventions include thousands of physicians. Feedback on prescription habits is usually organized at the level of groups of physician practices sharing a common electronic health record, or by health insurers based on billing data. Personalized feedback interventions are often part of multifaceted interventions . Mixed results were obtained, as some studies showed no reduction in antibiotic prescribing , while oQuality circles (QCs) are made up of 6 to 12 primary healthcare professionals who regularly meet to reflect on and improve their standard practice. Different types of QCs exist, e.g., with or without pharmacists. In Switzerland, the interprofessional QCs approach has shown potential to reduce antibiotic prescribing by GPs in private practice and in nursing homes . These pPublic campaigns are done in many countries to provide information on the appropriate use of antibiotics in outpatients. There is a wide variation in the intensity and type of campaign, some based on paper leaflets or simple internet messages to wide and expensive mass-media campaigns . Most caMost public campaigns seemed to reduce antibiotic use, but there is a lack of evidence demonstrating their impact as a stand-alone intervention ,77,78. MMany governments have national antimicrobial resistance actions plan, which can be very diverse. Seventeen government policy interventions have been described worldwide: most commonly public awareness campaign, guidelines, changing regulations around prescribing and reimbursement. Unfortunately, because of a lack of rigorous evaluations, their impact on antimicrobial use remains unclear. Most of these policies focus on changing the habits of physicians, rather than targeting other healthcare professionals or altering healthcare structures to reduce antibiotic consumption . HoweverStakeholders’ views on different government interventions are rarely described. Notably, a qualitative study in France indicated that GPs prefer government interventions not directly targeting prescribers. GPs rather preferred indirect interventions such as increasing the unit sales price of antibiotics, the restricted reporting of susceptibility tests, or the limitation of the number of molecules available in primary care .Governments should implement specific rules, funding and legislation for the antibiotic dispensing circuit, specifically of unit-dose antibiotics, in community and hospital pharmacies .Overall, this narrative review aimed to identify factors influencing the uptake of AMS activities in primary care. Indeed, several studies showed a low uptake of such activities, which jeopardizes their real-life impact on antibiotics consumption. Several individual, collective and policy AMS activities are usually combined, which makes them difficult to evaluate as a stand-alone intervention. Although there is a paucity of qualitative research on the uptake of AMS interventions in primary care, we still identified some recurrent patterns affecting the implementation of AMS activities. First, the main barrier to successful implementation of most AMS activities is the difficulty to reach primary care physicians. It is mainly due to the heterogeneity of GPs and to time constraints in their daily work, which jeopardizes the uptake of AMs activities. Quality circles seems to be a promising setting to enhance the uptake of AMS activities in primary care. Second, the tight workflow of GPs highlights the need of having flexible, easy-to-access and brief AMS activities. Third, several AMS activities are a unique opportunity of strengthening the physician-patients relationship, mainly through communication skills training and laboratory testing which reduce diagnostic uncertainty and can be used as a facilitator of implementation.We identified some elements affecting the implementation of AMS activities, main barriers being the difficulty to reach primary care physicians and the tight workflow of GPs, while a facilitator being the opportunity to strengthen physician-patients relationship.Failure to take these factors into account can result in stewardship activities resulting in little-to-no real-world impact. A thorough understanding of the individual and contextual factors that drive current behaviors and influence implementation of AMS activities is necessary to inform the systematic, tailored design of approaches to implement AMS in primary care setting.