Dear Sirs,Brain arterio-venous malformations (AVMs) are high-flow cerebral shunt systems that may cause severe intracranial bleeding . Often, Computed tomography (CT) angiography or time-of-flight (TOF) magnetic resonance angiography (MRA) are suited methods for primary diagnosis of brain AVMs. However, to reliably localize all vascular feeders and their architecture, digital subtraction angiography (DSA) is frequently required. DSA is also more sensitive in localizing venous drainage, an important criterion for estimating surgical risk based on the Spetzler–Martin grading system . FurtherIn patients with large brain AVMs, additional to the risk of bleeding, a blood steal phenomenon for the surrounding brain tissue is likely, possibly leading to border zone shifts , 6. ConvIn this case report, we seek to confirm high imaging concordance between DSA and 4D-sPACK for an illustrative case of a patient with a large right-hemispheric AVM. Furthermore, we aim to demonstrate that ssASL-based perfusion maps allow delineation and volumetric quantification of individual brain vascular territories. We herein introduce a novel approach for semi-automated segmentation of ssASL-based perfusion maps, yielding masks of individual perfusion territories that allow the quantification of shifts of perfusion territories.A 25-year-old man presented to the emergency department of a peripheral hospital with acute onset headache, paresthesia of the left hand, nausea and vomiting. The patient reported previous episodes of transient paresthesia of the left-hand and right-sided headaches. A non-contrast head CT and a cerebral CT angiography were performed, which showed a large space-occupying lesion at the level of the right basal ganglia with surrounding abnormal vascular structures. No signs of bleeding were found. The patient was transferred to our hospital for further diagnostic work-up and treatment. Upon arrival, the patient did not show any focal neurological deficits.For therapeutic planning, bi-planar DSA  was performed. Upon injection of an iodine-containing contrast agent into the right ICA, a brain AVM with a nidus of 4.5 × 6 × 8 cm was visualized Figs.  and 2.FiArterial feeders were identified, especially stemming from the right middle cerebral artery, but to a lesser extent also from the right anterior cerebral artery and posterior cerebral artery. The blood flow of the right ICA seemed to be limited to the marked steal of the AVM, contributing very little to the parenchymal perfusion of the right hemisphere. Evaluation of the left ICA by DSA was additionally performed, revealing extensive crossflow to the right-sided brain parenchyma via the Circle of Willis . Venous drainage occurred mostly via superficial veins into the dural sinuses. However, an ectatic deep cerebral drainage vein was also visualized Figs.  and 2.2*-weighted sequence. An extension of the standard protocol with 3D T1-weighted turbo field echo imaging and perfusion sequences was performed. Specifically, whole-brain pseudo-continuous ASL, ssASL, and 4D-sPACK sequences were acquired with labeling of the left and right ICA, using an automated label positioning tool based on TOF-MRA imaging at the neck level [Scanning by MRI was performed on a 3-Tesla system  using a 32-channel head coil and the Spinlab patch (Software release R5.6) to facilitate super-selective imaging. The standard protocol included a 3D fluid attenuated inversion recovery (FLAIR), TOF-MRA, diffusion-weighted, and Tck level . Parametck level , 13.FLAIR imaging revealed a space-occupying right-hemispheric lesion with densely packed central hypointense flow voids. No signs of bleeding were found on T2*-weighted imaging. The flow voids contained an arterial flow signal on TOF-MRA imaging Fig. . After sOverall, there was a high visual concordance between DSA and 4D-sPACK regarding the localization and extent of AVM feeders and the identification of the venous drainage Figs.  and 2. Fwww.fil.ion.ucl.ac.uk/spm; Wellcome Trust Centre, London, UK). Hemispherical shift of perfusion territories was calculated by the fractional volume shifted into the opposite hemisphere, normalized to the volume of the respective hemisphere.Quantitative evaluations of perfusion territory shifts were achieved by segmenting ssASL data using a two-step approach. First, ssASL data were segmented using VINCI . The proposed semi-automated approach comprised thresholding and manual corrections to derive masks of individual vascular perfusion territories based on the supply stemming from the left and right ICA, respectively Fig. . Second,In addition, hemispheric shifts of individual perfusion territory masks were compared against a vascular territory atlas of the frontal circulation , which wDue to its size and supposed functionally eloquent localization, the AVM was not considered for neurosurgical resection. An endovascular treatment approach was also deemed unsafe and with low probability of success. Thus, radiotherapy was recommended as the first-line treatment.In this case report, we show high agreement for the visualization of a large brain AVM between DSA and 4D-sPACK, and introduce an ssASL-based approach for segmentation of vascular territories with quantitative analysis. Perfusion maps derived from ssASL-based imaging may be used to create vessel-specific territorial perfusion masks, an application useful for visualizing border zones and border zone shifts in patients with cerebrovascular pathologies such as AVMs.In the presented case of a patient suffering from a large right-hemispheric AVM, 4D-sPACK was able to correctly identify the relevant arterial feeders and venous drainage of the complex AVM with functionally eloquent location. Furthermore, there was high concordance between DSA and 4D-sPACK regarding the identification of the high degree of shunting of the right ICA, leading to it contributing little to the physiological brain perfusion. As a potential advantage over DSA, 4D-sPACK precisely depicts the pathologically changed flow dynamics, whereas arterial flow in DSA may be potentially changed by the pressure of contrast injection.Owing to its vessel-selective approach together with the segmentation of individual perfusion territories, ssASL-based perfusion maps enable volumetric quantification of perfusion territories and border zones between vascular territories. In the presented case, a high degree of perfusion territory shift was visualized and confirmed by segmentation with quantitative verification in favor of the left ICA, due to the high degree of AVM-induced shunting regarding the right ICA. Thus, mere visual inspection of ssASL data could be markedly enhanced with the herein introduced semi-automated perfusion territory segmentation by allowing quantitative analyses on single-subject as well as group level. Accurate detection of border zone shifts using this technique is feasible and pending further study, even for pathologies where this effect could be less evident.Knowledge of individual perfusion territories is clinically relevant in surgical planning. For instance, the watershed areas known to exist in border zones between vascular territories are particularly prone to ischemic events, especially if they are located distal of an arterial stenosis . AlthougFuture clinical applications of ssASL and 4D-sPACK go beyond imaging of AVMs. Specifically, these MRI-based imaging techniques can be clinically used to follow up and monitor patients with Moyamoya disease without requiring repeated DSA. Furthermore, ssASL and 4D-sPACK could be used to monitor patients with in-stent stenosis after cerebral flow-diverter or stent implantation, providing information on potential consequences for brain perfusion in vessel-selective fashion. The herein proposed imaging methods may also be considered as alternatives to balloon occlusion testing, an endovascular procedure to determine safety of vessel sacrifice. All above-mentioned potential clinical applications of ssASL and 4D-sPACK may profit from the novel segmentation-based analysis approach introduced in this study, which allows objective quantification of changes in perfusion territories. In addition, automated labeling of the left and right ICA, as applied in the presented patient case, can support clinical feasibility and enhances the workflow during ssASL and 4D-sPACK acquisitions.Vessel-selective ASL-based perfusion imaging with segmentation of vascular perfusion territories and time-resolved angiography is a promising approach to evaluate cerebrovascular pathologies like AVMs. The techniques are feasible in their clinical implementation, are non-invasive, and do not require administration of contrast media. Moreover, arterial flow dynamics can be visualized and delineated without dependence on contrast injection pressure, as it is the case for DSA. Furthermore, ssASL-based perfusion imaging with segmentation and quantitative analysis allows reconstruction of vascular territories, an application previously not achievable with other techniques. This method shows promise also for the evaluation of other neurovascular pathologies.


Cancer-specific alternatively spliced events (ASE) play a role in cancer pathogenesis and can be targeted by immunotherapy, oligonucleotide therapy, and small molecule inhibition. However, identifying actionable ASE targets remains challenging due to the uncertainty of its protein product, structure impact, and proteoform (protein isoform) function. Here we argue that an integrated multi-omics profiling strategy can overcome these challenges, allowing us to mine this untapped source of targets for therapeutic development. In this review, we will provide an overview of current multi-omics strategies in characterizing ASEs by utilizing the transcriptome, proteome, and state-of-art algorithms for protein structure prediction. We will discuss limitations and knowledge gaps associated with each technology and informatics analytics. Finally, we will discuss future directions that will enable the full integration of multi-omics data for ASE target discovery. Alternative splicing is an integral transcriptional process that enables the production of multiple isoforms from a single gene , 2. The Cancer-specific isoforms are RNA transcript isoforms exclusively expressed in cancer cells. Because they originate from somatic events (or from genes regulated by a cancer-cell epigenetic program), they are typically not expressed in normal tissue , 20. Whi1. Chemotherapeutic inhibition of splicing factors, such as Sudemycin or other small molecules targeting SF3B1 –27.2. Oligonucleotide therapy to activate or inhibit certain splicing events. Whereas splice-switching oligonucleotides (SSOs) can block access of the RNA splicing machinery to a splice site , a decoy3. Identification of oncogene with splicing-associated therapeutic vulnerability See , lymphom4. Immunotherapy targeting of neoantigen derived from cancer-specific ASEs See  can be uGiven their therapeutic potential, identifying novel ASEs by high-throughput methods is critical to discovering new therapeutic targets for treating cancer patients.Currently, the mining of therapeutic targets is often limited to short-read RNA sequencing analysis of gene-level data without isoform considerations. This can limit the discovery process to a small number of targets, not considering either the protein isoform or its functional consequences. Thus, we posit that integrative multi-omics profiling will be necessary for the effective prioritization of ASE events as therapeutic targets as well as to understand their role in reprogramming the molecular network for cancer drug resistance  The polyadenylated (poly-A) protocol and 2) the Ribosome-depleted protocol . The poly-A method selects RNA transcripts with oligo (dT) primers , which eThere are several bioinformatics methods for analyzing short-read RNA sequencing data. For transcript-level bioinformatics analysis, the relative proportion of the full-length mRNA isoform can be estimated by RNAseq reads aligned to a given reference genome. The analysis can be divided into alignment-based algorithms and pseudo-alignment algorithms. Alignment-based algorithms include RSEM  and HISAHowever, these strategies assume a complete reference of transcript isoform annotation, and missing transcripts may impact the final estimation . Anothere.g., UniProt/SwissProt). Once assembled, these custom databases can be appended to standard  search databases, enabling the identification and quantification of non-standard peptides and proteins .While the RNA-based sequencing approach can identify novel transcripts, a significant portion (~2000) of the novel RNA isoforms lack proof of generating a protein product , 56. TheNumerous efforts have been made to resolve isoform-level protein quantification in human tissues –63. For Despite high splicing diversity reported by transcriptomic studies , 66, in A superior understanding of an exon’s structural properties and functional features of the encoded protein segment will be necessary to optimize therapeutic targets of alternatively spliced exons. Here, we review several computational methods and studies that attempted to perform functional characterization of protein isoforms. ASEs are often enriched in intrinsically disordered regions (IDRs) , regions1. Sequence scoring functions, using the additive and weighted functions to process the input protein sequences and sequence-derived information, such as FoldIndex  and IUPr2. Machine learning-based models trained from experimentally characterized IDRs, such as DisEMBL , DISOPRE3. Meta-predictors, using multiple disorder predictions as inputs to generate the new disorder prediction, such as metaPrDOS , MobiDB- For example, the unique isoforms and their IDRs  are typically enriched in tissue-specific isoforms, resulting in tissue-specific protein-protein interactions   for both long read sequencing and mass spectrometry analysis, obtaining deep coverage remains costly in terms of time and money. Additionally, technical challenges with coverage bias will also need to be addressed. To overcome this issue, the harmonization of different datasets may enhance our ability to discover novel targets. The assumption is a recurring candidate, while suboptimal for detection in certain settings, can then be rescreened by informatics processing or through a targeted mass spectrometry or re-sequencing effort. 2) algorithm prediction of proteoform function will need to be validated by experiments. While we have proposed that mass spectrometry data may be one mechanism to validate the PTMs, the oncogenic role of the region remains to be validated. For trans-acting variants in splicing factors, such as mutations in SF3B1, a genetic mouse model will be necessary to evaluate its effect on malignant transformation . For cisCancer-derived isoforms remains a dark matter in the context of cancer therapy. New omics dataset and structure prediction strategies will be critical for understanding the splicing function as well as optimizing therapeutic development priorities. Additional technologies, such as machine learning, will be essential to automate the integrative discovery platform from RNA-to-Protein-to-Structure-to-Function. In summary, the complete integration of these omics technologies will surely be necessary to facilitate the next generation of small molecule design, immunotherapeutic development, and to overcome therapeutic relapse and drug resistance.TS, BZ, YL, HW, LW, BM, PS, and AK contributed to the writing and review of the manuscript. TS, PS, and AK contributed to the conception of the review paper. All authors contributed to the article and approved the submitted version.The study was funded by American Cancer Society IRG-21-145-25, Moffitt Cancer Center Quantitative Science/Team Science, Moffitt Cancer Center Department of Biostatistics and Bioinformatics Pilot Award. This work has been supported in part by the Biostatistics and Bioinformatics Shared Resource at the H. Lee Moffitt Cancer Center & Research Institute, an NCI designated Comprehensive Cancer Center (P30-CA076292).TS reports a patent for EBD CAR US20220267425A1.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.


Chronic low-grade inflammation has been identified as a key pathway linking stress experience to human health. However, systematic evaluations on the relationship of work stress and immune function are scarce and predominantly based on cross-sectional studies. We performed a systematic review and meta-analysis of prospective studies on associations of working conditions and inflammatory biomarkers.In line with our previously established study protocol and the PRISMA-guidelines, we systematically searched electronic databases for prospective studies on working conditions as well as workplace interventions and inflammatory markers in employees. We classified studies  and performed rigorous risk-of-bias assessments. Studies were summarized qualitatively, and a meta-analysis was conducted.We identified 23 eligible studies (N=16 432) with a broad scope of working conditions and inflammatory markers. For interventional designs, we differentiated between individual-directed/behavioral  and organization-directed/structural interventions. Workplace physical exercise interventions were associated with a decrease in C-reactive protein . For other workplace interventions, ie, mental and organizational/structural, results were inconclusive. Concerning observational studies, dimensions of the job demand–control(–support) model were most frequently investigated, and results showed weak – if any – associations with inflammatory markers.The research base was heterogeneous and high-level evidence was limited. More prospective studies are needed with broader consideration of work stressors and inflammatory markers. For practical occupational health management, exercise interventions are effective measures to reduce chronic low-grade inflammation. Given the profound transformation of work in the age of digitalization, investigations into ramifications for employee health are of crucial importance. There is substantial evidence for associations between exposure to workplace-related stressors and risk of physical as well as mental morbidity, including cardiovascular diseases (CVD), metabolic conditions, depression, etc, and mortality –9. Over In general, the human stress response involves –besides the engagement of the main stress systems  – complex effects on the immune system most importantly with up-regulation of inflammatory pathways and down-regulation of cellular immunity –21. WhilReviews and meta-analyses in the field of psychoneuroimmunology demonstrate a large body of evidence that psychosocial distress affects immunological and inflammatory activity , 30–32. First, conclusive and systematic syntheses of the current knowledge base as well as quantitative aggregation of effects of work-related stress on employees’ chronic low-grade inflammation are scarce. Previous reviews and meta-analyses have focused on associations of psychosocial job stress , 38 and Secondly, collated evidence is lacking with regard to effects of other work exposures – besides the commonly studied psychosocial work factors – on employees’ immune function. With the ubiquitous and ever-increasing use of information and communication technologies (ICT) in the workplace, associated risks of professionals’ stress experience have become a phenomenon of growing scholarly interest. Human interaction with ICT at work is suggested as a potential source of negative psychological and biological sequelae for health and well-being , 41. YetA review based on prospective studies allows for conclusions on a higher level of evidence and for inferences concerning temporal order and direction of effects in the interplay of workplace stressors and inflammatory reactivity as a risk factor to serious long-term diseases . Beyond We conducted a systematic review and meta-analysis to determine the present evidence base on prospective associations between various working conditions and chronic low-grade inflammation in employees. More specifically, we aimed to (i) systematically summarize the current research base and establish quantitative estimations of associations. Furthermore, we sought to (ii) detect studies on ICT use at work and inflammatory markers. Lastly, we aimed to (iii) identify and evaluate workplace-related interventions to decrease inflammation.First, a systematic review protocol was developed and published . The revParticipants (P): adult employees/workers/professionals. Clinical samples with particular diagnoses as well as specific professional groups, like military personnel, athletes, artists, and students were excluded. Exposures/interventions (E/I): all kinds of working conditions and workplace-related interventions, including psychosocial, mental, and physical. There were no a-priori restrictions by type of workplace intervention, meaning all measures aiming at occupational health promotion or well-being on the job, conducted on or off site as well as during or outside working hours, were considered. Studies on environmental hazards, ie, chemical or biological agents and extreme heat, as well as nutritional, pharmaceutical, or nutraceutical interventions were not eligible. Furthermore, we excluded studies on shiftwork and exclusive shiftwork samples  as well as studies on socioeconomic status as exposure. A particular objective of our review were effects of work-related use of digital technologies and media, defined as all electronic devices (hardware), applications (software), and means of communication, such as computers, mobile phones, messaging systems, autonomous systems, etc. Comparators (C): workers not or exposed to a lower extent to working conditions/workplace interventions of interest. Outcomes: pre-defined biomarkers of inflammation within three main categories  measured in blood or saliva . Study design (S): prospective studies with at least one follow-up measure, ie, at least two consecutive measurements of the inflammation outcome. We included observational  and interventional studies, ie, randomized controlled trials (RCT) and non-randomized studies of interventions . Laboratory or simulation studies were not eligible. We included original research articles in the languages English or German published in peer-reviewed journals from 1982 until present. Conference proceedings, study protocols, and theses were excluded.We searched for studies on associations between working conditions and inflammation fulfilling the following PECOS/PICOS-criteria: As primary information source, we conducted a systematic search in five electronic databases, including PubMed/MEDLINE, Embase, PsycINFO, Web of Science, and Cochrane’s CENTRAL. Our search was finalized in November 2020. In addition, we performed citation searching of included studies in Google Scholar (forward search) and hand-searching of reference lists of included studies and relevant reviews (backward search).We developed a four-tier search string comprising a broad spectrum of terms related to the specified PECOS/PICOS elements . The four blocks were linked with the Boolean operator “AND” and within the blocks the terms were combined by “OR”. The screening procedure of retrieved records was conducted in Rayyan . Two revP=participants’ professional characteristics, age, gender, ethnicity, health-related variables, sample size, recruitment method, relevant inclusion/exclusion criteria; E/I and C=type and description of working condition/workplace intervention and comparators, theoretical foundation, and assessment; O=type and assessment of outcomes; statistical analyses, results, and moderators/control of confounders. Where reported, we extracted data from adjusted models for baseline biomarker levels and/or important covariates such as age or sex. After data extraction, professional samples were grouped into occupational settings based on the ILO classification of industries and sectors  extracted data of included studies in a pilot-tested Excel sheet (table S2) that was based on the Cochrane Consumers and Communication Group’s template . In case sectors .Two reviewers  performed standardized risk of bias (RoB) assessments, and systematic evaluations were established after consensus. For RCT, the updated version of the commonly used Cochrane risk-of-bias tool , and for NRSI, ROBINS-I was applied . Observa2 statistic. In case of low heterogeneity, additionally a fixed-effects model was applied. As the majority of studies were based on controlled designs with repeated measurements, we chose an effect size that accounts for pre-post changes in different groups. In particular, we calculated the recommended pretest-posttest-control group effect size dppc2, according to the following formula . Second, we provided a qualitative summary of all included studies in narrative and tabular format. In addition, main results were visualized by means of arrows indicating direction of effects. Third, where possible, we performed quantitative syntheses of sufficiently similar studies. Otherwise, results were summarized narratively for at least two studies within one cluster. A random-effects meta-analysis was conducted utilizing Meta-Essentials . Heterog formula , 57:T=treatment group; C=control group) for RCT and NRSI as well as of the quality of reporting assessment for observational studies are shown in table S3. All RCT were appraised to have “some concerns” regarding their overall RoB. Evaluations for NRSI are presented separately for controlled and uncontrolled studies and ranged from “serious” to “critical” overall RoB. For observational studies, on average 20 of the 33 checklist criteria were reported, leading to a mean summary score of 0.62 (range 0.41–0.70).In the following, results are described first for interventional and second for observational studies. For interventional designs, we further distinguished between individual/behavioral  and organizational/structural interventions.Physical Interventions. We found seven studies assessing effects of workplace physical activity/exercise interventions on inflammatory biomarkers, including five controlled (four RCT) and two uncontrolled studies. With regard to RCT, two studies examined effects of worksite aerobic exercise interventions in laboratory . An additional fixed-effects meta-analysis revealed similar results. Exclusion of one study appraised with “serious” RoB  and organization-directed/structural interventions as well as observational studies.physical interventions (k=7), the majority of studies reported reductions in inflammation-related plasma molecules. These interventions primarily aimed at changing individual behavior by adding physical exercises or activity into employees’ work routine (both on- and off-the-job) and were conducted among both sedentary and manual workers. The qualitative finding was corroborated in our meta-analysis demonstrating a medium to strong negative effect of physical exercise interventions  on employees’ CRP levels . This resonates well with a previous meta-analysis on studies in non-occupational settings showing that exercise training was associated with a decrease in CRP , the study base (k=5) was limited and inconclusive. However, there were indications for changes of inflammation-related processes on intra-cellular level, ie, gene expression . Despite indications of responsiveness of CRP and cytokines to organizational changes  was based on the JDC(-S) model. Results showed predominately null and/or weak associations. However, there were some indications for beneficial functions of job control and workplace social support as well as for sex-related effects. Conclusions of previous reviews are somewhat conflicting: Whereas Nakata  stress perception and regulation –103. ForOur findings should be interpreted in the light of some limitations. By defining the PECOS/PICOS components, we might have excluded relevant aspects. For example, shiftwork is an important risk factor for inflammation, and in our search, we found sound interventional studies in shift worker samples . However, as it is difficult to disentangle effects of working conditions from the effects of circadian misalignment per se on inflammatory markers , we deciFor occupational health management, a holistic approach integrating both individual/behavioral and organizational/structural measures may generate greater benefits for employee health . The repinteractions between both work environment, job content, organizational conditions and individual factors of the workers, such as capacities, needs etc., which may influence health and well-being , funded by the Bavarian Ministry of Science and Arts. MW and DN have been partly funded by the Munich Centre for Health Sciences . The funders were not involved in the study design, the collection, analysis and interpretation of the data, the writing of the report, and the decision to submit the paper for publication.The review is part of the research project “The authors declare no conflicts of interest.


Optoelectric biosensors measure the conformational changes of biomolecules and their molecular interactions, allowing researchers to use them in different biomedical diagnostics and analysis activities. Among different biosensors, surface plasmon resonance (SPR)-based biosensors utilize label-free and gold-based plasmonic principles with high precision and accuracy, allowing these gold-based biosensors as one of the preferred methods. The dataset generated from these biosensors are being used in different machine learning (ML) models for disease diagnosis and prognosis, but there is a scarcity of models to develop or assess the accuracy of SPR-based biosensors and ensure a reliable dataset for downstream model development. Current study proposed innovative ML-based DNA detection and classification models from the reflective light angles on different gold surfaces of biosensors and associated properties. We have conducted several statistical analyses and different visualization techniques to evaluate the SPR-based dataset and applied t-SNE feature extraction and min-max normalization to differentiate classifiers of low-variances. We experimented with several ML classifiers, namely support vector machine (SVM), decision tree (DT), multi-layer perceptron (MLP), k-nearest neighbors (KNN), logistic regression (LR) and random forest (RF) and evaluated our findings in terms of different evaluation metrics. Our analysis showed the best accuracy of 0.94 by RF, DT and KNN for DNA classification and 0.96 by RF and KNN for DNA detection tasks. Considering area under the receiver operating characteristic curve (AUC) (0.97), precision (0.96) and F1-score (0.97), we found RF performed best for both tasks. Our research shows the potentiality of ML models in the field of biosensor development, which can be expanded to develop novel disease diagnosis and prognosis tools in the future. Surface plasmon resonance (SPR)-based optical biosensors are clearly widely used biosensors, due to their affordability and precise binding affinity across biomolecules, allowing high-throughput results for downstream analysis2. SPR biosensors are widely being used in analyzing bio-molecules  and their molecular interactions3.Optical biosensors are getting popularity in multi-dimensional research, ranging from fundamental biological and bio-medical research, to environmental and agricultural monitoring programs4. e.g. diagnose pathogens in bacteremia patients5, leishmaniasis (skin diseases)6, chronic pulmonary aspergillosis7, urinary tract infections8 etc. Among different bindings, DNA hybridization is a significant biological procedure, where two single-stranded, complementary DNA bind together, by forming a double-helix DNA. Due to the ability to detect DNA hybridization in real-time and without any labeling, SPR-based biosensors have been deployed in detecting infectious components or associated DNA-mutations, resulting in hepatitis-B complex, cancer, and numerous congenital disorders9.The study of bio-molecules, especially, DNA, is directly linked over 400 diseases diagnosis and several diagnostic characterisation techniques has been developed based on DNA detection and their binding properties10, autism spectrum disorder11, cancer12, multiple sclerosis13, diabetes14 and mental health15. Vitor and Cleber16 developed an ML model to predict COVID-19 patients’ stay at special care facilities, based on physiological features resulting in a decision system, which showed potential to be applied in several different diseases, with low processing requirements. While most of these studies utilize patients’ physical and physiological data, derived from different biosensors, there are only a few handful studies, focused on developing a highly accurate biosensor. The probable reason can be the unavailability of a large and authentic dataset for developing and testing models with high confidence.While most of these detection and diagnosis works involve biomedical studies, it becomes hectic to work with large datasets, with numerous dependent and independent variables to work on. Integrating machine learning (ML) models will not only leverage the disease diagnosis procedure but also be helpful to develop precise biosensors, designing personalized medications and predicting disease prognostication for susceptible individuals. ML-models are being widely used for predicting numerous diseases, e.g. COVID-1917, used to develop SPR-based biosensors, suitable to develop an ML model for DNA detection and classification. The dataset consists of reflective angles of 632.8 nm light at different thicknesses of gold and DNA-attachment stages, with their corresponding permittivity and permeability scores. The hypothesis we were tested involves, is it possible to detect the presence and absence of DNA (DNA detection) from different reflective angles and associated independent variables and extend the model further to distinguish single and double-stranded DNA (DNA classification)? Generally, according to the law of light, a line perpendicular is drawn between the incident and reflective lights18, and the angles drawn between them are called incident and reflective angles. In the case of SPR, incident light is applied on various surfaces at definite angles and intervals , support vector machine (SVM), k-nearest neighbor (KNN), decision tree (DT), logistic regression (LR) and multilayer perceptron (MLP), and proposed the best-performing model for both tasks. The RF classifier is based on a decent number of decision trees constructed by random data selection24 and disease severity predictions as stated above, to best of our knowledge this current study, in our knowledge, is the first study which explored SPR-based dataset to detect and classify DNA—which open a new horizon at the field of biosensor development. The major contribution of the current study is to explore reflective angles on different gold surfaces of biosensors and utilize these parameters to classify DNA detection and classification tasks. While it is often a laborious and costly job to produce reflective angles for different surfaces and deposited biomolecules, current study set-up a baseline to develop innovative ML-based models for SPR-based bio-sensors, testing and predicting their performances at different parameter regimes. Our model for classifying and detecting DNA will ensure development of highly accurate biosensors, and assist in developing accurate classifiers for disease diagnosis and prognosis. Furthermore, current study wish to contribute to the tremendously expanding SPR-based dataset and relevant techniques, which needs investment from biosensor development communities.Even though, there are numerous studies on DNA detection17. The dataset was generated through SPR technology which is presented in Fig. The dataset, utilized in the present study is collected fromWe have done a few ablation studies to analyze the dataset for model development. We explored the distribution of gold thickness using a tree map and analyzed the variances of gold thickness with average reflective angles, in terms of the different dataset. The ablation study was helpful to detect the low variance within each data point and hyper-parameter optimisation of the models. Finally, a min-max normalization technique was applied for data normalization before applying feature selection approach.25 can be used, e.g. statistical dependency (SD), minimal-redundancy-maximum-relevance (MRMR), random subset feature selection (RSFS), sequential forward selection (SFS), SFFS  etc.26. The non-linear dimensionality reduction approach known as t-SNE is frequently used to visualize large datasets. Natural language processing (NLP)28, speech processing30 are a few of the key uses of t-SNE. In t-SNE feature extraction, Kullback–Leiber (KL) divergence is used to assess divergence within classifiers, followed by gradient descent to minimize the KL divergence. In current study, t-SNE was implemented for clustering the data according to their different stages or conditions.Several feature extraction techniquesAfter feature selection, we applied several ML models individually for each DNA detection and classification model. A flowchart of the work is presented in Fig. Model’s performance is usually measured using classifier’s performance indicators. Accuracy is the key findings, measuring the performance of a model which is the exact to total prediction. Precision and recall are also important parameters. Precision indicates actual positive outcome while recall indicates part of accurate prediction among the positive outcomes.Current research used accuracy, F1-score, AUC and precision scores to compare performances for different models. The area under the curve (AUC), which depends on sensitivity, is provided by the receiver operating characteristic (ROC) curve, which also provides a broad overview of the model. An improved classifier indicates by higher AUC value. We have conducted 10-fold cross-validation and calculated standard error which calculates standard deviation from average scores for a specific evaluation matrix. To visualise, a confusion matrix was constructed to show the outcomes of a classifier. As a result, the possibility of true-negative (TN), true-positive (TP), false-negative (FN), and false-positive (FP) outcomes can be extracted. For both DNA detection and classification model, confusion matrix was also constructed to visualise the performances.RF showed best performing model among all models. So, tuning of RF model was performed. As RF is a meta estimator, it utilizes averaging to increase predicted accuracy and reduce overfitting after fitting numerous decision tree classifiers to distinct dataset subsamples. Sklearn library’s RF model comprises 19 hyper-parameters. The most important hyper-parameters are n-estimators, max depth, max features and max samples. We have conducted experiments with different combinations of hyper-parameters, and found max-depth has significant effect on model performances and we optimised it for the best performance.The overall SPR dataset was subjected to exploratory statistical analysis. As permittivity, permeability and gold thickness play significant roles in SPR sensing, these parameters were taken into consideration and characterizations were done for evaluating SPR performance. The gold thickness of SPR biosensors plays an important role in reflective index measurements. In order to closely inspect the data, we have constructed a treemap in Fig. Further, we explored the gold thickness distribution at different DNA-deposition stages. From Fig. To address the problem of low-variance, we strategically sub-divided our data for two different models, namely DNA detection and classification models and observed the average reflective angle differences for both dataset. From Figs. We have utilised six-different supervised ML models and found RF and KNN achieved the best accuracy scores for both detection and classification models. For every model classes, the performances for DNA classification models were higher than that of DNA classification models. The highest accuracy 0.94 for DNA classification model was observed consistently across RF, DT and KNN models, while LR provides only 0.81 accuracy. the highest accuracy of 0.96 for DNA detection model was observed in both RF and KNN, while slightly low accuracy of 0.95 observed in DT and SVM, followed by 0.93 for MLP and 0.84 in LR. It is evident that, with 10-fold cross-validation, we have found a very low standard deviation (below 0.0001) for each model.In terms of F1-score and precision scores  performed better than several ML models, including neural network, for diabetes prediction39. For current dataset, we incrementally tested numerous ML models and found RF and LR are the best and worst performers respectively. The probable reason can be due to the complex tree-based architecture of RF model, rather than the simple linear model of LR, which leverages RF to work on sophisticated classifiers and a plethora of hyper-parameters. RF uses multiple decision trees to capture non-linear relationships in high-dimensional and imbalanced data, which allows this model to maintain good accuracies40. We also couldn't rule out the performances of other models, especially DT and KNN, which showed consistent performances across different models. For different bio-medical and biosensor-based datasets, DT showed over 0.94 classification accuracies42. So, further study and hyper-parameter optimization are required for both DT and KNN models. Further, after testing different models, we experimented with different hyper-parameters of RF model. We found max-depth parameter is crucial for overall performance, and subsequently adjusted it to get the best output. Hyper-parameter optimisation is often an obvious choice for ML-models to test and customise for different dataset43.Further, we have evaluated different ML models, and found RF’s superior performance compared to DT, SVM, KNN, MLP and LR. RF has been widely utilised in numerous dataset and showed well performances in terms of diabetes patient detectionOverall, all of the models, except LR, got accuracies above 0.89, with true positives and negatives above 0.92. It reflects the SPR data used in the current study is sufficiently large enough to train the models and our feature extraction method works well to differentiate each class. The high accuracy also gives an indication that average reflective angle data can be applicable for both DNA classification and detection problems for future predictive modelling. The reflective angles depend on numerous internal and external factors, e.g. gold thickness, permittivity and permeability, which needed to be considered for future modelling.There are a few limitations of the current study. While t-SNE based feature extraction methods worked well for DNA detection model, our future task urge using other feature extraction methods to get higher scores for DNA classification models as well. Moreover, the size of the current dataset restricted us using only shallow, classical ML models, whereas convolution neural network-based models are an obvious choice for future model development. Also, hyperparameter tuning of the tested models, such as DT, KNN and MLP might give more promising outcomes. Finally, due to lack of SPR-based dataset, we cannot test our model for its wider applicability. So, testing similar model architectures and feature selection models will be utilized on future SPR-based biosensor studies.In order to rank classifiers and feature subsets, we explored SPR-based dataset, using various statistical, machine learning, and visualisation techniques. Based on the available resources, we proposed two innovative models, namely DNA detection and classification models with a view to utilising reflective angles and associated features for high-throughput biosensor development. Our step-by-step experiments of ablation studies, t-SNE net based feature extraction, evaluation of different ML classifiers and proposed RF-based model showed applicability and potentiality of ML models in the field of optical biosensors. Although few limitations still exists, current study set up a baseline for future ML studies in the field of therapeutics and biomedical diagnostics.


As the human immunodeficiency virus (HIV) pandemic is far from over, whether there are subclinical macular changes in HIV-positive patients is something that should not be overlooked. We aimed to apply optical coherence tomography angiography (OCTA) to assess the macular structure and microvasculature changes in patients with HIV without infectious retinopathy.HIV-positive and -negative participants were included and classified into three groups: HIV-negative, HIV-positive, and HIV-positive with microvasculopathy. OCTA parameters regarding macular structure and microvasculature were analyzed.p < 0.05). No differences were found in OCTA parameters between the HIV-positive and HIV-positive with microvasculopathy groups. Retinal, retinal nerve fiber layer-ganglion cell layer-inner plexiform layer (RNFL-GCL-IPL), RNFL, GCL-IPL, and INL thickness showed a negative association with the duration of HIV diagnosis or antiretroviral therapy (ART) . All OCTA microvasculature parameters showed no association with HIV-related clinical variables .Compared with the HIV-negative group, the superficial retinal vessel density (VD) in the parafovea sectors and the whole Early Treatment of Diabetic Retinopathy Study (ETDRS) grid and the choroidal vascularity index (CVI) in the whole ETDRS grid were significantly decreased in the HIV-positive and HIV-positive with microvasculopathy groups (Subclinical macular changes existed in HIV-infected patients without clinical infectious retinopathy. Substructures from inner retinal layers might be associated with HIV infection or ART duration. The human immunodeficiency virus (HIV) pandemic has lasted four decades. Although remarkable achievements have been witnessed in it, it is still far from over . Since uThere were hypotheses about accelerated neuroretinal degeneration because of persistent HIV infection and ART or other factors . For patOptical coherence tomography angiography (OCTA) is a non-invasive instrument possessing an advantage to image retinal and choroidal blood flow and obtain quantitative thickness measurements , 13. BesWe sought to apply OCTA to assess the macular structure and microvasculature changes in patients with HIV, while there were no clinical infectious retinopathy or only asymptomatic cotton wool spots.This prospective cohort study was conducted between August 2019, and October 2019, and approved by the ethics committee of Beijing Youan Hospital, Capital Medical University.Patients with or without HIV infection were initially included. Patients with a history of diabetes mellitus and cardiovascular and cerebrovascular diseases were excluded. After a routine ocular examination of the anterior segment and fundus, eyes without visible ocular abnormalities or with HIV microvasculopathy were also included. HIV microvasculopathy was diagnosed as asymptomatic cotton wool spots by ophthalmoscopy, which could coexist with small intraretinal hemorrhages, microaneurysms, and telangiectasia , 24. TheThe main ocular examinations included slit-lamp biomicroscopy for anterior segment, fundus imaging by Optos Daytona, and axial length by Zeiss IOL Master 500.HIV infection was identified by self-reporting and a previous positive test. For patients with HIV infection, critical HIV-related parameters were collected, including the duration of infection, duration of ART, CD4/CD8, nadir CD4 counts, and blood HIV-RNA.The OCT device  used for macular examination was the same as described in previous studies , 25. It OCTA parameters were automatically calculated. For segmentation errors, manual corrections can be used to fix them. Macular sectors were performed according to the Early Treatment of Diabetic Retinopathy Study (ETDRS) grid, which was also described in our previous study . Within OCTA parameters were stratified into macular microvasculature and macular structural parameters. Microvasculature parameters included the foveal avascular zone (FAZ) from a 3 × 3 mm scan pattern, superficial retinal vessel density (VD), inner retinal VD, and choroidal vascularity index (CVI) from a 6 × 6 mm scan pattern, which also contained quantitative measurements of retinal and choroidal thicknesses. Besides, there were detailed measurements of the retinal nerve fiber layer (RNFL), ganglion cell layer (GCL), inner plexiform layer (IPL), RNFL, GCL-IPL, and INL from the inner retinal layer, as well as photoreceptor-retinal pigment epithelium (PR-RPE). The classification of each OCTA parameter was also described in our previous study .p < 0.05 was considered statistically significant.SPSS 25.0 software was used for all statistical analyses. Mann-Whitney test was used for HIV-related variables between HIV-positive and HIV-positive with microvasculopathy groups. Analysis of variance and chi-square test were used for continuous normal distributed and categorical variables, respectively. Differences in macular parameters between groups were tested by one-way ANOVA and Bonferroni tests. Multivariable linear regression analysis was performed between clinical variables and each OCTA parameter in the whole ETDRS grid. p > 0.05). The duration of HIV infection and blood HIV-RNA were not different between the HIV-positive and HIV-positive with microvasculopathy groups . The ART duration and CD4/CD8 and CD4 levels in the HIV-positive with microvasculopathy group were significantly different from those in the HIV-positive group  , 46 patients with HIV infection , and 20 patients with HIV infection with microvasculopathy . Age, gender, axial length (AL), and signal strength index was not significantly different between the groups (ctively) .p < 0.05), while CVI in the whole ETDRS grid was also significantly decreased in these two groups (p < 0.001)  .p > 0.05). However, the HIV microvasculopathy group showed a significantly higher INL thickness and lower PR-RPE thickness than those of the HIV-negative group   .p < 0.05). All OCTA microvasculature parameters  showed no association with HIV-related clinical variables, including CD4, CD4/CD8, blood HIV-RNA, duration of HIV, and duration of ART (p > 0.05)  .Macular damages in retinochoroid structure and microvasculature were identified by OCTA in patients with HIV infection who were free of infectious retinopathy. Both superficial retinal VD and CVI were significantly decreased in the HIV-positive group and HIV-positive with microvasculopathy groups. INL thickness was increased in the HIV microvasculopathy group. Macular substructures from the inner retinal layer, including RNFL-GCL-IPL, RNFL, GCL-IPL, and INL thickness, were associated with the duration of HIV diagnosis or ART.HIV microvasculopathy manifested as asymptomatic cotton wool spots (CWSs), which could also be called “HIV retinopathy” or “noninfectious retinopathy” , 24. TheThe HIV epidemic is still a challenge in China . OngoingRetinal structural assessments in patients with HIV infection using OCT had been conducted by various studies –37. TherIn this study, various substructures from the inner retinal layers showed a marked negative association with the duration of HIV infection or ART, indicating a degeneration tendency from the persistent HIV infection or ART. It provided a valuable clue for further studies about the relationships between retinal vascular changes and systemic vascular diseases in HIV-positive patients. Such presumed changes could help to monitor or predict systemic vascular diseases in these patients.There are limitations to this study. Although objective macular parameters regarding structure and microvasculature were ample in this study, data about visual function were not involved, such as color vision, contrast sensitivity, multifocal electroretinography, and visual fields. As HIV infection is turning into a type of chronic disease, the presence of many possible systemic confounders could contribute to observed changes. Further trials with longitudinal data and large sample size are warranted.With precise structure and microvasculature measurements, OCTA revealed decreased superficial retinal VD and CVI in patients with HIV infection with microvasculopathy or clinically normal fundus. HIV infection or ART duration could influence the inner retinal layer substructures. The application of OCTA was meaningful in the ocular observation of patients with HIV infection.The original contributions presented in the study are included in the article/The studies involving human participants were reviewed and approved by the Ethics Committee of Beijing Youan Hospital, Capital Medical University (LL-2018-150-K). The patients/participants provided their written informed consent to participate in this study.K-FD and W-BW contributed to the conception of the protocol. K-FD and CC obtained the dataset. K-FD, X-JH, L-YX, and W-JK analyzed the data. K-FD and X-JH contributed to writing the first draft of the manuscript and to the revision and editing of the present version of the manuscript. H-WD and W-BW commented on the manuscript and gave final approval of the version to be published. All authors contributed to the article and approved the submitted version.This study was supported by the Scientific Research Project of Beijing Youan Hospital, CCMU, 2018 (YNKTQN20180201), the National Science and Technology Major Project of China during the 13th Five-Year Plan Period (2017ZX10201101), and the Beijing Excellent Talent Plan (2018000021223ZK04).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.


The Special Issue, “Towards an Understanding of Retinal Diseases and Novel Treatment”, provides comprehensive information on retinal diseases such as glaucoma, age-related macular degeneration (AMD), diabetic retinopathy, retinitis pigmentosa (RP), and others. Globally, these diseases are, among uncorrected refractive errors and cataracts, leading causes of vision impairment. Especially in high-income countries, glaucoma, diabetic retinopathy, and AMD are, for example, more common reasons for vision impairment and blindness. The intention of this Special Issue is to provide insights into current research on these retinal diseases, which have complex pathomechanisms and are not fully understood yet.Glaucoma is one of the leading causes of blindness worldwide, and the aging of our society will lead to a further increase in the number of people affected in the coming years. Vision loss occurs in patients due to the loss of retinal ganglion cells (RGCs) and their optic nerve axons. The main risk factor is increased intraocular pressure (IOP), leading to mechanical stress on the peripapillary sclera and the optic nerve head. However, the exact pathomechanisms of this disease are not yet understood. Among other factors, ischemic processes, excitotoxicity, and immunological mechanisms seem to be involved.Decorin is a small leucine-rich proteoglycan and a natural inhibitor of several members of the transforming growth factor (TGF) family. This proteoglycan is present in the mouse optic nerve and is expressed by optic nerve astrocytes of mice as well as in the human optic nerve head. Decorin could attenuate the remodeling process in the optic nerve head in glaucoma and might be an interesting future therapeutic target . TGF-β2 Additionally, studies indicate an involvement of the WNT/β-catenin pathway, which, e.g., plays are role in cell proliferation and migration as well as apoptosis in glaucoma. The WNT/β-catenin pathway is considered to be involved in the pathophysiology of TM cells and might therefore affect IOP. Through activation of this pathway, cannabinoids could be an interesting therapeutic target for glaucoma in the future, affecting oxidative stress and inflammation .In a glaucoma model, where high pressure was induced by ocular injection of Ad5.MYOC, pattern electroretinography revealed deficits, while RGCs loss did not occur yet. The authors identified changes in glial activation, inceptive neuroinflammation, activation of the complement system, and synaptic loss in the retinae of these animals via RNA sequencing [The review by Al Hussein Al Awamlh et al. discusses if insulin signaling directly contributes to the pathogenesis of glaucoma or is rather a secondary response to the disease. There are trials for the use of intranasal insulin in other neurogenerative diseases. Due to the cross-talk insulin signaling and the various pathogenic events in glaucoma, which still need to be better understood, targeting insulin signaling might be a future therapy for glaucoma . A furthGlaucoma is also associated with glutamate-induced neurotoxicity. Intravitreal injection of N-methyl-D-aspartic acid (NMDA), a glutamate analog, leads to retinal degeneration in rats. Watanabe et al. investigated if this degeneration can be inhibited through metformin treatment. The study showed that metformin protected RGCs and amacrine cells against NMDA-induced excitotoxicity .Muscarinic acetylcholine receptors, belonging to the superfamily of G-protein-coupled receptors, have five subtypes. The review by Ruan and colleagues describes the distribution and role of the individual subtypes in the retina. These receptors could be novel treatment targets, e.g., in glaucoma. The M1 receptor is involved in retinal neuron survival and might be a promising treatment target .AMD is a progressive, neurodegenerative disease of the retina. The reason for the increasing vision loss is the accumulation of drusen between Bruch’s membrane and the retinal pigment epithelium (RPE). This results in undersupply (ischemia) and atrophy of the RPE, which ultimately leads to degeneration of the photoreceptor cells. Despite the knowledge gained so far, the exact pathogenesis of AMD is still unknown. Its complex pathology involves metabolic, functional, genetic, and environmental factors. Hypoxia, oxidative stress, and inflammation are among the contributing factors. Oxidative stress, an overproduction of reactive oxygen species, is involved in many neurodegenerative disorders, including retinal diseases such as AMD. Hence, antioxidants are an interesting treatment approach for AMD. In a study by Tosi et al., the possible anti-oxidative effect of N-acetyl-L-cysteine ethyl ester was analyzed in ARPE-19 cells and rats in comparison to N-acetyl-L-cysteine. In this study, N-acetyl-L-cysteine ethyl ester was more effective than N-acetyl-L-cysteine, e.g., it acted directly by reacting more rapidly with reactive oxygen species . As mentThe WWC protein family is an upstream regulator of the Hippo signaling pathway, and WWC2 plays a crucial role during embryonic and postnatal angiogenesis. In their current study, Brucher and colleagues noted that WWC2 is an essential regulator of ocular angiogenesis in mice, while WWC1 might not be that important in this context .With increasing age, lipofuscin accumulates in the RPE. The accumulation is mostly a result of the incomplete lysosomal digestion of photoreceptor outer segments, which contain high concentrations of docosahexaenoate. Products of docosahexaenoate oxidation exhibit photosensitizing properties, which can account for the photoreactivity of retinal lipofuscin .Diabetic retinopathy (DR) commonly occurs in patients with diabetes. It begins with the early stage of mild non-proliferative DR (NPDR), which is defined by microaneurysms, retinal hemorrhage, intraretinal microvascular abnormalities, and changes in venous diameter. It then progresses to the proliferative DR (PDR) stage, which is characterized by pathological preretinal neovascularization. Additionally, in DR, inflammation is involved, leading to the production of cytokines, such as interleukin-17A (IL-17A). Since the aryl hydrocarbon receptor can halt the production of pro-inflammatory cytokines, enhance the proliferation of T regulatory cells, and suppress Th17 cell differentiation, its agonist VAF347 was investigated in diabetic mice. Systemic application of the agonist led to ablation of pathogenic Th17 cells and IL-17A, making it a therapeutic candidate for diabetes and DR .In an in vitro blood–retinal barrier (BRB) model, based on human cells, treatment with high glucose in combination with the selective P2X7-receptor (P2X7R) agonist 2BzATP was investigated. This generated a BRB breakdown by enhancing barrier permeability. Increased expression of pro-inflammatory cytokines and P2X7R, vascular endothelial growth factor A (VEGF-A), and intercellular adhesion molecule (ICMA)-1 was noted as well as enhanced production of reactive oxygen species. The predicted P2X7R allosteric antagonist DHTS and the validated P2X7R antagonist JNJ47965567 significantly antagonized HG/BzATP-induced damage in this model .The review by Iyer et al. discusses methods of vitreous sampling in DR. The authors describe major proteins and lipid mediators that were detected in diabetic vitreous samples .Inherited retinal diseases are rare disorders caused by genetic anomalies. They include RP, Leber congenital amaurosis, early onset retinal dystrophy, and Usher syndrome. The RP leads to dystrophy of the photoreceptors. A novel mutation in gene PRF1 that causes RP could be identified using family-based whole-exome sequencing. In a Taiwanese family, six snps candidates were identified as RP disease-causing variants, one the novel pathogenic variant PRF1 . VariantSeveral animal models are available to study RP, but there is still a lack of human-representative animal models. In a new rat model for RP, a knockout at lecithin retinol acyltransferase (LRAT) is based on a patient group harboring a homologous homozygous frameshift mutation in the LRAT gene. Visual examination and further evaluations, such as optical coherence tomography measurements and electroretinography, showed characteristics of RP without debris in the subretinal space making this an interesting new model to study RP .All original articles and reviews of this Special Issue deal with various retinal diseases to contribute to a better understanding of pathogenesis and novel treatment options.


Our program implemented East EMWars, a year-long, longitudinal game that added competition to our existing curricular content. We surveyed residents to investigate the impact of gamification in emergency medicine residency training. We hypothesized that residents would report higher levels of motivation, engagement, and challenge with gamification compared to traditional didactics. Furthermore, we hypothesized that residents would exhibit generally positive perceptions about gamification as a learning tool and that it would translate to improved performance on the annual in-training examination.This was a single-center, prospective pre- and post-intervention survey study at a community-based emergency medicine residency program. Given the multiplicity of research questions and inherent nature of educational research, a mixed methods approach was utilized. We utilized nonparametric testing for quantitative data with paired responses pre- and post-intervention. We solicited comments on the post-intervention that were categorized under thematic approach and presented in complete and unedited form in the results.Eighteen (100%) of eligible residents in our program participated in both surveys. There were statistically significant increases in reported levels of motivation, engagement, and challenge with gamification compared to traditional didactic methods. Residents also reported overwhelmingly positive general perceptions about gamification and its broader generalizability and applicability. We did not reach statistical significance in determining if in-training exam scores were associated with our gamification initiative.This study was a first-of-its-kind look into a longitudinal game in an emergency medicine residency program. Although our results are encouraging, medical educators need further research to determine if this increase in motivation, engagement, and challenge will be associated with an increase in examination scores or, more importantly, healthcare outcomes. Theory-based, broader-scale, prospective studies are needed to further explore and help establish these associations and outcomes.The online version contains supplementary material available at 10.1186/s12245-022-00445-1. What really is gamification? Ten years ago, it was unheard of. Now, it is in the armory of many educators across the country. Gamification has been defined as “the use of game design elements in non-game contexts”  or “the Long before we had the terminology, gamification existed in popular culture. The American Airlines’ frequent flyer program began in 1981, rewarding loyal customers with incentives as they ascended the ranks of the program. More recently, gamification has made its way into most other areas of daily life; Starbucks is one example which gives users a platform to earn rewards and a mobile payment method all in one . DespiteAlthough emergency medicine may be leading the way, existing gamification projects are typically single sessions, narrowly focused on a particular content area, and heavily reliant on quiz-style gaming –15. InstThis was a prospective, observational research study designed to explore the perspective of emergency medicine residents in a newly implemented gamified graduate medical education curriculum. Given the multiplicity of research questions and inherent nature of educational research, a mixed methods approach was utilized. Surveys were administered to participants before and after their participation in a longitudinal game, held over a 10-month period between August 2020 and June 2021. The study was approved under exempt review by the AdventHealth Orlando Institutional Review Board.Our study took place in a single-center, community-based, 3-year emergency medicine program in the southeast. Participants were voluntarily enrolled from the pool of 18 emergency medicine residents currently in the program. Participation was 100%. Several measures were taken to avoid any type of coercion to participate in the study.Of note, the implementation of the game was an innovation to our existing curriculum, and as such, participation was required as a normal part of conference attendance and progression through our training program. Participation in this research study, by completing pre- and post-intervention surveys, was completely voluntary, and residents received no incentive to participate or consequence to not participate. For more information about game design, play mechanics, and scoring, see attached , held a Q and A session, and presented the letter of invitation to those residents that expressed interest in participating.Two surveys were distributed to all participants. The pre-intervention survey was distributed prior to the initiation of the game (August 2020) and included questions regarding perceived level of motivation, engagement, and challenge with the current (non-gamified) didactic curriculum as well as their perceptions about the implementation of gamification. The post-intervention survey was distributed after the completion of the game (June 2021) and included questions regarding perceived level of motivation, engagement, and challenge with the elements of gamification that had been added to the curriculum in comparison with the traditional didactic curriculum. This survey also solicited their perceptions about feasibility and generalization of gamification in graduate medical education.Paper surveys were distributed in sealed envelopes with each participant’s name printed on the front. Each survey was uniquely coded so that responses remained anonymous but were matched pre-intervention and post-intervention to the same participant for data analysis purposes. All 36 surveys were printed and sealed in envelopes prior to starting the study. This ensured we were able to pair responses between survey periods but maintain anonymity of each individual resident’s responses. Surveys are attached  and post-intervention (gamified curriculum) survey responses on a 5-point Likert scale. In-training examination percentile scores were self-reported via survey, where second- and third-year residents reported their 2020 ITE percentile score on the pre-intervention survey, while all respondents were asked to report their 2021 ITE percentile score on the post-intervention survey. Open-ended survey questions were summarized using qualitative methods, specifically thematic approach. Given this triangulation model of mixed methods data collection, there existed a possibility for contradiction in quantitative versus qualitative data . EducatiOf the 18 eligible participants, all 18 (100%) completed all required items on both pre-intervention and post-intervention surveys. Among the participants were 6 (33.3%) first-year residents, 6 (33.3%) second-year residents, and 6 (33.3%) third-year residents. The primary objective of the study was to evaluate resident motivation, engagement, and challenge with a longitudinal, gamified medical education curriculum. The secondary objective was to evaluate whether increased participation with this gamified curriculum translated to improved scores on the annual in-training examination.p-value = 0.0028), so we concluded there is a significant increase in resident motivation.For motivation, 13 (72.2%) respondents indicated increased motivation with a gamified curriculum compared to traditional didactics as evidenced by higher scores on a 5-point Likert scale post-intervention paired to pre-intervention responses; 3 (16.7%) reported no difference, and 2 (11.1%) had decreased motivation scores. The Wilcoxon signed-rank test supported rejection of the null hypothesis , so we concluded there is a significant increase in resident engagement.For engagement, 13 (72.2%) respondents indicated increased engagement with a gamified curriculum compared to traditional didactics as evidenced by higher scores on a 5-point Likert scale post-intervention paired to pre-intervention responses; 5 (27.8%) reported no difference, and none had decreased engagement scores. The Wilcoxon signed-rank test supported rejection of the null hypothesis , so we again concluded there is a significant increase in resident challenge. Descriptive statistics are displayed in Table For challenge, 10 (55.6%) respondents indicated increased challenge with a gamified curriculum compared to traditional didactics as evidenced by higher scores on a 5-point Likert scale post-intervention paired to pre-intervention responses, 7 (38.9%) reported no difference, and 1 (5.6%) had decreased challenge scores. The Wilcoxon signed-rank test supported rejection of the null hypothesis , failing to reject the null hypothesis. Subsequent power analysis (0.64) determined that the study was insufficiently powered to detect a difference if one exists. Unfortunately, we were limited by the number of eligible participants, not to mention a myriad of confounding factors that limit the usability of the obtained data.In-training exam percentile scores were self-reported by second- and third-year residents on both pre- and post-intervention surveys. Scores were reported in ranges of 10 percentile points and coded to a 10-point scale . There were a total of 12 respondents, 6 (50%) second-year residents and 6 (50%) third-year residents. Five (41.2%) respondents indicated an increase in ITE percentile scores after intervention, 2 (16.7%) respondents had no change in ITE percentile scores, and 5 (41.2%) respondents indicated an decrease in ITE percentile scores. A paired Lastly, we asked participants several questions regarding their perceptions of gamification on the post-intervention survey. Respondents were asked to rate their level of agreement with several statements on a 1 to 5 Likert scale, from “strongly disagree” to “strongly agree.” Results are summarized in Table Residents also provided summative qualitative feedback through open-ended comments on the post-intervention survey. Their complete responses are included in Table In this prospective, pre- and post-intervention survey, we determined that residents in an emergency medicine program perceive increased engagement, motivation, and challenge in a gamified curricular model compared to traditional, non-gamified, lecture-based didactics. Residents reported overwhelming agreement with positive statements regarding the implementation of gamification in their educational experiences, but our study was not powered to detect any significant difference in in-training examination scores before and after the intervention. Furthermore, there are a multitude of confounding variables affecting examination scores that were unable to be controlled for in this specific study.For over half a century, educational researchers have recognized the need for change in our approach to adult learning . TraditiOver the past decade, the implementation of gamification models into educational curriculum has been met with tremendous success in a variety of settings , 5. SystThe results supported our primary hypothesis that the game would increase learner motivation, engagement, and challenge. Selecting motivation as a measurable variable was important in that we believe it is a necessary precursor to self-directed learning. And the motivation we incur from gamification is the likely reason for its perceived success.Perhaps the most broadly researched macro-theory of motivation is self-determination theory. SDT describes the core incentives of human behavior to reside with the needs for competence, relatedness, and autonomy . Making Although our study was not powered to detect a significant difference in in-training examination scores, SDT supports that increasing levels of motivation, engagement, and challenge will lead to improved learning and outcomes. Further prospective, theory-based research is needed to determine whether gamification in emergency medicine residency programs can lead to improvements in both board examination scores and pass rates as well as patient outcomes.Medical education research, in general, suffers many methodological limitations . Our stuOur main limitation, however, was the lack of power to detect a significant difference in performance on the in-training exam, likely due to small sample size. Even still, there are multiple confounding variables that affect examination scores and would prove difficult to control across a 1-year period separating subsequent in-training exams: other curricular components, quality of conferences, clinical experiences, self-directed learning, and more. Furthermore, we only obtained data on percentile rankings for each respondent (not raw score) which vary from year to year over administration periods and are compared to scores of peers in the same level of training across the country. A future consideration would be to utilize raw scores of in-training exams or, more broadly, pass rates on the ABEM qualifying exam, as the latter is already used in program evaluation. This would likely require a much larger sample size to yield statistically significant differences across exam administration periods.Another potential limiting factor to the generalizability of our results is the intensive time requirement on the part of faculty to set up and conduct the game throughout the year. We estimate that a minimum of 30–50 faculty hours were volunteered throughout the game-play period to tabulate team scores, update leaderboards, and award prizes. This was in addition to time spent designing the game and performing the research component. Implementation of a large-scale game, like ours, would likely necessitate program support for protected faculty time and other resources. Despite these limitations, our study can be used as a basis to design future gamified curricular content and set up future prospective studies on its effectiveness.We performed a prospective, pre- and post-intervention survey study to explore the perspective of emergency medicine residents in a newly implemented gamified graduate medical education curriculum. Our results supported the hypothesis that residents reported increased levels of motivation, engagement, and challenge with gamification compared to traditional didactics. Furthermore, residents overwhelmingly indicated agreement with positively worded statements about the impact of gamification on their educational experience. The study produced insufficient evidence to determine whether in-training exam scores are influenced by exposure to gamification compared to a traditional didactic curriculum.Our study was a first-of-its-kind look into a longitudinal game in an emergency medicine residency program. Although our results are encouraging, medical educators need further research to determine if this increase in motivation, engagement, and challenge will be associated with an increase in examination scores or, more importantly, healthcare outcomes. Theory-based, broader-scale, and prospective studies are needed to further explore and help establish these associations and outcomes.Additional file 1: Appendix A. Game Design.Additional file 2: Appendix B. Play Mechanics and Scoring.Additional file 3: Appendix C. Pre-Intervention Survey.Additional file 4: Appendix D. Post-Intervention Survey.


Fc region-modified anti-CD154mAbs successfully prevent the thrombotic complications reported previously. In this review, we discuss (I) the optimal age of the islet-source pig, (ii) progress in genetic modification of pigs, (iii) the immunosuppressive regimen for pig islet xenotransplantation, and (iv) the reduction in the instant blood-mediated inflammatory reaction.Pig islet xenotransplantation is a potential treatment for patients with type 1 diabetes. Current efforts are focused on identifying the optimal pig islet source and overcoming the immunological barrier. The optimal age of the pig donors remains controversial since both adult and neonatal pig islets have advantages. Isolation of adult islets using GMP grade collagenase has significantly improved the quantity and quality of adult islets, but neonatal islets can be isolated at a much lower cost. Certain culture media and coculture with mesenchymal stromal cells facilitate neonatal islet maturation and function. Genetic modification in pigs affords a promising strategy to prevent rejection. Deletion of expression of the three known carbohydrate xenoantigens  will certainly be beneficial in pig organ transplantation in humans, but this is not yet proven in islet transplantation, though the challenge of the ‘4th xenoantigen’ may prove problematic in nonhuman primate models. Blockade of the CD40/CD154 costimulation pathway leads to long-term islet graft survival (of up to 965 days). Anti-CD40mAbs have already been applied in phase II clinical trials of islet  New altallotransplantation  the optimal age of the islet-source pig, (ii) the potential of genetic modification of the pig, (iii) the selection of the immunosuppressive regimen for pig-to-primate islet xenotransplantation, and (iv) potential steps to reduce the instant blood-mediated inflammatory reaction (IBMIR). We also briefly discuss the possible directions for future research.Based on previous studies of pig-to-NHP islet xenotransplantation, pigs can be divided into three age groups: adult (>12 weeks), neonatal (~first 14 days after birth), and fetal. Their characteristics are summarized in To date, adult pig islets transplanted into NHPs have displayed the longest survival time (965 days) and have always been considered the primary source for islet xenotransplantation due to their superior islet yield, immediate insulin response, lower Gal expression, and higher β-cell percentage compared with neonatal pigs Table 1.in vivo response to glucose after transplantation (that may be >4 weeks in NHPs), and so measuring islet loss is difficult  than adult pig islets (1.75 ± 0.60) , but othIn summary, a consensus on the optimal age for pig islet xenotransplantation has not been reached. Adult pig islets should be the primary option as better results have been achieved following transplantation into NHPs, but NICCs are regarded as a promising alternative islet source with several significant superiorities.The development of CRISPR/Cas9, an efficient genome editing technique, provides the capacity to produce pigs with multiple genetic modifications for xenotransplantation Table 3 A consensus has been reached that the three known carbohydrate xenoantigen genes  should be knocked-out for pig-to-human organ transplantation Table 4,th xenoantigen’ is exposed in TKO pig islets as it is in vascular endothelial cells. Whether TKO islets would provide an advantage in this regard remains uncertain.To our knowledge, the transplantation of TKO pig islets into NHPs has not been reported, and it remains unknown whether the ‘4Of relevance to this point, there were no statistically significant differences in human IgM and IgG binding to isolated islet cells from GTKO/hCD46 and GTKO/hCD46/NeuGcKO pigs . KnockouIf neonatal pigs are the source of islets , in which expression of Gal is considerable, the deletion of expression of Gal (and possibly of Neu5Gc and Sda) will be advantageous.Differences in N- and O-glycan profiles between human and porcine islets might prove to be the next gene modification sites. Novel xenoantigens include complex-type N-glycans with terminal neuraminic acid residues and high-mannose-type N-glycans with core fucosylation . Carbohyhttp://www.functionalglycomics.org/). The National Center for Functional Glycomics (NCFG) (https://ncfg.hms.harvard.edu/) offers a CFG mammalian-type glycan microarray, with 600 glycans present, that might be helpful in studying xenoantigens in the future.For future studies of the 4th xenoantigen(s), several sources might be helpful, e.g., the database of Glycomics  is required . therapy have proved inadequate, although islet graft survival of 222 days has been reported . In contTable 5, 70–76. . Samy et. Here weAnti-CD40mAbs are a chimeric form of Fab combined with IgG Fc fragments to prevent the stimulation of B and T cells through blockade of the CD40/CD154 pathway, which also participates in regulating thrombosis, tissue inflammation, and hematopoiesis . Unlike not in islet transplantation). These included bleselumab (ASKP1240), iscalimab (CFZ533), and BI 655064  without obvious complications  prevents rejection of heterotopic heart and life-supporting kidney allografts in monkeys . This regimen, or a modification of it, has not yet been tested in xenograft modelsOverall, although anti-CD40mAbs have proved successful in pig-to-NHP islet xenotransplantation, the new anti-CD154 agents may prove preferable for clinical trials Table 6 Bikhet et al. published an immunosuppressive regimen that has proved moderately successful in pig solid organ transplantation in NHPs , but sucin vitro , a substantial percentage of islets are lost in the immediate post-transplant period through an inflammatory response termed IBMIR. The loss is significantly greater if the islets are xeno-islets, e.g., pig islets into NHPs and pig islets to human blood in vitro –107. Coain vitro . Severalin vitro , and modin vitro , but nonin vitro , 111–115in vitro .allotransplantation study (NCT00789308)  . Nanopar0789308) , 122. Co0789308) . Islet-s0789308) .Cibinetide   also showed islet-protective effects by reducing IBMIR-induced platelet consumption . Based oKey factors in successfully developing pig islet xenotransplantation include determination of the optimal age of the islet-source pig , the optimal genetic modifications that should be made to the pig, and the optimal immunosuppressive regimen that should be administered to the recipient. Whether the ‘4th’ xenoantigen is problematic in the pig-to-NHP islet transplantation model needs to be clarified. More attention needs to be directed to genetic modifications that might reduce the instant blood-mediated inflammatory reaction and/or the adaptive immune response to pig islets. The advantages and disadvantages of immunosuppressive regimens based on anti-CD40 and anti-CD154 agents require clarification. Since the first case of successful pig-to-human kidney and heart transplantation had been reported recently , 127, weLM, DC, and ZP initiated the review. LM and GS wrote the manuscript. DC, YL, JC, SZ, JD, YH, YN, YZ, and ZC revised the manuscript. All authors contributed to the article and approved the submitted version.This work was supported by grants from the Shenzhen Foundation of Science and Technology (grant numbers GJHZ20200731095207021), the National Key R&D Program of China (2017YFC1103704) and from the Special Funds for the Construction of High Level Hospitals in Guangdong Province (2019).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.