In the past decades, highly innovative treatments in the field of diffuse large B-cell lymphoma (DLBCL) became available in clinical practice. The aim of this study was to assess the cost–benefit relation of third-line interventions in DLBCL from a German payer perspective.Clinical benefit of allogeneic stem cell transplantation , chimeric antigen receptor T cells therapy (CAR T) [tisagenlecleucel (tisa-cel) and axicabtagene ciloleucel (axi-cel)] and best supportive care (BSC) was assessed in terms of median overall survival (median OS) derived from a systematic literature review in PubMed. Real-world treatment costs were retrieved from the university hospitals Cologne and Hamburg-Eppendorf. The cost–benefit relation was analysed using the efficiency frontier concept.Median OS varied from 6.3 months in BSC to 23.5 months in CAR T (axi-cel), while median real-world treatment costs ranged likewise widely from €26,918 in BSC to €340,458 in CAR T (axi-cel). Shown by the efficiency frontier, alloSCT and axi-cel were found as most efficient interventions.The efficiency frontier supports the pricing of innovative therapies, such as third-line interventions in DLBCL, in relation to appropriate comparators. Yet, studies with longer follow-up periods are needed to include studies with unreached median OS and to reflect experiences gained with CAR T in clinical practice. Diffuse large B-cell lymphoma (DLBCL) is the most common malignant sub-type of the non-Hodgkin-lymphoma, reaching an incidence rate of 12.3 per 100,000 inhabitants in Germany in 2017 An enormous amount of research has been conducted in the field of DLBCL in the past decades, leading to highly innovative and promising treatment approaches for clinical practice. For instance allogeneic hematopoietic stem cell transplantation  and chimeric antigen receptor T cells therapy (CAR T) has shown promising clinical improvement for patients with progressive disease who had received two or more lines of systemic therapy before Previous international studies investigated and modelled cost-effectiveness of DLBCL third-line interventions We compared the costs and benefits of third-line interventions in DLBCL by applying the efficiency frontier as health economic evaluation method. This approach is recommended by the German Institute for Quality and Efficiency in Health Care (IQWiG) and intends to assess the relation of incurred treatment costs and clinical benefit for different interventions within one indication Based on the respective labels of the DLBCL third-line interventions approved by the European Medicines Agency (EMA), we identified the treatment options on 2021-03-05: alloSCT, two CAR T treatments (tisagenlecleucel (tisa-cel) [19]CAR T cell therapy, allogeneic stem cell transplantation and best supportive care were respectively combined with the search term diffuse large B-cell lymphoma by the Boolean Operator “AND”. Combinations of treatment options and indication were added by the operator “OR”. For each of the search terms, synonyms, similar concepts and different spellings were defined and inserted by the operator “OR”. If applicable, medical subject headings (MeSH terms) were used. Two researchers conducted the systematic literature review individually to avoid biases. Discrepancies were resolved by discussion.Clinical benefit was measured as median overall survival (median OS) as a primary patient-relevant outcome (PRO). We followed the official IQWiG guidelines for searching publications in bibliographic databases Articles were included if the indication was DLBCL in a third-line setting with EMA approval and guideline recommendation of the European Society for Medical Oncology To quantify treatment costs from a healthcare payers’ perspective, we analysed real-world data of each third-line intervention which patients incurred during the entire hospital stay of cell administration or palliative medical care. Costs were retrieved from the data warehouses of the University Hospital Cologne and the University Hospital Hamburg-Eppendorf. Thereby, we initially searched for the main diagnosis diffuse large B-cell lymphoma, specified by the International Statistical Classification of Diseases code (ICD-10-GM version 2021) C83.3. In a next step, third-line therapies were identified by the operating and procedure (OPS) codes shown in Table Due to the 1-year study period (discharge date from 01-06-2019 to 01-06-2020) and in accordance with the German recommendation on health economic evaluation and the General Methods of the IQWiG, no discounting of the treatment costs was applied In total, 1487 records were identified in PubMed via search term on the reporting date 2021-03-05  receiving third-line intervention in DLBCL were identified Table . The popThe casemix index (CMI), indicating the cases’ resource intensity, ranged from 1.70 in tisa-cel to 14.38 in alloSCT. Considering CAR T products, CMI, length of stay (LOS) and number of side diagnoses were lower in tisa-cel patients. Even though the median LOS was clearly the longest in BSC with 52 days, the maximum LOS of both CAR T products and BSC were comparably long. Median treatment costs  across all cases differed, ranging from € 26,918 in BSC to € 340,458 in axi-cel. The ranges of treatment costs for both CAR T products were largely overlapping. The case-based treatment costs are also displayed in Fig. Combining the results of clinical benefits as measured in median OS and treatment costs based on real-world data, the efficiency frontier for the third-line treatment interventions in DLBCL was plotted Fig. . The treThe medical benefit of the DLBCL third-line interventions has already been extensively investigated in previous studies As part of the treatment costs analysis, attention should be drawn to a particularity of the G-DRG system: The CMI for inpatient treatments reflects the complexity of a treatment and thus mostly treatment costs. In the case of the treatment with both CAR T products, tisa-cel and axi-cel, the low CMI in relation to the high treatment costs can be explained by the fact that the CMI only reflects diagnosis and OPS codes considered in the tariffs. Due to the adaptative nature of the G-DRG system, further cost data need to be acquired and evaluated to adequately consider CAR T in DRG tariffs. The manufacturing and administration process of CAR T, however, is currently remunerated by locally negotiated additional fees at the hospital level. Being reimbursed with around €265,000 (tisagenlecleucel) and €282,000 (axicabtagene ciloleucel) in 2022 The intervention of BSC differs from the other two interventions as not the disease itself is combated but accompanied symptoms or side effects shall be mitigated by a variety of patient-specific medical measures, often leading to a longer LOS. The wide LOS ranges in CAR T products were attributable to patients who received palliative medical care after cell administration.Even though the analysis was carried out in accordance with the highest health economic standards, differences in the underlying study population, previous therapies or allowance regarding bridging therapy have not been taken into account. In particular, the similarity of existing studies comparing CAR T treatments are currently focused by scientific discourse The herein conducted systemic literature review also builds the standard in research to identify, select and critically appraise available knowledge. Considering third-line interventions in patients with DLBCL, the availability of scientific articles reporting a median OS, is very limited for innovative treatments such as CAR T. Potentially leading to a time-lag bias, the review only considered articles as of 2017 to make results of different interventions more comparable. The median OS, however, may be imprecise as an indicator for the long-term survival of patients e.g. if the mortality rate is particularly high shortly after the start of therapy and flattens out in the further course of observation. To further increase the comparability and topicality in determining the median OS in this systematic literature review, only studies after the introduction of CAR T were considered. Real-life cost data of the third-line interventions were also retrieved from a timepoint when the reimbursement of CAR T was already firmly established. Several studies had to be excluded as the median OS has not been reached. Thus, comparative studies with longer follow-ups or updated Kaplan–Meier curves are indispensable to consider studies with currently unreached median OS. This would help to better reflect experience gained in clinical practice while potentially giving a better overview on potentially improved survival or a decrease in side effects. This in turn, may have an impact on resource consumption. In addition, an evaluation of the entire patient journey and across sector boundaries would enable a holistic cost analysis of the therapies and follow-up appointments of DLBCL patients.In the German healthcare context, the Act on the Reform of the Market for Medicinal Products  builds the legal obligation for the benefit assessment of new pharmaceutical in comparison to the appropriate comparative therapy. In case of third-line interventions in DLBCL, no appropriate comparator for CAR T were approved as both commercial CAR T products were granted almost simultaneously. The underlying efficiency frontier, however, can provide support for decision makers by indicating cost-covering and reimbursement in relation to the best currently available third-line intervention in DLBCL. For this purpose, it is also used by the Belgian Healthcare Knowledge Centre (KCE) and the Haute Autorité de Santé (HAS) in France  AND ) OR  AND ) OR  AND ).


Molecular targeting and immunotherapy provide durable responses for advanced lung cancer clinical therapy in many patients. However, the mechanisms of occurrence of progressive disease and resistance to targeted therapy and immunotherapy have not been elucidated. Herein, we report two cases of small cell transformation of non-small cell lung cancer (NSCLC) after targeted therapy or immunotherapy. The first case was a 63-year-old female patient presenting with cough and expectoration. Left lung invasive adenocarcinoma was diagnosed after left lung tumor biopsy. After epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) targeted therapy for almost 2 years, disease progression and symptom aggravation were observed. Pathological and immunohistochemical staining results after biopsy revealed small cell lung cancer (SCLC). The second case was a 75-year-old male patient diagnosed with stage IV squamous cell carcinoma of the lung, who received carboplatin/paclitaxel adjuvant chemotherapy and pembrolizumab treatment with partial response. Disease progression and metastasis occurred within 15 cycles of immunotherapy. Computed tomography revealed a lower left lung tumor. Cytological examination of lung lavage fluid and biopsy under thoracoscope revealed SCLC. In conclusion, histological transformation to SCLC is a potential mechanism of NSCLC resistance to targeted therapy or immunotherapy. During treatment, clinicians should monitor serum tumor markers or genome sequencing, particularly in patients with disease progression, as this may be beneficial for early detection of SCLC transformation. Repeated biopsy can be performed if necessary, and the therapeutic regimen can be adjusted in a timely manner according to the results of molecular pathological tests for personalization and whole-process management. EGFR) is one of the most common driving genes in NSCLC. Progress in molecular biology research has resulted in EGFR-tyrosine kinase inhibitors (EGFR-TKIs) emerging as the first-line treatment for patients with EGFR-mutant cancer. However, the majority of patients inevitably develop cross-acquired resistance after 9-13 months of treatment with first- and second-generation EGFR-TKIs through various mechanisms  level showed that they were abnormal and increased. By careful consultation, the patient had discontinued gefitinib for 5 months and switched to traditional Chinese medicine. By dialogue, resumption of targeted therapy usage with gefitinib was suggested. In August 2021, the patient presented with chest tightness and shortness of breath. CT scan results that were compared with previous 9-month images revealed partial enlargement of the left lung lesion and a large pleural effusion on the left side (A 63-year-old woman with no smoking history was admitted for cough and expectoration in June 2020. A computed tomography (CT) scan revealed a 3.3×2.5 cm mass in the lower left lung and multiple mediastinal lymph nodes  and EFGR-TKI targeted (Case 2) therapy. The common features of both cases were as follows: (1) before the transformation to SCLC, the lesions in both cases were multicentric and imaging results revealed enlarged lymph nodes; and (2) transformation to neuroendocrine SCLC occurred in both cases. Moreover, differences between the two cases were as follows: (1) the patients were of different gender and the male patient had a long history of smoking; (2) the histological types prior to transformation were distinct, whereby Case 1 was lung adenocarcinoma and Case 2 was squamous cell lung carcinoma; (3) different driving genes and molecular changes led to the use of different therapeutic drugs, whereby EGFR-TKI was used for Case 1 and a PD-1 inhibitor was used for Case 2; (4) the patients exhibited distinct therapeutic effects. Case 1 was not sensitive to targeted therapy and showed PD, while Case 2 was sensitive to immunotherapy and stable disease (SD).EGFR-activating mutations. The most common resistance mechanism is a Thr790Met mutation in EGFR, which has been reported in 50-60% of resistant tumor samples . EMT can antagonize the response to targeted therapy in lung cancer by inhibiting BIM  axis to promote the growth of adenocarcinoma with a higher degree of invasion  has been proposed as a mechanism of acquired resistance to EGFR-TKIs in several preclinical models. Deficiency in apoptosis leads to resistance of EGFR-TKIs in NSCLC with ting BIM . Furtherting BIM . In addi of SCLC , 30. Traen NSCLC . Geneticormation . Anotherutations . Moreoveinvasion .EGFR-sensitive mutations  can be used as a marker to predict the transformation from lung adenocarcinoma to SCLC ; Zhanjiang science and technology development special fund Competitive Allocation Project - key projects of disease prevention and control (2021A05145); Provincial Science and technology special fund (“College items+ task list”) project - special topic of basic and applied research (2021A05236).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.


Background: Under ideal circumstances, multiplex molecular respiratory panels can support early all discontinuation of unnecessary antibiotics by facilitating diagnosis of viral infection. Our goal was to identify clinic situations in which a positive respiratory panel was associated with antibiotic de-escalation. We focused on gram-negative antibiotics in recognition of the urgent threat posed by gram-negative resistance. Methods: The sample included hospitalized adults tested by respiratory panel while receiving gram-negative antibiotics at the University of Maryland Medical Center from 2015 to 2020. Only the first respiratory panel performed during hospitalization was included. The primary outcome was the combination of a positive result on respiratory panel indicating detection of a viral pathogen and de-escalation of gram-negative antibiotics. De-escalation was assessed based on antibiotics administered on day 3 after testing and was defined by discontinuation or switch to an agent with a narrower spectrum of activity. Least absolute shrinkage and selection operator (LASSO) regression was used to construct the multivariable logistic regression model. Classification and regression tree (CART) analysis was used to identify subgroups with a higher likelihood of the primary outcome. Results: Of 8,326 patients, 1,462 (17.6%) tested positive by respiratory panel. The most common pathogen was rhinovirus (7.9% of the sample). Gram-negative–targeted antibiotics were de-escalated in 4,456 cases (53.5% of the sample), including 887 patients with a positive result on respiratory panel indicating a viral pathogen . LASSO regression was used to select 12 variables (Table Conclusions: Targeted use of respiratory panel testing may increase the likelihood of an informative result that can drive decision making related to antibiotic use. Our exploratory analysis suggests that respiratory panel testing in the first 2 daysFunding: NoneDisclosures: None


Human consciousness is widely understood to be underpinned by rich and diverse functional networks, whose breakdown results in unconsciousness. Candidate neural correlates of anesthetic-induced unconsciousness include: (1) disrupted frontoparietal functional connectivity; (2) disrupted brain network hubs; and (3) reduced spatiotemporal complexity. However, emerging counterexamples have revealed that these markers may appear outside of the state they are associated with, challenging both their inclusion as markers of conscious level, and the theories of consciousness that rely on their evidence. In this study, we present a case series of three individuals in disorders of consciousness (DOC) who exhibit paradoxical brain responses to exposure to anesthesia. High-density electroencephalographic data were recorded from three patients with unresponsive wakefulness syndrome (UWS) while they underwent a protocol of propofol anesthesia with a targeted effect site concentration of 2 μg/ml. Network hubs and directionality of functional connectivity in the alpha frequency band (8–13 Hz), were estimated using the weighted phase lag index (wPLI) and directed phase lag index (dPLI). The spatiotemporal signal complexity was estimated using three types of Lempel-Ziv complexity (LZC). Our results illustrate that exposure to propofol anesthesia can paradoxically result in: (1) increased frontoparietal feedback-dominant connectivity; (2) posterior network hubs; and (3) increased spatiotemporal complexity. The case examples presented in this paper challenge the role of functional connectivity and spatiotemporal complexity in theories of consciousness and for the clinical evaluation of levels of human consciousness. The quest for a biological understanding of consciousness has emerged as one of the most fundamental scientific pursuits of the 21st century. Many prominent neuroscientific theories of consciousness posit that organized integration of cortical areas is central to the conscious state . In empiIn a recent study  we preseIn the current study, we present a case series of three examples of the paradoxical reconfigurations collected as part of these experiments  and demoThree individuals in a DOC were included in this case series. Individuals in a DOC were included following acquired brain injury . Patients were assessed by a trained experimenter using the Coma Recovery Scale-Revised (CRS-R)  on the dElectroencephalogram data were recorded from a 128-channel EGI Sensor Net using an Amps 400 amplifier , a sampling rate of 1 kHz and vertex reference. Electrode impedance was reduced to below 5 kΩ prior to data collection, as per manufacturer recommendation. Prior to functional connectivity analysis, the raw signal was average referenced and downsampled to 250 Hz. The data were high pass filtered at 0.1 Hz, low-pass filtered at 45 Hz and notch filtered at 60 Hz. Channels with an excessive level of noise were removed prior to average referencing. The signal was epoched into 10 s windows of continuous data and visually inspected by a trained investigator to manually reject signal sections containing non-physiological artifacts. Non-brain channels were removed from the subsequent analysis.All three participants in a DOC underwent an anesthetic protocol, described in our previous work . The firhttps://github.com/BIAPT/Python_Connectivity.We bandpass filtered the signal at the alpha bandwidth (8–13 Hz), as evidence from previous research has implicated this frequency band as providing discriminatory information in consciousness and awareness . Weighteis and js, weighted by the magnitude of the imaginary component of the cross-spectrum 𝒥(Cij):The strength of functional connectivity was estimated using the wPLI, a phase-based measure of functional connectivity . The wPLwhere E{.} denotes the expected value operator and sgn(.) refers to the sign function . The wPLis and js . Non-significant connections were set to 0 for wPLI and 0.5 for dPLI.Both estimates of functional connectivity were controlled for spurious connectivity by performing a surrogate data analysis. Twenty surrogate datasets were generated for each combination of channel pairs Signal complexity was calculated using LZC, which is an estimate of the compressibility and information-richness of a signal . Using tA 38-year-old female was admitted to the intensive care unit due to severe headache. The computer tomography scan presented severe intraventricular hemorrhage in lateral third and fourth ventricles with ventriculomegaly and hydrocephalus due to a ruptured right frontal periventricular arteriovenous malformation. Data acquisition was conducted 23 days post-injury, when the patient scored 5 on the Glasgow Coma Scale. At this time, the patient did not present new intra or extra-axial hemorrhage and did not show any significant interval change in the size and configuration of the supratentorial ventricular system. At the day of recording, the patient was classified to be in an UWS, determined by a CRS-R score of 5 . Within In response to propofol anesthesia, this participant exhibited a strong increase in global feedback-dominant connectivity in both hemispheres , as showA 75-year-old female was admitted to the intensive care unit due to severe headache. The computer tomography scan revealed a diffuse subarachnoid hemorrhage with predominant infratentorial distribution and intraventricular extension and hydrocephalus  due to a ruptured left posterior inferior cerebellar artery aneurysm. She also presented a mild right frontal subdural pneumocephalus and an unruptured saccular aneurysm of the cavernous segment of the left internal carotid artery. She suffered a right cerebellar infarct 5 days after admission. In addition to the brain injury, the patient suffered from hypertension and dyslipidemia. Data acquisition was conducted 10 days post-injury. The patient was classified to be in an UWS, determined by a CRS-R score of 5 . The patDue to a large bandage in the right frontocentral region, 24 channels were removed in addition to non-brain electrodes prior to analysis see . Due to In response to propofol anesthesia, this participant exhibited a strong increase in global feedback-dominant connectivity in the left hemisphere . FocusinDuring the post-anesthetic state, the anesthetic-induced changes in functional connectivity and complexity were reversed and reapproached the Baseline level see . InteresR = 6)  . Three mR = 6) .In response to propofol anesthesia, this participant exhibited a strong increase in global feedback-dominant connectivity in the left, but not the right hemisphere . FocusinMany attempts have been made to link neural mechanisms to different states of consciousness. Studies across multiple species and multiple laboratories have accumulated evidence that the disruption of large-scale network organization is associated with disrupted consciousness during sleep , generalTo date, the disruption of feedback-dominant connectivity  has been one of the top candidate biomarkers of anesthetic-induced unconsciousness . In contpost-hoc analysis on the directionality of network hubs , which has previously been implicated in studies of consciousness . It is pThird, this case series only includes female individuals in DOC. Whereas sex and gender is known to influence theoretical and practical pharmacokinetics of propofol , no workCumulatively, the paradoxical reconfiguration of brain networks of DOC patients undergoing exposure to anesthesia prompts a re-evaluation of two of the top candidate biomarkers of consciousness level: connectivity and complexity. Accounting for these counterexamples may be a source of insight and guidance toward the identification of generalizable biomarkers that underpin human consciousness.stefanie.blain-moraes@mcgill.ca.Data will be made available upon reasonable request. Requests to access these datasets should be directed to SB-M, The studies involving human participants were reviewed and approved by the McGill University Health Center Research Ethics Board (15-996-MP-CUSM). Written informed consent was not provided because participants were unresponsive at the time of data collection. Written informed consent was provided by their legal representative in accordance with the Declaration of Helsinki.CM, CD, and SB-M designed the research. CM performed the analysis. CM and SB-M wrote the manuscript. CD and SB-M provided feedback. All authors contributed to the article and approved the submitted version.


Molecular structure property modeling is an increasingly important tool for predicting compounds with desired properties due to the expensive and resource-intensive nature and the problem of toxicity-related attrition in late phases during drug discovery and development. Lately, the interest for applying deep learning techniques has increased considerably. This investigation compares the traditional physico-chemical descriptor and machine learning-based approaches through autoencoder generated descriptors to two different descriptor-free, Simplified Molecular Input Line Entry System (SMILES) based, deep learning architectures of Bidirectional Encoder Representations from Transformers (BERT) type using the Mondrian aggregated conformal prediction method as overarching framework. The results show for the binary CATMoS non-toxic and very-toxic datasets that for the former, almost equally balanced, dataset all methods perform equally well while for the latter dataset, with an 11-fold difference between the two classes, the MolBERT model based on a large pre-trained network performs somewhat better compared to the rest with high efficiency for both classes (0.93–0.94) as well as high values for sensitivity, specificity and balanced accuracy (0.86–0.87). The descriptor-free, SMILES-based, deep learning BERT architectures seem capable of producing well-balanced predictive models with defined applicability domains. This work also demonstrates that the class imbalance problem is gracefully handled through the use of Mondrian conformal prediction without the use of over- and/or under-sampling, weighting of classes or cost-sensitive methods. Drug discovery is an expensive and resource-intensive process that involves many challenges, not least within the area of toxicity . A growiMethods such as molecular structure property modeling have been used for decades in the pharmaceutical field in order to predict important properties, e.g., biological activity, solubility and toxicity, for prioritization of compounds with respect to potential toxicity issues and experimental testing . For a rDuring the last few years, deep learning (DL) techniques have successfully been applied in various domains, e.g., natural language processing  as well Many of the DL investigations have used the Simplified Molecular Input Line Entry System (SMILES) notation  as a stain silico predictive models for early prediction and verification of hazardous properties of new molecules or materials. This includes the development of molecular property models for predicting toxicity, e.g., acute toxicity.The Mistra SafeChem research program, financed by Mistra , has the overarching aims to create a sustainable chemical industry and reduce exposure to hazardous substances . One actThe well-known CATMoS benchmark dataset  has beenThe dataset can be downloaded from reference .The originally defined training and evaluation set in  were useThe training, validation and conformal prediction (CP) calibration sets were randomly selected from the original training set.Two different binary classification sets were investigated:The very toxic  and the non-toxic .The structures, represented as SMILES, were standardized using the “remove_salt_stereo” and “organic_filter” functions of the “preprocessing.py” script found in the Continuous and Data-Driven Descriptors (CDDD) GitHub repository , neutralA total of 96 different physiochemical descriptors were calculated using MolecularDescriptorCalculator in RDKit  list of.In total, 512 CDDD descriptors of length 512 were calculated using the CDDD GitHub repository code and model . The RNNThe binary classification models using RKDit and CDDD descriptors, respectively, were built using the Scikitlearn RandomForestClassifier  with defTwo different approaches were used for generating BERT models.The MolBERT binary classification models were fine-tuned using the MolBERT GitHub repository code  and the A second set of models where fine-tuned using a pre-trained model on 500 k randomly selected PubChem compounds with a validation set of 50 k compounds.The Molecular-graph-BERT binary classification models were fine-tuned using the Molecular-graph-BERT GitHub repository code  and a MoA conformal predictor (CP) is a member of a family called confidence predictors . These pThe nonconformist package , where sAn in-house script was employed to perform the conversion of predictions (scoring) from the models using BERT algorithms into results from conformal prediction. This conversion involves a calibration of the output for each compound and each label (class) in the evaluation set in relation to the output predictions for the compounds of the corresponding class in the calibration set.A CP binary classification problem can have four possible outcomes. A test  compound can be assigned a label for either of the two classes, assigned both labels (both classification) or none of the labels (empty classification). For a detailed description on how this calibration is performed, see Norinder and co-workers .A flow chart overview of the employed machine learning approaches and calibration is depicted in Validity and efficiency are two key measures in conformal prediction. Validity, for each class, is the percentage of correct predictions in Mondrian conformal prediction at a given significance level where the prediction contains the correct class. Thus, in binary classification the both classification is always correct (contains both available labels) while the empty classification is always erroneous (contains no labels). Models were considered valid when the resulting error rate does not exceed the set error rate (significance level) by more the 2.5%.Efficiency, for each class, is defined as the percentage of single label predictions (only one label), regardless of whether the prediction is correct or not, at a given significance level. High efficiency is therefore desirable since a larger number of predictions are single label predictions and more informative as they contain only one class.The aim of this study is to investigate how different molecular representations and algorithmic approaches may affect the predictive performance of the derived models. The present study therefore compares results from the traditionally used Random Forest/physico-chemical descriptor approach through an intermediate Random Forest/auto encoder representation to deep learning BERT/molecular-graph-based approaches.The results from the study are shown in From The class distribution (class “0”/class “1”) is ~1.6:1 for catmos_nt and ~11:1 for catmos_vt which means that the former data set is rather balanced while the latter is rather unbalanced. This imbalance may, in turn, cause some issues for ML algorithms to properly handle the minority class ,42,43.From the catmos_nt results presented in The catmos_vt results presented in The more balanced results from this investigation with respect to SE and SP are due to the independent handling of the two classes as part of the Mondrian conformal prediction calibration procedure; see The well-balanced SE/SP performance is of importance from the point of safety, i.e., not to err on the false negative side, when predicting a toxic compound to be non-toxic for the catmos_vt model. It is less of a problem for the catmos_nt model if a few more non-toxic compounds are predicted to be toxic from a safety perspective.All methods in Advantages of the BERT approaches over the RDKit descriptor-based approach is that they are descriptor-free in that SMILES are used as input without the need for explicit descriptor generation prior to modeling and that the results from the models can be projected back onto the atoms of the molecules through their attention mechanisms. The advantage of the RDKit and CDDD approaches is shorter computation costs and that smaller datasets can be modeled with acceptable outcomes compared to using deep learning in the form of BERT that usually requires larger training sets.The results show for the binary CATMoS non-toxic, almost equally balanced dataset that all methods perform equally well. The results also show that the MolBERT model based on a larger pre-trained network performs somewhat better compared to the rest for the binary CATMoS very-toxic dataset with an 11-fold difference between the two classes. The descriptor-free, SMILES based, deep learning BERT architectures seem capable of producing well-balanced predictive models with defined applicability domains. This work also demonstrates that the class imbalance problem is gracefully handled through the use of Mondrian conformal prediction without the use of over- and/or under-sampling, weighting of classes or cost-sensitive methods.


COVID-19 vaccinations have been prioritised for high risk individuals.Determine individual-level risk factors for care home residents testing positive for SARS-CoV-2.Longitudinal observational cohort study using individual-level linked data from the Secure Anonymised Information Linkage (SAIL) databank.Fourteen thousand seven hundred and eighty-six older care home residents (aged 65+) living in Wales between 1 September 2020 and 1 May 2021. Our dataset consisted of 2,613,341 individual-level daily observations within 697 care homes.We estimated odds ratios  using multilevel logistic regression models. Our outcome of interest was a positive SARS-CoV-2 PCR test. We included time-dependent covariates for the estimated community positive test rate of COVID-19, hospital inpatient status, vaccination status and frailty. Additional covariates were included for age, sex and specialist care home services.The multivariable regression model indicated an increase in age , community positive test rate , hospital inpatients , and residents in care homes with non-specialist dementia care  had an increased odds of a positive test. Having a positive test prior to the observation period  and either one or two doses of a vaccine  were associated with a decreased odds.Care providers need to remain vigilant despite the vaccination rollout, and extra precautions should be taken when caring for the most vulnerable. Minimising potential COVID-19 infection for care home residents when admitted to hospital should be prioritised. Increased community positive test rate had an increased likelihood of a positive SARS-CoV-2 PCR test.Hospital inpatients had an increased likelihood of a positive SARS-CoV-2 PCR test.One or two doses of vaccination indicated a decreased chance of a positive test.Care providers need to stay vigilant despite the vaccination rollout.Extra precautions should be taken when caring for the most vulnerable, especially in a hospital setting.et al., as ‘…a dynamic, specific and real threat to the health and well-being of older people’  [Care homes are a keystone of adult social care. They provide accommodation and care for those needing substantial help with personal care, but more than that, they are people’s homes , 2. In 20, p.10) . The impVaccinations for SARS-CoV-2 in the UK have been prioritised for those identified at higher risk including to older people living in care homes , 6. HoweThis is the first study we are aware of investigating this vulnerable sub-population at an individual-level with the inclusion of the community positive test rate of COVID-19, hospital admissions and vaccination status. Furthermore, we included information on previous positive SARS-CoV-2 polymerase chain reaction (PCR) tests, age, sex and frailty. As suggested in  we were We aimed to identify individual-level risk factors for SARS-CoV-2 infection amongst care home residents in Wales (UK) with the inclusion of community positive test rate of COVID-19, hospital admissions and vaccination status.Longitudinal observational cohort study using anonymised linked data from the SAIL Databank.Our cohort was 14,786 older care home residents (aged 65+) living in Wales between 1 September 2020 and 1 May 2021. Our dataset consisted of 2,613,341 individual-level daily observations for the same period within 697 care homes. Residents were included if they lived in a care home at any period during the observation window. Only residents with at least one PCR test (positive or negative) were included within the dataset. Residents were censored if they moved out of the care home or died. All data were collected retrospectively and linked anonymously within the SAIL Databank.We used linked longitudinal data from the SAIL Databank to create our dataset . We usedOur outcome of interest was a positive SARS-CoV-2 PCR test. The date of the positive test was recorded as the date when the specimen was collected. We used a binary variable to indicate the positive test dates for individuals.Exposure variables were daily COVID-19 community positive test rate estimates and a daily indicator for if a resident was a hospital inpatient. Daily inpatient status was identified using PEDW, residents were identified as being a hospital inpatient for all dates from the admission to the discharge date (inclusive) of a hospital spell.P(x), of at least one positive PCR test is the unobserved realisation of a spatially correlated stochastic process. Specifically, we used the total number of positive PCR tests in the 14-day window as the numerator, and total tests in the 14-day window as the denominator. The model has three parameters that determine the mean and variance of P(x) and the rate at which the correlation between the values of P(x) at two different locations decays with increasing distance between them, for more detail on the methodology see [The estimated COVID-19 community positive test rate was calculated by removing all tests for care home residents and creating a geospatial model for each daily observation. The model includes a spatial correlation term that decays with distance. In other words, areas that are close in proximity are likely to have a more similar estimate than those further away. This reduces the impact of artificial boundaries introduced by statistical geographies and provides a more realistic spatial distribution estimate of positive test rates in the communities surrounding a care home. The community test rate estimates were calculated for each LSOA using a 14-day lookback window for each date. The testing strategy in Wales in the observation period included asymptomatic testing of those caring for vulnerable people , and symptomatic testing for those in the community . Our comWe included the number of doses of a vaccine and a binary indicator for if an individual had a positive PCR test prior to the observation period as predictor variables. The number of vaccine doses received was time varying and was recorded as a categorical variable on each date . Additional variables included were specialist services provided by the care home where individuals were resident during the study period. This included: nursing care (yes/no), learning disability (yes/no), mental health (yes/no), dementia . Age (continuous), sex  and hospital frailty risk score  were included at the individual level. The Hospital Frailty Risk Score (HFRS) was developed using Hospital Episode Statistics (HES), a database containing details of all admissions, Emergency Department attendances and outpatient appointments at NHS hospitals in England, and validated on over 1-million older people using hospitals in 2014/15  (ICD-10)In the longitudinal dataset created for the analysis individuals have multiple daily observations. Each daily observation includes updated time-dependent covariates; the estimated community COVID-19 positive test rate, whether an individual was in hospital, and the number of vaccine doses received. All other variables were fixed at the value of entry into the study. Additionally, the dataset has anonymous individual and care home identifiers used to cluster the observations. For an example of the dataset design see Appendix 1 and 2.To help minimise selection bias we only included individuals who had at least one SARS-CoV-2 PCR test. Similarly, to minimise time interval bias we used a large observation period where we believe testing of care home residents was more consistent than at the start of the pandemic. We also included anonymous individual and care home level identifiers to account for correlation amongst repeated measures.Descriptive statistics included the demographic information for individuals at the start of their residency and stratifications for those who did and did not have a positive PCR test within the observation period. We produced time plots for the daily estimated community positive test rate, positive test rate of residents, numbers of PCR tests for residents and number of positive PCR tests for residents. For our analysis we used multilevel logistic regression models with a random intercept term for each care home. The regression was applied to the individual-level daily dataset. For sensitivity analysis we calculated null (intercept only) models with random effects at the individual level, care home level, and both (see Appendix 2). Observations with a missing LSOA were removed, and individuals were censored if they moved residence or died.We analysed 2,613,341 daily observations, consisting of 14,786 care home residents within 697 care homes. The reasons for exclusion from the analysis dataset are included in Cross-tabulations for the observations with and without positive tests whilst a hospital inpatient and the number of vaccine doses is displayed in The univariable and multivariable multilevel logistic regression results for positive SARS-CoV-2 PCR tests are presented in The random effects term indicated significant variance at the care home level in the multivariable model (intercept variance of 2.51 with standard error 0.178). We compared intercept only (null) models with random effects terms at the individual, care home, and individual and care home level and found most of the variability was accounted for at the care home level, see Appendix 3.An increase in the community positive test rate of COVID-19 led to an increase in the odds of care home residents testing positive, with an OR of 1.13  per percentage increase of community positive test rate. As the community positive test rate estimates ranged between 0% and 20% in the observed time period this suggests a potential 10-fold increase in the odds of a positive test in care home residents at the peak of community positive test rate. The association between community positive test rate and care home residents having a COVID-19 infection warrants further research with routes of potential ingress needing further exploration to provide evidence and develop robust policies including clear guidelines for staff and visitors. This should also take into consideration qualitative research investigating the overall well-being of care home residents , 22. OveWe found care home residents had a large increased odds of testing positive for SARS-CoV-2 whilst in hospital as an inpatient with an adjusted OR of 7.40 . This could be due to the type of hospital care home residents are admitted to, with an estimated 61.9% of COVID-19 infection being hospital acquired in residential community care hospitals, compared to 9.7% in hospitals providing acute and general care . ResidenIncreased age, sex  and increased frailty severity were associated with an increased odds of a positive test in the univariable models. These factors are individual-level characteristics that would possibly require an increased level of care, and therefore increased daily personal contact, potentially increasing the chance of transmission of COVID-19. In the multivariable model sex and frailty were statistically insignificant, suggesting a reduced importance compared to community positive test rate and hospital stays.Those testing positive for SARS-CoV-2 prior to the observation period and individuals with one and two doses of a vaccine were at a reduced odds of a positive PCR test, with adjusted odds ratios of 0.58 , 0.21 0.17,0.25) and 0.05 , respectively. Those who had previously tested positive may have built up immunity to COVID-19 in a similar way to receiving a vaccine  and 0.05. The croWe were able to create a large longitudinal dataset of care home residents and include linked individual-level data on hospital admissions, COVID-19 community positive test rate, vaccinations and demographic information. This was all possible using existing data from the SAIL Databank.We observed a change in the testing regime from February, where the total number of tests was reduced. We did not explicitly investigate the impact of the change in testing amongst care home residents but found the care home positive test rate remained consistent in shape and magnitude compared with the estimated community positive test rate. We were unable to link all care homes in Wales in the SAIL databank . We were unable to include details on care home staff who may have tested positive for SARS-CoV-2 as data on care home staff are currently cannot be linked to specific care homes. We restricted our cohort to older adults (aged 65+) living in care homes and those that did not move between care homes within the observation period (temporary residents) which may not be representative of all care home residents.Our findings indicate that measures taken to prevent COVID-19 from spreading to the most vulnerable in society are not completely effective. Although vaccination profoundly decreased the odds of testing positive for SARS-CoV-2, there was still an increased risk of infection for vaccinated individuals admitted to hospital. We also found that an increased community positive test rate of COVID-19 was associated with an increased odds of infection in care home residents. This may reflect higher risk from visitors or care staff living locally. Achieving and maintaining very high rates of vaccination in residents, health and care staff, and visitors is essential. We suggest that care providers need to stay vigilant despite the vaccination rollout, and extra precautions should be taken when caring for the most vulnerable. Follow-up research is needed to evaluate the impact of vaccine waning and changing prevalence of viral strains.aa-21-1799-File002_afac084Click here for additional data file.


Rigid origami, with applications ranging from nano-robots to unfolding solar sails in space, describes when a material is folded along straight crease line segments while keeping the regions between the creases planar. Prior work has found explicit equations for the folding angles of a flat-foldable degree-4 origami vertex and some cases of degree-6 vertices. We extend this work to generalized symmetries of the degree-6 vertex where all sector angles equal  What follows is a list of suggestions and open questions.et al. show in [We hope that the degree-6 folding angle relationships presented here will offer new tools to mechanical engineers and materials scientists looking for more origami methods to incorporate into their designs. As Tachi  show in , using t


Fragmentation patterns of cell-free DNA reflect the chromatin structure of the cells from which these fragments are derived. Nucleosomes protect the DNA from fragmentation, resulting in decreased sequencing coverage in regions of open chromatin. LIQUORICE is a user-friendly software tool that takes aligned whole-genome sequencing data as input and calculates bias-corrected coverage signatures for predefined, application-specific sets of genomic regions. The tool thereby enables a blood-based analysis of cell death in the body, and it provides a minimally invasive assessment of tumor chromatin states and cell-of-origin. With user-defined sets of regions that exhibit tissue-specific or disease-specific open chromatin, LIQUORICE can be applied to a wide range of detection, classification and quantification tasks in the analysis of liquid biopsies.http://liquorice.computational-epigenetics.org.LIQUORICE is freely and openly available as a Python package and command-line tool for UNIX-based systems from bioconda. Documentation, examples and usage instructions are provided at Bioinformatics Advances online. The analysis of cell-free DNA (cfDNA) from blood and other bodily fluids—commonly referred to as liquid biopsy—holds great promise for cancer diagnostics and other applications  and coveliquid biopsy regions-of-interest coverage estimation (‘LIQUORICE’). This method enables the analysis of chromatin accessibility in cfDNA samples based on whole-genome sequencing data. It calculates bias-corrected coverage signatures for sets of user-provided genomic regions—typically regions with tissue-specific or tumor-specific open chromatin that can be used to assess cell-of-origin and epigenetic cell states. We utilized this method to study patients with childhood cancers  and the Regulatory Elements Database (LIQUORICE takes four files as input: (i) an indexed BAM file containing aligned reads from paired-end whole-genome sequencing of a liquid biopsy sample; (ii) one or more BED files, each representing a set of regions of interest—such as DNase I hypersensitivity sites or enhancer regions specific for a tissue or tumor; (iii) the FASTA file of the reference genome; and (iv) a bigWig mappability file, which we provide for the hg19 and hg38 assemblies of the human genome and which is easily generated for other reference genomes. The selection of user-provided region sets depends on the application and typically includes regions of open chromatin of tissues or tumors that are relevant sources of cfDNA, which is obtained from blood plasma or other bodily fluids. Sets of tissue-specific and tumor-specific genomic regions can be obtained from public databases such as ENCODE  and quantifies the coverage signal . MoreoveA typical LIQUORICE analysis runs in <20 min per sample on a personal laptop , or in about 3 min per sample on a single compute node with 25 cores. Individual samples are processed independently and are readily parallelized on a compute cluster. LIQUORICE analysis profits from sequencing depths in the range of 5× to 15× genomic coverage, especially when the region sets of interest are relatively small (e.g. less than a thousand regions with an average length of a kilobase). Nevertheless, we have successfully used LIQUORICE with sequencing depths as low as 1× and fewer than 500 regions of interest .The following code shows how to install and run LIQUORICE, applying it to a cohort of patients with Ewing sarcoma as well as healthy controls . We expect that LIQUORICE will be broadly useful for liquid biopsy analysis in oncology and beyond, for example for detecting and quantifying tumor DNA in cancers with few genetic alterations (based on tumor-specific regions of open chromatin); assessing on-target and off-target effects of cancer drugs (based on tumor-specific and organ-specific regions of open chromatin); and monitoring acute organ damage, for example in patients with sepsis and recipients of organ transplants (based on organ-specific regions of open chromatin).vbac017_Supplementary_DataClick here for additional data file.


Plenty of literature reported the applicability and usefulness of telemedicine and teleassistance (TMTA) services in the management of diabetes and other chronic conditions. Specifically, TMTA proved to be effective for conditions that require radical lifestyle modifications, tailored pharmacological interventions, and periodic monitoring of clinical health status. The purpose of this study is to investigate the individual and contextual determinants of the perceived quality (PQ) of the telemedicine and teleassistance (TMTA) services and the willingness to continue (WC) with them among patients with diabetes using TMTA during the COVID-19 pandemic in one large region of Italy (Emilia-Romagna).A structured survey was administered to patients with type 1 and 2 diabetes who used TMTA services during the first wave of the COVID-19 pandemic. The questionnaire was comprised of questions on TMTA service experience and participants’ socio-demographic characteristics. Multiple regression models investigated the independent factors associated with PQ (score 1-100) and WC (yes/no).The final analysis included 569 patients with diabetes , with an average age of 58.1 years. TMTA services’ PQ and WC were high. A higher education  and being unemployed  were factors associated with an increased WC. Older age was negatively related to PQ . Perceived support from TMTA service was positively associated with PQ  and WC . Perceived increase in disease self-management was positively associated with PQ  and WC .Our study identified several determinants of PQ and WC. These socio-demographic and patient-perception related factors should be considered in the implementation of care pathways integrating in-person visits with TMTA services.• Socio-demographic factors play a crucial role in TMTA acceptance and should be taken into due consideration when implementing health pathways integrating in-person visits with TMTA services.• Health workers should always try to improve patients’ self-management skills and should always make patients feel supported. This is also true in the digital health era.


HIV-1 pre-exposure prophylaxis (PrEP) relies on inhibition of HIV-1 replication steps. To understand how PrEP modulates the immunological environment, we derived the plasma proteomic profile of men receiving emtricitabine-tenofovir (FTC-TDF) or emtricitabine-tenofovir alafenamide (FTC-TAF) during the CHAPS trial in South Africa and Uganda (NCT03986970). The CHAPS trial randomized 144 participants to one control and 8 PrEP arms, differing by drug type, number of PrEP doses and timing from final PrEP dose to sampling. Blood was collected pre- and post-PrEP. The inflammatory profile of plasma samples was analyzed using Olink (N=92 proteins) and Luminex (N=33) and associated with plasma drug concentrations using mass spectrometry. The proteins whose levels changed most significantly from pre- to post-PrEP were CCL4, CCL3 and TNF-α; CCL4 was the key discriminator between pre- and post-PrEP samples. CCL4 and CCL3 levels were significantly increased in post-PrEP samples compared to control specimens. CCL4 was significantly correlated with FTC drug levels in plasma. Production of inflammatory chemokines CCL4 and CCL3 in response to short-term PrEP indicates the mobilization of ligands which potentially block virus attachment to CCR5 HIV-1 co-receptor. The significant correlation between CCL4 and FTC levels suggests that CCL4 increase is modulated as an inflammatory response to PrEP. Pre-exposure prophylaxis (PrEP) with antiretroviral drugs (ARD) offers a unique opportunity for protection against HIV-1 infection. Daily oral administration of emtricitabine with tenofovir disoproxil fumarate (FTC-TDF) was shown to be efficacious in randomized controlled trials . GastroiData gaps remain over the adherence and dosing requirements for both on-demand and daily PrEP. Furthermore, it remains unclear whether dosing requirements are the same for insertive and receptive sex for either anal or vaginal sex. On demand PrEP has only been evaluated in a randomized controlled trial in men who have sex with men, which was unable to define a specific dosing regimen for insertive anal sex .ex vivo HIV-1 challenge of foreskin tissue after voluntary medical male circumcision (VMMC) from each participant. This approach may be useful to facilitate the efficient selection of effective candidates for more complex and expensive PrEP phase III trials.The CHAPS trial NCT03986970) was conducted to determine the optimal combination of drug, dose, time from dosing and acceptability for young men at risk of HIV-1 infection in Sub-Saharan Africa  are dysregulated and that inflammation markers remain elevated in individuals initiating anti-retroviral therapy (ART) at either the acute or chronic phase of infection , 8. The The mechanisms of action of different ARD classes are mediated by interference with the different stages of the HIV-1 life cycle. The nucleoside reverse-transcriptase inhibitors (NRTI) tenofovir (TFV) and emtricitabine (FTC) are phosphorylated intracellularly to produce an active triphosphate moiety which is incorporated into the viral DNA chain and ultimately terminates viral replication. Very few studies have addressed the possibility that PrEP administration may activate changes in the proteomic profile of treated subjects and that these changes may participate in reduction of HIV-1 spread. An induction of interferon (IFN)-related genes was reported in the gastrointestinal tract, but not PBMCs, of individuals receiving FTC-TDF as part of two HIV PrEP trials .As part of the CHAPS trial, plasma samples were collected pre- and post-PrEP in the randomized treatment and control arms and analyzed for a range of inflammatory proteins. The proteomic profile was assessed through two different methods, Olink and Luminex, and related to the circulating levels of TDF and FTC in individuals assigned to the different randomized groups. We found that the C-C Motif Chemokine Ligand 4 (CCL4), which binds to the CCR5 HIV-1 co-receptor, was increased as results of PrEP administration and correlated with NRTI plasma drug levels.The study design, methodology and recruitment pathways have been previously reported . Trial pBlood was collected at Chris Hani Baragwanath Academic Hospital in Soweto (South Africa) and Entebbe Regional Referral Hospital and the Uganda Virus Research Institute (UVRI) clinic (Uganda) pre- and post-PrEP administration. Blood was processed for mononuclear cells isolation by gradient centrifugation with Histopaque 1077 and plasma kept for proteomic analysis at -80 °C until transported in dry ice to the Karolinska Institutet.The samples from the two countries were analyzed together using the following assay modalities: 1) pre- and post-PrEP samples from the same individual were analyzed and compared  were analyzed at the Plasma Profiling National Facility, Science for Life Laboratory  for the presence of inflammation-related soluble proteins using the Olink Inflammation Panel, which probed for 92 different factors  . This plA total of thirty-three soluble immune proteins . Cytokin13C15N2) were added to plasma (100µL) and extracted using solid phase extraction cartridges . The eluents were evaporated to dryness under nitrogen at room temperature and reconstituted in water:acetonitrile  prior to injection onto the LC-MS autosampler. The analytes were chromatographically resolved using a reverse phase Synergi Polar C18 column . The calibration curve ranged between 1-1000 ng/ml (TFV) and 5-5000 ng/ml (FTC). Quantification was performed on an Sciex 4500 triple quadrupole mass spectrometer  operating in positive ionization mode.TFV and FTC concentrations in plasma were measured  using a validated liquid chromatography mass spectrometry (LC-MS) methodology. In brief, stable isotope labelled internal standards ; the Uganda Virus Research Institute research ethics committee (GC/127/18/12/680); Uganda National Council of Science and Technology (HS 2534); Uganda National Drug Authority (618/NDA/DPS/09/2019) and the London School of Hygiene and Tropical Medicine research ethics committee (Ref:17403). Informed written consent was collected from all participants. A subsequent amendment to ethics allowed the collection of plasma at randomization in Uganda and South Africa. The Swedish Ethics Review Authority approved the laboratory studies of the collected specimens at the Karolinska Institutet (2020–00941).2 > 0.7. Unless stated otherwise, a p-value < 0.01 was considered statistically significant.Raw normalized protein expression (NPx) data from the Olink text were transformed by scaling, yielding values with a mean of 0 and standard deviation of 1. NPx values were compared between plasma samples obtained pre- and post-PrEP from the same individual or between samples of individuals included in the different randomization arms using a non-parametric Wilcoxon rank-sum test with false discovery rate p-value adjustment. Random forest analysis to determine classification predictors was performed using recursive feature elimination with the random Forest package. All other analyses were performed in base R . CytokinTo establish the effect of the two PrEP drugs (FTC-TAF and FTC-TDF) on inflammation, we determined the protein levels of 63 inflammatory markers  Figure 2The analysis of paired samples (pre- and post-PrEP) was also conducted using a Luminex panel comprising 33 proteins. The comparison of paired samples divided into drug groups revealed that CCL4, as already detected in the Olink, was the protein which increased most post-PrEP, followed by two additional proteins CXCL10 and MCP-1, which were also among the highly significant proteins detected in Olink Figure 2The abundance of all significant proteins detected in Olink and Luminex when analyzing the pre- and post-PrEP samples is shown in Next, we performed random forest analysis to find out whether the pre- and post-PrEP samples could be differentiated using an unsupervised machine learning approach Figure 3All proteins detected in Olink were further analyzed for differences between the control arm and the PrEP arms and the significantly different proteins (p<0.01) are shown in Other proteins that differed between controls and PrEP arms fell in two categories  and emtricitabine (FTC) in plasma of the subjects included in the treated arms. TFV was detected in 88.9% and FTC in 89.6% of samples. In general, the attempt to correlate the proteins modulated by the drugs, shown in in vitro  and to a lesser extent CCL3 (MIP-1-α), with CCL4 significantly correlating with plasma concentrations of FTC. CCL4 is a CC inflammatory chemokine, ligand for CCR5, a co-receptor for HIV-1 infection. CCL4, together with CCL5 (RANTES) and CCL3, is produced by CD8+ T cells and is a suppressive factor for HIV-1 infection ; recombiin vitro , 15. Micin vitro . It is pTenofovir administration to individuals included in PrEP trials has been associated with several adverse events including gastrointestinal problems, 5% weight loss , lipid sWe provide unique evidence that an increase in CCL4 levels in plasma can be found in individuals receiving PrEP using two different sensitive platforms for proteomic characterization. CCL4 has not been previously associated with PrEP . One of Two additional chemokines CXCL9 and CXCL11, and to a lesser extent CXCL10, also showed an increased expression following PrEP initiation. Through their interaction with the chemokine receptor CXCR3, these chemokines mediate chemotaxis of leukocytes to the inflammatory sites but also differentiation of cytotoxic CD8+ T cells and NK cells; CXCL11 is primarily involved in directing the linage development of T-regulatory-1 cells (reviewed in ). In vieThe individuals included in our study are healthy young men  and it iThe pharmacokinetics of tenofovir and emtricitabine has been evaluated in the plasma of subjects included in the different CHAPS randomization arms. It is interesting that direct correlations between the levels of tenofovir and emtricitabine in plasma with CCL4 were found and this supports the notion that tenofovir based PrEP stimulates CCL4 production. Further research conducted on the mode of action of CCL4 upregulation during PrEP could instruct on how to potentiate CCL4 production to block HIV-1 binding to the CCR5 co-receptor. The major sources of CCL4 production are CD8+ T cells and macrophages and it is improbable that the CCL4 levels measured in plasma are the product of cellular mechanisms specifically taking place in the male genital tract. It is more likely that CD8+ T cells in the peripheral blood and in other organs may be activated by PrEP to produce CCL4; nonetheless CCL4 produced distally from the male genital tract may diffuse to this and additional organs thus contributing to prevention of HIV-1 infection. The field of PrEP is moving towards long-acting formulations and sustained drug delivery systems including injectable PrEP, vaginal rings in women and implants. It is of interest in the future to study whether PrEP administered as injections and implants will mobilize specific chemokines, as in our study, with the potential of blocking HIV-1 interaction with the CCR5 co-receptor. Our study highlights the interplay between PrEP and host immunity that uncovers possible mechanisms of ARV effectiveness.The original contributions presented in the study are included in the article/Ethical clearance to conduct the trial was obtained from the South African Health Products Regulatory Authority (20181004); the Uganda Virus Research Institute research ethics committee (GC/127/18/12/680); Uganda National Council of Science and Technology (HS 2534); Uganda National Drug Authority (618/NDA/DPS/09/2019) and the London School of Hygiene and Tropical Medicine research ethics committee (Ref:17403). Informed written consent was collected from all participants. A subsequent amendment to ethics allowed the collection of plasma at randomization in Uganda and South Africa. The Swedish Ethics Review Authority approved the laboratory studies of the collected specimens at the Karolinska Institutet (2020–00941). Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin.SP: conducted experiments, analyzed the data, created figures, and wrote the manuscript; CH: planned the study, conducted experiments, analyzed the data, and wrote the manuscript; LE: conducted experiments and analyzed the data; SM: sample processing and conducted experiments; PN: sample processing and conducted experiments; GO: sample processing and conducted experiments; DO: selected trial participants and obtained signed informed consent and clinical specimens; A-DP: sample processing and conducted experiments; TS: sample processing and conducted experiments; JS: conducted experiments and analyzed the data; AS: selected trial participants and obtained signed informed consent and clinical specimens; PK: planned the study; EW: analyzed data and wrote the paper; SK: planned study and analyzed specimens; LL: selected trial participants and obtained signed informed consent and clinical specimens; CG: planned the study and wrote the manuscript; NM: planned the study and wrote the manuscript; JF: planned the study and wrote the manuscript; FC: planned the study, analyzed the data, and wrote the manuscript. All authors contributed to the article and approved the submitted version.This study is part of the EDCTP2 programme supported by the European Union (grant number RIA2016MC-CHAPS). The study received support by a grant from the Swedish Research Council .The authors would like to acknowledge the technical support by the Plasma Profiling National Facility, Science for Life Laboratory, KTH-Royal Institute of Technology, Stockholm, Sweden. 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.


Background: Diabetic retinopathy (DR) afflicts more than 93 million people worldwide and is a leading cause of vision loss in working adults. While DR therapies are available, early DR development may go undetected without treatment due to the lack of sufficiently sensitive tools. Therefore, early detection is critically important to enable efficient treatment before progression to vision-threatening complications. A major clinical manifestation of early DR is retinal vascular leakage that may progress from diffuse to more localized focal leakage, leading to increased retinal thickness and diabetic macular edema (DME). In preclinical research, a hallmark of DR in mouse models is diffuse retinal leakage without increased thickness or DME, which limits the utility of optical coherence tomography and fluorescein angiography (FA) for early detection. The Evans blue assay detects diffuse leakage but requires euthanasia, which precludes longitudinal studies in the same animals. Methods: We developed a new modality of ratiometric fluorescence angiography with dual fluorescence (FA-DF) to reliably detect and longitudinally quantify diffuse retinal vascular leakage in mouse models of induced and spontaneous DR. Results: These studies demonstrated the feasibility and sensitivity of FA-DF in detecting and quantifying retinal vascular leakage in the same mice over time during DR progression in association with chronic hyperglycemia and age. Conclusions: These proof-of-concept studies demonstrated the promise of FA-DF as a minimally invasive method to quantify DR leakage in preclinical mouse models longitudinally. Diabetic retinopathy (DR) is a leading cause of vision loss in working adults. DR is caused by microvascular complications and neovascularization in the retina secondary to chronic exposure to diabetic hyperglycemia. Leakage of retinal vessels is an important early pathologic manifestation of DR that occurs before progression to more advanced complications associated with irreversible retinal damage that compromises vision. Early leakage in retinal vessels is typically widespread throughout the retina including in the periphery. If untreated, diffuse vascular leakage may progress to more localized leakage at focal lesions, leading to retinal thickness, diabetic macular edema (DME) or proliferative diabetic retinopathy (PDR), all of which threaten vision. Anti-angiogenic agents such as those against the vascular endothelial growth factor (VEGF) are used to treat various forms of DR, including DME and PDR ,2. As wiAnimal models are widely used in DR research. Mice and rats remain the preferred models due to their quick reproduction rates, short lifespan, and small size, which allow studying disease progression within reasonable timeframes . As earlNoninvasive retinal imaging is a key diagnostic tool for evaluating DR. Optical coherence tomography (OCT) can detect changes in retinal thickness and DME but cannot detect vascular leakage . OCT angIn this study, we developed a new modality of ratiometric fluorescence angiography with dual fluorescence (FA-DF) to reliably detect and longitudinally quantify diffuse leakage of retinal vessels. Conducting analyses in preclinical mouse models of induced and spontaneous DR, the current studies demonstrated the feasibility and sensitivity of FA-DF for detecting and quantifying diffuse leakage in retinas of the same mice over time during DR progression in association with chronic hyperglycemia and age. Therefore, this new noninvasive and longitudinal modality for detecting and quantifying retinal vascular leakage facilitates the preclinical investigation of DR and the development of therapeutics against it and has the potential to be further developed for clinical application.Akita/J; strain # 003548) mice were purchased from Jackson Laboratories  and housed under a 12/12 h of light/dark cycle with free access to food and water. B6 mice were made diabetic with streptozotocin  treatment as the induced DR model [C57BL/6  and AKITA  mice were fasted overnight for 8 to 12 h and injected with a freshly prepared solution of STZ in 1x citrate buffer  at a single dose of 150 mg/kg of body weight . Blood gThe EB assays were performed as previously described in detail . In brieFA-DF imaging was performed in mice under general anesthesia using a 10:1 ketamine–xylazine mixture as above with the eyes kept moist during imaging using balanced salt solution  to avoid corneal dryness. The pupils were dilated prior to anesthesia with tropicamide ophthalmic solution 0.5% . Baseline images were acquired in each mouse within 1 min before  intravenous injection of a mixture of two fluorescence-labelled dextrans of different molecular weight (M.W.) at 5 mg/mL each in saline solution, and time-lapse fluorescence imaging of the central and peripheral retina vasculature was initiated immediately after repositioning the mouse on the stage of a clinical-grade Heidelberg Engineering Multiline HRA + OCT SN 2884 imaging system . We used 500-kDa fluorescein isothiocyanate (FITC)-labeled dextran  and 2000-kDa tetramethyl rhodamine (TRITC)-labeled dextran . In each mouse, images were acquired every 5 min after the injection for 30 min. Image pairs  were acquired in the green and red channels by alternating between the appropriate instrument’s filter cubes (FA-PB for green and LWP542 for red) at every time-point. The same instrument settings  were used during each imaging session and throughout the longitudinal studies. After imaging, the mouse eyes were covered with a thin layer of antibiotic ophthalmic ointment  to prevent eye infection and drying of the cornea during recovery from anesthesia.Vascular Leakage”.Time-lapse images acquired from each mouse were exported as tiff files and imported into Volocity software version 6.3.1 , where the vascular leakage analysis was performed without image registration, as previously described . In brieWe performed initial studies to assess the feasibility of (a) acquiring composite fundus images A with a We performed longitudinal studies to assess the capability of FA-DF to detect and quantify the progression of induced DR in STZ-diabetic B6 mice. Diabetic mice were confirmed with severe hyperglycemia during the entire experiment A and eveWe next performed similar studies in diabetic AKITA mice using 500-kDa and 2000-kDa dextrans conjugated to FITC and TRITC as the leakage detection dye and non-leaking reference, respectively. These studies showed that the MFI of 500-kDa dextran initially increased in the EVS following the injection and progressively decreased after 10 min D.Leakage Index”, which is a measure of the vascular leakage of 500-kDa dextran normalized to the non-leaking reference 2000 kDa dextran at any given time after the injection relative to time zero. Because it is a ratiometric measurement based on the leaked dye relative to the non-leaking reference, the leakage index is insensitive to signal drifts observed in the leakage detection channel in association with dye excretion in the urine or other in vivo imaging artifacts, such as breathing movement and transient lens clouding during imaging or cataract development over time. We used the leakage index at 30 min (“Leakage Index (30)”) to compare longitudinal changes in the retinal leakage of aged STZ-diabetic mice imaged using the mixture of 500-kDa and 2000-kDa dextrans at different times after the STZ treatment and during DR progression. The leakage Index (30) increased significantly in these diabetic B6 mice at 124 days after the STZ treatment  in association with DR progression as the animals aged, and a significant difference was measured at 258 days of age in comparison to when they were 90 days old  and narrowing of retinal vessels occurs in association with DR ,25. To fIn this study, we developed a new technique of ratiometric FA-DF to quantify vascular leakage in the diabetic retina. Conventional FA uses only one dye, fluorescein-sodium, with a low M.W. (0.376 kDa) allowing it to diffuse freely through microcapillaries even in non-pathological conditions. This results in high background fluorescence signal, low signal-to-noise ratio, and poor sensitivity to reliably detect diffuse retinal leakage in early DR development. Conventional FA is also qualitative and does not allow quantitation of leakage, although, attempts have been made to quantify retinal permeability in FA images . CurrentIn contrast, the findings presented here demonstrated that FA-DF allows the longitudinal assessment of retinal vascular leakage in the same mice. The use of ratiometric measurements of the two fluorescent dyes also achieved reliable in vivo quantitation of early diffuse retinal leakage in two mouse models of induced and spontaneous DR. FA-DF measurements revealed a significant increase in retinal vascular leakage at time points consistent with the onset of DR in both models ,17. By uDespite its common use in the clinical setting, the qualitative and quantitative limitations of conventional FA are well recognized and, as such, the use of OCT/OCTA has become more common in ophthalmic practice. OCT and OCTA can reliably detect changes in retinal thickness, DME, PDR, as well occlusion/reperfusion in vessels of the retina; however, they cannot detect vascular leakage ,7. OtherAs demonstrated in the above presented findings, FA-DF enabled the detection and quantitation of retinal vascular leakage at various stages of DR in the employed mouse models. Future studies could also demonstrate its utility in other animal models. Vascular leakage occurs in DR and other retinal pathologies and is the result of increased permeability of blood vessels and compromised integrity of the blood–retina barrier, a structure that functions as a selective filter between blood vessels of the retina and the rest of the body vasculature. In general, the permeability of blood vessels depends on different factors, including the shape and size  of molecules and the vascular wall fenestration, which changes with inflammation and vascular endothelial cell pathologies ,36. In tMoreover, the current report demonstrated the relatively ready availability of FA-DF for preclinical studies and highlighted its utility as a minimally invasive intravital imaging method with superior capabilities compared to conventional FA. This also underscored its potential utility in the clinical setting. Although, future studies will be required to evaluate its sensitivity in detecting and assessing changes in retinal vascular leakage in DR patients with and without treatments. If proven successful in DR, FA-DF will also be applicable to other vasculopathies characterized by leakage, such as wet age-related macular degeneration (AMD) and central retinal vein occlusion . FurtherLeakage Index as a new ratiometric parameter to compare changes in the leakage of retinal vessels over time. We used here the leakage index at 30 min after injection of the labeled dextrans “Leakage Index (30)” to assess the progression of DR longitudinally in the same mice (Leakage Index (30) since it was the latest time point after the injection and when the leakage detection dye (500-kDa FITC-dextran) unambiguously reached a steady state in the EVS in the diabetic AKITA mice  that are more amenable for investigation by existing noninvasive modalities like FA, OCT, and OCTA. Our studies also demonstrate that FA-DF enables the longitudinal quantitation of DR leakage in the same animals. Thus, FA-DF may prove a valuable tool in the preclinical development and assessment of DR therapeutics. Future studies will also evaluate its feasibility and potential utility in the clinical setting to aid in the early diagnosis of DR and the assessment of treatment efficacy in conjunction with other imaging modalities available in the clinical toolkit.


Germinal centers (GC) are central places for the development of adaptive immune responses. While their development follows a first wave of extra-follicular activation, they uniquely contribute to the development of long-term responses including both diversified memory B cells with strong ability to activate and differentiate upon antigen re-challenge, and long-lived plasma cells producing high-affinity class-switched antibodies. They are central to protective immune responses against microbial or tumor-associated antigens, but they are also major sites where dealing with peripheral tolerance to autoantigens, programmed cell death and with the hazardous outcomes of DNA lesions inflicted to B-cells.There is currently no definitive model accounting for all the outcomes of GC formation, since these evolving structures integrate stimulating and inhibitory signals from multiple origins, including antigens, chemokines, cytokines, specific antibody level and they dynamically evolve so that the very same factors initially promoting GC development can later contribute to GC resolution.Trüb et al.). The cellular and molecular signals that govern the induction and the fate of TLS in such pathological situations are not well-understood, but are among the hotest research questions in this emerging TLS-Cancer topic with potential for new discovery in B cell focused immunotherapies. We therefore believe that classical GC and TLS structures are the two sides of the general mechanism of immune response and immune surveillance.In contexts of chronic local inflammation, “GC-like” tertiary lymphoid structures (TLS) have long been reported within some non-lymphoid tissues and they recently received strong attention for their contribution to tumor immunology. They share structural and functional characteristics with GC that form in the secondary lymphoid organs. TLS are induced by persistent infection, autoimmune disorders and cancer. Interestingly in many cancer types, a good correlation has been reported between richness in TLS in tumor site and prolonged patient survival  or plasma cells (PC). In this regard, Nakagawa and Calado review the conditions mediating positive selection of light zone(LZ) B-cells and instructing them to be selected as either PCs, MBCs or persistent GC-B cells reentering the dark zone. Santamaria et al. present new experimental data in human showing that this cell fate decision is also impacted by a threshold of IL-4/STAT6 signaling making GC LZ B cells either proliferate and transiently express a MYC-dependent transcriptional program or rather up-regulate BLIMP and instructively progress towards PC differentiation when CD23-dependent signaling becomes lower. Lemarié et al. also show induction of the unfolded protein response genes as a very early event at the pre-plasmablastic stage.In this Research Topic of Frontiers in Immunology, Lu and Craft review recent evidence about the contribution of Tfr cells in the balance between productive immune responses and B cell memory and the maintenance of homeostasis for avoiding immunopathology.Going deeper into the diversity of cell interactions, Rivas et al., analyzing both murine models and data from human DLBCL patients, provide new evidence about the caretaker role of the cohesin complex in genomic stability of GC B-cells. They notably show that cohesin ATPase subunit Smc3 haploinsufficiency favors malignant transformation, through gene repression and impaired enhancer-promoter interactions, with loss of epigenetic modifiers (TET2 or KMT2D) preventing B-cell exit from the GC reaction and their commitment to plasma cell differentiation.Schmiedel et al., using a Brg-1 deficient model, also provide data showing that ATPase, Brg1 and the BAF chromatin remodeling complex, are required for enhancer-promoter interactions which promote cell cycle-related gene expression during GC formation.Dauba and Khamlichi review into much details the long -range promoter-enhancer interactions within the IgH locus which promotes chromatin remodeling and synapses between target switch sequences in a B-cell stimulation-dependent manner in order to support CSR. Dalloul et al. additionally provide new evidence that the process of Cclass switch recombination (CSR) can initiate even in the absence of the activation induced cytidine deaminase (AID) enzyme as an intrinsic ability of an appropriately conformed IgH locus to undergo recombination, so that AID is rather catalyzing and boosting CSR rather than initiating CSR in GC B-cells. Fuertes et al. finally review the role of multiple microRNAs (miRNAs) in the regulation of Tfh and GC B-cell responses as well as in B cell neoplasia and GC response dysregulation.It altogether appears that cell fate decisions from GC B cells integrate both the quality and the cumulative amounts of signals that they have received from the Ag and their local microenvironment, following an instructive model, and their intrinsic ability to be committed to proliferation or differentiation, with cell death as another major fatal outcome of B cell activation and AID induction –4.All authors listed have made a substantial, direct, and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.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. 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Telemedicine use in nursing homes (NHs) expanded during the COVID-19 pandemic. The objectives of this study were to characterize plans to continue telemedicine among newly adopting NHs and identify factors limiting its use after COVID-19.Key informants from 9 Wisconsin NHs that adopted telemedicine during COVID-19 were recruited. Semi-structured interviews and surveys were employed to identify participant perceptions about the value of telemedicine, implementation challenges encountered, and plans and barriers to sustaining its delivery after COVID-19. Directed content analysis and a deductive thematic approach using the Systems Engineering Initiative for Patient Safety (SEIPS) model was used during analyses. Quantitative and qualitative data were integrated to identify participant views on the value of telemedicine and the tools and work system enhancements needed to make telemedicine easier and more effective.All participating NHs indicated a preference to continue telemedicine after COVID-19. Urgent assessments of resident change-in-condition and cognitively based sub-specialty consultations were identified as the encounter types most amenable to telemedicine. Reductions in resident off-site encounters and minimization of resident therapy interruptions were identified as major benefits of telemedicine. Twelve work system enhancements needed to better sustain telemedicine were identified, including improvements to: 1) equipment/IT infrastructure; 2) scheduling; 3) information exchange; and 4) telemedicine facilitators.NHs that adopted telemedicine during COVID-19 wish to continue its use. However, interventions that enhance the integration of telemedicine into NH and off-site clinic work systems require changes to existing regulations and reimbursement models to sustain its utilization after COVID-19.The online version contains supplementary material available at 10.1186/s12877-022-03046-y. Nursing homes (NHs) were the epicenter for the beginning of the coronavirus disease 2019 (COVID-19) pandemic in the United States . In an ePerhaps unsurprisingly, telemedicine activity expanded dramatically in U.S. NHs during the COVID-19 pandemic . While rOur research team recently embarked on a mixed-methods work system study in Wisconsin NHs that newly adopted or greatly expanded telemedicine services during the COVID-19 pandemic. The objectives of this study were to: 1) characterize facility plans for continuing telemedicine following COVID-19; 2) characterize staff and provider perspectives on the value and utility of telemedicine; and 3) identify the barriers NHs face with conducting telemedicine encounters. Based on the findings from this study, we discuss key work system changes that must occur to better sustain widespread utilization of telemedicine in NHs after COVID-19.We conducted a mixed methods convergent study  of telemData collection from members of the NH administrative staff was conducted using semi-structured interviews. These Interviews focused on the following areas: 1) facility experience and challenges with implementing and using telemedicine during COVID-19; 2) facility plans for using telemedicine after COVID-19; 3) the relative advantages and disadvantages of telemedicine versus face-to-face encounters; 4) the types of resident encounters most amenable to telemedicine; and 5) the tools and resources that can make telemedicine encounters easier and/or more effective. Data collection from APP and sub-specialty provider participants was achieved through a structured survey and semi-structured interviews, respectively. A survey approach was employed with APP participants as prior interviews with these providers had provided our team with a detailed understanding of how they conducted telemedicine encounters. In contrast, understanding of how sub-specialty providers prepared for and conducted telemedicine encounters was more limited and required a semi-structured interview data collection approach. Information collected from both provider groups focused on the advantages and disadvantages of telemedicine versus face-to-face encounters and the tools and resources that can make the conduct of telemedicine encounters easier and/or more effective.Participant interviews were recorded and transcribed. Interview transcripts were independently coded by the research team in teams of two . The coders met to discuss and resolve coding differences through consensus. The study PI (CC) resolved any coding discordance. Transcripts of interviews conducted with the nursing administrative staff participants were analyzed using two different qualitative methods. Coders utilized a structured checklist to enumerate data  during an = 3], Infectious Diseases [n = 3] and Wound Care [n = 1]). There was limited utilization of telemedicine in two of the participating NHs prior to COVID-19. In both cases, telemedicine was used in an ad hoc manner by mental health providers to address acute resident behavioral disorders. The other participating NHs had not employed telemedicine prior to COVID-19. All participating NHs expanded or implemented telemedicine services during COVID-19. All participating NHs reported encountering difficulties with one or more aspects of telemedicine expansion or implementation. Eight of 9 (78%) NHs experienced issues related to hardware and equipment availability/supply. Connectivity problems as well as software issues were experienced by 89% of NHs. Finally, all NHs experienced challenges with sufficient staff availability and different procedural tasks related to telemedicine . Many of the technological issues improved during the early implementation of telemedicine in NHs. However, many of the work system issues, as will be shown, remained a persistent problem in participating NHs. Despite these ongoing challenges, NH administrative staff endorsed positive feelings about continuing telemedicine services after COVID-19 albeit in a more limited fashion.A total of 27 individuals participated in this study, including 12 NH staff, 8 long-term care APPs and 7 sub-specialty care providers  encounters Table . ParticiNH administrative staff perceptions about the value of telemedicine for the conduct of sub-specialty encounters were positive Table . ParticiNH administrative staff participants identified a high number of work system factors impacting or impacted by telemedicine  providing NH staff and off-site clinicians with ready access to each other’s EHRs; 2) requiring that different health systems providing telemedicine services to the same NH agree to utilize a common telemedicine platform to reduce the number of platforms that NH staff must be familiar with; 3) development of protocols that standardize the content, structure and sharing of information between NH staff and providers involved in telemedicine encounters; 4) centralizing scheduling related tasks in the NHs and ensuring that these individuals are easily reachable by staff in off-site provider clinics; and 5) centralizing responsibility for facilitating telemedicine encounters to clinical NH staff who are given sufficient time to collect and prepare information needed before, during and after the conduct of an encounter. Further improvements will likely be realized through policies to promote greater inter-operability between NH and health system EHRs, greater adoption of technologies that allow cross-organizational scheduling and promote real-time communication between NH and off-site clinic staff responsible for resident scheduling decisions, and the introduction of novel funding mechanisms that provide NHs with the resources needed to adopt and sustain telemedicine technologies as well as hire and retain the staff needed to ensure its reliable delivery.Health care systems including providers, hospitals, and nursing homes could address the identified challenges by implementing enhancements needed to make nursing home telemedicine encounters easier and more effective . Some reThe project had several limitations. The interviews were conducted with a convenience sample of NH staff and providers and the viewpoints expressed may not be representative the experience of other NHs that adopted telemedicine during COVID-19 or the plans of other facilities for continuing its use after the pandemic. A statewide or national survey would provide insights from a more representative sample about prior experiences and future plans about telehealth utilization in nursing homes. Sub-specialist interviews were limited to a convenience sample of infectious disease, psychiatry, and wound care providers. The perceptions about telemedicine benefits and challenges are not generalizable to other nursing homes due to the study sample size especially for specific sub-specialists . Other sub-specialists may have different perspectives about the value of telemedicine and the enhancements needed to improve its delivery and effects. Future research should focus on conducting a wider sample of sub-specialist interviews including multiple perspectives within the same sub-speciality. Finally, the findings from the survey were limited to one long-term care APP group affiliated with a mid-western healthcare system. The survey should be replicated across a larger sample to determine the representativeness of the results observed in the current study.In conclusion, nursing staff and providers who deliver care in these facilities recognize that telemedicine is a valuable service modality that they feel should be continued. Many of the facilities participating in this study were able to enact changes to structure and process that enhanced their telemedicine workflows over time. However, facilities still face several ongoing internal and external environmental challenges that threaten the sustainability of telemedicine after COVID-19 recedes. Regulatory relief, new payment models and incentives to encourage greater collaboration between NHs and the healthcare systems that participate in facility telemedicine services will be needed to avoid a return to the pre-COVID-19 status quo.Additional file 1.