Driven by the rapid aging of the population, Japan introduced public long-term care insurance to reinforce healthcare services for the elderly in 2000. Precisely predicting future demand for long-term care services helps authorities to plan and manage their healthcare resources and citizens to prevent their health status deterioration.This paper presents our novel study for developing an effective model to predict individual-level future long-term care demand using previous healthcare insurance claims data. We designed two discriminative models and subsequently trained and validated the models using three learning algorithms with medical and long-term care insurance claims and enrollment records, which were provided by 170 regional public insurers in Gifu, Japan.The prediction model based on multiclass classification and gradient-boosting decision tree achieved practically high accuracy  for up to 12 months after the previous claims. The top important feature variables were indicators of current health status , risk factors to worsen future healthcare status , and preventive care services for improving future healthcare status .The intensive validation tests have indicated that the developed prediction method holds high robustness, even though it yields relatively lower accuracy for specific patient groups with health conditions that are hard to distinguish.  Quantitative estimation of future service demand is crucial to sustain and advance a sophisticated healthcare system. Several previous studies have aimed to predict future service demand –11. ThisJapan is experiencing a super-aging society previously unwitnessed by any country in the world. A universal public medical insurance system was established in Japan in 1961; however, it merely covers the treatment of injuries and diseases for citizens , 13. In Japan’s public long-term care insurance categorizes all individuals aged ≥65 years into eight eligibility levels according to their requirement for long-term care Table  14]. In. In14]. Certification of eligibility is provided by the local government authority on demand. An assigned official consults the elderly individual or their family to inspect their physical and psychological health status and cognitive ability in accordance with the eligibility criteria prescribed by the government. The official then decides their eligibility and designs a long-term care plan. Currently, the local government can only roughly predict future long-term care demand using population-based estimation techniques. Individual-level prediction based on medical and long-term care history is challenging but potentially improves the accuracy.Accurate prediction of future long-term care demand helps regional authorities to manage and plan their long-term care resources. Long-term care involves a package of various services that require human resources with different skills and licenses. For example, visiting nursing care is only conducted by licensed nurses by law. Therefore, enabling authorities to precisely estimate future demand  of specific services in an area of charge would allow them to act preemptively to secure the necessary workforce and would likely improve the stability and efficiency of regional long-term care resources.In addition, individual-level prediction can help to prevent the escalation of regional long-term care demand. Previous studies reported that preventive care, such as rehabilitation and exercise, could improve physical and cognitive function and thus prevent the deterioration of health status and the increase in individuals’ eligibility levels –26. EarlThe present study aimed to accurately predict individual-level future long-term care demand using information about medical services and long-term care services. Medical and long-term care services are separately managed in Japan’s healthcare system; however, their effects on each other are reciprocal. For example, onset of stroke or dementia are primary risk factors that worsen an individual’s future healthcare status and increase the demand for long-term care services , 27. In This paper presents an effective method to predict individual-level future long-term care demand using previous insurance claims data of medical and long-term care services. We designed two different discriminative models and trained them using three different learning algorithms with medical and long-term care insurance claims provided by 170 regional public insurers covering 42 local government areas in Gifu, Japan. Because different insurers separately manage medical insurance claims and long-term care insurance claims, this study utilized insurance enrollment history records that allow us to combine and integrate all the claims for each individual. Intensive comparative validation tests were performed to confirm the practical effectiveness. To our knowledge, this is the first study to use data from previous healthcare insurance claims to predict future long-term care demand. A preliminary version of this work has been reported elsewhere .We developed an effective prediction method to predict future long-term care demand using previous insurance claims data of medical and long-term care services at the individual level. We aimed to predict individuals’ long-term care eligibility levels at 3, 6, and 12 months using their insurance claims of medical services, and, if available, their long-term care services for the past 12 months.We employed a supervised machine learning approach. Figure Two different modeling approaches were employed in the design process of the discriminative models. The first approach was multiclass classification, in which all training datasets were directly employed to build a single predictor. Although multiclass classification was widely utilized, previous machine learning studies indicated that imbalanced learning samples resulted in low prediction accuracy –33. The The specific model design process can be divided into two steps as follows.Step 1: For the training dataset, we obtained medical and long-term care insurance claims from the past 12 months and future long-term care eligibility levels of 3, 6, and 12 subsequent months from the database, transformed them into a longitudinal form, and generated a feature variable vector for each individual. The feature variables included (1) the frequency of occurrence of each code for disease diagnosis, medical treatment, drug prescription, medical instrument, and long-term care service; (2) the medical expenditure of each month; (3) the number of claims in the last 1, 3, 6, and 12 months; (4) sex and age; (5) current eligibility levels; and (6) future long-term care eligibility levels for the following 3, 6, and 12 months.Step 2: In the multiclass classification approach, we applied a filtering technique using the F-value of the analysis of variance (ANOVA) to select 150 statistically representative codes –39. In tWe employed three major learning algorithms, namely, gradient-boosting decision tree (GBDT) , multilaWe applied the models to test datasets and clarified the prediction accuracy to evaluate the effectiveness of the designed and trained models. Precision, Recall, and F-measure  were used as evaluation indicators for the prediction of each eligibility group. In addition, (unweighted) averages and weighted averages of Precision, Recall, and F-measure over all the eligibility levels were obtained as overall evaluation indicators, in which the weighted average was calculated by (1) summing the indicator value multiplied by the number of individuals for each eligibility level and (2) dividing the total sum by the total number of individuals . We condWe used anonymized medical and long-term care insurance claims provided by 170 regional public insurers covering 42 local government areas in Gifu, Japan. The dataset included 49,991,060 medical insurance claims and 5,754,329 long-term insurance claims, which were recorded from April 2015 to March 2018. We also used anonymized insurance enrollment history information  to combine medical and long-term care insurance claims for each individual. The anonymization had been performed before being provided to us; personal identifiers  were removed or irreversibly converted and instead encoded identifiers  were appended. The dataset statistics are summarized in Table The anonymized medical and long-term care insurance claims were managed and processed on a specialized data platform . The ANOIntensive analyses were conducted to validate the accuracy of the proposed prediction method.First, we conducted a comparative study to compare two different modeling approaches: multiclass classification and class-specific classification. The designed prediction task was to predict future eligibility levels at 3 (June 2017), 6 (September 2017), and 12 (March 2018) months after previous insurance claims recorded from April 2016 to March 2017. Specifically, separate prediction models were designed for each approach, and the models were trained using the GBDT algorithm with insurance claims recorded from April 2016 to March 2017 and future eligibility levels recorded on June 2017, September 2017, and March 2018. We then evaluated the prediction accuracy of future eligibility levels of these three different timepoints  after the previous insurance claims (April 2016 to March 2017). We used insurance claims from 137,101 individuals randomly selected from the entire individual set for the model training and then used insurance claims of another 137,101 individuals for the prediction validation  Table . We perfWe further intensively evaluated the prediction method to verify its robustness for different time periods. We conducted the same tests as those presented in Table Table In the evaluation cases presented thus far, the prediction models were constructed from the training datasets of certain time periods, and the resulting prediction was evaluated using separate validation datasets for the same time periods. We separated the training and validation datasets to achieve fairness even though both were during the same time period. In contrast, the following analysis presents a scenario of “time-shift” prediction, where the training and validation were performed at different time periods with the same model.We constructed prediction models to predict eligibility levels at 3, 6, and 12 months after previous insurance claims from April 2016 to March 2017 and applied the prediction models for the following time periods: (1) April 2015 to March 2016; (2) July 2015 to 2016; (3) October 2015 to September 2016; and (4) January to December 2016. We then investigated the prediction accuracy of each test case.Table The findings of the present study can be summarized as follows. (1) The prediction model based on multiclass classification and GBDT achieved high accuracy up to 12-month prediction (weighted average F-measure = 0.873). (2) Direct indicators of current health status , risk factors that worsen future healthcare status , and preventive care services to improve future healthcare status  showed high importance in predicting future eligibility levels. (3) Contrary to our expectations, the multiclass classification approach consistently achieved significantly higher accuracy than the class-specific classification approach . (4) GBDT, MLP, and SVM performed comparably, but GBDT consistently achieved slightly higher accuracy than the other algorithms . (5) Intensive validation tests indicated that the prediction method was highly robust (weighted average F-measure ≥ 0.857).We also observed that the RS1 and RLTC1 groups showed lower prediction accuracy than the other groups. These findings require further investigation but may be because the individuals in the RS1 and RLTC1 groups had health conditions that were hard to distinguish from each other. Furthermore, the developed predictor occasionally failed to correctly predict sudden escalation of long-term care demand  and was particularly notable in long-range prediction cases. For example, in the case presented in Table A major limitation of the present study is the sample size of the dataset. We used a 3-year insurance claims dataset provided by 170 regional public insurers in Gifu, Japan. Extending the datasets, such as expanding the data period and adding more insurers, would strengthen our prediction method. Scaling up the population size would enable the prediction models to perform significantly better, particularly for the RS1 and RLTC1 groups. Currently, there are >2,000 public insurers working in Japan , and theOn another front, the prediction model proposed in this study has been defined as a classification problem, in which a temporal variable  is considered simply as a feature variable candidate but a temporal sequence property is not explicitly considered. Designing a time-series prediction model is an open problem; the time-series prediction model is useful for analyzing the temporal sequence property of future long-term care demands.The prediction method proposed in this study was verified only with the dataset provided by the regional public insurers in Gifu as above noted. We expect that the method can be directly applied for other prefectural areas in Japan to predict their future long-term care demands. A comparative verification study with multi-prefectural datasets is ongoing. In addition, the basic framework for future prediction presented in this study is potentially applied and extended to a wide spectrum of prediction scenarios for estimating other future demands  from past insurance claim records. However, extensive work for model tuning and verification should be necessary for each specific prediction scenario. For example, GBDT consistently performed better than other algorithms in this study, but this observation does not automatically mean that GBDT works similar for other scenarios. In addition, extensive data preparation work might be demanded by other tasks; longer-term datasets could be necessary to predict the future conditions of slowly-progressing diseases.Extending the future prediction to heterogeneous datasets is technically and socially interesting. For example, the association of lifestyle and psychosocial factors with a daily living competence is known . The insSeveral previous studies have examined prediction of future service demand –11. WongThe present study aimed to develop a novel and effective method to predict individual-level future long-term care demand using previous insurance claims data from medical and long-term care services. We designed discriminative models, which were then and trained and validated using medical and long-term care insurance claims and enrollment records provided by 170 regional public insurers covering 42 local government areas in Gifu, Japan. Overall analysis confirmed that the developed models offered a practically high accuracy for the prediction of individual-level future long-term care demand . In the future, we will aim to resolve the remaining issues by extending the training and validation datasets and refining learning algorithms/modeling approaches to further improve the prediction method.


Growing human population has put enormous pressure on the global food security. In the past few decades, the soybean yield has remained stagnant especially due to the use of conventional breeding technologies. In this regard, the research community were looking for the novel breeding technologies to revolutionize the soybean breeding. “Novel breeding technologies” are biotech-based approaches to modify plant characteristics fast and accurately. In this scenario, the current Research Topic “novel technologies for soybean improvement” is intended to collect articles on recent advances and future applications of the novel technologies in the soybean breeding to boost the soybean production. These approaches and technologies include molecular design breeding approaches, genome editing and transformation technology, RNA interference approach, Marker-Assisted and Genomics-Selection breeding approaches, machine learning and bioinformatics technology. Below we briefly highlight the applications and potential of the new approaches in the soybean breeding; and how they can be significant for increasing the yield and quality in soybean that are presented in a collection of 20 papers published in the special issue on the Research Topic: Novel Technologies for Soybean Improvement.Zhu et al. (2022) identified that among the various deep learning algorithms, the Faster R-CNN model performance was the best to phenotype soybean flowers and pods. The accuracy of Faster R-CNN model was 94.36 and 91% for detecting the flowers and pods, respectively. Furthermore, based on the Faster R-CNN model, they also proposed the fusion model for soybean flower and pod recognition and counting, that will greatly reduce labor intensity and improve efficiency.Soybean flower and pod drop are important determinants of soybean yield, thus use of advanced techniques that will increase the accuracy and speed for the flower and pod phenotyping has great influence on soybean breeding. In this regard, the computer vision techniques have recently emerged to phenotyping the flowers and pods in bulk at higher speed and accuracy. Wei et al. (2022) checked the performance of Terahertz frequency-domain spectroscopy and chemometrics for the identification of soybean varieties, and subsequently proposed the grey wolf optimizer-support vector machine (GWO-SVM) soybean variety identification model. These authors showed that combination of the Terahertz frequency-domain spectroscopy and GWO-SVM will identify the soybean varieties at higher accuracy and speed. Compared with discriminant partial least squares (DPLS) and particles swarm optimization support vector machine, GWO-SVM combined with the second derivative could establish a better soybean variety identification model.Identification of the soybean varieties with superior nutritional value and composition is an important goal of soybean researchers. In this regard, Amaral et al. (2022) argue that ANN can be used to categorize soybean genotypes from a population that has an either a wide genetic variability or a narrow genetic variability for the relative maturity character. The authors further demonstrated that ANN can be used to discriminate genotypes in early breeding generations. This would make it easier and to rapidly find high-performing cultivars with longer and shorter photoperiod amplitudes at a single selection site.Soybean possessing narrow genetic base makes it hard to screen the available genetic and phenotypic variability for identifying the superior genotypes. In this regard, “Artificial Neural Network” (ANN) is the type of machine learning process which is used to classify the narrow-range and pure lineage populations. Xing et al. (2022) used the computer vision (CV), machine learning based technology for the phenotypic analysis in soybean. They focused on expanding four different kinds of features disease traits, indoor test traits, field traits and soybean seed phenotypic traits. It was revealed that CV technology has the ability to collect highly accurate characteristics, and allows to provide large quantity of big data for breeding programs.Artificial intelligence (AI) technology has been expected to provide the accurate phenotypic data at high resolution and low cost in the plants. Toda et al. (2022) used the data collected by an Unmanned Aerial Vehicle (UAV) to predict genomic green fraction dynamics in soybean. In their study, the use of UAV remote sensing allowed the measurement of the longitudinal variations in the green fractions. Their study showed that the model effectively combined early growth data with training population phenotypic data for prediction. This prediction method could be applied to selection at an early growth stage in crop breeding, and could reduce the cost and time of field trials.Availability of high-throughput phenotyping systems has provided the opportunity to have an easy access to growth data. This will in turn allow to predict growth of cultivars with unknown performance by conducting the genomic prediction analysis. Till now, the genomic and growth modeling prediction have not been studied to the greater depth. In this regard, Zhang et al. (2022) reported the high-throughput and low-cost phenotypic platform that allows phenotyping whole plant including roots at uniform and controlled soil stress conditions. They concluded that responses of cotyledons can be used as non-destructive indicator for determining the salt tolerance of the seedlings based on their high-throughput multiple-phenotypic assays of 178 soybean cultivars.Salinity is one of the major environmental constraints effecting the crop yield. However, there is considerable lack of high throughput phenomics platforms that allow to record the salt stress responses of plants as well as the non-destructive collection method for root phenotypic data. Shamim et al. (2022) explored the underlying physiological mechanisms and genetic basis regulating the photosynthesis in the Japanese soybean germplasm by using the “MIC-100” system (high-throughput system for measuring the photosynthesis). They identified the signification association of single nucleotide polymorphism (SNPs) on Chromosome 17 with the light-saturated photosynthesis (Asat) trait based on genome wide association study (GWAS) analysis. The G protein alpha subunit 1 (GPA1) was identified as the strong candidate Asat gene. This study provides strong evidence to apply GWAS of plant germplasm for exploring the maximum potential of photosynthesis in soybean improvement.Photosynthesis is an important process that determines the plant growth and yield. Hence, it is prerequisite to more efficiently assay the photosynthetic capacity of crop plants in order to select the varieties with higher photosynthetic efficiency. Cheng et al. (2021) used an integrated transcriptomic approach for investigating the species-specific, starch-related, carbon metabolism-related and acyl-lipid-related genes in soybean and chickpea. They identified seven soybean-specific gene expression patterns, four of which are highly expressed at the middle- and late oil accumulation stages. They proposed the difference of metabolism pathways in seed oil and starch content between soybean and chickpea, this study has opened up the possibility of engineering other legumes for enhanced oil contents.Limited studies have been conducted to determine the molecular mechanism underlying the seed oil and starch content in soybean. These traits are important traits determining the yield and quality of soybean. Huang et al. (2022) used this approach to compare full-length transcriptomes in soybean genotype 09-138 infected with either soybean cyst nematode (Heterodera glycines) race 4 or race 5 that are avirulent and virulent, respectively to the soybean genotype. This study provides the insights about the soybean-nematode interaction which will serve as the basis for future research.Transcriptome sequencing of full-length transcripts by long sequencing read method is an efficient approach to reveal detailed information about the events at transcriptional or post-transcriptional levels. But this approach has not been used in the soybean till now. In this context, Shen and Chen (2022) used the weighted gene co-expression network analysis (WGCNA) for the first time to understand the global regulatory network of gene expression involved in soybean hypocotyl elongation and identified a crucial regulatory module. They reported two regulatory modules. GmPRE6s-EXPANSINs submodule and GmPIF1/GmPIF3-GmSAUR1/23-EXPANSINs submodule. This study provides important genes for developing molecular markers for breeding for soybean with enhanced hypocotyl length and seed vigor.Seed germination is regulated by the hypocotyl elongation, and it determines the vitality of seedlings; however, the mechanism underlying the hypocotyl elongation has remained largely unexplored. He et al. (2021) developed a highly sensitive approach for quantitatively detecting miRNAs by ligating ribonucleotide-modified DNA probes. This approach eliminates the use of complex reverse transcription-based quantitative approach. In the devised test, miR156b immediately hybridized two ribonucleotide-modified DNA probes, and amplification using universal primers followed the ligation process. The target miRNA could be detected at a 0.0001 amol level, and variations of a single base between miR156 family members could be recognized. The suggested quantitative technique works for overexpression-based genetically modified (GM) soybean. Ligation-based quantitative polymerase chain reaction (PCR) may be used to study miRNAs and investigate GM organisms.Role of the miRNAs in regulating biological processes as well as biotic and abiotic stress responses in plants has been well documented. Hence, identification of the miRNAs is essential to determine their role in the biological processes as well as for plant responses to environmental stresses in plants. Xu et al. (2022) identified the F-box gene family in soybean genome using the bioinformatic approach, and also elucidated the role of F-box-like gene GmFBL144 in adapting soybean to drought stress. They identified 507 F-box genes that were grouped into 11 subfamilies. The GmFBL144 showed higher expression in the roots and ectopic expression of the GmFBL144 in Arabidopsis enhanced the sensitivity to drought stress. Their study suggested that GmFBL144 protein negatively regulates drought stress through its interaction with small heat shock protein.In plants, the F-box gene family is one of the big gene families. F-box genes are involved in the regulation of plant growth as well as response to multiple environmental stresses, both biotic and abiotic. Qin et al. (2022) used the combined approach of linkage mapping and genome-wide association studies (GWAS) to identify the QTLs and genes underlying the seed protein in soybean. They identified major QTLs mapped to Chromosomes 6 and 20 in two segregating populations. Their study revealed the similar performance of both BL and rrBLUP, and the accuracy of GS was dependent on size of training population and SNP set. The protein-specific SNPs showed higher GS efficiency compared to that by all SNPs.To breed soybean varieties with high seed protein, it is a prerequisite to elucidate the genetic basis of high seed protein. Li et al. (2021) identified and validated the candidate genes regulating the three-seeded pod. They used the QTL mapping approach and identified 36 QTL. Four genes from 162 genes underlying these two QTL related to pod and seed set were identified as potential candidate genes controlling this trait. These four genes can be used as molecular markers for incorporating this trait into commercial soybean cultivars.The three-seeded pod number is an important trait that positively influences soybean yield. Soybean variety with increased three-seeded pod number contributes to the seed number/plant and higher yield. The candidate genes of the three-seeded pod may be the key for improving soybean yield. In this regard, Xu et al. (2022) reviewed the genetic transformation progress in soybean and reported the gain made in efficiency and genotype flexibility of soybean transformation over the last decade. Factors that influence the soybean transformation are proper explant selection, reagent selection, components of culture medium and the detailed understanding of the mechanism involved in the transformation.In the past four decades the genetic transformation was used to modify the genome of soybean for the trait improvement. However, the genetic transformation in soybean is still subjected to low transformation efficiency, genotype specificity as well as long and tedious transformation processes. Wang et al. (2021) demonstrated that mutation of GmAITR genes by CRISPR/Cas9 genome editing in soybean increased the salinity tolerance. The enhanced salt tolerance was observed both at the seed germination and seedlings stages of gmaitr mutants. Hence, the knockout of the GmAITR genes via CRISPR/Cas9 proved to be a powerful approach for inducing salt tolerance in soybean.CRISPR/Cas9 genome editing has emerged an efficient technique to breeding crop cultivars with enhance tolerance to environmental stresses. In Arabidopsis, knockout of the entire family of the abscisic acid (ABA)-induced transcription repressors (AITRs) enhanced drought and salinity tolerance without any fitness costs. Glyma.07g110300 (GmUGT) encodes an UDP-glycosyltransferase (UGT) which is the core enzyme that negatively regulates insect resistance. Zhang et al. (2022) demonstrated that CRISPR/Cas9-mediated mutagenesis/knockout of GmUGT enhanced soybean resistance to Helicoverpa armigera and Spodoptera litura. The ectopic expression of the GmUGT gene in Arabidopsis ugt72b1 mutant this mutant significantly reduced its resistance to H. armigera.Insect pests such as leaf-chewing insects are one of the major constraints in soybean production. In this context, the development of soybean varieties with increased resistance to leaf-chewing insects can greatly increase the soybean yield. The via host-induced gene silencing (HIGS) has potential to induce broad-spectrum resistance (BSR) to SMV. Jiang et al. (2021) employed the Nicotiana benthamiana-soybean mosaic virus (SMV) system to optimize the target SMV sequence for HIGS. They were able to use the information gained from the N. benthamiana-SMV system to develop transgenic soybean lines with effective HIGS to enhance SMV resistance in soybean.Soybean mosaic virus (SMV) is a prevalent soybean pathogen. Pyramiding multiple SMV-resistance genes into one individual is tedious and difficult. Targeting the viral genome Florigen is a key player regulating the balance between vegetative and reproductive growth. Hence, it regulates the crop yield; but the actual utilization of the florigen in improving the crop yield remains largely unexplored. In this context, Xu et al. (2021) developed a strategy to improve the yield of soybean significantly by using the florigen. They showed that fine-tuning of the florigen genes via RNAi can boosts soybean yield. The conserved functions of florigen in plants and use of the RNAi approach to manipulate the expression of this gene therefore may be used to increase crop productivity.via ultra-low temperature approach is an effective and safe way for long-term storage of plant germplasm resources. Jia et al. (2022) reported an efficient ultra-low temperature freezing approach to preserve soybean pollens. Soybean flowers collected at the fully-bloom stage were dried, frozen and stored at -196 or -80°C. They observed that 90% of the pollen grains remain viable based on in vitro tests. Their study documented that this technique of pollen preservation will break the spatiotemporal barrier of soybean hybridization and facilitate efficient soybean breeding.To maintain the crop germplasm for its use in future breeding programs, preservation of the pollen grains XF wrote the draft, DY and MB edited. All authors contributed to the article and approved the submitted version.This research was funded by National Nature Science Foundation of China (No. U21A20215).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.


We present a patient with severe tracheal stenosis resulting from a compression by the innominate artery 6 months after an arterial switch operation in a dextro-transposition of the great arteries. Segmentation and three-dimensional (3D) visualization were derived from a contrast-enhanced dual-source computed tomography and post-processing was performed using a dedicated open-source platform (3D Slicer). Post-processing allowed a comprehensible visualization of the relationship of the innominate artery to the trachea when compared to standard computer tomography reformations. Finally, the surgical approach to move the innominate artery anteriorly in order to relieve the tracheal obstruction was emphasized based on the improved 3D visualization of the actual pathology. An effective aortopexy could be performed and the postoperative result was confirmed by a second 3D visualization. About 3 months of follow-up, the patient is completely asymptomatic. Three-dimensional visualization offers excellent opportunities for diagnosis, treatment planning and follow-up in patients with a vascular-related tracheal stenosis in the context of congenital heart disease. Tracheal stenosis in paediatric patients may be related to a variety of aetiologies [1]. Tracheal stenosis in paediatric patients may be related to a variety of aetiologies . In case®, Siemens Healthineers AG, Erlangen, Germany) with high temporal (111 ms) and spatial resolution. The dedicated cardiac protocol [We present a 6-month-old patient with severe distal tracheal stenosis related to compression by a left-sided innominate artery (right aortic arch) after neonatal arterial switch operation. The patient developed progressive expiratory stridor and cyanotic spells. Biplane chest X-ray and echocardiography were unremarkable. Diagnosis of tracheal compression by the innominate artery was confirmed using a third-generation dual-source computer tomography scanner  image reconstruction was carried out by an open-source platform for analysis and display of medical imaging data (3D Slicer) . HounsfiThe applied 3D reconstruction provided excellent visualization of the pathology as well as the surrounding anatomy emphasizing the surgical approach to move the innominate artery anteriorly in order to relieve the tracheal obstruction (Fig. Tracheal stenosis in paediatric patients may be related to a variety of aetiologies . Apart fTypically, in complex anatomy cross-sectional imaging is the reference of standard . Post-prUnexpected postoperative findings in this setting, such as mild residual stridor, were verified by a second 3D reconstruction and clear visualization of the postoperative situs. While a tracheoscopy alone would have only allowed to evaluate the tracheal stenosis, the follow-up computer tomography not only visualized the decompression of the trachea but also the anteriorly extended aortic arch and the surrounding structures as well.However, 3D reconstruction may result in a loss of information: The connecting tissue between the aortic arch and the trachea was not segmented and, thus, is not displayed. As a result, the distance between these anatomic structures appears wider in 3D than in reality.In addition to the 3D visualization shown here, 3D printing is an alternative for preoperative planning. Improvements in the preoperative planning and postoperative outcomes could be shown .However, the here presented approach has the advantage of faster availability and lower costs due to the open-source platform.Three-dimensional visualization offers excellent opportunities for diagnosis, treatment planning and follow-up in patients with a vascular-related tracheal stenosis in the context of congenital heart disease.


Aim: This study compares the stage at the presentation of patients with colorectal cancer (CRC) before and after introducing COVID-19 restrictions and the mode of presentation.Methods: This is a retrospective cohort study comparing the incidence of CRC, TNM stage and mode of presentation in the pre-COVID and COVID cohorts at a single UK Trust. All patients discussed at the CRC multidisciplinary team (MDT) from March 2017 to March 2021 were included and split into two cohorts; the pre-COVID group from 01/03/2017 to 29/02/2020 and the COVID group from 01/03/2020 to 28/02/2021. Percentages were used for descriptive statistics. Student’s t-test was used for the comparison of demographic variables. Chi-squared test was used for the difference analysis for the categorical data, such as TNM and mode of presentation. P value ≤0.05 was significant.Results: In total, 1373 patients were diagnosed with CRC during the period from March 2017 to March 2021. The pre-COVID group (2017-2020) included 1104 CRC patients, compared to 269 patients in the COVID one (2020-2021). The mean age was higher in the pre-COVID group (p = 0.001). There was a statistically significant increase in the proportion of cases presenting with T4 disease (p = 0.023) and metastatic disease (p = 0.032) in the COVID group compared to the pre-COVID group. There was also a significant increase in the rate of emergency presentations (p < 0.0001).Conclusion: We observed a statistically significant increase in rates of locally advanced (T4) and metastatic (distant) CRC in patients presenting after introducing the COVID-19 lockdown. There was also an increase in emergency presentations. There was no observed difference in nodal status. This may reflect disruption to cancer diagnostic services and the reluctance of patients to access medical care during a pandemic, particularly the elderly. Colorectal cancer (CRC) is the fourth commonest cancer in the United Kingdom but has the second lowest five-year survival rate, after lung cancer . Early d2 deflation during laparoscopy [Because of the exponential surge in COVID-19 (SARS-CoV-2) infections rate, the UK implemented a nationwide lockdown in March 2020, which affected the presentation and management of patients with CRC ,8. The paroscopy .The mode of presentation of CRC also impacts prognosis. Compared to elective surgery, emergency surgery for CRC is more likely to result in postoperative morbidity and death, particularly when the procedure is associated with obstruction or perforation . This stThis was a retrospective cohort study performed at a large, multi-site NHS Foundation Trust. We studied two groups of CRC patients, one who presented before the COVID-19 pandemic (Pre-COVID: from March 2017 to February 2020) and the second who presented during the pandemic (COVID group: from March 2020 to March 2021). To avoid an odd percentage in the pre-pandemic year, the pre-COVID group included data from three preceding years (March 2017- February 2020) to more accurately reflect our practice. Study approval was gained from the audit department in our Trust as a retrospective audit on practice.All patients diagnosed with colonic or rectal adenocarcinoma between March 2017 and February 2021 were included. Patients’ data were identified retrospectively through the CRC multidisciplinary team (MDT) records. Data were collected from the Trust’s CRC dataset (Somerset Cancer Register). Patients with emergency or elective presentations were included, and the referral source was also identified. Any patient who needed to be evaluated by the on-call general surgery team was classified as an emergency presentation. Patient data were anonymized.Statistical analysis was performed. Percentages were used for descriptive statistics. Student’s t-test was used for the comparison of demographic variables. Chi-squared test was used for the difference analysis for the categorical data, such as TNM staging and mode of presentation. The statistics software program used was Jeffreys’s Amazing Statistics Program (JASP 0.16.1 2021).In total, 1373 patients were diagnosed with CRC during the period from March 2017 to March 2021. The pre-COVID group (2017-2020) included 1104 CRC patients, compared to 269 patients in the COVID one (2020-2021). The mean age was higher in the pre-COVID group (p = 0.001). There was also a 1.5-fold increase in the percentage of patients who presented in the emergency in 2020-2021 compared with 2017-2019 (p = 0.001)  the number of CRC patients that presented as an emergency increased during the COVID-19 pandemic (03/2020 to 02/2021) compared with the previous three years; (2) locally advanced (T4) and metastatic (M1) diseases’ percentages increased during the coronavirus widespread. Shinkwin et al. reported similar results with increasing numbers of CRC emergency presentations and T4 stages during the first wave of the 2020 lockdown . They hiLarge bowel blockage and intestinal perforation are the two most prevalent CRC presentations in emergencies . MorbidiDuring the pandemic, endoscopic CRC screening was disrupted because of cancellation and patient concerns apprehension over getting COVID-19. These service interruptions have resulted in delayed and advanced presentations of CRC patients with the potentially poorer clinical outcome than usual standards. We have no long-term follow-up yet to look at the effects of the pandemic on the long-term survival of patients with CRC; however, management of CRC in more advanced stage results in a poorer prognosis, as cited in many previous studies ,19.The COVID-19 pandemic reduced emergency surgical operations and overall admissions to emergency departments because of the widespread hospital fear and anxiety experienced by most patients during the peak of this outbreak. The causes of the sharp reduction of emergency surgical cases during the COVID-19 pandemic are multi-factorial, reflecting a dangerous situation with serious consequences for public health. According to the results of Mulita et al., patients in the COVID era presented to the hospital with delayed onset of symptoms, in comparison with those admitted before the COVID pandemic, because of their anxiety and fear of being infected with the coronavirus. As a result the hospital stay, as well as the operation duration of patients in the COVID era, was increased .We can learn lessons from the COVID-19 pandemic that will hopefully stand us in good stead for any future pandemics. Encourage patients to continue presenting to primary care with worrying symptoms in a timely fashion with support from public health campaigns and to ring-fence cancer services and diagnostics to prevent unnecessary delays.Developing resilient primary health care is mandatory for better management of future pandemics. We can use new policies in frontline health services such as reshaping the roles of community health professionals and giving them more room to deal with chronic patients (e.g. pharmacist prescription of regular medications). Community facilities also need to be equipped with innovative digital technology that delivers virtual care that could encourage the vulnerable population to present early and avoid any risky delays .The limitation of our study is that retrospective study that represents the COVID-19 effect on cancer service in a single NHS Trust. We have also not included survival analysis for the patients who presented after the coronavirus pandemic to examine the direct effect of a pandemic on CRC long-term oncological outcomes. Further multicentric trials with prolonged follow-up periods are mandatory to calculate these outcomes accurately.This study reported a significant rise in CRC emergency presentations, locally advanced (T4) and metastatic (remote) disease for patients who presented during the COVID-19 pandemic compared to those who presented pre-pandemic. The reported effects are most likely because of a pandemic’s impact on cancer diagnostic services and change in treatment pathways, but also a change in patients’ behaviour with a reluctance to seek medical care, especially the elderly.In pandemics similar to coronavirus, new public health strategies are required to primarily protect cancer services from unnecessary delays that result in advanced and emergent CRC presentations. People need more motivation to continue attending primary care once they express any concerning symptoms.


Tumour seeding along the needle tract following core needle biopsy of the parotid is a recognised complication. We present a unique case of mucoepidermoid carcinoma of the parotid in an 18-year-old patient with associated tumour seeding within the core needle biopsy tract. Tumour seeding was confirmed both histologically and radiologically on magnetic resonance imaging as early as 35 days post-biopsy. The patient was treated successfully with a combination of surgery and adjuvant proton beam therapy. This case also visually demonstrates a surgical approach to en-block excision of the mass and tract. Mucoepidermoid carcinoma represents the most common type of parotid malignancy . ClinicaThis report describes a confirmed case of core needle biopsy seeding from a mucoepidermoid carcinoma of the parotid in a young patient, including confirmatory imaging and histology. To the author's knowledge, there is no radiological evidence of tumour seeding post-core needle biopsy of the parotid in the published literature. This case also visually demonstrates a surgical approach to en-bloc excision of the mass and tract.An 18-year-old female presented with a six-month history of a non-painful fluctuant swelling overlying the right parotid gland, with no facial weakness. The patient was a non-smoker, had no other co-morbidities, and only took a combined contraceptive pill. An ultrasound of the neck demonstrated a 15x14x10 mm, well-defined, homogeneously hyperechoic lesion within the superficial portion of the right parotid gland, suggestive of a pleomorphic adenoma. A 6 mm diameter intra-parotid lymph node was noted lying superiorly within the superficial lobe, non-pathological in appearance. The rest of the parotid gland appeared normal. Two 1 cm long ultrasound-guided core biopsies were obtained using 18-gauge cutting biopsy needles. Histopathological testing and fluorescent in situ hybridisation (FISH) testing for the MAML2 gene was performed. Morphological features, supported by FISH findings, were those of mucoepidermoid carcinoma. There were no high-grade features in the biopsy.Subsequent contrast-enhanced computed tomography (CT) of the neck and chest and magnetic resonance imaging (MRI) of the neck were performed 35 days later. CT imaging demonstrated a 3 mm indeterminate subpleural nodule within the right lower lobe, unlikely to represent metastasis. Follow-up CT imaging in three months was recommended. The MRI demonstrated a 1.5 cm enhancing nodule in the superficial lobe of the right parotid gland and a linear tract to the skin surface, suspicious for local tumour extension  and fine-needle aspiration cytology (FNAC) remain established techniques in the diagnosis of head and neck masses . NumerouReviewing the literature, tract seeding is frequently reported in the assessment of breast malignancy and these commonly present late postoperatively with a palpable mass. A published case report shows MR imaging of a tract seeding preoperatively with breast malignancy . The casAdjuvant treatment in the form of proton beam therapy (PTB) can be used as an alternative to intensity-modulated radiation therapy (IMRT) for patients with salivary gland tumours, as was the case with this patient. Some studies including a 2020 study by Chuong et al. have recommended that PBT be strongly considered when ipsilateral radiation therapy is indicated for major salivary gland cancer due to a significantly lower incidence of acute toxicity and hence side effects .In conclusion, we highlight the importance of histological diagnosis in the investigation of patients presenting with parotid masses even in low-risk patients. This case demonstrates, to our knowledge, the only radiological evidence of tumour seeding post-core needle biopsy of a parotid mass. It also visually and neatly demonstrates a general principle of en-block oncological resection when two foci of diseases are involved.


A strictly intraglomerular two-photon laser-induced injury model was established. Labeled renin cells (RC) in transgenic renin reporter mice were fate-traced in healthy and injured glomeruli over several days by intravital microscopy and quantified via new three-dimensional image processing algorithms based on ray tracing. RC in healthy glomeruli demonstrated dynamic extraglomerular protrusions. Upon intraglomerular injury the corresponding RCN first increased in volume and then increased in area of dynamic migration up to threefold compared to their RCN. RC started migration reaching the site of injury within 3 hours and acquired a mesangial cell phenotype without losing physical RCN-contact. During intraglomerular repair only the corresponding RCN responded via stimulated neogenesis, a process of de novo differentiation of RC to replenish the RCN. Repeated continuous intravital microscopy provides a state-of-the-art tool to prove and further study the local intraglomerular RCN repair feedback system in individual glomeruli in vivo in a quantifiable manner.The juxtaglomerular niche occupied by renin cells (RCN) plays an important role in glomerular repair but the precise temporal and spatial interrelations remain unclear. This study proposes the hypothesis of a local intra-extraglomerular regenerative feedback system and establishes a new quantifiable system for RCN responses in individual glomeruli   Selectel biopsy  in the oThree-dimensional image processing and analysis was performed with Imaris 9.7.2 (Bitplane AG). Here, surface models of the renin cells and injured areas were created via intensity thresholds across all compared and normalized time points. Glomerular borders were generated by defining object edges in multiple z planes and then automatically extrapolated. The glomerular centers were also automatically calculated by volume statistics of the glomerular borders. All surface models and images were corrected for spatial drift and aligned to one another in image stacks.Novel automated three-dimensional image quantification was realized by the Marching Cubes algorithm. Image stacks were used to calculate a surface model described by triangles. The calculation of the directional thickness of the JGA in relation to the glomerular center in healthy glomeruli and to the injury center in laser-injured glomeruli was based on an adapted ray tracing method. A reference coordinate system was defined in these centers and used as the basis for further calculations. In this way, the point of origin 1, 2 see . The dirEq 4 see . For eacEq 5 see . By inseated see . Thus, Eated see  followedtrue see . The resThis novel image quantification method was applied to all intravitally imaged time points and the data was plotted in polar charts generated with the Python package plotly, as shown in two representations . The amoQuantifiable image deviation was calculated with the Python packages opencv-python and numpy and used as a representative surrogate parameter for glomerular perfusion. In brief, images were converted in two-dimensional float32 values, from which pixel standard deviations were calculated.r2 factors. D’Agostino-Pearson omnibus normality tests confirmed Gaussian distributions. Data was compared either with Student’s t test, or with two-way ANOVA tests and Tukey’s multiple comparisons test calculated individual mean ± SD values. P < 0.05 indicates statistical significance.Statistical data was calculated in GraphPad Prism 9.0. Linear regressions are indicated with corresponding All procedures were prospectively approved by the local authorities (TU Dresden and Landesdirektion Sachsen).TdTomato-labelled RC of the JGA in healthy glomeruli showed a constant flux of surface motion  amounted to an average of 76.71 ± 16.14% of RC from the first intravitally imaged time-point (100%) of the respective glomerulus. All underlying individual glomerular origin areas are listed in Comparison of pooled and time-matched RC migration based on the change of distance and volume revealed a decreased distance to the injured area  and an ip = 0.0352.To demonstrate that laser injury did not lead to global glomerular death but rather to a RC-mediated repair reaction, repetitive longitudinal perfusion of glomeruli was monitored. Healthy perfused glomerular capillaries were visible through Angiospark 680 (magenta) during intravital imaging before laser irradiation T0, . The AngEndpoint immunohistology with complementary pseudocolors further characterized RC migration with tdTomato positive RC (green) overlapping (white) with α8Integrin (magenta) in injured glomeruli but not in healthy glomeruli (de novo differentiated RC cluster can be observed via a gradient from tdTomato (non-RC) to GFP  to GFP (RC marker).Using constitutive transgenic mRenCre-mT/mG mice with GFP-expressing RC (green) and tdTomato-expressing non-RC (magenta), RC neogenesis important for maintaining the RCN can be identified over time, when in vivo migration model system using longitudinal intravital microscopy combined with an intraglomerular laser-induced injury model allows new insights in the physiological and regenerative behavior and regulation of the RCN at the JGA. With the development of this model system, it is possible to describe and quantify fast and continuing alterations of RC protrusions under physiological conditions associated with its juxta/extraglomerular position. Applying intraglomerular injury to this model system in individual glomeruli showed that a locally acting feedback system gives notice of intraglomerular injury specifically to the associated JGA, leading to a timely (one to 3 days) and spatially coordinated repair response via directed migration of juxtaglomerular RC towards the site of injury. Hereby, the migrating RC switch their phenotype towards mesangial cells by expressing α8Integrin and during process completion continuously keep contact to the niche of origin. This repair response is also evident in the return to a physiological state of glomerular perfusion after laser injury and RC migration. The demonstrated glomerular leakage of Angiospark 680 filled vascular fluid represents a mechanical consequence of the laser irradiation and disappears a certain time after the onset of the repair response.The presented novel quantifiable Besides the above described intra-/extraglomerular feedback repair mechanism, a second feedback system regulates specifically the maintenance of the RCN after intraglomerular RC migration. During the intraglomerular repair process by migrating RC, stimulation of their own replenishment via RC neogenesis was observed specifically in the corresponding RCN, a process in which nonRC switch their phenotype to a RC for the first time.With this novel model in hand, understanding of the intra-/extraglomerular as well as RCN feedback mechanisms and its mediators could now open the door for new treatments for glomerular disease via stimulating/regulating these important endogenous renal repair mechanisms.in vivo migration assay to study the hypothesis of a strictly locally regulated intraglomerular-extraglomerular feedback system for a RC-mediated response to injury in such a complex environment inflammatory response . Once thThis quantitative model system of the migratory response of RC after site-directed injury can now be used for comparative studies in different transgenic mouse strains or established therapies such as ACE-I, ARBs, SGLT-2 inhibitors, corticosteroids or may serve to find novel therapies.Starting from the introduced hypothesis of a feedback system, several mechanisms may be relevant to inform the JGA about intraglomerular injury . Either in vivo in a quantifiable manner. This model system may open the door for developing new treatments for glomerular disease via stimulating/regulating this important, endogenous renal repair mechanism.In conclusion, repeated continuous intravital microscopy provides a state-of-the-art tool to prove and further study the local intraglomerular injury - extraglomerular RCN repair feedback system in individual glomeruli


Extrachromosomal circular DNA (eccDNA) is a special class of DNA derived from linear chromosomes. It coexists independently with linear chromosomes in the nucleus. eccDNA has been identified in multiple organisms, including Homo sapiens, and has been shown to play important roles relevant to tumor progression and drug resistance. To date, computational tools developed for eccDNA detection are only applicable to bulk tissue. Investigating eccDNA at the single-cell level using a computational approach will elucidate the heterogeneous and cell-type-specific landscape of eccDNA within cellular context. Here, we performed the first eccDNA analysis at the single-cell level using data generated by single-cell Assay for Transposase-Accessible Chromatin with sequencing (scATAC-seq) in adult and pediatric glioblastoma (GBM) samples. Glioblastoma multiforme (GBM) is an aggressive tumor of the central nervous system with a poor prognosis. Our analysis provides an overview of cellular origins, genomic distribution, as well as the differential regulations between linear and circular genome under disease- and cell-type-specific conditions across the open chromatin regions in GBM. We focused on some eccDNA elements that are potential mobile enhancers acting in a trans-regulation manner. In summary, this pilot study revealed novel eccDNA features in the cellular context of brain tumor, supporting the strong need for eccDNA investigation at the single-cell level. However, there is a special class of DNA, extrachromosomal circular DNA (eccDNA), that is derived from linear chromosomes and exists independently in the nucleus Considering the diversity and functional importance of eccDNA in tumorigenesis, understanding of cellular regulation involving eccDNA is currently lacking due to the absence of comprehensive characterization at both bulk tissue and single-cell level. Apart from traditional imaging methods such as optical microscopy and electron microscopy, various computational tools have been developed to capture eccDNA using sequencing data. These tools include AmpliconArchitect Compared to the WGS approach, ATAC-seq presents a more convenient and effective way of building the eccDNA landscape at a larger scale. Despite the rapid advancement of eccDNA research, previous studies have mainly focused on using bulk sequencing, which cannot detect the cell-type-specific regulatory landscape of eccDNA during tumorigenesis. Here, we extended the scope of eccDNA research into the single-cell level, hoping to gain the true biological resolution to decode the additional cellular regulatory heterogeneity introduced by eccDNA, and to encourage eccDNA-centered studies in human cancers.22.1eccDNA has been observed in both ATAC-seq and WGS in glioblastoma multiforme (GBM) We collected and integrated scATAC-seq dataset for a total of nine GBM samples 2.2Our identified eccDNAs are largely overlapped with the intergenic regions. The relationship between these regions and distal regulators such as enhancers was thus investigated. We downloaded the ENCODE candidate cis-regulatory elements combined from all cell types 2.3To further elucidate the regulatory role of these eccDNAs, we increased the resolution of analysis to the single-cell level and successfully co-mapped the eccDNA reads to condition- and cell type-specific signatures. By integrating our in-house approach of cell-barcode tracing strategy as well as routine quality control of cell filtering An example of eccDNA  of a group of VDJ-recombination genes on the linear chromosome 2. As demonstrated in Altogether, this mosaic nature of eccDNA distribution across various cell types in human tumors cannot be elucidated using bulk sequencing data. More in-depth genome-wide studies characterizing eccDNA in human cancers are in pressing need to yield a better understanding of their contributions to tumorigenesis.2.4Our computational approach revealed the landscape of eccDNAs in human GBM. It not only demonstrated the feasibility of detecting and studying eccDNAs at the single-cell level, but also provided better biological resolution when compared to previous eccDNA studies using bulk RNA sequencing. By integrating scATAC-seq data from nine adult and pediatric GBM samples, we provided an atlas view of the cellular origin, the aligned linear genomic distribution as well as differential regulation between linear and circular form under disease- and cell-type-specific conditions across the open chromatin regions in GBM.Downstream analysis led us to further investigate the extensive overlap between eccDNA-mapped linear genomic locations and distal regulatory elements. Our results highlighted that the majority of our identified eccDNAs  originated from the linear chromosomes where at least one distal regulator was present, for example enhancers and/or insulators. Previous studies demonstrated that long eccDNAs  could harbor whole genes and, therefore, they act as an amplifier of oncogenes involving tumorigenesis and drug resistance Collectively, our results suggested that eccDNA might act as mobile regulators, thus contributing to the vast amount of heterogeneity observed in, but not limited to, human GBMs. Other forms of circular nucleic acid sequences  have been extensively studied for functional role in a variety of tissues and tumorigenesis. This study of eccDNA sheds light on their potential roles in cellular function and cancer biology By comparing the eccDNA distributions across multiple groups, we observed a higher number of locus-specific eccDNAs inferred in the adult than pediatric GBM samples. eccDNAs were previously reported as apoptotic products Remarkably, the eccDNAs identified in this study were largely mapped to intergenic regions, which is somewhat contradictory to the previous reports The vast majority of cells (67.77%) harboring eccDNAs were filtered out by the routine processing pipeline when considering all relevant quality control metrics. As eccDNAs are potentially generated during apoptotic process In addition, the low detection rate of eccDNAs in our study, as indicated that 67.77% of the cells harboring eccDNAs were filtered out during quality control, could also result from the high drop-out rates and data sparsity due to the loss of DNA material in the scATAC-seq protocol There are several main limitations in our study. The length of eccDNAs we identified was short in general, ranging from tens of kb to ∼150 kb, which was a potential bias of the eccDNA detection tool chosen in this study. When we learn more eccDNA structures and how they affect the mapping process of sequencing reads, better rules to define the circular structure will evolve accordingly. In particular, ecc_finder initially identifies eccDNAs longer than 1 Mb. However, the subsequent stringent quality control step that requires an even distribution of split and discordant reads accidently throws out all the long hits, because the middle portions of these eccDNAs lack sufficient reads to undergo peak naming with stringent false discovery rates. We propose a potential workaround in the future which loosens the above assumption by focusing on split reads only and manually tracking breakpoints spanning several Mb in the reference genome. Quality control measures remain to be further investigated, since candidate long eccDNAs tend to be false positives. In addition to algorithm limitations, representation of long-read eccDNAs in our study is limited by the current sequencing approach. A more efficient approach is to eliminate or reduce linear genomes in the library preparation using exonuclease, so that circular sequences will be enriched Taken together, our study presents a novel approach to study eccDNA at the single-cell level, which also yields novel insights into the potential regulatory roles of eccDNA in tumorigenesis from both disease- and cell-type-specific perspectives. This has the potential to expand our current understanding of eccDNA based on bulk sequencing approaches. Given the presence of eccDNA in both tumor and tumor microenvironment, and its role associated with tumorigenesis and drug resistance, we expect the refinement of currently available methods to extend eccDNA research. One promising avenue is the rapid development of new algorithms specifically targeting eccDNA at the single-cell level to gain more coverage in the near future.33.1A total of nine GBM samples profiled by scATAC-seq 3.2The algorithm ecc_finder 3.3Group-wise comparisons were carried out using non-parametric Wilcoxon Rank Sum and Signed Rank tests to calculate the statistical significance.10.13039/100000002National Institutes of Health (NIH) grant (R01LM012806) and the 10.13039/100004917Cancer Prevention and Research Institute of Texas grant (CPRIT RP180734 and RP170668). ZZ was also partially supported by 10.13039/100000002NIH grants R03AG077191 and R01DE030122. JK is a CPRIT Predoctoral Fellow in the Biomedical Informatics, Genomics and Translational Cancer Research Training Program (RP210045).This work was partially supported by HF and ZZ conceived the study. HF, JK, YD and JL collected the data, performed the data analysis and interpreted the results. JK, HF and ZZ wrote the manuscript. All authors read and approved the final manuscript.None.The authors declare no conflict of interest.


During the SARS-CoV-2 pandemic, genomic data generation has exceeded the capacities of existing analysis platforms, thereby crippling real-time analysis of viral evolution5. Here, we use a new phylogenomic method to search a nearly comprehensive SARS-CoV-2 phylogeny for recombinant lineages. In a 1.6 million sample tree from May 2021, we identify 589 recombination events, which indicate that around 2.7% of sequenced SARS-CoV-2 genomes have detectable recombinant ancestry. Recombination breakpoints are inferred to occur disproportionately in the 3' portion of the genome that contains the spike protein. Our results highlight the need for timely analyses of recombination for pinpointing the emergence of recombinant lineages with the potential to increase transmissibility or virulence of the virus. We anticipate that this approach will empower comprehensive real-time tracking of viral recombination during the SARS-CoV-2 pandemic and beyond.Accurate and timely detection of recombinant lineages is crucial for interpreting genetic variation, reconstructing epidemic spread, identifying selection and variants of interest, and accurately performing phylogenetic analyses A new phylogenomic method is developed that can detect recombinations in virus lineages in pandemic-scale datasets. By mixing genetic material from diverse genomes, recombination can produce new combinations of mutations that have potentially important phenotypic effects7. For example, recombination is thought to have played an important role in the recent evolutionary histories of Middle East respiratory syndrome8and severe acute respiratory syndrome coronavirus (SARS-CoV)12. Recombination might also have the potential to generate viruses with zoonotic potential in the future13. Therefore, accurate and timely characterization of recombination is foundational for understanding the evolutionary biology and infectious potential of established and emerging pathogens in human, agricultural and natural populations.Recombination is a primary contributor of new genetic variation in many prevalent pathogens, including betacoronaviruses14 and co-infection with different SARS-CoV-2 variants has been known to sometimes occur15, recombination is expected to be an important source of new genetic variation during the pandemic. Whether or not there is a detectable signal for recombination events in the SARS-CoV-2 genomes has been fiercely debated since the early days of the pandemic13. Nonetheless, several apparently genuine recombinant lineages have been identified using ad hoc approaches16 and semi-automated methods that cope with vast SARS-CoV-2 datasets by reducing the search space for possible pairs of recombinant ancestors17. Because of the importance of timely and accurate surveillance of viral genetic variation during the continuing SARS-CoV-2 pandemic, new approaches for detecting and characterizing recombinant haplotypes are needed to evaluate new variant genome sequences as quickly as they become available. Such rapid turnaround is essential for driving an informed and coordinated public health response to new SARS-CoV-2 variants.Now that substantial genetic diversity is present across SARS-CoV-2 populations18. RIPPLES exploits that signal by first identifying long branches on a comprehensive SARS-CoV-2 mutation-annotated tree20. RIPPLES then exhaustively breaks the potential recombinant sequence into distinct segments and replaces each onto a global phylogeny using maximum parsimony. RIPPLES reports the two parental nodes—hereafter termed donor and acceptor—that result in the highest parsimony score improvement relative to the original placement on the global phylogeny ) and other lineages, VOCs contribute slightly fewer spike protein mutations than non-VOC lineages on average . Third, recombinant clade size does not greatly differ from the remaining clade sizes, which would be expected if recombinant lineages experienced strong selection . Therefore, although natural selection on between-host transmission dynamics of recombinant lineages could also impact the observed distribution of recombinant breakpoint positions11, our data indicates that other biases shape the distribution of recombination events across the SARS-CoV-2 genome. These could include a neutral mechanistic bias affecting the distribution of recombination breakpoints.Several lines of evidence suggest that the skewed distribution of recombination breakpoint positions is not a consequence of positive selection at the level of between-host transmission dynamics. First, many of these recombinant clades have existed for a relatively short period of time, and might already be extinct. The mean timespan between the earliest and latest dates of observed descendants of detected recombinant nodes is just 37 days. Second, of the subset of recombination events that we inferred to occur between variants of concern  versus those resulting from de novo mutation (M), R/M, is commonly used to summarize the relative impacts of these two sources of variation22. Using our dataset of putative recombination events, we estimate that R/M = 0.00264 in SARS-CoV-2 (Supplementary Text R/M is estimated to be 0.25–0.31 (ref. 22)), which presumably reflects the extremely low genetic diversity among possible recombinant ancestors during the earliest phases of the pandemic and the conservative nature of our approach. As SARS-CoV-2 populations accumulate genetic diversity and co-infect hosts with other species of viruses, recombination will play an increasingly large role in generating functional genetic diversity and this ratio could increase24. RIPPLES is therefore poised to play a primary role in detecting new recombinant lineages and quantifying their impacts on viral genomic diversity as the pandemic progresses.Although not yet widespread among circulating SARS-CoV-2 genomes, recombination has measurably contributed to the genetic diversity in SARS-CoV-2 lineages. The ratio of variable positions contributed by recombination (25). RIPPLES discovers 223 recombination events within branches of the same Pango lineages. Our results also include 366 interlineage recombination events  (Supplementary Text RIPPLES uses the space-efficient data structure of mutation-annotated trees (MATs)https://github.com/bpt26/recombination/. These combinations yielded 2,000 total simulated recombinant lineages. We then measured the ability of RIPPLES to detect breakpoints as a function of the position of the breakpoint and the minimum genetic distance from the recombinant node to either parent  that comprised a recombinant event, we downloaded the consensus genome sequence for the nearest descendants of each node from COG-UK, GenBank, GISAID and the China National Center for Bioinformatics. We then aligned the sequences of all descendants for each trio using MAFFTWe also performed post hoc analysis using sample metadata to determine whether the ancestors of the recombinant nodes had higher spatial or temporal overlap than expected by chance. We computed geographic overlap as the joint probability of choosing a sample from the same country from the descendants of the donor and the acceptor nodes. For temporal overlap, we recorded intervals from the earliest to the most recent sample descended from the donor and acceptor, respectively, and calculated the minimum number of days separating the two intervals . We generated a null distribution for both categories by selecting, for each detected trio, two random internal nodes from the tree with a number of descendants equal to the real donor and acceptor respectively. We then calculated geographic and temporal overlap in the same way for this random set  Consortium.Supplementary Table 5This file contains acknowledgements recognizing originating and submitting laboratories for data from the National Center for Biotechnology Information database.


Human Mesenchymal Stem Cell (hMSC) immunotherapy has been shown to provide both anti-inflammatory and anti-microbial effectiveness in a variety of diseases. The clinical potency of hMSCs is based upon an initial direct hMSC effect on the pro-inflammatory and anti-microbial pathophysiology as well as sustained potency through orchestrating the host immunity to optimize the resolution of infection and tissue damage. Cystic fibrosis (CF) patients suffer from a lung disease characterized by excessive inflammation and chronic infection as well as a variety of other systemic anomalies associated with the consequences of abnormal cystic fibrosis transmembrane conductance regulator (CFTR) function. The application of hMSC immunotherapy to the CF clinical armamentarium is important even in the era of modulators when patients with an established disease still need anti-inflammatory and anti-microbial therapies. Additionally, people with CF mutations not addressed by current modulator resources need anti-inflammation and anti-infection management. Furthermore, hMSCs possess dynamic therapeutic properties, but the potency of their products is highly variable with respect to their anti-inflammatory and anti-microbial effects. Due to the variability of hMSC products, we utilized standardized in vitro and in vivo models to select hMSC donor preparations with the greatest potential for clinical efficacy. The models that were used recapitulate many of the pathophysiologic outcomes associated with CF. We applied this strategy in pursuit of identifying the optimal donor to utilize for the “First in CF” Phase I clinical trial of hMSCs as an immunotherapy and anti-microbial therapy for people with cystic fibrosis. The hMSCs screened in this study demonstrated significant diversity in antimicrobial and anti-inflammatory function using models which mimic some aspects of CF infection and inflammation. However, the variability in activity between in vitro potency and in vivo effectiveness continues to be refined. Future studies require and in-depth pursuit of hMSC molecular signatures that ultimately predict the capacity of hMSCs to function in the clinical setting. Cftr gene result in a wide range of phenotypes and severity in people with CF, but in most cases, patients suffer from severe pathophysiologic consequences and experience premature mortality. The advent of small molecule correctors and potentiators, known as modulators, have changed CF survival, and disabling morbidity . The negative controls included the basal medium used to grow the hMSCs, and gentamicin (50 μg/mL) served as the positive control. We had previously validated the sensitivity to gentamicin control and the dynamic range of potency [The hMSCs were evaluated for anti-microbial and antibiotic potency using a clinical isolate of mucoid fections ,22. The  potency .Cftr knockout (CF: tm1KthCftr) or wild type mice (WT: C57BL/6J). The inflammatory human target cell was an immortalized epithelial cell line obtained from a person with CF (Cftr−/−). Control consisted of a transformed cell transfected with an empty vector to provide paired Cftr−/− and Cftr+/+ controls. To correlate the murine in vivo modeling with in vitro potency assays, BMDM were obtained from Cftr−/− and Cftr+/+ mice and monitored for inflammatory response.These potency models utilized transformed epithelial cell lines from a CF patient (IB3 and S9 cells) or bone marrow-derived macrophages from The target cells  were cultured in the presence and absence of 100 µg/mL LPS to mimic pathogen-induced inflammation as seen in CF. Both non-treated and LPS-treated cellular target inflammatory responses were quantified for each of the 8 different hMSC-conditioned media at 1:1 of cell culture medium. Cultures were harvested after 24 h with supernatants harvested, centrifuged and aliquoted. Cell was harvested and processed for RNA using RNA easy for PCR analysis. The anti-inflammatory functional potency of the hMSCs was associated with the capacity to decrease inflammatory molecule production by immune cells. The controls included non-stimulated cell targets and stimulated cell targets cultured with the medium used to grow the parent hMSCs. The inflammatory profile of the target cells  was followed for pro-inflammatory cytokine gene expression  using RT-PCR ,31.Cftr-deficient mice (-tm1KthCftr) were utilized to provide a modeling system that would mimic the excessive inflammatory response to P. aeruginosa infection as seen in CF [Cftr−/− and Cftr+/+ mice were inoculated with a validated slurry of 106 CFUs of viable P. aeruginosa encased in agarose beads via the transtracheal route. Twenty-four hours after inoculation, hMSCs were infused at 106/100 µL through the retro-orbital sinus because of their efficient clearance into the lung. Each group of infected mice (WT and Cftr−/−) were subdivided into hMSC infusion or media control. The mice were monitored daily for 10 days for changes in body weight. Mice were euthanized and evaluated for pulmonary inflammation using bronchoalveolar lavage (BAL) and quantitative bacteriology of BAL fluid and whole lung homogenate.en in CF ,53,54,55LL-37, an antimicrobial peptide is produced by hMSCs . In prevP. aeruginosa and hMSCs as strata. Bacterial CFUs, white cell counts, and cytokine concentrations were log-transformed as necessary to compare between groups or conditions using one or two-way ANOVA, treating donors as experimental blocks. Prism Software (GraphPad Prism 9.0.2) was utilized for analyses. One-way ANOVA and Bartlett’s correction was incorporated to account for multiple variables and correction for data normalization when required. Non-parametric ANOVA analysis was utilized to analyze the correlation between LL-37 and functional antimicrobial activity. Significance was defined as p ≤ 0.05.The data underwent linear or log transformation and were utilized to compare between experimental conditions using unpaired T-tests and one-way ANOVA for statistical analysis ,57. In tIn these studies, eight different pre-GMP hMSC donor preparations were interrogated for anti-inflammatory and anti-microbial potency to identify the ideal hMSC sources for the First in CF Clinical Trial of hMSCs . Screening the eight pre-GMP donors using in vitro anti-inflammatory and anti-microbial assays aided in the selection of an hMSC donor with the potential to provide anti-inflammatory and anti-microbial efficacy in the clinical trial. The two most effective in vitro hMSC preparations were then evaluated in the in vivo murine model of CF lung infection and inflammation to select the potentially most efficacious donor preparation to be used in the clinical trial. The preparation of hMSC donor cells followed the requirements mandated by the FDA for human clinical trials. The systematic selection of the donor hMSC preparation for the CEASE trial was geared towards providing potency and potential efficacy against CF airway infection and inflammation. To directly correlate the in vitro and in vivo potency profiles of hMSCs to the clinical trial data in the Phase I study, future evaluations will be needed since the Phase I study only utilized one donor hMSC source.


Mycobacterium tuberculosis) and host directed drug-target interaction network led to new candidate drug targets for drug repurposing studies. The current work sheds light on host genes and pathways enriched in active tuberculosis disease and suggest potential drug repurposing targets for host-directed therapies.Multi-drug resistant tuberculosis still remains a major public health crisis globally. With the emergence of newer active tuberculosis disease, the requirement of prolonged treatment time and adherence to therapy till its completion necessitates the search of newer therapeutics, targeting human host factors. The current work utilized statistical meta-analysis of human gene transcriptomes of active pulmonary tuberculosis disease obtained from six public datasets. The meta-analysis resulted in the identification of 2038 significantly differentially expressed genes (DEGs) in the active tuberculosis disease. The gene ontology (GO) analysis revealed that these genes were major contributors in immune responses. The pathway enrichment analyses identified from various human canonical pathways are related to other infectious diseases. In addition, the comparison of the DEGs with the tuberculosis genome wide association study (GWAS) datasets revealed the presence of few genetic variants in their proximity. The analysis of protein interaction networks (human and  Mycobacterium tuberculosis (Mtb) is the main causative agent of TB. Around 10% of individuals develop TB when exposed to Mtb and 5% of the infected individuals develop TB within 1-2 years while the remaining 5% develop the disease at any other time (Tuberculosis (TB) is an infectious disease which remained throughout human history. her time . Active her time . Individth leading cause of death globally in 2020. Around 86% of new cases reported around the world in 2020 were contributed majorly by China, Indonesia, the Philippines, Pakistan, Nigeria, Bangladesh and South Africa with India leading the list  and also allow observations of such DEG upon vaccine/drug treatment. Further the DEG analysis also aids in understanding of regulatory mechanisms contributing to the functional consequences.In the current study, a statistical meta-analysis was carried out using whole blood expression profiles from infected TB patients to identify key human transcriptomics signatures characteristic of the disease. The study also utilized host genetic disease association and drug-repurposing analyses to further to prioritize the results. In addition, the gene ontology and pathway-based annotations identified genes and pathways significantly altered in the diseased condition.The whole-blood microarray gene expression profiles of patients with active pulmonary tuberculosis and healthy cohorts were retrieved from NCBI GEO . The datThe background corrected files were processed using limma package in R . The datStatistical meta-analysis was carried out using NetworkAnalyst . NetworkThe strength of the results obtained from meta-analysis was further validated by comparing the genes expressed in latent and control samples from the same datasets. Partial Least Square Discriminant Analysis (PLS-DA) was applied to the DEGs. Significant model was selected by 7-fold cross validation. The model performance was evaluated using the area under the Receiver Operating Characteristic (ROC) curve (AUC). All the above validation process was carried out using mixOmics package in R .Gene and pathway enrichment analysis was carried out using DAVID web server to identify significantly enriched Gene ontology (GO) biological processes (BPs) and KEGG pathways, which were ranked based on the hypergeometric test with FDR cutoff of 0.05 . DAVID wMtb protein interaction network (hmPPI).A comprehensive human protein-protein interaction network (hPPI) was constructed. High confidence, experimentally verified interactions extracted from STRING database was used for the construction . The STRThe highly interconnected components of the hPPI and hmPPI were identified using the Cytoscape plugin CytoHubba. CytoHubba is a user-friendly interface to explore important nodes in biological networks using various topological metrics. The hub genes of the networks were identified using the topological metrics degree and Maximal Clique Centrality (MCC) .The drug compounds interacting with DEG were retrieved from DrugBank Version 5 . DrugBanThe genetic differences between tuberculosis-affected and healthy individuals can give a mechanistic insight about the disease and functional implication of the affected gene. The single nucleotide polymorphisms (SNPs) proximal to the DEG were obtained from GRASP database  . GRASP dThe work plan and approaches implemented in the current study is illustrated in The database querying and filtering identified around 149 GEO microarray datasets for TB-related host response at the time of study (June 2022). Further, filtering based on the study inclusion criteria, a total of six datasets enclosing control and active TB samples were selected for next set analyses Table 1.The results of PCA indicated that the samples were clustered based on the observations of the study. However, after batch correction the samples were clustered based on the disease condition as active TB and control. We also observed a few samples outside the clusters before and after the batch correction procedure Figure 2When the datasets DEG were compared, we identified genes regulated in the same direction. The meta-analysis identified a total of 2038 DEG of which 861 genes were up-regulated and 1177 genes were down-regulated Table 2.via spliceosome, cytoplasmic translation and rRNA processing. Similarly for GO molecular function (MF) analyses, the DEGs were majorly enriched in RNA binding, protein binding and ATP binding. The GO CC analysis of down-regulated DEGs showed involvement in extracellular exosome, cytosol and lysosome. The GO BP analysis identified involvement in defense response to virus, innate immune response and response to virus. The GO MF analysis showed enriched functions such as protein binding, protease binding and MHC class I protein binding.The functional GO was carried out for the up and down regulated DEG identified by the meta-analysis Table 3.The pathway enrichment analysis implemented using DAVID identified various dysregulated pathways mediated by the DEGs Table 4.Salmonella infection, Hepatitis C and Epstein-Barr virus infection. We also observed the presence of Tuberculosis pathway in the list. The presence of tuberculosis pathway in our analysis indicated that the genes identified in the meta-analysis demonstrate their significant association with the disease.The down-regulated DEGs showed involvement in NOD-like receptor signaling pathway, Lysosome and other infectious disease pathways such as Influenza A, Identifying the physical interactions between the proteins will provide clues to combat infection. The mapping of 2038 DEGs along with their partners resulted in a network enclosing 325 nodes and 1460 edges Figure 5Mtb-host protein-protein interactions we did not observe any hub genes based on the topological metrics.The hmPPI interaction network enclosed 99 nodes and established 66 connections with an average of 1 connection between the neighbours Figure 6The DEGs from the study were queried against DrugBank to mine drugs targeting genes which may be used for repurposing against TB. A total of 22 drugs targeting 8 DEGs were obtained after the screening process Table 5.The detection of drug targets which has human genetic support by its involvement in the disease pathology may aid in success of the treatment against the disease by preventing late stage clinical failures. The evidence of involvement in the disease by the 2038 DEGs was retrieved from genome-wide association study (GWAS) datasets. A total of 483 TB-related SNPs were obtained from GRASP database. An overlap between the LD region with a SNP and the DEG location or the enhancer region suggest strong association between the SNP and that particular gene. A total of 33 genes showed association with TB-related SNPs Table 6.The meta-analysis of transcriptomes in this study identified S1PR1 as an up-regulated gene with highest fold change. S1PR1, by the detection of its ligand S1P in the blood and lymph, is crucial for naive lymphocytes to access the circulatory system. S1P-S1PR1 signaling is crucial for regulating immune cell development and function. S1P-S1PR1 signaling is needed for mature thymocytes to leave the thymus and for T/B cells to leave secondary lymphoid organs and enter the blood or lymph in both homeostatic and pathological situations . S1PR1 aMtb by controlling how Mtb moves through the lysosomes and stopping it from spreading in cells. Pre-mRNA splicing plays a crucial role in regulating gene expression and protein diversity. The Serine/Arginine rich (SR) proteins are the major components contributing to the selective splicing mechanism. The disruption in the RNA splicing mechanism can lead to crosstalk in the intricate network interactions. The Mtb infection alters the patterns of alternate splicing within the macrophages by affecting the expression of SR proteins -induced transmembrane (IFITM) gene family members. The IFITM members receive signals for their activation through type I and II IFN stimulation to preclude the establishment of productive infection. Ranjbar et al. show that IFITM proteins inhibit Mtb intracellular growth, indicating that they may contribute to host defense against intracellular bacterial infection  can be found in the article/Collected data, Implemented the analysis and Manuscript writing: NP. Conceived and designed the analysis: MA. All authors contributed to the article and approved the submitted version.This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.We would like to thank Vellore Institute of Technology (VIT) for providing computational facility.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.