Pneumocystis jirovecii pneumonia (PCP) has a substantial impact on the morbidity and mortality of patients, especially those with autoimmune disorders, thus requiring optimal dosing strategies of Trimethoprim–Sulfamethoxazole (TMP-SMX). Therefore, to ensure the safety of TMP-SMX, there is a high demand to review current evidence in PCP patients with a focus on dose optimization strategies; (2) Methods: Various databases were searched from January 2000 to December 2021 for articles in English, focusing on the dose optimization of TMP-SMX. The data were collected in a specific form with predefined inclusion and exclusion criteria. The quality of each article was evaluated using a Newcastle–Ottawa Scale (NOS) for retrospective studies, Joanna Briggs Institute (JBI) critical checklist for case reports, and Cochrane bias tool for randomized clinical trials (RCTs); (3) Results: Thirteen studies met the inclusion criteria for final analysis. Of the 13 selected studies, nine were retrospective cohort studies, two case reports, and two randomized controlled trials (RCT). Most of the studies compared the high-dose with low-dose TMP-SMX therapy for PCP. We have found that a low dose of TMP-SMX provides satisfactory outcomes while reducing the mortality rate and PCP-associated adverse events. This strategy reduces the economic burden of illness and enhances patients’ compliance to daily regimen plan; (4) Conclusions: The large-scale RCTs and cohort studies are required to improve dosing strategies to prevent initial occurrence of PCP or to prevent recurrence of PCP in immune compromised patients.(1) Background:  Pneumocystis carinii pneumonia (PCP), also known as Pneumocystis jirovecii pneumonia has remained the most frequent and highly morbid fungal infection for patients with autoimmune disorders [isorders ,2. The iisorders ,4. The cisorders . Therefoisorders . In the isorders .Co-trimoxazole is the combination of trimethoprim (TMP), which is a pyrimidine analog, and sulfamethoxazole (SMX), which is from the sulfonamide family . TMP-SMXAs with other antibiotics, TMP-SMX must be administered in an appropriate way to achieve adequate antimicrobial activity while reducing concentration-dependent toxicities, necessitating the avoidance of excessive dosage . SpecifiPneumocystis carinii pneumonia”, “Pneumocystis jirovecii pneumonia”, “HIV-patients”, “immune compromised patients” “Co-trimoxazole”, “trimethoprim/sulfamethoxazole”, and “TMP-SMX”. The systematic search assessing the dosing guidelines of TMP-SMX in patients was carried out using Preferred Items for Systematic Reviews and Meta-analysis (PRISMA) guidelines ,21. FreeThe studies reporting the dosing strategy of TMP-SMX in the patients for the occurrence or recurrence of PCP were included in this review. Based on the titles and abstracts, studies of all types with any data on clinical outcomes of TMP-SMX regimens were included for further screening. The retrospective studies, randomized controlled trials (RCTs), and case reports available in English were included in this study. The studies published before January 2000 or having inappropriate and incomplete information were excluded from the study. Titles and abstracts of all included articles collected through the search were screened by two reviewers independently. In case of uncertainty as to whether selected studies met inclusion criteria, they discussed with a third reviewer.Data extraction was performed using the predesigned data collection form for this review using Microsoft Word 2013. Information retrieved from the selected articles included author and year, design, sample size, characteristics of patients, dosing strategy, clinical outcomes, and findings. Data extraction was performed by one of the reviewers and reviewed by another co-author. Any discrepancies were resolved by a third reviewer.The quality of each article was evaluated using a Newcastle–Ottawa Scale (NOS) for retrospective studies, Joanna Briggs Institute, The University of Adelaide  critical checklist for case reports, and Cochrane bias tool for randomized clinical trials (RCTs) ,23,24. TN = 09), literature reviews (N = 04), inappropriate intervention (N = 03), no required data (N = 03), and non-English (N = 06). A total of 13 articles met the inclusion criteria for final analysis [In total, 857 related published studies were identified from grey literature as well as electronic databases. After the removal of the duplicate studies and other reasons, 163 studies were evaluated for eligibility criteria. Based on inclusion and exclusion criteria, 125 studies were excluded after the screening of the titles and abstracts. Most of the studies were identified through reference snowballing. Full-text articles were then screened for final analysis. Twenty-five articles were excluded due to the following reasons: no full-text  or one double-strength  . AlternaNowadays, patients with PCP differ from the patients that were treated 30–40 years ago . The staThis study does have several limitations. Firstly, the selected articles included patients from various countries, so the findings could have been affected by socio-demographic characteristics such as ethnicity, region, lifestyle, and environment. Secondly, the limited number of studies conducted after January 2000 forced us to include all studies that met inclusion criteria. Thirdly, although the quality of the majority of the included studies was good, due to small sample size, this may result in false-negative or false-positive findings.These findings provided the current dosing strategy of TMP-SMX for the prevention and treatment of PCP. The low dose of TMP-SMX provides satisfactory outcomes while reducing the mortality rate and PCP-associated AEs. This strategy also reduces overall economic burden and enhances patients’ compliance to daily regimen plan. For some patient populations, such as PCP emerging on TMP-SMX prophylaxis in HIV-patients, it may still warrant to start prophylactic treatment regimen. Due to limited data available on the optimal dose of TMP-SMX, these findings would support the conduct of large-scale, prospective RCTs and cohort studies to provide guidelines regarding the dosing strategies for the PCP.


Increasing numbers of children and adults with chronic disease status highlight the need for a value-based healthcare system. Patient-reported outcome measures (PROMs) are essential to value-based healthcare, yet it remains unclear how they relate to clinical outcomes such as health and daily functioning. We aimed to assess the added value of self-reported PROMs for health status (HS) and quality of life (QoL) in the long-term follow-up of children with foregut anomalies. We evaluated data of PROMs for HS and/or QoL among eight-year-olds born with congenital diaphragmatic hernia (CDH), esophageal atresia (EA), or congenital lung malformations (CLM), collected within the infrastructure of a multidisciplinary, longitudinal follow-up program. Clinical outcomes were categorized into different outcome domains, and their relationships with self-reported HS and QoL were assessed through multivariable linear regression analyses. A total of 220 children completed HS and/or QoL self-reports. In children with CDH and EA, lower cognition was significantly associated with lower self-reported HS. Due to the low number of cases, multivariable linear regression analysis was not possible in children with CLM. HS, QoL, and clinical outcomes represent different aspects of a child’s wellbeing and should be measured simultaneously to facilitate a more holistic approach to clinical decision making. Survival among children with congenital anomalies has greatly improved in recent decades. As a result, the numbers of children and adults living with a chronic condition are steadily increasing, causing significant financial strain on healthcare systems ,2,3. To Our tertiary level European center of expertise for children born with congenital anomalies offers all children born with congenital anatomical anomalies a standardized multidisciplinary long-term follow-up program . This unTo date, associations between clinical outcomes and self-perceived HS and QoL have never been investigated in children born with anatomical anomalies. Recognition of any relationship could serve to aid clinical decision making with respect to a given child’s own experiences. In time, this might help to optimize the value of healthcare from the perspectives of cost-effectiveness and patient compliance.We hypothesized that impairments in clinical outcomes would be associated with lower self-reported HS, but not with lower QoL. In this study, we aimed to assess the added value of PROMs for HS and QoL in the long-term follow-up of eight-year-old children with congenital foregut anomalies. Therefore, we studied the relationship between clinical outcomes and self-reported HS and QoL by evaluating clinical data from our longitudinal follow-up program.We analyzed available data from children born with congenital diaphragmatic hernia (CDH), esophageal atresia (EA), or congenital lung malformations (CLM) between January 1999 and December 2012, who joined our standardized prospective longitudinal follow-up program for children with congenital anomalies  and who For this study, we selected children who completed a Pediatric Quality of Life Inventory  and/or Dutch-Child-AZL-TNO-Quality-of-Life  questionnaire during follow-up assessments at eight years of age. Standardized assessments of health and daily functioning were performed as standard of care using previously described instruments and methods ,12,13,14Clinical outcomes were categorized into 14 domains , respiratory morbidity, daily use of medication for either a physical or psychological condition, presence of scoliosis, need for tube feeding, and need for home oxygen), modified from previous outcome classification tables ,15. AparDuring neuropsychological assessments, children filled in PedsQL and DUX-25 self-reports. Cognition was measured through intelligence testing. Emotional and behavioral problems (summarized as ‘behavior’) and daily executive functioning were evaluated using parent-proxy reports. Motor function was measured with the movement assessment battery for children. Maximum exercise capacity was evaluated using a treadmill test according to the Bruce protocol.Lung function was tested using spirometry. Presence of GER was routinely determined by 24 h pH-impedance testing in children with CDH and EA. Respiratory morbidity was defined as ≥3 lower respiratory tract infections in the past year requiring antibiotic therapy and/or hospitalization, or presence of vocal cord dysfunction or subglottic stenosis requiring regular follow-up. Treatment modalities affecting daily life, such as daily use of medication for physical and/or psychological conditions and the need for tube feeding and/or home oxygen, were registered. Scoliosis was defined as present when requiring regular follow-up and/or surgical intervention.The following background data were retrieved from electronic patient records: sex, gestational age, birth weight, type and specification of anomaly ,17, presData are presented as frequencies (%) or median (interquartile range). Background data were compared between participants and non-participants using Mann–Whitney-U tests or Fisher’s exact tests. Clinical outcomes were categorized as normal, borderline, or impaired , as normal or impaired , or as yes or no . See p-values. A p-value < 0.05 was considered to indicate significance. All data were analyzed using SPSS version 25.0 .To assess the relationship between the different domains and the PedsQL and DUX-25 scores, multivariable linear regression analyses were performed for each diagnostic group. All PedsQL and DUX-25 subscales were selected as dependent variables, whereas cognition, behavior, daily executive functioning, motor function, maximum exercise capacity, lung function, and GER were selected as candidate independent variables. Subsequently, multiple imputation using fully conditional specification was implemented for all candidate independent variables, using 30 iterations and 50 imputations. Results were pooled over imputed data sets according to Rubin’s rules. Variables with >35% missing values were excluded from the regression analysis. As a result, the regression models included the following independent variables: cognition, behavior, motor function, maximum exercise capacity, and lung function. For the final analyses, sex and MEL were added to the independent variables in order to rule out potential bias. Results are summarized as regression coefficients (B), 95% confidence intervals (CI), and We identified 563 children with CDH, EA, or CLM born between 1999 and 2012, of whom 83% had survived. Of the survivors, 15 children (3%) had syndromes with severe intellectual disability, 101 children with CLM had no surgery within 28 days after birth, and 1 child with CDH had an incidental finding of CLM, and was only analyzed in the CDH group, leaving 350 eligible children. A total of 220 children (63% of eligible number) completed the PedsQL and/or DUX-25 at 8 years of age  . ParticiThe majority of the 114 children with CDH scored normal on cognition, behavior, daily executive functioning, motor function, and lung function. Out of these these 114 children, 98 (86%) scored below normal scores for at least 1 of the 14 clinical outcome domains. Normal maximum exercise capacity was observed in 42% of children. GER was absent in the majority of children who underwent routine 24 h pH-impedance testing. See Lower cognition was significantly associated with lower self-reported HS . Figure The majority of the 93 children with EA scored normal on cognition, behavior, motor function, and lung function. Out of these 93 children, 83 (89%) had below normal scores for at least 1 of the 14 clinical outcome domains. Daily executive functioning was normal in 48% of children, and 46% had a normal exercise capacity. GER was absent in the majority of children who underwent routine 24 h pH-impedance testing. See Lower cognition and behavior were both significantly associated with lower self-reported HS . We founOut of the 13 children with CLM, 8 (62%) had below normal scores for at least one of the 14 clinical outcome domains. Due to the low number of cases, multivariable linear regression was not possible; therefore, the association between the health domains and self-perceived HS and QoL could not be evaluated in these children.In this cohort of eight-year-old children born with foregut anomalies, we evaluated the relationship between generic PROMs and clinical outcomes. The standardized assessments of health and daily functioning indicated favorable clinical outcomes overall. In total, 189 (86%) out of 220 children with CDH, EA, or CLM had below normal scores for at least 1 of the 14 clinical outcome domains. In children with CDH, only lower cognition was associated with lower patient-reported HS. In children with EA, lower cognition and behavior were both associated with lower HS; although, after correction for sex and MEL, only the association with behavior remained. We found no associations between clinical outcomes and patient-reported QoL. Our results suggest that disease-related morbidity is not associated with self-reported HS, nor with self-reported QoL in school-aged children.Determining what matters most to patients is essential to VBHC, the focus of which is improving quality of care while promoting cost-effectiveness . When evOur findings are similar to those previously reported by our research group for other critical conditions. A study in five-year-old neonatal extracorporeal membrane oxygenation (ECMO) survivors also found a relationship between cognition and HS, although proxy-reports were used instead of self-reports . AnotherIn the present study, we found an association between clinical outcome and patient-reported HS, but only for children with CDH and borderline cognition. The lack of relationships between other clinical outcomes and PROMs might be explained by several factors. First, it has been suggested that objective outcome measurements often focus on one physiological aspect, whereas PROMs are multifactorial . Second,The general disagreement between clinical outcomes and a child’s own experiences found in our study indicates that identification of long-term morbidities cannot be based solely on PROMs for HS and QoL. Given that clinical outcomes tend to deteriorate with age in children born with foregut anomalies ,28,29, tHere, we discuss the association between generic PROMs and clinical outcomes. Since condition-specific PROMs have been shown to provide more sensitive information than generic instruments , studiesStrengths of our study include the use of a relatively large and representative cohort of children with a rare congenital anomaly, whose data were collected within the infrastructure of a standardized longitudinal follow-up program. Several limitations need to be addressed. First, due to the small number of children with CLM who underwent surgery in the neonatal period, it was not feasible to assess the relationship between PROMs and clinical outcomes for these children. Nevertheless, we decided to show their data in order to cover the outcome results of a homogeneous group of children born with anatomical foregut anomalies. Second, a relatively large amount of data was missing for several clinical outcomes, notably behavior, daily executive functioning, and GER. These outcomes were added to our follow-up program at a later stage and might have caused confounding bias. Finally, our results may be subject to superiority bias. This tendency potentially disappears as children grow older. Longitudinal studies evaluating PROMs at 8 and 12 years of age are currently being performed by our department.In conclusion, apart from an association between lower cognition or behavioral problems and lower patient-reported HS, we found limited relationships between clinical outcomes and PROMs for HS and QoL in school-aged children with foregut anomalies. Based on these findings, HS, QoL and clinical outcomes should be regarded as different concepts that should be routinely measured in order to facilitate a more holistic approach to clinical decision making. PROMs may be useful to prioritize assessment and treatment of comorbidities in relation to a child’s experiences and, as such, can promote VBHC in children with chronic conditions.Our study provides a framework to assess the added value of PROMs in clinical decision making. Future studies should aim to broaden this perspective by including other chronic conditions and disease-specific PROMs. A clearer understanding of these relationships will help optimize healthcare systems while improving clinical outcomes, HS, and QoL in children with chronic disease status.


The Covid-19 pandemic has required nonpharmaceutical interventions, primarily physical distancing, personal hygiene and face mask use, to limit community transmission, irrespective of seasons. In fact, the seasonality attributes of this pandemic remain one of its biggest unknowns. Early studies based on past experience from respiratory diseases focused on temperature or humidity, with disappointing results. Our hypothesis that ultraviolet (UV) radiation levels might be a factor and a more appropriate parameter has emerged as an alternative to assess seasonality and exploit it for public health policies. Using geographical, socioeconomic and epidemiological criteria, we selected twelve North-equatorial-South countries with similar characteristics. We then obtained UV levels, mobility and Covid-19 daily incidence rates for nearly the entire 2020. Using machine learning, we demonstrated that UV radiation strongly associated with incidence rates, more so than mobility did, indicating that UV is a key seasonality indicator for Covid-19, irrespective of the initial conditions of the epidemic. Our findings can inform the implementation of public health emergency measures, partly based on seasons in the Northern and Southern Hemispheres, as the pandemic unfolds into 2021. Problem: One of the most crucial questions for the management of the Covid-19 pandemic has been whether the epidemic would exhibit a consistent seasonality pattern [ pattern . Yet, fr pattern ,4. Many  pattern ). The co pattern  and mist pattern .Background: As the pandemic has engulfed the globe, affecting human activities on an unprecedented level, and concerns that Covid-19 may become endemic in the entire population, as other coronavirus infections have [ons have , the issons have . Other fons have  or publions have . Yet, reons have , an elemons have . Furtherons have , there aons have . Finallyons have .Ultraviolet: The realization that ultraviolet (UV) radiation modulates the infection rate and therefore the spread of the epidemic has grown only recently, based on previous lab studies [ studies . This ha studies  or sophi studies . Detaile studies . These sCausality: Whether UV is just a factor that indicates seasons  or influences the biology of the virus (transmissibility in the open) or the host  is less relevant. There are multiple reports that link vitamin D with UV radiation [adiation , and theadiation  that canadiation . Correlaadiation  and drivMotivation: As the world needs nonpharmaceutical interventions (NPIs) to delay virus spread and contain outbreaks at the national, state or local level [al level , a carefal level . PolicymSeasonality: In this work, we examine Covid-19 incidence rates and attempt to correlate them with social behavior data and seasonal cycles of UV radiation levels across twelve countries. The selection of those countries was based on latitude, geography, comparable demographic or economic indices, epidemic surveillance statistics and availability of UV recordings over a yearly cycle. We showed that the UV level dominated the seasonal pattern of the epidemic and modulated the incidence rate for Covid-19, independently of other parameters, thus providing a well-defined factor to assess seasonality patterns of past, present or future epidemic outbreaks [utbreaks .A list of all world countries was the initial input for target country selection. Steps of data harvesting are listed below. Absolute and relative coastline measures were recorded (length and length/area), reflecting both size and exposure to ocean climate. Socioeconomic indices included literacy levels (percent of population) and density (population/area), as well as GDP in 2019 (per capita). Epidemic surveillance statistics included Covid-19 tests (absolute: tests and relative: tests per million people). We defined all the above features as “filter” parameters, in that they played no further role as inputs in the ensuing correlation and learning models. The structure of the input data is provided as an example, where the country features are recorded as filter parameter vectors and include geo- or socioeconomic indices, as above . DeployiSubsequently, 51 country filter parameter vectors were clustered using k-means (in R), to identify countries with similar statistics, and at least some pairwise similarity to one other country . We omitted countries with a large early spread of the epidemic, such as Italy or Spain. The final selection aimed at the detection of 12 target countries, based on the above criteria. The epidemiological statistics  and their potential relationship to UV radiation or mobility , which we defined as “model” parameters, did not play a role in country selection, other than data availability (for UV radiation rates). The only other element in the choice of target countries was the location across hemispheres, with six countries in the Northern Hemisphere, two countries near the equator and four countries in the Southern Hemisphere, a total of 12 target countries in three distinct, nonoverlapping groups , similarEpidemiological statistics were imported directly as reported, during the period 1 January 2020 to 30 November 2020 . While iMobility statistics were obtained by Apple Covid-19 mobility trends during the entire period examined (starting 21 January 2020). Apple Covid-19 mobility trends are reports published daily and reflect requests for directions in Apple Maps for driving and walking activities . A techn2. We opted to focus on those and not on the much simpler UV index that has a more limited numerical range.Ultraviolet (UV) radiation levels were downloaded from the Tropospheric Emission Monitoring Internet Service (TEMIS) archive . We direModel parameters were either correlated to each other as raw values in logarithmic scale or as dependent, normalized variables with the independent time variable, reported in days. In the latter case, feature scaling with min-max normalization was implemented for comparability and avoidance of incompatibility for extreme  values, as follows: x′ = (x − min(x))/(max(x) − min(x)); where x is m for mobility, p for cases or r for radiation. Empirical comparison of the distributions for p (cases) and r (radiation levels) was performed by computing the absolute difference Δ i = |p′i − r′i| for each country i. Finally, to obtain a single metric of descriptive statistics for each country case, sums of squares (SSQ) Δ i2 of the difference distributions were computed.We applied PCA analysis and analyzed data for the 12 countries on cases per 1 million population, mobility data related to driving and walking with UVDDF to find the correlation between incidence rates and UV radiation levels. Dimensionality reduction by PCA transformed the data by projecting to a set of orthogonal axes and visualized by heat map representations. We used ClustViz  with modhttps://github.com/interpretml/interpret-community/, accessed on 18 February 2020), an open-source Python package for training interpretable models and explain blackbox AI systems. Meta-explainers automatically select a suitable direct explainer and generate the best explanation based on the given model and data sets. In this case, we use the tabular mimic explainer lightGBM. A mimic explainer is based on training global surrogate models  to mimic blackbox models. A global surrogate model is an intrinsically interpretable model that trained to approximate the predictions of any blackbox model as accurately as possible. We interpreted the surrogate model to draw conclusions about the blackbox model, and in this case, to derive support for the hypothesis of UV levels as a seasonality index that modulates Covid-19 daily case dynamics over the reported period in 2020.We utilized automated machine learning (ML) for time series data to find the best model for predictions and extract evidence for feature importance. The ML optimization process, with a multitude of models that need to be tuned for optimal performance and a complex choice of hyperparameters, model types and configuration details, was executed on Microsoft Azure Automated ML for all target countries. Thus, the resulting optimization solution space could be reduced in complexity by the choice of few models among many potential solutions. Experiments for Greece, for instance, resulted in 1921 runs, and the best model was deemed one “voting ensemble”. The cloud service AzureML (“interpret”) was used to implement model interpretability techniques developed by Interpret-Community . OurWorldInData.org 2020: https://ourworldindata.org/coronavirus, accessed on 18 February 2020.Mobility: https://www.apple.com/covid19/mobility, accessed on 18 February 2020.UV radiation: http://www.temis.nl/uvradiation/UVindex.html, accessed on 18 February 2020.https://www.mhlw.go.jp/english/, accessed on 18 February 2020.Ministry of Health, Labour and Welfare. Available from: https://www.nzma.org.nz/journal-articles/new-zealands-elimination-strategy-for-the-covid-19-pandemic-and-what-is-required-to-make-it-work, accessed on 18 February 2020.New Zealand’s elimination strategy for the COVID-19 pandemic and what is required to make it work. Available from: https://blogs.otago.ac.nz/pubhealthexpert/2020/07/22/nzs-team-of-5-million-has-achieved-the-lowest-covid-19-death-rate-in-the-oecd-but-there-are-still-gaps-in-our-pandemic-response/, accessed on 18 February 2020.“Team of 5 million” has achieved the lowest COVID-19 death rate in the OECD—but there are still gaps in our pandemic response. Available from: https://www.stats.govt.nz/indicators/unemployment-rate, accessed on 18 February 2020.Unemployment rate. Available from: No patients were involved in this study.We investigated the reported Covid-19 incidence rates for twelve carefully selected countries with similar characteristics in terms of geography and socioeconomic factors, appropriately distributed around the globe , social behavior reflected in restrictive measures  and the reported Covid-19 incidence rates (collectively defined as “model parameters”), per-country snapshots were derived, in quotidian-independent or -dependent fashion. While regular variation would suggest a seasonality pattern for Covid-19, even exceptions to the rule can be illuminating, explained by historical records, and thus would not challenge the global picture.https://www.reuters.com/article/us-health-coronavirus-japan-idUSKCN24N0JE, accessed on 18 February 2020). Other factors might have also been at play.For example, in the case of Japan, if Covid-19 was seasonal, one would expect a relatively uniform distribution of incidence rates during July 2020: yet, cases started rising after mid-July. This acceleration resulted in more than 1000 daily cases reported nationwide, in 29–31 July . SpecifiAnother rather unique exception is represented by New Zealand: in this case, the country experienced significant outbreaks towards the end of the summer (early March 2020), and quickly shifted to mitigation measures, flattening the observed spread of the epidemic. At the same time, a combination of border controls, favorable geography and a stringent lockdown policy reduced community transmission during the winter period. As a result, New Zealand recorded one of the lowest COVID-19 mortality rates and limited economic damage compared to other high-income countries, so far. Similar success has been recorded in other countries with stringent lockdown policies, especially in Asia.The above examples, however, say little about seasonality trends throughout almost the entire year: we first examined these trends in a coarse-grained manner by selecting three periods in 2020 and associating UVDDF values with raw Covid-19 rates for the three groups of countries, where North vs. South could be equivalenced by a rotation  of the UV dimension (x-axis) .In the first group, Australia—another exceptional case, due to the reported early cruise ship outbreaks—exhibited a high incidence rate towards the end of the summer, i.e., until May 2020 a. In conhttps://uk.reuters.com/article/instant-article/idUKKBN21C0GY, accessed on 18 February 2020), due to regional outbreaks in that period. For Saudi Arabia, which experiences a wider variation of UV levels due to its higher latitude, some seasonality was observed, yet daily cases over the summer soared, most likely as a result of indoor, air-conditioned lifestyle in urban locations. As a matter of fact, both trends were independently confirmed by machine learning-based feature extraction (see below).In the second (control) group, in the two quasi-equatorial countries with similar filter parameters (see Methods), the seasonality pattern should be dampened during the yearly cycle: again, these patterns were somewhat convoluted and, not surprisingly, imperfect. Again, local incidents or social behavior explained exceptions and reinforced the model b. In theIn the third group, all countries followed a highly regular pattern, except New Zealand, for reasons mentioned above c. The reThe quotidian-independent observations, providing a coarse-grained view of the course of the epidemic on a per-country basis , needed Next, in order to demonstrate more clearly the seasonality parameter that UV radiation levels yield against daily Covid-19 cases, despite the restrictive measures imposed, we excluded mobility data as well as the UVDVF measure, focusing exclusively on the time-dependent quotidian-dependent course of the epidemic versus UVDDF levels . These sIt is evident that the observed patterns reflect a degree of seasonality seen here for the very first time over the year 2020. The achieved clarity is due to the availability of daily incidence data for the entire period of the Covid-19 epidemic and the normalization of the dependent model parameters (see Methods). The simple UV-daily case model implied an expected regular course of the pandemic that presented certain opportunities to optimize policy and use of NPIs until the situation is covered by extensive use of successful vaccines and pharmaceuticals. It can be argued that the most regular pattern was furnished by South Africa, as an “ideal”, hypothetical case for an epidemic curve that would be at a peak during the local winter of 2019, if such counts were hypothetically available .The composite signal between the two model parameter curves over time could be obtained with absolute difference curves, as the values were min-max normalized and became fully comparable (see Methods), to depict seasonality patterns and their fluctuating behaviors for the target countries . The difThe model received as input four model parameters, namely date, UVDDF level and mobility data as driving and walking statistics. The conditional parameter that needed to be predicted was Covid-19 daily case rate, using a multitude of potential machine learning models. Model optimization steps selected the best models that yielded the highest performance, i.e., minimal normalized square error. In our experience, the more effective models were the “voting ensemble”  and the “stack ensemble” (for the three countries mentioned). We have found that these robust predictions with respect to the rather simple model structure and rich, complete input data were able to assert the impact of UV levels as a seasonality indicator that was both reliable and highly regular, compared to any other environmental parameter. In some sense, the application of machine learning to substantiate the hypothesis and interpretation of seasonality for Covid-19 represents a software-based reverse engineering strategy that confirmed both our hypothesis  and simiMultiple factors are likely to influence Covid-19 incidence rates, first and foremost, human behavioral patterns as reflected either in individual mobility and social etiquette or in collective countermeasures such as business or school closures and other public health recommendations . These mviz. UV level) and not in the mobility factor, the latter being independent of any natural cycle for 2020. This was even more evident when only UV levels and incidence rates were compared for North-equatorial-South target countries (By selecting target countries which are, to a great extent, due to travel restrictions, self-contained “bubbles” where the epidemic unfolded, the geographical and socioeconomic affinities took out any demographic and cultural differences to a significant extent, as shown by the filter parameter step. Then, the seasonality effects of Covid-19 were exclusively dealt with using UV radiation levels and mobility information that reflected the period of the year and restrictive measures, respectively . It is tountries . Despiteountries .Our approach took advantage of open datasets about demographics, epidemiology, mobility and meteorology and benefits from a near real-time update of these dynamic datasets. The methodology is general enough that could be applicable to the assessment of seasonality for other respiratory infectious diseases. Both the quotidian-independent  and quotThe absolute difference distribution curves integrated UV level and Covid-19 data and unequivocally provided a strong argument for Covid-19 seasonal attributes, supported by machine learning, for very different countries and their restrictive measures, conditioned only on their location around the globe . As argu


The purpose of this study was to determine how Limb Deformity Modified Scoliosis Research Society (LD-SRS) scores differ between patients with different types and degrees of limb deformity compared with control subjects.Patients who were >17 years and scheduled for reconstructive surgery for limb lengthening or angular deformity with internal and/or external fixation and healthy control subjects were prospectively enrolled. Patients completed the LD-SRS preoperatively. Mechanical axis deviation (MAD) and leg length discrepancy (LLD) were recorded preoperatively. Participants were stratified into five groups based on their diagnosis. ANOVA was used to test for associations between LD-SRS scores and diagnosis as well as mechanical axis deviation.P < 0.001 for all). Patients with short stature reported significantly worse LD-SRS Self-Image/Appearance (P < 0.001) and total score compared with control subjects (P = 0.015). There was a significant correlation between LLD and LD-SRS Self-Image/Appearance in the LLD and angular deformity group .Patients with LLD, angular deformity, or combined LLD and angular deformity reported significantly worse scores than control subjects in LD-SRS Function/Activity, Pain, Self-Image/Appearance, and total score (Although LD-SRS scores were worse in patients with limb deformity compared to controls, LD-SRS scores were not related to the degree of deformity in most patients, indicating that patient self-perception may be a construct unrelated to objective radiographic or clinical findings. However, with recent advances in angular deformity correction and limb lengthening, surgical intervention can result in improved patient-reported and clinical outcomes.9Lower limb deformity can have a notable effect on quality of life.10 A modified version of the Scoliosis Research Society (SRS) outcome measure, the Limb Deformity Modified SRS (LD-SRS), is currently the only limb deformity-specific patient-reported outcome measure, which was designed to capture patient perceptions of their physical function, pain, mental health, and self-image.11 However, differences between patients with different types and degrees of lower limb deformities using the LD-SRS have not yet been established.One important indication for surgical intervention in lower limb deformity is its effect on the patient's quality of life. To assess this effect, patient-reported outcome measures (PROMs) are often used. To be useful and valid in the clinical setting, PROMs need to be multidimensional, addressing physical health and subjective perceptions of the patients.Before surgical intervention, it is important to understand how lower limb deformity affects patients' perceptions of their quality of life. The purpose of this study was to determine how LD-SRS scores differ between patients with LLD, angular deformity, short stature, and healthy control subjects. In addition, this study aimed to investigate if and how different degrees or severities of deformity affect patient-reported quality of life. The investigators hypothesized that patients with LLD, angular deformity and short stature would have worse LD-SRS scores than healthy control subjects.After obtaining IRB approval, patients scheduled for a limb deformity surgery at the study institution were prospectively enrolled. All included patients were at least 18 years of age and were scheduled for complex reconstructive surgery for limb lengthening or angular deformity with internal and/or external fixation. In addition, 30 healthy participants who were at least 18 years of age with no history of lower extremity surgery were enrolled prospectively through convenience sampling.11 The PROMIS domains are normalized to a mean of 50 with an SD of 10. Higher scores indicate more of the domain being measured .All participants completed the LD-SRS and a suite of PROMIS domains . Similar to the SRS instrument, the LD-SRS has four domains including Function/Activity, Pain, Mental Health, and Self Image/Appearance. Each domain is scored from 1 to 5, with a higher score indicating better quality of life . The total LD-SRS score is an average of the four domains, and a higher score indicates overall better outcomes. The minimum clinically important difference for the LD-SRS total score is 0.3.Participants were stratified into five groups based on their underlying conditions as follows: (1) short stature, (2) LLD, (3) angular deformity, (4) LLD with angular deformity, and (5) normal control subjects. Surgery for short stature was indicated for patients who presented at or below the fifth height percentile for their age after psychological assessment to rule out underlying psychological conditions . For all the noncontrol patient groups, mechanical axis deviation (MAD) and LLD were recorded from the medical chart. The greatest MAD was calculated between each leg for the patients, and this value was used in statistical analyses. The indication for surgery for LLD was a difference of greater than 18 mm between leg lengths, and for angular deformity, it was a deformity of greater than 5° with symptoms.P = 0.05.Descriptive statistics were reported for all demographic variables. Frequencies and percentages were reported for categorical variables, and means and SDs were reported for continuous variables. One-way ANOVA with Tukey post hoc pairwise analysis was used to determine statistically significant differences between groups of patients. Within the LLD group and the LLD with angular deformity group, Pearson correlation coefficients were calculated for LLD with the PROMs. In addition, within the angular deformity and angular deformity with LLD groups, Pearson correlations were calculated for the greatest MAD in each patient with the PROMs. All tests were two-tailed with significance threshold set at A total of 240 participants were included in this study, including 18 patients with short stature, 49 patients with an LLD, 94 patients with an angular deformity, 49 patients with a combined LLD and angular deformity, and 30 normal control subjects. Patients reported bilateral deformities in two LLD cases, 13 angular deformity cases, and two combined LLD and angular deformity cases. Demographic characteristics are provided in Table P < 0.001 for all domains; Table P < 0.001) and total score (P = 0.015) but were not significantly different in any of the other domains . All significant differences between groups on the LD-SRS total are shown in Figure P < 0.01 for all) but were not significantly different from other patient groups in Self-Image/Appearance. Patients with LLD with angular deformity reported significantly worse Mental Health outcomes than control subjects (P = 0.021), but there were no other significant differences between groups in the Mental Health domain.Patients with LLD, angular deformity, or LLD with combined angular deformity reported significantly worse scores than control subjects in LD-SRS Function/Activity, Pain, Self-Image/Appearance, and total score , but there were no other significant differences between groups in that domain.In the PROMIS domains, patients with LLD, angular deformity, or LLD with angular deformity reported significantly worse scores than control subjects and patients with short stature in Pain Interference, Physical Function, Pain Intensity, and Global Physical Health (P = 0.043). In addition, there was no correlation between MAD and LD-SRS scores in the angular deformity group or the LLD with angular deformity group.When assessing the degree of deformity within patient groups, no significant correlations were found between LLD and LD-SRS scores in the LLD group, but there was a significant correlation between LLD and LD-SRS Self-Image/Appearance in the LLD with combined angular deformity group  found that larger Cobb angles preoperatively were associated with worse pain, function, activity, and self-image.One of the strengths of this study includes the use of a lower limb deformity-specific validated patient-reported outcome measure. While more general Patient-Reported Outcomes Measurement Information System (PROMIS) scales did capture notable differences in outcomes between the patients with limb deformity and control subjects, the LD-SRS also reported differences in self-image/appearance and used fewer overall questions within the single questionnaire. There is currently no PROMIS domain for assessing deformity-related self-image. Therefore, the LD-SRS is an optimal way to assess presurgical lower limb deformity patient perceptions with low burden to the patient.19 In addition, patients were not categorized by underlying diagnosis or number of deformities. These factors might have an effect on self-perception as well. Future prospective studies with more participants should study how these diagnoses affect self-perception. Finally, the investigators selected a small convenience sample of healthy control subjects because of hospital COVID-19 restrictions on research. These control subjects may have had different demographic characteristics than the study population, which may have introduced bias of unclear clinical relevance. Future studies could investigate this further with more closely matched control subjects.In addition to its strengths, this study had several limitations. Because this was a retrospective analysis of prospectively collected data, no a priori power analysis was done. In addition, some patient groups had few participants, so some analyses may have been underpowered. Although this does not weaken the positive findings of this study, drawing conclusions using nonsignificant associations should be made with caution. In addition, several PROMIS forms were incomplete for the included participants, reducing the sample size for PROMIS analyses and therefore weakening conclusions related to PROMIS scores. Another limitation was the lack of an additional self-image or appearance-related scale with which to compare the LD-SRS scores. However, no applicable self-image domain currently exists for PROMIS.In conclusion, this study found notable differences in physical function, pain, and self-image between patients with lower limb deformities and healthy control subjects. This study further validates the LD-SRS as an objective and quantitative measure of patient perception and quality of life in patients with lower limb deformity. Radiographic measurements of degree/severity of deformity were not related to LD-SRS scores on average, indicating that patient self-perception may be unrelated to objective radiographic or clinical findings.


Normal tissue injury from accidental or therapeutic exposure to high-dose radiation can cause severe acute and delayed toxicities, which result in mortality and chronic morbidity. Exposure to single high-dose radiation leads to a multi-organ failure, known as acute radiation syndrome, which is caused by radiation-induced oxidative stress and DNA damage to tissue stem cells. The radiation exposure results in acute cell loss, cell cycle arrest, senescence, and early damage to bone marrow and intestine with high mortality from sepsis. There is an urgent need for developing medical countermeasures against radiation injury for normal tissue toxicity. In this review, we discuss the potential of applying secretory extracellular vesicles derived from mesenchymal stromal/stem cells, endothelial cells, and macrophages for promoting repair and regeneration of organs after radiation injury. The first report of detrimental effects of ionizing radiation on healthy normal tissues came to light after the atomic bomb explosions in 1945. This unprecedented event introduced to the world the lethal radiation poisoning or sickness, also known as acute radiation syndrome (ARS), where a relatively large number of people can be affected by sudden exposure to high amounts of irradiation over a short period of time due to nuclear power plant accidents or atomic war. The extent of damage to an organism depends on the duration and the dosage of radiation with very high mortality after a threshold dose. ARS is a multi-organ failure syndrome caused by a combination of radiation dose-dependent direct cytocidal effects of irradiation on tissue stem and progenitor cells and the supporting sinusoidal endothelial and mesenchymal cells of the stem cell niche, with subsequent neutropenia, anemia, and thrombocytopenia due to bone marrow failure. With higher doses, manifestations of gastro-intestinal (GI)-ARS with loss of the intestinal mucosal barrier, bacteremia, septic shock, and systemic inflammatory response syndrome ensues. In addition to such accidental exposures, normal tissues succumb to radiation during radiotherapy in cancer patients, which is often unavoidable . For insvia radiation-induced generation of reactive oxygen species (ROS), such as superoxide and hydroxide radicals from the radiolysis of intracellular water, which results in the oxidation of biological macromolecules. Radiation-induced single-strand (SSBs) and double-strand DNA breaks (DSBs) are considered the major events leading to cell death, cell cycle arrest, and senescence  are agents administered either as preventive or as mitigators post-exposure to radiation. The mitigators improve radiation-induced physiological damage such as cellular toxicity, apoptosis, and loss of stem cells. The radiation protectants prevent radiation-induced toxicity, for instance, by scavenging the free-radicals and reducing oxidative damage to cells. Several candidate MCMs were being identified and investigated . The cytin vitro and in vivo studies have shown that MSCs from bone marrow are relatively resistant to ionizing radiation and maintain their differentiation potential even exposure to high dose radiation  or iPSC-derived differentiated cells such as hepatocytes, ECs are being investigated for regenerative application   . TherefoEVs are the body's own nanoparticles that constitute a significant cell-to-cell communication system in multicellular organisms. According to the recent nomenclature stated by the International Society of Extracellular Vesicles (ISEV), EVs are broadly classified into plasma membrane-derived large microvesicles of 500–1,300 nm or endoplasmic reticulum-endosome derived small EVs of 30–200 nm . EV memb+/CD105+/CD29+), myeloid cells (CD45+/CD11b+), and ECs (CD34+/CD31+) improved the survival mice exposed to whole-body irradiation (WBI)  culture system enriched for MSCs (CD90on (WBI) . Howevervia EVs  were first reported by via extracellular matrix metalloproteinase inducer  derived EVs could rescue bone marrow hematopoietic cells from radiation damage in vitro and in vivo  is the parameter to determine the volume of mineralized bone per unit volume of the sample. The BV/TV of the non-irradiated mice was 67.6%, while it was decreased to 30.9% in the irradiated mice 28 days post-radiation. However, the BV/TV of the irradiated mice transplanted with BMMSCs and BMMSC-EVs was increased by 53 and 13%, respectively, compared to the irradiated mice without transplantation. Furthermore, incubation of BMMSC-EVs with 6 Gy irradiated BMMSCs in vitro showed a decrease in DSB as determined by γ-H2AX staining at 2, 4, and 12 h post-radiation; an increase in antioxidant effect via increasing the expression of superoxide dismutase (SOD) 1 and 2 at 12 h and 24 h post-radiation and activation of Wnt/β-catenin signaling in BMMSCs, which could be the mechanisms of improving bone loss observed in irradiated rats with BMMSC-EV treatment. This study also showed that BMMSC-EVs treatment restored the radiation-induced loss of BMMSCs differentiation potential as indicated by an increase in calcium deposition, Runx2 expression (osteogenic), and oil red O staining (adipogenic) compared to irradiated cells without treatment (reatment .+) stem cells at the crypt base via a Wnt signaling pathway   and PolyDuring radiotherapy for hepatocellular carcinoma, normal liver tissue is often exposed to radiation. This results in liver sinusoidal endothelial cell toxicity, atrophy of hepatocytes, and occlusion of veins, gradually leading to loss of liver function and progressing toward radiation-induced liver disease (RILD) or radiation hepatitis . An incrvia intra-splenic injection is shown to repair acute and late effects of RILD  accelerated liver regeneration in 70% hepatectomized rats , natural killer (NK) cells, Mɸ polarization, edema, and lung perfusion, eventually activating fibroblasts and myofibroblast differentiation to fibrosis in the irradiated lung . MSCs deHuman umbilical cord-MSCs could repair radiation-induced lung injury (RILI) by inhibiting myofibroblastic differentiation of human lung fibroblasts . Human avia arteries to capillaries and collects the blood via veins for waste removal and purification. The collective action of the blood vascular system and lymphatics maintains the fluid level in the body. All these vessels are lined by ECs that play an important role in vascular homeostasis. In addition, ECs mediate immune responses, involve in inflammation, coagulation, and angiogenesis  when stimulated with TNF-α . Similarin vitro  and mitogen-activated protein kinase (MAPK) signaling -cadherin. At 24 h after either 5 Gy (hematopoietic assays) or 8 Gy  WBI, 1.9 × 109 particles of EV were intravenously injected daily for four days. Irradiated mice with EC-EV treatment showed improved bone marrow cellularity, hematopoietic stem and progenitor cell content, preserved EC architecture, and showed a 50% survival advantage. Among the 48 cytokines tested, EC-EVs increased the expression level of tissue inhibitor of metalloproteinase 1, which is essential in vascular remodeling post-ischemia . This initiates an inflammatory cascade involving recruitment, proliferation, and activation of various hematopoietic and non-hematopoietic mediators , NK cells, B cells, T cells, fibroblasts, epithelial cells, ECs, and stem cells) which collectively work for tissue repair . Mɸs areMɸ-EVs are gaining a lot of interest as therapeutics. Tissue repair potential of Mɸ-EVs was reported in models of atherosclerosis , cardiac+/CD105+/CD29+), myeloid cells (CD45+/CD11b+), and ECs (CD34+/CD31+) (Porcnfl/fl), a gene essential for Wnt synthesis. These Porcn-depleted (null) mice have normal intestine but are hypersensitive to radiation injury compared to wild-type mice. The intestine in these mice showed loss of Lgr5+ cells, reduced crypt depth and number, and decreased survival after WBI (+/CD31+) . After t+/CD31+) . Mɸs act+/CD31+) . Wnt sigfter WBI . These mFurthermore, they have isolated EVs from the BMM-conditioned media using ultracentrifugation. These EVs were positive for TSG101, CD9, and Alix. When tested using T-cell factor/lymphoid enhancer factor (TCF/LEF)-luciferase reporter cell line, BMM-EVs showed activation of Wnt signaling. BMM-conditioned media improved the survival of mice post-18.5 Gy abdominal radiation. In contrast, the EV depleted media could not rescue, indicating that BMM-EVs carrying Wnt ligands that improve intestinal regeneration after radiation injury.The potent anti-inflammatory, anti-fibrotic characteristics of BMMSC-EVs, angiogenic and anti-inflammatory properties of EC- and Mɸ-EVs make them promising candidates for regenerative application. The summary of EVs derived from these three cell types that have been discussed in this review on interacting with various signaling molecules is depicted in in vivo is the crucial driver in the success of MCM and is influenced by the damage model, cell source of EVs, and route of administration. The route of administration of EVs affects biodistribution to various organs , growth factors, and extracellular proteases. This process collectively refers to as senescence-associate secretory phenotype (SASP)  are reported in ARS survivors. Irradiation injury causes DNA damage through free radicals, SSBs, and DSBs. Even though the DNA damage is repaired, the process is not always efficient. The defects in the DNA damage response pathway result in the development of either cell cycle arrest   or cell e (SASP) . The mole (SASP) . This pee (SASP) , oral mue (SASP) , cardiove (SASP) , hypo-sae (SASP) , hematope (SASP) . It has e (SASP) .Further SASP can lead to radiation-induced bystander effect (RIBE) . RIBE isin vivo study, the damaged DNA foci were significantly higher in non-irradiated mouse fibroblast cells when treated with the irradiated mouse's serum-EVs than non-irradiated mouse serum-EVs  ligand 2 (CCL2) and urokinase-type plasminogen activator (Plau/uPA). In this study, aortic MSC and BMMSCs were administered at 0.5 million cells 24 h after irradiation . SimilarEVs are classified based on immunolabeling of EV surface proteins such as tetraspanins, integrins, cell adhesion molecules, and growth factor receptors . The hetStudies that report specific cell-derived EVs should include the passage number of cells, seeding density, and culture conditions, which are essential parameters affecting the production and characteristics of EVs. Also, the dose of EVs used in pre-clinical models is often represented as micrograms of protein or particle number in some studies. Though particle concentration seems to be an ideal reference, such state-of-the-art facilities measuring particle number and concentration of EVs are not always available in every research laboratory. The number of EVs obtained from 1 million mouse BMMSCs under specific culture conditions might differ among each laboratory. Therefore, additional information as mentioned in EVs derived from specific cell types interact with distinct signaling molecules, as described in in vivo; however, ex vivo membrane labeling of EVs or labeled EVs (CD63-GFP) from reporter-cell lines or mice (in vivo and help design better dosing strategies to target radiation-induced injury.The small size and short life span of EVs make it more challenging to track them  or mice  are an aIn situations such as accidental exposure to radiation , the EVsin vitro culture conditions and treatments such as growth factors can influence the quantitative and qualitative production of EVs. For instance, serum-free culture or usage of exosome-free serum is often used to culture cells for EV production, which might alter the natural EV production threshold. On the other hand, parameters such as pH, temperature, and cryopreservation conditions can alter the EV uptake by cells (Biogenesis of EVs is a cellular response to physiological stimuli; therefore, by cells . Therefoin vitro and in vivo functionality assessment.Heterogeneity in the EV sub-populations poses them as challenging for developing as therapeutics. Simultaneously, their diversity in containing various protein, nucleic acid, and lipid in the cargo makes them attractive for targeted delivery with multi-functional activity. Thorough characterization of EVs according to MISEV guidelines and reproducibility in the generation of EVs of interest is essential. GMP grade methodologies for EV preparation need to be developed for clinical translation. To further develop EVs as radiation MCM, the quality of cell source, cell culture, and method of EV preparation need to be standardized and reproducible across respective fields, followed by the With the growing interest in the EVs for research and for clinical use, it is extremely important for universally acceptable methods and standards. ISEV community and its tools such as EV-TRACK and EV-METRIC are supporting EV-based research laboratories with centralized knowledge of EVs  Royo et. ResearcLarge-scale production of EVs is dependent on the type of cell source and method of isolation. Scaling up from T-flasks to bioreactors can increase the production of EVs. However, these changes in the culturing environment might influence cells and thereby change the characteristics of EVs. Therefore, these parameters need to be standardized in the scale-up process. The quality of EVs and their bioactivity need to be confirmed after the scale-up. Methods of EV isolation include but are not limited to differential/density-gradient ultracentrifugation, tangential flow filtration, bind/elute chromatography, size-specific separation, and immunolabeling-based EV selection. There is a unique principle and advantage for each of these methods. Ultracentrifugation is the most popularly used  and consAnother major challenge in EV research is their storage. Literature suggests storing EVs at +4°C for a few weeks. EVs can be stored long-term at −80°C either in PBS or with cryo-protectants such as DMSO, Trehalose , and glyin vivo microenvironment signals might influence viability, proliferation, and functionality of the transplanted cells. This might not be an issue in EV therapeutics since they do not replicate or differentiate and are short-lived. However, EVs that are naturally produced by a cell type are not necessarily specific to one tissue. Also, the multi-factorial bioactivity of EVs might cause side effects, a safety concern for targeted therapy. Another function of EVs secreted from the cells is a self-defense mechanism to maintain cellular homeostasis by removing the toxic molecules from the cells (In cell transplant studies, the he cells .To enhance a more targeted approach and minimize side effects, EVs can be locally administered to a specific tissue, such as intra-glandular injections at the site of injury. Alternately, engineered cells that produce tissue-specific EVs could be used.in vivo biodistribution, and bioactivity. Genetically engineered cell-derived EVs might carry oncogenic remnants; therefore, they need to be verified for immunogenic and tumorigenic side effects.EVs obtained from genetically engineered and immortalized cell lines might differ from naturally occurring EVs. Therefore, they need to be verified for purity, characterization, mechanism of action, There is a growing interest in using EVs as natural drug-delivery vehicles. Physical and chemical methods are used for drug-loading into the EVs. Hence, any immunogenic effect arising from these methodologies in the processing of EVs needs to be verified.Overall, EVs are no exception with respect to having safety regulations for therapeutic application. Aspects such as production methods, purification, dosing, storage, and administration are important to check points for safe EV therapeutics.An overview of the literature clearly indicates that research on EVs suffers from limitations. A number of technical points remain to be urgently clarified as summarized in The adaptability of BMMSC-, EC- and Mɸ-derived EVs exhibit a remarkable capacity to adapt to the requirements of the damaged tissue in which the vesicles integrate and provide a promising option to address for the medical field after radiation exposure and complication of radiotherapy in order to support a personalized treatment. MSC therapy has established an extensive safety profile in the clinical setting and compared to the other cell types such as the endothelial progenitor cells, they present important advantage related to their isolation, culture and high scale production. Thus, in case of a radiological or nuclear event, the use of MSC-EV approach will provide a promising option to address the unmet needs that are critically important in the medical management of mass casualties.


Literature describing children with COVID-19 infection and concurrent acute appendicitis is growing, and understanding these patients’ clinical picture is necessary for their proper treatment.We present a case series of six healthy children before they developed classic symptoms of appendicitis. At the same time, they were also found to have confirmed COVID-19. All patients had fever and right lower abdominal pain. Four of six children having Alvarado score above seven had surgical treatment, while the others only received systemic antibiotic and antiviral medication. Surgical results of two patients revealed perforated appendicitis. No mortality occurred among them.There is increasing recognition of gastrointestinal involvement in patients with COVID-19 and MIS-C. There are several postulates to explain appendicitis in COVID-19. First, inflammatory response is exaggerated in SARS-CoV-2 infected patients. Second, obstruction of the appendiceal lumen is caused by mesenteric adenopathy, which in turn, is caused by COVID-19 infection, not fecalith. Third, hyperinflammatory response in MIS-C triggers inflammation in appendix.Clinicians must recognize that abdominal pain with fever could be the presenting symptoms of COVID-19 with MIS-C. MIS-C, which has severe presentations with gastrointestinal manifestations and high mortality rate, should be considered as a differential diagnosis for a patient with appendicitis-like symptoms and a positive SARS-CoV-2 infection. •Inflammatory response is exaggerated in SARS-CoV-2 infected patients.•Obstruction of the appendiceal lumen is caused by mesenteric adenopathy.•Hyperinflammatory response in MIS-C triggers inflammation in appendix. Currently, several studies postulated that COVID-19 in children appears to be milder in clinical manifestations and the pediatric patients have a lower proportion of symptomatic infection than adults [In early May 2020, an increasing amount of evidence emerged in the United Kingdom (UK), the United States, and Europe regarding different manifestations of COVID-19 in pediatric patients, which are nowadays known as the multisystem inflammatory syndrome in children (MIS-C) . Organ , and the other was from feces (Klebsiella pneumoniae). All patients had sterile blood cultures. Two patients, who showed normal chest X-ray results, were either asymptomatic or had a mild degree of COVID-19 infection. The others had COVID-19 bilateral pneumonia with moderate to severe degree of COVID-19 infection.Hemoglobin levels were found normal, while levels of leucocyte, neutrophil, CRP, procalcitonin were elevated in all patients. All patients had tested positive for SARS-CoV-2, but none of them developed respiratory symptoms, and three of them had MIS-C. One of the three patients with MIS-C had increased levels of both aspartate and alanine transaminase. Positive culture was found in two patients; one result was from peritoneal fluid  identified that children with COVID-19 may present with clinical features suggestive of appendicitis. However, recent studies related to appendicitis and COVID-19 or MISC are still lacking in numbers. There are several postulates regarding appendicitis in COVID-19. First, in COVID-19 patients, the inflammatory response is exaggerated. ACE-2 receptors, a receptor for COVID-19, are widely distributed in both smooth muscle and endothelial cells of the small intestines and colon, as well as arterial and venous endothelial cells throughout the body. Second, obstruction of the appendiceal lumen is caused by mesenteric adenopathy, which in turn is caused by COVID-19 infection, not fecalith. Third, hyperinflammatory response in MIS-C triggers inflammation in appendix . No fecaAppendicitis in our patients were diagnosed based on their medical history, as well as physical and laboratory examination. All of those were applied to the Pediatric Alvarado score. Snyder et al. reported the probability of appendicitis was 80%, 20%, and 6% in patients whose scores were ≥8, 4–7, and <4 . In our The limitation of this study is that there was no confirming pathological examination in patients treated with conservative management. This study describes the presentation of cases found in one tertiary hospital, the description of cases in other hospitals and regions may show different results. A further multicenter study is needed to compare our findings.4Clinician must recognize that abdominal pain with fever could be the presenting symptoms of either COVID-19 or MIS-C. This case series emphasizes that further investigation of COVID-19 and MIS-C should be performed in patients with acute appendicitis. MIS-C, which can have severe manifestations with gastrointestinal involvement and high mortality rate, should be considered as a differential diagnosis for a patient with appendicitis-like symptoms with a positive SARS-CoV-2 infection. Reporting of these case series will help further our understanding of COVID-19 manifestations in children. Further research should be conducted to confirm these findings.The informed consent form was declared that patient data or samples will be used for educational or research purposes. Our institutional review board also do not provide an ethical approval in the form of case report.This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.Nurnaningsih, Gladys Indika Danudibroto, Desy Rusmawatiningtyas, Intan Fatah Kumara, Titis Widowati conceived the study and approved the final draft. Nurnaningsih, Desy Rusmawatiningtyas, Firdian Makrufardi, Intan Fatah Kumara and Titis Widowati drafted the manuscript, and critically revised the manuscript for important intellectual content. Nurnaningsih, Gladys Indika Danudibroto, Desy Rusmawatiningtyas, Intan Fatah Kumara, Firdian Makrufardi and Titis Widowati facilitated all project-related tasks.Written informed consent was obtained from the patient's parents for publication of this case report and accompanying images. A copy of the written consent forms is available for review by the Editor-in-Chief of this journal on request.researchregistry7479.Nurnaningsih.Not commissioned, externally peer reviewed.No potential conflict of interest relevant to this article was reported.


Point-of-care ultrasound (POCUS) devices are becoming more widely used in healthcare and have the potential to act as fomites. The objective of this project was to study the thoroughness of cleaning of POCUS machines before and after a quality improvement initiative. We designed a mixed-methods, pre/post study which took place over the course of one year at a university-affiliated health center. Cleaning rates of four ultrasound machines used by hospital medicine and critical care medicine services were evaluated using fluorescent marking. Interventions targeted physicians’ knowledge of best practices and improved access to cleaning supplies. Pre- and post-intervention cleaning rates were compared using a generalized linear model. The impact of the corona virus disease of 2019 (COVID-19) pandemic on baseline cleaning rates was also evaluated. Physicians’ attitudes and knowledge of cleaning practices were evaluated via unpaired pre/post surveys.p < 0.0001). There was no difference in baseline cleaning rates before  and after  the onset of the COVID-19 pandemic (p = 0.78). Post-intervention surveying found improved understanding of guideline-based cleaning practice, better performance on knowledge-based questions, and fewer reported barriers to machine cleaning.There was significant improvement in thoroughness of cleaning following intervention , Thoroughness of cleaning of POCUS machines can be improved with practical interventions that target knowledge and access to cleaning supplies.The online version contains supplementary material available at 10.1186/s13089-021-00244-4. Staphylococcus aureus, Pseudomonas aeruginosa and vancomycin-resistant enterococci [Over the past three decades, the use of point-of-care ultrasound (POCUS) has been increasingly integrated into routine clinical practice, including critical care and hospital medicine –3. Recenerococci –9. Additerococci . Thus, Perococci .Cleaning and disinfection of ultrasound equipment has been shown to significantly reduce microbiological burden , 13. MulA mixed-methods, pre/post-study of POCUS machine cleaning practices was conducted over a 1-year period at a 718-bed academic health center. This project was considered quality improvement by the local institutional review board and therefore did not require approval as human subject research. Two Philips SPARQ  and 2 GE Healthcare Venue  machines were included in the study. SPARQ machines were primarily used by hospital medicine (HM) faculty and internal medicine (IM) residents. One SPARQ machine was stored on an inpatient medical-surgery unit and the other was stored in a HM work room. Venue machines were used primarily by IM residents, pulmonary/critical care medicine (PCCM) fellows, and PCCM faculty, but they were also accessible to surgical and anesthesia critical care providers. One Venue was stored in a neuro-critical care unit and the other was stored in a medical critical care unit. All machines were outfitted with linear, convex, and phased array transducers.The IM residency program has approximately 95 house officers, including categorical, preliminary, primary care, and medicine-pediatric residents. The residency program has a longitudinal POCUS curriculum, including workshops, lectures, and a dedicated POCUS rotation. There are 13 PCCM fellows, who also have a dedicated POCUS curriculum and rotation. POCUS training and clinical use is variable for HM and PCCM faculty, with some high-frequency users and some non-users.Interventions were guided by a targeted needs assessment. To better educate physicians, we created a website that outlined best practices for POCUS cleaning and disinfection . The web® AF-3 disinfecting wipes were used throughout the study. This product is a quaternary ammonium-containing, low-level disinfectant, which is appropriate for disinfecting medical equipment that contacts intact skin [The educational intervention promoted a 2-step cleaning and disinfection process based on American Institute of Ultrasound in Medicine guidelines . First, act skin .We improved access to cleaning materials by ensuring that each machine always had a dedicated container of disinfecting wipes. Machines with inadequate space for wipes were equipped with commercially available canister caddies Fig. . Wipes wA fluorescent marking gel  was used to monitor POCUS machine cleaning practices. The use of these gels is a well-established method for monitoring environmental cleaning practices in infection control research , 30, 31.The pre-intervention data collection occurred from November 2019 to January 2020. Before implementation of the planned intervention, the COVID-19 pandemic spread rapidly across the United States. The heightened awareness around infection control during this time had the potential to confound post-intervention data. To address this possibility, the research team collected an additional 2 months of baseline data from May to June 2020. During this time only three of the four POCUS machines were available for data collection. Interventions were implemented in July 2020. Post-intervention data were collected from August to October 2020.A 16-question survey was created to assess participants’ demographics, POCUS experiences, and attitudes and knowledge towards machine cleaning  and marking site groups (transducers vs. cords vs. touch surfaces). Cleaning rates were analyzed using a generalized linear mixed model (GLMM) which accounted for the correlation of cleaning rates for each machine. Model-adjusted mean percentages and standard errors of cleaned components by phase were used to summarize the data. To account for occasions when a machine was not used between sampling periods, data were excluded when there was no documentation of use on the machine’s sign-out sheet and there was no evidence of use . This was done to avoid falsely under-estimating cleaning rates. Unpaired pre- and post-intervention survey results were compared using descriptive statistics. All analyses were done using SAS version 9.4, and a p = 0.78). These data were, therefore, combined to form an aggregate baseline cleaning rate that was used in the final analysis. Pre- and post-intervention cleaning rates are displayed in Table p < 0.0001), representing a 44% relative improvement. In a sub-group analysis, the HM-based machines  demonstrated greater improvement in the thoroughness of cleaning than the CCM-based machines . All surface category sub-groups showed significant improvement in cleaning rates , with probe cords showing the largest improvement  and after 0.61, SE 0.1) the COVID-19 outbreak (, SE 0.1 Table We were able to significantly improve the thoroughness of cleaning of POCUS machines by using targeted interventions. We identified lack of knowledge, inadequate access to cleaning supplies, and time constraint as primary barriers to guideline-based cleaning and disinfection. These findings support the concern raised by a global survey of ultrasound users which suggested that real-world cleaning practices are unsatisfactory and that users are unfamiliar with guidelines , 23. GivThe spread of the COVID-19 pandemic during our pre-intervention phase had the potential to confound our results, for example, through heightened provider awareness of disinfecting practices or system-level interventions. However, we did not find any difference in cleaning rates following the first spike of COVID-19-related admission in our region of the country . This fiWhen sub-grouped by area of use, the thoroughness of cleaning for machines used primarily for critical care approached, but did not meet, significance. These machines are shared with specialties that were not targeted by our interventions, providing a possible explanation for this finding. Of the surface category sub-groups, probe cords had the lowest baseline cleaning rate and the greatest relative improvement. While probe cords may not be thought of as a high-contact surface, they are frequently handled during exams or draped across the patient, and attention must be given to this area during cleaning. Though statistical analysis was not performed due to the unpaired nature of our survey, there was an improvement in all response categories following intervention, further supporting the efficacy of these actions.Although our intervention focused on POCUS users, we would like to highlight an opportunity for manufacturers to improve device design to promote easier cleaning and better infection control. Specifically, there should be efforts to reduce the number of difficult-to-clean crevices, such as we encountered around probe orientation markers. These are in the highest-contact region and we consistently found them to be poorly cleaned. Additional areas of concern include the keyboard and control knobs/wheels.There were limitations to our study. This was a single-center study, which may limit generalizability. The pre/post-intervention study design makes it possible that confounding variables influenced our findings. Our design allowed measurement of cleaning but not disinfection, although prior infection control studies demonstrated that improvement in thoroughness of cleaning correlated closely with microbiological assessment , 21, 26.Based on the results of this study, guideline-based cleaning practices and education have been integrated into our POCUS curriculum. This includes ongoing use of the online resources created for the project and improved access to dedicated disinfecting wipes on POCUS machines. In the future, we would like to work with our infection control division to update institutional cleaning and disinfection policies so that they are more easily interpretable with regards to POCUS. An internal sustainability study would add important information, and we would be interested in the findings of a similar project carried out at other sites. Finally, demonstrating a correlation between improvement in point-of-care ultrasound cleaning and decreased patient acquisition of microbes or healthcare-associated infection is desirable.We identified barriers to guideline-based cleaning and disinfection of point-of-care ultrasound machines: a lack of knowledge, time constraint, and inadequate access to cleaning supplies. By targeting these domains with practical interventions, we were able to significantly improve the thoroughness of cleaning of four point-of-care ultrasound machines used by internal medicine and critical care medicine providers.Additional file 1. Point-of-care ultrasound cleaning survey.


A cancer immune phenotype characterized by an active T-helper 1 (Th1)/cytotoxic response is associated with responsiveness to immunotherapy and favorable prognosis across different tumors. However, in some cancers, such an intratumoral immune activation does not confer protection from progression or relapse. Defining mechanisms associated with immune evasion is imperative to refine stratification algorithms, to guide treatment decisions and to identify candidates for immune-targeted therapy. Molecular alterations governing mechanisms for immune exclusion are still largely unknown. The availability of large genomic datasets offers an opportunity to ascertain key determinants of differential intratumoral immune response. We follow a network-based protocol to identify transcription regulators (TRs) associated with poor immunologic antitumor activity. We use a consensus of four different pipelines consisting of two state-of-the-art gene regulatory network inference techniques, regularized gradient boosting machines and ARACNE to determine TR regulons, and three separate enrichment techniques, including fast gene set enrichment analysis, gene set variation analysis and virtual inference of protein activity by enriched regulon analysis to identify the most important TRs affecting immunologic antitumor activity. These TRs, referred to as master regulators (MRs), are unique to immune-silent and immune-active tumors, respectively. We validated the MRs coherently associated with the immune-silent phenotype across cancers in The Cancer Genome Atlas and a series of additional datasets in the Prediction of Clinical Outcomes from Genomic Profiles repository. A downstream analysis of MRs specific to the immune-silent phenotype resulted in the identification of several enriched candidate pathways, including NOTCH1, TGF- Over the past decade, corroborations of the effects of antitumor immunity on tumor progression have piled up. The discovery of escape mechanisms of the tumor-immune system, including the identification of immune checkpoints, has led to major advancements in immunotherapy . DespiteIt is now accepted that a T-cell inflamed cancer phenotype is associated with responsiveness to immunotherapy and a favorable prognosis . In thisunctions , 60, 62.mentclasspt{minimaunctions . Howeverunctions .Using the TCGA database, pan-cancer analyses have sought to address this critical question by correlating the mutational status of driver cancer genes and/or the status of oncogenic signals with the degree of the intratumoral immune response , 49, 59.A necessary condition for tumor progression and drug resistance is transcriptional dysregulation , 32. A mHere we use the TCGA RNA-Seq data to discover key driver TRs, referred to as master regulators (MRs), for the immune-silent cancer phenotype. We utilize the RNA-Seq data for  active) , each harated in .There have been several methods in the literature , 38 thatAnother key component of MRA is to estimate enrichment/activity scores for TRs in a given sample, taking into consideration its regulon. This is essential to identify differentially enriched/activated TRs (MRs). While techniques such as RGBM utilize a simplistic difference in average expression of positively and negatively regulated targets to estimate the activity of a TR, methods such as virtual inference of protein-activity by enriched regulon analysis (VIPER)  and MARIIn recent literature, techniques such as Netfactor  and 24]24] take We investigate the MRs that are common across rognosis  and thusrognosis . Finallyrognosis  to discoFigure We wanted to select a wide list of candidates as TRs, looking for all the genes involved in the process that modulate the frequency, rate or extent of cellular DNA-templated transcription. Therefore, we selected all the genes annotated with the Gene Ontology (GO) term GO:0006355 (regulation of transcription) [ studies , 47, 48 Other interesting examples where hubs of the networks were focused on signal molecules (and not just TFs) include approach such as SigMaps  or surfaRNA-Seq data from the TCGA website were downloaded and processed using TCGA biolinks. RNA-Seq data for each cancer were represented as From the PRECOG repository, we selected 8 datasets, each corresponding to a different and the largest unique dataset available for a particular cancer type as the validation set. These included GEO Accession Id: GSE32894 for bladder urothelial carcinoma (BLCA), GSE3494 for breast invasive carcinoma (BRCA), GSE39582 for colon adenocarcinoma (COAD), GSE108474 for glioblastoma multiforme (GBM), GSE65858 for head and neck squamous cell carcinoma (HNSC), GSE72094 for lung adenocarcinoma (LUAD), GSE9891 for ovarian serous cystadenocarcinoma (OV) and GSE65904 for skin cutaneous melanoma (SKCM). Each of these lization , followened from .Gene signatures found in early studies on tumor rejection by immunotherapy strongly overlap with pathways that were upregulated during other instances of immune-mediated tissue rejection like graft versus host disease, allograft rejection or autoimmunity . This obWe previously classified the cancer samples in the TCGA using ICR classification  for eachustering  algorithCR genes  was applhttps://github.com/raghvendra5688/ICR_Analysis . Our objective was to compare the cancer samples with a highly active immune phenotype i.e. ICR-H with the immune-silent phenotype i.e. ICR-L. In [The cluster with the highest expression of ICR genes was termed ICR-H, while the cluster with the lowest ICR gene expression was termed ICR-L. All samples in the intermediate cluster were defined as ICR medium (ICR-M) for each cancer, cated in . The codCR-L. In , we emphCR-L. In .Given Given  in RGBM  and VIPE in RGBM . More deIn VIPER, a probabilistic framework that directly integrates the target mode of regulation i.e. whether targets are activated or repressed, confidence in regulator-target interactions and target overlap between different regulators (pleiotropy) is utilized to compute the normalized enrichment score of a TR’s regulon. Since VIPER expresses activity for all the TRs on the same scale i.e. NES, we can now perform differential analysis using a Bayesian statistical framework such as LIMMA   in both Figure As a first step, we aimed at identifying the most conserved MRs characterizing the two opposite immune phenotypes (ICR-H and ICR-L) within each prognostic cancer category (ICR-E and ICR-D). We then compare the lists of the identified MRs between the eight ICR-E and the four ICR-D cancers. We found This observation indicated that (1) the dominant features characterizing the two opposite immune phenotypes (ICR-H and ICR-L) resulted in the upregulation of MRs related to high immune activity rather than MRs capturing immune-exclusion and (2) these MRs are shared independently of the prognostic connotation of immunologic activity (ICR).We observed from Figure Figure We took a union of the MRs identified to be specific to the ICR-H for the eight ICR-E cancers as well as the four ICR-D cancers and considered only those MRs whose median activity in ICR-H cancer samples was We observed in Supplementary Table S9, a set of seven MRs with different median activity patterns between the ICR-EH and ICR-DH cancer samples across the eight ICR-E and the four ICR-D cancers of interest. Some of these MRs are not necessarily a TR in each of the ppressor  that inhSimilarly, we observed in Supplementary Table S10, a set of shown in .We additionally correlated the activity of MRs with survival outcomes. Interestingly four out of the six MRs with high median activity in ICR-DH  could segregate the entire cohort according to survival outcome , Cytokine Signaling in Immune System (R-HSA-1280215), Interferon Signaling R-HSA-913531), C-type lectin receptor signaling pathway (path:hsa04625), Interleukin-4 and Interleukin-13 signaling (WP4066), etc. as depicted in Supplementary Figure S8A. The MRs that were part of each enriched pathway were illustrated in Supplementary Figure S8B, where the intensity represents the median activity for that MR across all 12 cancer tissues of interest. Interestingly, we observed that the majority of the top significantly enriched pathways are hallmark pathways of immune engagement [13531, C-A similar analysis was performed for the The top significantly enriched pathways particular to the ICR-L phenotype include the Generic Transcription Pathway (R-HSA-212436), Transcriptional Regulation of TP53 R-HSA-3700989), NOTCH1 Intracellular Domain Regulates Transcription R-HSA-2122947), TNF-00989, NO47, TNF-0We performed a validation experiment by comapring the activity patterns of the consensus MRs determined by our framework to be specific to the ICR-H and ICR-L tumor samples in all ICR neutral (ICR-N) cancers. We performed hierarchical clustering of the MRs specific to ICR-L phenotype based on their activity patterns in ICR-N tumor samples. A similar hierarchical clustering was performed for the MRs specific to the ICR-H phenotype and the two dendrograms were assimilated together, as illustrated in Figure Moreover, an additional validation on the set of eight datasets  obtained from the PRECOG repository was conducted. For an MR whose gene expression is not available in a particular dataset, we considered its activity value to be in silico validations confirm that the MRs, which we determined using our consensus framework, were specific to the ICR-L phenotype and should likely be involved in immune exclusion functions. Thus, the enriched pathways associated with these MRs could potentially represent molecular mechanisms driving the immune-excluded cancer phenotype and could be targeted to design therapeutic strategies.These two In recent years, TR activities estimated from RNA-Seq data have attracted much attention in cancer research , 24, 42.Here, we designed and applied four different MRA pipelines using the TCGA RNA-Seq data to discover differentially activated TRs (MRs) w.r.t. the immunologic constant of rejection phenotype (ICR-H versus ICR-L). We took a consensus of the MRs identified by these varied MRA pipelines for our goal of identifying key driver MRs for the immune-silent (ICR-L) cancer phenotype. Our network-based framework led to the discovery of The primary goal of our work was to identify key driver genes and their associated mechanisms for an immune excluded cancer phenotype (ICR-L). The downstream analysis of MRs specific to ICR-L using ConsensusPathDB resulted in enrichment of the BRG1-associated protein complex, which has a known role in oncogenesis . Moreovemelanoma  and othemelanoma  and was melanoma .Similarly, TGF-ppressor  and its ppressor . The lisIt is noteworthy that the MRs, PABPC1L and SMO are the only MRs that have high positive median activity in ICR-DH tumors, whereas it has negative median activity in ICR-DL tumors. Moreover, it has negative activity in ICR-EH tumors while having high positive median activity across the majority of the ICR-EL tumors. Thus, PABPC1L and SMO are potential biomarkers that demand further investigation to better understand their prognostic role within the context of ICR.In silico validation of these consensus MRs was performed in ICR-N cancers and a set of eight different datasets collected from the PRECOG repository, suggesting that these MRs can be used as promising therapeutic markers. Finally, as the data were generated form bulk transcriptome, next steps would include the dissection of the origin of the identified MRs using single cell sequencing techniques and spatial transcriptomics [Briefly, our results demonstrate that TR activity profiles inferred from RNA-Seq data using RGBM + FGSEA, RGBM + GSVA, RGBM + Viper and ARACNE + Viper MRA pipelines can be used to discover key MRs associated with an immune excluded phenotype. iptomics , the coniptomics , the valiptomics  and theifinal_diff_bbab168Click here for additional data file.Reply_to_Reviewers_ICR_v3_bbab168Click here for additional data file.Supplementary_Information_for_Network_based_Identification_of_Key_MRs_for_ICR_bbab168Click here for additional data file.