Mycobacterium tuberculosis infection remains unclear. This is because studies related to the role of GM-CSF so far have been carried out in murine models of experimental TB, which is inherently susceptible to TB as compared to humans, who exhibit a resolution of infection in majority of cases. We found a significantly higher amount of GM-CSF production by human macrophages, compared to mouse macrophages, after infection with M. tuberculosis in vitro. The higher levels of GM-CSF produced by human macrophages were also directly correlated with their increased life span and ability to control M. tuberculosis infection. Other evidence from recent studies also support that M. tuberculosis infected human macrophages display heterogeneity in their antibacterial capacity, and cells with increased expression of genes involved in GM-CSF signaling pathway can control intracellular M. tuberculosis growth more efficiently. Collectively, these emerging evidence indicate that GM-CSF produced by lung resident macrophages could be vital for the host resistance against M. tuberculosis infection in humans. Identification of GM-CSF dependent key cellular pathways/processes that mediate intracellular host defense can lay the groundwork for the development of novel host directed therapies against TB as well as other intracellular infections.Although classically associated with myelopoiesis, granulocyte-macrophage colony-stimulating factor (GM-CSF) is being increasingly recognized for its potential role in innate resistance against tuberculosis (TB). While the GM-CSF is produced by a variety of host cells, including conventional and non-conventional T cells, macrophages, alveolar epithelial cells, the cell population that promotes GM-CSF mediated innate protection against  However, many population-based immunological and genetic studies support the belief that innate immunity is equally important in TB immunopathogenesis (Mycobacterium tuberculosis (MTB)  is primarily mediated by T cells, with CD4ogenesis –3. Macrois (MTB) . Currentis (MTB) –8. The id others , 10. Howd others . A recend others . While MMurine macrophages are also known to produce GM-CSF in the lung compartment during MTB infection . HoweverWe thus examined the level of GM-CSF produced by mouse MDMs (monocyte-derived macrophages) compared to human MDMs before and after infection with MTB . While iInterestingly, the viability decreased significantly, while the bacterial burden increased, with time in mouse MDMs . In contin vitro and examined their viability before and after MTB infection. As expected, the addition of GM-CSF to mouse MDMs significantly increased the longevity of both uninfected and MTB-infected mouse MDMs  upon overlay to infected human MDMs, indicating a role for GM-CSF in improving antigen processing and presentation . The GM-an cells . Remarkaan cells .These results indicate that GM-CSF helped macrophage control MTB by decreasing bacterial load, while also preventing host cell death. These findings on human vs. murine monocyte-derived macrophages also indicate that the ability of macrophages to contain MTB infection could be due to the dual role of GM-CSF in prolonging the host cell survival as well as stimulating intracellular anti-MTB effector functions. Though not studied during MTB infection, GM-CSF has been known to modulate the developmental as well as effector functions of different lung macrophage populations . GM-CSF in vivo . Ma. Main viin vivo  16). Di. Diin viin vivo  17–19).–19.in viin vivo  26). We. Wein viin vivo . It is trophages  11, 22), 22in virophages , 28, therophages –31. It iAll datasets generated for this study are included in the article/The studies involving human participants were reviewed and approved by UTHSC-Houston IRB. Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.AK designed the study. AM and VS led the project, conducted the experiments and analyzed the data. AK wrote the manuscript. SS edited the manuscript. CJ, JA, and RH provided the feedback on the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Overall, self-guided interventions showed small to medium effects in comparison to control groups. In particular, self-guided therapeutic approaches , selected positive psychology interventions, and multi-component and activity-based interventions  showed promising evidence for effectiveness. Overall, self-guided interventions on average did not show the same degree of effectiveness as traditional guided individual or group therapies. There was no consistent evidence of dose effects, baseline differences, and differential effectiveness of eHealth interventions. More research on the effectiveness of interventions in diverse cultural settings is needed.We conducted a rapid review and quantitative summary of meta-analyses that have examined interventions which can be used by individuals during quarantine and social distancing to manage anxiety, depression, stress, and subjective well-being. A literature search yielded 34 meta-analyses (total number of studies  Social distancing measures are effective in reducing viral spread in the current COVID-19 pandemic but have been shown to increase mental health burdens. These collateral effects are affecting large numbers of individuals globally, requiring urgent attention because of the strains on mental health providers struggling to provide adequate support for people in need. Although there are many self-help guidelines available online and via social media, it is unclear how effective these are. We provide a quantitative review of evidence-based practices that can be used by individuals at home or in confined physical environments during social distancing and quarantine to manage anxiety, depression, and stress. Given the likely continuation of social distancing measures in various parts of the world and shortages in mental health systems globally, our systematic review provides evidence on effective self-guided interventions, either as an initial stand-alone self-help intervention or while waiting for treatment.What strategies can an individual adopt to maintain good mental health and reduce anxiety and stress during quarantines and physical distancing? Quarantines are psychologically taxing  as an essential component of optimal mental health care  or experimental studies that evaluate the effectiveness of psychological interventions and strategies for a range of psychological outcomes. In particular, we focus on increasing subjective well-being , and decreasing anxiety, depression, or stress as key outcomes. Critically, we screened all meta-analyses identified by our search parameters, but only summarize evidence from those meta-analyses which included self-guided conditions which can be performed by individuals alone without the guidance of trained health professionals. Our meta-analysis is more inclusive in scope because previous meta-analyses have: (a) typically focused on either a specific type of intervention or compared a small number of interventions without considering the wider range of possible interventions that might be beneficial; or (b) did not specifically consider the relevance and evidence of self-guided practices that could be performed by individuals alone. Thus, our primary aim is to provide a comparative summary of the available evidence of diverse psychological strategies that can inform recommendations by public health workers and psychologists, as well as be made available to the larger public. The COVID-19 pandemic affects populations of all nations, but interventions are often conducted with Western, industrialized and individualistic samples , therefore, we use these metrics for plotting the effects. If no confidence interval was reported, only the mean effect size is displayed.We present the average effect size and 95% confidence interval as reported in the original meta-analyses in the form of a forest plot. The most common effect sizes are variations of the standardized mean difference . The majority of meta-analyses included general population samples, including working adults . The second largest sample group were mixed general population and clinical samples . Purely clinical samples were included in 4 meta-analyses and students were the primary population in 3 meta-analyses. All but one meta-analysis , medium (d = 0.5), and large (d = 0.8) effect sizes of standardized mean differences. This first allows us to assess the overall effectiveness of self-guided interventions compared to control interventions . Second, we compared the overall effect sizes of self-guided interventions against effectiveness benchmarks of traditional clinician-guided psychotherapy for reducing depression symptoms , with Cognitive-Behavioral Therapy (CBT) showing an effectiveness of g = 0.73 (k = 205); Behavioral Activation Theory g = 1.05 (k = 21), and third-wave therapies (including Acceptance and Commitment Therapy [ACT] and Mindfulness-Based Interventions [MBI]) an effectiveness of g = 0.85 (k = 19).We used two approaches to evaluate the relative effectiveness of self-guided interventions. First, we used Cohen  effect sk = 17), which encompass self-guided CBT, ACT, and MBI, as well as diverse Positive Psychology-based interventions (k = 8).A large number of studies used therapy-derived interventions including CBT, MBI, and ACT, and showed small to medium effect sizes for reducing anxiety, depression, and stress. Effects for subjective well-being in some analyses showed moderate to large effect sizes  attended to possible cultural differences in the effectiveness. Chu and Mak . We recoded effect sizes for anxiety, depression and stress so that positive numbers indicated a positive change (improvement) for the experimental group compared to the control group. As z-transformed r, the average effect size r was comparable across the four outcome variables, for anxiety r = 0.19 ; depression r = 0.17 ; subjective well-being r = 0.19 , and stress r = 0.19 . Effect sizes computed for comparisons with active control groups yielded smaller effects, but this difference was only significant for depression .After converting standardized mean differences to b = 0.19 [95% CI: 0.05–0.55]; p = 0.009). For subjective well-being, we found a trend for mindfulness interventions to show larger effect sizes compared to CBT . For depression, we first controlled for active control group comparisons (see the results reported above). Expressive writing exercises had a significantly smaller effect size on average compared to CBT/ACT based interventions . We did not find statistically significant differences in the effectiveness of different interventions for stress.When examining differences between types of interventions, we used therapeutic approaches  as the reference category. For positive psychology interventions, we separated gratitude and expressive writing interventions from other positive interventions. Due to the small number of effect size summaries available, we included activity-based interventions  with other/multicomponent interventions. For anxiety, we found a significantly larger effect for mixed, multicomponent and other interventions compared to CBT and ACT  and, to some extent, active controls . In particular, self-guided therapy-derived interventions , mindfulness-based practices, positive psychology interventions, and activity-based interventions  appear effective in reducing anxiety, depression, stress, and in increasing subjective well-being compared to both active and inactive control groups. However, dose effects were largely inconsistent. Therefore, we cannot recommend specific intervals or durations for any of the intervention categories. Baseline effects were largely absent, implying that even individuals with elevated stress or psychological problems can use these practices at home without supervision, however we strongly recommend contacting health professionals if an individual is experiencing distress.The unique context created by social distancing and quarantine necessitates reflection on the way self-guided interventions might be used. Although expressive writing interventions showed effectiveness compared to control groups, effectiveness was consistently lower compared to CBT and ACT-based interventions. Expressive writing about concerns or worries (including detailed reflections of difficult or traumatic events) may not be appropriate without adequate clinical support or guidance . Given the current strain on the mental health system and the likelihood of further restrictions in the near future, it is important to provide widely available evidence-based practices to avoid negative collateral effects on mental health at the population level (see Brooks et al., At the same time, the review clearly highlights blind spots in our understanding of evidence-based practices. More highly controlled research on self-guided and home-based interventions is needed to inform public health decision-making during pandemics that require quarantine and social distancing over potentially long periods of time. What are the ideal levels of compliance for self-guided mental health interventions beyond an initial lockdown period and how can mental health be maintained? When should self-guided interventions first be implemented or recommended to populations at risk and how long should these practices be maintained after the immediate lifting of more restrictive lockdowns? What are the effects of repeated lockdowns: should recommended self-guided interventions be switched or rotated? The meta-analyses summarize studies that were not specifically geared toward evaluating interventions that are focused on home practices during lockdown.We isolate three main limitations of the current evidence-base to guide future research. A first gap, especially in the current context of global pandemic, is the lack of attention to culturally transferable interventions. Most studies have been conducted with samples from high-income, highly educated, and mainly Western nations. Given the greater population density and living arrangements in non-Western environments, these conditions may make effective mental health interventions even more urgent. We need further national and international multi-center research that includes diverse groups of participants to better understand whether interventions developed for autonomous individuals socialized into societies that emphasize individuality and self-reliance are as applicable and as effective in more community oriented contexts (Smith et al., Finally, it is worth considering the broader role of psychologists in responding to the COVID-19 pandemic. A major concern for individuals, groups, organizations, and nations is the economic impact of the current pandemic. The mental health impact of quarantine is more dramatic for lower income groups (see Reynolds et al., In summary, the current evidence suggests that a number of self-guided interventions suitable for at-home practice during lockdown and physical distancing are effective to for improving mental health. Specifically, we recommend interventions based on cognitive behavioral therapy, mindfulness, and acceptance-based activities, selected positive psychology activities, physical exercise, and music as useful first-line mental health interventions. However, these activities are not as effective as in-person and group based therapeutic interventions, and so they should not replace clinician-guided interventions for individuals and groups in need. Many of these interventions are now available via smartphone and web-based applications. In order to provide broad access to such evidence-based interventions to mitigate the negative side-effects of social distancing measures, this article includes an online supplement with selected exercises and further information to help individuals cope with the mental health challenges of physical distancing and quarantine.https://osf.io/fpx4s/.Publicly available datasets were analyzed in this study. This data can be found at: RF conceptualized the study and wrote the first draft. RF, TB, JK, MZ, KR, AR, and LG conducted the literature search and data extraction. RF, JK, and TB conducted the analyses and created the visualizations. DW coordinated the activity material collation. DW, TN, BI, and MC collated and summarized the activity material. PM provided feedback and advice. All authors approved the final version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The within-group analysis revealed no significant effect of SSG-based training interventions  or HIIT-based training interventions  on RSA. The meta-comparison revealed that neither SSGs nor HIIT-based interventions were effective in improving RSA in soccer players, and no differences were found between the two types of training. This suggests that complementary training may be performed to improve the effects of SSGs and HIIT. It also suggests that different forms of HIIT can be used because of the range of opportunities that such training affords.This systematic review with a meta-analysis was conducted to compare the effects of small-sided games (SSGs)-based interventions with the effects of running-based high-intensity interval training (HIIT) interventions on soccer players’ repeated sprint ability (RSA). The data sources utilized were Web of Science, Scopus, SPORTDiscus, and PubMed. The study eligibility criteria were: (i) parallel studies (SSG-based programs vs. running-based HIIT) conducted in soccer players with no restrictions on age, sex, or competitive level; (ii) isolated intervention programs  with no restrictions on duration; (iii) a pre–post outcome for RSA; (iv) original, full-text, peer-reviewed articles written in English. An electronic search yielded 513 articles, four of which were included in the present study. There was no significant difference between the effects of SSG-based and HIIT-based training interventions on RSA (effect size (ES) = 0.30;  Small-sided games (SSGs) are adjusted formats of play that are often used in soccer training to develop a specific tactical/technical attribute  while inUsually, SSGs are prescribed with regimens similar to those recommended for long-interval high-intensity interval training (HIIT) sessions . The intSoccer is characterized by its intermittent nature, in which low-to-moderate intensity activities are interspaced with highly demanding activities in which explosive actions and repeated high exertion occur based on the context of the game . Among oIn soccer, the optimization of the training time is crucial. Therefore, it is important to understand whether drill-based exercises  can develop RSA to a similar extent as other forms of exercise  and whether they are significantly beneficial for soccer players. Such an understanding (SSG vs. running-based HIIT) will help define a practical application for the soccer field.Additionally, a systematic review and meta-analysis will help summarize the main training protocols and parallel studies that have compared SSGs and running-based HIIT, with a focus on their effects on RSA. Although two meta-analyses of SSGs have been carried out recently ,14, one This study followed the Cochrane Collaboration guidelines  and the A comprehensive computerized search of the following electronic databases was performed: (i) Web of Science; (ii) Scopus; (iii) SPORTDiscus; (iv) PubMed. The searching process for relevant publications had no restriction regarding the year of publication and included articles retrieved until 1 September 2020. The following search strings were employed:  AND  AND (“sprint”).The following inclusion criteria were established: (i) parallel randomized studies (SSG-based programs vs. running-based HIIT) conducted in soccer players with no restriction of age, sex, or competitive level; (ii) isolated intervention programs  with no restrictions on duration; (iii) a pre–post outcome for RSA; (iv) original, peer-reviewed articles written in English that provided the full text.Studies were excluded on the basis that they (i) were observational analytic designs; (ii) included other sports; (iii) used SSG or running-based HIIT combined with other training methods or between them ; (iv) were conducted in recreational soccer  or physical education contexts; (iv) were review articles, letters to the editor, errata, invited commentaries, or conference abstracts.An Excel spreadsheet was designed  to process the data extraction . Two of The outcomes chosen for this systematic review and meta-analysis included RSA measured through field-based tests. The RSA was collected based on the mean time (s), mean power (W), or total time (s) in a series of multiple sprints. Additionally, the following information was extracted from the included studies: (i) the number of participants (n), age (years), competitive level (if available), and sex; (ii) the SSGs format and pitch size (if available); (iii) the period of intervention (number of weeks) and number of sessions per week (n/w); (iv) the regimen of intervention .To assess the methodological quality of the included articles, the methodological index for non-randomized studies (MINORS) was used . Twelve g effect size (ES). The inverse-variance random-effects model for meta-analyses was used because it allocates a proportionate weight to trials based on the size of their individual standard errors [The analysis and interpretation of results in this systematic review and meta-analysis were conducted only in the case that at least three study groups provided baseline and follow-up data for RSA ,20,21. Md errors  and enabd errors . The ESsd errors : <0.2, tI2 statistic [I2 values of <25%, 25–75%, and >75%, respectively [To estimate the degree of heterogeneity between the included studies, the percentage of total variation across the studies due to heterogeneity was used to calculate the tatistic . Low, moectively .The extended Egger’s test  was usedTM X9 . Duplicates (249) were subsequently removed, either automatically or manually. The remaining 264 articles were screened for their relevance based on titles and abstracts, resulting in the removal of a further 242 studies. The full texts of the remaining 22 articles were examined diligently. After reading the full texts, a further 18 studies were excluded due to a number of reasons . There was no significant difference between SSG-based compared to HIIT-based training interventions on the effect over RSA . In a well-known pair of published articles ,32, HIIT2 [2 [Typically, SSGs are prescribed as a part of long-interval regimens . Among the studies included in this meta-analysis, SSG duration varied between 45 s  and 4 mi2  and a ma2 [2  per play2 [2 ,34, as w2 [2 ,36. TherThe absence of significant differences between groups might be related to the range of running-based HIIT. Among the training regimens, one involved short intervals (15 s–15 s work–rest) , one invInterestingly, the within-group changes also revealed no significant effects of training interventions in RSA. This was somewhat unexpected, mainly in the running-based HIIT group. The particularly detrimental results obtained in a long-interval HIIT intervention  could exOne of the limitations of the current systematic review and meta-analysis is that only English articles from the Web of Science, Scopus, SPORTDiscus, and PubMed were included, thus potentially overlooking other relevant publications. Another limitation is the reduced number of included articles. However, this serves to highlight the need for more research on this topic. For instance, the absence of research on women and professional players was apparent. The results might change based on such moderators or other factors, such as baseline levels or even the volume of training completed beyond the interventions. Future research on this topic should apply the same training regimen to SSGs and running-based HIIT to homogenize the methodological process. Future research should also account for the responding profile of players to determine which type of profile is more responsive to the interventions.As for practical applications, this meta-analysis highlights the importance of including complementary training methods that may help to develop RSA. Among other training regimens, combining SSG with running-based HIIT ,40 or wiThe current meta-analytical comparison revealed no significant changes in the effects of SSG-based and running-based HIIT interventions on soccer players’ RSA. Additionally, among the included parallel studies, the within-group analysis revealed no significant improvements after SSG or running-based HIIT interventions. Despite the limited number of studies included in the present analysis, the findings should be carefully considered as practical applications. Specifically, the results indicate that complementary training methods  could help to improve RSA due to their multifactor-dependent quality. Finally, more research comparing SSG and running-based HIIT is needed; no studies on women or professional players were found in the present analysis.


Substance use is common among people living with HIV and has been associated with suboptimal HIV treatment outcomes. Integrating substance use services into HIV care is a promising strategy to improve patient outcomes.International epidemiology Databases to Evaluate AIDS (IeDEA) consortium. HIV care and treatment sites participating in IeDEA are primarily public-sector health facilities and include both academic and community-based hospitals and health facilities. A total of 286 sites in 45 countries participated in the 2014–2015 survey and 237 sites in 44 countries participated in the 2017 survey. We compared changes over time for 147 sites that participated in both surveys.We report on substance use education, screening, and referral practices from two surveys of HIV care and treatment sites participating in the In 2014–2015, most sites (75%) reported providing substance use-related education on-site . Approximately half reported on-site screening for substance use (52%) or referrals for substance use treatment (51%). In 2017, the proportion of sites providing on-site substance use-related education, screening, or referrals increased by 9%, 16%, and 8%, respectively. In 2017, on-site substance use screening and referral were most commonly reported at sites serving only adults  and at sites in high-income countries .Although there have been increases in the proportion of sites reporting substance use education, screening, and referral services across IeDEA sites, gaps persist in the integration of substance use services into HIV care, particularly in relation to screening and referral practices, with reduced availability for children/adolescents and those receiving care within resource-constrained settings. Substance use, in the form of the consumption of alcohol, tobacco, and other injection and non-injection drugs, is common among people living with HIV (PLWH) and has been associated with suboptimal HIV treatment access and outcomes, including higher lost to follow-up rates and mortality , 2. In mEvidence-based approaches to screen and treat substance use are associated with reduced substance use and improved health outcomes –32. SubsInternational epidemiology Databases to Evaluate AIDS (IeDEA) consortium from 2014 to 2017 and identify research priorities related to the integration of substance use and HIV treatment services.In this paper we assess the availability and changes in availability of substance use-related education, screening and referral to treatment at HIV treatment centers participating in the The IeDEA consortium is a global research consortium established in 2006 of HIV treatment sites in seven geographic regions: Central Africa; East Africa; South Africa; West Africa; the Asia-Pacific; the Caribbean, Central, and South America (CCASAnet); and North America (NA-ACCORD) –39. IeDEIeDEA site assessment surveys are conducted approximately every two years. To be eligible to participate, sites must be active HIV treatment clinics and contribute data to IeDEA at the time of the survey. Research-only, non-clinical sites that do not contribute data to IeDEA are excluded. For the current analyses, we compared data from the two most recent site surveys. Data were collected between September 2014 and January 2015 and between June and December 2017. Both surveys collected information on characteristics of participating IeDEA sites including facility characteristics  and HIV treatment services.In addition, sites were asked if they provided the following services to enrolled HIV patients:any education on high-risk substance use behaviors and harm reduction practices;any screening for drug or alcohol use; andany referral for substance use treatment.Availability of substance use-related education, screening, and referral were categorized as occurring either on-site or off-site. On-site provision of substance use-related education, screening, or referral was defined as providing that service at the HIV clinic or the same health facility. Off-site provision of substance-use education, screening, or referral was defined as providing that service at another health facility or organization. The survey asked broadly about whether any substance use-related education, screening, or referral services were provided to HIV patients at each site. Thus, education-related services likely included a range of activities including one-on-one education and counseling as well as group-based educational programming. Similarly, screening services likely included a range of activities including the use of validated screening tools, assessment of biomarkers, and clinical inquiry.English and French versions of the surveys were available online and in paper form. The online version was implemented using REDCap, a secure web-based data collection application hosted at Vanderbilt University . Sites wDescriptive statistics summarized the prevalence of any substance use-related education, any screening, and any referral to treatment at IeDEA clinics in 2014–2015 and 2017. Analyses between site-level characteristics and substance use-related education, screening, and referral were conducted using chi-squared or Fisher’s exact tests, as appropriate. Income designation of the country in which the site was located was based on World Bank Income Designation of the country (as of July 2017). For sites that participated in both site assessments, McNemar’s tests were used to compare reported availability of substance use-related education, screening, and referral to treatment. A p-value of <0.05 guided interpretation of statistical significance. Analyses were performed using SAS Version 9.4.Among the 287 sites that were eligible to participate in the 2014–2015 site assessment and completed the survey, substance use-related data were available from 286 (99.6%) sites in 45 countries . Among tIn 2014–2015, most sites  reported providing education to patients on substance use behaviors or harm reduction on-site  and half reported on-site substance use screening  or providing referrals for substance use treatment  . Forty-oIn 2017, 84% (RAR 68–93%) of sites reported providing on-site education, 68% (RAR 29–90%) reported on-site substance use screening, and 59% (RAR 29–81%) reported providing referrals for substance use treatment. Approximately half (49%) of sites reported that on-site substance use-related education, screening, and referral to treatment were all available on-site. Substance use-related education was reported to be unavailable (either on-site or off-site) in 12% (RAR 5–26%) of sites. Substance use screening was reported to be unavailable in 26% (RAR 5–50%) of sites, and referrals for substance use treatment were unavailable in 24% (RAR 2–50%) of sites. Eleven percent of sites surveyed in 2017 reported that education, screening, and referral were all unavailable on-site.Among sites with data from both surveys (n = 147), the proportion of sites reporting on-site availability of substance use-related education, screening, or referral to treatment was compared between site assessments in 2014–2015 and 2017. Approximately 80% of sites reported on-site substance use-related education at both time points . There was a significant increase in the proportion of sites reporting on-site screening for substance use from 61% of sites in 2014–2015 to 74% of sites in 2017 (p<0.01) . The proAmong sites with data from both surveys, substance use-related education was reported to be available on-site at both time points at 76% of sites and unavailable at both time points in 5% of sites . Twelve A minority of sites reported having discontinued on-site substance use-related education , screening , or referrals  between 2014–2015 and 2017. Sites that discontinued substance use-related education were distributed across IeDEA regions, facility levels , types of patient populations served , and were located in both urban and rural areas. All sites that discontinued on-site substance use-related education were publicly funded. Sites that reported having discontinued on-site substance use screening represented 6 IeDEA regions , rural and urban sites, various levels of health care facilities, and all types of patient populations served. Most (n = 10) sites that discontinued substance use screening were publicly funded. Similarly, sites that reported having discontinued substance use referrals were distributed across 6 IeDEA regions,  and served a variety of patient populations. Most sites that reported having discontinued on-site substance use referrals were regional, provincial, or teaching hospitals (n = 12), in urban areas (n = 12), and were publicly funded (n = 16).Overall, reported availability of on-site substance use-related education was common among sites surveyed. Availability of on-site substance use-related education was significantly associated with level of care of the health facility and with the geographic region in which the health facility was located . On-siteOn-site substance use screening was least commonly reported at sites serving only children or adolescents and most commonly reported at sites serving only adults . On-site substance use screening was also least commonly reported at sites located in low-income countries as compared to sites in middle- or high-income countries. On-site screening was significantly associated with geographic region, with North American sites most commonly reporting screening and West African sites least commonly reporting screening.On-site referral to substance use treatment was most commonly reported by sites that serve adults only as compared to sites that serve children or adolescents only or both adults and children or adolescents. On-site referral to substance use treatment was least commonly reported in sites located in low-income countries and most commonly reported in sites located in high-income countries. The majority of sites located in North America (80%) reported on-site referral to substance use treatment compared to 29% of sites located in West Africa.Substantial gaps persist in the integration of substance use services into HIV care settings, particularly in relation to screening and referral to treatment, with reduced availability seen for younger patients  and persons receiving HIV care within resource-constrained settings. Future context-specific research should investigate potentially modifiable, multilevel barriers and facilitators to the integration of substance use-related screening and referral, including barriers and facilitators at patient, provider, health facility, and policy levels. For example, U.S.-based research has demonstrated that the clinical care system for PLWH has not viewed substance use as a top priority and that U.S.-based HIV primary care providers have had limited knowledge and training to address alcohol use disorder , 43. In Sites participating in IeDEA change over time, creating a challenge to conducting longitudinal research in real-world service settings. Analysis of the clinics for which 2014–2015 and 2017 data were available found a statistically significant increase in reported availability of on-site substance use screening between 2014–2015 and 2017. While this increase is encouraging, data on factors associated with this increase are limited, and the site surveys did not explore details on how screening was conducted, the quality of screening, the specific substances for which screening was conducted, or who conducted screening. Future assessments of substance use service integration should include more nuanced, culturally-specific questions capable of assessing such aspects of screening and treatment services. A literature review examining substance use screening approaches in HIV care settings found that the use of validated substance use screening tools was limited and providers’ reliance on self-reported substance use was common . UnderreA minority of clinics that reported availability of substance use-related education, screening, or referral in 2014–2015 reported that these services were no longer available in 2017. It is unclear what led to the discontinuation of these services. Substance use service discontinuation was reported by clinics across all IeDEA regions, patient populations, and by clinics in rural and urban areas. On-site referral to substance abuse treatment was the service most commonly reported to have been discontinued between 2014–2015 and 2017. Such findings highlight the need for longitudinal studies of service integration to better understand the feasibility and sustainability of service integration strategies. Little is known about factors associated with discontinuation of service integration. Research with clinics that implemented but discontinued service integration models can yield important insights into the development, refinement, and sustainability of integration models and strategies.Reported availability of substance use-related screening and referral varied by geographical region and by income level of the country in which the clinic was located. These services were least commonly reported at clinics within low-income countries and most commonly reported at clinics located within high-income countries. This distribution may reflect the context, including the prevalence of substance use among PLWH as well as the overall  lack of available services and staff trained to screen and treat substance use disorders and other mental health conditions, which is more prominent in resource-restricted countries . BarrierImplementation research to integrate substance use treatment within HIV care settings should consider the context-specific burden of substance use as well as context-specific variability in substances used due to the need for specific treatment interventions for different substances. Medications, for example, are an important component of treatment for opioid use disorder and alcohol use disorder and have been successfully implemented in U.S.-based HIV clinics , 49, 50.Several promising strategies for integrating substance use and HIV care in the U.S. have been evaluated and could be adapted for resource-limited settings. Two systematic reviews have focused on studies that evaluated integrated substance use and HIV care , 52. TheSubstance use screening and referral were less commonly reported at HIV programs that treat only children or adolescents as compared to programs that treat only adults. This is consistent with data from adolescents and young adults in the general population. In the U.S., it has been estimated that fewer than half of pediatricians screen adolescents for substance use or substance use disorders and less than 10% of adolescents with a substance use disorder are referred to substance use treatment –63. CompThis study has several key limitations. Data were collected from HIV treatment sites participating in the IeDEA consortium and may not be representative of all HIV treatment centers in a particular country or region. Because IeDEA sites have the capacity to routinely contribute electronic data, these sites may function at a higher level than sites in that country that are not participating in IeDEA. As such, integration of substance use-related services may be more common at IeDEA sites as compared to HIV treatment centers that are not participating in IeDEA. Information on clinic setting  was missing for 28 sites and information on level of care  was missing for 27 sites in the 2017 survey. This analysis relied on reports from health facility staff regarding substance use-related services at participating clinics. Availability of reported services was not independently verified, and specific approaches to substance use-related services or services related to individual substances were not assessed. Wide variability likely exists in the quality and procedures related to substance use-related services in HIV clinics in IeDEA.This study reports the availability of substance use-related education, screening, and referrals in HIV treatment centers across global settings. Gaps persist particularly in relation to the integration of substance use screening and referral into HIV care settings, especially for pediatric populations and those receiving care within low-income countries. Future research should assess how screening is conducted, for which substances screening is conducted, the use of validated screening tools, how referral or treatment is conducted in response to positive screening, and the quality and consistency of such procedures. Potentially modifiable, multilevel barriers and facilitators to sustainable integration should be identified and promising implementation strategies which address these barriers and facilitators should be developed, implemented, and evaluated. Longitudinal research into the sustainability of various integration models is needed.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file.


Rituximab (R) is increasingly incorporated in reduced intensity conditioning (RIC) regimens for allogeneic hematopoietic cell transplantation  in patients with B-cell malignancies, not only to improve disease control, but also to prevent graft-versus-host disease (GVHD). There are no randomized prospective data to validate this practice, although single center data and the CIBMTR analysis have shown promising results. We aimed at validation of these findings in a large registry study. We conducted a retrospective analysis using the EBMT registry of 3,803 adult patients with B-cell malignancies undergoing alloHCT (2001–2013) with either rituximab (R-RIC-9%) or non-rituximab (RIC-91%) reduced intensity regimens respectively. Median age and median follow up were 55 years (range 19.1–77.3) and 43.2 months (range 0.3–179.8), respectively. There was no difference in transplant outcomes (R-RIC vs RIC), including 1-year overall survival (69.9% vs 70.7%), 1-year disease-free survival (64.4% vs 62.2%), 1-year non-relapse mortality (21% vs 22%), and day-100 incidence of acute GVHD 2-4° (12% vs 12%). In summary, we found that addition of rituximab in RIC regimens for B-cell malignancies had no significant impact on major transplant outcome variables. Of note, data on chronic GVHD was not available, limiting the conclusions that can be drawn from the present study. An optimal outcome after allogeneic hematopoietic cell transplantation , includes not only cure of the underlying malignancy, but minimizing the incidence of GVHD, both acute and chronic. Reduced intensity conditioning (RIC) allows patients in higher age groups and/or with significant comorbidities to access alloHCT, while minimizing transplant related mortality, and preserving graft-versus-tumor effect , 2.RIC regimens have historically been designed to be unrelated to underlying disease, but at the same time, choice of agents used often has a scientific rationale for offering tumor-specific disease control , 4.RIC regimens containing rituximab, an anti-CD20 antibody with anti-neoplastic activity, have been included for targeting B-cell malignancies and single center studies suggest a superior alloHCT outcome compared to center-specific historical cohorts –6. RecenWe analyzed 3,803 patients with B cell lymphoid malignancies , who received a RIC allo-HCT between 2001 and 2013 and were reported to the registry of the EBMT. Patients undergoing myeloablative HCT  or alemtuzumab containing regimens were excluded. Umbilical cord and haploidentical HCT were excluded. All RIC or R-RIC regimens were included and were stratified as fludarabine (Flu), busulfan (Bu2), other FluBu regimens, other Flu containing regimens or other regimens.Patients undergoing alloHCT often receive anti-T-cell globulin (ATG) as a part of the conditioning regimen for GVHD prophylaxis. Thus, regimens were stratified for ATG or non-ATG regimens, within each group of RIC or R-RIC, for analyses. Details of ATG dosing and schedule were not available. Similarly, the details of the dosing and schedule of rituximab were not available in the EBMT registry. GVHD prophylaxis details were captured as cyclosporine (CSA), mycophenolate mofetil (MMF), CSA plus MMF, CSA plus methotrexate (MTX), or other regimens.Baseline demographics including follow-up, age at transplant, gender, diagnosis, disease status at alloHCT, year of alloHCT, donor type, recipient-donor gender match or mismatch, source of stem cells were obtained from the registry.Maximum acute GVHD grade within the first 100 days was available in the majority of patients. In contrast, chronic GVHD status was available in a small minority of the patients and was not analyzed. Transplant outcomes of overall survival (OS), relapse incidence, and non-relapse mortality were available.P values less than 0.05 were considered statistically significant. All statistical analyses were performed using R software by the EBMT statistical team.Comparison of patient and transplant characteristics was conducted using the Mann-Whitney test for continuous variables and the chi-squared test for categorical variables. Overall survival, progression-free survival, and GVHD-free/relapse-free survival (GRFS) were estimated using the Kaplan-Meier method. Multivariable prediction models for continuous and binary outcomes were made using standard and logistic regression, respectively. Based on comparison of patient and transplant characteristics, age at HCT, year of HCT, preparative regimen, donor type, and GVHD prophylaxis were included in the multivariate analyses. Cumulative incidence functions were calculated using Gray’s Method . CumulatGiven the differential impact of preparative regimens on outcomes as reported by the CIBMTR , we studHistorically, preparative regimens have remained unrelated to the underlying disease –3. The gIn this study, we report that the addition of rituximab to RIC regimen had no incremental impact on outcomes of alloHCT in patients with B-cell malignancies. This study is in contrast to the CIBMTR analyses showing that the addition of rituximab was associated with improved PFS . It is iIn a separate CIBMTR study confined only to patients with follicular lymphoma, comparing the two most commonly used RIC approaches – fludarabine and busulfan (FluBu) versus fludarabine, cyclophosphamide, and rituximab (FCR) reported survival outcomes  were not significantly different . The onlIn our study, data on chronic GVHD incidence and severity in the context of the NIH classification system was not available, limiting the conclusions that can be drawn.Rituximab was used in the treatment of chronic GVHD and studied as a pre-emptive strategy in post-HCT setting for prevention of chronic GVHD , 11–13. The above data may suggest that the effect of rituximab in modulating outcome is modest at the best and confined to regimens which are inherently associated with a slower rate of converting to full donor chimerism (non-busulfan containing regimens) , 7, 10. Finally, what is the role of anti-thymocyte globulin in rituximab containing preparative regimens? The cumulative incidence of grade 3–4 acute GVHD, relapse, non-relapse mortality, disease-free, and overall survival did not show any significant differences in patients who did not receive ATG in the preparative regimen.However, it does not answer the question if ATG can be safely omitted without compromising outcomes when rituximab containing preparative regimens are used for unrelated donor transplants. As preparative regimens are often “bundled” together with certain GVHD prophylaxis, it is challenging to study the differential impact of the regimen versus the GVHD prophylaxis. In our study, given the large sample size, we could adjust for the GVHD prophylaxis and did not see any interaction.In conclusion, we could not demonstrate the benefit of adding rituximab to RIC regimens for B-cell malignancies on HCT outcomes. Although our study was limited by the absence of chronic GVHD data, the larger sample size in contrast to the positive the CIBMTR study, suggests that the effect of rituximab needs to be systematically re-examined. Many centers have adopted this institutional practice, despite the lack of well conducted mechanistic studies to optimize the dosage and schedule. The cost of rituximab is not trivial, and the transplant community needs to take a step back to re-examine the role of B-cell inhibitors in the preparative regimens. It is unlikely that a phase 3 study will ever be done to address this question. However, with the advent of more targeted B-cell receptor or signaling pathway inhibitors it is critical, that the transplant community should follow the conventional pathway of drug development with carefully conducted phase I studies, prior to accepting agents approved for other indications and integrating it into the preparative regimens.The original contributions presented in the study are included in the article/AT and MJ contributed equally to writing this manuscript. EB analyzed the data. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Salmonella (NTS) are a frequent cause of invasive infections in sub-Saharan Africa. They are frequently multidrug resistant , and resistance to third-generation cephalosporin and fluoroquinolone non-susceptibility have been reported. Third-generation cephalosporins and fluoroquinolones are often used to treat invasive NTS infections, but azithromycin might be an alternative. However, data on antibiotic treatment efficacy in invasive NTS infections are lacking. In this study, we aimed to assess the spatiotemporal distribution of antimicrobial resistance in invasive NTS infections in sub-Saharan Africa and to describe the available evidence and recommendations on antimicrobial treatment.Non-typhoidal We conducted a systematic review of all available literature on antimicrobial resistance and treatment in invasive NTS infections. We performed a random effects meta-analysis to assess the temporal distribution of multidrug resistance, third-generation cephalosporin resistance, and fluoroquinolone non-susceptibility. We mapped these data to assess the spatial distribution. We provided a narrative synthesis of the available evidence and recommendations on antimicrobial treatment.Since 2001, multidrug resistance was observed in 75% of NTS isolates from all sub-Saharan African regions . Third-generation cephalosporin resistance emerged in all sub-Saharan African regions and was present in 5%  after 2010. Fluoroquinolone non-susceptibility emerged in all sub-Saharan African regions but did not increase over time. Azithromycin resistance was reported in DR Congo. There were no reports on carbapenem resistance. We did not find high-quality evidence on the efficacy of antimicrobial treatment. There were no supranational guidelines. The “Access group” antibiotics ampicillin, trimethoprim-sulfamethoxazole, and chloramphenicol and “Watch group” antibiotics ceftriaxone, cefotaxime, and ciprofloxacin were recommended as the first-choice antibiotics in national guidelines or reviews. These also recommended (a switch to) oral fluoroquinolones or azithromycin.In addition to the widespread multidrug resistance in invasive NTS infections in sub-Saharan Africa, resistance to third-generation cephalosporins and fluoroquinolone non-susceptibility was present in all regions. There was a lack of data on the efficacy of antimicrobial treatment in these infections, and supranational evidence-based guidelines were absent. Salmonella (NTS) are an important cause of foodborne and self-limiting enteritis . In. In80]. To treat invasive NTS infections, both the “Access group” antibiotics ampicillin, trimethoprim-sulfamethoxazole, and chloramphenicol, and C3G (ceftriaxone or cefotaxime) or fluoroquinolones (ciprofloxacin) from the “Watch group” antibiotics were often recommended as the first-choice antibiotics Table .Some authors had concerns about the use of chloramphenicol to treat invasive NTS infections due to a possible lack of bactericidal activity , 99, higThe need for clinical efficacy data and clinical trials on fluoroquinolones and C3G was already stated in 1986 and 1988 , 122. FoFluoroquinolones other than ciprofloxacin and C3G other than ceftriaxone and cefotaxime were generally listed as second-choice antimicrobial treatment options  Typhi and NTS or invasive and intestinal infections. As such, we may have missed some data on AMR and treatment. We could include only four studies that reported susceptibility data from NTS isolated before 1991, and from some sub-Saharan African countries, we could not include a single study. In addition, the temporal resolution was affected by compiled antibiotic susceptibility data from studies that reported in long time periods. Moreover, only nine from the 16 treatment efficacy studies reported data from sub-Saharan Africa. As the Salmonella Typhimurium and Enteritidis strains circulating in sub-Saharan Africa have a more invasive genotype and phenotype, data from NTS causing invasive infections in high-risk patients or transient bacteremia in other settings may not be generalizable to invasive NTS infections in sub-Saharan Africa.This review had some limitations. Firstly, we had to exclude studies that did not report separate data for Secondly, it is likely that publication and reporting bias favoring studies that reported resistant NTS occurred, particularly during the early years. However, MDR and FQNS will also have been underreported. The definition of MDR is a recent convention , 23, 24,Finally, a careful interpretation of the pooled proportions of MDR, C3G resistance, and FQNS is warranted. The heterogeneity was high in all meta-analyses. Although subgroup analysis per time period reduced the heterogeneity, the residual heterogeneity remained high. By meta-regression, we aimed to further reduce the heterogeneity, but we could not identify other study characteristics as effect moderators. It is however plausible that the study designs differed in too many aspects to identify the effect of specific characteristics. In addition, we decided to exclude the data from studies reporting susceptibility data for < 50 NTS, although these studies might still be representative for their study population, and when these studies were included in the meta-analysis, a trend in increasing FQNS was observed. Finally, we excluded studies with a grade E quality from the meta-analyses due to uncertainty or inconsistency in some of their reported data, while (most of) their data might be correct. By contrast, we included studies with a grade D quality in the meta-analysis, while the quality of these data cannot be fully assured neither.Salmonella Typhi in sub-Saharan Africa in these two reviews [Salmonella Typhi, while they reported more FQNS than our observations in NTS [Salmonella Typhi and non-typhi infections in sub-Saharan Africa, without differentiating between intestinal and invasive infections. They reported similar pooled MDR and FQNS proportions. They also reported more MDR in Salmonella Typhimurium than in Salmonella Enteritidis, but reported more FQNS in Salmonella Enteritidis than in Salmonella Typhimurium [Salmonella infections and enteric fever [This study is the first systematic review and meta-analysis on AMR and antimicrobial therapy in invasive NTS infections. While recently two systematic reviews have been published on the emergence of AMR in enteric fever , 130, th reviews , 130. Hos in NTS , 130. Anhimurium . Finallyic fever , 132–135gyrA mutation). Gatifloxacin has been withdrawn from the market in many countries due to dysglycemia in elderly and diabetic patients, but this patient profile is uncommon in invasive NTS infections in sub-Saharan Africa [The widespread presence of MDR and subsequent emergence of C3G resistance and FQNS in invasive NTS infections in sub-Saharan Africa seriously challenge treatment. Due to the high burden of invasive NTS infections in sub-Saharan Africa  and the n Africa . Finallyn Africa . These cSecondly, the global action plan against antimicrobial resistance highlights the need to optimize the use of antibiotics . HoweverThirdly, and again in line with the global action plan against antimicrobial resistance, the emerging resistance in and the lack of data on the optimal treatment of invasive NTS infections highlight the need for preventive measures to decrease the disease incidence . CurrentIn this systematic review and meta-analysis, we demonstrated the widespread presence of MDR and the emergence of C3G resistance and FQNS in invasive NTS infections in all sub-Saharan African regions. In addition, we highlighted the lack of data on the efficacy and safety of antimicrobial treatment of these infections, which was reflected in the absence of supranational evidence-based guidelines. To contain the emergence of AMR in invasive NTS infections, close monitoring and harmonized reporting of surveillance data, including data on the presence of combined resistance patterns, are essential. Some of the current recommendations list azithromycin as a second-choice antibiotic to treat invasive NTS infections. However, to allow its rational use, interpretative criteria for its susceptibility testing are needed. Lastly, clinical studies must be organized to provide the much needed data on the treatment efficacy of different antibiotic regimens, including regimens with early switch to oral administration. These data will finally allow the development of evidence-based and internationally harmonized guidelines and facilitate antibiotic stewardship.Additional file 1: Table S1. Prisma checklist. List S2. Search strategy used for MEDLINE and Ovid Embase database searches. Table S3. In- and exclusion criteria for both research questions. List S4. Published articles from which no full text was found. Figure S5. Sensitivity analysis for multidrug resistance, third generation cephalosporins & fluoroquinolone non-susceptibility. Figure S6. Meta-analysis and forest plots of multidrug resistance, third generation cephalosporin resistance and fluoroquinolone non-susceptibility according to NTS serotype. Table S7. Meta-regression to identify moderators of the pooled proportions of multidrug resistance (MDR), third generation cephalosporin resistance (C3G-resistance) and fluoroquinolone non-susceptibility (FQNS).Additional file 2. Data extraction database and datasets for meta-analysis.Additional file 3. Dataset from risk of bias assessment.


Deubiquitinating enzymes regulate various cellular processes, particularly protein degradation, localization, and protein–protein interactions. The dysregulation of deubiquitinating enzyme (DUB) activity has been linked to several diseases; however, the function of many DUBs has not been identified. Therefore, the development of methods to assess DUB activity is important to identify novel DUBs, characterize DUB selectivity, and profile dynamic DUB substrates. Here, we review various methods of evaluating DUB activity using cell lysates or purified DUBs, as well as the types of probes used in these methods. In addition, we introduce some techniques that can deliver DUB probes into the cells and cell-permeable activity-based probes to directly visualize and quantify DUB activity in live cells. This review could contribute to the development of DUB inhibitors by providing important information on the characteristics and applications of various probes used to evaluate and detect DUB activity in vitro and in vivo. Post-translational modifications (PTMs) contribute to the dynamic regulation of cellular processes by changing the structure and properties of proteins through the covalent modification of proteins. Ubiquitination is a versatile PTM and it is involved in various cellular processes such as protein degradation, protein–protein interaction, and cellular localization. Ubiquitination is carried out by the cascade of three enzymes, i.e., E1 (ubiquitin-activating enzyme), which mediates the activation step, E2 (ubiquitin-conjugating enzyme), which mediates the conjugation step, and E3 (ubiquitin ligase), which mediates the ligation step. These three enzymes catalyze the isopeptide bond between the Lys (K) residue on a substrate protein and the Gly residue on the C-terminus of ubiquitin, resulting in ubiquitination of a substrate . The mosUbiquitination can be reversed via the cleavage of ubiquitin from the substrate mediated by a group of enzymes known as deubiquitinating enzymes (DUBs). Approximately 100 DUBs have been identified in the human genome and are grouped in seven different families: ubiquitin C-terminal hydrolases (UCHs), ubiquitin-specific proteases (USPs), ovarian tumor proteases (OTUs), Machado–Joseph domain proteases (MJDs), JAB1/MPN/Mov34 metalloenzymes , and zinc finger with UFM1-specific peptidases (ZUFSPs), and MIU-containing novel DUB family (MINDY). Members of the JAMM/MPN + family are zinc metalloproteases, but the others are cysteine proteases also known as thiol proteases ,6 and brTo elucidate the biological roles of DUB or to evaluate the effect of DUB inhibitors, assay systems that can provide a precise assessment of the degree of ubiquitination are essential. Particularly, high-throughput screening (HTS) -based deubiquitination assay is required for more efficient screening of compounds targeting DUBs, and the global demand for new probes and methods applicable to HTS is growing as the importance of DUBs as a drug target increases. Here, we review the currently available deubiquitination assay systems. Escherichia coli are limited due to lack of PTMs such as phosphorylation [Deubiquitination assay typically measures the degree of ubiquitination, thus, requiring ubiquitinated substrate and DUBs. For the assays, purified recombinant DUBs are usually used; however, immunoprecipitated DUBs from cell lysates could also be used. In a typical process of determining the activity of DUBs or DUB inhibitors, deubiquitination assays with purified recombinant DUBs are performed first, followed by deubiquitination assays with immunoprecipitated DUBs or deubiquitination assays with mammalian cells to assess and confirm intracellular activity. The use of immunoprecipitated DUBs for deubiquitination assays including ubiquitin chain cleavage assays allows the analysis of DUBs as an intracellular moiety. Recombinant proteins extracted from rylation . Howeverrylation ,26. In cUbiquitin chain cleavage assay can allow for the visualization of DUB activity based on the mono-ubiquitin band by SDS-PAGE including Coomassie blue staining, silver staining, or western blotting using ubiquitin antibodies. DUBs can disassemble the ubiquitin chain, forming mono-ubiquitin. Therefore, DUB activity can be measured by examining the mono-ubiquitin band on the gel. As the deubiquitinating reaction progresses, more mono-ubiquitin bands appear. To quantify the results, the band intensity of mono-ubiquitin should be analyzed by a program such as ImageJ. Ubiquitin chains in this assay are purified recombinant protein substrates and are reacted with either purified recombinant DUBs or immunoprecipitated DUBs. Ubiquitin chains can be used for various experimental purposes. These chains have different lengths depending on the types of ubiquitin, e.g., di-Ub, tetra-Ub, and hexa-Ub. Furthermore, ubiquitin chains can be classified according to the type of ubiquitin linkage. The different types of ubiquitin chains allow the analysis of ubiquitin-linkage specificity, e.g., K11, K48, or K63 linkage chains. The linkage specificity of DUBs can be determined by performing ubiquitin chain cleavage assay using different types of linkage chains. For example, MINDY-1 is a specific DUB for K48-linked ubiquitin chains . USP35 mAs mentioned above, ubiquitin substrate and DUB are needed for deubiquitination assays. In addition to ubiquitin chains, ubiquitin probes are used as ubiquitinated substrates for deubiquitination assays. Ubiquitin probes can measure and quantify the activity of DUB through various detection methods, such as fluorescence, luminescence, mass spectrometry, HPLC, SDS-PAGE, western blotting, and MALDI-TOF assay. Traditionally, the ubiquitination of target proteins has been monitored using ubiquitin that is either epitope-tagged or radiolabeled, thus requiring laborious detection methods. Currently, fluorescent or luminescent-labeled ubiquitin is frequently used for examining ubiquitination in vitro because this approach is rapid. Here, we summarize the various ubiquitin probes commonly used.Assays with Ub-AMC can visualize the DUB activity by measuring fluorescence. Ub-AMC is the most frequently used fluorescent ubiquitin substrate whose C-terminal derivative is AMC . This deAssays with Ub-AML can visualize DUB activity by measuring the luminescence. Ub-AML is a ubiquitin reporter substrate whose C-terminal derivative is AML. Luciferin is released from Ub-AML via DUB activity. Subsequently, ATP and luciferase are added to produce a luminescent signal in proportion to DUB activity . Therefo2 can visualize the DUB activity by measuring fluorescence. Ub-PLA2 is a ubiquitin reporter substrate whose C-terminal is fused to PLA2. PLA2 requires a free amino terminus to be catalytically active. Active PLA2 cleaves the 2-acyl linkage of 3-sn-phosphoglycerides in a Ca2+-dependent reaction and is dose-dependently detected by the release of the fluorophore 7-nitrobenz-2-oxa-1,3-diazole (NBD) from 2-amino)hexanoyl-1-hexadecanoyl-sn-glycero-3-phosphocholine (NBD C6-HPC) [2 to give a free amino terminus for catalytically active PLA2. Reaction monitoring can be performed using 460 nm excitation and 534 nm emission wavelengths. In a previous study, the nonselective isopeptidase inhibitor NSC 632839 did not inhibit the PLA2 reporter enzyme, indicating that the reported inhibition was selective for isopeptidases but not for the PLA2 reporter reaction [2 is not an isopeptide bond shown in the natural ubiquitin chain; and PLA2 is just covalently linked to ubiquitin. A deubiquitination assay with Ub-PLA2 demonstrated that the DUB inhibitor P22077 inhibited USP7 and USP47, which have high similarity. In contrast, another DUB inhibitor, PR-619, exhibited a limited inhibitory effect on multiple DUBs [Assays with Ub-PLA C6-HPC) . Thereforeaction . Howeverple DUBs .Assays with Ub-Rho110 can visualize DUB activity by measuring fluorescence. Ub-Rho110 is a ubiquitin substrate whose C-terminal derivatives are Rho110. While rhodamine is in the Ub-Rho110 moiety, it is di-substituted thereby quenching the intrinsic fluorescence. However, mono-substituted rhodamine, which exhibits intense fluorescence, is released by the DUBs, thus allowing the real-time monitoring of the sensitivity or activity of DUBs. This monitoring can be performed using 485 nm excitation and 535 nm emission wavelengths . With thFP is a technique based on the difference between the polarized fluorescent light emitted when a fluorescent-labeled molecule is highly excited by polarized light and the less polarized fluorescent light emitted when a fluorescent-labeled molecule is cleaved by DUBs. Therefore, the signal is inversely proportional to DUB activity. The higher the DUB activity, the lower the emitted fluorescence. FP assays are readily adaptable to HTS, which would be useful for the screening of DUB inhibitors ,42,43. Assays with Ub-Lys-TAMRA-Gly can visualize DUB activity by measuring fluorescence. TAMRA is a fluorophore, and Ub-TAMRA is a ubiquitin substrate whose derivative is TAMRA. The Lys-Gly sequence is modified by TAMRA and is linked to ubiquitin through an isopeptide bond with a lysine side-chain, thus mimicking ubiquitinated substrates. It is useful for studying C-terminal ubiquitin hydrolytic activity over a continuous period at longer wavelengths. Ub-Lys-TAMRA-Gly has been prepared by chemical synthesis, and this probe allows the detection of ubiquitination based on fluorescence . The act3+-complexes (the donor). This modified assay showed good sensitivity and selectivity for monitoring the effect of the UCH-L1 inhibitor [Similar to ubiquitin chain cleavage assays, deubiquitination assays with di-Ub probe allow a better understanding of the DUB linkage-type preference . The ubinhibitor . 2 assay is better suited for drug discovery than that of the Ub-AMC assay. However, in an experiment screening commercial libraries for USP7 inhibitors using this assay, it could not be distinguished whether the inhibitors found inhibited USP7 or PLA2, which limits its broader applicability [Fluorescence-based deubiquitination assays have been found to have a few limitations. The most common and widely used Ub-AMC has a relatively narrow wavelength range compared to other Ub-based probes, and the excitation wavelength is in the UV range. Therefore, it is not suitable for drug discovery because it can excite many screening compounds and cause false positives . Unfortucability . Unlike cability .Deubiquitination assays with mammalian cells are performed using mammalian cell lysates or live cells. This allows the monitoring of DUB activity within the intracellular environment. In cell lysates, DUB retains its native form, either bound to interacting proteins required for proper folding or multimeric assembly, or modified by “authentic” human-like PTMs such as phosphorylation . TherefoK, e.g., only the Lys48 residue is replaced with Arg and all Lys residues are replaced with Arg except Lys48 [Nickel pull-down assays can visualize the ubiquitination of the substrate protein by western blotting using anti-substrate antibodies. In these assays, the substrate, DUB, and His-tagged ubiquitin are overexpressed in cells, and ubiquitinated cellular proteins are extracted by Ni-NTA agarose and analyzed by western blotting. Ni-NTA agarose is a nickel-charged affinity resin for purifying proteins tagged with a polyhistidine (6 × His) sequence. His residues of the His-tagged protein bind to the immobilized nickel ions with high specificity and affinity , and othpt Lys48 . Nickel pt Lys48 . In anotpt Lys48 . Deubiquitination assays with mammalian cells and ABPs can confirm DUB activity by SDS-PAGE using anti-DUB antibodies or antibodies to tag the probes. Activity-based DUB probes (DUB ABPs) include various electrophiles, substrates, and N-terminal tags. Propargylamide (PA or Prg), vinylmethyl ester (VME), and vinylsulfone (VS) are typical electrophiles and their classification is summarized in When ABPs are incubated with mammalian cell lysates, they form a covalent bond with the active site of DUBs. Monoubiquitin probes are widely used; thus, a DUB bound to the probes is shifted to a higher molecular weight position on the gel. This upper band suggests that the active DUB is well labeled with the probes. As the extent of probe labeling depends on the activity of the DUB, the more intense the probe-bound band, the higher the DUB activity. When the DUB is inhibited by a DUB inhibitor, the upper band disappears or shows a lower band intensity ,63. In cA number of probe designs have been reported since the development of the first DUB-related ABP; however, none of them showed cell permeability, which may be attributed to the large size of the recognition element in the probes . ABPs foPreviously, pore-forming toxin and electroporation were used to deliver DUB ABPs that could not pass through the cell membrane into cells. Claessen et al. were the first to use perfringolysin O (PFO), a pore-forming toxin that binds to cholesterol in the plasma membrane and forms a large pore complex, to deliver their developed catch-and-release ubiquitin ABP, which is a Ub-VME derivative with a cleavable linker (release motif) attached to a biotin affinity handle (catch part), into cells . When thRecently, ABPs that can cross the cell membrane have been developed. Attaching a CPP to DUB ABPs allows the probes to pass through the cell membrane. CPPs have been reported as versatile delivery vehicles for cell-impermeable molecules . Gui et As mentioned above, the requirement of a full-length ubiquitin as a recognition element is the main reason why DUB ABPs cannot pass through cell membrane. To address this problem, the use of small molecule-based DUB ABPs has been considered. The idea of using small molecule-based DUB ABPs was proposed during the development of DUB inhibitors. Small molecules covalently bound to DUBs have been modified by the addition of a reporter group e.g., fluorescent molecules and used for competitive activity-based protein profiling (ABPP).A small molecule-based DUB ABP, based on a chloroacetylpyrrole scaffold, was first introduced by Ward et al., which had an inhibitory effect on USP4 and USP11 . This inSmall-molecule covalent inhibitors of ubiquitin carboxy-terminal hydrolases (UCHs), another DUB subfamily, are useful as novel cell-permeable ABPs for identifying and quantifying target protein in cells. Panyain et al. developed a cyanamide-containing inhibitor for UCHL1. This inhibitor and its alkyne-tagged analog, IMP1720, labeled the catalytic Cys residue of UCHL1 in an activity-dependent manner and provided a new tool to examine UCHL1 activity in several cell types . AnotherThere have been limited reports of effective small-molecule DUB ABPs. Nevertheless, the development of small-molecule DUB ABPs is regarded as vital for the application of DUB ABPs at low concentrations (nanomolar levels) in cells. However, strong off-target effects and toxicity may limit the utility of these probes in living systems. If these problems are addressed, small-molecule-based ABPs will be a potent cell-permeable tool with selective DUB activity for target identification in cells.All DUBs bind to ubiquitin adjacent to their catalytic site. Therefore, ABPs for DUBs typically include full-length ubiquitin as a substrate with a Cys reactive electrophilic group. However, some reports revealed that DUB ABPs also label various non-DUB proteins ,76,77. IThe use of CPPs with ABPs to pass through the cell membrane has also been reported to substantially disrupt cell membrane integrity, leading to the formation of non-physiological subcellular compartments . Indeed,Small-molecule-based ABPs have strong off-target effects, affecting proteins such as non-DUB proteins, which could induce cellular toxicity. For example, b-AP15, a known inhibitor of USP14 and UCH-37 , and itsThe profiling of DUB activity using various probes is important for characterizing novel DUBs and identifying DUB inhibitors. Since each probe has different reactivities and specific applications, it is necessary to understand their features and efficacy. We have summarized deubiquitination assays according to the detection methods  and purp


Case Presentation. A 20-year-old male presented with a one-week history of worsening nonpruritic erythematous maculopapular skin rash (resembling EM) which appeared on the face and subsequently spread to the trunk, arms, legs, palms, and soles. There was no mucosal involvement. At the onset of rash, the patient reported headaches, associated with photosensitivity and intermittent fevers. Workup for viral meningitis yielded negative results. Laboratory investigation revealed mild anemia, elevated inflammatory markers, a positive ANA with speckled pattern, a positive anti-Ro/SSA, anti-La/SSB antibodies, and a positive rheumatoid factor (RF). Lupus anticoagulant antibody was positive along with a low-positive anticardiolipin IgM antibody and a negative beta-2 glycoprotein antibody. Anti-dsDNA, anti-Smith, anti-Jo-1, anti-centromere, and anti-Scl-70 antibodies were negative. Hepatitis serologies, herpes simplex virus 1 and 2, mycoplasma, Epstein–Barr virus, HIV, and parvovirus B19 were negative. Left arm skin biopsy demonstrated vacuolar interface dermatitis and positive colloidal iron stain suggestive of dermal mucin deposition, favoring the diagnosis of cutaneous collagen vascular disease. Cutaneous lesions improved with administration of oral prednisolone.  Rowell's syndrome is comprised of the presentation of erythema multiforme- (EM-) like lesions in association with lupus erythematosus (LE), along with serologies of speckled antinuclear antibodies (ANAs), positive rheumatoid factor (RF), positive anti-La/anti-Ro, and the clinical finding of chilblains. As per the redefined criteria by Zeitouni et al., three major criteria in addition to at least 1 minor criterion are necessary for diagnosis.  Rowell's syndrome should be considered in patients who present with cutaneous LE and lesions resembling EM. Further serological markers should be pursued in the absence of obvious EM-precipitating factors. Rowell's syndrome describes the constellation of erythema multiforme- (EM-) like lesions in association with lupus erythematosus (LE), along with the findings of speckled ANA, positive rheumatoid factor (RF), positive anti-La/anti-Ro, and the clinical finding of chilblains.The coexistence of EM-like lesions and LE was first described by Scholtz in 1922; however, the distinct entity of Rowell's syndrome was then described in 1963 by Rowell et al. who recognized particular serologic markers of patients with this overlap [Ever since its proposal in 1963, there have been numerous reports of cases relating their findings to Rowell's syndrome, many of which were later found to lack the criteria originally described by Rowell. This led to a revision of diagnostic criteria, which were redefined by Zeitouni et al. in the year 2000 to account for the inconsistency of certain criteria as compared to others. As redefined, major criteria include the coexistence of lupus erythematosus (SLE or discoid lupus erythematosus) or subacute cutaneous lupus erythematosus, with EM-like lesions with or without mucosal involvement and finally speckled pattern of ANA. Minor criteria include clinical finding of chilblains, positive anti-Ro or anti-La antibodies, and lastly positive rheumatoid factor (RF). In order to make the diagnosis, all three major criteria, as well as at least 1 minor criterion, must be met [Whether Rowell's syndrome exists has remained a matter of debate among physicians with some believing it to be a mere coincidental occurrence of EM in patients with LE while some believing it to be a cutaneous manifestation of lupus.We report the case of a healthy 20-year-old male who presented with EM-like lesions, positive RF, positive ANA with a speckled pattern, and positive anti-Ro antibody titer consistent with diagnosis of Rowell's syndrome.A 20-year-old male with no prior medical diagnoses presented with a one-week history of worsening nonpruritic erythematous maculopapular skin rash which initially appeared on his face  and subsOne month later, he presented once again with worsening of the same nonpruritic rash, associated with neck pain, photophobia, headache, low-back pain, fatigue, anorexia, as well as intermittent fevers with a peak temperature of 102F. Examination revealed generalized well-defined nonpruritic, nonblanching confluent targetoid papules and plaques (resembling EM) in various stages of healing. The lesions were located primarily on the torso, back, arms, legs, palms, soles, and lips. Newer lesions appeared erythematous, while older lesions appeared darker. The lesions located on the forearms and face were noted to exhibit small areas of fine scaling. There was no evidence of mucosal involvement.In addition to the initial workup for infectious etiology and viral as well as bacterial meningitis , laboratory investigation revealed mild anemia (Hb 12.6 g/dL), elevated erythrocyte sedimentation rate (ESR) of 75 mm/h, C-reactive protein (CRP) of 11.05 mg/L, a positive ANA titer of >1 : 1280 with speckled pattern, a positive anti-Ro/SSA, anti-La/SSB titers of >8.0, and a positive RF of 34 IU/ml. Lupus anticoagulant antibody was positive (>44.4 sec), along with a low-positive anticardiolipin IgM antibody (>13 U/ml) and a negative beta-2 glycoprotein antibody. Anti-dsDNA, anti-Smith, anti-Jo-1, anti-centromere, and anti-Scl-70 antibodies were negative. Serum chemistry and urinalysis were within normal limits. Hepatitis B virus (HBV), hepatitis C virus (HCV), herpes simplex virus (HSV) 1 and 2, mycoplasma, Epstein–Barr virus (EBV), human immunodeficiency virus (HIV), and parvovirus B19 were negative.The patient then underwent a left arm skin biopsy, which demonstrated vacuolar interface dermatitis and positive colloidal iron stain suggestive of dermal mucin deposition, favoring the diagnosis of cutaneous collagen vascular disease.Cutaneous lesions improved with administration of oral prednisolone.This case highlights the importance of maintaining a diagnostic suspicion for Rowell's syndrome in LE patients who present with EM-like lesions. Lupus anticoagulant antibody was positive in our patient, but he did not meet criteria for antiphospholipid syndrome (APS). It should be noted, however, that a rare association between APS and Rowell's syndrome has been described. RS in association with LE and APS could be a distinct entity, or RS could be a spectrum of clinical and immunologic phenomenon of which APS may be one. Further characterization requires analysis of coagulation parameters in patients .The important learning points from this case are twofold. First and foremost, although Rowell's syndrome is a recognized syndrome in patients with a diagnosis of LE who present with a certain correlation of clinical symptoms and findings, a degree of suspicion should be maintained in those who do not have a prior diagnosis of LE, such as in our patient.Patients with cutaneous LE may develop coincidental EM. However, if characteristic serological abnormalities are present and there is no obvious precipitating event, the association is known as Rowell's syndrome.Since the first report of RS, not more than 71 cases have been reported in the English literature; nonetheless, a recent review demonstrated that most of the reported cases did not fulfill all the diagnostic criteria of Rowell's original description, especially the presence of RF and anti-La antibody .The current diagnostic criteria help us distinguish RS from other causes of EM which include drugs and infections. Specifically, in patients with LE, possible causes for EM in the presence of an inciting event could be NSAIDs if used for management of arthritis. Use of immunosuppressants can predispose patients to infections  which are independent triggers for EM .Other possible differentials for EM include urticaria, Stevens–Johnson syndrome (SJS), fixed drug eruption, and cutaneous small vasculitis . These cOur patient met 3 major and 2 minor criteria thus establishing a diagnosis of RS . A detaiDiagnosis of this condition is prudent for further management and appropriate monitoring of possible complications of LE. Most reports show a successful treatment using systemic steroids and additional immunosuppressive drugs such as azathioprine and cyclosporine. Some studies also reported the use of topical steroids in the management of RS with variable response , 9. ZeitRowell's syndrome should be considered in patients who present with cutaneous LE and lesions resembling EM. Further serological markers should be pursued in the absence of obvious EM-precipitating factors. This presentation is unique since Rowell's syndrome has been described rarely in men; most of the cases reported in the literature are predominantly middle-aged women.


Alzheimer’s disease (AD) has been a devastating public health with the development of global aging. Approaches for reducing the current AD epidemic are becoming a primary focus of human healthcare due to the lack of achieved lasting and complete remission strategies to treat AD with the characteristics of heterogeneity and complexity. Exosomes, which is the new emerging approach to intercellular communication, provide novel perspective on identified therapeutic strategies of AD. Mesenchymal stem cell-derived exosomes (MSC-exos) are emerging to be an appealing therapeutic tool for AD, with the donor-derived properties and the characteristics of minimal immunogenicity, effortless storage, nature delivery vehicles, and low risks of tumor formation based on the previous researches. In this review, we elaborate the mechanism of MSC-exos in the treatment of AD and discuss limitations in the clinical application. Alzheimer’s disease (AD) is the most common cause of dementia in the elderly, which contributes to 60–80% of total dementia population . What isAD is characterized by increased deposition of β-amyloid peptides and aggregation of hyperphosphorylated tau in neurofibrillary tangles (NFT) . HoweverIn recent years, mesenchymal stem cells (MSCs) have attracted much attention as the potential cell-based therapeutic tools due to its ability of migrating and mediating damage repair. MSCs facilitate neurological recovery and neo-angiogenesis through the secretion of neurotrophins and angiogenesis regulatory factors –10. The Exosomes ) are considered as a subtype of extracellular microvesicles with a diameter of 30–100 nm, including lipid bilayer membrane structure, and released by variety of cell types , 20. InvHerein we outline the role of exosomes in AD treatment. Additionally, we elaborate the therapeutic properties of MSC-exos in AD and discuss the advantages and challenges of MSC-exos as a novel cell-free therapeutic agent.Enormous efforts were invested to clarify the mechanism of exosome biogenesis, secretion, and uptake Fig. . OriginaAlthough the process of exosome release remains elusive, it is widely accepted that Rab GTPases, soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs), and cytoskeleton are involved in the regulation process . Rab GTPIt has been reported that endocytosis is the primary pathway for exosome uptake. In addition, exosomes can deliver cargo to recipient cells through direct membrane fusion and receptor-ligand binding . The steTo date, the exact etiology of AD is not particularly clear. Amyloid cascade hypothesis plays dominant role in explaining the genesis and progression of AD . HoweverFirstly, brain-derived exosomes in peripheral blood have shown great potential to be an ideal “liquid biopsy” for AD Table . ExosomeSecondly, exosomes are considered as a potential vehicle for drug delivery in AD. As a natural biological agent, exosomes possess homing capabilities to transport active molecules between cells with favorable biocompatibility, and transiently modulate functions of targeted cells . It has Additionally, exosomes are involved in the process of pathogenic protein clearance. Beta-amyloid peptide and tau, two symbolic pathological proteins in AD, have been suggested to be associated with the neuronal damage and death, leading to gradual decline in memory and cognitive impairment . PreviouFinally, exosomes play a role in promoting neurogenesis and reducing cognitive impairment. However, the specific cellular and molecular mechanisms of these processes are still unclear. It has been reported that NDEs are able to regulate the number of AMPA receptors for glutamate transmission or induce synaptic pruning by the overexpression complement component 3 of microglia cells, indicating that NDEs are involved in the process of synaptic plasticity. Additionally, neuronal EVs are involved in the regulation of miRNA content and neuronal excitability , 80. SevMSCs are a class of adult multipotent stromal cells with self-renewal capabilities. They can be isolated from a variety of sources, including bone marrow (BM-MSCs), adipose tissue (AD-MSCs), umbilical cord (UC-MSCs), amniotic fluid, placenta, and peripheral blood –91. The Given the heterogeneity and complexity of exosomes, different isolation methods have been discovered Table . (1) UltSeveral studies indicate that the storage temperature and freeze-thaw times are able to affect the therapeutic utility of exosomes. After comparing, researchers suggest that storage temperature at − 80 °C is the most suitable condition for long-term preservation of exosomes . RepeateTo date, the administration route and dosage of MSC-exos are still inconclusive. Experimental results indicate that the route or schedule of administration may significantly influence the dosage of therapeutic MSC-exos . In addiMSC-derived microvesicles were initially proposed to promote cell proliferation and enhance anti-apoptotic ability of tubular epithelial cells in acute tubular injury model in 2009 . AccumulIn the previous studies, β-amyloid is considered as the production of proteolytic cleavage of the amyloid precursor protein (APP) by β- and γ-secretases . Aβ monoIt is widely indicated that the pathogenesis of AD is closely related to the immune system. To our knowledge, classical neuroimmune cell-to-cell communication is interacted with membrane . MSCs plNeuroinflammation is emerging as a central pathological process in AD . ExcessiIn addition, MSC-exos are able to suppress inflammatory response through regulating enzyme activity. Aβ induce nitric oxide synthase (NOS) in glial cells and then release high levels of nitric oxide (NO). NO induces neurotoxicity via inhibition of mitochondrial respiration, resulting in neuronal cell death . In thisSeveral previous studies suggested that MSC-exos affect post-transcriptional gene expression and ensue protein expression in the target cells via the delivery of miRNAs. miRNAs, small noncoding RNAs, play a key role in regulating several biological processes such as growth, inflammation, and angiogenesis. Exosomes containing miRNAs released by MSCs can inhibit the activity of immune cells and enable their phenotypic conversion into anti-inflammatory. For example, microglia plays the role of first innate immune defensive line in brain. When activated, microglia exhibit two different polarized phenotypes namely M1/M2 , 148. InSynapse dysfunction, another pathologic hallmark, generally appears in the early stage of AD, which is directly related to cognitive impairment. As suggested by recent findings, hMSC-EVs protect hippocampal neurons via blocking oxidative stress and synapse damage exposed to amyloid beta oligomers (AβOs) . AdditioGiven different cell sources, the characters of exosomes vary according to content of cargos, which are affected by the physiological situation and extracellular environment . Hence, www.clinicaltrials.gov utilizing MSC-exos. Researchers from the Shanghai Jiao Tong University School of Medicine decide to evaluate the safety and effectiveness of MSC-exos in patients with mild to moderate dementia (NCT04388982). This clinical trial plans to recruit 9 patients to participate in the study. Patients will be given three doses of low, medium, and high  MSC-exos twice a week, respectively, for 12 weeks via nasal drip as planned. In the primary stage of trails, patients will be measured for the functions of liver or kidney and treatment-related adverse events. Cognitive function, quality of life, and neuroimaging will be evaluated during the secondary stage of trails. Although the clinical trials of MSC-exos in the treatment of AD have not yet been done, promising results have been confirmed in other diseases . It is encouraging that MSC-exos can ameliorate inflammation and improve kidney function of grade III-IV chronic kidney disease (CKD) patients in a phase II/III clinical pilot study. Of note, during the 1 year follow-up, no adverse events related to the administration of MSC-exos were found in subjects [Despite the results of previous experiment are promising, few on-going or completed clinical studies have explored the potential role of MSC-exos in clinical trials . After csubjects . In addiAccumulating evidence suggest that MSC-exos possess superior safety profile, anti-inflammatory effects, minimal immunogenicity, and low risks of tumor formation , 157. UnAs a therapeutic agent, MSC-exos still face great challenges. The main reasons may be attributed to the following points. (1) The isolation, storage, and purification protocols of MSC-exos still need continuous majorization and standardization to enhance the comparability and reproducibility. Since the conventional isolated methods mainly depend on density and size, some substances  may overlap with their characteristics, resulting in incomplete removal. Clinical applications depend on time-saving, low-cost, and convenient methods, but the existing separation methods obviously do not meet these conditions. In addition, in order to promote the development of effective biomarkers for exosomes, sensitive, accurate, and rapid quantitative methods are essential. (2) The therapeutic effects of MSC-exos in promoting neurite growth may vary depending on the source of MSCs . HoweverAlthough much effort has been invested in AD, the achievements seem to be unsatisfactory . The pre


E. coli K-12 for 0–20 s, leading to time- and dose-dependent reductions in colony-forming units (CFU) and morphological changes. Both membrane lipids and cytoplasmic metabolites of the control and IPL-treated E. coli were examined by the liquid chromatography-mass spectrometry (LC-MS)-based metabolomic fingerprinting. The results from multivariate modeling and marker identification indicate that the metabolites in electron transport chain (ETC), redox response, glycolysis, amino acid, and nucleotide metabolism were selectively affected by the IPL treatments. The time courses and scales of these metabolic changes, together with the biochemical connections among them, revealed a cascade of events that might be initiated by the degradation of quinone electron carriers and then followed by oxidative stress, disruption of intermediary metabolism, nucleotide degradation, and morphological changes. Therefore, the degradations of membrane quinones, especially the rapid depletion of menaquinone-8 (MK-8), can be considered as a triggering event in the IPL-elicited metabolic changes in E. coli.Intense pulsed light (IPL) is becoming a new technical platform for disinfecting food against pathogenic bacteria. Metabolic changes are deemed to occur in bacteria as either the causes or the consequences of IPL-elicited bactericidal and bacteriostatic effects. However, little is known about the influences of IPL on bacterial metabolome. In this study, the IPL treatment was applied to  Escherichia coli is a Gram-negative and facultative anaerobic bacterium widely distributed in the intestinal tracts of animals and humans + = m/z 609.2812) for non-polar fraction. Chromatograms were acquired and processed by MassLynxTM . Tandem MS (MSMS) fragmentation with a collision energy ramp of 10–50 eV was used to assist in determining the structure of metabolites. The performance of LC-MS analysis was monitored by injecting a pooled sample three times  in each run as quality controls (QC) (https://www.metabolomicsworkbench.org/ (accessed on 1 January 2021)) with their study IDs as ST001657, ST001663, and ST001664.The workflow of LC-MS analysis was described previously ,60. In bols (QC) . The LC-m/z), and ion abundance were included in the matrix. The relative intensity of each ion was calculated by normalizing the single ion counts (SIC) versus the total ion counts (TIC) in the whole chromatogram, while setting the TIC arbitrarily to 10,000. The processed data matrix was further exported into SIMCA-P+TM software , transformed by Pareto scaling, and then processed by principal components analysis (PCA). The contribution of samples to the principal components were described in a scores scatter plot of a multivariate model. The IPL-responsive metabolites were identified by analyzing the ions contributing to the principal components in a loadings scatter plot. The chemical identities of metabolite markers were determined by elemental composition analysis, database search ; ECMDB, http://ecmdb.ca/ (accessed on 16 January 2021); HMDB, www.hmdb.ca (accessed on 16 January 2021); and KEGG, https://www.genome.jp/kegg/ (accessed on 16 January 2021)), fragmentation, and comparison with authentic standards if possible. Elemental composition analysis was conducted using the respective module in MassLynx  software, which calculates the molecular formula based on both accurate mass (within 5 ppm of exact mass) and the fitness value based on the similarity between the measured abundances of isotopic peaks and their theoretical abundances.In total, 497 features were captured by MarkerLynx software  and incorporated into a multivariate data matrix after centroiding, deisotoping, filtering, peak recognition, and integration. Sample name, ion feature  for comparing the relative abundances of metabolites across different treatment groups.