To conduct a bibliographic review on tuberculosis (TB) disease in children and adolescents with rheumatic diseases, being managed with biologic therapy.tuberculosis”] AND ([“children”] OR [“adolescent”]) AND [“rheumatic diseases”] AND , between January 2010 and October 2021.An integrative review with a search in the U.S. National Library of Medicine and the National Institutes of Health (PubMed) using the following descriptors and Boolean operators: , 80 cases of pulmonary tuberculosis (PTB), and four of extrapulmonary tuberculosis (EPTB). The main rheumatic disease was juvenile idiopathic arthritis. Among LTBI cases, most were diagnosed at screening and none progressed to TB disease during follow-up. Of the TB cases using biologics, most used tumor necrosis factor-alpha inhibitors (anti-TNFα) drugs. There was only one death.The study revealed a low rate of active TB in pediatric patients using biologic therapy. Screening for LTBI before initiating biologics should be done in all patients, and treatment, in cases of positive screening, plays a critical role in preventing progression to TB disease. It is estimated that 10 million people became ill due to TB in 2019 and that there were 1.2 million deaths in individuals with TB not infected with the human immunodeficiency virus (HIV) that same year.Despite advances in the diagnosis and treatment of tuberculosis (TB), the disease remains among the ten leading causes of death in the world. comprising more than a fifth of TB patients in high-incidence countries and accounting for 8–20% of TB-related deaths in those countries.Children under the age of 15 accounted for 12% of reported TB cases in 2019,Rheumatic diseases are a heterogeneous group of diseases characterized by alterations of the musculoskeletal system and systemic manifestations, including autoimmune connective tissue diseases, auto-inflammatory diseases, and vasculitis. Patients with rheumatic diseases exhibit a higher frequency of TB than the general population (2–10 times in adults), which may be associated with immunosuppression linked to the underlying disease, as well as due to the use of immunosuppressive medications, such as corticosteroids and biologics.The objective of this study was to conduct a bibliographic review, referring to TB disease in children and adolescents with rheumatic diseases, being managed on biologic agents.tuberculosis”] AND ([“children”] OR [“adolescent”]) AND [“rheumatic diseases”] AND . The PubMed database was adopted because it is the main source of consultation in the medical field.For the bibliographic review, referring to TB in children and adolescents with rheumatic diseases, a search was performed in the U.S. National Library of Medicine and the National Institutes of Health (PubMed) using the following descriptors and Boolean operators:  and extrapulmonary TB (EPTB) in children and adolescents aged 0–19 years with rheumatologic diseases using biologic drugs. The time period of the bibliographic search was from January 2010 to October 2021. Exclusion criteria were studies that 1) addressed only latent tuberculosis infection (LTBI) and 2) included a narrative literature review.Initially, the titles and abstracts were evaluated. Following the initial screening, the selected articles were read in full, and the studies to be included in the review were finally chosen. After the complete reading of the articles, a direct search of the bibliographic references was performed by author and title in the PubMed database in order to find articles omitted from the initial search and relevant to the subject. To minimize biases, the search, evaluation, and selection of studies were carried out by two independent reviewers. When there was disagreement, a third reviewer was consulted and, after a consensus discussion, with the agreement of all, the articles to be included on the review were selected.In the present review, some items of the articles were evaluated to assess their quality: objective; adequate methodology for the proposed objective; detailed data collection; rigor in data analysis; presentation and discussion of results; and research contributions and limitations. Articles that fulfilled all the criteria were able to be included in the review. From the bibliographic references of the works read in full, two articles were included through a direct search by author and title in the PubMed database. Thirty-seven studies were included.Among the 37 references, most studies were prospective (15/37) and retrospective (10/37), followed by case reports (4/37).The total number of patients investigated was 36,198, with 30,246 pediatric rheumatologic patients using biologic drugs. There were 81 cases of LTBI, 80 cases of PTB, and four cases of EPTB. In 34 studies, the disease evaluated was juvenile idiopathic arthritis (JIA), and in three, in addition to JIA, systemic lupus erythematosus (SLE), juvenile dermatomyositis (JDM), familial Mediterranean fever, spondyloarthritis, vasculitis, collagen diseases and auto-inflammatory syndromes. shows the authors, year of publication, type of studies — with the number of pediatric rheumatologic patients using biologic drugs — and population — age and rheumatic diseases.Among the 81 cases of LTBI described, most (73/81) were diagnosed at screening, before therapy with a biologic agent was initiated, and no case progressed to TB disease during follow-up. Only 7/81 of the cases were diagnosed during treatment with biologics: 5/7 treated with etanercept (ETA), 1/7 with infliximab (IFX), and 1/7 with sequential use of IFX, adalimumab (ADA), and ETA. All had been screened before the start of the biologic agent. One case of LTBI occurred in a patient using methotrexate (MTX), with previous therapy with ETA. In this case, there is no report of pre-biologic screening.Among the cases of PTB, 2/80 were diagnosed during the initial screening and 52/80 were in pediatric patients not using biologic drugs . The other 26/80 cases of PTB were in patients using biologic agents. Among the 26, 21 were being treated with tumor necrosis factor-alpha inhibitors (anti-TNFα), two were using canakinumab, and three were using unspecified medications outside the anti-TNFα class.Of the four EPTB cases, 1/4 was miliary, central nervous system, and articular; 1/4, central nervous system and gastrointestinal tract; 1/4, pulmonary and ophthalmic; and 1/4, pulmonary and pleural. Three of the patients were being managed with anti-TNFα and one was sequentially using ETA, ADA, and abatacept, with a diagnosis of EPTB while using abatacept. The latter had a fatal outcome after complications of central nervous system involvement and was the only death reported in all the included studies.  Most (10/17) were studies with anti-TNFα medications. There were only eight specific papers on TB in children with rheumatic diseases using biologics. One was a case-control study that evaluated the risk of TB in children with JIA in Taiwan, two were retrospective studies that evaluated the safety of anti-TNFα/biologics in relation to TB, one was a prospective study that evaluated TB during treatment with biologic agents in a population vaccinated with bacillus Calmette-Guérin (BCG), and four were case reports.Studies that evaluated the safety of biologic drugs and reported their findings on TB were included. Seventeen articles  did not report any cases of active TB or LTBI and also pointed to JIA as the most frequent disease. Of the 30 cases of TB disease , 8/30 occurred in patients who had been screened before biologic therapy and 22/30 in those not screened. Of the eight specific studies on TB, screening was conducted in six; of the two studies in which there was no report on screening for TB, one is the case that progressed to death.Screening for TB before starting biologic therapy was performed in just over half (19/37) of the studies. This system evaluates clinical, radiological, and epidemiological data and does not involve bacteriological confirmation, which is known to be challenging in children. The difficulty of bacteriological isolation in children occurs because the disease is paucibacillary in this age group and also because of the difficulty in collecting respiratory specimens.In Brazil, the diagnosis of PTB in children and adolescents is based on a scoring system of the Ministry of Health — Brazil.On chest radiography, signs suggestive of active TB are considered: mediastinal and/or lymph node enlargement, alveolar opacifications, pleural effusion, miliary nodules, and cavitations.Mycobacterium tuberculosis infection or latent TB infection (LTBI) as a state of persistent immune response to stimulation by Mycobacterium tuberculosis (M. tb) antigens without evidence of clinical manifestations of active TB.The World Health Organization defines latent  If LTBI is not diagnosed and treated appropriately, activation occurs in 5–10% of cases.In cases of LTBI, despite the absence of symptoms, there is a risk that patients with rheumatic diseases will develop TB disease, especially in the first two years after the primary infection.M. tb or other mycobacteria, and vaccination with BCG. TST may, therefore, produce a false-positive result because it is not specific to M. tb. IGRAs are more specific tests because they detect the immune response against M. tb antigens that are not present in BCG or other mycobacteria. However, IGRAs are costlier than the TST and must be performed in a laboratory.There are two tests available for the diagnosis of LTBI, the tuberculin skin test (TST), and the interferon-gamma release assay tests (IGRAs). TST is performed by intradermal injection, using the Mantoux method, whereby purified protein derivative (PPD) detects previous infection with  Elevated levels of tumor necrosis factor-alpha (TNFα) in rheumatologic diseases reduces the cytokine response, with recovery after initiation of anti-TNFα therapy. One study reported conversion of TST to positive one year after the first screening, in 30% of patients who started treatment with anti-TNFα, which could be explained by the restoration of suppressed immune reactivity against TB antigens, with a decrease in the underlying disease activity. Children below five years of age may also have undetermined IGRAs.A limitation associated with TST and IGRAs is related to the immunological status of patients. TST and IGRAs can be affected by severe immunosuppression in patients before starting treatment. Biologic drugs are derived from living organisms produced using molecular biology techniques, which act as monoclonal antibodies or receptor or cytokine antagonists, hence the term “biologic” or “immunobiologic”.From 1975 onwards, there has been the development of biologic drugs, which are medications that act on cell signaling or interaction processes, resulting in the activation and/or regulation of the immune response. For example, children with JIA or inflammatory bowel disease exhibit high levels of pro-inflammatory cytokines in peripheral blood, synovial fluid, or gastrointestinal mucosa. Anti-TNFα drugs prevent excessive inflammation and the consequent tissue damage.In chronic inflammatory diseases, high concentrations of TNFα are produced, leading to excessive inflammation and damage to the body.Among the biologic drugs, those that act on TNFα were developed first and tested in adults with rheumatoid arthritis, with positive results. This encouraged their application in other diseases, eventually expanding their use to pediatrics, particularly in cases that are refractory to traditional medications. ADA and tocilizumab (TCZ) were the second and third biologics, respectively, used in patients with JIA.ETA was the first anti-TNFα drug approved for the treatment of JIA in 1998.M. tb, particularly in the formation and maintenance of granuloma integrity. TNFα works synergistically with interferon-gamma, increases the expression of intercellular adhesion molecules , and stimulates the production of the bactericidal compound from intermediates of nitrogen and oxygen. Therefore, the granuloma, composed of differentiated macrophages and lymphocytes, restricts the growth and spread of M. tb, resulting in a dynamic balance between pathogen and host and the induction of LTBI. Blocking this cytokine impedes the immune system’s ability to contain M. tb within granulomas.TNFα is a pro-inflammatory cytokine secreted by monocytes, macrophages, and T lymphocytes and develops an important role in the immune response against  In some cases, extrapulmonary manifestations or disseminated forms of TB develop.Exposure to these drugs has been associated with LTBI reactivation, even after discontinuation of treatment, with progression to TB disease. Before starting biologic drugs, serious infections such as hepatitis B and C, HIV, and TB (active and LTBI) should be excluded or adequate therapeutic planning and treatment of these conditions undertaken whenever possible, before initiating biological therapy.Since the emergence of biologic agents, there has been an increase in the risk of infections, and patients should be kept under infection surveillance.Cakan et al. showed that screening with TST and chest X-ray, associated with treatment for LTBI with isoniazid for six months, in cases of TST ≥5 mm, would be sufficient to protect against active TB before initiating the biologic agent. This difference may occur due to the differences in affinity of the receptors of the drugs; IFX has affinity for the TNF 1 receptor and ETA for the TNF 2 receptor. The TNF 2 receptor develops a less significant role in defending against TB. IFX and ADA reduce gamma interferon production by 65–70%, in contrast to ETA, which causes almost no change in gamma interferon.Studies suggest that IFX and ADA increase the risk of TB compared to ETA.M. tb. However, this does not play a fundamental role in infection control, which may explain why there is minimal or no risk of developing TB in patients treated with anti-IL1 drugs.In turn, interleukin-1 (IL1) appears to be involved in late hypersensitivity to  The American guideline recommends screening for all patients scheduled to initiate therapy with biologics, such as IGRAs or TST. The American College of Rheumatology recommends that patients with JIA be screened for LTBI before initiating biologics.Each country determines its guidelines for LTBI screening; some guide the use of TST and IGRAs .The evaluation of LTBI in Brazil includes the performance of TST, chest X-ray, and epidemiological history of close contact with TB, which should be performed before starting biological therapy. In patients with no clinical-radiological signs of active TB and TST ≥5 mm or history of contact with TB, LTBI treatment should be performed, and a biologic drug should be initiated one month after the start of the LTBI treatment, in adults. Physicians and patients should be aware of the risks related to TB in order to initiate investigation for the disease as early as possible in case of suggestive signs/symptoms or an epidemiological history of contact with TB. In the study by Atikan et al., screening for TB is strongly recommended before starting therapy with biologics, as well as annually, with chest radiography, TST, and/or IGRAs, in addition to clinical history, for all patients who continue to be managed on biologic therapy.Screening for LTBI and administering treatment before starting biologics have proven to be effective in preventing the TB activation.However, the periodicity of screening for TB in the pediatric population using biologic therapy requires evaluation in further prospective studies and in countries with high rates of TB. However, in countries with a low rate of the disease, systematic repeat screening for TB in children using anti-TNFα drugs, after an initial negative test, may not be necessary.Among the included studies, the only case of death occurred in a patient sequentially using ETA (for seven years), ADA, and ABA (for three months each). The diagnosis of EPTB was made when using ABA, a fully humanized fusion protein, the activity of which occurs on antigen 4 associated with cytotoxic T lymphocytes. This drug is safe and is associated with a low rate of adverse effects. In this case, the fact that the patient used anti-TNFα therapy for a considerable time before starting ABA may be related to the severity of the TB infection because in patients using anti-TNFα drugs, TB cases can be more severe.The present review of literature demonstrates the low rate of TB in pediatric patients using biologic therapy, which can be related to the screening and appropriate treatment of LTBI cases before initiating use of a biologic drug.Biologics are drugs with tremendous value in the control of rheumatologic diseases in pediatric patients, either alone or in combination with other immunosuppressive medications, resulting in improved quality of life and prognosis.The articles included in the discussion demonstrate the relevance of the theme. Most of them were prospective, but had a small sample, which can be considered as a limitation. The source of the articles was PubMed, the main source of consultation in the medical field; however, other databases were not searched, which is another limitation of our study. In addition, this was an integrative review, not including statistical methods. A systematic literature review is suggested to facilitate a statistical and broader assessment of TB cases in pediatric patients treated with biologic agents.As a conclusion, the study revealed a low rate of active TB in pediatric rheumatic patients using biologic therapy, which may be associated with screening and effective therapy for LTBI before the initiation of biologic agents. Screening for LTBI before initiating biologic therapy should be conducted in all patients, particularly in endemic areas for TB, such as Brazil. The treatment administered in cases of positive screening plays a critical role in preventing progression to TB disease.


After further adjusting for sodium, saturated fatty acid, and fiber intake, the results remained significant for total plant-based protein intake . In conclusion, a higher NEAP or PRAL score may be associated with increased odds of MS, while a higher intake of plant-based protein instead of animal-based protein may be protective.This study aimed to investigate the association between dietary acid load (DAL) and multiple sclerosis (MS), through the potential renal acid load (PRAL) and net endogenous acid production (NEAP) scores. In a hospital-based case–control study of 109 patients with MS and 130 healthy individuals, a validated 168-item semi-quantitative food frequency questionnaire and a logistic regression model were used to evaluate the association between the DAL and MS. After adjusting for age (years), gender , body mass index (Kg/m Multiple sclerosis (MS) is an inflammatory autoimmune disease of the central nervous system (CNS), with an unknown cause . MS typiInflammation is a common feature throughout all the stages of MS, with the acute phases exhibiting more apparent inflammation than the chronic phases. At the onset of the disease, peripheral immune cells, such as macrophages and CD8+ T cells, invade the CNS, due to disruption of the blood–brain barrier. As the disease progresses, the relative proportion of invading B cells and plasma cells increases. Over time, the inflammation within the CNS becomes more structured, and fewer invading cells are detected in lesions as the disease progresses. The formation of tertiary lymphoid structures in the meninges has been observed in secondary progressive MS. These inflammatory aggregates may cause additional demyelination and tissue damage in the later stages of the disease .The typical lifespan of a person with MS is slightly reduced, but many people with relapsing–remitting MS (RRMS) live for decades without persistent disability. However, about half of RRMS patients will progress to secondary progressive MS within 10 years of the disease onset , which uSeveral retrospective studies have reported an inverse relationship between MS risk and healthy dietary patterns, such as the vegetarian-based  and MediThe dietary acid load can have a significant impact on the functioning of the immune system. High acid levels can lead to a decreased white blood cell function, increased inflammation, reduced antibody production, and impaired T-cell function . AdditioThe Institutional Review Board of the National Institute for Medical Research Development (NIMAD) (Research number = 962667), the research ethics committee of NIMAD, and the research ethics board of the Sina MS research center at Tehran University of Medical Sciences (Ethics code: IR.NIMAD.REC.1396.320) approved the study’s protocol. The research adhered to the Helsinki Declaration of 1964 and its subsequent revisions. Each participant provided written consent to participate in the research.2, left more than 70 items blank on the food frequency questionnaire (FFQ), or reported an energy intake lower than 800 Kcal or higher than 4200 Kcal.This hospital-based case–control study was conducted in Tehran, Iran, between January 2018 and January 2021. The sampling and methods are detailed in our previous study . BrieflyThe demographic and anthropometric data measurements are provided in detail . Data coA verified nutrition specialist conducted a personal interview with the participants to gather data on their usual diet over the preceding year. This was done using a Willett-format semi-quantitative FFQ, consisting of 168 food items. The reliability and validity of the questionnaire have been reported for the Iranian population in studies ,18,19. TThe three distinct methodologies for measuring the dietary acid load are referred to as the PRAL, NEAP, and protein/potassium ratio. All have been validated in previous studies ,22 and a2) and total calories . Furthermore, model 2 was adjusted for the intake of sodium (mg/day), saturated fatty acids (gr/day), and fiber (gr/day). Statistical significance was attributed to p-values less than 0.05.The data analysis was carried out using SPSS software (v.26). The normal distribution of the quantitative variables was assessed separately in each group, using skewness, histogram charts, Q–Q plots, stem-and-leaf plots, box plots, and the Kolmogorov–Smirnov test. The independent two-sample t-test was used to compare the quantitative data between the case and control groups. The Mann–Whitney U test was employed to examine data that were not normally distributed. The chi-squared test was used to compare the qualitative variables between the cases and controls. To explore the relationship between MS and the dietary acid load components, the variables were split into tertiles based on the control intakes. The odds ratio (OR) with a 95% CI for each tertile was calculated using logistic regression models, with the first tertile serving as the reference group. The basic binary logistic regression models contained age and gender as variables. To address potential confounding factors, binary logistic regression model 1 was employed, incorporating the BMI . Additionally, the mean ± SD of the BMI in the control group was significantly higher than in the MS patients . The total intake of calories and macro/micronutrients in the patients with MS and the healthy controls are represented in p-value = 0.645), PRAL , and NEAP  scores  and 130 healthy controls  were included. The median (Q1–Q3) age in the control group was significantly higher than that of the case group  and 33 , respectively; ) scores .The total intake of calories and micro/macronutrients according to the PRAL and NEAP tertiles is provided in p-value = 0.013), and about five times higher in the last tertile of the PRAL score  and NEAP score model , compared to the first tertile. In model 1, additionally adjusted for BMI (Kg/m2) and total calories , the MS odds were 92% lower for those in the last tertile of total plant-based protein , and about four times higher for those in the last tertile of the PRAL  and NEAP score  than in the first tertile. In model 2, additionally adjusted for sodium (mg/day), saturated fatty acids (gr/day), and fiber (gr/day) intake, the patients in the last tertile of total plant-based protein had 93% lower odds of MS . A significant direct association was observed between protein/potassium ratio and odds of MS in both the base  and the first model  (In the base model adjusted for age (years) and gender , the odds of MS were 2.28 times higher for those in the last tertile of total animal-based protein  .In the current study, after adjusting for age, gender, BMI, and total calories, a higher dietary acid load, defined by a higher NEAP or PRAL score, was correlated with increased odds of MS. Moreover, the MS odds were increased in individuals with a higher protein/potassium ratio. In addition, a higher total plant-based protein intake was associated with reduced odds of MS, which was the only variable that remained statistically significant after further adjustment for sodium, saturated fatty acids, and fiber intake. Food items rich in sodium and saturated fatty acids, compared to those rich in fiber, are shown to have a higher dietary acid load, so their adjustment in the last model may have affected the results observed in the previous models.Foods that increase a diet’s acidity levels include meat, fish, grains, and cheese, while foods that increase a diet’s alkalinity levels include fruit, vegetables, milk, and yogurt . With reHigher levels of dietary acid load are associated with an increased insulin resistance , and redAn acidic diet can lead to an imbalance in the body’s pH levels, which can lead to a decrease in white blood cell production. Moreover, certain specific foods can further decrease white blood cell function, such as processed foods and meat, which can reduce the number of circulating white blood cells, due to their high acidity . A decreAn increase in the dietary acid load can trigger an autoreactive T-cell response . UsuallyA higher dietary acid load increases the number of fat cells, and consequently leads to obesity . A systeMoreover, an investigation into the comorbidities of MS has shown that individuals with MS have a lower bone mineral density, and a greater incidence of osteoporosis than those of a similar age and gender who do not have MS. This may contribute to the disability experienced by these patients . AccordiThe present study had several strengths. We used a validated FFQ, and individuals with an energy intake lower than 800 or higher than 4200 Kcal were excluded. In order to attain a causal interpretation, and diminish recall bias, newly diagnosed MS patients were included. The participation rates were high in both individuals with MS, and healthy controls. The dietician who specialized in the field was blinded to the diagnostic results while conducting the interview using questionnaires. Nevertheless, we encountered certain limitations pertaining to measurement bias and recall bias. The cultural and religious prohibition of alcohol and opium in Iran hindered our ability to gather data on these variables. Moreover, the inflammatory biomarkers associated with the examined serum samples were not analyzed, along with the serum and urinary pH.After accounting for various factors, the study found that a higher dietary acid load, as indicated by a higher NEAP or PRAL score, was associated with an increased likelihood of MS. Additionally, individuals with a higher protein/potassium ratio had an increased likelihood of MS. On the other hand, a higher intake of plant-based protein was linked to a reduced likelihood of MS, even after further adjustments for sodium, saturated fatty acids, and fiber intake. Further studies with other designs, such as cohorts and clinical trials, are needed in order to clarify the effects observed in this study.


High sodium intake is a crucial risk factor for the development and progression of chronic kidney disease (CKD). However, the latest global spatiotemporal patterns of CKD burden attributable to high sodium intake still remain unclear. We aimed to evaluate the level and trends of the CKD burden associated with high sodium intake according to sex, age, socio-demographic index (SDI), region, and country from 1990 to 2019.Data on CKD burden attributable to high sodium intake from 1990 to 2019 were extracted from the Global Burden of Disease (GBD) Study 2019. The CKD-related deaths, disability-adjusted life years (DALYs), age-standardized mortality rate (ASMR), and age-standardized DALYs rate (ASDR) attributable to high sodium intake were estimated by age, sex, SDI, region, and country. The estimated annual percentage change (EAPC) was calculated to evaluate the secular trends of ASMR and ASDR of CKD attributable to high sodium intake from 1990 to 2019. We further explored the associations of SDI with the ASMR and ASDR of CKD attributable to high sodium intake.Globally, the number of CKD-related deaths and DALYs attributable to high sodium intake were 45,530  and 1.32 million (95% UI: 0.43 to 2.8) in 2019, both twice as many as those in 1990. However, the ASMR and ASDR slightly grew, with an EAPC of 0.22 (95% CI: 0.16 to 0.28) and 0.10 (95% CI: 0.04 to 0.16), respectively. The age-specific numbers and rates of deaths, as well as DALYs of CKD attributable to high sodium intake, rose with age and were greater in males than in females. The rates of deaths and DALYs peaked in the >95 age group for both females and males in 2019. From 1990 to 2019, the trends of both age-specific rates of mortality and DALYs of CKD attributable to high sodium intake were down in people under 60, while in people over 60, the trends were the opposite. The burden of CKD attributable to high sodium intake in 2019 and its temporal trends from 1990 to 2019 varied greatly by SDI quintile and geographic location. The ASMR or ASDR showed a non-linear negative correlation with SDI at the regional level. The EAPC in ASMR or ASDR showed a markedly negative correlation with ASMR or ASDR in 1990, with a coefficient of −0.40. Nevertheless, the EAPC in ASMR rather than ASDR was positively correlated with SDI in 2019, with a coefficient of 0.18.Our findings suggest that there are significant sexual and geographic variations in the burden of CKD attributable to high sodium intake and its temporal trends. Globally, the high sodium intake-caused CKD burden continues to elevate, posing a major challenge to public health. In response to this, strengthened and tailored approaches for CKD prevention and sodium intake management are needed, especially for elderly populations, males, and the population in the middle SDI regions. Chronic kidney disease (CKD) has been recognized as a major global public health concern, affecting nearly one in ten individuals. The Global Burden of Disease (GBD) Study 2019 estimated that there are approximately 697 million CKD cases globally, with 41.5 million disability-adjusted life years (DALYs) resulting from CKD . The lifIn recent decades, modifiable risk factors, e.g., impaired fasting plasma glucose, hypertension, obesity, and smoking, have been regarded as the major causes of CKD DALYs and deaths , 11. EffAlthough behavior changes interventions such as awareness campaigns and health education programs are implemented to reduce sodium intake, the average sodium intake in CKD patients from salt per day [approximately 9–12 g (150–200 mmol sodium)] far exceeds the recommended level , 18. Groper capita, educational attainment for those aged 15 and above, and the total fertility rate among females aged below 25  based on a serum creatinine measurement and/or an elevated urine albumin-to-creatinine ratio (ACR) . This deThe general methods of the GBD and the specific methods for estimating the burden of CKD attributable to high sodium intake have been elucidated elsewhere , 25, 27.Data on high sodium intake exposure were extracted from multiple sources, including nationally and subnationally representative nutrition surveys, household budget surveys, accounts of national sales from the Euromonitor, and availability data from the United Nations Food and Agriculture Organization Supply and Utilization Accounts. Ninety two original data sources from 53 countries of high sodium intake exposure were used in this dietary risk modeling in GBD 2019.asgtPAF was the PAF for CKD burden attributable to high sodium intake for age group a, sex s, location g, and year t. ast (x)RR was the relative risks between exposure level x (from l to u) of high sodium intake and CKD for age group a, sex s, and year t; and asgt(x)P was the proportion of the population exposed to high sodium intake at the level x for age group a, sex s, location g, and year t. TMREL for age group a, and sex s. The sixth was to account for the potential mediating effect. Last, the YLLs, YLDs, and deaths for CKD were multiplied by the high sodium intake risk factor PAF to estimate the CKD burden attributable to high sodium intake.A comparative risk assessment framework was used to calculate the fraction of disease burden of CKD attributable to high sodium intake exposure , 11. Thex denoted the calendar year. Then, EAPC could be obtained from the model p value of less than 0.05 was considered statistically significant.Data on deaths, DALYs, ASMR, and ASDR were reported as numbers with 95% uncertainty intervals (UIs) based on the 2.5th and 97.5th percentiles of the ordered 1,000 estimations . The ASRs 95% CI . If the s 95% CI , 31. All3 and 1318.81 × 103, representing 6.6% (1.4 to 15.7%) and 6.1% (1.5 to 14.0%) of all CKD-related deaths and DALYs, respectively  and ASDR  of CKD attributable to high sodium intake in 1990, respectively  and the EAPC in ASDR presented no correlation with SDI in 2019   ,D.This study provides up-to-date estimates of the global spatial and temporal trends of the high sodium intake-caused CKD burden, covering 204 countries and territories worldwide from 1990 to 2019. Our results showed that in 2019, the global number of deaths and DALYs from CKD attributable to high sodium intake nearly doubled in both sexes compared to 1990. However, after age-standardization, the deaths and DALYs showed only a slight increase in males, while the corresponding ASMR was stable and the corresponding ASDR slightly decreased in females. The trends in the absolute number of CKD deaths and DALYs attributable to high sodium intake can be partly attributed to population growth and aging. Additionally, the global CKD deaths and DALYs attributable to high sodium intake were greater in males than in females aged below 90, which was contrary to the condition in groups aged above 90. Furthermore, the spatiotemporal trends of the burden of CKD attributable to high sodium intake were not homogeneous, showing a complex association with sociodemographic factors.Sodium is an essential dietary mineral and nutrient and is significant for cell function normalization, nerve impulse transmission, acid–base balance, and plasma volume maintenance. The daily minimum sodium intake for maintaining normal physiological function is about 200–500 mg , 33. How+ homeostasis during acclimation to high sodium intake challenges, making them much less vulnerable to the adverse influence of high sodium intake exposure , the Scientific research project of Hunan Health Commission (Grant Nos. 202102041763 and 202203014949), the Changsha Municipal Natural Science Foundation (No. kq2014267), and the Hunan Engineering Research Center of intelligent prevention and control for drug induced organ injury (No. 40).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Background: Burst suppression (BS) is an electroencephalography (EEG) pattern in which there are isoelectric periods interspersed with bursts of cortical activity. Targeting BS through anaesthetic administration is used as a tool in the neuro-intensive care unit but its relationship with cerebral blood flow (CBF) and cerebral autoregulation (CA) is unclear. We performed a systematic scoping review investigating the effect of BS on CBF and CA in animals and humans.Methods: We searched MEDLINE, BIOSIS, EMBASE, SCOPUS and Cochrane library from inception to August 2022. The data that were collected included study population, methods to induce and measure BS, and the effect on CBF and CA.Results: Overall, there were 66 studies that were included in the final results, 41 of which examined animals, 24 of which examined humans, and 1 of which examined both. In almost all the studies, BS was induced using an anaesthetic. In most of the animal and human studies, BS was associated with a decrease in CBF and cerebral metabolism, even if the mean arterial pressure remained constant. The effect on CA during periods of stress  was variable.Discussion: BS is associated with a reduction in cerebral metabolic demand and CBF, which may explain its usefulness in patients with brain injury. More evidence is needed to elucidate the connection between BS and CA. One metO2) and thereby can be protective during times of cerebral stress such as during cardiac surgery, cerebral aneurysm surgery, and carotid surgery  . As cereO2 and cerebral metabolic rate for glucose (CMRglu) and there are proportional increases in CBF . To measure autoregulation, it is essential to measure changes in CBF while keeping all other factors such as partial pressure of CO2 and cerebral metabolic rate (CMR) constant.CBF is tightly controlled through a variety of mechanisms . The ters in CBF . CMRO2 cO2 responsible for the decrease in CBF through flow-metabolism coupling or the direct effect of the anaesthetic on the cerebral vasculature? Further, it is unclear whether anaesthetics effect autoregulation in addition to the CBF. We sought to conduct a systematic scoping review on the effect that BS, induced primarily by anaesthesia, has on CBF and autoregulation in animals and humans.Many anaesthetics decrease CBF, but it remains unclear if it is the anaesthetic itself or the BS that causes a reduction in CBF; that is to say, is the depression of CMRUsing the methodology outlined in the Cochrane Handbook for Systematic Reviews, we performed a systematically conducted scoping review of the available literature . ReportiThe question evaluated for this systematic review was as follows: What is the effect of BS on CBF and CA in animals and humans?BS was defined as an EEG pattern characterized by alternating periods of isoelectricity and active oscillations or “spikes” (2). Note, for the present study, “isoelectricity” was considered synonymous with BS as they both indicate a state of profound cerebral inactivity. BS could be induced by anaesthesia or by brain injury.The measures of CBF that were included in the search were the following: measuring blood flow in the superior sagittal sinus, Xenon133 clearance, MS techniques , laser Doppler flowmetry, thermal diffusion flowmetry, positron emission tomography (PET) imaging, perfusion weighted magnetic resonance imaging (PW-MRI), arterial spin labeling magnetic resonance imaging (ASL-MRI), functional magnetic resonance imaging (fMRI) and blood-oxygen-level-dependent (BOLD), perfusion computed tomography (CTP), xenon-enhanced perfusion computed tomography (Xe-CT), single-photon emission computed tomography (SPECT), near infrared spectroscopy (NIRS), and transcranial doppler (TCD). The primary outcome of interest was the quantifiable effect that the induction of BS has on these measures of CBF. Animal studies included all studies performed in non-human animals including but not limited to rats, canines, swine, and non-human primates. Human studies included adults as well as pediatric populations.Both continuous and intermittent methods of measures of CA were included in the search, a complete list of which can be found in Inclusion criteria for this study were as follows: BS measured by EEG in some form ; animal subjects, human participants, original full-length papers, use of some form of CBF measurement including the above, and/or a measurement of CA. Exclusion criteria included: non-English language and non-original studies.MEDLINE, BIOSIS, Cochrane library, EMBASE, Global Health, and SCOPUS were all searched from their inceptions to August 2022 using relevant search terms. An example of the used search strategy can be found in A two-step review of all articles returned by our search was conducted using two reviewers (A.Z.S. and L.F.). In the first filtering phase, reviewers independently screened all titles and abstracts for possible inclusion. Next, full texts of articles that passed the first filtering phase were assessed to confirm that they met all inclusion criteria. Any discrepancies between the two reviewers were resolved by the senior author (F.A.Z.). Finally, the reference lists for each included article were screened for any missed articles.The following data was extracted from each of the final included articles and is presented in Due to the heterogenous nature of the study designs and results of the relevant literature, no formal meta-analysis was conducted.Given the overall goal of this review, to perform a comprehensive systematic review of the literature, a formal bias assessment was not conducted.The overall search and filtration results have been summarized in animal and human sections. The animal studies are described in Overall, there were 66 studies that were included in the final results, 41 of which examined animals , 24 of wOf the 42 studies that examined animals, six used propofol , 13 usedIn terms of the subjects that were used in the studies, 12 used different species of dogs  , 18 usedr = 0.86; p < 0.05), and that cortical cerebral vascular resistance (CVR) and PI were significantly correlated .Out of the six studies that used propofol to induce BS, two measured CBF by using the Laser Doppler Flowmetry Method , one by Overall, there was only one paper that was found that examined an established measure of CA . Werner O2 in addition to CBF  and Werner et al., found a 70% decrease in CMRO2 to match the 70% decrease in CBF(24). In summary, most of the evidence suggests that BS induced by propofol reduces CBF, but it remains unclear if the decrease in CBF induced by BS is associated with a concurrent decrease in CMRO2. Overall, there is a lack of evidence regarding the effect of BS induced by propofol on CA as assessed by well-established methods of CA measurement. However, given that CBF is disproportionately affected compared with MAP, there is some evidence to suggest that CA impairs autoregulation and the Lassen Curve  . The amount of reduction in CBF ranged from 23%  to 80%  in the s < 0.05) . However < 0.05) .O2  and a strong negative correlation between CVR and CMRO2   . One not < 0.05) .There were two studies that did not demonstrate a reduction in CBF with induction of BS . One useO2 implying that it may be due to reduced brain metabolism. Overall, there is a lack of evidence regarding the effect of BS induced by propofol on CA as assessed by well-established methods of CA measurement. However, again given the connection between CBF and CA, there may be some impairment of autoregulation.In summary, like the propofol studies, almost all of the studies demonstrated that BS causes a significant reduction in CBF. The reduction in CBF is paralleled by a decrease in CMROut of the 18 studies that used inhalational anaesthetics to induce BS, four measured blood flow in the superior sagittal sinus , two useO2 , and oxygenated hemoglobin (oxyHgb) significantly increased , and the amount of deoxygenated hemoglobin (deoxyHgb) significantly decreased   .In summary, there still exists heterogeneity in the literature regarding the effects that BS induced by inhalational anaesthetics has on CBF and autoregulation. However, there is evidence to suggest that there is tight neurovascular coupling in the brain and spikes on EEG during the BS state are associated with concurrent spikes in CBF. Like the other anaesthetics, there is a paucity of literature on the effect of BS on CA measures.glu with pentobarbital or isoflurane. Another study used a barbiturate and propofol (p < 0.001). This may suggest greater decrease in cerebral metabolism with propofol compared with isoflurane.Four studies used multiple methods to induce BS. Two used a barbiturate and inhalational anaesthetic  but in opropofol  to inducpropofol  in an isvice versa, limiting the conclusions that can be drawn.One study also used hemorrhagic hypotension to induce BS in dogs  but the In summary, there is mild evidence to suggest that barbiturates and propofol may have stronger effects on CBF compared with inhalational anaesthetics, but more studies are needed comparing the different anaesthetics. The effect on CA remains unclear.Of the 24 studies that examined humans, six used propofol , four usIn terms of the participants that were used in the studies, five involved adults undergoing cardiac surgery  , five inOut of the six studies that used propofol to induce BS, one measured CBF using the Laser Doppler Flowmetry Method , one by p < 0.0001) and it was achieved in adult patients undergoing orthopedic procedures (p < 0.05), seen in adult patients undergoing cardiac surgery  (p < 0.05), hypothermia , and during rewarming ; CMRO2 followed the same pattern (p < 0.001) suggesting that patients with risk factors may already have altered CA. Overall, there is a lack of evidence regarding the effect of BS induced by propofol on CA as assessed by well-established methods of CA measurement.Out of the six studies that used propofol to induce BS, five directly looked at CBF and all of them found a significant decrease in CBF . The higocedures  and the  groups) . In one  pattern . In all  pattern . The “hiin lieu of arterial CBF and there was no significant change found   .Out of the 4 studies that used barbiturates, one uses althesin , one usep < 0.05) which was also accompanied by a decrease in CMRO2 and in Young et al., there was a 48% significant decrease in CBF (p < 0.01). In the former, there was a 10% decrease in MAP and in the latter, phenylephrine was used to maintain MAP. In the other two studies, CBF was either not measured at baseline without BS, limiting the conclusions that can be drawn  but no change in CVR with isoflurane, and 32% reduction in CBF velocity and 41% increase in CVR (p < 0.05) without a significant reduction in CPP with sevoflurane. There was only a 10% reduction in MAP in the isoflurane group and no reduction in MAP in the sevoflurane group. p < 0.05) but more interestingly, that this was also accompanied by a 13% decrease in cerebral oxygen extraction (COE) (p < 0.05). Further, under BS, there was a steeper positive slope of relationship between CBF velocity and CPP and a steeper negative slope between CPP and COE (p < 0.05), which may suggest the effect of sevoflurane was through vasodilation.Out of the six studies that used inhalational anaesthetics, three used sevoflurane , one usep < 0.05) (p < 0.001)   or a low< 0.001) .In summary, inhalational anaesthetics may reduce CBF but more evidence is needed to determine what effect that these anaesthetics have on CVR and cerebral metabolism. Further, much like the studies with the other anaesthetics, there is a lack of evidence regarding the evidence regarding BS induced by inhalational anaesthetics and CA.O2 (p < 0.05)  .O2 was 21% lower in the thiopental group and 34% lower in the isoflurane group compared to control (p < 0.05). This difference was 34% and 29% respectively for thiopental and isoflurane in the hypothermic condition (p < 0.05). Taken together, this may mean that inhalational anaesthetics, while having a significant effect on cerebral metabolic demand, do not have as significant effect on CBF and autoregulation but more comparative studies are needed.Finally, one study used FDG-PET with a smaller and more heterogenous sample size did not look at CBF directly . HoweverOverall, more research is needed regarding the differential effects of anaesthetics on CBF as well as how different anesthetics effect CA.p = 0.21 R2 = 0.569, total hemoglobin p = 0.034 R2 = 0.403); when BS was induce there was an increase in oxygenated hemoglobin and total hemoglobin following onset of bursts in representative hemodynamic response functions , which are not as applicable to humans and is a weakness of the literature. The largest sample sized used in the small-animal models was 61  and invoγ-aminobutyric acid (GABA) agonist with a rapid terminal half life that readily crosses the blood brain barrier  (O2 (75% vs. 54%), the anaesthetic used to induce BS was etomidate and CBF plateaued before BS was even reached  . In anot reached . As ment reached . Etomiday by 40% . Therefobarbital . This wabarbital . There wbarbital  and one barbital . The lacbarbital .p < 0.05), the visual cortex did not see any significant change, and the extrapyramidal system (140%–170%) and limbic system (87%–120%) saw a significant increase, and finally, with 2.0 MAC, all regions saw a significant increase in CBF . Compared to inhalational anaesthetics, barbiturates seem to have a higher effect on reducing CBF, which was seen in an animal  as well O2  and determine the effect size that BS has on CBF or even on clinical outcomes.in vivo methods of monitoring CBF such as NIRS in neurologically ill patients to determine the effect of BS in this population. The NIRS technique uses the infrared light, which can penetrate into deep tissues, and be absorbed by oxygenated and deoxygenated hemoglobin (in-vivo methods of neuromonitoring such as NIRS.Future work should use moglobin . The tecmoglobin . Cerebramoglobin . When COmoglobin . In patiIn this systematic review of the effect of BS on CBF and autoregulation in animals and humans, we identified 67 studies. Most of the literature involved animal models, which largely involved small animals. In the human studies, most were conducted on patients undergoing surgery or who were critically ill, as expected. There was significant evidence to suggest that propofol and barbiturates decrease CBF in both animals and humans. However, the evidence regarding inhalational anaesthetics, comparative effects, and clinical implications is lacking. More large animal models as well as clinically relevant human studies are needed to further elucidate the mechanisms behind BS and CBF and what implications this has on the future of care in the neurological intensive care unit.


In glucose metabolism, the pentose phosphate pathway (PPP) is the major metabolic pathway that plays a crucial role in cancer growth and metastasis. Although it has been pointed out that blockade of the PPP is a promising approach against cancer, in the clinical setting, effective anti-PPP agents are still not available. Dysfunction of the G6PD enzyme in this pathway leads to cancer development as this enzyme possesses oncogenic activity. In the present study, an attempt was made to identify bioactive compounds that can be developed as potential G6PD inhibitors. In the present study, 11 natural compounds and a controlled drug were taken. The physicochemical and toxicity properties of the compounds were determined via ADMET and ProTox-II analysis. In the present study, the findings of docking studies revealed that staurosporine was the most effective compound with the highest binding energy of −9.2 kcal/mol when docked against G6PD. Homology modeling revealed that 97.56% of the residues were occupied in the Ramachandran-favored region. The modeled protein gave a quality Z-score of −10.13 by ProSA tool. iMODS server provided significant insights into the mobility, stability and flexibility of the G6PD protein that described the collective functional protein motion. In the present study, the physical and functional interactions between proteins were determined by STRING. CASTp server determined the topological and geometric properties of the G6PD protein. The findings of the present study revealed that staurosporine could be developed as a potential G6PD inhibitor; however, further in vivo and in vitro studies are needed for further validation of these results. In pentose phosphate pathway (PPP), a key enzyme is the glucose-6-phosphate dehydrogenase (G6PD) that converts glucose-6-phosphate into 6-phosphogluconate in addition to nicotine adenosine dinucleotide phosphate (NADPH) and nucleotide precursors . In all Cancer is the most common disease and leading cause of death in the world every year. This disease is multifactorial and shares many features with other cancers, and one of the common hallmarks is reprogrammed energy metabolism . In the The efficacy of cancer drugs is affected by G6PD, therefore leading to drug resistance. Computational drug studies have been continuously increased with high success rates as the in silico analysis has become well correlated and more realistic with the actual in vitro studies. In the near future, computational tools will be a fundamental part of the drug discovery studies. Hence, the present study was designed with the aim of finding new bioactive G6PD inhibitors after knowing the dearth of approved clinical medications. Continued efforts to develop therapies and treatments against cancer other than chemotherapy and radiotherapy are being made. However, these treatments further deteriorate health of patients and make them suffer from a lot of stress . SubsequThe recent development in computational field has developed a rationale for identifying potential bioactives that can target G6PD protein. Computational approaches could be potentially used to repurpose natural bioactives for drug designing. Here, in this study, we have used Staurosporine, Withanoside II, Zingerone, Zerumbone, Polydatin, Naringenin, Isosilychristin, Eugenol, Gingerenone and Syringic acid molecules, which could act as potential G6PD inhibitors by using computational methods. In this study, ADMET  analysis of plant bioactives and the control drug was performed. Further, the present study also aimed to determine docking analysis of the G6PD protein with natural compounds, toxicity analysis in addition to homology modeling, molecular dynamics, protein-protein interactions (PPIs) and topological features of human G6PD protein.https://pubchem.ncbi.nlm.nih.gov/compound; last accessed on 5 December 2022), was used for 2D ligand structure retrieval in a simple data format (SDF).In the present study, ligands such as Staurosporine, Withanoside II, Zingerone, Zerumbone, Polydatin, Naringenin, Isosilychristin, Eugenol, Gingerenone and Syringic acid were selected along with the control drug (Lapatinib). An online server, PubChem .The ligands’ smiles retrieved from PubChem server were used to convert ligands from SDF into PDB format by using an online web server , was used to determine ADMET properties of natural compounds and drug molecules [logP should be <5. To determine ADMET  of molecules is very important for being used as drug. SWISSADME, an online webserver  was used to evaluate compound toxicity.ProTox-II an online web tool  with the PDB ID of 6JYU. Auto Dock tools  performed the docking analysis of G6PD protein in this study. The docking method was performed as per the protocol mentioned by [The three-dimensional structure of G6PD was retrieved from the protein data bank  was determined from binding energy (ΔG) by using the following formula:https://swissmodel.expasy.org/; accessed on 5 December 2022), was used [In the structure building of protein, SWISS-MODEL (version 8.9), an easily available online tool  [The overall quality and validity of the modeled protein structure was determined by ProSAweb tool (er 2022) .https://saves.mbi.ucla.edu/; accessed on 5 December 2022) [The accuracy of the G6PD protein model was assessed by PROCHECK tool, and the findings were presented in Ramachandran plot (er 2022) ,32.https://imods.chaconlab.org/; accessed on 5 December 2022). This tool computes vibrational modes from various chains of nucleic acids and proteins by using NMA coordinates.An easily available online web server that performs normal mode analysis (NMA) on both proteins and nucleic acids . This tool provides scores wherein predicted confidence scores in edges of each network possess values between 0 and 1, respectively. In the present study, this server was used to assess network summary, gene neighborhood, gene co-occurrence and gene fusion.The protein–protein interactions (PPIs) were predicted by an easily available biological database (STRING)  measured the geometric and topological features of human G6PD protein. The surface pockets, cross channels and interior cavities in the protein structure are located, delineated and measured by the CASTp server.An online web tool  and Withanoside V (−8.9), respectively, when docked against human G6PD. Least binding affinity was shown by Zingerone . The results obtained from docking experiments are illustrated in The 2D interaction of G6PD protein with four best docked compounds and a standard drug (Lapatinib) is graphically shown in In the present study, In the present study, the top four compounds that showed best results for docking were further analyzed for ADMET studies. In the present study, physiochemical and drug-like properties of phytocompounds were evaluated based on Lipinski’s rule of five parameters. The phytocompounds that yielded good LOGP/lead likeness/bioavailability, synthetic accessibility, high gastrointestinal absorption, blood–brain barrier (BBB) permeability were selected. In the present study, withanoside II and withanoside IV did not follow Lipinski’s rule and showed three violations, i.e., molecular weight greater than 500 g/mol, number of hydrogen bond acceptors (>10) and donors (>5). In this study, Polydatin and Staurosporine followed Lipinski’s rule and withanoside II and withanoside IV did not. The ADMET results are shown in 50), prediction of toxicity class, hepatotoxicity, cytotoxicity, immunotoxicity, mutagenicity and carcinogenicity. Levels of toxicity were categorized as classes 1 and 2 , class 3 , class 4 , class 5  and class 6 (non-toxic). The toxicity of the target compounds predicted is mainly based on 15 different targets from the Novartis in vitro safety panels of the target proteins associated to adverse drug reactions. The calculated toxicity targets information and the results are displayed as toxicity radar plots compares the average confidence score of the active compounds in given model training group to that of the taken input compound. The prediction of hepatotoxicity is estimated via data collected from drug induced liver injury and national institute of health liver toxicity database. Similarly data for prediction of carcinogenicity is obtained from carcinogenic potency database (CPDB). Moreover the data is collected from Ames test for prediction of mutagenicity and Chemical European Biology Laboratory (ChEMBL) for cytotoxicity. Further for immunotoxicity prediction, information is retrieved from U.S National Cancer Institute’s (NCI) database. Based on respective LD50 (mg/kg), active toxicity was shown by compounds for several toxicities, i.e., Polydatin (immunotoxicity), Withanoside II, Withanoside IV and Staurosporine . Moreover, the standard drug Lapatinib showed active toxicity for hepatotoxicity, cytotoxicity and immunotoxicity. The organ toxicity of Polydatin is predicted with a confidence score of 0.85 and is predicted to be active for hepatotoxicity. Similarly, the organ toxicity for Withanoside II, Withanoside IV and Staurosporine was predicted to have a confidence score of 0.55, 0.50 and 0.79 and predicted to be active for cytotoxicity and confidence score of 0.99 and 0.92 and predicted to be active for immunotoxicity. Moreover, for the drug Lapatinib, the predicted confidence score of 0.85, and 0.74 was found and hence reported to have active toxicity for hepatotoxicity, cytotoxicity and immunotoxicity. The results of toxicity obtained from ProTox-II are shown in Different factors were considered for studying toxicity, such as lethal dose 50 , additionally allowed (yellow), generously allowed  and disallowed (white) . The overall quality and structure validity of p53 protein were determined by a ProSA tool, which showed overall model quality Z-score of −10.13. The Z-score predicts the complete energy deviation of a protein structure as compared to the distributed energy attained randomly.The Z-score measures the protein structures total energy deviation in comparison to energy distribution acquired from random conformations. The findings obtained from ProSA server are illustrated in In this study, iMODS server was used to study deformity, B-factor, eigenvalues, correlation map and variance, and the results obtained are illustrated in −5 (The peaks in the graph represent the areas with deformability (main-chain deformity) in the protein A. In the−5 C. Higher−5 E.In this study, STRING database was used to predict associations between proteins, including functional as well as physical interactions. In the present study, Log10 (observed/expected) describes the magnitude of the enrichment effect. It is the ratio of the number of proteins in the network that are annotated with a term to the number of proteins in a random network of the same size that is expected to be annotated with this term. The false discovery rate describes the significance of the enrichment and Benjamini-Hochberg procedure is used to correct p-values for multiple testing within each category.The findings of functional enrichments of human G6PD protein as generated by string server are illustrated in In the present study, the exact area, volume and well as mouth size were determined. Clinically, in the world, the most prevalent X-linked enzymopathy is the G6PD deficiency. G6PD is a typical housekeeping enzyme, and its severe deficiency in animal models has been found intolerant for growth and development ,34,35,36In the development of new generation inhibitors, molecular docking plays an important role. Prominent insights about protein structure and dynamics are provided by computational approaches in addition to identifying novel therapeutic agents. In the present study, molecular docking analysis revealed that Staurosporine was the most effective compound that showed highest binding affinity of −9.2 kcal/mol against human G6PD protein. This can be explained by the fact that Staurosporine is a competitive inhibitor of protein kinases that binds in the binding pockets of target kinases by competing with ATP molecule . SimilarIn the present study, natural bioactives such as Polydatin, Withanoside II, Withanoside IV and Staurosporine were selected. In the process of drug discovery, a great interest lies in the identification, synthesis and purification of compounds. The physiochemical properties of phytocompounds were determined by ADMET analysis. The evaluation of drug-like properties are particularly important as they are associated with dissolution and intestinal permeability ,61,62. BIn the drug development process, it is particularly very important to determine the compound toxicities, and, in such a scenario, ProTox-II is an important computational tool . The preIn the present study, homology modeling was used to estimate 3D structure of human G6PD from its amino acid sequence. In the drug discovery process, homology modeling has been applied successfully as drugs interact with the receptor (protein) as this approach has made a significant contribution in lowering the gap between the known sequences of protein and the experimental structures. The Ramachandran plot is important as it predicts the correctness and quality of the protein structures. The observations of the present study depict the protein model to be stable and stereochemically possible. In homology and comparative studies, modeling methods have proven to be a potential acquaintance to the structural studies when the experimental results are unavailable . In SWISIn the present study, the iMODS tool determined the molecular dynamics of the protein molecule using NMA procedure. The deformable areas in the protein model were not vital, and hence the structure remained quite stable. B-factors indicate the flexible areas/binding sites and are related to atom packing in the protein molecule ,72,73. TIn the present study, CASTp revealed the topological and geometric properties that are particularly important for protein structures to carry out their functions, such as DNA interaction, ligand binding and enzymatic activity. The determination of topological properties is significant in understanding the structure–function association of proteins, developing therapeutics against targets and manufacturing proteins with desired characteristics ,76,77.G6PD is the most prominent enzyme-linked deficiency whose activity is elevated in different types of cancer. Due to adverse effects caused by medications, natural bioactives have shown immense potential because of their diverse composition. Molecular docking was used to determine the ability of plant bioactives to reveal their inhibitory activity against human G6PD. Our study revealed that Staurosporine was the most effective molecule against G6PD. The physiochemical properties of the natural compounds were determined by ADMET studies. The toxicity of the compounds was estimated by ProTox-II, with many compounds showing active activity for various classes of toxicities. Molecular modeling revealed the quality of protein model, with 97.56% of the residues occupying the Ramachandran-favored region. MolProbity score predicted the quality and correctness of the modeled G6PD protein structure. In cellular systems, protein–protein interactions play a significant role, and the string server determined the physical and functional associations. The molecular dynamic approach was used to reveal the behavior of G6PD protein. CASTp determined the topological properties in the G6PD protein structure as significant for the protein molecule to perform its function. The present study demonstrated that Staurosporine could be an effective bioactive molecule to tackle G6PD-related diseases. However, in vivo and in vitro studies are further required to validate the role of this compound to diseased states. Thus, based on the observations, we can conclude that Staurosporine can prove a potent and efficacious compound that interacts with G6PD protein and can be further repurposed as a drug candidate.


Shigella species and demographic and geographic factors, we used multivariate negative binomial regression to analyse national case notifications of shigellosis from 2001 to 2019.Shigellosis is an increasing cause of gastroenteritis in Australia, with prolonged outbreaks reported in remote Aboriginal and Torres Strait Islander (hereafter “First Nations”) communities and among men who have sex with men (MSM) in major cities. To determine associations between S. sonnei, 29%  were S. flexneri, 1%  were S. boydii, less than 1%  were S. dysenteriae, and species information was unknown for 28%  of cases. Males accounted for 54%  of cases, and the highest proportion of cases were in children aged 0–4 years . Crude annual notification rates ranged from 2.2 cases per 100,000 in 2003 and 2011 to 12.4 cases per 100,000 in 2019. Nationally, notification rates increased from 2001 to 2019 with yearly notification rate ratios of 1.04 (95% CI 1.02–1.07) for S. boydii and 1.05 (95% CI 1.04–1.06) for S. sonnei. Children aged 0–4 years had the highest burden of infection for S. flexneri, S. sonnei and S. boydii; and males had a higher notification rate for S. sonnei . First Nations Australians were disproportionately affected by shigellosis, with the notification rate in this population peaking in 2018 at 92.1 cases per 100,000 population. Over the study period, we also observed a shift in the testing method used to diagnose shigellosis, with culture independent diagnostic testing (CIDT) increasing from 2014; this also coincided with an increase in notifications of untyped Shigella. This change in testing methodology may have contributed to the observed increase in shigellosis notifications since 2014, with CIDT being more sensitive than culture dependent testing methods.Between 2001 and 2019, Australian states and territories reported 18,363 shigellosis cases to the National Notifiable Diseases Surveillance System (NNDSS), of which age, sex and organism information were available for >99%  of cases. Of the cases included in our analysis, 42%  were The findings of this study provide important insights into the epidemiological characteristics of shigellosis in Australia, including identification of high-risk groups. This can be used to inform public health prevention and control strategies, such as targeted communication programs in First Nations communities and places with high levels of interaction between young children, such as childcare centres. Our study findings also highlight the implications of culture independent testing on shigellosis surveillance, particularly a reduction in the availability of species level information. This emphasises the continued importance of culture dependant testing for national surveillance of shigellosis. Shigella spp. causes gastroenteritis and is responsible for significant morbidity and mortality worldwide. In Australia, largescale shigellosis outbreaks have been reported in First Nations communities and among men who have sex with men in recent years. We undertook an analysis of the trends in shigellosis cases in Australia over a 19-year period to understand the epidemiology of the disease, including which population groups were primarily impacted, and when and where cases were occurring. We also analysed trends in the diagnostic methods used to detect shigellosis over time, to determine whether changing detection methods had any impact on the surveillance of shigellosis cases at the national level. We found that notification rates of two species of Shigella, i.e. S. boydii and S. sonnei, significantly increased over the study period; the highest notification rate of shigellosis was in young children; and that First Nations Australians were disproportionately impacted by shigellosis. We also observed a shift in the testing methods used to diagnose shigellosis, with culture independent methods increasing from 2014. Our findings will assist in targeting prevention and control interventions to the most impacted groups and highlight the continued importance of culture dependant testing for national surveillance of shigellosis. Shigella spp. bacteria, which is primarily transmitted through the faecal-oral route by direct or indirect contact with faecal matter  and the ACT Health Human Research Ethics Committee’s Low Risk Sub-Committee [2018/ETH/00158]. No participants were recruited for this study and all data used was previously collected during public health investigations. Given that de-identified data was collected, representing infections occurring many years ago, it was not be feasible to seek individual consent, nor to inform participants about the use of data in this study.We analysed shigellosis case notifications made to the National Notifiable Diseases Surveillance System (NNDSS) from 2001 to 2019 by all Australian states and territories. Australia is comprised of eight jurisdictions: New South Wales (NSW), Queensland (QLD), South Australia (SA), Tasmania (TAS), Victoria (VIC) and Western Australia (WA); and two territories: the Australian Capital Territory (ACT) and Northern Territory (NT).Shigella species isolated; or that Shigella is detected by nucleic acid testing in combination with epidemiological evidence [Shigella by nucleic acid testing only, was implemented from 1 July 2018; prior to this, only confirmed cases were required to be notified. In this study, we did not include ‘probable’ cases. However, it should be noted, prior to the implementation of the new case definition in July 2018, testing and notification practices varied by jurisdiction. The old shigellosis case definition defined laboratory definitive evidence as ‘isolation or detection of Shigella species’. Based on this, most states interpreted a PCR positive result as not a detection and waited for a positive result from reflex culture before making a notification. However, a few states sent all these case notifications to the NNDSS marked as ‘probable’, which were recorded by the NNDSS system as a ‘confirmed’ case. This primarily occurred from late 2017 until the implementation of the new case definition in mid- 2018. As not all these ‘probable’ cases can be identified in the NNDSS dataset, they were not excluded from this study. In addition to differences in the application of the case definition, the testing methodologies used to detect Shigella also varied by jurisdiction and were implemented inconsistently between states and territories over time.Shigellosis is a nationally notifiable disease in Australia, and states and territories report all confirmed and probable cases to the Australian Government Department of Health through the NNDSS. The national case definition requires that a confirmed case of shigellosis has evidence . The addNew South Wales–Australia’s most populous state–was the last Australian jurisdiction to make shigellosis reportable, which occurred in 2001. Therefore, we included de-identified data on all confirmed shigellosis case notifications reported to the NNDSS from 2001 to 2019 with the following variables: state; sex; 5 year age-group; month and year of diagnosis; country of acquisition; organism; biotype/subtype; Indigenous status and laboratory diagnosis method. NNDSS data were provided by the Office of Health Protection, Department of Health, on behalf of the Communicable Diseases Network Australia. The month and year of diagnosis was calculated from the diagnosis date: a derived field representing the onset date of the illness or the earliest of the specimen collection date, the date of report, or the date of receipt at the state or territory health authority. The data fields in the laboratory diagnosis method variable were consolidated into the following categories depending on whether culture and nucleic acid testing were reported: PCR; culture; PCR and culture; unknown (including blanks); and other .The primary aim of the analysis was to examine the trend in notification rates of shigellosis over time. Cases where sex, age-group and organism were unknown were excluded from the analysis. We calculated rates of illness per 100,000 population using mid-year residential population estimates from the Australian Bureau of Statistics (ABS) for the years 2001 to 2019.S. dysenteriae in ACT, NT and TAS, these states were removed from the regression model for S. dysenteriae. StataCorp Stata 15 and Microsoft Excel 2016 were used for analysis.We used a multivariate negative binomial regression model to estimate notification rate ratios (NRR) by sex, age and state and territory, with significance defined as a p-value of less than 0.05. An interaction term was included in the model to analyse the trend over time for each state and territory, with diagnosis date included as a continuous variable. As NSW is the most populous Australian jurisdiction, we applied it as the reference group for the jurisdictional analysis in the model. Due to low (<5) or no cases of S. sonnei, 29%  were S. flexneri, 1%  were S. boydii, and less than 1%  were S. dysenteriae  of cases. Of the cases included in the analysis, 42%  were enteriae . SpeciesS. flexneri did not fit the data well, which is likely due to outbreaks occurring in the latter years of surveillance in specific states. The results of the regression model for S. flexneri are available in the supplementary material (S. boydii and S. sonnei with yearly notification rate ratios of 1.04 (95% CI 1.02–1.07) and 1.05 (95% CI 1.04–1.06), respectively .Nationally, crude annual notification rates ranged from 2.2 cases per 100,000 in 2003 and 2011 to 12.4 cases per 100,000 in 2019 . Rates rmaterial . Nationaectively . The NRRS. sonnei ; while there was no significant difference between the sexes for the other species  of cases. Over the 19-year period, the overall crude notification rate of shigellosis was higher in males than females in the eastern states ; while the western states and territories experienced higher rates in females . In the  species . For S. th sexes .S. boydii, S. flexneri and S. sonnei were highest in children aged 0–4 years . Comparing the two most prevalent species, in those aged 25–59 years, the notification rate of S. sonnei was significantly higher than S. flexneri, while there was no significant difference in the notification rate of these two species in the other age groups . By species, notification rates of –4 years . The hige groups .S. sonnei were significantly higher in the states and territories in the west of Australia, that is NT , WA  and SA , compared to the south eastern continental states and territories. The notification rate of this species in TAS was significantly lower than the other jurisdictions . For S. dysenteriae and S. boydii, there was considerable uncertainty in the results due to low case numbers, making it difficult to conclude there was a difference in the notification rate between states and territories  . After tritories . The NT,S. sonnei significantly increased in NSW, QLD and VIC , although results were imprecise. There was no significant change in the notification rates of S. dysenteriae for any state or territory over the 19 year period  of cases included in the analysis. There were higher rates of shigellosis among First Nations Australians when compared to the non-Indigenous population . BetweenShigella, with >63-fold increase in cases of untyped Shigella from 2013 (n = 30) to 2019 . At a jurisdictional level, all states and territories experienced a dramatic increase in untyped Shigella in the past few years . Between 2001 and 2013, most nationally notified cases of shigellosis were reported to be diagnosed using culture only, peaking at 95% (786/829) of cases in 2008. From 2014 to 2019 there was a decrease in the proportion of cases diagnosed via culture methods, ranging from 54%  in 2014 to 27%  in 2019, due to increasing use of CIDT methods. This led to increased reporting of untyped ew years .Country of acquisition data was available for 53%  of cases. Due to the poor completeness of this field, no further analysis was undertaken using this variable.S. dysenteriae. The most common species in Australia was S. sonnei , followed by S. flexneri (29% of cases), consistent with the reported global burden of shigellosis [S. flexneri is traditionally linked to low and middle income countries and S. sonnei to high income countries, in recent years the expansion of S. sonnei has been documented in many economically transitional regions in Asia, Latin America and the Middle East [S. boydii is limited, with this species being predominantly endemic in the Indian Subcontinent [S. dysenteriae in Australia were very low compared to S. sonnei and S. flexneri, with S. dysenteriae rarely being isolated in current surveillance globally [S. dysenteriae serotype 1, which is the only Shigella species and serotype that expresses the Shiga toxin gene, has high epidemic potential and has been identified as a cause of several large scale outbreaks of dysentery, particularly in settings associated with civil unrest [S. dysenteriae is distinct from the other species of Shigella, with epidemics tending to disappear and then reappear years later, with few sporadic cases reported in between outbreaks [S. dysenteriae serotype 1 were prevalent in the second half of the twentieth century in Asia, Africa and Central America, there has been a drastic decline in the incidence of S. dysenteriae in the last few decades, which remains largely unexplained [Notification rates of shigellosis have increased considerably in Australia over the past 19 years (2001–2019), particularly since the introduction of CIDT. The reported rate of all species increased over the time period, except for gellosis . While Sdle East . Globallglobally . S. dysel unrest –34. Thisl unrest ,36. The utbreaks ,38. Whilxplained ,34,39.Shigella species in Australia varied by jurisdiction, with western jurisdictions, particularly the Northern Territory (NT) having higher rates across all years. The NT is unique demographically, with this jurisdiction having the highest proportion of its residents living in remote and very remote areas (approximately 40%), compared to the other states and territories [S. flexneri 2b that occurred among First Nations communities in central Australia, predominantly in the NT [Shigella affecting First Nations Australians [The relative importance of ritories ,42. In aritories ,44. In An the NT . Antimictralians . An estitralians . Overcrotralians . A multiShigella results in person-to-person transmission, which often occurs between children in childcare settings through close contact [Consistent with global trends, the burden of shigellosis in Australia was highest in children aged less than five years ,48. Chil contact ,51. Tran contact ,51. HighS. sonnei in males than females, particularly in those aged 30 to 64 years, which is the species responsible for several of the recent outbreaks among MSM in Australian communities. The eastern jurisdictions of Australia have a higher estimated proportion of adult gay men in the population compared to the western states and territories, with gay men tending to form more highly populated geospatial communities in urban areas compared to rural and regional areas [S. sonnei, resistant to all recommended oral antibiotics, which has been reported among MSM in NSW and VIC [The eastern states of Australia had higher notification rates of shigellosis in males than females. This is likely to reflect ongoing transmission among MSM in these states, with outbreaks occurring over several years in three of the most populous states (Victoria (VIC), New South Wales (NSW) and Queensland) ,52–54. Ial areas . It is v and VIC ,57.Shigella, coinciding with increased use of culture independent testing methods for shigellosis diagnosis. Several studies have demonstrated the increased capability of CIDT in identifying the aetiology of diarrhoeal patients when compared to culture dependent methods, with estimates ranging from two to four times when using CIDT [Shigella and EIEC, which are closely related and genetically constitute the same species. Whole genome sequencing (WGS) allows public health agencies to obtain detailed epidemiological data for surveillance and investigation. WGS-based characterisation of Shigella species and serotypes provides higher-resolution and more accurate data than conventional biochemical and serological testing methods. The process to obtain this information is faster as WGS does not require the application of multiple subtyping methodologies [Shigella and EIEC, which have many biochemical properties and virulence genes in common [The availability of species level information on shigellosis cases was strongly influenced by changes in diagnostic testing methodologies over time. Since 2014, there was an increase in cases of untyped ing CIDT –60. Thising CIDT . In addidologies . Addition common ,63. It iShigella spp. is not available. However, in Australia, only a limited number of nucleic acid tests for faecal pathogens are approved for use in diagnostic laboratories [Shigella spp.) to be used in Australian laboratories, the tests generally require accreditation from a national accreditation authority for each laboratory undertaking testing. This ensures compliance with international best practice and means that only high-quality tests are used in Australian laboratories.Differences between states and territories in the reporting of data presents some limitations for this study, particularly in making accurate comparisons between jurisdictions. For instance, the laboratory diagnostic method field in the NNDSS was applied inconsistently between jurisdictions, with some of these applications potentially resulting in an underestimation of cases diagnosed using CIDT and others resulting in an overestimate of cases. As the data used in this study is collected for surveillance purposes, detailed information on the protocols used by each laboratory for the detection of ratories . Furtherisolation or detection of Shigella species’. The broad nature of this wording led a few jurisdictions to transmit cases that were detected using nucleic acid testing only to NNDSS, which, prior to the inclusion of a ‘probable’ category in the case definition in 2018, was recorded by the system as a ‘confirmed’ case. This primarily occurred from late 2017 until the implementation of the new case definition in mid-2018. Therefore, the observed increase in shigellosis cases between 2017 and mid-2018 may be in part attributable to the inadvertent classification of cases that were diagnosed using nucleic acid testing only as a ‘confirmed’ case of shigellosis. The differing testing practices between jurisdictions is evident when looking at the prevalence of untyped Shigella. For instance, VIC, NT and Tasmania all had a pronounced increase in the proportion of untyped Shigella from 2014 onwards, which is attributable to the uptake of PCR testing in these jurisdictions. The differential use of CIDT in jurisdictions, yielding a higher proportion of unknown Shigella species, will also impact the observed notification ratio of known species, particularly the two most prevalent species, S. sonnei and S. flexneri. For instance, in 2014, the use of CIDT in NSW was less than that in NT, which means that the contribution of the NT to notifications of S. flexneri is reduced compared to NSW due to a higher number of notifications of untyped Shigella in the NT.Inconsistent interpretation of the surveillance case definition by some states presents another limitation of this study and may have particularly impacted the trends observed in the latter years of this study. Throughout the majority of the study period, the surveillance case definition for a ‘confirmed’ case of shigellosis was ‘Another limitation of the study was the lack of clinical information on cases. The nature of the data used in this study , meant that clinical information was not available for analysis, thereby limiting our ability to comment on clinical characteristics, such as the presence of symptoms, treatments, disease progression and severity.Returning travellers are a high risk group for shigellosis . To examS. sonnei, potentially reflecting transmission among MSM, which has been widely reported in Australia. The study observed a considerable increase in untyped Shigella in recent years which coincided with an increased use of CIDT. This highlights the implications of culture independent testing on shigellosis surveillance, with CIDT leading to a reduction in the availability of species level information. Enhancements in surveillance and laboratory practices, such as through the application of the revised surveillance case definition and use of WGS, will provide further insights into the epidemiological features of shigellosis in Australia, thereby enabling the implementation of targeted and efficient prevention and control interventions.Since 2001, notification rates of shigellosis have increased in Australia. This study has contributed to improving the understanding of the characteristics of shigellosis activity, including identifying high risk groups such as young children and First Nations Australians. Higher rates of transmission were also observed in males than females for S1 Fig(DOCX)Click here for additional data file.S2 Fig(DOCX)Click here for additional data file.S3 Fig(DOCX)Click here for additional data file.S4 Fig(DOCX)Click here for additional data file.S5 Fig(DOCX)Click here for additional data file.S6 Fig(DOCX)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.


Mesenchymal stromal cell (MSC) therapy is a promising treatment that allows for drug minimization in clinical kidney transplantation. While it is thought that MSCs rapidly go into apoptosis after infusion, clinical evidence for this is scarce since methods to detect cell death of infused cells in vivo are lacking. Cell-free DNA (cfDNA) has recently gained attention as a biomarker for cell death. In this study, we longitudinally measured cfDNA in plasma samples of the recipient, kidney donor, and allogeneic third-party MSC in the context of the Neptune study. cfDNA levels were measured at several time points before and after allogeneic MSC infusion in the 10 recipients who participated in the Neptune study. cfDNA ratios between the recipient, kidney graft, and MSC were determined.We observed a peak in MSC-derived cfDNA 4 h after the first and second infusions, after which MSC-derived cfDNA became undetectable. Generally, kidney graft-derived cfDNA remained in the baseline-level range. Our results support preclinical data that MSC are short-lived after infusion, also in a clinical in vivo setting, and are relevant for further research into the mechanism of action of MSC therapy. MEJ Reinders was affiliated with Leiden University Medical Center at the time of the trial and is currently affiliated with Erasmus University Medical Center.Mesenchymal stromal cell (MSC) therapy has gained significant interest in kidney transplantation in recent years. MSC therapy has been investigated in several clinical research settings, either as induction therapy, treatment of acute rejection, or support of maintenance therapy allowing weaning of immunosuppressive drugs –5. In thMSCs are thought to promote immunological tolerance after transplantation and to have immune-modulatory and anti-inflammatory properties , 9, 10. de novo donor-specific antibody (DSA) development  has been identified as an interesting biomarker for rejection in solid organ transplantation . The preelopment . More reelopment . This stelopment .in vivo. Since we used third-party MSC, this study is the first to separate cfDNA for three genomes: the recipient, the kidney donor, and the MSC donor. Our results confirm the short life span of MSC in humans upon infusion.In the human setting, the fate and working mechanism of MSC remain unclear. In the current study, we measured patient, kidney donor, and MSC donor-specific cfDNA levels at several time points after MSC infusion as an indirect measure of allogeneic MSC cell death ®), and everolimus. Six months after kidney transplantation (weeks 25 and 26), patients received two doses of allogeneic MSC with a target of 1–2 × 106 cells/kg of body weight. These MSCs were specifically selected to have no repeated HLA mismatches with the kidney donor for HLA-A, HLA-B, HLA-DR, and HLA-DQ, to minimize the risk of antidonor immune response against the kidney graft. For the complete characteristics and study design, we refer to previous papers regarding the Neptune study . The final time points 5 and 6 were 1 and 8 weeks after the second infusion (Single-nucleotide polymorphism (SNP) typing to identify the three genomes was performed on genomic DNA. cfDNA was measured using the AlloSeq cfDNA assay (CareDx Inc.), which is a multiplexed polymerase chain reaction (PCR)-based method to prepare libraries for sequencing from cfDNA extracted from plasma. Following cfDNA extraction, the cfDNA was amplified using PCR primer pairs covering 202 SNP loci. Amplification was performed on a SimpliAmp (Thermo Fisher), followed by a short library preparation, consisting of a double bead purification and two ethanol washes. The resulting PCR products were sequenced on an Illumina MiniSeq instrument  with the high-output flow cell in batches of a maximum of 24 samples. The sequencing data were analyzed using CareDx AlloSeq cfDNA software, where the read counts of amplified sequences were used to measure the amount of donor-derived cfDNA relative to the total amount of cfDNA in the plasma sample.R2 of 0.91362 for clinical samples, 0.945799 for analytical samples near LoD/LoQ, and 0.999138 in linearity analysis (tested up to 70%)  and showed agreement with orthogonal results with an  to 70%) , 28.To validate the ability to accurately determine the relative levels of three sources of cfDNA, we first prepared several genomic DNA mixtures from donor/recipient couple 7 and the corresponding MSC product that was infused. Different percentages of genomic DNA derived from donor, recipient, and MSC products were tested, with donor and MSC product cfDNA as low as 0.5%. As shown in Next, we analyzed the plasma samples from all patients included in the Neptune trial.In 6 MSC/kg. The highest absolute number of MSC  was infused in recipient 10 during the second infusion, at week 26. The lowest number, 98.8 × 106 in total, was infused in patient No. 1 during the first infusion at week 25  to 93.3 kg (recipient No. 7). The mean of the number of infused MSC was 1.5 × 10in vivo. We first investigated the capacity and accuracy of the applied technique to separate three different genomes by cfDNA measurement. Until now, studies have described the separation of two different genomes, for example, derived from recipients and kidney allografts  and decline 1 week after infusion suggest that the MSC died within 7 days, which is in line with preclinical studies –12. HoweAnother important result from this study is that cfDNA derived from the kidney graft was consistently low over time, in line with the results from the DART trial (median cfDNA of 0.3% of the group without ABMR) . Only at6 cells/kg of body weight), so we assume that the variation in percentage MSC-derived cfDNA is due to patient factors rather than reflecting the absolute amount of degraded MSCs. In addition, as mentioned before, there is a possibility that not all MSCs have died and a subset survives. However, 7 days after infusion, no MSC-derived cfDNA was detectable in all patients. Future research with a combination of cfDNA measurement and cell labeling would probably help to address this point. Besides the fate of MSC, more insight into the actual working mechanism is interesting. Previously, it has been shown that apoptosis, dependent on IDO activity, seems to be an important factor for the immunomodulatory effect of MSC (The study has several limitations. The amount of MSC-derived cfDNA is expressed as a percentage of total cfDNA and not as an absolute amount. This means that we were not able to investigate the relative number of MSCs that died. However, the amount of MSCs infused was comparable in all patients (1.5 × 10t of MSC –34. ThisSince this is the first study to separate three genomes with cfDNA measurement, this sets the stage for further research in transplantation, especially in studies using third-party products. Furthermore, this study shows that in the setting of clinical kidney transplantation, MSCs perish shortly after infusion, suggesting that their mechanism of action is an indirect process. It is important to further investigate this process, as it may open the possibility of developing therapies specifically directed at the downstream processes. This may ultimately result in the development of cell-free treatments with the same results but without the risks associated with cellular therapies.The datasets presented in this article are not readily available due to storage restrictions of the original data from the Neptune study, following the rules of the CCMO. Requests to access the datasets should be directed to the principal investigator of the study.The studies involving humans were approved by the local ethics committee and by the Central Committee on Research involving Human Subjects. The studies were conducted in accordance with the local legislation and institutional requirements. The participants provided their written informed consent to participate in this study.GD: analyzed the data and wrote the manuscript. JD: performed the experiments. JF, CK, MR, and SH: designed the study and co-wrote the manuscript. All authors contributed to the article and approved the submitted version.