The development of reliable predictive models for individual cancer cell lines to identify an optimal cancer drug is a crucial step to accelerate personalized medicine, but vast differences in cancer cell lines and drug characteristics make it quite challenging to develop predictive models that result in high predictive power and explain the similarity of cell lines or drugs. Our study proposes a novel network-based methodology that breaks the problem into smaller, more interpretable problems to improve the predictive power of anti-cancer drug responses in cell lines. For the drug-sensitivity study, we used the GDSC database for 915 cell lines and 200 drugs. The theory of optimal mass transport was first used to separately cluster cell lines and drugs, using gene-expression profiles and extensive cheminformatic drug features, represented in a form of data networks. To predict cell-line specific drug responses, random forest regression modeling was separately performed for each cell-line drug cluster pair. Post-modeling biological analysis was further performed to identify potential biological correlates associated with drug responses. The network-based clustering method resulted in 30 distinct cell-line drug cluster pairs. Predictive modeling on each cell-line-drug cluster outperformed alternative computational methods in predicting drug responses. We found that among the four drugs top-ranked with respect to prediction performance, three targeted the PI3K/mTOR signaling pathway. Predictive modeling on clustered subsets of cell lines and drugs improved the prediction accuracy of cell-line specific drug responses. Post-modeling analysis identified plausible biological processes associated with drug responses. Importantly, the genomic and transcriptomic profiles of all cancer cell lines employed in GDSC were extensively characterized as a part of the COSMIC cell line project . These resources have the potential to link anti-cancer drug sensitivity to detailed genomic information and facilitate the discovery of relevant molecular biomarkers when coupled with powerful analytical tools to cope with the high-dimensionality and complexity of these datasets.Recent significant advances in investigating drug sensitivity have been driven by advances in high-throughput technologies that can generate large amounts of biological data at low cost. Pioneers of such datasets include the NCI-60 database , GenomicA variety of approaches have been proposed for investigating drug sensitivity in cancer cell lines. One of the first models was developed by Staunton et al., which employed a weighted voting classification model for anti-cancer drug sensitivity based on NCI-60 gene-expression data . Recent http://www.hprd.org, accessed on 9 December 2021). [In the present study, we demonstrate that cell-line and drug clustering prior to machine learning modeling can significantly improve the accuracy of cell-line drug-sensitivity prediction. We first represented genomic profiles of cell lines and chemical features of drugs in the form of separate feature networks. Several network-based papers for drug-sensitivity prediction have been previously published. For instance, Wang et al. proposed a heterogeneous network model of cell lines, drugs, and targets ,18. Zhanr 2021). . This rer 2021). ,24,25. Tr 2021). ,26. It wIn summary, the heterogeneity of pan-cancer cell lines and structurally diverse drugs in large-scale pharmacogenomic databases makes prediction of drug sensitivity challenging. In this work, we propose a novel computation method for clustering cell lines and drugs in an unsupervised way, followed by a supervised prediction-modeling of drug responses. Our results demonstrate that modeling on homogeneous data significantly improves the prediction accuracy. Moreover, clustering increases the focus for understanding potential biomarkers and mechanisms of drug sensitivity.Hierarchical clustering of the cell lines resulted in six clusters with the highest average silhouette score. The numbers of cell lines in each cluster were 149, 113, 130, 174, 208, and 141, respectively, labeled clusters 1 through 6. 2) values were computed for the predicted and observed log(IC50) values. 2 of the predicted and observed log(IC50) values in the 30 paired clusters of cell lines and drugs.For each of the 30 paired clusters (six clusters for cell lines and five clusters for drugs), random forest regression models were trained and validated, using 635 genes and 165 cheminformatic features. A three-fold cross-validation approach was employed, such that in each cross validation, 2/3 of the data were used for training, and 1/3 of the data were used for validation of the model. After performing the three-fold cross validation in each paired cluster, correlation (R) and coefficient of determination  obtained via random forest on the whole dataset  using a three-fold cross validation scheme. Further, R and R2 in the best and worst paired clusters with respect to prediction accuracies were (R = 0.96 and R2 = 0.93) and (R = 0.79 and R2 = 0.62), respectively  values for all the 30 clusters and then calculated R and R2 . For comectively . The celectively . In addiAfter applying the modeling pipeline, we investigated the prediction accuracy for individual cell lines and drugs. t-test, differentially expressed genes were further identified. For example, we investigated a paired cluster: cell-line cluster 4 and drug cluster 1, which is one of the highest performing cluster pairs. Initially, the top 200 genes were selected based on the importance score in random forest modeling, and 70 out of the 200 genes met a Bonferroni corrected p-value < 0.05. For these 70 genes, gene ontology enrichment analysis was performed using MetaCore software to discover significant biological correlates. −20. The hypergeometric distribution was used to compute unadjusted p-values. For further insight, a protein–protein interaction (PPI) network with direct connections among the set of 70 gene products was constructed as shown in To identify significant genes, we employed a two-step approach: (1) the importance score for each gene was derived based on its contribution to the random forest accuracy  and (2) In this study, we developed a network-based method for predicting the drug sensitivity of pan-cancer cell lines in the GDSC database. Several studies have proposed network-based methods for drug response prediction on single omics data ,21, wherBCL6 gene, was initially discovered as an oncogene in B-cell lymphomas, driving a malignant phenotype via the repression of DNA damage and proliferation checkpoints [BCL6 has also been implicated in an expanding spectrum of solid and hematologic tumors, including leukemia, breast cancer, and NSCLC [BCL6 expression has been implicated in the modulation of apoptotic responses of malignant cells to chemotherapeutic reagents, suggesting the development of BCL6 inhibitors as a potential therapeutic option [In the application of machine-learning techniques to biology, interpretability is very important. Clustered cell lines and drugs, and the resulting random forest models in individual paired clusters, can be deeply interrogated to gain further insights into the determinants of cell-line drug effectiveness. We have demonstrated that post-modeling analysis using bioinformatics techniques enables the identification of plausible biological correlates. For example, we investigated the pair of cell-line cluster 4 and drug cluster 1 see A. Cell-lckpoints . BCL6 hand NSCLC . Additioc option .A limitation of this study is that using IC50 as the measure of drug sensitivity may be biased, due to different growth rates of cancer cells growing in culture . MoreoveWe used the anti-cancer drug-response data from the GDSC database. GDSC is a publicly available large-scale pharmacogenomic database that includes drug-screening data for more than a thousand human pan-cancer cell lines. The dataset consists of 265 compounds, including cytotoxic chemotherapeutics as well as targeted therapeutics. GDSC drug responses are given as log-transformed IC50 values  and the area under the curve (AUC) for a fitted model. We used log(IC50) as the degree of drug responses. Genomic mRNA expression profiles (Affymetrix Human Genome U219 Array) of the cell lines within GDSC were obtained from the CCLP database. A protein–protein interaction (PPI) network was obtained from the HPRD database. Cell lines with missing data for more than 80% of the drugs were removed, leaving 915 cell lines .http://oncokb.org/, accessed on 9 December 2021) that consists of 1019 genes. Among those 1019 genes, 796 genes were common to both CCLP and HPRD. In the HPRD network with those 796 genes, the largest connected network component consisted of 635 genes, which we focused on in this study  and downloaded the SMILES (Simplified Molecular Input Line Entry Specification) string of 241 drugs, for which the PubChem ID was provided in the GDSC database. Two-hundred drugs had response values for more than half of the cell lines, resulting in 183,000 cell-line drug pairs . The descriptors included functional groups, fragment counts, and estimated chemical properties as well as simple atomic descriptors. The overview of data analysis is shown in The CCLP and HPRD datasets had 8483 genes in common. Even though our method is applicable to large scale data, in this study, we wanted to focus on genes of known relevance in cancer. Thus, we used a smaller set of genes from the OncoKB (Precision Oncology Knowledge Base) database (gej) . By normIf we let this stochastic signal-passing process proceed from an initial state based on gene expressions, in repeated steps according to these probabilities , it can be shown that the system reaches a stationary distribution, implying that the system is invariant under a right multiplication by e., πp=π . SolvingWe initially extracted molecular descriptors of the 200 drugs from Dragon software. The following descriptors were removed: descriptors that are constant or near constant and descriptors with missing values, yielding 1500 features. We further removed many highly correlated features via unsupervised clustering, using the Spearman’s correlation between the features. We selected a representative feature from each cluster, which had the highest average correlation to all other intra-cluster features. This further reduced the number of features to 500. We then constructed a network of these cheminformatic features via the graphical LASSO, which suppressed unimportant feature connections to build a sparse network (see the W1 Wasserstein distance (EMD) metric  metric . OMT is The resultant pair-wise Wasserstein distance matrix was input to a hierarchical agglomerative clustering method, resulting in a set of clusters of cell lines or drugs. To find the optimal number of clusters, the silhouette score was used . The silmtry parameter was set to p/3, where p is the number of features [Predictive modeling of drug responses was conducted in each cell-line drug cluster, employing random forest regression on the associated gene-expression profiles and cheminformatic features. We chose the number of decision trees to be 100. For all other parameters, the default settings were used: the minimum number of samples in the terminal nodes was set to 5, and the features . Figure features , which ifeatures . We asseThis study proposed a novel network-based clustering method based on OMT theory for drug response prediction. Clustering was performed for cell lines and drugs using gene-expression profiles and cheminformatic drug features, respectively, represented in the form of data networks. Random forest modeling was then performed for each cell-line drug cluster pair. Prediction modeling on clustered homogeneous data is likely to improve the prediction accuracy for drug sensitivity, as well as enhance the biological interpretability compared to modeling using all the data together. We plan to apply the proposed approach to several biomedical problems with multi-modal data, including genomics and medical imaging.


Acute-on-chronic liver failure (ACLF) is considered a main prognostic event in patients with chronic liver disease (CLD). We analyzed the 28-day and 90-day mortality in ACLF patients with or without underlying cirrhosis enrolled in the ACLF Research Consortium (AARC) database.A total of 1,621 patients were prospectively enrolled and 637 (39.3%) of these patients had cirrhosis. Baseline characteristics, complications and mortality were compared between patients with and without cirrhosis.p < 0.0001), while non-alcoholic fatty liver disease/cryptogenic CLD  and chronic HBV reactivation  were more common in non-cirrhosis. Only 0.8% of patients underwent liver transplantation. Overall, 28-day and 90-day mortality rates were 39.3% and 49.9%, respectively. Patients with cirrhosis had a greater chance of survival compared to those without cirrhosis both at 28-day  and 90-day , respectively. In alcohol CLD, non-cirrhosis patients had a higher 28-day  and 90-day  mortality rate than cirrhosis patients. ACLF patients with cirrhosis had longer mean survival than non-cirrhosis patients (25.5 vs. 18.8 days at 28-day and 65.2 vs. 41.2 days at 90-day). Exaggerated systemic inflammation might be the reason why non-cirrhosis patients had a poorer prognosis than those with cirrhosis after ACLF had occurred.Alcohol consumption was more common in cirrhosis than non-cirrhosis (66.4% vs. 44.2%, The 28-day and 90-day mortality rates of ACLF patients without cirrhosis were significantly higher than those with cirrhosis in alcoholic CLD. The presence of cirrhosis and its stage should be evaluated at baseline to guide for management. Thai Clinical Trials Registry, TCTR20191226002.The online version contains supplementary material available at 10.1007/s12072-021-10266-8. Acute-on-chronic liver failure (ACLF) is a distinct syndrome that is described in patients with chronic liver disease (CLD) who have acute deterioration of liver function after precipitated by certain factors and results in severe hepatic dysfunction with or without extra-hepatic organ failure, which leads to high short-term mortality . The preA number of literatures have tried to identify factors that could be utilized as prognostic predictors. Unfortunately, results from various studies are inconsistent and cannot be concluded. The CLIF-SOFA score, based on the types and number of organ failure, is regarded as the best tool for diagnostic accuracy to predict ACLF mortality , 3. HoweIn the initial ACLF definitions, European Association for the Study of the Liver (EASL) and the American Association for the Study of Liver Disease (AASLD), emphasized only patients with cirrhosis. In contrast, the Asia Pacific Association for the Study of the Liver (APASL) definition included both cirrhosis and non-cirrhosis patients. The APASL ACLF Research Consortium (AARC) data demonstrated that non-cirrhotic CLD patients with ACLF have a high 4-week mortality rate (above 33%) . There ahttp://www.aclf.in/), we prospectively collected information from the hospitalized patients. The study period was from October 2009 to April 2016. All adult patients age above 18 years who were hospitalized from the emergency room or outpatient clinical due to having ACLF, were included in the study. Liver cirrhosis was diagnosed based on the radiological imaging findings of liver nodularity and/or portal hypertension. Histopathological study was performed in selected cases. A diagnosis of ACLF was defined according to the APASL criteria: jaundice (serum bilirubin ≥ 5 mg/dl) and coagulopathy (INR ≥ 1.5 or prothrombin activity < 40%), followed by ascites and/or encephalopathy within 4 weeks in patients with prior diagnosed or undiagnosed chronic liver disease or cirrhosis [On behalf of the APASL ACLF Research Consortium (AARC) . This study was registered in the Thai Clinical Trials Registry, ID TCTR20191226002. All authors had access to the study data, and they reviewed and approved the final manuscript.Categorical variables were described as counts and percentages and were compared using Fisher's exact test. Continuous variables were demonstrated as means and standard deviations. The independent sample t-test and Mann–Whitney (Wilcoxon rank) test were used to compare groups with and without normal distribution. Primary and secondary outcomes were 28-day and 90-day mortality rates. Kaplan–Meier method was used in survival analysis. The log-rank (Mantel-Cox) test was used to evaluate survival of cirrhosis and non-cirrhosis groups, which followed a Chi-square distribution. Crude analysis was demonstrated by the Kaplan–Meier curves and compared by the mean survival time between each group. Multivariate analysis by Cox regression was performed to assess the relationship between potential factors that might affect mortality. The parameters in the multivariate analyses were considered by two types according to the onset of ACLF development; (1) pre-ACLF parameters including age, sex, CLD etiologies and precipitating factors, and (2) post-ACLF parameters including the initial laboratory profiles, CTP and MELD scores, the presence of hepatic encephalopathy, ascites and number of organ failure. The Hazard ratio was used to estimate the direction and degree of each variable to the prognosis of ACLF at the 28-day and 90-day periods.Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) flowchart of patient enrolment is shown in Fig. p < 0.0001) and liver insult . Regarding cryptogenic CLD cases, they were more common in non-cirrhosis than cirrhosis . In addition, chronic HBV infection and reactivation was significantly more common in non-cirrhosis than cirrhosis .Alcohol consumption shared a greater number in cirrhosis than non-cirrhosis in terms of both CLD etiology .Baseline hematological findings , and platelet count) and total bilirubin level from both groups were similar. Non-cirrhosis CLD patients had a statistically higher level of serum sodium, creatinine, albumin, alanine aminotransferase, and INR than cirrhosis CLD patients. Baseline CTP and MELD scores were significantly higher in non-cirrhosis than cirrhosis group .Most patients had single organ failure (41.3% in cirrhosis and 33.7% in non-cirrhosis patients), multiple organ failure was more common in non-cirrhosis group . Liver failure was the most common type of organ failure in patients with cirrhosis (79.3%) and non-cirrhosis (80.2%). In contrast, cerebral failure was the most common extra-hepatic organ failure in those with cirrhosis (38.9%) and non-cirrhosis (51.9%). ACLF patients without cirrhosis had more proportion of cerebral, coagulation, renal and circulatory failure compared to those with cirrhosis (Table p < 0.0001) and day 90  (Table p < 0.001) and 90-day  mortality rate than cirrhosis patients. Characteristics of ACLF patients with alcohol-related liver disease with or without cirrhosis were shown in Supplementary Table 1. Non-cirrhotic patients had a higher white blood cell count than cirrhotic patients. In addition, cerebral, coagulation, renal, and circulatory failure and more than two organ failures were more frequently found in ACLF patients without cirrhosis than patients with cirrhosis (Supplementary Table 1).Only 0.8% of patients underwent liver transplantation. Overall, 28-day and 90-day mortality rates were 39.3% and 49.9%, respectively. Non-cirrhosis patients had a significantly higher mortality rate than cirrhosis patients, both at day 28 .The survival analyses according to the presence of cirrhosis in alcoholic CLD are demonstrated in Fig. 2p < 0.0001) and 0.56 , respectively. Among the pre-ACLF parameters, age was a variable associated with a poorer prognosis, while acute injury from alcohol was independently associated with lower 28-day mortality. Sex and etiologies of CLD were not independently associated with 28-day and 90-day mortality. Regarding the post-ACLF parameters, defined as parameters presented at the time of ACLF diagnosis, we found that total WBC count, MELD scores, number of organ failures, and baseline hepatic encephalopathy were significantly associated with more significant mortalities at both 28-day and 90-day.The potential risk factors associated with the 28-day and 90-day mortality were analyzed using univariate and multivariate analysis Tables  and 4. TThis study analyzed the information from the AARC registry, the largest ACLF patients database in the Asia–Pacific region. In comparison with the previous information published by the APASL in 2014 . The curSeveral variables that influence ACLF prognosis has been discovered; however, there were conflicting associations between each parameter to the outcome. Apart from the presence of cirrhosis, aging and acute insult from alcohol were the pre-ACLF factors that influence ACLF mortality, while other parameters, including sex and types of CLD etiologies, which were found potentially related with prognosis by univariate analysis. These parameters were found insignificant to mortality after being adjusted with the presence of cirrhosis by multivariate analysis. Shi et al.  showed tThe 28-day and 90-day mortality rates in cirrhosis patients were significantly lower than in non-cirrhosis patients. Both univariate and multivariate analyses confirmed that the presence of cirrhosis significantly impacted the prognosis among ACLF patients. Cirrhosis ACLF patients had about a 50% greater chance to survive at both 28-days and 90-days in comparison to those without cirrhosis. To compare our results with other literature, only a few studies had evaluated the effects of cirrhosis on mortality. Three studies evaluated CLD patients from any cause. They did not find noteworthy differences between the presence of cirrhosis and short-term (28-days) and long-term (after 28-days) mortalities , 21, 22.Although most literature did not find significant effects of the presence of cirrhosis on prognosis, which contrasted results from our study, we should take into account the fact that these results were not the primary objective of their studies, so the number of the study population might be too small to discriminate the effect of cirrhosis on mortality. The significant impact of the presence of cirrhosis was consistently observed from every evaluation in our study, in which the study protocol was specifically generated. Moreover, our database was relatively large and enrolled diverse participants from various countries and ethnicities. This was the strength of our study, reassuring that the presence of cirrhosis influenced ACLF prognosis particularly in patients who had alcohol-related liver disease.Systemic inflammatory response after liver injury is a major contributing mechanism in the pathogenesis of ACLF. There are two types of triggers that are able to stimulate inflammatory responses. Exogenous triggers, such as bacterial infection stimulate different types of inflammatory cells and cytokine pathways that give rise to inflammation. Endogenous triggers originating from necrotic hepatocytes and products from extracellular matrix breakdown induce inflammation to promote tissue restoration. Two types of inducers act synergistically in response to the liver injury to stimulate inflammatory cascade and repair mechanisms; however, excessive inflammation could adversely bring about organ failure and death . BesidesTwo possible reasons might explain why patients with non-cirrhosis had a worse prognosis than patients with cirrhosis after ACLF development in alcohol-related liver disease. First, non-cirrhosis had a more inappropriate and exaggerated immune response than cirrhosis. Although ACLF patients did not have cirrhosis, they still developed systemic inflammation, which repetitively or continuously provoked an immune response. The study showed that white blood cell count was higher in alcoholic CLD patients with non-cirrhosis, demonstrating a higher degree of systemic inflammatory response. Additionally, patients with non-cirrhosis had more cerebral, coagulation, renal, and circulatory failure than patients with cirrhosis. Systemic inflammation is a distinct characteristic of ACLF, and it is significantly more prevalent in alcoholic liver disease-ACLF –27. An aOur study demonstrated that although non-cirrhosis CLD patients had a lower chance of liver-related complications, they were equally or at higher risk of poor outcome in cases with complications than patients with cirrhosis. While most physicians focused their concerns on cirrhosis patients, we recommended increasing the level of attention to CLD patients particularly alcoholic CLD. At ACLF presentation, non-cirrhosis patients should be concerned that they might have serious systemic inflammation. We encourage including the presence of cirrhosis with the type and number of organ failures at an initial evaluation for severity grading.n = 13) underwent liver transplantation, resulting in difficulty in analyzing its relation with prognostic outcomes. Second, we did not collect and compare inflammatory biomarkers between cirrhosis and non-cirrhosis ACLF patients to confirm our hypothesis. Hence, we suggest collecting inflammatory biomarkers for future research, which might help establish the hypothesis of the exaggerated inflammation and identify correlations with prognosis.Our study had some limitations. First, there was marked heterogeneity in treatment protocols between each institution. We did not include treatment effects, particularly liver transplantation, in prognosis assessment. In addition, only a small number of patients Below is the link to the electronic supplementary material.


Graft versus Host Disease (GVHD) typically affects the ocular surface, with a presentation resembling Dry Eye Disease (DED). Although the etiopathology is not completely known, the conjunctiva might be a key site of T-cell activation. The differential diagnosis might be tricky at early stages, because of the lack of dedicated clinical and laboratory tests. To meet these needs, we evaluated the suitability of ocular surface matrix metalloproteinase-9 (MMP-9) clinical test.  Consecutive GVHD patients, referred to IRCCS San Raffaele Scientific Institute, were recruited. DED patients served as controls. MMP-9 was tested through InflammaDry immunoassay kit in both groups; Ocular Surface Disease Index (OSDI) questionnaire, tear osmolarity, fluorescein Tear Break-up Time (TBUT), corneal and conjunctival staining, and Schirmer test I were also collected. Parametric and nonparametric statistical tests were used to analyze the intergroup differences; Receiver Operating Characteristics (ROC) curve analysis was carried out to perform sensitivity and specificity evaluations. p ≤ 0.001). Corneal and conjunctival staining scores resulted worse in GVHD than in DED . No significant differences regarded the other collected parameters. GVHD group was characterized by positive correlations between MMP-9 and conjunctival staining  and between MMP-9 and OSDI ; a faint inverse correlation was found between MMP-9 and Schirmer test .  Forty-five GVHD and 40 DED patients were included. MMP-9 expression was significantly higher in GVHD group than in DED (84.4% vs 33%,  MMP-9 has a role in physiologic cellular remodeling; when a proinflammatory stimulus occurs, MMP-9 molecules are overreleased in the extracellular matrix. The positive expression of MMP-9 in GVHD may be interpreted as the consequence of a T-cell aggression against self-antigens and may be considered a reliable biomarker to detect ocular surface inflammation in GVHD, even in early stages of the disease. Ocular chronic graft versus host disease (GVHD) often affects patients undergoing allogeneic hematopoietic stem-cell transplantation  –4.The advancements in human leukocyte antigen (HLA) matching techniques and the improvements in posttransplant treatments made allo-HSCT the gold-standard cure for life-threatening hematologic malignancies , 6. HoweThe risk of occurrence of ocular GVHD turned out to be higher in the first/second year since the allo-HSCT, depending on several factors, either following or preceding the systemic manifestations . The mosDifferent from the multifactorial etiology of dry eye disease (DED),  the pathMatrix metalloproteinases (MMPs) belong to the family of zinc- and calcium-dependent endopeptidases, playing a role in several intercellular activities. Among them, matrix metalloproteinase-9 (MMP-9) is present at the level of the ocular surface, being involved in cellular remodeling pathways; when pathologic stimuli occur, the MMP-9 molecules are overreleased in the extracellular matrix, accelerating the cellular turnover and maintaining the proinflammatory status. For all these reasons, MMP-9 is considered crucial for the induction and the perpetuation of ocular surface damage –13. PrevIn the present study, we measured the expression of ocular surface MMP-9 in early GVHD compared with non-GVHD mild DED patients, and we evaluated its sensitivity and specificity for the ocular surface diagnostic workup of GVHD population. We also evaluated further ocular surface clinical features in the two groups. Finally, we assessed the relationship among MMP-9 expression and the other diagnostic evaluations in both groups of patients.In this cross-sectional, observational study, we enrolled consecutive GVHD patients admitted to the Ocular Immunopathology Service of the Department of Ophthalmology, IRCCS San Raffaele Scientific Institute, Milan, Italy. They were compared with a group of non-Sjogren DED patients. DED diagnosis was made following the Tear Film and Ocular Surface Society (TFOS) Dry Eye Workshop (DEWS II) criteria .The inclusion criteria for the study group were the presence of early ocular chronic GVHD, according to the National Institutes of Health (NIH) consensus criteria, , 17 treaExclusion criteria were severe ocular surface involvement, topical treatments administered in the 6 months before the inclusion into the study, including topical steroids, cyclosporine, and therapeutic contact lenses use, evidence or history of ocular surface disorders other than ocular GVHD, the inability to complete the Ocular Surface Disease Index (OSDI) questionnaire, the presence of other ocular diseases, including ocular hypertension or glaucoma and retinal disorders, any ocular surgery other than uneventful cataract surgery performed at least 6 months before the study visit, the coexistence of systemic disorders, or the assumption of drugs potentially influencing the ocular surface status, with the exception of immunosuppressants for systemic GVHD.For the control group, the inclusion criteria were the diagnosis of mild-moderate DED according to the current guidelines , 18 and The study was conducted according to the tenets of the declaration of Helsinki for research involving human subjects and had the approval of the ethic committee of San Raffaele Scientific Institute. All the patients signed an informed consent before the inclusion in the study.Demographic characteristics, primary hematopoietic disease, and systemic treatments received after allo-HSCT were recorded.Ocular examination consisted in best-corrected visual acuity measurement, anterior segment slit-lamp evaluation, Goldmann applanation tonometry, and dilated fundus ophthalmoscopy.Ocular surface examination included the following tests, performed with at least five-minute interval in between: OSDI questionnaire, tear film osmolarity, assessment of MMP-9 ocular surface expression, tear film break-up time (TBUT), and corneal and conjunctival staining with fluorescein using the modified Oxford Scale and Schirmer test without anesthesia . The objSubjective symptoms were evaluated with the OSDI questionnaire, which is a 12-item questionnaire designed to provide rapid assessment of symptoms consistent with DED. The OSDI answers were used to calculate a score through the following formula:OSDI higher scores represent more severe DED according to the following classification: normal = 0 to 12, mild disease = 13 to 22, moderate disease = 23 to 32, severe disease = 33 to 100 .Tear osmolarity was evaluated with the noninvasive TearLabTM Osmolarity System , which is a “lab-on a-chip” system collecting and analyzing a 50 nL tear sample from the inferior lateral meniscus with a single-use test card. After instrument calibration, tears are collected directly from the eye, and tear osmolarity is analyzed according to the instruction of the producer. An osmolarity level of ≥308 mOsm/L is considered as a sensitive cutoff value to identify DED . All meaThe measurement of MMP-9 expression was carried out through the InflammaDry immunoassay , using direct sampling microfiltration technology. The test was performed following the manufacturer procedures. The eyelid was lowered to expose the tarsal conjunctiva, and the sampling probe was applied on the external, middle, and internal thirds. The test was assembled placing the probe of the collector into the transfer window of the test cassette body. The cassette body presented an adsorbent tip that was then immersed for 20 seconds into a buffer vial, containing specific antibodies against MMP-9. Then, the test was placed flat for 10 minutes, waiting for the appearance of the first line in the control zone of the result window of the cassette. A positive reaction was detected when MMP-9 concentration was above 40 ng/ml, corresponding to the appearance of a second line, in the result window after further 10 minutes .After waiting 15 minutes, we performed a TBUT test, using a fluorescein strip  wetted with a saline solution drop , Italy). For each test, the time of the first break-up was measured in seconds. The test was performed three times and then averaged to provide a final value, considered for statistical purposes. The severity grades for TBUT were as follows: ≥10 seconds = normal, 5 to 9 seconds = mild to moderate, and <5 seconds = severe tear film instability .A = absent staining, B = minimal, C = mild, D = moderate, E = marked, >E = severe) to be compared with patients' clinical picture. To adapt this grading score with the statistical analysis, we replaced the letters with numbers  [Corneal and conjunctival staining were evaluated at the slit lamp, 3 min after fluorescein instillation, under blue light and yellow filter, in accordance with a modified Oxford scale, separately considering nasal and temporal sectors, both for the cornea and the conjunctiva . The staining severity was graded according to Bron and coauthors grading system, which provided panels defined by letters ( >E = 5) , 24.After waiting at least 20 minutes, we performed Schirmer test without topical anesthesia, using standardized filter strips  for 5 minutes with the patients' eyes closed. Schirmer test grading was performed as follows: >10 mm normal, 10–6 mm mild to moderate, and ≤5 mm severe impairment of tear production .Primary outcome measure was the frequency of the expression of MMP-9 in GVHD compared with DED group. Secondary outcomes included the possible correlations among MMP-9 expression and the other clinical evaluations in both groups and the differences among the other clinical tests between the two groups.For each participant, only one eye was included into the statistical analysis, chosen considering the eye with the worst tear osmolarity, or, in case of similar osmolarity values, the right eye .t-test and the Mann–Whitney U test, as appropriate. For the correlation analysis, Kendall's Tau test was performed. The Receiver Operating Characteristic (ROC) curve was calculated for sensitivity and specificity of MMP-9 test in GVHD diagnosis. All data were expressed as mean ± standard deviation (SD), and p < 0.05 was considered statistically significant.The statistical analysis was carried out using MedCalc (version 13.1.2.0), through parametric and nonparametric tests. The calculations were performed using chi-square tests, and statistical differences were reported according to Fisher's exact test. For intergroup difference analysis, we used Student's p=0.9). Sex distribution was homogeneous within the groups  (p=0.21). Best-corrected visual acuity was 20/20 in all the analyzed eyes; anterior chamber slit lamp evaluation, Goldmann applanation tonometry, and dilated fundus ophthalmoscopy were within normal limits. The period lasting from allo-HSCT to the occurrence of ocular GVHD was of 21.4 ± 12.2 months; no statistical significance was found between MMP-9 positive and MMP-9 negative patients .Forty-five eyes of 45 consecutive GVHD patients were enrolled in the study. Forty eyes of 40 consecutive DED patients were also included as control group. The mean age of GVHD group was 51.5 ± 10.6 years, while the mean age of DED group was 51.2 ± 13.5 years (p ≤ 0.001). The ROC curve analysis demonstrated an area under the curve equal to 0.897  .The frequency of the expression of MMP-9 resulted in significantly higher GVHD than in DED  . Both cop > 0.05). All data are shown in OSDI score, tear osmolarity, TBUT, and Schirmer test values results were similar between the two groups , and between MMP-9 and OSDI score ; moreover, it showed a weak inverse correlation between MMP-9 and Schirmer test I . Tear osmolarity showed a positive correlation with OSDI  and an inverse correlation with Schirmer test I . TBUT positively correlated with Schirmer test I  and negatively correlated with corneal staining  and with OSDI . Schirmer test I showed a positive correlation with TBUT  and inverse correlations with MMP-9 , OSDI , tear osmolarity , and corneal staining . Corneal staining showed a positive correlation with OSDI  and inverse correlations with TBUT  and Schirmer test I . Conjunctival staining showed a positive correlation with MMP-9 .The correlation analysis of GVHD patients showed positive correlations between MMP-9 and conjunctival staining . Corneal staining showed positive correlations with MMP-9 , tear osmolarity , and conjunctival staining . Conjunctival staining showed a positive correlation with corneal staining . All the correlation data are shown in In DED group, we found a positive correlation between MMP-9 and corneal staining , developing from coexisting conditions.In ocular GVHD, the pathogenic mechanism is peculiar, based on the reaction of allogeneic T-cells against ocular surface self-antigens, triggering the cascade of events leading to the development of the vicious circle sustaining the inflammatory process. Previous literature data proved that conjunctiva holds a key role in the GVHD-related immune aggression. Conjunctival epithelium has been described as an important immunological hub: in healthy subjects, immune cells are organized in patches, resulting in the conjunctiva-associated lymphoid tissue (CALT). CALT is mainly composed by antigen presenting cells that are responsible for the beginning of the immune response . MoreoveThe family of MMPs actively intervenes in processes of tissues remodeling and degradation, being responsible of several intercellular exchanges . From thSince the increased release of MMP-9 is associated with epithelial damage, it might be an early spy of potential fibrosis occurring in the latest phases of GVHD. Fibrosis of the tissues is known as a dangerous sight-threatening complication, potentially leading to opacity and conjunctivalization of the cornea. Nevertheless, we acknowledge that this issue was outside the aim of our observations and does need further studies to be proved. InflammaDry immunoassay is a commercially available tool that has been previously proved effective in evaluating the expression of conjunctival and tears MMP-9,  with gooIn our evaluations, carried out in patients with early GVHD and mild-moderate dry eye, we found that the positive expression of MMP-9 was significantly more frequent in GVHD than in DED; moreover, the ROC curve analysis demonstrated high sensitivity and specificity of the positivity of the InflammaDry test, also considering this mild stage of GVHD. This result may be interpreted as the consequence of the intense conjunctival inflammation that is a key element of ocular GVHD pathogenesis.The significantly higher corneal and conjunctival staining scores may be interpreted as the consequence of the chronic surface inflammation characterizing GVHD.The reported symptoms, assessed by means of OSDI questionnaire, and the clinical tests performed  were similar between groups. This is explicable with the apparent clinical similarity between the two diseases in the early stages, and for this reason, the identification of more precise biomarkers would be useful to make the differential diagnosis.With respect to the correlation analysis between MMP-9 and the other clinical tests in the GVHD group, the positive correlation with conjunctival staining is worth noticing. This finding may be interpreted as a further demonstration of the reliability of MMP-9 in evaluating conjunctival inflammation in GVHD. Moreover, if we consider patients characterized by a positive InflammaDry test and a relatively low conjunctival staining score, we may speculate about the role of MMP-9 as a biomarker of inflammation even in a subclinical stage of the disease; consequently, MMP-9 can be considered as a highly sensitive biomarker for the detection of conjunctival inflammation.The positive correlation with OSDI questionnaire score may be related with the discomfort symptoms causes by ocular surface inflammation, even in the absence of clinically relevant corneal epithelium alterations. Furthermore, the inverse correlation with Schirmer test could provide evidence for the parallel damage of the lacrimal gland also occurring in ocular GVHD manifestations .A limitation of the study might be the dichotomic response of the MMP-9 test (positive/negative). A deeper quantification of the MMP-9 expression might be more accurate and might provide clinically relevant cut-offs for the risk assessment of severe progression of disease. Furthermore, the diagnostic test does not allow distinguishing the differences in the nature of the inflammation .In conclusion, the significant development of ocular surface inflammation in GVHD is well known. Previous studies have demonstrated the important role of conjunctival self-antigens aggression in the pathogenetic mechanisms at the basis of GVHD. In this study, we found higher frequency of MMP-9 expression in GVHD than in DED patients, with a positive correlation with conjunctival staining, even at an early stage of the disease. For this reason, MMP-9 can be considered a reliable biomarker of ocular surface involvement in GVHD and might be useful to make a differential diagnosis with other forms of DED in transplanted patients, even in case of poor clinical objectivity. MMP-9 might be a useful biomarker also to facilitate the decision of starting anti-inflammatory treatments and to monitor their efficacy.


Direct oral anticoagulants (DOAC) including factor Xa inhibitors are associated with bleeding events which can lead to severe morbidity and mortality. Reversal agents like andexanet alfa (AA) and 4F-PCC (Four-factor prothrombin concentrate complex) are available for treating bleeding that occurs with DOAC therapy but a comparison on their efficacy is lacking. In this study, we analyzed the efficacy and safety of patients treated with andexanet alfa for bleeding events from DOAC. Databases were searched for relevant studies where AA was used to determine efficacy and safety in bleeding patients who were on factor Xa inhibitors. Published papers were screened independently by two authors. RevMan 5.4  was used for data synthesis. Odds ratio (OR) and mean difference (MD) was used to estimate the outcome with a 95% confidence interval (CI). Among 1245 studies were identified after a thorough database search and three studies were included for analysis. AA resulted in lower odds of mortality compared to 4F- PCC  among patients with intracerebral hemorrhage. There was no difference in thrombotic events between patients receiving AA and 4F-PCC . No differences in length of hospital stay and intensive care unit (ICU) stay were seen between patients receiving AA and 4F-PCC. In conclusion, andexanet alfa reduced in-hospital mortality in patients who had bleeding due to factor Xa inhibitors compared to 4F-PCC. However, there were no differences in thrombotic events, length of ICU, and hospital stay between patients treated with AA and 4F-PCC. Direct oral anticoagulants (DOAC) have been increasingly used in patients for the prevention of systemic embolization in atrial fibrillation as well as treatment and prevention of deep vein thrombosis (DVT) and venous thromboembolism (VTE). As a result, the indications of DOAC have significantly expanded in the last decade -5. PrediBefore the introduction of andexanet alfa (AA), off-label use of 4 factor-prothrombin concentrate complex (4F-PCC) was advised and was used in the situation of life-threatening bleeding . ProthroMethodsWe used Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) guidelines for the systematic review of available literature . The stuLiterature searchWe searched PubMed, PubMed Central, Scopus, Embase, and Cochrane library for relevant studies published till February 2021. Searches were conducted using the keywords like "andexanet alfa", "andexanet", "andexanet alpha", "bleeding", "factor Xa inhibitor," and "factor Xa inhibitors" and appropriate boolean operators. Details of the search strategy are available in Supplementary Material 1.Selection of studiesA. Types of StudiesWe included studies done to determine the efficacy and safety of andexanet alfa in patients who had bleeding in the setting of factor Xa inhibitor use. As randomized controlled trials were not available, we included prospective and retrospective studies and case series with more than ten patients. AA was used to determine efficacy and safety in bleeding patients on factor Xa inhibitors in qualitative analysis. In addition, the studies with both treatment and control groups were included in the quantitative synthesis.B. Types of ParticipantsThe studies required patients to be more than 18 years of age and had bleeding in the setting of Factor Xa inhibitor use.C. Types of InterventionsAndexanet alfa was taken in the treatment arm, while 4F-PCC or other blood products were included in the control arm.D. Types of Outcome MeasuresOur outcome of interest was hemostatic efficacy, mortality within 30 days, the incidence of thrombotic events, and length of hospital and ICU stay following treatment with AA or other blood products. We excluded types of studies with the following characteristics: meta-analysis, reviews, in-vitro studies, studies done on healthy subjects, case reports, editorials, opinions, letters, protocols, abstracts/presentations, dissertation, and animal studies. Case series with fewer than ten patients, articles where full-text articles were not available, ongoing studies, and studies with incomplete data were also excluded.Data extraction and managementTitles, abstracts, and full texts were screened for study and report characteristics that matched eligibility criteria. Studies were independently screened by two reviewers (AA and SS) using Covidence  and data were extracted for both quantitative and qualitative synthesis. The conflicts were resolved by taking the opinion of the third reviewer (NK). The data extraction sheet was created using Microsoft Excel software. One reviewer collected the data from all articles; the second reviewer verified the data for accuracy and highlighted discrepancies; the third reviewer resolved any disagreements and carried out a thorough evaluation to ensure that only the outcomes of interest were taken into account. The following variables were included: first author, type of design, site of study, year of publication, sample size, mean age, percentage of male and female, indication for anticoagulation, hemostatic efficacy, mortality within 30 days, length of hospital stay, length of ICU stay and incidence of thrombotic events. Risk of BiasWe used the Joanna Briggs Institute (JBI) critical appraisal checklist for cohorts and case series for quality and risk of bias assessment Tables -2.Statistical AnalysisRevMan 5.4  was used for statistical analysis. Odds ratio (OR) and mean difference (MD) was used to estimate the outcome with a 95% confidence interval (CI). Assessment of Heterogeneity2 test based on previously recommended stratifications. In the case of heterogeneity, we used the invariance and random-effect finally, well. Finally, we evaluated the sensitivity by rerunning the analysis to assess any unrevealed differences.The statistical heterogeneity among the studies was calculated and assessed with the IResultsA total of 1245 studies were identified after thorough database searching, and 351 duplicates were removed. Title and abstracts of 894 studies were screened, and 860 irrelevant studies were excluded. The full-text eligibility of 34 studies was assessed, and 24 studies were excluded for definite reasons      receiving andexanet alfa across ten studies. In contrast, the overall mortality rate in a recent 4F-PCC meta-analysis in FXa inhibitor bleeding was 18% compared to 12.13% in our analysis and 14% in the ANNEXA-4 trial . The stuWe found no difference in the incidence of thrombotic events caused by AA in comparison to 4F-PCC for the reversal of bleeding caused by factor Xa inhibitor. A recent meta-analysis of seven studies including 240 patients showed thrombotic events of 4% with the use of 4F-PCC . In contThe definition of hemostatic efficacy and time since AA administration for determining efficacy was different in between studies. Hemostatic efficacy was measured and reported as good/excellent or poor following criteria used by Sarode et al in five studies which include the ANNEXA-4 study. The Ammar et al. study used different values for hematoma expansion, the Culbreth et al. 2018 study reported repeat CT as stable or worsening while the Culbreth et al. 2019 study which included patients requiring emergent surgery reported hemostatic effectiveness as per surgeon ,22,23. CClinical benefit of AA use was observed in bleeding due to factor Xa inhibitors in our analysis; however, the cost of stocking AA in most hospitals might be prohibitive for the immediate use for reversible DOAC related life-threatening bleeding. The median projected cost of andexanet alfa was $22,120/patient compared to $5670/patient for 4F-PCC. 4F-PCC currently is more widely available and less expensive, but that may change if the cost for AA comes down in the future . 4F-PCC Limitations of the studyMost of the studies included were case series and retrospective observational studies. Only one prospective study, the ANNEXA-4 trial, was included. There were control groups in only three of our studies which were all retrospective. The sample size was less in our studies. Therefore, there was a moderate to high risk of bias in our studies. ANNEXA-4 trial had wide exclusion criteria: planned surgery within 12 hours after andexanet alfa administration, ICH with GCS less than 7, hematoma volume more than 60 cc, expected survival less than one month, use of VKA, dabigatran, PCC, WB, or plasma in last seven days. Giovino’s study also excluded patients with GCS less than 7 and hematoma volume >60 ml . HoweverAndexanet alfa reduced in-hospital mortality in patients who had bleeding due to factor Xa inhibitors compared to 4F-PCC. There was no difference in thrombotic events, length of ICU, and hospital stay between andexanet alfa and 4F-PCC. Thus, AA is a promising therapeutic agent for the reversal of factor Xa-associated bleeding. However, the cost of stocking AA in most hospitals might be prohibitive for the immediate use for reversible of DOAC related life-threatening bleeding. 4F-PCC currently is more widely available and less expensive, but that may change when the cost for AA decreases. More studies are required in the future to determine the effect of AA as compared to 4F-PCC in patients with DOAC-related bleeding other than intracranial bleeding.


Lactobacillus paracasei IJH-SONE68, prevent and ameliorate allergic reaction on contact in dermatitis model mice. In the present study, we conducted a clinical trial using a capsule containing spray-dried powder from pineapple juice broth fermented with the LAB strain as an experimental diet. The clinical trial was conducted as a double-blind and placebo-controlled randomized comparative study from May 2019 to July 2021. Males and females between the ages of 21 and 70 who experience chronic allergies participated in the study. Sixty subjects were instructed to orally take a capsule containing the IJH-SONE68 powder or placebo, every day for 12 weeks. After the clinical trial was over, the scores based on subjects’ self-assessment of allergic status were significantly improved in the intervention group, as compared with the placebo group. Some serum biochemicals associated with inflammation response were also significantly improved by intake of the experimental diet. In conclusion, the IJH-SONE68-derived EPS improves chronic allergy status in humans and is expected to decrease their inconvenience.We have recently demonstrated that the exopolysaccharides (EPSs) produced by a plant-derived lactic acid bacterium,  Allergic diseases are immunoglobulin E (IgE)-mediated type I inflammatory disorders, including allergic rhinitis ,2. The mLactic acid bacteria (LABs) is a generic name given to Gram-positive bacteria that produce a large amount of lactic acid . The LABWe have isolated many kinds of LABs from natural sources, especially from raw plants, including fruits and medicinal herbs. Through randomized clinical trials, we have found in our established plant-derived LAB library of more than 1000 strains, some useful ones for preventive medicine, such as for improving constipation, obesity, and hepatic dysfunction ,12,13. FLactobacillus (Lb.) paracasei IJH-SONE68, was isolated from a fig leaf. Our previous study revealed that the IJH-SONE68-derived neutral EPS has a novel structure, which mainly consists of α-1,6-linked chains made of N-acetylglucosamine [One of the EPS-producing strains, cosamine . In addicosamine ,17,24,25cosamine  and by rcosamine , respectHealthy volunteers who live in the Hiroshima area of Japan and who are between 20 and 70 years old, were recruited through a series of advertisements. The inclusion criteria were that subjects must be healthy males or females who have at least once (1) a score of 8 to 20 on the self-assessment questionnaire for allergic symptoms  and (2) 5 cells, was used to fill four separate capsules. The placebo capsule contained the same amount of dextrin.We delegated the production of placebo and test capsules to SAKURAO Brewery and Distillery Co., Ltd., Japan. The test capsules contained heat-killed IJH-SONE68-strain powder and dextrin, whereas the placebo capsules contained dextrin only. The IJH-SONE68 strain was cultured in pineapple juice and then the cultured broth was heat-treated before applying a spray dryer. In total, 1040 mg of IJH-SONE68 powder, which contained approximately 5.0 × 10This trial was conducted at Hiroshima University  from May 2019 to July 2021 using a double-blind, randomized, placebo-controlled parallel-group study. Eligible subjects who met the criteria were enrolled and stratified according to their score on the self-assessment questionnaire (12 ≤ or less) and serum IgE concentration (251 ≤ IU/mL or less), then assigned to the IJH-SONE68 or placebo group using the blocked randomization method in a 1:1 allocation ratio with a block size of 4. The allocation table was generated using Microsoft Excel software. Randomization assignments were carried out by non-clinical staff, with no analytical involvement in the present trial. The subjects and outcome assessors were blinded to the assignment information.The subjects were instructed to take 4 capsules of either the IJH-SONE68 or placebo daily at any time within the day for 12 weeks. They were also directed to keep their ordinary dietary habitats and not to donate blood during the trial period. The subjects were provided with daily dated record forms throughout the trial period, to make a record of their consumption of capsules and their health conditions. The forms also contained a daily answer sheet for a self-assessment questionnaire and VAS value. Adverse events that newly emerged or worsened after intervention were evaluated when those grades shifted higher, according to the Common Terminology Criteria for Adverse Events version 5.0 (CTCAE v5.0).The primary outcomes of the present study were, changes in the self-assessment questionnaire and VAS value, and the secondary outcomes were, changes in the serum total IgE and specific IgE  levels. The subjects visited Hiroshima University for physical examinations (every 4 weeks), serum total IgE measurements (every 4 weeks), answering the self-assessment questionnaire and assigning a VAS value (every 4 weeks), and serum specific IgE and biochemical measurements and urinalysis (at 12 weeks). Blood samples were obtained after a 9 h overnight fast. Body fat percentage was measured using a body composition analyzer . Blood pressure was measured according to the Japanese Society of Hypertension Guidelines for the Management of Hypertension 2019 (JSH2019) .The protocol of the present clinical trial was approved by the Ethics Committee of Hiroshima University  and performed according to the guidelines of the Helsinki Declaration. This trial was registered in the University Hospital Medical Information Network Clinical Trials Registry (UMIN-CTR), ID: UMIN000036317, on 11 March 2019.t-test. The difference and S.D. for sample size estimation were performed on the basis of the data from our previous animal study [t-test. Statistical analysis for changes in each item on the questionnaire was done using the Mann–Whitney U test, since the results did not show a normal distribution. All statistical analyses were performed using IBM SPSS Statistics 17.0 J for Windows .The sample size in each group in the present study was calculated as 46, to detect 10% difference with an estimated S.D. of 20% for total IgE, assuming 80% power and a two-sided significance level of 0.05 using two-sample al study . The obtal study . For eacRecruitment for the present trial is summarized in n = 100). The baseline characteristics of the subjects are summarized in Based on the scheduled deadline and the limited financial resources, it was difficult to recruit subjects in a short period of time with prevention of the spread of COVID-19. Therefore, this study was unavoidably completed before the original registration goal was achieved (p = 0.008). Likewise, the average VAS value in both groups decreased considerably, but changes in the value were not significant (p = 0.380).The changes in primary outcomes, which were the self-assessment questionnaire scores and the VAS values , during p < 0.05).The score changes in each item of the self-assessment questionnaire were also compared . AlthougThe serum total IgE decreased slightly in the IJH-SONE68 group; however, there was no significant difference between the two groups for changes in the total IgE . AlthougPhysical examinations and other serum parameters were also monitored to detect adverse effects of IJH-SONE68 intake. According to the CTCAE v5.0, there were no significant differences in adverse events possibly related to the study design, between the IJH-SONE68 and placebo groups . In addiDifferent from seasonal allergies like pollinosis, which is caused by pollen allergens, perennial allergies cause chronic symptoms that appear year-round or intermittently. Therefore, perennial allergies considerably decrease the QOL for those patients. Common allergens that account for perennial allergy symptoms are indoor allergens, such as house dust mites (mostly), animal dander, cockroaches, and fungi ,32. A nup = 0.077), indicating that the result partially corresponds to data obtained from the animal experiment [Previously, we showed that the IJH-SONE68-derived EPSs significantly decrease the accelerated serum IgE level and the expression of IL-4 mRNA, in the contact dermatitis model mice . As a prperiment .Our recent study also showed that the IJH-SONE68-derived EPS prevents and ameliorates inflammatory responses in the DSS-induced ulcerative colitis model mice with significant repression of MIP-2, which is a functional analogue of human IL-8 . The infThere were only a few subjects with specific IgE-positive results; therefore, a statistical analysis could not be conducted. As shown in The amelioration of the self-assessment questionnaire scores seems to be due to the anti-inflammatory effect of the IJH-SONE68-derived EPSs confirmed in previous studies ,31,33, ip = 0.058). Our preliminary study suggested that IJH-SONE68 also has an anti-obesity effect, and therefore, lipid metabolism and fatty liver might be improved during the trial, resulting in the decrease of ALT and ChE. The present study indicated that the IJH-SONE68 strain can be expected to help persons with perennial allergies, to decrease their inconvenience.ChE catalyzes the hydrolyzation of choline esters like acetylcholine and the enzyme is synthesized mainly in hepatocytes. A decrease in the ChE level is observed in patients with liver dysfunction, such as cirrhosis; whereas a high serum ChE level has been reported to be associated with obesity and metabolic syndrome ,41,42. ILb. paracasei IJH-SONE68, has been shown to significantly improve the scores based on subjects’ self-assessment of allergic status in the present clinical study. The present outcome demonstrates that the IJH-SONE68 strain can be expected to help persons with perennial allergies and decrease inconvenience.The oral administration of the spray-dried powder derived from the culture broth of


REThink is an online therapeutic game designed as a stand-alone prevention tool, aiming to increase resilience in healthy children and adolescents in a Rational Emotive Behavioral Therapy framework . The aim of this pilot study was to investigate the validity of in-game performance measurements or scores as indicators of the game effectiveness in building real life emotion-regulation abilities. We analyzed how scores of different game levels (addressing different skills) are associated with improvements in mental health and emotion regulation abilities. Our preliminary results suggest that in-game performance at some levels (scores) consistently reflect improvements in psychological functioning, while in-game performance at other levels are less associated with changes in real life self-reported psychological functioning. These results offer important information about which levels can be used as preliminary indicators of psychological improvements, and which levels need to be revised in terms of task or scoring. Overall, results of our study offer preliminary validation of REThink's game scoring system, while also suggesting the elements to be refined.Therapeutic or serious games are considered innovative ways of delivering psychological interventions especially suited for children and adolescents, which can have a positive impact on mental health, while also being fun and easily accessible online. While most serious games for children and adolescents address specific issues, such as anxiety or depression, preventive measures received less attention.  In the past years, researchers‘ attempts to bring psychological interventions closer to individuals in need have led to the development of online therapeutic or serious games. Therapeutic games are designed to be attractive, fun, and motivating, while incorporating elements which trigger behavior and attitude change, pursue therapeutic goals, or train different skills , 2. A syAs Brezinka  noticed,Treasure Hunt,  save the Earth from the negative character . In each level, players have a specific mission through which they can conquer the Earth territories which were previously occupied by Irrationalizer. At the end of each level, players have to win the key which will allow them to access the next territory (level). Players‘ performance is registered by both number of errors (vs. correct actions), and by a total score, which is computed by different algorithms depending on the task for each level  will be correlated with higher gains in self-reported mood  and emotion-regulation abilities (measured by ERICA).Higher game-based skills in recognizing irrational processes  and changing them (Higher scores at Level 4) will be correlated with higher improvements (gains) in self-reported irrational/rational beliefs (measured by CASI).Higher game based problem-solving skills (Higher scores at Level 5) will be correlated with higher improvements in self-reported problem-solving (measured by VAS).Higher game based relaxation skills (Higher scores at Level 6) will be correlated with higher improvements in self-reported negative mood and stress (measured by FD-CMS).REThink game are associated with real life self-reported improvements in in mental health and emotion-regulation skills. Thus, we expected that improvements in skills at each level (higher scores) are related to self-reported real life improvements in mental health and emotion regulation skills.Our secondary aim was to investigate if gains in performance from consecutively playing the N = 54) assigned to the REThink condition in the clinical trial by David et al. (N = 36), and their age ranged between 10 and 16 years, with a mean age of 13 years (SD = 2.05). Considering the small sample size our study is underpowered but being a pilot study can offer preliminary evidence for the effect of the game on self-reported improvements in real life. Written informed consent was obtained from the parents and the school management and the study was approved by the ethical committee of the institution.Children and adolescents , Ponderancer (representing non-awfulizing beliefs), Toleraser (representing high frustration tolerance beliefs), Acceptableizer  and Optimizer (representing happiness). The four irrational servants of Irrationalizer symbolize irrational beliefs: Necessitizer (representing demandingness beliefs), Awfulizer (representing awfulizing beliefs), Frustralizer (representing low frustration tolerance beliefs) and Discourager .REThink has seven levels which focus on objectives based on the REBT model: Level 1: identifying the emotional reactions, differentiating between basic emotions, complex emotions and functional and dysfunctional emotions, Level 2: identifying cognitive processes, Level 3: identifying the relation between cognitive processes, emotions and behavioral reactions, Level 4: changing irrational cognitions into rational cognitions, Level 5: building problem solving skills, Level 6: developing relaxation skills, and Level 7: consolidation of previous skills and building happiness skills  session before the actual level begun. For the actual game play, performance indicators were automatically registered during each level, and participants had to restart a level/sublevel if they did not finish 50% of the level, or had consecutive errors for 25% of the entire level/sublevel.REThink and the effectiveness of the game , and a post-intervention assessment after the modules were completed. For the objective of the current study, we analyzed only the changes in psychological symptoms from the first to the last assessment.Strengths and Difficulties Questionnaire—child version  is a 25-items self-report instrument measuring prosocial behavior as a psychological strength, and emotional symptoms, conduct problems, hyperactivity/inattention, and peer relationship problems as psychological difficulties. Higher scores for this instrument are representative for higher levels of psychological strength/difficulties. Internal consistencies found in the main study for SDQ are α = 0.75 for emotional symptoms subscale, α = 0.80 for the total level of psychological difficulties, α = 0.65 for conduct problems subscale, α = 0.65 for hyperactivity-attention subscale, α = 0.63 for peer problems subscale, and α = 0.67 for prosocial behavior subscale , low frustration tolerance for work (LFTW), low frustration tolerance for rules (LFT-R), and the total irrationality score. Children and adolescents rated their agreement to the 28 sentences on a 5-point Likert scale, so that higher scores indicate high levels of each dimension. Internal consistency reported in the study regarding the mechanisms of change responsible for the effect of REThink was α = 0.65 for low frustration tolerance for work, α = 0.80 for low frustration tolerance of rules, and α = 0.80 for CASI total score. The demandingness subscale showed lower reliability, α = 0.27 and was excluded from all analyses  will be correlated with higher improvements in self-reported mood  and emotion-regulation (ERICA).r(37) = 0.27, p = 0.04] and peer relations problems , as well as with improvements in the total score for strengths and difficulties experienced   = 0.29, p = 0.03]. Changes in attention, fear, and inhibitory control were not significantly associated with game-based emotion recognition skills (Level 1 performance). However, participants with higher emotion recognitions skills (Level 1 scores) reported experiencing significantly more positive emotions, as measured by FD-CMS . Improvements in negative emotions , and emotion regulation were not significantly associated with game-based emotion recognition skills.Our results show that higher scores at Level 1 (game based emotion recognition skills) were correlated with improvements in mental health (SDQ) related conduct problems [.03] see , 2. ConcHigher game-based skills in recognizing irrational processes  and changing them (Level 4) will be correlated with higher improvements in self-reported irrational/rational beliefs (CASI).r(34) = −0.29, p = 0.03], and increased their frustration tolerance for work , measured by CASI.Higher game-based skills in recognizing and changing irrational processes were not reflected significantly in improvements in self-reported total irrational beliefs see . ParticiHigher game based problem-solving skills (Higher scores at Level 5) will be correlated with higher improvements in self-reported problem-solving (VAS).r(37) = 0.14, p = 0.19] (see Our results suggest that self-reported problem solving was not significantly associated with the game based problem-solving skills [.19] see .Higher game-based relaxation skills (Higher scores at Level 6) will be correlated with higher improvements in self-reported negative mood and stress.r(33) = 0.42, p = 0.006] and lower emotional difficulties , as measured by the SDQ (see r(33) = 0.22, p = 0.09] and situation responsiveness , measured by ERICA.Participants with higher scores at Level 6 reported significantly less functional negative emotions (FD-CMS) [ SDQ see , 6. Morep = 0.036  .036 see .REThink). Specifically, we aimed to investigate if these in-game performance indicators (scores for each level) are associated with improvements in real life self-reported mental health and emotion regulation skills. Demonstrating these associations could support the use of in-game scoring gains as marker of changes in psychological symptoms, thus allowing for a certain level of progress monitoring even outside the research lab, and without specialized psychological assessments.The objective of the present pilot study was to analyze the in-game performance indicators of a preventive therapeutic game for children and adolescents  consistently reflect improvements  in psychological functioning, namely improvements—in total mental health, increased tolerance for rules, positive emotions, emotional control while in-game performance at other levels are less associated with changes in psychological functioning, namely reduced depressive mood, and lower emotional difficulties improvements.Results obtained showed that improvements in-game scores at Level 1, which trains emotion recognition, are associated with self-reported improvements in youth's general mental health, as hypothesized. More specifically, higher level 1 game scores were associated with improvements in depressed mood, conduct problems and significantly with higher levels peer relationship problems and positive emotions. The association of emotion recognition in-game gains with real-life self-reported improvements in emotional abilities was in line with our expectation. We did not expect however specific significant improvements in conduct problems and peer relationships, which were surprising for us. It might be that by improving their emotional awareness and recognizing emotions in peers, children and adolescents were able to improve their aggressive behavior and their relationship problems. Future studies need to investigate if this is indeed the mechanism thought which emotional game abilities produced real life changes.In terms of our second hypothesis regarding game scores at levels involving the recognition and change of irrational processes , results obtained only partially confirmed our expectations. Children and adolescents who were better at identifying the connection between thinking and feeling based on their scores at Level 2, reported higher improvements in their irrational beliefs areas—more specifically in low frustration tolerance for work and rules. This is an important finding considering that the role of these specific irrational beliefs has been documented in relation to emotional difficulties and academic performance in children and adolescents . ResultsOur third hypothesis regarding improvements in scores obtained al Level 5 which trained problem solving skills being related to self-reported problem-solving abilities did not received support in our study. This might be due to the nature of the self-report measure which was a VAS type item. In future studies we need to employ more rigorous measures of problem solving abilities. Also, it might be that the tasks of the Level 5 need to be revised in order to better support problem-solving skills consolidation.Results obtained provide support for our fourth hypothesis concerning positive associations between gains in game scores at Level 6, which trains relaxation abilities, and self-reported mood and stress. We found that children and adolescents that registered higher improvements in their in-game scores at this level, reported lower emotional difficulties, lower negative emotions and better emotion-regulation skills.REThink game scoring system. Third, future studies need to use more complex analyses in order to draw firm conclusions about the relationship between in-game scores and real-life improvements. Another limitation of the study are the measurements. We used self-report measures and some questionnaires, even though we found that they have good internal consistency on our sample, are not validated on the Romanian population. Future studies need to use other-report measures in addition to the self-report ones and use questionnaires that are validated for the population used.This study is not without limitations. First, due to technical difficulties we were not able to analyze if gains in the scores at Level 7 are related to real life improvements reported by children and adolescents. Future studies will need to investigate if scores obtained at this level are associated with mental health and positive emotionality. Second, our results need to be interpreted cautiously due to the small sample, lack of statistical power and gender disproportion which limited us in performing further analyses. Future studies need to include a larger sample in order to be able to delineate specific associations and draw clear conclusions regarding the predictive validity of the REThink game is effective in promoting mental health by improving dysfunctional thinking mechanisms. Results of this study offer promising preliminary validation for the REThink's game scoring and suggest that higher scores will reflect real-life changes in children and adolescents' mental health.In sum, our preliminary results showed that improvements in game scores are associated with improvements in self-reported mental health. More specifically, game scoring gains are associated with improved negative and positive emotions, conduct problems, peer relationship problems and emotion-regulation . We have also found that better scores at Level 2 in recognizing the connection between thinking and feeling was associated with improvements in irrational thinking. These findings are in line with the findings from our clinical trial showing that the The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Ethical Committee of Babes-Bolyai University of Cluj-Napoca. Written informed consent to participate in this study was provided by the participants' legal guardian/next of kin.OD contributed to the conception and design of the study. SM organized the database and performed the statistical analysis. OD, SM, and CT wrote the manuscript. All authors contributed to manuscript revision, read, and approved the submitted version.ClinicalTrials.gov, NCT03308981.This work was supported by two grants awarded to OD from the Romanian National Authority for Scientific Research, CNCS—UEFISCDI (Grant Numbers PN-II-PT-PCCA-2013-4-1937 and PN-III-P4-ID-PCE-2020-2170). Trial Registration 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.


Ageing displays clear sexual dimorphism, evident in both morbidity and mortality. Ageing is also associated with changes in DNA methylation, but very little focus has been on the sex chromosomes, potential biological contributors to the observed sexual dimorphism. Here, we sought to identify DNA methylation changes associated with ageing in the Y and X chromosomes, by utilizing datasets available in data repositories, comprising in total of 1240 males and 1191 females, aged 14–92 years.In total, we identified 46 age-associated CpG sites in the male Y, 1327 age-associated CpG sites in the male X, and 325 age-associated CpG sites in the female X. The X chromosomal age-associated CpGs showed significant overlap between females and males, with 122 CpGs identified as age-associated in both sexes. Age-associated X chromosomal CpGs in both sexes were enriched in CpG islands and depleted from gene bodies and showed no strong trend towards hypermethylation nor hypomethylation. In contrast, the Y chromosomal age-associated CpGs were enriched in gene bodies, and showed a clear trend towards hypermethylation with age.Significant overlap in X chromosomal age-associated CpGs identified in males and females and their shared features suggest that despite the uneven chromosomal dosage, differences in ageing-associated DNA methylation changes in the X chromosome are unlikely to be a major contributor of sex dimorphism in ageing. While age-associated CpGs showed good replication across datasets in the present study, only a limited set of previously reported age-associated CpGs were replicated. One contributor to the limited overlap are differences in the age range of individuals included in each data set. Further study is needed to identify biologically significant age-associated CpGs in the sex chromosomes.The online version contains supplementary material available at 10.1186/s13072-021-00407-6. Ageing displays clear sexual dimorphism. Females have a longevity advantage, which can be observed across nationalities and in historical data, where and when reliable demographic information is available , 2. DespSex chromosomes are one of the biological factors contributing to sexual dimorphism in humans and other mammals . The Y cAgeing-associated changes in DNA methylation have been widely studied  in the Y chromosome in four datasets. The number of age-CpGs varied from 2 to 90 across datasets Fig. . All Y cMajority, 76%, of the Y chromosomal age-CpGs identified in two or more datasets were hypermethylated. Their chromosomal distribution in relation to CpG islands was not different to what would be expected by chance. Regarding gene regions, we detected an enrichment of age-CpG sites in gene bodies and a depletion in intergenic regions  and hypomethylated 53%) with age. The age-CpGs were enriched in CpG islands and depleted from open sea and island shelfs. Regarding gene regions, the age-CpGs were enriched in 1st Exon and 5’UTR but depleted from gene body, 3’UTR and from intragenic regions . The age-CpGs were enriched in CpG islands and depleted in open sea and CpG island shelfs. In relation to genes, the age-CpGs were enriched in TSS200 and depleted in gene body .The identified age-CpGs were both hyper- 54%) and hypomethylated (46%) with age. As compared to males, the proportion of hypermethylated age-CpGs was higher and this difference was statistically significant . Four CpGs  were identified in four datasets in both females and males , when X chromosomal genes that were represented by one or more probes in the Illumina HumanMethylation450 BeadChip were used as background.Of the age-CpGs identified in both sexes, 57% were hypermethylated with age. The age-CpGs common for males and females were depleted from open sea as well as from gene bodies  and TAF7L (TATA-box-binding protein associated factor 7 like) (cg01538344), are mainly expressed in testes and have been implicated to have a role in spermatogenesis [TAF7L is also classified as a cancer-testis antigen (CTAg), belonging to a group of genes typically expressed only in the germline but also in malignant tumours [TAF7L were also identified as age-CpGs in at least two datasets in both females and males included in this study  (cg04532200) is highly expressed in the brain and plays a role in axon guidance [TSC22D3 (TSC22 domain family member 3) (cg06461462) regulates T-cell activation and has immunosuppressive and anti-inflammatory effects [GO term enrichment analysis of the sex-chromosomal age-CpGs did not associate these CpGs with functions that are known to be strongly associated with the molecular mechanisms of ageing . The indogenesis , 31. TAF tumours . Two add1210 cg1398184 andguidance . TSC22D3A1210 cg198184 and effects .PRKY (cg20401549) and NLGN4Y (cg27214488). One additional CpG site located in PRKY and seven additional CpG sites located in NLGN4Y were also identified as age-CpGs in at least two datasets included in this study  , a gene that is highly expressed in the prostate and shares great similarity with TBL1X in the X chromosome. We also identified two additional a-CpGs located in TBL1Y in the present study   reportedTBL1Y, is replicated in our study in three datasets .One study only has previously focused specifically on ageing-associated DNA methylation changes in the Y chromosome  . In totaELOVL2, FHL2, and EDARADD, have been replicated in majority of studies [In genome-wide DNA methylation studies on autosomes, at the level of individual CpGs, the results have not shown great replication from study to study. Only a small number of CpG sites, for example those located in  studies , 48, 52.Specifically, age distribution within and between datasets likely influences the replication of age-CpGs. In our analysis, the number of age-CpGs was dependent on the age distribution of the dataset, with higher the IQR for age, the higher the power was to detect significant age-CpGs Fig. . PreviouTo decipher to what extent data processing methods could explain the limited overlap between our results and those reported by Li et al. (2020) , we repeThe sex chromosomes can also undergo more large-scale changes with age, which could affect the observed DNA methylation pattern. Both loss of Y (LOY) and loss of X (LOX) have been associated with ageing , 55. EspA limitation of our study is that as we used data from a data repository, we had only limited phenotypic information that could be adjusted for in the analysis. Adjusting for confounding factors such as disease status or lifestyle factors could increase the sensitivity of the analysis to identify age-CpGs. We did adjust for cell-type proportions, as these can be estimated from the methylation data itself, and they have been shown to explain majority of variation in DNA methylation .Taken together, we report here ageing-associated changes in DNA methylation in both X and Y chromosomes. Our results show the similarities in age-associated DNA methylation changes in females and males and suggest that they share features with DNA methylation changes observed in the autosomes. Significant overlap in X chromosomal age-CpGs identified in males and females and their shared features suggest that despite the uneven chromosomal dosage, differences in ageing-associated DNA methylation changes in the X chromosome are unlikely to be a major contributor of sex dimorphism in ageing. Age-associated DNA methylation changes in the Y chromosome appear to differ from those observed in the autosomes, but due to technical reasons, the results should be interpreted with caution.Very little has been published regarding the age-associated DNA methylation changes in the sex chromosomes and the published results show very limited overlap. Future study is needed to pinpoint analysis methods that yield biologically the most significant results. In addition, functionality of the observed DNA methylation changes in the sex chromosomes and more broadly throughout the genome requires more study. Analysis strategies, including for example pathway and enrichment analysis, that allow the combination of results from autosomes and sex chromosomes could possibly produce the most accurate picture of these changes. In addition, and especially for the Y chromosome, DNA methylation analysis with, for example, a bisulphite-sequencing method with better coverage could help to differentiate true biological phenomenon from technical bias.Methylation data were downloaded from GEO NCBI database  using thGSE61496 consists of monozygotic twin pairs from Danish Twin Registry, who are disconcordant for birth weight. GSE73103 consists of healthy individuals recruited in Uppsala, Sweden. GSE87571 is a sample of The Northern Sweden Population Health Study in Sweden. GSE111629 is a sample of a population-based case–control study of Parkinson’s disease in California, USA. GSE128235 is comprised of depressed and control subjects who were recruited at the Max Planck Institute of Psychiatry, Germany. Detailed information can be found under accession numbers at GEO NCBI database [For each dataset, male and female samples were preprocessed separately using R software and minfi therein. Visual inspection of the data  and one male sample from both GSE61496 and GSE111629. Individual methylation level values were excluded when detection p value was more than 0.05. After preprocessing, the number of probes remaining for statistical testing across datasets was 9505–9513 for female X, 9717–9807 for male X, and 296–298 for male Y.p value < 0.05.Methylation sites associated with age in X and Y chromosomes were identified within each data set using linear multivariate modeling. Methylation level, ß-value, was set as the dependent variable and the model was adjusted for blood cell composition. The blood cell composition was estimated using function EstimateCellCounts. Both twins in GSE61496 were included and considered as singletons in the analysis. Threshold for statistical significance within each data set was Benjamini–Hochberg-adjusted Overlap between datasets in this study and other studies were visualized using webtool Venn . ChromosAdditional file 1: Y chromosomal age-CpGs. Contains lists of Y chromosomal age-CpGs identified in each individual dataset (A–D), list of age-CpGs identified in multiple datasets (E) and location of identified Y chromosomal age-CpGs in relation to CpG islands and gene regions (F).Additional file 2: X chromosomal age-CpGs identified in males. Contains lists of X chromosomal age-CpGs identified in males in each individual dataset (A–E), list of age-CpGs identified in multiple datasets (F), location of identified X chromosomal age-CpGs in relation to CpG islands and gene regions (G) and enriched GO terms for X chromosomal age-CpGs in males (H).Additional file 3: X chromosomal age-CpGs identified in females. Contains lists of X chromosomal age-CpGs identified in females in each individual dataset (A–E), list of age-CpGs identified in multiple datasets (F) and location of identified X chromosomal age-CpGs in relation to CpG islands and gene regions (G).Additional file 4: X chromosomal age-CpGs identified in both females and males. Contains list of X chromosomal age-CpGs identified in both females and males (A) and location of those age-CpGs in relation to CpG islands and gene regions (B).Additional file 5: Figures and Supplementary analysis.