The Intestine Harbors Functionally Distinct Homeostatic Tissue-Resident and Inflammatory Th17 Cells.

T helper 17 (Th17) cells are pathogenic in many inflammatory diseases, but also support the integrity of the intestinal barrier in a non-inflammatory manner. It is unclear what distinguishes inflammatory Th17 cells elicited by pathogens and tissue-resident homeostatic Th17 cells elicited by commensals. Here, we compared the characteristics of Th17 cells differentiating in response to commensal bacteria (SFB) to those differentiating in response to a pathogen (Citrobacter rodentium). Homeostatic Th17 cells exhibited little plasticity towards expression of inflammatory cytokines, were characterized by a metabolism typical of quiescent or memory T cells, and did not participate in inflammatory processes. In contrast, infection-induced Th17 cells showed extensive plasticity towards pro-inflammatory cytokines, disseminated widely into the periphery, and engaged aerobic glycolysis in addition to oxidative phosphorylation typical for inflammatory effector cells. These findings will help ensure that future therapies directed against inflammatory Th17 cells do not inadvertently damage the resident gut population.

Normalization of RNA-Seq data using adaptive trimmed mean with multi-reference.

The normalization of RNA sequencing data is a primary step for downstream analysis. The most popular method used for the normalization is the trimmed mean of M values (TMM) and DESeq. The TMM tries to trim away extreme log fold changes of the data to normalize the raw read counts based on the remaining non-deferentially expressed genes. However, the major problem with the TMM is that the values of trimming factor M are heuristic. This paper tries to estimate the adaptive value of M in TMM based on Jaeckel's Estimator, and each sample acts as a reference to find the scale factor of each sample. The presented approach is validated on SEQC, MAQC2, MAQC3, PICKRELL and two simulated datasets with two-group and three-group conditions by varying the percentage of differential expression and the number of replicates. The performance of the present approach is compared with various state-of-the-art methods, and it is better in terms of area under the receiver operating characteristic curve and differential expression.

STAT3 Decoy Oligodeoxynucleotides Suppress Liver Inflammation and Fibrosis in Liver Cancer Cells and a DDC-Induced Liver Injury Mouse Model.

Liver damage caused by various factors results in fibrosis and inflammation, leading to cirrhosis and cancer. Fibrosis results in the accumulation of extracellular matrix components. The role of STAT proteins in mediating liver inflammation and fibrosis has been well documented; however, approved therapies targeting STAT3 inhibition against liver disease are lacking. This study investigated the anti-fibrotic and anti-inflammatory effects of STAT3 decoy oligodeoxynucleotides (ODN) in hepatocytes and liver fibrosis mouse models. STAT3 decoy ODN were delivered into cells using liposomes and hydrodynamic tail vein injection into 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC)-fed mice in which liver injury was induced. STAT3 target gene expression changes were verified using qPCR and Western blotting. Liver tissue fibrosis and bile duct proliferation were assessed in animal experiments using staining techniques, and macrophage and inflammatory cytokine distribution was verified using immunohistochemistry. STAT3 decoy ODN reduced fibrosis and inflammatory factors in liver cancer cell lines and DDC-induced liver injury mouse model. These results suggest that STAT3 decoy ODN may effectively treat liver fibrosis and must be clinically investigated.

NSrp70 is a lymphocyte-essential splicing factor that controls thymocyte development.

Alternative pre-mRNA splicing is a critical step to generate multiple transcripts, thereby dramatically enlarging the proteomic diversity. Thus, a common feature of most alternative splicing factor knockout models is lethality. However, little is known about lineage-specific alternative splicing regulators in a physiological setting. Here, we report that NSrp70 is selectively expressed in developing thymocytes, highest at the double-positive (DP) stage. Global splicing and transcriptional profiling revealed that NSrp70 regulates the cell cycle and survival of thymocytes by controlling the alternative processing of various RNA splicing factors, including the oncogenic splicing factor SRSF1. A conditional-knockout of Nsrp1 (NSrp70-cKO) using CD4Cre developed severe defects in T cell maturation to single-positive thymocytes, due to insufficient T cell receptor (TCR) signaling and uncontrolled cell growth and death. Mice displayed severe peripheral lymphopenia and could not optimally control tumor growth. This study establishes a model to address the function of lymphoid-lineage-specific alternative splicing factor NSrp70 in a thymic T cell developmental pathway.

Current therapeutic targets and multifaceted physiological impacts of caffeine.

Caffeine, which shares consubstantial structural similarity with purine adenosine, has been demonstrated as a nonselective adenosine receptor antagonist for eliciting most of the biological functions at physiologically relevant dosages. Accumulating evidence supports caffeine's beneficial effects against different disorders, such as total cardiovascular diseases and type 2 diabetes. Conversely, paradoxical effects are also linked to caffeine ingestion in humans including hypertension-hypotension and tachycardia-bradycardia. These observations suggest the association of caffeine action with its ingested concentration and/or concurrent interaction with preferential molecular targets to direct explicit events in the human body. Thus, a coherent analysis of the functional targets of caffeine, relevant to normal physiology, and disease pathophysiology, is required to understand the pharmacology of caffeine. This review provides a broad overview of the experimentally validated targets of caffeine, particularly those of therapeutic interest, and the impacts of caffeine on organ-specific physiology and pathophysiology. Overall, the available empirical and epidemiological evidence supports the dose-dependent functional activities of caffeine and advocates for further studies to get insights into the caffeine-induced changes under specific conditions, such as asthma, DNA repair, and cancer, in view of its therapeutic applications.

Rifaximin-α reduces gut-derived inflammation and mucin degradation in cirrhosis and encephalopathy: RIFSYS randomised controlled trial.

BACKGROUND & AIMS: Rifaximin-α is efficacious for the prevention of recurrent hepatic encephalopathy (HE), but its mechanism of action remains unclear. We postulated that rifaximin-α reduces gut microbiota-derived endotoxemia and systemic inflammation, a known driver of HE. METHODS: In a placebo-controlled, double-blind, mechanistic study, 38 patients with cirrhosis and HE were randomised 1:1 to receive either rifaximin-α (550 mg BID) or placebo for 90 days. PRIMARY OUTCOME: 50% reduction in neutrophil oxidative burst (OB) at 30 days. SECONDARY OUTCOMES: changes in psychometric hepatic encephalopathy score (PHES) and neurocognitive functioning, shotgun metagenomic sequencing of saliva and faeces, plasma and faecal metabolic profiling, whole blood bacterial DNA quantification, neutrophil toll-like receptor (TLR)-2/4/9 expression and plasma/faecal cytokine analysis. RESULTS: Patients were well-matched: median MELD (11 rifaximin-α vs. 10 placebo). Rifaximin-α did not lead to a 50% reduction in spontaneous neutrophil OB at 30 days compared to baseline (p = 0.48). However, HE grade normalised (p = 0.014) and PHES improved (p = 0.009) after 30 days on rifaximin-α. Rifaximin-α reduced circulating neutrophil TLR-4 expression on day 30 (p = 0.021) and plasma tumour necrosis factor-α (TNF-α) (p <0.001). Rifaximin-α suppressed oralisation of the gut, reducing levels of mucin-degrading sialidase-rich species, Streptococcus spp, Veillonella atypica and parvula, Akkermansia and Hungatella. Rifaximin-α promoted a TNF-α- and interleukin-17E-enriched intestinal microenvironment, augmenting antibacterial responses to invading pathobionts and promoting gut barrier repair. Those on rifaximin-α were less likely to develop infection (odds ratio 0.21; 95% CI 0.05-0.96). CONCLUSION: Rifaximin-α led to resolution of overt and covert HE, reduced the likelihood of infection, reduced oralisation of the gut and attenuated systemic inflammation. Rifaximin-α plays a role in gut barrier repair, which could be the mechanism by which it ameliorates bacterial translocation and systemic endotoxemia in cirrhosis. CLINICAL TRIAL NUMBER: ClinicalTrials.gov NCT02019784. LAY SUMMARY: In this clinical trial, we examined the underlying mechanism of action of an antibiotic called rifaximin-α which has been shown to be an effective treatment for a complication of chronic liver disease which effects the brain (termed encephalopathy). We show that rifaximin-α suppresses gut bacteria that translocate from the mouth to the intestine and cause the intestinal wall to become leaky by breaking down the protective mucus barrier. This suppression resolves encephalopathy and reduces inflammation in the blood, preventing the development of infection.

Anti-Fibrotic Effect of Synthetic Noncoding Decoy ODNs for TFEB in an Animal Model of Chronic Kidney Disease.

Despite emerging evidence suggesting that autophagy occurs during renal interstitial fibrosis, the role of autophagy activation in fibrosis and the mechanism by which autophagy influences fibrosis remain controversial. Transcription factor EB (TFEB) is a master regulator of autophagy-related gene transcription, lysosomal biogenesis, and autophagosome formation. In this study, we examined the preventive effects of TFEB suppression on renal fibrosis. We injected synthesized TFEB decoy oligonucleotides (ODNs) into the tail veins of unilateral ureteral obstruction (UUO) mice to explore the regulation of autophagy in UUO-induced renal fibrosis. The expression of interleukin (IL)-1ß, tumor necrosis factor-α (TNF-α), and collagen was decreased by TFEB decoy ODN. Additionally, TEFB ODN administration inhibited the expression of microtubule-associated protein light chain 3 (LC3), Beclin1, and hypoxia-inducible factor-1α (HIF-1α). We confirmed that TFEB decoy ODN inhibited fibrosis and autophagy in a UUO mouse model. The TFEB decoy ODNs also showed anti-inflammatory effects. Collectively, these results suggest that TFEB may be involved in the regulation of autophagy and fibrosis and that regulating TFEB activity may be a promising therapeutic strategy against kidney diseases.

A Genome-Wide Screen Reveals That Endocytic Genes Are Important for Pma1p Asymmetry during Cell Division in Saccharomyces cerevisiae.

An asymmetry in cytosolic pH between mother and daughter cells was reported to underlie cellular aging in the budding yeast Saccharomyces cerevisiae; however, the underlying mechanism remains unknown. Preferential accumulation of Pma1p, which pumps cytoplasmic protons out of cells, at the plasma membrane of mother cells, but not of their newly-formed daughter cells, is believed to be responsible for the pH increase in mother cells by reducing the level of cytoplasmic protons. This, in turn, decreases the acidity of vacuoles, which is well correlated with aging of yeast cells. In this study, to identify genes that regulate the preferential accumulation of Pma1p in mother cells, we performed a genome-wide screen using a collection of single gene deletion yeast strains. A subset of genes involved in the endocytic pathway, such as VPS8, VPS9, and VPS21, was important for Pma1p accumulation. Unexpectedly, however, there was little correlation between deletion of each of these genes and the replicative lifespan of yeast, suggesting that Pma1p accumulation in mother cells is not the key determinant that underlies aging of mother cells.

Bee Venom and Its Major Component Melittin Attenuated Cutibacterium acnes- and IGF-1-Induced Acne Vulgaris via Inactivation of Akt/mTOR/SREBP Signaling Pathway.

Acne vulgaris is the most common disease of the pilosebaceous unit. The pathogenesis of this disease is complex, involving increased sebum production and perifollicular inflammation. Understanding the factors that regulate sebum production is important in identifying novel therapeutic targets for the treatment of acne. Bee Venom (BV) and melittin have multiple effects including antibacterial, antiviral, and anti-inflammatory activities in various cell types. However, the anti-lipogenic mechanisms of BV and melittin have not been elucidated. We investigated the effects of BV and melittin in models of Insulin-like growth factor-1 (IGF-1) or Cutibacterium acnes (C. acnes)-induced lipogenic skin disease. C. acnes or IGF-1 increased the expression of sterol regulatory element-binding protein-1 (SREBP-1) and proliferator-activated receptor gamma (PPAR-γ), transcription factors that regulate numerous genes involved in lipid biosynthesis through the protein kinase B (Akt)/mammalian target of rapamycin (mTOR)/SREBP signaling pathway. In this study using a C. acnes or IGF-1 stimulated lipogenic disease model, BV and melittin inhibited the increased expression of lipogenic and pro-inflammatory factor through the blockade of the Akt/mTOR/SREBP signaling pathway. This study suggests for the first time that BV and melittin could be developed as potential natural anti-acne agents with anti-lipogenesis, anti-inflammatory, and anti-C. acnes activity.

Synthetic Non-Coding RNA for Suppressing mTOR Translation to Prevent Renal Fibrosis Related to Autophagy in UUO Mouse Model.

The global burden of chronic kidney disease is increasing, and the majority of these diseases are progressive. Special site-targeted drugs are emerging as alternatives to traditional drugs. Oligonucleotides (ODNs) have been proposed as effective therapeutic tools in specific molecular target therapies for several diseases. We designed ring-type non-coding RNAs (ncRNAs), also called mTOR ODNs to suppress mammalian target rapamycin (mTOR) translation. mTOR signaling is associated with excessive cell proliferation and fibrogenesis. In this study, we examined the effects of mTOR suppression on chronic renal injury. To explore the regulation of fibrosis and inflammation in unilateral ureteral obstruction (UUO)-induced injury, we injected synthesized ODNs via the tail vein of mice. The expression of inflammatory-related markers (interleukin-1ß, tumor necrosis factor-α), and that of fibrosis (α-smooth muscle actin, fibronectin), was decreased by synthetic ODNs. Additionally, ODN administration inhibited the expression of autophagy-related markers, microtubule-associated protein light chain 3, Beclin1, and autophagy-related gene 5-12. We confirmed that ring-type ODNs inhibited fibrosis, inflammation, and autophagy in a UUO mouse model. These results suggest that mTOR may be involved in the regulation of autophagy and fibrosis and that regulating mTOR signaling may be a therapeutic strategy against chronic renal injury.

Anti-Fibrotic Effect of Synthetic Noncoding Oligodeoxynucleotide for Inhibiting mTOR and STAT3 via the Regulation of Autophagy in an Animal Model of Renal Injury.

Renal fibrosis is a common process of various kidney diseases. Autophagy is an important cell biology process to maintain cellular homeostasis. In addition, autophagy is involved in the pathogenesis of various renal disease, including acute kidney injury, glomerular diseases, and renal fibrosis. However, the functional role of autophagy in renal fibrosis remains poorly unclear. The mammalian target of rapamycin (mTOR) plays a negative regulatory role in autophagy. Signal transducer and activator of transcription 3 (STAT3) is an important intracellular signaling that may regulate a variety of inflammatory responses. In addition, STAT3 regulates autophagy in various cell types. Thus, we synthesized the mTOR/STAT3 oligodeoxynucleotide (ODN) to regulate the autophagy. The aim of this study was to investigate the beneficial effect of mTOR/STAT3 ODN via the regulation of autophagy appearance on unilateral ureteral obstruction (UUO)-induced renal fibrosis. This study showed that UUO induced inflammation, tubular atrophy, and tubular interstitial fibrosis. However, mTOR/STAT3 ODN suppressed UUO-induced renal fibrosis and inflammation. The autophagy markers have no statistically significant relation, whereas mTOR/STAT3 ODN suppressed the apoptosis in tubular cells. These results suggest the possibility of mTOR/STAT3 ODN for preventing renal fibrosis. However, the role of mTOR/STAT3 ODN on autophagy regulation needs to be further investigated.

Precise Characterization of Genetic Interactions in Cancer via Molecular Network Refining Processes.

Genetic interactions (GIs), such as the synthetic lethal interaction, are promising therapeutic targets in precision medicine. However, despite extensive efforts to characterize GIs by large-scale perturbation screening, considerable false positives have been reported in multiple studies. We propose a new computational approach for improved precision in GI identification by applying constraints that consider actual biological phenomena. In this study, GIs were characterized by assessing mutation, loss of function, and expression profiles in the DEPMAP database. The expression profiles were used to exclude loss-of-function data for nonexpressed genes in GI characterization. More importantly, the characterized GIs were refined based on Kyoto Encyclopedia of Genes and Genomes (KEGG) or protein-protein interaction (PPI) networks, under the assumption that genes genetically interacting with a certain mutated gene are adjacent in the networks. As a result, the initial GIs characterized with CRISPR and RNAi screenings were refined to 65 and 23 GIs based on KEGG networks and to 183 and 142 GIs based on PPI networks. The evaluation of refined GIs showed improved precision with respect to known synthetic lethal interactions. The refining process also yielded a synthetic partner network (SPN) for each mutated gene, which provides insight into therapeutic strategies for the mutated genes; specifically, exploring the SPN of mutated BRAF revealed ELAVL1 as a potential target for treating BRAF-mutated cancer, as validated by previous research. We expect that this work will advance cancer therapeutic research.

A Systems Biology Approach to Investigating the Interaction between Serotonin Synthesis by Tryptophan Hydroxylase and the Metabolic Homeostasis.

Obesity has become a global public health and economic problem. Obesity is a major risk factor for a number of complications, such as type 2 diabetes, cardiovascular disease, fatty liver disease, and cancer. Serotonin (5-hydroxytryptamine [5-HT]) is a biogenic monoamine that plays various roles in metabolic homeostasis. It is well known that central 5-HT regulates appetite and mood. Several 5-HT receptor agonists and selective serotonin receptor uptake inhibitors (SSRIs) have shown beneficial effects on appetite and mood control in clinics. Although several genetic polymorphisms related to 5-HT synthesis and its receptors are strongly associated with obesity, there is little evidence of the role of peripheral 5-HT in human metabolism. In this study, we performed a systemic analysis of transcriptome data from the Genotype-Tissue Expression (GTEX) database. We investigated the expression of 5-HT and tryptophan hydroxylase (TPH), the rate-limiting enzyme of 5-HT biosynthesis, in the human brain and peripheral tissues. We also performed differential gene expression analysis and predicted changes in metabolites by comparing gene expressions of tissues with high TPH expression to the gene expressions of tissues with low TPH expression. Our analyses provide strong evidence that serotonin plays an important role in the regulation of metabolic homeostasis in humans.

Ameliorating Fibrotic Phenotypes of Keloid Dermal Fibroblasts through an Epidermal Growth Factor-Mediated Extracellular Matrix Remodeling.

Keloid and hypertrophic scars are skin fibrosis-associated disorders that exhibit an uncontrollable proliferation of fibroblasts and their subsequent contribution to the excessive accumulation of extracellular matrix (ECM) in the dermis. In this study, to elucidate the underlying mechanisms, we investigated the pivotal roles of epidermal growth factor (EGF) in modulating fibrotic phenotypes of keloid and hypertrophic dermal fibroblasts. Our initial findings revealed the molecular signatures of keloid dermal fibroblasts and showed the highest degree of skin fibrosis markers, ECM remodeling, anabolic collagen-cross-linking enzymes, such as lysyl oxidase (LOX) and four LOX-like family enzymes, migration ability, and cell-matrix traction force, at cell-matrix interfaces. Furthermore, we observed significant EGF-mediated downregulation of anabolic collagen-cross-linking enzymes, resulting in amelioration of fibrotic phenotypes and a decrease in cell motility measured according to the cell-matrix traction force. These findings offer insight into the important roles of EGF-mediated cell-matrix interactions at the cell-matrix interface, as well as ECM remodeling. Furthermore, the results suggest their contribution to the reduction of fibrotic phenotypes in keloid dermal fibroblasts, which could lead to the development of therapeutic modalities to prevent or reduce scar tissue formation.

TCSBN: a database of tissue and cancer specific biological networks.

Biological networks provide new opportunities for understanding the cellular biology in both health and disease states. We generated tissue specific integrated networks (INs) for liver, muscle and adipose tissues by integrating metabolic, regulatory and protein-protein interaction networks. We also generated human co-expression networks (CNs) for 46 normal tissues and 17 cancers to explore the functional relationships between genes as well as their relationships with biological functions, and investigate the overlap between functional and physical interactions provided by CNs and INs, respectively. These networks can be employed in the analysis of omics data, provide detailed insight into disease mechanisms by identifying the key biological components and eventually can be used in the development of efficient treatment strategies. Moreover, comparative analysis of the networks may allow for the identification of tissue-specific targets that can be used in the development of drugs with the minimum toxic effect to other human tissues. These context-specific INs and CNs are presented in an interactive website http://inetmodels.com without any limitation.

LncRNAs Act as a Link between Chronic Liver Disease and Hepatocellular Carcinoma.

Long non-coding RNAs (lncRNAs) are emerging as important contributors to the biological processes underlying the pathophysiology of various human diseases, including hepatocellular carcinoma (HCC). However, the involvement of these molecules in chronic liver diseases, such as nonalcoholic fatty liver disease (NAFLD) and viral hepatitis, has only recently been considered in scientific research. While extensive studies on the pathogenesis of the development of HCC from hepatic fibrosis have been conducted, their regulatory molecular mechanisms are still only partially understood. The underlying mechanisms related to lncRNAs leading to HCC from chronic liver diseases and cirrhosis have not yet been entirely elucidated. Therefore, elucidating the functional roles of lncRNAs in chronic liver disease and HCC can contribute to a better understanding of the molecular mechanisms, and may help in developing novel diagnostic biomarkers and therapeutic targets for HCC, as well as in preventing the progression of chronic liver disease to HCC. Here, we comprehensively review and briefly summarize some lncRNAs that participate in both hepatic fibrosis and HCC.

Pyruvate kinase L/R is a regulator of lipid metabolism and mitochondrial function.

The pathogenesis of non-alcoholic fatty liver disease (NAFLD) and hepatocellular carcinoma (HCC) has been associated with altered expression of liver-specific genes including pyruvate kinase liver and red blood cell (PKLR), patatin-like phospholipase domain containing 3 (PNPLA3) and proprotein convertase subtilisin/kexin type 9 (PCSK9). Here, we inhibited and overexpressed the expression of these three genes in HepG2 cells, generated RNA-seq data before and after perturbation and revealed the altered global biological functions with the modulation of these genes using integrated network (IN) analysis. We found that modulation of these genes effects the total triglycerides levels within the cells and viability of the cells. Next, we generated IN for HepG2 cells, identified reporter transcription factors based on IN and found that the modulation of these genes affects key metabolic pathways associated with lipid metabolism (steroid biosynthesis, PPAR signalling pathway, fatty acid synthesis and oxidation) and cancer development (DNA replication, cell cycle and p53 signalling) involved in the progression of NAFLD and HCC. Finally, we observed that inhibition of PKLR lead to decreased glucose uptake and decreased mitochondrial activity in HepG2 cells. Hence, our systems level analysis indicated that PKLR can be targeted for development efficient treatment strategy for NAFLD and HCC.

Network analyses identify liver-specific targets for treating liver diseases.

We performed integrative network analyses to identify targets that can be used for effectively treating liver diseases with minimal side effects. We first generated co-expression networks (CNs) for 46 human tissues and liver cancer to explore the functional relationships between genes and examined the overlap between functional and physical interactions. Since increased de novo lipogenesis is a characteristic of nonalcoholic fatty liver disease (NAFLD) and hepatocellular carcinoma (HCC), we investigated the liver-specific genes co-expressed with fatty acid synthase (FASN). CN analyses predicted that inhibition of these liver-specific genes decreases FASN expression. Experiments in human cancer cell lines, mouse liver samples, and primary human hepatocytes validated our predictions by demonstrating functional relationships between these liver genes, and showing that their inhibition decreases cell growth and liver fat content. In conclusion, we identified liver-specific genes linked to NAFLD pathogenesis, such as pyruvate kinase liver and red blood cell (PKLR), or to HCC pathogenesis, such as PKLR, patatin-like phospholipase domain containing 3 (PNPLA3), and proprotein convertase subtilisin/kexin type 9 (PCSK9), all of which are potential targets for drug development.

Dysregulated signaling hubs of liver lipid metabolism reveal hepatocellular carcinoma pathogenesis.

Hepatocellular carcinoma (HCC) has a high mortality rate and early detection of HCC is crucial for the application of effective treatment strategies. HCC is typically caused by either viral hepatitis infection or by fatty liver disease. To diagnose and treat HCC it is necessary to elucidate the underlying molecular mechanisms. As a major cause for development of HCC is fatty liver disease, we here investigated anomalies in regulation of lipid metabolism in the liver. We applied a tailored network-based approach to identify signaling hubs associated with regulation of this part of metabolism. Using transcriptomics data of HCC patients, we identified significant dysregulated expressions of lipid-regulated genes, across many different lipid metabolic pathways. Our findings, however, show that viral hepatitis causes HCC by a distinct mechanism, less likely involving lipid anomalies. Based on our analysis we suggest signaling hub genes governing overall catabolic or anabolic pathways, as novel drug targets for treatment of HCC that involves lipid anomalies.

Predicting unintended effects of drugs based on off-target tissue effects.

Unintended effects of drugs can be caused by various mechanisms. Conventional analysis of unintended effects has focused on the target proteins of drugs. However, an interaction with off-target tissues of a drug might be one of the unintended effect-related mechanisms. We propose two processes to predict a drug's unintended effects by off-target tissue effects: 1) identification of a drug's off-target tissue and; 2) tissue protein - symptom relation identification (tissue protein - symptom matrix). Using this method, we predicted that 1,177 (10.7%) side-effects were related to off-target tissue effects in 11,041 known side-effects. Off-target tissues and unintended effects of successful repositioning drugs were also predicted. The effectiveness of relations of the proposed tissue protein - symptom matrix were evaluated by using the literature mining method. We predicted unintended effects of drugs as well as those effect-related off-target tissues. By using our prediction, we are able to reduce drug side-effects on off-target tissues and provide a chance to identify new indications of drugs of interest.