VSTM2L is a promising therapeutic target and a prognostic soluble-biomarker in cholangiocarcinoma.

The aim of the present study is to provide a rational background for silencing the V-set and transmembrane domain containing 2 like (VSTM2L) in consort with recognising soluble VSTM2L against cholangiocarcinoma. A therapeutic target against cholangiocarcinoma was selected using iterative patient partitioning (IPP) calculation, and it was verified by in vitro and in silico analyses. VSTM2L was selected as a potential therapeutic target against cholangiocarcinoma. Silencing the VSTM2L expression significantly attenuated the viability and survival of cholangiocarcinoma cells through blockade of the intracellular signalling pathway. In silico analysis showed that VSTM2L affected the positive regulation of cell growth in cholangiocarcinoma. Liptak's z value revealed that the expression of VSTM2L worsened the prognosis of cholangiocarcinoma patients. In addition, soluble VSTM2L was significantly detected in the whole blood of cholangiocarcinoma patients compared with that of healthy donors. Our report reveals that VSTM2L might be the potential therapeutic target and a soluble prognostic biomarker against cholangiocarcinoma. [BMB Reports 2024; 57(7): 324-329].

Fibronectin Type III Domain Containing 3B as a Potential Prognostic and Therapeutic Biomarker for Glioblastoma.

Glioblastoma (GBM) is a representative malignant brain tumor characterized by a dismal prognosis, with survival rates of less than 2 years and high recurrence rates. Despite surgical resection and several alternative treatments, GBM remains a refractory disease due to its aggressive invasiveness and resistance to anticancer therapy. In this report, we explore the role of fibronectin type III domain containing 3B (FNDC3B) and its potential as a prognostic and therapeutic biomarker in GBM. GBM exhibited a significantly higher cancer-to-normal ratio compared to other organs, and patients with high FNDC3B expression had a poor prognosis (p < 0.01). In vitro studies revealed that silencing FNDC3B significantly reduced the expression of Survivin, an apoptosis inhibitor, and also reduced cell migration, invasion, extracellular matrix adhesion ability, and stem cell properties in GBM cells. Furthermore, we identified that FNDC3B regulates PTEN/PI3K/Akt signaling in GBM cells using MetaCore integrated pathway bioinformatics analysis and a proteome profiler phospho-kinase array with sequential western blot analysis. Collectively, our findings suggest FNDC3B as a potential biomarker for predicting GBM patient survival and for the development of treatment strategies for GBM.

Discovering the anti-cancer phytochemical rutin against breast cancer through the methodical platform based on traditional medicinal knowledge.

A number of therapeutic drugs have been developed from functional chemicals found in plants. Knowledge of plants used for medicinal purposes has historically been transmitted by word of mouth or through literature. The aim of the present study is to provide a systemic platform for the development of lead compounds against breast cancer based on a traditional medical text. To verify our systematic approach, integrating processes consisted of text mining of traditional medical texts, 3-D virtual docking screening, and in vitro and in vivo experimental validations were demonstrated. Our text analysis system identified rutin as a specific phytochemical traditionally used for cancer treatment. 3-D virtual screening predicted that rutin could block EGFR signaling. Thus, we validated significant anticancer effects of rutin against breast cancer cells through blockade of EGFR signaling pathway in vitro. We also demonstrated in vivo anti-cancer effects of rutin using the breast cancer recurrence in vivo models. In summary, our innovative approach might be proper for discovering new phytochemical lead compounds designing for blockade of malignant neoplasm including breast cancer. [BMB Reports 2023; 56(11): 594-599].

Differential dependency of human glioblastoma cells on vascular endothelial growth factor­A signaling via neuropilin­1.

Despite the high expression of neuropilin­1 (NRP­1) in human glioblastoma (GB), the understanding of its function as a co­receptor of vascular endothelial growth factor receptors (VEGFRs) in angiogenesis is currently limited. Therefore, the aim of the present study was to elucidate the non­classical function of NRP­1 expression in human GB. Expression patterns of NRP­1 and VEGF­A were determined by sandwich ELISA, western blot analysis, or immunohistochemistry. Differential dependency of GB cells following ablation of VEGF­A signaling was validated in vitro and in vivo. Cellular mechanism responsible for distinct response to VEGF­A signaling was evaluated by western blotting and immunoprecipitation analysis. Prognostic implications were assessed using IHC analysis. GB cells exhibited differing sensitivity to silencing of vascular endothelial growth factor (VEGF)­A signaling, which resulted in a distinct expression pattern of wild­type or chondroitin­sulfated NRP­1. VEGF­A­sensitive GB exhibited the physical interaction between wild­type NRP­1 and FMS related receptor tyrosine kinase 1 (Flt­1) whereas VEGF­A­resistant GB exhibited chondroitin­sulfated NRP­1 without interaction with Flt­1. Eliminating the chondroitin sulfate modification in NRP­1 led to re­sensitization to VEGF­A signaling, and chondroitin sulfate modification was found to be associated with an adverse prognosis in patients with GB. The present study identified the distinct functions of NRP­1 in VEGF­A signaling in accordance with its unique expression type and interaction with Flt­1. The present research is expected to provide a strong basis for targeting VEGF­A signaling in patients with GB, with variable responses.

Soluble TGFBI aggravates the malignancy of cholangiocarcinoma through activation of the ITGB1 dependent PPARγ signalling pathway.

BACKGROUND: Cholangiocarcinoma is a devastating cancer with a poor prognosis. Previous reports have presented conflicting results on the role of transforming growth factor-ß-induced protein (TGFBI) in malignant cancers. Currently, our understanding of the role of TGFBI in cholangiocarcinoma is ambiguous. The aim of the present study was to investigate the role of TGFBI in human cholangiocarcinoma. METHODS: Iterative patient partitioning (IPP) scoring and consecutive elimination methods were used to select prognostic biomarkers. mRNA and protein expression levels were determined using Gene Expression Omnibus (GEO), Western blot and ELISA analyses. Biological activities of selected biomarkers were examined using both in vitro and in vivo assays. Prognostic values were assessed using Kaplan-Meier and Liptak's z score analyses. RESULTS: TGFBI was selected as a candidate cholangiocarcinoma biomarker. GEO database analysis revealed significantly higher TGFBI mRNA expression levels in cholangiocarcinoma tissues compared to matched normal tissues. TGFBI protein was specifically detected in a soluble form in vitro and in vivo. TGFBI silencing evoked significant anti-cancer effects in vitro. Soluble TGFBI treatment aggravated the malignancy of cholangiocarcinoma cells both in vitro and in vivo through activation of the integrin beta-1 (ITGB1) dependent PPARγ signalling pathway. High TGFBI expression was associated with a poor prognosis in patients with cholangiocarcinoma. CONCLUSIONS: Our data suggest that TGFBI may serve as a promising prognostic biomarker and therapeutic target for cholangiocarcinoma.

Diagnostic performance of automated, streamlined, daily updated exome analysis in patients with neurodevelopmental delay.

BACKGROUND: The diagnostic yield of whole-exome sequencing (WES) varies from 30%-50% among patients with mild to severe neurodevelopmental delay (NDD)/intellectual disability (ID). Routine retrospective reanalysis of undiagnosed patients has increased the total diagnostic yield by 10-15%. Here, we performed proband-only WES of 1065 patients with NDD/ID and applied a prospective, daily reanalysis automated pipeline to patients without clinically significant variants to facilitate diagnoses. METHODS: The study included 1065 consecutive patients from 1056 nonconsanguineous unrelated families from 10 multimedical centers in South Korea between April 2018 and August 2021. WES data were analyzed daily using automatically updated databases with variant classification and symptom similarity scoring systems. RESULTS: At the initial analysis, 402 patients from 1056 unrelated families (38.0%, 402/1,056 families) had a positive genetic diagnosis. Daily prospective, automated reanalysis resulted in the identification of 34 additional diagnostic variants in 31 patients (3%), which increased our molecular diagnostic yield to 41% (433/1056 families). Among these 31 patients, 26 were diagnosed with 23 different diseases that were newly discovered after 2019. The time interval between the first analysis and the molecular diagnosis by reanalysis was 1.2 ± 0.9 years, which was shorter in the patients enrolled during the latter part of the study period. CONCLUSION: Daily updated databases and reanalysis systems enhance the diagnostic performance in patients with NDD/ID, contributing to the rapid diagnosis of undiagnosed patients by applying the latest molecular genetic information.

An in vitro evaluation of luffa cylindrica stem sap in preadipocytes and dermal fibroblasts.

BACKGROUND: Luffa cylindrica stem sap (LuCS) has been ethnopharmacologically used as a cosmetic ingredients to improve the facial condition in Asians, but there is no scientific proof about the advantages of LuCS as a supplement for skin elasticity inducer. PURPOSE: Presently, we have validated the beneficial effect of LuCS in human preadipocyte and fibroblast. METHODS: In vitro activities of LuCS on expression of cellular elastin and collagen type I were validated using Western blot analysis in human fibroblasts. Effect of LuCS on preadipocyte development was performed using MDI medium containing isobutyl-methylxanthine, dexamethasone, and insulin and then evaluated using oil red O staining. RESULTS: Treatment of LuCS stimulated the expression of cellular elastin and type I procollagen in human skin fibroblasts. Exposure to LuCS induced lipid accumulation of preadipocytes via activation of CEBP/α signaling pathway in preadipocytes. Expression of collagen I, elastin, or CEBP/α mRNA was decreased by age. 3-bromo-3-methylisoxazol-5-amine enhanced the synthesis of cellular lipid in preadipocytes. CONCLUSIONS: Collectively, these results suggest the rationale of LuCS treatment in enhancing the skin condition.

Extra-domain B of fibronectin as an alternative target for drug delivery and a cancer diagnostic and prognostic biomarker for malignant glioma.

Extra-domain B of fibronectin (EDB-FN) is an alternatively spliced form of fibronectin with high expression in the extracellular matrix of neovascularized tissues and malignant cancer cells. In this study, we evaluated the practicality of using EDB-FN as a biomarker and therapeutic target for malignant gliomas (MGs), representative intractable diseases involving brain tumors. Methods: The microarray- and sequence-based patient transcriptomic database 'Oncopression' and tissue microarray of MG patient tissue samples were analyzed. EDB-FN data were extracted and evaluated from 23,344 patient samples of 17 types of cancer to assess its effectiveness and selectivity as a molecular target. To strengthen the results of the patient data analysis, the utility of EDB-FN as a molecular marker and target for MG was verified using active EDB-FN-targeting ultrasmall lipidic micellar nanoparticles (~12 nm), which had a high drug-loading capacity and were efficiently internalized by MG cells in vitro and in vivo. Results: Brain tumors had a 1.42-fold cancer-to-normal ratio (p < 0.0001), the second highest among 17 cancers after head and neck cancer. Patient tissue microarray analysis showed that the EDB-FN high-expression group had a 5.5-fold higher risk of progression than the EDB-FN low-expression group (p < 0.03). By labeling docetaxel-containing ultrasmall micelles with a bipodal aptide targeting EDB-FN (termed APTEDB-DSPE-DTX), we generated micelles that could specifically bind to MG cells, leading to superior antitumor efficacy of EDB-FN-targeting nanoparticles compared to nontargeting controls. Conclusions: Taken together, these results show that EDB-FN can be an effective drug delivery target and biomarker for MG.

Diagnostic yield and clinical utility of whole exome sequencing using an automated variant prioritization system, EVIDENCE.

EVIDENCE, an automated variant prioritization system, has been developed to facilitate whole exome sequencing analyses. This study investigated the diagnostic yield of EVIDENCE in patients with suspected genetic disorders. DNA from 330 probands (age range, 0-68 years) with suspected genetic disorders were subjected to whole exome sequencing. Candidate variants were identified by EVIDENCE and confirmed by testing family members and/or clinical reassessments. EVIDENCE reported a total 228 variants in 200 (60.6%) of the 330 probands. The average number of organs involved per patient was 4.5 ± 5.0. After clinical reassessment and/or family member testing, 167 variants were identified in 141 probands (42.7%), including 105 novel variants. These variants were confirmed as being responsible for 121 genetic disorders. A total of 103 (61.7%) of the 167 variants in 95 patients were classified as pathogenic or probably to be pathogenic before, and 161 (96.4%) variants in 137 patients (41.5%) after, clinical assessment and/or family member testing. Factor associated with a variant being regarded as causative includes similar symptom scores of a gene variant to the phenotype of the patient. This new, automated variant interpretation system facilitated the diagnosis of various genetic diseases with a 42.7% diagnostic yield.

Association of Jagged1 expression with malignancy and prognosis in human pancreatic cancer.

PURPOSE: Pancreatic cancer is one of the most aggressive cancers. Preclinical and clinical data indicate that Notch 1 ligand jagged1 (JAG1) plays a pro-oncogenic role in several malignant cancers. As yet, however, the role of JAG1 in pancreatic cancer is poorly understood. The objective of the present study was to investigate JAG1 as a therapeutic target in human pancreatic cancer. METHODS: Expression levels of Notch signaling molecules were assessed using GEO datasets and Western blot analysis, respectively. Anti-tumor effects following JAG1 silencing were evaluated using in vitro and in vivo assays. Prognostic implications were assessed using GEO datasets. RESULTS: Using GEO datasets and Western blot analysis we detected significantly higher JAG1 mRNA and protein expression levels in pancreatic cancer compared to normal pancreatic tissues. JAG1 silencing significantly restrained the growth, migration and invasion of pancreatic cancer cells through the induction of apoptosis and blockade of various kinases independent of the Notch1 pathway. Combined JAG1 silencing and gemcitabine treatment showed synergistic anti-viability effects in human pancreatic cancer cells. JAG1 silencing also resulted in significant anti-cancer effects in vivo and high JAG1 expression was found to be associated with an adverse prognosis in pancreatic cancer patients. CONCLUSIONS: From our data we conclude that JAG1 may be a promising therapeutic target in pancreatic cancer.

Calcium Channels as Novel Therapeutic Targets for Ovarian Cancer Stem Cells.

Drug resistance in epithelial ovarian cancer (EOC) is reportedly attributed to the existence of cancer stem cells (CSC), because in most cancers, CSCs still remain after chemotherapy. To overcome this limitation, novel therapeutic strategies are required to prevent cancer recurrence and chemotherapy-resistant cancers by targeting cancer stem cells (CSCs). We screened an FDA-approved compound library and found four voltage-gated calcium channel blockers (manidipine, lacidipine, benidipine, and lomerizine) that target ovarian CSCs. Four calcium channel blockers (CCBs) decreased sphere formation, viability, and proliferation, and induced apoptosis in ovarian CSCs. CCBs destroyed stemness and inhibited the AKT and ERK signaling pathway in ovarian CSCs. Among calcium channel subunit genes, three L- and T-type calcium channel genes were overexpressed in ovarian CSCs, and downregulation of calcium channel genes reduced the stem-cell-like properties of ovarian CSCs. Expressions of these three genes are negatively correlated with the survival rate of patient groups. In combination therapy with cisplatin, synergistic effect was shown in inhibiting the viability and proliferation of ovarian CSCs. Moreover, combinatorial usage of manidipine and paclitaxel showed enhanced effect in ovarian CSCs xenograft mouse models. Our results suggested that four CCBs may be potential therapeutic drugs for preventing ovarian cancer recurrence.

Silencing Delta-like 1 Expression Induces Migratory Features in Pancreatic Cancer Cells Through Stimulation of Src and p38 Signalling Pathway.

BACKGROUND/AIM: The prognosis of pancreatic cancer has not improved due to its migratory feature and refractory potential to chemo-resistance with absence of effective diagnosis. Despite continuous efforts, its underlying mechanisms of malignant nature remain ambiguous. The objective of this study was to investigate delta-like 1 (DLL1) as a tumor suppressor in the metastasic ability of human pancreatic cancer cells. MATERIALS AND METHODS: Cellular expression of DLL1 was demonstrated using the GEO public database and western blot analysis. The biological function of DLL1 was validated by biological behavior analysis. Prognosis to DLL1 expression was demonstrated using analysis of the GEO public database. RESULTS: Analysis using the GEO database and western blotting showed higher DLL1 mRNA and protein expression levels in pancreatic cancer compared to those in normal pancreas. DLL1 was uniquely expressed in seven human pancreatic cancer cell lines compared to human pancreatic duct epithelial H6c7 cells. Ablation of DLL1 expression stimulated migration and invasion by activating Src and p38 phosphorylation, but not viability and chemo-resistance of human pancreatic cancer cells. In addition, expression of DLL1 was correlated with migratory features of pancreatic cancer in vivo. Moreover, high DLL1 expression was associated with a favorable prognosis in pancreatic cancer patients. CONCLUSION: DLL1 is a potent suppressor of pancreatic cancer metastasis. Understanding correlation between expression and function of DLL1 might contribute to our knowledge of the complicated mechanism of pancreatic cancer metastasis.

Differential Dependency of Human Pancreatic Cancer Cells on Targeting PTEN via PLK 1 Expression.

Even though the tumour suppressive role of PTEN is well-known, its prognostic implications are ambiguous. The objective of this study was to further explore the function of PTEN expression in human pancreatic cancer. The expression of PTEN has been dominant in various human cancers including pancreatic cancer when compared with their matched normal tissues. The pancreatic cancer cells have been divided into PTEN blockade-susceptible and PTEN blockade-impassible groups dependent on targeting PTEN by altering intracellular signaling. The expression of PTEN has led to varying clinical outcomes of pancreatic cancer based on GEO Series (GSE) data analysis and Liptak's z analysis. Differential dependency to PTEN blockade has been ascertained based on the expression of polo-like kinase1 PLK1 in pancreatic cancer cells. The prognostic value of PTEN also depends on PLK1 expression in pancreatic cancer. Collectively, the present study provides a rationale for targeting PTEN as a promising therapeutic strategy dependent on PLK1 expressions using a companion biomarker discovery platform.

Identification of Matrix Metalloproteinase 11 as a Prognostic Biomarker in Pancreatic Cancer.

BACKGROUND/AIM: The aim of this study was to investigate matrix metalloproteinase 11 (MMP11) as a promising biomarker in human pancreatic cancer. MATERIALS AND METHODS: A consecutive eliminating method was used to select biomarker candidates in pancreatic cancer. mRNA and protein expression levels of candidates were determined in tissues and whole blood samples of healthy donors and pancreatic cancer patients. The prognostic value of MMP11 was determined using various data-sets and Liptak's Z analysis. RESULTS: Analysis using Gene Expression Omnibus (GEO) database showed significantly higher MMP11 mRNA expression in pancreatic cancer tissues compared to that in various normal tissues. MMP11 protein was specifically expressed in pancreatic cancer tissues, but not in various normal or other cancer tissues. Secreted MMP11 levels could be measured using easily accessible techniques and whole blood samples of pancreatic cancer. In addition, high levels of MMP11 were associated with poor prognosis of pancreatic cancer patients. CONCLUSION: MMP11 may be a promising prognostic biomarker for pancreatic cancer patients.

Scattered DUSP28 is a novel biomarker responsible for aggravating malignancy via the autocrine and paracrine signaling in metastatic pancreatic cancer.

Pancreatic cancer remains one of the most dangerous cancers with a grave prognosis. We have reported that dual specificity phosphatise 28 (DUSP28) could be secreted in pancreatic cancer cells. However, its biological function is poorly understood. Here, we distinguish the function of scattered DUSP28 in human pancreatic cancer. DUSP28 was specifically secreted to cultured medium in metastatic pancreatic cancer cells. Treatment with recombinant DUSP28 significantly increased the migration, invasion, and viability of metastatic pancreatic cancer cells through the activation of CREB, AKT, and ERK1/2 signaling pathways. In addition, administration of recombinant DUSP28 elicited pro-angiogenic effects in human umbilical vein endothelial cells. Injection of recombinant DUSP28 also produced tumor growth in vivo. Of interest, DUSP28 formed an autocrine loop with integrin α1 (ITGα1) by transcriptional regulation and recombinant DUSP28 acted as an oncogenic reagent through the interaction with ITGα1. Notably, scattered DUSP28 could be detected in whole blood samples of pancreatic cancer patients by accessible immunoassay. These results provide the basis for DUSP28 as a promising therapeutic target and a biomarker for metastatic pancreatic cancer.

Blockade of integrin α3 attenuates human pancreatic cancer via inhibition of EGFR signalling.

The prognosis of pancreatic cancer remains dismal despite continuous and considerable efforts. Integrins (ITGs) are highly expressed in various malignant cancers. However, very few studies investigated the role of integrin α3 (ITGα3) in malignant cancers. Here, we determined the functional role of ITGα3 in pancreatic cancer. Analysis of public microarray databases and Western blot analysis indicated a unique expression of ITGα3 in human pancreatic cancer. Silencing ITGα3 expression significantly inhibited the viability and migration of human pancreatic cancer cells. Notably, ablation of ITGα3 expression resulted in a significant decrease of epidermal growth factor receptor (EGFR) expression compared with transfection of control-siRNA through an increased number of leucine-rich repeats and immunoglobulin-like domain protein 1 (LRIG1) expression. In addition, ablating ITGα3 inhibited tumour growth via blockade of EGFR signalling in vivo. Furthermore, the highly expressed ITGα3 led to a poor prognosis of pancreatic cancer patients. Our results provide novel insights into ITGα3-induced aggressive pancreatic cancer.

Oleanolic Acids Inhibit Vascular Endothelial Growth Factor Receptor 2 Signaling in Endothelial Cells: Implication for Anti-Angiogenic Therapy.

Angiogenesis must be precisely controlled because uncontrolled angiogenesis is involved in aggravation of disease symptoms. Vascular endothelial growth factor (VEGF)/VEGF receptor 2 (VEGFR-2) signaling is a key pathway leading to angiogenic responses in vascular endothelial cells (ECs). Therefore, targeting VEGF/VEGFR-2 signaling may be effective at modulating angiogenesis to alleviate various disease symptoms. Oleanolic acid was verified as a VEGFR-2 binding chemical from anticancer herbs with similar binding affinity as a reference drug in the Protein Data Bank (PDB) entry 3CJG of model A coordination. Oleanolic acid effectively inhibited VEGF-induced VEGFR-2 activation and angiogenesis in HU-VECs without cytotoxicity. We also verified that oleanolic acid inhibits in vivo angiogenesis during the development and the course of the retinopathy of prematurity (ROP) model in the mouse retina. Taken together, our results suggest a potential therapeutic benefit of oleanolic acid for inhibiting angiogenesis in proangiogenic diseases, including retinopathy.

Blood flow characteristics of diabetic patients with complications detected by optical measurement.

BACKGROUND: Diabetes mellitus (DM) is one of the most common diseases worldwide. Uncontrolled and prolonged hyperglycemia can cause diabetic complications, which reduce the quality of life of patients. Diabetic complications are common in DM patients. Because it is impossible to completely recover from diabetic complications, it is important for early detection. In this study, we suggest a novel method of determining blood flow characteristics based on fluorescence image analysis with indocyanine green and report that diabetic complications have unique blood flow characteristics. METHODS: We analyzed time-series fluorescence images obtained from controls, DM patients, and DM patients with complications. The images were segmented into the digits and the dorsum of the feet and hands, and each part has been considered as arterial and capillary flow. We compared the blood flow parameters in each region among the three groups. RESULTS: The DM patients with complications showed similar blood flow parameters to the controls, except the area under the curve and the maximum intensity, which indicate the blood flow volume. These parameters were significantly decreased in DM patients with complications. Although some blood flow parameters in the feet of DM patients with complications were close to normal blood flow, the vascular response of the macrovessels and microvessels to stimulation of the hands was significantly reduced, which indicates less reactivity in DM patients with complications. CONCLUSIONS: Our results suggest that DM patients, and DM patients with complications, have unique peripheral blood flow characteristics.

Fermented Extraction of Citrus unshiu Peel Inhibits Viability and Migration of Human Pancreatic Cancers.

Pancreatic cancer is one of the most dangerous cancers with high mortality rates. Despite continuous efforts, there has been limited improvement in its prognosis. In this study, we prepared fermented extract of Citrus unshiu peel (fCUP) from the by-product after juice processing and then examined the anticancer effects of fCUP on human pancreatic cancer cells. Treatment with fCUP inhibited the growth of human pancreatic cancer cells through induction of caspase-3 cleavage both in vitro and in vivo. Treatment with fCUP also blocked the migration of human pancreatic cancer cells through activation of intracellular signaling pathways such as MKK3/6 and P38. In contrast, treatment with fCUP did not inhibit growth and migration of human umbilical vein endothelial cells. In addition, we found that fCUP mainly consisted of aboriginal compounds, narirutin and hesperidin, as well as newly generated compounds, naringenin and hesperetin. In silico analysis showed that naringenin and hesperetin were the unique modules related to anticancer effect. Furthermore, fCUP exhibited the anticancer effects in in vivo xenograft models. Collectively, these results suggest that fCUP might have the potential to be developed into an effective anticancer drug for pancreatic cancers without causing adverse side-effects.