Examining the expression of low-density lipoprotein receptor (LDLR) and low-density lipoprotein receptor-related protein 6 (LRP6) genes in breast cancer cell lines.

Cholesterol and the Wnt/ß-catenin pathway have an effective role in the proliferation, survival, drug resistance, immune exhaustion, and metastasis of all types of cancer cells. Considering the role of LDLR and LRP6 proteins in cholesterol uptake by cells and activation of Wnt/ß-catenin pathway, this study aims to examine the gene expression of LDLR and LRP6 in cell lines of breast cancer. Human breast cancer cell lines MCF7, MD468 and SKBR3 were cultured in suitable conditions and after extracting total RNA from them, real-Time PCR was used to measure the levels of gene expression for LDLR and LRP6. Our results showed that the expression of LDLR and LRP6 genes is significantly increased in MCF7 and MD468 cells compared to SKBR3 cells. These results suggest that LRP6 and LDLR can be considered as a therapeutic target in tumors that have a genetic profile similar to MCF7 and MD468 cells.

Suppression of Growth Differentiation Factor 15 Gene Expression by Curcumin in Patients with Beta-Thalassemia Intermedia.

BACKGROUND: ß-thalassemia is an inherited disorder caused by defects in the synthesis of the beta-globin chain. One of the significant clinical complications in ß-thalassemia intermedia is iron overload toxicity, which may be attributed to reduced levels of hepcidin. This reduction in hepcidin leads to increased absorption of iron in the intestines, ultimately resulting in iron overload. The objective of this study was to assess the impact of curcumin on the expression of growth differentiating factor-15 (GDF-15) and hepcidin genes in patients with beta-thalassemia intermedia. METHODS: This study was designed as a randomized controlled double-blind clinical trial. Prior to and after the intervention period with curcumin, a blood sample of 5 mL was collected from both the placebo and curcumin-treated groups for the assessment of hepcidin and growth differentiating factor-15 gene expression. RESULTS: This study revealed a significant reduction in the expression of growth differentiating factor-15 in the curcumin group compared to the placebo group during the 3-month treatment period. Furthermore, curcumin supplementation led to a remarkable 10.1-fold increase in the levels of hepcidin in the curcumin group compared to the placebo group. CONCLUSIONS: The results of this study show that curcumin administration increases the mRNA levels of hepcidin in whole blood of thalassemia intermedia patients and supports the idea that curcumin could be a potential treatment to reduce suppression of hepcidin in thalassemias and other iron-loading anemias. CONCLUSIONS: The results of this study show that curcumin administration increases the mRNA levels of hepcidin in whole blood of thalassemia intermedia patients and supports the idea that curcumin could be a potential treatment to reduce suppression of hepcidin in thalassemias and other iron-loading anemias.

An Expert Overview on Therapies in Non-Transfusion-Dependent Thalassemia: Classical to Cutting Edge in Treatment.

The thalassemia issue is a growing worldwide health concern that anticipates the number of patients suffering from the disease will soon increase significantly. Patients with ß-thalassemia intermedia (ß-TI) manifest mild to intermediate levels of anemia, which is a reason for it to be clinically located between thalassemia minor and ß-thalassemia major (ß-TM). Notably, the determination of the actual rate of ß-TI is more complicated than ß-TM. The leading cause of this illness could be partial repression of ß-globin protein production; accordingly, the rate of ß-globin gene repression is different in patients, and the gene repression intensity creates a different clinical status. This review article provides an overview of functional mechanisms, advantages, and disadvantages of the classic to latest new treatments for this group of patients, depending on the disease severity divided into the typical management strategies for patients with ß-TI such as fetal hemoglobin (Hb) induction, splenectomy, bone marrow transplantation (BMT), transfusion therapy, and herbal and chemical iron chelators. Recently, novel erythropoiesis-stimulating agents have been added. Novel strategies are subclassified into molecular and cellular interventions. Genome editing is one of the efficient molecular therapies for improving hemoglobinopathies, especially ß-TI. It encompasses high-fidelity DNA repair (HDR), base and prime editing, clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 procedure, nuclease-free strategies, and epigenetic modulation. In cellular interventions, we mentioned the approach pattern to improve erythropoiesis impairments in translational models and patients with ß-TI that involve activin II receptor traps, Janus-associated kinase 2 (JAK2) inhibitors, and iron metabolism regulation.

Epigenetic regulation of autophagy in gastrointestinal cancers.

The development of novel therapeutic approaches is necessary to manage gastrointestinal cancers (GICs). Considering the effective molecular mechanisms involved in tumor growth, the therapeutic response is pivotal in this process. Autophagy is a highly conserved catabolic process that acts as a double-edged sword in tumorigenesis and tumor inhibition in a context-dependent manner. Depending on the stage of malignancy and cellular origin of the tumor, autophagy might result in cancer cell survival or death during the GICs' progression. Moreover, autophagy can prevent the progression of GIC in the early stages but leads to chemoresistance in advanced stages. Therefore, targeting specific arms of autophagy could be a promising strategy in the prevention of chemoresistance and treatment of GIC. It has been revealed that autophagy is a cytoplasmic event that is subject to transcriptional and epigenetic regulation inside the nucleus. The effect of epigenetic regulation (including DNA methylation, histone modification, and expression of non-coding RNAs (ncRNAs) in cellular fate is still not completely understood. Recent findings have indicated that epigenetic alterations can modify several genes and modulators, eventually leading to inhibition or promotion of autophagy in different cancer stages, and mediating chemoresistance or chemosensitivity. The current review focuses on the links between autophagy and epigenetics in GICs and discusses: 1) How autophagy and epigenetics are linked in GICs, by considering different epigenetic mechanisms; 2) how epigenetics may be involved in the alteration of cancer-related phenotypes, including cell proliferation, invasion, and migration; and 3) how epidrugs modulate autophagy in GICs to overcome chemoresistance.

Evidence of histone modification affecting ARID1A expression in colorectal cancer cell lines.

Aim: The current study aimed to focus on the role of histone deacetylation in reduced ARID1A expression in colorectal cancer cell lines. Background: ARID1A, a subunit of the switch/sucrose nonfermentable chromatin remodeling complex, has emerged as a bona fide tumor suppressor and is frequently downregulated and inactivated in multiple human cancers. Epigenetic modifications play an important role in dysregulation of gene expression in cancer. DNA methylation has been reported as an important regulator of ARID1A expression in colorectal cancer cell lines; however, the histone modification role in ARID1A suppression in colorectal cancer remains unclear. Methods: The expression levels of ARID1A mRNA were determined using real-time quantitative PCR in colorectal cancer cell lines including HCT116, SW48, HT29, SW742, LS180, and SW480. To evaluate the effect of histone deacetylation on ARID1A expression, all cell lines were treated with trichostatin A (TSA), a histone deacetylase inhibitor. SPSS software (Version 23) and GraphPad Prism (Version 6.01) were applied for data analysis using one-way ANOVA, followed by Tukey's multiple comparison tests. Results: Treatment of colorectal cancer cell lines with TSA increased ARID1A expression in a cell line-dependent manner, suggesting that histone deacetylation is at least one factor contributing to ARID1A downregulation in colorectal cancer. Conclusion: Histone deacetylase inhibitors might provide a strategy to restore ARID1A expression and may bring benefits to the colorectal cancer patients with a broader range of genetic backgrounds.

The Effect of Curcumin on Iron Overload in Patients with Beta-Thalassemia Intermedia.

BACKGROUND: ß-thalassemia is an inherited disorder that stems from a defect in beta-globin chain synthesis. Iron overload toxicity is one of the major clinical complications in ß-thalassemia that may be due to a reduction in the hepcidin level. As a result, intestinal iron absorption increases and finally iron overload occurs. The current study aimed to investigate the effect of curcumin on serum iron status, ferritin, and transferrin in patients with ß-thalas-semia intermedia. METHODS: This study was a randomized, controlled, double-blind clinical trial. Before and after the intervention period with curcumin, 5 ml blood was taken for the measurement of the entire index related to iron status. RESULTS: Our results demonstrated the levels of serum iron (p-value < 0.001), ferritin (p-value = 0.002), and transferrin saturation (p-value < 0.001) significantly decreased in the curcumin group compared to placebo. CONCLUSIONS: The data presented in this article show that curcumin supplementation would be effective in alleviating iron overload in patients with ß-thalassemia intermedia.

An overview of the biological and multifunctional roles of IL-38 in different infectious diseases and COVID-19.

Undoubtfully, the normal immune system can make a potential response to variable pathogens and neutralize or kill them depending on the type of infection through innate and acquired immunity. Cytokines have poly-peptide nature and are considered as signaling molecules that could amplify or alleviate immune responses besides their other biological functions. Interleukin 38 (IL-38) is a member of the IL-1 family cytokine that, however, its anti-inflammatory role has been observed in different autoimmune diseases like systemic lupus erythematosus (SLE), psoriasis, and Sjogren's syndrome; there is a controversy about the cytokine pro-inflammatory function. In the current review, we skimmed IL-38 structure, signaling mechanism, and its immunological functions, IL-38-producing immune cells. Also, we argued about the role of this cytokine in viral infections including hepatitis B (HBV), hepatitis C (HCV), influenza (Flu), and COVID-19. Also, it illustrated the IL-38 protective effects on sepsis. Moreover, we explained the modulatory role of IL-38 in the COVID-19 cytokine storm.

Up-regulated lncRNA-PVT1 expression in peripheral blood mononuclear cells of patients with coronary artery disease is correlated with decreased interleukin-10 production.

OBJECTIVES: Plasmacytoma variant translocation 1 (PVT1) is a newly discovered long non-coding RNA, which has not been previously studied in the inflammatory responses of the peripheral blood mononuclear cells (PBMCs) of patients with coronary artery disease (CAD). MATERIALS AND METHODS: This cross-sectional study was conducted on 15 CAD patients and 15 non-CAD (NCAD) individuals. The PVT1 expression was assessed in the PBMCs of the participants using a real-time polymerase chain reaction. Interleukin (IL)-10, IL-22, and matrix metalloproteinase-9 (MMP-9) were measured in the plasma and supernatant of cultured PBMCs in the presence or absence of lipopolysaccharide (LPS) using flow cytometry and enzyme-linked immunosorbent assay. RESULTS: An increased expression of PVT1 was observed in the untreated PBMCs of CAD patients, compared to the NCAD group. The PVT1 was significantly up-regulated after LPS treatment in the PBMCs of both groups. Plasma MMP-9 levels were found to be higher in CAD patients than in the control individuals. The level of IL-10 and IL-22 production by the non-treated PBMCs of CAD cases was significantly lower than the NCAD group. Overall, in the examined population, PVT1 expression was negatively correlated with IL-10 secretion. Moreover, the results showed a significant negative correlation between PVT1 expression and IL-10 production by untreated cells. CONCLUSIONS: The PVT1 expression augmented in the PBMCs of CAD patients, which could be associated with the decreased IL-10 generation by the PBMCs of these patients.

Expression and methylation status of BTG2, PPP1CA, and PEG3 genes in colon adenocarcinoma cell lines: promising treatment targets.

Aim: This study investigated the association between methylation status and expression levels of BTG2, PPP1CA, and PEG3 genes in colon cancer. Background: Aberrant DNA methylation is one of the most important epigenetic modifications in the development of cancer. Evidence indicates that hypermethylation of various tumor suppressor genes could be a potential mechanism of colon tumorigenesis. Methods: The expression levels of BTG2, PPP1CA, and PEG3 genes were evaluated in HT-29/219, HCT116, SW48, SW742, SW480, and LS180 cell lines using quantitative Real-Time PCR. The methylation status of BTG2 and PPP1CA was determined by methylation-specific PCR (MSP) method, and the methylation pattern of PEG3 was evaluated by bisulfite sequencing PCR (BSP). To investigate the effect of methylation on the expression of these genes, all colon cancer cell lines were treated by 5-Azacitidine (5-Aza) and/or Trichostatin A (TSA). Results: The expression levels of BTG2, PPP1CA, and PEG3 were highly heterogeneous and quantitatively correlated to their promoter methylation status in the studied colon cancer cell lines. Treatment by 5-Aza and/or TSA increased the expression of the above-named genes in colon cancer cell lines. Conclusion: Overall, it seems that BTG2, PPP1CA, and PEG3 act as tumor suppressor genes in colon cancer, and methylation is a potential mechanism for their loss of expression. Therefore, these genes may be considered as suitable targets for demethylation approaches and, eventually, colon cancer treatment. Combined treatment by 5-Aza and TSA may be a promising therapeutic strategy for colon cancer treatment. Further studies may contribute to confirm these results.

ARID1A regulates E-cadherin expression in colorectal cancer cells: a promising candidate therapeutic target.

BACKGROUND: Metastasis is a major cause of death in Colorectal cancer (CRC) patients, and the Epithelial-mesenchymal transition (EMT) has been known to be a crucial event in cancer metastasis. Downregulated expression of AT-rich interaction domain-containing protein 1A (ARID1A), a bona fide tumor suppressor gene, plays an important role in promoting EMT and CRC metastasis, but the underlying molecular mechanisms remain poorly understood. Here, we evaluated the impact of ARID1A knockdown and overexpression on the expression of EMT­related genes, E-cadherin and ß-catenin, in human CRC cells. METHODS AND RESULTS: The expression levels of ARID1A, E-cadherin and ß-catenin in CRC cell lines were detected via real-time quantitative PCR (qPCR) and western blot. ARID1A overexpression and shRNA-mediated knockdown were performed to indicate the effect of ARID1A expression on E-cadherin and ß-catenin expression in CRC cell lines. The effect of ARID1A knockdown on the migration ability of HCT116 cells was assessed using wound-healing assay. We found that the mRNA and protein expression of adhesive protein E-cadherin was remarkably downregulated in response to shRNA-mediated ARID1A knockdown in HCT116 and HT29 cells. Conversely, overexpression of ARID1A in SW48 cells significantly increased E-cadherin expression. In addition, ARID1A silencing promoted the migration of HCT116 cells. ARID1A knockdown and overexpression did not alter the level of ß-catenin expression. CONCLUSIONS: Our study demonstrates that E-cadherin levels were closely correlated with ARID1A expression. Thus, ARID1A downregulation may promote CRC metastasis through decreasing EMT­related protein E-cadherin and promoting epithelial cell movement. ARID1A could represent a promising candidate therapeutic target for CRC.

Antioxidative and antidiabetic effects of Capparis spinosa fruit extract on high-fat diet and low-dose streptozotocin-induced type 2 diabetic rats.

Capparis spinosa (CS) is known as a hypoglycemic medication in many countries. This study was designed to reveal the protective effects of the hydro-ethanolic extract of CS (HECS) fruit against diabetes and oxidative stress in type 2 diabetic rats (T2D). T2D was induced in 4 groups of adult male Sprague Dawley rats, using high fat diet (HFD) and low dose of streptozotocin (STZ). The four groups of diabetic rats were orally gavaged with HECS (200 & 400 mg/kg), metformin (50 mg/kg) or vehicle for 28 days. Two non-diabetic groups were assigned as normal control and HECS treated ones (400 mg/kg). The glucose intolerance, HOMA-IR score, HbA1c level, antioxidative status and expression of genes involved in hepatic gluconeogenesis and lipogenesis were determined. Although HECS had no significant effect on decreasing of HOMA-IR score and HbA1c, it significantly decreased glucose intolerance as well as oxidative stress by reduction of hepatic lipid peroxidation and increase of antioxidant enzymes levels in diabetic rats. Also, HECS treated diabetic rats showed a significant enhanced dyslipidemia, increased weight gain and sera insulin level. In addition, HECS significantly decreased hepatic phosphoenolpyruvate carboxykinase (PEPCK), increased acetyl CoA carboxylase and non-significantly decreased hepatocyte nuclear factor-4α (HNF-4α) as a transactivator of PEPCK at mRNA expression level in diabetic rats. This study indicated the anti-oxidative and anti-diabetic effects of C. spinosa fruit extract and confirmed its traditional usage as a remedy for T2D.

The effect of curcumin on serum copper, zinc, and zinc/copper ratio in patients with ß-thalassemia intermedia: a randomized double-blind clinical trial.

Thalassemia intermedia is a subgroup of ß-thalassemia which originates from mutations in the beta-globin gene. Zinc and copper play important roles in the metabolism. Due to its significant therapeutic effects, curcumin has led many studies to focus on curcumin. In a double-blind clinical trial study, 30 patients with beta-thalassemia intermedia with an age range of 20 to 35 years were randomly selected 1:1 to receive either curcumin or placebo for 3 months. Before and after the intervention period, 5 ml of blood was taken to determine the serum levels of zinc and copper. The laboratory tests were checked at baseline and at the end of the treatment. While the serum levels of zinc and zinc/copper significantly increased, the serum levels of copper decreased after 3 months of curcumin intake. In addition, on the basis of baseline characteristics, a negative correlation was found between zinc and body mass index and positive correlations were identified between copper with triglyceride and high-density lipoprotein. Also, the level of ferritin protein in the curcumin group compared to the placebo group showed a significant decrease after 3 months of curcumin use. Therefore, it could be concluded that curcumin might exert a net protective effect on copper toxicity in thalassemia intermedia patients. The investigation also implicated that curcumin represents an approach to regulating zinc homeostasis and may be useful as a complementary treatment of patients with thalassemia intermedia, especially in patients with zinc deficiency or low serum zinc/copper ratio. Clinical Trial Registration Number: IRCT20190902044668N1.

Stevia rebaudiana extract attenuate metabolic disorders in diabetic rats via modulation of glucose transport and antioxidant signaling pathways and aquaporin-2 expression in two extrahepatic tissues.

Today, plant-based therapies have been attracted attention to overcome diabetes complications. This study was an attempt to evaluate whether antidiabetic and nephroprotective effects of Stevia Rebaudiana Bertoni (SRB) can be exerted via upregulation of GLUT-4, SNAP23, and Stx4 in skeletal muscles or modulation of AQP2 mRNA expression and antioxidant signaling pathway activity (Nrf2/Keap1) in kidneys. To achieve this aim, diabetes was induced via STZ-nicotinamide (STZ-NA). Diabetes increased the level of Blood Urea Nitrogen (BUN), serum creatinine, Fasting Blood Sugar (FBS), and Keap1 mRNA expression, which was coincide with reduction in mRNA levels of Nrf2, GLUT4, SNAP23, and Stx4. SRB and metformin compensate mentioned variables. However, SRB extract was more effective than metformin to increase the levels of GLUT4 and Nrf2 mRNA. It seems that SRB might attenuate the diabetic complications via manipulating the glucose uptake components in peripheral tissues and might exert the nephroprotective effects by modulation of AQP2, and Nrf2/Keap1 mRNA expression. PRACTICAL APPLICATIONS: Synthetic antidiabetic drugs have been only partially successful in controlling the diabetic complications. Moreover, use of these drugs is associated with a number of adverse effects. Over the past few years, a renewed attention has been paid to the prevention and treatment of diabetes using medicinal plants and functional foods. SRB that have been known as natural sweetener for centuries, is a such natural agent that has high source of various phytochemicals with antidiabetic, renal protective, antitumor, and antioxidant properties. In the current study, possible molecular mechanisms of insulin-mimetic and nephroprotective effects of SRB extract was evaluated in diabetic rats. Due to powerful antihyperglycemic and nephroprotective effects of SRB extract that were showed in this study and previous studies, hence the fact that SRB is to be highlighted for future research as a new therapeutic agent for diabetes.

Altered ARID1A expression in colorectal cancer.

BACKGROUND: ARID1A has been described as a tumor suppressor gene, participating in chromatin re-modeling, epithelial-mesenchymal-transition and many other cellular and molecular processes. It has been cited as a contribute in tumorigenesis. The role of ARID1A in CRC is not yet defined. AIM: To investigate the role of ARID1A methylation and CNV in its expression in CRC cell lines and to examine the relationship between ARID1A status with survival and clinicopathologic characteristics in patients with CRC. METHODS: We used RT-PCR to determine both CNV and expression of ARID1A from six CRC cell lines. We used MSP to evaluate methylation of ARID1A. IHC was used to assess ARID1A protein expression. We also evaluated MSI and EMAST status in 18 paired CRC and adjacent normal tissues. 5AzadC was used to assess effect of DNA demethylation on ARID1A expression. Statistical analysis was performed to establish correlations between ARID1A expression and other parameters. RESULTS: Among the 18 CRC tumors studied, 7 (38.8%) and 5 tumors (27.7%) showed no or low ARID1A expression, respectively. We observed no significant difference in ARID1A expression for overall patient survival, and no difference between clinicopathological parameters including MSI and EMAST. However, lymphatic invasion was more pronounced in the low/no ARID1A expression group when compared to moderate and high expression group (33% VS. 16.6% respectively. ARID1A promoter methylation was observed in 4/6 (66%) cell lines and correlated with ARID1A mRNA expression level ranging from very low in SW48, to more pronounced in HCT116 and HT-29/219. Treatment with the methyltransferase inhibitor 5-Azacytidine (5-aza) resulted in a 25.4-fold and 6.1-fold increase in ARID1A mRNA expression in SW48 and SW742 cells, respectively, while there was no change in SW480 and LS180 cells. No ARID1A CNV was observed in the CRC cell lines. CONCLUSION: ARID1A expression is downregulated in CRC tissues which correlates with it being a tumor suppressor protein. This finding confirms ARID1A loss of expression in CRC development. Our in-vitro results suggest high methylation status associates with reduced ARID1A expression and contributes to CRC tumorigenesis. However, there was no significant association between ARID1A loss of expression and clinicopathological characteristics. Future in-vivo analysis is warranted to further establish ARID1A role in colorectal neoplastic transformation.

BDNF Val66Met genetic variation and its plasma level in patients with morbid obesity: A case-control study.

Obesity is a major public health concern worldwide. Genetic, behavioral, and environmental factors contribute to the multifactorial etiology of obesity. Evidence suggests an association between human Brain-Derived Neurotrophic Factor (BDNF) Val66Met single nucleotide polymorphism (SNP) and obesity. Reduced plasma BDNF levels have also been reported in patients with eating disorders and obesity. We aimed to evaluate the BDNF Val66Met (rs6265) SNP and also plasma BDNF levels in morbidly obese patients compared with healthy normal controls in southern Iran. One hundred morbidly obese patients and one hundred eight healthy normal controls were enrolled. Blood-derived DNA samples were genotyped using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method and confirmed by DNA sequencing. Plasma BDNF levels were evaluated using a commercially available sandwich enzyme-linked immunosorbent assay (ELISA) kit for human BDNF. Data analysis was performed by SPSS software, version 18.0. Genotype distribution was not significantly different between obese patients and controls. However, plasma BDNF levels were significantly lower in obese patients compared with controls. Interestingly, a significant association was found between BDNF Val66Met SNP and plasma BDNF levels. No relationship was observed between BDNF Val66Met SNP and all assessed demographic and clinical characteristics of obese patients. It seems that plasma BDNF levels were associated with both obesity and BDNF Val66Met SNP. However, this association was not found between BDNF Val66Met SNP and obesity. Further studies with larger sample sizes are needed for more detailed assessment of this genetic variation as a potential biomarker for obesity.

The effect of decitabine on the expression and methylation of the PPP1CA, BTG2, and PTEN in association with changes in miR-125b, miR-17, and miR-181b in NALM6 cell line.

Precursor B-cell acute lymphoblastic leukemia (B-ALL) is the most prevalent pediatric cancer. DNA methylation and changes in the microRNAs (miRNAs) expression are known to be important causes of B-ALL. Decitabine as a DNA methyltransferase inhibitor agent is able to induce hypomethylation in several tumor suppressor genes. Much evidence has proven BTG2, PPP1CA, and PTEN act as tumor suppressor genes in many malignancies. In this case control study, the messenger RNA (mRNA) expression of PPP1CA, BTG2, and PTEN genes using quantitative real-time polymerase chain reaction (rRT-PCR) in Nalm6 cell line and five patients suffer from ALL with mean age 5.6 years were determined in compare with seven normal healthy donors age and sex matched. qRT-PCR analysis revealed that the expression levels of PPP1CA, BTG2, and PTEN genes were significantly decreased in Nalm6 ([FC] = 0.46, [FC] = 0.046, [FC] = 0.54) and according to the Methylation-specific PCR (MSP) analysis, these genes were hypermethylated in Nalm6. In next step, the effects of decitabine treatment on the methylation and expression of these genes in association with changes in miR-125b, miR-17, and miR-181b expression levels were evaluated in optimal concentration 2.5 µM of decitabine. Our data showed that decitabine is able to restore the expression levels of aforementioned genes and downregulate expression levels of oncomiRs; including miR-125b, miR-17, and miR-181b in Nalm6 cell line. Therefore, it seems that decitabine can be used as a potential drug for the first line treatment of patients with B-ALL, but further in vivo investigation is necessary.