scholarly journals Sulfasalazine Modifies Metabolic Profiles and Enhances Cisplatin Chemosensitivity on Cholangiocarcinoma Cells in in vitro and in vivo models

2020 ◽  
Author(s):  
Malinee Thanee ◽  
Sureerat Padthaisong ◽  
Manida Suksawat ◽  
Hasaya Dokduang ◽  
Jutarop Phetcharaburanin ◽  
...  
Keyword(s):  
Redox Regulation ◽  
Treated Group ◽  
Control Group ◽  
High Recurrence Rate ◽  
Nucleic Acid Metabolism ◽  
In Vivo Models ◽  
Tryptophan Degradation ◽  
Xenograft Mice

Abstract Background: Sulfasalazine (SSZ) is widely known as an xCT inhibitor suppressing CD44v9 expressed cancer stem like cells (CSCs) being related to redox regulation. Cholangiocarcinoma (CCA) has a high recurrence rate and no effective chemotherapy. A recent report revealed high levels of CD44v9 positive cells in CCA patients. Therefore, a combination of drugs could prove a suitable strategy for CCA treatment via individual metabolic profiling. Methods: We examined the effect of xCT-targeted CD44v9-CSCs using sulfasalazine combined with cisplatin (CIS) or gemcitabine in CCA in vitro and in vivo model and did NMR-based metabolomics of xenograft mice tumor tissues. Results: Our findings suggest that combined SSZ and CIS leads to a higher inhibition of cell proliferation and induction of cell death than CIS alone in both in vitro and in vivo models. Xenograft mice showed that the CD44v9-CSC marker and CK-19-CCA proliferative marker were reduced in the combination treatment. Interestingly, different metabolic signatures and the significant metabolites were observed in the drug treated group compared with the control group that revealed the cancer suppression mechanisms. Conclusions: Taken together, SSZ could improve CCA therapy by sensitization to CIS through killing CD44v9-positive cells and modifying the metabolic pathways, in particular tryptophan degradation (i.e. kynurenine pathway, serotonin pathway) and nucleic acid metabolism.

scholarly journals Sulfasalazine modifies metabolic profiles and enhances cisplatin chemosensitivity on cholangiocarcinoma cells in in vitro and in vivo models

2021 ◽  
Vol 9 (1) ◽  
Author(s):  
Malinee Thanee ◽  
Sureerat Padthaisong ◽  
Manida Suksawat ◽  
Hasaya Dokduang ◽  
Jutarop Phetcharaburanin ◽  
...  
Keyword(s):  
Redox Regulation ◽  
Treated Group ◽  
Control Group ◽  
High Recurrence Rate ◽  
Nucleic Acid Metabolism ◽  
In Vivo Models ◽  
Tryptophan Degradation ◽  
Xenograft Mice

Abstract Background Sulfasalazine (SSZ) is widely known as an xCT inhibitor suppressing CD44v9-expressed cancer stem-like cells (CSCs) being related to redox regulation. Cholangiocarcinoma (CCA) has a high recurrence rate and no effective chemotherapy. A recent report revealed high levels of CD44v9-positive cells in CCA patients. Therefore, a combination of drugs could prove a suitable strategy for CCA treatment via individual metabolic profiling. Methods We examined the effect of xCT-targeted CD44v9-CSCs using sulfasalazine combined with cisplatin (CIS) or gemcitabine in CCA in vitro and in vivo models and did NMR-based metabolomics analysis of xenograft mice tumor tissues. Results Our findings suggest that combined SSZ and CIS leads to a higher inhibition of cell proliferation and induction of cell death than CIS alone in both in vitro and in vivo models. Xenograft mice showed that the CD44v9-CSC marker and CK-19-CCA proliferative marker were reduced in the combination treatment. Interestingly, different metabolic signatures and significant metabolites were observed in the drug-treated group compared with the control group that revealed the cancer suppression mechanisms. Conclusions SSZ could improve CCA therapy by sensitization to CIS through killing CD44v9-positive cells and modifying the metabolic pathways, in particular tryptophan degradation (i.e., kynurenine pathway, serotonin pathway) and nucleic acid metabolism.

Cold Plasma Treatment Promotes Full-thickness Healing of Skin Wounds in Murine Models

2021 ◽  
pp. 153473462110021
Author(s):  
Joon M. Jung ◽  
Hae K. Yoon ◽  
Chang J. Jung ◽  
Soo Y. Jo ◽  
Sang G. Hwang ◽  
...  
Keyword(s):  
Wound Healing ◽  
Plasma Treatment ◽  
Cold Plasma ◽  
Smooth Muscle Actin ◽  
Treated Group ◽  
Control Group ◽  
Alpha Smooth Muscle Actin ◽  
Skin Wound Healing

Cold plasma can be beneficial for promoting skin wound healing and has a high potential of being effectively used in treating various wounds. Our aim was to verify the effect of cold plasma in accelerating wound healing and investigate its underlying mechanism in vitro and in vivo. For the in vivo experiments, 2 full-thickness dermal wounds were created in each mouse (n = 30). While one wound was exposed to 2 daily plasma treatments for 3 min, the other wound served as a control. The wounds were evaluated by imaging and histological analyses at 4, 7, and 11 days post the wound infliction process. Immunohistochemical studies were also performed at the same time points. In vitro proliferation and scratch assay using HaCaT keratinocytes and fibroblasts were performed. The expression levels of wound healing–related genes were analyzed by real-time polymerase chain reaction and western blot analysis. On day 7, the wound healing rates were 53.94% and 63.58% for the control group and the plasma-treated group, respectively. On day 11, these rates were 76.05% and 93.44% for the control and plasma-treated groups, respectively, and the difference between them was significant ( P = .039). Histological analysis demonstrated that plasma treatment promotes the formation of epidermal keratin and granular layers. Immunohistochemical studies also revealed that collagen 1, collagen 3, and alpha-smooth muscle actin appeared more abundantly in the plasma-treated group than in the control group. In vitro, the proliferation of keratinocytes was promoted by plasma exposure. Scratch assay showed that fibroblast exposure to plasma increased their migration. The expression levels of collagen 1, collagen 3, and alpha-smooth muscle actin were elevated upon plasma treatment. In conclusion, cold plasma can accelerate skin wound healing and is well tolerated.

scholarly journals Anti-Obesity and Anti-Adipogenic Effects of Chitosan Oligosaccharide (GO2KA1) in SD Rats and in 3T3-L1 Preadipocytes Models

Molecules ◽  
2021 ◽  
Vol 26 (2) ◽  
pp. 331
Author(s):  
Jung-Yun Lee ◽  
Tae Yang Kim ◽  
Hanna Kang ◽  
Jungbae Oh ◽  
Joo Woong Park ◽  
...  
Keyword(s):  
Gene Expression ◽  
Body Weight ◽  
Density Lipoprotein ◽  
Treated Group ◽  
Control Group ◽  
Chitosan Oligosaccharide ◽  
Sd Rats ◽  
Adipogenic Gene

Excess body weight is a major risk factor for type 2 diabetes (T2D) and associated metabolic complications, and weight loss has been shown to improve glycemic control and decrease morbidity and mortality in T2D patients. Weight-loss strategies using dietary interventions produce a significant decrease in diabetes-related metabolic disturbance. We have previously reported that the supplementation of low molecular chitosan oligosaccharide (GO2KA1) significantly inhibited blood glucose levels in both animals and humans. However, the effect of GO2KA1 on obesity still remains unclear. The aim of the study was to evaluate the anti-obesity effect of GO2KA1 on lipid accumulation and adipogenic gene expression using 3T3-L1 adipocytes in vitro and plasma lipid profiles using a Sprague-Dawley (SD) rat model. Murine 3T3-L1 preadipocytes were stimulated to differentiate under the adipogenic stimulation in the presence and absence of varying concentrations of GO2KA1. Adipocyte differentiation was confirmed by Oil Red O staining of lipids and the expression of adipogenic gene expression. Compared to control group, the cells treated with GO2KA1 significantly decreased in intracellular lipid accumulation with concomitant decreases in the expression of key transcription factors, peroxisome proliferator-activated receptor gamma (PPARγ) and CCAAT/enhancer-binding protein alpha (CEBP/α). Consistently, the mRNA expression of downstream adipogenic target genes such as fatty acid binding protein 4 (FABP4), fatty acid synthase (FAS), were significantly lower in the GO2KA1-treated group than in the control group. In vivo, male SD rats were fed a high fat diet (HFD) for 6 weeks to induced obesity, followed by oral administration of GO2KA1 at 0.1 g/kg/body weight or vehicle control in HFD. We assessed body weight, food intake, plasma lipids, levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) for liver function, and serum level of adiponectin, a marker for obesity-mediated metabolic syndrome. Compared to control group GO2KA1 significantly suppressed body weight gain (185.8 ± 8.8 g vs. 211.6 ± 20.1 g, p < 0.05) with no significant difference in food intake. The serum total cholesterol, triglyceride, and low-density lipoprotein (LDL) levels were significantly lower in the GO2KA1-treated group than in the control group, whereas the high-density lipoprotein (HDL) level was higher in the GO2KA1 group. The GO2KA1-treated group also showed a significant reduction in ALT and AST levels compared to the control. Moreover, serum adiponectin levels were significantly 1.5-folder higher than the control group. These in vivo and in vitro findings suggest that dietary supplementation of GO2KA1 may prevent diet-induced weight gain and the anti-obesity effect is mediated in part by inhibiting adipogenesis and increasing adiponectin level.

scholarly journals In Vivo Antiviral Effects of U18666A Against Type I Feline Infectious Peritonitis Virus

Pathogens ◽  
2020 ◽  
Vol 9 (1) ◽  
pp. 67 ◽  
Author(s):  
Tomoyoshi Doki ◽  
Tomoyo Tarusawa ◽  
Tsutomu Hohdatsu ◽  
Tomomi Takano
Keyword(s):  
Clinical Symptoms ◽  
Treated Group ◽  
Control Group ◽  
Type I ◽  
Feline Infectious Peritonitis Virus ◽  
Feline Infectious Peritonitis ◽  
Amphiphilic Drug ◽  
Antiviral Effects

Background: The cationic amphiphilic drug U18666A inhibits the proliferation of type I FIPV in vitro. In this study, we evaluated the in vivo antiviral effects of U18666A by administering it to SPF cats challenged with type I FIPV. Methods: Ten SPF cats were randomly assigned to two experimental groups. FIPV KU-2 were inoculated intraperitoneally to cats. The control group was administered PBS, and the U18666A-treated group was administered U18666A subcutaneously at 2.5 mg/kg on day 0, and 1.25 mg/kg on days 2 and 4 after viral inoculation. Results: Two of the five control cats administered PBS alone developed FIP. Four of the five cats administered U18666A developed no signs of FIP. One cat that temporarily developed fever, had no other clinical symptoms, and no gross lesion was noted on an autopsy after the end of the experiment. The FIPV gene was detected intermittently in feces and saliva regardless of the development of FIP or administration of U18666A. Conclusions: When U18666A was administered to cats experimentally infected with type I FIPV, the development of FIP might be suppressed compared with the control group. However, the number of animals with FIP is too low to establish anti-viral effect of U18666A in cats.

scholarly journals An Explanation of the Underlying Mechanisms for the In Vitro and In Vivo Antiurolithic Activity of Glechoma longituba

2016 ◽  
Vol 2016 ◽  
pp. 1-11 ◽  
Author(s):  
Qiang Liang ◽  
Xiaoran Li ◽  
Wangning Zhou ◽  
Yu Su ◽  
Shenbao He ◽  
...  
Keyword(s):  
Kidney Injury ◽  
Positive Control ◽  
Potassium Citrate ◽  
Positive Control Group ◽  
Control Group ◽  
Protective Effects ◽  
In Vivo Models ◽  
Glechoma Longituba

Purpose. To use in vitro and in vivo models to evaluate Glechoma longituba extract to provide scientific evidence for this extract’s antiurolithic activity. Materials and Methods. Potassium citrate was used as a positive control group. Oxidative stress (OS) markers and the expression of osteopontin (OPN) and kidney injury molecule-1 (KIM-1) were measured to assess the protective effects of Glechoma longituba. Multiple urolithiasis-related biochemical parameters were evaluated in urine and serum. Kidneys were harvested for histological examination and the assessment of crystal deposits. Results. In vitro and in vivo experiments demonstrated that treatment with Glechoma longituba extract significantly decreased calcium oxalate- (CaOx-) induced OPN expression, KIM-1 expression, and OS compared with the positive control group (P<0.05). Additionally, in vivo rats that received Glechoma longituba extract exhibited significantly decreased CaOx deposits and pathological alterations (P<0.05) compared with urolithic rats. Significantly lower levels of oxalate, creatinine, and urea and increased citrate levels were observed among rats that received Glechoma longituba (P<0.05) compared with urolithic rats. Conclusion. Glechoma longituba has antiurolithic effects due to its possible combined effects of increasing antioxidant levels, decreasing urinary stone-forming constituents and urolithiasis-related protein expression, and elevating urinary citrate levels.

scholarly journals Blockade of NF-κB and MAPK pathways by ulinastatin attenuates wear particle-stimulated osteoclast differentiation in vitro and in vivo

2016 ◽  
Vol 36 (5) ◽  
Author(s):  
Jiang-Ying Ru ◽  
Hai-Dong Xu ◽  
Dai Shi ◽  
Jun-Bo Pan ◽  
Xiao-Jin Pan ◽  
...  
Keyword(s):  
Cathepsin K ◽  
Treated Group ◽  
Receptor Activation ◽  
Raw264.7 Cells ◽  
Control Group ◽  
Nuclear Factor ◽  
Trabecular Thickness ◽  
Tnf Α

Ulinastatin, a urinary trypsin inhibitor (UTI), is widely used to clinically treat lipopolysaccharide (LPS)-related inflammatory disorders recently. Adherent pathogen-associated molecular patterns (PAMPs), of which LPS is the best-studied and classical endotoxin produced by Gram-negative bacteria, act to increase the biological activity of osteopedic wear particles such as polymethyl-methacrylate (PMMA) and titanium particles in cell culture and animal models of implant loosening. The present study was designed to explore the inhibitory effect of UTI on osteoclastogenesis and inflammatory osteolysis in LPS/PMMA-mediated Raw264.7 cells and murine osteolysis models, and investigate the potential mechanism. The in vitro study was divided into the control group, LPS-induced group, PMMA-stimulated group and UTI-pretreated group. UTI (500 or 5000 units/ml) pretreatment was followed by PMMA (0.5 mg/ml) with adherent LPS. The levels of inflammatory mediators including tumour necrosis factor-α (TNF-α), matrixmetallo-proteinases-9 (MMP-9) and interleukin-6 (IL-6), receptor activation of nuclear factor NF-κB (RANK), and cathepsin K were examined and the amounts of phosphorylated I-κB, MEK, JNK and p38 were measured. In vivo study, murine osteolysis models were divided into the control group, PMMA-induced group and UTI-treated group. UTI (500 or 5000 units/kg per day) was injected intraperitoneally followed by PMMA suspension with adherent LPS (2×108 particles/25 μl) in the UTI-treated group. The thickness of interfacial membrane and the number of infiltrated inflammatory cells around the implants were assessed, and bone mineral density (BMD), trabecular number (Tb.N.), trabecular thickness (Tb.Th.), trabecular separation (Tb.Sp.), relative bone volume over total volume (BV/TV) of distal femur around the implants were calculated. Our results showed that UTI pretreatment suppressed the secretion of proinflammatory cytokines including MMP-9, IL-6, TNF-α, RANK and cathepsin K through down-regulating the activity of nuclear factor kappa B (NF-κB) and MAPKs partly in LPS/PMMA-mediated Raw264.7 cells. Finally, UTI treatment decreased the inflammatory osteolysis reaction in PMMA-induced murine osteolysis models. In conclusion, these results confirm the anti-inflammatory potential of UTI in the prevention of particle disease.

scholarly journals Neuroprotective Effect and Mechanism of Thiazolidinedione on Dopaminergic Neurons In Vivo and In Vitro in Parkinson’s Disease

PPAR Research ◽  
2017 ◽  
Vol 2017 ◽  
pp. 1-12 ◽  
Author(s):  
Yanqin Wang ◽  
Weilin Zhao ◽  
Ge Li ◽  
Jinhu Chen ◽  
Xin Guan ◽  
...  
Keyword(s):  
Dopaminergic Neurons ◽  
Neuroprotective Effect ◽  
Oral Treatment ◽  
Treated Group ◽  
Control Group ◽  
Peroxisome Proliferator ◽  
Peroxisome Proliferator Activated Receptor ◽  
Nuclear Respiratory Factor

The aim of the present study was to gain insight into the neuroprotection effects and mechanism of thiazolidinedione pioglitazone in both in vitro and in vivo MPP+/MPTP induced PD models. In vivo experimental results showed that oral treatment of pioglitazone resulted in significant improvements in behavior symptoms damaged by MPTP and increase in the survival of TH positive neurons in the pioglitazone intervention groups. In addition, oral treatment of pioglitazone increased the expression of peroxisome proliferator-activated receptor-γ coactivator of 1α (PGC-1α) and increased the number of mitochondria, along with an observed improvement in mitochondrial ultrastructure. From in vitro studies, 2,4-thiazolidinedione resulted in increased levels of molecules regulated function of mitochondria, including PGC-1α, nuclear respiratory factor 1 (NRF1), NRF2, and mitochondria fusion 2 (Mfn2), and inhibited mitochondria fission 1 (Fis1). We show that protein levels of Bcl-2 and ERK were reduced in the MPP+-treated group compared with the control group. This effect was observed to be reversed upon treatment with 2,4-thiazolidinedione, as Bcl-2 and ERK expression levels were increased. We also observed that levels of the apoptotic protein Bax showed opposite changes compared to Bcl-2 and ERK levels. The results from this study confirm that pioglitazone/2,4-thiazolidinedione is able to activate PGC-1α and prevent damage of dopaminergic neurons and restore mitochondria ultrastructure through the regulation of mitochondria function.

scholarly journals Inhibition of pyruvate dehydrogenase kinase influence microbiota and metabolomic profile in pancreatic cancer xenograft mice

2020 ◽  
Author(s):  
Kaarel Adamberg ◽  
Raivo Vilu ◽  
Valerio Pazienza
Keyword(s):  
Pancreatic Cancer ◽  
Treatment Options ◽  
Xenograft Model ◽  
Treated Group ◽  
Pyruvate Dehydrogenase Kinase ◽  
Control Group ◽  
Metabolomic Profile ◽  
Mouse Xenograft Model ◽  
Xenograft Mice

Abstract Objective Despite recent advances in treatment options, pancreatic cancer remains the most deadly major cancer. Targeting metabolism represents an emerging anti-cancer strategy. Results Metagenomic 16S analysis was employed to explore the effect of Dichloroacetate (DCA) on the composition of the fecal microbiota and metabolomic profile was assessed on in vivo pancreatic cancer mouse xenograft model. Pancreatic cancer xenograft mice displayed a shift of microbiota’ profile as compared to control mice without DCA treatment and a significant decrease of the purine bases inosine xanthine together with their metabolically-related compound hypoxanthine were observed in the DCA treated group as compared to the control group. Two aminoacids methionine and aspartic acid resulted decreased and increased respectively. DCA affects tumor environment and studies are needed in order to understand whether DCA supplementation could be supportive as synergistic approach to enhance the efficacy of existing cancer treatments in pancreatic cancer patients.

scholarly journals Biotherapic of Trypanosoma cruzi 17d increases apoptosis in experimentally infected mice

2021 ◽  
Vol 10 (36) ◽  
pp. 119-124
Author(s):  
Patrícia Flora Sandri ◽  
Gislaine Janaina Sanchez Falkowski ◽  
Luzmarina Hernandes ◽  
Márcia Machado de Oliveira Dalálio ◽  
Denise Lessa Aleixo ◽  
...  
Keyword(s):  
Trypanosoma Cruzi ◽  
Controlled Trial ◽  
Basic Research ◽  
Treated Group ◽  
Oral Route ◽  
Control Group ◽  
Phagocytic Cells ◽  
Number Of Cells

Introduction: the mechanism of action of ultradiluted medicines has not yet been established[1,3]. Many basic research studies have focused on isopathic models using in vitro or in vivo designs [4,5]. Recent studies indicate that an ultradiluted (isopathic) antigen can transfer signals to the immune system and modulate its response when an organism is challenged against this same antigen [6]. Some studies on experimental infection of mice by T. cruzi identified apoptotic cells and showed that the increase of their number is associated with an increase also in the number of parasites in the blood of the infected animals, while blockage of apoptosis can be the target of therapeutic intervention [7,8]. Aim: to evaluate the development of apoptosis in mice treated with biotherapic of Trypanosoma cruzi in dilution 17d through in situ detection of fragmented DNA. Method: in a blind randomized controlled trial, 36 male Swiss mice age 4 or 8 weeks were distributed in groups control - treated with 7% hydroalcoholic solution(CI-4=9 animals or CI-8=9 animals); and treated with biotherapic 17d (BIOT-4=9 animals or BIOT-8=9 animals). Infection was performed with 1,400 trypomastigotes T. cruzi-strain Y via intraperitoneal. Biotherapic 17d was prepared through the addition of 0.9ml of concentrated T. cruzi (10E+7 trypomastigotes/ml) to 9.1 ml of distilled water. The following dilutions were prepared in 86% hydroalcoholic solution until dilution 16d. Dilution 17d was prepared with 7% hydroalcoholic solution. It was performed microbiological control and biological risk in vivo. Treatment: 0.2 ml in 3 consecutive days, oral route, from the moment infection was verified. Animals were sacrificed on the 3rd day of treatment in a chamber saturated with ether. The liver and spleen were removed and fixated in 4% paraformaldehyde for 24 hours and then included in paraffin. Apoptosis was evaluated through DNA fragmentation – TUNEL technique (TdT dUTP-biotin Nick End Labeling (ApopTag® Peroxidade-Chemicon). For statistical analysis software Statistica 8.0 was used. This study was approved by the Ethics Committee for Animal Experimentation of UEM. Results and Discussion: in the samples of liver of animals age 4 and 8 weeks either treated or not with biotherapic 17d it was found cells parasitized by amastigotes of T. cruzi with apoptotic bodies, or phagocytic cells with phagocytic vacuole with apoptotic marked material inside them. The number of cells in apoptosis in animals age 4 weeks was not significantly (p=0.03) larger in treated group BIOT-C4 than in control group CI-4 (Figure 1). In animals age 8 weeks, the number of cells in apoptosis was significantly (p

scholarly journals In Vitro and In Vivo Activities of Newer Fluoroquinolones against Vibrio vulnificus

2002 ◽  
Vol 46 (11) ◽  
pp. 3580-3584 ◽  
Author(s):  
Hung-Jen Tang ◽  
Ming-Chung Chang ◽  
Wen-Chien Ko ◽  
Kun-Yen Huang ◽  
Chih-Lung Lee ◽  
...  
Keyword(s):  
Vibrio Vulnificus ◽  
Survival Rates ◽  
Treated Group ◽  
Inhibitory Effect ◽  
Clinical Strain ◽  
Control Group ◽  
Dilution Method ◽  
Agar Dilution Method

ABSTRACT The MICs of six fluoroquinolones as well as minocycline and cefotaxime for 46 clinical isolates of Vibrio vulnificus were determined by the agar dilution method. All the drugs tested had good activities against all isolates, with the MICs at which 90% of the isolates tested were inhibited (MIC90s) by five of the fluoroquinolones ranging between 0.03 and 0.06 μg/ml. The MIC90 of lomefloxacin, on the other hand, was 0.12 μg/ml. Time-kill studies were conducted with these agents and a clinical strain of V. vulnificus, VV5823. When approximately 5 × 105 CFU of V. vulnificus/ml was incubated with any one of the above-mentioned six fluoroquinolones at concentrations of two times the MIC, there was an inhibitory effect on V. vulnificus that persisted for more than 48 h with no noted regrowth. The efficacies of the fluoroquinolones were further evaluated in vivo in the mouse model of experimental V. vulnificus infection and compared to the efficacy of a combination therapy using cefotaxime plus minocycline. With an inoculum of 1.5 × 107 CFU, 28 (87.5%) of 32 mice in the cefotaxime-minocycline-treated group survived and 29 (91%) of the 32 mice in the moxifloxacin-treated group survived while none of the 32 mice in the control group did. With an inoculum of 3.5 × 107 CFU, the difference in survival rates among groups of 15 mice treated with levofloxacin (13 of 15), moxifloxacin (10 of 15), gatifloxacin (10 of 15), sparfloxacin (11 of 15), ciprofloxacin (12 of 15), or lomefloxacin (10 of 15) was not statistically significant while none of the 15 mice treated with saline survived. We concluded that the newer fluoroquinolones as single agents are as effective as the cefotaxime-minocycline combination in inhibiting V. vulnificus both in vitro and in vivo.

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