Medicinal Chemistry and Pharmaceutics Commons^™

Fluorescence Detection Of Malachite Green In Fish Based On Aptamer And Sybr Green I, Jia Wang, Chengyi Hong, Zhengzhong Lin, Zhi-Yong Huang

Journal of Food and Drug Analysis

A simple fluorescence method to quantitatively monitor malachite green (MG) was introduced based on the conformation switch effect of MG-aptamer (Apt) and the fluorochrome of SYBR Green I (SGI). SGI can embed in the stem–loop structure of Apt to produce fluorescence. However, the fluorescence will be quenched when MG is introduced because of the specific binding of Apt to MG. Under the optimal conditions, the fluorescence method displayed excellent linear relationship between the fluorescence quenching efficiencies and the MG concentrations ranging from 0.02 μmol L⁻¹ to 1.0 μmol L⁻¹. The detection limit of the method was 0.67 …

Bromelain Activates The Amp-Activated Protein Kinase-Autophagy Pathway To Alleviate Hepatic Lipid Accumulation, Po-An Hu, Man-Chen Hsu, Szu-Han Chen, Chia-Hui Chen, Yu Ru Kou, Jenq-Wen Huang, Tzong-Shyuan Lee

Journal of Food and Drug Analysis

Bromelain, a cysteine protease found in pineapple, is known to exert protective effects against non-alcoholic fatty liver disease (NAFLD); however, the underlying mechanism is unclear. In this study, we aimed to investigate the molecular mechanisms underlying the beneficial effects of bromelain using in vivo and in vitro models. C57BL/6 mice were fed a high-fat diet (HFD) with or without bromelain (20 mg/kg/day) for 12 weeks. We found that treatment with bromelain alleviated hepatic lipid accumulation accompanied by the activation of AMP-activated protein kinase (AMPK) and autophagy flux, as evidenced by the elevated levels of phosphorylated AMPK, ATG5, ATG7, LC3-II, and …

Metabolism-Involved Drug Interactions With Traditional Chinese Medicines In Cardiovascular Diseases, Ren-Jong Liang, Shu-Hao Hsu, Tien-Yu Chang, Tzu-Yi Chiang, Hong-Jaan Wang, Yune-Fang Ueng

Journal of Food and Drug Analysis

Herbal medicines have been widely used for the past millennia. Traditional Chinese medicine (TCM) is a major modality in Chinese medical care and has garnered global attention owing to its pharmacological effects and multi-targeted actions. The increased incidence of sequential or concurrent use of herbs and drugs in patients forces us to consider herb-drug interactions (HDIs) in this modern era. One of the main causes of HDIs is modulation of drug metabolism, in which cytochrome P450 (CYP), UDP-glucuronosyltransferase (UGT), and transporters play primary roles. In this review, we focus on in vivo studies of HDIs, particularly in the treatment of …

Real-World Evidence Of The Herb-Drug Interactions, Hsueh-Yi Pan, Li-Wei Wu, Pin-Chun Wang, Ping-Hung Chiu, Meng-Ting Wang

Journal of Food and Drug Analysis

Herbal medicine (HM) is a type of medicine that uses active ingredients made from plants to treat diseases and maintain health and wellbeing. Due to its increasing worldwide usage, the possibility of HMs and conventional drugs being concurrently used is high, potentially leading to adverse events resulting from herb-drug interactions. Despite the safety concerns regarding such interactions, few studies have been conducted for assessing clinical consequences of using HMs with conventional drugs in real-world settings. As clinical trials are not forthcoming rapidly enough to provide the evidence for herb-drug interactions, observational studies are considered as an alternative approach. The present …

Neuropilin-2 In Advanced Prostate Cancer And Its Targeting With Novel Small Molecule Inhibitors, Ridwan Islam

Theses & Dissertations

Despite the development of new therapies, clinical management of advanced metastatic castrationresistant prostate cancer (mCRPC) still remains challenging owing to the emergence of therapy resistance. Resistance to the therapies is driven by both AR and non-AR pathways. Non-AR pathway is characterized by a lineage switch that leads to the development of neuroendocrine-like (NE-like) prostate cancer (PCa), which has a fulminant clinical course due to the lack of effective treatment strategy. Therefore, novel molecular targets need to be identified to develop effective therapeutic regimen to improve the outcomes in advanced PCa patients including NE-like PCa. Recent investigation on advanced PCa patients …

Preclinical Development Of Novel Chemotherapeutic Agent Ac1lpszg: Formulation Optimization, In Vitro Characterization, And In Vivo Pharmacokinetics, Ritu Gupta

Dissertations (2016-Present)

Preclinical development of novel chemotherapeutic agent AC1LPSZG, a mammalian target of rapamycin (mTOR) inhibitor, involved development of sensitive reverse-phase ultra-performance liquid chromatography (UPLC) and LC-MS/MS methods for quantification of AC1LPSZG in in vitro study samples and rat plasma, respectively. Pharmacokinetic studies were done in SD rats after intravenous injection of cosolvent formulations. The resulting pharmacokinetic parameters were analyzed using non-compartmental analysis (NCA) and two-compartmental modeling. Poly (D, L-lactic-co-glycolic acid) (PLGA) is most used biodegradable synthetic polymer for nano drug delivery due to its non-toxic and biodegradable nature, and tunable release properties. PLGA nanoparticles (NPs) were prepared by ‘nanoprecipitation’ technique using …

Identification And Quantitation Of Unspecified Impurities Discovered In Novel Oral Irinotecan By Lc-Ms/Ms And Uhplc, Laura B. Miller

Forensic Science Master's Projects

Irinotecan is a cancer medication approved for medical use in the United States in 1996. It is currently administered via intravenous injection. However, intravenous dosing has disadvantages such as requirement of administration by a trained professional in a medical facility, possible severe adverse effects, pain at the injection site, and possible hemolysis if injected too rapidly. Previously oral administration was not feasible because the p-glycoprotein (p-GP) on the cell membrane of stomach cells acts as a defense mechanism against harmful substances by pumping the irinotecan back into the intestinal lumen for excretion. With the discovery of the p-GP inhibitor encequidar …

Phytochemicals, Acute Toxicity And Genotoxicity Of Chewing Sticks From Zanthoxylum Zanthoxyloides Root And Distemonanthus Benthamianus Stem, Chinwe S. Alaribe, Akolade R. Oladipupo, Anjola T. Adebesin, Omolayomi Kaska, Emuejevoke T. Toye

Bulletin of Faculty of Pharmacy Cairo University

Dental caries has been reported to touch almost half of the world’s population, making it one of the most prevalent disease conditions in the world. Studies have shown that chewing sticks could be useful in cleaning of teeth and could offer protection against dental conditions, including caries. Given the increasing usage of chewing sticks and their unstandardized nature, in this study we investigated the acute toxicity, genotoxic effects on liver and kidney, and phytochemical constituents of chewing sticks from Zanthoxylum zanthoxyloides root and Distemonanthus benthamianus stem, widely used in West Africa. Chemical tests showed the presence of saponins, alkaloids, flavonoids, …

A Conserved Mechanism For Hormesis In Molecular Systems, Sharon N. Greenwood, Regina G. Belz, Brian P. Weiser

Rowan-Virtua School of Osteopathic Medicine Departmental Research

Hormesis refers to dose-response phenomena where low dose treatments elicit a response that is opposite the response observed at higher doses. Hormetic dose-response relationships have been observed throughout all of biology, but the underlying determinants of many reported hormetic dose-responses have not been identified. In this report, we describe a conserved mechanism for hormesis on the molecular level where low dose treatments enhance a response that becomes reduced at higher doses. The hormetic mechanism relies on the ability of protein homo-multimers to simultaneously interact with a substrate and a competitor on different subunits at low doses of competitor. In this …

Electrospray Deposition Of Polyvinylidene Fluoride (Pvdf) Microparticles: Impact Of Solvents And Flow Rate, Akinwunmi Joaquim, Omari Paul, Michael Ibezim, Dewayne Johnson, April Falconer, Ying Wu, Frances Williams, Richard Mu

TIGER Institute Faculty Research

Polymeric microparticles have been shown to have great impacts in the area of drug delivery, biosensing, and tissue engineering. Electrospray technology, which provides a simple yet effective technique in the creation of microparticles, was utilized in this work. In addition, altering the electrospray experimental parameters such as applied voltage, flow rate, collector distance, solvents, and the polymer-solvent mixtures can result in differences in the size and morphology of the produced microparticles. The effects of the flow rate at (0.15, 0.3, 0.45, 0.6, 0.8, and 1 mL/h) and N, N-Dimethylformamide (DMF)/acetone solvent ratios (20:80, 40:60, 60:40, 80:20, 100:0 v/v) in the …

Biomimetic Strategies To Control Therapeutic Release From Novel Dna Nanoparticles, Robert J. Mosley

Theses and Dissertations

The inherent chemical, mechanical, and structural properties of nucleic acids make them ideal candidates for the formulation of tunable, personalized drug nanocarriers. However, none so far have exploited these properties for the controlled release of therapeutic drugs. In this dissertation, a biomimetic approach to controlling drug release is exhibited by specifically manipulating the architecture of novel, DNA nanoparticles to take advantage of drug binding mechanisms of action. Rationally designed DNA strands were immobilized on gold surfaces via a terminal thiol modification. Immobilized monomers can be manipulated to form distinct monolayer architectures including flat, folded, coiled, or stretched structures. Increasing the …

An Investigation Into Use Of Natural Mistletoe Extracts And Their Formulations As Potential Anti-Cancer Treatments In Human Carcinomas, Rebecca Healy, Anne M. Friel

SURE Journal: Science Undergraduate Research Experience Journal

Cancer is a prevalent disease that is responsible for around 10 million deaths a year. Cases and deaths will continue to rise daily if nothing is done to find the most effective treatment. Mistletoe is an evergreen, semi-parasitic plant that grows on trees. This plant contains a number of biologically active compounds that contribute to its anti-cancer properties e.g., lectins. Mistletoe is obtained from nature and has been shown to be safe, potent, and effective in treating cancer. Herein we review the biological effects of mistletoe on cancer cells, formulations that can be used in cancer treatment and discuss the …

Biowill - Characterising Willow Bark Bio-Actives For Skin Therapies, Arnold Marisa

ORBioM (Open Research BioSciences Meeting)

Willow bark is considered as a disposable by-product when processing willow for biomass. Willow (Salix) is known to contain high value bioactive compounds which include salicin and its derivatives, and other phytochemicals of interest such as polyphenols and flavonoids. The plant is historically known as the primary source of salicylates to which the well-known drug aspirin is derived from. The work forms part of the Interreg project BioWILL, which is focused on integrated “Zero Waste” biorefinery utilising all fractions of willow feedstock for the production of biochemicals and renewable energy. This project aims to investigate the crude and …

Corrigendum To “The Impact Of The Rare Disease And Orphan Drug Act In Taiwan” [J Food Drug Anal 29 (2021) 717-725]

Journal of Food and Drug Analysis

No abstract provided.

The Study Of Distribution Of Ingested Terbinafine On Skin With Ambient Ionization Tandem Mass Spectrometry, Yi-Tzu Cho, Hung Su, Ching-Ying Wu, Jingyueh Jeng, Chi-Wei Lee, Deng‐Chyang Wu, Tiao-Lai Huang, Jentaie Shiea

Journal of Food and Drug Analysis

We aim to develop an efficient and non-invasive strategy for monitoring of drugs and their metabolites via human skin. Probe sampling was combined with thermal desorption-electrospray ionization tandem mass spectrometry (TD-ESI/MS/MS) to characterize trace terbinafine, which was secreted on patient’s skin after ingesting terbinafine tablets. The terbinafine ion signals were directly detected in the samples collected from different skin regions and the signals were monitored for 8 weeks. The detection and location of terbinafine via the skin suggest that the methods are useful in rapidly and noninvasively collecting the molecular information of the ingested drug on skin for pharmacokinetic studies.

Chemiluminescence Of Lucigenin-Tetracycline And Itsapplication For Sensitive Determination Of Procyanidin, Shuai Quan, Kaixiang Ji, Fangshuo Liu, Tesfaye Hailemariam Barkae, Mohamed Ibrahim Halawa, Saima Hanif, Baohua Lou, Jianping Li, Guobao Xu

Journal of Food and Drug Analysis

Tetracycline was reported to trigger intense lucigenin chemiluminescence (CL). Based on its significant quenching to lucigenin-tetracycline CL, procyanidin was detected. The mechanism of the lucigenin-tetracycline CL and its quenching by procyanidin were proposed. Under the optimum conditions, the calibration curve is linear in the range of 10-1000 nM for procyanidin with a detection limit of 4.4 nM. The RSD for nine replicate determinations of procyanidin was less than 1.7%. Moreover, this method was satisfactorily applied to the determination of procyanidin in human urine samples and health food capsule of grape seed.

Quantitation Of Mycophenolic Acid And Metabolites By Uplc-Ms/Ms In Renal Transplant Patients, Yun-Fen Huang, Ya-Ching Huang, Hsiao-Chen Ning, Chia-Ni Lin

Journal of Food and Drug Analysis

Mycophenolate mofetil (MMF) and enteric-coated mycophenolate sodium (EC-MPS), specific inhibitors of lymphocyte proliferation, are commonly used as adjuvant therapy with calcineurin inhibitor agents after kidney transplantation. After administration, MMF and EC-MPS are hydrolyzed to mycophenolic acid (MPA), the active form of the drug, which must be monitored due to its narrow therapeutic window, drug–drug interactions, and large intra-and inter-individual pharmacokinetic variability despite a fixed dose. Monitoring plasma MPA level is recommended to maintain the drug within the therapeutic window, optimize its efficacy, and minimize side effects. This study aims to develop a method for quantifying MPA and its major metabolites …

Camphorataimide B Suppresses The Metastasis Of Human Colorectal Cancer Cell By Inhibiting Smad/Fak/Akt Axis And Promoting Degradation Of Snail/Bmp4 Complex, Chi-Chou Huang, Chia-Hung Hung, Yi-Ju Lee, Tsui-Hwa Tseng, Yean-Jang Lee, Shao-Hsuan Kao, Chau-Jong Wang

Journal of Food and Drug Analysis

Camphorataimide B (CamB) has anticancer activities against several tumors. Here, we aimed to investigate the mechanism(s) by which CamB inhibits metastasis of colorectal cancer (CRC) cells. Low-dose CamB did not affect the cell viability and cell cycle progression of CRC cells, but significantly inhibited the metastatic potentials of CRC cells. Mechanically, CamB reduced the protein and mRNA expression of BMP4, and inhibited Smad and FAK/Src/Akt signaling. CamB also decreased Snail levels by promoting its degradation via proteasome and thereafter reduced Snail-mediated BMP4 transcription. Moreover, CamB considerably inhibited the in vivo metastasis of DLD-1 cells in xenograft mice.

Orally Delivered Perilla (Perilla Frutescens) Leaf Extract Effectively Inhibits Sars-Cov-2 Infection In A Syrian Hamster Model, Yuan-Fan Chin, Wen-Fan Tang, Yu-Hsiu Chang, Tein-Yao Chang, Wen-Chin Lin, Chia-Yi Lin, Chuen-Mi Yang, Hsueh-Ling Wu, Ping-Cheng Li, Jun-Ren Sun, Shu-Chen Hsu, Chia-Ying Lee, Hsuan-Ying Lu, Jia-Yu Chang, Jia-Rong Jheng, Cheng Cheung Chen, Jyh-Hwa Kau, Chih-Heng Huang, Cheng-Hsun Chiu, Yi-Jen Hung, Hui-Ping Tsai, Jim-Tong Horng

Journal of Food and Drug Analysis

On analyzing the results of cell-based assays, we have previously shown that perilla (Perilla frutescens) leaf extract (PLE), a food supplement and orally deliverable traditional Chinese medicine approved by the Taiwan Food and Drug Administration, effectively inhibits SARS-CoV-2 by directly targeting virions. PLE was also found to modulate virus-induced cytokine expression levels. In this study, we explored the anti-SARS-CoV-2 activity of PLE in a hamster model by examining viral loads and virus-induced immunopathology in lung tissues. Experimental animals were intranasally challenged with different SARS-CoV-2 doses. Jugular blood samples and lung tissue specimens were obtained in the acute disease stage (3−4 …

Assessing The Fate Of Fatty Acid Esters Of Hydroxy Fatty Acids, Diglycerides And Monoacetyldiacylglycerides In Grilled Ruminant Meats Marinated With Unfiltered Beer-Based Marinades, Charles F. Manful, Thu H. Pham, Evan Wheeler, Muhammad Nadeem, Oludoyin A. Adigun, Nicole Walsh, Raymond H. Thomas

Journal of Food and Drug Analysis

Ruminant meats contain functional lipids including fatty acid esters of hydroxy fatty acids (FAHFA), diglycerides (DG), monoacetyldiglycerides (MAcDG) and medium chain triglycerides (McTG) whose consumption in the normal diet can confer benefits for consumer health. However, very little is known concerning how meat processing techniques such as marination and grilling affect the quantity and quality of these functional lipids in ruminant meats. We used ultra high-performance liquid chromatography coupled to high resolution accurate mass tandem mass spectrometry (UHPLC-HRAM-MS/MS) to show how grilling following marination with either India or Wheat ale unfiltered beer-based marinades affected the quantity and quality of these …

Exosomal Proteomics In Kidney Disease: From Technical Approaches To Clinical Applications, Ageng Brahmadhi, Yung-Kun Chuang, San-Yuan Wang, Chih-Chin Kao, I-Lin Tsai

Journal of Food and Drug Analysis

Exosomes are small extracellular vesicles (sEVs) secreted from cells and have a general diameter ranging from 30~150 nm. It was reported that exosomes have essential roles in intercellular communication and can be targeted as biomarkers of disease or as therapeutic agents. Among the different techniques used for exosome investigation, the mass spectrometry-based proteomics approach has accelerated the unraveling of the molecular composition of exosomes and has contributed to improved knowledge of molecular processes in various diseases. In this review, we focused on proteomics-based studies of exosomes and clinical applications in kidney diseases. A general introduction of exosomes, isolation and characterization …

Differential Scanning Calorimetry For Authentication Of Edible Fats And Oils - What Can We Learn From The Past To Face The Current Challenges?, Mahbuba Islam, Liliana Bełkowska, Piotr Konieczny, Emilia Fornal, Jolanta Tomaszewska-Gras

Journal of Food and Drug Analysis

Fats and oils authentication has become an important issue recently, due to the growing interest in consumption of cold-pressed oils. Therefore, it is becoming more and more difficult to maintain official control over the growing assortment of new cold-pressed oils. Authenticity of plant oils is also an important issue for religious or cultural reasons.This review article focuses on the application of differential scanning calorimetry (DSC) in the field of assessing authenticity of various fats and oils (e.g. olive oil, palm oil, confectionery fats, butter). Extra virgin olive oil (EVOO) is the most comprehensively tested oil by means of the DSC …

Trends And Applications Of Food Protein-Origin Hydrolysates And Bioactive Peptides, Yi-Hsieng Samuel Wu, Yi-Chen Chen

Journal of Food and Drug Analysis

It was reported that protein hydrolysates or derived peptides have more functionalities than their parent protein. Most functional protein hydrolysates or peptides are identified from various food products, including plant, fish, and land-animal protein sources. Within a few decades, the application of food protein-origin functional hydrolysates or peptides could be divided into two main categories according to their applied intentions: 1) preservatives and bioactive packing materials; 2) nutraceutical ingredients. According to the literature, the applications of food protein-origin functional hydrolysates or peptides on food preservative and nutraceutical ingredients have attracted much attention. However, the approach method should be changed. Multi-activities, …

A Bioaffinity Self-Assembling Gel For Localized Delivery Of Immunotherapeutics, Ngoc Pham

Electronic Theses and Dissertations

Renal cell carcinoma (RCC) is a type of cancer in which malignant cells develop in the kidney tubules. Immune checkpoint inhibitors (ICI), including anti-PD-1 antibodies (aPD1), as monotherapies or in combination settings have advanced to the front-line treatment for patients diagnosed with RCC. Systemic infusion of ICI may render subtherapeutic concentrations in the lesions and increase risks of immune-related adverse events. To overcome these limitations, locoregional drug delivery has gained substantial interests as a modality in immuno-oncology. However, conventional intratumoral injections may be ineffective because the high interstitial fluid pressure in tumors resists drug retention and results in rapid local …

Hyperbranched Polyester-Based Drug Delivery System For The Optical Imaging And Treatment Of Cancer, Truptiben Patel

Electronic Theses & Dissertations

Hyperbranched polymers are well known to be a promising new class of drug delivery system for biomedical application. In this research, a novel hyperbranched polyester polymer was synthesized by following melt polymerization technique utilizing our proprietary A₂B monomer, triethylene glycol and diethylenetriaminepentaacetic acid (DTPA). This polymerization process was catalyzed by p-toluene sulfonic acid and was monitored by analyzing the polymer sample in regular time intervals. The final polymer was purified using the solvent precipitation method and characterized using MALDI-TOF, NMR, and GPC, DSC, TGA, FT-IR. The solvent diffusion method was used to formulate polymeric nanoparticle and doxorubicin drug …

Polymeric Nanocarriers For Delivery Of Small Molecules Inhibiting Tubulin Polymerization For The Treatment Of Pancreatic Cancer And Lung Metastatic Melanoma, Rajan Sharma Bhattarai

Theses & Dissertations

The aim of this thesis is to develop delivery systems for the novel small molecules which inhibit tubulin polymerization. One of the small molecules was modified to lipid conjugate to increase the lipophilicity of the molecules which in turn drastically improved the drug loading in amphiphilic polymeric system. The second molecule was conjugated to the amphiphilic polymeric backbone with pH sensitive Schiff’s linker for the tumor site specific delivery in lung metastatic melanoma model.

Chapter 1 discusses the tumor microenvironment for the solid tumor especially focusing on Pancreatic Ductal Adenocarcinoma (PDAC). Further the drug delivery system currently researched for addressing …

The Use Of Nebulizer Medications As A Possible Treatment For Covid-19, Jacob Kaufman

Honors College Theses

For the past few years, the COVID-19 pandemic has been the focal point in healthcare and research. This disease has permanently changed daily life and left a historic impact on the world. Most people have felt the effects of this pandemic either directly, via infection, or indirectly, via change in workflow, financial impact, etc. The main question for this virus still remains today; how do we treat this illness effectively? While many ideas are being tested and suggested, a definite answer has yet to be procured. Vaccine rates are climbing on a daily basis, serving as the first and most …

Finding The Optimal Ionic Liquid To Target White Blood Cell Subpopulations Using Nanoparticles, Meghan Gorniak

Honors Theses

Disorders and diseases of the immune system have become more prevalent in recent decades and can have life-threatening effects on those afflicted. Granulocyte disorders disproportionately affect infants and children, while chronic lymphocytic leukemia is the most common type of leukemia in adults. Unfortunately, current treatments have many drawbacks such as everyday injection, short-lived efficacy, and unknown levels of safety and effectiveness. It is imperative to find more effective treatment options that could allow for easier drug delivery to specific populations of white blood cells (WBCs). Nanoscale medicine shows promising application, but complications remain in reaching the desired target site and …

The Racial Divide: A Follow Up Study On Racial Disparity Amongst Covid-19 Survivors In An Urban Community, Christopher Millet, Emily Racoosin, Spandana Narvaneni, George Horani, Sherif Roman, Alisa Farokhian, Arslan Chaudhry, Sohail Chaudhry, Yezin Shamoon, Humberto Jimenez, Patrick Michael, Jin Suh

Journal of Community Hospital Internal Medicine Perspectives

Background Studies have shown that COVID-19 has had a disproportionate effect on minority groups in both the clinical and social settings in America. We conducted a follow up study on patients previously diagnosed with COVID-19 one year ago in an urban community in New Jersey. The purpose of the study was to evaluate the socioeconomic impact of COVID-19 as well as assess for receptiveness towards COVID-19 vaccination amongst various ethnic groups.

Methods This was a prospective cohort study consisting of patients who had recovered from COVID-19 one year prior. The patients included in the study had a confirmed COVID-19 diagnosis …

Identification Of Translesion Synthesis Inhibitors That Target Rev7/Rev3 Protein-Protein Interactions, Seema Patel

University Scholar Projects

Translesion synthesis (TLS) is a cellular mechanism utilized by cancer cells to tolerate DNA damage caused by chemotherapeutics, like cisplatin, by replicating past unrepaired lesions. This increases the rate of mutations, which leads to the emergence of drug-resistant cancer cells. Preliminary studies have shown that disrupting the protein-protein interactions (PPI) in the TLS heteroprotein complex increases cells’ sensitivity to first-line genotoxic chemotherapy, illustrating how inhibiting TLS assembly and function can significantly increase cancer cell death. These results underscore the therapeutic potential of targeting TLS PPI. My thesis focuses on identifying inhibitors capable of disrupting the Rev7/Rev3 TLS PPI. This study …