Digital Commons Network^™

The Impact Of Maternal Environmental Exposures On The Immune Landscape Of The Maternal-Fetal Interface, Heather E. True

Theses and Dissertations--Pharmacy

Maternal environmental exposures and inflammation are known to impact fetal development in utero as well as increase the risk for chronic diseases later in life. Therefore, it is essential that we understand how the maternal environment alters placental structure and immune function.

Therefore, we isolated fetal (chorionic villous) tissues from the placentas of healthy pregnant participants (control) and those with inflammatory conditions, including SARS-COV-2 infection, pregravid obesity, and opioid use disorder. Isolated placental leukocytes were subjected to a series of assays to uncover the phenotypic, functional, and transcriptional (single-cell RNA-sequencing) landscape of placental immune cells. Additional placental tissues were subjected …

Simplified Molecular Diagnostics Methods For Environmental Testing Of Pathogens And Other Applications, Mohammad Dehghan Banadaki

Theses and Dissertations--Mechanical and Aerospace Engineering

Environmental Surveillance (ES) has emerged as a powerful tool to monitor the emergence and persistence of a pathogen within a facility or community using wastewater/water analysis. Over the past four years, implementation of ES has experienced an explosive acceleration, driven by efforts to monitor the spread of SARS-CoV-2, which has made ES a routinely used public health tool in many locations. We can now use ES to measure the emergence/presence/abundance of different viruses (e.g., SARS-CoV-2, influenza, mpox), bacteria (e.g., E. coli, V. cholerae, antibiotic-resistant strains), fungi (e.g., C. auris), and other biological markers indicative of infectious diseases. However, its broad …

Engineering Novel Biomimetic Models Of Equine Host-Microbe Interactions, Lynn M. Leedhanachoke

Theses and Dissertations--Veterinary Science

One major limitation to the study of equine disease is the lack of in vitro models that accurately reflect the dynamic physiology of the horse. Thus, there is a pressing need to develop in vitro systems that recapitulate diverse equine tissue and organ microenvironments. To overcome this deficiency, we developed multiple biomimetic equine organoid systems that can be employed for a variety of investigations aimed at resolving outstanding questions in infectious disease pathogenesis, therapeutic development and drug metabolism, host defense mechanisms, immune-modulated disease, and equine developmental biology. Our innovative platform incorporates novel equine intestinal enteroid and airway organoid models derived …

“What We Say Matters”: Reducing Drug Use Stigmatization By Healthcare Professionals Through A Narrative-Based Awareness Campaign, Adam M. Traugott

DNP Projects

Background: Stigma towards substance use has been identified as a contributor to poor care and health outcomes amongst people who use drugs (PWUD), a vulnerable population. Healthcare worker stigma negatively affects the outcomes of individual patients and may have greater public health implications. Methods of addressing drug-use stigma have previously been implemented in a variety of clinical settings. Presenting the lived experience of PWUD to healthcare workers via emotionally compelling narratives has been found to be an effective means of stigma reduction.

Purpose: This study utilized two psychometrically validated tools to measure (1) the baseline prevalence of drug use stigma …

Outcomes Of A Pilot Randomized Clinical Trial Testing Brief Interventions To Increase Hiv Pre-Exposure Prophylaxis Uptake Among Rural People Who Inject Drugs Attending Syringe Services Programs, Hilary L. Surratt, Sarah Brown, Abby L. Burton, Will Cranford, Laura C. Fanucchi, Christie Green, Stephanie M. Mersch, Rebecca Rains, Philip M. Westgate

Biostatistics Faculty Publications

Background: Kentucky is one of seven states with high, sustained rural HIV transmission tied to injection drug use. Expanding access to pre-exposure prophylaxis (PrEP) has been endorsed as a key HIV prevention strategy; however, uptake among people who inject drugs (PWID) has been negligible in rural areas. Syringe services programs (SSPs) have been implemented throughout Kentucky’s Appalachian region, providing an important opportunity to integrate PrEP services. Objectives: The primary objective was to examine preliminary efficacy and effect sizes of the study interventions on PrEP initiation among HIV-negative PWID. Design: Parallel group randomized controlled trial. Methods: Eighty participants were enrolled from …

How Patient, Infection, And Cysticercus Characteristics Impact The Evolution Of Taenia Solium Larva In The Human Brain, Hongbin Zhang, Meghana G. Shamsunder, Pryanka Bawa, Arturo Carpio, W. Allen Hauser, Karina Quinde-Herrera, Alex Jaramillo, Elizabeth A. Kelvin

Biostatistics Faculty Publications

Neurocysticercosis is a poorly understood infection of the central nervous system with Taenia solium larva and treatment often fails to kill all the parasitic larva. Most research on this infection has used patient-level data, looking at summaries of the encysted parasitic cysticercus burden. Cyst-level analysis is needed to identify factors that impact individual cyst trajectories and how that may vary based on characteristics of the patient, infection, and cyst being followed. We disaggregated data on 221 cysts from 117 patients who participated in a trial evaluating the impact of albendazole treatment to identify factors that impact cyst evolution over time …

Characterization Of Municipal Solid Waste Using Machine Learning Based Imaging And Chemical Analysis, Enyonam Ahadzi

Theses and Dissertations--Biosystems and Agricultural Engineering

According to the U.S. Department of Energy’s 2023-billion-ton report, the United States generates nearly 300 million tons of municipal solid waste every year. More than 50% of that waste is underutilized and ends up in landfills. This poses serious public health and environmental risks. But beyond the environmental and health implications, this is also a wasted opportunity. The U.S. Energy Information Administration estimates that, for every 100 lbs of waste we throw away, about 85% can be used as fuel or for electricity generation. However, its complex and heterogeneous nature poses challenges for efficient sorting and valorization in waste to …

Covid-19 Impacts On Refugee-Serving Ngos In Clarkston And The Atlanta Metropolitan Area, Georgia, United States, Ghazal Khaksari

Theses and Dissertations--Anthropology

This study provides a unique anthropological perspective on how technology, language, NGO staff, and culture intersect during emergency times, which can affect NGOs’ abilities to reach and help communities. Findings of this study emphasize the importance of equality in digital access and of available and culturally competent interpreters in reducing the gap between NGOs and refugee communities. The digital divide has played a fundamental role during the pandemic, often leading to other problems for both NGOs and refugees. This issue has taken a variety of forms in terms of access to education, language classes, community cohesion, and communication with and …

Intracellular Survival Of Staphylococcus Aureus In Macrophages During Osteomyelitis, William A. Lathram, Christopher D. Radka

Microbiology, Immunology, and Molecular Genetics Faculty Publications

Staphylococcus aureus, traditionally viewed as an extracellular pathogen, is increasingly recognized for its ability to persist intracellularly, particularly within macrophages. This intracellular lifestyle is central to osteomyelitis, a chronic bone infection characterized by persistent inflammation, bone destruction, and impaired repair. Within bone, S. aureus exploits macrophage plasticity by driving a shift from pro-inflammatory, bactericidal M1-like states to anti-inflammatory, tissue-reparative M2-like phenotypes. This polarization suppresses immune clearance and promotes an environment conducive to bacterial survival and dissemination. Additional strategies – including biofilm formation, small colony variants, and inhibition of phagolysosomal killing – further enhance persistence and immune evasion. While these …

Breaking Ground On Self-Efficacy: A Qualitative Exploration Of Intimate Partner Violence Survivors’ Experiences With Therapeutic Horticulture, Rachel Marie Reeves

Theses and Dissertations--Sociology

This dissertation explores how participation in a Therapeutic Horticulture program may support the self-efficacy of survivors of Intimate Partner Violence. Through a thematic analysis of interviews with 45 women living at a domestic violence shelter in Lexington, Kentucky, this study investigates whether and how gardening activities contributed to participants’ belief in their abilities to shape outcomes in their lives.

Findings are organized around Bandura’s four sources of self-efficacy: enactive mastery experiences, vicarious experience, verbal persuasion, and physiological/affective states. These insights contribute to feminist criminology and trauma-informed care literature by illuminating the transformative potential of nature-assisted healing programs.

Examining Associations Between Sociodemographic Characteristics And Ever Breastfed Children, Nhanes 1999–2020, Jessica Amezcua, Lindsey M. West, Camelia Malkami, Marlo M. Vernon, Elinita Pollard, Justin X. Moore

Markey Cancer Center Faculty Publications

Although breastfeeding provides health benefits to both mother and child, this study aimed to explore whether disparities in breastfeeding continue to exist, particularly among non-Hispanic Black (NHB) mothers and children. We performed a cross-sectional analysis among 19,830 children in the United States (US) using the National Health and Nutrition Examination Survey (NHANES) data from 1999 to 2020. Breastfeeding initiation and duration rates increased overall from 1999 to 2020. Children who were ever breastfed were more likely to have higher body weight, older mothers, mothers who did not smoke during pregnancy, a higher family poverty–income ratio (PIR), food security, mothers in …

Bacteria-Engineered Vesicles For Cancer Immunotherapy: From Immunomodulation In Vitro To Anti-Tumor Effects In Melanoma Models, Lan Li

Theses and Dissertations--Chemistry

Bacterial vesicles hold immense potential in various biomedical fields. Among these, outer membrane vesicles (OMVs) produced by Gram-negative bacteria are the most extensively studied. While the exact mechanism of OMV production remains unclear, numerous environmental factors have been shown to influence both their yield and composition. In this study, we investigated the effect of three different antimicrobial families on OMV production by E. coli. Interestingly, antimicrobials within the same family did not provide the same effects on OMV yield, suggesting that OMV production may not directly correlate with the antimicrobial mechanism of action. OMVs have demonstrated tumor-inhibitory activity in multiple …

Responses To Tobacco Use Screening And Tobacco Cessation Assistance Offers Among Rural And Ubran Cancer Survivors At An Nci-Designated Comprehensive Cancer, Abigayle Feather

Theses and Dissertations--Psychology

Smoking after cancer diagnosis is associated with poor outcomes while smoking cessation can improve quality of life and promote survival. Although there are calls for equitable tobacco treatment delivery in cancer and other healthcare settings, disparities in tobacco-related disease burden persist. In the context of an outpatient cancer center’s population-based tobacco treatment program, this study examines the association between cancer survivors’ rural and Appalachian residence and 1) current tobacco use status, 2) decision to decline cessation assistance, and 3) reason for declining assistance. A cross-sectional, retrospective analysis was conducted using electronic health record data from 16,839 adults (64.04% female, 88.49% …

The Effects Of Plx 3397 On Fentanyl Escalation In Male And Female Sprague-Dawley Rats, Jakob Shaykin

Theses and Dissertations--Psychology

Purpose: A hallmark characteristic of opioid use disorder (OUD) is a persons’ ‘loss of control’, which leads to the compulsion to seek and obtain opioids, resulting in increased consumption. This phenomenon is known as ‘escalation of drug intake’. Chronic opioid use causes microglial reactivity and subsequent release of proinflammatory cytokines leading to neuroinflammation, weakening one’s executive control over drug taking and ultimately leading to greater use. Previous research in our laboratory has explored heterogeneity of fentanyl intake in extended access (6-hr) sessions and identified distinct endophenotypes of fentanyl intake. Moreover, we have observed a positive correlation between Treml1 expression …

A Method For Teaching Biostatisticians To Write And Critique Specific Aims, Jesse D. Troy, Gregory P. Samsa, Megan L. Neely, Steven C. Grambow, Sarah Peskoe, Emily Slade, Gina Maria Pomann

Biostatistics Faculty Publications

This article describes the design of a single instructional module on writing and critiquing the Specific Aims (SA) section of a grant proposal for a biomedical research study. It begins by discussing the goals and learning objectives for the module. Then, it discusses the typical structure of a SA section to orient readers to the core content. This method is rooted in using an established framework for scientific writing, which is presented in brief, along with examples of how it applies to writing the SA section. Then, the structure of the instructional module is described, including the exercises for participants. …

Using Public Funeral And Obituary Listings To Identify Spikes In Excess Mortality In One Appalachian County, Allen Archer, Melissa White, Megan Quinn, Randy Wykoff

Journal of Appalachian Health

Introduction: Delays (10-22 months) in availability of official state and county-level mortality data could have significant public health consequences. The COVID-19 pandemic illuminated the need for health officials to access timely death data to identify unexpected increases in mortality in their communities.

Purpose: The purpose of this study is to determine if funeral home listings and/or newspaper obituaries could help identify excess mortality on the local level, prior to the availability of official death records.

Methods: To calculate excess mortality, four years (2017–2020) of data were collected from three sources: the state health department, online funeral home listings, and newspaper …

Bacillus Thuringiensis Cry14a Family Proteins As Novel Anthelmintics Against Gastrointestinal Nematode Parasites, Duy Hoang, Kelly Flanagan, Qian Ding, Nicholas R. Cazeault, Hanchen Li, Stefani Díaz-Valerio, Florentina Rus, Esther A. Darfour, Elizabeth Kass, Katherine H. Petersson, Martin Krarup Nielsen, Heiko Liesegang, Gary R. Ostroff, Raffi V. Aroian

Veterinary Science Faculty Publications

Bacillus thuringiensis crystal (Cry) proteins have been expressed in commercial transgenic crops for nearly 30 years, providing safe and effective control of insect pests and significantly reducing the application of hazardous chemical pesticides. B. thuringiensis crystal proteins have also been shown to target parasitic nematodes, including plant parasitic nematodes. Recently, transgenic soybean crops expressing Cry14Ab have been shown to provide control against the soybean cyst nematode Heterodera glycines, marking the first time a crystal protein is being commercialized in transgenic crops for control of a nematode pest. However, apart from H. glycines and the free-living nematode, Caenorhabditis elegans, the breadth …

Lessons Learned From Developing Dashboards To Support Decision-Making For Community Opioid Response By Community Stakeholders: Mixed Methods And Multisite Study., Naleef Fareed, Ramona G. Olvera, Yiting Wang, Michael Hayes, Elizabeth Liz Larimore, Peter Balvanz, Ronald Langley, Corinna A. Noel, Peter J. Rock, Daniel Redmond, Jessica Neufeld, Sarah Kosakowski, Daniel R. Harris, Marc Larochelle, Timothy R. Huerta, Lashawn Glasgow, Emmanuel Oga, Jennifer Villani, Elwin Wu

Center on Drug and Alcohol Research Faculty Publications

BACKGROUND: Data dashboards are published tools that present visualizations; they are increasingly used to display data about behavioral health, social determinants of health, and chronic and infectious disease risks to inform or support public health endeavors. Dashboards can be an evidence-based approach used by communities to influence decision-making in health care for specific populations. Despite widespread use, evidence on how to best design and use dashboards in the public health realm is limited. There is also a notable dearth of studies that examine and document the complexity and heterogeneity of dashboards in community settings.

OBJECTIVE: Community stakeholders engaged in the …

Buprenorphine Prescribing Characteristics Following Relaxation Of X-Waiver Training Requirements., Paul J. Christine, Rouba A. Chahine, Simeon D. Kimmel, Nicole Mack, Christian Douglas, Thomas J. Stopka, Katherine Calver, Laura C. Fanucchi, Svetla Slavova, Michelle R. Lofwall, Daniel J. Feaster, Michael Lyons, Jerel M. Ezell, Marc R. Larochelle

Center on Drug and Alcohol Research Faculty Publications

IMPORTANCE: Local-level data are needed to understand whether the relaxation of X-waiver training requirements for prescribing buprenorphine in April 2021 translated to increased buprenorphine treatment.

OBJECTIVE: To assess whether relaxation of X-waiver training requirements was associated with changes in the number of clinicians waivered to and who prescribe buprenorphine for opioid use disorder and the number of patients receiving treatment.

DESIGN, SETTING, AND PARTICIPANTS: This serial cross-sectional study uses an interrupted time series analysis of 2020-2022 data from the HEALing Communities Study (HCS), a cluster-randomized, wait-list-controlled trial. Urban and rural communities in 4 states (Kentucky, Massachusetts, New York, and Ohio) …

Antimicrobial Resistance In Equines: A Growing Threat To Horse Health And Beyond-A Comprehensive Review., Ajran Kabir, Bibek Lamichhane, Tasmia Habib, Alexis Adams, Hossam El-Sheikh Ali, Nathan M Slovis, Mats H. T. Troedsson, Yosra A. Helmy

Veterinary Science Faculty Publications

The equine industry holds substantial economic importance not only in the USA but worldwide. The occurrence of various infectious bacterial diseases in horses can lead to severe health issues, economic losses, and restrictions on horse movement and trade. Effective management and control of these diseases are therefore crucial for the growth and sustainability of the equine industry. While antibiotics constitute the primary treatment strategy for any bacterial infections in horses, developing resistance to clinically important antibiotics poses significant challenges to equine health and welfare. The adverse effects of antimicrobial overuse and the escalating threat of resistance underscore the critical importance …

Association Of Circulating Markers Of Microbial Translocation And Hepatic Inflammation With Liver Injury In Patients With Type 2 Diabetes, Leila Gobejishvili, Vatsalya Vatsalya, Diana V. Avila, Yana B. Feygin, Craig J. Mcclain, Sriprakash Mokshagundam, Shirish Barve

Biostatistics Faculty Publications

Background: Virtually the entire spectrum of liver disease is observed in association with type 2 diabetes mellitus (T2DM); indeed, T2DM is now the most common cause of liver disease in the U.S. We conducted a pilot study to investigate the relevance of increased microbial translocation and systemic inflammation in the development of liver injury in patients with T2DM. Methods: Patients with T2DM (n = 17) and non-diabetic controls (NDC; n = 11) aged 25–80 yrs. participated in this study. Serum levels of endotoxin, calprotectin, soluble CD14 and CD163, and several inflammatory cytokines were measured. In addition to standard liver injury …

Plant Model Of Α-Synucleinopathy: Expression Of Α-Synuclein A53t Variant In Hairy Root Cultures Leads To Proteostatic Stress And Dysregulation Of Iron Metabolism, Jasmina Kurepa, Kristen A. Bruce, Greg A. Gerhardt, Jan A. Smalle

Neurology Faculty Publications

Synucleinopathies, typified by Parkinson’s disease (PD), entail the accumulation of α- synuclein (αSyn) aggregates in nerve cells. Various αSyn mutants, including the αSyn A53T variant linked to early-onset PD, increase the propensity for αSyn aggregate formation. In addition to disrupting protein homeostasis and inducing proteostatic stress, the aggregation of αSyn in PD is associated with an imbalance in iron metabolism, which increases the generation of reactive oxygen species and causes oxidative stress. This study explored the impact of αSyn A53T expression in transgenic hairy roots of four medicinal plants (Lobelia cardinalis, Artemisia annua, Salvia miltiorrhiza, and Polygonum multiflorum). In all …

A Multi-Ancestry Genetic Study Of Pain Intensity In 598,339 Veterans., Sylvanus Toikumo, Rachel Vickers-Smith, Zeal Jinwala, Heng Xu, Divya Saini, Emily E Hartwell, Mirko Pavicic, Kyle A Sullivan, Ke Xu, Daniel A Jacobson, Joel Gelernter, Christopher T Rentsch, Million Veteran Program, Eli Stahl, Martin Cheatle, Hang Zhou, Stephen G Waxman, Amy C Justice, Rachel L Kember, Henry R Kranzler

Epidemiology and Environmental Health Faculty Publications

Chronic pain is a common problem, with more than one-fifth of adult Americans reporting pain daily or on most days. It adversely affects the quality of life and imposes substantial personal and economic costs. Efforts to treat chronic pain using opioids had a central role in precipitating the opioid crisis. Despite an estimated heritability of 25-50%, the genetic architecture of chronic pain is not well-characterized, in part because studies have largely been limited to samples of European ancestry. To help address this knowledge gap, we conducted a cross-ancestry meta-analysis of pain intensity in 598,339 participants in the Million Veteran Program, …

Relative Adrenal Insufficiency Is A Risk Factor For Pediatric Sepsis: A Proof-Of-Concept Study, Dan Hao, Ling Guo, Qian Wang, Misa Ito, Bin Huang, Chieko Mineo, Philip W. Shaul, Xiang-An Li

Markey Cancer Center Faculty Publications

Glucocorticoid (GC) therapy had been strongly recommended for pediatric sepsis (grade 1A). However, the recommendation was changed to grade 2C in 2020 due to weak evidence. About 32.8% of patients with pediatric septic develop relative adrenal insufficiency (RAI). But whether GC therapy should be determined by RAI status is controversial. This study utilized 21-day- old SF1CreSRBIfl/fl mice as the first pediatric RAI mouse model to assess the pathogenesis of RAI and evaluate GC therapy. RAI mice exhibited a substantially higher mortality rate in cecal ligation and puncture and cecal slurry–induced sepsis. These mice featured persistent inflammatory responses and were effectively …

Interferons In Viral Infections, Pracheta Sengupta, Saurabh Chattopadhyay

Markey Cancer Center Faculty Publications

No abstract provided.

Wastewater Surveillance For Identifying Sars-Cov-2 Infections In Long-Term Care Facilities, Kentucky, Usa, 2021–2022, James W. Keck, Reuben Adatorwovor, Matthew Liversedge, Blazan Mijotavich, Cullen Olsson, William D. Strike, Atena Amirsoleimani, Ann Noble, Soroosh Torabi, Alexus Lori Rockward, Mohammad Dehghan Banadaki, Ted Smith, Parker Lacy, Scott M. Berry

UK CARES Faculty Publications

Persons living in long-term care facilities (LTCFs) were disproportionately affected by COVID-19. We used wastewater surveillance to detect SARS-CoV-2 infection in this setting by collecting and testing 24-hour compos- ite wastewater samples 2–4 times weekly at 6 LTCFs in Kentucky, USA, during March 2021–February 2022. The LTCFs routinely tested staff and symptomatic and exposed residents for SARS-CoV-2 using rapid antigen tests. Of 780 wastewater samples analyzed, 22% (n = 173) had detectable SARS-CoV-2 RNA. The LTCFs reported 161 positive (of 16,905) SARS-CoV-2 clinical tests. The wastewater SARS-CoV-2 signal showed vari- able correlation with clinical test data; we observed the strongest …

Staphylococcus Aureus Oleate Hydratase Produces Ligands That Activate Host Ppara, Christopher D. Radka, Matthew W. Frank, Tyler S. Simmons, Cydney N. Johnson, Jason W. Rosch, Charles O. Rock

Markey Cancer Center Faculty Publications

Commensal gut bacteria use oleate hydratase to release a spectrum of hydroxylated fatty acids using host-derived unsaturated fatty acids. These compounds are thought to attenuate the immune response, but the underlying signaling mechanism(s) remain to be established. The pathogen Staphylococcus aureus also expresses an oleate hydratase and 10- hydroxyoctadecanoic acid (h18:0) is the most abundant oleate hydratase metabolite found at Staphylococcal skin infection sites. Here, we show h18:0 stimulates the transcription of a set of lipid metabolism genes associated with the activation of peroxisome proliferator activated receptor (PPAR) in the RAW 264.7 macrophage cell line and mouse primary bone marrow-derived …

Applying The Concepts Of Animal Husbandry To Human Health, John Madany

IGC Proceedings (1977-2023)

Instead of a new paradigm, we need to modify an old paradigm. We must focus on restoring soil health and reviving human health. Ruminants can be used to heal the land and the people. Pastoralists, by managing ruminants, can preserve their own health while promoting soil health.

Forage Plant Secondary Metabolites At The Intersection Of Animal Health, Productivity, And Sustainability, Michael D. Flythe

IGC Proceedings (1977-2023)

The nutritive value of forages and other feeds have long been studied. However, the roles of secondary metabolites produced by forage-plants and their endophytes are an emerging field. Historically, the term secondary metabolite was used for compounds that were less abundant in plant tissue than the primary metabolites, such as fiber, sugars, and protein. It has become clear that secondary metabolites play important roles in the ecology of forage plants, and that both the detrimental and beneficial impacts of secondary metabolites to forage-animals should not be underestimated. This thematic session will consider fescue toxicosis, a detrimental outcome of ergot alkaloids …

Simple Sars-Cov-2 Concentration Methods For Wastewater Surveillance In Low Resource Settings, Mohammad Dehghan Banadaki, Soroosh Torabi, Alexus Lori Rockward, William Strike, Ann Noble, James W. Keck, Scott M. Berry

UK CARES Faculty Publications

Wastewater-based epidemiology (WBE) measures pathogens in wastewater to monitor infectious disease prevalence in communities. Due to the high dilution of pathogens in sewage, a concentration method is often required to achieve reliable biomarker signals. However, most of the current concentration methods rely on expensive equipment and labor-intensive processes, which limits the application of WBE in low-resource settings. Here, we compared the performance of four inexpensive and simple concentration methods to detect SARS-CoV-2 in wastewater samples: Solid Fraction, Porcine Gastric Mucin-conjugated Magnetic Beads, Calcium Flocculation- Citrate Dissolution (CFCD), and Nanotrap® Magnetic Beads (NMBs). The NMBs and CFCD methods yielded the highest …