Virology Commons^™

Interactions Of The Sars-Cov-2 Viral Genome 3’-Untranslated Region With Viral And Host Rnas, Caleb Frye, Mihaela Rita Mihailescu

Electronic Theses and Dissertations

This dissertation focuses on the characterization of RNA-RNA interactions within the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genome and with host microRNAs. As the causative agent of coronavirus disease 2019 (COVID-19), SARS-CoV-2 has evolved rapidly since its appearance. This has warranted prompt characterization of the virus particularly of its single stranded RNA (ssRNA) genome. By using a combination of bioinformatics, biophysics, and/or biological assays, we analyzed the SARS-CoV-2 viral genomic RNA and uncovered interactions of genomic RNA with host RNAs, highlighting an underutilized method of targeting RNA viruses. We showed here that the conserved elements in the viral genomic …

Multi-Antigen Elephant Endotheliotropic Herpesvirus (Eehv) Mrna Vaccine Induces Humoral And Cell-Mediated Responses In Mice, Jessica R Watts, Jennifer L Spencer Clinton, Jeroen Pollet, Rongsheng Peng, Jie Tan, Paul D Ling

Faculty, Staff and Students Publications

Background/Objectives: Elephant endotheliotropic herpesvirus (EEHV) causes lethal hemorrhagic disease (HD) in Asian and African elephants in human care and the wild. It is the leading cause of death for young Asian elephants in North American and European zoos despite sensitive diagnostic tests and improved treatments. Thus, there is a critical need to develop an effective vaccine to prevent severe illness and reduce mortality from EEHV-HD. We generated a multi-antigenic EEHV mRNA vaccine to address this need that encodes the EEHV1A-subtype glycoproteins gB, gH, gL, and gO. These conserved proteins are the entry machinery for several herpesviruses in the betaherpesvirus subfamily …

The Rise, Fall, And Resurgence Of Phage Therapy For Urinary Tract Infection, Jacob J Zulk, Kathryn A Patras, Anthony W Maresso

Faculty, Staff and Students Publications

In the face of rising antimicrobial resistance, bacteriophage therapy, also known as phage therapy, is seeing a resurgence as a potential treatment for bacterial infections including urinary tract infection (UTI). Primarily caused by uropathogenic Escherichia coli, the 400 million UTI cases annually are major global healthcare burdens and a primary cause of antibiotic prescriptions in the outpatient setting. Phage therapy has several potential advantages over antibiotics including the ability to disrupt bacterial biofilms and synergize with antimicrobial treatments with minimal side effects or impacts on the microbiota. Phage therapy for UTI treatment has shown generally favorable results in recent …

Elucidating The Mechanism Of Interleukin-17a Signaling In The Pathogenesis Of West Nile Virus, Farzana Nazneen

Dissertations

West Nile Virus (WNV), a neurotropic flavivirus transmitted by mosquitoes, is a leading cause of viral encephalitis in the United States and remains a significant public health concern. Unfortunately, no vaccine or therapeutic intervention is available against WNV infection. Previous studies, including ours, demonstrated that interleukin-17A (IL-17A) signaling increases the cytotoxic potential of CD8⁺ T cells to facilitate WNV and parasite clearance; however, the molecular mechanism is not understood. IL-17 receptor C (IL-17RC) is an obligatory co-receptor with IL-17 receptor A (IL-17RA) for signaling induced by IL-17A, IL-17A/F, and IL-17F. In this study, we found that IL-17RC deficient ( …

Enhancement Of Zika Virus Infection By West Nile And Dengue Virus Antibody Positive Cord-Blood Samples From Mothers In El Paso-Texas, Jeanette Orbegozo

Open Access Theses & Dissertations

The recent Zika virus (ZIKV) outbreak and its association with microcephaly cases and other neurological syndromes are important public health concerns. A phenomenon known as antibody-dependent enhancement (ADE) involves preexisting flavivirus antibodies to a specific flavivirus that enhances the severity of a second infection with a different flavivirus. Since ZIKV is related to flaviviruses like West Nile virus (WNV) and dengue virus (DENV), prior exposure to these viruses could elicit cross-reactive antibodies that may enhance a subsequent ZIKV infection. Therefore, this study aimed to determine if WNV and DENV antibodies from cord blood enhanced ZIKV infection in vitro. Initially, a …

Implementing The Methodology To Generate Diagnostic And Therapeutic Antibodies, Andrea Mariana Carlin

Open Access Theses & Dissertations

This research aimed to establish the methodology for developing monoclonal antibodies at The University of Texas at El Paso. Monoclonal antibodies are indispensable across multiple fields, including diagnostics, therapeutics, and biotechnology, yet there is a shortage of commercially available antibodies targeting specific proteins or possessing specialized characteristics. To address this gap, three models were selected: a peptide derived from the intracellular protein Schlafen 8 and two cell surface protein receptors: mouse vascular endothelial growth factor receptor 2 (VEGFR2) and human melanocortin receptor 4 (MC4R). These proteins present unique challenges in antibody generation, including issues of accessibility, expression, and specificity.

Throughout …

Infant-Derived Human Nasal Organoids Exhibit Relatively Increased Susceptibility, Epithelial Responses, And Cytotoxicity During Rsv Infection, Gina M Aloisio, Divya Nagaraj, Ashley M Murray, Emily M Schultz, Trevor Mcbride, Letisha Aideyan, Erin G Nicholson, David Henke, Laura Ferlic-Stark, Anubama Rajan, Amal Kambal, Hannah L Johnson, Elina Mosa, Fabio Stossi, Sarah E Blutt, Pedro A Piedra, Vasanthi Avadhanula

Faculty, Staff and Students Publications

Background: Respiratory syncytial virus (RSV) causes significant morbidity and mortality, especially in young children. Why RSV infection in children is more severe compared to healthy adults is not fully understood.

Methods: We used ex-vivo human nasal organoid platforms from infants and adults to investigate the underlying mechanism of this disease disparity at the initial site of RSV replication, the nasal epithelium.

Results: Infant-derived human nasal organoid-air liquid interface (HNO-ALIs) lines were more susceptible to early RSV replication. Moreover, infant-derived HNO-ALIs elicited a statistically significant greater overall cytokine response, enhanced mucous production, and greater cellular damage compared to their adult counterparts. …

Host Dna Depletion On Frozen Human Respiratory Samples Enables Successful Metagenomic Sequencing For Microbiome Studies, Minsik Kim, Raymond C Parrish, Michael J Tisza, Viral S Shah, Thi Tran, Matthew Ross, Juwan Cormier, Aribah Baig, Ching-Ying Huang, Laura Brenner, Isabel Neuringer, Katrine Whiteson, J Kirk Harris, Amy D Willis, Peggy S Lai

Faculty, Staff and Students Publications

Most respiratory microbiome studies use amplicon sequencing due to high host DNA. Metagenomics sequencing offers finer taxonomic resolution, phage assessment, and functional characterization. We evaluated five host DNA depletion methods on frozen nasal swabs from healthy adults, sputum from people with cystic fibrosis (pwCF), and bronchoalveolar lavage (BAL) from critically ill patients. Median sequencing depth was 76.4 million reads per sample. Untreated nasal, sputum, and BAL had 94.1%, 99.2%, and 99.7% host reads, respectively. Host depletion effects varied by sample type, generally increasing microbial reads, species and functional richness; this was mediated by higher effective sequencing depth. Rarefaction curves showed …

Nuclear Polyhedrosis Virus (Npv) Motivates Wandering Behavior In The Gulf Fritillary Caterpillar, \Emph{Dione Vanillae}, Kayla Lizaola, Tiffany Bresnan, Arietta Fleming-Davies

Annual Symposium on Biomathematics and Ecology Education and Research

No abstract provided.

Gene Discovery From Microbial Gene Libraries I: Protection Against Reactive Oxygen Species-Driven Dna Damage, John C Taylor, Carmen Gu Liu, James D Chang, Brianna E Thompson, Anthony W Maresso

Faculty, Staff and Students Publications

Reactive oxygen species (ROS) pose a lethal risk for all life forms by causing damage to cell processes, genome-wide DNA damage-driving mutation, replicative instability, and death. Thus, the development of mechanisms to resist or repair ROS-induced DNA damage is critical for the reliable replication of nucleic acids. DNA repair and protection mechanisms have been discovered in all forms of life. However, the vast array of microbes that may harbor novel repair or protection mechanisms, especially bacterial viruses, have not been adequately assessed. Here, we screened a microbial gene library composed primarily of phage open reading frames (ORFs) to uncover elements …

A Cross-Systems Primer For Synthetic Microbial Communities, Elijah C Mehlferber, Gontran Arnault, Bishnu Joshi, Laila P Partida-Martinez, Kathryn A Patras, Marie Simonin, Britt Koskella

Faculty, Staff and Students Publications

The design and use of synthetic communities, or SynComs, is one of the most promising strategies for disentangling the complex interactions within microbial communities, and between these communities and their hosts. Compared to natural communities, these simplified consortia provide the opportunity to study ecological interactions at tractable scales, as well as facilitating reproducibility and fostering interdisciplinary science. However, the effective implementation of the SynCom approach requires several important considerations regarding the development and application of these model systems. There are also emerging ethical considerations when both designing and deploying SynComs in clinical, agricultural or environmental settings. Here we outline current …

Construction And Characterization Of Dna Libraries From Cultured Phages And Environmental Viromes, Carmen Gu Liu, Brianna E Thompson, James D Chang, Lorna Min, Anthony W Maresso

Faculty, Staff and Students Publications

Despite many efforts to understand and leverage the functional potential of environmental viromes, most bacteriophage genes are largely uncharacterized. To explore novel biology from uncultivated microbes like phages, metagenomics has emerged as a powerful tool to directly mine new genes without the need to culture the diverse microbiota and the viruses within. When a pure computational approach cannot infer gene function, it may be necessary to create a DNA library from environmental genomic DNA, followed by the screening of that library for a particular function. However, these screens are often initiated without a metagenomic analysis of the completed DNA library …

Corrigendum: Sars-Cov-2 Seroprevalence In Pregnant Women In Kilifi, Kenya From March 2020 To March 2022, Angela Koech, Geoffrey Omuse, Joseph Mutunga, Moses Mukhanya, Onesmus Wanje, Grace Mwashigadi, Geoffrey Katana, Rachel Craik, Alex Mugo, Marleen Temmerman

Pathology, East Africa

Background: Seroprevalence studies are an alternative approach to estimating the extent of transmission of SARS-CoV-2 and the evolution of the pandemic in different geographical settings. We aimed to determine the SARS-CoV-2 seroprevalence from March 2020 to March 2022 in a rural and urban setting in Kilifi County, Kenya.

Methods: We obtained representative random samples of stored serum from a pregnancy cohort study for the period March 2020 to March 2022 and tested for antibodies against the spike protein using a qualitative SARS-CoV-2 ELISA kit (Wantai, total antibodies). All positive samples were retested for anti-SARS-CoV-2 anti-nucleocapsid antibodies (Euroimmun, ELISA kits, NCP, …

Influenza C Virus In Us Children With Acute Respiratory Infection 2016–2019, Bethany K Sederdahl, Geoffrey A Weinberg, Angela P Campbell, Rangaraj Selvarangan, Jennifer E Schuster, Joana Y Lively, Samantha M Olson, Julie A Boom, Pedro A Piedra, Natasha B Halasa, Laura Stewart, Peter G Szilagyi, G K Balasubramani, Theresa Sax, Judith M Martin, Robert W Hickey, Marian G Michaels, John V Williams, New Vaccine Surveillance Network

Faculty, Staff and Students Publications

Influenza C virus (ICV) is an orthomyxovirus related to influenza A and B, yet due to few commercial assays, epidemiologic studies may underestimate incidence of ICV infection and disease. We describe the epidemiology and characteristics of ICV within the New Vaccine Surveillance Network (NVSN), a Centers for Disease Control and Prevention (CDC)-led network that conducts population-based surveillance for pediatric acute respiratory illness (ARI). Nasal or/combined throat swabs were collected from emergency department (ED) or inpatient ARI cases, or healthy controls, between 12/05/2016-10/31/2019 and tested by molecular assays for ICV and other respiratory viruses. Parent surveys and chart review were used …

Rotavirus Vaccine Effectiveness Against Severe Acute Gastroenteritis: 2009-2022, Alpha Oumar Diallo, Mary E Wikswo, Iddrisu Sulemana, Leila C Sahni, Julie A Boom, Sasirekha Ramani, Rangaraj Selvarangan, Mary E Moffatt, Christopher J Harrison, Natasha Halasa, James Chappell, Laura Stewart, Mary Allen Staat, Elizabeth Schlaudecker, Christina Quigley, Eileen J Klein, Janet A Englund, Danielle M Zerr, Geoffrey A Weinberg, Peter G Szilagyi, Christina Albertin, Samantha H Johnston, John V Williams, Marian G Michaels, Robert W Hickey, Aaron T Curns, Michelle Honeywood, Slavica Mijatovic-Rustempasic, Mathew D Esona, Michael D Bowen, Umesh D Parashar, Rashi Gautam, Sara A Mirza, Jacqueline E Tate

Faculty, Staff and Students Publications

Background: Rotavirus was the leading cause of acute gastroenteritis among US children until vaccine introduction in 2006, after which, substantial declines in severe rotavirus disease occurred. We evaluated rotavirus vaccine effectiveness (VE) over 13 years (2009-2022).

Methods: We analyzed data from the New Vaccine Surveillance Network using a test-negative case-control design to estimate rotavirus VE against laboratory-confirmed rotavirus infections among children seeking care for acute gastroenteritis (≥3 diarrhea or ≥1 vomiting episodes within 24 hours) in the emergency department (ED) or hospital. Case-patients and control-patients were children whose stool specimens tested rotavirus positive or negative, respectively, by enzyme immunoassay or …

The Dengue Vaccine Initiative: A Bibliometric Analysis Of Research Publications, Festus Mulakoli, Abednego Ongeso, Horatius Musembi

School of Nursing & Midwifery, East Africa

Dengue fever has become a significant public health problem in tropical and subtropical regions and affects millions of people annually. Approximately 390 million people worldwide are estimated to be infected with dengue every year, of which 96 million develop dengue fever. Recently, there has been a noticeable increase in dengue cases, primarily in areas with tropical and subtropical climates. Vaccines have been developed to prevent or reduce the severity of the disease. This study reviews the number of publications on dengue vaccines and related topics worldwide. We aimed to evaluate the research output and create a narrative on global vaccine …

Sequencing-Based Detection Of Avian Influenza A(H5n1) Virus In Wastewater In Ten Cities, Michael J Tisza, Blake M Hanson, Justin R Clark, Li Wang, Katelyn Payne, Matthew C Ross, Kristina D Mena, Anna Gitter, Sara J Javornik Cregeen, Juwan Cormier, Vasanthi Avadhanula, Austen Terwilliger, John Balliew, Fuqing Wu, Janelle Rios, Jennifer Deegan, Pedro A Piedra, Joseph F Petrosino, Eric Boerwinkle, Anthony W Maresso

Faculty, Staff and Students Publications

No abstract provided.

Disruption Of Cochlear Development By Murine Cytomegalovirus Infection And Therapeutic Approaches That Prevent Hearing Loss, Matthew Smith

All ETDs from UAB

Human cytomegalovirus (HCMV) is a ubiquitous virus, infecting greater than 50% of most populations worldwide. Although rarely causing symptomatic infection in immunocompetent individuals, HCMV can cause life-threatening and severe end-organ disease in immunocompromised hosts including the developing fetus. HCMV can cross the placenta and is the most common viral infection of the developing fetus. Most newborn infants with congenital HCMV infection (cCMV) exhibit no clinical evidence of infection; however, up to 15% will develop long term neurodevelopmental sequelae including sensorineural hearing loss (SNHL). No prophylactic vaccines or effective antiviral treatments exist for cCMV infection, which may in part be secondary …

Fcrider: A Recombinant Fc Nanoparticle With Endogenous Adjuvant Activities For Hybrid Immunization, Changchuin Mao, Karen Eberle, Xiaojie Chen, Yiming Zhou, Jun Li, Hong Xin, Wenda Gao

School of Graduate Studies Faculty Publications

Active immunization (vaccination) induces long-lasting immunity with memory, which takes weeks to months to develop. Passive immunization (transfer of neutralizing antibodies) provides immediate protection, yet with high cost and effects being comparatively short-lived. No currently approved adjuvants are compatible with formulations to combine active and passive immunizations, not to mention their huge disparities in administration routes and dosage. To solve this, we engineered the Fc fragment of human IgG1 into a hexamer nanoparticle and expressed its afucosylated form in Fut8−/− CHO cells, naming it “FcRider.” FcRider is highly soluble with long-term stability, easily produced at high levels equivalent to those …

Bean Yellow Mosaic Virus And Its Management In Lupins, Department Of Primary Industries And Regional Development, Western Australia

Grains and other field crops factsheets

Bean yellow mosaic virus (BYMV) is an aphid-transmitted virus that commonly causes necrotic symptoms that kills lupin plants when infected early before pod set. When plants are infected after pod set, black pods develop (known as black pod syndrome).

BYMV is found predominantly in high rainfall areas of the Western Australian (WA) grainbelt. This page outlines the symptoms, diagnosis, factors favouring disease risk and spread, yield and quality impacts, and management for BYMV in lupin crops in WA

Cucumber Mosaic Virus And Its Management In Narrow Leafed Lupins, Department Of Primary Industries And Regional Development, Western Australia

Grains and other field crops factsheets

Cucumber mosaic virus (CMV) is a seed- and aphid-borne virus that infects narrow-leafed lupins. Western Australian (WA) grainbelt regions most at risk are the high rainfall zones of the northern and central agricultural region and the south coastal region.

The principal infection source for lupin crops is sowing infected lupin seed.

Complete Genome Sequence Of An Indian Outbreak Strain Of Chikungunya Virus, Eshna Laha, Deepak Jena, Viplov K. Biswas, Sharad Singh, Sunil K. Raghav, Asit K. Pattnaik, Soma Chattopadhyay

Nebraska Center for Virology: Faculty Publications

Here, we report the complete genome sequence of an Indian strain of chikungunya virus isolated from an infected patient from Hyderabad, Andhra Pradesh, India, during a massive outbreak in 2005–2006. The genome length spans 11,811 nucleotides and has a poly(A) tail of 29 residues at the 3′ end.

Wheat Streak Mosaic Virus And Wheat Curl Mite And Their Management, Department Of Primary Industries And Regional Development, Western Australia

Grains and other field crops factsheets

Wheat streak mosaic virus (WSMV) is a seed and mite-borne virus that infects wheat, causing severe leaf symptoms and reduced yields. The symptoms and damage are more severe in wheat than other hosts, in which infection is symptomless.

Wheat streak mosaic virus can cause crop failure when widespread infection occurs in seedling wheat, particularly when a heavily WSMV-infected green bridge is not controlled before sowing an early crop, and warm temperatures favour mites.

Wheat curl mite (Aceria tosichella) is the only known vector of WSMV. It can transmit the virus in its nymph and adult stages.

Tulane Virus Persistence And Microbial Stability In 3d Food Ink Under Various Storage Conditions: A Pre- And Post-Printing Analysis, Allyson N. Hamilton, Kristen E. Gibson

Food Science Faculty Publications and Presentations

3D food printers facilitate novel customization of the physicochemical properties of food. This study aimed to investigate the impact of storage conditions on the inactivation of the human norovirus surrogate, Tulane virus (TuV), within 3D printed foods. TuV-inoculated protein cookie food ink (∽ 4 log PFU/g) was distributed into 18 3D food printer capsules (50 g each); half immediately underwent extrusion. Storage of the capsules and printed food products at 20 °C (0, 6, 12, and 24 h), 4 °C (0, 1, 3, and 5d), and − 18 °C (0, 1, 3, and 5d) was completed before analysis for TuV …

Enterococcus Faecalis-Derived Adenine Enhances Enterohaemorrhagic Escherichia Coli Type 3 Secretion System-Dependent Virulence, Fernando H Martins, Thibaut Rosay, Anubama Rajan, Hannah E Carter, Tayah Turocy, Andres Mejia, Jason M Crawford, Anthony W Maresso, Vanessa Sperandio

Faculty, Staff and Students Publications

Interactions between microbiota and enteric pathogens can promote colonization resistance or enhance pathogenesis. The pathobiont Enterococcus faecalis increases enterohaemorrhagic E. coli (EHEC) virulence by upregulating Type 3 Secretion System (T3SS) expression, effector translocation, and attaching and effacing (AE) lesion formation on enterocytes, but the mechanisms underlying this remain unknown. Using co-infection of organoids, metabolomics, supplementation experiments and bacterial genetics, here we show that co-culture of EHEC with E. faecalis increases the xanthine-hypoxanthine pathway activity and adenine biosynthesis. Adenine or E. faecalis promoted T3SS gene expression, while transcriptomics showed upregulation of adeP expression, which encodes an adenine importer. Mechanistically, adenine relieved …

A Case Of Persistent Intra-Abdominal Stenotrophomonas Maltophilia Infection Despite Bacteriophage Therapy, Grace D Cullen, Keiko Christine Salazar, Austen Lee Terwilliger, Saima Aslam, Justin R Clark, Anthony W Maresso, Paul L Bollyky, Jenny R Aronson

Faculty, Staff and Students Publications

Multidrug resistant infections are a challenge in the health care setting and a cause of patient morbidity and mortality. Bacteriophages (phages) are viruses that target and kill bacteria and have been used in patients to treat bacterial infections. We present a case of disseminated Stenotrophomonas maltophilia infection, with pulmonary, intra-abdominal and bloodstream involvement. The patient was treated with a combination of antibiotics and personalized phage therapy, administered daily for 12 days both intravenously as well as via intra-abdominal drains. Phage therapy was well-tolerated, the patient cleared S. maltophilia from their bloodstream and their intra-abdominal abscesses were stable or decreased in …

Sequence Specificity Of Non-Structural Protein 15 From Sars-Cov-2, John Vincent Mcguire

Electronic Theses and Dissertations

The SARS-CoV-2 virus was recently able to cause a global pandemic, largely due to the asymptomatic period that occurs during initial infection. During this period, patients are infectious but do not exhibit symptoms, resulting in the unintentional spread of the virus. This occurs because the virus effectively avoids detection by the immune system during the early stages of infection, and as symptoms are the result of immune system activation, they never manifest. Non-structural Protein 15 (Nsp15) is one of the tools the virus uses to remain undetected, via its degradation of Pathogen-Associated Molecular Patterns (PAMPs) that would otherwise be recognized …

Investigating The Roles Of Sirtuins During Cvb3 Infection And Innate Immunity, Bridget M. Hulsebosch

Master's Theses

Coxsackievirus B3 (CVB3) is an enterovirus that chronically infects heart tissue, leading to dilated cardiomyopathy. However, no vaccines or antivirals exist for CVB3 infection or to treat CVB3-induced dilated cardiomyopathy, except for a heart transplant. Sirtuins (SIRTs), class III lysine deacetylases, have diverse and complex roles in cellular functions, contributing to energy production, NF-κB signaling, cell cycle regulation, and inflammation. Previous studies showed that SIRTs are essential for hepatitis B and A viral replication and regulate the IFN response during viral infection. However, the role of SIRT1 and 2 during CVB3 infection is entirely unknown. CVB3-infected SIRT1 and SIRT2 knockout …

Rna Secondary Structures: Structure-Function Studies In Viral And Neurodegenerative Diseases, Caylee Cunningham

Electronic Theses and Dissertations

The studies within this dissertation highlight the importance of RNA structure and function, highlighting potential mechanisms in the pathogenesis of the SARS-CoV-2 virus and hallmarks of ALS. Specifically, the s2m, a 41-nt structure found within the hyper variable region of the 3’ UTR of the SARS-CoV-2 virus, was investigated as this element has no definitive role in viruses but its high conservation in several viral families warrants investigation. We used various biophysical techniques to determine if a palindromic sequence in the terminal loop could participate in dimerization activities, which may have implications in recombination and the longevity of the virus. …

Identification And Characterization Of A Skin Microbiome On Caenorhabditis Elegans Suggests Environmental Microbes Confer Cuticle Protection, Nadia B Haghani, Robert H Lampe, Buck S Samuel, Sreekanth H Chalasani, Molly A Matty

Faculty, Staff and Students Publications

In the wild, C. elegans are emersed in environments teeming with a veritable menagerie of microorganisms. The C. elegans cuticular surface serves as a barrier and first point of contact with their microbial environments. In this study, we identify microbes from C. elegans natural habitats that associate with its cuticle, constituting a simple “skin microbiome.” We rear our animals on a modified CeMbio, mCeMbio, a consortium of ecologically relevant microbes. We first combine standard microbiological methods with an adapted micro skin-swabbing tool to describe the skin-resident bacteria on the C. elegans surface. Furthermore, we conduct 16S rRNA gene sequencing studies …