Digital Commons Network^™

Integrating Tropical Research Into Biology Education Is Urgently Needed, Ann E. Russell, T. Mitchell Aide, Elizabeth Braker, Carissa N. Ganong, Rebecca D. Hardin, Karen D. Holl, Sara C. Hotchkiss, Jeffrey A. Klemens, Erin K. Kuprewicz, Deedra Mcclearn, George Middendorf, Rebecca Ostertag, Jennifer S. Powers, Sabrina E. Russo, Jennifer L. Stynoski, Ursula Valdez, Charles G. Willis

School of Biological Sciences: Faculty Publications

Understanding tropical biology is important for solving complex problems such as climate change, biodiversity loss, and zoonotic pandemics, but biology curricula view research mostly via a temperatezone lens. Integrating tropical research into biology education is urgently needed to tackle these issues.

The tropics are engines of Earth systems that regulate global cycles of carbon and water, and are thus critical for management of greenhouse gases. Compared with higher-latitude areas, tropical regions contain a greater diversity of biomes, organisms, and complexity of biological interactions. The tropics house the majority of the world’s human population and provide important global commodities from species …

Functional Response Of Harmonia Axyridis To The Larvae Of Spodoptera Litura: The Combined Effect Of Temperatures And Prey Instars, Yasir Islam, Farhan Mahmood Shah, Ali Güncan, John Paul Delong, Xingmiao Zhou

School of Biological Sciences: Faculty Publications

Functional responses are central to predator–prey dynamics and describe how predation varies with prey abundance. Functional responses often are measured without regard to prey size (i.e., body mass) or the temperature dependence of feeding rates. However, variation in prey size within populations is ubiquitous, and predation rates are often both size and temperature-dependent. Here, we assessed functional responses of larvae and adult Harmonia axyridis on the 1st, 2nd, and 3rd instars of the prey Spodoptera litura across a range of temperatures (i.e., 15, 20, 25, 30, and 35◦C). The type and parameters of the functional responses were determined using logistic …

Pea Aphid Winged And Wingless Males Exhibit Reproductive, Gene Expression, And Lipid Metabolism Differences, Omid Saleh Ziabari, Qingyi Zhong, Swapna R. Purandare, Joel Reiter, Anthony J. Zera, Jennifer A. Brisson

School of Biological Sciences: Faculty Publications

Alternative, intraspecific phenotypes offer an opportunity to identify the mechanistic basis of differences asso- ciated with distinctive life history strategies. Wing dimorphic insects, in which both flight-capable and flight- incapable individuals occur in the same population, are particularly well-studied in terms of why and how the morphs trade offflight for reproduction. Yet despite a wealth of studies examining the differences between fe- male morphs, little is known about male differences, which could arise from different causes than those acting on females. Here we examined reproductive, gene expression, and biochemical differences between pea aphid ( Acyrthosiphon pisum ) winged and wingless …

Social Networks In The Single Cell, Moira Rodriguez, Ana Martinez-Hottovy, Alan C. Christensen

School of Biological Sciences: Faculty Publications

Plant mitochondrial DNA (mtDNA) can become damaged in many ways. A major repair mechanism is homologous recombination, which requires an undamaged DNA template. Presumably, this template comes from a different mitochondrion in the same cell. Plant mitochondria undergo fission and fusion to form transient networks which could allow the exchange of genetic information. To test this hypothesis, Chustecki et al. (2022) used msh1 mutants with defective DNA repair, and showed that mitochondrial interactions increased, revealing a link between the physical and genetic behavior of mitochondria.

Late Pleistocene Megafauna Extinction Leads To Missing Pieces Of Ecological Space In A North American Mammal Community, Felisa A. Smith, Emma A. Elliott Smith, Amelia Villaseñor, Catalina P. Tomé, S. Kathleen Lyons, Seth D. Newsome

School of Biological Sciences: Faculty Publications

The conservation status of large-bodied mammals is dire. Their decline has serious consequences because they have unique ecological roles not replicated by smaller-bodied animals. Here, we use the fossil record of the megafauna extinction at the terminal Pleistocene to explore the consequences of past biodiversity loss. We characterize the isotopic and body-size niche of a mammal community in Texas before and after the event to assess the influence on the ecology and ecological interactions of surviving species (>1 kg). Preextinction, a variety of C₄ grazers, C₃ browsers, and mixed feeders existed, similar to modern African savannas, with …

Fishing Regulations, Sexual Dimorphism, And The Life History Of Harvest, Lyndsie S. Wszola, Zachary S. Feiner, Christopher J. Chizinski, Jamilynn B. Poletto, John P. Delong

School of Biological Sciences: Faculty Publications

Freshwater recreational fisheries regulations are a vital tool for achieving social and ecological fisheries objectives. However, angler behavior and fish biology may interact to influence regulation efficacy in unexpected ways. We combined models of fish growth and angler behavior to explore how angler behavior interacts with fish life history to shape the probability of fish harvest given capture across ages, life stages, and sexes of walleye (Sander vitreus). Compared to females, males grew more quickly as juveniles, matured earlier, and reached smaller maximum sizes. Male walleye were therefore vulnerable to harvest for more of their reproductive lives than …

Forage Database: A Compilation Of Functional Responses For Consumers And Parasitoids, Stella Uiterwaal, Ian T. Lagerstrom, Shelby R. Lyon, John Delong

School of Biological Sciences: Faculty Publications

Functional responses, the relationships between consumer foraging rate and resource (prey) density, provide key insights into consumer–resource interactions while also being a major driver of population dynamics and food web structure. We present a global database of 2598 standardized functional responses and parameters extracted from the published literature. We refit the functional responses with a Type II model using standardized methods and report the fitted parameters along with data on experimental conditions, consumer and resource taxonomy and type, as well as the habitat and dimensionality of the foraging interaction. The consumer and resource species covered here are taxonomically diverse, from …

An Empiricist’S Guide To Using Ecological Theory, Tess N. Grainger, Athmanathan Senthilnathan, Po-Ju Ke, Matthew A. Barbour, Natalie T. Jones, John Delong, Sarah P. Otto, Mary I. O'Connor, Kyle E. Coblentz, Nikunj Goel, Jawad Sakarchi, Megan C. Szojka, Jonathan M. Levine, Rachel M. Germain

School of Biological Sciences: Faculty Publications

A scientific understanding of the biological world arises when ideas about how nature works are formalized, tested, refined, and then tested again. Although the benefits of feedback between theoretical and empirical research are widely acknowledged by ecologists, this link is still not as strong as it could be in ecological research. This is in part because theory, particularly when expressed mathematically, can feel inaccessible to empiricists who may have little formal training in advanced math. To address this persistent barrier, we provide a general and accessible guide that covers the basic, step-by-step process of how to approach, understand, and use …

Towards An Integrative View Of Virus Phenotypes, John P. Delong, Maitham A. Al-Sammak, Zeina Al-Ameeli, David Dunigan, Kyle F. Edwards, Jeffry J. Fuhrmann, Jason P. Gleghorn, Hanqun Li, Kona Haramoto, Amelia O. Harrison, Marcia F. Marston, Ryan M. Moore, Shawn W. Polson, Barbra D. Ferrell, Miranda E. Salsbery, Christopher R. Schvarcz, Jasmine Shirazi, Grieg F. Steward, James L. Van Etten, K. Eric Wommack

School of Biological Sciences: Faculty Publications

Understanding how phenotypes emerge from genotypes is a foundational goal in biology. As challenging as this task is when considering cellular life, it is further complicated in the case of viruses. During replication, a virus as a discrete entity (the virion) disappears and manifests itself as a metabolic amalgam between the virus and the host (the virocell). Identifying traits that unambiguously constitute a virus’s phenotype is straightforward for the virion, less so for the virocell. Here, we present a framework for categorizing virus phenotypes that encompasses both virion and virocell stages and considers functional and performance traits of viruses in …

Predator Functional Responses And The Biocontrol Of Aphids And Mites, John Delong, Stella Uiterwaal

School of Biological Sciences: Faculty Publications

Biocontrol with predators is a key tool for controlling agricultural pests and preserving the productive efficiency of crops. Determining which predators to use for biocontrol often involves measuring their functional response—the relationship between foraging rate and prey abundance, yet comparisons of functional responses across predators are complicated by differences in experimental procedures. Here we use a compilation of functional responses standardized for time and space units to illustrate key sources of variation in functional responses for predators being tested for control of aphids and mites. Our results show that arena size (as a proxy for habitat structure) is a crucial …

What Happens When Transition Metal Trichalcogenides Are Interfaced With Gold?, Archit Dhingra, Dmitri E. Nikonov, Alexey Lipatov, Alexander Sinitskii, Peter Dowben

Peter Dowben Publications

Transition metal trichalcogenides (TMTs) are two-dimensional (2D) systems with quasi-one-dimensional (quasi-1D) chains. These 2D materials are less susceptible to undesirable edge defects, which enhances their promise for low-dimensional optical and electronic device applications. However, so far, the performance of 2D devices based on TMTs has been hampered by contact-related issues. Therefore, in this review, a diligent effort has been made to both elucidate and summarize the interfacial interactions between gold and various TMTs, namely, In₄Se₃, TiS₃, ZrS₃, HfS₃, and HfSe₃. X-ray photoemission spectroscopy data, supported by the results …

A Biological And Chemical Approach To Restoring Water Quality: A Case Study In An Urban Eutrophic Pond, Levi J. Mckercher, Tiffany L. Messer, Aaron R. Mittelstet, Steve D. Comfort

School of Natural Resources: Faculty Publications

Efforts to improve water quality of eutrophic ponds often involve implementing changes to watershed management practices to reduce external nutrient loads. While this is required for long-term recovery and prevention, eutrophic conditions are often sustained through the recycling of internal nutrients already present within the waterbody. In particular, internal phosphorus bound to organic material and adsorbed to sediment has the potential to delay lake recovery for decades. Thus, pond and watershed management techniques are needed that not only reduce external nutrient loading but also mitigate the effects of internal nutrients already present. Therefore, our objective was to demonstrate a biological …

Phenotypic And Genomic Diversification In Complex Carbohydrate-Degrading Human Gut Bacteria, Nicholas A. Pudlo, Karthik Urs, Ryan Crawford, Ali Pirani, Todd Atherly, Roberto Jimenez, Nicolas Terrapon, Bernard Henrissat, Daniel Peterson, Cherie Ziemer, Evan Snitkin, Eric C. Martens

Department of Food Science and Technology: Faculty Publications

Symbiotic bacteria are responsible for the majority of complex carbohydrate digestion in the human colon. Since the identities and amounts of dietary polysaccharides directly impact the gut microbiota, determining which microorganisms consume specific nutrients is central for defining the relationship between diet and gut microbial ecology. Using a custom phenotyping array, we determined carbohydrate utilization profiles for 354 members of the Bacteroidetes, a dominant saccharolytic phylum. There was wide variation in the numbers and types of substrates degraded by individual bacteria, but phenotype-based clustering grouped members of the same species indicating that each species performs characteristic roles. The ability …

Experimental Evaluation Of Ecological Principles To Understand And Modulate The Outcome Of Bacterial Strain Competition In Gut Microbiomes, Rafael R. Segura Munoz, Sara Mantz, Ines Martinez, Fuyong Li, Robert J. Schmaltz, Nicholas A. Pudlo, Karthik Urs, Eric C. Martens, Jens Walter, Amanda Ramer-Tait

Department of Food Science and Technology: Faculty Publications

It is unclear if coexistence theory can be applied to gut microbiomes to understand their characteristics and modulate their composition. Through experiments in gnotobiotic mice with complex microbiomes, we demonstrated that strains of Akkermansia muciniphila and Bacteroides vulgatus could only be established if microbiomes were devoid of these species. Strains of A. muciniphila showed strict competitive exclusion, while B. vulgatus strains coexisted but populations were still influenced by competitive interactions. These differences in competitive behavior were reflective of genomic variation within the two species, indicating considerable niche overlap for A. muciniphila strains and a broader niche space for B. vulgatus …

Large-Scale Analyses Of Heat Shock Transcription Factors And Database Construction Based On Whole-Genome Genes In Horticultural And Representative Plants, Tong Yu, Yun Bai, Zhuo Liu, Zhiyuan Wang, Qihang Yang, Tong Wu, Shuyan Feng, Yu Zhang, Shaoqin Shen, Quang Li, Liqiang Gu, Xiaoming Song

Department of Food Science and Technology: Faculty Publications

Heat shock transcription factor (Hsf) plays a critical role in regulating heat resistance. Here, 2950 Hsf family genes were identified from 111 horticultural and representative plants. More Hsf genes were detected in higher plants than in lower plants. Based on all Hsf genes, we constructed a phylogenetic tree, which indicated that Hsf genes of each branch evolved independently after species differentiation. Furthermore, we uncovered the evolutionary trajectories of Hsf genes by motif analysis. There were only six motifs (M1–M6) in lower plants, and then four novel motifs (M7–M10) appeared in higher plants. However, the motifs of some Hsf genes were …

Thermal Inactivation Of Salmonella On Chicken Wings Cooked In Domestic Convection And Air Fryer Ovens, Carmen Cano, Xinyao Wei, Cyril A. Etaka, Byron D. Chaves

Department of Food Science and Technology: Faculty Publications

Chicken wings are among the most popular poultry products for home and foodservice consumption. Poultry products must be handled and cooked safely to decrease the risk of foodborne salmonellosis for consumers. This study aims to validate the use of domestic appliances (convection and air fryer ovens) for the thermal inactivation of Salmonella on chicken wings. Wings (n = 3, 46.5 ± 4.3 g) were inoculated with a five-strain cocktail of Salmonella (ca. 8 log₁₀ CFU/wing) and cooked in a convection oven (179.4ºC) or an air fryer (176, 190, or 204ºC) for 2, 5, 10, 15, 20, 22, or 25 …

Recent Advances In Biosensors For Detection Of Covid-19 And Other Viruses, Shobhit K. Patel, Jaymit Surve, Juveriya Parmar, Kawsar Ahmed, Francis M. Bui, Fahad Ahmed Al-Zahrani

Department of Mechanical and Materials Engineering: Faculty Publications

This century has introduced very deadly, dangerous, and infectious diseases to humankind such as the influenza virus, Ebola virus, Zika virus, and the most infectious SARS-CoV-2 commonly known as COVID-19 and have caused epidemics and pandemics across the globe. For some of these diseases, proper medications, and vaccinations are missing and the early detection of these viruses will be critical to saving the patients. And even the vaccines are available for COVID-19, the new variants of COVID-19 such as Delta, and Omicron are spreading at large. The available virus detection techniques take a long time, are costly, and complex and …

A Robot-Assisted Acoustofluidic End Effector, Jan Durrer, Prajwal Agrawal, Ali Ozgul, Stephen C.F. Neuhauss, Nitesh Nama, Daniel Ahmed

Department of Mechanical and Materials Engineering: Faculty Publications

Liquid manipulation is the foundation of most laboratory processes. For macroscale liquid handling, both do-it-yourself and commercial robotic systems are available; however, for microscale, reagents are expensive and sample preparation is difficult. Over the last decade, lab-on-a-chip (LOC) systems have come to serve for microscale liquid manipulation; however, lacking automation andmulti-functionality. Despite their potential synergies, each has grown separately and no suitable interface yet exists to link macro-level robotics with micro-level LOC or microfluidic devices. Here, we present a robot-assisted acoustofluidic end effector (RAEE) system, comprising a robotic arm and an acoustofluidic end effector, that combines robotics and microfluidic functionalities.We …

An Examination Of Ethical Attitudes Towards Wild Pig (Sus Scrofa) Toxicants In The United States, Keith Carlisle, Erin E. Harper, Stephanie A. Shwiff

United States Department of Agriculture Wildlife Services: Staff Publications

This research aims to understand ethical attitudes of the U.S. public towards the use of a toxicant to control wild pigs (Sus scrofa), a destructive invasive species whose population growth is proving difficult to control through conventional management methods. Using a nationwide self-administered survey with 2,186 completed and returned questionnaires, we found that among six different lethal control methods, toxicant usage was the only method that a majority of respondents (51%) found to be unethical, with no significant differences between rural and urban respondents or between respondents from counties with wild pigs and counties where the species is absent. The …

Why Neighborhoods (And How We Study Them) Matter For Adolescent Development, T.D. Warner, R.A. Settersten

Department of Sociology: Faculty Publications

Adolescence is a sensitive developmental period marked by significant changes that unfold across multiple contexts. As a central context of development, neighborhoods capture—in both physical and social space—the stratification of life chances and differential distribution of resources and risks. For some youth, neighborhoods are springboards to opportunities; for others, they are snares that constrain progress and limit the ability to avoid risks. Despite abundant research on “neighborhood effects,” scant attention has been paid to how neighborhoods are a product of social stratification forces that operate simultaneously to affect human development. Neighborhoods in the United States are the manifestation of three …

Replicating Or Franchising A Stem Afterschool Program Model: Core Elements Of Programmatic Integrity, Nikolaus Stevenson, Amie S. Sommers, Neal Grandgenett, William Tapprich, Julia Mcquillan, Michelle Phillips, Rachael Jensen, Christine Cutucache

Department of Sociology: Faculty Publications

Background: Designed in 2012 with a first implementation in 2013, NE STEM 4U is a professional development program for post-secondary students/undergraduates, and serves as a source of outreach, content knowledge generation, and STEM literacy for youth in grades kindergarten through 8th grade (ages 5–14). The model empowers post-secondary students as facilitators of inquiry-based learning within the context of an out-of-school time program. This study investigated the potential for replicating or ‘franchising’ this model by evaluating on the following: (1) Is the model replicable? And, if so, (2) what core elements are necessary for program fidelity? And (3) is there …

Is Perceived Inability To Procreate Associated With Life Satisfaction? Evidence From A German Panel Study, Julia Mcquillan, Jasmin Passet-Wittig, Arthur L. Greil, Martin Bujard

Department of Sociology: Faculty Publications

Most studies of the psychosocial consequences of infertility have focused on those who seek medical treatment, leaving a research gap regarding the psychosocial consequences of perceived inability to procreate in the general population. Moreover, most studies are cross-sectional and the results are thus likely affected by omitted variable bias. Inspired by aspects of the Theory of Conjunctural Action, this study analysed 10 waves of data from the German Family Panel (pairfam) for women and men using fixed effects panel regression and including time-varying control variables suggested by theory and research. This study found that both women and men experienced lower …

The Third International Hackathon For Applying Insights Into Large-Scale Genomic Composition To Use Cases In A Wide Range Of Organisms, Kimberly Walker, Divya Kalra, Rebecca Lowdon, Guangyi Chen, Fritz J. Sedlazeck, Ben Busby, Chia-Sin Liew, Et Al.

Nebraska Center for Biotechnology: Faculty and Staff Publications

In October 2021, 59 scientists from 14 countries and 13 U.S. states collaborated virtually in the Third Annual Baylor College of Medicine & DNANexus Structural Variation hackathon. The goal of the hackathon was to advance research on structural variants (SVs) by prototyping and iterating on open-source software. This led to nine hackathon projects focused on diverse genomics research interests, including various SV discovery and genotyping methods, SV sequence reconstruction, and clinically relevant structural variation, including SARS-CoV-2 variants. Repositories for the projects that participated in the hackathon are available at https://github.com/collaborativebioinformatics.

Direct Observation Of Ferroelectric Switching In Two-Dimensional Mos2, A. Lipatov, P. Chaudhary, Z. Guan, H. Lu, G. Li, O. Crégut, K. D. Dorkenoo, R. Proksch, Ding-Fu Shao, Evgeny Y. Tsymbal, J. Íñiguez, A. Sinitskii, Alexei Gruverman

Evgeny Tsymbal Publications

No abstract provided.

Improving Taxonomic Delimitation Of Fungal Species In The Age Of Genomics And Phenomics, Ashley Stengel, Kimberly M. Stanke, Amanda C. Quattrone, Joshua R. Herr

Department of Plant Pathology: Faculty Publications

Species concepts have long provided a source of debate among biologists. These lively debates have been important for reaching consensus on how to communicate across scientific disciplines and for advancing innovative strategies to study evolution, population biology, ecology, natural history, and disease epidemiology. Species concepts are also important for evaluating variability and diversity among communities, understanding biogeographical distributions, and identifying causal agents of disease across animal and plant hosts. While there have been many attempts to address the concept of species in the fungi, there are several concepts that have made taxonomic delimitation especially challenging. In this review we discuss …

Determining The Novel Pathogen Neodothiora Populina As The Causal Agent Of The Aspen Running Canker Disease In Alaska, Loretta M. Winton, Gerard C. Adams, Roger W. Ruess

Department of Plant Pathology: Faculty Publications

Neodothiora populina Crous, G.C. Adams & Winton was determined to be a new pathogen of trembling aspen (Populus tremuloides) growing in Alaska, based on completion of Koch's Postulates in replicated forest and growth chamber inoculation trials. The pathogen is responsible for severe damage and widespread rapid mortality of sapling to mature aspen (≥ 80 years) in the boreal forests of interior Alaska, due to large diffuse annual (1–2 years) cankers. Isolation of the pathogen was challenging, and identification based on cultural characters was difficult. Fruiting bodies were not found on wild diseased trees, but erumpent pycnidia were found …

Dynamic Causal Modeling Of Neural Responses To An Orofacial Pneumotactile Velocity Array, Yingying Wang, Rebecca Custead, Hyuntaek Oh, Steven M. Barlow

Department of Special Education and Communication Disorders: Faculty Publications

The effective connectivity of neuronal networks during orofacial pneumotactile stimulation with different velocities is still unknown. The present study aims to characterize the effectivity connectivity elicited by three different saltatory velocities (5, 25, and 65 cm/s) over the lower face using dynamic causal modeling on functional magnetic resonance imaging data of twenty neurotypical adults. Our results revealed the contralateral SI and SII as the most likely sources of the driving inputs within the sensorimotor network for the pneumotactile stimuli, suggesting parallel processing of the orofacial pneumotactile stimuli. The 25 cm/s pneumotactile stimuli modulated forward interhemispheric connection from the contralateral SII …

Ankrd24 Organizes Triobp To Reinforce Stereocilia Insertion Points, Jocelyn F. Krey, Chang Liu, Inna A. Belyantseva, Michael Bateschell, Rachel A. Dumont, Jennifer Goldsmith, Paroma Chatterjee, Rachel S. Morrill, Lev M. Fedorov, Sarah Foster, Jinkyung Kim, Alfred L. Nuttal, Sherri M. Jones, Dongseok Choi, Thomas B. Friedman, Anthony J. Ricci, Bo Zhao, Peter G. Barr-Gillespie

Department of Special Education and Communication Disorders: Faculty Publications

The stereocilia rootlet is a key structure in vertebrate hair cells, anchoring stereocilia firmly into the cell’s cuticular plate and protecting them from overstimulation. Using superresolution microscopy, we show that the ankyrin-repeat protein ANKRD24 concentrates at the stereocilia insertion point, forming a ring at the junction between the lower and upper rootlets. Annular ANKRD24 continues into the lower rootlet, where it surrounds and binds TRIOBP-5, which itself bundles rootlet F-actin. TRIOBP-5 is mislocalized in Ankrd24^KO/KO hair cells, and ANKRD24 no longer localizes with rootlets in mice lacking TRIOBP-5; exogenous DsRed–TRIOBP-5 restores endogenous ANKRD24 to rootlets in these mice. Ankrd24 …

Psychological And Physical Intimate Partner Aggression Are Associated With Broad And Specific Internalizing Symptoms During Pregnancy, Gabriela R. Perez, Sara M. Stasik-O’Brien, Lauren M. Laifer, Rebecca L. Brock

Department of Psychology: Faculty Publications

Background: Intimate partner violence (IPV) has serious consequences, particularly during high-risk periods such as pregnancy, which poses a significant risk to maternal mental health. However, it is unclear whether IPV presents a broad risk for psychopathology or is specific to distinct diagnoses or symptom dimensions (e.g., panic, social anxiety). Further, the relative impact of physical versus psychological aggression remains unclear. Methods: One hundred and fifty-nine pregnant couples completed surveys assessing psychological and physical intimate partner aggression unfolding in the couple relationship, as well as a range of internalizing symptoms. Results: Psychological and physical aggression were each associated with broad negative …

Attachment Anxiety And Avoidance Predict Postnatal Partner Support Through Impaired Affective Communication, Frances C. Calkins, Rebecca L. Brock

Department of Psychology: Faculty Publications

Objective: The purpose of the present study was to investigate perceived difficulties in affective communication as a key mechanism linking attachment anxiety and avoidance during pregnancy to the quality of postpartum support received by partners.

Background: During the postpartum period, partner support has the potential to promote family well-being by mitigating stress related to changes experienced during this transition. Attachment security is one of the most robust predictors of intimate relationship processes and impacts partner communication and support dynamics.

Method: Heterosexual couples (N = 159) completed surveys and semi-structured interviews to obtain measures of attachment security, perceived difficulties in …