Trustees Of Indiana University

NIH Research Projects · FY 2022 · 2021-09

Our research will investigate how critical axonal microtubules are in the pathogenesis of glaucoma. The axons of the retinal ganglion cells (RGCs) are gradually lost in glaucoma, but little is known about the mechanistic link between ocular hypertension, which is a major risk factor, and the loss of RGC axons. Our overarching hypothesis is that RGC microtubules are involved in a reversible stage of the disease and may provide a molecular substrate for the RGC’s sensitivity to the elevated intraocular pressure triggering the pathogenic pathway. Recently, we demonstrated that axonal microtubules degrade more rapidly than the loss of RGC axons, resulting in microtubule deficit, and microtubule deficit is spatially correlated with axonal atrophy. It suggests that microtubule deficit is a new pathology of early glaucoma, which shares a common pathogenic origin as the loss of RGC axons. Here we will investigate microtubule deficit in depth. The potential mechanism and therapeutic significance will be studied using DBA mice as a model of inherited glaucoma. In aim 1, we will investigate the possible relationship between the posttranslational modifications of axonal microtubules and microtubule deficit. Also known as the tubulin code, various posttranslational modifications modulate the interaction with microtubule-associated proteins to regulate the stability and function of microtubules. We will examine how the tubulin code is altered during glaucoma and how it is correlated with other glaucoma pathologies such as microtubule deficit. In aim 2, we will test microtubule deficit as an endpoint to evaluate the efficacy of therapeutic treatments. Microtubule deficit will be measured of the DBA retinas receiving an increased level of nicotinamide adenine dinucleotide, which is known to protect the RGC axons and promote the RGC viability. The recovery of microtubules will suggest a crucial causal relationship with metabolic stress. Upon completion, our research will yield new insights into the mechanism of microtubule deficit toward improved glaucoma therapy. The notion that the tubulin codes might play a role in glaucoma pathogenesis is innovative. Also, new types of data will be obtained due to a novel retinal imaging, i.e., intrinsic second-harmonic generation (SHG) microscopy. Consequently, our research will open a new field of questions and can be paradigm-shifting in glaucoma therapy.

NIH Research Projects · FY 2025 · 2021-08

PROJECT SUMMARY Polymicrobial communities are ubiquitous in the human body and their behaviors are critical drivers of both health and disease. Bacteria are social organisms; thus, the behavior of the group is driven not only by the composition of the community, but also by interactions between the constituents and their surrounding environment. A key principle shared among all communities, is that space matters. Groups organize to maximize acquisition of goods, minimize exposure to toxic compounds, and optimize communication. Our recent data reveal bacteria can communicate with members of distant species and respond by changing how they spatially structure their communities. My laboratory seeks to understand how bacteria communicate between species by observing them in their native environment, tracking their movements, and listening to and decoding their languages. We utilize Pseudomonas aeruginosa, the most notoriously problematic opportunistic pathogen in multiple types of polymicrobial infections, including chronic wounds and lung infections in cystic fibrosis (CF) patients. We recently reported that P. aeruginosa establishes infections with other important pathogens in the lungs of patients with CF for decades, remaining unresponsive to intense antibiotic therapies and causing lung decline and early death. In this proposal, we begin by visualizing the spatial landscape of polymicrobial communities in situ, in transplanted lungs from CF patients and animal models of polymicrobial infection. By combining next-generation tissue clearing and fluorescent in situ hybridization, we are able to visualize the spatial positioning of species on a range of spatial scales, in relation to each other and host structures. This will yield an unprecedented view of microbes during infection and provide a platform for visualizing communities in other organs rich in polymicrobial communities, such as the gastrointestinal tract. Next, we will systematically decode the interspecies signaling language. We designed high-throughput screens to identify genes necessary for signaling and the repertoire of signaling molecules. Using live-imaging techniques pioneered by my lab, we visualize and track the movement of bacterial cells, gene expression, and dynamics of motility appendages, upon modulation of the identified pathways. Initial studies reveal P. aeruginosa responds by activating multiple signaling systems, which tune the direction of movement and activate virulence systems. Collectively, these studies will construct a comprehensive picture of interspecies communication and enlighten how interactions exacerbate disease.

NIH Research Projects · FY 2025 · 2021-08

Gene expression is a fundamental problem at the core of life science. All cells in an adult organism contain an identical set of genes, but their expression varies widely depending on the context, such as cell type and environmental queues. Transcription is the critical first step in gene expression, which is intricately controlled by complex regulations and the disruption is pathogenic for many diseases. Much knowledge about the mechanism has been gained using fixed cells and sequencing assays. Nonetheless, we still do not understand how the variability of transcription is regulated in living mammals, dynamically in space and time, as the animal performs development, physiology, and cognitive tasks such as learning. In particular, the role of the cis- regulatory elements (i.e., enhancers) plays in the context-dependent transcriptional dynamics is obscure. Here we will develop technology for addressing the current bottleneck. Over the past years, our laboratory has advanced methods for visualizing transcriptional dynamics in the brain of live mouse (‘intravital MS2 technology’). Single molecules of mRNA of a gene of interest have been imaged in a specific cell type by intravital multiphoton microscopy (MPM), revealing previously unknown properties of nascent transcripts in vivo. For the next five years, we will demonstrate more versatile intravital MS2 technology geared toward the functional annotation of the genome. Taking inspirations from the prior success of illuminating the structure and function of the neuronal network in vivo, we will fuse intravital MPM, smart molecular sensors, and genomic engineering to dissect the function of the transcriptional regulatory network in live murine brains. Our research will lay a foundation for unraveling the origin of diverse cell types, plasticity, and neurodegenerative disorders of the mammalian brain.

NIH Research Projects · FY 2024 · 2021-08

Project Summary / Abstract Health policies, educational programming, and health-related services require accurate and up- to-date data on the sexual behaviors, attitudes, and experiences of the population. We propose to conduct Wave 8 of the National Survey of Sexual Health and Behavior (NSSHB) in order to support such public health efforts. Sexual health is an important part of human health. Since 2014, sexually transmitted infection (STI) rates have increased annually. STIs challenge the health of individuals and newborns, and cost billions of dollars each year. Unintended pregnancies have decreased overall, though adolescent and young adult women are at greater risk for unintended pregnancy as are sexual minority women. Among adolescents aged 15-19, 3 of 4 pregnancies are unintended. Sexual health has been conceptualized by the World Health Organization (WHO) as a state of physical, emotional, mental and social well-being in relation to sexuality; it refers not only to the absence of disease but also to the possibility of pleasurable and safe sexual experiences that are free of coercion, discrimination, and violence. From adolescence through old age, people have diverse sexual health needs. These sexual health needs include choosing when to become sexually active with a partner, preventing unintended pregnancy, reducing risks of HIV and sexually transmitted infection (STI), accessing and using condoms/contraception, sexual identity development, patient/provider sexual health conversations, painful sex, vaginal dryness, sexual coercion and assault, sexual function, as well as the development and maintenance of satisfying relationships. Since 2009, the NSSHB has served as the nation's only U.S. nationally representative probability survey that is focused on sexual health, assesses diverse sexual behaviors and attitudes, and samples individuals from adolescence through advanced age (often ages 14 to 94). We have a unique opportunity to develop the NSSHB-Wave 8 so as to maximize cross-national comparisons with other nationally representative probability surveys of sexual health being conducted in a similar time frame in the U.K., France, and Australia. We aim to: (1) Design and field NSSHB-Wave 8, surveying 9500+ individuals; (2) Demonstrate the scientific and public health importance of NSSHB-Wave 8 by updating population-prevalence of sexual health behaviors, establishing the population-prevalence of understudied sexual behaviors, and examining associations between certain sexual behaviors and reproductive coercion and sexual coercion; and (3) work closely with an Advisory Board of sexual and reproductive health scientists, educators, and key leaders to disseminate findings and to facilitate data availability to other scientists.

NIH Research Projects · FY 2025 · 2021-07

Modified Project Summary/Abstract Section Over the past 40 years, incarceration in the US has increased. More than 9 million Americans are incarcerated in jail (facilities housing individuals awaiting trial and serving short sentences) each year. Of these individuals, most are racial and ethnic minorities and have low socioeconomic status. Among individuals incarcerated in a rural, county jail, there are high rates of anxiety, hypertension, and poor sleep quality while incarcerated. Physical activity (PA) can mitigate these outcomes and be immediate. A single bout of moderate-to-vigorous PA improves anxiety symptoms, decreases blood pressure, and improves sleep on the day it is performed. Despite these benefits, over 75% of individuals incarcerated in jail do not attend yard time, a structured time dedicated for PA, outside. Of those who attended yard time, over half were sedentary and no interventions have been conducted during yard time to promote PA. The scientific objective of the proposed research is to develop and test the feasibility and preliminary impact of a structured physical activity program among individuals while incarcerated in jail. We hypothesize that there will be higher yard time attendance, PA levels during yard time, self-efficacy, expectations and values of PA, perceived behavior of others, sleep quality, as well as lower stress, anxiety, and depression symptoms when a structured PA program is offered during yard time compared to no structured PA program. We will identify essential theoretical constructs of Social Cognitive Theory (SCT, i.e., self-efficacy, social support, self-regulation, behavioral capability, outcome expectations, environment), as well as behavioral and cultural attributes of PA among individuals incarcerated to enhance the relevance and effectiveness of a PA program through focus groups and key informant interviews. From formative research, input from jail administration on feasibility of implementation, and involvement from individuals incarcerated on acceptability, we will develop a robust PA program to promote yard time attendance and increase PA among individuals incarcerated. Once developed, we will determine feasibility and preliminary impact of the PA program using a pre-post intervention design. Study outcomes include yard time attendance, PA levels at yard time, self-efficacy, expectations and values of PA, perceived behavior of others, sleep quality, stress, anxiety, and depression. The potential individual gains from even one bout of PA are substantial. Providing individuals opportunities to attend and be physically active during yard time may improve health conditions in correctional institutions. This research plan is complemented by a training plan that builds on the applicant’s background in epidemiology that includes new training in (1) qualitative and mixed methods, (2) intervention development, implementation, and evaluation, and (3) clinical trials. The combined research and training plans will prepare the applicant for a successful, independent research career focused on identifying approaches to promote PA and reduce related health disparities among incarcerated populations.

NIH Research Projects · FY 2024 · 2021-07

ABSTRACT Asthma is a lung disease caused by exaggerated lung inflammation leading to airway obstruction and compromised airflow. Despite significant advances in its diagnosis and treatment, asthma continues to be a significant health problem affecting more than 25 million patients in the US, and over 300 million around the world. Well-characterized sex and gender differences in asthma have been reported, with changes in morbidity throughout life. Starting around puberty and peaking during mid-life, women have increased asthma prevalence and higher rates of asthma exacerbations than men. Causes of these disparities remain unclear; however, studies have shown that sex-specific inflammatory mechanisms controlled by hormones contribute to differences in airway reactivity in response to environmental stimuli. Despite this, experimental models of asthma have not explored the contributions of sex hormones to inflammatory mechanisms in the female and male lung, and no studies have explored the effects of feminizing hormone therapy with estrogen in the lungs of trans women. Prior studies from our laboratory using mouse models have reported sex differences and influences of the estrous cycle and circulating sex hormones in the inflammatory response to environmental exposures. Based on these findings, we hypothesized that female sex hormones, specifically estrogens, contribute to asthma phenotypes in the lung via activation of inflammatory mechanisms mediated by estrogen receptors. In the proposed study, we will test this hypothesis by determining the mechanisms by which estrogen mediates sex and gender influences in asthma. In Aim 1, we will determine the contributions of sex chromosome complement (XX vs. XY) vs. gonadal hormones in asthma phenotypes, by developing a mouse house dust mite (HDM) asthma model on the four core genotypes (FCG) model. In Aim 2, we will study the contributions of estrogens to HDM-induced asthma outcomes using male and female gonadectomized mice treated with estradiol, as well as bronchial epithelial cells from male and female healthy and asthma patients to exposed to HDM in the presence/absence of estrogen receptor agonists/antagonists. In Aim 3, we will determine the roles of nuclear (ER) and membrane- bound (GPER-1) estrogen receptors in estrogen-mediated mechanisms of inflammation in HDM-induced asthma, using ER and GPER1 knockout mice. Our studies will be the first to characterize estrogen-mediated mechanisms of inflammation in asthma phenotypes in the male and female lung, contributing to the characterization of sex- and gender-specific factors accounting for inter-individual differences, as well as the effects of feminizing hormone therapy in lung pathobiology. We expect that our studies would serve to develop potential sex- and gender-specific treatments and recommendations for dosage of therapeutic agents to treat and prevent asthma in cis and transgender women.

NIH Research Projects · FY 2025 · 2021-06

Project Summary We are developing micro- and nanofluidic devices to probe virus capsids, bacteria, and extracellular vesicles at the single-particle level with improved spatial and temporal resolution. Single-particle (or digital) measurements provide not only improved sensitivity but also insight into population heterogeneity. Information content is further enhanced by performing these assays in a high-throughput, multiplexed format, where individual events are tracked, but population statistics are also obtained. We are targeting rare or infrequent events, which can significantly impact the overall function or fate of a system, because these events are often obscured when measurements are made on bulk samples. In the first project, we are studying virus capsid assembly and disassembly. To monitor reactions with capsids, we are designing in-plane nanofluidic devices with multiple nanopores in series for resistive-pulse sensing, which is a label-free, nondestructive sizing technique. Resistive-pulse sensing detects events in real time and has sufficient sensitivity to monitor assembly at biologically relevant concentrations and over a range of reaction conditions. With these nanofluidic devices, we are evaluating how assembly effectors and chaotropes impact the assembly process and produce a variety of particle morphologies, including kinetically trapped intermediates and aberrant structures. In the second project, we are tracking development and aging of bacteria with microfluidic devices that have integrated nanochannel arrays to physically trap bacteria. The nanochannels confine growth of bacteria in one dimension, and when coupled with fluorescence microscopy, image analysis is reduced from a three-dimensional to one-dimensional problem and greatly simplified. Growth and division rates, subcellular functions, epigenetic effects, and antibiotic response are easily tracked for extended periods of time and across multiple generations. In the third project, we are profiling N- and O-glycans derived from serum, urine, and ascites fluid, and their extracellular vesicles. For thorough characterization of these glycans, we are combining chemical labeling strategies to neutralize and differentiate sialyl linkage isomers with analysis by microfluidic capillary electrophoresis and capillary electrophoresis-mass spectrometry. Differences in glycan sizes, degrees of fucosylation and sialylation, and ratios of sialyl linkage isomers are quantified in these samples. We are using single-particle techniques to characterize the physical and chemical properties of extracellular vesicles to correlate these properties with their glycan profiles.

NIH Research Projects · FY 2025 · 2021-05

Abstract Circadian clocks orchestrate myriad molecular, physiological, and behavioral processes to insure internal temporal order and optimal daily timing. In animals, the master clock resides deep within the brain where it relies on complex neural networks to ensure a robust internal sense of time that can readily synchronize with 24-h environmental cycle. There is growing consensus that the operation of our master circadian clock under modern light and social environments contributes significantly to a troubling array of health challenges. Understanding the neural mechanisms underlying circadian timekeeping and the synchronization of the master pacemaker with environmental cycles (i.e., entrainment) is therefore critical. A significant barrier to our understanding of the central circadian clock is the complexity of its constituent neural networks, a complexity compounded by the fact that clock-containing neurons employ multiple neurochemical signals that act via distinct signaling mechanisms. Critical clock neurons in both mammals and insects express multiple transmitters – including peptide co- transmitters - some of which function as local signals across defined synapses while others act as diffusible signals that act over large distances. Peptide co-release, though a common feature of nervous systems, is not well understood. Likewise, how clock neurons employ both local and paracrine signals to mediate circadian timekeeping and entrainment remains enigmatic. Here we propose to study key peptidergic clock neurons in Drosophila as a model to examine how two neuropeptides released from the same neuron can mediate distinct behavioral and physiological functions to support robust circadian timekeeping and entrainment. Our work will not only inform our understanding of circadian timekeeping in the mammalian brain but will also be relevant to the mechanism of peptide co-release generally.

NIH Research Projects · FY 2025 · 2021-04

Project Summary The organization and segregation of replicated chromosomes are fundamental to living systems. Structural maintenance of chromosomes (SMC) complexes play central roles in these processes in all domains of life. These ring-shaped ATPases share common structures and inter-subunit contacts, consistent with a common mechanism of action. Over the last five years, studies in Bacillus subtilis and eukaryotes have provided compelling in vivo and in vitro evidence that SMC complexes utilize ATP hydrolysis to extrude DNA loops. In the case of B. subtilis, SMC condensin complexes are loaded at centromeric parS sites near the replication origin, then translocate down the left and right chromosome arms, tethering them together. In this way, condensins generate a single chromosome loop centered on the origin that draws sister chromosomes in on themselves and away from each other. This elegantly simple loop-extrusion model provides a unifying mechanism to explain how eukaryotic SMC cohesin complexes form topologically associating domains (TADs) in interphase, how eukaryotic SMC condensin complexes compact DNA into rod-shaped sister chromatids, and how bacterial SMC condensins resolve newly replicated origins. However, this model raises an important question: how do SMC complexes extrude DNA loops when the chromosome is coated by numerous proteins and acted upon by replication and transcription machineries? And how are the topologically loaded complexes released from the chromosome? The goal of this proposed research is to understand the mechanism of condensin action in the context of cellular activities, taking advantage of the many molecular and cytological tools we have developed. First, we will determine how condensins act when they encounter the replisome or other condensin molecules. Second, we will characterize how condensins are released from the chromosome when they reach the terminus region. Finally, we will explore condensin’s role in the organization and dynamics of a multipartite bacterial genome that contains both a circular and a linear chromosome. Taken together, the proposed work has the potential to provide the general principles of chromosome folding and compaction in all organisms.

NIH Research Projects · FY 2025 · 2021-03

Project Summary According to the World Health Organization, age-related lens pathologies are the leading cause of visual impairment in the world. Cataracts, defined as any opacity in the eye lens, remain the leading cause of blindness in the world. Presbyopia is caused by a reduction in the lens’ ability to change shape during focusing (accommodation), and, by extension, the need for reading glasses. Unaddressed presbyopia is the leading cause of visual impairment globally. Decades of study have focused on congenital lens pathologies, and thus, very little is known about the underlying cellular and molecular mechanisms that facilitate lifelong lens homeostasis. Recent studies have reported that mutations of the EphA2 receptor or the ephrin-A5 ligand are associated with variable congenital and age-related cataracts in humans and mice, and these bidirectional signaling molecules are key components for regulating lens cell organization and stability. Our mouse models reveal that loss of EphA2 leads to age-related cortical cataracts similar to human patients with EphA2 dysfunction. We will evaluate cataract progression in our mouse line to study the cellular and molecular changes that occur during cortical cataract formation. We hypothesize that loss of EphA2 results in changes in cytoskeletal structures or cell-cell adhesion of peripheral fiber cells leading to optical discontinuities in the lens cortex. Our new data show that the lens utilizes both canonical ligand-mediated EphA2 bidirectional signaling and non-canonical ligand-independent EphA2 signaling pathways. We hypothesize that canonical EphA2 signaling is required in equatorial epithelial cells while non-canonical EphA2 activation is required for fiber cell differentiation and maturation and that this segregation of receptor activity may explain the large variety of human congenital and age-related cataracts caused by EphA2 mutations. We will evaluate the activation pattern of EphA2 spatially and temporally and determine the activity of known downstream pathways in different lens cell populations. We will apply EphA2 agonist and antagonist peptides to primary culture mouse lens epithelial cells to generate mini lenses as an in vitro model for lens development. Increased size and stiffness of the lens center or nucleus has been hypothesized to be a key factor for not only age-related increases in overall lens stiffness and presbyopia, but also poor nutrient and waste transport leading to age-related nuclear cataracts. Our new preliminary data shows that loss of EphA2 leads to softer, smaller lens nuclei. We hypothesize that Eph-ephrin signaling is required for normal cell-cell adhesion and cytoskeleton rearrangement that drives nuclear fiber cell membrane re-organization and compaction. This data will provide a better understanding of coordinated signaling mechanisms for maintaining homeostasis during normal aging and in lenses with changes in transparency and biomechanical properties, which may lead to the development of new non-surgical approaches to delay or prevent age-related lens pathologies.

NIH Research Projects · FY 2025 · 2020-12

PROJECT SUMMARY/ABSTRACT Every year, nearly 2.5 million U.S. high school athletes participate in contact sports. Each of these athletes sustains an average of 650 subconcussive head impacts (SHI) in a single season, with some athletes exceeding 1,000 hits from, for example, football tackles and soccer headings. A subconcussive head impact is defined as an impact that does not trigger the clinically detectable signs and symptoms of concussion. However, these mechanical forces, if applied repeatedly, can trigger subclinical cellular and molecular disruptions in brain cells. Adolescence is an especially vulnerable time for neurodevelopment, because of (a) arborization of white matter tracts within the prefrontal cortex (PFC) and between the PFC and limbic structures and (b) synaptic pruning. Our clinical studies suggest that both acute and chronic exposure to SHI can impair neuro-ophthalmologic functions, increase the levels of brain-derived proteomic (NF-L, Tau, UCH-L1, GFAP, S100B) and exosome (7 neuron/glia-specific exosomes) biomarkers in blood, and trigger changes in the microstructural integrity of white matter. Despite these serious public health implications, no empirical basis exists for establishing a safety protocol or predicting who may develop cumulative pathologic sequelae from SHI, and to what extent, during a 4-year high school football career. The overall goal of this study is to determine the longitudinal effects of SHI on neural integrity and function in adolescents throughout their high school football careers and to identify the dose and intensity of SHI that induce chronic, progressive neurodegeneration. Our central hypothesis is that SHIs in adolescents will gradually degrade neuronal cellular and functional integrity across multiple football seasons in a head-impact-dependent manner. There are three related, hypothesis-driven aims. We hypothesize (1) that a panel of brain-derived proteomic (NF-L, Tau, UCH-L1, GFAP) and exosome markers will increase in blood in response to SHI; (2) that chronic exposure to SHI will disrupt neuro-ophthalmologic function, as reflected in increased variability in smooth eye pursuit and slower King-Devick performance; and (3) that repetitive SHI will lead to disruption in white matter microstructure and changes in resting-state fMRI connectivity. We further hypothesize that with repetitive SHI these changes may not return to baseline and may carry over and accumulate from one season to the next. By tracking SHI exposure and neurologic health in the same athletes for 4 years, the proposed study will help to establish safety guidelines for adolescent athletes. The long-term goal is to prevent neurocognitive deficits in competitive sports athletes.

NIH Research Projects · FY 2024 · 2020-09

PROJECT SUMMARY Social and lifestyle interventions are a promising innovation for delaying the onset of Alzheimer’s disease (AD) and related dementias. Extensive work has shown that maintaining social connectedness through personal social networks (the group of family members, friends, and other acquaintances in which individuals are socially embedded) confers resilience to cognitive decline and AD. The current proposal uses an interdisciplinary and multi-method approach to elucidate the mechanism by which this occurs. To date, existing research has focused on the implications of social networks for one neurocognitive pathway – general cognitive ability. The current proposal tests the novel prediction that a different pathway – social cognitive function – is a key component underlying the linkages between social networks, general cognitive function, and AD resilience. Social cognitive function – the process by which people understand, store, and apply information about others – is essential for successfully navigating social interactions, and declines over the lifespan. The proposed work explores the prediction that exercising social cognitive abilities through complex social interactions within their personal social networks stimulates older adults’ general cognitive function, thereby improving their resilience. In Aim 1, we examine the relationship between older adults’ social networks and their social cognitive function, as well as the possibility that having better social cognitive function may be protective for general cognitive function. Aim 2 uses a novel neuroimaging approach to identify the neurobiological mechanisms that give rise to the relationship between social networks and social cognitive function. Finally, Aim 3 uses a longitudinal design to gain insight into the causal relationship between social network dynamics, social cognitive decline, and general cognitive decline. The proposed study is interdisciplinary, combining cutting-edge methods from the social and biomedical sciences, and leveraging the resources of funded centers for AD, neuroimaging, and network science. The long-term goal of this project is to improve the clinical course of AD, and reduce the prevalence and public health impact of dementia. By increasing our understanding of the links between biological and social processes, this project may help identify novel targets for intervention to reduce the burden of AD on individuals, families, and the health care system.

NIH Research Projects · FY 2026 · 2020-09

PROJECT SUMMARY By 2050, over 75% of Alzheimer’s disease and related dementia (ADRD) cases are projected to occur in low- and middle-income countries, such as South Africa. Government cash transfer programs as a form of social protection may increase individual and household income, with implications for ADRD risk. In the original project period of R01AG069128, we linked data from a cohort study of aging (HAALSI) and a randomized cash transfer trial (HPTN 068) to their shared sampling frame, a longitudinal regional census (AHDSS) in rural South Africa. We identified a protective causal effect of a randomized controlled cash transfer on later-life memory decline, dementia probability, and mortality risk, and causal relationships between expansions of two South African government cash transfer programs and the later-life cognitive health of their targeted recipients. Now, there is a critical need to determine how to optimize cash transfer program design to most effectively leverage cash transfer income to protect against ADRD. South Africa is an ideal setting for this research given its robust and expanding government portfolio of cash transfer programs. Our overarching goal for the proposed renewal of R01AG069128 is to expand our successful data linkage platform to identify the optimal amounts, durations, target recipients, and adult life course periods whereby cash transfer income may protect against later-life ADRD. Our central hypothesis is that greater accumulation of individual-level and household-level cash transfers across the adult life course will result in slower memory decline, lower incidence of ADRD, and greater cognitive reserve, and will also lead to investments in cognitively stimulating household resources. To test this hypothesis, we will newly map historical eligibility for multiple sources of cash transfer income for all HAALSI participants and their household members over a 23-year exposure period (1993-2014/15) and link these eligibility histories to new follow-up data on memory decline and ADRD in HAALSI over 13 years (2014/15-2027). In addition, we will add cognitive reserve as a new study outcome, operationalized from neuroimaging and neuropsychological data in the HAALSI Dementia Study (n=700; 2024/25). We aim to 1) determine the role of access to cash transfers accumulated across the adult life course at the individual- and household-levels in slowing later-life memory decline, reducing ADRD risk, and promoting cognitive reserve; 2) determine the relative importance of adult life course timing and trajectories of household access to cash transfers for memory decline, ADRD risk, and cognitive reserve; and, 3) compare later-life asset ownership and expenditures on cognitively stimulating resources across households according to their accumulated access to cash transfers. Over 130 countries use cash transfers as a form of social protection, including the United States, and more are considering or piloting cash transfer programs. The ADRD impacts of these programs are unknown, and actionable evidence is needed for them to optimally support the health and longevity of the rapidly aging populations that they serve.

NIH Research Projects · FY 2024 · 2020-09

Accelerated Sample Preparation for Microbial Pathogen Detection in Large Number of Pet Food Samples Abstract We are submitting a proposal for Discipline A: Microbiology, track 3 for Animal Food Product testing with Dog treat food/Salmonella as a hazard to Commodity pairs. Here we combine different expertise from 2 laboratories, Laboratory of Renewable Resources Engineering (LORRE) and the Office of Indiana State Chemist (OICS), to assemble a proposal consisting in animal food sample collection, accelerated food sample preparation to bring microorganisms of interest to detection levels, FDA approved loop mediated isothermal amplification (LAMP) for Salmonella screening in animal food, and whole genome sequencing as a method for confirmation of pathogen isolates. We have further optimized our sample preparation approach, as a follow up to 2015 FDA challenge award, and we now can simultaneously concentrate and recovery microorganisms of interest in 4 samples, bringing their numbers from initially not detectable, due to be present at initial levels of 1CFU/g or lower, to detection levels. This accelerated processing of samples is complete in a time frame of about 4 hours. It consists of a microbial short enrichment in selective medium combined with an enzyme treatment, followed by a pre-filtration step to remove large particles and microfiltration to concentrate and recovery selected microorganisms to detection level. The enzyme treatment helps to break down possible biofilms formation and particles that may clog pores in the hollow fiber membrane during microfiltration. Enzymes selected are based on food matrices. In the case of the samples proposed here, proteases possible combined with lipases will be used. We have shown the enzymes do not affect microbial viability, when in the presence of their respective substrates. While we have focused on Salmonella as a microorganism of interest, our developed approach can also be applied to other microorganisms. The Office of Indiana State Chemist (OISC) microbiology laboratory has major expertise in sample collection for animal food testing. It has also many years of experience in microbiological method development, testing for antibiotic concentration, and bacteria enumeration and identification, including several years of pathogen screening for Salmonella, Listeria monocytogenes, Campylobacter and Escherichia coli (STEC) in animal feed. Last year, the OISC microbiology laboratory contributed for testing in a national wide Salmonella outbreak in pet treat pig ear in Indiana. The proposed number of samples to be tested here is 250 samples pairing dog treat/Salmonella. Expected outcomes are accelerated sample preparation, increased speed and sensitivity of sample testing, and initial insights of potential recovering and detection of difficult to culture microorganisms.

NIH Research Projects · FY 2025 · 2020-08

Project Summary / Abstract Organisms rarely live in isolation. They live in complex multi-species communities where soluble chemical factors mediate cell-cell signaling, cooperation, and competition. Mutualist microbes exchange metabolites and signals to benefit their hosts in myriad ways, while parasites release virulence factors that harm their hosts. Microbes also exhibit similar chemically mediated benefits and challenges to one another in environmental and host- associated contexts. From a cellular perspective, even a multicellular organism can be considered a complex symbiosis of phenotypically distinct cells using chemicals to cooperate for the overall benefit of the ‘community’. My laboratory of chemists, biochemists, and biologists aims to discover the chemical languages that shape interactions within microbial communities and between microbes and their hosts. Our proposed work focuses on chemical signaling in three distinct areas. First, we aim to uncover chemical signaling processes that regulated key transitions in the origin of the first multicellular animals (e.g., formation of multicellular bodies and differentiation of cell types). We pursue this goal by identifying the chemical signals and mechanisms that regulate multicellular behaviors in some of the closest living relatives of animals—the unicellular holozoans Capsaspora owczarzaki, Ministeria vibrans, Salpingoeca rosetta, and Corallochytrium limacisporum. This work may reveal how the first animals evolved and may inform how the diverse cells within healthy animals develop and function. Second, we aim to uncover chemical signaling processes that enable the protozoan snail symbiont Capsaspora owczarzaki to colonize its snail host. This symbiont can kill the parasitic schistosomes that mature in snails before spreading to humans where they cause the neglected tropical disease schistosomiasis. Therefore, understanding Capsaspora’s ability to persist in snails and hunt schistosomes may improve its development as a biocontrol agent to curtail the spread of schistosomes through snails near human populations. Finally, we aim to uncover new chemical signals that regulate bacterial behaviors necessary for pathogenesis. By arresting these behaviors, we can disarm pathogens. Overall, our work will reveal previously unrecognized ways that symbionts and cells within animals communicate and compete on a chemical level. This work may be applied to reveal the most fundamental mechanisms of regulated multicellularity in animals, and it also may be applied to inform efforts to curtail the transmission and virulence of infectious diseases.

NIH Research Projects · FY 2025 · 2019-09

Peptidoglycan (PG) is the mesh-like scaffolding that determines the shape and size of bacterial cells and protects them from osmotic shock. In Gram-positive bacteria, thick PG provides the outer cellular layer to which wall- teichoic acids, capsule, and extracellular proteins are covalently attached. The human respiratory pathogen Streptococcus pneumoniae (pneumococcus) has emerged as a leading model for PG synthesis and its regulation in ovoid-shaped Gram-positive bacteria. Pneumococcal PG synthesis shows fundamental differences from PG synthesis in rod-shaped and spherical bacteria. Most notably, PG synthesis is zonal and carried out by two separable PG synthase nanomachines confined to the midcell of dividing pneumococcal cells. The septal PG synthase (bPBP2x:FtsW) locates to the leading edge of the closing septal annulus, while the elongasome PG synthase (bPBP2b:RodA with RodZ, MreCD) locates to the outer rim of the septal annulus and pushes peripheral PG outward. Both the septal and elongasome PG synthases move circumferentially at midcell driven by PG synthesis, but not by FtsZ treadmilling. This project builds on previous findings to address the most important current questions about PG synthesis in S. pneumoniae. One set of questions centers on the functions and regulation of the three Class A PBP PG synthases (aPBP1a, aPBP1b, aPBP2a) in exponentially growing and stressed pneumococcal cells, about which relatively little is known. By using a comprehensive approach that includes innovative assays of septal and elongation PG synthesis rates in live cells, single-molecule (sm) motion dynamics, and high-resolution microscopy, we will determine the contributions of the aPBPs to PG synthesis and how their dynamics and localization are altered by mutational changes and cell-wall stress. Unbiased and directed approaches will be used to identify aPBP interactors at different stages of division and how these interactions regulate aPBP functions. A second set of questions center on the mechanisms that organize and regulate the septal and elongasome PG synthase nanomachines. We will determine mechanisms that organize circumferential motion and nodal distributions of PG synthases at midcell. We will also determine the assembly pathway of the septal divisome in early-divisional cells and the mechanisms by which the septal and elongasome PG synthesis machines separate and are regulated later in division. We will continue studies of newly discovered mechanisms that link PG synthase functions to the availability of PG precursor metabolites and to second messengers. A third set of questions concerns the roles, interactions, and regulation of the FtsEX:PcsB hydrolase in PG remodeling and the MpgB and MpgA muramidases in PG-chain release in septal and elongasome PG synthesis, respectively. Altogether, this project will fill in major knowledge gaps about the dynamics, functions, and regulation of aPBPs, about the organization and regulation of septal and elongasome PG synthesis, and about PG remodeling and hydrolysis in S. pneumoniae, add significantly to the PG synthesis field in general, and reveal additional vulnerabilities and targets for new antibiotic and vaccine development.

NIH Research Projects · FY 2023 · 2019-09

PROJECT SUMMARY / ABSTRACT CANDIDATE: Austin T. Robinson, Ph.D is a Postdoctoral Associate at the University of Delaware (UD). Dr. Robinson aims to study racial differences in cardiovascular responses to high dietary sodium (Na+). He recently published data demonstrating that compared to white individuals, 1) black individuals have augmented increases in serum Na+ to a hypertonic saline infusion; and 2) exhibit higher blood pressure (BP) for a given serum Na+. In this proposal, he will translate these findings by comprehensively assessing neurovascular responses to acute (single meal) and chronic (10 days of a controlled feeding) high dietary Na+. The central hypothesis is that high dietary Na+ influences sympathetic nerve activity similarly in black and white individuals; however, diminished vasodilator capacity and augmented sympathetic transduction (vasoconstrictor responses to sympathetic nerve bursts) contribute to exaggerated BP dysregulation in black individuals. He will also determine the role of lifestyle factors (i.e., sleep, physical activity, and nutrition) on potential baseline racial differences in CV physiology. CAREER DEVELOPMENT PLAN: Dr. Robinson proposes to enhance his career development by: 1) Acquiring new skills in the assessment of sleep, assessment of (nitric oxide) NO. bioavailability, and assessment of reactive oxygen species (ROS). 2) Advanced training in epidemiology to analyze large data sets. Specifically, he will perform secondary data of samples collected for the Multi-Ethnic Study of Atherosclerosis (MESA) under the guidance of advisor Dr. Norrina Allen, an expert epidemiologist. 3) Refining his professional skills though formal course work, attendance and presentations at weekly journal clubs, and at national/international scientific meetings. ENVIRONMENT: Dr. Robinson will train in an outstanding research environment supported by a multi- disciplinary team of mentors. The primary mentor, Dr. Farquhar, is an NHLBI-funded full professor at UD with a record of successful mentorship. He is an expert in autonomic control of circulation, and executing controlled salt feeding studies. Co-mentor Dr. Edwards is also at UD and a leading expert in vascular physiology function and executing controlled dietary salt feeding studies. Co-mentor Dr. Brown is an expert in lifestyle as a preventive and treatment strategy for hypertension and vascular function in black individuals. Advisors Dr. Zimmerman and Dr. Poole are experts in ROS and NO. signaling, respectively. Advisor Dr. Buxton is a recognized expert in sleep and health consequences of sleep deficiency, especially cardiometabolic outcomes. Advisor Dr. Wright is a professor in Nursing at UD and has expertise in neuropsychology and racial disparities. RESEARCH: Black individuals are more prone to salt-sensitive elevations in BP and adverse cardiovascular conditions associated with chronic high dietary Na+ intake compared to other racial groups. Racial disparities in vascular function and, autonomic control of BP in black individuals under basal conditions also exist. There is a critical need to elucidate the physiology underlying these racial disparities and to determine if these racial disparities are attributable to lifestyle differences.

NIH Research Projects · FY 2025 · 2019-07

Thirty-two collaborative training faculty from the Departments of Chemistry, Biology and Molecular and Cellular Biochemistry, and interdepartmental programs in Cell, Molecular and Cancer Biology (CMCB) and Neuroscience, request funding to renew our predoctoral training program at the chemistry-biology interface (CBI) in Quantitative and Chemical Biology (QCB) at Indiana University, Bloomington. Our training mission is to transform graduate education in the molecular sciences on our campus by facilitating interdisciplinary and collaborative research training in the chemical and biological sciences to address important problems in biology and medicine. We outline an evolving, innovative and forward- looking program that takes our trainees from mere technical proficiency in their chosen disciplines to the development of critical thinking skills across disciplinary boundaries and professional communication and leadership skills that will allow our trainees to thrive in the next stage of their careers. We couple a core didactic curriculum in chemical and physical biology through two 1.5 credit core courses, Introduction to Quantitative Biology and Measurement (CHEM C680) and Introduction to Chemical Biology I (CHEM C681) and QCB Journal Club (CHEM C689) to a topical collection of extracurricular programmatic elements that are organized nearly exclusively by QCB trainees under the leadership of two rotating QCB ambassadors. These programmatical activities include QCB Student-invited Seminar Series and QCB Evenings, a “super-group” research seminar series, and the annual Watanabe Symposium in Chemical Biology, which brings scientists to Bloomington from academia and industry to network with QCB trainees. Innovative programmatic elements include a trainee-run off-campus retreat and an e- learning/”flipped” classroom instructional approach used to bring prospective trainees “up to speed” on core didactic material and to significantly augment Plans for the Instruction in Methods for Enhancing Reproducibility and the Responsible Conduct of Research. We also outline an internship program for third-year trainees to help prepare them for a wide range of career paths after graduation. The program is directed by a Program Director and a Co-Program Director, each with complementary expertise in physical and chemical biology, and is overseen by a Steering Committee that includes preceptors from all five participating departments and programs. The Recruitment Committee is charged with all aspects of identifying and recruiting training grant-eligible students for support by the program in training years 2 and 3 or year 3 only. Our program enjoys strong financial support from the College of Arts and Sciences and the University Graduate School who have committed fifteen (15) matching slots over the five-year project period and significant matching funds for programmatic elements.

NIH Research Projects · FY 2026 · 2019-05

PROJECT SUMMARY/ABSTRACT Wolbachia pipientis is an obligate intracellular alpha-proteobacterium that infects 40-60% of insect species on the planet. Wolbachia infection inhibits RNA virus replication in insects, a phenomenon known as pathogen blocking. Therefore, Wolbachia infected mosquitos are being released in many parts of the world to control the spread of human diseases. Importantly, although the mechanism behind Wolbachia’s virus inhibition is not known, Wolbachia must colonize the host and be efficiently maternally transmitted in order for pathogen blocking to work. Our long-term goals are to identify the mechanisms used by Wolbachia to establish infection. To that end, we focus on the type IV secretion system (T4SS), a molecular nanomachine used by Wolbachia to inject proteins, termed effectors, into the host cellular environment. Via these secreted effectors, the host cell is modified, allowing Wolbachia to invade and persist. Our previous work identified and characterized the first secreted effector in Wolbachia (WalE1) and established that this effector disrupts host endocytosis. We developed a live assay for visualizing Wolbachia infection and showed that the actin cytoskeleton must be intact for this to occur. We identified important Wolbachia effectors upregulated during infection using proteomics and show that the T4SS is upregulated upon host internalization. In this proposed renewal, we will continue or work to identify the Wolbachia secretome across strains, within both Drosophila and Aedes, and identify pathways important for Wolbachia infection of both host species. We will also further identify how the important effector WalE1 functions across strains and determine the conservation and function of important domains for this protein. Guided by strong preliminary data, we propose to pursue three Specific Aims to identify and characterize Wolbachia effectors, host pathways important for infection by the microbe, and WalE1 function. We will (1) Determine and characterize the Wolbachia secretome, across strains, (2) Identify host pathways important for colonization of hosts by different Wolbachia strains, and (3) Characterize the molecular biology and evolution of Wolbachia’s WalE1. Studies of Wolbachia - host interactions are still in their infancy despite the recognized contributions of endosymbiotic associations to insect reproduction and evolution, and the ability to alter vector competence. These proposed studies will significantly advance our understanding of how Wolbachia employs its effectors to establish infection, a necessary prerequisite to pathogen blocking.

NIH Research Projects · FY 2026 · 2019-04

ABSTRACT Many bacteria are motile by synthesizing corkscrew-like flagella which when rotated propel bacteria through the environment. Each bacterium synthesizes a species-specific number of flagella and inserts the flagella in a species-specific pattern on the cell surface. Flagella are complex nanomachines assembled from dozens of different proteins and how each bacterial species controls flagellar number and patterning is poorly- understood. Moreover, the number of flagella per cell increases when cells come into contact a solid surface to initiate a form of surface motility called swarming. The Kearns lab uses classical forward genetics, super- resolution microscopy, and biochemistry to study flagellar biosynthesis and swarming motility of the Gram positive bacterium Bacillus subtilis. The goals of the project are to understand flagellar biosynthesis in the context of growing cell architecture. First, we will determine how flagellar number is controlled by the poorly- understood master regulator of flagellar biosynthesis SwrA and a response regulator DegU. Second, we will explore how the surface contact response is transduced to inhibit the adaptor-mediated regulatory proteolysis of SwrA and increase flagellar number. Third, flagella are synthesized in a grid-like pattern and we will study how flagellar patterning is interpreted and updated in time during cell growth, and coordinated with peptidoglycan insertion. Fourth, we will study how flagellar assembly is integrated with two other envelope- associated machines, the cell elongasome and the divisome controlling cell growth and division, respectively. Ultimately, we want to achieve a holistic understanding of how a cell dynamically governs the initiation of flagellar biosynthesis at specific locations to insert the machine through the cell wall. Our basic research is fundamental to how cells self-organize and is applicable to the spatiotemporal control of the assembly of transenvelope nanomachines involved in pathogenesis including flagella, pili and protein secretion apparati.

NIH Research Projects · FY 2026 · 2019-04

Project Summary/Abstract: The invention of new methods to access chiral organic molecules is a critical objective in modern organic chemistry as it is essential for the efficient synthesis of pharmaceutical agents. This is especially relevant as the pharmaceutical industry is making efforts to increase the 3D complexity of drug candidates. Despite substantial progress in the field of stereoselective chemical synthesis, many structures remain challenging to prepare in useful quantities. Therefore, development of new methods and strategies for the chemical synthesis of stereochemically and topologically complex molecules is of contemporary interest. The long-term goals of our research program are to introduce general and efficient strategies for the stereoselective synthesis of difficult- to-access molecular frameworks found in important bioactive molecules. Towards this end, we are interested in the conversion of abundant and readily available alkenes to more complex structures through difunctionalization reactions. This approach is attractive because the rapid buildup of complexity can be achieved as two new bonds and two new stereocenters are generated in a single operation. The studies described in this application focus on three distinct programs. The first is the development of stereoselective cross-coupling reactions of Csp3- nucleophiles that are catalytically generated in situ from simple alkenes. Our rationale for development of these reactions is that widely available alkenes, diboron reagents, and organohalides are converted to synthetically versatile intermediates. We will develop new Pd/Cu-catalyzed and Ni-catalyzed systems with a particular emphasis placed on saturated heterocycle synthesis. In addition, with the advent of new catalytic systems we will engage Csp3-electrophiles for the generation of multiple stereogenic centers. In the second program of research, we are developing methods for the stereoselective synthesis of cyclobutanes by [2+2] cycloadditions of alkenes that are enabled by boron. Our rationale for the development of these reactions is that due to the unique feature of boron, a broad range of borylated cyclobutanes can be prepared. Finally, we are introducing new classes of novel building blocks to enable drug discovery, such as bicyclo[2.2.0]hexanes as isosteres for meta-substituted aromatic rings. Within this program, we are also developing novel strain release reactions initiated by energy transfer. Overall, these studies in reaction development will introduce new concepts and strategies, as well as provide access to new building blocks for chemical synthesis by exploring new cross- coupling paradigms and cycloaddition reactions.

NIH Research Projects · FY 2025 · 2018-09

The goal of this renewal of the Social Networks and Alzheimer’s Disease study (R01 AG057739) is to understand the social and biological mechanisms underlying the role of social connectedness in mild cognitive impairment (MCI) and Alzheimer’s disease and related dementias (ADRD). This longitudinal study leverages a cohort of older adults with early stage ADRD, MCI, and age-matched cognitively normal controls (N=609) established in 2015 and followed annually through 2023. The SNAD study features high dimensional characterization of personal social networks, relationships, environments, and activities obtained via in-home interviews, integrating these data with clinical consensus diagnosis, extensive cognitive testing, genotyping, and functional and structural neuroimaging data collected annually through the NIA-sponsored Indiana Alzheimer’s Disease Research Center. Findings from SNAD reveal robust and extensive cognitive and neurological benefits of social bridging, or access to an expansive and diverse set of loosely connected individuals. As a form of cognitive enrichment, social bridging may be protective of cognitive decline through the development of cognitive reserve (CR), or cognitive adaptability that buffers the impact of brain pathology on cognitive function. In addition to producing novel insights, SNAD raised new research questions and revealed data limitations that necessitate a second project period and additional longitudinal follow up. In the next phase of the study, we propose to increase sample size and extend the follow-up period, increase population representativeness in the cohort, and collect additional social and biomarker data (i.e., DNA methylation) to test novel mechanisms through stress pathways. Aim 1 is to conduct long-term follow-ups of the SNAD cohort to examine longitudinal relationships between personal social network dynamics, neurodegenerative changes, and clinical conversion to MCI or ADRD. Aim 2 is to expand the SNAD cohort to determine whether observed associations between social network characteristics and clinical cognitive decline are replicable in communities at high risk for ADRD. Aim 3 is to explore mediating and moderating relationships between social network characteristics, stress exposures, biological aging, and cognitive decline. The SNAD study is interdisciplinary, combining leading-edge methods from the social and biomedical sciences, and leveraging the resources of funded centers for ADRD, neuroimaging, and sociomedical sciences. By increasing our understanding of the links between biological and social processes, this project may help identify novel targets for intervention to reduce the burden of ADRD on individuals, families, and the health care system.

NIH Research Projects · FY 2025 · 2018-09

Project Summary The Dalia lab studies the regulation and mechanisms of horizontal gene transfer by natural transformation (NT) using Vibrio cholerae as a model system. While this bacterium is the causative agent of the diarrheal disease cholera, we do not seek to study bacterial pathogenesis. Instead, we leverage this well- established model system and the genetic tools we have developed to characterize NT in a physiologically relevant context. NT contributes to the rapid spread of antibiotic resistance determinants and virulence factors in bacterial pathogens. Thus, characterizing the regulation and mechanisms of NT may uncover novel approaches to combat diverse clinically relevant infections. The genes required for NT are tightly regulated, and only induced when V. cholerae forms biofilms on the chitinous shells of crustacean zooplankton in the aquatic environment. The formation of chitin biofilms is also important for the survival of this facultative pathogen in its environmental reservoir, and promotes the waterborne transmission of cholera. Vibrio-chitin interactions can be easily studied in a lab setting, which provides a physiologically relevant and highly tractable model system for studying NT. In the next five years, we will focus on three major areas. First, V. cholerae uses dynamic surface appendages called type IV pili to bind to chitinous surfaces and to take up DNA for NT. The mechanisms that regulate the dynamic extension and retraction of these appendages, however, remains poorly characterized. Using tools we helped develop to label type IV pili in live cells, we will address how motor ATPases, minor pilins, and other environmental cues regulate pilus dynamic activity. Second, we will study how two membrane-embedded DNA-binding transcription factors, ChiS and TfoS, coordinate to activate the chitin regulon in V. cholerae. Membrane- embedded regulators are understudied, and this work will help elucidate the dynamics and constraints for the DNA-binding activity of membrane-embedded vs soluble transcription factors. Third, we will use a combination of cell biological and genetic approaches to study horizontal gene transfer by natural transformation. Namely, we will address the spatiotemporal dynamics of natural transformation within chitin biofilms; and the impact of neighbor predation on these dynamics. Also, we will formally test the long-standing, yet untested, hypothesis that DNA uptake during natural transformation plays an important nutritional role. Together, our basic research extends a fundamental understanding of a number of critical and conserved processes (e.g. pili, signal transduction, horizontal gene transfer) that are shared by diverse microbial species, including many pathogens.

NIH Research Projects · FY 2025 · 2018-08

This project catalyzes research on brain networks by developing computational methods and software tools for analysis of diffusion MRI (dMRI) data and validating them in vivo. Understanding brain networks and their relation to neural computation is a major challenge in contemporary neuroscience. The proper function of brain networks is also inextricably tied to neurological, cognitive and psychiatric health. The networks that connect distinct regions in the brain are composed of large bundles that contain the axons of millions of neurons. DMRI is the only currently available method to measure the trajectory and physical properties of these bundles in the human brain non-invasively and a large body of research using dMRI has substantially contributed to our understanding of the way in which differences in brain connections contribute to individual differences across a spectrum of behaviors and clinical conditions. Progress in research and methods development has also translated into increasing use of dMRI in clinical applications. The project is led by the founders of the Diffusion Imaging in Python (DIPY) software project who have been working to invigorate the neuroimaging user and developer community by developing, implementing, disseminating, and maintaining important software tools. The proposed project pursues a next generation phase of development, in which the overall objective is to generate a platform to better utilize dMRI data in accordance with BRAIN 2.0 targets. To address current barriers to progress, we plan to address the following aims in the current proposal: Aim 1 will develop new tractometry methods that enhance the interpretability of dMRI data; Aim 2 will introduce new pre-processing algorithms including methods for susceptibility correction; Aim 3 will focus on improving computational performance using parallel computing within a single node (e.g., via use of graphical processing units) and across nodes (i.e., distributed computing); Aim 4 will focus on the validation of DIPY methods using publicly available human and non-human primate data. Overall, this work will enable impactful brain research and will facilitate subsequent clinical adoption of advanced computational methods using dMRI data.

NIH Research Projects · FY 2025 · 2017-09

Project Summary Bacteria communicate using the cell-cell signaling system called quorum sensing to collectively alter gene expression in response to changes in population density and composition. Quorum sensing controls behaviors that benefit the group for adaption and survival, including biofilm formation, motility, bioluminescence, and toxin production and secretion. A more comprehensive understanding of how cell-cell signaling regulates virulence and impacts bacteria in their environmental niches can lead to the development of anti-microbial molecules that modulate quorum sensing to mitigate pathogenesis. Despite advances in elucidating the quorum signaling inputs, comparatively less is known about the output – the transcriptional regulation program that controls group behaviors and development in bacteria. The objective of the proposed research is to define how bacteria use quorum sensing signaling to control virulence gene expression, using Vibrio bacteria as established quorum sensing model systems and relevant pathogens. In Vibrio species, LuxR is the master transcription factor and the conserved core regulator of quorum sensing genes and virulence. Previous work identified important and highly conserved biochemical, biophysical, and genetic features of LuxR and Vibrio quorum signaling systems that govern gene expression. Yet, the influence of quorum sensing on numerous developmental pathways varies even among closely related Vibrio strains through means that are not understood. The proposed research will expand upon these findings to examine gene regulation by LuxR at the mechanistic level and then more broadly connect this information to the conservation and impact of quorum signaling networks across Vibrio species. First, the proposed research will determine the connections between spatial organization of the chromosome and LuxR regulation based on established findings that nucleoid structuring proteins impinge on quorum sensing gene expression in several Vibrio species. Second, to more broadly examine the influence of signals from “self” (quorum sensing autoinducers) and “other” (environment), quorum sensing gene expression will be assessed at the single-cell and population- wide level in response to variations in autoinducer signaling and nutrient availability. The proposed microfluidics, cell culture, and host infection experiments combined with comparative genomics will provide key links between quorum sensing signaling and Vibrio adaptation to environmental and host signals. Third, the van Kessel lab recently developed thiophenesulfonamide inhibitors that specifically block LuxR protein function in Vibrio bacteria. These molecules are key tools that will guide our understanding of LuxR function and inform structure-activity modeling and inhibitor design for potential therapeutic compounds. Collectively, the proposed research will provide fundamental data critical to understanding quorum signaling and how it impacts bacterial pathogenesis to contribute to future advances in vibriosis disease treatment.