Now showing items 21-40 of 1921

    • From Data to Decisions: Utilizing Pharmacometrics to Optimize Clinical Therapeutics and Drug Development in Neuropsychiatry

      Kalaria, Shamir; Gopalakrishnan, Mathangi (2020)
      At least 50% of clinical trials of neuropsychiatric compounds fail due to an unclear understanding of disease pathophysiology and drug pharmacology. Further, lack of dosing information in special patient populations for approved neuropsychiatric drugs could contribute to suboptimal outcomes. The current research highlights the role of pharmacometrics in (i) optimizing therapeutics in patients receiving antiepileptics and continuous renal replacement therapy (CRRT) and (ii) informing efficient trial design for binge eating disorder (BED). Currently, no dosing recommendations exist for CRRT patients receiving antiepileptics. Real-world clinical studies were conducted to characterize the pharmacokinetics of levetiracetam and lacosamide in patients (N=18) receiving CRRT at the University of Maryland Medical Center. Major determinants for drug clearance were drug-specific extraction coefficient (EC) approximated to fraction unbound (levetiracetam: 0.89, lacosamide: 0.80), effluent flow rate, and preserved non-renal clearance. Ex-vivo models of CRRT were developed using human plasma and normal saline containing albumin solutions. The developed ex-vivo in-vivo correlation model demonstrated an average bias of <15% in predicting in-vivo CRRT clearance for levetiracetam and lacosamide. Similarity in ECs justified the ability to bridge dosing information across CRRT modalities. This research, in combination with a priori knowledge of drug pharmacokinetics, confirms the use of ex-vivo CRRT models to establish dosing recommendations and alleviate the need for CRRT pharmacokinetic studies. The development of BED therapies are challenged by high placebo response and high dropout rates in clinical trials. A comprehensive disease-drug-trial (DDT) model was developed using data from 12 different investigator-led BED clinical trials (N = 578; 6 to 16-week duration) to inform optimal clinical trial design elements. Baseline BED severity metrics were predictors for placebo response and dropouts. Stimulants and anticonvulsants demonstrated 1.8 times higher effect differences as compared to antidepressants. Among the clinical trial designs (placebo run-in, drug run-in, sequential parallel comparison design) evaluated in-silico, placebo-controlled trial of shorter (6-week) duration with model-based analysis demonstrated superior trial design properties (40% lower sample size with 50% lower dropouts) as compared to current 12-week registration trials for BED. The proposed DDT framework can inform efficient trial design and potentially increase the number of therapeutic options for BED.
    • Whole-genome analysis of Plasmodium falciparum isolates to understand allele-specific immunity to malaria

      Shah, Zalak; Takala-Harrison, Shannon (2020)
      After repeated P. falciparum infections, individuals in high-transmission areas acquire clinical immunity to malaria. However, the genes important in determining allele-specific immunity are not entirely known. Previous genome-wide approaches explored signatures of selection in the parasite genome to identify targets of clinical immunity; however, these approaches did not account for individual level allele-specific immunity. Here we take a whole-genome approach to identify genes that may be involved in acquisition of allele-specific immunity to malaria by analyzing parasite genomes collected from infected individuals in Malawi. However, obtaining whole genome sequence data from clinical samples is one of the major hurdles in the field of malaria genomics. In order to obtain whole genome sequence data from non-leukocyte depleted, low parasitemia samples, we optimized a selective-whole genome amplification (sWGA) by filtering the DNA prior to sWGA, to generate high coverage, whole genome sequence data from P. falciparum clinical samples with low amounts of parasite DNA. Using this optimized approach, we successfully performed whole-genome sequencing on 202 parasite isolates. We compared parasite genomes from individuals with varying levels of clinical immunity, defined using an individual’s proportion of symptomatic infections during the course of the study, hypothesizing that individuals with higher immunity become symptomatically ill due to infection with parasites with less common alleles. Using FST, we identified 161 SNPs to be genetically differentiated between the two groups and the median allele frequency was significantly lower at these sites in individuals in higher immunity group compared to the lower immunity group. We also examined pairs of parasites collected at different time points from the same individuals and identified 225 loci in 174 genes that vary within same individuals more often than expected by chance. Using both of these approaches, we identified 25 genes that encode likely targets of immunity, including a known antigen, CLAG8. Further analysis of clag8 global diversity showed evidence of immune selection in the C-terminal region, supporting the use of this approach in identification of new vaccine targets. Identifying and further analyzing these genomic regions will provide insights into mechanisms involved in allele-specific acquired immunity.
    • Placebo Analgesia in Neuropathic Pain: A Translational Investigative Approach from Rodents to Humans

      Akintola, Titilola; Colloca, Luana (2020)
      Pain is a complex phenomenon which can be influenced by various factors. Placebo analgesia (PA) is the experience of pain relief after the administration of a physiologically inert intervention via the expectation of benefit. However, adequate animal models of PA in chronic neuropathic pain were unavailable to determine how PA occurs in neuropathic pain. Neuropathic pain (NP) is a chronic pain condition characterized by a dysfunction of the peripheral or central nervous system. There is still limited progress in translating the findings of preclinical studies to address the clinical burden of chronic pain. This is thought to partly reflect difficulties in reliably assessing pain in animals. Hence, we employ a translational approach in both rodents and humans to explore the occurrence of PA in chronic NP. First, I tested the hypothesis that the facial grimace scale is a useful metric of spontaneous pain in rodents. We performed a chronic constriction injury of the infraorbital nerve (CCI-ION) and tested for changes in mechanical hypersensitivity and grimace scores. Results showed rodents with CCI-ION had significantly higher grimace scores and lower mechanical withdrawal thresholds compared to controls. These changes were reversed by an opioid, indicating the grimace scale as a sensitive metric for assessing ongoing pain in CCI-ION. Secondly, I tested the hypothesis that pharmacological conditioning with fentanyl would produce PA in a rat model of CCI-ION. Rats were pharmacologically conditioned with or without contextual cues. We administered a placebo and found marginally significant PA effect via the grimace scale but not in mechanical sensitivity. These findings suggest that PA may be more challenging to induce in rodents. Finally, in humans, I investigated how NP-like symptoms in Temporomandibular Joint Disorder alter PA. The effect of NP on PA is yet to be fully understood. I tested the hypothesis that the presence of NP-like symptoms would decrease PA in TMD. NP assessment was carried out both in the orofacial region and across the whole body using validated screening tools. Our results showed that the presence of co-occurring NP-like symptoms increased PA in TMD. We also show that this effect is mediated by reinforced expectation.
    • Pharmacometric Approaches to Precision Therapeutic Management for Antimicrobials

      Wang, Hechuan; Ivaturi, Vijay (2020)
      Antimicrobials have been widely used for decades in the treatment of various types of bacterial infections and their properties have been thoroughly characterized in pediatric and adult patients. However, high variability and unpredictability of antimicrobials’ pharmacokinetics (PK) in patients still exist, which reinforces the value of precision dosing. The research in this thesis highlights the role of pharmacometrics in precision therapeutic management of two prototype antimicrobials, gentamicin and rifampin. The first project developed a Bayesian forecasting algorithm for precision dosing of gentamicin in pediatrics. We developed the first population PK model for gentamicin across the whole pediatric age spectrum ranging from 1-day-old newborns to 19-year-old young adults. The model utilized physiologically plausible covariate parameterization driven by principles of allometric scaling. Renal function changes manifested by glomerular filtration were described by postmenstrual age, and serum creatinine was standardized by age. The model was used as a prior in the subsequent full Bayesian analyses in pediatric patients. A full Bayesian analysis-based model-informed precision dosing (MIPD) was introduced for gentamicin dosing in pediatric patients. With a predefined probability of target attainment (PTA) criteria of 70% for both maximum and trough concentrations, the dosing regimens recommended by the empirical dosing guideline NeoFax could achieve the predefined criteria in about 5% of the 1013 patients, in comparison with 90% of the patients when the initial dosing recommendation from the MIPD approach was used. Finally, a workflow was designed for a new patient in a clinical scenario to provide MIPD for initial dosing recommendation and dosing adjustment after TDM level becomes available via a full Bayesian approach. The second project focuses on dose optimization of rifampin in adult patients with tuberculosis through dynamic positron emission tomography (PET) scans. A semi-mechanistic PK-lung-biodistribution model was developed based on plasma and intralesional drug concentration data measured by PET scans. The model could well predict the mass spectrometry data from therapeutic dose and PET data from 11C-labled micro-dose. The developed model was externally validated through exposure predictions in the therapeutic range of 10-35 mg/kg. Based on the projected drug exposure in the cavity walls at higher rifampin doses, the bacterial killing curves obtained from hollow fiber systems were used to predict the clinical cure rates in humans for higher rifampin doses (>600mg). Standard oral rifampin dosing of 10 mg/kg would achieve a 95% probability of cure in 6-9 months of treatment. Similarly, an oral rifampin dose of at least 35 mg/kg would be needed to cure patients in 4 months.
    • Identifying pathogenic mechanisms and new therapeutic targets for Gaucher disease using induced pluripotent stem cells

      Srikanth, Manasa; Feldman, Ricardo A. (2020)
      Gaucher Disease (GD), the most common lysosomal storage disorder, is caused by mutations in the GBA1 gene, which codes for the lysosomal enzyme β-glucocerebrosidase (GCase). GCase breaks down sphingolipids but when it is mutated, it causes the accumulation of glucosylceramide (GlcCer) and glucosylsphingosine (GlcSph). The common manifestations of GD include hepatosplenomegaly, anemia, thrombocytopenia, skeletal disease, and in case of severe mutations, there are also fatal neurological manifestations. The conventional treatment is not effective in managing the skeletal or neurological manifestations. Hence, a better understanding of the underlying mechanisms that cause GD pathology is required for development of effective therapeutic strategies. The goal of this thesis was to identify the molecular mechanisms responsible for phenotypic alterations in osteoblasts and neuronal cells from GD patients, thereby pinpoint molecular targets for therapeutic intervention. Our laboratory utilizes patient-specific induced pluripotent stem cells (iPSCs) harboring GBA1 mutations to model GD. We have previously differentiated these iPSCs to various cell types and have shown that we can recapitulate the pathologic hallmarks of GD. Thus, in this study, we generated GD-iPSC derived osteoblasts and neuronal cells and found that mutations in GBA1 disrupt the canonical Wnt signaling and lysosomal compartment in these cell types. The phenotypic consequence of this was observed in the form of defective osteoblast differentiation and maturation as well as loss of midbrain/hindbrain neuronal progenitors in the respective cell types. Due to the known lysosomal dysregulation in GD, we then explored the mTOR pathway which is upstream of lysosomal biogenesis. We found hyperactivation of mTOR in GD neuronal cells was mediated by the significant accumulation of GlcSph, a lysolipid of GlcCer. In addition, when we blocked the conversion of GlcCer to GlcSph using acid ceramidase inhibitors, we were able to reverse mTOR hyperactivation and restore lysosomal expression, suggesting that GlcSph is partly, if not fully, responsible for the lysosomal abnormalities observed in GD. In conclusion, our study reveals that activation of canonical Wnt pathway or suppression of mTOR pathway ameliorates the phenotypic abnormalities in GD and identifies b-catenin, mTOR and acid ceramidase as potential therapeutic targets for GD.
    • Adult Patients’ Experience Using Patient Portal: The Impact of Perceived Usability on Portal Use Behavior

      Son, Hyojin; Nahm, Eun-Shim (2020)
      Background: Patient portals (PPs) are a robust tool that can engage patients into their care. PPs can be especially helpful for older adults who have complex healthcare conditions. Usability of PPs is a major influencing factor for PP use. PP usability is more important for older adults who tend to be less familiar with technologies and may need additional support for using PPs. Currently, there has been a lack of studies that examined PP usability perceived by patients after PP implementation in healthcare settings. Objectives: The primary aim of the study was to test a modified PP Acceptance Model that explains factors affecting patients’ PP use. The secondary aim was to compare the difference in PP usability, PP self-efficacy, and PP use between older adults recruited from community settings and older adults recruited from hospital settings. Methods: To test the primary aim, an anonymous cross-sectional online survey was conducted with adult patients in an integrated healthcare system. Data from 743 patients who used PPs in the past 12 months were subject to structural equation modeling (SEM). For the secondary aim, a secondary data analysis was performed using descriptive statistics and content analysis (272 community-residing older adults). From this sample, those who used PPs (n=126) were compared with hospital/clinic-based older adults (n=174) by conducting regression analyses. Results: Among 743, about two-thirds were White and female (mean age, 53.1; range, 18-92). Mean PP usability was 36.6 (range, 6-42). The SEM revealed that the final model fit the data: CFI=.983, RMSEA=.059. PP self-efficacy and privacy/security concerns had a direct impact on PP use. PP use was indirectly influenced by PP usefulness, PP ease of use, eHealth literacy, education, and age. The secondary data analysis indicated that older adults recognized PP benefits and were willing to use PPs. However, their PP use was limited due to several challenges. The relationship between PP usability and PP use was stronger in the community sample. Conclusion: Findings suggest a strong potential for using PPs to engage patients in healthcare and strategies to improve patients’ PP use. Further studies need to include more diverse populations in various settings.
    • Expression patterns of the metastasis suppressor NME1 in metastatic melanoma

      Snyder, Devin; Kaetzel, David M. (2020)
      Localized melanoma is a curable form of skin cancer, with a 98% chance for survival. However, for individuals diagnosed with metastatic melanoma, the five-year survival rate is reduced down to 23%. To enhance patient survival, it remains imperative to fully differentiate melanoma metastasis mechanisms. Our motivation lies on depicting underlying patterns accompanying metastatic inhibition. Here, we examine inhibition of melanoma metastasis through analysis of the metastasis suppressor protein, NME1. NME1 is a known inhibitor of melanoma metastasis. Overall loss of NME1 protein in melanoma tumors is associated with reduced survival. Yet, not all melanoma tumors are the same. Melanomas are heterogenous and exhibit distinct differences across patients and across individual primary tumor cells. The prognostic implications of heterogenous NME1 expression within melanomas has not previously been defined. Our focus lies in characterizing how expression of NME1 across patients and across primary tumor cells may impact melanoma prognosis and overall metastatic incidence. We utilized a NME1-based approach to identify a mRNA expression signature associated with metastasis suppression in melanoma. Bioinformatic analysis of our metastasis suppression signature in a melanoma patient dataset exposed a subset of melanoma patients with altered survival. We were also interested in analyzing the heterogenous expression of NME1 protein within melanoma cells. Flow cytometry analysis revealed that melanoma cell lines contain a rare population of cells with low levels of NME1 protein. We utilized multiple methods to characterize the impact of NME1 loss on cell behavior. Experiments designed to mimic loss of NME1 through shNME1 produced results which suggest that NME1 is associated with melanoma spheroid formation. We further utilized an innovative approach, which relied on CRISPR technology, to study endogenous NME1 expression. Characterization of endogenous NME1 expression patterns lead to the identification of a previously unidentified group of cells, which have a neural crest-like phenotype and contain enhanced metastatic capacity. Overall, the expression pattern of NME1 within tumor cells and across patients provides an advantageous tool for identifying metastatic susceptibility.
    • Evaluation of skin tape stripping in healthy human volunteers as a methodology for quantifying local drug bioavailability from dermal products

      Shukla, Sagar; Stinchcomb, Audra L.; Hassan, Hazem (2020)
      Stratum corneum (SC) tape stripping is a valuable methodology that has been used for quantifying bioavailability (BA) of topical drug products at the site of action. Although the Food and Drug Administration (FDA) tape stripping guidance was withdrawn several years ago due to variable results, with an appropriate study design, tape stripping procedures can be a reproducible BA method. Therefore, the objective of this work was to investigate the use of tape stripping to quantify BA and evaluate in vitro/in vivo correlations (IVIVC) of two model compounds (lidocaine and diclofenac). These compounds were selected for their differing physicochemical properties and skin permeation rates. Two healthy human volunteer pharmacokinetic and tape stripping studies were conducted to quantify the BA in the SC and measure the elimination rate constant through the skin (kesc). Investigator variability from the in vivo tape stripping study was also examined, and the method variability potentially induced by the investigator can be mitigated by the quality by design (QBD) approach of using transepidermal water loss (TEWL) for determining when most of the SC has been removed in each individual volunteer. TEWL readings assisted the investigator by representing the SC tape stripping endpoint, and the SC masses removed from each volunteer were similar for the two investigators. Harmonized IVPT studies were also conducted and key parameter estimates were determined (absolute bioavailability (F) and (kesc)). These parameter estimates were used to simulate in vivo SC drug concentrations. The error in the SC drug concentration predictions from both in vivo studies was usually less than 20% compared to observed values, which demonstrates the predictive power of carefully harmonized IVPT studies. IVPT studies require less time and expense than human studies, and therefore these models play an important role in the early stages of drug development to predict in vivo SC drug concentrations, and absorption of drug through the skin. In this study, in vivo kesc for a quickly permeating drug (lidocaine) appears to be well predicted by IVPT; however, further work needs to be done to predict a slowly permeating drug’s (diclofenac) SC drug concentrations and kesc.
    • Development and Application of Vertebrate Models to Investigate the Risk of Defense-Relevant Chemicals

      Narizzano, Allison; Pereira, Edna F. R.; Quinn, Michael J., Jr. (2020)
      Adverse effects from unintended exposure to chemicals have been widely described and have provided the basis for many chemical and environmental management regulations and policies that are intended to protect humans, animals, and/or the environment. The present work focuses on two classes of chemicals that are tested and/or used in training activities at military sites: insensitive munitions (IMs) and per- and polyfluoroalkyl substances (PFAS). As groundwater is a common source of drinking water, accurate risk assessments are critically needed to ensure protection of the environment and public health. IMs are more resistant to accidental, unintentional or incidental detonation than traditional explosives. Incomplete detonation may deposit munition components and by-products on impacted installations and, importantly, mobilize those constituents into groundwater. One component of several IM mixtures is 3-nitro-1,2,4-triazol-5-one (NTO). NTO is especially worrisome because it is highly water soluble and, thus, mobile in the environment. Additionally, laboratory tests with rodents have identified the testes and epididymides as targets of NTO. PFAS are also prevalent and problematic, nearly universal contaminants. PFAS are manufactured for use in paints, cleaning agents, non-stick cookware and food containers, water-impermeable products, and Aqueous Film Forming Foams (AFFFs). PFAS have attracted increased regulatory scrutiny because of their resistance to degradation, ability to bioaccumulate, and growing evidence of toxicity in animals. A considerable body of work has examined the effects of NTO in laboratory rodents. However, there is still uncertainty in the derivation of a safe workplace environmental exposure level (WEEL) for NTO. The present studies evaluate the effects of NTO in the Japanese quail (Coturnix japonica) and in Sprague Dawley rats. In concert, these toxicity data on NTO will improve risk estimation efforts, provide data to explore taxa read-across, and fill a regulatory needed data-gap. The toxicity of PFAS will be evaluated in the native mammalian species, Peromyscus leucopus. Given that the serum elimination half-lives within this class of chemicals can vary greatly from hours to years based on species, sex, and dose, extrapolating between species is difficult and inaccurate. As such, wild mammalian-specific data is novel and valuable for risk estimation in taxa that are directly site-specific.
    • Use of a Functional Chewing Gum in Reduction of Gingival Inflammation

      Merati, Arash; Shiau, Harlan (2020)
      Control of plaque biofilm is central to prevention of gingivitis. In addition to professional care, effective oral hygiene measures are known to improve gingival health. The objective of this study was to investigate the effect of chitosan/blackberry-extract, delivered in a chewing gum, as an adjunct to oral hygiene, on gingival inflammation and plaque biofilm accumulation. In this 12-week randomized controlled study, the use of chitosan/blackberry-extract gum was compared to placebo in 34 subjects (17/17). Plaque index(mPI) and gingival index(mGI) were the main outcome measures followed at baseline, week 2, 4, 8, and 12. Measured patient compliance and mPI had no significant difference between experimental and control at any interval during the study. mGI was significantly lower for the experimental group compared to control at 12 weeks(P<0.005). Chitosan/blackberry-extract chewing gum may be beneficial in reduction of clinical signs of gingival inflammation and has potential as an adjunct to routine oral hygiene.
    • Inducing DNA Repair Deficiencies in Triple Negative Breast Cancers Through Pharmacologic Stimulation of Innate Immune Signaling

      McLaughlin, Lena; Rassool, Feyruz V. (2020)
      Poly (ADP-ribose) polymerase inhibitors (PARPi) are FDA approved in a subset of patients with ovarian cancer or metastatic breast cancers who harbor BRCA gene mutations. These mutations generate homologous recombination deficiencies (HRD) and are the main predictor to PARPi sensitivity. Unfortunately, responses to therapy have not been durable and have failed for the majority of sporadic triple negative breast cancers (TNBC). We previously reported that DNA methyltransferase inhibitor (DNMTi) azacytidine (Aza) improves the efficacy of a new generation of PARPi, Talazoparib (Tal), through increased trapping of cytotoxic PARP-DNA complexes in both BRCA-mutant and -proficient TNBC. These trapped complexes lead to increased and persistent levels of lethal double strand breaks (DSBs), suggesting that DSB repair may also be impaired with this treatment. In the present study, we show that Aza/Tal treatment in BRCA-proficient TNBC cell lines significantly downregulates expression of HR and Fanconi Anemia (FA) genes, notably FANCD2, and decreases HR activity, thus generating HRD. DNMTi have also been established to induce a viral mimicry response which upregulate Type I interferon (IFN) signaling and production of inflammatory cytokines. We now link Aza/Tal facilitated HRD and induction of innate immune and inflammatory related genes, mediated in part through a STING dependent mechanism. Gene set enrichment analysis of RNA-Seq data derived from mono- and combination-treatments, reveal enrichment of innate immune and cytosolic DNA sensing pathways with significant increases of TNFα/NF-κB and IFNαβ gene sets. Overlap between HRD and immune related signaling was evaluated using the STRING database, which reveals a significant interaction specifically between FA pathway and TNFα/NF-κB and IFNαβ pathway genes. This inverse relationship was also validated in both METABRIC TNBC dataset and other TCGA data sets suggesting broad applicability of this observed transcriptional program independent of pharmacologic intervention. Additionally, Tal driven cytosolic DNA as well as an Aza augmentation in STING protein expression, emerges as the key node in Aza/Tal induced innate immune signaling to drive HRD. Induction of what we define as a pathogen mimicry response to drive HRD mechanism suggests that DNMTi-PARPi therapy strategies can expand the therapeutic scope of PARPi to encompass treatment of BRCA-proficient cancers.
    • Elucidating the Localization of Estrogen and Estrogen-related Receptors in the Inner Ear

      Lipford, Erika; Hertzano, Ronna P. (2020)
      Hearing loss is the most common sensory impairment, affecting hundreds of millions of people worldwide. Although both men and women are impacted by hearing loss, the incidence rate differs between the two sexes, with the prevalence of bilateral high-frequency hearing loss reported to be 2.7 times higher in males. Recent studies have implicated estrogen as having a protective effect against hearing loss in females. While the localization of the estrogen receptors within the cochlea is known, the roles of estrogen-related receptors in auditory function require further investigation. The aim of this study is to elucidate the localization of estrogen receptors 1 and 2 and estrogen-related receptors α, β, and γ within the inner ear of adult mice using RNAscope. Our results give insight into the molecular mechanism through which estrogen receptors and estrogen-related receptors may grant protection against hearing loss and contribute to the functionality of the inner ear.
    • The Effect of ZSCAN4 on Telomere Chromatin Remodeling

      Lin, Phyo Nay; Zalzman, Michal (2020)
      Telomeres are repetitive sequences at the ends of chromosomes that protect the coding regions of DNA. Telomeres shorten with every cell division and therefore operate as a biological clock. Thus, factors regulating telomeric chromatin impact cell replicative lifespan, tumor formation and growth. The murine Zinc Finger and SCAN Domain Containing 4 (mZscan4) promotes telomere homeostasis and genomic stability in mouse embryonic stem cells (mESCs). A transient expression of mZscan4 was shown to correlate with chromatin de-repression in mESCs. However, the function of human ZSCAN4 in its contribution to the epigenetic landscape changes at telomeric chromatin remains to be determined. In this study, we defined the effect of ZSCAN4 on histone 3 and 4 hyperacetylation at the telomere region which is associated with telomere extension. Understanding the mechanism by which ZSCAN4 affects the telomeric chromatin is important for designing new therapeutic approaches to target cancer cell replicative lifespan.
    • The Interrelationships of Adverse Childhood Experiences (ACEs), Alcohol use in Adulthood, and Intimate Partner Violence (IPV) Perpetration Among Black Men in the United States

      Lee, Kerry-Ann; Bright, Charlotte Lyn (2020)
      Historically, Black men have been found to perpetrate IPV at higher rates in comparison to other ethnic groups; however, studies of IPV perpetration have largely focused on samples of White individuals. There is a paucity of empirical research related to the interrelationships among ACEs, IPV perpetration, and alcohol use among Black men. This study used data from Wave 2 of the National Epidemiologic Survey of Alcohol and Related Conditions. Study aims were: (1) to examine the interrelationships of ACEs, alcohol use, and IPV perpetration; to investigate whether alcohol use moderates the relationship between ACEs and IPV perpetration; and whether interpersonal social support moderates the relationships between ACEs and IPV perpetration and between alcohol use and IPV perpetration; (2) to determine if higher cumulative ACE score is associated with increased alcohol use and IPV perpetration; (3) to determine if racial discrimination is significantly associated with increased alcohol use and IPV perpetration; and (4) to identify how subgroups of ACEs (individually or in combination) are associated with alcohol use and IPV perpetration among Black men in adulthood. The sample consisted of 2,326 Black men aged ≥ 20 years. Bivariate results indicated an interrelationship among ACEs, alcohol use, and IPV perpetration. Regression analyses showed that alcohol use exacerbated the ACEs and IPV perpetration relationship for men with no ACEs; but for men who had a history of ACEs, the effect of alcohol on IPV was less substantial. Contrary to study hypotheses, higher interpersonal social support was found to buffer the relationship between ACEs and IPV perpetration for men with three ACEs, but exacerbated the alcohol use and IPV perpetration relationship. Higher cumulative ACE score was significantly associated with alcohol use and IPV perpetration. Racial discrimination was not significantly associated with study outcomes. Latent class analysis yielded three classes. Membership in classes 1 and 2 was associated with IPV perpetration; however, only class 1 membership was associated with alcohol use when compared to class 3 membership. Findings revealed factors that may contribute to IPV perpetration among a sample of Black men. Future research with Black men should aid in the development of culturally-appropriate interventions.
    • Understanding the Role of Small Ankryin 1 in Calicum Regulation in Excitable Cells

      Labuza, Amanda; Bloch, Robert J. (2020)
      Small Ankryin 1 (sAnk1) is a 17kD transmembrane protein that plays a role in stabilizing the network sarcoplasmic reticulum in skeletal muscle (Ackermann et al., 2011). Recent studies have shown that sAnk1 can bind to and regulate sarco(endo)plasmic reticulum Ca2+-ATPase1 (SERCA1) activity (Desmond et al., 2015). SERCA1 transports Ca2+ against its gradient into the SR after muscle contraction. SERCA is inhibited by sarcolipin (SLN) in fast twitch skeletal muscle and atrial cardiac muscle and by phospholamban (PLN) in slow twitch muscle and ventricular cardiac muscle. Like SLN and PLN, sAnk1 also interacts with SERCA at least in part through its transmembrane domain (Asahi et al., 2003; Hutter et al., 2002; Desmond et al., 2015). The interaction of SERCA with SLN and PLN has been studied individually and together, but the effects of sAnk1 and its regulatory activity have only recently started to be addressed (Desmond et al., 2015, 2017). Here I show that sAnk1 can interact with PLN or SLN independently of SERCA1. sAnk1 forms a three-way complex with SLN and SERCA1 that ablates SLN inhibition (Desmond et al., 2017). sAnk1 can also form a three-way complex with PLN and SERCA1 that abolishes all inhibition. I show that the complexes that sAnk1 forms with SLN or PLN and SERCA1 are distinct, suggesting unique roles for each protein in SERCA regulation. I also examined sAnk1 and SERCA in several CNS tissues, and found that sAnk1 is not expressed in neurons, but that it is expressed in astrocytes, where it has the potential to bind and regulate SERCA2B. Studying the multi-protein complex of SERCA, sAnk1, SLN, and/or PLN can help us better understand physiological SERCA regulation. This knowledge can lead to better treatment for diseases related to misregulation of calcium, including muscular dystrophies and potentially some neuropathies.
    • Cryo-Electron Microscopy Structure Determination of the Anthrax Toxin Protective Antigen Bound to its Lethal and Edema Factors

      Hardenbrook, Nathan; Krantz, Bryan A. (2020)
      Protein translocation is an essential function within all living cells. Translocons are dedicated protein translocation machinery, responsible for the unfolding and translocation of proteins. Due to the thermostability of most proteins in their native states, these translocons utilize various different forms of energy to drive the translocation of their substrates. This process is mediated by polypeptide clamps responsible for catalyzing the unfolding and translocation of the protein. Using lipid nanodiscs and cryo-electron microscopy (cryoEM), we have determined structures of heptameric anthrax lethal toxin and edema toxin channels to 4.6 and 3.2-Å resolution, respectively. Additionally, using cryoEM we have determined the first atomic structures of PA8 prechannel bound to full-length EF and LF to 3.3 and 3.7-Å resolution, respectively. In this pre-translocation state, the first α helix and β strand of LF and EF unfold and the α clamp, which resides at the interface of two PA subunits. The α clamp-helix interactions exhibit structural plasticity when comparing the structures of lethal and edema toxins, supporting previous work indicating that the α-clamp engages substrate α-helices repeatedly during translocation. A PA loop in the binding interface is displaced between the prechannel and channel. This results in the loss of a salt bridge and leading to the weakening of the binding interface prior to translocation in the PA7EF structure. Lastly, EF undergoes a largescale conformational rearrangement when forming the complex with PA, compared the solution structure of EF bound to calmodulin. Recruitment to the PA prechannel exposes an originally buried β strand and enables domain organization of EF. Many interactions are formed on domain interfaces in both PA prechannel-bound EF and LF, leading to toxin compaction prior to translocation. This work has resulted in the first structures of PA bound by edema factor, as well as the first structures of PA bound to a full-length substrate. These structures have provided insight into important biophysical steps occurring in preparation for translocation They reveal structural plasticity within the binding α-clamp binding site, allowing the translocating substrate to be engaged multiple times. This provides a greater understanding of how anthrax toxin can invade the host cytosol.
    • Therapeutic Effect of Anti-Progranulin/GP88 Antibody AG01 in Triple Negative and Letrozole Resistant ER+ Breast Cancer Cells

      Guha, Rupa; Serrero, Ginette (2020)
      Progranulin (GP88, PCDGF, granulin/epithelin precursor, acrogranin) is a secreted autocrine growth/survival glycoprotein that functions as a biological driver of tumor cell proliferation, tumorigenesis, survival, invasiveness and drug resistance in several cancers, including breast cancer. Progranulin is found in the serum of breast cancer patients at higher levels than in healthy subjects and pathological studies have shown that in ER+ tumor biopsies, progranulin/GP88 is an independent prognostic factor of recurrence. Although TNBC represents a small percentage (15-20%) of breast cancer diagnoses, it is clinically important because of its highly aggressive nature and the fact that the disease progresses to metastasis within an exceedingly shorter period. Higher progranulin levels have also been shown to be associated with TNBC cases. Progranulin represents a therapeutic and diagnostic target in breast cancer. We have characterized a recombinant neutralizing anti-human progranulin/GP88 monoclonal antibody AG01 that inhibits progranulin biological effect in vitro and in vivo. Since GP88 is associated with poor outcomes in BC patients, we have investigated the effect of AG01 to inhibit proliferation and enhance letrozole responsiveness of letrozole resistance breast cancer cell lines as well as inhibit proliferation and migration of TNBC cells, two breast cancer areas with unmet medical needs for targeted therapy. We found that progranulin levels were sharply elevated in letrozole resistant cells as compared to the parent cell lines. Simultaneously, TNBC cells showed an increase in progranulin expression while it is undetectable in normal mammary cells. This emphasized the importance of targeting PGRN to treat letrozole resistance in ACLRTUSM as well as provide a therapeutic agent in TNBC cells. We report here that treatment of ACLRTUSM with anti-PGRN antibody (AG01) not only reduced their proliferation but increased the sensitivity of ACLRTUSM cells towards letrozole treatment. In several TNBC models, AG01 treatment reduced cell proliferation, migration, and invasion. Taken together, the research work discussed here provides new information to better understand the targeting progranulin and the effectiveness of AG01 as a potential therapeutic agent in breast cancer. Future work continuing characterization of AG01 will provide further insight into its role in regulating cancer biology.
    • The Accuracy and Efficiency of a Dynamic 3D Navigation System for Negotiating Calcified Canals

      dianat, omid; Chand, Priya (2020)
      The purpose of this study is to compare the use of a dynamic navigation system (DNS) to a freehand (FH) method for locating calcified canals. Sixty single-rooted teeth with obliterations were selected. In the DNS group, access preparation was made under navigation and in the FH group without any guidance. Linear and angular deviations and reduced dentin thickness were measured. Furthermore, efficiency was evaluated. The mean linear and angular measurements showed significantly less deviations in the DNS group compared to the FH group. Reduced dentin thickness, at both levels, was significantly less in the DNS group. Furthermore, DNS was faster and more successful than FH method. The DNS group showed only one unsuccessful attempt, compared to five perforations and three large transportations in the FH group. Within the limitation of this study, the dynamic navigation system allowed for more accurate and efficient negotiation of the calcified root canal system.
    • Quantitative Analysis of Compartments in the Leg and Implications for Trauma Surgery

      Agandi, Lorreen; Puche, Adam C. (2020)
      Compartment syndrome is characterized as an excess in swelling leading to an increase in pressure in a limited space and a lower extremity fasciotomy is performed to mitigate the effects. A two-incision fasciotomy is performed on the medial and lateral sides of the leg, accessing the posterior superficial, deep superficial, anterior and lateral compartments respectively. Ongoing studies have shown the lateral compartment is commonly decompressed incorrectly. This error has led to the hypothesis that there is variability in septum position and that using the fibula as a landmark can lead to erroneous incision placement in patients. CTA scans were analyzed to assess septum position. Findings indicate that the septum position shifts anteriorly progressing distally down the leg, indicating variability at different points in the leg. If surgeons do not take septum variability into consideration when decompressing the lateral compartment, this can lead to incorrect decompression of the lateral compartments.
    • Insertional Mutants of Chlamydia caviae Display Altered Virulence

      Filcek, Kimberly; Bavoil, Patrik M. (2020)
      The genus Chlamydia encompasses multiple bacterial species capable of colonizing and causing infection in a diverse range of host organisms. The type III secretion system and secreted effector proteins of this genus are important subjects for elucidating chlamydial biology. One important group of secreted proteins is the Inc family which are integral membrane proteins of the chlamydial inclusion found in all Chlamydia. Another novel protein, SinC, is a type III secreted effector initially described in Chlamydia psittaci, a zoonotic pathogen, but whose role in pathogenesis remains largely unknown. Characterization of specific genes in Chlamydia has been uniquely difficult due to its genetic intractability, but recent advances in chlamydial genetics have provided us the opportunity to generate targeted, stable mutants. The Chlamydia caviae GPIC strain has been used to model ocular and genital tract infection in guinea pigs. However recent C. caviae isolates were implicated in zoonotic respiratory infection in humans. The GPIC strain carries the two genes of interest, sinC and incA. Unlike for C. psittaci, we were able to perform mutagenesis in C. caviae without the constraints of BSL-3 containment, and with the benefit of well-characterized animal models. Here I engineered site-specific insertional mutations in sinC and incA, the first instances of targeted mutagenesis in the C. caviae lineage, with the aim of investigating the role of these genes in chlamydial pathogenesis.