2019 SURP Participants

The purpose of this project was to determine how neural circuits are affected during warming by examining sensory neurons, the neuromuscular junction, and the cardiac function and behavior of the commercially important crustacean species, the red swamp crayfish (Procambarus clarkii). Rapid inactivation of neural function in crustaceans prior to slaughter is important in order to limit exposure to noxious stimuli, thus improving animal welfare. In this study we demonstrated that as the animal is warmed at 1°C/min the heartbeat stops at 44°C. When temperature is rapidly increased, at 44°C synaptic transmission at the neuromuscular junction ceases and primary sensory neurons stop functioning. Even though animals do not respond to stimuli after being warmed to 44°C, if sensory neurons are returned to 20°C saline after two minutes they may regain function. Conversely, the neuromuscular junction does not regain function after two minutes in 44°C saline. Examining behavior and heart rate while warming at 1°C/min, 12°C/min, or 46°C/min to 80°C indicated that at approximately 40°C the heart rate is altered. Within 10 seconds at 80°C the heart stops with the highest heating rate. Directly placing crayfish in boiling water stopped the heart quickest, within 10 seconds, which likely represents denaturing of the tissue by heat. Using an impedance measure to detect heart rate may also be influenced by movements in the denaturing process of the tissue. A rapid increase in the temperature of the crayfish above 44°C is key to limit its exposure to noxious stimuli.

Spermatid individualization is the little-researched process by which spermatids become segregated from each other during spermatogenesis. This is performed by the formation of an actin meshwork called individualization complexes around spermatid nuclei that will move down the spermatids and remove excess cytoplasm. Previous research has determined that JAK/STAT signaling in the somatic cyst cells is necessary for successful individualization to occur, so this project seeks to ascertain the specific genes which regulate spermatid individualization via effect of the JAK/STAT biochemical signaling pathway in Drosophila melanogaster. To accomplish this, RNA Interference knock-downs of candidate genes were performed to determine whether they have functions in individualization. Knock-downs of somatic cyst cell expression of scpr-A, scpr-B, and GP150 were each significantly higher in the number of ICs produced than their negative controls, with scpr-A also yielding an elevated ratio of ICs to nuclei clusters. The other two exhibiting proportional increases in nuclei clusters. These results indicate that the identified genotypes function in the regulation of D. melanogaster spermatid individualization but are not required for the process to occur. With continued research, such identifications will contribute to a more complete understanding of reproductive function by characterizing the little-documented biochemical mechanism for somatic-germline interaction, as well as lay the groundwork for future human-focused investigations based upon the similarities between Drosophila and mammalian spermatogenesis.

CHARGE syndrome is a congenital disorder characterized by ocular coloboma, heart defects, choanal atresia, retardation of growth and development, genital hypoplasia, and ear abnormalities. Mutations in CHD7, a chromatin remodeler, have been identified in the majority of patients with CHARGE syndrome. However, recently a patient with no CHD7 mutations, but rather a Sox11 mutation was clinically diagnosed with CHARGE syndrome. 1 Sox11, is a transcription factor, required for the proper development of ocular structures and a downstream target of CHD7 in neural stem cells. 2. The Morris lab has also shown that knocking down Sox11 in zebrafish results in CHARGE syndrome phenotypes. 3 In addition, Sox11 is expressed in cranial neural crest cells during ocular development. This leads to the hypothesis that disruption of Sox11 expression may result in neural crest cell defects resulting in the ocular phenotypes observed in CHARGE syndrome. In this study, we will test this hypothesis by knocking down Sox11 in zebrafish using morpholino oligonucleotides in the transgenic reporter line, Sox10:RFP, a neural crest cell marker. After knockdown, we will image and analyze neural crest cells and investigate any developmental defects. This work will allow for a better understating of the potential role of Sox11 in the pathogenesis of CHARGE syndrome.

 Ear pinna injury in spiny mice (Acomys) activates cell cycle re-entry and resident cell proliferation to replace injured tissue during regenerative healing. An identical injury in outbred laboratory mice (Mus) induces limited cell cycle progression and fibrotic repair. Cell cycle progression is at least partly regulated by factors that induce cellular senescence. Our in vivo data showed a disproportionately high number of resident cells with nuclear localization of p21 and p27 concomitant with fewer proliferating cells during scarring in Mus. This was accompanied by high levels of local reactive oxygen species (ROS) during regeneration and scarring. Our in vitro data demonstrated that fibroblasts from highly regenerative mammals were refractory to ROS-induced senescence while fibroblasts from non-regenerating mammals showed senesce in response to the ROS stressor, hydrogen peroxide. To extend our in vitro results and test if local injury produces a differential induction of cellular senescence associated factors during regeneration and fibrotic repair, we analyzed healing tissue for the DNA damage response marker (γ-H2AX), tumor suppressor proteins (p21, p53, p16 and p19) and the proliferative marker (EdU). We used immunohistochemistry to detect proteins on paraffin sections from healing tissues at D10 and D20 from Acomys and Mus. Our preliminary data shows that the fraction of γ-H2AX+, p21+ and p53+ cells was proportionately lower in regenerating tissue from spiny mice compared to Mus. These results require additional validation to understand the functional consequences of ROS-induced cellular senescence as a regulator of tissue healing.

The Protein Tyrosine Phosphate 4a (PRL) family has been found to be over-expressed in various tumor types, however, their exact function and pathways are still undetermined. In this report, we use Zebrafish models to study the phenotypic effect of the ablation of these genes. Zebrafish are excellent models as approximately 70% of human genes have a Zebrafish ortholog, which is true for the PTP4a genes (two orthologs for PTP4a2). The CRISPRCas9 method of gene knockout is used via injection of RNP complexes, as well as a fluorescent dye, into single-cell Zebrafish embryos. Random samples were selected to genotype by the T7 Endonuclease method, and embryos were screened for phenotypes at 4 dpf. Based upon our results, we assume that all four ptp4a orthologs were functionally disrupted, which will later be confirmed by sequencing, and an increase in the frequency of pericardial edemas was apparent.

Diabetes mellitus is a world epidemic which affects ten percent of Americans. This percentage is expected to rise in the coming years. Diabetes has been well-studied.  However, there are still many aspects that are not well-understood, like the roles of various genes in pancreatic development and how mutations in these can lead to diabetes.  One of these genes of interest is Sox4, a transcription factor from the SoxC subfamily of genes. In zebrafish, sox4b, an ortholog to human Sox4, is expressed in endocrine pancreatic cells and thought to play a role in islet cell determination. To further understand the role of sox4b in pancreatic development, I plan to inject and image an insulin CFP-NTR transgenic line with a sox4b translation blocking morpholino to knockdown expression.  Furthermore, I plan to perform glucose assays and a whole-mount immunohistochemistry (IHC) for insulin on sox4b maternal zygotic mutant larvae to see if the absence of sox4b alters insulin expression or contributes to hyperglycemia. Current research aims to study pancreatic development in hopes of finding a cure to diabetes through generating new, healthy, and functioning pancreatic cells in affected individuals. My project will add to the overall picture through studying sox4, a gene whose overall contribution involved in pancreatic development is currently unknown.

Eye development within zebrafish is a complex process that involves the differentiation and proliferation of the cells that comprise the highly specified retina. Failure of this process due to mutations in key factors can result in congenital retinal diseases. Currently, there is little-to no treatment to prevent vision loss among humans with congenital retinal diseases. In an attempt to better understand the physiological changes associated with congenital blinding disorders my project aims to investigate the role of post-translational modifications during retinal development. In particular, I am interested in the mechanism of adding polyubiquitin chains to specific molecules so they can be targeted for proteasomal degradation. Siah, an E3 ubiquitin ligase, previously shown to be involved in fruit fly eye development and more recently zebrafish optic fissure closure is the primary focus of my project. As an E3 enzyme, Siah directly selects targets for polyubiquitination. Previous work in the lab identified a protein CDHR1a as a target of Siah. CDHR1a is a cadherinrelated family member known to be necessary for photoreceptor disk maturation. I hypothesize that Siah is directly responsible for modulating the stability of CDHR1a through proteasomal degradation during photoreceptor disk maturation. The goal of my KBRIN summer project is to determine if Siah does bind to CDHR1a and if this binding is required for proper photoreceptor development. To test whether Siah and CDHR1a are co-expressed, 2-color fluorescence wholemount in situ hybridization was be conducted in wild-type embryos. This assay revealed co-expression from 3 dpf until 7dpf. Furthermore, we also tested newly generated zebrafish specific antibodies against Siah1 and CDHR1a using immunohistochemistry. My data indicates that Siah1 and CDHR1a protein are both found in the photoreceptor layer of developing zebrafish retinas. Taken together, my results indicate that siah1 and CDHR1a are expressed in the developing photoreceptors, and therefore, may function to regulate photoreceptor development.

In this project, Kiahra Burns will grow and transform competent E. coli and transform with a plasmid containing the gene of interest (in this case MLII).  The DNA will be isolated from the bacteria and sequenced for verification before transformation of Agrobacterium tumefaciens, which naturally infects tobacco plants. The transformed Agrobacterium will be used to infiltrate tobacco plants, and after a few days of plant growth, the infected leaves will be harvested, crushed and filtered for protein purification. The plant extract will be separated by fast protein liquid chromatography (FPLC), and the resulting fractions will be assessed by SDS polyacrylamide gel electrophoresis (PAGE). If enough pure protein is obtained, bioactivity tests, for example sugar binding properties, will be determined to evaluate the potential anticancer activity of the whole protein in general and its B-subunit in particular.   

Alcohol abuse is quite common in patients that have bipolar, or manic-depressive, illness.  The common comorbidity of the two conditions suggests a biological underpinning.  We have demonstrated that ethanol levels that are achieved in intoxicated humans can have an effect similar to lithium (a medication known to be effective in the treatment of mania) in cultured olfactory neuroepithelial precursors that we obtained from subjects with bipolar illness and thus have the genetic heritage of the illness.  This is potentially an important finding in understanding both bipolar illness and alcohol abuse in patients with bipolar illness.  

Spinal cord injury results in sensory and motor deficits. The extent of these deficits is dependent upon the location and areal extent of the damage. To better understand the impact of spinal cord injury on gait function and recovery, we use experimental animal models of spinal cord injury. Specific to this project, is the use of a thoracic dorsal hemisection injury that disrupts primarily ascending sensory information to the rostral spinal cord, brainstem and cerebral cortex. Dependent upon the extent of the dorsal hemisection, it may or may not involve two descending pathways implicated in voluntary gait. Gait performance is tested using treadmills and overground runways. The former is used to focus on circuitry in the lumbosacral spinal cord associated with the basic, alternating stepping pattern between the hind limbs which is a consistent feature of normal gait. This circuitry includes a central pattern generator which lies below the thoracic lesion. Voluntary locomotion is tested using runways and, although it also requires the contributions of the central pattern generator, is dependent upon descending supraspinal control. Preliminary angular data captured and generated using a 3-D camera and software system (Vicon) shows that stepping ability is disrupted more greatly on the treadmill than during overground tasks following dorsal hemisection. These initial findings suggesting that ascending sensory information is part of a multi-synaptic feedback system which impacts function of the caudal spinal circuitry during gait tasks thought to be dependent upon only spinal circuitry. Understanding how dorsal injuries impact gait function will help us better understand the circuitry involved in gait and how we may design better rehabilitation approaches for those with spinal cord injuries.

Lung cancer is the leading cause of cancer mortality and further occurs at a higher incidence rate than even breast or colorectal cancer. Early diagnosis and intervention of any cancer type is paramount to successful treatment. Dysregulation of environments proliferated with cancer results in an output of metabolites from the dysregulated environment. This provides an interesting area for noninvasive clinical diagnosis of cancer in regards to taking simplistic breath samples for diagnosis. But the body produces a large number of microscopic metabolites from simple respiration, these compounds also readily dissipate within normal environments outside the body further complicating diagnostic methods. With the use of microchip preconcentrators and specialized collection methods a look into the viability of using metabolite markers to diagnose. There is reasonable data to support further inquiry into exhaled metabolite diagnostic methods but, reasonable care must be given to overcome previous and further complications of dealing with such volatile and small compounds to ensure diagnostic accuracy.  

B cells and antibodies are critical for both innate and adaptive immunity in mammals. Mouse models are commonly used to study immune-related disease, including those that involve B cell mediated processes. However, antibody diversity among biomedically-relevant mouse strains remains poorly characterized, and thus the role of antibodies in mouse models for disease is likely underappreciated. To address this knowledge gap, we are characterizing germline immunoglobulin heavy (IGH), kappa (IGK), and lambda (IGL) chain repertoires in 18 different mouse strains predicted to broadly represent IGH/K/L genetic diversity among >100 strains for which whole-genome data is available. In order to do this, RNA was extracted from the spleens of these mouse strains. cDNA was then generated using 5’RACE and V (variable) regions were amplified during two rounds of PCR. DNA was purified using AMPure beads and libraries were quantified via Qubit and bioanalyzer. Sequencing libraries were then pooled and sequenced at the UofL genomics core on MiSeq 2x300 bp. Our data are currently being processed using our previously developed analysis pipeline; results are forthcoming. This data will allow us to characterize and compare V genes for IGH, IGK, and IGL loci for all 18 strains. We expect these data will lead to improvements of existing germline databases and nomenclature systems, both of which are necessary for the analysis and interpretation of expressed antibody repertoire sequencing experiments. This will be critical if we are to more fully understand how genetic diversity within the IG gene regions contributes to variation in B cell mediated immunity.

The appearance of Zika Virus (ZIKV) in the central nervous system (CNS) correlates with various neurological complications such as encephalitis and Guillain-Barré syndrome. However, the method by which ZIKV gains entry into the CNS is not fully understood. Previous work in the Chung Lab determined that prior to infection in the CNS, ZIKV is found in the cerebral-spinal fluid, choroid plexus, and meninges. Further work showed ZIKV is localized in pericytes surrounding the endothelial cells in the vascular layer of the choroid plexus. This suggests that ZIKV gains entry into the CNS by crossing the blood-CSF barrier instead of the blood brain barrier. To gain a better understanding the role pericytes have in facilitating ZIKV into the CNS, a time course infection of primary human brain vascular pericytes (HBVP) was conducted. The concentration of HBVPs infected was measured using flow cytometry and the amount of virus produced measured in a viral titer. The study indicates that HBVP cells are both susceptible and permissive to ZIKV.

Heart disease is the leading cause of death among both men and women in the United States. This is due to the lack of proliferative activity of cardiomyocytes. However, with the use of a Non-integrating Lentivirus researchers are able to deliver a gene cocktail composed of 4 cell cycle genes; cyclin dependent kinase-1, cyclin dependent kinase-4, cyclin B1, and cyclin D1 (collectively known as 4F) along with a Troponin T promoter to make the treatment cardiomyocyte specific; to infarcted areas of the heart to induce cardiomyocyte proliferation. This treatment is also transiently expressed to avoid excessive proliferation due to the non-integrating lentivirus. The NIL-4F treatment effectively decreased the scar size by 30%, and also presented an increase of a bout 50% of newly synthesized DNA on the cardiomyocytes surrounding the infarcted area, along with a noticeable increase in stoke volume and ejection fraction after ischemia reperfusion. In conclusion, the new gene therapy using non integrating lentivirus encoding the 4F is a promising therapeutic for heart failure. Further experiments in large animals and humans are needed to validate the efficiency.

The endoplasmic reticulum (ER) is an organelle in which secreted proteins are synthesized, and folded. In addition, ER contributes to Ca storage and lipid synthesis. Disruption of ER function results in an ER stress response that is transduced by the conserved ER stress signaling pathways including the unfolded protein response (UPR) and the integrated stress response (ISR). High rates of protein synthesis make cells more vulnerable to ER stress and the ISR-driven apoptosis. Following a traumatic spinal cord injury, ER stress is induced in the damaged spinal cord tissue leading to ISR-mediated apoptosis of oligodendrocytes (OLs) and neurons. The rates of protein synthesis are linked to the oscillations of the circadian rhythm through the transcription/translation factor Brain and Muscle ARNT-Like 1 (BMAL1) and its phosphorylation. BMAL1 promotes high levels of protein synthesis acting as a transcription factor in the nucleus. After phosphorylation by the mTOR pathway, BMAL1 acts as a translation factor that is associated with ribosomes. OLs and OL precursor r cells (OPCs) are highly sensitive to ER stress. However, role of BMAL1 in OL’s ER stress has never been investigated. Therefore, we have studied expression of BMAL1 in rat OPCs or OPC-derived OLs following pharmacological induction of ER stress with tunicamycin (TM) or thapsigargin (TG). Our preliminary western blot analysis revealed trends towards ER stress-mediated downregulation of total BMAL1 protein levels both in OPCs and OLs. However, at least in OLs such a response appeared to be transient reaching a maximum at 6 h and BMAL1 levels recovering at 12 h of TM or TG treatment. Therefore, BMAL1 is present in ER stressed OLs and OPCs and may participate in setting sensitivity of those cells to ER stress-induced apoptosis.