Browsing by Author "Al-Ubaidi, Muayyad R."
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Item Ablation of Retbindin, the Riboflavin Binding Protein, Exacerbates Retinal Degenerative Phenotypes in Mouse Models of Human Retinal Diseases(2020-05) Genc, Ayse Mine; Naash, Muna I.; Al-Ubaidi, Muayyad R.; Conley, Shannon M.; Frishman, Laura J.; Roh, JinsookRetbindin (Rtbdn) is a retina-specific, riboflavin binding protein, expressed only by the rod photoreceptor cells. Riboflavin is the precursor of flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) which are essential co-factors for enzymes involved in wide-range of metabolic processes. Since flavins are directly linked to the metabolism and metabolic dysregulation is a hallmark of degeneration, we hypothesized that Rtbdn, as a riboflavin binding protein, plays a role in modulating the degenerative process. In support of our hypothesis, we found that Rtbdn is significantly upregulated in the retinas of mouse models of late-onset cone rod dystrophy (Prph2R172W), retinitis pigmentosa (RhoP23H/+) and pattern dystrophy (Prph2Y141C/+). We investigated the effect of Rtbdn ablation in these retinal degenerative models in order to elucidate the function of this novel protein in retinal health and disease. Although, the ablation of Rtbdn alone (Rtbdn-/-) had no deleterious effects on retinal function up to postnatal day (P) 120, eliminating Rtbdn in the models led to significant reductions in both scotopic and photopic electroretinographic amplitudes, compared to the single mutants. Histologic assessments revealed severe thinning of diseased retinas upon elimination of Rtbdn. While the outer nuclear layer (ONL) cell counts in wild type (WT) and Rtbdn-/- were not significantly different from each other, we observed significant loss of ONL cells in the Prph2R172W/Rtbdn-/-, RhoP23H/+/Rtbdn-/-, and Prph2Y141C/+/Rtbdn-/- in comparison to the single mutants. Ultrastructural analyses revealed shorter, malformed and disorganized photoreceptor outer segments, swollen inner segments and dilated Bruch’s membrane in the retinas of degenerative models in absence of Rtbdn. In addition, elimination of Rtbdn in the diseased retina led to vascular pathologies and appearance of neovascular tufts secondary to cell loss. We observed model-dependent alterations in the riboflavin, FAD, and FMN levels. Our data demonstrate the potential protective role of Rtbdn, making it a strong candidate as a therapeutic target in retinal degenerative diseases. Our future work will focus on investigating that potential by studying the effects of overexpression of Rtbdn in slowing the retinal degenerative process in models of human retinal degeneration.Item Flavin Homeostasis in the Mouse Retina During Aging and Diabetes as a Marker for Retina Health(2017) Sinha, Tirthankar; Al-Ubaidi, Muayyad R.; Naash, Muna I.Nutritive and therapeutic intervention to restore the retina energy metabolism can lead to delayed onset and progression of blindness because of aging and type I and II diabetes.Item Identification and Application of Novel Therapeutic Targets for PRPH2-Associated Disorders(2023-08) Rutan Woods, Christian Tyler; Al-Ubaidi, Muayyad R.; Naash, Muna I.; Wu, Tianfu; Hurley, James B.; Guo, BinMutations in the photoreceptor specific tetraspanin Peripherin-2 (PRPH2) are some of the most pervasive pathogenic inherited retinal disease mutations with over 200 identified. Lack of an FDA approved treatment, numerous low prevalence mutations, and complex pathogenic mechanisms make it imperative to identify a ubiquitous therapeutic target. We attempted to identify pan-mutation therapeutic strategies by targeting OS structural abnormalities associated with Prph2 mutations as well as investigating disruptions in the metabolic symbiosis between the retina and RPE. In attempts to ameliorate OS structural abnormalities, we investigated reducing rhodopsin (RHO) levels as a ubiquitous therapeutic target in Prph2K153Δ /+ and Prph2Y141C/+ mice. We hypothesized increased ratios of RHO to PRPH2 play a role in PRPH2-associated pathogenesis as mutations decrease functional PRPH2 levels. Proof-of-concept studies utilized partial genetic ablation to reduce RHO and improve this ratio in favor of PRPH2. Reduction improved OS ultrastructure resulting in increased photoreceptor physiological responses in a mutation-independent manner across multiple time points. Investigating the clinical relevance of this strategy, we employed an antisense oligonucleotide (ASO), mRho ASO1, to reduce RHO levels in Prph2Y141C /+ mice. ASO intervention at multiple time points was able to successfully recapitulate the previously observed photoreceptor functional and ultrastructural improvements. However, no differences were seen in the inner retina b-wave functional responses. Strikingly, we observed increased photoreceptor survival following early intervention and reduced immune cell infiltration following late intervention. These results show modulation of OS structural abnormalities to be a viable option for amelioration of retinal degeneration. In parallel studies, we investigated the role of metabolic dysregulation in Prph2-associated disease pathogenesis. Despite the fact PRPH2 is photoreceptor specific, mutations result in secondary RPE defects. We performed untargeted metabolomics analysis on retinas and eyecups from Prph2K153Δ /+ and Prph2Y141C /+ mice to evaluate changes in metabolite steady-state levels. The retina and RPE are known to possess a complementary metabolic relationship imperative to their health. We report disease stage dependent perturbations in metabolite levels essential to this homeostasis. These findings present multiple targets for future evaluation to better understand the role of metabolic abnormalities in Prph2-associated disease progression and secondary RPE defects.Item Investigating Phenotypic Changes in Response to Drug Treatments in Epithelial Ovarian Cancer Using a Novel Si-TiB2 Micropatterned Substrate(2022-05-10) Eisenbrandt, Margaret Ruth; Merchant, Fatima Aziz; Majd, Sheereen; Zagozdzon-Wosik, Wanda; Al-Ubaidi, Muayyad R.Epithelial ovarian cancer (EOC) is one of the most prevalent cancers in women worldwide, with nearly 80% of cases diagnosed in late-stage of disease and an overall survival rate of less than 50%. 3D culture is necessary in order to study the in vivo response of cancer cells to therapeutic agents, in systems that better mimic innate cell-cell and cell-ECM interactions. This study evaluates the potential of a novel micropatterned substrate, fabricated using photolithography to deposit TiB2 micropatterns onto a Si wafer to study the response of EOC to epigenetic and chemotherapeutic drugs. Previous work has validated that the Si-TiB2 substrate enables selective deposition of growth factors and self-assembly of cells onto the TiB2 pattern through differences in stiffness, roughness, wetness, and charge gradient. For EOC cell lines, OVCAR3 (low invasiveness) and SKOV3 (high invasiveness), the Si-TiB2 micropatterned substrate supported cell proliferation and maintained viability, and 3D aggregation for SKOV3 cells. Treating SKOV3 aggregates and OVCAR3 monolayers on patterned substrates, with an epigenetic drug, vorinostat, also known as suberoylanilide hydroxamic acid (SAHA), resulted in decreased diameter and thickness, however viability of the remaining cells was unchanged. Genomic analysis of the treated aggregates suggests a change in phenotype of SKOV3 after treatment with SAHA. While studies show that SAHA alone may not be a sufficient treatment for EOC, it does have the potential to augment the treatment of some cancers when used in combination with chemotherapeutic agents. Treatment of SKOV3 aggregates with a combination of SAHA and Paclitaxel was shown to decrease proliferation and halt growth for an extended period after treatment however these results are not statistically different from the Paclitaxel treatment group, which agrees with a clinical trial of SAHA and Paclitaxel combinatorial treatment. Collectively, data support use of the micropatterned substrate for investigation of potential drug therapies for cancer treatment and cellular changes in response to drug treatment.Item NOVEL 3D CELL CULTURE TECHNIQUE UTILIZING A SILICON TITANIUM DIBORIDE MICROPATTERNED SUBSTRATE FOR DIFFERENTIATING MESENCHYMAL STEM CELLS INTO INSULIN PRODUCING CELLS(2023-12) Friguglietti, Jefferson; Merchant, Fatima Aziz; Akay, Yasemin M.; Wu, Tianfu; Al-Ubaidi, Muayyad R.; Sabek, Omaima3D cell culture techniques are increasingly used in stem cell tissue engineering since they better mimic the in vivo environments compared to conventional 2D culture. Current 3D culture methods include less complex suspension methods such as hanging well and ultra-low attachment plate, to more advance methods incorporating scaffold designs to support cell-cell and cell-extracellular matrix interaction (ECM). In this study we investigate a novel microfabricated silicon-titanium diboride (Si-TiB2) substrate’s efficacy in increasing function of differentiated mesenchymal stem cells (MSCs) into insulin producing cells (IPCs). This substrate provides a 3D microenvironment (aggregates) and geometric (pattern shape), mechanical (stiffness gradients) and biochemical (selectively adsorbed proteins) cues that are critical for cell differentiation. Differentiated IPCs are essential, providing an alternative tissue bank for future transplantation into diabetic patients. Utilizing photolithography technique, e-beam deposited TiB2 layers were fabricated on Si, wherein differences in their surface properties (hardness, stiffness, wetness, and electrical charge), enable selective adsorption of specific proteins on the micropatterns. MSCs from adult bone marrow were cultured on the substrate, and following a seven-day culture period subjected to a differentiation protocol. Functional analysis included quantification of c-peptide following a glucose stimulated insulin response (GSIR) assay. Morphological analysis of the cytoskeleton through immunofluorescence staining (f-actin, green) revealed increased rounded morphology of cells within the multicellular aggregates confirming a 3D culture environment on the SiTiB2 substrate. Additionally, immunofluorescence staining for biomarkers (n - cadherin, red) showed increased expression within the rounded core indicating cell-cell interaction with the aggregates. Further BioAFM analysis indicated cells with higher elastic modulus in the center of aggregates compared to cells on the edges of the micropatterns (p < 0.05). GSIR assay showed MSCs differentiated on the Si-TiB2 substrate had a better response to high glucose stimulation when compared to those differentiated in 2D monolayers in tissue culture plates (p < 0.05). Finally, co – culture with HUVEC and hBM-MSCs revealed unique formation of aggregation with HUVEC located primarily in the center of the aggregate. The TiB2 substrate provides a unique culture platform to better understand differentiation of MSCs into insulin producing cells when compared to ULP and traditional 2D flask.Item Optimization of DNA Nanoparticle Mediated Gene Therapy for Inherited Ocular Diseases(2019) Rodriguez, DevonIn this study, DNA NPs containing the correct gene sequence were subretinally and vitreally injected and later analyzed. This project was completed with contributions from Mashal Kakakhel from Sam Houston State University.Item Reducing Oxidative Stress in Retinitis Pigmentosa Mouse Model P23H(2018-10-18) Nguyen, KevinRetinitis Pigmentosa is a genetic disorder that can disrupt our vision through the deterioration of rods cells and, eventually, cone cells. A study found that patients with RP have higher oxidative stress levels than a control group. Patients who exhibit higher levels of reactive oxygen species would not be able to adequately compensate with normal levels of antioxidant enzymes. Furthermore, previous studies have shown that SOD3 was essential for reducing the oxidative damage done to the injured tissue. Through this study, we hope to provide a method to minimize the damage done by RP and to establish the importance of the extracellular matrix in photoreceptors’ health. Transgenic heterozygous SOD3OE were crossed with a knock-in gain of function heterozygous RhoP23H/+ to produce our target genotype for this experiment, SOD3OE/RhoP23H/+. At post-natal day 30 and 60, the mice’s retinal functional ability was tested using ERG. One retina from each mouse was collected for Western blot for protein analysis, while the other was embedded in paraffin for structural analysis. The results show that the SOD3OE/RhoP23H/+ has higher ERG at P30, but not statistically significant. At P60, the ERG’s were reduced, indicating that SOD3OE cannot mitigate the effects of RhoP23H/+. The mutation overwhelms the effectivity of SOD3, and, as a result, is not a viable treatment plan for this mutation. Although the overexpression of the anti-oxidant has not entirely counteracted RhoP23H/+, this study advances the understanding of the importance of extracellular enzymes and the possibility of delaying the onset of RP in patients.Item Riboflavin and the Riboflavin Binding Protein, Retbindin, are Essential for the Metabolic Homeostasis of the Retina(2020-05) Sinha, Tirthankar; Naash, Muna I.; Al-Ubaidi, Muayyad R.; Mohan, Chandra; Widger, William R.; Hurley, James B.Retbindin is a novel retina specific riboflavin binding protein, ablation of which leads to degeneration. Using a combination of untargeted steady state metabolomics and targeted flux metabolomics, we here investigated the mechanism behind this degeneration. Furthermore, the neural retina (NR) and retinal pigment epithelium (RPE) maintain a symbiotic metabolic relationship, disruption of which leads to debilitating vision loss. In the current study we have identified the differences in the steady state metabolite levels and the pathways functioning between bona fide NR and RPE. Moreover, involvement of key metabolic cofactors, flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) in cellular homeostasis has been well established for tissues other than the retina. Here, we present an optimized method to effectively extract and quantify FAD and FMN from a single neural retina and its corresponding RPE. We also show that in absence of retbindin there is significant reduction of flavins in both the neural retina and RPE, causing an imbalance in their symbiosis. However, there is no report on how the retina responds to riboflavin deficiency. Thus, here we developed a diet induced model of riboflavin deficiency and identified how that results in metabolic dysfunction in both the neural retina and RPE. We have further shown that these metabolic abnormalities can eventually compromise the electrophysiological response of both the neural retina and the RPE. Thus, we provide evidence how riboflavin and its binding protein retbindin can help in regulating retinal homeostasis.Item The Spatiotemporal Dynamics of Mycobacterial Infection(2019-08) Stolley, Danielle L.; May, Elebeoba E.; Al-Ubaidi, Muayyad R.; Mohan, Chandra; Zhang, Yingchun; Lin, YingHost response to Mycobacterium tuberculosis (Mtb) is distinctive in the use of a spatial immunological response to limit the progression of infection. This results in the formation granulomas, aggregations of immune cells that isolate invading microbes, a hallmark of the adaptive immune response to Mtb infection. Traditional in vivo studies have investigated the mature granuloma, but relatively fewer studies investigate how these structures form during the early stages of infection nor how spatial organization impacts control, resolution, or dissemination of the bacterium. Research has shown that initial aggregation of macrophages during innate immune response influences the progression of disease and formation of granulomas during adaptive immunity. However, current experimental methods for studying cellular interactions during early stages of infection are ill adapted for concurrent spatial and temporal quantification of host-pathogen dynamics, which is necessary for a quantitative understanding of the innate spatial immune response to Mtb and to inform the development of accurate computational models of tuberculosis disease. To address this, we developed a three-dimensional (3D) ex vivo model of mycobacterium infected macrophages cultured in reconstituted basement membrane and characterized the structural impact of 3D structure on infection dynamics in comparison to standard two-dimensional (2D) infection models. We quantified temporal immune response using standard biological sampling methodologies and long-term time-lapse confocal imaging to quantify the early spatiotemporal dynamics of macrophage response to mycobacterium infection. Our studies using Mycobacterium smegmatis indicate that the 3D environment induces a shift in dynamics. In 3D we see significantly higher cellular velocities in infected conditions as compared to control non-infected conditions, whereas the converse occurs in 2D. This may impact computational models that utilize 2D assumptions. We developed a data analysis pipeline to quantify macrophage state with respect to infection and cellular microenvironment. Results show non-infected and non-active macrophages within infected environments present dynamics comparable to controls, while infected and activated macrophages exhibit comparable spatiotemporal dynamics in 2D and 3D. Using the more virulent Mycobacterium bovis BCG, we observe a greater distinction between control and infected conditions and preliminary evidence of a more distinct 3D immune response resulting in increased cell death and extracellular bacteria.Item Transient Metabolic Alterations Induced by Chemotherapeutics in Cancer Persisters(2022-05-12) Karki, Prashant; Orman, Mehmet A.; Willson, Richard C.; Varadarajan, Navin; Şen, Mehmet; Al-Ubaidi, Muayyad R.Acquired drug tolerance has been a major challenge in cancer therapy. Recent evidence has revealed the existence of slow-cycling persister cells that survive drug treatments and give rise to multi-drug tolerant mutants in cancer. The mechanisms associated with persister phenotypes are highly diverse and complex, and many aspects of persister cell physiology remain to be explored. In this study, we aim to characterize the metabolic profiles of cancer persister cells mediated by cancer therapeutics, as epigenetic changes induced by drugs can lead to a transient metabolic rewiring of persister cells that can be associated with the phenotypic switch between normal and persister cell state. Determining the metabolic mechanisms underlying persister cell survival and maintenance will facilitate the development of novel treatment strategies that target persisters and enhance cancer therapy. In our first project, we treated melanoma cells with various conventional chemotherapeutic agents and showed that melanoma persister cells are not necessarily preexisting dormant cells. In fact, our data indicate they may be induced by cancer chemotherapeutics. Furthermore, with the use of untargeted metabolomics and phenotype microarrays, we demonstrated a transient upregulation in Krebs cycle metabolism in persister cells. We verified that targeting mitochondrial activity can significantly reduce melanoma persister levels. The reported metabolic remodeling feature seems to be a conserved characteristic of melanoma persistence, as it has been observed in various melanoma persister subpopulations derived from a diverse range of chemotherapeutics. In the next project, we explored metabolic alterations in melanoma cells mediated by Vemurafenib (VEM), a BRAF inhibitor. Co-treatment with BRAF inhibitors is a common treatment strategy for melanoma cancer. However, how a BRAF inhibitor itself alters melanoma cell metabolism and mediates persister survival is not well understood. Our findings demonstrate that metabolites associated with phospholipid synthesis, pyrimidine, one-carbon metabolism, and branched-chain amino acid metabolism are significantly altered in vemurafenib persister cells when compared to the bulk cancer population. Our data also show that vemurafenib persisters have higher lactic acid consumption rates as well as higher cell viability in a medium with lactate as the primary carbon source compared to control cells, further validating the existence of a unique metabolic reprogramming in these drug-tolerant cells. In the final project, we aim to elucidate the signaling pathways that link the therapeutic treatments to the observed metabolic reprogramming in melanoma persister cells. Using a high throughput assay with commercially available antibody arrays and western blotting, we identified that the cJUN pathway was transiently upregulated in cultures treated with chemotherapeutic agents. We further show that co-treatment with a cJUN inhibitor, JNK-In-8, resulted in an increased survival rate in cancer cells in the presence of chemotherapeutic agents. Furthermore, we highlighted that the phenomenon associated with cJUN was predominantly active in cultures treated with antimetabolites that act as a nucleoside analog for deoxycytidine. Overall, these results lead us to believe that cJUN, which is at the crossroads for both cell survival and apoptotic pathways, plays a significant role in persister physiology.Item Understanding The Visual And Auditory Defect In Ush2a Mouse Model(2023-05-08) Crane, Ryan; Naash, Muna I.; Al-Ubaidi, Muayyad R.; Groves, Andrew K.; Yang, Jun; Porter, Jason; Romero-Ortega, Mario I.Usher syndrome (USH) is the most common form of dual deafness and irreversible vision loss found in patients worldwide. USH2 is the most prevalently occurring sub type, accounting for ~50 to 75% of USH clinical cases. Patients with USH2 suffer from congenital hearing loss and progressive vision loss beginning from adolescence. Mutations in USH2A (usherin) account for ~80% of USH2 patients; making it the most common genetically mutated gene among USH patients. Usherin has been detected in the photoreceptors of the retina and in the developing inner ear cell stereocilia. The USH2A gene is very large and attempting gene therapy with conventional viral delivery is not easily attainable due to limited payload capacity of commonly used Adeno-associated viruses (AAV) ( <4.7 kbp.) To understand the mechanism underlying hearing and visual impairments, a knock in (KI) mouse model (Ush2adelG/delG) was developed using one of the most prevalent human mutations of USH2A, 2299delG. While other models for Ush2a exist, no other KI model has been able to successfully mimic the genetic mutations found in humans. This model will be valuable to investigate possible delivery methods for USH2A. Retinal phenotype in the KI model was found to follow similar progression as patients, with a notable gradual loss of vison that became apparent at older ages, along with concomitant photoreceptor degeneration. More in-depth analysis found that this was due to the mislocalization of the KI mutant protein and a subsequent mislocalization of its other USH2 interacting partners. Along with patients exhibiting phenotypes associated with homozygous mutations of USH2A, there is also a prevalence of cases involving heterozygous mutations of USH2A in combination with mutations in other USH or non-USH genes that results in varying degrees of phenotype. Backcrossing of Ush2adelG/delG with three knockouts of photoreceptor-specific proteins, rhodopsin (Rho-/-), ABCA4-/-, and ROM1-/-, resulted in double heterozygous of digenic mutants with a mixture of retinal phenotype. The combination of heterozygous USH2A with rhodopsin (Rho+/-/Ush2adelG/+) led to a surprising protective effect of retinal phenotype. We next evaluated the cochlear phenotype of the KI model, showing congenital hearing loss in the lower frequency range, particularly at 8 and 11 kHz, in the auditory brainstem response (ABR) tone tests. Associated stereocilia of the inner hair cells in the apical portion of the cochlea were correspondingly found to be disorganized. Like the retina, the Ush2adelG/delG mutant protein was found to be mislocalized while the associated proteins were found to be properly localized to the stereocilia of the hair cells. Following the analysis and verification of the KI model as a viable model for therapeutic testing, the next step was to develop a therapeutic approach for both the retina and cochlea. With one major challenge of therapy for USH2 being its very large gene, development of an alternative, non-viral delivery, method is needed and an example of such an approach is by encapsulating the therapeutic gene using hyaluronic acid nanospheres (NSs). Delivery approaches for the eye, specifically targeting the retina, using NSs in conjunction with a small molecule, sulfotyrosine, have shown great promise. Preliminary studies using NSs filled with plasmid DNA with GFP expression cassette under the control of ubiquitous promotion (chicken beta actin with CMV enhance, CAG) led to noticeable GFP expression in retinal cells following intravitreal injections. No GFP was detected with the plasmid DNA alone. Along with the retina, delivery to the inner ear cells of the cochlea was also a goal. Preliminary injections of the NSs in P0-P1 round window injections into the cochlea showed no functional differences between the injected and non-injected (contralateral) ears, indicating no major toxic effect caused by the NSs. Future work will look more in depth at the possibility of delivering native USH2A genes into the retina and cochlea as a potential therapeutic options.