Browsing by Department "Molecular And Cellular Medicine"

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Advanced Methods for Mimicking the Osteogenic Niche to Heal Bone
Molecular And Cellular Medicine; TAMHSC; https://hdl.handle.net/20.500.14641/638; DHHS-NIH-National Institute of Arthritis and Musculoskeletal and Skin Diseases
DESCRIPTION (provided by applicant): Of the 13 million yearly fractures that occur in the United States, about 10% fail to repair and in extreme cases this can result in immobility or amputation. While autologous bone grafts are the most effective method to heal complex defects, the available graft material is limited, and the procedure involves additional surgery known to cause chronic donor-site pain in many patients. Human mesenchymal stem cells (hMSCs) have been intensely investigated for their ability to promote bone healing, but results have been variable and disappointing. This is partly due to variability in cell preparations that occurs through donor variation or through inconsistent culture methodologies. Our work has also indicated that hMSCs are not retained at the site of injury for sufficient time to achieve engraftment and promote repair. In an attempt to solve these problems, we recently demonstrated that acceleration of canonical Wnt signaling with the small molecule PPAR¿ inhibitor GW9662 produces osteogenically enhanced hMSCs (OEhMSCs). Further, these OEhMSCs produce extracellular matrix (hMatrix) that dramatically increases OEhMSC retention and bone repair in calvarial and femoral defects. Our central hypothesis for this proposal is that an injectable microsphere vehicle co-administering GW9662, hMatrix and hMSCs will promote osteo- repair through a mechanism that involves extended hMSC retention, trophic factor secretion and paracrine activation of the host stroma. Our hypothesis will be tested via three Specific Aims. Aim 1: Optimize the delivery of OEhMSCs, hMatrix and GW9662 via collagen/poly (lactide-co-glycolide) capsules in vitro. Aim 2: Evaluate bone repair in immune-compromised murine models of calvarial and femoral trauma. Aim 3: Delineate the mechanisms by which the hMatrix components collagen types VI and XII and their cognate integrins increase OEhMSC retention and expression of osteoinductive and angiogenic paracrine factors. These studies will lay the groundwork for translating this novel hMSC-based method for osteo-repair to the clinic. Successful completion of this project could lead to a revolutionary new method for bone repair that could effectively dismiss the need for autologus bone graft in regenerative orthopedics. Our multi-disciplinary team of stem cell biologists, biochemists, bioengineers, orthopedic clinicians and commercialization experts are highly equipped to achieve the goals of the project.
Bacterial Export of Folded Proteins: Transport Mechanism of the Tat Translocon
Molecular And Cellular Medicine; TAMHSC; https://hdl.handle.net/20.500.14641/549; National Institutes of Health
DESCRIPTION (provided by applicant): Since proteins are found in every membrane and aqueous compartment within cells and yet are primarily synthesized on cytoplasmic ribosomes, protein targeting and transport across or into lipid membranes is a fundamental process in all organisms. Many distinct types of translocation systems exist that allow large protein molecules to cross membranes without compromising the membranes' role as a permeability barrier to ions, metabolites, and macromolecules. While many protein transport systems translocate 'linearized polypeptides', the twin-arginine translocation (Tat) system transports fully-folded and assembled proteins without collapsing ion gradients. The absence of a functional Tat system in bacteria often leads to growth defects and occasionally death. The Tat machinery is also responsible for the export of proteins important for bacterial virulence in humans. Since animals, including humans, do not contain homologues of Tat machinery proteins, inhibitors of Tat transport could potentially find use as novel antibiotics. Our long-term goal is to decipher the mechanism of protein transport by the bacterial Tat machinery at a molecular level. In past research, numerous in vitro assays that enable biochemical and biophysical investigations of the Escherichia coli Tat transport mechanism have been developed. Recent work indicates that the Tat machinery catalyzes insertion of a signal peptide hairpin into the membrane in an energy-independent manner, and that full translocation of the C-terminal end of the signal peptide requires a proton motive force. These results establish that the signal peptide's binding interactions and its membrane translocation are critical for directly promoting mature domain transport. However, the structural and oligomeric nature of the translocation pore and how leakage is prevented remain major unsolved problems. Characterization of the translocation pore is particularly challenging because it disassembles in the absence of a proton motive force. We will examine structural and dynamic properties of the Tat machinery, and test hypotheses generated by our Hairpin-Hinge Model of transport. Our Specific Aims are: (1) to identify the signal peptide binding site on the TatBC receptor complex, and determine the arrangement of TatBC heterodimers in the TatBC oligomer; (2) to characterize translocon and cargo dynamics during Tat transport using real-time kinetic approaches; and (3) to identify the conformational and environmental changes that TatA experiences during transport. We will use crosslinking and real-time fluorescence approaches, including single molecule studies, to probe binding interactions and the dynamic transitions between important structural intermediates in the translocation cycle. This work will clearly establish the manner in which signal peptides interact with the Tat machinery, and the structural, dynamic, and oligomeric properties of the translocation pore. In total, this study will significantly advance our understanding of the Tat transport mechanism, which will in turn provide a sound framework for understanding bacterial growth and virulence, and for the development of antibiotics and biotechnological tools.
Context-specific Functions of CDK8
Molecular And Cellular Medicine; TAMHSC; https://hdl.handle.net/20.500.14641/229; DHHS-NIH-National Institute of General Medical Science
Title: Context-specific functions of CDK8 Project Summary: The long-term goal of this project is to elucidate the function and regulation of the CDK8 module, a key component of the transcription cofactor Mediator complex, in the versatile model organism Drosophila melanogaster. The four subunits of the CDK8 module – CDK8, CycC, MED12, and MED13 – are either mutated or amplified in cardiovascular diseases and a number of human cancers, such as melanoma and colorectal cancers. Elucidating the function and regulation of the CDK8 module in different biological contexts is essential to understanding the pathological consequences of CDK8 module misregulation, which is important for the design of clinical strategies to treat these diseases. Studies in the previous funding cycle of this project have demonstrated that CDK8-CycC serves as a critical regulatory node linking nutrient intake to fat metabolism and developmental timing in Drosophila development. These studies have shown that CDK8-CycC is a direct inhibitor of SREBP (sterol response element binding protein)-dependent gene expression, and that CDK8-CycC positively regulates ecdysone receptor-activated gene expression. The overall objective of this project is to unravel the function and regulation of CDK8 in different developmental contexts. Studies in the previous funding cycle illustrate that Drosophila is an ideal and powerful experimental system to achieve this long-term goal. Two new aims are proposed in this funding cycle. Aim 1 will determine the role of CDK8 in regulating the expression of telomeric retrotransposons and telomere biology in Drosophila. Our RNA-seq analyses revealed a specific upregulation of telomeric retrotransposon expression and a significant increase in telomere length in cdk8 and cycC mutants. We have also discovered similar deregulation of telomeric retrotransposon expression and telomere length in med7 and Scalloped (Sd) mutants. The transcription factor Sd functions downstream of the conserved Hippo pathway. Thus we propose to examine the unexplored functions of the CDK8 module and Sd in regulating the expression of telomeric retrotransposons and telomere length in Drosophila. Aim 2 of the proposal will identify and validate upstream regulators and downstream effectors of CDK8 in Drosophila. We have established novel and robust wing phenotypes caused by specific alterations of CDK8 activities, allowing us to perform a dominant modifier genetic screen to identify factors interacting with CDK8 in vivo. We have identified 26 genomic loci whose haploinsufficiency modifies these CDK8-specific phenotypes; further genetic analysis led us to identify genetic interactions between CDK8 and the components of the epidermal growth factor receptor (EGFR) and Dpp/TGF? signaling pathways, as well as several specific genes. In parallel, we have performed immunoprecipitation coupled to mass spectrometry (IP- MS) analyses to identify proteins that can interact with CDK8. Combination of these biochemical and genetic screens puts us in a unique position to systematically identify and validate upstream regulators and down- stream effectors of CDK8 in vivo, which will impact our understanding of the context-specific functions of CDK8.
Context-specific Functions of CDK8
Molecular And Cellular Medicine; TAMHSC; https://hdl.handle.net/20.500.14641/229; DHHS-NIH-National Institute of General Medical Science
Title: Context-specific functions of CDK8 Project Summary: The long-term goal of this project is to elucidate the function and regulation of the CDK8 module, a key component of the transcription cofactor Mediator complex, in the versatile model organism Drosophila melanogaster. The four subunits of the CDK8 module – CDK8, CycC, MED12, and MED13 – are either mutated or amplified in cardiovascular diseases and a number of human cancers, such as melanoma and colorectal cancers. Elucidating the function and regulation of the CDK8 module in different biological contexts is essential to understanding the pathological consequences of CDK8 module misregulation, which is important for the design of clinical strategies to treat these diseases. Studies in the previous funding cycle of this project have demonstrated that CDK8-CycC serves as a critical regulatory node linking nutrient intake to fat metabolism and developmental timing in Drosophila development. These studies have shown that CDK8-CycC is a direct inhibitor of SREBP (sterol response element binding protein)-dependent gene expression, and that CDK8-CycC positively regulates ecdysone receptor-activated gene expression. The overall objective of this project is to unravel the function and regulation of CDK8 in different developmental contexts. Studies in the previous funding cycle illustrate that Drosophila is an ideal and powerful experimental system to achieve this long-term goal. Two new aims are proposed in this funding cycle. Aim 1 will determine the role of CDK8 in regulating the expression of telomeric retrotransposons and telomere biology in Drosophila. Our RNA-seq analyses revealed a specific upregulation of telomeric retrotransposon expression and a significant increase in telomere length in cdk8 and cycC mutants. We have also discovered similar deregulation of telomeric retrotransposon expression and telomere length in med7 and Scalloped (Sd) mutants. The transcription factor Sd functions downstream of the conserved Hippo pathway. Thus we propose to examine the unexplored functions of the CDK8 module and Sd in regulating the expression of telomeric retrotransposons and telomere length in Drosophila. Aim 2 of the proposal will identify and validate upstream regulators and downstream effectors of CDK8 in Drosophila. We have established novel and robust wing phenotypes caused by specific alterations of CDK8 activities, allowing us to perform a dominant modifier genetic screen to identify factors interacting with CDK8 in vivo. We have identified 26 genomic loci whose haploinsufficiency modifies these CDK8-specific phenotypes; further genetic analysis led us to identify genetic interactions between CDK8 and the components of the epidermal growth factor receptor (EGFR) and Dpp/TGFβ signaling pathways, as well as several specific genes. In parallel, we have performed immunoprecipitation coupled to mass spectrometry (IP- MS) analyses to identify proteins that can interact with CDK8. Combination of these biochemical and genetic screens puts us in a unique position to systematically identify and validate upstream regulators and down- stream effectors of CDK8 in vivo, which will impact our understanding of the context-specific functions of CDK8.
Diet-NFkB interactions in the transcriptional regulation of metabolic homeostasis
Molecular And Cellular Medicine; TAMHSC; https://hdl.handle.net/20.500.14641/388; DHHS-NIH-National Institute of Diabetes and Digestive and Kidney Disorders
Project Summary/Abstract: Metabolic and innate immune responses, two primitive systems critical for the long-term homeostasis of multi- cellular organisms, have evolved to promote cooperative, adaptive responses against diverse environmental challenges. Unfortunately, as humans appear evolutionarily unprepared for modern diets, over-nutrition and dietary imbalances often leads to mis-regulation of these responses and the development of metabolic dysfunction. Uncovering the integration of these ancestral metabolic and innate immune systems thus advances both understanding of basic physiology and the complex etiology associated with metabolic disease. An over- arching goal of the research proposed here is to establish a framework of innate immune-metabolic signaling networks employing invertebrate models. Utilizing Drosophila, a powerful integrative physiology model, new insights derived from previous studies have revealed a role for the innate immune transcription factor NF-kB in modulating metabolic target gene expression during adaptation to dietary changes. NF-kB transcription factors, evolutionarily conserved regulators of innate immunity, are emerging as a critical node in the bidirectional communication and coordination of metabolic and innate immune signaling pathway interactions. It was uncovered that NF-kB antagonism of Foxo function (a key nutrient-responsive transcription factor) is crucial to influence metabolic target genes in diverse cell types to shape distinctive aspects of lipid metabolism (largely linked to catabolism - usage, breakdown, and mobilization). This antagonism subsequently balances energy homeostasis with diet-dependent nutrient supply and promotes metabolic adaptation. These findings highlight a critical need to explore the distinct molecular and cellular mechanisms, governed by ancient innate immune signaling pathways, that may shape the equilibrium between normal physiology and pathology associated with diet-mediated disruptions in lipid metabolism. To this end, it is possible that diet- and NF-kB-dependent antagonism of metabolic transcription factor function may be central to the integration of innate immune- metabolic signaling networks. Drosophila provide an invaluable, genetically tractable model to characterize such mechanisms; as these signaling networks are conserved from insects to humans, and many ancestral insect tissues combine functions of nutrient and pathogen sensing organs, highlighting the inherent association between metabolic state and innate immune function. There are three specific aims to this proposal: (i) to explore interactions between NF-kB and histone deacetylases in the control of diet-dependent chromatin remodeling and lipid metabolism, (ii) to determine whether unique signaling mechanisms direct diet- and NF-kB-dependent transcriptional attenuation (vs activation) of metabolic target genes, and (ii) to characterize NF-kB-modulated gene regulatory networks shaped by dietary imbalances and chromatin remodeling. Exploiting Drosophila to explore the origin of innate immune-metabolic interactions holds promise for an enhanced rate of uncovering novel mechanisms that underly lipid-metabolic imbalances and metabolic dysfunction.
Melatonin for Reversing Brain Dysfunction in Gulf War Illness
Molecular And Cellular Medicine; TAMHSC; https://hdl.handle.net/20.500.14641/473; DOD-Army-Medical Research and Materiel Command
Multiple persisting health issues including brain dysfunction exemplify Gulf War Illness (GWI), which afflicts 25%-32% of the 700,000 Persian Gulf War 1 (PGW-1) Veterans. The most conspicuous brain-related impairments comprise learning difficulties, inability to make new memories or recall recent memories, depression, and sleep problems. A multitude of potential causes have been suggested for this ailment. A thorough report by the Department of Veterans Affairs Research Advisory Committee on GWI implied that the symptoms exhibited by a major percentage of PGW-1 Veterans are likely due to exposures to a mixture of chemicals that includes pyridostigmine bromide (PB) and pesticides such as DEET and permethrin (PM) during the war. These exposures were thought to have happened because of the following circumstances. The drug PB was given to Service personnel as a prophylactic treatment against possible nerve gas attack by the enemy. The pesticides such as DEET and PM were widely used by troops on skin and uniforms to combat insects and rodents in the desert. An exposure to chemical weapons is likely also the cause of GWI in some Veterans particularly those who were posted close to the chemical weapon depot demolitions. Nonetheless, it is widely believed that the neurological symptoms in a significant fraction of Gulf War Veterans is owed to an interaction of chemicals PB, DEET, and PM or interaction of one or more of these chemicals with war-related stress. Indeed, experiments performed in our laboratory using a rat model demonstrated that combined exposure to low doses of chemicals PB, DEET, and PM with mild or moderate stress for 4 weeks causes persistent dysfunction in the hippocampus, a region of the brain vital for making new memories and maintaining normal mood function. Dysfunction of this region was typified by learning difficulties, inability to make new memories or recall recently formed memories and anxiety or depressive-like behavior. Interestingly, these alterations were allied with persistently higher levels of reactive oxygen species, mitochondrial abnormalities, chronic low-level inflammation and greatly waned production of new neurons in the hippocampus. Greatly reduced hippocampal neurogenesis at least partly underlies memory and mood impairments observed in this GWI model because regular addition of newly born neurons to the hippocampus circuitry is a vital process that aids in the formation of new memories and preservation of normal mood. Since both oxidative stress and inflammation can adversely influence memory and mood function either directly or indirectly through reducing neurogenesis in the hippocampus, it is likely that persistently augmented oxidative stress and chronic low-level inflammation are among the major reasons behind memory and mood dysfunction in GWI. From these perspectives, drugs that are proficient for easing increased oxidative stress and chronic inflammation appear useful for improving both hippocampal neurogenesis and memory and mood function in GWI. Using a well-established rat model of GWI, we offer to investigate the effectiveness of oral administration of low to moderate doses of melatonin (MEL; an over-the-counter dietary supplement having robust antioxidant, anti-inflammatory, and sleep promoting properties) for improving, learning, memory, and mood function. Earlier studies have shown that MEL is efficacious for: (i) providing protection against developing Alzheimer’s disease and Parkinson’s disease, (ii) reducing the size of infarct area in stroke, (iii) minimizing brain swelling and dysfunction after a head injury, and (iv) improving the lifespan. Furthermore, MEL intake can promote better sleep, which is important since sleep disturbances is one of the major brain-related issues in GWI. Our preliminary studies in a rat model support the idea that MEL treatment has potential to alleviate several brain-related impairments seen in GWI. Therefore, using a rat model of chronic GWI, we propose to rigorously test the usefulness of low to moderate doses of MEL for improving memory and mood function along with suppression of oxidative stress and inflammation in the hippocampus. Taken together, the proposed studies are extremely pertinent towards developing a treatment that improves cognition, memory, and mood function in PGW-1 Veterans.
Modulation of peptidergic neurons by the gluconeogenic enzyme Glucose-6-Phosphatase
Molecular And Cellular Medicine; TAMHSC; https://hdl.handle.net/20.500.14641/238; National Institutes of Health
Neuropeptides (NPs) play crucial roles in behavior and physiology. NPs are short proteins that are stored and released from Large Dense Core Vesicles of peptidergic neurons. Peptidergic neurons activate neural circuits that regulate and modulate a range of physiological processes and impact an array of behaviors, including feeding, social interactions, circadian behavior and others. Some peptidergic neurons can express multiple NPs, and most contain also small Synaptic Vesicles that release classic neurotransmitters. An additional level of complexity is imparted by various degrees of NP processing, which can occur prior or after their release (i.e. regulated transport, localization, processing etc.). Thus, peptidergic neurons are multimodal that can convey information within the nervous system. In addition, some NPs act also as neurohormones and can signal to tissues other than the CNS. Even though the functions of many NPs have been characterized and their receptors have been identified, little is known about how their activity is modulated. In the fruit fly Drosophila, cell fate of many peptidergic neurons is dependent on the transcriptional activator Dimmed (DIMM). The recent discovery that many DIMM positive neurons express G6P-GAL4, a marker for cells expressing the highly conserved enzyme Glucose-6-phosphatase (G6P), implies that G6P has a non-canonical role in Drosophila. G6P is better known for its role in gluconeogenesis, a process restricted to the liver and kidney of mammals, for the generation of glucose from non-carbohydrate precursors to maintain blood glucose homeostasis when animals are food-deprived. Initial characterization of Drosophila G6P has established that G6P-expressing peptidergic neurons have gluconeogenic capacity and are able to use alanine as a substrate to generate glucose. Moreover, G6P is necessary for NP accumulation in neural processes, and preliminary data presented in this application reveal that G6P is required in these neurons to generate appropriately sized Golgi complexes. Based on these data, G6P (and by inference gluconeogenesis) are proposed to play a critical role in peptidergic neurons, presumably in the biogenesis of Golgi and/or LDCV structures, to affect NP release, thereby modulating physiological and behavioral processes. Importantly, mammals harbor, three G6P genes, only one of which (G6PC1) is involved in hepatic gluconeogenesis. The other two genes, G6PC2 and G6PC3, are expressed in secretory cells of other tissues, including the brain and CNS. The function of these non-canonical mammalian G6P genes is not known, but given their similarities to Drosophila G6P with regard to expression and cellular context, it is intriguing to posit that the G6PC2 and G6PC3 enzymes have similar roles in mammals as the single G6P enzyme in the fly CNS. !
Modulation of peptidergic neurons by the gluconeogenic enzyme Glucose-6-Phosphatase
Molecular And Cellular Medicine; TAMHSC; https://hdl.handle.net/20.500.14641/238; National Institutes of Health
Neuropeptides (NPs) play crucial roles in behavior and physiology. NPs are short proteins that are stored and released from Large Dense Core Vesicles of peptidergic neurons. Peptidergic neurons activate neural circuits that regulate and modulate a range of physiological processes and impact an array of behaviors, including feeding, social interactions, circadian behavior and others. Some peptidergic neurons can express multiple NPs, and most contain also small Synaptic Vesicles that release classic neurotransmitters. An additional level of complexity is imparted by various degrees of NP processing, which can occur prior or after their release (i.e. regulated transport, localization, processing etc.). Thus, peptidergic neurons are multimodal that can convey information within the nervous system. In addition, some NPs act also as neurohormones and can signal to tissues other than the CNS. Even though the functions of many NPs have been characterized and their receptors have been identified, little is known about how their activity is modulated. In the fruit fly Drosophila, cell fate of many peptidergic neurons is dependent on the transcriptional activator Dimmed (DIMM). The recent discovery that many DIMM positive neurons express G6P-GAL4, a marker for cells expressing the highly conserved enzyme Glucose-6-phosphatase (G6P), implies that G6P has a non-canonical role in Drosophila. G6P is better known for its role in gluconeogenesis, a process restricted to the liver and kidney of mammals, for the generation of glucose from non-carbohydrate precursors to maintain blood glucose homeostasis when animals are food-deprived. Initial characterization of Drosophila G6P has established that G6P-expressing peptidergic neurons have gluconeogenic capacity and are able to use alanine as a substrate to generate glucose. Moreover, G6P is necessary for NP accumulation in neural processes, and preliminary data presented in this application reveal that G6P is required in these neurons to generate appropriately sized Golgi complexes. Based on these data, G6P (and by inference gluconeogenesis) are proposed to play a critical role in peptidergic neurons, presumably in the biogenesis of Golgi and/or LDCV structures, to affect NP release, thereby modulating physiological and behavioral processes. Importantly, mammals harbor, three G6P genes, only one of which (G6PC1) is involved in hepatic gluconeogenesis. The other two genes, G6PC2 and G6PC3, are expressed in secretory cells of other tissues, including the brain and CNS. The function of these non-canonical mammalian G6P genes is not known, but given their similarities to Drosophila G6P with regard to expression and cellular context, it is intriguing to posit that the G6PC2 and G6PC3 enzymes have similar roles in mammals as the single G6P enzyme in the fly CNS.
Nanoengineered bone repair scaffolds generated from stem cells and their secreted products for improved spinal fusion
Molecular And Cellular Medicine; TAMU; https://hdl.handle.net/20.500.14641/638; DHHS-NIH-National Institute of Arthritis and Musculoskeletal and Skin Diseases
Degenerative disc disease is an epidemic, ultimately resulting in untenable pain and immobility. In advanced cases, spinal fusion is performed where vertebrae are surgically fixed with a mechanical device and an osteogenic material (bone substitute) is bridged between them in an attempt to induce fusion. Of the 600,000 yearly procedures performed in the US, the most common is posterolateral lumbar arthrodesis but the failure rate can reach 25-40% with standard commercial bone substitutes. The reason for failure rests in part with limited biocompatibility of synthetic bone substitutes, inconsistencies with processed cadaveric bone substitutes and in some cases, health complications caused by supraphysiologic doses of bone morphogenic protein. Autologous bone grafts are much more effective, but the approach is associated with donor site morbidity and the volume of available graft is limited. Compromising strategies that employ bone substitutes and bone marrow aspirate (BMA) are becoming common, but efficacy continues to be limited by the bone substitute. The fact that spine related disability is a growing global problem and standard of care interventions have an unacceptable failure rate clearly demonstrates the need for implants that safely and effectively promote bone fusion. The successes and failures of past spinal fusion strategies indicate that a bone substitute that mimics autograft will meet this need. Therefore, the goal of this proposal is to develop a 3D printed biomimetic bone graft substitute (the scaffold) by an innovative combination of stem cell biology, matrix biology, and biomedical engineering. The scaffold will consist of a tough, porous and flexible nanoengineered hydrogel consisting of gelatin methacrylate (gel-MA) coated with extracellular matrix (ECM) purified from osteogenically enhanced human mesenchymal stem cells derived from induced pluripotent stem cells (OEihMSCs). By mimicking the composition of bone matrix, OEihMSC-derived ECM is highly osteogenic. The gel-MA will be further enhanced by addition of novel silicate nanoparticles that impart stiffness and further stimulate osteogenesis. The scaffold will be designed to drive fusion with efficacy equivalent to autograft, but it will be manufactured from a standardized and sustainable source of materials. To achieve this goal, we will: optimize methodology for the generation of various forms of scaffold with a range of gel-MA, nanosilicate and ECM formulations with variations in macroporosity and stiffness (Aim1), optimize attachment, distribution, viability and osteogenesis of cells on the scaffold using in vitro 3D cell culture assays based on rotating wall bioreactor technology (Aim2) and finally, test the optimized scaffold with human BMA in a rodent posterolateral fusion model, incorporating imaging and biomechanical testing approaches (Aim 3). The rationale for this approach is that it has the capacity to satisfy a need for safe and effective autologous bone repair scaffolds for a rapidly growing population. With the current disposition of the FDA favoring autologous and minimally manipulated cytotherapeutic preparations, this strategy is well suited to clinical translation.
Nanoparticles Made from iPS Cell-Derived Mesenchymal Stem Cells as Standardized Natural Platforms for Targeted Treatment of Drug-Resistant Prostate Cancer
Molecular And Cellular Medicine; TAMHSC; https://hdl.handle.net/20.500.14641/319; DOD-Army-Medical Research and Materiel Command
Chemotherapy is the first-line treatment for advanced prostate cancer (PCa), but the early onset of resistance to chemotherapy drugs is common. Nanoparticles are promising platforms for treatment of drug-resistant PCa. Currently, most nanoparticles target tumors passively due to the variable leakage of newly formed blood vessels and the lack of effective lymphatic drainage in tumors; hence, their accumulation in tumors is limited. Nanoghosts (NGs) are a novel type of actively tumor-targeting nanoparticles readily reconstructed from the entire cell membrane (ghost cells) of mesenchymal stem cells (MSC), adult stem cells present in multiple tissues such as bone marrow and fat, and appear safe to use in human by hundreds of clinical trials worldwide. MSC-NGs retain targeting capabilities of MSCs to PCa cells, do not trigger immune response, and are cleared from healthy blood-filtering organs rapidly in a mouse model after intraperitoneal infusion. However, the yield of NGs and the efficiency of loading anti-PCa agents into NGs are very low, and a huge amount of MSCs is needed for preparing enough NGs to study such PCa therapy even in mouse. A similar but simpler method was used to produce nanovesicles (NVs) containing cytoplasm components from other cells with much higher yield and showed superior efficacy of delivering anti-cancer agent via intravenous route than mainstream nanoparticles. But conventional tissue-derived MSCs contain many pro-tumor cytoplasmic factors; hence, the efficacy of NVs made from these MSC to deliver anti-tumor agents might be compromised. For further research of MSC NGs and NVs in large-animal models and future clinical application, a huge amount of standardized MSCs is required. However, conventional tissue-derived MSCs from different donors vary considerably and cannot be expanded extensively, and their tumor-targeting property decreases after prolonged expansion, which severely hinders the research and application of MSC NGs/NVs for cancer therapy. To circumvent these issues of tissue-derived MSCs, my laboratory derived MSCs from human induced pluripotent stem cells (iPSCs) that are originated from normal adult cells but can give rise to any kind of cells in the body and have almost unlimited expandability. Our derivation protocol can be readily scaled up to produce an enormous amount of standardized MSCs. Our iPSC-MSCs maintained the expression of tumor targeting membrane proteins during prolonged expansion and in different derivation batches, whereas the expression of multiple pro-tumor factors in our iPSC-MSCs is remarkably lower than that in tissue-derived MSCs. NGs and NVs reconstructed from our iPSC-MSCs did not trigger immune response and exhibited selective binding toward PCa cells over non-tumor cells. This research addresses the Prostate Cancer Research Program overarching challenge: "Develop effective treatments and address mechanisms of resistance for men with high-risk or metastatic prostate cancer." This research will help drug-resistant PCa patients by providing a nature nanoparticle platform with unlimited supply and uniform biological properties for tumor-targeted delivery of various anti-PCa agents. The potential clinical applications of iPSC-MSC NGs/NVs include tumor-targeted delivery of chemotherapy drugs, anti-PCa proteins, and/or cDNAs encoding these proteins. The anti-PCa proteins can be either recombinant soluble proteins or membrane proteins expressed by iPSC-MSCs. The potential benefits of iPSCMSC NGs/NVs mediated PCa therapy is the uniform PCa-targeting capacity, the decreased pro-tumor risk, and the ease of safe and efficient genetic engineering in immortal iPSCs to enhance penetration of NGs/NVs through endothelium and tumor, which will ultimately help overcoming the resistance of advanced PCa to chemotherapy. No significant potential risks of iPSC-MSC NGs/NVs are expected since MSCs showed no significant risk in hundreds of clinical trials worldwide and NGs made from bone marrow MSCs showed no significant risk in mouse models. The projected time to achieve a patient-related outcome is about 12 years. The first 3 years, with the support of current applied grant, will provide the proof of principle in mouse models. The following 9 years will optimize the payload and genetic membrane engineering of iPSC-MSCs, scale up the production of iPSC-MSCs and NGs/NVs made from them, confirm the anti-PCa efficacy of NG/NV-mediated therapy in clinically relevant large-animal models, and then start a Phase I clinical trial to test the safety of iPSC-MSCs NG/NVs. The likely contributions of this study to advancing the field of prostate cancer research is to provide a standardized natural tumor-targeting platform for delivering various anti-PCa agents including chemotherapy drugs to improve the treatment of drug-resistant metastatic PCa.
NFkb: Integrator of Host-Diet-Microbiota Interactions
Molecular And Cellular Medicine; TAMHSC; https://hdl.handle.net/20.500.14641/388; DHHS-NIH-National Institute of Diabetes and Digestive and Kidney Disorders
Project Summary: Nutrition and dietary adaptation shape all aspects of animal physiology across taxa, including the composition and maintenance of intestinal microbiota, which, in turn, influence host animal metabolic responses. The reciprocal interactions between diet, host signaling networks, and microbiota define a physiological rheostat that governs host metabolism. Importantly, when these interactions are misregulated, the result is often metabolic dysfunction and disease. Thus, there is a critical need to explore the distinct cellular and molecular host signaling mechanisms that promote diet-microbe interactions and influence host physiology. The overall goal in this proposal is to investigate diet-dependent host signaling mechanisms, driven by the evolutionarily conserved, innate immune signaling pathway transcription factor NFkB, that influence intestinal microbiota homeostasis. I provide evidence that NFkB transcription factor function in the Drosophila intestine can govern microbiota maintenance and metabolic signaling pathway activity in response to specific changes in dietary macronutrients, putatively influencing microbiota-regulated aspects of host health and dietary adaptation. More specifically, I find that, in response to high carbohydrate / low protein dietary macronutrient ratios, NFkB activity can modulate the function of the 4EBP/TOR signaling pathway, a conserved regulator of physiology that couples nutrition, cellular energy homeostasis and mRNA translation. I hypothesize that intestinal NFkB function is required to maintain microbiota homeostasis in response to dietary changes through influencing host 4EBP/TOR signaling pathways. To address this hypothesis, I aim to utilize the fruit fly model, Drosophila melanogaster, to; (i) assess NFkB/4EBP/TOR integration by characterizing the cellular interactions of these signaling pathways and their impact on microbiota maintenance during dietary adaptation. A result of disruption of these signaling interactions is a significant change in mRNA translation within the intestine, thus I also plan to; (ii) explore the influence of intestinal translational on microbiota maintenance. Lastly, I anticipate that the changes I see in microbiota composition during dietary adaptation are impacting host physiology. Specifically, based upon current literature, I aim to; (iii) investigate the effect of NFkB-and- diet-dependent changes in microbiota maintenance on host proteolytic activity and amino acid homeostasis. Exploiting Drosophila to explore evolutionarily conserved host signaling mechanisms involved in maintaining commensal microbes under changing dietary conditions holds promise for developing integrative diet- microbiota interaction frameworks that are useful for optimizing human health.
NIK Promotes a Leader Cell Phenotype in Glioma
Molecular And Cellular Medicine; TAMHSC; https://hdl.handle.net/20.500.14641/390; National Institutes of Health
PROJECT SUMMARY: No cure currently exists for high grade gliomas, which have a mean survival time of approximately 1 year from diagnosis. These tumors are highly aggressive and invasive, despite current treatment protocols. Noncanonical NF-¿B signaling has been linked to glioma progression, but the downstream consequences of activation of this pathway have not been completely defined. Thus, a better understanding of the key molecular signals that regulate the invasive nature of glioma could aid in intervening in these clinical situations. This proposal will examine whether the NF-¿B-inducing kinase (NIK) enhances the leader cell phenotype in glioma to increase collective cell migration, which facilitates tumor invasion of surrounding normal tissue. Based on previous and preliminary findings, we hypothesize that NIK and noncanonical NF-¿B signaling promotes the acquisition of invasive potential in leader cells responsible for tumor dissemination through upregulation of the a11 integrin subunit (ITGA11) and MT1-MMP activity. The enclosed aims will test whether noncanonical NF-¿B signaling enhances ITGA11 expression and pseudopodial localization, and also determine the functional significance of noncanonical NF-¿B controlled ITGA11 expression on glioma cell invasion. We will use complementary approaches that involve the use of primary human brain tumor lines in vitro and in vivo. These studies are expected to show that NIK and noncanonical NF-¿B signaling simultaneously enhances ECM recognition and degradation via ITGA11/MT1-MMP complexes in the mesenchymal subtype of glioma to facilitate tumor dissemination. These studies will contribute to our long-term goal of an improved understanding of the molecular signals that initiate cell migration in three- dimensions and glioma progression.
Role of Alpha-Catenin and Wnt Signaling in Regulating Lipid Homeostasis
Molecular And Cellular Medicine; TAMHSC; https://hdl.handle.net/20.500.14641/229; DHHS-NIH-National Institute of General Medical Science
Wnt signaling, normally limited to embryogenesis, stem cell renewal and wound healing, is inappropriately re- employed in a variety of human cancers, such as hepatocellular carcinoma and colorectal cancer, as well as other diseases. Aberrant Wnt signaling and altered lipid metabolism are both signs of oncogenesis, and recent data suggest that Wnt control of adipogenesis and lipid metabolism may occur through separate mechanisms. Currently, the mechanisms remain poorly understood, and so remain outside of our ability to monitor, mitigate, prevent, or correct. It has been impossible to clearly delineate separate functions of Wnt in adipogenesis, lipid anabolism, and lipid catabolism, because these processes are inextricably interconnected in mammals. To circumvent this limitation, we use Drosophila as a primary experimental system, which provides unparalleled sophistication in manipulating Wnt (Wingless in Drosophila) activity in vivo. More importantly, the unique temporal separation of adipogenesis, lipogenesis, lipolysis, and fatty acid ?-oxidation during the Drosophila life cycle allows us to precisely monitor and manipulate these fundamental processes. Our genetic analyses of Axin and Alpha-catenin, two components of the Wnt signaling pathway, have revealed that Wnt signaling regulates lipid homeostasis during the late larval stage, separately from adipogenesis completed during embryogenesis. We have confirmed that the phenotypes of Axin mutants are caused by a gain of the canonical Wnt activity, elevated expression of Beta-catenin target genes, and altered expression of genes encoding enzymes involved in lipid catabolism. By screening a library of diverse FDA-approved drugs, we discovered that both the defective lipid homeostasis and the hyperactive Wnt signaling are potently suppressed by peptide boronic acids, a class of proteasome inhibitors. The suppressive effects of these inhibitors are dependent on Alpha-catenin. Despite the important role of Alpha-catenin in Wnt signaling, the precise mechanisms that normally regulate the stability of Alpha-catenin remain unclear. Thus the objective of this proposal is to determine how Alpha-catenin stability in particular, and Wnt signaling in general, regulates lipid catabolism. We will identify the molecular and cellular mechanisms that control the stability of Alpha-catenin in Drosophila by analyzing fat deposition and lipid accumulation. Our investigations will define the molecular mechanism(s) that control the stability of Alpha-catenin and reveal how Wnt signaling regulates lipid mobilization and lipid catabolism, thereby advancing our understanding of the tumor suppressive effects of Alpha-catenin and how Wnt signaling regulates lipid homeostasis.
The MENTORS (Model Education Networks to Optimize Rural Science) Project
Molecular And Cellular Medicine; TAMHSC; https://hdl.handle.net/20.500.14641/211; DHHS-NIH-National Institute of General Medical Science
DESCRIPTION (provided by applicant): The goal of the MENTORS (Model Education Networks to Optimize Rural Science) Project is to actualize an exportable model of development and dissemination of culturally relevant and innovative programs that stimulate interest in and enhance preparation for careers in science, technology, engineering and medicine. Integrated components that will provide mentoring and career preparation include: summer research internships, science exploration camp, Field Experiences (FEs), and innovative classroom activities for students, and a rigorous professional development (PD) program for K-12 teachers. Our project will build strong partnerships between biomedical, engineering, public health and education researchers at Texas A&M University and K-12 teachers, school/district administrators, students and parents. MENTORS focuses on school districts that are rural and have a high proportion of students who are underserved and/or underrepresented in science and medicine. Such districts often lack programs to optimally prepare students for STEM careers. The multi-disciplinary Project team, which includes experts in biomedical, health disparities and biomedical engineering research as well as K-12 educational theory, curriculum development and evaluation, will work with classroom teachers to develop innovative, career based, educational modules focused on 21st century skills. The Specific Aims of the MENTORS Project are: 1. To provide authentic research and field experiences (FEs) for elementary, middle and high school students, designed to enhance STEM education and stimulate interest in and pursuit of a broad variety of science, technology, engineering and health-related careers. 2. To engage a network of K-12 teachers and biomedical, public health, biomedical engineering and educational researchers to develop learner-centered, career focused educational modules that will attract and motivate students, particularly those who are traditionally underrepresented in science, medicine and technology. 3. To provide rigorous professional development for K-12 teachers in TX, through an established and impactful Summer Institute (SI). All activities and products will be extensively evaluated using a mixed methods approach, with a variety of instruments designed to collect and analyze the most pertinent data. These will assess the effectiveness of the model as a whole, and of the components, in achieving the objectives of enhancing student interest and developing the skills needed for educational and career success.
The MENTORS (Model Education Networks to Optimize Rural Science) Project
Molecular And Cellular Medicine; TAMHSC; https://hdl.handle.net/20.500.14641/211; DHHS-NIH-National Institute of General Medical Science
DESCRIPTION (provided by applicant): The goal of the MENTORS (Model Education Networks to Optimize Rural Science) Project is to actualize an exportable model of development and dissemination of culturally relevant and innovative programs that stimulate interest in and enhance preparation for careers in science, technology, engineering and medicine. Integrated components that will provide mentoring and career preparation include: summer research internships, science exploration camp, Field Experiences (FEs), and innovative classroom activities for students, and a rigorous professional development (PD) program for K-12 teachers. Our project will build strong partnerships between biomedical, engineering, public health and education researchers at Texas A&M University and K-12 teachers, school/district administrators, students and parents. MENTORS focuses on school districts that are rural and have a high proportion of students who are underserved and/or underrepresented in science and medicine. Such districts often lack programs to optimally prepare students for STEM careers. The multi-disciplinary Project team, which includes experts in biomedical, health disparities and biomedical engineering research as well as K-12 educational theory, curriculum development and evaluation, will work with classroom teachers to develop innovative, career based, educational modules focused on 21st century skills. The Specific Aims of the MENTORS Project are: 1. To provide authentic research and field experiences (FEs) for elementary, middle and high school students, designed to enhance STEM education and stimulate interest in and pursuit of a broad variety of science, technology, engineering and health-related careers. 2. To engage a network of K-12 teachers and biomedical, public health, biomedical engineering and educational researchers to develop learner-centered, career focused educational modules that will attract and motivate students, particularly those who are traditionally underrepresented in science, medicine and technology. 3. To provide rigorous professional development for K-12 teachers in TX, through an established and impactful Summer Institute (SI). All activities and products will be extensively evaluated using a mixed methods approach, with a variety of instruments designed to collect and analyze the most pertinent data. These will assess the effectiveness of the model as a whole, and of the components, in achieving the objectives of enhancing student interest and developing the skills needed for educational and career success.
Therapeutic Effects of MSC-Derived Extracellular Vesicles on SjÃ¶grens Syndrome
Molecular And Cellular Medicine; TAMHSC; https://hdl.handle.net/20.500.14641/319; DHHS-NIH-National Institute of Dental and Craniofacial Research
Abstract: Sjögren's syndrome is a chronic inflammatory autoimmune disease affecting mainly salivary glands and lacrimal glands with an incidence of about 1% in the general population and up to 3% in people above the age of 50, with women accounting for more than 90% of diagnosed cases. Hypofunction of salivary glands or dry mouth is a major symptom of Sjögren's syndrome and severely impairs the quality of life of patients. No effective treatment of Sjögren's syndrome is available now. Mesenchymal stem/stromal cells (MSCs) are conventionally isolated from tissues such as bone marrow, are capable of inhibiting immune responses and inflammation, and have shown therapeutic efficacies in Sjögren's syndrome. However, there are many limitations of using tissue-derived MSCs directly for therapies including their limited expandability, considerable donor variations and variations caused by different expansion methods, and the lack of standard assays for testing therapeutic efficacy of the To overcome these limitations, we have derived MSCs efficiently from human induced pluripotent stem cells (iPSCs) with almost unlimited expandability using a modified protocol that can be easily scaled up to produce very large amounts of standardized MSCs. Our iPSC-derived MSCs showed anti-inflammatory and immunosuppressive effects comparable to tissue-derived MSCs. Nevertheless, there are still many difficulties in clinical application of MSCs such as the need for frozen storage and shipping and to thaw frozen cells, the high cost, and the dynamic changes of live cells. Extracellular vesicles (EVs) are nano- sized particles released from cells spontaneously. EVs carry bioactive molecules similar to their originating cells, and are much safer and more feasible for clinical application compared to their originating cells. MSC EVs showed immunosuppressive activities similar to MSCs in vitro. Our preliminary study indicated that systemically injected iPSC-MSCs or their EVs inhibited the inflammation of salivary gland in a mouse model of Sjögren's syndrome. This proposal aims to determine the potentials and mechanisms of iPSC-MSC EVs in preventing the progression of Sjögren's syndrome using a more faithful mouse model. First, we will determine effects of systemic infusion of iPSC-MSC EVs on preventing progression of Sjögren's syndrome and related changes in immune cells and salivary gland cells. Second, we will determine components of iPSC-MSC EVs essential for preventing progression of Sjögren's syndrome. The success of the proposed research will provide the proof-of-concept for a novel and clinically feasible therapy for Sjögren's syndrome, and will also encourage further research on MSC EVs for improving treatment of a broad range of disorders that have low therapeutic alternatives but share similar inflammatory and/or immune backgrounds including rheumatoid arthritis, inflammatory bowel diseases, graft versus host disease, and rejections of transplants.
Tolerance Mechanisms Through Innovative Population Analysis
Molecular And Cellular Medicine; TAMHSC; https://hdl.handle.net/20.500.14641/681; DOD-Advanced Research Projects Agency
Salmonella typhimurium Overall, 32 CC lines were screened in the initial 1-week experiments. Of those 32 lines, 14 were susceptible and succumbed to infection before 7 days and 18 had >50% survival and were infected over a 3-week period. 6 of these lines displayed delayed susceptibility (succumbed to infection after 1 week but before 3 weeks), 4 were characterized as tolerant (CC002, CC017, CC043, and CC072), and 5 had resistant phenotypes (CC015, CC024, CC051, CC057, CC058). We were unable to determine the phenotype on the remaining 3 lines due to insufficient number of mice. Tolerance was defined as surviving to 3 weeks and maintaining at least 104 CFU in spleen and liver. Resistant mice had less than 104 and/or significantly reduced the bacterial load in spleen and liver from 1 week to 3 weeks. (Figures 1-4). Resistant mice also appeared to repair tissue damage by 3 weeks while tolerant did not (Figure 5). Tolerance appears to be heterogenous – there is not necessarily one pathway that determines tolerance across all lines. Some possible theories include preventing tissue damage (CC043) or having much more productive healthy cells in spleen and liver to make up for damaged cells (CC072). CC072 is the most interesting tolerant line because the mice significantly increase their colonization at day 21 and show a trend toward increasing tissue damage while surviving. Each line appears to have a unique CBC and cytokines profile, which speaks to the heterogeneity of response to infection. However, this relationship needs to be probed more. NRAMP was not a great determinant of survival as previously thought: 10/14 susceptible lines from the 1-week experiments had a wild type allele and CC045 had a mutated allele and survived to day 7. Mutated NRAMP has historically led to susceptibility before day 7 while wild type has led to resistance. Mutated NRAMP lines include CC021, CC031, CC032 (heterozygous), CC042, CC045, and CC061. Histologically, there is extraordinarily little tissue damage in the gut (ileum, cecum, colon) and most of the damage is in the systemic organs (liver and spleen). This suggests that the systemic phase of infection is what is determining if mice survive or not. The mice also do not display the typical gastrointestinal symptoms, which further supports this. There is a sex difference found in the response to infections in two of the lines. CC027 females make it to day 7, while none of the males do and CC013 females make it to day 6 while males only make it to day 4. One line (CC013) get a head tilt after infection, which suggests there may be some salmonella in the brain, which is not a usual place for salmonella to travel There is a significant QTL peak when aspiration is put in. The CC017 is the most prone to aspiration, but only when gavaged with salmonella, never when gavaged with PBS or heavy metals. This suggests that certain CC lines are susceptible to pulmonary Salmonella infections and/or may have reflux.
Yr. 2 IHSFC Voucher- A Novel Mouse Model of Immunodeficiency to Evaluate the Impact of Environmental Toxicants on Mitochondrial Dysfunction.
Molecular And Cellular Medicine; TAMU; https://hdl.handle.net/20.500.14641/497; DHHS-NIH-National Institute of Environmental Health Sciences
Overall Project Description and Goals Mitochondrial metabolic reprogramming is critical for the differentiation and function of hematopoietic stem cells and immune cell progenitors, particularly macrophages and T-cells. Indeed, patients with immunodeficiencies (IDs) are increasingly recognized to exhibit perturbation of mitochondrial function. Likewise, patients with primary mitochondrial diseases (PMDs) exhibit aberrant immune and inflammatory responses. In addition to these primary genetic diseases, environmental exposures such as l-methyl-4phenyl-l, 2, 3, 6-tetra-hydropyridine (MPTP), and pesticides such as rotenone and paraquat (PQ2+) target mitochondrial functions, causing oxidative stress, de-regulated innate immune responses and inflammatory disease. However, the molecular mechanisms responsible for orchestrating immuno-metabolic switches in macrophages and T- cells, and the potential synergistic contribution of environmental toxicants to mitochondrial dysfunction in IDs, remain largely unknown. Moreover, the significance of environmental toxicant exposures as potential triggers of immunodeficiency in PMDs has not been systematically studied. While Nuclear Factor ?B (NF-?B) signaling is critical for innate immune responses, a role for NF-?B in regulating metabolic reprogramming or mitochondrial function in innate immune cells has not yet been investigated in the context of IDs and PMDs. Preliminary data demonstrated that NF-?B-inducing kinase (NIK) is required for the characteristic metabolic switching that occurs during myeloid differentiation, macrophage M1 and M2 polarization, acquisition of immune effector function, and normal hematopoietic differentiation. Our overarching hypothesis is that mitochondrial metabolic dysregulation is a key underlying cause of NIK-mediated immunodeficiency. This pilot project seeks to develop a novel, genetically engineered, humanized mouse model of ID caused by NIK loss-of-function. Specifically, we proposed to use CRISPR/Cas9 synthetic gRNAs to generate a C57Bl6 knock-in transgenic mouse with a known NIKCys-306-Val*2 mutation that was recently identified in ID patients and to use these mice to investigate how environmental toxicants that target mitochondrial functions synergize with de-regulated innate immune responses to impact human health and susceptibility to infectious disease.