Biology subjects

Kennedy, L.

Publications and source records attributed to Kennedy, L..

8 recordsLinked to original sources

bioRxiv2024

OPA1 mediates cardiac function and metabolism: in silico and in vivo evidence

OPA1 is an inner mitochondrial membrane protein that mediates diverse signaling processes. OPA1 is important for cardiac function and protects against cardiac insults such as ischemia reperfusion injury. We sought to further assess OPA1 in human and mouse cardiac pathologies, hypothesizing that OPA1 may also function in a protective manner in chronic heart failure. Bioinformatic analyses of histological and transcript data from the GTEx database indicated that OPA1 expression levels vary in the human heart, where elevated OPA1 transcript levels were correlated with fatty acid, branch chain amino acid and contractile gene signatures. To experimentally assess these correlations, mice with a 1.5-fold whole body OPA1 overexpression (OPA1-OE) were subjected to transverse aortic constriction surgery and displayed improved 2D and 4D cardiac functional parameters compared to WT mice. OPA1-OE mice had no induction of fibrotic transcript markers and displayed sustained transcript levels of fatty acid, branch chain amino acid and contractile markers. Maximal oxidative capacity was sustained in both WT and OPA1-OE cardiac myofibers post-TAC. These results further demonstrate the important role of OPA1 in mediating cardiac function and highlight protective signaling pathways.

biochemistry↗

bioRxiv2022

Development of Scaffold-free 3D Cholangiocyte Organoids to Study the Progression of Primary Sclerosing Cholangitis

Organoids are novel in vitro models to study intercellular crosstalk between the different types of cells in the pathophysiology of disease. To better understand the underlying mechanisms driving the progression of primary sclerosing cholangitis (PSC), we developed scaffold-free multi-cellular 3D cholangiocyte organoids (3D-CHO) using primary liver cell lines derived from normal and PSC patients. Human liver samples from healthy donors and late-stage PSC patients were used to isolate primary cholangiocytes (EPCAM+/CK-19+), liver endothelial cells (LECs, CD31+), and hepatic stellate cells (HSCs, CD31-/CD68-/Desmin+/Vitamin A+). 3D-CHOs were formed using cholangiocytes:HSCs:LECs and kept viable for up to 1 month. Isolated primary cell lines and 3D-CHOs were further characterized by immunofluorescence (IF), qRT-PCR, and transmission electron microscopy. Gene expressions for cholangiocytes (SOX9, CFTR, EpCAM, AE, SCT, SCTR), fibrosis (ACTA2, COL1A1, DESMIN, TGF{beta}1), angiogenesis (PECAM, VEGF, CDH5, vWF), and inflammation (IL-6, TNF-) confirmed PSC phenotypes of 3D-CHOs. Since cholangiocytes develop a neuroendocrine phenotype and express neuromodulators, confocal-IF demonstrated that neurokinin-1 receptor (NK-1R, expressed by cholangiocytes and upregulated in PSC), was localized within CK-19+ cholangiocytes. Moreover, 3D-CHOs from PSC patients confirmed PSC phenotypes with upregulated NK-1R, tachykinin precursor 1, and downregulated membrane metalloendopeptidase. Our viable scaffold-free multiple-cell 3D-CHOs showed superiority as an in vitro model in mimicking PSC in vivo phenotypes compared to 2D cell culture, which can be used in PSC disease-related research.

bioengineering↗

bioRxiv2022

Establishing Novel Doxorubicin-Loaded Polysaccharide Hydrogel for Controlled Drug Delivery for Treatment of Pediatric Brain Tumors

According to the National Cancer Institute, of the more than 10 million cancer survivors alive in the United States at least 270,000 were originally diagnosed under the age of 21. While the 5-year survival rates for most childhood cancers appear very promising, the long-term survival rates are still very dismal. There is significant long-term morbidity and mortality associated with treatment of childhood cancer, and the risk of these effects continues to increase years after completion of therapy. Among childhood cancer survivors the cumulative incidence of a chronic health condition is 73.4% 30 years after the original cancer diagnosis, with a cumulative incidence of 42.4% for severe, disabling, life-threatening, or death due to a chronic condition caused by the chemotherapy used to treat the initial malignancy. Brain tumors are the most prevalent solid tumor diagnosed in children, and account for 20 percent of all childhood cancer deaths. The efficacy of all chemotherapy agents can be limited by their toxicity, their instability, and their ability to be formulated into practical drug products for use in the clinical setting To address this gap, our group has developed a novel carbohydrate-based hydrogel, Amygel, that is capable of being loaded with drugs and injected directly into the site of disease. Local drug delivery using Amygel has potential to improve childhood cancer treatment outcomes and prevent the devastating effects of systemic chemotherapy exposure. Development of Amygel for clinical use has three focus areas including: increasing drug concentration at the target site; improving chemotherapy penetration through tumor tissue, and; establishing chemotherapy dosage forms for pediatric use. For this study, we formulated Amygel with dimethyl sulfoxide and integrated the chemotherapy doxorubicin (DOX). High-performance liquid chromatography (HPLC) was used to confirm the quality of DOX after hydrogel synthesis, rheology and syringability tests to characterize the mechanical properties, and performed an in vitro cytotoxicity test against the pediatric medulloblastoma cell line DAOY. On HPLC, we found that after integrating DOX into the Amygel matrix the drug maintained a strong band on the chromatograph at the same point with the same intensity as the control free drug, indicating there were no changes in the structural properties of DOX. The mechanical tests showed that there was a proportionate increase in the storage modulus of the drug-loaded hydrogels as the concentration of amylopectin increased from 3 wt% to 20 wt%, but even at 20 wt% the hydrogel remained below the medical standard for injectables that the burst force should not exceed 40 N and the sliding force below 20 N. Correlating with the rheology findings, as the concentration of amylopectin increased, and therefore the strength of the hydrogel, there was an increase in the magnitude of force required for gel injection. These mechanical studies additionally provide evidence that the mechanical stability of the gel is not dampened by the incorporation of DOX. Drug release and cytotoxicity studies demonstrated a sufficient release of DOX from the hydrogels, and that the DOX released was able to achieve significant (p<0.01) cell death.

bioengineering↗

bioRxiv2022

Development of an In Vitro Test for the Optimization of Drug Diffusion in Pediatric Solid Tumors

There is significant long-term morbidity and mortality associated with the treatment of childhood cancer, and the risk of these effects continues to increase years after completion of therapy. Among childhood cancer survivors the cumulative incidence of a chronic health condition is 99% within 50 years of the original cancer diagnosis. There is a high risk for severe, disabling, or life-threatening chronic condition caused by the chemotherapy used to treat the initial malignancy. Current standards for determining chemotherapy dosage to treat solid tumor malignancies of pediatric patients is based on several factors, including the patients surface area, age, weight, and height. To reduce the long-term effects of chemotherapy in pediatric patients our group is focused on developing novel local drug delivery systems to treat solid tumors and minimize systemic effects. The aim of the current study is to develop an in vitro method to quantify drug diffusion through tumor tissue that will allow us to optimize the dose required to treat solid tumor malignancies in vivo. We hope by modeling the significant parameters that influence drug penetration of chemotherapy drugs, we can facilitate the development of innovative drug delivery methods and more effective administration of anticancer agents to better treat pediatric malignancies and improve both short-term and long-term outcomes for childhood cancer.

bioengineering↗

bioRxiv2022

Unravelling specific diet and gut microbial contributions to inflammatory bowel disease

Inflammatory bowel diseases (IBDs) are chronic conditions characterized by periods of spontaneous intestinal inflammation and are increasing in industrialized populations. Combined with host genetics, diet and gut bacteria are thought to contribute prominently to IBDs, but mechanisms are still emerging. In mice lacking the IBD-associated cytokine, Interleukin-10, we show that low dietary fiber promotes bacterial erosion of colonic mucus, leading to lethal colitis. A fiber-free exclusive enteral nutrition diet also induces mucus erosion but inhibits inflammation by simultaneously increasing an anti-inflammatory bacterial metabolite, isobutyrate. Diet-induced inflammation is driven by Th1 immune responses, which increase in the presence of mucin-degrading bacteria and are preceded by expansion of natural killer cells and altered immunoglobulin-A coating of some bacteria. Inflammation occurs first in intestinal regions with thinner mucus. Our work underscores the importance of focusing on microbial functions--not taxa--contributing to IBDs and some diet-mediated functions block those that promote disease.

microbiology↗

bioRxiv2020

ALKBH7 mediates necrosis via rewiring of glyoxal metabolism

Alkb homolog 7 (ALKBH7) is a mitochondrial -ketoglutarate dioxygenase required for necrotic cell death in response to DNA alkylating agents, but its physiologic role within tissues remains unclear. Herein, we show that ALKBH7 plays a key role in the regulation of dialdehyde metabolism, which impacts cardiac survival in response to ischemia-reperfusion (IR) injury. Using a multi-omics approach, we do not find evidence that ALKBH7 functions as a prolyl-hydroxylase. However, we do find that mice lacking ALKBH7 exhibit a significant increase in glyoxalase I (GLO-1), a dialdehyde detoxifying enzyme. Consistent with increased dialdehyde production, metabolomics analysis reveals rewiring of metabolic pathways related to the toxic glycolytic by-product methylglyoxal (MGO), as well as accelerated glycolysis and elevated levels of MGO protein adducts, in mice lacking ALKBH7. Consistent with roles for both necrosis and glycative stress in cardiac IR injury, hearts from male but not female Alkbh7-/- mice are protected against IR, although somewhat unexpectedly this protection does not appear to involve modulation of the mitochondrial permeability transition pore. Highlighting the importance of MGO metabolism for the observed protection, removal of glucose as a metabolic substrate or pharmacologic inhibition of GLO-1 both abrogate cardioprotection in ALKBH7 deficient mice. Integrating these observations, we propose that ALKBH7 plays a role in the regulation of glyoxal metabolism, and that protection against necrosis and IR injury bought on by ALKBH7 deficiency originates from hormetic signaling in response to elevated MGO stress.

cell biology↗

bioRxiv2018

A model of repetitive mild brain injury without symptoms – risk for Parkinson’s disease with aging?

Objectives: To test the hypothesis that repetitive mild traumatic brain injury in early life may be a potential risk factor for Parkinson's disease. Methods: A closed-head momentum exchange model was used to produce one or three mild concussions in young adult male rats as compare to non-injured, age and weight-matched controls. Six-seven weeks post-injury, rats were studied for deficits in cognitive and motor function Changes in brain anatomy and function were evaluated through analysis of resting state functional connectivity, diffusion weighted imaging with quantitative anisotropy and immunohistochemistry for neuroinflammation. Results: Head injuries occurred without skull fracture or signs of intracranial bleeding or contusion. There were no significant differences in cognitive or motors behaviors between experimental groups. Rats concussed three times showed altered diffusivity in white matter tracts, basal ganglia, central amygdala, brainstem, and cerebellum. With a single concussion, the affected areas were limited to the caudate/putamen and central amygdala. Disruption of functional connectivity was most pronounced with three concussions as the midbrain dopamine system, hippocampus and brainstem/cerebellum showed hypoconnectivity. The suprachiasmatic nucleus was isolated from all functional connections. Interestingly, rats exposed to one concussion showed enhanced functional connectivity (or hyperconnectivity) across brain sites, particularly between the olfactory system and the cerebellum. Immunostaining for microglia activation showed inflammation in striatum and substantia nigra with three concussions but not with one. Interpretation: Neuroradiological and immunohistochemical evidence of altered brain structure and function, particularly in striatal and midbrain dopaminergic areas, persists long after mild repetitive head injury. These changes may be long lasting and serve as early biomarkers of neurodegeneration and risk for Parkinson's disease with aging.

neuroscience↗

bioRxiv2017

Recapitulating Early Development Of Mouse Musculoskeletal Precursors Of The Paraxial Mesoderm In Vitro

In vertebrates, body skeletal muscles and axial skeleton derive from the paraxial mesoderm which flanks the neural tube and notochord. The paraxial mesoderm forms in the posterior region of the embryo as presomitic mesoderm (PSM), which generates the embryonic segments called somites. Here, we characterized gene signatures identified using microarray series from the mouse PSM and compared the PSM transcriptome dynamics to that of the developing neural tube. In contrast to the PSM where an abrupt transcriptome reorganisation occurs at the level of the determination front, we show that transcriptome changes are progressive during parallel stages of neural tube differentiation. We show that these early differentiation stages of the paraxial mesoderm can be efficiently recapitulated in monolayer culture in vitro using murine Embryonic Stem (ES) cells. We describe a serum-containing protocol which parallels in vivo tissue maturation allowing differentiation of ES cells towards a paraxial mesoderm fate. We show that R-spondin treatment or Wnt activation alone can induce posterior PSM markers in both mouse and human ES/iPS cells but acquisition of a committed posterior PSM fate requires BMP inhibition to prevent induced cells to drift to a lateral plate mesoderm identity. We show that posterior PSM-like cells induced from mouse ES cells can be further differentiated in vitro to acquire an anterior PSM Pax3-positive identity. When grafted into injured adult muscle, these induced PSM-like precursors generated large numbers of immature muscle fibers. We further show that exposing ES-derived PSM-like cells to a brief FGF inhibition step followed by culture in horse serum-containing medium allows efficient recapitulation of the myogenic program. Differentiating ES cells first produce mononucleated embryonic myocytes and subsequently multinucleated myotubes, as well as Pax7-positive cells. The protocol described here results in improved differentiation and maturation of mouse muscle fibers differentiated in vitro over serum-free protocols. It provides an efficient system for the study of myogenic processes otherwise difficult to study in vivo such as fusion or satellite cell differentiation.

developmental biology↗