- Browse by Author
Browsing by Author "Anatomy, Cell Biology and Physiology, School of Medicine"
Now showing 1 - 10 of 279
Results Per Page
Sort Options
Item 3058 – Sars-Cov-2 Binding in Hematopoietic Stem and Progenitor Cells Under Low Oxygen Conditions(Elsevier, 2021) Dausinas, Paige; Hartman, Melissa; Allman, Lauren; O'Leary, Heather; Anatomy, Cell Biology and Physiology, School of MedicineThe SARS-CoV-2 pandemic highlighted a need for in-depth understanding of interaction/identification of receptors and mechanisms/functional consequences of viral binding/entry. SARS-CoV-2 spike protein (SBP) facilitates viral entry via ACE2 and/or NRP1 binding, with DPP4 as a potential co-receptor. These binding partners are expressed on various cell types including hematopoietic stem and progenitor (HSC/HSPC) cells [1-3]. HSC/HSPCs generate blood cells and reside in the low oxygen (lowO2, 1-4%) bone marrow niches that provide critical signals for maintenance, self-renewal, and differentiation. To investigate aspects of SARS-CoV-2 interactions with HSC/HSPC such as endogenous receptor expression, SPB binding and subsequent functional alterations in native low O2, we performed transcriptional and phenotypic/functional analysis. In lowO2, we identified increased surface expression of ACE2, DPP4 and NRP1, and enhanced binding of SBP to HSC/HSPC populations which amplified proliferation of SBP bound in lowO2. ACE2 and DPP4 surface expression were ∼2-fold higher in HSPCs (p=0.017, p=0.001) and HSCs (p=0.010, p=0.03), and NRP1 was ∼1.5-fold (p=0.002) higher in HSPCs in lowO2 compared to air. Interestingly, in lowO2, overall SBP binding was enhanced in HSPC (2.2-fold, p<.001) and HSC (2.6-fold, p=.018). Although not all cells expressing ACE2/DPP4/NRP1 bind SBP (∼50%), all cells exhibiting SBP binding in HSC/HSPC populations are triple positive for ACE2, NRP1, and DPP4. Additionally, we observed greater than a 2-fold increase in proliferation of SBP bound vs unbound cells in replating assays in lowO2 (p<.001). These data impart compelling evidence that SBP binding/functional outcomes are unique in low O2, providing a foundation that may have potential clinical implications for COVID19 treatment and expanding our baseline understanding of SARS-CoV-2 viral binding implications.Item 4539 Building a Translational Science pipeline: The Indiana CTSI STEM K-12 Program(Cambridge University Press, 2020-07-29) Sanders, Elmer; Barth, Vanessa; Cruz, Leigh-Ann; Sherrer, Ilesha; Olson, Jacob; Speidell, Emily; Solis, Elvia; Harrison, Sharon; Hinshaw, Amy; McAteer, James A.; Anatomy, Cell Biology and Physiology, School of MedicineOBJECTIVES/GOALS: Develop strong network of science teachers interested in promoting scientific research to their students. Place students in an immersive summer research internship that, when possible, matches their career interests. Expose students to the numerous career paths within the STEM field. METHODS/STUDY POPULATION: The program recruits socio-economically disadvantaged students and provides them a stipend, and also accepts students who can participate unpaid. Local school teachers are engaged in a summer fellowship to learn biotechnologies and research. In Spring these teachers help recruit students and during the subsequent Fall help students with college and scholarship applications. Students are placed in a variety of laboratories within the Schools of Medicine, Science, Dentistry, Public Health, Informatics, Health and Human Sciences, Engineering and Technology, especially in biomedical engineering. Students are also placed in industry laboratories such as Eli Lilly and the Indiana Bioscience Research Institute. Long-term program follow-up is done through post-internship surveys to assess impact on graduate and professional school admission. RESULTS/ANTICIPATED RESULTS: Since the Indiana CTSI was established in 2008, 872 students have participated in the summer internship. 71% of past interns are underrepresented minorities in science or classified as disadvantaged by NIH criteria. 17% of students interned during grade 10, 72% during grade 11, and 11% during grade 12. 21% of students engage in the program for more than one year. 100% of past interns are currently enrolled in or have graduated college. Over 60% of those with a bachelors degree proceed to graduate and professional schools and over 80% stay in STEM related fields. These rates are equal for interns from underrepresented minorities or those classified as disadvantaged by NIH criteria. DISCUSSION/SIGNIFICANCE OF IMPACT: Students engaged in the Indiana CTSI STEM program are progressing through the translational science pipeline based on their graduating from college and remaining in the STEM field.Item A Barth Syndrome Patient-Derived D75H Point Mutation in TAFAZZIN Drives Progressive Cardiomyopathy in Mice(MDPI, 2024-07-27) Snider, Paige L.; Sierra Potchanant, Elizabeth A.; Sun, Zejin; Edwards, Donna M.; Chan, Ka-Kui; Matias, Catalina; Awata, Junya; Sheth, Aditya; Pride, P. Melanie; Payne, R. Mark; Rubart, Michael; Brault, Jeffrey J.; Chin, Michael T.; Nalepa, Grzegorz; Conway, Simon J.; Anatomy, Cell Biology and Physiology, School of MedicineCardiomyopathy is the predominant defect in Barth syndrome (BTHS) and is caused by a mutation of the X-linked Tafazzin (TAZ) gene, which encodes an enzyme responsible for remodeling mitochondrial cardiolipin. Despite the known importance of mitochondrial dysfunction in BTHS, how specific TAZ mutations cause diverse BTHS heart phenotypes remains poorly understood. We generated a patient-tailored CRISPR/Cas9 knock-in mouse allele (TazPM) that phenocopies BTHS clinical traits. As TazPM males express a stable mutant protein, we assessed cardiac metabolic dysfunction and mitochondrial changes and identified temporally altered cardioprotective signaling effectors. Specifically, juvenile TazPM males exhibit mild left ventricular dilation in systole but have unaltered fatty acid/amino acid metabolism and normal adenosine triphosphate (ATP). This occurs in concert with a hyperactive p53 pathway, elevation of cardioprotective antioxidant pathways, and induced autophagy-mediated early senescence in juvenile TazPM hearts. However, adult TazPM males exhibit chronic heart failure with reduced growth and ejection fraction, cardiac fibrosis, reduced ATP, and suppressed fatty acid/amino acid metabolism. This biphasic changeover from a mild-to-severe heart phenotype coincides with p53 suppression, downregulation of cardioprotective antioxidant pathways, and the onset of terminal senescence in adult TazPM hearts. Herein, we report a BTHS genotype/phenotype correlation and reveal that absent Taz acyltransferase function is sufficient to drive progressive cardiomyopathy.Item A Bisphosphonate With a Low Hydroxyapatite Binding Affinity Prevents Bone Loss in Mice After Ovariectomy and Reverses Rapidly With Treatment Cessation(Wiley, 2021-03-03) Coffman, Abigail A.; Basta-Pljakic, Jelena; Guerra, Rosa M.; Ebetino, Frank H.; Lundy, Mark W.; Majeska, Robert J.; Schaffler, Mitchell B.; Anatomy, Cell Biology and Physiology, School of MedicineBisphosphonates (BPs) are a mainstay of osteoporosis treatment; however, concerns about bone health based on oversuppression of remodeling remain. Long‐term bone remodeling suppression adversely affects bone material properties with microdamage accumulation and reduced fracture toughness in animals and increases in matrix mineralization and atypical femur fractures in patients. Although a “drug holiday” from BPs to restore remodeling and improve bone quality seems reasonable, clinical BPs have long functional half‐lives because of their high hydroxyapatite (HAP) binding affinities. This places a practical limit on the reversibility and effectiveness of a drug holiday. BPs with low HAP affinity and strong osteoclast inhibition potentially offer an alternative approach; their antiresorptive effect should reverse rapidly when dosing is discontinued. This study tested this concept using NE‐58025, a BP with low HAP affinity and moderate osteoclast inhibition potential. Young adult female C57Bl/6 mice were ovariectomized (OVX) and treated with NE‐58025, risedronate, or PBS vehicle for 3 months to test effectiveness in preventing long‐term bone loss. Bone microarchitecture, histomorphometry, and whole‐bone mechanical properties were assessed. To test reversibility, OVX mice were similarly treated for 3 months, treatment was stopped, and bone was assessed up to 3 months post‐treatment. NE‐58025 and RIS inhibited long‐term OVX‐induced bone loss, but NE‐58025 antiresorptive effects were more pronounced. Withdrawing NE‐58025 treatment led to the rapid onset of trabecular resorption with a 200% increase in osteoclast surface and bone loss within 1 month. Cessation of risedronate treatment did not lead to increases in resorption indices or bone loss. These results show that NE‐58025 prevents OVX‐induced bone loss, and its effects reverse quickly following cessation treatment in vivo. Low‐HAP affinity BPs may have use as reversible, antiresorptive agents with a rapid on/off profile, which may be useful for maintaining bone health with long‐term BP treatment.Item A crystallin mutant cataract with mineral deposits(Elsevier, 2023) Minogue, Peter J.; Gao, Junyuan; Mathias, Richard T.; Williams, James C., Jr.; Bledsoe, Sharon B.; Sommer, Andre J.; Beyer, Eric C.; Berthoud, Viviana M.; Anatomy, Cell Biology and Physiology, School of MedicineConnexin mutant mice develop cataracts containing calcium precipitates. To test whether pathologic mineralization is a general mechanism contributing to the disease, we characterized the lenses from a nonconnexin mutant mouse cataract model. By cosegregation of the phenotype with a satellite marker and genomic sequencing, we identified the mutant as a 5-bp duplication in the γC-crystallin gene (Crygcdup). Homozygous mice developed severe cataracts early, and heterozygous animals developed small cataracts later in life. Immunoblotting studies showed that the mutant lenses contained decreased levels of crystallins, connexin46, and connexin50 but increased levels of resident proteins of the nucleus, endoplasmic reticulum, and mitochondria. The reductions in fiber cell connexins were associated with a scarcity of gap junction punctae as detected by immunofluorescence and significant reductions in gap junction-mediated coupling between fiber cells in Crygcdup lenses. Particles that stained with the calcium deposit dye, Alizarin red, were abundant in the insoluble fraction from homozygous lenses but nearly absent in wild-type and heterozygous lens preparations. Whole-mount homozygous lenses were stained with Alizarin red in the cataract region. Mineralized material with a regional distribution similar to the cataract was detected in homozygous lenses (but not wild-type lenses) by micro-computed tomography. Attenuated total internal reflection Fourier-transform infrared microspectroscopy identified the mineral as apatite. These results are consistent with previous findings that loss of lens fiber cell gap junctional coupling leads to the formation of calcium precipitates. They also support the hypothesis that pathologic mineralization contributes to the formation of cataracts of different etiologies.Item A four-grating interferometer for x-ray multi-contrast imaging(Wiley, 2024) Miao, Houxun; Williams, James C., Jr.; Josell, Daniel; Anatomy, Cell Biology and Physiology, School of MedicineBackground: X-ray multi-contrast imaging with gratings provides a practical method to detect differential phase and dark-field contrast images in addition to the x-ray absorption image traditionally obtained in laboratory or hospital environments. Systems have been developed for preclinical applications in areas including breast imaging, lung imaging, rheumatoid arthritis hand imaging and kidney stone imaging. Purpose: Prevailing x-ray interferometers for multi-contrast imaging include Talbot-Lau interferometers and universal moiré effect-based phase-grating interferometers. Talbot-Lau interferometers suffer from conflict between high interferometer sensitivity and large field of view (FOV) of the object being imaged. A small period analyzer grating is necessary to simultaneously achieve high sensitivity and large FOV within a compact imaging system but is technically challenging to produce for high x-ray energies. Phase-grating interferometers suffer from an intrinsic fringe period ranging from a few micrometers to several hundred micrometers that can hardly be resolved by large area flat panel x-ray detectors. The purpose of this work is to introduce a four-grating x-ray interferometer that simultaneously allows high sensitivity and large FOV, without the need for a small period analyzer grating. Methods: The four-grating interferometer consists of a source grating placed downstream of and close to the x-ray source, a pair of phase gratings separated by a fixed distance placed downstream of the source grating, and an analyzer grating placed upstream of and close to the x-ray detector. The object to be imaged is placed upstream of and close to the phase-grating pair. The distance between the source grating and the phase-grating pair is designed to be far larger than that between the phase-grating pair and the analyzer grating to promote simultaneously high sensitivity and large FOV. The method was evaluated by constructing a four-grating interferometer with an 8 µm period source grating, a pair of phase gratings of 2.4 µm period, and an 8 µm period analyzer grating. Results: The fringe visibility of the four-grating interferometer was measured to be ≈24% at 40 kV and ≈18% at 50 kV x-ray tube operating voltage. A quartz bead of 6 mm diameter was imaged to compare the theoretical and experimental phase contrast signal with good agreement. Kidney stone specimens were imaged to demonstrate the potential of such a system for classification of kidney stones. Conclusions: The proposed four-grating interferometer geometry enables a compact x-ray multi-contrast imaging system with simultaneously high sensitivity and large FOV. Relaxation of the requirement for a small period analyzer grating makes it particularly suitable for high x-ray energy applications such as abdomen and chest imaging.Item A moderate spinal contusion injury in rats alters bone turnover both below and above the level of injury with sex-based differences apparent in long-term recovery(Elsevier, 2024-04-10) Metzger, Corinne E.; Moore, Robert C.; Pirkle, Alexander S.; Tak, Landon Y.; Rau, Josephina; Bryan, Jessica A.; Stefanov, Alexander; Allen, Matthew R.; Hook, Michelle A.; Anatomy, Cell Biology and Physiology, School of MedicineSpinal cord injury (SCI) leads to significant sublesional bone loss and high fracture rates. While loss of mechanical loading plays a significant role in SCI-induced bone loss, animal studies have demonstrated mechanical loading alone does not fully account for loss of bone following SCI. Indeed, we have shown that bone loss occurs below the level of an incomplete moderate contusion SCI, despite the resumption of weight-bearing and stepping. As systemic factors could also impact bone after SCI, bone alterations may also be present in bone sites above the level of injury. To examine this, we assessed bone microarchitecture and bone turnover in the supralesional humerus in male and female rats at two different ages following a moderate contusion injury in both sub-chronic (30 days) and chronic (180 days) time points after injury. At the 30-day timepoint, we found that both young and adult male SCI rats had decrements in trabecular bone volume at the supralesional proximal humerus (PH), while female SCI rats were not different from age-matched shams. At the 180-day timepoint, there were no statistical differences between SCI and sham groups, irrespective of age or sex, at the supralesional proximal humerus. At the 30-day timepoint, all SCI rats had lower BFR and higher osteoclast-covered trabecular surfaces in the proximal humerus compared to age-matched sham groups generally matching the pattern of SCI-induced changes in bone turnover seen in the sublesional proximal tibia. However, at the 180-day timepoint, only male SCI rats had lower BFR at the supralesional proximal humerus while female SCI rats had higher or no different BFR than their age-matched counterparts. Overall, this preclinical study demonstrates that a moderate contusion SCI leads to alterations in bone turnover above the level of injury within 30-days of injury; however male SCI rats maintained lower BFR in the supralesional humerus into long-term recovery. These data further highlight that bone loss after SCI is not driven solely by disuse. Additionally, these data allude to potential systemic factors exerting influence on bone following SCI and highlight the need to consider treatments for SCI-induced bone loss that impact both sublesional and systemic factors.Item A novel decellularized matrix of Wnt signaling-activated osteocytes accelerates the repair of critical-sized parietal bone defects with osteoclastogenesis, angiogenesis, and neurogenesis(Elsevier, 2022-08-16) Wang, Xiaofang; Ma, Yufei; Chen, Jie; Liu, Yujiao; Liu, Guangliang; Wang, Pengtao; Wang, Bo; Taketo, Makoto M.; Bellido, Teresita; Tu, Xiaolin; Anatomy, Cell Biology and Physiology, School of MedicineCell source is the key to decellularized matrix (DM) strategy. This study compared 3 cell types, osteocytes with/without dominant active Wnt/β-catenin signaling (daCO and WTO) and bone marrow stromal cells (BMSCs) for their DMs in bone repair. Decellularization removes all organelles and >95% DNA, and retained >74% collagen and >71% GAG, maintains the integrity of cell basement membrane with dense boundaries showing oval and honeycomb structure in osteocytic DM and smooth but irregular shape in the BMSC-DM. DM produced higher cell survival rate (90%) and higher proliferative activity. In vitro, daCO-DM induces more and longer stress fibers in BMSCs, conducive to cell adhesion, spreading, and osteogenic differentiation. 8-wk after implantation of the critical-sized parietal bone defect model, daCO-DM formed tight structures, composed of a large number of densely-arranged type-I collagen under polarized light microscope, which is similar to and integrated with host bone. BV/TV (>54%) was 1.5, 2.9, and 3.5 times of WTO-DM, BMSC-DM, and none-DM groups, and N.Ob/T.Ar (3.2 × 102/mm2) was 1.7, 2.9, and 3.3 times. At 4-wk, daCO-DM induced osteoclastogenesis, 2.3 times higher than WTO-DM; but BMSC-DM or none-DM didn't. daCO-DM increased the expression of RANKL and MCSF, Vegfa and Angpt1, and Ngf in BMSCs, which contributes to osteoclastogenesis, angiogenesis, and neurogenesis, respectively. daCO-DM promoted H-type vessel formation and nerve markers β3-tubulin and NeuN expression. Conclusion: daCO-DM produces metabolic and neurovascularized organoid bone to accelerate the repair of bone defects. These features are expected to achieve the effect of autologous bone transplantation, suitable for transformation application.Item A novel murine model of combined insulin-dependent diabetes and chronic kidney disease has greater skeletal detriments than either disease individually(Elsevier, 2022-12) Damrath, John G.; Metzger, Corinne E.; Allen, Matthew R.; Wallace , Joseph M.; Anatomy, Cell Biology and Physiology, School of MedicineDiabetes and chronic kidney disease (CKD) consistently rank among the top ten conditions in prevalence and mortality in the United States. Insulin-dependent diabetes (IDD) and CKD each increase the risk of skeletal fractures and fracture-related mortality. However, it remains unknown whether these conditions have interactive end-organ effects on the skeleton. We hypothesized that combining IDD and CKD in mice would cause structural and mechanical bone alterations that are more deleterious compared to the single disease states. Female C57BL6/J mice were divided into four groups: 1) N=12 Control (CTRL), 2) N=10 Streptozotocin-induced IDD (STZ), 3) N=10 Adenine diet-induced CKD (AD), and 4) N=9 Combination (STZ+AD). STZ administration resulted in significantly higher blood glucose, HbA1c (p<0.0001), and glucose intolerance (p<0.0001). AD resulted in higher blood urea nitrogen (p=0.0002) while AD, but not STZ+AD mice, had high serum parathyroid hormone (p<0.0001) and phosphorus (p=0.0005). STZ lowered bone turnover (p=0.001). Trabecular bone volume was lowered by STZ (p<0.0001) and increased by AD (p=0.003). Tissue mineral density was lowered by STZ (p<0.0001) and AD (p=0.02) in trabecular bone but only lowered by STZ in cortical bone (p=0.002). Cortical porosity of the proximal tibia was increased by AD, moment of inertia was lower in both disease groups, and most cortical properties were lower in all groups vs CTRL. Ultimate force, stiffness, toughness, and total displacement/strain were lowered by STZ and AD. Fracture toughness was lower by AD (p=0.003). Importantly, Cohen’s D indicated that STZ+AD most strongly lowered bone turnover and mechanical properties. Taken together, structural and material-level bone properties are altered by STZ and AD while their combination resulted in greater detriments, indicating that improving bone health in the combined disease state may require novel interventions.Item A Reproducible Cartilage Impact Model to Generate Post-Traumatic Osteoarthritis in the Rabbit(MyJove Corporation, 2023-11-21) Dilley, Julian; Noori-Dokht, Hessam; Seetharam, Abhijit; Bello, Margaret; Nanavaty, Aaron; Natoli, Roman M.; McKinley, Todd; Bault, Zachary; Wagner, Diane; Sankar, Uma; Anatomy, Cell Biology and Physiology, School of MedicinePost-traumatic osteoarthritis (PTOA) is responsible for 12% of all osteoarthritis cases in the United States. PTOA can be initiated by a single traumatic event, such as a high-impact load acting on articular cartilage, or by joint instability, as occurs with anterior cruciate ligament rupture. There are no effective therapeutics to prevent PTOA currently. Developing a reliable animal model of PTOA is necessary to better understand the mechanisms by which cartilage damage proceeds and to investigate novel treatment strategies to alleviate or prevent the progression of PTOA. This protocol describes an open, drop tower-based rabbit femoral condyle impact model to induce cartilage damage. This model delivered peak loads of 579.1 ± 71.1 N, and peak stresses of 81.9 ± 10.1 MPa with a time-to-peak load of 2.4 ± 0.5 ms. Articular cartilage from impacted medial femoral condyles (MFCs) had higher rates of apoptotic cells (p = 0.0058) and possessed higher Osteoarthritis Research Society International (OARSI) scores of 3.38 ± 1.43 compared to the non-impacted contralateral MFCs (0.56 ± 0.42), and other cartilage surfaces of the impacted knee (p < 0.0001). No differences in OARSI scores were detected among the non-impacted articular surfaces (p > 0.05).