Biomedical Problem Solving and Learning Community Building

Problem-based learning (PBL) has long been recognized as a valuable pedagogical tool. The Biomedical Masters in Science (BMS) program is uniquely suited to promote active learning through the use of discussion based PBLs, but also foster a supportive community of learners by incorporating volunteer facilitators that are BMS alums and current medical students at Marian University. PBLs extend our Medical Physiology and Pharmacology curriculum, but also provide an opportunity for different learners to connect material across graduate and medical curriculums. PBL exercises are graded via a group rubric, while debriefing and reflection occurs via the video response system FlipGrid

Signaling mechanisms that suppress the anabolic response of osteoblasts and osteocytes to fluid shear stress

Indiana University-Purdue University Indianapolis (IUPUI)Bone is a dynamic organ that responds to its external environment. Cell signaling cascades are initiated within bone cells when changes in mechanical loading occur. To describe these molecular signaling networks that sense a mechanical signal and convert it into a transcriptional response, we proposed the mechanosome model. “GO” and “STOP” mechansomes contain an adhesion-associated protein and a nucleocytoplasmic shuttling transcription factor. “GO” mechanosomes functions to promote the anabolic response of bone to mechanical loading, while “STOP” mechanosomes function to suppress the anabolic response of bone to mechanical loading. While much work has been done to describe the molecular mechanisms that enhance the anabolic response of bone to loading, less is known about the signaling mechanisms that suppress bone’s response to loading. We studied two adhesion-associated proteins, Src and Pyk2, which may function as “STOP” mechanosomes. Src kinase is involved in a number of signaling pathways that respond to changes in external loads on bone. An inhibition of Src causes an increase in the expression of the anabolic bone gene osteocalcin. Additionally, mechanical stimulation of osteoblasts and osteocytes by fluid shear stress further enhanced expression of osteocalcin when Src activity was inhibited. Importantly, fluid shear stress stimulated an increase in nuclear Src activation and activity. The mechanism by which Src participates in attenuating anabolic gene transcription remains unknown. The studies described here suggest Src and Pyk2 increase their association in response to fluid shear stress. Pyk2, a protein-tyrosine kinase, exhibits nucleocytoplasmic shuttling, increased association with methyl-CpG-binding protein 2 (MBD2), and suppression of osteopontin expression in response to fluid shear stress. MBD2, known to be involved in DNA methylation and interpretation of DNA methylation patterns, may aid in fluid shear stress-induced suppression of anabolic bone genes. We conclude that both Src and Pyk2 play a role in regulating bone mass, possibly through a complex with MBD2, and function to limit the anabolic response of bone cells to fluid shear stress through the suppression of anabolic bone gene expression. Taken together, these data support the hypothesis that “STOP” mechanosomes exist and their activity is simulated in response to fluid shear stress

Hypophosphatemic rickets: Revealing Novel Control Points for Phosphate Homeostasis

Rapid and somewhat surprising advances have recently been made towards understanding the molecular mechanisms causing heritable disorders of hypophosphatemia. The results of clinical, genetic, and translational studies have interwoven novel concepts underlying the endocrine control of phosphate metabolism, with far-reaching implications for treatment of both rare, Mendelian diseases as well as common disorders of blood phosphate excess such as chronic kidney disease (CKD). In particular, diseases caused by changes in the expression and proteolytic control of the phosphaturic hormone Fibroblast growth factor-23 (FGF23) have come to the forefront in terms of directing new models explaining mineral metabolism. These hypophosphatemic disorders, as well as others resulting from independent defects in phosphate transport or metabolism, will be reviewed herein, and implications for emerging therapeutic strategies based upon these new findings will be discussed

Novel functions of circulating Klotho

A significant portion of the key biological functions of αKlotho (αKL) and its cognate ligand Fibroblast growth factor-23 (FGF23) have been revealed through the study of rare diseases of mineral metabolism. These findings have far reaching implications for common disorders such as chronic kidney disease-mineral bone disorder (CKD-MBD). αKL’s predominant effect on mineral homeostasis is through its actions in the kidney as a co-receptor for FGF23, however emerging data has shed light on its capacity to act as a circulating factor through the cleavage of the transmembrane form of αKL (‘mKL’) to produce ‘cleaved KL’ or ‘cKL’. This review summarizes new findings from studies using extended delivery of cKL to mouse models with phenotypes reflecting those arising in CKD-MBD

Monitoring Biosensor Activity in Living Cells with Fluorescence Lifetime Imaging Microscopy

Live-cell microscopy is now routinely used to monitor the activities of the genetically encoded biosensor proteins that are designed to directly measure specific cell signaling events inside cells, tissues, or organisms. Most fluorescent biosensor proteins rely on Förster resonance energy transfer (FRET) to report conformational changes in the protein that occur in response to signaling events, and this is commonly measured with intensity-based ratiometric imaging methods. An alternative method for monitoring the activities of the FRET-based biosensor proteins is fluorescence lifetime imaging microscopy (FLIM). FLIM measurements are made in the time domain, and are not affected by factors that commonly limit intensity measurements. In this review, we describe the use of the digital frequency domain (FD) FLIM method for the analysis of FRET signals. We illustrate the methods necessary for the calibration of the FD FLIM system, and demonstrate the analysis of data obtained from cells expressing “FRET standard” fusion proteins. We then use the FLIM-FRET approach to monitor the changes in activities of two different biosensor proteins in specific regions of single living cells. Importantly, the factors required for the accurate determination and reproducibility of lifetime measurements are described in detail

Family Conflict In Eat, Pray, Love Movie: A Sociological Approach

This study aims to examine family conflicts that occur in the film Eat, Pray, Love and to explain why these conflicts occur. This type of research is a descriptive qualitative study using a sociological approach. The data of the research are in the form of observations on the Eat, Pray, Love movie which are equipped with the stories in the movie. The data collection technique uses the following steps: Reading, Browsing and reading the related articles, Taking notes of the important related with the study, Identifying the conflict problem, Making conclusion and its suggestion. The scope of this research is family conflict in the Eat, Pray, Love movie and used a sociological approach. Here, a sociological approach is used to analyze the conflict in the Eat, Pray, Love movie. However, in this study the researchers only analyzed what problems occurred in the Eat, Pray, Love movie and the factors that caused these problems. The results of this study are: First, what causes family conflict to occur, namely: failed in marriage, moved to a new house, traveling long distance to personal pleasure, never ending divorced issues, a heartbreak caused by much expectations. Any factors that influence conflict in a family are dishonesty, the threat, and war. And how to deal with family conflicts are try to communicate life clearly and honestly in Italy, try to stay calm in India, try to put the emotions in Indonesia

Developing an In Vitro Model of CKD-MBD Induced αKlotho Suppression

Chronic Kidney Disease (CKD) affects approximately 1 in 10 Americans. Diabetic nephropathy is also associated with the development of chronic kidney disease-mineral bone disorder (CKD-MBD). CKD-MBD disrupts the normal bone-kidney endocrine axis responsible for regulating mineral metabolism, and hyperphosphatemia develops in late stage disease. Important clinical hallmarks of the CKD-MBD progression include elevated bioactive Fibroblast growth factor-23 (FGF23) and suppression of FGF23’s co-receptor, αKlotho (αKL). In healthy individuals the hormone FGF23, primarily produced by bone, and aKL aid in maintaining normal phosphate and vitamin D homeostasis. It is currently unknown what drives the suppression of αKL expression, however increasing αKL expression in CKD-MBD models is being investigated as a novel therapeutic. Our study sought to develop a novel in vitro model of one of the clinical hallmarks of the progression of CKD-MBD, αKL suppression, to investigate both possible stimuli of its repression and downstream signaling events. The Human Embryonic Kidney (HEK) cell line was used to determine if changes in fluid shear stress, similar to those that occur in diabetic nephropathy, could lead to reduced αKL expression. HEK cells were plated and exposed to oscillatory fluid shear stress (OFSS) for intervals between 0-60 min to examine protein expression or 0-2 hours to assess gene expression. HEK cells were sensitive to mechanical stimulation as pathways including increased ERK phosphorylation occurred in response to OFSS. In response to longer bouts of OFSS αKL expression was significantly (p\u3c0.05) reduced. Dramatic changes in fluid shear stress may serve as a stimulus for reduced αKL expression in CKD-MBD. Further studies are underway to investigate downstream signaling events related to αKL suppression. Understanding both the stimuli of αKL suppression and related downstream signaling events could provide novel therapeutic targets for the treatment of CKD-MBD

The metabolic bone disease associated with the Hyp mutation is independent of osteoblastic HIF1α expression

Fibroblast growth factor-23 (FGF23) controls key responses to systemic phosphate increases through its phosphaturic actions on the kidney. In addition to stimulation by phosphate, FGF23 positively responds to iron deficiency anemia and hypoxia in rodent models and in humans. The disorder X-linked hypophosphatemia (XLH) is characterized by elevated FGF23 in concert with an intrinsic bone mineralization defect. Indeed, the Hyp mouse XLH model has disturbed osteoblast to osteocyte differentiation with altered expression of a wide variety of genes, including FGF23. The transcription factor Hypoxia inducible factor-1α (HIF1α) has been implicated in regulating FGF23 production and plays a key role in proper bone cell differentiation. Thus the goals of this study were to determine whether HIF1α activation could influence FGF23, and to test osteoblastic HIF1α production on the Hyp endocrine and skeletal phenotypes in vivo. Treatment of primary cultures of osteoblasts/osteocytes and UMR-106 cells with the HIF activator AG490 resulted in rapid HIF1α stabilization and increased Fgf23 mRNA (50-100 fold; p < 0.01-0.001) in a time- and dose-dependent manner. Next, the Phex gene deletion in the Hyp mouse was bred onto mice with a HIF1α/Osteocalcin (OCN)-Cre background. Although HIF1α effects on bone could be detected, FGF23-related phenotypes due to the Hyp mutation were independent of HIF1α in vivo. In summary, FGF23 can be driven by ectopic HIF1α activation under normal iron conditions in vitro, but factors independent of HIF1α activity after mature osteoblast formation are responsible for the disease phenotypes in Hyp mice in vivo

Osteocyte-Specific Deletion of the α2δ1 Auxiliary Voltage Sensitive Calcium Channel Subunit

Context: Skeletal unloading due to disuse, disease, or aging increases bone loss and the risk of skeletal fracture. Conversely, mechanical loading is anabolic to the skeleton, promoting skeletal integrity through increased bone formation. As calcium influx is the first measurable response of bone cells to mechanical stimuli, voltage sensitive calcium channels (VSCCs) play a critical role in bone formation. Given VSCC activity is influenced by its auxiliary α2δ1 subunit, regulating the gating kinetics of the channel’s pore-forming (α1) subunit and forward trafficking of VSCCs to cell membranes, the α2δ1 subunit may govern anabolic bone responses. Objective & Design: We hypothesized that osteocyte-specific α2δ1 deletion in a mouse model would impair skeletal development, decrease bone formation and mechanosensitivity. Methods: Generation of an osteocyte-specific α2δ1 knockout was accomplished by crossing mice (C57BL/6) harboring LoxP sequences flanking Cacna2d1, the gene encoding α2δ1, with mice expressing Cre recombinase under the control of the Dmp1 (10Kb) promoter (Cacna2d1fl/fl, Dmp1-Cre+). To assess skeletal phenotype and mechanosensitivity, longitudinal whole body and site-specific DXA, in vivo μCT (10wk old), and two weeks of tibial loading (16wks) will be conducted before femurs are collected at 20 wks for mechanical testing, ex vivo μCT, and quantitative histomorphometry. Results & Conclusion: Preliminary analyses show no differences in whole body or site-specific BMD and BMC values between mice over time, suggesting osteocyte-specific α2δ1 deletion may not influence skeletal development. However, key differences in mechanosensitivity following tibial loading are expected given the potential role of α2δ1 in mechanically-induced bone formation