Characterization of Type I Collagen and Osteoblast Response to Mechanical Loading

Bone is a composite material made up of an inorganic (hydroxyapatite mineral) phase, a proteinaceous organic phase, and water. Comprising 90% of bones organic phase, type I collagen is the most abundant protein in the human body. Both hydroxyapatite and collagen contribute to bone mechanical properties, and because bone is a hierarchical material, changes in properties of either phase can influence bulk mechanical properties of the tissue and bone structure. Type I collagen in bone is synthesized by osteoblasts as a helical structure formed from three polypeptide chains of amino acids. These molecules are staggered into an array and the resulting collagen fibrils are stabilized by crosslinks. Enzymatic crosslinking can be limited by compounds such as -aminopropionitrile (BAPN) and result in a crosslink deficiency characterizing a disease known as lathyrism. BAPN acts by irreversibly binding to the active site of the lysyl oxidase enzyme, blocking the formation of new crosslinks and the maturation of pre-existing immature crosslinks. Understanding how changes in bone properties on a cellular level transcend levels of bone hierarchy provides an opportunity to detect or diagnose bone disease before disease-related changes are expressed at the organ or tissue level. This dissertation studies the in vitro effect of BAPN-induced enzymatic crosslink reduction on osteoblast-produced collagen nanostructure, mechanical properties, crosslink ratio, and expression of genes related to type I collagen synthesis and crosslinking. The work also explores the effect of mechanical loading via applied substrate strain on these properties to investigate its potential compensatory impact

β-Aminopropionitrile-induced reduction in enzymatic crosslinking causes in vitro changes in collagen morphology and molecular composition

Type I collagen morphology can be characterized using fibril D-spacing, a metric which describes the periodicity of repeating bands of gap and overlap regions of collagen molecules arranged into collagen fibrils. This fibrillar structure is stabilized by enzymatic crosslinks initiated by lysyl oxidase (LOX), a step which can be disrupted using β-aminopropionitrile (BAPN). Murine in vivo studies have confirmed effects of BAPN on collagen nanostructure and the objective of this study was to evaluate the mechanism of these effects in vitro by measuring D-spacing, evaluating the ratio of mature to immature crosslinks, and quantifying gene expression of type I collagen and LOX. Osteoblasts were cultured in complete media, and differentiated using ascorbic acid, in the presence or absence of 0.25mM BAPN-fumarate. The matrix produced was imaged using atomic force microscopy (AFM) and 2D Fast Fourier transforms were performed to extract D-spacing from individual fibrils. The experiment was repeated for quantitative reverse transcription polymerase chain reaction (qRT-PCR) and Fourier Transform infrared spectroscopy (FTIR) analyses. The D-spacing distribution of collagen produced in the presence of BAPN was shifted toward higher D-spacing values, indicating BAPN affects the morphology of collagen produced in vitro, supporting aforementioned in vivo experiments. In contrast, no difference in gene expression was found for any target gene, suggesting LOX inhibition does not upregulate the LOX gene to compensate for the reduction in aldehyde formation, or regulate expression of genes encoding type I collagen. Finally, the mature to immature crosslink ratio decreased with BAPN treatment and was linked to a reduction in peak percent area of mature crosslink hydroxylysylpyridinoline (HP). In conclusion, in vitro treatment of osteoblasts with low levels of BAPN did not induce changes in genes encoding LOX or type I collagen, but led to an increase in collagen D-spacing as well as a decrease in mature crosslinks. © 2016 Canelón, Wallace. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

Substrate strain mitigates effects of β-aminopropionitrile-induced reduction in enzymatic crosslinking

Enzymatic crosslinks stabilize type I collagen and are catalyzed by lysyl oxidase (LOX), a step interrupted through β-aminopropionitrile (BAPN) exposure. This study evaluated dose-dependent effects of BAPN on osteoblast gene expression of type I collagen, LOX, and genes associated with crosslink formation. The second objective was to characterize collagen produced in vitro after exposure to BAPN, and to explore changes to collagen properties under continuous cyclical substrate strain. To evaluate dose-dependent effects, osteoblasts were exposed to a range of BAPN dosages (0–10 mM) for gene expression analysis and cell proliferation. Results showed significant upregulation of BMP-1, POST, and COL1A1and change in cell proliferation. Results also showed while the gene encoding LOX was unaffected by BAPN treatment, other genes related to LOX activation and matrix production were upregulated. For the loading study, the combined effects of BAPN and mechanical loading were assessed. Gene expression was quantified, atomic force microscopy was used to extract elastic properties of the collagen matrix, and Fourier Transform infrared spectroscopy was used to assess collagen secondary structure for enzymatic crosslinking analysis. BAPN upregulated BMP-1 in static samples and BAPN combined with mechanical loading downregulated LOX when compared to control-static samples. Results showed a higher indentation modulus in BAPN-loaded samples compared to control-loaded samples. Loading increased the mature to immature crosslink ratios in control samples, and BAPN increased the height ratio in static samples. In summary, effects of BAPN (upregulation of genes involved in crosslinking, mature/immature crosslinking ratios, upward trend in collagen elasticity) were mitigated by mechanical loading

Uncovering Patterns for Adverse Pregnancy Outcomes with Spatial Analysis: Evidence from Philadelphia

We introduce a spatial model for analyzing patient-specific and neighborhood risks of stillbirth and preterm birth in Philadelphia. Using electronic health records and census tract data, we find that both patient-level characteristics (e.g. self-identified race/ethnicity) and neighborhood-level characteristics (e.g. violent crime) are associated with patients' odds of stillbirth or preterm birth. Census tracts with higher rates of women in poverty or on public assistance have greater neighborhood risk for these outcomes, whereas census tracts with higher rates of college-educated women or women in the labor force have lower risk. Our findings could be useful for targeted individual and neighborhood interventions.Comment: 27 pages, 7 figures, 9 table

Ten simple rules to host an inclusive conference.

Conferences are spaces to meet and network within and across academic and technical fields, learn about new advances, and share our work. They can help define career paths and create long-lasting collaborations and opportunities. However, these opportunities are not equal for all. This article introduces 10 simple rules to host an inclusive conference based on the authors' recent experience organizing the 2021 edition of the useR! statistical computing conference, which attracted a broad range of participants from academia, industry, government, and the nonprofit sector. Coming from different backgrounds, career stages, and even continents, we embraced the challenge of organizing a high-quality virtual conference in the context of the Coronavirus Disease 2019 (COVID-19) pandemic and making it a kind, inclusive, and accessible experience for as many people as possible. The rules result from our lessons learned before, during, and after the organization of the conference. They have been written mainly for potential organizers and selection committees of conferences and contain multiple practical tips to help a variety of events become more accessible and inclusive. We see this as a starting point for conversations and efforts towards building more inclusive conferences across the world. * Translated versions of the English abstract and the list of rules are available in 10 languages in S1 Text: Arabic, French, German, Italian, Japanese, Korean, Portuguese, Spanish, Tamil, and Thai

Representative 3.5 μm x 3.5 μm collagen AFM image.

<p>AFM images of collagen in its native state were coupled with Fourier transform analysis to measure the periodic fibril D-spacing of collagen synthesized <i>in vitro</i> by osteoblasts.</p

Medication-Wide Association Study Using Electronic Health Record Data of Prescription Medication Exposure and Multifetal Pregnancies: Retrospective Study

BackgroundMedication-wide association studies (MWAS) have been applied to assess the risk of individual prescription use and a wide range of health outcomes, including cancer, acute myocardial infarction, acute liver failure, acute renal failure, and upper gastrointestinal ulcers. Current literature on the use of preconception and periconception medication and its association with the risk of multiple gestation pregnancies (eg, monozygotic and dizygotic) is largely based on assisted reproductive technology (ART) cohorts. However, among non-ART pregnancies, it is unknown whether other medications increase the risk of multifetal pregnancies. ObjectiveThis study aimed to investigate the risk of multiple gestational births (eg, twins and triplets) following preconception and periconception exposure to prescription medications in patients who delivered at Penn Medicine. MethodsWe used electronic health record data between 2010 and 2017 on patients who delivered babies at Penn Medicine, a health care system in the Greater Philadelphia area. We explored 3 logistic regression models: model 1 (no adjustment); model 2 (adjustment for maternal age); and model 3—our final logistic regression model (adjustment for maternal age, ART use, and infertility diagnosis). In all models, multiple births (MBs) were our outcome of interest (binary outcome), and each medication was assessed separately as a binary variable. To assess our MWAS model performance, we defined ART medications as our gold standard, given that these medications are known to increase the risk of MB. ResultsOf the 63,334 distinct deliveries in our cohort, only 1877 pregnancies (2.96%) were prescribed any medication during the preconception and first trimester period. Of the 123 medications prescribed, we found 26 (21.1%) medications associated with MB (using nominal P values) and 10 (8.1%) medications associated with MB (using Bonferroni adjustment) in fully adjusted model 3. We found that our model 3 algorithm had an accuracy of 85% (using nominal P values) and 89% (using Bonferroni-adjusted P values). ConclusionsOur work demonstrates the opportunities in applying the MWAS approach with electronic health record data to explore associations between preconception and periconception medication exposure and the risk of MB while identifying novel candidate medications for further study. Overall, we found 3 novel medications linked with MB that could be explored in further work; this demonstrates the potential of our method to be used for hypothesis generation

Representative mature and immature crosslink peak fittings underneath the FTIR spectral curve.

<p>A decrease in the 1654cm^-1 peak area is evident in the BAPN-treated sample relative to control. The BAPN-treated and control samples were plotted on different axes to visually highlight this difference. The black solid and dashed lines correspond to the full spectra of control and BAPN-treated samples, respectively.</p

Collagen D-spacing obtained from the D-spacing measurements in each group (n = 4).

<p>A clear shift towards higher D-spacing values in the BAPN-treated group is evident in the (a) histogram, (b) cumulative distribution function (CDF), and mean, indicated by the diamond marks on the CDF.</p

Collagen structure and organization.

<p>Collagen molecules self-assemble in a quarter-staggered array into microfibrils to form collagen fibrils with characteristic periodic D-spacing.</p