The only temple completed by Mormonism's founder, Joseph Smith Jr., the Kirtland Temple in Kirtland, Ohio, receives 30,000 Mormon pilgrims every year. The site's religious significance and the space ...itself are contested by distinct Mormon denominations: its owner, the relatively liberal Community of Christ, and the larger Church of Jesus Christ of Latter-day Saints. David J. Howlett sets the biography of Kirtland Temple against the backdrop of this religious rivalry. The two sides have long contested the temple's ownership, purpose, and significance in both the courts and Mormon literature. Yet members of each denomination have occasionally cooperated to establish periods of co-worship, host joint tours, and create friendships. Howlett uses the temple to build a model for understanding what he calls parallel pilgrimage--the set of dynamics of disagreement and alliance by religious rivals at a shared sacred site. At the same time, he illuminates social and intellectual changes in the two main branches of Mormonism since the 1830s, providing a much-needed history of the lesser-known Community of Christ.
Shear flow is indirectly implicated in amyloid formation in vitro. Despite the association between amyloid fibrils and disease, and the prevalence of flow in physiological systems, the effect of this ...parameter is uncharacterized. We designed a novel Couette cell to quantitatively investigate shear exposure during fibrillogenesis. Amyloid formation by β-lactoglobulin was monitored in situ with real-time fluorescence measurements across a range of shear rates. We demonstrate shear-induced aggregation of spheroidal seed-like species. These seeds enhance fibril formation in native β-lactoglobulin, thereby demonstrating that shear flow generates an amyloidogenic precursor. Furthermore, preformed fibrils are degraded by exposure to high shear rates. Our results have implications for the mechanism of amyloid formation in physiological flow conditions.
Background: Successful percutaneous nephrolithotomy (PCNL) relies on a technically challenging, precise needle puncture of the renal collecting system. We aimed to compare, in an ex vivo model, the ...use of a real time image guidance system (the SabreSource™) and a mechanical stabilising device with conventional manual techniques for the accuracy of needle placement.Methods: The SabreSource™ system (Minrad International Inc.; New York, USA) is a real time image guidance system. The system platform is mounted on a C-arm fluoroscope. It employs targeting cross hairs on the fluoroscopic image that can be easily positioned to target the desired renal calyx. The system directs a visible laser beam onto the patient which is precisely aligned with the cross hairs on the fluoroscopic image. This provides the correct “bull’s-eye” angle of approach to the calyx, even after the x-ray source is turned off. The locator then stabilises the needle in the “bull’s-eye” position so that only screening for depth is required. Objective assessment using a simulated PCNL puncture was performed by 7 urologic trainees on a kidney phantom with and without using the SabreSource™. Fluoroscopy screening time (FST) and amount of radiation (mGy) used to achieve successful puncture were compared.Results: Simulated PCNL puncture was quicker and resulted in reduced radiation exposure when the apparatus was used. The mean FST for traditional “bull’s-eye” vs SabreSource™ puncture was 17 vs 5 seconds (p = 0.01), and the mean radiation exposure to puncture was 0.7 vs 0.2 mGy (p = 0.03), respectively.Conclusion: The SabreSource™ is a novel assistant to achieving successful PCNL puncture. In combination with “the locator” the preliminary in vitro testing suggests that the device reduces fluoroscopy exposure and is quicker. The device warrants further evaluation in the clinical setting.
Phosphoinositide 3‐kinase (PI3K) is aberrantly activated in head and neck squamous cell carcinomas (HNSCC) and plays a pivotal role in tumorigenesis by driving Akt signaling, leading to cell survival ...and proliferation. Phosphorylation of Akt Thr308 by PI3K‐PDK1 and Akt Ser473 by mammalian target of rapamycin complex 2 (mTORC2) activates Akt. Targeted inhibition of PI3K is a major area of preclinical and clinical investigation as it reduces Akt Thr308 phosphorylation, suppressing downstream mTORC1 activity. However, inhibition of mTORC1 releases feedback inhibition of mTORC2, resulting in a resurgence of Akt activation mediated by mTORC2. While the role of PI3K‐activated Akt signaling is well established in HNSCC, the significance of mTORC2‐driven Akt signaling has not been thoroughly examined. Here we explore the expression and function of mTORC2 and its obligate subunit RICTOR in HNSCC primary tumors and cell lines. We find RICTOR to be overexpressed in a subset of HNSCC tumors, including those with PIK3CA or EGFR gene amplifications. Whereas overexpression of RICTOR reduced susceptibility of HNSCC tumor cells to PI3K inhibition, genetic ablation of RICTOR using CRISPR/Cas9 sensitized cells to PI3K inhibition, as well as to EGFR inhibition and cisplatin treatment. Further, mTORC2 disruption led to reduced viability and colony forming abilities of HNSCC cells relative to their parental lines and induced loss of both activating Akt phosphorylation modifications (Thr308 and Ser473). Taken together, our findings establish RICTOR/mTORC2 as a critical oncogenic complex in HNSCC and rationalize the development of an mTORC2‐specific inhibitor for use in HNSCC, either combined with agents already under investigation, or as an independent therapy.
While the role of phosphoinositide 3‐kinase (PI3K)‐activated Akt signalling is well established in head/neck squamous cell carcinogenesis, the significance of mammalian target of rapamycin complex 2 (mTORC2)‐driven Akt signalling has not been thoroughly examined. Our study reveals an oncogenic role for mTORC2 and its obligate subunit RICTOR. Disruption of RICTOR/mTORC2 signalling impairs cellular viability and enhances the efficacy of PI3K targeted inhibition. Collectively, these findings rationalize the development of an mTORC2‐specific inhibitor.
Advances in next-generation sequencing (NGS) have facilitated parallel analysis of multiple genes enabling the implementation of cost-effective, rapid, and high-throughput methods for the molecular ...diagnosis of multiple genetic conditions, including the identification of BRCA1 and BRCA2 mutations in high-risk patients for hereditary breast and ovarian cancer. We clinically validated a NGS pipeline designed to replace Sanger sequencing and multiplex ligation-dependent probe amplification analysis and to facilitate detection of sequence and copy number alterations in a single test focusing on a BRCA1 / BRCA2 gene analysis panel. Our custom capture library covers 46 exons, including BRCA1 exons 2, 3, and 5 to 24 and BRCA2 exons 2 to 27, with 20 nucleotides of intronic regions both 5′ and 3′ of each exon. We analyzed 402 retrospective patients, with previous Sanger sequencing and multiplex ligation-dependent probe amplification results, and 240 clinical prospective patients. One-hundred eighty-three unique variants, including sequence and copy number variants, were detected in the retrospective ( n = 95) and prospective ( n = 88) cohorts. This standardized NGS pipeline demonstrated 100% sensitivity and 100% specificity, uniformity, and high-depth nucleotide coverage per sample (approximately 7000 reads per nucleotide). Subsequently, the NGS pipeline was applied to the analysis of larger gene panels, which have shown similar uniformity, sample-to-sample reproducibility in coverage distribution, and sensitivity and specificity for detection of sequence and copy number variants.
Apolipoprotein A-I (apoA-I) is the major protein component of HDL, where it plays an important role in cholesterol transport. The deposition of apoA-I derived amyloid is associated with various ...hereditary systemic amyloidoses and atherosclerosis; however, very little is known about the mechanism of apoA-I amyloid formation. Methionine residues in apoA-I are oxidized via several mechanisms in vivo to form methionine sulfoxide (MetO), and significant levels of methionine oxidized apoA-I (MetO-apoA-I) are present in normal human serum. We investigated the effect of methionine oxidation on the structure, stability, and aggregation of full-length, lipid-free apoA-I. Circular dichrosim spectroscopy showed that oxidation of all three methionine residues in apoA-I caused partial unfolding of the protein and decreased its thermal stability, reducing the melting temperature (Tm) from 58.7 °C for native apoA-I to 48.2 °C for MetO-apoA-I. Analytical ultracentrifugation revealed that methionine oxidation inhibited the native self association of apoA-I to form dimers and tetramers. Incubation of MetO-apoA-I for extended periods resulted in aggregation of the protein, and these aggregates bound Thioflavin T and Congo Red. Inspection of the aggregates by electron microscopy revealed fibrillar structures with a ribbon-like morphology, widths of approximately 11 nm, and lengths of up to several microns. X-ray fibre diffraction studies of the fibrils revealed a diffraction pattern with orthogonal peaks at spacings of 4.64 Å and 9.92 Å, indicating a cross-β amyloid structure. This systematic study of fibril formation by full-length apoA-I represents the first demonstration that methionine oxidation can induce amyloid fibril formation.
Human apolipoprotein (apo) C‐II is one of several plasma apolipoproteins that form amyloid deposits in vivo and is an independent risk factor for cardiovascular disease. Lipid‐free apoC‐II readily ...self‐assembles into twisted‐ribbon amyloid fibrils but forms straight, rod‐like amyloid fibrils in the presence of low concentrations of micellar phospholipids. Charge mutations exerted significantly different effects on rod‐like fibril formation compared to their effects on twisted‐ribbon fibril formation. For instance, the double mutant, K30D‐D69K apoC‐II, readily formed twisted‐ribbon fibrils, while the rate of rod‐like fibril formation in the presence of micellar phospholipid was negligible. Structural analysis of rod‐like apoC‐II fibrils, using hydrogen–deuterium exchange and NMR analysis showed exchange protection consistent with a core cross‐β structure comprising the C‐terminal 58–76 region. Molecular dynamics simulations of fibril arrangements for this region favoured a parallel cross‐β structure. X‐ray fibre diffraction data for aligned rod‐like fibrils showed a major meridional spacing at 4.6 Å and equatorial spacings at 9.7, 23.8 and 46.6 Å. The latter two equatorial spacings are not observed for aligned twisted‐ribbon fibrils and are predicted for a model involving two cross‐β fibrils in an off‐set antiparallel structure with four apoC‐II units per rise of the β‐sheet. This model is consistent with the mutational effects on rod‐like apoC‐II fibril formation. The lipid‐dependent polymorphisms exhibited by apoC‐II fibrils could determine the properties of apoC‐II in renal amyloid deposits and their potential role in the development of cardiovascular disease.
Human apolipoprotein (apo) C‐II can form two distinct types of amyloid fibrils, depending on the presence and nature of lipid in solution. We have investigated the structure of ‘rod‐like’ apoC‐II amyloid fibrils using biophysical and computational methods, and propose a model for the core cross‐β structure of the fibrils.
Diabetes affects select organs such as the eyes, kidney, heart, and brain. Our recent studies show that diabetes also enhances adipogenesis in the bone marrow and reduces the number of ...marrow-resident vascular regenerative stem cells. In the current study, we have performed a detailed spatio-temporal examination to identify the early changes that are induced by diabetes in the bone marrow. Here we show that short-term diabetes causes structural and molecular changes in the marrow, including enhanced adipogenesis in tibiae of mice, prior to stem cell depletion. This enhanced adipogenesis was associated with suppressed transforming growth factor-beta (TGFB) signaling. Using human bone marrow-derived mesenchymal progenitor cells, we show that TGFB pathway suppresses adipogenic differentiation through TGFB-activated kinase 1 (TAK1). These findings may inform the development of novel therapeutic targets for patients with diabetes to restore regenerative stem cell function.
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