Best Practices for Conducting Observational Research to Assess the Relation between Nutrition and Bone: An International Working Group Summary

Diet is a modifiable factor that can affect bone strength and integrity, and the risk of fractures. Currently, a hierarchy of scientific evidence contributes to our understanding of the role of diet on bone health and fracture risk. The strength of evidence is generally based on the type of study conducted, the quality of the methodology employed, the rigor and integrity of the data collected and analysis plan, and the transparency and completeness of the results. Randomized controlled trials (RCTs) are considered to be the gold standard from a clinical research paradigm, but there is a dearth of high-quality diet-related intervention trials with bone as the primary outcome, forcing the use of observational research to inform research and clinical practices. However, for observational research to be of the most utility, standardization and optimization of the study design, accurate and reliable measurement of key variables, and appropriate data analysis and data reporting are paramount. Although there have been recommendations made in relation to RCTs in the field of nutrition, no clear rubric exists for best practices in conducting observational research with regard to nutrition and bone health. Therefore, the purpose of this paper is to describe the best practices and considerations for designing, conducting, analyzing, interpreting, and reporting observational research specifically for understanding the role of nutrition in bone health, amassed by a global panel of scientific experts with strengths in bone, nutrition epidemiology, physical activity, public health, clinical and translational trials, and observational study methods. The global panel of scientific experts represents the leadership and selected participants from the 10th annual International Symposium for the Nutritional Aspects of Osteoporosis. The topics selected and best practices presented reflect expert opinion and areas of scientific expertise of the authors rather than a systematic or comprehensive literature review or professional reporting guidelines

Serum microRNA screening for DICER1-associated pleuropulmonary blastoma.

CT chest scanning has been recommended to screen for pleuropulmonary blastoma (PPB) in babies and young children known to harbor germline DICER1 mutations. However, only a minority of these patients will develop PPB, and the use of CT scans is associated with risks such as secondary malignancy. Recently, we identified a panel of microRNAs that were highly abundant in the serum of a patient with a germline DICER1-mutated PPB, but present at normal levels in healthy relatives carrying the same germline mutation. Consequently, we advocate the addition of serum microRNA profiling to this programme of radiological surveillance, in order to establish its clinical utility as a PPB biomarker. If validated, this blood-based screening-tool may reduce our reliance on CT imaging.This is the accepted manuscript. The final version is available at http://onlinelibrary.wiley.com/doi/10.1002/pbc.25209/abstract

Biallelic somatic SMARCA4 mutations in small cell carcinoma of the ovary, hypercalcemic type (SCCOHT).

Small cell carcinoma of the ovary, hypercalcemic type (SCCOHT) is a rare, aggressive tumor that primarily affects young women. SCCOHT has recently been identified as a monogenic disorder caused by germline and/or somatic SMARCA4 mutations. We describe a 15-year-old Caucasian female with a SCCOHT harboring a previously unreported somatic mutation in the SMARCA4 gene (c.1757delA; p.K586.fs) with loss of heterozygosity. No germline mutation was identified. Subsequent immunohistochemical staining confirmed loss of SMARCA4 protein. These molecular findings will aid with SCCOHT diagnosis through immunohistochemical staining for SMARCA4 and in the future may have implications for the management of this disease.This is the accepted manuscript. The final version is available at http://onlinelibrary.wiley.com/doi/10.1002/pbc.25279/abstract

Solid tumors of childhood display specific serum microRNA profiles.

BACKGROUND: Serum biomarkers for diagnosis and risk stratification of childhood solid tumors would improve the accuracy/timeliness of diagnosis and reduce the need for invasive biopsies. We hypothesized that differential expression and/or release of microRNAs (miRNAs) by such tumors may be detected as altered serum miRNA profiles. METHODS: We undertook global quantitative reverse transcription PCR (qRT-PCR) miRNA profiling (n = 741) on RNA from 53 serum samples, representing 33 diagnostic cases of common childhood cancers plus 20 controls. Technical confirmation was performed in a subset of 21 cases, plus four independent samples. RESULTS: We incorporated robust quality control steps for RNA extraction, qRT-PCR efficiency and hemolysis quantification. We evaluated multiple methods to normalize global profiling data and identified the 'global mean' approach as optimal. We generated a panel of six miRNAs that were most stable in pediatric serum samples and therefore most suitable for normalization of targeted miRNA qRT-PCR data. Tumor-specific serum miRNA profiles were identified for each tumor type and selected miRNAs underwent confirmatory testing. We identified a panel of miRNAs (miR-124-3p/miR-9-3p/miR-218-5p/miR-490-5p/miR-1538) of potential importance in the clinical management of neuroblastoma, as they were consistently highly overexpressed in MYCN-amplified high-risk cases (MYCN-NB). We also derived candidate miRNA panels for noninvasive differential diagnosis of a liver mass (hepatoblastoma vs. combined MYCN-NB/NB), an abdominal mass (Wilms tumor vs. combined MYCN-NB/NB), and sarcoma subtypes. CONCLUSIONS: This study describes a pipeline for robust diagnostic serum miRNA profiling in childhood solid tumors, and has identified candidate miRNA profiles for prospective testing. IMPACT: We propose a new noninvasive method with the potential to diagnose childhood solid tumors.RCUK, OtherThis is the Author Accepted Manuscript. The final version is available from AACR at http://cebp.aacrjournals.org/content/24/2/350.lon

PDX1 dynamically regulates pancreatic ductal adenocarcinoma initiation and maintenance

Aberrant activation of embryonic signaling pathways is frequent in pancreatic ductal adenocarcinoma (PDA), making developmental regulators therapeutically attractive. Here we demonstrate diverse functions for pancreatic and duodenal homeobox 1 (PDX1), a transcription factor indispensable for pancreas development, in the progression from normal exocrine cells to metastatic PDA. We identify a critical role for PDX1 in maintaining acinar cell identity, thus resisting the formation of pancreatic intraepithelial neoplasia (PanIN)-derived PDA. Upon neoplastic transformation, the role of PDX1 changes from tumor-suppressive to oncogenic. Interestingly, subsets of malignant cells lose PDX1 expression while undergoing epithelial-to-mesenchymal transition (EMT), and PDX1 loss is associated with poor outcome. This stage-specific functionality arises from profound shifts in PDX1 chromatin occupancy from acinar cells to PDA. In summary, we report distinct roles of PDX1 at different stages of PDA, suggesting that therapeutic approaches against this potential target need to account for its changing functions at different stages of carcinogenesis. These findings provide insight into the complexity of PDA pathogenesis and advocate a rigorous investigation of therapeutically tractable targets at distinct phases of PDA development and progression

Improving burst wave lithotripsy effectiveness for small stones and fragments by increasing frequency: theoretical modeling and ex vivo study

Introduction and Objective: In clinical trial NCT03873259, a 2.6-mm lower pole stone was treated transcutaneously and ex vivo with 390-kHz burst wave lithotripsy (BWL) for 40 minutes and failed to break. The stone was subsequently fragmented with 650-kHz BWL after a 4-minute exposure. This study investigated how to fragment small stones and why varying BWL frequency may more effectively fragment stones to dust. Methods: A linear elastic model was used to calculate the stress created inside stones from shock wave lithotripsy (SWL) and different BWL frequencies mimicking the stone’s size, shape, lamellar structure, and composition. To test model predictions about the impact of BWL frequency, matched pairs of stones (1-5 mm) were treated at 1) 390 kHz, 2) 830 kHz, and 3) 390 kHz followed by 830 kHz. The mass of fragments greater than 1 and 2 mm was measured over 10 minutes of exposure. Results: The linear elastic model predicts that the maximum principal stress inside a stone increases to more than 5.5 times the pressure applied by the ultrasound wave as frequency is increased, regardless of composition tested. The threshold frequency for stress amplification is proportionate to the wave speed divided by the stone diameter. Thus, smaller stones may be likely to fragment at higher frequency, but not lower frequency below a limit. Unlike with SWL, this amplification in BWL occurs consistently with spherical and irregularly shaped stones. In water tank experiments, stones smaller than the threshold size broke fastest at high frequency (p=0.0003), whereas larger stones broke equally well to sub-millimeter dust at high, low, or mixed frequency. Conclusions: For small stones and fragments, increasing frequency of BWL may produce amplified stress in the stone causing the stone to break. Using the strategies outlined here, stones of all sizes may be turned to dust efficiently with BWL

Targeting oncogenic microRNAs in malignant germ cell tumours.

Malignant germ cell tumours (mGCTs) are a heterogenous group of tumours that occur primarily in the testes and ovaries but also in other anatomical sites, and affect people of all ages. It was established by the Coleman group through microRNA profiling in 2010 that all mGCTs, regardless of tumour site, patient age or histological subtype, display common microRNA profiles that segregate them from non-malignant controls. In particular, two clusters of miRNAs, miR-371~3 and miR-302/367 were found to be markedly over-expressed in mGCTs. MicroRNAs are short, non-protein-coding RNAs that regulate gene expression through binding of the microRNA seed region to the 3’untranslated region (3’UTR) of messenger RNAs, primarily resulting in mRNA degradation. Six of the eight main microRNAs from the two over-expressed clusters share an identical seed region ‘AAGUGC’, that has been found to down-regulate a range of functionally significant genes. This common seed region is associated with cisplatin resistance in GCT cells and has dose-dependent effects, with higher levels found in GCT subtypes with inferior outcomes. My hypothesis was that targeting these over-expressed microRNAs would confirm their functional significance, identify the pathways in which they function in mGCTs, and identify them as potential therapeutic targets. Multiple strategies were employed in the attempt to target these microRNAs, including gene deletion through the CRISPR/Cas9 system, targeting the primary transcript (pri-miRNA) of each cluster using locked nucleic acid (LNA)-based gapmeRs and targeting the mature miRNAs with peptide nucleic acid and LNA-based inhibitors. Targeting the mature microRNAs using LNA-based inhibitors resulted in a significant and reproducible negative growth phenotype. Gene expression profiling was performed, and although there was no de-repression of individual mRNAs, Sylamer analysis revealed significant enrichment of mRNAs containing the seed complementary region amongst upregulated genes. Pathway analysis of the upregulated mRNA targets demonstrated that these targets were primarily involved in cell cycle regulation. This was functionally validated through cell cycle analysis using flow cytometry. This demonstrated that inhibiting the key, over-expressed microRNAs with LNA-based inhibitors results in cell cycle arrest of mGCT cell lines

Factors Influencing Fluoroscopy use During Ureteroscopy at a Residency Training Program.

INTRODUCTION: Ionizing radiation is used throughout urologic surgery and is known to cause a greater cancer risk with increasing exposure. The ICRP states that it is the control of radiation dose that is important, no matter the source. However, there are few reports on the amount of radiation used by Urology residents during ureteroscopy (URS). We present the largest database evaluating fluoroscopy (fluoro) use during URS at a resident training program. Our objective is to assess the amount of fluoro use at varying levels of experience and to identify factors that lead to increased fluoro use. METHODS: Retrospective data from 242 URS performed at two resident training sites were collected. 105 surgeries were done by 2 attending physicians without and 137 with residents (Uro1-Uro3). Patient data were collected from the EMR. Statistical analyses included ANOVA, Spearman correlations, and multiple linear regression (MLR). RESULTS: Comparisons between Yr1 and Yr2 revealed significantly (p\u3c0.05) decreased fluoro time (20.0 s) and operative time (12.2 m) for the Yr2 resident. Total operative time was significantly (p\u3c0.05) decreased (11.1 m) for attending physicians operating on their own compared to a Yr1 resident. Significant (p\u3c0.05) correlations with fluoro time were demonstrated for operative time, stone size, ureteral dilation, ureteral access sheath use, presence of a preoperative stent, resident year, and resident month. Operative time, ureteral dilation and a preoperative stent placement were significant predictors of fluoro time on MLR (p\u3c0.05). CONCLUSION: Fluoro time during retrograde URS was significantly reduced as residents gained more experience in the operating room. An increase in fluoro time was also associated with ureteral dilation, access sheath use, increasing stone size, and lack of pre-stenting. With knowledge of these factors, emphasis can be placed on using and teaching techniques that limit radiation exposure