Easy-to-Use HPLC Method to Measure Intracellular Ascorbic Acid Levels in Human Peripheral Blood Mononuclear Cells and in Plasma

Given the growing interest in ascorbic acid (AA), there is a need for a reliable and reproducible method to measure AA status in the human body. Serum AA concentrations do not correlate well with tissue levels, but AA levels in leukocytes do. However, a standard method for clinical application is lacking. This present study describes a method to measure AA in the peripheral blood mononuclear cells (PBMCs) with hydrophilic interaction liquid chromatography (HILIC). The method can also be used in plasma and other leukocyte subsets. The measurements of AA in PBMCs and plasma were performed with HPLC with HILIC separation and UV detection. The sample preparation involved the isolation of PBMCs and lysis and precipitation with acetonitrile. European Medicine Agency guidelines for bioanalytic method validation were followed for the evaluation. A highly precise execution of the method was found with intra- and inter-assay variations at a maximum of 7.8%. In 40 healthy donors, a mean intracellular AA concentration of 7.9 microgram/108 cells was found in PBMCs. A correlation between plasma and PBMC AA concentration was not present (r = 0.22). In conclusion, we developed a convenient, reliable, and reproducible method for the quantitative determination of AA within PBMCs and plasma from human blood.

Optimized Cell Mixing Facilitates the Reproducible Bioprinting of Constructs with High Cell Viability

Bioprinting with cell-laden hydrogels (bioink) requires the careful mixing of cells with the hydrogel carrier to ensure that the bioink is homogeneous and functional, and the printing results are reproducible. Bioink preparation is therefore a critical process step that must accommodate the specific rheological properties of different bioinks. Here, we developed a reproducible method for the optimized mixing of cells and hydrogel carriers that can be integrated into current bioprinting processes. First, we tested and optimized different mixing devices for their effect on bioink homogeneity and rheological properties, resulting in a low-shear process for the preparation of homogenous bioinks. Based on these findings, we evaluated the impact of different cell densities on the rheological profile of bioinks according to shear and temperature, and estimated the impact of shear stress intensity and duration on 1.1B4 cells. Finally, we integrated the optimized mixing method into a current printing process and monitored the printed construct for 14 days to confirm cell viability. We found that the cell viability in the printed cell-laden constructs remained in excess of 91% after 14 days.

A Network-based Analysis of Ingredients Lists in Nutrient Profile Systems

It is well-established that diseases can be prevented or mitigated through dietary intervention, yet proper nutrition is one aspect consumers struggle to manage. Recent studies have shown that there are barriers to understanding the nutrition facts component of a food label which can be linked to dietary choices. In this work, we demonstrate reproducibility and replicability of a network-based method for automating the analysis of ingredients on a food product label en masse using the Open Food Facts Database and the USDA FoodData Central Branded Foods database in February 2020. Our results, which analyze the co-occurrence of 72,754 ingredients across show some consensus in labeling across FALCPA-regulated ingredients in food product labels across databases but highlight potential areas for discrepancy in consumer understanding and labeling practices for terms not subject to strict regulations. The key findings or contributions of this work include the provision of a reproducible method for quantifying the ingredients of packaged food in the United States across two nutritional profiling systems, and have identified 17 total ingredients that appear in the top 20 most co-occurring ingredients for both databases examined. We compare how of 8 FALCPA-regulated ingredients are represented in ingredients lists versus a common, but non-FALCPA regulated ingredient (corn), to demonstrate how one could examine differences between ingredient labeling between products. These findings suggest more research is needed in developing information systems to increase information available for consumers.

Introduction of loxP sites by electroporation in the mouse genome; a simple approach for conditional allele generation in complex targeting loci.

Background: The discovery of the CRISPR-Cas9 system and its applicability in mammalian embryos has revolutionized the way we generate genetically engineered animal models. To date, models harbouring conditional alleles (i.e.: two loxP sites flanking an exon or a critical DNA sequence of interest) remain the most challenging to generate as they require simultaneous cleavage of the genome using two guides in order to properly integrate the repair template. In the current manuscript, we describe a modification of the sequential electroporation procedure described by Horii et al (2017). We demonstrate production of conditional allele mouse models for eight different genes via one of two alternative strategies: either by consecutive sequential electroporation (strategy A) or non-consecutive sequential electroporation (strategy B). Results: By using strategy A, we demonstrated successful generation of conditional allele models for three different genes (Icam1, Lox, and Sar1b), with targeting efficiencies varying between 5 to 13%. By using strategy B, we generated five conditional allele models (Loxl1, Pard6a, Pard6g, Clcf1, and Mapkapk5), with targeting efficiencies varying between 3 to 25%. Conclusion: Our modified electroporation-based approach, involving one of the two alternative strategies, allowed the production of conditional allele models for eight different genes via two different possible paths. This reproducible method will serve as another reliable approach in addition to other well-established methodologies in the literature for conditional allele mouse model generation.

Sonography-Based Determination of Hip Joint Anterior Alpha-Angle: A Reliable and Reproducible Method

Purpose Femoroacetabular impingement (FAI) is a known risk factor for hip osteoarthritis. The gold standard for diagnostics is X-ray and MRI. The accuracy of hip joint alpha angle measurements obtained using sonography is equal to measurements in MRI for patients with cam impingement of the hip joint. Materials and Methods Patients with hip pain and MRI and sonography were evaluated between January 2015 and December 2019 in a single center. Musculoskeletal sonography was performed according to the DEGUM guidelines by ultrasound-certified specialists. Measurements were repeated three times by two independent investigators. Results 285 patients were screened, and 110 patients (49 females, 61 males) met the inclusion criteria. The mean age at time of investigation of 54 left and 56 right hip joints was 54.2 years. 1320 measurements were performed. The mean alpha angle was 50.7° in MRI and 50.4° in sonography with a mean difference of 0.28° (p>0.05). Conclusion Determining hip alpha angle using sonography is a safe and reproducible method. No statistically significant differences between results in MRI and sonography could be seen. Although this is a retrospective, single-center study including only Caucasian mid-Europeans and with the known limitations of ultrasound imaging, it nevertheless shows that sonography can be used as a simple, cheap, and fast technique to assess the hip alpha angle without losing diagnostic quality.

Construction of a Retrospective Cohort to Observe 10-Year Urologic Cancer Treatment Trends at the Biggest Medical Center of South Korea

Purpose: To construct a urologic cancer database using a standardized, reproducible method, and to assess preliminary characteristics of this cohort.Materials and Methods: Patients with prostate, bladder, and kidney cancers who were enrolled with diagnostic codes in the electronic medical record (EMR) at Asan Medical Center from 2007–2016 were included. Research Electronic Data Capture (REDCap) was used to design the Asan Medical Center-Urologic Cancer Database (AMC-UCD). The process included developing a data dictionary, applying branching logic, mapping clinical data warehouse structures, alpha testing, clinical record summary testing, creating “standards of procedure,” importing data, and entering data. Descriptive statistics were used to identify rates of surgeries and numbers of patients.Results: Clinical variables (n=407) were selected to develop a data dictionary from REDCap. In total, 20,198 urologic cancer patients visited our institution from 2007–2016 (bladder cancer, 4,616; kidney cancer, 5,750; prostate cancer, 10,330). The overall numbers of patients and surgeries increased over time, with robotic surgeries rapidly growing over a decade. The most common treatment for urologic cancer was surgery, followed by chemotherapy and radiation therapy.Conclusions: Using a standardized method, the AMC-UCD fosters multidisciplinary research. This constructed database provides access to clinical statistics to effectively assist research. Preliminary data should be refined through EMR chart review. The successful organization of data from 2007–2016 provides a framework for future periods of investigation and prospective models.

Characterization and Filtration Efficiency of Sustainable PLA Fibers Obtained via a Hybrid 3D-Printed/Electrospinning Technique

The enormous world demand for personal protective equipment to face the current SARS-CoV-2 epidemic has revealed two main weaknesses. On one hand, centralized production led to an initial shortage of respirators; on the other hand, the world demand for single-use equipment has had a direct and inevitable effect on the environment. Polylactide (PLA) is a biodegradable, biocompatible, and renewable thermoplastic polyester, mainly derived from corn starch. Electrospinning is an established and reproducible method to obtain nano- and microfibrous materials with a simple apparatus, characterized by high air filtration efficiencies. In the present work, we designed and optimized an open-source electrospinning setup, easily realizable with a 3D printer and using components widely available, for the delocalized production of an efficient and sustainable particulate matter filter. Filters were realized on 3D-printed PLA support, on which PLA fibers were subsequently electrospun. NaCl aerosol filtration tests exhibited an efficiency greater than 95% for aerosol having an equivalent diameter greater than 0.3 μm and a fiber diameter comparable to the commercially available FFP2 melt-blown face mask. The particulate entrapped by the filters when operating in real environments (indoors, outdoors, and working scenario) was also investigated, as well as the amount of heavy metals potentially released into the environment after filtration activity.

Landslide Susceptibility Mapping by Comparing GIS-Based Bivariate Methods: A Focus on the Geomorphological Implication of the Statistical Results

Landslide susceptibility is one of the main topics of geomorphological risk studies. Unfortunately, many of these studies applied an exclusively statistical approach with little coherence with the geomorphodynamic models, resulting in susceptibility maps that are difficult to read. Even if many different models have been developed, those based on statistical techniques applied to slope units (SUs) are among the most promising. SU segmentation divides terrain into homogenous domains and approximates the morphodynamic response of the slope to landslides. This paper presents a landslide susceptibility (LS) analysis at the catchment scale for a key area based on the comparison of two GIS-based bivariate statistical methods using the landslide index (LI) approach. A new simple and reproducible method for delineating SUs is defined with an original GIS-based terrain segmentation based on hydrography. For the first time, the morphometric slope index (MSI) was tested as a predisposing factor for landslides. Beyond the purely statistic values, the susceptibility maps obtained have strong geomorphological significance and highlight the areas with the greatest propensity to landslides. We demonstrate the efficiency of the SU segmentation method and the potential of the proposed statistical methods to perform landslide susceptibility mapping (LSM).

Enhancing the stability and crystallinity of CsPbIBr2 through antisolvent engineering

All inorganic lead-based perovskites containing bromine-iodine alloys, such as CsPbIBr2, have arisen as one of the most attractive candidates for absorber layers in solar cells. That said, there remains a large gap when it comes to film and crystal quality between the inorganic and hybrid perovskites. In this work, antisolvent engineering is employed as a simple and reproducible method for improving CsPbIBr2 thin films. We found that both the antisolvent used and the conditions under which it was applied have a measurable impact on both the quality and stability of the final product. We arrived at this conclusion by characterising the samples using scanning electron microscopy, X-ray diffraction, UV–visible and photoluminescence measurements, as well as employing a novel system to quantify stability. Our findings, and the application of our novel method for quantifying stability, demonstrate the ability to significantly enhance CsPbIBr2 samples, produced via a static one-step spin coating method, by applying isopropanol 10 s after commencing the spin programme. The antisolvent quenched CsPbIBr2 films demonstrate both improved crystallinity and an extended lifespan.