Assessing non-adjunctive CGM safety at home and in new markets (ANSHIN).

INTRODUCTION: Continuous glucose monitoring (CGM) can guide treatment for people with type 1 (T1D) and type 2 diabetes (T2D). The ANSHIN study assessed the impact of non-adjunctive CGM use in adults with diabetes using intensive insulin therapy (IIT). MATERIALS AND METHODS: This single-arm, prospective, interventional study enrolled adults with T1D or T2D who had not used CGM in the prior 6 months. Participants wore blinded CGMs (Dexcom G6) during a 20-day run-in phase, with treatment based on fingerstick glucose values, followed by a 16-week intervention phase and then a randomized 12-week extension phase with treatment based on CGM values. The primary outcome was change in HbA1c. Secondary outcomes were CGM metrics. Safety endpoints were the number of severe hypoglycaemic (SH) and diabetic ketoacidosis (DKA) events. RESULTS: Of the 77 adults enrolled, 63 completed the study. Those enrolled had mean (SD) baseline HbA1c of 9.8% (1.9%), 36% had T1D, and 44% were ≥65 years old. Mean HbA1c decreased by 1.3, 1.0 and 1.0 percentage points for participants with T1D, T2D or age ≥65, respectively (p < .001 for each). CGM-based metrics including time in range also improved significantly. SH events decreased from the run-in period (67.3 per 100 person-years) to the intervention period (17.0 per 100 person-years). Three DKA events unrelated to CGM use occurred during the total intervention period. CONCLUSIONS: Non-adjunctive use of the Dexcom G6 CGM system improved glycaemic control and was safe for adults using IIT.

Measuring DNA modifications with the comet assay: a compendium of protocols.

The comet assay is a versatile method to detect nuclear DNA damage in individual eukaryotic cells, from yeast to human. The types of damage detected encompass DNA strand breaks and alkali-labile sites (e.g., apurinic/apyrimidinic sites), alkylated and oxidized nucleobases, DNA-DNA crosslinks, UV-induced cyclobutane pyrimidine dimers and some chemically induced DNA adducts. Depending on the specimen type, there are important modifications to the comet assay protocol to avoid the formation of additional DNA damage during the processing of samples and to ensure sufficient sensitivity to detect differences in damage levels between sample groups. Various applications of the comet assay have been validated by research groups in academia, industry and regulatory agencies, and its strengths are highlighted by the adoption of the comet assay as an in vivo test for genotoxicity in animal organs by the Organisation for Economic Co-operation and Development. The present document includes a series of consensus protocols that describe the application of the comet assay to a wide variety of cell types, species and types of DNA damage, thereby demonstrating its versatility.

Longitudinal analysis of users transitioning from the Dexcom G5 to the G6 RT-CGM system in Germany, Sweden and the United Kingdom (2018-2020).

AIMS: Regional variations in the adoption of diabetes technology may be reflected in population-level metrics of glycaemic control. In this observational study, we aimed to assess the glycaemic impacts of transitioning from the Dexcom G5 Real-Time Continuous Glucose Monitoring (RT-CGM) System to the Dexcom G6 in three European countries. METHODS: Anonymised RT-CGM data (uploaded to the Dexcom Clarity app) were from users in Germany, Sweden, and the United Kingdom (UK) who transitioned from G5 to G6 between 9-12 months after G6 launched in 2018. Primary endpoints were percent time in hypoglycaemia, percent time in range (TIR), user retention rates, device utilisation, and urgent low soon (ULS) alert utilisation. Metrics were computed for 3-month intervals in the 2-year study window. RESULTS: In all three countries, the transition from G5 to G6 was associated with a clear decrease in hypoglycaemia. In months 0-3 after transitioning, the median percent time 〈3 mmol/L (54 mg/dL) and 〈3.9 mmol/L (70 mg/dL) decreased by [0.12-0.28] and [0.40-0.43] percentage points, respectively, with another [0.11-0.21] and [0.34-0.65] percentage point decrease in months 3-6 in the three countries analysed. TIR and CGM utilisation were sustained or improved slightly across all countries. At the end of the study window, the retention rate was [88.8-94.8%] and ULS utilization was [83.9-86.9%] in the three countries analysed. CONCLUSIONS: Similar RT-CGM trends were observed across Germany, Sweden, and the UK. Improvements in hypoglycaemia occurred in all countries. The high retention of users may lead to sustained glycaemic benefits associated with RT-CGM use.

Integrated Genotoxicity Testing of three anti-infective drugs using the TGx-DDI transcriptomic biomarker and high-throughput CometChip® assay in TK6 cells.

Genotoxicity testing relies on the detection of gene mutations and chromosome damage and has been used in the genetic safety assessment of drugs and chemicals for decades. However, the results of standard genotoxicity tests are often difficult to interpret due to lack of mode of action information. The TGx-DDI transcriptomic biomarker provides mechanistic information on the DNA damage-inducing (DDI) capability of chemicals to aid in the interpretation of positive in vitro genotoxicity data. The CometChip® assay was developed to assess DNA strand breaks in a higher-throughput format. We paired the TGx-DDI biomarker with the CometChip® assay in TK6 cells to evaluate three model agents: nitrofurantoin (NIT), metronidazole (MTZ), and novobiocin (NOV). TGx-DDI was analyzed by two independent labs and technologies (nCounter® and TempO-Seq®). Although these anti-infective drugs are, or have been, used in human and/or veterinary medicine, the standard genotoxicity testing battery showed significant genetic safety findings. Specifically, NIT is a mutagen and causes chromosome damage, and MTZ and NOV cause chromosome damage in conventional in vitro tests. Herein, the TGx-DDI biomarker classified NIT and MTZ as non-DDI at all concentrations tested, suggesting that NIT's mutagenic activity is bacterial specific and that the observed chromosome damage by MTZ might be a consequence of in vitro test conditions. In contrast, NOV was classified as DDI at the second highest concentration tested, which is in line with the fact that NOV is a bacterial DNA-gyrase inhibitor that also affects topoisomerase II at high concentrations. The lack of DNA damage for NIT and MTZ was confirmed by the CometChip® results, which were negative for all three drugs except at overtly cytotoxic concentrations. This case study demonstrates the utility of combining the TGx-DDI biomarker and CometChip® to resolve conflicting genotoxicity data and provides further validation to support the reproducibility of the biomarker.

With or Without Residual C-Peptide, Patients with Type 2 Diabetes Realize Glycemic Benefits from Real-Time Continuous Glucose Monitoring.

Real-time continuous glucose monitoring (RT-CGM) is superior to blood glucose monitoring (BGM) for adults with insulin-treated type 2 diabetes (T2D); however, the utility of C-peptide levels for predicting the magnitude of the glycemic benefits is controversial. Data were from a subset of 147 participants in the MOBILE study who were treated with basal-only insulin and who had baseline C-peptide levels ≥0.5 ng/mL. Participants were randomized to treatment with either RT-CGM (n = 100) or BGM (n = 47). Between-group differences in hemoglobin A1c (HbA1c) and time in range (TIR) changes were assessed. The between-group difference in HbA1c favored the RT-CGM group (by 0.58 percentage points, P = 0.004 at 3 months and by 0.42 percentage points, P = 0.04 at 8 months). TIR was 16% higher, and time >180 mg/dL was 16% lower, in the RT-CGM group at 8 months (P = 0.002 for each). In T2D managed with basal insulin, RT-CGM benefits occur for those with residual insulin secretory capacity. Clinical Trial Identifier: NCT03566693.

SpheroidChip: Patterned Agarose Microwell Compartments Harboring HepG2 Spheroids are Compatible with Genotoxicity Testing.

Three-dimensional tissue culture models are emerging as effective alternatives to animal testing. They are especially beneficial for liver toxicity studies, enabling hepatocytes to display improved levels of liver-specific functions. One common model is hepatocyte spheroids, which are spontaneously formed cell aggregates. Techniques for spheroid formation include the use of ultralow attachment plates and the hanging drop method, both of which are technically challenging and relatively low throughput. Here, we describe a simple-to-use platform that improves spheroid production and is compatible with genotoxicity testing by the comet assay. To achieve this, we created a chip containing a microwell array where dozens of spheroids are contained within a single well of a 96-well plate. The microwells are made from agarose, a nontoxic material suitable for cell growth and spheroid formation. HepG2 cells loaded into customizable microwells formed spheroids through agarose-assisted aggregation within one to two days. In addition, the agarose matrix allows the same platform to be used in DNA damage analysis. Specifically, the comet assay enables quantification of DNA breaks based on the increased migration of damaged DNA through agarose during electrophoresis. Here, we developed a modified comet assay and show that intact HepG2 spheroids cultured in microwells can be electrophoresed to reveal the extent of DNA damage following exposure to inflammatory chemicals (H2O2 and SIN-1). With this SpheroidChip analysis method, we detected a dose-dependent increase in DNA damage and observed rapid repair of H2O2-induced DNA damage. In summary, we utilized an agarose microarray to condense what had required an entire 96-well plate into a single well, enabling analysis techniques that were cumbersome or impossible under conditions of a single spheroid per well of a 96-well plate.

Applications of CometChip for Environmental Health Studies.

Environmental exposures have long been known to impact public health and safety. For example, exposures to airborne particulates, heavy metals in water, or certain industrial chemicals can contribute to aging and to risk of developing cancer and other diseases. Environmental factors can impact health in a variety of ways, but a key concern is DNA damage, which can lead to mutations that cause cancer. Cancer can take years to develop following chemical exposure; however, one way to predict carcinogenicity in a more practical time frame is by studying the chemical's ability to induce DNA damage. The comet assay (or single-cell gel electrophoresis assay) has been used successfully for genotoxicity testing. The comet assay allows for the detection of DNA strand breaks via analysis of DNA migration during electrophoresis. Previously, the Engelward laboratory, in collaboration with the Bhatia laboratory, developed the CometChip for measurements of DNA damage and repair. The CometChip is a high-throughput comet assay that improves user reproducibility and significantly shortens total assay time. Here, we describe how the high-throughput CometChip platform can be used to measure DNA damage in established cell lines, animal models, and human samples. We also discuss technical challenges associated with these studies and provide recommendations on how to achieve optimal results for researchers interested in adopting this assay.