Benchmarking informatics approaches for virus discovery: caution is needed when combining in silico identification methods.

Understanding the ecological impacts of viruses on natural and engineered ecosystems relies on the accurate identification of viral sequences from community sequencing data. To maximize viral recovery from metagenomes, researchers frequently combine viral identification tools. However, the effectiveness of this strategy is unknown. Here, we benchmarked combinations of six widely used informatics tools for viral identification and analysis (VirSorter, VirSorter2, VIBRANT, DeepVirFinder, CheckV, and Kaiju), called "rulesets." Rulesets were tested against mock metagenomes composed of taxonomically diverse sequence types and diverse aquatic metagenomes to assess the effects of the degree of viral enrichment and habitat on tool performance. We found that six rulesets achieved equivalent accuracy [Matthews Correlation Coefficient (MCC) = 0.77, Padj ≥ 0.05]. Each contained VirSorter2, and five used our "tuning removal" rule designed to remove non-viral contamination. While DeepVirFinder, VIBRANT, and VirSorter were each found once in these high-accuracy rulesets, they were not found in combination with each other: combining tools does not lead to optimal performance. Our validation suggests that the MCC plateau at 0.77 is partly caused by inaccurate labeling within reference sequence databases. In aquatic metagenomes, our highest MCC ruleset identified more viral sequences in virus-enriched (44%-46%) than in cellular metagenomes (7%-19%). While improved algorithms may lead to more accurate viral identification tools, this should be done in tandem with careful curation of sequence databases. We recommend using the VirSorter2 ruleset and our empirically derived tuning removal rule. Our analysis provides insight into methods for in silico viral identification and will enable more robust viral identification from metagenomic data sets. IMPORTANCE: The identification of viruses from environmental metagenomes using informatics tools has offered critical insights in microbial ecology. However, it remains difficult for researchers to know which tools optimize viral recovery for their specific study. In an attempt to recover more viruses, studies are increasingly combining the outputs from multiple tools without validating this approach. After benchmarking combinations of six viral identification tools against mock metagenomes and environmental samples, we found that these tools should only be combined cautiously. Two to four tool combinations maximized viral recovery and minimized non-viral contamination compared with either the single-tool or the five- to six-tool ones. By providing a rigorous overview of the behavior of in silico viral identification strategies and a pipeline to replicate our process, our findings guide the use of existing viral identification tools and offer a blueprint for feature engineering of new tools that will lead to higher-confidence viral discovery in microbiome studies.

Orthopaedic Manifestations of Thanatophoric Dwarfism: A Case Report.

CASE: We report the rare case of a 3-year-old male patient with thanatophoric dwarfism, a fatal skeletal dysplasia that arises from an autosomal dominant mutation in the fibroblast growth factor receptor 3 gene. The role of the orthopaedic surgeon in the in the management of this disease is discussed. CONCLUSION: We advocate for the close monitoring of disease progression by the orthopaedic surgery team and offer a potential surgical intervention that may help prevent cardiorespiratory demise.

A Novel Method to Achieve Precision and Reproducibility in Exposure Parameters for Low-Frequency Pulsed Magnetic Fields in Human Cell Cultures.

The effects of extremely low-frequency electromagnetic field (ELF-MF) exposure on living systems have been widely studied at the fundamental level and also claimed as beneficial for the treatment of diseases for over 50 years. However, the underlying mechanisms and cellular targets of ELF-MF exposure remain poorly understood and the field has been plagued with controversy stemming from an endemic lack of reproducibility of published findings. To address this problem, we here demonstrate a technically simple and reproducible EMF exposure protocol to achieve a standardized experimental approach which can be readily adopted in any lab. As an assay system, we chose a commercially available inflammatory model human cell line; its response to magnetic fields involves changes in gene expression which can be monitored by a simple colorimetric reporter gene assay. The cells were seeded and cultured in microplates and inserted into a custom-built, semi-automated incubation and exposure system which accurately controls the incubation (temperature, humidity, CO2) and magnetic-field exposure conditions. A specific alternating magnetic field (<1.0% spatial variance) including far-field reduction provided defined exposure conditions at the position of each well of the microplate. To avoid artifacts, all environmental and magnetic-field exposure parameters were logged in real time throughout the duration of the experiment. Under these extensively controlled conditions, the effect of the magnetic field on the cell cultures as assayed by the standardized operating procedure was highly reproducible between experiments. As we could fully define the characteristics (frequency, intensity, duration) of the pulsed magnetic field signals at the position of the sample well, we were, for the first time, able to accurately determine the effect of changing single ELF-MF parameters such as signal shape, frequency, intensity and duty cycle on the biological response. One signal in particular (10 Hz, 50% duty cycle, rectangular, bipolar, 39.6µT) provided a significant reduction in cytokine reporter gene expression by 37% in our model cell culture line. In sum, the accuracy, environmental control and data-logging capacity of the semi-automated exposure system should greatly facilitate research into fundamental cellular response mechanisms and achieve the consistency necessary to bring ELF-MF/PEMF research results into the scientific mainstream.

Microbial subnetworks related to short-term diel O2 fluxes within geochemically distinct freshwater wetlands.

Oxygen (O2) concentrations often fluctuate over diel timescales within wetlands, driven by temperature, sunlight, photosynthesis and respiration. These daily fluxes have been shown to impact biogeochemical transformations (e.g. denitrification), which are mediated by the residing microbial community. However, little is known about how resident microbial communities respond to diel physical and chemical fluxes in freshwater wetland ecosystems. In this study, total microbial (bacterial and archaeal) community structure was significantly related to diel time points in just one out of four distinct freshwater wetlands sampled. This suggests that daily environmental shifts may influence wetlands differentially based upon the resident microbial community and specific physical and chemical conditions of a freshwater wetland. When exploring the microbial communities within each wetland at finer resolutions, subcommunities of taxa within two wetlands were found to correspond to fluctuating O2 levels. Microbial taxa that were found to be susceptible to fluctuating O2 levels within these subnetworks may have intimate ties to metabolism and/or diel redox cycles. This study highlights that freshwater wetland microbial communities are often stable in community structure when confronted with short-term O2 fluxes; however, specialist taxa may be sensitive to these same fluxes.

Comparison of HER2 Dual-Color and Fluorescence In Situ Hybridization in Breast Cancer: A Cohort Study Emphasizing Equivocal Cases.

OBJECTIVES: Human epidermal growth factor receptor 2 (HER2; ERBB2 gene) is of prognostic and predictive significance in breast carcinoma. Both fluorescence in situ hybridization (FISH) and dual-color in situ hybridization (DISH) methods are available. DISH and FISH are highly concordant in validation studies, but differences may be more prevalent in the equivocal range. Our goal was to compare FISH and DISH on a cohort enriched for equivocal cases, with respect to HER2 determination. METHODS: The cohort was enriched for equivocal (2+) cases. DISH and FISH were evaluated using standard protocols and the results compared with respect to HER2 status, HER2 copy number, and HER2/chromosome 17 (Chr17) ratio. RESULTS: In total, 109 cases were identified. The agreement rate of DISH with FISH was 74%. The mean ± SD HER2/Chr17 ratio by DISH was 1.63 ± 0.08 vs 1.59 ± 0.26 by FISH (P = .45). The mean ± SD HER2 copy number by DISH was 4.56 ± 0.45 vs 4.75 ± 1.08 by FISH (P = .004). Individual signals were more easily resolved using FISH in cases with higher copy numbers. CONCLUSIONS: In our cohort enriched for equivocal cases, the numerical values of HER2 copy number were significantly lower using DISH, resulting in discordances. Although DISH is a valid method, variations with FISH may be expected in high-equivocal cases and in quality assurance activities.

Utility of p16(ink4a) immunocytochemistry in liquid-based cytology specimens from women treated for high-grade squamous intraepithelial lesions.

OBJECTIVE: To examine whether p16(ink4a) immunocytochemical (ICC) expression detected intraepithelial disease in liquid-based cytology (LBC) specimens from women with high-grade squamous intraepithelial lesions (HSIL), whose specimen was labeled negative for intraepithelial lesion or malignany (NILM). STUDY DESIGN: Residual LBC specimens from women treated for HSIL (n = 21), whose LBC test was interpreted as NILM including marked benign inflammatory changes (BCC) were used. The control (n = 25) consisted of residual LBC specimens from women with documented HSIL. ICC for p16p(16k4a) was performed on a second ThinPrep (ThinPrep 2000, Cylyl Corporation, Boxborough, Massachusetts, U.S.A.) preparation; the percentage ofpositive cells and intensity of immunostaining were recorded. Standard LBC preparations for p16(ink4a) ICC-positive and ICC-negative control cases were reviewed. RESULTS: Twenty-four of 25 (96%) of the HSIL control group were ICC p16(ink4a) positive. In the NILM/BCC group, 2 of 21 with adequate LBC residua were ICC p16(ink4a) positive; on review both were reclassified as epithelial abnormality--1 HSIL and 1 atypical squamous cells cannot exclude HSIL. In both, subsequent colposcopic biopsy yielded HSIL. CONCLUSION: p16(ink4a) ICC positivity on NILM/BCC LBC residua from patients with HSIL may identify cases that merit cytologic review and possible reclassification. The utility of p16(ink4a) ICC in this situation requires further study.