A Multi-Center Clinical Study to Harvest and Characterize Circulating Tumor Cells from Patients with Metastatic Breast Cancer Using the Parsortix® PC1 System.

Circulating tumor cells (CTCs) captured from the blood of cancer patients may serve as a surrogate source of tumor material that can be obtained via a venipuncture (also known as a liquid biopsy) and used to better understand tumor characteristics. However, the only FDA-cleared CTC assay has been limited to the enumeration of surface marker-defined cells and not further characterization of the CTCs. In this study, we tested the ability of a semi-automated device capable of capturing and harvesting CTCs from peripheral blood based on cell size and deformability, agnostic of cell-surface markers (the Parsortix® PC1 System), to yield CTCs for evaluation by downstream techniques commonly available in clinical laboratories. The data generated from this study were used to support a De Novo request (DEN200062) for the classification of this device, which the FDA recently granted. As part of a multicenter clinical trial, peripheral blood samples from 216 patients with metastatic breast cancer (MBC) and 205 healthy volunteers were subjected to CTC enrichment. A board-certified pathologist enumerated the CTCs from each participant by cytologic evaluation of Wright-Giemsa-stained slides. As proof of principle, cells harvested from a concurrent parallel sample provided by each participant were evaluated using one of three additional evaluation techniques: molecular profiling by qRT-PCR, RNA sequencing, or cytogenetic analysis of HER2 amplification by FISH. The study demonstrated that the Parsortix® PC1 System can effectively capture and harvest CTCs from the peripheral blood of MBC patients and that the harvested cells can be evaluated using orthogonal methodologies such as gene expression and/or Fluorescence In Situ Hybridization (FISH).

Stacking Machine Learning Algorithms for Biomarker-Based Preoperative Diagnosis of a Pelvic Mass.

OBJECTIVE: To identify the most predictive parameters of ovarian malignancy and develop a machine learning (ML) based algorithm to preoperatively distinguish between a benign and malignant pelvic mass. METHODS: Retrospective study of 70 predictive parameters collected from 140 women with a pelvic mass. The women were split into a 3:1 "training" to "testing" dataset. Feature selection was performed using Gini impurity through an embedded random forest model and principal component analysis. Nine unique ML classifiers were assessed across a variety of model-specific hyperparameters using 25 bootstrap resamples of the training data. Model predictions were then combined into an ensemble stack by LASSO regression. The final ensemble stack and individual classifiers were then applied to the testing dataset to assess model performance. RESULTS: Feature selection identified HE4, CA125, and transferrin as three predictive parameters of malignancy. Assessment of the ensemble stack on the testing dataset outperformed all individual ML classifiers in predicting malignancy. The ensemble stack demonstrated an accuracy of 97.1%, a receiver operating characteristic (ROC) area under the curve (AUC) of 0.951, and a sensitivity of 93.3% with a specificity of 100%. CONCLUSIONS: Combining the measurement of three distinct biomarkers with the stacking of multiple ML classifiers into an ensemble can provide valuable preoperative diagnostic predictions for patients with a pelvic mass.

Volatile organic compounds at two oil and natural gas production well pads in Colorado and Texas using passive samplers.

UNLABELLED: A pilot study was conducted in application of the U.S. Environmental Protection Agency (EPA) Methods 325A/B variant for monitoring volatile organic compounds (VOCs) near two oil and natural gas (ONG) production well pads in the Texas Barnett Shale formation and Colorado Denver-Julesburg Basin (DJB), along with a traffic-dominated site in downtown Denver, CO. As indicated in the EPA method, VOC concentrations were measured for 14-day sampling periods using passive-diffusive tube samplers with Carbopack X sorbent at fenceline perimeter and other locations. VOCs were significantly higher at the DJB well pad versus the Barnett well pad and were likely due to higher production levels at the DJB well pad during the study. Benzene and toluene were significantly higher at the DJB well pad versus downtown Denver. Except for perchloroethylene, VOCs measured at passive sampler locations (PSs) along the perimeter of the Barnett well pad were significantly higher than PSs farther away. At the DJB well pad, most VOC concentrations, except perchloroethylene, were significantly higher prior to operational changes than after these changes were made. Though limited, the results suggest passive samplers are precise (duplicate precision usually ≤10%) and that they can be useful to assess spatial gradients and operational conditions at well pad locations over time-integrated periods. IMPLICATIONS: Recently enacted EPA Methods 325A/B use passive-diffusive tube samplers to measure benzene at multiple fenceline locations at petrochemical refineries. This pilot study presents initial data demonstrating the utility of Methods 325A/B for monitoring at ONG facilities. Measurements revealed elevated concentrations reflective of production levels and spatial gradients of VOCs relative to source proximity at the Barnett well pad, as well as operational changes at the DJB well pad. Though limited, these findings indicate that Methods 325A/B can be useful in application to characterize VOCs at well pad boundaries.

Regulation of plasmid-encoded isoprene metabolism in Rhodococcus, a representative of an important link in the global isoprene cycle.

Emissions of biogenic volatile organic compounds (VOCs) form an important part of the global carbon cycle, comprising a significant proportion of net ecosystem productivity. They impact atmospheric chemistry and contribute directly and indirectly to greenhouse gases. Isoprene, emitted largely from plants, comprises one third of total VOCs, yet in contrast to methane, which is released in similar quantities, we know little of its biodegradation. Here, we report the genome of an isoprene degrading isolate, Rhodococcus sp. AD45, and, using mutagenesis shows that a plasmid-encoded soluble di-iron centre isoprene monooxygenase (IsoMO) is essential for isoprene metabolism. Using RNA sequencing (RNAseq) to analyse cells exposed to isoprene or epoxyisoprene in a substrate-switch time-course experiment, we show that transcripts from 22 contiguous genes, including those encoding IsoMO, were highly upregulated, becoming among the most abundant in the cell and comprising over 25% of the entire transcriptome. Analysis of gene transcription in the wild type and an IsoMO-disrupted mutant strain showed that epoxyisoprene, or a subsequent product of isoprene metabolism, rather than isoprene itself, was the inducing molecule. We provide a foundation of molecular data for future research on the environmental biological consumption of this important, climate-active compound.

Characterization of a feedback-resistant mevalonate kinase from the archaeon Methanosarcina mazei.

The mevalonate pathway is utilized for the biosynthesis of isoprenoids in many bacterial, eukaryotic, and archaeal organisms. Based on previous reports of its feedback inhibition, mevalonate kinase (MVK) may play an important regulatory role in the biosynthesis of mevalonate pathway-derived compounds. Here we report the purification, kinetic characterization, and inhibition analysis of the MVK from the archaeon Methanosarcina mazei. The inhibition of the M. mazei MVK by the following metabolites derived from the mevalonate pathway was explored: dimethylallyl diphosphate (DMAPP), geranyl pyrophosphate (GPP), farnesyl pyrophosphate (FPP), isopentenyl monophosphate (IP), and diphosphomevalonate. M. mazei MVK was not inhibited by DMAPP, GPP, FPP, diphosphomevalonate, or IP, a proposed intermediate in an alternative isoprenoid pathway present in archaea. Our findings suggest that the M. mazei MVK represents a distinct class of mevalonate kinases that can be differentiated from previously characterized MVKs based on its inhibition profile.

Facility fence-line monitoring using passive samplers.

In 2009, the U.S. Environmental Protection Agency (EPA) executed a year-long field study at a refinery in Corpus Christi, TX, to evaluate the use of passive diffusive sampling technology for assessing time-averaged benzene concentrations at the facility fence line. The purpose of the study was to investigate the implementation viability and performance of this type of monitoring in a real-world setting as part of EPA's fence-line measurement research program. The study utilized 14-day, time-integrated Carbopack X samplers deployed at 18 locations on the fence line and at two nearby air monitoring sites equipped with automated gas chromatographs. The average fence-line benzene concentration during the study was 1075 parts per trillion by volume (pptv) with a standard deviation of 1935 pptv. For a 6-month period during which wind direction was uniform, the mean concentration value for a group of downwind sites exceeded the mean value of a similar upwind group by 1710 pptv. Mean value differences for these groups were not statistically significant for the remaining 6-month time period when wind directions were mixed. The passive sampling approach exhibited acceptable performance with a data completeness value of 97.1% (n = 579). Benzene concentration comparisons with automated gas chromatographs yielded an r2 value of 0.86 and a slope of 0.90 (n = 50). A linear regression of duplicate pairs yielded an r2 of 0.97, unity slope, and zero intercept (n = 56). In addition to descriptions of technique performance and general results, time-series analyses are described, providing insight into the utility of 2-week sampling for source apportionment under differing meteorological conditions. The limitations of the approach and recommendations for future measurement method development work are also discussed.

Hydrologic disturbance reduces biological integrity in urban streams.

The impact of urbanization on stream ecosystems is linked by land cover changes to the alteration of the natural hydrology and subsequent physical disruption of stream biota and habitat. Seasonal floods are part of the natural disturbance regime of many streams, but urbanization increases their frequency and magnitude. This study evaluated the impact of hydrologic disturbance on fish and aquatic macroinvertebrates in 81 (56 urban/25 reference) Ohio streams. Hydrologic variables included annual and monthly 24-h rainfall maxima and computed annual peak discharge, with computation supported by GIS-based drainage area delineation and land cover characterization. Ohio biological criteria for fish and macroinvertebrates measured during the late spring and summer were negatively impacted by annual peak discharge in urban streams as compared to reference streams. Results support the application of stormwater best management practices as part of stream restoration efforts to mitigate urbanization impacts to fish and macroinvertebrates.

A bistable switch and anatomical site control Vibrio cholerae virulence gene expression in the intestine.

A fundamental, but unanswered question in host-pathogen interactions is the timing, localization and population distribution of virulence gene expression during infection. Here, microarray and in situ single cell expression methods were used to study Vibrio cholerae growth and virulence gene expression during infection of the rabbit ligated ileal loop model of cholera. Genes encoding the toxin-coregulated pilus (TCP) and cholera toxin (CT) were powerfully expressed early in the infectious process in bacteria adjacent to epithelial surfaces. Increased growth was found to co-localize with virulence gene expression. Significant heterogeneity in the expression of tcpA, the repeating subunit of TCP, was observed late in the infectious process. The expression of tcpA, studied in single cells in a homogeneous medium, demonstrated unimodal induction of tcpA after addition of bicarbonate, a chemical inducer of virulence gene expression. Striking bifurcation of the population occurred during entry into stationary phase: one subpopulation continued to express tcpA, whereas the expression declined in the other subpopulation. ctxA, encoding the A subunit of CT, and toxT, encoding the proximal master regulator of virulence gene expression also exhibited the bifurcation phenotype. The bifurcation phenotype was found to be reversible, epigenetic and to persist after removal of bicarbonate, features consistent with bistable switches. The bistable switch requires the positive-feedback circuit controlling ToxT expression and formation of the CRP-cAMP complex during entry into stationary phase. Key features of this bistable switch also were demonstrated in vivo, where striking heterogeneity in tcpA expression was observed in luminal fluid in later stages of the infection. When this fluid was diluted into artificial seawater, bacterial aggregates continued to express tcpA for prolonged periods of time. The bistable control of virulence gene expression points to a mechanism that could generate a subpopulation of V. cholerae that continues to produce TCP and CT in the rice water stools of cholera patients.

p300 (histone acetyltransferase) biomarker predicts prostate cancer biochemical recurrence and correlates with changes in epithelia nuclear size and shape.

BACKGROUND: p300 impacts the transcription of several genes involved in key pathways critical to PCa progression. Therefore, we evaluated the prognostic value of p300 expression and its correlation with nuclear alterations seen in tumor cells in men with long-term follow-up after radical prostatectomy (RP). METHODS: NCI Cooperative Prostate Cancer Tissue Resource tissue microarray cores of 92 RP cases (56 non-recurrences and 36 PSA recurrences) were utilized for the study. p300 expression was assessed by quantitative immunohistochemistry and nuclear alterations in Feulgen-stained nuclei were evaluated by digital image analysis using the AutoCyte Pathology Workstation. Cox proportional hazards regression, Spearman's rank correlation, and Kaplan-Meier plots were employed to analyze the data. RESULTS: p300 expression significantly correlated with nuclear alterations seen in tumor cells; specifically with circular form factor (P = 0.012) and minimum feret (P = 0.048). p300 expression in high grade tumors (Gleason score >or=7) was significantly higher compared to low grade tumors (Gleason score <7) [17.7% versus 13.7%, respectively, P = 0.03]. TNM stage, Gleason score, and p300 expression were univariately significant in the prediction of PCa biochemical recurrence-free survival (P or=7 and p300 expression >24% showed the highest risk for PCa biochemical recurrence (P = 0.002). CONCLUSIONS: p300 expression correlates with nuclear alterations seen in tumor cells and has prognostic value in predicting long-term PCa biochemical recurrence-free survival.

vpsA- and luxO-independent biofilms of Vibrio cholerae.

The natural life cycle of Vibrio cholerae involves the transitioning of cells between different environmental surfaces such as the chitinous shell of Crustaceae and the epithelial layer of the human intestine. Previous studies using static biofilm systems showed a strict dependence of biofilm formation on the vps and lux genes, which are essential for exopolysaccharide formation and cell-cell signaling, respectively. The authors' report here that in biofilms grown under hydrodynamic conditions, DeltavpsA and DeltaluxO mutants of V. cholerae do form pronounced, three-dimensional biofilms that resemble all aspects of wild-type biofilms. By genetic experiments, it was shown that in hydrodynamically grown biofilms this independence of vpsA is due to the expression of rpoS, which is a negative regulator of vpsA expression. Biofilms also underwent substantial dissolution after 96 h that could be induced by a simple stop of medium flow. The studies indicate that metabolic conditions control the reversible attachment of cells to the biofilm matrix and are key in regulating biofilm cell physiology via RpoS. Furthermore, the results redefine the roles of vps and quorum-sensing in V. cholerae biofilms.

Significant variations in nuclear structure occur between and within Gleason grading patterns 3, 4, and 5 determined by digital image analysis.

BACKGROUND: Alterations in nuclei structure and DNA content captured from Gleason grading patterns 3, 4 and 5 of radical prostatectomy (RP) cases were determined by a computer-assisted microscope. Quantitative Nuclear Morphometry (QNM) profiles were created to evaluate variability in nuclear structure within each of these grades. METHODS: A tissue microarray (TMA) was constructed using RP cases and the prostate cancer (PCa) TMA cores prepared from 20 GG-3, 9 GG-4, 10 GG-5 patterns, and 20 benign cancer-adjacent cases from RP archival paraffin blocks. Feulgen-stained nuclei were captured from 0.6 mm spots using the AutoCyte system. Pools of 1100 nuclei captured from each test group were used to calculate Multivariate Logistic Regression (MLR) models that generated predictive indices and predictive probabilities (PP) to make comparisons between and within each set of pooled nuclei. RESULTS: A single QNM profiles yielded areas of receiver operator characteristic curves (ROC) that distinguished differences among benign cancer-adjacent nuclei and GG-3 (ROC-AUC = 0.78); GG-4 (ROC-AUC = 0.86) and GG-5 (ROC-AUC = 0.88) with accuracies of 73%, 78% and 80% respectively. Applying PP plots generated from MLR models of GG 3, 4, and 5 nuclei clearly demonstrated marked heterogeneity within each of these three GG patterns. CONCLUSIONS: QNM signatures illustrate alterations in nuclei structure, based upon nuclear morphometry within each of these three GG patterns, and might signify potential variations in PCa disease risk of progression outcomes. In the future a modified system of Gleason grading that considers not only glandular architecture but also quantitative nuclear grade may ensure accuracy in prognosis.

Genomic and phenotypic diversity of coastal Vibrio cholerae strains is linked to environmental factors.

Studies of Vibrio cholerae diversity have focused primarily on pathogenic isolates of the O1 and O139 serotypes. However, autochthonous environmental isolates of this species routinely display more extensive genetic diversity than the primarily clonal pathogenic strains. In this study, genomic and metabolic profiles of 41 non-O1/O139 environmental isolates from central California coastal waters and four clinical strains are used to characterize the core genome and metabolome of V. cholerae. Comparative genome hybridization using microarrays constructed from the fully sequenced V. cholerae O1 El Tor N16961 genome identified 2,787 core genes that approximated the projected species core genome within 1.6%. Core genes are almost universally present in strains with widely different niches, suggesting that these genes are essential for persistence in diverse aquatic environments. In contrast, the dispensable genes and phenotypic traits identified in this study should provide increased fitness for certain niche environments. Environmental parameters, measured in situ during sample collection, are correlated to the presence of specific dispensable genes and metabolic capabilities, including utilization of mannose, sialic acid, citrate, and chitosan oligosaccharides. These results identify gene content and metabolic pathways that are likely selected for in certain coastal environments and may influence V. cholerae population structure in aquatic environments.