Detection of Circulating Tumor Cells Using the Attune NxT

Circulating tumor cells (CTCs) have been detected in many patients with different solid malignancies. It has been reported that presence of CTCs correlates with worse survival in patients with multiple types of cancer. Several techniques have been developed to detect CTCs in liquid biopsies. Currently, the only method for CTC detection that is approved by the Food and Drug Administration is CellSearch. Due to low abundance of CTCs in certain cancer types and in early stages of disease, its clinical application is currently limited to metastatic colorectal cancer, breast cancer and prostate cancer. Therefore, we aimed to develop a new method for the detection of CTCs using the Attune NxT—a flow cytometry-based application that was specifically developed to detect rare events in biological samples without the need for enrichment. When healthy donor blood samples were spiked with variable amounts of different EpCAM+EGFR+ tumor cell lines, recovery yield was on average 75%. The detection range was between 1000 and 10 cells per sample. Cell morphology was confirmed with the Attune CytPix. Analysis of blood samples from metastatic colorectal cancer patients, as well as lung cancer patients, demonstrated that increased EpCAM+EGFR+ events were detected in more than half of the patient samples. However, most of these cells showed no (tumor) cell-like morphology. Notably, CellSearch analysis of blood samples from a subset of colorectal cancer patients did not detect CTCs either, suggesting that these blood samples were negative for CTCs. Therefore, we anticipate that the Attune NxT is not superior to CellSearch in detection of low amounts of CTCs, although handling and analysis of samples is easier. Moreover, morphological confirmation is essential to distinguish between CTCs and false positive events.     

High Acetate Concentration Protects Intestinal Barrier and Exerts Anti-Inflammatory Effects in Organoid-Derived Epithelial Monolayer Cultures from Patients with Ulcerative Colitis

Short-chain fatty acids as well as their bacterial producers are of increasing interest in inflammatory bowel diseases. Although less studied compared to butyrate, acetate might also be of interest as it may be less toxic to epithelial cells, stimulate butyrate-producing bacteria by cross-feeding, and have anti-inflammatory and barrier-protective properties. Moreover, one of the causative factors of the probiotic potency of Saccharomyces cerevisae var. boulardii is thought to be its high acetate production. Therefore, the objective was to preclinically assess the effects of high acetate concentrations on inflammation and barrier integrity in organoid-based monolayer cultures from ulcerative colitis patients. Confluent organoid-derived colonic epithelial monolayers (n = 10) were exposed to basolateral inflammatory stimulation or control medium. After 24 h, high acetate or control medium was administered apically for an additional 48 h. Changes in TEER were measured after 48 h. Expression levels of barrier genes and inflammatory markers were determined by qPCR. Pro-inflammatory proteins in the supernatant were quantified using the MSD platform. Increased epithelial resistance was observed with high acetate administration in both inflamed and non-inflamed conditions, together with decreased expression levels of IL8 and TNFα and CLDN1. Upon high acetate administration to inflamed monolayers, upregulation of HIF1α, MUC2, and MKI67, and a decrease of the majority of pro-inflammatory cytokines was observed. In our patient-derived human epithelial cell culture model, a protective effect of high acetate administration on epithelial resistance, barrier gene expression, and inflammatory protein production was observed. These findings open up new possibilities for acetate-mediated management of barrier defects and inflammation in IBD.     

A simulator study on the impact of traffic calming measures in urban areas on driving behavior and workload

This study examined the impact of traffic calming measures (TCM) on major roads in rural and urban areas. More specifically we investigated the effect of gate constructions located at the entrance of the urban area and horizontal curves within the urban area on driving behavior and workload. Forty-six participants completed a 34 km test-drive on a driving simulator with eight thoroughfare configurations, i.e., 2 (curves: present, absent) × 2 (gates: present, absent) × 2 (peripheral detection task (PDT): present, absent) in a within-subject design.     

Application of culture-dependent and culture-independent methods for the identification of Lactobacillus kefiranofaciens in microbial consortia present in kefir grains

The biological and technological characteristics of kefiran as well as its importance in grain integrity led us to analyze the microbial kefir grain consortium with focus on Lactobacillus kefiranofaciens. The presence of L. kefiranofaciens in the nine kefir grains studied was demonstrated by denaturing gradient gel electrophoresis. By culture dependent methods applying a methodology focused on the search of this species, 22 isolates with typical morphology were obtained and identified applying a combination of SDS-PAGE of whole cell proteins, (GTG)₅-PCR and sequence analysis of the housekeeping gene encoding the α-subunit of bacterial phenylalanyl-tRNA synthase (pheS). This polyphasic approach allowed the reliable identification of 11 L. kefiranofaciens, 5 Lactobacillus paracasei, 4 Lactobacillus kefiri and 2 Lactobacillus parakefiri isolates. Isolated L. kefiranofaciens strains produced polysaccharide in strain-dependent concentrations and EPS produced by them also differed in the degree of polymerization. The isolation and accurate identification of L. kefiranofaciens is relevant taking into account the important role of this microorganism in the grain ecosystem as well as its potential application as starter in food fermentations.     

Transfer of pyrrolizidine alkaloids from various herbs to eggs and meat in laying hens

To investigate the potential transfer of pyrrolizidine alkaloids (PAs), laying hens were fed for 14 days with diets containing 0.5% of dried common ragwort, common groundsel, narrow-leaved ragwort or viper’s bugloss, or 0.1% of common heliotrope. This resulted in total PA levels in feed of respectively 5.5, 11.1, 53.1, 5.9 and 21.7 mg kg^–1, with varying composition. PAs were transferred to eggs, in particular yolk, with steady-state levels of respectively 12, 21, 216, 2 and 36 µg kg^–1. Overall transfer rates for the sum of PAs were estimated between 0.02% and 0.23%, depending on the type of PAs in the feed. In animals slaughtered shortly after the last exposure, levels in meat were slightly lower than those in eggs, levels in livers somewhat higher. When switched to clean feed, levels in eggs gradually decreased, but after 14 days were still above detection limits in the hens exposed to higher PA levels. Similar was the case for meat and especially kidneys and livers. It is concluded that the intake of PA containing herbs by laying hens may result in levels in eggs and meat that could be of concern for consumers, and as such should be avoided.     

A field study to determine the prevalence,dairy herd management systems,and fresh cow clinical conditions associated with ketosis in western European dairy herds

The aim of this study was to determine the prevalence, major management systems, and fresh cow clinical conditions associated with ketosis in western European dairy herds. A total of 131 dairies were enrolled in Germany, France, Italy, the Netherlands, and the United Kingdom during 2011 to 2012. A milk-based test for ketones (Keto-Test; Sanwa Kagaku Kenkyusho Co. Ltd., Nagoya, Japan; distributed by Elanco Animal Health, Antwerp, Belgium) was used for screening cows between d 7 and 21 after calving and ketosis was defined as a Keto-Test ≥100 µmol/L. Study cows were observed for clinical disease up to 35 d postcalving. Multivariate analysis (generalized estimating equation logistic regression) was performed to determine country, farm, management, feed, and cow factors associated with ketosis and to determine associations between ketosis and fresh cow diseases. Thirty-nine percent of the cows were classified as having ketosis. The herd average of ketosis was 43% in Germany, 53% in France, 31% in Italy, 46% in the Netherlands, and 31% in the United Kingdom. Of the 131 farms, 112 (85%) had 25% or more of their fresh cows resulting as positive for ketosis. Clinical ketosis was not reported in most farms and the highest level of clinical ketosis reported was 23%. The risks of ketosis were significantly lower in Italy and the United Kingdom compared with France, the Netherlands, and Germany. Larger herd size was associated with a decreased risk of ketosis. The farms that fed partially mixed rations had 1.5 times higher odds of ketosis than those that fed total mixed rations. Cows that calved in April to June had the highest odds of ketosis, with about twice as high odds compared with cows that calved in July to September. The cows that calved in January to March tended to have 1.5 times higher risk of ketosis compared with cows that calved in July to September. The odds of ketosis in parity 2 and parity 3 to 7 was significantly higher (1.5 and 2.8 times higher, respectively) than the odds of ketosis in parity 1. The odds of ketosis was significantly smaller in parity 2 compared with parity 3 to 7. Ketosis was associated with significantly higher odds of all common fresh cow conditions: metritis, mastitis, displaced abomasum, clinical ketosis, lameness, and gastrointestinal disorders. Odds of ketosis in cows having had twins or dystocia were not increased, whereas higher odds of ketosis were observed in cows with milk fever or retained placenta.     

Guest Editorial: Special Issue on Global Hydrological Datasets for Local Water Management Applications

Water Resources Management -