Characterizing the tumor immune microenvironment of ependymomas using targeted gene expression profiles and RNA sequencing.

BACKGROUND: Defining the tumor immune microenvironment (TIME) of patients using transcriptome analysis is gaining more popularity. Here, we examined and discussed the pros and cons of using RNA sequencing for fresh frozen samples and targeted gene expression immune profiles (NanoString) for formalin-fixed, paraffin-embedded (FFPE) samples to characterize the TIME of ependymoma samples. RESULTS: Our results showed a stable expression of the 40 housekeeping genes throughout all samples. The Pearson correlation of the endogenous genes was high. To define the TIME, we first checked the expression of the PTPRC gene, known as CD45, and found it was above the detection limit in all samples by both techniques. T cells were identified consistently using the two types of data. In addition, both techniques showed that the immune landscape was heterogeneous in the 6 ependymoma samples used for this study. CONCLUSIONS: The low-abundant genes were detected in higher quantities using the NanoString technique, even when FFPE samples were used. RNA sequencing is better suited for biomarker discovery, fusion gene detection, and getting a broader overview of the TIME. The technique that was used to measure the samples had a considerable effect on the type of immune cells that were identified. The limited number of tumor-infiltrating immune cells compared to the high density of tumor cells in ependymoma can limit the sensitivity of RNA expression techniques regarding the identification of the infiltrating immune cells.

(1) H MRS in the human spinal cord at 7 T using a dielectric waveguide transmitter, RF shimming and a high density receive array.

Multimodal MRI is the state of the art method for clinical diagnostics and therapy monitoring of the spinal cord, with MRS being an emerging modality that has the potential to detect relevant changes of the spinal cord tissue at an earlier stage and to enhance specificity. Methodological challenges related to the small dimensions and deep location of the human spinal cord inside the human body, field fluctuations due to respiratory motion, susceptibility differences to adjacent tissue such as vertebras and pulsatile flow of the cerebrospinal fluid hinder the clinical application of (1) H MRS to the human spinal cord. Complementary to previous studies that partly addressed these problems, this work aims at enhancing the signal-to-noise ratio (SNR) of (1) H MRS in the human spinal cord. To this end a flexible tight fit high density receiver array and ultra-high field strength (7 T) were combined. A dielectric waveguide and dipole antenna transmission coil allowed for dual channel RF shimming, focusing the RF field in the spinal cord, and an inner-volume saturated semi-LASER sequence was used for robust localization in the presence of B1 (+) inhomogeneity. Herein we report the first 7 T spinal cord (1) H MR spectra, which were obtained in seven independent measurements of 128 averages each in three healthy volunteers. The spectra exhibit high quality (full width at half maximum 0.09 ppm, SNR 7.6) and absence of artifacts and allow for reliable quantification of N-acetyl aspartate (NAA) (NAA/Cr (creatine) 1.31 ± 0.20; Cramér-Rao lower bound (CRLB) 5), total choline containing compounds (Cho) (Cho/Cr 0.32 ± 0.07; CRLB 7), Cr (CRLB 5) and myo-inositol (mI) (mI/Cr 1.08 ± 0.22; CRLB 6) in 7.5 min in the human cervical spinal cord. Thus metabolic information from the spinal cord can be obtained in clinically feasible scan times at 7 T, and its benefit for clinical decision making in spinal cord disorders will be investigated in the future using the presented methodology. Copyright © 2016 John Wiley & Sons, Ltd.

Comparative accessory gene fingerprinting of surface water Escherichia coli reveals genetically diverse naturalized population.

AIMS: To utilize comparative accessory gene fingerprinting to discriminate between naturalized and faecal Escherichia coli, with particular emphasis on strains from phylogroup B1. METHODS AND RESULTS: Fourteen accessory genes that were potentially ecotype-specific were selected on the basis of comparative genomic DNA sequence analysis between faecal and environmental strains and also using a literature-based strategy. PCR assays were designed for each gene, and used to screen 107 faecal strains from various hosts and 106 environmental strains from surface water and sediment. While none of the 14 accessory genes were ecotype-specific, six of the genes were ecotype-enriched. Specifically, toxin-antitoxin system genes were more abundant among faecal strains, whereas genes involved in iron acquisition, complement resistance/surface exclusion, and biofilm formation were more abundant among environmental strains. These six genes were used to form composite fingerprints which revealed the presence of several ecotype-specific and -enriched fingerprints. Notably, some of the environmental strain-specific or -enriched fingerprints consisted of strains putatively belonging to clade ET-1, which has been previously recognized as a naturalized subpopulation. CONCLUSIONS: Unlike single genes which did not reliably distinguish between faecal and naturalized phylogroup B1 E. coli strains, composite fingerprints of ecotype-enriched accessory genes may offer a novel method for distinguishing between these two populations. SIGNIFICANCE AND IMPACT OF THE STUDY: Accessory gene fingerprinting may have important practical implications for improving the specificity of methods that are widely used for quantifying and identifying the sources of faecal contamination in surface water.

Traumatic brain injury promotes neurogenesis at the cost of astrogliogenesis in the adult hippocampus of male mice.

Traumatic brain injury (TBI) can result in long-lasting changes in hippocampal function. The changes induced by TBI on the hippocampus contribute to cognitive deficits. The adult hippocampus harbors neural stem cells (NSCs) that generate neurons (neurogenesis), and astrocytes (astrogliogenesis). While deregulation of hippocampal NSCs and neurogenesis have been observed after TBI, it is not known how TBI may affect hippocampal astrogliogenesis. Using a controlled cortical impact model of TBI in male mice, single cell RNA sequencing and spatial transcriptomics, we assessed how TBI affected hippocampal NSCs and the neuronal and astroglial lineages derived from them. We observe an increase in NSC-derived neuronal cells and a concomitant decrease in NSC-derived astrocytic cells, together with changes in gene expression and cell dysplasia within the dentate gyrus. Here, we show that TBI modifies NSC fate to promote neurogenesis at the cost of astrogliogenesis and identify specific cell populations as possible targets to counteract TBI-induced cellular changes in the adult hippocampus.

High-resolution MRI of the carotid arteries using a leaky waveguide transmitter and a high-density receive array at 7 T.

A setup for 7T MRI of the carotid arteries in the neck was designed and constructed. Separate dedicated arrays were used for transmit and receive. For the transmit array, single-side adapted dipole antennas were mounted on a dielectric pillow, which was shown to serve as a leaky waveguide, efficiently distributing B1 into the neck. Risk assessment was performed by simulations. Phantom measurements were performed to establish optimal positions of the antennas on the pillow. Using two antennas, a dual transmit setup was created. In vivo B1 (+) maps with different shim configurations were acquired to assess transmit performance. This effective transmit array was used in combination with a dedicated 30 channel small element receive coil. High-resolution in vivo turbo spin echo images were acquired to demonstrate the excellent performance of the setup.

Current status of clinical magnetic resonance imaging for plaque characterisation in patients with carotid artery stenosis.

OBJECTIVE: The article aims to provide an overview of the literature that assessed the agreement between magnetic resonance imaging (MRI) and histology for specific carotid plaque characteristics associated with vulnerability in terms of sensitivity and specificity. METHODS: A systematic search strategy was conducted in MEDLINE and EMBASE databases resulting in 1084 articles. Finally, we included 17 papers. Due to variation in presentation, especially in MRI and histology methods, a pooled analysis could not be performed. RESULTS: Two studies were performed on a 3.0-T MRI scanner; all other studies were performed on a 1.5-T scanner. Most performed sequences were two-dimensional (2D) and three-dimensional (3D) T1-weighted and all histology protocols varied slightly. Our results indicate that calcification, fibrous cap, intraplaque haemorrhage and lipid-rich necrotic cores can be identified with moderate-to-good sensitivity and specificity. CONCLUSIONS: Based on current literature, it appears premature for routine application of MRI as an imaging modality to assess carotid plaque characteristics associated with plaque vulnerability. Although MRI still holds promise, clinical application for plaque characterisation would require consensus regarding MRI settings and confirmation by histology. Predefined protocols for histology and MR imaging need to be established.

Investigation of an Escherichia coli environmental benchmark for waterborne pathogens in agricultural watersheds in Canada.

Canada's National Agri-Environmental Standards Initiative sought to develop an environmental benchmark for low-level waterborne pathogen occurrence in agricultural watersheds. A field study collected 902 water samples from 27 sites in four intensive agricultural watersheds across Canada from 2005 to 2007. Four of the sites were selected as reference sites away from livestock and human fecal pollution sources in each watershed. Water samples were analyzed for Campylobacter spp., Salmonella spp., Escherichia coli O157:H7, Cryptosporidium spp., Giardia spp., and the water quality indicator E. coli. The annual mean number of pathogen species was higher at agricultural sites (1.54 ± 0.07 species per water sample) than at reference sites (0.75 ± 0.14 species per water sample). The annual mean concentration of E. coli was also higher at agricultural sites (491 ± 96 colony-forming units [cfu] 100 mL(-1)) than at reference sites (53 ± 18 cfu 100 mL(-1)). The feasibility of adopting existing E. coli water quality guideline values as an environmental benchmark was assessed, but waterborne pathogens were detected at agricultural sites in 80% of water samples with low E. coli concentrations (<100 cfu 100 mL(-1)). Instead, an approach was developed based on using the natural background occurrence of pathogens at reference sites in agricultural watersheds to derive provisional environmental benchmarks for pathogens at agricultural sites. The environmental benchmarks that were derived were found to represent E. coli values lower than geometric mean values typically found in recreational water quality guidelines. Additional research is needed to investigate environmental benchmarks for waterborne pathogens within the context of the "One World, One Health" perspective for protecting human, domestic animal, and wildlife health.

Autologous dendritic cells pulsed with allogeneic tumour cell lysate induce tumour-reactive T-cell responses in patients with pancreatic cancer: A phase I study.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is notorious for its poor prognosis even after curative resection. Responses to immunotherapy are rare and related to inadequate T-cell priming. We previously demonstrated the potency of allogeneic lysate-dendritic cell (DC) vaccination in a preclinical model. Here we translate this concept to patients. METHODS: In this phase I study, patients with resected PDAC were included when they demonstrated no radiologic signs of recurrence after standard-of-care treatment. Allogeneic tumour lysate-loaded autologous monocyte-derived DCs were injected at weeks 0, 2, 4 and at months 3 and 6. Objectives are feasibility, safety and immunogenicity of allogeneic tumour-DCs. The presence of tumour antigens shared between the vaccine and patient tumours was investigated. Immunological analyses were performed on peripheral blood, skin and tumour. RESULTS: Ten patients were included. DC production and administration were successful. All patients experienced a grade 1 injection-site and infusion-related reaction. Two patients experienced a grade 2 fever and 1 patient experienced a grade 3 dyspnoea. No vaccine-related serious adverse events were observed. Shared tumour antigens were found between the vaccine and patient tumours. All evaluated patients displayed a vaccine-induced response indicated by increased frequencies of Ki67+ and activated PD-1+ circulating T-cells. In addition, treatment-induced T-cell reactivity to autologous tumour of study patients was detected. Seven out of ten patients have not experienced disease recurrence or progression at a median follow-up of 25 months (15-32 months). CONCLUSION: Allogeneic tumour lysate-DC treatment is feasible, safe and induces immune reactivity to PDAC expressed antigens.

The distribution of Salmonella enterica serovars and subtypes in surface water from five agricultural regions across Canada.

Serovar prevalence of the zoonotic pathogen, Salmonella enterica, was compared among 1624 surface water samples collected previously from five different Canadian agricultural watersheds over multiple years. Phagetyping, pulsed field gel electrophoresis (PFGE), and antimicrobial resistance subtyping assays were performed on serovars Enteritidis, Typhimurium, and Heidelberg. Serovars and subtypes from surface water were compared with those from animal feces, human sewage, and serovars reported to cause salmonellosis in Canadians. Sixty-five different serovars were identified in surface water; only 32% of these were isolated from multiple watersheds. Eleven of the 13 serovars most commonly reported to cause salmonellosis in Canadians were identified in surface water; isolates of these serovars constituted >40% of the total isolates. Common phagetypes and PFGE subtypes of serovars associated with illness in humans such as S. Enteritidis and S. Typhimurium were also isolated from surface water and animal feces. Antimicrobial resistance was generally low, but was highest among S. Typhimurium. Monitoring of these rivers helps to identify vulnerable areas of a watershed and, despite a relatively low prevalence of S. enterica overall, serovars observed in surface water are an indication of the levels of specific S. enterica serovars present in humans and animals.

Prognostic factors in human primary breast cancer: comparison of c-myc and HER2/neu amplification.

Amplification of oncogenes in primary tumours may have prognostic and/or therapeutic significance for patients with breast cancer. We have studied HER2/neu and c-myc amplification together with steroid receptors in human primary breast tumours and related the outcome with (relapse-free) survival. A strong inverse correlation was found between HER2/neu amplification and the presence of oestrogen and progesterone receptors. Actuarial 5-years survival showed that breast cancer patients with c-myc amplification in their primary tumours experience a shorter relapse-free survival, especially in node-negative and in receptor-positive tumours, whereas HER2/neu amplification may be of prognostic value for overall survival in receptor-negative tumours. Overall, in our hands, c-myc amplification appeared to be a more potent prognosticator than HER2/neu amplification in human primary breast cancer.

NADH-Regulated metabolic model for growth of Methylosinus trichosporiumOB3b. Cometabolic degradation of trichloroethene and optimization of bioreactor system performance.

A metabolic model describing growth of Methylosinus trichosporium OB3b and cometabolic contaminant conversion is used to optimize trichloroethene (TCE) conversion in a bioreactor system. Different process configurations are compared: a growing culture and a nongrowing culture to which TCE is added at both constant and pulsed levels. The growth part of the model, presented in the preceding article, gives a detailed description of the NADH regeneration required for continued TCE conversion. It is based on the metabolic pathways, includes Michaelis-Menten type enzyme kinetics, and uses NADH as an integrating and controlling factor. Here the model is extended to include TCE transformation, incorporating the kinetics of contaminant conversion, the related NADH consumption, toxic effects, and competitive inhibition between TCE and methane. The model realistically describes the experimentally observed negative effects of the TCE conversion products, both on soluble methane monooxygenase through the explicit incorporation of the activity of this enzyme and on cell viability through the distinction between dividing and nondividing cells. In growth-based systems, the toxicity of the TCE conversion products causes rapid cell death, which leads to wash-out of suspended cultures at low TCE loads (below microM inlet concentrations). Enzyme activity, which is less sensitive, is hardly affected by the toxicity of the TCE conversion products and ensures high conversions (>95%) up to the point of wash-out. Pulsed addition of TCE (0.014-0.048 mM) leads to a complete loss of viability. However, the remaining enzyme activity can still almost completely convert the subsequently added large TCE pulses (0.33-0.64 mM). This emphasizes the inefficient use of enzyme activity in growth-based systems. A comparison of growth-based and similar non-growth-based systems reveals that the highest TCE conversions per amount of cells grown can be obtained in the latter. Using small amounts of methane (negligible compared to the amount needed to grow the cells), NADH limitation in the second step of this two-step system can be eliminated. This results in complete utilization of enzyme activity and thus in a very effective treatment system.

NADH-Regulated metabolic model for growth of Methylosinus trichosporium OB3b. Model presentation, parameter estimation, and model validation.

A biochemical model is presented that describes growth of Methylosinus trichosporium OB3b on methane. The model, which was developed to compare strategies to alleviate NADH limitation resulting from cometabolic contaminant conversion, includes (1) catabolism of methane via methanol, formaldehyde, and formate to carbon dioxide; (2) growth as formaldehyde assimilation; and (3) storage material (poly-beta-hydroxybutyric acid, PHB) metabolism. To integrate the three processes, the cofactor NADH is used as central intermediate and controlling factor-instead of the commonly applied energy carrier ATP. This way a stable and well-regulated growth model is obtained that gives a realistic description of a variety of steady-state and transient-state experimental data. An analysis of the cells' physiological properties is given to illustrate the applicability of the model. Steady-state model calculations showed that in strain OB3b flux control is located primarily at the first enzyme of the metabolic pathway. Since no adaptation in V(MAX) values is necessary to describe growth at different dilution rates, the organism seems to have a "rigid enzyme system", the activity of which is not regulated in response to continued growth at low rates. During transient periods of excess carbon and energy source availability, PHB is found to accumulate, serving as a sink for transiently available excess reducing power.

Detection of fetal structural malformations by ultrasound in insulin-dependent pregnant women.

A total of 136 insulin-dependent pregnancies was referred to our ultrasound unit for an anomaly scan. In 94 cases the scan was requested between 17 and 22 weeks of gestation (median 20 weeks), in the remaining 42 cases the patient was not referred until after 24 weeks (median 27 weeks), which is too late for termination of pregnancy to be exercised. From the available data, optimal metabolic control was attempted in at least 80% of cases between 4-8 weeks after conception. The incidence of structural anomalies was 8.1%, the majority comprising cardiac and central nervous system malformations. Prenatal ultrasound detection of fetal structural defects was only feasible in seven (64%) out of 11 affected pregnancies. The missed structural anomalies were of cardiac origin in three cases and of gastrointestinal origin in the remaining case. Maternal obesity was present in 72% of our patients and was responsible for false negative results in three out of four cases with a fetal structural defect. This diagnostic limitation should be discussed with the parents when counselling takes place. The high incidence of structural defects (8.1%) and subsequent high perinatal mortality rate (64%) emphasizes the importance of preconceptional metabolic control in insulin-dependent diabetic women.

PLACD-7T Study: Atherosclerotic Carotid Plaque Components Correlated with Cerebral Damage at 7 Tesla Magnetic Resonance Imaging.

INTRODUCTION: In patients with carotid artery stenosis histological plaque composition is associated with plaque stability and with presenting symptomatology. Preferentially, plaque vulnerability should be taken into account in pre-operative work-up of patients with severe carotid artery stenosis. However, currently no appropriate and conclusive (non-) invasive technique to differentiate between the high and low risk carotid artery plaque in vivo is available. We propose that 7 Tesla human high resolution MRI scanning will visualize carotid plaque characteristics more precisely and will enable correlation of these specific components with cerebral damage. STUDY OBJECTIVE: The aim of the PlaCD-7T study is 1: to correlate 7T imaging with carotid plaque histology (gold standard); and 2: to correlate plaque characteristics with cerebral damage ((clinically silent) cerebral (micro) infarcts or bleeds) on 7 Tesla high resolution (HR) MRI. DESIGN: We propose a single center prospective study for either symptomatic or asymptomatic patients with haemodynamic significant (70%) stenosis of at least one of the carotid arteries. The Athero-Express (AE) biobank histological analysis will be derived according to standard protocol. Patients included in the AE and our prospective study will undergo a pre-operative 7 Tesla HR-MRI scan of both the head and neck area. DISCUSSION: We hypothesize that the 7 Tesla MRI scanner will allow early identification of high risk carotid plaques being associated with micro infarcted cerebral areas, and will thus be able to identify patients with a high risk of periprocedural stroke, by identification of surrogate measures of increased cardiovascular risk.

Molecular subtypes of Campylobacter spp., Salmonella enterica, and Escherichia coli O157:H7 isolated from faecal and surface water samples in the Oldman River watershed, Alberta, Canada.

Campylobacter spp., Salmonella enterica, and Escherichia coli O157:H7 isolated from 898 faecal, 43 sewage, and 342 surface water samples from the Oldman River were characterized using bacterial subtyping methods in order to investigate potential sources of contamination of the watershed. Among these pathogens, Campylobacter spp. were the most frequently isolated from faecal, sewage, and surface water samples (266/895, 11/43, and 91/342, respectively), followed by Salmonella (67/898, 8/43, and 29/342, respectively), and E. coli O157:H7 (16/898, 2/43, and 8/342, respectively). Salmonella Rubislaw was the most common serovar isolated from water. This serovar was also isolated from two wild bird species. Most other serovars isolated from water were either not isolated from animals or were isolated from multiple species. E. coli O157:H7 was predominantly isolated from cattle. The most common phage-types of this pathogen from cattle were also the most common among water isolates, and there were exact pulsed field gel electrophoresis and comparative genomic fingerprint matches between cattle, sewage, and water isolates. Campylobacters were commonly isolated from surface waters and faeces from most animal species. Restriction fragment length polymorphism of the Campylobacter flaA gene identified several location and host species-specific (cattle, goose, pig) fingerprints. Molecular subtyping of these bacterial pathogens shows considerable promise as a tool for determining the sources of faecal pollution of water.