Quantitative Analysis of Viral Load per Haploid Genome Revealed the Different Biological Features of Merkel Cell Polyomavirus Infection in Skin Tumor

Merkel cell polyomavirus (MCPyV) has recently been identified in Merkel cell carcinoma (MCC), an aggressive cancer that occurs in sun-exposed skin. Conventional technologies, such as polymerase chain reaction (PCR) and immunohistochemistry, have produced conflicting results for MCPyV infections in non-MCC tumors. Therefore, we performed quantitative analyses of the MCPyV copy number in various skin tumor tissues, including MCC (n = 9) and other sun exposure-related skin tumors (basal cell carcinoma [BCC, n = 45], actinic keratosis [AK, n = 52], Bowen’s disease [n = 34], seborrheic keratosis [n = 5], primary cutaneous anaplastic large-cell lymphoma [n = 5], malignant melanoma [n = 5], and melanocytic nevus [n = 6]). In a conventional PCR analysis, MCPyV DNA was detected in MCC (9 cases; 100%), BCC (1 case; 2%), and AK (3 cases; 6%). We then used digital PCR technology to estimate the absolute viral copy number per haploid human genome in these tissues. The viral copy number per haploid genome was estimated to be around 1 in most MCC tissues, and there were marked differences between the MCC (0.119–42.8) and AK (0.02–0.07) groups. PCR-positive BCC tissue showed a similar viral load as MCC tissue (0.662). Immunohistochemistry with a monoclonal antibody against the MCPyV T antigen (CM2B4) demonstrated positive nuclear localization in most of the high-viral-load tumor groups (8 of 9 MCC and 1 BCC), but not in the low-viral-load or PCR-negative tumor groups. These results demonstrated that MCPyV infection is possibly involved in a minority of sun-exposed skin tumors, including BCC and AK, and that these tumors display different modes of infection

Neuroimaging and clinical outcomes of oral anticoagulant-associated intracerebral hemorrhage

Objective Methods Whether intracerebral hemorrhage (ICH) associated with non-vitamin K antagonist oral anticoagulants (NOAC-ICH) has a better outcome compared to ICH associated with vitamin K antagonists (VKA-ICH) is uncertain. We performed a systematic review and individual patient data meta-analysis of cohort studies comparing clinical and radiological outcomes between NOAC-ICH and VKA-ICH patients. The primary outcome measure was 30-day all-cause mortality. All outcomes were assessed in multivariate regression analyses adjusted for age, sex, ICH location, and intraventricular hemorrhage extension. Results Interpretation We included 7 eligible studies comprising 219 NOAC-ICH and 831 VKA-ICH patients (mean age = 77 years, 52.5% females). The 30-day mortality was similar between NOAC-ICH and VKA-ICH (24.3% vs 26.5%; hazard ratio = 0.94, 95% confidence interval [CI] = 0.67-1.31). However, in multivariate analyses adjusting for potential confounders, NOAC-ICH was associated with lower admission National Institutes of Health Stroke Scale (NIHSS) score (linear regression coefficient = -2.83, 95% CI = -5.28 to -0.38), lower likelihood of severe stroke (NIHSS > 10 points) on admission (odds ratio [OR] = 0.50, 95% CI = 0.30-0.84), and smaller baseline hematoma volume (linear regression coefficient = -0.24, 95% CI = -0.47 to -0.16). The two groups did not differ in the likelihood of baseline hematoma volume <30cm(3) (OR = 1.14, 95% CI = 0.81-1.62), hematoma expansion (OR = 0.97, 95% CI = 0.63-1.48), in-hospital mortality (OR = 0.73, 95% CI = 0.49-1.11), functional status at discharge (common OR = 0.78, 95% CI = 0.57-1.07), or functional status at 3 months (common OR = 1.03, 95% CI = 0.75-1.43). Although functional outcome at discharge, 1 month, or 3 months was comparable after NOAC-ICH and VKA-ICH, patients with NOAC-ICH had smaller baseline hematoma volumes and less severe acute stroke syndromes. Ann Neurol 2018;84:702-712Peer reviewe

Non-invasive diagnostic tests for Helicobacter pylori infection

BACKGROUND: Helicobacter pylori (H pylori) infection has been implicated in a number of malignancies and non-malignant conditions including peptic ulcers, non-ulcer dyspepsia, recurrent peptic ulcer bleeding, unexplained iron deficiency anaemia, idiopathic thrombocytopaenia purpura, and colorectal adenomas. The confirmatory diagnosis of H pylori is by endoscopic biopsy, followed by histopathological examination using haemotoxylin and eosin (H & E) stain or special stains such as Giemsa stain and Warthin-Starry stain. Special stains are more accurate than H & E stain. There is significant uncertainty about the diagnostic accuracy of non-invasive tests for diagnosis of H pylori. OBJECTIVES: To compare the diagnostic accuracy of urea breath test, serology, and stool antigen test, used alone or in combination, for diagnosis of H pylori infection in symptomatic and asymptomatic people, so that eradication therapy for H pylori can be started. SEARCH METHODS: We searched MEDLINE, Embase, the Science Citation Index and the National Institute for Health Research Health Technology Assessment Database on 4 March 2016. We screened references in the included studies to identify additional studies. We also conducted citation searches of relevant studies, most recently on 4 December 2016. We did not restrict studies by language or publication status, or whether data were collected prospectively or retrospectively. SELECTION CRITERIA: We included diagnostic accuracy studies that evaluated at least one of the index tests (urea breath test using isotopes such as13C or14C, serology and stool antigen test) against the reference standard (histopathological examination using H & E stain, special stains or immunohistochemical stain) in people suspected of having H pylori infection. DATA COLLECTION AND ANALYSIS: Two review authors independently screened the references to identify relevant studies and independently extracted data. We assessed the methodological quality of studies using the QUADAS-2 tool. We performed meta-analysis by using the hierarchical summary receiver operating characteristic (HSROC) model to estimate and compare SROC curves. Where appropriate, we used bivariate or univariate logistic regression models to estimate summary sensitivities and specificities. MAIN RESULTS: We included 101 studies involving 11,003 participants, of which 5839 participants (53.1%) had H pylori infection. The prevalence of H pylori infection in the studies ranged from 15.2% to 94.7%, with a median prevalence of 53.7% (interquartile range 42.0% to 66.5%). Most of the studies (57%) included participants with dyspepsia and 53 studies excluded participants who recently had proton pump inhibitors or antibiotics.There was at least an unclear risk of bias or unclear applicability concern for each study.Of the 101 studies, 15 compared the accuracy of two index tests and two studies compared the accuracy of three index tests. Thirty-four studies (4242 participants) evaluated serology; 29 studies (2988 participants) evaluated stool antigen test; 34 studies (3139 participants) evaluated urea breath test-13C; 21 studies (1810 participants) evaluated urea breath test-14C; and two studies (127 participants) evaluated urea breath test but did not report the isotope used. The thresholds used to define test positivity and the staining techniques used for histopathological examination (reference standard) varied between studies. Due to sparse data for each threshold reported, it was not possible to identify the best threshold for each test.Using data from 99 studies in an indirect test comparison, there was statistical evidence of a difference in diagnostic accuracy between urea breath test-13C, urea breath test-14C, serology and stool antigen test (P = 0.024). The diagnostic odds ratios for urea breath test-13C, urea breath test-14C, serology, and stool antigen test were 153 (95% confidence interval (CI) 73.7 to 316), 105 (95% CI 74.0 to 150), 47.4 (95% CI 25.5 to 88.1) and 45.1 (95% CI 24.2 to 84.1). The sensitivity (95% CI) estimated at a fixed specificity of 0.90 (median from studies across the four tests), was 0.94 (95% CI 0.89 to 0.97) for urea breath test-13C, 0.92 (95% CI 0.89 to 0.94) for urea breath test-14C, 0.84 (95% CI 0.74 to 0.91) for serology, and 0.83 (95% CI 0.73 to 0.90) for stool antigen test. This implies that on average, given a specificity of 0.90 and prevalence of 53.7% (median specificity and prevalence in the studies), out of 1000 people tested for H pylori infection, there will be 46 false positives (people without H pylori infection who will be diagnosed as having H pylori infection). In this hypothetical cohort, urea breath test-13C, urea breath test-14C, serology, and stool antigen test will give 30 (95% CI 15 to 58), 42 (95% CI 30 to 58), 86 (95% CI 50 to 140), and 89 (95% CI 52 to 146) false negatives respectively (people with H pylori infection for whom the diagnosis of H pylori will be missed).Direct comparisons were based on few head-to-head studies. The ratios of diagnostic odds ratios (DORs) were 0.68 (95% CI 0.12 to 3.70; P = 0.56) for urea breath test-13C versus serology (seven studies), and 0.88 (95% CI 0.14 to 5.56; P = 0.84) for urea breath test-13C versus stool antigen test (seven studies). The 95% CIs of these estimates overlap with those of the ratios of DORs from the indirect comparison. Data were limited or unavailable for meta-analysis of other direct comparisons. AUTHORS' CONCLUSIONS: In people without a history of gastrectomy and those who have not recently had antibiotics or proton ,pump inhibitors, urea breath tests had high diagnostic accuracy while serology and stool antigen tests were less accurate for diagnosis of Helicobacter pylori infection.This is based on an indirect test comparison (with potential for bias due to confounding), as evidence from direct comparisons was limited or unavailable. The thresholds used for these tests were highly variable and we were unable to identify specific thresholds that might be useful in clinical practice.We need further comparative studies of high methodological quality to obtain more reliable evidence of relative accuracy between the tests. Such studies should be conducted prospectively in a representative spectrum of participants and clearly reported to ensure low risk of bias. Most importantly, studies should prespecify and clearly report thresholds used, and should avoid inappropriate exclusions

Structuring Plant-Based Foods by Selective Pre-Processing of Raw Materials: Evaluation of the Endogenous Structure-Determining Potential of Tomato, Carrot and Broccoli

Dispersed plant-based foods, such as soups, sauces and smoothies, represent a category of ready-to-eat foods which contain one or more fruit and/or vegetable sources. Owing to their convenience next to their perception as natural products, these foods may convince present-day consumers to a higher consumption of fruits and vegetables. In fruits and vegetables, the cell wall polysaccharide pectin is important for tissue structure. Different types of naturally-present pectin-modifying enzymes (i.e., pectinases) affect pectin nanostructure, changing the functional properties (i.e., texture, rheology) of fruit- and vegetable-based products. Among different fruit and vegetable sources, the differences in pectin structure and variation in endogenous pectinases offer opportunities for structuring plant-based foods. Additionally, thermal and high-pressure processing can be used as techniques for (selective) inactivation of the pectinases. In the context of food products comprising multiple fruit- and/or vegetable sources, this natural or process-modified variety of pectin and pectinases can fully be exploited using split-stream processing. Split-stream processing is a technique in which the individual ingredients, prior to being combined, are individually subjected to a sequence of specific unit operations directed to specific end-product functionalities. The present research work aimed at evaluating the potential of the split-stream processing concept for structuring dispersed plant-based foods consisting of multiple fruit and/or vegetable sources, targeting the individual pre-treatments of each of the raw materials at (selective) inactivation of pectinases. As the first step, the pectin structure was investigated for the selected raw materials (i.e., tomato, carrot and broccoli). Subsequently, the presence as well as the thermal and high-pressure stability of selected pectinases in tomato, carrot and broccoli purées was studied. The pectinases pectinmethylesterase, polygalacturonase, ß-D-galactosidase and alfa-L-arabinofuranosidase were selected. Pectinmethylesterase and polygalacturonase affect linear homogalacturonan, the most abundant pectin subdomain. Pectinmethylesterase demethoxylates homogalacturonan, changing the pectin degree of methoxylation while polygalacturonase depolymerizes homogalacturonan. ß-D-galactosidase and alfa-L-arabinofuranosidase degrade galactose- and arabinose-containing side chain structures of the pectin rhamnogalacturonan I subdomain, respectively. Finally, as a split-stream processing case-study, endogenous tomato pectinases were used to change the consistency of tomato-carrot purées. The pectin structure of tomato was clearly different from the pectin structures of broccoli and carrot. Tomato pectin showed, in comparison, the broadest range in degree of methoxylation, the highest molar mass, the highest overall linearity and the lowest extent of rhamnogalacturonan I branching. Unlike broccoli and carrot, tomato thus contains particularly long, linear pectin.Thermal and high-pressure inactivation data were obtained for pectinmethylesterase, ß-D-galactosidase andalfa-L-arabinofuranosidase in broccoli, carrot and tomato purées, and for polygalacturonase in tomato purée. These inactivation data allowed for the identification of processing conditions resulting in specific enzyme populations. By applying a thermal treatment to tomato purée, pectinmethylesterase and polygalacturonase catalytic activities could be largely maintained while ß-D-galactosidase was completely inactivated and alfa-L-arabinofuranosidase activity was largely reduced. High-pressure treatment of tomato purée allowed for selective inactivation of polygalacturonase, ß-D-galactosidase and alfa-L-arabinofuranosidase, resulting in an endogenous enzyme population exclusively comprising catalytically active pectinmethylesterase. In case of carrot purée, thermal treatment allowed to largely inactivate ß-D-galactosidase and alfa-L-arabinofuranosidase while pectinmethylesterase activity was mainly maintained. High-pressure treatment of carrot and broccoli purées could maintain high levels of pectinmethylesterase activity while reducing the ß-D-galactosidase and alfa-L-arabinofuranosidase activity levels considerably.For the split-stream processing case-study, raw tomatoes were used as source of pectinases while carrots were thermally pre-treated at two different intensities, i.e. blanching and cooking (both inactivating all endogenous pectinases), in order to obtain different levels of thermosolubilized pectin. After mechanical disintegration of tomato and carrot into tomato-carrot purées, stimulation of enzyme action at medium temperature level allowed tomato pectinmethylesterase and polygalacturonase action on both carrot and tomato pectin. Carrot pectin, when present in a purée mix of tomato and blanched carrot, was both solubilized and depolymerized by tomato polygalacturonase while mainly enzymatic depolymerization of the thermosolubilized carrot pectin was observed in the tomato-carrot purée containing cooked carrot. The final serum pectin properties were however similar for both types of tomato-carrot purée. Carrot contributed more to the consistency of the purée mix compared to tomato. Stimulating the catalytic activity of the tomato pectinases resulted in loss of this contribution, leading to a consistency reduction of the purée mix. This liquefaction of purée was larger for the tomato-carrot purée containing blanched instead of cooked carrots. Based on the obtained results, it is suggested that the liquefying effect is related to solubilization and degradation of pectin that is counteracted by a reduction in particle size. In this respect, the purée mix containing cooked carrot showed smaller particle sizes than the purée mix containing blanched carrot. The present work showed the potential of split-stream processing for structuring dispersed plant-based foods composed of multiple fruit and/or vegetable sources by the interaction of pectin and pectinases of selectively pre-processed streams of raw materials. This processing principle is promising for structuring diverse products within this category towards consumer s preferences, making use of the particular set of pectic polysaccharides and pectinases belonging to each of the composing plant sources.status: publishe

Comparative study of the cell wall composition of broccoli, carrot, and tomato: Structural characterization of the extractable pectins and hemicelluloses

This study delivers a comparison of the pectic and hemicellulosic cell wall polysaccharides between the commonly used vegetables broccoli (stem and florets separately), carrot, and tomato. Alcohol-insoluble residues were prepared from the plant sources and sequentially extracted with water, cyclohexane-trans-1,2-diamine tetra-acetic acid, sodium carbonate, and potassium hydroxide solutions, to obtain individual fractions, each containing polysaccharides bound to the cell wall in a specific manner. Structural characterization of the polysaccharide fractions was conducted using colorimetric and chromatographic approaches. Sugar ratios were defined to ameliorate data interpretation. These ratios allowed gaining information concerning polysaccharide structure from sugar composition data. Structural analysis of broccoli revealed organ-specific characteristics: the pectin degree of methoxylation (DM) of stem and florets differed, the sugar composition data inferred differences in polymeric composition. On the other hand, the molar mass (MM) distribution profiles of the polysaccharide fractions were virtually identical for both organs. Carrot root displayed a different MM distribution for the polysaccharides solubilized by potassium hydroxide compared to broccoli and tomato, possibly due to the high contribution of branched pectins to this otherwise hemicellulose-enriched fraction. Tomato fruit showed the pectins with the broadest range in DM, the highest MM, the greatest overall linearity and the lowest extent of branching of rhamnogalacturonan I, pointing to particularly long, linear pectins in tomato compared with the other vegetable organs studied, suggesting possible implications toward functional behavior.status: publishe

Study of mango endogenous pectinases as a tool to engineer mango purée consistency

The objective of this work was to evaluate the possibility of using mango endogenous pectinases to change the viscosity of mango purée. Hereto, the structure of pectic polysaccharide and the presence of sufficiently active endogenous enzymes of ripe mango were determined. Pectin of mango flesh had a high molecular weight and was highly methoxylated. Pectin methylesterase showed a negligible activity which is related to the confirmed presence of a pectin methylesterase inhibitor. Pectin contained relatively high amounts of galactose and considerable β-galactosidase (β-Gal) activity was observed. The possibility of stimulating β-Gal activity during processing (temperature/pressure, time) was investigated. β-Gal of mango was rather temperature labile but pressure stable relatively to the temperature and pressure levels used to inactivate destructive enzymes in industry. Creating processing conditions allowing endogenous β-Gal activity did not substantially change the consistency of mango purée.status: publishe

Kinetics of thermal and high-pressure inactivation of avocado polygalacturonase

Despite high-pressure (HP) processed avocado products are nowadays commercialized, there is no information about the effect of pressure on avocado polygalacturonase (PG) yet. In the present study, PG inactivation kinetics in crude avocado extract was investigated under isobaric conditions at 20°C. Moreover, PG inactivation kinetics under isothermal conditions at atmospheric pressure was also included to make comparisons with the processing technique usually employed in the industry. Both temperature and pressure inactivation of avocado PG, either at 60–70°C/0.1MPa or at 20°C/350–500MPa, could be mathematically described by first-order inactivation models. No thermal or pressure resistant fractions were detected. Thus, avocado PG appeared a rather labile enzyme which could be completely inactivated after relatively mild thermal or pressure treatments (10min/70°C/0.1MPa or 15min/20°C/450MPa). PG inactivation models developed for PG in crude avocado extract could predict PG inactivation in avocado purée relatively well.High-pressure processed avocado products are, nowadays, commercially available, but no data exist about the effect of high-pressure on pectinases yet. This paper provides, for the first time in the literature, scientific data about kinetics of thermal and high-pressure inactivation of avocado polygalacturonase. Moreover, first-order inactivation models are issued to mathematically describe inactivation. All these data should be of high interest for the food industry since polygalacturonase can affect the texture of avocado halves and chunks and the rheological properties of guacamole, avocado purée and sauces during the storage.status: publishe

Thermal and high-pressure stability of pectinmethylesterase, polygalacturonase, β-galactosidase and α-arabinofuranosidase in a tomato matrix: Towards the creation of specific endogenous enzyme populations through processing

The thermal and pressure stability of tomato pectinmethylesterase (PME), polygalacturonase (PG), β-galactosidase (β-Gal), and α-arabinofuranosidase (α-Af) were investigated in situ. Enzyme inactivation by thermal and high-pressure processing (respectively 5 min at 25-95 °C at 0.1 MPa and 10 min at 0.1-800 MPa at 20 °C) was monitored by measuring the residual activity in crude enzyme extracts of treated tomato purée samples. PME was completely inactivated after a 5-min treatment at 75 °C. Only 30 % of the pressure stable PME was inactivated after a treatment at 800 MPa (20 °C, 10 min). A 5-min treatment at 95 °C and a treatment at 550 MPa (20 °C, 10 min) caused complete PG inactivation. β-Gal and α-Af activities were already reduced significantly by thermal treatments at 42.5-52.5 °C and 45-60 °C, respectively. These enzymes were, however, rather pressure resistant: treatments at respectively 700 and 600 MPa were necessary to reduce the activity below 10 % of the initial value. Assuming that first-order, fractional conversion or biphasic inactivation models could be applied to the respective enzyme inactivation data, inactivation rate constants and their temperature or pressure dependence for the different enzymes were determined. Based on differences in process stability of the enzymes, possibilities for the creation of specific "enzyme populations" in tomato purée by selective enzyme inactivation were identified. For industrially relevant process conditions, the enzyme inactivation data obtained for tomato purée were shown to be transferable to intact tomato tissue. © 2012 Springer Science+Business Media New York.status: publishe