Deletion of Glutathione Peroxidase-2 Inhibits Azoxymethane-Induced Colon Cancer Development

The selenoprotein glutathione peroxidase-2 (GPx2) appears to have a dual role in carcinogenesis. While it protected mice from colon cancer in a model of inflammation-triggered carcinogenesis (azoxymethane and dextran sodium sulfate treatment), it promoted growth of xenografted tumor cells. Therefore, we analyzed the effect of GPx2 in a mouse model mimicking sporadic colorectal cancer (azoxymethane-treatment only). GPx2-knockout (KO) and wild-type (WT) mice were adjusted to an either marginally deficient (−Se), adequate (+Se), or supranutritional (++Se) selenium status and were treated six times with azoxymethane (AOM) to induce tumor development. In the −Se and ++Se groups, the number of tumors was significantly lower in GPx2-KO than in respective WT mice. On the +Se diet, the number of dysplastic crypts was reduced in GPx2-KO mice. This may be explained by more basal and AOM-induced apoptotic cell death in GPx2-KO mice that eliminates damaged or pre-malignant epithelial cells. In WT dysplastic crypts GPx2 was up-regulated in comparison to normal crypts which might be an attempt to suppress apoptosis. In contrast, in the +Se groups tumor numbers were similar in both genotypes but tumor size was larger in GPx2-KO mice. The latter was associated with an inflammatory and tumor-promoting environment as obvious from infiltrated inflammatory cells in the intestinal mucosa of GPx2-KO mice even without any treatment and characterized as low-grade inflammation. In WT mice the number of tumors tended to be lowest in +Se compared to −Se and ++Se feeding indicating that selenium might delay tumorigenesis only in the adequate status. In conclusion, the role of GPx2 and presumably also of selenium depends on the cancer stage and obviously on the involvement of inflammation.     

Evaluation of Mitotic Activity Index in Breast Cancer Using Whole Slide Digital Images

Introduction

Mitotic Activity Index (MAI) is an important independent prognostic factor and an integral part of the breast cancer grading system. Thus, correct estimation of this prognostically relevant feature is essential for guiding treatment decision and assessing patient prognosis.The aim of this study was to validate the use of high resolution Whole Slide Images (WSI) in estimating MAI in breast cancer specimens.

Methods

MAI was evaluated in 100 consecutive breast cancer specimens by three observers on two occasions, microscopically and on WSI with a wash out period of 4 months. MAI was also translated to mitotic scores as in grading. Inter- and intra-observer agreement between microscopic and digital MAI counts and scores was measured.

Results

Almost perfect inter-observer agreements were obtained from counting MAI using a conventional microscope (intra-class correlation coefficient (ICCC) 0.879) as well as on WSI (ICCC 0.924). K coefficients reflected good inter-observer agreements among observers'' microscopic mitotic scores (average kappa 0.642). Comparable results were also observed among digital mitotic scores (average kappa 0.635). There was strong to perfect intra-observer agreements between MAI counts and mitotic scores for the two diagnostic modalities (ICCC 0.716–0.863, kappa 0.506–0.617). There were no significant differences in mitotic scores using both diagnostic modalities.

Conclusion

Scoring mitoses using WSI in breast cancer seems to be just as reliable and reproducible as when using a microscope. Further development of software and image quality will definitely encourage the use of WSI in routine pathology practice.     

Real-Time Analysis and Visualization for Single-Molecule Based Super-Resolution Microscopy

Accurate multidimensional localization of isolated fluorescent emitters is a time consuming process in single-molecule based super-resolution microscopy. We demonstrate a functional method for real-time reconstruction with automatic feedback control, without compromising the localization accuracy. Compatible with high frame rates of EM-CCD cameras, it relies on a wavelet segmentation algorithm, together with a mix of CPU/GPU implementation. A combination with Gaussian fitting allows direct access to 3D localization. Automatic feedback control ensures optimal molecule density throughout the acquisition process. With this method, we significantly improve the efficiency and feasibility of localization-based super-resolution microscopy.     

Estimation of Parameters Influencing Waterborne Transmission of Infectious Hematopoietic Necrosis Virus (IHNV) in Atlantic Salmon (Salmo salar)

Understanding how pathogenic organisms spread in the environment is crucial for the management of disease, yet knowledge of propagule dispersal and transmission in aquatic environments is limited. We conducted empirical studies using the aquatic virus, infectious hematopoietic necrosis virus (IHNV), to quantify infectious dose, shedding capacity, and virus destruction rates in order to better understand the transmission of IHN virus among Atlantic salmon marine net-pen aquaculture. Transmission of virus and subsequent mortality in Atlantic salmon post-smolts was initiated with as low as 10 plaque forming units (pfu) ml⁻¹. Virus shedding from IHNV infected Atlantic salmon was detected before the onset of visible signs of disease with peak shed rates averaging 3.2×10⁷ pfu fish⁻¹ hour⁻¹ one to two days prior to mortality. Once shed into the marine environment, the abundance of free IHNV is modulated by sunlight (UV A and B) and the growth of natural biota present in the seawater. Virus decayed very slowly in sterilized seawater while rates as high as k =  4.37 d⁻¹ were observed in natural seawater. Decay rates were further accelerated when exposed to sunlight with virus infectivity reduced by six orders of magnitude within 3 hours of full sunlight exposure. Coupling the IHNV transmission parameter estimates determined here with physical water circulation models, will increase the understanding of IHNV dispersal and provide accurate geospatial predictions of risk for IHNV transmission from marine salmon sites.     

Genetic variability and demographic history of Anguilla mossambica (Peters, 1852) from continental Africa and Madagascar

In this study, population genetic and demographic parameters were inferred using sequence data from 151 individuals of Anguilla mossambica originating from continental south and south-east Africa and Madagascar. The analyses were based on a 532 bp segment of the mitochondrial cytochrome b gene. The absence of genetic structuring was observed thereby supporting the hypothesis of panmixia for the endemic A. mossambica. The overall nucleotide diversity π = 0.002 and the haplotype diversity reached h = 0.691. Significant negative values from several tests of neutrality and mismatch analysis pointed to fluctuating historical population sizes. Bayesian averaging resulted in higher support for population growth models vs. a constant population-size model. Population decline and subsequent growth most likely predated the last glacial and were probably related to extended periods of extreme drought followed by wetter and more stable hydroclimate between 150 and 75,000 years before present (kBP). According to this scenario the female effective population size has increased since 110 kBP by c. two orders of magnitude to a recent level of about 650,000 (219,317–2,292,000).     

Fossil liberation: a model to explain high biodiversity in the Triassic Cassian Formation

With 1429 animal species, the Triassic Cassian Formation in the Dolomites, Southern Alps (Italy), yields the highest species richness reported from any spatially constrained pre-Quaternary formation known to science. The high preserved diversity is partly attributable to a high primary diversity governed by the tropical setting, increasing alpha diversity, and the breadth of habitats spurring beta diversity. More important is the excellent preservation of fossils and the ease with which they can be extracted from the poorly lithified sediments. We propose the term ‘liberation Lagerstätten’ to capture this preservational window. In contrast to conservation Lagerstätten, liberation Lagerstätten like the Cassian Formation originate from normal marine conditions but low-grade diagenesis. Molluscs contribute substantially to species richness, comprising 67% of all invertebrate species in the Cassian Formation. The gastropod dominance (39% of all species) is nearly as great as in Recent tropical settings, contradicting the concept of a substantial Cenozoic rise.     

Seed size,number and strategies in annual plants: a comparative functional analysis and synthesis

Background and AimsPlants depend fundamentally on establishment from seed. However, protocols in trait-based ecology currently estimate seed size but not seed number. This can be rectified. For annuals, seed number should simply be a positive function of vegetative biomass and a negative function of seed size.MethodsUsing published values of comparative seed number as the ‘gold standard’ and a large functional database, comparative seed yield and number per plant and per m² were predicted by multiple regression. Subsequently, ecological variation in each was explored for English and Spanish habitats, newly calculated C-S-R strategies and changed abundance in the British flora.Key ResultsAs predicted, comparative seed mass yield per plant was consistently a positive function of plant size and competitive ability, and largely independent of seed size. Regressions estimating comparative seed number included, additionally, seed size as a negative function. Relationships differed numerically between regions, habitats and C-S-R strategies. Moreover, some species differed in life history over their geographical range. Comparative seed yield per m² was positively correlated with FAO crop yield, and increasing British annuals produced numerous seeds. Nevertheless, predicted values must be viewed as comparative rather than absolute: they varied according to the ‘gold standard’ predictor used. Moreover, regressions estimating comparative seed yield per m² achieved low precision.ConclusionsFor the first time, estimates of comparative seed yield and number for >800 annuals and their predictor equations have been produced and the ecological importance of these regenerative traits has been illustrated. ‘Regenerative trait-based ecology’ remains in its infancy, with work needed on determinate vs. indeterminate flowering (‘bet-hedging’), C-S-R methodologies, phylogeny, comparative seed yield per m² and changing life history. Nevertheless, this has been a positive start and readers are invited to use estimates for >800 annuals, in the Supplementary data, to help advance ‘regenerative trait-based ecology’ to the next level.     

Side-by-side comparability of batch and continuous downstream for the production of monoclonal antibodies

Continuous processing is the future production method for monoclonal antibodies (mAbs). A fully continuous, fully automated downstream process based on disposable equipment was developed and implemented inside the MoBiDiK pilot plant. However, a study evaluating the comparability between batch and continuous processing based on product quality attributes was not conducted before. The work presented fills this gap comparing both process modes experimentally by purifying the same harvest material (side-by-side comparability). Samples were drawn at different time points and positions in the process for batch and continuous mode. Product quality attributes, product-related impurities, as well as process-related impurities were determined. The resulting polished material was processed to drug substance and further evaluated regarding storage stability and degradation behavior. The in-process control data from the continuous process showed the high degree of accuracy in providing relevant process parameters such as pH, conductivity, and protein concentration during the entire process duration. Minor differences between batch and continuous samples are expected as different processing conditions are unavoidable due to the different nature of batch and continuous processing. All tests revealed no significant differences in the intermediates and comparability in the drug substance between the samples of both process modes. The stability study of the final product also showed no differences in the stability profile during storage and forced degradation. Finally, online data analysis is presented as a powerful tool for online-monitoring of chromatography columns during continuous processing.     

Human immune response against salivary antigens of Simulium damnosum s.l.: A new epidemiological marker for exposure to blackfly bites in onchocerciasis endemic areas

BackgroundSimulium damnosum sensu lato (s.l.) blackflies transmit Onchocerca volvulus, a filarial nematode that causes human onchocerciasis. Human landing catches (HLCs) is currently the sole method used to estimate blackfly biting rates but is labour-intensive and questionable on ethical grounds. A potential alternative is to measure host antibodies to vector saliva deposited during bloodfeeding. In this study, immunoassays to quantify human antibody responses to S. damnosum s.l. saliva were developed, and the salivary proteome of S. damnosum s.l. was investigated.Methodology/Principal findingsBlood samples from people living in onchocerciasis-endemic areas in Ghana were collected during the wet season; samples from people living in Accra, a blackfly-free area, were considered negative controls and compared to samples from blackfly-free locations in Sudan. Blackflies were collected by HLCs and dissected to extract their salivary glands. An ELISA measuring anti-S. damnosum s.l. salivary IgG and IgM was optimized and used to quantify the humoral immune response of 958 individuals. Both immunoassays differentiated negative controls from endemic participants. Salivary proteins were separated by gel-electrophoresis, and antigenic proteins visualized by immunoblot. Liquid chromatography mass spectrometry (LC–MS/MS) was performed to characterize the proteome of S. damnosum s.l. salivary glands. Several antigenic proteins were recognized, with the major ones located around 15 and 40 kDa. LC–MS/MS identified the presence of antigen 5-related protein, apyrase/nucleotidase, and hyaluronidase.Conclusions/SignificanceThis study validated for the first time human immunoassays that quantify humoral immune responses as potential markers of exposure to blackfly bites. These assays have the potential to facilitate understanding patterns of exposure as well as evaluating the impact of vector control on biting rates. Future studies need to investigate seasonal fluctuations of these antibody responses, potential cross-reactions with other bloodsucking arthropods, and thoroughly identify the most immunogenic proteins.