Very different performance of the power Doppler modalities of several ultrasound machines ascertained by a microvessel flow phantom

Introduction

In many patients with rheumatoid arthritis (RA) subclinical disease activity can be detected with ultrasound (US), especially using power Doppler US (PDUS). However, PDUS may be highly dependent on the type of machine. This could create problems both in clinical trials and in daily clinical practice. To clarify how the PDUS signal differs between machines we created a microvessel flow phantom.

Methods

The flow phantom contained three microvessels (150, 1000, 2000 microns). A syringe pump was used to generate flows. Five US machines were used. Settings were optimised to assess the lowest detectable flow for each US machine.

Results

The minimal detectable flow velocities showed very large differences between the machines. Only two of the machines may be able to detect the very low flows in the capillaries of inflamed joints. There was no clear relation with price. One of the lower-end machines actually performed best in all three vessel sizes.

Conclusions

We created a flow phantom to test the sensitivity of US machines to very low flows in small vessels. The sensitivity of the power Doppler modalities of 5 different machines was very different. The differences found between the machines are probably caused by fundamental differences in processing of the PD signal or internal settings inaccessible to users. Machines considered for PDUS assessment of RA patients should be tested using a flow phantom similar to ours. Within studies, only a single machine type should be used.     

Rapid and Scalable Plant-based Production of a Cholera Toxin B Subunit Variant to Aid in Mass Vaccination against Cholera Outbreaks

Introduction

Cholera toxin B subunit (CTB) is a component of an internationally licensed oral cholera vaccine. The protein induces neutralizing antibodies against the holotoxin, the virulence factor responsible for severe diarrhea. A field clinical trial has suggested that the addition of CTB to killed whole-cell bacteria provides superior short-term protection to whole-cell-only vaccines; however, challenges in CTB biomanufacturing (i.e., cost and scale) hamper its implementation to mass vaccination in developing countries. To provide a potential solution to this issue, we developed a rapid, robust, and scalable CTB production system in plants.

Methodology/Principal Findings

In a preliminary study of expressing original CTB in transgenic Nicotiana benthamiana, the protein was N-glycosylated with plant-specific glycans. Thus, an aglycosylated CTB variant (pCTB) was created and overexpressed via a plant virus vector. Upon additional transgene engineering for retention in the endoplasmic reticulum and optimization of a secretory signal, the yield of pCTB was dramatically improved, reaching >1 g per kg of fresh leaf material. The protein was efficiently purified by simple two-step chromatography. The GM1-ganglioside binding capacity and conformational stability of pCTB were virtually identical to the bacteria-derived original B subunit, as demonstrated in competitive enzyme-linked immunosorbent assay, surface plasmon resonance, and fluorescence-based thermal shift assay. Mammalian cell surface-binding was corroborated by immunofluorescence and flow cytometry. pCTB exhibited strong oral immunogenicity in mice, inducing significant levels of CTB-specific intestinal antibodies that persisted over 6 months. Moreover, these antibodies effectively neutralized the cholera holotoxin in vitro.

Conclusions/Significance

Taken together, these results demonstrated that pCTB has robust producibility in Nicotiana plants and retains most, if not all, of major biological activities of the original protein. This rapid and easily scalable system may enable the implementation of pCTB to mass vaccination against outbreaks, thereby providing better protection of high-risk populations in developing countries.     

Promoter hypermethylation using 24-gene array in early head and neck cancer: Better outcome in oral than in oropharyngeal cancer

Silencing of tumor suppressor genes (TSGs) by DNA promoter hypermethylation is an early event in carcinogenesis and a potential target for personalized cancer treatment. In head and neck cancer, little is known about the role of promoter hypermethylation in survival. Using methylation specific multiplex ligation-dependent probe amplification (MS-MLPA) we investigated the role of promoter hypermethylation of 24 well-described genes (some of which are classic TSGs), which are frequently methylated in different cancer types, in 166 HPV-negative early oral squamous cell carcinomas (OSCC), and 51 HPV-negative early oropharyngeal squamous cell carcinomas (OPSCC) in relation to clinicopathological features and survival. Early OSCC showed frequent promoter hypermethylation in RARB (31% of cases), CHFR (20%), CDH13 (13%), DAPK1 (12%), and APC (10%). More hypermethylation (≥ 2 genes) independently correlated with improved disease specific survival (hazard ratio 0.17, P = 0.014) in early OSCC and could therefore be used as prognostic biomarker. Early OPSCCs showed more hypermethylation of CDH13 (58%), TP73 (14%), and total hypermethylated genes. Hypermethylation of two or more genes has a significantly different effect on survival in OPSCC compared with OSCC, with a trend toward worse instead of better survival. This could have a biological explanation, which deserves further investigation and could possibly lead to more stratified treatment in the future.     

Personalized Oncology Suite: integrating next-generation sequencing data and whole-slide bioimages

Background

Cancer immunotherapy has recently entered a remarkable renaissance phase with the approval of several agents for treatment. Cancer treatment platforms have demonstrated profound tumor regressions including complete cure in patients with metastatic cancer. Moreover, technological advances in next-generation sequencing (NGS) as well as the development of devices for scanning whole-slide bioimages from tissue sections and image analysis software for quantitation of tumor-infiltrating lymphocytes (TILs) allow, for the first time, the development of personalized cancer immunotherapies that target patient specific mutations. However, there is currently no bioinformatics solution that supports the integration of these heterogeneous datasets.

Results

We have developed a bioinformatics platform – Personalized Oncology Suite (POS) – that integrates clinical data, NGS data and whole-slide bioimages from tissue sections. POS is a web-based platform that is scalable, flexible and expandable. The underlying database is based on a data warehouse schema, which is used to integrate information from different sources. POS stores clinical data, genomic data (SNPs and INDELs identified from NGS analysis), and scanned whole-slide images. It features a genome browser as well as access to several instances of the bioimage management application Bisque. POS provides different visualization techniques and offers sophisticated upload and download possibilities. The modular architecture of POS allows the community to easily modify and extend the application.

Conclusions

The web-based integration of clinical, NGS, and imaging data represents a valuable resource for clinical researchers and future application in medical oncology. POS can be used not only in the context of cancer immunology but also in other studies in which NGS data and images of tissue sections are generated. The application is open-source and can be downloaded at http://www.icbi.at/POS.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2105-15-306) contains supplementary material, which is available to authorized users.     

Theoretical prediction of the basic helix types in alpha,beta-hybrid peptides

This study provides a complete overview on all possible helical- folding patterns, their stabilities, and their detailed molecular structure in the novel foldamer class of alpha,beta-hybrid peptides on the basis of ab initio molecular orbital (MO) theory. The results indicate a considerable intrinsic potential of backbone folding. As found for other peptide foldamers, representatives of mixed or beta-helices are most stable in more apolar media, whereas polar environments favor the helices with the hydrogen bonds pointing in only one direction. The theoretical results confirm the hydrogen-bonding patterns found in the first experimental studies on these hybrid peptides. Selecting special backbone substitution patterns, the secondary structure potential of the alpha,beta-hybrid peptides could be of great importance for a rational peptide and protein design.     

Impact of olfactory non-host predator cues on aggregation behaviour and activity in <Emphasis Type="Italic">Polymorphus minutus</Emphasis> infected <Emphasis Type="Italic">Gammarus pulex</Emphasis>

Parasites with a complex life cycle are supposed to influence the behaviour of their intermediate host in such a way that the transmission to the final host is enhanced, but reduced to non-hosts. Here, we examined whether the trophically transmitted bird parasite Polymorphus minutus increases the antipredator response of its intermediate host, the freshwater amphipod Gammarus pulex to fish cues, i.e. non-host cues (‘increased host abilities hypothesis’). Aggregation behaviour and reduced activity are assumed to decrease the predation risk of gammarids by fishes. Uninfected G. pulex are known to aggregate in the presence of a fish predator. In the present study, gammarids were allowed to choose either to join a group of conspecifics or to stay solitary (experiment 1) or between two groups differing in infection status (experiment 2), both in the presence or absence of fish odour. The perception of the groups was limited to mainly olfactory cues. Contrary to the ‘increased host abilities hypothesis’, in infected gammarids of experiment 1, fish cues induced similar aggregation behaviour as in their uninfected conspecifics. In experiment 2, uninfected as well as infected gammarids did not significantly discriminate between infected and uninfected groups. Although only uninfected gammarids reduced their activity in the presence of predator cues, infected G. pulex were generally less active than uninfected conspecifics. This might suggest that P. minutus manipulates rather the general anti-predator behaviour than the plastic response to predation risk.     

Evolution of dark-spored Myxomycetes (slime-molds): molecules versus morphology

The Myxomycetes are a major component of soil amoebae, displaying a complex life cycle that terminates in the formation of often macroscopic fruiting bodies. The classification of Myxomycetes is controversial and strongly depends on the weight given by different authors to morphological and developmental characters. We used a molecular approach to establish the phylogenetic relationships in the dark-spored orders Stemonitales and Physarales. Twenty-five small subunit ribosomal RNA gene sequences were obtained, with focus on two Stemonitales genera, Lamproderma and Comatricha. Unexpectedly, our results show that Stemonitales are paraphyletic with Physarales arising from within a Lamproderma clade. The genus Lamproderma itself is polyphyletic and can be divided into two distinct clades. Additionally, we found that Comatricha nigricapillitia comprises two cryptic species, both related to Enerthenema. Our study allows the reappraisal of morphological and developmental characters in the light of molecular data and sets foundations for a new classification of Myxomycetes.     

Evolution of nonstop,no-go and nonsense-mediated mRNA decay and their termination factor-derived components

Background  

Members of the eukaryote/archaea specific eRF1 and eRF3 protein families have central roles in translation termination. They are also central to various mRNA surveillance mechanisms, together with the eRF1 paralogue Dom34p and the eRF3 paralogues Hbs1p and Ski7p. We have examined the evolution of eRF1 and eRF3 families using sequence similarity searching, multiple sequence alignment and phylogenetic analysis.     

Phylogenetic analysis of the UDP-glycosyltransferase multigene family of Arabidopsis thaliana

A class of UDP-glycosyltransferases (UGTs) defined by the presence of a C-terminal consensus sequence is found throughout the plant and animal kingdoms. Whereas mammalian enzymes use UDP-glucuronic acid, the plant enzymes typically use UDP-glucose in the transfer reactions. A diverse array of aglycones can be glucosylated by these UGTs. In plants, the aglycones include plant hormones, secondary metabolites involved in stress and defense responses, and xenobiotics such as herbicides. Glycosylation is known to regulate many properties of the aglycones such as their bioactivity, their solubility, and their transport properties within the cell and throughout the plant. As a means of providing a framework to start to understand the substrate specificities and structure-function relationships of plant UGTs, we have now applied a molecular phylogenetic analysis to the multigene family of 99 UGT sequences in Arabidopsis. We have determined the overall organization and evolutionary relationships among individual members with a surprisingly high degree of confidence. Through constructing a composite phylogenetic tree that also includes all of the additional plant UGTs with known catalytic activities, we can start to predict both the evolutionary history and substrate specificities of new sequences as they are identified. The tree already suggests that while the activities of some subgroups of the UGT family are highly conserved among different plant species, others subgroups shift substrate specificity with relative ease.