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Abstract Background Conventional fluorescent proteins, such as GFP, its derivatives and flavin mononucleotide based fluorescent proteins (FbFPs) are often used as fusion tags for detecting recombinant proteins during cultivation. These reporter tags are state-of-the-art; however, they have some drawbacks, which can make on-line monitoring challenging. It is discussed in the literature that the large molecular size of proteins of the GFP family may stress the host cell metabolism during production. In addition, fluorophore formation of GFP derivatives is oxygen-dependent resulting in a lag-time between expression and fluorescence detection and the maturation of the protein is suppressed under oxygen-limited conditions. On the contrary, FbFPs are also applicable in an oxygen-limited or even anaerobic environment but are still quite large (58% of the size of GFP). Results As an alternative to common fluorescent tags we developed five novel tags based on clustered tryptophan residues, called W-tags. They are only 5-11% of the size of GFP. Based on the property of tryptophan to fluoresce in absence of oxygen it is reasonable to assume that the functionality of our W-tags is also given under anaerobic conditions. We fused these W-tags to a recombinant protein model, the anti-CD30 receptor single-chain fragment variable antibody (scFv) Ki-4(scFv) and the anti-MucI single-chain fragment variable M12(scFv). During cultivation in Microtiter plates, the overall tryptophan fluorescence intensity of all cultures was measured on-line for monitoring product formation via the different W-tags. After correlation of the scattered light signal representing biomass concentration and tryptophan fluorescence for the uninduced cultures, the fluorescence originating from the biomass was subtracted from the overall tryptophan signal. The resulting signal, thus, represents the product fluorescence of the tagged and untagged antibody fragments. The product fluorescence signal was increased. Antibodies with W-tags generated stronger signals than the untagged construct. Conclusions Our low-molecular-weight W-tags can be used to monitor the production of antibody fragments on-line. The binding specificity of the recombinant fusion protein is not affected, even though the binding activity decreases slightly with increasing number of tryptophan residues in the W-tags. Thus, the newly designed W-tags offer a versatile and generally applicable alternative to current fluorescent fusion tags.

Background Myocardial fibrosis (MF) and necrosis of the left heart has been reported as CF-related complication, especially in postmortem case series (Witt et al. 2000). Discussion We report a 9-year-old, pancreatic insufficient, malnourished CF-boy [F508del/CFTRdele2,3(21kb)], who presented with dyspnoea, cough and cardiac arrhythmia without pulmonary exacerbation. He had a normal lung function (FEV1%pred 86%) and no chronic Pseudomonas (Ps.) infection. Ventricular tachycardia and isolated left heart failure with dilatation of the left ventricle (ejection fraction 28%) were diagnosed. Myocarditis or inherited cardiomyopathy were made unlikely. He underwent a pulmonary banding, received an implantable cardioverter-defibrillator and was listed for heart transplantation. Under anticongestive therapy he remained stable until an uncomplicated orthotopic heart transplantation at the age of 12 years. Histological examination of explanted heart showed no inflammation, no malignoma – but obvious interstitial MF. Immunosuppression included tacrolimus, mycophenolate and initial prednisone. No acute or chronic rejection occurred over 3.5 years. Intermittent infection with Ps. aeruginosa was treated by eradication therapy. Under long-term antibiotic prophylaxis against Staphylococcus his lung function remained stable (last FEV1%pred 79%). His height increased (<3. to 10. percentile), but underweight remained (<3. percentile over time). Conclusion Isolated left heart failure is a rare, but life-threatening complication in CF. Isolated heart transplantation could be performed successfully without negative impact on lung disease due to extensive immunosuppression. [ABSTRACT FROM AUTHOR]

Abstract Background Genetic markers and linkage mapping are basic prerequisites for marker-assisted selection and map-based cloning. In the case of the key grassland species Lolium spp., numerous mapping populations have been developed and characterised for various traits. Although some genetic linkage maps of these populations have been aligned with each other using publicly available DNA markers, the number of common markers among genetic maps is still low, limiting the ability to compare candidate gene and QTL locations across germplasm. Results A set of 204 expressed sequence tag (EST)-derived simple sequence repeat (SSR) markers has been assigned to map positions using eight different ryegrass mapping populations. Marker properties of a subset of 64 EST-SSRs were assessed in six to eight individuals of each mapping population and revealed 83% of the markers to be polymorphic in at least one population and an average number of alleles of 4.88. EST-SSR markers polymorphic in multiple populations served as anchor markers and allowed the construction of the first comprehensive consensus map for ryegrass. The integrated map was complemented with 97 SSRs from previously published linkage maps and finally contained 284 EST-derived and genomic SSR markers. The total map length was 742 centiMorgan (cM), ranging for individual chromosomes from 70 cM of linkage group (LG) 6 to 171 cM of LG 2. Conclusions The consensus linkage map for ryegrass based on eight mapping populations and constructed using a large set of publicly available Lolium EST-SSRs mapped for the first time together with previously mapped SSR markers will allow for consolidating existing mapping and QTL information in ryegrass. Map and markers presented here will prove to be an asset in the development for both molecular breeding of ryegrass as well as comparative genetics and genomics within grass species.

Abstract Background Chemoresistance acquisition may influence cancer cell biology. Here, bioinformatics analysis of gene expression data was used to identify chemoresistance-associated changes in neuroblastoma biology. Results Bioinformatics analysis of gene expression data revealed that expression of angiogenesis-associated genes significantly differs between chemosensitive and chemoresistant neuroblastoma cells. A subsequent systematic analysis of a panel of 14 chemosensitive and chemoresistant neuroblastoma cell lines in vitro and in animal experiments indicated a consistent shift to a more pro-angiogenic phenotype in chemoresistant neuroblastoma cells. The molecular mechanims underlying increased pro-angiogenic activity of neuroblastoma cells are individual and differ between the investigated chemoresistant cell lines. Treatment of animals carrying doxorubicin-resistant neuroblastoma xenografts with doxorubicin, a cytotoxic drug known to exert anti-angiogenic activity, resulted in decreased tumour vessel formation and growth indicating chemoresistance-associated enhanced pro-angiogenic activity to be relevant for tumour progression and to represent a potential therapeutic target. Conclusion A bioinformatics approach allowed to identify a relevant chemoresistance-associated shift in neuroblastoma cell biology. The chemoresistance-associated enhanced pro-angiogenic activity observed in neuroblastoma cells is relevant for tumour progression and represents a potential therapeutic target.

Abstract Background RAI3 is an orphan G-protein coupled receptor (GPCR) that has been associated with malignancy and may play a role in the proliferation of breast cancer cells. Although its exact function in normal and malignant cells remains unclear and evidence supporting its role in oncogenesis is controversial, its abundant expression on the surface of cancer cells would make it an interesting target for the development of antibody-based therapeutics. To investigate the link with cancer and provide more evidence for its role, we carried out a systematic analysis of RAI3 expression in a large set of human breast cancer specimens. Methods We expressed recombinant human RAI3 in bacteria and reconstituted the purified protein in liposomes to raise monoclonal antibodies using classical hybridoma techniques. The specific binding activity of the antibodies was confirmed by enzyme-linked immunosorbent assay (ELISA), western blot and immunocytochemistry. We carried out a systematic immunohistochemical analysis of RAI3 expression in human invasive breast carcinomas (n = 147) and normal breast tissues (n = 44) using a tissue microarray. In addition, a cDNA dot blot hybridisation assay was used to investigate a set of matched normal and cancerous breast tissue specimens (n = 50) as well as lymph node metastases (n = 3) for RAI3 mRNA expression. Results The anti-RAI3 monoclonal antibodies bound to recombinant human RAI3 protein with high specificity and affinity, as shown by ELISA, western blot and ICC. The cDNA dot blot and immunohistochemical experiments showed that both RAI3 mRNA and RAI3 protein were abundantly expressed in human breast carcinoma. However, there was no association between RAI3 protein expression and prognosis based on overall and recurrence-free survival. Conclusion We have generated a novel, highly-specific monoclonal antibody that detects RAI3 in formaldehyde-fixed paraffin-embedded tissue. This is the first study to report a systematic analysis of RAI3 expression in normal and cancerous human breast tissue at both the mRNA and protein levels.

The phase stability, nonstoichiometry, point defects, and magnetoresistance (MR) of polycrystalline Sr2FeMoO6-δ (SFMO) were studied. Thermogravimety at 1200 °C in combination with x-ray diffraction shows that single-phase SFMO exists between -10.2≤log pO2≤-13.7 at 1200 °C. At lower oxygen partial pressure mass loss signals reductive decomposition; at higher pO2 a mass gain indicates oxidative decomposition into SrMoO4 and SrFeO3-x. The nonstoichiometry δ at 1200 °C was measured as function of pO2 and oxygen vacancies were found to represent majority defects. The vacancy concentration increases with decreasing pO2; a maximum nonstoichiometry of δ=0.086 is observed close to the lower phase boundary. Samples with different δ were prepared at 1200 °C and various pO2. The variation of structural parameters, magnetization, and MR is discussed in relation to oxygen nonstoichiometry δ. Maximum MR=6.5% at 298 K and 1 T is observed for samples with large δ. [ABSTRACT FROM AUTHOR]

Determination of B concentrations in natural fresh and saline waters by ICP-OES may be strongly affected by matrix effects resulting in a deviation (shift to apparently lower values) in the order of 10 to 20% (if working without matrix-matched standard solutions), at variance with the NTIMS (Negative Thermal Ionization Mass Spectrometry) isotope dilution technique which is virtually unaffected by such effects. NTIMS isotope dilution is a highly sensitive, precise and effective technique which requires only trace amounts of boron (1–10 ng B) and allows boron concentrations in natural waters to be analyzed with an analytical uncertainty of ± 2% (2 σ). [ABSTRACT FROM AUTHOR]

Abstract Background In screening of libraries derived by expression cloning, expression of active proteins in E. coli can be limited by formation of inclusion bodies. In these cases it would be desirable to enrich gene libraries for coding sequences with soluble gene products in E. coli and thus to improve the efficiency of screening. Previously Wilkinson and Harrison showed that solubility can be predicted from amino acid composition (Biotechnology 1991, 9(5):443–448). We have applied this analysis to members of the alpha/beta hydrolase fold family to predict their solubility in E. coli. alpha/beta hydrolases are a highly diverse family with more than 1800 proteins which have been grouped into homologous families and superfamilies. Results The predicted solubility in E. coli depends on hydrolase size, phylogenetic origin of the host organism, the homologous family and the superfamily, to which the hydrolase belongs. In general small hydrolases are predicted to be more soluble than large hydrolases, and eukaryotic hydrolases are predicted to be less soluble in E. coli than prokaryotic ones. However, combining phylogenetic origin and size leads to more complex conclusions. Hydrolases from prokaryotic, fungal and metazoan origin are predicted to be most soluble if they are of small, medium and large size, respectively. We observed large variations of predicted solubility between hydrolases from different homologous families and from different taxa. Conclusion A comprehensive analysis of all alpha/beta hydrolase sequences allows more efficient screenings for new soluble alpha/beta hydrolases by the use of libraries which contain more soluble gene products. Screening of hydrolases from families whose members are hard to express as soluble proteins in E. coli should first be done in coding sequences of organisms from phylogenetic groups with the highest average of predicted solubility for proteins of this family. The tools developed here can be used to identify attractive target genes for expression using protein sequences published in databases. This analysis also directs the design of degenerate, family- specific primers to amplify new members from homologous families or superfamilies with a high probability of soluble alpha/beta hydrolases.

Abstract Background Common oral diseases and dental caries can be prevented effectively by passive immunization. In humans, passive immunotherapy may require the use of humanized or human antibodies to prevent adverse immune responses against murine epitopes. Therefore we generated human single chain and diabody antibody derivatives based on the binding characteristics of the murine monoclonal antibody Guy's 13. The murine form of this antibody has been used successfully to prevent Streptococcus mutans colonization and the development of dental caries in non-human primates, and to prevent bacterial colonization in human clinical trials. Results The antibody derivatives were generated using a chain-shuffling approach based on human antibody variable gene phage-display libraries. Like the parent antibody, these derivatives bound specifically to SAI/II, the surface adhesin of the oral pathogen S. mutans. Conclusions Humanization of murine antibodies can be easily achieved using phage display libraries. The human antibody fragments bind the antigen as well as the causative agent of dental caries. In addition the human diabody derivative is capable of aggregating S. mutans in vitro, making it a useful candidate passive immunotherapeutic agent for oral diseases.

In a molecular Auger spectrum information on the decaying state is implicitly ensemble-averaged. For a repulsive core-ionized state, for example, contributions from all parts of its potential curve are superimposed in the Auger spectrum. Using carbon tetrafluoride (CF4, tetrafluoromethane), we demonstrate for the first time that these contributions can be disentangled by recording photoelectron–Auger electron coincidence spectra with high energy resolution. For the F K-VV spectrum of CF4, there are significant differences in the Auger decay at different intermediate state (single core hole) geometries. With the help of calculations, we show that these differences result primarily from zero-point fluctuations in the neutral molecular ground state, but are amplified by the nuclear dynamics during Auger decay. [ABSTRACT FROM AUTHOR]

Background: RAI3 is an orphan G-protein coupled receptor (GPCR) that has been associated with malignancy and may play a role in the proliferation of breast cancer cells. Although its exact function in normal and malignant cells remains unclear and evidence supporting its role in oncogenesis is controversial, its abundant expression on the surface of cancer cells would make it an interesting target for the development of antibody-based therapeutics. To investigate the link with cancer and provide more evidence for its role, we carried out a systematic analysis of RAI3 expression in a large set of human breast cancer specimens.Methods: We expressed recombinant human RAI3 in bacteria and reconstituted the purified protein in liposomes to raise monoclonal antibodies using classical hybridoma techniques. The specific binding activity of the antibodies was confirmed by enzyme-linked immunosorbent assay (ELISA), western blot and immunocytochemistry. We carried out a systematic immunohistochemical analysis of RAI3 expression in human invasive breast carcinomas (n = 147) and normal breast tissues (n = 44) using a tissue microarray. In addition, a cDNA dot blot hybridisation assay was used to investigate a set of matched normal and cancerous breast tissue specimens (n = 50) as well as lymph node metastases (n = 3) for RAI3 mRNA expression.Results: The anti-RAI3 monoclonal antibodies bound to recombinant human RAI3 protein with high specificity and affinity, as shown by ELISA, western blot and ICC. The cDNA dot blot and immunohistochemical experiments showed that both RAI3 mRNA and RAI3 protein were abundantly expressed in human breast carcinoma. However, there was no association between RAI3 protein expression and prognosis based on overall and recurrence-free survival.Conclusion: We have generated a novel, highly-specific monoclonal antibody that detects RAI3 in formaldehyde-fixed paraffin-embedded tissue. This is the first study to report a systematic analysis of RAI3 expression in normal and cancerous human breast tissue at both the mRNA and protein levels. [ABSTRACT FROM AUTHOR]

Extending nanostructures into the third dimension has become a major research avenue in modern magnetism, superconductivity, and spintronics, because of geometry-, curvature-, and topology-induced phenomena. Here, we introduce Co–Fe nanovolcanoes—nanodisks overlaid by nanorings—as purpose-engineered 3D architectures for nanomagnonics, fabricated by focused electron beam-induced deposition. We use both perpendicular spin-wave resonance measurements and micromagnetic simulations to demonstrate that the rings encircling the volcano craters harbor the highest-frequency eigenmodes, while the lower-frequency eigenmodes are concentrated within the volcano crater, due to the non-uniformity of the internal magnetic field. By varying the crater diameter, we demonstrate the deliberate tuning of higher-frequency eigenmodes without affecting the lowest-frequency mode. Thereby, the extension of 2D nanodisks into the third dimension allows one to engineer their lowest eigenfrequency by using 3D nanovolcanoes with 30% smaller footprints. The presented nanovolcanoes can be viewed as multi-mode microwave resonators and 3D building blocks for nanomagnonics. [ABSTRACT FROM AUTHOR]

Following growers' reports of herbicide control problems, populations of 30 wild oats, Avena fatua, were collected from the south-east main arable counties of Ireland in 2016 and investigated for the occurrence and potential for herbicide resistance to acetyl-CoA carboxylase (ACCase) inhibitors pinoxaden, propaquizafop and cycloxydim, as well as acetolactate synthase (ALS) inhibitor mesosulfuron + iodosulfuron. Plant survival ≥20% was considered as the discriminating threshold between resistant and susceptible populations, when plants were treated with full recommended field rates of ACCase/ALS inhibitors. Glasshouse sensitivity screens revealed 2 out of 30 populations were cross-resistant to all three ACCase inhibitors. While three populations were cross-resistant to both pinoxaden and propaquizafop, and additionally, two populations were resistant to propaquizafop only. Different degree of resistance and cross-resistance between resistant populations suggest the involvement of either different point mutations or more than one resistance mechanism. Nevertheless, all populations including the seven ACCase-resistant populations were equally susceptible to ALS inhibitor. An integrated weed management (cultural/non-chemical control tactics and judicious use of herbicides) approach is strongly recommended to minimize the risk of herbicide resistance evolution. [ABSTRACT FROM AUTHOR]

Despite being known of for decades, the actual realization of memory devices based on the memristive effect is progressing slowly, due to processing requirements and the need for exotic materials which are not compatible with today's complementary-metal-oxide-semiconductor (CMOS) technology. Here, we report an experimental study on a Ge quantum wire device featuring distinct signatures of memristive behavior favorable for integration in CMOS platform technology. Embedding the quasi-1D Ge quantum wire into an electrostatically modulated back-gated field-effect transistor, we demonstrate that individual current transport channels can be addressed directly by controlling the surface trap assisted electrostatic gating. The resulting quantization of the current represents the ultimate limit of memristors with practically zero off-state current and low footprint. In addition, the proposed device has the advantage of non-destructive successive reading cycles capability. Importantly, our findings provide a framework towards fully CMOS compatible ultra-scaled Ge based memristors. [ABSTRACT FROM AUTHOR]

Festulolium are hybrids between species within the Lolium and Festuca genera. They are attractive candidates for hybridisation due to their complementary characteristics in the context of forage production and environmental challenges, with Lolium exhibiting greater digestibility and Festuca possessing superior resilience to abiotic stress. To establish the extent of the representation of these characteristics in different Festulolium hybrids, a five-year field experiment was undertaken in a mild Atlantic climate. Four alternative species combinations and six pure species (controls) were evaluated for dry matter yield and persistency from 2014 to 2018, and various nutritional parameters in the first silage cut were measured from 2015 to 2017. Festulolium hybrids derived from L. multiflorum were found to group with both Lolium and Festuca pure species for a mean ground score to evaluate persistency (P < 0.05). Hybrid types of the L. perenne lineage were found to have ground score values more alike to their Lolium component and distinct from the F. pratensis pure species. In terms of dry matter yield, the majority of L. multiflorum hybrids observed formed a higher yielding group with the tetraploid L. multiflorum pure species and distinct from diploid F. pratensis pure species, whereas all other hybrid types and controls shared groups in common. For the nutrient content parameters including dry matter digestibility, water soluble sugars, and buffering capacity, Lolium pure species exhibited more favourable values, whereas hybrids were largely found to display intermediate to low values. Festuca pure species consistently exhibited poorer values with the exception of crude protein content where F. pratensis displayed the highest values and also the 2017 measurement of buffering capacity. In conclusion, the Festulolium hybrids shared characteristics with both parental types, but they often showed a greater likeness to the Lolium component. [ABSTRACT FROM AUTHOR]