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Infection with Respiratory Syncytial Virus (RSV) causes both upper and lower respiratory tract disease in humans, leading to significant morbidity and mortality in both young children and older adults. Currently, there is no licensed vaccine available, and therapeutic options are limited. During the infection process, the type I viral fusion (F) glycoprotein on the surface of the RSV particle rearranges from a metastable prefusion conformation to a highly stable postfusion form. In people naturally infected with RSV, most potent neutralizing antibodies are directed to the prefusion form of the F protein. Therefore, an engineered RSV F protein stabilized in the prefusion conformation (DS-Cav1) is an attractive vaccine candidate. Long-term stability at 4°C or higher is a desirable attribute for a commercial subunit vaccine antigen. To assess the stability of DS-Cav1, we developed assays using D25, an antibody which recognizes the prefusion F-specific antigenic site Ø, and a novel antibody 4D7, which was found to bind antigenic site I on the postfusion form of RSV F. Biophysical analysis indicated that, upon long-term storage at 4°C, DS-Cav1 undergoes a conformational change, adopting alternate structures that concomitantly lose the site Ø epitope and gain the ability to bind 4D7. [ABSTRACT FROM AUTHOR]

Abstract: Over the past decade, microfluidic techniques have been established as a versatile platform to perform live cell experiments under well-controlled conditions. To investigate the directional responses of cells, stable concentration profiles of chemotactic factors can be generated in microfluidic gradient mixers that provide a high degree of spatial control. However, the times for built-up and switching of gradient profiles are in general too slow to resolve the intracellular protein translocation events of directional sensing of eukaryotes. Here, we review an example of a conventional microfluidic gradient mixer as well as the novel flow photolysis technique that achieves an increased temporal resolution by combining the photo-activation of caged compounds with the advantages of microfluidic chambers. [Copyright &y& Elsevier]

Advanced microscopy and the corresponding image analysis have been developed in recent years into a powerful tool for studying molecular and morphological events in cells and tissues. Cell-based high-content screening (HCS) is an upcoming methodology for the investigation of cellular processes and their alteration by multiple chemical or genetic perturbations. Multiparametric characterization of responses to such changes can be analyzed using intact live cells as reporter. These disturbances are screened for effects on a variety of molecular and cellular targets, including subcellular localization and redistribution of proteins. In contrast to biochemical screening, they detect the responses within the context of the intercellular structural and functional networks of normal and diseased cells, respectively. As cell-based HCS of small-molecule libraries is applied to identify and characterize new therapeutic lead compounds, large pharmaceutical companies are major drivers of the technology and have already shown image-based screens using more than 100000 compounds. [Copyright &y& Elsevier]

Abstract: Butterflies of the genus Heliconius are well known for their peculiar habits of utilizing pollen as a source of amino acids. Saliva plays a major role in the process of extracting amino acids and proteins from the pollen grains. In this investigation, we obtained samples of saliva from adult Heliconius melpomene by placing pumpkin pollen or fine glass-beads on the proboscis, which stimulates the butterflies to release saliva. Proteolytic activity was determined in the saliva by an insoluble protein-dye that turns blue when cleaved by proteases. Its extinction value was measured with a spectrophotometer at 595nm. Both the saliva sampled with pollen and the saliva obtained from inert glass-beads exhibit proteolytic activity demonstrating that the saliva contains proteases. The proteolytic activity of the pollen/saliva samples was higher than that of the glass-bead/saliva samples, which we attribute to the stimulating effects of pollen, such as taste, smell, and texture, and not to proteases which might have been liberated from the pollen. This is indicated by the fact that pollen samples without saliva showed only a negligible indication for proteolytic activity. In general, females exhibit higher proteolytic activities than males, presumably due to their greater amino acid investment in reproduction. We present here first evidence for the existence of proteases in the saliva of a butterfly species and suggest that these enzymes are crucial for the use of amino acids and proteins from pollen in Heliconius butterflies. [Copyright &y& Elsevier]

The intracellular location of the membrane-bound ammonia monooxygenase (AMO) in all genera of ammonia oxidizing bacteria ( Nitrosomonas, Nitrosococcus and Nitrosospira) was determined by electron microscopic immunocytochemistry. Polyclonal antibodies recognizing the two subunits, AmoA- and AmoB-proteins, were used for post-embedding labeling. Ultrathin sections revealed that the AmoB-protein was located in all genera on the cytoplasmic membrane. In cells of Nitrosomonas and Nitrosococus additional but less AmoB-labeling was found on the intracytoplasmic membrane (ICM). In contrast to the detection of AmoB-protein, the AmoA-antibodies failed to detect the AmoA-protein. Based on quantitative immunoblots the extent of ICM in Nitrosomonas eutropha was correlated with the amount of AmoA in the cells. The highest extent of ICM and amount of AmoA was found at low ammonium substrate concentrations. [ABSTRACT FROM AUTHOR]

Spider silks are some of the strongest materials found in nature. Achieving the high tensile strength and elasticity of the dragline of orb-weaving spiders, such as Nephila clavipes, is a principal goal in biomimetics research. The dragline has a composite nature and is predominantly made up by two proteins, the major ampullate spidroins 1 and 2 (refs 3,6,7), which can be considered natural block copolymers. On the basis of their molecular structures both spidroins are thought to contribute, in different ways, to the mechanical properties of dragline silk. The spinning process itself is also considered important for determining the observed features by shaping the hierarchical structure of the fibre. Here we study the heterogeneous distribution of proteins along the radial axis of the fibre. This heterogeneity is generated during the conversion of the liquid spinning dope into solid fibre. Whereas spidroin 1 is distributed almost uniformly within the fibre core, spidroin 2 is missing in the periphery and is tightly packed in certain core areas. Our findings suggest that the role of spidroin 2 in the spinning process could be to facilitate the formation of fibrils and contribute directly to the elasticity of the silk. [ABSTRACT FROM AUTHOR]

Modern methods of high-throughput molecular biology render it possible to generate time series of metabolite concentrations and the expression of genes and proteins in vivo. These time profiles contain valuable information about the structure and dynamics of the underlying biological system. This information is implicit and its extraction is a challenging but ultimately very rewarding task for the mathematical modeler. Using a well-suited modeling framework, such as Biochemical Systems Theory (BST), it is possible to formulate the extraction of information as an inverse problem that in principle may be solved with a genetic algorithm or nonlinear regression. However, two types of issues associated with this inverse problem make the extraction task difficult. One type pertains to the algorithmic difficulties encountered in nonlinear regressions with moderate and large systems. The other type is of an entirely different nature. It is a consequence of assumptions that are often taken for granted in the design and analysis of mathematical models of biological systems and that need to be revisited in the context of inverse analyses. The article describes the extraction process and some of its challenges and proposes partial solutions. [ABSTRACT FROM AUTHOR]

Abstract: N→O acyl migrations at serine or threonine residues in peptides or proteins have previously been observed upon treatment with strong acids. Here we show that the extent of such N→O shifts depends on the peptide sequence and even in the presence of moderately acidic trifluoroacetic acid, as during Fmoc or Bsmoc-based solid phase peptide synthesis, may give rise to large amounts of depsipeptide by-products. [Copyright &y& Elsevier]

The diffraction pattern of native protein crystals of F420-dependent methylenetetrahydro-methanopterin dehydrogenase from Methanopyrus kandleri shows weak additional reflections compared with the selenomethionine-labelled protein crystals, indicating a doubled c unit-cell parameter. These reflections indicate small reorientations of the hexameric structural units, breaking the translational symmetry. TLS refinement of the selenomethionine-labelled protein structure at 1.55 Å resolution revealed an anisotropic rigid-body libration of the hexameric units. The anisotropy is consistent with the static reorientation in the native protein crystals. These results are discussed as related to the crystal packing. The relation between the two structures suggests an analogy to structural changes during certain kinds of phase transitions that have been well studied in inorganic structural chemistry. [ABSTRACT FROM AUTHOR]

Research on the export of proteins and nucleic acids from the nucleus to the cytoplasm has greatly gained from the discovery that the actinobacterial toxin leptomycin B (LMB) specifically inactivates the export receptor chromosomal region maintenance 1 (CRM1). Recently, it was shown that myxobacterial cytotoxins, named ratjadones (RATs), also bind to CRM1 and inhibit nuclear export. However, the reaction mechanism of RATs was not resolved. Here, we show that LMB and RAT A employ the same molecular mechanism to inactivate CRM1. Alkylation of residue Cys528 of CRM1 determines both LMB and RAT sensitivity and prevents nuclear export of CRM1 cargo proteins. [Copyright &y& Elsevier]

Novel high-throughput techniques are capable of producing dense time series of many simultaneous in vivo measurements of the expression of genes, concentrations of metabolites, the prevalence and activation states of proteins, and of other biomarkers characterizing the physiological state of a cell or organism. These data contain enormous information, much of which, however, is not explicit but must be extracted with mathematical and computational means. The magnitude of high-throughput data generation and the potential and demands that these data entail will dramatically affect strategies for modeling, analyzing, and optimizing pathways and usher in a new era of metabolic systems analysis. [Copyright &y& Elsevier]

A more detailed characterization of particulate organic matter in wastewater streams is needed to improve solid–liquid separation and biological processes for wastewater treatment. The objective of this paper was to evaluate particle size distributions and the associated chemical composition for municipal, industrial, and agricultural waste streams. Most of the organic matter in these wastewaters was larger than a molecular weight of 103 amu and therefore would require extracellular hydrolysis before any bacterial metabolism. Particle size distributions were significantly different for the studied waste streams. In municipal wastewater, the organic matter was evenly distributed in all eight size fractions ranging from 103 amu up to 63 μm. The industrial and agricultural wastewaters, however, contained mainly soluble organic matter (<103 amu) and larger particles (>1.2 μm for the industrial and >10 μm for the agricultural waste) leaving a gap in the size range of large macromolecules and colloids. The relative protein and carbohydrate concentrations varied for the different size fractions compared to the measured chemical oxygen demand (COD) in the corresponding size fraction. Thus, the design of the solid–liquid separation at a treatment plant could be used to purposefully modify the overall chemical composition of the organic matter before further biological treatment. Particle size distributions will influence design and operation of biological nutrient removal processes such as denitrification or biological phosphorus removal that may be carbon limited if a large fraction of the organic matter is composed of large particles with slow hydrolysis rates. Measured particle size distributions for the different waste streams in this study (municipal, industrial, agricultural) were significantly different requiring specific approaches for treatment plant design. [Copyright &y& Elsevier]

Covalent lipid attachments are essential co- and posttranslational modifications for signalling proteins. Gαs, the α-subunit of the heterotrimeric G protein that activates adenylyl cyclase, is known to be palmitoylated at the third N-terminal amino acid, a cysteine. Palmitoylation is involved in anchoring Gαs to the membrane by increasing its intrinsic hydrophobicity. We identified by mass spectrometry a second, functionally even more important, covalent modification. It consists of another palmitoyl residue attached to the preceding glycine (Gly²). Palmitoylation at this position has profound consequences for levels of signal transduction. It sensitizes the cell up to 200-fold for adenylyl cyclase-stimulating agents. The inhibitory inputs mediated by Gαi are downregulated to <10%. Thereby, Gly2-palmitoylation of Gαs relieves cellular stimulation at the level of adenylyl cyclase whereas it renders the inhibitory modulation via Gαi more difficult. [ABSTRACT FROM AUTHOR]

Pax5 (BSAP) functions as both a transcriptional activator and repressor during midbrain patterning, B-cell development and lymphomagenesis. Here we demonstrate that Pax5 exerts its repression function by recruiting members of the Groucho corepressor family. In a yeast two-hybrid screen, the groucho-related gene product Grg4 was identified as a Pax5 partner protein. Both proteins interact cooperatively via two separate domains: the N-terminal Q and central SP regions of Grg4, and the octapeptide motif and C-terminal transactivation domain of Pax5. The phosphorylation state of Grg4 is altered in vivo upon Pax5 binding. Moreover, Grg4 efficiently represses the transcriptional activity of Pax5 in an octapeptide-dependent manner. Similar protein interactions resulting in transcriptional repression were also observed between distantly related members of both the Pax2/5/8 and Groucho protein families. In agreement with this evolutionary conservation, the octapeptide motif of Pax proteins functions as a Groucho-dependent repression domain in Drosophila embryos. These data indicate that Pax proteins can be converted from transcriptional activators to repressors through interaction with corepressors of the Groucho protein family. [ABSTRACT FROM AUTHOR]

Reports on the energetic consequences of a cavity introduced by Leu/Ala substitution at the tightly packed dimeric interface and how addition of 30% glycerol affects the folding thermodynamics of the engineered dimeric leucine zipper A2 and the cavity mutants. Indication that mutational and solvent effects on protein stability can be thermodynamically complex.

The T 7gene-4 protein has been purified to near homogeneity using a complementation assay in vitro, and it is designated T7 DNA-priming protein (DNA primase). The purified enzyme enables T7 DNA polymerase to initiate DNA synthesis on various circular single-stranded DNA templates by a mechanism which involves the synthesis of a very short RNA primer. The oligoribonucleotide, which is linked to the product DNA via a 3' : 5'-phosphodiester bond, starts with pppA-C and terminates predominantly with AMP. When only ATP and CTP are precursors, the RNA primer is found to be primarily a tetranucleotide of the sequence pppA-C-C-A. Using oligoribonucleotides in place of ribonucleoside triphosphates as chain initiators, T7 DNA-priming protein drastically increases the efficiency with which T7 DNA polymerase can utilize particular tetranucleotide primers containing A and C residues. T7 DNA-priming protein also enables T7 DNA polymerase to make use of native or nicked duplex T7 DNA as template-primer. This reaction does not require ribonucleoside triphosphates, although their addition enhances DNA synthesis 2-4-fold. The product formed in their absence is covalently attached to the template DNA and is found to contain a few long branches when examined by electron microscopy. In the presence of ribonucleoside triphosphates most of the newly made product arises from initiation of DNA chains tie novo. Incubation of three proteins: T7 DNA-priming protein, T7 DNA polymerase, and T7 DNA- binding protein, with ribonucleoside and deoxyribonucleoside triphosphates, and with φX174 DNA as template leads to the generation of 'rolling circle-dike' structures as visualized in the electron microscope. Single-stranded regions at the tail-circle junction indicate that initiations can occur tie novo on the displaced complementary strand. This is consistent with a discontinuous mode of 'lagging' strand synthesis and suggests that the same proteins may also be responsible for fork propagation in vivo. [ABSTRACT FROM AUTHOR]

At 77 K the electron spin resonance (ESR) spectra of the NO derivatives of the mutant haemoglobins Hb M Iwate and Hb Zürich as well as of the isolated chains of normal haemoglobin were studied. Two types of ESR spectra differing in the g-value and the hyperfine splitting at gzz were observed. The type II spectrum is characterized by a hyperfine structure at gzz = 2.005 with a splitting constant of ΔH = 23 G (14NO) or 32 G (15NO), respectively. In the type I spectrum the splitting constant of the hyperfine structure at gzz = 2.009 amounts to AH = 18 G (14NO) or 23 G (15NO), respectively. In some cases this hyperfine structure is coincident with another one at gxx = 2.064 with nearly identical splitting constant. In addition, the type I spectrum is characterized by an increased ESR absorption at gxx = 2.064. At neutral pH the NO derivatives of the isolated chains as well as of the mutant haemoglobins give rise to a type II spectrum. In correspondence with previous results gained with normal NO haemoglobin, the ESR spectra of the NO-α chains and NO-Hb Zürich show a transition to type I in the acid region. This transition is favoured by binding of 2,3-bisphosphoglycerate. On the other hand, the ESR spectra of the NO-β chains and NO-Hb M Iwate are of the type II also at acid pH. The NO-β chains show a transition of the ESR spectrum from type II to type I only at alkaline pH. These results indicate that in the tetrameric NO haemoglobin only the α chains are responsible for the transition of the ESR spectrum from type II to type I in the acid region. The two types of ESR spectra are interpreted in terms of two kinds of haem-NO complexes differing in the iron-NO and iron-imidazole distances. The type II spectrum is attributed to a complex with a relatively short iron-imidazole distance which is responsible for a weakened σ-bond in trans position. The type I spectrum arises then from a complex with a larger iron-imidazole bond leading to an approach of the NO molecule to the iron. The influence of the protein conformation upon the iron-imidazole bond length is discussed with regard to the ESR spectra of the mutant NO haemoglobins and considering the influence of agents modifying the protein structure. [ABSTRACT FROM AUTHOR]

A site-specific and strand-specific nick, introduced into the RSF1010 plasmid origin of transfer (oriT), initiates unidirectional DNA transfer during bacterial conjugation. We have previously reproduced this nicking at the duplex oriT in vitro purified preparations of the three known RSF1010-mobilization proteins: MobA (78-kDa form of RSF1010 primase). MobB and MobC (Scherzinger, E., Lurz, R., Otto. S & Dobrinski, B. (1992) Nucleic Acids Res. 20, 41-481. In this study we report the purification of MobA to apparent bomogeneity and demonstrate that this 78-kDa protein by itself is capable of creating the oriT-specific nick if the DNA is present in the single-stranded form. By studying the cleavage of sets of oligodeoxyribonucleotides varying successively by sing nucleotides at the 5' or 3' end, the minimal substrate for cleavage has been defined. The results identify the MobA recognition sequence within the 11-residue oligonucleotide AAGTGCGCCCT which is cleaved at the 3' side of the G at position 7. During the cleavage reaction, MobA becomes covalently linked to the 5'-phosphate end of each broken DNA molecule and retains its activity for the rejoining reaction. It can transfer the attached DNA to an incoming acceptor strand provided that the DNA molecule contains at its 3' end at least the seven nucleotides upstream of the nick site. The covalent MobA-DNA linkage has been determined by two-dimensional thin-layer electrophoresis to be a phosphate. Extensive digestion of the 32P-labeled MobA-oligonucleotide complex with lysine carboxypeptidase yielded a single DNA-bound peptide which was purified and sequenced. The resulting peptide sequence consists of amino acid residues at positions 22-30 in the MobA sequence and identifies Tyr24 as the residue linked to DNA in the covalent complex. [ABSTRACT FROM AUTHOR]

A sensitive and rapid assay of Ca2+ binding to proteins was developed, based on the competition C2+ binding to the protein of interest and fluo-3, a fluorescent Ca2+ indicator. Ca2+ binding to fluo-3 and bovine α-lactalbumin was analyzed at ten different pH values and a range of Na+ and K+ concentrations. We demonstrate that the binding constants of α-lactalbumin, determined by means of the competition assay and using intrinsic protein fluorescence, are the same within experimental error. The dissociation constant of the α-lactalbumin-Ca2+ complex in 50 mM Hepes containing 150 mM Na2+ at pH 7.4 and 25 °C, was found to be 123 ± 2 nM and 103 ± 43 nM when determined by the competition assay and intrinsic protein fluorescence, respectively. Binding of Ca2+ to α-lactalbumin did not depend on pH in the range 6.6–8.4 and was differently affected by Na2+ and K+. EDTA-agarose, a chelating chromatography material, was synthesized and used to remove Ca2+ from buffer and protein solutions. The total concentration of Ca2+ in 50 mM Hepes, containing 150 mM Na2+ at pH 7.4, was lowered to 119 ± 13 nM and the number of Ca2+ bound/molecule α-lactalbumin was lowered to 0.069 ± 0.006. No interaction between fluo-3 and α-lactalbumin could be discerned from spectral analysis and fluorescence anisotropy measurements. [ABSTRACT FROM AUTHOR]

Recent information obtained, mainly by recombinant cDNA technology, on structural heterogeneity of hormone and transmitter receptors, of GTP-binding proteins (G-proteins) and, especially, of G-protein-linked receptors is reviewed and the implications of structural heterogeneity for diversity of hormone and transmitter actions is discussed. For the future, three-dimensional structural analysis of membrane proteins participating in signal transmission and transduction pathways is needed in order to understand the molecular basis of allosteric regulatory mechanisms governing the interactions between these proteins including hysteretic properties and cell cybernetic aspects. [ABSTRACT FROM AUTHOR]

50 S ribosomal subunits of Escherichia coli obtained by zonal centrifugation in B XV rotors were treated with various concentrations of LiCl in the absence and presence of urea. This treatment resulted in three protein fractions, each of which was subjected to CM-cellulose chromatography. Gel filtration in Sephadex G-100 (and preparative polyacrylamide electrophoresis) were used for further separation. The purity of the isolated proteins was better than 95% as shown by four methods. Proteins were isolated in yields of 6-90 mg depending mainly on the number and kind of purification steps. Instead of fractionation with LiCl prior to column chromatography, precipitation of extracted proteins at various concentrations of ammonium sulfate was tested. This method is useful if a given protein, e.g. from mutants is wanted in large quantity. [ABSTRACT FROM AUTHOR]

The sixth (C′6), seventh (C′7) and eighth (C′8) components of human complement were separated from each other by chromatography on hydroxyl apatite and obtained in functionally pure form. Density gradient ultracentrifugation revealed that C′6 and C′7 interact with the fifth component of complement (C′5) to form a reversible protein-protein complex. Evidence was obtained that this complex occurs also in unfractionated, fresh human serum. Kinetic analysis of the reaction sequence in immune haemolysis disclosed that C′5, C′6 and C′7 act, in this order, subsequent to the third component (C′3) and prior to C′8. Exploration of the mode of action of C′5, C′6 and C′7 led to the formulation of two alternative hypotheses. The three components act either sequentially or as one functional unit. Their fluid phase interaction, the shape of dose-response curves and the encountered difficulty to isolate intermediate reaction products favour the concept that they act as a functional unit. [ABSTRACT FROM AUTHOR]

Background: The study of Onchocerca volvulus has been limited by its host range, with only humans and non-human primates shown to be susceptible to the full life cycle infection. Small animal models that support the development of adult parasites have not been identified. Methodology/Principal findings: We hypothesized that highly immunodeficient NSG mice would support the survival and maturation of O. volvulus and alteration of the host microenvironment through the addition of various human cells and tissues would further enhance the level of parasite maturation. NSG mice were humanized with: (1) umbilical cord derived CD34+ stem cells, (2) fetal derived liver, thymus and CD34+ stem cells or (3) primary human skeletal muscle cells. NSG and humanized NSG mice were infected with 100 O. volvulus infective larvae (L3) for 4 to 12 weeks. When necropsies of infected animals were performed, it was observed that parasites survived and developed throughout the infection time course. In each of the different humanized mouse models, worms matured from L3 to advanced fourth stage larvae, with both male and female organ development. In addition, worms increased in length by up to 4-fold. Serum and urine, collected from humanized mice for identification of potential biomarkers of infection, allowed for the identification of 10 O. volvulus-derived proteins found specifically in either the urine or the serum of the humanized O. volvulus-infected NSG mice. Conclusions/Significance: The newly identified mouse models for onchocerciasis will enable the development of O. volvulus specific biomarkers, screening for new therapeutic approaches and potentially studying the human immune response to infection with O. volvulus. [ABSTRACT FROM AUTHOR]

The article offers information regarding a study on the effect of plasma protein adsorption on protein excretion in kidney-transplant recipients with recurrent nephrotic syndrome conducted by researchers including J. Dantal and E. Bigot. The study has revealed that the removal of serum proteins by adsorption to protein A Sepharose leads to remission of proteinuria in patients with recurrences of the nephrotic syndrome after transplantation. The study also concluded that despite the presence of focal segmental glomerulosclerosis, the renal prognosis is relatively good if urinary protein excretion is below 3 grams per 24 hours.

Background: 3D domain swapping is an oligomerization process in which structural elements get exchanged between subunits. This mechanism grasped interest of many researchers due to its association with neurodegenerative diseases like Alzheimer's disease, spongiform encephalopathy etc. Despite the biomedical relevance, very little is known about understanding this mechanism. The quest for ruling principles behind this curious phenomenon that could enable early prediction provided an impetus for our bioinformatics studies. Methodology: A novel method, HIDE, has been developed to find non-domain-swapped homologues and to identify hinge from domain-swapped oligomers. Non-domain-swapped homologues were identified from the protein structural databank for majority of the domain-swapped entries and hinge boundaries could be recognised automatically by means of successive superposition techniques. Different sequence and structural features in domain-swapped proteins and related proteins have also been analysed. Conclusions: The HIDE algorithm was able to identify hinge region in 83% cases. Sequence and structural analyses of hinge and interfaces reveal amino acid preferences and specific conformations of residues at hinge regions, while comparing the domain-swapped and non-domain-swapped states. Interactions differ significantly between regular dimeric interfaces and interface formed at the site of domain-swapped examples. Such preferences of residues, conformations and interactions could be of predictive value. [ABSTRACT FROM AUTHOR]

Abstract: In order to keep the anaerobic process stably and uniformly producing biogas it needs to be supplied with either an even amount of substrate of stable quality or varying amounts according to variations in quality. Feeding amounts are usually adjusted manually as a reaction to changing rates of biogas production. Continuous information about the actual substrate quality is not available and feedstuff analyses are costly. Aim of this study was to assess the feasibility of near infrared spectroscopic (NIRS) online monitoring of substrate quality in order to find ways towards more exact control of biogas plant feeding. A NIRS sensor system was designed, constructed and calibrated for continuous monitoring of (RMSECV in brackets) dry matter (DM) (0.75%fresh matter (FM)), volatile solids (0.74%FM), crude fat (0.09%FM), crude protein (0.22%FM), crude fiber (1.50%DM) and nitrogen-free extracts (0.93%FM) of maize silage. [Copyright &y& Elsevier]

Periplasmic membrane-bound [NiFe]-hydrogenases undergo a complex maturation pathway, including cofactor incorporation, subunit assembly, and finally twin-arginine-dependent membrane translocation (Tat). In this study, the role of the two accessory proteins HoxO and HoxQ in the maturation of the membrane-bound [NiFe]-hydrogenase (MBH) of Ralstonia eutropha H16 was investigated. MBH activity was absent in soluble as well as membrane fractions of cells with deletions in the respective genes. The absence of HoxO and HoxQ led to degradation of the small subunit precursor (preHoxK) of the MBH. The two accessory proteins directly interacted with preHoxK prior to assembly of active MBH dimer in the cytoplasm. MBH mutants with modified Tat signal peptides were disrupted in preHoxK/HoxO/HoxQ complex formation. Isolated HoxO and HoxQ proteins formed a complex in vitro with the chemically synthesized HoxK Tat signal peptide. Two functions of the two chaperones are discussed: (i) protection of the Fe–S cluster containing HoxK subunit under oxygenic conditions, and (ii) avoidance of HoxK export prior to dimerization with the large MBH subunit HoxG. [ABSTRACT FROM AUTHOR]

The article presents a study on whether region 2.1 of the Escherichia coli heat-shock sigma factor RpoH is necessary for degradation by the FtsH protease. The cellular level of E. coli heat-shock sigma factor RpoH is negatively controlled by chaperone-mediated proteolysis through the essential metalloprotease FtsH. Hybrid proteins were constructed between E. coli RpoH and Bradyrhizobium japonicum to assess the importance of this turnover element. Results of the study show that region 2.1 is involved in direct interaction with FtsH.

Background: The current recommendations for establishing intra-laboratory reference limits (RLs) cannot be fulfilled by most laboratories because of the expense involved. In the current study, a bimodal method was developed to derive RLs from data stored in a laboratory information system without any assumption concerning the distribution of the diseased subgroup. Methods: A smoothed kernel density function (Dmix) was estimated for the distribution of combined data for non-diseased and diseased adult subjects. It was assumed that the “central” part of the distribution represents the non-diseased population, which was defined and used to estimate a Gaussian distribution of either the original values or Box-Cox transformed data. This normal distribution was now considered the distribution of the non-diseased subgroup (Dnd). Percentiles were calculated to obtain retrospective RLs. The density function of the diseased subgroup (Dd) was calculated by subtracting the non-diseased density function from Dmix (Dd=Dmix–Dnd). The intersection point of the Dnd and Dd curves identified the RL with the highest diagnostic efficiency. Results: The model was applied to catalytic activity concentrations of several enzymes with data from different laboratories. The RLs obtained were similar to recently published consensus values. Differences between laboratories were small but significant. Gender stratification was necessary for alanine aminotransferase (ALT), aspartate aminotransferase (AST), and γ-glutymaltransferse (γ-GT), not significant for lipase and amylase and inconsistent among the laboratories for alkaline phosphatase (AP) and lactate dehydrogenase (LDH). Age stratification was only tested for two groups (18–49 and ≥50 years) and was significant for AST (females only), γ-GT and lipase, not significant for amylase and inconsistent for AP, LDH and ALT. For γ-GT, further stratification for age in decades was necessary for males. Creatine kinase MB (CK-MB) values were not stratified owing to the low number of data available. Conclusions: Retrospective RLs derived from intra-laboratory data pools for the catalytic activity concentration of enzymes using a modified procedure plausibly agreed with published consensus values. However, most RLs varied significantly among laboratories, thus supporting the “old” plea for intra-laboratory RLs. Clin Chem Lab Med 2007;45:1043–57. [ABSTRACT FROM AUTHOR]

Problem The presence of antisperm antibodies (ASA) in semen may impair sperm function leading to immunological infertility. The aim of the study was to identify the evolutionary conserved antigens on mouse sperm surface that react with human ASA in order to study the mechanism of autoimmune infertility. Methods of study The binding of human ASA to mouse sperm was investigated by means of indirect immunofluorescence. 2D-electrophoresis was applied to separate the biotin-labelled mouse membrane proteins using isoelectric focusing followed by polyacrylamide gel electrophoresis. Cognate antigens of ASA from seminal plasma of infertile patients were analysed by Western blotting. Performing avidin-blots it was detected which of the proteins recognized were sperm surface proteins. The spots of interest were analysed by means of mass spectrometry. Results ASA bound most frequently (36%) to the post-acrosomal region and to the midpiece of mouse spermatozoa. About 30% of ASA recognized apo lactate dehydrogenase (LDHC4) as a cognate antigen, 30% voltage-dependent anion channel (VDAC2). ASA of 20% bound to outer dense fibre protein and 20% of samples recognized glutathione S-transferase mu5. Conclusions Human ASA bound to specific cognate antigens of mouse spermatozoa, offering the possibility to study their functional relevance in the mouse model. [ABSTRACT FROM AUTHOR]

Coat components localize to specific membrane domains, where they sort selected transmembrane proteins. To study how clathrin coats are stabilized on such domains and to identify the protein networks involved, we combined proteomic screens and in vitro liposome-based assays that recapitulate the fidelity of protein sorting in vivo. Our study identifying ≈40 proteins on AP-1A-coated liposomes revealed that AP-1A coat assembly triggers the concomitant recruitment of Rac1, its effectors, and the Wave/Scar complex as well as that of Rab11 and Rab14. The coordinated recruitment of these different machineries requires a mosaic of membrane components comprising the GTPase ADP-ribosylation factor 1, sorting signals in selected transmembrane proteins, and phosphatidylinositol 4-phosphate. These results demonstrate that the combinatorial use of low-affinity binding sites present on the same membrane domain accounts not only for a selective coat assembly but also for the coordinated assembly of selected machineries required for actin polymerization and subsequent membrane fusion. [ABSTRACT FROM AUTHOR]

Quorum sensing (QS) in Sinorhizobium meliloti, the N-fixing bacterial symbiont of Medicago host plants, involves at least half a dozen different N-acyl homoserine lactone (AHL) signals and perhaps an equal number of AHL receptors. The accumulation of 55 proteins was found to be dependent on SinI, the AHL synthase, and/or on ExpR, one of the AHL receptors. Gas chromatography-mass spectrometry and electrospray ionization tandem mass spectrometry identified 3-oxo-C14-homoserine lactone (3-oxo-C14-HSL), C16-HSL, 3-oxo-C16-HSL, C16:1-HSL, and 3-oxo-C16:1-HSL as the sinI-dependent AHL QS signals accumulated by the 8530 expR+ strain under the conditions used for proteome analysis. The 8530 expR+ strain secretes additional, unidentified QS-active compounds. Addition of 200 nM C14-HSL or C16.1-HSL, two of the known SinI AHLs, affected the levels of 75% of the proteins, confirming that their accumulation is QS regulated. A number of the QS-regulated proteins have functions plausibly related to symbiotic interactions with the host, including ExpE6, IdhA, MocB, Gor, PckA, LeuC, and AglE. Seven of 10 single-crossover β-glucuronidase (GUS) transcriptional reporters in genes corresponding to QS-regulated proteins showed significantly different activities in the sinI and expR mutant backgrounds and in response to added SinI AHLs. The sinI mutant and several of the single-crossover strains were significantly delayed in the ability to initiate nodules on the primary root of the host plant, Medicago truncatula, indicating that sinI-dependent QS regulation and QS-regulated proteins contribute importantly to the rate or efficiency of nodule initiation. The sinI and expR mutants were also defective in surface swarming motility. The sinI mutant was restored to normal swarming by 5 nM C16:1-HSL. [ABSTRACT FROM AUTHOR]

Background: Splicing variants of human cathepsinB primary transcripts (CB(-2,3)) result in an expression product product which lacks the signal peptide and parts of the propeptide. This naturally truncated Δ51CB is thus unable to follow the regular CB processing and sorting pathway. It is addressed to the mitochondria through an activated N-terminal mitochondrial targeting signal instead. Although Δ51CB is supposed to be devoid of the typical CB enzymatic activity, it might play a role in malignancies and trigger cell death/apoptosis independent from the function of the regular enzyme. Cytoplasmic presence of the mature CB might occur as a result of lysosomal damage. Results: We investigated such "aberrant" proteins by artificial CB-GFP chimeras covering various sequence parts in respect to their enzymatic activity, their localization in different cell types, and the effects on the cell viability. Unlike the entire full length CB form, the artificial single chain form was not processed and did not reveal typical enzymatic CB activity during transient overexpression in large cell lung carcinoma cells. Δ51CB was found predominantly in mitochondria. In contrast, the shorter artificial CB constructs localized in the cytoplasm, inside the cell nucleus, and in the midbodies of dividing cells. Bleaching experiments revealed both mobile and immobile fractions of these constructs in the nucleus. Nuclear accumulation of artificially truncated CB variants led to disintegration of nuclei, followed by cell death. Conclusion: We propose that cell death associated with CB is not necessarily triggered by its regular enzymatic activity but alternatively by a yet unknown activity profile of truncated CB. Cytoplasmic CB might be able to enter the cell nucleus. According to a mutational analysis, the part of CB that mediates its nuclear import is a signal patch within its heavy chain domain. The results suggest that besides the N-terminal signal peptide also other CB domains contain patterns which are responsible for a differentiated targeting of the molecule, e.g. to the mitochondria, to the nucleus, or to vesicles. We propose a hierarchy of targeting signals depending on their strength and availability. This implies other possible transport mechanisms besides the usual trafficking via the mannose-6- pathway. [ABSTRACT FROM AUTHOR]

The vesicular amine transporters (VATs) are expressed as integral proteins of the lipid bilayer membrane of secretory vesicles in neuronal and endocrine cells. Their function is to allow the transport of acetylcholine (by the vesicular acetylcholine transporter VAChT; SLC18A3) and biogenic amines (by the vesicular monoamine transporters VMAT1 and VMAT2; SLC18A1 and SLC18A2) into secretory vesicles, which then discharge them into the extracellular space by exocytosis. Transport of positively charged amines by members of the SLC18 family in all cases utilizes an electrochemical gradient across the vesicular membrane established by proton pumping into the vesicle via a vacuolar ATPase; the amine is accumulated in the vesicle at the expense of the proton gradient, at a ratio of one translocated amine per two translocated protons. The members of the SLC18 family have become important histochemical markers for chemical coding in neuroendocrine tissues and cells. The structural basis of their remarkable ability to transport positively charged amines against a very large concentration gradient, as well as potential disease association with impaired transporter function and expression, are under intense investigation. [ABSTRACT FROM AUTHOR]

We investigated the association of polymorphisms of drug-metabolizing enzymes and susceptibility to oral cavity cancer. Polymerase chain reaction (PCR)-based analyses were performed on genomic DNA of 94 Caucasian patients in Germany and 92 healthy German controls to determine genotypes of polymorphisms in CYP1A1, GSTM1 and NAT2. For CYP1A1, the homozygous mutant genotype Val/Val did not occur. The heterozygous genotype Ile/Val (6.5% cases versus 4.3% controls) and the homozygous wild-type Ile/Ile (95.7% cases versus 93.5% controls) showed no statistically significant differences between groups (X2=0.47; P=0.534, Fisher''s exact test, two-sided). The GSTM1 homozygous null genotype occurred more frequently in cancer patients (59.6%) compared to controls (53.3%) but this difference remained insignificant in X2-analysis (X2=1.07; P=0.587). Almost identical genotype distributions between cases and controls were found for all three NAT2 acetylators. Hence, these three genetic polymorphisms are unlikely to be associated with oral cavity cancer in the population studied. [ABSTRACT FROM AUTHOR]

Examines the supramolecular structure of the green plant photosystem I (PSI) pigment-protein complex solubilized by the mild detergent n-dodecyl-alpha-D-maltoside by electron microscopy. Immunoblot analyses; Comparison of monomeric and dimeric PSI-200 complexes with cyanobacterial PSI; Classification of trimeric aggregates of PSI-200.

Background: Shunt obstruction is a common cause of shunt failure in the treatment of hydrocephalus. Valve occlusion is traditionally believed to originate from elevated CSF protein or cellular components, although detailed evidence is scarce and contradictory. Therefore, this study aimed to examine CSF protein and cell count as risk factors for valve obstruction. Methods: We retrospectively examined 274 patients who underwent shunt placement for hydrocephalus between 2009 and 2018 and had at least 1 year follow-up. Age, aetiology of hydrocephalus, valve type, occurrence of revision, reason for revision and CSF protein and cell count at the time of shunt insertion and revision surgery were analysed. Results: Thirty-two of 274 patients (11.7%) required revision surgery due to valve occlusion. Mean time to revision was 143 days. CSF white blood cell (WBC) count but not protein was associated with valve occlusion overall. Of all obstructed valve patients, 25% showed CSF protein level within the normal range, whereas 13.6% of the patients overall showed greatly elevated CSF protein level without evidence of valve obstruction. Persistently elevated CSF protein level at the time of shunt revision was significantly associated with valve obstruction within 90 days of initial insertion (early occlusion). Children with congenital malformations and post-haemorrhagic patients were significantly overrepresented in the occlusion group, particularly in the early occlusion group. Conclusion: Pathological CSF values such as WBC count and persistently elevated protein level serves as a risk factor for early valve obstruction. Late obstruction occurs independent of normal CSF values. Infants are particularly prone to early and late valve obstructions. CSF protein level at shunt insertion is not predictive of valve occlusion. [ABSTRACT FROM AUTHOR]

We describe a new technique in protein synthesis that extends the existing repertoire of methods for protein modification: A chemoselective reaction that induces reactivity for a subsequent bioconjugation. An azide‐modified building block reacts first with an ethynylphosphonite through a Staudinger‐phosphonite reaction (SPhR) to give an ethynylphosphonamidate. The resulting electron‐deficient triple bond subsequently undergoes a cysteine‐selective reaction with proteins or antibodies. We demonstrate that ethynylphosphonamidates display excellent cysteine‐selective reactivity combined with superior stability of the thiol adducts, when compared to classical maleimide linkages. This turns our technique into a versatile and powerful tool for the facile construction of stable functional protein conjugates. [ABSTRACT FROM AUTHOR]

We describe a new technique in protein synthesis that extends the existing repertoire of methods for protein modification: A chemoselective reaction that induces reactivity for a subsequent bioconjugation. An azide‐modified building block reacts first with an ethynylphosphonite through a Staudinger‐phosphonite reaction (SPhR) to give an ethynylphosphonamidate. The resulting electron‐deficient triple bond subsequently undergoes a cysteine‐selective reaction with proteins or antibodies. We demonstrate that ethynylphosphonamidates display excellent cysteine‐selective reactivity combined with superior stability of the thiol adducts, when compared to classical maleimide linkages. This turns our technique into a versatile and powerful tool for the facile construction of stable functional protein conjugates. [ABSTRACT FROM AUTHOR]

BACKGROUND: Worldwide, populations are ageing with significant impact on health patterns. Studies have shown that low protein, high carbohydrate diets are associated with more favourable outcomes. Plant-based diets have also been shown to have a positive impact on cardiometabolic health, weight loss and prevention of all causes of mortality. OBJECTIVE: The aim of the current study is to assess the main and interactive effects of two macronutrient interventions in a 2×2 factorial dietary design to determine their effects on appetite and health outcomes in older individuals. METHODS: Individuals aged 65 to 75 with a BMI between 20 and 35 kg/m2 were recruited. Participants were provided with ad libitum access to one of four diets for four weeks, designed as a 2×2 factorial. Each dietary treatment provided 14% of energy as protein, which was either omnivorous or semi-vegetarian in origin. The remaining complement of macronutrient energy (80–82%) was either relatively high in fat (37–41% of energy) and low in carbohydrates (41–43%), or vice versa (28–29% fat with 53% carbohydrate). Study diets were provided via a meal delivery company. Self-completed questionnaires, biospecimen and clinical assessments were collected before and after intervention to assess cardio-metabolic, oral and gut health, body composition and physical performance. RESULTS: Trial status is currently ongoing (sample and data analyses). CONCLUSIONS: This study will help determine whether protein-source (plant vs animal) and fat to carbohydrate ratio have an impact on predictors of healthy ageing. These findings will also create a sound foundation on which to base nutritional interventions in older individuals to support healthy ageing. [ABSTRACT FROM AUTHOR]

In the search for novel therapeutic targets, RNA interference screening has become a valuable tool. High-throughput technologies are now broadly accessible but their assay development from baseline remains resource-intensive and challenging. Focusing on this assay development process, we here describe a target discovery screen using pooled shRNA libraries and next-generation sequencing (NGS) deconvolution in a cell line model of Ewing sarcoma. In a strategy designed for comparative and synthetic lethal studies, we screened for targets specific to the A673 Ewing sarcoma cell line. Methods, results and pitfalls are described for the entire multi-step screening procedure, from lentiviral shRNA delivery to bioinformatics analysis, illustrating a complete model workflow. We demonstrate that successful studies are feasible from the first assay performance and independent of specialized screening units. Furthermore, we show that a resource-saving screen depth of 100-fold average shRNA representation can suffice to generate reproducible target hits despite heterogeneity in the derived datasets. Because statistical analysis methods are debatable for such datasets, we created ProFED, an analysis package designed to facilitate descriptive data analysis and hit calling using an aim-oriented profile filtering approach. In its versatile design, this open-source online tool provides fast and easy analysis of shRNA and other count-based datasets to complement other analytical algorithms. [ABSTRACT FROM AUTHOR]

In north-eastern France, red deer (Cervus elaphus L.) populations were rebuilt from a few hundred individuals, which have subsisted in remote valleys of the Vosges mountains, and to a lesser extent from individuals escaped from private enclosures; at present times, this species occupies large areas, mainly in the Vosges Mountains. In this study, we examined the population dynamics of red deer in the Vosges Mountains using ancient and contemporary mitochondrial DNA (mtDNA) from 140 samples (23 ancient + 117 modern) spanning the last 7’000 years. In addition, we reconstructed the feeding habits and the habitat of red deer since the beginning of agriculture applying isotopic analyses in order to establish a basis for current environmental management strategies. We show that past and present red deer in the Vosges Mountains belong to mtDNA haplogroup A, suggesting that they originated from the Iberian refugium after the last glacial maximum (LGM). Palaeogenetic analysis of ancient bone material revealed the presence of two distinct haplotypes with different temporal distributions. Individuals belonging to the two haplotype groups apparently occupied two different habitats over at least 7’000 years. AM6 correlates with an ecological type that feeds in densely forested mountain landscapes, while AM235 correlates with feeding in lowland landscapes, composed of a mixture of meadows and riverine, herb-rich woodlands. Our results suggest that red deer of north-eastern France was able to adapt, over the long term, to these different habitat types, possibly due to efficient ethological barriers. Modern haplotype patterns support the historical record that red deer has been exposed to strong anthropogenic influences as a major game species. [ABSTRACT FROM AUTHOR]

Introduction: Bipolar disorder (BD) is a chronic psychiatric disease which can take most different and unpredictable courses. It is accompanied by unspecific brainstructural changes and cognitive decline. The neurobiological underpinnings of these processes are still unclear. Emerging evidence suggests that tryptophan catabolites (TRYCATs), which involve all metabolites of tryptophan towards the kynurenine (KYN) branch, are involved in the etiology as well as in the course of BD. They are proposed to be mediators of immune-inflammation and neurodegeneration. In this study we measured the levels of KYN and its main catabolites consisting of the neurotoxic hydroxykynurenine (3-HK), the more neuroprotective kynurenic acid (KYNA) and anthranilic acid (AA) and evaluated the ratios between end-products and substrates as proxies for the specific enzymatic activity (3-HK/KYN, KYNA/KYN, AA/KYN) as well as 3-HK/KYNA as a proxy for neurotoxic vs. neuroprotective end-product relation in individuals with BD compared to healthy controls (HC). Methods: We took peripheral TRYCAT blood levels of 143 euthymic to mild depressive BD patients and 101 HC. For statistical analyses MANCOVA’s controlled for age, sex, body mass index, cardiovascular disease and smoking were performed. Results: The levels of KYNA (F = 5,579; p <.05) were reduced in BD compared to HC. The enzymatic activity of the kynurenine-3-monooxygenase (KMO) reflected by the 3-HK/KYN ratio was increased in BD individuals compared to HC (F = 5,394; p <.05). Additionally the ratio of 3-HK/KYNA was increased in individuals with BD compared to healthy controls (F = 11,357; p <.01). Discussion: In conclusion our findings subserve the concept of KYN -pathway alterations in the pathophysiology of BD. We present evidence of increased breakdown towards the neurotoxic branch in KYN metabolism even in a euthymic to mild depressive state in BD. From literature we know that depression and mania are accompanied by inflammatory states which should be capable to produce an even greater imbalance due to activation of key enzymes in the neurotoxic direction of KYN -conversion. These processes could finally be involved in the development of unspecific brain structural changes and cognitive deficits which are prevalent in BD. Further research should focus on state dependent changes in TRYCATs and its relation to cognition, brain structure and staging parameters. [ABSTRACT FROM AUTHOR]

Background: Chikungunya virus (CHIKV) is a mosquito-transmitted alphavirus that causes high fever, rash, and recurrent arthritis in humans. It has efficiently adapted to Aedes albopictus, which also inhabits temperate regions, including Europe and the United States of America. In the past, CHIKV has mainly affected developing countries, but has recently caused large outbreaks in the Caribbean and Latin America. No treatment or licensed CHIKV vaccine exists. Methodology/Principal Findings: Here, we have identified determinants in the CHIKV cell-attachment protein E2 that facilitate cell binding. The extracellular part of the E2 gene is subdivided into the three domains, A, B, and C. These domains were expressed in E. coli and as Fc-fusion proteins generated from HEK293T cells and used for cell-binding assays. Domains A and B bound to all cells tested, independently of their permissiveness to CHIKV infection. Domain C did not bind to cells at all. Furthermore, CHIKV cell entry was promoted by cell-surface glycosaminoglycans (GAGs) and domain B interacted exclusively with GAG-expressing cells. Domain A also bound, although only moderately, to GAG-deficient cells. Soluble GAGs were able to inhibit CHIKV infection up to 90%; however, they enhanced the transduction rate of CHIKV Env pseudotyped vectors in GAG-negative cells. Conclusion/Significance: These data imply that CHIKV uses at least two mechanisms to enter cells, one GAG-dependent, via initial attachment through domain B, and the other GAG-independent, via attachment of domain A. These data give indications that CHIKV uses multiple mechanisms to enter cells and shows the potential of GAGs as lead structures for developing antiviral drugs. [ABSTRACT FROM AUTHOR]

Background: Cardiac inwardly rectifying Kir current (IK1) mediates terminal repolarisation and is critical for the stabilization of the diastolic membrane potential. Its predominant molecular basis in mammalian ventricle is heterotetrameric assembly of Kir2.1 and Kir2.2 channel subunits. It has been shown that PKC inhibition of IK1 promotes focal ventricular ectopy. However, the underlying molecular mechanism has not been fully elucidated to date. Methods and Results: In the Xenopus oocyte expression system, we observed a pronounced PKC-induced inhibition of Kir2.2 but not Kir2.1 currents. The PKC regulation of Kir2.2 could be reproduced by an activator of conventional PKC isoforms and antagonized by pharmacological inhibition of PKCβ. In isolated ventricular cardiomyocytes (rat, mouse), pharmacological activation of conventional PKC isoforms induced a pronounced inhibition of IK1. The PKC effect in rat ventricular cardiomyocytes was markedly attenuated following co-application of a small molecule inhibitor of PKCβ. Underlining the critical role of PKCβ, the PKC-induced inhibition of IK1 was absent in homozygous PKCβ knockout-mice. After heterologous expression of Kir2.1-Kir2.2 concatemers in Xenopus oocytes, heteromeric Kir2.1/Kir2.2 currents were also inhibited following activation of PKC. Conclusion: We conclude that inhibition of cardiac IK1 by PKC critically depends on the PKCβ isoform and Kir2.2 subunits. This regulation represents a potential novel target for the antiarrhythmic therapy of focal ventricular arrhythmias. [ABSTRACT FROM AUTHOR]

Background: Podocalyxin-like 1 (PODXL) is an anti-adhesive transmembrane protein that has been demonstrated to be an independent factor of poor prognosis in colorectal cancer (CRC). The gene encoding PODXL is located to chromosome 7, which also harbours the gene for the epidermal growth factor receptor (EGFR). The aim of this study was to examine the associations between PODXL and EGFR expression in CRC in vitro and in vivo.Methods: EGFR expression was analysed in tumours from three independent patient cohorts; cohort 1 (n = 533), cohort 2 (n = 259) and cohort 3 (n = 310), previously analysed for immunohistochemical PODXL expression and KRAS and BRAF mutations (cohort 1 and 3). Levels of EGFR and PODXL were determined by western blot in six different CRC cell lines.Results: High expression of PODXL was significantly associated with high EGFR expression (p < 0.001) in all three cohorts, and with BRAF mutation (p < 0.001) in cohort 1 and 3. High EGFR expression correlated with BRAF mutation (p < 0.001) in cohort 1. High EGFR expression was associated with adverse clinicopathological factors and independently predicted a reduced 5-year overall survival (OS) in cohort 1 (HR 1.77; 95 % CI 1.27-2.46), cohort 2 (HR 1.58; 95 % CI 1.05-2.38) and cohort 3 (HR 1.83; 95 % CI 1.19-2.81). The highest risk of death within 5 years was observed in patients with tumours displaying high expression of both EGFR and PODXL in cohort 1 and 3 (HR 1.97; 95 % CI 1.18-3.28 and HR 3.56; 95 % CI 1.75-7.22, respectively). Western blot analysis showed a uniform expression of PODXL and EGFR in all six examined CRC cell lines.Conclusions: The results from this study demonstrate that high expression of EGFR is an independent factor of poor prognosis in CRC. Moreover, strong links have been uncovered between expression of the recently proposed biomarker candidate PODXL with EGFR expression in CRC in vivo and in vitro, and with BRAF mutation in vivo. High expression of both PODXL and EGFR may also have a synergistic adverse effect on survival. These findings suggest a potential functional link in CRC between PODXL, EGFR and BRAF, all originating from chromosome 7, which may be highly relevant in the clinical setting and therefore merit future in-depth study. [ABSTRACT FROM AUTHOR]

Background: Gastric and esophageal adenocarcinomas are major global cancer burdens. These cancer forms are characterized by a poor prognosis and a modest response to chemo- radio- and targeted treatment. Hence there is an obvious need for further enhanced diagnostic and treatment strategies. The aim of this study was to examine the expression and prognostic impact of human epidermal growth factor receptor 1 (HER1/EGFR) and 3 (HER3), as well as the occurrence of EGFR and KRAS mutations in gastric and esophageal adenocarcinoma. Methods: Immunohistochemical expression of EGFR and HER3 was analysed in all primary tumours and a subset of lymph node metastases in a consecutive cohort of 174 patients with adenocarcinoma of the stomach, cardia and esophagus. The anti-HER3 antibody used was validated by siRNA-mediated knockdown, immunohistochemistry and quantitative real-time PCR. EGFR and KRAS mutation status was analysed by pyrosequencing tecchnology. Results and Discussion: High EGFR expression was an independent risk factor for shorter overall survival (OS), whereas high HER3 expression was associated with a borderline significant trend towards a longer OS. KRAS mutations were present in only 4% of the tumours and had no prognostic impact. All tumours were EGFR wild-type. These findings contribute to the ongoing efforts to decide on the potential clinical value of different HERs and druggable mutations in gastric and esophageal adenocarcinomas, and attention is drawn to the need for more standardised investigational methods. [ABSTRACT FROM AUTHOR]

The purpose of this study is to characterize the spectrum of cerebrospinal fluid (CSF) findings in patients with posterior reversible encephalopathy syndrome (PRES) and determine its associations with severity of edema. In this retrospective cross-sectional study electronic medical reports were screened for patients with PRES. Clinical and laboratory data closest to onset of clinical symptoms were collected. Neuroimaging reports from all patients were categorized according to the presence of vasogenic edema, cytotoxic edema and contrast enhancement. From Jan 1999 to Feb 2015, 87 patients with PRES and CSF findings were included. Mean total protein levels were 793 ± 929 mg/l. Median cell counts were 2/µl (IQR 1-4). Eight patients had cell counts >6/µl with a maximum of 41/µl. In 31 patients with extended CSF analysis, mean CSF/serum albumin quotient (QAlb) was 10.1 (IQR 6-17.3). An elevated QAlb (age-adjusted) was found in 74 % of these 31 patients. PRES patients with severe edema had higher mean total protein levels in CSF (mean difference = 407 mg/l; 95 % CI 187-628). Significant correlations were found for edema severity with levels of CSF protein ( r = 0.48, p < 0.001) as well with QAlb ( r = 0.44, p = 0.013). This study shows that total protein in CSF and QAlb are elevated and correlate with severity of edema in PRES patients, whereas pleocytosis was rare in our cohort. This supports the theory that PRES is caused by a dysfunctional blood brain barrier. [ABSTRACT FROM AUTHOR]

Protein modification by interferon-stimulated gene 15 (ISG15), an ubiquitin-like modifier, affects multiple cellular functions and represents one of the major antiviral effector systems. Covalent linkage of ISG15 to proteins was previously reported to be counteracted by ubiquitin-specific protease 18 (USP18). To date, analysis of the molecular properties of USP18 was hampered by low expression yields and impaired solubility. We established high-yield expression of USP18 in insect cells and purified the protease to homogeneity. USP18 binds with high affinity to ISG15, as shown by microscale thermophoresis with a Kd of 1.3 ± 0.2 μ m. The catalytic properties of USP18 were characterized by a novel assay using ISG15 fused to a fluorophore via an isopeptide bond, giving a Km of 4.6 ± 0.2 μ m and a kcat of 0.23 ± 0.004 s−1, respectively, at pH 7.5. Furthermore, the recombinant enzyme cleaves efficiently ISG15 but not ubiquitin from endogenous cellular substrates. In line with these data, USP18 exhibited neither cross-reactivity with an ubiquitin isopeptide fluorophore substrate, nor with a ubiquitin vinyl sulfone, showing that the enzyme is specific for ISG15. Structured digital abstract ● and by () ● by () [ABSTRACT FROM AUTHOR]