Overexpression of juvenile hormone binding protein in bacteria and Pichia pastoris

Galleria mellonella juvenile hormone binding protein (JHBP) is a single chain glycoprotein with two disulfide bonds and a molecular mass of 25,880 Da. This report describes the expression of JHBP in bacteria and yeast cells (Pichia pastoris). The expression in bacteria was low and the protein was rapidly degraded upon cell lysis. The expression of His8-tagged rJHBP (His8-rJHBP) in P. pastoris was high and the non-degraded protein was purified to homogeneity with high yield in a one-step immobilized Ni++ affinity chromatography. His8-rJHBP from P. pastoris contains one JH III binding site with KD of 3.7 +/- 1.3x10(-7) M. The results suggest that P. pastoris is the preferred system for expression of His8-rJHBP in non-degraded fully active form.     

In situ quantification of aberrant p53 in colorectal neoplasia

Aberrant p53 protein accumulation was measured immunohistologically in 342 colorectal paraffin-embedded tissue sections from 115 patients (24 with adenocarcinoma, 59 with adenoma and 32 'hospital controls'). Subjective scoring was compared with quantitative cell imaging, including dichotomous (p53⁺/p53^-) status, ng p53^mut mg^-1 enterocyte protein, and tumour burden and patient body 'burden' of aberrant p53. A total of 62.5% cancer patients, 23.7% adenoma patients and 3.1% hospital controls were accorded p53⁺ status on the basis of p53 quantification. Quantitative p53⁺/p53^- assignment had a stronger inverse association with survival (χ²=6.17, p=0.013, Kaplan-Meier test) than subjective 'visual estimation' (χ²=0.57, p=0.449). There was a strong inverse relationship between the p53 'body burden' and the months of post-diagnosis survival (hazard ratio=1.42, p=0.0004, Cox proportional hazards). Absolute quantification for inactivated p53 permits objective and reproducible scoring, adjusts for intra-laboratory immunostaining 'batch effects', corrects for fixation artefacts, and standardizes for inter-laboratory differences in fixation, antibody selection and staining method. Clinically, in situ quantification of p53 will permit more accurate survival prognoses and will inform therapy selection and dose. Ultimately, accurate quantitative tissue/blood p53 correlations may provide a minimally invasive and systemic surrogate measure for these same clinical purposes.     

Racial differences in genome-wide methylation profiling and gene expression in breast tissues from healthy women

Breast cancer is more common in European Americans (EAs) than in African Americans (AAs) but mortality from breast cancer is higher among AAs. While there are racial differences in DNA methylation and gene expression in breast tumors, little is known whether such racial differences exist in breast tissues of healthy women. Genome-wide DNA methylation and gene expression profiling was performed in histologically normal breast tissues of healthy women. Linear regression models were used to identify differentially-methylated CpG sites (CpGs) between EAs (n = 61) and AAs (n = 22). Correlations for methylation and expression were assessed. Biological functions of the differentially-methylated genes were assigned using the Ingenuity Pathway Analysis. Among 485 differentially-methylated CpGs by race, 203 were hypermethylated in EAs, and 282 were hypermethylated in AAs. Promoter-related differentially-methylated CpGs were more frequently hypermethylated in EAs (52%) than AAs (27%) while gene body and intergenic CpGs were more frequently hypermethylated in AAs. The differentially-methylated CpGs were enriched for cancer-associated genes with roles in cell death and survival, cellular development, and cell-to-cell signaling. In a separate analysis for correlation in EAs and AAs, different patterns of correlation were found between EAs and AAs. The correlated genes showed different biological networks between EAs and AAs; networks were connected by Ubiquitin C. To our knowledge, this is the first comprehensive genome-wide study to identify differences in methylation and gene expression between EAs and AAs in breast tissues from healthy women. These findings may provide further insights regarding the contribution of epigenetic differences to racial disparities in breast cancer.     

Mice deficient in small leucine-rich proteoglycans: novel in vivo models for osteoporosis,osteoarthritis, Ehlers-Danlos syndrome,muscular dystrophy,and corneal diseases

Small leucine-rich proteoglycans (SLRPs) are extracellular molecules that bind to TGFbetas and collagens and other matrix molecules. In vitro, SLRPs were shown to regulate collagen fibrillogenesis, a process essential in development, tissue repair, and metastasis. To better understand their functions in vivo, mice deficient in one or two of the four most prominent and widely expressed SLRPs (biglycan, decorin, fibromodulin, and lumican) were recently generated. All four SLRP deficiencies result in the formation of abnormal collagen fibrils. Taken together, the collagen phenotypes demonstrate a cooperative, sequential, timely orchestrated action of the SLRPs that altogether shape the architecture and mechanical properties of the collagen matrix. In addition, SLRP-deficient mice develop a wide array of diseases (osteoporosis, osteoarthritis, muscular dystrophy, Ehlers-Danlos syndrome, and corneal diseases), most of them resulting primarily from an abnormal collagen fibrillogenesis. The development of these diseases by SLRP-deficient mice suggests that mutations in SLRPs may be part of undiagnosed predisposing genetic factors for these diseases. Although the distinct phenotypes developed by the different singly deficient mice point to distinct in vivo function for each SLRP, the analysis of the double-deficient mice also demonstrates the existence of rescuing/compensation mechanisms, indicating some functional overlap within the SLRP family.     

The Diversity of Yellow-Related Proteins in Sand Flies (Diptera: Psychodidae)

Yellow-related proteins (YRPs) present in sand fly saliva act as affinity binders of bioamines, and help the fly to complete a bloodmeal by scavenging the physiological signals of damaged cells. They are also the main antigens in sand fly saliva and their recombinant form is used as a marker of host exposure to sand flies. Moreover, several salivary proteins and plasmids coding these proteins induce strong immune response in hosts bitten by sand flies and are being used to design protecting vaccines against Leishmania parasites. In this study, thirty two 3D models of different yellow-related proteins from thirteen sand fly species of two genera were constructed based on the known protein structure from Lutzomyia longipalpis. We also studied evolutionary relationships among species based on protein sequences as well as sequence and structural variability of their ligand-binding site. All of these 33 sand fly YRPs shared a similar structure, including a unique tunnel that connects the ligand-binding site with the solvent by two independent paths. However, intraspecific modifications found among these proteins affects the charges of the entrances to the tunnel, the length of the tunnel and its hydrophobicity. We suggest that these structural and sequential differences influence the ligand-binding abilities of these proteins and provide sand flies with a greater number of YRP paralogs with more nuanced answers to bioamines. All these characteristics allow us to better evaluate these proteins with respect to their potential use as part of anti-Leishmania vaccines or as an antigen to measure host exposure to sand flies.     

Differences in rhizome aeration of Phragmites australis in a constructed wetland

Waterlogged soils are a challenging environment for plants due to anoxic conditions and enrichment of phytotoxic substances in the rhizosphere. High contents of organic matter in the substrate as present in constructed wetlands (CW) may amplify these effects. The influence of substrate organic matter on the rhizome aeration of Phragmites australis (common reed) was investigated in a CW for sewage treatment on two sites with different organic concentrations: (a) inflow part of the bed with a high organic charge (high-organic site) and (b) outflow zone of the bed with a lower organic charge (low-organic site). Several diurnal oxygen (O₂) courses were recorded inside the rhizomes using micro-optodes. Maximum O₂ percentages in the rhizomes were generally the same at both sites, but minimum O₂ values were lower in the low-organic site than in the high-organic site, leading to higher amplitudes at the low-organic site. The results suggest that oxygen release from the roots is hampered under high-organic compared to low-organic conditions. This observation might be explained by changes in gas conductivity of the roots, but also by alterations in microbial oxygen demand, under different organic burden.     

Phylogenetic and morphological diversity of novel soil cercomonad species with a description of two new genera (Nucleocercomonas and Metabolomonas)

Cercomonads are important components of microbial food webs in soils and aquatic sediments. Here, we investigated the general morphology, behaviour, life cycle and 18S rDNA phylogeny of cercomonad cultures from a German grassland soil habitat. We describe ten new species including two new genera from 23 strains. Three Cercomonas, two Eocercomonas and three Paracercomonas species are described. Based on large phylogenetic distance and distinct morphology, we erect two novel clade B genera near the root of the cercomonad tree. Nucleocercomonas nov. gen. bears a number of characters unusual for cercomonads: Its anterior flagellum is extremely long, it mostly does not glide, and in its most frequent life stage the cell body does not attach to the substratum, but produces unattached pseudopodia. Furthermore, it has a unique nucleus with a peripheral nucleolus that attaches to the nuclear envelope opposite the basal body connection. Metabolomonas nov. gen. is extremely metabolic. It is characterized by a very high beating frequency of the anterior flagellum, fast gliding, rapid changes in shape and strong cytoplasmic streams. A new genus Brevimastigomonas is erected for the previously described species Paracercomonas anaerobica. The general morphology of cercomonad species often does not correspond with their phylogenetic position: closely related species may have a very different morphology.     

MGluRs regulate the expression of neuronal calcium sensor proteins NCS-1 and VILIP-1 and the immediate early gene arg3.1/arc in the hippocampus in vivo

The metabotropic glutamate receptor (mGluR) agonist (R,S)-3,5-dihydroxyphenylglycine (DHPG) is involved in several forms of hippocampal synaptic plasticity. DHPG application can induce slow-onset potentiation, a form of long-term potentiation (LTP), in the dentate gyrus and in the CA1 region in vivo. The induction of LTP correlates with increased expression levels of neuronal calcium sensor (NCS), considered as key elements for plasticity. In this study we investigated mGluR- and time-dependent changes in the expression of two different NCS proteins. Following DHPG application in vivo NCS-1 and VILIP-1 expression increased, with significant levels reached after 8 and 24h. The effect was attenuated by treatment with the group I mGluR specific antagonist S-4-carboxyphenylglycine. The immediate early gene (IEG) arg3.1/arc showed highest expression levels 2h after DHPG-treatment. Therefore, mGluRs at concentrations which induce synaptic plasticity regulate the expression of IEGs and NCS proteins in different time frames and thus contribute to late phases of synaptic plasticity.     

Serotypes and Clonal Diversity of Streptococcus pneumoniae Causing Invasive Disease in the Era of PCV13 in Catalonia,Spain

The aim of this study was to study the serotypes and clonal diversity of pneumococci causing invasive pneumococcal disease in Catalonia, Spain, in the era of 13-valent pneumococcal conjugate vaccine (PCV13). In our region, this vaccine is only available in the private market and it is estimated a PCV13 vaccine coverage around 55% in children. A total of 1551 pneumococcal invasive isolates received between 2010 and 2013 in the Molecular Microbiology Department at Hospital Sant Joan de Déu, Barcelona, were included. Fifty-two serotypes and 249 clonal types—defined by MLST—were identified. The most common serotypes were serotype 1 (n = 182; 11.7%), 3 (n = 145; 9.3%), 19A (n = 137; 8.8%) and 7F (n = 122; 7.9%). Serotype 14 was the third most frequent serotype in children < 2 years (15 of 159 isolates). PCV7 serotypes maintained their proportion along the period of study, 16.6% in 2010 to 13.4% in 2013, whereas there was a significant proportional decrease in PCV13 serotypes, 65.3% in 2010 to 48.9% in 2013 (p<0.01). This decrease was mainly attributable to serotypes 19A and 7F. Serotype 12F achieved the third position in 2013 (n = 22, 6.4%). The most frequent clonal types found were ST306 (n = 154, 9.9%), ST191 (n = 111, 7.2%), ST989 (n = 85, 5.5%) and ST180 (n = 80, 5.2%). Despite their decrease, PCV13 serotypes continue to be a major cause of disease in Spain. These results emphasize the need for complete PCV13 vaccination.