Location of glycine mutations within a bacterial collagen protein affects degree of disruption of triple-helix folding and conformation

The hereditary bone disorder osteogenesis imperfecta is often caused by missense mutations in type I collagen that change one Gly residue to a larger residue and that break the typical (Gly-Xaa-Yaa)(n) sequence pattern. Site-directed mutagenesis in a recombinant bacterial collagen system was used to explore the effects of the Gly mutation position and of the identity of the residue replacing Gly in a homogeneous collagen molecular population. Homotrimeric bacterial collagen proteins with a Gly-to-Arg or Gly-to-Ser replacement formed stable triple-helix molecules with a reproducible 2 °C decrease in stability. All Gly replacements led to a significant delay in triple-helix folding, but a more dramatic delay was observed when the mutation was located near the N terminus of the triple-helix domain. This highly disruptive mutation, close to the globular N-terminal trimerization domain where folding is initiated, is likely to interfere with triple-helix nucleation. A positional effect of mutations was also suggested by trypsin sensitivity for a Gly-to-Arg replacement close to the triple-helix N terminus but not for the same replacement near the center of the molecule. The significant impact of the location of a mutation on triple-helix folding and conformation could relate to the severe consequences of mutations located near the C terminus of type I and type III collagens, where trimerization occurs and triple-helix folding is initiated.     

Hemiarthroplasty of hip joint: An experimental validation using porcine acetabulum

Biphasic properties of articular cartilage allow it to be an excellent bearing material and have been studied through several simplified experiments as well as finite element modelling. However, three-dimensional biphasic finite element (FE) models of the whole joint are rare. The current study was carried out to experimentally validate FE methodology for modelling hemiarthroplasty. Material properties such as equilibrium elastic modulus and permeability of porcine acetabular cartilage were initially derived by curve-fitting an experimental deformation curve with that obtained using FE. These properties were then used in the hemiarthroplasty hip joint modelling. Each porcine acetabular cup was loaded with 400N using a 34mm diameter CoCr femoral head. A specimen-specific FE model of each acetabular cup was created using μCT and a series of software processes. Each model was analysed under conditions similar to those tested experimentally. Contact stresses and contact areas predicted by the model, immediately after loading, were then compared with the corresponding experimentally measured values. Very high peak contact stresses (maximum experimental: 14.09MPa) were recorded. A maximum difference of 12.42% was found in peak contact stresses. The corresponding error for contact area was 20.69%. Due to a fairly good agreement in predicted and measured values of contact stresses and contact areas, the integrated methodology developed in this study can be used as a basis for future work. In addition, FE predicted total fluid load support was around 80% immediately after loading. This was lower than that observed in conforming contact problems involving biphasic cartilage and was due to a smaller local contact area and variable clearance making fluid exudation easier.     

Genome Sequence of the ethene- and vinyl chloride-oxidizing actinomycete Nocardioides sp. strain JS614

Nocardioides sp. strain JS614 grows on ethene and vinyl chloride (VC) as sole carbon and energy sources and is of interest for bioremediation and biocatalysis. Sequencing of the complete genome of JS614 provides insight into the genetic basis of alkene oxidation, supports ongoing research into the physiology and biochemistry of growth on ethene and VC, and provides biomarkers to facilitate detection of VC/ethene oxidizers in the environment. This is the first genome sequence from the genus Nocardioides and the first genome of a VC/ethene-oxidizing bacterium.     

Genome sequence of Victivallis vadensis ATCC BAA-548, an anaerobic bacterium from the phylum Lentisphaerae, isolated from the human gastrointestinal tract

Victivallis vadensis ATCC BAA-548 represents the first cultured representative from the novel phylum Lentisphaerae, a deep-branching bacterial lineage. Few cultured bacteria from this phylum are known, and V. vadensis therefore represents an important organism for evolutionary studies. V. vadensis is a strictly anaerobic sugar-fermenting isolate from the human gastrointestinal tract.     

U1A Regulates 3′ Processing of the Survival Motor Neuron mRNA

Insufficient expression of the survival motor neuron (SMN) protein causes spinal muscular atrophy, a neurodegenerative disease characterized by loss of motor neurons. Despite the importance of maintaining adequate SMN levels, little is known about factors that control SMN expression, particularly 3′ end processing of the SMN pre-mRNA. In this study, we identify the U1A protein as a key regulator of SMN expression. U1A, a component of the U1 snRNP, is known to inhibit polyadenylation upon direct binding to mRNA. We show that U1A binds directly and with high affinity and specificity to the SMN 3′-UTR adjacent to the polyadenylation site, independent of the U1 snRNP (U1 small nuclear ribonucleoprotein). Binding of U1A inhibits polyadenylation of the SMN pre-mRNA by specifically inhibiting 3′ cleavage by the cleavage and polyadenylation specificity factor. Expression of U1A in excess of U1 snRNA causes inhibition of SMN polyadenylation and decreases SMN protein levels. This work reveals a new mechanism for regulating SMN levels and provides new insight into the roles of U1A in 3′ processing of mRNAs.     

Crumbs3 Is Essential for Proper Epithelial Development and Viability

First identified in Drosophila, the Crumbs (Crb) proteins are important in epithelial polarity, apical membrane formation, and tight junction (TJ) assembly. The conserved Crb intracellular region includes a FERM (band 4.1/ezrin/radixin/moesin) binding domain (FBD) whose mammalian binding partners are not well understood and a PDZ binding motif that interacts with mammalian Pals1 (protein associated with lin seven) (also known as MPP5). Pals1 binds Patj (Pals1-associated tight-junction protein), a multi-PDZ-domain protein that associates with many tight junction proteins. The Crb complex also binds the conserved Par3/Par6/atypical protein kinase C (aPKC) polarity cassette that restricts migration of basolateral proteins through phosphorylation. Here, we describe a Crb3 knockout mouse that demonstrates extensive defects in epithelial morphogenesis. The mice die shortly after birth, with cystic kidneys and proteinaceous debris throughout the lungs. The intestines display villus fusion, apical membrane blebs, and disrupted microvilli. These intestinal defects phenocopy those of Ezrin knockout mice, and we demonstrate an interaction between Crumbs3 and ezrin. Taken together, our data indicate that Crumbs3 is crucial for epithelial morphogenesis and plays a role in linking the apical membrane to the underlying ezrin-containing cytoskeleton.     

Investigating the Effects of Probiotics on Pneumococcal Colonization Using an In Vitro Adherence Assay

Adherence of Streptococcus pneumoniae (the pneumococcus) to the epithelial lining of the nasopharynx can result in colonization and is considered a prerequisite for pneumococcal infections such as pneumonia and otitis media. In vitro adherence assays can be used to study the attachment of pneumococci to epithelial cell monolayers and to investigate potential interventions, such as the use of probiotics, to inhibit pneumococcal colonization. The protocol described here is used to investigate the effects of the probiotic Streptococcus salivarius on the adherence of pneumococci to the human epithelial cell line CCL-23 (sometimes referred to as HEp-2 cells). The assay involves three main steps: 1) preparation of epithelial and bacterial cells, 2) addition of bacteria to epithelial cell monolayers, and 3) detection of adherent pneumococci by viable counts (serial dilution and plating) or quantitative real-time PCR (qPCR). This technique is relatively straightforward and does not require specialized equipment other than a tissue culture setup. The assay can be used to test other probiotic species and/or potential inhibitors of pneumococcal colonization and can be easily modified to address other scientific questions regarding pneumococcal adherence and invasion.     

Recombinant Human Interferon Alpha 2b Prevents and Reverses Experimental Pulmonary Hypertension

Pulmonary hypertension (PH) is a progressive and fatal disease with no cure. Vascular remodeling in PH involves intraluminal growth of endothelial and smooth muscle cells, leading to obliterative vascular lesions. Cell growth in these lesions is quasi-neoplastic, with evidence of monoclonality, apoptosis resistance and cancer-like metabolic derangements. Herein we tested the effect of human interferon alpha 2b (IFNα), a pleiotropic cytokine and anti-cancer therapeutic, on the development and progression of PH in the rat SU5416/hypoxia (SUH) model and mouse hypoxia model of the disease. In both models IFNα attenuated the development of PH and reversed established PH as assessed by measuring right ventricular systolic pressure and right ventricular hypertrophy. The effect of IFNα was dependent on the type I interferon receptor (IFNAR) since mice lacking a subunit of the IFNAR were not protected by IFNα. Morphometric analysis of pulmonary aterioles from hypoxic mice or SUH rats showed that IFNα inhibited pulmonary vascular remodeling in both models and that IFNα reversed remodeling in SUH rats with established disease. Immunohistochemical staining revealed that IFNα decreased the number of PCNA and Tunel positive cells in the wall of pulmonary arterioles. In vitro, IFNα inhibited proliferation of human pulmonary artery smooth muscle cells and as well as human pulmonary artery endothelial cell proliferation and apoptosis. Together these findings demonstrate that IFNα reverses established experimental PH and provide a rationale for further exploration of the use of IFNα and other immunotherpies in PH.