Recognition of the neural chemoattractant Netrin-1 by integrins alpha6beta4 and alpha3beta1 regulates epithelial cell adhesion and migration

Netrins, axon guidance cues in the CNS, have also been detected in epithelial tissues. In this study, using the embryonic pancreas as a model system, we show that Netrin-1 is expressed in a discrete population of epithelial cells, localizes to basal membranes, and specifically associates with elements of the extracellular matrix. We demonstrate that alpha6beta4 integrin mediates pancreatic epithelial cell adhesion to Netrin-1, whereas recruitment of alpha6beta4 and alpha3beta1 regulate the migration of CK19+/PDX1+ putative pancreatic progenitors on Netrin-1. These results provide evidence for the activation of epithelial cell adhesion and migration by a neural chemoattractant, and identify Netrin-1/integrin interactions as adhesive/guidance cues for epithelial cells.     

Induced refolding of a temperature denatured llama heavy-chain antibody fragment by its antigen

In a previous study we have shown that llama VHH antibody fragments are able to bind their antigen after a heat shock of 90 degrees C, in contrast to the murine monoclonal antibodies. However, the molecular mechanism by which antibody:antigen interaction occurs under these extreme conditions remains unclear. To examine in more detail the structural and thermodynamic aspects of the binding mechanism, an extensive CD, ITC, and NMR study was initiated. In this study the interaction between the llama VHH -R2 fragment and its antigen, the dye Reactive Red-6 (RR6) has been explored. The data show clearly that most of the VHH-R2 population at 80 degrees C is in an unfolded conformation. In contrast, CD spectra representing the complex between VHH-R2 and the dye remained the same up to 80 degrees C. Interestingly, addition of the dye to the denatured VHH-R2 at 80 degrees C yielded the spectrum of the native complex. These results suggest an induced refolding of denatured VHH-R2 by its antigen under these extreme conditions. This induced refolding showed some similarities with the well established "induced fit" mechanism of antibody-antigen interactions at ambient temperature. However, the main difference with the "induced fit" mechanism is that at the start of the addition of the antigen most of the VHH molecules are in an unfolded conformation. The refolding capability under these extreme conditions and the stable complex formation make VHHs useful in a wide variety of applications.     

Intronic Variants in the NFKB1 Gene May Influence Hearing Forecast in Patients with Unilateral Sensorineural Hearing Loss in Meniere's Disease

Meniere''s disease is an episodic vestibular syndrome associated with sensorineural hearing loss (SNHL) and tinnitus. Patients with MD have an elevated prevalence of several autoimmune diseases (rheumatoid arthritis, systemic lupus erythematosus, ankylosing spondylitis and psoriasis), which suggests a shared autoimmune background. Functional variants of several genes involved in the NF-κB pathway, such as REL, TNFAIP3, NFKB1 and TNIP1, have been associated with two or more immune-mediated diseases and allelic variations in the TLR10 gene may influence bilateral affectation and clinical course in MD. We have genotyped 716 cases of MD and 1628 controls by using the ImmunoChip, a high-density genotyping array containing 186 autoimmune loci, to explore the association of immune system related-loci with sporadic MD. Although no single nucleotide polymorphism (SNP) reached a genome-wide significant association (p<10⁻⁸), we selected allelic variants in the NF-kB pathway for further analyses to evaluate the impact of these SNPs in the clinical outcome of MD in our cohort. None of the selected SNPs increased susceptibility for MD in patients with uni or bilateral SNHL. However, two potential regulatory variants in the NFKB1 gene (rs3774937 and rs4648011) were associated with a faster hearing loss progression in patients with unilateral SNHL. So, individuals with unilateral MD carrying the C allele in rs3774937 or G allele in rs4648011 had a shorter mean time to reach hearing stage 3 (>40 dB HL) (log-rank test, corrected p values were p = 0.009 for rs3774937 and p = 0.003 for rs4648011, respectively). No variants influenced hearing in bilateral MD. Our data support that the allelic variants rs3774937 and rs4648011 can modify hearing outcome in patients with MD and unilateral SNHL.     

Oocyte apoptosis is suppressed by disruption of the acid sphingomyelinase gene or by sphingosine-1-phosphate therapy

The time at which ovarian failure (menopause) occurs in females is determined by the size of the oocyte reserve provided at birth, as well as by the rate at which this endowment is depleted throughout post-natal life. Here we show that disruption of the gene for acid sphingomyelinase in female mice suppressed the normal apoptotic deletion of fetal oocytes, leading to neonatal ovarian hyperplasia. Ex vivo, oocytes lacking the gene for acid sphingomyelinase or wild-type oocytes treated with sphingosine-1-phosphate resisted developmental apoptosis and apoptosis induced by anti-cancer therapy, confirming cell autonomy of the death defect. Moreover, radiation-induced oocyte loss in adult wild-type female mice, the event that drives premature ovarian failure and infertility in female cancer patients, was completely prevented by in vivo therapy with sphingosine-1-phosphate. Thus, the sphingomyelin pathway regulates developmental death of oocytes, and sphingosine-1-phosphate provides a new approach to preserve ovarian function in vivo.     

Effect of reline material and denture base surface treatment on the impact strength of a denture base acrylic resin

doi:10.1111/j.1741‐2358.2009.00292.x
Effect of reline material and denture base surface treatment on the impact strength of a denture base acrylic resin Objective: In this study, the effect of relining and surface treatment on the impact strength (IS) of a heat‐polymerising denture base acrylic resin (Lucitone 550‐L) was evaluated. Materials and methods: Rectangular bars of L were made (60 × 6 × 2 mm) and relined (2 mm) with the relining resins Ufi Gel Hard (UH) and Tokuso Rebase Fast (TR). Specimens relined with L and intact L, TR and UH specimens were also made (60 × 6 × 4 mm), for comparison. Before relining, the L surface was left untreated or wetted with methyl methacrylate monomer and/or the bonding agents (BA) supplied by manufacturers of the reline resins. V‐notches were machined at the midpoint of the length of all specimens. The notches were made either across the width (Nw) or across the thickness of the specimens (Nth). The Charpy impact test was performed using a 0.5‐J pendulum, which had been specially designed and constructed. Data were analysed separately for each notch position using one‐way analysis of variance and Tukey honestly significant difference post‐hoc test (p = 0.05). Results: The IS of L was similar to that of L/L. For the Nw notch, treating the denture base L with TR BA and relining with TR reline material produced the highest IS. Conclusion: The IS of specimens made from heat polymerising acrylic resin Lucitone 550 was increased after relining using the hard chairside reline resin TR with its proprietary BA.     

Characterization of single chain antibody targets through yeast two hybrid

Background  

Due to their unique ability to bind their targets with high fidelity, antibodies are used widely not only in biomedical research, but also in many clinical applications. Recombinant antibodies, including single chain variable fragments (scFv), are gaining momentum because they allow powerful in vitro selection and manipulation without loss of function. Regardless of the ultimate application or type of antibody used, precise understanding of the interaction between the antibody's binding site and its specific target epitope(s) is of great importance. However, such data is frequently difficult to obtain.     

The effects of nutrient addition and pH manipulation in bag experiments on the phytoplankton of a small acidic lake in West Virginia,USA

In situ bag experiments were performed during summer and autumn in a small acidic lake, Tibbs Run Lake, West Virginia, USA. The objective was to evaluate phytoplankton responses to pH manipulation and nutrient addition. Increasing the pH from below 4.5 to over 6.3 resulted in great declines in phytoplankton biovolume. There was also a succession from dinoflagellates (Peridinium inconspicuum to small chlorophytes. The trend was more rapid where phosphorus (P) additions were made along with pH enhancement. During summer, P limitation was indicated, while nitrogen (N) appeared to limit production in autumn. In both seasons, nutrient additions greatly altered the phytoplankton composition in high pH treatments, but had no discernable effects at (the natural) low pH. A low pH, P addition treatment in autumn was the single exception. When N was subsequently added, phytoplankton composition changed dramatically, probably because the proceeding P additions caused severe secondary N-limitation. In general, however, the results supported the view that phytoplankton compositional responses to nutrient additions are suppressed in low pH, relative to high pH lake water.     

Obesity and body fat classification in the metabolic syndrome: Impact on cardiometabolic risk metabotype

Objective:

Obesity is a key factor in the development of the metabolic syndrome (MetS), which is associated with increased cardiometabolic risk. We investigated whether obesity classification by BMI and body fat percentage (BF%) influences cardiometabolic profile and dietary responsiveness in 486 MetS subjects (LIPGENE dietary intervention study).

Design and Methods:

Anthropometric measures, markers of inflammation and glucose metabolism, lipid profiles, adhesion molecules, and hemostatic factors were determined at baseline and after 12 weeks of four dietary interventions (high saturated fat (SFA), high monounsaturated fat (MUFA), and two low fat high complex carbohydrate (LFHCC) diets, one supplemented with long chain n‐3 polyunsaturated fatty acids (LC n‐3 PUFAs)).

Results:

About 39 and 87% of subjects classified as normal and overweight by BMI were obese according to their BF%. Individuals classified as obese by BMI (≥30 kg/m²) and BF% (≥25% (men) and ≥35% (women)) (OO, n = 284) had larger waist and hip measurements, higher BMI and were heavier (P < 0.001) than those classified as nonobese by BMI but obese by BF% (NOO, n = 92). OO individuals displayed a more proinflammatory (higher C reactive protein (CRP) and leptin), prothrombotic (higher plasminogen activator inhibitor‐1 (PAI‐1)), proatherogenic (higher leptin/adiponectin ratio) and more insulin resistant (higher HOMA‐IR) metabolic profile relative to the NOO group (P < 0.001). Interestingly, tumor necrosis factor‐α (TNF‐α) concentrations were lower post‐intervention in NOO individuals compared with OO subjects (P < 0.001).

Conclusions:

In conclusion, assessing BF% and BMI as part of a metabotype may help to identify individuals at greater cardiometabolic risk than BMI alone.     

Mice expressing the alpha(1B)-adrenergic receptor induces a synucleinopathy with excessive tyrosine nitration but decreased phosphorylation

We had previously reported that systemic overexpression of the alpha(1B)-adrenergic receptor (AR) in a transgenic mouse induced a neurodegenerative disease that resembled the parkinsonian-like syndrome called multiple system atrophy (MSA). We now report that our mouse model has cytoplasmic inclusion bodies that colocalize with oligodendrocytes and neurons, are positive for alpha-synuclein and ubiquitin, and therefore may be classified as a synucleinopathy. Alpha-synuclein monomers as well as multimers were present in brain extracts from both normal and transgenic mice. However, similar to human MSA and other synucleinopathies, transgenic mice showed an increase in abnormal aggregated forms of alpha-synuclein, which also increased its nitrated content with age. However, the same extracts displayed decreased phosphorylation of alpha-synuclein. Other traits particular to MSA such as Purkinje cell loss in the cerebellum and degeneration of the intermediolateral cell columns of the spinal cord also exist in our mouse model but differences still exist between them. Interestingly, long-term therapy with the alpha(1)-AR antagonist, terazosin, resulted in protection against the symptomatic as well as the neurodegeneration and alpha-synuclein inclusion body formation, suggesting that signaling of the alpha(1B)-AR is the cause of the pathology. We conclude that overexpression of the alpha(1B)-AR can cause a synucleinopathy similar to other parkinsonian syndromes.     

ALFY-Controlled DVL3 Autophagy Regulates Wnt Signaling,Determining Human Brain Size

Primary microcephaly is a congenital neurodevelopmental disorder of reduced head circumference and brain volume, with fewer neurons in the cortex of the developing brain due to premature transition between symmetrical and asymmetrical cellular division of the neuronal stem cell layer during neurogenesis. We now show through linkage analysis and whole exome sequencing, that a dominant mutation in ALFY, encoding an autophagy scaffold protein, causes human primary microcephaly. We demonstrate the dominant effect of the mutation in drosophila: transgenic flies harboring the human mutant allele display small brain volume, recapitulating the disease phenotype. Moreover, eye-specific expression of human mutant ALFY causes rough eye phenotype. In molecular terms, we demonstrate that normally ALFY attenuates the canonical Wnt signaling pathway via autophagy-dependent removal specifically of aggregates of DVL3 and not of Dvl1 or Dvl2. Thus, autophagic attenuation of Wnt signaling through removal of Dvl3 aggregates by ALFY acts in determining human brain size.