Enamel Formation Genes Influence Enamel Microhardness Before and After Cariogenic Challenge

There is evidence for a genetic component in caries susceptibility, and studies in humans have suggested that variation in enamel formation genes may contribute to caries. For the present study, we used DNA samples collected from 1,831 individuals from various population data sets. Single nucleotide polymorphism markers were genotyped in selected genes (ameloblastin, amelogenin, enamelin, tuftelin, and tuftelin interacting protein 11) that influence enamel formation. Allele and genotype frequencies were compared between groups with distinct caries experience. Associations with caries experience can be detected but they are not necessarily replicated in all population groups and the most expressive results was for a marker in AMELX (p = 0.0007). To help interpret these results, we evaluated if enamel microhardness changes under simulated cariogenic challenges are associated with genetic variations in these same genes. After creating an artificial caries lesion, associations could be seen between genetic variation in TUFT1 (p = 0.006) and TUIP11 (p = 0.0006) with enamel microhardness. Our results suggest that the influence of genetic variation of enamel formation genes may influence the dynamic interactions between the enamel surface and the oral cavity.     

Implication of C-type natriuretic peptide-3 signaling in glycosaminoglycan synthesis and chondrocyte hypertrophy during TGF-β1 induced chondrogenic differentiation of chicken bone marrow-derived mesenchymal stem cells

This study investigated the involvement of CNP-3, chick homologue for human C-type natriuretic peptide (CNP), in TGF-β1 induced chondrogenic differentiation of chicken bone marrow-derived mesenchymal stem cells (MSCs). Chondrogenic differentiation of MSCs in pellet cultures was induced by TGF-β1. Chondrogenic differentiation and glycosaminoglycan synthesis were analyzed on the basis of basic histology, collagen type II expression, and Alcian blue staining. Antibodies against CNP and NPR-B were used to block their function during these processes. Results revealed that expression of CNP-3 and NPR-B in MSCs were regulated by TGF-β1 in monolayer cultures at mRNA level. In pellet cultures of MSCs, TGF-β1 successfully induced chondrogenic differentiation and glycosaminoglycan synthesis. Addition of CNP into the TGF-β1 supplemented chondrogenic differentiation medium further induced the glycosaminoglycan synthesis and hypertrophy of differentiated chondrocytes in these pellets. Pellets induced with TGF-β1 and treated with antibodies against CNP and NPR-B, did show collagen type II expression, however, Alcian blue staining showing glycosaminoglycan synthesis was significantly suppressed. In conclusion, CNP-3/NPR-B signaling may strongly be involved in synthesis of glycosaminoglycans of the chondrogenic matrix and hypertrophy of differentiated chondrocytes during TGF-β1 induced chondrogenic differentiation of MSCs.     

A Rapid Simultaneous Saccharification and Fermentation (SSF) Technique to Determine Ethanol Yields

We have developed a relatively simple simultaneous saccharification and fermentation (SSF) technique to determine the ethanol production potential for large sets of biomass samples. The technique is based on soaking approximately 0.5 grams of a biomass sample in aqueous ammonia at room temperature and at atmospheric pressure for 24 h, then fermenting with Saccharomyces cerevisiae D₅A for 24 h using Spezyme CP, for enzymatic hydrolysis of structural polysaccharides. We have tested the technique on a set of corn stover samples representing much of the genetic variability in the commercial corn hybrid population. The samples were weighed into modified Ankom filter bags (F57) before soaking to avoid biomass loss during the process. Fermentation samples were analyzed for ethanol after 24 h by HPLC. Percentages of theoretical maximum ethanol yields of the samples ranged between 44.9 and 73%. We observed that percentages of theoretical maximum ethanol yields were highly correlated (r ²?=?0.90) with acid detergent lignin concentration while a low correlation was observed between cellulose concentration and ethanol yield. Near infrared spectra of corn stover samples were also examined. The coefficient of determination (r ²) from regression of predicted versus measured percent theoretical maximum ethanol yield was 0.96. This result suggests that using NIRS is a promising method for predicting ethanol yield, but larger calibration sets are necessary for obtaining improved accuracy for larger sample populations. We conclude that the developed SSF technique could be applied to large numbers of biomass samples to rapidly estimate ethanol yields and to compare different biomass samples in terms of ethanol yields.     

Antagonism of microRNA-122 in mice by systemically administered LNA-antimiR leads to up-regulation of a large set of predicted target mRNAs in the liver

MicroRNA-122 (miR-122) is an abundant liver-specific miRNA, implicated in fatty acid and cholesterol metabolism as well as hepatitis C viral replication. Here, we report that a systemically administered 16-nt, unconjugated LNA (locked nucleic acid)-antimiR oligonucleotide complementary to the 5′ end of miR-122 leads to specific, dose-dependent silencing of miR-122 and shows no hepatotoxicity in mice. Antagonism of miR-122 is due to formation of stable heteroduplexes between the LNA-antimiR and miR-122 as detected by northern analysis. Fluorescence in situ hybridization demonstrated uptake of the LNA-antimiR in mouse liver cells, which was accompanied by markedly reduced hybridization signals for mature miR-122 in treated mice. Functional antagonism of miR-122 was inferred from a low cholesterol phenotype and de-repression within 24 h of 199 liver mRNAs showing significant enrichment for miR-122 seed matches in their 3′ UTRs. Expression profiling extended to 3 weeks after the last LNA-antimiR dose revealed that most of the changes in liver gene expression were normalized to saline control levels coinciding with normalized miR-122 and plasma cholesterol levels. Combined, these data suggest that miRNA antagonists comprised of LNA are valuable tools for identifying miRNA targets in vivo and for studying the biological role of miRNAs and miRNA-associated gene-regulatory networks in a physiological context.     

Conversion of xylose to ethanol by a novel phenol-tolerant strain of Enterobacteriaceae isolated from olive mill wastewater

A xylose-fermenting bacterium of the family Enterobacteriaceae was isolated from olive mill wastewater. It converted xylose to ethanol with a yield of 0.19 g ethanol g^–1 xylose. Although phenolic compounds normally inhibit pentose-utilizing microorganisms, this isolate was tolerant to phenol. Both the yield and the productivity of xylose fermentation decreased by 30% when phenol was added at a final concentration of 0.8 g phenol l^–1. Xylose (23 g l^–1) was totally fermented to ethanol (4.3 g l^–1) within 48 h in the absence of phenol; however, in the presence of 0.8 g phenol l^–1, only 3.3 g ethanol l^–1 was obtained from the same starting concentration of xylose after 70 h.     

Construct optimization for protein NMR structure analysis using amide hydrogen/deuterium exchange mass spectrometry

Disordered or unstructured regions of proteins, while often very important biologically, can pose significant challenges for resonance assignment and three‐dimensional structure determination of the ordered regions of proteins by NMR methods. In this article, we demonstrate the application of ¹H/²H exchange mass spectrometry (DXMS) for the rapid identification of disordered segments of proteins and design of protein constructs that are more suitable for structural analysis by NMR. In this benchmark study, DXMS is applied to five NMR protein targets chosen from the Northeast Structural Genomics project. These data were then used to design optimized constructs for three partially disordered proteins. Truncated proteins obtained by deletion of disordered N‐ and C‐terminal tails were evaluated using ¹H‐¹⁵N HSQC and ¹H‐¹⁵N heteronuclear NOE NMR experiments to assess their structural integrity. These constructs provide significantly improved NMR spectra, with minimal structural perturbations to the ordered regions of the protein structure. As a representative example, we compare the solution structures of the full length and DXMS‐based truncated construct for a 77‐residue partially disordered DUF896 family protein YnzC from Bacillus subtilis, where deletion of the disordered residues (ca. 40% of the protein) does not affect the native structure. In addition, we demonstrate that throughput of the DXMS process can be increased by analyzing mixtures of up to four proteins without reducing the sequence coverage for each protein. Our results demonstrate that DXMS can serve as a central component of a process for optimizing protein constructs for NMR structure determination. Proteins 2009. © 2009 Wiley‐Liss, Inc.     

Effect of high nitrate concentration on PHB storage in sequencing batch reactor under anoxic conditions

The study investigated effect of high influent nitrate concentration on poly-beta-hydroxybutyrate, (PHB), storage in a sequencing batch reactor, (SBR), under anoxic conditions. Acetate was fed as pulse during anoxic phase, sustained with external nitrate feeding. SBR operation involved three runs at steady state with COD/N ratios of 3.84, 2.93 and 1.54 gCOD/gN, where external nitrate concentrations gradually increased from 50 mg N/l to 114 mg N/l and 226 mg N/l, in 1st, 2nd and 3rd runs, respectively. In 1st run, acetate was fully converted into PHB with the storage yield value of 0.57-0.59 gCOD/gCOD, calculated both in terms of PHB formation and NO(X) utilization, confirming storage was the sole substrate utilization mechanism. In the following runs, PHB formation was reduced and the storage yield based on PHB dropped down to 0.40 and 0.33 gCOD/gCOD with increasing influent nitrate concentrations, indicating that higher portions of acetate were diverted to simultaneous direct growth. The observations suggested that nitrite accumulation detected at low COD/N ratios was responsible for inhibition of PHB storage.     

The Effects Of Short Time Right Ventricular Pacing On Left Atrial Mechanical Functions

Objectives

Left atrium (LA) plays an important role in left ventricular filling. It is well known that right ventricular apical pacing has unfavorable effects on ventricular systolic and diastolic performance. The aim of this study is to evaluate the LA mechanical functions with 2D echocardiography in patients with a permanent pacemaker after short time ventricular pacing.

Design

Echocardiographic examination was performed in 38 patients (mean age 63.0± 10.9, 18 female) with dual chamber pacemakers or defibrillators (< 20% ventricular pacing within previous 6 months, all of them on sinus rhythm) before and after 4 hours > 90% ventricular pacing at 70 beats per minute in DDD mode with an optimal AV interval. Left atrial volumes (LAV) including at the time of mitral valve opening (Vmax), at closure (Vmin), and at the onset of atrial systole (Volp) were measured. The passive emptying, conduit, active emptying and total emptying volume, stroke volumes were also calculated.

Results

No significant differences were noted at baseline and after pacing for absolute Vmax, Volp, passive emptying, conduit, active emptying, total emptying volumes as well as the volumes indexed to body surface area (p >0.05).

Conclusions

Short - time RV pacing seems to have no acute effects on left atrial mechanical functions.     

Association of putative concave protein-binding sites with the fluctuation behavior of residues

Here, we propose a binding site prediction method based on the high frequency end of the spectrum in the native state of the protein structural dynamics. The spectrum is obtained using an elastic network model (GNM). High frequency vibrating (HFV) residues are determined from the fastest modes dynamics. HFV residue clusters and the associated surface patch residues are tested for their likelihood to locate at the binding interfaces using two different data sets, the Benchmark Set of mainly enzymes and antigen/antibodies and the Cluster Set of more diverse structures. The binding interface is identified to be within 7.5 A of the HFV residue clusters in the Benchmark Set and Cluster Set, for 77% and 70% of the structures, respectively. The success rate increases to 88% and 84%, respectively, by using the surface patches. The results suggest that concave binding interfaces, typically those of enzyme-binding sites, are enriched by the HFV residues. Thus, we expect that the association of HFV residues with the interfaces is mostly for enzymes. If, however, a binding region has invaginations and cavities, as in some of the antigen/antibodies and in cases in the Cluster data set, we expect it would be detected there too. This implies that binding sites possess several (inter-related) properties such as cavities, high packing density, conservation, and disposition for hotspots at binding surfaces. It further suggests that the high frequency vibrating residue-based approach is a potential tool for identification of regions likely to serve as protein-binding sites. The software is available at http://www.prc.boun.edu.tr/PRC/software.html.