WDR12, a Member of Nucleolar PeBoW-Complex,Is Up-Regulated in Failing Hearts and Causes Deterioration of Cardiac Function

Aims

In a recent genome-wide association study, WD-repeat domain 12 (WDR12) was associated with early-onset myocardial infarction (MI). However, the function of WDR12 in the heart is unknown.

Methods and Results

We characterized cardiac expression of WDR12, used adenovirus-mediated WDR12 gene delivery to examine effects of WDR12 on left ventricular (LV) remodeling, and analyzed relationship between MI associated WDR12 allele and cardiac function in human subjects. LV WDR12 protein levels were increased in patients with dilated cardiomyopathy and rats post-infarction. In normal adult rat hearts, WDR12 gene delivery into the anterior wall of the LV decreased interventricular septum diastolic and systolic thickness and increased the diastolic and systolic diameters of the LV. Moreover, LV ejection fraction (9.1%, P<0.05) and fractional shortening (12.2%, P<0.05) were declined. The adverse effects of WDR12 gene delivery on cardiac function were associated with decreased cellular proliferation, activation of p38 mitogen–activated protein kinase (MAPK)/heat shock protein (HSP) 27 pathway, and increased protein levels of Block of proliferation 1 (BOP1), essential for ribosome biogenesis. Post-infarction WDR12 gene delivery decreased E/A ratio (32%, P<0.05) suggesting worsening of diastolic function. In human subjects, MI associated WDR12 allele was associated significantly with diastolic dysfunction and left atrial size.

Conclusions

WDR12 triggers distinct deterioration of cardiac function in adult rat heart and the MI associated WDR12 variant is associated with diastolic dysfunction in human subjects.     

Macoma balthica in the White and Barents Seas: properties of a widespread marine hybrid swarm (Mollusca: Bivalvia)

A main molecular subdivision in the circumpolar Macoma balthica complex has been described between Atlantic and Pacific taxa. In NE Europe, the clams of the White and Barents Seas, however, show deviant genetic structures. Using allozyme and mitochondrial DNA data, we explore the hypothesis that these deviations result from hybridization between an Atlantic (M. b. rubra) and an invading Pacific (M. b. balthica) lineage. A practically pure Atlantic Macoma extends from France north to the Varanger Peninsula (NE Norway), whereas populations farther east have genetic compositions intermediate between true Atlantic and true Pacific. Admixture estimates range from 32 to 90% Pacific contribution, with a notable deviation in a nearly pure Atlantic outpost in the Mezen Bay (NE White Sea). The pattern of variation is not one of a simple collinear mixing however. Different characters exhibit different degrees of introgression, and the relative introgression varies regionally. Yet, there are practically no interlocus genotypic disequilibria between the diverged loci, which brings out the White Sea-Barents Sea M. balthica as the best-documented marine animal hybrid swarms so far, arisen through amalgamation of genomes previously isolated since pre-Pleistocene times. On top of the main admixture pattern, strong geographical structuring is also seen in characters unrelated to the principal systematic distinction. The persistence of the regional patterns indicates restricted gene flow at the present time, despite the high dispersal potential of the species. The causes of this structuring could be in a complex history of colonization events and features of local hydrography enhancing isolation and divergence of populations.     

Array CGH using whole genome amplification of fresh-frozen and formalin-fixed, paraffin-embedded tumor DNA

The ability to utilize formalin-fixed, paraffin-embedded (FFPE) archival specimens reliably for high-resolution molecular genetic analysis would be of immense practical application in the study of human disease. We have evaluated the ability of the GenomePlex whole genome amplification (WGA) kit to amplify frozen and FFPE tissue for use in array CGH (aCGH). GenomePlex gave highly representative data compared with unamplified controls both from frozen material (Pearson's R(2) = 0.898) and from FFPE (R(2) = 0.883). Artifactual amplification observed using DOP-PCR at chromosomes 1p, 3, 13q, and 16p was not seen with GenomePlex. Highly reproducible aCGH profiles were obtained using as little as 5 ng starting material from FFPE (R(2) = 0.918). This WGA method should readily lend itself to the determination of DNA copy number alterations from small fresh-frozen and FFPE clinical tumor specimens, although some care must be taken to optimize the DNA extraction procedure.     

Evaluation of Antimicrobial Therapy of Blood Culture Positive Healthcare-Associated Infections in Children

Aim

Knowledge of the quality of antimicrobial therapy (AMT) used for invasive healthcare-associated infections (HAIs) in paediatrics is scarce. Influence of the final information about the isolated pathogen on the subsequent targeted AMT was investigated in our study.

Methods

Data on 149 children (0–17 years) with blood culture positive HAIs were collected. The causative microbes under investigation were Staphylococcus aureus, Staphylococcus epidermidis, streptococci, Gram negative rods, and mixed infections were likewise included. For adjusting the antimicrobial regimen, an expert panel evaluated the quality of the targeted AMT and the delay of 72 hours after final microbiology results. AMT was regarded as inappropriate if the pathogen was totally resistant to the used antimicrobials (i) or if the chosen therapy was of not optimal efficacy against the pathogen (ii).

Results

17% of the patients received inappropriate AMT. Half of these infections 13/26 (50%) were treated with an antimicrobial to which the isolate was resistant. Three (3/13, 23%) of these patients received antimicrobials which were totally ineffective according to in vitro data. Suboptimal or too broad spectrum AMT was administered to 13/26 (50%) patients. The most common causes of inappropriate use were the use of beta-lactams in oxacillin-resistant Staphylococcus epidermidis infections and vancomycin given in oxacillin-sensitive Staphylococcus aureus infections.

Conclusion

Approximately 17% of the selected cohort received inappropriate AMT. More attention should be paid to the appropriate use of antimicrobials, and training of prescribers should be urgently provided.     

Factors influencing the natural reproduction decline in the beluga (Huso huso,Linnaeus, 1758), Russian sturgeon (Acipenser gueldenstaedtii,Brandt & Ratzeburg, 1833), and stellate sturgeon (A. stellatus,Pallas, 1771) of the Volga–Caspian basin: A review

The review describes the changes in natural reproduction of three important sturgeon species in the Volga–Caspian basin: (a) the beluga (Huso huso Linneaus, 1758), (b) the Russian sturgeon (Acipenser gueldenstaedtii Brandt &Ratzeburg, 1833), and (c) the stellate sturgeon (Acipenser stellatus Pallas, 1771). Since the past 60 years, these species responded to severe influences of natural and anthropogenic factors. On the basis of original and published data, an analysis has been made of (a) the numbers of larvae migrating from spawning sites (according to plankton net survey), (b) fecundity and histological anomalies in gonad development, (c) the numbers of adult sturgeons in the Caspian Sea and of spawners migrating to the Volga River (according to trawl and beach seine survey), and (d) foraging resources for the sturgeons. The results show that their natural reproduction in the Volga–Caspian basin has declined drastically during the past decades under the impact of (a) fluctuations of the Caspian Sea level and flow discharge from the Volga River, (b) blockage of sturgeon migration routes and loss of spawning sites because of dam construction, (c) water pollution in the lower reaches of the Volga River and in the Caspian Sea, and (d) intensive and selective illegal and unreported fishing. The relative significance of these factors has been changing during the study period.     

Structure of an acidic polysaccharide from the agar-decomposing marine bacterium Pseudoalteromonas atlantica strain IAM 14165 containing 5,7-diacetamido-3,5,7,9-tetradeoxy-L-glycero-L-manno-non-2-ulosonic acid

The structure of an acidic polysaccharide from Pseudoalteromonas atlantica strain 14165 containing 5,7-diacetamido-3,5,7,9-tetradeoxy-L-glycero-L-manno-non-2-ulosonic acid (di-N-acetylpseudaminic acid, Pse5Ac7Ac) has been elucidated. The polysaccharide was studied by 1H and 13C NMR spectroscopy, including 2D experiments, along with sugar and methylation analyses. After a selective hydrolysis a modified polysaccharide devoid of its side chain could be isolated. It was found that the polysaccharide has pentasaccharide repeating units with following structure: [structure: see text].     

Changes in end-to-end interactions of tropomyosin affect mouse cardiac muscle dynamics

The ends of striated muscle tropomyosin (TM) are integral for thin filament cooperativity, determining the cooperative unit size and regulating the affinity of TM for actin. We hypothesized that altering the alpha-TM carboxy terminal overlap end to the beta-TM counterpart would affect the amino-terminal association, which would alter the end-to-end interactions of TM molecules in the thin filament regulatory strand and affect the mechanisms of cardiac muscle contraction. To test this hypothesis, we generated transgenic (TG) mouse lines that express a mutant form of alpha-TM in which the first 275 residues are from alpha-TM and the last nine amino acids are from beta-TM (alpha-TM9aaDeltabeta). Molecular analyses show that endogenous alpha-TM mRNA and protein are nearly completely replaced with alpha-TM9aaDeltabeta. Working heart preparations data show that the rates of contraction and relaxation are reduced in alpha-TM9aaDeltabeta hearts. Left ventricular pressure and time to peak pressure are also reduced (-12% and -13%, respectively). The ratio of maximum to minimum first derivatives of change in left ventricular systolic pressure with respect to time (ratio of +dP/dt to -dP/dt, respectively) is increased, but tau is not changed significantly. Force-intracellular calcium concentration ([Ca2+]i) measurements from intact papillary fibers demonstrate that alpha-TM9aaDeltabeta TG fibers produce less force per given [Ca2+]i compared with nontransgenic fibers. Taken together, the data demonstrate that the rate of contraction is primarily affected in TM TG hearts. Protein docking studies show that in the mutant molecule, the overall carbon backbone is perturbed about 1.5 A, indicating that end-to-end interactions are altered. These results demonstrate that the localized flexibility present in the coiled-coil structures of TM isoforms is different, and that plays an important role in interacting with neighboring thin filament regulatory proteins and with differentially modulating the myofilament activation processes.     

Novel Microbiological and Spatial Statistical Methods to Improve Strength of Epidemiological Evidence in a Community-Wide Waterborne Outbreak

Failures in the drinking water distribution system cause gastrointestinal outbreaks with multiple pathogens. A water distribution pipe breakage caused a community-wide waterborne outbreak in Vuorela, Finland, July 2012. We investigated this outbreak with advanced epidemiological and microbiological methods. A total of 473/2931 inhabitants (16%) responded to a web-based questionnaire. Water and patient samples were subjected to analysis of multiple microbial targets, molecular typing and microbial community analysis. Spatial analysis on the water distribution network was done and we applied a spatial logistic regression model. The course of the illness was mild. Drinking untreated tap water from the defined outbreak area was significantly associated with illness (RR 5.6, 95% CI 1.9–16.4) increasing in a dose response manner. The closer a person lived to the water distribution breakage point, the higher the risk of becoming ill. Sapovirus, enterovirus, single Campylobacter jejuni and EHEC O157:H7 findings as well as virulence genes for EPEC, EAEC and EHEC pathogroups were detected by molecular or culture methods from the faecal samples of the patients. EPEC, EAEC and EHEC virulence genes and faecal indicator bacteria were also detected in water samples. Microbial community sequencing of contaminated tap water revealed abundance of Arcobacter species. The polyphasic approach improved the understanding of the source of the infections, and aided to define the extent and magnitude of this outbreak.     

Attachment of LcrV from Yersinia pestis at dual binding sites to human TLR-2 and human IFN-gamma receptor

The virulence antigen (V-antigen, LcrV) of Yersinia pestis, the causative agent of bubonic plague, is an established protective antigen known to regulate, target, and mediate type III translocation of cytotoxic yersiniae outer proteins termed Yops; LcrV also prompts TLR2-dependent upregulation of anti-inflammatory IL-10. In this study, we determined the parameters of specific interaction of LcrV with TLR2 expressed on human transfected HEK293 cells (TLR2+/CD14-), VTEC2.HS cells (TLR2+/CD14-), primary monocytes (TLR2+/CD14+), and THP-1 cells (TLR2+/CD14+). The IRRL314-317 motif of the extracellular domain of human and mouse TLR2 accounted for high-affinity binding of LcrV. The CD14 co-receptor did not influence this interaction. LcrV did not bind to human U937 (TLR2-/CD14-) and alveolar macrophages (TLR2-/CD14+) in the absence of receptor-bound human IFN-gamma or a synthetic C-terminal fragment (hIFN-gamma132-143). The latter, but not mouse IFN-gamma (or synthetic control peptides), shared a GRRA138-141 site necessary for high-affinity specific binding. LcrV of Y. pestis shares the N-terminal LEEL32-35 binding site of Yersinia enterocolitica and also has an exposed internal DEEI203-206 binding site. Comparison of binding constants and consideration of steric restrictions indicate that binding is not cooperative and only the internal site binds LcrV to target cells. Both the LEEL32-35 and DEEI203-206 binding sites are removed by five amino acids from DKN residues associated with biological activity of bound LcrV. LcrV of Y. pestis promoted both TLR2/CD14-dependent and TLR2/CD14-independent amplification of IL-10 and concomitant downregulation of TNF-alpha in human target cells. The ability of LcrV to utilize human IFN-gamma (a major inflammatory effector of innate immunity) to minimize inflammation is insidious and may account in part for the severe symptoms of plague in man.