Selective circular oligonucleotide probes improve detection of point mutations in DNA

The synthesis of disulfide-cross-linked circular oligonucleotides, employing two different approaches, was accomplished. Several circular oligomers, which bear a C(5)-aminoalkyl-tethered thymidine unit, were labeled with photoluminescent europium(III) chelates. All circular structures were thoroughly characterized with denaturing PAGE and electrospray-ionization mass spectrometry. It was demonstrated that the disulfide cross-linking, resulting in circularization, considerably increases the enzymatic stability of phosphodiester oligonucleotides. In addition, UV melting experiments, followed, where possible, by extraction of thermodynamic parameters, revealed that several circular oligomers appear to be more selective towards their complementary targets than their corresponding linear precursors. Finally, the mixed-phase hybridization experiments have demonstrated that use of circular probes indeed improves the selectivity in the detection of DNA point mutations.     

Effective anomerisation of 2'-deoxyadenosine derivatives during disaccharide nucleoside synthesis

The formation of a disaccharide nucleoside (11) by O3'-glycosylation of 5'-O-protected 2'-deoxyadenosine or its N6-benzoylated derivative has been observed to be accompanied by anomerisation to the corresponding alpha-anomeric product (12). The latter reaction can be explained by instability of the N-glycosidic bond of purine 2'-deoxynucleosides in the presence of Lewis acids. An independent study on the anomerisation of partly blocked 2'-deoxyadenosine has been carried out. Additionally, transglycosylation has been utilized in the synthesis of 3'-O-beta-D-ribofuranosyl-2'deoxyadenosines and its alpha-anomer.     

The three-dimensional structures of antagonistic and agonistic forms of the glucocorticoid receptor ligand-binding domain: RU-486 induces a transconformation that leads to active antagonism

Here we describe the three-dimensional crystal structures of human glucocorticoid receptor ligand-binding domain (GR-LBD) in complex with the antagonist RU-486 at 2.3 A resolution and with the agonist dexamethasone ligand together with a coactivator peptide at 2.8 A. The RU-486 structure was solved in several different crystal forms, two with helix 12 intact (GR1 and GR3) and one with a protease-digested C terminus (GR2). In GR1, part of helix 12 is in a position that covers the co-activator pocket, whereas in the GR3, domain swapping is seen between the crystallographically identical subunits in the GR dimer. An arm consisting of the end of helix 11 and beyond stretches out from one molecule, and helix 12 binds to the other LBD, partly blocking the coactivator pocket of that molecule. This type of GR-LBD dimer has not been described before but might be an artifact from crystallization. Furthermore, the subunits of the GR3 dimers are covalently connected via a disulfide bond between the Cys-736 residues in the two molecules. All three RU-486 GR-LBD structures show that GR has a very flexible region between the end of helix 11 and the end of helix 12.     

Preferences of farmed blue foxes for different floor types

In the first experiment, farmed blue foxes were allowed to choose for 1 week between four standard farm cages equipped with different floor materials: plastic-coated wire mesh, dry wood, dry sand and wet (summer) or icy (winter) sand. Resting consisted of 10-15 separate bouts/day occupying 50-60% of the total 24-h. There were no other differences in the use of the cages except that the time spent on, and the duration and number of resting bouts were lower on wet or icy sand, resting periods being more affected than activity. In the second experiment, two cages were connected with a 1.2 m tunnel. One cage was always elevated (50 cm) compared to that one which was lower. One cage of each pair had a wire floor whereas the other cage either had sand or wire floor. Sand floor was preferred for active behaviours and wire floor for resting if these were on elevated level. Of two identical wire-floored cages, the elevated one had the priority. Foxes preferred to rest on the same floor where they had finished their previous resting bout, often exclusively and independently of floor material or floor level.     

Hsp40 interacts directly with the native state of the yeast prion protein Ure2 and inhibits formation of amyloid-like fibrils

Ure2 is the protein determinant of the [URE3] prion phenotype in Saccharomyces cerevisiae and consists of a flexible N-terminal prion-determining domain and a globular C-terminal glutathione transferase-like domain. Overexpression of the type I Hsp40 member Ydj1 in yeast cells has been found to result in the loss of [URE3]. However, the mechanism of prion curing by Ydj1 remains unclear. Here we tested the effect of overexpression of Hsp40 members Ydj1, Sis1, and Apj1 and also Hsp70 co-chaperones Cpr7, Cns1, Sti1, and Fes1 in vivo and found that only Ydj1 showed a strong curing effect on [URE3]. We also investigated the interaction of Ydj1 with Ure2 in vitro. We found that Ydj1 was able to suppress formation of amyloid-like fibrils of Ure2 by delaying the process of fibril formation, as monitored by thioflavin T binding and atomic force microscopy imaging. Controls using bovine serum albumin, Sis1, or the human Hsp40 homologues Hdj1 or Hdj2 showed no significant inhibitory effect. Ydj1 was only effective when added during the lag phase of fibril formation, suggesting that it interacts with Ure2 at an early stage in fibril formation and delays the nucleation process. Using surface plasmon resonance and size exclusion chromatography, we demonstrated a direct interaction between Ydj1 and both wild type and N-terminally truncated Ure2. In contrast, Hdj2, which did not suppress fibril formation, did not show this interaction. The results suggest that Ydj1 inhibits Ure2 fibril formation by binding to the native state of Ure2, thus delaying the onset of oligomerization.     

Analysis of 8-hydroxy-2'-deoxyguanosine in urine using high-performance liquid chromatography-electrospray tandem mass spectrometry

8-hydroxy-2'-deoxyguanosine (8-OHdG) is a widely used biomarker of oxidative stress in research related to DNA, protein damage as well as lipid peroxidation. HPLC-MS/MS with electrospray ionization (ESI) and the use of isotopically labelled 8-OHdG as an internal standard allows a simple quantification of 8-OHdG in urine samples. HPLC separation utilized the peak cutting technique and a 1.5 mmx120 mm analytical anion exchange column. Novel method entails only minimal sample handling including the addition of a buffer and an internal standard followed by centrifugation before the samples are ready for analysis. The levels of 8-OHdG in human urine samples (n=246) varied from 0.16 to 16.48 microg/L and the corresponding creatinine-normalized values were ranged from 0.49 to 14.27 microg of 8-OHdG/g creatinine. The correlation between the developed HPLC-MS/MS method and the existing HPLC-EC method was good with an R2 value of 0.8707.     

Ethanol induced NF-{kappa}B activation protects against cell injury in cultured rat gastric mucosal epithelium

Ethanol is a well-established irritant inducing inflammation in gastric mucosa, but the effects at the cellular level remain unclear. This study investigates NF-kappaB activation in gastric mucosal cells by ethanol and assesses the effects of heat shock pretreatment in this ulcerogenic situation. Rat gastric mucosal epithelia were exposed to ethanol for different time periods. Heat shock was induced by incubating the cells at 42 degrees C for 1 h prior to the experiments. For evaluation of NF-kappaB activation, the nuclear fraction of the cell lysates was analyzed with an EMSA or an ELISA-based assay. Caspase-3 (a promoter of apoptosis) activity was measured with a time-resolved fluorescence based assay, cell viability with a tetrazolium assay, and cell membrane integrity with a LDH assay. Ethanol (1-5%) induced NF-kappaB activation, reaching a maximum after 3 h, and also led to moderately increased COX-2 expression. Heat shock pretreatment and the intracellular calcium chelator BAPTA were able to inhibit ethanol-induced NF-kappaB activation. Heat shock pretreatment decreased ethanol-induced caspase-3 activation, decreased cell membrane damage, and retained cellular viability. Inhibition of NF-kappaB activation by NEMO-binding peptide, by decreasing RelA expression, or by inhibiting COX-2 activity by CAY-14040 promoted the effects of ethanol, such as increased caspase-3 activity and decreased cell viability. In conclusion, ethanol induces NF-kappaB activation via a calcium-dependent pathway and induces COX-2 expression. Inhibition of the NF-kappaB activation or COX-2 activity potentiates apoptosis and cell damage induced by ethanol, suggesting a protective role for NF-kappaB activation and COX-2 expression.     

Podocalyxin Is a Marker of Poor Prognosis in Pancreatic Ductal Adenocarcinoma

Aim of the Study

Podocalyxin-like 1 is a transmembrane glyco-protein whose overexpression associates in many cancers with poor prognosis and unfavorable clinicopathological characteristics. Until now, its prognostic value has never been studied in pancreatic ductal adenocarcinoma (PDAC). The aim of this study was to investigate podocalyxin expression in PDAC by a novel monoclonal antibody and a commercially available polyclonal antibody.

Patients and Materials

With tissue microarrays and immuno-histochemistry, podocalyxin expression evaluation involved 168 PDAC patients. The associa-tions of the podocalyxin tumor expression with clinicopathological variables were explored by Fisher’s exact test and the linear-by-linear test. Survival analyses were by Kaplan-Meier anal-ysis and the Cox proportional hazard model.

Results

The polyclonal antibody revealed membranous podocalyxin expression in 73 (44.0%) specimens and the monoclonal antibody was highly expressed in 36 (21.8%) cases. Membranous expression by the polyclonal antibody was associated with T classification (p=0.045) and perineural invasion (p=0.005), and high expression by the mono-clonal antibody with poor differentiation (p=0.033). High podocalyxin expression associated significantly with higher risk of death from PDAC by both the polyclonal antibody (hazard ratio (HR) = 1.62; 95% confidence interval (CI) 1.12-2.33; p=0.01) and the monoclonal antibody (HR = 2.10, 95% CI 1.38-3.20; p<0.001). The results remained significant in multivariate analysis, adjusted for age, gender, stage, lymph node ratio (≥/< 20%), and perivascular invasion (respectively as HR = 2.03; 95% CI 1.32-3.13, p=0.001; and as HR = 2.36; 95% CI 1.47-3.80, p<0.001).

Conclusion

We found podocalyxin to be an independent factor for poor prognosis in PDAC. To our knowledge, this is the first such report of its prognostic value.     

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.