Culture and PCR analysis of joint fluid in the diagnosis of prosthetic joint infection

This prospective study compared PCR and culture techniques in the diagnosis of prosthetic joint infection (PJI). We obtained joint fluid samples (JFS; n=115) from patients who had failed total joint arthroplasty between January 2003 and June 2005; 49 were positive for PJI according to established strict criteria. JFS were analyzed by PCR (n=35; control n=66) or culture (n=46, control n=48). PCR was positive in 71% of PJI cases, resulting in sensitivity, specificity, accuracy, positive predictive value, negative predictive value, and likelihood ratio for positive results as follows: 0.71; 0.97; 0.88; 0.93; 0.87 and 23.6, respectively. Culture was positive in 44% of PJI samples. Corresponding statistics were 0.44; 0.94; 0.69; 0.87; 0.63 and 7.0, respectively. Significantly higher sensitivity, accuracy and negative predictive values were calculated for PCR versus culture, and there was 83% concordance between the results of intraoperative culture and PCR detection of causative bacteria. Therefore, we conclude that PCR analysis of synovial fluid increases the utility of pre-operative aspiration for patients who require revision total joint surgery.     

Algorithm for identification of fusion proteins via mass spectrometry

Identification of fusion proteins has contributed significantly to our understanding of cancer progression, yielding important predictive markers and therapeutic targets. While fusion proteins can be potentially identified by mass spectrometry, all previously found fusion proteins were identified using genomic (rather than mass spectrometry) technologies. This lack of MS/MS applications in studies of fusion proteins is caused by the lack of computational tools that are able to interpret mass spectra from peptides covering unknown fusion breakpoints (fusion peptides). Indeed, the number of potential fusion peptides is so large that the existing MS/MS database search tools become impractical even in the case of small genomes. We explore computational approaches to identifying fusion peptides, propose an algorithm for solving the fusion peptide identification problem, and analyze the performance of this algorithm on simulated data. We further illustrate how this approach can be modified for human exons prediction.     

The first survey of Cystobasidiomycete yeasts in the lichen genus Cladonia; with the description of Lichenozyma pisutiana gen. nov., sp. nov.

The view of lichens as a symbiosis only between a mycobiont and a photobiont has been challenged by discoveries of diverse associated organisms. Specific basidiomycete yeasts in the cortex of a range of macrolichens were hypothesized to influence the lichens' phenotype. The present study explores the occurrence and diversity of cystobasidiomycete yeasts in the lichen genus Cladonia. We obtained seven cultures and 56 additional sequences using specific primers from 27 Cladonia species from all over Europe and performed phylogenetic analyses based on ITS, LSU and SSU rDNA loci. We revealed yeast diversity distinct from any previously reported. Representatives of Cyphobasidiales, Microsporomycetaceae and of an unknown group related to Symmetrospora have been found. We present evidence that the Microsporomycetaceae contains mainly lichen-associated yeasts. Lichenozyma pisutiana is circumscribed here as a new genus and species. We report the first known associations between cystobasidiomycete yeasts and Cladonia (both corticate and ecorticate), and find that the association is geographically widespread in various habitats. Our results also suggest that a great diversity of lichen associated yeasts remains to be discovered.     

The zinc-binding motif of human RECQ5beta suppresses the intrinsic strand-annealing activity of its DExH helicase domain and is essential for the helicase activity of the enzyme

RecQ family helicases, functioning as caretakers of genomic integrity, contain a zinc-binding motif which is highly conserved among these helicases, but does not have a substantial structural similarity with any other known zinc-finger folds. In the present study, we show that a truncated variant of the human RECQ5beta helicase comprised of the conserved helicase domain only, a splice variant named RECQ5alpha, possesses neither ATPase nor DNA-unwinding activities, but surprisingly displays a strong strand-annealing activity. In contrast, fragments of RECQ5beta including the intact zinc-binding motif, which is located immediately downstream of the helicase domain, exhibit much reduced strand-annealing activity but are proficient in DNA unwinding. Quantitative measurements indicate that the regulatory role of the zinc-binding motif is achieved by enhancing the DNA-binding affinity of the enzyme. The novel intramolecular modulation of RECQ5beta catalytic activity mediated by the zinc-binding motif may represent a universal regulation mode for all RecQ family helicases.     

Nitric Oxide Upregulates Proteasomal Protein Degradation in Neurons

Nitric oxide (NO) is involved in many neuronal functions such as neuromodulation and intracellular signaling. Recent studies have demonstrated that nitric oxide is involved in regulation of proteasomal protein degradation. However, its role in neuronal protein degradation still remains unclear. In our study, we investigated the influence of endogenous nitric oxide production in this process. We have shown that nitric oxide synthase blockade prevents decline of the Ub^G76V-GFP fluorescence (GFP-based proteasomal protein degradation reporter) in neuronal processes of the cultured hippocampal neurons. It suggests that nitric oxide may regulate ubiquitin-dependent proteasomal protein degradation in neurons. Also, we have confirmed that the NO synthesis blockade alone significantly impairs long-term potentiation, and demonstrated for the first time that simultaneous blockade of the NO and proteins synthesis leads to the long-term potentiation amplitude rescue to the control values. Obtained results suggest that nitric oxide is involved in the protein degradation in proteasomes in physiological conditions.     

Cyclic nucleotide-dependent protein kinases inhibit binding of 14-3-3 to the GTPase-activating protein Rap1GAP2 in platelets

GTPase-activating proteins are required to terminate signaling by Rap1, a small guanine nucleotide-binding protein that controls integrin activity and cell adhesion. Recently, we identified Rap1GAP2, a GTPase-activating protein of Rap1 in platelets. Here we show that 14-3-3 proteins interact with phosphorylated serine 9 at the N terminus of Rap1GAP2. Platelet activation by ADP and thrombin enhances serine 9 phosphorylation and increases 14-3-3 binding to endogenous Rap1GAP2. Conversely, inhibition of platelets by endothelium-derived factors nitric oxide and prostacyclin disrupts 14-3-3 binding. These effects are mediated by cGMP- and cAMP-dependent protein kinases that phosphorylate Rap1GAP2 at serine 7, adjacent to the 14-3-3 binding site. 14-3-3 binding does not change the GTPase-activating function of Rap1GAP2 in vitro. However, 14-3-3 binding attenuates Rap1GAP2 mediated inhibition of cell adhesion. Our findings define a novel crossover point of activatory and inhibitory signaling pathways in platelets.     

Genome mining reveals high incidence of putative lipopeptide biosynthesis NRPS/PKS clusters containing fatty acyl‐AMP ligase genes in biofilm‐forming cyanobacteria

Cyanobacterial lipopeptides have antimicrobial and antifungal bioactivities with potential for use in pharmaceutical research. However, due to their hemolytic activity and cytotoxic effects on human cells, they may pose a health issue if produced in substantial amounts in the environment. In bacteria, lipopeptides can be synthesized via several well‐evidenced mechanisms. In one of them, fatty acyl‐AMP ligase (FAAL) initiates biosynthesis by activation of a fatty acyl residue. We have performed a bioinformatic survey of the cyanobacterial genomic information available in the public databases for the presence of FAAL‐containing non‐ribosomal peptide synthetase/polyketide synthetase (NRPS/PKS) biosynthetic clusters, as a genetic basis for lipopeptide biosynthesis. We have identified 79 FAAL genes associated with various NRPS/PKS clusters in 16% of 376 cyanobacterial genomic assemblies available, suggesting that FAAL is frequently incorporated in NRPS/PKS biosynthetases. FAAL was present either as a stand‐alone protein or fused either to NRPS or PKS. Such clusters were more frequent in derived phylogenetic lineages with larger genome sizes, which is consistent with the general pattern of NRPS/PKS pathways distribution. The putative lipopeptide clusters were more frequently found in genomes of cyanobacteria that live attached to surfaces and are capable of forming microbial biofilms. While lipopeptides are known in other bacterial groups to play a role in biofilm formation, motility, and colony expansion, their functions in cyanobacterial biofilms need to be tested experimentally. According to our data, benthic and terrestrial cyanobacteria should be the focus of a search for novel candidates for lipopeptide drug synthesis and the monitoring of toxic lipopeptide production.     

Catalytically Active Monomers of E. coli Glyceraldehyde-3-Phosphate Dehydrogenase

Monomeric forms of E. coli glyceraldehyde-3-phosphate dehydrogenase have been prepared using two different experimental approaches: (1) covalent immobilization of a tetramer on a solid support via a single subunit with subsequent dissociation of non-covalently bound subunits in the presence of urea, and (2) entrapment of monomeric species into reversed micelles of Aerosol OT in octane. Isolated monomers were shown to be catalytically active, exhibiting K _M values close to the parameters characteristic of the tetrameric forms. Like tetramers, isolated monomers did not use NADP7 as a coenzyme.     

Redox regulation of Cdc25C

The Cdc25 family of dual specific phosphatases are critical components of cell cycle progression and checkpoint control. Certain stresses such as ultraviolet light stimulate the rapid and selective destruction of Cdc25A protein through a Chk1 protein kinase-dependent pathway. We demonstrate that in contrast to cellular stresses previously examined, hydrogen peroxide exposure affects Cdc25C but not Cdc25A levels. Pharmacological inhibition of Chk1 activity or a mutant of Cdc25C that lacks the Chk1 phosphorylation site still undergoes degradation in response to oxidants. We also demonstrate that in vitro hydrogen peroxide stimulates an intramolecular disulfide bond between the active site cysteine at position 377 and another invariant cysteine at position 330. The in vivo stability of Cdc25C is substantially reduced by the mutation of either of these two cysteine residues. In contrast, a double (C2) mutant of both cysteine 330 and cysteine 377 results in a protein that is more stable than wild type Cdc25C and is resistant to oxidative stress-induced degradation. In addition, the C2 mutant, which is unable to form an intramolecular disulfide bond, has reduced binding to 14-3-3 in vitro and in vivo. These results suggest that oxidative stress may induce cell cycle arrest in part through the degradation of Cdc25C.