Proportion and predictors of FVC decline in patients with interstitial lung disease.

RATIONALE: The proportion of patients who develop progressive pulmonary fibrosis (PPF), along with risk factors for progression remain poorly understood. OBJECTIVES: To examine factors associated with an increased risk of developing PPF among patients at a referral center. METHODS: We identified patients with a diagnosis of interstitial lung disease (ILD) seen within the Cleveland Clinic Health System. Utilizing a retrospective observational approach we estimated the risk of developing progression by diagnosis group and identified key clinical predictors using the FVC component of both the original progressive fibrotic interstitial lung disease (PFILD) and the proposed PPF (ATS) criteria. RESULTS: We identified 5934 patients with a diagnosis of ILD. The cumulative incidence of progression over the 24 months was similar when assessed with the PFILD and PPF criteria (33.1 % and 37.9 % respectively). Of those who met the ATS criteria, 9.5 % did not meet the PFILD criteria. Conversely, 4.3 % of patients who met PFILD thresholds did not achieve the 5 % absolute FVC decline criteria. Significant differences in the rate of progression were seen based on underlying diagnosis. Steroid therapy (HR 1.46, CI 1.31-1.62) was associated with an increased risk of progressive fibrosis by both PFILD and PPF criteria. CONCLUSION: Regardless of the definition used, the cumulative incidence of progressive disease is high in patients with ILD in the 24 months following diagnosis. Some differences are seen in the risk of progression when assessed by PFILD and PPF criteria. Further work is needed to identify modifiable risk factors for the development of progressive fibrosis.

TET-TDG Active DNA Demethylation at CpG and Non-CpG Sites.

In mammalian genomes, cytosine methylation occurs predominantly at CG (or CpG) dinucleotide contexts. As part of dynamic epigenetic regulation, 5-methylcytosine (mC) can be erased by active DNA demethylation, whereby ten-eleven translocation (TET) enzymes catalyze the stepwise oxidation of mC to 5-hydroxymethylcytosine (hmC), 5-formylcytosine (fC), and 5-carboxycytosine (caC), thymine DNA glycosylase (TDG) excises fC or caC, and base excision repair yields unmodified cytosine. In certain cell types, mC is also enriched at some non-CG (or CH) dinucleotides, however hmC is not. To provide biochemical context for the distribution of modified cytosines observed in biological systems, we systematically analyzed the activity of human TET2 and TDG for substrates in CG and CH contexts. We find that while TET2 oxidizes mC more efficiently in CG versus CH sites, this context preference can be diminished for hmC oxidation. Remarkably, TDG excision of fC and caC is only modestly dependent on CG context, contrasting its strong context dependence for thymine excision. We show that collaborative TET-TDG oxidation-excision activity is only marginally reduced for CA versus CG contexts. Our findings demonstrate that the TET-TDG-mediated demethylation pathway is not limited to CG sites and suggest a rationale for the depletion of hmCH in genomes rich in mCH.

Defining the Role of Nucleotide Flipping in Enzyme Specificity Using 19F NMR.

A broad range of proteins employ nucleotide flipping to recognize specific sites in nucleic acids, including DNA glycosylases, which remove modified nucleobases to initiate base excision repair. Deamination, a pervasive mode of damage, typically generates lesions that are recognized by glycosylases as being foreign to DNA. However, deamination of 5-methylcytosine (mC) generates thymine, a canonical DNA base, presenting a challenge for damage recognition. Nevertheless, repair of mC deamination is important because the resulting G·T mispairs cause C → T transition mutations, and mC is abundant in all three domains of life. Countering this threat are three types of glycosylases that excise thymine from G·T mispairs, including thymine DNA glycosylase (TDG). These enzymes must minimize excision of thymine that is not generated by mC deamination, in A·T pairs and in polymerase-generated G·T mispairs. TDG preferentially removes thymine from DNA contexts in which cytosine methylation is prevalent, including CG and one non-CG site. This remarkable context specificity could be attained through modulation of nucleotide flipping, a reversible step that precedes base excision. We tested this idea using fluorine NMR and DNA containing 2'-fluoro-substituted nucleotides. We find that dT nucleotide flipping depends on DNA context and is efficient only in contexts known to feature cytosine methylation. We also show that a conserved Ala residue limits thymine excision by hindering nucleotide flipping. A linear free energy correlation reveals that TDG attains context specificity for thymine excision through modulation of nucleotide flipping. Our results provide a framework for characterizing nucleotide flipping in nucleic acids using 19F NMR.

Nucleosomes and the three glycosylases: High, medium, and low levels of excision by the uracil DNA glycosylase superfamily.

Human cells express the UDG superfamily of glycosylases, which excise uracil (U) from the genome. The three members of this structural superfamily are uracil DNA glycosylase (UNG/UDG), single-strand selective monofunctional uracil DNA glycosylase (SMUG1), and thymine DNA glycosylase (TDG). We previously reported that UDG is efficient at removing U from DNA packaged into nucleosome core particles (NCP) and is minimally affected by the histone proteins when acting on an outward-facing U in the dyad region. In an effort to determine whether this high activity is a general property of the UDG superfamily of glycosylases, we compare the activity of UDG, SMUG1, and TDG on a U:G wobble base pair using NCP assembled from Xenopus laevis histones and the Widom 601 positioning sequence. We found that while UDG is highly active, SMUG1 is severely inhibited on NCP and this inhibition is independent of sequence context. Here we also provide the first report of TDG activity on an NCP, and found that TDG has an intermediate level of activity in excision of U and is severely inhibited in its excision of T. These results are discussed in the context of cellular roles for each of these enzymes.

The Helicobacter pylori adhesin protein HopQ exploits the dimer interface of human CEACAMs to facilitate translocation of the oncoprotein CagA.

Helicobacter pylori infects half of the world's population, and strains that encode the cag type IV secretion system for injection of the oncoprotein CagA into host gastric epithelial cells are associated with elevated levels of cancer. CagA translocation into host cells is dependent on interactions between the H. pylori adhesin protein HopQ and human CEACAMs. Here, we present high-resolution structures of several HopQ-CEACAM complexes and CEACAMs in their monomeric and dimeric forms establishing that HopQ uses a coupled folding and binding mechanism to engage the canonical CEACAM dimerization interface for CEACAM recognition. By combining mutagenesis with biophysical and functional analyses, we show that the modes of CEACAM recognition by HopQ and CEACAMs themselves are starkly different. Our data describe precise molecular mechanisms by which microbes exploit host CEACAMs for infection and enable future development of novel oncoprotein translocation inhibitors and H. pylori-specific antimicrobial agents.

Intravenous hemostatic nanoparticles increase survival following blunt trauma injury.

Trauma is the leading cause of death for people ages 1-44, with blood loss comprising 60-70% of mortality in the absence of lethal CNS or cardiac injury. Immediate intervention is critical to improving chances of survival. While there are several products to control bleeding for external and compressible wounds, including pressure dressings, tourniquets, or topical materials (e.g., QuikClot, HemCon), there are no products that can be administered in the field for internal bleeding. There is a tremendous unmet need for a hemostatic agent to address internal bleeding in the field. We have developed hemostatic nanoparticles (GRGDS-NPs) that reduce bleeding times by ~50% in a rat femoral artery injury model. Here, we investigated their impact on survival following administration in a lethal liver resection injury in rats. Administration of these hemostatic nanoparticles reduced blood loss following the liver injury and dramatically and significantly increased 1 h survival from 40 and 47% in controls (inactive nanoparticles and saline, respectively) to 80%. Furthermore, we saw no complications following administration of these nanoparticles. We further characterized the nanoparticles' effect on clotting time (CT) and maximum clot firmness (MCF) using rotational thromboelastometry (ROTEM), a clinical measurement of whole-blood coagulation. Clotting time is significantly reduced, with no change in MCF. Administration of these hemostatic nanoparticles after massive trauma may help staunch bleeding and improve survival in the critical window following injury, and this could fundamentally change trauma care.