Transamidation by transglutaminase 2 transforms S100A11 calgranulin into a procatabolic cytokine for chondrocytes

In osteoarthritis (OA), low-grade joint inflammation promotes altered chondrocyte differentiation and cartilage catabolism. S100/calgranulins share conserved calcium-binding EF-hand domains, associate noncovalently as homodimers and heterodimers, and are secreted and bind receptor for advanced glycation end products (RAGE). Chondrocyte RAGE expression and S100A11 release are stimulated by IL-1beta in vitro and increase in OA cartilage in situ. Exogenous S100A11 stimulates chondrocyte hypertrophic differentiation. Moreover, S100A11 is covalently cross-linked by transamidation catalyzed by transglutaminase 2 (TG2), itself an inflammation-regulated and redox stress-inducible mediator of chondrocyte hypertrophic differentiation. In this study, we researched mouse femoral head articular cartilage explants and knee chondrocytes, and a soluble recombinant double point mutant (K3R/Q102N) of S100A11 TG2 transamidation substrate sites. Both TG2 and RAGE knockout cartilage explants retained IL-1beta responsiveness. The K3R/Q102N mutant of S100A11 retained the capacity to bind to RAGE and chondrocytes but lost the capacity to signal via the p38 MAPK pathway or induce chondrocyte hypertrophy and glycosaminoglycans release. S100A11 failed to induce hypertrophy, glycosaminoglycan release, and appearance of the aggrecanase neoepitope NITEGE in both RAGE and TG2 knockout cartilages. We conclude that transamidation by TG2 transforms S100A11 into a covalently bonded homodimer that acquires the capacity to signal through the p38 MAPK pathway, accelerate chondrocyte hypertrophy and matrix catabolism, and thereby couple inflammation with chondrocyte activation to potentially promote OA progression.     

Cel9D, an atypical 1,4-beta-D-glucan glucohydrolase from Fibrobacter succinogenes: characteristics, catalytic residues, and synergistic interactions with other cellulases

The increasing demands of renewable energy have led to the critical emphasis on novel enzymes to enhance cellulose biodegradation for biomass conversion. To identify new cellulases in the ruminal bacterium Fibrobacter succinogenes, a cell extract of cellulose-grown cells was separated by ion-exchange chromatography and cellulases were located by zymogram analysis and identified by peptide mass fingerprinting. An atypical family 9 glycoside hydrolase (GH9), Cel9D, with less than 20% identity to typical GH9 cellulases, was identified. Purified recombinant Cel9D enhanced the production of reducing sugar from acid swollen cellulose (ASC) and Avicel by 1.5- to 4-fold when mixed separately with each of four other glucanases, although it had low activity on these substrates. Cel9D degraded ASC and cellodextrins with a degree of polymerization higher than 2 to glucose with no apparent endoglucanase activity, and its activity was restricted to beta-1-->4-linked glucose residues. It catalyzed the hydrolysis of cellulose by an inverting mode of reaction, releasing glucose from the nonreducing end. Unlike many GH9 cellulases, calcium ions were not required for its function. Cel9D had increased kcat/Km values for cello-oligosaccharides with higher degrees of polymerization. The kcat/Km value for cellohexaose was 2,300 times higher than that on cellobiose. This result indicates that Cel9D is a 1,4-beta-D-glucan glucohydrolase (EC 3.2.1.74) in the GH9 family. Site-directed mutagenesis of Cel9D identified Asp166 and Glu612 as the candidate catalytic residues, while Ser168, which is not present in typical GH9 cellulases, has a crucial structural role. This enzyme has an important role in crystalline cellulose digestion by releasing glucose from accessible cello-oligosaccharides.     

Development of an RSA calibration system with improved accuracy and precision

In this study, a new radiostereometric analysis (RSA) calibration cage was developed with the aim of improving the accuracy and precision of RSA. This development consisted of three steps: a numerical simulation technique was first used to design the new cage; a synthetic imaging method was then implemented to predict the performance of the designed cage before it was actually fabricated; and an experimental phantom test was finally conducted to verify the actual performance of the new cage and compare with two currently widely used cages. Accuracy was calculated as the 95% prediction intervals from regression analyses between the measured and actual displacements, and precision was defined as the standard deviation of repeated measurements. The final experimental phantom tests showed that the accuracy and precision of the new calibration cage were improved by about 40% over an existing biplanar cage and by about 70% compared to a uniplanar cage design. This new cage can be used with any skeletal joints, in either static or kinematic examination, which is helpful for the standardization of the RSA application.     

Structure and folding of disulfide-rich miniproteins: insights from molecular dynamics simulations and MM-PBSA free energy calculations

The fold of small disulfide-rich proteins largely relies on two or more disulfide bridges that are main components of the hydrophobic core. Because of the small size of these proteins and their high cystine content, the cysteine connectivity has been difficult to ascertain in some cases, leading to uncertainties and debates in the literature. Here, we use molecular dynamics simulations and MM-PBSA free energy calculations to compare similar folds with different disulfide pairings in two disulfide-rich miniprotein families, namely the knottins and the short-chain scorpion toxins, for which the connectivity has been discussed. We first show that the MM-PBSA approach is able to discriminate the correct knotted topology of knottins from the laddered one. Interestingly, a comparison of the free energy components for kalata B1 and MCoTI-II suggests that cyclotides and squash inhibitors, although sharing the same scaffold, are stabilized through different interactions. Application to short-chain scorpion toxins suggests that the conventional cysteine pairing found in many homologous toxins is significantly more stable than the unconventional pairing reported for maurotoxin and for spinoxin. This would mean that native maurotoxin and spinoxin are not at the lowest free energy minimum and might result from kinetically rather than thermodynamically driven oxidative folding processes. For both knottins and toxins, the correct or conventional disulfide connectivities provide lower flexibilities and smaller deviations from the initial conformations. Overall, our work suggests that molecular dynamics simulations and the MM-PBSA approach to estimate free energies are useful tools to analyze and compare disulfide bridge connectivities in miniproteins.     

Adiposity measures and oxidative stress among police officers

Our objective was to investigate associations between adiposity measures (BMI, waist circumference, waist-to-hip ratio, waist-to-height ratio, and abdominal height) and biomarkers of oxidative stress (glutathione (GSH), GSH peroxidase (GSH-Px), vitamin C, thiobarbituric acid reactive substances (TBARS), and trolox equivalent antioxidant capacity (TEAC)) among police officers. This cross-sectional study included randomly selected police officers (43 policewomen; 67 policemen) from Buffalo, New York. Adiposity measures were performed using standardized methods. Biomarkers were measured on fasting blood specimens. An oxidative stress score (OSS) was created as a composite of the biomarkers. ANOVAs were used to compare mean levels of biomarkers across tertiles of the adiposity measures. Officers were 26- to 61-years old. GSH was inversely associated with waist circumference (trend P = 0.030) and waist-to-hip ratio (trend P = 0.026). GSH-Px was inversely associated with BMI (trend P = 0.004) and with waist-to-height ratio (trend P = 0.017). No associations were observed for TEAC, TBARS, or OSS with any adiposity measure. Significant interactions were observed by physical activity status for GSH with waist circumference and waist-to-hip ratio and for vitamin C with waist circumference, waist-to-hip and waist-to-height ratios. The above associations were inversely related only among officers who reported engaging in physical activity. Inverse associations were observed for BMI and waist circumference with GSH, but only among women; the interaction with gender was significant. Larger indices of adiposity were associated with increased levels of oxidative stress and decreased levels of antioxidant defense.     

The dental calculus metabolome in modern and historic samples

Introduction

Dental calculus is a mineralized microbial dental plaque biofilm that forms throughout life by precipitation of salivary calcium salts. Successive cycles of dental plaque growth and calcification make it an unusually well-preserved, long-term record of host-microbial interaction in the archaeological record. Recent studies have confirmed the survival of authentic ancient DNA and proteins within historic and prehistoric dental calculus, making it a promising substrate for investigating oral microbiome evolution via direct measurement and comparison of modern and ancient specimens.

Objective

We present the first comprehensive characterization of the human dental calculus metabolome using a multi-platform approach.

Methods

Ultra performance liquid chromatography-tandem mass spectrometry (UPLC–MS/MS) quantified 285 metabolites in modern and historic (200 years old) dental calculus, including metabolites of drug and dietary origin. A subset of historic samples was additionally analyzed by high-resolution gas chromatography–MS (GC–MS) and UPLC–MS/MS for further characterization of metabolites and lipids. Metabolite profiles of modern and historic calculus were compared to identify patterns of persistence and loss.

Results

Dipeptides, free amino acids, free nucleotides, and carbohydrates substantially decrease in abundance and ubiquity in archaeological samples, with some exceptions. Lipids generally persist, and saturated and mono-unsaturated medium and long chain fatty acids appear to be well-preserved, while metabolic derivatives related to oxidation and chemical degradation are found at higher levels in archaeological dental calculus than fresh samples.

Conclusions

The results of this study indicate that certain metabolite classes have higher potential for recovery over long time scales and may serve as appropriate targets for oral microbiome evolutionary studies.