Forward and reverse degradomics defines the proteolytic landscape of human knee osteoarthritic cartilage and the role of the serine protease HtrA1.

OBJECTIVES: Proteolytic destruction of articular cartilage, a major pathogenic mechanism in osteoarthritis (OA), was not previously investigated by terminomics strategies. We defined the degradome of human knee OA cartilage and the contribution therein of the protease HtrA1 using Terminal Amine Isotopic Labeling of Substrates (TAILS). DESIGN: Proteins from OA cartilage taken at knee arthroplasty (n = 6) or separately, from healthy cartilage incubated in triplicate with/without active HtrA1, were labeled at natural and proteolytically cleaved N-termini by reductive dimethylation, followed by trypsin digestion, enrichment of N-terminally labeled/blocked peptides, tandem mass spectrometry and positional peptide annotation to identify cleavage sites. Biglycan proteolysis by HtrA1 was validated biochemically and Amino-Terminal Oriented Mass Spectrometry of Substrates (ATOMS) was used to define the HtrA1 cleavage sites. RESULTS: We identified 10,155 unique internal peptides from 2,162 proteins, suggesting at least 10,797 cleavage sites in OA cartilage. 7,635 internal peptides originated in 371 extracellular matrix/secreted components, many undergoing extensive proteolysis. Rampant ragging of protein termini suggested pervasive exopeptidase activity. HtrA1, the most abundant protease in OA cartilage, experimentally generated 323 cleavages in 109 cartilage proteins, accounting for 171 observed cleavages in the OA degradome. ATOMS identified HtrA1 cleavage sites in a selected substrate, biglycan, whose direct cleavage by HtrA1 was thus orthogonally validated. CONCLUSIONS: OA cartilage demonstrates widespread proteolysis by endo- and exopeptidases. HtrA1 contributes broadly to cartilage proteolysis. Forward degradomics of OA cartilage together with reverse degradomics of proteases active in OA, e.g., HtrA1, can potentially fully annotate OA proteolytic pathways and provide new biomarkers.

High mobility group box 1 (HMGB1) protein in human uterine fluid and its relevance in implantation.

STUDY QUESTION: Does a differential abundance of high mobility group box 1 (HMGB1) protein in uterine fluid (UF) have a functional significance? SUMMARY ANSWER: In rats, an excess of HMGB1 in UF during the receptive phase is detrimental to pregnancy. WHAT IS KNOWN ALREADY: The identification of constituents of the human uterine secretome has been a subject of renewed interest, due to the advent of high throughput proteomic technologies. Proteomic-based investigations of human UF have revealed the presence of several proteins such as mucins, host defense proteins S100, heat shock protein 27 and haptoglobin, etc. The present study reports on the presence of HMGB1, a nuclear protein, in human UF. Activated macrophages/monocytes, natural killer cells, mature dendritic cells, pituicytes and erythroleukemic cells are also known to secrete HMGB1. Existing data suggest that extracellular HMGB1 plays a role in inflammation. STUDY DESIGN, SIZE, DURATION: The human part of this study was cross-sectional in design. UF and endometrial tissues were collected from regularly cycling women in the early secretory (i.e. pre-receptive phase, Day 2 post-ovulation, n = 7) or secretory phase (i.e. receptive phase, Day 6 post-ovulation, n = 7) of their menstrual cycles. Samples were also collected from cycling rats in the proestrous (n = 8) or metestrous (n = 8) phase of their estrous cycles. Uteri were also collected from HMGB1-treated pregnant (n = 7) and untreated pseudo-pregnant (n = 7) rats and from pregnant rats at Day 3-5 post-coitum (p.c.) (n = 18, 3 each for six-time points). PARTICIPANTS/MATERIALS, SETTING, METHODS: In each group of human samples, four samples were used for isobaric tag for relative and absolute quantification (iTRAQ) analysis and three samples were used for immunoblotting experiments to determine the abundance of HMGB1 in pre-receptive and receptive phase UF samples. HMGB1 levels in rat UF and endometrial tissue samples were estimated by ELISA and immunohistochemical studies, respectively. The expression of inflammation-associated molecules, such as nuclear factor kappa B (NFκB), receptor for advanced glycation end products (RAGEs), tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6), was analyzed by immunohistochemistry in HMGB1-treated and pseudo-pregnant rats. MAIN RESULTS AND THE ROLE OF CHANCE: HMGB1 was identified as one of the differentially abundant proteins in the list generated by 8-plex iTRAQ analysis of receptive and pre-receptive phase UF samples. In both humans and rats, secreted and cellular levels of HMGB1 showed a similar pattern, i.e. significantly (P < 0.05) lower abundance in the receptive phase compared with that in the pre-receptive phase. A significant (P < 0.05) decline was also observed in the endometrial expression of HMGB1 on the day of implantation in pregnant rats. Exogenous administration of recombinant HMGB1, on Day 3 p.c., led to pregnancy failure, whereas administration of recombinant leukemia inhibitory factor or saline had no effect on pregnant rats. Further investigations revealed morphological changes in the endometrium, an increase in the expression of luminal epithelial NFκB and significantly (P < 0.05) higher expression levels of endometrial RAGE, TNF-α and IL-6 in HMGB1-treated rats, compared with untreated pseudo-pregnant rats. LIMITATIONS, REASONS FOR CAUTION: The mechanisms, contributing to a decline in the cellular and extracellular levels of HMGB1 during the receptive phase, remain to be ascertained. WIDER IMPLICATIONS OF THE FINDINGS: An excess of HMGB1 in the UF may be associated with infertility in women.

Intrauterine cocaine and crack exposure: neonatal outcome.

OBJECTIVE: Adverse health effects are associated with intrauterine cocaine exposure. The purpose of this study was to investigate the impact of fetal cocaine and crack exposure on neonatal outcome. STUDY DESIGN: We enrolled 386 mother-infant pairs, including 130 matched control pairs, in the study. The course of pregnancy and delivery was followed and neonatal outcome was assessed by physical and neurologic examination, as well as by the Brazelton Neonatal Behavioral Assessment Scale and the Neonatal Stress Scale. RESULTS: The cocaine-exposed neonates had significantly more adverse effects than the matched control infants. Birth weight, length, and head circumference were significantly lower in the cocaine- and crack-exposed infants (p < or = 0.001). There were significantly more premature infants (p < or = 0.007) in this group. They demonstrated significant abnormalities on the neurologic examination (p < or = 0.001), inferior performance on the Brazelton Neonatal Behavioral Assessment Scale (p < or = 0.001), and higher scores on the Neonatal Stress Scale (p < or = 0.001). Predictors of negative neonatal outcome were maternal age (p < or = 0.02), poor paternal relationship with the mother (p < or = 0.002), crack use (p < or = 0.004), cocaine use (p < or = 0.009), and marijuana use (p < or = 0.05). CONCLUSION: The single most important predictor of neonatal outcome is the frequency, quantity, and type of cocaine used.