Variants in ALDH1A2 reveal an anti-inflammatory role for retinoic acid and a new class of disease-modifying drugs in osteoarthritis

More than 40% of individuals will develop osteoarthritis (OA) during their lifetime, yet there are currently no licensed disease-modifying treatments for this disabling condition. Common polymorphic variants in ALDH1A2, which encodes the key enzyme for synthesis of all-trans retinoic acid (atRA), are associated with severe hand OA. Here, we sought to elucidate the biological significance of this association. We first confirmed that ALDH1A2 risk variants were associated with hand OA in the U.K. Biobank. Articular cartilage was acquired from 33 individuals with hand OA at the time of routine hand OA surgery. After stratification by genotype, RNA sequencing was performed. A reciprocal relationship between ALDH1A2 mRNA and inflammatory genes was observed. Articular cartilage injury up-regulated similar inflammatory genes by a process that we have previously termed mechanoflammation, which we believe is a primary driver of OA. Cartilage injury was also associated with a concomitant drop in atRA-inducible genes, which were used as a surrogate measure of cellular atRA concentration. Both responses to injury were reversed using talarozole, a retinoic acid metabolism blocking agent (RAMBA). Suppression of mechanoflammation by talarozole was mediated by a peroxisome proliferator–activated receptor gamma (PPARγ)–dependent mechanism. Talarozole was able to suppress mechano-inflammatory genes in articular cartilage in vivo 6 hours after mouse knee joint destabilization and reduced cartilage degradation and osteophyte formation after 26 days. These data show that boosting atRA suppresses mechanoflammation in the articular cartilage in vitro and in vivo and identifies RAMBAs as potential disease-modifying drugs for OA

Dietary Heterogeneity among Western Industrialized Countries Reflected in the Stable Isotope Ratios of Human Hair

Although the globalization of food production is often assumed to result in a homogenization of consumption patterns with a convergence towards a Western style diet, the resources used to make global food products may still be locally produced (glocalization). Stable isotope ratios of human hair can quantify the extent to which residents of industrialized nations have converged on a standardized diet or whether there is persistent heterogeneity and glocalization among countries as a result of different dietary patterns and the use of local food products. Here we report isotopic differences among carbon, nitrogen and sulfur isotope ratios of human hair collected in thirteen Western European countries and in the USA. European hair samples had significantly lower δ13C values (−22.7 to −18.3‰), and significantly higher δ15N (7.8 to 10.3‰) and δ34S (4.8 to 8.3‰) values than samples from the USA (δ13C: −21.9 to −15.0‰, δ15N: 6.7 to 9.9‰, δ34S: −1.2 to 9.9‰). Within Europe, we detected differences in hair δ13C and δ34S values among countries and covariation of isotope ratios with latitude and longitude. This geographic structuring of isotopic data suggests heterogeneity in the food resources used by citizens of industrialized nations and supports the presence of different dietary patterns within Western Europe despite globalization trends. Here we showed the potential of stable isotope analysis as a population-wide tool for dietary screening, particularly as a complement of dietary surveys, that can provide additional information on assimilated macronutrients and independent verification of data obtained by those self-reporting instruments

Tracking Cats: Problems with Placing Feline Carnivores on δ18O, δD Isoscapes

Several felids are endangered and threatened by the illegal wildlife trade. Establishing geographic origin of tissues of endangered species is thus crucial for wildlife crime investigations and effective conservation strategies. As shown in other species, stable isotope analysis of hydrogen and oxygen in hair (δD(h), δ(18)O(h)) can be used as a tool for provenance determination. However, reliably predicting the spatial distribution of δD(h) and δ(18)O(h) requires confirmation from animal tissues of known origin and a detailed understanding of the isotopic routing of dietary nutrients into felid hair.We used coupled δD(h) and δ(18)O(h) measurements from the North American bobcat (Lynx rufus) and puma (Puma concolor) with precipitation-based assignment isoscapes to test the feasibility of isotopic geo-location of felidae. Hairs of felid and rabbit museum specimens from 75 sites across the United States and Canada were analyzed. Bobcat and puma lacked a significant correlation between H/O isotopes in hair and local waters, and also exhibited an isotopic decoupling of δ(18)O(h) and δD(h). Conversely, strong δD and δ(18)O coupling was found for key prey, eastern cottontail rabbit (Sylvilagus floridanus; hair) and white-tailed deer (Odocoileus virginianus; collagen, bone phosphate).Puma and bobcat hairs do not adhere to expected pattern of H and O isotopic variation predicted by precipitation isoscapes for North America. Thus, using bulk hair, felids cannot be placed on δ(18)O and δD isoscapes for use in forensic investigations. The effective application of isotopes to trace the provenance of feline carnivores is likely compromised by major controls of their diet, physiology and metabolism on hair δ(18)O and δD related to body water budgets. Controlled feeding experiments, combined with single amino acid isotope analysis of diets and hair, are needed to reveal mechanisms and physiological traits explaining why felid hair does not follow isotopic patterns demonstrated in many other taxa

Preparative HPLC separation of underivatized amino acids for isotopic analysis

Single-compound analysis of stable or radioactive isotopes has found application in a number of fields ranging from archaeology to forensics. Often, the most difficult part of these analyses is the development of a method for isolating the compound(s) of interest, which can derive from a wide range of sample types including the hair, nails, and bone. Here we describe three complementary preparative HPLC techniques suitable for separating and isolating amino acids from bone collagen and hair keratin. Using preparative reversed-phase, ion-pair, or mixed-mode chromatography in aqueous carbon-free mobile phases, or those from which carbon can easily be removed, underivatized single amino acids can be isolated and further analyzed using mass spectrometric techniques.</p

Preparative HPLC separation of underivatized amino acids for isotopic analysis

Single-compound analysis of stable or radioactive isotopes has found application in a number of fields ranging from archaeology to forensics. Often, the most difficult part of these analyses is the development of a method for isolating the compound(s) of interest, which can derive from a wide range of sample types including the hair, nails, and bone. Here we describe three complementary preparative HPLC techniques suitable for separating and isolating amino acids from bone collagen and hair keratin. Using preparative reversed-phase, ion-pair, or mixed-mode chromatography in aqueous carbon-free mobile phases, or those from which carbon can easily be removed, underivatized single amino acids can be isolated and further analyzed using mass spectrometric techniques

Bioassay-guided isolation of cytotoxic flavonoids from aerial parts of Coronopus didymus

Ethnopharmacological relevance: Coronopus didymus Linn. (Brassicaceae) is a medicinal plant used traditionally as antipyretic, expectorant, to purify blood and for alleviating symptoms of pain, inflammations, malaria, wounds and cancer. Aim of the study: The present study was designed to isolate and identify the cytotoxic compounds responsible for anticancer activity from this traditionally useful medicinal plant. Materials and methods: Bioassay-guided fractionation of the ethanolic extract of aerial parts of C. didymus allowed the isolation of compounds responsible for anticancer activity. Their structures were elucidated by UV Spectroscopy (with shift reagents), ESI-MS and NMR spectral data. Preliminary anticancer activity of ethanolic extract, different fractions and isolated compounds was assessed through MTT in vitro cytotoxicity assay in a dose dependent manner against human cancer cell lines (HeLa and LN18) and normal 293T cells. Results: Three flavonoids namely 5,7,4′-trihydroxy-3′-methoxyflavone-4′-O-β-D-glucoside (1), 5,7,4′-trihydroxy-3′-methoxyflavone-4′-O-(6''-acetyl)-β-D-glucoside (2) and 5,7,4′-trihydroxy-3′-methoxy flavone (3), were isolated from aerial parts. Compound 1 was identified for the first time from the genus Coronopus. All the compounds 1–3 showed promising activity against HeLa cells with IC50 values of 43.50, 0.63 and 3.67 µM , respectively. Significant result was also obtained with compound 3 against LN18 cells with IC50 value of 46.63 µM . Conclusion: The cytotoxic activity of the crude extract and fractions which may largely be due to its major isolated constituents, flavonoids 1–3, against both HeLa and LN18 cells provides a scientific basis for the ethnopharmacological use of C. didymus as anticancer agent

Bioassay-guided isolation of cytotoxic flavonoids from aerial parts of Coronopus didymus

Ethnopharmacological relevance: Coronopus didymus Linn. (Brassicaceae) is a medicinal plant used traditionally as antipyretic, expectorant, to purify blood and for alleviating symptoms of pain, inflammations, malaria, wounds and cancer. Aim of the study: The present study was designed to isolate and identify the cytotoxic compounds responsible for anticancer activity from this traditionally useful medicinal plant. Materials and methods: Bioassay-guided fractionation of the ethanolic extract of aerial parts of C. didymus allowed the isolation of compounds responsible for anticancer activity. Their structures were elucidated by UV Spectroscopy (with shift reagents), ESI-MS and NMR spectral data. Preliminary anticancer activity of ethanolic extract, different fractions and isolated compounds was assessed through MTT in vitro cytotoxicity assay in a dose dependent manner against human cancer cell lines (HeLa and LN18) and normal 293T cells. Results: Three flavonoids namely 5,7,4′-trihydroxy-3′-methoxyflavone-4′-O-β-D-glucoside (1), 5,7,4′-trihydroxy-3′-methoxyflavone-4′-O-(6''-acetyl)-β-D-glucoside (2) and 5,7,4′-trihydroxy-3′-methoxy flavone (3), were isolated from aerial parts. Compound 1 was identified for the first time from the genus Coronopus. All the compounds 1–3 showed promising activity against HeLa cells with IC50 values of 43.50, 0.63 and 3.67 µM , respectively. Significant result was also obtained with compound 3 against LN18 cells with IC50 value of 46.63 µM . Conclusion: The cytotoxic activity of the crude extract and fractions which may largely be due to its major isolated constituents, flavonoids 1–3, against both HeLa and LN18 cells provides a scientific basis for the ethnopharmacological use of C. didymus as anticancer agent

Polyphenols: bioavailability, microbiome interactions and cellular effects on health in humans and animals

Polyphenolic compounds have a variety of functions in plants including protecting them from a range of abiotic and biotic stresses such as pathogenic infections, ionising radiation and as signalling molecules. They are common constituents of human and animal diets, undergoing extensive metabolism by gut microbiota in many cases prior to entering circulation. They are linked to a range of positive health effects, including anti-oxidant, anti-inflammatory, antibiotic and disease-specific activities but the relationships between polyphenol bio-transformation products and their interactions in vivo are less well understood. Here we review the state of knowledge in this area, specifically what happens to dietary polyphenols after ingestion and how this is linked to health effects in humans and animals; paying particular attention to farm animals and pigs. We focus on the chemical transformation of polyphenols after ingestion, through microbial transformation, conjugation, absorption, entry into circulation and uptake by cells and tissues, focusing on recent findings in relation to bone. We review what is known about how these processes affect polyphenol bioactivity, highlighting gaps in knowledge. The implications of extending the use of polyphenols to treat specific pathogenic infections and other illnesses is explored

RADIOCARBON DATING OF INDIVIDUAL AMINO ACIDS FROM ARCHAEOLOGICAL BONE COLLAGEN

Since the development of accelerator mass spectrometry (AMS) for radiocarbon dating in the late 1970s, its ability to date small samples of bone has been of huge importance in archaeology and Quaternary paleoecology. The conventional approach to sample preparation has been to extract and gelatinize protein, which is then combusted and graphitized for analysis. However, this "bulk protein" can contain a heterogeneous mixture of non-collagenous molecules, including humic acids and other soil components that may be of a different age than the bone and therefore affect the accuracy of its 14C date. Sample pretreatment methods have been an important area of development in recent years but still show inadequacies for the dating of severely contaminated bone. The idea of isolating and dating individual compounds such as single amino acids, to improve dating accuracy, has been discussed in the literature since the 1960s. Hydroxyproline, for example, makes up over 10% of bone collagen but is extremely rare in most other animal proteins, increasing the chances of its presence being endogenous to the individual being dated. Its successful isolation has therefore been considered a potential "gold standard" for dating archaeological bone; however, extracting and suitably purifying single amino acids from bone has proved a challenging task. This paper presents a novel method for the compound-specific 14C dating of individual amino acids, including hydroxyproline, from archaeological bone protein. It is based on a preparative, mixed-mode liquid chromatography separation of underivatized amino acids, entirely in aqueous solution and free of organic solvents. The method is presented here in detail including application to standard bone samples establishing its accuracy and background carbon contribution. Results from 14C dating hydroxyproline and other individual amino acids, from both historical and archaeological bone, are shown to provide AMS dates that are statistically indistinguishable from those of the bulk protein. © 2010 by the Arizona Board of Regents on behalf of the University of Arizona