Moving Towards Common Data Elements and Core Outcome Measures in Frailty Research.

With aging populations around the world, frailty is becoming more prevalent increasing the need for health systems and social systems to deliver optimal evidence based care. However, in spite of the growing number of frailty publications, high-quality evidence for decision making is often lacking. Inadequate descriptions of the populations enrolled including frailty severity and frailty conceptualization, lack of use of validated frailty assessment tools, utilization of different frailty instruments between studies, and variation in reported outcomes impairs the ability to interpret, generalize and implement the research findings. The utilization of common data elements (CDEs) and core outcome measures (COMs) in clinical trials is increasingly being adopted to address such concerns. To catalyze the development and use of CDEs and COMs for future frailty studies, the Canadian Frailty Network (www.cfn-nce.ca; CFN), a not-for-profit pan-Canadian nationally-funded research network, convened an international group of experts to examine the issue and plan the path forward. The meeting was structured to allow for an examination of current frailty evidence, ability to learn from other COMs and CDEs initiatives, discussions about specific considerations for frailty COMs and CDEs and finally the identification of the necessary steps for a COMs and CDEs consensus initiative going forward. It was agreed at the onset of the meeting that a statement based on the meeting would be published and herein we report the statement.

Proceedings of the Canadian Frailty Network Workshop: Identifying Biomarkers of Frailty to Support Frailty Risk Assessment, Diagnosis and Prognosis. Toronto, January 15, 2018.

The Canadian Frailty Network (CFN), a pan-Canadian not-for-profit organization funded by the Government of Canada through the Networks of Centres of Excellence Program, is dedicated to improving the care of older Canadians living with frailty. The CFN has partnered with the Canadian Longitudinal Study on Aging (CLSA) to measure potential frailty biomarkers in biological samples (whole blood, plasma, urine) collected in over 30,000 CLSA participants. CFN hosted a workshop in Toronto on January 15 2018, bringing together experts in the field of biomarkers, aging and frailty. The overall objectives of the workshop were to start building a consensus on potential frailty biomarker domains and identify specific frailty biomarkers to be measured in the CLSA biological samples. The workshop was structured with presentations in the morning to frame the discussions for the afternoon session, which was organized as a free-flowing discussion to benefit from the expertise of the participants. Participants and speakers were from Canada, Italy, Spain, United Kingdom and the United States. Herein we provide pertinent background information, a summary of all the presentations with key figures and tables, and the distillation of the discussions. In addition, moving forward, the principles CFN will use to approach frailty biomarker research and development are outlined. Findings from the workshop are helping CFN and CLSA plan and conduct the analysis of biomarkers in the CLSA samples and which will inform a follow-up data access competition.

Dry stacking of wastewater treatment sludges.

Drying pans are used during wastewater treatment (WWT) to store, stabilise and dry residual solids. The pans are filled with sludge that dries via exposure to sunshine and wind. We propose that drying pans be operated based on dry stacking principles, a technique with proven success in the mineral processing industry. The implementation of the dry stacking technique requires very little in the way of additional engineering beyond a conventional drying pan. By applying the sludge in thin layers, the sludge naturally forms its own stack with an angle that is dependent on the consistency of the material. The benefits of dry stacking are that the slope allows instantaneous run-off of rainfall and supernatant, allowing operation throughout the year rather than seasonally. The layering approach also maximises the evaporation achieved in the available deposition area compared to filling the pans sequentially. A series of laboratory tests were carried out on samples from Melbourne Water's Western Treatment Plant in Werribee, Australia, to provide validation of the dry stacking concept for WWT sludges. Rheological tests showed that samples had appropriate flow properties to form stacks. Drying and re-wetting tests on the samples indicated that a sloped, partially dry sludge sheds rainfall, depending on the slope, cake dryness and amount of rainfall. Local rainfall data was used to estimate a potential increase in pan throughput of 65%-140% due to dry stacking. The greatest improvements were predicted to occur during wetter years. In combination, the results indicated that dry stacking has the potential to dramatically improve the performance of WWT sludge drying pans.

Winery wastewater inhibits seed germination and vegetative growth of common crop species.

The ability to reuse winery wastewater would be of significant benefit to the wine industry, as it could potentially be a cost-effective method of wastewater management, whilst at the same time providing a valuable water resource. This study investigated the effects of different dilutions of a semi-synthetic winery wastewater on the growth and germination of four common crop species in a glasshouse study; barley (Hordeum vulgare), millet (Pennisetum glaucum), lucerne (Medicago sativa) and phalaris (Phalaris aquatica). The wastewater caused a significant delay in the germination of lucerne, millet and phalaris, although overall germination percentage of all species was not affected. Vegetative growth was significantly reduced in all species, with millet being the most severely affected. The germination index of barley correlated very highly (r(2)=0.99) with barley biomass, indicating that barley seed germination bioassays are highly relevant to plant growth, and therefore may be of use as a bioassay for winery wastewater toxicity.

Overexpression of human RAD51 and RAD52 reduces double-strand break-induced homologous recombination in mammalian cells.

Double-strand breaks (DSBs) can be repaired by homologous recombination (HR) in mammalian cells, often resulting in gene conversion. RAD51 functions with RAD52 and other proteins to effect strand exchange during HR, forming heteroduplex DNA (hDNA) that is resolved by mismatch repair to yield a gene conversion tract. In mammalian cells RAD51 and RAD52 overexpression increase the frequency of spontaneous HR, and one study indicated that overexpression of mouse RAD51 enhances DSB-induced HR in Chinese hamster ovary (CHO) cells. We tested the effects of transient and stable overexpression of human RAD51 and/or human RAD52 on DSB-induced HR in CHO cells and in human cells. DSBs were targeted to chromosomal recombination substrates with I-SceI nuclease. In all cases, excess RAD51 and/or RAD52 reduced DSB-induced HR, contrasting with prior studies. These distinct results may reflect differences in recombination substrate structures or different levels of overexpression. Excess RAD51/RAD52 did not increase conversion tract lengths, nor were product spectra otherwise altered, indicating that excess HR proteins can have dominant negative effects on HR initiation, but do not affect later steps such as hDNA formation, mismatch repair or the resolution of intermediates.

Clinical features of Ota's naevus in Koreans and its treatment with Q-switched alexandrite laser.

Ota's naevus is a fairly common pigmentary disorder in Asians. Recently, encouraging results in the treatment of Ota's naevus have been obtained, but most of these concerned the white skins of Caucasian patients. Our purpose was to examine the clinical features of Ota's naevus in Koreans and to assess the clinical outcomes and histological changes induced by a Q-switched alexandrite laser at 755 nm. Eighty-seven Koreans with Ota's naevus were studied; the peak age of onset was during the first decade and adolescence. The infraorbital area was the most frequent site and black or dark brown colours predominated. Improvements were achieved in 52 patients (77%). Better results were obtained in unilateral lesions and patients who received a greater number of treatments. Mild hyperpigmentation after treatment was noticed in 14 patients and mild hypopigmentation in eight patients. However, all of these were reversed in time. Hypertrophic scarring or secondary infection did not occur. The histology of laser-irradiated lesions showed selective thermal damage of melanocytes in the upper dermis and the elimination of upper dermal pigmentation. Our clinical data demonstrate the usefulness of the Q-switched alexandrite laser for the treatment of Ota's naevus in brown skin.

D-amino acids as putative neurotransmitters: focus on D-serine.

Of the twenty amino acids in the mammalian body, only serine and aspartate occur in D-configuration as well as L-configuration in significant amount. D-serine is selectively concentrated in the brain, localized to protoplasmic astrocytes that ensheath synapses and distributed similarly to N-methyl-D-aspartate (NMDA) subtype of glutamate receptors. D-serine has been found to function as an endogenous ligand for the "glycine" site of the NMDA receptor. Evidences for this include the greater potency of D-serine to activate this site than glycine, and D-amino acid oxidase, which degrades D-serine as well as other neutral D-amino acids, markedly attenuates NMDA neurotransmission. D-serine is also formed by serine racemase, a recently cloned enzyme that converts L-serine to D-serine. Thus, in many ways D-serine fulfills criteria for defining its functionality as a neurotransmitter and challenges the dogma relating to neurotransmission, for it is the "unnatural" isomeric form of an amino acid derived from glia rather than neurons.

Investigation of the tobacco-specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone for in vivo and iIn vitro murine embryopathy and embryonic ras mutations.

The teratological potential of the carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is unknown. In vivo, NNK (100 mg/kg i.p.) was administered to pregnant CD-1 mice during organogenesis, with or without pretreatment with the P450 inducer phenobarbital (60 mg/kg i.p.). With NNK alone, 3 of 374 fetuses had open eye and one had a cleft palate, which were not observed in 160 controls. With phenobarbital plus NNK, two fetuses had a cleft palate, two had exencephaly and one had a kinky tail, although phenobarbital controls showed no anomalies (P <.05). NNK-initiated fetal postpartum lethality was enhanced by phenobarbital pretreatment. There were no fetal skeletal anomalies or alterations in resorptions or fetal body weight in any group. In embryo culture, gestational day 9.5 embryos exposed to 10 microM NNK had decreases in yolk sac diameter, crown-rump length and somite development (P <.05), and 100 microM NNK decreased anterior neuropore closure and crown-rump length (P <.05). Embryos exposed to 100 microM NNK were assessed for K-ras codon 12 mutations and none were detected. This is the first evidence for NNK teratogenicity and embryotoxicity, the molecular mechanism of which appears to differ from that for its carcinogenicity.

Peroxidase-dependent bioactivation and oxidation of DNA and protein in benzo[a]pyrene-initiated micronucleus formation.

Micronucleus formation initiated by benzo[a]pyrene (B[a]P) and related xenobiotics is widely believed to reflect potential carcinogenic initiation, yet neither a dependence upon bioactivation nor the critical enzymes have been demonstrated. Using rat skin fibroblasts, protein oxidation (carbonyl formation) and content of prostaglandin H synthase (PHS) and cytochrome P4501A1 (CYP1A1) protein were determined by Western blot/immunodetection with enhanced chemiluminescence. DNA oxidation as 8-hydroxy-2'-deoxyguanosine formation was quantified using high-performance liquid chromatography with electrochemical detection. Fibroblast CYP1A1 activity assessed as ethoxyresorufin-O-deethylase was not detectable, and even CYP1A1 protein was measurable only after induction with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). However, TCDD additionally induced prostaglandin H synthase (PHS), which also was detectable constitutively. B[a]P 10 microM initiated the oxidation of DNA and protein, and the formation of micronuclei, all of which were enhanced over 2-fold by the dual CYP1A1/PHS inducer TCDD 10 nM, as well as by other PHS inducers, 12-O-tetradecanoylphorbol-13-acetate 1 microM and interleukin-1alpha 0.625 or 1.25 ng/ml, that do not induce CYP1A1 (p < .05). Conversely, B[a]P target oxidation and micronucleus formation were abolished by 1-aminobenzotriazole 1 mM (p < .05), which was a potent inhibitor of both peroxidases and P450. These results provide the first direct evidence that B[a]P-initiated micronucleus formation, like carcinogenic initiation, requires enzymatic bioactivation, and that peroxidase-dependent, reactive oxygen species-mediated oxidation of DNA, and possibly protein, constitutes a molecular mechanism of initiation in uninduced cells. Induction of either CYP1A1 or peroxidases such as PHS substantially enhances this genotoxic initiation, which may reflect cancer risk.

UDP-glucuronosyltransferase-mediated protection against in vitro DNA oxidation and micronucleus formation initiated by phenytoin and its embryotoxic metabolite 5-(p-hydroxyphenyl)-5-phenylhydantoin.

UDP-Glucuronosyltransferases (UGTs) are important in the elimination of most xenobiotics, including 5-(p-hydroxyphenyl)-5-phenylhydantoin (HPPH), the major, reputedly nontoxic, metabolite of the anticonvulsant drug phenytoin. However, HPPH alternatively may be bioactivated by peroxidases, such as prostaglandin H synthase, to a reactive intermediate that initiates DNA oxidation (reflected by 8-hydroxy-2'-deoxyguanosine), genotoxicity (reflected by micronuclei) and embryopathy. This hypothesis was evaluated in skin fibroblasts cultured from heterozygous (+/j) and homozygous (j/j) UGT-deficient Gunn rats and in mouse embryo culture, with confirmation of direct NG-glucuronidation of phenytoin in Gunn rats in vivo. HPPH (80 microM) increased micronuclei by 2.0-, 4.8- and 4.6-fold in +/+ UGT-normal cells (P = .03) and +/j and j/j UGT-deficient cells (P = .0001), respectively. HPPH-initiated micronucleus formation was increased 3.0- and 3.4-fold in +/j (P = .02) and j/j (P = .04) UGT-deficient cells, respectively, vs. +/+ UGT-normal cells. Micronuclei were not initiated by 10 microM HPPH in +/+ UGT-normal cells but were increased by 4- and 3.8-fold in +/j and j/j UGT-deficient cells (P = .0001), respectively, and were increased 2.7- and 3.0-fold in +/j (P = .007) and j/j (P = .0002) UGT-deficient cells, respectively, vs. +/+ UGT-normal cells. 8-Hydroxy-2'-deoxyguanosine was increased in j/j UGT-deficient but not +/+ UGT-normal cells treated with 80 microM HPPH (P < .05). The embryopathic potency of 80 microM HPPH in embryo culture, reflected by decreases in anterior neuropore closure, turning, yolk sac diameter and crown-rump length (P < .05), was equivalent to that reported for phenytoin. Phenytoin (80 microM) enhanced micronucleus formation 1.7-, 4.4- and 3.8-fold in +/+ cells (P = .03) and +/j and j/j UGT-deficient cells (P = .0001), respectively. Phenytoin-initiated micronucleus formation was increased about 4-fold in both +/j (P = .006) and j/j (P = .009) UGT-deficient cells vs. +/+ UGT-normal cells, providing the first evidence that the bioactivation and oxidative toxicity of phenytoin itself may be avoided by direct N-glucuronidation, which was confirmed by tandem mass spectrometry. These results further indicate that, with UGT deficiencies, HPPH potentially is a potent mediator of phenytoin-initiated genotoxicity and embryopathy, which may be relevant to teratogenesis and other adverse effects of phenytoin.

Oxidative damage in chemical teratogenesis.

The teratogenicity of many xenobiotics is thought to depend at least in part upon their bioactivation by embryonic cytochromes P450, prostaglandin H synthase (PHS) and lipoxygenases (LPOs) to electrophilic and/or free radical reactive intermediates that covalently bind to or oxidize cellular macromolecules such as DNA, protein and lipid, resulting in in utero death or teratogenesis. Using as models the tobacco carcinogens benzo[a]pyrene (B[a]P) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), the anticonvulsant drug phenytoin, structurally related anticonvulsants (e.g. mephenytoin, nirvanol, trimethadione, dimethadione) and the sedative drug thalidomide, we have examined the potential teratologic relevance of free radical-initiated, reactive oxygen species (ROS)-mediated oxidative molecular target damage, genotoxicity (micronucleus formation) and DNA repair in mouse and rabbit models in vivo and in embryo culture, and in vitro using purified enzymes or cultured rat skin fibroblasts. These teratogens were bioactivated by PHS and LPOs to free radical reactive intermediary metabolites, characterized by electron spin resonance spectrometry, that initiated ROS formation, including hydroxyl radicals, which were characterized by salicylate hydroxylation. ROS-initiated oxidation of DNA (8-hydroxy-2'-deoxyguanosine formation), protein (carbonyl formation), glutathione (GSH) and lipid (peroxidation), and embryotoxicity were shown for phenytoin, its major hydroxylated metabolite 5-(p-hydroxyphenyl)-5-phenylhydantoin [HPPH], thalidomide, B[a]P and NNK in vivo and/or in embryo culture, the latter indicating a teratologically critical role for embryonic, as distinct from maternal, processes. DNA oxidation and teratogenicity of phenytoin and thalidomide were reduced by PHS inhibitors. Oxidative macromolecular lesions and teratogenicity also were reduced by the free radical trapping agent phenylbutylnitrone (PBN), and the antioxidants caffeic acid and vitamin E. In embryo culture, addition of superoxide dismutase (SOD) to the medium enhanced embryonic SOD activity, and SOD or catalase blocked the oxidative lesions and embryotoxicity initiated by phenytoin and B[a]P, suggesting a major contribution of ROS, as distinct from covalent binding, to the teratologic mechanism. In in vivo studies, other antioxidative enzymes like GSH peroxidase, GSH reductase and glucose-6-phosphate dehydrogenase (G6PD) were similarly protective. Even untreated G6PD-deficient mice had enhanced embryopathies, indicating a teratological role for endogenous oxidative stress. In cultured fibroblasts, B[a]P, NNK, phenytoin and HPPH initiated DNA oxidation and micronucleus formation, which were inhibited by SOD. Oxidation of DNA may be particularly critical, since transgenic mice with +/- or -/- deficiencies in the p53 tumor suppressor gene, which facilitates DNA repair, are more susceptible to phenytoin and B[a]P teratogenicity. Even p53-deficient mice treated only with normal saline showed enhanced embryopathies, suggesting the teratological importance of endogenous oxidative stress, as observed with G6PD deficiency. These results suggest that oxidative macromolecular damage may play a role in the teratologic mechanism of xenobiotics that are bioactivated to a reactive intermediate, as well in the mechanism of embryopathies occurring in the absence of xenobiotic exposure.

Genoprotection by UDP-glucuronosyltransferases in peroxidase-dependent, reactive oxygen species-mediated micronucleus initiation by the carcinogens 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone and benzo[a]pyrene.

UDP-glucuronosyltransferases (UGTs) catalyze the glucuronidation and elimination of putative tobacco carcinogens such as benzo[a]pyrene (B[a]P) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), which may reduce competing bioactivation and toxicity. B[a]P-initiated cytotoxicity and micronucleus formation, believed to reflect carcinogenic initiation, are enhanced in UGT-deficient rat fibroblasts, and UGTs may provide similar genoprotection against NNK. Using skin fibroblasts from wild-type UGT-normal (+/+) and congenic heterozygous (+/j) and homozygous (j/j) UGT-deficient rats, this study evaluated NNK in relation to B[a]P with respect to the mechanism of genotoxicity, evidenced by micronucleus formation, and genoprotection++ by UGTs. Molecular mechanisms were determined by changes in B[a]P- and NNK-initiated micronucleus formation when cells were incubated with the antioxidative enzyme superoxide dismutase (1680 IU/ml), inhibitors of cytochrome P450 (1 mM 1-aminobenzotriazole) and peroxidases (1-aminobenzotriazole; 40 microM eicosatetraynoic acid), and inducers of CYP1A1/2(10 nM 2,3,7,8-tetrachlorodibenzo-p-dioxin) and peroxidases [2,3,7,8-tetrachlorodibezo-p-dioxin; 0.625 ng/ml (0.0367 nM) interleukin 1alpha; 1 microM 12-0-tetradecanoylphorbol-13-acetate]. In +/+ fibroblasts, NNK and B[a]P initiated concentration-dependent, respective maximum 2.7-fold and 1.7-fold increases over DMSO controls in micronucleus formation (P < 0.05), with 10 microM NNK being 2.4-fold more genotoxic than B[a]P (P < 0.05). In both +/j and j/j UGT-deficient cells, micronuclei initiated by NNK and B[a]P each were over 2-fold higher than that in +/+ UGT normal cells (P < 0.05). Both NNK- and B[a]P-initiated micronuclei were decreased by superoxide dismutase and cytochrome P450/peroxidase inhibitors, while only that initiated by B[a]P was enhanced, up to 2.4-fold, by inducers, of which only interleukin 1alpha was effective in all UGT phenotypes (P < 0.05). These results provide the first evidence that: (a) UGTs may be genoprotective for NNK, with even heterozygous UGT deficiencies being toxicologically critical; and (b) peroxidase-catalyzed bioactivation, reactive oxygen species, and molecular target oxidation may contribute differentially to the genotoxicity of both NNK and B[a]P.

Phenytoin-initiated hydroxyl radical formation: characterization by enhanced salicylate hydroxylation.

Bioactivation of phenytoin and related teratogens by peroxidases such as prostaglandin H synthase (PHS) may initiate hydroxyl radical (.OH) formation that is teratogenic. Salicylate is hydroxylated by .OH at the third and fifth carbon atoms, forming 2,3- and 2,5-dihydroxybenzoic acids (DHBA). In vivo salicylate metabolism produces only the 2,5-isomer, so 2,3-DHBA formation may reflect .OH production. In the present study, we validated the salicylate assay using the known .OH generator paraquat and evaluated .OH production by phenytoin. Female CD-1 mice were treated with paraquat (30 mg/kg, intraperitoneally) given 30 min after acetylsalicylic acid (ASA) (200 mg/kg, intraperitoneally). Blood was collected at 5, 15, and 30 min and 1 and 2 hr after paraquat, and plasma was analyzed for DHBA isomers and glucuronide conjugates by high performance liquid chromatography with electrochemical detection. Paraquat increased 2,3-DHBA formation 19.2-fold, with substantial inter-individual variability in the time of maximal formation (p = 0.0001). The 2,3-DHBA glucuronide conjugates in vivo and in hepatic microsomal studies amounted to approximately 11% and 0.43%, respectively, of total 2,3-DHBA equivalents. To investigate putative .OH production initiated via PHS-catalyzed phenytoin bioactivation, ASA was given 30 min before phenytoin (65 or 100 mg/kg, intraperitoneally), resulting in respective 7.6-fold (p = 0.02) and 14.2-fold (p = 0.003) increases in phenytoin-initiated maximal 2,3-DHBA formation. Maximal 2,3-DHBA formation was 2.1-fold higher when ASA was administered after rather than before the same dose (65 mg/kg) of phenytoin (p = 0.03), indicating ASA inhibition of PHS-catalyzed phenytoin bioactivation. Urinary analysis was much less sensitive, and the 2,5-isomer reflected enzymatic rather than .OH-mediated hydroxylation. The paraquat studies demonstrate the importance of timing in accurately quantifying 2,3-DHBA formation and suggest that glucuronidation does not interfere. The substantial, dose-dependent initiation of 2,3-DHBA formation by phenytoin, and its inhibition by ASA, provide the first in vivo evidence that PHS-dependent .OH formation could contribute to the molecular mechanism of phenytoin teratogenesis.

A eukaryotic DNA glycosylase/lyase recognizing ultraviolet light-induced pyrimidine dimers.

Cyclobutane pyrimidine dimers (CPDs) are the predominant product of photodamage in DNA after exposure of cells to ultraviolet light and are cytotoxic, mutagenic and carcinogenic in a variety of cellular and animal systems. In prokaryotes, enzymes and protein complexes have been characterized that remove or reverse CPDs in DNA. Micrococcus luteus and T4 phage-infected Escherichia coli contain a specific N-glycosylase/apurinic-apyrimidinic lyase that catalyses a two-step DNA incision process at sites of CPDs, thus initiating base excision repair of these lesions. It is well established that CPDs are recognized and removed from eukaryotic DNA by excision repair processes but very little information exists concerning the nature of the proteins involved in CPD recognition and DNA incision events. We report here that an enzyme functionally similar to the prokaryotic N-glycosylase/apurinic-apyrimidinic lyases exists in Saccharomyces cerevisiae. To our knowledge, this is the first time such an activity has been found in a eukaryote and is also the first example of an organism having both direct reversal and base excision repair pathways for the removal of CPDs from DNA.