Geographic variation of mutagenic exposures in kidney cancer genomes.

International differences in the incidence of many cancer types indicate the existence of carcinogen exposures that have not yet been identified by conventional epidemiology make a substantial contribution to cancer burden1. In clear cell renal cell carcinoma, obesity, hypertension and tobacco smoking are risk factors, but they do not explain the geographical variation in its incidence2. Underlying causes can be inferred by sequencing the genomes of cancers from populations with different incidence rates and detecting differences in patterns of somatic mutations. Here we sequenced 962 clear cell renal cell carcinomas from 11 countries with varying incidence. The somatic mutation profiles differed between countries. In Romania, Serbia and Thailand, mutational signatures characteristic of aristolochic acid compounds were present in most cases, but these were rare elsewhere. In Japan, a mutational signature of unknown cause was found in more than 70% of cases but in less than 2% elsewhere. A further mutational signature of unknown cause was ubiquitous but exhibited higher mutation loads in countries with higher incidence rates of kidney cancer. Known signatures of tobacco smoking correlated with tobacco consumption, but no signature was associated with obesity or hypertension, suggesting that non-mutagenic mechanisms of action underlie these risk factors. The results of this study indicate the existence of multiple, geographically variable, mutagenic exposures that potentially affect tens of millions of people and illustrate the opportunities for new insights into cancer causation through large-scale global cancer genomics.

Biobanking in the COVID-19 Era and Beyond: Part 2. A Set of Tool Implementation Case Studies.

The COVID-19 era has brought about a number of novel challenges for the global biobanking community. An array of diverse tools (e.g., standards, best practices, and plans) exists to support quality and fitness-for-purpose in biobank operations. The International Society for Biological and Environmental Repositories (ISBER) COVID-19 Response Task Force has set out to identify needs and gaps in these tools and make recommendations for the next generation of available tools, having closely examined the COVID-19-related challenges. While conducting this work to examine the relationships between tools and biobank adaptability, a subgroup of the task force conducted a parallel effort to develop and describe individual COVID-19 era case studies based on a number of operating biobanks. Each case study presents a different combination of implemented tools. Observations and lessons learned from these case studies are provided, and experiences with tool implementation are discussed. This information is supplemented by data relating to tool usefulness that was obtained through an ISBER survey discussed in a companion article. The knowledge gained from this study will be combined with other task force efforts to make recommendations to better position the biobanking community in their response to future emergencies.

Biobanking in the COVID-19 Era and Beyond: Part 1. How Early Experiences Can Translate into Actionable Wisdom.

The era of COVID-19 has brought about a number of novel challenges for the global biobanking community. To better position the biobanking community to cope with current and future challenges, the International Society for Biological and Environmental Repositories (ISBER) COVID-19 Response Task Force was convened to identify needs and gaps in biobanking tools (existing resources that support good practice), for example, standards, best practices, business, etc. and to make recommendations to benefit the community. Toward these goals, the Task Force assembled a set of questions to explore individual biobanks' experiences, with emphasis on identification of key challenges and approaches, including tools employed. A survey was designed with the use of these questions and administered by ISBER. This article presents a summary of the aggregated data obtained from the survey responses, illustrating some of the major issues encountered and identifying which tools the survey respondents found most useful. In particular, this article focuses on the challenges identified during the early months of the COVID-19 era. Recommendations are provided to support biobank emergency preparedness for the future, address lessons learned, and propose solutions to bridge identified gaps. The analysis and the complete survey dataset will also inform the larger Task Force goal to develop specific tool recommendations.

RNA and DNA Integrity Remain Stable in Frozen Tissue After Long-Term Storage at Cryogenic Temperatures: A Report from the Ontario Tumour Bank.

Background: It is widely assumed that the integrity of tissue specimens remains stable indefinitely if preserved at cryogenic temperatures. With biobanking reaching a level of maturity where samples are increasingly stored for 10 years and beyond, this assumption of prolonged stability should be tested. Data from such an assessment are critical to verify if samples stored for extended durations remain "fit for purpose" or if there is need to reconsider the utility of samples stored beyond a certain timeframe. The Ontario Tumour Bank has been collecting samples since 2004, and assesses a random selection of frozen samples each year for RNA and DNA integrity as a part of ongoing quality control (QC) practices. This historical quality assessment data provide a unique opportunity to assess the impact of extended storage on nucleic acid integrity using replicate samples that remain in the bank in the present day as comparators. Methods: To examine the stability of fresh-frozen tumor tissue stored at cryogenic temperatures, RNA was extracted and analyzed from 87 cases over 14 disease sites stored long term in vapor-phase liquid nitrogen (LN2) (approximately -180°C). Historical QC data were compared against new data after re-extraction of replicate samples to determine the effect of extended storage on RNA quality. In addition, DNA was extracted from a subselection of samples (n = 20) to determine the effect of prolonged storage on DNA integrity. Results: No time-dependent decrease in tissue RNA or DNA quality, as measured by RNA integrity number (RIN) and DNA integrity number, was observed over an 11-year period. As a secondary observation, RNA integrity was not predictive of DNA integrity: DNA quality may still be very good, and as such RIN scores should not be used as a substitute indicator for evaluating DNA. Conclusions: Extended cryogenic storage beyond 2-11 years remains a viable option for maintaining the high quality of specimens in biobanks.

Visual detection of affordances for aperture negotiation in people with Parkinson disease.

BACKGROUND: An essential requirement for the guidance of action in cluttered environments is that people can accurately perceive what actions are afforded by particular surroundings given the person's action capabilities. Research has shown that healthy young individuals turn their shoulders when walking through a doorway when the aperture is less than a certain percentage of their shoulder width and that they are able to detect this critical width with visual inspection. These findings imply that movements are constrained by perception of the environment in body-scaled unit. OBJECTIVES: The present work examined whether the visual affordance of doorway passability is altered in people with Parkinson disease (PD). METHODS: People with PD, healthy age-matched controls, and young adults (16 participants per group) walked through a series of apertures scaled to shoulder width. Participants also had to visually judge a series of apertures to determine if they could walk through the gap with their normal gait pattern. Finally, participants had to estimate their eye height. RESULTS: Statistical analysis revealed that people with PD initiated shoulder turning to go through the doorway at larger apertures (A) relative to their shoulder (S) width (A/S = 1.61) in comparison to healthy age-matched participants (A/S = 1.41) and young adults (A/S = 1.26). In comparison to healthy participants, People with PD also judged wider apertures as impassable. Individuals with PD were less accurate in their estimation of eye height (Error = 10.1%) than the healthy older (Error = 6.29%) and younger adults (Error = 4.79%). CONCLUSIONS: PD significantly impacted the affordances for aperture negotiation. Such altered perceptual affordances may contribute to gait pattern changes in people with PD when walking through doorways. These findings suggest that some of the motor symptoms in PD might have a perceptual underpinning.

Biobank bootstrapping: is biobank sustainability possible through cost recovery?

BACKGROUND: The pre-eminent goal of biobanks is to accelerate scientific discovery and support improvements in healthcare through the supply of high quality biospecimens to enable excellent science. Despite the need for retrospective future-proofed cancer repositories, they are presented with significant fiscal challenges. While it was once thought that biobanks could recover most, if not all, operational costs through distribution fees, biobanks have been consistently unable to fully realize this dream. METHODS: Using data from three mature Canadian cancer biobanks, common attributes and assumptions related to cost recovery were evaluated. The values were entered into a simple financial model to determine the cost recovery potential for biobanks. RESULTS: Over a 5-year period analyzed, aliquots from almost 40% (8990) of 23055 cases collected have been distributed in whole or in part to researchers. The financial modeling demonstrates that, based on values derived from the real life experiences of three major Canadian biobanks, full cost recovery through distribution is not feasible. A more realistic, experience based, expectation of cost recovery from distribution fees is in the range of 5%-25%, and this range is lower if only academic research is supported as opposed to also supporting industry researchers. CONCLUSIONS: Biobanks are expensive and, to mitigate costs, are frequently challenged to operate under "self-sustainable" financial models. However, the only possible route to self-sustainability through distribution fees in today's market would require an almost exclusive targeting of commercial researchers and, even then, evidence suggests this is an impossible goal to attain. Support for biobanks should recognize that they exist to further development of personalized treatments and diagnostics essential for precision medicine. For biobanks to continue to achieve this goal, pro bono publicum, funders need to be aware of the full funding requirements of biobanks and create appropriate funding streams.

Generating a comprehensive set of standard operating procedures for a biorepository network-The CTRNet experience.

Despite the integral role of biorepositories in fueling translational research and the advancement of medicine, there are significant gaps in harmonization of biobanking practices, resulting in variable biospecimen collection, storage, and processing. This significantly impacts accurate downstream analysis and, in particular, creates a problem for biorepository networks or consortia. The Canadian Tumour Repository Network (CTRNet; www.ctrnet.ca ) is a consortium of Canadian tumor biorepositories that aims to enhance biobanking capacity and quality through standardization. To minimize the issue of variable biobanking practices throughout its network, CTRNet has developed and maintained a comprehensive set of 45 standard operating procedures (SOPs). There were four key elements to the CTRNet SOP development process: 1) an SOP development team was formed from members across CTRNet to co-produce each SOP; 2) a principal author was appointed with responsibility for overall coordination of the SOP development process; 3) the CTRNet Management Committee (composed of principal investigators for each member biorepository) reviewed/revised each SOP completed by the development team; and 4) external expert reviewers provided feedback and recommendations on each SOP. Once final Management Committee approval was obtained, the ratified SOP was published on the CTRNet website for public access. Since the SOPs were first published on the CTRNet website (June 2008), there have been approximately 15,000 downloads of one or more CTRNet SOPs/Policies by users from over 60 countries. In accordance with biobanking best practices, CTRNet performs an exhaustive review of its SOPs at set intervals, to coincide with each granting cycle. The last revision was completed in May 2012.

Mode of action, in vitro activity, and in vivo efficacy of AFN-1252, a selective antistaphylococcal FabI inhibitor.

The mechanism of action of AFN-1252, a selective inhibitor of Staphylococcus aureus enoyl-acyl carrier protein reductase (FabI), which is involved in fatty acid biosynthesis, was confirmed by using biochemistry, macromolecular synthesis, genetics, and cocrystallization of an AFN-1252-FabI complex. AFN-1252 demonstrated a low propensity for spontaneous resistance development and a time-dependent reduction of the viability of both methicillin-susceptible and methicillin-resistant S. aureus, achieving a ≥2-log(10) reduction in S. aureus counts over 24 h, and was extremely potent against clinical isolates of S. aureus (MIC(90), 0.015 µg/ml) and coagulase-negative staphylococci (MIC(90), 0.12 µg/ml), regardless of their drug resistance, hospital- or community-associated origin, or other clinical subgroup. AFN-1252 was orally available in mouse pharmacokinetic studies, and a single oral dose of 1 mg/kg AFN-1252 was efficacious in a mouse model of septicemia, providing 100% protection from an otherwise lethal peritoneal infection of S. aureus Smith. A median effective dose of 0.15 mg/kg indicated that AFN-1252 was 12 to 24 times more potent than linezolid in the model. These studies, demonstrating a selective mode of action, potent in vitro activity, and in vivo efficacy, support the continued investigation of AFN-1252 as a targeted therapeutic for staphylococcal infections.

Radical-induced oxidation of trans-resveratrol.

trans-Resveratrol (RVT) (3,5,4'-trihydroxystilbene), a polyphenolic constituent of red wine, is thought to be beneficial in reducing the incidence of cardiovascular diseases, partly via its antioxidant properties. However, the mechanism of action by which trans-resveratrol displays its antioxidant effect has not been totally unravelled. This study aimed at establishing a comprehensive scheme of the reaction mechanisms of the direct scavenging of HO(*) and O(2)(*-) radicals generated by water gamma radiolysis. Aerated aqueous solutions of trans-RVT (from 10 to 100µmolL(-1)) were irradiated with increasing radiation doses (from 25 to 400Gy) and further analyzed by UV-visible absorption spectrophotometry for detection of trans-RVT oxidation products. Separation and quantification of RVT and its four oxidation products previously identified by mass spectrometry, i.e., piceatannol (PCT), 3,5-dihydroxybenzoic acid (3,5-DHBA), 3,5-dihydroxybenzaldehyde (3,5-DHB) and para-hydroxybenzaldehyde (PHB), were performed by HPLC/UV-visible spectrophotometry. Determination of the radiolytic yields of trans-RVT consumption and oxidation product formation has allowed us to establish balance between trans-RVT disappearance and the sum of oxidation products formation. Under our conditions, O(2)(-) radicals seemed to poorly initiate oxidation of trans-RVT, whereas the latter, whatever its initial concentration, quantitatively reacted with HO() radicals, via a dismutation mechanism. Two reaction pathways involving HO()-induced trans-RVT primary radicals have been proposed to explain the formation of the oxidation end-products of trans-RVT.

Melatonin protects PLPC liposomes and LDL towards radical-induced oxidation.

This study investigated the in vitro protective effects of melatonin against oxidation of 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphocholine (PLPC) liposomes [(PLPC) = 250 µm] and low-density lipoproteins (LDL, 3 g/L total concentration) by hydroxyl radicals produced by water gamma radiolysis. Conjugated dienes (CD) and hydroperoxides from cholesteryl esters (CEOOH) and phospholipids (PCOOH) were measured as indices of lipid peroxidation. Protein (apoB) oxidation in LDL was assessed by carbonyl groups. Two LDL antioxidants (vitamin E and ß-carotene) were monitored as a function of the radiation dose. Three concentrations of melatonin were studied in PLPC liposomes, i.e., 20, 50 and 100 µm, and one in LDL, i.e., 100 µm. Melatonin consumption was also followed up in both lipid models upon irradiation, together with the residual PLPC concentration in liposomes. In PLPC liposomes, scavenging of lipid-derived peroxyl radicals was not the only phenomenon to explain the protective properties of melatonin towards lipid peroxidation. Indeed, melatonin also reacted with hydroxyl radicals generated in aqueous phase, which led us to suggest that hydroxyl radicals reacted relatively slowly with PLPC. Melatonin was efficient in lowering lipid peroxidation in LDL, as shown by the decrease in the formation of CDs and in hydroperoxides. Moreover, melatonin clearly slowed radio-induced apolipoprotein B carbonylation and protected α-tocopherol and ß-carotene in LDL.

Reaction mechanism of melatonin oxidation by reactive oxygen species in vitro.

Melatonin (N-acetyl-5-hydroxytryptamine) is a pineal hormone widely known for its antioxidant properties, both in vivo and by direct capture of free radicals in vitro. Although some metabolites and oxidation products of melatonin have been identified, the molecular mechanism by which melatonin exerts its antioxidant properties has not been totally unravelled. This study investigated the reaction mechanism of oxidation of melatonin by radio-induced reactive oxygen species, generated by gamma radiolysis of water for aqueous solutions of melatonin (from 20 to 200 µm), in the presence or absence of molecular oxygen. The hydroxyl radical was found to be the unique species able to initiate the oxidation process, leading to three main products, e.g. N(1)-acetyl-N(2)-formyl-5-methoxykynurenin (AFMK), N(1)-acetyl-5-methoxykynurenin (AMK) and hydroxymelatonin (HO-MLT). The generation of AFMK and HO-MLT strongly depended on the presence of molecular oxygen in solution: AFMK was the major product in aerated solutions (84%), whereas HO-MLT was favoured in the absence of oxygen (86%). Concentrations of AMK remained quite low, and AMK was proposed to result from a chemical hydrolysis of AFMK in solution. A K-value of 1.1 × 10(-4) was calculated for this equilibrium. Both hydrogen peroxide and superoxide dismutase had no effect on the radio-induced oxidation of melatonin, in good accordance for the second case with the poor reactivity of the superoxide anion towards melatonin. Finally, a reaction mechanism was proposed for the oxidation of melatonin in vitro.

Experimental evidence of the reciprocal oxidation of Bovine Serum Albumin and Linoleate in aqueous solution, initiated by HO* free radicals.

An investigation of radiation-induced oxidation of aqueous bovine serum albumin (BSA) in the presence of linoleate (LH) at pH 10.5 has been carried out in order to better understand the respective oxidative processes involved in both lipid and protein phases. Solutions containing BSA (15 micromol L(-1)) and linoleate (15-600 micromol L(-1)) below the critical micellar concentration (cmc=2000 micromol L(-1)), have been irradiated by gamma-rays (137Cs) at radiation doses ranging from 10 to 400 Gy (dose rate 9.5 Gy min(-1)). It can be noticed that, in the absence of BSA, the main hydroperoxides formed from HO*-induced linoleate oxidation below the cmc, do not exhibit a conjugated dienic structure. This was also verified in the presence of BSA. Selected chemical markers of oxidation have been monitored: non-conjugated dienic hydroperoxides and conjugated dienes (without hydroperoxide function) for linoleate oxidation, and carbonyl groups for BSA oxidation. We have shown that for the lowest linoleate concentration (15 micromol L(-1)) in the presence of BSA (15 micromol L(-1)), the formation of conjugated dienes was not observed, meaning that LH was not exposed to HO* radicals attack. However, non-conjugated dienic lipid hydroperoxides were simultaneously detected, indicating that LH was secondarily oxidised by BSA oxidised species. Moreover, the oxidation of linoleate was found to be enhanced by the presence of BSA. For the highest linoleate concentration (600 micromol L(-1)), the expected protection of BSA by LH was not observed, even if LH monomers were responsible for the total scavenging of HO* radicals. In this latter case, the formation of non-conjugated dienic lipid hydroperoxides was lower than expected. Those results showed that BSA was not oxidised by the direct action of HO* radicals but was undergoing a secondary oxidation by non-dienic lipid hydroperoxides and/or lipid radical intermediates, coming from the HO*-induced linoleate oxidation.

Interaction between non-anionic phospholipids and cytochrome c induced by reactive oxygen species.

The oxidative interaction of cytochrome c (Cyt c) with liposomes of Palmitoyl Linoleyl Phosphatidyl Choline (PLPC) initiated by radio-induced free radicals was investigated. Results showed that the peroxidation of PLPC is decreased in the presence of Cyt c, meaning that this latter is the preferential target of hydroxyl radicals. In addition, when Cyt c was incubated with peroxidized PLPC, it was found to be able to decompose hydroperoxides of PLPC into hydroxides. The peroxidase activity of Cyt c proceeded via the opening of the tertiary structure of Cyt c, as suggested by the loss of the sixth coordination bond of the heme-iron. Even if it is known to preferentially interact with cardiolipin, this work shows that Cyt c is also able to interact with hydroperoxide species of non-anionic phospholipids.

Liquid chromatographic/electrospray ionization mass spectrometric identification of the oxidation end-products of trans-resveratrol in aqueous solutions.

trans-Resveratrol (3,5,4'-trihydroxystilbene) is a natural polyphenolic compound that exhibits antioxidant properties. Our study aimed at studying the HO*-induced oxidation of resveratrol (100 micromol.L(-1)) in aerated aqueous solutions. Gamma radiolysis of water was used to generate HO*/O(2)(*-) free radicals (I = 10 Gy.min(-1), dose = 400 Gy). Oxidation products were identified by direct infusion mass spectrometry and high-performance liquid chromatography/mass spectrometry. For each product, structural elucidation was based on simple mass spectra, fragmentation spectra and deuterium/hydrogen exchange spectra; the comparison with mass spectra of synthetic products provided valuable information allowing the complete identification of the oxidation products. Four products resulting from the direct attack of HO* radicals towards resveratrol were identified respectively as piceatannol (trans-3,5,3',4'-tetrahydroxystilbene), 3,5-dihydroxybenzoic acid, 3,5-dihydroxybenzaldehyde and 4-hydroxybenzaldehyde.

Radiolytic yield of cardiolipin peroxidation by gamma rays in large unilamellar vesicles of phosphatidylcholine.

Large unilamellar vesicles of 1-hexanoyl-2-(9Z-12Z-octadecadienoyl)-sn-glycero-3-phosphocholine (PLPC) have been used as model membrane to investigate the effect of increasing amount of cardiolipin (1',3'-bis-[1,2-Di-(9Z-12Z-octadecadienoyl)-sn-glycero-3-phospho]-sn-glycerol, CL) on the peroxidizability of the lipid phase. Hydroxyl radicals generated by gamma radiolysis of water initiated the lipid peroxidation. Both peroxidation products (conjugated dienes and hydroperoxides of PLPC, mono- and dihydroperoxides of CL) and disappearance of CL and PLPC were assessed as a function of the radiation dose (25 to 400 Gy, I = 10 Gy min(-1)). Our results show that the addition of 5% to 15% CL to large unilamellar vesicles (concentration ratio) produces almost complete inhibition of PLPC peroxidation. Thus, for 15% CL (known to be the proportion of CL in the inner mitochondrial membrane), the radiolytic yield of formation of PLPC hydroperoxides is reduced to zero, whereas it is equal to (3.1 +/- 0.2) x 10(-7) mol J(-1) for CL hydroperoxides, showing the importance of the targeted CL. For this concentration ratio (CL/ PLPC 15%), we have established the balance equation between the consumption of CL [G(-CL) = (2.8 +/- 0.1) x 10(-7) mol J(-1)] and the formation of CL hydroperoxides [G(CLOOH(T)) = (3.1 +/- 0.2) x 10(-7) mol J(-1)]. In addition, the radiolytic yields of disappearance of PLPC and CL have been determined [(1.5 +/- 0.1) x 10(-7) mol J(-1) and (2.8 +/- 0.1) x 10(-7) mol J(-1), respectively], their sum [(4.3 +/- 0.2) x 10(-7) mol J(-1)] being higher than G(HO.) (2.8 x 10(-7) mol J(-1)). However, there is no balance between the radiolytic yield of formation of PLPC hydroperoxides [G (PCOOH(T)) approximately 0] and the yield of disappearance of PLPC [(1.5 +/- 0.1) x 10(-7) mol J(-1)], likely because lipid fragments (not measured in this work) could be generated from HO(.) reaction on the polar head of PLPC. These results have been interpreted by assuming that the hydroxyl radicals attack in competition both lipid targets, i.e. PLPC and CL, with a higher sensitivity to CL oxidation. It can be concluded that a little amount of CL (10-15% CL/ PLPC concentration ratio) may exert a strong protective effect against the HO(.)-induced peroxidation of PLPC.

High-resolution array CGH identifies novel regions of genomic alteration in intermediate-risk prostate cancer.

Approximately one-third of prostate cancer patients present with intermediate risk disease. Interestingly, while this risk group is clinically well defined, it demonstrates the most significant heterogeneity in PSA-based biochemical outcome. Further, the majority of candidate genes associated with prostate cancer progression have been identified using cell lines, xenograft models, and high-risk androgen-independent or metastatic patient samples. We used a global high-resolution array comparative genomic hybridization (CGH) assay to characterize copy number alterations (CNAs) in intermediate risk prostate cancer. Herein, we show this risk group contains a number of alterations previously associated with high-risk disease: (1) deletions at 21q22.2 (TMPRSS2:ERG), 16q22-24 (containing CDH1), 13q14.2 (RB1), 10q23.31 (PTEN), 8p21 (NKX3.1); and, (2) amplification at 8q21.3-24.3 (containing c-MYC). In addition, we identified six novel microdeletions at high frequency: 1q42.12-q42.3 (33.3%), 5q12.3-13.3 (21%), 20q13.32-13.33 (29.2%), 22q11.21 (25%), 22q12.1 (29.2%), and 22q13.31 (33.3%). Further, we show there is little concordance between CNAs from these clinical samples and those found in commonly used prostate cancer cell models. These unexpected findings suggest that the intermediate-risk category is a crucial cohort warranting further study to determine if a unique molecular fingerprint can predict aggressive versus indolent phenotypes.

Online H/D exchange liquid chromatography as a support for the mass spectrometric identification of the oxidation products of melatonin.

The hydrogen-deuterium exchange of protonated melatonin and its in vitro oxidation end-products have been examined by liquid chromatography coupled with ion-trap mass spectrometry. Specific H/D scrambling of protons in the C2 and C4 positions of the indole ring during gas-phase fragmentation process was observed for both melatonin and its oxidation products. Collision-induced dissociation spectra showed losses of variably deuterated NH(3), H(2)O and CH(3)CONH(2). In addition, a similar H/D scrambling behaviour was observed for the oxidation products, obtained from the opening of the indole ring by oxidative attack. Fragmentation pathways are proposed and H/D scrambling has been employed as a fingerprint, allowing identification of N(1)-acetyl-5-methoxykynurenin (AMK), N(1)-acetyl-N(2)-formyl-5-methoxykynurenin (AFMK), dehydro-AFMK and hydroxymelatonin as the oxidation products of melatonin in vitro.