3,N4-Etheno-5-methylcytosine blocks TET1-3 oxidation but is repaired by ALKBH2, 3 and FTO.

5-Methyldeoxycytidine (5mC) is a major epigenetic marker that regulates cellular functions in mammals. Endogenous lipid peroxidation can convert 5mC into 3,N4-etheno-5-methylcytosine (ϵ5mC). ϵ5mC is structurally similar to the mutagenic analog 3,N4-ethenocytosine (ϵC), which is repaired by AlkB family enzymes in the direct reversal repair (DRR) pathway and excised by DNA glycosylases in the base excision repair (BER) pathway. However, the repair of ϵ5mC has not been reported. Here, we examined the activities against ϵ5mC by DRR and BER enzymes and TET1-3, enzymes that modify the 5-methyl group in 5mC. We found that the etheno modification of 5mC blocks oxidation by TET1-3. Conversely, three human homologs in the AlkB family, ALKBH2, 3 and FTO were able to repair ϵ5mC to 5mC, which was subsequently modified by TET1 to 5-hydroxymethylcytosine. We also demonstrated that ALKBH2 likely repairs ϵ5mC in MEF cells. Another homolog, ALKBH5, could not repair ϵ5mC. Also, ϵ5mC is not a substrate for BER glycosylases SMUG1, AAG, or TDG. These findings indicate DRR committed by ALKBH2, 3 and FTO could reduce the detrimental effects of ϵ5mC in genetics and epigenetics and may work together with TET enzymes to modulate epigenetic regulations.

New psychoactive Substances: A Canadian perspective on emerging trends and challenges for the clinical laboratory.

The production and use of New Psychoactive Substances (NPS) has skyrocketed over the last decade, causing major challenges for government authorities, public health agencies, and laboratories across the world. NPS are designed to mimic the psychoactive effects of unregulated or controlled drugs, while constantly being modified to evade drug control regulation. Hence, they are referred to as "legal highs", as they are technically legal to sell, possess, and use. NPS can be classified by their pharmacological mechanism of action and include cannabimimetic, depressants, dissociatives, hallucinogens, opioids, and stimulants. There is significant structural diversity within each NPS class, leading to variable detection using traditional clinical laboratory testing and complicating the interpretation of results. In this article, we review each of the NPS classes and summarize their associated mechanism of action, common structures, and metabolic pathways, and provide examples of recent drugs and emerging threats with a focus on Canadian drug trends. We also explore the current analytical advantages and limitations commonly faced by the clinical laboratory and provide insight on how toxicosurveillance can improve detection of NPS in the ever-changing NPS landscape.

A half century of exploring DNA excision repair in chromatin.

DNA in eukaryotic cells is packaged into the compact and dynamic structure of chromatin. This packaging is a double-edged sword for DNA repair and genomic stability. Chromatin restricts the access of repair proteins to DNA lesions embedded in nucleosomes and higher order chromatin structures. However, chromatin also serves as a signaling platform in which post-translational modifications of histones and other chromatin-bound proteins promote lesion recognition and repair. Similarly, chromatin modulates the formation of DNA damage, promoting or suppressing lesion formation depending on the chromatin context. Therefore, the modulation of DNA damage and its repair in chromatin is crucial to our understanding of the fate of potentially mutagenic and carcinogenic lesions in DNA. Here, we survey many of the landmark findings on DNA damage and repair in chromatin over the last 50 years (i.e., since the beginning of this field), focusing on excision repair, the first repair mechanism studied in the chromatin landscape. For example, we highlight how the impact of chromatin on these processes explains the distinct patterns of somatic mutations observed in cancer genomes.

North and South: Exploring isotopic analysis of bone carbonates and collagen to understand post-medieval diets in London and northern England.

OBJECTIVES: We evaluate the potential of paired isotopic analysis of bone carbonate and collagen to examine the diet of post-medieval human and animal populations from England (17th-19th c.), including, for the first time, manufacturing towns in northern England. The potential for identifying C4 crop consumption is explored alongside regional and local patterning in diet by sex and socioeconomic status. MATERIALS AND METHODS: Humans (n = 216) and animals (n = 168) were analyzed from sites in London and northern England for both carbon and nitrogen isotopes of bone collagen (𝛿13 Ccoll , 𝛿15 Ncoll ). Isotopic analysis of bone carbonates (𝛿13 Ccarb , 𝛿18 Ocarb ) was carried out on all humans and 27 animals, using Fourier transform infrared spectroscopy-attenuated total reflectance to assess diagenesis. RESULTS: Variations in diet were observed between and within different populations by geographical location and socioeconomic status. Three pigs and one cow consumed C4 resources, indicating the availability of C4 -fed animal protein. Londoners consumed more animal and marine protein and C4 resources. Middle- and upper-class populations from both London and northern populations also had greater access to these foods compared to those of lower status in the same regions. DISCUSSION: This substantial multi-isotope dataset deriving from bone carbonate and collagen combined from diverse post-medieval urban communities enabled, for the first time, the biomolecular identification of the dynamics of C4 consumption (cane sugar/maize) in England, providing insight into the dynamics of food globalization during this period. We also add substantially to the animal dataset for post-medieval England, providing further insight into animal management during a key moment of agricultural change.

A Novel Enzymatic Hydrolysis Method for Urine Aldosterone Quantification: A Case for Reassessing Clinical Cut-Offs of Primary Aldosteronism.

BACKGROUND: Primary aldosteronism (PA) is a common endocrine cause of secondary hypertension. The aldosterone/renin ratio is an important tool for PA screening, and dynamic testing in serum or urine is used to confirm the diagnosis. While LC-MS/MS is considered the gold standard for testing, there is significant interlaboratory variability between the extraction procedures, which can impact diagnostic interpretation. To help overcome this, we present a simple and accurate LC-MS/MS method for the quantification of both serum and urine aldosterone using a novel enzymatic hydrolysis procedure. METHODS: Serum and urine aldosterone was extracted and measured by LC-MS/MS. Urine-conjugated aldosterone glucuronide was hydrolyzed using a genetically modified glucuronidase enzyme. The assay precision, accuracy, limit of quantification, recovery, and carryover were evaluated and the new assay cut-offs were proposed. RESULTS: The liquid chromatography method allowed for adequate separation of the aldosterone peak from closely eluting peaks. Significant in vitro aldosterone loss was observed during acid-catalyzed hydrolysis of urine, which was corrected with the addition of the internal standard to the urine before the hydrolysis step. Glucuronidase catalyzed hydrolysis of urine aldosterone glucuronide displays good correlation with the corrected acid-catalyzed hydrolysis. Serum aldosterone showed good agreement with reference values and the consensus range reported for external quality assessment specimens. CONCLUSION: A simple, fast, and highly accurate method for the detection of serum and urine aldosterone has been developed. The proposed novel enzymatic procedure allows for short hydrolysis time and compensates for urine aldosterone loss during the hydrolysis step.

More than what we eat: Investigating an alternative pathway for intact starch granules in dental calculus using Experimental Archaeology.

Starch granules and other plant tissues are commonly found as part of the microdebris assemblage analysed within dental calculus. These are often interpreted as evidence of past diets. However, many of the starch granules extracted from dental calculus are intact, and do not show evidence of alterations as a result of being processed for consumption. This research examines if plant material can accidently enter the mouth while being processed for a meal, with a focus on starch granules. Grinding experiments were performed on three types of cereal grains (wheat, oat and millet). We compare the presence of intact and altered starch granules in mouthwash samples (in place of dental calculus samples) from individuals involved in grinding and also from samples in the environment surrounding the grinding activity. This experiment is a proof of concept aimed to expand experimental research in the field of dental calculus analysis and to encourage the exploration of pathways beyond direct and deliberate consumption.

The Present and Future of Lipid Testing in Cardiovascular Risk Assessment.

BACKGROUND: Lipids play a central role in the pathogenesis of cardiovascular disease (CVD), a leading cause of morbidity and mortality worldwide. Plasma lipids and lipoproteins are routinely measured to help identify individuals at high risk of developing CVD and to monitor patients' response to therapy. The landscape of lipid testing is rapidly changing, including new ways to estimate traditional lipid parameters (e.g., low-density lipoprotein-cholesterol [LDL-C] calculations) and new lipid parameters that show superiority for risk prediction (e.g., non-high-density lipoprotein-cholesterol [non-HDL-C], apolipoprotein B [apoB], and lipoprotein a [Lp(a)]). CONTENT: Various national guidelines for managing dyslipidemia to prevent CVD are available, which primarily focus on LDL-C for identifying those at high risk and setting thresholds for optimal response to therapy. However, LDL-C can be calculated and measured in various ways, each with advantages and disadvantages. Importantly, the recently established Sampson-NIH LDL-C equation appears to be superior to preceding calculations, as is clear from the literature and in guidelines. There is now a shift towards using lipid parameters other than LDL-C, such as non-HDL-C, apoB, and Lp(a), to identify high-risk patients and/or establish treatment targets. SUMMARY: The goal of this review is to discuss the present and future of lipid testing for CVD risk assessment through describing various national clinical guidelines, critically reviewing methods to calculate and measure LDL-C and discussing the clinical utility of additional lipid parameters.

Ionic strength modulates excision of uracil by SMUG1 from nucleosome core particles.

Ionic strength affects many cellular processes including the packaging of genetic material in eukaryotes. For example, chromatin fibers are compacted in high ionic strength environments as are the minimal unit of packaging in chromatin, nucleosome core particles (NCPs). Furthermore, ionic strength is known to modulate several aspects of NCP dynamics including transient unwrapping of DNA from the histone protein core, nucleosome gaping, and intra- and internucleosomal interactions of the N-terminal histone tails. Changes in NCP structure may also impact interactions of transcriptional, repair, and other cellular machinery with nucleosomal DNA. One repair process, base excision repair (BER), is impacted by NCP structure and may be further influenced by changes in ionic strength. Here we examine the effects of ionic strength on the initiation of BER using biochemical assays. Using a population of NCPs containing uracil (U) at dozens of geometric locations, excision of U by single-strand selective monofunctional uracil DNA glycosylase (SMUG1) is assessed at higher and lower ionic strengths. SMUG1 has increased excision activity in the lower ionic strength conditions. On duplex DNA, however, SMUG1 activity is largely unaffected by ionic strength except at short incubation times, suggesting that changes in SMUG1 activity are likely due to alterations in NCP structure and dynamics. These results allow us to further understand the cellular role of SMUG1 in a changing ionic environment and broadly contribute to the understanding of BER on chromatin and genomic stability.

Drug checking services as a surveillance tool for clinical laboratories: Examining trends in the unregulated fentanyl supply.

OBJECTIVES: Timely assessment and understanding of drug trends is essential for clinical laboratories to effectively respond to the overdose epidemic. In this proof-of-concept study, we sought to determine whether information obtained through Toronto's Drug Checking Services (DCS) and cross-provincial urine drug testing (UDT) data can be used as a surveillance tool for clinical laboratories and discuss the value of collaboration between the clinical laboratory, clinicians, and community partners to optimize patient care. DESIGN & METHODS: Mass spectrometry-based UDT data from LifeLabs Ontario (n = 127,529) and British Columbia (n = 14,848), and drug checking data from Toronto DCS (n = 3,308 drugs or used paraphernalia) was collected between August 2020 and October 2021. Fentanyl co-positivity with toxic adulterants such as benzodiazepine-related drugs and fentanyl analogues were examined. RESULTS: The percent co-positivity of fentanyl with etizolam, flualprazolam, flubromazolam, carfentanil, and acetylfentanyl in both Ontario UDT and DCS drugs/used paraphernalia showed similar trends. Regional differences in co-positivity with etizolam and fentanyl analogues were noted between Ontario and British Columbia UDT with patterns consistent over the entire 15-month collection period. CONCLUSIONS: Clinical laboratories should connect with their local DCS, if available, to understand and monitor unregulated drug trends. These data can be used as an important tool to help clinical laboratories tailor their UDT menus and thereby provide a community-focused service to improve patient care.

Conceptualisations of COPD self-management: A narrative review of the research literature.

AIM: To examine how self-management is conceptualised in the research literature on chronic obstructive pulmonary disease (COPD). METHODS: A narrative review was undertaken to search the research literature on COPD self-management. Ten databases (2000-2021) were searched for published texts. Sixty-two articles met the inclusion criteria. A thematic analysis was conducted of the literature. RESULTS: Three conceptualisations of COPD self-management were identified: 1) a dominant medicocentric conceptualisation which represented self-management as medical in focus; 2) a less dominant experiential conceptualisation that viewed it as arising from the experiences of people living with COPD; and 3) a smaller body of literature that attempted to integrate medicocentric and experiential conceptualisations of self-management. DISCUSSION: The dominance of the medicocentric conceptualisation of self-management and the polarisation of medicocentric and experiential perspectives were striking. An integrated conceptualisation of self-management has the potential to unite these competing perspectives and promote collaborative relationships between individuals and professionals, so long as the underlying values informing it are made explicit. However, there is a dearth of literature on this approach and it would benefit from more attention. Methods such as Co-production and the Personal Outcomes Approach offer the potential to support an integrated perspective in clinical practice.

Shooting hoops: globetrotting plasmids spreading more than just antimicrobial resistance genes across One Health.

Our study provides novel insights into the global nature of antimicrobial resistance (AMR) plasmids across the food chain. We provide compelling evidence of the globetrotting nature of AMR plasmids and the need for surveillance to sequence plasmids with a template of analyses for others to expand these data. The AMR plasmids analysed were detected in 63 countries and in samples from humans, animals and the environment. They contained a combination of known and novel AMR genes, metal resistance genes, virulence factors, phage and replicon types.

Formative Research to Inform Market-Based Interventions to Increase Egg Purchase and Consumption in Tigray, Ethiopia.

Animal source foods (ASFs) have a demonstrated ability to improve child health yet are underutilized by many communities faced with malnutrition. Recognizing that improving knowledge about the benefits of consuming ASFs alone is not adequate to change behavior, the Studying Animal Food Markets in Rural Areas (SAFIRA) pilot project planned to test a market-based intervention to increasing the intake of ASFs by children 6-23 months in rural Tigray, Ethiopia. Our process of designing in-market behavior change strategies involved identifying the project's target ASF, cocreating and testing marketing interventions, and understanding barriers and enablers driving key retailer behaviors. Qualitative research methods including focus group discussions, key informant interviews, trials of improved practices, and transect walks were used throughout 2 rounds of formative research. The first round of formative research led the project to focus on eggs, and the second round resulted in an improved understanding of the Tigrayan local food markets and egg consumption. Consumers were receptive to nutrition messaging from trusted community members and consider eggs to be healthy and affordable relative to other ASFs. Despite a willingness on the part of egg retailers in Tigrayan markets to try new practices to market eggs to consumers, formative research revealed that retailers function primarily as aggregators, moving eggs toward urban markets, correcting a foundational assumption that households routinely purchase eggs at local markets. These findings demonstrate the importance of formative research to inform design-especially in the development of context-specific behavior change interventions situated within local marketplaces.

Histone variants H3.3 and H2A.Z/H3.3 facilitate excision of uracil from nucleosome core particles.

At the most fundamental level of chromatin organization, DNA is packaged as nucleosome core particles (NCPs) where DNA is wound around a core of histone proteins. This ubiquitous sequestration of DNA within NCPs presents a significant barrier to many biological processes, including DNA repair. We previously demonstrated that histone variants from the H2A family facilitate excision of uracil (U) lesions by DNA base excision repair (BER) glycosylases. Here, we consider how the histone variant H3.3 and double-variant H2A.Z/H3.3 modulate the BER enzymes uracil DNA glycosylase (UDG) and single-strand selective monofunctional uracil DNA glycosylase (SMUG1). Using an NCP model system with U:G base pairs at a wide variety of geometric positions we generate the global repair profile for both glycosylases. Enhanced excision of U by UDG and SMUG1 is observed with the H3.3 variant. We demonstrate that these H3.3-containing NCPs form two species: (1) octasomes, which contain the full complement of eight histone proteins and (2) hexasomes which are sub-nucleosomal particles that contain six histones. Both the octasome and hexasome species facilitate excision activity of UDG and SMUG1, with the largest impacts observed at sterically-occluded lesion sites and in terminal regions of DNA of the hexasome that do not closely interact with histones. For the double-variant H2A.Z/H3.3 NCPs, which exist as octasomes, the global repair profile reveals that UDG but not SMUG1 has increased U excision activity. The enhanced glycosylase activity reveals potential functions for these histone variants to facilitate BER in packaged DNA and contributes to our understanding of DNA repair in chromatin and its significance regarding mutagenesis and genomic integrity.

Obstacles and opportunities for base excision repair in chromatin.

Most eukaryotic DNA is packaged into chromatin, which is made up of tandemly repeating nucleosomes. This packaging of DNA poses a significant barrier to the various enzymes that must act on DNA, including DNA damage response enzymes that interact intimately with DNA to prevent mutations and cell death. To regulate access to certain DNA regions, chromatin remodeling, variant histone exchange, and histone post-translational modifications have been shown to assist several DNA repair pathways including nucleotide excision repair, single strand break repair, and double strand break repair. While these chromatin-level responses have been directly linked to various DNA repair pathways, how they modulate the base excision repair (BER) pathway remains elusive. This review highlights recent findings that demonstrate how BER is regulated by the packaging of DNA into nucleosome core particles (NCPs) and higher orders of chromatin structures. We also summarize the available data that indicate BER may be enabled by chromatin modifications and remodeling.

Bias in IGF-1 concentrations and interpretation across three different clinical laboratory assays.

In this study, we compared the DiaSorin LiaisonXL IGF-1 immunoassay to both the Roche Elecsys IGF-1 immunoassay and to the liquid chromatography-high resolution mass spectrometry (LC-MS) IGF-1 assay. Our study shows a constant positive bias in DiaSorin compared to the Roche immunoassay (mean 42 µg/L, 24%), and a proportional positive bias in DiaSorin compared to the LC-MS method (mean 49 µg/L, 29%). Further, we demonstrate the potential clinical impact of this bias by evaluating 43 adult samples, collected over a 2-month period, which were shown to be discrepant based on a chart review. Despite the positive analytical bias in the Diasorin assay compared to the LC-MS assay, the Diasorin assay upper reference limits were lower than those of the LC-MS assay. This effect caused nine out of forty-three samples to show falsely elevated results when they were clinically diagnosed as negative for acromegaly. Discussed in the context of previous literature, our findings emphasize the importance of adjusting reference intervals for IGF-1 assays based on the clinical needs of a patient population.

Kinetic Analysis of the Effect of N-Terminal Acetylation on Thymine DNA Glycosylase.

Thymine DNA glycosylase (TDG) is tasked with initiating DNA base excision repair by recognizing and removing T, U, the chemotherapeutic 5-fluorouracil (5-FU), and many other oxidized and halogenated pyrimidine bases. TDG contains a long, unstructured N-terminus that contains four known sites of acetylation: lysine (K) residues 59, 83, 84, and 87. Here, K to glutamine (Q) mutants are used as acetyl-lysine (AcK) analogues to probe the effect of N-terminal acetylation on the kinetics of TDG. We find that mimicking acetylation affects neither the maximal single-turnover rate kmax nor the turnover rate kTO, indicating that the steps after initial binding, through chemistry and product release, are not affected. Under subsaturating conditions, however, acetylation changes the processing of U substrates. Subtle differences among AcK analogues are revealed with 5-FU in single-stranded DNA. We propose that the subtleties observed among the AcK analogues may be amplified on the genomic scale, leading to regulation of TDG activity. N-terminal acetylation, though, may also play a structural, rather than kinetic role in vivo.

The North American opioid epidemic: opportunities and challenges for clinical laboratories.

Since 1999, the opioid epidemic in North America has resulted in over 1 million deaths, and it continues to escalate despite numerous efforts in various arenas to combat the upward trend. Clinical laboratories provide drug testing to support practices such as emergency medicine, substance use disorder treatment, and pain management; increasingly, these laboratories are collaborating in novel partnerships including drug-checking services (DCS) and multidisciplinary treatment teams. This review examines drug testing related to management of licit and illicit opioid use, new technologies and test strategies employed by clinical laboratories, barriers hindering laboratory response to the opioid epidemic, and areas for improvement and standardization within drug testing. Literature search terms included combinations of "opioid," "opiate," "fentanyl," "laboratory," "epidemic," "crisis," "mass spectrometry," "immunoassay," "drug screen," "drug test," "guidelines," plus review of PubMed "similar articles" and references within publications. While immunoassay (IA) and point-of-care (POC) test options for synthetic opioids are increasingly available, mass spectrometry (MS) platforms offer the greatest flexibility and sensitivity for detecting novel, potent opioids. Previously reserved as a second-tier application in most drug test algorithms, MS assays are gaining a larger role in initial screening for specific patients and DCS. However, there are substantial differences among laboratories in terms of updating test menus, algorithms, and technologies to meet changing clinical needs. While some clinical laboratories lack the resources and expertise to implement MS, many are also slow to adopt available IA and POC tests for newer opioids such as fentanyl. MS-based testing also presents challenges, including gaps in available guidance for assay validation and ongoing performance assessment that contribute to a dramatic lack of standardization among laboratories. We identify opportunities for improvement in laboratory operations, reporting, and interpretation of drug test results, including laboratorian and provider education and laboratory-focused guidelines. We also highlight the need for collaboration with providers, assay and instrument manufacturers, and national organizations to increase the effectiveness of clinical laboratory and provider efforts in preventing morbidity and mortality associated with opioid use and misuse.

Comparative Analyses of Three Point-of-Care Urine Drug Test Devices' Performance Characteristics for Use in Ambulatory Clinic Settings.

BACKGROUND: Urine drug testing (UDT) is a standard practice used for monitoring controlled and illicit substances in ambulatory care patients. Point-of-care (POC) UDTs are useful tools that allow for drug identification within minutes, providing rapid and objective diagnostic assistance for clinicians. The objective of this study was to evaluate the performance characteristics of 3 different POC UDT devices compared to reference methods. METHODS: A total of 106 residual urine specimens were collected to evaluate the 3 POC UDT devices: the Profile®-V MEDTOX Scan® drugs of abuse test, Quidel Triage® TOX Drug screen, and Quidel Triage Rapid OXY-BUP-MDMA panel. Device performance was assessed by their ability to identify drug classes/compounds compared to manufacturer and reference method (mass spectrometry) cutoffs. RESULTS: The results from quantitative mass spectrometry showed that 77% (84/106) of the samples were positive for one or more drugs. Each device had variable performance across each drug class. Overall, the specificity of the Profile-V MEDTOX Scan test was 90.1%, while the Quidel Triage TOX Drug Screen and Rapid OXY-BUP-MDMA devices had specificities of 89.0% and 50.0% using their respective manufacturer-stated cutoffs. Overall sensitivity was determined to be 98.6%, 97.0%, and 100% for the Profile-V MEDTOX Scan, Quidel Triage TOX Drug Screen, and Rapid OXY-BUP-MDMA, respectively. CONCLUSIONS: Of the 3 POC UDT devices evaluated, the Profile-V MEDTOX Scan demonstrated the best overall sensitivity and specificity compared to reference methods. False positive and negative results are possible with UDTs, ultimately the best device may depend on patient population and drugs of interest.