DNA-m6A calling and integrated long-read epigenetic and genetic analysis with fibertools.

Long-read DNA sequencing has recently emerged as a powerful tool for studying both genetic and epigenetic architectures at single-molecule and single-nucleotide resolution. Long-read epigenetic studies encompass both the direct identification of native cytosine methylation as well as the identification of exogenously placed DNA N6-methyladenine (DNA-m6A). However, detecting DNA-m6A modifications using single-molecule sequencing, as well as coprocessing single-molecule genetic and epigenetic architectures, is limited by computational demands and a lack of supporting tools. Here, we introduce fibertools, a state-of-the-art toolkit that features a semisupervised convolutional neural network for fast and accurate identification of m6A-marked bases using PacBio single-molecule long-read sequencing, as well as the coprocessing of long-read genetic and epigenetic data produced using either PacBio or Oxford Nanopore sequencing platforms. We demonstrate accurate DNA-m6A identification (>90% precision and recall) along >20 kilobase long DNA molecules with a ~1,000-fold improvement in speed. In addition, we demonstrate that fibertools can readily integrate genetic and epigenetic data at single-molecule resolution, including the seamless conversion between molecular and reference coordinate systems, allowing for accurate genetic and epigenetic analyses of long-read data within structurally and somatically variable genomic regions.

A pilot study investigating plasma pharmacokinetics and tolerance of oral capecitabine in carcinoma-bearing dogs.

BACKGROUND: Capecitabine is an oral prodrug of the active metabolite 5-fluorouracil, which has been used effectively in human colorectal, head and neck, and mammary carcinomas. Capecitabine has several properties that make it an attractive treatment option for dogs: (i) it is relatively inexpensive, (ii) it has a short half-life in humans, allowing for rapid plasma concentration changes to be achieved with dosage adjustments, (iii) it is effective for treating carcinomas in humans, for which there are no widely-effective oral chemotherapy options in dogs, and (iv) it is thought to preferentially target cancer cells due to different expression of thymidine phosphorylase, thereby decreasing the risk of off-target side effects. However, capecitabine has not been widely explored as a chemotherapy agent for dogs. The goal of this study was to determine the plasma disposition of capecitabine in dogs following a single oral dose and to document any adverse events associated with capecitabine administration over the course of 5 weeks. RESULTS: Capecitabine was well tolerated throughout the 5-week study period when administered to 5 dogs with naturally occurring carcinomas at 750 mg/m[Formula: see text] by mouth once daily for 14 consecutive days in a 3-week cycle. No dogs withdrew from the study due to adverse events or other causes. The median AUC[Formula: see text] was 890 h[Formula: see text]ng/ml (range 750-1100 h[Formula: see text]ng/ml); however, the maximum blood concentration and time to reach that concentration of capecitabine was highly variable after a single dose. CONCLUSIONS: Capecitabine appears well-tolerated as an oral chemotherapy agent for dogs with carcinomas, although individualized dosing may be necessary, and further studies are warranted.

Perampanel's forgiveness factor in a variable medication adherence paradigm in a rat model of chronic epilepsy.

BACKGROUND: Poor medication adherence contributes to increased morbidity and mortality in patients with epilepsy and may be under-addressed in clinical practice. Ethical concerns make it impossible to study the impact of medication nonadherence in clinical trials, but our previous work emphasizes the importance of using preclinical approaches to address these questions. With over 30 clinically available antiseizure medicines (ASM's), it remains an important question to understand the relationship between poor adherence and seizure incidence across mechanistically distinct ASM's, including the broad-spectrum ASM, perampanel (PER). METHODS: We formulated PER into chow pellets to deliver to rats in a 100% fully adherent or 50% variable nonadherent paradigm via our novel automated medication-in-food delivery system. Chronic oral dosing was initiated in male rats with chronic epilepsy while monitoring 24/7 for videoEEG evidence of seizures during a 4-week placebo baseline and 4-week treatment phase. PER concentrations were monitored in plasma at 1-week intervals and correlated with degree of seizure control. The relationship between missed doses and extended patterns of nonadherence were correlated with breakthrough seizures. RESULTS: Fully adherent rats demonstrated a median reduction in seizure frequency of 50%, whereas nonadherent rats had a median increase of 54%. Plasma concentrations of PER were stable over the 4-week treatment period in both fully adherent and nonadherent groups, with levels being twice as high in fully adherent animals. There was no correlation between a single missed dose or series of missed doses and the incidence of breakthrough seizures. However, those animals in the nonadherent group that received PER for every meal during a 24-h period had a reduced likelihood of seizure incidence. CONCLUSIONS: If our preclinical data is supported in the clinic, PER's favorable pharmacokinetic profile in humans, combined with a lowered risk of breakthrough seizures suggests that it may provide a certain forgiveness factor if a dose is missed within a 24-h window.

Do Extremely Low Gestational Age Neonates Regulate Iron Absorption via Hepcidin?

OBJECTIVES: To evaluate whether extremely preterm infants regulate iron status via hepcidin. STUDY DESIGN: In this retrospective analysis of infants from the Preterm Epo Neuroprotection (PENUT) Trial, urine hepcidin (Uhep) normalized to creatinine (Uhep/UCr) was evaluated among infants randomized to erythropoietin (Epo) or placebo. RESULTS: The correlation (r) between Uhep/UCr and serum markers of iron status (ferritin and zinc protoporphyrin-to-heme ratio [ZnPP/H]) and iron dose was assessed. A total of 243 urine samples from 76 infants born at 24-276/7 weeks gestation were analyzed. The median Uhep/UCr concentration was 0.3, 1.3, 0.4, and 0.1 ng/mg at baseline, 2 weeks, 4 weeks, and 12 weeks, respectively, in placebo-treated infants. The median Uhep/UCr value in Epo-treated infants were not significantly different, with the exception of the value at the 2-week time point (median Uhep/UCr, 0.1 ng/mg; P < .001). A significant association was seen between Uhep/UCr and ferritin at 2 weeks (r = 0.63; P < .001) and at 4 weeks (r = 0.41; P = .01) and between Uhep/UCr and ZnPP/H at 2 weeks (r = -0.49; P = .002). CONCLUSIONS: Uhep/UCr values correlate with serum iron markers. Uhep/UCr values vary over time and are affected by treatment with Epo, suggesting that extremely preterm neonates can regulate hepcidin and therefore their iron status. Uhep is suppressed in extremely preterm neonates, particularly those treated with Epo.

Who should get the scarce ICU bed? The US public's view on triage in the time of COVID-19.

OBJECTIVE: To determine the relative importance members of the US public place on different patient attributes in triage decisions about who should receive the last available intensive care unit (ICU) bed. METHODS: A discrete choice experiment was conducted with a nationally representative sample of 2000 respondents from the YouGov internet panel of US households. Respondents chose which of three hypothetical patients with COVID-19 should receive an ICU bed if only one were available. The three patients differed in age, gender, Alzheimer's-like disability and probability of survival if the patient received the ICU bed. An experimental design varied the values of the four attributes of the three hypothetical patients with COVID-19 that a respondent saw in four choice tasks. RESULTS: The most important patient attribute to respondents was the probability the patient survives COVID-19 if they get the ICU bed (OR CI: 4.41 to 6.91). There was heterogeneity among different age groups of respondents about how much age of the patient mattered. Respondents under 30 years of age were more likely to choose young patients and old patients, and less likely to select patients aged 40-60 years old. For respondents in the age group 30-49 years old, as the age of the patient declined, their preference for saving the patient declined modestly in a linear fashion. CONCLUSIONS: Respondents favoured giving the last ICU bed available to the patient with the highest probability of surviving COVID-19. Public opinion suggests a simple guideline for physician choices based on likelihood of survival as opposed to the number of life-years saved. There was heterogeneity among respondents of different age groups for allocating the last ICU bed, as well as to the importance of the patient having an Alzheimer's-like disability (where religion of the respondent is important) and the gender of the patient (where the gender and racial identity are important).

Repeated isolation of an antibiotic-dependent and temperature-sensitive mutant of Pseudomonas aeruginosa from a cystic fibrosis patient.

BACKGROUND: Bacteria adapt to survive and grow in different environments. Genetic mutations that promote bacterial survival under harsh conditions can also restrict growth. The causes and consequences of these adaptations have important implications for diagnosis, pathogenesis, and therapy. OBJECTIVES: We describe the isolation and characterization of an antibiotic-dependent, temperature-sensitive Pseudomonas aeruginosa mutant chronically infecting the respiratory tract of a cystic fibrosis (CF) patient, underscoring the clinical challenges bacterial adaptations can present. METHODS: Respiratory samples collected from a CF patient during routine care were cultured for standard pathogens. P. aeruginosa isolates recovered from samples were analysed for in vitro growth characteristics, antibiotic susceptibility, clonality, and membrane phospholipid and lipid A composition. Genetic mutations were identified by whole genome sequencing. RESULTS: P. aeruginosa isolates collected over 5 years from respiratory samples of a CF patient frequently harboured a mutation in phosphatidylserine decarboxylase (psd), encoding an enzyme responsible for phospholipid synthesis. This mutant could only grow at 37°C when in the presence of supplemented magnesium, glycerol, or, surprisingly, the antibiotic sulfamethoxazole, which the source patient had repeatedly received. Of concern, this mutant was not detectable on standard selective medium at 37°C. This growth defect correlated with alterations in membrane phospholipid and lipid A content. CONCLUSIONS: A P. aeruginosa mutant chronically infecting a CF patient exhibited dependence on sulphonamides and would likely evade detection using standard clinical laboratory methods. The diagnostic and therapeutic challenges presented by this mutant highlight the complex interplay between bacterial adaptation, antibiotics, and laboratory practices, during chronic bacterial infections.

Quantitative analysis of chondroitin sulfate disaccharides from human and rodent fixed brain tissue by electrospray ionization-tandem mass spectrometry.

Chondroitin sulfates (CS) are long, negatively charged, unbranched glycosaminoglycan (GAG) chains attached to CS-proteoglycan (CSPG) core proteins that comprise the glycan component in both loose interstitial extracellular matrices (ECMs) and in rigid, structured perineuronal net (PNN) scaffolds within the brain. As aberrant CS-PNN formations have been linked to a range of pathological states, including Alzheimer's disease (AD) and schizophrenia, the analysis of CS-GAGs in brain tissue at the disaccharide level has great potential to enhance disease diagnosis and prognosis. Two mass-spectrometry (MS)-based approaches were adapted to detect CS disaccharides from minute fixed tissue samples with low picomolar sensitivity and high reproducibility. The first approach employed a straightforward, quantitative direct infusion (DI)-tandem mass spectrometry (MS/MS) technique to determine the percentages of Δ4S- and Δ6S-CS disaccharides within the 4S/6S-CS ratio, while the second used a comprehensive liquid chromatography (LC)-MS/MS technique to determine the relative percentages of Δ0S-, Δ4S-, Δ6S-, Δ4S6S-CS and Δ2S6S-CS disaccharides, with internal validation by full chondroitin lyase activity. The quantitative accuracy of the five primary biologically relevant CS disaccharides was validated using a developmental time course series in fixed rodent brain tissue. We then analyzed the CS disaccharide composition in formalin-fixed human brain tissue, thus providing the first quantitative report of CS sulfation patterns in the human brain. The ability to comprehensively analyze the CS disaccharide composition from fixed brain tissue provides a means with which to identify alterations in the CS-GAG composition in relation to the onset and/or progression of neurological diseases.

Influence of Stereochemistry on the Bioactivation and Glucuronidation of 4-Ipomeanol.

A potential CYP4B1 suicide gene application in engineered T-cell treatment of blood cancers has revived interest in the use of 4-ipomeanol (IPO) in gene-directed enzyme prodrug therapy, in which disposition of the administered compound may be critical. IPO contains one chiral center at the carbon bearing a secondary alcohol group; it was of interest to determine the effect of stereochemistry on 1) CYP4B1-mediated bioactivation and 2) (UGT)-mediated glucuronidation. First, (R)-IPO and (S)-IPO were synthesized and used to assess cytotoxicity in HepG2 cells expressing rabbit CYP4B1 and re-engineered human CYP4B1, where the enantiomers were found to be equipotent. Next, a sensitive UPLC-MS/MS assay was developed to measure the IPO-glucuronide diastereomers and product stereoselectivity in human tissue microsomes. Human liver and kidney microsomes generated (R)- and (S)-IPO-glucuronide diastereomers in ratios of 57:43 and 79:21, respectively. In a panel of 13 recombinantly expressed UGTs, UGT1A9 and UGT2B7 were the major isoforms responsible for IPO glucuronidation. (R)-IPO-glucuronide diastereoselectivity was apparent with each recombinant UGT, except UGT2B15 and UGT2B17, which favored the formation of (S)-IPO-glucuronide. Incubations with IPO and the UGT1A9-specific chemical inhibitor niflumic acid significantly decreased glucuronidation in human kidney, but only marginally in human liver microsomes, consistent with known tissue expression patterns of UGTs. We conclude that IPO glucuronidation in human kidney is mediated by UGT1A9 and UGT2B7. In human liver, it is mediated primarily by UGT2B7 and, to a lesser extent, UGT1A9 and UGT2B15. Overall, the lack of pronounced stereoselectivity for IPO's bioactivation in CYP4B1-transfected HepG2 cells, or for hepatic glucuronidation, suggests the racemate is an appropriate choice for use in suicide gene therapies.

Structure-Activity Relationships for CYP4B1 Bioactivation of 4-Ipomeanol Congeners: Direct Correlation between Cytotoxicity and Trapped Reactive Intermediates.

Cytochrome P450 4B1 (CYP4B1) has been explored as a candidate enzyme in suicide gene systems for its ability to bioactivate the natural product 4-ipomeanol (IPO) to a reactive species that causes cytotoxicity. However, metabolic limitations of IPO necessitate discovery of new "pro-toxicant" substrates for CYP4B1. In the present study, we examined a series of synthetically facile N-alkyl-3-furancarboxamides for cytotoxicity in HepG2 cells expressing CYP4B1. This compound series maintains the furan warhead of IPO while replacing its alcohol group with alkyl chains of varying length (C1-C8). Compounds with C3-C6 carbon chain lengths showed similar potency to IPO (LD50 ≈ 5 µM). Short chain analogs (<3 carbons) and long chain analogs (>6 carbons) exhibited reduced toxicity, resulting in a parabolic relationship between alkyl chain length and cytotoxicity. A similar parabolic relationship was observed between alkyl chain length and reactive intermediate formation upon trapping of the putative enedial as a stable pyrrole adduct in incubations with purified recombinant rabbit CYP4B1 and common physiological nucleophiles. These parabolic relationships reflect the lower affinity of shorter chain compounds for CYP4B1 and increased ω-hydroxylation of the longer chain compounds by the enzyme. Furthermore, modest time-dependent inhibition of CYP4B1 by N-pentyl-3-furancarboxamide was completely abolished when trapping agents were added, demonstrating escape of reactive intermediates from the enzyme after bioactivation. An insulated CYP4B1 active site may explain the rarely observed direct correlation between adduct formation and cell toxicity reported here.

Fatty-acyl chain profiles of cellular phosphoinositides.

Phosphoinositides are rapidly turning-over phospholipids that play key roles in intracellular signaling and modulation of membrane effectors. Through technical refinements we have improved sensitivity in the analysis of the phosphoinositide PI, PIP, and PIP2 pools from living cells using mass spectrometry. This has permitted further resolution in phosphoinositide lipidomics from cell cultures and small samples of tissue. The technique includes butanol extraction, derivatization of the lipids, post-column infusion of sodium to stabilize formation of sodiated adducts, and electrospray ionization mass spectrometry in multiple reaction monitoring mode, achieving a detection limit of 20pg. We describe the spectrum of fatty-acyl chains in the cellular phosphoinositides. Consistent with previous work in other mammalian primary cells, the 38:4 fatty-acyl chains dominate in the phosphoinositides of the pineal gland and of superior cervical ganglia, and many additional fatty acid combinations are found at low abundance. However, Chinese hamster ovary cells and human embryonic kidney cells (tsA201) in culture have different fatty-acyl chain profiles that change with growth state. Their 38:4 lipids lose their dominance as cultures approach confluence. The method has good time resolution and follows well the depletion in <20s of both PIP2 and PIP that results from strong activation of Gq-coupled receptors. The receptor-activated phospholipase C exhibits no substrate selectivity among the various fatty-acyl chain combinations.

PhoPQ regulates acidic glycerophospholipid content of the Salmonella Typhimurium outer membrane.

Gram-negative bacteria have two lipid membranes separated by a periplasmic space containing peptidoglycan. The surface bilayer, or outer membrane (OM), provides a barrier to toxic molecules, including host cationic antimicrobial peptides (CAMPs). The OM comprises an outer leaflet of lipid A, the bioactive component of lipopolysaccharide (LPS), and an inner leaflet of glycerophospholipids (GPLs). The structure of lipid A is environmentally regulated in a manner that can promote bacterial infection by increasing bacterial resistance to CAMP and reducing LPS recognition by the innate immune system. The gastrointestinal pathogen, Salmonella Typhimurium, responds to acidic pH and CAMP through the PhoPQ two-component regulatory system, which stimulates lipid A remodeling, CAMP resistance, and intracellular survival within acidified phagosomes. Work here demonstrates that, in addition to regulating lipid A structure, the S. Typhimurium PhoPQ virulence regulators also regulate acidic GPL by increasing the levels of cardiolipins and palmitoylated acylphosphatidylglycerols within the OM. Triacylated palmitoyl-PG species were diminished in strains deleted for the PhoPQ-regulated OM lipid A palmitoyltransferase enzyme, PagP. Purified PagP transferred palmitate to PG consistent with PagP acylation of both lipid A and PG within the OM. Therefore, PhoPQ coordinately regulates OM acidic GPL with lipid A structure, suggesting that GPLs cooperate with lipid A to form an OM barrier critical for CAMP resistance and intracellular survival of S. Typhimurium.

Species Differences in Microsomal Oxidation and Glucuronidation of 4-Ipomeanol: Relationship to Target Organ Toxicity.

4-Ipomeanol (IPO) is a model pulmonary toxicant that undergoes P450-mediated metabolism to reactive electrophilic intermediates that bind to tissue macromolecules and can be trapped in vitro as the NAC/NAL adduct. Pronounced species and tissue differences in IPO toxicity are well documented, as is the enzymological component of phase I bioactivation. However, IPO also undergoes phase II glucuronidation, which may compete with bioactivation in target tissues. To better understand the organ toxicity of IPO, we synthesized IPO-glucuronide and developed a new quantitative mass spectrometry-based assay for IPO glucuronidation. Microsomal rates of glucuronidation and P450-dependent NAC/NAL adduct formation were compared in lung, kidney, and liver microsomes from seven species with different target organ toxicities to IPO. Bioactivation rates were highest in pulmonary and renal microsomes from all animal species (except dog) known to be highly susceptible to the extrahepatic toxicities induced by IPO. In a complementary fashion, pulmonary and renal IPO glucuronidation rates were uniformly low in all experimental animals and primates, but hepatic glucuronidation rates were high, as expected. Therefore, with the exception of the dog, the balance between microsomal NAC/NAL adduct and glucuronide formation correlate well with the risk for IPO-induced pulmonary, renal, and hepatic toxicities across species.

Sphingosine, a modulator of human translesion DNA polymerase activity.

Translesion (TLS) DNA polymerases are specialized, error-prone enzymes that synthesize DNA across bulky, replication-stalling DNA adducts. In so doing, they facilitate the progression of DNA synthesis and promote cell proliferation. To potentiate the effect of cancer chemotherapeutic regimens, we sought to identify inhibitors of TLS DNA polymerases. We screened five libraries of ∼ 3000 small molecules, including one comprising ∼ 600 nucleoside analogs, for their effect on primer extension activity of DNA polymerase η (Pol η). We serendipitously identified sphingosine, a lipid-signaling molecule that robustly stimulates the activity of Pol η by ∼ 100-fold at low micromolar concentrations but inhibits it at higher concentrations. This effect is specific to the Y-family DNA polymerases, Pols η, κ, and ι. The addition of a single phosphate group on sphingosine completely abrogates this effect. Likewise, the inclusion of other sphingolipids, including ceramide and sphingomyelin to extension reactions does not elicit this response. Sphingosine increases the rate of correct and incorrect nucleotide incorporation while having no effect on polymerase processivity. Endogenous Pol η activity is modulated similarly as the recombinant enzyme. Importantly, sphingosine-treated cells exhibit increased lesion bypass activity, and sphingosine tethered to membrane lipids mimics the effects of free sphingosine. Our studies have uncovered sphingosine as a modulator of TLS DNA polymerase activity; this property of sphingosine may be associated with its known role as a signaling molecule in regulating cell proliferation in response to cellular stress.

Functional genetic screen of human diversity reveals that a methionine salvage enzyme regulates inflammatory cell death.

Genome-wide association studies can identify common differences that contribute to human phenotypic diversity and disease. When genome-wide association studies are combined with approaches that test how variants alter physiology, biological insights can emerge. Here, we used such an approach to reveal regulation of cell death by the methionine salvage pathway. A common SNP associated with reduced expression of a putative methionine salvage pathway dehydratase, apoptotic protease activating factor 1 (APAF1)-interacting protein (APIP), was associated with increased caspase-1-mediated cell death in response to Salmonella. The role of APIP in methionine salvage was confirmed by growth assays with methionine-deficient media and quantitation of the methionine salvage substrate, 5'-methylthioadenosine. Reducing expression of APIP or exogenous addition of 5'-methylthioadenosine increased Salmonellae-induced cell death. Consistent with APIP originally being identified as an inhibitor of caspase-9-dependent apoptosis, the same allele was also associated with increased sensitivity to the chemotherapeutic agent carboplatin. Our results show that common human variation affecting expression of a single gene can alter susceptibility to two distinct cell death programs. Furthermore, the same allele that promotes cell death is associated with improved survival of individuals with systemic inflammatory response syndrome, suggesting a possible evolutionary pressure that may explain the geographic pattern observed for the frequency of this SNP. Our study shows that in vitro association screens of disease-related traits can not only reveal human genetic differences that contribute to disease but also provide unexpected insights into cell biology.

Effect of travel distance on household demand for typhoid vaccines: implications for planning.

Typhoid fever causes millions of illnesses and hundreds of thousands of deaths yearly. Vaccinations would mitigate this problem, but the users would probably have to pay some or most of the cost. Several willingness-to-pay studies have assessed the effect of price on private demand to provide a basis for financial planning of campaigns, but the effect of travel distance, which is a potentially important determinant of demand, has not been studied. This paper thus has two objectives: (i) conduct a willingness-to-pay survey to assess the effects of distance, price and other variables on the private demand for typhoid vaccinations in a rural township of China where a campaign is under consideration; and (ii) embed the demand function in a mathematical model to address three planning questions; should each village have its own clinic, would one clinic be best or should the number of clinics be something in-between? Private vaccine demand was found to depend on and be inelastic with respect to both price and travel distance. A 1-km increase in distance caused the number of vaccinations demanded to decrease the same as a $0.5 increase in price. Thus, the marginal rate of substitution was $0.5 per km. A single clinic would be best for the township only if diseconomies of scale in supplying vaccinations exceeded the marginal rate of substitution. Otherwise, multiple clinics close to users would be optimal. Thus, deciding the number, location and capacities of clinics for vaccination planning is as important as deciding what price(s) to charge.

P2X7 receptor-dependent increase in endocannabinoid 2-arachidonoyl glycerol production by neuronal cells in culture: Dynamics and mechanism.

BACKGROUND AND PURPOSE: Neurotransmission and neuroinflammation are controlled by local increases in both extracellular ATP and the endocannabinoid 2-arachidonoyl glycerol (2-AG). While it is known that extracellular ATP stimulates 2-AG production in cells in culture, the dynamics and molecular mechanisms that underlie this response remain poorly understood. Detection of real-time changes in eCB levels with the genetically encoded sensor, GRABeCB2.0, can address this shortfall. EXPERIMENTAL APPROACH: 2-AG and arachidonoylethanolamide (AEA) levels in Neuro2a (N2a) cells were measured by LC-MS, and GRABeCB2.0 fluorescence changes were detected using live-cell confocal microscopy and a 96-well fluorescence plate reader. KEY RESULTS: 2-AG and AEA increased GRABeCB2.0 fluorescence in N2a cells with EC50 values of 81 and 58 nM, respectively; both responses were reduced by the cannabinoid receptor type 1 (CB1R) antagonist SR141617 and absent in cells expressing the mutant-GRABeCB2.0. ATP increased only 2-AG levels in N2a cells, as measured by LC-MS, and induced a transient increase in the GRABeCB2.0 signal within minutes primarily via activation of P2X7 receptors (P2X7R). This response was dependent on diacylglycerol lipase ß activity, partially dependent on extracellular calcium and phospholipase C activity, but not controlled by the 2-AG hydrolysing enzyme, α/ß-hydrolase domain containing 6 (ABHD6). CONCLUSIONS AND IMPLICATIONS: Considering that P2X7R activation increases 2-AG levels within minutes, our results show how these molecular components are mechanistically linked. The specific molecular components in these signalling systems represent potential therapeutic targets for the treatment of neurological diseases, such as chronic pain, that involve dysregulated neurotransmission and neuroinflammation.

Support for regulation versus compliance: Face masks during COVID-19.

Objectives: Wearing masks could still be one of the few non-pharmaceutical interventions for controlling the pandemic. There are people who wear them and people who don't, but this framing is overly simplistic. We aim to chart the contradictions in attitudes and behavior regarding mask wearing and describe the messaging challenge that these generate. Study design: Our data come from a survey administered to a nationally representative sample of 2000 respondents from the YouGov panel of US households in August-September 2020. Methods: Respondents were asked whether they wear a facemask when they go outside their home since the COVID-19 epidemic began and whether they support or oppose your municipal government passing mask wearing regulation. We also collected respondents' demographic and economic characteristics, knowledge regarding the facts of COVID-19 and political ideology. Results: A substantial majority of Americans (60%) both favor a masking requirement and are themselves wearing masks, while 13% oppose a mask mandate and do not wear masks. In contrast, 17% of Americans oppose a mask mandate but are currently wearing one, while 10% do not wear a mask but favor a mask mandate. These two groups are distinctively different from one another and the other groups in their socioeconomic characteristics, risk perception and political beliefs. Conclusions: Our study offers a better understanding of the mismatch between mask wearing behavior and attitude toward the mask mandate, which will help the public health authorities to devise policies regarding mask wearing as an effective intervention to manage the pandemic.

How Does the Public Evaluate Vaccines for Low-Incidence, Severe-Outcome Diseases? A General-Population Choice Experiment.

BACKGROUND: Because immunizing large numbers of healthy people could be required to reduce a relatively small number of infections, disease incidence has a large impact on cost effectiveness, even if the infection is associated with very serious health outcomes. In addition to cost effectiveness, the US Advisory Committee on Immunization Practices requires evidence of stakeholders' values and preferences to help inform vaccine recommendations. This study quantified general-population preferences for vaccine trade-offs among disease severity, disease incidence, and other vaccine features. METHODS: We developed a best-practice discrete choice experiment survey and administered it to 1185 parents of children aged 12-23 years and 1203 young adults aged 18-25 years from a national opt-in consumer panel. The data were analyzed using exploded-logit latent-class analysis. RESULTS: Latent-class analysis identified two classes with similar relative-importance weights in both samples. One of the two classes represented about half the samples and had preferences consistent with well-structured, logically ordered, and acceptably precise stated-preference utility. Preferences for the other half of the samples were poorly defined over the ranges of vaccine and disease attributes evaluated. Both parents and young adults in the first class evaluated protection from a disease with 1 in 100 incidence and full recovery at home as having statistically the same preference utility as a disease with 1 in 1 million incidence requiring hospitalization and resulting in permanent deafness. CONCLUSIONS: The results suggest that vaccines that protect against low-incidence, severe-outcome diseases, provide 'peace of mind' benefits not captured by standard health-outcome metrics. The fact that half the respondents had poorly defined vaccine preferences is a reminder of the challenges of implementing patient-centric vaccine decision making.

The Effects of Pregnancy on Amino Acid Levels and Nitrogen Disposition.

Limited data are available on the effects of pregnancy on the maternal metabolome. Therefore, the objective of this study was to use metabolomics analysis to determine pathways impacted by pregnancy followed by targeted confirmatory analysis to provide more powerful conclusions about metabolic alterations during pregnancy. Forty-seven pregnant women, 18-50 years of age were included in this study, with each subject serving as their own control. Plasma samples were collected between 25 and 28 weeks gestation and again ≥3 months postpartum for metabolomics analysis utilizing an HILIC/UHPLC/MS/MS assay with confirmatory targeted specific concentration analysis for 10 of the significantly altered amino acids utilizing an LC/MS assay. Principle component analysis (PCA) on metabolomics data clearly separated pregnant and postpartum groups and identified outliers in a preliminary assessment. Of the 980 metabolites recorded, 706 were determined to be significantly different between pregnancy and postpartum. Pathway analysis revealed three significantly impacted pathways, arginine biosynthesis (p = 2 × 10-5 and FDR = 1 × 10-3), valine, leucine, and isoleucine metabolism (p = 2 × 10-5 and FDR = 2 × 10-3), and xanthine metabolism (p = 4 × 10-5 and FDR = 4 × 10-3). Of these we focused analysis on arginine biosynthesis and branched-chain amino acid (BCAA) metabolism due to their clinical importance and interconnected roles in amino acid metabolism. In the confirmational analysis, 7 of 10 metabolites were confirmed as significant and all 10 confirmed the direction of change of concentrations observed in the metabolomics analysis. The data support an alteration in urea nitrogen disposition and amino acid metabolism during pregnancy. These changes could also impact endogenous nitric oxide production and contribute to diseases of pregnancy. This study provides evidence for changes in both the ammonia-urea nitrogen and the BCAA metabolism taking place during pregnancy.

DNA-m6A calling and integrated long-read epigenetic and genetic analysis with fibertools.

Long-read DNA sequencing has recently emerged as a powerful tool for studying both genetic and epigenetic architectures at single-molecule and single-nucleotide resolution. Long-read epigenetic studies encompass both the direct identification of native cytosine methylation as well as the identification of exogenously placed DNA N6-methyladenine (DNA-m6A). However, detecting DNA-m6A modifications using single-molecule sequencing, as well as co-processing single-molecule genetic and epigenetic architectures, is limited by computational demands and a lack of supporting tools. Here, we introduce fibertools, a state-of-the-art toolkit that features a semi-supervised convolutional neural network for fast and accurate identification of m6A-marked bases using PacBio single-molecule long-read sequencing, as well as the co-processing of long-read genetic and epigenetic data produced using either PacBio or Oxford Nanopore sequencing platforms. We demonstrate accurate DNA-m6A identification (>90% precision and recall) along >20 kilobase long DNA molecules with a ~1,000-fold improvement in speed. In addition, we demonstrate that fibertools can readily integrate genetic and epigenetic data at single-molecule resolution, including the seamless conversion between molecular and reference coordinate systems, allowing for accurate genetic and epigenetic analyses of long-read data within structurally and somatically variable genomic regions.