Experimental infection of pigs and ferrets with "pre-pandemic," human-adapted, and swine-adapted variants of the H1N1pdm09 influenza A virus reveals significant differences in viral dynamics and pathological manifestations.

Influenza A viruses are RNA viruses that cause epidemics in humans and are enzootic in the pig population globally. In 2009, pig-to-human transmission of a reassortant H1N1 virus (H1N1pdm09) caused the first influenza pandemic of the 21st century. This study investigated the infection dynamics, pathogenesis, and lesions in pigs and ferrets inoculated with natural isolates of swine-adapted, human-adapted, and "pre-pandemic" H1N1pdm09 viruses. Additionally, the direct-contact and aerosol transmission properties of the three H1N1pdm09 isolates were assessed in ferrets. In pigs, inoculated ferrets, and ferrets infected by direct contact with inoculated ferrets, the pre-pandemic H1N1pdm09 virus induced an intermediary viral load, caused the most severe lesions, and had the highest clinical impact. The swine-adapted H1N1pdm09 virus induced the highest viral load, caused intermediary lesions, and had the least clinical impact in pigs. The human-adapted H1N1pdm09 virus induced the highest viral load, caused the mildest lesions, and had the least clinical impact in ferrets infected by direct contact. The discrepancy between viral load and clinical impact presumably reflects the importance of viral host adaptation. Interestingly, the swine-adapted H1N1pdm09 virus was transmitted by aerosols to two-thirds of the ferrets. Further work is needed to assess the risk of human-to-human aerosol transmission of swine-adapted H1N1pdm09 viruses.

Does offering small financial incentives to smokers at the time of being wait-listed for surgery increase smoking cessation by the day of surgery? A randomised feasibility trial.

OBJECTIVE: This study aims to assess whether offering small financial incentives to smokers on elective surgery wait-lists is feasible and increases quitting before surgery. DESIGN: Randomised controlled trial, prospective, double-blinded. SETTING: Single-centre, Australian metropolitan public hospital. PARTICIPANTS: 620 adult smokers (≥10 cigarettes per day) were randomised on being wait-listed for elective surgery and 404 underwent operations (28 January 2021-31 July 2022) at the hospital (65.2%) by trial's end. INTERVENTION: Intervention participants were offered at wait-listing an $A70 supermarket voucher for verified abstinence on the day of surgery, provided they registered an intention to quit before surgery. Registrants intending to quit were also referred to Quitline. Neither intervention was offered to control participants (usual care). Smokers wait-listed from 17 May 2021 were offered an increased incentive of $A140. MAIN OUTCOME MEASURES: Primary outcome, quitting at least 24 hours before surgery, verified by exhaled carbon monoxide testing. Feasibility outcomes were the proportion taking up offers, ease of patient contact and disputes about quit status. RESULTS: Of 620 randomised participants (control 312, intervention 308), 404 had surgery at the hospital during the trial (control 214, intervention 190), which was lower than expected (for COVID-19 reasons). Offering $A70 resulted in 21.9% registering to quit, increasing to 32.6% with $A140. Telephone calls were the most effective means to gain registrations. The proportion of intervention group patients verified quit at least 24 hours before surgery was similar to controls (9.5% vs 8.9%, OR 1.1, 95% CI 0.5 to 2.2). Quitline contact was higher in the intervention group (13.2% vs 2.3%, OR 6.3, 95% CI 2.3 to 21.6). Disputes over test results did not occur, but 17.4% of intervention participants claiming quit failed verification. CONCLUSION: A single offer of financial rewards for perioperative cessation was feasible, without achieving clinically important quit differences. TRIAL REGISTRATION NUMBER: ACTRN12620000130965.

CellBiAge: Improved single-cell age classification using data binarization.

Aging is a major risk factor for many diseases. Accurate methods for predicting age in specific cell types are essential to understand the heterogeneity of aging and to assess rejuvenation strategies. However, classifying organismal age at single-cell resolution using transcriptomics is challenging due to sparsity and noise. Here, we developed CellBiAge, a robust and easy-to-implement machine learning pipeline, to classify the age of single cells in the mouse brain using single-cell transcriptomics. We show that binarization of gene expression values for the top highly variable genes significantly improved test performance across different models, techniques, sexes, and brain regions, with potential age-related genes identified for model prediction. Additionally, we demonstrate CellBiAge's ability to capture exercise-induced rejuvenation in neural stem cells. This study provides a broadly applicable approach for robust classification of organismal age of single cells in the mouse brain, which may aid in understanding the aging process and evaluating rejuvenation methods.

Epidemiology and molecular characterization of avian influenza A viruses H5N1 and H3N8 subtypes in poultry farms and live bird markets in Bangladesh.

Avian influenza virus (AIV) remains a global threat, with waterfowl serving as the primary reservoir from which viruses spread to other hosts. Highly pathogenic avian influenza (HPAI) H5 viruses continue to be a devastating threat to the poultry industry and an incipient threat to humans. A cross-sectional study was conducted in seven districts of Bangladesh to estimate the prevalence and subtypes (H3, H5, and H9) of AIV in poultry and identify underlying risk factors and phylogenetic analysis of AIVs subtypes H5N1 and H3N8. Cloacal and oropharyngeal swab samples were collected from 500 birds in live bird markets (LBMs) and poultry farms. Each bird was sampled by cloacal and oropharyngeal swabbing, and swabs were pooled for further analysis. Pooled samples were analyzed for the influenza A virus (IAV) matrix (M) gene, followed by H5 and H9 molecular subtyping using real-time reverse transcription-polymerase chain reaction (rRT-PCR). Non-H5 and Non-H9 influenza A virus positive samples were sequenced to identify possible subtypes. Selected H5 positive samples were subjected to hemagglutinin (HA) and neuraminidase (NA) gene sequencing. Multivariable logistic regression was used for risk factor analysis. We found that IAV M gene prevalence was 40.20% (95% CI 35.98-44.57), with 52.38%, 46.96%, and 31.11% detected in chicken, waterfowl, and turkey, respectively. Prevalence of H5, H3, and H9 reached 22%, 3.4%, and 6.9%, respectively. Waterfowl had a higher risk of having AIV (AOR: 4.75), and H5 (AOR: 5.71) compared to chicken; more virus was detected in the winter season than in the summer season (AOR: 4.93); dead birds had a higher risk of AIVs and H5 detection than healthy birds, and the odds of H5 detection increased in LBM. All six H5N1 viruses sequenced were clade 2.3.2.1a-R1 viruses circulating since 2015 in poultry and wild birds in Bangladesh. The 12 H3N8 viruses in our study formed two genetic groups that had more similarity to influenza viruses from wild birds in Mongolia and China than to previous H3N8 viruses from Bangladesh. The findings of this study may be used to modify guidelines on AIV control and prevention to account for the identified risk factors that impact their spread.

Single-cell transcriptomics of peripheral blood in the aging mouse.

Compositional and transcriptional changes in the hematopoietic system have been used as biomarkers of immunosenescence and aging. Here, we use single-cell RNA-sequencing to study the aging peripheral blood in mice and characterize the changes in cell-type composition and transcriptional profiles associated with age. We identified 17 clusters from a total of 14,588 single cells. We detected a general upregulation of antigen processing and presentation and chemokine signaling pathways and a downregulation of genes involved in ribosome pathways with age. In old peripheral blood, we also observed an increased percentage of cells expressing senescence markers (Cdkn1a, and Cdkn2a). In addition, we detected a cluster of activated T cells exclusively found in old blood, with lower expression of Cd28 and higher expression of Bcl2 and Cdkn2a, suggesting that the cells are senescent and resistant to apoptosis.

Which smokers enroll in a hospital based smoking cessation trial? Survey of smoking related behaviors, quit attempts, and motivation to quit.

BACKGROUND: Understanding smoking behaviors in hospital patients who smoke may improve inpatient cessation treatments. This study aimed to describe smoking-related behaviors, past-quit attempts, and self-reported difficulties experienced in quitting among those who enrolled in a smoking cessation trial of varenicline. METHODS: Baseline data were obtained from adult hospitalized smokers (average ≥ 10 cigarettes/day in 4-weeks prior to hospitalization) who enrolled in a randomized, placebo-controlled trial of varenicline ± nicotine lozenges at five Australian public hospitals. A logistic regression model tested the association between participant characteristics and quitting in the previous 12 months. RESULTS: Participants' (n = 320; 57% male, 52.5 ± 12.1 years old) motivation and confidence in quitting were high. A total of 120 participants (37.5%) had attempted quitting in the previous 12-months. Prior hospitalization (P = .008) and employment status (P = .015) were significantly associated with past quit attempts. No statistically significant differences were noted in the reason for hospitalization or the level of nicotine dependence between participants who attempted quitting in the previous 12 months and their counterparts. Smoking cessation pharmacotherapy was used by 55% of those attempting to quit; nicotine replacement therapy (65.2%) and varenicline (16.7%) most common. Stress or anxiety, urges to smoke and a lack of motivation were the difficulties experienced in past quit attempts. CONCLUSIONS: Those who had a prior hospitalization and were unemployed had significantly greater odds of reporting past quit attempts. Further research is needed to investigate the degree of adherence among inpatient smokers with the smoke-free hospital policies and the frequency of NRT provision and uptake on admission.

Human exposures to Phytolacca americana in Kentucky.

Phytolacca americana, known more commonly as "pokeweed", is a large perennial plant found ubiquitously throughout the United States. Despite known toxicities, characterization of pokeweed exposure demographics, symptoms, treatments, and outcomes is currently limited. The objective of this study is to describe human pokeweed exposures, treatments, and outcomes, in the state of Kentucky, between 2000 and 2019. The National Poison Database System was queried for all Phytolacca americana exposures in the state of Kentucky between 2000 and 2019. After the removal of non-human cases, investigators independently reviewed data to ensure all coding was appropriate per the standards set forth by NPDS. The primary objective of this study was to describe pokeweed exposure demographics within the state of Kentucky during the previously established timeframe. Secondary objectives included characterizing pokeweed treatment trends and evaluating their affiliated medical outcomes. 1669 cases of human pokeweed exposure were reported. Patients were predominantly young in age, with a median age of 3 years reported. The majority of patients were male (54.9%), with unintentional exposures representing most exposure reasons (97.2%). Oral ingestion of plant material represented the bulk of the exposure route (98.3%), with pokeberries most often implicated in these cases (93.9%). Exposures were generally well tolerated. 239 total adverse events were noted during the timeframe. Abdominal pain, nausea, vomiting, and diarrhea were most common. Dermal exposures resulted in cutaneous edema, pain, and swelling. Treatments were mainly supportive, with no deaths reported during the study timeframe. In conclusion, Phytolacca americana is commonly encountered in the United States. In this observational study, patients most heavily implicated in pokeweed exposures are young males. Oral ingestion was most commonly reported, with berries most often implicated. Exposures are generally well tolerated, with gastrointestinal symptoms most frequently reported. Cutaneous exposures represent an underappreciated exposure route. Treatments are largely supportive in nature.

Stem cell phenotype predicts therapeutic response in glioblastomas with MGMT promoter methylation.

A growing body of evidence supports the presence of a population of cells in glioblastoma (GBM) with a stem cell-like phenotype which shares certain biological markers with adult neural stem cells, including expression of SOX2, CD133 (PROM1), and NES (nestin). This study was designed to determine the relationship between the expression of these stem cell markers and the clinical outcome in GBM patients. We quantified the intensity of expression of the proteins CD133 and SOX2 by immunohistochemistry (IHC) in a cohort of 86 patients with IDH-wildtype GBM, and evaluated patient outcomes using Kaplan-Meier and Cox proportional hazards analysis. In our patients, MGMT promoter methylation status and age were predictors of overall survival and progression free survival. The levels of SOX2 and CD133 were not associated with outcome in univariate analysis; however, stratification of tumors based on low or high levels of CD133 or SOX2 expression revealed that MGMT methylation was a predictor of progression-free survival and overall survival only for tumors with high levels of expression of CD133 or SOX2. Tumors with low levels of expression of CD133 or SOX2 did not show any relationship between MGMT methylation and survival. This relationship between MGMT and stem cell markers was confirmed in a second patient cohort, the TCGA dataset. Our results show that stratification of GBM by the level of expression of CD133 and SOX2 improved the prognostic power of MGMT promoter methylation status, identifying a low-expressing group in which the clinical outcome is not associated with MGMT promoter methylation status, and a high-expressing group in which the outcome was strongly associated with MGMT promoter methylation status. These findings support the concept that the presence of a high stem cell phenotype in GBM, as marked by expression of SOX2 or CD133, may be associated with the clinical response to treatment.

Understanding the aging hypothalamus, one cell at a time.

The hypothalamus is a brain region that integrates signals from the periphery and the environment to maintain organismal homeostasis. To do so, specialized hypothalamic neuropeptidergic neurons control a range of processes, such as sleep, feeding, the stress response, and hormone release. These processes are altered with age, which can affect longevity and contribute to disease status. Technological advances, such as single-cell RNA sequencing, are upending assumptions about the transcriptional identity of cell types in the hypothalamus and revealing how distinct cell types change with age. In this review, we summarize current knowledge about the contribution of hypothalamic functions to aging. We highlight recent single-cell studies interrogating distinct cell types of the mouse hypothalamus and suggest ways in which single-cell 'omics technologies can be used to further understand the aging hypothalamus and its role in longevity.

Single-cell analysis of the aging female mouse hypothalamus.

Alterations in metabolism, sleep patterns, body composition, and hormone status are all key features of aging. While the hypothalamus is a well-conserved brain region that controls these homeostatic and survival-related behaviors, little is known about the intrinsic features of hypothalamic aging. Here, we perform single nuclei RNA-sequencing of 40,064 hypothalamic nuclei from young and aged female mice. We identify cell type-specific signatures of aging in neuronal subtypes as well as astrocytes and microglia. We uncover changes in cell types critical for metabolic regulation and body composition, and in an area of the hypothalamus linked to cognition. Our analysis also reveals an unexpected female-specific feature of hypothalamic aging: the master regulator of X-inactivation, Xist, is elevated with age, particularly in hypothalamic neurons. Moreover, using machine learning, we show that levels of X-chromosome genes, and Xist itself, can accurately predict cellular age. This study identifies critical cell-specific changes of the aging hypothalamus in mammals, and uncovers a potential marker of neuronal aging in females.

Offering mailed nicotine replacement therapy and Quitline support before elective surgery: a randomised controlled trial.

OBJECTIVE: To assess whether offering free mailed nicotine replacement therapy (NRT) and telephone counselling to smokers on elective surgery waiting lists increases quitting before surgery. DESIGN, SETTING: Randomised, controlled trial at Frankston Hospital, a public tertiary referral hospital in Melbourne. PARTICIPANTS: Adult smokers added to elective surgery waiting lists for operations at least ten days in the future, 1 April 2019 - 3 April 2020. INTERVENTION: In addition to normal care, intervention participants received a brochure on the risks of low frequency smoking, an offer of Quitline call-back registration, and an offer of mailed NRT according to reported daily smoking: 1-9 cigarettes/day, 2 mg lozenges; 10-15/day, 7-14 mg patches [three weeks] and 2 mg lozenges; > 15/day, 7-21 mg patches [five weeks] and 2 mg lozenges. MAIN OUTCOME MEASURES: Primary outcome: quitting at least 24 hours before surgery, verified by exhaled carbon monoxide testing. SECONDARY OUTCOMES: quitting at least four weeks before surgery, adverse events, and (for those who had quit before surgery) abstinence three months after surgery. RESULTS: Of 748 eligible participants (control, 363; intervention, 385), 516 (69%) had undergone elective surgery when the trial was terminated early (for COVID-19-related reasons) (intervention group, 274; control group, 242). 122 of the 385 intervention participants (32%) had accepted the offer of cessation support. The proportions of intervention participants who quit at least 24 hours before surgery (18% v 9%; odds ratio [OR], 1.97; 95% CI, 1.22-3.15) or at least four weeks before surgery (9% v 4%; OR, 2.20; 95% CI, 1.08-4.50) were larger than for the control group. Three months after surgery, 27 of 58 intervention (47%) and 12 of 25 control participants (48%) who quit before surgery reported not smoking in the preceding seven days. No major adverse events were reported. CONCLUSION: Uptake of free mailed NRT and Quitline support by smokers on elective surgery waiting lists was good, and offering additional support was associated with higher proportions of smokers quitting before surgery. TRIAL REGISTRATION: Australian New Zealand Clinical Trials Registry, ACTRN12619000032156 (prospective).

Intravenous phytonadione administered orally in reducing warfarin-related coagulopathy.

INTRODUCTION: The cost of phytonadione tablets has increased markedly and is significantly higher than the intravenous formulation. The intravenous formulation given orally is a potential alternative but has not been directly evaluated in comparison to the commercially available tablet. The objective of this study was to evaluate the efficacy of phytonadione intravenous solution given orally compared to commercially available phytonadione tablets for reversal of coagulopathy related to warfarin. METHODS: We conducted a retrospective, observational study of adult patients who received phytonadione tablets and the IV formulation orally for warfarin-related coagulopathy. The international normalized ratio (INR) was measured before and after phytonadione administration. The primary outcome was INR <1.5 at 24 h after phytonadione administration. RESULTS: From January 1, 2015 to August 1, 2018 a total of 200 patients were identified. In total, 58% (n = 116) patients received IV phytonadione solution given orally and 42% (n = 84) patients received the tablets. The primary outcome of INR <1.5 at 24 h was not significantly different between groups (p = 0.321). DISCUSSION: The IV phytonadione solution given by mouth and the tablet formulation performed similarly.

Adverse effects associated with bupropion therapeutic errors in adults reported to four United States Poison Centers.

CONTEXT: Bupropion is a frequently used medication. Excessive doses may cause altered mental status, seizures, and dysrhythmias. There is a need for accurate estimate of seizure risk with therapeutic errors and determination if minor symptoms are harbingers of more severe effects. METHODS: A retrospective review of adult, acute, unintentional therapeutic error, single substance bupropion ingestions with known outcome reported to four poison centers from January 1, 2004 to December 31, 2016. Data included age, gender, single error dose, total bupropion dose over 18 h, prior history of seizure, management site, observation time, occurrence of an out-of-hospital adverse event, "jittery"/anxious/agitated, tachycardia/palpitations, seizures, and dysrhythmias. We recorded the total bupropion dose over 18 h if known; otherwise, we used the single error dose. We compared means for parametric data. We used Fisher's exact test and Mann-Whitney for nonparametric data. RESULTS: We identified 754 potential cases, of which 637 met inclusion criteria after case review. Median age was 42 years, and 76.1% were female. Cases were predominantly managed at home (56.2%). Outcomes were no effect (50.1%), minor (45.5%) and moderate (4.4%). The reported dose with no effect/minor outcome was 694 (±297) mg, and for moderate outcome was 1250 (±815) mg (p < 0.0001). Seizures occurred in four patients with median onset time of 7 h [range 2-21.5 h]. The median reported dose in patients who seized was 900 mg [range 600-3000 mg]. Of patients who developed a seizure and/or an out-of-hospital adverse event, 83% were "jittery"/anxious/agitated whereas "jittery"/anxious/agitated was present in 27% of cases that did not (p = 0.008). Tachycardia/palpitations was reported in 12% of cases; more serious dysrhythmias were not reported. CONCLUSIONS: Outcomes from single unintentional ingestions of bupropion in adults are overall mild and appear to be dose related. Home management may be an option with doses up to 900 mg in an appropriate patient population.

Corticotropin Releasing Factor Binding Protein as a Novel Target to Restore Brain Homeostasis: Lessons Learned From Alcohol Use Disorder Research.

Stress is well-known to contribute to the development of many psychiatric illnesses including alcohol and substance use disorder (AUD and SUD). The deleterious effects of stress have also been implicated in the acceleration of biological age, and age-related neurodegenerative disease. The physio-pathology of stress is regulated by the corticotropin-releasing factor (CRF) system, the upstream component of the hypothalamic-pituitary-adrenal (HPA) axis. Extensive literature has shown that dysregulation of the CRF neuroendocrine system contributes to escalation of alcohol consumption and, similarly, chronic alcohol consumption contributes to disruption of the stress system. The CRF system also represents the central switchboard for regulating homeostasis, and more recent studies have found that stress and aberrations in the CRF pathway are implicated in accelerated aging and age-related neurodegenerative disease. Corticotropin releasing factor binding protein (CRFBP) is a secreted glycoprotein distributed in peripheral tissues and in specific brain regions. It neutralizes the effects of CRF by sequestering free CRF, but may also possess excitatory function by interacting with CRF receptors. CRFBP's dual role in influencing CRF bioavailability and CRF receptor signaling has been shown to have a major part in the HPA axis response. Therefore, CRFBP may represent a valuable target to treat stress-related illness, including: development of novel medications to treat AUD and restore homeostasis in the aging brain. This narrative review focuses on molecular mechanisms related to the role of CRFBP in the progression of addictive and psychiatric disorders, biological aging, and age-related neurodegenerative disease. We provide an overview of recent studies investigating modulation of this pathway as a potential therapeutic target for AUD and age-related neurodegenerative disease.

Altered Chromatin States Drive Cryptic Transcription in Aging Mammalian Stem Cells.

A repressive chromatin state featuring trimethylated lysine 36 on histone H3 (H3K36me3) and DNA methylation suppresses cryptic transcription in embryonic stem cells. Cryptic transcription is elevated with age in yeast and nematodes, and reducing it extends yeast lifespan, though whether this occurs in mammals is unknown. We show that cryptic transcription is elevated in aged mammalian stem cells, including murine hematopoietic stem cells (mHSCs) and neural stem cells (NSCs) and human mesenchymal stem cells (hMSCs). Precise mapping allowed quantification of age-associated cryptic transcription in hMSCs aged in vitro. Regions with significant age-associated cryptic transcription have a unique chromatin signature: decreased H3K36me3 and increased H3K4me1, H3K4me3, and H3K27ac with age. Genomic regions undergoing such changes resemble known promoter sequences and are bound by TBP even in young cells. Hence, the more permissive chromatin state at intragenic cryptic promoters likely underlies increased cryptic transcription in aged mammalian stem cells.

Changing and stable chromatin accessibility supports transcriptional overhaul during neural stem cell activation and is altered with age.

Neural stem cells (NSCs) in the adult and aged brain are largely quiescent, and require transcriptional reprogramming to re-enter the cell cycle. However, the mechanisms underlying these changes and how they are altered with age remain undefined. Here, we identify the chromatin accessibility differences between primary neural stem/progenitor cells in quiescent and activated states. These distinct cellular states exhibit shared and unique chromatin profiles, both associated with gene regulation. Accessible chromatin states specific to activation or quiescence are active enhancers bound by key pro-neurogenic and quiescence factors. In contrast, shared sites are enriched for core promoter elements associated with translation and metabolism. Unexpectedly, through integrated analysis, we find that many sites that become accessible during NSC activation are linked to gene repression and associated with pro-quiescence factors, revealing a novel mechanism that may preserve quiescence re-entry. Furthermore, we report that in aged NSCs, chromatin regions associated with metabolic and transcriptional functions bound by key pro-quiescence transcription factors lose accessibility, suggesting a novel mechanism of age-associated NSC dysfunction. Together, our findings reveal how accessible chromatin states regulate the transcriptional switch between NSC quiescence and activation, and how this switch is affected with age.