Aortic tissue stiffness and tensile strength are correlated with density changes following proteolytic treatment.

INTRODUCTION: Dissection or rupture of the aorta is accompanied by high mortality rates, and there is a pressing need for better prediction of these events for improved patient management and clinical outcomes. Biomechanically, these events represent a situation wherein the locally acting wall stress exceed the local tissue strength. Based on recent reports for polymers, we hypothesized that aortic tissue failure strength and stiffness are directly associated with tissue mass density. The objective of this work was to test this novel hypothesis for porcine thoracic aorta. METHODS: Three tissue specimens from freshly harvested porcine thoracic aorta were treated with either collagenase or elastase to selectively degrade structural proteins in the tissue, or with phosphate buffer saline (control). The tissue mass and volume of each specimen were measured before and after treatment to allow for density calculation, then mechanically tested to failure under uniaxial extension. RESULTS: Protease treatments resulted in statistically significant tissue density reduction (sham vs. collagenase p = 0.02 and sham vs elastase p = 0.003), which in turn was significantly and directly correlated with both ultimate tensile strength (sham vs. collagenase p = 0.02 and sham vs elastase p = 0.03) and tangent modulus (sham vs. collagenase p = 0.007 and sham vs elastase p = 0.03). CONCLUSIONS: This work demonstrates for the first time that tissue stiffness and tensile strength are directly correlated with tissue density in proteolytically-treated aorta. These findings constitute an important step towards understanding aortic tissue failure mechanisms and could potentially be leveraged for non-invasive aortic strength assessment through density measurements, which could have implications to clinical care.

Molecular classification of metastatic and recurrent endometrial endometrioid carcinoma: prognostic relevance among low- and high-stage tumours.

AIMS: Molecular classification according to The Cancer Genome Atlas (TCGA) improves endometrial endometrioid carcinoma (EEC) prognostication and has specific treatment implications; however, original data were skewed towards low-grade and low-stage tumours. Herein, we molecularly classify EECs metastatic at the time of diagnosis or with subsequently documented recurrent/metastatic disease to examine correlation with clinical outcomes. METHODS: TCGA categories include POLE-mutated, microsatellite instability (MSI), p53 abnormal (p53 abnl) and no specific molecular profile (NSMP). POLE targeted sequencing at exons 9, 11, 13 and 14 and immunohistochemistry (IHC) for PMS2, MSH6 and p53 were performed to establish molecular classification. RESULTS: The distribution in our cohort of 141 EECs was similar to that generally reported in EEC, with nine POLE-mutated (6%), 45 MSI (32%), 16 p53 abnl (11%) and 71 NSMP (50%), with similar distributions between low- and high-stage cohorts. We demonstrate that when stratified by molecular subtype, disease-specific survival from the time of high-stage (stages III-IV) presentation or time of recurrence in low-stage (stages I-II) disease among metastatic and/or recurrent EEC is strongly associated with TCGA classification (high-stage P = 0.02, low-stage P = 0.017). Discordant molecular classification between primary and metastatic/recurrent tumours occurred in four of 105 (3.8%) patients, two related to PMS2/MSH6 IHC and two related to p53 IHC. CONCLUSIONS: We demonstrate that molecular classification is prognostically relevant not only at the time of diagnosis, but also at the time of recurrence and in the metastatic setting. Rare subclonal alterations occur and suggest a role for confirming TCGA classification in recurrent/metastatic tumours.

Dietary glycine supplementation activates mechanistic target of rapamycin signaling pathway in tissues of pigs with intrauterine growth restriction.

The mechanistic target of rapamycin (mTOR) cell signaling pathway serves as the central mechanism for the regulation of tissue protein synthesis and growth. We recently reported that supplementing 1% glycine to corn- and soybean meal-based diets enhanced growth performance between weaning and market weights in pigs with intrauterine growth restriction (IUGR). Results of recent studies have revealed an important role for glycine in activating mTOR and protein synthesis in C2C12 muscle cells. Therefore, the present study tested the hypothesis that dietary glycine supplementation enhanced the mTOR cell signaling pathway in skeletal muscle and other tissues of IUGR pigs. At weaning (21 d of age), IUGR pigs and litter mates with normal birth weights (NBW) were assigned randomly to one of the two groups: supplementation with either 1% glycine or 1.19% l-alanine (isonitrogenous control) to a corn- and soybean meal-based diet. Tissues were obtained from the pigs within 1 wk after the feeding trial ended at 188 d of age to determine the abundances of total and phosphorylated forms of mTOR and its two major downstream proteins: eukaryotic initiation factor 4E-binding protein-1 (4EBP1) and ribosomal protein S6 kinase-1 (p70S6K). Results showed that IUGR decreased (P < 0.05) the abundances of both total and phosphorylated mTOR, 4EBP1, and p70S6K in the gastrocnemius muscle and jejunum. In the longissimus lumborum muscle of IUGR pigs, the abundances of total mTOR did not differ (P > 0.05) but those for phosphorylated mTOR and both total and phosphorylated 4EBP1 and p70S6K were downregulated (P < 0.05), when compared to NBW pigs. These adverse effects of IUGR in the gastrocnemius muscle, longissimus lumborum muscle, and jejunum were prevented (P < 0.05) by dietary glycine supplementation. Interestingly, the abundances of total or phosphorylated mTOR, 4EBP1, and p70S6K in the liver were not affected (P > 0.05) by IUGR or glycine supplementation. Collectively, our findings indicate that IUGR impaired the mTOR cell signaling pathway in the tissues of pigs and that adequate glycine intake was crucial for maintaining active mTOR-dependent protein synthesis for the growth and development of skeletal muscle.

Soybean meal is the major source of dietary protein for growing pigs in many regions of the world, including North America, South America, and Asia. However, this conventional feedstuff is recognized to be severely deficient in glycine (the most abundant amino acid in the bodies of animals, including pigs). Compared to pigs with normal birth weights (NBW), pigs with intrauterine growth restriction (IUGR) have a lower ability to synthesize glycine and reduced growth performance between weaning and market weights. Results of recent studies with cultured muscle cells have revealed that glycine stimulates the mechanistic target of rapamycin (mTOR) cell signaling pathway (the master activator of initiation of protein synthesis in tissues) to promote protein synthesis and cell growth. There is also evidence that the mTOR pathway is impaired in the skeletal muscle of young IUGR pigs. Thus, dietary glycine supplementation may play an important role in maintaining an active mTOR cell signaling pathway for the growth of tissues, particularly skeletal muscle. Results of this study indicated that market-weight IUGR pigs had lower abundances of both total and phosphorylated mTOR, as well as its downstream target proteins in the gastrocnemius muscle, longissimus lumborum muscle, and jejunum, when compared with NBW pigs. In contrast, neither IUGR nor glycine supplementation affected the mTOR cell signaling pathway in the liver of pigs. Taken together, these novel findings indicate that IUGR pigs have insufficient cell signaling via the mTOR cell pathway in a tissue-specific manner during their growing-finishing phases of development and that this negative impact of IUGR can be mitigated by supplementing corn- and soybean meal-based diets with 1% glycine.

Dietary glycine supplementation enhances glutathione availability in tissues of pigs with intrauterine growth restriction.

This study tested the hypothesis that dietary supplementation with glycine enhances the synthesis and concentrations of glutathione (GSH, a major antioxidant) in tissues of pigs with intrauterine growth restriction (IUGR). At weaning (21 d of age), IUGR pigs and litter mates with normal birth weights (NBW) were assigned randomly to one of two groups, representing supplementation with 1% glycine or 1.19% l-alanine (isonitrogenous control) to a corn- and soybean meal-based diet. Blood and other tissues were obtained from the pigs within 1 wk after the feeding trial ended at 188 d of age to determine GSH, oxidized GSH (GSSG), and activities of GSH-metabolic enzymes. Results indicated that concentrations of GSH + GSSG or GSH in plasma, liver, and jejunum (P < 0.001) and concentrations of GSH in longissimus lumborum and gastrocnemius muscles (P < 0.05) were lower in IUGR pigs than in NBW pigs. In contrast, IUGR increased GSSG/GSH ratios (an indicator of oxidative stress) in plasma (P < 0.001), jejunum (P < 0.001), both muscles (P < 0.05), and pancreas (P = 0.001), while decreasing activities of γ-glutamylcysteine synthetase and GSH synthetase in liver (P < 0.001) and jejunum (P < 0.01); and GSH reductase in jejunum (P < 0.01), longissimus lumborum muscle (P < 0.01), gastrocnemius muscle (P < 0.05), and pancreas (P < 0.01). In addition, IUGR pigs had greater (P < 0.001) concentrations of thiobarbituric acid reactive substances (TBARS; an indicator of lipid peroxidation) in plasma, jejunum, muscles, and pancreas than NBW pigs. Compared with isonitrogenous controls, dietary glycine supplementation increased concentrations of GSH plus GSSG and GSH in plasma (P < 0.01), liver (P < 0.001), jejunum (P < 0.001), longissimus lumborum muscle (P = 0.001), and gastrocnemius muscle (P < 0.05); activities of GSH-synthetic enzymes in liver (P < 0.01) and jejunum (P < 0.05), while reducing GSSG/GSH ratios in plasma (P < 0.001), jejunum (P < 0.001), longissimus lumborum muscle (P < 0.001), gastrocnemius muscle (P = 0.01), pancreas (P < 0.05), and kidneys (P < 0.01). Concentrations of GSH plus GSSG, GSH, and GSSG/GSH ratios in kidneys were not affected (P > 0.05) by IUGR. Furthermore, glycine supplementation reduced (P < 0.001) TBARS concentrations in plasma, jejunum, muscles, and pancreas. Collectively, IUGR reduced GSH availability and induced oxidative stress in pig tissues, and these abnormalities were prevented by dietary glycine supplementation in a tissue-specific manner.

Pigs have the highest rate of intrauterine growth restriction (IUGR) among livestock species. These pigs, which have low birth weights (<1.1 kg) and account for ~15% to 20% of newborn pigs, are often culled after birth because they have lower growth performance and feed efficiency due to multiple factors (including oxidative stress in tissues), when compared with litter mates with normal birth weights (NBW). Much evidence shows that glutathione, which is a tripeptide synthesized from glutamate, glycine, and cysteine via enzymes (biological catalysts, γ-glutamylcysteine synthetase, and glutathione synthetase), is a major low-molecular-weight antioxidant in animal cells. Based on the findings of our recent study that dietary glycine supplementation enhanced the growth performance of IUGR pigs from weaning to market weight, the current study tested the hypothesis that this nutritional strategy increased the synthesis and availability of glutathione in their tissues. Our results indicated that the key organs of the digestive system (the small intestine, liver, and pancreas) as well as both longissimus lumborum and gastrocnemius muscles of IUGR pigs had lower concentrations of glutathione as compared with NBW pigs, due to reductions in both the activities of glutathione-synthetic enzymes and the availability of glycine. Dietary supplementation with 1% glycine prevented these metabolic deficiencies in tissues of IUGR pigs. Our findings support the notion that IUGR pigs fed conventional corn- and soybean meal-based diets do not synthesize adequate glutathione and that dietary glycine supplementation plays an important role in enhancing the availability of glutathione and mitigating oxidative stress to improve health and growth in these compromised animals.

Laryngological Complaint Prevalence in Hypermobile Ehlers-Danlos or Hypermobility Spectrum Disorders.

OBJECTIVE: The aim was to study laryngological complaints in patients with hypermobile Ehlers-Danlos syndrome (hEDS) or hypermobility spectrum disorders (HSD). METHODS: A total of 363 patients met inclusion for the study by completing questions related to voice, upper airway, and swallowing between July 7, 2020 and July 13, 2022. Demographic data, voice-related questions, and hypermobility diagnosis were analyzed retrospectively. From those, 289 patients were diagnosed with hEDS or HSD with 74 that did not meet the diagnostic criteria for either diagnosis serving as controls. RESULTS: There were no statistically significant differences between patients with hEDS and HSD regarding Voice Handicap Index (VHI-10) scores, voice, upper airway, or swallow complaints. However, more hEDS/HSD patients answered positively to the laryngeal dysfunction question versus controls (p = 0.031). 22.5% of hEDS/HSD patients (n = 65) reported hoarseness, of which 52.3% reported hoarseness >2 days/month. 33.9% (n = 98) with hEDS/HSD reported symptoms of dysphagia, and 27.0% (n = 78) reported laryngeal dysfunction symptoms. Controls demonstrated 20.3% prevalence of hoarseness, of which 46.7% reported hoarseness >2 days/month. 24.3% of controls had dysphagia and 14.9% laryngeal dysfunction symptoms. Of the 363 patients, VHI-10 scores >11 were more likely in patients reporting >2 days of hoarseness/month (p = 0.001) versus those with <2 days of hoarseness/month. There was an increased prevalence of voice, upper airway, and dysphagia symptoms in hEDS/HSD patients compared with previously reported prevalence data in the general population. CONCLUSION: A significant proportion of patients diagnosed with hypermobility due to hEDS or HSD were found to have voice, upper airway, and dysphagia symptoms. These rates are higher than those previously reported in the general population. LEVEL OF EVIDENCE: 3 Laryngoscope, 134:773-778, 2024.

Dietary glycine supplementation enhances postweaning growth and meat quality of pigs with intrauterine growth restriction.

Pigs with intrauterine growth restriction (IUGR) have suboptimum growth performance and impaired synthesis of glycine (the most abundant amino acid in the body). Conventional corn- and soybean meal-based diets for postweaning pigs contain relatively low amounts of glycine and may not provide sufficient glycine to meet requirements for IUGR pigs. This hypothesis was tested using 52 IUGR pigs and 52 litter mates with normal birth weights (NBW). At weaning (21 d of age), IUGR or NBW pigs were assigned randomly to one of two nutritional groups: supplementation of a corn-soybean meal-based diet with either 1% glycine plus 0.19% cornstarch or 1.19% L-alanine (isonitrogenous control). Feed consumption and body weight (BW) of pigs were recorded daily and every 2 or 4 wks, respectively. All pigs had free access to their respective diets and clean drinking water. Within 1 wk after the feeding trial ended at 188 d of age, blood and other tissue samples were obtained from pigs to determine concentrations of amino acids and meat quality. Neither IUGR nor glycine supplementation affected (P > 0.05) feed intakes of pigs per kg BW. The final BW, gain:feed ratio, carcass dressing percentages, and four-lean-cuts percentages of IUGR pigs were 13.4 kg, 4.4%, 2%, and 15% lower (P < 0.05) for IUGR pigs than NBW pigs, respectively. Compared with pigs in the alanine group, dietary glycine supplementation increased (P < 0.05) final BW, gain:feed ratio, and meat a* value (a redness score) by 3.8 kg, 11%, and 10%, respectively, while reducing (P < 0.05) backfat thickness by 18%. IUGR pigs had lower (P < 0.05) concentrations of glycine in plasma (-45%), liver (-25%), jejunum (-19%), longissimus dorsi muscle (-23%), gastrocnemius muscle (-26%), kidney (-15%), and pancreas (-6%), as compared to NBW pigs. In addition, dietary glycine supplementation increased (P < 0.05) concentrations of glycine in plasma and all analyzed tissues. Thus, supplementing 1% of glycine to corn-soybean meal-based diets improves the growth performance, feed efficiency, and meat quality of IUGR pigs.

About 15­20% of pigs are born naturally with low birth weights (<1.1 kg) due to intrauterine growth restriction (IUGR). These pigs are often culled after birth because they have lower growth performance and feed efficiency during the production period from weaning to market weight, compared with litter mates with normal birth weights (NBW). In many countries and regions (including North America, South America, and Asia), postweaning pigs are generally fed corn- and soybean meal-based diets that contain relatively a low amount of glycine. Glycine is the most abundant amino acid in the plasma and tissue proteins of pigs but may not be formed adequately from other amino acids in the body, particularly IUGR pigs that are now known to have an impaired ability for glycine synthesis. Results of the present study indicate that IUGR pigs fed conventional corn-SBM-based diets had lower concentrations of glycine in plasma and tissues (including skeletal muscle), compared with NBW litter mates. Dietary supplementation with 1% glycine improved the growth performance, feed efficiency, and meat quality of IUGR pigs. This simple nutritional means is expected to enhance the productivity of the global swine industry.

A local-scale One Health genomic surveillance of Clostridioides difficile demonstrates highly related strains from humans, canines, and the environment.

Although infections caused by Clostridioides difficile have historically been attributed to hospital acquisition, growing evidence supports the role of community acquisition in C. difficile infection (CDI). Symptoms of CDI can range from mild, self-resolving diarrhoea to toxic megacolon, pseudomembranous colitis, and death. In this study, we sampled C. difficile from clinical, environmental, and canine reservoirs in Flagstaff, Arizona, USA, to understand the distribution and transmission of the pathogen in a One Health framework; Flagstaff is a medium-sized, geographically isolated city with a single hospital system, making it an ideal site to characterize genomic overlap between sequenced C. difficile isolates across reservoirs. An analysis of 562 genomes from Flagstaff isolates identified 65 sequence types (STs), with eight STs being found across all three reservoirs and another nine found across two reservoirs. A screen of toxin genes in the pathogenicity locus identified nine STs where all isolates lost the toxin genes needed for CDI manifestation (tcdB, tcdA), demonstrating the widespread distribution of non-toxigenic C. difficile (NTCD) isolates in all three reservoirs; 15 NTCD genomes were sequenced from symptomatic, clinical samples, including two from mixed infections that contained both tcdB+ and tcdB- isolates. A comparative single nucleotide polymorphism (SNP) analysis of clinically derived isolates identified 78 genomes falling within clusters separated by ≤2 SNPs, indicating that ~19 % of clinical isolates are associated with potential healthcare-associated transmission clusters; only symptomatic cases were sampled in this study, and we did not sample asymptomatic transmission. Using this same SNP threshold, we identified genomic overlap between canine and soil isolates, as well as putative transmission between environmental and human reservoirs. The core genome of isolates sequenced in this study plus a representative set of public C. difficile genomes (n=136), was 2690 coding region sequences, which constitutes ~70 % of an individual C. difficile genome; this number is significantly higher than has been published in some other studies, suggesting that genome data quality is important in understanding the minimal number of genes needed by C. difficile. This study demonstrates the close genomic overlap among isolates sampled across reservoirs, which was facilitated by maximizing the genomic search space used for comprehensive identification of potential transmission events. Understanding the distribution of toxigenic and non-toxigenic C. difficile across reservoirs has implications for surveillance sampling strategies, characterizing routes of infections, and implementing mitigation measures to limit human infection.

Photodynamic Therapy Minimally Affects HEMA-DMAEMA Hydrogel Viscoelasticity.

Soft matter implants are a rapidly growing field in medicine for reconstructive surgery, aesthetic treatments, and regenerative medicine. Though these procedures are efficacious, all implants carry risks associated with microbial infection which are often aggressive. Preventative and responsive measures exist but are limited in applicability to soft materials. Photodynamic therapy (PDT) presents a means to perform safe and effective antimicrobial treatments in proximity to soft implants. HEMA-DMAEMA hydrogels are prepared with the photosensitizer methylene blue included at 10 and 100 µM in solution used for swelling over 2 or 4 days. Thirty minutes or 5 h of LED illumination at 9.20 m W c m 2 $9.20\frac{{mW}}{{c{m}^2}}$ is then used for PDT-induced generation of reactive oxygen species in direct contact with hydrogels to test viable limits of treatment. Frequency sweep rheological measurements reveal minimal overall changes in terms of loss modulus and loss factor but a statistically significant drop in storage modulus for some PDT doses, though within the range of controls and biological variation. These mild impacts suggest the feasibility of PDT application for infection clearing in proximity to soft implants. Future investigation with additional hydrogel varieties and current implant models will further detail the safety of PDT in implant applications.

Biting midges (Diptera: Ceratopogonidae) as putative vectors of zoonotic Onchocerca lupi (Nematoda: Onchocercidae) in northern Arizona and New Mexico, southwestern United States.

Onchocerca lupi (Rodonaja, 1967) is an understudied, vector-borne, filarioid nematode that causes ocular onchocercosis in dogs, cats, coyotes, wolves, and is also capable of infecting humans. Onchocercosis in dogs has been reported with increasing incidence worldwide. However, despite the growing number of reports describing canine O. lupi cases as well as zoonotic infections globally, the disease prevalence in endemic areas and vector species of this parasite remains largely unknown. Here, our study aimed to identify the occurrence of O. lupi infected dogs in northern Arizona, New Mexico, and Utah, United States and identify the vector of this nematode. A total of 532 skin samples from randomly selected companion animals with known geographic locations within the Navajo Reservation were collected and molecularly surveyed by PCR for the presence of O. lupi DNA (September 2019-June 2022) using previously published nematode primers (COI) and DNA sequencing. O. lupi DNA was detected in 50 (9.4%) sampled animals throughout the reservation. Using positive animal samples to target geographic locations, pointed hematophagous insect trapping was performed to identify potential O. lupi vectors. Out of 1,922 insects screened, 38 individual insects and 19 insect pools tested positive for the presence of O. lupi, all of which belong to the Diptera family. This increased surveillance of definitive host and biological vector/intermediate host is the first large scale prevalence study of O. lupi in companion animals in an endemic area of the United States, and identified an overall prevalence of 9.4% in companion animals as well as multiple likely biological vector and putative vector species in the southwestern United States. Furthermore, the identification of these putative vectors in close proximity to human populations coupled with multiple, local zoonotic cases highlight the One Health importance of O. lupi.

Minimal/Measurable Residual Disease Monitoring in Patients with Lymphoid Neoplasms by High-Throughput Sequencing of the T-Cell Receptor.

High-throughput sequencing of the T-cell receptor beta (TRB) and gamma (TRG) loci is increasingly utilized due to its high sensitivity, specificity, and versatility in the diagnosis of various T-cell malignancies. Application of these technologies for tracking disease burden can be valuable in detecting recurrence, determining response to therapy, guiding future management of patients, and establishing endpoints for clinical trials. In this study, the performance of the commercially available LymphoTrack high-throughput sequencing assay was assessed for determining residual disease burden in patients with various T-cell malignancies. A custom bioinformatics pipeline and database was also developed to facilitate minimal/measurable residual disease analysis and clinical reporting. This assay demonstrated excellent test performance characteristics, achieving a sensitivity of 1 of 100,000 T-cell equivalents for the DNA inputs evaluated and high concordance with orthogonal testing methods. This assay was further utilized to correlate disease burden in several patients, demonstrating its potential utility for monitoring patients with T-cell malignancies.

Effect of Chronic Cough on Voice Measures in Patients With Dysphonia.

INTRODUCTION: Measuring the impact of chronic cough on voice quality can be difficult and challenging in daily practice. Evidence about its potential effects on diagnostic tools used in voice evaluation is lacking. We hypothesized that the presence of chronic cough plays a role in patients' perception of dysphonia severity, leading to a mismatch between the subjective, objective, and perceptual evaluations. METHODS: A retrospective chart review involving patients with a diagnosis of dysphonia and a complete speech voice evaluation was performed. A total of 311 patients were stratified into two different groups according to the presence of chronic cough. A total of 151 patients were assigned to the dysphonia and chronic cough group, while 160 patients were assigned to the dysphonia only group. During the initial evaluation, patients completed the Voice Handicap Index (VHI)-30, Glottal Function Index (GFI), and Reflux Symptoms Index (RSI). Voice evaluation also included aerodynamic/acoustic measures and the application of the GRBAS scale by a speech-language specialist. A paired t test and a linear regression analysis were used to compare subjective, perceptual, and aerodynamic/acoustic measures in both groups. RESULTS: The mean VHI-30 and GFI were elevated in both groups but significantly lower among patients with dysphonia and chronic cough when compared to patients with dysphonia only (P= 0.01). Additionally, a significantly higher RSI was found among patients with dysphonia and chronic cough (P< 0.01). No difference in aerodynamic/acoustic measures was found between groups (P> 0.05). Our linear regression model demonstrated a significant effect of the presence of chronic cough on the VHI-30, RSI, and GFI questionnaires (P< 0.05). Our model also found that the VHI-30 is a significant predictor for the (G), (B), (A), and (S) components of the GRBAS scale (P< 0.05). CONCLUSION: The presence of chronic cough has a significant impact on the different patient-reported outcome measures, including VHI-30, RSI, and GFI. The use of VHI-30 as a predictor for the GRBAS scale reinforces the importance of subjective and perceptual assessment among patients with voice disorders and establishes a new area for exploration.

Characterising advertising strategies and expenditures for conventional and newer smokeless tobacco products.

OBJECTIVE: Nicotine pouch products are an emerging and rapidly growing smokeless tobacco (ST) category in the USA. Little is known about the promotional strategies and media channels used to advertise this ST category or the extent to which the marketing strategies differ from strategies used to promote 'conventional' smokeless products (eg, snuff). We describe the nature, timing of and expenditures related to conventional, snus and newer ST product advertising on print, broadcast and internet media. METHODS: Advertising expenditures were collected using Kantar Media's 'Stradegy' tool, which provides advertising data including dollars spent promoting specific products across various media channels, including print magazines and newspapers, broadcast television and radio, outdoor posters and billboards, and internet. We identified 306 smokeless products within Kantar database and collected ad expenditures retrospectively for January 2018-April 2020. Promotional expenditures were aggregated by product category, by month and by designated market area (DMA). RESULTS: Kantar data analysis returned 28 conventional ST, 22 oral nicotine and 3 snus products (53 total) advertised during the period of observation, with over $71 million spent collectively on ST promotion. Across categories, more advertising dollars were spent on conventional ST products (63%) than newer oral nicotine products (25%) or snus (12%). However, during the later 9-month period from August 2019 to April 2020, oral nicotine products accounted for the majority of monthly ad spending. Most ad spending was placed in the national market ($66.5 million), with Atlanta ($1.1 million), Houston ($1 million) and Las Vegas ($0.8 million) as the top three local DMAs for expenditures. DISCUSSION: Advertising expenditures for nicotine pouches have recently exceeded conventional ST product advertising and nicotine pouches are being promoted nationally. Marketing surveillance as well as understanding consumer appeal, perceptions and consumption are critical next steps in tracking potential uptake of these new products.

LupiQuant: A real-time PCR based assay for determining host-to-parasite DNA ratios of Onchocerca lupi and host Canis lupus from onchocercosis samples.

Onchocerca lupi is a filarial nematode that causes ocular onchocercosis in canines globally including North America and areas of Europe, North Africa, and the Middle East. Reported incidence of this parasite in canines has continued to steadily escalate since the early 21st century and was more recently documented in humans. Whole genome sequencing (WGS) of this parasite can provide insight into gene content, provide novel surveillance targets, and elucidate the origin and range expansion. However, past attempts of whole genome sequencing of other Onchocerca species reported a substantial portion of their data unusable due to the variable over-abundance of host DNA in samples. Here, we have developed a method to determine the host-to-parasite DNA ratio using a quantitative PCR (qPCR) approach that relies on two standard plasmids each of which contains a single copy gene specific to the parasite genus Onchocerca (major body wall myosin gene, myosin) or a single copy gene specific to the canine host (polycystin-1 precursor, pkd1). These plasmid standards were used to determine the copy number of the myosin and pkd1 genes within a sample to calculate the ratio of parasite and host DNA. Furthermore, whole genome sequence (WGS) data for three O. lupi isolates were consistent with our host-to-parasite DNA ratio results. Our study demonstrates, despite unified DNA extraction methods, variable quantities of host DNA within any one sample which will likely affect downstream WGS applications. Our quantification assay of host-to-parasite genome copy number provides a robust and accurate method of assessing canine host DNA load in an O. lupi specimen that will allow informed sample selection for WGS. This study has also provided the first whole genome draft sequence for this species. This approach is also useful for future focused WGS studies of other parasites.

Refractory Methemoglobinemia Secondary to Topical Dapsone With Subsequent Autoimmune Hemolytic Anemia.

We present a case of refractory methemoglobinemia with subsequent autoimmune hemolytic anemia in a young female after two days of topical dapsone use. A 15-year-old female with no known genetic risk factors was found to have a methemoglobinemia concentration of 37.1% after presenting with cyanosis, dyspnea, tachycardia, and oxygen saturation of 88% on 15 L of oxygen via a non-rebreather mask. Despite treatment with methylene blue, her methemoglobin concentrations continued to spike, requiring additional doses of methylene blue in addition to ascorbic acid and cimetidine. After discharge on the fourth day, she presented to another hospital with similar symptoms and was again found to have methemoglobinemia before developing autoimmune hemolytic anemia. This patient had no known underlying risk factors, including a normal BMI, normal renal function, two negative glucose-6-phosphate-dehydrogenase (G6PD) deficiency tests, and surprisingly a negative Coombs test. Although rare, particularly in the setting of topical dapsone use, methemoglobinemia remains an important consideration in the differential diagnosis of cyanosis and hypoxia, with early recognition by the emergency medicine physician being imperative for good patient outcomes.

Coccidioidomycosis in Northern Arizona: an Investigation of the Host, Pathogen, and Environment Using a Disease Triangle Approach.

Coccidioides immitis and Coccidioides posadasii are the etiological agents of coccidioidomycosis (Valley fever [VF]). Disease manifestation ranges from mild pneumonia to chronic or extrapulmonary infection. If diagnosis is delayed, the risk of severe disease increases. In this report, we investigated the intersection of pathogen, host, and environment for VF cases in Northern Arizona (NAZ), where the risk of acquiring the disease is much lower than in Southern Arizona. We investigated reported cases and assessed pathogen origin by comparing genomes of NAZ clinical isolates to isolates from other regions. Lastly, we surveyed regional soils for presence of Coccidioides. We found that cases of VF increased in NAZ in 2019, and Coccidioides NAZ isolates are assigned to Arizona populations using phylogenetic inference. Importantly, we detected Coccidioides DNA in NAZ soil. Given recent climate modeling of the disease that predicts that cases will continue to increase throughout the region, and the evidence presented in this report, we propose that disease awareness outreach to clinicians throughout the western United States is crucial for improving patient outcomes, and further environmental sampling across the western U.S. is warranted. IMPORTANCE Our work is the first description of the Valley fever disease triangle in Northern Arizona, which addresses the host, the pathogen, and the environmental source in the region. Our data suggest that the prevalence of diagnosed cases rose in 2019 in this region, and some severe cases necessitate hospitalization. We present the first evidence of Coccidioides spp. in Northern Arizona soils, suggesting that the pathogen is maintained in the local environment. Until disease prevention is an achievable option via vaccination, we predict that incidence of Valley fever will rise in the area. Therefore, enhanced awareness of and surveillance for coccidioidomycosis is vital to community health in Northern Arizona.

Sox9 and Lef1 Regulate the Fate and Behavior of Airway Glandular Progenitors in Response to Injury.

Cartilaginous airways of larger mammals and the mouse trachea contain at least 3 well-established stem cell compartments, including basal cells of the surface airway epithelium (SAE) and ductal and myoepithelial cells of the submucosal glands (SMG). Here we demonstrate that glandular Sox9-expressing progenitors capable of SAE repair decline with age in mice. Notably, Sox9-lineage glandular progenitors produced basal and ciliated cells in the SAE, but failed to produce secretory cells. Lef1 was required for glandular Sox9 lineage contribution to SAE repair, and its deletion significantly reduced proliferation following injury. By contrast, in vivo deletion of Sox9 enhanced proliferation of progenitors in both the SAE and SMG shortly following injury, but these progenitors failed to proliferate in vitro in the absence of Sox9, similar to that previously shown for Lef1 deletion. In cystic fibrosis ferret airways, Sox9 expression inversely correlated with Ki67 proliferative marker expression in SMG and the SAE. Using in vitro and ex vivo models, we demonstrate that Sox9 is extinguished as glandular progenitors exit ducts and proliferate on the airway surface and that Sox9 is required for migration and proper differentiation of SMG, but not surface airway, progenitors. We propose a model whereby Wnt/Lef1 and Sox9 signals differentially regulate the proliferative and migratory behavior of glandular progenitors, respectively.

"I must do this!": A latent profile analysis approach to understanding the role of irrational beliefs and motivation regulation in mental and physical health.

In the research concerning rational emotive behaviour therapy (REBT) in sport and exercise, irrational beliefs are proposed as a risk factor for health. Concurrent to this, researchers have also indicated that autonomous and controlled motivation, as proposed in organismic integration theory could, together with irrational beliefs, determine individual health. However, research is yet to align irrational beliefs and motivation, and explore how this alignment relates to mental health. The present two study paper identifies individual subgroups, drawn from data concerning irrational beliefs, motivation, and health (psychological distress, and physical health), in a sample of exercisers (study 1) and student athletes (study 2). We examined the latent profile structure of irrational beliefs and motivation, and how these latent profiles relate to psychological distress (studies 1 and 2), and physical health (study 2). Results indicate a two class profile whereby class 1 is characterised by high irrational beliefs, low self-determined motivation, and poor health outcomes. Class 2 is characterised by low irrational beliefs, high self-determined motivation, and better health outcomes. The findings are discussed in relation to the theoretical implications for REBT and organismic integration theory, and the practical implications for key stakeholders in the health of exercise participants and athletes.

Identification of novel, cryptic Clostridioides species isolates from environmental samples collected from diverse geographical locations.

Clostridioides difficile is a pathogen often associated with hospital-acquired infection or antimicrobial-induced disease; however, increasing evidence indicates infections can result from community or environmental sources. Most genomic sequencing of C. difficile has focused on clinical strains, although evidence is growing that C. difficile spores are widespread in soil and water in the environment. In this study, we sequenced 38 genomes collected from soil and water isolates in Flagstaff (AZ, USA) and Slovenia in an effort targeted towards environmental surveillance of C. difficile. At the average nucleotide identity (ANI) level, the genomes were divergent to C. difficile at a threshold consistent with different species. A phylogenetic analysis of these divergent genomes together with Clostridioides genomes available in public repositories confirmed the presence of three previously described, cryptic Clostridioides species and added two additional clades. One of the cryptic species (C-III) was almost entirely composed of Arizona and Slovenia genomes, and contained distinct sub-groups from each region (evidenced by SNP and gene-content differences). A comparative genomics analysis identified multiple unique coding sequences per clade, which can serve as markers for subsequent environmental surveys of these cryptic species. Homologues to the C. difficile toxin genes, tcdA and tcdB, were found in cryptic species genomes, although they were not part of the typical pathogenicity locus observed in C. difficile, and in silico PCR suggested that some would not amplify with widely used PCR diagnostic tests. We also identified gene homologues in the binary toxin cluster, including some present on phage and, for what is believed to be the first time, on a plasmid. All isolates were obtained from environmental samples, so the function and disease potential of these toxin homologues is currently unknown. Enzymatic profiles of a subset of cryptic isolates (n=5) demonstrated differences, suggesting that these isolates contain substantial metabolic diversity. Antimicrobial resistance (AMR) was observed across a subset of isolates (n=4), suggesting that AMR mechanisms are intrinsic to the genus, perhaps originating from a shared environmental origin. This study greatly expands our understanding of the genomic diversity of Clostridioides. These results have implications for C. difficile One Health research, for more sensitive C. difficile diagnostics, as well as for understanding the evolutionary history of C. difficile and the development of pathogenesis.