A Regional Study to Evaluate the Impact of Coal-fired Power Plants on Lung Cancer Incident Rates.

Background: Lung cancer is the leading cause of cancer related deaths. In Kansas, where coal-fired power plants account for 34% of power, we investigated whether hosting counties had higher age-adjusted lung cancer incidence rates. We also examined demographics, poverty levels, percentage of smokers, and environmental conditions using spatial analysis. Methods: Data from the Kansas Health Matters, and the Behavioral Risk Factor Surveillance System (2010-2014) for 105 counties in Kansas were analyzed. Multiple Linear Regression (MLR) assessed associations between potential risk factors and age-adjusted lung cancer incidence rates while Geographically Weighted Regression (GWR) examined regional risk factors. Results: Moran's I test confirmed spatial autocorrelation in age-adjusted lung cancer incidence rates (p<0.0003). MLR identified percentage of smokers, population size, and proportion of elderly population as significant predictors of age-adjusted lung cancer incidence rates (p<0.05). GWR showed positive associations between percentage of smokers and age-adjusted lung cancer incidence rates in over 50% of counties. Conclusion: Contrary to our hypothesis, proximity to a coal-fired power plant was not a significant predictor of age-adjusted lung cancer incidence rates. Instead, percentage of smokers emerged as a consistent global and regional risk factor. Regional lung cancer outcomes in Kansas are influenced by wind patterns and elderly population.

Multichannel bridges and NSC synergize to enhance axon regeneration, myelination, synaptic reconnection, and recovery after SCI.

Regeneration in the injured spinal cord is limited by physical and chemical barriers. Acute implantation of a multichannel poly(lactide-co-glycolide) (PLG) bridge mechanically stabilizes the injury, modulates inflammation, and provides a permissive environment for rapid cellularization and robust axonal regrowth through this otherwise inhibitory milieu. However, without additional intervention, regenerated axons remain largely unmyelinated (<10%), limiting functional repair. While transplanted human neural stem cells (hNSC) myelinate axons after spinal cord injury (SCI), hNSC fate is highly influenced by the SCI inflammatory microenvironment, also limiting functional repair. Accordingly, we investigated the combination of PLG scaffold bridges with hNSC to improve histological and functional outcome after SCI. In vitro, hNSC culture on a PLG scaffold increased oligodendroglial lineage selection after inflammatory challenge. In vivo, acute PLG bridge implantation followed by chronic hNSC transplantation demonstrated a robust capacity of donor human cells to migrate into PLG bridge channels along regenerating axons and integrate into the host spinal cord as myelinating oligodendrocytes and synaptically integrated neurons. Axons that regenerated through the PLG bridge formed synaptic circuits that connected the ipsilateral forelimb muscle to contralateral motor cortex. hNSC transplantation significantly enhanced the total number of regenerating and myelinated axons identified within the PLG bridge. Finally, the combination of acute bridge implantation and hNSC transplantation exhibited robust improvement in locomotor recovery. These data identify a successful strategy to enhance neurorepair through a temporally layered approach using acute bridge implantation and chronic cell transplantation to spare tissue, promote regeneration, and maximize the function of new axonal connections.

Effects of Short-term Electronic(e)-Cigarette Aerosol Exposure in the Mouse Larynx.

OBJECTIVES: The effects of electronic cigarettes (e-cigarettes) on the larynx are relatively unknown. This study examined the short-term effects of e-cigarette inhalation on cellular and inflammatory responses within the mouse laryngeal glottic and subglottic regions after exposure to pod-based devices (JUUL). METHODS: Male C57BL6/J mice (8-9 weeks) were assigned to control (n = 9), JUUL flavors Mint (JMi; n = 10) or Mango (JMa; n = 10). JUUL mice were exposed to 2 h/day for 1, 5, and 10 days using the inExpose inhalation system. Control mice were in room air. Vocal fold (VF) epithelial thickness, cell proliferation, subglandular area and composition, inflammatory cell infiltration, and surface topography were evaluated in the harvested larynges. Mouse body weight and urinary nicotine biomarkers were also measured. Chemical analysis of JUUL aerosols was conducted using selective ion flow tube mass spectrometry. RESULTS: JUUL-exposed mice had reduced body weight after day 5. Urinary nicotine biomarker levels indicated successful JUUL exposure and metabolism. Quantitative analysis of JUUL aerosol indicated that chemical constituents differ between JMi and JMa flavors. VF epithelial thickness, cellular proliferation, glandular area, and surface topography remained unchanged after JUUL exposures. Acidic mucus content increased after 1 day of JMi exposure. VF macrophage and T-cell levels slightly increased after 10 days of JMi exposures. CONCLUSIONS: Short-term e-cigarette exposures cause minimal flavor- and region-specific cellular and inflammatory changes in the mouse larynx. This work provides a foundation for long-term studies to determine if these responses are altered with multiple e-cigarette components and concentrations. LEVEL OF EVIDENCE: N/A Laryngoscope, 134:1316-1326, 2024.

Method for Collecting Single Epithelial Cells from the Mouse Larynx.

OBJECTIVE: The larynx is lined by specialized epithelial cell populations. Studying molecular changes occurring in individual epithelial cell types requires a reliable method for removing these cells from the larynx. Our objective was to develop a method to harvest individual epithelial cells from the mouse larynx while minimizing contamination from non-laryngeal sites and non-epithelial laryngeal cells. METHODS: Mice were euthanized, and the larynx was carefully exposed and separated from non-laryngeal sites. A small dental brush was inserted into the laryngeal inlet and rotated to obtain epithelial cells. Cells were transferred to collection media, counted, and cytospin preparations stained for laryngeal epithelial (i.e., Pan-Keratin, EpCAM, NGFR, p63, K5, ß-tubulin, MUC5AC) and non-epithelial (i.e., vimentin) cell markers. Histopathology was completed on brushed laryngeal tissue sections to evaluate the depth of cell collection. Preliminary Single-cell RNA sequencing (scRNA-seq) was performed to confirm this method can capture diverse laryngeal cell types. RESULTS: We collected 6000-8000 cells from a single larynx and 35000-40000 cells from combining brushings from three tissues. Histopathology demonstrated brushing removed the epithelial layer of the larynx and some underlying tissue. Immunofluorescence staining demonstrated the phenotype of harvested cells was primarily epithelial. Preliminary scRNA-seq was successfully conducted and displayed nine unique cell clusters. CONCLUSION: We developed a reliable method of harvesting individual epithelial cells from the mouse larynx. This method will be useful for collection of laryngeal cells for a variety of downstream cellular and molecular assays, including scRNA-seq, protein analyses, and cell-culture-based experiments, following laryngeal injury. LEVEL OF EVIDENCE: NA Laryngoscope, 134:786-794, 2024.

Treatment and Control of Hypertension Among Adults With Chronic Kidney Disease, 2011 to 2019.

BACKGROUND: Hypertension frequently accompanies chronic kidney disease (CKD) as etiology and sequela. We examined contemporary trends in hypertension treatment and control in a national sample of adults with CKD. METHODS: We evaluated 5% cross-sectional samples of adults with CKD between 2011 and 2019 in the Veterans Health Administration. We defined CKD as a sustained estimated glomerular filtration rate value <60 mL/min per 1.73 m2 or a urine albumin-to-creatinine ratio ≥30 mg/g. The main outcomes were blood pressure (BP) control, defined as a systolic BP <140 mm Hg and a diastolic BP <90 mm Hg based on the mean of monthly BP measurements, and prescriptions for antihypertensive medications. RESULTS: The annual samples ranged between n=22 110 and n=33 039 individuals, with a mean age of 72 years, 96% of whom were men. Between 2011 and 2014, the age-adjusted proportion of adults with controlled BP declined from 78.0% to 72.2% (P value for linear trend, <0.001), reached a nadir of 71.0% in 2015, and then increased to 72.9% by 2019 (P value for linear trend, <0.001). Among adults with BP above goal, the age-adjusted proportion who did not receive antihypertensive treatment increased throughout the decade from 18.8% to 21.6%, and the age-adjusted proportion who received ≥3 antihypertensive medications decreased from 41.8% to 36.3%. Prescriptions for first-line antihypertensive agents also decreased. CONCLUSIONS: Among adults with CKD treated in the Veterans Health Administration, the proportion with controlled BP declined between 2011 and 2015 followed by a modest increase, coinciding with fewer prescriptions for antihypertensive medications.

Multichannel bridges and NSC synergize to enhance axon regeneration, myelination, synaptic reconnection, and recovery after SCI.

Regeneration in the injured spinal cord is limited by physical and chemical barriers. Acute implantation of a multichannel poly(lactide-co-glycolide) (PLG) bridge mechanically stabilizes the injury, modulates inflammation, and provides a permissive environment for rapid cellularization and robust axonal regrowth through this otherwise inhibitory milieu. However, without additional intervention, regenerated axons remain largely unmyelinated (<10%), limiting functional repair. While transplanted human neural stem cells (hNSC) myelinate axons after spinal cord injury (SCI), hNSC fate is highly influenced by the SCI inflammatory microenvironment, also limiting functional repair. Accordingly, we investigated the combination of PLG scaffold bridges with hNSC to improve histological and functional outcome after SCI. In vitro, hNSC culture on a PLG scaffold increased oligodendroglial lineage selection after inflammatory challenge. In vivo, acute PLG bridge implantation followed by chronic hNSC transplantation demonstrated a robust capacity of donor human cells to migrate into PLG bridge channels along regenerating axons and integrate into the host spinal cord as myelinating oligodendrocytes and synaptically integrated neurons. Axons that regenerated through the PLG bridge formed synaptic circuits that connected ipsilateral forelimb muscle to contralateral motor cortex. hNSC transplantation significantly enhanced the total number of regenerating and myelinated axons identified within the PLG bridge. Finally, the combination of acute bridge implantation and hNSC transplantation exhibited robust improvement in locomotor recovery vs. control and hNSC transplant alone. These data identify a successful novel strategy to enhance neurorepair through a temporally layered approach using acute bridge implantation and chronic cell transplantation to spare tissue, promote regeneration, and maximize the function of new axonal connections.

Cell-autonomous and differential endocannabinoid signaling impacts the development of presynaptic retinal ganglion cell axon connectivity in vivo.

Cannabis exposure during gestation evokes significant molecular modifications to neurodevelopmental programs leading to neurophysiological and behavioral abnormalities in humans. The main neuronal receptor for Δ9-tetrahydrocannabinol (THC) is the type-1 cannabinoid receptor CB1R, one of the most abundant G-protein-coupled receptors in the nervous system. While THC is the major psychoactive phytocannabinoid, endocannabinoids (eCBs) are the endogenous ligands of CB1R and are known to act as retrograde messengers to modulate synaptic plasticity at different time scales in the adult brain. Accumulating evidence indicates that eCB signaling through activation of CB1R plays a central role in neural development. During development, most CB1R localized to axons of projection neurons, and in mice eCB signaling impacts axon fasciculation. Understanding of eCB-mediated structural plasticity during development, however, requires the identification of the precise spatial and temporal dynamics of CB1R-mediated modifications at the level of individual neurons in the intact brain. Here, the cell-autonomous role of CB1R and the effects of CB1R-mediated eCB signaling were investigated using targeted single-cell knockdown and pharmacologic treatments in Xenopus. We imaged axonal arbors of retinal ganglion cells (RGCs) in real time following downregulation of CB1R via morpholino (MO) knockdown. We also analyzed RGC axons with altered eCB signaling following treatment with URB597, a selective inhibitor of the enzyme that degrades Anandamide (AEA), or JZL184, an inhibitor of the enzyme that blocks 2-Arachidonoylglycerol (2-AG) hydrolysis, at two distinct stages of retinotectal development. Our results demonstrate that CB1R knockdown impacts RGC axon branching at their target and that differential 2-AG and AEA-mediated eCB signaling contributes to presynaptic structural connectivity at the time that axons terminate and when retinotectal synaptic connections are made. Altering CB1R levels through CB1R MO knockdown similarly impacted dendritic morphology of tectal neurons, thus supporting both pre- and postsynaptic cell-autonomous roles for CB1R-mediated eCB signaling.

Effects of Electronic (E)-cigarette Vapor and Cigarette Smoke in Cultured Vocal Fold Fibroblasts.

OBJECTIVE: The public use of electronic-cigarettes (e-cigs) is rapidly growing. When heated, e-cigs produce a vapor that is inhaled. The vocal folds are among the first tissues exposed to this insult. However, the impact of e-cigs on vocal fold health is almost entirely unknown. Our objective was to evaluate the effects of e-cig vapor on cultured human vocal fold fibroblasts (hVFFs), the primary cell type of the lamina propria. We compared the cellular effects of e-cig vapor without and with nicotine and conventional cigarette smoke. STUDY DESIGN: In vitro. METHODS: E-cig vapor extract (EVE) and cigarette smoke extract (CSE) were created by bubbling vapor and smoke, respectively, into the cell culture medium. hVFFs were exposed to EVE without or with nicotine or CSE for 24 hours. Untreated cells were used as a control group. Cells were harvested, and cytotoxicity, extracellular matrix and inflammatory gene expression, and DNA damage were assessed. RESULTS: Undiluted EVE without and with nicotine reduced the viability of hVFFs to a cytotoxic level. CSE reduced hVFFs viability to a greater extent than EVE and induced DNA damage as measured by DNA double-strand breaks. No changes in gene expression were observed following EVE or CSE exposure. CONCLUSION: EVE induces cytotoxicity in hVFFs. However, cellular responses were greater following exposure to CSE, suggesting cigarette smoke may induce more harm, at least in the short term. Findings from this investigation improve our understanding of responses of hVFFs to e-cigs and form the basis for an in vitro methodology to study the vocal fold responses to these products. LEVEL OF EVIDENCE: NA Laryngoscope, 133:139-146, 2023.

Modulation of mouse laryngeal inflammatory and immune cell responses by low and high doses of mainstream cigarette smoke.

Cigarette smoking is a major risk factor for laryngeal diseases. Despite well-documented cigarette smoke (CS) induced laryngeal histopathological changes, the underlying immunopathological mechanisms remain largely unexplored. The goal of this study was to evaluate inflammatory and immune cell responses in a CS-exposed larynx. Specifically, we used a 4-week subacute whole-body CS inhalation mouse model to assess these responses in the laryngeal mucosa upon exposure to low (LD; 1 h/day) and high dose (HD; 4 h/day) CS. Laryngeal tissues were harvested and evaluated using a 254-plex NanoString inflammation panel and neutrophil/macrophage/T-cell immunohistochemistry (IHC). NanoString global and differential gene expression analysis revealed a unique expression profile only in the HD group, with 26 significant differentially expressed genes (DEGs). StringDB KEGG pathway enrichment analysis revealed the involvement of these DEGs with pro-inflammatory pathways including TNF/TNFα and IL-17. Furthermore, inflammatory responses remained inhibited in conjunction with predicted activated states of anti-inflammatory regulators like PPARγ and NFE2L2 upon Ingenuity Pathway Analysis (IPA). Subglottic T-cell levels remained significantly inhibited as corroborated by IPA predictions. Overall, our key findings are consistent with HD exposures being anti-inflammatory and immunosuppressive. Furthermore, the identification of important regulatory genes and enriched pathways may help improve clinical interventions for CS-induced laryngeal diseases.

Short-term whole body cigarette smoke exposure induces regional differences in cellular response in the mouse larynx.

The larynx is an essential organ in the respiratory tract and necessary for airway protection, respiration, and phonation. Cigarette smoking is a significant risk factor associated with benign and malignant laryngeal diseases. Despite this association, the underlying mechanisms by which cigarette smoke (CS) drives disease development are not well elucidated. In the current study, we developed a short-term murine whole body inhalation model to evaluate the first CS-induced cellular responses in the glottic [i.e. vocal fold (VF)] and subglottic regions of the larynx. Specifically, we investigated epithelial cell proliferation, cell death, surface topography, and mucus production, at various time points (1 day, 5 days, 10 days) after ∼ 2 h exposure to 3R4F cigarettes (Delivered dose: 5.6968 mg/kg per cigarette) and following cessation for 5 days after a 5 day CS exposure (CSE). CSE elevated levels of BrdU labeled proliferative cells and p63 labeled epithelial basal cells on day 1 in the VF. CSE increased proliferative cells in the subglottis at days 5, 10 and following cessation in the subglottis. Cleaved caspase-3 apoptotic activity was absent in VF at all time points and increased at day 1 in the subglottis. Evaluation of the VF surface by scanning electron microscopy (SEM) revealed significant epithelial microprojection damage at day 10 and early signs of necrosis at days 5 and 10 post-CSE. SEM visualizations additionally indicated the presence of deformed cilia at days 5 and 10 after CSE and post-cessation in the respiratory epithelium lined subglottis. In terms of mucin content, the impact of short-term CSE was observed only at day 10, with decreasing acidic mucin levels and increasing neutral mucin levels. Overall, these findings reveal regional differences in murine laryngeal cellular responses following short-term CSE and provide insight into potential mechanisms underlying CS-induced laryngeal disease development.

Mainstream Cigarette Smoke Impacts the Mouse Vocal Fold Epithelium and Mucus Barrier.

OBJECTIVES/HYPOTHESIS: Cigarette smoke (CS) is a primary risk factor for the development of numerous benign and malignant laryngeal diseases. The epithelium and mucus lining the vocal folds (VF) are the first barriers against CS. The primary objective of this study was to investigate epithelial and mucus barrier changes in the mouse laryngeal mucosa upon exposure to subacute CS. The secondary objective was to compare mucus barrier changes in mice and human smokers and nonsmokers. Study Design Animal model. METHODS: Mice were exposed to CS for 4 weeks for 4 hours (N = 12, high dose [HD]) or 1 hour (N = 12, low dose [LD]) per day. Air-exposed mice were used as a control group (N = 10). Larynges were harvested and VF epithelial barrier integrity was evaluated including cellular proliferation and expression of cell junctions. We also investigated mucus production by examining mucus cell area and mucin expression in mice and human smokers and nonsmokers. RESULTS: HD CS increased VF epithelial cellular proliferation but did not alter the expression of cell junctions. HD CS also induced hypertrophy of the mucus-producing submucosal glands. However, only LD CS increased MUC5AC gene expression. MUC5AC staining appeared elevated in laryngeal specimens from smokers, but this was not significant as compared to nonsmokers. CONCLUSIONS: These findings help us identify potential adaptive mechanisms to CS exposure as well as set the foundation for further study of key aspects of epithelial and mucus barrier integrity that may be implicated in laryngeal disease development following prolonged smoking. LEVEL OF EVIDENCE: NA Laryngoscope, 131:2530-2539, 2021.

Cigarette smoke-induced changes in the murine vocal folds: a Raman spectroscopic observation.

Raman spectroscopic methods are being projected as novel tools to study the early invisible molecular level changes in a label-free manner. In the present study, we have used Raman spectroscopy to explore the earliest biochemical changes in murine vocal folds in response to time-bound cigarette smoke exposure. Mice were exposed to cigarette smoke for 2 or 4-weeks through a customized smoke inhalation system. The larynx was collected and initial evaluations using standard methods of analysis such as histopathology and immunofluorescence was performed. Concurrent unstained sections were used for Raman imaging. Two common pathological features of vocal fold disorders including alterations in collagen content and epithelial hypercellularity, or hyperplasia, were observed. The mean spectra, principal component analysis, and Raman mapping also revealed differences in the collagen content and hypercellularity in the smoke exposed tissues. The differences in 2-week exposed tissues were found to be more prominent as compared to 4-week. This was attributed to adaptive responses and the already reported biphasic effects, which suggest that collagen synthesis is significantly reduced at higher cigarette smoke concentrations. Overall findings of the study are supportive of the prospective application of Raman imaging in monitoring changes due to cigarette smoke in the vocal folds.

Floodplain farm fields provide novel rearing habitat for Chinook salmon.

When inundated by floodwaters, river floodplains provide critical habitat for many species of fish and wildlife, but many river valleys have been extensively leveed and floodplain wetlands drained for flood control and agriculture. In the Central Valley of California, USA, where less than 5% of floodplain wetland habitats remain, a critical conservation question is how can farmland occupying the historical floodplains be better managed to improve benefits for native fish and wildlife. In this study fields on the Sacramento River floodplain were intentionally flooded after the autumn rice harvest to determine if they could provide shallow-water rearing habitat for Sacramento River fall-run Chinook salmon (Oncorhynchus tshawytscha). Approximately 10,000 juvenile fish (ca. 48 mm, 1.1 g) were reared on two hectares for six weeks (Feb-March) between the fall harvest and spring planting. A subsample of the fish were uniquely tagged to allow tracking of individual growth rates (average 0.76 mm/day) which were among the highest recorded in fresh water in California. Zooplankton sampled from the water column of the fields were compared to fish stomach contents. The primary prey was zooplankton in the order Cladocera, commonly called water fleas. The compatibility, on the same farm fields, of summer crop production and native fish habitat during winter demonstrates that land management combining agriculture with conservation ecology may benefit recovery of native fish species, such as endangered Chinook salmon.

The Challenges of Implementing Next Generation Sequencing Across a Large Healthcare System, and the Molecular Epidemiology and Antibiotic Susceptibilities of Carbapenemase-Producing Bacteria in the Healthcare System of the U.S. Department of Defense.

OBJECTIVE: We sought to: 1) provide an overview of the genomic epidemiology of an extensive collection of carbapenemase-producing bacteria (CPB) collected in the U.S. Department of Defense health system; 2) increase awareness of the public availability of the sequences, isolates, and customized antimicrobial resistance database of that system; and 3) illustrate challenges and offer mitigations for implementing next generation sequencing (NGS) across large health systems. DESIGN: Prospective surveillance and system-wide implementation of NGS. SETTING: 288-hospital healthcare network. METHODS: All phenotypically carbapenem resistant bacteria underwent CarbaNP® testing and PCR, followed by NGS. Commercial (Newbler and Geneious), on-line (ResFinder), and open-source software (Btrim, FLASh, Bowtie2, an Samtools) were used for assembly, SNP detection and clustering. Laboratory capacity, throughput, and response time were assessed. RESULTS: From 2009 through 2015, 27,000 multidrug-resistant Gram-negative isolates were submitted. 225 contained carbapenemase-encoding genes (most commonly blaKPC, blaNDM, and blaOXA23). These were found in 15 species from 146 inpatients in 19 facilities. Genetically related CPB were found in more than one hospital. Other clusters or outbreaks were not clonal and involved genetically related plasmids, while some involved several unrelated plasmids. Relatedness depended on the clustering algorithm used. Transmission patterns of plasmids and other mobile genetic elements could not be determined without ultra-long read, single-molecule real-time sequencing. 80% of carbapenem-resistant phenotypes retained susceptibility to aminoglycosides, and 70% retained susceptibility to fluoroquinolones. However, among the CPB-confirmed genotypes, fewer than 25% retained susceptibility to aminoglycosides or fluoroquinolones. CONCLUSION: Although NGS is increasingly acclaimed to revolutionize clinical practice, resource-constrained environments, large or geographically dispersed healthcare networks, and military or government-funded public health laboratories are likely to encounter constraints and challenges as they implement NGS across their health systems. These include lack of standardized definitions and quality control metrics, limitations of short-read sequencing, insufficient bandwidth, and the current limited availability of very expensive and scarcely available sequencing platforms. Possible solutions and mitigations are also proposed.