Identifying FUS amyotrophic lateral sclerosis disease signatures in patient dermal fibroblasts.

Amyotrophic lateral sclerosis (ALS) is a rapidly progressing, highly heterogeneous neurodegenerative disease, underscoring the importance of obtaining information to personalize clinical decisions quickly after diagnosis. Here, we investigated whether ALS-relevant signatures can be detected directly from biopsied patient fibroblasts. We profiled familial ALS (fALS) fibroblasts, representing a range of mutations in the fused in sarcoma (FUS) gene and ages of onset. To differentiate FUS fALS and healthy control fibroblasts, machine-learning classifiers were trained separately on high-content imaging and transcriptional profiles. "Molecular ALS phenotype" scores, derived from these classifiers, captured a spectrum from disease to health. Interestingly, these scores negatively correlated with age of onset, identified several pre-symptomatic individuals and sporadic ALS (sALS) patients with FUS-like fibroblasts, and quantified "movement" of FUS fALS and "FUS-like" sALS toward health upon FUS ASO treatment. Taken together, these findings provide evidence that non-neuronal patient fibroblasts can be used for rapid, personalized assessment in ALS.

Stem cells, cell therapies, and bioengineering in lung biology and diseases 2023.

Repair and regeneration of a diseased lung using stem cells or bioengineered tissues is an exciting therapeutic approach for a variety of lung diseases and critical illnesses. Over the past decade, increasing evidence from preclinical models suggests that mesenchymal stromal cells, which are not normally resident in the lung, can be used to modulate immune responses after injury, but there have been challenges in translating these promising findings to the clinic. In parallel, there has been a surge in bioengineering studies investigating the use of artificial and acellular lung matrices as scaffolds for three-dimensional lung or airway regeneration, with some recent attempts of transplantation in large animal models. The combination of these studies with those involving stem cells, induced pluripotent stem cell derivatives, and/or cell therapies is a promising and rapidly developing research area. These studies have been further paralleled by significant increases in our understanding of the molecular and cellular events by which endogenous lung stem and/or progenitor cells arise during lung development and participate in normal and pathological remodeling after lung injury. For the 2023 Stem Cells, Cell Therapies, and Bioengineering in Lung Biology and Diseases Conference, scientific symposia were chosen to reflect the most cutting-edge advances in these fields. Sessions focused on the integration of "omics" technologies with function, the influence of immune cells on regeneration, and the role of the extracellular matrix in regeneration. The necessity for basic science studies to enhance fundamental understanding of lung regeneration and to design innovative translational studies was reinforced throughout the conference.

Beta-defensin index: A functional biomarker for oral cancer detection.

There is an unmet clinical need for a non-invasive and cost-effective test for oral squamous cell carcinoma (OSCC) that informs clinicians when a biopsy is warranted. Human beta-defensin 3 (hBD-3), an epithelial cell-derived anti-microbial peptide, is pro-tumorigenic and overexpressed in early-stage OSCC compared to hBD-2. We validate this expression dichotomy in carcinoma in situ and OSCC lesions using immunofluorescence microscopy and flow cytometry. The proportion of hBD-3/hBD-2 levels in non-invasively collected lesional cells compared to contralateral normal cells, obtained by ELISA, generates the beta-defensin index (BDI). Proof-of-principle and blinded discovery studies demonstrate that BDI discriminates OSCC from benign lesions. A multi-center validation study shows sensitivity and specificity values of 98.2% (95% confidence interval [CI] 90.3-99.9) and 82.6% (95% CI 68.6-92.2), respectively. A proof-of-principle study shows that BDI is adaptable to a point-of-care assay using microfluidics. We propose that BDI may fulfill a major unmet need in low-socioeconomic countries where pathology services are lacking.

Two-pore potassium channel TREK-1 (K2P2.1) regulates NLRP3 inflammasome activity in macrophages.

Because of the importance of potassium efflux in inflammasome activation, we investigated the role of the two-pore potassium (K2P) channel TREK-1 in macrophage inflammasome activity. Using primary alveolar macrophages (AMs) and bone marrow-derived macrophages (BMDMs) from wild-type (wt) and TREK-1-/- mice, we measured responses to inflammasome priming [using lipopolysaccharide (LPS)] and activation (LPS + ATP). We measured IL-1ß, caspase-1, and NLRP3 via ELISA and Western blot. A membrane-permeable potassium indicator was used to measure potassium efflux during ATP exposure, and a fluorescence-based assay was used to assess changes in membrane potential. Inflammasome activation induced by LPS + ATP increased IL-1ß secretion in wt AMs, whereas activation was significantly reduced in TREK-1-/- AMs. Priming of BMDMs using LPS was not affected by either genetic deficiency or pharmacological inhibition of TREK-1 with Spadin. Cleavage of caspase-1 following LPS + ATP treatment was significantly reduced in TREK-1-/- BMDMs. The intracellular potassium concentration in LPS-primed wt BMDMs was significantly lower compared with TREK-1-/- BMDMs or wt BMDMs treated with Spadin. Conversely, activation of TREK-1 with BL1249 caused a decrease in intracellular potassium in wt BMDMs. Treatment of LPS-primed BMDMs with ATP caused a rapid reduction in intracellular potassium levels, with the largest change observed in TREK-1-/- BMDMs. Intracellular K+ changes were associated with changes in the plasma membrane potential (Em), as evidenced by a more depolarized Em in TREK-1-/- BMDMs compared with wt, and Em hyperpolarization upon TREK-1 channel opening with BL1249. These results suggest that TREK-1 is an important regulator of NLRP3 inflammasome activation in macrophages.NEW & NOTEWORTHY Because of the importance of potassium efflux in inflammasome activation, we investigated the role of the two-pore potassium (K2P) channel TREK-1 in macrophage inflammasome activity. Using primary alveolar macrophages and bone marrow-derived macrophages from wild-type and TREK-1-/- mice, we measured responses to inflammasome priming (using LPS) and activation (LPS + ATP). Our results suggest that TREK-1 is an important regulator of NLRP3 inflammasome activation in macrophages.

Deficiency of Acute-Phase Serum Amyloid A Exacerbates Sepsis-Induced Mortality and Lung Injury in Mice.

Serum amyloid A (SAA) is a family of proteins, the plasma levels of which may increase >1000-fold in acute inflammatory states. We investigated the role of SAA in sepsis using mice deficient in all three acute-phase SAA isoforms (SAA-TKO). SAA deficiency significantly increased mortality rates in the three experimental sepsis mouse models: cecal ligation and puncture (CLP), cecal slurry (CS) injection, and lipopolysaccharide (LPS) treatments. SAA-TKO mice had exacerbated lung pathology compared to wild-type (WT) mice after CLP. A bulk RNA sequencing performed on lung tissues excised 24 h after CLP indicated significant enrichment in the expression of genes associated with chemokine production, chemokine and cytokine-mediated signaling, neutrophil chemotaxis, and neutrophil migration in SAA-TKO compared to WT mice. Consistently, myeloperoxidase activity and neutrophil counts were significantly increased in the lungs of septic SAA-TKO mice compared to WT mice. The in vitro treatment of HL-60, neutrophil-like cells, with SAA or SAA bound to a high-density lipoprotein (SAA-HDL), significantly decreased cellular transmigration through laminin-coated membranes compared to untreated cells. Thus, SAA potentially prevents neutrophil transmigration into injured lungs, thus reducing exacerbated tissue injury and mortality. In conclusion, we demonstrate for the first time that endogenous SAA plays a protective role in sepsis, including ameliorating lung injury.

Sex gaps in perception of tobacco conversations between adult patients who now smoke cigarettes and oral health care providers: National Health and Nutrition Examination Survey 2017-March 2020 prepandemic.

BACKGROUND: Smoking cessation is difficult. A potential gap based on sex exists in the tobacco cessation aid that dental care professionals provide to patients. The purpose of this research was to examine whether there is a sex difference in dental patients' reports of having a direct conversation about the benefits of giving up cigarettes or other types of tobacco products with their oral health care provider. METHODS: National Health and Nutrition Examination Survey 2017-March 2020 prepandemic data were used in this cross-sectional study for participants 18 years and older who reported that they "now smoke cigarettes," had a dental visit within the previous year, self-reported their sex, and responded whether their oral health care provider had a direct conversation about the benefits of giving up cigarettes or other types of tobacco products to improve dental health at their last visit (n = 582). Multivariable logistic regression analysis was conducted to compare data according to sex. RESULTS: Overall, 50.7% of patients (59.2% of men, 42.9% of women; P = .0037) reported having a conversation about tobacco cessation at their dental visit. The odds of women reporting having no such discussion were twice those of men (odds ratio, 2.17; 95% CI, 1.10 to 4.28; P = .0270). CONCLUSIONS: One-half of the participants reported having no tobacco cessation conversation about the benefits of giving up cigarettes or other types of tobacco with their dental care provider. Women were twice as likely to report no such discussion. PRACTICAL IMPLICATIONS: Oral health care providers need to ensure that primary and secondary prevention information and intervention programs about the benefits of giving up cigarettes or other types of tobacco products are provided equitably to all patients.

Activation of TREK-1 (K2P2.1) potassium channels protects against influenza A-induced lung injury.

Influenza-A virus (IAV) infects yearly an estimated one billion people worldwide, resulting in 300,000-650,000 deaths. Preventive vaccination programs and antiviral medications represent the mainstay of therapy, but with unacceptably high morbidity and mortality rates, new targeted therapeutic approaches are urgently needed. Since inflammatory processes are commonly associated with measurable changes in the cell membrane potential (Em), we investigated whether Em hyperpolarization via TREK-1 (K2P2.1) K+ channel activation can protect against influenza-A virus (IAV)-induced pneumonia. We infected mice with IAV, which after 5 days caused 10-15% weight loss and a decrease in spontaneous activity, representing a clinically relevant infection. We then started a 3-day intratracheal treatment course with the novel TREK-1 activating compounds BL1249 or ML335. We confirmed TREK-1 activation with both compounds in untreated and IAV-infected primary human alveolar epithelial cells (HAECs) using high-throughput fluorescent imaging plate reader (FLIPR) assays. In mice, TREK-1 activation with BL1249 and ML335 counteracted IAV-induced histological lung injury and decrease in lung compliance and improved BAL fluid total protein levels, cell counts, and inflammatory IL-6, IP-10/CXCL-10, MIP-1α, and TNF-α levels. To determine whether these anti-inflammatory effects were mediated by activation of alveolar epithelial TREK-1 channels, we studied the effects of BL1249 and ML335 in IAV-infected HAEC, and found that TREK-1 activation decreased IAV-induced inflammatory IL-6, IP-10/CXCL10, and CCL-2 secretion. Dissection of TREK-1 downstream signaling pathways and construction of protein-protein interaction (PPI) networks revealed NF-κB1 and retinoic acid-inducible gene-1 (RIG-1) cascades as the most likely targets for TREK-1 protection. Therefore, TREK-1 activation may represent a novel therapeutic approach against IAV-induced lung injury.

Targeting the Microbiome to Improve Gut Health and Breathing Function After Spinal Cord Injury.

Spinal cord injury (SCI) is a devastating condition characterized by impaired motor and sensory function, as well as internal organ pathology and dysfunction. This internal organ dysfunction, particularly gastrointestinal (GI) complications, and neurogenic bowel, can reduce the quality of life of individuals with an SCI and potentially hinder their recovery. The gut microbiome impacts various central nervous system functions and has been linked to a number of health and disease states. An imbalance of the gut microbiome, i.e., gut dysbiosis, contributes to neurological disease and may influence recovery and repair processes after SCI. Here we examine the impact of high cervical SCI on the gut microbiome and find that transient gut dysbiosis with persistent gut pathology develops after SCI. Importantly, probiotic treatment improves gut health and respiratory motor function measured through whole-body plethysmography. Concurrent with these improvements was a systemic decrease in the cytokine tumor necrosis factor-alpha and an increase in neurite sprouting and regenerative potential of neurons. Collectively, these data reveal the gut microbiome as an important therapeutic target to improve visceral organ health and respiratory motor recovery after SCI. Research Highlights: Cervical spinal cord injury (SCI) causes transient gut dysbiosis and persistent gastrointestinal (GI) pathology.Treatment with probiotics after SCI leads to a healthier GI tract and improved respiratory motor recovery.Probiotic treatment decreases systemic tumor necrosis factor-alpha and increases the potential for sprouting and regeneration of neurons after SCI.The gut microbiome is a valid target to improve motor function and secondary visceral health after SCI.

Au naturale: use of biologically derived cyclic di-nucleotides for cancer immunotherapy.

Cyclic di-nucleotides (CDNs) are widespread second messenger signalling molecules that regulate fundamental biological processes across the tree of life. These molecules are also potent modulators of the immune system, inducing a Type I interferon response upon binding to the eukaryotic receptor STING. Such a response in tumours induces potent immune anti-cancer responses and thus CDNs are being developed as a novel cancer immunotherapy. In this review, I will highlight the use, challenges and advantages of using naturally occurring CDNs to treat cancer.

The ever-expanding world of bacterial cyclic oligonucleotide second messengers.

Cyclic dinucleotide (cdN) second messengers are essential for bacteria to sense and adapt to their environment. These signals were first discovered with the identification of 3'-5', 3'-5' cyclic di-GMP (c-di-GMP) in 1987, a second messenger that is now known to be the linchpin signaling pathway modulating bacterial motility and biofilm formation. In the past 15 years, three more cdNs were uncovered: 3'-5', 3'-5' cyclic di-AMP (c-di-AMP) and 3'-5', 3'-5' cyclic GMP-AMP (3',3' cGAMP) in bacteria and 2'-5', 3'-5' cyclic GMP-AMP (2',3' cGAMP) in eukaryotes. We now appreciate that bacteria can synthesize many varieties of cdNs from every ribonucleotide, and even cyclic trinucleotide (ctN) second messengers have been discovered. Here we highlight our current understanding of c-di-GMP and c-di-AMP in bacterial physiology and focus on recent advances in 3',3' cGAMP signaling effectors, its role in bacterial phage response, and the diversity of its synthase family.

Cyclic di-GMP-Mediated Regulation of Extracellular Mannuronan C-5 Epimerases Is Essential for Cyst Formation in Azotobacter vinelandii.

The genus Azotobacter, belonging to the Pseudomonadaceae family, is characterized by the formation of cysts, which are metabolically dormant cells produced under adverse conditions and able to resist desiccation. Although this developmental process has served as a model for the study of cell differentiation in Gram-negative bacteria, the molecular basis of its regulation is still poorly understood. Here, we report that the ubiquitous second messenger cyclic dimeric GMP (c-di-GMP) is critical for the formation of cysts in Azotobacter vinelandii Upon encystment induction, the levels of c-di-GMP increased, reaching a peak within the first 6 h. In the absence of the diguanylate cyclase MucR, however, the levels of this second messenger remained low throughout the developmental process. A. vinelandii cysts are surrounded by two alginate layers with variable proportions of guluronic residues, which are introduced into the final alginate chain by extracellular mannuronic C-5 epimerases of the AlgE1 to AlgE7 family. Unlike in Pseudomonas aeruginosa, MucR was not required for alginate polymerization in A. vinelandii Conversely, MucR was necessary for the expression of extracellular alginate C-5 epimerases; therefore, the MucR-deficient strain produced cyst-like structures devoid of the alginate capsule and unable to resist desiccation. Expression of mucR was partially dependent on the response regulator AlgR, which binds to two sites in the mucR promoter, enhancing mucR transcription. Together, these results indicate that the developmental process of A. vinelandii is controlled through a signaling module that involves activation by the response regulator AlgR and c-di-GMP accumulation that depends on MucR.IMPORTANCEA. vinelandii has served as an experimental model for the study of the differentiation processes to form metabolically dormant cells in Gram-negative bacteria. This work identifies c-di-GMP as a critical regulator for the production of alginates with specific contents of guluronic residues that are able to structure the rigid laminated layers of the cyst envelope. Although allosteric activation of the alginate polymerase complex Alg8-Alg44 by c-di-GMP has long been recognized, our results show a previously unidentified role during the polymer modification step, controlling the expression of extracellular alginate epimerases. Our results also highlight the importance of c-di-GMP in the control of the physical properties of alginate, which ultimately determine the desiccation resistance of the differentiated cell.

Adolescents' Perceived Risk of Harm Due to Smoking: The role of extracurricular activities.

Purpose: Tobacco use initiated during adolescence often leads to continued use in adulthood. There are multiple factors influencing initiation, including low perceived risk of harm. Adolescents involved in school-based extracurricular activities have opportunities to interact with coaches, leaders, and group supervisors who may influence their perception of risk. The purpose of this study was to examine the role of extracurricular activities and adolescents' perceived risks of harm of tobacco use, utilizing an existing dataset.Methods: The 2016 National Survey on Drug Use and Health (NSDUH) dataset was used for a cross-sectional study of youth, ages 12 to <18 years. Adolescents involved in one or more school-based, extracurricular activities were compared with adolescents involved in no activities. The key variable was the response to the NSDUH question regarding perceived risk of harm from daily smoking (≥1 packs of cigarettes). Chi-square tests and multinomial logistic regression were used to analyze the data.Results: At the time of the 2016 NSDUH study period, among the adolescents aged 12 to <18 years (n=4,308), 17.5% indicated that they did not participate in any extracurricular activities and 10.6% reported no/slight perceived risk of harm associated with tobacco use. Adolescents who did not participate in extracurricular activities were more likely to report no/slight risk of harm from smoking (Adjusted Odds Ratio, AOR= 2.21 [95%CI: 1.62, 3.02]) as opposed to the perception of great risk of harm.Conclusion: Adolescents who are not involved in extracurricular activities are more likely to endorse the perception that cigarettes have no/slight risk of harm. School-based extracurricular activities may provide unintended benefits to adolescents; health care professionals, including dental hygienists, should be aware of this associated health benefit.

Nitroxide-enhanced MRI of cardiovascular oxidative stress.

BACKGROUND: In vivo imaging of oxidative stress can facilitate the understanding and treatment of cardiovascular diseases. We evaluated nitroxide-enhanced MRI with 3-carbamoyl-proxyl (3CP) for the detection of myocardial oxidative stress. METHODS: Three mouse models of cardiac oxidative stress were imaged, namely angiotensin II (Ang II) infusion, myocardial infarction (MI), and high-fat high-sucrose (HFHS) diet-induced obesity (DIO). For the Ang II model, mice underwent MRI at baseline and after 7 days of Ang II (n = 8) or saline infusion (n = 8). For the MI model, mice underwent MRI at baseline (n = 10) and at 1 (n = 8), 4 (n = 9), and 21 (n = 8) days after MI. For the HFHS-DIO model, mice underwent MRI at baseline (n = 20) and 18 weeks (n = 13) after diet initiation. The 3CP reduction rate, Kred , computed using a tracer kinetic model, was used as a metric of oxidative stress. Dihydroethidium (DHE) staining of tissue sections was performed on Day 1 after MI. RESULTS: For the Ang II model, Kred was higher after 7 days of Ang II versus other groups (p < 0.05). For the MI model, Kred , in the infarct region was significantly elevated on Days 1 and 4 after MI (p < 0.05), whereas Kred in the noninfarcted region did not change after MI. DHE confirmed elevated oxidative stress in the infarct zone on Day 1 after MI. After 18 weeks of HFHS diet, Kred was higher in mice after diet versus baseline (p < 0.05). CONCLUSIONS: Nitroxide-enhanced MRI noninvasively quantifies tissue oxidative stress as one component of a multiparametric preclinical MRI examination. These methods may facilitate investigations of oxidative stress in cardiovascular disease and related therapies.

The Association of Sleep Duration and the Use of Electronic Cigarettes, NHANES, 2015-2016.

Introduction. The unknown effects of electronic cigarettes are public health concerns. One potential effect of electronic cigarette fluid constituents, such as nicotine, may influence sleep. The purpose of this study is to determine if there is an association between sleep duration and electronic cigarette use. METHODS: A retrospective, cross-sectional study was conducted using National Health and Nutrition Examination Survey (NHANES) 2015-2016. Variables of interest included responses to questions concerning electronic cigarette use, hours of sleep, and other variables associated with sleep. Data analyses were conducted with the Rao-Scott chi square test and logistic regression. RESULTS: This study was conducted on 2889 participants, aged 18-65 years, of whom 50.7% were female. Using a bivariate analyses of electronic cigarette usage and sleep duration, participants who never used an electronic cigarette were more likely to have appropriate sleep durations as compared with participants who were currently using electronic cigarettes (P < 0.0001). After adjusting for sociodemographic variables and cigarette smoking, current electronic cigarette use was associated with higher odds of less sleep duration (adjusted odds ratio = 1.82; 95% CI: 1.18, 2.79; P < 0.0001). After adjusting for sociodemographic variables and cigarette smoking, current electronic cigarette use was associated with higher odds of less sleep duration (adjusted odds ratio = 1.82; 95% CI: 1.18, 2.79. CONCLUSIONS: Participants currently using electronic cigarettes are more likely to have less sleep as compared to participants who have never used electronic cigarettes. Implications. With sleep time duration being a major factor for proper body function and repair, this study can serve as confirmation that the use of electronic cigarettes is not a harmless health behavior.

Hydrogels Embedded With Melittin and Tobramycin Are Effective Against Pseudomonas aeruginosa Biofilms in an Animal Wound Model.

We demonstrate that the antimicrobial peptide, melittin, is effective alone and in combination with the aminoglycosides tobramycin to kill Pseudomonas aeruginosa growing as biofilms both in vitro and in vivo. Melittin and tobramycin show enhanced in vitro activity in combination at micromolar concentrations, resulting in a 2-log10 reduction in the number of cells within mature PAO1 P. aeruginosa biofilms after 6-h of treatment. Alternatively, either agent alone resulted in half-a-log10 reduction. Time-killing assays demonstrated that the combination of melittin and tobramycin was effective at 2-h whereas tobramycin was not effective until after 6-h of treatment. We also found the combination was more effective than tobramycin alone against biofilms of 7 P. aeruginosa cystic fibrosis clinical isolates, resulting in a maximum 1.5-log10 cellular reduction. Additionally, melittin alone was effective at killing biofilms of 4 Staphylococcus aureus isolates, resulting in a maximum 2-log10 cellular reduction. Finally, melittin in combination with tobramycin embedded in an agarose-based hydrogel resulted in a 4-fold reduction in bioluminescent P. aeruginosa colonizing mouse wounds by 4-h. In contrast, tobramycin or melittin treatment alone did not cause a statistically significant reduction in bioluminescence. These data demonstrate that melittin in combination with tobramycin embedded in a hydrogel is a potential treatment for biofilm-associated wound infections.

Dental Care Utilization among Veterans by Smoking Status.

BACKGROUND: Given the critical importance of dental care utilization among veterans and the overall health consequences of tobacco use in all populations, the purpose of this research is to examine smoking as a risk factor for poor dental care utilization among United States Veterans. METHODS: A secondary data analysis of cross-sectional data from the National Survey of Veterans was conducted. The primary outcome was dental care utilization (Yes, No). Frequency, chi-square analyses, and multivariate logistic regression statistical tests were performed while adjusting for confounding factors. RESULTS: There were 6,308 veterans in the study. Veterans who were current smokers were less likely to have dental care utilization within the previous six months than former smokers or never smokers. In unadjusted logistic regression analysis, current smokers had an odds ratio of 2.83 [95% CI: 2.36, 3.40] as compared with never smokers. The adjusted odds ratio for current smoking on dental care utilization was 1.71 [95% CI: 1.40, 2.09] as compared with never smoking. CONCLUSIONS: Since veterans who smoked are less likely to have dental care utilization within the previous six months, they are at higher risk for later diagnosis of dental problems. Veterans who smoke should be specifically targeted with interventions to ensure frequent dental visits, so future problems may be averted or managed early in their development.

The ionophore oxyclozanide enhances tobramycin killing of Pseudomonas aeruginosa biofilms by permeabilizing cells and depolarizing the membrane potential.

OBJECTIVES: To assess the ability of oxyclozanide to enhance tobramycin killing of Pseudomonas aeruginosa biofilms and elucidate its mechanism of action. METHODS: Twenty-four hour biofilms formed by the P. aeruginosa strain PAO1 and cystic fibrosis (CF) isolates were tested for susceptibility to oxyclozanide and tobramycin killing using BacTiter-Glo™ and cfu. Biofilm dispersal was measured using crystal violet staining. Membrane potential and permeabilization were quantified using DiOC2(3) and TO-PRO-3, respectively. RESULTS: Here we show that the ionophore anthelmintic oxyclozanide, combined with tobramycin, significantly increased killing of P. aeruginosa biofilms over each treatment alone. This combination also significantly accelerated the killing of cells within biofilms and stationary phase cultures and it was effective against 4/6 CF clinical isolates tested, including a tobramycin-resistant strain. Oxyclozanide enhanced the ability of additional aminoglycosides and tetracycline to kill P. aeruginosa biofilms. Finally, oxyclozanide permeabilized cells within the biofilm, reduced the membrane potential and increased tobramycin accumulation within cells of mature P. aeruginosa biofilms. CONCLUSIONS: Oxyclozanide enhances aminoglycoside and tetracycline activity against P. aeruginosa biofilms by reducing membrane potential, permeabilizing cells and enhancing tobramycin accumulation within biofilms. We propose that oxyclozanide counteracts the adaptive resistance response of P. aeruginosa to aminoglycosides, increasing both their maximum activity and rate of killing. As oxyclozanide is widely used in veterinary medicine for the treatment of parasitic worm infections, this combination could offer a new approach for the treatment of biofilm-based P. aeruginosa infections, repurposing oxyclozanide as an anti-biofilm agent.

Apoptosis signal-regulating kinase-1 promotes inflammasome priming in macrophages.

We previously showed that mice deficient in apoptosis signal-regulating kinase-1 (ASK1) were partially protected against ventilator-induced lung injury. Because ASK1 can promote both cell death and inflammation, we hypothesized that ASK1 activation regulates inflammasome-mediated inflammation. Mice deficient in ASK1 expression (ASK1-/-) exhibited significantly less inflammation and lung injury (as measured by neutrophil infiltration, IL-6, and IL-1ß) in response to treatment with inhaled lipopolysaccharide (LPS) compared with wild-type (WT) mice. To determine whether this proinflammatory response was mediated by ASK1, we investigated inflammasome-mediated responses to LPS in primary macrophages and bone marrow-derived macrophages (BMDMs) from WT and ASK1-/- mice, as well as the mouse alveolar macrophage cell line MH-S. Cells were treated with LPS alone for priming or LPS followed by ATP for activation. When macrophages were stimulated with LPS followed by ATP to activate the inflammasome, we found a significant increase in secreted IL-1ß from WT cells compared with ASK1-deficient cells. LPS priming stimulated an increase in NOD-like receptor 3 (NLRP3) and pro-IL-1ß in WT BMDMs, but expression of NLRP3 was significantly decreased in ASK1-/- BMDMs. Subsequent ATP treatment stimulated an increase in cleaved caspase-1 and IL-1ß in WT BMDMs compared with ASK1-/- BMDMs. Similarly, treatment of MH-S cells with LPS + ATP caused an increase in both cleaved caspase-1 and IL-1ß that was diminished by the ASK-1 inhibitor NQDI1. These results demonstrate, for the first time, that ASK1 promotes inflammasome priming.