Crystalline silica-induced pulmonary inflammation and autoimmunity in mature adult NZBW/f1 mice: age-related sensitivity and impact of omega-3 fatty acid intervention.

OBJECTIVE: Occupational exposure to respirable crystalline silica (cSiO2) has been linked to lupus development. Previous studies in young lupus-prone mice revealed that intranasal cSiO2 exposure triggered autoimmunity, preventable with docosahexaenoic acid (DHA). This study explores cSiO2 and DHA effects in mature lupus-prone adult mice, more representative of cSiO2-exposed worker age. METHODS: Female NZBWF1 mice (14-week old) were fed control (CON) or DHA-supplemented diets. After two weeks, mice were intranasally instilled saline (VEH) or 1 mg cSiO2 weekly for four weeks. Cohorts were then analyzed 1- and 5-weeks postinstillation for lung inflammation, cell counts, chemokines, histopathology, B- and T-cell infiltration, autoantibodies, and gene signatures, with results correlated to autoimmune glomerulonephritis onset. RESULTS: VEH/CON mice showed no pathology. cSiO2/CON mice displayed significant ectopic lymphoid tissue formation in lungs at 1 week, increasing by 5 weeks. cSiO2/CON lungs exhibited elevated cellularity, chemokines, CD3+ T-cells, CD45R + B-cells, IgG + plasma cells, gene expression, IgG autoantibodies, and glomerular hypertrophy. DHA supplementation mitigated all these effects. DISCUSSION: The mature adult NZBWF1 mouse used here represents a life-stage coincident with immunological tolerance breach and one that more appropriately represents the age (20-30 yr) of cSiO2-exposed workers. cSiO2-induced robust pulmonary inflammation, autoantibody responses, and glomerulonephritis in mature adult mice, surpassing effects observed previously in young adults. DHA at a human-equivalent dosage effectively countered cSiO2-induced inflammation/autoimmunity in mature mice, mirroring protective effects in young mice. CONCLUSION: These results highlight life-stage significance in this preclinical lupus model and underscore omega-3 fatty acids' therapeutic potential against toxicant-triggered autoimmune responses.

Toward personalized care for insomnia in the US Army: a machine learning model to predict response to cognitive behavioral therapy for insomnia.

STUDY OBJECTIVES: The standard of care for military personnel with insomnia is cognitive behavioral therapy for insomnia (CBT-I). However, only a minority seeking insomnia treatment receive CBT-I, and little reliable guidance exists to identify those most likely to respond. As a step toward personalized care, we present results of a machine learning (ML) model to predict CBT-I response. METHODS: Administrative data were examined for n = 1,449 nondeployed US Army soldiers treated for insomnia with CBT-I who had moderate-severe baseline Insomnia Severity Index (ISI) scores and completed 1 or more follow-up ISIs 6-12 weeks after baseline. An ensemble ML model was developed in a 70% training sample to predict clinically significant ISI improvement (reduction of at least 2 standard deviations on the baseline ISI distribution). Predictors included a wide range of military administrative and baseline clinical variables. Model accuracy was evaluated in the remaining 30% test sample. RESULTS: 19.8% of patients had clinically significant ISI improvement. Model area under the receiver operating characteristic curve (standard error) was 0.60 (0.03). The 20% of test-sample patients with the highest probabilities of improvement were twice as likely to have clinically significant improvement compared with the remaining 80% (36.5% vs 15.7%; χ21 = 9.2, P = .002). Nearly 85% of prediction accuracy was due to 10 variables, the most important of which were baseline insomnia severity and baseline suicidal ideation. CONCLUSIONS: Pending replication, the model could be used as part of a patient-centered decision-making process for insomnia treatment. Parallel models will be needed for alternative treatments before such a system is of optimal value. CITATION: Gabbay FH, Wynn GH, Georg MW, et al. Toward personalized care for insomnia in the US Army: a machine learning model to predict response to cognitive behavioral therapy for insomnia. J Clin Sleep Med. 2024;20(6):921-931.

Dietary docosahexaenoic acid supplementation inhibits acute pulmonary transcriptional and autoantibody responses to a single crystalline silica exposure in lupus-prone mice.

Introduction: Workplace exposure to respirable crystalline silica (cSiO2) has been epidemiologically linked to lupus. Consistent with this, repeated subchronic intranasal cSiO2 instillation in lupus-prone NZBWF1 mice induces inflammation-/autoimmune-related gene expression, ectopic lymphoid tissue (ELT), autoantibody (AAb) production in the lung within 5 to 13 wk followed systemic AAb increases and accelerated onset and progression of glomerulonephritis within 13 to 17 wk. Interestingly, dietary docosahexaenoic acid (DHA) supplementation suppresses these pathologic effects, but the underlying molecular mechanisms remain unclear. Methods: This study aimed to test the hypothesis that dietary DHA supplementation impacts acute transcriptional and autoantibody responses in the lungs of female NZBWF1 mice 1 and 4 wk after a single high-dose cSiO2 challenge. Groups of mice were initially fed a control (Con) diet or a DHA-containing diet (10 g/kg). Cohorts of Con- and DHA-fed were subjected to a single intranasal instillation of 2.5 mg cSiO2 in a saline vehicle (Veh), while a Con-fed cohort was instilled with Veh only. At 1 and 4 wk post-instillation (PI), we compared cSiO2's effects on innate-/autoimmune-related gene expression and autoantibody (AAb) in lavage fluid/lungs of Con- and DHA-fed mice and related these findings to inflammatory cell profiles, histopathology, cell death, and cytokine/chemokine production. Results: DHA partially alleviated cSiO2-induced alterations in total immune cell and lymphocyte counts in lung lavage fluid. cSiO2-triggered dead cell accumulation and levels of inflammation-associated cytokines and IFN-stimulated chemokines were more pronounced in Con-fed mice than DHA-fed mice. Targeted multiplex transcriptome analysis revealed substantial upregulation of genes associated with autoimmune pathways in Con-fed mice in response to cSiO2 that were suppressed in DHA-fed mice. Pathway analysis indicated that DHA inhibited cSiO2 induction of proinflammatory and IFN-regulated gene networks, affecting key upstream regulators (e.g., TNFα, IL-1ß, IFNAR, and IFNγ). Finally, cSiO2-triggered AAb responses were suppressed in DHA-fed mice. Discussion: Taken together, DHA mitigated cSiO2-induced upregulation of pathways associated with proinflammatory and IFN-regulated gene responses within 1 wk and reduced AAb responses by 4 wk. These findings suggest that the acute short-term model employed here holds substantial promise for efficient elucidation of the molecular mechanisms through which omega-3 PUFAs exert protective effects against cSiO2-induced autoimmunity.

An experimental evaluation of a stopping rule aimed at maximizing cost-quality trade-offs in surveys.

Surveys face difficult choices in managing cost-error trade-offs. Stopping rules for surveys have been proposed as a method for managing these trade-offs. A stopping rule will limit effort on a select subset of cases to reduce costs with minimal harm to quality. Previously proposed stopping rules have focused on quality with an implicit assumption that all cases have the same cost. This assumption is unlikely to be true, particularly when some cases will require more effort and, therefore, more costs than others. We propose a new rule that looks at both predicted costs and quality. This rule is tested experimentally against another rule that focuses on stopping cases that are expected to be difficult to recruit. The experiment was conducted on the 2020 data collection of the Health and Retirement Study (HRS). We test both Bayesian and non-Bayesian (maximum-likelihood or ML) versions of the rule. The Bayesian version of the prediction models uses historical data to establish prior information. The Bayesian version led to higher-quality data for roughly the same cost, while the ML version led to small reductions in quality with larger reductions in cost compared to the control rule.

Visible Cash, a Second Incentive, and Priority Mail? An Experimental Evaluation of Mailing Strategies for a Screening Questionnaire in a National Push-to-Web/Mail Survey.

In push-to-web surveys that use postal mail to contact sampled cases, participation is contingent on the mail being opened and the survey invitations being delivered. The design of the mailings is crucial to the success of the survey. We address the question of how to design invitation mailings that can grab potential respondents' attention and sway them to be interested in the survey in a short window of time. In the household screening stage of a national survey, the American Family Health Study, we experimentally tested three mailing design techniques for recruiting respondents: (1) a visible cash incentive in the initial mailing, (2) a second incentive for initial nonrespondents, and (3) use of Priority Mail in the nonresponse follow-up mailing. We evaluated the three techniques' overall effects on response rates as well as how they differentially attracted respondents with different characteristics. We found that all three techniques were useful in increasing the screening response rates, but there was little evidence that they had differential effects on sample subgroups that could help to reduce nonresponse biases.

Methods for improving participation rates in national self-administered web/mail surveys: Evidence from the United States.

In the United States, increasing access to the internet, the increasing costs of large-scale face-to-face data collections, and the general reluctance of the public to participate in intrusive in-person data collections all mean that new approaches to nationally representative surveys are urgently needed. The COVID-19 pandemic accelerated the need for faster, higher-quality alternatives to face-to-face data collection. These trends place a high priority on the evaluation of innovative web-based data collection methods that are convenient for the U.S. public and yield scientific information of high quality. The web mode is particularly appealing because it is relatively inexpensive, it is logistically flexible to implement, and it affords a high level of privacy and confidentiality when correctly implemented. With this study, we aimed to conduct a methodological evaluation of a sequential mixed-mode web/mail data collection protocol, including modular survey design concepts, which was implemented on a national probability sample in the U.S. in 2020-2021. We implemented randomized experiments to test theoretically-informed hypotheses that 1) the use of mail and increased incentives to follow up with households that did not respond to an invitation to complete a household screening questionnaire online would help to recruit different types of households; and 2) the use of modular survey design, which involves splitting a lengthy self-administered survey up into multiple parts that can be completed at a respondent's convenience, would improve survey completion rates. We find support for the use of mail and increased incentives to follow up with households that have not responded to a web-based screening questionnaire. We did not find support for the use of modular design in this context. Simple descriptive analyses also suggest that attempted telephone reminders may be helpful for the main survey.

Deriving Priors for Bayesian Prediction of Daily Response Propensity in Responsive Survey Design: Historical Data Analysis Versus Literature Review.

Responsive survey design (RSD) aims to increase the efficiency of survey data collection via live monitoring of paradata and the introduction of protocol changes when survey errors and increased costs seem imminent. Daily predictions of response propensity for all active sampled cases are among the most important quantities for live monitoring of data collection outcomes, making sound predictions of these propensities essential for the success of RSD. Because it relies on real-time updates of prior beliefs about key design quantities, such as predicted response propensities, RSD stands to benefit from Bayesian approaches. However, empirical evidence of the merits of these approaches is lacking in the literature, and the derivation of informative prior distributions is required for these approaches to be effective. In this paper, we evaluate the ability of two approaches to deriving prior distributions for the coefficients defining daily response propensity models to improve predictions of daily response propensity in a real data collection employing RSD. The first approach involves analyses of historical data from the same survey, and the second approach involves literature review. We find that Bayesian methods based on these two approaches result in higher-quality predictions of response propensity than more standard approaches ignoring prior information. This is especially true during the early-to-middle periods of data collection, when survey managers using RSD often consider interventions.

Toward personalized care for insomnia in the US Army: development of a machine-learning model to predict response to pharmacotherapy.

STUDY OBJECTIVES: Although many military personnel with insomnia are treated with prescription medication, little reliable guidance exists to identify patients most likely to respond. As a first step toward personalized care for insomnia, we present results of a machine-learning model to predict response to insomnia medication. METHODS: The sample comprised n = 4,738 nondeployed US Army soldiers treated with insomnia medication and followed 6-12 weeks after initiating treatment. All patients had moderate-severe baseline scores on the Insomnia Severity Index (ISI) and completed 1 or more follow-up ISIs 6-12 weeks after baseline. An ensemble machine-learning model was developed in a 70% training sample to predict clinically significant ISI improvement, defined as reduction of at least 2 standard deviations on the baseline ISI distribution. Predictors included a wide range of military administrative and baseline clinical variables. Model accuracy was evaluated in the remaining 30% test sample. RESULTS: 21.3% of patients had clinically significant ISI improvement. Model test sample area under the receiver operating characteristic curve (standard error) was 0.63 (0.02). Among the 30% of patients with the highest predicted probabilities of improvement, 32.5.% had clinically significant symptom improvement vs 16.6% in the 70% sample predicted to be least likely to improve (χ21 = 37.1, P < .001). More than 75% of prediction accuracy was due to 10 variables, the most important of which was baseline insomnia severity. CONCLUSIONS: Pending replication, the model could be used as part of a patient-centered decision-making process for insomnia treatment, but parallel models will be needed for alternative treatments before such a system is of optimal value. CITATION: Gabbay FH, Wynn GH, Georg MW, et al. Toward personalized care for insomnia in the US Army: development of a machine-learning model to predict response to pharmacotherapy. J Clin Sleep Med. 2023;19(8):1399-1410.

Lipidome modulation by dietary omega-3 polyunsaturated fatty acid supplementation or selective soluble epoxide hydrolase inhibition suppresses rough LPS-accelerated glomerulonephritis in lupus-prone mice.

Introduction: Lipopolysaccharide (LPS)-accelerated autoimmune glomerulonephritis (GN) in NZBWF1 mice is a preclinical model potentially applicable for investigating lipidome-modulating interventions against lupus. LPS can be expressed as one of two chemotypes: smooth LPS (S-LPS) or rough LPS (R-LPS) which is devoid of O-antigen polysaccharide sidechain. Since these chemotypes differentially affect toll-like receptor 4 (TLR4)-mediated immune cell responses, these differences may influence GN induction. Methods: We initially compared the effects of subchronic intraperitoneal (i.p.) injection for 5 wk with 1) Salmonella S-LPS, 2) Salmonella R-LPS, or 3) saline vehicle (VEH) (Study 1) in female NZBWF1 mice. Based on the efficacy of R-LPS in inducing GN, we next used it to compare the impact of two lipidome-modulating interventions, ω-3 polyunsaturated fatty acid (PUFA) supplementation and soluble epoxide hydrolase (sEH) inhibition, on GN (Study 2). Specifically, effects of consuming ω-3 docosahexaenoic acid (DHA) (10 g/kg diet) and/or the sEH inhibitor 1-(4-trifluoro-methoxy-phenyl)-3-(1-propionylpiperidin-4-yl) urea (TPPU) (22.5 mg/kg diet ≈ 3 mg/kg/day) on R-LPS triggering were compared. Results: In Study 1, R-LPS induced robust elevations in blood urea nitrogen, proteinuria, and hematuria that were not evident in VEH- or S-LPS-treated mice. R-LPS-treated mice further exhibited kidney histopathology including robust hypertrophy, hyperplasia, thickened membranes, lymphocytic accumulation containing B and T cells, and glomerular IgG deposition consistent with GN that was not evident in VEH- or SLPS-treated groups. R-LPS but not S-LPS induced spleen enlargement with lymphoid hyperplasia and inflammatory cell recruitment in the liver. In Study 2, resultant blood fatty acid profiles and epoxy fatty acid concentrations reflected the anticipated DHA- and TPPU-mediated lipidome changes, respectively. The relative rank order of R-LPS-induced GN severity among groups fed experimental diets based on proteinuria, hematuria, histopathologic scoring, and glomerular IgG deposition was: VEH/CON< R-LPS/DHA ≈ R-LPS/TPPU<<< R-LPS/TPPU+DHA ≈ R-LPS/CON. In contrast, these interventions had modest-to- negligible effects on R-LPS-induced splenomegaly, plasma antibody responses, liver inflammation, and inflammation-associated kidney gene expression. Discussion: We show for the first time that absence of O-antigenic polysaccharide in R-LPS is critical to accelerated GN in lupus-prone mice. Furthermore, intervention by lipidome modulation through DHA feeding or sEH inhibition suppressed R-LPS-induced GN; however, these ameliorative effects were greatly diminished upon combining the treatments.

Investigation into the use of gamma irradiated Cytodex-1 microcarriers to produce a human cytomegalovirus (HCMV) vaccine candidate in epithelial cells.

V160 is a viral vaccine candidate against human cytomegalovirus (HCMV) that is manufactured using Adult Retinal Pigment Epithelial cells (ARPE-19) grown on Cytodex-1 microcarriers. The microcarriers are generally hydrated, washed, and autoclaved prior to use, which can be limiting at large production scales. To minimize microcarrier preparation and sterilization, the use of gamma irradiated Cytodex-1 was investigated. Similar ARPE-19 cell growth was observed on heat-sterilized and gamma irradiated Cytodex-1; however, significantly reduced virus production was observed in cultures exposed to gamma irradiated Cytodex-1. Additional experiments suggest that infection inhibition is not exclusive to ARPE-19 but is most directly linked to HCMV V160, as evidenced by similar inhibition of V160 with Vero cells and no inhibition of Measles virus with either cell type. These observations suggest a putative impact on HCMV infection from the presence of extractable(s)/leachable(s) in the gamma irradiated microcarriers. Thorough aseptic rinsing of gamma irradiated Cytodex-1 prior to use can mitigate this impact and enable comparable process performance to heat-sterilized Cytodex-1. Though not fully a "ready-to-use" product for the HCMV V160 production process, utilization of Cytodex-1 microcarriers was possible without requiring heat sterilization, suggesting a potential path forward for large scale production of V160.

An Experimental Evaluation of Two Approaches for Improving Response to Household Screening Efforts in National Mail/Web Surveys.

Survey researchers have carefully modified their data collection operations for various reasons, including the rising costs of data collection and the ongoing Coronavirus disease (COVID-19) pandemic, both of which have made in-person interviewing difficult. For large national surveys that require household (HH) screening to determine survey eligibility, cost-efficient screening methods that do not include in-person visits need additional evaluation and testing. A new study, known as the American Family Health Study (AFHS), recently initiated data collection with a national probability sample, using a sequential mixed-mode mail/web protocol for push-to-web US HH screening (targeting persons aged 18-49 years). To better understand optimal approaches for this type of national screening effort, we embedded two randomized experiments in the AFHS data collection. The first tested the use of bilingual respondent materials where mailed invitations to the screener were sent in both English and Spanish to 50 percent of addresses with a high predicted likelihood of having a Spanish speaker and 10 percent of all other addresses. We found that the bilingual approach did not increase the response rate of high-likelihood Spanish-speaking addresses, but consistent with prior work, it increased the proportion of eligible Hispanic respondents identified among completed screeners, especially among addresses predicted to have a high likelihood of having Spanish speakers. The second tested a form of nonresponse follow-up, where a subsample of active sampled HHs that had not yet responded to the screening invitations was sent a priority mailing with a $5 incentive, adding to the $2 incentive provided for all sampled HHs in the initial screening invitation. We found this approach to be quite valuable for increasing the screening survey response rate.

Key Attributes of a Medical Learning Community Mentor at One Medical School: The Mentee Perspective.

To assess the elements necessary to be a successful learning community (ClinCalc) mentor to medical students from the mentee's perspective. Few such studies have utilized the in-depth and richness of detail obtained in qualitative studies. This qualitative study analyzed four focus group discussions lasting 45-90 min conducted at the University of Texas Southwestern Medical School, which has an established LC, in the year 2018. The groups included 14 pre-clerkship and 8 clerkship students. Investigators evaluated transcriptions of the focus group discussions using ATLAS.ti software. Three overarching categories of discussion emerged from the group discussions: (1) Relationship Competence, (2) Teaching Competence, and (3) Ethical and Compassionate Medical Practice Competence. Relationship Competence themes included "walk with me," relationship is most important, and one-on-one. Teaching Competence themes included above and beyond, recognize and address mentor limitations, and safe and enriching environment. Ethical and Compassionate Medical Practice Competence themes included ethical decision making and compassionate care for diverse patient populations. Mentees focused on various aspects of the mentor-mentee relationship as the single most essential competence. Themes mentees discussed as important qualities of a successful mentor may denote qualities to be prioritized in faculty development and mentor recruitment. Future studies could investigate how the LC environment informs former medical students and promotes patient outcomes.

Comparative effects of human-equivalent low, moderate, and high dose oral prednisone intake on autoimmunity and glucocorticoid-related toxicity in a murine model of environmental-triggered lupus.

Autoimmune diseases can be triggered by environmental toxicants such as crystalline silica dust (cSiO2). Here, we characterized the dose-dependent immunomodulation and toxicity of the glucocorticoid (GC) prednisone in a preclinical model that emulates onset and progression of cSiO2-triggered lupus. Two cohorts of 6-wk-old female NZBWF1 mice were fed either control AIN-93G diet or one of three AIN-93G diets containing prednisone at 5, 15, or 50 mg/kg diet which span human equivalent oral doses (HED) currently considered to be low (PL; 5 mg/d HED), moderate (PM; 14 mg/d HED), or high (PH; 46 mg/d HED), respectively. At 8 wk of age, mice were intranasally instilled with either saline vehicle or 1 mg cSiO2 once weekly for 4 wk. The experimental plan was to 1) terminate one cohort of mice (n=8/group) 14 wk after the last cSiO2 instillation for pathology and autoimmunity assessment and 2) to maintain a second cohort (n=9/group) to monitor glomerulonephritis development and survival. Mean blood concentrations of prednisone's principal active metabolite, prednisolone, in mice fed PL, PM, and PH diets were 27, 105, 151 ng/ml, respectively, which are consistent with levels observed in human blood ≤ 12 h after single bolus treatments with equivalent prednisone doses. Results from the first cohort revealed that consumption of PM, but not PL diet, significantly reduced cSiO2-induced pulmonary ectopic lymphoid structure formation, nuclear-specific AAb production, inflammation/autoimmune gene expression in the lung and kidney, splenomegaly, and glomerulonephritis in the kidney. Relative to GC-associated toxicity, PM diet, but not PL diet, elicited muscle wasting, but these diets did not affect bone density or cause glucosuria. Importantly, neither PM nor PL diet improved latency of cSiO2-accelerated death. PH-fed mice in both cohorts displayed robust GC-associated toxicity including body weight loss, reduced muscle mass, and extensive glucosuria 7 wk after the final cSiO2 instillation requiring their early removal from the study. Taken together, our results demonstrate that while moderate doses of prednisone can reduce important pathological endpoints of cSiO2-induced autoimmunity in lupus-prone mice, such as upstream ectopic lymphoid structure formation, these ameliorative effects come with unwanted GC toxicity, and, crucially, none of these three doses extended survival time.

Rapid In-Process Measurement of Live Virus Vaccine Potency Using Laser Force Cytology: Paving the Way for Rapid Vaccine Development.

Vaccinations to prevent infectious diseases are given to target the body's innate and adaptive immune systems. In most cases, the potency of a live virus vaccine (LVV) is the most critical measurement of efficacy, though in some cases the quantity of surface antigen on the virus is an equally critical quality attribute. Existing methods to measure the potency of viruses include plaque and TCID50 assays, both of which have very long lead times and cannot provide real time information on the quality of the vaccine during large-scale manufacturing. Here, we report the evaluation of LumaCyte's Radiance Laser Force Cytology platform as a new way to measure the potency of LVVs in upstream biomanufacturing process in real time and compare this to traditional TCID50 potency. We also assess this new platform as a way to detect adventitious agents, which is a regulatory expectation for the release of commercial vaccines. In both applications, we report the ability to obtain expedited and relevant potency information with strong correlation to release potency methods. Together, our data propose the application of Laser Force Cytology as a valuable process analytical technology (PAT) for the timely measurement of critical quality attributes of LVVs.

Neuroinflammatory and Neurometabolomic Consequences From Inhaled Wildfire Smoke-Derived Particulate Matter in the Western United States.

Utilizing a mobile laboratory located >300 km away from wildfire smoke (WFS) sources, this study examined the systemic immune response profile, with a focus on neuroinflammatory and neurometabolomic consequences, resulting from inhalation exposure to naturally occurring wildfires in California, Arizona, and Washington in 2020. After a 20-day (4 h/day) exposure period in a mobile laboratory stationed in New Mexico, WFS-derived particulate matter (WFPM) inhalation resulted in significant neuroinflammation while immune activity in the peripheral (lung, bone marrow) appeared to be resolved in C57BL/6 mice. Importantly, WFPM exposure increased cerebrovascular endothelial cell activation and expression of adhesion molecules (VCAM-1 and ICAM-1) in addition to increased glial activation and peripheral immune cell infiltration into the brain. Flow cytometry analysis revealed proinflammatory phenotypes of microglia and peripheral immune subsets in the brain of WFPM-exposed mice. Interestingly, endothelial cell neuroimmune activity was differentially associated with levels of PECAM-1 expression, suggesting that subsets of cerebrovascular endothelial cells were transitioning to resolution of inflammation following the 20-day exposure. Neurometabolites related to protection against aging, such as NAD+ and taurine, were decreased by WFPM exposure. Additionally, increased pathological amyloid-beta protein accumulation, a hallmark of neurodegeneration, was observed. Neuroinflammation, together with decreased levels of key neurometabolites, reflect a cluster of outcomes with important implications in priming inflammaging and aging-related neurodegenerative phenotypes.

Combined adjuvant effects of ambient vapor-phase organic components and particulate matter potently promote allergic sensitization and Th2-skewing cytokine and chemokine milieux in mice: The importance of mechanistic multi-pollutant research.

Although exposure to ambient particulate matter (PM) is linked to asthma, the health effects of co-existing vapor-phase organic pollutants (vapor) and their combined effects with PM on this disease are poorly understood. We used a murine asthma model to test the hypothesis that exposure to vapor would enhance allergic sensitization and this effect would be further strengthened by co-existing PM. We found that vapor and PM each individually exerted adjuvant effects on OVA sensitization. Co-exposure to vapor and PM during sensitization further enhanced allergic lung inflammation and OVA-specific antibody production which was accompanied by pulmonary cytokine/chemokine milieu that favored T-helper 2 immunity (i.e. increased IL-4, downregulation of Il12a and Ifng, and upregulation of Ccl11 and Ccl8). TNFα, IL-6, Ccl12, Cxcl1 and detoxification/antioxidant enzyme responses in the lung were pollutant-dependent. Inhibition of lipopolysaccharide-induced IL-12 secretion from primary antigen-presenting dendritic cells correlated positively with vapor's oxidant potential. In conclusion, concurrent exposure to vapor and PM led to significantly exaggerated adjuvant effects on allergic lung inflammation which were more potent than that of each pollutant type alone. These findings suggest that the effects of multi-component air pollution on asthma may be significantly underestimated if research only focuses on a single air pollutant (e.g., PM).

Gamma-tocopherol, a major form of vitamin E in diets: Insights into antioxidant and anti-inflammatory effects, mechanisms, and roles in disease management.

γ-Tocopherol (γT) is a major form of vitamin E in the US diet and the second most abundant vitamin E in the blood and tissues, while α-tocopherol (αT) is the predominant vitamin E in tissues. During the last >25 years, research has revealed that γT has unique antioxidant and anti-inflammatory activities relevant to disease prevention compared to αT. While both compounds are potent lipophilic antioxidants, γT but not αT can trap reactive nitrogen species by forming 5-nitro-γT, and appears to show superior protection of mitochondrial function. γT inhibits ionophore-stimulated leukotrienes by blocking 5-lipoxygenase (5-LOX) translocation in leukocytes, decreases cyclooxygenase-2 (COX-2)-catalyzed prostaglandins in macrophages and blocks the growth of cancer cells but not healthy cells. For these activities, γT is stronger than αT. Moreover, γT is more extensively metabolized than αT via cytochrome P-450 (CYP4F2)-initiated side-chain oxidation, which leads to formation of metabolites including 13'-carboxychromanol (13'-COOH) and carboxyethyl-hydroxychroman (γ-CEHC). 13'-COOH and γ-CEHC are shown to be the predominant metabolites found in feces and urine, respectively. Interestingly, γ-CEHC has natriuretic activity and 13'-COOH inhibits both COX-1/-2 and 5-LOX activity. Consistent with these mechanistic findings of γT and metabolites, studies show that supplementation of γT mitigates inflammation and disease symptoms in animal models with induced inflammation, asthma and cancer. In addition, supplementation of γT decreased inflammation markers in patients with kidney diseases and mild asthma. These observations support that γT may be useful against inflammation-associated diseases.

Sorting for secreted molecule production using a biosensor-in-microdroplet approach.

Sorting large libraries of cells for improved small molecule secretion is throughput limited. Here, we combine producer/secretor cell libraries with whole-cell biosensors using a microfluidic-based screening workflow. This approach enables a mix-and-match capability using off-the-shelf biosensors through either coencapsulation or pico-injection. We demonstrate the cell type and library agnostic nature of this workflow by utilizing single-guide RNA, transposon, and ethyl-methyl sulfonate mutagenesis libraries across three distinct microbes (Escherichia coli, Saccharomyces cerevisiae, and Yarrowia lipolytica), biosensors from two organisms (E. coli and S. cerevisiae), and three products (triacetic acid lactone, naringenin, and L-DOPA) to identify targets improving production/secretion.

Genome Engineering of Yarrowia lipolytica with the PiggyBac Transposon System.

A mutant excision+/integration- piggyBac transposase can be used to seamlessly excise a chromosomally integrated, piggyBac-compatible selection marker cassette from the Yarrowia lipolytica genome. This piggyBac transposase-based genome engineering process allows for both positive selection of targeted homologous recombination events and scarless or footprint-free genome modifications after precise marker recovery. Residual non-native sequences left in the genome after marker excision can be minimized (0-4 nucleotides) or customized (user-defined except for a TTAA tetranucleotide). Both of these options reduce the risk of unintended homologous recombination events in strains with multiple genomic edits. A suite of dual positive/negative selection marker pairs flanked by piggyBac inverted terminal repeats (ITRs) have been constructed and are available for precise genome engineering in Y. lipolytica using this method. This protocol specifically describes the split marker homologous recombination-based disruption of Y. lipolytica ADE2 with a piggyBac ITR-flanked URA3 cassette, followed by piggyBac transposase-mediated excision of the URA3 marker to leave a 50 nucleotide synthetic barcode at the ADE2 locus. The resulting ade2 strain is auxotrophic for adenine, which enables the use of ADE2 as a selectable marker for further strain engineering.