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Improving Health Outcomes for LGBTQ+ Youth Through Provider Education

Author: Simko Marissa
Publication venue: USF Scholarship: a digital repository @ Gleeson Library | Geschke Center
Publication date: 18/05/2023
Field of study

Background and Problem: LGBTQ+ adolescents and young adults (ages 12-25 years) are known to have higher rates of physical and mental health concerns compared to heterosexual and cisgender youth. Within the LGBTQ+ youth community, rates of suicidality, substance misuse, homelessness, and STIs are higher than in the general population. LGBTQ+ youth have greater challenges accessing healthcare and higher rates of healthcare discrimination. This paper presents a quality improvement project focused on improving evidence-based practices in LGBTQ+ healthcare through the development and implementation of a LGBTQ+ youth centered educational workshop for healthcare providers. Methods: This project was organized in collaboration with stakeholders at Adolescent Health Working Group (AHWG) based on identified needs of the organization. The design and education of the workshop was supported by current literature and evaluation was performed. The theory of cultural humility informed the development of the project and outcome measures. Interventions: An educational workshop was provided to 11 providers within the AHWG network. A pre- and post-workshop evaluation was provided to assess learning outcomes. Additionally, a toolkit drive was provided with educational resources for providers and educational handouts for youth and caregivers. Results: Providers were assessed on knowledge, confidence in providing LGBTQ+ centered youth care, and attitudes toward LGBTQ+ youth within the pre- and post-evaluations. For knowledge-centered questions, the highest attainable score increased by 29.9%. For confidence-centered questions, the highest attainable score increased by 26.8%. And the attitudes-centered highest attainable score increased by 6.0%. Overall, this led to an average improvement in pre- and post-workshop scores of 20.9%. Conclusion: There is a gap in healthcare provider knowledge around LGBTQ+ youth centered care. Providing education on LGBTQ+ youth health needs can help address the knowledge gap in healthcare providers. With increased education, healthcare providers will have improved abilities in providing equitable LGBTQ+ youth care. Keywords: lgbt*, sensitive or humility or competent, education or care or training, access or engagement, young adult or youth or adoles

University of San Francisco

The MDT-15 Subunit of Mediator Interacts with Dietary Restriction to Modulate Longevity and Fluoranthene Toxicity in Caenorhabditis elegans

Dietary restriction (DR), the limitation of calorie intake while maintaining proper nutrition, has been found to extend life span and delay the onset of age-associated disease in a wide range of species. Previous studies have suggested that DR can reduce the lethality of environmental toxins. To further examine the role of DR in toxin response, we measured life spans of the nematode Caenorhabditis elegans treated with the mutagenic polyaromatic hydrocarbon, fluoranthene (FLA). FLA is a direct byproduct of combustion, and is one of U.S. Environmental Protection Agency's sixteen priority environmental toxins. Treatment with 5 µg/ml FLA shortened the life spans of ad libitum fed nematodes, and DR resulted in increased sensitivity to FLA. To determine the role of detoxifying enzymes in the toxicity of FLA, we tested nematodes with mutations in the gene encoding the MDT-15 subunit of mediator, a transcriptional coactivator that regulates genes involved in fatty acid metabolism and detoxification. Mutation of mdt-15 increased the life span of FLA treated animals compared to wild-type animals with no difference observed between DR and ad libitum fed mdt-15 animals. We also examined mutants with altered insulin-IGF-1-like signaling (IIS), which is known to modulate life span and stress resistance in C. elegans independently of DR. Mutation of the genes coding for the insulin-like receptor DAF-2 or the FOXO-family transcription factor DAF16 did not alter the animals' susceptibility to FLA compared to wild type. Taken together, our results suggest that certain compounds have increased toxicity when combined with a DR regimen through increased metabolic activation. This increased metabolic activation appears to be mediated through the MDT-15 transcription factor and is independent of the IIS pathway

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PubMed Central

Transaldolase inhibition impairs mitochondrial respiration and induces a starvation-like longevity response in Caenorhabditis elegans

<div>Mitochondrial dysfunction can increase oxidative stress and extend lifespan in Caenorhabditis elegans. Homeostatic mechanisms exist to cope with disruptions to mitochondrial function that promote cellular health and organismal longevity. Previously, we determined that decreased expression of the cytosolic pentose phosphate pathway (PPP) enzyme transaldolase activates the mitochondrial unfolded protein response (UPRmt) and extends lifespan. Here we report that transaldolase (tald-1) deficiency impairs mitochondrial function in vivo, as evidenced by altered mitochondrial morphology, decreased respiration, and increased cellular H2O2 levels. Lifespan extension from knockdown of tald-1 is associated with an oxidative stress response involving p38 and c-Jun N-terminal kinase (JNK) MAPKs and a starvation-like response regulated by the transcription factor EB (TFEB) homolog HLH-30. The latter response promotes autophagy and increases expression of the flavin-containing monooxygenase 2 (fmo-2). We conclude that cytosolic redox established through the PPP is a key regulator of mitochondrial function and defines a new mechanism for mitochondrial regulation of longevity.</div

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Transaldolase deficiency causes a starvation-like response that decreases animal fat content and rewires lipid metabolism gene expression.

(A) Intestinal fat staining decreases from RNAi knockdown of tald-1 or cco-1. Oil Red O (ORO) staining was performed on day 3 from hatching animals propagated at 20°C. Scale bar, 50 μm. (B) Quantification of ORO staining within anterior intestine (N = 2 independent experiments, pooled individual worm values, error bars indicate s.e.m., student’s t-test with Bonferroni’s correction). (C) RNAi knockdown of tald-1 causes an increase in adipose triglyceride lipase ATGL-1 protein levels. Scale bar, 200 μm. (D) Mean relative fluorescence of ATGL-1::GFP signal in animals grown on tald-1(RNAi) or cco-1(RNAi). Fluorescence is calculated relative to EV(RNAi) controls (N = 4 independent experiments, pooled individual worm values, error bars indicate s.e.m., student’s t-test with Bonferroni’s correction). (E) RNAi knockdown of tald-1 or cco-1 causes a decrease in stearoyl-CoA desaturase fat-7p::gfp reporter expression. Scale bar, 200 μm. (F) Mean relative fluorescence of fat-7p::gfp reporter animals grown on tald-1(RNAi) or cco-1(RNAi). Fluorescence is calculated relative to EV(RNAi) controls (N = 3 independent experiments, pooled individual worm values, error bars indicate s.e.m., student’s t-test with Bonferroni’s correction). (G) Gene expression of starvation-responsive lipid metabolism genes is altered in tald-1(RNAi) animals. Log2 fold change calculated to emphasize the increases and decreases in gene expression levels from RNAi treatments (N = 6–8 independent experiments, error bars indicate s.e.m., paired student’s t-tests with Bonferroni’s correction). (H) RNAi knockdown of tald-1 does not robustly extend lifespan of BD animals. N2 fed EV(RNAi) (mean 18.2±0.2 days, n = 161), N2 fed tald-1(RNAi) (mean 20.4±0.2 days, n = 151), BD animals developed on EV(RNAi) (mean 20.2±0.2 days, n = 123), BD animals developed on tald-1(RNAi) (mean 21.5±0.3 days, n = 150). Lifespans were performed at 25°C, with one experiment shown. (I) RNAi knockdown of cco-1 extends lifespan dissimilar from BD. N2 fed EV(RNAi) (mean 18.2±0.2 days, n = 161), N2 fed cco-1(RNAi) (mean 24±0.3 days, n = 156), BD animals developed on EV(RNAi) (mean 20.2±0.2 days, n = 123), BD animals developed on cco-1(RNAi) (mean 27.1±0.3 days, n = 148). Lifespans were performed at 25°C, with one experiment shown. (J) RNAi knockdown of tald-1 does not require NHR-49 for lifespan extension. N2 fed EV(RNAi) (mean 17.5±0.1 days, n = 366), N2 fed tald-1(RNAi) (mean 20.1±0.1 days, n = 397), nhr-49(nr2041) fed EV(RNAi) (mean 11.5±0.1 days, n = 310), nhr-49(nr2041) fed tald-1(RNAi) (mean 12.9±0.1 days, n = 333). Lifespans were performed at 25°C, with pooled data from three independent experiments shown. (K) RNAi knockdown of cco-1 does not require NHR-49 for lifespan extension. N2 fed EV(RNAi) (mean 17±0.1 days, n = 532), N2 fed cco-1(RNAi) (mean 22.6±0.2 days, n = 344), nhr-49(nr2041) fed EV(RNAi) (mean 11.1±0.1 days, n = 495), nhr-49(nr2041) fed cco-1(RNAi) (mean 15±0.1 days, n = 489). Lifespans were performed at 25°C, with pooled data from four independent experiments shown. Lifespans in this figure are indicated as mean±s.e.m. and statistical analysis is provided in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006695#pgen.1006695.s011" target="_blank">S1 Table</a>. In this figure, statistics are displayed as: * p<0.05, ** p<0.01, *** p<0.001.</p

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Inhibition of the pentose phosphate pathway activates the UPRmt and extends lifespan.

(A) Diagram of both the oxidative and non-oxidative branches of the PPP. The oxidative branch produces NADPH, while the non-oxidative branch produces ribose-5-P and interconverts sugar carbon backbones. The white boxes contain enzyme names with the human gene listed above the C. elegans homolog. (B) PPP gene knockdown increases hsp-6p::gfp reporter expression. (C) Mean relative fluorescence of hsp-6p::gfp animals grown on PPP RNAi. Fluorescence is calculated relative to EV(RNAi) controls (N = 4 independent experiments, pooled individual worm values, error bars indicate s.e.m., student’s t-test with Bonferroni’s correction). (D) RNAi knockdown of PPP genes extends C. elegans lifespan. N2 fed EV(RNAi) (mean 17.4±0.1 days, n = 455), N2 fed tald-1(RNAi) (mean 19.9±0.2 days, n = 391), N2 fed tkt-1(RNAi) (mean 18.4±0.1 days, n = 461), N2 fed T25B9.9(RNAi) (mean 18.8±0.2 days, n = 311). Lifespans were performed at 25°C, with pooled data from four independent experiments shown. (E) RNAi knockdown of tald-1 extends lifespan independently of the UPRmt. N2 fed EV(RNAi) (mean 19.3±0.2 days, n = 192), N2 fed tald-1(RNAi) (mean 22.1±0.2 days, n = 251), atfs-1(tm4525) fed EV(RNAi) (mean 19.6±0.2 days, n = 230), atfs-1(tm4525) fed tald-1(RNAi) (mean 24.5±0.3 days, n = 228), atfs-1(tm4525);gcn-2(ok871) fed EV(RNAi) (mean 18.9±0.2 days, n = 205), atfs-1(tm4525);gcn-2(ok871) fed tald-1(RNAi) (mean 23.1±0.3 days, n = 220). Lifespans were performed at 20°C, with pooled data from two independent experiments shown. (F) RNAi knockdown of cco-1 extends lifespan independently of the UPRmt. N2 fed EV(RNAi) (mean 19.3±0.2 days, n = 192), N2 fed cco-1(RNAi) (mean 32.3±0.5 days, n = 187), atfs-1(tm4525) fed EV(RNAi) (mean 19.6±0.2 days, n = 230), atfs-1(tm4525) fed cco-1(RNAi) (mean 29±0.6 days, n = 194), atfs-1(tm4525);gcn-2(ok871) fed EV(RNAi) (mean 18.9±0.2 days, n = 205), atfs-1(tm4525);gcn-2(ok871) fed cco-1(RNAi) (mean 32.6±0.5 days, n = 228). Lifespans were performed at 20°C, with pooled data from two independent experiments shown. (G) RNAi knockdown of tald-1 extends lifespan only when knockdown occurs during development. N2 fed EV(RNAi) (mean 14.2±0.1 days, n = 361), N2 fed tald-1(RNAi) from hatching (mean 16.4±0.2 days, n = 468), N2 fed tald-1(RNAi) from L4 (mean 14.4±0.1 days, n = 330). Lifespans were performed at 25°C, with pooled data from three independent experiments shown. Lifespans in this figure are indicated as mean±s.e.m. and statistical analysis is provided in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006695#pgen.1006695.s011" target="_blank">S1 Table</a>. In this figure, statistics are displayed as: * p<0.05, ** p<0.01, *** p<0.001.</p

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Transaldolase deficiency alters mitochondrial morphology and decreases in vivo mitochondrial respiration.

(A) Diagram depicting the posterior intestinal cells that were visualized for mitochondrial morphology. (B) Intestinal mitochondrial morphology is altered by tald-1(RNAi) and cco-1(RNAi). The top panel represents a single 0.34 μm slice imaged using confocal microscopy, with a magnified area displayed in a white dotted box to highlight morphology differences. The bottom panel consists of a max intensity projection of five z-slices to emphasize mitochondrial content in these cells. Scale bar, 10 μm. (C) Quantification of percent mitochondrial area per cell. (N = 2 independent experiments, error bars indicate s.e.m., student’s t-test with Bonferroni’s correction). (D) Mitochondrial morphology changes from tald-1(RNAi) are regulated by DRP-1. RNAi treatments include EV(RNAi), tald-1(RNAi) [50:50 with EV(RNAi)], drp-1(RNAi) [50:50 with EV(RNAi)], and tald-1(RNAi) [50:50 with drp-1(RNAi)]. Scale bar, 10 μm. (E) Oxygen consumption rate decreases with tald-1(RNAi) and cco-1(RNAi). OCR was measured using the Seahorse XF Analyzer and normalized to animal number (N = 6 independent experiments, error bars indicate s.e.m., student’s t-test with Bonferroni’s correction). (F) P/O ratio (the ATP produced per oxygen atom reduced), (G) respiratory control index (State 3:State 4 rates), (H) malate-driven respiration (Complex I-IV), succinate-driven respiration (Complex II-IV), and TMPD/ascorbate-driven respiration (Complex IV) were measured using the OXPHOS assay on isolated mitochondria from RNAi treated animals. Respiratory rates were measured as rate of disappearance of oxygen (nmol[O2]) per minute per mg protein (N = 4 independent experiments, error bars indicate s.e.m., student’s t-test with Bonferroni’s correction). Also, in this figure, color coating of bars and lines reflect the legend in (C).</p

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Lifespan extension from tald-1(RNAi) or cco-1(RNAi) requires stress-activated MAPKs.

(A) RNAi knockdown of tald-1 extends lifespan through the JNK MAPK JNK-1. N2 fed EV(RNAi) (mean 17.2±0.1 days, n = 506), N2 fed tald-1(RNAi) (mean 20.4±0.1 days, n = 500), jnk-1(gk7) fed EV(RNAi) (mean 17±0.1 days, n = 582), jnk-1(gk7) fed tald-1(RNAi) (mean 18.1±0.1 days, n = 488). Lifespans were performed at 25°C, with pooled data from five independent experiments shown. (B) RNAi knockdown of cco-1 extends lifespan partially through the JNK MAPK JNK-1. N2 fed EV(RNAi) (mean 16.9±0.1 days, n = 494), N2 fed cco-1(RNAi) (mean 22.7±0.2 days, n = 431), jnk-1(gk7) fed EV(RNAi) (mean 16.1±0.1 days, n = 594), jnk-1(gk7) fed cco-1(RNAi) (mean 19.9±0.2 days, n = 408). Lifespans were performed at 25°C, with pooled data from four independent experiments shown. (C) RNAi knockdown of tald-1 extends lifespan through the JNK MAPK KGB-1. N2 fed EV(RNAi) (mean 15±0.1 days, n = 630), N2 fed tald-1(RNAi) (mean 18.7±0.1 days, n = 657), kgb-1(um3) fed EV(RNAi) (mean 13.1±0.1 days, n = 580), kgb-1 fed tald-1(RNAi) (mean 11.9±0.1 days, n = 600). Lifespans were performed at 25°C, with pooled data from four independent experiments shown. (D) RNAi knockdown of cco-1 extends lifespan partially through the JNK MAPK KGB-1. N2 fed EV(RNAi) (mean 15±0.1 days, n = 630), N2 fed cco-1(RNAi) (mean 23.2±0.2 days, n = 511), kgb-1(um3) fed EV(RNAi) (mean 13.1±0.1 days, n = 580), kgb-1 fed cco-1(RNAi) (mean 15.8±0.2 days, n = 501). Lifespans were performed at 25°C, with pooled data from four independent experiments shown. (E) RNAi knockdown of tald-1 extends lifespan through the p38 MAPK PMK-1. N2 fed EV(RNAi) (mean 16.8±0.1 days, n = 494), N2 fed tald-1(RNAi) (mean 19.3±0.1 days, n = 460), pmk-1(km25) fed EV(RNAi) (mean 14.3±0.1 days, n = 514), pmk-1(km25) fed tald-1(RNAi) (mean 14±0.1 days, n = 525). Lifespans were performed at 25°C, with pooled data from four independent experiments shown. (F) RNAi knockdown of cco-1 does not require the p38 MAPK PMK-1 for lifespan extension. N2 fed EV(RNAi) (mean 16±0.1 days, n = 575), N2 fed cco-1(RNAi) (mean 22.3±0.2 days, n = 448), pmk-1(km25) fed EV(RNAi) (mean 13.8±0.1 days, n = 609), pmk-1(km25) fed cco-1(RNAi) (mean 18.7±0.1 days, n = 535). Lifespans were performed at 25°C, with pooled data from four independent experiments shown. (G) RNAi knockdown of tald-1 extends lifespan through the MAP3K NSY-1. N2 fed EV(RNAi) (mean 14.6±0.1 days, n = 542), N2 fed tald-1(RNAi) (mean 17.2±0.1 days, n = 599), nsy-1(ag3) fed EV(RNAi) (mean 14.9±0.1 days, n = 473), nsy-1(ag3) fed tald-1(RNAi) (mean 14.4±0.1 days, n = 508). Lifespans were performed at 25°C, with pooled data from four independent experiments shown. (H) RNAi knockdown of cco-1 extends lifespan partially through the MAP3K NSY-1. N2 fed EV(RNAi) (mean 14.6±0.1 days, n = 542), N2 fed cco-1(RNAi) (mean 22.5±0.2 days, n = 454), nsy-1(ag3) fed EV(RNAi) (mean 14.9±0.1 days, n = 473), nsy-1(ag3) fed cco-1(RNAi) (mean 18.5±0.2 days, n = 458). Lifespans were performed at 25°C, with pooled data from four independent experiments shown. Lifespans in this figure are indicated as mean±s.e.m. and statistical analysis is provided in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006695#pgen.1006695.s011" target="_blank">S1 Table</a>.</p

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The flavin-containing monooxygenase FMO-2 is upregulated in a HLH-30 and PMK-1 dependent fashion and regulates the lifespan extension from tald-1(RNAi).

(A)fmo-2p::mCherry reporter expression is increased by tald-1(RNAi) or BD in a HLH-30 and PMK-1 dependent fashion. BD animals were starved for 24 hours on FUDR plates prior to imaging. Scale bar, 200 μm. (B) Mean relative fluorescence of fmo-2p::mCherry reporter animals in the context of the hlh-30(tm1978) mutation. Fluorescence is calculated relative to N2 EV(RNAi) controls (N = 3 independent experiments, pooled individual worm values, error bars indicate s.e.m., ANOVA with Bonferroni’s post-hoc). (C) Mean relative fluorescence of fmo-2p::mCherry reporter animals in the context of the pmk-1(km25) mutation. Fluorescence is calculated relative to N2 EV(RNAi) controls (N = 5 independent experiments, pooled individual worm values, error bars indicate s.e.m., ANOVA with Bonferroni’s post-hoc). (D) Gene expression of fmo-2 is upregulated by tald-1(RNAi) or cco-1(RNAi) (N = 11 biological replicates, error bars indicate s.e.m., student’s t-test with Bonferroni’s correction). (E) Gene expression of fmo-2 is upregulated by tald-1(RNAi) in a HLH-30 and PMK-1 dependent fashion (N = 3–6 biological replicates, error bars indicate s.e.m., ANOVA with Bonferroni’s post-hoc). (F) Percent of animals displaying HLH-30 nuclear localization. BD animals were starved for 8 hours on FUDR plates prior to imaging (N = 5 independent experiments, error bars indicate s.e.m., ANOVA with Bonferroni’s post-hoc). (G) FMO-2 is required for the lifespan extension from tald-1(RNAi). N2 fed EV(RNAi) (mean 15.3±0.1 days, n = 341), N2 fed tald-1(RNAi) (mean 17.8±0.1 days, n = 353), fmo-2(ok2147) fed EV(RNAi) (mean 18±0.2 days, n = 314), fmo-2(ok2147) fed tald-1(RNAi) (mean 17.4±0.2 days, n = 382). Lifespans were performed at 25°C, with pooled data from three independent experiments shown. (H) FMO-2 is partially required for the lifespan extension from cco-1(RNAi). N2 fed EV(RNAi) (mean 15.7±0.1 days, n = 562), N2 fed cco-1(RNAi) (mean 23.3±0.2 days, n = 616), fmo-2(ok2147) fed EV(RNAi) (mean 18.3±0.1 days, n = 474), fmo-2(ok2147) fed cco-1(RNAi) (mean 20.5±0.2 days, n = 473). Lifespans were performed at 25°C, with pooled data from five independent experiments shown. (I) Lifespan extension from fmo-2 overexpression is not additive with tald-1(RNAi). N2 fed EV(RNAi) (mean 16.5±0.1 days, n = 453), N2 fed tald-1(RNAi) (mean 20.6±0.1 days, n = 421), eft-3p::fmo-2 fed EV(RNAi) (mean 18.2±0.1 days, n = 439), eft-3p::fmo-2 fed tald-1(RNAi) (mean 19.1±0.1 days, n = 435). Lifespans were performed at 25°C, with pooled data from three independent experiments shown. (J) Lifespan extension from fmo-2 overexpression is additive with cco-1(RNAi). N2 fed EV(RNAi) (mean 16.5±0.1 days, n = 453), N2 fed cco-1(RNAi) (mean 23.3±0.2 days, n = 259), eft-3p::fmo-2 fed EV(RNAi) (mean 18.2±0.1 days, n = 439), eft-3p::fmo-2 fed cco-1(RNAi) (mean 25.5±0.2 days, n = 352). Lifespans were performed at 25°C, with pooled data from three independent experiments shown. Lifespans in this figure are indicated as mean±s.e.m. and statistical analysis is provided in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006695#pgen.1006695.s011" target="_blank">S1 Table</a>. In this figure, statistics are displayed as: * p<0.05, ** p<0.01, *** p<0.001.</p

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Model of transaldolase deficiency mediated longevity.

Reduced activity of the pentose phosphate pathway enzyme transaldolase has several consequences, including inhibition of mitochondrial respiration, induction of a mitochondrial stress response, alterations in redox homeostasis, and activation of a starvation-like metabolic response. Lifespan extension in response to transaldolase deficiency appears to be mediated by both MAPK signaling and HLH-30 mediated induction of autophagy and activation of FMO-2.</p

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Alkaline gradient (Run time 11 min).

Alkaline gradient (Run time 11 min).</p

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Improving Health Outcomes for LGBTQ+ Youth Through Provider Education

The MDT-15 Subunit of Mediator Interacts with Dietary Restriction to Modulate Longevity and Fluoranthene Toxicity in Caenorhabditis elegans

Transaldolase inhibition impairs mitochondrial respiration and induces a starvation-like longevity response in <i>Caenorhabditis elegans</i>

Transaldolase deficiency causes a starvation-like response that decreases animal fat content and rewires lipid metabolism gene expression.

Inhibition of the pentose phosphate pathway activates the UPR<sup>mt</sup> and extends lifespan.

Transaldolase deficiency alters mitochondrial morphology and decreases <i>in vivo</i> mitochondrial respiration.

Lifespan extension from <i>tald-1(RNAi)</i> or <i>cco-1(RNAi)</i> requires stress-activated MAPKs.

The flavin-containing monooxygenase FMO-2 is upregulated in a HLH-30 and PMK-1 dependent fashion and regulates the lifespan extension from <i>tald-1(RNAi)</i>.

Model of transaldolase deficiency mediated longevity.

Alkaline gradient (Run time 11 min).