Gender-Sexuality Alliance meeting experiences predict weekly variation in hope among LGBTQ+ youth.

Hope is considered a marker of resilience among youth facing oppression, including LGBTQ+ youth. This 8-week weekly diary study among 94 LGBTQ+ youth (ages 14-19; Mage  = 15.91, 46% youth of color, 44% transgender or nonbinary) in 2021 considered whether a youth's meeting-to-meeting experiences in Gender-Sexuality Alliances (GSAs; LGBTQ+ affirming school clubs) predicted subsequent hope from week to week. Youth reported greater hope on days following meetings where they felt more group support, greater advisor responsiveness, and had taken on more leadership responsibilities. Group support and advisor responsiveness were stronger predictors of a youth's hope on days closer to GSA meetings; leadership's effect was stronger when more days had elapsed. Findings suggest how GSAs may cultivate hope among LGBTQ+ youth.

GSA Advocacy Predicts Reduced Depression Disparities Between LGBQ+ and Heterosexual Youth in Schools.

OBJECTIVE: Depression disparities between heterosexual youth and lesbian, gay, bisexual, queer, and other non-heterosexual (LGBQ+) youth are robust and linked to discrimination in schools. Advocacy by school-based Gender-Sexuality Alliances (GSAs) to raise awareness of LGBQ+ issues and to counteract discrimination may reduce these disparities within schools, yet has not been investigated schoolwide. We considered whether GSA advocacy over the school year moderated sexual orientation differences in depressive symptoms at the school year's end for students in the general school population (i.e., students who were not members of the GSA). METHOD: Participants were 1,362 students (Mage = 15.68; 89% heterosexual; 52.6% female; 72.2% White) in 23 Massachusetts secondary schools with GSAs. Participants reported depressive symptoms at the beginning and end of the school year. Separately, GSA members and advisors reported their GSA's advocacy activities during the school year and other GSA characteristics. RESULTS: LGBQ+ youth reported higher depressive symptoms than heterosexual youth at the school year's beginning. However, after adjusting for initial depressive symptoms and multiple covariates, sexual orientation was a weaker predictor of depressive symptoms at the school year's end for youth in schools whose GSAs engaged in more advocacy. Depression disparities were significant in schools whose GSAs reported lower advocacy, but were statistically non-significant in schools whose GSAs reported higher advocacy. CONCLUSION: Advocacy could be a means by which GSAs achieve school-wide impacts, benefiting LGBQ+ youth who are not GSA members. GSAs may therefore be a key resource for addressing the mental health needs of LGBQ+ youth.

Engagement in Gender-Sexuality Alliances Predicts Youth's Positive and Negative Affect: An 8-Week Weekly Diary Study.

Gender-Sexuality Alliances (GSAs) are school clubs for LGBTQ + youth and peer allies to support one another. This 8-week weekly diary study considered whether a youth's positive and negative affect during a given week could be predicted by experiences in their most recently attended GSA meeting. Ninety-nine GSA members (Mage = 15.90, SD = 1.33; 79% LGBQ + ; 41% trans/non-binary; 59% youth of color) in 11 states completed weekly surveys between January and May 2021. On average, some youth reported higher positive and negative affect than others. Youth also varied notably in their own positive and negative affect from week to week. Youth reported relatively higher positive affect on days following GSA meetings where they were more engaged than in other meetings and had spent time socializing in the meeting. Youth reported relatively higher negative affect on days following GSA meetings where they had discussed personal concerns, and relatively lower negative affect on days following meetings where they were more engaged and perceived greater advisor responsiveness. These findings offer a dynamic portrayal of youth's varied experiences across GSA meetings and the more immediate predictive effects of GSA experiences.

Adjuvant abemaciclib combined with endocrine therapy for high-risk early breast cancer: updated efficacy and Ki-67 analysis from the monarchE study.

BACKGROUND: Adjuvant abemaciclib combined with endocrine therapy (ET) previously demonstrated clinically meaningful improvement in invasive disease-free survival (IDFS) and distant relapse-free survival (DRFS) in hormone receptor-positive, human epidermal growth factor receptor 2-negative, node-positive, high-risk early breast cancer at the second interim analysis, however follow-up was limited. Here, we present results of the prespecified primary outcome analysis and an additional follow-up analysis. PATIENTS AND METHODS: This global, phase III, open-label trial randomized (1 : 1) 5637 patients to adjuvant ET for ≥5 years ± abemaciclib for 2 years. Cohort 1 enrolled patients with ≥4 positive axillary lymph nodes (ALNs), or 1-3 positive ALNs and either grade 3 disease or tumor ≥5 cm. Cohort 2 enrolled patients with 1-3 positive ALNs and centrally determined high Ki-67 index (≥20%). The primary endpoint was IDFS in the intent-to-treat population (cohorts 1 and 2). Secondary endpoints were IDFS in patients with high Ki-67, DRFS, overall survival, and safety. RESULTS: At the primary outcome analysis, with 19 months median follow-up time, abemaciclib + ET resulted in a 29% reduction in the risk of developing an IDFS event [hazard ratio (HR) = 0.71, 95% confidence interval (CI) 0.58-0.87; nominal P = 0.0009]. At the additional follow-up analysis, with 27 months median follow-up and 90% of patients off treatment, IDFS (HR = 0.70, 95% CI 0.59-0.82; nominal P < 0.0001) and DRFS (HR = 0.69, 95% CI 0.57-0.83; nominal P < 0.0001) benefit was maintained. The absolute improvements in 3-year IDFS and DRFS rates were 5.4% and 4.2%, respectively. Whereas Ki-67 index was prognostic, abemaciclib benefit was consistent regardless of Ki-67 index. Safety data were consistent with the known abemaciclib risk profile. CONCLUSION: Abemaciclib + ET significantly improved IDFS in patients with hormone receptor-positive, human epidermal growth factor receptor 2-negative, node-positive, high-risk early breast cancer, with an acceptable safety profile. Ki-67 index was prognostic, but abemaciclib benefit was observed regardless of Ki-67 index. Overall, the robust treatment benefit of abemaciclib extended beyond the 2-year treatment period.

An Assessment of Earth's Climate Sensitivity Using Multiple Lines of Evidence.

We assess evidence relevant to Earth's equilibrium climate sensitivity per doubling of atmospheric CO2, characterized by an effective sensitivity S. This evidence includes feedback process understanding, the historical climate record, and the paleoclimate record. An S value lower than 2 K is difficult to reconcile with any of the three lines of evidence. The amount of cooling during the Last Glacial Maximum provides strong evidence against values of S greater than 4.5 K. Other lines of evidence in combination also show that this is relatively unlikely. We use a Bayesian approach to produce a probability density function (PDF) for S given all the evidence, including tests of robustness to difficult-to-quantify uncertainties and different priors. The 66% range is 2.6-3.9 K for our Baseline calculation and remains within 2.3-4.5 K under the robustness tests; corresponding 5-95% ranges are 2.3-4.7 K, bounded by 2.0-5.7 K (although such high-confidence ranges should be regarded more cautiously). This indicates a stronger constraint on S than reported in past assessments, by lifting the low end of the range. This narrowing occurs because the three lines of evidence agree and are judged to be largely independent and because of greater confidence in understanding feedback processes and in combining evidence. We identify promising avenues for further narrowing the range in S, in particular using comprehensive models and process understanding to address limitations in the traditional forcing-feedback paradigm for interpreting past changes.

Effect of cycloheximide on development of methotrexate resistance of Chinese hamster ovary cells treated with inhibitors of DNA synthesis.

We examined the effects of 18 h of incubation of Chinese hamster ovary (CHO K1) cells with cycloheximide, hydroxyurea, and aphidicolin. Treatment of cells with cycloheximide alone at a concentration adequate to inhibit DNA synthesis to less than 10% of control was significantly less cytotoxic and clastogenic than treatment with hydroxyurea or aphidicolin, did not induce unbalanced cellular growth, and had no effect on the frequency of resistant cells in methotrexate selections compared with control cells. When combined with hydroxyurea or aphidicolin and compared with the effects of either drug alone, cycloheximide blocked the induction of unbalanced growth during drug treatment, reduced the frequency of chromosomal aberrations in recovering cell populations, and decreased cell killing. In addition, the increased frequency of methotrexate-resistant cells observed after treatment with hydroxyurea or aphidicolin was eliminated when cycloheximide was present during drug treatment.

Transient inhibition of DNA synthesis results in increased dihydrofolate reductase synthesis and subsequent increased DNA content per cell.

We examined the role that blockage of cells in the cell cycle may play in the stimulation of gene amplification and enhancement of drug resistance. We found that several different inhibitors of DNA synthesis, which were each able to block cells at the G1-S-phase boundary, induced an enhanced cycloheximide-sensitive synthesis of an early S-phase cell cycle-regulated enzyme, dihydrofolate reductase, and of other proteins as well. This response was specific, in that blockage at the G2 phase did not result in overproduction of the enzyme. When the cells were released from drug inhibition, DNA synthesis resumed, resulting in a cycloheximide-sensitive elevation in DNA content per cell. We speculate that the excess DNA synthesis (which could contribute to events detectable later as gene amplification) is a consequence of the accumulation of S-phase-specific proteins in the affected cells, which may then secondarily influence the pattern of DNA replication.

Quantitative model for prediction of hydrodynamic size of nonionic reverse micelles.

The sizes of nonionic reverse micelles were investigated as a function of the molecular structure of the surfactant, the type of oil, the total concentration of surfactant [NP], the ratio of surfactant to total surfactant (r), the water to surfactant molar ratio (omega), temperature, salt concentration, and polar phase. The basis of our investigation was a mixture of nonylphenol polyethoxylates--NP4 and NP7, various polar phases, and several oils. Micelle sizes were determined using dynamic light scattering (DLS). A central composite experimental design was used to quantitatively model micelle size as a function of omega, surfactant concentration, and r. The model has demonstrated the capability of predicting the mean diameter of micelles from 4 to 13 with a precision of +/-2 nm as measured by DLS. This quantitative correlation between the size of reverse micelles and the synthetic variables provides the foundation for choosing experimental conditions to control reverse micelle size. In turn, this allows control of the size of nanoparticles synthesized within them.

Interaction of hyperthermia and metabolic inhibitors on the induction of chromosome damage in Chinese hamster ovary cells.

We have examined the chromosomal effects of heating asynchronously growing Chinese hamster ovary (CHO K1) cells in the presence of actinomycin D or cycloheximide. Actinomycin D was found to strongly potentiate the chromosome damaging effects of heat shock, an effect correlated with a strong nonadditive reduction in cell survival. In contrast, cycloheximide treatment reduced heat shock induced chromosome damage and resulted in a significant nonadditive increase in cell survival following heat shock. The different effects of these two inhibitors on chromosomal damage and cell survival are correlated in part with their effects on the rate of DNA synthesis during heat shock. The results suggest that an important aspect of the interaction of heat and metabolic inhibitors involves changes in cell cycle phase distribution of and/or progression through the S phase of the cell cycle induced by drug treatment prior to and during heat shock. The data indicate that the protective effect of cycloheximide in heat shocked cells may involve altered cell cycle progression and/or phase distribution of cells during hyperthermia.

Flow cytometric characterization of antifolate resistance in cultured mammalian cells using fluoresceinated methotrexate and daunorubicin.

We have studied antifolate-resistant rodent cell lines with respect to dihydrofolate reductase gene expression and expression of the "classic" multidrug resistance phenotype by flow cytometry. Using a series of antifolate-resistant and colchicine-resistant Chinese hamster ovary cell lines obtained by single-step and stepwise selection protocols, we show that viable cell staining with fluoresceinated methotrexate and daunorubicin correlates well with drug resistance and expression of dihydrofolate reductase protein and the "classic" MDR phenotype in these cell lines. We show that flow cytometric analysis makes it possible to rapidly assess the potentially complex drug resistance phenotype(s) of cells selected with hydrophilic and lipophilic antifolates.

Cytotoxic effects of cell cycle phase specific agents: result of cell cycle perturbation.

Although agents which act in a cell cycle phase specific manner are commonly used in the clinic and in basic research, it is as yet unclear why these agents are cytotoxic. In this paper, we examine the cellular events associated with the cytotoxicity of aphidicolin and vincristine in CHO strain AA8 cells. Cell killing resulting from aphidicolin treatment was found to require a period of inhibition-free growth following removal of the drug and was associated with characteristic aberrant mitotic processes. The cytotoxic effects of aphidicolin could be antagonized by the concomitant inhibition of protein synthesis with cycloheximide in the period of DNA synthesis inhibition. Cell killing resulting from treatment with vincristine was associated with the aberrant segregation of nuclear material and the formation of multiple partial nuclei. Vincristine cytotoxicity was found to be antagonized by concomitant administration of cycloheximide or cytochalasin D. These data support a hypothesis that the cytotoxic effects of cell cycle phase specific agents do not derive directly from their biochemical actions per se. We propose that cell death results from processes that are evoked by dissociation of normally integrated cell cycle events, and that dissociation involves replicative/mitotic events in the case of aphidicolin and karyokinetic/nuclear reformation events in the case of vincristine.

Heterogeneity in the mitotic checkpoint control of BALB/3T3 cells and a correlation with gene amplification propensity.

Chinese hamster ovary (and many rodent cell lines) transiently delay mitosis and progress into a second cell cycle without undergoing cytokinesis when treated with Colcemid, whereas HeLaS3 (and most human cell lines) arrest permanently in mitosis. We have discussed these differences and their consequences for cell survival under cell cycle-perturbing conditions within the context of mitotic checkpoint control (Schimke et al., Cold Spring Harbor Symp. Quant. Biol., 56: 417-425, 1991). Here, we report studies with mouse BALB/3T3 cell populations which, by the criterion of response to Colcemid, constitute a heterogeneous population with respect to mitotic checkpoint control. Clonal and subclonal populations retain population heterogeneity but with a bias for enrichment of cell populations that respond as do HeLaS3 cells. We have analyzed clones for their propensity for gene amplification as assessed by a stepwise increment selection protocol in methotrexate and report that there are significant differences in amplification propensities that correlate with differences in mitotic checkpoint control properties.

Requirement for p38 and p44/p42 mitogen-activated protein kinases in RAGE-mediated nuclear factor-kappaB transcriptional activation and cytokine secretion.

Advanced glycation end product (AGE) activation of the signal-transducing receptor for AGE (RAGE) has been linked to a proinflammatory phenotypic change within cells. However, the precise intracellular signaling pathways involved have not been elucidated. We demonstrate here that human serum albumin modified with N(epsilon)-(carboxymethyl)lysine (CML), a major AGE adduct that progressively accumulates with aging, diabetes, and renal failure, induced nuclear factor (NF)-kappaB-driven reporter gene expression in human monocytic THP-1 cells. The NF-kappaB response was blocked with a synthetic peptide corresponding to the putative ligand-binding domain of RAGE, with anti-RAGE antiserum, and by coexpression of truncated receptors lacking the intracellular domain. Signal transduction from RAGE to NF-kappaB involved the generation of reactive oxygen species, since reporter gene expression was blocked with the antioxidant N-acetyl-L-cysteine. CML-modified albumin produced rapid transient activation of tyrosine phosphorylation, extracellular signal-regulated kinase 1 and 2, and p38 mitogen-activated protein kinase (MAPK), but not c-Jun NH(2)-terminal kinase. RAGE-mediated NF-kappaB activation was suppressed by the selective p38 MAPK inhibitor SB203580 and by coexpression of a kinase-dead p38 dominant-negative mutant. Activation of NF-kappaB by CML-modified albumin increased secretion of proinflammatory cytokines (tumor necrosis factor-alpha, interleukin-1beta, and monocyte chemoattractant protein-1) severalfold, and inhibition of p38 MAPK blocked these increases. These results indicate that p38 MAPK activation mediates RAGE-induced NF-kappaB-dependent secretion of proinflammatory cytokines and suggest that accelerated inflammation may be a consequence of cellular activation induced by this receptor.

Evaluation of hereditary risk in a mammography population.

PURPOSE: BRCA1 and BRCA2 mutations significantly increase a women's lifetime risk of breast and ovarian cancer. Because several management options have shown promise in decreasing morbidity and mortality for these women, identifying potential mutation carriers is increasingly important. We have developed a large-scale method to collect family histories in a population of unaffected women presenting for mammography. We then applied current risk-assessment models to determine the prevalence of women at risk for hereditary breast and ovarian cancer. MATERIALS AND METHODS: We performed a retrospective review of family histories using data collected on all unaffected women presenting for mammography over a 14-week period. The Claus, Myriad II, and Hartmann models for hereditary risk assessment were applied to the survey results. RESULTS: The questionnaire was completed by 5736 women, 695 of whom were excluded because of a personal history of breast or ovarian cancer. Family histories of the remaining 5041 women were evaluated. Totals of 5.9%, 5.2%, and 3.3% of patients, respectively, met criteria for increased risk according to the Hartmann, Myriad II, and Claus models, corresponding to 3.5, 3.1, and 1.9 patients per day. Although 9.2% of patients met criteria for >/=1 model, only 1.4% met criteria for all 3. CONCLUSIONS: Application of available models to a screening population classifies a larger than expected number of women at high risk for a BRCA1 or BRCA2 mutation. New approaches to risk assessment and counseling are needed to apply our knowledge of hereditary risk to a broad population in a practical manner.

Secondary morbidity among the recently bereaved.

Discussions of the health consequences of bereavement have appeared with increasing frequency in the literature in recent years. Capitalizing on one of the largest samples of bereaved subjects to date, the authors analyzed National Hospice Study bereavement interview data regarding the rate of medical care use and short-term secondary morbidity. Results suggest that physician visit rates were somewhat higher but hospitalization rates lower among the recently bereaved than age- and sex-adjusted national norms. Multivariate analyses revealed that previous health problems and having been married to the deceased were consistently the strongest predictors of morbidity and health care use.

UCP4, a novel brain-specific mitochondrial protein that reduces membrane potential in mammalian cells.

Uncoupling proteins (UCPs) are a family of mitochondrial transporter proteins that have been implicated in thermoregulatory heat production and maintenance of the basal metabolic rate. We have identified and partially characterized a novel member of the human uncoupling protein family, termed uncoupling protein-4 (UCP4). Protein sequence analyses showed that UCP4 is most related to UCP3 and possesses features characteristic of mitochondrial transporter proteins. Unlike other known UCPs, UCP4 transcripts are exclusively expressed in both fetal and adult brain tissues. UCP4 maps to human chromosome 6p11.2-q12. Consistent with its potential role as an uncoupling protein, UCP4 is localized to the mitochondria and its ectopic expression in mammalian cells reduces mitochondrial membrane potential. These findings suggest that UCP4 may be involved in thermoregulatory heat production and metabolism in the brain.

A novel method for measuring CTL and NK cell-mediated cytotoxicity using annexin V and two-color flow cytometry.

An assay based on two-color flow cytometry has been developed to measure CTL and NK cell-mediated cytotoxicity. After effector/target cells are incubated together, CTL or NK populations are stained with an effector cell specific PE-conjugated mAb. Subsequently, annexin V-FITC binds to cells expressing phosphatidylserine (an early marker of apoptosis) on the cell surface. Target cells are gated upon as PE-negative and quantified with respect to their annexin V positivity. The shift from annexin Vneg to annexin Vhi is a discrete event such that all target cells fall within discernible populations with respect to annexin V. There is a strong correlation between cytotoxicity measured with our assay and a standard 51Cr release assay (r2 = 0.989). The PE/annexin V assay shows increased sensitivity at early timepoints after target/effector cell mixing. In addition, this method allows for analysis of target cells at the single cell level. Therefore, we have described a promising new technique to measure in vitro cell-mediated cytotoxicity. It avoids the potential difficulties of working with radioactive isotopes, and offers increased sensitivity and versatility.

CNS germinoma: disease control and long-term functional outcome for 12 children treated with craniospinal irradiation.

PURPOSE: To provide evidence that radiation therapy alone in the form of craniospinal irradiation (CSI) and a boost to the primary site of disease provides effective disease control and limited additional morbidity for patients with CNS germinoma. METHODS AND MATERIALS: Twelve patients with a median age of 12 years (range 9-16 years) with CNS germinoma were treated with CSI (median 25.6 Gy, range 23.4-32 Gy) and a boost to the primary site of disease (50.4 Gy, range 45-54 Gy) between January 1987 and June 1998. All patients were biopsied prior to radiation therapy and none received chemotherapy. No patients were lost to follow-up and the majority had long-term (> 45 month) pre- and postirradiation endocrine and psychology assessment. RESULTS: All 12 patients are alive and no failures have occurred with a median follow-up of 69 months (range 14-143 months). Preirradiation endocrine deficiencies were present in 6 of 6 suprasellar tumors and 1 of 6 pineal tumors; with follow-up there was no substantial difference between age and gender adjusted pre- and postirradiation stature and weight. With long-term follow-up, there were no significant differences between pre- and postirradiation full-scale, verbal, and performance IQ scores. CONCLUSIONS: This study confirms the ability of radiation therapy alone to achieve disease control with a high rate of success in pediatric patients and demonstrates that the treatment toxicity faced by these patients may be less than anticipated. Because these patients present with substantial preexisting morbidity at diagnosis and may be of an age where the potential for radiation-related side effects is relatively small, the superiority of treatment alternatives may be difficult to prove.

Beta-galactosidase histochemistry and telomere loss in senescent retinal pigment epithelial cells.

PURPOSE: To investigate the relation of senescence-related beta-galactosidase activity and telomere shortening to replicative senescence in cultured human retinal pigment epithelial (RPE) cells. METHODS: A human RPE cell line was serially passaged until 80% of cells were nondividing in a 72-hour 5-bromo-2'-deoxyuridine (BrdU) labeling study. Early- and late-passage cells were double-stained for BrdU and senescence-related beta-galactosidase activity (pH 6). The average chromosomal telomere length at several population doublings was estimated by Southern blot analysis after double digestion of DNA with RsaI and HinfI and using a telomere-specific probe. RESULTS: BrdU-beta-galactosidase double-staining revealed an inverse correlation between the number of BrdU-labeled nuclei and beta-galactosidase-labeled cells as a function of population doubling level (PDL). At PDL 58, only 20% of all cells labeled for BrdU, whereas 57% stained for beta-galactosidase. The mean terminal restriction fragment length (TRF) was reduced from 10 kb in early (PDL 12) cultures to 4 kb in late (PDL 57) cultures. CONCLUSIONS: Senescence-related beta-galactosidase activity and mean TRF length may prove useful in studying the senescence of RPE cells in vitro. These techniques may be valuable in determining senescence of the retinal pigment epithelium in vivo, where senescent RPE cells could be involved in the development of age-related maculopathy and age-related macular degeneration.