The Association between Cardiorespiratory Fitness and the Risk of Breast Cancer in Women.

INTRODUCTION: Studies have shown an inverse association between the risk of breast cancer in women and physical activity. However, information on the association between cardiorespiratory fitness (CRF) assessed objectively by a standardized test and the risk of developing breast cancer is limited. PURPOSE: To examine the CRF-breast cancer risk association in healthy females. METHODS: This retrospective study was derived from the ETHOS cohort (n = 750,302). Female participants (n = 44,463; mean age ± SD; 55.1 ± 8.9 years) who completed an exercise treadmill (ETT) test evaluation (Bruce protocol) at the Veterans Affairs (VA) Medical Centers nationwide from 1999 to 2020 were studied. The cohort was stratified into four age-specific CRF categories (Least-fit, Low-fit, Moderate-fit, and Fit), based on the peak metabolic equivalents (METs) achieved during the ETT. RESULTS: During 438,613 person-years of observation, 994 women developed breast cancer. After controlling for covariates, the risk of breast cancer was inversely related to exercise capacity. For each 1-MET increase in CRF the risk of cancer was 7% lower (HR 0.93; 95% CI, 0.90-0.95; P < .001). When risk was assessed across CRF categories with the Least-fit group as the referent, the risk was 18% lower for Low-fit women (HR 0.82; 95% CI, 0.70-0.96; P = .013), 31% for Moderate-fit (HR 0.69; 95% CI, 0.58-0.82; P < .001) and 40% for Fit (HR 0.60; 95% CI, 0.47-0.75; P < .001). CONCLUSIONS: We observed an inverse and graded association between CRF and breast cancer risk in women. Thus, encouraging women to improve CRF may help attenuate the risk of developing breast cancer.

Neurotoxicity assessment of QoI strobilurin fungicides azoxystrobin and trifloxystrobin in human SH-SY5Y neuroblastoma cells: Insights from lipidomics and mitochondrial bioenergetics.

Strobilurin fungicides are quinone outside inhibitors (QoI) used to treat fungal pathogens for agricultural and residential use. Here, we compared the potential for neurotoxicity of the widely used strobilurins, azoxystrobin (AZS) and trifloxystrobin (TFS), in differentiated human SH-SY5Y cells. Fungicides did not include cytotoxicity up to 200 µM but both induced loss of cell viability at 48 h, with TFS showing slightly higher toxicity that AZS. Caspase 3/7 activity was induced in SH-SY5Y cells by both fungicides at 48 h (50 µM for AZS and 25 µM for TFS). ATP levels were reduced following a 24-hour exposure to > 25 µM AZS and > 6.25 µM TFS and both fungicides rapidly impaired oxidative respiration (~12.5 µM for AZS and ~3.125 µM TFS) and decreased oligomycin-induced ATP production, maximal respiration, and mitochondrial spare capacity. AZS at 100 µM showed a continual impairment of mitochondrial membrane potential (MMP) between 4 and 48 h while TFS at > 50 µM decreased MMP at 24 h. Taken together, TFS exerted higher mitochondrial toxicity at lower concentrations compared to AZS in SH-SY5Y cells. To discern toxicity mechanisms of strobilurin fungicides, lipidomics was conducted in SH-SY5Y cells following exposure to 6.25 µM and 25 µM AZS, and a total of 1595 lipids were detected, representing 49 different lipid classes. Lipid classes with the largest proportion of lipids detected in SH-SY5Y cells included triglycerides (17%), phosphatidylethanolamines (8%), ether-linked triglycerides (8%), phosphatidylcholines (7%), ether-linked phosphatidylethanolamines (6%), and diacylglycerols (5%). Together, these 5 lipid classes accounted for over 50% of the total lipids measured in SH-SY5Y cells. Lipids that were increased by AZS included acyl carnitine, which plays a role in long chain fatty acid utilization for mitochondrial ß-oxidation, as well as non-modified, ether linked, and oxidized triacylglycerols, suggesting compensatory upregulation of triglyceride biosynthesis. The ceramide HexCer-NS, linked to neurodegenerative diseases, was decreased in abundance following AZS exposure. In summary, strobilurin fungicides rapidly inhibit mitochondrial oxidative respiration and alter the abundance of several lipids in neuronal cells, relevant for understanding environmental exposure risks related to their neurotoxicity.

Disease network data for the pesticide fipronil in rat dopamine cells.

Transcriptome data were collected in rat dopamine cells exposed to fipronil for 24 h using microarray analysis. Fipronil is a phenylpyrazole pesticide that acts to inhibit gamma-aminobutyric acid (GABA), blocking inhibitory synaptic transmission in the central nervous system. Transcriptome data were subjected to pathway analysis and subnetwork enrichment analysis. We report that 25 µM fipronil altered transcriptional networks in dopamine-synthesizing cells that are associated with Alzheimer's Disease, Huntington Disease, and Schizophrenia. Data analysis revealed that nerve fibre degeneration, nervous system malformations, neurofibrillary tangles, and neuroinflammation were all disease processes related to the transcriptome profile observed in the rat neuronal cells. Other disease networks altered by fipronil exposure at the transcript level were associated with the mitochondria, including mitochondrial DNA depletion syndrome and mitochondrial encephalomyopathies. These data, along with those presented in Souders et al. (2021), are significant because they increase understanding into the molecular mechanisms underlying human disease following exposures to neuroactive pesticides. These data can be reused to inform adverse outcome pathways for neurotoxic pesticides.

Metabolic profiling in human SH-SY5Y neuronal cells exposed to perfluorooctanoic acid (PFOA).

Perfluorooctanoic acid (PFOA) is an abundant per- and polyfluoroalkyl substance (PFAS) detected in both indoor and outdoor environments. While studies suggest exposure concerns for humans, studies investigating PFOA-induced neurotoxicity are lacking. To address this gap, we exposed differentiated human SH-SY5Y cells to PFOA (0.1 µM up to 500 µM) at different time points (4, 24, 48, and 72 h) and measured cell viability, Casp3/7 activity, ATP levels, ATP synthase enzyme activity, mitochondrial membrane potential, reactive oxygen species (ROS), oxygen consumption rates for mitochondrial stress test (XFe24 Flux analyzer), glucose utilization, and global metabolome profiles to assess the potential for PFOA-induced neurotoxicity. Treatment with 10 or 100 µM PFOA did not compromise cell viability nor induce cytotoxicity to SH-SY5Y cells over a 48-hour exposure period. However, >250 µM PFOA compromised cell viability, induced cytotoxicity, and induced caspase 3/7 activity at 48 h. ATP levels were reduced in cells treated with 400 µM PFOA for 24 and 48 h, and with 100 µM PFOA and higher at 72 h. ATP synthase activity was inhibited by 250 µM PFOA but was unchanged by PFOA treatment at 200 µM or less. Conversely, mitochondrial membrane potential was reduced by >10 µM PFOA after 24 h. Total ROS was increased with 100 µM PFOA and higher after 4 h of exposure. Several mitochondria-related endpoints (basal respiration, ATP production, maximum respiration) were negatively affected at 250 µM PFOA at both 24- and 48-hour exposure, but were unaltered at concentrations of 100 µM PFOA or less. One exception was mitochondrial spare capacity, which was reduced by 100 µM PFOA after 24-hour exposure. Similarly, glycolysis, glycolytic capacity, and glycolytic reserve of SH-SY5Y cells were not altered by 10 nor 100 µM PFOA. Nontargeted metabolomics was conducted in cells treated with either 10 or 100 µM PFOA for 48 h, as these two concentrations were not cytotoxic and 28 metabolites differed among treatments. Notable was that 10 µM PFOA had little effect on the SH-SY5Y metabolome, and the metabolic profile was not statistically different from media nor solvent controls. On the other hand, 100 µM PFOA shifted the metabolic signature of the neuronal cells, leading to reduced abundance of ATP-related metabolites (adenine, nicotinamide), neurotransmitter precursors (DL-tryptophan, l-tyrosine), and metabolites that protect mitochondria during oxidative stress (betaine, orotic acid, and l-acetyl carnitine). We hypothesize that this metabolic signature may be associated with the reduced mitochondrial membrane potential observed at lower PFOA concentrations. Metabolic shifts appear to precede compromised cell viability, cytotoxicity, and apoptosis. This study generates mechanistic knowledge regarding PFOA-induced neurotoxicity, focusing on mitochondrial oxidative respiration and the neuronal metabolome.

Mitochondrial and transcriptome responses in rat dopaminergic neuronal cells following exposure to the insecticide fipronil.

The phenylpyrazole fipronil is an insecticide that inhibits γ -amino-butyric acid (GABA) ionotropic receptors in the central nervous system. Experimental evidence suggests that fipronil acts as a neurotoxin and it is implicated in neurodegenerative diseases; however, the mechanisms of neurotoxicity are not fully elucidated. The objective of this study was to quantify mechanisms of fipronil-induced neurotoxicity in dopamine cells. Rat primary immortalized mesencephalic dopaminergic cells (N27) were treated with fipronil (0.25 up to 500 µM depending on the assay). We measured endpoints related to mitochondrial bioenergetics, mitophagy, mitochondrial membrane potential, and ATP production in addition to discerning transcriptome responses to the pesticide. Fipronil reduced cell viability at 500 µM after 24 h exposure and caspase 3/7 activity was significant increased after 6 and 12 h by 250 and 500 µM fipronil. Subsequent endpoints were thus assessed at concentrations that were below cytotoxicity. We measured oxidative respiration of N27 cells following a 24 h exposure to one dose of either 0.25, 2.5, 25, or 50 µM fipronil. Oxygen consumption rates (OCR) were not different between vehicle-control and 0.25 or 2.5 µM fipronil treatments, but there was a ∼40-60 % reduction in basal respiration, as well as reduced oligomycin-induced ATP production at 50 µM. The reduction in OCR is hypothesized to be related to lower mitochondrial mass due to mitophagy. Mitochondrial membrane potential was also sensitive to fipronil, and it was compromised at concentrations of 2.5 µM and above. To further elucidate the mechanisms linked to neurotoxicity, we conducted transcriptomics in dopamine cells following treatment with 25 µM fipronil. Fipronil suppressed transcriptional networks associated with mitochondria (damage, depolarization, permeability, and fission), consistent with its effects on mitochondrial membrane potential. Altered gene networks also included those related to Alzheimer disease, inflammatory disease, nerve fiber degeneration, and neurofibrillary tangles. This study clarifies molecular targets of fipronil-induced neurotoxicity and supports, through multiple lines of evidence, that fipronil acts as a mitochondrial toxicant in dopamine cells. This is relevant to neurodegenerative diseases like Parkinson's disease as exposure to fipronil is associated with the progressive loss of nigrostriatal dopaminergic neurons in rodents.

Novel Myh11 Dual Reporter Mouse Model Provides Definitive Labeling and Identification of Smooth Muscle Cells-Brief Report.

OBJECTIVE: Myh11 encodes a myosin heavy chain protein that is specifically expressed in smooth muscle cells (SMCs) and is important for maintaining vascular wall stability. The goal of this study is to generate a Myh11 dual reporter mouse line for definitive visualization of MYH11+ SMCs in vivo. Approach and Results: We generated a Myh11 knock-in mouse model by inserting LoxP-nlacZ-4XpolyA-LoxP-H2B-GFP-polyA-FRT-Neo-FRT reporter cassette into the Myh11 gene locus. The nuclear (n) lacZ-4XpolyA cassette is flanked by 2 LoxP sites followed by H2B-GFP (histone 2B fused green fluorescent protein). Upon Cre-mediated recombination, nlacZ-stop cassette is removed thereby permitting nucleus localized H2B-GFP expression. Expression of the nuclear localized lacZ or H2B-GFP is under control of the endogenous Myh11 promoter. Nuclear lacZ was expressed specifically in SMCs at embryonic and adult stages. Following germline Cre-mediated deletion of nuclear lacZ, H2B-GFP was specifically expressed in the nuclei of SMCs. Comparison of nuclear lacZ expression with Wnt1Cre and Mef2cCre mediated-H2B-GFP expression revealed heterogenous origins of SMCs from neural crest and second heart field in the great arteries and coronary vessels adjacent to aortic root. CONCLUSIONS: The Myh11 knock-in dual reporter mouse model offers an exceptional genetic tool to visualize and trace the origins of SMCs in mice.

Prospective Endovascular Treatment in Acute Ischemic Stroke Evaluating Non-Contrast Head CT versus CT Perfusion (PLEASE No CTP).

BACKGROUND: Studies have shown a lack of agreement of computed tomography perfusion (CTP) in the selection of acute ischemic stroke (AIS) patients for endovascular treatment. PURPOSE: To demonstrate whether non-contrast computed tomography (CT) within 8 h of symptom onset is comparable to CTP imaging. METHODS: Prospective study of consecutive anterior circulation AIS patients with a National Institute of Health Stroke Scale (NIHSS) score > 7 presenting within 8 h of symptom onset with endovascular treatment. All patients had non-contrast CT, CT angiography, and CTP. The neuro-interventionalist was blinded to the results of the CTP and based the treatment decision using the Alberta Stroke Program Early CT score (ASPECTS). Baseline demographics, co-morbidities, and baseline NIHSS scores were collected. Outcomes were modified Rankin scale (mRS) score at discharge and in-hospital mortality. Good outcomes were defined as a mRS score of 0-2. RESULTS: 283 AIS patients were screened for the trial, and 119 were enrolled. The remaining patients were excluded for: posterior circulation stroke, no CTP performed, could not obtain consent, and NIHSS score < 7. Mean -NIHSS score at admission was 16.8 ± 3, and mean ASPECTS was 8.4 ± 1.4. There was no statistically significant correlation with CTP penumbra and good outcomes: 50 versus 47.8% with no penumbra present (p = 0.85). In patients without evidence of CTP penumbra, there was 22.5% mortality compared to 22.1% mortality in patients with a CTP penumbra. If ASPECTS ≥7, 64.6% had good outcome versus 13.3% if ASPECTS < 7 (p < 0.001). Patients with an ASPECTS ≥7 had 10% mortality versus 51.4% in patients with an ASPECTS < 7 (p < 0.001). CONCLUSIONS: CTP penumbra did not identify patients who would benefit from endovascular treatment when patients were selected with non-contrast CT ASPECTS ≥7. There is no correlation of CTP penumbra with good outcomes or mortality. Larger prospective trials are warranted to justify the use of CTP within 6 h of symptom onset.

Neurotoxicity assessment of triazole fungicides on mitochondrial oxidative respiration and lipids in differentiated human SH-SY5Y neuroblastoma cells.

Indiscriminate overuse or occupational exposure to agricultural chemicals can lead to neurotoxicity. Many pesticides act to impair mitochondrial function which can lead to exacerbation of neurodegeneration. Triazole fungicides are applied to grain, fruit, and vegetable crops to combat mold and fungi and their use is increasing worldwide. Here, we assessed the in vitro toxicity of two widely used triazole fungicides, propiconazole and tebuconazole, to mitochondria using differentiated SH-SY5Y neuroblastoma cells as an in vitro cell model used in Parkinson's disease research. Cell viability (based on ATP levels), mitochondrial membrane potential, oxidative respiration, and reactive oxygen species (ROS) were measured following fungicide treatments. Cell viability was decreased with 100 µM propiconazole after 24 and 48 h, while tebuconazole required higher doses to affect viability (-200 µM at 24 h). Mitochondrial membrane potential (MMP) was reduced with 50 µM propiconazole after 24 h while 200 µM tebuconazole reduced MMP. Oxidative respiration of SH-SY5Y cells was then measured using a XFe24 Flux analyzer and 100 µM propiconazole reduced basal respiration, oligomycin-induced ATP production, and FCCP-induced maximum respiration by -40-50%, while tebuconazole did not affect mitochondrial bioenergetics at the concentrations tested. Acute exposure to 100 µM propiconazole over 4 h did not immediately affect oxidative respiration in SH-SY5Y cells. ROS were not induced by propiconazole and tebuconazole up to 100 and 300 µM respectively. Based on these results, we focused our lipidomics investigations on SH-SY5Y exposed only to propiconazole, as lipid dysregulation is associated with mitochondrial dysfunction. Both 50 and 100 µM propiconazole altered the abundance of some ceramides, specifically reducing glucosylceramide non-hydroxyfatty acid-sphingosine (HexCer-NS) and increasing N-stearoyl-phytosphingosine (CerNP). Moreover, a recently discovered bioactive lipid called fatty acid ester of hydroxy fatty acid (FAHFA) was increased 5-fold, hypothesized to be a neuroprotective mechanism that has been demonstrated in other studies of human diseases. Additional lipids reduced in abundance included oxidized phosphatidylcholine (OxPC) and oxidized phosphatidylethanolamine (OxPE). There were no changes in cellular triacylglycerols nor total lipids with exposure to propiconazole. Taken together, this study provides insight into the toxicity of triazole fungicides in neuronal cells, which has implications for neurodegenerative diseases that involve the mitochondria such as Parkinson's disease.

Endovascular treatment outcomes using the Stroke Triage Education, Procedure Standardization, and Technology (STEPS-T) program.

Background "Door to treatment" time affects outcomes of acute ischemic stroke (AIS) patients undergoing endovascular treatment (EVT). However, the correlation between staff education and accessible technology with stroke outcomes has not been demonstrated. Objective The objective of this paper is to demonstrate the five-year impact of the Stroke Triage Education, Procedure Standardization, and Technology (STEPS-T) program on time-to-treat and clinical outcomes. Methods The study analyzed a prospectively maintained database of AIS patients who benefited from EVT through implementation of STEPS-T. Demographics, clinical characteristics, and modified Rankin Score at three months were analyzed. Thrombolysis in Cerebral Infarction (TICI) scale was used to grade pre- and post-procedure angiographic recanalization. Using electronic hemodynamic recording, stepwise workflow times were collected for door time (TD), entering angiography suite (TA), groin puncture (TG), first DSA (TDSA), microcatheter placement (TM), and final recanalization (TR). Median intervention time (TA to TR) and recanalization time (TG to TR) were compared through Year 1 to Year 5. Results A total of 230 individuals (age 74 ± 12, between 30 to 95) were enrolled. Median intervention and recanalization times were significantly reduced, from 121 minutes to 52 minutes and from 83 minutes to 36 minutes respectively from Year 1 to Year 5, ( p < 0.001). Across the study period, annual recruitment went up from 12 to 66 patients, and modified Rankin Score between 0 and 2 increased from 36% to 59% ( p = 0.024). Conclusions STEPS-T improved time-to-treat in patients undergoing mechanical thrombectomy for AIS. During the observation period, clinical outcomes significantly improved.

Does Hospitalist Directed Care for Acute Ischemic Stroke Patients Improve Adherence to "Get with the Guidelines"?

BACKGROUND AND PURPOSE: Hospitalist directed care is associated with improved outcomes in several medical conditions. The hospitalist effect has not been studied in acute ischemic stroke (AIS) patients. We compare length of stay (LOS), outcome, and adherence to "Get with the Guidelines" (GWTG) stroke quality measures among AIS patients admitted under a hospitalist with three other specialties (internist, family practice, or specialist). METHODS: We collected demographics, risk factors and discharge outcomes (modified Rankin Scale (mRS)) for consecutive AIS patients over 4-year period (2010-2014). We categorized all stroke admissions according to admitting physicians. We compared rates of adherence with all of the GWTG Stroke inpatient quality measures between the four groups. RESULTS: A total of 1584 patients [mean age ( ± SD) 68.6 ± 13.7 years; 55.6% men] were admitted with AIS. There was no statistically significant difference in LOS between the four groups (p=0.4). There was significant difference in the GWTG inpatient quality measures with the hospitalist group having lowest rates of any nonadherence observed in 5% of admissions (p=0.03), and the internists had the highest rate of nonadherence observed in 16% of admissions (p=0.01). The most common deficiency was not prescribing statin at discharge (56% of total fallouts). There was no difference in rates of poor outcomes on discharge (mRS 3-6) (p=0.2). CONCLUSIONS: There was a significantly higher rate of adherence to GWTG inpatient stroke measures when AIS patients were admitted under the care of a hospitalist. Prospective studies are required to evaluate if higher rates of adherence lead to better long term outcomes.

How Compliance Measures, Behavior Modification, and Continuous Quality Improvement Led to Routine HIV Screening in an Emergency Department in Brooklyn, New York.

OBJECTIVE: New York State adopted a new HIV testing law in 2010 requiring medical providers to offer an HIV test to all eligible patients aged 13-64 years during emergency room or ambulatory care visits. Since then, Wyckoff Heights Medical Center (WHMC) in Brooklyn, New York, began implementing routine HIV screening organization-wide using a compliance, behavior-modification, and continuous quality-improvement process. METHODS: WHMC first implemented HIV screening in the emergency department (ED) and evaluated progress with the following monthly indicators: HIV tests offered, HIV tests accepted, HIV tests ordered (starting in December 2013), HIV tests administered, positive HIV tests, and linkage to HIV care. Compliance with the delivery of HIV testing was determined by the proportion of patients who, after accepting a test, received one. RESULTS: During August 2013 through July 2014, of 57,852 eligible patients seen in the WHMC ED, a total of 31,423 (54.3%) were offered an HIV test. Of those, 8,229 (26.2%) patients accepted a test. Of those, 6,114 (74.3%) underwent a test. A total of 26 of the 6,114 patients tested (0.4%) had a positive test, and 24 of the 26 HIV-positive patients were linked to HIV medical care. By July 2014, the monthly proportion of patients offered a test was 62%; the proportion of those offered a test who had a test ordered was 98%, and the proportion of those with a test ordered who were tested was 81%. Testing compliance increased substantially at the WHMC ED, from 77% in December 2013 to >98% in July 2014. CONCLUSION: Using compliance-monitoring, behavior-modification, and continuous quality-improvement processes produced substantial increases in offers and HIV test completion. WHMC is replicating this approach across departments, and other hospitals implementing routine HIV screening programs should consider this approach as well.

"That word, cancer": breast care behavior of Hispanic women in new Mexico background and literature review.

Despite international efforts, national and ethnic disparities in utilization of breast cancer (BC) screenings prevail. In the United States, Hispanic women have one of the lowest BC screening rates. The purpose of our study was to examine how Hispanic women in New Mexico described their breast care behavior (BCB; BC screening practices, motivation to act, and breast care information behavior). Analysis of focus groups revealed five types of approaches to BCB. These findings have global implications for health care practitioners in directing attention toward the complexity of BC preventive behavior. Implications for other ethnic groups are discussed.

Hispanic women's preferences for breast health information: subjective cultural influences on source, message, and channel.

The purpose of this study was to investigate the influence of 3 subjective cultural variables--self-construals (independence and interdependence), ethnic identity (bicultural, assimilation, traditional, and marginal), and cultural health attributions (equity and behavioral-environmental attributions)--on source, message, and channel preferences for receiving breast health information by Hispanic women age 35 or older. Subjective cultural variables collectively accounted for 2% to 28% of the variance in communication preferences. In addition, several associations were discovered: (a) having an interdependent self-construal was associated positively with preferences for significant other as a source, family sources, fear messages, media channels, and face-to-face channels; (b) having a bicultural identity was associated positively with preferences for family sources and media channels, but negatively with a desire for no information; and (c) having a marginal identity and equity attributions were associated positively with preferences for fear messages and a desire for no information, but negatively with preferences for expert sources. These findings are discussed in the context of tailoring breast health information to Hispanic women using computer technology and entertainment-education.

Brn-3b enhances the pro-apoptotic effects of p53 but not its induction of cell cycle arrest by cooperating in trans-activation of bax expression.

The Brn-3a and Brn-3b transcription factor have opposite and antagonistic effects in neuroblastoma cells since Brn-3a is associated with differentiation whilst Brn-3b enhances proliferation in these cells. In this study, we demonstrate that like Brn-3a, Brn-3b physically interacts with p53. However, whereas Brn-3a repressed p53 mediated Bax expression but cooperated with p53 to increase p21cip1/waf1, this study demonstrated that co-expression of Brn-3b with p53 increases trans-activation of Bax promoter but not p21cip1/waf1. Consequently co-expression of Brn-3b with p53 resulted in enhanced apoptosis, which is in contrast to the increased survival and differentiation, when Brn-3a is co-expressed with p53. For Brn-3b to cooperate with p53 on the Bax promoter, it requires binding sites that flank p53 sites on this promoter. Furthermore, neurons from Brn-3b knock-out (KO) mice were resistant to apoptosis and this correlated with reduced Bax expression upon induction of p53 in neurons lacking Brn-3b compared with controls. Thus, the ability of Brn-3b to interact with p53 and modulate Bax expression may demonstrate an important mechanism that helps to determine the fate of cells when p53 is induced.