An External Validation Study for Automated Segmentation of Vestibular Schwannoma.

OBJECTIVE: To validate how an automated model for vestibular schwannoma (VS) segmentation developed on an external homogeneous dataset performs when applied to internal heterogeneous data. PATIENTS: The external dataset comprised 242 patients with previously untreated, sporadic unilateral VS undergoing Gamma Knife radiosurgery, with homogeneous magnetic resonance imaging (MRI) scans. The internal dataset comprised 10 patients from our institution, with heterogeneous MRI scans. INTERVENTIONS: An automated VS segmentation model was developed on the external dataset. The model was tested on the internal dataset. MAIN OUTCOME MEASURE: Dice score, which measures agreement between ground truth and predicted segmentations. RESULTS: When applied to the internal patient scans, the automated model achieved a mean Dice score of 61% across all 10 images. There were three tumors that were not detected. These tumors were 0.01 ml on average (SD = 0.00 ml). The mean Dice score for the seven tumors that were detected was 87% (SD = 14%). There was one outlier with Dice of 55%-on further review of this scan, it was discovered that hyperintense petrous bone had been included in the tumor segmentation. CONCLUSIONS: We show that an automated segmentation model developed using a restrictive set of siloed institutional data can be successfully adapted for data from different imaging systems and patient populations. This is an important step toward the validation of automated VS segmentation. However, there are significant shortcomings that likely reflect limitations of the data used to train the model. Further validation is needed to make automated segmentation for VS generalizable.

Surgical Considerations in Inner Ear Gene Therapy from Human Temporal Bone Anatomy.

OBJECTIVE(S): Recently directed methods of inner ear drug delivery underscore the necessity for understanding critical anatomical dimensions. This study examines anatomical measurements of the human middle and inner ear relevant for inner ear drug delivery studied with three different imaging modalities. METHODS: Post-mortem human temporal bones were analyzed using human temporal bone histopathology (N = 24), micro computerized tomography (µCT; N = 4), and synchrotron radiation phase-contrast imaging (SR-PCI; N = 7). Nine measurements involving the oval and round windows were performed when relevant anatomical structures were visualized for subsequent age-controlled analysis, and comparisons were made between imaging methods. RESULTS: Combined human temporal bone histopathology showed the mean distance to the saccule from the center of the stapes footplate (FP) was 2.07 ± 0.357 mm and the minimum distance was 1.23 mm. The mean distance from the round window membrane (RWM) to the osseous spiral lamina (OSL) was 1.75 ± 0.199 mm and the minimum distance was 1.43 mm. Instruments inserted up to 1 mm past the center of the FP are unlikely to cause saccular damage, provided there are no endolymphatic hydrops. Similarly, instruments inserted up to 1 mm through the RWM in the trajectory toward the OSL are unlikely to cause OSL damage. CONCLUSION: The combined analyses of inner-ear dimensions of age-controlled groups and imaging modalities demonstrate critical dimensions of importance to consider when inserting delivery vehicles into the human cochlea. LEVEL OF EVIDENCE: N/A Laryngoscope, 134:2879-2888, 2024.

Evaluating the Feasibility of a Novel Firearm Injury Prevention Program for Pre-adolescent Children Through Health Care and Community-Based Partnerships: The Future Healers Program Pilot Study.

BACKGROUND: Firearm violence is an American public health crisis that negatively impacts children and disproportionately affects Black youth. Few firearm injury prevention programs have been described in pre-adolescent children. The Future Healers Program is a novel collaboration constructed via partnership between the medical school, trauma center, academic surgery department, and local non-profit community organization. Our study sought to evaluate if (1) partnering with community organizations facilitated recruitment of children with prior exposure to firearm violence and (2) the health care community was a potential trusted partner appropriate for program delivery. METHODS: Children aged 4-13 were recruited to join the program via news outlets and social media and in partnership with a local non-profit organization. Of the children and parents participating in the program, 48% (44/92) and 59% (38/64), respectively, completed an IRB-approved survey study. Pearson's chi-square, percentages, and 95% confidence intervals evaluated differences between children and caregivers on sociodemographic characteristics, firearm exposure (FE), firearm violence exposure (FVE), and perception of health care. Participant's residence was geocoded in relationship to incidents of firearm injury (2008-2021) in the same region. RESULTS: Caregivers (95%) and children (84%) reported substantial exposure to firearm violence and resided in areas with frequent firearm injury incidents. Notably, 82% of caregivers and 66% of children reported having a family member injured by gunfire. A high percentage of caregivers (79%) and children (91%) self-reported trust in the health care system. CONCLUSION: Partnerships between community organizations and health care systems can develop prevention programs that effectively recruit and engage pre-adolescent children impacted by firearm violence.

Identification of immune-related candidate biomarkers in plasma of patients with sporadic vestibular schwannoma.

Vestibular schwannoma (VS) is an intracranial tumor arising from neoplastic Schwann cells and typically presenting with hearing loss. The traditional belief that hearing deficit is caused by physical expansion of the VS, compressing the auditory nerve, does not explain the common clinical finding that patients with small tumors can have profound hearing loss, suggesting that tumor-secreted factors could influence hearing ability in VS patients. We conducted profiling of patients' plasma for 66 immune-related factors in patients with sporadic VS (N > 170) and identified and validated candidate biomarkers associated with tumor size (S100B) and hearing (MCP-3). We further identified a nine-biomarker panel (TNR-R2, MIF, CD30, MCP-3, IL-2R, BLC, TWEAK, eotaxin, and S100B) with outstanding discriminatory ability for VS. These findings revealed possible therapeutic targets for VS, providing a unique diagnostic tool that may predict hearing change and tumor growth in VS patients, and may inform the timing of tumor resection to preserve hearing.

Perceptions of General Surgery Program Leadership and Residents on Research Sabbatical.

INTRODUCTION: The incorporation of a 1-y- or 2-y research period during general surgery residency is increasingly common in many academic programs, yet often, it is heterogeneously structured and ill-defined. This survey-based observational study aimed to characterize the perceptions of general surgery program directors (PDs) and residents regarding an in-training, dedicated research sabbatical. METHODS: Two surveys were conducted using Qualtrics software. One survey was sent to general surgery residency PDs, and the other was sent to general surgery residents currently on a research sabbatical. The primary aim of the survey was to assess the PDs' and research residents' perceptions of the research sabbatical. RESULTS: Seven hundred and fifty-two surveys were analyzed, of which 120 were from PDs and 632 from research residents. Among the residents, 44.1% felt that the research time delayed their surgical training. Regarding research funding, 46.7% of the responding residents said that their residency program funded their research, 30.9% said they acquired funding independently, and 19.1% said that it came from a combination of residency program and independent funding. Finally, regarding how residents found their research opportunity, 42.7% said they found it independently and 53.3% said their program provided it. CONCLUSIONS: Research sabbaticals during residency may be considered essential to academic development. However, in this survey-based study, perceptions of research time and its structure varied greatly between PDs and residents. An intentional push toward developing guidelines for research sabbaticals may benefit residency program leadership and residents.

Immune cells are increased in normal breast tissues of BRCA1/2 mutation carriers.

PURPOSE: Breast cancer risk is elevated in pathogenic germline BRCA 1/2 mutation carriers due to compromised DNA quality control. We hypothesized that if immunosurveillance promotes tumor suppression, then normal/benign breast lobules from BRCA carriers may demonstrate higher immune cell densities. METHODS: We assessed immune cell composition in normal/benign breast lobules from age-matched women with progressively increased breast cancer risk, including (1) low risk: 19 women who donated normal breast tissue to the Komen Tissue Bank (KTB) at Indiana University Simon Cancer Center, (2) intermediate risk: 15 women with biopsy-identified benign breast disease (BBD), and (3) high risk: 19 prophylactic mastectomies from women with germline mutations in BRCA1/2 genes. We performed immunohistochemical stains and analysis to quantitate immune cell densities from digital images in up to 10 representative lobules per sample. Median cell counts per mm2 were compared between groups using Wilcoxon rank-sum tests. RESULTS: Normal/benign breast lobules from BRCA carriers had significantly higher densities of immune cells/mm2 compared to KTB normal donors (all p < 0.001): CD8 + 354.4 vs 150.9; CD4 + 116.3 vs 17.7; CD68 + 237.5 vs 57.8; and CD11c + (3.5% vs 0.4% pixels positive). BBD tissues differed from BRCA carriers only in CD8 + cells but had higher densities of CD4 + , CD11c + , and CD68 + immune cells compared to KTB donors. CONCLUSIONS: These preliminary analyses show that normal/benign breast lobules of BRCA mutation carriers contain increased immune cells compared with normal donor breast tissues, and BBD tissues appear overall more similar to BRCA carriers.

New hyaluronan-based biomatrix for 3-D follicle culture yields functionally competent oocytes.

BACKGROUND: Encapsulation of follicles within a biomatrix is one approach to maintaining 3-D follicle architecture during culture. Hyaluronan is one component of the natural extracellular matrix (ECM) that provides support to cells in vivo. This report describes the application of a novel tyramine-linked hyaluronan for 3-D in vitro follicle culture and the production of developmentally competent metaphase II oocytes. MATERIALS AND METHODS: Enzymatically isolated mouse preantral follicles or follicle clusters (FL-C) from fresh or vitrified ovaries were encapsulated in 3 mg/ml of hyaluronan gel (HA). Follicle growth, antrum formation and meiotic maturation to metaphase II oocytes was monitored. Chromatin staining was used to assess GV oocyte progression towards meiotic competence. Functional competence of in vitro matured (IVM) oocytes was evaluated by in vitro fertilization and ability to develop to blastocyst. Modifying the HA gel by inclusion of laminin (HA-LM), mouse sarcoma extracellular matrix (Matrigel;HA-MG) or placental extracellular matrix (HA-PM) was also tested to see if this might further enhance IVM outcomes. RESULTS: A total of 402 preantral follicles were cultured in HA gel. After hCG trigger, 314 oocyte-cumulus complexes ovulated from the embedded follicles. Meiotic maturation rate to the metaphase II stage was 73% (228/314). After insemination 83% (188/228) of IVM oocytes fertilized with a subsequent blastulation rate of 46% (87/188). A pilot transfer study with 3 recipient mice resulted in the birth of a single pup. HA gel supported individually isolated follicles as well ovarian tissue fragments containing clusters of 6-8 preantral follicles. Meiotic maturation was lower with FL-clusters from vitrified versus fresh ovaries (34% and 55%, respectively; p < 0.007). Modification of the HA gel with ECMs or laminin affected antrum formation and follicle retention. Maturation rates to the metaphase II stage were however not significantly different: 74% for HA gel alone as compared to HA-LM (67%), HA-MG (56%) and HA-PM (58%). CONCLUSION: Hyaluronan gel is an effective and versatile extracellular matrix based biomaterial for 3-D culture of ovarian follicles. This culture model allowed ovulation of functionally competent metaphase II oocytes, capable of fertilization, genomic activation and blastulation. Future testing with human follicles that require longer in vitro culture times should be considered.

Reversible contrast enhancement for visualization of human temporal bones using micro computed tomography.

Sensorineural hearing loss (SNHL), which typically arises from the inner ear, is the most common sensory deficit worldwide. The traditional method for studying pathophysiology underlying human SNHL involves histological processing of the inner ear from temporal bones collected during autopsy. Histopathological analysis is destructive and limits future use of a given specimen. Non-destructive strategies for the study of the inner ear are urgently needed to fully leverage the utility of each specimen because access to human temporal bones is increasingly difficult and these precious specimens are required to uncover disease mechanisms and to enable development of new devices. We highlight the potential of reversible iodine staining for micro-computed tomography imaging of the human inner ear. This approach provides reversible, high-resolution visualization of intracochlear structures and is becoming more rapid and accessible.

Association Between Implementation of a Geriatric Trauma Clinical Pathway and Changes in Rates of Delirium in Older Adults With Traumatic Injury.

Importance: Older adults (age ≥65 years) are at risk for high rates of delirium and poor outcomes; however, how to improve outcomes is still being explored. Objective: To assess whether implementation of a geriatric trauma clinical pathway was associated with reduced rates of delirium in older adults with traumatic injury. Design, Setting, and Participants: A retrospective case-control study of electronic health records of patients aged 65 years or older with traumatic injury from 2018 to 2020 was conducted at a single level I trauma center. Eligible patients were age 65 years or older admitted to the trauma service and who did not undergo an operation. Intervention: The implementation of a clinical pathway based on geriatric best practices, which included order sets, guidelines, automated consultations, and escalation pathways executed by a multidisciplinary team. Main Outcomes and Measures: The primary outcome was delirium. The secondary outcome was hospital length of stay. Process measures for pathway compliance were also assessed. Results: Of the 859 eligible patients, 712 patients were included in the analysis (442 [62.1%] in the baseline group; 270 [37.9%] in the postimplementation group; mean [SD] age: 81.4 [9.1] years; 394 [55.3%] were female). The mechanism of injury was not different between groups, with 247 in the baseline group (55.9%) and 162 in the postimplementation group (60.0%) (P = .43) experiencing a fall. Injuries were minor or moderate in both groups (261 in baseline group [59.0%] and 168 in postimplementation group [62.2%]; P = .87). The adjusted odds ratio for delirium in the postimplementation cohort was 0.54 (95% CI, 0.37-0.80; P < .001). Goals of care documentation improved significantly in the postimplementation cohort vs the baseline cohort with regard to documented goals of care notes (53.7% in the postimplementation cohort [145 of 270] vs 16.7% in the baseline cohort [74 of 442]; P < .001) and a shortened time to discussion from presenting to the emergency department (36 hours in the postimplementation cohort vs 50 hours in the baseline cohort; P = .03). Conclusions and Relevance: In this study, implementation of a multidisciplinary clinical pathway for injured older adults at a single level I trauma center was associated with improved care and clinical outcomes. Interventions such as these may have utility in this vulnerable population, and findings should be confirmed across multiple centers.

Sporadic Vestibular Schwannoma Size and Location Do not Correlate With the Severity of Hearing Loss at Initial Presentation.

Vestibular schwannoma (VS) is a non-malignant intracranial neoplasm arising from the vestibular branch of the 8th cranial nerve; sensorineural hearing loss (SNHL) is the most common associated symptom. Understanding whether VS imaging characteristics at the time of VS diagnosis can be associated with severity of VS-induced SNHL can impact patient counseling and define promising areas for future research. Patients diagnosed with VS at Massachusetts Eye and Ear (MEE) from 1994 through 2018 were analyzed if magnetic resonance imaging at VS presentation and sequential audiometry were available. Results were compared with original studies available in PubMed, written in English, on VS imaging characteristics and their impact on hearing in patients. A total of 477 patients with unilateral VS from the MEE database demonstrated no significant correlation between any features of tumor imaging at the time of VS diagnosis, such as VS size, impaction or location, and any hearing loss metric. Twenty-three published studies on the impact of VS imaging characteristics on patient hearing met inclusion criteria, with six solely involving NF2 patients and three including both sporadic and NF2-related VS patients. Fifteen studies reported a significant relationship between SNHL and at least one VS imaging characteristic; however, these trends were universally limited to NF2 patients or involved small patient populations, and were not reproduced in larger studies. Taken together, SNHL in sporadic VS patients is not readily associated solely with any tumor imaging characteristics. This finding motivates future studies to define how VS microenvironment and secreted molecules influence VS-induced SNHL.

Lipotoxicity reduces DDX58/Rig-1 expression and activity leading to impaired autophagy and cell death.

Nonalcoholic fatty liver disease (NAFLD) is the leading cause of chronic liver disease globally. NAFLD is a consequence of fat accumulation in the liver leading to lipotoxicity. Increasing evidence has demonstrated the critical role of autophagy in NAFLD. This study uncovers the unexpected role of immune surveillance protein DDX58/Rig-1 (DExD/H box helicase 58) in activating macroautophagy/autophagy and protecting from lipotoxicity associated with NAFLD. Here we show for the first time that DDX58 protein is significantly reduced in nonalcoholic steatohepatitis (NASH) mouse model, an aggressive form of NAFLD characterized by inflammation and fibrosis of the liver. In addition to decreased expression of DDX58, we found that DDX58 activity can be attenuated by treatments with palmitic acid (PA), a saturated fatty acid. To investigate whether PA inhibition of DDX58 is harmful to the cell, we characterized DDX58 function in hepatocytes when exposed to high doses of PA in the presence and/or absence of DDX58. We show that siRNA knockdown of DDX58 promotes apoptosis. Importantly, we show that stable overexpression of DDX58 is protective against toxic levels of PA and stimulates autophagy. This study begins to demonstrate the regulation of the autophagy receptor protein SQSTM1/p62 through DDX58. DDX58 expression directly influences SQSTM1 mRNA and protein levels. This work proposes a model in which activating DDX58 increases an autophagic response and this aids in clearing toxic lipid inclusion bodies, which leads to inflammation and apoptosis. Activating a DDX58-induced autophagy response may be a strategy for treating NAFLD.Abbreviations:5'pppdsRNA: 5' triphosphate double-stranded RNA; CDAHFD: choline-deficient, L-amino acid defined high-fat diet; CEBPB: CCAAT/enhancer binding protein (C/EBP), beta; CQ: chloroquine; DDX58/retinoic acid inducible gene 1/Rig-1: DExD/H box helicase 58; h: hours; IFIH1/MDA5: interferon induced with helicase C domain 1; IFNB/IFN-ß: interferon beta 1, fibroblast; KO: knockout; MAVS: mitochondrial antiviral signaling protein; NAFLD: nonalcoholic fatty liver disease; NASH: nonalcoholic steatohepatitis; NFKB/NF-κB: nuclear factor of kappa light polypeptide gene enhancer in B cells; PA: palmitic acid; poly:IC: polyinosinic:polycytidylic acid; PRR: pattern recognition receptors; PSR: picrosirus red; RAP: rapamycin; RLR: RIG-I-like receptor; SQSTM1/p62: sequestosome 1; STING1: stimulator of interferon response cGAMP interactor 1; TBK1: TANK-binding kinase 1.

Losartan prevents tumor-induced hearing loss and augments radiation efficacy in NF2 schwannoma rodent models.

Hearing loss is one of the most common symptoms of neurofibromatosis type 2 (NF2) caused by vestibular schwannomas (VSs). Fibrosis in the VS tumor microenvironment (TME) is associated with hearing loss in patients with NF2. We hypothesized that reducing the fibrosis using losartan, an FDA-approved antihypertensive drug that blocks fibrotic and inflammatory signaling, could improve hearing. Using NF2 mouse models, we found that losartan treatment normalized the TME by (i) reducing neuroinflammatory IL-6/STAT3 signaling and preventing hearing loss, (ii) normalizing tumor vasculature and alleviating neuro-edema, and (iii) increasing oxygen delivery and enhancing efficacy of radiation therapy. In preparation to translate these exciting findings into the clinic, we used patient samples and data and demonstrated that IL-6/STAT3 signaling inversely associated with hearing function, that elevated production of tumor-derived IL-6 was associated with reduced viability of cochlear sensory cells and neurons in ex vivo organotypic cochlear cultures, and that patients receiving angiotensin receptor blockers have no progression in VS-induced hearing loss compared with patients on other or no antihypertensives based on a retrospective analysis of patients with VS and hypertension. Our study provides the rationale and critical data for a prospective clinical trial of losartan in patients with VS.

Muscle-specific deletion of the vitamin D receptor in mice is associated with diaphragm muscle weakness.

Diseases or conditions where diaphragm muscle (DIAm) function is impaired, including chronic obstructive pulmonary disease, cachexia, asthma, and aging, are associated with an increased risk of pulmonary symptoms, longer duration of hospitalizations, and increasing requirements for mechanical ventilation. Vitamin D deficiency is associated with proximal muscle weakness that resolves following therapy with vitamin D3. Skeletal muscle expresses the vitamin D receptor (VDR), which responds to the active form of vitamin D, 1,25-dihydroxyvitamin D3 by altering gene expression in target cells. In knockout mice without skeletal muscle VDRs, there is marked atrophy of muscle fibers and a change in skeletal muscle biochemistry. We used a tamoxifen-inducible skeletal muscle Cre recombinase in Vdrfl/fl mice (Vdrfl/fl actin.iCre+) to assess the role of muscle-specific VDR signaling on DIAm-specific force, fatigability, and fiber type-dependent morphology. Vdrfl/fl actin.iCre+ mice treated with vehicle and Vdrfl/fl mice treated with tamoxifen served as controls. Seven days following the final treatment, mice were euthanized, the DIAm was removed, and isometric force and fatigue were assessed in DIAm strips using direct muscle stimulation. The proportion and cross-sectional areas of DIAm fiber types were evaluated by immunolabeling with myosin heavy chain antibodies differentiating type I, IIa and IIx, and/or IIb fibers. We show that in mice with skeletal muscle-specific VDR deletion, maximum specific force and residual force following fatigue are impaired, along with a selective atrophy of type IIx and/or IIb fibers. These results show that the VDR has a significant biological effect on DIAm function independent of systemic effects on mineral metabolism.NEW & NOTEWORTHY Vitamin D deficiency and vitamin D receptor (VDR) polymorphisms are associated with adverse pulmonary and diaphragm muscle (DIAm)-associated respiratory outcomes. We used a skeletal muscle-specific tamoxifen-inducible VDR knockout to investigate DIAm dysfunction following reduced VDR signaling. Marked DIAm weakness and atrophy of type IIx and/or IIb fibers are present in muscle-specific tamoxifen-induced VDR knockout mice compared with controls. These results show that the VDR has a significant biological effect on DIAm function independent of systemic effects on mineral metabolism.

Evolution of pediatric gastrointestinal ulcer disease: Is acute surgical intervention relevant?

BACKGROUND: There is a lack of contemporary data about pediatric gastrointestinal ulcer disease. We hypothesized that ulcers found in immunosuppressed children were more likely to require surgical intervention. METHODS: All children <21 years (n = 129) diagnosed with ulcers at a quaternary hospital from 1990 to 2019 were retrospectively reviewed. Clinical findings and pertinent information were collected. RESULTS: Of 129 cases, 19 (14.7%) were immunosuppressed. Eight were post-transplant; four were diagnosed with post-transplant lymphoproliferative disease (PTLD).  Eight were associated with cancer. Three were both.  Three of 19 immunosuppressed and 28/110 immunocompetent patients were taking acid suppression therapy. Nine immunosuppressed patients required surgical intervention, including all PTLD cases, compared to 14 immunocompetent (47.3% vs 16.4%, p < 0.01). Five patients had duodenal perforation, two had multiple small bowel perforations, and two had uncontrolled bleeding. Of 9/19 immunosuppressed patients, surgical complications included bleeding (n = 7), sepsis (n = 2), ostomy reoperation/readmissions (n = 2), and death within 30 days (n = 2). Two/eighteen immunocompetent patients had bleeding complications. CONCLUSION: Surgical treatment for ulcers remains relevant for pediatric patients. Immunosuppressed patients have more complications, longer hospital stays, and are more likely to need surgical intervention. Efforts should be made for ulcer prophylaxis with a low threshold to investigate epigastric pain in these complex patients. LEVEL OF EVIDENCE: Prognosis Study Level III Evidence.

The 50 Most Cited Articles on Meniscus Injuries and Surgery from 2000 to 2019 Focus on Arthroscopic Repair or Removal, Originate from Institutions Within the United States and Were Published Before 2010.

PURPOSE: To identify the 50 most cited original articles on meniscus injury and surgery from 2000 to 2019, and to perform a bibliometric analysis of the identified articles. METHODS: A Clarivate Web of Science search, completed in June 2020, generated a list of the most cited articles related to meniscus research. Articles were sorted by number of times cited, and review articles or those unrelated to the meniscus were removed. Articles were classified as basic science or assigned the appropriate level of evidence. Extracted data included title, authors, journal, year of publication, country/institution of origin, total number of citations, and number of citations per year. RESULTS: The final list of 50 included articles with a range of 106 to 490 citations and a mean of 162.34 total or 11.91 citations per year. The most cited articles appeared in 8 of the most influential journals in the field per the Journal Citation Index. Twenty-nine (58%) originated from institutions within the United States, and only 13 (26%) were published in 2010 or later. Overall, 25 (50%) were classified as therapeutic, only 5 (10%) were therapeutic randomized controlled studies, and 17 (34%) were basic science. "Arthroscopic meniscal repair or meniscectomy" appeared most frequently, with 16 (32%) falling into this subclassification. CONCLUSION: This study of the most cited meniscus articles showed a strong predominance for therapeutic studies, studies generated and published within the United States, and studies focused on topics of arthroscopic repair or removal. Overwhelmingly, included articles were published before 2010, affirming the criticism that bibliometric analysis favors older articles. CLINICAL RELEVANCE: This study provides information about which articles are driving the field relating to meniscus injuries and meniscus surgery in the last two decades.

Carbon monoxide concentration in mainstream E-cigarette emissions measured with diode laser spectroscopy.

The e-fluid heated in electronic cigarettes (e-cigarettes) is largely composed of organic compounds, specifically propylene glycol, vegetable glycerin and flavouring compounds. When heated, as it is in an e-cigarette, the chemical species in this fluid have the potential to oxidise into carbon monoxide (CO) and other species. Using diode laser spectroscopy, the concentration of CO in e-cigarette mainstream effluent as a function of e-cigarette power and flavour was measured. Carbon monoxide concentration was found to be a direct function of the power of the resistive heating. At the highest powers testable using commercial e-cigarette components, the maximum CO concentration measured was over 180 ppm. The flavouring compounds in the e-fluid also had an effect on the concentration of carbon monoxide present in the effluent.

STAT3 signaling stimulates miR-21 expression in bovine cumulus cells during in vitro oocyte maturation.

MicroRNAs are potent regulators of gene expression that have been widely implicated in reproduction and embryo development. Recent studies have demonstrated that miR-21, a microRNA extensively studied in the context of disease, is important in multiple facets of reproductive biology including folliculogenesis, ovulation, oocyte maturation and early mammalian development. Surprisingly, little is known about the mechanisms that regulate miR-21 and no studies have characterized these regulatory pathways in cumulus-oocyte complexes (COCs). We therefore investigated miR-21 in an in vitro model of bovine oocyte maturation. Levels of the primary transcript of miR-21 (pri-miR-21) and mature miR-21 increased markedly in COCs over the maturation period. Cloning of the bovine pri-miR-21 gene and promoter by 5'3'RACE (rapid amplification of cDNA ends) revealed a highly conserved region immediately upstream of the transcription start site and two alternatively-spliced variants of pri-miR-21. The promoter region contained several putative transcription factor binding sites, including two for signal transducer and activator of transcription 3 (STAT3). Mutation of these sites significantly decreased both the intrinsic activity of pri-miR-21 promoter-luciferase constructs and the response to leukemia inhibitory factor (LIF) (a STAT3 activator) in cultured MCF7 cells. In COCs, treatment with a STAT3 pathway inhibitor markedly decreased pri-miR-21 expression and prevented cumulus expansion. Pri-miR-21 expression was also inhibited by the protein synthesis inhibitor cycloheximide, suggesting that a protein ligand or signaling cofactor synthesized during maturation is necessary for transcription. Together these studies represent the first investigation of signaling pathways that directly influence miR-21 expression in bovine oocytes and cumulus cells.