fmo-4 promotes longevity and stress resistance via ER to mitochondria calcium regulation in C. elegans.

Flavin-containing monooxygenases (FMOs) are a conserved family of xenobiotic enzymes upregulated in multiple longevity interventions, including nematode and mouse models. Previous work supports that C. elegans fmo-2 promotes longevity, stress resistance, and healthspan by rewiring endogenous metabolism. However, there are five C. elegans FMOs and five mammalian FMOs, and it is not known whether promoting longevity and health benefits is a conserved role of this gene family. Here, we report that expression of C. elegans fmo-4 promotes lifespan extension and paraquat stress resistance downstream of both dietary restriction and inhibition of mTOR. We find that overexpression of fmo-4 in just the hypodermis is sufficient for these benefits, and that this expression significantly modifies the transcriptome. By analyzing changes in gene expression, we find that genes related to calcium signaling are significantly altered downstream of fmo-4 expression. Highlighting the importance of calcium homeostasis in this pathway, fmo-4 overexpressing animals are sensitive to thapsigargin, an ER stressor that inhibits calcium flux from the cytosol to the ER lumen. This calcium/ fmo-4 interaction is solidified by data showing that modulating intracellular calcium with either small molecules or genetics can change expression of fmo-4 and/or interact with fmo-4 to affect lifespan and stress resistance. Further analysis supports a pathway where fmo-4 modulates calcium homeostasis downstream of activating transcription factor-6 ( atf-6 ), whose knockdown induces and requires fmo-4 expression. Together, our data identify fmo-4 as a longevity- promoting gene whose actions interact with known longevity pathways and calcium homeostasis.

Constraints to counting bioluminescence producing cells by a commonly used transgene promoter and its implications for experimental design.

It is routine to genetically modify cells to express fluorescent or bioluminescent reporter proteins to enable tracking or quantification of cells in vitro and in vivo. Herein, we characterized the stability of luciferase reporter systems in C4-2B prostate cancer cells in mono-culture and in co-culture with bone marrow-derived mesenchymal stem/stromal cells (BMSC). An assumption made when employing the luciferase reporter is that the luciferase expressing cell number and bioluminescence signal are linearly proportional. We observed instances where luciferase expression was significantly upregulated in C4-2B cell populations when co-cultured with BMSC, resulting in a significant disconnect between bioluminescence signal and cell number. We subsequently characterized luciferase reporter stability in a second C4-2B reporter cell line, and six other cancer cell lines. All but the single C4-2B reporter cell population had stable luciferase reporter expression in mono-culture and BMSC co-culture. Whole-genome sequencing revealed that relative number of luciferase gene insertions per genome in the unstable C4-2B reporter cell population was lesser than stable C4-2B, PC3 and MD-MBA-231 luciferase reporter cell lines. We reasoned that the low luciferase gene copy number and genome insertion locations likely contributed to the reporter gene expression being exquisitely sensitive BMSC paracrine signals. In this study, we show that it is possible to generate a range of stable and reliable luciferase reporter prostate- and breast- cancer cell populations but advise not to assume stability across different culture conditions. Reporter stability should be validated, on a case-by-case basis, for each cell line and culture condition.

Bone marrow-derived stem/stromal cells (BMSC) 3D microtissues cultured in BMP-2 supplemented osteogenic induction medium are prone to adipogenesis.

Bone marrow-derived mesenchymal stem/stromal cells (BMSC) may facilitate bone repair through secretion of factors that stimulate endogenous repair processes or through direct contribution to new bone through differentiation into osteoblast-like cells. BMSC microtissue culture and differentiation has been widely explored recently, with high-throughput platforms making large-scale manufacture of microtissues increasingly feasible. Bone-like BMSC microtissues could offer an elegant method to enhance bone repair, especially in small-volume non-union defects, where small diameter microtissues could be delivered orthoscopically. Using a high-throughput microwell platform, our data demonstrate that (1) BMSC in 3D microtissue culture result in tissue compaction, rather than growth, (2) not all mineralised bone-like matrix is incorporated in the bulk microtissue mass and (3) a significant amount of lipid vacuole formation is observed in BMSC microtissues exposed to BMP-2. These factors should be considered when optimising BMSC osteogenesis in microtissues or developing BMSC microtissue-based therapeutic delivery processes.

The Microwell-mesh: A high-throughput 3D prostate cancer spheroid and drug-testing platform.

Treatment following early diagnosis of Prostate cancer (PCa) is increasingly successful, whilst the treatment of advanced and metastatic PCa remains challenging. A major limitation in the development of new therapies is the prediction of drug efficacy using in vitro models. Classic in vitro 2-dimensional (2D) cell monolayer cultures are hypersensitive to anti-cancer drugs. As a result, there has been a surge in the development of platforms that enable three dimensional (3D) cultures thought to better replicate natural physiology and better predict drug efficacy. A deficiency associated with most 3D culture systems is that their complexity reduces the number of replicates and combination therapies that can be feasibly evaluated. Herein, we describe the use of a microwell platform that utilises a nylon mesh to retain 3D micro-tumours in discrete microwells; termed the Microwell-mesh. The Microwell-mesh enables the manufacture of ~150 micro-tumours per well in a 48-well plate, and response to anti-tumour drugs can be readily quantified. Our results demonstrate that 3D micro-tumours, unlike 2D monolayers, are not hypersensitive to Docetaxel or Abiraterone Acetate, providing a superior platform for the evaluation of sequential drug treatment. In summary, the Microwell-mesh provides an efficient 3D micro-tumour platform for single and sequential drug screening.

A kilonova as the electromagnetic counterpart to a gravitational-wave source.

Gravitational waves were discovered with the detection of binary black-hole mergers and they should also be detectable from lower-mass neutron-star mergers. These are predicted to eject material rich in heavy radioactive isotopes that can power an electromagnetic signal. This signal is luminous at optical and infrared wavelengths and is called a kilonova. The gravitational-wave source GW170817 arose from a binary neutron-star merger in the nearby Universe with a relatively well confined sky position and distance estimate. Here we report observations and physical modelling of a rapidly fading electromagnetic transient in the galaxy NGC 4993, which is spatially coincident with GW170817 and with a weak, short γ-ray burst. The transient has physical parameters that broadly match the theoretical predictions of blue kilonovae from neutron-star mergers. The emitted electromagnetic radiation can be explained with an ejected mass of 0.04 ± 0.01 solar masses, with an opacity of less than 0.5 square centimetres per gram, at a velocity of 0.2 ± 0.1 times light speed. The power source is constrained to have a power-law slope of -1.2 ± 0.3, consistent with radioactive powering from r-process nuclides. (The r-process is a series of neutron capture reactions that synthesise many of the elements heavier than iron.) We identify line features in the spectra that are consistent with light r-process elements (atomic masses of 90-140). As it fades, the transient rapidly becomes red, and a higher-opacity, lanthanide-rich ejecta component may contribute to the emission. This indicates that neutron-star mergers produce gravitational waves and radioactively powered kilonovae, and are a nucleosynthetic source of the r-process elements.

No differences in early results of a hybrid glenoid compared with a pegged implant.

BACKGROUND: Glenoid component loosening after total shoulder arthroplasty is one of the most common causes of failure. A hybrid glenoid that uses peripherally cemented pegs and a central press-fit post may improve implant longevity. QUESTIONS/PURPOSES: We asked, compared with polyethylene pegged glenoid implants, do hybrid glenoid implants with a titanium post provide (1) better ingrowth with fewer radiolucencies, (2) better outcome and pain scores, and (3) lower risk of complications and revisions? METHODS: Between 2009 and 2010, 126 patients underwent primary total shoulder arthroplasty for osteoarthritis. Patients were included in this retrospective study if they consented for inclusion in a shoulder arthroplasty registry, had complete baseline and 2-year data, and had complete radiographs. Eighty-three (67%) were available at an average followup of 3.2 years (range, 24-45 months). Forty received a conventional all-polyethylene pegged glenoid and 43 received a hybrid component. During the period in question, four of the participating surgeons used only one implant, and four used only the other; there was one high-volume surgeon in each of the study groups. Radiographs were taken at the 2-year followup and analyzed for radiolucent lines. CT scans were obtained randomly for 10 patients with hybrid glenoid implants to assess bone ongrowth. American Shoulder and Elbow Surgeons score, VAS score, complications and revisions were recorded. RESULTS: At final followup, radiolucent lines between the two study groups were not different (hybrid, 1.0 ± 0.4; pegged, 1.6 ± 0.3; mean difference, 0.6; 95% CI, 0.85-1.72; p = 0.323). Final VAS pain scores were not different (hybrid, 1.2 ± 0.2; pegged, 1.5 ± 0.3; p = 0.056). Change in American Shoulder and Elbow Surgeons scores were not different (hybrid, 33.7 ± 7.3; pegged, 35.5 ± 8.2; p = 0.283). There were no differences in complication risk (hybrid, one of 43 [2.3%]; pegged, three of 40 [7.5%]; relative risk, 2.3; 95% CI, 0.82-3.12; p = 0.061). CONCLUSIONS: With the numbers available and at early followup, there were no differences between the hybrid and pegged glenoids in terms of fixation, functional outcome, pain scores, and complications. CT scans confirmed bone ongrowth on the porous titanium post in a small subcohort of patients. Further studies are needed to determine how this new implant will perform with time. Until then, its use should be initiated with caution. LEVEL OF EVIDENCE: Level III, therapeutic study.

Stellar dynamics. The fastest unbound star in our Galaxy ejected by a thermonuclear supernova.

Hypervelocity stars (HVSs) travel with velocities so high that they exceed the escape velocity of the Galaxy. Several acceleration mechanisms have been discussed. Only one HVS (US 708, HVS 2) is a compact helium star. Here we present a spectroscopic and kinematic analysis of US 708. Traveling with a velocity of ~1200 kilometers per second, it is the fastest unbound star in our Galaxy. In reconstructing its trajectory, the Galactic center becomes very unlikely as an origin, which is hardly consistent with the most favored ejection mechanism for the other HVSs. Furthermore, we detected that US 708 is a fast rotator. According to our binary evolution model, it was spun-up by tidal interaction in a close binary and is likely to be the ejected donor remnant of a thermonuclear supernova.

An ultraviolet-optical flare from the tidal disruption of a helium-rich stellar core.

The flare of radiation from the tidal disruption and accretion of a star can be used as a marker for supermassive black holes that otherwise lie dormant and undetected in the centres of distant galaxies. Previous candidate flares have had declining light curves in good agreement with expectations, but with poor constraints on the time of disruption and the type of star disrupted, because the rising emission was not observed. Recently, two 'relativistic' candidate tidal disruption events were discovered, each of whose extreme X-ray luminosity and synchrotron radio emission were interpreted as the onset of emission from a relativistic jet. Here we report a luminous ultraviolet-optical flare from the nuclear region of an inactive galaxy at a redshift of 0.1696. The observed continuum is cooler than expected for a simple accreting debris disk, but the well-sampled rise and decay of the light curve follow the predicted mass accretion rate and can be modelled to determine the time of disruption to an accuracy of two days. The black hole has a mass of about two million solar masses, modulo a factor dependent on the mass and radius of the star disrupted. On the basis of the spectroscopic signature of ionized helium from the unbound debris, we determine that the disrupted star was a helium-rich stellar core.

In vitro primary cell culture as a physiologically relevant method for preclinical testing of human oncolytic adenovirus.

Ad[I/PPT-E1A] is an oncolytic adenovirus that specifically kills prostate cells via restricted replication by a prostate-specific regulatory element. Off-target replication of oncolytic adenoviruses would have serious clinical consequences. As a proposed ex vivo test, we describe the assessment of the specificity of Ad[I/PPT-E1A] viral cytotoxicity and replication in human nonprostate primary cells. Four primary nonprostate cell types were selected to mimic the effects of potential in vivo exposure to Ad[I/PPT-E1A] virus: bronchial epithelial cells, urothelial cells, vascular endothelial cells, and hepatocytes. Primary cells were analyzed for Ad[I/PPT-E1A] viral cytotoxicity in MTS assays, and viral replication was determined by hexon titer immunostaining assays to quantify viral hexon protein. The results revealed that at an extreme multiplicity of infection of 500, unlikely to be achieved in vivo, Ad[I/PPT-E1A] virus showed no significant cytotoxic effects in the nonprostate primary cell types apart from the hepatocytes. Transmission electron microscopy studies revealed high levels of Ad[I/PPT-E1A] sequestered in the cytoplasm of these cells. Adenoviral green fluorescent protein reporter studies showed no evidence for nuclear localization, suggesting that the cytotoxic effects of Ad[I/PPT-E1A] in human primary hepatocytes are related to viral sequestration. Also, hepatocytes had increased amounts of coxsackie adenovirus receptor surface protein. Active viral replication was only observed in the permissive primary prostate cells and LNCaP prostate cell line, and was not evident in any of the other nonprostate cells types tested, confirming the specificity of Ad[I/PPT-E1A]. Thus, using a relevant panel of primary human cells provides a convenient and alternative preclinical assay for examining the specificity of conditionally replicating oncolytic adenoviruses in vivo.

Outcomes of laparoscopic-assisted transhiatal esophagectomy for adenocarcinoma of the esophagus and esophago-gastric junction.

Esophagectomy is associated with substantial morbidity and mortality, yet it is the only modality that offers the possibility of cure for esophageal and gastroesophageal junction (E-GEJ) adenocarcinoma. Several minimally invasive techniques have been developed to decrease the morbidity of the operation, but to date, the results have not led to its wide adoption in part due to their complexity. We developed a technique of laparoscopic-assisted transhiatal esophagectomy (LA-THE) with the idea of preserving some of the advantages of the minimally invasive approach while eliminating the degree of complexity and the time required to complete the operation solely using laparoscopy. The course of all patients who underwent LA-THE for E-GEJ adenocarcinoma at the University of Washington Medical Center was determined by analysis of all hospital records to determine perioperative variables, complications, and survival. Patients were also given a follow-up survey in order to assess long-term health-related quality of life (Gastrointestinal Quality of Life Index or GIQLI). Seventy-two patients underwent LA-THE between 1995 and 2007. Median age was 64 years (range, 42-83 years), and the median body mass index was 28 (range 17-35). Twenty-eight tumors (39%) were categorized as Siewert I, 41 (57%) as Siewert II, and 3 (4%) as Siewert III. Median operative time was 299min (range, 212-700min). All the resections were R-0. The median number of lymph nodes harvested was 11 (range, 2-32). Using the Dindo-Clavien classification of surgical complication, we had a total of 48 postoperative complications in 37 patients: 26 (53%) grade I, 20 (41%) grade II, 1 (2%) grade IIIb, 1 (2%) grade IVb, and 1 (2%) grade V complications. Median length of hospital stay was 9 days (range, 7-58 days). One patient (1.4%) died within 30 days. Overall, 3- and 5-year survival (calculated Kaplan-Meier) was 68% and 63%, respectively. Forty-nine patients (90% of those still alive) answered the GIQLI survey. Median follow-up was 26 months (range, 6-144 months). The mean GIQLI score was 108 (range, 74-138) from a maximum possible value of 144. Our study shows that LA-THE is feasible, safe, and effective in the treatment of adenocarcinoma of the esophagus and GEJ and should probably be considered an alternative to open esophagectomy and other minimally invasive techniques in the treatment of this disease.

Requirement for chromatin-remodeling complex in novel tumor suppressor HIC1-mediated transcriptional repression and growth control.

HIC1 is a newly discovered tumor suppressor and transcriptional repressor that is frequently silenced in human tumors. HIC1 protein expression has been linked to better outcomes in breast cancers. The molecular mechanism underlying HIC1-mediated transcriptional and growth suppression, and the relevant targets of HIC1-mediated transcriptional modulation, is currently unclear. We have identified an HIC1 DNA-binding site in E2F-responsive gene promoters and demonstrate that HIC1 targets E2F-responsive genes for transcriptional regulation and growth suppression. We and others have recently discovered that Brg1, a central component of the SWI/SNF chromatin-remodeling family, is required for the transcriptional regulation of multiple cell cycle control-related genes, including E2F-responsive promoters. We studied HIC1 interactions with, and dependence upon, Brg1 activity, and found that HIC1 can recruit Brg1 to E2F-responsive promoters and that its transcriptional repression of these genes is dependent upon Brg1. These data indicate that HIC1 is a central molecule in a novel mechanism controlling cell growth and that the disruption of this HIC1-mediated pathway may lead to abnormal cell proliferation and, ultimately, cancer.

Polarized fluid movement and not cell death, creates luminal spaces in adult prostate epithelium.

There are two predominant theories for lumen formation in tissue morphogenesis: cavitation driven by cell death, and membrane separation driven by epithelial polarity. To define the mechanism of lumen formation in prostate acini, we examined both theories in several cell lines grown in three-dimensional (3D) Matrigel culture. Lumen formation occurred early in culture and preceded the expression of cell death markers for apoptosis (active caspase 3) and autophagy (LC-3). Active caspase 3 was expressed by very few cells and inhibition of apoptosis did not suppress lumen formation. Despite LC-3 expression in all cells within a spheroid, this was not associated with cell death. However, expression of a prostate-secretory protein coincided with lumen formation and subsequent disruption of polarized fluid movement led to significant inhibition of lumen formation. This work indicates that lumen formation is driven by the polarized movement of fluids and proteins in 3D prostate epithelial models and not by cavitation.

Reprogramming of the SWI/SNF complex for co-activation or co-repression in prohibitin-mediated estrogen receptor regulation.

The SWI/SNF complex participates as a co-activator in the transcriptional regulation of certain genes. Conversely, we and others have recently established that Brg1 and Brm, the central components of SWI/SNF, act instead as co-repressors for E2F-mediated transcriptional repression, and for the transcription of certain other promoters. We report here that Brg-1 and Brm can switch their mode of function at same promoter between activation and repression by ligand-directed differential coordination with BAF155, BAF170, HDAC1, p300 and prohibitin. This ligand and context-dependent reprogramming of the SWI/SNF complex allows it to differentially serve as either a co-repressor or a co-activator of transcription at the same promoter.

Diagnostic accuracy of self collected vaginal specimens for human papillomavirus compared to clinician collected human papillomavirus specimens: a meta-analysis.

BACKGROUND/OBJECTIVES: Providing summary recommendations regarding self collection of vaginal specimens for human papillomavirus (HPV) testing is difficult owing to the wide range of published estimates for the diagnostic accuracy of this approach. To determine summary estimates from analyses of reported findings of the sensitivity, specificity and summary receiver operating characteristic curves (SROC) for self collected vaginal specimens for HPV testing compared to the reference standard, clinician collected HPV specimens. METHODS: Standard search criteria for a diagnostic systematic review were employed. Eligible studies were combined using a random effects model and summary ROC curves were derived for overall and for specific subgroups. RESULTS: Summary measures were determined from 12 studies. Six studies where patients used Dacron or cotton swabs or cytobrushes to obtain samples were pooled and had an overall sensitivity of 0.74 (95% CI 0.61 to 0.84) and specificity of 0.88 (95% CI 0.83 to 0.92), with diagnostic odds ratio of 22.3 and an area under the curve of 0.91. Self specimens using Dacron or cotton swabs or cytobrushes collected by women enrolled at referral clinics had an overall sensitivity of 0.81 (95% CI 0.65 to 0.91) and specificity of 0.90 (95% CI 0.80 to 0.95). Sensitivity and specificity of tampons ranged from 0.67-0.94 and 0.80-0.85 respectively. CONCLUSIONS: Our findings indicate that the combined sensitivity for HPV-DNA is more than 70% when patients use Dacron swabs, cotton swabs, or cytobrushes to obtain their own vaginal specimens for HPV-DNA evaluation. Self collected HPV-DNA swabs may be an appropriate alternative for low resource settings or in patients reluctant to undergo pelvic examinations.

Disruption of MAP kinase activation and nuclear factor binding to the IL-12 p40 promoter in HIV-infected myeloid cells.

Progressive immunodeficiency in HIV infection is paralleled by a decrease in IL-12 production, a cytokine crucial for cellular immune function. Here we examine the molecular mechanisms by which HIV infection suppresses IL-12 p40 expression. HIV infection of THP-1 myeloid cells resulted in decreased LPS-induced nuclear factor binding to the NF-kappaB, AP-1, and Sp1 sites of the IL-12 p40 promoter. By site-directed mutagenesis we determined that each of these sites was necessary for transcriptional activation of the IL-12 p40 promoter. Binding of NF-kappaB p50, c-Rel, p65, Sp1, Sp3, c-Fos, and c-Jun proteins to their cognate nuclear factor binding sites was somewhat impaired by HV infection, although a role for other as yet unidentified factors cannot be dismissed. The cellular levels of these transcription factors were unaffected by HIV infection, with the exception of a decrease in expression of NF-kappaB p65, consistent with the observed decrease in its binding to the IL-12 p40 promoter following HIV infection. Analysis of regulation of upstream LPS-induced MAP kinases demonstrated impaired phosphorylation of JNK and p38 MAPK, and suppressed phosphorylation and degradation of IkappaBalpha following HIV infection. These results suggest that alterations in nuclear factor binding to numerous sites in the IL-12 p40 promoter, together may contribute to the suppression in IL-12 p40 transcription previously reported. These effects on nuclear factor binding may be a direct effect of HIV infection on the IL-12 p40 promoter, or may occur indirectly as a consequence of altered MAP kinase activation.

Are interpersonal skills ratings influenced by gender in a clinical skills assessment using standardized patients?

PURPOSE: The purpose of this study was to explore possible performance differences in interpersonal skills (IPS) ratings as a function of candidate and standardized patient (SP) gender. METHODOLOGY: The IPS scores and SP characteristics for 79,999 patient encounters were studied. This included 18,325 (20.36%) female candidate to female SP, 26,872 (29.86%) male candidate to female SP, 18,281 (20.31%) female candidate to male SP, and 16,521 (29.47%) male candidate to male SP interactions. RESULTS: The analysis did not reveal a significant candidate gender by SP gender effect. There were no meaningful differences in IPS scores as a function of SP or candidate gender. CONCLUSIONS: The non-significant interaction between SP gender and candidate gender provides some evidence that male and female candidates are being assessed equivalently by male and female SPs. This result, combined with the extremely weak relationship between gender (candidate or SP) and IPS ratings, provides additional support for the fairness and defensibility of the IPS measures.

Urine test for the assessment of smoking status.

A simple, quick and inexpensive test for smoking status would be useful in a variety of settings. The non-polar barbituric acid derivative 1,3-dibutyl-2-thiobarbituric acid (DBTB) is described as a novel derivatisation reagent for nicotine and its metabolites in the König reaction to assess smoking status. The relative performance of qualitative methods for assessing smoking status using DBTB and the previously employed derivatisation reagent 1,3-diethyl-2-thiobarbituric acid (DETB), as well as quantitatively-based methods for determining 'total nicotine metabolites' (TNMs) using these two reagents, were evaluated against a cotinine-based radioimmunoassay (RIA) as the 'gold standard'. Clinical sensitivity and specificity for all the approaches studied were in excess of 94%. Simple qualitative assessment by eye was superior to quantitatively-based measures of smoking status. Correlation between estimation of nicotine metabolites using DBTB, DETB and RIA were good. The most efficient and convenient method to distinguish between smokers and non-smokers was the simple qualitative method using the more lipophilic reagent DBTB.

Quantitative polymerase chain reaction analysis of DNA from noninvasive samples for accurate microsatellite genotyping of wild chimpanzees (Pan troglodytes verus).

Noninvasive samples are useful for molecular genetic analyses of wild animal populations. However, the low DNA content of such samples makes DNA amplification difficult, and there is the potential for erroneous results when one of two alleles at heterozygous microsatellite loci fails to be amplified. In this study we describe an assay designed to measure the amount of amplifiable nuclear DNA in low DNA concentration extracts from noninvasive samples. We describe the range of DNA amounts obtained from chimpanzee faeces and shed hair samples and formulate a new efficient approach for accurate microsatellite genotyping. Prescreening of extracts for DNA quantity is recommended for sorting of samples for likely success and reliability. Repetition of results remains extensive for analysis of microsatellite amplifications beginning from low starting amounts of DNA, but is reduced for those with higher DNA content.