Characteristics of Fentanyl Overdose - Massachusetts, 2014-2016.

Opioid overdose deaths in Massachusetts increased 150% from 2012 to 2015 (1). The proportion of opioid overdose deaths in the state involving fentanyl, a synthetic, short-acting opioid with 50-100 times the potency of morphine, increased from 32% during 2013-2014 to 74% in the first half of 2016 (1-3). In April 2015, the Drug Enforcement Agency (DEA) and CDC reported an increase in law enforcement fentanyl seizures in Massachusetts, much of which was believed to be illicitly manufactured fentanyl (IMF) (4). To guide overdose prevention and response activities, in April 2016, the Massachusetts Department of Public Health and the Office of the Chief Medical Examiner collaborated with CDC to investigate the characteristics of fentanyl overdose in three Massachusetts counties with high opioid overdose death rates. In these counties, medical examiner charts of opioid overdose decedents who died during October 1, 2014-March 31, 2015 were reviewed, and during April 2016, interviews were conducted with persons who used illicit opioids and witnessed or experienced an opioid overdose. Approximately two thirds of opioid overdose decedents tested positive for fentanyl on postmortem toxicology. Evidence for rapid progression of fentanyl overdose was common among both fatal and nonfatal overdoses. A majority of interview respondents reported successfully using multiple doses of naloxone, the antidote to opioid overdose, to reverse suspected fentanyl overdoses. Expanding and enhancing existing opioid overdose education and prevention programs to include fentanyl-specific messaging and practices could help public health authorities mitigate adverse effects associated with overdoses, especially in communities affected by IMF.

Effects of species differences on oocyte regulation of granulosa cell function.

The aims were to investigate whether oocyte-secreted growth factors from a high (i.e. rat) and low (i.e. sheep) ovulation rate species could stimulate (3)H-thymidine incorporation in granulosa cells (GC) from antral follicles from the same or across species. Denuded oocytes (DO) were co-incubated with GC with or without specific antibodies to growth differentiating factor 9 (GDF9) or bone morphogenetic protein 15 (BMP15). Co-incubations of DO-GC from the same or across species significantly increased thymidine incorporation in GC with increasing numbers of DO. GDF9 immuno-neutralisation reduced thymidine incorporation in rat GC co-incubated with either rat or ovine DO and in ovine GC co-incubated with ovine or rat DO. BMP15 immuno-neutralisation only reduced thymidine incorporation when ovine DO were co-incubated with either ovine or rat GC. Western blotting of oocytes co-incubated with GC identified GDF9 and BMP15 proteins for sheep and GDF9 protein for rats in oocyte lysates and incubation media. With respect to rat BMP15, a promature protein was identified in the oocyte lysate but not in media. Expression levels of GDF9 relative to BMP15 mRNA in DO co-incubated with GC were highly correlated (R (2)=0.99) within both species. However, the expression ratios were markedly different for the rat and sheep (4.3 vs 1.0 respectively). We conclude that during follicular development, rat oocytes secrete little, if any, BMP15 and that GDF9 without BMP15 can stimulate proliferation of rat and ovine GC. In contrast, ovine oocytes secrete both BMP15 and GDF9, and both were found to stimulate proliferation in ovine and rat GC.

The ratio of growth differentiation factor 9: bone morphogenetic protein 15 mRNA expression is tightly co-regulated and differs between species over a wide range of ovulation rates.

Recent evidence suggests that the species-specific ovulation-rate phenotypes may be influenced by differences in the expression levels of bone morphogenetic protein 15 (BMP15) and growth differentiation factor 9 (GDF9) mRNA and protein. The aim of this study was to compare GDF9 and BMP15 mRNA levels in individual denuded oocytes (DO) from a range of single (i.e. cow, red deer), single-to-triple (i.e. sheep) and high (i.e. pig, mouse, rat) ovulation-rate species. Compared to all other species studied, GDF9 mRNA levels were lower in DO of cows and deer, whilst BMP15 levels were highest in DO of pigs. There was no detectable expression of either GDF9 or BMP15 mRNA in CC from any species. The ratio of GDF9:BMP15 mRNA expression was highly correlated (R(2)>0.80) within each species but differed markedly between species (P<0.01). Thus, we conclude that the ratio of GDF9:BMP15 mRNA is species-specific across a wide range of ovulation-rate phenotypes.

Peloruside- and laulimalide-resistant human ovarian carcinoma cells have ßI-tubulin mutations and altered expression of ßII- and ßIII-tubulin isotypes.

Peloruside A and laulimalide are potent microtubule-stabilizing natural products with a mechanism of action similar to that of paclitaxel. However, the binding site of peloruside A and laulimalide on tubulin remains poorly understood. Drug resistance in anticancer treatment is a serious problem. We developed peloruside A- and laulimalide-resistant cell lines by selecting 1A9 human ovarian carcinoma cells that were able to grow in the presence of one of these agents. The 1A9-laulimalide resistant cells (L4) were 39-fold resistant to the selecting agent and 39-fold cross-resistant to peloruside A, whereas the 1A9-peloruside A resistant cells (R1) were 6-fold resistant to the selecting agent while they remained sensitive to laulimalide. Neither cell line showed resistance to paclitaxel or other drugs that bind to the taxoid site on ß-tubulin nor was there resistance to microtubule-destabilizing drugs. The resistant cells exhibited impaired peloruside A/laulimalide-induced tubulin polymerization and impaired mitotic arrest. Tubulin mutations were found in the ßI-tubulin isotype, R306H or R306C for L4 and A296T for R1 cells. This is the first cell-based evidence to support a ß-tubulin-binding site for peloruside A and laulimalide. To determine whether the different resistance phenotypes of the cells were attributable to any other tubulin alterations, the ß-tubulin isotype composition of the cells was examined. Increased expression of ßII- and ßIII-tubulin was observed in L4 cells only. These results provide insight into how alterations in tubulin lead to unique resistance profiles for two drugs, peloruside A and laulimalide, that have a similar mode of action.

Oocytes in sheep homozygous for a mutation in bone morphogenetic protein receptor 1B express lower mRNA levels of bone morphogenetic protein 15 but not growth differentiation factor 9.

The aim of this study was to test the hypothesis that the high ovulation rate in ewes (BB) homozygous for a mutation in the bone morphogenetic protein receptor type 1B (BMPR1B) gene is linked to lower BMP15 and/or GDF9 mRNA in oocytes compared with those in wild-type (++) ewes. Cumulus cell-oocyte complexes (COC) and granulosa cells (GC) were recovered from ≥1 mm diameter follicles of BB and ++ ewes during a prostaglandin-induced follicular phase. Expression levels of GDF9 and BMP15 were measured by multiplex qPCR from individual COC. The gonadotropin-induced cAMP responses of the GC from each non-atretic follicle were measured following treatment with FSH or human chorionic gonadotropin. In a separate validation experiment, GDF9 and BMP15 expression was present only in oocytes and not in cumulus cells. There was no effect of follicular diameter on oocyte-derived GDF9 or BMP15 mRNA levels. The mean expression levels of BMP15, but not GDF9, were significantly lower in all non-atretic follicles, including the subsets containing either FSH- or LH-responsive GC in BB, compared with ++, ewes. No genotype effects were noted for FSH-induced cAMP production by GC either with respect to dose of, or number of follicles responding to, FSH. However, ovaries from BB ewes contained significantly more follicles responsive to LH, with respect to cAMP production in GC. We propose that these findings are consistent with the hypothesis that the higher ovulation rate in BB sheep is due, at least in part, to lower oocyte-derived BMP15 mRNA levels together with the earlier onset of LH-responsiveness in GC.

The hypothalamic-pituitary-ovarian axis and manipulations of the oestrous cycle in the brushtail possum.

The main purpose of this review is to provide a comprehensive update on what is known about the regulatory mechanisms of the hypothalamic-pituitary-ovarian axis in the brushtail possum, and to report on the outcomes of attempts made to manipulate by hormonal means, these processes in the possum. Over the last 15 years, several unique features of possum reproductive physiology have been discovered. These include an extended follicular phase despite elevated concentrations of FSH during the luteal phase, and early expression of LH receptors on granulosa cells of small antral follicles, suggesting a different mechanism for the selection of a dominant follicle. The use of routine synchronisation protocols that are effective in eutherians has failed to be effective in possums, and so the ability to reliably synchronise oestrus in this species remains a challenge.

Neonatal estrogenic effects upon the male rat pituitary: early gonadotrophin attenuation precedes long-term recovery.

Neonatal exposure to potent estrogenic compounds can affect multiple components of the male reproductive system causing impaired development of the epithelium and overgrowth of stromal tissue in the epididymis, vas deferens, seminal vesicles, and prostate. However, very little is known about the direct effects of estrogenic compounds on the anterior pituitary gland. In this study we have investigated the effects of neonatal estrogenic exposure upon the anterior pituitary. Both the early- and late-stage effects of exposure to a synthetic estrogenic agent, diethylstilbestrol (DES), upon pituitary gonadotroph cell function were assessed. We administered either a high dose (10 microg) or a low dose (0.1 microg) of DES to male rats during their neonatal period (P2-12). Gonadotroph function, cell number and morphology shortly after DES treatment (P18) and during adulthood (P90) were assessed. At P18 there was a significant decrease in follicle stimulating hormone (FSH) immunoreactivity in the pituitary gonadotroph cells in the high DES dose treated rats compared to control animals. No significant change in luteinizing hormone (LH) was observed at either DES dose. In adulthood (P90), there was no significant difference in FSH or LH gonadotroph immunoreactivity between control rats and any dose of DES-treated rats. Therefore, despite acute and selective ablation of FSH expression the gonadotrophs were able to recover in adulthood, suggesting that perinatal estrogenic exposure was only temporarily deleterious.

A simple solid phase mass tagging approach for quantitative proteomics.

New mass-tagging reagents for quantitative proteomics measurements have been designed using solid phase peptide synthesis technology. The solid phase mass tags have been used to accurately measure the relative amounts of cysteine-containing peptides in model peptide mixtures as well as in mixtures of tryptic digests in the femtomol range. Measurements were made using both matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) and online reversed-phase capillary liquid chromatography coupled through a nanoelectrospray interface to an ion trap mass spectrometer (capillary LC/ESI-MS). Results of mass-tagging experiments obtained from these two mass spectrometry techniques and their relative advantages and disadvantages for identification and quantitation of mass tagged peptides are compared. These reagents provide a simple, rapid and cost-effective alternative to currently available mass tagging technologies.