Impact of a controlled culture temperature gradient on mouse embryo development and morphokinetics.

RESEARCH QUESTION: Temperature control within the IVF laboratory is an important aspect of a quality control system, helping to reduce environmental stress and ensure good-quality embryo development. Temperature fluctuations are probably more common than expected and the optimal temperature for embryo culture is not known. Modern incubators offer the opportunity to examine the impact of culture temperature on preimplantation embryo development while controlling for other variables within the system. The purpose of this study was to examine the effect of a range of temperatures during extended embryo culture on resulting mouse embryo development and morphokinetic timings. METHODS: Using a single time-lapse incubator with six individual chambers, frozen-thawed one-cell mouse embryos were cultured individually at temperatures adjusted by 0.5°C between chambers to cover the range of 35.0-37.5°C. Resulting blastocyst formation and embryo morphokinetic timings were recorded and compared. RESULTS: Changes in culture temperature had a significant impact on mouse blastocyst development and morphokinetic timings (P < 0.05). Under the conditions used in this study, blastocyst development was best at 37.0°C. Mouse preimplantation embryo mitotic cell divisions were generally slower at cooler temperatures and accelerated as the temperature increased from 35.0°C to 37.5°C. CONCLUSION: Incubator culture temperature must be carefully controlled, as even slight variations of 0.5°C in the temperature used for extended embryo culture can have significant impacts on embryo development and mitotic cell divisions. These data have potential implications for application of universal morphokinetic selection algorithms and may help explain differences in mitotic errors/embryo mosaicism between laboratories.

Temporary Percutaneous Mechanical Circulatory Support in Advanced Heart Failure.

Cardiogenic shock is severe, refractory heart failure caused by significant myocardial dysfunction in the setting of adequate preload that is accompanied by systemic hypoperfusion. Progressive end-organ dysfunction is a hallmark of persistent cardiogenic shock and necessitates intervention to overcome altered hemodynamics and restore end-organ perfusion. Temporary percutaneous mechanical circulatory support is an established modality in the treatment of cardiogenic shock and is increasingly used in patients with cardiogenic shock as a bridge to recovery or further definitive therapy. This article reviews the current devices, their effects on left ventricular hemodynamics, and the evidence supporting their continued use.

The effects of H-bonding and sterics on the photoreactivity of a trimethyl butyrophenone derivative.

The irradiation of ester 1 in methanol and chloroform does not yield any photoproducts, whereas the photolysis of 1 in dry argon-saturated benzene produces cyclobutanol 4, which is converted to lactone 5 by the addition of HCl. Laser-flash photolysis of ester 1 demonstrates that 1 undergoes intramolecular H-atom abstraction to form the biradical 2 (λ(max)∼ 310 nm, τ = 200 ns, benzene), which intersystem crosses to photoenols, Z-3 (λ(max)∼ 380 nm, τ = 30-60 µs, benzene) and E-3 (λ(max)∼ 380 nm, τ = 11 ms, benzene). Density functional theory calculations were performed to support the proposed mechanism for forming cyclobutanol 4 and to explain how steric demand facilitates photoenol E-3 to form cyclobutanol 4 rather than lactone 5.

Short-term mechanical management of cardiogenic shock.

OPINION STATEMENT: Cardiogenic shock (CS), a state of cardiac dysfunction that results in systemic hypoperfusion and end-organ dysfunction, is associated with high in-hospital mortality. Various forms of mechanical circulatory support have been used to treat CS. First employed in the 1960s, the intra-aortic balloon pump (IABP) has been a mainstay in the treatment of acute CS. However, the IABP is unable to provide adequate support in many patients, and newer technologies, including extracorporeal membrane oxygenation and percutaneous ventricular assist devices, appear to be more effective in reversing CS. These devices are also useful for supporting patients during complex percutaneous coronary intervention. Perhaps most importantly, they can be used as a bridge to decision or definitive therapy in CS patients who are potential candidates for surgical ventricular assist devices or cardiac transplantation.

Conditional loss of ERK1 and ERK2 results in abnormal placentation and delayed parturition in the mouse.

Extracellular-signal-regulated kinases (ERK) 1 and 2 regulate many aspects of the hypothalamic-pituitary-gonadal axis. We sought to understand the role of ERK1/2 signaling in cells expressing a Cre allele regulated by the endogenous GnRHR promoter (GRIC-ERKdko). Adult female GRIC-ERKdko mice were hypogonadotropic and anovulatory. Gonadotropin administration and mating led to pregnancy in one-third of the ERKdko females. Litters from ERKdko females and pup weights were reduced coincident with delayed parturition and 100% neonatal mortality. Based on this, we examined Cre expression in implantation sites as a potential mechanism. GnRHR mRNA levels at e10.5 and e12.5 were comparable to pituitary levels from adult female mice at proestrus and GnRHR mRNA in decidua was enriched compared to whole implantation site. In vivo studies confirmed recombination in decidua, and GRIC-ERKdko placentas showed reduced ERK2 expression. Histopathology revealed abnormalities in placental architecture in the GRIC-ERKdko animals. Regions of apoptosis at the decidual/uterine interface at e18.5 were observed in control animals but apoptotic tone in these regions was reduced in ERKdko animals. These studies support a potential model of ERK-dependent signaling within the implantation site leading to loss of placental architecture and mis-regulation of apoptotic events at parturition occurring coincident with prolonged gestation and neonatal mortality.

(1-Bromo-naphthalen-2-yl)acetonitrile.

The title compound, C(12)H(8)BrN, was prepared as a starting material for a Suzuki cross-coupling reaction with a pinacol ester. The torsion angle about the ring-methylene C-C bond is 30.7 (3)°, such that the N atom is displaced by 1.174 (4) Šfrom the plane of the naphthalene ring system.

Sex- and Age-Specific Impact of ERK Loss Within the Pituitary Gonadotrope in Mice.

Extracellular signal-regulated kinase (ERK) signaling regulates hormone action in the reproductive axis, but specific mechanisms have yet to be completely elucidated. In the current study, ERK1 null and ERK2 floxed mice were combined with a gonadotropin-releasing hormone receptor (GnRHR)-internal ribosomal entry site-Cre (GRIC) driver. Female ERK double-knockout (ERKdko) animals were hypogonadotropic, resulting in anovulation and complete infertility. Transcript levels of four gonadotrope-specific genes (GnRHR and the three gonadotropin subunits) were reduced in pituitaries at estrus in ERKdko females, and the postcastration response to endogenous GnRH hyperstimulation was blunted. As females aged, they exhibited abnormal ovarian histology, as well as increased body weight. ERKdko males were initially less affected, showing moderate subfertility, up to 6 months of age. Male ERKdko mice also displayed a blunted response to endogenous GnRH following castration. By 12 months of age, ERKdko males had reduced testicular weights and sperm production. By 18 months of age, the ERKdko males displayed reduced testis and seminal vesicle weights, marked seminiferous tubule degeneration, and a 77% reduction in sperm production relative to controls. As the GRIC is also active in the male germ line, we examined the specific role of ERK loss in the testes using the stimulated by retinoic acid 8 (Stra8)-Cre driver. Whereas ERK loss in GRIC and Stra8 males resulted in comparable losses in sperm production, seminiferous tubule histological degeneration was only observed in the GRIC-ERKdko animals. Our data suggest that loss of ERK signaling and hypogonadotropism within the reproductive axis impacts fertility and gonadal aging.

Novel Insights into Gonadotropin-Releasing Hormone Action in the Pituitary Gonadotrope.

The hypothalamic-pituitary-gonadal axis controls reproduction via a series of hormones regulating gonadal function through interconnected feedback loops. Secretion of hypothalamic-derived gonadotropin-releasing hormone (GnRH) integrates inputs from higher brain centers to coordinate the activity of the pituitary gonadotrope and the biosynthesis and secretion of the gonadotropins which ultimately regulate gonadal function. Failure of GnRH to serve as the central integrator of this system has been associated with hypogonadotropic-hypogonadism and clinical infertility, while pharmacological application of GnRH analogs and gonadotropins have important implications of the treatment of such infertility. Furthermore, the GnRH-GnRH receptor system has been characterized in several types of cancer and may offer therapeutic possibilities in their treatment. Given the central role of GnRH action in the control of fertility, it is of paramount importance to understand the molecular basis of control of GnRH action in the pituitary gonadotrope, including new and novel alternate ways to modulate GnRH action and gonadotropin secretion. The goal of this review is to discuss several new findings in this field focusing on novel regulators of GnRH action.

The F0F1 ATP Synthase Complex Localizes to Membrane Rafts in Gonadotrope Cells.

Fertility in mammals requires appropriate communication within the hypothalamic-pituitary-gonadal axis and the GnRH receptor (GnRHR) is a central conduit for this communication. The GnRHR resides in discrete membrane rafts and raft occupancy is required for signaling by GnRH. The present studies use immunoprecipitation and mass spectrometry to define peptides present within the raft associated with the GnRHR and flotillin-1, a key raft marker. These studies revealed peptides from the F0F1 ATP synthase complex. The catalytic subunits of the F1 domain were validated by immunoprecipitation, flow cytometry, and cell surface biotinylation studies demonstrating that this complex was present at the plasma membrane associated with the GnRHR. The F1 catalytic domain faces the extracellular space and catalyzes ATP synthesis when presented with ADP in normal mouse pituitary explants and a gonadotrope cell line. Steady-state extracellular ATP accumulation was blunted by coadministration of inhibitory factor 1, limiting inorganic phosphate in the media, and by chronic stimulation of the GnRHR. Steady-state extracellular ATP accumulation was enhanced by pharmacological inhibition of ecto-nucleoside triphosphate diphosphohydrolases. Kisspeptin administration induced coincident GnRH and ATP release from the median eminence into the hypophyseal-portal vasculature in ovariectomized sheep. Elevated levels of extracellular ATP augmented GnRH-induced secretion of LH from pituitary cells in primary culture, which was blocked in media containing low inorganic phosphate supporting the importance of extracellular ATP levels to gonadotrope cell function. These studies indicate that gonadotropes have intrinsic ability to metabolize ATP in the extracellular space and extracellular ATP may serve as a modulator of GnRH-induced LH secretion.

Patterned changes in urge ratings with tic suppression in youth with chronic tic disorders.

BACKGROUND: Premonitory urges are central to emerging behavioral models of chronic tic disorders (CTD). Urge reduction has been proposed as a behavioral explanation for tic maintenance and exacerbation as well as the efficacy of behavioral treatments. Prior investigations have produced inconsistent findings despite common methodologies. The current study evaluated the possibility that data aggregation obscures distinct and meaningful patterns of change in urge ratings when tics are freely expressed versus suppressed. METHOD: Participants (n = 12) included children with moderate-to-marked tic severity and noticeable premonitory urges. Tic frequencies and urge ratings were obtained at 15 s and 10-s intervals, respectively, across an alternating sequence of 10-min tic freely and 40-min tic suppression conditions. Patterns were established using a two step approach. RESULTS: Five distinct patterns of urge rating change emerged, suggesting data aggregation may obscure meaningful patterns in the urge-tic relationship when tics are completed versus suppressed. LIMITATIONS: Eligibility criteria may have unintentionally excluded younger affected children and included older participants with more severe tic disorders than commonly seen. Additional research with less stringent eligibility criteria and a larger sample size will help validate the results. CONCLUSIONS: The relationship between urges and tics is much more complex than previously theorized. Investigations that rely on global assessments of urge and tic severity and/or assume uniformity when aggregating participant data may obscure meaningful differences in the urge-tic relationship. Future investigations should examine the possibility that individual differences and/or developmental considerations modulate the functional urge-tic relationship.

Identifying Regulators of Morphogenesis Common to Vertebrate Neural Tube Closure and Caenorhabditis elegans Gastrulation.

Neural tube defects including spina bifida are common and severe congenital disorders. In mice, mutations in more than 200 genes can result in neural tube defects. We hypothesized that this large gene set might include genes whose homologs contribute to morphogenesis in diverse animals. To test this hypothesis, we screened a set of Caenorhabditis elegans homologs for roles in gastrulation, a topologically similar process to vertebrate neural tube closure. Both C. elegans gastrulation and vertebrate neural tube closure involve the internalization of surface cells, requiring tissue-specific gene regulation, actomyosin-driven apical constriction, and establishment and maintenance of adhesions between specific cells. Our screen identified several neural tube defect gene homologs that are required for gastrulation in C. elegans, including the transcription factor sptf-3. Disruption of sptf-3 in C. elegans reduced the expression of early endodermally expressed genes as well as genes expressed in other early cell lineages, establishing sptf-3 as a key contributor to multiple well-studied C. elegans cell fate specification pathways. We also identified members of the actin regulatory WAVE complex (wve-1, gex-2, gex-3, abi-1, and nuo-3a). Disruption of WAVE complex members reduced the narrowing of endodermal cells' apical surfaces. Although WAVE complex members are expressed broadly in C. elegans, we found that expression of a vertebrate WAVE complex member, nckap1, is enriched in the developing neural tube of Xenopus. We show that nckap1 contributes to neural tube closure in Xenopus. This work identifies in vivo roles for homologs of mammalian neural tube defect genes in two manipulable genetic model systems.

Do opioids evoke the release of serotonin in the spinal cord? An in vivo microdialysis study of the regulation of extracellular serotonin in the rat.

This study investigated the regulation of serotonin (5-HT) and its major metabolite 5-hydroxyindoleacetic acid (5-HIAA) in the dorsal spinal cord of awake, freely moving rats, using microdialysis coupled to HPLC with electrochemical detection and tested the hypothesis that opioids exert their analgesic effect in part through the increased release of 5-HT in the dorsal horn. A dialysis tube was placed transversely at the L4 segment of the dorsal spinal cord and the basal concentration of 5-HT in the dialysate was characterized by infusion of a variety of substances through the dialysis probe: tetrodotoxin (TTX), KCl, imipramine, fluoxetine and amphetamine (AMPH). To evaluate the contribution of opioids, we also studied the effects of either systemic or intracerebroventricular (i.c.v.) injection of morphine or DAMGO. Extracellular concentrations of 5-HT and 5-HIAA were partially and reversibly reduced by TTX. In the presence of KCl, imipramine, fluoxetine or AMPH, 5-HT levels significantly increased. Under these conditions, extracellular 5-HIAA levels usually decreased. By contrast, the effects of opioids on 5-HT concentrations were highly variable. Low doses of morphine administered systemically increased 5-HT concentrations in only 3 of 6 rats. This was paralleled by a decrease in 5-HIAA. Higher doses of morphine, alone or in the presence of fluoxetine, did not change 5-HT concentrations. Intracerebroventricular injection of morphine or DAMGO increased the extracellular concentrations of 5-HT in only about one third of the animals. After intracerebroventricular opioid injection, extracellular concentrations of 5-HIAA either decreased by about 20% or did not change.(ABSTRACT TRUNCATED AT 250 WORDS)

Assessing the co-occurring capability of mental health treatment programs: the Dual Diagnosis Capability in Mental Health Treatment (DDCMHT) Index.

The Dual Diagnosis Capability in Mental Health Treatment (DDCMHT) Index was developed to assess the capability of mental health programs to provide substance abuse and co-occurring treatment services. The DDCMHT is an objective scale rated following a site visit that includes semi-structured interviews with staff at all levels, review of program documents and client charts, and ethnographic observation of the milieu and setting. Using data from 67 mental health programs across six states, this study found that the DDCMHT had excellent total score reliability, variable subscale reliability, high inter-rater reliability (n = 18), and moderate construct validity (n = 22). Results also suggest that many mental health programs are at a relatively low level of capability for the delivery of care to individuals with co-occurring disorders. Results from this important new benchmark measure, the DDCMHT, can be used with programs in implementation planning and with treatment systems, states, or national organizations to guide policy change.

Congenital craniopharyngioma and hypercalcemia induced by parathyroid hormone-related protein.

OBJECTIVE: To report a case of congenital craniopharyngioma and parathyroid hormone-related protein (PTHrP)-associated humoral hypercalcemia. METHODS: Details of this unusual case are reviewed, from detection of fetal hydrocephalus and a brain tumor, through cesarean delivery at 36 weeks of gestation, to subsequent laboratory studies, management, and confirmation of the diagnosis. RESULTS: Although PTHrP has been well documented as a cause of humoral hypercalcemia of malignancy (HHM) in adult patients with cancer, HHM is uncommon in children. In addition, HHM has rarely been ascribed to nonmalignant tumors. To the best of our knowledge, we report the first case of a neonate with congenital craniopharyngioma and refractory hypercalcemia (peak ionized calcium level of 1.92 mmol/L; normal, 1.05 to 1.3) attributed to an elevated PTHrP value of 8.6 pmol/L (normal, less than 4.7). Intact parathyroid hormone was appropriately undetectable (less than 10 pg/mL; normal, 15 to 65). Despite calcitonin treatment, the hypercalcemia persisted. Although pamidronate infusion stabilized the serum calcium level, the baby did not survive. CONCLUSION: The diagnosis of craniopharyngioma was confirmed at autopsy, and immunohistochemical studies substantiated that the craniopharyngioma produced PTHrP.

Multidimensional Outcomes Analysis of Intrathecal, Oral Opioid, and Behavioral-Functional Restoration Therapy for Failed Back Surgery Syndrome: A Retrospective Study With 4 Years' Follow-up.

Study Design. A retrospective design comparing three matched groups was used to evaluate the application of a multidimensional approach to outcomes analysis using a variety of disease-specific and generic outcome measures to assess three treatments for failed back surgery syndrome. Objective. The objective of this study was to explore the use of a multidimensional analysis of outcomes to compare and contrast the effects of three different treatments: 1) intrathecal therapy using an implantable drug administration system (DAS), 2) standard medical therapy emphasizing the use of oral opioids (OO), and 3) residential pain and rehabilitation program (RPRP) for the treatment of chronic low back pain. Summary of Background Data. The incidence of low back pain in patients with prior back surgery remains significant. Treatments for failed back surgery syndrome include interventional, pharmacologic, and functional restorative therapies. Outcome studies have rarely compared these three treatments over an extended follow-up period using both disease-specific and generic outcome measures. This study examined three groups of patients completing treatment in clinical setting with a 4-year follow-up. Methods. Patients were selected from three different clinical treatment populations: DAS, OO, and RPRP. The three groups consisted of 40-50 patients each, matched on a number of demographic and pain variables. The average duration of follow-up for each group was about 4 years. Data were collected on disease-specific outcome variables using Numerical Pain Ratings, the McGill Pain Questionnaire, the Beck Depression Inventory, the Oswestry Disability Scale, medication utilization, and other generic outcomes such as SF-36, Quality of Well-Being, Overall Improvement in Pain, and Patient Satisfaction. Information was obtained via chart review and telephone interviews with patients by a disinterested third party. Analysis of variance, chi-square test, and t-test were performed on the various dependent measures. Results. The DAS group appeared to be superior to the OO and RPRP groups on 8 of 11 dependent measures, but only one dependent measure, the posttreatment numerical pain rating, was statistically significant. The RPRP group reported the highest Quality of Well-Being score, while the OO group had the highest level of patient satisfaction. Overall, each of the groups fell well below the "normal" range on generic measures, even when compared to other clinical populations. Measures of depression, subjective disability, and pain remained in the "moderate" range with little evidence of improved functioning or quality of life (QoL). Despite these findings, patients rated their overall improvement at 50-60% and reported high levels of satisfaction with their treatment. Conclusions. The interpretation as to the most effective treatment depended on the particular outcome measure emphasized. There appeared to be a "disconnect" between ratings of pain, disability, mood, and QoL. The use of a multidimensional outcomes approach revealed a number of inconsistencies in the data, which could have been overlooked using only pain ratings and patient satisfaction data. No one treatment emerged as the most effective across all of the disease-specific and generic measures. However, patients in the DAS group tended to report greater improvement. Overall, although generally "satisfied" with treatment, they were generally "satisfied" with treatment despite continuing to report significant levels of pain, disability, and impaired QoL.

PTEN blocks insulin-mediated ETS-2 phosphorylation through MAP kinase, independently of the phosphoinositide 3-kinase pathway.

The tumor suppressor PTEN possesses lipid and protein phosphatase activities. It has been well established that the lipid phosphatase activity is essential for its tumor-suppressive function via the phosphoinositide 3-kinase (PI3K) and Akt pathways. The precise role of the protein phosphatase activity is still unclear. In the current study, we demonstrate that overexpression of wild-type PTEN in the MCF-7 breast cancer line results in phosphatase activity-dependent decreases in the phosphorylation of ETS-2, which is a transcription factor whose DNA-binding ability is controlled by phosphorylation. Exposure of MCF-7 cells to insulin, insulin-like growth factor 1 (IGF-1) or epidermal growth factor (EGF) can lead to the phosphorylation of ETS-2, Akt and ERK1/2. The MEK inhibitor PD590089 abrogates insulin-stimulated phosphorylation of ETS-2. In contrast, the PI3K inhibitor LY492002 has no effect on insulin-stimulated phosphorylation of ETS-2, despite the fact that it diminishes insulin-stimulated phosphorylation of Akt. Interestingly, overexpression of PTEN in MCF-7 leads to blockade of insulin-stimulated, but not EGF-stimulated, phosphorylation of ERK, accompanied by dramatic decreases in ETS-2 phosphorylation. We further show that the relationship of PTEN and ETS-2 has functional significance by demonstrating that PTEN abrogates activation of the uPA Ras-responsive enhancer, a target of ETS-2 action, in a phosphatase-dependent manner, irrespective of the presence or absence of insulin. Our observations, therefore, suggest that PTEN blocks insulin-stimulated ETS-2 phosphorylation through inhibition of the ERK members of the MAP kinase family independently of PI3K, and that the PTEN effect on the phosphorylation status of ETS-2 may be mediated through PTEN's protein phosphatase activity.

Zebrafish pronephros: a model for understanding cystic kidney disease.

The embryonic kidney of the zebrafish is the pronephros. The ease of genetic analysis and experimentation in zebrafish, coupled with the simplicity of the pronephros, make the zebrafish an ideal model system for studying kidney development and function. Several mutations have been isolated in zebrafish genetic screens that result in cyst formation in the pronephros. Cloning and characterization of these mutations will provide insight into kidney development but may also provide understanding of the molecular basis of cystic kidney diseases. In this review, we focus on the zebrafish as a model for understanding cystic kidney disease and the links between cystic kidney disease and left-right patterning.