Gcn2 rescues reprogramming in the absence of Hog1/p38 signaling in C. neoformans during thermal stress.

The fungus Cryptococcus neoformans is an opportunistic pathogen of people that reprograms its translatome to facilitate adaptation and virulence within the host. We studied the role of Hog1/p38 in reprogramming translation during thermal stress adaptation, and found that this pathway acts on translation via crosstalk with the Gcn2 pathway, a well-studied regulator of general translation control. Using a combination of molecular assays and phenotypic analysis, we show that increased output from the Gcn2 pathway in a Hog1 deletion mutant is associated with rescue of thermal stress adaptation at both molecular and phenotypic scales. We characterize known outputs of the Hog1 pathway during thermal stress as either Gcn2-dependent or Gcn2-independent, and demonstrate that Hog1 activation regulates the Gcn2 pathway even in the absence of thermal stress. Finally, we implicate this phenomenon in another Hog1-regulated process, morphogenesis, and recapitulate Hog1-Gcn2 crosstalk in the distantly related fungal pathogen, Candida albicans. Our results point to an important link between the stress response machinery and translation control, and clarify the etiology of phenotypes associated with Hog1 deletion. More broadly, this study highlights complex interplay between core conserved signal transduction pathways and the utility of molecular assays to better understand how these pathways are connected.

Characterization of a Cleavable Fusion of Human CYP24A1 with Adrenodoxin Reveals the Variable Role of Hydrophobics in Redox Partner Binding.

The improper maintenance of the bioactivated form of vitamin-D (1α,25(OH)2D) may result in vitamin-D insufficiency and therefore compromise the absorption of dietary calcium. A significant regulator of vitamin-D metabolism is the inactivating function of the mitochondrial enzyme cytochrome P450 24A1 (CYP24A1). In humans, CYP24A1 carries out hydroxylation of carbon-23 (C23) or carbon-24 (C24) of the 1α,25(OH)2D side chain, eventually resulting in production of either an antagonist of the vitamin-D receptor (C23 pathway) or calcitroic acid (C24 pathway). Despite its importance to human health, the human isoform (hCYP24A1) remains largely uncharacterized due in part to the difficulty in producing the enzyme using recombinant means. In this study, we utilize a cleavable fusion with the cognate redox partner, human Adx (hAdx), to stabilize hCYP24A1 during production. The subsequent cleavage and isolation of active hCYP24A1 allowed for an investigation of substrate and analog binding, enzymatic activity, and redox partner recognition. We demonstrate involvement of a nonpolar contact involving Leu-80 of hAdx and a nonconserved proximal surface of hCYP24A1. Interestingly, shortening the length of this residue (L80V) results in enhanced binding between the CYP-Adx complex and 1α,25(OH)2D yet unexpectedly results in decreased catalysis. The same mutation has a negligible effect on rat CYP24A1 (a C24-hydroxylase), indicating the presence of a species-specific requirement that may correlate with differences in regioselectivity of the reaction. Taken together, this work presents an example of production of a challenging human CYP as well as providing details regarding hydrophobic modulation of a CYP-Adx complex that is critical to human vitamin-D metabolism.

Neoadjuvant treatment of endometrial cancer using anastrozole: a randomised pilot study.

OBJECTIVE: Excessive oestrogenic stimulation is a well-known risk factor for the development and progression of endometrial cancer. Aromatase is the key enzyme which catalyses the conversion of androgens to oestrogens in postmenopausal women. Inhibition of aromatase may therefore be a useful strategy in the management of endometrial cancer. A pilot study was designed to assess the feasibility of a neoadjuvant model and understand the biological effects of anastrozole, an aromatase inhibitor, in the treatment of endometrial cancer. METHODS: Patients with endometrial cancer who consented to participate in the study were randomised to receive anastrozole or placebo for a minimum of 14 days prior to definitive surgery. Endometrial samples were obtained before and after treatment. Immunohistochemistry was performed to ascertain the expression of oestrogen receptor alpha (ERα), progesterone receptor (PR), androgen receptor (AR), ki-67 and Bcl2 before and after treatment in glands and stroma of the endometrium. RESULTS: A total of 16 patients were randomised to the anastrozole arm and 8 to the placebo arm (2:1 randomisation). A significant decrease in the glandular expression of ERα and AR was observed in the anastrozole arm. There was no significant change in the expression of PR or Bcl2. Expression of ki-67, a proliferation marker, also decreased significantly following treatment with anastrozole. CONCLUSIONS: Treatment with anastrozole caused a significant decrease in proliferation as demonstrated by decreased ki-67 expression. A large randomised controlled trial is warranted to fully assess the role of anastrozole in the neoadjuvant treatment of endometrial cancer.

A robust RNA integrity-preserving staining protocol for laser capture microdissection of endometrial cancer tissue.

Laser capture microdissection of frozen tissue sections allows homogeneous cell populations to be isolated for expression profiling. However, this requires striking a balance between retaining adequate morphology for accurate microdissection and maintaining RNA integrity. Various staining protocols were applied to frozen endometrial carcinoma tissue sections. Although alcohol-based methods were superior to aqueous stains for maintaining RNA integrity, they suffered from irreproducible staining intensity. We developed a modified alcohol-based, buffered cresyl violet staining protocol that provides reproducible staining with minimal RNA degradation suitable for tissues with moderate to high levels of intrinsic RNase activity.

A lower incidence of gynecologic adverse events and interventions with anastrozole than with tamoxifen in the ATAC trial.

OBJECTIVE: Results of the Arimidex, Tamoxifen, Alone or in Combination (ATAC) trial have shown that tamoxifen is associated with a significantly higher incidence of gynecologic adverse events than anastrozole. STUDY DESIGN: This was a retrospective analysis of all gynecologic adverse events and interventions conducted in patients receiving anastrozole or tamoxifen in the main ATAC trial database. RESULTS: Women taking tamoxifen experienced significantly more gynecologic adverse events than those taking anastrozole (34.2% vs 20.5%; P < .0001) and this led to more diagnostic and/or therapeutic interventions, including an almost 4-fold increase in the number of hysterectomies (5.1% vs 1.3%; P < .0001). The majority of the gynecologic adverse events with tamoxifen occurred during the first 2.5 years. CONCLUSION: The lower incidence of gynecologic adverse events and interventions with anastrozole and the early occurrence of these events provide further support for using anastrozole as the initial adjuvant treatment for early hormone receptor-positive breast cancer.

Patient satisfaction with outpatient hysteroscopy performed by nurse hysteroscopists.

OBJECTIVE: To investigate the effect of the introduction of nurse hysteroscopists on patient satisfaction at an outpatient hysteroscopy clinic in the United Kingdom. METHODS: Satisfaction with outpatient hysteroscopy performed in a University Teaching Hospital was measured using an anonymous structured questionnaire in 2000 and 2005. The unpaired t test, Mann-Whitney U test, or chi(2) test was used depending on the level of measurement. RESULTS: A total of 102 women surveyed in 2005 were compared with 139 women surveyed in 2000. Age, ethnicity, perceived health status, previous satisfaction with outpatient appointments, and expectations of the appointment did not differ between the groups. Waiting time for an appointment and once at the clinic fell during the study period (P<0.001); satisfaction increased with the former (P<0.001), but not the latter (P=0.25). Satisfaction with the professional skills of healthcare providers and overall satisfaction was 95% or greater in both years. CONCLUSION: High levels of patient satisfaction with outpatient hysteroscopy were maintained after the introduction of nurse hysteroscopists.

An in vitro uterine perfusion model for investigating endometrial cryoablation.

STUDY OBJECTIVES: To build a simple in vitro uterine perfusion model for investigating the clinical effectiveness of endometrial ablation. DESIGN: Comparative laboratory and in vivo study (Canadian Task Force classification II-2). SETTING: University teaching hospital. PATIENTS: Women undergoing hysterectomy for menorrhagia with uteri of normal shape and size. INTERVENTIONS: A single 5-minute freeze, followed by an active thaw was applied to the endometrial cavity of uteri in vivo and in the in vitro perfusion model. MEASUREMENTS AND MAIN RESULTS: Endometrial/myometrial temperature change was measured continuously during the cryosurgical procedure. Depth of cell death was measured using nicotinamide adenine dinucleotide diaphorase enzyme assay. There was no significant difference in temperature change and depth of cell death in endometrial/myometrial tissue between in vivo and in vitro perfusion experiments. CONCLUSIONS: The in vitro perfusion model described is a useful tool for investigating endometrial cryoablation and has potential for investigating and developing other intrauterine surgical modalities.

Susceptibility of uterine myomas and endometrium to cryosurgery using a carbon dioxide cryosurgical probe in vitro.

STUDY OBJECTIVE: To determine the effect of a 9-mm diameter carbon dioxide cryoprobe, the Endocryo, on myomas and endometrial/myometrial tissue in vitro. DESIGN: Comparative laboratory study (Canadian Task Force classification II-2). SETTING: University laboratory. PATIENTS: Women with and without myomas, undergoing hysterectomy. INTERVENTION: A single 5-minute freeze followed by an active thaw was applied to uterine myomas and endometrial/myometrial tissue in vitro. MEASUREMENTS AND MAIN RESULTS: Endometrial/myometrial and uterine myoma temperature change was measured continuously during the cryosurgical procedure. Depth of cell death was measured using nicotinamide adenine dinucleotide diaphorase enzyme assay. There was no significant difference in temperature change and depth of cell death between myomas and endometrial/myometrial tissue in vitro. CONCLUSIONS: The Endocryo produces the same cryosurgical effect on both uterine myomas and endometrial/myometrial tissue in vitro, an important principal for future development of a clinically effective cryosurgical device for the treatment of menorrhagia in the presence of submucous myomas.

Standard setting for outpatient gynaecology procedures: a multidisciplinary framework for implementation.

This chapter describes the changing cultural background of health care from which any service is delivered. In particular, the authors hope to outline cultural, educational, technical and environmental changes that have been used as opportunities to develop a quality-assessed outpatient hysteroscopic service. Examined within the chapter will be the roles and limitations of evaluation and audit, research and the multidisciplinary team. The importance of process, relationships and collaborative working within organizations will be explored, and outpatient hysteroscopy will be used as a working example of how these inform a model of practice development.

Molecular markers in the endometrium at baseline of postmenopausal patients with early breast cancer in the ATAC (Arimidex, tamoxifen, alone, or in combination) trial.

OBJECTIVE: This study was undertaken to assess baseline endometrial molecular events in the ATAC (Arimidex, tamoxifen, alone, or in combination) trial of breast cancer adjuvant therapy. STUDY DESIGN: Estrogen receptor (ER) and progesterone receptor (PR) levels and markers of cell proliferation (Ki67) and apoptosis ( Bcl -2) were assessed in 93 patients at baseline. RESULTS: An inactive/atrophic endometrium was found in 63 patients, 5 had a proliferative endometrium, and 12 had a secretory endometrium. Thirteen endometrial polyps were analyzed. Inactive endometrium showed high levels of ER in the glandular epithelium, whereas in more than 50% of samples, PR expression was negative or low (+) in the glandular epithelium, and stroma. Ki67 expression was low in both the glandular epithelium and the stroma of the inactive endometrium, whereas Bcl -2 expression was mostly high or very high (+++/++++) in the glandular epithelium. Bcl -2 was strongly expressed (+++/++++) in the glandular epithelium of polyps. CONCLUSION: Although all patients were asymptomatic, some had endometrial pathology.

The differential expression of oestrogen receptors, progesterone receptors, Bcl-2 and Ki67 in endometrial polyps.

OBJECTIVE: To obtain a greater understanding of the pathogenesis of endometrial polyps and to gain insight into which factors play a pivotal role in their growth. DESIGN: Retrospective analysis of archived paraffin-embedded specimens. SETTING: St James's University Hospital. SAMPLE: Thirty secretory phase endometrial samples, 10 secretory phase endometrial polyps, 8 proliferative phase endometrial samples and 10 proliferative phase endometrial polyps. METHODS: Immunohistochemistry was used to characterise the expression of oestrogen and progesterone receptors, Bcl-2 and Ki67 in cycling endometrium and phase-matched endometrial polyps. Patterns of expression were compared between the polyps and the endometrium. MAIN OUTCOME MEASURE: The expression of oestrogen receptors, progesterone receptors, Bcl-2 and Ki67. RESULTS: Three significant differences were found between the endometrium and the polyps. Polyps taken from the proliferative phase of the cycle displayed significantly elevated expression of Bcl-2 and weak or no expression of progesterone receptors. Secretory phase polyps displayed an elevated expression of oestrogen receptors. CONCLUSION: A localised increase in Bcl-2 expression and consequential decline or cessation of apoptosis is an important mechanism underlying the pathogenesis of endometrial polyps. Elevated Bcl-2 expression results in failure of the polyp tissue from undergoing normal cyclical apoptosis during the late secretory phase. This may mean the polyp is not shed along with the rest of the endometrium during menstruation.