Endocrine responses induced in anestrous goats by the administration of different hormones after a fluorogestone acetate treatment

This study was designed to investigate the endocrinological variations induced in anestrous goats by means of different hormonal stimulations. Twenty goats were divided into four groups and, after treatment for 21 days with fluorogestone acetate (FGA) in vaginal sponges, were treated as follows: (1) vehicle; (2) 500 LU. pregnant mare serum gonadotropin (PMSG) 48 h before sponge removal (s.r.); (3) 500 LU. PMSG 48 h before s.r. and 1 μg gonadotropin-releasing hormone (GnRH) every 3 h for 8 times beginning 3 h before s.r. and (4) an ampoule of human menopausal gonadotropin (HMG) equivalent to 300 LU. luteinizing hormone (LH)-like and 300 LU. follicle-stimulating hormone (FSH)-like activity at s.r. Progesterone, estradiol 17β (E2), LH, FSH and prolactin (PRL) plasma variations were analyzed by validated radioimmunoassays. The stimulation of anestrous goats with FGA alone was inadequate to induce either behavioural estrus or variations in the endocrine pattern. All the other treatments (PMSG, PMSG+ GnRH, HMG) induced an increased in estradiol 17β concentration; the highest E2 levels were induced by PMSG + GnRH treatment. The E2 peaks were followed by LH and FSH surges, which occurred at different times depending on the treatments: the LH peak was significantly (P < 0.001) delayed in HMG-treated does compared with PMSG-GnRH-treated animals. Administration of PMSG alone was not adequate to induce a satisfactory synchronization of the LH peak. No relationship seems to exist between PRL plasma variations and estrus-related endocrine patterns.     

Measurement of Lipid Accumulation in Chlorella vulgaris via Flow Cytometry and Liquid-State 1H NMR Spectroscopy for Development of an NMR-Traceable Flow Cytometry Protocol

In this study, we cultured Chlorella vulgaris cells with a range of lipid contents, induced via nitrogen starvation, and characterized them via flow cytometry, with BODIPY 505/515 as a fluorescent lipid label, and liquid-state ¹H NMR spectroscopy. In doing so, we demonstrate the utility of calibrating flow cytometric measurements of algal lipid content using triacylglyceride (TAG, also known as triacylglycerol or triglyceride) content per cell as measured via quantitative ¹H NMR. Ensemble-averaged fluorescence of BODIPY-labeled cells was highly correlated with average TAG content per cell measured by bulk NMR, with a linear regression yielding a linear fit with r ² = 0.9974. This correlation compares favorably to previous calibrations of flow cytometry protocols to lipid content measured via extraction, and calibration by NMR avoids the time and complexity that is generally required for lipid quantitation via extraction. Flow cytometry calibrated to a direct measurement of TAG content can be used to investigate the distribution of lipid contents for cells within a culture. Our flow cytometry measurements showed that Chlorella vulgaris cells subjected to nitrogen limitation exhibited higher mean lipid content but a wider distribution of lipid content that overlapped the relatively narrow distribution of lipid content for replete cells, suggesting that nitrogen limitation induces lipid accumulation in only a subset of cells. Calibration of flow cytometry protocols using direct in situ measurement of TAG content via NMR will facilitate rapid development of more precise flow cytometry protocols, enabling investigation of algal lipid accumulation for development of more productive algal biofuel feedstocks and cultivation protocols.     

Phase 1 trial of the oral AKT inhibitor MK-2206 plus carboplatin/paclitaxel, docetaxel, or erlotinib in patients with advanced solid tumors

Background

Inhibition of AKT with MK-2206 has demonstrated synergism with anticancer agents. This phase 1 study assessed the MTD, DLTs, PK, and efficacy of MK-2206 in combination with cytotoxic and targeted therapies.

Methods

Advanced solid tumor patients received oral MK-2206 45 or 60 mg (QOD) with either carboplatin (AUC 6.0) and paclitaxel 200 mg/m² (arm 1), docetaxel 75 mg/m² (arm 2), or erlotinib 100 or 150 mg daily (arm 3); alternative schedules of MK-2206 135-200 mg QW or 90-250 mg Q3W were also tested.

Results

MTD of MK-2206 (N?=?72) was 45 mg QOD or 200 mg Q3W (arm 1); MAD was 200 mg Q3W (arm 2) and 135 mg QW (arm 3). DLTs included skin rash (arms 1, 3), febrile neutropenia (QOD, arms 1, 2), tinnitus (Q3W, arm 2), and stomatitis (QOD, arm 3). Common drug-related toxicities included fatigue (68%), nausea (49%), and rash (47%). Two patients with squamous cell carcinoma of the head and neck (arm 1; Q3W) demonstrated a complete and partial response (PR); additional PRs were observed in patients (1 each) with melanoma, endometrial, neuroendocrine prostate, NSCLC, and cervical cancers. Six patients had stable disease ≥6 months.

Conclusion

MK-2206 plus carboplatin and paclitaxel, docetaxel, or erlotinib was well-tolerated, with early evidence of antitumor activity.

Trial registration

ClinicalTrials.gov: NCT00848718.     

Knowledge-based computational methods for identifying or designing novel,non-homologous antimicrobial peptides

We describe computational approaches for identifying promising lead candidates for the development of peptide antibiotics, in the context of quantitative structure–activity relationships (QSAR) studies for this type of molecule. A first approach deals with predicting the selectivity properties of generated antimicrobial peptide sequences in terms of measured therapeutic indices (TI) for known antimicrobial peptides (AMPs). Based on a training set of anuran AMPs, the concept of sequence moments was used to construct algorithms that could predict TIs for a second test set of natural AMPs and could also predict the effect of point mutations on TI values. This approach was then used to design peptide antibiotics (adepantins) not homologous to known natural or synthetic AMPs. In a second approach, many novel putative AMPs were identified from DNA sequences in EST databases, using the observation that, as a rule, specific subclasses of highly conserved signal peptides are associated exclusively with AMPs. Both anuran and teleost sequences were used to elucidate this observation and its implications. The predicted therapeutic indices of identified sequences could then be used to identify new types of selective putative AMPs for future experimental verification.     

Temperature, oxygen, and salt-sensing neurons in C. elegans are carbon dioxide sensors that control avoidance behavior

Homeostatic control of body fluid CO(2) is essential in animals but is poorly understood. C.?elegans relies on diffusion for gas exchange and avoids environments with elevated CO(2). We show that C.?elegans temperature, O(2), and salt-sensing neurons are also CO(2) sensors mediating CO(2) avoidance. AFD thermosensors respond to increasing CO(2) by a fall and then rise in Ca(2+) and show a Ca(2+) spike when CO(2) decreases. BAG O(2) sensors and ASE salt sensors are both activated by CO(2) and remain tonically active while high CO(2) persists. CO(2)-evoked Ca(2+) responses in AFD and BAG neurons require cGMP-gated ion channels. Atypical soluble guanylate cyclases mediating O(2) responses also contribute to BAG CO(2) responses. AFD and BAG neurons together stimulate turning when CO(2) rises and inhibit turning when CO(2) falls. Our results show that C.?elegans senses CO(2) using functionally diverse sensory neurons acting homeostatically to minimize exposure to elevated CO(2).     

Multi-Purpose Utility of Circulating Plasma DNA Testing in Patients with Advanced Cancers

Tumor genomic instability and selective treatment pressures result in clonal disease evolution; molecular stratification for molecularly targeted drug administration requires repeated access to tumor DNA. We hypothesized that circulating plasma DNA (cpDNA) in advanced cancer patients is largely derived from tumor, has prognostic utility, and can be utilized for multiplex tumor mutation sequencing when repeat biopsy is not feasible. We utilized the Sequenom MassArray System and OncoCarta panel for somatic mutation profiling. Matched samples, acquired from the same patient but at different time points were evaluated; these comprised formalin-fixed paraffin-embedded (FFPE) archival tumor tissue (primary and/or metastatic) and cpDNA. The feasibility, sensitivity, and specificity of this high-throughput, multiplex mutation detection approach was tested utilizing specimens acquired from 105 patients with solid tumors referred for participation in Phase I trials of molecularly targeted drugs. The median cpDNA concentration was 17 ng/ml (range: 0.5–1600); this was 3-fold higher than in healthy volunteers. Moreover, higher cpDNA concentrations associated with worse overall survival; there was an overall survival (OS) hazard ratio of 2.4 (95% CI 1.4, 4.2) for each 10-fold increase in cpDNA concentration and in multivariate analyses, cpDNA concentration, albumin, and performance status remained independent predictors of OS. These data suggest that plasma DNA in these cancer patients is largely derived from tumor. We also observed high detection concordance for critical ‘hot-spot’ mutations (KRAS, BRAF, PIK3CA) in matched cpDNA and archival tumor tissue, and important differences between archival tumor and cpDNA. This multiplex sequencing assay can be utilized to detect somatic mutations from plasma in advanced cancer patients, when safe repeat tumor biopsy is not feasible and genomic analysis of archival tumor is deemed insufficient. Overall, circulating nucleic acid biomarker studies have clinically important multi-purpose utility in advanced cancer patients and further studies to pursue their incorporation into the standard of care are warranted.     

Use of inert gas jets to measure the forces required for mechanical gene transfection

Background

Abnormal blood glucose (BG) concentrations have been associated with increased morbidity and mortality in both critically ill adults and infants. Furthermore, hypoglycaemia and glycaemic variability have both been independently linked to mortality in these patients. Continuous Glucose Monitoring (CGM) devices have the potential to improve detection and diagnosis of these glycaemic abnormalities. However, sensor noise is a trade-off of the high measurement rate and must be managed effectively if CGMs are going to be used to monitor, diagnose and potentially help treat glycaemic abnormalities.

Aim

To develop a tool that will aid clinicians in identifying unusual CGM behaviour and highlight CGM data that potentially need to be interpreted with care.

Methods

CGM data and BG measurements from 50 infants at risk of hypoglycaemia were used. Unusual CGM measurements were classified using a stochastic model based on the kernel density method and historical CGM measurements from the cohort. CGM traces were colour coded with very unusual measurements coloured red, highlighting areas to be interpreted with care. A 5-fold validation of the model was Monte Carlo simulated 25 times to ensure an adequate model fit.

Results

The stochastic model was generated using ~67,000 CGM measurements, spread across the glycaemic range ~2-10?mmol/L. A 5-fold validation showed a good model fit: the model 80% confidence interval (CI) captured 83% of clinical CGM data, the model 90% CI captured 91% of clinical CGM data, and the model 99% CI captured 99% of clinical CGM data. Three patient examples show the stochastic classification method in use with 1) A stable, low variability patient which shows no unusual CGM measurements, 2) A patient with a very sudden, short hypoglycaemic event (classified as unusual), and, 3) A patient with very high, potentially un-physiological, glycaemic variability after day 3 of monitoring (classified as very unusual).

Conclusions

This study has produced a stochastic model and classification method capable of highlighting unusual CGM behaviour. This method has the potential to classify important glycaemic events (e.g. hypoglycaemia) as true clinical events or sensor noise, and to help identify possible sensor degradation. Colour coded CGM traces convey the information quickly and efficiently, while remaining computationally light enough to be used retrospectively or in real-time.