Ultra-high throughput screen of two-million-member combinatorial compound collection in a miniaturized, 1536-well assay format

Results of a complete survey of the more than 2-million-member Pharmacopeia compound collection in a 1536-well microvolume screening assay format are reported. A complete technology platform, enabling the performance of ultra-high throughput screening in a miniaturized 1536-well assay format, has been assembled and utilized. The platform consists of tools for performing microvolume assays, including assay plates, liquid handlers, optical imagers, and data management software. A fluorogenic screening assay for inhibition of a protease enzyme target was designed and developed using this platform. The assay was used to perform a survey screen of the Pharmacopeia compound collection for active inhibitors of the target enzyme. The results from the survey demonstrate the successful implementation of the ultra-high throughout platform for routine screening purposes. Performance of the assay in the miniaturized format is equivalent to that of a standard 96-well assay, showing the same dependence on kinetic parameters and ability to measure enzyme inhibition. The survey screen identified an active class of compounds within the Pharmacopeia compound collection. These results were confirmed using a standard 96-well assay.     

Can Willingness and Hands-on Work Together? Teaching Biological Evolution and Dealing with Barriers

This qualitative research aims at first defining how pre-service science teachers think regarding the theory of evolution (TOE) with a particular hands-on and minds-on activity (The article by O’Brien (Am Biol Teach 62(8):578–582, 2000) entitled A toilet paper timeline of evolution: 5 E cycle on the concept of scale is modified and applied in the present research as an intervention process.) Finally, specific suggestions in instructional techniques are offered based on a geological time scale. At the end of intervention, the research follows how participants respond to what they found out or came across. If participants had common misconceptions regarding the TOE, these are revealed. The present research was conducted in two spring semesters of 2009 and 2010 in a geology course. The participant number of the study was 129. The research lasted three weeks. Research results showed that participants were generally misinformed regarding conceptual knowledge of the TOE. Approximately half of the participants did not believe (the term believing in or acceptance of the TOE does not affect research findings) in the TOE. Mostly, they thought that there is no place for human evolution in Islam. Few of them could reconcile scientific information with their own Islamic point of view. Even though they did not believe in the TOE, most of them accepted that they did not have adequate knowledge regarding the TOE. They mostly said they were shocked about the first appearance of human beings in the geological time scale, which is the main issue in the present research. After intervention, conceptual change was considerable, though this is a secondary concern. The results also show that even though students were quite prejudiced regarding the TOE, they were ready to explore it deeply. Such concrete activities might be helpful to push students to think over the TOE. However, there are numerous barriers such as ill-prepared science textbooks and websites, unwillingness of instructors, and official policy of the Ministry of National Education.     

Effects of Prenatal and Postnatal Exposure to GSM-Like Radiofrequency on Blood Chemistry and Oxidative Stress in Infant Rabbits, an Experimental Study

We aimed to investigate the potential hazardous effects of prenatal and/or postnatal exposure to 1800 MHz GSM-like radiofrequency radiation (RFR) on the blood chemistry and lipid peroxidation levels of infant rabbits. A total of 72 New Zealand female and male white rabbits aged 1-month were used. Thirty-six female and 36 male were divided into four groups which were composed of nine infants: (i) Group 1 were the sham exposure (control), (ii) Group 2 were exposed to RFR, 15 min daily for 7 days in the prenatal period (between 15th and 22nd days of the gestational period) (prenatal exposure group). (iii) Group 3 were exposed to RFR 15 min/day (14 days for male, whereas 7 days for female) after they reached 1-month of age (postnatal exposure group). (iv) Group 4 were exposed to RFR for 15 min daily during 7 days in the prenatal period (between 15th and 22nd days of the gestational period) and 15 min/day (14 days for male, whereas 7 days for female) after they reached 1-month of age (prenatal and postnatal exposure group). Results showed that serum lipid peroxidation level in both female and male rabbits changed due to the RFR exposure. However, different parameters of the blood biochemistry were affected by exposure in male and female infants. Consequently, the whole-body 1800 MHz GSM-like RFR exposure may lead to oxidative stress and changes on some blood chemistry parameters. Studies on RFR exposure during prenatal and postnatal periods will help to establish international standards for the protection of pregnants and newborns from environmental RFR.     

Autologous rabbit adipose tissue-derived mesenchymal stromal cells for the treatment of bone injuries with distraction osteogenesis

Background aimsAdipose tissue-derived mesenchymal stromal cells (MSCs) have a higher capacity for proliferation and differentiation compared with other cell lineages. Although distraction osteogenesis is the most important therapy for treating bone defects, this treatment is restricted in many situations. The aim of this study was to examine the therapeutic potential of adipose tissue-derived MSCs and osteoblasts differentiated from adipose tissue-derived MSCs in the treatment of bone defects.MethodsBone defects were produced in the tibias of New Zealand rabbits that had previously undergone adipose tissue extraction. Tibial osteotomy was performed, and a distractor was placed on the right leg of the rabbits. The rabbits were placed in control (group I), stem cell (group II) and osteoblast-differentiated stem cell (group III) treatment groups. The rabbits were sacrificed, and the defect area was evaluated by radiologic, biomechanical and histopathologic tests to examine the therapeutic effects of adipose tissue-derived MSCs.ResultsRadiologic analyses revealed that callus density and the ossification rate increased in group III compared with group I and group II. In biomechanical tests, the highest ossification rate was observed in group III. Histopathologic studies showed that the quality of newly formed bone and the number of cells active in bone formation were significantly higher in group III rabbits compared with group I and group II rabbits.ConclusionsThese data reveal that osteoblasts differentiated from adipose tissue-derived MSCs shorten the consolidation period of distraction osteogenesis. Stem cells could be used as an effective treatment for bone defects.     

Evaluation of the WHO 2010 Grading and AJCC/UICC Staging Systems in Prognostic Behavior of Intestinal Neuroendocrine Tumors

Background

The increasing incidence and heterogeneous behavior of intestinal neuroendocrine tumors (iNETs) pose a clinicopathological challenge. Our goal was to decribe the prognostic value of the new WHO 2010 grading and the AJCC/UICC TNM staging systems for iNETs. Moreover, outcomes of patients treated with somatostatin analogs were assessed.

Methods

We collected epidemiological and clinicopathological data from 93 patients with histologically proven iNETs including progression and survival outcomes. The WHO 2010 grading and the AJCC/UICC TNM staging systems were applied for all cases. RECIST criteria were used to define progression. Kaplan-Meier analyses for progression free survival (PFS) and overall survival (OS) were performed.

Results

Mean follow-up was 58.6 months (4–213 months). WHO 2010 grading yielded PFS and disease-specific OS of 125.0 and 165.8 months for grade 1 (G1), 100.0 and 144.2 months for G2 and 15.0 and 15.8 months for G3 tumors (p = 0.004 and p = 0.001). Using AJCC staging, patients with stage I and II tumors had no progression and no deaths. Stage III and IV patients demonstrated PFS of 138.4 and 84.7 months (p = 0.003) and disease-specific OS of 210.0 and 112.8 months (p = 0.017). AJCC staging also provided informative PFS (91.2 vs. 50.0 months, p = 0.004) and OS (112.3 vs. 80.0 months, p = 0.005) measures with somatostatin analog use in stage IV patients.

Conclusion

Our findings underscore the complementarity of WHO 2010 and AJCC classifications in providing better estimates of iNETS disease outcomes and extend the evidence for somatostatin analog benefit in patients with metastatic disease.     

A Yeast-Based Chemical Screen Identifies a PDE Inhibitor That Elevates Steroidogenesis in Mouse Leydig Cells via PDE8 and PDE4 Inhibition

A cell-based high-throughput screen (HTS) was developed to detect phosphodiesterase 8 (PDE8) and PDE4/8 combination inhibitors. By replacing the Schizosaccharomyces pombe PDE gene with the murine PDE8A1 gene in strains lacking adenylyl cyclase, we generated strains whose protein kinase A (PKA)-stimulated growth in 5-fluoro orotic acid (5FOA) medium reflects PDE8 activity. From our previously-identified PDE4 and PDE7 inhibitors, we identified a PDE4/8 inhibitor that allowed us to optimize screening conditions. Of 222,711 compounds screened, ∼0.2% displayed composite Z scores of >20. Additional yeast-based assays using the most effective 367 compounds identified 30 candidates for further characterization. Among these, compound BC8-15 displayed the lowest IC₅₀ value for both PDE4 and PDE8 inhibition in in vitro enzyme assays. This compound also displays significant activity against PDE10A and PDE11A. BC8-15 elevates steroidogenesis in mouse Leydig cells as a single pharmacological agent. Assays using BC8-15 and two structural derivatives support a model in which PDE8 is a primary regulator of testosterone production by Leydig cells, with an additional role for PDE4 in this process. BC8-15, BC8-15A, and BC8-15C, which are commercially available compounds, display distinct patterns of activity against PDE4, PDE8, PDE10A, and PDE11A, representing a chemical toolkit that could be used to examine the biological roles of these enzymes in cell culture systems.