Comprehensive mutational scanning of the p53 coding region by two- dimensional gene scanning

A comprehensive mutational scanning test for the p53 coding region based on multiplex PCR and two-dimensional DNA electrophoresis was designed and evaluated. In a 2-step multiplex PCR, the p53 coding region (exons 2-11) was amplified as a single 8646-bp fragment by long- distance PCR in step one. This fragment served as a template for the subsequent co-amplification of the individual exons in two multiplex groups in step two. The multiplex products were then separated, first on the basis of size in non-denaturant polyacrylamide gels and then on the basis of sequence by denaturing gradient gel electrophoresis (DGGE). Primers for optimal PCR, melting behavior and 2-D gel distribution were designed using a recently developed computer program. The resulting two-dimensional gene scanning (TDGS) test was evaluated by screening, in a blinded fashion, 29 coded DNA samples from Li- Fraumeni syndrome patients with previously identified germline mutations. All mutations were correctly detected. This assay provides an accurate, cost-effective and non-radioactive method for simultaneous mutational scanning of all p53 coding exons.      

Absence of 185delAG mutation of the BRCA1 gene and 6174delT mutation of the BRCA2 gene in Ashkenazi Jewish men with prostate cancer.

Epidemiological studies have demonstrated a clustering of breast and prostate cancers in some families. Moreover, there is an increase in the number of cases of prostate cancer in families with inherited mutations of the breast cancer susceptibility gene BRCA1. We assessed the role of BRCA1 and BRCA2 in prostate cancer. We tested for the BRCA1 185delAG frameshift mutation, found in 0.9% of Ashkenazi Jews, and the BRCA2 6174delT mutation, found in 1% of Ashkenazi Jews, in Ashkenazi Jewish men with prostate cancer. We studied 60 Ashkenazi men with prostate cancer. A family history was obtained by interview or a self-report questionnaire. Histological confirmation of diagnosis was obtained for all subjects. Ethnic background was confirmed for all subjects by self-report or interview. Mutations of BRCA1 and BRCA2 were detected by amplification of lymphocyte DNA from peripheral blood according to standard polymerase chain reaction (PCR) and dot blot procedures. Patients'' ages ranged from 55 to 80 years (mean +/- s.d. 70 +/- 5.25). There were six men with a family history of prostate cancer; three of these had a father with prostate cancer. Five of the men had a family history of breast cancer, in a mother, a sister or an aunt. None of the men had a family history of both breast and prostate cancer. None of the 60 men carried the 185delAG BRCA1 or 6174delT BRCA2 mutations. Of 268 Ashkenazi Jewish women with sporadic breast cancer, tested in an unrelated study, 16 carried either the 185delAG mutation of BRCA1 or the 6174delT mutation of BRCA2. There was a significant difference in the incidence of the BRCA1 and BRCA2 mutations in the breast and prostate cancer cases (P = 0.05, two-tailed Fisher''s exact test). The contribution of germline BRCA1 and BRCA2 mutations to prostate cancer incidence is probably small and could be limited to specific subgroups.     

Response of quiescent and total tumor cells in solid tumors to neutrons with various cadmium ratios

Purpose: Response of quiescent (Q) and total tumor cells in solid tumors to neutron irradiation with three different cadmium (Cd) ratios was examined. The role of Q cells in tumor control was also discussed.Methods and Materials: C3H/He mice bearing SCC VII tumors received continuous administration of 5-bromo-2′-deoxyuridine (BrdU) for 5 days using implanted mini-osmotic pumps to label all proliferating (P) cells. Thirty minutes after intraperitoneal injection of sodium borocaptate-¹⁰B (BSH), or 3 h after oral administration of dl-p-boronophenylalanine-¹⁰B (BPA), the tumors were irradiated with neutrons, or those without ¹⁰B-compounds were irradiated with gamma rays. This neutron irradiation was performed using neutrons with three different cadmium (Cd) ratios. The tumors were then excised, minced, and trypsinized. The tumor cell suspensions were incubated with cytochalasin-B (a cytokinesis-blocker), and the micronucleus (MN) frequency in cells without BrdU labeling (Q cells) was determined using immunofluorescence staining for BrdU. The MN frequency in total (P + Q) tumor cells was determined from tumors that were not pretreated with BrdU. The sensitivity to neutrons was evaluated in terms of the frequency of induced micronuclei in binuclear tumor cells (MN frequency).Results: Without ¹⁰B-compounds, the MN frequency in Q cells was lower than that in the total cell population. The sensitivity difference between total and Q cells was reduced by neutron irradiation. Relative biological effectiveness (RBE) of neutrons compared with gamma rays was larger in Q cells than in total cells, and the RBE values for low-Cd-ratio neutrons tended to be larger than those for high-Cd-ratio neutrons. With ¹⁰B-compounds, MN frequency for each cell population was increased, especially for total cells. This increase in MN frequency was marked when high-Cd-ratio neutrons were used. BPA increased the MN frequency for total tumor cells more than BSH. Nevertheless, the sensitivity of Q cells treated with BPA was lower than that in BSH-treated Q cells. This tendency was clearly observed in high-Cd-ratio neutrons.Conclusion: From the viewpoint of enhancing the Q-cell sensitivity, tumors should be irradiated with high-Cd-ratio neutrons after BSH administration. However, normal tissue reaction remains to be examined because of its low tumor-to-normal tissue and tumor-to-blood biodistribution ratios.     

Target-dependent axonal sprouting following vagal-hypoglossal nerve anastomosis in cats

Purpose: To investigate the relationships between the axonal sprouting and target neurotization by central neurons after nerve heterocon-nection. Methods: Unilateral (right) vagal-hypoglossal nerve anastomosis (VHA) was performed in adult cats. Following 3-315 days postoperation (dpo), quantitative analyses and ultrastructural changes in the proximal portion of the vagal-hypoglossal heteroconnected nerve as well as the time course of neuronal regeneration were studied. Along with this, horseradish peroxidase (HRP) retrograde tracing technique was used to label the neurons of dorsal motor vagal nucleus (DMV) and nucleus ambiguus (NA) to ascertain if target neurotization was established. Results: The contralateral (left) intact vagus nerve proximal to the level of ansa cervicalis showed an average of 33 +/- 1 myelinated and 74 +/- 4 unmyelinated axons in 727 &mgr;m(2) sectional area of the nerve. In the heteroconnected nerve at the corresponding level just proximal to the anastomosis site, there was a marked increase in the number of small axons sprouting from the unmyelinated nerve fibers between 18 and 25 dpo. The number of these axonal sprouts appeared to decline at 32 dpo but its increase of 131 % was sustained until the late regeneration stage at 315 dpo when compared with the contralateral nerve serving as a control. The mean number of myelinated axons per area unit (727 &mgr;m(2)) was reduced to 18 at 3 dpo but was immediately restored to the normal range at 7 dpo. The retrograde labelling of neurons in both the DMV and NA was first detected at 22 dpo and was progressively increased peaking by about 67 dpo. Conclusions: We conclude that compared with the unmyelinated axons, the myelinated axons may acquire a superior interaction with the new target. Furthermore, the postoperative neurotization of tongue muscles may initiate and facilitate the retraction of the redundant axonal sprouts.     

金银花药材中抗呼吸道病毒感染的咖啡酰奎宁酸类成分的定量研究

目的:寻找金银花中的抗呼吸道病毒感染的成分,并进行定量研究。方法:采用各种柱层析和制备薄层层析等方法分离、细胞病变法进行抗病毒作用的研究,采用RP-HPLC方法进行定量方法的研究。结果:分离得到了13种咖啡酰奎宁酸类化合物和咖啡酸、咖啡酸甲酯,确认咖啡酰奎宁酸类成分具有较强的抗呼吸道病毒作用,并采用HPLC方法测定了金银花中的 6种咖啡酰奎宁酸类成分和咖啡酸的含量。结论:咖啡酰奎宁酸类成分为金银花中的有效成分,定量方法准确、重复性好。     

Interpretation of Emergency Department radiographs: a comparison of emergency medicine physicians with radiologists, residents with faculty, and film with digital display

OBJECTIVE: We determined the relative value of teleradiology and radiology resident coverage of the emergency department by measuring and comparing the effects of physician specialty, training level, and image display method on accuracy of radiograph interpretation. MATERIALS AND METHODS: A sample of four faculty emergency medicine physicians, four emergency medicine residents, four faculty radiologists, and four radiology residents participated in our study. Each physician interpreted 120 radiographs, approximately half containing a clinically important index finding. Radiographs were interpreted using the original films and high-resolution digital monitors. Accuracy of radiograph interpretation was measured as the area under the physicians' receiver operating characteristic (ROC) curves. RESULTS: The area under the ROC curve was 0.15 (95% confidence interval [CI], 0.10-0.20) greater for radiologists than for emergency medicine physicians, 0.07 (95% CI, 0.02-0.12) greater for faculty than for residents, and 0.07 (95% CI, 0.02-0.12) greater for films than for video monitors. Using these results, we estimated that teleradiology coverage by faculty radiologists would add 0.09 (95% CI, 0.03-0.15) to the area under the ROC curve for radiograph interpretation by emergency medicine faculty alone, and radiology resident coverage would add 0.08 (95% CI, 0.02-0.14) to this area. CONCLUSION: We observed significant differences between the interpretation of radiographs on film and on digital monitors. However, we observed differences of equal or greater magnitude associated with the training level and physician specialty of each observer. In evaluating teleradiology services, observer characteristics must be considered in addition to the quality of image display.     

Oleoylethanolamide inhibits food intake in free-feeding rats after oral administration.

Oleoylethanolamide (OEA) is an endogenous lipid that contributes in important ways to the peripheral regulation of food intake. When administered intraperitoneally, OEA is a potent satiety-inducing anorexiant in rats and mice [Nature 414 (2001) 209; Neuropsycopharmacology 28 (2003) 1311; Nature 425 (2003) 90]. In the present study, we show that oral administration of OEA in pH-sensitive enteric-coated capsules produces a profound and long-lasting inhibition of food intake in free-feeding rats. This effect is accompanied by a marked elevation in OEA levels in the small intestine, but not in brain or muscle.     

Large‐scale bone mineral histomorphometry – report of a simplified technique

Karantzoulis V, Liapi C & Papaggelopoulos P
(2012) Histopathology
Large‐scale bone mineral histomorphometry – report of a simplified technique Aims: The aim of this study was the development of a simplified technique for bone mineral histomorphology on large undecalcified bone samples. Established techniques, such as undecalcified bone thin sectioning, ultrathin grinding, surface‐stained block grinding and micro‐computerized tomography (CT), are expensive, time‐consuming and put very high demands on equipment, safety standards, personnel and laboratory facilities. Methods and results: The method is based on the surface‐stained block‐grinding principle; however, its novelty lies in the selection of user‐friendly, safe and low‐cost materials, equipment and digitization techniques. We describe in detail the relevant steps, as well as many practical tips for their successful implementation: accurate bone cutting in thin sections with a customized arrangement on a commercial bandsaw, defatting with sodium hypochlorite, embedding in epoxy resin blocks at room temperature, silicon carbide paper grinding, von Kossa staining, flatbed scanner digitization and image processing. Conclusion: We believe that the proposed methodology could contribute to the expansion of the study of bone tissue, as it enables the rapid examination of bone specimens on a large scale with minimal laboratory requirements and consumables costs.     

High-Fidelity Simulation Training in Advanced Resuscitation for Pharmacy Residents

Objective. To assess the impact of high-fidelity patient simulation on pharmacy resident knowledge, confidence, and competency with advanced resuscitation algorithms and interventions.Design. An overview of the institutional cardiopulmonary arrest algorithm and a review of pertinent medications and calculations were presented to postgraduate year 1 (PGY1) pharmacy residents, followed by participation in 3 simulated clinical scenarios using a high-fidelity mannequin.Assessment. An improvement of pharmacy resident knowledge, confidence, and competency with advanced resuscitation skills was observed. In addition, pharmacy residents demonstrated high performance levels with skills requiring advanced competency and proactive interactions with the cardiac arrest team.Conclusion. Incorporating high-fidelity patient simulation into an advanced resuscitation training program can help pharmacy residents achieve competency through the active learning of practical skills.