Identification of MEN1 gene mutations in sporadic carcinoid tumors of the lung

Lung carcinoids occur sporadically and rarely in association with multiple endocrine neoplasia type 1 (MEN1). There are no well defined genetic abnormalities known to occur in these tumors. We studied 11 sporadic lung carcinoids for loss of heterozygosity (LOH) at the locus of the MEN1 gene on chromosome 11q13, and for mutations of the MEN1 gene using dideoxy fingerprinting. Additionally, a lung carcinoid from a MEN1 patient was studied. In four of 11 (36%) sporadic tumors, both copies of the MEN1 gene were inactivated. All four tumors showed the presence of a MEN1 gene mutation and loss of the other allele. Observed mutations included a 1 bp insertion, a 1 bp deletion, a 13 bp deletion and a single nucleotide substitution affecting a donor splice site. Each mutation predicts truncation or potentially complete loss of menin. The remaining seven tumors showed neither the presence of a MEN1 gene mutation nor 11q13 LOH. The tumor from the MEN1 patient showed LOH at chromosome 11q13 and a complex germline MEN1 gene mutation. The data implicate the MEN1 gene in the pathogenesis of sporadic lung carcinoids, representing the first defined genetic alteration in these tumors.      

High throughput parallel analysis of hundreds of patient samples for more than 100 mutations in multiple disease genes

As more mutations are identified in genes of known sequence, there is a crucial need in the areas of medical genetics and genome analysis for rapid, accurate and cost-effective methods of mutation detection. We have developed a multiplex allele-specific diagnostic assay (MASDA) for analysis of large numbers of samples (> 500) simultaneously for a large number of known mutations (> 100) in a single assay. MASDA utilizes oligonucleotide hybridization to interrogate DNA sequences. Multiplex DNA samples are immobilized on a solid support and a single hybridization is performed with a pool of allele-specific oligonucleotide (ASO) probes. Any probes complementary to specific mutations present in a given sample are in effect affinity purified from the pool by the target DNA. Sequence-specific band patterns (fingerprints), generated by chemical or enzymatic sequencing of the bound ASO(s), easily identify the specific mutation(s). Using this design, in a single diagnostic assay, we tested samples for 66 cystic fibrosis (CF) mutations, 14 beta-thalassemia mutations, two sickle cell anemia (SCA) mutations, three Tay-Sachs mutations, eight Gaucher mutations, four mutations in Canavan disease, four mutations in Fanconi anemia, and five mutations in BRCA1. Each mutation was correctly identified. Finally, in a blinded study of 106 of these mutations in > 500 patients, all mutations were properly identified. There were no false positives or false negatives. The MASDA assay is capable of detecting point mutations as well as small insertion or deletion mutations. This technology is amenable to automation and is suitable for immediate utilization for high-throughput genetic diagnostics in clinical and research laboratories.      

Mosquitocidal properties of Calotropis gigantea (Family: Asclepiadaceae) leaf extract and bacterial insecticide, Bacillus thuringiensis, against the mosquito vectors

Calotropis gigantea leaf extract and Bacillus thuringiensis were tested first to fourth-instar larvae and pupae of Anopheles stephensi, Aedes aegypti, and Culex quinquefasciatus. The medicinal plants were collected from the area around Bharathiar University, Coimbatore, India. Calotropis gigantea leaf was washed with tap water and shade-dried at room temperature. An electrical blender powdered the dried plant materials (leaves). The powder 500 g of the leaf was extracted with 1.5 L of organic solvents of methanol for 8 h using a Soxhlet apparatus and filtered. The crude leaf extracts were evaporated to dryness in a rotary vacuum evaporator. The plant extract showed larvicidal and pupicidal effects after 24 h of exposure; no mortality was observed in the control group. For Calotropis gigantea, the median lethal concentration values (LC(50)) observed for the larvicidal and pupicidal activities against mosquito vector species Anopheles stephensi I to IV larval instars and pupae were 73.77, 89.64, 121.69, 155.49, and 213.79 ppm; Aedes aegypti values were 92.27, 106.60, 136.48, 164.01, and 202.56 ppm; and Culex quinquefasciatus values were 104.66, 127.71, 173.75, 251.65, and 314.70 ppm, respectively. For B. thuringiensis, the LC(50) values of I to IV larval instars and pupae of Anopheles stephensi were 37.24, 45.41, 57.82, 80.09, and 98.34 ppm; Aedes aegypti values were 42.38, 51.90, 71.02, 96.17, and 121.59 ppm; and Culex quinquefasciatus values were 55.85, 68.07, 94.11, 113.35, and 133.87 ppm, respectively. The study proved that the methanol leaf extract of Calotropis gigantea and bacterial insecticide B. thuringiensis has mosquitocidal property and was evaluated as target species of mosquito vectors. This is an ideal ecofriendly approach for the control of vector control programs.     

Laboratory and field evaluation of medicinal plant extracts against filarial vector, Culex quinquefasciatus Say (Diptera: Culicidae)

The present study explored the effects of Jatropha curcas, Hyptis suaveolens, Abutilon indicum, and Leucas aspera tested against third instar larvae of filarial vector, Culex quinquefasciatus. The dried plant materials were powdered by an electrical blender. From each sample, 500 g powder was macerated with 1.5 L of hexane, chloroform, ethyl acetate, and methanol 8h, using Soxhlet apparatus, and filtered. The extracts were concentrated at reduced temperature on a rotary evaporator and stored at a temperature of 4°C. The yield of crude extract was 11.4, 12.2, 10.6, and 13.5 g in hexane, chloroform, ethyl acetate, and methanol, respectively. The hexane, chloroform, ethyl acetate, and methanol extract of J. curcas with LC(50) values of 230.32, 212.85, 192.07, and 113.23 ppm; H. suaveolens with LC(50) values of 213.09, 217.64, 167.59, and 86.93 ppm; A. indicum with LC(50) values of 204.18, 155.53, 166.32, and 111.58 ppm; and L. aspera with LC(50) values of 152.18, 118.29, 111.43, and 107.73 ppm, respectively, against third instar larvae of C. quinquefasciatus. The larval mortality was observed after 24 h of exposure. Maximum larvicidal activity was observed in the methanolic extract followed by ethyl acetate, chloroform, and hexane extract. No mortality was observed in the control. The observed mortality were statistically significant at P?相似文献   

Mannheimia haemolytica leukotoxin-induced cytolysis of ovine (Ovis aries) leukocytes is mediated by CD18, the beta subunit of beta2-integrins

Mannheimia (Pasteurella) haemolytica causes severe pneumonia in cattle, sheep and goats. Leukotoxin (Lkt) is the most important virulence determinant produced by this organism. Previously, we identified CD18, the beta subunit of beta(2)-integrins, as the receptor for Lkt on bovine leukocytes. Since Lkt is specific for leukocytes of cattle, sheep and goats, we hypothesized that Lkt utilizes CD18 as its receptor on ovine leukocytes as well. Therefore, the objective of this study was to transfect an Lkt-resistant murine cell line (P815) with cDNA encoding ovine CD18, and to determine the susceptibility of the transfectants to Lkt-induced cytolysis. cDNA for ovine CD18 cloned from polymorphonuclear leukocytes was transfected into P815 cells. Flow cytometric analysis of the transfectants revealed surface expression of ovine CD18, and Lkt binding. In a cytotoxicity assay, the transfectants were lysed by Lkt in a concentration-dependent manner, whereas the parent cells were not. Pre-incubation of Lkt with an anti-Lkt neutralizing antibody and pre-incubation of transfectants with an anti-CD18 antibody resulted in inhibition of cytolysis confirming the interaction between Lkt and CD18. Taken together, these results indicate that CD18 on ovine leukocytes serves as a receptor for Lkt, and that CD18 is sufficient to mediate Lkt-induced cytolysis of ovine leukocytes.     

A Comparative Study on Morphochemical Properties and Osteogenic Cell Differentiation within Bone Graft and Coral Graft Culture Systems

The objective of this study was to compare the morphological and chemical composition of bone graft (BG) and coral graft (CG) as well as their osteogenic differentiation potential using rabbit mesenchymal stem cells (rMSCs) in vitro. SEM analysis of BG and CG revealed that the pores in these grafts were interconnected, and their micro-CT confirmed pore sizes in the range of 107-315 µm and 103-514 µm with a total porosity of 92% and 94%, respectively. EDS analysis indicated that the level of calcium in CG was relatively higher than that in BG. FTIR of BG and CG confirmed the presence of functional groups corresponding to carbonyl, aromatic, alkyl, and alkane groups. XRD results revealed that the phase content of the inorganic layer comprised highly crystalline form of calcium carbonate and carbon. Atomic force microscopy analysis showed CG had better surface roughness compared to BG. In addition, significantly higher levels of osteogenic differentiation markers, namely, alkaline phosphatase (ALP), Osteocalcin (OC) levels, and Osteonectin and Runx2, Integrin gene expression were detected in the CG cultures, when compared with those in the BG cultures. In conclusion, our results demonstrate that the osteogenic differentiation of rMSCs is relatively superior in coral graft than in bone graft culture system.     

Education initiative improves the evidence‐based use of metoclopramide following morphine administration in the emergency department

Objective: We aimed to evaluate a multifaceted education initiative designed to reduce the prophylactic use of metoclopramide. Methods: This was a pre‐ and post‐intervention trial undertaken in a single ED. All ED doctors and nurses were targeted. The intervention comprised a specifically designed, 19‐slide ‘e‐learning module’, accessible via the ED intranet, supplemented by in‐service training and a range of reminder techniques (posters, emails and drug room flyers). The primary end‐point was the proportion of patients administered metoclopramide prophylactically with their initial morphine dose. Data were collected on random samples of patients who received morphine, using explicit medical chart review. Results: Both pre‐ and post‐intervention periods were of 3 month duration. The charts of 146 cases were reviewed in each period. In the post‐intervention period: ? The proportion of patients administered metoclopramide prophylactically decreased from 22.6% to 4.1% (difference 18.5% [95% CI 10.3–26.7], P < 0.001) ? The proportion of patients administered metoclopramide appropriately (for known morphine sensitivity, established nausea and rescue anti‐emesis) rose marginally from 28.8% to 32.9% (difference 4.1% [95% CI ?7.2–15.4], P = 0.53) ? There was a 12.7% decrease in the number of ampoules of metoclopramide issued to the ED without a concurrent rise in the issue of other anti‐emetic drugs Conclusion: The education initiative resulted in a significant improvement in the evidence‐based use of metoclopramide.