Rapid detection of beta-globin gene (HBB) mutations coupling heteroduplex and primer-extension analysis by DHPLC

Beta-thalassemia is a common inherited disease, resulting from one or more of a total of more than 200 different mutations in the beta-globin gene (HBB). Efficient and reliable mutation-screening methods are essential in order to establish appropriate prevention programs for at-risk populations based upon a molecular diagnosis. We have developed a rapid and highly-specific mutation screening test for the diagnosis of beta-thalassemia by coupling heteroduplex and primer-extension analysis based on the denaturing high performance liquid chromatography (DHPLC) system. A total of 161 healthy heterozygous Taiwanese carriers featuring 10 different HBB mutations and 30 patients exhibiting 12 different compound heterozygous or homozygous HBB mutations were subjected to DHPLC. The elution profile for the heteroduplex analysis of DHPLC could be successfully used to identify the common disease-causing mutations of HBB. To further confirm the sequence variants, we developed a technique combining multiplex primer-extension analysis coupled with DHPLC for the genotyping of eight common disease-causing mutations in the HBB gene. Overall, by coupling heteroduplex and primer-extension analysis based upon DHPLC, we were able to unambiguously identify the most-common beta-thalassemia mutations corresponding to more than 99% of HBB alleles among the Taiwanese population. In conclusion, compared to classic approaches to mutation screening for this malady, we suggest that DHPLC is an excellent technique to be applied to the genetic screening of prenatal and postnatal individuals as a part of a diagnosis program for beta-thalassemia and provides a more-efficient, economic, and sensitive means to undertake such a screening program.     

Phase II study of oral ridaforolimus in women with recurrent or metastatic endometrial cancer

Objective

The phosphatidylinositol-3 kinase/serine–threonine kinase PI3K/AKT pathway is postulated to be central to cancer cell development. Activation of this pathway is believed to promote angiogenesis, protein translation and cell cycle progression. A large percentage of endometrial carcinomas have demonstrated mutations within this regulation pathway which result in constitutional activation. The downstream effector protein mammalian target of rapamycin (mTOR) acts as a critical checkpoint in cancer cell cycling and is a logical target for drug development. The efficacy and tolerability of the oral mTOR inhibitor ridaforolimus were evaluated in this study.

Methods

This phase II study evaluated the single agent tolerability and activity of oral ridaforolimus administered at a dose of 40 mg for 5 consecutive days followed by a 2 day break, in women with recurrent or metastatic endometrial carcinoma who had received no chemotherapy in the metastatic setting.

Results

31 of 34 patients were evaluable. Three partial responses (8.8%) were observed with response duration ranging between 7.9 and 26.5 months. An additional 18 patients showed disease stabilization (52.9%) for a median duration of 6.6 months. Response rates were not affected by previous chemotherapy exposure. No correlation was found between response and mutation status.

Conclusion

Oral ridaforolimus was reasonably tolerated and demonstrated modest activity in women with recurrent or metastatic endometrial cancers. Potential synergy between mTOR inhibition, angiogenesis and hormonal pathways warrants ongoing evaluation.     

Multiple spurting test in microsurgical reconstruction of the lower extremities

Appropriate recipient artery is essential for the success of free flap reconstruction, however the selection could be difficult in a traumatized lower extremity. To detect unnoticed damage of the recipient artery, vascular integrity should be verified. For that purpose, we propose a simple and effective evaluation method, called Multiple Spurting Test (MST) this test esteems the dynamic status of the recipient arteries by measuring the length of arterial spurting. All the microsurgeons usually do something like MST: but it is not a standardized test yet.     

Serologic Response to Lower-Molecular-Weight Proteins of H. pylori Is Related to Clinical Outcome of H. pylori Infection in Taiwan

The study aimed to examine the serum serological response among H. pylori-infected patients with various upper gastrointestinal diagnoses; to ascertain whether it could be predictive to the diagnostic outcome of dyspepsia. One hundred seventy H. pylori-infected patients with dyspeptic symptoms but without previous treatment were enrolled, including those with duodenal ulcer disease (N = 47), gastric ulcer (N = 23), nonulcer dyspepsia (N = 60), gastric cancer (N = 34), and MALToma (N = 6). Sera from dyspeptic patients without H. pylori infection (N = 33) were used as controls. During endoscopy, gastric biopsies were taken for CLO-test, histology, and culture for the detection of H. pylori infection, defined by a positive culture or positive results of both CLO-test and histology. Total H. pylori IgG antibody was tested by an ELISA method. Antibody responses to specific H. pylori proteins were tested by a western blotting system. Of patients with H. pylori-infected gastroduodenal diseases, 76.5%, 42.9%, 23.6%, 46.7%, 84.1%, 76.5%, 82.9%, and 32.4% on average, showed responses to the 116-kDa (CagA), 89-kDa (VacA), 60-kDa, 45-kDa, 35-kDa, 30-kDa, 26.5-kDa, and 19.5-kDa H. pylori-specific proteins, respectively. A significant association was found between the serological response to 19.5-kDa and 26.5-kDa proteins and malignant outcome of H. pylori infection (P < 0.02). Among patients without malignancy, the absence of a band at 19.5 kDa was statistically associated with the presence of an ulcer (P < 0.05). The presence of serum antibody against CagA is not different between patients with ulcer and with malignancy in clinical diagnosis. The serum test for detecting antibodies against lower-molecular-weight proteins of H. pylori, such as those of 19.5 and 26.5 kDa, could be useful to identify H. pylori-infected patients at risk of peptic ulcer or malignancy.     

Melatonin prevents pigment gallstone formation induced by bile duct ligation in guinea pigs

Free radical-mediated oxidative stress has been implicated in the genesis of gallstone in vitro. This study was designed to examine the oxidative stress changes during pigment gallstone formation and to investigate whether melatonin (MLT) could act as a chemopreventive agent for cholelithiasis in a guinea pig model. The common bile duct of guinea pigs was ligated with or without MLT pretreatment. Animals were studied on day 7, 9, 12, and 14 after surgery. Stone and/or sludge developed in ligated guinea pigs without MLT. Fourier transform infrared spectra of the sludge showed the presence of calcium bilirubinate, whose peak height per milligram of sludge gradually increased with time after ligation. Total antioxidant activity (TAA) in bile of guinea pigs at day 14 after ligation reduced to one third of the level in sham-operated controls (P <.001). In addition, the bile of ligated guinea pigs had increased pH (P <.001), bile salts (P <.01), and malondialdehyde (MDA) (P <.05), compared to sham controls. Pretreatment of guinea pigs with MLT at a dose of 1,000 microg/kg significantly decreased the incidence of pigment gallstone formation at day 14 after ligation, as compared to no pretreatment (0/7 vs. 8/10). MLT also reverted the ligation-induced changes in biliary bile salts, pH, MDA, and TAA to control levels. These in vivo findings support a causative role of oxidative stress in the bile duct ligation-induced pigment gallstone formation. Antioxidants may prove useful in preventing pigment gallstone formation in humans.     

Morphology of the seagull's inner ear

The morphology of the seagull ear was investigated and found to be generally similar to that of other Aves. Several specific outer middle and inner ear features were found to be sufficiently different as to be remarkable. Most notably, the dual membranes of the external auditory meatus: one, a collecting (and possibly amplifying and reflecting) parabolic membrane chamber situated immediately anterior to the slightly deeper tympanic membrane. The convex tympanic membrane attaches a cartilagenous extracolumella and a thick osseous columella, which is less thin and fragile than that observed in domestic fowl of comparable body size. The columella footplate and round window are completely covered with a second middle ear membrane which apparently serves as an H2O seal and hydrostatic pressure matcher. The short, hard seagull cochlea houses an elaborate basilar papilla which contains thousands of short and tall hair cells, each with a systematic arrangement of stereocilia bundles. In the seagull, the length of the basilar papilla and the length, width, and number of stereocilia, features which affect the transduction process and frequency resolution, are different from those reported for certain other Aves.