Preimplantation genetic diagnosis using combined strategies on a breast cancer patient with a novel genomic deletion in BRCA2

Purpose

To perform Preimplantation Genetic Diagnosis (PGD) on a paternal Brca2 unknown mutation carrier with early-onset breast cancer, whose paternal grandmother and mother had breast cancer at 60s.

Method

Elucidating the linkage via single sperm haplotyping on patient''s carrier brother, and identifying the genomic deletion via BLAST followed by PCR screening. PGD was subsequently conducted.

Result

The mutant allele was found by using 4 microsatellite and 2 intragenic SNP markers. Recombination was detected in 8 % of sperms. BLAST was utilized to locate putative hairpin structure(s), followed by PCR screening with seven sets of primers. A novel 2,596 bp deletion containing exon 15 ~ 16 was identified. Due to the severity of phenotype and the integrity of exon 11 encoding RAD51 binding domain, and the fact that the patient''s mother also had breast cancer at her 60s, we speculate a possible coexistence of maternal breast cancer risk allele(s). Embryo biopsy was performed on day 3. Unaffected morula and blastocyst were replaced on day 5, resulting in a singleton livebirth. A breast lump appeared in the patient after delivery without the presence of malignant cells.

Conclusion

Concerning the assisted reproductive option for breast cancer patients, the possibility of coexistence of multiple familial risk alleles and the significance of each mutation to the phenotype should be evaluated. To eliminate misdiagnosis resulting from recombination and/or allelic drop-out, both direct mutation detection and linkage analysis approaches may be necessary. BLAST is a very useful and cost-effective tool for identifying large genomic deletion.     

Use of Modeling and Simulation Tools for Understanding the Impact of Formulation on the Absorption of a Low Solubility Compound: Ciprofloxacin

This study explored the utility of mechanistic absorption models to describe the in vivo performance of a low solubility/low permeability compound in normal healthy subjects. Sixteen healthy human volunteers received three oral formulations and an intravenous infusion in a randomized crossover design. Plasma ciprofloxacin concentrations were estimated by HPLC. In vitro ciprofloxacin release from the oral tablets was tested under a variety of conditions. A mechanistic model was used to explore in vivo dissolution and intestinal absorption. Although dissolution rate influenced the location of drug release, absorption challenges appeared to be associated with permeability limitations in the lower small intestine and colon. The apparent relationship between drug solubilization within the upper small intestinal and formulation overall bioavailability suggested the presence of an intestinal absorption window in many individuals. Failure to absorb drug within this window appeared to be linked with the likelihood of in vivo drug precipitation. Challenges encountered during this modeling exercise included large intersubject variability in product in vivo dissolution and the apparent limitations in ciprofloxacin absorption. Although transporter activity was not included as a model parameter, this evaluation demonstrated how identifying the location of drug absorption across several formulations provided an opportunity to identify factors to consider when formulating similar low solubility/low permeability compounds. The use of mechanistic models was invaluable for our understanding of in vivo product performance and for the assessment of individual profiles rather than means. The latter was essential for understanding the potential challenges that may be encountered when introducing a formulation into a patient population.     

Characterization of vascular complications in experimental model of fructose-induced metabolic syndrome

Vascular dysfunction is an important complication associated with metabolic syndrome (MS). Here we fully characterized vascular complications in a rat model of fructose-induced MS. MS was induced by adding fructose (10%) to drinking water to male Wistar rats of 6 weeks age. Blood pressure (BP) and isolated aorta responses phenylephrine (PE), KCl, acetylcholine (ACh), and sodium nitroprusside (SNP) were recorded after 6, 9, and 12 weeks of fructose administration. In addition, serum levels of glucose, insulin, uric acid, tumor necrosis factor α (TNFα), lipids, advanced glycation end products (AGEs), and arginase activity were determined. Furthermore, aortic reactive oxygen species (ROS) generation, hemeoxygenase-1 expression, and collagen deposition were examined. Fructose administration resulted in a significant hyperinslinemia after 6 weeks which continued for 12 weeks. It was also associated with a significant increase in BP after 6 weeks which was stable for 12 weeks. Aorta isolated from MS animals showed exaggerated contractility to PE and KCl and impaired relaxation to ACh compared with control after 6 weeks which were clearer at 12 weeks of fructose administration. In addition, MS animals showed significant increases in serum levels of lipids, uric acid, AGEs, TNFα, and arginase enzyme activity after 12 weeks of fructose administration. Furthermore, aortae isolated from MS animals were characterized by increased ROS generation and collagen deposition. In conclusion, adding fructose (10%) to drinking water produces a model of MS with vascular complications after 12 weeks that are characterized by insulin resistance, hypertension, disturbed vascular reactivity and structure, hyperuricemia, dyslipidemia, and low-grade inflammation.     

马兜铃酸的倍半萜的酯类化合物VI.绵毛马兜铃中马兜铃酸萜酯I的结构鉴定

从绵毛马兜铃(Aristolocha mannisima Hance)中分得一个马兜铃酸倍半萜的酯类化合物,经红外、紫外、高分辩质谱和多种一维和二维核磁共振谱鉴定,确定了其骨架结构及顺反构型,命名为马兜铃酸萜酯I。     

Investigation of nitrogen balance in dairy cows and steers nourished by intragastric infusion. Effects of submaintenance energy input with or without protein

Two dairy cows were maintained by intragastric infusion of volatile fatty acids and casein. Except when fasting, the casein-nitrogen was held constant, while total gross energy supply was varied from zero during fasting to 650 kJ/kg body-weight (W)0 . 75. One cow was estimated to attain zero N balance at an energy intake of 255 kJ/kg W0 . 75 and the other at 307 kJ/kg W0 . 75, which was calculated to be substantially below the estimated energy required for zero energy balance. When the cows were later given an N-free infusion for a period preceding the trial, N balance occurred at 98 kJ/kg W0 . 75 for one cow and 115 kJ/kg W0 . 75 for the other. Four steers were similarly nourished by intragastric infusion and the energy nutrient increased from 0 at fasting to 450 kJ/kg W0 . 75. The protein was held constant at 1 g N/kg W0 . 75 except at fasting. The energy level at which N balance occurred was 154 (SE 38) kJ/kg W0 . 75 or approximately equal to the energy content of the protein. The practical implications of these findings are discussed.     

Chrysin and Luteolin Attenuate Diabetes‐Induced Impairment in Endothelial‐Dependent Relaxation: Effect on Lipid Profile,AGEs and NO Generation

Chrysin and luteolin are two important plant flavonoids. In the present study, we hypothesized that they protect against deleterious vascular effects of diabetes. Diabetes was induced in rats by streptozotocin (STZ) injection, while chrysin and luteolin were administered two weeks after STZ administration for 6 weeks. Then, blood pressure (BP) and serum levels of glucose, advanced glycation end products (AGEs), triglycerides (TGs), total cholesterol and low density lipoprotein–cholesterol (LDL‐C) were determined. Concentration response curves for KCl, phenylephrine (PE), acetylcholine (ACh) and ACh‐induced NO generation were obtained in isolated aorta. Compared with control, diabetes increased diastolic and systolic BP, while chrysin and luteolin attenuated diastolic BP elevation without affecting the developed hyperglycemia. Diabetes increased contractile response of aorta to KCl, PE, decreased relaxation response to ACh, while chrysin and luteolin prevented the impaired response to ACh. In addition, diabetes was accompanied by elevated levels of TGs, total and LDL cholesterol, while both chrysin and luteolin prevented this dyslipidemia. Furthermore, chrysin decreased the elevated AGEs level in serum of diabetic animals, while luteolin abrogated the impaired NO generation in diabetic aorta. Collectively, chrysin and luteolin attenuate diabetes‐evoked impairment in endothelial‐dependent relaxation possibly via ameliorating detrimental changes in lipid profile, AGEs and NO generation. Copyright © 2013 John Wiley & Sons, Ltd.