Thermal performance of quartz capillaries for vitrification

In this paper we report the thermal behavior of a new approach for vitrification. Thermal performance of traditional open pulled straws is compared with a new technique based on the combined use of quartz capillaries with slush nitrogen. This new method of vitrification achieved ultrafast cooling rates of 250,000 °C/min. As a result, a much lower concentration of cryoprotectant was needed to reach vitrification. In fact, a cryoprotectant solution typically used in oocyte slow freezing protocols was shown to remain transparent after cooling to liquid nitrogen temperatures indicating apparent “vitrification”. This approach offers a new and very promising technique for vitrification of cells using low levels of cryoprotectants.     

Regulating cytoskeleton-based vesicle motility

During vesicular transport, the assembly of the coat complexes and the selection of cargo proteins must be coordinated with the subsequent translocation of vesicles from the donor to an acceptor compartment. Here, we review recent progress toward uncovering the molecular mechanisms that connect transport vesicles to the protein machinery responsible for cytoskeleton-mediated motility. An emerging theme is that vesicle cargo proteins, either directly or through binding interactions with coat proteins, are able to influence cytoskeletal dynamics and motor protein function. Hence, a vesicle's cargo composition may help direct its intracellular motility and targeting.     

Isolation and characterization of Saccharomyces cerevisiae mutants defective in chromosome transmission in an undergraduate genetics research course

An upper-level genetics research course was developed to expose undergraduates to investigative science. Students are immersed in a research project with the ultimate goal of identifying proteins important for chromosome transmission in mitosis. After mutagenizing yeast Saccharomyces cerevisiae cells, students implement a genetic screen that allows for visual detection of mutants with an increased loss of an ADE2-marked yeast artificial chromosome (YAC). Students then genetically characterize the mutants and begin efforts to identify the defective genes in these mutants. While engaged in this research project, students practice a variety of technical skills in both classical and molecular genetics. Furthermore, students learn to collaborate and gain experience in sharing scientific findings with others in the form of written papers, poster presentations, and oral presentations. Previous students indicated that, relative to a traditional laboratory course, this research course improved their understanding of scientific concepts and technical skills and helped them make connections between concepts. Moreover, this course allowed students to experience scientific inquiry and was influential for students as they considered future endeavors.     

Effect of inositol hexakisphosphate kinase 2 on transforming growth factor beta-activated kinase 1 and NF-kappaB activation

We previously showed that inositol hexakisphosphate kinase 2 (IHPK2) functions as a growth-suppressive and apoptosis-enhancing kinase during cell stress. Overexpression of IHPK2 sensitized ovarian carcinoma cell lines to the growth-suppressive and apoptotic effects of interferon beta (IFN-beta), IFN-alpha2, and gamma-irradiation. Expression of a kinase-dead mutant abrogated 50% of the apoptosis induced by IFN-beta. Because the kinase-dead mutant retained significant response to cell stressors, we hypothesized that a portion of the death-promoting function of IHPK2 was independent of its kinase activity. We now demonstrate that IHPK2 binds to tumor necrosis factor (TNF) receptor-associated factor (TRAF) 2 and interferes with phosphorylation of transforming growth factor beta-activated kinase 1 (TAK1), thereby inhibiting NF-kappaB signaling. IHPK2 contains two sites required for TRAF2 binding, Ser-347 and Ser-359. Compared with wild type IHPK2-transfected cells, cells expressing S347A and S359A mutations displayed 3.5-fold greater TAK1 activation following TNF-alpha. This mutant demonstrated a 6-10-fold increase in NF-kappaB DNA binding following TNF-alpha compared with wild type IHPK2-expressing cells in which NF-kappaB DNA binding was inhibited. Cells transfected with wild type IHPK2 or IHPK2 mutants that lacked S347A and S359A mutations displayed enhanced terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling staining following TNF-alpha. We believe that IHPK2-TRAF2 binding leads to attenuation of TAK1- and NF-kappaB-mediated signaling and is partially responsible for the apoptotic activity of IHPK2.     

Acquisition and voxelwise analysis of multi-subject diffusion data with tract-based spatial statistics

There is much interest in using magnetic resonance diffusion imaging to provide information on anatomical connectivity in the brain by measuring the diffusion of water in white matter tracts. Among the measures, the most commonly derived from diffusion data is fractional anisotropy (FA), which quantifies local tract directionality and integrity. Many multi-subject imaging studies are using FA images to localize brain changes related to development, degeneration and disease. In a recent paper, we presented a new approach, tract-based spatial statistics (TBSS), which aims to solve crucial issues of cross-subject data alignment, allowing localized cross-subject statistical analysis. This works by transforming the data from the centers of the tracts that are consistent across a study's subjects into a common space. In this protocol, we describe the MRI data acquisition and analysis protocols required for TBSS studies of localized change in brain connectivity across multiple subjects.     

Simultaneous quantification of malonyl-CoA and several other short-chain acyl-CoAs in animal tissues by ion-pairing reversed-phase HPLC/MS

Malonyl-CoA is a key intermediate involved in lipid synthesis and lipid oxidation. Here, we report on a novel method for the quantification of malonyl-CoA and seven other short-chain acyl-CoAs in various rat and mouse tissues using ion-pairing reversed-phase HPLC/MS. This method is capable of measuring malonyl-CoA, free coenzyme A (CoASH), acetyl-CoA, beta-hydroxyl-butyryl-CoA (HB-CoA), 3-hydroxy-3-methyl-glutaryl-CoA (HMG-CoA), propionyl-CoA, succinyl-CoA, and isobutyryl-CoA simultaneously with a dynamic linear range over two orders of magnitude in a 7.0 min HPLC gradient run. The lower limit of quantification (LLOQ) was 0.225 pmol for all acyl-CoAs studied, except for HMG-CoA which had a higher LLOQ of 0.90 pmol. The interference of HB-CoA on the quantification of malonyl-CoA in animal tissues was also explored for the first time.     

Determination of ciclosporin A and its six main metabolites in isolated T-lymphocytes and whole blood using liquid chromatography-tandem mass spectrometry

A specific and sensitive method for determination of intracellular ciclosporin A (CsA) and its six main metabolites AM1, AM9, AM1c, AM1c9, AM19 and AM4N, in isolated T-lymphocytes and whole blood is described. T-lymphocytes were separated from whole blood using Prepacyte. The analytes were extracted and purified from isolated lymphocytes and whole blood by protein precipitation followed by solid-phase extraction (SPE). The analytes and the internal standard, ciclosporin C (CsC), were separated on a reversed phase C8 column (30 mm x 2.1mm, 3 microm) with a 10 mm x 2 mm, 5 microm Drop-In Guard Cartridge, using gradient elution chromatography and tandem ion trap mass spectrometry detection. The method has been validated in accordance with FDA guidelines and showed linear range from 0.25 to 500 ng/mL for CsA, 0.5 to 500 ng/mL for AM1, AM9 and AM19, 1 to 500 ng/mL for AM4N, AM1c and AM1c9 in intracellular matrix, and 2.5 to 3000 ng/mL for all analytes in whole blood. The applicability of the method is shown on patient samples.     

A mutation in the myostatin gene increases muscle mass and enhances racing performance in heterozygote dogs

Double muscling is a trait previously described in several mammalian species including cattle and sheep and is caused by mutations in the myostatin (MSTN) gene (previously referred to as GDF8). Here we describe a new mutation in MSTN found in the whippet dog breed that results in a double-muscled phenotype known as the “bully” whippet. Individuals with this phenotype carry two copies of a two-base-pair deletion in the third exon of MSTN leading to a premature stop codon at amino acid 313. Individuals carrying only one copy of the mutation are, on average, more muscular than wild-type individuals (p = 7.43 × 10⁻⁶; Kruskal-Wallis Test) and are significantly faster than individuals carrying the wild-type genotype in competitive racing events (Kendall's nonparametric measure, τ = 0.3619; p ≈ 0.00028). These results highlight the utility of performance-enhancing polymorphisms, marking the first time a mutation in MSTN has been quantitatively linked to increased athletic performance.     

Genetic variation in the mitochondrial enzyme carbamyl-phosphate synthetase I predisposes children to increased pulmonary artery pressure following surgical repair of congenital heart defects: a validated genetic association study

Increased pulmonary artery pressure (PAP) can complicate the postoperative care of children undergoing surgical repair of congenital heart defects. Endogenous NO regulates PAP and is derived from arginine supplied by the urea cycle. The rate-limiting step in the urea cycle is catalyzed by a mitochondrial enzyme, carbamoyl-phosphate synthetase I (CPSI). A well-characterized polymorphism in the gene encoding CPSI (T1405N) has previously been implicated in neonatal pulmonary hypertension. A consecutive modeling cohort of children (N=131) with congenital heart defects requiring surgery was prospectively evaluated to determine key factors associated with increased postoperative PAP, defined as a mean PAP>20 mmHg for at least 1h during the 48h following surgery measured by an indwelling pulmonary artery catheter. Multiple dimensionality reduction (MDR) was used to both internally validate observations and develop optimal two-variable through five-variable models that were tested prospectively in a validation cohort (N=41). Unconditional logistic regression analysis of the modeling cohort revealed that age (OR=0.92, p=0.01), CPSI T1405N genotype (AC vs. AA: OR=4.08, p=0.04, CC vs. AA: OR=5.96, p=0.01), and Down syndrome (OR=5.25, p=0.04) were independent predictors of this complex phenotype. MDR predicted that the best two-variable model consisted of age and CPSI T1405N genotype (p<0.001). This two-variable model correctly predicted 73% of the outcomes from the validation cohort. A five-variable model that added race, gender and Down's syndrome was not significantly better than the two-variable model. In conclusion, the CPSI T1405N genotype appears to be an important new factor in predicting susceptibility to increased PAP following surgical repair of congenital cardiac defects in children.