Flow injection analysis electrospray ionization mass spectrometry for high-throughput screening of a gene library for amidase activity

In high-throughput screening of gene and mutant libraries, high analysis speeds and short method development times are important factors. Mass spectrometry (MS) is considered to be a generic analytical technique with a relatively short development time. Furthermore, when applying flow injection analysis (FIA) for sample introduction, the requirements for high throughput are met. In this work, the use of a single quadrupole electrospray MS instrument for assaying amidase activity in a gene library is demonstrated. The desired selectivity for measuring the amino acid, the reaction product of the amidase reaction, in the presence of high concentrations of the corresponding amino acid amide substrate was obtained by selective ionization of the amino acid in negative ion mode electrospray. The only sample preparation required was a 200-fold dilution of the reaction mixture. For obtaining quantitative results, a complementary calibration procedure was set up to correct for the change in ionization suppression as a function of conversion. This approach was used to screen a Mycobacterium neoaurum gene library consisting of 11,520 clones with α-methylleucine amide as substrate within 24 h. Conversion was measured on the [M−H]⁻ species of the corresponding α-methylleucine (m/z 144). Five positive clones were detected with a conversion ranging from 0.2% to 3.4%.     

Effect of high contents of dietary animal-derived protein or carbohydrates on canine fecal microbiota

ABSTRACT: BACKGROUND: Considerable evidence suggests that food impacts both the gastro-intestinal (GI) function and the microbial ecology of the canine GI tract. The aim of this study was to evaluate the influence of high-carbohydrate (HC), high-protein (HP) and dry commercial (DC) diets on the canine colonic microbiota in Beagle dogs. Diets were allocated according to the Graeco-Latin square design. For this purpose, microbial DNA was isolated from faecal samples and separated by density gradient centrifugation, resulting in specific profiling based on the guanine-cytosine content (%G+C). In addition, 16S rRNA gene amplicons were obtained from the most abundant %G+C peaks and analysed by sequence analysis, producing a total of 720 non-redundant sequences (240 sequences per diet). RESULTS: The DC diet sample showed high abundance of representatives of the orders Clostridiales, Lactobacillales, Coriobacteriales and Bacteroidales. Sequence diversity was highest for DC diet samples and included representatives of the orders Lactobacillales and Bacteroidales, which were not detected in samples from the HP and HC diets. These latter two diets also had reduced levels of representatives of the family Lachnospiraceae, specifically Clostridial cluster XIVa. The HC diet favoured representatives of the order Erysipelotrichales, more specifically the Clostridial cluster XVIII, while the HP diet favoured representatives of the order Fusobacteriales. CONCLUSIONS: This study detected Coriobacteriales in dog faeces, possibly due to the non-selective nature of the %G+C profiling method used in combination with sequencing. Moreover, our work demonstrates that the effect of diet on faecal microbiota can be explained based on the metabolic properties of the detected microbial taxa.     

Geometrical models for cardiac MRI in rodents: comparison of quantification of left ventricular volumes and function by various geometrical models with a full-volume MRI data set in rodents

MRI has been proven to be an accurate method for noninvasive assessment of cardiac function. One of the current limitations of cardiac MRI is that it is time consuming. Therefore, various geometrical models are used, which can reduce scan and postprocessing time. It is unclear how appropriate their use is in rodents. Left ventricular (LV) volumes and ejection fraction (EF) were quantified based on 7.0 Tesla cine-MRI in 12 wild-type (WT) mice, 12 adipose triglyceride lipase knockout (ATGL(-/-)) mice (model of impaired cardiac function), and 11 rats in which we induced cardiac ischemia. The LV volumes and function were either assessed with parallel short-axis slices covering the full volume of the left ventricle (FV, gold standard) or with various geometrical models [modified Simpson rule (SR), biplane ellipsoid (BP), hemisphere cylinder (HC), single-plane ellipsoid (SP), and modified Teichholz Formula (TF)]. Reproducibility of the different models was tested and results were correlated with the gold standard (FV). All models and the FV data set provided reproducible results for the LV volumes and EF, with interclass correlation coefficients ≥0.87. All models significantly over- or underestimated EF, except for SR. Good correlation was found for all volumes and EF for the SR model compared with the FV data set (R(2) ranged between 0.59-0.95 for all parameters). The HC model and BP model also predicted EF well (R(2) ≥ 0.85), although proved to be less useful for quantitative analysis. The SP and TF models correlated poorly with the FV data set (R(2) ≥ 0.45 for EF and R(2) ≥ 0.29 for EF, respectively). For the reduction in acquisition and postprocessing time, only the SR model proved to be a valuable method for calculating LV volumes, stroke volume, and EF.     

Association of LIN28B with Adult Adiposity-Related Traits in Females

Context

Pubertal timing is under strong genetic control and its early onset associates with several adverse health outcomes in adulthood, including obesity, type 2 diabetes and cardiovascular disease. Recent data indicate strong association between pubertal timing and genetic variants near LIN28B, but it is currently unknown whether the gene contributes to the association between puberty and adult disease.

Objective

To elucidate the putative genetic link between early puberty and adult disease risk, we examined the association of two genetic variants near LIN28B with adult body size and metabolic profiles in randomly ascertained adult Finnish males and females.

Methods

Two single nucleotide polymorphisms (SNPs), rs7759938, the lead SNP previously associated with pubertal timing and height, and rs314279, previously also associated with menarcheal age but only partially correlated with rs7759938 (r² = 0.30), were genotyped in 26,636 study subjects participating in the Finnish population survey FINRISK. Marker associations with adult height, weight, body mass index (BMI), hip and waist circumference, blood glucose, serum insulin and lipid/lipoprotein levels were determined by linear regression analyses.

Results

Both rs7759938 and rs314279 associated with adult height in both sexes (p = 2×10⁻⁶ and p = 0.001). Furthermore, rs314279 associated with increased weight in females (p = 0.001). Conditioned analyses including both SNPs in the regression model verified that rs314279 independently associates with adult female weight, BMI and hip circumference (p<0.005). Neither SNP associated with glucose, lipid, or lipoprotein levels.

Conclusion

Genetic variants near the puberty-associated gene LIN28B associate with adult weight and body shape in females, suggesting that the gene may tag molecular pathways influencing adult adiposity-related traits.     

Genetic profiling using genome-wide significant coronary artery disease risk variants does not improve the prediction of subclinical atherosclerosis: the Cardiovascular Risk in Young Finns Study, the Bogalusa Heart Study and the Health 2000 Survey--a meta-analysis of three independent studies

Background

Genome-wide association studies (GWASs) have identified a large number of variants (SNPs) associating with an increased risk of coronary artery disease (CAD). Recently, the CARDIoGRAM consortium published a GWAS based on the largest study population so far. They successfully replicated twelve already known associations and discovered thirteen new SNPs associating with CAD. We examined whether the genetic profiling of these variants improves prediction of subclinical atherosclerosis – i.e., carotid intima-media thickness (CIMT) and carotid artery elasticity (CAE) – beyond classical risk factors.

Subjects and Methods

We genotyped 24 variants found in a population of European ancestry and measured CIMT and CAE in 2001 and 2007 from 2,081, and 2,015 subjects (aged 30–45 years in 2007) respectively, participating in the Cardiovascular Risk in Young Finns Study (YFS). The Bogalusa Heart Study (BHS; n = 1179) was used as a replication cohort (mean age of 37.5). For additional replication, a sub-sample of 5 SNPs was genotyped for 1,291 individuals aged 46–76 years participating in the Health 2000 population survey. We tested the impact of genetic risk score (GRS_24SNP/CAD) calculated as a weighted (by allelic odds ratios for CAD) sum of CAD risk alleles from the studied 24 variants on CIMT, CAE, the incidence of carotid atherosclerosis and the progression of CIMT and CAE during a 6-year follow-up.

Results

CIMT or CAE did not significantly associate with GRS_24SNP/CAD before or after adjusting for classical CAD risk factors (p>0.05 for all) in YFS or in the BHS. CIMT and CAE associated with only one SNP each in the YFS. The findings were not replicated in the replication cohorts. In the meta-analysis CIMT or CAE did not associate with any of the SNPs.

Conclusion

Genetic profiling, by using known CAD risk variants, should not improve risk stratification for subclinical atherosclerosis beyond conventional risk factors among healthy young adults.     

Coordination of tooth morphogenesis and neuronal development through tissue interactions: Lessons from mouse models

In addition to being an advantageous model to investigate general molecular mechanisms of organ formation, the tooth is a distinct target organ for peripheral nerve innervation. These nerves are required for the function and protection of the teeth and, as shown in fish, also for their regeneration. This review focuses on recent findings of the local tissue interactions and molecular signaling mechanisms that regulate the early nerve arrival and patterning of mouse mandibular molar tooth sensory innervation.     

Betaine aids in the osmoregulation of duodenal epithelium of broiler chicks, and affects the movement of water across the small intestinal epithelium in vitro.

In Experiment 1, the water holding capacity of broiler chick intestinal tissue was studied in vitro. The chicks were fed with corn-based diets with or without a 0.2% betaine supplementation in the drinking water. Slices from duodenum and jejunum were incubated in iso-osmotic (300 mM) or hyperosmotic saline (600 mM) with or without 10 mM betaine. The water volume of tissue slices was studied by adding tritiated water in the incubation medium while [14C]inulin was used to correct for the adherent water. After 30 min of incubation, by which time the steady-state of tritium influx had been achieved, the 3H and 14C-activities of the tissue slices were measured. The ileal and duodenal tissues incubated in the hyperosmotic saline accumulated less tritium than those incubated in iso-osmotic saline. Duodenal slices incubated in hyperosmotic saline with the presence of betaine showed a tritium content similar to slices incubated in iso-osmotic saline. The data suggest that the presence of betaine helped the duodenal, but not jejunal, epithelium to maintain water balance in hyperosmotic conditions. The dietary betaine supplementation diminished the differences between the incubation treatments in duodenal, but not in ileal tissue. In Experiment 2, the same double labeling method, but with shorter incubation times, was used to assess the rate of water flux from the incubation medium to duodenal or jejunal slices. The dietary treatments (as in Experiment 1) had little effect on the results. Betaine in the hyperosmotic saline significantly decreased the rate of tritium accumulation into the tissue slices, indicating that betaine slowed down the influx of water to the epithelium. We suggest that betaine affects the movement of water across the intestinal epithelium and has a role in the osmoregulation of small intestine of broiler chicks.