Identification of the facilitative glucose transporter 12 gene Glut12 in mouse preimplantation embryos

In this study we report the cloning and characterisation of the mouse Glut12 gene and examine for the first time its expression pattern in the earliest stages of development. Mouse Glut12 (mGlut12) was cloned from preimplantation embryos by 5'RACE RT-PCR using primers designed from an EST clone corresponding to a human GLUT12 antigenic sequence after positive immunoreactivity was observed in mouse two-cell embryos by western immunoblotting. The mGlut12 gene contains an open reading frame of 1869 base pairs, potentially encoding a polypeptide of 622 amino acids. The predicted mGLUT12 protein bears all the hallmarks of the SLC2A family of hexose transporters and shares an 83% sequence homology to human GLUT12. Consistent with its human homolog mGlut12 mRNA is found highly expressed in skeletal and cardiac muscle and fat. Additionally, it was also found in the uterus and during early embryogenesis. During early development in the mouse, Glut12 expression is clearly apparent in ovulated oocytes and two-cell embryos but declines in day 3 morulae. With the exception of some Glut12 expression apparent in blastocysts, Glut12 mRNA remains at low to undetectable levels until E11.     

Chirally dimorphic male genitalia in praying mantids (Ciulfina: Liturgusidae)

Although male polymorphisms occur widely in nature and have received considerable recent attention from studies of alternative mating strategies, male genital polymorphisms are less well known. Here, we describe a dimorphism in the orientation of the male genitalic complex of the praying mantid genus Ciulfina. Populations of Ciulfina species vary in the proportion of males with dextral (right‐oriented) and sinistral (left‐oriented) genitalia, ranging from directional asymmetry (single orientation only) to apparent antisymmetry (equal proportions of both orientations). The proportion of dextral males varied between species (C. baldersoni: 46%; C. rentzi: 24%; C. klassi: 100%; C. biseriata: 83%) and between populations. We used elliptic Fourier analysis to quantify shape and size variation between the genitalia of dextral and sinistral males and determined that the two forms were mirror images of one another in two species. We found that the level of mechanical reproductive isolation between heterospecific populations of opposite genital orientation was no greater than that between heterospecific populations with the same orientation or of mixed orientation. Genital orientation therefore did not influence premating isolation between these species, despite complete postmating isolation. The geographic proximity of populations to heterospecifics also showed no particular pattern with respect to genital orientation. These results suggest that reversible trait asymmetry in Ciulfina is not driven by reproductive isolation, and add to the growing evidence against the species isolation hypothesis for rapid genital evolution. J. Morphol. 271:1176–1184, 2010. © 2010 Wiley‐Liss, Inc.     

Dissection of the nuclear genome of barley by chromosome flow sorting

Isolation of mitotic chromosomes using flow cytometry is an attractive way to dissect nuclear genomes into their individual chromosomal components or portions of them. This approach is especially useful in plants with complex genomes, where it offers a targeted and hence economical approach to genome analysis and gene cloning. In several plant species, DNA of flow-sorted chromosomes has been used for isolation of molecular markers from specific genome regions, for physical mapping using polymerase chain reaction (PCR) and fluorescence in situ hybridization (FISH), for integration of genetic and physical maps and for construction of chromosome-specific DNA libraries, including those cloned in bacterial artificial chromosome vectors. Until now, chromosome analysis and sorting using flow cytometry (flow cytogenetics) has found little application in barley (2n = 14, 1C ∼ 5,100 Mbp) because of the impossibility of discriminating and sorting individual chromosomes, except for the smallest chromosome 1H and some translocation chromosomes with DNA content significantly different from the remaining chromosomes. In this work, we demonstrate that wheat–barley ditelosomic addition lines can be used to sort any arm of barley chromosomes 2H–7H. Thus, the barley genome can be dissected into fractions representing only about 6–12% of the total genome. This advance makes the flow cytogenetics an attractive tool, which may greatly facilitate genome analysis and gene cloning in barley.     

G4 motifs affect origin positioning and efficiency in two vertebrate replicators

DNA replication ensures the accurate duplication of the genome at each cell cycle. It begins at specific sites called replication origins. Genome‐wide studies in vertebrates have recently identified a consensus G‐rich motif potentially able to form G‐quadruplexes (G4) in most replication origins. However, there is no experimental evidence to demonstrate that G4 are actually required for replication initiation. We show here, with two model origins, that G4 motifs are required for replication initiation. Two G4 motifs cooperate in one of our model origins. The other contains only one critical G4, and its orientation determines the precise position of the replication start site. Point mutations affecting the stability of this G4 in vitro also impair origin function. Finally, this G4 is not sufficient for origin activity and must cooperate with a 200‐bp cis‐regulatory element. In conclusion, our study strongly supports the predicted essential role of G4 in replication initiation.     

Role of histidine for charge regulation of unstructured peptides at interfaces and in bulk

Histidine‐rich, unstructured peptides adsorb to charged interfaces such as mineral surfaces and microbial cell membranes. At a molecular level, we investigate the adsorption mechanism as a function of pH, salt, and multivalent ions showing that (1) proton charge fluctuations are—in contrast to the majority of proteins—optimal at neutral pH, promoting electrostatic interactions with anionic surfaces through charge regulation and (2) specific zinc(II)‐histidine binding competes with protons and ensures an unusually constant charge distribution over a broad pH interval. In turn, this further enhances surface adsorption. Our analysis is based on atomistic molecular dynamics simulations, coarse grained Metropolis Monte Carlo, and classical polymer density functional theory. This multiscale modeling provides a consistent picture in good agreement with experimental data on Histatin 5, an antimicrobial salivary peptide. Biological function is discussed and we suggest that charge regulation is a significant driving force for the remarkably robust activity of histidine‐rich antimicrobial peptides. Proteins 2014; 82:657–667. © 2013 Wiley Periodicals, Inc.     

Medication Monitoring in a Nurse-Led Respiratory Outpatient Clinic: Pragmatic Randomised Trial of the West Wales Adverse Drug Reaction Profile

Objective

To assess the clinical effect of medication monitoring using the West Wales Adverse Drug Reaction (ADR) Profile for Respiratory Medicine.

Design

Single-site parallel-arm pragmatic trial using stratified randomisation.

Setting

Nurse-led respiratory outpatient clinic in general hospital in South Wales.

Participants

54 patients with chronic respiratory disease receiving bronchodilators, corticosteroids or leukotriene receptor antagonists.

Intervention

Following initial observation of usual nursing care, we allocated participants at random to receive at follow up: either the West Wales ADR Profile for Respiratory Medicine in addition to usual care (‘intervention arm’ with 26 participants); or usual care alone (‘control arm’ with 28 participants).

Main Outcome Measures

Problems reported and actions taken.

Results

We followed up all randomised participants, and analysed data in accordance with treatment allocated. The increase in numbers of problems per participant identified at follow up was significantly higher in the intervention arm, where the median increase was 20.5 [inter-quartile range (IQR) 13–26], while that in the control arm was −1 [−3 to +2] [Mann-Whitney U test: z = 6.28, p<0.001]. The increase in numbers of actions per participant taken at follow up was also significantly higher in the intervention arm, where the median increase was 2.5 [1]–[4] while that in the control arm was 0 [−1.75 to +1] [Mann-Whitney U test: z = 4.40, p<0.001].

Conclusion

When added to usual nursing care, the West Wales ADR Profile identified more problems and prompted more nursing actions. Our ADR Profile warrants further investigation as a strategy to optimise medication management.

Trial Registration

Controlled-trials.com ISRCTN10386209     

Decrease in Urinary Creatinine Excretion in Early Stage Chronic Kidney Disease

Background

Little is known about muscle mass loss in early stage chronic kidney disease (CKD). We used 24-hour urinary creatinine excretion rate to assess determinants of muscle mass and its evolution with kidney function decline. We also described the range of urinary creatinine concentration in this population.

Methods

We included 1072 men and 537 women with non-dialysis CKD stages 1 to 5, all of them with repeated measurements of glomerular filtration rate (mGFR) by ⁵¹Cr-EDTA renal clearance and several nutritional markers. In those with stage 1 to 4 at baseline, we used a mixed model to study factors associated with urinary creatinine excretion rate and its change over time.

Results

Baseline mean urinary creatinine excretion decreased from 15.3±3.1 to 12.1±3.3 mmol/24 h (0.20±0.03 to 0.15±0.04 mmol/kg/24 h) in men, with mGFR falling from ≥60 to <15 mL/min/1.73 m², and from 9.6±1.9 to 7.6±2.5 (0.16±0.03 to 0.12±0.03) in women. In addition to mGFR, an older age, diabetes, and lower levels of body mass index, proteinuria, and protein intake assessed by urinary urea were associated with lower mean urinary creatinine excretion at baseline. Mean annual decline in mGFR was 1.53±0.12 mL/min/1.73 m² per year and that of urinary creatinine excretion rate, 0.28±0.02 mmol/24 h per year. Patients with fast annual decline in mGFR of 5 mL/min/1.73 m² had a decrease in urinary creatinine excretion more than twice as big as in those with stable mGFR, independent of changes in urinary urea as well as of other determinants of low muscle mass.

Conclusions

Decrease in 24-hour urinary creatinine excretion rate may appear early in CKD patients, and is greater the more mGFR declines independent of lowering protein intake assessed by 24-hour urinary urea. Normalizing urine analytes for creatininuria may overestimate their concentration in patients with reduced kidney function and low muscle mass.