Selective protein removal and desalting using microchip CE

This paper describes the on-line sample pretreatment and analysis of proteins and peptides with a poly(methylmethacrylate) (PMMA) microfluidic device (IonChip). This chip consists of two hyphenated electrophoresis channels with integrated conductivity detectors. The first channel can be used for sample preconcentration and sample clean-up, while in the second channel the selected compounds are separated. Isotachophoresis (ITP) combined with zone electrophoresis (CZE) was used to preconcentrate a myoglobin sample by a factor of about 65 before injection into the second dimension and to desalt a mixture of six proteins with 100 mM NaCl. However, ITP-CZE could not be used for the removal of two proteins from a protein/peptide sample since the protein zone in the ITP step was too small to remove certain compounds. Therefore, we used CZE-CZE for the removal of proteins from a protein/peptide mixture, thereby injecting only the peptides into the second CZE separation channel.     

Sugar-induced increases in trehalose 6-phosphate are correlated with redox activation of ADPglucose pyrophosphorylase and higher rates of starch synthesis in Arabidopsis thaliana

Tre6P (trehalose 6-phosphate) is implicated in sugar-signalling pathways in plants, but its exact functions in vivo are uncertain. One of the main obstacles to discovering these functions is the difficulty of measuring the amount of Tre6P in plant tissues. We have developed a highly specific assay, using liquid chromatography coupled to MS-Q3 (triple quadrupole MS), to measure Tre6P in the femto-picomole range. The Tre6P content of sucrose-starved Arabidopsis thaliana seedlings in axenic culture increased from 18 to 482 pmol x g(-1) FW (fresh weight) after adding sucrose. Leaves from soil-grown plants contained 67 pmol x g(-1) FW at the end of the night, which rose to 108 pmol x g(-1)FW after 4 h of illumination. Even greater changes in Tre6P content were seen after a 6 h extension of the dark period, and in the starchless mutant, pgm. The intracellular concentration of Tre6P in wild-type leaves was estimated to range from 1 to 15 microM. It has recently been reported that the addition of Tre6P to isolated chloroplasts leads to redox activation of AGPase (ADPglucose pyrophosphorylase) [Kolbe, Tiessen, Schluepmann, Paul, Ulrich and Geigenberger (2005) Proc. Natl. Acad. Sci. U.S.A. 102, 11118-11123]. Using the new assay for Tre6P, we found that rising sugar levels in plants are accompanied by increases in the level of Tre6P, redox activation of AGPase and the stimulation of starch synthesis in vivo. These results indicate that Tre6P acts as a signalling metabolite of sugar status in plants, and support the proposal that Tre6P mediates sucrose-induced changes in the rate of starch synthesis.     

Scaling of offspring number and mass to plant and animal size: model and meta-analysis

The scaling of reproductive parameters to body size is important for understanding ecological and evolutionary patterns. Here, we derived allometric relationships for the number and mass of seeds, eggs and neonates from an existing model on population production. In a separate meta-analysis, we collected 79 empirical regressions on offspring mass and number covering different taxa and various habitats. The literature review served as a validation of the model, whereas, vice versa, consistency of isolated regressions with each other and related ecological quantities was checked with the model. The total offspring mass delivered in a reproductive event scaled to adult size with slopes in the range of about 3/4 to 1. Exponents for individual seed, egg and neonate mass varied around 1/2 for most heterotherms and between 3/4 and 1 for most homeotherms. The scaling of the progeny number released in a sowing, clutch or litter was opposite to that of their size. The linear regressions fitted into a triangular envelope where maximum offspring mass is limited by the size of the adult. Minimum seed and egg size scaled with weight exponents of approximately 0 up to 1/4. These patterns can be explained by the influence of parents on the fate of their offspring, covering the continuum of r-strategists (pelagic–aquatic, arial, most invertebrates, heterotherms) and K-strategists (littoral–terrestrial, some invertebrates, homeotherms).     

Protein tyrosine phosphatases: functional inferences from mouse models and human diseases

Some 40-odd genes in mammals encode phosphotyrosine-specific, 'classical' protein tyrosine phosphatases. The generation of animal model systems and the study of various human disease states have begun to elucidate the important and diverse roles of protein tyrosine phosphatases in cellular signalling pathways, development and disease. Here, we provide an overview of those findings from mice and men, and indicate several novel approaches that are now being exploited to further our knowledge of this fascinating enzyme family.     

Implementation of a Bundle of Care to Reduce Surgical Site Infections in Patients Undergoing Vascular Surgery

Background

Surgical site infections (SSI’s) are associated with severe morbidity, mortality and increased health care costs in vascular surgery.

Objective

To implement a bundle of care in vascular surgery and measure the effects on the overall and deep-SSI’s rates.

Design

Prospective, quasi-experimental, cohort study.

Methods

A prospective surveillance for SSI’s after vascular surgery was performed in the Amphia hospital in Breda, from 2009 through 2011. A bundle developed by the Dutch hospital patient safety program (DHPSP) was introduced in 2009. The elements of the bundle were (1) perioperative normothermia, (2) hair removal before surgery, (3) the use of perioperative antibiotic prophylaxis and (4) discipline in the operating room. Bundle compliance was measured every 3 months in a random sample of surgical procedures and this was used for feedback.

Results

Bundle compliance improved significantly from an average of 10% in 2009 to 60% in 2011. In total, 720 vascular procedures were performed during the study period and 75 (10.4%) SSI were observed. Deep SSI occurred in 25 (3.5%) patients. Patients with SSI’s (28,5±29.3 vs 10.8±11.3, p<0.001) and deep-SSI’s (48.3±39.4 vs 11.4±11.8, p<0.001) had a significantly longer length of hospital stay after surgery than patients without an infection. A significantly higher mortality was observed in patients who developed a deep SSI (Adjusted OR: 2.96, 95% confidence interval 1.32–6.63). Multivariate analysis showed a significant and independent decrease of the SSI-rate over time that paralleled the introduction of the bundle. The SSI-rate was 51% lower in 2011 compared to 2009.

Conclusion

The implementation of the bundle was associated with improved compliance over time and a 51% reduction of the SSI-rate in vascular procedures. The bundle did not require expensive or potentially harmful interventions and is therefore an important tool to improve patient safety and reduce SSI’s in patients undergoing vascular surgery.     

Liver glycogen phosphorylase is upregulated in glioblastoma and provides a metabolic vulnerability to high dose radiation

Channelling of glucose via glycogen, known as the glycogen shunt, may play an important role in the metabolism of brain tumours, especially in hypoxic conditions. We aimed to dissect the role of glycogen degradation in glioblastoma (GBM) response to ionising radiation (IR). Knockdown of the glycogen phosphorylase liver isoform (PYGL), but not the brain isoform (PYGB), decreased clonogenic growth and survival of GBM cell lines and sensitised them to IR doses of 10–12 Gy. Two to five days after IR exposure of PYGL knockdown GBM cells, mitotic catastrophy and a giant multinucleated cell morphology with senescence-like phenotype developed. The basal levels of the lysosomal enzyme alpha-acid glucosidase (GAA), essential for autolysosomal glycogen degradation, and the lipidated forms of gamma-aminobutyric acid receptor-associated protein-like (GABARAPL1 and GABARAPL2) increased in shPYGL U87MG cells, suggesting a compensatory mechanism of glycogen degradation. In response to IR, dysregulation of autophagy was shown by accumulation of the p62 and the lipidated form of GABARAPL1 and GABARAPL2 in shPYGL U87MG cells. IR increased the mitochondrial mass and the colocalisation of mitochondria with lysosomes in shPYGL cells, thereby indicating reduced mitophagy. These changes coincided with increased phosphorylation of AMP-activated protein kinase and acetyl-CoA carboxylase 2, slower ATP generation in response to glucose loading and progressive loss of oxidative phosphorylation. The resulting metabolic deficiencies affected the availability of ATP required for mitosis, resulting in the mitotic catastrophy observed in shPYGL cells following IR. PYGL mRNA and protein levels were higher in human GBM than in normal human brain tissues and high PYGL mRNA expression in GBM correlated with poor patient survival. In conclusion, we show a major new role for glycogen metabolism in GBM cancer. Inhibition of glycogen degradation sensitises GBM cells to high-dose IR indicating that PYGL is a potential novel target for the treatment of GBMs.Subject terms: Cancer metabolism, CNS cancer     

How allometric scaling relates to soil abiotics

For most species, the logarithm of their average body mass is negatively related to the logarithm of their relative population density, i.e. the numerical abundance. In this way, the allometric scaling (both mass–abundance regressions and body–size spectra) becomes useful in ecological theory to build and explain food webs. Using empirical evidence derived from 145 Dutch sites, a hypothesis is formulated to explain how soil microbivores, detritivores and predators react to increasing resource availability. Shifts in size distribution, and subsequently changes in soil food‐web structure, are further discussed in the perspective of Holling's sequential interactions between basic system functions. We show that the allometric scaling and the averages of the (log‐transformed) prey:predator body‐mass ratios are reliable predictors for assessing faunal responses to nutrient availability. We view this work as a first attempt toward an extensive comparison of ecological processes in different soil systems.     

A Phase 1b Randomized,Controlled, Double-Blinded Dosage-Escalation Trial to Evaluate the Safety,Reactogenicity and Immunogenicity of an Adenovirus Type 35 Based Circumsporozoite Malaria Vaccine in Burkinabe Healthy Adults 18 to 45 Years of Age

Background

Ad35.CS.01 is a pre-erythrocytic malaria candidate vaccine. It is a codon optimized nucleotide sequence representing the P. falciparum circumsporozoite (CS) surface antigen inserted in a replication deficient Adenovirus 35 backbone. A Phase 1a trial has been conducted in the USA in naïve adults and showed that the vaccine was safe. The aim of this study is to assess the safety and immunogenicity of ascending dosages in sub Saharan Africa.

Methods

A double blind, randomized, controlled, dose escalation, phase Ib trial was conducted in a rural area of Balonghin, the Saponé health district (Burkina Faso). Forty-eight healthy adults aged 18-45 years were randomized into 4 cohorts of 12 to receive three vaccine doses (day 0, 28 and 84) of 10⁹, 10¹⁰, 5X10¹⁰, 10¹¹ vp of Ad35.CS.01 or normal saline by intra muscular injection. Subjects were monitored carefully during the 14 days following each vaccination for non serious adverse events. Severe and serious adverse events were collected throughout the participant study duration (12 months from the first vaccination). Humoral and cellular immune responses were measured on study days 0, 28, 56, 84, 112 and 140.

Results

Of the forty-eight subjects enrolled, forty-four (91.7%) received all three scheduled vaccine doses. Local reactions, all of mild severity, occurred in thirteen (27.1%) subjects. Severe (grade 3) laboratory abnormalities occurred in five (10.4%) subjects. One serious adverse event was reported and attributed to infection judged unrelated to vaccine. The vaccine induced both antibody titers and CD8 T cells producing IFNγ and TNFα with specificity to CS while eliciting modest neutralizing antibody responses against Ad35.

Conclusion

Study vaccine Ad35.CS.01 at four different dose levels was well-tolerated and modestly immunogenic in this population. These results suggest that Ad35.CS.01 should be further investigated for preliminary efficacy in human challenge models and as part of heterologous prime-boost vaccination strategies.

Trial Registration

ClinicalTrials.gov NCT01018459 http://clinicaltrials.gov/ct2/show/NCT01018459