Electric impedance sensing in cell-substrates for rapid and selective multipotential differentiation capacity monitoring of human mesenchymal stem cells

Biosensor systems which enable impedance measurements on adherent cell layers under label-free conditions are considered powerful tools for monitoring specific biological characteristics. A radio frequency identification-based sensor platform was adopted to characterize cultivation and differentiation of human bone marrow-derived multipotent stem cells (bmMSC) over periods of up to several days and weeks. Electric cell-substrate impedance sensing was achieved through fabrication of sensitive elements onto glass substrates which comprised two comb-shaped interdigitated gold electrodes covering an area of 1.8 mm×2 mm. The sensing systems were placed into the wells of a 6-well tissue culture plate, stacked onto a reader unit and could thus be handled and operated under sterile conditions. Continuous measurements were carried out with a sinusoidal voltage of 35 mV at a frequency of 10 kHz. After seeding of human bmMSC, this sensor was able to trace significant impedance changes contingent upon cell spreading and adhesion. The re-usable system was further proven suitable for live examination of cell-substrate attachment or continuous cell monitoring up to several weeks. Induction of either osteogenic or adipogenic differentiation could be validated in bmMSC cultures within a few days, in contrast to state-of-the-art protocols, which require several weeks of cultivation time. In the context of medical cell production in a GMP-compliant process, the here presented interdigitated electric microsensor technology allows the documentation of MSC quality in a fast, efficient and reliable fashion.     

Detection of Coxiella burnetii DNA on small-ruminant farms during a Q fever outbreak in the Netherlands

During large Q fever outbreaks in the Netherlands between 2007 and 2010, dairy goat farms were implicated as the primary source of human Q fever. The transmission of Coxiella burnetii to humans is thought to occur primarily via aerosols, although available data on C. burnetii in aerosols and other environmental matrices are limited. During the outbreak of 2009, 19 dairy goat farms and one dairy sheep farm were selected nationwide to investigate the presence of C. burnetii DNA in vaginal swabs, manure, surface area swabs, milk unit filters, and aerosols. Four of these farms had a positive status during the Coxiella burnetii bulk milk monitoring program in 2009 and additionally reported abortion waves in 2008 or 2009. Eleven farms were reported as having positive bulk milk only, and five selected (control) farms had a bulk milk-negative status in 2009 and no reported Q fever history. Screening by quantitative PCR (qPCR) revealed that on farms with a history of abortions related to C. burnetii and, to a lesser extent, on farms positive by bulk milk monitoring, generally higher proportions of positive samples and higher levels of C. burnetii DNA within positive samples were observed than on the control farms. The relatively high levels of C. burnetii DNA in surface area swabs and aerosols sampled in stables of bulk milk-positive farms, including farms with a Q fever-related abortion history, support the hypothesis that these farms can pose a risk for the transmission of C. burnetii to humans.     

Low plasma carnosinase activity promotes carnosinemia after carnosine ingestion in humans

A polymorphism in the carnosine dipeptidase-1 gene (CNDP1), resulting in decreased plasma carnosinase activity, is associated with a reduced risk for diabetic nephropathy. Because carnosine, a natural scavenger/suppressor of ROS, advanced glycation end products, and reactive aldehydes, is readily degraded in blood by the highly active carnosinase enzyme, it has been postulated that low serum carnosinase activity might be advantageous to reduce diabetic complications. The aim of this study was to examine whether low carnosinase activity promotes circulating carnosine levels after carnosine supplementation in humans. Blood and urine were sampled in 25 healthy subjects after acute supplementation with 60 mg/kg body wt carnosine. Precooled EDTA-containing tubes were used for blood withdrawal, and plasma samples were immediately deproteinized and analyzed for carnosine and β-alanine by HPLC. CNDP1 genotype, baseline plasma carnosinase activity, and protein content were assessed. Upon carnosine ingestion, 8 of the 25 subjects (responders) displayed a measurable increase in plasma carnosine up to 1 h after supplementation. Subjects with no measurable increment in plasma carnosine (nonresponders) had ～2-fold higher plasma carnosinase protein content and ～1.5-fold higher activity compared with responders. Urinary carnosine recovery was 2.6-fold higher in responders versus nonresponders and was negatively dependent on both the activity and protein content of the plasma carnosinase enzyme. In conclusion, low plasma carnosinase activity promotes the presence of circulating carnosine upon an oral challenge. These data may further clarify the link among CNDP1 genotype, carnosinase, and diabetic nephropathy.     

Maturation of Pichia pastoris-derived recombinant pro-Der p 1 induced by deglycosylation and by the natural cysteine protease Der p 1 from house dust mite.

The mature cysteine protease from Dermatophgoides pteronyssinus, Der p 1, is a major house dust mite allergen. Its enzymatic activity has been shown to have pro-inflammatory effects that could also negatively influence efficacy of allergen-specific immunotherapy. The aim of this study was to express recombinant pro-Der p 1 (rpro-Der p 1) in the yeast Pichia pastoris and to study its maturation. Expression was achieved at a concentration ranging from 45 mg.L-1 (methanol-induced expression) to 168 mg.L-1 (constitutive expression). No significant spontaneous maturation of the secreted proenzyme was observed. rpro-Der p 1 with a sequence-based molecular mass of 34 kDa was hyperglycosylated by the yeast, migrating at 50-60 kDa on SDS/PAGE. Compared with its natural counterpart (nDer p 1), the recombinant proenzyme demonstrated decreased IgE reactivity, resulting in a 30-fold lower capacity to induce histamine release from human basophils. Decreased immunoreactivity was also shown by competitive RIA and sandwich ELISA with Der p 1-specific antibody reagents. CD spectra of rpro-Der p 1 and nDer p 1 revealed significant structural differences. Deglycosylation of rpro-Der p 1 with endoglycosidase H resulted in a decrease in apparent molecular mass from 50 kDa to 34 kDa, but did not affect nDer p 1. On removal of N-glycans from rpro-Der p 1, which harbours two putative N-glycosylation sites in both propeptide and mature sequence, the mature rDer p 1 appeared. This suggests that hyperglycosylation hampers spontaneous maturation. Maturation of the recombinant pro-enzyme was also achieved by addition of the active natural cysteine protease, nDer p 1. In conclusion, high-level expression of rpro-Der p 1 in P. pastoris results in a stable hypoallergenic proenzyme with potential for use in allergen-specific immunotherapy.     

Discovery and optimization of highly ligand-efficient oxytocin receptor antagonists using structure-based drug design

A novel oxytocin antagonist was identified by ‘scaffold-hopping’ using Cresset FieldScreen molecular field similarity searching. A single cycle of optimization driven by an understanding of the key pharmacophoric elements required for activity led to the discovery of a potent, selective and highly ligand-efficient oxytocin receptor antagonist. Selectivity over vasopressin receptors was rationalized based on differences in the structure of the natural ligands.     

A Qualitative Study Exploring Barriers Related to Use of Footwear in Rural Highland Ethiopia: Implications for Neglected Tropical Disease Control

Background

The role of footwear in protection against a range of Neglected Tropical Diseases (NTDs) is gaining increasing attention. Better understanding of the behaviors that influence use of footwear will lead to improved ability to measure shoe use and will be important for those implementing footwear programs.

Methodology/Principal Findings

Using the PRECEDE-PROCEED model we assessed social, behavioral, environmental, educational and ecological needs influencing whether and when children wear shoes in a rural highland Ethiopian community endemic for podoconiosis. Information was gathered from 242 respondents using focus groups, semi-structured interviews and extended case studies. Shoe-wearing norms were said to be changing, with going barefoot increasingly seen as ‘shameful’. Shoes were thought to confer dignity as well as protection against injury and cold. However, many practical and social barriers prevented the desire to wear shoes from being translated into practice. Limited financial resources meant that people were neither able to purchase more than one pair of shoes to ensure their longevity nor afford shoes of the preferred quality. As a result of this limited access, shoes were typically preserved for special occasions and might not be provided for children until they reached a certain age. While some barriers (for example fit of shoe and fear of labeling through use of a certain type of shoe) may be applicable only to certain diseases, underlying structural level barriers related to poverty (for example price, quality, unsuitability for daily activities and low risk perception) are likely to be relevant to a range of NTDs.

Conclusions/Significance

Using well established conceptual models of health behavior adoption, we identified several barriers to shoe wearing that are amenable to intervention and which we anticipate will be of benefit to those considering NTD prevention through shoe distribution.