A regenerable flow-through affinity sensor for label-free detection of proteins and DNA

Label-free monitoring of biomolecular reactions in real-time is of great interest since it can provide valuable information about binding kinetics and equilibrium constants. In this report, a sensor based on White Light Reflectance Spectroscopy (WLRS) is presented that is capable of real-time monitoring of biomolecular reactions taking place on top of a polymer covered silicon dioxide reflective surface. The optical set-up consists of a visible–near infrared light source, a bifurcated optical fiber and a spectrometer. The outer part of the optical fiber guides the light vertically onto the surface where the biomolecular reactions occur, whereas the reflected light is driven from the central part of the fiber to the spectrometer. A microfluidic module in combination with a pump supplies the reagents at a constant rate. The biomolecular interactions are monitored as shifts of the wavelength of the interference minimum. The proposed methodology was applied for real-time and label-free monitoring mouse gamma-globulins binding onto immobilized anti-mouse IgG antibody. Mouse gamma-globulins at concentrations down to 150 pM were detected in reaction times of 1-min. Regeneration of immobilized antibody was accomplished up to seven times without loss of its activity. In addition, real-time monitoring of hybridization reaction between complementary oligonucleotides was accomplished. The proposed sensor provides a simple, fast, low cost approach for label-free monitoring of biomolecular interactions and therefore it should by suitable for a wide range of analytical applications.     

Visualization of the membrane engineering concept: evidence for the specific orientation of electroinserted antibodies and selective binding of target analytes

Membrane engineering is a generic methodology for increasing the selectivity of a cell biosensor against a target molecule, by electroinserting target‐specific receptor‐like molecules on the cell surface. Previous studies have elucidated the biochemical aspects of the interaction between various analytes (including viruses) and their homologous membrane‐engineered cells. In the present study, purified anti‐biotin antibodies from a rabbit antiserum along with in‐house prepared biotinylated bovine serum albumin (BSA) were used as a model antibody‐antigen pair of molecules for facilitating membrane engineering experiments. It was proven, with the aid of fluorescence microscopy, that (i) membrane‐engineered cells incorporated the specific antibodies in the correct orientation and that (ii) the inserted antibodies are selectively interacting with the homologous target molecules. This is the first time the actual working concept of membrane engineering has been visualized, thus providing a final proof of the concept behind this innovative process. In addition, the fluorescence microscopy measurements were highly correlated with bioelectric measurements done with the aid of a bioelectric recognition assay. Copyright © 2013 John Wiley & Sons, Ltd.     

Single-cell genomics reveal low recombination frequencies in freshwater bacteria of the SAR11 clade

Background

The SAR11 group of Alphaproteobacteria is highly abundant in the oceans. It contains a recently diverged freshwater clade, which offers the opportunity to compare adaptations to salt- and freshwaters in a monophyletic bacterial group. However, there are no cultivated members of the freshwater SAR11 group and no genomes have been sequenced yet.

Results

We isolated ten single SAR11 cells from three freshwater lakes and sequenced and assembled their genomes. A phylogeny based on 57 proteins indicates that the cells are organized into distinct microclusters. We show that the freshwater genomes have evolved primarily by the accumulation of nucleotide substitutions and that they have among the lowest ratio of recombination to mutation estimated for bacteria. In contrast, members of the marine SAR11 clade have one of the highest ratios. Additional metagenome reads from six lakes confirm low recombination frequencies for the genome overall and reveal lake-specific variations in microcluster abundances. We identify hypervariable regions with gene contents broadly similar to those in the hypervariable regions of the marine isolates, containing genes putatively coding for cell surface molecules.

Conclusions

We conclude that recombination rates differ dramatically in phylogenetic sister groups of the SAR11 clade adapted to freshwater and marine ecosystems. The results suggest that the transition from marine to freshwater systems has purged diversity and resulted in reduced opportunities for recombination with divergent members of the clade. The low recombination frequencies of the LD12 clade resemble the low genetic divergence of host-restricted pathogens that have recently shifted to a new host.     

Biocompatible photolithographic process for the patterning of biomolecules

A new approach for the patterning of biomolecule layers is introduced based on the design of a new photoresist material with biocompatible lithographic processing requirements. The photoresist is based on poly(t-butyl acrylate), which allows positive imaging with very dilute basic solutions, tolerable by selected biomolecules used in immunoanalysis. Sensitivity at lambda>300 nm is obtained using a suitable sulfonium salt photoacid generator. Thermal steps also take place under conditions tolerable by biomolecules. Lithographic results on Si wafer substrates show resolution capabilities for equal lines/spaces, down to the range of 5-10 microm under biocompatible conditions. The process is also used on substrates of different geometries, including inner capillary surfaces. The patterning of the inner surface of a polystyrene capillary with mouse IgG is reported to demonstrate the principles of the above approach.     

Molecular Evolution of GII-4 Norovirus Strains

BACKGROUND: Human Noroviruses (NoV) are the major cause of acute nonbacterial gastroenteritis and the leading cause of outbreaks of gastroenteritis worldwide. Genotype II-4 (GII-4) NoV has been shown to spread rapidly and is the most commonly detected strain worldwide, particularly in association with outbreaks. Previously, we have shown that circulating GII-4 NoV strains exist as populations of selectively neutral variants, and that the emergence of epidemic GII-4 NoV strains correlated with mutations in at least two key sites (Sites A and B) within the P2 domain of the surface exposed major capsid protein (VP1). METHODOLOGY: We developed a rapid pyrosequencing method for screening of the two Sites A and B and a homology based modelling system was used to predict the effects of amino acid substitutions at these sites on the antigenic properties of the virus (defined as surface motif types). PRINCIPLE FINDING/CONCLUSION: Here, we describe the characterisation of amino acid diversity at Sites A and B for 1062 GII-4 NoV strains from clinical specimen associated with outbreak of gastroenteritis (2000-2011) and 250 GII-4 NoV sequences from Genbank. Our data identified a high diversity of different Site A and B site combinations at amino acid level and amino acid diversity was higher at Site B than Site A. Site A motifs could be grouped into 3 clusters based on similar surface motif types. We predict that Site A is a major epitope on the virus surface, responsible for defining the antigenic profile, and a more subtle role for Site B, maintaining minor antigenic variation within the virus population.     

Detectability of the global weed Hypochaeris radicata is influenced by species,environment and observer characteristics

Aims To determine the detectability of a global weedy perennial weed Hypochaeris radicata and its relationship with five common observer, species and environmental variables.Methods Trained independent observers conducted time-limited repeat surveys of H. radicata during autumn in an endangered grassy box-gum woodland ecosystem in south-east Australia. Single-species single-season site-occupancy modelling was used to determine if detectability of H. radicata was altered by five covariates, observer, litter height, grazing, maximum plant height and flowering state.Important findings Detectability for H. radicata varied significantly with observer, litter height, plant maximum height and flowering state, but not with grazing. Despite significant observer-specific variation, there was a consistent increase in detectability with plant height and when plants are in flower for all observers. Detectability generally decreased as litter height increases. Perfect or constant detection rates cannot be assumed in plant surveys, even for easily recognizable plants in simple survey conditions. Understanding how detectability is influenced by common survey variables can help improve the efficacy of plant monitoring programs by quantifying the extent of uncertainty in inferences made from survey data, or by determining optimal survey conditions to increase the reliability of collected data. For plants with traits similar to H. radicata, surveying when most plants are at maximum height or in flower, increasing search intensity when litter levels are high and minimizing observer-related heterogeneity are potentially simple and effective ways to reduce detection errors. We speculate that detection rates may be lower, more variable and involve additional covariates when surveying during the peak flowering spring season with the presence of more warm season and taller annual species.     

The genomic determinants of alcohol preference in mice

Searches for the identity of genes that influence the levels of alcohol consumption by humans and other animals have often been driven by presupposition of the importance of particular gene products in determining positively or negatively reinforcing effects of ethanol. We have taken an unbiased approach and performed a meta-analysis across three types of mouse populations to correlate brain gene expression with levels of alcohol intake. Our studies, using filtering procedures based on QTL analysis, produced a list of eight candidate genes with highly heritable expression, which could explain a significant amount of the variance in alcohol preference in mice. Using the Allen Brain Atlas for gene expression, we noted that the candidate genes' expression was localized to the olfactory and limbic areas as well as to the orbitofrontal cortex. Informatics techniques and pathway analysis illustrated the role of the candidate genes in neuronal migration, differentiation, and synaptic remodeling. The importance of olfactory cues, learning and memory formation (Pavlovian conditioning), and cortical executive function, for regulating alcohol intake by animals (including humans), is discussed.     

Bioelectrical impedance assay to monitor changes in cell shape during apoptosis

Apoptosis is a strictly regulated and genetically encoded cell 'suicide' that may be triggered by cytokines, depletion of growth factors or certain chemicals. It is morphologically characterized by severe alterations in cell shape like cell shrinkage and disintegration of cell-cell contacts. We applied a non-invasive electrochemical technique referred to as electric cell-substrate impedance sensing (ECIS) in order to monitor the apoptosis-induced changes in cell shape in an integral and quantitative fashion with a time resolution in the order of minutes. In ECIS the cells are grown directly on the surface of small gold-film electrodes (d = 2 mm). From readings of the electrical impedance of the cell-covered electrode, performed with non-invasive, low amplitude sensing voltages, it is possible to deduce alterations in cell-cell and cell-substrate contacts. To improve the sensitivity of this impedance assay we used endothelial cells derived from cerebral micro-vessels as cellular model systems since these are well known to express electrically tight intercellular junctions. Apoptosis was induced by cycloheximide (CHX) and verified by biochemical and cytological assays. The time course of cell shape changes was followed with unprecedented time resolution by impedance readings at 1 kHz and correlated with biochemical parameters. From impedance readings along a broad frequency range of 1-10(6) Hz we could assign the observed impedance changes to alterations on the subcellular level. We observed that disassembly of barrier-forming tight junctions precedes changes in cell-substrate contacts and correlates strongly with the time course of protease activation.