Direct in vivo screening of intrabody libraries constructed on a highly stable single-chain framework

Single-chain Fv antibody fragments (scFv) represent a convenient antibody format for intracellular expression in eukaryotic or prokaryotic cells. These so-called intrabodies have great potential in functional genomics as a tool to study the function of newly identified proteins in vivo, for example by binding-induced modulation of their activity or by blocking interactions with other proteins. However, the intracellular expression and activity of many single-chain Fvs are limited by their instability and folding efficiency in the reducing intracellular environment, where the highly conserved intrachain disulfide bonds do not form. In the present work, we used an in vivo selection system to isolate novel antigen-binding intrabodies. We screened two intrabody libraries carrying a randomized third hypervariable loop onto the heavy chain of a stable framework, which had been further optimized by random mutagenesis for better behavior in the selection system, and we biophysically characterized the selected variants to interpret the outcome of the selection. Our results show that single-framework intrabody libraries can be directly screened in vivo to rapidly select antigen-specific intrabodies.     

The chemical synthesis of oligoribonucleotides with selectively placed 2'-O-phosphates

A phosphoramidite, solid support method for the chemical synthesis of oligoribonucleotides containing 2'-O-phosphate at a selected position is presented. Synthesis of these oligoribonucleotides is based on uridine- and adenosine-(2'-O-phosphate)-3'-phosphoramidites, and a new condition for removal of 2'-O-phosphate protecting groups, which does not cleave internucleotide bonds. The structure of oligoribonucleotides with 2'-O-phosphate has been proven by enzymatic digestions and dephosphorylation by yeast 2'-phosphotransferase.     

Predicting the performance of a genetic testing service for cancer susceptibility

A genetic testing service can determine which members of a population might benefit most from cancer prevention. The eligibility criteria will affect the number of people who use a service and the proportion who test positive. This affects both the service's costs and benefits. The goal of this study was to create computer software that predicts the effect of eligibility restrictions on the performance of a genetic testing service. The software allows eligibility restrictions based on age, gender, and family history of disease. As performance measures, we considered the sensitivity and specificity of eligibility criteria to identify people with genetic cancer susceptibility, the likelihood of genetic susceptibility among people who are eligible for the service, and the likelihood of genetic susceptibility among people who are ineligible. We compared the performance predicted by our model with the observed performance of the Hereditary Cancer Program at the BC Cancer Agency, and studied the effects of changes to model parameters. There was good agreement between model predictions and observed outcomes, however, performance measures were affected by changes to the underlying model parameters. Computer software to predict the performance of a genetic testing service for cancer susceptibility is implemented on the website http://142.103.207.51:8080/gtsim.     

Performance of a mobile mechanical screen to improve the commercial quality of wood chips for energy

The study analyzed the performance of a mobile screening device for upgrading coarse wood chips to residential user standards, by removing oversize particles and fines. The machine was designed for transportation to forest landings, logistic terminals and plant chip yards. Average productivity was 1.9 oven-dry tons (odt) h⁻¹, corresponding to a screening cost of 28.5 € odt⁻¹. This figure was lower than the price increase obtained by upgrading industrial chips to residential user standards. Hence, screening offered a profit of 4.7 € odt⁻¹, or 16% of the original screening cost. The screening process was capable of upgrading chips from industrial to residential specifications, by reducing the incidence of oversize particles below the 1% critical threshold. Screening also allowed a substantial reduction in the content of fines. A similar effect was not verified for crushed wood, which failed to meet the specifications for residential fuel.     

IL-33 induces granzyme C expression in murine mast cells via an MSK1/2-CREB-dependent pathway

Granzymes comprise a group of proteases involved in the killing of infected or cancerous cells by the immune system. Although best studied in T cells and natural killer (NK) cells, they are also expressed in some innate immune cells. Granzymes B and C are encoded in the mouse chymase locus that also encodes a number of mast cell-specific proteases. In line with this, mast cells can express granzyme B, although how this is regulated and their ability to express other granzymes is less well studied. We therefore examined how IL-33, a cytokine able to activate mast cells but not induce degranulation, regulated granzyme B and C levels in mast cells. Granzyme C, but not B, mRNA was strongly up-regulated in bone marrow-derived mast cells following IL-33 stimulation and there was a corresponding increase in granzyme C protein. These increases in both granzyme C mRNA and protein were blocked by a combination of the p38α/β MAPK inhibitor VX745 and the MEK1/2 inhibitor PD184352, which blocks the activation of ERK1/2. ERK1/2 and p38α activate the downstream kinases, mitogen and stress-activated kinases (MSK) 1 and 2, and IL-33 stimulated the phosphorylation of MSK1 and its substrate CREB in an ERK1/2 and p38-dependent manner. The promoter for granzyme C contains a potential CREB-binding site. Bone marrow-derived mast cells from either MSK1/2 double knockout or CREB Ser133Ala knockin mice were unable to up-regulate granzyme C. Together these results indicate that IL-33-induced granzyme C expression in mast cells is regulated by an MSK1/2-CREB-dependent pathway.     

A portable surface plasmon resonance sensor system for real-time monitoring of small to large analytes

Many environmental applications exist for biosensors capable of providing real-time analyses. One pressing current need is monitoring for agents of chemical- and bio-terrorism. These applications require systems that can rapidly detect small organics including nerve agents, toxic proteins, viruses, spores and whole microbes. A second area of application is monitoring for environmental pollutants. Processing of grab samples through chemical laboratories requires significant time delays in the analyses, preventing the rapid mapping and cleanup of chemical spills. The current state of development of miniaturized, integrated surface plasmon resonance (SPR) sensor elements has allowed for the development of inexpensive, portable biosensor systems capable of the simultaneous analysis of multiple analytes. Most of the detection protocols make use of antibodies immobilized on the sensor surface. The Spreeta 2000 SPR biosensor elements manufactured by Texas Instruments provide three channels for each sensor element in the system. A temperature-controlled two-element system that monitors for six analytes is currently in use, and development of an eight element sensor system capable of monitoring up to 24 different analytes will be completed in the near future. Protein toxins can be directly detected and quantified in the low picomolar range. Elimination of false positives and increased sensitivity is provided by secondary antibodies with specificity for different target epitopes, and by sensor element redundancy. Inclusion of more than a single amplification step can push the sensitivity of toxic protein detection to femtomolar levels. The same types of direct detection and amplification protocols are used to monitor for viruses and whole bacteria or spores. Special protocols are required for the detection of small molecules. Either a competition type assay where the presence of analyte inhibits the binding of antibodies to surface-immobilized analyte, or a displacement assay, where antibodies bound to analyte on the sensor surface are displaced by free analyte, can be used. The small molecule detection assays vary in sensitivity from the low micromolar range to the high picomolar.

     

Human bites of the eyelid

Five patients with traumatic colobomas of the eyelid secondary to human bites were surgically repaired with retrieved autogenous tissue. All patients were treated with prophylactic intravenous antibiotics. Surgical repair consisted of debridement of the autograft, meticulous layered closure of the autograft to the wound, and placement of a lid margin suture. In two of the patients, mild upper eyelid retraction was noted, and two patients had loss of cilia.