RT-PCR analysis and stress response capacity of transgenic gshI-poplar clones (Populus x canescens) in response to paraquat exposure

Stress response capacity (Fv/Fm at 690 nm and F690/F735 at Fmax) of untransformed hybrid poplar, Populus x canescens (P tremula x P alba), and two transgenic lines overexpressing gamma-ECS (gamma-glutamylcysteine synthetase) either in the cytosol (cyt-ECS) or in the chloroplast (chl-ECS) was studied in response to the herbicide paraquat (4.0 x 10(-9) to 4.0 x 10(-6) M) for 21 days. Significant differences at sublethal (4.0 x 10(-7) M) and bleaching (4.0 x 10(-6) M) concentrations of paraquat were observed with about a two-fold and eight-fold decrease in the photosynthetic activity (Fv/Fm at 690 nm and F690/F735 at Fmax), respectively. None of the gshI transgenic lines (cyt-ECS, chl-ECS) with elevated GSH content exhibited significant tolerance to paraquat. Semiquantitative RT-PCR of the cyt-ECS clone was used for gene expression analysis of the nuclear encoded rbcS gene and the stress responsive gst gene. Expression of the constitutively expressed 26SrRNA ribosomal gene was probed as a control for all RT-PCR reactions. The relative intensities of gene expressions normalized to the level of 26SrRNA intensity showed a 50% decrease in the nuclear encoded rbcS expression and a 120% increase in the stress responsive gst gene expression of the paraquat treated (4.0 x 10(-7) M) samples of the transgenic poplar line (cyt-ECS).     

A role for dendritic cells in the dissemination of mycobacterial infection

The ability of mycobacteria to disseminate from the initial site of infection has an important role in immune priming and in the seeding of disease in multiple organs. To study this phenomenon, we used flow cytometry to analyse the distribution of green fluorescent protein-labelled BCG amongst different populations of antigen-presenting cells in the lungs of mice following intranasal infection, and monitored appearance of live bacteria in the draining mediastinal lymph nodes. BCG predominantly infected alveolar macrophages (CD11c(+)/CD11b(-)) and dendritic cells (CD11c(+)/CD11b(+)) in the lungs. The bacteria that disseminated to the lymph node were found in dendritic cells. The results are consistent with a model in which mycobacterial dissemination from the lung is initiated by the migration of infected dendritic cells to the draining lymph nodes.     

Molecular detection of QTLs for agronomic and quality traits in a doubled haploid population derived from two Canadian wheats (Triticum aestivum L.)

Development of high-yielding wheat varieties with good end-use quality has always been a major concern for wheat breeders. To genetically dissect quantitative trait loci (QTLs) for yield-related traits such as grain yield, plant height, maturity, lodging, test weight and thousand-grain weight, and for quality traits such as grain and flour protein content, gluten strength as evaluated by mixograph and SDS sedimentation volume, an F₁-derived doubled haploid (DH) population of 185 individuals was developed from a cross between a Canadian wheat variety “AC Karma” and a breeding line 87E03-S2B1. A genetic map was constructed based on 167 marker loci, consisting of 160 microsatellite loci, three HMW glutenin subunit loci: Glu-A1, Glu-B1 and Glu-D1, and four STS-PCR markers. Data for investigated traits were collected from three to four environments in Manitoba, Canada. QTL analyses were performed using composite interval mapping. A total of 50 QTLs were detected, 24 for agronomic traits and 26 for quality-related traits. Many QTLs for correlated traits were mapped in the same genomic regions forming QTL clusters. The largest QTL clusters, consisting of up to nine QTLs, were found on chromosomes 1D and 4D. HMW glutenin subunits at Glu-1 loci had the largest effect on breadmaking quality; however, other genomic regions also contributed genetically to breadmaking quality. QTLs detected in the present study are compared with other QTL analyses in wheat.     

TLR2 and TLR4 differentially regulate B7-1 resulting in distinct cytokine responses to the mycoplasma superantigen MAM as well as to disease induced by Mycoplasma arthritidis

Mycoplasma arthritidis mitogen (MAM) is a superantigen secreted by M. arthritidis, an agent of murine arthritis and toxicity. We previously demonstrated that C3H mouse sub-strains differing in expression of Toll-like receptor 4 (TLR4), differed in immune reactivity to MAM due to differential engagement of TLR2 and TLR4. Here we examine the role of B7 co-stimulatory molecules in immune outcome and disease manifestations resulting from these different MAM/TLR2 and MAM/TLR4 interactions. Injections of MAM into C3H/HeJ mice upregulated expression of B7-1 but not B7-2 on peritoneal adherent cells, whereas B7-1 expression was lower on cells from C3H/HeSnJ mice. Anti-B7-1 antibody but not anti-B7-2, injected in vivo, changed the type 1 cytokines in MAM-injected C3H/HeJ mice to a type 2 cytokines and, conversely, the type 2 response in C3H/HeSnJ mice injected with anti-B7-1 shifted to a type 1 pattern. Whereas anti-B7-2 exerted no effect on disease in either mouse strain, anti-B7-1 significantly delayed the lethal toxicity of M. arthritidis in C3H/HeJ mice but enhanced arthritis in C3H/HeSnJ mice. Thus, TLR-mediated regulation of B7-1 results in diverse cytokine profiles in C3H sub-strains, and that the interaction of MAM with different TLR(s) may differentially affect cytokine responses and ultimately, M. arthritidis disease.     

Relative stabilities of IgG1 and IgG4 Fab domains: Influence of the light-heavy interchain disulfide bond architecture

The stability of therapeutic antibodies is a prime pharmaceutical concern. In this work we examined thermal stability differences between human IgG1 and IgG4 Fab domains containing the same variable regions using the thermofluor assay. It was found that the IgG1 Fab domain is up to 11°C more stable than the IgG4 Fab domain containing the same variable region. We investigated the cause of this difference with the aim of developing a molecule with the enhanced stability of the IgG1 Fab and the biological properties of an IgG4 Fc. We found that replacing the seven residues, which differ between IgG1 C(H) 1 and IgG4 C(H) 1 domains, while retaining the native IgG1 light-heavy interchain disulfide (L-H) bond, did not affect thermal stability. Introducing the IgG1 type L-H interchain disulfide bond (DSB) into the IgG4 Fab resulted in an increase in thermal stability to levels observed in the IgG1 Fab with the same variable region. Conversely, replacement of the IgG1 L-H interchain DSB with the IgG4 type L-H interchain DSB reduced the thermal stability. We utilized the increased stability of the IgG1 Fab and designed a hybrid antibody with an IgG1 C(H) 1 linked to an IgG4 Fc via an IgG1 hinge. This construct has the expected biophysical properties of both the IgG4 Fc and IgG1 Fab domains and may therefore be a pharmaceutically relevant format.     

Valid and reliable instruments for arm-hand assessment at ICF activity level in persons with hemiplegia: a systematic review

Background

Loss of arm-hand performance due to a hemiparesis as a result of stroke or cerebral palsy (CP), leads to large problems in daily life of these patients. Assessment of arm-hand performance is important in both clinical practice and research. To gain more insight in e.g. effectiveness of common therapies for different patient populations with similar clinical characteristics, consensus regarding the choice and use of outcome measures is paramount. To guide this choice, an overview of available instruments is necessary. The aim of this systematic review is to identify, evaluate and categorize instruments, reported to be valid and reliable, assessing arm-hand performance at the ICF activity level in patients with stroke or cerebral palsy.

Methods

A systematic literature search was performed to identify articles containing instruments assessing arm-hand skilled performance in patients with stroke or cerebral palsy. Instruments were identified and divided into the categories capacity, perceived performance and actual performance. A second search was performed to obtain information on their content and psychometrics.

Results

Regarding capacity, perceived performance and actual performance, 18, 9 and 3 instruments were included respectively. Only 3 of all included instruments were used and tested in both patient populations. The content of the instruments differed widely regarding the ICF levels measured, assessment of the amount of use versus the quality of use, the inclusion of unimanual and/or bimanual tasks and the inclusion of basic and/or extended tasks.

Conclusions

Although many instruments assess capacity and perceived performance, a dearth exists of instruments assessing actual performance. In addition, instruments appropriate for more than one patient population are sparse. For actual performance, new instruments have to be developed, with specific focus on the usability in different patient populations and the assessment of quality of use as well as amount of use. Also, consensus about the choice and use of instruments within and across populations is needed.     

Breast cancer risk and 6q22.33: combined results from Breast Cancer Association Consortium and Consortium of Investigators on Modifiers of BRCA1/2

Recently, a locus on chromosome 6q22.33 (rs2180341) was reported to be associated with increased breast cancer risk in the Ashkenazi Jewish (AJ) population, and this association was also observed in populations of non-AJ European ancestry. In the present study, we performed a large replication analysis of rs2180341 using data from 31,428 invasive breast cancer cases and 34,700 controls collected from 25 studies in the Breast Cancer Association Consortium (BCAC). In addition, we evaluated whether rs2180341 modifies breast cancer risk in 3,361 BRCA1 and 2,020 BRCA2 carriers from 11 centers in the Consortium of Investigators of Modifiers of BRCA1/2 (CIMBA). Based on the BCAC data from women of European ancestry, we found evidence for a weak association with breast cancer risk for rs2180341 (per-allele odds ratio (OR)?=?1.03, 95% CI 1.00-1.06, p?=?0.023). There was evidence for heterogeneity in the ORs among studies (I(2)?=?49.3%; p?=?<0.004). In CIMBA, we observed an inverse association with the minor allele of rs2180341 and breast cancer risk in BRCA1 mutation carriers (per-allele OR?=?0.89, 95%CI 0.80-1.00, p?=?0.048), indicating a potential protective effect of this allele. These data suggest that that 6q22.33 confers a weak effect on breast cancer risk.     

Response to genomic selection: The Bulmer effect and the potential of genomic selection when the number of phenotypic records is limiting

Background

Over the last ten years, genomic selection has developed enormously. Simulations and results on real data suggest that breeding values can be predicted with high accuracy using genetic markers alone. However, to reach high accuracies, large reference populations are needed. In many livestock populations or even species, such populations cannot be established when traits are difficult or expensive to record, or when the population size is small. The value of genomic selection is then questionable.

Methods

In this study, we compare traditional breeding schemes based on own performance or progeny information to genomic selection schemes, for which the number of phenotypic records is limiting. Deterministic simulations were performed using selection index theory. Our focus was on the equilibrium response obtained after a few generations of selection. Therefore, we first investigated the magnitude of the Bulmer effect with genomic selection.

Results

Results showed that the reduction in response due to the Bulmer effect is the same for genomic selection as for selection based on traditional BLUP estimated breeding values, and is independent of the accuracy of selection. The reduction in response with genomic selection is greater than with selection based directly on phenotypes without the use of pedigree information, such as mass selection. To maximize the accuracy of genomic estimated breeding values when the number of phenotypic records is limiting, the same individuals should be phenotyped and genotyped, rather than genotyping parents and phenotyping their progeny. When the generation interval cannot be reduced with genomic selection, large reference populations are required to obtain a similar response to that with selection based on BLUP estimated breeding values based on own performance or progeny information. However, when a genomic selection scheme has a moderate decrease in generation interval, relatively small reference population sizes are needed to obtain a similar response to that with selection on traditional BLUP estimated breeding values.

Conclusions

When the trait of interest cannot be recorded on the selection candidate, genomic selection schemes are very attractive even when the number of phenotypic records is limited, because traditional breeding requires progeny testing schemes with long generation intervals in those cases.