Re-establishment of Calluna vulgaris (L.) Hull in an eight-year grazing experiment on upland acid grassland

Upland heathland is an internationally important habitat but a large area in the UK has been degraded to acid grassland by intensive livestock grazing. Re-establishment of dwarf shrubs, particularly Calluna vulgaris (L.) Hull, is a key objective for restoring heathland on these sites. A replicated plot-scale experiment was set up to examine effects of disturbance and seed addition on C. vulgaris establishment in a Nardus stricta L. grassland under three grazing regimes: sheep only (1.5 ewes ha^?1 for 10 months per year); cattle only (0.5 heifers ha^?1 in summer only); and, the cattle regime combined with sheep (1.0 ewes ha^?1 for 10 months per year). Early results of the experiment have been reported previously but it was not known if these results were an indication of the longer-term restoration success. Here we evaluate the success of the restoration methods (disturbance, seeding treatments and grazing regime) eight years after the treatments began. In seeded plots, young C. vulgaris plants had greatest above-ground height, dry weight and shoot length if grazing was excluded or the cattle-only regime was applied. C. vulgaris cover was greatest, and increased most, in plots that had been disturbed, seeded and ungrazed or subjected to the cattle-only regime. The vegetation in these plots also became more similar to reference sites with 50% or more cover of C. vulgaris. The invasive Juncus effusus L. was more frequent in disturbed and grazed plots but less frequent in plots with C. vulgaris established from added seed. Previous results that showed the benefits of disturbance and seeding treatments were still valid but changes in the vegetation composition were still occurring and longer-term studies will be needed to determine when grazing regimes including sheep might be reintroduced.     

Comparison and Validation of Methods To Quantify Cry1Ab Toxin from Bacillus thuringiensis for Standardization of Insect Bioassays

Standardization of toxin preparations derived from Bacillus thuringiensis (Berliner) used in laboratory bioassays is critical for accurately assessing possible changes in the susceptibility of field populations of target pests. Different methods were evaluated to quantify Cry1Ab, the toxin expressed by 80% of the commercially available transgenic maize that targets the European corn borer, Ostrinia nubilalis (Hübner). We compared three methods of quantification on three different toxin preparations from independent sources: enzyme-linked immunosorbent assay (ELISA), sodium dodecyl sulfate-polyacrylamide gel electrophoresis and densitometry (SDS-PAGE/densitometry), and the Bradford assay for total protein. The results were compared to those obtained by immunoblot analysis and with the results of toxin bioassays against susceptible laboratory colonies of O. nubilalis. The Bradford method resulted in statistically higher estimates than either ELISA or SDS-PAGE/densitometry but also provided the lowest coefficients of variation (CVs) for estimates of the Cry1Ab concentration (from 2.4 to 5.4%). The CV of estimates obtained by ELISA ranged from 12.8 to 26.5%, whereas the CV of estimates obtained by SDS-PAGE/densitometry ranged from 0.2 to 15.4%. We standardized toxin concentration by using SDS-PAGE/densitometry, which is the only method specific for the 65-kDa Cry1Ab protein and is not confounded by impurities detected by ELISA and Bradford assay for total protein. Bioassays with standardized Cry1Ab preparations based on SDS-PAGE/densitometry showed no significant differences in LC₅₀ values, although there were significant differences in growth inhibition for two of the three Cry1Ab preparations. However, the variation in larval weight caused by toxin source was only 4% of the total variation, and we conclude that standardization of Cry1Ab production and quantification by SDS-PAGE/densitometry may improve data consistency in monitoring efforts to identify changes in insect susceptibility to Cry1Ab.     

A Genotype-First Approach for the Molecular and Clinical Characterization of Uncommon De Novo Microdeletion of 20q13.33

Background

Subtelomeric deletions of the long arm of chromosome 20 are rare, with only 11 described in the literature. Clinical features of individuals with these microdeletions include severe limb malformations, skeletal abnormalities, growth retardation, developmental and speech delay, mental retardation, seizures and mild, non-specific dysmorphic features.

Methodology/Principal Findings

We characterized microdeletions at 20q13.33 in six individuals referred for genetic evaluation of developmental delay, mental retardation, and/or congenital anomalies. A comparison to previously reported cases of 20q13.33 microdeletion shows phenotypic overlap, with clinical features that include mental retardation, developmental delay, speech and language deficits, seizures, and behavior problems such as autistic spectrum disorder. There does not appear to be a clinically recognizable constellation of dysmorphic features among individuals with subtelomeric 20q microdeletions.

Conclusions/Significance

Based on genotype-phenotype correlation among individuals in this and previous studies, we discuss several possible candidate genes for specific clinical features, including ARFGAP1, CHRNA4 and KCNQ2 and neurodevelopmental deficits. Deletion of this region may play an important role in cognitive development.     

A Single Amino Acid Substitution Prevents Recognition of a Dominant Human Aquaporin-4 Determinant in the Context of HLA-DRB1*03:01 by a Murine TCR

BackgroundAquaporin 4 (AQP4) is considered a putative autoantigen in patients with Neuromyelitis optica (NMO), an autoinflammatory disorder of the central nervous system (CNS). HLA haplotype analyses of patients with NMO suggest a positive association with HLA-DRB1* 03:01. We previously showed that the human (h) AQP4 peptide 281–300 is the dominant immunogenic determinant of hAQP4 in the context of HLA-DRB1*03:01. This immunogenic peptide stimulates a strong Th₁ and Th₁₇ immune response. AQP4_281-300-specific encephalitogenic CD4⁺ T cells should initiate CNS inflammation that results in a clinical phenotype in HLA-DRB1*03:01 transgenic mice.MethodsControlled study with humanized experimental animals. HLA-DRB1*03:01 transgenic mice were immunized with hAQP4_281-300, or whole-length hAQP4 protein emulsified in complete Freund’s adjuvant. Humoral immune responses to both antigens were assessed longitudinally. In vivo T cell frequencies were assessed by tetramer staining. Mice were followed clinically, and the anterior visual pathway was tested by pupillometry. CNS tissue was examined histologically post-mortem. Flow cytometry was utilized for MHC binding assays and to immunophenotype T cells, and T cell frequencies were determined by ELISpot assay.ResultsImmunization with hAQP4_281-300 resulted in an in vivo expansion of antigen-specific CD4⁺ T cells, and an immunoglobulin isotype switch. HLA-DRB1*03:01 TG mice actively immunized with hAQP4_281-300, or with whole-length hAQP4 protein were resistant to developing a neurological disease that resembles NMO. Experimental mice show no histological evidence of CNS inflammation, nor change in pupillary responses. Subsequent analysis reveals that a single amino acid substitution from aspartic acid in hAQP4 to glutamic acid in murine (m)AQP4 at position 290 prevents the recognition of hAQP4_281-300 by the murine T cell receptor (TCR).ConclusionInduction of a CNS inflammatory autoimmune disorder by active immunization of HLA-DRB1*03:01 TG mice with human hAQP4_281-300 will be complex due to a single amino acid substitution. The pathogenic role of T cells in this disorder remains critical despite these observations.     

Modeling Relapsing Disease Dynamics in a Host-Vector Community

Vector-borne diseases represent a threat to human and wildlife populations and mathematical models provide a means to understand and control epidemics involved in complex host-vector systems. The disease model studied here is a host-vector system with a relapsing class of host individuals, used to investigate tick-borne relapsing fever (TBRF). Equilibrium analysis is performed for models with increasing numbers of relapses and multiple hosts and the disease reproduction number, R₀, is generalized to establish relationships with parameters that would result in the elimination of the disease. We show that host relapses in a single competent host-vector system is needed to maintain an endemic state. We show that the addition of an incompetent second host with no relapses increases the number of relapses needed for maintaining the pathogen in the first competent host system. Further, coupling of the system with hosts of differing competencies will always reduce R₀, making it more difficult for the system to reach an endemic state.     

The Perinatal Risk Index: Early Risks Experienced by Domestic Adoptees in the United States

We aimed to assess comprehensively the prevalence of perinatal risks experienced by a potentially high-risk yet understudied population of children domestically adopted in the United States. Data are from participant report and medical records from mothers (n = 580) who completed a domestic adoption placement with nonrelatives at or near birth (Mean placement age = 7 days). We describe a comprehensive measure of perinatal risks, including divergences from previous assessment tools and the incorporation of multiple reporters, and report the prevalence of various types of perinatal risks. The prevalence of each specific risk factor was generally low, although several risks were more prevalent in this sample than estimates from nationally representative publicly available data. Nearly the entire sample (99%) experienced some type of risk exposure. Birth mothers who placed their children for adoption domestically in the US experience higher levels of perinatal risks than the national average, but not for all specific types of risk. Thus, the developmental trajectories of children adopted domestically may systematically differ from the general population to the extent that these specific perinatal risks impact development.     

Differentiation of nonbeating embryonic stem cells into beating cardiomyocytes is dependent on downregulation of PKC beta and zeta in concert with upregulation of PKC epsilon

Cardiomyocyte differentiation overall has been analyzed in vivo and in vitro at the molecular level by homologous recombination, gene mutation studies, and by transgenics; however, the roles of many signal transduction mechanisms that drive this differentiation process are still not fully understood. One set of signal transduction components that has been studied in detail in mature, differentiated cardiomyocytes is the PKC isotype superfamily. However, while the function of each isotype is slowly being uncovered in adult cardiomyocytes, limited information persists concerning their function in the differentiation process of cardiomyocytes. To begin analyzing the function of specific PKC isotypes in the differentiation process, we employed an established model for differentiating ES cells into cardiomyocyte-positive embryoid bodies (EBs) in vitro. RT-PCR, Western analyses, and confocal microscopy all showed that the expression of specific PKC isotypes was significantly changed as ES cells differentiated into cardiomyocytes. More importantly, by using antagonists specific for each isotype we found that this change was a final step in the differentiation process. PKC beta and zeta downregulation served to promote differentiation (beating), while upregulation of PKC epsilon appeared to amplify differentiation (beating). Finally, melding classical tools (i.e., ionic exchange glass beads) with recently developed methods for differentiating ES cells creates a possible novel technique for investigating differentiation of ES cells into cardiomyocytes as well as other cell types.