Maturation and trafficking markers on rotavirus-specific B cells during acute infection and convalescence in children

We have previously studied B cells, from people and mice, that express rotavirus-specific surface immunoglobulin (RV-sIg) by flow cytometry with recombinant virus-like particles that contain green fluorescent protein. In the present study we characterized circulating B cells with RV-sIg in children with acute and convalescent infection. During acute infection, circulating RV-sIgD(-) B cells are predominantly large, CD38(high), CD27(high), CD138(+/-), CCR6(-), alpha4beta7(+), CCR9(+), CCR10(+), cutaneous lymphocyte antigen-negative (CLA(-)), L-selectin(int/-), and sIgM(+), sIgG(-), sIgA(+/-) lymphocytes. This phenotype likely corresponds to gut-targeted plasma cells and plasmablasts. During convalescence the phenotype switches to small and large lymphocytes, CD38(int/-), CD27(int/-), CCR6(+), alpha4beta7(+/-), CCR9(+/-) and CCR10(-), most likely representing RV-specific memory B cells with both gut and systemic trafficking profiles. Of note, during acute RV infection both total and RV-specific murine IgM and IgA antibody-secreting cells migrate efficiently to CCL28 (the CCR10 ligand) and to a lesser extent to CCL25 (the CCR9 ligand). Our results show that CCR10 and CCR9 can be expressed on IgM as well as IgA antibody-secreting cells in response to acute intestinal infection, likely helping target these cells to the gut. However, these intestinal infection-induced plasmablasts lack the CLA homing receptor for skin, consistent with mechanisms of differential CCR10 participation in skin T versus intestinal plasma cell homing. Interestingly, RV memory cells generally lack CCR9 and CCR10 and instead express CCR6, which may enable recruitment to diverse epithelial sites of inflammation.     

Sensitivity of methods for estimating breeding values using genetic markers to the number of QTL and distribution of QTL variance

The objective of this simulation study was to compare the effect of the number of QTL and distribution of QTL variance on the accuracy of breeding values estimated with genomewide markers (MEBV). Three distinct methods were used to calculate MEBV: a Bayesian Method (BM), Least Angle Regression (LARS) and Partial Least Square Regression (PLSR). The accuracy of MEBV calculated with BM and LARS decreased when the number of simulated QTL increased. The accuracy decreased more when QTL had different variance values than when all QTL had an equal variance. The accuracy of MEBV calculated with PLSR was affected neither by the number of QTL nor by the distribution of QTL variance. Additional simulations and analyses showed that these conclusions were not affected by the number of individuals in the training population, by the number of markers and by the heritability of the trait. Results of this study show that the effect of the number of QTL and distribution of QTL variance on the accuracy of MEBV depends on the method that is used to calculate MEBV.     

The genetic rescue of the Florida panther

We examine the consequences of panthers introduced from Texas into south Florida, an area housing a small, isolated, inbred and distinct subspecies ( Puma concolor coryi ). Once part of a continuous, widespread population, panthers became isolated in south Florida more than a century ago. Numbers declined and the occurrence of genetic defects increased. Hoping to reverse the genetic damage, managers introduced eight female panthers from Texas into south Florida in the mid-1990s. This action was highly controversial and we explain the arguments for and against the intervention. We synthesized data systematically collected on the Florida panthers from before, during and after this management intervention. These data include information on movements, breeding, mortality, survivorship and range. There is no evidence that purebred Florida females produce fewer kittens at a later age or less often than do hybrid cats (i.e. those with a Texas ancestor). Hybrid kittens have about a three times higher chance of becoming adults as do purebred ones. Hybrid adult females survive better than purebred females; there is no obvious difference between the males. Males die younger than females, are more often killed by other males and are more likely to disperse longer distances into habitats that are dangerous to them. Hybrids are expanding the known range of habitats panthers occupy and use.     

Antiviral and regulatory T cell immunity in a patient with stromal interaction molecule 1 deficiency

Stromal interaction molecule 1 (STIM1) deficiency is a rare genetic disorder of store-operated calcium entry, associated with a complex syndrome including immunodeficiency and immune dysregulation. The link from the molecular defect to these clinical manifestations is incompletely understood. We report two patients with a homozygous R429C point mutation in STIM1 completely abolishing store-operated calcium entry in T cells. Immunological analysis of one patient revealed that despite the expected defect of T cell proliferation and cytokine production in vitro, significant antiviral T cell populations were generated in vivo. These T cells proliferated in response to viral Ags and showed normal antiviral cytotoxicity. However, antiviral immunity was insufficient to prevent chronic CMV and EBV infections with a possible contribution of impaired NK cell function and a lack of NKT cells. Furthermore, autoimmune cytopenia, eczema, and intermittent diarrhea suggested impaired immune regulation. FOXP3-positive regulatory T (Treg) cells were present but showed an abnormal phenotype. The suppressive function of STIM1-deficient Treg cells in vitro, however, was normal. Given these partial defects in cytotoxic and Treg cell function, impairment of other immune cell populations probably contributes more to the pathogenesis of immunodeficiency and autoimmunity in STIM1 deficiency than previously appreciated.     

Ecophysiological analysis of moss-dominated biological soil crusts and their separate components from the Succulent Karoo, South Africa

Biological soil crusts, formed by an association of soil particles with cyanobacteria, lichens, mosses, fungi and bacteria in varying proportions, live in or directly on top of the uppermost soil layer. To evaluate their role in the global carbon cycle, gas exchange measurements were conducted under controlled conditions. Moss-dominated soil crusts were first analyzed as moss tufts on soil, then the mosses were removed and the soil was analyzed separately to obtain the physiological response of both soil and individual moss stems. Net photosynthetic response of moss stems and complete crusts was decreased by insufficient and excess amounts of water, resulting in optimum curves with similar ranges of optimum water content. Light saturation of both sample types occurred at high irradiance, but moss stems reached light compensation and saturation points at lower values. Optimum temperatures of moss stems ranged between 22 and 27°C, whereas complete crusts reached similar net photosynthesis between 7 and 27°C. Under optimum conditions, moss stems reached higher net photosynthesis (4.0 vs. 2.8 μmol m^?2 s^?1) and lower dark respiration rates (?0.9 vs. ?2.4 μmol m^?2 s^?1). Respiration rates of soil without moss stems were high (up to ?2.0 μmol m^?2 s^?1) causing by far lower absolute values of NP/DR ratios of soil crusts as compared to moss stems. In carbon balances, it therefore has to be clearly distinguished between measurements of soil crust components versus complete crusts. High rates of soil respiration may be caused by leaching of mosses, creating high-nutrient microsites that favor microorganism growth.     

Induction of adhesion-dependent signals using low-intensity ultrasound

In multicellular organisms, cell behavior is dictated by interactions with the extracellular matrix. Consequences of matrix-engagement range from regulation of cell migration and proliferation, to secretion and even differentiation. The signals underlying each of these complex processes arise from the molecular interactions of extracellular matrix receptors on the surface of the cell. Integrins are the prototypic receptors and provide a mechanical link between extracellular matrix and the cytoskeleton, as well as initiating some of the adhesion-dependent signaling cascades. However, it is becoming increasingly apparent that additional transmembrane receptors function alongside the integrins to regulate both the integrin itself and signals downstream. The most elegant of these examples is the transmembrane proteoglycan, syndecan-4, which cooperates with α(5)β(1)-integrin during adhesion to fibronectin. In vivo models demonstrate the importance of syndecan-4 signaling, as syndecan-4-knockout mice exhibit healing retardation due to inefficient fibroblast migration. In wild-type animals, migration of fibroblasts toward a wound is triggered by the appearance of fibronectin that leaks from damaged capillaries and is deposited by macrophages in injured tissue. Therefore there is great interest in discovering strategies that enhance fibronectin-dependent signaling and could accelerate repair processes. The integrin-mediated and syndecan-4-mediated components of fibronectin-dependent signaling can be separated by stimulating cells with recombinant fibronectin fragments. Although integrin engagement is essential for cell adhesion, certain fibronectin-dependent signals are regulated by syndecan-4. Syndecan-4 activates the Rac1 protrusive signal, causes integrin redistribution, triggers recruitment of cytoskeletal molecules, such as vinculin, to focal adhesions, and thereby induces directional migration. We have looked for alternative strategies for activating such signals and found that low-intensity pulsed ultrasound (LIPUS) can mimic the effects of syndecan-4 engagement. In this protocol we describe the method by which 30 mW/cm(2), 1.5 MHz ultrasound, pulsed at 1 kHz (Fig. 1) can be applied to fibroblasts in culture (Fig. 2) to induce Rac1 activation and focal adhesion formation. Ultrasound stimulation is applied for a maximum of 20 minutes, as this combination of parameters has been found to be most efficacious for acceleration of clinical fracture repair. The method uses recombinant fibronectin fragments to engage α(5)β(1)-integrin, without engagement of syndecan-4, and requires inhibition of protein synthesis by cycloheximide to block deposition of additional matrix by the fibroblasts. The positive effect of ultrasound on repair mechanisms is well documented, and by understanding the molecular effect of ultrasound in culture we should be able to refine the therapeutic technique to improve clinical outcomes.     

Maize histone H2B-mCherry: a new fluorescent chromatin marker for somatic and meiotic chromosome research

Cytological studies of fluorescent proteins are rapidly yielding insights into chromatin structure and dynamics. Here we describe the production and cytological characterization of new transgenic maize lines expressing a fluorescent histone fusion protein, H2B-mCherry. The transgene is expressed under the control of the maize ubiquitin1 promoter, including its first exon and intron. Polymerase chain reaction-based genotyping and root-tip microscopy showed that most of the lines carrying the transgene also expressed it, producing bright uniform staining of nuclei. Further, plants showing expression in root tips at the seedling stage also showed expression during meiosis, late in the life cycle. Detailed high-resolution three-dimensional imaging of cells and nuclei from various somatic and meiotic cell types showed that H2B-mCherry produced remarkably clear images of chromatin and chromosome fiber morphology, as seen in somatic, male meiotic prophase, and early microgametophyte cells. H2B-mCherry also yielded distinct nucleolus staining and was shown to be compatible with fluorescence in situ hybridization. We found several instances where H2B-mCherry was superior to DAPI as a generalized chromatin stain. Our study establishes these histone H2B-mCherry lines as new biological reagents for visualizing chromatin structure, chromosome morphology, and nuclear dynamics in fixed and living cells in a model plant genetic system.     

The Revised Classification of Eukaryotes

This revision of the classification of eukaryotes, which updates that of Adl et al. [J. Eukaryot. Microbiol. 52 (2005) 399], retains an emphasis on the protists and incorporates changes since 2005 that have resolved nodes and branches in phylogenetic trees. Whereas the previous revision was successful in re‐introducing name stability to the classification, this revision provides a classification for lineages that were then still unresolved. The supergroups have withstood phylogenetic hypothesis testing with some modifications, but despite some progress, problematic nodes at the base of the eukaryotic tree still remain to be statistically resolved. Looking forward, subsequent transformations to our understanding of the diversity of life will be from the discovery of novel lineages in previously under‐sampled areas and from environmental genomic information.