Bordetella pertussis Strain Lacking Pertactin and Pertussis Toxin

A Bordetella pertussis strain lacking 2 acellular vaccine immunogens, pertussis toxin and pertactin, was isolated from an unvaccinated infant in New York State in 2013. Comparison with a French strain that was pertussis toxin–deficient, pertactin wild-type showed that the strains carry the same 28-kb deletion in similar genomes.     

Cell-surface residence of sphingosine 1-phosphate receptor 1 on lymphocytes determines lymphocyte egress kinetics

The sphingosine 1-phosphate receptor 1 (S1P₁) promotes lymphocyte egress from lymphoid organs. Previous work showed that agonist-induced internalization of this G protein–coupled receptor correlates with inhibition of lymphocyte egress and results in lymphopenia. However, it is unclear if S1P₁ internalization is necessary for this effect. We characterize a knockin mouse (S1p1r^S5A/S5A) in which the C-terminal serine-rich S1P₁ motif, which is important for S1P₁ internalization but dispensable for S1P₁ signaling, is mutated. T cells expressing the mutant S1P₁ showed delayed S1P₁ internalization and defective desensitization after agonist stimulation. Mutant mice exhibited significantly delayed lymphopenia after S1P₁ agonist administration or disruption of the vascular S1P gradient. Adoptive transfer experiments demonstrated that mutant S1P₁ expression in lymphocytes, rather than endothelial cells, facilitated this delay in lymphopenia. Thus, cell-surface residency of S1P₁ on T cells is a primary determinant of lymphocyte egress kinetics in vivo.Sphingosine 1-phosphate (S1P), a multifunctional lipid mediator that signals via five G protein–coupled receptors (GPCRs), regulates vascular maturation, permeability, and angiogenesis (Hla, 2004; Cyster, 2005). Recently, interest in the roles of S1P and its receptors in the immune system has been prompted in part by the identification of the immunomodulator FTY720 (Brinkmann et al., 2002; Mandala et al., 2002; Chiba, 2005), which upon phosphorylation by Sphk2 to FTY720-P (Sanchez et al., 2003; Zemann et al., 2006) acts as a strong agonist for four out of five S1P receptors (Brinkmann et al., 2004). FTY720 induces profound lymphopenia by inhibiting the egress of lymphocytes from the thymus, peripheral lymph nodes, and Peyer’s patches (Chiba, 2005). Indeed, it is now appreciated that S1P signaling modulates the trafficking of not only naive and central memory T cells, but also B cells, dendritic cells, NK cells, osteoclasts, and hematopoietic progenitor cells (Allende and Proia, 2002; Kabashima et al., 2006; Massberg et al., 2007; Schwab and Cyster, 2007; Walzer et al., 2007; Ledgerwood et al., 2008; Rivera et al., 2008; Sebzda et al., 2008; Ishii et al., 2009). These studies suggest that S1P regulates hematopoietic and immune cell trafficking under homeostatic and disease conditions; however, it is unclear precisely how S1P receptor signaling modulates cellular responses to egress cues.The mechanism of how S1P regulates T cell trafficking has been intensively investigated; T cell–specific deletion of S1p1r or hematopoietic reconstitution using S1p1r^−/− fetal liver cells resulted in profound lymphopenia, suggesting that the T cell–intrinsic S1P receptor 1 (S1P₁) is essential for their egress from the thymus and secondary lymph nodes (Allende et al., 2004; Matloubian et al., 2004). This observation, coupled with the finding that FTY720-P induces the loss of cell-surface S1P₁ from lymphocytes in an irreversible manner (Gräler and Goetzl, 2004; Matloubian et al., 2004), suggests that functional antagonism of S1P₁ in the lymphocyte compartment is essential for the inhibition of T cell egress.However, other studies have led to the proposal of an alternative mechanism by which S1P₁ regulates lymphocyte egress. Immunofluorescence microscopy demonstrated high expression levels of S1P₁ in endothelial cells, whereas staining of lymphocytes was weaker (Singer et al., 2005; Sinha et al., 2009). Moreover, administration of SEW2971, a selective S1P₁ agonist, does not induce irreversible receptor loss from the cell surface but causes significant lymphopenia in vivo (Jo et al., 2005). Two-photon microscopy of explanted lymph nodes containing labeled lymphocytes suggested that S1P₁ agonists may modulate barrier function and closure of vascular portals in the medulla, through which T cells egress into efferent lymphatics (Wei et al., 2005). Thus, this alternative proposal favors endothelial cells as the primary target cell type for S1P₁ agonists to inhibit lymphocyte egress (Rosen et al., 2008).Close interactions between immune and vascular cells may underlie the ability of S1P₁ to promote lymphocyte egress. In lymph node cortical sinuses, egress of T and B cells required S1P₁-dependent transendothelial traverse (Grigorova et al., 2009; Sinha et al., 2009). Indeed, competing chemotactic signaling between the egress-promoting S1P–S1P₁ system and the retention-promoting CXCL21–CCR7 chemokine receptor system of T cells appears to determine the rate and extent of their egress from secondary lymphoid organs (Pham et al., 2008). Whether S1P₁ signaling in lymphocytes, endothelial compartments, or both is important in the process of egress is not known.S1P₁ is a type I GPCR that is rapidly phosphorylated upon agonist stimulation. Although several protein kinases are involved in the phosphorylation of S1P₁ (Lee et al., 2001), phosphorylation at the C-terminal domain is particularly relevant to receptor desensitization and internalization (Hla, 2001). Because FTY720-P is degraded less efficiently than S1P by S1P lyase and S1P phosphatases (Bandhuvula et al., 2005; Mechtcheriakova et al., 2007; Yamanaka et al., 2008), its ligation likely induces sustained receptor activation kinetics. Presumably, this underlies the FTY720-P–induced irreversible internalization and proteosomal degradation of S1P₁ and resultant lymphopenia (Oo et al., 2007). The GRK-2 enzyme is capable of phosphorylating the serine-rich motif in the C-terminal tail of S1P₁ (Watterson et al., 2002), and we recently demonstrated that mutation of the five serines in the C terminus of S1P₁ to nonphosphorylatable alanines inhibited S1P- and FTY720-P–induced receptor internalization in transfected HEK293 cells (Oo et al., 2007). Although previous studies of GPCR signaling and chemotaxis have provided some insights into the role of internalization in these processes, the results appear to be receptor specific. For example, a CXCR4 superagonist induced greater chemotaxis than the native ligand stromal cell–derived factor–1α (SDF-1α) with no perceptible receptor internalization (Sachpatzidis et al., 2003). Conversely, mutations in the C terminus of CXCR2 resulted in defective receptor internalization concomitant with impaired chemotaxis (Sachpatzidis et al., 2003). In the case of S1P₁, it is unknown whether internalization is required for lymphocyte egress and recirculation.To address the role of S1P₁ internalization in the control of lymphocyte egress during homeostasis and FTY720 treatment, we developed a mouse model in which WT S1P₁ is replaced by the internalization-deficient mutant (S5A-S1P₁). We show that although T cell trafficking under homeostasis is unaltered, S1p1r^S5A/S5A mice display kinetic resistance to lymphopenia induced by the S1P₁ modulator (FTY720-P) or disruption of the S1P gradient. Adoptive transfer of S1p1r^WT/WT and S1p1r^S5A/S5A lymphocytes and S1P₁ surface staining of lymph node endothelial cells demonstrate that the T cell S1P₁, and not endothelial cell S1P₁ expression, regulates the rate of lymphocyte egress in vivo. These data support a T cell–intrinsic model of S1P₁ signaling in egress kinetics wherein the internalization of S1P₁ is a crucial modulator of the cues for T cell migration.     

Characterization of 107 genomic DNA reference materials for CYP2D6, CYP2C19, CYP2C9, VKORC1, and UGT1A1: a GeT-RM and Association for Molecular Pathology collaborative project

Pharmacogenetic testing is becoming more common; however, very few quality control and other reference materials that cover alleles commonly included in such assays are currently available. To address these needs, the Centers for Disease Control and Prevention's Genetic Testing Reference Material Coordination Program, in collaboration with members of the pharmacogenetic testing community and the Coriell Cell Repositories, have characterized a panel of 107 genomic DNA reference materials for five loci (CYP2D6, CYP2C19, CYP2C9, VKORC1, and UGT1A1) that are commonly included in pharmacogenetic testing panels and proficiency testing surveys. Genomic DNA from publicly available cell lines was sent to volunteer laboratories for genotyping. Each sample was tested in three to six laboratories using a variety of commercially available or laboratory-developed platforms. The results were consistent among laboratories, with differences in allele assignments largely related to the manufacturer's assay design and variable nomenclature, especially for CYP2D6. The alleles included in the assay platforms varied, but most were identified in the set of 107 DNA samples. Nine additional pharmacogenetic loci (CYP4F2, EPHX1, ABCB1, HLAB, KIF6, CYP3A4, CYP3A5, TPMT, and DPD) were also tested. These samples are publicly available from Coriell and will be useful for quality assurance, proficiency testing, test development, and research.     

Mitotic catastrophe cell death induced by heat shock protein 90 inhibitor in BRCA1-deficient breast cancer cell lines

Heat shock protein 90 (Hsp90) is a molecular chaperone involved in folding, assembly, maturation, and stabilization of the client proteins that regulate survival of malignant cells. As previous reports correlate high Hsp90 expression with decreased survival in breast cancer, Hsp90 may be a favorable target for investigational therapy in breast cancer. In our study, we have examined the response of a panel of both BRCA1-null (UACC 3199, HCC 1937, and MBA-MD-436) and BRCA1-wt breast cancer cell lines (MCF-7, MBA-MD-157, and Hs578T) to determine the proteins governing response to Hsp90 inhibitor 17-allyloamino-17-demethoxy-geldanamycin. On treatment with the drug, cells arrested at G(2)-M phase and entered aberrant mitosis in a BRCA1-dependent manner. Failure to arrest the cells at or before mitosis resulted in formation of micronucleated cells, aberrant segregation of chromosomes, microtubule misalignment, and multicentrosomes, leading in eventual mitotic catastrophe cell death. Our observations show that BRCA1 mediates G(2)-M transition mainly through chek1 on 17-allyloamino-17-demethoxy-geldanamycin treatment.     

Functional rarity and evenness are key facets of biodiversity to boost multifunctionality

The functional traits of organisms within multispecies assemblages regulate biodiversity effects on ecosystem functioning. Yet how traits should assemble to boost multiple ecosystem functions simultaneously (multifunctionality) remains poorly explored. In a multibiome litter experiment covering most of the global variation in leaf trait spectra, we showed that three dimensions of functional diversity (dispersion, rarity, and evenness) explained up to 66% of variations in multifunctionality, although the dominant species and their traits remained an important predictor. While high dispersion impeded multifunctionality, increasing the evenness among functionally dissimilar species was a key dimension to promote higher multifunctionality and to reduce the abundance of plant pathogens. Because too-dissimilar species could have negative effects on ecosystems, our results highlight the need for not only diverse but also functionally even assemblages to promote multifunctionality. The effect of functionally rare species strongly shifted from positive to negative depending on their trait differences with the dominant species. Simultaneously managing the dispersion, evenness, and rarity in multispecies assemblages could be used to design assemblages aimed at maximizing multifunctionality independently of the biome, the identity of dominant species, or the range of trait values considered. Functional evenness and rarity offer promise to improve the management of terrestrial ecosystems and to limit plant disease risks.

Biodiversity is of pivotal importance for maintaining ecosystem functions, such as primary productivity, litter decomposition, or soil nutrient cycling, and for preventing disease risks (1–4). Despite the important advances in our understanding of the role of biodiversity in natural and managed ecosystems, we still ignore how the physiological, morphological, and biochemical characteristics of species—their functional traits—should assemble to boost multiple functions simultaneously [multifunctionality (5)]. Uncovering the trait assemblages that promote high multifunctionality is critical to identify baselines that track the consequences of biodiversity loss on ecosystems, to undertake effective restoration actions, or to engineer the species assemblages of managed ecosystems that promote biodiversity and high multifunctionality in a changing world.The relationship between functional traits and multifunctionality has been shown to vary from positive to negative depending on the ecosystem, species pool, and biogeographical context considered (6–8). Such a high context dependency may largely depend on how functional traits are assembled within communities (9). While the traits of dominant species (hereafter referred to as functional dominance) can strongly determine individual ecosystem functions (10), their role becomes less clear when considering multifunctionality (7, 11). This is so because in an ecosystem, species that are functionally different from the dominant ones—functional diversity—may contribute more to certain key functions than their lower abundance would suggest (7, 11, 12). High functional diversity—through the dispersion of trait values (hereafter referred to as functional dispersion) or the presence of species with infrequent trait values (hereafter referred to as functional rarity)—for instance, in the case of keystone species—may enhance multifunctionality (9) if functionally dissimilar species exploit or release contrasting resources or the same resources but at different spatial or temporal scales (1). However, if species become too dissimilar, this could lead to strong negative effects on ecosystems (e.g., in the case of invasive species adding a new set of trait values) (6, 7, 13). In the later case, higher evenness among functionally dissimilar species (hereafter referred to as functional evenness) could promote synergistic interactions and counteract such negative biodiversity effects on multifunctionality (6, 7). However, functional dominance, dispersion, rarity, and evenness often covary in real-world ecosystems (14), hindering the evaluation of their individual effect on multifunctionality (6, 14, 15). A manipulative study revealing which trait assemblages could boost positive biodiversity effects on multifunctionality across multiple ecosystems is yet lacking.The distribution of trait values (hereafter referred to as trait distribution) within complex, multispecies assemblages often deviates from the symmetric normal distribution, classically assumed in ecological studies (14, 15). While the mean and the variance allow for characterization of the functional dominance and dispersion of a normal distribution, the skewness and kurtosis offer insights on the shape of the complex trait distributions encountered in naturally assembled communities (6, 14, 15). The skewness represents the asymmetry of the distributions. High negative or positive values of skewness occur when trait distributions are strongly left or right tailed as a result of rare species with infrequent trait values compared with the bulk of the distribution: a definition of functional rarity. Kurtosis represents the relative peakiness of trait distribution, where a low kurtosis value reflects functionally even distributions. Investigating complex trait distributions thus offers a unique opportunity to decipher the interplay of functional dominance, dispersion, rarity, and evenness in determining multifunctionality and represents a fundamental step toward the design and management of species assemblages that could maximize biodiversity effects on ecosystems.Here, we present results from a multibiome experiment examining how the functional dominance, dispersion, evenness, and rarity of plant litter assemblages influence multifunctionality and soil microbial communities. We manipulated complex trait distributions to disentangle the influence of the four biodiversity attributes, while species richness (n = 15 species each) and total litter biomass (1 g) were kept constant among litter assemblages. We assembled 570 experimental leaf litter mixtures and monocultures using 90 species from six biomes covering a wide range of the global variability of two key plant functional traits (Specific Leaf Area [SLA] and lignin content) (16, 17) and tracked changes in multifunctionality and soil microbial communities as litter decomposed (Fig. 1; see also Methods and SI Appendix, Tables S1 and S2 and Figs. S1 and S2). We used a single decomposition environment (i.e., one soil type and controlled climatic condition) to avoid variations due to differences in decomposer communities, soil parameters, and climate. Leaf litter assemblages were set up using a set of 120,000 simulated functional trait distributions (see Methods and SI Appendix, Figs. S3 and S4). Then, we selected a subset of 570 assemblages that covered the entire range of values that functional dominance and diversity could take while minimizing their correlations within and across biomes (SI Appendix, Table S3 and Fig. S4). We calculated multifunctionality using nine litter and soil functions related to carbon (C), nitrogen (N), and phosphorus (P) cycling (see Methods and SI Appendix, Fig. S5). We also addressed the relative abundance (fungal trophic modes) and diversity of soil bacteria and fungi. Monitoring changes in litter decomposition, soil processes, and microbial communities thus allowed us to consider a part—albeit a functionally important part—of whole ecosystem functioning. We tested the core hypothesis that functionally dispersed and highly even trait distributions are the litter trait assemblages to maximize multifunctionality.Open in a separate windowFig. 1.The experimental and analytical framework to test the effects of dominant species and their traits (dominance) and of functional diversity (dispersion, rarity, and evenness) on multifunctionality. (A) The SLA and leaf litter lignin content of 90 species from six biomes covering a wide array of the global variation in the traits observed (see also SI Appendix, Table S2). (B) Disentangling functional dominance, dispersion, rarity, and evenness by manipulating the mean, variance, skewness, and kurtosis of trait-abundance distributions. (C) The microcosms containing the leaf litter communities (photographs by J.H.C.C, L.D., N.G., N.S., and R.M.).     

Reference intervals of aortic pulse wave velocity assessed with an oscillometric device in healthy children and adolescents from Argentina

Age-related reference intervals (RIs) of aortic pulse wave velocity (Ao-PWV) obtained from a large healthy population are lacking in South America. The aims of this study were to determine Ao-PWV RIs in a cohort of healthy children and adolescents from Argentina and to generate year-to-year percentile curves.

Ao-PWV was measured in 1000 healthy subjects non-exposed to traditional cardiovascular risk factors (Age: 10–22 y. o., 56% males). First, we evaluated if RIs for males and females were necessaries (correlation and covariate analysis). Second, mean (M) and standard deviation (SD) age-related equations were obtained for cf-PWV, using parametric regression methods based on fractional polynomials. Third, age-specific (year to year) percentiles curves (for all, males and females children and adolescents) were generated using the standard normal distribution. They were, age-specific 1st, 2.5th, 5th, 10th, 25th, 50th, 75th, 90th, 95th, 97.5th and 99th percentile curves and values.

After covariate analysis (i.e., adjusting by age, jugulum-symphysis distance, body weight and height), specific RIs for males and females of children and adolescents were evidenced as necessaries. The equations were

For all subjects:

Ao-PWV_Mean = 4.98 + 12.86x10^?5 Age³.

Ao-PWV_SD = 0.47 + 21.00x10^?6Age³.

For girls:

Ao-PWV_Mean = 5.07 + 10.23x10^?5Age³.

Ao-PWV_SD = 0.50 + 10.00x10^?6Age³.

For boys:

Ao-PWV_Mean = 4.87 + 15.81x10^?5Age³.

Ao-PWV_SD = 0.46 + 22.34x10^?6Age³.

Our study provides the largest database to-date concerning Ao-PWV in healthy children and adolescents in Argentina. Age-related equations (M and SD values) for Ao-PWV are reported by the first time. Specific RIs and percentiles of Ao-PWV are now available according to age and sex for an Argentinian population.     

Chronic treatment with either dexfenfluramine or sibutramine in diet-switched diet-induced obese mice

Dexfenfluramine (DEX) and sibutramine (SIB) are effective antiobesity agents. Their effects on weight control and hormone profile have not been previously studied in diet-switched diet-induced obese (DIO) mice, in which treatment is initiated upon cessation of a low-fat diet and resumption of a high-fat diet. Furthermore, their effects on circulating ghrelin in obese humans or in animal models of obesity have not yet been reported. Male C57BI/6J DIO mice after 16 wk on a high-fat diet (HF, 60 kcal% fat) were switched to a low-fat diet (LF, 10 kcal% fat) for 50 d. HF diet resumed concurrently with treatment for 28 d with DEX 3 and 10 mg/kg, twice a day (BID); SIB 5 mg/kg BID; or vehicle. Rapid weight regain ensued in vehicle-treated DIO mice. DEX or SIB treatment significantly blunted the body weight gain. Caloric intake was decreased acutely by DEX or SIB vs vehicle during the first 2 d treatment, but returned to control after 5 d. At the end of study, epididymal fat weight and whole body fat mass determined by DEXA scan were decreased by DEX 10 mg/kg, and whole body lean mass decreased with DEX 3 mg/kg treatment. Circulating ghrelin on d 28 was increased with either DEX 3 or 10 mg/kg treatment, while growth hormone and insulin were decreased. Leptin was also decreased in the DEX 10 mg/kg group. SIB did not significantly affect fat mass, ghrelin, growth hormone, insulin, or leptin. Mice chronically fed LF diet maintained a lower caloric intake, gained less weight and fat mass than diet-switched mice, and had higher ghrelin and lower insulin and leptin. In summary, weight regain in diet-switched DIO mice is delayed with either DEX or SIB treatment. DEX treatment of diet-switched DIO mice decreased growth hormone, insulin, leptin, fat mass, lean mass, and increased ghrelin, while SIB only decreased body weight.     

Long-term modulation of Na+ and K+ channels by TGF-��1 in neonatal rat cardiac myocytes

Previous work shows that transforming growth factor-β1 (TGF-β1) promotes several heart alterations, including atrial fibrillation (AF). In this work, we hypothesized that these effects might be associated with a potential modulation of Na(+) and K(+) channels. Atrial myocytes were cultured 1-2?days under either control conditions, or the presence of TGF-β1. Subsequently, Na(+) (I(Na)) and K(+) (I(K)) currents were investigated under whole-cell patch-clamp conditions. Three K(+) currents were isolated: inward rectifier (I(Kin)), outward transitory (I(to)), and outward sustained (I(Ksus)). Interestingly, TGF-β1 decreased (50%) the densities of I(Kin) and I(Ksus) but not of I(to). In addition, the growth factor reduced by 80% the amount of I(Na) available at -80?mV. This effect was due to a significant reduction (30%) in the maximum I(Na) recruited at very negative potentials or I(max), as well as to an increased fraction of inactivated Na(+) channels. The latter effect was, in turn, associated to a -7?mV shift in V(1/2) of inactivation. TGF-β1 also reduced by 60% the maximum amount of intramembrane charge movement of Na(+) channels or Q(max), but did not affect the corresponding voltage dependence of activation. This suggests that TGF-β1 promotes loss of Na(+) channels from the plasma membrane. Moreover, TGF-β1 also reduced (50%) the expression of the principal subunit of Na(+) channels, as indicated by western blot analysis. Thus, TGF-β1 inhibits the expression of Na(+) channels, as well as the activity of K(+) channels that give rise to I(Ksus) and I(Kin). These results may contribute to explaining the previously observed proarrhythmic effects of TGF-β1.