Towards greater realism in inclusive fitness models: the case of worker reproduction in insect societies

The conflicts over sex allocation and male production in insect societies have long served as an important test bed for Hamilton''s theory of inclusive fitness, but have for the most part been considered separately. Here, we develop new coevolutionary models to examine the interaction between these two conflicts and demonstrate that sex ratio and colony productivity costs of worker reproduction can lead to vastly different outcomes even in species that show no variation in their relatedness structure. Empirical data on worker-produced males in eight species of Melipona bees support the predictions from a model that takes into account the demographic details of colony growth and reproduction. Overall, these models contribute significantly to explaining behavioural variation that previous theories could not account for.     

In Vivo T Cell Activation Induces the Formation of CD209+ PDL-2+ Dendritic Cells

Two critical functions of dendritic cells (DC) are to activate and functionally polarize T cells. Activated T cells can, in turn, influence DC maturation, although their effect on de novo DC development is poorly understood. Here we report that activation of T cells in mice, with either an anti-CD3 antibody or super antigen, drives the rapid formation of CD209⁺CD11b⁺CD11c⁺ MHC II⁺ DC from monocytic precursors (Mo-DC). GM-CSF is produced by T cells following activation, but surprisingly, it is not required for the formation of CD209⁺ Mo-DC. CD40L, however, is critical for the full induction of Mo-DC following T cell activation. T cell induced CD209⁺ Mo-DC are comparable to conventional CD209^- DC in their ability to stimulate T cell proliferation. However, in contrast to conventional CD209^- DC, CD209⁺ Mo-DC fail to effectively polarize T cells, as indicated by a paucity of T cell cytokine production. The inability of CD209⁺ Mo-DC to polarize T cells is partly explained by increased expression of PDL-2, since blockade of this molecule restores some polarizing capacity to the Mo-DC. These findings expand the range of signals capable of driving Mo-DC differentiation in vivo beyond exogenous microbial factors to include endogenous factors produced following T cell activation.     

An Intriguing Shift Occurs in the Novel Protein Phosphatase 1 Binding Partner,TCTEX1D4: Evidence of Positive Selection in a Pika Model

T-complex testis expressed protein 1 domain containing 4 (TCTEX1D4) contains the canonical phosphoprotein phosphatase 1 (PPP1) binding motif, composed by the amino acid sequence RVSF. We identified and validated the binding of TCTEX1D4 to PPP1 and demonstrated that indeed this protein is a novel PPP1 interacting protein. Analyses of twenty-one mammalian species available in public databases and seven Lagomorpha sequences obtained in this work showed that the PPP1 binding motif ₉₀RVSF₉₃ is present in all of them and is flanked by a palindromic sequence, PLGS, except in three species of pikas (Ochotona princeps, O. dauurica and O. pusilla). Furthermore, for the Ochotona species an extra glycosylation site, motif ₉₆NLS₉₈, and the loss of the palindromic sequence were observed. Comparison with other lagomorphs suggests that this event happened before the Ochotona radiation. The d_N/d_S for the sequence region comprising the PPP1 binding motif and the flanking palindrome highly supports the hypothesis that for Ochotona species this region has been evolving under positive selection. In addition, mutational screening shows that the ability of pikas TCTEX1D4 to bind to PPP1 is maintained, although the PPP1 binding motif is disrupted, and the N- and C-terminal surrounding residues are also abrogated. These observations suggest pika as an ideal model to study novel PPP1 complexes regulatory mechanisms.     

Role of Cystatin C in Amyloid Precursor Protein-induced Proliferation of Neural Stem/Progenitor Cells

The amyloid precursor protein (APP) is well studied for its role in Alzheimer disease. However, little is known about its normal function. In this study, we examined the role of APP in neural stem/progenitor cell (NSPC) proliferation. NSPCs derived from APP-overexpressing Tg2576 transgenic mice proliferated more rapidly than NSPCs from the corresponding background strain (C57Bl/6xSJL) wild-type mice. In contrast, NSPCs from APP knock-out (APP-KO) mice had reduced proliferation rates when compared with NSPCs from the corresponding background strain (C57Bl/6). A secreted factor, identified as cystatin C, was found to be responsible for this effect. Levels of cystatin C were higher in the Tg2576 conditioned medium and lower in the APP-KO conditioned medium. Furthermore, immunodepletion of cystatin C from the conditioned medium completely removed the ability of the conditioned medium to increase NSPC proliferation. The results demonstrate that APP expression stimulates NSPC proliferation and that this effect is mediated via an increase in cystatin C secretion.     

3D representation and analysis of enthesis morphology

This comparison of methods for assessing the development of muscle insertion sites, or entheses, suggests that three‐dimensional (3D) quantification of enthesis morphology can produce a picture of habitual muscle use patterns in a past population that is similar to one produced by ordinal scores for describing enthesis morphology. Upper limb skeletal elements (humeri, radii, and ulnae) from a sample of 24 middle‐aged adult males from the Pottery Mound site in New Mexico were analyzed for both fibrous and fibrocartilaginous enthesis development with three different methods: ordinal scores, two‐dimensional (2D) area measurements, and 3D surface areas. The methods were compared using tests for asymmetry and correlations among variables in each quantitative data set. 2D representations of enthesis area did not agree as closely as ordinal scores and 3D surface areas did regarding which entheses were significantly asymmetrical. There was significant correlation between 3D and 2D data, but correlation coefficients were not consistently high. Intraobserver error was also assessed for the 3D method. Cronbach's alpha values fell between 0.68 and 0.73, and error rates for all entheses fell between 10% and 15%. Marginally acceptable intraobserver error and the analytic versatility of 3D images encourage further investigation of using 3D scanning technology for quantifying enthesis development. Am J Phys Anthropol 152:417–424, 2013. © 2013 Wiley Periodicals, Inc.     

Reduced Tonoplast Fast-Activating and Slow-Activating Channel Activity Is Essential for Conferring Salinity Tolerance in a Facultative Halophyte,Quinoa

Halophyte species implement a “salt-including” strategy, sequestering significant amounts of Na⁺ to cell vacuoles. This requires a reduction of passive Na⁺ leak from the vacuole. In this work, we used quinoa (Chenopodium quinoa) to investigate the ability of halophytes to regulate Na⁺-permeable slow-activating (SV) and fast-activating (FV) tonoplast channels, linking it with Na⁺ accumulation in mesophyll cells and salt bladders as well as leaf photosynthetic efficiency under salt stress. Our data indicate that young leaves rely on Na⁺ exclusion to salt bladders, whereas old ones, possessing far fewer salt bladders, depend almost exclusively on Na⁺ sequestration to mesophyll vacuoles. Moreover, although old leaves accumulate more Na⁺, this does not compromise their leaf photochemistry. FV and SV channels are slightly more permeable for K⁺ than for Na⁺, and vacuoles in young leaves express less FV current and with a density unchanged in plants subjected to high (400 mm NaCl) salinity. In old leaves, with an intrinsically lower density of the FV current, FV channel density decreases about 2-fold in plants grown under high salinity. In contrast, intrinsic activity of SV channels in vacuoles from young leaves is unchanged under salt stress. In vacuoles of old leaves, however, it is 2- and 7-fold lower in older compared with young leaves in control- and salt-grown plants, respectively. We conclude that the negative control of SV and FV tonoplast channel activity in old leaves reduces Na⁺ leak, thus enabling efficient sequestration of Na⁺ to their vacuoles. This enables optimal photosynthetic performance, conferring salinity tolerance in quinoa species.The increasing problem of global land salinization (Flowers, 2004; Rengasamy, 2006) and its associated multibillion dollar losses in agricultural production require a better understanding of the key physiological mechanisms that confer salinity tolerance in crops. One effective way of gaining such knowledge comes from studying halophytes (Glenn et al., 1999; Flowers and Colmer, 2008; Shabala and Mackay, 2011).One of the prominent features of halophytes is their ability to efficiently sequester cytosolically toxic Na⁺ to the cell vacuole. The classic view is that this sequestration is achieved by tonoplast Na⁺/H⁺ antiporters (Barkla et al., 1995; Flowers and Colmer, 2008), a process energized by both vacuolar H⁺ pumps: ATPase (Ayala et al., 1996; Vera-Estrella et al., 1999; Wang et al., 2001) and pyrophosphatase (Parks et al., 2002; Vera-Estrella et al., 2005; Guo et al., 2006; Krebs et al., 2010). However, recent studies have added more complexity to the relationship between Na⁺/H⁺ antiporters and vacuolar Na⁺ sequestration, assigning a role to the transporter in the regulation of K⁺ and H⁺ homeostasis (for review, see Rodríguez-Rosales et al., 2009; Jiang et al., 2010; Bassil et al., 2011). Vacuolar Na⁺/H⁺ antiporters encoded by NHX genes have been shown to also act as K⁺/H⁺ antiporters, with a relatively weak selectivity between Na⁺ and K⁺. The Na⁺/K⁺ selectivity ratio, in turn, is regulated by vacuolar calmodulin in a pH- and Ca²⁺-dependent manner (Yamaguchi et al., 2005). Consequently, other transporters, in addition to and different from NHX, are likely to be involved in vacuolar Na⁺ sequestration. In addition, salt-induced up-regulation of Na⁺/H⁺ antiporter expression levels has been observed in leaves but not in roots (Cosentino et al., 2010), suggesting the importance of Na⁺ exclusion and intracellular sequestration, primarily in photosynthesizing cells. Thus, tissue- and species-specific differences in the respective mechanisms should be considered as well.Whatever the actual mechanisms are for intracellular Na⁺ sequestration, efficient Na⁺ pumping into vacuole is only one side of the coin. To confer salinity tolerance, toxic Na⁺ ions must be prevented from leaking back into the cytosol. Indeed, given the at least 4- to 5-fold concentration gradient between the vacuole and the cytosol (Shabala and Mackay, 2011) and a zero or slightly negative cytosol-to-vacuole voltage difference across the tonoplast, Na⁺ leakage from the vacuole is thermodynamically favorable. Thus, to avoid energy-consuming futile Na⁺ cycling between the cytosol and the vacuole, and to achieve efficient vacuolar sequestration of toxic Na⁺, passive tonoplast Na⁺ conductance has to be kept to an absolute minimum. This implies strict and efficient control over Na⁺-permeable tonoplast channels.Two major types of Na⁺-permeable channels are present in the tonoplast, the slow-activating (SV) and fast-activating (FV) vacuolar channels. The SV channel is permeable to both monovalent and divalent cations and is activated by cytosolic Ca²⁺ and positive vacuolar voltage (Hedrich and Neher, 1987; Ward and Schroeder, 1994; Pottosin et al., 1997, 2001). The FV channel is permeable for monovalent cations only, is activated by large voltages of either sign, and is inhibited by divalent cations from either side of the membrane (Tikhonova et al., 1997; Brüggemann et al., 1999a, 1999b). In Arabidopsis (Arabidopsis thaliana), SV channels are shown to be encoded by a TPC1 (for two-pore channel1) protein (Peiter et al., 2005; Pottosin and Schönknecht, 2007; Hedrich and Marten, 2011). Importantly, recent studies on mammalian two-pore channels have suggested that endolysosomal TPCs are, in fact, Na⁺-selective channels (Wang et al., 2012). In contrast, the molecular identity of FV channels remains elusive. Both SV and FV channels are ubiquitous and abundant (up to several copies per μm²) in plant tissues, including mesophyll cell vacuoles (Pottosin and Muñiz, 2002; Pottosin and Schönknecht, 2007). SV and FV channel activity is strongly controlled at physiologically attainable conditions (physiological tonoplast voltages and vacuolar and cytosolic divalent and polyvalent cation concentrations). Importantly, even with 0.1% to 1% of the total population of channels open at any one time, impressive monovalent cation currents in the range of tens of pA per vacuole can be conducted. This is equivalent to a current mediated by the whole vacuole population of H⁺ pumps (Hedrich et al., 1988). Thus, under saline conditions, SV and FV channel activity probably needs to be further reduced.Early attempts to unravel any dramatic differences between the properties of tonoplast cation channels in salt-tolerant and salt-sensitive plants did not yield a clear outcome. Ivashikina and Hedrich (2005) studied the voltage dependence of the SV channels in vacuoles from Arabidopsis cell culture and found that an increase in luminal Na⁺/K⁺ ratio, mimicking the accumulation of Na⁺ in vacuoles during salt stress, shifted the threshold for SV activation to positive potentials, reducing SV channel open probability under saline conditions. Maathuis and coworkers (1992) found significant SV channel activity in leaf vacuoles isolated from the extreme halophyte Suaeda maritima, even when plants were grown under high (200 mm) NaCl conditions. The estimated activity of the transporter at physiologically relevant cytosolic Ca²⁺ levels and relatively small transmembrane voltage differences was low. Thus, the authors suggested that, rather than possessing some specific salt-induced control over the SV channel, the transporter’s low activity would mean that even under highly saline conditions, it would consume only about 30% of the H⁺-ATPase-generated power. Further studies from this laboratory demonstrated that voltage gating, unitary conductance, and Na⁺/K⁺ selectivity (P_K = P_Na) of SV channels from roots of Plantago media (salt sensitive) and Plantago maritima (salt tolerant) were essentially the same (Maathuis and Prins, 1990). However, when both species were grown under saline conditions, the SV channel activity greatly diminished. Yet, based on the original data of this study, it is not possible to decipher whether the SV channel activity in the two species was the same or different under control conditions and whether it was a statistically significant difference between the salt-induced decrease in the open probability of SV channels between P. media and P. maritima. As for FV channels, we are not aware of a single study on their properties/expression in relation to the salt tolerance.While the total number of halophytic species is relatively small compared with glycophytes, it still amounts to at least several thousand species (Glenn et al., 1999; Flowers et al., 2010). Moreover, halophytes are present in about one-half of higher plant families (Flowers and Colmer, 2008). These species possess a wide range of anatomical and morphological features that may potentially enable their superior performance under saline conditions (Shabala and Mackay, 2011). Nonetheless, the extent to which the above considerations could be extrapolated to all halophytes remains to be assessed. In this work, we used quinoa (Chenopodium quinoa) mesophyll leaf vacuoles to address some of these issues. Quinoa is a facultative halophyte species that originates from the Andean region of South America and was domesticated for human consumption some 3,000 to 4,000 years ago. It can grow under extreme saline conditions with a soil electrical conductivity exceeding 40 dS m⁻¹, approximately 500 mm NaCl (Jacobsen et al., 2003; Razzaghi et al., 2011). Optimal plant growth is usually observed at NaCl concentrations of around 100 mm (Hariadi et al., 2011), but this may be genotype specific (Adolf et al., 2012). Quinoa possesses some degree of leaf succulence as well as epidermal bladder cells (EBC), so it has the potential to employ two different sequestration strategies for cytosolic Na⁺ exclusion: internal (e.g. vacuolar sequestration) and external (sequestration in EBC). This makes quinoa an excellent model species to investigate the role of vacuolar Na⁺ sequestration in the overall salinity tolerance in this crop plant as well as to determine the contribution of SV and FV channels in this process. Here, we report a highly significant difference in SV and FV channel activity between old and young leaves of quinoa plants, a difference that is further enhanced under saline conditions. We conclude that the ability of quinoa plants to control ion leak via SV and FV tonoplast channels is essential for conferring salinity tolerance in this species. The possible implications of these findings for crop breeding for salinity tolerance are discussed.     

Inflammation in disseminated lesions: an analysis of CD4+, CD20+, CD68+, CD31+ and vW+ cells in non-ulcerated lesions of disseminated leishmaniasis

Disseminated leishmaniasis (DL) differs from other clinical forms of the disease due to the presence of many non-ulcerated lesions (papules and nodules) in non-contiguous areas of the body. We describe the histopathology of DL non-ulcerated lesions and the presence of CD4-, CD20-, CD68-, CD31- and von Willebrand factor (vW)-positive cells in the inflamed area. We analysed eighteen biopsies from non-ulcerated lesions and quantified the inflamed areas and the expression of CD4, CD20, CD68, CD31 and vW using Image-Pro software (Media Cybernetics). Diffuse lymphoplasmacytic perivascular infiltrates were found in dermal skin. Inflammation was observed in 3-73% of the total biopsy area and showed a significant linear correlation with the number of vW⁺ vessels. The most common cells were CD68⁺ macrophages, CD20⁺ B-cells and CD4⁺ T-cells. A significant linear correlation between CD4⁺ and CD20⁺ cells and the size of the inflamed area was also found. Our findings show chronic inflammation in all DL non-ulcerated lesions predominantly formed by macrophages, plasmacytes and T and B-cells. As the inflamed area expanded, the number of granulomas and extent of the vascular framework increased. Thus, we demonstrate that vessels may have an important role in the clinical evolution of DL lesions.     

Alginate esters via chemoselective carboxyl group modification

Alginates are (1 → 4) linked linear copolysaccharides composed of β-d-mannuronic acid (M) and its C-5 epimer, α-l-guluronic acid (G). Several strategies for synthesis of carboxyl modified alginate derivatives exist in the literature. Most of these however employ aqueous chemistries, such as carbodiimide coupling reactions. Based on our recently discovered method for homogeneous dissolution of tetrabutylammonium (TBA)-alginate, we now describe use of tetrabutylammonium fluoride (TBAF)-based two component solvent systems as media for synthesis of carboxyl-modified alginate esters. Partially and fully esterified benzyl, butyl, ethyl, and methyl alginates were synthesized via reaction with the corresponding alkyl halides. The newly synthesized derivatives were soluble in polar aprotic solvents without the addition of TBAF. Saponification was performed to demonstrate that alkylation was completely regioselective for carboxylate groups in preference to hydroxyl groups to form esters. We demonstrate the utility of these alginate esters to enhance aqueous solubility of the flavonoid naringenin by formation of solid dispersions.     

Characterization of the GGPP synthase gene family in Arabidopsis thaliana

Geranylgeranyl diphosphate (GGPP) is a key precursor of various isoprenoids that have diverse functions in plant metabolism and development. The annotation of the Arabidopsis thaliana genome predicts 12 genes to encode geranylgeranyl diphosphate synthases (GGPPS). In this study we analyzed GGPPS activity as well as the subcellular localization and tissue-specific expression of the entire protein family in A. thaliana. GGPPS2 (At2g18620), GGPPS3 (At2g18640), GGPPS6 (At3g14530), GGPPS7 (At3g14550), GGPPS8 (At3g20160), GGPPS9 (At3g29430), GGPPS10 (At3g32040) and GGPPS11 (At4g36810) showed GGPPS activity in Escherichia coli, similar to activities reported earlier for GGPPS1 (At1g49530) and GGPPS4 (At2g23800) (Zhu et al. in Plant Cell Physiol 38(3):357–361, 1997a; Plant Mol Biol 35(3):331–341, b). GGPPS12 (At4g38460) did not produce GGPP in E. coli. Based on DNA sequence analysis we propose that GGPPS5 (At3g14510) is a pseudogene. GGPPS–GFP (green fluorescent protein) fusion proteins of the ten functional GGPP synthases localized to plastids, mitochondria and the endoplasmic reticulum, with the majority of the enzymes located in plastids. Gene expression analysis using quantitative real time-PCR, GGPPS promoter-GUS (β-glucuronidase) assays and publicly available microarray data revealed a differential spatio-temporal expression of GGPPS genes. The results suggest that plastids and mitochondria are key subcellular compartments for the synthesis of ubiquitous GGPP-derived isoprenoid species. GGPPS11 and GGPPS1 are the major isozymes responsible for their biosynthesis. All remaining paralogs, encoding six plastidial isozymes and two cytosolic isozymes, were expressed in specific tissues and/or at specific developmental stages, suggesting their role in developmentally regulated isoprenoid biosynthesis. Our results show that of the 12 predicted GGPPS encoded in the A. thaliana genome 10 are functional proteins that can synthesize GGPP. Their specific subcellular location and differential expression pattern suggest subfunctionalization in providing GGPP to specific tissues, developmental stages, or metabolic pathways.