Discrete Subpopulations Research Articles

Neuronal localization of M2 muscarinic receptor immunoreactivity in the rat amygdala

Muscarinic cholinergic neurotransmission in the amygdala is critical for memory consolidation in emotional/motivational learning tasks, but little is known about the neuronal distribution of different receptor subtypes. Immunohistochemistry was used in the present investigation to localize the m2 receptor (M2R). Differential patterns of M2R-immunoreactivity (M2R-ir) were observed in the somata and neuropil of the various amygdalar nuclei. Neuropilar M2R-ir was strongest in rostral portions of the basolateral nuclear complex (BLC). M2R-positive (M2R+) somata were seen in low numbers in all nuclei of the amygdala. Most M2R+ neurons associated with the BLC were in the lateral nucleus and external capsule. These cells were nonpyramidal neurons that contained glutamatic acid decarboxylase (GAD), somatostatin (SOM), and neuropeptide Y (NPY), but not parvalbumin (PV), calretinin (CR), or cholecystokinin (CCK). Little or no M2R-ir was observed in GAD+, PV+, CR+, or CCK+ axons in the BLC, but it was seen in some SOM+ axons and many NPY+ axons. M2R-ir was found in a small number of spiny and aspiny neurons of the central nucleus that were mainly located along the lateral and ventral borders of its lateral subdivision. Many of these cells contained SOM and NPY. M2R+ neurons were also seen in the medial nucleus, including a distinct subpopulation of neurons that surrounded its anteroventral subdivision. The latter neurons were negative for all neuronal markers analyzed. The intercalated nuclei (INs) were associated with two types of large M2R+ neurons, spiny and aspiny. The small principal neurons of the INs were M2R-negative. The somata and dendrites of the large spiny neurons, which were actually found in a zone located just outside of the rostral INs, expressed SOM and NPY, but not GAD. These findings indicate that acetylcholine can modulate a variety of discrete neuronal subpopulations in various amygdalar nuclei via M2Rs, especially neurons that express SOM and NPY.

Functionally defined substates within the human embryonic stem cell compartment

Human embryonic stem (ES) cells can undergo spontaneously differentiation in standard culture conditions, demonstrating that the undifferentiated state is relatively unstable. The heterogeneous expression of SSEA3 observed within human ES colonies, provides a means to examine undifferentiated stem cell substates. Through functional testing of single cells we have shown that undifferentiated ES cells can be segregated into functionally discrete subpopulations on the basis of SSEA3 expression: SSEA3 High, SSEA Low and SSEA3 Negative. Human ES subpopulations were found to be interconvertible, but they possess distinct properties when challenged to differentiate along the neural lineage. These data suggest that ES cells with pluripotent/self-renewal capacities can exhibit different responses to induction of differentiation.

Abstract 483: CXCR6, a newly defined biomarker of tissue-specific stem cell asymmetric self-renewal, identifies more aggressive human melanoma cancer stem cells

Abstract Background- A fundamental problem in cancer research is identifying the cell type that is capable of sustaining neoplastic growth and its origin from normal tissue cells. Recent investigations of a variety of tumor types have shown that phenotypically identifiable and isolable subfractions of cells possess the tumor-forming ability. In the present paper, using two lineage-related human melanoma cell lines, primary melanoma line IGR39 and its metastatic derivative line IGR37, two main contributes are reported. The first one is the first phenotypic evidence to support the origin of melanoma CSCs from mutated tissue-specific stem cells and the second one is the identification of a more aggressive subpopulation CXCR6+ in melanoma. Methods/Findings- We defined CXCR6 as a new biomarker for tissue-specific stem cell asymmetric self-renewal. Thus, the relationship between melanoma formation and ABCG2 and CXCR6 expression was investigated. Consistent with their non-metastatic character, unsorted IGR39 cells formed significantly smaller tumors than unsorted IGR37 cells. In addition, ABCG2+ tumors gave a 2-fold greater mass than tumors from unsorted cells or ABCG2- cells. Furthermore, CXCR6+ cells produced more aggressive tumors. These findings show that CXCR6 identifies a more discrete subpopulation of cultured human melanoma cells with a more aggressive melanoma cancer stem cell phenotype than cells selected on the basis of ABCG2, and CXCR6+/ABCG2+ expression. Conclusions/Significance- The association of more aggressive tumor phenotype with asymmetric self-renewal phenotype reveals a previously unrecognized aspect of tumor cell physiology. Namely, the retention of some tissue-specific stem cell attributes, like the ability to preserve self-phenotype, impacts the natural history of human tumor development. Knowledge of this new aspect of tumor progression may provide new targets for cancer prevention and treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 483. doi:10.1158/1538-7445.AM2011-483

Changes in the structures of motile sperm subpopulations in dog spermatozoa after both cryopreservation and centrifugation on PureSperm ® gradient

The aims of the present study were to: (1) determine if discrete motile sperm subpopulations exist and their incidence in fresh dog ejaculates, (2) evaluate the effects of cryopreservation on the distribution of spermatozoa within the different subpopulations, and (3) determine the effect of the discontinuous PureSperm ® gradient on the sperm subpopulation structure of frozen–thawed dog spermatozoa. Semen from 5 dogs were collected and cryopreserved following a standard protocol. After thawing, semen samples were selected by centrifugation on PureSperm ®. Sperm motility (assessed by computerized-assisted semen analysis, CASA) was assessed before freezing, just after thawing and after preparation on the PureSperm ® gradients. Cryopreservation had a significant ( P < 0.001) effect on CASA-derived parameters. PureSperm ® centrifugation yielded sperm suspensions with improved motility ( P < 0.01). A multivariate clustering procedure separated 19414 motile spermatozoa into four subpopulations: Subpopulation 1 consisting of poorly active and non-progressive spermatozoa (20.97%), Subpopulation 2 consisting of slow and low-linear spermatozoa (18.24%), Subpopulation 3 consisting of highly active but non-progressive spermatozoa (20.75%), and Subpopulation 4 consisting of high speed and progressive spermatozoa (40.03%). Although, cryopreservation had a significant ( P < 0.001) effect on both the frequency distribution of spermatozoa within subpopulations and the motion characteristics of each subpopulation, the sperm subpopulation structure was perfectly maintained after freezing and thawing. The selected sperm samples was enrich in Subpopulation 4, reaching a proportion of 31.9% of the present spermatozoa, in contrast with the unselected sperm samples, where this sperm subpopulation accounted for 24.9% of the total. From these results, we concluded that four well-defined motile sperm subpopulations were present either in fresh semen, in unselected sperm samples or in selected preparations from dogs. The discontinuous PureSperm ® gradient is a simple method to improve the quality of canine frozen–thawed semen samples, since Subpopulation 4 (high-speed and progressive spermatozoa) was more frequently observed after preparation on the gradient. Finally, this study also demonstrated that the general motile sperm structure present in dog remains constant despite the effect caused by either cryopreservation or separation on PureSperm ® gradient.

Cell fate takes a slug in BRCA1-associated breast cancer

Understanding why BRCA1 mutation carriers have a predilection for developing clinically aggressive basal-like breast tumors could inform the development of targeted treatment or prevention strategies. Analysis of both mouse and human mammary epithelial cells has identified a role for BRCA1 in orchestrating differentiation. The ability to isolate discrete epithelial subpopulations from mammary tissue has recently directed attention to luminal progenitor cells - the descendants of mammary stem cells - as the likely 'cells-of-origin' in BRCA1-associated breast cancer. A new publication has confirmed the importance of aberrant luminal cells as key culprits and provided insights on how BRCA1 haploinsufficiency biases luminal cells toward a basal-like fate through aberrant expression of the transcription factor SLUG.

Incorporating Spatial Structure in Stock Assessment: Movement Modeling in Marine Fish Population Dynamics

Investigations into population structure have been at the forefront of fisheries research for decades, yet it is generally ignored in stock assessment models. As the complex nature of marine population structure has been uncovered, models have attempted to accurately portray it through the development of spatially explicit assessments that allow for movement between sub-populations. Although current tag-integrated movement models are highly complex, many arose from the relatively simple models of Beverton and Holt (1957). This article traces the historical development of these models and compares their features. Originally estimation of movement utilized only tag-recapture models, but now tag-integrated assessment models incorporate several sources of fishery, survey, and tag-recapture information to inform movement estimates. As spatial management measures become more widely used, it is increasingly important that assessment models include the spatial complexities of population structure and patterns of fishery removals, in order for more reliable monitoring of population rebuilding to take place. A generalized metapopulation model is proposed for use in fisheries stock assessment, which allows for adult movement among spatially discrete sub-populations. The input requirements for the model include region-specific catch-at-age, a tag-recapture dataset, and auxiliary information, such as a fishery-independent abundance index.

Evidence for discrete subpopulations of sea perch (Helicolenus ercoides) across four fjords in Fiordland, New Zealand

In coastal populations of invertebrates and fishes, the distribution of discrete subpopulations is influenced by adult and larval dispersal, as well as by the effects of habitat heterogeneity on site fidelity or connectivity. Here, we examine evidence for spatial structure of sea perch, Helicolenus percoides, populations among four fjords in the Fiordland region of southwestern New Zealand. We examine patterns in adult morphology, length-at-age, δ13C and δ15N of muscle tissue, and trace elemental composition of whole otoliths as proxies for population isolation among the four inner fjord regions. A multivariate analysis of morphometrics reveals significant differences among populations from each of the four sites, suggesting existence of four distinct subpopulations. These patterns are consistent with observed differences in δ13C and δ15N, and length-at-age estimates among the four subpopulations. Differences in whole otolith concentrations of Sr, Ba, Mg and Li, and high classification scores based on the whole otolith elemental fingerprint are also consistent with significant subdivision among areas. Patterns across all four markers are consistent with discrete subpopulation structure of adult sea perch among the four study sites. These data indicate that the newly implemented network of marine protected areas in Fiordland is likely to contain discrete populations of sea perch.

Characterization of Lymphocyte Subsets in Patients with Common Variable Immunodeficiency Reveals Subsets of Naive Human B Cells Marked by CD24 Expression

Increased proportions of naive B cell subset and B cells defined as CD27(neg)CD21(neg)CD38(neg) are frequently found in patients with common variable immunodeficiency (CVID) syndrome. Current methods of polychromatic flow cytometry and PCR-based detection of κ deletion excision circles allow for fine definitions and replication history mapping of infrequent B cell subsets. We have analyzed B cells from 48 patients with CVID and 49 healthy controls to examine phenotype, frequency, and proliferation history of naive B cell subsets. Consistent with previous studies, we have described two groups of patients with normal (CVID-21norm) or increased (CVID-21lo) proportions of CD27(neg)CD21(neg)CD38(neg) B cells. Upon further analyses, we found two discrete subpopulations of this subset based on the expression of CD24. The B cell subsets showed a markedly increased proliferation in CVID-21lo patients as compared with healthy controls, suggesting developmental arrest rather than increased bone marrow output. Furthermore, when we analyzed CD21(pos) naive B cells, we found two different subpopulations based on IgM and CD24 expression. They correspond to follicular (FO) I and FO II cells previously described in mice. FO I subset is significantly underrepresented in CVID-21lo patients. A comparison of the replication history of naive B cell subsets in CVID patients and healthy controls implies refined naive B cell developmental scheme, in which human transitional B cells develop into FO II and FO I. We propose that the CD27(neg)CD21(neg)CD38(neg) B cells increased in some of the CVID patients originate from the two FO subsets after loss of CD21 expression.

Isolation of the Stromal/Vascular Fraction of Murine Bone Marrow Dramatically Enhances MSC Yield, Allows Isolation of Marrow Endothelial Cells and Reveals Multiple Subpopulations of Stromal Cells.

AbstractAbstract 3861The bone marrow is the organ of residence of a population of multipotent progenitor cells most commonly referred to as mesenchymal stem cells (MSC) based upon their multilineage differentiation potential into bone, cartilage and adipose tissue. The capacity for MSC to contribute to tissue repair demonstrated by numerous previous reports has engendered considerable interest in their application to a broad range of cellular therapies. It follows that a robust reproducible methodology for obtaining high yields of MSC from preclinical animal models, such as rodents, would greatly facilitate the development of these various MSC-based cellular therapies. The plastic-adherent, clonogenic progenitors termed colony forming unit-fibroblast (CFU-F) originally identified by Freidenstein and colleagues that initiate MSC cultures are a rare population in the marrow of all mammalian species so far examined. This is particularly so in the case of the mouse where reported incidences of CFU-F are typically in the range of 1/200,000 bone marrow BM cells. The low incidence of CFU-F significantly complicates the isolation of homogeneous populations of MSC from mouse BM, a common problem being contamination with hematopoietic cells. Seeking to develop an improved methodology to harvest MSC from mouse using methods based on plastic adherent bone marrow, we took advantage of burgeoning evidence demonstrating the perivascular location of MSC not only in the bone marrow, but also in multiple tissues. We hypothesized that a potential reason for the low yield of mMSC from mBM is the flushing of the marrow used to remove single cells suspensions and the consequent destruction of the marrow vasculature, which may adversely affect recovery of MSC physically associated with the abluminal surface of blood vessels. Herein, we describe a simple methodology based on preparation of intact marrow plugs that yields distinct populations of both stromal and endothelial cells. BM plugs are subjected to 3 sequential rounds of digestion in collagenase/dispase and each fraction assayed for content of CFU-F. The recovery of CFU-F obtained by pooling the product of each digestion (1643+199) reproducibly exceeds that obtained using the standard BM flushing technique (13.3+1.9) by at least 2 orders of magnitude (P=<0.001; N = 8) with an accompanying 196-fold enrichment of CFU-F frequency. Purified BM stromal cell populations devoid of hematopoietic contamination are readily obtained by FACS at P0 and these demonstrate robust multilineage differentiation into bone, adipose and chondrogenic progeny using standard in vitro bioassays. A detailed immunophenotypic analysis of the P0 cultures demonstrated the existence of multiple stromal cell subpopulations many of the markers analyzed, including Sca-1, CD90, CD105, CD146 and PDGFRa, which was progressively lost with serial passaging. Discrete subpopulations of stromal cells identified at P0, in many cases had phenotypically identical counterparts in the BM cell suspensions prepared by serial digestion and we are in the process of quantitatively analyzing the evolution of selected phenotypes in vitro to provide clues as to the identity of the founder population of stromal cells that gives rise to ‘MSC’ in vitro. Finally, the phenotypic analysis of P0 cultures also revealed a discrete population of CD105BrightPDGFRaNeg cells representing a mean of 26.7% of hematopoietic lineage-negative cells. Upon isolation and serial propagation, the cells maintain expression of all of the vascular endothelial markers examined including CD31, CD105, VCAM-1, CD144 and MECA32 and also demonstrate inducible expression of E-selectin upon treatment with TNF-a. In conclusion, we describe a simple and robust methodology that, for the first time, allows the simultaneous isolation of both the stromal and vascular components of mouse BM. Secondly, the yield of ‘MSC’ afforded by this technique far exceeds that reported in any previous study. Thirdly, this technique reveals a level of stromal cell heterogeneity not apparent in previous analyses of mouse BM-derived MSC that more realistically reflects the likely complexity of stromal cell populations in vivo and represents a platform for the eventual prospective isolation of specific subpopulations. These studies will greatly enhance experimental strategies designed to analyze not only MSC identity but also the function of the vascular hematopoietic niche. Disclosures:No relevant conflicts of interest to declare.

Identification of a Discrete Subpopulation of T-Cells Over-Expressing HDAC11 with a TH17 Phenotype

AbstractAbstract 588We recently defined a novel role of histone deacetylase 11 (HDAC11), the newest member of the HDAC family, as a negative regulator of IL-10 gene transcription in antigen-presenting cells (APCs).1 To better understand the role of HDAC11 gene expression in immune cells in vivo, we have utilized a BAC (Bacterial artificial chromosome) transgenic mouse in which the EGFP reporter gene was inserted downstream of the HDAC11 promoter region but immediately upstream of the HDAC11 coding sequence (TgHDAC11-EGFP mice).2 In the steady-state, macrophages and B-cells isolated from spleen of TgHDAC11-EGFP mice express low levels of HDAC11 as evidenced by a slight shift in EGFP fluorescence from background. In sharp contrast, we identified a discrete population (11.9%) of T-cells over-expressing HDAC11 as demonstrated both by flow cytometry for EGFP and by qRT-PCR for HDAC11, a majority of which were CD4+ T-cells. Sorting of this EGFP+, CD4+ T-cell population confirmed that the increased EGFP expression correlated with an increased HDAC11mRNA expression. Reminiscent of our prior data in APCs, the increased expression of HDAC11 in T-cells was also inversely correlated with IL-10mRNA expression. Further analyses revealed that in the absence of any stimulation or T-cell polarizing conditions, this EGFP positive population expressed significantly elevated levels of ROR-γt and IL-17 mRNA, markers specific for the TH17 subpopulation. Polarization of wild type CD4+ T-cells into functional TH17 cells was associated with reduction of HDAC11 expression, suggesting a potential role for HDAC11 in regulating T-cell function and/or activation, in particular within the TH17 subset. Further support for this regulatory role of HDAC11 has been provided by our additional findings that T-cells devoid of HDAC11 are indeed hyper-reactive in vitro and in in vivo models.1. Villagra A, et al. Nat Immunol. 2009 Jan;10(1):92-100.2. Gong S, et al. Nature. 2003 Oct 30;425(6961):917-25. Disclosures:No relevant conflicts of interest to declare.

Population genetic structure and landscape connectivity of the Eastern Yellowbelly Racer (Coluber constrictor flaviventris) in the contiguous tallgrass prairie of northeastern Kansas, USA

The tallgrass prairie of North America has undergone widespread habitat loss and fragmentation ( 100 km), but because of sufficient gene flow between locations, we were unable to define discrete subpopulations using Bayesian clustering analyses. Resistance distance, which considers the permeability of habitats, did not explain significant variation in genetic distance beyond Euclidean distance alone, suggesting that racers are not currently influenced by landscape composition. In northeastern Kansas, racers appear to be an abundant and continuously distributed snake that perceives the landscape as well connected with no cover type currently impeding snake dispersal or gene flow.

Stem cells and colon cancer: The questionable cancer stem cell hypothesis

The fine-tuning between cell proliferation and differentiation of self-renewing stem cells and pluripotent progenitors in gastric glands and colon epithelial crypts is coordinated by the mechanisms that regulate colon epithelial cell migration and guidance along the crypt axis. This leads to the acquisition of specialized cellular functions and the exfoliation of desquamated senescent and apoptotic epithelial cells at the apical mucosa interface with the gut lumen. Self-renewing stem cells and pluripotent progenitors are involved in the clonal and polyclonal growth of digestive tumors. Several lines of evidence support the existence of a subpopulation of cancer cells with stem cell-like (SCL) phenotypes in solid tumors of breast and digestive system. Consistently, epithelial cancer cell lines in long-term culture are phenotypically and functionally heterogeneous. It is suggested that only a small proportion of transformed cells are clonogenic in vivo and ex vivo to form colonies and to initiate tumor growth in immunodeficient mice. A discrete subpopulation of tumor -initiating SCL cancer cells are highly competent to survive, propagate and spread through the invasive and metastatic cascade. A better understanding of the mechanisms driving the plasticity and pluripotency of stem cells, their derived progenitors and SCL colon cancer initiating cells during tumor progression will open new avenues for the early detection and treatment of local and distant tumors of the digestive tract.

BENEFITS AND RISKS OF INCREASED SPATIAL RESOLUTION IN THE MANAGEMENT OF FISHERY METAPOPULATIONS UNDER UNCERTAINTY

Stock assessments and harvest guidelines are typically based on the concept of a “fish stock,” which may encompass a very large area. The presence of discrete subpopulations within managed fish stocks presents risks and opportunities for fishery management. Failure to manage catch at the same scale as the true population structure can lead to extirpation of discrete subpopulations and to declines in the productivity of the larger metapopulation. However, it may be difficult and costly to assess and manage stocks at a finer spatial scale, and there is likely greater uncertainty about the size of substocks than about the aggregate stock. We use a two-area simulation model to compare the performance of fishery management at different spatial resolutions when there is uncertainty about growth, the size of the total population, and the relative size of the subpopulations. We show that relative benefits of finer scale management, in terms of profits and risks of depleting subpopulations, depend on a number of biological, technical, and economic factors. In some cases it may be both less risky and more profitable to manage the fishery with a single total allowable catch, even when there are biologically separate fish populations in the two areas.

Demographic Structure of Zanzibar Red Colobus Populations in Unprotected Coral Rag and Mangrove Forests

More than half of the global population of the endangered Zanzibar red colobus (Procolobus kirkii) live outside the single major protected area on Zanzibar Island. We present data on the 2 largest, discrete subpopulations living in unprotected areas at extremes of the species’ range. We compare the size and structure of 11 groups, specifically 6 core groups inhabiting interior, mature forest with 5 peripheral groups living in disturbed/degraded edge habitats. Groups living in southern mangrove forest—a species-poor but more productive and less seasonal habitat than coral rag thicket—had larger group sizes and more heterogeneous age structure, were more stable, and had higher rates of infant survival than did groups in northern coral rag. Group size ranged from 5.5 ± 1.6 SD (the smallest reported for this species) in edge coral rag to 31.2 + 1.9 SD in core mangroves. Edge groups were significantly smaller than core groups in northern coral rag while in the south, where all groups had access to mangroves, we found no significant difference in mean group size between edge and core areas. Groups using mangroves exhibited frequent social play, an indicator of habitat quality, and had a higher ratio of births per female per year. We suggest that mangroves are an important refuge and possibly source habitat for Zanzibar red colobus. We urge the conservation of mangrove and remaining coral rag in the unprotected areas described here in an effort to sustain this endemic species throughout its range.

Identification of sperm subpopulations with defined motility characteristics in ejaculates from Florida goats

The aims of this study were to test the presence of discrete sperm subpopulations in Florida goat ejaculates using a computer-assisted sperm analysis (CASA) system and to establish the relationship between the distribution of the subpopulations found and individual buck, total motility, and sperm concentration. Clustering methods and discriminant analysis were applied to identify motile sperm subpopulations within the semen samples. Principal component analysis revealed that three principal components represented more than the 88% of the variance. After the cluster analysis was performed four motile sperm subpopulations were identified. Subpopulation 1 consisted of rapid and linear sperm (39.84%), Subpopulation 2 consisted of slow but linear spermatozoa (33.23%), Subpopulation 3 consisted of rapid, high ALH but non-linear spermatozoa (14.63%), and Subpopulation 4 consisted of slow and non-linear spermatozoa (12.31%). There were significant differences in the distribution of the four subpopulations (P < 0.001) as well as in the percentage of total motility and the overall sperm concentration (P < 0.05) in fresh ejaculates among the four bucks tested. In conclusion, four well-defined motile sperm subpopulations were identified in Florida goat ejaculates. The relationship between the distribution of the sperm subpopulations and individual buck, total motility, and sperm concentration shows that the spermatozoa of each have different motility patterns. Therefore, the study of discrete subpopulations of motile spermatozoa could lead to a substantial increase in information acquired during caprine semen analysis.

IL-4Ralpha-independent expression of mannose receptor and Ym1 by macrophages depends on their IL-10 responsiveness.

IL-4Rα-dependent responses are essential for granuloma formation and host survival during acute schistosomiasis. Previously, we demonstrated that mice deficient for macrophage-specific IL-4Rα (LysMcreIl4ra−/lox) developed increased hepatotoxicity and gut inflammation; whereas inflammation was restricted to the liver of mice lacking T cell-specific IL-4Rα expression (iLckcreIl4ra−/lox). In the study presented here we further investigated their role in liver granulomatous inflammation. Frequencies and numbers of macrophage, lymphocyte or granulocyte populations, as well as Th1/Th2 cytokine responses were similar in Schistosoma mansoni-infected LysMcreIl4ra−/lox liver granulomas, when compared to Il4ra−/lox control mice. In contrast, a shift to Th1 responses with high IFN-γ and low IL-4, IL-10 and IL-13 was observed in the severely disrupted granulomas of iLckcreIl4ra−/lox and Il4ra−/− mice. As expected, alternative macrophage activation was reduced in both LysMcreIl4ra−/lox and iLckcreIl4ra−/lox granulomas with low arginase 1 and heightened nitric oxide synthase RNA expression in granuloma macrophages of both mouse strains. Interestingly, a discrete subpopulation of SSChighCD11b+I-A/I-EhighCD204+ macrophages retained expression of mannose receptor (MMR) and Ym1 in LysMcreIl4ra−/lox but not in iLckcreIl4ra−/lox granulomas. While aaMφ were in close proximity to the parasite eggs in Il4ra−/lox control mice, MMR+Ym1+ macrophages in LysMcreIl4ra−/lox mice were restricted to the periphery of the granuloma, indicating that they might have different functions. In vivo IL-10 neutralisation resulted in the disappearance of MMR+Ym1+ macrophages in LysMcreIl4ra−/lox mice. Together, these results show that IL-4Rα-responsive T cells are essential to drive alternative macrophage activation and to control granulomatous inflammation in the liver. The data further suggest that in the absence of macrophage-specific IL-4Rα signalling, IL-10 is able to drive mannose receptor- and Ym1-positive macrophages, associated with control of hepatic granulomatous inflammation.

Inferring population structure and genetic diversity of broad range of wild diploid alfalfa (Medicago sativa L.) accessions using SSR markers

Diversity analyses in alfalfa have mainly evaluated genetic relationships of cultivated germplasm, with little known about variation in diploid germplasm in the M. sativa-falcata complex. A collection of 374 individual genotypes derived from 120 unimproved diploid accessions from the National Plant Germplasm System, including M. sativa subsp. caerulea, falcata, and hemicycla, were evaluated with 89 polymorphic SSR loci in order to estimate genetic diversity, infer the genetic bases of current morphology-based taxonomy, and determine population structure. Diploid alfalfa is highly variable. A model-based clustering analysis of the genomic data identified two clearly discrete subpopulations, corresponding to the morphologically defined subspecies falcata and caerulea, with evidence of the hybrid nature of the subspecies hemicycla based on genome composition. Two distinct subpopulations exist within each subsp. caerulea and subsp. falcata. The distinction of caerulea was based on geographical distribution. The two falcata groups were separated based on ecogeography. The results show that taxonomic relationships based on morphology are reflected in the genetic marker data with some exceptions, and that clear distinctions among subspecies are evident at the diploid level. This research provides a baseline from which to systematically evaluate variability in tetraploid alfalfa and serves as a starting point for exploring diploid alfalfa for genetic and breeding experiments.

Regional Heterogeneity and Diversity in Cytokine and Chemokine Production by Astroglia: Differential Responses to HIV-1 Tat, gp120, and Morphine Revealed by Multiplex Analysis

HIV-infected individuals who abuse opiates show a faster progression to AIDS and higher incidence of encephalitis. The HIV-1 proteins Tat and gp120 have been shown to cause neurodegenerative changes either in vitro or when injected or expressed in the CNS, and we have shown that opiate drugs can exacerbate neurotoxic effects in the striatum through direct actions on pharmacologically discrete subpopulations of mu-opioid receptor-expressing astroglia. Opiate coexposure also significantly enhances release of specific inflammatory mediators by astroglia from the striatum, and we theorize that astroglial reactivity may underlie aspects of HIV neuropathology. To determine whether astroglia from different regions of the central nervous system have distinct, intrinsic responses to HIV-1 proteins and opiates, we used multiplex suspension array analyses to define and compare the inflammatory signature of cytokines released by murine astrocytes grown from cerebral cortex, cerebellum, and spinal cord. Results demonstrate significant regional differences in baseline secretion patterns, and in responses to viral proteins. Of importance for the disease process, astrocytes from all regions have very limited inflammatory response to gp120 protein, as compared to Tat protein, either in the presence or absence of morphine. Overall, the chemokine/cytokine release is higher from spinal cord and cortical astroglia than from cerebellar astroglia, paralleling the relatively low incidence of HIV-related neuropathology in the cerebellum.

Green-to-red photoconvertible fluorescent proteins: tracking cell and protein dynamics on standard wide-field mercury arc-based microscopes

BackgroundGreen fluorescent protein (GFP) and other FP fusions have been extensively utilized to track protein dynamics in living cells. Recently, development of photoactivatable, photoswitchable and photoconvertible fluorescent proteins (PAFPs) has made it possible to investigate the fate of discrete subpopulations of tagged proteins. Initial limitations to their use (due to their tetrameric nature) were overcome when monomeric variants, such as Dendra, mEos, and mKikGR were cloned/engineered.ResultsHere, we report that by closing the field diaphragm, selective, precise and irreversible green-to-red photoconversion (330-380 nm illumination) of discrete subcellular protein pools was achieved on a wide-field fluorescence microscope equipped with standard DAPI, Fluorescein, and Rhodamine filter sets and mercury arc illumination within 5-10 seconds. Use of a DAPI-filter cube with long-pass emission filter (LP420) allowed the observation and control of the photoconversion process in real time. Following photoconversion, living cells were imaged for up to 5 hours often without detectable phototoxicity or photobleaching.ConclusionsWe demonstrate the practicability of this technique using Dendra2 and mEos2 as monomeric, photoconvertible PAFP representatives fused to proteins with low (histone H2B), medium (gap junction channel protein connexin 43), and high (α-tubulin; clathrin light chain) dynamic cellular mobility as examples. Comparable efficient, irreversible green-to-red photoconversion of selected portions of cell nuclei, gap junctions, microtubules and clathrin-coated vesicles was achieved. Tracking over time allowed elucidation of the dynamic live-cycle of these subcellular structures. The advantage of this technique is that it can be performed on a standard, relatively inexpensive wide-field fluorescence microscope with mercury arc illumination. Together with previously described laser scanning confocal microscope-based photoconversion methods, this technique promises to further increase the general usability of photoconvertible PAFPs to track the dynamic movement of cells and proteins over time.

Population structure of carite ( Scomberomorus brasiliensis) in waters offshore of Trinidad and northern Venezuela

Population structure of carite (Serra Spanish mackerel, Scomberomorus brasiliensis) in waters offshore of Trinidad and northern Venezuela was assessed by analysis of allele and genotype distributions at 16 nuclear-encoded microsatellites and mitochondrial (mt)DNA haplotype distribution among individuals from five localities in the southern Caribbean Sea. Tests of homogeneity (microsatellites) revealed a genetically discrete subpopulation (stock) of carite in the Cariaco Basin along the northern coast of Venezuelan mainland. This subpopulation contained a small, highly divergent clade of four mtDNA haplotypes that possibly represent recent admixture of formerly isolated subpopulations. Spatial autocorrelation analysis of the microsatellite data was consistent with the notion that carite from Isla de Margarita (Venezuela) and Trinidad may warrant management as distinct stocks, while spatial autocorrelation analysis and tests of selective neutrality of mtDNA variation suggested that carite off the south coast of Trinidad differ from carite along the west and north coasts. The implication of these findings is that there may be a number of spawning stocks of carite in the southern Caribbean Sea.