Subcapsular sinus macrophage fragmentation and CD169+ bleb acquisition by closely associated IL-17-committed innate-like lymphocytes

Subcapsular sinus macrophages (SSMs) in lymph nodes are rapidly exposed to antigens arriving in afferent lymph and have a role in their capture and display to B cells. In tissue sections SSMs exhibit long cellular processes and express high amounts of CD169. Here, we show that many of the cells present in lymph node cell suspensions that stain for CD169 are not macrophages but lymphocytes that have acquired SSM-derived membrane blebs. The CD169 bleb(+) lymphocytes are enriched for IL-17 committed IL-7Rα(hi)CCR6(+) T cells and NK cells. In addition, the CD169 staining detected on small numbers of CD11c(hi) dendritic cells is frequently associated with membrane blebs. Counter intuitively the CD169 bleb(+) lymphocytes are mostly CD4 and CD8 negative whereas many SSMs express CD4. In situ, many IL-7Rα(hi) cells are present at the subcapsular sinus and interfollicular regions and migrate in close association with CD169(+) macrophages. These findings suggest SSMs undergo fragmentation during tissue preparation and release blebs that are acquired by closely associated cells. They also suggest an intimate crosstalk between SSMs and IL-17 committed innate-like lymphocytes that may help provide early protection of the lymph node against lymph-borne invaders.     

Design,synthesis and pharmacological characterization of coumarin-based fluorescent analogs of excitatory amino acid transporter subtype 1 selective inhibitors,UCPH-101 and UCPH-102

The excitatory amino acid transporters (EAATs) play a pivotal role in regulating the synaptic concentration of glutamate in the mammalian central nervous system. To date, five different subtypes have been identified, named EAAT15 in humans (and GLAST, GLT-1, EAAC1, EAAT4, and EAAT5, respectively, in rodents). Recently, we have published and presented a structure–activity relationship (SAR) study of a novel class of selective inhibitors of EAAT1 (and GLAST), with the analogs UCPH-101 (IC₅₀ = 0.66 μM) and UCPH-102 (IC₅₀ = 0.43 μM) being the most potent inhibitors in the series. In this paper, we present the design, synthesis and pharmacological evaluation of six coumarin-based fluorescent analogs of UCPH-101/102 as subtype-selective inhibitors at EAAT1. Analogs 1114 failed to inhibit EAAT1 function (IC₅₀ values >300 μM), whereas analogs 15 and UCPH-102F inhibited EAAT1 with IC₅₀ values in the medium micromolar range (17 μM and 14 μM, respectively). Under physiological pH no fluorescence was observed for analog 15, while a bright blue fluorescence emission was observed for analog UCPH-102F. Regrettably, under confocal laser scanning microscopy selective visualization of expression of EAAT1 over EAAT3 was not possible due to nonspecific binding of UCPH-102F.     

Root aeration via aerenchymatous phellem: three-dimensional micro-imaging and radial O2 profiles in Melilotus siculus

? Internal root aeration enables waterlogging-tolerant species to grow in anoxic soil. Secondary aerenchyma, in the form of aerenchymatous phellem, is of importance to root aeration in some dicotyledonous species. Little is known about this type of aerenchyma in comparison with primary aerenchyma. ? Micro-computed tomography was employed to visualize, in three dimensions, the microstructure of the aerenchymatous phellem in roots of Melilotus siculus. Tissue porosity and respiration were also measured for phellem and stelar tissues. A multiscale, three-dimensional, diffusion-respiration model compared the predicted O(2) profiles in roots with those measured using O(2) microelectrodes. ? Micro-computed tomography confirmed the measured high porosity of aerenchymatous phellem (44-54%) and the low porosity of stele (2-5%) A network of connected gas spaces existed in the phellem, but not within the stele. O(2) partial pressures were high in the phellem, but fell below the detection limit in the thicker upper part of the stele, consistent with the poorly connected low porosity and high respiratory demand. ? The presented model integrates and validates micro-computed tomography with measured radial O(2) profiles for roots with aerenchymatous phellem, confirming the existence of near-anoxic conditions at the centre of the stele in the basal parts of the root, coupled with only hypoxic conditions towards the apex.     

The Caenorhabditis elegans NR4A nuclear receptor is required for spermatheca morphogenesis

The gene nhr-6 encodes the Caenorhabditis elegans ortholog of the NR4A nuclear receptor. We determined the biological functions of NHR-6 through the isolation and characterization of a deletion allele of nhr-6, lg6001. We demonstrate that nhr-6 has an essential role in the development of the C. elegans somatic gonad. Specifically, nhr-6 is required for the development of the hermaphrodite spermatheca, a somatic gonad organ that serves as the site of sperm storage and oocyte fertilization. Using a variety of spermatheca cell markers, we have determined that loss of nhr-6 function causes severe morphological defects in the spermatheca and associated spermathecal valves. This appears to be due to specific requirements for nhr-6 in regulating cell proliferation and cell differentiation during development of these structures. The improper development of these structures in nhr-6(lg6001) mutants leads to defects in ovulation and significantly reduced fecundity of C. elegans hermaphrodites. The phenotypes of nhr-6(lg6001) mutants are consistent with a role for nhr-6 in organogenesis, similar to the functions of its mammalian homologs.     

Anuran occupancy of created wetlands in the Central Appalachians

Evaluating the adequacy of created wetlands to replace the functions of lost natural wetlands is important because wetland mitigation is a major tool used to offset wetland losses. However, measurements such as vegetative cover and presence of wildlife may not provide sufficient evidence that created wetlands are functioning properly and thus examining the ecology of wetland biota such as that of amphibians may be a more useful surrogate for function. The objectives of this study were to compare the occupancy and detection of calling anurans in created wetlands relative to beaver-created wetlands. Five-, 10-min, and broadcast call surveys were performed at 24 wetlands throughout the Central Appalachians once every month from March through August of 2009 and 2010. Occupancy modeling was used to estimate the occupancy and detection of individual species, incorporating relevant environmental variables. The occupancy of anurans did not differ between human-created and beaver (Castor canadensis)-created wetlands. Detection of anurans was largely unaffected by call survey type, but several environmental covariates had a significant effect on the detectability of calling anurans. Our results suggest that the function of providing adequate chorusing habitat for adult anurans is being fulfilled by the created wetlands that we examined.     

Role of MAdCAM-1 and its ligand on the homing of transplanted hematopoietic cells in irradiated mice

We examined the expression of VCAM-1 and MAdCAM-1 after bone marrow transplantation (BMT). We also examined the influence of alpha(4)beta(7) integrin blockade on the homing of cells to the bone marrow and spleen. The expression of VCAM-1 and MAdCAM-1 by endothelial cells in the spleen and bone marrow was examined by immunoelectron microscopy using colloidal gold and was analyzed semiquantitatively. To examine the role of alpha(4)beta(7) integrin in donor cells, a homing assay was conducted following alpha(4)beta(7) integrin blockade in bone marrow-derived hematopoietic cells or spleen colony cells. Immediately after BMT, the expression of VCAM-1 and MAdCAM1 markedly decreased, but expression recovered significantly between 12 and 24 h after BMT. VCAM-1 recovered more acutely than MAdCAM-1 from 12 h onward. In the group transplanted with anti-alpha(4)beta(7) integrin antibody-treated bone marrow cells, the numbers of homing cells in the spleen and bone marrow were significantly decreased in an antibody dose-dependent manner. However, the number of homing cells was not different in either the spleen or bone marrow between anti-alpha(4)beta(7) integrin antibody treated and untreated spleen colony cells. It has been reported that alpha(4)beta(1) integrin and its receptor VCAM-1 play major roles in the homing of hematopoietic cells to bone marrow. Our study indicates the importance of MAdCAM-1 and its ligand, alpha(4)beta(7) integrin, in the homing of bone marrow-derived hematopoietic cells, but not spleen colony-derived cells, to both the spleen and bone marrow.     

Porphyrin Homeostasis Maintained by ABCG2 Regulates Self-Renewal of Embryonic Stem Cells

Background

Under appropriate culture conditions, undifferentiated embryonic stem (ES) cells can undergo multiple self-renewal cycles without loss of pluripotency suggesting they must be equipped with specific defense mechanisms to ensure sufficient genetic stability during self-renewal expansion. The ATP binding cassette transporter ABCG2 is expressed in a wide variety of somatic and embryonic stem cells. However, whether it plays an important role in stem cell maintenance remains to be defined.

Methodology/Principal Findings

Here we provide evidence to show that an increase in the level of ABCG2 was observed accompanied by ES colony expansion and then were followed by decreases in the level of protoporphyrin IX (PPIX) indicating that ABCG2 plays a role in maintaining porphyrin homoeostasis. RNA-interference mediated inhibition of ABCG2 as well as functional blockage of ABCG2 transporter with fumitremorgin C (FTC), a specific and potent inhibitor of ABCG2, not only elevated the cellular level of PPIX, but also arrest the cell cycle and reduced expression of the pluripotent gene Nanog. Overexpression of ABCG2 in ES cells was able to counteract the increase of endogenous PPIX induced by treatment with 5-Aminolevulinic acid suggesting ABCG2 played a direct role in removal of PPIX from ES cells. We also found that excess PPIX in ES cells led to elevated levels of reactive oxygen species which in turn triggered DNA damage signals as indicated by increased levels of γH2AX and phosphorylated p53. The increased level of p53 reduced Nanog expression because RNA- interference mediated inhibition of p53 was able to prevent the downregulation of Nanog induced by FTC treatment.

Conclusions/Significance

The present work demonstrated that ABCG2 protects ES cells from PPIX accumulation during colony expansion, and that p53 and γH2AX acts as a downstream checkpoint of ABCG2-dependent defense machinery in order to maintain the self-renewal of ES cells.     

Large-scale identification and mapping of nuclear matrix-attachment regions in the distal imprinted domain of mouse chromosome 7.

Mammalian imprinted genes, which are expressed from only one of the parental alleles, have a tendency to form clusters and are regulated by long-range mechanisms. Nuclear matrix-attachment regions (MARs), the anchor points of loop domains, are involved in coordination of gene expression and could play a role in regulation of imprinted domains. We have identified and mapped a total of 52 MARs in a 1-Mb imprinted domain on mouse distal chromosome 7 using our cosmid contigs and an in vitro MAR assay. We find two MAR clusters (comprising 20 and 19 MARs), one of which is mapped in the Th-Ins2 intergenic region, coincident with the boundary between the two imprinted subdomains. However, the imprinted/non-imprinted boundaries are not associated with a MAR. Based on the sequence information, we find that many of the MARs are rich in long interspersed nuclear elements. In addition, comparisons of the results obtained with several MAR-prediction software programs reveal good performance of ChrClass in terms of both sensitivity and specificity. This study presents the first large-scale mapping of MARs in an imprinted domain and provides a platform for understanding the roles of MARs in imprinting.     

Methods for the analysis of cannabinoids in biological materials: a review

Various methods for the analysis of cannabinoids in biological materials, including plant and human body materials, are reviewed. Chromatographic methods, such as TLC, GC and HPLC, and non-chromatographic methods, mainly immunoassays, are discussed and compared. Chromatography is most commonly used in the analysis of plant material, with GC apparently offering the most advantages. Immunoassays, such as radioimmunoassay and fluorescence polarisation immunoassay, and enzyme immunoassay methods, such as enzyme multiplied immunoassay technique and enzyme-linked immunosorbent assay, can be used for human body materials; however, GC-MS is still necessary for confirmation and accurate quantification. Preferred methods are suggested for various specific purposes.