[Genetic variant analysis and prenatal diagnosis for Chinese pedigrees affected with cblC methylmalonic acidemia].

OBJECTIVE: To detect variants of the MMACHC gene among 110 ethnic Han Chinese pedigrees affected with metabolic deficiency methylmalonic acidemia (MMA) of cobalamin C (cblC). METHODS: Peripheral blood samples were collected from the probands and their parents. Following DNA extraction, the coding regions of the MMACHC gene were subjected to PCR amplification, Sanger sequencing and quantitative PCR assaying. For 48 pedigrees, chorionic villus samples were taken for prenatal genetic diagnosis. RESULTS: Thirty five types of variants were detected among the 110 pedigrees, which included missense, nonsense, frameshifting, splicing variants and exonic deletions. Most variants have occurred in exons 4 (73.18%). The detection rate for c.609G>A (p.Trp203Ter) variant was the highest (33.64%), followed by c.658_660delAAG (12.27%), c.567dupT (9.09%) and c.80A>G (6.82%). Two variants, namely c.57_58insT (p.Gly20Trpfs*14) and c.505_506delAT (p.Ile169Argfs*12), were unreported previously and both were of frameshifting types. For the 48 pedigrees undergoing prenatal diagnosis, 14 fetuses were found to be normal, 24 have carried heterozygous variants, the remaining 10 have carried compound heterozygous or homozygous variants. CONCLUSION: The discovery of the two novel variants has expanded the spectrum of the MMACHC gene variants among ethnic Han population. Above finding has provide a basis for the prenatal diagnosis and genetic counseling for the affected pedigrees.

CeO2 Nanowire-BODIPY-Adenosine Triphosphate Fluorescent Sensing Platform for Highly Specific and Sensitive Detection of Arsenate.

Effective and sensitive monitoring of arsenate in drinking water is significant for risk management of public health. Here, we demonstrated that a CeO2 nanowire acted as an efficient quencher for small fluorescent molecules with a phosphate group, BODIPY-adenosine triphosphate (BODIPY-ATP) and riboflavin-5'-phosphate (Rf-P), and developed a CeO2 nanowire-BODIPY-ATP platform for highly selective and sensitive detection of arsenate. The response strategy was based on the competitive coordination chemistry of CeO2 nanowire between arsenate and phosphate group of BODIPY-ATP. Arsenate displaced adsorbed BODIPY-ATP to enhance fluorescence, allowing detection of arsenate down to 7.8 nM, which is lower than the WHO-defined limit of 130 nM. An excellent linear range of 20-150 and 150-1000 nM was obtained. Importantly, this system was simple in design and convenient in operation. Also, the platform exhibited excellent selectivity for arsenate without the interference of phosphate ions. Finally, the proposed method had been successfully employed for determination of arsenate in real water samples.

Highly selective and ratiometric fluorescent nanoprobe for the detection of cysteine and its application in test strips.

Cysteine (Cys) is a bithiol that plays a vital role in many physiological processes. However, it is difficult to discriminate Cys from homocysteine (Hcy) and glutathione (GSH), due to their similar chemical structures and reactivity. Herein, we have developed a polymeric nanoprobe, nanoHFA, for ratiometric, highly selective, and sensitive detection of Cys based on 7-hydroxycoumarin-3-carboxylic acid (HC) and fluorescein isothiocyanate (FITC)-acrylate (FITC-A) group-functionalized lipopolymer DSPE-PEG. The probe nanoHFA showed a strong fluorescence emission peak centered at 450 nm attributed to HC and a weak fluorescence emission peak centered at 520 nm due to the photoinduced electron transfer (PET) process of FITC induced by acrylate group. In the presence of Cys, the fluorescence signal at 520 nm could be lit up and the ratio of F520nm/F450nm showed a good linear relationship in the range of 5-60 µM with a low detection limit of 0.37 µM. The probe also displayed excellent water solubility and high selectivity to Cys over other biothiols such as Hcy and GSH. Moreover, we further used probe nanoHFA to detect Cu2+ ions in the range of 100-550 nM with a detection limit of 77 nM. The nanoprobe was successfully applied for the quantitative detection of Cys in fetal bovine serum, and fluorescent strips were developed for facile and visual detection of Cys and Cu2+ ions. Graphical abstract ᅟ.

A fast, highly sensitive and selective assay of iodide ions with single-stranded DNA-templated copper nanoparticles as a fluorescent probe for its application in Kunming mice samples.

The development of fast, sensitive, selective and flexible methods for the detection of iodide is highly demanded and is of great significance. In this work, single-stranded DNA-templated copper nanoparticles (ssDNA-CuNPs) generated by sodium ascorbate reduction of Cu2+ along the single-stranded DNA of poly-T were utilized as a fluorescent probe for the determination of iodide ions (I-). The detection scheme is based on the instant quenching of the fluorescence of ssDNA-CuNPs by iodide ions. I- can be quantified in the concentration range from 0.050 to 40 µM and from 40 to 80 µM, and the limit of detection is as low as 15 nM. This method provides a simple and convenient strategy for the biochemical assay of I-, which is also helpful for early diagnosis of related diseases. The establishment of a low cost and fast detection method would be particularly important in developing countries where medical supplies are lacking.

Differential DARC/ACKR1 expression distinguishes venular from non-venular endothelial cells in murine tissues.

BACKGROUND: Intravascular leukocyte recruitment in most vertebrate tissues is restricted to postcapillary and collecting venules, whereas capillaries and arterioles usually support little or no leukocyte adhesion. This segmental restriction is thought to be mediated by endothelial, rather than hemodynamic, differences. The underlying mechanisms are largely unknown, in part because effective tools to distinguish, isolate, and analyze venular endothelial cells (V-ECs) and non-venular endothelial cells (NV-ECs) have been unavailable. We hypothesized that the atypical chemokine receptor DARC (Duffy Antigen Receptor for Chemokines, a.k.a. ACKR1 or CD234) may distinguish V-ECs versus NV-ECs in mice. METHODS: We generated a rat-anti-mouse monoclonal antibody (MAb) that specifically recognizes the erythroid and endothelial forms of native, surface-expressed DARC. Using this reagent, we characterized DARC expression and distribution in the microvasculature of murine tissues. RESULTS: DARC was exquisitely restricted to post-capillary and small collecting venules and completely absent from arteries, arterioles, capillaries, veins, and most lymphatics in every tissue analyzed. Accordingly, intravital microscopy showed that adhesive leukocyte-endothelial interactions were restricted to DARC+ venules. DARC was detectable over the entire circumference of V-ECs, but was more concentrated at cell-cell junctions. Analysis of single-cell suspensions suggested that the frequency of V-ECs among the total microvascular EC pool varies considerably between different tissues. CONCLUSIONS: Immunostaining of endothelial DARC allows the identification and isolation of intact V-ECs from multiple murine tissues. This strategy may be useful to dissect the mechanisms underlying segmental microvascular specialization in healthy and diseased tissues and to characterize the role of EC subsets in tissue-homeostasis, immune surveillance, infection, inflammation, and malignancies.

Purification and characterization of novel extracellular cholesterol esterase from Acinetobacter sp.

CHE4-1, a bacterial strain that belongs to the genus Acinetobacter and expresses high level of inducible extracellular cholesterol esterase (CHE), was isolated from feces of carnivore Panthera pardus var. The cholesterol esterase of the strain CHE4-1 was purified by ultrafiltration followed with DEAE-Sepharose FF chromatography and Phenyl-Sepharose CL-4B chromatography, and then by Sephadex G-50 gel filtration. Different from other known microbial cholesterol esterase, the purified CHE from CHE4-1 strain is a monomer with molecular weight of 6.5 kD and has high activity to both long-chain and short-chain cholesterol ester. Enzymatic activity was enhanced in the presence of metal ion Ca(2+), Zn(2+) and boracic acid, and was not significantly affected by several detergents including sodium cholate, Triton X100 and Tween-80. The enzyme was found to be stable during long-term aqueous storage at 4 °C, indicating its potential as a clinical diagnostic reagent. To the best of our knowledge, this is the first report regarding purification and characterization of CHE from Acinetobacter sp. The results demonstrated that this particular CHE is a novel cholesterol esterase.

Distamycin A inhibits HMGA1-binding to the P-selectin promoter and attenuates lung and liver inflammation during murine endotoxemia.

BACKGROUND: The architectural transcription factor High Mobility Group-A1 (HMGA1) binds to the minor groove of AT-rich DNA and forms transcription factor complexes ("enhanceosomes") that upregulate expression of select genes within the inflammatory cascade during critical illness syndromes such as acute lung injury (ALI). AT-rich regions of DNA surround transcription factor binding sites in genes critical for the inflammatory response. Minor groove binding drugs (MGBs), such as Distamycin A (Dist A), interfere with AT-rich region DNA binding in a sequence and conformation-specific manner, and HMGA1 is one of the few transcription factors whose binding is inhibited by MGBs. OBJECTIVES: To determine whether MGBs exert beneficial effects during endotoxemia through attenuating tissue inflammation via interfering with HMGA1-DNA binding and modulating expression of adhesion molecules. METHODOLOGY/PRINCIPAL FINDINGS: Administration of Dist A significantly decreased lung and liver inflammation during murine endotoxemia. In intravital microscopy studies, Dist A attenuated neutrophil-endothelial interactions in vivo following an inflammatory stimulus. Endotoxin induction of P-selectin expression in lung and liver tissue and promoter activity in endothelial cells was significantly reduced by Dist A, while E-selectin induction was not significantly affected. Moreover, Dist A disrupted formation of an inducible complex containing NF-kappaB that binds an AT-rich region of the P-selectin promoter. Transfection studies demonstrated a critical role for HMGA1 in facilitating cytokine and NF-kappaB induction of P-selectin promoter activity, and Dist A inhibited binding of HMGA1 to this AT-rich region of the P-selectin promoter in vivo. CONCLUSIONS/SIGNIFICANCE: We describe a novel targeted approach in modulating lung and liver inflammation in vivo during murine endotoxemia through decreasing binding of HMGA1 to a distinct AT-rich region of the P-selectin promoter. These studies highlight the ability of MGBs to function as molecular tools for dissecting transcriptional mechanisms in vivo and suggest alternative treatment approaches for critical illness.

Distinct roles for LFA-1 affinity regulation during T-cell adhesion, diapedesis, and interstitial migration in lymph nodes.

During the course of homing to lymph nodes (LNs), T cells undergo a multistep adhesion cascade that culminates in a lymphocyte function-associated antigen 1 (LFA-1)-dependent firm adhesion to the luminal surface of high endothelial venules (HEVs). The importance of LFA-1 affinity regulation in supporting T-cell arrest on HEVs has been well established, however, its importance in the postadhesion phase, which involves intraluminal crawling and diapedesis to the extravascular space, remains elusive. Here we have shown that LFA-1 affinity needs to be appropriately regulated to support these essential steps in the homing cascade. Genetically engineered T cells that were unable to properly down-regulate LFA-1 affinity underwent enhanced, chemokine-independent arrest in HEVs but showed perturbed intravascular crawling to transmigration sites and compromised diapedesis across HEVs. By contrast, the extravascular migration of T cells was insensitive to the affinity-enhancing LFA-1 mutation. These results highlight the requirement for balanced LFA-1 affinity regulation in intravascular and transvascular, but not extravascular, T-cell migration in LNs.

Genetic perturbation of the putative cytoplasmic membrane-proximal salt bridge aberrantly activates alpha(4) integrins.

alpha(4) integrins play a pivotal role in leukocyte migration and tissue-specific homing. The ability of integrins to bind ligand is dynamically regulated by activation-dependent conformational changes triggered in the cytoplasmic domain. An NMR solution structure defined a putative membrane-proximal salt bridge between the alpha(IIb)beta(3) integrin cytoplasmic tails, which restrains integrins in their low-affinity state. However, the physiological importance of this salt bridge in alpha(4) integrin regulation remains to be elucidated. To address this question, we disrupted the salt bridge in murine germ line by mutating the conserved cytoplasmic arginine R(GFFKR) in alpha(4) integrins. In lymphocytes from knock-in mice (alpha(4)-R/A(GFFKR)), alpha(4)beta(1) and alpha(4)beta(7) integrins exhibited constitutively up-regulated ligand binding. However, transmigration of these cells across VCAM-1 and MAdCAM-1 substrates, or across endothelial monolayers, was reduced. Perturbed detachment of the tail appeared to cause the reduced cell migration of alpha(4)-R/A(GFFKR) lymphocytes. In vivo, alpha(4)-R/A(GFFKR) cells exhibited increased firm adhesion to Peyer patch venules but reduced homing to the gut. Our results demonstrate that the membrane-proximal salt bridge plays a critical role in supporting proper alpha(4) integrin adhesive dynamics. Loss of this interaction destabilizes the nonadhesive conformation, and thereby perturbs the properly balanced cycles of adhesion and deadhesion required for efficient cell migration.

Resolvin E1, an EPA-derived mediator in whole blood, selectively counterregulates leukocytes and platelets.

Resolvin E1 (RvE1) is an omega-3 eicosapentaenoic acid (EPA)-derived lipid mediator generated during resolution of inflammation and in human vasculature via leukocyte-endothelial cell interactions. RvE1 possesses anti-inflammatory and proresolving actions. Here, we report that RvE1 in human whole blood rapidly regulates leukocyte expression of adhesion molecules. RvE1 in the 10- to 100-nM range stimulated L-selectin shedding, while reducing CD18 expression in both neutrophils and monocytes. When added to whole blood, RvE1 did not stimulate reactive oxygen species by either neutrophils or monocytes, nor did it directly stimulate cytokine/chemokine production in heparinized blood. Intravital microscopy (IVM) demonstrated that RvE1 rapidly reduced leukocyte rolling (approximately 40%) in venules of mice. In human platelet-rich plasma (PRP), RvE1 selectively blocked both ADP-stimulated and thromboxane receptor agonist U46619-stimulated platelet aggregation in a concentration-dependent manner. In contrast, Delta 6,14-trans-RvE1 isomer was inactive. RvE1 did not block collagen-stimulated aggregation, and regulation of ADP-induced platelet aggregation was not further enhanced with aspirin treatment. These results indicate RvE1 is a potent modulator of leukocytes as well as selective platelet responses in blood and PRP, respectively. Moreover, the results demonstrate novel agonist-specific antiplatelet actions of RvE1 that are potent and may underlie some of the beneficial actions of EPA in humans.

Aberrant activation of integrin alpha4beta7 suppresses lymphocyte migration to the gut.

Integrin adhesion molecules mediate lymphocyte migration and homing to normal and inflamed tissues. While the ligand-binding activity of integrins is known to be modulated by conformational changes, little is known about how the appropriate balance of integrin adhesiveness is maintained in order to optimize the migratory capacity of lymphocytes in vivo. In this study we examined the regulation of the gut homing receptor alpha4beta7 integrin by manipulating at the germline level an integrin regulatory domain known as adjacent to metal ion-dependent adhesion site (ADMIDAS). ADMIDAS normally serves to raise the activation threshold of alpha4beta7, thereby stabilizing it in the default nonadhesive state. Lymphocytes from knockin beta7 (D146A) mice, which harbor a disrupted ADMIDAS, not only expressed an alpha4beta7 integrin that persistently adhered to mucosal addressin cell adhesion molecule-1 (MAdCAM-1), but also exhibited perturbed cell migration along MAdCAM-1 substrates resulting from improper de-adhesion of the lymphocyte trailing edge. In vivo, aberrantly activated alpha4beta7 enhanced adhesion to Peyer's patch venules, but suppressed lymphocyte homing to the gut, diminishing the capacity of T cells to induce colitis. Our results underscore the importance of a proper balance in the adhesion and de-adhesion of the alpha4beta7 integrin, both for lymphocyte trafficking to the gut and for colitis progression.

Activation of bone marrow-resident memory T cells by circulating, antigen-bearing dendritic cells.

Dendritic cells (DCs) carry antigen from peripheral tissues via lymphatics to lymph nodes. We report here that differentiated DCs can also travel from the periphery into the blood. Circulating DCs migrated to the spleen, liver and lung but not lymph nodes. They also homed to the bone marrow, where they were retained better than in most other tissues. Homing of DCs to the bone marrow depended on constitutively expressed vascular cell adhesion molecule 1 and endothelial selectins in bone marrow microvessels. Two-photon intravital microscopy in bone marrow cavities showed that DCs formed stable antigen-dependent contacts with bone marrow-resident central memory T cells. Moreover, using this previously unknown migratory pathway, antigen-pulsed DCs were able to trigger central memory T cell-mediated recall responses in the bone marrow.

Reciprocal and dynamic control of CD8 T cell homing by dendritic cells from skin- and gut-associated lymphoid tissues.

T cell activation by intestinal dendritic cells (DC) induces gut-tropism. We show that, reciprocally, DC from peripheral lymph nodes (PLN-DC) induce homing receptors promoting CD8 T cell accumulation in inflamed skin, particularly ligands for P- and E-selectin. Differential imprinting of tissue-tropism was independent of Th1/Th2 cytokines and not restricted to particular DC subsets. Fixed PLN-DC retained the capacity to induce selectin ligands on T cells, which was suppressed by addition of live intestinal DC. By contrast, fixed intestinal DC failed to promote gut-tropism and instead induced skin-homing receptors. Moreover, the induction of selectin ligands driven by antigen-pulsed PLN-DC could be suppressed "in trans" by adding live intestinal DC, but PLN-DC did not suppress gut-homing receptors induced by intestinal DC. Reactivation of tissue-committed memory cells modified their tissue-tropism according to the last activating DC's origin. Thus, CD8 T cells activated by DC acquire selectin ligands by default unless they encounter fixation-sensitive signal(s) for gut-tropism from intestinal DC. Memory T cells remain responsive to these signals, allowing for dynamic migratory reprogramming by skin- and gut-associated DC.

Tumor necrosis factor-alpha-converting enzyme (ADAM17) mediates GPIbalpha shedding from platelets in vitro and in vivo.

Interaction of the platelet receptor glycoprotein (GP) Ib-V-IX with von Willebrand factor exposed at a site of vascular injury is an essential step in the initiation of a hemostatic plug. Proteolytic cleavage (shedding) of the GPIbalpha subunit was first described >25 years ago, the protease mediating this event as well as its physiological function, however, have not been elucidated. We reported recently that shedding of GPIbalpha induced by platelet storage or mitochondrial injury involves a platelet-derived metalloproteinase(s). Here we show that GPIbalpha shedding in response to mitochondrial injury or physiological activation is inhibited in platelets obtained from chimeric mice, which express inactive tumor necrosis factor-alpha converting enzyme (TACE(DeltaZn/DeltaZn)) in blood cells only. Shedding was also inhibited in mouse and human platelets in the presence of 2 potent TACE inhibitors: TAP1 and TMI-1. Our data further suggest that TACE is important in the regulation of GPIbalpha expression in vivo because we observed an approximately 90% reduction in soluble GPIbalpha (glycocalicin) in plasma of TACE(DeltaZn/DeltaZn) chimeras as well as significantly increased levels of GPIbalpha on circulating platelets. In contrast, shedding of P-selectin from activated platelets was not affected by the mutation in TACE. Damaged TACE(DeltaZn/DeltaZn) platelets were further characterized by a markedly improved post-transfusion recovery and hemostatic function in mice. In conclusion, our data demonstrate that TACE is expressed in platelets and that it is the key enzyme mediating shedding of GPIbalpha.

A novel endothelial L-selectin ligand activity in lymph node medulla that is regulated by alpha(1,3)-fucosyltransferase-IV.

Lymphocytes home to peripheral lymph nodes (PLNs) via high endothelial venules (HEVs) in the subcortex and incrementally larger collecting venules in the medulla. HEVs express ligands for L-selectin, which mediates lymphocyte rolling. L-selectin counterreceptors in HEVs are recognized by mAb MECA-79, a surrogate marker for molecularly heterogeneous glycans termed peripheral node addressin. By contrast, we find that medullary venules express L-selectin ligands not recognized by MECA-79. Both L-selectin ligands must be fucosylated by alpha(1,3)-fucosyltransferase (FucT)-IV or FucT-VII as rolling is absent in FucT-IV+VII(-/-) mice. Intravital microscopy experiments revealed that MECA-79-reactive ligands depend primarily on FucT-VII, whereas MECA-79-independent medullary L-selectin ligands are regulated by FucT-IV. Expression levels of both enzymes paralleled these anatomical distinctions. The relative mRNA level of FucT-IV was higher in medullary venules than in HEVs, whereas FucT-VII was most prominent in HEVs and weak in medullary venules. Thus, two distinct L-selectin ligands are segmentally confined to contiguous microvascular domains in PLNs. Although MECA-79-reactive species predominate in HEVs, medullary venules express another ligand that is spatially, antigenically, and biosynthetically unique. Physiologic relevance for this novel activity in medullary microvessels is suggested by the finding that L-selectin-dependent T cell homing to PLNs was partly insensitive to MECA-79 inhibition.