CD4+ T Cells Support Production of Simian Immunodeficiency Virus Env Antibodies That Enforce CD4-Dependent Entry and Shape Tropism In Vivo

CD4⁺ T cells rather than macrophages are the principal cells infected by human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV) in vivo. Macrophage tropism has been linked to the ability to enter cells through CCR5 in conjunction with limiting CD4 levels, which are much lower on macrophages than on T cells. We recently reported that rhesus macaques (RM) experimentally depleted of CD4⁺ T cells before SIV infection exhibit extensive macrophage infection as well as high chronic viral loads and rapid progression to AIDS. Here we show that early-time-point and control Envs were strictly CD4 dependent but that, by day 42 postinfection, plasma virus of CD4⁺ T cell-depleted RM was dominated by Envs that mediate efficient infection using RM CCR5 independently of CD4. Early-time-point and control RM Envs were resistant to neutralization by SIV-positive (SIV⁺) plasma but became sensitive if preincubated with sCD4. In contrast, CD4-independent Envs were highly sensitive to SIV⁺ plasma neutralization. However, plasma from SIV-infected CD4⁺ T cell-depleted animals lacked this CD4-inducible neutralizing activity and failed to neutralize any Envs regardless of sCD4 pre-exposure status. Enhanced sensitivity of CD4-independent Envs from day 42 CD4⁺ T cell-depleted RM was also seen with monoclonal antibodies that target both known CD4-inducible and other Env epitopes. CD4 independence and neutralization sensitivity were both conferred by Env amino acid changes E84K and D470N that arose independently in multiple animals, with the latter introducing a potential N-linked glycosylation site within a predicted CD4-binding pocket of gp120. Thus, the absence of CD4 T cells results in failure to produce antibodies that neutralize CD4-independent Envs and CD4-pretriggered control Envs. In the absence of this constraint and with a relative paucity of CD4⁺ target cells, widespread macrophage infection occurs in vivo accompanied by emergence of variants carrying structural changes that enable entry independently of CD4.     

Myosin-cross-reactive antigens from four different lactic acid bacteria are fatty acid hydratases

The 67 kDa myosin-cross-reactive antigen (MCRA) is a member of the MCRA family of proteins present in a wide range of bacteria and was predicted to have fatty acid isomerase function. We have now characterised the catalytic activity of MCRAs from four LAB stains, including Lactobacillus rhamnosus LGG, L. plantarum ST-III, L. acidophilus NCFM and Bifidobacterium animalis subsp. lactis BB-12. MCRA genes from these strains were cloned and expressed in Escherichia coli, and the recombinant protein function was analysed with lipid profiles by GC–MS. The four MCRAs catalysed the conversion of linoleic acid and oleic acid to their respective 10-hydroxy derivatives, which suggests that MCRA proteins catalyse the first step in conjugated linoleic acid production. This is the first report of MCRA from L. rhamnosus with such catalytic function.     

Nanoparticle-tethered NAD+ with in situ cofactor regeneration

A new and simple route for the preparation of immobilized NAD⁺ on carboxyl-activated silica nanoparticles activated by γ-aminpropyltriethoxysilane and glutaric anhydride was developed. In addition, formate dehydrogenase, keto-reductase and the silica nanoparticle-attached NAD⁺ were applied to catalyze the coupled reactions for production of l-lactate with the cofactor regenerated within the reaction cycle. As indicated by thermogravimetric analysis and FT-IR, the silica nanoparticles were successfully activated and the loading of carboxyl groups was 0.53 mmol g^?1 particle. The amount of immobilized NAD⁺ on the support was 73 mg g^?1 particle. With 0.2 M pyruvate and 3 M formate, 0.16 M l-lactate was produced after the coupled reactions. The immobilized system showed excellent efficiency and stabilities in recycling, and it retained 60 % residual activity even after six reuses.     

Synergistic roles of leaf boron and calcium during the growing season in affecting sugar and starch accumulation in ripening apple fruit

Fruit sugar content is one of the most important flavor quality traits in the fresh market. Minerals, such as boron (B) and calcium (Ca), are associated with fruit sugar and starch accumulation in many plant species. To better understand the roles of B and Ca in affecting sugar and starch accumulation in apples, 2 g L^?1 Na₂B₄O₇·10H₂O or 10 g L^?1 CaCl₂ was supplied by foliar spray to 20-year-old ‘Fuji’ (Malus domestica Borkh. cv. Fuji) trees at four developmental stages (fruit set, onset of rapid fruit growth, rapid fruit growth and the end of rapid fruit growth), in 2010–2011. The most effective treatment significantly increasing soluble sugar and starch levels in ripening fruit was the foliar application of 2 g L^?1 Na₂B₄O₇·10H₂O during rapid fruit growth, and the robustness of the effects was confirmed for two cultivars, ‘Fuji’ and ‘Orin’, at three orchards in 2011. Foliar applications of B during the onset of rapid fruit growth and rapid fruit growth, as well as the foliar application of Ca at fruit set, significantly increased the soluble sugar content in ripening fruit. In addition, the B application was effective in increasing the fruit starch content, but Ca was not. Both B and Ca treatments significantly increased the leaf concentrations of the other element at least transiently. However, B and Ca effects on fruit sugar/starch did not seem to depend on higher leaf B or Ca levels. In conclusion, B and Ca interact in enhancing fruit sugar and starch contents at the fruit ripening stage.     

CmPEX6, a Gene Involved in Peroxisome Biogenesis,Is Essential for Parasitism and Conidiation by the Sclerotial Parasite Coniothyrium minitans

Coniothyrium minitans is a sclerotial parasite of the plant-pathogenic fungus Sclerotinia sclerotiorum, and conidial production and parasitism are two important aspects for commercialization of this biological control agent. To understand the mechanism of conidiation and parasitism at the molecular level, we constructed a transfer DNA (tDNA) insertional library with the wild-type strain ZS-1. A conidiation-deficient mutant, ZS-1TN22803, was uncovered through screening of this library. This mutant could produce pycnidia on potato dextrose agar (PDA), but most were immature and did not bear conidia. Moreover, this mutant lost the ability to parasitize or rot the sclerotia of S. sclerotiorum. Analysis of the tDNA flanking sequences revealed that a peroxisome biogenesis factor 6 (PEX6) homolog of Saccharomyces cerevisiae, named CmPEX6, was disrupted by the tDNA insertion in this mutant. Targeted gene replacement and gene complementation tests confirmed that a null mutation of CmPEX6 was responsible for the phenotype of ZS-1TN22803. Further analysis showed that both ZS-1TN22803 and the targeted replacement mutants could not grow on PDA medium containing oleic acid, and they produced much less nitric oxide (NO) and hydrogen peroxide (H₂O₂) than wild-type strain ZS-1. The conidiation of ZS-1TN22803 was partially restored by adding acetyl-CoA or glyoxylic acid to the growth media. Our results suggest that fatty acid β-oxidation, reactive oxygen and nitrogen species, and possibly other unknown pathways in peroxisomes are involved in conidiation and parasitism by C. minitans.     

Novel technique for internal structure and elemental distribution analyses of granular sludge from reactors for wastewater treatment

A novel technique for internal structure and elemental distribution analyses of granular sludge is presented. Sludge samples were freeze-dried and embedded in epoxy resin to form a module, which were then ground and polished to obtain sequential cross-sections. The cross-sections were analyzed by scanning electron microscopy (SEM) and energy dispersive X-ray spectroscopy (EDX). SEM observations showed that one granule was formed having several cores with different inorganic minerals, rather than a single core. EDX results indicate that the main elements of the granules are O, Ca, Mg, and P. In addition, the distribution areas of calcium and magnesium in the granule do not coincide.     

Iodine Excess Induces Hepatic Steatosis Through Disturbance of Thyroid Hormone Metabolism Involving Oxidative Stress in BaLB/c Mice

Iodine excess is emerging as a new focus. A better understanding of its hazardous effects on the liver will be of great benefit to health. The aim of this study is to illustrate the effects of iodine excess on hepatic lipid homeostasis and explore its possible mechanisms. One hundred twenty BaLB/c mice were given iodine at different levels (0, 0.3, 0.6, 1.2, 2.4, and 4.8 mg I/L) in drinking water for 1 or 3 months. Lipid parameters and serum thyroid hormones were measured. Hepatic type 1 deiodinase activity and oxidative stress parameters were evaluated. The mRNA expression of sterol regulatory element-binding protein-1c (SREBP-1c) and fatty acid synthase (FAS) was detected by real-time polymerase chain reaction. Dose-dependent increase of hepatic triglyceride content was detected (r?=?0.680, P?<?0.01) in iodine-loaded groups. Evident hepatic steatosis was observed in 2.4 and 4.8 mg I/L iodine-loaded groups. The activities of antioxidant enzymes (glutathione peroxidase and superoxide dismutase) were decreased, and the malondialdehyde level was increased by excessive iodine in both serum and liver in a dose-dependent manner, accompanying the decrease of hepatic D1 activity. That resulted in the increase of serum total thyroxine and the decrease of serum total triiodothyronine in iodine-loaded groups. The mRNA expression of SREBP-1c and FAS was increased in iodine-loaded groups in response to the change of serum triiodothyronine. Present findings demonstrated that iodine excess could dose dependently induce hepatic steatosis. Furthermore, our data suggested that the disturbance of thyroid hormone metabolism involving oxidative stress may play a critical role in iodine excess-induced hepatic steatosis.     

Effect of CdTe Quantum Dots Size on the Conformational Changes of Human Serum Albumin: Results of Spectroscopy and Isothermal Titration Calorimetry

Quantum dots (QDs) are recognized as some of the most promising candidates for future applications in biomedicine. However, concerns about their safety have delayed their widespread application. Human serum albumin (HSA) is the main protein component of the circulatory system. It is important to explore the interaction of QDs with HSA for the potential in vivo application of QDs. Herein, using spectroscopy and isothermal titration calorimetry (ITC), the effect of glutathione-capped CdTe quantum dots of different sizes on the HSA was investigated. After correction for the inner filter effect, the fluorescence emission spectra and synchronous fluorescence spectra showed that the microenvironment of aromatic acid residues in the protein was slightly changed when the glutathione (GSH)–cadmium telluride (CdTe) QDs was added, and GSH–CdTe QDs with larger particle size exhibited a much higher effect on HSA than the small particles. Although a ground-state complex between HSA and GSH–CdTe QDs was formed, the UV–vis absorption and circular dichroism spectroscopic results did not find appreciable conformational changes of HSA. ITC has been used for the first time to characterize the binding of QDs with HSA. The ITC results revealed that the binding was a thermodynamically spontaneous process mainly driven by hydrophobic interactions, and the binding constant tended to increase as the GSH–CdTe QDs size increased. These findings are helpful in understanding the bioactivities of QDs in vivo and can be used to assist in the design of biocompatible and stable QDs.     

Centrosome repositioning in T cells is biphasic and driven by microtubule end-on capture-shrinkage

T cells rapidly reposition their centrosome to the center of the immunological synapse (IS) to drive polarized secretion in the direction of the bound target cell. Using an optical trap for spatial and temporal control over target presentation, we show that centrosome repositioning in Jurkat T cells exhibited kinetically distinct polarization and docking phases and required calcium flux and signaling through both the T cell receptor and integrin to be robust. In “frustrated” conjugates where the centrosome is stuck behind the nucleus, the center of the IS invaginated dramatically to approach the centrosome. Consistently, imaging of microtubules during normal repositioning revealed a microtubule end-on capture-shrinkage mechanism operating at the center of the IS. In agreement with this mechanism, centrosome repositioning was impaired by inhibiting microtubule depolymerization or dynein. We conclude that dynein drives centrosome repositioning in T cells via microtubule end-on capture-shrinkage operating at the center of the IS and not cortical sliding at the IS periphery, as previously thought.