Quantitatively Lighting up the Spatial Organization of CD47/SIRPα Immune Checkpoints on the Cellular Membrane with Single-Molecule Localization Microscopy.

Immunotherapy including immune checkpoint inhibition has reinvigorated the current cancer treatment field. The development of efficient cancer immunotherapies depends on a thorough understanding of the status of immune checkpoints and how they interact. However, the distribution and spatial organization changes of immune checkpoints during their interactions at the single-molecule level remain difficult to directly visualize due to the lack of in situ imaging techniques with appropriate spatial and stoichiometric resolution. Herein, we report the direct visualization and quantification of the spatial distribution and organization of CD47 on the bladder tumor cell membrane and SIRPα on the macrophage membrane by using a single-molecule localization imaging technique called quantitative direct stochastic optical reconstruction microscopy (QdSTORM). Results showed that a portion of CD47 and SIRPα was present on cell membranes as heterogeneous clusters of varying sizes and densities prior to activation. Quantitative analyses of the reconstructed super-resolution images and theoretical simulation revealed that CD47 and SIRPα were reorganized into larger clusters upon binding to each other. Furthermore, we found that blocking the immune checkpoint interaction with small-molecule inhibitors or antibodies significantly impacted the spatial clustering behavior of CD47 on bladder tumor cells, demonstrating the promise of our QdSTORM strategy in elucidating the molecular mechanisms underlying immunotherapy. This work offers a promising strategy to advance our understanding of immune checkpoint state and interactions while also contributing to the fields including signal regulation and cancer therapy.

Redox signaling-driven modulation of microbial biosynthesis and biocatalysis.

Microbial communication can drive coordinated functions through sensing, analyzing and processing signal information, playing critical roles in biomanufacturing and life evolution. However, it is still a great challenge to develop effective methods to construct a microbial communication system with coordinated behaviors. Here, we report an electron transfer triggered redox communication network consisting of three building blocks including signal router, optical verifier and bio-actuator for microbial metabolism regulation and coordination. In the redox communication network, the Fe3+/Fe2+ redox signal can be dynamically and reversibly transduced, channeling electrons directly and specifically into bio-actuator cells through iron oxidation pathway. The redox communication network drives gene expression of electron transfer proteins and simultaneously facilitates the critical reducing power regeneration in the bio-actuator, thus enabling regulation of microbial metabolism. In this way, the redox communication system efficiently promotes the biomanufacturing yield and CO2 fixation rate of bio-actuator. Furthermore, the results demonstrate that this redox communication strategy is applicable both in co-culture and microbial consortia. The proposed electron transfer triggered redox communication strategy in this work could provide an approach for reducing power regeneration and metabolic optimization and could offer insights into improving biomanufacturing efficiency.

In-situ synthesis of biochar modified PbMoO4: An efficient visible light-driven photocatalyst for tetracycline removal.

For highly efficient photocatalytic remediation of organic pollutants, broad-spectrum light response and effective charge separation are two key goals. To achieve these goals, a novel biochar (BC) modified PbMoO4 composite catalyst was successfully synthesized in situ by combining coprecipitation with pyrolysis treatment of poplar sawdust and the technical feasibility of degradation of tetracycline (TC) with compound photocatalyst prepared from recovered agricultural and forestry residues was preliminarily demonstrated. The characterization demonstrated that the presence of BC narrowed the bandgap, enhanced visible light absorption as well as facilitated charge separation. Three composites (with the mass ratio of PbMoO4 to BC = 1:4; 1:1; and 4:1, respectively) displayed higher activity than pure PbMoO4. The results showed that the composite with the PbMoO4 to BC ratio of 1:4 exhibited the best photocatalytic activity, for 150 mg L-1 TC the removal rate was 61.0%, and the rate constant was 8.1 × 10-3 min-1, while the photocatalytic activity of PbMoO4 was 26.0% and 3.9 × 10-3 min-1. The reactions in the presence of radical quenchers indicated that holes (h+) and superoxide radicals (O2-) were the dominant active species for photodegradation. In different water matrices, for 150 mg L-1 TC solution the photocatalytic activity of optimal photocatalyst decreased as follows: ultrapure water > artificial sewage > farm sewage > municipal sewage. Moreover, the catalyst exhibited good stability over five cycles. Therefore, BC doped PbMoO4 provides a useful strategy for improving the photocatalytic ability of PbMoO4-based photocatalysts and offers a promising method for water purification.

Novel magnetic Fe3O4/g-C3N4/MoO3 nanocomposites with highly enhanced photocatalytic activities: Visible-light-driven degradation of tetracycline from aqueous environment.

In the present work, a series of magnetically separable Fe3O4/g-C3N4/MoO3 nanocomposite catalysts were prepared. The as-prepared catalysts were characterized by XRD, EDX, TEM, FT-IR, UV-Vis DRS, TGA, PL, BET and VSM. The photocatalytic activity of photocatalytic materials was evaluated by catalytic degradation of tetracycline solution under visible light irradiation. Furthermore, the influences of weight percent of MoO3 and scavengers of the reactive species on the degradation activity were investigated. The results showed that the Fe3O4/g-C3N4/MoO3 (30%) nanocomposites exhibited highest removal ability for TC, 94% TC was removed during the treatment. Photocatalytic activity of Fe3O4/g-C3N4/MoO3 (30%) was about 6.9, 5, and 19.9-fold higher than those of the MoO3, g-C3N4, and Fe3O4/g-C3N4 samples, respectively. The excellent photocatalytic performance was mainly attributed to the Z-scheme structure formed between MoO3 and g-C3N4, which enhanced the efficient separation of the electron-hole and sufficient utilization charge carriers for generating active radials. The highly improved activity was also partially beneficial from the increase in adsorption of the photocatalysts in visible range due to the combinaion of Fe3O4. Superoxide ions (·O2-) was the primary reactive species for the photocatalytic degradation of TC, as degradation rate were decreased to 6% in solution containing benzoquinone (BQ). Data indicate that the novel Fe3O4/g-C3N4/MoO3 was favorable for the degradation of high concentrations of tetracycline in water.

Synergistic adsorption-photocatalytic degradation effect and norfloxacin mechanism of ZnO/ZnS@BC under UV-light irradiation.

Norfloxacin (NOF) is an environmentally harmful and ubiquitous aquatic pollutant with extensive production and application. In this study, a novel composition named carbon-based composite photocatalytic material of zinc oxide and zinc sulphide (ZnO/ZnS@BC) was successfully obtained by the impregnation-roasting method to remove NOF under UV-light. Scanning electron microscopy, X-ray photoelectron spectroscopy, transmission electron microscopy and energy dispersive spectrometer characterised the composition. ZnO/ZnS was successfully decorated on the surface of biochar (BC). The pH, the ZnSO4/PS ratio, and ions and quenchers, were investigated. High removal efficiency was obtained with a pH of 7 and a ZnSO4/PS ratio of 1:1, and the removal ratio of NOF reached 95% within three hours; the adsorption and degradation ratios reached 46% and 49%, respectively. Fe2+ promoted the degradation of NOF, whereas other ions inhibited it, with NO3- showing the strongest inhibitory effect. Three reactive species (tert-butanol, quinone, and ammonium oxala) were identified in the catalytic system. The decreasing order of the contribution of each reactive species was: O2- > ·OH- > h+. Additionally, a recycling experiment demonstrated the stability of the catalyst; the catalytic degradation ratio of NOF reached 78% after five successive runs. Therefore, ZnO/ZnS@BC possessed strong adsorption capacity and high ultraviolet photocatalysis ability.

Investigation on gene transfer from genetically modified corn (Zea mays L.) plants to soil bacteria.

Knowledge about the prevalence and diversity of antibiotic resistance genes in soil bacteria communities is required to evaluate the possibility and ecological consequences of the transfer of these genes carried by genetically modified (GM) plants to soil bacteria. The neomycin phosphotransferase gene (nptII) conferring resistance to kanamycin and neomycin is one of the antibiotic resistance genes commonly present in GM plants. In this study, we investigated kanamycin-resistant (Km(R)) and neomycin-resistant (Nm(R)) soil bacterial populations in a 3-year field trial using a commercial GM corn (Zea mays L.) carrying the nptII gene and its near isogenic line. The results showed that a portion (2.3 - 15.6 %) of cultivable soil bacteria was naturally resistant to kanamycin or neomycin. However, no significant difference in the population level of Km(R) or Nm(R) soil bacteria was observed between the GM and non-GM corn fields. The nptII gene was not detected in any of the total 3000 Km(R) or Nm(R) isolates screened by PCR. Further, total soil bacterial cells were collected through Nycodenz gradient centrifugation and bacterial community DNA was subjected to PCR. Detection limit was about 500 cells per gram of fresh soil. Our study suggests that the nptII gene was relatively rare in the soil bacterial populations and there was no evidence of gene transfer from a GM corn plant to soil bacteria based on the data from total soil bacterial communities.

CD4+ T cell acquisition of the bystander pMHC I colocalizing in the same immunological synapse comprising pMHC II and costimulatory CD40, CD54, CD80, OX40L, and 41BBL.

We previously showed that CD4+ T cells acquired peptide/major histocompatibility complex (pMHC) I and costimulatory molecules by dendritic cell (DC) activation. However, the molecular mechanism for pMHC I acquisition is unclear. In this study, by using a panel of engineered DC2.4 cells or incubation of these cells with Con A-stimulated CD4+ T cells, we conducted capping and synapse formation assay and examined them by confocal fluorescence microscopy. We demonstrated that (i) CD54 and CD80 colocalized with pMHC I/II in the same lipid rafts, whereas CD40, OX40L, and 41BBL localized in the lipid rafts but separately from pMHC I/II, and (ii) MHC I/II colocalized with the costimulatory molecules in the same synapse formed between a DC and a CD4+ T cell, leading to expression of the acquired bystander pMHC I on CD4+ T cells via internalization/recycling pathway. These results provide some useful information in composition and dynamics of immunological synapses.

Novel exosome-targeted CD4+ T cell vaccine counteracting CD4+25+ regulatory T cell-mediated immune suppression and stimulating efficient central memory CD8+ CTL responses.

T cell-to-T cell Ag presentation is increasingly attracting attention. In this study, we demonstrated that active CD4+ T (aT) cells with uptake of OVA-pulsed dendritic cell-derived exosome (EXO(OVA)) express exosomal peptide/MHC class I and costimulatory molecules. These EXO(OVA)-uptaken (targeted) CD4+ aT cells can stimulate CD8+ T cell proliferation and differentiation into central memory CD8+ CTLs and induce more efficient in vivo antitumor immunity and long-term CD8+ T cell memory responses than OVA-pulsed dendritic cells. They can also counteract CD4+25+ regulatory T cell-mediated suppression of in vitro CD8+ T cell proliferation and in vivo CD8+ CTL responses and antitumor immunity. We further elucidate that the EXO(OVA)-uptaken (targeted)CD4+ aT cell's stimulatory effect is mediated via its IL-2 secretion and acquired exosomal CD80 costimulation and is specifically delivered to CD8+ T cells in vivo via acquired exosomal peptide/MHC class I complexes. Therefore, EXO-targeted active CD4+ T cell vaccine may represent a novel and highly effective vaccine strategy for inducing immune responses against not only tumors, but also other infectious diseases.

Bidirectional membrane molecule transfer between dendritic and T cells.

The acquisition of dendritic cell (DC) molecules by T cells has been previously reported. However, it remains unclear whether the transfer is only mono- or bidirectional. In this study, we incubated CMFDA-labeled ovalbumin (OVA)-pulsed DC2.4 (DC2.4(OVA)) cells with Dil-labeled OT II CD4(+) T cells and analyzed the potential bidirectional molecule transfer. We also assessed the distribution of internalized membrane using two engineered DC2.4/Ia(b)GFP and MF4/TCRCFP DC lines. Our findings showed that membrane molecule transfer is bidirectional. CD4(+) T cells acquired Ia(b), CD11c, CD40, and CD80 from DC2.4(OVA) cells, and conversely DC2.4(OVA) cells took up CD4, CD25, CD69, and T cell receptor from T cells. The internalized molecules acquired by T cells and DCs mostly localized in endosomes and lysosomes, respectively. Taken together, this study demonstrated a novel phenomenon of bidirectional membrane molecule transfer between DCs and T cells.

Interferon gamma stimulates cellular maturation of dendritic cell line DC2.4 leading to induction of efficient cytotoxic T cell responses and antitumor immunity.

Dendritic cells (DCs) are the most potent antigen-presenting cells (APCs) for the initiation of antigen (Ag)-specific immune responses. In most studies, mature DCs are generated from bone marrow cells or peripheral monocytes; in either case, the harvested cells are then cultured in medium containing recombinant GM-CSF, IL-4 and TNF-alpha for 7-10 days and stimulated with lipopolysaccharide (LPS). However, this approach is time-consuming and expensive. There is another less cost approach of using immobilized DC cell lines, which can easily grow in the medium. A disadvantage with the immobilized DC cell lines, however, is that they are immature DCs and lack expression of MHC class II and costimulatory CD40 and CD80 molecules. This, therefore, limits their capacity for inducing efficient antitumor immunity. In the current study, we investigated the possible efficacy of various stimuli (IL-1beta,IFN-gamma, TNF-alpha, CpG and LPS) in converting the immature dendritic cell line DC2.4 to mature DCs. Our findings were quite interesting since we demonstrated for the first time that IFN-gamma was able to stimulate the maturation of DC2.4 cells. The IFN-gamma-activated ovalbumin (OVA)-pulsed DC2.4 cells have capacity to upregulate MHC class II, CD40, CD80 and CCR7, and to more efficiently stimulate in vitro and in vivo OVA-specific CD8+ T cell responses and antitumor immunity. Therefore, IFN-gamma-activated immortal DC2.4 cells may prove to be useful in the study of DC biology and antitumor immunity.