Porous graphene possesses great potential as an anode material for sodium-ion batteries. Due to the porosity of the graphene, the increase in surface area allows for shorter diffusion length for electrolyte ions. Herein, the porous graphene oxide is created using the breath figure method, and then reduced by chemical, electrochemical, or thermal means. Sodium storage performance of the devised porous graphene is investigated and compared. Reduced graphene oxide shows altered hierarchical porous structures, which are highly dependent on reduction method and conditions. Of the three reduction methods, thermal reduction shows a superior sodium storage performance, yielding a maximum discharge capacity of 187[Formula: see text]mAh/g and charge capacity of 63[Formula: see text]mAh/g at a current rate of 50[Formula: see text]mA/g.

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Engineering cellular therapeutics by programming T cells has great potential in immunology. The primary mechanism employed by T cells for the specific transfer of proteins at the immunological synapse is via the lysosomal perforin pathway that facilitates the transfer of cytotoxic granzymes leading to apoptosis in target cells. Facilitating the delivery of non-cytotoxic proteins through perforin oligomers will dramatically expand the range of protein cargos that T cells can traffic to the target cells. Here, we have identified the intralysosomal protein, NPC2, as a chaperone that can facilitate the delivery of T-cell derived reporter proteins through perforin pores at the immunological synapse. Structural and biophysical considerations suggested that NPC2 could traverse through perforin pores and in vitro experiments confirmed the transport of purified NPC2 through perforin pores on cell membranes. To characterize the ability of NPC2 to facilitate the transfer of payloads in T cells, we constructed NPC2-mCherry fusion proteins in T cells. Using confocal microscopy and flow cytometry, we confirmed the colocalization of the NPC2 fused protein with lytic granules and the transfer of the fluorescent protein payload from T cells to target cells in co-culture experiments. The NPC2 fusion enabled the localization of mCherry to secretory lysosomes in mouse TCR CD8+ T cells and human CD4+ and CD8+ chimeric antigen receptor (CAR) T cells. These results illustrate that by using NPC2 as a molecular chaperone, the NPC2-perforin pathway can be exploited as a programmable molecular delivery system for cell-based therapies.

Background Chimeric antigen receptor (CAR) T cell therapies show remarkable progress in treating liquid tumors, with a complete remission rate of over 57%. 1 Translating the success of CAR T cells to solid tumors will need an understanding of the key mechanisms responsible for the cytotoxicity of CAR T cells. The primary factors contributing to tumor resistance against CAR T therapies are widely contested 2 , therefore, we seek to explore the impact of different CAR T cell killing mechanisms of tumors. Methods We examine CAR T cell killing of a leukemic cell line, NALM6, and an ovarian cancer cell line, SkOV3-CD19, in the presence of Granzyme B inhibitors and a Fas ligand inhibitor. We develop a fluorescent membrane reporter that translocates to the nucleus upon specific proteolytic cleaving by Granzyme A and B. Results . Overexpressing native Granzyme B inhibitor, ProteaseInhibitor-9 (PI-9), in NALM6 and SkOV3-CD19 does not affect killing frequencies in CAR(19-41BBz and 19-28z) T cell cytotoxicity assays. . Treating 19-41BBz with a small molecule inhibitor of Granzyme B does not impact killing frequencies in cytotoxicity assays against NALM6 and SkOV3-CD19. . Overexpressing PI-9 in NALM6 and SkOV3-CD19 does not affect 19-41BBz CAR T killing frequencies or killing kinetics in single cell time-lapse assays. . Inhibition of Fas ligand on 19-41BBz CAR T cells does not impact killing frequencies against NALM6 and SkOV3-CD19.Conclusions Our findings suggest that suppressing Granzyme B activity with small molecules or native proteins does not impair killing frequencies of 19-41BBz CAR T cells en masse or at the single cell level. We hypothesize that Granzyme A facilitates CAR T killing in the absence of Granzyme B, implying redundancy in granzyme expression. This study provides a comprehensive understanding of the main mechanisms associated with CAR T cell-mediated killing.

Extracellular vesicles (EVs) regulate the tumor microenvironment by facilitating transport of biomolecular cargo including RNA, protein, and metabolites. The biological effects of EV-mediated transport have been studied using supra-physiological concentrations of EVs, but the cells that are responsible for EV secretion and the mechanisms that support EV secretion are not well characterized. We developed an integrated method based on arrays of nanowells to identify individual cells with differences in EV secretion and used an automated robot to perform linked single-cell RNA-sequencing on cloned single cells from the metastatic breast cancer cell line, MDAMB231. Gene expression profiles of clonal cells with differences in EV secretion were analyzed, and a four-gene signature of breast cancer EV secretion was identified: HSP90AA1, HSPH1, EIF5, and DIAPH3. We functionally validated this gene signature by testing it across different cell lines with different metastatic potential demonstrating that the signature correlated with levels of EV secretion. Analysis of the TCGA and METABRIC datasets showed that this signature is associated with poor survival, more invasive breast cancer types, and reduced CD8+ T cell infiltration in human tumors. We anticipate that our method for directly identifying the molecular determinants of EV secretion will have broad applications across cell types and diseases.