The functions of novel supramolecular structure assembly in enzymes are not fully understood. Analyzing these structures may provide insight into how they regulate enzyme activity as well as any novel roles they serve in the cell. For example, identifying the triggers of GDH1 polymerization could explain how mutations in GLUD1, the human ortholog, cause familial hypoglycemia. Similarly, understanding how PRPS1 filaments form in the nucleus could explain why gain and loss of function mutations have overlapping disease phenotypes. Both PRPS1 and GDH1 were tagged with GFP and HA and transfected into yeast to examine any adverse effects on polymerization. No changes in supramolecular structure integrity were observed in GDH1 strains containing either tag whereas PRPS1 required immunostaining for natural polymerization. GDH3 and GLT1 yeast knockout strains exhibited increased puncta frequency suggesting pathway epistasis. Human PRPS1, HA tagged yeast orthologues PRS3 and PRS5, and PRPS1 superactivity mutations were transformed into yeast to examine the effects on filament formation. PRS3, PRS5, and PRPS1 filaments localized into the cytosol unlike PRPS1 filament formation in the nucleus of human fibroblasts. This suggests that the nuclear localization machinery is unique to mammalian cells. The L129I mutant’s filament frequency and expression did not change. The H193Q mutation led to decreased filament frequency during postdiauxic shift, but no changes in expression level. The A190V, D52H, D183H, and N114S mutations led to both an increase in filament frequency and expression levels. However, all PRPS1 mutants display deformed and enlarged PRPS1 filaments suggesting that they affect filament integrity.

Glioblastoma (GBM) is the most common malignant tumor in the central nervous system and has poor patient survival rates. Unlike other cancers, immune checkpoint therapies, such as PD-1 checkpoint blockade, have been largely ineffective in GBMs for several reasons: an immunosuppressive tumor microenvironment, a lack of suitable neoantigens, and poor intratumoral T cell infiltration and activity. However, there is evidence that using anti-PD-1 therapy in the neoadjuvant setting may generate a more robust anti-tumor immune response, though characterizing how the GBM tumor microenvironment changes with such therapy is incomplete. As such, we performed high dimensional analysis using CyTOF mass cytometry and single-cell RNAsequencing to study the intratumoral immune populations in GBM patients treated with or without neoadjuvant anti-PD-1 therapy. Characterizing PD-1 expressing tumor infiltrating T cell populations showed that PD-1 was associated with markers of T cell activation and dysfunction regardless of treatment and that this association existed less strongly in peripheral PD-1 expressing T cells. We studied the effects of neoadjuvant anti-PD-1 therapy on a large patient cohort of tumor infiltrating immune cells and found that neoadjuvant anti-PD-1 therapy significantly increased the proportion of several intratumoral T cell sub-populations, including a TCF7-expressing progenitor exhausted population. Downstream effects of neoadjuvant anti-PD-1 therapy on T cells, namely increased production of IFN-g, included transcriptionally altering the myeloid and dendritic cell populations to be more immune suppressive but also potentially more vulnerable to other immune checkpoint therapies, specifically anti-TIGIT and anti-CTLA-4 therapies. Due to the impact of neoadjuvant anti-PD-1 therapy on intratumoral T cell populations, we examined whether we could detect tumor-reactive T cells by cloning TCRs from transcriptionally defined populations in a patient treated with neoadjuvant anti-PD-1 and testing reactivity to the patient-derived gliomasphereline. Among TCRs cloned, we discovered that T cells arising from the activated and exhausted population showed tumor reactivity, suggesting that utilizing transcriptional phenotypes can guide selection of potential tumor-reactive TCRs in GBM patients treated with neoadjuvant anti-PD-1 therapy. In conclusion, neoadjuvant anti-PD-1 therapies alters the immune landscape in these tumors and can be potentially used in combination with other immunotherapies to more effectively treat this malignancy.

Aureococcus anophagefferens, a harmful bloom-forming alga responsible for brown tides in estuaries of the Middle Atlantic U.S., has been investigated by atomic force microscopy for the first time, using probes functionalized with a monoclonal antibody specific for the alga. The rupture force between a single monoclonal antibody and the surface of A. anophagefferens was experimentally found to be 246±11 pN at the load rate of 12 nN/s. Force histograms for A. anophagefferens and other similarly-sized algae are presented and analyzed. The results illustrate the effects of load rates, and demonstrate that force-distance measurements can be used to build biosensors with high signal-to-noise ratios for A. anophagefferens. The methods described in this paper can be used, in principle, to construct sensors with single-cell resolution for arbitrary cells for which monoclonal antibodies are available.

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