Circadian tumor infiltration and function of CD8+ T cells dictate immunotherapy efficacy.

The quality and quantity of tumor-infiltrating lymphocytes, particularly CD8+ T cells, are important parameters for the control of tumor growth and response to immunotherapy. Here, we show in murine and human cancers that these parameters exhibit circadian oscillations, driven by both the endogenous circadian clock of leukocytes and rhythmic leukocyte infiltration, which depends on the circadian clock of endothelial cells in the tumor microenvironment. To harness these rhythms therapeutically, we demonstrate that efficacy of chimeric antigen receptor T cell therapy and immune checkpoint blockade can be improved by adjusting the time of treatment during the day. Furthermore, time-of-day-dependent T cell signatures in murine tumor models predict overall survival in patients with melanoma and correlate with response to anti-PD-1 therapy. Our data demonstrate the functional significance of circadian dynamics in the tumor microenvironment and suggest the importance of leveraging these features for improving future clinical trial design and patient care.

A Quantitative Approach to Determine Hydrophobe Content of Associating Polyacrylamide Using a Fluorescent Probe.

Hydrophobically associating polymers have found widespread applications in many domains due to their unique rheological behavior, which is primarily dictated by the hydrophobe content. However, the low fraction of hydrophobic monomers in polymers makes this parameter's precise and straightforward measurement difficult. Herein, a variety of hydrophobically associating polyacrylamides (HAPAM) with different alkyl chain lengths (L) and hydrophobic contents ([H]) were prepared by post-modification and accurately characterized by 1H NMR spectroscopy. The maximal fluorescence emission intensity (I) of 8-anilino-1-naphthalenesulfonic acid, which is sensitive to hydrophobic environments, was then detected in those polymer solutions and shown as a ratio to that in the polymer-free solution (I0). It was found that I/I0 for 0.5 wt% HAPAM can be scaled versus CH, which is a variate related to both L and [H], as I/I0 = 1.15 + 1.09 × 108CH3.42, which was also verified to be applicable for hydrophobic associating hydrolyzed polyacrylamide (HHAPAM). This relationship provides a handy method for determining the hydrophobic content of hydrophobically associating polymers, particularly for field applications.

Acid-Base Interactions of Polystyrene Sulfonic Acid in Amorphous Solid Dispersions Using a Combined UV/FTIR/XPS/ssNMR Study.

This study investigates the potential drug-excipient interactions of polystyrene sulfonic acid (PSSA) and two weakly basic anticancer drugs, lapatinib (LB) and gefitinib (GB), in amorphous solid dispersions. Based on the strong acidity of the sulfonic acid functional group, PSSA was hypothesized to exhibit specific intermolecular acid-base interactions with both model basic drugs. Ultraviolet (UV) spectroscopy identified red shifts, which correlated well with the color change observed in lapatinib-PSSA solutions. Fourier transform infrared (FTIR) spectra suggest the protonation of the quinazoline nitrogen atom in both model compounds, which agrees well with data from the crystalline ditosylate salt of lapatinib. X-ray photoelectron spectroscopy (XPS) detected increases in binding energy of the basic nitrogen atoms in both lapatinib and gefitinib, strongly indicating protonation of these nitrogen atoms. (15)N solid-state NMR spectroscopy provided direct spectroscopic evidence for protonation of the quinazoline nitrogen atoms in both LB and GB, as well as the secondary amine nitrogen atom in LB and the tertiary amine nitrogen atom in GB. The observed chemical shifts in the LB-PSSA (15)N spectrum also agree very well with the lapatinib ditosylate salt where proton transfer is known. Additionally, the dissolution and physical stability behaviors of both amorphous solid dispersions were examined. PSSA was found to significantly improve the dissolution of LB and GB and effectively inhibit the crystallization of LB and GB under accelerated storage conditions due to the beneficial strong intermolecular acid-base interaction between the sulfonic acid groups and basic nitrogen centers.

Dynamic single-cell regulomes characterize human peripheral blood innate lymphoid cell subpopulations.

Innate lymphoid cells (ILCs) are plastic immune cells divided into 3 main subsets, characterized by distinct phenotypic and functional profiles. Using single cell approaches, heightened heterogeneity of mouse ILCs has been appreciated, imprinted by tissue signals that shape their transcriptome and epigenome. Intra-subset diversity has also been observed in human ILCs. However, combined transcriptomic and epigenetic analyses of single ILCs in humans are lacking. Here, we show high transcriptional and epigenetic heterogeneity among human circulating ILCs in healthy individuals. We describe phenotypically distinct subclusters and diverse chromatin accessibility within main ILC populations, compatible with differentially poised states. We validate the use of this healthy donor-based analysis as resource dataset to help inferring ILC changes occurring in disease conditions. Overall, our work provides insights in the complex human ILC biology. We anticipate it to facilitate hypothesis-driven studies in patients, without the need to perform single cell OMICs using precious patients' material.

Lsd1 safeguards T-cell development via suppressing endogenous retroelements and interferon responses.

The histone demethylase Lsd1 has been shown to play multiple essential roles in mammalian biology. However, its physiological functions in thymocyte development remain elusive. We observed that the specific deletion of Lsd1 in thymocytes caused significant thymic atrophy and reduced peripheral T cell populations with impaired proliferation capacity. Single-cell RNA sequencing combined with strand-specific total RNA-seq and ChIP-seq analysis revealed that ablation of Lsd1 led to the aberrant derepression of endogenous retroelements, which resulted in a viral mimicry state and activated the interferon pathway. Furthermore, the deletion of Lsd1 blocked the programmed sequential down-regulation of CD8 expression at the DP→CD4+CD8lo stage and induced an innate memory phenotype in both thymic and peripheral T cells. Single-cell TCR sequencing revealed the kinetics of TCR recombination in the mouse thymus. However, the preactivation state after Lsd1 deletion neither disturbed the timeline of TCR rearrangement nor reshaped the TCR repertoire of SP cells. Overall, our study provides new insight into the function of Lsd1 as an important maintainer of endogenous retroelement homeostasis in early T-cell development.

Single-Cell Sequencing Reveals the Novel Role of Ezh2 in NK Cell Maturation and Function.

Natural killer (NK) cells are lymphocytes primarily involved in innate immunity and exhibit important functional properties in antimicrobial and antitumoral responses. Our previous work indicated that the enhancer of zeste homolog 2 (Ezh2) is a negative regulator of early NK cell differentiation and function through trimethylation of histone H3 lysine 27 (H3K27me3). Here, we deleted Ezh2 from immature NK cells and downstream progeny to explore its role in NK cell maturation by single-cell RNA sequencing (scRNA-seq). We identified six distinct NK stages based on the transcriptional signature during NK cell maturation. Conditional deletion of Ezh2 in NK cells resulted in a maturation trajectory toward NK cell arrest in CD11b SP stage 5, which was clustered with genes related to the activating function of NK cells. Mechanistically, we speculated that Ezh2 plays a critical role in NK development by activating AP-1 family gene expression independent of PRC2 function. Our results implied a novel role for the Ezh2-AP-1-Klrg1 axis in altering the NK cell maturation trajectory and NK cell-mediated cytotoxicity.

Histone methyltransferase Ezh2 negatively regulates NK cell terminal maturation and function.

NK cells are innate lymphoid cells that play important roles in tumor eradication and viral clearance. We previously found that deletion or inhibition of the histone methyltransferase Ezh2 (enhancer of zeste homolog 2) in hematopoietic stem and progenitor cells (HSPCs) from both mice and humans enhanced the commitment and cytotoxicity of NK cells to tumor cells. This study tested the hypothesis that inhibiting Ezh2, especially in NK lineage cells, could also affect NK cell development and function. We crossed Ezh2fl/fl mice with Ncr1iCre mice to delete the Ezh2 gene in immature NK cells and downstream progeny. Ezh2 deficiency increased the total number of NK cells and promoted NK cell terminal differentiation, as the percentages of the most mature CD27- CD11b+ subsets were increased. The NK cell cytotoxicity against tumor cells in vitro was enhanced, with increased degranulation and IFN-γ production. In addition, during the process of human NK cells differentiating from HSPCs , inhibiting EZH2 catalytic activity at day 14 (when NK lineage commitment began) also resulted in increased proportions of mature NK cells and cytotoxicity. Furthermore, RNA-seq and CUT&RUN-qPCR assays showed that the effects of Ezh2 may be based on its direct modulation of the expression of the transcription factor Pbx1 (pre-B-cell leukemia transcription factor 1), which has been reported to promote NK cell development. In summary, we demonstrate that Ezh2 is a negative regulator of NK cell terminal maturation and function.

Synthesis of 1, 2, 4-triazole benzoyl arylamine derivatives and their high antifungal activities.

Two series of novel 1, 2, 4-triazole benzoyl arylamine derivatives were prepared and screened for their activities against three pathogens of Gaeumannomyces graminis var.tritici, Sclerotinia sclerotiorum and Fusarium graminearum using the mycelium growth inhibition method in vitro. The results indicated that most of the synthesized derivatives displayed antifungal activities. Compounds 6c-d and 6f-g exhibited lower EC50s against all the three pathogens. Among of them, the compound 6g displayed the most potent antifungal activities with EC50 values of 0.01, 0.19 and 0.12 µg mL-1 respectively. The structure and activity relationship showed that election-withdrawing group at pata-position of aniline was favorable for high activities, and the preferred groups were alkoxy carbonyls. These results proposed that the compound 6g can be a lead compound for development of novel fungicide.

Acid-base interactions in amorphous solid dispersions of lumefantrine prepared by spray-drying and hot-melt extrusion using X-ray photoelectron spectroscopy.

This study investigates drug-excipient interactions in amorphous solid dispersions (ASDs) of the model basic compound lumefantrine (LMN), with five acidic polymers. X-ray photoelectron spectroscopy (XPS) was used to measure the extent of the protonation of the tertiary amine in LMN by the five acidic polymers. The extent/efficiency of protonation of the ASDs was assessed a function of polymer type, manufacturing process (hot-melt extrusion vs. spray drying), and drug loading (DL). The most strongly acidic polymer, polystyrene sulfonic acid (PSSA) was found to be the most efficient polymer in protonating LMN, independently of manufacturing method and DL. The rank order for the protonation extent of LMN by each polymer is roughtly the same for both manufacturing processes. However, protonation efficiency of polymers of similar acidic strength ranged from ∼0% to 75% (HPMCAS and Eudragit L100-55, respectively), suggesting an important role of molecular/mixing effects. For some polymers, including Eudragit L100 55 and HPMCP, spray-drying resulted in higher protonation efficiency compared to hot-melt extrusion. This result is attributable to a more favorable encounter between acid and base groups, when exposed to each other in solution phase. Increasing DL led to decreased protonation efficiency in most cases, particularly for polyacrylic acid, despite having the highest content of acidic groups per unit mass. These results indicate that the combined effects of acid strength and mixing phenomena regulate the efficiency of acid-base interactions in the ASDs.

Crystalline, liquid crystalline, and isotropic phases of sodium deoxycholate in water.

Sodium deoxycholate (NaDC) is an important example of bile salts, representing systems with complex phase behavior involving both crystalline and mesophase structures. In this study, properties of NaDC-water mixtures were evaluated as a function of composition and temperature via X-ray diffraction with synchrotron (sXRD) and laboratory radiation sources, water sorption, polarized light, hot-stage microscopy, and freezing-point osmometry. Several phases were detected depending on the composition and temperature, including isotropic solution phase, liquid crystalline (LC) phase, crystalline hydrate, and ice. The LC phase was identified as hexagonal structure by sXRD, with up to 14 high-order reflections detected. The crystalline phase was found to be nonstoichiometric hydrate, based on XRD and water sorption data. The phase diagram of NaDC-water system has been refined based on both results of this study and other reports in literature.