Protocol for PPP1R15A-inhibited mouse model establishment with subcutaneous B16F1 tumor and single-cell analysis.

Here, we present a protocol for exploring the effects of PPP1R15A inhibitor, Sephin1, on antitumor immunity of B16F1 subcutaneous tumor in mice. We describe steps for constructing single-cell transcriptome and TCR libraries, sequencing, and using sequencing data for the integration of expression and TCR data. We then detail procedures for gene differentiation, regulon and cell-cell communication analysis, and validation of single-cell analysis results. For complete details on the use and execution of this protocol, please refer to Wang et al.1.

Single-cell RNA sequencing reveals the suppressive effect of PPP1R15A inhibitor Sephin1 in antitumor immunity.

Protein phosphatase 1 regulatory subunit 15A (PPP1R15A) is an important factor in the integrated stress response (ISR) in mammals and may play a crucial role in tumorigenesis. In our studies, we found an inhibitor of PPP1R15A, Sephin1, plays a protumorigenic role in mouse tumor models. By analyzing the single-cell transcriptome data of the mouse tumor models, we found that in C57BL/6 mice, Sephin1 treatment could lead to higher levels of ISR activity and lower levels of antitumor immune activities. Specifically, Sephin1 treatment caused reductions in antitumor immune cell types and lower expression levels of cytotoxicity-related genes. In addition, T cell receptor (TCR) repertoire analysis demonstrated that the clonal expansion of tumor-specific T cells was inhibited by Sephin1. A special TCR + macrophage subtype in tumor was identified to be significantly depleted upon Sephin1 treatment, implying its key antitumor role. These results suggest that PPP1R15A has the potential to be an effective target for tumor therapy.

PI3Kα inhibitor CYH33 triggers antitumor immunity in murine breast cancer by activating CD8+T cells and promoting fatty acid metabolism.

BACKGROUND: The phosphatidylinositol 3-kinase (PI3K) is frequently hyperactivated in cancer and plays important roles in both malignant and immune cells. The effect of PI3Kα inhibitors on the tumor microenvironment (TME) remains largely unknown. Here, we investigated the modulation of the TME by a clinical PI3Kα-specific inhibitor CYH33. METHODS: The activity of CYH33 against a panel of murine tumors in the immune-competent context or athymic mice was detected. Single-cell RNA sequencing and multi-parameter flow cytometry were performed to determine the immune profiling of TME. The effect of CYH33 on immune cells was conducted with primary murine cells. RESULTS: CYH33 exhibited more potent antitumor activity in immune-competent context. CYH33 enhanced the infiltration and activation of CD8+T and CD4+T cells, while attenuating M2-like macrophages and regulatory CD4+T cells. Increase in memory T cells was confirmed by the induction of long-term immune memory on CYH33 treatment. Mechanistically, CYH33 relieved the suppressed expansion of CD8+T cells via preferential polarization of the macrophages to the M1 phenotype. CYH33 promoted fatty acid (FA) metabolism in the TME, while FA enhanced the activity of CD8+T cells in vitro. The combination of CYH33 with the FA synthase (FASN) inhibitor C75 synergistically inhibited tumor growth with enhanced host immunity. CONCLUSIONS: CYH33 induces immune activation and synergizes with FASN inhibitor to further promote the antitumor immunity, which gains novel insights into how PI3K inhibitors exert their activity by modulating TME and provides a rationale for the concurrent targeting of PI3K and FASN in breast cancer treatment.

Construction of H4x K2x Sn2-x S4+x /TiO2 nanocomposites with enhanced visible light-driven photocatalytic performance.

In this paper, a new photocatalyst with TiO2 nanospheres decorated on ultrathin layered thiostannate H4x K2x Sn2-x S4+x (X = 0.5-0.6, HKTS) nanosheets was successfully synthesized by a facile solvothermal method combined with the hydrolysis of tetrabutyl titanate and it was denoted as HKTS/TiO2. By adjusting the content of tetrabutyl titanate, composites with different Sn/Ti molar ratios were prepared. The composites were applied for RhB degradation under visible light irradiation, and the optimum proportion of HKTS/TiO2 was obtained. The results of X-ray diffraction, Raman spectroscopy, Fourier transform infrared spectroscopy and scanning electron microscopy confirmed that TiO2 was successfully decorated on HKTS nanosheets. The combination of TiO2 and HKTS extended the absorption wavelength of TiO2 from UV to visible light range, and the separation efficiency of photoexcited electron-hole pairs was also enhanced. The photocatalytic degradation rate of RhB over HKTS/TiO2-1.0 was almost 97.9% after 60 min illumination, which was higher than those of HKTS and pure TiO2. The photocatalyst exhibited excellent reusability and stability as the degradation rate of RhB was 95.7% even after three cycles. The photocatalytic mechanism experiment indicated that ·O2 - and h+ played a dominant role in the photocatalytic process. All these results indicate that the newly fabricated HKTS/TiO2 composites provide a high-performance photocatalyst for waste water treatment, and the application of thiostannate can be extended to the field of photocatalytic materials.

Drivers of nitrous oxide accumulation in denitrification biofilters with low carbon:nitrogen ratios.

Heterotrophic denitrification is usually inhibited by insufficient carbon sources; however, the underlying mechanisms responsible for nitrous oxide (N2O) accumulation within denitrification at low carbon:nitrogen (C/N) ratios have not been quantified from a molecular level. In this study, five denitrification biofilters were developed and exhibited efficiency (total nitrogen: 18.5%-92.2%; nitrate nitrogen: 42.9%-99.5%; chemical oxygen demand: 50.5%-93.7%) in remediating micro-polluted water with C/N ratios ranging between 0.65 and 3.0. A combined analysis revealed that the coupling of anaerobic ammonium oxidation (ANAMMOX) and denitrification accounted for N2O accumulation in the biofilters, and the key drivers of the N2O accumulation rates were qnorB/nirK, nosZ/(narG + napA), amx/(nirS + nirK), narG/bacteria and qnorB/bacteria. Our study demonstrated that genetic association was indicative of microbial processes relative to nitrogen cycling and reflected N2O flux within denitrification biofilters at low C/N ratios.

Genetic associations as indices of nitrogen cycling rates in an aerobic denitrification biofilter used for groundwater remediation.

An aerobic denitrification biofilter (ADB) for groundwater remediation was developed with high removal efficiencies (total nitrogen (TN): 82.3-95.8%; NO3(-)-N: 93.2-98.2%). Nitrate (NO3(-)-N) transformation rates stabilized between 21.0 and 23.4 g/(m(3) h), whereas nitrite (NO2(-)-N) and ammonium (NH4(+)-N) transformation rates remained less than 6.0 g/(m(3) h) as the dissolved oxygen (DO) level increased from 1.0 mg/L to 6.0 mg/L. Nitric oxide (NO) and nitrous oxide (N2O) accumulated with great fluctuations (NO: 0-1.6×10(-3) g/(m(3) h); N2O: 0.1-1.1g/(m(3)h)) throughout the experiment. This study suggested that gene associations reflect quantitative relationships with aerobic denitrification rates and can provide useful information regarding aerobic denitrification processes in groundwater. Especially, the qnorB/nosZ ratio acts as the main driver for NO3(-)-N and NH4(+)-N transformation, while the qnorB/nosZ ratio followed by the (nirS+nirK)/nosZ ratio serve a dominant role in the accumulation of N2O and NO.

Sludge granulation and performance of a low superficial gas velocity sequencing batch reactor (SBR) in the treatment of prepared sanitary wastewater.

A sequencing batch reactor (SBR) employing a low superficial gas velocity was used to produce aerobic granular sludge for wastewater treatment. At a gas velocity of 0.0056 m s(-1) sludge containing a mixture of light yellow and black granules with similar functional groups was quickly formed. The black granules contained crystals of CaCO(3), FeS, and Fe(2)O(3) as well as filamentous bacteria that strengthened the particles and reduced the mass transfer resistance. No inorganic crystals were detected in the yellow sludge granules, and their structure was maintained through cohesion mediated by extracellular polymeric substances (EPS). The light yellow granules were denser and offered better settling performance than the black granules, enhancing the settling properties of the mixed sludge. During a 12-h cycle, the maximum reductions in chemical oxygen demand (COD), NH(3)-N, and total nitrogen (TN) occurred at 240, 480, and 360 min with removal efficiencies of 90%, 90%, and 54%. When the cycle time was limited to 480 min, self-dissolution of the granules was avoided while sill maintaining removal efficiencies for COD, NH(3)-N, and TN of 88%, 90%, and 53%.