Identification and functional study of a novel variant of PAX9 causing tooth agenesis.

OBJECTIVES: To search for pathogenic gene of a family with non-syndromic tooth agenesis, and explore the possible pathogenesis. MATERIALS AND METHODS: A Chinese family with non-syndromic tooth agenesis was recruited and screened for the pathogenic variants by whole exome sequencing technology and co-segregation analysis. The subcellular localization of wild-type and mutant protein was detected by immunofluorescence assay. Cycloheximide chase assay was performed to examine the difference in degradation rate between mutant protein and wild-type one. Dual-luciferase reporter assays were conducted to explore the alterations of mutant protein in the regulation of downstream target genes. RESULTS: A novel missense variant of PAX9 (c.296C>A:p.A99D) was found in this family. Bioinformatics software showed ß-return and the random coil were shortened in the p.A99D. The variant did not affect the subcellular localization of PAX9, but the degradation rate of p.A99D was accelerated (p < 0.05). p.A99D inhibited the activation of downstream target gene BMP4 (p < 0.05). CONCLUSIONS: This novel variant expands the pathogenic gene spectrum. The variant impaired the protein structure, accelerated the degradation of protein, and inhibited the activation of the downstream target gene BMP4, an upstream molecule in the TGF-ß/BMP pathway, which may contribute to tooth agenesis in this family.

Cystine deprivation triggers CD36-mediated ferroptosis and dysfunction of tumor infiltrating CD8+ T cells.

Cancer cells develop multiple strategies to evade T cell-mediated killing. On one hand, cancer cells may preferentially rely on certain amino acids for rapid growth and metastasis. On the other hand, sufficient nutrient availability and uptake are necessary for mounting an effective T cell anti-tumor response in the tumor microenvironment (TME). Here we demonstrate that tumor cells outcompete T cells for cystine uptake due to high Slc7a11 expression. This competition induces T-cell exhaustion and ferroptosis, characterized by diminished memory formation and cytokine secretion, increased PD-1 and TIM-3 expression, as well as intracellular oxidative stress and lipid-peroxide accumulation. Importantly, either Slc7a11 deletion in tumor cells or intratumoral cystine supplementation improves T cell anti-tumor immunity. Mechanistically, cystine deprivation in T cells disrupts glutathione synthesis, but promotes CD36 mediated lipid uptake due to dysregulated cystine/glutamate exchange. Moreover, enforced expression of glutamate-cysteine ligase catalytic subunit (Gclc) promotes glutathione synthesis and prevents CD36 upregulation, thus boosting T cell anti-tumor immunity. Our findings reveal cystine as an intracellular metabolic checkpoint that orchestrates T-cell survival and differentiation, and highlight Gclc as a potential therapeutic target for enhancing T cell anti-tumor function.

Fueling T-cell Antitumor Immunity: Amino Acid Metabolism Revisited.

T cells are the key players in eliminating malignant tumors. Adoptive transfer of tumor antigen-specific T cells and immune checkpoint blockade has yielded durable antitumor responses in the clinic, but not all patients respond initially and some that do respond eventually have tumor progression. Thus, new approaches to enhance the utility of immunotherapy are needed. T-cell activation and differentiation status are tightly controlled at the transcriptional, epigenetic, and metabolic levels. Amino acids are involved in multiple steps of T-cell antitumor immunity, including T-cell activation, proliferation, effector function, memory formation as well as functional exhaustion. In this review, we briefly discuss how amino acid metabolism is linked to T-cell fate decisions and summarize how amino acid deprivation or accumulation of certain amino acid metabolites within the tumor microenvironment diminishes T-cell functionality. Furthermore, we discuss potential strategies for immunotherapy via modulating amino acid metabolism either in T cells intrinsically or extrinsically to achieve therapeutic efficacy.

The intrinsic role and mechanism of tumor expressed-CD38 on lung adenocarcinoma progression.

It has been recently reported that CD38 expressed on tumor cells of multiple murine and human origins could be upregulated in response to PD-L1 antibody therapy, which led to dysfunction of tumor-infiltrating CD8+ T immune cells due to increasing the production of adenosine. However, the role of tumor expressed-CD38 on neoplastic formation and progression remains elusive. In the present study, we aimed to delineate the molecular and biochemical function of the tumor-associated CD38 in lung adenocarcinoma progression. Our clinical data showed that the upregulation of tumor-originated CD38 was correlated with poor survival of lung cancer patients. Using multiple in vitro assays we found that the enzymatic activity of tumor expressed-CD38 facilitated lung cancer cell migration, proliferation, colony formation, and tumor development. Consistently, our in vivo results showed that inhibition of the enzymatic activity or antagonizing the enzymatic product of CD38 resulted in the similar inhibition of tumor proliferation and metastasis as CD38 gene knock-out or mutation. At biochemical level, we further identified that cADPR, the mainly hydrolytic product of CD38, was responsible for inducing the opening of TRPM2 iron channel leading to the influx of intracellular Ca2+ and then led to increasing levels of NRF2 while decreasing expression of KEAP1 in lung cancer cells. These findings suggested that malignant lung cancer cells were capable of using cADPR catalyzed by CD38 to facilitate tumor progression, and blocking the enzymatic activity of CD38 could be represented as an important strategy for preventing tumor progression.

Quantification of crustacean tropomyosin in foods using high-performance liquid chromatography-tandem mass spectrometry method.

BACKGROUND: Allergic reactions to crustacean products have been increasing owing to the rising consumption. Tropomyosin (TM) is the main crustacean allergen; it has a coiled-coil structure, which shows stability to various food processing methods. Crustacean processed products have been used in several food products, thereby causing greater difficulties in detecting TM in these products. We aimed to develop an assay based on high-performance liquid chromatography-tandem mass spectrometry for the accurate and reproducible quantification of crustacean TM in foods. RESULTS: The three peptides IQLLEEDLER, LAEASQAADESER, and IVELEEELR were selected as peptide markers, and the peptide IVELEEELR was selected as the quantitative marker. Extraction conditions and enzymatic digestion conditions were completely optimized. The extraction solution of Tris-hydrochloric acid buffer (50 mmol L-1 , pH 7.4) containing 1 mol L-1 potassium chloride and the enzymatic treatment at 1:15 ratio (enzyme/protein, m/m) for 13 h showed excellent efficiency. The method exhibited a good linear relationship, with the qualified coefficient of determination (R2  = 0.9994) in the wide range of 1 to 1000 µg L-1 . The accuracy was validated based on spiked recovery at three spiking levels (12.5, 25.0, and 50.0 µg kg-1 , TM/matrix) in blank matrices that included chicken sausages, beef balls, and egg-milk biscuits. The recoveries ranged from 91% to 109% with qualified relative standard deviations <15% with the limit of quantification (of 1.6 mg kg-1 , TM/matrix). CONCLUSION: This new approach can be used for the qualitative and quantitative detection of crustacean TM in various food matrices. © 2021 Society of Chemical Industry.

PCC0208018 exerts antitumor effects by activating effector T cells.

Poor prognosis is associated with melanoma due to immunosuppression profiles, suggesting that immune alterations have an important role in the occurrence, growth, and metastasis of melanoma. Here, we found that PCC0208018, a small-molecule compound, enhanced T cell proliferation and activation to release interferon gamma (IFN-γ) and interleukin-2 (IL-2) without blocking the programmed cell death 1 (PD-1)/programmed cell death-ligand 1 (PD-L1) binding and did not directly affect tumor cell viability in vitro. Furthermore, PCC0208018 increased the phosphorylation of protein kinase B (PKB/AKT) as well as extracellular regulated protein kinases (ERK) in human peripheral blood mononuclear cells (PBMCs) in vitro. The secretion of cytokines induced by PCC0208018 was significantly suppressed by the PI3K inhibitor GDC-0941. In B16-F10 melanoma-harboring mice, PCC0208018 significantly inhibited tumor growth as well as increasing CD3+, CD3+CD4+, and CD3+CD8+ T cell abundance in tumors without affecting PD-L1 expression. This study showed that PCC0208018 potentially increased PBMCs proliferation and function by activating the phosphatidylinositol 3 kinase (PI3K)/AKT and mitogen-activated protein kinase (MEK)/ERK pathways to exert antitumor effects.

Development of a method for the quantification of fish major allergen parvalbumin in food matrix via liquid chromatography-tandem mass spectrometry with multiple reaction monitoring.

The availability of analytical methods for quantification of allergens is crucial for the correct assessment and labeling of products in order to protect allergic consumers. For this purpose, a simple, sensitive and accurate technique was developed based on liquid chromatography-tandem mass spectrometry and multiple reaction monitoring mass spectrometry (LC-MRM-MS/MS). The proposed method uses a simple purification with heat and a completely optimized tryptic digestion. This method has been validated according to the requirements defined by ICH (Q2 [R1]), having a linear range from 0.10 to 1179.36 nM with r > 0.999. The parvalbumin beta in flounder (Paralichthys olivaceus) has been quantified at a low level down to 0.10 µg/g with satisfactory precision (RSD < 18.35%) and accuracy (<13.3%). The new approach was successfully applied for the determination of parvalbumin beta in the other food matrices.

PCC0208009 enhances the anti-tumor effects of temozolomide through direct inhibition and transcriptional regulation of indoleamine 2,3-dioxygenase in glioma models.

Indoleamine 2,3-dioxygenase (IDO), which is highly expressed in human glioblastoma and involved in tumor immune escape and resistance to chemotherapy, is clinically correlated with tumor progression and poor clinical outcomes, and is a promising therapeutic target for glioblastoma. IDO inhibitors are marginally efficacious as single-agents; therefore, combination with other therapies holds promise for cancer therapy. The aim of this study was to investigate the anti-tumor effects and mechanisms of the IDO inhibitor PCC0208009 in combination with temozolomide. The effects of PCC0208009 on IDO activity inhibition, and mRNA and protein expression in HeLa cells were observed. In the mouse glioma GL261 heterotopic model, the effects of PCC0208009 on l-kynurenine/tryptophan (Kyn/Trp), tumor growth, flow cytometry for T cells within tumors, and immunohistochemistry for IDO and Ki67 were examined. In the rat glioma C6 orthotopic model, animal survival, flow cytometry for T cells within tumors, and immunohistochemistry for proliferating cell nuclear antigen (PCNA) and IDO were examined. The results show that PCC0208009 is a highly effective IDO inhibitor, not only directly inhibiting IDO activity but also participating in the gene regulation of IDO expression at the transcription and translation levels. PCC0208009 significantly enhanced the anti-tumor effects of temozolomide in GL261 and C6 models, by increasing the percentages of CD3+, CD4+, and CD8+ T cells within tumors and suppressing tumor proliferation. These findings indicate that PCC0208009 can potentiate the anti-tumor efficacy of temozolomide and suggest that combination of IDO inhibitor-based immunotherapy with chemotherapy is a potential strategy for brain tumor treatment.