USP8-governed GPX4 homeostasis orchestrates ferroptosis and cancer immunotherapy.

Ferroptosis is an iron-dependent type of regulated cell death resulting from extensive lipid peroxidation and plays a critical role in various physiological and pathological processes. However, the regulatory mechanisms for ferroptosis sensitivity remain incompletely understood. Here, we report that homozygous deletion of Usp8 (ubiquitin-specific protease 8) in intestinal epithelial cells (IECs) leads to architectural changes in the colonic epithelium and shortens mouse lifespan accompanied by increased IEC death and signs of lipid peroxidation. However, mice with heterozygous deletion of Usp8 in IECs display normal phenotype and become resistant to azoxymethane/dextran sodium sulfate-induced colorectal tumorigenesis. Mechanistically, USP8 interacts with and deubiquitinates glutathione peroxidase 4 (GPX4), leading to GPX4 stabilization. Thus, USP8 inhibition destabilizes GPX4 and sensitizes cancer cells to ferroptosis in vitro. Notably, USP8 inhibition in combination with ferroptosis inducers retards tumor growth and enhances CD8+ T cell infiltration, which potentiates tumor response to anti-PD-1 immunotherapy in vivo. These findings uncover that USP8 counteracts ferroptosis by stabilizing GPX4 and highlight targeting USP8 as a potential therapeutic strategy to boost ferroptosis for enhancing cancer immunotherapy.

UFL1 ablation in T cells suppresses PD-1 UFMylation to enhance anti-tumor immunity.

UFMylation is an emerging ubiquitin-like post-translational modification that regulates various biological processes. Dysregulation of the UFMylation pathway leads to human diseases, including cancers. However, the physiological role of UFMylation in T cells remains unclear. Here, we report that mice with conditional knockout (cKO) Ufl1, a UFMylation E3 ligase, in T cells exhibit effective tumor control. Single-cell RNA sequencing analysis shows that tumor-infiltrating cytotoxic CD8+ T cells are increased in Ufl1 cKO mice. Mechanistically, UFL1 promotes PD-1 UFMylation to antagonize PD-1 ubiquitination and degradation. Furthermore, AMPK phosphorylates UFL1 at Thr536, disrupting PD-1 UFMylation to trigger its degradation. Of note, UFL1 ablation in T cells reduces PD-1 UFMylation, subsequently destabilizing PD-1 and enhancing CD8+ T cell activation. Thus, Ufl1 cKO mice bearing tumors have a better response to anti-CTLA-4 immunotherapy. Collectively, our findings uncover a crucial role of UFMylation in T cells and highlight UFL1 as a potential target for cancer treatment.

Cognitive Deficits and Alzheimer's Disease-Like Pathologies in the Aged Chinese Tree Shrew.

Alzheimer's disease (AD) is the most common chronic progressive neurodegenerative disease in the elderly. It has an increasing prevalence and a growing health burden. One of the limitations in studying AD is the lack of animal models that show features of Alzheimer's pathogenesis. The tree shrew has a much closer genetic affinity to primates than to rodents and has great potential to be used for research into aging and AD. In this study, we aimed to investigate whether tree shrews naturally develop cognitive impairment and major AD-like pathologies with increasing age. Pole-board and novel object recognition tests were used to assess the cognitive performance of adult (about 1 year old) and aged (6 years old or older) tree shrews. The main AD-like pathologies were assessed by Western blotting, immunohistochemical staining, immunofluorescence staining, and Nissl staining. Our results showed that the aged tree shrews developed an impaired cognitive performance compared to the adult tree shrews. Moreover, the aged tree shrews exhibited several age-related phenotypes that are associated with AD, including increased levels of amyloid-ß (Aß) accumulation and phosphorylated tau protein, synaptic and neuronal loss, and reactive gliosis in the cortex and the hippocampal tissues. Our study provides further evidence that the tree shrew is a promising model for the study of aging and AD.

Targeting METTL3 reprograms the tumor microenvironment to improve cancer immunotherapy.

The tumor microenvironment (TME) is a heterogeneous ecosystem containing cancer cells, immune cells, stromal cells, cytokines, and chemokines which together govern tumor progression and response to immunotherapies. Methyltransferase-like 3 (METTL3), a core catalytic subunit for RNA N6-methyladenosine (m6A) modification, plays a crucial role in regulating various physiological and pathological processes. Whether and how METTL3 regulates the TME and anti-tumor immunity in non-small-cell lung cancer (NSCLC) remain poorly understood. Here, we report that METTL3 elevates expression of pro-tumorigenic chemokines including CXCL1, CXCL5, and CCL20, and destabilizes PD-L1 mRNA in an m6A-dependent manner, thereby shaping a non-inflamed TME. Thus, inhibiting METTL3 reprograms a more inflamed TME that renders anti-PD-1 therapy more effective in several murine lung tumor models. Clinically, NSCLC patients who exhibit low-METTL3 expression have a better prognosis when receiving anti-PD-1 therapy. Collectively, our study highlights targeting METTL3 as a promising strategy to improve immunotherapy in NSCLC patients.

Coexistence of Multiple Functional Variants and Genes Underlies Genetic Risk Locus 11p11.2 of Alzheimer's Disease.

BACKGROUND: Genome-wide association studies have identified dozens of genetic risk loci for Alzheimer's disease (AD), yet the underlying causal variants and biological mechanisms remain elusive, especially for loci with complex linkage disequilibrium and regulation. METHODS: To fully untangle the causal signal at a single locus, we performed a functional genomic study of 11p11.2 (the CELF1/SPI1 locus). Genome-wide association study signals at 11p11.2 were integrated with datasets of histone modification, open chromatin, and transcription factor binding to distill potentially functional variants (fVars). Their allelic regulatory activities were confirmed by allele imbalance, reporter assays, and base editing. Expressional quantitative trait loci and chromatin interaction data were incorporated to assign target genes to fVars. The relevance of these genes to AD was assessed by convergent functional genomics using bulk brain and single-cell transcriptomic, epigenomic, and proteomic datasets of patients with AD and control individuals, followed by cellular assays. RESULTS: We found that 24 potential fVars, rather than a single variant, were responsible for the risk of 11p11.2. These fVars modulated transcription factor binding and regulated multiple genes by long-range chromatin interactions. Besides SPI1, convergent evidence indicated that 6 target genes (MTCH2, ACP2, NDUFS3, PSMC3, C1QTNF4, and MADD) of fVars were likely to be involved in AD development. Disruption of each gene led to cellular amyloid-ß and phosphorylated tau changes, supporting the existence of multiple likely causal genes at 11p11.2. CONCLUSIONS: Multiple variants and genes at 11p11.2 may contribute to AD risk. This finding provides new insights into the mechanistic and therapeutic challenges of AD.

ERK and USP5 govern PD-1 homeostasis via deubiquitination to modulate tumor immunotherapy.

The programmed cell death protein 1 (PD-1) is an inhibitory receptor on T cells and plays an important role in promoting cancer immune evasion. While ubiquitin E3 ligases regulating PD-1 stability have been reported, deubiquitinases governing PD-1 homeostasis to modulate tumor immunotherapy remain unknown. Here, we identify the ubiquitin-specific protease 5 (USP5) as a bona fide deubiquitinase for PD-1. Mechanistically, USP5 interacts with PD-1, leading to deubiquitination and stabilization of PD-1. Moreover, extracellular signal-regulated kinase (ERK) phosphorylates PD-1 at Thr234 and promotes PD-1 interaction with USP5. Conditional knockout of Usp5 in T cells increases the production of effector cytokines and retards tumor growth in mice. USP5 inhibition in combination with Trametinib or anti-CTLA-4 has an additive effect on suppressing tumor growth in mice. Together, this study describes a molecular mechanism of ERK/USP5-mediated regulation of PD-1 and identifies potential combinatorial therapeutic strategies for enhancing anti-tumor efficacy.

Functional genomics identify causal variant underlying the protective CTSH locus for Alzheimer's disease.

Alzheimer's disease (AD) is the most prevalent age-related neurodegenerative disease, which has a high heritability of up to 79%. Exploring the genetic basis is essential for understanding the pathogenic mechanisms underlying AD development. Recent genome-wide association studies (GWASs) reported an AD-associated signal in the Cathepsin H (CTSH) gene in European populations. However, the exact functional/causal variant(s), and the genetic regulating mechanism of CTSH in AD remain to be determined. In this study, we carried out a comprehensive study to characterize the role of CTSH variants in the pathogenesis of AD. We identified rs2289702 in CTSH as the most significant functional variant that is associated with a protective effect against AD. The genetic association between rs2289702 and AD was validated in independent cohorts of the Han Chinese population. The CTSH mRNA expression level was significantly increased in AD patients and AD animal models, and the protective allele T of rs2289702 was associated with a decreased expression level of CTSH through the disruption of the binding affinity of transcription factors. Human microglia cells with CTSH knockout showed a significantly increased phagocytosis of Aß peptides. Our study identified CTSH as being involved in AD genetic susceptibility and uncovered the genetic regulating mechanism of CTSH in pathogenesis of AD.

Direct evidence of CRISPR-Cas9-mediated mitochondrial genome editing.

Pathogenic mitochondrial DNA (mtDNA) mutations can cause a variety of human diseases. The recent development of genome-editing technologies to manipulate mtDNA, such as mitochondria-targeted DNA nucleases and base editors, offer a promising way for curing mitochondrial diseases caused by mtDNA mutations. The CRISPR-Cas9 system is a widely used tool for genome editing; however, its application in mtDNA editing is still under debate. In this study, we developed a mito-Cas9 system by adding the mitochondria-targeted sequences and 3' untranslated region of nuclear-encoded mitochondrial genes upstream and downstream of the Cas9 gene, respectively. We confirmed that the mito-Cas9 system was transported into mitochondria and enabled knockin of exogenous single-stranded DNA oligonucleotides (ssODNs) into mtDNA based on proteinase and DNase protection assays. Successful knockin of exogenous ssODNs into mtDNA was further validated using polymerase chain reaction-free third-generation sequencing technology. We also demonstrated that RS-1, an agonist of RAD51, significantly increased knockin efficiency of the mito-Cas9 system. Collectively, we provide direct evidence that mtDNA can be edited using the CRISPR-Cas9 system. The mito-Cas9 system could be optimized as a promising approach for the treatment of mitochondrial diseases caused by pathogenic mtDNA mutations, especially those with homoplasmic mtDNA mutations.

Synthesis of Azepinoindoles via Pd-Catalyzed C(sp2)-H Imidoylative Cyclization Reactions.

An efficient and convenient method for the construction of diverse free (N-H)-benzazepinoindoles by Pd-catalyzed C(sp2)-H imidoylative cyclization of 3-(2-isocyanobenzyl)-1H-indoles was developed. The reaction shows a wide substrate scope and can be scaled up, providing a practical route to valuable bioactive azepinoindoles.

USP8 inhibition reshapes an inflamed tumor microenvironment that potentiates the immunotherapy.

Anti-PD-1/PD-L1 immunotherapy has achieved impressive therapeutic outcomes in patients with multiple cancer types. However, the underlined molecular mechanism(s) for moderate response rate (15-25%) or resistance to PD-1/PD-L1 blockade remains not completely understood. Here, we report that inhibiting the deubiquitinase, USP8, significantly enhances the efficacy of anti-PD-1/PD-L1 immunotherapy through reshaping an inflamed tumor microenvironment (TME). Mechanistically, USP8 inhibition increases PD-L1 protein abundance through elevating the TRAF6-mediated K63-linked ubiquitination of PD-L1 to antagonize K48-linked ubiquitination and degradation of PD-L1. In addition, USP8 inhibition also triggers innate immune response and MHC-I expression largely through activating the NF-κB signaling. Based on these mechanisms, USP8 inhibitor combination with PD-1/PD-L1 blockade significantly activates the infiltrated CD8+ T cells to suppress tumor growth and improves the survival benefit in several murine tumor models. Thus, our study reveals a potential combined therapeutic strategy to utilize a USP8 inhibitor and PD-1/PD-L1 blockade for enhancing anti-tumor efficacy.

Positron Emission Tomography/Magnetic Resonance Imaging Radiomics in Predicting Lung Adenocarcinoma and Squamous Cell Carcinoma.

OBJECTIVE: To investigate the diagnostic value of positron emission tomography (PET)/magnetic resonance imaging (MRI) radiomics in predicting the histological classification of lung adenocarcinoma and lung squamous cell carcinoma. METHODS: PET/MRI radiomics and clinical data were retrospectively collected from 61 patients with lung cancer. According to the pathological results of surgery or fiberscope, patients were divided into two groups, lung adenocarcinoma and squamous cell carcinoma group, which were set as positive for adenocarcinoma (40 cases) and negative for squamous cell carcinoma (21 cases). The radiomics characteristics most related to lung cancer classification were calculated and selected using radiomics software, and the two lung cancer groups were randomly assigned into a training set (70%) and a test set (30%). Maximum relevance and minimum redundancy (mRMR) and least absolute shrinkage and selection operator (LASSO) methods in the uAI Research Portal software (United Imaging Intelligence, China) were used to select the desired characteristics from 2600 features extracted from MRI and PET. Eight optimal features were finally retained through 5-fold cross-validation, and a PET/MRI fusion model was constructed. The predictive ability of this model was evaluated by the difference in area under the curve (AUC) obtained from the receiver operating characteristic (ROC) curve. RESULTS: AUC of PET/MRI model for the training group and test group were 0.886 (0.787-0.985) and 0.847 (0.648-1.000), respectively. PET/MRI radiomics features revealed different degrees of correlation with the classification of lung adenocarcinoma and squamous cell carcinoma, with significant differences. CONCLUSION: The prediction model constructed based on PET/MRI radiomics features can predict the preoperative histological classification of lung adenocarcinoma and squamous cell carcinoma without seminality and repeatability. It can also provide an objective basis for accurate clinical diagnosis and individualized treatment, thus having important guiding significance for clinical treatment.

Characterization, antioxidant, and anticancer activities of a neutral polysaccharide from Duchesnea indica (Andr.) Focke.

A neutral polysaccharide (DIP-1) from Duchesnea indica (Andr.) Focke was obtained by hot water extraction, ethanol precipitation and chromatographic separation (DEAE-52 cellulose anion-exchange column and Sephadex G-100 gel column). The physicochemical properties of DIP-1 were elucidated by gel permeation chromatography, monosaccharide composition, Fourier transform infrared spectrometry, UV-visible spectrophotometry, scanning electron microscope and Congo red test. The results indicated that DIP-1 was consisted of mannose, glucosamine, glucose, galactose and arabinose in a ratio of 1.00:0.42:18.36:14.17:0.81, and its molecular weight was 218.3 kDa. Meanwhile, DIP-1 presented a straight hexahedron structure, but no triple-helical conformation. In antioxidant activity tests, DIP-1 exhibited powerful scavenging activities on hydroxyl, DPPH, ABTS radicals and reducing power in a dose-dependent manner. Especially, DIP-1 demonstrated high inhibitory activities against SKOV-3 and Hep-G2 cells in vitro, with IC50 values of 1.42 and 1.23 mg/ml, respectively. PRACTICAL APPLICATIONS: D. indica has been used for a long time as a Chinese medicine for therapy of many diseases, including cancer, inflammation, leprosy, fever, bleeding and so on. At present, polysaccharides have attracted comprehensive attention because of a large range of pharmacological and biological properties, including antitumor, antidiabetic, antioxidant and immunomodulatory activity. In the present study, we purified and characterized a neutral polysaccharide from D. indica for the first time. Moreover, the neutral polysaccharide exhibits significant antioxidant and antitumor activities. Therefore, the present study laid a foundation for the high-value application of D. indica polysaccharides in functional food and pharmaceutical industries.

Synthesis and luminescence properties of brick-shaped lanthanum-organic frameworks with mesoporous and macroporous architectures.

Generally, metal-organic frameworks (MOFs) are made up from kinds of repeating microporous structure. Here, a series of Eu3+ ions activated terephthalate-based lanthanum-organic frameworks (La-MOFs) was synthesized by a hydrothermal reaction. By controlling the reaction time, we obtained some unique brick-shaped La-MOFs in a micron scale size range, and these La-MOFs showed tunable mesoporous and macroporous architectures. It is speculated that the change in the composition and structure of building units results in the formation of this mesoporous and macroporous heterogeneous architectures. Powder X-ray diffraction patterns and Eu3+ luminescence behavior support the speculation.