The phosphatase PAC1 acts as a T cell suppressor and attenuates host antitumor immunity.

Cancer cells subvert immune surveillance through inhibition of T cell effector function. Elucidation of the mechanism of T cell dysfunction is therefore central to cancer immunotherapy. Here, we report that dual specificity phosphatase 2 (DUSP2; also known as phosphatase of activated cells 1, PAC1) acts as an immune checkpoint in T cell antitumor immunity. PAC1 is selectively upregulated in exhausted tumor-infiltrating lymphocytes and is associated with poor prognosis of patients with cancer. PAC1hi effector T cells lose their proliferative and effector capacities and convert into exhausted T cells. Deletion of PAC1 enhances immune responses and reduces cancer susceptibility in mice. Through activation of EGR1, excessive reactive oxygen species in the tumor microenvironment induce expression of PAC1, which recruits the Mi-2ß nucleosome-remodeling and histone-deacetylase complex, eventually leading to chromatin remodeling of effector T cells. Our study demonstrates that PAC1 is an epigenetic immune regulator and highlights the importance of targeting PAC1 in cancer immunotherapy.

Structural insights into the ubiquitylation strategy of the oligomeric CRL2FEM1B E3 ubiquitin ligase.

Cullin-RING E3 ubiquitin ligase (CRL) family members play critical roles in numerous biological processes and diseases including cancer and Alzheimer's disease. Oligomerization of CRLs has been reported to be crucial for the regulation of their activities. However, the structural basis for its regulation and mechanism of its oligomerization are not fully known. Here, we present cryo-EM structures of oligomeric CRL2FEM1B in its unneddylated state, neddylated state in complex with BEX2 as well as neddylated state in complex with FNIP1/FLCN. These structures reveal that asymmetric dimerization of N8-CRL2FEM1B is critical for the ubiquitylation of BEX2 while FNIP1/FLCN is ubiquitylated by monomeric CRL2FEM1B. Our data present an example of the asymmetric homo-dimerization of CRL. Taken together, this study sheds light on the ubiquitylation strategy of oligomeric CRL2FEM1B according to substrates with different scales.

The tumor suppressor PTEN has a critical role in antiviral innate immunity.

The gene encoding PTEN is one of the most frequently mutated tumor suppressor-encoding genes in human cancer. While PTEN's function in tumor suppression is well established, its relationship to anti-microbial immunity remains unknown. Here we found a pivotal role for PTEN in the induction of type I interferon, the hallmark of antiviral innate immunity, that was independent of the pathway of the kinases PI(3)K and Akt. PTEN controlled the import of IRF3, a master transcription factor responsible for IFN-ß production, into the nucleus. We further identified a PTEN-controlled negative phosphorylation site at Ser97 of IRF3 and found that release from this negative regulation via the phosphatase activity of PTEN was essential for the activation of IRF3 and its import into the nucleus. Our study identifies crosstalk between PTEN and IRF3 in tumor suppression and innate immunity.

The phosphatase DUSP2 controls the activity of the transcription activator STAT3 and regulates TH17 differentiation.

Deregulation of the TH17 subset of helper T cells is closely linked with immunological disorders and inflammatory diseases. However, the mechanism by which TH17 cells are regulated remains elusive. Here we found that the phosphatase DUSP2 (PAC1) negatively regulated the development of TH17 cells. DUSP2 was directly associated with the signal transducer and transcription activator STAT3 and attenuated its activity through dephosphorylation of STAT3 at Tyr705 and Ser727. DUSP2-deficient mice exhibited severe susceptibility to experimental colitis, with enhanced differentiation of TH17 cells and secretion of proinflammatory cytokines. In clinical patients with ulcerative colitis, DUSP2 was downregulated by DNA methylation and was not induced during T cell activation. Our data demonstrate that DUSP2 is a true STAT3 phosphatase that modulates the development of TH17 cells in the autoimmune response and inflammation.

Acetylation of PHF5A Modulates Stress Responses and Colorectal Carcinogenesis through Alternative Splicing-Mediated Upregulation of KDM3A.

Alternative pre-mRNA-splicing-induced post-transcriptional gene expression regulation is one of the pathways for tumors maintaining proliferation rates accompanying the malignant phenotype under stress. Here, we uncover a list of hyperacetylated proteins in the context of acutely reduced Acetyl-CoA levels under nutrient starvation. PHF5A, a component of U2 snRNPs, can be acetylated at lysine 29 in response to multiple cellular stresses, which is dependent on p300. PHF5A acetylation strengthens the interaction among U2 snRNPs and affects global pre-mRNA splicing pattern and extensive gene expression. PHF5A hyperacetylation-induced alternative splicing stabilizes KDM3A mRNA and promotes its protein expression. Pathologically, PHF5A K29 hyperacetylation and KDM3A upregulation axis are correlated with poor prognosis of colon cancer. Our findings uncover a mechanism of an anti-stress pathway through which acetylation on PHF5A promotes the cancer cells' capacity for stress resistance and consequently contributes to colon carcinogenesis.

RNA-binding protein PTENα blocks RIG-I activation to prevent viral inflammation.

A timely inflammatory response is crucial for early viral defense, but uncontrolled inflammation harms the host. Retinoic acid-inducible gene I (RIG-I) has a pivotal role in detecting RNA viruses, yet the regulatory mechanisms governing its sensitivity remain elusive. Here we identify PTENα, an N-terminally extended form of PTEN, as an RNA-binding protein with a preference for the CAUC(G/U)UCAU motif. Using both in vivo and in vitro viral infection assays, we demonstrated that PTENα restricted the host innate immune response, relying on its RNA-binding capacity and phosphatase activity. Mechanistically, PTENα directly bound to viral RNA and enzymatically converted its 5'-triphosphate to 5'-monophosphate, thereby reducing RIG-I sensitivity. Physiologically, brain-intrinsic PTENα exerted protective effects against viral inflammation, while peripheral PTENα restricted host antiviral immunity and, to some extent, promoted viral replication. Collectively, our findings underscore the significance of PTENα in modulating viral RNA- and RIG-I-mediated immune recognition, offering potential therapeutic implications for infectious diseases.

PTENε suppresses tumor metastasis through regulation of filopodia formation.

PTEN is one of the most frequently mutated genes in malignancies and acts as a powerful tumor suppressor. Tumorigenesis is involved in multiple and complex processes including initiation, invasion, and metastasis. The complexity of PTEN function is partially attributed to PTEN family members such as PTENα and PTENß. Here, we report the identification of PTENε (also named as PTEN5), a novel N-terminal-extended PTEN isoform that suppresses tumor invasion and metastasis. We show that the translation of PTENε/PTEN5 is initiated from the CUG816 codon within the 5'UTR region of PTEN mRNA. PTENε/PTEN5 mainly localizes in the cell membrane and physically associates with and dephosphorylates VASP and ACTR2, which govern filopodia formation and cell motility. We found that endogenous depletion of PTENε/PTEN5 promotes filopodia formation and enhances the metastasis capacity of tumor cells. Overall, we identify a new isoform of PTEN with distinct subcellular localization and molecular function compared to the known members of the PTEN family. These findings advance our current understanding of the importance and diversity of PTEN functions.

FAM69C functions as a kinase for eIF2α and promotes stress granule assembly.

Stress granules are dynamic cytoplasmic ribonucleoprotein granules that assemble in response to cellular stress. Aberrant formation of stress granules has been linked to neurodegenerative diseases. However, the molecular mechanisms underlying the initiation of stress granules remain elusive. Here we report that the brain-enriched protein kinase FAM69C promotes stress granule assembly through phosphorylation of eukaryotic translation initiation factor 2 (eIF2α). FAM69C physically interacts with eIF2α and functions as a stress-specific kinase for eIF2α, leading to stress-induced protein translation arrest and stress granule assembly. Primary microglia derived from Fam69c knockout mice exhibit aberrant stress granule assembly in response to oxidative stress and ATP. Defective stress granule assembly in microglia correlates with the formation of ASC specks and NLRP3 inflammasome activation, whereas induction of stress granule precludes inflammasome formation. Consistently, increased NLRP3 levels, caspase-1 cleavage and Il18 expression corroborate microglia-associated neuroinflammation in aged Fam69c knockout mice. Our study demonstrates that FAM69C is critical for stress granule assembly and suggests its role in the regulation of microglia function.

Platelet-Rich Plasma in Young and Elderly Humans Exhibits a Different Proteomic Profile.

As people age, their ability to resist injury and repair damage decreases significantly. Platelet-rich plasma (PRP) has demonstrated diverse therapeutic effects on tissue repair. However, the inconsistency of patient outcomes poses a challenge to the practical application of PRP in clinical practice. Furthermore, a comprehensive understanding of the specific impact of aging on PRP requires a systematic investigation. We derived PRP from 6 young volunteers and 6 elderly volunteers, respectively. Subsequently, 95% of high-abundance proteins were removed, followed by mass spectrometry analysis. Data are available via ProteomeXchange with the identifier PXD050061. We detected a total of 739 proteins and selected 311 proteins that showed significant differences, including 76 upregulated proteins in the young group and 235 upregulated proteins in the elderly group. Functional annotation and enrichment analysis unveiled upregulation of proteins associated with cell apoptosis, angiogenesis, and complement and coagulation cascades in the elderly. Conversely, IGF1 was found to be upregulated in the young group, potentially serving as the central source of enhanced cell proliferation ability. Our investigation not only provides insights into standardizing PRP preparation but also offers novel strategies for augmenting the functionality of aging cells or tissues.

A Deep Learning-Based System Trained for Gastrointestinal Stromal Tumor Screening Can Identify Multiple Types of Soft Tissue Tumors.

The accuracy and timeliness of the pathologic diagnosis of soft tissue tumors (STTs) critically affect treatment decision and patient prognosis. Thus, it is crucial to make a preliminary judgement on whether the tumor is benign or malignant with hematoxylin and eosin-stained images. A deep learning-based system, Soft Tissue Tumor Box (STT-BOX), is presented herein, with only hematoxylin and eosin images for malignant STT identification from benign STTs with histopathologic similarity. STT-BOX assumed gastrointestinal stromal tumor as a baseline for malignant STT evaluation, and distinguished gastrointestinal stromal tumor from leiomyoma and schwannoma with 100% area under the curve in patients from three hospitals, which achieved higher accuracy than the interpretation of experienced pathologists. Particularly, this system performed well on six common types of malignant STTs from The Cancer Genome Atlas data set, accurately highlighting the malignant mass lesion. STT-BOX was able to distinguish ovarian malignant sex-cord stromal tumors without any fine-tuning. This study included mesenchymal tumors that originated from the digestive system, bone and soft tissues, and reproductive system, where the high accuracy of migration verification may reveal the morphologic similarity of the nine types of malignant tumors. Further evaluation in a pan-STT setting would be potential and prospective, obviating the overuse of immunohistochemistry and molecular tests, and providing a practical basis for clinical treatment selection in a timely manner.

COVID-19 increases extracorporeal coagulation during hemodialysis associated with upregulation of vWF/FBLN5 signaling in patients with severe/critical symptoms.

BACKGROUND: COVID-19 has been shown to increase the risk of extracorporeal coagulation during hemodialysis in patients, but the underlying mechanism remains unclear. This study aimed to investigate the effect and mechanism of COVID-19 on the risk of extracorporeal coagulation in patients with chronic kidney disease undergoing hemodialysis. METHODS: A retrospective analysis of the extracorporeal coagulation status of 339 hemodialysis patients at our center before and after COVID-19 infection was performed, including subgroup analyses. Post-infection blood composition was analyzed by protein spectrometry and ELISA. RESULTS: Compared to the pre-COVID-19 infection period, COVID-19-induced extracorporeal coagulation predominantly occurred in patients with severe/critical symptoms. Further proteomic analysis demonstrated that in patients with severe/critical symptoms, the coagulation cascade reaction, platelet activation, inflammation, and oxidative stress-related pathways were significantly amplified compared to those in patients with no/mild symptoms. Notably, the vWF/FBLN5 pathway, which is associated with inflammation, vascular injury, and coagulation, was significantly upregulated. CONCLUSIONS: Patients with severe/critical COVID-19 symptoms are at a higher risk of extracorporeal coagulation during hemodialysis, which is associated with the upregulation of the vWF/FBLN5 signaling pathway. These findings highlight the importance of early anticoagulant therapy initiation in COVID-19 patients with severe/critical symptoms, particularly those undergoing hemodialysis. Additionally, vWF/FBLN5 upregulation may be a novel mechanism for virus-associated thrombosis/coagulation.

Discovery of 2,4-diarylaminopyrimidine derivatives bearing sulfonamide moiety as novel FAK inhibitors.

Two series of 2,4-diarylaminopyrimidine derivatives containing sulfonamide moiety were designed and synthesized for screening as inhibitors of focal adhesion kinase (FAK). Most compounds significantly inhibited the enzymatic activities of FAK, and the best compound was 7b (IC50 = 0.27 nM). A majority of aminoethyl sulfonamide derivatives could effectively inhibit the proliferation of human cancer cell lines (HCT116, A549, MDA-MB-231 and Hela) expressing high levels of FAK. Particularly, compounds 7b, 7c, and 7o exhibited more significant efficacy against all of four cancer cell lines within concentrations of 1.5 µM. Furthermore, these three compounds displayed higher selectivity of cancer cells over normal cells (SI value > 14), compared to the positive control TAE226 (SI value = 1.63). Interestingly, introduction of dithiocarbamate moiety to the aminoethyl sulfonamide derivatives can indeed improve the antiproliferative activities against A549 cells. Especially, compound 8d demonstrated most significant cytotoxicity activity against A549 cells with an IC50 value of 0.08 µM, which is 20-fold superior to parent compound 7k. Additionally, compound 7b, which display the best anti-FAK potency, can inhibit the clone formation and migration of HCT-116 cells, and cause cell cycle arrest at G2/M phase, inducing apoptosis by promoting ROS production. Overall, these results suggest that 7b is a valuable FAK inhibitor that deserves further optimization to improve its druggability.

Evaluation of Small Molecules in Blank EDTA Plasma Tubes and Optimization of Metabolomic Workflow for Biomarker Studies Using Plasma Samples.

As the first step of metabolomic analysis in biomarker identification studies, various types of blood collection tubes are used in clinical practice. However, little attention is paid to potential contamination caused by the blank tube itself. Here, we evaluated small molecules in blank EDTA plasma tubes through LC-MS-based untargeted metabolomic analysis and identified small molecules with markedly varied levels among different production batches or specifications. Our data demonstrate possible contamination and data interference caused by blank EDTA plasma tubes when employing large clinical cohorts for biomarker identification. Therefore, we propose a workflow of filtering metabolites in blank tubes prior to statistical analysis to improve the fidelity of biomarker identification.

Citric acid of ovarian cancer metabolite induces pyroptosis via the caspase-4/TXNIP-NLRP3-GSDMD pathway in ovarian cancer.

Malignant tumors display profound changes in cellular metabolism, yet how these altered metabolites affect the development and growth of tumors is not fully understood. Here, we used metabolomics to analyze the metabolic profile differences in ovarian cancer and found that citric acid (CA) is the most significantly downregulated metabolite. Recently, CA has been reported to inhibit the growth of a variety of tumor cells, but whether it is involved in pyroptosis of ovarian cancer and its potential molecular mechanisms still remains to be further investigated. Here, we demonstrated that CA inhibits the growth of ovarian cancer cells in a dose-dependent manner. RNA-seq analysis revealed that CA significantly promoted the expression of thioredoxin interacting protein (TXNIP) and caspase-4 (CASP4). Morphologic examination by transmission electron microscopy indicated that CA-treated ovarian cancer cells exhibited typical pyroptosis characteristics. Further mechanistic analyses showed that CA facilitates pyroptosis via the CASP4/TXNIP-NLRP3-Gesdermin-d (GSDMD) pathway in ovarian cancer. This study elucidated that CA induces ovarian cancer cell death through classical and non-classical pyroptosis pathways, which may be beneficial as an ovarian cancer therapy.

Associations between visceral adipose index and stress urinary incontinence among US adult women: a cross-sectional study.

OBJECTIVE: Visceral adipose index (VAI) is a novel parameter for the evaluation of visceral obesity. The present study aimed to investigate the association between VAI levels and stress urinary incontinence (SUI) in a nationally representative population. MATERIALS AND METHODS: The National Health and Nutrition Examination Survey (NHANES) women population aged > 20 years were analyzed from 2001 to 2018. SUI was determined by self-reported questions. VAI was calculated using physical examination data and laboratory tests. Survey-weighted logistic regression models were used to analyze the correlation between SUI and VAI. RESULTS: The final analysis included 9709 women. Among them, 4032 (41.53%) were any SUI, 1130 (11.64%) were at least weekly SUI, and 506 (5.21%) were at least daily SUI. In multivariate analysis, the odds ratio (OR) for overall SUI increased slightly after full adjustment (OR 1.06, 95% CI 1.03-1.10, P = 0.001). Similar results were observed in weekly (OR 1.04, 95% CI 1.00-1.08, P = 0.0327) and daily (OR 1.04, 95% CI 1.00-1.09, P = 0.0702) SUI. The analysis of VAI categorized showed an increased OR of any, weekly, and daily SUI in the highest compared to the lowest tertile (OR 1.44, 95% CI 1.26-1.65, P < 0.0001 for trend, OR 1.38, 95% CI 1.07-1.78, P = 0.0153 for trend, OR 1.33, 95% CI 0.94-1.87, P = 0.094 for trend). CONCLUSION: This study revealed a significant association between SUI and VAI among US adult women. VAI is an easily applicable index for the evaluation of visceral fat dysfunction, which might be useful for the calculation of SUI risk.

The deubiquitinase OTUD1 enhances iron transport and potentiates host antitumor immunity.

Although iron is required for cell proliferation, iron-dependent programmed cell death serves as a critical barrier to tumor growth and metastasis. Emerging evidence suggests that iron-mediated lipid oxidation also facilitates immune eradication of cancer. However, the regulatory mechanisms of iron metabolism in cancer remain unclear. Here we identify OTUD1 as the deubiquitinase of iron-responsive element-binding protein 2 (IREB2), selectively reduced in colorectal cancer. Clinically, downregulation of OTUD1 is highly correlated with poor outcome of cancer. Mechanistically, OTUD1 promotes transferrin receptor protein 1 (TFRC)-mediated iron transportation through deubiquitinating and stabilizing IREB2, leading to increased ROS generation and ferroptosis. Moreover, the presence of OTUD1 promotes the release of damage-associated molecular patterns (DAMPs), which in turn recruits the leukocytes and strengthens host immune response. Reciprocally, depletion of OTUD1 limits tumor-reactive T-cell accumulation and exacerbates colon cancer progression. Our data demonstrate that OTUD1 plays a stimulatory role in iron transportation and highlight the importance of OTUD1-IREB2-TFRC signaling axis in host antitumor immunity.

Development of a modularized aptamer targeting the nuclear T-cell suppressor PAC1.

Aptamers associated with cancer targeting therapy are commonly focused on cell membrane proteins; however, the study of intracellular, particularly, nuclear proteins is limited. The nuclear phosphatase PAC1 has been reported to be a potential T cell-related immunotherapeutic target. Here, we identified an aptamer, designated as PA5, with high affinity and specificity for PAC1 through the systematic evolution of ligands by exponential enrichment (SELEX) procedure. We then developed a dual-module aptamer PAC1-AS consisting of a cell-internalizing module and a targeting module, which can recognize PAC1 in the nucleus under physiological conditions. This modularized aptamer raises the possibility of manipulating endosomes and provides insights into the exploration and development of an efficient cancer immunotherapy approach.

Carbon dots-mediated synthesis of gold nanodendrites with extended absorption into NIR-II window for in vivo photothermal therapy.

BACKGROUND: Photothermal therapy (PTT) in the second near-infrared (NIR-II) window has attracted extensive attention due to the benefits in high maximum permissible exposure and penetration depth. Current photothermal agents generally show a broadband absorption accompanied by a gradual attenuation of absorption in the NIR-II window, leading to poor effect of PTT. It remains a great challenge to gain photothermal agents with strong and characteristic absorption in NIR-II regions. To overcome this problem, based on carbon dots (CDs)-mediated growth strategy, we proposed a simple and feasible approach to prepare plasmonic gold nanodendrites (AuNDs) with NIR-II absorption to enhance the therapeutic effect of PTT. RESULTS: By rationally regulating the size and branch length of AuNDs, the AuNDs exhibited a broadband absorption from 300 to 1350 nm, with two characteristic absorption peaks located at 1077 and 1265 nm. The AuNDs demonstrated desired optical photothermal conversion efficiency (38.0%), which was further applied in NIR-II photoacoustic imaging (PAI) and PTT in human colon cancer cells (HCT 116)-tumor-bearing mice model. The tumor cells could be effectively eliminated in vivo under 1064 nm laser irradiation by the guidance of PAI. CONCLUSIONS: We reported a simple but powerful synthetic method to obtain the unique AuNDs with strong and characteristic absorption peaks in the NIR-II window. This study provides a promising solution to tuning the growth of nanoparticles for bioimaging and phototherapy in the NIR-II window.

Transcranial Ultrasonic Focusing by a Phased Array Based on Micro-CT Images.

In this paper, we utilize micro-computed tomography (micro-CT) to obtain micro-CT images with a resolution of 60 µm and establish a micro-CT model based on the k-wave toolbox, which can visualize the microstructures in trabecular bone, including pores and bone layers. The transcranial ultrasound phased array focusing field characteristics in the micro-CT model are investigated. The ultrasonic waves are multiply scattered in skull and time delays calculations from the transducer to the focusing point are difficult. For this reason, we adopt the pulse compression method and the linear frequency modulation Barker code to compute the time delay and implement phased array focusing in the micro-CT model. It is shown by the simulation results that ultrasonic loss is mainly caused by scattering from the microstructures of the trabecular bone. The ratio of main and side lobes of the cross-correlation calculation is improved by 5.53 dB using the pulse compression method. The focusing quality and the calculation accuracy of time delay are improved. Meanwhile, the beamwidth at the focal point and the sound pressure amplitude decrease with the increase in the signal frequency. Focusing at different depths indicates that the beamwidth broadens with the increase in the focusing depth, and beam deflection focusing maintains good consistency in the focusing effect at a distance of 9 mm from the focal point. This indicates that the phased-array method has good focusing results and focus tunability in deep cranial brain. In addition, the sound pressure at the focal point can be increased by 8.2% through amplitude regulation, thereby enhancing focusing efficiency. The preliminary experiment verification is conducted with an ex vivo skull. It is shown by the experimental results that the phased array focusing method using pulse compression to calculate the time delay can significantly improve the sound field focusing effect and is a very effective transcranial ultrasound focusing method.

Fermentation of Robinia pseudoacacia flower for improving the antioxidation: optimized conditions, active composition, mechanism, and biotransformation process.

Robinia pseudoacacia flower is a natural product with many biological activities, including antioxidation. To further develop its antioxidation, the extract was fermented by Aspergillus niger FFCC 3112 in the medium with carbon to nitrogen ratio of 1.4:1 and initial pH of 4.2 for 3.5 days to form the best antioxidant activity of the fermentation product by strain screening, single factor optimization, and response surface methodology. Further analysis, isolation and activity determination showed that a main chemical component, kaempferol-3-O-α-L-rhamnopyranosyl-(1→6)-ß-D-galactopyranosyl-7-O-α-L-rhamnopyranoside, in the extract was completely hydrolyzed to kaempferol-7-O-α-L-rhamnopyranoside and kaempferol with better antioxidant activity through biotransformation, which was the basis for improving the antioxidant activity of fermentation products. Moreover, the mechanism of antioxidant and the contribution of phenolic hydroxyl groups were investigated by density functional theory. The result indicated that the antioxidant capacity of kaempferol-7-O-α-L-rhamnopyranoside and kaempferol increased with the increase of solvent polarity. In high-polarity solvents, they mainly scavenge free radicals through single electron transfer followed by proton transfer.