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 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.

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.

LORF9 of Marek's disease virus is involved in the early cytolytic replication of B lymphocytes and can act as a target for gene deletion vaccine development.

IMPORTANCE: Marek's disease virus (MDV) is a highly infectious and oncogenic virus that can induce severe T cell lymphomas in chickens. MDV encodes more than 100 genes, most of which have unknown functions. This work indicated that the LORF9 gene is necessary for MDV early cytolytic replication in B lymphocytes. In addition, we have found that the LORF9 deletion mutant has a comparative immunological protective effect with CVI988/Rispens vaccine strain against very virulent MDV challenge. This is a significant discovery that LORF9 can be exploited as a possible target for the development of an MDV gene deletion vaccine.

Improved photosynthetic performance under unilateral weak light conditions in a wide-narrow-row intercropping system is associated with altered sugar transport.

Intercropping improves resource utilization. Under wide-narrow-row maize (Zea mays) intercropping, maize plants are subjected to weak unilateral illumination and exhibit high photosynthetic performance. However, the mechanism regulating photosynthesis under unilateral weak light remains unknown. We investigated the relationship between photosynthesis and sugar metabolism in maize under unilateral weak light. Our results showed that the net photosynthetic rate (Pn) of unshaded leaves increased as the level of shade on the other side increased. On the contrary, the concentration of sucrose and starch and the number of starch granules in the unshaded leaves decreased with increased shading due to the transfer of abundant C into the grains. However, sink loss with ear removal reduced the Pn of unshaded leaves. Intense unilateral shade (40% to 20% normal light), but not mild unilateral shade (60% normal light), reduced grain yield (37.6% to 54.4%, respectively). We further found that in unshaded leaves, Agpsl, Bmy, and Mexl-like expression significantly influenced sucrose and starch metabolism, while Sweet13a and Sut1 expression was crucial for sugar export. In shaded leaves, expression of Sps1, Agpsl, and Sweet13c was crucial for sugar metabolism and export. This study confirmed that unshaded leaves transported photosynthates to the ear, leading to a decrease in sugar concentration. The improvement of photosynthetic performance was associated with altered sugar transport. We propose a narrow-row spacing of 40 cm, which provides appropriate unilateral shade and limits yield reduction.

Identifying potential breath biomarkers for early diagnosis of papillary thyroid cancer based on solid-phase microextraction gas chromatography-high resolution mass spectrometry with metabolomics.

INTRODUCTION: Thyroid cancer incidence rate has increased substantially worldwide in recent years. Fine needle aspiration biopsy (FNAB) is currently the golden standard of thyroid cancer diagnosis, which however, is invasive and costly. In contrast, breath analysis is a non-invasive, safe and simple sampling method combined with a promising metabolomics approach, which is suitable for early cancer diagnosis in high volume population. OBJECTIVES: This study aims to achieve a more comprehensive and definitive exhaled breath metabolism profile in papillary thyroid cancer patients (PTCs). METHODS: We studied both end-tidal and mixed expiratory breath, solid-phase microextraction gas chromatography coupled with high resolution mass spectrometry (SPME-GC-HRMS) was used to analyze the breath samples. Multivariate combined univariate analysis was applied to identify potential breath biomarkers. RESULTS: The biomarkers identified in end-tidal and mixed expiratory breath mainly included alkanes, olefins, enols, enones, esters, aromatic compounds, and fluorine and chlorine containing organic compounds. The area under the curve (AUC) values of combined biomarkers were 0.974 (sensitivity: 96.1%, specificity: 90.2%) and 0.909 (sensitivity: 98.0%, specificity: 74.5%), respectively, for the end-tidal and mixed expiratory breath, indicating of reliability of the sampling and analysis method CONCLUSION: This work not only successfully established a standard metabolomic approach for early diagnosis of PTC, but also revealed the necessity of using both the two breath types for comprehensive analysis of the biomarkers.

Infection phase-dependent dynamics of the viral and host N6-methyladenosine epitranscriptome in the lifecycle of an oncogenic virus in vivo.

Dynamic alteration of the epitranscriptome exerts regulatory effects on the lifecycle of oncogenic viruses in vitro. However, little is known about these effects in vivo because of the general lack of suitable animal infection models of these viruses. Using a model of rapid-onset Marek's disease lymphoma in chickens, we investigated changes in viral and host messenger RNA (mRNA) N6-methyladenosine (m6 A) modification during Marek's disease virus (MDV) infection in vivo. We found that the expression of major epitranscriptomic proteins varies among viral infection phases, reprogramming both the viral and the host epitranscriptomes. Specifically, the methyltransferase-like 3 (METTL3)/14 complex was suppressed during the lytic and reactivation phases of the MDV lifecycle, whereas its expression was increased during the latent phase and in MDV-induced tumors. METTL3/14 overexpression inhibits, whereas METTL3/14 knockdown enhances, MDV gene expression and replication. These findings reveal the dynamic features of the mRNA m6 A modification program during viral replication in vivo, especially in relation to key pathways involved in tumorigenesis.

E3 ubiquitin ligase NEURL3 promotes innate antiviral response through catalyzing K63-linked ubiquitination of IRF7.

Interferon regulatory factor 7 (IRF7), as the interferon-stimulated gene, maximally drives type I interferon (IFN) production. However, the mechanisms by which the biological function of IRF7 is regulated remain elusive. In this study, we found that IRF7 selectively interacted with the neuralized E3 ubiquitin-protein ligase 3 (NEURL3). In concomitant with IRF7 induction, NEURL3 is upregulated by NF-κB signaling in the late phase of viral infection. Moreover, NEURL3 augmented the host antiviral immune response through ubiquitinating IRF7. A mechanistic study revealed that NEURL3 triggered K63-linked poly-ubiquitination on IRF7 lysine 375, which in turn epigenetically enhanced the transcription of interferon-stimulated genes (ISGs) through disruption of the association of IRF7 with Histone Deacetylase 1 (HDAC1), consequently augmenting host antiviral immune response. Accordingly, Neurl3-/- mice produced less type I IFNs and exhibited increased susceptibility to viral infection. Taken together, our findings identify NEURL3 as an E3 ubiquitin ligase of IRF7 and shed new light on the positive regulation of IRF7 in host antiviral immune signaling.

Identification of a suppressor for the wheat stripe rust resistance gene Yr81 in Chinese wheat landrace Dahongpao.

KEY MESSAGE: Combined with BSE-Seq analysis and multiple genetic populations, three genes involved in stripe rust resistance were identified in Chinese wheat landrace Dahongpao, including a novel suppressor on 2BS. Dahongpao (DHP), a landrace of hexaploid wheat in China, exhibits a high degree of stripe rust resistance in the field for many years. In this study, bulked segregant analysis coupled with exome capture sequencing (BSE-Seq) was used to identify genes encoding stripe rust resistance in multiple genetic populations from the cross between DHP and a susceptible hexaploid Australian cultivar, Avocet S (AvS). The most effective QTL in DHP was Yr18, explaining up to 53.08% of phenotypic variance in the F2:3 families. To identify additional genes, secondary mapping populations SP1 and SP2 were produced by crossing AvS with two resistant lines derived from F2:3 families lacking Yr18. An all-stage resistance gene, Yr.DHP-6AS, was identified via BSE-Seq analysis of SP1. Combined the recombinant plants from both SP1 and SP2, Yr.DHP-6AS was located between KP6A_1.66 and KP6A_8.18, corresponding to the same region as Yr81. In addition, secondary mapping populations SP3 and SP4 were developed by selfing a segregating line from F2:3 families lacking Yr18. A novel suppressor gene on chromosome 2BS was identified from DHP for effectively suppressing the resistance of Yr.DHP-6AS in the SP3 and SP4. As a result, the wheat lines carrying both Yr18 and Yr.DHP-6AS show higher level of stripe rust resistance than DHP, providing an effective and simple combination for developing new wheat cultivars with ASR and APR genes. Further, the newly developed KASP markers, KP6A_1.99 and KP6A_5.22, will facilitate the application of Yr.DHP-6AS in wheat breeding via marker-assisted selection.

circPTEN1, a circular RNA generated from PTEN, suppresses cancer progression through inhibition of TGF-ß/Smad signaling.

BACKGROUND: PTEN is one of the most frequently mutated genes in human cancer. Although the roles of canonical PTEN protein and PTEN isoforms have been extensively explored, the current understanding of PTEN family members cannot fully illustrate the diversity of their roles in biological processes and tumor development. Notably, the function of noncoding RNAs arising from PTEN has been less elucidated. METHODS: We searched circBase and circInteractome to analyze the potential of PTEN for generating circRNAs. Then, Sanger sequencing, RNase R and Actinomycin D assays were used to verify the ring structure of circPTEN1. In situ hybridization and qRT-PCR were used to determine the level of circPTEN1 in peritumor and tumor tissues of colorectal cancer (CRC). Furthermore, functional experiments, including Transwell assay, 3D multicellular tumor spheroid invasion assay and metastasis models, were performed using circPTEN1 knockdown and overexpression cell lines in vitro and in vivo to investigate the effects of circPTEN1 on tumor metastasis in CRC. Mechanistically, luciferase reporter assay, fluorescence in situ hybridization, electrophoretic mobility shift assay, RNA immunoprecipitation, RNA pull-down and mass spectrometry were executed. RESULTS: We identified a circular RNA generated from the PTEN gene, designated circPTEN1, that is frequently downregulated in colorectal cancer, and decreased expression of circPTEN1 predicts poor survival. Low expression of circPTEN1 promotes metastasis in PDX models in vivo and accelerates cancer cell invasion in vitro, whereas overexpression of circPTEN1 reveals opposite roles. Mechanically, we found that circPTEN1 is capable of binding the MH2 domain of Smad4 to disrupt its physical interaction with Smad2/3, which reduces the formation and subsequent nucleus translocation of Smad complexes and consequently suppresses the expression of its downstream genes associated with epithelial-mesenchymal transition upon TGF-ß stimulation. Furthermore, we found that eIF4A3 suppresses the cyclization of circPTEN1 by directly binding to the circPTEN1 flanking region. CONCLUSIONS: Our study uncovered a novel PTEN gene-generated circRNA with a tumor suppression function, and further revealed the mechanism of circPTEN1 in CRC metastasis mediated by TGF-ß. The identification of circPTEN1 provides a new direction for PTEN investigation, and elucidation of circPTEN1/TGF-ß/Smad signaling may pave the way for the development of a potential therapeutic strategy for the suppression of cancer progression.

CAFU: a Galaxy framework for exploring unmapped RNA-Seq data.

A widely used approach in transcriptome analysis is the alignment of short reads to a reference genome. However, owing to the deficiencies of specially designed analytical systems, short reads unmapped to the genome sequence are usually ignored, resulting in the loss of significant biological information and insights. To fill this gap, we present Comprehensive Assembly and Functional annotation of Unmapped RNA-Seq data (CAFU), a Galaxy-based framework that can facilitate the large-scale analysis of unmapped RNA sequencing (RNA-Seq) reads from single- and mixed-species samples. By taking advantage of machine learning techniques, CAFU addresses the issue of accurately identifying the species origin of transcripts assembled using unmapped reads from mixed-species samples. CAFU also represents an innovation in that it provides a comprehensive collection of functions required for transcript confidence evaluation, coding potential calculation, sequence and expression characterization and function annotation. These functions and their dependencies have been integrated into a Galaxy framework that provides access to CAFU via a user-friendly interface, dramatically simplifying complex exploration tasks involving unmapped RNA-Seq reads. CAFU has been validated with RNA-Seq data sets from wheat and Zea mays (maize) samples. CAFU is freely available via GitHub: https://github.com/cma2015/CAFU.

Tunable optofluidic microbubble lens.

Optofluidic microlenses are one of the crucial components in many miniature lab-on-chip systems. However, many optofluidic microlenses are fabricated through complex micromachining and tuned by high-precision actuators. We propose a kind of tunable optofluidic microbubble lens that is made by the fuse-and-blow method with a fiber fusion splicer. The optical focusing properties of the microlens can be tuned by changing the refractive index of the liquid inside. The focal spot size is 2.8 µm and the focal length is 13.7 µm, which are better than those of other tunable optofluidic microlenses. The imaging capability of the optofluidic microbubble lens is demonstrated under a resolution test target and the imaging resolution can reach 1 µm. The results indicate that the optofluidic microbubble lens possesses good focusing properties and imaging capability for many applications, such as cell counting, optical trapping, spatial light coupling, beam shaping and imaging.

Ultrasensitive optofluidic coupled Fabry-Perot capillary sensors.

Refractive index (RI) measurements are pertinent in concentration and biomolecular detection. Accordingly, an ultrasensitive optofluidic coupled Fabry-Perot (FP) capillary sensor based on the Vernier effect for RI sensing is proposed. Square capillaries integrated with the coupled FP microcavity provide multiple microfluidic channels while reducing the complexity of the fabrication process. The incoherent light source and spectrometer used during measurement facilitate the development of a low-cost sensing system. An ultrahigh RI sensitivity of 51709.0 nm/RIU and detection limit of 2.84 × 10-5 RIU are experimentally demonstrated, indicating acceptable RI sensing performance. The proposed sensor has significant potential for practical and low-cost applications such as RI, concentration, or biomolecular sensing.

PPP2R1B is modulated by ubiquitination and is essential for spermatogenesis.

The serine-threonine protein phosphatase 2A (PP2A) is a heterotrimeric enzyme complex that regulates many fundamental cellular processes. PP2A is involved in tumorigenesis because mutations in the scaffold subunit, PPP2R1B, were found in several types of cancers. However, the biological function of PPP2R1B remains largely unknown. We report here that homozygous deletion of Ppp2r1b in Mus musculus impairs meiotic recombination and causes meiotic arrest in spermatocytes. Consistently, male mice lacking Ppp2r1b are characterized with infertility. Furthermore, heterozygous missense mutations in the Homo sapiens PPP2R1B gene, which encodes PPP2R1B, are identified in azoospermia patients with meiotic arrest. We found that PPP2R1B mutants are susceptible to degradation by an E3 ligase CRL4ADCAF6 , and resistant to de-polyubiquitylation by ubiquitin-specific protease 5 (USP5). In addition, heterozygous mutations in PPP2R1B reduce stability of the wild-type PPP2R1B. Our results demonstrate an essential role of PPP2R1B in spermatogenesis and identify upstream regulators of PPP2R1B.

PTEN arginine methylation by PRMT6 suppresses PI3K-AKT signaling and modulates pre-mRNA splicing.

Arginine methylation is a ubiquitous posttranslational modification that regulates critical cellular processes including signal transduction and pre-mRNA splicing. Here, we report that the tumor-suppressor PTEN is methylated by protein arginine methyltransferase 6 (PRMT6). Mass-spectrometry analysis reveals that PTEN is dimethylated at arginine 159 (R159). We found that PTEN is mutated at R159 in cancers, and the PTEN mutant R159K loses its capability to inhibit the PI3K-AKT cascade. Furthermore, PRMT6 is physically associated with PTEN, promotes asymmetrical dimethylation of PTEN, and regulates the PI3K-AKT cascade through PTEN R159 methylation. In addition, using transcriptome analyses, we found that PTEN R159 methylation is involved in modulation of pre-mRNA alternative splicing. Our results demonstrate that PTEN is functionally regulated by arginine methylation. We propose that PTEN arginine methylation modulates pre-mRNA alternative splicing and influences diverse physiologic processes.

Exome Sequencing from Bulked Segregant Analysis Identifies a Gene for All-Stage Resistance to Stripe Rust on Chromosome 1AL in Chinese Wheat Landrace 'Xiaohemai'.

Stripe rust caused by Puccinia striiformis f. sp. tritici is one of the most destructive diseases of wheat. Identifying novel resistance genes applicable for developing disease-resistant cultivars is important for the sustainable control of wheat stripe rust. Chinese wheat landrace 'Xiaohemai' ('XHM') is an elite germplasm line with all-stage resistance (ASR) effective against predominant Chinese P. striiformis f. sp. tritici races. In this study, we performed a bulked segregant analysis coupled with exome capture sequencing (BSE-seq) to identify a candidate genomic region strongly associated with stripe rust resistance on chromosome 1AL in 173 F2:3 lines derived from the cross 'XHM' × 'Avocet S'. The gene, designated as YrXH-1AL, was validated by a conventional quantitative trait locus analysis using newly developed Kompetitive allele-specific PCR (KASP) markers, explaining up to 48.50% of the phenotypic variance. By testing a secondary mapping population comprising 144 lines from the same cross at the seedling stage with prevalent P. striiformis f. sp. tritici race CYR34, YrXH-1AL was identified as a single Mendelian factor in a 1.5-cM interval flanked by KASP markers KP1A_484.33 and KP1A_490.09. This region corresponded to a 5.76-Mb genomic interval on 'Chinese Spring' chromosome 1AL. Furthermore, two cosegregating KASP markers showed high polymorphisms among 130 Chinese wheat cultivars and could be used for marker-assisted selection. Because no other Yr genes for ASR that originated from common wheat have been detected on chromosome 1AL, YrXH-1AL is likely a novel gene that can be incorporated into modern breeding materials to develop wheat cultivars with enhanced stripe rust resistance.

Direct Amination of Benzene with Molecular Nitrogen Enabled by Plasma-Liquid Interactions.

Nitrogen fixation is industrially realized by mass production of ammonia, the principal intermediate nitrogen source for N-containing organic molecules. Instead, direct C-N bond formation from dinitrogen (N2 ) is of great interest but remains a challenge. Here, by virtue of unique plasma-liquid interactions, we developed an environmentally benign one-pot approach to directly couple benzene and N2 , two naturally abundant yet chemically inert molecules, into value-added arylamines. Under the optimal conditions, an amination yield of 45 % was rapidly achieved, far better than the reported benzene amination efficiency using ammonia. A tentative reaction mechanism was proposed involving the long-lived N2 (A3 Σ u + ) and N2 + species, as evidenced by the key intermediates detected. With a deeper mechanistic understanding and by further optimizing the plasma reactor, the realization of cost-effective electrical amination of benzene with N2 could become reality.

Phosphorylated LASS2 inhibits prostate carcinogenesis via negative regulation of Wnt/ß-catenin signaling.

LASS2 is a novel tumor-suppressor gene and has been characterized as a ceramide synthase, which synthesizes very-long acyl chain ceramides. However, LASS2 function and pathway-related activity in prostate carcinogenesis are still largely unexplored. Here, we firstly report that LASS2 promotes ß-catenin degradation through physical interaction with STK38, SCYL2, and ATP6V0C via the ubiquitin-proteasome pathway, phosphorylation of LASS2 is essential for ß-catenin degradation, and serine residue 248 of LASS2 is illustrated to be a key phosphorylation site. Furthermore, we find that dephosphorylation of LASS2 at serine residue 248 significantly enhances prostate cancer cell growth and metastasis in vivo, indicating that phosphorylated LASS2 inhibits prostate carcinogenesis through negative regulation of Wnt/ß-catenin signaling. Thus, our findings implicate LASS2 as a potential biomarker and therapeutic target of prostate cancer.