GAS41 modulates ferroptosis by anchoring NRF2 on chromatin.

YEATS domain-containing protein GAS41 is a histone reader and oncogene. Here, through genome-wide CRISPR-Cas9 screenings, we identify GAS41 as a repressor of ferroptosis. GAS41 interacts with NRF2 and is critical for NRF2 to activate its targets such as SLC7A11 for modulating ferroptosis. By recognizing the H3K27-acetylation (H3K27-ac) marker, GAS41 is recruited to the SLC7A11 promoter, independent of NRF2 binding. By bridging the interaction between NRF2 and the H3K27-ac marker, GAS41 acts as an anchor for NRF2 on chromatin in a promoter-specific manner for transcriptional activation. Moreover, the GAS41-mediated effect on ferroptosis contributes to its oncogenic role in vivo. These data demonstrate that GAS41 is a target for modulating tumor growth through ferroptosis. Our study reveals a mechanism for GAS41-mediated regulation in transcription by anchoring NRF2 on chromatin, and provides a model in which the DNA binding activity on chromatin by transcriptional factors (NRF2) can be directly regulated by histone markers (H3K27-ac).

PHLDA2-mediated phosphatidic acid peroxidation triggers a distinct ferroptotic response during tumor suppression.

Although the role of ferroptosis in killing tumor cells is well established, recent studies indicate that ferroptosis inducers also sabotage anti-tumor immunity by killing neutrophils and thus unexpectedly stimulate tumor growth, raising a serious issue about whether ferroptosis effectively suppresses tumor development in vivo. Through genome-wide CRISPR-Cas9 screenings, we discover a pleckstrin homology-like domain family A member 2 (PHLDA2)-mediated ferroptosis pathway that is neither ACSL4-dependent nor requires common ferroptosis inducers. PHLDA2-mediated ferroptosis acts through the peroxidation of phosphatidic acid (PA) upon high levels of reactive oxygen species (ROS). ROS-induced ferroptosis is critical for tumor growth in the absence of common ferroptosis inducers; strikingly, loss of PHLDA2 abrogates ROS-induced ferroptosis and promotes tumor growth but has no obvious effect in normal tissues in both immunodeficient and immunocompetent mouse tumor models. These data demonstrate that PHLDA2-mediated PA peroxidation triggers a distinct ferroptosis response critical for tumor suppression and reveal that PHLDA2-mediated ferroptosis occurs naturally in vivo without any treatment from ferroptosis inducers.

Epidemiology, genetic characteristics, and association with meteorological factors of human metapneumovirus infection in children in southern China: A 10-year retrospective study.

OBJECTIVES: This study aimed to determine the epidemiological and genetic features of human metapneumovirus (HMPV) infection in children in southern China, and the effect of meteorological factors on infection. METHODS: 14,817 children (≤14 years) with acute respiratory tract infections from 2010 to 2019 were examined for HMPV and other respiratory viruses by real-time quantitative polymerase chain reaction. Full-length F gene of 54 positive samples were sequenced and subjected to phylogenetic analysis. The correlation between the HMPV-positive rate and meteorological factors was analyzed by linear regression analysis. RESULTS: HMPV was detected in 524 (3.5%) children, who were mostly younger than 1 year. The seasonal peak of HMPV prevalence mainly occurred in spring. Respiratory syncytial virus was the most common virus coinfected with HMPV (5.3%). Phylogenetic analysis revealed that the sequenced HMPV strains belonged to four sublineages, including A2b (1.9%), A2c (31.5%), B1 (50.0%), and B2 (16.7%). After adjusting for all meteorological factors, sunshine duration was inversely correlated with the HMPV-positive rate. CONCLUSION: HMPV is an important respiratory pathogen that causes acute respiratory tract infections in children in southern China, particularly in children ≤5 years old. The prevalence peak of HMPV in this area appeared in spring, and the predominant subtype was B1. Meteorological factors, especially long sunshine duration, might decrease the HMPV prevalence.

SHFL inhibits enterovirus A71 infection by triggering degradation of viral 3Dpol protein via the ubiquitin-proteasome pathway.

Enterovirus A71 (EV-A71) is a highly contagious virus that poses a major threat to global health, representing the primary etiological agent for hand-foot and mouth disease (HFMD) and neurological complications. It has been established that interferon signaling is critical to establishing a robust antiviral state in host cells, mainly mediated through the antiviral effects of numerous interferon-stimulated genes (ISGs). The host restriction factor SHFL is a novel ISG with broad antiviral activity against various viruses through diverse underlying molecular mechanisms. Although SHFL is widely acknowledged for its broad-spectrum antiviral activity, it remains elusive whether SHFL inhibits EV-A71. In this work, we validated that EV-A71 triggers the upregulation of SHFL both in cell lines and in a mouse model. Knockdown and overexpression of SHFL in EVA71-infected cells suggested that this factor could markedly suppress EV-A71 replication. Our findings further revealed an intriguing mechanism of SHFL that it could interact with the nonstructural proteins 3Dpol of EV-A71 and promoted the degradation of 3Dpol through the ubiquitin-proteasome pathway. Furthermore, the zinc-finger domain and the 36 amino acids (164-199) of SHFL were crucial to the interaction between SHFL and EV-A71 3Dpol . Overall, these findings broadened our understanding of the pivotal roles of SHFL in the interaction between the host and EV-A71.

MDM2-Targeting Reassembly Peptide (TRAP) Nanoparticles for p53-Based Cancer Therapy.

Gene mutations and functional inhibition are the major obstacles for p53-mediated oncotherapy. For p53-wild-type tumors, the underlying mechanisms of functional inhibition of p53 during oncogenesis are unknown. The results reveal that the expression of the MDM2 inhibitor ARF is inhibited in p53-wild-type tumors, indicating that the restoration of ARF could be a potential oncotherapy strategy for p53-wild-type tumors. Therefore, ARF-mimetic MDM2-targeting reassembly peptide nanoparticles (MtrapNPs) for p53-based tumor therapy is developed. The results elucidated that the MtrapNPs respond to and form a nanofiber structure with MDM2. By trapping MDM2, the MtrapNPs stabilize and activate p53 for the inhibition of p53-wild-type tumors. In most cases, reactivated mutant p53 is inhibited and degraded by MDM2. In the present study, MtrapNPs are used to load and deliver arsenic trioxide, a p53 mutation rescuer, for p53-mutated tumor treatment in both orthotopic and metastatic models, and they exhibit significant therapeutic effects. Therefore, the study provides evidence supporting a link between decreased ARF expression and tumor development in patients with p53-wild-type tumors. Thus, the MDM2-trap strategy, which addresses both the inhibition and mutations of p53, is an efficient strategy for the treatment of p53-mutated tumors.

Host factor DUSP5 potently inhibits dengue virus infection by modulating cytoskeleton rearrangement.

Cytoskeleton has been reported to play an essential role in facilitating the viral life cycle. However, whether the host can exert its antiviral effects by modulating the cytoskeleton is not fully understood. In this study, we identified that host factor DUSP5 was upregulated after dengue virus (DENV) infection. In addition, we demonstrated that overexpression of DUSP5 remarkably inhibited DENV replication. Conversely, the depletion of DUSP5 led to an increase in viral replication. Moreover, DUSP5 was found to restrain viral entry into host cells by suppressing F-actin rearrangement via negatively regulating the ERK-MLCK-Myosin IIB signaling axis. Depletion of dephosphorylase activity of DUSP5 abolished its above inhibitory effects. Furthermore, we also revealed that DUSP5 exhibited broad-spectrum antiviral effects against DENV and Zika virus. Taken together, our studies identified DUSP5 as a key host defense factor against viral infection and uncovered an intriguing mechanism by which the host exerts its antiviral effects through targeting cytoskeleton rearrangement.

Ring Finger Protein 215 Negatively Regulates Type I IFN Production via Blocking NF-κB p65 Activation.

Germline-encoded pattern recognition receptors (PRRs) recognize molecules frequently found in pathogens (pathogen-associated molecular patterns [PAMPs]) during viral infection. This process induces production of IFNs, leading to expression of IFN-stimulated genes to establish a cellular antiviral state against viral infection. However, aberrant activation of the IFN system may cause immunopathological damage and systemic autoimmune diseases such as systemic lupus erythematosus. Stringent control of IFN signaling activation is critical for maintaining homoeostasis of the immune system; yet, the mechanisms responsible for its precise regulation remain to be elucidated. In this study, we identified that ring finger protein 215 (RNF215), a zinc finger protein, was upregulated by viral infection in human macrophages. In addition, we demonstrated that RNF215 inhibited the production of type I IFNs at least in part via interacting with p65, a subunit of NF-κB, and repressed the accumulation of NF-κB in the promoter region of IFNB1. Moreover, we found that the expression of RNF215 negatively correlated with type I IFNs in patients with systemic lupus erythematosus, indicating that RNF215 plays an important role in the pathogenesis of autoimmune diseases. Collectively, our data identified RNF215 as a key negative regulator of type I IFNs and suggested RNF215 as a potential target for intervention in diseases with aberrant IFN production.

iPLA2ß-mediated lipid detoxification controls p53-driven ferroptosis independent of GPX4.

Here, we identify iPLA2ß as a critical regulator for p53-driven ferroptosis upon reactive oxygen species (ROS)-induced stress. The calcium-independent phospholipase iPLA2ß is known to cleave acyl tails from the glycerol backbone of lipids and release oxidized fatty acids from phospholipids. We found that iPLA2ß-mediated detoxification of peroxidized lipids is sufficient to suppress p53-driven ferroptosis upon ROS-induced stress, even in GPX4-null cells. Moreover, iPLA2ß is overexpressed in human cancers; inhibition of endogenous iPLA2ß sensitizes tumor cells to p53-driven ferroptosis and promotes p53-dependent tumor suppression in xenograft mouse models. These results demonstrate that iPLA2ß acts as a major ferroptosis repressor in a GPX4-independent manner. Notably, unlike GPX4, loss of iPLA2ß has no obvious effect on normal development or cell viability in normal tissues but iPLA2ß plays an essential role in regulating ferroptosis upon ROS-induced stress. Thus, our study suggests that iPLA2ß is a promising therapeutic target for activating ferroptosis-mediated tumor suppression without serious toxicity concerns.

The deubiquitinase USP11 regulates cell proliferation and ferroptotic cell death via stabilization of NRF2 USP11 deubiquitinates and stabilizes NRF2.

The transcription factor nuclear factor (erythroid-derived 2)-like 2 (NRF2) plays a key role in cancer progression and is tightly regulated by the proteasome pathway. E3 ligases that mediate NRF2 ubiquitination have been widely reported, but the mechanism of NRF2 deubiquitination remains largely unclear. Here, we identified ubiquitin-specific-processing protease 11 (USP11) in NRF2 complexes and confirmed an interaction between these two proteins. We further found that USP11 deubiquitinates NRF2; this modification stabilizes NRF2. Functionally, USP11 depletion contributes to the suppression of cell proliferation and induction of ferroptotic cell death due to ROS-mediated stress, which can be largely abrogated by overexpression of NRF2. Finally, immunohistochemical staining of USP11 and NRF2 was performed using a lung tissue microarray, which revealed that USP11 is highly expressed in patients with NSCLC and positively correlated with NRF2 expression. Together, USP11 stabilizes NRF2 and is thus an important player in cell proliferation and ferroptosis.

Total synthesis and a systematic structure-activity relationship study of WAP-8294A2.

WAP-8294A2 is a cyclic peptide antibiotic with novel structure and excellent activity against Gram-positive pathogens. Herein, we report the total synthesis of complex macrocyclic peptide WAP-8294A2 (W1), ent-analogue W2, deoxy analogue W3 and de-methyl analogue W4 using a solid-phase synthetic route followed by a final stage solution-phase cyclization reaction. Exploitation of this process allowed the synthesis of eleven alanine-scanning analogues and eight lysine-scanning analogues. The antimicrobial activity of these analogues was evaluated in vitro against Gram-positive bacteria. Based on the MIC results, a primary systematic structure-activity relationship has been established.

Convergent Synthesis of Calcium-Dependent Antibiotic CDA3a and Analogues with Improved Antibacterial Activity via Late-Stage Serine Ligation.

A convergent synthesis via the late-stage serine ligation of naturally occurring calcium-dependent antibiotic CDA3a and its analogues has been developed, which allowed us to readily synthesize the analogues with the variation on the lipid tail. Some analogues were found to show 100-500-fold higher antimicrobial activity than the natural compound CDA3a against drug resistant bacteria. This study will enhance our understanding of CDA3a and provide valuable antibacterial lead candidates for further development.

Tumour-associated macrophages as a novel target of VEGI-251 in cancer therapy.

Tumour-associated macrophages (TAMs), which possess M2-like characters and are derived from immature monocytes in the circulatory system, represent a predominant population of inflammatory cells in solid tumours. TAM infiltration in tumour microenvironment can be used as an important prognostic marker in many cancer types and is a potential target for cancer prevention or treatment. VEGI-251 not only is involved in the inhibition of tumour angiogenesis, but also participates in the regulation of host immunity. This work aimed to investigate the involvement of VEGI-251 in the regulation of specific antitumour immunity. We found that recombinant human VEGI-251(rhVEGI-251) efficiently mediated the elimination of TAMs in tumour tissue in mice, and induced apoptosis of purified TAMs in vitro. During this process, caspase-8 and caspase-3 were activated, leading to PARP cleavage and apoptosis. Most importantly, we further elucidated the mechanism underlying VEGI-251-triggered TAM apoptosis, which suggests that ASK1, an intermediate component of the VEGI-251, activates the JNK pathway via TRAF2 in a potentially DR3-dependent manner in the process of TAM apoptosis. Collectively, our findings provide new insights into the basic mechanisms underlying the actions of VEGI-251 that might lead to future development of antitumour therapeutic strategies using VEGI-251 to target TAMs.

Improved total synthesis of the antibiotic A54145B.

A54145B is a calcium-dependent cyclic lipodepsipeptide antibiotic that is active against Gram-positive pathogens. Herein, we report an improved synthetic route toward A54145B in terms of the yield and time required. The key changes include using a pre-assembled minimalist tetradepsipeptide building block to solve the difficult on-resin esterification from our previous synthetic route, and a new macrocyclization site to avoid the peptide self-cleavage problem.

N6-Methyladenosine Modulates Nonsense-Mediated mRNA Decay in Human Glioblastoma.

The N6-methyladenosine (m6A) modification influences various mRNA metabolic events and tumorigenesis, however, its functions in nonsense-mediated mRNA decay (NMD) and whether NMD detects induced carcinogenesis pathways remain undefined. Here, we showed that the m6A methyltransferase METTL3 sustained its oncogenic role by modulating NMD of splicing factors and alternative splicing isoform switches in glioblastoma (GBM). Methylated RNA immunoprecipitation-seq (MeRIP-seq) analyses showed that m6A modification peaks were enriched at metabolic pathway-related transcripts in glioma stem cells (GSC) compared with neural progenitor cells. In addition, the clinical aggressiveness of malignant gliomas was associated with elevated expression of METTL3. Furthermore, silencing METTL3 or overexpressing dominant-negative mutant METTL3 suppressed the growth and self-renewal of GSCs. Integrated transcriptome and MeRIP-seq analyses revealed that downregulating the expression of METTL3 decreased m6A modification levels of serine- and arginine-rich splicing factors (SRSF), which led to YTHDC1-dependent NMD of SRSF transcripts and decreased SRSF protein expression. Reduced expression of SRSFs led to larger changes in alternative splicing isoform switches. Importantly, the phenotypes mediated by METTL3 deficiency could be rescued by downregulating BCL-X or NCOR2 isoforms. Overall, these results establish a novel function of m6A in modulating NMD and uncover the mechanism by which METTL3 promotes GBM tumor growth and progression. SIGNIFICANCE: These findings establish the oncogenic role of m6A writer METTL3 in glioblastoma stem cells.

Total Synthesis and Structural Establishment/Revision of Antibiotics A54145.

A54145 is a family of antibacterial cyclic lipodepsipeptides structurally resembling daptomycin. Since its discovery in 1990, only the ambiguous structures of the methoxy-aspartic acid (MeO-Asp) and the hydroxy-asparagine (HO-Asn) have been reported. We have developed efficient routes to obtain the fully protected l-MeO-Asp and l-HO-Asn building blocks compatible with Fmoc-SPPS, and a total synthesis of A54145 that enabled us to establish its structure, consisting of l-3S-HO-Asn and l-3R-MeO-Asp, revising the wrongly proposed structure of l-3S-MeO-Asp.

ALOX12 is required for p53-mediated tumour suppression through a distinct ferroptosis pathway.

It is well established that ferroptosis is primarily controlled by glutathione peroxidase 4 (GPX4). Surprisingly, we observed that p53 activation modulates ferroptotic responses without apparent effects on GPX4 function. Instead, ALOX12 inactivation diminishes p53-mediated ferroptosis induced by reactive oxygen species stress and abrogates p53-dependent inhibition of tumour growth in xenograft models, suggesting that ALOX12 is critical for p53-mediated ferroptosis. The ALOX12 gene resides on human chromosome 17p13.1, a hotspot of monoallelic deletion in human cancers. Loss of one Alox12 allele is sufficient to accelerate tumorigenesis in Eµ-Myc lymphoma models. Moreover, ALOX12 missense mutations from human cancers abrogate its ability to oxygenate polyunsaturated fatty acids and to induce p53-mediated ferroptosis. Notably, ALOX12 is dispensable for ferroptosis induced by erastin or GPX4 inhibitors; conversely, ACSL4 is required for ferroptosis upon GPX4 inhibition but dispensable for p53-mediated ferroptosis. Thus, our study identifies an ALOX12-mediated, ACSL4-independent ferroptosis pathway that is critical for p53-dependent tumour suppression.

ARF-NRF2: A new checkpoint for oxidative stress responses?

NRF2 (nuclear factor erythroid 2-related factor 2) is a transcription factor which plays a major role in oxidative stress responses by regulating antioxidant gene expression. We have recently identified the ARF tumor suppressor as a key regulator of NRF2. ARF can significantly inhibit NRF2 transcriptional activities, and the ARF-NRF2 interaction may function as a novel checkpoint for oxidative stress responses.

NRF2 Is a Major Target of ARF in p53-Independent Tumor Suppression.

Although ARF can suppress tumor growth by activating p53 function, the mechanisms by which it suppresses tumor growth independently of p53 are not well understood. Here, we identified ARF as a key regulator of nuclear factor E2-related factor 2 (NRF2) through complex purification. ARF inhibits the ability of NRF2 to transcriptionally activate its target genes, including SLC7A11, a component of the cystine/glutamate antiporter that regulates reactive oxygen species (ROS)-induced ferroptosis. As a consequence, ARF expression sensitizes cells to ferroptosis in a p53-independent manner while ARF depletion induces NRF2 activation and promotes cancer cell survival in response to oxidative stress. Moreover, the ability of ARF to induce p53-independent tumor growth suppression in mouse xenograft models is significantly abrogated upon NRF2 overexpression. These results demonstrate that NRF2 is a major target of p53-independent tumor suppression by ARF and also suggest that the ARF-NRF2 interaction acts as a new checkpoint for oxidative stress responses.

Effects of raw material extrusion and steam conditioning on feed pellet quality and nutrient digestibility of growing meat rabbits.

This study was conducted to investigate the effects of raw material extrusion and steam conditioning on feed pellet quality and nutrient digestibility of growing meat rabbits, in order to determine appropriate rabbit feed processing methods and processing parameters. In Exp. 1, an orthogonal design was adopted. Barrel temperature, material moisture content and feed rate were selected as test factors, and acid detergent fiber (ADF) content was selected as an evaluation index to research the optimum extrusion parameters. In Exp. 2, a two-factor design was adopted. Four kinds of rabbit feeds were processed and raw material extrusion adopted optimum extrusion parameters of Exp. 1. A total of 40 healthy and 42-day-old rabbits with similar weight were used in a randomized design, which consisted of 4 groups and 10 replicates in each group (1 rabbits in each replicate). The adaptation period lasted for 7 d, and the digestion trial lasted for 4 d. The results showed as follows: 1) ADF was significantly affected by barrel temperature (P < 0.05); the optimum extrusion parameters were barrel temperature 125 °C, moisture content 16% and feed rate 9 Hz. 2) Raw material extrusion and steam conditioning both significantly decreased powder percentage, pulverization ratio and protein solubility (P < 0.05), significantly improved hardness and starch gelatinization degree of rabbit feed (P < 0.05). They both had significant interaction effects on the processing quality of rabbit feed (P < 0.05). 3) Extrusion significantly improved the apparent digestibility of dry matter and total energy (P < 0.05). Extrusion and steam conditioning both significantly improved the apparent digestibility of crude fiber (CF), ADF and NDF (P < 0.05), but they had no interaction effects on the apparent digestibility of rabbit feed. Thus, using extrusion and steam conditioning technology at the same time in the weaning rabbits feed processing can improve the pellet quality and nutrient apparent digestibility of rabbit feed.