Identification of COP9 signalosome (CSN) subunits and antiviral function analysis of CSN5 in shrimp.

The constitutive photomorphogenesis 9 (COP9) signalosome (CSN) typically composing of eight subunits (CSN1-8) mediates the process of deneddylation and deubiquitination. The fifth subunit of COP9 signalosome, CSN5, has special characteristics compared with the other seven subunits, and plays vital roles in the deneddylation activity and diverse cellular processes. However, the role of CSN5 in antiviral immunity is not clear. In this study, we identified 8 subunits (CSN1-8) of COP9 signalosome in shrimp Marsupenaeus japonicus. CSN1-6 were existed in all tested tissues, but CSN7-CSN8 were not detected in hepatopancreas. After WSSV challenged, the expression level of Csn1 to Csn4, and Csn6 to Csn8 were highly decreased, but the expression level of Csn5 was conspicuously increased in shrimp challenged by white spot syndrome virus (WSSV). The CSN5 was recombinantly expressed in Escherichia coli and its polyclonal antibody was prepared. The expression level of CSN5 was conspicuously increased at RNA and protein levels in the shrimp challenged by WSSV. After knockdown of Csn5 by RNA interference, the WSSV replication was obviously increased in shrimp. When injected the recombinant protein of CSN5 with the membrane penetrating peptide into shrimp, WSSV replication was inhibited and the survival rate of shrimp was significantly improved compared with control. We further analyzed the expression of antimicrobial peptides (AMPs) in Csn5-RNAi shrimp, and the results showed that the expression of several AMPs was declined significantly. These results indicate that CSN5 inhibits replication of WSSV via regulating expression of AMPs in shrimp, and the recombinant CSN5 might be used in shrimp aquaculture for the white spot syndrome disease control.

Discovery of a Promising CBP/p300 Degrader XYD129 for the Treatment of Acute Myeloid Leukemia.

The epigenetic target CREB (cyclic-AMP responsive element binding protein) binding protein (CBP) and its homologue p300 were promising therapeutic targets for the treatment of acute myeloid leukemia (AML). Herein, we report the design, synthesis, and evaluation of a class of CBP/p300 PROTAC degraders based on our previously reported highly potent and selective CBP/p300 inhibitor 5. Among the compounds synthesized, 11c (XYD129) demonstrated high potency and formed a ternary complex between CBP/p300 and CRBN (AlphaScreen). The compound effectively degraded CBP/p300 proteins and exhibited greater inhibition of growth in acute leukemia cell lines compared to its parent compound 5. Furthermore, 11c demonstrated significant inhibition of tumor growth in a MOLM-16 xenograft model (TGI = 60%) at tolerated dose schedules. Our findings suggest that 11c is a promising lead compound for the treatment of AML.

Discovery and pharmacological characterization of 1,2,3,4-tetrahydroquinoline derivatives as RORγ inverse agonists against prostate cancer.

The retinoic acid receptor-related orphan receptor γ (RORγ) is regarded as an attractive therapeutic target for the treatment of prostate cancer. Herein, we report the identification, optimization, and evaluation of 1,2,3,4-tetrahydroquinoline derivatives as novel RORγ inverse agonists, starting from high throughput screening using a thermal stability shift assay (TSA). The representative compounds 13e (designated as XY039) and 14a (designated as XY077) effectively inhibited the RORγ transcriptional activity and exhibited excellent selectivity against other nuclear receptor subtypes. The structural basis for their inhibitory potency was elucidated through the crystallographic study of RORγ LBD complex with 13e. Both 13e and 14a demonstrated reasonable antiproliferative activity, potently inhibited colony formation and the expression of AR, AR regulated genes, and other oncogene in AR positive prostate cancer cell lines. Moreover, 13e and 14a effectively suppressed tumor growth in a 22Rv1 xenograft tumor model in mice. This work provides new and valuable lead compounds for further development of drugs against prostate cancer.

Discovery of Highly Potent and Efficient CBP/p300 Degraders with Strong In Vivo Antitumor Activity.

The transcriptional coactivator cAMP response element binding protein (CREB)-binding protein (CBP) and its homologue p300 have emerged as attractive therapeutic targets for human cancers such as acute myeloid leukemia (AML). Herein, we report the design, synthesis, and biological evaluation of a series of cereblon (CRBN)-recruiting CBP/p300 proteolysis targeting chimeras (PROTACs) based on the inhibitor CCS1477. The representative compounds 14g (XYD190) and 14h (XYD198) potently inhibited the growth of AML cells with low nanomolar IC50 values and effectively degraded CBP and p300 proteins in a concentration- and time-dependent manner. Mechanistic studies confirmed that 14g and 14h can selectively bind to CBP/p300 bromodomains and induce CBP and p300 degradation in bromodomain family proteins in a CRBN- and proteasome-dependent manner. 14g and 14h displayed remarkable antitumor efficacy in the MV4;11 xenograft model (TGI = 88% and 93%, respectively). Our findings demonstrated that 14g and 14h are useful lead compounds and deserve further optimization and activity evaluation for the treatment of human cancers.

A Polymer-Based Antigen Carrier Activates Two Innate Immune Pathways for Adjuvant-Free Subunit Vaccines.

The activation of multiple Pattern Recognition Receptors (PRRs) has been demonstrated to trigger inflammatory responses and coordinate the host's adaptive immunity during pathogen infections. The use of PRR agonists as vaccine adjuvants has been reported to synergistically induce specific humoral and cellular immune responses. However, incorporating multiple PRR agonists as adjuvants increases the complexity of vaccine design and manufacturing. In this study, we discovered a polymer that can activate both the Toll-like receptor (TLR) pathway and cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway. The polymer was then conjugated to protein antigens, creating an antigen delivery system for subunit vaccines. Without additional adjuvants, the antigen-polymer conjugates elicited strong antigen-specific humoral and cellular immune responses. Furthermore, the antigen-polymer conjugates, containing the Receptor Binding Domain (RBD) of the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Spike Protein or the Monkeypox Antigen M1R as the antigens, were found to induce potent antigen-specific antibodies, neutralizing antibodies, and cytotoxic T cells. Immunization with M1R-polymer also resulted in effective protection in a lethal challenge model. In conclusion, this vaccine delivery platform offers an effective, safe, and simple strategy for inducing antigen-specific immunity against infectious diseases.

KAT8 is upregulated and recruited to the promoter of Atg8 by FOXO to induce H4 acetylation for autophagy under 20-hydroxyecdysone regulation.

Selective gene expression in cells in physiological or pathological conditions is important for the growth and development of organisms. Acetylation of histone H4 at K16 (H4K16ac) catalyzed by histone acetyltransferase 8 (KAT8) is known to promote gene transcription; however, the regulation of KAT8 transcription and the mechanism by which KAT8 acetylates H4K16ac to promote specific gene expression are unclear. Using the lepidopteran insect Helicoverpa armigera as a model, we reveal that the transcription factor FOXO promotes KAT8 expression and recruits KAT8 to the promoter region of autophagy-related gene 8 (Atg8) to increase H4 acetylation at that location, enabling Atg8 transcription under the steroid hormone 20-hydroxyecdysone (20E) regulation. H4K16ac levels are increased in the midgut during metamorphosis, which is consistent with the expression profiles of KAT8 and ATG8. Knockdown of Kat8 using RNA interference results in delayed pupation and repression of midgut autophagy and decreases H4K16ac levels. Overexpression of KAT8-GFP promotes autophagy and increases H4K16ac levels. FOXO, KAT8, and H4K16ac colocalized at the FOXO-binding region to promote Atg8 transcription under 20E regulation. Acetylated FOXO at K180 and K183 catalyzed by KAT8 promotes gene transcription for autophagy. 20E via FOXO promotes Kat8 transcription. Knockdown or overexpression of FOXO appeared to give similar results as knockdown or overexpression of KAT8. Therefore, FOXO upregulates KAT8 expression and recruits KAT8 to the promoter region of Atg8, where the KAT8 induces H4 acetylation to promote Atg8 transcription for autophagy under 20E regulation. This study reveals the mechanism that KAT8 promotes transcription of a specific gene.

RUNX1/miR-429 feedback loop promotes growth, metastasis, and epithelial-mesenchymal transition in oral squamous cell carcinoma by targeting ITGB1.

This study aimed to explore the role of miR-429 on the progression of oral squamous cell carcinoma (OSCC). OSCC cell lines were transfected with miR-429 mimic, pcDNA3.1-RUNX1, or pcDNA3.1-ITGB1, and their cell viability, apoptosis, migration, and invasion abilities were analyzed by cell counting, terminal deoxynucleotidyl transferase dUTP nick-end labeling staining, wound healing, and transwell assays, respectively. Furthermore, luciferase reporter assay, RNA pull-down, and ChIP were used to assess the regulation of miR-429, RUNX1, and ITGB1 expression in OSCC. Lastly, the biological role of the RUNX1/miR-429 feedback loop was explored in nude mice. The results revealed that miR-429 level was down-regulated, while RUNX1 and ITGB1 levels were up-regulated in OSCC tissues and that miR-429 was negatively correlated with RUNX1 and ITGB1 in OSCC tissues. Transfection of miR-429 mimic suppressed OSCC cell proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT). Moreover, we found that miR-429 participated in OSCC progression by directly targeting ITGB1. Additionally, we found that RUNX1 negatively regulated miR-429 expression by binding to its promoter. Our results also revealed that the RUNX1/miR-429 feedback loop regulated ITGB1 expression and that RUNX1 overexpression rescued the inhibitory effects of miR-429 mimic on OSCC cells. In addition, miR-429 mimic significantly suppressed tumor growth, inflammatory cell infiltration, EMT, and ITGB1 expression in vivo, which were inhibited by RUNX1 overexpression. Altogether, these results indicate that the RUNX1/miR-429 feedback loop promoted growth, metastasis, and EMT in OSCC by targeting ITGB1.

Discovery of Novel Phenoxyaryl Pyridones as Bromodomain and Extra-Terminal Domain (BET) Inhibitors with High Selectivity for the Second Bromodomain (BD2) to Potentially Treat Acute Myeloid Leukemia.

Bromodomain-selective BET inhibition has emerged as a promising strategy to improve the safety profiles of pan-BET inhibitors. Herein, we report the discovery of potent phenoxyaryl pyridones as highly BD2-selective BET inhibitors. Compound 23 (IC50 = 2.9 nM) exhibited a comparable BRD4 BD2 inhibitory activity relative to 10 (IC50 = 1.0 nM) and remarkably improved selectivity over BRD4 BD1 (23: 2583-fold; 10: 344-fold). This lead compound significantly inhibited the proliferation of acute myeloid leukemia (AML) cell lines through induction of G0/G1 arrest and apoptosis in vitro. Excellent in vivo antitumor efficacy with 23 was achieved in an MV;411 mouse xenograft model. Pleasingly, compound 23 (hERG IC50 > 30 µM) mitigated the inhibition of the human ether-à-go-go-related gene (hERG) ion channel compared with 10 (hERG IC50 = 2.8 µM). This work provides a promising BD2-selective lead for the development of more effective and safe BET inhibitors as anticancer agents.

White spot syndrome virus hijacks host PP2A-FOXO axes to promote its propagation.

Viruses have developed superior strategies to escape host defenses or exploit host components and enable their infection. The forkhead box transcription factor O family proteins (FOXOs) are reportedly utilized by human cytomegalovirus during their reactivation in mammals, but if FOXOs are exploited by viruses during their infection remains unclear. In the present study, we found that the FOXO of kuruma shrimp (Marsupenaeus japonicus) was hijacked by white spot syndrome virus (WSSV) during infection. Mechanistically, the expression of leucine carboxyl methyl transferase 1 (LCMT1) was up-regulated during the early stages of WSSV infection, which activated the protein phosphatase 2A (PP2A) by methylation, leading to dephosphorylation of FOXO and translocation into the nucleus. The FOXO directly promoted transcription of the immediate early gene, wsv079 of WSSV, which functioned as a transcriptional activator to initiate the expression of viral early and late genes. Thus, WSSV utilized the host LCMT1-PP2A-FOXO axis to promote its replication during the early infection stage. We also found that, during the late stages of WSSV infection, the envelope protein of WSSV (VP26) promoted PP2A activity by directly binding to FOXO and the regulatory subunit of PP2A (B55), which further facilitated FOXO dephosphorylation and WSSV replication via the VP26-PP2A-FOXO axis in shrimp. Overall, this study reveals novel viral strategies by which WSSV hijacks host LCMT1-PP2A-FOXO or VP26-PP2A-FOXO axes to promote its propagation, and provides clinical targets for WSSV control in shrimp aquaculture.

Can the implementation of household waste classification mitigate greenhouse gas emissions in Beijing? A comprehensive analysis of recent trends and future scenarios.

Household waste contributes significantly to global greenhouse gas (GHG) emissions, and waste classification is crucial for reducing emissions. This study focuses on Beijing and utilizes life cycle assessment (LCA) and material flow analysis (MFA) to calculate GHG emissions in waste management systems and quantify emission reduction potential of different measures. The results show that net emissions from the classification system in 2021 are 116.77 kg CO2-eq/t waste, reducing 61.82 % compared to the traditional mixed collection and transportation system. Waste volume, classification efficiency, and treatment strategies are the primary factors affecting emissions in classification systems. Recycling is identified as effective treatment methods. Three scenarios are designed to explore emission pathway of the system toward 2060. In the business-as-usual (BAU) Scenario, emissions will continue to grow to 108.57 × 104 t CO2-eq/yr in 2060. In the Classification Efficiency Scenario and the Comprehensive Scenario, emissions in 2060 will be cut to -177.26 × 104 t CO2-eq/yr and -702.00 × 104 t CO2-eq/yr, respectively. These results underscore the critical role of waste classification and recycling in mitigating the negative impacts of increasing waste volume. By 2060, combining waste classification with recycling can offset emissions by 803.51 × 104 t CO2-eq/yr, contributing 99 % to emission reduction potential. Improving classification efficiency and recycling ratio are key measures for achieving this reduction goal. Meanwhile, treatment methods and technologies should prioritize classification and recycling. Aiming at carbon neutrality, the study proposes several recommendations to improve classification systems, including enhancing classification efficiency, optimizing treatment facilities and strategies, and establishing recycling and utilization systems, etc.

Laser induced high temperature thermal-mechanical-oxygen coupling experimental system and method.

In this paper, a thermal-mechanical-oxidation coupling experimental system based on laser heating is developed, containing two modes of Gaussian and flat-top lasers, which has a series of advantages such as high temperature range, rapid heating rate, and convenient observation. The system adopts active illumination and an optical filter to solve the problem where it is difficult for traditional digital-image correlation technology to image clearly under laser heating. A biaxial mechanical test machine is used to simulate the complex load by applying biaxial tension or compression loads on the material. Combined with the radiation temperature measurement and controllable flow field device, the thermal-mechanical-oxygen coupling experiment of high temperature resistant materials under aerobic environment can be carried out. The maximum uniform heat flux output density is 27.2 kW/cm2, and the maximum Gaussian heat flux output density is 105 kW/cm2. The thermal-mechanical-oxygen coupling experimental system and method are of great significance to the development of new high temperature resistant materials and thermal barrier coatings.

Boosting with an aerosolized Ad5-nCoV elicited robust immune responses in inactivated COVID-19 vaccines recipients.

Introduction: The SARS-CoV-2 Omicron variant has become the dominant SARS-CoV-2 variant and exhibits immune escape to current COVID-19 vaccines, the further boosting strategies are required. Methods: We have conducted a non-randomized, open-label and parallel-controlled phase 4 trial to evaluate the magnitude and longevity of immune responses to booster vaccination with intramuscular adenovirus vectored vaccine (Ad5-nCoV), aerosolized Ad5-nCoV, a recombinant protein subunit vaccine (ZF2001) or homologous inactivated vaccine (CoronaVac) in those who received two doses of inactivated COVID-19 vaccines. Results: The aerosolized Ad5-nCoV induced the most robust and long-lasting neutralizing activity against Omicron variant and IFNg T-cell response among all the boosters, with a distinct mucosal immune response. SARS-CoV-2-specific mucosal IgA response was substantially generated in subjects boosted with the aerosolized Ad5-nCoV at day 14 post-vaccination. At month 6, participants boosted with the aerosolized Ad5-nCoV had remarkably higher median titer and seroconversion of the Omicron BA.4/5-specific neutralizing antibody than those who received other boosters. Discussion: Our findings suggest that aerosolized Ad5-nCoV may provide an efficient alternative in response to the spread of the Omicron BA.4/5 variant. Clinical trial registration: https://www.chictr.org.cn/showproj.html?proj=152729, identifier ChiCTR2200057278.

A Rift Valley fever mRNA vaccine elicits strong immune responses in mice and rhesus macaques.

Rift Valley fever virus (RVFV) is listed as a priority pathogen by the World Health Organization (WHO) because it causes serious and fatal disease in humans, and there are currently no effective countermeasures. Therefore, it is urgent to develop a safe and efficacious vaccine. Here, we developed six nucleotide-modified mRNA vaccines encoding different regions of the Gn and Gc proteins of RVFV encapsulated in lipid nanoparticles, compared their ability to induce immune responses in mice and found that mRNA vaccine encoding the full-length Gn and Gc proteins had the strongest ability to induce cellular and humoral immune responses. IFNAR(-/-) mice vaccinated with mRNA-GnGc were protected from lethal RVFV challenge. In addition, mRNA-GnGc induced high levels of neutralizing antibodies and cellular responses in rhesus macaques, as well as antigen-specific memory B cells. These data demonstrated that mRNA-GnGc is a potent and promising vaccine candidate for RVFV.

Single-cell analysis of lizard blastema fibroblasts reveals phagocyte-dependent activation of Hedgehog-responsive chondrogenesis.

Lizards cannot naturally regenerate limbs but are the closest known relatives of mammals capable of epimorphic tail regrowth. However, the mechanisms regulating lizard blastema formation and chondrogenesis remain unclear. Here, single-cell RNA sequencing analysis of regenerating lizard tails identifies fibroblast and phagocyte populations linked to cartilage formation. Pseudotime trajectory analyses suggest spp1+-activated fibroblasts as blastema cell sources, with subsets exhibiting sulf1 expression and chondrogenic potential. Tail blastema, but not limb, fibroblasts express sulf1 and form cartilage under Hedgehog signaling regulation. Depletion of phagocytes inhibits blastema formation, but treatment with pericytic phagocyte-conditioned media rescues blastema chondrogenesis and cartilage formation in amputated limbs. The results indicate a hierarchy of phagocyte-induced fibroblast gene activations during lizard blastema formation, culminating in sulf1+ pro-chondrogenic populations singularly responsive to Hedgehog signaling. These properties distinguish lizard blastema cells from homeostatic and injury-stimulated fibroblasts and indicate potential actionable targets for inducing regeneration in other species, including humans.

Small ponds have stronger potential for net nitrogen removal: Insight from direct dissolved N2 measurement.

Growing demands for watershed nitrogen (N) removal have called attention to abundant small bodies of water such as ponds, which have long been heralded as efficient storage and processing systems. Although pond conservation, restoration, and creation have been widely implemented to mitigate N pollution, information is limited regarding the impact of size-that is, whether N removal potential and efficiency are dependent upon pond size. We investigated the dynamics of N removal rates in 56 ponds from a hilly watershed by studying their bimonthly N2 concentrations and fluxes. Our results showed that smaller ponds performed better in net N removal. This can be discerned from the areal N2 fluxes, which were the highest in small ponds (< 4, 000 m2). The corresponding N2 fluxes (4.73 ± 4.53 mmol N2 m-2 d-1) were 2 to 14 times greater than those observed in larger ponds. The N removal efficiency, a metric used to describe the portions of the substrates released as N2, was also significantly higher in the small ponds (∼8.7 %) than in the larger ponds (∼5.0 %). Further regression analysis showed that both areal N2 flux and N removal efficiency were negatively correlated with pond area. The underlying mechanisms behind the size effects of N removal could be attributed to small ponds having larger sediment contact area to water volume ratios. Thus, smaller ponds allow more opportunities for N to interact with bioactive sediments than larger ponds. Overall, our findings contribute to the understanding of the distal role of pond size in affecting N removal. This research also provides a strong rationale for considering the effects of system size when implementing management practices dedicated to maximizing N removal.

Precise modification of the surface charge of antigen enhances vaccine immunogenicity.

Aluminum (alum) adjuvant is the most extensively used protein subunit vaccine adjuvant, and its effectiveness and safety have been widely recognized. The surface charge of the antigen determines its electrostatic adsorption to alum adjuvant, which directly affects the immune efficacy of the protein vaccine. In our study, we precisely modified its surface charge by inserting charged amino acids into the flexible region of the SARS-CoV-2 receptor-binding domain (RBD), achieving electrostatic adsorption and a site-specific anchor between the immunogen and alum adjuvant. This innovative strategy extended the bioavailability of the RBD and directionally displayed the neutralizing epitopes, thereby significantly enhancing humoral and cellular immunity. Furthermore, the required dose of antigen and alum adjuvant was greatly reduced, which improved the safety and accessibility of the protein subunit vaccine. On this basis, the wide applicability of this novel strategy to a series of representative pathogen antigens such as SARS-RBD, MERS-RBD, Mpox-M1, MenB-fHbp, and Tularemia-Tul4 was further confirmed. Charge modification of antigens provides a straightforward approach for antigenicity optimization of alum-adjuvanted vaccines, which has great potential to be adopted as a global defense against infectious diseases.

Scavenger receptor B2, a type III membrane pattern recognition receptor, senses LPS and activates the IMD pathway in crustaceans.

The immune deficiency (IMD) pathway is critical for elevating host immunity in both insects and crustaceans. The IMD pathway activation in insects is mediated by peptidoglycan recognition proteins, which do not exist in crustaceans, suggesting a previously unidentified mechanism involved in crustacean IMD pathway activation. In this study, we identified a Marsupenaeus japonicus B class type III scavenger receptor, SRB2, as a receptor for activation of the IMD pathway. SRB2 is up-regulated upon bacterial challenge, while its depletion exacerbates bacterial proliferation and shrimp mortality via abolishing the expression of antimicrobial peptides. The extracellular domain of SRB2 recognizes bacterial lipopolysaccharide (LPS), while its C-terminal intracellular region containing a cryptic RHIM-like motif interacts with IMD, and activates the pathway by promoting nuclear translocation of RELISH. Overexpressing shrimp SRB2 in Drosophila melanogaster S2 cells potentiates LPS-induced IMD pathway activation and diptericin expression. These results unveil a previously unrecognized SRB2-IMD axis responsible for antimicrobial peptide induction and restriction of bacterial infection in crustaceans and provide evidence of biological diversity of IMD signaling in animals. A better understanding of the innate immunity of crustaceans will permit the optimization of prevention and treatment strategies against the arising shrimp diseases.

20-hydroxyecdysone reprograms amino acid metabolism to support the metamorphic development of Helicoverpa armigera.

Amino acid metabolism is regulated according to nutrient conditions; however, the mechanism is not fully understood. Using the holometabolous insect cotton bollworm (Helicoverpa armigera) as a model, we report that hemolymph metabolites are greatly changed from the feeding larvae to the wandering larvae and to pupae. Arginine, alpha-ketoglutarate (α-KG), and glutamate (Glu) are identified as marker metabolites of feeding larvae, wandering larvae, and pupae, respectively. Arginine level is decreased by 20-hydroxyecdysone (20E) regulation via repression of argininosuccinate synthetase (Ass) expression and upregulation of arginase (Arg) expression during metamorphosis. α-KG is transformed from Glu by glutamate dehydrogenase (GDH) in larval midgut, which is repressed by 20E. The α-KG is then transformed to Glu by GDH-like in pupal fat body, which is upregulated by 20E. Thus, 20E reprogrammed amino acid metabolism during metamorphosis by regulating gene expression in a stage- and tissue-specific manner to support insect metamorphic development.