Siglec-15/sialic acid axis as a central glyco-immune checkpoint in breast cancer bone metastasis.

Immunotherapy is a promising approach for treating metastatic breast cancer (MBC), offering new possibilities for therapy. While checkpoint inhibitors have shown great progress in the treatment of metastatic breast cancer, their effectiveness in patients with bone metastases has been disappointing. This lack of efficacy seems to be specific to the bone environment, which exhibits immunosuppressive features. In this study, we elucidate the multiple roles of the sialic acid-binding Ig-like lectin (Siglec)-15/sialic acid glyco-immune checkpoint axis in the bone metastatic niche and explore potential therapeutic strategies targeting this glyco-immune checkpoint. Our research reveals that elevated levels of Siglec-15 in the bone metastatic niche can promote tumor-induced osteoclastogenesis as well as suppress antigen-specific T cell responses. Next, we demonstrate that antibody blockade of the Siglec-15/sialic acid glyco-immune checkpoint axis can act as a potential treatment for breast cancer bone metastasis. By targeting this pathway, we not only aim to treat bone metastasis but also inhibit the spread of metastatic cancer cells from bone lesions to other organs.

An artificially evolved gene for herbicide-resistant rice breeding.

Discovering and engineering herbicide-resistant genes is a crucial challenge in crop breeding. This study focuses on the 4-hydroxyphenylpyruvate dioxygenase Inhibitor Sensitive 1-Like (HSL) protein, prevalent in higher plants and exhibiting weak catalytic activity against many ß-triketone herbicides (ß-THs). The crystal structures of maize HSL1A complexed with ß-THs were elucidated, identifying four essential herbicide-binding residues and explaining the weak activity of HSL1A against the herbicides. Utilizing an artificial evolution approach, we developed a series of rice HSL1 mutants targeting the four residues. Then, these mutants were systematically evaluated, identifying the M10 variant as the most effective in modifying ß-THs. The initial active conformation of substrate binding in HSL1 was also revealed from these mutants. Furthermore, overexpression of M10 in rice significantly enhanced resistance to ß-THs, resulting in a notable 32-fold increase in resistance to methyl-benquitrione. In conclusion, the artificially evolved M10 gene shows great potential for the development of herbicide-resistant crops.

Retinoic acid receptor α activity in proximal tubules prevents kidney injury and fibrosis.

Chronic kidney disease (CKD) is characterized by a gradual loss of kidney function and affects ~13.4% of the global population. Progressive tubulointerstitial fibrosis, driven in part by proximal tubule (PT) damage, is a hallmark of late stages of CKD and contributes to the development of kidney failure, for which there are limited treatment options. Normal kidney development requires signaling by vitamin A (retinol), which is metabolized to retinoic acid (RA), an endogenous agonist for the RA receptors (RARα, ß, γ). RARα levels are decreased in a mouse model of diabetic nephropathy and restored with RA administration; additionally, RA treatment reduced fibrosis. We developed a mouse model in which a spatiotemporal (tamoxifen-inducible) deletion of RARα in kidney PT cells of adult mice causes mitochondrial dysfunction, massive PT injury, and apoptosis without the use of additional nephrotoxic substances. Long-term effects (3 to 4.5 mo) of RARα deletion include increased PT secretion of transforming growth factor ß1, inflammation, interstitial fibrosis, and decreased kidney function, all of which are major features of human CKD. Therefore, RARα's actions in PTs are crucial for PT homeostasis, and loss of RARα causes injury and a key CKD phenotype.

Classical swine fever virus non-structural protein 5B hijacks host METTL14-mediated m6A modification to counteract host antiviral immune response.

Classical Swine Fever (CSF), caused by the Classical Swine Fever Virus (CSFV), inflicts significant economic losses on the global pig industry. A key factor in the challenge of eradicating this virus is its ability to evade the host's innate immune response, leading to persistent infections. In our study, we elucidate the molecular mechanism through which CSFV exploits m6A modifications to circumvent host immune surveillance, thus facilitating its proliferation. We initially discovered that m6A modifications were elevated both in vivo and in vitro upon CSFV infection, particularly noting an increase in the expression of the methyltransferase METTL14. CSFV non-structural protein 5B was found to hijack HRD1, the E3 ubiquitin ligase for METTL14, preventing METTL14 degradation. MeRIP-seq analysis further revealed that METTL14 specifically targeted and methylated TLRs, notably TLR4. METTL14-mediated regulation of TLR4 degradation, facilitated by YTHDF2, led to the accelerated mRNA decay of TLR4. Consequently, TLR4-mediated NF-κB signaling, a crucial component of the innate immune response, is suppressed by CSFV. Collectively, these data effectively highlight the viral evasion tactics, shedding light on potential antiviral strategies targeting METTL14 to curb CSFV infection.

AMPK Attenuation of ß-Adrenergic Receptor-Induced Cardiac Injury via Phosphorylation of ß-Arrestin-1-ser330.

BACKGROUND: ß-adrenergic receptor (ß-AR) overactivation is a major pathological cue associated with cardiac injury and diseases. AMPK (AMP-activated protein kinase), a conserved energy sensor, regulates energy metabolism and is cardioprotective. However, whether AMPK exerts cardioprotective effects via regulating the signaling pathway downstream of ß-AR remains unclear. METHODS: Using immunoprecipitation, mass spectrometry, site-specific mutation, in vitro kinase assay, and in vivo animal studies, we determined whether AMPK phosphorylates ß-arrestin-1 at serine (Ser) 330. Wild-type mice and mice with site-specific mutagenesis (S330A knock-in [KI]/S330D KI) were subcutaneously injected with the ß-AR agonist isoproterenol (5 mg/kg) to evaluate the causality between ß-adrenergic insult and ß-arrestin-1 Ser330 phosphorylation. Cardiac transcriptomics was used to identify changes in gene expression from ß-arrestin-1-S330A/S330D mutation and ß-adrenergic insult. RESULTS: Metformin could decrease cAMP/PKA (protein kinase A) signaling induced by isoproterenol. AMPK bound to ß-arrestin-1 and phosphorylated Ser330 with the highest phosphorylated mass spectrometry score. AMPK activation promoted ß-arrestin-1 Ser330 phosphorylation in vitro and in vivo. Neonatal mouse cardiomyocytes overexpressing ß-arrestin-1-S330D (active form) inhibited the ß-AR/cAMP/PKA axis by increasing PDE (phosphodiesterase) 4 expression and activity. Cardiac transcriptomics revealed that the differentially expressed genes between isoproterenol-treated S330A KI and S330D KI mice were mainly involved in immune processes and inflammatory response. ß-arrestin-1 Ser330 phosphorylation inhibited isoproterenol-induced reactive oxygen species production and NLRP3 (NOD-like receptor protein 3) inflammasome activation in neonatal mouse cardiomyocytes. In S330D KI mice, the ß-AR-activated cAMP/PKA pathways were attenuated, leading to repressed inflammasome activation, reduced expression of proinflammatory cytokines, and mitigated macrophage infiltration. Compared with S330A KI mice, S330D KI mice showed diminished cardiac fibrosis and improved cardiac function upon isoproterenol exposure. However, the cardiac protection exerted by AMPK was abolished in S330A KI mice. CONCLUSIONS: AMPK phosphorylation of ß-arrestin-1 Ser330 potentiated PDE4 expression and activity, thereby inhibiting ß-AR/cAMP/PKA activation. Subsequently, ß-arrestin-1 Ser330 phosphorylation blocks ß-AR-induced cardiac inflammasome activation and remodeling.

Two novel compounds inhibit Flavivirus infection in vitro and in vivo by targeting lipid metabolism.

Flavivirus infection capitalizes on cellular lipid metabolism to remodel the cellular intima, creating a specialized lipid environment conducive to viral replication, assembly, and release. The Japanese encephalitis virus (JEV), a member of the Flavivirus genus, is responsible for significant morbidity and mortality in both humans and animals. Currently, there are no effective antiviral drugs available to combat JEV infection. In this study, we embarked on a quest to identify anti-JEV compounds within a lipid compound library. Our research led to the discovery of two novel compounds, isobavachalcone (IBC) and corosolic acid (CA), which exhibit dose-dependent inhibition of JEV proliferation. Time-of-addition assays indicated that IBC and CA predominantly target the late stage of the viral replication cycle. Mechanistically, JEV nonstructural proteins 1 and 2A (NS1 and NS2A) impede 5'-adenosine monophosphate (AMP)-activated protein kinase (AMPK) activation by obstructing the liver kinase B1 (LKB1)-AMPK interaction, resulting in decreased p-AMPK expression and a consequent upsurge in lipid synthesis. In contrast, IBC and CA may stimulate AMPK by binding to its active allosteric site, thereby inhibiting lipid synthesis essential for JEV replication and ultimately curtailing viral infection. Most importantly, in vivo experiments demonstrated that IBC and CA protected mice from JEV-induced mortality, significantly reducing viral loads in the brain and mitigating histopathological alterations. Overall, IBC and CA demonstrate significant potential as effective anti-JEV agents by precisely targeting AMPK-associated signaling pathways. These findings open new therapeutic avenues for addressing infections caused by Flaviviruses. IMPORTANCE: This study is the inaugural utilization of a lipid compound library in antiviral drug screening. Two lipid compounds, isobavachalcone (IBC) and corosolic acid (CA), emerged from the screening, exhibiting substantial inhibitory effects on the Japanese encephalitis virus (JEV) proliferation in vitro. In vivo experiments underscored their efficacy, with IBC and CA reducing viral loads in the brain and mitigating JEV-induced histopathological changes, effectively shielding mice from fatal JEV infection. Intriguingly, IBC and CA may activate 5'-adenosine monophosphate (AMP)-activated protein kinase (AMPK) by binding to its active site, curtailing the synthesis of lipid substances, and thus suppressing JEV proliferation. This indicates AMPK as a potential antiviral target. Remarkably, IBC and CA demonstrated suppression of multiple viruses, including Flaviviruses (JEV and Zika virus), porcine herpesvirus (pseudorabies virus), and coronaviruses (porcine deltacoronavirus and porcine epidemic diarrhea virus), suggesting their potential as broad-spectrum antiviral agents. These findings shed new light on the potential applications of these compounds in antiviral research.

Knockdown of LAP2α inhibits adipogenesis of human adipose-derived stem cells and ameliorates high-fat diet-induced obesity.

Adipogenesis, a pivotal cellular process involving the differentiation of mesenchymal stem cells (MSCs) to mature adipocytes, plays a significant role in various physiological functions. Dysregulation of adipogenesis is implicated in conditions such as obesity. However, the complete molecular understanding of adipogenesis remains elusive. This study aimed to uncover the novel role of lamina-associated polypeptide 2 alpha (LAP2α) in human adipose-derived stem cells (hASCs) adipogenesis and its impact on high-fat diet (HFD)-induced obesity and associated metabolic disturbances. LAP2α expression was assessed during the adipogenic differentiation of hASCs using RT-qPCR and western blotting. The functional role of LAP2α in adipogenesis was explored both in vitro and in vivo through loss- and gain-of-function studies. Moreover, mice with HFD-induced obesity received lentivirus injection to assess the effect of LAP2α knockdown on fat accumulation. Molecular mechanisms underlying LAP2α in adipogenic differentiation were investigated using RT-qPCR, Western blotting, immunofluorescence staining, and Oil Red O staining. LAP2α expression was upregulated during hASCs adipogenic differentiation. LAP2α knockdown hindered adipogenesis, while LAP2α overexpression promoted adipogenic differentiation. Notably, LAP2α deficiency resisted HFD-induced obesity, improved glucose intolerance, mitigated insulin resistance, and prevented fatty liver development. Mechanistically, LAP2α knockdown attenuated signal transducer and activator of transcription 3 (STAT3) activation by reducing the protein level of phosphorylated STAT3. A STAT3 activator (Colivelin) counteracted the negative impact of LAP2α deficiency on hASCs adipogenic differentiation. Taken together, our current study established LAP2α as a crucial regulator of hASCs adipogenic differentiation, unveiling a new therapeutic target for obesity prevention.

Engineering small-molecule and protein drugs for targeting bone tumors.

Bone cancer is common and severe. Both primary (e.g., osteosarcoma, Ewing sarcoma) and secondary (e.g., metastatic) bone cancers lead to significant health problems and death. Currently, treatments such as chemotherapy, hormone therapy, and radiation therapy are used to treat bone cancer, but they often only shrink or slow tumor growth and do not eliminate cancer completely. The bone microenvironment contributes unique signals that influence cancer growth, immunogenicity, and metastasis. Traditional cancer therapies have limited effectiveness due to off-target effects and poor distribution on bones. As a result, therapies with improved specificity and efficacy for treating bone tumors are highly needed. One of the most promising strategies involves the targeted delivery of pharmaceutical agents to the site of bone cancer by introduction of bone-targeting moieties, such as bisphosphonates or oligopeptides. These moieties have high affinities to the bone hydroxyapatite matrix, a structure found exclusively in skeletal tissue, and can enhance the targeting ability and efficacy of anticancer drugs when combating bone tumors. This review focuses on the engineering of small molecules and proteins with bone-targeting moieties for the treatment of bone tumors.

Anticoagulation Versus Antiplatelets in Spontaneous Cervical Artery Dissection: A Systematic Review and Meta-Analysis.

BACKGROUND: It is uncertain whether antiplatelets or anticoagulants are more effective in preventing early recurrent stroke in patients with cervical artery dissection. Following the publication of the observational Antithrombotic for STOP-CAD (Stroke Prevention in Cervical Artery Dissection) study, which has more than doubled available data, we performed an updated systematic review and meta-analysis comparing antiplatelets versus anticoagulation in cervical artery dissection. METHODS: The systematic review was registered in PROSPERO (CRD42023468063). We searched 5 databases using a combination of keywords that encompass different antiplatelets and anticoagulants, as well as cervical artery dissection. We included relevant randomized trials and included observational studies of dissection unrelated to major trauma. Where studies were sufficiently similar, we performed meta-analyses for efficacy (ischemic stroke) and safety (major hemorrhage, symptomatic intracranial hemorrhage, and death) outcomes using relative risks. RESULTS: We identified 11 studies (2 randomized trials and 9 observational studies) that met the inclusion criteria. These included 5039 patients (30% [1512] treated with anticoagulation and 70% [3527]) treated with antiplatelets]. In meta-analysis, anticoagulation was associated with a lower ischemic stroke risk (relative risk, 0.63 [95% CI, 0.43 to 0.94]; P=0.02; I2=0%) but higher major bleeding risk (relative risk, 2.25 [95% CI, 1.07 to 4.72]; P=0.03, I2=0%). The risks of death and symptomatic intracranial hemorrhage were similar between the 2 treatments. Effect sizes were larger in randomized trials. There are insufficient data on the efficacy and safety of dual antiplatelet therapy or direct oral anticoagulants. CONCLUSIONS: In this study of patients with cervical artery dissection, anticoagulation was superior to antiplatelet therapy in reducing ischemic stroke but carried a higher major bleeding risk. This argues for an individualized therapeutic approach incorporating the net clinical benefit of ischemic stroke reduction and bleeding risks. Large randomized clinical trials are required to clarify optimal antithrombotic strategies for management of cervical artery dissection.

Predicting 5-year recurrence risk in colorectal cancer: development and validation of a histology-based deep learning approach.

BACKGROUND: Accurate estimation of the long-term risk of recurrence in patients with non-metastatic colorectal cancer (CRC) is crucial for clinical management. Histology-based deep learning is expected to provide more abundant information for risk stratification. METHODS: We developed and validated a weakly supervised deep-learning model for predicting 5-year relapse-free survival (RFS) to stratify patients with different risks based on histological images from three hospitals of 614 cases with non-metastatic CRC. A deep prognostic factor (DL-RRS) was established to stratify patients into high and low-risk group. The areas under the curve (AUCs) were calculated to evaluate the performances of models. RESULTS: Our proposed model achieves the AUCs of 0.833 (95% CI: 0.736-0.905) and 0.715 (95% CI: 0.647-0.776) on validation cohort and external test cohort, respectively. The 5-year RFS rate was 45.7% for high DL-RRS patients, and 82.5% for low DL-RRS patients respectively in the external test cohort (HR: 3.89, 95% CI: 2.51-6.03, P < 0.001). Adjuvant chemotherapy was associated with improved RFS in Stage II patients with high DL-RRS (HR: 0.15, 95% CI: 0.06-0.38, P < 0.001). CONCLUSIONS: DL-RRS has a good predictive performance of 5-year recurrence risk in CRC, and will better serve the clinical decision-making.

G protein-coupled estrogen receptor activates PI3K/AKT/mTOR signaling to suppress ferroptosis via SREBP1/SCD1-mediated lipogenesis.

BACKGROUND: Lung cancer is the leading cause of cancer-related death worldwide. The sex differences in the occurrence and fatality rates of non-small cell lung cancer (NSCLC), along with its association with estrogen dependence, suggest that estrogen receptors (ERs) contribute to the development of NSCLC. However, the influence of G protein-coupled estrogen receptor (GPER1) on NSCLC remains to be determined. Escape from ferroptosis is one of the hallmarks of tumor discovered in recent years. In this context, the present study evaluated whether GPER1 promotes NSCLC progression by preventing ferroptosis, and the underlying mechanism through which GPER1 protects against ferroptosis was also explored. METHODS: The effects of GPER1 on the cytotoxicity of H2O2, the ferroptosis inducer RSL3, and Erastin were assessed using the CCK8 assay and plate cloning. Lipid peroxidation levels were measured based on the levels of MDA and BODIPY™581/591C11. GPER1 overexpression and knockdown were performed and G1 was used, and the expression of SCD1 and PI3K/AKT/mTOR signaling factors was measured. Immunofluorescence analysis and immunohistochemistry were performed on paired specimens to measure the correlation between the expression of GPER1 and SCD1 in NSCLC tissues. The effect of GPER1 on the cytotoxicity of cisplatin was measured in vitro using the CCK8 assay and in vivo using xenograft tumor models. RESULTS: GPER1 and G1 alleviated the cytotoxicity of H2O2, reduced sensitivity to RSL3, and impaired lipid peroxidation in NSCLC tissues. In addition, GPER1 and G1 promoted the protein and mRNA expression of SCD1 and the activation of PI3K/AKT/mTOR signaling. GPER1 and SCD1 expression were elevated and positively correlated in NSCLC tissues, and high GPER1 expression predicted a poor prognosis. GPER1 knockdown enhanced the antitumor activity of cisplatin in vitro and in vivo. CONCLUSION: GPER1 prevents ferroptosis in NSCLC by promoting the activation of PI3K/AKT/mTOR signaling, thereby inducing SCD1 expression. Therefore, treatments targeting GPER1 combined with cisplatin would exhibit better antitumor effects.

Nobiletin enhances mitochondrial function by regulating SIRT1/PGC-1α signaling in porcine oocytes during in vitro maturation.

Nobiletin is a natural flavonoid found in citrus fruits with beneficial effects, including anti-inflammatory, anti-cancer and anti-oxidation effects. The aim of this study was to investigate whether nobiletin improves mitochondrial function in porcine oocytes and examine the underlying mechanism. Oocytes enclosed by cumulus cells were cultured in TCM-199 for 44 h with 0.1% dimethyl sulfoxide (control), or supplemented with 5, 10, 25, and 50 µM of nobiletin (Nob5, Nob10, Nob25, and Nob50, respectively). Oocyte maturation rate was significantly enhanced in Nob10 (70.26 ± 0.45%) compared to the other groups (control: 60.12 ± 0.47%; Nob5: 59.44 ± 1.63%; Nob25: 63.15 ± 1.38%; Nob50: 46.57 ± 1.19%). The addition of nobiletin reduced the levels of reactive oxygen species and increased glutathione levels. Moreover, Nob10 promoted mitochondrial biogenesis by upregulating the protein levels of sirtuin 1 (SIRT1) and peroxisome proliferator-activated receptor-gamma coactivator 1α (PGC-1α). This resulted in an increase in the number of active mitochondria, mitochondrial DNA copy number, mitochondrial membrane potential, and ATP production, thereby enhancing mitochondrial function. The protein level of p53 decreased, followed by the phosphorylation of B-cell lymphoma 2, suggesting a reduction in mitochondria-mediated apoptosis in the Nob10 group. Additionally, the release of cytochrome c from the mitochondria was significantly diminished along with a decrease in the protein expression of caspase 3. Thus, nobiletin has a great potential to promote the in vitro maturation of porcine oocytes by suppressing oxidative stress and promoting mitochondrial function through the upregulation of the SIRT1/PGC-1α signaling pathway.

Transcriptome analysis of Gossypium hirsutum cultivar Zhongzhimian No.2 uncovers the gene regulatory networks involved in defense against Verticillium dahliae.

BACKGROUND: Cotton is globally important crop. Verticillium wilt (VW), caused by Verticillium dahliae, is the most destructive disease in cotton, reducing yield and fiber quality by over 50% of cotton acreage. Breeding resistant cotton cultivars has proven to be an efficient strategy for improving the resistance of cotton to V. dahliae. However, the lack of understanding of the genetic basis of VW resistance may hinder the progress in deploying elite cultivars with proven resistance. RESULTS: We planted the VW-resistant Gossypium hirsutum cultivar Zhongzhimian No.2 (ZZM2) in an artificial greenhouse and disease nursery. ZZM2 cotton was subsequently subjected to transcriptome sequencing after Vd991 inoculation (6, 12, 24, 48, and 72 h post-inoculation). Several differentially expressed genes (DEGs) were identified in response to V. dahliae infection, mainly involved in resistance processes, such as flavonoid and terpenoid quinone biosynthesis, plant hormone signaling, MAPK signaling, phenylpropanoid biosynthesis, and pyruvate metabolism. Compared to the susceptible cultivar Junmian No.1 (J1), oxidoreductase activity and reactive oxygen species (ROS) production were significantly increased in ZZM2. Furthermore, gene silencing of cytochrome c oxidase subunit 1 (COX1), which is involved in the oxidation-reduction process in ZZM2, compromised its resistance to V. dahliae, suggesting that COX1 contributes to VW resistance in ZZM2. CONCLUSIONS: Our data demonstrate that the G. hirsutum cultivar ZZM2 responds to V. dahliae inoculation through resistance-related processes, especially the oxidation-reduction process. This enhances our understanding of the mechanisms regulating the ZZM2 defense against VW.

Anti-PD-1 antibody in combination with radiotherapy as first-line therapy for unresectable intrahepatic cholangiocarcinoma.

BACKGROUND: Unresectable intrahepatic cholangiocarcinoma (iCCA) has a poor prognosis despite treatment with standard combination chemotherapy. We aimed to evaluate the efficacy and safety of radiotherapy in combination with an anti-PD-1 antibody in unresectable iCCA without distant metastases. METHODS: In this phase II study, patients with histopathologically confirmed unresectable primary or postoperative recurrent iCCA without distant metastases were enrolled. Patients received external radiotherapy with a dose of ≥45 Gy (2-2.5 Gy per fraction), followed by anti-PD-1 immunotherapy (camrelizumab 200 mg once, every 3 weeks) initiated within 7 days after completion of radiotherapy as first-line therapy. The primary endpoint was 1-year progression-free survival (PFS) rate. The secondary end points included safety, objective response rate (ORR), disease control rate (DCR), and overall survival (OS). RESULTS: From December 2019 to March 2021, 36 patients completed radiotherapy and at least one cycle of immunotherapy and were included in efficacy and safety analyses. The median follow-up was 19.0 months (IQR 12.0-24.0), and the one-year PFS rate was 44.4% (95% CI, 30.8-64.0). The median PFS was 12.0 months (95% CI, 7.5-not estimable); the median OS was 22.0 months (95% CI, 15.0-not estimable). The ORR was 61.1% and the DCR was 86.1%. Seventeen of 36 (47.2%) patients experienced treatment-related adverse effects (AEs) of any grade. The most common AE was reactive cutaneous capillary endothelial proliferation (25.0%). Five (13.9%) patients experienced grade ≥3 treatment-related AEs, including decreased lymphocyte (5.6%), bullous dermatitis (2.8%), decreased platelet count (2.8%), and deep-vein thrombosis (2.8%). CONCLUSIONS: External radiotherapy plus camrelizumab, as first-line therapy, met its primary endpoint and showed antitumor activity and low toxicity levels in patients with unresectable iCCA without distant metastases, warranting further investigation. TRIAL REGISTRATION: NCT03898895. Registered 2 April 2019.

SARS-CoV-2 spike protein acts as a ß-adrenergic receptor agonist: A potential mechanism for cardiac sequelae of long COVID.

BACKGROUND: Currently, pathophysiological mechanisms of post-acute sequelae of coronavirus disease-19-cardiovascular syndrome (PASC-CVS) remain unknown. METHODS AND RESULTS: Patients with PASC-CVS exhibited significantly higher circulating levels of severe acute respiratory syndrome-coronavirus-2 spike protein S1 than the non-PASC-CVS patients and healthy controls. Moreover, individuals with high plasma spike protein S1 concentrations exhibited elevated heart rates and normalized low frequency, suggesting cardiac ß-adrenergic receptor (ß-AR) hyperactivity. Microscale thermophoresis (MST) assay revealed that the spike protein bound to ß1- and ß2-AR, but not to D1-dopamine receptor. These interactions were blocked by ß1- and ß2-AR blockers. Molecular docking and MST assay of ß-AR mutants revealed that the spike protein interacted with the extracellular loop 2 of both ß-ARs. In cardiomyocytes, spike protein dose-dependently increased the cyclic adenosine monophosphate production with or without epinephrine, indicating its allosteric effects on ß-ARs. CONCLUSION: Severe acute respiratory syndrome-coronavirus-2 spike proteins act as an allosteric ß-AR agonist, leading to cardiac ß-AR hyperactivity, thus contributing to PASC-CVS.

Direct Observation of Z-Scheme Route in Cu31S16/ZnxCd1-xS Heteronanoplates for Highly Efficient Photocatalytic Hydrogen Evolution.

Although photocatalytic hydrogen production from water holds great potential as a renewable and sustainable energy alternative, the practical application of the technology demands cost-effective, simple photocatalytic systems with high efficiency in hydrogen evolution reaction (HER). Herein, the synthesis and characterization of Cu31S16/ZnxCd1-xS heterostructured nanoplates (Cu31S16/ZnCdS HNPs) as a high photocatalytic system are reported. The cost-effective, hierarchical structures are easily prepared using the Cu31S16 NPs as the seed by the epitaxial growth of the ZnCdS nanocrystals (NCs). The Cu31S16/ZnCdS without the noble metal cocatalyst exhibits a high HER rate of 61.7 mmol g-1 h-1, which is 8,014 and 17 times higher than that of Cu31S16 and ZnCdS, respectively, under visible light irradiation. The apparent quantum yield (AQY) of Cu31S16/ZnCdS reaches 67.9% at 400 nm with the highest value so far in the reported ZnCdS-based photocatalysts. The excellent activity and stability of the Cu31S16/ZnCdS are attributed to the formation of a strong internal electric field (IEF) and the Z-scheme pathway. The comprehensive experiments and theoretical calculations provide the direct evidences of the Z-scheme route. This work may offer a way for the design and development of efficient photocatalysts to achieve solar-to-chemical energy conversion at a practically useful level.

Biosynthesis of Halogenated Tryptophans for Protein Engineering using Genetic Code Expansion.

Genetic Code Expansion technology offers significant potential in incorporating noncanonical amino acids into proteins at precise locations, allowing for the modulation of protein structures and functions. However, this technology is often limited by the need for costly and challenging-to-synthesize external noncanonical amino acid sources. In this study, we address this limitation by developing autonomous cells capable of biosynthesizing halogenated tryptophan derivatives and introducing them into proteins using Genetic Code Expansion technology. By utilizing inexpensive halide salts and different halogenases, we successfully achieve the selective biosynthesis of 6-chloro-tryptophan, 7-chloro-tryptophan, 6-bromo-tryptophan, and 7-bromo-tryptophan. These derivatives are introduced at specific positions with corresponding bioorthogonal aminoacyl-tRNA synthetase/tRNA pairs in response to the amber codon. Following optimization, we demonstrate the robust expression of proteins containing halogenated tryptophan residues in cells with the ability to biosynthesize these tryptophan derivatives. This study establishes a versatile platform for engineering proteins with various halogenated tryptophans.

Characterization of the MIKCC-type MADS-box gene family in blueberry and its possible mechanism for regulating flowering in response to the chilling requirement.

MAIN CONCLUSION: The expression peak of VcAP1.4, VcAP1.6, VcAP3.1, VcAP3.2, VcAG3, VcFLC2, and VcSVP9 coincided with the endo-dormancy release of flower buds. Additionally, GA4+7 not only increased the expression of these genes but also promoted flower bud endo-dormancy release. The MIKCC-type MADS-box gene family is involved in the regulation of flower development. A total of 109 members of the MIKCC-type MADS-box gene family were identified in blueberry. According to the phylogenetic tree, these 109 MIKCC-type MADS-box proteins were divided into 13 subfamilies, which were distributed across 40 Scaffolds. The results of the conserved motif analysis showed that among 20 motifs, motifs 1, 3, and 9 formed the MADS-box structural domain, while motifs 2, 4, and 6 formed the K-box structural domain. The presence of 66 pairs of fragment duplication events in blueberry suggested that gene duplication events contributed to gene expansion and functional differentiation. Additionally, the presence of cis-acting elements revealed that VcFLC2, VcAG3, and VcSVP9 might have significant roles in the endo-dormancy release of flower buds. Meanwhile, under chilling conditions, VcAP3.1 and VcAG7 might facilitate flower bud dormancy release. VcSEP11 might promote flowering following the release of endo-dormancy, while the elevated expression of VcAP1.7 (DAM) could impede the endo-dormancy release of flower buds. The effect of gibberellin (GA4+7) treatment on the expression pattern of MIKCC-type MADS-box genes revealed that VcAP1.4, VcAP1.6, VcAP3.1, VcAG3, and VcFLC2 might promote flower bud endo-dormancy release, while VcAP3.2, VcSEP11, and VcSVP9 might inhibit its endo-dormancy release. These results indicated that VcAP1.4, VcAP1.6, VcAP1.7 (DAM), VcAP3.1, VcAG3, VcAG7, VcFLC2, and VcSVP9 could be selected as key regulatory promoting genes for controlling the endo-dormancy of blueberry flower buds.

IRF8 deficiency-induced myeloid-derived suppressor cell promote immune evasion in lung adenocarcinoma.

BACKGROUND: Patients with lung adenocarcinoma (LUAD) have a low response rate to immune checkpoint blockade. It is highly important to explore the tumor immune escape mechanism of LUAD patients and expand the population of patients who may benefit from immunotherapy. METHODS: Based on 954 bulk RNA-seq data of LUAD patients and 15 single-cell RNA-seq data, the relationships between tumor immune dysfunction and exclusion (TIDE) scores and survival prognosis in each patient were calculated and evaluated, and the immune escape mechanism affecting the independent prognosis of LUAD patients was identified. Functional enrichment analysis explored the antitumour immune response and biological behavior of tumor cells among different LUAD groups. Single-cell annotation and pseudotemporal analysis were used to explore the target molecules and immune escape mechanisms of LUAD. RESULTS: Myeloid-derived suppressor cells (MDSCs) and IRF8 were identified as risk and protective factors for the independent prognosis of LUAD patients, respectively. In the tumor microenvironment of patients with high infiltration of MDSCs, the antitumor immune response is significantly suppressed, while tumor cell division, proliferation, and distant metastasis are significantly enhanced. Single-cell RNA-seq analysis revealed that IRF8 is an important regulator of MDSC differentiation in LUAD myeloid cells. In addition, IRF8 may regulate the differentiation of MDSCs through the IL6-JAK-STAT3 signalling pathway. CONCLUSIONS: IRF8 deficiency impairs the normal development of LUAD myeloid cells and induces their differentiation into MDSCs, thereby accelerating the immune escape of LUAD cells. IRF8-targeted activation to inhibit the formation of MDSCs may be a new target for immunotherapy in LUAD.

PASTICCINO2 interacting with Golgi Anti-Apoptotic Proteins resists endoplasmic reticulum stress dependent on very long-chain fatty acids.

The endoplasmic reticulum (ER) is crucial for maintaining cell homeostasis because it is the primary site for synthesizing secreted and transmembrane proteins and lipids. The unfolded protein response (UPR) is activated to restore ER homeostasis under ER stress. However, the relationship between lipids and the ER stress response in plants is not well understood. Arabidopsis Golgi anti-apoptotic proteins (GAAPs) are involved in resisting ER stress. To elucidate the function of GAAPs, PASTICCINO2 (PAS2), involved in very long-chain fatty acid (VLCFA) synthesis, was found to interact with GAAPs and IRE1. Single pas2 and gaap1/gaap2pas2 double mutants exhibited increased seedling damage and impaired UPR response under chronic ER stress. Site mutation combined with genetic analysis revealed that the role of PAS2 in resisting ER stress depended on its VLCFA synthesis domain. VLCFA contents were upregulated under ER stress, which required GAAPs. Exogenous VLCFAs partially restored the defect in UPR upregulation caused by PAS2 or GAAP mutations under chronic ER stress. These findings demonstrate that the association of PAS2 with GAAPs confers plant resistance to ER stress by regulating VLCFA synthesis and the UPR. This provides a basis for further studies on the connection between lipids and cell fate decisions under stress.