Conserved class B GPCR activation by a biased intracellular agonist.

Class B G-protein-coupled receptors (GPCRs), including glucagon-like peptide 1 receptor (GLP1R) and parathyroid hormone 1 receptor (PTH1R), are important drug targets1-5. Injectable peptide drugs targeting these receptors have been developed, but orally available small-molecule drugs remain under development6,7. Here we report the high-resolution structure of human PTH1R in complex with the stimulatory G protein (Gs) and a small-molecule agonist, PCO371, which reveals an unexpected binding mode of PCO371 at the cytoplasmic interface of PTH1R with Gs. The PCO371-binding site is totally different from all binding sites previously reported for small molecules or peptide ligands in GPCRs. The residues that make up the PCO371-binding pocket are conserved in class B GPCRs, and a single alteration in PTH2R and two residue alterations in GLP1R convert these receptors to respond to PCO371. Functional assays reveal that PCO371 is a G-protein-biased agonist that is defective in promoting PTH1R-mediated arrestin signalling. Together, these results uncover a distinct binding site for designing small-molecule agonists for PTH1R and possibly other members of the class B GPCRs and define a receptor conformation that is specific only for G-protein activation but not arrestin signalling. These insights should facilitate the design of distinct types of class B GPCR small-molecule agonist for various therapeutic indications.

Spatial proteomic landscape of primary and relapsed hepatocellular carcinoma reveals immune escape characteristics in early relapse.

BACKGROUND AIMS: Surgical resection serves as the principal curative strategy for hepatocellular carcinoma (HCC), yet the incidence of postoperative recurrence remains alarmingly high. However, the spatial molecular structural alterations contributing to postoperative recurrence in HCC are still poorly understood. APPROACH RESULTS: We employed imaging mass cytometry to profile the in-situ expression of 33 proteins within 358,729 single cells of 92 clinically annotated surgical specimens from 46 patients who were treated with surgical resections for primary and relapsed tumors. We revealed the recurrence progression of HCC was governed by the dynamic spatial distribution and functional interplay of diverse cell types across adjacent normal, tumor margin, and intratumor regions. Our exhaustive analyses revealed an aggressive, immunosuppression-related spatial ecosystem in relapsed HCC. Additionally, we illustrated the prominent implications of the TME of tumor margins in association with relapse HCC. Moreover, we identified a novel subpopulation of dendritic cells (PDL1+CD103+ DCs) enriched in the peritumoral area that correlated with early postoperative recurrence, which was further validated in an external cohort. Through the analysis of scRNA-seq data, we found the interaction of PDL1+CD103+ DCs with regulatory T cells and exhausted T cells enhanced immunosuppression and immune escape via multiple ligand-receptor pathways. CONCLUSIONS: We comprehensively depicted the spatial landscape of single-cell dynamics and multicellular architecture within primary and relapsed HCC. Our findings highlight spatial organization is a prominent determinant of HCC recurrence and provide a valuable insight into the immune evasion mechanisms driving recurrence.

STING-targeted PET tracer for early assessment of tumor immunogenicity in colorectal cancer after chemotherapy.

PURPOSE: To optimize chemotherapy regimens and improve the effectiveness of chemotherapy combined with immunotherapy, a PET tracer specifically targeting the stimulator of interferon genes (STING), denoted as [18F]FBTA was used to monitor the early changes in tumor immunogenicity after chemotherapy in colorectal cancer (CRC) mice. METHODS: The toluene sulfonate precursor was labeled with 18F to produce the STING targeted probe-[18F]FBTA. [18F]FBTA-PET imaging and biodistribution were performed using CRC mice treated with oxaliplatin (OXA) or cisplatin (CDDP). CRC mice were also treated with low (CDDP-LD: 1 mg/kg) or medium (CDDP-MD: 2.5 mg/kg) doses of CDDP, and subjected to PET imaging and biodistribution. The effects of different chemotherapeutic agents and different doses of CDDP on tumor innate immunity were verified by flow cytometry and immunohistochemistry. RESULTS: PET imaging of CRC mice exhibited notably enhanced tumor uptake in the early phase of chemotherapy with treatment with OXA (3.09 ± 0.25%ID/g) and CDDP (4.01 ± 0.18%ID/g), especially in the CDDP group. The PET-derived tumor uptake values have strong correlations with STING immunohistochemical score. Flow cytometry showed both agents led to DCs and macrophages infiltration in tumors. Compared with OXA, CDDP treatment recruits more DCs and macrophages in CRC tumors. Both CDDP-LD and CDDP-MD treatment elevated uptake in CRC tumors, especially in CDDP-MD group. Immunohistochemistry and flow cytometry confirmed CDDP-MD treatment recruits more DCs and macrophages than CDDP-LD treatment. CONCLUSION: Overall, the STING-targeted tracer-[18F]FBTA was demonstrated to monitor early changes in tumor immunogenicity in CRC mice after chemotherapy. Besides, the STING-targeted strategy may help to select the appropriate chemotherapy regimen, including chemotherapeutic agents and doses, which further improve clinical decision making for combination immunotherapy after chemotherapy for CRC.

DDX24 Is Essential for Cell Cycle Regulation in Vascular Smooth Muscle Cells During Vascular Development via Binding to FANCA mRNA.

BACKGROUND: The DEAD-box family is essential for tumorigenesis and embryogenesis. Previously, we linked the malfunction of DDX (DEAD-box RNA helicase)-24 to a special type of vascular malformation. Here, we aim to investigate the function of DDX24 in vascular smooth muscle cells (VSMCs) and embryonic vascular development. METHODS: Cardiomyocyte (CMC) and VSMC-specific Ddx24 knockout mice were generated by crossing Tagln-Cre mice with Ddx24flox/flox transgenic mice. The development of blood vessels was explored by stereomicroscope photography and immunofluorescence staining. Flow cytometry and cell proliferation assays were used to verify the regulation of DDX24 on the function of VSMCs. RNA sequencing and RNA immunoprecipitation coupled with quantitative real-time polymerase chain reaction were combined to investigate DDX24 downstream regulatory molecules. RNA pull-down and RNA stability experiments were performed to explore the regulation mechanism of DDX24. RESULTS: CMC/VSMC-specific Ddx24 knockout mice died before embryonic day 13.5 with defects in vessel formation and abnormal vascular remodeling in extraembryonic tissues. Ddx24 knockdown suppressed VSMC proliferation via cell cycle arrest, likely due to increased DNA damage. DDX24 protein bound to and stabilized the mRNA of FANCA (FA complementation group A) that responded to DNA damage. Consistent with the function of DDX24, depletion of FANCA also impacted cell cycle and DNA repair of VSMCs. Overexpression of FANCA was able to rescue the alterations caused by DDX24 deficiency. CONCLUSIONS: Our study unveiled a critical role of DDX24 in VSMC-mediated vascular development, highlighting a potential therapeutic target for VSMC-related pathological conditions.

Targeting inflammatory macrophages rebuilds therapeutic efficacy of DOT1L inhibition in hepatocellular carcinoma.

Epigenetic reprogramming is a promising therapeutic strategy for aggressive cancers, but its limitations in vivo remain unclear. Here, we showed, in detailed studies of data regarding 410 patients with human hepatocellular carcinoma (HCC), that increased histone methyltransferase DOT1L triggered epithelial-mesenchymal transition-mediated metastasis and served as a therapeutic target for human HCC. Unexpectedly, although targeting DOT1L in vitro abrogated the invasive potential of hepatoma cells, abrogation of DOT1L signals hardly affected the metastasis of hepatoma in vivo. Macrophages, which constitute the major cellular component of the stroma, abrogated the anti-metastatic effect of DOT1L targeting. Mechanistically, NF-κB signal elicited by macrophage inflammatory response operated via a non-epigenetic machinery to eliminate the therapeutic efficacy of DOT1L targeting. Importantly, therapeutic strategy combining DOT1L-targeted therapy with macrophage depletion or NF-κB inhibition in vivo effectively and successfully elicited cancer regression. Moreover, we found that the densities of macrophages in HCC determined malignant cell DOT1L-associated clinical outcome of the patients. Our results provide insight into the crosstalk between epigenetic reprogramming and cancer microenvironments and suggest that strategies to influence the functional activities of inflammatory cells may benefit epigenetic reprogramming therapy.

A robust method for simultaneous determination of eight B vitamins in human serum by liquid chromatography tandem mass spectrometry.

The level of vitamin B group in human serum is an important index of human health. Among B vitamins, cyanocobalamin in serum is unstable and its content is extremely low. Rapid and simultaneous detection of multiple B vitamins including cyanocobalamin is a challenge. Herein, we have developed a rapid and stable method that can realize the determination of thiamine, riboflavin, nicotinamide, pantothenic acid, pyridoxic acid, biotin, 5-methyltetrahydrofolate, and cyanocobalamin simultaneously in 6 min. The method was established based on protein precipitation with methanol and then chromatographic separation was achieved using Waters acquity ultra-high-performance liquid chromatography high strength silica T3 column, which was stable and sensitive especially for cyanocobalamin. Limit of quantification, precision, trueness, and matrix effect were validated according to the European Medicines Agency and United States Food and Drug guidelines and Clinical and Laboratory Standards Institute guidelines on bioanalytical method. The limit of quantification for thiamine, riboflavin, nicotinamide, pantothenic acid, pyridoxic acid, biotin, 5-methyltetrahydrofolate, and cyanocobalamin was 0.4, 0.4, 0.8, 2.0, 0.4, 0.1, 0.4, and 0.04 ng/mL separately, respectively. Intra- and interday precisions were 1.1%-12.4% and 2.0%-13.5%, respectively. The relative errors were between 0.3% and 13.3%, and the matrix effects were between 2.6% and 10.4%.

Computer-aided diagnosis of distal metastasis in non-small cell lung cancer by low-dose CT based radiomics and deep learning signatures.

BACKGROUND: This study aimed to develop and validate radiomics and deep learning (DL) signatures for predicting distal metastasis (DM) of non-small cell lung cancer (NSCLC) in low-dose computed tomography (LDCT). METHODS: Images and clinical data were retrospectively collected for 381 NSCLC patients and prospectively collected for 114 patients at the Fifth Affiliated Hospital of Sun Yat-Sen University. Additionally, we enrolled 179 patients from the Jiangmen Central Hospital to externally validate the signatures. Machine-learning algorithms were employed to develop radiomics signature while the DL signature was developed using neural architecture search. The diagnostic efficiency was primarily quantified with the area under receiver operating characteristic curve (AUC). We interpreted the reasoning process of the radiomics signature and DL signature by radiomics voxel mapping and attention weight tracking. RESULTS: A total of 674 patients with pathologically-confirmed NSCLC were included from two institutions, with 143 of them having DM. The radiomics signature achieved AUCs of 0.885, 0.854, and 0.733 in the internal validation, prospective validation, and external validation while those for DL signature were 0.893, 0.786, and 0.780. The proposed signatures achieved a promising performance in predicting the DM of NSCLC and outperformed the approaches proposed in previous studies. Interpretability analysis revealed that both radiomics and DL signatures could detect the variations among voxels inside tumors, which helped in identifying the DM of NSCLC. CONCLUSIONS: Our study demonstrates the potential of LDCT-based radiomics and DL signatures for predicting DM in NSCLC. These signatures could help improve lung cancer screening regarding further diagnostic tests and treatment strategies.

Clinical Characteristics of Coronavirus Disease 2019 in China.

BACKGROUND: Since December 2019, when coronavirus disease 2019 (Covid-19) emerged in Wuhan city and rapidly spread throughout China, data have been needed on the clinical characteristics of the affected patients. METHODS: We extracted data regarding 1099 patients with laboratory-confirmed Covid-19 from 552 hospitals in 30 provinces, autonomous regions, and municipalities in mainland China through January 29, 2020. The primary composite end point was admission to an intensive care unit (ICU), the use of mechanical ventilation, or death. RESULTS: The median age of the patients was 47 years; 41.9% of the patients were female. The primary composite end point occurred in 67 patients (6.1%), including 5.0% who were admitted to the ICU, 2.3% who underwent invasive mechanical ventilation, and 1.4% who died. Only 1.9% of the patients had a history of direct contact with wildlife. Among nonresidents of Wuhan, 72.3% had contact with residents of Wuhan, including 31.3% who had visited the city. The most common symptoms were fever (43.8% on admission and 88.7% during hospitalization) and cough (67.8%). Diarrhea was uncommon (3.8%). The median incubation period was 4 days (interquartile range, 2 to 7). On admission, ground-glass opacity was the most common radiologic finding on chest computed tomography (CT) (56.4%). No radiographic or CT abnormality was found in 157 of 877 patients (17.9%) with nonsevere disease and in 5 of 173 patients (2.9%) with severe disease. Lymphocytopenia was present in 83.2% of the patients on admission. CONCLUSIONS: During the first 2 months of the current outbreak, Covid-19 spread rapidly throughout China and caused varying degrees of illness. Patients often presented without fever, and many did not have abnormal radiologic findings. (Funded by the National Health Commission of China and others.).

Radiomics Nomogram with Added Nodal Features Improves Treatment Response Prediction in Locally Advanced Esophageal Squamous Cell Carcinoma: A Multicenter Study.

OBJECTIVE: We aimed to develop and validate a radiomics nomogram and determine the value of radiomic features from lymph nodes (LNs) for predicting pathological complete response (pCR) to neoadjuvant chemoradiotherapy (NCRT) in patients with locally advanced esophageal squamous cell carcinoma (ESCC). METHODS: In this multicenter retrospective study, eligible participants who had undergone NCRT followed by radical esophagectomy were consecutively recruited. Three radiomics models (modelT, modelLN, and modelTLN) based on tumor and LN features, alone and combined, were developed in the training cohort. The radiomics nomogram was developed by incorporating the prediction value of the radiomics model and clinicoradiological risk factors using multivariate logistic regression, and was evaluated using the receiver operating characteristic curve, validated in two external validation cohorts. RESULTS: Between October 2011 and December 2018, 116 patients were included in the training cohort. Between June 2015 and October 2020, 51 and 27 patients from two independent hospitals were included in validation cohorts 1 and 2, respectively. The radiomics modelTLN performed better than the radiomics modelT for predicting pCR. The radiomics nomogram incorporating the predictive value of the radiomics modelTLN and heterogeneous after NCRT outperformed the clinicoradiological model, with an area under the curve (95% confidence interval) of 0.833 (0.765-0.894) versus 0.764 (0.686-0.833) [p = 0.088, DeLong test], 0.824 (0.718-0.909) versus 0.692 (0.554-0.809) [p = 0.012], and 0.902 (0.794-0.984) versus 0.696 (0.526-0.857) [p = 0.024] in all three cohorts. CONCLUSIONS: Radiomic features from LNs could provide additional value for predicting pCR in ESCC patients, and the radiomics nomogram provided an accurate prediction of pCR, which might aid treatment decision.

Preclinical evaluation of a novel EGFR&c-Met bispecific near infrared probe for visualization of esophageal cancer and metastatic lymph nodes.

PURPOSE: This study aimed to establish a near infrared fluorescent (NIRF) probe based on an EGFR&c-Met bispecific antibody for visualization of esophageal cancer (EC) and metastatic lymph nodes (mLNs). METHODS: EGFR and c-Met expression were assessed by immunohistochemistry. EGFR&c-Met bispecific antibody EMB01 was labeled with IRDye800cw. The binding of EMB01-IR800 was assessed by enzyme linked immunosorbent assay, flow cytometry, and immunofluorescence. Subcutaneous tumors, orthotopic tumors, and patient-derived xenograft (PDX) were established for in vivo fluorescent imaging. PDX models using lymph nodes with or without metastasis were constructed to assess the performance of EMB01-IR800 in differential diagnosis of lymph nodes. RESULTS: The prevalence of overexpressing EGFR or c-Met was significantly higher than single marker either in EC or corresponding mLNs. The bispecific probe EMB01-IR800 was successfully synthesized, with strong binding affinity. EMB01-IR800 showed strong cellular binding to both Kyse30 (EGFR overexpressing) and OE33 (c-Met overexpressing) cells. In vivo fluorescent imaging showed prominent EMB01-IR800 uptake in either Kyse30 or OE33 subcutaneous tumors. Likewise, EMB01-IR800 exhibited superior tumor enrichment in both thoracic orthotopic esophageal squamous cell carcinoma and abdominal orthotopic esophageal adenocarcinoma models. Moreover, EMB01-IR800 produced significantly higher fluorescence in patient-derived mLNs than in benign lymph nodes. CONCLUSION: This study demonstrated the complementary overexpression of EGFR and c-Met in EC. Compared to single-target probes, the EGFR&c-Met bispecific NIRF probe can efficiently depict heterogeneous esophageal tumors and mLNs, which greatly increased the sensitivity of tumor and mLN identification.

Ankylosing spondylitis PET imaging and quantifications via P2X7 receptor-targeting radioligand [18F]GSK1482160.

PURPOSE: Ankylosing spondylitis (AS) is a chronic inflammatory disease of the axial spine; however, the quantitative detection of inflammation in AS remains a challenge in clinical settings. We aimed to investigate the feasibility of using a specific P2X7R-targeting 18F-labeled tracer [18F]GSK1482160 for positron emission tomography (PET) imaging and the quantification of AS. METHODS: The radioligand [18F]GSK1482160 was obtained based on nucleophilic aliphatic substitution. Dynamic [18F]GSK1482160 and [18F]FDG micro-PET/CT imaging were performed on AS mice (n = 8) and age-matched controls (n = 8). Tracer kinetics modeling was performed using Logan's graphical arterial input function analysis to quantify the in vivo expression of P2X7R. The post-PET tissues were collected for hematoxylin-eosin (H&E), immunohistochemical (IHC), and immunofluorescence (IF) staining. RESULTS: [18F]GSK1482160 PET/CT imaging revealed that the specific binding in the ankle joint and sacroiliac joint (SIJ) of the AS at 8 weeks group (BPNDankle-AS-8W (non-displaceable binding potential of the ankle) 3.931 ± 0.74; BPND SIJ-AS-8W (BPBD of the SIJ) 4.225 ± 0.84) were significantly higher than the controls at 8 weeks group (BPNDankle-Ctr-8W 0.325 ± 0.15, BPNDSJJ-Ctr-8W 0.319 ± 0.17) respectively, and the AS at 14 weeks group (BPNDankle-AS-14W 12.212 ± 2.25; BPNDSJJ-AS-14W 13.389 ± 3.60) were significantly higher than the controls at 14 weeks group (BPNDankle-Ctr-14W 0.204 ± 0.16, BPNDSJJ-Ctr-14W 0.655 ± 0.35) respectively. The four groups had no significant difference in the [18F]FDG uptake of ankle and SIJ. IHC and IF staining revealed that the overexpression of P2X7R was colocalized with activated macrophages from the ankle synovium and spinal endplate in mice with AS, indicating that quantification of P2X7R may contribute to the understanding of the pathogenesis of inflammation in human AS. CONCLUSION: This study developed a novel P2X7R-targeting PET tracer [18F]GSK1482160 to detect the expression of P2X7R in AS mouse models and provided powerful non-invasive PET imaging and quantification for AS.

Application of Dynamic 18F-FDG PET/CT for Distinguishing Intrapulmonary Metastases from Synchronous Multiple Primary Lung Cancer.

It has been a big challenge to distinguish synchronous multiple primary lung cancer (sMPLC) from primary lung cancer with intrapulmonary metastases (IPM). We aimed to assess the clinical application of dynamic 18F-FDG PET/CT in patients with multiple lung cancer nodules. We enrolled patients with multiple pulmonary nodules who had undergone dynamic 18F-FDG PET/CT and divided them into sMPLC and IPM groups based on comprehensive features. The SUVmax, fitted K i value based on dynamic scanning, and corresponding maximum diameter (D max) from the two largest tumors were determined in each patient. We determined the absolute between-tumor difference of SUVmax/D max and K i /D max (ΔSUVmax/D max; ΔK i /D max) and assessed the between-group differences. Further, the diagnostic accuracy was evaluated by ROC analysis and the correlation between ΔSUVmax/D max and ΔK i /Dmax from all groups was determined. There was no significant difference for ΔSUVmax/D max between the IPM and sMPLC groups, while the IPM group had a significantly higher ΔK i /Dmax than the sMPLC group. The AUC of ΔK i /D max for differentiating sMPLC from IPM was 0.80 (cut-off value of K i = 0.0059, sensitivity 79%, specificity 75%, p < 0.001). There was a good correlation (Pearson r = 0.91, 95% CI: 0.79-0.96, p < 0.0001) between ΔSUVmax/D max and ΔK i /D max in the IPM group but not in the sMPLC group (Pearson r = 0.45, p > 0.05). Dynamic 18F-FDG PET/CT could be a useful tool for distinguishing sMPLC from IPM. K i calculation based on Patlak graphic analysis could be more sensitive than SUVmax in discriminating IPM from sMPLC in patients with multiple lung cancer nodules.

A Positron Emission Tomography Tracer Targeting the S2 Subunit of SARS-CoV-2 in Extrapulmonary Infections.

Tracking the pathogen of coronavirus disease 2019 (COVID-19) in live subjects may help estimate the spatiotemporal distribution of SARS-CoV-2 infection in vivo. This study developed a positron emission tomography (PET) tracer of the S2 subunit of spike (S) protein for imaging SARS-CoV-2. A pan-coronavirus inhibitor, EK1 peptide, was synthesized and radiolabeled with copper-64 after being conjugated with 1,4,7-triazacyclononane-1,4,7-triyl-triacetic acid (NOTA). The in vitro stability tests indicated that [64Cu]Cu-NOTA-EK1 was stable up to 24 h both in saline and in human serum. The binding assay showed that [64Cu]Cu-NOTA-EK1 has a nanomolar affinity (Ki = 3.94 ± 0.51 nM) with the S-protein of SARS-CoV-2. The cell uptake evaluation used HEK293T/S+ and HEK293T/S- cell lines that showed that the tracer has a high affinity with the S-protein on the cellular level. For the in vivo study, we tested [64Cu]Cu-NOTA-EK1 in HEK293T/S+ cell xenograft-bearing mice (n = 3) and pseudovirus of SARS-CoV-2-infected HEK293T/ACE2 cell bearing mice (n = 3). The best radioactive xenograft-to-muscle ratio (X/Nxenograft 8.04 ± 0.99, X/Npseudovirus 6.47 ± 0.71) was most evident 4 h postinjection. Finally, PET imaging in the surrogate mouse model of beta-coronavirus, mouse hepatic virus-A59 infection in C57BL/6 J mice showed significantly enhanced accumulation in the liver than in the uninfected mice (1.626 ± 0.136 vs 0.871 ± 0.086 %ID/g, n = 3, P < 0.05) at 4 h postinjection. In conclusion, our experimental results demonstrate that [64Cu]Cu-NOTA-EK1 is a potential molecular imaging probe for tracking SARS-CoV-2 in extrapulmonary infections in living subjects.

Cooperation of ATF4 and CTCF promotes adipogenesis through transcriptional regulation.

Adipogenesis is a multi-step process orchestrated by activation of numerous TFs, whose cooperation and regulatory network remain elusive. Activating transcription factor 4 (ATF4) is critical for adipogenesis, yet its regulatory network is unclarified. Here, we mapped genome-wide ATF4 binding landscape and its regulatory network by Chip-seq and RNA-seq and found ATF4 directly modulated transcription of genes enriching in fat cell differentiation. Motifs of TFs especially CTCF were found from ATF4 binding sites, suggesting a direct role of ATF4 in regulating adipogenesis associated with CTCF and other TFs. Deletion of CTCF attenuated adipogenesis while overexpression enhanced adipocyte differentiation, indicating CTCF is indispensable for adipogenesis. Intriguingly, combined analysis of Chip-seq data of these two TFs showed that ATF4 co-localized with CTCF in the promoters of key adipogenic genes including Cebpd and PPARg and co-regulated their transactivation. Moreover, ATF4 directly regulated CTCF expression and interacted with CTCF in differentiated 3T3-L1 cells. In vivo, downregulation of ATF4 suppressed the expression of CTCF, Cebpd, and PPARg, leading to reduced adipose tissue expansion in refeeding mice. Consistently, mRNA expression of ATF4 and CTCF was positively correlated with each other in human subcutaneous adipose tissue and inversely associated with BMI, indicating a possible involvement of these two TFs in adipose development. Taken together, our data propose for the first time that ATF4 and CTCF work cooperatively to control adipogenesis and adipose development via orchestrating transcription of adipogenic genes. Our findings reveal novel therapeutic targets in obesity treatment.

Critical role of PAFR/YAP1 positive feedback loop in cardiac fibrosis.

Aberrant activation of cardiac fibroblasts is the main cause and character of cardiac fibrosis, and inhibition of cardiac fibrosis becomes a promising treatment for cardiac diseases. Platelet-activating factor (PAF) and Hippo pathway is recently recognized as key signaling mechanisms in cardiovascular diseases. In this study we explored the potential roles of PAF and Hippo signaling pathway in cardiac fibrosis. Myocardial infarction (MI) was induced in mice by left anterior descending artery ligation. After 28 days, the mice were sacrificed, and the hearts were collected for analyses. We showed that PAF receptor (PAFR) and yes-associated protein 1 (YAP1, a key effector in the Hippo pathway) were significantly increased in the heart of MI mice. Increased expression of PAFR and YAP1 was also observed in angiotensin II (Ang II)-treated mouse cardiac fibroblasts. In mouse cardiac fibroblasts, forced expression of YAP1 increased cell viability, resulted in collagen deposition and promoted fibroblast-myofibroblast transition. We showed that PAF induced fibrogenesis through activation of YAP1 and promoted its nuclear translocation via interacting with PAFR, while YAP1 promoted the expression of PAFR by binding to and activating transcription factor TEAD1. More importantly, silencing PAFR or YAP1 by shRNA, or using transgenic mice to induce the conditional deletion of YAP1 in cardiac fibroblasts, impeded cardiac fibrosis and improved cardiac function in MI mice. Taken together, this study elucidates the role and mechanisms of PAFR/YAP1 positive feedback loop in cardiac fibrosis, suggesting a potential role of this pathway as novel therapeutic targets in cardiac fibrosis.

Hypoxic Hepatocellular Carcinoma Cells Acquire Arsenic Trioxide Resistance by Upregulating HIF-1α Expression.

BACKGROUND: Although arsenic trioxide (ATO) is used in the treatment of advanced hepatocellular carcinoma (HCC) in clinical trials, it is not satisfactory in terms of improving HCC patients' overall survival. Intratumoral hypoxia and overexpression of hypoxia-inducible factor-1α (HIF-1α) may result in ATO resistance and tumor progression. AIMS: We investigated the mechanisms involving HIF-1α expression and acquired ATO chemoresistance in HCC cells and mice. METHODS: The therapeutic effects of ATO in normoxic and hypoxic HCC cells were assessed using cell viability and apoptosis assays in vitro and a xenograft model in vivo. mRNA and protein expression of HIF-1α, P-glycoprotein, and VEGF were measured by qRT-PCR and western blotting. HIF-1α inhibition was performed to investigate the mechanism of ATO resistance. VEGF secretion was tested using ELISA and tube formation assays. RESULTS: Compared to normoxic cells, hypoxic HCC cells were more resistant to ATO, with higher IC50 values and less apoptosis, and upregulated HIF-1α protein expression, accompanied with the enhancement of P-glycoprotein and VEGF synthesis after ATO treatment. VEGF secretion was elevated in the supernatant of ATO-treated HCC cells, and this change can potentiate angiogenesis in vitro. HIF-1α inhibition attenuated ATO resistance and angiogenesis and promoted the anticancer effects of ATO both in vitro and in vivo by downregulating therapy-induced P-glycoprotein and VEGF overexpression. CONCLUSIONS: Hypoxic HCC cells acquire ATO resistance by upregulating HIF-1α levels; thus, combining ATO with a HIF-1α-targeting agent may lead to enhanced antitumor effects in HCC.

Near-infrared fluorescence imaging-guided lymphatic mapping in thoracic esophageal cancer surgery.

PURPOSE: Identifying the lymphatic drainage pathway is important for accurate lymph node (LN) dissection in esophageal cancer (EC). This study aimed to assess lymphatic drainage mapping in thoracic EC using near-infrared fluorescent (NIRF) imaging with indocyanine green (ICG) and identify its feasibility for intraoperative LN drainage visualization and dissection. METHODS: From November 2019 to August 2020, esophagectomy was performed using intraoperative NIRF navigation with ICG injected into the esophageal submucosa by endoscopy. All LNs were divided into four groups according to the NIRF status and presence of metastasis: NIRF+LN+, NIRF+LN-, NIRF-LN+, and NIRF-LN-. RESULTS: Regional LNs were detected in all 84 enrolled patients with thoracic EC. A total of 2164 LNs were removed, and the mean number of dissected LNs was 25.68 ± 12.00. NIRF+ LNs were observed in all patients and distributed at 19 LN stations, which formed lymphatic drainage maps. The top five LN stations of NIRF+ probability in upper thoracic EC were No. 7, 106ecR, 107, 1, and 106recL; in middle thoracic EC, they were No. 107, 7, 110, 1, and 105; and in lower thoracic EC, they were No. 107, 7, 110, 106recR, and 1. There were no cases of ICG-related adverse events or chylothorax. The 30-day mortality rate was 0%. Major complications included anastomotic fistula (7.14%), pneumonia (4.76%), pleural effusion (13.10%), atelectasis (3.75%), hoarseness (8.33%), and arrhythmia (4.76%). CONCLUSION: Regional LN mapping of thoracic EC was performed using ICG/NIRF imaging, which showed different preferred LN drainage stations in various anatomical locations of the thoracic esophagus. ICG/NIRF imaging is feasible for intraoperative LN drainage visualization and dissection. CLINICAL TRIAL REGISTRATION: The clinical trial registration number is NCT04173676 ( http://www. CLINICALTRIALS: gov/ ).

Loss-of-function Mutations K11E or E271K Lead to Novel Tumor Suppression, Implicate Nucleolar Helicase DDX24 Oncogenicity.

Purpose: Mutations (K11E or E271K) of DEAD-box RNA helicase 24 (DDX24) were related to multi-organ venous lymphatic malformation syndrome (MOVLD). However, the relationship between these mutations and DDX24-function still remains unknown. Understanding whether K11E and E271K cause "loss-of-function" or "gain-of-function" for DDX24 is significant for related diseases. DDX24 was reported to be related to tumors closely, thus this study aims to explore how K11E and E271K affect DDX24-function in tumor proliferation. Methods: Cell lines stably expressing wild-type DDX24, K11E-DDX24, E271K-DDX24, along with vector only based on Chinese hamster ovary cells (CHO) and Balb/c tumor-bearing mice models were constructed. Then immunofluorescence staining, proliferation assay and colony formation assay in vitro and 18F-FDG PET/CT-scan were performed. Finally, the tumor tissues were collected to perform transcriptome sequencing to predict the potential mechanism. Results: Contrasted with CHO-WT-DDX24, CHO-K11E-DDX24 or CHO-E271K-DDX24 showed a decreased number of nucleoli, a slower proliferation rate and a lower colony formation rate significantly. Moreover, mice, inoculated with CHO-K11E-DDX24 or CHO-E271K-DDX24 cells, showed lower tumor formation rate, slower tumor growth rate, better prognosis, reduced standard uptake value and Ki of glucose in subcutaneous tumors. Sequencing indicated CHO-K11E-DDX24 or CHO-E271K-DDX24 caused increasing expression of TNF or chemokines and alteration in immune-related signal pathways. Conclusion: K11E or E271K mutation could lead to "loss-of-function" of DDX24 in cell proliferation and tumor bearing mice, which may be acted by non-specific immune killing to inhibit tumor growth.

First Report of Red Leaf Midrib lesions on Sugarcane Caused by Lasiodiplodia theobromae in China.

Sugarcane (Saccharum officinarum) is an economically important crop and is extensively planted across China. In August 2020, leaf midribs with red lesions were observed on cultivar 'Yunzhe 081609' in Kaiyuan (103.27°E, 23.71°N), Yunnan, Southwestern China. In July to August 2021, similar symptoms were observed on cultivar 'Liucheng 05-136' in Hechi (108.48°E, 24.47°N), Guangxi, and on cultivars 'Yingyu 91-59' and 'Yunzhe 081609' in Lingcang (99.45°E, 23.33°N), Yunnan. Initially symptoms appeared as red spots on the leaf midribs, which gradually expanded, forming elongated red lesions. At high severity, the leaves broke and hung down. Disease incidence of leaves was estimated at 30 to 50% across the locations. To identify the etiology of this disease, three symptomatic leaves were collected from cultivars 'Liucheng 05-136', 'Yingyu 91-59', and 'Yunzhe 081609', respectively. Symptomatic leaf midribs were cut to small fragments (3 × 5 mm), surface sterilized with 70% ethanol for 30 s followed by 1% NaClO for 1 min, rinsed with sterilized distilled water three times, air dried on sterile filter paper, plated on potato dextrose agar (PDA), and incubated at 28°C in the dark. Ten isolates with similar morphological characteristics were obtained. Colonies on PDA were white to grayish-white with aerial mycelium growing initially upward and then forming clusters. After 10 days, mycelia turned to grayish black. Immature conidia were initially hyaline, aseptate, and ellipsoid. Mature conidia became dark brown, septate, longitudinal striate, and measured 21.2 to 25.8 × 11.4 to 16.4 µm (n = 30). Morphologically, the isolates were identified as Lasiodiplodia theobromae (Alves et al. 2008). For molecular identification, genomic DNA of four representative isolates (LTGX1, LTGX2, LTYN1 and LTYN2) was extracted using the Ezup Column Fungi Genomic DNA Purification kit. The internal transcribed spacer (ITS) region of rDNA, translation elongation factor 1-alpha (TEF-1α) gene, and ß-tubulin (TUB) gene were amplified with primer pairs ITS1/ITS4 for ITS, EF1-728F/EF1-986R for TEF-1α, and Bt2a/Bt2b for TUB, respectively (Glass and Donaldson 1995; Carbone and Kohn 1999; White et al. 1990), and then sequenced. The ITS (ON533336-ON533339), TEF-1α (ON939550-ON939553) and TUB (OP747306-OP747309) sequences were deposited in GenBank. BLAST searches showed >99% nucleotide identity to the sequences of ex-type isolate CBS 164.96 of L. theobromae (ITS, 99.8% to AY640255; TEF-1α, 99.9% to AY640258; TBU, 100% to EU673110). Phylogenetic analysis using maximum likelihood based on the combined ITS, TEF-1α, and TUB sequences of the isolates and reference sequences of Lasiodiplodia spp. downloaded from the GenBank indicated the isolates obtained in this study formed a clade strongly supported based on bootstrap values (100%) to the ex-type isolate CBS 164.96 sequences of L. theobromae. For pathogenicity tests, three healthy 6-month-old potted sugarcane leaf midribs of cultivar 'Yunzhe 081609' were wounded with a sterile needle, then inoculated using 8-mm mycelial agar plugs from a 10-day-old culture of strain LTYN1, and covered with wet cotton to maintain high relative humidity. Sterile PDA plugs were used as controls. Plants were placed in a greenhouse at 28 to 32°C. The test was conducted twice. Five days after inoculation, red lesions appeared on the inoculated leaf midribs. These symptoms were similar to those observed in the field. The leaves used for negative controls remained symptomless. The same fungus (L. theobromae) was re-isolated from all inoculated-symptomatic tissues; and isolates had the same morphological traits mentioned above. The DNA sequence data of these isolates was also similar than the original isolates. The association of L. theobromae with S. officinarum was recorded earlier in Cuba (Urtiaga, 1986), Myanmar (Thaung, 2008) and the Philippines (Reinking, 1919). Leaf midribs with red lesions caused by Colletotrichum falcatum has already been described around the world (Costa et al. 2021; Hossain et al. 2021; Xie et al. 2019). All together, this information indicates that L. theobromae is one of the causal agent of the red lesions symptoms on the sugarcane leaf midribs. To our knowledge, this is the first report of L. theobromae causing red lesions on leaf midribs of sugarcane in China. Further research will focus on developing management strategies to control this disease effectively.

Preclinical Evaluation of [64Cu]NOTA-CP01 as a PET Imaging Agent for Metastatic Esophageal Squamous Cell Carcinoma.

Targeting metastatic esophageal squamous cell carcinoma (ESCC) has been a challenge in clinical practice. Emerging evidence demonstrates that C-X-C chemokine receptor 4 (CXCR4) highly expresses in ESCC and plays a pivotal role in the process of tumor metastasis. We developed a copper-64 (t1/2 = 12.7 h, 19% beta+) labeling route of NOTA-CP01 derived from LY2510924, a cyclopeptide-based CXCR4 potent antagonist, in an attempt to noninvasively visualize CXCR4 expression in metastatic ESCC. Precursor NOTA-CP01 was designed by modifying the C-terminus of LY2510925 with bis-t-butyl NOTA via a butane-1,4-diamine linker. The radiolabeling process was finished within 15 min with high radiochemical yield (>95%), radiochemical purity (>99%), and specific activity (10.5-21 GBq/µmol) (non-decay-corrected). The in vitro solubility and stability tests revealed that [64Cu]NOTA-CP01 had a high water solubility (log P = -3.44 ± 0.12, n = 5) and high stability in saline and fetal bovine serum. [64Cu]NOTA-CP01 exhibited CXCR4-specific binding with a nanomolar affinity (IC50 = 1.61 ± 0.96 nM, Kd = 0.272 ± 0.14 nM) similar to that of the parental LY2510924. The in vitro cell uptake assay indicated that the [64Cu]NOTA-CP01-selective accumulation in EC109 cells was CXCR4-specific. Molecular docking of the CXCR4/NOTA-CP01 complex suggested that the Lys, Arg, and NOTA of this ligand have a strong polar interaction with the key residues of CXCR4, which explains the tight affinity of [64Cu]NOTA-CP01 for CXCR4. To test the target engagement in vivo, prolonged-time positron emission computed tomography (PET) imaging was performed at 0.5, 4, 6, 8, 12, 16, and 24 h postinjection of [64Cu]NOTA-CP01 to the EC109 tumor-bearing mice. The EC109 tumors were most visible with high contrast to the contralateral background at 6 h postinjection. The tracer revealed receptor-specific tumor accumulation, which was illustrated by effective blocking via coinjection with a blocking dose of LY2510924. Quantification analysis of the prolonged-time images showed that there was obvious radioactivity accumulation in the tumor (1.27 ± 0.19%ID/g) with the best tumor-to-blood ratio (4.79 ± 0.06) and tumor-to-muscle ratio (15.44 ± 2.94) at 6 h postinjection of the probe. The immunofluorescence and immunohistochemistry confirmed the positive expression of CXCR4 in the EC109 tumor and ESCC and metastatic lymph nodes of patients, respectively. We concluded that [64Cu]NOTA-CP01 possessed a very high target engagement for CXCR4-positive ESCC and could be a potential candidate in the clinical detection of metastatic ESCC.