Nanoparticle Vaccines Based on the Receptor Binding Domain (RBD) and Heptad Repeat (HR) of SARS-CoV-2 Elicit Robust Protective Immune Responses.

Various vaccine strategies have been proposed in response to the global COVID-19 pandemic, each with unique strategies for eliciting immune responses. Here, we developed nanoparticle vaccines by covalently conjugating the self-assembled 24-mer ferritin to the receptor binding domain (RBD) and/or heptad repeat (HR) subunits of the Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) spike (S) protein. Compared to monomer vaccines, nanoparticle vaccines elicited more robust neutralizing antibodies and cellular immune responses. RBD and RBD-HR nanoparticle vaccinated hACE2 transgenic mice vaccinated with RBD and/or RBD-HR nanoparticles exhibited reduced viral load in the lungs after SARS-CoV-2 challenge. RBD-HR nanoparticle vaccines also promoted neutralizing antibodies and cellular immune responses against other coronaviruses. The nanoparticle vaccination of rhesus macaques induced neutralizing antibodies, and T and B cell responses prior to boost immunization; these responses persisted for more than three months. RBD- and HR-based nanoparticles thus present a promising vaccination approach against SARS-CoV-2 and other coronaviruses.

The utility of P-I-R classification in predicting the on-treatment histological and clinical outcomes of patients with hepatitis B and advanced liver fibrosis.

BACKGROUND AND AIMS: The predominantly progressive, indeterminate, and predominantly regressive (P-I-R) classification extends beyond staging and provides information on dynamic changes of liver fibrosis. However, the prognostic implication of P-I-R classification is not elucidated. Therefore, in the present research, we investigated the utility of P-I-R classification in predicting the on-treatment clinical outcomes. APPROACH AND RESULTS: In an extension study on a randomized controlled trial, we originally enrolled 1000 patients with chronic hepatitis B and biopsy-proven histological significant fibrosis, and treated them for more than 7 years with entecavir-based therapy. Among the 727 patients with a second biopsy at treatment week 72, we compared P-I-R classification and Ishak score changes in 646 patients with adequate liver sections for the histological evaluation. Progressive, indeterminate, and regressive cases were observed in 70%, 17%, and 13% of patients before treatments and 20%, 14%, and 64% after 72-week treatment, respectively, which could further differentiate the histological outcomes of patients with stable Ishak scores. The 7-year cumulative incidence of HCC was 1.5% for the regressive cases, 4.3% for the indeterminate cases, and 22.8% for the progressive cases ( p <0.001). After adjusting for age, treatment regimen, platelet counts, cirrhosis, Ishak fibrosis score changes, and Laennec staging, the posttreatment progressive had a HR of 17.77 (vs. posttreatment regressive; 95% CI: 5.55-56.88) for the incidence of liver-related events (decompensation, HCC, and death/liver transplantation). CONCLUSIONS: The P-I-R classification can be a meaningful complement to the Ishak fibrosis score not only in evaluating the histological changes but also in predicting the clinical outcomes.

MicroRNA-9 promotes axon regeneration of mauthner-cell in zebrafish via her6/ calcium activity pathway.

MicroRNA (miRNA), functioning as a post-transcriptional regulatory element, plays a significant role in numerous regulatory mechanisms and serves as a crucial intrinsic factor influencing axon regeneration. Prior investigations have elucidated the involvement of miRNA-9 in various processes, however, its specific contribution to axon regeneration in the central nervous system (CNS) remains uncertain. Hence, the zebrafish Mauthner axon regeneration model was employed to manipulate the expression of miRNA-9 in single cells, revealing that upregulation of miRNA-9 facilitated axon regeneration. Additionally, her6, a downstream target gene of miRNA-9, was identified as a novel gene associated with axon regeneration. Suppression of her6 resulted in enhanced Mauthner axon regeneration, as evidenced by the significantly improved regenerative capacity observed in her6 knockout zebrafish. In addition, modulation of her6 expression affects intracellular calcium levels in neurons and promoting her6 expression leads to a decrease in calcium levels in vivo using the new NEMOf calcium indicator. Moreover, the administration of the neural activity activator, pentylenetetrazol (PTZ) partially compensated for the inhibitory effect of her6 overexpression on the calcium level and promoted axon regeneration. Taken together, our study revealed a role for miRNA-9 in the process of axon regeneration in the CNS, which improved intracellular calcium activity and promoted axon regeneration by inhibiting the expression of downstream target gene her6. In our study, miRNA-9 emerged as a novel and intriguing target in the intricate regulation of axon regeneration and offered compelling evidence for the intricate relationship between calcium activity and the facilitation of axon regeneration.

Macrophage KLF15 prevents foam cell formation and atherosclerosis via transcriptional suppression of OLR-1.

BACKGROUND: Macrophage-derived foam cells are a hallmark of atherosclerosis. Scavenger receptors, including lectin-like oxidized low-density lipoprotein (LDL) receptor-1 (OLR-1), are the principal receptors responsible for the uptake and modification of LDL, facilitating macrophage lipid load and the uptake of oxidized LDL by arterial wall cells. Krüppel-like factor 15 (KLF15) is a transcription factor that regulates the expression of genes by binding to the promoter during transcription. Therefore, this study aimed to investigate the precise role of macrophage KLF15 in atherogenesis. METHODS: We used two murine models of atherosclerosis: mice injected with an adeno-associated virus (AAV) encoding the Asp374-to-Tyr mutant version of human PCSK9, followed by 12 weeks on a high-fat diet (HFD), and ApoE-/-- mice on a HFD. We subsequently injected mice with AAV-KLF15 and AAV-LacZ to assess the role of KLF15 in the development of atherosclerosis in vivo. Oil Red O, H&E, and Masson's trichome staining were used to evaluate atherosclerotic lesions. Western blots and RT-qPCR were used to assess protein and mRNA levels, respectively. RESULTS: We determined that KLF15 expression was downregulated during atherosclerosis formation, and KLF15 overexpression prevented atherosclerosis progression. KLF15 expression levels did not affect body weight or serum lipid levels in mice. However, KLF15 overexpression in macrophages prevented foam cell formation by reducing OLR-1-meditated lipid uptake. KLF15 directly targeted and transcriptionally downregulated OLR-1 levels. Restoration of OLR-1 reversed the beneficial effects of KLF15 in atherosclerosis. CONCLUSION: Macrophage KLF15 transcriptionally downregulated OLR-1 expression to reduce lipid uptake, thereby preventing foam cell formation and atherosclerosis. Thus, our results suggest that KLF15 is a potential therapeutic target for atherosclerosis.

Engeletin alleviates depressive-like behaviours by modulating microglial polarization via the LCN2/CXCL10 signalling pathway.

Microglial polarization and associated inflammatory activity are the key mediators of depression pathogenesis. The natural Smilax glabra rhizomilax derivative engeletin has been reported to exhibit robust anti-inflammatory activity, but no studies to date have examined the mechanisms through which it can treat depressive symptoms. We showed that treatment for 21 days with engeletin significantly alleviated depressive-like behaviours in chronic stress social defeat stress (CSDS) model mice. T1-weighted imaging (T1WI), T2-weighted imaging (T2WI) imaging revealed no significant differences between groups, but the bilateral prefrontal cortex of CSDS mice exhibited significant increases in apparent diffusion coefficient and T2 values relative to normal control mice, with a corresponding reduction in fractional anisotropy, while engeletin reversed all of these changes. CSDS resulted in higher levels of IL-1ß, IL-6, and TNF-a production, enhanced microglial activation, and greater M1 polarization with a concomitant decrease in M2 polarization in the mPFC, whereas engeletin treatment effectively abrogated these CSDS-related pathological changes. Engeletin was further found to suppress the LCN2/C-X-C motif chemokine ligand 10 (CXCL10) signalling axis such that adeno-associated virus-induced LCN2 overexpression ablated the antidepressant effects of engeletin and reversed its beneficial effects on the M1/M2 polarization of microglia. In conclusion, engeletin can alleviate CSDS-induced depressive-like behaviours by regulating the LCN2/CXCL10 pathway and thereby altering the polarization of microglia. These data suggest that the antidepressant effects of engeletin are correlated with the polarization of microglia, highlighting a potential avenue for future design of antidepressant strategies that specifically target the microglia.

Metabolic pathway-based subtyping reveals distinct microenvironmental states associated with diffuse large B-cell lymphoma outcomes.

Diffuse large B-cell lymphoma (DLBCL) is a biologically and clinically heterogeneous disease that requires personalized clinical treatment. Assigning patients to different risk categories and cytogenetic abnormality and genetic mutation groups has been widely applied for prognostic stratification of DLBCL. Increasing evidence has demonstrated that dysregulated metabolic processes contribute to the initiation and progression of DLBCL. Metabolic competition within the tumor microenvironment is also known to influence immune cell metabolism. However, metabolism- and immune-related stratification has not been established. Here, 1660 genes involved in 84 metabolic pathways were selected and tested to establish metabolic clusters (MECs) of DLBCL. MECs established based on independent lymphoma datasets distinguished different survival outcomes. The CIBERSORT algorithm and EcoTyper were applied to quantify the relative abundance of immune cell types and identify variation in cell states for 13 lineages comprising the tumor micro environment among different MECs, respectively. Functional characterization showed that MECs were an indicator of the immune microenvironment and correlated with distinctive mutational characteristics and oncogenic signaling pathways. The novel immune-related MECs exhibited promising clinical prognostic value and potential for informing DLBCL treatment decisions.

Association between non-high-density lipoprotein cholesterol to high-density lipoprotein cholesterol ratio (NHHR) and kidney stone: evidence from NHANES 2007-2018.

BACKGROUND: As an innovative lipid parameter, NHHR (the ratio of non-high-density lipoprotein cholesterol to high-density lipoprotein cholesterol) can serve as a valuable tool for assessing cardiovascular disease risk. Nevertheless, the relationship between NHHR and the risk of kidney stones remains unexplored. METHODS: A cross-sectional survey utilized data from the National Health and Population Survey (NHANES) database in the United States spanning from 2007 to 2018. Distinct statistical analyses were applied, including weighted logistic regression, stratified and interaction analysis and restricted cubic spline curve (RCS) models, to examine the correlation between NHHR and the incidence of kidney stones. RESULTS: This analysis encompassed 24,664 participants, with 9.63% reporting incidents of kidney stones. Following multivariate logistic regression and comprehensive adjustments, participants in NHHR quartile 4 (OR 1.34; 95% CI 1.12, 1.60, P < 0.01) exhibited a significantly increased risk of kidney stones compared to those in NHHR quartile 1 (Q1). The RCS result further illustrated a non-linear correlation between NHHR and the incidence of kidney stones. The result of subgroup analysis manifested that participants without diabetes had a higher risk of kidney stones when measured high NHHR levels compared those with diabetes (p for interaction < 0.05). CONCLUSION: Elevated NHHR levels were found to be associated with an increased risk of kidney stones. Based on these findings, NHHR appears to be a promising predictive indicator for the occurrence of kidney stones.

The ORF8 protein of SARS-CoV-2 mediates immune evasion through down-regulating MHC-Ι.

COVID-19, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has become a global pandemic and has claimed over 2 million lives worldwide. Although the genetic sequences of SARS-CoV and SARS-CoV-2 have high homology, the clinical and pathological characteristics of COVID-19 differ significantly from those of SARS. How and whether SARS-CoV-2 evades (cellular) immune surveillance requires further elucidation. In this study, we show that SARS-CoV-2 infection leads to major histocompability complex class Ι (MHC-Ι) down-regulation both in vitro and in vivo. The viral protein encoded by open reading frame 8 (ORF8) of SARS-CoV-2, which shares the least homology with SARS-CoV among all viral proteins, directly interacts with MHC-Ι molecules and mediates their down-regulation. In ORF8-expressing cells, MHC-Ι molecules are selectively targeted for lysosomal degradation via autophagy. Thus, SARS-CoV-2-infected cells are much less sensitive to lysis by cytotoxic T lymphocytes. Because ORF8 protein impairs the antigen presentation system, inhibition of ORF8 could be a strategy to improve immune surveillance.

A novel member of miR169 family negatively regulates maize resistance against Bipolaris maydis.

MicroRNAs (miRNAs) have been confirmed to play important roles in plant defense response. However, the key maize miRNAs involved in the defense response against Bipolaris maydis are very limited. In this study, a novel member of the miR169 family in response to B. maydis, named zma-miR169s, was discovered and investigated. The expression levels of pre-miR169s and zma-miR169s were significantly repressed during B. maydis infection. CRISPR/Cas9-induced zma-miR169s mutant exhibited more resistance against B. maydis, whereas overexpression zma-miR169s enhanced susceptibility, supporting that zma-miR169s might play a negative role in maize resistance. Moreover, RNA-seq and GO analysis showed that differentially expressed genes were highly enriched in the oxidation-reduction process and plant hormone pathway. Hence, reactive oxygen species (ROS) and plant hormone levels were further investigated. ROS detection confirmed that zma-miR169s mutant accumulated more ROS, while less ROS was detected in transgenic maize OE-miR169s. Furthermore, more remarkable changes in PR-1 expression levels and salicylic acid (SA) contents were detected in zma-miR169s mutant compared to wild-type and transgenic maize during B. maydis infection. Additionally, nuclear transcription factors (NF-YA1 and NF-YA13) were identified as targets regulated by zma-miR169s through the agrobacterium-mediated transient expression method. Overexpression of ZmNF-YA13 enhanced Arabidopsis resistance to Pseudomonas syringae pv. tomato DC3000. Taken together, our results suggest that zma-miR169s negatively regulate maize defense responses by influencing ROS accumulation and the SA-dependent signaling pathway.

CDKN2B-AS1 mediates proliferation and migration of vascular smooth muscle cells induced by insulin.

Excessive proliferation and migration of vascular smooth muscle cells (VSMCs) contribute to the intimal hyperplasia in type 2 diabetes mellitus (T2DM) patients after percutaneous coronary intervention. We aimed to investigate the role of lncRNA cyclin-dependent kinase inhibitor 2B antisense RNA 1 (CDKN2B-AS1) in VSMC proliferation and migration, as well as the underlying mechanism. T2DM model mice with carotid balloon injury were used in vivo and mouse aortic vascular smooth muscle cells (MOVAS) stimulated by insulin were used in vitro to assess the role of CDKN2B-AS1 in VSMC proliferation and migration following vascular injury in T2DM state. To investigate cell viability and migration, MTT assay and Transwell assay were conducted. To elucidate the underlying molecular mechanisms, the methylation-specific polymerase chain reaction, RNA immunoprecipitation, RNA-pull down, co-immunoprecipitation, and chromatin immunoprecipitation were performed. In vivo, CDKN2B-AS1 was up-regulated in common carotid artery tissues. In vitro, insulin treatment increased CDKN2B-AS1 level, enhanced MOVAS cell proliferation and migration, while the promoting effect was reversed by CDKN2B-AS1 knockdown. CDKN2B-AS1 forms a complex with enhancer of zeste homolog 2 (EZH2) and DNA methyltransferase (cytosine-5) 1 (DNMT1) to regulate smooth muscle 22 alpha (SM22α) methylation levels. In insulin-stimulated cells, SM22α knockdown abrogated the inhibitory effect of CDKN2B-AS1 knockdown on cell viability and migration. Injection of lentivirus-sh-CDKN2B-AS1 relieved intimal hyperplasia in T2DM mice with carotid balloon injury. Up-regulation of CDKN2B-AS1 induced by insulin promotes cell proliferation and migration by targeting SM22α through forming a complex with EZH2 and DNMT1, thereby aggravating the intimal hyperplasia after vascular injury in T2DM.

Chemoradiotherapy for untreated Masaoka-Koga stage IVB thymic carcinoma: a single-center retrospective study.

BACKGROUND: Thymic carcinoma (TC) is a rare type of a malignant tumor. The optimal treatment for Masaoka-Koga stage IVB TC patients is controversial due to the rarity of the disease. Chemotherapy is still the preferred option, but the outcomes are unsatisfactory. Whether radiotherapy combined with chemotherapy could improve prognosis remains unclear. METHODS: Untreated stage IVB TC patients who have received first-line chemotherapy were included in the present study. The patients who have undergone surgery were excluded. The primary outcomes were objective response rate (ORR) and progression-free survival (PFS). RESULTS: Sixty-seven patients were included in the study. A total of 31 patients received chemoradiotherapy (ChemoRT cohort), and the remaining 36 patients only received chemotherapy (Chemo cohort). The median follow-up period was 40.3 months. The ORR for the ChemoRT and Chemo cohorts was 61.3 and 27.8%, respectively (P = 0.006). Furthermore, PFS (P = 0.003) and OS (P = 0.046) were significantly superior in the ChemoRT cohort. Radiotherapy maintained a significant favorable effect on PFS in multivariate analysis (P = 0.014), but the effect on OS was insignificant (P = 0.249). There was no advantage in PFS (P = 0.302) in the ChemoRT cohort in patients who received < 4 cycles of chemotherapy. In contrast, radiotherapy significantly improved PFS (P = 0.005) in patients who received ≥ 4 cycles of chemotherapy. CONCLUSIONS: Chemoradiotherapy used as the first-line treatment improved ORR and PFS in Masaoka-Koga stage IVB TC patients. Patients receiving more cycles of chemotherapy may have a better chance to benefit from chemoradiotherapy.

Improved method to stratify lymphoma patients with risk of secondary central nervous system involvement: A multicenter retrospective analysis.

Secondary central nervous system (SCNS) involvement is an infrequent but universally fatal event in diffused large B-cell lymphoma. The occurrence rate of SCNS involvement is approximately 5% but comes with a poor prognosis ever after. However, existing risk models to predict the incidence and prognosis of these patients with SCNS involvement lack both efficiency and accuracy. Controversy has also been reported regarding which risk factor may best identify the population with a high CNS relapse rate. In this study, we retrospectively analyzed 831 patients with diffused large B-cell lymphoma, diagnosed between March 2008 and June 2018 in Tianjin Medical University Cancer Institute and Hospital, Beijing Cancer Hospital, and Cancer Hospital of The University of Chinese Academy of Science. Risk factors and nomogram were identified and established based on Fine and Gray's competing risk analysis. Among these patients, 55 (6.6%) of them eventually developed SCNS involvement. The 1- and 2-year incidence for SCNS involvement were 3.9% and 4.7%, respectively. The median time from de novo diagnosis to CNS relapse was 8 months, and the median overall survival of these patients was 28 months. Considering the competing mortality before SCNS involvement, Fine and Gray's competing risk model was performed to analyze the characteristics related to SCNS involvement, and identified risk factors as the multiple extranodal involvements, elevated LDH and AMC level, and the involvement of breast, adrenal gland/kidney, pulmonary and bone. Corresponding factors were integrated into the competing nomogram for SCNS involvement (c-index = 0.778). In conclusion, we present the first predictive nomogram to evaluate the risk to develop SCNS involvement in de novo DLBCL patients, which may help in both prognostic evaluation and clinical decision for this subgroup.

GPC3-IL7-CCL19-CAR-T primes immune microenvironment reconstitution for hepatocellular carcinoma therapy.

BACKGROUND: Chimeric antigen receptor (CAR)-T-cell therapy is a revolutionary treatment that has become a mainstay of advanced cancer treatment. Conventional glypican-3 (GPC3)-CAR-T cells have not produced ideal clinical outcomes in advanced hepatocellular carcinoma (HCC), and the mechanism is unclear. This study aims to investigate the clinical utility of novel GPC3-7-19-CAR-T cells constructed by our team and to explore the mechanisms underlying their antitumor effects. METHODS: We engineered a novel GPC3-targeting CAR including an anti-GPC3 scFv, CD3ζ, CD28 and 4-1BB that induces co-expression of IL-7 at a moderate level (500 pg/mL) and CCL19 at a high level (15000 pg /mL) and transduced it into human T cells. In vitro, cell killing efficacy was validated by the xCELLigence RTCA system, LDH nonradioactive cytotoxicity assay and was confirmed in primary HCC organoid models employing a 3D microfluid chip. In vivo, the antitumor capacity was assessed in a humanized NSG mouse xenograft model. Finally, we initiated a phase I clinical trial to evaluate the safety and effect of GPC3-7-19-CAR-T cells in the clinic. RESULTS: GPC3-7-19-CAR-T cells had 1.5-2 times higher killing efficiency than GPC3-CAR-T cells. The tumor formation rates in GPC3-7-19-CAR-T cells treated model were reduced (3/5vs.5/5), and the average tumor volumes were 0.74 cm3 ± 1.17 vs. 0.34 cm3 ± 0.25. Of note, increased proportion of CD4+ TEM and CD8+ TCM cells was infiltrated in GPC3-7-19-CAR-T cells group. GPC3-7-19-CAR-T cells obviously reversed the immunosuppressive tumor microenvironment (TME) by reducing polymorphonuclear (PMN)-myeloid-derived suppressor cells (MDSCs) and regulatory T (Treg) cells infiltration and recruiting more dendritic cells (DCs) to HCC xenograft tumor tissues. In one patient with advanced HCC, GPC3-7-19-CAR-T-cell treatment resulted in tumor reduction 56 days after intravenous infusion. CONCLUSIONS: In conclusion, GPC3-7-19-CAR-T cells achieved antitumor effects superior to those of conventional GPC3-CAR-T cells by reconstructing the TME induced by the dominant CD4+ TEM and CD8+ TCM cell subsets. Most importantly, GPC3-7-19-CAR-T cells exhibited good safety and antitumor efficacy in HCC patients in the clinic. ► Novel GPC3-7-19-CAR-T cells designed with mediate level of IL-7 secretion and high level of CCL19 secretion, which could recruit more mature DCs to assist killing on GPC3+HCCs. ►DC cells recruited by CCL19 could interact with CD4+ T cells and promote the differentiation of CD4+TEFF cells into CD4+TEM and CD8+TCM subsets, leading a better anti-tumor effect on GPC3+HCCs. ►Compared with conventional GPC3-CAR-T, GPC3-7-CCL19-CAR-T cells could reverse tumor immunosuppressive microenvironment by reducing PMN-MDSC and Treg cell infiltration.

Predicting the efficacy of immune checkpoint inhibitors monotherapy in advanced non-small cell lung cancer: a machine learning method based on multidimensional data.

Immunotherapy has improved the prognosis of patients with advanced non-small cell lung cancer (NSCLC), but only a small subset of patients achieved clinical benefit. The purpose of our study was to integrate multidimensional data using a machine learning method to predict the therapeutic efficacy of immune checkpoint inhibitors (ICIs) monotherapy in patients with advanced NSCLC. We retrospectively enrolled 112 patients with stage IIIB-IV NSCLC receiving ICIs monotherapy. The random forest (RF) algorithm was used to establish efficacy prediction models based on five different input datasets, including precontrast computed tomography (CT) radiomic data, postcontrast CT radiomic data, a combination of the two CT radiomic data, clinical data, and a combination of radiomic and clinical data. The 5-fold cross-validation was used to train and test the random forest classifier. The performance of the models was assessed according to the area under the curve (AUC) in the receiver operating characteristic curve. Survival analysis was performed to determine the difference in progression-free survival (PFS) between the two groups with the prediction label generated by the combined model. The radiomic model based on the combination of precontrast and postcontrast CT radiomic features and the clinical model produced an AUC of 0.92±0.04 and 0.89±0.03, respectively. By integrating radiomic and clinical features together, the combined model had the best performance with an AUC of 0.94±0.02. The survival analysis showed that the two groups had significantly different PFS times (p<0.0001). The baseline multidimensional data including CT radiomic and multiple clinical features were valuable in predicting the efficacy of ICIs monotherapy in patients with advanced NSCLC.

Deletion of the chd7 Hinders Oligodendrocyte Progenitor Cell Development and Myelination in Zebrafish.

CHD7, an encoding ATP-dependent chromodomain helicase DNA-binding protein 7, has been identified as the causative gene involved in CHARGE syndrome (Coloboma of the eye, Heart defects, Atresia choanae, Retardation of growth and/or development, Genital abnormalities and Ear abnormalities). Although studies in rodent models have expanded our understanding of CHD7, its role in oligodendrocyte (OL) differentiation and myelination in zebrafish is still unclear. In this study, we generated a chd7-knockout strain with CRISPR/Cas9 in zebrafish. We observed that knockout (KO) of chd7 intensely impeded the oligodendrocyte progenitor cells' (OPCs) migration and myelin formation due to massive expression of chd7 in oilg2+ cells, which might provoke upregulation of the MAPK signal pathway. Thus, our study demonstrates that chd7 is critical to oligodendrocyte migration and myelination during early development in zebrafish and describes a mechanism potentially associated with CHARGE syndrome.

[Panax notoginseng saponins improve monocrotaline-induced pulmonary arterial hypertension in rats by inhibiting ADAM10/Notch3 signaling pathway].

In this study, we investigated the effects of Panax notoginseng saponins (PNS) on pulmonary vascular remodeling and ADAM10/Notch3 pathway in pulmonary arterial hypertension (PAH). PAH rat model was established, and male Sprague Dawley (SD) rats were randomly divided into control group, monocrotaline (MCT) group and MCT+PNS group, with 10 rats in each group. Rats in the control group were intraperitoneally injected with equal volume of normal saline. Rats in the MCT group was injected intraperitoneally with 60 mg/kg MCT on the first day, and then with the same volume of normal saline every day. Rats in the MCT+PNS group was injected intraperitoneally with 60 mg/kg MCT on the first day, and then with 50 mg/kg PNS every day. The modeling time of each group lasted for 21 days. After the model was established, the mean pulmonary artery pressure (mPAP) was measured by right heart catheterization technique, the right ventricular hypertrophy index (RVHI) was calculated, the microscopic morphology and changes of pulmonary vascular wall were observed by HE and Masson staining, and the expressions of ADAM10, Notch3, Hes-1, P27, PCNA, Caspase-3 proteins and mRNA in pulmonary vascular tissue of rats were detected by Western blot and qPCR. The expression and localization of Notch3 and α-SMA were detected by immunofluorescence staining. The protein expression of ADAM10 was detected by immunohistochemical staining. The results showed that compared with the control group, mPAP, RVHI, pulmonary vessels and collagen fibers in the MCT group were significantly increased, the expressions of ADAM10, Notch3, Hes-1, and PCNA protein and mRNA were significantly increased, while the expressions of P27 and Caspase-3 protein and mRNA were decreased significantly. Compared with the MCT group, mPAP and RVHI were significantly decreased, pulmonary vessels were significantly improved and collagen fibers were significantly reduced, the expressions of protein and mRNA of ADAM10, Notch3, Hes-1, and PCNA were decreased in MCT+PNS group, but the expressions of protein and mRNA of P27 and Caspase-3 were increased slightly. The results of immunofluorescence showed that Notch3 and α-SMA staining could overlap, which proved that Notch3 was expressed in smooth muscle cells. The expression of Notch3 in the MCT group was increased significantly compared with that in the control group, while PNS intervention decreased the expression of Notch3. Immunohistochemical staining showed that compared with the control group, the amount of ADAM10 in the MCT group was increased significantly, and the expression of ADAM10 in the MCT+PNS group was decreased compared with the MCT group. These results indicate that PNS can improve the PAH induced by MCT in rats by inhibiting ADAM10/Notch3 signaling pathway.

[Acacetin protects rats from cerebral ischemia-reperfusion injury by regulating TLR4/NLRP3 signaling pathway].

This study aims to investigate the mechanism of acacetin in protecting rats from cerebral ischemia-reperfusion injury via the Toll-like receptor 4(TLR4)/NOD-like receptor protein 3(NLRP3) signaling pathway. Wistar rats were randomized into sham, model, low-and high-dose acacetin, and nimodipine groups, with 10 rats in each group. The rat model of middle cerebral artery occlusion(MCAO) was established with the improved suture method in other groups except the sham group. The neurological deficit score and cerebral infarction volume of each group were evaluated 24 h after modeling. Enzyme-linked immunosorbent assay(ELISA) was employed to measure the levels of interleukin-1ß(IL-1ß), IL-6, tumor necrosis factor-α(TNF-α), malondialdehyde(MDA), supe-roxide dismutase(SOD), and glutathione(GSH). Western blot was employed to determine the expression levels of B-cell lymphonoma-2(Bcl-2), Bcl-2-associated X protein(Bax), and TLR4/NLRP3 signaling pathway-related proteins(TLR4, p-NF-κB/NF-κB, NLRP3, pro-caspase-1, cleaved caspase-1, pro-IL-1ß, and cleaved IL-1ß) in the rat brain tissue. Hematoxylin-eosin(HE) staining was employed to reveal the histopathological changes in the ischemic area. Compared with the sham group, the modeling of MCAO increased the neurological deficit score and cerebral infarction volume, elevated the IL-1ß, IL-6, TNF-α, and MDA levels and lowered the SOD and GSH levels in the brain tissue(P<0.05). Compared with the MCAO model group, low-and high-dose acacetin and nimodipine decreased the neurological deficit score and cerebral infarction volume, lowered the IL-1ß, IL-6, TNF-α, and MDA levels and elevated the SOD and GSH levels in the brain tissue(P<0.05). Compared with the sham group, the model group showed up-regulated protein levels of Bax, TLR4, p-NF-κB/NF-κB, NLRP3, pro-caspase-1, cleaved caspase-1, pro-IL-1ß, and cleaved IL-1ß and down-regulated protein level of Bcl-2 in the brain tissue(P<0.05). Compared with the MCAO model group, the acacetin and nimodipine groups showed down-regulated protein levels of Bax, TLR4, p-NF-κB/NF-κB, NLRP3, pro-caspase-1, cleaved caspase-1, pro-IL-1ß, and cleaved IL-1ß and up-regulated protein level of Bcl-2 in the brain tissue(P<0.05). In conclusion, acacetin regulates the TLR4/NLRP3 signaling pathway to inhibit neuroinflammatory response and oxidative stress, thus exerting the protective effect on cerebral ischemia-reperfusion injury in rats.

Construction of full-length infectious cDNA clones of two Korean isolates of turnip mosaic virus breaking resistance in Brassica napus.

In this work, two new turnip mosaic virus (TuMV) strains (Canola-12 and Canola-14) overcoming resistance in canola (Brassica napus) were isolated from a B. napus sample that showed typical TuMV-like symptoms and was collected in the city of Gimcheon, South Korea, in 2020. The complete genome sequence was determined and an infectious clone was made for each isolate. Phylogenetic analysis indicated that the strains isolated from canola belonged to the World-B group. Both infectious clones, which used 35S and T7 promoters to drive expression, induced systemic symptoms in Nicotiana benthamiana and B. napus. To our knowledge, this is the first report of TuMV infecting B. napus in South Korea.

Construction of full-length infectious clones of turnip mosaic virus isolates infecting Perilla frutescens and genetic analysis of recently emerged strains in Korea.

Perilla is an annual herb with a unique aroma and taste that has been cultivated in Korea for hundreds of years. It has been widely cultivated in many Asian and European countries as a food and medicinal crop. Recently, several viruses have been reported to cause diseases in perilla in Korea, including turnip mosaic virus (TuMV), which is known as a brassica pathogen due to its significant damage to brassica crops. In this study, we determined the complete genome sequences of two new TuMV isolates originating from perilla in Korea. Full-length infectious cDNA clones of these two isolates were constructed, and their infectivity was tested by agroinfiltration of Nicotiana benthamiana and sap inoculation of Chinese cabbage and radish plants. In addition, we analyzed the phylogenetic relationship of six new Korean TuMV isolates to members of the four major groups. We also used RDP4 software to conduct recombination analysis of recent isolates from Korea, which provided new insight into the evolutionary relationships of Korean isolates of TuMV.

Clinical outcomes of EGFR-TKIs in advanced squamous cell lung cancer.

We aimed to explore the treatment efficacy of first-generation epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) for lung squamous cell carcinoma (SCC) patients and identify potential beneficial subgroups of EGFR-mutated lung SCC patients in this study. Between February 1st, 2013 and December 1st, 2021, 657 advanced lung SCC patients were enrolled at Zhejiang Cancer Hospital. Amplification refractory mutation system PCR or next-generation sequencing were used to detect gene abnormality. Clinicopathological features were analyzed by chi-square test and the clinical results of lung SCC patients who received first-generation EGFR-TKI were analyzed by the Kaplan-Meier method. Lung SCC patients harboring EGFR mutation accounted for 11.0% in this study. Of 657 lung SCC patients, the median PFS and OS of 116 patients who received targeted therapy were 3.6 months and 16.2 months, patients treated with targeted therapy had similar OS to patients without targeted therapy (p=0.839). Of 110 lung SCC patients who received first-generation EGFR-TKI, EGFR-mutated patients had long PFS (p=0.000) but similar OS (p=0.472) than patients with EGFR wide type. EGFR-mutated SCC patients who received first-generation EGFR-TKI as a first-line benefit are equal to patients who received first-generation EGFR-TKI as the second line or beyond according to similar PFS (p=0.311) and OS (p=0.721) between them. In addition, there was also no significant difference in PFS (p=0.376) and OS (p=0.205) between patients with exon 19 deletion and L858R point mutation. Lung SCC patients harboring EGFR mutation received first-generation EGFR-TKI had better clinical survival than patients with EGFR wide type.