Hyperspectral imaging combined with GA-SVM for maize variety identification.

The demand for identification of maize varieties has increased dramatically due to the phenomenon of mixed seeds and inferior varieties pretending to be high-quality varieties continuing to occur. It is urgent to solve the problem of efficient and accurate identification of maize varieties. A hyperspectral image acquisition system was used to acquire images of maize seeds. Regions of interest (ROI) with an embryo size of 10 × 10 pixel were extracted, and the average spectral information in the range of 949.43-1709.49 nm was intercepted for the subsequent study in order to eliminate random noise at both ends. Savitzky-Golay (SG) smoothing algorithm and multiple scattering correction (MSC) were used to pretreat the full-band spectrum. The feature wavelengths were screened by successive projection algorithms (SPA), competitive adaptive reweighted sampling (CARS) single screening, and two combinations of CARS-SPA and CARS + SPA, respectively. Support vector machines (SVMs) and models optimized based on genetic algorithm (GA), particle swarm optimization (PSO) were established by using full bands (FB) and feature bands as the model input. The results showed that the MSC-(CARS-SPA)-GA-SVM model had the best performance with 93.00% of the test set accuracy, 8 feature variables, and a running time of 24.45 s. MSC pretreatment can effectively eliminate the scattering effect of spectral data, and the feature wavelengths extracted by CARS-SPA can represent all wavelength information. The study proved that hyperspectral imaging combined with GA-SVM can realize the identification of maize varieties, which provided a theoretical basis for maize variety classification and authenticity identification.

A Fruit-Expressed MYB Transcription Factor Regulates Anthocyanin Biosynthesis in Atropa belladonna.

Anthocyanins are water-soluble flavonoid pigments that play a crucial role in plant growth and metabolism. They serve as attractants for animals by providing plants with red, blue, and purple pigments, facilitating pollination and seed dispersal. The fruits of solanaceous plants, tomato (Solanum lycopersicum) and eggplant (Solanum melongena), primarily accumulate anthocyanins in the fruit peels, while the ripe fruits of Atropa belladonna (Ab) have a dark purple flesh due to anthocyanin accumulation. In this study, an R2R3-MYB transcription factor (TF), AbMYB1, was identified through association analysis of gene expression and anthocyanin accumulation in different tissues of A. belladonna. Its role in regulating anthocyanin biosynthesis was investigated through gene overexpression and RNA interference (RNAi). Overexpression of AbMYB1 significantly enhanced the expression of anthocyanin biosynthesis genes, such as AbF3H, AbF3'5'H, AbDFR, AbANS, and Ab3GT, leading to increased anthocyanin production. Conversely, RNAi-mediated suppression of AbMYB1 resulted in decreased expression of most anthocyanin biosynthesis genes, as well as reduced anthocyanin contents in A. belladonna. Overall, AbMYB1 was identified as a fruit-expressed R2R3-MYB TF that positively regulated anthocyanin biosynthesis in A. belladonna. This study provides valuable insights into the regulation of anthocyanin biosynthesis in Solanaceae plants, laying the foundation for understanding anthocyanin accumulation especially in the whole fruits of solanaceous plants.

Discovering a mitochondrion-localized BAHD acyltransferase involved in calystegine biosynthesis and engineering the production of 3ß-tigloyloxytropane.

Solanaceous plants produce tropane alkaloids (TAs) via esterification of 3α- and 3ß-tropanol. Although littorine synthase is revealed to be responsible for 3α-tropanol esterification that leads to hyoscyamine biosynthesis, the genes associated with 3ß-tropanol esterification are unknown. Here, we report that a BAHD acyltransferase from Atropa belladonna, 3ß-tigloyloxytropane synthase (TS), catalyzes 3ß-tropanol and tigloyl-CoA to form 3ß-tigloyloxytropane, the key intermediate in calystegine biosynthesis and a potential drug for treating neurodegenerative disease. Unlike other cytosolic-localized BAHD acyltransferases, TS is localized to mitochondria. The catalytic mechanism of TS is revealed through molecular docking and site-directed mutagenesis. Subsequently, 3ß-tigloyloxytropane is synthesized in tobacco. A bacterial CoA ligase (PcICS) is found to synthesize tigloyl-CoA, an acyl donor for 3ß-tigloyloxytropane biosynthesis. By expressing TS mutant and PcICS, engineered Escherichia coli synthesizes 3ß-tigloyloxytropane from tiglic acid and 3ß-tropanol. This study helps to characterize the enzymology and chemodiversity of TAs and provides an approach for producing 3ß-tigloyloxytropane.

Significant response to pembrolizumab plus lenvatinib in Epstein-Barr-virus-associated intrahepatic cholangiocarcinoma: a case report.

BACKGROUND: The prognosis for advanced intrahepatic cholangiocarcinoma (iCCA) is poor, and there remains an urgent need to develop efficient systemic therapy. The efficacy of Pembrolizumab immunotherapy combined with lenvatinibin in iCCA is still unclear. The role of Epstein-Barr-virus (EBV) as a biomarker in iCCA for response to immunotherapy needs further exploration. CASE PRESENTATION: We report a case of a 60-year-old female with EBV-associated advanced iCCA (EBVaiCCA) who progressed after first-line therapy. She accomplished an available response to the combination therapy of pembrolizumab with lenvatinib, with overall survival of 20 months. CONCLUSIONS: As far as we know, this is the first case report about the application of Pembrolizumab with lenvatinib for EBVaiCCA patients. This case indicates that the combination of immunotherapy and antiangiogenic therapy provides a glimmer of hope for advanced EBVaiCCA patients.

Biosynthesis of Scopoletin in Sweet Potato Confers Resistance against Fusarium oxysporum.

Fusarium wilt is a severe fungal disease caused by Fusarium oxysporum in sweet potato. We conducted transcriptome analysis to explore the resistance mechanism of sweet potato against F. oxysporum. Our findings highlighted the role of scopoletin, a hydroxycoumarin, in enhancing resistance. In vitro experiments confirmed that scopoletin and umbelliferone had inhibitory effects on the F. oxysporum growth. We identified hydroxycoumarin synthase genes IbF6'H2 and IbCOSY that are responsible for scopoletin production in sweet potatoes. The co-overexpression of IbF6'H2 and IbCOSY in tobacco plants produced the highest scopoletin levels and disease resistance. This study provides insights into the molecular basis of sweet potato defense against Fusarium wilt and identifies valuable genes for breeding wilt-resistant cultivars.

KCTD15 acts as an anti-tumor factor in colorectal cancer cells downstream of the demethylase FTO and the m6A reader YTHDF2.

Potassium Channel Tetramerization Domain Containing 15 (KCTD15) participates in the carcinogenesis of several solid malignancies; however, its role in colorectal cancer (CRC) remains unclear. Here we find that KCTD15 exhibits lower expression in CRC tissues as compared to para-carcinoma tissues. Tetracycline (tet)-induced overexpression and knockdown of KCTD15 confirms KCTD15 as an anti-proliferative and pro-apoptotic factor in CRC both in vitro and in xenografted tumors. N6-methyladenosine (m6A) is known to affect the expression, stabilization, and degradation of RNAs with this modification. We demonstrate that upregulation of fat mass and obesity-associated protein (FTO), a classical m6A eraser, prevents KCTD15 mRNA degradation in CRC cells. Less KCTD15 RNA is recognized by m6A 'reader' YTH N6-Methyladenosine RNA Binding Protein F2 (YTHDF2) in FTO-overexpressed cells. Moreover, KCTD15 overexpression decreases protein expression of histone deacetylase 1 (HDAC1) but increases acetylation of critical tumor suppressor p53 at Lys373 and Lys382. Degradation of p53 is delayed in CRC cells post-KCTD15 overexpression. We further show that the regulatory effects of KCTD15 on p53 are HDAC1-dependent. Collectively, we conclude that KCTD15 functions as an anti-growth factor in CRC cells, and its expression is orchestrated by the FTO-YTHDF2 axis. Enhanced p53 protein stabilization may contribute to KCTD15's actions in CRC cells.

Identifying an AML Prognostic Model Using 10 Marker Genes from Single-Cell Transcriptome and Bulk Transcriptome Analysis.

Fanconi anemia (FA) is the predominant hereditary syndrome of bone marrow failure (BMF), distinguished by impairments in DNA repair mechanisms. The deficiency in the FANC pathway, which governs DNA repair and replication rescue, results in aberrant responses to DNA damage in individuals with FA. The objective of this study is to examine the involvement of the FANC core complex in BMF and ascertain nucleolar homeostasis-related genes by conducting transcriptome analysis on primary hematopoietic stem cells obtained from FA patients with FANCA and FANCC variants. In the present study, we analyzed scRNA-seq data obtained from both healthy donors and individuals diagnosed with FA in order to investigate the phenomenon of cell-cell communication. Through the implementation of trajectory analysis, the differentiation pathways of several progenitor cell types, such as HSC cells transitioning into LMPP, N, M, B-prog, and E cells, were elucidated. Moreover, by scrutinizing the inferred interactions, notable disparities in cell-cell communication were observed between FA patients and their healthy counterparts. Our analysis has unveiled heightened interactions involving TNFSF13B, MIF, IL16, and COL4A2 in patients with FA. Furthermore, we have developed a prognostic model for predicting the outcome of acute myeloid leukemia (AML) which has exhibited remarkable predictive precision, with an AUC exceeding 0.83 at the 3- and 5-year intervals following the baseline assessment. In summary, the prognostic model that has been developed provides a valuable instrument for forecasting outcomes of AML by utilizing the genes identified through both single-cell and bulk transcriptome analyses.

Preparation methods and research progress of super-hydrophobic anti-icing surface.

The problem of surface icing poses a serious threat to people's economy and safety, especially in the fields of aerospace, wind power generation and circuit transmission. Super-hydrophobic has excellent anti-icing performance, so it has been widely studied. As the most promising anti-icing technology, superhydrophobic anti-icing surface should not only be simple to prepare, but also have excellent comprehensive performance, which can meet the anti-icing task under harsh working conditions and long-term durability. This paper summarizes the basic performance requirements of superhydrophobic surface for anti-icing operation, and then summarizes the preparation methods and existing problems of superhydrophobic surface in recent years. Finally, the future development trend of superhydrophobic anti-icing surface is prospected and discussed, hoping to provide certain technical guidance for the subsequent research of high-performance superhydrophobic anti-icing surface.

The Enrichment of miRNA-Targeted mRNAs in Translationally Less Active over More Active Polysomes.

miRNAs moderately inhibit the translation and enhance the degradation of their target mRNAs via cognate binding sites located predominantly in the 3'-untranslated regions (UTR). Paradoxically, miRNA targets are also polysome-associated. We studied the polysome association by the comparative translationally less-active light- and more-active heavy-polysome profiling of a wild type (WT) human cell line and its isogenic mutant (MT) with a disrupted DICER1 gene and, thus, mature miRNA production. As expected, the open reading frame (ORF) length is a major determinant of light- to heavy-polysome mRNA abundance ratios, but is rendered less powerful in WT than in MT cells by miRNA-regulatory activities. We also observed that miRNAs tend to target mRNAs with longer ORFs, and that adjusting the mRNA abundance ratio with the ORF length improves its correlation with the 3'-UTR miRNA-binding-site count. In WT cells, miRNA-targeted mRNAs exhibit higher abundance in light relative to heavy polysomes, i.e., light-polysome enrichment. In MT cells, the DICER1 disruption not only significantly abrogated the light-polysome enrichment, but also narrowed the mRNA abundance ratio value range. Additionally, the abrogation of the enrichment due to the DICER1 gene disruption, i.e., the decreases of the ORF-length-adjusted mRNA abundance ratio from WT to MT cells, exhibits a nearly perfect linear correlation with the 3'-UTR binding-site count. Transcription factors and protein kinases are the top two most enriched mRNA groups. Taken together, the results provide evidence for the light-polysome enrichment of miRNA-targeted mRNAs to reconcile polysome association and moderate translation inhibition, and that ORF length is an important, though currently under-appreciated, transcriptome regulation parameter.

Revalidation of the ATTRACTION-4 study in a real-world setting: a multicenter, retrospective propensity score matching study in China.

Background: Immune-checkpoint inhibitors (ICIs) combined with chemotherapy have been successfully used in clinical trials to treat advanced gastric cancer. However, the efficacy and safety of first-line immunotherapy combined with chemotherapy in Chinese patients are unknown. Methods: This multicenter retrospective study included patients with human epidermal growth factor receptor-2 (HER-2) negative advanced gastric cancer treated with first-line chemotherapy or chemotherapy with an ICI between January 2019 and December 2022. Propensity score matching was used to compare progression-free survival (PFS), overall survival, objective response rates, and adverse reactions between cohorts. Results: After propensity score matching, 138 patients, who had balanced baseline characteristics, were included in the chemotherapy and combination treatment groups. The median follow-up duration was 16.90 months, and the median PFS was 8.53 months (95% confidence interval [CI] 7.77-9.28) in the combination treatment group and 5.97 months (95% CI 4.56-7.37) in the chemotherapy group. The median survival duration was 17.05 months (95% CI 14.18-19.92) in the combination treatment group and 16.46 months (95% CI 12.99-19.93) in the chemotherapy group. The PFS subgroup analysis revealed that age ≥65 years, women, Eastern Cooperative Oncology Group performance status of 1, non-signet ring cell carcinoma, esophagogastric junction, liver metastasis, peritoneal metastasis, no massive ascites, only one metastatic organ, and combined platinum-based chemotherapy correlated with treatment benefit. The incidences of adverse events above grade 3 were comparable between groups. Conclusions: Our study confirmed the ATTRACTION-4 trial results. Compared with chemotherapy, first-line ICIs combined with chemotherapy prolonged PFS but did not improve overall survival in patients with HER-2-negative advanced gastric cancer.

Hyperspectral imaging combined with CNN for maize variety identification.

Introduction: As the third largest food crop in the world, maize has wide varieties with similar appearances, which makes identification difficult. To solve the problem of identification of hybrid maize varieties, a method based on hyperspectral image technology combined with a convolutional neural network (CNN) is proposed to identify maize varieties. Methods: In this study, 735 maize seeds from seven half-parent hybrid maize varieties were regarded as the research object. The maize seed images in the range of 900 ~ 1700nm were obtained by hyperspectral image acquisition system. The region of interest (ROI) of the embryo surface was selected, and the spectral reflectance of maize seeds was extracted. After Savitzky-Golay (SG) Smoothing pretreatment, Maximum Normalization (MN) pretreatment was performed. The 56 feature wavelengths were selected by Competitive Adaptive Reweighting Algorithm (CARS) and Successive Projection Algorithm (SPA). And the 56 wavelengths were mapped to high-dimensional space by high-dimensional feature mapping and then reconstructed into three-dimensional image features. A five-layer convolution neural network was used to identify three-dimensional image features, and nine (SG+MN)-(CARS+SPA)-CNN maize variety identification models were established by changing the input feature dimension and the depth factor size of the model layer. Results and Discussion: The results show that the maize variety classification model works best, when the input feature dimension is 768 and the layer depth factor d is 1.0. At this point, the model accuracy of the test set is 96.65% and the detection frame rate is1000 Fps/s in GPU environment, which can realize the rapid and effective non-destructive detection of maize varieties. This study provides a new idea for the rapid and accurate identification of maize seeds and seeds of other crops.

Long-term efficacy and safety of different corticosteroid courses plus mycophenolate mofetil for autoimmune encephalitis with neuronal surface antibodies without tumor.

Objective: To compare the efficacy and safety of different-course corticosteroids plus mycophenolate mofetil (MMF) as maintenance therapy in autoimmune encephalitis (AE) with neuronal surface antibodies (NSAbs) without tumor and explore the optimal course of corticosteroids. Methods: Fifty-five patients with definite AE without tumor were enrolled consecutively between June 2015 and November 2020 and retrospectively divided three groups according to the course of treatment with corticosteroid, i.e., a group of patients with a course of 3-6 months (Group 3-6mo), 6-12 months (Group 6-12mo), and >12 months (Group >12mo). Demographic data, clinical manifestation and ancillary tests results were recorded. The dosage and courses of corticosteroid treatment, the recovery of neurological function, the occurrence of adverse effects, and relapses were followed up. Results: A total of 55 patients were included in the final analysis. The numbers of patients in Group 3-6 mo, Group 6-12 mo, and Group >12 mo was 14, 17, and 24, respectively. A significantly higher proportion of patients in Group >12 mo showed a decreased level of consciousness at the onset (12, 50%) than in Group 3-6 mo and Group 6-12 mo (2,14.3%; 3, 17.6%) (p = 0.033). The incidence of MRI abnormalities was significantly higher in Group 6-12 mo and Group >12 mo (10, 58.8%; 16, 66.7%) than in Group 3-6 mo (3, 21.4%) (P=0.023). Ordinal regression analysis indicated that decreased level of consciousness was associated with the course of corticosteroid (OR=3.838, 95% CI: 1.103-13.323, P=0.035). No significant difference was observed between the three groups regarding the cumulative dose of corticosteroids administered during the first three months of long-term treatment (P>0.05). Additionally, no significant difference in the cumulative dosage of corticosteroids was found between patients in Group 6-12 months and Group >12 months during the first 6 months after beginning long-term treatment. The mRS scores of the three groups were not statistically significant before and after first-line treatment or at the last follow-up. Bonferroni multiple comparison test indicated that the mRS scores of patients in Group 6-12 months and Group >12 months were not statistically significant at 3 months and 12 months after the start of long-term treatment. During the follow-up, 50 (90.9%) patients achieved satisfactory neurological function (mRS score ≤2). Five patients (9.1%) experienced a first relapse and 2 of them were overlapped with both anti-NMDA receptor and glial antibodies. The incidence of adverse effects was significantly higher in Group >12 mo (17, 70.8%) than in Group 3-6 mo (3, 21.4%) and Group 6-12 mo (5, 29.4%) (P=0.003). Conclusions: The beneficial effects of oral corticosteroid treatment may do not persist beyond 12 months and may even contribute to an increased incidence of adverse effects. In order to optimize the effectiveness and safety of treatment, we recommend a corticosteroid course of 3-12 months. Patients with reduced levels of consciousness may be more inclined to choose longer courses of corticosteroids for long-term treatment. Patients with an "overlapping syndrome" may require more intense immunotherapy to prevent relapse.

The quality of life in nasopharyngeal carcinoma radiotherapy: A longitudinal study.

Objective: This article aims to longitudinally compare nasopharyngeal carcinoma (NPC) patients' quality of life (QoL) during radiotherapy (RT) and identify QoL correlates. Methods: This study included 98 patients, with 85 completing full follow-up. Data were collected at baseline (T1), midpoint of RT (T2), and RT completion (T3), between October 2021 and November 2022. QoL was assessed using the European Organization for Research and Treatment of Cancer Quality of Life Questionnaire-Core 30 (EORTC QLQ-C30). RIOM severity was evaluated by the toxicity criteria of Radiation Therapy Oncology Group (RTOG). The nutritional status was evaluated using the Nutritional Risk Screening 2002 (NRS 2002), body mass index (BMI), and the Patient-Generated Subjective Global Assessment (PG-SGA). The generalized estimating equation described the QoL evolution and correlated it with RIOM, nutritional status, and other influential factors. Results: Significant deterioration was observed in various subscales of EORTC QLQ-C30 during RT, including global health status (GHS), physical function, role function, emotional function, fatigue, nausea/vomiting, pain, insomnia, appetite loss, and constipation (all P â€‹< â€‹0.05). Substantial deterioration was also observed in RIOM, nutritional status, and part of hematological indexes (all P â€‹< â€‹0.05). The decline of QoL was associated with gender, age, education level, chemotherapy regimen, Karnofsky performance status (KPS) score, RIOM severity, NRS 2002 score, PG-SGA score, and lymphocyte level (all P â€‹< â€‹0.05). Conclusions: QoL declined during RT and were associated with certain factors. Healthcare professionals should focus on alleviating treatment-related complications and identifying individuals at high risk of malnutrition early to improve outcomes for patients with NPC.

HGF-modified human umbilical cord mesenchymal stem cells rescue impaired ovarian reserve function in chemotherapy-induced POI rats by improving angiogenesis while decreasing apoptosis and fibrosis in the ovary.

Complications caused by Primary ovarian insufficiency (POI), including infertility, osteoporosis, cardiovascular diseases and depression, severely affect the life quality of female patients. Although hormone replacement therapy (HRT) can alleviate some long-term complications, there is still no standard treatment for the restoration of ovarian reserve function. Currently, human umbilical cord mesenchymal stem cells (HUCMSC) transplantation showed considerable treatment effect for POI in both rat model and clinic. To improve the effectiveness of naïve HUCMSC (HUCMSC-Null) treatments on POI, an exogenous gene hepatocyte growth factor (HGF) which promotes follicular angiogenesis in POI ovaries was used to modify HUCMSC. Subsequently, HGF-overexpressed HUCMSC (HUCMSC-HGF) was transplanted into the ovaries of chemotherapy-induced POI Sprague-Dawley (SD) rats to observe the effectiveness on POI improvement and its related mechanisms. Our results showed that when compared with POI and HUCMSC-Null treatment group, HUCMSC-HGF significantly improved ovarian reserve function in POI group, which might be attributed to the decrease of ovarian tissue fibrosis and granulosa cells (GCs) apoptosis, and the increase of ovarian angiogenesis mediated by HGF over-expression. The findings suggest that HGF-modified HUCMSC may present a more superior capacity than HUCMSC alone for the rescue of ovarian reserve function in POI.

Effects of Si-Miao-Yong-An decoction on myocardial I/R rats by regulating gut microbiota to inhibit LPS-induced TLR4/NF-κB signaling pathway.

BACKGROUND: Coronary Artery Disease (CAD) is primarily caused by inflammation which is closely linked to the gut microbiota. Si-Miao-Yong-An (SMYA) decoction is a traditional Chinese herbal formula with anti-inflammatory properties that found to be effective against CAD. However, it is still unclear whether SMYA can modulate gut microbiota and whether it contributes to the improvement of CAD by reducing inflammation and regulating the gut microbiota. METHODS: The identification of components in the SMYA extract was conducted using the HPLC method. A total of four groups of SD rats were orally administered with SMYA for 28 days. The levels of inflammatory biomarkers and myocardial damage biomarkers were measured through ELISA, while echocardiography was used to assess heart function. Histological alterations in the myocardial and colonic tissues were examined following H&E staining. Western blotting was performed to evaluate protein expression, whereas alterations in gut microbiota were determined by 16 s rDNA sequencing. RESULTS: SMYA was found to enhance cardiac function and decrease the expression of serum CK-MB and LDH. SMYA was also observed to inhibit the TLR4/NF-κB signaling pathway by downregulating the protein expression of myocardial TLR4, MyD88, and p-P65, leading to a reduction in serum pro-inflammatory factors. SMYA modified the composition of gut microbiota by decreasing the Firmicutes/Bacteroidetes ratio, modulating Prevotellaceae_Ga6A1 and Prevotellaceae_NK3B3 linked to the LPS/TLR4/NF-κB pathway, and increasing beneficial microbiota such as Bacteroidetes, Alloprevotella, and other bacterial species. Moreover, SMYA was found to safeguard the intestinal mucosal and villi structures, elevate the expression of tight junction protein (ZO-1, occludin), and reduce intestinal permeability and inflammation. CONCLUSIONS: The results indicate that SMYA has the potential to modulate the gut microbiota and protect the intestinal barrier, thereby reducing the translocation of LPS into circulation. SMYA was also found to inhibit the LPS-induced TLR4/NF-κB signaling pathway, leading to a decrease in the release of inflammatory factors, which ultimately mitigated myocardial injury. Hence, SMYA holds promise as a therapeutic agent for the management of CAD.

Pretreatment high cholesterol and low neutrophils predict complete pathological response after neoadjuvant short­course radiotherapy followed by chemotherapy and immunotherapy in locally advanced rectal cancer.

The present study was aimed at looking for hematological indicators that could predict pathological complete response (pCR) in patients with locally advanced rectal cancer (LARC) treated with short-course radiotherapy (SCRT) followed by chemotherapy and immunotherapy. A total of 171 patients were enrolled in this observational retrospective study. Pretreatment values of albumin, total cholesterol, lactate dehydrogenase, neutrophil, platelet and lymphocytes were available. Univariate and multivariate logistics analyses were used to determine the prognostic factor for pCR. SCRT followed by chemotherapy and immunotherapy was demonstrated to double the pCR rate (50.5%) compared with long-course chemoradiotherapy. For the former group, baseline high platelet to lymphocyte ratio (P=0.047), high cholesterol (P=0.026) and low neutrophils (P=0.012) level were associated with high pCR rate and baseline high cholesterol (P=0.016) and low neutrophils (P=0.020) level were the independent prognostic factors for pCR. In conclusion, pretreatment high cholesterol and low neutrophils were the independent prognostic predictors of pCR in patients with LARC treated with SCRT followed by chemotherapy and immunotherapy. Clinical trial no. NCT04928807, June 16, 2021.

Network pharmacology-based approach to research the effect and mechanism of Si-Miao-Yong-An decoction against thromboangiitis obliterans.

BACKGROUND: Si-Miao-Yong-An decoction (SMYAD) is a conventional therapeutic formula for treat thromboangiitis obliterans (TAO), consisting of four Chinese herbs: Lonicerae japonicae Thunb. (Jinyinhua), Scrophularia ningpoensis Hemsl. (Xuanshen), Angelica sinensis (Oliv.) Diels (Danggui) and Glycyrrhiza uralensis Fisch. (Gancao). However, the mechanism of SMYAD in TAO treatment remains unclear. METHODS: Components, as well as potential targets of SMYAD in TAO therapy, were downloaded from Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP). Subsequently, with the Database for Annotation, Visualization, and Integrated Discovery (DAVID) server, the gene ontology (GO) biological processes and the Kyoto encyclopedia of genes and genomes (KEGG) signalling pathways of the targets enrichment were performed. Next, based on STRING online database, the protein interaction network of vital targets was built and analysed. Molecular docking and calculation of the binding affinity were performed using AutoDock. The PyMOL software was employed to observe docking outcomes of active compounds and protein targets. Based on the predicted outcomes of network pharmacology, in vivo and in vitro tests were performed for validation. In vivo experiment, the TAO rats model was established using sodium laurate injection into the femoral artery. The symptoms as well as pathological changes of the femoral artery were observed. Besides, the predicted targets were verified by the RT-qPCR, in vitro experiment. The cell viability in LPS-induced human umbilical vein endothelial cells (HUVECs) was detected using CCK-8 kit, and the predicted targets were also verified by the RT-qPCR. RESULTS: In the network pharmacology analysis, we obtained 105 chemical components in SMYAD and 24 therapeutic targets. We found that the mechanism SMYAD in TAO therapy was primarily associated with inflammation and angiogenesis by constructing multiple networks. Quercetin, vestitol and beta-sitosterol were important compounds, and interleukin-6 (IL6), MMP9, and VEGFA were key targets. According to molecular docking, active compounds (quercetin, vestitol and beta-sitosterol) and targets (IL6, MMP9 and VEGFA) showed good binding interactions. In in vivo experiment, SMYAD ameliorated the physical signs and pathological changes, inhibited the expression of IL6 and MMP9, and enhanced the expression of VEGFA. In an in vitro experiment, SMYAD increased the cell viability of LPS-induced HUVECs and the expression of VEGFA, and reduced the expression of IL6 and MMP9. CONCLUSIONS: This study showed that SMYAD improves TAO symptoms and inhibits the development of TAO. The mechanism could be associated with anti-inflammatory and therapeutic angiogenesis.

Broad Sarbecovirus Neutralizing Antibodies Obtained by Computational Design and Synthetic Library Screening.

Members of the Sarbecovirus subgenus of Coronaviridae have twice caused deadly threats to humans. There is increasing concern about the rapid mutation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which has evolved into multiple generations of epidemic variants in 3 years. Broad neutralizing antibodies are of great importance for pandemic preparedness against SARS-CoV-2 variants and divergent zoonotic sarbecoviruses. Here, we analyzed the structural conservation of the receptor-binding domain (RBD) from representative sarbecoviruses and chose S2H97, a previously reported RBD antibody with ideal breadth and resistance to escape, as a template for computational design to enhance the neutralization activity and spectrum. A total of 35 designs were purified for evaluation. The neutralizing activity of a large proportion of these designs against multiple variants was increased from several to hundreds of times. Molecular dynamics simulation suggested that extra interface contacts and enhanced intermolecular interactions between the RBD and the designed antibodies are established. After light and heavy chain reconstitution, AI-1028, with five complementarity determining regions optimized, showed the best neutralizing activity across all tested sarbecoviruses, including SARS-CoV, multiple SARS-CoV-2 variants, and bat-derived viruses. AI-1028 recognized the same cryptic RBD epitope as the parental prototype antibody. In addition to computational design, chemically synthesized nanobody libraries are also a precious resource for rapid antibody development. By applying distinct RBDs as baits for reciprocal screening, we identified two novel nanobodies with broad activities. These findings provide potential pan-sarbecovirus neutralizing drugs and highlight new pathways to rapidly optimize therapeutic candidates when novel SARS-CoV-2 escape variants or new zoonotic coronaviruses emerge. IMPORTANCE The subgenus Sarbecovirus includes human SARS-CoV, SARS-CoV-2, and hundreds of genetically related bat viruses. The continuous evolution of SARS-CoV-2 has led to the striking evasion of neutralizing antibody (NAb) drugs and convalescent plasma. Antibodies with broad activity across sarbecoviruses would be helpful to combat current SARS-CoV-2 mutations and longer term animal virus spillovers. The study of pan-sarbecovirus NAbs described here is significant for the following reasons. First, we established a structure-based computational pipeline to design and optimize NAbs to obtain more potent and broader neutralizing activity across multiple sarbecoviruses. Second, we screened and identified nanobodies from a highly diversified synthetic library with a broad neutralizing spectrum using an elaborate screening strategy. These methodologies provide guidance for the rapid development of antibody therapeutics against emerging pathogens with highly variable characteristics.