Establishment of an Efficient Agrobacterium-Mediated Genetic Transformation System to Enhance the Tolerance of the Paraquat Stress in Engineering Goosegrass (Eleusine Indica L.).

Eleusine indica (goosegrass) is a problematic weed worldwide known for its multi-herbicide tolerance/resistance biotype. However, a genetic transformation method in goosegrass has not been successfully established, making a bottleneck for functional genomics studies in this species. Here, we report a successful Agrobacterium-mediated transformation method for goosegrass. Firstly, we optimized conditions for breaking seed dormancy and increasing seed germination rate. A higher callus induction rate from germinated seeds was obtained in N6 than in MS or B5 medium. Then the optimal transformation efficiency of the gus reporter gene was obtained by infection with Agrobacterium tumefaciens culture of OD600 = 0.5 for 30 min, followed by 3 days of co-cultivation with 300 µmol/L acetosyringone. Concentrations of 20 mg L-1 kanamycin and 100 mg L-1 timentin were used to select the transformed calli. The optimal rate of regeneration of the calli was generated by using 0.50 mg L-1 6-BA and 0.50 mg L-1 KT in the culture medium. Then, using this transformation method, we overexpressed the paraquat-resistant EiKCS gene into a paraquat-susceptible goosegrass biotype MZ04 and confirmed the stable inheritance of paraquat-resistance in the transgenic goosegrass lines. This approach may provide a potential mechanism for the evolution of paraquat-resistant goosegrass and a promising gene for the manipulation of paraquat-resistance plants. This study is novel and valuable in future research using similar methods for herbicide resistance.

Effects of Biological Nitrogen Metabolism on Glufosinate-Susceptible and -Resistant Goosegrass (Eleusine indica L.).

Glufosinate is a broad-spectrum herbicide used to control most weeds in agriculture worldwide. Goosegrass (Eleusine indica L.) is one of the top ten malignant weeds across the world, showing high tolerance to glufosinate via different mechanisms that are not yet fully understood. This study revealed that nitrogen metabolism could be a target-resistant site, providing clues to finally clarify the mechanism of glufosinate resistance in resistant goosegrass populations. Compared to susceptible goosegrass (NX), the resistant goosegrass (AUS and CS) regarding the stress of glufosinate showed stronger resistance with lower ammonia contents, higher target enzyme GS (glutamine synthetase) activity, and lower GOGAT (glutamine 2-oxoglutarate aminotransferase) activity. The GDH (glutamate dehydrogenase) activity of another pathway increased, but its gene expression was downregulated in resistant goosegrass (AUS). Analyzing the transcriptome and proteome data of goosegrass under glufosinate stress at 36 h showed that the KEGG pathway of the nitrogen metabolism was enriched in glufosinate-susceptible goosegrass (NX), but not in glufosinate-resistant goosegrass (CS and AUS). Several putative target genes involved in glufosinate stress countermeasures were identified. This study provides specific insights into the nitrogen metabolism of resistant goosegrass, and gives a basis for future functional verification of glufosinate-tolerance genes in plants.

Exploring C-to-G and A-to-Y Base Editing in Rice by Using New Vector Tools.

CRISPR/Cas9-based cytosine base editors (CBEs) and adenine base editors (ABEs) can efficiently mediate C-to-T/G-to-A and A-to-G/T-to-C substitutions, respectively; however, achieving base transversions (C-to-G/C-to-A and A-to-T/A-to-C) is challenging and has been rarely studied in plants. Here, we constructed new plant C-to-G base editors (CGBEs) and new A-to-Y (T/C) base editors and explored their base editing characteristics in rice. First, we fused the highly active cytidine deaminase evoFENRY and the PAM-relaxed Cas9-nickase variant Cas9n-NG with rice and human uracil DNA N-glycosylase (rUNG and hUNG), respectively, to construct CGBE-rUNG and CGBE-hUNG vector tools. The analysis of five NG-PAM target sites showed that these CGBEs achieved C-to-G conversions with monoallelic editing efficiencies of up to 27.3% in T0 rice, with major byproducts being insertion/deletion mutations. Moreover, for the A-to-Y (C or T) editing test, we fused the highly active adenosine deaminase TadA8e and the Cas9-nickase variant SpGn (with NG-PAM) with Escherichia coli endonuclease V (EndoV) and human alkyladenine DNA glycosylase (hAAG), respectively, to generate ABE8e-EndoV and ABE8e-hAAG vectors. An assessment of five NG-PAM target sites showed that these two vectors could efficiently produce A-to-G substitutions in a narrow editing window; however, no A-to-Y editing was detected. Interestingly, the ABE8e-EndoV also generated precise small fragment deletions in the editing window from the 5'-deaminated A base to the SpGn cleavage site, suggesting its potential value in producing predictable small-fragment deletion mutations. Overall, we objectively evaluated the editing performance of CGBEs in rice, explored the possibility of A-to-Y editing, and developed a new ABE8e-EndoV tool, thus providing a valuable reference for improving and enriching base editing tools in plants.

Photoredox-Catalyzed Generation of Sulfamyl Radicals: Sulfonamidation of Enol Silyl Ether with Chlorosulfonamide.

A novel and practical photoredox-catalyzed generation of sulfamyl radicals followed by radical sulfonamidation of enol silyl ether has been described. Diverse functionalized ß-ketosulfonamides were prepared in modest to excellent yields under mild and economic reaction conditions through the present catalytic protocol. Furthermore, the methodology developed provides an efficient and convenient approach to the synthesis of the antiseizure drug Zonisamide.

Intratumoral Microbiota Composition Regulates Chemoimmunotherapy Response in Esophageal Squamous Cell Carcinoma.

Neoadjuvant chemoimmunotherapy (NACI) has shown promise in the treatment of resectable esophageal squamous cell carcinoma (ESCC). The microbiomes of patients can impact therapy response, and previous studies have demonstrated that intestinal microbiota influences cancer immunotherapy by activating gut immunity. Here, we investigated the effects of intratumoral microbiota on the response of patients with ESCC to NACI. Intratumoral microbiota signatures of ß-diversity were disparate and predicted the treatment efficiency of NACI. The enrichment of Streptococcus positively correlated with GrzB+ and CD8+ T-cell infiltration in tumor tissues. The abundance of Streptococcus could predict prolonged disease-free survival in ESCC. Single-cell RNA sequencing demonstrated that responders displayed a higher proportion of CD8+ effector memory T cells but a lower proportion of CD4+ regulatory T cells. Mice that underwent fecal microbial transplantation or intestinal colonization with Streptococcus from responders showed enrichment of Streptococcus in tumor tissues, elevated tumor-infiltrating CD8+ T cells, and a favorable response to anti-PD-1 treatment. Collectively, this study suggests that intratumoral Streptococcus signatures could predict NACI response and sheds light on the potential clinical utility of intratumoral microbiota for cancer immunotherapy. SIGNIFICANCE: Analysis of intratumoral microbiota in patients with esophageal cancer identifies a microbiota signature that is associated with chemoimmunotherapy response and reveals that Streptococcus induces a favorable response by stimulating CD8+ T-cell infiltration. See related commentary by Sfanos, p. 2985.

JNK-dependent Atg4 upregulation mediates asperphenamate derivative BBP-induced autophagy in MCF-7 cells.

N-Benzoyl-O-(N'-(1-benzyloxycarbonyl-4-piperidiylcarbonyl)-D-phenylalanyl)-D-phenylalaninol (BBP), a novel synthesized asperphenamate derivative with the increased solubility, showed growth inhibitory effect on human breast carcinoma MCF-7 cells in a time- and concentration-dependent manner. The growth inhibitory effect of BBP was associated with induction of autophagy, which was demonstrated by the development of acidic vesicular organelles, cleavage of LC3 and upregulation of Atg4 in BBP-treated MCF-7 cells. Since the application of Atg4 siRNA totally blocked the cleavage of LC3, we demonstrated a central role of Atg4 in BBP-induced autophagy. The further studies showed that BBP increased the levels of reactive oxygen species (ROS), and pretreatment with NAC effectively blocked the accumulation of ROS, autophagy and growth inhibition triggered by BBP. Moreover, BBP induced the activation of JNK, and JNK inhibitor SP600125 reversed autophagy, the increase of Atg4 levels, conversion of LC3 and growth inhibition induced by BBP. Knockdown of JNK by siRNA efficiently inhibited ROS production and autophagy, but antioxidant NAC failed to block JNK activation induced by BBP, indicating that JNK activation may be a upstream signaling of ROS and should be a core component in BBP-induced autophagic signaling pathway. These results suggest that BBP produces its growth inhibitory effect through induction of the autophagic cell death in MCF-7 cells, which is modulated by a JNK-dependent Atg4 upregulation involving ROS production.

UV-H2O2 degradation of methyl orange catalysed by H3PW12O40/activated clay.

A catalyst consisting of phosphotungstic acid (H3PW12O40) combined with activated clay was prepared by the impregnation method, and an experiment was carried out to evaluate the catalytic activity of the H3PW12O40/activated clay for the degradation of methyl orange (MO) in the UV-H2O2 process. The degradation ratio of MO can be affected by H2O2 concentration, reaction time, catalyst dosage, pH and temperature. The reaction temperature should be controlled at less than 70 degrees C, and the catalyst has a wide applicable pH range in the UV-H2O2 process. Hydroxyl radicals were generated in the UV-H2O2 system under the action of H3PW12O40/activated clay, and MO was degraded by hydroxyl radicals. Compared with traditional catalysts used in UV-H2O2 systems, H3PW12O40/activated clay has certain advantages for its practical application.

Periosteum-inspired in situ CaP generated nanocomposite hydrogels with strong bone adhesion and superior stretchability for accelerated distraction osteogenesis.

BACKGROUND: Distraction osteogenesis (DO) is an efficacious but lengthy procedure to reconstruct segmental bone defects under the principle of tension-stress, during which the periosteum-mediated mechanical stimulation plays a pivotal role. Inspired by the dynamic process of DO and the mechanical stimulation of periosteum, a new design of bionic periosteum was developed to simulate the mechanical transduction of natural periosteum for the application in DO procedure. METHODS: In this study, an injectable organic-inorganic hybrid hydrogel was developed based on a novel combination of the PEGylated poly (glycerol sebacate) (PEGS) polymer network and in situ formed CaP nanoparticles (ICPNs). Rat bone marrow mesenchymal stem cells (rBMSCs) and human umbilical vein endothelial cells (HUVECs) were cultured and tested in vitro to evaluate biocompatibility, cell adhesion, proliferation, and pro-osteogenic and pro-angiogenic activity. In vivo experiments were conducted in the rat tibial model of distraction osteogenesis. RESULTS: The developed nanocomposite hydrogels exhibited excellent injectability, robust bone adhesion, superior stretchability, and enhanced osteogenic activity. The results of in vitro and in vivo studies showed that PEGS/ICPN hydrogels could promote new bone formation and mineralization during the dynamic distraction process through the synergistic effects of angiogenesis and osteogenesis. CONCLUSIONS: This periosteum-inspired nanocomposite hydrogel represents a mechanobiology approach for effectively restoring large bone defects through the dynamic DO process.

Overexpression of EiKCS confers paraquat-resistance in rice (Oryza sativa L.) by promoting the polyamine pathway.

BACKGROUND: Paraquat is used widely as one of the bipyridine herbicides, which generates reactive oxygen species to cause cell death. With a growing number of paraquat-resistant weeds, the mechanism of paraquat-resistance in plants remains unclear. This research verified the functions of a previously confirmed putative paraquat-resistant gene, EiKCS, from paraquat-resistant goosegrass by genetic engineering in a single overexpressing line in rice. RESULTS: Overexpression of EiKCS improved paraquat resistance in transgenic rice (KCSox). Pre-applied (12 h) exogenous spermidine (1.5 mmol L-1 ), alleviated the injury of paraquat in rice. Paraquat induced injury in KCSox was 19.57%, which was lower than 32.22% injury it induced in wild-type (WT) rice. The paraquat-resistant mechanism was through the increased activity of antioxidant enzymes and the overproduction of endogenous polyamines. The spermine content in KCSox was more than 30 µg mL-1 , while that in WT rice was less than 5 µg mL-1 . Quantitative proteomics showed that ß-ketoacyl-coenzyme A (CoA) synthase (51.81 folds) encoded by the transgenic EiKCS gene promoted the synthesis of the proteins involved with the polyamine pathway. The synthesized putrescine was promoted by the arginine decarboxylase (ADC) pathway. The spermidine synthase I (1.10-fold) and three eceriferum cofactors (CERs) were responsive to the paraquat stress. We validated putrescine (C18 H20 N2 O2 ) spermidine (C28 H31 N3 O3 ), and spermine (C38 H42 N4 O4 ) in this study. CONCLUSION: EiKCS encoding ß-ketoacyl-CoA synthase from goosegrass has been shown as an ideal candidate gene for engineering genetically modified organism (GMO) crops, as its overexpression does not only bring paraquat-resistance, but also have potential benefits without decreasing yield and rice grain quality. © 2021 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

A comprehensive study on the combustion kinetic modeling of typical electronic plastic waste-television set (TV) plastic shell.

Electronic waste is the fastest growing waste stream and one of the most significant constituents is electronic plastics. In this study, the combustion kinetic of typical electronic plastic waste-television set (TV) plastic shell-was investigated using two basic kinetic methods. The reaction mechanism and kinetic compensation effect were probed as well. The thermogravimetric analysis (TGA) revealed that its degradation process can be divided into four stages, namely, reaction initiation stage (20-300 °C), major reaction stage (300-450 °C), minor reaction stage (450-600 °C), and reaction cessation stage (600-1,000 °C). The activation energy (E) were calculated and indicated that, the kinetic parameters from six model-free methods gradually decreased with α increasing from 0.1 to 0.35, and then slightly increased. The Flynn--Wall--Ozawa (FWO) method was more reliable and E values decreased from 155.0 to 147.51 kJ/mol with α range of 0.1-0.35, then gradually increased to 165.21 kJ/mol. Within the Coats--Redfern method, the first-order (F1) model had higher coefficient of determination (R2) and comparable E values with that from FWO method. The result of kinetic compensation effect confirmed that the compensation effect existed between E and A during the plastic waste combustion. A linear relationship lnA = 0.183E-3.11 (R2 = 0.991) was obtained. The pre-exponential factors (A) were also determined as 7.67 × 1010 min-1 based on the F1 reaction model and FWO method.Implications: Municipal solid waste (MSW) is a complex mixture of different components and the plastic takes up a significant portion in total MSW. Understanding the combustion process of typical electronic plastic waste and further probing its combustion kinetic are significant. Through this study, it will be significant for the reactor designing and optimizing in practice.

MMS22L Expression as a Predictive Biomarker for the Efficacy of Neoadjuvant Chemoradiotherapy in Oesophageal Squamous Cell Carcinoma.

Long-term survival in oesophageal squamous cell carcinoma (ESCC) is related with pathological response after neoadjuvant chemoradiotherapy (NCRT) followed by surgery. However, effective biomarkers to predict the pathologic response are still lacking. Therefore, a systematic analysis focusing on genes associated with the efficacy of chemoradiotherapy in ESCC will provide valuable insights into the regulation of molecular processes. By screening publications deposited in PubMed, we collected genes associated with the efficacy of chemoradiotherapy. A specific subnetwork was constructed using the Steiner minimum tree algorithm. Survival analysis in Kaplan-Meier Plotter online resources was performed to explore the relationship between gene mRNA expression and the prognosis of patients with ESCC. Quantitative real-time polymerase chain reaction (qRT-PCR), Western blotting, and immunohistochemical staining (IHC) were used to evaluate the expression of key genes in cell lines and human samples. The areas under the receiver operating characteristic (ROC) curves (AUCs) were used to describe performance and accuracy. Transwell assays assessed cell migration, and cell viability was detected using the Cytotoxicity Assay. Finally, we identified 101 genes associated with efficacy of chemoradiotherapy. Additionally, specific molecular networks included some potential related genes, such as CUL3, MUC13, MMS22L, MME, UBC, VAPA, CYP1B1, and UGDH. The MMS22L mRNA expression level showed the most significant association with the ESCC patient outcome (p < 0.01). Furthermore, MMS22L was downregulated at both the mRNA (p < 0.001) and protein levels in tumour tissues compared with that in normal tissues. Lymph node metastasis was significantly associated with low MMS22L expression (p < 0.01). MMS22L levels were inversely correlated with the NCRT response in ESCC (p < 0.01). The resulting area under the ROC curve was 0.847 (95% CI: 0.7232 to 0.9703; p < 0.01). In conclusion, low expression of MMS22L is associated with poor response to NCRT, worse survival, lymph node metastasis, and enhanced migration of tumour cells in ESCC.

The impact of adjuvant therapy on survival for node-negative esophageal squamous cell carcinoma: a propensity score-matched analysis.

BACKGROUND: At present, the primary treatment of esophageal cancer is surgery-based comprehensive treatment, including adjuvant therapy such as chemotherapy and/or radiotherapy. However, the role of adjuvant therapy for esophageal squamous cell carcinoma (ESCC) with pathologically node-negative (pN0) disease is controversial. This study aimed to evaluate the impact of postoperative adjuvant therapy on survival in patients with pN0 ESCC. METHODS: Patients with ESCC who underwent R0 esophagectomy in the Department of Thoracic Surgery of Sichuan Cancer Hospital from January 2008 to December 2013 were enrolled. Patients were divided into two groups: a surgery alone (Group S) group or a surgery + adjuvant therapy (Group S + A) group. The primary outcomes were overall survival (OS) and disease-free survival (DFS), and every consecutive case was followed up until death or the last follow-up. RESULTS: A total of 387 patients with ESCC patients who had pN0 were enrolled in the study. After propensity score matching (PSM), each group consisted of 150 patients. In the overall cohort, the 5-year OS (75.6% vs. 69.7%; P=0.004) and 5-year DFS (64.9% vs. 48.2%; P=0.003) rates were higher in Group S + A than in Group S. In the matched samples, the same outcomes were observed (5-year OS: 75.6% vs. 69.7%, P=0.026; 5-year DFS: 67.6% vs. 69.6%, P=0.036). Multivariate regression analysis indicated that postoperative chemotherapy was associated with longer OS [hazard ratio (HR): 0.622, 95% confidence interval (CI): 0.416-0.928; P=0.02] and DFS (HR: 0.571, 95% CI: 0.390-0.836; P=0.004); in contrast, T3 stage tumors (HR: 1.953, 95% CI: 1.238-3.082; P=0.004) and <15 lymph node dissections (HR: 1.81; 95% CI: 1.238-2.648; P = 0.002) were found to be independent risk factors for pN0 ESCC. CONCLUSIONS: Adjuvant therapy, especially chemotherapy, prolonged OS and DFS for patients with ESCC who had pN0 disease. Fewer lymph node dissections and T3 stage tumors were independent risk factors for OS and DFS.

The SOX9-MMS22L Axis Promotes Oxaliplatin Resistance in Colorectal Cancer.

BACKGROUND: Colorectal cancer (CRC) is estimated to be one of the most common cancers and the leading cause of cancer-related death worldwide. SOX9 is commonly overexpressed in CRC and participates in drug resistance. In addition, DNA damage repair confers resistance to anticancer drugs. However, the correlation between DNA damage repair and high SOX9 expression is still unclear. In this study, we aimed to investigate the function and the specific underlying mechanism of the SOX9-dependent DNA damage repair pathway in CRC. METHODS: The expression levels of SOX9 and MMS22L in CRC were examined by immunohistochemistry (IHC) and TCGA analysis. RNA sequencing was conducted in RKO SOX9-deficient cells and RKO shControl cells. Mechanistic studies were performed in CRC cells by modulating SOX9 and MMS22L expression, and we evaluated drug sensitivity and DNA damage repair signaling events. In addition, we investigated the effect of oxaliplatin in tumors with SOX9 overexpression and low expression of MMS22L in vivo. RESULTS: Our study showed that SOX9 has a higher expression level in CRC tissues than in normal tissues and predicts poor prognosis in CRC patients. Overexpression and knockdown of SOX9 were associated with the efficacy of oxaliplatin. In addition, SOX9 activity was enriched in the DNA damage repair pathway via regulation of MMS22L expression and participation in DNA double-strand break repair. SOX9 was upregulated and formed a complex with MMS22L, which promoted the nuclear translocation of MMS22L upon oxaliplatin treatment. Moreover, the xenograft assay results showed that oxaliplatin abrogated tumor growth from cells with MMS22L downregulation in mice. CONCLUSIONS: In CRC, activation of the SOX9-MMS22L-dependent DNA damage pathway is a core pathway regulating oxaliplatin sensitivity. Targeting this pathway in oxaliplatin-resistant CRC cells is a promising therapeutic option.

Differences of endogenous polyamines and putative genes associated with paraquat resistance in goosegrass (Eleusine indica L.).

BACKGROUND: Paraquat is one of the most effective herbicides used to control weeds in agricultural management, while the pernicious weed goosegrass (Eleusine indica) has evolved resistance to herbicides, including paraquat. Polyamines provide high-level paraquat resistance in many plants. In the present study, we selected three polyamines, namely, putrescine, spermidine, and spermine, as putative genes to investigate their correlation with paraquat resistance by using paraquat-resistant (R) and paraquat-susceptible (S) goosegrass populations. RESULTS: There was no significant difference in the putrescine nor spermine content between the R and S biotypes. However, 30 and 90 min after paraquat treatment, the spermidine concentration was 346.14-fold and 421.04-fold (P < 0.001) higher in the R biotype than in the S biotype, but the spermidine concentration was drastically reduced to a marginal level after 90 min. Since the transcript level of PqE was low while the spermidine concentration showed a transient increase, the PqE gene was likely involved in the synthesis of the paraquat resistance mechanism, regulation of polyamine content, and synthesis of spermidine and spermine. PqTS1, PqTS2, and PqTS3 encode transporter proteins involved in the regulation of paraquat concentration but showed different transcription patterns with synchronous changes in polyamine content. CONCLUSION: Endogenous polyamines (especially spermidine) play a vital role in paraquat resistance in goosegrass. PqE, PqTS1, PqTS2, and PqTS3 were speculated on the relationship between polyamine metabolism and paraquat resistance. To validate the roles of PqE, PqTS1, PqTS2, and PqTS3 in polyamine transport systems, further research is needed.