CsMLO8/11 are required for full susceptibility of cucumber stem to powdery mildew and interact with CsCRK2 and CsRbohD.

Powdery mildew (PM) is one of the most destructive diseases that threaten cucumber production globally. Efficient breeding of novel PM-resistant cultivars will require a robust understanding of the molecular mechanisms of cucumber resistance against PM. Using a genome-wide association study, we detected a locus significantly correlated with PM resistance in cucumber stem, pm-s5.1. A 1449-bp insertion in the CsMLO8 coding region at the pm-s5.1 locus resulted in enhanced stem PM resistance. Knockout mutants of CsMLO8 and CsMLO11 generated by CRISPR/Cas9 both showed improved PM resistance in the stem, hypocotyl, and leaves, and the double mutant mlo8mlo11 displayed even stronger resistance. We found that reactive oxygen species (ROS) accumulation was higher in the stem of these mutants. Protein interaction assays suggested that CsMLO8 and CsMLO11 could physically interact with CsRbohD and CsCRK2, respectively. Further, we showed that CsMLO8 and CsCRK2 competitively interact with the C-terminus of CsRbohD to affect CsCRK2-CsRbohD module-mediated ROS production during PM defense. These findings provide new insights into the understanding of CsMLO proteins during PM defense responses.

The clinical utility of integrative genomics in childhood cancer extends beyond targetable mutations.

We conducted integrative somatic-germline analyses by deeply sequencing 864 cancer-associated genes, complete genomes and transcriptomes for 300 mostly previously treated children and adolescents/young adults with cancer of poor prognosis or with rare tumors enrolled in the SickKids Cancer Sequencing (KiCS) program. Clinically actionable variants were identified in 56% of patients. Improved diagnostic accuracy led to modified management in a subset. Therapeutically targetable variants (54% of patients) were of unanticipated timing and type, with over 20% derived from the germline. Corroborating mutational signatures (SBS3/BRCAness) in patients with germline homologous recombination defects demonstrates the potential utility of PARP inhibitors. Mutational burden was significantly elevated in 9% of patients. Sequential sampling identified changes in therapeutically targetable drivers in over one-third of patients, suggesting benefit from rebiopsy for genomic analysis at the time of relapse. Comprehensive cancer genomic profiling is useful at multiple points in the care trajectory for children and adolescents/young adults with cancer, supporting its integration into early clinical management.

Oral spatial-to-point cascade targeting "sugar-coated bullets" for precise and safe chemotherapy by intervention Warburg effect.

Glycolysis plays a vital role in the development and progression of tumors. Inhibiting glycolysis via smart and safe methods serves as a promising target for cancer therapy. Here, an oral "sugar-coated bullet" aiming at intervening Warburg effect is designed by coating colloidal mesoporous silica nanoparticles (CMS) encapsulating glycolysis inhibitor shikonin (SHK) with dextran, namely DCMS/SHK. The solubility and drug-loading capacity of SHK were enhanced by the special structure of CMS. Besides, the tempting bullets possess the spatial-to-point cascade targeting ability in delivering SHK from the colonic lumen to colon cancer cells and finally to PKM2. After DCMS/SHK reaches the colon, the dextran is hydrolyzed by dextranase especially existing in the colon site to glucose and the carriers become glucose-coated nanoparticles. The glucose-cloak nanoparticles would be largely endocytosed by tumor cells and complete the efficient delivery of SHK. The encapsulated SHK can prevent the glycolysis of cancer cells and thus inhibit tumor growth effectively. This work presents an ingenious cascade colon-targeting strategy to treat colon cancer by destroying cell energy metabolism.

Cross-Modal Contrastive Hashing Retrieval for Infrared Video and EEG.

It is essential to estimate the sleep quality and diagnose the clinical stages in time and at home, because they are closely related to and important causes of chronic diseases and daily life dysfunctions. However, the existing "gold-standard" sensing machine for diagnosis (Polysomnography (PSG) with Electroencephalogram (EEG) measurements) is almost infeasible to deploy at home in a "ubiquitous" manner. In addition, it is costly to train clinicians for the diagnosis of sleep conditions. In this paper, we proposed a novel technical and systematic attempt to tackle the previous barriers: first, we proposed to monitor and sense the sleep conditions using the infrared (IR) camera videos synchronized with the EEG signal; second, we proposed a novel cross-modal retrieval system termed as Cross-modal Contrastive Hashing Retrieval (CCHR) to build the relationship between EEG and IR videos, retrieving the most relevant EEG signal given an infrared video. Specifically, the CCHR is novel in the following two perspectives. Firstly, to eliminate the large cross-modal semantic gap between EEG and IR data, we designed a novel joint cross-modal representation learning strategy using a memory-enhanced hard-negative mining design under the framework of contrastive learning. Secondly, as the sleep monitoring data are large-scale (8 h long for each subject), a novel contrastive hashing module is proposed to transform the joint cross-modal features to the discriminative binary hash codes, enabling the efficient storage and inference. Extensive experiments on our collected cross-modal sleep condition dataset validated that the proposed CCHR achieves superior performances compared with existing cross-modal hashing methods.

Fine mapping a quantitative trait locus underlying seedling resistance to gummy stem blight using a residual heterozygous lines-derived strategy in cucumber.

Gummy stem blight (GSB), caused by Didymella bryoniae, is one of the most devastating diseases that severely reduces cucumber production. Developing resistant varieties would be an effective strategy to control GSB. Although several GSB-resistant QTLs have been reported, causal genes for GSB resistance have not yet been identified in cucumber. A novel loci gsb3.1 for seedling GSB resistance from the "PI 183967" genotype was previously identified in a 1.7-Mb interval on chromosome 3. In this study, we developed a residual heterozygous line-derived strategy from Recombinant Inbred Lines to perform fine mapping, and with this approach, the gsb3.1 locus was narrowed to a 38 kb interval. There were six predicted genes at the gsb3.1 locus, four of which differed in expression in the GSB-resistant compared to the susceptible lines after fungal inoculation. These candidate genes (Csa3G020050, Csa3G020060, Csa3G020090, and Csa3G020590) within the gsb3.1 locus could be helpful for the genetic study of GSB resistance and marker-assisted selection in cucumber. Phylogenetic analyses indicated that the resistant gsb3.1 allele may uniquely exist in the wild species present in the Indian group, and that nucleotide diversity was significantly reduced in cultivated accessions. Therefore, the gsb3.1 allele could be introgressed into existing commercial cultivars and combined with other resistance QTLs to provide broad-spectrum and robust GSB resistance in cucumber.

A Large-Scale Genomic Association Analysis Identifies the Candidate Genes Regulating Salt Tolerance in Cucumber (Cucumis sativus L.) Seedlings.

Salt stress seriously restricts plant growth and development, affects yield and quality, and thus becomes an urgent problem to be solved in cucumber stress resistance breeding. Mining salt tolerance genes and exploring the molecular mechanism of salt tolerance could accelerate the breeding of cucumber germplasm with excellent salt stress tolerance. In this study, 220 cucumber core accessions were used for Genome-Wide Association Studies (GWAS) and the identification of salt tolerance genes. The salinity injury index that was collected in two years showed significant differences among the core germplasm. A total of seven loci that were associated with salt tolerance in cucumber seedlings were repeatedly detected, which were located on Chr.2 (gST2.1), Chr.3 (gST3.1 and gST3.2), Chr.4 (gST4.1 and gST4.2), Chr.5 (gST5.1), and Chr.6 (gST6.1). Within these loci, 62 genes were analyzed, and 5 candidate genes (CsaV3_2G035120, CsaV3_3G023710, CsaV3_4G033150, CsaV3_5G023530, and CsaV3_6G009810) were predicted via the functional annotation of Arabidopsis homologous genes, haplotype of extreme salt-tolerant accessions, and qRT-PCR. These results provide a guide for further research on salt tolerance genes and molecular mechanisms of cucumber seedlings.

Fine mapping and candidate gene analysis of gummy stem blight resistance in cucumber stem.

KEY MESSAGE: Two candidate genes (Csa6G046210 and Csa6G046240) were identified by fine-mapping gsb-s6.2 for gummy stem blight resistance in cucumber stem. Gummy stem blight (GSB) is a serious fungal disease caused by Didymella bryoniae, that affects cucumber yield and quality worldwide. However, no GSB-resistant genes have been identified in cucumber cultivars. In this study, the wild cucumber accession 'PI 183967' was used as a source of resistance to GSB in adult stems. An F2 population was mapped using resistant line 'LM189' and susceptible line 'LM6' derived from a cross between 'PI 183967' and '931'. By developing InDel and SNP markers, the gsb-s6.2 QTL on Chr. 6 was fine-mapped to a 34 kb interval harboring six genes. Gene Expression analysis after inoculation showed that two candidate genes (Csa6G046210 and Csa6G046240) were induced and differentially expressed between the resistant and susceptible parents, and may be involved in disease defense. Sequence alignment showed that Csa6G046210 encodes a multiple myeloma tumor-associated protein, and it harbored two nonsynonymous SNPs and one InDel in the third and the fourth exons, and two InDels in the TATA-box of the basal promoter region. Csa6G046240 encodes a MYB transcription factor with six variants in the AP2/ERF and MYB motifs in the promoter. These two candidate genes lay the foundation for revealing the mechanism of GSB resistance and may be useful for marker-assisted selection in cucumber disease-resistant breeding.

A mutually beneficial macrophages-mediated delivery system realizing photo/immune therapy.

The accumulation of nanomedicines in tumor tissues determines their therapeutic efficacy. We herein exploit the tropism of macrophages to improve the accumulation and retention time of nanomedicine at tumors. Interestingly, macrophages are not merely as transporters, but killers activated by nanomedicine. The system(M@C-HA/ICG) was established by decorating macrophages with hyaluronic acid-modified hollow mesoporous carbon (C) nanoparticles loading indocyanine green (ICG). Notably, C nanoparticles with superior photothermal conversion capability not merely guarantee the efficient delivery of ICG through high drug loading efficiency and inhibiting the premature leaky, but effectually activate the polarization of macrophages. The results exhibited that those activated macrophages could release pro-inflammatory cytokines (NO, TNF-α, IL-12), while M@C-HA/ICG afforded about 2-fold higher tumor accumulation compared with pure nanoparticle C-HA/ICG and produced heat and singlet oxygen (1O2) under irradiation of an 808 nm laser, realizing the combination of photodynamic therapy (PDT), photothermal therapy (PTT) and cytokines-mediated immunotherapy. Specially, we also investigated the relationship of singlet oxygen (1O2) or temperature and tumor-killing activity for understanding the specific effectual procedure of PDT/PTT synergistic therapy. Overall, we firstly established an "all active" delivery system integrating the features of nanomedicine with biological functions of macrophages, providing a novel insight for cell-mediated delivery platform and tumor targeted multimodality anti-cancer therapy.

A biomimetic nanocomposite made of a ginger-derived exosome and an inorganic framework for high-performance delivery of oral antibodies.

For inflammatory bowel disease (IBD) therapy, systemic exposure of anti-TNF-α antibodies brought by current clinical injection always causes serious adverse effects. Colon-targeted delivery of anti-TNF-α antibodies through the oral route is of great importance but remains a formidable challenge. Here, we reported a biomimetic nanocomposite made of a ginger-derived exosome and an inorganic framework for this purpose. A large mesoporous silicon nanoparticle (LMSN) was uniquely customized for the antibody (infliximab, INF) to load it at high levels up to 61.3 wt% and prevent its aggregation. Exosome-like nanovesicles were isolated from ginger (GE) with a high-level production (17.5 mg kg-1). Then, ultrasound was used to coat GE onto the LMSN to obtain the biomimetic nanocomposite LMSN@GE. As expected, LMSN@GE showed advantages in the oral delivery of INF: stability in the gastrointestinal tract, colon-targeted delivery and high intestinal epithelium permeability. Amazingly, GE also presented an anti-inflammatory effect by blocking the NLRP3 inflammasome in addition to its delivery value. As a result, INF/LMSN@GE showed a significantly higher efficacy in colitis mice compared to the intravenously administered INF. This work provides new insights into colon-targeted delivery of anti-TNF-α antibodies via the oral route. Moreover, it puts forward a novel strategy for drug delivery using one therapeutic agent (herb-derived exosomes).

Tolerable upper intake level of iron damages the liver of weaned piglets.

Iron is one of the essential trace elements, which is often supplemented as an additive to meet the growing needs of toddlers and young animals. Recommended nutrient intake (RNI) and tolerable upper intake levels (UL) are always set when the iron is supplemented. The purpose of this study was to evaluate the subacute (28 days) toxicity of UL iron to weaned piglet liver. Thirty 23-day-old weaned piglets were divided into three groups and, respectively, supplemented with 100, 300 or 3000 (UL) mg/kg iron. UL iron caused significant weight loss in 4th week (p < 0.05). Divalent metal transporter 1(DMT1) decreased significantly, ferroportin 1 and ferritin increased significantly in the liver of UL iron group (p < 0.05). Although there was no significant effect on liver morphology, UL iron significantly increased hepatic iron, reactive oxygen species (ROS), malondialdehyde (MDA) and protein carbonyl (p < 0.05). UL iron significantly reduced glutathione peroxidase (GSH-Px), superoxide dismutase (SOD), catalase (CAT) and total anti-oxidation capacity (T-AOC) in the liver (p < 0.05). Nuclear factor erythroid 2-related factor 2 (Nrf2) activated subunits of glutamate cysteine ligase (Gclc) and glutathione S-transferase A1 (Gsta1) upregulation in the UL iron group liver, thereby increasing resistance to oxidative stress. In conclusion, UL iron supplementation altered iron metabolism, generated free radicals, reduced antioxidant enzyme activity and activated Nrf2 signalling pathway in the weaned piglet liver.

Tolerable upper intake level of iron damages the intestine and alters the intestinal flora in weaned piglets.

Iron supplementation has been an intervention to improve iron storage and prevent iron deficiency anemia in weaned piglets and the recommended nutrient intake (RNI) and tolerable upper intake levels (UL) of iron have been established. The purpose of this study is to investigate the potential harm of UL iron to the gut and microbes of weaned piglets. Thirty 23 day old weaned piglets were assigned to three dietary treatments: a basal diet supplemented with 100 (RNI), 300, and 3000 (UL) mg FeSO4 per kg diet for 28 days. Then, we used the intestinal porcine epithelial cell line (IPEC-1) as a cell model to study the effect of UL iron on the gut of weaned piglets. Weaned piglets showed a significant decrease in villus height after feeding on a UL iron diet (P < 0.05). The protein levels of DMT1 and Zip14 decreased, and the protein levels of ferritin increased in the duodenal mucosa (P < 0.05) of UL iron fed weaned piglets. Moreover, UL iron also increased the content of ROS and malondialdehyde and decreased the activity of superoxide dismutase in the duodenal mucosa of weaned piglets (P < 0.05). The addition of UL iron to the diet significantly reduced the expression of tight junction proteins Claudin-1, Occludin, and ZO-1 in the duodenal mucosa of weaned piglets (P < 0.05). In the IPEC-1 cell model, iron induced the production of cytosolic and mitochondrial ROS and reduced the mitochondrial membrane potential, which in turn led to cellular vacuolation and fibrosis. Furthermore, UL iron significantly altered the cecum flora of weaned piglets, and the relative abundance of Clostridiales, Faecalibacterium, and Prevotellaceae decreased significantly (P < 0.05), while the relative abundance of Desulfovibrio and Anaerovibrio increased significantly (P < 0.05). In conclusion, UL iron caused damage to the intestinal villi, induced oxidative stress, reduced iron absorption protein, damaged the intestinal barrier, and modified the intestinal microbial structure in weaned piglets.

Creation of Early Flowering Germplasm of Soybean by CRISPR/Cas9 Technology.

Soybean is an important economic crop and a typical short-day crop, sensitive to photoperiod, and has narrow geographical adaptative region, which limit the creation of transgenic materials and reduce the breeding efficiency of new varieties. In addition, the genetic transformation efficiency of soybean is lower than that of many other crops, and the available receptor genotypes are limited. In this study, Agrobacterium-mediated transformation were used to introduce the CRISPR/Cas9 expression vector into soybean cultivar Jack and generated targeted mutants of E1 gene controlling soybean flowering. We obtained two novel types of mutations, 11 bp and 40 bp deletion at E1 coding region, respectively, and frameshift mutations produced premature translation termination codons and truncated E1 proteins, causing obvious early flowering under long day condition. In addition, no off-target effects were observed by predicting and analyzing the potential off-target sites of E1 targets. Significant decreased E1 gene expression of two novel mutants showed that the truncated E1 protein disinhibited GmFT2a/5a and increasing GmFT2a/5a gene expressions resulted obvious early flowering. Homozygous trans-clean mutants without T-DNA elements were also obtained and showed early flowering under long day condition. The photo-insensitive soybean transformation receptor we created laid a foundation for breeding excellent transgenic receptors suitable for high latitudes.

QTL mapping pod dehiscence resistance in soybean (Glycine max L. Merr.) using specific-locus amplified fragment sequencing.

KEY MESSAGE: We constructed a high-density genetic linkage map comprising 4,593 SLAF markers using specific-locus amplified fragment sequencing and identified six quantitative trait loci for pod dehiscence resistance in soybean. Pod dehiscence is necessary for propagation in wild soybean (Glycine soja). It is a major component causing yield losses in cultivated soybean, however, and thus, cultivated soybean varieties have been artificially selected for resistance to pod dehiscence. Detecting quantitative trait loci (QTLs) related to pod dehiscence is required for molecular marker-assisted selection for breeding new varieties with pod dehiscence resistance. In this study, we constructed a high-density genetic linkage map using 260 recombinant inbred lines derived from the cultivars of Heihe 43 (pod-indehiscent) (ZDD24325) and Heihe 18 (pod-dehiscent) (ZDD23620). The map contained 4953 SLAF markers spanning 1478.86 cM on 20 linkage groups with an average distance between adjacent markers of 0.53 cM. In total, six novel QTLs related to pod dehiscence were mapped using inclusive composite interval mapping, explaining 7.22-24.44% of the phenotypic variance across 3 years, including three stable QTLs (qPD01, qPD05-1 and qPD08-1), that had been validated by developing CAPS/dCAPS markers. Based on the SNP/Indel and significant differential expression analyses of two parents, seven genes were selected as candidate genes for future study. The high-density map, three stable QTLs and their molecular markers will be helpful for map-based cloning of pod dehiscence resistance genes and marker-assisted selection of pod dehiscence resistance in soybean breeding.

The effect of enzymes on release of trace elements in feedstuffs based on in vitro digestion model for monogastric livestock.

BACKGROUND: This experiment was conducted to study the effect of different feed enzymes (phytase, xylanase, ß-glucanase) on release rate of trace elements (Fe, Cu, Mn and Zn) in 6 commonly used feedstuffs (corn, wheat, barley, soybean meal, wheat bran, wheat middlings) by using an in vitro model, simulating the digestive processes in stomach for 2 h and then in small intestine for 6 h at 39 °C. RESULTS: Phytase raised (P < 0.05) the release rate of Cu and Zn in corn, Cu, Zn and Mn in wheat, Cu in barley, Cu, Zn and Mn in soybean meal, Zn, Fe in wheat bran and Zn, Fe, Mn in wheat middlings. The release rate of various trace elements in feedstuffs was increased after xylanase addition. Compared with the control group, the release rate of soluble Cu in corn, wheat, barley and soybean meal, soluble Zn in corn, wheat and wheat middlings and soluble of Mn in corn, wheat, barley and wheat bran increased (P < 0.05) after xylanase treatment. After the treatment of ß-glucanase, the release rate of soluble Cu in corn, wheat and wheat bran, soluble Fe in barley, soybean meal and wheat bran and soluble Mn in corn and wheat bran all increased (P < 0.05) compared with the control group. In each feedstuff, after corresponding enzyme treatment, the contents of phytic acid, xylan and ß-glucan were significantly lower than those of the control group (P < 0.05). CONCLUSIONS: Results showed that bound trace elements in feedstuffs can be released by feed enzymes. It may be necessary to take the trace elements in feedstuffs into account in the actual feed preparation including feed enzymes.

Effects of intracellular iron overload on cell death and identification of potent cell death inhibitors.

Iron overload causes many diseases, while the underlying etiologies of these diseases are unclear. Cell death processes including apoptosis, necroptosis, cyclophilin D-(CypD)-dependent necrosis and a recently described additional form of regulated cell death called ferroptosis, are dependent on iron or iron-dependent reactive oxygen species (ROS). However, whether the accumulation of intracellular iron itself induces ferroptosis or other forms of cell death is largely elusive. In present study, we study the role of intracellular iron overload itself-induced cell death mechanisms by using ferric ammonium citrate (FAC) and a membrane-permeable Ferric 8-hydroxyquinoline complex (Fe-8HQ) respectively. We show that FAC-induced intracellular iron overload causes ferroptosis. We also identify 3-phosphoinositide-dependent kinase 1 (PDK1) inhibitor GSK2334470 as a potent ferroptosis inhibitor. Whereas, Fe-8HQ-induced intracellular iron overload causes unregulated necrosis, but partially activates PARP-1 dependent parthanatos. Interestingly, we identify many phenolic compounds as potent inhibitors of Fe-8HQ-induced cell death. In conclusion, intracellular iron overload-induced cell death form might be dependent on the intracellular iron accumulation rate, newly identified cell death inhibitors in our study that target ferroptosis and unregulated oxidative cell death represent potential therapeutic strategies against iron overload related diseases.