Key message
CRISPR/Cas9-based multiplexed editing of SlHyPRP1 resulted in precise deletions of its functional motif(s), thereby resulting in salt stress-tolerant events in cultivated tomato.
Crop ...genetic improvement to address environmental stresses for sustainable food production has been in high demand, especially given the current situation of global climate changes and reduction of the global food production rate/population rate. Recently, the emerging clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas)-based targeted mutagenesis has provided a revolutionary approach to crop improvement. The major application of CRISPR/Cas in plant genome editing has been the generation of indel mutations via error-prone nonhomologous end joining (NHEJ) repair of DNA DSBs. In this study, we examined the power of the CRISPR/Cas9-based novel approach in the precise manipulation of protein domains of tomato hybrid proline-rich protein 1 (HyPRP1), which is a negative regulator of salt stress responses. We revealed that the precise elimination of SlHyPRP1 negative-response domain(s) led to high salinity tolerance at the germination and vegetative stages in our experimental conditions. CRISPR/Cas9-based domain editing may be an efficient tool to engineer multidomain proteins of important food crops to cope with global climate changes for sustainable agriculture and future food security.
Glioblastoma (GBM), the most severe and common brain tumor in adults, is characterized by multiple somatic mutations and aberrant activation of inflammatory responses. Immune cell infiltration and ...subsequent inflammation cause tumor growth and resistance to therapy. Somatic loss-of-function mutations in the gene encoding tumor suppressor protein p53 (TP53) are frequently observed in various cancers. However, numerous studies suggest that TP53 regulates malignant phenotypes by gain-of-function (GOF) mutations. Here we demonstrate that a TP53 GOF mutation promotes inflammation in GBM. Ectopic expression of a TP53 GOF mutant induced transcriptomic changes, which resulted in enrichment of gene signatures related to inflammation and chemotaxis. Bioinformatics analyses revealed that a gene signature, upregulated by the TP53 GOF mutation, is associated with progression and shorter overall survival in GBM. We also observed significant correlations between the TP53 GOF mutation signature and inflammation in the clinical database of GBM and other cancers. The TP53 GOF mutant showed upregulated C-C motif chemokine ligand 2 (CCL2) and tumor necrosis factor alpha (TNFA) expression via nuclear factor kappa B (NFκB) signaling, consequently increasing microglia and monocyte-derived immune cell infiltration. Additionally, TP53 GOF mutation and CCL2 and TNFA expression correlated positively with tumor-associated immunity in patients with GBM. Taken together, our findings suggest that the TP53 GOF mutation plays a crucial role in inflammatory responses, thereby deteriorating prognostic outcomes in patients with GBM.
Summary
Genome editing via the homology‐directed repair (HDR) pathway in somatic plant cells is very inefficient compared with error‐prone repair by nonhomologous end joining (NHEJ). Here, we ...increased HDR‐based genome editing efficiency approximately threefold compared with a Cas9‐based single‐replicon system via the use of de novo multi‐replicon systems equipped with CRISPR/LbCpf1 in tomato and obtained replicon‐free but stable HDR alleles. The efficiency of CRISPR/LbCpf1‐based HDR was significantly modulated by physical culture conditions such as temperature and light. Ten days of incubation at 31 °C under a light/dark cycle after Agrobacterium‐mediated transformation resulted in the best performance among the tested conditions. Furthermore, we developed our single‐replicon system into a multi‐replicon system that effectively increased HDR efficiency. Although this approach is still challenging, we showed the feasibility of HDR‐based genome editing of a salt‐tolerant SlHKT1;2 allele without genomic integration of antibiotic markers or any phenotypic selection. Self‐pollinated offspring plants carrying the HKT1;2 HDR allele showed stable inheritance and germination tolerance in the presence of 100 mm NaCl. Our work may pave the way for transgene‐free editing of alleles of interest in asexually and sexually reproducing plants.
This study looks at foreign aid as a channel to transfer clean technologies to developing countries, including those with smaller economies. The focus is on the technological knowledge transfer ...through the technical cooperation of foreign aid. As transferred knowledge accumulates, the recipient country's technological capacity increases. To better understand this accumulating capacity, this study explores the effect of foreign aid as a channel of technology transfer on the recipients’ technological capacity in the renewable energy sector. To investigate the effect of hands-on assistance, the empirical model estimates the long-term effects of foreign-aid disbursements for non-hydro renewable energy (NHRE) projects. I find that foreign aid for technical cooperation in NHRE projects catalyzes investments in NHRE capacity in low-income countries with a long incubation period. The findings confirm that hands-on cooperation, as emphasized by developing countries, contributes to their capacity building, although only in countries with low capacity to begin with. This study contributes to the ongoing discussions on the effectiveness of technology transfer channels for clean energy.
•Tacit knowledge transfer in aid benefits only the low-income recipient countries.•Technical cooperation needs at least 6 years to realize its full effect.•Lower-middle-income aid recipient countries benefit more from FDI than from aid.
Efficient delivery of tumor antigens and immunostimulatory adjuvants into lymph nodes is crucial for the maturation and activation of antigen-presenting cells (APCs), which subsequently induce ...adaptive antitumor immunity. A dissolving microneedle (MN) has been considered as an attractive method for transcutaneous immunization due to its superior ability to deliver vaccines through the stratum corneum in a minimally invasive manner. However, because dissolving MNs are mostly prepared using water-soluble sugars or polymers for their rapid dissolution in intradermal fluid after administration, they are often difficult to formulate with poorly water-soluble vaccine components. Here, we develop amphiphilic triblock copolymer-based dissolving MNs in situ that generate nanomicelles (NMCs) upon their dissolution after cutaneous application, which facilitate the efficient encapsulation of poorly water-soluble Toll-like receptor 7/8 agonist (R848) and the delivery of hydrophilic antigens. The sizes of NMCs range from 30 to 40 nm, which is suitable for the efficient delivery of R848 and antigens to lymph nodes and promotion of cellular uptake by APCs, minimizing systemic exposure of the R848. Application of MNs containing tumor model antigen (OVA) and R848 to the skin of EG7-OVA tumor-bearing mice induced a significant level of antigen-specific humoral and cellular immunity, resulting in significant antitumor activity.
We previously reported that the ethyl acetate (EtOAc) fraction of a 70% ethanol extract of
(ESE) inhibits varicella-zoster virus (VZV) and human cytomegalovirus (HCMV) replication in vitro. PGG ...(1,2,3,4,6-penta-O-galloyl-ß-D-glucose) is a major chemical constituent of the EtOAc fraction of ESE that inhibits VZV but not HCMV replication. In this study, we comprehensively screened the chemical compounds identified in the EtOAc fraction of ESE for potential antiviral properties. Among the examined compounds, quercetin and isoquercitrin displayed potent antiviral activities against both VZV and HCMV with no significant cytotoxic effects. Both compounds strongly suppressed the expression of VZV and HCMV immediate-early (IE) genes. Our collective results indicated that, in addition to PGG, quercetin and isoquercitrin are bioactive compounds in the EtOAc fraction of ESE that effectively inhibit human herpesvirus replication.
2'‐Fucosyllactose (2'‐FL) is the most abundant oligosaccharide in human milk and one of the most actively studied human milk oligosaccharides (HMOs). When 2'‐FL is produced through biological ...production using a microorganism, like Escherichia coli, d‐lactose is often externally fed as an acceptor substrate for fucosyltransferase (FT). When d‐glucose is used as a carbon source for the cell growth and d‐lactose is transported by lactose permease (LacY) in lac operon, d‐lactose transport is under the control of catabolite repression (CR), limiting the supply of d‐lactose for FT reaction in the cell, hence decreasing the production of 2'‐FL. In this study, a remarkable increase of 2'‐FL production was achieved by relieving the CR from the lac operon of the host E. coli BL21 and introducing adequate site‐specific mutations into α‐1,2‐FT (FutC) for enhancement of catalytic activity and solubility. For the host engineering, the native lac promoter (Plac) was substituted for tac promoter (Ptac), so that the lac operon could be turned on, but not subjected to CR by high d‐glucose concentration. Next, for protein engineering of FutC, family multiple sequence analysis for conserved amino acid sequences and protein–ligand substrate docking analysis led us to find several mutation sites, which could increase the solubility of FutC and its activity. As a result, a combination of four mutation sites (F40S/Q150H/C151R/Q239S) was identified as the best candidate, and the quadruple mutant of FutC enhanced 2'‐FL titer by 2.4‐fold. When the above‐mentioned E. coli mutant host transformed with the quadruple mutant of futC was subjected to fed‐batch culture, 40 g l−1 of 2'‐FL titer was achieved with the productivity of 0.55 g l−1 h−1 and the specific 2'‐FL yield of 1.0 g g−1 dry cell weight.
The increased production of 2'‐Fucosyllactose in Escherichia coli was achieved through the engineering of lactose operon for relieving catabolite repression from the operon, and the engineering of α‐1,2‐fucosyltransferase for increasing its solubility.
Obesity and metabolic disorders caused by alterations in lipid metabolism are major health issues in developed, affluent societies. Adipose tissue is the only organ that stores lipids and prevents ...lipotoxicity in other organs. Mature adipocytes can affect themselves and distant metabolism‐related tissues by producing various adipokines, including adiponectin and leptin. The engulfment adaptor phosphotyrosine‐binding domain‐containing 1 (GULP1) regulates intracellular trafficking of glycosphingolipids and cholesterol, suggesting its close association with lipid metabolism. However, the role of GULP1 in adipocytes remains unknown. Therefore, this study aimed to investigate the function of GULP1 in adipogenesis, glucose uptake, and the insulin signaling pathway in adipocytes. A 3T3‐L1 cell line with Gulp1 knockdown (shGulp1) and a 3T3‐L1 control group (U6) were established. Changes in shGulp1 cells due to GULP1 deficiency were examined and compared to those in U6 cells using microarray analysis. Glucose uptake was monitored via insulin stimulation in shGulp1 and U6 cells using a 2‐NBDG glucose uptake assay, and the insulin signaling pathway was investigated by western blot analysis. Adipogenesis was significantly delayed, lipid metabolism was altered, and several adipogenesis‐related genes were downregulated in shGulp1 cells compared to those in U6 cells. Microarray analysis revealed significant inhibition of peroxisome proliferator‐activated receptor signaling in shGulp1 cells compared with U6 cells. The production and secretion of adiponectin as well as the expression of adiponectin receptor were decreased in shGulp1 cells. In particular, compared with U6 cells, glucose uptake via insulin stimulation was significantly decreased in shGulp1 cells through the disturbance of ERK1/2 phosphorylation. This is the first study to identify the role of GULP1 in adipogenesis and insulin‐stimulated glucose uptake by adipocytes, thereby providing new insights into the differentiation and functions of adipocytes and the metabolism of lipids and glucose, which can help better understand metabolic diseases.
Cytochrome P450s (CYPs), heme-containing monooxygenases, play important roles in a wide variety of metabolic processes important for development as well as biotic/trophic interactions in most living ...organisms. Functions of some CYP enzymes are similar across organisms, but some are organism-specific; they are involved in the biosynthesis of structural components, signaling networks, secondary metabolisms, and xenobiotic/drug detoxification. Fungi possess more diverse CYP families than plants, animals, or bacteria. Various fungal CYPs are involved in not only ergosterol synthesis and virulence but also in the production of a wide array of secondary metabolites, which exert toxic effects on humans and other animals. Although few studies have investigated the functions of fungal CYPs, a recent systematic functional analysis of CYP genes in the plant pathogen
identified several novel CYPs specifically involved in virulence, asexual and sexual development, and degradation of xenobiotics. This review provides fundamental information on fungal CYPs and a new platform for further metabolomic and biochemical studies of CYPs in toxigenic fungi.
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