An efficient and specific CRISPR-Cas9 genome editing system targeting soybean phytoene desaturase genes.

BACKGROUND: Genome editing by CRISPR/Cas9 has become a popular approach to induce targeted mutations for crop trait improvement. Soybean (Glycine max L. Merr.) is an economically important crop worldwide. Although gene editing has been demonstrated in soybean, its utilization in stably transformed plants through whole plant regeneration is still not widespread, largely due to difficulties with transformation or low mutation efficiencies. RESULTS: We sought to establish a simple, efficient, and specific CRISPR/Cas9 system to induce heritable mutations in soybean through stable transformation. We targeted phytoene desaturase (PDS) genes due to the distinctive dwarf and albino phenotypes of the loss of function mutant. To evaluate gene editing efficiency and specificity, three constructs targeting each of the two homologous soybean PDS genes specifically, as well as two constructs targeting both simultaneously with one guide RNA were created. Instead of using cotyledonary nodes from germinated seedlings, we used 'half-seed' explants derived from imbibed seeds for Agrobacterium-mediated transformation of cultivar Williams 82. Transformed plants for all five constructs were recovered. Dwarf and albino phenotypes were observed in transgenic plants harboring the constructs targeting both PDS genes. Gene editing at the desired loci was detected in the majority of T0 transgenic plants, with 75-100% mutation efficiencies. Indel frequencies varied widely among plants (3-100%), with those exhibiting visible mutant phenotypes showing higher frequencies (27-100%). Deletion was the predominant mutation type, although 1-nucleotide insertion was also observed. Constructs designed to target only one PDS gene did not induce mutation in the other homologous counterpart; and no mutation at several potential off-target loci was detected, indicating high editing specificity. Modifications in both PDS genes were transmitted to T1 progenies, including plants that were negative for transgene detection. Strong mutant phenotypes were also observed in T1 plants. CONCLUSIONS: Using simple constructs containing one guide RNA, we demonstrated efficient and specific CRISPR/Cas9-mediated mutagenesis in stably transformed soybean plants, and showed that the mutations could be inherited in progenies, even in plants that lost transgenes through segregation. The established system can be employed to edit other genes for soybean trait improvement.

Differential Gene Expression of Brachypodium distachyon Roots Colonized by Gluconacetobacter diazotrophicus and the Role of BdCESA8 in the Colonization.

Alternatives to synthetic nitrogen fertilizer are needed to reduce the costs of crop production and offset environmental damage. Nitrogen-fixing bacterium Gluconacetobacter diazotrophicus has been proposed as a possible biofertilizer for monocot crop production. However, the colonization of G. diazotrophicus in most monocot crops is limited and deep understanding of the response of host plants to G. diazotrophicus colonization is still lacking. In this study, the molecular response of the monocot plant model Brachypodium distachyon was studied during G. diazotrophicus root colonization. The gene expression profiles of B. distachyon root tissues colonized by G. diazotrophicus were generated via next-generation RNA sequencing, and investigated through gene ontology and metabolic pathway analysis. The RNA sequencing results indicated that Brachypodium is actively involved in G. diazotrophicus colonization via cell wall synthesis. Jasmonic acid, ethylene, gibberellin biosynthesis. nitrogen assimilation, and primary and secondary metabolite pathways are also modulated to accommodate and control the extent of G. diazotrophicus colonization. Cellulose synthesis is significantly downregulated during colonization. The loss of function mutant for Brachypodium cellulose synthase 8 (BdCESA8) showed decreased cellulose content in xylem and increased resistance to G. diazotrophicus colonization. This result suggested that the cellulose synthesis of the secondary cell wall is involved in G. diazotrophicus colonization. The results of this study provide insights for future research in regard to gene manipulation for efficient colonization of nitrogen-fixing bacteria in Brachypodium and monocot crops.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Development of a Csy4-processed guide RNA delivery system with soybean-infecting virus ALSV for genome editing.

BACKGROUND: A key issue for implementation of CRISPR-Cas9 genome editing for plant trait improvement and gene function analysis is to efficiently deliver the components, including guide RNAs (gRNAs) and Cas9, into plants. Plant virus-based gRNA delivery strategy has proven to be an important tool for genome editing. However, its application in soybean which is an important crop has not been reported yet. ALSV (apple latent spherical virus) is highly infectious virus and could be explored for delivering elements for genome editing. RESULTS: To develop a ALSV-based gRNA delivery system, the Cas9-based Csy4-processed ALSV Carry (CCAC) system was developed. In this system, we engineered the soybean-infecting ALSV to carry and deliver gRNA(s). The endoribonuclease Csy4 effectively releases gRNAs that function efficiently in Cas9-mediated genome editing. Genome editing of endogenous phytoene desaturase (PDS) loci and exogenous 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) sequence in Nicotiana. benthamiana (N. benthamiana) through CCAC was confirmed using Sanger sequencing. Furthermore, CCAC-induced mutagenesis in two soybean endogenous GW2 paralogs was detected. CONCLUSIONS: With the aid of the CCAC system, the target-specific gRNA(s) can be easily manipulated and efficiently delivered into soybean plant cells by viral infection. This is the first virus-based gRNA delivery system for soybean for genome editing and can be used for gene function study and trait improvement.

HD2-type histone deacetylases: unique regulators of plant development and stress responses.

Plants have developed sophisticated and complex epigenetic regulation-based mechanisms to maintain stable growth and development under diverse environmental conditions. Histone deacetylases (HDACs) are important epigenetic regulators in eukaryotes that are involved in the deacetylation of lysine residues of histone H3 and H4 proteins. Plants have developed a unique HDAC family, HD2, in addition to the RPD3 and Sir2 families, which are also present in other eukaryotes. HD2s are well conserved plant-specific HDACs, which were first identified as nucleolar phosphoproteins in maize. The HD2 family plays important roles not only in fundamental developmental processes, including seed germination, root and leaf development, floral transition, and seed development but also in regulating plant responses to biotic and abiotic stresses. Some of the HD2 members coordinate with each other to function. The HD2 family proteins also show functional association with RPD3-type HDACs and other transcription factors as a part of repression complexes in gene regulatory networks involved in environmental stress responses. This review aims to analyse and summarise recent research progress in the HD2 family, and to describe their role in plant growth and development and in response to different environmental stresses.

Characterization of the Role of SPL9 in Drought Stress Tolerance in Medicago sativa.

Extreme environmental conditions, such as drought, are expected to increase in frequency and severity due to climate change, leading to substantial deficiencies in crop yield and quality. Medicago sativa (alfalfa) is an important crop that is relied upon as a staple source of forage in ruminant feed. Despite its economic importance, alfalfa production is constrained by abiotic stress, including drought. In this report, we investigate the role of Squamosa Promoter Binding Protein-Like 9 (SPL9), a target of miR156, in drought tolerance. Transgenic alfalfa plants with RNAi-silenced MsSPL9 (SPL9-RNAi) were compared to wild-type (WT) alfalfa for phenotypic changes and drought tolerance indicators. In SPL9-RNAi plants, both stem thickness and plant height were reduced in two- and six-month-old alfalfa, respectively; however, yield was unaffected. SPL9-RNAi plants showed less leaf senescence and had augmented relative water content under drought conditions, indicating that SPL9-RNAi plants had greater drought tolerance potential than WT plants. Interestingly, SPL9-RNAi plants accumulated more stress-alleviating anthocyanin compared to WT under both drought and well-watered control conditions, suggesting that MsSPL9 may contribute to drought tolerance in alfalfa, at least in part, by regulating anthocyanin biosynthesis. The results suggest that targeting MsSPL9 is a suitable means for improving alfalfa resilience towards drought conditions.

Identification and molecular characterization of the Brachypodium distachyon NRT2 family, with a major role of BdNRT2.1.

The small monocot plant Brachypodium distachyon is rapidly emerging as a powerful model system to study questions unique to the monocot crops. An extensive BLAST search was carried to identify putative orthologues of the Arabidopsis NRT2 genes in the fully sequenced Brachypodium genome. Seven genes encoding putative high-affinity nitrate transporters (BdNRT2) were identified. Transcriptional analysis of individual BdNRT2 gene under various nitrogen sources and levels in the wild-type and a T-DNA mutant of BdNRT2.1 were performed. A transgenic approach was taken to complement the bdnrt2.1 mutant. BdNRT2.1 and BdNRT2.2 were strongly induced by nitrate resupply to nitrogen-starved plants and were classified as inducible genes. BdNRT2.5 was found to be repressed by nitrate resupply whereas other members were constitutively expressed in the root. Interestingly, higher ammonium concentrations also triggered similar gene expression regulation, suggesting BdNRT2 gene expression was also governed by internal nitrogen status, not just external nitrate concentrations. In bdnrt2.1 mutant, the high-affinity transporter system (HATS) was reduced by 30% and BdNRT2.2 and BdNRT2.6 were differentially regulated. This pioneering research demonstrates that genes in the BdNRT2 family have diverse roles, differing from the Arabidopsis AtNRT2 family, in response to various nitrogen conditions. BdNRT2.1 serves as a key member of the family.

C-C motif chemokine 22 ligand (CCL22) concentrations in sera of gastric cancer patients are related to peritoneal metastasis and predict recurrence within one year after radical gastrectomy.

BACKGROUND: Gastric cancer is a common cancer with a poor prognosis. Chemokines play important roles in the tumor microenvironments to support tumor growth and metastasis. The effects of C-C motif chemokine ligand 22 (CCL22) in gastric cancer remain unclear. MATERIALS AND METHODS: Between January 1, 2014 and April 31, 2014, a total of 298 gastric cancer patients were recruited to this study. Circulating concentrations of CCL22 were measured in gastric cancer patients before surgery, at discharged and during follow-up visits. The expression of CCL22 in gastric cancer tumor beds was measured by immunohistochemistry. The proportion of CD3+CD4+CD25+Foxp3+ regulatory T cells in tumor sites was assessed by flow cytometry. RESULTS: Gastric cancer patients had higher serum CCL22 levels compared to healthy controls (P < 0.001). Immunohistochemistry indicated that the gastric cancer tumor beds were the source of serum CCL22, as gastric cancer patients had an increased proportion of strong expression of CCL22 (P < 0.01), and immunohistochemistry scores were positively correlated with levels of circulating CCL22 (P < 0.001). Gastric cancer tissue harbored a higher percentage of regulatory T cells compared to normal tumor-free stomach margins (P < 0.001), and this abundance of regulatory T cells was positively correlated with circulating levels of CCL22 (P < 0.001). Gastric cancer patients with peritoneal metastasis showed increased levels of circulating CCL22 before surgery compared to metastasis-free patients (P < 0.001). Gastric cancer patients with the recurrence within the first year after surgery had elevated serum CCL22 concentrations at different time points compared to those of recurrence-free patients (P < 0.001). Logistic regression analysis indicated that high CCL22 circulating levels before surgery is a risk factor for peritoneal metastasis and an independent risk factor for an early recurrence after surgery. CONCLUSIONS: CCL22 plays an important role in supporting gastric cancer development presumably by increasing the percentage of regulatory T cells in the tumor microenvironments. CCL22 levels in sera have a predictive value for gastric cancer peritoneal metastasis and the early recurrence. Therefore, CCL22 may be a therapeutic target for gastric cancer.

Ectopic expression of miR156 represses nodulation and causes morphological and developmental changes in Lotus japonicus.

The effects of microRNA156 overexpression on general plant architecture, branching, flowering time and nodulation were investigated in the model legume, Lotus japonicus. We cloned an miR156 homolog, LjmiR156a, from L. japonicus, and investigated its SQUAMOSA PROMOTER BINDING PROTEIN LIKE (SPL) genes and its biological function at enhancing vegetative biomass yield, extending flowering time, and its impact on nodulation. Thirteen potential targets for LjmiR156 were identified in vitro and their expression profiles were determined in aerial and underground parts of mature plants, including genes coding for eight SPLs, one WD-40, one RNA-directed DNA polymerase, two transport proteins, and one histidine-phosphotransfer protein. Two SPL and one WD-40 cleavage targets for LjmiR156-TC70253, AU089191, and TC57859-were identified. Transgenic plants with ectopic expression of LjmiR156a showed enhanced branching, dramatically delayed flowering, underdeveloped roots, and reduced nodulation. We also examined the transcript levels of key genes involved in nodule organogenesis and infection thread formation to determine the role of miR156 in regulating symbiosis. Overexpression of LjmiR156a led to repression of several nodulation genes during the early stages of root development such as three ENOD genes, SymPK, POLLUX, CYCLOPS, Cerberus, and Nsp1, and the stimulation of NFR1. Our results show that miR156 regulates vegetative biomass yield, flowering time and nodulation by silencing downstream target SPLs and other genes, suggesting that the miR156 regulatory network could be modified in forage legumes (such as alfalfa and trefoils) and in leafy vegetables (like lettuce and spinach) to positively impact economically valuable crop species.

Evaluation of somatic embryos of alfalfa for recombinant protein expression.

KEY MESSAGE: Somatic embryos of alfalfa can accumulate higher levels of recombinant proteins comparing to vegetative organs. Somatic embryos may be explored as a new system for new protein production for plants. Plants have been explored via genetic engineering as an inexpensive system for recombinant protein production. However, protein expression levels in vegetative tissues have been low, which limits the commercial utilization of plant expression systems. Somatic embryos resemble zygotic embryos in many aspects and may accumulate higher levels of proteins as true seed. In this study, somatic embryo of alfalfa (Medicago sativa L.) was investigated for the expression of recombinant proteins. Three heterologous genes, including the standard scientific reporter uid that codes for ß-glucuronidase and two genes of interest: ctb coding for cholera toxin B subunit (CTB), and hIL-13 coding for human interleukin 13, were independently introduced into alfalfa via Agrobacterium-mediated transformation. Somatic embryos were subsequently induced from transgenic plants carrying these genes. Somatic embryos accumulated approximately twofold more recombinant proteins than vegetative organs including roots, stems, and leaves. The recombinant proteins of CTB and hIL-13 accumulated up to 0.15 and 0.18 % of total soluble protein in alfalfa somatic embryos, respectively. The recombinant proteins expressed in somatic embryos also exhibited biological activities. As somatic embryos can be induced in many plant species and their production can be scaled up via different avenues, somatic embryos may be developed as an efficient expression system for recombinant protein production.

An Arabidopsis homolog of importin ß1 is required for ABA response and drought tolerance.

The import of proteins into the nucleus in response to drought is critical for mediating the reprogramming of gene expression that leads to drought tolerance. However, regulatory mechanisms involved in nuclear protein import remain largely unknown. Here, we have identified an Arabidopsis gene (AtKPNB1) as a homolog of human KPNB1 (importin ß1). AtKPNB1 was expressed in multiple organs, and the protein was localized in the cytoplasm and nucleus. AtKPNB1 was able to facilitate nuclear import of a model protein. Null mutation of AtKPNB1 delayed development under normal growth conditions and increased sensitivity to abscisic acid (ABA) during seed germination and cotyledon development. Inactivation of AtKPNB1 increased stomatal closure in response to ABA, reduced the rate of water loss, and substantially enhanced drought tolerance. AtKPNB1 interacted with several importin α proteins, nucleoporin AtNUP62, and the Arabidopsis Ran proteins. Inactivation of AtKPNB1 did not affect the ABA responsiveness or the expression level or subcellular localization of ABI1, ABI2 or ABI5, key regulators of the ABA signaling pathway. Moreover, phenotypic analysis of epistasis revealed that AtKPNB1 modulates the ABA response and drought tolerance through a pathway that is independent of ABI1 and ABI5. Collectively, our results show that AtKPNB1 is an Arabidopsis importin ß that functions in ABA signaling.

Expression analysis of histone acetyltransferases in rice under drought stress.

Histone acetylation is one of the vital reversible modifications of chromatin structure that regulates gene expression in eukaryotes. Histone acetyltransferases (HATs) and histone deacetylases (HDACs) maintain the homeostasis of histone acetylation. Studies in Arabidopsis have revealed that HATs are involved in plant responses to various stresses including light, temperature, salt and ABA. Drought stress, a very common environmental stress, could cause a range of physiological and biochemical responses in plants involving HATs. Eight HATs in four different families (CBP, GNAT, MYST, and TAF(II)250 family) are known in rice. In this research, four OsHATs, one from each family, were chosen based on in silico domain and promoter analysis for their response under drought conditions. Drought stress was introduced to two-leaf-stage rice seedlings. The effectiveness of drought treatment was confirmed by the measurement of relative water content (RWC). Real-time quantitative polymerase chain reaction analysis demonstrated that drought stress caused a significant increase in the expression of four HATs (OsHAC703, OsHAG703, OsHAF701 and OsHAM701) in rice plants. Additionally, the Western-blot analysis showed that the acetylation level on certain lysine sites of H3 (lysine 9, lysine 18 and lysine 27) and H4 (lysine 5) increased with OsHATs expression. The significant increase in the transcript levels of OsHATs and the acetylation level of lysine residues on Histone H3 and H4 suggest that OsHATs are involved in drought stress responses in rice.

AtHD2D is involved in regulating lateral root development and participates in abiotic stress response in Arabidopsis.

Roots are essential to terrestrial plants, as their growth and morphology are crucial for plant development. The growth of the roots is affected and regulated by several internal and external environmental signals and metabolic pathways. Among them, chromatin modification plays an important regulatory role. In this study, we explore the potential roles of the histone deacetylase AtHD2D in root development and lay the foundation for further research on the biological processes and molecular mechanisms of AtHD2D in the future. Our study indicates that AtHD2D affects the root tip microenvironment homeostasis by affecting the gene transcription levels required to maintain the root tip microenvironment. In addition, we confirmed that AtHD2D is involved in regulating Arabidopsis lateral root development and further explained the possible role of AtHD2D in auxin-mediated lateral root development. AtHD2D can effectively enhance the resistance of Arabidopsis thaliana to abiotic stress. We believe that AtHD2D is involved in coping with abiotic stress by promoting the development of lateral roots. Overexpression of AtHD2D promotes the accumulation of reactive oxygen species (ROS) in roots, indicating that AtHD2D is also involved in developing lateral roots mediated by ROS. Previous studies have shown that the overexpression of AtHD2D can effectively enhance the resistance of Arabidopsis thaliana to abiotic stress. Based on our data, we believe that AtHD2D participates in the response to abiotic stress by promoting the development of lateral roots. AtHD2D-mediated lateral root development provides new ideas for studying the mechanism of HDAC protein in regulating root development.

Biological activity of the tzs gene of nopaline Agrobacterium tumefaciens GV3101 in plant regeneration and genetic transformation.

Agrobacterium tumefaciens has been widely used in plant genetic transformation. Hormone-encoding genes residing in the T-DNA region have been removed, resulting in disarmed Agrobacterium strains that are used in various transformation experiments. Nopaline Agrobacterium strains, however, carry another hormone gene, trans-zeatin synthesizing (tzs), that codes for trans-zeatin in the virulence region of the tumor-inducing plasmids. We investigated the activity and function of the tzs gene of a nopaline Agrobacterium sp. strain GV3101 in plant in vitro regeneration. Leaf explants of tobacco and Nicotiana benthamiana co-cultured with strain GV3101 exhibited active shoot regeneration in media without added plant growth regulators. On medium without plant growth regulators, transgenic shoots were also induced from explants co-cultured with GV3101 containing a binary vector. Enzyme-linked immunosorbent assay showed that cell-free extracts of Agrobacterium sp. strain GV3101 culture contained the trans-zeatin at 860 ng/liter. Polymerase chain reaction using tzs-specific primers showed that the tzs gene was present in strain GV3101 but not in other Agrobacterium strains. The study showed that the tzs gene in GV3101 was actively expressed, and that trans-zeatin produced in the Agrobacterium strain can promote plant shoot regeneration.

Histone acetyltransferases in rice (Oryza sativa L.): phylogenetic analysis, subcellular localization and expression.

BACKGROUND: Histone acetyltransferases (HATs) play an important role in eukaryotic transcription. Eight HATs identified in rice (OsHATs) can be organized into four families, namely the CBP (OsHAC701, OsHAC703, and OsHAC704), TAFII250 (OsHAF701), GNAT (OsHAG702, OsHAG703, and OsHAG704), and MYST (OsHAM701) families. The biological functions of HATs in rice remain unknown, so a comprehensive protein sequence analysis of the HAT families was conducted to investigate their potential functions. In addition, the subcellular localization and expression patterns of the eight OsHATs were analyzed. RESULTS: On the basis of a phylogenetic and domain analysis, monocotyledonous CBP family proteins can be subdivided into two groups, namely Group I and Group II. Similarly, dicotyledonous CBP family proteins can be divided into two groups, namely Group A and Group B. High similarities of protein sequences, conserved domains and three-dimensional models were identified among OsHATs and their homologs in Arabidopsis thaliana and maize. Subcellular localization predictions indicated that all OsHATs might localize in both the nucleus and cytosol. Transient expression in Arabidopsis protoplasts confirmed the nuclear and cytosolic localization of OsHAC701, OsHAG702, and OsHAG704. Real-time quantitative polymerase chain reaction analysis demonstrated that the eight OsHATs were expressed in all tissues examined with significant differences in transcript abundance, and their expression was modulated by abscisic acid and salicylic acid as well as abiotic factors such as salt, cold, and heat stresses. CONCLUSIONS: Both monocotyledonous and dicotyledonous CBP family proteins can be divided into two distinct groups, which suggest the possibility of functional diversification. The high similarities of protein sequences, conserved domains and three-dimensional models among OsHATs and their homologs in Arabidopsis and maize suggested that OsHATs have multiple functions. OsHAC701, OsHAG702, and OsHAG704 were localized in both the nucleus and cytosol in transient expression analyses with Arabidopsis protoplasts. OsHATs were expressed constitutively in rice, and their expression was regulated by exogenous hormones and abiotic stresses, which suggested that OsHATs may play important roles in plant defense responses.

HD2A and HD2C co-regulate drought stress response by modulating stomatal closure and root growth in Arabidopsis.

Histone deacetylase 2 (HD2) is a unique family of histone deacetylases (HDACs) in plants. Despite evidence that certain HD2 family HDACs play an important role in plant growth and stress response, the coordination of HD2s in these processes remains largely unknown. We found that HD2-type, HD2A and HD2C coordinate to play a role in drought stress response in Arabidopsis. We showed that the hd2a.hd2c double mutant (Mac16) exhibit decreased drought survival and increased water loss as compared to the single mutants, hd2a and hd2c. Gene expression analysis showed that the ABI1 and ABI2 genes were upregulated and SLAC1 was downregulated which led to the modified stomatal functioning in the Mac16 as compared to the single mutants. Overexpression of HD2A and HD2C showed enhanced drought survival and decreased water loss. We also showed that the GA2ox1 and GA2ox2 genes, which are involved in the catabolism of bioactive gibberellic acids, were upregulated in the Mac16 as compared to the single mutants, which led to a decreased root growth in the Mac16. Furthermore, we showed that HD2A and HD2C can physically interact and increased genome-wide H3K9 acetylation was observed in the Mac16, compared to the single mutants. Overall, our investigation revealed that HD2A and HD2C coordinate to play a cumulative role in drought stress response and root growth in Arabidopsis.

Arabidopsis DNA methyltransferase AtDNMT2 associates with histone deacetylase AtHD2s activity.

DNA methyltransferase2 (DNMT2) is always deemed to be enigmatic, because it contains highly conserved DNA methyltransferase motifs but lacks the DNA methylation catalytic capability. Here we show that Arabidopsis DNA methyltransferase2 (AtDNMT2) is localized in nucleus and associates with histone deacetylation. Bimolecular fluorescence complementation and pull-down assays show AtDNMT2 interacts with type-2 histone deacetylases (AtHD2s), a unique type of histone deacetylase family in plants. Through analyzing the expression of AtDNMT2: ss-glucuronidase (GUS) fusion protein, we demonstrate that AtDNMT2 has the ability to repress gene expression at transcription level. Meanwhile, the expression of AtDNMT2 gene is altered in athd2c mutant plants. We propose that AtDNMT2 possibly involves in the activity of histone deacetylation and plant epigenetic regulatory network.

BdHD1, a histone deacetylase of Brachypodium distachyon, interacts with two drought-responsive transcription factors, BdWRKY24 and BdMYB22.

Histone deacetylases (HDACs) play an important role in plant stress response. In Brachypodium distachyon, which is model species for molecular biology research on monocot plants, the histone deacetylase BdHD1, homologous to AtHDAC1 of the RPD3/HDA1 class, functions as a positive regulator in the plant drought stress response. AtHDAC1 has been found to interact with transcription factors to regulate gene expression. However, the drought-responsive transcription factors that interact with BdHD1 have not been identified yet. Previously, we identified BdWRKY24 and BdMYB22 as drought responsive transcription factors in Brachypodium. In this study, we used yeast two-hybrid (Y2 H) and bimolecular fluorescence complementation (BiFC) assays to show that BdHD1 interacts with BdWRKY24 and BdMYB22. Our findings provides a base to investigate BdHD1-transcription factor complexes in the context of drought stress response in Brachypodium.

Roxatidine inhibits fibrosis by inhibiting NF­κB and MAPK signaling in macrophages sensing breast implant surface materials.

Capsular contracture is an important complication after silicone mammary implant surgery. Fibroblasts and macrophages play critical roles in the pathogenesis of capsular contracture, making these two cell types therapeutic targets. It has been reported that inhibiting histamine receptors results attenuates fibrosis, but the role of roxatidine (a histamine receptor 2 inhibitor) in preventing fibrosis caused by breast implant materials remains unknown. The aim of the present study was to assess the hypothesis that roxatidine might have a prophylactic effect in capsular contracture induced by implant material. Inflammation induced by breast implant materials was mimicked by co­culturing macrophages or fibroblasts with these materials in vitro. Capsular contracture was modeled in mice by planting breast implant materials in a subcutaneous pocket. Roxatidine was added in the culture medium or administered to mice bearing breast implant materials. By co­culturing macrophages or fibroblasts with common breast implant materials (micro­textured or smooth breast implants), the present study demonstrated that macrophages respond to these materials by producing pro­inflammatory cytokines, a process that was abolished by addition of roxatidine to the culture medium. Although fibroblasts did not respond to implant surface materials in the same way as macrophages, the conditioned media of macrophages induced proliferation of fibroblasts. Mechanistically, administration of roxatidine inhibited activation of NF­κB and p38/mitogen­activated protein kinase (MAPK) signaling in macrophages. Furthermore, treatment with roxatidine in implant­bearing mice reduced serum concentrations of transforming growth factor­ß and the abundance of fibroblasts around the implant. The present study concluded that roxatidine plays an important role in preventing fibrosis by inhibiting activation of NF­κB and p38/MAPK signaling in macrophages.

Therapy With Carboplatin and Anti-PD-1 Antibodies Before Surgery Demonstrates Sustainable Anti-Tumor Effects for Secondary Cancers in Mice With Triple-Negative Breast Cancer.

Patients with triple-negative breast cancer (TNBC) suffer an unfavorable prognosis. Carboplatin (CBDCA) as a cytotoxic reagent has been widely administered to patients with cancer including TNBC. Programmed cell death protein 1 (PD-1) is an immune checkpoint, blockade of which unleashes T cell functions that kill cancer cells. However, the efficacy of CBDCA combined with anti-PD-1 antibodies in TNBC has not been determined. Patient-derived xenografts (PDX) were implanted to immune-deficient mice. Three mouse TNBC cell lines (4T1, EMT6, and E0771) were seeded to immune-competent mice. Tumor volumes and survival rates were monitored. CBDCA and anti-PD-1 antibodies were administered by intra-peritoneal injection at designated time points. Total CD8+ T cells, memory CD8+ T cells, and CD103+ dendritic cells (DC) in the tumor were measured by flow cytometry. Tumor-specific CD8+ T cells were quantified by the ELISpot assay. Administration of CBDCA to PDX-bearing mice induced increased levels of tumor cell necrosis and reduced tumor size. Treatment with CBDCA and anti-PD-1 antibodies reduced TNBC tumor volumes and slightly improved survival rates. More importantly, therapy with CBDCA and anti-PD-1 antibodies before surgery showed a remarkably improved, sustainable protection against a secondary tumor after surgery by a CD8+- T-cell-dependent manner, which required CCL4 expressed in the tumor and subsequently CD103+ DC recruited to the tumor microenvironment. Immunochemotherapy with CBDCA and anti-PD-1 antibodies before surgery improves the outcome of a secondary tumor after surgery via increasing the number of tumor-specific CD8+ T cells in the tumor microenvironment of murine TNBC. These results highlight the possibility to utilize this regimen in clinical practice.

Regulation of oleosin expression in developing peanut (Arachis hypogaea L.) embryos through nucleosome loss and histone modifications.

Nucleosome loss and histone modifications are important mechanisms for transcriptional regulation. Concomitant changes in chromatin structures of two peanut (Arachis hypogaea L.) oleosin genes, AhOleo17.8 and AhOleo18.5, were examined in relation to transcriptional activity. Spatial and temporal expression analyses showed that both AhOleo17.8 and AhOleo18.5 promoters can adopt three conformational states, an inactive state (in vegetative tissues), a basal activated state (in early maturation embryos), and a fully activated state (in late maturation embryos). Chromatin immunoprecipitation assays revealed an increase of histone H3 acetylation levels at the proximal promoters and coding regions of AhOleo17.8 and AhOleo18.5 associated with basal transcription in early maturation embryos. Meanwhile, a decrease of histone H3K9 dimethylation levels at coding regions of oleosins was observed in early maturation embryos. However, a dramatic decrease in the histone acetylation signal was observed at the core promoters and the coding regions of the two oleosins in the fully activated condition in late maturation embryos. Although a small decrease of histone H3 levels of oleosins chromatin was detected in early maturation embryos, a significant loss of histone H3 levels occurred in late maturation embryos. These analyses indicate that the histone eviction from the proximal promoters and coding regions is associated with the high expression of oleosin genes during late embryos maturation. Moreover, the basal expression of oleosins in early maturation embryos is accompanied by the increase of histone H3 acetylation and decrease of histone H3K9me2.