FgCWM1 modulates TaNDUFA9 to inhibit SA synthesis and reduce FHB resistance in wheat.

BACKGROUND: Fusarium head blight (FHB) significantly impacts wheat yield and quality. Understanding the intricate interaction mechanisms between Fusarium graminearum (the main pathogen of FHB) and wheat is crucial for developing effective strategies to manage and this disease. Our previous studies had shown that the absence of the cell wall mannoprotein FgCWM1, located at the outermost layer of the cell wall, led to a decrease in the pathogenicity of F. graminearum and induced the accumulation of salicylic acid (SA) in wheat. Hence, we propose that FgCWM1 may play a role in interacting between F. graminearum and wheat, as its physical location facilitates interaction effects. RESULTS: In this study, we have identified that the C-terminal region of NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 9 (NDUFA9) could interact with FgCWM1 through the yeast two-hybrid assay. The interaction was further confirmed through the combination of Co-IP and BiFC analyses. Consistently, the results of subcellular localization indicated that TaNDUFA9 was localized in the cytoplasm adjacent to the cell membrane and chloroplasts. The protein was also detected to be associated with mitochondria and positively regulated complex I activity. The loss-of-function mutant of TaNDUFA9 exhibited a delay in flowering, decreased seed setting rate, and reduced pollen fertility. However, it exhibited elevated levels of SA and increased resistance to FHB caused by F. graminearum infection. Meanwhile, inoculation with the FgCWM1 deletion mutant strain led to increased synthesis of SA in wheat. CONCLUSIONS: These findings suggest that TaNDUFA9 inhibits SA synthesis and FHB resistance in wheat. FgCWM1 enhances this inhibition by interacting with the C-terminal region of TaNDUFA9, ultimately facilitating F. graminearum infection in wheat. This study provides new insights into the interaction mechanism between F. graminearum and wheat. TaNDUFA9 could serve as a target gene for enhancing wheat resistance to FHB.

Genome-Wide Screening for Pathogenic Proteins and microRNAs Associated with Parasite-Host Interactions in Trypanosoma brucei.

Tsetse flies are a type of blood-sucking insect living in diverse locations in sub-Saharan Africa. These insects can transmit the unicellular parasite Trypanosoma brucei (T. brucei) which causes African trypanosomiasis in mammals. There remain huge unmet needs for prevention, early detection, and effective treatments for this disease. Currently, few studies have investigated the molecular mechanisms of parasite-host interactions underlying African trypanosomiasis, mainly due to a lack of understanding of the T. brucei genome. In this study, we dissected the genomic and transcriptomic profiles of T. brucei by annotating the genome and analyzing the gene expression. We found about 5% of T. brucei proteins in the human proteome, while more than 80% of T. brucei protein in other trypanosomes. Sequence alignment analysis showed that 142 protein homologs were shared among T. brucei and mammalian genomes. We identified several novel proteins with pathogenic potential supported by their molecular functions in T. brucei, including 24 RNA-binding proteins and six variant surface glycoproteins. In addition, 26 novel microRNAs were characterized, among which five miRNAs were not found in the mammalian genomes. Topology analysis of the miRNA-gene network revealed three genes (RPS27A, UBA52 and GAPDH) involved in the regulation of critical pathways related to the development of African trypanosomiasis. In conclusion, our work opens a new door to understanding the parasite-host interaction mechanisms by resolving the genome and transcriptome of T. brucei.

High Concentration Crystalline Silk Fibroin Solution for Silk-Based Materials.

As a functional biomaterial, silk fibroin has been widely used in drug release, cell encapsulation and tissue regeneration. To meet the requirements of these applications, the properties of silk fibroin-based materials should be finely tunable. Many useful properties of biomaterials emerge from the collective interactions among ordered and disordered domains. Thus, increasing subtle control of silk hierarchical structures is required. As a characteristic of ordered silk fibroin, crystalline silk fibroin (CSF) is an important part of silk fibroin-based biomaterials, but the preparation of CSF solution, especially high concentration CSF solution, remains a challenge. Here, a solution composed of ß-sheet-rich silk fibroin is reported. These CSF were obtained by the sonication of silk fibroin hydrogel, destroying the hydrogel network, and turning silk fibroin hydrogels into CSF solution. These ß-sheet-rich CSF solutions were stable enough for several days or even weeks. In addition, they were typically ordered crystalline domains, which could be mixed with disordered domains and fabricated into porous scaffolds, films, hydrogels and other silk fibroin-based scaffolds with different properties.

High-cytocompatible semi-IPN bio-ink with wide molecular weight distribution for extrusion 3D bioprinting.

The development of 3D printing has recently attracted significant attention on constructing complex three-dimensional physiological microenvironments. However, it is very challenging to provide a bio-ink with cell-harmless and high mold accuracy during extrusion in 3D printing. To overcome this issue, a technique improving the shear-thinning performance of semi-IPN bio-ink, which is universally applicable to all alginate/gelatin-based materials, was developed. Semi-IPN bio-ink prepared by cyclic heating-cooling treatment in this study can reduce the cell damage without sacrificing the accuracy of the scaffolds for its excellent shear-thinning performance. A more than 15% increase in post-printing Cell viability verified the feasibility of the strategy. Moreover, the bio-ink with low molecular weight and wide molecular weight distribution also promoted a uniform cell distribution and cell proliferation in clusters. Overall, this strategy revealed the effects of molecular parameters of semi-IPN bio-inks on printing performance, and the cell activity was studied and it could be widely applicable to construct the simulated extracellular matrix with various bio-inks.

Identification of a Pm4 Allele as a Powdery Mildew Resistance Gene in Wheat Line Xiaomaomai.

Powdery mildew, caused by Blumeria graminis f. sp. tritici (Bgt), is one of the most destructive foliar diseases of wheat. In this study, we combined the bulked segregant RNA sequencing (BSR-seq) and comparative genomics analysis to localize the powdery mildew resistance gene in Chinese landrace Xiaomaomai. Genetic analysis of F1 plants from a crossing of Xiaomaomai × Lumai23 and the derived F2 population suggests that a single recessive gene, designated as pmXMM, confers the resistance in this germplasm. A genetic linkage map was constructed using the newly developed SNP markers and pmXMM was mapped to the distal end of chromosome 2AL. The two flanking markers 2AL15 and 2AL34 were closely linked to pmXMM at the genetic distance of 3.9 cM and 1.4 cM, respectively. Using the diagnostic primers of Pm4, we confirmed that Xiaomaomai carries a Pm4 allele and the gene function was further validated by the virus-induced gene silencing (VIGS). In addition, we systematically analyzed pmXMM in comparison with the other Pm4 alleles. The results suggest that pmXMM is identical to Pm4d and Pm4e at sequence level. Pm4b is also not different from Pm4c according to their genome/amino acid sequences. Only a few nucleotide variances were detected between pmXMM and Pm4a/b, which indicate the haplotype variation of the Pm4 gene.

Transcriptomic profiling of wheat stem during meiosis in response to freezing stress.

Low temperature injury in spring has seriously destabilized the production and grain quality of common wheat. However, the molecular mechanisms underlying spring frost tolerance remain elusive. In this study, we investigated the response of a frost-tolerant wheat variety Zhongmai8444 to freezing stress at the meiotic stage. Transcriptome profiles over a time course were subsequently generated by high-throughput sequencing. Our results revealed that the prolonged freezing temperature led to the significant reductions in plant height and seed setting rate. Cell wall thickening in the vascular tissue was also observed in the stems. RNA-seq analyses demonstrated the identification of 1010 up-regulated and 230 down-regulated genes shared by all time points of freezing treatment. Enrichment analysis revealed that gene activity related to hormone signal transduction and cell wall biosynthesis was significantly modulated under freezing. In addition, among the identified differentially expressed genes, 111 transcription factors belonging to multiple gene families exhibited dynamic expression pattern. This study provided valuable gene resources beneficial for the breeding of wheat varieties with improved spring frost tolerance.

Effects of Chinese herbal medicines on lifespan in Drosophila.

Food ingredients have shown beneficial effect in delaying aging and extend lifespan. There are Chinese herbal medicines in the category of "homology of medicine and food". In order to find out whether these herbal medicines can act as food component to slow aging, this study selected 12 Chinese herbal medicines containing strong antioxidant components, Canarium album, Amomum villosum, Elsholtzia splendens, Foeniculum vulgare, Fructus hordei germinatus, stir-baked Fructus hordei germinatus, Lilium brownie, Citrus medica, Sophora japonica, Myristica fragrans, Herba houttuyniae, Carthamus tinctoriu, and examined the effects on lifespan using Drosophila melanogaster as the model organism. Our results show that the extracts of the 12 Chinese herbal medicines have various effects on longevity. Some reduced the lifespan in both sexes. Some only shortened the lifespan in one sex. Some have no significant effect in both sexes. There are two herbal medicine extended lifespan, but only in females. The present results suggest that herbal medicines may provide potential candidates for anti-aging ingredients.

Viscoelastic Silk Fibroin Hydrogels with Tunable Strength.

Hydrogels are often used as synthetic extracellular matrices (ECMs) for biomedical applications. Natural ECMs are viscoelastic and exhibit partial stress relaxation. However, commonly used hydrogels are typically elastic. Hydrogels developed from ECM-based proteins are viscoelastic, but they often have weak mechanical properties. Here, biocompatible viscoelastic hydrogels with excellent mechanical performance are fabricated by an all aqueous process at body or room temperature. These hydrogels offer obvious stress relaxation and tunable mechanical properties and gelation kinetics. Their compressive modulus can be controlled between 2 kPa and 1.2 MPa, covering a significant portion of the properties of native tissues. Investigation of the gelation mechanism revealed that silk fibroin gelation is caused by the synergistic effects of hydrophobic interaction and hydrogen bonding between silk fibroin molecules. Newly formed crystals serve as the cross-link sites and form a network endowing the hydrogel with stable structure, and the flexible noncrystalline silk nanofibers connect disparate silk fibroin crystals, endowing hydrogels with viscoelastic properties. The all aqueous gelation process avoids complex chemical and physical treatments and is beneficial for encapsulating cells or biomolecules. Encapsulation of chondrocytes results in high initial survival rate (95% ± 1%). These silk fibroin-based viscoelastic hydrogels, combined with superior biocompatible and tunable mechanical properties, represent an exciting option for tissue engineering and regenerative medicine.

Arabidopsis sucrose synthase localization indicates a primary role in sucrose translocation in phloem.

Sucrose synthase (SuSy) is one of two enzyme families capable of catalyzing the first degradative step in sucrose utilization. Several earlier studies examining SuSy mutants in Arabidopsis failed to identify obvious phenotypic abnormalities compared with wild-type plants in normal growth environments, and as such a functional role for SuSy in the previously proposed cellulose biosynthetic process remains unclear. Our study systematically evaluated the precise subcellular localization of all six isoforms of Arabidopsis SuSy via live-cell imaging. We showed that yellow fluorescent protein (YFP)-labeled SuSy1 and SuSy4 were expressed exclusively in phloem companion cells, and the sus1/sus4 double mutant accumulated sucrose under hypoxic conditions. SuSy5 and SuSy6 were found to be parietally localized in sieve elements and restricted only to the cytoplasm. SuSy2 was present in the endosperm and embryo of developing seeds, and SuSy3 was localized to the embryo and leaf stomata. No single isoform of SuSy was detected in developing xylem tissue of elongating stem, the primary site of cellulose deposition in plants. SuSy1 and SuSy4 were also undetectable in the protoxylem tracheary elements, which were induced by the vascular-related transcription factor VND7 during secondary cell wall formation. These findings implicate SuSy in the biological events related to sucrose translocation in phloem.

Effect of Electrospun Silk Fibroin-Silk Sericin Films on Macrophage Polarization and Vascularization.

Biomaterial implantation is followed by an inflammatory cascade dominated by the macrophages, which polarized to the proinflammation M1 phenotype or prohealing M2 phenotype. Generally, silk sericin (SS) is considered to be of high immunogenicity associated with native silk fibers. The blends of silk fibroin (SF) and SS in different mass ratios might elicit different host immune responses and induce macrophage phenotype switch. The objective of this study was to assess the effects of electrospun SF-SS fibrous films with different mass ratios (10:0, 9:1, 8:2, and 7:3) on the macrophage phenotypes and explore the optimal ratio of SF and SS for angiogenesis. Our results indicated that the macrophages were activated by the addition of SS. When the mass ratio of SF and SS reached 7:3, the film displayed the highest degree of vascularization. The macrophages were induced to secrete more M1 and M2 cytokines accompanying with high M2/M1 ratio. Taken together, this study provided a perspective to promote neovascularization by modulating appropriate host response and macrophage phenotypes in tissue engineering field.

Simultaneous nano- and microscale structural control of injectable hydrogels via the assembly of nanofibrous protein microparticles for tissue regeneration.

Injectable hydrogels are advantageous as tissue regeneration scaffolds, as they can be delivered through a minimally invasive injection and seamlessly integrate with the target tissues. However, an important shortcoming of current injectable hydrogels is the lack of simultaneous control over their micro- and nanoscale structures. In this article, the authors report a strategy for developing injectable hydrogels that integrate a fibrous nanostructure and porous microstructure. The hydrogels are prepared by using novel nanofibrous microparticles as the building blocks. The protein based nanofibrous microparticles, fabricated by a spray freezing technology, can be injected through a syringe-needle system. A cell-compatible photocuring process can be deployed to connect the microparticles and form a mechanically robust hydrogel scaffold. The inter-particle voids combined to form the interconnected micropores and the diameter of the nanofibers (100-300 nm) closely mimics that of the native extracellular matrix. Compared to the non-porous hydrogels and non-fibrous hydrogels, the microparticle annealed nanofibrous (MANF) hydrogels potently enhance the osteogenic-marker expression (ALP, Runx2, OCT and BSP) and mineralization of human mesenchymal stem cells in vitro. MANF hydrogels also facilitate cell infiltration and enhance neovasculization in a subcutaneous implantation model in vivo. The capacity of MANF hydrogels to promote bone regeneration is investigated in a calvarial bone repair model. MANF hydrogels demonstrate significant higher bone regeneration after 8 weeks, indicating the significant role of microporosity and nanofibrous architecture in bone regeneration.

Surface Modification of Multiple Bioactive Peptides to Improve Endothelialization of Vascular Grafts.

Endothelialization is an effective approach to prevent thrombus formation and enhance vascular graft survival. Surface modification of biomolecules has been proved to be effective in regulating endothelial cell behaviors. In this study, several peptides including YIGSR, RGD, and REDV sequences are covalently immobilized on the surface of electrospun silk fibroin scaffolds and the effects of combined application of these peptides on cell behaviors are studied. The results show that, compared with the scaffolds modified with single peptides, the scaffolds modified with dual peptides (YIGSR+RGD) could significantly enhance the proliferation of human umbilical vein endothelial cells (HUVECs). However, the combination of REDV+RGD or YIGSR+REDV does not promote the adhesion or proliferation of HUVECs. Notably, YIGSR-modified scaffolds improved HUVEC migration significantly in comparison to REDV- or RGD-modified groups. Moreover, its combination with either of these two peptides also presents excellent effect on cell migration. Thus, all the data suggest that the combined application of peptides might be a promising method to enhance the endothelialization of small-diameter vascular grafts.

Growth factor-free salt-leached silk scaffolds for differentiating endothelial cells.

Recently, controllable kinetic assembly was introduced into the salt-leaching process with silk proteins to form scaffolds, which achieved improvement in tuning the micro-structural and mechanical properties. Here, more control of the kinetic assembly of silk in the process was integrated into salt-leaching process, resulting in significant mechanical modification of the scaffolds generated. Both glycerol additions and treatment to concentrate the protein were used to tune hydrophilic interactions during aqueous solution processing and to reduce beta-sheet formation during the salt-leaching process. These new scaffolds showed gradient changes in elastic modulus in the range of 0.9 to 7.9 kPa. Bone marrow mesenchymal stem cells grew well and showed endothelial differentiation behavior on the scaffolds with optimized stiffness. These results indicated that the introduction of silk kinetic assembly provides an additional option for the control of porous silk scaffold properties.

A methodology for detecting the wound state sensing in terms of its colonization of pathogenic bacteria.

A methodology for wound state sensing in terms of its colonization with pathogenic bacteria such as Staphylococcus aureus or Pseudomonas aeruginosa has been developed. Here we report polydiacetylene (PDA) liposomes containing self-quenched carboxyfluorescein dye are only sensitive to toxins/enzymes secreted by Pathogenic bacteria but not by non-pathogenic species of Escherichia coli (DH5α). The basis of the detection assay is that at high concentration, carboxyfluorescein is non-fluorescent. Following breakdown of the bilayer of liposome containers by bacterial toxins, the dye becomes diluted and "switches on". The methodology can be easily adapted to evaluate the release of payloads from PDA liposomes in terms of fluorescence intensity and in addition to detect the potential interaction mechanism of biomimetic bilayer and pathogenic bacteria. •Self-quenched when encapsulated at high concentration, while fluorescence when diluted in solution•Easy quantification by measuring fluorescence intensity•Simple measurement procedure required (plate reading fluorimeter).

Facile incorporation of REDV into porous silk fibroin scaffolds for enhancing vascularization of thick tissues.

Rapid neovascularization within scaffolds is critical for the regeneration of thick complex tissues. The surface immobilization of peptides and other active molecules have been explored to improve the vascularization capacity of implants. However, the rapid degradation of these molecules, the reaction conditions and cross-linking usually result in decreased vascularization capability. Here, we introduced a temperate, all-aqueous process to achieve bulk porous silk fibroin (SF) scaffolds. A temperature controlled process was used to induce the water stable structure by SF self-assembly. Arg-Glu-Asp-Val (REDV) peptides were added into SF solution and fixed within SF scaffolds during the self-assembly process. The results showed that the functionalized scaffolds markedly promoted the adhesion of endothelial cells in vitro. Moreover, the in vivo studies indicated enhanced cell infiltration in the bulk functionalized SF scaffolds and impressive vascularization at 4 weeks post-implantation. The functionalized scaffolds demonstrated excellent vascularization capability, providing an exciting biomaterial option for thick tissue regeneration.

Enhancing neural differentiation of induced pluripotent stem cells by conductive graphene/silk fibroin films.

Nerve regeneration and function recovery remain challenges for tissue engineering. The application of suitable scaffold in tissue engineering has been demonstrated to be able to enhance nerve regeneration and differentiation. However, a desired scaffold must meet the requirements of good cytocompatibility and high electrical conductivity simultaneously. In this study, a conductive film composed of SF and graphene was successfully fabricated, which was applied to evaluate its effect on the neural differentiation of iPSCs. The conductive film was found to enhance the differentiation of iPSCs toward neurons. In addition, the differentiation was enhanced with graphene contents and highest value was obtained at graphene content of 4%. Thus, the results in this study suggested that 4% G/SF film might be a suitable biomaterial scaffold for application in neural regeneration. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 2973-2983, 2018.

Bacteria-responsive intelligent wound dressing: Simultaneous In situ detection and inhibition of bacterial infection for accelerated wound healing.

The evolved resistance of antibiotics exhibited by some dreaded clinical pathogens and formation of biofilms has caused life-threatening problems for patients with burns and other wounds. Here, in order to avoid antibiotic overuse, and thus decreasing the occurrence of antimicrobial-resistant bacteria, a theranostic wound dressing, composed of biocompatible UV-photocrosslinkable methacrylated gelatin (GelMA) encapsulating both antimicrobial and fluorescent vesicles, has been developed. The system can respond to the microbiological environment of the wound via a simple color change and antimicrobials release only when require and this is in essence passive as they do not respond to their local environments and benign bacteria, and only operates when pathogenic bacteria exist. Both in Vitro and in Vivo study demonstrated that the proposed wound dressing was able to kill/inhibit the growth of methicillin-resistant S. aureus and P. aeruginosa, whilst providing a visual warning of infection, due to vesicle bilayer membrane lysed by toxins secreted by the two strains of pathogens but not by a non-pathogenic Escherichia coli species. The strategy of microbiologically responsive wound dressing proposed here could also be used as a general methodology for the design and fabrication of bacterial responsive functional biomaterials that offer opportunities to combat drug-resistant bacterial infections.

Silk fibroin scavenges hydroxyl radicals produced from a long-term stored water-soluble fullerene system.

Fullerene has been investigated for use in intratracheal instillation and inhalation. However, its use may be compromised due to the formation of reactive oxygen species (ROS) from a long-term stored water-soluble fullerene system, which will result in pulmonary injury. In this study, we investigated the ability of different concentrations of silk fibroin (SF) to scavenge hydroxyl radicals (OH˙) produced by a water-soluble fullerene system. In addition, we elaborated the mechanism of OH˙ formation and scavenging, the degradation of water-soluble fullerene (WSF), and the effects of OH˙ and WSF on the viability of endothelial cells (ECs). WSF was found to rapidly degrade when incubated with SF at 4 °C, which suggested that OH˙ and the deposition of WSF over 5 half-lives might be reduced by mixing WSF with SF. Moreover, it was observed that OH˙ and WSF could generate adverse effects on EC viability, and OH˙ produced by the WSF system on day 55 could be scavenged by SF. Overall, this study indicated that SF as an antioxidant was capable of scavenging OH˙ and accelerating the degradation of WSF, which provides further insight into the application of WSF in intratracheal instillation and inhalation.

Fabrication of Silk Scaffolds with Nanomicroscaled Structures and Tunable Stiffness.

Detailed control of nano- and microstructures in porous biomaterial scaffold systems is important for control of interfacial and biological functions. Self-assembled silk protein nanostructured building blocks were incorporated into salt-leached scaffolds to control these features. Controllable concentration and pH were used to induce the formation of amorphous silk nanofibers in solution and also to reduce ß-sheet transformation during the more traditional salt-leaching process. These new scaffolds showed nanofibrous-microporous structures, reduced ß-sheet content, and tunable mechanical properties. Bone marrow mesenchymal stem cells grew better and showed differentiation behavior on these nanofibrous scaffolds, suggesting cytocompatibility and support for tunable differentiation via the scaffolds. These results suggested a new strategy of designing bioactive silk scaffolds by combining traditional scaffold formation processes with the controllable self-assembly of silk.

Regulating Coupling Efficiency of REDV by Controlling Silk Fibroin Structure for Vascularization.

Controlled and rapid vascularization of engineered tissues remains one of the main challenges for tissue engineering. The immobilization of peptides and other bioactive molecules on the scaffolds has been demonstrated to be able to improve vascularization. However, the density of peptides modified on the scaffold surface is an important factor influencing vascularization. Thus, regulating the coupling efficiency of peptides may be an effective way to adjust vascularization. In this study, two-dimensional (2D) silk fibroin (SF) films and three-dimensional (3D) porous SF scaffolds with different secondary structure were prepared and coupled with Arg-Glu-Asp-Val (REDV) peptide. Compared with the high crystalline scaffolds, more peptides were bound on the scaffolds with low crystalline both in 2D and 3D forms with the result that more endothelial cells adhered on the low crystalline SF scaffolds. In addition, the in vivo angiogenic assays demonstrated that the low crystalline scaffolds showed higher blood vessel density after 28 days of implantation, which was 1.4-times as much as that of the high crystalline group. The results indicated that the peptide density could be controlled by SF structure and that the low crystalline SF scaffolds modified with REDV peptide could be a potential candidate for inducing angiogenesis in tissue engineering applications.