Characterization of Transgenic Silkworm Yielded Biomaterials with Calcium-Binding Activity.
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ABSTRACT:
SUBMITTER: Wang S
PROVIDER: S-EPMC4944971 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Silk is a naturally occurring biopolymer formed into fibers composed primarily of fibroin and sericin proteins. The outstanding mechanical properties of silk fibroin (SF) provides numerous applications for silk-based biomaterials. However, the canonical approaches for fabricating silk-based biomaterials typically involve degumming to remove the silk sericin (SS) to avoid adverse biological effects. Meanwhile, sericin has multiple biological functions including outstanding hydrophilicity, promoting cell attachment that are useful to exploit in new materials, inspiring the use of sericin-based biomaterials for biomedical applications. However, compared to fibroin, sericin is not a structural protein, thus sericin-based materials do not provide robust mechanical properties. To address this problem, we report an effective method for fabricating silk fibroin-sericin protein (SS-SF) composites directly from whole cocoons, negating the traditional extraction step to remove the sericin. This approach combines the material features from both fibroin as a structural unit and sericin as a biological functional unit, to achieve advantages regarding processing and materials properties, not only simplifying processing and maintaining the mechanical properties of the fibroin by avoiding degumming, but also endowing these SS-SF composite materials with enhanced hydrophilicity and cell adhesion performance to promote cell growth and proliferation. In addition, these protein composites could be fabricated into a variety of materials formats (e.g. films, sponges, monoliths) to fit different biomedical applications.
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Project description:Intracellular Ca2+ signaling regulates cellular activities during embryogenesis and in adult organisms. We generated stable Tg[βactin2:GCaMP6s]stl351 and Tg[ubi:GCaMP6s]stl352 transgenic lines that combine the ubiquitously-expressed Ca2+ indicator GCaMP6s with the transparent characteristics of zebrafish embryos to achieve superior in vivo Ca2+ imaging. Using the Tg[βactin2:GCaMP6s]stl351 line featuring strong GCaMP6s expression from cleavage through gastrula stages, we detected higher frequency of Ca2+ transients in the superficial blastomeres during the blastula stages preceding the midblastula transition. Additionally, GCaMP6s also revealed that dorsal-biased Ca2+ signaling that follows the midblastula transition persisted longer during gastrulation, compared with earlier studies. We observed that dorsal-biased Ca2+ signaling is diminished in ventralized ichabod/β-catenin2 mutant embryos and ectopically induced in embryos dorsalized by excess β-catenin. During gastrulation, we directly visualized Ca2+ signaling in the dorsal forerunner cells, which form in a Nodal signaling dependent manner and later give rise to the laterality organ. We found that excess Nodal increases the number and the duration of Ca2+ transients specifically in the dorsal forerunner cells. The GCaMP6s transgenic lines described here enable unprecedented visualization of dynamic Ca2+ events from embryogenesis through adulthood, augmenting the zebrafish toolbox.
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Project description:BackgroundThe ATP-binding cassette (ABC) transporter superfamily is the largest transporter gene family responsible for transporting specific molecules across lipid membranes in all living organisms. In insects, ABC transporters not only have important functions in molecule transport, but also play roles in insecticide resistance, metabolism and development.ResultsFrom the genome of the silkworm, Bombyx mori, we have identified 51 putative ABC genes which are classified into eight subfamilies (A-H) by phylogenetic analysis. Gene duplication is very evident in the ABCC and ABCG subfamilies, whereas gene numbers and structures are well conserved in the ABCD, ABCE, ABCF, and ABCH subfamilies. Microarray analysis revealed that expression of 32 silkworm ABC genes can be detected in at least one tissue during different developmental stages, and the expression patterns of some of them were confirmed by quantitative real-time PCR. A large number of ABC genes were highly expressed in the testis compared to other tissues. One of the ABCG genes, BmABC002712, was exclusively and abundantly expressed in the Malpighian tubule implying that BmABC002712 plays a tissue-specific role. At least 5 ABCG genes, including BmABC005226, BmABC005203, BmABC005202, BmABC010555, and BmABC010557, were preferentially expressed in the midgut, showing similar developmental expression profiles to those of 20-hydroxyecdysone (20E)-response genes. 20E treatment induced the expression of these ABCG genes in the midgut and RNA interference-mediated knockdown of USP, a component of the 20E receptor, decreased their expression, indicating that these midgut-specific ABCG genes are 20E-responsive.ConclusionIn this study, a genome-wide analysis of the silkworm ABC transporters has been conducted. A comparison of ABC transporters from 5 insect species provides an overview of this vital gene superfamily in insects. Moreover, tissue- and stage-specific expression data of the silkworm ABCG genes lay a foundation for future analysis of their physiological function and hormonal regulation.
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Project description:The CRISPR/Cas9-mediated genome engineering has been shown to efficiently suppress infection by disrupting genes of the pathogen. We recently constructed transgenic lines expressing CRISPR/Cas9 and the double sgRNA target Bombyx mori nucleopolyhedrovirus (BmNPV) immediate early-1 (ie-1) gene in the silkworm, respectively, and obtained four transgenic hybrid lines by G1 generation hybridization: Cas9(-)/sgRNA(-), Cas9(+)/sgRNA(-), Cas9(-)/sgRNA(+), and Cas9(+)/sgRNA(+). We demonstrated that the Cas9(+)/sgRNA(+) transgenic lines effectively edited the target site of the BmNPV genome, and large fragment deletion was observed after BmNPV infection. Further antiviral analysis of the Cas9(+)/sgRNA(+) transgenic lines shows that the median lethal dose (LD50) is 1,000-fold higher than the normal lines after inoculation with occlusion bodies. The analysis of economic characters and off-target efficiency of Cas9(+)/sgRNA(+) transgenic hybrid line showed no significant difference compared with the normal lines. Our findings indicate that CRISPR/Cas9-mediated genome engineering more effectively targets the BmNPV genomes and could be utilized as an insect antiviral treatment.
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Project description:The state-of-the-art of biocomposites and hybrid biomaterials based on calcium orthophosphates that are suitable for biomedical applications is presented in this review. Since these types of biomaterials offer many significant and exciting possibilities for hard tissue regeneration, this subject belongs to a rapidly expanding area of biomedical research. Through successful combinations of the desired properties of matrix materials with those of fillers (in such systems, calcium orthophosphates might play either role), innovative bone graft biomaterials can be designed. Various types of biocomposites and hybrid biomaterials based on calcium orthophosphates, either those already in use or being investigated for biomedical applications, are extensively discussed. Many different formulations, in terms of the material constituents, fabrication technologies, structural and bioactive properties as well as both in vitro and in vivo characteristics, have already been proposed. Among the others, the nanostructurally controlled biocomposites, those containing nanodimensional compounds, biomimetically fabricated formulations with collagen, chitin and/or gelatin as well as various functionally graded structures seem to be the most promising candidates for clinical applications. The specific advantages of using biocomposites and hybrid biomaterials based on calcium orthophosphates in the selected applications are highlighted. As the way from the laboratory to the hospital is a long one, and the prospective biomedical candidates have to meet many different necessities, this review also examines the critical issues and scientific challenges that require further research and development.
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