Transgenic PDGF-BB sericin hydrogel potentiates bone regeneration of BMP9-stimulated mesenchymal stem cells through a crosstalk of the Smad-STAT pathways

Silk as a natural biomaterial is considered as a promising bone substitute in tissue regeneration.Sericin and fibroin are the main components of silk and display unique features for their programmable mechanical properties,biocompatibility,biodegradability and morphological plasticity.It has been reported that sericin recombinant growth factors(GFs)can support cell proliferation and induce stem cell differentiation through cross-talk of signaling pathways during tissue regeneration.The transgenic technology allows the productions of bioactive heterologous GFs as fusion proteins with sericin,which are then fabricated into solid matrix or hydrogel format.Herein,using an injectable hydrogel derived from transgenic platelet-derived GF(PDGF)-BB silk sericin,we demonstrated that the PDGF-BB sericin hydrogel effectively augmented osteogenesis induced by bone morphogenetic protein(BMP9)-stimulated mesenchymal stem cells(MSCs)in vivo and in vitro,while inhibiting adipogenic differentiation.Further gene expression and protein-protein interactions studies demonstrated that BMP9 and PDGF-BB synergistically induced osteogenic differentiation through the cross-talk between Smad and Stat3 pathways in MSCs.Thus,our results provide a novel strategy to encapsulate osteogenic factors and osteoblastic progenitors in transgenic sericin-based hydrogel for robust bone tissue engineering.

破译pH值和金属离子编程的家蚕纺丝机制

Silk is one of the toughest fibrous materials known despite spun at ambient temperature and pressure with water as a solvent.It is a great challenge to reproduce high-performance artificial fibers comparable to natural silk by bionic for the incomplete understanding of silkworm spinning in vivo.Here,we found that amphipol and digitonin stabilized the structure of natural silk fibroin(NSF)by a large-scale screening in vitro,and then studied the close-to-native ultrastructure and hierarchical assembly of NSF in the silk gland lumen.Our study showed that NSF formed reversible flexible nanofibrils mainly composed of random coils with a sedimentation coefficient of 5.8 S and a diameter of about 4 nm,rather than a micellar or rod-like structure assembled by the aggregation of globular NSF molecules.Metal ions were required for NSF nanofibril formation.The successive p H decrease from posterior silk gland(PSG)to anterior silk gland(ASG)resulted in a gradual increase in NSF hydrophobicity,thus inducing the sol-gelation transition of NSF nanofibrils.NSF nanofibrils were randomly dispersed from PSG to ASG-1,and self-assembled into anisotropic herringbone patterns at ASG-2 near the spinneret ready for silkworm spinning.Our findings reveal the controlled self-assembly mechanism of the multi-scale hierarchical architecture of NSF from nanofibrils to herringbone patterns programmed by metal ions and p H gradient,which provides novel insights into the spinning mechanism of silk-secreting animals and bioinspired design of high-performance fibers.

Targeted DNA N^(6)-methyladenine editing by dCas9 fused to METTL4 in the lepidopteran model insect Bombyx mori

Dear Editor,N^(6)-Methyladenine(6mA)DNA modification is an important epigenetic mechanism with roles in regulating gene expression,nucleosome positioning,DNA damage repair,and cell cycle progression(Heyn&Esteller,2015;Luo et al.,2015;Boulias&Greer,2022).Despite progress in understanding the biological functions of 6mA,the contribution of individual 6mA installations on site-specific target genes is largely unknown,and therefore deciphering the molecular mechanism of 6mA in target gene expression has been difficult.

Genome-wide identification of target genes for transcription factor BR-C in the silkworm,Bombyx mori

Transcription factor Broad Complex(BR-C)is an ecdysone primary response gene in insects and participates in the regulation of insect growth and development.In this study,we performed a genome-wide identification of BR-C target genes in silkworm(Bombyx mori)using chromatin immunoprecipitation followed by high-throughput sequencing(ChIP-seq).As a result,a total of 1006 BR-C ChIP peaks were identified,and 15%of peaks were located in the promoter regions of 133 protein-coding genes.Functional annotation revealed that these ChIP peak-associated genes,as potential BR-C targets,were enriched in pathways related to biosynthetic process,metabolic process,and development.Transcriptome analysis and quantitative real-time polymerase chain reaction(PCR)examination revealed that developmental changes in expression patterns of a portion of potential BR-C targets,including HR96 and GC-otl,were similar to those of BR-C.ChIP-PCR examination confirmed that BR-C could directly bind to the promoters of potential targets.Further,dual luciferase assays demonstrated that HR96 promoter activity was significantly upregulated following BR-C overexpression,and this upregu-lation was abolished when the binding motif in the promoter was truncated.This study will be helpful for deciphering the regulatory roles of BR-C during insect growth and development.

Homeodomain proteins POU-M2, antennapedia and abdominal-B are involved in regulation of the segment-specific expression of the clip-domain serine protease gene CLIP13 in the silkworm, Bombyx mori

Clip-domain serine proteases (CLIPs) play important roles in insect innate immunity and development. Our previous studies indicated that CLIP13, an epidermis-specific gene, was involved in cuticle remodeling during molting and metamorphosis in the silkworm, Bombyx mori. However, the transcriptional regulatory mechanism and regulatory pathways of CLIP13 remained unclear. In the present study, we investigated CLIP13 expression and the regulation pathway controlled by 20-hydroxyecdysone (20E) in the silkworm. At the transcriptional level, expression of CLIP13 exhibited pronounced spatial and temporal specificity in different regions of the epidermis;homeodomain transcription factors POU-M2, antennapedia (Antp), and abdominal-B (Abd-B) showed similar expression change trends as CLIP13 in the head capsule, thorax, and abdomen, respectively. Furthermore, results of cell transfection assays, electrophoretic mobility shift assays, and chromatin immunoprecipitation demonstrated that POU-M2, Antp, and Abd-B were involved in the transcriptional regulation of CLIP13 by directly binding to their cis-response elements in CLIP13 promoter. RNA interference-mediated silencing of POU-M2, Antp, and Abd-B led to a decrease of CLIP13 expression in the head capsule, the epidermis of the 1st to 3rd thoracic segments and the 7th to 10th abdominal segments, respectively. Consistent with CLIP13, 20E treatment significantly upregulated expression of POU-M2, Antp, and Abd-B in the silkworm epidermis. Taken together, these data suggest that 20E positively regulates transcription of CLIP13 via homeodomain proteins POU-M2, Antp, and Abd-B in different regions of the silkworm epidermis during metamorphosis, thus affecting the molting process. Our findings provide new insight into the functions of homeodomain transcription factors in insect molting.

Recent Advances of DNA Hydrogels in Biomedical Applications

DNA hydrogel is one of DNA-based nanomaterials with unique advantages such as precise self-assembly,programmability,addressability,high stability,excellent biocompatibility and biodegradability,and tunable versatility.These features have greatly promoted the development of DNA hydrogels in various applications,especially molecular diagnostics,biosensing,drug delivery,and cancer therapy.In this review,we briefly review the history of DNA hydrogels,the latest advances of DNA hydrogels in biomedical applications especially in biosensing,drug delivery and cancer therapy,and prospect the key challenges and future directions.

Genetically engineered FGF1-sericin hydrogel material treats intrauterine adhesion and restores fertility in rat

Endometrial injury can cause intrauterine adhesions(IUA)and induce the formation of endometrial fibrosis,leading to infertility and miscarriage.At present,there is no effective treatment method for severe IUA and uterine basal injury with adhesion area larger than one-third of the uterus.In this study,we prepared FGF1 silk sericin hydrogel material(FGF1-SS hydrogel)to treat endometrial injury and prevent endometrial fibrosis.Compared with the silk sericin hydrogel material(WT-SS hydrogel),FGF1-SS hydrogel significantly promotes the cell migration and infiltration ability of endometrial stromal cells(ESCs).More importantly,FGF1-SS hydrogel can release FGF1 stably for a long time and inhibit the ESCs injury model forms fibrosis through the TGF-β/Smad pathway.In the IUA rat model,FGF1-SS hydrogel treatment effectively restored the number of uterine glands and uterine wall thickness in rats,with a fertility rate of 65.1%66.4%.The results show that FGF1-SS hydrogel is expected to be a candidate to prevent IUA.