Improving hard metal implant and soft tissue integration by modulating the“inflammatory-fibrous complex”response

Soft tissue integration is one major difficulty in the wide applications of metal materials in soft tissue-related areas.The inevitable inflammatory response and subsequent fibrous reaction toward the metal implant is one key response for metal implant-soft tissue integration.It is of great importance to modulate this inflammatory-fibrous response,which is mainly mediated by the multidirectional interaction between fibroblasts and macrophages.In this study,macrophages are induced to generate M1 and M2 macrophage immune microenvironments.Their cytokine profiles have been proven to have potentially multi-regulatory effects on fibroblasts.The multi-reparative effects of soft tissue cells(human gingival fibroblasts)cultured on metal material(titanium alloy disks)in M1 and M2 immune microenvironments are then dissected.Fibroblasts in the M1 immune microenvironment tend to aggravate the inflammatory response in a pro-inflammatory positive feedback loop,while M2 immune microenvironment enhances multiple functions of fibroblasts in soft tissue integration,including soft tissue regeneration,cell adhesion on materials,and contraction to immobilize soft tissue.Enlighted by the close interaction between macrophages and fibroblasts,we propose the concept of an“inflammatory-fibrous complex”to disclose possible methods of precisely and effectively modulating inflammatory and fibrous responses,thus advancing the development of metal soft tissue materials.

Generating homozygous mutant populations of barley microspores by ethyl methanesulfonate treatment

Induced mutations are important for genetic research and breeding.Mutations induced by physical or chemical mutagenesis are usually heterozygous during the early generations.However,mutations must be fixed prior to phenotyping or field trials,which requires additional rounds of self-pollination.Microspore culture is an effective method to produce double-haploid(DH)plants that are fixed homozygotes.In this study,we conducted ethyl methanesulfonate(EMS)-induced mutagenesis of microspore cultures of barley(Hordeum vulgare)cultivar‘Hua30’and landrace‘HTX’.The EMS concentrations were negatively correlated with the efficiency of callus induction and the frequency of mutant plant regeneration.The two genotypes showed different regeneration efficiencies.The phenotypic variation of the regenerated M1 plants and the presence of genome-wide nucleotide mutations,revealed by whole-genome sequencing,highlight the utility of EMS-induced mutagenesis of isolated microspore cultures for developing DH mutants.Genome-wide analysis of the mutation frequency in the regenerated plants revealed that a considerable proportion of mutations resulted from microspore culture(somaclonal variation)rather than EMS-induced mutagenesis.In addition to producing a population of 1972 homozygous mutant lines that are available for future field trials,this study lays the foundation for optimizing the regeneration efficiency of DH plants and the richness of mutations(mainly by fine-tuning the mutagen dosage).

A reference-guided TILLING by amplicon-sequencing platform supports forward and reverse genetics in barley

Barley is a diploid species with a genome smaller than those of other members of the Triticeae tribe,making it an attractive model for genetic studies in Triticeae crops.The recent development of barley genomics has created a need for a high-throughput platform to identify genetically uniform mutants for gene function investigations.In this study,we report an ethyl methanesulfonate(EMS)-mutagenized population consisting of 8525M_(3) lines in the barley landrace“Hatiexi”(HTX),which we complement with a high-quality de novo assembly of a reference genome for this genotype.The mutation rate within the population ranged from 1.51 to 4.09 mutations per megabase,depending on the treatment dosage of EMS and the mutation discrimination platform used for genotype analysis.We implemented a three-dimensional DNA pooling strategy combined with multiplexed amplicon sequencing to create a highly efficient and cost-effective TILLING(targeting induced locus lesion in genomes)platform in barley.Mutations were successfully identified from 72 mixed amplicons within a DNA pool containing 64 individual mutants and from 56 mixed amplicons within a pool containing 144 individuals.We discovered abundant allelic mutants for dozens of genes,including the barley Green Revolution contributor gene Brassinosteroid insensitive 1(BRI1).As a proof of concept,we rapidly determined the causal gene responsible for a chlorotic mutant by following the MutMap strategy,demonstrating the value of this resource to support forward and reverse genetic studies in barley.