Genetic testing of PAX8 mutations associated with thyroid dysgenesis in Chinese congenital hypothyroidism patients

Introduction: Thyroid dysgenesis (TD) is the main cause of congenital hypothyroidism (CH), affecting nearly 1 in 2000–3000 newborns worldwide, as the most common neonatal endocrine disorder. Paired box gene 8 (PAX8), expressed during all stages of thyroid follicular cell, plays a key role in thyroid morphogenesis by a complex regulatory network. In conclusion, the genetic mechanism of PAX8 mutant in TD is still ambiguous; therefore, further research is needed. Material and methods: Blood samples were collected from 289 TD patients in Shandong Province, China. Genomic DNA was extracted from peripheral blood. All the exons of PAX8 along with their exon-intro boundaries were amplified by PCR and analysed by Sanger sequencing. Results: We identified three novel PAX8 nonsense mutations in three patients by sequence analysis of PAX8: Patient 1 (c.285C>G, p.Tyr95Ter), Patient 2 (c.747T>G, p.Tyr249Ter), and Patient 3 (c.786C>A, p.Tyr262Ter). All the three patients carrying PAX8 variants had obvious clinical phenotypes of thyroid anomaly, such as hypoplasia and athyreosis. Conclusion: We conducted the largest worldwide PAX8 mutation screening so far in TD patients. Three presumably pathogenic PAX8 mutations were detected in 289 TD cases for the first time, showing the mutation rate of PAX8 is 1.04% in Chinese TD patients. In addition, our study expands the gene mutation spectrum of TD

A chromosome-scale genome sequence of sudangrass (Sorghum sudanense) highlights the genome evolution and regulation of dhurrin biosynthesis

Sudangrass [ Sorghum sudanense (Piper) Stapf] is a hybrid between grain sorghum and its wild relative S. bicolor ssp. verticilliflorum and is grown as a forage crop due to its high biomass production and low dhurrin content compared to sorghum. In this study, we sequenced the sudangrass genome and showed that the assembled genome was 715.95 Mb with 35,243 protein-coding genes. Phylogenetic analysis with whole genome proteomes demonstrated that the sudangrass genome was more similar to US commercial sorghums than to its wild relatives and cultivated sorghums from Africa. We confirmed that at seedling stage, sudangrass accessions contained significantly lower dhurrin as measured by hydrocyanic acid potential (HCN-p) than cultivated sorghum accessions. Genome-wide association study identified a QTL most tightly associated with HCN-p and the linked SNPs were located in the 3’ UTR of Sobic.001G012300 which encodes CYP79A1, the enzyme that catalyzes the first step of dhurrin biosynthesis. As in other grasses such as maize and rice, we also found that copia/gypsy long terminal repeat retrotransposons were more abundant in cultivated than in wild sorghums, implying that crop domestication in the grasses was accompanied by increased copia/gypsy LTR retrotransposon insertions in the genomes

Building osteogenic microenvironments with a double-network composite hydrogel for bone repair

The critical factor determining the in vivo effect of bone repair materials is the microenvironment, which greatly depends on their abilities to promote vascularization and bone formation. However, implant materials are far from ideal candidates for guiding bone regeneration due to their deficient angiogenic and osteogenic microenvironments. Herein, a double-network composite hydrogel combining vascular endothelial growth factor (VEGF)-mimetic peptide with hydroxyapatite (HA) precursor was developed to build an osteogenic microenvironment for bone repair. The hydrogel was prepared by mixing acrylated β-cyclodextrins and octacalcium phosphate (OCP), an HA precursor, with gelatin solution, followed by ultraviolet photo-crosslinking. To improve the angiogenic potential of the hydrogel, QK, a VEGF-mimicking peptide, was loaded in acrylated β-cyclodextrins. The QK-loaded hydrogel promoted tube formation of human umbilical vein endothelial cells and upregulated the expression of angiogenesis-related genes, such as Flt1 , Kdr , and VEGF , in bone marrow mesenchymal stem cells. Moreover, QK could recruit bone marrow mesenchymal stem cells. Furthermore, OCP in the composite hydrogel could be transformed into HA and release calcium ions facilitating bone regeneration. The double-network composite hydrogel integrated QK and OCP showed obvious osteoinductive activity. The results of animal experiments showed that the composite hydrogel enhanced bone regeneration in skull defects of rats, due to perfect synergistic effects of QK and OCP on vascularized bone regeneration. In summary, improving the angiogenic and osteogenic microenvironments by our double-network composite hydrogel shows promising prospects for bone repair

Microfluidics-based fabrication of cell-laden hydrogel microfibers for potential applications in tissue engineering

Fibrous hydrogel scaffolds have recently attracted increasing attention for tissue engineering applications. While a number of approaches have been proposed for fabricating microfibers, it remains difficult for current methods to produce materials that meet the essential requirements of being simple, flexible and bio-friendly. It is especially challenging to prepare cell-laden microfibers which have different structures to meet the needs of various applications using a simple device. In this study, we developed a facile two-flow microfluidic system, through which cell-laden hydrogel microfibers with various structures could be easily prepared in one step. Aiming to meet different tissue engineering needs, several types of microfibers with different structures, including single-layer, double-layer and hollow microfibers, have been prepared using an alginate-methacrylated gelatin composite hydrogel by merely changing the inner and outer fluids. Cell-laden single-layer microfibers were obtained by subsequently seeding mouse embryonic osteoblast precursor cells (MC3T3-E1) cells on the surface of the as-prepared microfibers. Cell-laden double-layer and hollow microfibers were prepared by directly encapsulating MC3T3-E1 cells or human umbilical vein endothelial cells (HUVECs) in the cores of microfibers upon their fabrication. Prominent proliferation of cells happened in all cell-laden single-layer, double-layer and hollow microfibers, implying potential applications for them in tissue engineering

Three-dimensional biofabrication of nanosecond laser micromachined nanofibre meshes for tissue engineered scaffolds

There is a high demand for bespoke grafts to replace damaged or malformed bone and cartilage tissue. Three-dimensional (3D) printing offers a method of fabricating complex anatomical features of clinically relevant sizes. However, the construction of a scaffold to replicate the complex hierarchical structure of natural tissues remains challenging. This paper reports a novel biofabrication method that is capable of creating intricately designed structures of anatomically relevant dimensions. The beneficial properties of the electrospun fibre meshes can finally be realised in 3D rather than the current promising breakthroughs in two-dimensional (2D). The 3D model was created from commercially available computer-aided design software packages in order to slice the model down into many layers of slices, which were arrayed. These 2D slices with each layer of a defined pattern were laser cut, and then successfully assembled with varying thicknesses of 100 μm or 200 μm. It is demonstrated in this study that this new biofabrication technique can be used to reproduce very complex computer-aided design models into hierarchical constructs with micro and nano resolutions, where the clinically relevant sizes ranging from a simple cube of 20 mm dimension, to a more complex, 50 mm-tall human ears were created. In-vitro cell-contact studies were also carried out to investigate the biocompatibility of this hierarchal structure. The cell viability on a micromachined electrospun polylactic-co-glycolic acid fibre mesh slice, where a range of hole diameters from 200 μm to 500 μm were laser cut in an array where cell confluence values of at least 85% were found at three weeks. Cells were also seeded onto a simpler stacked construct, albeit made with micromachined poly fibre mesh, where cells can be found to migrate through the stack better with collagen as bioadhesives. This new method for biofabricating hierarchical constructs can be further developed for tissue repair applications such as maxillofacial bone injury or nose/ear cartilage replacement in the future

Biofabrication : reappraising the definition of an evolving field

Biofabrication is an evolving research field that has recently received significant attention. In particular, the adoption of Biofabrication concepts within the field of Tissue Engineering and Regenerative Medicine has grown tremendously, and has been accompanied by a growing inconsistency in terminology. This article aims at clarifying the position of Biofabrication as a research field with a special focus on its relation to and application for Tissue Engineering and Regenerative Medicine. Within this context, we propose a refined working definition of Biofabrication, including Bioprinting and Bioassembly as complementary strategies within Biofabrication

CircFKBP5 Suppresses Apoptosis and Inflammation and Promotes Osteogenic Differentiation

ABSTRACT: Objectives: Dental pulp stem cells (DPSCs) are a type of mesenchymal stem cell possessing self-renewal and multilineage differentiation capabilities. The dysfunction of DPSCs is related to the pathologic process of pulpitis. The participation of circular RNAs (circRNAs) in DPSC differentiation has been identified. This work focussed on exploring the functions and mechanism of circFKBP5 in DPSC dysfunction evoked by lipopolysaccharide (LPS). Materials and methods: The viability and apoptosis of human DPSCs (hDPSCs) were determined using Cell Counting Kit-8 assay and flow cytometry. Inflammation was analysed by measuring the release of inflammatory cytokines. The osteogenic differentiation of hDPSCs was investigated by performing alkaline phosphatase (ALP) staining and alizarin red S staining and detecting the changes of ALP and runt-related transcription factor 2 (RUNX2) proteins. The dual-luciferase reporter, RNA immunoprecipitation (RIP), and pull-down assays were used to confirm the binding between miR-708-5p and circFKBP5 or G-protein-coupled receptor (GPCR)–kinase interacting protein 2 (GIT2). Results: CircFKBP5 expression was decreased in hDPSCs and, functionally, reexpression of circFKBP5 attenuated LPS-induced apoptosis, inflammation, and inhibition of proliferation ability and osteogenic differentiation in hDPSCs. Mechanistically, circFKBP5 acted as a sponge for miR-708-5p, which was verified to target GIT2. LPS induced miR-708-5p expression in hDPSCs, and knockdown of miR-708-5p protected against LPS-evoked hDPSC dysfunction. Besides, GIT2 expression was decreased in hDPSCs after LPS treatment. Rescue experiments showed that GIT2 could mediate the protective functions of circFKBP5 on hDPSCs under LPS treatment. Conclusions: CircFKBP5 could protect against LPS-induced apoptosis, inflammation, and osteogenic differentiation inhibition in hDPSCs via the miR-708-5p/GIT2 axis

A passively Q-switched Ho:YVO4 Laser at 2.05 μm with Graphene Saturable Absorber

We report a passively Q-switched Ho:YVO4 laser pumped at 1.94 µm with multilayer graphene as a saturable absorber. At the absorbed pump power of 9.3 W, the maximum average output power of 2.2 W was obtained in Ho:YVO4 laser with minimum pulse width of 265.2 ns and pulse repetition rate of 131.6 kHz at 2052.1 nm. In addition, a beam quality factor of M2~1.7 was measured at the maximum output level. This is, as far as we know, the first time that graphene has been used in a passively Q-switched Ho:YVO4 laser

Targeted regions sequencing identified four novel PNPLA1 mutations in two Chinese families with autosomal recessive congenital ichthyosis

Abstract Background Autosomal recessive congenital ichthyosis (ARCI) is a rare genetically heterogeneous cutaneous disease predominantly characterized by erythroderma, generalized abnormal scaling of the whole body and a collodion membrane at birth. Numerous causative genes have been demonstrated to be responsible for ARCI including PNPLA1 which can cause ARCI type 10. The objectives of this study are to describe clinical features of three ARCI patients from two Chinese unrelated families and to identify the underlying causative mutations. Methods Genomic DNA was extracted from peripheral venous blood obtained from the two Chinese ARCI families in Shandong province. Subsequently, targeted regions sequencing (TRS) followed by Sanger sequencing was conducted to identify and validate the likely pathogenic mutations of the ARCI families. Results Genetic analyses revealed four novel PNPLA1 variants that are predicted to be probably to lead to ARCI in three patients of two families. Patient 1 in one family was in compound heterozygous status for c.604delC/p.Arg202Glyfs*27 and c.820dupC/p.Arg274Profs*15, whereas c.738_742delinsCCCACAGATCCTGC/ p.Gly247_Tyr248delinsProGlnIleLeuHis, and c.816dupC/p.Arg274Profs*15 were found in patient 2 and 3 of the other family. In addition, these variants cosegregate in the two pedigrees and are all within highly conserved regions of the PNPLA1 protein, which indicate that the four mutations are likely pathogenic. Conclusion Our findings not only broaden the mutational spectrum of PNPLA1, but also contribute to establishing genotype–phenotype correlations for different forms of ARCI