Age-related changes in the ratio of Type I/III collagen and fibril diameter in mouse skin.

The content of type I collagen (COL-I) and type III collagen (COL-III) and the ratio between them not only affect the skin elasticity and mechanical strength, but also determine the fibril diameter. In this research, we investigated the age-related changes in COL-I/COL-III ratio with their formed fibril diameter. The experimental result was obtained from high performance liquid chromatography-mass spectrometer, hydroxyproline determination, picrosirius red staining and transmission electron microscopes (TEM), respectively. The result indicated that the COL-I/COL-III ratio in mouse skin increased with aging. From the 0th to 9th week, the COL-I/COLIII ratio increased from 1.3:1 to 4.5:1. From the 9th to the 18th week, it remained between 4.5:1 and 4.9:1. The total content of COL-I and COL-III firstly increased and then decreased with aging. The TEM result showed that the fibril diameter increased with aging. From the 0th to 9th week, the average fibril diameter increased from 40 to 112 nm; From the 9th to 18th weeks, it increased from 112 to 140 nm. After the 9th week. The fibril diameter showed obvious uneven distribution. Thus, the COL-I/COLIII ratio was proportional to the fibril diameter, but inversely proportional to the uniformity of fibril diameter.

Genome-Wide Identification of DOF Gene Family and the Mechanism Dissection of SbDof21 Regulating Starch Biosynthesis in Sorghum.

Starch is one of the main utilization products of sorghum (Sorghum bicolor L.), the fifth largest cereal crop in the world. Up to now, the regulation mechanism of starch biosynthesis is rarely documented in sorghum. In the present study, we identified 30 genes encoding the C2-C2 zinc finger domain (DOF), with one to three exons in the sorghum genome. The DOF proteins of sorghum were divided into two types according to the results of sequence alignment and evolutionary analysis. Based on gene expressions and co-expression analysis, we identified a regulatory factor, SbDof21, that was located on chromosome 5. SbDof21 contained two exons, encoding a 36.122 kD protein composed of 340 amino acids. SbDof21 co-expressed with 15 genes involved in the sorghum starch biosynthesis pathway, and the Pearson correlation coefficients (PCCs) with 11 genes were greater than 0.9. The results of qRT-PCR assays indicated that SbDof21 is highly expressed in sorghum grains, exhibiting low relative expression levels in the tissues of roots, stems and leaves. SbDOF21 presented as a typical DOF transcription factor (TF) that was localized to the nucleus and possessed transcriptional activation activity. Amino acids at positions 182-231 of SbDOF21 formed an important structure in its activation domain. The results of EMSA showed that SbDOF21 could bind to four tandem repeats of P-Box (TGTAAAG) motifs in vitro, such as its homologous proteins of ZmDOF36, OsPBF and TaPBF. Meanwhile, we also discovered that SbDOF21 could bind and transactivate SbGBSSI, a key gene in sorghum amylose biosynthesis. Collectively, the results of the present study suggest that SbDOF21 acts as an important regulator in sorghum starch biosynthesis, exhibiting potential values for the improvement of starch contents in sorghum.

Profiling of transcriptional regulators associated with starch biosynthesis in sorghum (Sorghum bicolor L.).

Starch presents as the major component of grain endosperm of sorghum (Sorghum bicolor L.) and other cereals, serving as the main energy supplier for both plants and animals, as well as important industrial raw materials of human beings, and was intensively concerned world widely. However, few documents focused on the pathway and transcriptional regulations of starch biosynthesis in sorghum. Here we presented the RNA-sequencing profiles of 20 sorghum tissues at different developmental stages to dissect key genes associated with sorghum starch biosynthesis and potential transcriptional regulations. A total of 1,708 highly expressed genes were detected, namely, 416 in grains, 736 in inflorescence, 73 in the stalk, 215 in the root, and 268 genes in the leaf. Besides, 27 genes encoded key enzymes associated with starch biosynthesis in sorghum were identified, namely, six for ADP-glucose pyrophosphorylase (AGPase), 10 for starch synthases (SSs), four for both starch-branching enzymes (SBE) and starch-debranching enzymes (DBEs), two for starch phosphorylases (SPs), and one for Brittle-1 (BT1). In addition, 65 transcription factors (TFs) that are highly expressed in endosperm were detected to co-express with 16 out of 27 genes, and 90 cis-elements were possessed by all 27 identified genes. Four NAC TFs were cloned, and the further assay results showed that three of them could in vitro bind to the CACGCAA motif within the promoters of SbBt1 and SbGBSSI, two key genes associated with starch biosynthesis in sorghum, functioning in similar ways that reported in other cereals. These results confirmed that sorghum starch biosynthesis might share the same or similar transcriptional regulations documented in other cereals, and provided informative references for further regulatory mechanism dissection of TFs involved in starch biosynthesis in sorghum.

Quantitation of Collagen Type V in Tissues by High-Performance Liquid Chromatography Coupled to Mass Spectrometry.

A method for quantifying the bovine collagen type V (Col. V) was established based on high-performance liquid chromatography coupled to mass spectrometry by the marker peptide external standard. High-purity Col. V was extracted by the acid-enzyme hydrolysis process, and the marker peptide of Col. V was identified by LCQ mass spectrometry as GPAGPMGLTGR. A broad linear range (0.01-5.00 µg/mL) with a correlation coefficient of 0.9984 was achieved, and the limit of detection and limit of quantification were found to be 3.00 × 10-3 and 6.25 × 10-3 µg/mL, respectively. The method precision was 1.49%. The recovery rate was determined as 97.1-109.6% with a relative standard deviation less than 5%. The proposed method was successfully applied for the determination of Col. V contents in the bovine heart, lung, and cornea, which were 0.72 ± 0.01%, 0.23 ± 0.01%, and 2.89 ± 0.00%, respectively. The results show that the proposed method is more suitable for measuring the content of Col. V in tissue samples compared with the enzyme-linked immunosorbent assay. The marker peptide method has high accuracy and great reproducibility, and will lay a foundation for the extraction and application of Col. V. Impact statement The accurate quantitative method for collagen type V (Col. V) is particularly important in scientific research, disease diagnosis and treatment, and industrial production. In this article, we proposed a high-performance liquid chromatography coupled to mass spectrometry method based on the external standard marker peptide to quantify bovine Col. V. This method shows a higher accuracy and recovery rate than enzyme-linked immunosorbent assay (ELISA), indicating that it is more suitable for measuring the content of Col. V in tissue samples than ELISA. The establishment of this method has laid a solid foundation for the extraction and application of Col. V.

[Changes of collagen and MMP-1 in liver, lung and kidney during growth of mice].

The high performance liquid chromatography (HPLC) and enzyme-linked immunoassay (ELISA) were used to investigate the changes of collagen and matrix metalloproteinase-1 (MMP-1) in liver, lung and kidney during growth process of mice. The mice from 0 to 18 weeks were used as the research objects. The contents and proportions of hydroxyproline (Hyp), which were used to calculate the collagen contents, in liver, lung and kidney of different weeks were analyzed with HPLC. The contents and activity of MMP-1 in liver, lung and kidney of different weeks were analyzed with ELISA. The results showed that the collagen contents in liver, lung, and kidney were different (Lung(COL)>Kidney(COL)>Liver(COL)), and they all increased first and then decreased with weeks. The collagen contents in liver, lung, and kidney reached the highest level in the ninth (5.52 ng/mg), sixth (54.10 ng/mg) and ninth (19.20 ng/mg) week, respectively. Then it declined slowly from 9 to 18 weeks. The result of ELISA showed that the MMP-1 contents in liver, lung and kidney decreased first and then increased with weeks, and the trend of MMP-1 activity was opposite. It indicated that the increase of collagen contents in the tissues will inhibit the secretion of MMP-1.

Detection of Type I and III collagen in porcine acellular matrix using HPLC-MS.

Acellular matrix (ACM) has been widely used as a biomaterial. As the main component of ACM, collagen type and content show influence on the material properties. In this research, the collagen in ACM from different tissues of pig were determined by detection of marker peptides. The marker peptides of Type I and III collagen were identified from the digested collagen standards using ions trap mass spectrometry (LCQ). The relationship between the abundance of marker peptide and collagen concentration was established using triple quadrupole mass spectrometer (TSQ). The contents of Type I and III collagen in ACM from different tissues were determined. The method was further verified by hydroxyproline determination. The results showed that, the sum of Type I and III collagen contents in the ACM from small intestinal submucosa, dermis and Achilles tendon of pig were about 87.59, 81.41 and 61.13%, respectively, which were close to the total collagen contents in these tissues. The results proved that this method could quantitatively detect the collagen with different types in the ACM of various tissues.

Comparative evaluation of the physicochemical properties of nano-hydroxyapatite/collagen and natural bone ceramic/collagen scaffolds and their osteogenesis-promoting effect on MC3T3-E1 cells.

The use of various types of calcium phosphate has been reported in the preparation of repairing materials for bone defects. However, the physicochemical and biological properties among them might be vastly different. In this study, we prepared two types of calcium phosphates, nano-hydroxyapatite (nHA) and natural bone ceramic (NBC), into 3D scaffolds by mixing with type I collagen (CoL), resulting in the nHA/CoL and NBC/CoL scaffolds. We then evaluated and compared the physicochemical and biological properties of these two calcium phosphates and their composite scaffold with CoL. Scanning electron microscopy (SEM), X-ray photoelectron spectroscopy (XPS), Fourier-transform infrared spectroscopy (FTIR), X-ray diffraction spectroscopy (XRD) and compressive tests were used to, respectively, characterize the morphology, composition, distribution and the effect of nHA and NBC to collagen. Next, we examined the biological properties of the scaffolds using cytotoxicity testing, flow cytometry, immunofluorescence staining, biocompatibility testing, CCK-8 assays and RT-PCR. The results reflected that the Ca2+ released from nHA and NBC could bind chemically with collagen and affect its physicochemical properties, including the infrared absorption spectrum and compression modulus, among others. Furthermore, the two kinds of scaffolds could promote the expression of osteo-relative genes, but showed different gene induction properties. In short, NBC/CoL could promote the expression of early osteogenic genes, while nHA/CoL could upregulate late osteogenic genes. Conclusively, these two composite scaffolds could provide MC3T3-E1 cells with a biomimetic surface for adhesion, proliferation and the formation of mineralized extracellular matrices. Moreover, nHA/CoL and NBC/CoL had different effects on the period and extent of MC3T3-E1 cell mineralization.

Effect of in vitro collagen fibrillogenesis on Langmuir-Blodgett (LB) deposition for cellular behavior regulation.

Collagen fibrillogenesis is of special significance for the maintenance of collagen scaffold's mechanical stability and biological performance. Comprehensive information about the mechanism of collagen fibrillogenesis in vitro, as well as the effect of fibrillogenesis on deposited layers of ordered collagen molecules for cellular behavior regulation is thus crucial. In the current study, the pH, phosphate ion as well as reconstitution time impacting on the in vitro fibrillogenesis was systematically investigated, including the zeta potential and turbidity measurement. Furthermore, the fibrillogenesis impacting on the π-a isotherms of collagen assembly at the air/water interface was then fully evaluated. By applying LB technique, collagen fibril-assembling arrays structure can be successfully transferred to form surface deposition onto the mica and glass substrate. The morphology and collagen content were subsequently assessed by atomic force microscopy (AFM) and hydrolyzing examination respectively. Effect of collagen LB deposition on the adhesion and proliferation of SD rat bone marrow mesenchymal stem cells were estimated by Rhodamine Phalloidin/DIPI staining and CCK8 proliferation assays. The results show that highly oriented and collagen-abundant thin film can further facilitate cell adhesion and proliferation, indicating an innovative direction for tissue engineering.

GhWRKY25, a group I WRKY gene from cotton, confers differential tolerance to abiotic and biotic stresses in transgenic Nicotiana benthamiana.

WRKY transcription factors are involved in various processes, ranging from plant growth to abiotic and biotic stress responses. Group I WRKY members have been rarely reported compared with group II or III members, particularly in cotton (Gossypium hirsutum). In this study, a group I WRKY gene, namely, GhWRKY25, was cloned from cotton and characterized. Expression analysis revealed that GhWRKY25 can be induced or deduced by the treatments of abiotic stresses and multiple defense-related signaling molecules. Overexpression of GhWRKY25 in Nicotiana benthamiana reduced plant tolerance to drought stress but enhanced tolerance to salt stress. Moreover, more MDA and ROS accumulated in transgenic plants after drought treatment with lower activities of SOD, POD, and CAT. Our study further demonstrated that GhWRKY25 overexpression in plants enhanced sensitivity to the fungal pathogen Botrytis cinerea by reducing the expression of SA or ET signaling related genes and inducing the expression of genes involved in the JA signaling pathway. These results indicated that GhWRKY25 plays negative or positive roles in response to abiotic stresses, and the reduced pathogen resistance may be related to the crosstalk of the SA and JA/ET signaling pathways.