Neutrophils are involved in early bone formation during midpalatal expansion.

OBJECTIVE: Midpalatal expansion (MPE) is routinely employed to treat transverse maxillary arch deficiency. Neutrophils are indispensable for recruiting bone marrow stromal cells (BMSCs) at the initial stage of bone regeneration. This study aimed to explore whether neutrophils participate in MPE and how they function during bone formation under mechanical stretching. MATERIALS AND METHODS: The presence and phenotype of neutrophils in the midpalatal suture during expansion were detected by flow cytometry and immunofluorescence staining. The possible mechanism of neutrophil recruitment and polarization was explored in vitro by exposing vascular endothelial cells (VECs) to cyclic tensile strain. RESULTS: The number of neutrophils in the distracted suture peaked on Day 3, and N2-type neutrophils significantly increased on Day 5 after force application. The depletion of circulatory neutrophils reduced bone volume by 43.6% after 7-day expansion. The stretched VECs recruited neutrophils via a CXCR2 mechanism in vitro, which then promoted BMSC osteogenic differentiation through the VEGFA/VEGFR2 axis. Consistently, these neutrophils showed higher expression of canonical N2 phenotype genes, including CD206 and Arg1. CONCLUSIONS: These results suggested that neutrophils participated in early bone formation during MPE. Based on these findings, we propose that stretched VECs recruited and polarized neutrophils, which, in turn, induced BMSC osteogenic differentiation.

Spatial distribution and migration of lead and zinc and the influence of parent materials in typical paddy soils of Hunan Province, China.

This study aimed to investigate the distribution and migration characteristics of lead (Pb) and zinc (Zn) in paddy soils in Hunan Province, China. A total of 343 soil samples from 63 profiles were collected from typical regions. The concentration, spatial distribution, and migration behaviors of Pb and Zn in the paddy soils were examined. The results showed that (1) the concentration ranges of Pb and Zn in the surface layer were 17.62-114.07 mg/kg and 44.98-146.84 mg/kg, respectively. (2) The content was higher in the middle and lower reaches of the Xiangjiang River basin horizontally and exhibited shallow enrichment characteristics vertically. (3) Pb migration was weaker than Zn migration, and the parent material had the most significant influence on Pb and Zn content in the bottom soil layer. The research results will clarify the characteristics of Pb and Zn contents in paddy soils in Hunan Province, further understand the horizontal distribution and vertical migration and transformation characteristics of Pb and Zn contents in paddy soils, and provide basic data for scientific rice cultivation and safe food production.

Matrix mechanics regulate the polarization state of bone marrow-derived neutrophils through the JAK1/STAT3 signaling pathway.

Matrix mechanics regulate essential cell behaviors through mechanotransduction, and as one of its most important elements, substrate stiffness was reported to regulate cell functions such as viability, communication, migration, and differentiation. Neutrophils (Neus) predominate the early inflammatory response and initiate regeneration. The activation of Neus can be regulated by physical cues; however, the functional alterations of Neus by substrate stiffness remain unknown, which is critical in determining the outcomes of engineered tissue mimics. Herein, a three-dimensional (3D) culture system made of hydrogels was developed to explore the effects of varying stiffnesses (1.5, 2.6, and 5.7 kPa) on the states of Neus. Neus showed better cell integrity and viability in the 3D system. Moreover, it was shown that the stiffer matrix tended to induce Neus toward an anti-inflammatory phenotype (N2) with less adhesion molecule expression, less reactive oxygen species (ROS) production, and more anti-inflammatory cytokine secretion. Additionally, the aortic ring assay indicated that Neus cultured in a stiffer matrix significantly increased vascular sprouting. RNA sequencing showed that a stiffer matrix could significantly activate JAK1/STAT3 signaling in Neus and the inhibition of JAK1 ablated the stiffness-dependent increase in the expression of CD182 (an N2 marker). Taken together, these results demonstrate that a stiffer matrix promotes Neus to shift to the N2 phenotype, which was regulated by JAK1/STAT3 pathway. This study lays the groundwork for further research on fabricating engineered tissue mimics, which may provide more treatment options for ischemic diseases and bone defects. STATEMENT OF SIGNIFICANCE.

Senescence-Specific Expression of RAmy1A Accelerates Non-structural Carbohydrate Remobilization and Grain Filling in Rice (Oryza sativa L.).

Remobilization of pre-anthesis NSCs (non-structural carbohydrates) is significant for effective grain filling in rice (Oryza sativa L.). However, abundant starch particles as an important component of NSCs are still present in the leaf sheath and stem at the late stage of grain filling. There are no studies on how bioengineering techniques can be used to improve the efficiency of NSC remobilization. In this study, RAmy1A was expressed under the senescence-specific promoter of SAG12, which was designed to degrade starch in the leaf sheath and stem during grain filling. RAmy1A mRNA successfully accumulated in the leaf, stem, and sheath of transgenic plants after anthesis. At the same time, the starch and total soluble sugar content in the leaf, stem, and leaf sheath were obviously decreased during the grain-filling period. The photosynthetic rate of transgenic lines was higher than that of the wild types by an average of 4.0 and 9.9%, at 5 and 10 days after flowering, respectively. In addition, the grain-filling rate of transgenic lines was faster than that of the wild types by an average of 26.09%. These results indicate an enhanced transport efficiency of NSCs from source tissues in transgenic rice. Transgenic rice also displayed accelerated leaf senescence, which was hypothesized to contribute to decreased grain weight.

Resequencing of 1,143 indica rice accessions reveals important genetic variations and different heterosis patterns.

Obtaining genetic variation information from indica rice hybrid parents and identification of loci associated with heterosis are important for hybrid rice breeding. Here, we resequence 1,143 indica accessions mostly selected from the parents of superior hybrid rice cultivars of China, identify genetic variations, and perform kinship analysis. We find different hybrid rice crossing patterns between 3- and 2-line superior hybrid lines. By calculating frequencies of parental variation differences (FPVDs), a more direct approach for studying rice heterosis, we identify loci that are linked to heterosis, which include 98 in superior 3-line hybrids and 36 in superior 2-line hybrids. As a proof of concept, we find two accessions harboring a deletion in OsNramp5, a previously reported gene functioning in cadmium absorption, which can be used to mitigate rice grain cadmium levels through hybrid breeding. Resource of indica rice genetic variation reported in this study will be valuable to geneticists and breeders.

Cyclic Digestion and Ligation-Mediated PCR Used for Flanking Sequence Walking.

Ligation-mediated PCR (LM-PCR) is a classical method for isolating flanking sequences; however, it has a common limitation of reduced success rate owing to the circularization or multimerization of target restriction fragments including the known sequence. To address this limitation, we developed a novel LM-PCR method, termed Cyclic Digestion and Ligation-Mediated PCR (CDL-PCR). The novelty of this approach involves the design of new adapters that cannot be digested after being ligated with the restriction fragment, and cyclic digestion and ligation may be manipulated to block the circularization or multimerization of the target restriction fragments. Moreover, to improve the generality and flexibility of CDL-PCR, an adapter precursor sequence was designed, which could be digested to prepare 12 different adapters at low cost. Using this method, the flanking sequences of T-DNA insertions were obtained from transgenic rice and Arabidopsis thaliana. The experimental results demonstrated that CDL-PCR is an efficient and flexible method for identifying the flanking sequences in transgenic rice and Arabidopsis thaliana.

Genome-wide analysis of sulfotransferase genes and their responses to abiotic stresses in Chinese cabbage (Brassica rapa L.).

Sulfotransferases (SOTs; EC 2.8.2.-), which are widespread from prokaryotes to eukaryotes, constitute a multi-protein family that plays crucial roles in plant growth, development and stress adaptation. However, this family has not been systemically investigated in Brassica rapa. Here, a genome-wide systemic analysis of SOT genes in B. rapa subsp. pekinensis, a globally cultivated vegetable, were conducted. We identified 56 SOT genes from the whole B. rapa genome using Arabidopsis SOT sequences as queries and classified them into nine groups, rather than the eight groups of previous research. 56 B. rapa SOT genes (BraSOTs) were distributed on all 10 chromosomes except for chromosome 5. Of these, 27 BraSOTs were distributed in seven clusters on five chromosomes (ChrA01, ChrA02, Chr03, ChrA07, and Chr09). Among the BraSOT proteins, 48 had only one SOT_1 domain and 6 had two, while 2 had one SOT_3 domain. Additionally, 47 BraSOT proteins contained only known SOT domains. The remaining nine proteins, five in group-VIII and two in group-IX, contained additional transmembrane domains. Specific motif regions I and IV for 3'-phosphoadenosine 5'-phosphosulfate binding were found in 41 BraSOT proteins. Introns were present in only 18 BraSOT genes, and all seven BraSOT genes in groups VIII and IX had more than three introns. To identify crucial SOTs mediating the response to abiotic stress in B. rapa, expression changes in 56 BraSOT genes were determined by quantitative RT-PCR after drought, salinity, and ABA treatments, and some BraSOT genes were associated with NaCl, drought and ABA stress, e.g. Bra017370, Bra009300, Bra027880.

In Vitro Seamless Stack Enzymatic Assembly of DNA Molecules Based on a Strategy Involving Splicing of Restriction Sites.

The standard binary enzymatic assembly, which operates by inserting one DNA fragment into a plasmid, has a higher assembly success rate than the polynary enzymatic assembly, which inserts two or more fragments into the plasmid. However, it often leaves a nucleotide scar at the junction site. When a large DNA molecule is assembled stepwise into a backbone plasmid in a random piecewise manner, the scars will damage the structure of the original DNA sequence in the final assembled plasmids. Here, we propose an in vitro Seamless Stack Enzymatic Assembly (SSEA) method, a novel binary enzymatic assembly method involving a seamless strategy of splicing restriction sites via a stepwise process of multiple enzymatic reactions that does not leave nucleotide scars at the junction sites. We have demonstrated the success and versatility of this method through the assembly of 1) a 4.98 kb DNA molecule in the 5' → 3' direction using BamHI to generate the sticky end of the assembly entrance, 2) a 7.09 kb DNA molecule in the 3' → 5' direction using SmaI to generate the blunt end of the assembly entrance, and 3) an 11.88 kb DNA molecule by changing the assembly entrance.

QTL analysis and dissection of panicle components in rice using advanced backcross populations derived from Oryza Sativa cultivars HR1128 and 'Nipponbare'.

Panicle traits are among the most important agronomic characters which directly relate to yield in rice. Grain number (GN), panicle length (PL), primary branch number (PBN), and secondary branch number (SBN) are the major components of rice panicle structure, and are all controlled by quantitative trait loci (QTLs). In our research, four advanced backcross overlapping populations (BIL152, BIL196a, BIL196b, and BIL196b-156) carrying introgressed segments from chromosome 6 were derived from an indica/japonica cross that used the super-hybrid rice restorer line HR1128 and the international sequenced japonica cultivar 'Nipponbare' as the donor and recurrent parents, respectively. The four panicle traits, GN, PL, PBN, and SBN, were evaluated for QTL effects using the inclusive composite interval mapping (ICIM) method in populations over two years at two sites. Results showed that a total of twelve QTLs for GN, PL, PBN, and SBN were detected on chromosome 6. Based on marker loci physical positions, the QTLs were found to be tightly linked to three important chromosomal intervals described as RM7213 to RM19962, RM20000 to RM20210, and RM412 to RM20595. Three QTLs identified in this study, PL6-5, PBN6-1, and PBN6-2, were found to be novel compared with previous studies. A major QTL (PL6-5) for panicle length was detected in all four populations at two locations, and its position was narrowed down to a 1.3Mb region on chromosome 6. Near isogenic lines (NILs) carrying PL6-5 will be developed for fine mapping of the QTL, and our results will provide referable information for gene excavation of panicle components in rice.

[The effect of Foxc2 overexpression on the osteogenic properties of C3H10T1/2 cells].

PURPOSE: To investigate the effect of Foxc2 overexpression on osteogenic and adipogenic differentiation of C3H10T1/2 cells. METHODS: C3H10T1/2 cells were transfected with plenti-Foxc2 and selected with puromycin for stable clones. The expression of Foxc2 was determined by real-time PCR and Western blot. Cell proliferation was detected by CCK-8 kit. Cell cycle and apoptosis were detected by flow cytometry. The level of osteogenic biomarkers Runx2, OPN, OCN and adipogenic biomarker PPARγ were quantified by real-time PCR and Western blot. Alkaline phosphatase (ALP) staining and oil red staining were conducted to evaluate the effect of Foxc2 overexpression on osteogenic and adipogenic differentiation. Statistical analysis was performed using SPSS 17.0 software package. RESULTS: C3H10T1/2-Foxc2 cell line was successfully constructed and verified by direct sequencing and Foxc2 overexpression in vitro. Cell proliferation was reduced and cell cycle was blocked in G1/G0 phase. Enhanced ALP staining and reduced oil red staining were observed in C3H10T1/2-Foxc2 cells as compared with the control. Foxc2 overexpression up-regulated Runx2, OPN, OCN during osteogenic differentiation and down-regulated PPARγduring adipogenic differentiation. CONCLUSIONS: C3H10T1/2 cell line stably expressing Foxc2 gene was successfully established, cell proliferation was reduced, osteogenesis biomarkers were up-regulated during the osteogenesis by overexpression Foxc2, PPARγwas down-regulated during adipogenesis.

[The effect of estrogen on adipogenic differentiation of rat bone mesenchymal stem cells].

PURPOSE: In this study, 10⁻9 mol/L 17 ß-estradiol (E2) was applied in the adipogenic differentiation of rat bone mesenchymal stem cells (rBMSCs) and the effect of E2 was explored. METHODS: Rat BMSCs were obtained from the femurs and tibias of SD rats. 10⁻9 mol/L E2 was involved in the adipogenic differentiation of rBMSCs. Oil red staining, real time PCR and Western blot were carried out to detect the effect of 10⁻9 mol/L E2 on adipogenic differentiation of rBMSCs. The data was statistically analyzed using SPSS 19.0 software package. RESULTS: The use of 10⁻9 mol/L E2 decreased the amount of lipid droplets in rBMSCs and weakened the expression of adipogenic related genes and proteins like C/EBP α, C/EBP ß, PPAR γ, aP2, and ARDP, which were significantly lower than the adipogenic induced group. CONCLUSIONS: The use of 10⁻9 mol/L E2 inhibited adipogenic differentiation of rBMSCs significantly in vitro.

[Changes of the microarchitecture of alveolar bone due to different duration of ovariectomy: a Micro-CT study in rats].

PURPOSE: To study the changes of microarchitecture of alveolar bone due to different duration of ovariectomy in rats. METHODS: Twenty-four virgin Sprague-Dawley rats were randomly assigned to ovariectomy group (OVX) or sham-ovariectomy group (sham). OVX rats were subjected to bilateral ovariectomy and sham-ovariectomy was conducted in sham rats. Six rats of each group were sacrificed respectively 3 months and 6 months after surgery. The right semi-maxilla of all rats were scanned by Micro-CT, and the inter-radicular alveolar bone of the maxillary first molar was analyzed. Statistical analysis was carried out with SPSS 16.0 software package. RESULTS: Two-dimensional and three-dimensional reconstructed images of the alveolar bone showed porotic changes in rats both 3 months and 6 months after ovariectomy, including thinner, looser trabeculae and expanded bone marrow. When compared with corresponding sham rats, BMD, BV/TV and Tb.Th of alveolar bone significantly decreased in OVX rats both 3 months and 6 months after ovariectomy (P<0.05). Tb.N and Tb.Sp significantly increased in both OVX groups (P<0.05). When compared with 3 months after ovariectomy, the rats 6 months after ovaroectomy shared deceased BMD, BV/TV and Tb.Th (P<0.05) and increased Tb.N (P<0.05). CONCLUSIONS: Bone loss and deterioration of trabeculae of alveolar bone aggravates with the extended duration of ovariectomy in OVX rats.

[The effect of estrogen on proliferation and osteogenic differentiation of rat bone marrow mesenchymal stem cells].

PURPOSE: Different concentrations of 17 ß-estradiol (E2) were applied in the osteogenic differentiation of rat bone mesenchymal stem cells (BMSCs), and the proliferation and apoptosis of BMSCs were explored. METHODS: BMSCs were obtained from the femurs and tibias of SD rats. The proliferation curve was conducted to rBMSCs in culture medium containing 0, 10(-9), and 10(-7) mol/L 17 ß-estradiol by CCK-8 for 7 days. Annexin V and PI for flow cytometry were applied to detect the impact of E2 on apoptosis of rBMSCs. After 1, 7, 11 and 14 days of osteogenic induction, the activity of alkaline phosphatase (ALP) was assayed; ALP staining was performed on day 7 and day 14; Alizarin red staining for calcium deposits was carried out on day 21. Concentrations of 0, 10(-9), and 10(-7) mol/L 17 ß-estradiol were administrated to rBMSCs for real-time PCR of osteogenic related genes on day 1, 3, 5, 7, 14, and day 21. The data was statistically analyzed using SPSS 19.0 software package. RESULTS: The effect of 17 ß-estradiol on proliferation and apoptosis of rBMSCs was not obvious. However, after osteogenic induction, the ALP activity and Alizarin red staining were significantly stronger in the groups containing 17 ß-estradiol. Especially, the use of 17 ß-estradiol with the concentration of 10(-9) mol/L enhanced the expression of osteogenic related genes like RUNX2, ALP, COL I, and OCN, which was significantly higher than other groups. CONCLUSIONS: 17 ß-estradiol promotes osteogenic differentiation of BMSCs in a dose-dependent pattern in vitro.