Molecular analysis of the chloroplast Cu/Zn-SOD gene(AhCSD2) in peanut

Superoxide dismutase(SOD, EC 1.15.1.1) plays a key role in response to drought stress, and differences in SOD activity changes among cultivars are important under drought conditions. We obtained the full-length DNA of the chloroplast Cu/Zn-SOD gene(Ah CSD2)from 11 allotetraploid cultivars and 5 diploid wild species in peanut. BLAST search against the peanut genome showed that the Ah CSD2 genes g CSD2-1 and g CSD2-2 are located at the tops of chromosome A03(A genome) and B03(B genome), respectively, and both contain 8exons and 7 introns. Nucleotide sequence analyses indicated that g CSD2-2 sequences were identical among all the tested cultivars, while g CSD2-1 sequences showed allelic variations.The amino acid sequences deduced from g CSD2-1 and g CSD2-2 both contain a chloroplast transit peptide and are distinguished by 6 amino acid(aa) residue differences. The other 2aa residue variations in the mature peptide regions give rise to three-dimensional structure changes of the protein deduced from the genes g CSD2-1 and g CSD2-2. Sequences analyses of cultivars and wild species showed that g CSD2-2 of Arachis hypogaea and g Aip CSD2(Arachis ipaensis) are identical, and despite the abundant polymorphic loci between g CSD2-1 of A.hypogaea and sequences from A genome wild species, the deduced amino acid sequence of Ah CSD2-1(A. hypogaea) is identical to that of Adu CSD2(Arachis duranensis), whereas Aco CSD2(Arachis correntina) and Aca CSD2(Arachis cardenasii) both have 2 aa differences in the transit peptide region compared with Ah CSD2-1(A. hypogaea). Based on the Peanut Genome Project, promoter prediction revealed many stress-related cis-acting elements within the potential promoter regions(pp-A and pp-B). pp-A contains more binding sites for drought-associated transcriptional factors than pp-B. We hypothesize that the marked changes in SOD activity in different cultivars under drought stress are tightly regulated by transcription factors through transcription and expression of Ah CSD2 genes.

An improved high-oleic line created by chemical mutagen of ‘Huayu 40’

By using chemical mutagenesis, ‘Huayu 40’, a normal-oleic(NO) large-seed high yielding peanut cultivar with wide adaptability was transformed into mutants with desirable high-oleic(HO) quality traits, comparable productivity and adaptability. Of the 3 chemical treatments, viz. 5 mM ethyl methane sulfonate(EMS), 25 mM EMS and 15 mM sodium azide(NaN_3), only NaN_3 produced HO M_2 mutant plants. High oleate phenotype was initially identified by near infrared spectroscopy(NIRS) screening, and was further confirmed by gas spectrometry(GC) analysis. Sequence analysis showed that HO plant had a 448 G>A mutation in FAD2 A and a 441_442 insA mutation in FAD2 B, respectively. Although the direct use of high yielding and stress resistant cultivars/lines to induce quality mutations might be a judicious choice to accelerate breeding, our results showed that chemical mutagenesis could quickly create HO peanut materials and expand the genetic bases of HO peanuts.

Macrophage exosomes modified by miR-365-2-5p promoted osteoblast osteogenic differentiation by targeting OLFML1

In the bone immune microenvironment,immune cells can regulate osteoblasts through a complex communication network.Macrophages play a central role in mediating immune osteogenesis,exosomes derived from them have osteogenic regulation and can be used as cariers in bone tissue engineering.However,there are problems with exosomal therapy alone,such as poor targeting,and the content of loaded molecules cannot reach the therapeutic concentration.In this study,macrophage-derived exosomes modified with miR-365-2-5p were developed to accelerate bone healing.MC3T3-E1 cells were incubated with the culture supermatants of Mo,M1 and M2 macrophages,and it was found that the culture medium of M2 macrophages had the most significant effects in contributing to osteogenesis.High-throughput sequencing identified that miR-365-2-5p was significantly expressed in exosomes derived from M2 macrophages.We incubated MC3T3-E1 with exosomes overexpressing or kmocking down miR-365-2-5p to examine the biological function of exosome miR-365-2-5p on MC3T3-E1 differentiation.These findings suggested that miR-365-2-5p secreted by exosomes increased the osteogenesis of MC3T3-E1.Moreover,miR-365-2-5p had a direct influence over osteogenesis for MC3T3-Ei.Sequencing analysis combined with dual luciferase detection indicated that miR-365-2-5p binded to the 3'-UTR of OLFML1.In summary,exosomes secreted by M2 macrophages targeted OLFML1 through miR-365-2-5p to facilitate osteogenesis.

CaP-coated Zn-Mn-Li alloys regulate osseointegration via influencing macrophage polarization in the osteogenic environment

Immune response is an important factor in determining the fate of bone replacement materials,in which macrophages play an important role.It is a new idea to design biomaterials with immunomodulatory function to reduce inflammation and promote bone integration by regulating macrophages polarization.In this work,the immunomodulatory properties of CaP Zn-Mn-Li alloys and the specific mechanism of action were investigated.We found that the CaP Zn0.8Mn0.1Li alloy promoted the polarization of macrophages toward M2 and reduced inflammation,which could effectively upregulate osteogenesis-related factors and promote new bone formation,indicating the important role of macrophages polarization in biomaterial induction of osteogenesis.In vivo studies further demonstrated that CaP Zn0.8Mn0.1Li alloy could stimulate osteogenesis better than other Zn-Mn-Li alloys implantations by regulating macrophages polarization and reducing inflammation.In addition,transcriptome results showed that CaP Zn0.8Mn0.1Li played an important regulatory role in the life process of macrophages,activating Toll-like receptor signaling pathway,which participated in the activation and attenuation of inflammation,and accelerated bone integration.Thus,by preparing CaP coatings on the surface of Zn-Mn-Li alloys and combining the bioactive ingredient with controlled release,the biomaterial will be imbibed with beneficial immunomodulatory properties that promote bone integration.

MicroRNA-21a-5p-modified macrophage exosomes as natural nanocarriers promote bone regeneration by targeting GATA2

Bone immune responses based on macrophages are critical in the osteogenesis of bone abnormalities.In general,M2 macrophage facilitate the promotion of osteogenesis,as well,M1 macrophage play an important role in early bone healing,as confirmed by previous studies.However,it is not clear how M1 macrophage are involved in the bone immune response.MiR-21a-5p is a highly expressed microRNA in M1 macrophage in contrast to M2.Therefore,the current work sought to ascertain the influence of M1 macrophage on bone healing via exosomal miR-21a-5p and the probable mechanism.We discovered that injecting M1 macrophage exosomes overexpressing miR-21a-5p into bone defect locations enhanced bone regeneration in vivo.Furthermore,by directly targeting GATA2,miR-21a-5p accelerated MC3T3-E1 osteogenic differentiation.Our findings showed that exosomal miR-21a-5p from M1 macrophage may be transported to osteoblasts and target GATA2 to enhance bone defect healing.

Establishment and evaluation of a peanut association panel and analysis of key nutritional traits

Breeding programs aim to improve the yield and quality of peanut （Arachis hypogaea L.）; using association mapping to identify genetic markers linked to these quantitative traits could facilitate selection efficiency. A peanut association panel was established consisting of 268 lines with extensive phenotypic and genetic variation, meeting the requirements for associa- tion analysis. These lines were grown over 3 years and the key agronomic traits, including protein and oil content were examined. Population structure （Q） analysis showed two subpopulations and clustering analysis was consistent with Q-based membership assignment and closely related to botanical type. Relative Kinship （K） indicated that most of the panel members have no or weak familial related- ness, with 52.78% of lines showing K=o. Linkagedisequilibrium （LD） analysis showed a high level of LD occurs in the panel. Model comparisons indicated false positives can be effectively controlled by taking Q and K into consideration and more false positives were generated by K than Q. A preliminary association analysis using a Q＋ K model found markers significantly associated with oil, protein, oleic acid, and linoleic acid, and identified a set of alleles with positive and negative effects. These results show that this panel is suitable for association analysis, providing a resource for marker-assisted selection for peanut improvement.

Determining the critical size of a rabbit rib segmental bone defect model

In order to establish and standardize the rabbit rib segmental bone defect model,it is of vital importance to determine rabbit rib critical size defect(CSD).According to the general time needed for spontaneous long-bone regeneration,three-month observation period was set to determine the CSD.The rabbit rib segmental bone defects with different sizes from 1 to 5cm with or without periosteum were performed in the eighth rib of 4-month-old male New Zealand rabbits and underwent Xray examinations at the 4th,8th and 12th weeks postoperatively.The gross and histological examinations at postoperative week 12 were evaluated,which showed that the critical sizes in the rabbit rib models with and without periosteum were 5 and 2 cm,respectively.This study provides prerequisite data for establishing rabbit rib CSD model and evaluating bonematerials using this model.

Integrin α_(v)β_(3)-targeted polydopamine-coated gold nanostars for photothermal ablation therapy of hepatocellular carcinoma

Photothermal therapy(PTT)has emerged as a promising cancer therapeutic method.In this study,Arg-Gly-Asp(RGD)peptide-conjugated polydopamine-coated gold nanostars(Au@PDA-RGD NPs)were prepared for targeting PTT of hepatocellular carcinoma(HCC).A polydopamine(PDA)shell was coated on the surface of gold nanostars by the oxidative self-polymerization of dopamine(termed as Au@PDA NPs).Au@PDA NPs were further functionalized with polyethylene glycol and RGD peptide to improve biocompatibility as well as selectivity toward the HCC cells.Au@PDARGD NPs showed an intense absorption at 822 nm,which makes them suitable for near-infraredexcited PTT.Our results indicated that the Au@PDA-RGD NPs were effective for the PTT therapy of the α_(v)β_(3) integrin receptor-overexpressed HepG2 cells in vitro.Further antitumor mechanism studies showed that the Au@PDA-RGD NPs-based PTT induced human liver cancer cells death via the mitochondrial-lysosomal and autophagy pathways.In vivo experiments showed that Au@PDARGD NPs had excellent tumor treatment efficiency and negligible side effects.Thus,our study showed that Au@PDA-RGD NPs could offer an excellent nanoplatform for PTT of HCC.

Tapered Optical Fiber-Based LSPR Biosensor for Ascorbic Acid Detection

The ascorbic acid(AA)is a biomarker that can be used to detect the symptoms of severe disorders such as scurvy,Parkinson’s,Alzheimer’s,and cardiovascular diseases.In this work,a simple and effective sensor model is developed to diagnose the presence of AA samples.To develop the sensor,a tapered single-mode optical fiber has been used with the well-known phenomenon of localized surface plasmon resonance(LSPR).For LSPR,the tapered region is immobilized with synthesized gold nanoparticles(AuNPs)and zinc oxide nanoparticles(ZnO-NPs)whose absorbance peak wavelengths appear at 519nm and 370nm,respectively.On the basis of nanoparticles(NPs)configurations,two different biosensor probes are developed.In the first one,the sensing region is immobilized with AuNPs and named Probe I.In the second probe,the immobilized layer of AuNPs is further coated with a layer of ZnO-NPs,and a resultant probe is termed as Probe II.The characterizations of synthesized AuNPs and developed fiber probes are done by the ultraviolet-visible(UV-vis)spectrophotometer,high-resolution transmission electron microscope(HR-TEM),atomic force microscopy(AFM),and scanning electron microscope(SEM).To enhance the selectivity,a sensing region of probes is functionalized with ascorbate oxidase enzyme that oxidizes the AA in the presence of oxygen.The response of developed sensor probes is authenticated by sensing the samples of AA in the range from 500 nM to 1 mM,which covers the range of AA found in human bodies,i.e.,40μM-120μM.The performance analysis of the developed sensor probes has been done in terms of their stability,reproducibility,reusability,and selectivity.To observe the stability of AA,a pH-test has also been done that results in a better solubility of AA molecules in phosphate-buffered saline(PBS)solution.

Novel pre-vascularized tissue-engineered dermis based on stem cell sheet technique used for dermis-defect healing

Insufficient donor dermis and the shortage of three-dimensional vascular networks are the main limitations in the tissue-engineered dermis(TED).To solve these problems,we initially constructed pre-vascularized bone marrow mesenchymal stem cell sheet(PBMCS)and pre-vascularized fibroblasts cell sheet(PFCS)by cell sheet technology,and then superimposed or folded them together to construct a pre-vascularized TED(PTED),aiming to mimic the real dermis structure.The constructed PTED was implanted in nude mice dorsal dermis-defect wound and the wound-healing effect was quantified at Days 1,7 and 14 via the methods of histochemistry and immunohistochemistry.The results showed that PTED could rapidly promote the wound closure,especially at Day 14,and the wound-healing rate of three-layer PTED could reach 97.2%(P<0.01),which was faster than the blank control group(89.1%),PBMCS(92.4%),PFCS(93.8%)and six-layer PTED(92.3%).In addition,the vessel density in the PTED group was higher than the other groups on the 14th day.Taken together,it is proved that the PTED,especially three-layer PTED,is more conducive to the fullthickness dermis-defect repair and the construction of the three-dimensional vascular networks,indicating its potential application in dermis-defect repair.