Exosomes combined with biosynthesized cellulose conduits improve peripheral nerve regeneration.

Peripheral nerve injury is one of the more common forms of peripheral nerve disorders, and the most severe type of peripheral nerve injury is a defect with a gap. Biosynthetic cellulose membrane (BCM) is a commonly used material for repair and ligation of nerve defects with gaps. Meanwhile, exosomes from mesenchymal stem cells can promote cell growth and proliferation. We envision combining exosomes with BCMs to leverage the advantages of both to promote repair of peripheral nerve injury. Prepared exosomes were added to BCMs to form exosome-loaded BCMs (EXO-BCM) that were used for nerve repair in a rat model of sciatic nerve defects with gaps. We evaluated the repair activity using a pawprint experiment, measurement and statistical analyses of sciatica function index and thermal latency of paw withdrawal, and quantitation of the number and diameter of regenerated nerve fibers. Results indicated that EXO-BCM produced comprehensive and durable repair of peripheral nerve defects that were similar to those for autologous nerve transplantation, the gold standard for nerve defect repair. EXO-BCM is not predicted to cause donor site morbidity to the patient, in contrast to autologous nerve transplantation. Together these results indicate that an approach using EXO-BCM represents a promising alternative to autologous nerve transplantation, and could have broad applications for repair of nerve defects.

Construction of Exosomes that Overexpress CD47 and Evaluation of Their Immune Escape.

Our general purpose was to provide a theoretical and practical foundation for the use of exosomes (EXOs) that have high levels of CD47 as stable and efficient drug carriers. Thus, we prepared EXOs from adipose tissue-derived mesenchymal stromal cells (ADMSCs) that had high levels of CD47 (EXOsCD47) and control EXOs (without CD47), and then compared their immune escape in vivo and their resistance to phagocytosis in vitro. Nanoflow cytometry was used to determine the CD47 level in these EXOs, and the amount of EXOsCD47 that remained in rat plasma at 3 h after intraperitoneal injection. Phagocytosis of the EXOs was also determined using in vitro rat macrophage bone marrow (RMA-BM) experiments. Our in vitro results showed that macrophages ingested significantly more control EXOs than EXOsCD47 (p < 0.01), with confirmation by ultra-high-definition laser confocal microscopy. Consistently, our in vivo results showed that rats had 1.377-fold better retention of EXOsCD47 than control EXOs (p < 0.01). These results confirmed that these engineered EXOsCD47 had improved immune escape. Our results therefore verified that EXOsCD47 had increased immune evasion relative to control EXOs, and have potential for use as drug carriers.

Investigating the effect of coal particle size on spontaneous combustion and oxidation characteristics of coal.

As a key parameter, the particle size of residual coal contributes greatly to its oxidation characteristics, so it is a significant and far-reaching topic to explore the role of different particle sizes in coal spontaneous combustion disaster. In this work, temperature-programmed system (TPS) was applied to analyze the oxygen consumption rate and CO and C2H4 production rules of six groups of coal samples with different particle sizes in the process of oxidation heating. The critical temperature (CT) and xerochasy temperature (XT) of different coal samples were obtained, and the coal oxidation process was divided into three stages (S1, slow oxidation stage; S2, fast oxidation stage; and S3, combustion stage). Then, the apparent activation energy (E) and pre-exponential factor (A) in three stages were regressed combined with Arrhenius formula. The results show that the smaller the coal particle size is, the larger the specific surface area is, the stronger the adsorption capacity of coal molecules and oxygen molecules is, resulting in the larger oxygen consumption rate. The values of CT and XT with particle size of 0.125-0.18 mm and 2-4 mm are the smallest and largest. For coal samples with the same particle size, the maximum values of E and A occur in stage S3 and the minimum values appear in stage S1. This is mainly due to the higher temperature of stage S3, which allows the activation of functional groups with higher apparent activation energy, stronger collisions between activated molecules, and more intense oxidation reactions.

Involvement of abscisic acid-responsive element-binding factors in cassava (Manihot esculenta) dehydration stress response.

Cassava (Manihot esculenta) is a major staple food, animal feed and energy crop in the tropics and subtropics. It is one of the most drought-tolerant crops, however, the mechanisms of cassava drought tolerance remain unclear. Abscisic acid (ABA)-responsive element (ABRE)-binding factors (ABFs) are transcription factors that regulate expression of target genes involved in plant tolerance to drought, high salinity, and osmotic stress by binding ABRE cis-elements in the promoter regions of these genes. However, there is little information about ABF genes in cassava. A comprehensive analysis of Manihot esculenta ABFs (MeABFs) described the phylogeny, genome location, cis-acting elements, expression profiles, and regulatory relationship between these factors and Manihot esculenta betaine aldehyde dehydrogenase genes (MeBADHs). Here we conducted genome-wide searches and subsequent molecular cloning to identify seven MeABFs that are distributed unevenly across six chromosomes in cassava. These MeABFs can be clustered into three groups according to their phylogenetic relationships to their Arabidopsis (Arabidopsis thaliana) counterparts. Analysis of the 5'-upstream region of MeABFs revealed putative cis-acting elements related to hormone signaling, stress, light, and circadian clock. MeABF expression profiles displayed clear differences among leaf, stem, root, and tuberous root tissues under non-stress and drought, osmotic, or salt stress conditions. Drought stress in cassava leaves and roots, osmotic stress in tuberous roots, and salt stress in stems induced expression of the highest number of MeABFs showing significantly elevated expression. The glycine betaine (GB) content of cassava leaves also was elevated after drought, osmotic, or salt stress treatments. BADH1 is involved in GB synthesis. We show that MeBADH1 promoter sequences contained ABREs and that MeBADH1 expression correlated with MeABF expression profiles in cassava leaves after the three stress treatments. Taken together, these results suggest that in response to various dehydration stresses, MeABFs in cassava may activate transcriptional expression of MeBADH1 by binding the MeBADH1 promoter that in turn promotes GB biosynthesis and accumulation via an increase in MeBADH1 gene expression levels and MeBADH1 enzymatic activity. These responses protect cells against dehydration stresses by preserving an osmotic balance that enhances cassava tolerance to dehydration stresses.

Taxonomy and Broad-Spectrum Antifungal Activity of Streptomyces sp. SCA3-4 Isolated From Rhizosphere Soil of Opuntia stricta.

Actinobacteria are important producers of bioactive compounds. Extreme ecosystems cause evolution of novel secondary metabolic pathways of Actinobacteria and increase the possible discovery of new biological functions of bioactive compounds. Here, we isolated 65 Actinobacteria from rhizosphere soil samples of Opuntia stricta. An Actinobacteria strain (named SCA3-4) was screened against Fusarium oxysporum f. sp. cubense Tropical Race 4 (Foc TR4, ATCC 76255). The strain produced pink-white aerial mycelia and brown substrate mycelium on Gause No. 1 agar. Biverticillate chains of cylindrical spores were observed by scanning electron microscopy (SEM). Based on alignment of 16S rRNA sequences, a constructed phylogenetic tree showed that strain SCA3-4 shared a 99.54% similarity with Streptomyces lilacinus NRRL B-1968T. The morphological, biochemical, physiological, and molecular characteristics further indicated that strain SCA3-4 belongs to the Streptomyces sp. It can grow well on medium with the following antibiotics chloramphenicol, streptomycin, penicillin-G, gentamicin, erythromycin, nystatin or neomycin sulfate. The polymerase chain reaction (PCR) amplification of types I and II polyketide synthase genes (PKS-I and PKS-II) suggested its bioactive potential. Under treatment with 100 µg/ml of ethyl acetate extracts isolated from Streptomyces sp. SCA3-4, growth of Foc TR4 was inhibited and cell membrane was destroyed. Crude extracts also showed a broad-spectrum antifungal activity against 13 phytopathogenic fungi including Foc TR4 and displayed the lowest minimum inhibitory concentration (MIC) (0.781 µg/ml) against Colletotrichum fragariae (ATCC 58718). A total of 21 different compounds identified by gas chromatography-mass spectrometry (GC-MS) were composed of phenolic compound, pyrrolizidine, hydrocarbons, esters, and acids. Besides the known active compounds, Streptomyces sp. SCA3-4 possesses antimicrobial or other biological activities. Further attention will be paid on other compounds with no functional annotation, aiming at the discovery of new bioactive substances.

Deciphering microbial diversity associated with Fusarium wilt-diseased and disease-free banana rhizosphere soil.

BACKGROUND: Fusarium wilt of banana (Musa spp.) caused by the fungal pathogen Fusarium oxysporum f. sp. cubense (Foc) is a typical soilborne disease, that severely devastates the banana industry worldwide, and soil microbial diversity is closely related to the spread of Fusarium wilt. To understand the relationship between microbial species and Fusarium wilt, it is important to understand the microbial diversity of the Fusarium wilt-diseased and disease-free soils from banana fields. RESULTS: Based on sequencing analysis of the bacterial 16S rRNA genes and fungal internal transcribed spacer (ITS) sequences, Foc abundance, fungal or bacterial richness and diversity were higher in the diseased soils than in the disease-free soils. Although Ascomycota and Zygomycota were the most abundant fungi phyla in all soil samples, Ascomycota abundance was significantly reduced in the disease-free soils. Mortierella (36.64%) was predominant in the disease-free soils. Regarding bacterial phyla, Proteobacteria, Acidobacteria, Chloroflexi, Firmicutes, Actinobacteria, Gemmatimonadetes, Bacteroidetes, Nitrospirae, Verrucomicrobia and Planctomycetes were dominant phyla in all soil samples. In particular, Firmicutes contributed 16.20% of the total abundance of disease-free soils. At the bacterial genus level, Bacillus, Lactococcus and Pseudomonas were abundant in disease-free soils with abundances of 8.20, 5.81 and 2.71%, respectively; lower abundances, of 4.12, 2.35 and 1.36%, respectively, were found in diseased soils. The distribution characteristics of fungal and bacterial genera may contribute to the abundance decrease of Foc in the disease-free soils. CONCLUSION: Unique distributions of bacteria and fungi were observed in the diseased and disease-free soil samples from banana fields. These specific genera are useful for constructing a healthy microbial community structure of soil.

Bioinformatic identification and expression analysis of banana microRNAs and their targets.

MicroRNAs (miRNAs) represent a class of endogenous non-coding small RNAs that play important roles in multiple biological processes by degrading targeted mRNAs or repressing mRNA translation. Thousands of miRNAs have been identified in many plant species, whereas only a limited number of miRNAs have been predicted in M. acuminata (A genome) and M. balbisiana (B genome). Here, previously known plant miRNAs were BLASTed against the Expressed Sequence Tag (EST) and Genomic Survey Sequence (GSS), a database of banana genes. A total of 32 potential miRNAs belonging to 13 miRNAs families were detected using a range of filtering criteria. 244 miRNA:target pairs were subsequently predicted, most of which encode transcription factors or enzymes that participate in the regulation of development, growth, metabolism, and other physiological processes. In order to validate the predicted miRNAs and the mutual relationship between miRNAs and their target genes, qRT-PCR was applied to detect the tissue-specific expression levels of 12 putative miRNAs and 6 target genes in roots, leaves, flowers, and fruits. This study provides some important information about banana pre-miRNAs, mature miRNAs, and miRNA target genes and these findings can be applied to future research of miRNA functions.

The MaASR gene as a crucial component in multiple drought stress response pathways in Arabidopsis.

Abscisic acid (ABA)-, stress-, and ripening-induced (ASR) proteins are involved in abiotic stress responses. However, the exact molecular mechanism underlying their function remains unclear. In this study, we report that MaASR expression was induced by drought stress and MaASR overexpression in Arabidopsis strongly enhanced drought stress tolerance. Physiological analyses indicated that transgenic lines had higher plant survival rates, seed germination rates, and leaf proline content and lower water loss rates (WLR) and malondialdehyde (MDA) content. MaASR-overexpressing lines also showed smaller leaves and reduced sensitivity to ABA. Further, microarray and chromatin immunoprecipitation-based sequencing (ChIP-seq) analysis revealed that MaASR participates in regulating photosynthesis, respiration, carbohydrate and phytohormone metabolism, and signal transduction to confer plants with enhanced drought stress tolerance. Direct interactions of MaASR with promoters for the hexose transporter and Rho GTPase-activating protein (RhoGAP) genes were confirmed by electrophoresis mobility shift array (EMSA) analysis. Our results indicate that MaASR acts as a crucial regulator of photosynthesis, respiration, carbohydrate and phytohormone metabolism, and signal transduction to mediate drought stress tolerance.

Evaluation of different methods of protein extraction and identification of differentially expressed proteins upon ethylene-induced early-ripening in banana peels.

BACKGROUND: Banana peels (Musa spp.) are a good example of a plant tissue where protein extraction is challenging due to the abundance of interfering metabolites. Sample preparation is a critical step in proteomic research and is critical for good results. RESULTS: We sought to evaluate three methods of protein extraction: trichloroacetic acid (TCA)-acetone precipitation, phenol extraction, and TCA precipitation. We found that a modified phenol extraction protocol was the most optimal method. SDS-PAGE and two-dimensional gel electrophoresis (2-DE) demonstrated good protein separation and distinct spots of high quality protein. Approximately 300 and 550 protein spots were detected on 2-DE gels at pH values of 3-10 and 4-7, respectively. Several spots were excised from the 2-DE gels and identified by mass spectrometry. CONCLUSIONS: The protein spots identified were found to be involved in glycolysis, the tricarboxylic acid cycle, and the biosynthesis of ethylene. Several of the identified proteins may play important roles in banana ripening.