miR-497-5p promoted neuronal injury in ischemic stroke by inhibiting the BDNF/TrkB/PI3K/Akt pathway.

The aim of this study was to investigate the molecular mechanism by which miR-497-5p regulates neuronal injury after ischemic stroke through the BDNF/TrkB/Akt signaling pathway. PC12 cells were used to construct a stroke injury model by oxygen-glucose deprivation/reoxygenation (OGD/R). The expression level of miR-497-5p was measured by RT-qPCR. CCK-8 kit was used to detect cell viability. Cell apoptosis and reactive oxygen species (ROS) were detected by flow cytometry. MDA and SOD detection kits were used to detect MDA content and SOD activity. A double luciferase reporter system was used to verify the targeting relationship between miR-497-5p and BDNF. The expression of BDNF, TrkB, p-TrkB, Akt and p-Akt was detected by Western blot. We have found that miR-497-5p expression was inhibited after treatment with OGD/R. Simultaneously, cell apoptosis, MDA content and ROS were upregulated, while cell viability and SOD were significantly decreased in PC12 cells. The effects of OGD/R on PC12 cells were reversed with the downregulation of miR-497-5p. A double luciferase reporter assay demonstrated that miR-497-5p negatively targets BDNF. BDNF inhibited cell apoptosis and oxidative stress injury in PC12 cells. These findings suggest that miR-497-5p aggravates neuronal injury in experimental model of ischemic stroke by inhibiting the BDNF/TrkB/PI3K/Akt signaling pathway.

MiR-10b-5p Regulates Neuronal Autophagy and Apoptosis Induced by Spinal Cord Injury Through UBR7.

This study aimed to explore the effects of miR-10b-5p on autophagy and apoptosis in neuronal cells after spinal cord injury (SCI) and the molecular mechanism. Bioinformatics was used to analyze the differentially expressed miRNAs. The expression of related genes and proteins were detected by real-time fluorescence quantitative polymerase chain reaction (RT-qPCR) and Western blot, respectively. Cell proliferation was detected by 5-ethynyl-2'-deoxyuridine (EdU), and apoptosis was detected by flow cytometry or terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay (TUNEL). Coimmunoprecipitation confirmed the interaction between UBR7 and Wnt1 or Beclin1. Autophagy was detected by the dansylcadaverine (MDC). The Basso Beattie Bresnahan (BBB) score was used to evaluate motor function, and hematoxylin-eosin (H&E) and Nissl staining were used to detect spinal cord tissue repair and neuronal changes. The result shows that the expression of miR-10b-5p was downregulated in the SCI models, and transfection of a miR-10b-5p mimic inhibited neuronal cell apoptosis. MiR-10b-5p negatively regulated the expression of UBR7, and the inhibitory effect of the miR-10b-5p mimic on neuronal cell apoptosis was reversed by overexpressing UBR7. In addition, UBR7 can regulate apoptosis by affecting the Wnt/ß-catenin pathway by promoting Wnt1 ubiquitination. Treatment with the miR-10b-5p mimic effectively improved motor function, inhibited neuronal cell apoptosis, and promoted spinal cord tissue repair in SCI rats. Overall, miR-10b-5p can alleviate SCI by downregulating UBR7 expression, inhibiting Wnt/ß-catenin signaling pathway ubiquitination to reduce neuronal apoptosis, or inhibiting Beclin 1 ubiquitination to promote autophagy.

Blue Light Enhances Health-Promoting Sulforaphane Accumulation in Broccoli (Brassica oleracea var. italica) Sprouts through Inhibiting Salicylic Acid Synthesis.

As a vegetable with high nutritional value, broccoli (Brassica oleracea var. italica) is rich in vitamins, antioxidants and anti-cancer compounds. Glucosinolates (GLs) are one of the important functional components widely found in cruciferous vegetables, and their hydrolysate sulforaphane (SFN) plays a key function in the anti-cancer process. Herein, we revealed that blue light significantly induced the SFN content in broccoli sprouts, and salicylic acid (SA) was involved in this process. We investigated the molecular mechanisms of SFN accumulation with blue light treatment in broccoli sprouts and the relationship between SFN and SA. The results showed that the SFN accumulation in broccoli sprouts was significantly increased under blue light illumination, and the expression of SFN synthesis-related genes was particularly up-regulated by SA under blue light. Moreover, blue light considerably decreased the SA content compared with white light, and this decrease was more suppressed by paclobutrazol (Pac, an inhibitor of SA synthesis). In addition, the transcript level of SFN synthesis-related genes and the activity of myrosinase (MYR) paralleled the trend of SFN accumulation under blue light treatment. Overall, we concluded that SA participates in the SFN accumulation in broccoli sprouts under blue light.

Necroptosis-mediated HMGB1 secretion of keratinocytes as a key step for inflammation development in contact hypersensitivity.

Keratinocyte necroptosis (with proinflammatory characteristic) is required for epidermal damage in contact hypersensitivity (CHS). In DNCB-induced CHS mice model, we observed the aggravated keratinocyte death and increased phosphorylation level of MLKL, RIPK3 and RIPK1. However, CHS skin lesion did not present in keratinocyte-specific Mlkl knockout mice. We validated that MLKL-mediated keratinocyte necroptosis is required for epidermal damage in response to immune microenvironment in CHS. Moreover, MLKL-mediated necroptosis deficiency or inhibition resulted in blocking recruitment and activation of inflammatory cells in CHS via reducing HMGB1 release in keratinocytes. This study suggests that MLKL-mediated keratinocyte necroptosis functions as a self-amplified actor in inflammatory responses and could be considered as an effective therapeutic target. It proposes an innovative prospective that inhibiting keratinocyte necroptosis can prevent the development of epidermal damage in CHS.

Waxy is an important factor for grain fissure resistance and head rice yield as revealed by a genome-wide association study.

Head rice yield (HRY) is an essential quality trait, and is sensitive to environmental stresses during the grain-filling, harvest, and postharvest stages. It is therefore important for rice production and global food security to select for superior HRY traits; however, the molecular basis of this trait remains unknown. Using diverse rice germplasm material, we performed a genome-wide association study of grain fissure resistance (GFR), the phenotype most associated with HRY, and found that the granule-bound starch synthase I gene Waxy is an important gene controlling GFR. Analysis of near-isogenic lines demonstrated that genetic variations in Waxy conferred different levels of tolerance to fissuring in grains. The null allele wx resulted in the highest GFR, while alleles that increased amylose synthesis reduced GFR. Increases in amylose content led to increases in the ratio of the widths of the amorphous layer to the semi-crystalline layer of the starch granules, and also to increased occurrence of chalkiness. The layer structure determined GFR by affecting the degree of swelling of granules in response to moisture, and chalkiness acted as an accelerator of moisture infiltration to rapidly increase the number of swelling granules. Our study reveals the molecular basis of GFR and HRY, thus opening the door for further understanding of the molecular networks of GFR and HRY.

The phosphoinositide-specific phospholipase C1 modulates flowering time and grain size in rice.

MAIN CONCLUSION: Preferential expression of OsPLC1 is detected at the heading stage of rice, OsPLC1 overexpression results in early flowering, increased-grain size and yield; however, opposing phenotypes produced in the osplc1 mutants. Abstract: The importance of phospholipase C (PLC) in plant development has been demonstrated in several studies. OsPLC1, a member of PI-PLC in rice, although its role in the response to salt stress of rice seedlings has been reported, its functions in the growth and development of rice is elusive. Here, we report that OsPLC1 expression could be detectable in various tissues throughout the developmental stages of rice, and the highest expression level of OsPLC1 was detected at the heading stage. OsPLC1 overexpression (OE) produced rice plants with early flowering, whereas OsPLC1 loss-of-function led to delay in flowering. The expression levels of subset genes, which are involved in the control of flowering time in rice, were altered in the plants of OE and osplc1. In addition, the enlargement of grain size was observed in OE plants, however, the reduction of grain size was noticed in osplc1 mutants. The increase in the grain size and the grain yield of OE lines were associated with the improvement of cell length and expression levels of a set of genes related to cell expansion, contrarily, the decrease in osplc1 mutant grain size and yield were linked to declined cell length and expression levels of related genes. No significant differences, in terms of the grain quality of mature seeds, were found in OE and osplc1 mutants, with compared to those in Nipponbare (Nip). In summary, our study suggests that OsPLC1 could modulate rice flowering time and grain size.

The F-Box/DUF295 Brassiceae specific 2 is involved in ABA-inhibited seed germination and seedling growth in Arabidopsis.

To bear harsh environmental threats, plants have developed complex protection mechanisms involving phytohormones, counting abscisic acid (ABA). The function of the F-Box family containing the Domain of Unknown Function 295 (DUF295) has not yet been comprehensively characterized in Arabidopsis (Arabidopsis thaliana). In this study, we evaluated the function of a putative member of the F-Box/DUF295 family in Arabidopsis, F-box/DUF295 Brassiceae specific 2 (FDB2). We found that FDB2 expression was suppressed by ABA and abiotic stresses. FDB2 overexpression (OE) reduced ABA sensitivity during seed germination and seedling growth, but enhanced ABA-sensitivity of seed germination and seedling growth in fdb2 mutants was scored. When treated with ABA, expressions of ABI3, ABI4 and ABI5 showed decreased in OE lines but increased in fdb2 mutants. In addition, ABA-induced FDB2 degradation exhibited sensitive to MG132, suggesting that FDB2 degradation by ABA might be mediated by the ubiquitin-26S proteasome system. Moreover, ABA-induced significant over-accumulation of reactive oxygen species (ROS) at the root tips of fdb2 mutants was observed, this phenomenon was correlated to reduced activities of a set of ROS scavengers in fdb2 mutants relative to Col-0. In summary, our results suggest that Arabidopsis FDB2 is involved in ABA-mediated inhibition of seed germination, seedling growth including modulation of ROS homeostasis in roots.

The phytomelatonin receptor PMTR1 regulates seed development and germination by modulating abscisic acid homeostasis in Arabidopsis thaliana.

Melatonin is known to involve multiple physiological actions in plants. Herein, we found that exogenous melatonin inhibited the Arabidopsis seedling growth through the elevated abscisic acid (ABA) levels, and the elevated ABA was ascribed to the upregulation of 9-cis-epoxycarotenoid dioxygenase genes (NCEDs) in the ABA biosynthesis pathway. We also found that the overexpression lines of the melatonin receptor gene PMTR1 (also known as Cand2) yielded smaller seeds and germinated slower than the wild type, whereas PMTR1-knockout mutants produced larger seeds and germinated faster than the wild type. During the seed development, the accumulation peak of ABA was higher in the PMTR1-knockout mutant, while it was lower in the PMTR1-overexpression line than that in the wild type. In the dry seeds and imbibed seeds, the PMTR1-overexpression line accumulated higher ABA levels, while the PMTR1-knockout contained less ABA than the wild type. In summary, our findings suggest that PMTR1 is involved in ABA-mediated seed development and germination in Arabidopsis.

The rice phosphoinositide-specific phospholipase C3 is involved in responses to osmotic stresses via modulating ROS homeostasis.

Four members of phosphoinositide-specific phospholipase C (PI-PLC) are predicted in rice genome. Although the involvement of OsPLC1 and OsPLC4 in the responses of rice to salt and drought stresses has been documented, the role of OsPLC3 in which, yet, is elusive. Here, we report that OsPLC3 was ubiquitously expressed in various tissues during the development of rice. The expression of YFP-tagged OsPLC3 was observed at the plasma membrane (PM), cytoplasm and nucleus of rice protoplasts, onion epidermal cells and tobacco leaves. The catalytic activity of OsPLC3 was measured using the thin-layer chromatography (TLC) method. The inhibition of OsPLC3 expression was detected in the treatments of NaCl and mannitol. Overexpression (OE) of OsPLC3 produced plants showing more sensitive to osmotic stresses when they were compared to the wild-type (HJ) and osplc3 mutants, the phenomena such as decreased plant fresh weight and increased water loss rate (WLR) were observed. Under the treatment of NaCl or mannitol, expressions of a subset osmotic stress-related genes were altered, in both OE and osplc3 mutant lines. In addition, the expressions and the enzyme activities of reactive oxygen species (ROS) scavengers were significantly decreased in OE lines, leading to over-accumulation of ROS together with less osmotic adjustment substances including proline, soluble sugars and soluble proteins in OE plants which caused the growth inhibition. Thus, our results suggested that, via modulating ROS homeostasis, OsPLC3 is involved in responses to the osmotic stress in rice.

Inhibition of lncRNA H19/miR-370-3p pathway mitigates neuronal apoptosis in an in vitro model of spinal cord injury (SCI).

BACKGROUND: Spinal cord injury (SCI) is the most serious complication of spinal injury, often leading to severe dysfunction of the limbs below the injured segment. Conventional therapy approaches are becoming less and less effective, and gene therapy is a new research direction by now. METHODS: The Sprague-Dawley rats were haphazardly assigned to two groups, namely sham group and SCI model group, and lncRNA H19 and miR-370-3p levels were investigated using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Correlation between lncRNA H19 and miR-370-3p was ascertained by luciferase report assay and RT-qPCR. After transfection with si-H19, miR-370-3p inhibitor, negative controls (NC), or both, primary spinal neurons were subjected to the simulation of lipopolysaccharide (LPS) for inducing in vitro model of SCI. Cell viability, apoptotic rate, caspase-3 activity, Bax and Bcl-2 protein, ROS generation, TNF-α, IL-1ß, and IL-6 protein, as well as IκBα and p65 phosphorylation ratio were evaluated adopting 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), apoptosis, caspase-3 activity, ROS generation, and western blot assays, thereby searching for the specific action mechanism on LPS-induced spinal never injury. RESULTS: SCI resulted in lncRNA H19 higher expression and miR-370-3p lower expression. LPS simulation raised a series of cellular biological changes, such as decreased viability, promoted apoptosis, generated ROS, and released inflammatory factors. lncRNA H19 inhibition reversed above LPS-induced changes. Besides, as the downstream target of lncRNA H19, miR-370-3p was oppositely regulated by lncRNA H19. The above biological changes induced by lncRNA H19 inhibition were reversed by miR-370-3p upregulation. Moreover, lncRNA H19 inhibition could block NF-κB pathway through miR-370-3p upregulation. CONCLUSION: Inhibition of lncRNA H19/miR-370-3p mitigated spinal neuron apoptosis in an in vitro model of SCI. This provided the possibility for clinical use of gene therapy.

ABA-INDUCED expression 1 is involved in ABA-inhibited primary root elongation via modulating ROS homeostasis in Arabidopsis.

To endure environmental stresses, plants have evolved complex regulatory mechanisms involving phytohormones, including abscisic acid (ABA). The function of the plant-specific AT-rich sequence zinc-binding protein (PLATZ) family has not yet been extensively characterized in Arabidopsis (Arabidopsis thaliana). In this report, we evaluated the function of a putative member of the PLATZ family in Arabidopsis, ABA-INDUCED expression 1 (AIN1). We determined that AIN1 expression was induced by ABA and abiotic stresses. AIN1 overexpression (OE) enhanced ABA sensitivity and inhibited primary root elongation, but reduced expression of AIN1 in RNA interference (RNAi) plants produced roots less sensitive to ABA. When treated with ABA, we observed a reduction of meristem size and over-accumulation of reactive oxygen species (ROS) at the root tips of OE lines, demonstrating the importance of AIN1 in plant responses to ABA. A set of ROS scavenger genes showed reduced expression in the OE lines but improved in the RNAi plants relative to Col-0. In addition, we report that exogenous application of reduced glutathione (GSH) rescued the root growth defects seen in AIN1 overexpression lines treated with ABA. In summary, our results suggest that Arabidopsis AIN1 is involved in ABA-mediated inhibition of root elongation by modulating ROS homeostasis.

Multiplex Detection of Viral DNAs in Blood by Colorimetrically Identifying Polymerase Chain Reaction Amplicons with Serial Invasive Reaction Assisted Gold Nanoparticle Probes Assembling.

Detection of blood-borne pathogenic viruses is essential for blood transfusion, and has great significance for epidemiology, as well as clinical practices. Common blood-borne viruses causing infectious diseases include Hepatitis B virus (HBV), Hepatitis C virus (HCV), Human immunodeficiency virus (HIV) and Treponema pallidum (TP). Therefore, multiplex detection of these viruses is more in the line with the needs of clinical testing. Although real-time PCR-based multiplex nucleic acid testing (NAT) was developed for pathogen detection, however, the requirement of multichannel realtime PCR machine increases the instrumental cost and is not suitable for use in resource-limited areas. In this study, we proposed a multiplex and colorimetric assay for detecting viral nucleic acids in blood by using serial invasive reaction assisted gold nanoparticle (AuNPs) probes assembling to identify multiplex PCR amplicons. As low as 2 copies per reaction of HIV and TP targets, and 20 copies per reaction of HBV and HCV targets can be detected. The results can be observed by naked eyes; thus, just a standard PCR machine is required. In addition, the hairpin probe and the AuNPs for signal read out are universal for all the targets, reducing the detection cost. About 20 DNA samples remaining after clinical HBV testing were successfully detected, and the results were consistent with that of commercially available real-time PCR based kit, indicating that this method has a potential for clinical applications.

Evaluation of Mogroside V as a Promising Carrier in Drug Delivery: Improving the Bioavailability and Liver Distribution of Silybin.

The objective of this work was to investigate the capacity of mogroside V (MOG-V), a food additive, as a novel carrier to improve the bioavailability and liver distribution of silybin (SLY). Solid dispersion particles (SDPs) of SLY/MOG-V were prepared utilizing the solvent evaporation method. The physicochemical characterizations of SDPs were evaluated by using dynamic light scattering (DLS), differential scanning calorimetry (DSC), and powder X-ray diffraction (PXRD) measurements. DLS results demonstrated the formation of nanoparticles (206 nm) of SDPs in water. DSC and PXRD analysis revealed that SLY was in amorphous form or molecularly dispersed in SDPs. SDPs also exhibited a major increase in both dissolution rate and saturation solubility, as evidenced by a 1931-fold improvement (2201 µg/mL) in solubility compared with pure SLY (1.14 µg/mL). The pharmacokinetic study in rats showed that oral absorption of SLY/MOG-V SDPs was dramatically increased. The mean value of AUC until 12 h for SLY/MOG-V SDPs (27,481 ng·min/mL) was 24.5-fold higher than that of pure SLY (1122 ng·min/mL). In vivo tissue distribution experiment in mice confirmed that the major distribution tissue was changed from lungs to liver after SLY was loaded into MOG-V. In addition, even orally administrated to mice at a high dose (4.2 g/kg), MOG-V exhibited no undesirable effect on the plasma glucose concentrations. Thus, MOG-V may have the applicability to serve as an ideal excipient for solubilization or as a novel liver targeting carrier for the delivery of SLY.

The Rice Eukaryotic Translation Initiation Factor 3 Subunit f (OseIF3f) Is Involved in Microgametogenesis.

Microgametogenesis is the post-meiotic pollen developmental phase when unicellular microspores develop into mature tricellular pollen. In rice, microgametogenesis can influence grain yields to a great degree because pollen abortion occurs more easily during microgametogenesis than during other stages of pollen development. However, our knowledge of the genes involved in microgametogenesis in rice remains limited. Due to the dependence of pollen development on the regulatory mechanisms of protein expression, we identified the encoding gene of the eukaryotic translation initiation factor 3, subunit f in Oryza sativa (OseIF3f). Immunoprecipitation combined with mass spectrometry confirmed that OseIF3f was a subunit of rice eIF3, which consisted of at least 12 subunits including eIF3a, eIF3b, eIF3c, eIF3d, eIF3e, eIF3f, eIF3g, eIF3h, eIF3i, eIF3k, eIF3l, and eIF3m. OseIF3f showed high mRNA levels in immature florets and is highly abundant in developing anthers. Subcellular localization analysis showed that OseIF3f was localized to the cytosol and the endoplasmic reticulum in rice root cells. We further analyzed the biological function of OseIF3f using the double-stranded RNA-mediated interference (RNAi) approach. The OseIF3f-RNAi lines grew normally at the vegetative stage but displayed a large reduction in seed production and pollen viability, which is associated with the down-regulation of OseIF3f. Further cytological observations of pollen development revealed that the OseIF3f-RNAi lines showed no obvious abnormalities at the male meiotic stage and the unicellular microspore stage. However, compared to the wild-type, OseIF3f-RNAi lines contained a higher percentage of arrested unicellular pollen at the bicellular stage and a higher percentage of arrested unicellular and bicellular pollen, and aborted pollen at the tricellular stage. These results indicate that OseIF3f plays a role in microgametogenesis.

Strong association between HLA-B*1502 and carbamazepine-induced Stevens-Johnson syndrome and toxic epidermal necrolysis in mainland Han Chinese patients.

PURPOSE: The purpose of this study is to examine the association of HLA-B*1502 allele with CBZ-induced SJS/TEN in the mainland Han Chinese population. METHODS: HLA-B*1502 genotyping with sequence-specific primer polymerase chain reaction (PCR-SSP) and PCR-sequencing based typing (PCR-SBT) was performed on 17 CBZ-induced SJS/TEN patients, 21 CBZ-tolerant controls, and 185 healthy controls recruited during 2008-2010. RESULTS: HLA-B*1502 allele was present in 94.1% (16/17) of CBZ-SJS/TEN patients, 9.5% (2/21) of CBZ-tolerant patients, and 9.2% (17/185) of healthy controls. The risk of CBZ-induced SJS/TEN was significantly higher (P < 0.01) in the patients with HLA-B*1502. One CBZ-induced SJS patient tested negative for HLA-B*1502, and the test result showed HLA-B*3503/B*4601. CONCLUSIONS: We found a strong association between HLA-B*1502 and CBZ-induced SJS/TEN in the Han Chinese population from central and northern China. Combined with previous studies of the southern Han Chinese subpopulation, our results suggest that HLA-B*1502 is strongly associated with CBZ-induced SJS/TEN in the whole Han Chinese population.

Rice OsRAD21-2 is expressed in actively dividing tissues and its ectopic expression in yeast results in aberrant cell division and growth.

Rad21 and its meiotic counterpart Rec8, the key components of the cohesin complex, are essential for sister chromatid cohesion and chromosome segregation in mitosis and meiosis, respectively. In contrast to yeast and vertebrates, which have only two RAD21/REC8 genes, the rice genome encodes four Rad21/Rec8 proteins. Here, we report on the cloning and characterization of OsRAD21-2 from rice (Oryza sativa L.). Phylogenetic analysis of the full-length amino acids showed that OsRad21-2 was grouped into the plant-specific Rad21 subfamily. Semi-quantitative reverse transcription-polymerase chain reaction revealed OsRAD21-2 preferentially expressed in premeiotic flowers. Further RNA in situ hybridization analysis and promoter::ß-glucuronidase staining indicated that OsRAD21-2 was mainly expressed in actively dividing tissues including premeiotic stamen, stem intercalary meristem, leaf meristem, and root pericycle. Ectopic expression of OsRAD21-2 in fission yeast resulted in cell growth delay and morphological abnormality. Flow cytometric analysis revealed that the OsRAD21-2-expressed cells were arrested in G2 phase. Our results suggest that OsRad21-2 functions in regulation of cell division and growth.

RA68 is required for postmeiotic pollen development in Oryza sativa.

Postmeiotic development is a unique characteristic of flowering plants. During the development, microspores undergo two cycles of mitosis (PMI and PMII) and a subsequent maturation process to finally produce the mature pollen, but the mechanism underlying the development is still largely unknown. Here, we report on the roles of a novel gene, RA68, in postmeiotic pollen development in Oryza sativa. RA68 was expressed preferentially in shoots and flowers. In flowers, the transcript persisted from the floral organ differentiation to the mature pollen stages and showed preferential accumulation in male meiocytes, developing pollen and tapetal cells. RA68-deficient RNAi lines showed reduced seed setting and pollen viability but not an aberrant phenotype in vegetative organs. Knockdown of RA68 led to arrested PMI, smaller pollen grains with little or no starch, and aborted pollen but not severely distruped male meiosis. Additionally, no abnormality of anther wall development was observed in RA68-RNAi lines. RA68 may be required for postmeiotic pollen development by affecting PMI and starch accumulation.