100 MHz large bandwidth preamplifier and record-breaking 50 kHz scanning rate quantum point contact mode probe microscopy imaging with atomic resolution.

The high-bandwidth preamplifier is a vital component designed to increase the scanning speed of a high-speed scanning tunneling microscope (STM). However, the bandwidth is limited not only by the characteristic GΩ feedback resistor RF but also by the characteristic unity-gain-stable operational amplifier (UGS-OPA) in the STM preamplifier. Here, we report that paralleling a resistor with the tunneling junction (PRTJ) can break both limitations. Then, the UGS-OPA can be replaced by a higher rate, higher antinoise ability, decompensated OPA. By doing so, a bandwidth of more than 100 MHz was achieved in the STM preamplifier with decompensated OPA657, and a higher bandwidth is possible. High-clarity atomic resolution STM images were obtained under about 10 MHz bandwidth and quantum point contact microscopy mode with a record-breaking line rate of 50 k lines/s and a record-breaking frame rate of 250 frames/s. Both the PRTJ method and the decompensated OPA will pave the way for higher scanning speeds and play a key role in the design of high-performance STMs.

OsmiR528 regulates rice-pollen intine formation by targeting an uclacyanin to influence flavonoid metabolism.

The intine, the inner layer of the pollen wall, is essential for the normal development and germination of pollen. However, the composition and developmental regulation of the intine in rice (Oryza sativa) remain largely unknown. Here, we identify a microRNA, OsmiR528, which regulates the formation of the pollen intine and thus male fertility in rice. The mir528 knockout mutant aborted pollen development at the late binucleate pollen stage, significantly decreasing the seed-setting rate. We further demonstrated that OsmiR528 affects pollen development by directly targeting the uclacyanin gene OsUCL23 (encoding a member of the plant-specific blue copper protein family of phytocyanins) and regulating intine deposition. OsUCL23 overexpression phenocopied the mir528 mutant. The OsUCL23 protein localized in the prevacuolar compartments (PVCs) and multivesicular bodies (MVBs). We further revealed that OsUCL23 interacts with a member of the proton-dependent oligopeptide transport (POT) family of transporters to regulate various metabolic components, especially flavonoids. We propose a model in which OsmiR528 regulates pollen intine formation by directly targeting OsUCL23 and in which OsUCL23 interacts with the POT protein on the PVCs and MVBs to regulate the production of metabolites during pollen development. The study thus reveals the functions of OsmiR528 and an uclacyanin during pollen development.

Laccase-13 Regulates Seed Setting Rate by Affecting Hydrogen Peroxide Dynamics and Mitochondrial Integrity in Rice.

Seed setting rate is one of the most important components of rice grain yield. To date, only several genes regulating setting rate have been identified in plant. In this study, we showed that laccase-13 (OsLAC13), a member of laccase family genes which are known for their roles in modulating phenylpropanoid pathway and secondary lignification in cell wall, exerts a regulatory function in rice seed setting rate. OsLAC13 expressed in anthers and promotes hydrogen peroxide production both in vitro and in the filaments and anther connectives. Knock-out of OsLAC13 showed significantly increased seed setting rate, while overexpression of this gene exhibited induced mitochondrial damage and suppressed sugar transportation in anthers, which in turn affected seed setting rate. OsLAC13 also induced H2O2 production and mitochondrial damage in the root tip cells which caused the lethal phenotype. We also showed that high abundant of OsmiR397, the suppressor of OsLAC13 mRNA, increased the seed setting rate of rice plants, and restrains H2O2 accumulation in roots during oxidative stress. Our results suggested a novel regulatory role of OsLAC13 gene in regulating seed setting rate by affecting H2O2 dynamics and mitochondrial integrity in rice.

Circular RNAs roll into the regulatory network of plants.

As a novel class of endogenous non-coding RNAs, circular RNAs (circRNAs) have become a new research hotspot in recent years. The wide distribution of circRNAs in different plant species has been proven. Furthermore, circRNAs show significant tissue-specific expression patterns in plant development and are responsive to a variety of biotic and abiotic stresses, indicating that circRNAs might have important biological functions in plant development. Here, we summarize the current knowledge of plant circRNAs in recent years and discuss views and perspectives on the possible regulatory roles of plant circRNAs, including the function of miRNA sponges, regulating the expression of their parental genes or linear mRNAs, translating into peptides or proteins and responses to different stresses. These advances have sculpted a framework of plant circRNAs and provide new insights for functional RNA regulation research in the future.

Large-scale rewiring of innate immunity circuitry and microRNA regulation during initial rice blast infection.

Rice blast is a recurrent fungal disease, and resistance to fungal infection is a complex trait. Therefore, a comprehensive examination of rice transcriptome and its variation during fungal infection is necessary to understand the complex gene regulatory networks. In this study, adopting Next-Generation Sequencing we profiled the transcriptomes and microRNAomes of rice varieties, one susceptible and the other resistant to M. oryzae, at multiple time points during the fungal infection. Our results revealed a substantial variation in the plant transcriptome and microRNAome as well as change to rice innate immunity during fungal infection. A number of putative R gene candidates were identified from a perturbed rice transcriptome analysis. The expression of genes and non-coding RNA molecules changed in both fungal resistant and susceptible plants during M. oryzae invasion discovered distinct pathways triggered in the susceptible and resistant plants. In addition, a number of fungus genes in the susceptible and resistant plants were constantly expressed at different time points, suggesting that they were likely to be the potential AVR genes. Our results revealed large-scale rewiring of innate immunity circuitry and microRNA regulation during initial rice blast infection, which would help to develop more robust blast-resistant rice plants.

Genome-wide screening and functional analysis identify a large number of long noncoding RNAs involved in the sexual reproduction of rice.

BACKGROUND: Long noncoding RNAs (lncRNAs) play important roles in a wide range of biological processes in mammals and plants. However, the systematic examination of lncRNAs in plants lags behind that in mammals. Recently, lncRNAs have been identified in Arabidopsis and wheat; however, no systematic screening of potential lncRNAs has been reported for the rice genome. RESULTS: In this study, we perform whole transcriptome strand-specific RNA sequencing (ssRNA-seq) of samples from rice anthers, pistils, and seeds 5 days after pollination and from shoots 14 days after germination. Using these data, together with 40 available rice RNA-seq datasets, we systematically analyze rice lncRNAs and definitively identify lncRNAs that are involved in the reproductive process. The results show that rice lncRNAs have some different characteristics compared to those of Arabidopsis and mammals and are expressed in a highly tissue-specific or stage-specific manner. We further verify the functions of a set of lncRNAs that are preferentially expressed in reproductive stages and identify several lncRNAs as competing endogenous RNAs (ceRNAs), which sequester miR160 or miR164 in a type of target mimicry. More importantly, one lncRNA, XLOC_057324, is demonstrated to play a role in panicle development and fertility. We also develop a source of rice lncRNA-associated insertional mutants. CONCLUSIONS: Genome-wide screening and functional analysis enabled the identification of a set of lncRNAs that are involved in the sexual reproduction of rice. The results also provide a source of lncRNAs and associated insertional mutants in rice.

Dopamine D2 receptor stimulation inhibits angiotensin II-induced hypertrophy in cultured neonatal rat ventricular myocytes.

1. Myocardial hypertrophy is a common pathological change that accompanies cardiovascular disease. Dopamine D2 receptors have been demonstrated in cardiovascular tissues. However, the pathophysiological involvement of D2 receptors in myocardial hypertrophy is unclear. Therefore, the effects of the D2 receptor agonist bromocriptine and the D2 receptor antagonist haloperidol on angiotensin (Ang) II- or endothelin (ET)-1-induced hypertrophy of cultured neonatal rat ventricular myocytes were investigated in the present study. 2. Protein content and protein synthesis, determined by examining [(3)H]-leucine uptake, were used as estimates of cardiomyocyte hypertrophy. The expression of D2 receptor protein in neonatal rat ventricular myocytes was determined using western blotting. Changes in [Ca(2+)](i) in cardiomyocytes were observed by laser scanning confocal microscopy. 3. Angiotensin II and ET-1, both at 10 nmol/L, induced myocyte hypertrophy, as demonstrated by increased protein content and synthesis, [Ca(2+)](i) levels, protein kinase C (PKC) activity and phosphorylation of extracellular signal-regulated kinase, c-Jun N-terminal kinase and mitogen-activated protein kinase (MAPK) p38 (p38). Concomitant treatment of cells with 10 nmol/L AngII plus 10 micromol/L bromocriptine significantly inhibited cardiomyocyte hypertrophy, MAPK phosphorylation and PKC activity in the membrane, as well as [Ca(2+)](i) signalling pathways, compared with the effects of AngII alone. In addition, 10 micromol/L bromocriptine significantly inhibited cardiomyocyte hypertrophy induced by 10 nmol/L ET-1. However, pretreatment with haloperidol (10 micromol/L) had no significant effects on cardiomyocyte hypertrophy induced by either AngII or ET-1. 4. In conclusion, D2 receptor stimulation inhibits AngII-induced hypertrophy of cultured neonatal rat ventricular myocytes via inhibition of MAPK, PKC and [Ca(2+)](i) signalling pathways.

[The role of calcium-sensing receptor on ischemia/reperfusion-induced rat cardiomyocyte apoptosis].

OBJECTIVE: To investigate the role of calcium-sensing receptor (CaR) on ischemia/reperfusion-induced rat cardiomyocyte apoptosis. METHODS: The isolated rat hearts were subjected to 40 min ischemia followed by 2h of reperfusion with or without CaR agonist GdCl3 at the beginning of reperfusion. Control hearts (without ischemia) and ischemic hearts (40 min ischemia without reperfusion) served as controls. The protein expressions of CaR, Bcl-2 and cyt C were detected by Western blot. Cardiomyocyte apoptosis was detected by TUNEL. Mitochondrial potential (Deltaphim) was detected by laser confocal microscopy. RESULTS: Compared to controls groups, the expressions of CaR and apoptotic cells were significantly increased, Deltaphim and expressions of mitochondria cyt C and Bcl-2 were significantly reduced in ischemia/reperfusion hearts with or without GdCl3. CONCLUSION: CaR was involved in the induction of cardiomyocyte apoptosis during ischemia/reperfusion via mitochondrial pathway.

Role of polyamines in myocardial ischemia/reperfusion injury and their interactions with nitric oxide.

Polyamines (putrescine, spermidine, and spermine) are present in all higher eukaryotic cells and are essential for cell growth, differentiation and apoptosis. Sharing common precursor with polyamines, nitric oxide (NO) is associated with myocardial ischemia/reperfusion injury by the generation of peroxynitrite. Although polyamines have been implicated in tissue ischemia injury, their metabolism and interactions with NO in myocardial ischemia/reperfusion injury have not been fully understood. In our experiment, when Langendorff perfused rat hearts were subjected to 40 min ischemia without reperfusion, both ornithine decarboxylase (ODC) and Spermidine/spermine N(1)-acetyltransferase (SSAT) activities were up-regulated and putrescine accumulated. While after reperfusion, ODC activity decreased and SSAT activity increased, resulting in putrescine accumulation and decreased spermidine and spermine. Meanwhile NO content was increased. In addition, sodium nitroprusside (SNP, a NO donor) decreased ODC activity in cardiac tissue homogenate but increased SSAT activity in a dose-dependent manner. Pre-treatment of isolated heart with N(omega)-nitro-L-arginine methyl ester hydrochloride (L-NAME, an inhibitor of NO synthase) increased ODC activity. Exogenous spermine (1 mM) administration prior to ischemia prevented spermine decrease, reduced cardiac myocyte necrosis and apoptosis, and promoted the recovery of cardiac function after ischemia/reperfusion. These results suggest that acute heart ischemia activates myocardial polyamine stress response characterized by increased ODC and SSAT activities and accumulation of putrescine. Ischemia/reperfusion disturbs polyamine metabolism, and the loss of spermine might be associated with NO increase and thereby influences myocardial cell viability. Exogenous spermine may protect the hearts from myocardial ischemia/reperfusion injury.

[Association between calcium-sensing receptor protein expression and rat cardiomyocyte apoptosis].

OBJECTIVE: To investigate the relationship between calcium-sensing receptor protein (CaSR) expression and rat cardiomyocyte apoptosis and related signal transduction pathways. METHODS: The CaSR, BCl2, Caspase3 protein and ERK1/2 phosphorylation or non-phosphorylation were detected by Western blot. Cardiomyocyte apoptosis was detected by flow cytometry and immunofluorescence. RESULTS: CaSR protein was detected in rat cardiac tissue and CaSR activator gadolinium (GdCl3) induced cardiomyocyte apoptosis and increased ERK1/2 phosphorylation and expression of BCl2 and activated Caspase3. The selective mitogen-activated protein kinase (MAPK) inhibitor PD98059 abolished gadolinium -induced ERK1/2 activation and BCl2 expression, further increased the activation of Caspase3 and cardiomyocyte apoptosis. CONCLUSION: Our results demonstrate the CaSR existence in cardiomyocytes and CaSR activation by gadolinium can induce myocyte apoptosis by activating Caspase3 and tyrosine protein kinase pathway.

Involvement of calcium-sensing receptor in ischemia/reperfusion-induced apoptosis in rat cardiomyocytes.

The calcium-sensing receptor (CaR) is a seven-transmembrane G-protein coupled receptor, which activates intracellular effectors, for example, it causes inositol phosphate (IP) accumulation to increase the release of intracellular calcium. Although intracellular calcium overload has been implicated in the cardiac ischemia/reperfusion (I/R)-induced apoptosis, the role of CaR in the induction of apoptosis has not been fully understood. This study tested the hypothesis that CaR is involved in I/R cardiomyocyte apoptosis by increasing [Ca2+]i. The isolated rat hearts were subjected to 40-min ischemia followed by 2 h of reperfusion, meanwhile GdCl3 was added to reperfusion solution. The expression of CaR increased at the exposure to GdCl3 during I/R. By laser confocal microscopy, it was observed that the intracellular calcium was significantly increased and exhibited a Deltapsim, as monitored by 5,5',6,6'-tetrachloro-1,1',3,3'- tetraethylbenzimidazolcarbocyanine iodide (JC-1) during reperfusion with GdCl3. Furthermore, the number of apoptotic cells was significantly increased as shown by TUNEL assay. Typical apoptotic cells were observed with transmission electron microscopy in I/R with GdCl3 but not in the control group. The expression of cytosolic cytochrome c and activated caspase-9 and caspase-3 was significantly increased whereas the expression of mitochondrial cytochrome c significantly decreased in I/R with GdCl3 in comparison to the control. In conclusion, these results suggest that CaR is involved in the induction of cardiomyocyte apoptosis during ischemia/reperfusion through activation of cytochrome c-caspase-3 signaling pathway.