Positional cloning and characterization reveal the role of a miRNA precursor gene ZmLRT in the regulation of lateral root number and drought tolerance in maize.

Lateral roots play essential roles in drought tolerance in maize (Zea mays L.). However, the genetic basis for the variation in the number of lateral roots in maize remains elusive. Here, we identified a major quantitative trait locus (QTL), qLRT5-1, controlling lateral root number using a recombinant inbred population from a cross between the maize lines Zong3 (with many lateral roots) and 87-1 (with few lateral roots). Fine-mapping and functional analysis determined that the candidate gene for qLRT5-1, ZmLRT, expresses the primary transcript for the microRNA miR166a. ZmLRT was highly expressed in root tips and lateral root primordia, and knockout and overexpression of ZmLRT increased and decreased lateral root number, respectively. Compared with 87-1, the ZmLRT gene model of Zong3 lacked the second and third exons and contained a 14 bp deletion at the junction between the first exon and intron, which altered the splicing site. In addition, ZmLRT expression was significantly lower in Zong3 than in 87-1, which might be attributed to the insertions of a transposon and over large DNA fragments in the Zong3 ZmLRT promoter region. These mutations decreased the abundance of mature miR166a in Zong3, resulting in increased lateral roots at the seedling stage. Furthermore, miR166a post-transcriptionally repressed five development-related class-III homeodomain-leucine zipper genes. Moreover, knockout of ZmLRT enhanced drought tolerance of maize seedlings. Our study furthers our understanding of the genetic basis of lateral root number variation in maize and highlights ZmLRT as a target for improving drought tolerance in maize.

Mechanisms of peripheral neurotoxicity associated with four chemotherapy drugs using human induced pluripotent stem cell-derived peripheral neurons.

The awareness of the long-term toxicities of cancer survivors after chemotherapy treatment has been gradually strengthened as the population of cancer survivors grows. Generally, chemotherapy-induced peripheral neurotoxicity (CIPN) is studied by animal models which are not only expensive and time-consuming, but also species-specific differences. The generation of human induced pluripotent stem cells (hiPSCs) and differentiation of peripheral neurons have provided an in vitro model to elucidate the risk of CIPN. Here, we developed a drug-induced peripheral neurotoxicity model using hiPSC-derived peripheral neurons (hiPSC-PNs) to study the mechanisms of different chemotherapeutic agents on neuronal viability using LDH assay, a cell apoptosis assay determined by caspase 3/7 activation, neurite outgrowth, ion channel expression and neurotransmitter release following treatment of cisplatin, bortezomib, ixabepilone, or pomalidomide. Our data showed that the multiple endpoints of the hiPSC-PNs model had different sensitivity to various chemotherapeutic agents. Furthermore, the chemotherapeutics separated cell viability from the decrease in neurite lengthand changed levels of ion channels and neurotransmitters to a certain extent. Thus, we study the mechanisms of peripheral neurotoxicity induced by chemotherapeutic agents through changes in these indicators.

Bufalin-induced cardiotoxicity: new findings into mechanisms.

Bufalin is one of the main pharmacological and toxicological components of Venenum Bufonis and many traditional Chinese medicine preparations. The cardiotoxicity clearly limits its application to patients living in countries. Hence, an investigation of its toxicological mechanism is helpful for new drug development and treatment of the related clinical adverse reactions. We investigate the cardiotoxicity of bufalin using human induced pluripotent stem cells-derived cardiomyocytes (hiPSC-CMs) (0.003-0.1 µmol·L-1), human induced pluripotent stem cells-derived cardiomyocytes (hiPSC-CMs) (0.03-0.3 µmol·L-1) and eight human cardiac ion channel currents (0.01-100 µmol·L-1) combined with an impedance-based bioanalytical and patch clamp method. Biphasic effect of bufalin on the contractility in hiPSC-CMs, which has been shown to strengthen myocardial contractility, accelerate conduction, and increase beating rate at the earlier stage of administration, whereas weakened myocardial contractility, abolished conduction, and ceased beating rate at the later stage of administration. Bufalin decreased the action potential duration (Action potential duration at 30%, 50% and 90% repolarization), cardiac action potential amplitude, and maximal depolarization rate and depolarized the resting membrane potential of hiPSC-CMs. Spontaneous beating rates of hiPSC-CMs were markedly increased at 0.03 µmol·L-1, while were weakened at 0.3 µmol·L-1 after application. Bufalin blocks INav1.5 in a concentration-dependent manner with half maximal inhibitory concentration of 74.5 µmol·L-1. Bufalin respectively increased the late sodium current and Na+-Ca2+ exchange current with a concentration for 50% of maximal effect of 2.48 and 66.06 µmol·L-1 in hiPSC-CMs. Whereas, bufalin showed no significant effects on other cardiac ion channel currents. The enhancement of the late sodium current is one of the main mechanism for cardiotoxicity of bufalin.

A fusion algorithm of passive sound source localization based on the two-plane four-element cross array.

In this work, a fusion algorithm is proposed for improving the accuracy and stability of passive sound source localization. Different from the traditional algorithm that contains a single-plane cross array, here, the fusion algorithm is used to overcome the position blur in the process of localization. First, the two-plane four-element cross array model is established. Based on this model, the method is defined to judge the position where the sound source is located. According to the localization principle, we derive the calculation formula of the sound source position, based on a single four-element planar array. Then, the elevation angle sine value is introduced into the coordinate formula as the weighted coefficient by analyzing the indirect measurement error, and the fusion algorithm is employed to conduct the sound source localization, based on the two-plane four-element cross array. Finally, the relationships are obtained, between the time delay estimation error, the elevation angle, the horizontal angle, and the localization performance. Besides, the validity of this algorithm is validated by measuring the ranging and direction-finding accuracy. The results show that the distance error rate is within 2%, and the angle error rate is within 3%, which means a good localization effect. The proposed algorithm is expected to be widely used in thunderstorm cloud detection for its quick measurement and high precision.

Building Extraction from High-Resolution Remote Sensing Images by Adaptive Morphological Attribute Profile under Object Boundary Constraint.

A novel adaptive morphological attribute profile under object boundary constraint (AMAP-OBC) method is proposed in this study for automatic building extraction from high-resolution remote sensing (HRRS) images. By investigating the associated attributes in morphological attribute profiles (MAPs), the proposed method establishes corresponding relationships between AMAP-OBC and building characteristics in HRRS images. In the preprocessing step, the candidate object set is extracted by a group of rules for screening of non-building objects. Second, based on the proposed adaptive scale parameter extraction and object boundary constraint strategies, AMAP-OBC is conducted to obtain the initial building set. Finally, a further identification strategy with adaptive threshold combination is proposed to obtain the final building extraction results. Through experiments of multiple groups of HRRS images from different sensors, the proposed method shows outstanding performance in terms of automatic building extraction from diverse geographic objects in urban scenes.

Doubling the coexisting attractors.

When the offset boosting technique is introduced into a chaotic system for attractor shifting, the number of coexisting attractors in the system can be doubled under the application of the employed absolute-value function. Consequently, the offset booster becomes a doubling parameter determining the distance between the two coexisting attractors, and therefore can polymerize these attractors to become a pseudo-multi-scroll attractor. This paper demonstrates that the attractor doubling operation can be applied to any dimension of the system and can also be nested at any time leading to the geometric growth of the coexisting attractors. Furthermore, various regimes of coexistence can be merged and composed together to reproduce an integrated attractor in the system.

MicroRNA-29a activates a multi-component growth and invasion program in glioblastoma.

BACKGROUND: Glioblastoma is a malignant brain tumor characterized by rapid growth, diffuse invasion and therapeutic resistance. We recently used microRNA expression profiles to subclassify glioblastoma into five genetically and clinically distinct subclasses, and showed that microRNAs both define and contribute to the phenotypes of these subclasses. Here we show that miR-29a activates a multi-faceted growth and invasion program that promotes glioblastoma aggressiveness. METHODS: microRNA expression profiles from 197 glioblastomas were analyzed to identify the candidate miRNAs that are correlated to glioblastoma aggressiveness. The candidate miRNA, miR-29a, was further studied in vitro and in vivo. RESULTS: Members of the miR-29 subfamily display increased expression in the two glioblastoma subclasses with the worst prognoses (astrocytic and neural). We observed that miR-29a is among the microRNAs that are most positively-correlated with PTEN copy number in glioblastoma, and that miR-29a promotes glioblastoma growth and invasion in part by targeting PTEN. In PTEN-deficient glioblastoma cells, however, miR-29a nevertheless activates AKT by downregulating the metastasis suppressor, EphB3. In addition, miR-29a robustly promotes invasion in PTEN-deficient glioblastoma cells by repressing translation of the Sox4 transcription factor, and this upregulates the invasion-promoting protein, HIC5. Indeed, we identified Sox4 as the most anti-correlated predicted target of miR-29a in glioblastoma. Importantly, inhibition of endogenous miR-29a decreases glioblastoma growth and invasion in vitro and in vivo, and increased miR-29a expression in glioblastoma specimens correlates with decreased patient survival. CONCLUSIONS: Taken together, these data identify miR-29a as a master regulator of glioblastoma growth and invasion.

Intraglandular transplantation of adipose-derived stem cells combined with platelet-rich fibrin extract for the treatment of irradiation-induced salivary gland damage.

The aim of the present study was to determine the effect of adipose-derived mesenchymal stem cells (ADSCs) combined with heterologous platelet-rich fibrin extract (PRFe) on irradiation-induced salivary gland (SG) damage. ADSCs were isolated from C3H mice, whereas PRFe was obtained from New Zealand rabbits. Twelve weeks post irradiation, the ADSCs or PRFe or their combination were transplanted into the submandibular glands of C3H mice with irradiation-induced damage. The salivary flow rate (SFR) was determined and histopathological analysis was performed at 12 weeks post transplantation. Immunofluorescence, microvessel density measurements and transmission electron microscopy were performed to assess α-amylase (AMY) production, apoptosis and microstructural changes in the cells. The administration of ADSCs combined with PRFe increased the SFR at 12 weeks post transplantation, whereas ADSCs alone or PRFe alone failed to do so. The ADSCs+PRFe-treated, irradiated SGs had fewer damaged and atrophied acinar cells, higher AMY levels and an increased microvessel density compared with the untreated irradiated SGs. Moreover, SG tissue from the ADSCs+PRFe group also showed decreased apoptotic and increased proliferative activity compared to that from the irradiated group. In conclusion, ADSCs or PRFe alone did not restore permanent, irradiation-induced damage of SG tissue when used alone, but when used together, they provided effective treatment outcomes.

Association between shunt-responsive idiopathic normal pressure hydrocephalus and alcohol.

OBJECTIVE Idiopathic normal pressure hydrocephalus (iNPH) is characterized by ventriculomegaly, gait difficulty, incontinence, and dementia. The symptoms can be ameliorated by CSF drainage. The object of this study was to identify factors associated with shunt-responsive iNPH. METHODS The authors reviewed the medical records of 529 patients who underwent shunt placement for iNPH at their institution between July 2001 and March 2015. Variables associated with shunt-responsive iNPH were identified using bivariate and multivariate analyses. Detailed alcohol consumption information was obtained for 328 patients and was used to examine the relationship between alcohol and shunt-responsive iNPH. A computerized patient registry from 2 academic medical centers was queried to determine the prevalence of alcohol abuse among 1665 iNPH patients. RESULTS Bivariate analysis identified associations between shunt-responsive iNPH and gait difficulty (OR 4.59, 95% CI 2.32-9.09; p < 0.0001), dementia (OR 1.79, 95% CI 1.14-2.80; p = 0.01), incontinence (OR 1.77, 95% CI 1.13-2.76; p = 0.01), and alcohol use (OR 1.98, 95% CI 1.23-3.16; p = 0.03). Borderline significance was observed for hyperlipidemia (OR 1.56, 95% CI 0.99-2.45; p = 0.054), a family history of hyperlipidemia (OR 3.09, 95% CI 0.93-10.26, p = 0.054), and diabetes (OR 1.83, 95% CI 0.96-3.51; p = 0.064). Multivariate analysis identified associations with gait difficulty (OR 3.98, 95% CI 1.81-8.77; p = 0.0006) and alcohol (OR 1.94, 95% CI 1.10-3.39; p = 0.04). Increased alcohol intake correlated with greater improvement after CSF drainage. Alcohol abuse was 2.5 times more prevalent among iNPH patients than matched controls. CONCLUSIONS Alcohol consumption is associated with the development of shunt-responsive iNPH.

Protective efficacy of intravenous transplantation of adipose-derived stem cells for the prevention of radiation-induced salivary gland damage.

OBJECTIVE: High-dose radiation therapy in the head and neck area can lead to irreversible damage to salivary glands (SGs) with consequent xerostomia. Adipose-derived stem cells (ADSCs) have been shown to repair or rescue damaged SGs. Thus, we investigated the protective efficacy of ADSCs in the prevention of SG damage induced by high dose radiation. METHODS: Third-passage ADSCs (1×10(6)) were transplanted by intravenous infusion into the tail-vein of 8-week-old C57BL/6 mice, immediately after local irritation at a dose of 18Gy. The process was repeated twice a week during a period of six consecutive weeks. Eight weeks after radiation, functional evaluations were conducted by measuring salivary flow rate (SFR). Histological, immunohistochemical and transmission electron microscopic (TEM) examinations were performed to analyze microstructural and ultrastructural changes, microvessel density, amylase production, apoptosis, and proliferation activity. RESULTS: Intravenously administrated ADSCs could home to irradiated SGs within 24h after infusion, significantly increasing SG weights, improving SFR, and preserving the microscopic morphologies of SGs eight weeks post-radiation. More functional acini, higher amylase production levels, and higher microvessel densities were observed in ADSC-treated SGs than in irradiated SGs. Additionally, enhanced cell proliferation activity and reduced radiation-induced SG apoptosis was observed in the ADSC-treated group when compared with the irradiated group. CONCLUSION: Systemic administration of ADSCs immediately after radiation at a dose of 18Gy can protect both the morphology and function of SGs eight weeks after radiation in mice, and can be used as a protective measure for the prevention of SG damage induced by high-dose radiation.

A major role for microRNAs in glioblastoma cancer stem-like cells.

Studies have demonstrated that miRNAs contribute to the maintenance and phenotype of in several cancer types. This review will focus on the roles of a few well studied miRNAs in cancer stem-like cells of glioblastoma.

Maize (Zea mays L.) seedling leaf nuclear proteome and differentially expressed proteins between a hybrid and its parental lines.

To better understand the underlying molecular basis of leaf development in maize, a reference map of nuclear proteins in basal region of seedling leaf was established using a combination of 2DE and MALDI-TOF-MS. In total, 441 reproducible protein spots in nuclear proteome of maize leaf basal region were detected with silver staining in a pH range of 3-10, among which 203 spots corresponding to 163 different proteins were identified. As expected, proteins implicated in RNA and protein-associated functions were overrepresented in nuclear proteome. Remarkably, a high percentage (10%) of proteins was identified to be involved in cell division and growth. In addition, comparative nuclear proteomic analysis in leaf basal region of highly heterotic hybrid Mo17/B73 and its parental lines was also performed and 52 of 445 (11.69%) detected protein spots were differentially expressed between the hybrid and its parental lines, among which 16 protein spots displayed nonadditively expressed pattern. These results indicated that hybridization between two parental lines can cause changes in the expression of a variety of nuclear proteins, which may be responsible for the observed leaf size heterosis.

Numb regulates glioma stem cell fate and growth by altering epidermal growth factor receptor and Skp1-Cullin-F-box ubiquitin ligase activity.

Glioblastoma contains a hierarchy of stem-like cancer cells, but how this hierarchy is established is unclear. Here, we show that asymmetric Numb localization specifies glioblastoma stem-like cell (GSC) fate in a manner that does not require Notch inhibition. Numb is asymmetrically localized to CD133-hi GSCs. The predominant Numb isoform, Numb4, decreases Notch and promotes a CD133-hi, radial glial-like phenotype. However, upregulation of a novel Numb isoform, Numb4 delta 7 (Numb4d7), increases Notch and AKT activation while nevertheless maintaining CD133-hi fate specification. Numb knockdown increases Notch and promotes growth while favoring a CD133-lo, glial progenitor-like phenotype. We report the novel finding that Numb4 (but not Numb4d7) promotes SCF(Fbw7) ubiquitin ligase assembly and activation to increase Notch degradation. However, both Numb isoforms decrease epidermal growth factor receptor (EGFR) expression, thereby regulating GSC fate. Small molecule inhibition of EGFR activity phenocopies the effect of Numb on CD133 and Pax6. Clinically, homozygous NUMB deletions and low Numb mRNA expression occur primarily in a subgroup of proneural glioblastomas. Higher Numb expression is found in classical and mesenchymal glioblastomas and correlates with decreased survival. Thus, decreased Numb promotes glioblastoma growth, but the remaining Numb establishes a phenotypically diverse stem-like cell hierarchy that increases tumor aggressiveness and therapeutic resistance.

Genome-wide identification and expression profiling of auxin response factor (ARF) gene family in maize.

BACKGROUND: Auxin signaling is vital for plant growth and development, and plays important role in apical dominance, tropic response, lateral root formation, vascular differentiation, embryo patterning and shoot elongation. Auxin Response Factors (ARFs) are the transcription factors that regulate the expression of auxin responsive genes. The ARF genes are represented by a large multigene family in plants. The first draft of full maize genome assembly has recently been released, however, to our knowledge, the ARF gene family from maize (ZmARF genes) has not been characterized in detail. RESULTS: In this study, 31 maize (Zea mays L.) genes that encode ARF proteins were identified in maize genome. It was shown that maize ARF genes fall into related sister pairs and chromosomal mapping revealed that duplication of ZmARFs was associated with the chromosomal block duplications. As expected, duplication of some ZmARFs showed a conserved intron/exon structure, whereas some others were more divergent, suggesting the possibility of functional diversification for these genes. Out of these 31 ZmARF genes, 14 possess auxin-responsive element in their promoter region, among which 7 appear to show small or negligible response to exogenous auxin. The 18 ZmARF genes were predicted to be the potential targets of small RNAs. Transgenic analysis revealed that increased miR167 level could cause degradation of transcripts of six potential targets (ZmARF3, 9, 16, 18, 22 and 30). The expressions of maize ARF genes are responsive to exogenous auxin treatment. Dynamic expression patterns of ZmARF genes were observed in different stages of embryo development. CONCLUSIONS: Maize ARF gene family is expanded (31 genes) as compared to Arabidopsis (23 genes) and rice (25 genes). The expression of these genes in maize is regulated by auxin and small RNAs. Dynamic expression patterns of ZmARF genes in embryo at different stages were detected which suggest that maize ARF genes may be involved in seed development and germination.

PEST sequences mediate heat shock factor 2 turnover by interacting with the Cul3 subunit of the Cul3-RING ubiquitin ligase.

Cullin-RING ubiquitin ligases promote the polyubiquitination and degradation of many important cellular proteins, which previous studies indicated can be targeted for degradation via interaction with BTB domain-containing subunits of this E3 ligase complex. PEST domains are known to promote the degradation of proteins that contain them. However, the molecular mechanism by which PEST sequences promote degradation of these proteins is not understood. Here we show that the PEST sequences of a short-lived protein called HSF2 interact with Cullin3, a subunit of a Cullin-RING E3 ubiquitin ligase, and that this interaction mediates the Cul3-dependent ubiquitination and degradation of HSF2. These results indicate how, at the molecular level, PEST sequences can promote the proteolysis of proteins that contain them. They also expand understanding of the mechanisms by which substrates can be recruited to Cullin-RING E3 ubiquitin ligases to include interactions between PEST sequences and Cul3.

Chromosome-wide analysis of protein binding and modifications.

In order to fully understand the functions of a DNA-binding protein it is necessary to identify all of its binding sites in chromosomes and assess the role of each site in the overall biological function of the factor. An approach ChIP-on-Chip which combines the chromatin immunoprecipitation technique with chromosomal DNA microarray analysis, has proven to be a powerful means for the chromosome-wide identification of protein binding sites. This approach can also be used to characterize chromosome-wide variations in patterns of post-translational protein modifications, for example histone modifications. This chapter presents methodologies for the ChIP-on-Chip analysis, using as an example the identification of chromosome-wide binding sites for the TATA-binding protein in mitotic cells.

The TBP-PP2A mitotic complex bookmarks genes by preventing condensin action.

To maintain phenotypes of cell lineages, cells must 'remember' which genes were active before mitosis entry and transmit this information to their daughter cells so that expression patterns can be faithfully re-established in G1. This phenomenon is called gene bookmarking. However, during mitosis transcription ceases, most sequence-specific proteins dissociate from DNA and the chromatin is tightly compacted, making it difficult to understand how gene activity 'memory' is maintained through this stage of the cell cycle. A feature of gene bookmarking is that in mitotic cells, the promoters of formerly active genes lack compaction, but how compaction of these regions is inhibited is unknown. Here we show that during mitosis, TATA-binding protein (TBP), which remains bound to DNA during mitosis, recruits PP2A. TBP also interacts with condensin to allow efficient dephosphorylation and inactivation of condensin near these promoters to inhibit their compaction. Further, ChIP-on-chip data show that TBP is bound to many chromosomal sites during mitosis, and is higher in transcribed regions but low in regions containing pseudogenes and genes whose expression is tissue-restricted. These results suggest that TBP is involved not only in gene transcription during interphase but also in preserving the memory of gene activity through mitosis to daughter cells.

HSF1-TPR interaction facilitates export of stress-induced HSP70 mRNA.

Stress conditions inhibit mRNA export, but mRNAs encoding heat shock proteins continue to be efficiently exported from the nucleus during stress. How HSP mRNAs bypass this stress-associated export inhibition was not known. Here, we show that HSF1, the transcription factor that binds HSP promoters after stress to induce their transcription, interacts with the nuclear pore-associating TPR protein in a stress-responsive manner. TPR is brought into proximity of the HSP70 promoter after stress and preferentially associates with mRNAs transcribed from this promoter. Disruption of the HSF1-TPR interaction inhibits the export of mRNAs expressed from the HSP70 promoter, both endogenous HSP70 mRNA and a luciferase reporter mRNA. These results suggest that HSP mRNA export escapes stress inhibition via HSF1-mediated recruitment of the nuclear pore-associating protein TPR to HSP genes, thereby functionally connecting the first and last nuclear steps of the gene expression pathway, transcription and mRNA export.

Identification of the PP2A-interacting region of heat shock transcription factor 2.

Previous work in our laboratory demonstrated the existence of an association between heat shock transcription factor 2 (HSF2) and the serine/threonine phosphatase 2A, which is mediated by interaction between HSF2 and the A subunit (also called PR65) of this protein phosphatase. In light of the importance of HSF2-PP2A association for HSF2 cellular function, in this study, we have sought to dissect the sequences within HSF2 that are important for interaction with the A subunit of PP2A. The results of these experiments indicate that the HSF2 region comprising amino acids 343-363 is important for A subunit interaction. This region includes part of the C-terminal leucine zipper motif of HSF2 called heptad repeat C (HR-C). The results of transfection/immunoprecipitation experiments also show that deletion of the 6 amino acids from 343 to 348 from HSF2 (HSF2 (delta343-348)), is sufficient to prevent HSF2 from interacting with PP2A. These data provide insight into a new functional domain of HSF2, the PP2A A subunit-interacting region.

Increased pathogen resistance and yield in transgenic plants expressing combinations of the modified antimicrobial peptides based on indolicidin and magainin.

Reverse peptide of indolicidin (Rev4), a 13-residue peptide based on the sequence of indolicidin, has been shown to possess both strong antimicrobial and protease inhibitory activities in vitro. To evaluate its efficacy in vivo, we produced and evaluated transgenic tobacco (Nicotiana tabacum L.) and Arabidopsis thaliana [(L.) Heynh.] plants expressing Rev4 with different signal peptide sequences for pathogen resistance. All transgenic plants showed normal growth and development, an indication of no or low cytotoxicity of the peptide. Furthermore, the transgenic plants exhibited elevated resistance to three bacterial and two oomycete pathogens. Interestingly, tobacco plants expressing Rev4 displayed enhanced yield compared to the control as indicated by an increased biomass production by as much as 34% in two field trials. When Rev4 was coexpressed with another antimicrobial peptide, Myp30, the disease resistance levels in the transgenic Arabidopsis were enhanced. These findings suggest the potential of using these peptides to protect plants from microbial pathogens and to enhance yield.