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Efficient isolation of Magnolia protoplasts and the application to subcellular localization of MdeHSF1
Yamei Shen1,2 na1,
Dong Meng2 na1,
Kim McGrouther3,
Junhong Zhang2,4 &
Lailiang Cheng2
Magnolia is a woody ornamental plant, which is widely used in urban landscaping. However, its lengthy juvenile period and recalcitrance to regeneration impedes functional characterization of its genes.
We developed an efficient protoplast isolation and transient expression system for Magnolia denudata × Magnolia acuminata 'Yellow River'. The highest yield of protoplasts was obtained from young leaves digested in 3% Cellulase R10, 0.8% Macerozyme R10, 0.04% pectinase and 0.4 M mannitol enzymolysis solution for 6 h. For transfection of protoplasts, 20% PEG4000 for 5 min was optimal. To verify the protoplast system and begin to understand heat tolerance in Magnolia, a heat shock transcription factor MdeHSF1 was cloned from 'Yellow River', which belongs to the HSF subfamily A and has significant homology with AtHSFA1A. Subcellular localization analysis indicated that MdeHSF1 was expressed in the cell nucleus. Furthermore, qPCR analysis of the MdeHSF1 transcript level in response to high temperature stress suggested that MdeHSF1 might be involved in regulating heat stress tolerance in 'Yellow River'.
The described protocol provides a simple and straightforward method for isolating protoplast and exploring gene subcellular localization of MdeHSF1 in Magnolia. This expands the new research of protoplast isolation and transfection in Magnolia.
Magnolia is among the best known ornamental plant used in landscaping. The majority of Magnolia species are distributed in America and East Asia, including relict plants, some of which have survived for hundreds of millions of years [1]. Many species of Magnolia growing in the wild are endangered, and some face extinction. Several factors have contributed to this situation, including threats to their habitats, limited genetic diversity, and difficulties associated with breeding. Efforts are being made to protect, develop, and spread magnolias. Grafting and seeding are the main methods for propagating Magnolia. However, Magnolia has a long juvenile period (4–5 years or more) and is recalcitrant to regeneration. Currently, lack of any transformation system has become a limiting step for functional characterization of its genes including subcellular localization, protein–protein interaction, DNA–protein interaction, and transgenic studies [2,3,4]. Thus, transformation methods are urgently needed.
The plant protoplast can serve as a versatile cell-based experimental system to analyze gene expression during a transient time period [3, 5, 6]. Macromolecules, such as DNA, RNA and proteins, can be delivered into protoplasts using various methods, e.g., PEG-calcium fusion [7], electroporation [7] and microinjection [8]. Signal transduction and metabolic pathways as well as the transcription and translation machinery can be transiently manipulated to investigate cell-autonomous regulation and responses [9]. In addition, protoplasts can be used as a method for breeding. Plant protoplast isolation was first reported half a century ago using the root tip of tomato seedlings [10]. Subsequently, mesophyll cells [11,12,13], fruits [14, 15], stem [16], seed [17] and calluses [18] have been used as materials to isolate the protoplast. In recent years, protoplast isolation and transfection was well established for Arabidopsis [3, 4, 9, 19], Populus spp. [20, 21] and Bambusa vulgaris [22], etc. However, no published protocol for protoplast isolation and transfection is available for Magnolia.
As temperatures continue to rise globally, plants are increasingly exposed to heat stress. The major heat shock factors regulating heat stress response are heat shock transcription factors (HSFs) [23], which are important regulators for sensing and signaling heat stress [24]. HSFs have been characterized in Brassica rapa spp. pekinensis [23], Helianthus annuus [25], Arabidopsis [26], Zea mays [27], Glycine max [28] and Pyrus bretschneideri [24]. As most species of Magnolia appear to be particularly sensitive to heat stress, characterizing HSFs in Magnolia is of great significance to understanding and eventually improving their tolerance to heat stress.
In this study, we selected one classical species of Magnolia, Magnolia denudata × M. acuminata 'Yellow River' ('Yellow River'), as the plant material to develop an efficient protoplast isolation and transfection protocol. 'Yellow River' is a hybrid of M. denudata × M. acuminata (Fig. 1). They have characteristic yellow flowers and are widely used in urban landscaping in China. As 'Yellow River' performs better than M. × soulangeana (Additional file 1: Table S1) in terms of heat stress tolerance, the molecular mechanism responsible for higher heat-stress resistance in 'Yellow River' is worth elucidating. The transient transformation system in protoplast is a cell-based functional genomics tool to explore this molecular mechanism. Therefore, we cloned its heat shock transcription factor MdHSF1 and characterized its subcellular localization using protoplast transfection in this paper, and the results confirm that the transient transformation system is viable. The method reported here for protoplast isolation and transfection of Magnolia 'Yellow River' lays a good foundation for the breeding of Magnolia, and expands the functional gene research in the field of Magnolia.
Different tissues for protoplast isolation in 'Yellow River'. The shape of 'Yellow River' (a), mature leaves of 'Yellow River' (b), young leaves of 'Yellow River' (c), 'cut-off' leaves from 'Yellow River' (d, e), Protoplasts isolated from the young leaves (f, g), bar 50 μm
Plant material and growth conditions
M. denudata × M. acuminata 'Yellow River' was grown on the campus of Cornell University in Ithaca, New York, USA (Fig. 1a) was approximately 20 years old, with the diameter at breast height (DBH) of 11.0 cm. Mature leaves were sampled on July 7, 2014 (Fig. 1b). The annual branches with leaf buds were collected on February 16, 2015 (Fig. 1c). The annual branches were taken into the lab, and cultured in water at 23 °C. After one week, the sprouted leaves were used to isolate protoplasts. Meanwhile, the buds were also collected. All these materials were taken from the annual branches and they all faced in one direction. After being detached, mature leaves, young leaves and buds were washed twice with sterile water, and then sterilized with 70% ethanol for 5 s first and then with 0.1% HgCl2 for 5 min. Finally, the materials were used to isolate protoplast after being washed 4 times with sterile water (Fig. 1d–g).
Protoplast isolation
Protoplast isolation was performed following the methods of Wu [3], Zhai [29], Rezazadeh [11] and Guo [20], with some modifications. 2 g of leaves (Mature leaves or young leaves) were sliced to 1–2 mm width in a sterilized petri dish with a fresh razor blade (2 g of buds was also sliced to fragments with a razor blade), then rapidly transferred into 100 mL sterilized beaker which contained 10 mL enzymatic solution (The enzymatic solution was placed at 55 °C for 10 min, cooled to room temperature, and sterilized using a 0.45 µm filter prior to use).
Then three methods were used to treat samples for choosing the optimal way of protoplast isolation: Method 1: Four concentrations mannitol/sorbitol (0.2, 0.4, 0.6 or 0.8 M) were used to treat the samples for getting optimal enzymatic solution (replicated three times each experiment); Method 2: Six combinations with different concentrations of Cellulase R10 and Macerozyme R10 (a) 1% (w/v) Cellulase R10, 0.4% (w/v) Macerozyme R10, 0.04% (w/v) Pectinase; (b) 1.5% (w/v) Cellulase R10, 0.5% (w/v) Macerozyme R10, 0.04% (w/v) Pectinase; (c) 2% (w/v) Cellulase R10, 0.6% (w/v) Macerozyme R10, 0.04% (w/v) Pectinase; (d) 2.5% (w/v) Cellulase R10, 0.7% (w/v) Macerozyme R10, 0.04% (w/v) Pectinase; (e) 3% (w/v) Cellulase R10, 0.8% (w/v) Macerozyme R10, 0.04% (w/v) Pectinase; (f) 3.5% (w/v) Cellulase R10, 0.9% (w/v) Macerozyme R10, and 0.04% (w/v) Pectinase). Each combination was prepared to the appropriate concentration using a solution containing 20 mM KCl; 20 mM MES-KOH (pH 5.7); 10 mM CaCl2; 10 mM β-mercaptoethanol and 0.1% (w/v) bovine serum albumin (replicated three times each experiment). The third method was based on the results from the methods 1 and 2, and involved vacuum-infiltrating leaf strips for 30 min and incubated in the enzymatic solution at 25 °C in darkness with rotation (60 rpm) for about 2, 4, 6, 8, 10 or 12 h (the beaker with the leaf strips was covered with parafilm and aluminum foil) (replicated three times each experiment). After digestion, an equal volume of W5 solution (154 mM NaCl, 125 mM CaCl2, 5 mM KCl, 2 mM MES-KOH (pH 5.7), sterilized using a 0.45-mm filter) was added. Next, the solution was filtered through a sterilized nylon membrane (200 µm mesh size) and transferred into a 50 mL sterilized centrifuge tube. Then the nylon membrane was washed twice with 10 mL W5 solution and all the filtrate was transferred to a 50 mL centrifuge tube.
Finally, the filtrate was centrifuged at 100g for 10 min, and the supernatant was carefully removed using a pipettor. The protoplasts were washed twice with W5 solution and re-suspended to a final concentration of 1 × 106 protoplasts/mL for transfection.
The protoplast yield of different tissue types (mature leaves, young leaves and bud) was also compared. The viability of the isolated protoplasts was also determined with FDA (fluorescein diacetate) dye [30]. The FDA stock solution was added into the isolated protoplasts at a final concentration of 0.01%. After being incubated in darkness for 5 min and washed three times with W5 solution, protoplast viability was checked under a fluorescence microscope.
Protoplast transfection
The isolated protoplasts were placed on ice, and an equal volume of PEG solution (20% w/v polyethylene glycol, PEG4000, 0.3 M mannitol and 100 mM CaCl2) (with 10% PEG4000 as a control, replicated three times) was added. In order to select the optimal concentration of PEG4000, concentrations of 10, 15, 20, 25 and 30% PEG4000, w/v were tested. Next, 20 µg of the pEZS-NL plasmid, which contained a 35S promoter and the coding sequence fused to EGFP and OCS 3′ terminator which can used for transient expression [31] (Isolated by PureLink® Quick Plasmid Miniprep Kit, Invitrogen, cat. no. K210010), 100 µL of isolated protoplasts and 120 µL of the PEG solution were combined and mixed gently in a 2 mL microfuge tube. The mixture was incubated at room temperature for 5, 10, 15 and 30 min in triplicate (to determine the optimum transfection time) before 220 µL of W5 solution was added to terminate the reaction. The mixture was centrifuged at 120g for 5 min, and the supernatant was carefully removed with a pipettor. The protoplasts were re-suspended overnight at room temperature in 2 mL of incubation buffer (0.4 mM mannitol, 4 mM MES-KOH (pH 5.7), 20 mM KCl). Finally, the protoplasts were centrifuged at 120g for 2 min and re-suspended with an appropriate amount of WI solution (0.5 M mannitol, 4 mM MES-KOH (pH 5.7), 20 mM KCl). A Zeiss LSM 710 fluorescence microscope was used to visualize the EGFP signal. The transformation efficiency was determined by (%) = (fluorescent protoplast number in view/total protoplast number in view) × 100%.
All data were analyzed by SPSS.19. The data of transfection rate was analyzed through the square root of the arcsine difference.
RNA isolation and cloning of MdeHSF1 and construction of its expression vector
RNA was extracted from leaves of 'Yellow River' according to a modified CTAB method and digested with DNase I. RNA was detected by electrophoresis. Total RNA was used to synthesize first-strand cDNA using the SuperScript reverse transcriptase with Oligo-dT primer. The cDNA was then used as templates for PCR amplification.
Our preliminary data obtained on 'Yellow River' at Hangzhou, Zhejiang Province of China (Additional file 1: Table S1) showed that 'Yellow River' has stronger tolerance to heat stress than M. × soulangana. We also determined the transcript profile of 'Yellow River' leaves using RNA-seq in response to heat treatment. Based on the transcriptome data of M. sinostellata, the primers were designed, F: 5′-ATGGACGGTGCTCATGGCAGCA-3′, R: 5′-CAGCCTTTGTTTTCTGATGTAAGAA-3′, for homology-based cloning of MdeHSF1 via PCR. The PCR products were cloned into the pMD19-T simple vector using T4 DNA Ligase, Then the vector was transferred into E. coli. DH5a competent cells and grown on the LB medium with Ampicillin. Positive bacterial colonies were detected by PCR using the M13 primer shown in the pMD19-T vector user manual; then sequenced at Cornell's Biotechnology Resources Center. Finally, MdeHSF1 was analyzed from the website of http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi, the protein structure was analyzed using the smart website http://smart.embl-heidelberg.de/, the protein prediction was from the website https://www.predictprotein.org/, and MdeHSF1 was compared with HSF of Arabidopsis thaliana.
The correct clone of MdeHSF1 was cloned into the transient expression vector pEZS-NL through double enzyme digestion (BamHI and XhoI), then the vector was transferred into the DH5a competent cells. The correct clone was detected by PCR using the 35S promoter primer F and HSF primer R. The clone was cultured on LB liquid medium (Ampicillin) at 37 °C with rotation (120 rpm) for 12 h. The plasmid was isolated using PureLink® Quick Plasmid Miniprep Kit.
RNA extraction and quantitative real-time PCR (qRT-PCR)
'Yellow River' and M. × soulangeana leaf samples were finely grounded in liquid nitrogen, and RNA was extracted using the CTAB method as described by Gasic et al. [32]. After treatment with RQ1 DNase, RNA was quantified using a NanoDrop spectrophotometer, and the RNA integrity was confirmed by agarose gel electrophoresis. One milligram of total RNA was reverse-transcribed into cDNA using the iScript cDNA Synthesis kit. Actin gene served as the internal control. Every qRT-PCR was performed in 3 replicates on an Icycler iQ5 (BioRad) using the SYBR Green Supermix kit (Bio-Rad) according to the instruction manual. Data were analyzed using iQ5 2.0 software (Bio-Rad) with the ddCT method.
The sources of all relative reagents were showed in Additional file 1: Table S2.
An efficient method of protoplast isolation in 'Yellow River'
The yield of protoplast in the treatment of mannitol was significantly higher than that under sorbitol treatment (p < 0.01). The optimal concentration was 0.4 M mannitol, which yielded 1.89 × 105 protoplasts/g FW (fresh weight), the highest yield of all the treatments (p < 0.01) (Fig. 2a).
The protoplast yield under different treatments. In response to a range (0.2, 0.4, 0.6 and 0.8 M) of mannitol and sorbitol concentrations in digestion solution with 3% Cellulase R10, 0.8% Macerozyme R10, and 0.4% Pectinase (a), protoplast yield as affected by different concentrations of digestion enzymes (b): (a) 1% (w/v) Cellulase R10, 0.4% (w/v) Macerozyme R10, 0.04% (w/v) Pectinase; (b) 1.5% (w/v) Cellulase R10, 0.5% (w/v) Macerozyme R10, 0.04% (w/v) Pectinase; (c) 2% (w/v) Cellulase R10, 0.6% (w/v) Macerozyme R10, 0.04% (w/v) Pectinase; (d) 2.5% (w/v) Cellulase R10, 0.7% (w/v) Macerozyme R10, 0.04% (w/v) Pectinase; (e) 3% (w/v) Cellulase R10, 0.8% (w/v) Macerozyme R10, 0.04% (w/v) Pectinase; (f) 3.5% (w/v) Cellulase R10, 0.9% (w/v) Macerozyme R10, and 0.04% (w/v) Pectinase, protoplast yield as affected by digestion time (2, 4, 6, 8, 10 or 12 h) (c), protoplast yield as affected by tissue type (buds, young leaves and mature leaves) (d)
Furthermore, we tested the effects of different concentration combinations of cell wall-degrading enzymes on protoplast yield. Via the addition of various concentrations of the enzymes, the protoplast yield tended to increase until the concentration reached 3% (w/v) Cellulase (R10), 0.8% (w/v) Macerozyme (R10) and 0.04% (w/v) Pectinase. The protoplast yield in this combination was significantly higher than in other combinations (p < 0.01) (Fig. 2b). Effects of enzymolysis time on protoplast yield were also tested. As digestion time increased from 2 to 6 h, protoplast yield increased, but with further increases in digestion time, protoplast yield decreased significantly (Fig. 2c). These results indicated that the optimal digestion time was 6 h.
Finally, we examined the effect of three tissue types (buds, young leaves and mature leaves) on protoplast yield and found that protoplast yield obtained from young leaves was significantly higher than from the other two types (p < 0.01) (Fig. 2d). In summary, the optimal method for protoplast isolation in 'Yellow River' is to digest strips of young leaves in a 3% (w/v) Cellulase (R10), 0.8% Macerozyme (R10), 0.04% (w/v) Pectinase, and 0.4 M mannitol enzymolysis solution for 6 h.
Establishment of a 'Yellow River' protoplast transient expression system
We established a PEG-mediated transient expression system for 'Yellow River' by testing a range of PEG4000 concentrations and transfection time. The transient expression vector pEZS-NL harboring an EGFP-tag [31] was used to study the transformation efficiency in response to PEG concentrations and transfection time (Fig. 3). The results showed that 20% w/v of PEG4000 was the optimal to get the high transformation efficiency (p < 0.01) (Fig. 3a). The transformation efficiency was significantly higher after 10 min and 15 min than after 30 min (p < 0.01), and did not differ significantly from efficiency after 5 min (Fig. 3b). Therefore, 5 min was taken as the optimal transfection time.
The protoplast transfection ratio and transient expression. The ratio of transfection at 10, 15, 20, 25 and 30% (w/v) PEG4000 concentration (a), the ratio of transfection after 5, 10, 15 and 20 min (b), images of GFP and chlorophyll autofluorescence (Chl) as well as bright field images of protoplasts captured 12 h after transfection (c, d). Bar in c, 50 μm. Bar in d, 10 μm
Cloning a heat shock transcription factor MdeHSF1 and its expression pattern under heat stress in 'Yellow River'
The complete sequence of MdeHSF1 gene was cloned from 'Yellow River'. The length of ORF was 1521 bp, coding 506 AA, with PI value of 4.97. The gene contained a conserved high HSF-DNA-binding domain at N terminal, belonging to the heat shock transcription factor family (Fig. 4). The phylogenetic tree showed that MdeHSF1 had the highest homology to AtHSFA1A (Fig. 5a). Furthermore, the subcellular localization of MdeHSF1 was also predicted to be in the cell nucleus, a typical characteristic of transcription factors (Fig. 5b).
The protein of MdeHSF1 and three AtHSF proteins were compared using DNAman6.0 software. AtHSFA1A is At4g17750; AtHSFB1 is At4g36990; AtHSFC1 is At3g24520
The HSF DNA binding domain is underlined (a). Phylogenetic relationship between MdeHSF1 and AtHSF proteins drawn using MEGA6.0 (b), the conserved domain analysis of the MdeHSF1 and AtHSF proteins using the MEME website (c)
Using expression changes of MdeHSF1 as a proxy, we estimated the difference in the heat-stress resistance of two species in Magnolia, 'Yellow River' and M. × soulangeana. MdeHSF1 shared similar expression patterns in two species, exemplified by the increase in expression levels increased with the longer duration of heat stress. The highest expression of MdeHSF1 in both species occurred after 5 h of heat stress. However, the expression of MdeHSF1 was more induced by heat stress in 'Yellow River' than in M. × soulangeana. Higher expression of MdeHSF1 was consistent with the phenomenon that 'Yellow River' exhibited higher heat-stress resistance in nature, than that in M. × soulangeana (Fig. 6).
qPCR analysis of the MdeHSF1 expression level under high temperature (42 °C) from 0 to 5 h in 'Yellow River' and M. × soulangeana leaves
Subcellular localization analysis of MdeHSF1 in 'Yellow River' protoplast
To determine the subcellular localization of MdeHSF1, an MdeHSF1-EGFP fusion protein vector, pESZ-NL-MdeHSF1, was constructed. Protoplast isolation and transformation was performed as previously described, and the MdeHSF1 transient expression vector was successfully expressed in the 'Yellow River' protoplast (Fig. 7a). These results confirmed that MdeHSF1 was expressed in the cell nucleus (Fig. 7b), and demonstrated the utility of the protoplast isolation and transient expression system we developed.
Plasmids can be transferred to protoplasts for transient expression of MdeHSF1. The protected picture of the subcellular location of MdeHSF1 (a), the subcellular location of MdeHSF1 in 'Yellow River' protoplast (b) Bar 10 μm
Protoplast isolation and transient expression is widely used in plant molecular research to examine gene function, subcellular position and protein–protein interactions. In Arabidopsis, the protoplast transient expression system is very mature, and some rapid and simple methods have been developed. For example, the Tape Sandwich system, allows for rapid isolation of high-quality protoplasts from Arabidopsis leaves [3]. There are also efficient protoplast isolation and transient expression systems developed for other model plants and economically important plants, such as Nicotiana tabacum [33], Oryza sativa [16, 34], Malus × domestica [35] and Fragaria × ananassa [36]. These protoplast transient expression systems have been used in a variety of experimental studies and have facilitated the advancement of gene function research [16, 34] In contrast, there are a limited number of reports on protoplast isolation and transient expression in ornamental plants. In this study, we developed an efficient protoplast isolation and transient expression system for 'Yellow River' after testing different tissue types, enzymatic combinations, enzymolysis solution osmotic potentials, and enzymolysis time. Similar to previous reports on other plants, the effective concentration of PEG solution for the transient expression system is approximately 20%. Under optimal condition, the protoplast yield of 'Yellow River' young leaves can reach approximately 1.89 × 105 protoplasts/g FW. This value was higher than the protoplast yield of Arabidopsis, Oryza sativa and Populus [3, 20, 29, 34]. Compared with mature leaves, the young leaves grown in vitro or in a lab, are more suitable to isolate protoplasts.
With continued rise in temperatures globally, plants are increasingly exposed to high temperatures. This is more of a problem for Magnolia as it is often used in urban landscaping. Compared with other Magnolia species, such as M. × soulangeana, 'Yellow River' has higher tolerance to heat stress, but the molecular mechanism has remained unclear. Several studies showed that heat-shock transcription factors (HSF) are central regulators of heat-stress responsive genes. The plant HSF family shows a striking multiplicity, showing a strong diversification of expression pattern and function within the family [37]. In plants, there are three classes in the HSF protein family (classes A, B, and C), which are differentiated by peculiarities of their flexible linkers and HR-A/B regions [38]. And many researchers showed that AtHSFA1A was overexpressed in the heat shock response [39,40,41]. HSFs trigger the expression of Heat-shock Protein (HSP) in response to various stresses in living organisms, and makes HSP complex [40]. In the present paper, one HSF transcription factor, MdeHSF1 was cloned, and we found that the expression level of MdeHSF1 was much higher in 'Yellow River' than that of M. × soulangeana under high temperature stress. This finding indicates that MdeHSF1 might be an important gene regulator controlling heat tolerance in 'Yellow River'. Furthermore, the protein–protein interaction prediction results suggest that MdeHSF1 exhibits homology with AtHSFA1A and plays an important role in the cell signal network.
These results not only verify the efficiency of the protoplast transient expression system but also provide important clues to understanding heat tolerance in 'Yellow River'. In future studies, responses of proteins to the expression level of MdeHSF1 should be explored in more detail and the use of the protoplast system for detecting protein–protein interactions via bimolecular fluorescence complementation assays, could be investigated.
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von Koskull-Döring P, Scharf K-D, Nover L. The diversity of plant heat stress transcription factors. Trends Plant Sci. 2007;12(10):452–7.
Nover L, Bharti K, Döring P, Mishra SK, Ganguli A, Scharf K-D. Arabidopsis and the heat stress transcription factor world: how many heat stress transcription factors do we need? Cell Stress Chaperon. 2001;6(3):177–89.
H-C Liu, Y-Y Charng. Common and distinct functions of Arabidopsis class A1 and A2 heat shock factors in diverse abiotic stress responses and development. Plant Physiol. 2013;163(1):276–90.
Liu Y, Zhang C, Chen J, Guo L, Li X, Li W, Yu Z, Deng J, Zhang P, Zhang K. Arabidopsis heat shock factor HsfA1a directly senses heat stress, pH changes, and hydrogen peroxide via the engagement of redox state. Plant Physiol Bioch. 2013;64:92–8.
Qian J, Chen J, Liu Y, Yang L, Li W, Zhang L. Overexpression of Arabidopsis HsfA1a enhances diverse stress tolerance by promoting stress-induced Hsp expression. Genet Mol Res. 2014;13(1):1233–43.
Conceived and designed the experiments: YS, DM, and LC. Performed the experiments: YS and DM. Data analyses: YS, DM and JZ. Contributed to the writing of the manuscript: YS, DM, LC, JZ and KM. All authors read and approved the final manuscript.
We thank the Olena Vatamaniuk Lab for sharing a Zeiss LSM 710 fluorescence microscope and Jiapei Yan and Yutian Dong for assistance with the microscope. Tianzhong Li lab provided the pEZS-NL plasmid.
Availability of data and material
All data generated in this study are included in this article and additional files. Material is available from the corresponding author on reasonable request.
This work was supported in part by the National Natural Science Foundation of China (No. 31400599), the 13th Five-Year-Plan Projects for Floriculture Special Breeding of Zhejiang Province, China (No. 2016C02056-12), the Public Welfare Forestry Industry Project of State Forestry Administration, China (No. 201504322), the Science Foundation of Zhejiang A & F University (KF201323).
Yamei Shen and Dong Meng have contributed equally to this work
School of Landscape and Architecture, Zhejiang A & F University, Lin'an, 311300, Zhejiang, China
Yamei Shen
Department of Horticulture, Cornell University, Ithaca, NY, 14853, USA
Yamei Shen, Dong Meng, Junhong Zhang & Lailiang Cheng
Scion, Private Bag 3020, Rotorua, 3046, New Zealand
Kim McGrouther
State Key Laboratory of Subtropical Silviculture, Zhejiang A & F University, Lin'an, 311300, Zhejiang, China
Junhong Zhang
Dong Meng
Lailiang Cheng
Correspondence to Lailiang Cheng.
Additional file
Additional file 1: Table S1
. Injury degree of landscape plants in the parks and roads in Hang Zhou, China under nature high-temperature. Table S2. The sources of all relative reagents
Shen, Y., Meng, D., McGrouther, K. et al. Efficient isolation of Magnolia protoplasts and the application to subcellular localization of MdeHSF1 . Plant Methods 13, 44 (2017). https://doi.org/10.1186/s13007-017-0193-3
Heat shock transcription factor
Subcellular localization
Plant Genome Editing


text:
What's all the fuss? It's just a cat, says woman seen on CCTV shoving tabby in wheelie bin
By Claire Ellicott for the Daily Mail
Updated: 09:13 EST, 25 August 2010
[caption]
A woman who enraged animal lovers across the world by dumping a cat in a wheelie bin said today she 'thought it would be funny'.
But Mary Bale, 45, has received death threats, and a Facebook page set up to track her down has attracted more than 18,000 members.
Miss Bale was caught on video picking up four-year-old tabby cat Lola before throwing her into the bin and closing the lid.
But she claimed the outcry had been blown out of all proportion.
She said: 'I don't know what the fuss is about. It's just a cat.'
The unmarried bank worker said she was just walking home on Saturday when she saw the cat and decided to play with it. But she told the Sun that she 'suddenly thought it would be funny' to put it in the bin.
'I did it as a joke because I thought it would be funny. I never thought it would be trapped, I expected it to wriggle out,' she said.
Referring to the CCTV footage, she told how she could not believe it was being seen around the world and was afraid it would upset her family.
She said: 'I don't know what my relatives will think but to be honest I think everyone is overreacting a bit.'
While Miss Bale admitted she should not have done it, she said she did not deserve to be 'hated' by everyone and assumed the cat would find its own way out the bin.
Her mother also insisted the unmarried bank clerk 'loved cats' - and had even kept them as pets as a child.
Celia Bale, 78, said: 'She's my daughter and as far as I'm concerned she is the most caring person you could ever wish to meet.
'She had cats when she was a little girl and has never shown any cruelty to animals before in her life - she loves animals.
'I talked to her on the phone today and she can't explain why she did it or what got into her head, only that she really regrets it.'
The mother-of-three added that her husband Maurice, Miss Bale's father, was in a critical condition after falling down the stairs last week, damaging his pelvis and vertebrae.
She said of her daughter: 'She's been under a great deal of strain these past two weeks.
'Her father had a nasty fall and has been in a critical condition in hospital ever since.
'She's very close to her father and I think it's had a real impact on her.
Reunited: Lola back with owner Darryl Mann yesterday. He posted footage of the incident on YouTube in a bid to catch the culprit, now identified as Miss Bale (pictured above at her church choir's annual dinner)
'She's been soldiering on but I think it has got to her more than I realised.
'I'm awfully worried about her because people have been saying some dreadful things about her and making threats.'
She added that she believed her daughter might keep animals if she didn't work.
'I know that sounds ironic considering what has happened, but she does love cats,' she said.
It was also revealed last night that Miss Bale, who lives in Coventry and works as a cashier at RBS in nearby Rugby, was once a member of the Birmingham Bach Choir.
Miss Bale seems harmless as she strokes four-year-old cat, Lola, as it walks along a wall on a street in Coventry. But she then grabs it by the scruff of its neck, puts it in a wheelie bin and casually walks away
Miss Bale was caught on camera in Coventry as she threw the cat into the wheelie bin this weekend.
Lola was found 15 hours later when owner Darryl Mann, 26, heard her mewing from inside the bin.
Members of a Facebook group set up by Lola's owners to identify the woman in the CCTV footage have posted death threats against Miss Bale. Some have been posted from as far away as Australia.
Hundreds of thousands around the globe have seen the YouTube video they also posted, 100,000 in the UK alone.
One wrote 'throw her in the bin and see how she likes it', while others called for her to be killed. Last night, owner Stephanie Andrews-Mann, 24, said she was glad the culprit had been found and hoped she'd be brought to justice.
She said: 'All I wanted was a name so I could pass it on to the police.
'It's terrible that her dad's in hospital, but to be totally honest, it's not my cat's problem.
'Though what she did was totally wrong, I don't want her to be too petrified to go out.'
West Midlands Police were keeping watch outside Miss Bale's terraced home yesterday and a spokesman said she had been offered advice on how to stay safe.
Neighbours described her as a reclusive woman who rarely had visitors. One, who asked not to be named, said: 'She is a very nice lady, though very quiet.
The police and RSPCA investigation is continuing.
Cat bin woman Mary Bale: What's all the fuss? It's just a cat
Bengal Tiger bites off safari car's bumper in southern India

redpajama_set_name: RedPajamaCommonCrawl


text:
/**
 @file
 @author  Nicholas Gillian <ngillian@media.mit.edu>
 @version 1.0

 @brief This is the main base class that all GRT PreProcessing algorithms should inherit from.

 A large number of the functions in this class are virtual and simply return false as these functions must be overwridden by the inheriting class.
 */

/**
 GRT MIT License
 Copyright (c) <2012> <Nicholas Gillian, Media Lab, MIT>

 Permission is hereby granted, free of charge, to any person obtaining a copy of this software
 and associated documentation files (the "Software"), to deal in the Software without restriction,
 including without limitation the rights to use, copy, modify, merge, publish, distribute, sublicense,
 and/or sell copies of the Software, and to permit persons to whom the Software is furnished to do so,
 subject to the following conditions:

 The above copyright notice and this permission notice shall be included in all copies or substantial
 portions of the Software.

 THE SOFTWARE IS PROVIDED "AS IS", WITHOUT WARRANTY OF ANY KIND, EXPRESS OR IMPLIED, INCLUDING BUT NOT
 LIMITED TO THE WARRANTIES OF MERCHANTABILITY, FITNESS FOR A PARTICULAR PURPOSE AND NONINFRINGEMENT.
 IN NO EVENT SHALL THE AUTHORS OR COPYRIGHT HOLDERS BE LIABLE FOR ANY CLAIM, DAMAGES OR OTHER LIABILITY,
 WHETHER IN AN ACTION OF CONTRACT, TORT OR OTHERWISE, ARISING FROM, OUT OF OR IN CONNECTION WITH THE
 SOFTWARE OR THE USE OR OTHER DEALINGS IN THE SOFTWARE.
 */

#ifndef GRT_PRE_PROCESSING_HEADER
#define GRT_PRE_PROCESSING_HEADER

#include "MLBase.h"

namespace GRT{

class PreProcessing : public MLBase
{
public:
    /**
     Default Constructor
     */
	PreProcessing(void);

    /**
     Default Destructor
     */
	virtual ~PreProcessing(void);

    /**
     This is the base deepCopyFrom function for the PreProcessing modules. This function should be overwritten by the derived class.

     @param const PreProcessing *preProcessing: a pointer to the PreProcessing base class, this should be pointing to another instance of a matching derived class
     @return returns true if the deep copy was successfull, false otherwise (the PreProcessing base class will always return flase)
     */
    virtual bool deepCopyFrom(const PreProcessing *rhs){ return false; }

    /**
     This copies the PreProcessing variables from preProcessing to the instance that calls the function.

     @param const PreProcessing *preProcessing: a pointer to a pre processing module from which the values will be copied
     @return returns true if the copy was successfull, false otherwise
     */
    bool copyBaseVariables(const PreProcessing *preProcessingModule);

    /**
     This is the main processing interface for all the pre processing modules and should be overwritten by the inheriting class.

     @param const VectorDouble &inputVector: a vector containing the data that should be processed
     @return returns true if the pre processing was successfull, false otherwise
     */
    virtual bool process(const VectorDouble &inputVector){ return false; }

    /**
     This is the main reset interface for all the GRT preprocessing modules. This should be overwritten by the derived class.

     @return returns true if the derived class was reset succesfully, false otherwise (the base class always returns true)
     */
    virtual bool reset();

    /**
     This is the main clear interface for all the GRT preprocessing modules. This should be overwritten by the derived class.
     It will completely clear the module, removing any IO values setting and all the base variables to their default values.

     @return returns true if the derived class was cleared succesfully, false otherwise
     */
    virtual bool clear();

    /**
     This saves the pre processing settings to a file.
     This function should be overwritten by the derived class.

     @param string filename: the name of the file to save the settings to
     @return returns true if the settings were saved successfully, false otherwise (the base class always returns false)
     */
    virtual bool saveSettingsToFile(string filename) const{ return false; }

    /**
     This loads the pre processing settings from a file.
     This function should be overwritten by the derived class.

     @param string filename: the name of the file to load the settings from
     @return returns true if the settings were loaded successfully, false otherwise (the base class always returns false)
     */
    virtual bool loadSettingsFromFile(string filename){ return false; }

    /**
     This saves the pre processing settings to a file.
     This function should be overwritten by the derived class.

     @param fstream &file: a reference to the file to save the settings to
     @return returns true if the settings were saved successfully, false otherwise (the base class always returns false)
     */
    virtual bool saveSettingsToFile(fstream &file) const{ return false; }

    /**
     This loads the pre processing settings from a file.
     This function should be overwritten by the derived class.

     @param fstream &file: a reference to the file to load the settings from
     @return returns true if the settings were loaded successfully, false otherwise (the base class always returns false)
     */
    virtual bool loadSettingsFromFile(fstream &file){ return false; }

    /**
     @return returns the pre processing type as a string, e.g. LowPassFilter
     */
	string getPreProcessingType() const;

    /**
     Returns the size of the input vector expected by the pre processing module.

     @return returns the size of the input vector expected by the pre processing module
     */
    UINT getNumInputDimensions() const;

    /**
     Returns the size of the vector that will be computed by the pre processing module.

     @return returns the size of the vector that will be computed by the pre processing module
     */
    UINT getNumOutputDimensions() const;

    /**
     Returns true if the pre processing module has been initialized correctly.

     @return returns true if the pre processing module has been initialized succesfully, false otherwise
     */
    bool getInitialized() const;

    /**
     @return returns a VectorDouble containing the most recent processed data
     */
	VectorDouble getProcessedData() const;

    /**
     This typedef defines a map between a string and a PreProcessing pointer.
     */
	typedef std::map< string, PreProcessing*(*)() > StringPreProcessingMap;

    /**
     This static function will dynamically create a new PreProcessing instance from a string.

     @param string const &preProcessingType: the name of the PreProcessing class you want to dynamically create
     @return a pointer to the new PreProcessing instance that was created
     */
    static PreProcessing* createInstanceFromString(string const &preProcessingType);

    /**
     This static function will dynamically create a new PreProcessing instance based on the type of this instance
    */
    PreProcessing* createNewInstance() const;

protected:
    /**
     Initializes the base preprocessing module, this will resize the processedData vector and get the instance ready for preprocessing new data.
     The inheriting class must first initialize the module before calling this function.

     @return returns true if the module was initialized, false otherwise
     */
    bool init();

    /**
     Saves the core preprocessing settings to a file.

     @return returns true if the base settings were saved, false otherwise
     */
    bool savePreProcessingSettingsToFile(fstream &file) const;

    /**
     Loads the core preprocessing settings from a file.

     @return returns true if the base settings were loaded, false otherwise
     */
    bool loadPreProcessingSettingsFromFile(fstream &file);

    string preProcessingType;
    bool initialized;
    UINT numInputDimensions;
    UINT numOutputDimensions;
    VectorDouble processedData;

	static StringPreProcessingMap *getMap() {
        if( !stringPreProcessingMap ){ stringPreProcessingMap = new StringPreProcessingMap; }
        return stringPreProcessingMap;
    }

private:
    static StringPreProcessingMap *stringPreProcessingMap;
    static UINT numPreProcessingInstances;
};

//These two functions/classes are used to register any new PreProcessing Module with the PreProcessing base class
template< typename T >  PreProcessing *newPreProcessingModuleInstance() { return new T; }

template< typename T >
class RegisterPreProcessingModule : PreProcessing {
public:
    RegisterPreProcessingModule(string const &newPreProcessingModuleName) {
        getMap()->insert( std::pair<string, PreProcessing*(*)()>(newPreProcessingModuleName, &newPreProcessingModuleInstance< T > ) );
    }
};

} //End of namespace GRT

#endif // GRT_POST_PROCESSING_HEADER

redpajama_set_name: RedPajamaGithub


text:
KEVIN HART WITHDRAWS AS HOST OF 2019 OSCARS
By Nkonkonsa.com , December 10, 2018 5:24 pm January 8, 2019 0 257
Actor and comedian Kevin Hart announced on Friday he would back out of hosting the Academy Awards after homophobic tweets he posted several years ago surfaced.
The Academy Awards, also known as the Oscars, are the world's most important annual film awards.
Hart, who starred in Ride Along and Jumanji: Welcome to the Jungle, said he stepped down because he did not want to be a distraction during the event and he apologised to the LGBTQ community.
The 39-year old also said he had grown as a person since then and is now a different man.
"I have made the choice to step down from hosting this year's Oscar's….this is because I do not want to be a distraction on a night that should be celebrated by so many amazing talented artists. I sincerely apologize to the LGBTQ community for my insensitive words from my past".
"I'm sorry that I hurt people.. I am evolving and want to continue to do so. My goal is to bring people together not tear us apart. Much love & appreciation to the Academy. I hope we can meet again", Kevin Hart tweeted.
Before he withdrew, Hart had previously said he did not want to apologise after the Academy of Motion Picture Arts and Sciences, which organises Hollywood's annual Oscar awards ceremony, asked him to.
In a video posted on Instagram, Hart had said he had grown since he posted the controversial tweets.
"The reason why I passed is I've addressed this several times… I've said where the rights and wrongs were. I've said who I am now versus who I was then. I've done it, I'm not going to continue to go back, " Hart said.
"We feed on Internet trolls and we reward them. I'm not going to do it. I'm going to stand my ground," he added.
"Regardless Academy, I'm thankful for the opportunity. If it goes away, no harm, no foul"
In the tweets stemming from 2009 and 2011, Hart made several homophobic comments, including one in which he said he would not accept it if his son said he was gay.
"Yo if my son comes home & try's 2 play with my daughters doll house I'm going 2 break it over his head & say n my voice 'stop that's gay," Hart wrote in the tweet.
Another tweet read "Why does @wayne215 have so many pictures of me in his phone!!! What ru some type of FAT FAG that takes pic of small black men all day?".
Hart used the homophobic language until 2011, the year he had his mainstream breakthrough.
The Academy announced Hart as the host of the 2019 Oscars on Tuesday but after the tweets surfaced a campaign was started by LGBTQ groups for the Academy to rescind the invitation to host.
The actor deleted some of the tweets, but refused to apologise, eventually leading to his stepping down as host.
The Oscars have attracted stinging criticism in recent years over a lack of diversity among nominees and winners, with critics arguing that the awards do not reward the accomplishments of women and people of colour enough.
After that controversy, which took on the hashtag #OscarsSoWhite, the Academy pledged to double its membership of women and minorities by 2020.
Hart's withdrawal is not the first time a Hollywood celebrity has had to step down over old tweets. Earlier this year, Guardians of the Galaxy director James Gunn was fired by Disney after several social media posts surfaced in which he joked about the 9/11 attacks, the Holocaust and rape.
Following his dismissal, Gunn said he had since "developed as a person" and apologised for his comments.
IDRIS ELBA NAMED 2018 SEXIEST MAN ALIVE
By Nkonkonsa.com , November 8, 2018 11:56 am January 10, 2019 0 520
Idris Elba has won the title of People's Sexiest Man Alive for 2018.
The actor, best known for his roles in The Wire, Luther, and Beasts of No Nation, made an appearance on The Tonight Show with Jimmy Fallon via satellite from London, in order to reveal his newly crowned status.
"I was like, 'Come on, no way. Really?'" he told the magazine. "Looked in the mirror, I checked myself out. I was like, 'Yeah, you are kind of sexy today.' But to be honest, it was just a nice feeling. It was a nice surprise — an ego boost for sure."
Elba returned to his role as Heimdall for this year's Avengers: Infinity War and starred as Walter in Sky's sitcom In the Long Run, while also making his directorial debut with Yardie. He also performs on the side under the name DJ Big Driis and has his own clothing line.
He's set to star in the fifth series of Luther, alongside appearances in Hobbs and Shaw and Netflix's Turn Up Charlie. He will also star as Macavity in the film adaptation of Cats.
The actor told People about his childhood growing up in East London, as the only child of Sierra Leonean and Ghanian parents. He said that his parents were strict and he was often picked on while attending an all-boys school despite his enthusiasm for sports including football, basketball, cricket, hockey and rugby.
"I was very tall and skinny," said Elba. "And my name was Idrissa Akuna Elba, OK? I got picked on a little bit. But again, as soon as I could grow a moustache, I was the coolest kid on the block. Grew a moustache, had some muscles, bonkers."
Past winners have included Dwayne Johnson, David Beckham, Chris Hemsworth, Adam Levine, Channing Tatum and Blake Shelton.
UGANDAN POP-STAR POLITICIAN BOBI WINE REVEALS HOW HE WAS TORTURED BY SOLDIERS
By Nkonkonsa.com , September 4, 2018 2:07 pm September 20, 2018 0 414
The Ugandan politician Robert Kyagulanyi Ssentamu, better known as pop star Bobi Wine, has detailed for the first time the beatings he says he received from security officers after his arrest last month.
In a lengthy statement posted on Facebook, Kyagulanyi wrote that he wanted to "set the record straight".
The popular 36-year-old musician, who entered Uganda's parliament as an independent MP in 2017, was arrested last month and charged with treason after some of his supporters allegedly stoned President Yoweri Museveni's car.
"They beat me, punched me and kicked me with their boots. No part of my body was spared," he said from the US where he is seeking medical treatment for injuries he says were sustained during his arrest and detention.
"They wrapped me in a thick piece of cloth and bundled me into a vehicle," he wrote of his arrest in the north-western town of Arua. "Those guys did to me unspeakable things in that vehicle! They pulled my manhood and squeezed my testicles while punching me with objects I didn't see," Kyagulanyi said, adding the officers stole his wallet and phone before knocking him unconscious.
Kyagulanyi, who was charged with treason last week and released on bail, was re-arrested while trying to leave the country to seek medical treatment. He was subsequently allowed to depart.
The young MP has become a lightning rod for opposition to the long rule of the country's 74-year-old president who critics say is out of touch with a youthful population.
His case has drawn international attention with scores of musicians, activists and politicians signing an open letter condemning his treatment by the government.
Monday's 3,500-word Facebook post was Kyagulanyi's first public statement since his arrest. "I was shocked on how they tried to downplay the atrocities committed by security agencies on innocent citizens," he said.
MICHAEL JACKSON'S FATHER DIES AT THE AGE OF 89
By Nkonkonsa.com , June 27, 2018 6:09 pm June 27, 2018 0 513
Michael Jackson's father Joe Jackson has died aged 89, just days after being hospitalized with stage four-pancreatic cancer.
Joe Jackson passed away on Wednesday at the age of 89.
DailyMail.com revealed last week that the King of Pop's father was on his deathbed and later hospitalized as he battled stage four pancreatic cancer.
Joe was being treated in Las Vegas as recently as last week, but TMZ reports that he passed away in Los Angeles at around 3:30am with his wife Katherine by his side.
It was later confirmed that he had in fact died while in hospice care in Las Vegas.
Jackson and his wife had been living apart for the past few years following a tumultuous 69-year relationship during which Jackson fathered a love child, had multiple affairs and was accused of abusing the couple's children.
The music manager and father to 11 children, including pop superstar Michael and Janet Jackson, was being cared for in a hospital in Las Vegas. Jackson was said to be in the final stages of terminal cancer. He had previously suffered a stroke in São Paulo in 2015, along with a number of heart attacks. His grandson, Taj Jackson, confirmed the news on Twitter.
His fourth child, Jermaine, told the Daily Mail before Jackson's death that his father had attempted to prevent visits from his family, withholding information about his location and diagnosis. They were permitted to see him on 19 June. "No one knew what was going on," said Jackson. "We shouldn't have to beg, plead and argue to see our own father, especially at a time like this. We have been hurting."
On 24 June, Joe Jackson appeared to tweet from his personal account: "I have seen more sunsets than I have left to see. The sun rises when the time comes and whether you like it or not the sun sets when the time comes." The tweet was accompanied by a photograph of Jackson looking at the horizon.
However, Paris Jackson, Michael's daughter, tweeted her doubts that it had come from her grandfather: "This is a beautiful tweet. Though it upsets me to see whoever is in charge of this account taking advantage of it. My grandfather did not tweet this. I'm not sure if he's ever used this account."
Michael Jackson's family has also released a statement:
"We are deeply saddened by Mr. Jackson's passing and extend our heartfelt condolences to Mrs. Katherine Jackson and the family," said John Branca and John McClain, co-executors of the estate. "Joe was a strong man who acknowledged his own imperfections and heroically delivered his sons and daughters from the steel mills of Gary, Indiana to worldwide pop superstardom."
Joe Jackson's legacy is the shaping and promotion of the Jackson 5, comprising his sons Jackie, Tito, Jermaine, Marlon and Michael, and later Randy. The young family band from Gary, Indiana, became a phenomenon in the late 1960s and 70s, with their first four singles, I Want You Back in 1969, and ABC, The Love You Save and I'll Be There in 1970, topping the Billboard charts.
But Jackson's legacy is also one of cruelty. In 2003, he told the BBC that he whipped Michael as a child. In 2010, he told Oprah Winfrey that he beat his children with a strap and didn't regret doing so. "It kept them out of jail and kept them right," he said. In the 2003 ITV documentary Living With Michael Jackson, the singer told interviewer Martin Bashir that his father would oversee the Jackson 5's intense rehearsals "with a belt in his hand" and "tear you up, really get you" if one of the brothers missed a step.
"I just remember hearing my mother scream, 'Joe you're gonna kill him, you're gonna kill him, stop it,'" Michael recalled. "I was so fast he couldn't catch me half the time, but when he would catch me, oh my god it was bad, it was really bad." Michael also said his father would taunt him about his "fat nose", and later told Winfrey he was so afraid of his father that he would sometimes vomit when he saw him. In 1979, Michael fired his father as his manager and took control of his career himself. The rest of the Jackson 5 followed suit in 1983.
BLACK PANTHER JUST $1 OFF $1.3BN WORLDWIDE GROSS
By Nkonkonsa.com , April 9, 2018 4:12 pm April 9, 2018 0 406
Walt Disney's Black Panther earned another $8.4 million in its eighth weekend of domestic release, dropping just 26% and snagging the 11th-biggest eighth weekend (sans inflation) and bringing its 52-day domestic cume to $665.4m.
It is now past the $659m lifetime gross of Titanic (again, sans inflation) and is now the third-biggest domestic earner of all time. And on this specific list, that's where the journey will likely end.
No, I don't see the Chadwick Boseman superhero movie finding another $95m and sneaking past the $760m domestic cume of Avatar and the once-in-a-lifetime $937m gross of Star Wars: The Force Awakens is out of the question.
As mentioned yesterday, while it's important to note higher ticket prices and various upcharges for PLF screens, D-Box, 3D and the like, the theatrical experience becomes less and less of a dominant entertainment option every year.
Yes, ticket prices are a lot higher than they were in 1998 or 1968, but there were a heck of a lot less distractions from the theatrical experience (DVD, YouTube, Hulu, etc.) than there are today.
That Black Panther has sold around 72 million tickets over seven weeks in a "Netflix-and-Chill" world an astonishing achievement, especially for a film that so explicitly bucks conventional wisdom about what makes a hit movie.
Black Panther is currently the 34th-biggest inflation-adjusted grosser. It has already sold more tickets than any Sony/Columbia/TriStar release, any Lionsgate/Summit flick and anything out of DreamWorks Animation. If it gets past The Dark Knight ($534 million in 2008/$684m adjusted), it will A) be the second-biggest comic book flick behind The Avengers ($623m in 2012/$706m adjusted) and B) have sold more tickets than any Warner Bros./Time Warner Inc. offering save for The Exorcist.
It's it passes that arbitrary milestone, it'll be the 30th-biggest "adjusted for inflation" grosser and T'Challa will be able to brag that he's bigger than Batman and bigger than James Bond (sorry, Thunderball).
Ryan Coogler and Joe Robert Cole's action drama just missed the $1.3 billion worldwide mark, ending the frame with (eye-roll) $1.2999b. It is the 10th-biggest global grosser, between Frozen ($1.277b in 2013/2014) and Star Wars: The Last Jedi ($1.33b in 2017/2018).
It just might get past the last Star Wars film (if not the last Harry Potter flick, which earned $1.43b in 2011). Considering the inexplicably negative press that The Last Jedi got for "only" grossing $1.33b, it might not be in Disney's interest for the Star Wars flick to be taken down a notch. Moreover, the higher Black Panther gets, the more Infinity War must gross to avoid negative press.
Yes, this is insane, but that's where Disney is right now. Black Panther doing as well as it has further puts Star Wars on the defensive while in turn upping the expectations for Avengers: Infinity War to a greater degree than if the Boseman flick had "only" earned $800 million worldwide.
That's not exactly a problem, but even if it is there are worse problems for a studio to have. On the plus side, it means a genuine whiff like A Wrinkle in Time, made with the best intentions and primed to become a future generational kid-friendly "classic" (think Hook or Labyrinth), doesn't sting that much.
Featured, World Celebs
DRAKE WINS 13 AWARDS AT BILLBOARD MUSIC AWARDS 2017
By admin , May 22, 2017 11:15 am May 22, 2017 Off 497
The Canadian rapper picked up 13 prizes, beating Adele by one, at the event in Las Vegas including best artist.
He was joined on the stage by Nicki Minaj, Lil Wayne and his father.
"I just want to say hold tight Adele because when a new ting drops you will crawl your way back to get the record back," said Drake accepting the award.
Adele set the former record at the 2012 awards when she won prizes including top artist, top female artist and top pop artist.
Drake also won top male artist and top Billboard 200 album for Views after getting 22 nominations at the event as well as performing Gyalchester inside the famous Bellagio Fountains in Las Vegas.
Zayn Malik won best new artist but didn't collect the prize in person, telling fans he was busy finishing new songs.
Twenty One Pilots picked up top duo/group and top rock artist with Metallica winning in the rock album category.
Beyonce's Lemonade was picked as the best R&B album while The Chainsmokers won top dance/electronic artist and top Hot 100 Song for Closer featuring Halsey.
K-pop group BTS beat the likes of Justin Bieber and Selena Gomez to win the top social artist award.
The ceremony also saw Cher handed an icon award.
The star, who turned 71 the day before, said her success was "mostly luck" as she collected the honour from Gwen Stefani.
Audioslave and Soundgarden singer Chris Cornell was called a "true innovator" after his death last week.
Ahead of a moment's silence, Imagine Dragons singer Dan Reynolds paid tribute to Cornell as a "prolific songwriter, a legendary performer, a singer who had a voice for the ages and a philanthropist".
Most of the categories were decided by song sales over the past year, streaming, radio airplay, touring and social media interactions as tracked by Billboard.
Halsey performed new single Now or Never on stage in an outfit which apparently took two days to make.
Celine Dion performed My Heart Will Go On to honour the 20th anniversary of the movie – Titanic.
There were also performances from Nicki Minaj, Bruno Mars, Lorde, The Chainsmokers, Camila Cabello, John Legend, Miley Cyrus, Imagine Dragons and country music stars Florida Georgia Line and Sam Hunt plus Selena Gomez songwriter Julia Michaels.
Meanwhile, Sean "Diddy" Combs celebrated Notorious B.I.G. on what would have been the hip-hop legend's 45th birthday.
He was killed in a drive-by shooting in 1997.
The rapper's son CJ Wallace told the audience: "I know my father's looking down on all of us tonight.
"My sister and I will continue to carry on his memory with tremendous pride and live my life by his words."
Vanessa Hudgens and Ludacris hosted the event.
WRONG FILM NAMED BEST PICTURE AT 2017 OSCARS
By admin , February 27, 2017 1:39 pm February 27, 2017 Off 442
Moonlight has won best picture at the Oscars – but only after Faye Dunaway initially said La La Land had won.
Warren Beatty was handed the previous winner's envelope, containing a card saying "Emma Stone, La La Land", resulting in the error.
The team from La La Land was in the middle of its acceptance speeches when the mistake was discovered.
Stone said backstage afterwards: "Is that the craziest Oscar moment of all time? Cool! It's going to be history."
Musical La La Land won six Oscars including best director and best actress. Moonlight also won adapted screenplay and best supporting actor.
Damien Chazelle, director of La La Land, became the youngest filmmaker to win a best director Oscar at the age of 32.
Bonnie and Clyde stars Beatty and Dunaway announced La La Land had taken the best picture prize – but then the musical's producer Jordan Horowitz announced there had been a mistake.
He said: "This is not a joke. Moonlight is best picture," and showed the camera the card that revealed the winner.
Two La La Land producers had already given their acceptance speeches before the mistake was revealed.
Host Jimmy Kimmel joked that he knew he would mess up compering the ceremony, and added: "Personally I blame Steve Harvey for this," referring to Harvey's mistake in announcing the Miss Universe winner in 2015.
'In my dreams'
Beatty had opened the envelope but hesitated to read out what it said in side, leaving it to Dunaway to announce La La Land.
He returned to the microphone to say: "I want to tell you what happened. I opened the envelope and it said 'Emma Stone, La La Land', and that is why I took such a long look at Faye and at you.
"I wasn't trying to be funny. This is Moonlight for best picture."
The film's director Barry Jenkins said: "Very clearly even in my dreams this can't be true. But to hell with it because this is true. It's true, its not fake."
Paying tribute to the team behind La La Land for the way they handled the mistake, he said: "We have been on the road with these guys and it was so gracious and so generous of them."
Emma Stone, who won the coveted best actress prize, said backstage.
"I'm so excited for Moonlight. Of course it was an amazing thing to hear La La Land and I would love to win best picture. But we are so excited for Moonlight. I think it is one of the best films of all time.
"Is that the craziest Oscar moment of all time? Cool! It's going to be history."
She also said that she still had the envelope for best actress in her hand, although there are always two envelopes (one for back-up), which might explain why the mistake occurred.
La La Land also picked up best cinematography, best score and best original song for City of Stars.
It also won best production design.
Manchester by the Sea won two awards – one of them was for best original screenplay.
Acting winners
Viola Davis – best supporting actress, Fences
Mahershala Ali – best supporting actor, Moonlight
Emma Stone – best actress, La La Land
Casey Affleck – best actor, Manchester by the Sea
Davis, who had been nominated twice previously for Doubt and The Help, also thanked her "captain", Washington.
Host Jimmy Kimmel put on a strong performance, with much of his patter unsurprisingly relating to the current US political climate.
In a reference to Donald Trump's Twitter attack on Meryl Streep following her criticism of him at the Golden Globes, Kimmel joked that Streep was "uninspiring… mediocre… underwhelming and lacklustre".
He asked the audience to give a round of applause for "the highly overrated Meryl Streep. Great dress, is that Ivanka?"
Kimmel also joked that Hollywood doesn't discriminate – apart from "on age and weight".
The first political speech of the night came from Alessandro Bertolazzi, one of the make-up and hairstyling winners for Suicide Squad, who said: "I'm an immigrant, I'm from Italy, this is for all the immigrants."
Colleen Atwood won an impressive fourth Oscar for costume design for Fantastic Beasts and Where to Find Them, having won previously in 2011 for Alice in Wonderland, in 2006 for Memoirs of a Geisha and in 2003 for Chicago.
Kevin O'Connell finally picked up an Oscar after 21 nominations for Hacksaw Ridge. He was first nominated back in 1984 for Terms of Endearment.
The film also picked up best editing.
Iranian film director Asghar Farhadi's The Salesman was named best foreign film.
Farhadi chose to stay away from Sunday's ceremony following President Trump's attempt to ban travel from Iran and six other mainly Muslim countries.
Zootopia won best animation, beating films such as Kubo and the Two Strings and Moana.
Other winners included British film The White Helmets, which picked up best documentary short.
The US denied entry to 21-year-old Syrian Khaled Khatib behind the film because of problems with his documents.
The a 40-minute Netflix documentary follows volunteers who risk their lives to rescue civilians in Syria.
Featured, Trending, World Celebs
BEYONCE IS PREGNANT WITH TWINS
By admin , February 1, 2017 7:52 pm February 1, 2017 Off 409
American Singer/Performer Beyonce has confirmed with a baby bump photo that she is pregnant–with twins.
The singer who already has a daughter-Blue Ivy with rapper husband-Jay Z made the shock announcement on Wednesday on Instagram.
She wrote: "We would like to share our love and happiness. We have been blessed two times over. We are incredibly grateful that our family will be growing by two, and we thank you for your well wishes. -The Carters," she added.
Beyoncé is due to headline April's Coachella festival. Assuming the announcement has come after the traditional 12-week pregnancy waiting period, she will be at least five and a half months pregnant when she takes to the stage.
Beyoncé and husband Jay Z's daughter Blue Ivy was born in 2012. She revealed her pregnancy with Blue Ivy on the red carpet at the MTV music awards in 2011, before showing off her bump after a performance of Love on Top.
Beyoncé, 35, is due to make an appearance at the Grammy awards on 12 February, where she is nominated in nine categories for her Lemonade album last year, the most nominations of any artist this year.
KIM KARDASHIAN & KANYE AGREE TO FIGHT FOR THEIR MARRIAGE
By admin , December 20, 2016 7:08 pm December 20, 2016 Off 444
Kim Kardashian and Kanye West have decided to fight for their marriage 'for the sake of their kids', North, 3, and one-year-old Saint, DailyMail.com can reveal.
The couple's four-year relationship has become the subject of intense speculation, after it was reported that Kardashian, 36, was considering a divorce from the rapper, 39.
But following a 'heart-to-heart' over the weekend, the pair have now decided to get counselling in an effort to mend their marriage, which was rocked by West's breakdown last month.
A glum-looking West, who spent part of last week with President-Elect Donald Trump in New York, was pictured with his equally gloomy wife on Sunday night, leaving a Santa Monica restaurant.
However a source close to the couple insisted that, contrary to appearances, things have begun to improve. 'They're actually doing better,' said the insider.
'It was obviously a bit tough when everything [West's psychotic breakdown] went down and it was confusing with the stuff going on. But they're doing better now.'
Kardashian, who was left 'conflicted' over the future of her marriage following West's hospitalization on November 21, now wants to give the relationship a chance.
'Kim is so in love with him and love can be blinding sometimes – she just adores him and the family loves him as well,' said the friend of the couple's.
'It's a situation that she's giving another shot, a second chance. She feels he's been under a lot of pressure, so she feels like he deserves another shot.'
'They had a heart-to-heart about it and Kim is still deeply in love with him.'
Kardashian is also making her two children with West a priority and, having thought hard about her situation, has concluded that making the marriage work would be better for them.
The insider told DailyMail.com: 'Despite the fact that logic tells her the best thing to do would be to move on, the other side of that is that she has children with him now.
'Kim told me, "It's not just about me, it's about the kids". So she wants to make it work. The only issue right now is he needs more time to recover and get better so she wants to wait and see how it goes.'
Source: DailyMail.com
KANYE WEST HOSPITALIZED
By admin , November 22, 2016 10:46 am November 22, 2016 Off 517
Kanye West is reportedly receiving treatment and was placed under assessment in hospital, just a day after cancelling his Life of Saint Pablo Tour.
The rapper scrapped a concert at The Forum in Inglewood, California on Sunday night at late notice, and on Monday his representatives announced his remaining dates had been axed.
It has been reported that Kanye is being treated for sleep deprivation and exhaustion, according to People magazine.
"He is exhausted and currently dealing with sleep deprivation," a source told the magazine.
Reports suggest Los Angeles police responded to a medical welfare call at 1.20pm local time, and West was hospitalised for his own health and safety.
It is unclear if Kanye remains in hospital, however the rapper's concert promoter, Live Nation, confirmed that the 39-year-old will not be performing the remaining dates of his Saint Pablo Tour.
Kanye's friends fear the star is reportedly suffering from "nervous exhaustion" Mirror reports.


Now that the poorly old tooth I have been stubbornly trying to save for the last couple of years has bitten (sorry) the dust, how clear it has become to me that I should have given up long ago. I was ingesting too much paracetamol for too long, and insisting that it was a perfectly reasonable objective to hang on to the beast for another ten years or so.
Good old hindsight. And well done dentist for being more patient than his patient.
Not only I am free of pain and paracetamol, I think my brain is waking up; slowly.
I can now plan things without forgetting what the first thing was I started from and where I put the list when the plan was completed.
I can read more and faster and better.
But I can't seem to write yet. Or promote what I've written. It looks as if energy levels need to be much higher for these creative works to restart.
Do the nerves from your back teeth have some sort of hotline to the writing areas of the brain?


text:
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}  // namespace views


text:
Imports SistFoncreagro.BussinessEntities
Imports System.Data
Imports System.Data.SqlClient
Public Class TipoDocumentoRepository : Inherits MasterDataAccess : Implements ITipoDocumentoRepository
    Private Function SelectObjectFactory(ByVal command As SqlCommand) As List(Of TipoDocumento)
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                lista.Add(_TipoDocumento)
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        Return lista
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        Dim command As SqlCommand = MyBase.CreateSPCommand("DeleteTIPODOCUMENTO")
        command.Parameters.AddWithValue("IdSubdiario", IdTipoDocumento)
        MyBase.ExecuteNonQuery(command)
    End Sub

    Public Function GetAllFromTIPODOCUMENTO() As System.Collections.Generic.List(Of BussinessEntities.TipoDocumento) Implements ITipoDocumentoRepository.GetAllFromTIPODOCUMENTO
        Dim command As SqlCommand = MyBase.CreateSPCommand("GetAllFromTIPODOCUMENTO")
        Return SelectObjectFactory(command)
    End Function

    Public Function GetTIPODOCUMENTOByIdTipoDocumento(ByVal IdTipoDocumento As Integer) As BussinessEntities.TipoDocumento Implements ITipoDocumentoRepository.GetTIPODOCUMENTOByIdTipoDocumento
        Dim command As SqlCommand = MyBase.CreateSPCommand("GetTIPODOCUMENTOByIdTipoDocumento")
        command.Parameters.AddWithValue("IdTipoDocumento", IdTipoDocumento)
        Return SelectObjectFactory(command).SingleOrDefault
    End Function

    Public Sub SaveTIPODOCUMENTO(ByVal _TipoDocumento As BussinessEntities.TipoDocumento) Implements ITipoDocumentoRepository.SaveTIPODOCUMENTO
        Dim command As SqlCommand = MyBase.CreateSPCommand("SaveTIPODOCUMENTO")
        command.Parameters.AddWithValue("Codigo", _TipoDocumento.Codigo)
        command.Parameters.AddWithValue("IdTipoDocumento", _TipoDocumento.IdTipoDocumento)
        command.Parameters.AddWithValue("Nombre", _TipoDocumento.Nombre)
        command.Parameters.AddWithValue("SujetoRetencion", _TipoDocumento.SujetoRetencion)
        MyBase.ExecuteNonQuery(command)
    End Sub

    Public Sub UpdateEstadoTIPODOCUMENTO(ByVal IdTipoDocumento As Integer) Implements ITipoDocumentoRepository.UpdateEstadoTIPODOCUMENTO
        Dim command As SqlCommand = MyBase.CreateSPCommand("UpdateEstadoTIPODOCUMENTO")
        command.Parameters.AddWithValue("IdTipoDocumento", IdTipoDocumento)
        MyBase.ExecuteNonQuery(command)
    End Sub
End Class


In his TED talk, Scott McCloud goes in search of "durable mutations" in the world of comics as they move toward the web.One of the things he notes in his talk, and this is developed in his book Understanding Comics, is the relationship between time and space in the graphical representations of comics. On a certain level, we can think of film in the same way as a series of frames going by really fast (though that isn't technically the case for digital film, even if digital video can be represented as frames like in iMovie). The difference with comics, as McCloud notes is that we are asked to fill in temporal gaps between frames in a way that is more consciously active than the gap filling we do with film or video. We also control the spatial-temporal flow in a way we don't with film (rewind and fast-forward notwithstanding).
As such, we might imagine the screen as a window into a digital scholarly ecology where authorship is downplayed and the intersections are highlighted (in comparison to print), where knowledge is offered to us in a variety of media, but most importantly where we are able to recapture the intellectual conversation that we never really had but have always pursued.
And this is where I come back to McCloud, who envisions web comics as comics only more so, who sees emerging technologies as enabling comic artists to pursue their aesthetic goals more fully beyond the limitations of print. If we recognize that fundamentally scholarship is about communicating and building knowledge with our colleagues, that it is about the conversation, then we can recognize the durable mutations that print technologies offered in the past, and we can recognize the limits of print and the ways in which digital media networks might allow us to pursue our ongoing intellectual goals beyond the limitations of print.


One of the best things to do in Okinawa is explore its many parks and recreational areas—and the island is certainly full of them! Okinawan parks are often distinguished by their tell-tale faux wooden fence posts and stair steps, and are commonly located near (but not limited to) community centers, elementary schools, beach scenic areas, or tucked away in local neighborhoods. One just has to develop a keen eye to spot the fence posts and be brave enough to get out of the car to take a good look around!
That being said, some of the best parks to be found on island happen to be located near and around a handful of dams that collect and channel fresh water throughout Okinawa's extensive reservoir system. Kanna Dam Park, located just a few miles north of Camp Hansen along Highway 329, is one such area that has managed to incorporate a rather impressive public park within its setting.
Although Kanna Dam Park does not offer playground equipment on its grounds, there are plenty of paved walking and biking trails; scenic vantage points overlooking the reservoir; picnic and gazebo areas; a lily pond; and a mini museum showcasing the flora and fauna of the Kanna area.
Entrance to the museum is free, however, there is a ledger located at the museum's kiosk window that visitors should sign into. Your last name and the number of people in your party should suffice.
There are two bathrooms located in the park. One is located in the building attached to the museum, while the other is located within the Kanna Lake Park area. Vending machines can be found within the park as well, but be mindful that there are no garbage cans or recycling receptacles available on the grounds. Whatever trash visitors bring or accumulate during their visit must be taken and disposed of at home.
Hours: 6:00 a.m. – 5:30 p.m.
Directions: Exit the 8 Okinawa Expressway toll road and travel north along Highway 329 passing Camp Hansen. As you travel through the towns of Kin and Ginoza, be on the lookout for a tall Taurus II Restaurant Mansion building on your right (this is your first landmark). Once you see the Taurus II Restaurant Mansion there will be a noticeable curve to the main road and you will see a farmer's market within a large parking lot off to your left (this is your second landmark). As you pass the farmer's market, you will immediately see a beige-colored building (Kanna Drive-Thru Restaurant) next to a Kijimuna statue (Kijimuna is the mythical red headed Okinawan sprite.). Turn left onto that corner and make a right onto the street marked by a blue sign with white lettering pointing to Kanna Dam. There are two more signs along this road that lead the way to two separate park entrances. The first one takes you to the museum and dam side, while the other takes you to the lakeside. Once on the park grounds, either area can be accessed by way of a long bridge connecting them by foot, bike, or car.


Israel first rehearsal | ESCKAZ.com live from Lisbon!
It took quite a lot for this performance to get started, so we are probably getting behind schedule here while the looper and other items were being adjusted.
So, first run-through, very first impression.
Netta is wearing a Japanese-style dress again like one she wears in the video and promo photos as well, however, this time in purple/red/white/black. The Japanese theme is continued in the yellow panels behind her which on a closer look feature many 'maneki-neko' (yes I had to look that up) - Japanese fortune cats.
There's a lot going on on stage, of course there's the aforementioned looper and the three dancers/backing vocals that start on the bridge on the side and later join her in the middle of the stage. In the last part of the song Netta walks all over the stage and ring in front of it.
It sounds and looks very confident, they're not gone yet by far.
We got two runs more which show that there is still some work to do - but of course it's only a first rehearsal. Some missed camera's, some wrong shots by the director, but overall very solid. The last run revealed a lot of smoke pyro's - in yellow and purple effects, it does complete the performance.


Image Title: Ikea Broom Closet Full Image For Ergonomic Intended Organizer Designs 15. Post Title: Broom Closet Organizer. Filename: ikea-broom-closet-full-image-for-ergonomic-intended-organizer-designs-15.jpg. Image Dimension: 659 x 985 pixels. Images Format: jpg/jpeg. Publisher/Author: Darrion Bogisich. Uploaded Date: Monday - September 17th. 2018 12:36:24 PM. Category: Architecture. Image Source: digsdigs.com. Broom Closet Organizer Uxstudent Club Throughout Plan 18. Broom Closet Organizer Pic Wardrobe Design With Prepare 7. Broom Closet Organizer Ideas Regarding Remodel 19. Broom Storage Ideas Amazing Closet With Regard To Organizer Designs 11. Cleaning Closet Organization Ideas Broom In Organizer 12. Broom Closet Ideas Storage Organizers With Organizer Decor 4. Broom Closet Organizers Awesome Organizer 16 Best Hide The For 8 With Regard To Ideas 3. 7 Broom Closet Storage Solutions For Kitchens Of Any Size Kitchn Intended Organizer Designs 9. Broom Closet Organizer And Mop Intended For Ideas 6. Broom Closet Organizers Awesome Organizer 16 Best Hide The For 8 Within Remodel.


Fire, Earthquakes and other natural disasters can occur at any time – how will you know who was on-site?
Vistab can help in many ways.
All evacuation management begins when planning for a disaster. Capturing the correct information is vital for an evacuation to be successful. VisTab has the tools to obtain exactly what you need to know.
When and where visitors, contractors, staff signed in from.
Head counts from any mobile phone, internet capable device, or the VisTab tablet.
Inform which visitors require help to evacuate.


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text:
Biggest freshman class in 35 years pushes enrollment past 25K
September 8, 2014 · 4 min read
by Steve Smith | University Communication
UNL's overall enrollment has grown by 15.4 percent since 2005.
Powered by its largest freshman class in 35 years, UNL's total enrollment reached 25,006 students in fall 2014 – a 2.3 percent increase over last year and the second-highest enrollment in the university's history.
The 4,652 first-time freshmen represent an increase of 232 students, or 5.2 percent, in the number of freshmen from fall 2013. That's the second-largest freshman class in UNL's history and the biggest since 1979, when 4,702 first-time students were enrolled.
"It has been a campus-wide priority to increase enrollment and it is pleasing to see the continuing results of these efforts," said Chancellor Harvey Perlman, who has set a goal for UNL to raise its enrollment to 30,000 students by 2020. "Our growth as a university is a reflection of our upward trajectory not only in enrollment but in energy, optimism and ambition."
UNL's overall enrollment has grown by 15.4 percent since 2005. The all-time high for overall enrollment is 25,075 in 1982.
This fall, the university also saw an overall increase in undergraduates over last year – 19,979 total, or an increase of 603 students (3.1 percent).
The College of Engineering led all UNL colleges in the highest percentage of undergraduate enrollment growth, increasing by 8.5 percent. Engineering was followed by the College of Journalism and Mass Communications (8.0 percent), the College of Agricultural Sciences and Natural Resources (6.6 percent) and the College of Business Administration (4.8 percent).
"It is particularly gratifying to see increases at the College of Engineering because of the demand for this talent in Nebraska," Perlman said.
Alan Cerveny, dean of enrollment management, said that crossing the 25,000-student threshold was noteworthy but the university remains focused on its long-term enrollment goals.
"The growing national attention UNL is receiving is rewarding and I am particularly pleased by the increased interest we've seen this year from Nebraska students, from Scottsbluff to Omaha," he said. This fall, first-time freshman Nebraska residents number 3,497 – a 5.4 percent climb from last year.
"We take our role in preparing the next generation seriously, so it is gratifying to see so many prospective students choosing to attend UNL," said Amber Williams, director of admissions. "Our first-year class is a reflection of the university's academic programs and our success in attracting resident and non-resident students."
Other highlights from the official 2014 UNL census, which was reported today to the University of Nebraska Board of Regents:
UNL's enrollment of 2,523 minority undergraduate students – 12.6 percent of the undergraduate total – makes this fall's undergraduate student body the most diverse in school history.
"As demographics continue to shift, it is important UNL reflects the growing diversity of Nebraska and the country," Williams said. "We're pleased our efforts to recruit students from all backgrounds has led to a more diverse campus."
The university welcomed 2,485 international students this fall – an increase of 359 students (16.9 percent). There are 1,515 undergraduate and 970 graduate and professional students from other countries.
Overall, nonresident enrollments increased by 8.6 percent, including 14.2 percent at the undergraduate level. Nonresident students now account for 29 percent of all students at UNL.
In addition to traditionally strong out-of-state markets in South Dakota and western Iowa, the university experienced increases of students from Minnesota (19.1 percent), California (10.8 percent) and Illinois (25.1 percent).
Though larger than last year's, UNL's new freshman class maintains a strong academic profile. This year's average freshman ACT score of 25.4 ties the record for the highest average in school history, set in 2012.
Graduate enrollment of 4,517 represented a slight dip (0.8 percent) from 2013.
The average credit-hour load for undergraduates is 13.8, consistent with last year.
The fall semester registrations are reported annually to the provost of the NU system, based on six-day census figures from each of the four NU campuses' Institutional Research and Planning offices. For more details, see http://www.nebraska.edu.
Freshman Class
Agricultural Sciences and Natural Resources
Craig Chandler | University Communications
UNL's class of 2018 gathered at Memorial Stadium the week before the start of fall semester.