The plant circadian clock orchestrates 24-hour rhythms in internal physiological processes to coordinate these activities with daily and seasonal changes in the environment. The circadian clock has a profound impact on many aspects of plant growth and development, including biomass accumulation and flowering time. Despite recent advances in understanding the circadian system of the model plant Arabidopsis thaliana, the contribution of the circadian oscillator to important agronomic traits in Zea mays and other cereals remains poorly defined. To address this deficit, this study investigated the transcriptional landscape of the maize circadian system.
Since transcriptional regulation is a fundamental aspect of circadian systems, genes exhibiting circadian expression were identified in the sequenced maize inbred B73. Of the over 13,000 transcripts examined, approximately 10 percent displayed circadian expression patterns. The majority of cycling genes had peak expression at subjective dawn and dusk, similar to other plant circadian systems. The maize circadian clock organized co-regulation of genes participating in fundamental physiological processes, including photosynthesis, carbohydrate metabolism, cell wall biogenesis, and phytohormone biosynthesis pathways.
Circadian regulation of the maize genome was widespread and key genes in several major metabolic pathways had circadian expression waveforms. The maize circadian clock coordinated transcription to be coincident with oncoming day or night, which was consistent with the circadian oscillator acting to prepare the plant for these major recurring environmental changes. These findings highlighted the multiple processes in maize plants under circadian regulation and, as a result, provided insight into the important contribution this regulatory system makes to agronomic traits in maize and potentially other C4 plant species.
Plants match their physiology to daily and seasonal environmental changes through the circadian clock, an internal timekeeping mechanism that regulates a wide range of plant behavior. Overt circadian rhythms in plants include rhythmic leaf movements, stomatal conductance, and growth . Rhythms are maintained with a period of approximately 24 hours in the absence of environmental cues and over the normal range of ambient temperatures. The circadian system enables plants to anticipate and synchronize their physiology to the recurring environmental changes brought on by day and night. The consequence of proper clock and environment synchronization is optimized fitness [2, 3]. Beyond the daily control of plant biology, circadian rhythms also allow plants to track seasonal change according to day length, or photoperiod [4, 5]. The interplay of the circadian clock and photoperiod allows photoperiod sensitive plants to initiate floral development in accordance with the season [6, 7]. Thus, the circadian clock is an endogenous timer that maintains normal plant biology on both daily and seasonal timescales.
The circadian clock is best described in the model plant Arabidopsis thaliana. Arabidopsis mutants with impaired clock function show reduced fitness arising from mismatch between internal rhythms and external environmental conditions [8, 9]. In addition, many circadian clock mutants exhibit alterations in flowering time associated with defects in day length perception . At the molecular level, Arabidopsis circadian physiology requires the products of so-called core clock genes, whose mutation widely disrupts circadian physiology . The core clock proteins regulate expression of one another in a network of feedback loops . The myb-like transcription factors CCA1 and LHY directly control TOC1, PRR7, and PRR9 expression [13, 14]. In turn, these pseudoresponse regulators define the expression patterns of CCA1 and LHY. In addition, the TCP transcription factor CHE serves as a transcriptional regulator of CCA1 . Additionally, the clock-specific photoreceptor and F-box protein ZTL controls TOC1 function through 26S proteasome-mediated protein degradation of TOC1 at night [16, 17]. The core circadian oscillator also requires the activity of ELF3 and LUX in the evening [18, 19]. This regulatory network generates phase-specific 24-hour oscillations in each core clock gene, with the state of the overall system reflecting time of day.
Orthologs of circadian clock components in plants outside of Arabidopsis have been best characterized in rice  and Lemna . The rice genome encodes single orthologs of CCA1 and GI, but two potential orthologs of ZTL. In addition, rice possesses five unique PRR orthologs, including a clear ortholog of TOC1 . Overexpression of these rice orthologs in Arabidopsis modifies circadian rhythms, which is consistent with the function of these proteins being conserved between rice and Arabidopsis . In addition, rice TOC1 and PRR7 partially complement the corresponding Arabidopsis toc1 and prr7 mutants . Circadian clock related genes have also been described in two species of the monocot Lemna, including LHY, GI, ELF3, TOC1, and the other PRRs . Knockdown and overexpression of LHY, ELF3, and GI in Lemna suggest the topology of the Lemna circadian system is conserved with Arabidopsis and rice .
Genome-wide expression assays have revealed details behind circadian clock regulation of overt plant physiology. In Arabidopsis, the core oscillator genes are only a fraction of the genes showing cyclic expression in constant conditions. Whole genome transcriptional profiling demonstrates that the steady state transcripts of ~30% of Arabidopsis genes cycle every 24 hours in constant conditions [25, 26]. Clock controlled phasing of these cyclic genes throughout a day activates or represses metabolic and signal transduction pathways, thereby yielding macroscopic circadian rhythms in plant physiology. Genes involved in related pathways share timing of peak expression; for example, genes encoding enzymes in secondary metabolite biosynthesis, nutrient assimilation, and hormone signaling are co-regulated by the circadian clock [27–29].
Approximately half of the world's grass species employ C4 photosynthesis, including the food crops maize, sugarcane, and sorghum, as well as the potential biofuel crops switchgrass and Miscanthus. C4 plants capture CO2 as the 4-carbon compound oxaloacetate in specialized mesophyll cells and the newly captured carbon is then transported into adjacent bundle sheath cells to enter the Calvin cycle through the action of Rubisco. Primary carbon fixation in C3 plants like Arabidopsis occurs in mesophyll cells through Rubisco-mediated incorporation of CO2 into the 3-carbon compound 3-phosphoglycerate. Direct CO2 capture by Rubisco reduces the photosynthetic efficiency of C3 plants because photosynthetic rate is limited both by CO2 diffusion from the atmosphere and by photorespiration that increases at low CO2 concentrations and high temperatures. The physical partitioning of CO2 capture and the Calvin cycle in C4 plants improves photosynthetic efficiency under low CO2 concentrations and reduces photorespiration by Rubisco at high temperatures and low CO2 concentrations. As a consequence, C4 crop plants assimilate biomass more efficiently than C3 plants at the high temperatures typical of agricultural settings .
Previous studies have shown the circadian clock serves to coordinate expression of genes encoding the photosynthesis apparatus in plants that carry out C3 photosynthesis . The circadian system of C4 plants remains uncharacterized. Therefore, examination of the maize circadian system is fundamental to understanding the impact of circadian regulation on C4 photosynthesis, as well as the many other areas of maize metabolism where circadian rhythms are important. In this study, transcriptional profiling revealed the maize circadian transcriptome and this provided an initial characterization of the aspects of maize physiology under circadian clock influence.
To map the maize circadian transcriptome, mRNA levels in the aerial tissues of week-old maize B73 plants were determined by transcriptional profiling with the Affymetrix GeneChip® Maize Genome Array. The B73 inbred is widely used and is the source material for the complete maize genome sequence . Young plants were chosen as the experimental model because at this developmental stage all the tissues of the plant were easily sampled at once, unlike older maize plants that were too large to sample whole. Plants were exposed to 12 h light:12 h dark photocycles to set the circadian clock and then transferred to LL conditions for 48 additional hours. While in LL, aerial tissue was harvested every 4 hours and transcript levels in three pooled replicate samples were determined for the 13,339 maize genes represented on the microarray. The genes on this microarray account for approximately 41% of the entire maize genome, as the maize genome is predicted to have over 32,540 protein-encoding genes . Consequently, the genes with circadian expression identified here may represent nearly half of the number of predicted maize genes with rhythmic expression.
Following preprocessing and normalization of the raw hybridization values, genes on the array exhibiting circadian waveforms were identified by two well-established methods known as COSOPT and HAYSTACK [26, 32]. COSOPT is a method that fits gene expression profiles to a series of modified cosine models, with the agreement between the experimental data and the model reported by the MMC-β value . Lower values of MMC-β indicate a better fit to the cosine model. 952 genes were considered to exhibit a circadian waveform based on a MMC-β cutoff value of ≤ 0.05 (Figure 1A), which is a cutoff established by previously published reports for analogous circadian datasets in Arabidopsis and mouse [25, 26, 28, 33, 34]. The HAYSTACK tool identifies rhythmic waveforms by determining the agreement between expression profiles and a collection of models, which represent different types of potential circadian expression patterns . The degree of correlation between model and experimental waveforms is determined by Pearson's correlation coefficient, where an exact match corresponds to PCC = 1.0. Maize transcripts with expression patterns matching a model with a PCC value of 0.7 or greater were considered to have a circadian rhythm. Based on this criterion, HAYSTACK analysis called 702 transcripts as exhibiting a circadian rhythm (Figure 1A). PCC values of 0.7 or greater consistently selected good matches between models and experimental waveforms, but this cutoff is more relaxed than the 0.8 value applied previously for a large collection of Arabidopsis time courses .
A large fraction of maize genes exhibited circadian expression patterns and expression occurred at all phases. A) Venn diagram of the overlap between transcripts found to have circadian waveforms by HAYSTACK (blue) and COSOPT (green). Union is shown in black. B) Waveforms of overlapping (black bars) and unique (white bars) cycling probe sets identified by HAYSTACK compared to those found by COSOPT. C) The majority of maize transcripts were preferentially expressed at morning (ZT0) or midnight (ZT12).
Comparing the genes called rhythmic by COSOPT and HAYSTACK revealed that the two methods had 210 genes in common (Figure 1A), a proportion similar to that described for Arabidopsis circadian expression datasets . COSOPT identified almost twice as many unique rhythmic transcripts as found by HAYSTACK. This outcome was unexpected, since HAYSTACK has the potential to match a larger diversity of waveforms  and HAYSTACK has been shown to identify a larger proportion of rhythmically expressed transcripts in Arabidopsis datasets, most notably those with expression patterns in the spike class . Not surprisingly, HAYSTACK appeared to favor maize genes showing the spike and box waveforms over other waveform classes, like cosine that COSOPT was designed to find (Figure 1B). Thus, under these standard cutoffs COSOPT was more sensitive than HAYSTACK in finding transcripts that match a cosine waveform. Furthermore, the cosine waveform appeared to be the dominant pattern of expression in maize seedlings under LL conditions.
Collectively, COSOPT and HAYSTACK indicated 1,444 transcripts, or ~10% of the expressed genes on the microarray showed rhythmic expression (Additional file 1 Table S1); therefore, a substantial part of the maize transcriptome is subject to regulation by the circadian clock. Genes in this collection included known maize circadian clock-regulated transcripts such as putative maize flowering time genes gi1A and conz1 , the well-established circadian clock-regulated gene cat3 , and several lhcb genes  (Additional file 2 Figure S1). Assuming a maize genome of 32,540 genes , the full circadian transcriptome of maize potentially represents a minimum of 3,254 genes. This degree of clock regulation is comparable to that observed in Arabidopsis with a partial genome array  and in other model systems like Drosophila melanogaster, Neurospora crassa, and Mus musculus .
A fundamental role of the clock is to anticipate day and night; as a result, the majority of circadian gene expression in Arabidopsis, rice, and poplar is timed, or phased, to precede or coincide with these recurring environmental shifts [26, 28]. To determine whether the maize circadian transcriptome was similarly organized, the cycling maize transcripts were sorted into six phase bins based on expression waveform. The six phase bins were distributed in 4-hour intervals throughout the 24-hour subjective day: dawn (ZT0 hours), midday (ZT4), late day (ZT8), dusk (ZT12), midnight (ZT16), and early morning (ZT20). K-means clustering successfully placed all transcripts into one of these six phase bins (Additional file 3 Figure S2), with the exception of four outliers. As expected, rhythmically expressed transcripts were preferentially phased to the transitions into or out of subjective day (Figure 1C). This distribution was significantly different from that expected by chance, as assessed by χ2 testing (χ2 = 109.83, P(χ2) = 4.45 × 10-22, df = 5, α = .05). The single largest phase bin was ZT12 with 335 of cycling genes, followed by ZT0 encompassing 314 transcripts (Figure 1C). The early morning phase bin was also predominant, containing 249 genes with peak expression at ZT20. The remaining transcripts were distributed almost equally over the remaining phase bins. This analysis indicated the maize circadian oscillator parses gene expression in accordance with predictable environmental changes associated with day and night.
The genes on the array were annotated to identify the maize cellular processes under clock control. The annotation process involved matching the probe sets on the array to maize genes and then extracting the GO Slim classification for each transcript provided by the Maize Sequence Consortium (full method is described in "Methods" and outlined in Additional file 4 Figure S3). Of the cycling transcripts, 655 were assigned to GO Slim classifications for Cellular Component, Biological Process, and Molecular Function (Additional file 5 Table S2). The cycling dataset was evaluated for overrepresentation of GO Slim categories to determine whether the maize circadian clock preferentially regulated specific plant processes. Applying hypergeometric distribution analysis to the entire cycling dataset showed the Molecular Function GO Slim terms "catalytic activity" (GO: 0003824), "binding" (GO: 0005488), and "transporter activity" (GO: 0005215) were overrepresented in this group of transcripts (Table 1). Each of these GO Slim terms indicates transcripts encoding enzymes, receptors, and proteins involved in movement of macromolecules, small molecules, and ions are an important aspect of the maize circadian transcriptome. In the Biological Process classification, genes annotated with the GO Slim terms ""generation of precursor metabolites and energy" (GO: 0006091), "carbohydrate metabolic process" (GO: 0005975), and "photosynthesis" (GO: 0015979) were enriched in the circadian expressed gene set (Table 1). Together, these three GO Slim terms suggested the maize circadian clock makes an important contribution to fitness by regulating energy and carbohydrate metabolism. Similar GO Slim terms were also found to be overrepresented in the Arabidopsis circadian transcriptome , which demonstrates the involvement of the plant circadian clock in these processes is highly conserved.
1Overrepresentation of GO Slim terms was calculated using hypergeometric distribution followed by a modified Bonfferoni correction for multiple testing as implemented in GeneMerge , where the e-score is comparable to a P-value. Overrepresented GO terms were those with e-scores < 0.05.
2Each phase group was analyzed separately to identify GO terms overrepresented at a specific phase.
Hypergeometric distribution analysis further tested whether expression of genes in particular GO Slim terms showed a time-of-day specific enrichment. The photosynthesis GO Slim term was enriched in the genes phased to ZT4 (Table 1). The bias toward photosynthesis genes at ZT4 is clear when the abundance of these genes is plotted as a function of phase (Figure 2). The Arabidopsis circadian clock similarly organizes photosynthesis-associated genes to reach peak expression early in the day . Therefore, the circadian clock prepares maize plants for increasing illumination by up-regulating photosynthesis genes early in the light period. A phasing preference was also clear for the expression of genes associated with "cellular amino acid and derivative metabolic process" (GO: 0006519), which showed overrepresentation of genes with peak levels at ZT20 (Table 1, Figure 2). These results indicate maize protein synthesis may also have a pre-dawn phase. Interestingly, Arabidopsis genes associated with protein synthesis are preferentially expressed at around dawn when plants are exposed to thermocycles, but at midday in photocycles . Since the maize plants used here did not experience thermocycles, maize plants may organize proteins synthesis at a time of day distinct from Arabidopsis. These findings show the maize circadian clock coordinates the expression of major cellular processes to occur at defined times within a normal 24-hour day. The specific physiological processes where the circadian clock plays a large role in coordinating transcription are explored in more depth below.
The maize circadian system directed phase-specific expression of genes involved in biosynthesis. Fraction of all genes in each phase cluster with GO Slim terms GO: 0015979 "photosynthesis" (PS, green), GO: 0006519 "cellular amino acid and derivative metabolic process" (AM, blue), and GO: 0009058 "biosynthetic process" (BP, yellow) compared to all genes on the microarray (AG, red). Hypergeometric distribution analysis from Table 2 indicated GO: 0015979 "photosynthesis" genes are overrepresented at midday (ZT4), whereas GO: 0006519 "cellular amino acid and derivative metabolic process" and GO: 0009058 "biosynthetic process" were enriched at early morning (ZT20).
The maize proteins encoded by the transcripts represented on the array were given a functional annotation by matching amino acid sequences to likely orthologs in rice and Arabidopsis (Additional file 6 Table S3). The functional annotation grouped genes into a high confidence set and a low confidence set based on the criteria described in "Methods" and outlined in Additional file 4 Figure S3. Importantly, the high confidence genes represent those genes where orthologs were identified in both Arabidopsis and rice. The genes in this high confidence set were used to explore the influence of the circadian clock on maize physiology, in particular maize metabolic and signaling pathways.
The contribution of the maize circadian clock to photosynthesis was of special interest for its potential to provide insight into circadian regulation of C4 photosynthesis. Many key enzymes involved in C4 photosynthesis were under circadian clock control and expression of these were generally phased around dawn (Table 2; Figure 3), similar to Arabidopsis C3 photosynthesis . A gene encoding PEPC, the C4 enzyme that initially captures carbon for photosynthesis  (Figure 3), showed early morning phasing (Table 2). This PEPC is a strong candidate for a genuine C4 enzyme, as its amino acid sequence is highly homologous to a PEPC from sorghum and the genome regions around the maize and sorghum genes are syntenic (data not shown). Circadian regulation of a maize pepc gene is a novel observation. However, the true impact of the circadian oscillator on PEPC protein abundance remains to be determined, as the rhythms in transcription may not be translated into changes in protein levels. Interestingly, the Arabidopsis transcript encoding a homologous PEPC has a nearly identical phase of expression as the maize pepc transcript (Table 2). A maize ppdk gene, which encodes the enzyme that furnishes PEPC substrates  (Figure 3), and a ca gene both reach peak expression at dawn (Table 2). Expression of an Arabidopsis homolog of PPDK also has a phase matching the maize ppdk gene. Therefore, the circadian regulatory networks in C4 and C3 plants similarly organize expression of photosynthesis components. Logically, the expression of pepc, ppdk, and ca immediately precedes stomatal opening and the onset of light that stimulates C4 metabolism . Other components of photosynthesis displayed similar anticipatory expression, including transcripts encoding RBCS3B, RCA, and several LHCB proteins, as well as GAPD and CS, which contribute to chlorophyll synthesis (Table 2). Similarly, a large proportion of the enzymes of the Calvin cycle on the array were circadian clock-regulated such that peak expression preceded or occurred at dawn (Figure 3, Table 2). Consistent with the apparently conserved nature of the circadian network structure, the phase of expression for this set of photosynthesis genes was essentially invariant between maize and Arabidopsis (Table 2). Thus, the maize system has incorporated the initial CO2 capture of C4 photosynthesis into the overall coordination of photosynthesis that is shared with C3 photosynthesis.
1Phase of Arabidopsis transcript expression from the DIURNAL web tool , where phase is determined with HAYSTACK . "nc" indicates noncycling transcript with a waveform assigned a PCC < 0.7 by HAYSTACK analysis.
2Function of maize protein inferred from the function of the Arabidopsis ortholog according to Kyoto Encyclopedia of Genes and Genomes .
The circadian clock organized expression of maize photosynthesis, carbohydrate metabolism, and cell wall synthesis genes. Maize genes encoding enzymes associated with the CO 2 capture steps of C4 photosynthesis, which take place in mesophyll cells, showed coordinated expression phased to morning (ZT20 and ZT0) and midday (ZT4). Expression of genes encoding enzymes of the Calvin cycle, which occurs in bundle sheath cells, was generally phased to early morning (ZT16 and ZT20). Expression of genes encoding enzymes involved in cell wall synthesis, which are illustrated in a separate cell for clarity, occurred in the morning (ZT20 and ZT0). Large light blue shapes represent cells and green boxes within cells represent chloroplasts. Small dark blue boxes indicate genes on the array and red boxes are genes absent from the array. White clock faces next to enzymes indicate the circadian expression of the gene encoding that enzyme and the number within the clock face shows the expression phase in ZT.
The state of the maize circadian system observed here is likely an incomplete representation of the network in field grown mature plants, since young maize seedlings are developing their C4 photosynthetic capacity. Burris and de Veau showed that while 9 day-old maize seedlings exhibited a 3 times higher rate of photorespiration than 3 month-old maize plants, consistent with limited C4 photosynthesis in seedlings compared to the mature plants, the rate of photorespiration in these maize seedlings was 5 to 7 times lower than mature wheat plants performing C3 photosynthesis . Their analysis demonstrates young maize seedlings have the capacity for C4 photosynthesis, albeit a modified version with a higher rate of photorespiration than mature plants. Therefore, 7 day-old maize seedlings represent a suitable model of C4 photosynthesis.
Circadian expression was evident for genes encoding enzymes involved in biosynthesis of carotenoids including PSY, BCH, and ZEO (also known as ABA DEFICIENT 1 in Arabidopsis) (Table 2, Figure 3). Carotenoids serve as photoprotective pigments and are also important structural components of light harvesting complexes. PSY catalyzes the first and rate-limiting step in the production of carotenoids and both BCH and ZEO lie downstream of PSY [44, 45]. Expression of these genes was phased to either early morning (ZT20) or midday (ZT4), clearly showing the maize circadian system orchestrates expression of upstream and downstream enzymes for carotenoid production to coincide with the time when these compounds are needed for photosynthesis and non-photochemical quenching. PSY is also circadian regulated in Arabidopsis , with a phase matching that found here for maize (Table 2), which again suggests strong conservation of the regulatory networks underlying the maize transcriptome. Protection from ROS produced by photosynthesis is provided by the action of catalase and superoxide dismutase enzymes. Genes encoding maize CAT3 and an ortholog of MSD1 exhibited circadian expression in maize seedlings (Table 2). Consistent with their role in scavenging ROS arising from photosynthesis, expression of the cat3 and msd1 genes reached peak levels during late day (ZT8) when ROS is more likely to accumulate (Figure 3). Overall, these findings show the circadian clock organizes maize C4 photosynthesis to ensure the plant makes maximal use of light energy available during the day.
The maize clock also regulates genes contributing to carbohydrate metabolism and carbon flux (Figure 3, Table 1). For example, the circadian clock controlled expression of two PFK encoding genes and peak expression for each occurred around dawn (Figure 3). Note that the annotation matched two different maize transcripts to the same Arabidopsis gene (PFK3), which explains the different phase values shown in Table 2. PFK enzymes mediate the central regulatory step of glycolysis. Two glycolysis enzymes of the FBAase class, which reversibly convert fructose bisphosphate to triose phosphate, were clock regulated such that one peaked in the morning (FBAase2) and another expressed in the evening (FBAase1). As in energy generation, the clock regulates genes involved in energy storage. AGPase is the major regulatory enzyme in starch biosynthesis, where it converts glucose 6-phosphate to ADP-glucose, the substrate for starch synthase . The transcript encoding AGPL, a subunit of AGPase, reached peak levels near dawn at ZT20 (Figure 3). Early morning accumulation of AGPase may be an anticipatory strategy to prepare the system for midday when photosynthate is in excess and starch biosynthesis commences. Similarly, two ss transcripts encoding starch synthases were maximally expressed early in the day (ZT0 and ZT4) like their Arabidopsis orthologs (Table 2). Thus, the maize circadian clock anticipated the need for carbon metabolism components and up-regulated expression of these genes so that enzymes would be present at the time when photosynthate would be available for energy production and storage. Together, the coordinated regulation of carbohydrate metabolism and C4 photosynthesis indicates that the circadian clock in maize organizes gene expression to ensure efficient and maximal energy production, use, and storage throughout the day.
Cell walls are a major constituent of plant biomass, and their enlargement exhibits a biological rhythm [47, 48]. Consistent with rhythmic growth, the transcripts of several enzymes involved in cell wall biosynthesis were found to be circadian-regulated in maize (Table 2, Figure 3). CCR and CAD catalyze two of the final steps in the conversion of phenylpropanoid into monolignins to achieve wall hardening . Transcripts encoding both these enzymes were found to have cyclic expression peaking at dawn (Figure 3). The maize CAD is orthologous to Arabidopsis CAD4. Arabidopsis CAD4 is a class II CAD enzyme that acts on sinapaldehyde instead of coniferaldehyde ; therefore, the substrate for the maize CAD is likely to be sinapaldehyde (Figure 3). 4-CL acts early in phenylpropanoid synthesis and a maize gene encoding an ortholog of Arabidopsis 4-CL3 is rhythmically expressed with peak expression at ZT16, instead of ZT0 like the ccr and cad transcripts. Since the Arabidopsis 4-CL3 enzyme participates in flavonoid biosynthesis instead of contributing to lignin production , this maize 4-CL is likely not involved in lignin biosynthesis but flavonoid biosynthesis instead. The similar expression waveforms of ccr and cad in maize and Arabidopsis suggests the timing of this aspect of lignin biosynthesis is conserved (Table 2) . Several cellulose synthase genes shared nearly the same dawn phasing as the ccr and cad transcripts (Figure 3). Similarly, transcripts for two endoglucanases and a cellulase cycled at dawn in the maize dataset (Figure 3). Circadian dawn expression of cell wall-related enzymes correlates with the time of maximal plant growth rate reported for Arabidopsis [49, 51]; therefore, the maize circadian clock seems to regulate daily cell wall construction so that it coincides with growth trigged by phytohormone signaling.
Several recent studies have shown a fundamental role of the Arabidopsis circadian clock is to indirectly control growth and development through transcriptional regulation of genes involved in phytohormone biosynthesis and response [25, 27, 29]. Placement of the maize cycling genes in the context of metabolic pathways revealed that the maize circadian clock also regulates genes involved in the synthesis of phytohormones fundamental to plant growth (Table 2).
GAs and ethylene are important positive regulators of plant cell growth. GAs promote longitudinal expansion of cells and ethylene promotes transverse cell expansion . Daily rhythmic growth exhibited by Arabidopsis is, in part, controlled through diurnal transcriptional control of genes encoding enzymes for synthesis of GAs and ethylene . For GAs, GA 12 biosynthesis is a prerequisite for the production of active GAs. In Arabidopsis, GA1, which encodes ENT-COPALYL DIPHOSPHATE SYNTHASE, and KAO2 act in the biosynthetic pathways that provide the GA 12 precursor of GA synthesis [53, 54]. Maize orthologs of GA1 and KAO2 were among the proteins encoded by circadian expressed transcripts (Table 2). Maize ga1 reached peak expression at subjective midday (Figure 4A), consistent with the phasing reported for GA synthesis genes in Arabidopsis seedlings under constant light conditions ; however, Arabidopsis GA1 does not exhibit circadian expression (Table 2). Maize kao2 showed peak circadian expression at ZT16, which was 12 hours later than maize ga1 and 8 hours later than its Arabidopsis counterpart (Figure 4A). Nevertheless, the robust circadian expression of ga1 and kao1 indicates the participation of the maize circadian clock in the coordination of maize growth through regulation of phytohormone biosynthesis. In support of this possibility, two putative ethylene biosynthesis genes also showed circadian expression: orthologs of Arabidopsis MTO3 and ACS6. MTO and ACS enzymes act sequentially to produce ACC, which is the precursor to ethylene . The dawn-phased expression of the maize acs6 and mto3 genes was in contrast to the non-circadian expression for their Arabidopsis orthologs (Table 2). The lack of correspondence in the phasing of GA and ethylene synthesis between maize and Arabidopsis may be a consequence of the constant light condition used here. Along with the circadian clock, diurnal light and dark cues serve an important role in determining the phase of phytohormone synthesis genes and ultimately the timing of cellular growth [26, 51]. Therefore, evaluation of maize expression in diurnal conditions will be important to fully comprehend the molecular basis for rhythmic growth in maize.
Genes associated with phytohormone biosynthesis, flowering time, phototropism, and the circadian clock exhibited rhythmic expression. A) Expression waveforms of high confidence cycling biosynthesis genes for ethylene (green open symbols), ABA (blue closed symbols) and GA (red open symbols). B) Expression waveforms of high confidence cycling genes for phototropism (blue closed squares), circadian clock (green closed symbols), and flowering time (red open symbols). Shaded regions represent subjective night. For each gene, the normalized expression values from the array were further normalized to the highest expression value for that gene.
The plant stress hormone ABA antagonizes the growth promoting effects of GA and ethylene  and, like GAs and ethylene, ABA biosynthesis genes are part of a transcriptional regulatory network that exerts daily control over plant growth . The carotenoid biosynthesis pathway supplies precursors for the biosynthesis of ABA . As noted above, the transcript encoding PSY, the enzyme governing the first committed step in carotenoid synthesis, was under circadian control with peak expression in the early morning (Table 2, Figure 4A). Several genes downstream of PSY that are involved in ABA synthesis also showed dawn expression (Table 2, Figure 4A), which is consistent with ABA synthesis taking place in the early morning. Comparable circadian regulation of PSY and genes encoding downstream components of this pathway has been noted in Arabidopsis . The conserved circadian regulation of maize carotenoid biosynthesis genes observed here suggests that synthesis of ABA in maize leaves is under circadian control, which likely impacts the key role ABA plays in antagonizing cell growth and in stimulating stomatal closure . The latter role of ABA is associated higher-water use efficiencies in crop plants, since closure of stomata reduces water loss. Therefore, the maize circadian system is likely a critically important, but under appreciated, contributor to maize productivity in the field.
Several orthologs of Arabidopsis circadian clock and flowering time genes were present in the maize cycling gene set; however, the array had limited overall representation of genes in these categories. Included in the rhythmic collection was the gene encoding CONZ1, which the annotation matched to Arabidopsis COL1 (Table 2, Figure 4B). CONZ1 is a strong candidate for the functional ortholog of Arabidopsis CONSTANS and rice Heading date 1 . CONSTANS and Heading date 1 serve in control of flowering time to regulate the expression of the FT class of proteins. FT proteins act as florigen molecules that promote floral development at the shoot apical meristem . gi1A and gi1B are paralogous flowering time and circadian clock genes identified in the same study as conz1 . A probe set for gi1A was on the array and this transcript exhibited a waveform and phase similar to that of Arabidopsis GI expression (Figure 4B, Table 2) . Orthologs of Arabidopsis LDL1 and FCA were two other potential flowering time transcripts with rhythmic expression in the maize dataset (Figure 4B). LDL1 and FCA are known to act in the Arabidopsis autonomous flowering pathway . LDL1 is involved in modification of chromatin and, therefore, is not likely to be strictly involved in flowering time . Similarly, FCA plays a role in RNA-mediated silencing through DNA methylation . Circadian clock components present on the array included orthologs of Arabidopsis PRR7 and LUX (Table 2, Figure 4B). Expression of maize prr7 and lux exhibited the same phasing as their Arabidopsis orthologs (Table 2), indicating the conserved nature of the core maize circadian clock. Finally, the maize phot1 transcript, encoding a blue light photoreceptor of the phototropin family likely to be involved in phototropism, was robustly rhythmic with the identical phase as its Arabidopsis counterpart (Figure 4B). This restricted sampling of flowering time, circadian clock, and phototropism genes suggests that maize relies on conserved signaling networks for these key processes.
Identification of maize genes under circadian clock regulation and the predicted contribution of these genes to metabolism, growth, and development indicates the maize circadian clock plays an important role in coordinating the overall physiology of this C4 crop plant. In general, circadian regulation of the enzymes mediating C4 photosynthesis was predictable based on the Arabidopsis C3 model and without any apparent large-scale changes to accommodate the specialized C4 anatomy and enzymology. Recent investigation of the global effects of the circadian system on Arabidopsis physiology demonstrated that rhythms are critical to plant fitness and optimal growth . A more complete appreciation of the maize circadian clock will reveal where the circadian system impacts maize growth and development, as well as highlight novel approaches for optimization of crop production through targeted modification of the circadian system.
Seeds of inbred B73 were obtained from the Maize Genetics Cooperation Center. B73 is widely used and is the source material for the complete maize genome sequence . Seedlings were germinated and grown in 57 × 30 × 6 cm flats filled with Scotts Supersoil (scotts.com) inside a Conviron (conviron.com) growth chamber at constant 26°C and constant 70% humidity. High intensity cool white fluorescent bulbs provided illumination at a fluence rate of 200 μmoles m-2 s-1. Seedlings were grown for seven days under 12 hours of light followed by 12 hours of dark before being transferred to LL for two days at the same temperature and humidity.
Tissue harvest began following transfer of seedlings to LL on the 8th day. The entire aerial portion (corresponding to all tissue above the prop roots) of five seedlings was harvested beginning at dawn and every 4 hours thereafter for the next 48 hours. Samples were immediately frozen in liquid nitrogen. Three separate experimental replicates were collected in this way. Tissue was ground to a fine powder under liquid nitrogen and total RNA isolated by Trizol extraction (Invitrogen; invitrogen.com) followed by Qiagen RNeasy columns and treatment with RNase-free DNase I (Qiagen; qiagen.com). cRNA was generated from total RNA of three pooled replicate samples with the GeneChip® One-Cycle Target Labeling kit according to the manufacturer's recommendations (Affymetrix, affymetrix.com). The University of California, Berkeley Functional Genomics Laboratory  hybridized samples to Affymetrix GeneChip® Maize Genome Arrays and scanned the washed arrays as suggested by manufacturer. Present, Marginal, and Absent calls for each probe set were determined with MAS5 analysis  and these are presented in Additional file 7 Table S4. Probe sets called "Absent" at more than nine time points were removed from the downstream analysis . Raw hybridization intensities were normalized across all twelve arrays using RMAExpress in Perfect Match mode [64, 65]. The raw microarray data is available online as experiment ZM28 at the Plant Expression Database .
Rhythmic expression waveforms within the normalized dataset were identified by analysis with the COSOPT curve fitting algorithm and the pattern matching function HAYSTACK. The COSOPT algorithm is a cosine waveform fitting method that identifies transcripts with circadian expression by first performing an arithmetic linear-regression detrend of the normalized time series data for each transcript followed by testing detrended data for a fit to 101 cosine test models . An approximate goodness of fit value produced by COSOPT analysis is MMC-β, where lower values correspond to a better fit to an ideal cosine curve. Based on previous studies in Arabidopsis [25, 26, 28, 33] and our empirical tests, a MMC-β threshold of ≤ 0.05 was chosen to call a given probe set circadian. HAYSTACK is a model-based, pattern-matching tool that identifies rhythmic expression patterns by aligning time-series microarray data to a set of discrete diurnal and circadian models . HAYSTACK conducts a least square linear regression for each gene against all the models and calculates the best-fit model, phase-of-expression, and p-value for each gene. HAYSTACK was run on the normalized maize dataset with the following parameters: fold cutoff = 2, p-value cutoff = 0.05, background cutoff = 50, and PCC = 0.7 with the 552 model set described in Michael et al. 2008 . In general, goodness of fit between the model and experimental traces is revealed by the PCC value. We empirically determined that a PCC ≥ 0.7 produced good matches between model and experimental data. This cutoff was less stringent than that applied in previous studies investigating Arabidopsis circadian expression [26, 29].
Probe sets with similar expression waveforms were grouped together by K-means clustering. The RMAExpress normalized values for probe sets with MMC-β ≤ 0.05 and those with PCC ≥ 0.7 were converted to normalized values with the following calculation: value = [(expression at single time point) - mean(all time points)]/[standard deviation(all time points)]. These values were used to fit the waveforms to six clusters representing each ZT phase with the K-means clustering algorithm built into the TIGR Multiexpression Viewer tool . Clustering ran for a total of 50 iterations and was considered complete when probe sets were no longer exchanged between clusters. In two independent trials, no greater than 7 iterations were required to build the clusters.
Additional file 4 Figure S3 outlines the annotation scheme used to match probe sets to genes in the maize genome, identify GO Slim terms for maize genes, and functionally annotate maize genes. Transcripts represented on the Affymetrix GeneChip® Maize array were mapped to maize transcripts with a custom Perl script that used the DNA sequences provided by Affymetrix and BLASTn to query the ZmB73 4.53a filtered CDS sequences . After the maize gene ID was identified for each probe set, GO Slim terms and associated PlantCyc pathways  were extracted from the Maize Genome Consortium website . Maize gene IDs for each gene were used with a local implementation of BLASTp to identify likely rice orthologs in the Michigan State University Rice Pseudomolecule and Genome Annotation release 6.1 and likely Arabidopsis orthologs in TAIR9_pep_20090619 sequences. The output of these queries were separated into high and low confidence lists (Additional file 6 Table S3). Criteria for the high confidence list were amino acid identity of 40% or greater, Highest Scoring Segment Pair (HSP) of 50 amino acids or greater, and a match to putative orthologs in both Arabidopsis and rice. The low confidence list contains the remaining genes that encode proteins not meeting these three criteria. Putative functions of matched orthologs were derived from Kyoto Encyclopedia of Genes and Genomes . Phase of expression for Arabidopsis orthologs shown in Table 2 was determined with the DIURNAL tool from the "LL12" dataset and matches between model and data considered significant at PCC values ≥ 0.7 . Overrepresentation of GO Slim terms was calculated with hypergeometric distribution followed by a modified Bonfferoni correction for multiple testing as implemented in GeneMerge . GeneMerge produced an e-score comparable to a P-value that was used to determine significance of overrepresentation. GO Slim terms for the entire array were the population set and the indicated collection of cycling genes served as the study set. Overrepresented GO Slim terms were considered those with e-scores < 0.05.
We thank Dr. Hector Candela-Anton, James Schnable, and Imran N. Amirani for writing perl scripts, as well as Dr. Robin Buell for help and advice on annotation with rice sequences. We also thank Bryan Thines for critical reading of the manuscript. This work is supported by United States Department of Agriculture grant CRIS 5335-21000-026-00D to FGH.
SK carried out the microarray analysis, annotation, comparative analysis, and drafted the manuscript. SCR helped draft the manuscript and created figures. FGH conceived the study, participated in its design and coordination, created figures, and drafted the manuscript. All authors read and approved the final manuscript.


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Tension over Europe's security has grown in recent years due to instability in the Middle East and Africa and Russia's annexation of Ukraine. These threats come at a time when European defence budgets are under increasing pressure, making it hard for EU Member States to raise spending on their capabilities. Moreover, the United States, an essential guarantor of European security through NATO, has been complaining that Europeans are not doing enough themselves in defence.
The result has been a series of initiatives to bolster European defence, which are finally giving momentum to European defence cooperation.
Firstly, Member States are starting to develop common capabilities in order to reduce costs. The European Commission has actively led this agenda, which it sees as an important contribution to a competitive European defence industry.
Secondly, the EU has developed its first substantial defence policy. Deepened security and defence cooperation has been at the heart of official thinking on the future of the EU, as it is a component of the Union's ability to survive crises. In 2016, the Global Strategy for Foreign and Security Policy translated threats to European security into policies and instruments extending beyond crisis management. It also shifted the focus of security and defence policy closer to the EU's territory and borders.
Thirdly, Member States are no longer split along NATO and non-NATO lines in their views on the EU's role. This has been helped by the EU-NATO joint declaration in 2016, where the organizations agreed to step up cooperation in areas including hybrid threats, capacity building, cyber defence and maritime security. As a result, most EU members now seem convinced that there is a role for both the EU and NATO in European security politics.
This new dynamism in EU defence policy has met with an apparently firm consensus by the member states. However, there are differences over the details and the contribution expected from the EU.
Concerned by Russia's aggressive foreign policy, most East and Central European members now support the EU becoming prepared more concretely to implement its mutual defence clause – Article 42.7 of the Lisbon Treaty, which was activated for the first time in by France after the Paris terrorist attacks in 2015.
Of the three Nordic EU members, Finland has been most constructive. As a small nation with a territorial defence system, it expects the EU to add concrete value to the management of its multiple threats. Denmark and Sweden have been more reserved, and are still contemplating their positions for the post-Brexit era. Denmark's opt-out from EU defence cooperation limits its participation. Sweden is concerned about new supranational elements in security and defence and about the implications of procurement cooperation for the Swedish defence industry.
Despite changing policy on security and defence, Germany is not yet at ease with questions of military security in the EU context. Germany has thus focused on the new capability agenda and the EU's comprehensive approach, instead of promoting debate on the military needs for the Union's territorial security. Defence cooperation has also formed one of the core topics on the Franco-German agenda as the countries seek consensus over the EU's future. Germany is working with France in Permanent Structured Cooperation (PESCO), a mechanism enabled by the Lisbon Treaty for groups of Member States to launch joint projects in defence cooperation.
The Franco-German consensus does not, however, extend to the level of detail, and old dividing lines between the countries have delayed an agreement. France had wanted PESCO to become an instrument for EU operative efficiency that would provide capabilities for demanding military tasks. But Germany, loyal to its principles, wanted to safeguard EU unity by making the enhanced cooperation as inclusive as possible. A compromise has now been reached, and a framework should be ready for the December European Council meeting.
The renewed ambitions in security and defence are also meant to supersede some less-successful projects. The EU's Battle Groups reached operational capacity as early as 2007, but have not yet been used, reflecting a major divide among Member States over the Union's strategic role. Now though, ongoing efforts to increase their funding through a common budget might facilitate their use. Similarly, the European Defence Agency (EDA) was facing difficulties in obtaining funding to enhance common projects in research and technology within the European defence industry, but it now benefits from the European Defence Fund.
There is now every reason to hope that the EU will make a success of its new agenda on security and defence. The contemporary Union with its strengthened internal interdependencies and common external borders is much more vulnerable than in the early years of this millennium. And the world around it doesn't seem to be much more peaceful.


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Game design is the real heart of a videogame, the most creative, innovative and complex part of game development.
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text:
Military Vets Take Biden Administration to Court Over New Gun Restriction
Ivy League Lawyer Who Firebombed Cop Car Will Spend a Year and a Day in Prison
Dealers Report Surge in Gun Ownership Among Blacks
Survey shows that African Americans helped drive record gun sales in 2020
Stephen Gutowski
African Americans have been a major driver of 2020's record-setting growth in new gun ownership, according to an industry survey.
Gun dealers reported a 58.2 percent increase in black customers in 2020—the most rapid growth of any ethnic group, according to a survey from the National Shooting Sports Foundation (NSSF), which represents gun makers and dealers. More than eight million guns have been sold in 2020, according to background-check records, and many purchasers are first-time gun owners.
"It has been well known inside the industry that purchasers of guns and ammunition have long had diverse demographic backgrounds," the NSSF said in a statement on the survey. "The highest overall firearm sales increase comes from black men and women who show a 58.2 percent increase in purchases during the first six months of 2020 versus the same period last year."
The numbers indicate firearms ownership in the United States continues to diversify. The changing demographics of gun ownership could have a significant impact on the politics surrounding guns—especially with millions of Americans becoming new gun owners in the months that followed the coronavirus outbreak. The growth in gun ownership was not limited to black customers. The survey found white customers increased by 51.9 percent, Hispanic customers by 49.4 percent, and Asian customers by 42.9 percent.
"Throw out the stereotypes on American gun ownership," Larry Keane, spokesman and general counsel of NSSF, said in a statement. "Against the backdrop of historically high firearm sales, one major theme is shattering misconceptions that America's gun owners are 'old white men.' A surge in gun buyers across the country in 2020, more than 2.5 million since March alone, has boosted the diversity of the firearm-owning population."
The NSSF found that minorities made up a disproportionately high share of gun store customers in the first half of 2020. The survey found that white Americans accounted for 72.4 percent of gun buyers, despite making up 76.3 percent of the population, according to U.S. Census Bureau estimates.
The new NSSF survey comes after the National African American Gun Association saw a record-breaking surge in membership after George Floyd was killed by a white Minneapolis police officer in May. NSSF said the surge was not surprising, as the industry has tracked rising gun ownership among women and minority groups for years. A survey from the group conducted between 2008 and 2012 found that new shooters "are younger, female and more urban dwelling when compared to established target shooters, or those participating for more than five years."
Keane said the gun industry's attempts to appeal to new shooters over the past several years, combined with the desire by many Americans from all different backgrounds to protect themselves during uncertain times, have led to a more representative gun-owning population.
"Today's gun buyer looks more like the rest of America," Keane said in the statement. "They represent all walks of life and those buying firearms today increasingly are women, minorities, and more urban than in previous generations."
Published under: Black Lives Matter , George Floyd , Second Amendment

redpajama_set_name: RedPajamaCommonCrawl


text:
Filharmonie Brno to stage more concerts than ever before in its 66th season, with a fully-fledged repertoire prepared with no pandemic compromises.
NewsPress releases
PRESS RELEASE Brno, May 27th 2021
A year which has affected every single person in a way nobody could ever have previously imagined. A world which has been turned upside down in so many regards. At a time when we appear to spy light at the end of the tunnel, Filharmonie Brno is unveiling its 66th season. It carries with it the consequences of all of the above: the repeated, or more precisely postponed, first performances of certain projects and concerts, uncertainty regarding auditorium capacity, concern over whether worry or the desire for live music will prevail amongst our audience, and above all our great desire to meet up again. None of the restrictions, however, have impacted the planning of the season in terms of making compromises in regard to artistic and dramaturgy demands and intentions, combining the already played and the newly planned, or in cancelling projects under way. Thus, Filharmonie Brno is unveiling a fully-fledged and compromise-free programme for the next eleven months.
The orchestra will be staging more concerts than ever before during the upcoming season. "Based on feedback and response, we can see that the desire to listen to live music has gone up significantly after more than half a year without it. I must say this is true not just for our audience, but also for our musicians. That's why we have prepared more concerts and also launched a new chamber evening subscription," says Filharmonie Brno Managing Director, Marie Kučerová. Stars who have enjoyed extraordinary success amongst the audience are returning to Brno – including Elisabeth Leonskaja, Martina Janková, Maki Namekawa, Matt Haimovitz, Emanuele Arciuli and others… Artists co-operating with the orchestra for the first time will also be performing: world-renowned violinist Arabella Steinbacher, composer and conductor Joe Hisaishi who will be conducting his own work, Berliner Philharmoniker oboist Dominik Wollenweber, bassoonist Guilhaume Santana and legendary jazz pianist Brad Mehldau.
Brno Filharmonie Chief Conductor Dennis Russell Davies perceives the upcoming season with a particular focus on some important aspects. We as an orchestra with a tremendous organization behind us are dedicated to two important, even vital, responsibilities: Firstly, the preservation and expansion of our great classical music tradition, and secondly, to make the live performances of this music available to the widest possible audiences. These ideals have motivated and guided us during the past year as we have navigated through hard choices involving how to maintain the integrity of our work while keeping our audience, musicians, and staff as safe as possible," says Davies.
As a result, amongst other performances audiences will finally be able to enjoy Wagner's The Ring Without Words alongside Schubert's Unfinished Symphony, Kabeláč and Shostakovich's Fifth Symphonies, and a Michal Nejtek world premiere, a programme incorporating works by Respighi, Rautavaara and Mendelssohn and a concert originally planned for the sixtieth birthday of conductor Leoš Svárovský (Berlioz, Weber and Roussel).
As is tradition, a range of music proven over the centuries has been prepared alongside contemporary works. The orchestra will be returning to celebrations of the anniversary of Beethoven's birth, for example, honouring him through composers inspired by Beethoven: Liszt, Hindemith, Franck. Some of the classical composers we will be performing include Schumann, Brahms, Berlioz, Mahler and Mozart. The season will begin with a classic of national classical music: Smetana's Má vlast. More recent works will be represented by Sumera, Vasks, Jan Novák, Kančeli and Bernstein amongst others.
"As programme organiser, I have always enjoyed looking for connections not just within individual programmes, but also across the entire season – between concerts, series and festivals – and even between seasons. This is how two programmes communicate together for example, when always incorporating one Brahms piano concert combined with one of the early symphonies – the Second or Fourth – of Brahms' friend and philosophical kindred artist, Antonín Dvořák," says Filharmonie Brno Programme Director, Vítězslav Mikeš. He says that two Beethoven concerts recall each other in a similar way: "Honour Beethoven's" at Janáček Theatre, and the performance of the Violin Concerto by Beethoven himself at Besední dům a week earlier.
"I'm personally most looking forward to Elisabeth Leonskaja, Martina Janková and Brad Mehldau. Also to Lautching the Season with Má vlast and The Ring Without Words," says Kučerová. Davies usually doesn't like to say what he is most looking forward to, but this time he is clear. "To the audience. Definitely," says.
With the Czech premieres of pieces by Lithuanian composer Kutavičius, Latvian composer Vasks and Estonian composer Sumera, Filharmonie Brno is professing to its tradition of performing works by Baltic composers. Similarly, it is performing Jan Novák, an author closely linked to Brno for whom celebrations of the 100th anniversary of his birth will reach a climax at this year's Moravian Autumn festival. "Frequent visitors to our concerts are also sure to register that we are continuing in our performances of the works of Alfred Schnittke – by performing his Faust Cantata "Seid nüchten und wachet…" we are commemorating the fact that it was when playing this piece four years ago at Prague Spring that the orchestra became acquainted with its current Chief Conductor, Davies. In a nutshell: we are inviting you not just to savour the experience of our individual concerts, but also to experience the adventure of the discovery tour called the Brno Filharmonie 2021/22 Season," adds Mikeš.
Besides concerts, Brno Filharmonie will also be significantly building on its educational activities for children, schools, young professionals and adults. Its holiday workshops will be complemented by joint events for children and parents, programmes and concerts for schools and masterclass courses, as well as educational courses for adults. "Behind us is a year in which we met up with children five times a week in the on-line space, and so we are really looking forward to meeting up in person," says Filharmonie Brno's teacher for educational activities, Kristýna Drášilová.
The orchestra has also undertaken many international guest spots in prestigious halls in Austria, Germany and Poland. "We trust that we will be setting out on our big autumn tour of Great Britain, during which we will be performing the work of mainly Czech authors, Janáček, Dvořák, Martinů and Korngold, at nine concerts in nine cities," says Orchestra Manager, Pavel Šindelář.
The launch of subscription ticket sales begins on 7 June, both in person and online. Places for subscriptions not renewed will be released two weeks later, i.e. on 21 June, when music lovers can also purchase tickets for individual concerts and festivals, plus the popular Season of Your Choice ticket where subscribers are able to set up and choose their own portfolio of concerts to attend.
Media Contact Kateřina Konečná, Head of PR and Marketing, Filharmonie Brno
THE AUDITION for THE POSITION OF SECOND CLARINET WITH E FLAT CLARINET DUTY
Filharmonie Brno Will Perform In Leipzig To Celebrate 50 Years of Twinning Agreement
Filharmonie Brno is setting off on an American tour. They will play the New York premiere of Glass' Symphony No. 12, "Lodger" in Carnegie Hall, with the author personally participating
Benjamin Yusupov to celebrate his birthday milestone in Brno. With three concerts and five premieres
Online: Concerts
Sale in Besední Street (downstairs from the courtyard of the Besední dům). Opening hours: Mon – Fri 12 PM - 6 PM (except public holidays).
At the concert venue, it is no sooner than ¾ hours before the concert starts.


text:
Q: Appending a TR to Table Not Formatting Correctly My PHP script is sending back an array:
$currentStatus = ( isset( $status ) ) ? "1" : "0";
$html = "
" . $email . "	" . ( ( $status->type == 0 ) ? "View Only" : ( ( $status->type == 1 ) ? "View & Mark" : "View, Mark & Edit" ) ) . "	Invited
";

echo json_encode( array( $html, $currentStatus ) );

Then my jquery is appending this to the table:
...
success: function( result ) {

   var resultArray = eval( result );

   $( "#myTable tr:last").append( resultArray[0] );
   ...
}

Problem is, when it prints, it prints the following extra marks:
name@email.com<\/td>    View Only<\/td> Invited<\/td><\/tr>","1"]

If I only echo the HTML it appends to table just fine, but I cant do that because I need the $currentStatus back too to do something with it.
Thanks!

A: Allowing \/ helps when embedding JSON in a

redpajama_set_name: RedPajamaStackExchange


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There is increased patient compliance with nasal cannulae as patients can still speak, eat and drink.
The soft and ergonomically shaped nose piece of the UN810 nasal cannula reduces mucosa dehydration risk and the flexible, kink resistant tubing is an easy-to-use design.


It is important for any person who is thinking about beginning cricket or simply taking it up as their sports hobby to find out a bit more about the game before actually committing to the sport and playing it. To begin with, unless you are playing cricket in an extremely casual manner where you don't really play in the proper ground, you will need to get the right equipment for the job. Cricket is one of those sports where there is a pretty proper way of getting ready for the game. To play the game you will need a proper kit of shin and knee pads, a helmet, batting gloves, cricket sneakers, wickets, protective gear, a few cricket balls, and a cricket bat. All of these things can easily be found at a store that sells cricket equipment like Meulemans mega store in Joondalup. The game is considered to be the gentleman's game because of the elegance of the game, the discipline and grace of the players, and most importantly because of the sportsmanship and respect the game teaches you.
Now once you have the correct equipment and spirit of the game you must understand how it works. There are basically two teams of 11 who aim to make a bigger amount of 'runs' than the other team. The teams take turns batting and bowling to make and stop each other from making runs, and that is called an innings. You win the game by having more runs at the end of the match which is decided by a preset number of times bowling will happen or when the other team has no more batsman to score. It is a game that requires good hand eye coordination, quick reflexes, strength and accuracy when bowling and batting, and stamina to play the game.


Nerdy Alerty - Page 3 of 15 - Just a girl with a neon sign above her head blinking "Nerd Alert"
Has anyone ever tried bullet journaling? One of my friends wants to try it, and as I've been looking into it, it seems rather interesting. I decided to troll through Pinterest in an effort to find out more about it.
BOY! Did I discover a lot about Bullet Journaling!
Feel free to follow this board to see what ideas I come up with and/or find!
First thing I noticed is: it's basically an art form. Like seriously, some of the things people put into these journals are works of art! With pretty ribbons, and crazy amounts of organization! I'm really impressed with some things that I've seen. A lot of the organization of everything reminds me of what I've seen of Passion Planners which my friend Christa over at ObsessiveGirlFan swears by! So here's my question, if I combine the two, and make a Passion Planner part of my bullet journaling, do I level up in my organization skills?
My next step, after seeing a VERY general idea (*salutes* General Idea!) of how to accomplish bullet journaling was to FIND a journal. Here's the thing, I'm a bit picky sometimes when it comes to what I carry around with me on a daily basis. My Supernatural Purse was something I poured my heart into, I always have my phone, my iPad and a book in my purse, so if I was going to add some more stuff to it, I needed to figure out what I wanted added in my bad.
GUYS! LOOK AT HOW PRETTY IT IS!!!! It's so pretty and I want it!
With Bullet Journaling, it appears the best journals for it are ones that are unlined or just dots (most commonly in a Leuchtturm 1917 notebook). This Zelda one is unlined….. so that means I should get it right? Chances are for my first foray into bullet journaling, I'll most likely go for a very standard Leuchtturm 1917 notebook, just until I know what I'm looking for.
Have you ever tried bullet journaling? What has worked for you? What hasn't?
This is a new beginning for me (A New Beginning, Same Nerd… get it?), but I didn't want to forget where I started, so I brought all my posts with me! It took me far too long to come up with this title. I was torn for a really long time between this title or some lyrics from Jekyll and Hyde (the musical). This fit the theme a bit more.
Raise a glass to new beginnings!
Though I imported all of my posts from WordPress.com, this is my first official post on my brand new shiny site. I'm a little scared about taking this new step in my blogging universe, but I'm equally excited.
I love Hope Floats, don't you?!?
In January I talked about how I wanted to move to my own site, and after a couple months of researching and procrastinating (lets be real, it was mostly procrastinating), it's finally here! It's a bit minimal, but I love it just the same. Here you'll find the same things you found on my previous blog, but with a newfound sense of purpose and a little bit more heart!
First up, a recap of the Comic Swap I did with my Geek Girls x Bloggers group!
I'm going to attempt to keep up with more of the weekly features I used to do (Thursday and Thrilled, Funko Friday and 5FF), while also trying to schedule some time to do some more monthly things. I will be returning to my Rewind articles again, I loved doing those. I also started a list of all the Funkos I own… not for any particular reason, I just wanted to.
But for now, I'm just making sure everything is in it's proper spot and functions well. If you have any advice for a newbie in hosting, please send them my way! I'll take all I can get.
What are some of the things you learned when switch to your very own hosted site?


It's 2AM and as Julie's 18th birthday party winds down, her closest relationships are tested until they are completely changed.
It's the story of Julie, presented by Foul Play written by Holly Brindley who adapted Strindberg's 1888 play Miss Julie.
She spoke with Tara Nash about it.
29 September – 14 October at Adelaide Botanic Gardens.


PhilosophyENT.com | Philosophy Entertainment, LLC is an independent rap/hip-hop label. That specialize in music releases, videos, artist signing and artist development. Also distribution, book publishing, movie treatments and promotion merchandise.
Philosophy Entertainment focus predominantly on rap / hip – hop music distribution as a label. Further executions are signing and developing acts, book releases and promotional merchandise.
On October 22, 1982 Bill and Naomi Perkins, who later divorced, gave birth to a son named Derek Perkins in a small college town called Charlottesville, Virginia. Derek has a younger sister, as well as had an older brother who died in 1993 at the age of 19 from a mysterious Four Wheel ATV accident.
Derek managed to graduate from Monticello High School in Charlottesville in 2001. During the years there, he suffered lots of verbal abuse from the teaching staff in front of and behind his back as well as verbal and physical confrontation with fellow classmates. This led to in-and –out of school suspensions.
To escape the madness, Derek would unwind in his music collection, which later became his curriculum and inspiration to want to express himself writing down his feelings about what he was going through. Prior to many failed attempts on creating a writing structure, a day before his 17th birthday it all came together on paper. The start of honing his craft. He started out writing poems in his own style that he later named "poetic memoirs" and later converted to writing raps that he studied and learned how to do. Always being a fan of rap/hip-hop, he made it his personal mission to study the street art form and it's history.
Wanting to have the skill set of a typical New York City M.C. lyricist, Derek felt the need to come up with a distinct identity that captured who he was and is. C-zar Wisemen is what he researched and created. Later in April 3, 2015 he went to court to legally have his name changed from stage name to his daily life name. The name stands for ruler of wisdom wit personalities of 7. Each letter in the last name "Wisemen" describe a personality trait that the artist possesses. W stands for warrior, I stands for intelligent, S stands for smooth, E stands for educated, M stands for mean, E stands for emperor and N stands for notorious. The second E for emperor relates back to the original meaning for his first name "Czar" with or without the hyphen. The letter G is the seventh letter in the alphabet, which is the first letter of the spelling of the word of "God", which C-zar feels he is on a daily basis feeling liberated and completed. His first name is taken from the Russian noun. The last name is biblical. To help further his identity, he has his hair in dreadlocks, showing that it is his crown. His personal logo is a lion, which was designed specifically for him by his father.
While jumping from one job to the next , being in and out of the local jail in Charlottesville ( due to his temper ), along with few demos made he managed to complete his first album after networking with local rappers and producers. The LP is called "Tha New Testament". It was released on July 2005. The album is under his label, "Philosophy Entertainment, LLC". Ten months earlier, C-zar released the first single off the album entitled " I Hold My Own". The album also featured his group Tha Philosophers, which featured himself, Jaco Sage King and Polaris.
C-zar always had a passion for acting, first doing some drama in high school, then doing one day of extra work on the set of "Evan Almighty" then joining an acting class which was taught by David Webster of Webster Studios in Charlottesville, VA. Training in improv led to his performance in two short films and one indie feature. The first is a comedy short in 2007 called "Super Dog" directed by David Webster. The second in the same year is called "Wetwork" a short action film directed by Jason Shifflett and indie feature called " Ever Seen The Rain?" also in 2007 directed by David Webster that was never completed, roles acted all under his given name.
C-zar self – published his first poetry book entitled "Poetic Philosophy" under his given name released on September 2007. C-zar wanted to present Tha Philosophers in a sole project. In November 2008, narrowed down to a duo C-zar and Polaris teamed up again and released a single called "Abortion". The single was supposed to have been off an indie dark comedy film about abortion that was never made.
Now living in Los Angeles,CA C-zar completed his second LP while being in the city of angles called "Real Niggaz" which was released in November 2012. The first single off it was " I'm Down 4 Whatever" featuring Renate' Page. Originally released on May 2007 for the album, but re-released again 2012. In 2015 C-zar released another single called "Everywhere I Go" , which was taken off the track list for the forthcoming third LP but will not appear on it. Ten year's after the release of the poetry book "Poetic Philosophy" C-zar released the sequel "Revolutionary" in March 2018 through "Philosophy Entertainment" in his new legal name as the author. That same month C-zar released his EP titled "Revolution" featuring the single "50 States", along with another current single "Heat" on July, 2018 from the forthcoming third studio album "White Amerikkka'z Nightmare" out now. C-zar Wisemen's main goal is make an incredible impact in rap/hip-hop music and help along with other's within his race to make a revolution for black people forever.
Poetic Philosophy is a collection of poetry that is based on feelings and things that I have gone through as a hip hop artist, a businessman, up and coming actor, and poet. As well as tackling issues that are going on in this country and around the world that needs a change for the better.


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Republicans are obfuscating the debate by offering a false choice between gender equality and the public financing of elections, when really the two issues are deeply linked.
Needless to say, public financing of elections is not about drafting citizens to endorse particular politicians. Rather, devoting tax dollars to funding campaigns is a smart and urgent way to dilute the influence of big money, which warps our politics and makes citizens bystanders as the public coffers are drained to reward private donors. But there's a larger lesson here.
As we've seen since Cuomo reached the Governor's Mansion, there's a big difference between what the governor backs on paper, and what he's willing to put his considerable political muscle behind (more often than not, it's "social issues" where Cuomo chooses to champion progressivism). Perhaps the governor believed all along (incorrectly) that he only had the power to pass one of these landmark pieces of legislation, and chose to put them both out there, thereby creating competition between the movements backing each. But we need campaign finance reform, and we need a Women's Equality Act that takes on domestic violence, sexual harassment, workplace discrimination, pay inequity, and attacks on reproductive choice.
This situation offers a challenge to progressives, who too often allow themselves to be placed into single-issue silos, and thus pitted against each other. We should know by now that regressive policies usually hit women the hardest, and single moms are often the most acutely affected of all. When we treat "women's issues" as distinct from clean elections, or labor, or foreign policy, we endanger women and hold back progress.
Defeat or delay for clean elections means fewer women in office. There is a ton of data that women candidates win at the same rates as men. The problem is, they don't run at the same rates. And the #1 issue given by prospective women candidates who consider but then do not run is that they don't think they can raise the necessary money. New York's public financing proposal will, in fact, enable enormous numbers of women to run.
Just look at Arkansas, where last year the Democratic Party lost the state house for the first time since Reconstruction. Culprits include Koch brothers cash. Once elected, Republicans quickly passed a twelve-week abortion ban, a frontal assault on the lives and autonomy of women across the state. That blatantly unconstitutional law will also have to be defended in court. In austerity-era Arkansas, that will no doubt sap needed resources from already-hamstrung programs that women and children count on. For progressives, there may be single-issue campaigns, but there are rarely just single-issue defeats.
There is an alternative. Governor Cuomo has the chance to mount a real fight for a bolder agenda now. That includes centralizing women's equality in his agenda, and pairing it with public financing, because they go hand in hand if we care about economic justice and social progress. In the meantime, it falls to the rest of us to build deeper ties, and a less compromised and compromising political movement—demanding a government that respects and reflects women's dignity and autonomy at the workplace, the doctor's office, and the ballot box. As history has taught us, an injury to one is an injury to all.
Governor Cuomo wants to get rid of that pesky Working Families Party, Katrina vanden Heuvel writes.


"Free Press" an Urban Myth? - Fake News or Real News.
"Free Press" an Urban Myth?
Posted bymihirs	 June 16, 2018 August 23, 2018 Leave a comment on "Free Press" an Urban Myth?
Is the term 'Free Press' a Reality in modern times?
A body of book publishers, news media, etc., not controlled or restricted by government censorship in political or ideological matters.
If a country has a free press, its newspapers, magazines, and television and radio stations are able to express any opinions they want. Even if these criticize the government and other organizations.
So now we know how the world defines 'Free Press'.
Fake news is a type of yellow journalism or propaganda that consists of deliberate misinformation or hoaxes. Spread via traditional print and broadcast news media or online social media.
False stories that appear to be news. Spread on the internet or using other media. Usually created to influence political views or as a joke.
Now we also know what is fake news and function of fake news.
Can the free press be the source of fake news?
Is the free press a premier participant of the Fake News?
You must have often heard of the terms "Russian Propaganda" and the "Communist Propaganda". That is if You are from one of the European or North American countries.
These terms have been seeded into your brains and have been defined and justified by the western media.
You must wonder, how come the moment any country tries to defend itself from the pressure tactics. And bullying from western countries. You start hearing about how the Government of that country is spreading propaganda against the western democratic governments. Often when a country is governed by people the westerners are not confident in. The western media demonizes that government. And the country due to that government is a threat world peace.
The reality is far from the truth.
Well… if you go to the country being demonized by the western media, you will realize how the Western media is the prime example of a Propagating fake news and hatred towards any institution that defies the authority of the western countries.
To give you a context of what I am saying lets follow the example of what happened in the World War II, the cold war, Iraq war and the Syrian War.
America and United Kingdoms are the only True Heroes.
After the world war II, we saw how the role of Russia in enabling the fall of Nazi Germany was downplayed by the western media and in-fact the utter destruction and killing of civilians at the hands of United States and United Kingdom being virtually erased with the help of war-time western propaganda.
During the cold war, despite of the fact that the United States was literally bullying every other country in middle east for Oil and the European countries for technology. And to do so USA had been setting up military and nuclear bases in the Pacific and in Europe. But the moment Russia tries to set up a base near United State. The Western media starts to blame Russia for aggravating the situation and creating war type environment.
United States, to fulfill its goal of stealing oil from the middle eastern countries. It has illegally invaded Iraq. Under the pretense of disarming the nuclear sites in Iraq. And freeing the Iraqi people from Saddam. During these illegal invasion, the Western Media started its propaganda. Being in its full swing to ensure that United States and its allies do not face any flack over it.
Invasion of Iraq was the first piece of the domino effect that has led to what today is a destabilized middle east and death of millions of civilians and soldiers.
American Gun Manufacturers, manufacture guns and wars.
America has often invaded other countries and several times brought violence to the region of its interest.
Al Qaeda, the blood child of the United States.
Even when the fact is that United States armed and gave birth to Al-Qaeda during illegal Russian occupation of Afghanistan. the Western media has not hesitated in distancing the western countries from these facts.
So whom should we trust for news?
Pic sourced from the internet.
Not all is lost when it comes to the news. The above examples were just to prove how mainstream media, "Free Press", even in a Democratic country is controlled by its rich owners and the government. The state of media in Russia and China is far more worse. In-fact in China every piece of media is controlled, manipulated and distorted by its government. At-least in comparison there are several media outlets in Europe that try their best to stick to the facts.
Verifying and confirming the news.
It is our duty to verify and confirm whether the news we are getting is true or no. Ensuring whether the news we receive is being told from every perspective is our duty too.
Acting up-on the one sided news can be harmful to your understanding of the world.
Calling the current institutions of media the free press in any country will be irresponsible.
I'd say that the free press, i.e. news outlets today are being swayed by the politics and the mood of the country. Instead of being free of all the binding nature of a country's emotions, political and global diplomatic stance. Today's main stream media horribly fails at showcasing the position and perspective of both the favored and opposed point of view.
To conclude, with a heavy heart, I confirm that the Free Press is just an urban myth. Although there are many flag bearers who are trying to portray the facts and stories from all the perspective. Be it via a documentary or a blog. They are shouting at the face of the main stream media. No matter what you can not suppress the truth.


Emerson is a Top Ten Winner in our summer 2017 national art contest for her artwork "Emerson's Still Life".
Hello, my name is Emerson Moore and I'm going to talk a little bit about myself. In the future I would like to be a singer, an artist, and an author and illustrator. I also want to be an art teacher. Art really runs in my family, my sister and my dad are artists. My great grandma was an artist too. I got inspired by them. Art has always been a part of me ever since I was born. I am proud that my chalk pastel, "Emerson's Still Life", is a top ten winner.
Anastasiya-Stefaniya is a Top Ten Winner in our summer 2017 national art contest for her piece "Lake View".
My name is Anastasiya-Stefaniya Vyazmina. I am a 9 years old and live in St Albert, Alberta, Canada. I was born in Ukraine and moved to Canada 4 years ago. I have been taking art classes for two years at Alberta Arts academy, Edmonton with amazing teacher Svetlana Kanyo.
The true inspiration behind my painting "Lake View " was my family trip to Jasper National park.
My 3rd year in art school I want to express myself developing drawing techniques and trying in oil painting. I hope I can get into the top ten again next year.
Oishee is a Top Ten Winner in our summer 2017 national art contest for her artwork "House in the Woods".
I'm Oishee and I'm currently a freshman at Monta Vista High School. I love art and I go to Mr. Armando's every week usually on Saturdays or Fridays. I love trying out new mediums and styles of art.
I was inspired to create "House in the Woods" because it was a beautiful landscape that I really wanted to attempt to capture. It was my second oil painting, and I wanted to try out everything new I had learned from my first one into this piece.
My goals for the future are to just continue doing art because it makes me happy and helps me express myself without the constraint of words.
Inspiration: These old decrepit tracks are one of my favourite places for an afternoon walk. Many of the boards are broken, some nearly falling apart, and have plants growing in the cracks. The spikes are rusted, bent and show what years of exposure to the elements can do. It is nature taking itself back. Perhaps they are plain looking to some. However, in the fall the maples that line the sides completely transform the railway. They burst into a brilliant assortment of colours, rich reds, golden yellows and bright oranges that are not fully represented in the picture, as fall was just beginning. I hoped to show train tracks as this, as a place of colour, beauty and history, as opposed to lifeless grey veins branching through the land.
Goals: In the future, I hope to continue to create and evolve my skills. I hope to pursue a career that allows me to use these skills as often as possible.
Information: I am a 13 year old who enjoys art in any form but specifically pottery and painting. My other passions lie in music, particularly piano and guitar. I also practice photography. These tracks are one of my most frequent subjects.
Sophie Fu is currently an art teacher affiliated to Hua Xia Chinese School, NJ. She has extensive experience in art education. After graduating from the School of Fine Arts at Nanjing Normal University in China, Mrs. Fu has been in art education field ever since.
Teacher Fu's students range from age 5 to 18. In teacher Fu's eye, each child is unique. Teacher Fu is a reputable art teacher who can help kids finding their own inner talent and passion in art. She inspires each kid based on their own personality and strength. A few of her past students, who are in art major and fashion design major in college now, can not say enough "thank you" for teacher Fu's inspiration, encouragement and confidence in them.
According to teacher Fu, child art education is not only teaching different painting skills and techniques, but also a means of intellectual development. Cultivating an interest in art may facilitate children to develop and to enhance their imagination, creativity, aesthetic ability and practical skills. Below are selected art pieces of teacher Fu's current and past students.
Teacher Fu's Studio located in Manalapan, Plainsboro and West Windsor, New Jersey. She can be reach at sophiaws63@gmail.com or 908-692-1915 or wechat scan code below.
"Gilded Peacock", Angela Espino, Top Ten Winner in summer 2017 national art contest.
My name is Elizabeth M. Ibarra. I am a professional artist who has had the privilege of working as an ASES Art Teacher for 5 successful school years at Desert Garden Elementary School. It is an honor to be a Celebrating Art Top Ten Art Teacher. The recognition each of my published art students has received is a source of pride and excitement among my students, their parents and colleagues alike. Having a Top Ten winner published in the Summer 2017 anthology is validation of the young talent in my classes and of the effort they put into crafting their remarkable works of art.
There are many factors which contribute to success in the classroom; not the least of which is the willingness of my students to actively participate in their own development as young artists. My teaching style is influenced by having been homeschooled and later attending a prestigious art school for a year. My own art education was very self-motivated. I had the support of my family; especially my parents as I embarked on my artistic journey. They signed me up for classes with professional artists during my elementary and high school years. These experiences have now come full circle as I am a professional artist, motivating my students to take their artistic abilities seriously. I feel that family participation and recognition of the work they create is vital to their early success. I submit student works in our annual district art festival, local, regional and national contests with Celebrating Art as a focus. The process of student participation in Celebrating Art directly involves their families as I request permission to enter art created by their children. The familial joy and enthusiasm involved in these contests is a motivation for my students to consider themselves artists and to take pride in crafting their works of art.
Another factor of success I have had in the classroom is working with the abilities of each student, which are as varied as their personalities. I create lesson plans which cover a lot of artistic styles and media, giving support to the needs of each of my students. Creating impressions of famous artwork and discussing art history is always engaging and fun for my students. We spend time in-between elaborate projects practicing drawing skills with nature photographs. Developing hand-eye coordination is greatly important as it gives students more confidence while approaching complex projects. In class recognition of their hard work and success is another great motivating tool. It is my goal to boost student enthusiasm for their own works and those of their classmates.
Remember that we as artists and art teachers are helping keep the arts alive in our schools. Art education is important. Best of luck to you in your art teaching endeavors.
My piece was inspired by our family vacation at Cape May, NJ. My family visits Cape May, NJ every summer. We stay on the beach front hotel and relax. We love to watch the sunrise and sunset. Sailing boat in Sunset is one of the scenes in my memory about Cape May, NJ.
When I grow up, I would like to be a fashion designer. I am proud of my art piece and I am passionate in art.


text:
Woman convicted of money laundering
A 50-year-old woman was convicted of money laundering offence at the District Court today (September 4). The case was adjourned to September 18 for sentence.
The court heard that between August 2002 and March 2004, a company had inflated its sales revenue by $300 million by false overseas sales invoices in order to be listed on the Main Board. The defendant was the then accounting manager of the company when a circulation of fund amounting to $68 million was channelled via her personal bank account to generate false sales.
She was convicted of dealing with property known or believed to represent proceeds of an indictable offence and the case was adjourned to September 18 for sentence.
Police appeal to the public to refrain from lending their accounts for any illegal purposes, thus contravening "Dealing with property known or believed to represent proceeds of an indictable offence" under the Organized and Serious Crimes Ordinance (section 25 of Cap 455).
Police Report No. 306