1) (Theiling et al., 2000). However, within this reach, the area upstream of Lock and Dam 6 has experienced exceptional island growth, beginning in the 1960s (Fremling et al., 1973). Improving the hydrologic and sediment regime, floodplain function, ecological functions, and current river management practices are often described as the desired outcomes of restoration (Ward

et al., 2001, Buijse et al., 2002 and Palmer et al., 2005). However, the scale and costs of restoration can combine to make large river restorations contentious and controversial (Ward et al., 2001 and Palmer et al., 2005). On the UMRS, restoration and habitat enhancement efforts have been undertaken by the US Army Corps of Engineers (USACE). These projects have received over $241 million in federal funding since 1985 (USACE, B-Raf inhibitor drug 2010). Since 1986, 54 projects have been completed BKM120 in UMRS Pools 1–10, including dredging backwaters to enhance aquatic habitat, bank and island stabilization to limit future erosion, and periodic drawdowns to permit seed germination. More than 30 islands have been created in Pools 5, 5A, 7, 8, and 9 (http://www.mvr.usace.army.mil/Missions/EnvironmentalProtectionandRestoration/UpperMississippiRiverRestoration.aspx).

The goal of the project was to identify factors that make sites geomorphically favorable for island restoration in the UMRS or other large, engineered rivers with shallow pooled areas. To this end, we quantified and evaluated effects of river management on island growth, persistence, and loss in Pool 6 of the UMRS, and contrasted the setting of Pool 6 to other parts of the UMRS. Pool 6 of the UMR spans 22.5 km (river miles 714–728) between Lock and Dam 5a in Winona, Minnesota and Lock and

Dam 6 at Trempealeau, Wisconsin (Fig. 1). Pool 6 of the UMRS drains approximately 153,327 km2 at US Geological Survey (USGS) gage 05378500 at Winona. The islands and surrounding aquatic environments within Pool 6 are part of the U.S. Fish and Wildlife Service’s Upper Mississippi River Fish and Wildlife Refuge and Trempealeau National Wildlife Refuge. Pool 6 is located in the Driftless Area, a region that remained unglaciated for much of the Pleistocene. The UMR functioned as a principal southern drainage for glacial meltwater and sediments. Bluffs, 180 m high, flank the river and its floodplain, constricting the width Dichloromethane dehalogenase of the UMRS’s floodplain in places and reducing the channel’s ability to migrate (Knox, 2008). Following European settlement in the mid 1800s, conversion of forests to intensive agriculture resulted in dramatic hillslope erosion, sediment fluxes, and floodplain sedimentation, which declined only with the onset of erosion control practices in the 1930s (Knox, 1977, Knox, 1987, Knox, 2001, Trimble, 1983 and Trimble, 1999). Most of the sediments transported to Pool 6 are quartz sands from the Chippewa River, which enters the Mississippi River ∼39 km upstream (Rose, 1992).

Some researchers assume that inter-fluvial forests were not occupied extensively and thus not altered by people (Bush and Silman, 2007, Denevan, 1996, McMichael et al., 2012 and Steege et al., 2013). But many of the documented cultural forests are indeed in interfluves away from the mainstream (Balee, 1989, Balee, 2013, Balick, 1984, Goulding and Smith, 2007, Levis et al., 2012, Politis, 2007 and Smith http://www.selleckchem.com/products/PF-2341066.html et al., 2007). My surveys along the Curua River in the middle Xingu interfluves also encountered anthropic forests at current

and former villages and at archeological sites (Fig. 13) (Roosevelt et al., 2009:465–466). Many researchers depict oligarchic forests as “uninhabited” (Pitman et al., 2001 and Steege et al.,

2013) and assume they are a natural phenomenon, without conducting research to exclude a human influence, however. Amazonian forests in different regions differ significantly from one another in topography, climate, geology, hydrology, structure, seasonality, and history, but, nonetheless, they often resemble each other in having this pattern of unexpected dominance and density of a small group of plant species. This pattern has been found wherever Amazon Selleck MDV3100 forests have been inventoried and has yet to be explained by natural factors. The diverse regional and local forests of Amazonia are in essence united by these dominants, most of which have an association with humans. The so-called oligarchs (from Greek for “rule by few”) in the Amazon forests are a group of more than 200 predominant species that make up only 1.4% of all the Amazon forest species but almost half of the trees in any given forest

(Steege et al., 2013). Traditionally, Amazonian tropical forests are considered to be taxonomically very diverse floras in which individuals of most species are locally rare and widely separated from one another, limiting the intensity of exploitation possible in any one place (Longman and Jenik, 1987:115–123; Junk et al., 2010, Pires, 1984, Whitmore and Prance, 1987 and Whitmore, 2010:149–152). Therefore, where a small group of species are significantly more common than the others, in contrast to this pattern, and no natural reason has been suggested, these groupings may not be Interleukin-3 receptor a solely natural product but a partly human one. Researchers recognize that trees and shrubs are much affected by numerous faunal species, so it’s hardly a reach to consider human effects. The dominant tree species tend to be ones valued and actively managed by Amazonian people today, or ones that benefit from the effects of human occupation. People influence them variously: planting them, concentrating or dispersing their propagules, clearing around them, protecting them, attracting or eliminating their animal predators, and/or fertilizing them with their refuse.

Funding for our research has been provided by our home institutions and grants from the National Science Foundation, National Geographic Society, Wenner Gren Foundation, and other sources. We thank the editors, Todd Braje, and two anonymous reviewers for help in CX-5461 supplier the review and production of this manuscript. “
“The Northwest China Upper Paleolithic site of Shuidonggou, and related sites in Ukraine, the

Central Russian Plain, Mongolia, Siberia, and Korea confirm that after about 40,000 cal BP technologically sophisticated and socially well-organized hunting-gathering populations of anatomically modern humans were widely present across northeastern Eurasia (Milisauskas, 2011 and Morgan et al., 2014). find protocol Extensive biological, geological, and archeological research shows that warming climate and rising sea levels in final Pleistocene and early Holocene times greatly increased the biodiversity and productivity of natural landscapes throughout East Asia, and substantial pollen records from Japan document a gradual northward spread of broadleaf oak and beech woodlands from southerly Pleistocene refugia between about 20,000 and 8500 cal BP (Aikens and Akazawa, 1996, Aikens and Higuchi, 1982 and Tsukada

et al., 1986). The return of a rich mid-latitude biota fed growing human population densities. All animals affect the environments they occupy, but humans are uniquely creative both intellectually and technologically. To a much greater degree than other animals,

humans are able to create and modify their own ecological niche because their large brains support an ability to learn quickly, anticipate the future, and share detailed knowledge and experience through highly specific linguistic communication. Their long legs and sturdy feet Thymidine kinase help them travel efficiently and routinely over long distances in the course of earning their living, and their deft hands and binocular vision enable them to create highly detailed and refined objects using a variety of tools. Humans also are omnivorous and able to thrive in a broad range of environmental settings. As humans became ever more numerous in East Asia during the final Pleistocene and Holocene, the landscapes they occupied took on an increasingly “anthropogenic” character. Natural scientists seeking to define a new human-centered epoch of earth history suggest that human effects on the climates and environments of earth are now so powerful and pervasive as to warrant the recognition of a new “Anthropocene” epoch of earth history. As recently proposed by Foley et al. (2014), the anthropogenic developments treated in this paper might well be seen as belonging to a “Paleoanthropocene” prelude – belonging to an interval when the human capabilities and actions that are now becoming decisive factors in planet Earth’s climatic and geological history were just beginning to ramp up.

We used adult individuals of L. terrestris obtained from two commercial suppliers (R. Pechmann, Langenzersdorf, Austria; Denu’s Würmer Stuttgart, Germany), with a mean initial biomass of 3684 ± 365 mg. Adult and semi-adult individuals of A. caliginosa, with a mean initial biomass of 705 ± 54 mg were collected by hand-sorting from a garden soil south of Vienna, Austria in March 2008. After four days in the labelled soil, earthworms were transferred into new boxes containing 200 g unlabelled and sterilized moist soil. Boxes were again stored in the

dark at 15 °C and re-randomized daily. On days 1, 3, 7, 14 and 21 after transferring the earthworms into unlabelled soil, a pooled sample Compound Library of casts (a small portion of all casts present in a box taken with a laboratory scoop’s point) and one earthworm were collected from each replicate and analysed (see below). Since we planned to use labelled

find protocol casts of L. terrestris for a subsequent experiment, we wanted to test how the isotopic enrichment would be affected by storage. Therefore, after the last worm was taken out of the boxes on day 21 of the above described sampling period, labelled L. terrestris casts from treatment “once + incub” were stored in two different ways. First, three boxes containing the labelled casts were stored in the dark at 15 °C in a conditioning cabinet with no additional moisture being added throughout the storage period. Second, six cast samples from each box were packed separately in plastic tissue capsules with grid openings on each side (volume ca. 3 ml;

Histosette I, Simport, Beloeil, QC, Canada) and buried at a depth of 30 cm in a pot filled with field soil (volume 40 l) in a greenhouse (mean temperature during storing period: 14.5 ± 3.1 °C). A pooled cast sample of each box and a plastic tissue capsule corresponding to each box were taken every two weeks over Inositol oxygenase a period of 105 days and prepared for analyses. The earthworm cast samples were dried at 60° for 24 h and homogenized with a ball mill. The earthworms taken from the boxes were rinsed individually with water, dried on tissue paper, weighed and deep-frozen (−20 °C). Later on they were dissected and cleaned of internal organs including intestines by rinsing with a fine stream of distilled water. Only the anterior 15 segments of the frozen earthworms were used to avoid contamination from intestinal contents. Earthworm tissue was dried for 24 h at 60 °C and pulverized manually using a mortar and pestle. Earthworm casts and earthworm tissues were analysed for 13C and 15N by continuous flow isotope ratio mass spectrometry (CF-IRMS). For calculations, isotopic enrichment was expressed in atom % excess (APE), where APE is the difference in atom % between the sample and the natural abundance level of 13C and 15N in the worm tissue or casts from control treatments (L. terrestris: tissue 1.080 ± 0.002 at.% 13C, 0.369 ± 0.0004 at.% 15N, casts 1.096 ± 0.001 at.% 13C, 0.379 ± 0.006 at.% 15N; A.

Cada endoscópio deve possuir um código

único de identificação, e deve ser implementado um sistema específico para endoscópios vindos do exterior. O sistema de rastreabilidade deve ser avaliado regularmente (pelo menos uma vez por ano) para se assegurar da sua efetividade. O material (escovas, escovilhões, etc.) utilizado para a limpeza deve ser preferencialmente de uso único. Caso contrário, deve ser descontaminado após cada utilização de acordo com as indicações do fabricante. Cat IC e Cat II 1, 6, 8 and 9 A limpeza ultrassónica dos acessórios endoscópicos reutilizáveis e componentes dos endoscópios, com uma frequência superior a 30 kHz, deve ser utilizada para remover sujidade e material orgânico de áreas de difícil limpeza de acordo com as indicações dos fabricantes. Cat II 1 and 14 As pinças de biopsia e outros acessórios que têm a Quizartinib in vivo indicação para uso único devem ser descartados após a utilização. As pinças de biopsia e outros acessórios reutilizáveis que violam a barreira mucosa C59 wnt supplier devem ser submetidos a uma limpeza mecânica com detergente enzimático e esterilizados (a desinfeção de nível elevado não é suficiente). Cat. IA 1, 5, 10, 11, 12, 14 and 20 Os frascos de água e os tubos conectores,

devem ser esterilizados ou submetidos a desinfeção de nível elevado de acordo com as indicações do fabricante. Os frascos de água devem ser esterilizados após cada sessão de endoscopia. A água utilizada nos frascos deve ser estéril. Cat. IB1, 8, 9, 10, 11 and 21 Deve existir um registo da manutenção preventiva e das reparações dos endoscópios de acordo com as instruções

do fabricante. Deve existir um registo da manutenção preventiva e reparações do RAE de acordo com as instruções do fabricante. O RAE deve ter um plano de manutenção. Deve existir um registo específico do plano de manutenção e desinfeção de qualquer sistema de purificação de água do RAE. Deve existir um registo da manutenção preventiva e reparações da tina ultrassónica de acordo com as instruções do fabricante. Deve existir um registo de higienização periódica do sistema do RAE. Deve existir um registo da manutenção preventiva e reparações dos armários de armazenamento de Sitaxentan acordo com as instruções do fabricante. Deve existir evidência de que o RAE foi validado na instalação de acordo com as normas internacionais aplicáveis. O RAE deve ser revalidado se houver introdução de um desinfetante novo. Um profissional deve ser responsável pelos controlos diários, semanais, trimestrais e anuais de acordo com as normas europeias. Deve existir um profissional responsável pela análise regular dos resultados obtidos e a implementação de ações de melhoria quando indicado. Deve existir um plano de intervenção que define as medidas corretivas.

De novo sequence mutations and CNVs appear to be independent risk factors – ASD subjects carrying large, gene-rich CNVs presumed or documented to affect risk, have a lower de novo rate than ASD subjects without

them [ 80]. All three studies support an earlier estimate, based on the distribution of de novo CNVs [ 20•• and 38•], that hundreds of genes are involved in the ASD phenotype, possibly more [ 80, 81 and 82]. Moreover, the de novo events, about one per exome even in control subjects, highlight many new ASD candidate genes, especially when mutations recur in brain-expressed genes. While case–control analyses have proven more challenging, we believe that with larger samples, auxiliary data (e.g., concerning CNVs, de novo events, recessive and compound heterozygous inheritance, protein–protein interactions) and new analytical techniques, these http://www.selleckchem.com/products/z-vad-fmk.html exomes will yield evidence of ASD risk genes. Indeed we anticipate that thousands of ASD subjects’ genomes will be sequenced two years hence, and more than 100 novel ASD genes identified (Autism Sequencing Consortium,

unpublished). Some will fall in (or near) CNV regions like 16p11.2 and 1q21.1 ( Table 1), which so far have resisted identification of specific ASD genes. A challenge for the future will be to relate genetic variation and ASD genes to relevant clinical phenotypes. Evidence is tenuous for individual common variants that affect risk of ASD. check details Three large, independent

genome-wide association studies (GWAS) have been reported thus far (Table 2). Two assayed half-million single nucleotide polymorphisms (SNPs) each and reported significant association at two different loci: 5p14.1 [83] and 5p15.2 [84]. Subsequently, by assaying one million SNPs, Anney et al. [ 85] identified a single, significant association: for rs4141463 at 20p12.1. None of these studies was based on large sample size ( Table 2) relative to most GWAS, and perhaps for that reason their results are not complementary: results in Anney et al. [ 85] do Rapamycin supplier not support the earlier associations. Further, a newly published study targeting rs4141463 found no support for its association with ASD [ 86]. An unpublished follow-up study by the Autism Genome Project (Anney et al., unpublished) reporting on 2705 families, found no single SNP significantly associated with ASD. Yet, by deriving an allele-score [ 87] from their Stage 1 data, and showing it predicts pattern in their independent Stage 2 data, they do find evidence that common variants, en masse, affect risk. These findings comport with earlier analyses of results in Devlin et al. [ 88], which predict that few if any common variants have a substantial impact on risk (odds ratio >1.2), but many common variants could have a more modest impact.

In contrast, 5 patients with mutant cfDNA had no corresponding mutations in matched tumor tissue. This phenomenon has also been reported and could

be explained by tumor heterogeneity: these biopsied tumor tissue samples may not carry the EGFR mutations detected in blood, because these mutations come from different parts of the tumor [25], [26] and [27]. However, 4 of these 5 patients received EGFR-TKIs and had a comparable PFS with those who exhibited Galunisertib clinical trial wild type in both blood and tumor tissue, suggesting that these mutations detected in blood could be false positive results. There have been a limited number of studies on the correlation between EGFR mutation status in cfDNA and efficacy of EGFR-TKIs [28], [29], [30], [31] and [32]. Though the researchers tend to agree that EGFR activating mutations in cfDNA may be predictive of better response to EGFR-TKIs, they are still uncertain whether EGFR mutation status in cfDNA can predict survival benefit from EGFR-TKIs. In a subgroup analysis of IPASS, ORR was 75.0% (18/24) and 27.1% (19/70) with gefitinib in patients with or without EGFR mutant cfDNA, respectively.

PFS was significantly longer with see more gefitinib than carboplatin/paclitaxel in the cfDNA mutant subgroup (hazard ratio [HR], 0.29; 95% CI, 0.14-0.60; P < 0.001) but not in the cfDNA wild-type subgroup (HR, 0.88; 95% CI, 0.61-1.28; P = 0.50) [22]. Xu et al. reported that an significant correlation between EGFR mutations status in plasma and tumor response to gefitinib was observed using ARMS but not denaturing high-performance liquid chromatography (DHPLC), whereas no association between EGFR mutation status

in plasma and PFS or overall survival (OS) was observed no matter using ARMS or DHPLC [33]. Bai et al. detected EGFR mutations in plasma using DHPLC and found that about 62.2% of patients with EGFR mutations responded to gefitinib, whereas 37.8% of patients with wild-type EGFR also responded. They noted that patients with EGFR mutant cfDNA had a significantly aminophylline longer PFS than those with wild-type cfDNA (11.1 months versus 5.9 months, P = 0.044), though no difference in OS was seen [25]. In the current study, patients with EGFR activating mutations in tumor tissue had significantly greater ORR and longer PFS with EGFR-TKIs, which accords with the finding of previous clinical trials [4], [5], [6], [7] and [8]. Patients harboring EGFR activating mutations in cfDNA also had significantly higher ORR, which was consistent to that of patients with mutant tumors. In addition, patients with mutant cfDNA tended to have longer PFS than those with wild-type cfDNA, though the difference was not significant. These data suggest that EGFR activating mutations detected in blood may be predictive of improved tumor response and survival benefit from EGFR-TKIs.

EGFR mutation rate was significantly higher in tumor tissue than in plasma (46.5% versus 25.5%, P < 0.001) and serum (46.5% versus 22.2%, P < 0.001). The correlation between EGFR mutation status and patients’ clinicopathologic characteristics was summarized in Table 3. In tumor tissue, EGFR mutation status was correlated with patients’ gender, smoking history and histology. EGFR mutation rate was significantly higher in females than in males (60.0% versus 36.6%, P = 0.006), in never smokers than in smokers (55.4% versus 36.8%, P = 0.026) and in patients with adenocarcinoma

than in those with other histology (53.7% versus 23.5%, P = 0.002). In blood samples, EGFR mutation status was check details only associated with histology. Patients with adenocarcinoma had significantly higher mutation rate than Inhibitor Library those with other histology in both plasma (30.0% versus 9.7%, P = 0.022) and serum (26.7% versus 4.5%, P = 0.024). Plasma versus Tumor Tissue T790M was detected in 14 (8.5%) patients. Among them, one patient exhibited T790M concurrent with 19Del in matched plasma, serum and tumor tissue, whereas 10 patients had discrepant results between blood and tumor tissue. In 68 patients who received EGFR-TKIs, the correlation between EGFR

mutation status and response to EGFR-TKIs was analyzed ( Table 5). For tumor tissue, objective response rate (ORR) of patients with or without EGFR activating mutations was 68.4% (26/38) and 10.5% (2/19), respectively (P < 0.001). For plasma samples, ORR of patients with or without EGFR activating mutations was 68.4% (13/19) and 38.9% (14/36), respectively (P = 0.037). For serum samples, ORR of EGFR activating mutation positive and negative patients was 75.0% (12/16) and 39.5% (15/38), respectively (P = 0.017). ORR of patients with EGFR mutant tumor was consistent to that of patients with EGFR mutant cfDNA in plasma (P = 1.000) and serum (P = 0.751), whereas ORR of patients with wild-type Non-specific serine/threonine protein kinase tumor was significantly lower than that of patients with wild-type cfDNA in plasma (P = 0.028) and serum (P = 0.024). Of 17

patients who provided samples after PD to EGFR-TKIs, 9 (52.9%) exhibited T790M concurrent with an EGFR activating mutation. In addition, one patient with L858R in tumor tissue but T790M in plasma before EGFR-TKIs treatment directly experienced PD after 1.4 months. The correlation between EGFR mutation status and median PFS time in patients treated with EGFR-TKIs was assessed. For tumor tissue, PFS for patients with or without EGFR activating mutations was 13.6 months (95% confidence interval [CI], 9.9 to 17.3) and 2.1 months (95% CI, 0.8 to 3.4), respectively. The difference was statistically significant (P < 0.001, Figure 1A). For plasma samples, patients with EGFR activating mutations had a PFS of 7.9 months (95% CI, 1.6 to 14.1) compared with 6.1 months (95% CI, 2.7 to 9.6) for patients with wild-type EGFR (P = 0.953, Figure 1B).

Systems combining phosphorothioate and bridging oxygen-substitutions (Table 3, entry 7) have demonstrated potential as therapeutics against Alzheimer’s disease owing to their metal GSI-IX chemical structure ion chelation properties [47 and 48]. The use of sulfur-based analogues in the determination of mechanism has been reviewed recently [49]. Recent synthetic advances have also given (easier) access

to: azido-phosphonate dNTPs, where bridging O-atoms have been replaced by CHN3 groups (Table 3, entry 8), and these analogues can be isolated as separate diastereomers [50]; and oxymethyl analogues (CH2 insertion between O and P within anhydride linkages) for following ApnA and NpnN degradation and metabolism (Table 3, entry 9) [51]. Phosphonate NDP-sugar analogues, where the C1-oxygen of the glycosyl group has been replaced by methylene, have given insight into the mechanism of UDP-apiose/UDP-xylose synthase (Table 3,entry 10) [52], and bis-α,β-β,γ-CF2-NTPs offer sterically undemanding mimics that do not hydrolyse while maintaining comparable polarity properties to their natural NTP progenitors (Table 3,entry 11) [53]. Multi-faceted approaches

combining several experimental techniques and/or computational methods are currently giving some of the clearest pictures of phosphoryl transfer strategies. Most of these approaches have, in principle, been available for some time, however, experimental difficulties have precluded their exploitation. Synthesis MK-2206 solubility dmso of analogues remains a substantial obstacle, with many ‘obvious’ analogues only becoming

accessible through painstaking development of challenging routes. This is particularly true of the phosphoanhydride systems. Fortunately, several groups are working towards more convenient methodologies for the preparation of phosphoesters, anhydrides and their analogues, and details of these efforts can be accessed elsewhere [55, 56•, 57, 58, 59, 60•, 61, 62, 63•, 64, 65•, 66, 67, 68•• and 69]. Heavy isotope kinetic studies have proven extremely enlightening, however, the measurement of these extremely small effects (even in best case scenarios) remains the preserve of a few specialist groups. Combinations selleck of experimental approaches with computational methods are also allowing more rigorous, quantitative assessment of observed kinetic data, where interpretations of kinetic results can often be complex. In summary, synthetic methodology, in tandem with kinetic measurements and computational dissection are providing enzymologists with an enhanced toolbox for the determination of phosphoryl transfer mechanisms. Papers of particular interest, published within the period of review, have been highlighted as: • of special interest HJK was funded by a postdoctoral grant from the Jenny and Antti Wihuri Foundation. LPC was funded by a PhD studentship from EPSRC.

1). Ears at 10, 15, 20, 22, 25 and 30 days after pollination (DAP) were collected from plants grown under standard greenhouse

conditions. Kernels were dissected from the ears, immediately frozen in liquid nitrogen, and stored at − 80 °C until RNA extraction. Isolated total RNA was size-fractionated on a 15% Tris–borate–EDTA (TBE) urea polyacrylamide gel to enrich molecules of 15–30 nt. The small RNA was ligated with adapters (5′-GTCTCTAGCCTGCAGGATCGATG-3′) Maraviroc solubility dmso and (5′-AAAGATCCTGCAGGTGCGTCA-3′), and (5′-GTCTCTAGCCTGCAGGATCGATG-3′) and (5′-AAAGATCCTGCAGGTGCGTCA-3′) using T4 RNA ligase and size-fractionated on a 15% TBE urea polyacrylamide gel. The resultant RNA was reversely transcribed to cDNA with a small RNA RT-primer (5′-CAAGCAGAAGACGGCATACGA-3′), and the cDNA was then directly subcloned into vector pMD18-T (TaKaRa). These tandem cDNA fragments were transformed into Escherichia Selleckchem Epacadostat coli strain DH5 by electroporation. Colony PCR was performed using 5′ and 3′ primers, and clones with lengths of 60–80 bp were used for sequencing according to the manufacturer’s protocols (Colony PCR Made Easy,

http://www.lucigen.com/colonyPCR). Small RNAs (200 nt) were isolated with the mirVana PARIS Kit (Ambion) according to the manufacturer’s instructions. For reverse transcription (RT), 1 μg of small RNA was treated with the miScript Reverse Transcription Kit (Qiagen) at 37 °C for 60 min and a final incubation at 95 °C for 5 min. Real-time PCR of miRNAs was carried out Thiamine-diphosphate kinase using the miScript SYBR Green PCR kit (Qiagen) in an Applied Biosystems 7500 real-time PCR machine (ABI). PCR was conducted at 95 °C for 15 min, followed by 40 cycles of incubation at 94 °C for 15 s, 55 °C for 30 s, and then 70 °C for 30 s. Each PCR was repeated at least three times. All samples were normalized to 5S rRNA expression and fold change expression was calculated according to the 2− ΔΔCt method as described previously [39]. High-quality small RNA reads larger than 18 nt were extracted from the raw reads and mapped to maize genome sequences (http://www.maizesequence.org) using SOAPaligner/soap2

(http://soap.genomics.org.cn/soapaligner.html) [40]. Matched sequences were then queried against non-coding RNAs from the Rfam database (http://www.sanger.ac.uk/Software/Rfam) and the ncRNA database (http://www.ncrna.org/frnadb/blast/fRNAdb). Most non-miRNAs, non-siRNAs and mRNA degradation fragments were removed by a BLASTn search of the NCBI GenBank database (http://www.ncbi.nlm.nih.gov/blast/Blast.cgi) [41]. Any small RNAs with exact matches to these sequences were excluded from further analysis. miRNAs were predicted with Mireap (https://sourceforge.net/projects/mireap/). Secondary structures of the predicted miRNAs were confirmed using the RNAfold online tool (http://rna.tbi.univie.ac.at/cgi-bin/RNAfold.cgi).