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Real-time quantitative polymerase chain reaction (qPCR) has become widely used as a method to compare gene transcript levels across different conditions. However, selection of suitable reference genes to normalize qPCR data is required for accurate transcript level analysis. Recently, Marchantia polymorpha has been adopted as a model for the study of liverwort development and land plant evolution. Identification of appropriate reference genes has therefore become a necessity for gene expression studies. In this study, transcript levels of eleven candidate reference genes have been analyzed across a range of biological contexts that encompass abiotic stress, hormone treatment and different developmental stages. The consistency of transcript levels was assessed using both geNorm and NormFinder algorithms, and a consensus ranking of the different candidate genes was then obtained. MpAPT and MpACT showed relatively constant transcript levels across all conditions tested whereas the transcript levels of other candidate genes were clearly influenced by experimental conditions. By analyzing transcript levels of phosphate and nitrate starvation reporter genes, we confirmed that MpAPT and MpACT are suitable reference genes in M. polymorpha and also demonstrated that normalization with an inappropriate gene can lead to erroneous analysis of qPCR data.
Citation: Saint-Marcoux D, Proust H, Dolan L, Langdale JA (2015) Identification of Reference Genes for Real-Time Quantitative PCR Experiments in the Liverwort Marchantia polymorpha. PLoS ONE 10(3): e0118678. https://doi.org/10.1371/journal.pone.0118678
Academic Editor: Rogerio Margis, Universidade Federal do Rio Grande do Sul, BRAZIL
Received: November 27, 2014; Accepted: January 8, 2015; Published: March 23, 2015
Copyright: © 2015 Saint-Marcoux et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Data Availability: All relevant data are within the paper and its Supporting Information files.
Funding: This work was funded by ERC Advanced Investigator grants to JAL (EDIP) and LD (EVO500). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Real-time quantitative polymerase chain reaction (qPCR) is a sensitive and reproducible technique that is used to measure and compare transcript accumulation levels for genes in many organisms. Correct interpretation of qPCR results, however, is subject to an understanding of several parameters (e.g. the amount and integrity of initial RNA, variations between batches of reagents, and intrinsic stochastic variability in the biochemical reactions). In an attempt to standardize procedures and thus allow comparability between experiments, guidelines establishing a checklist for qPCR experiments were published several years ago [1–3]. Notably, the use of an appropriate normalization strategy is perhaps the most important point to consider for accurate transcript level comparison, because normalization can compensate for experimental variations between different samples. Normalization in qPCR experiments is most commonly carried out by comparing transcript levels of the gene of interest with transcript levels of a 'constitutively expressed' reference gene. As such, qPCR experiments can only accurately report transcript levels for a gene of interest, if there are suitable reference genes available for use with the biological samples under investigation [4,5].
Marchantia polymorpha is a liverwort that belongs to the early divergent bryophyte grade of land plants [6,7]. Although the phylogenetic relationship between the bryophyte lineages of liverworts, mosses and hornworts is still debated, the position of liverworts as one of (if not) the earliest land plant lineage(s) is undisputed [8]. As such, studies of liverwort biology are important for understanding the evolution of development processes in land plants. Over the last few years, M. polymorpha has become the liverwort species of choice, not least because it is easy to propagate, mutate and genetically transform [9–11]. The adoption of M. polymorpha as a model [12–14], has led to the need to develop quantitative methods for analyzing mRNA levels. As such, reference genes for which transcript levels are relatively constant across a range of routine conditions must now be identified. The identification of suitable genes for normalization of qPCR analyses in M. polymorpha will facilitate gene expression studies in a representative of one of the earliest land plant lineages.
Specialized tools have been developed over the years to help identify good reference genes for qPCR analyses [15–18], and a number of studies reporting suitable genes have been published in a diverse range of organisms [4,5,18–28]. Notably, studies suggested that commonly used reference genes such as ACTIN, TUBULIN, UBIQUITIN or GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE are not the most appropriate [4,5,20,24,25], and a number of studies have suggested that genes such as SAND [20], EUKARYOTIC ELONGATION FACTOR 5 [28], or CULLIN [24] are better alternatives. Here, we report the analysis of transcript levels for eleven genes in M. polymorpha across different growth conditions, a range of developmental stages, and under a variety of abiotic stress and hormone treatments. We identify MpAPT and MpACT as a good pair of reference genes for normalization of qPCR assays in M. polymorpha and demonstrate that MpGAPC1, MpTUB8 and MpUBQ10 are unsuitable. Finally, using phosphate and nitrate starvation reporter genes, we exemplify how choosing suitable reference genes is crucial for the accurate analysis of qPCR data.
Materials and Methods
Plant material and culture conditions
Gemmae from female Marchantia polymorpha (accession Takaragaike-2) [11] were propagated on Petri dishes filled with ½ Gamborg's medium pH 5.6 (# G0210, Duchefa) supplemented with 1% agar, or in pots filled with a soil-based compost (Levingston M2) under a 16h light:8h dark photoperiod, respectively at 23°C and a light intensity of 56 μE.m−2.s−1, or at 18°C and a light intensity of 150 μE.m−2.s−1. Male and female sexual organs were obtained from 2-month-old plants (accession Takaragaike-1 and Takaragaike-2 respectively) cultivated in soil conditions supplemented with far-red light [29,30].
For cold treatment, 17 day old plants were transferred to 4°C for 24 hours. Hormone treatments were carried out on ½ Gamborg's supplemented with, abscisic acid (ABA), 1-naphthaleneacetic acid (NAA) or GR24 at a final concentration of 2 μM, 750 nM and 1 μM respectively. Hormones were initially dissolved in 100% acetone then dilutions to 1/10000th were prepared to supplement the medium. Medium for starvation experiments was prepared by modulating phosphate or nitrate concentration in ½ Johnson's medium [31]. Low phosphate medium was prepared by adding only 1 mM of (NH4)2SO4 instead of 1 mM of NH4H2PO4. For both low and high nitrate media, 2 mM of CaCl2 was added to the medium instead of 2 mM of Ca(NO3). Low nitrate medium was prepared by adding 1 mM KNO3 instead of 3 mM KNO3, and 2 mM of KH2PO4 as phosphate source. High nitrate medium was prepared by adding 5 mM of NH4NO3, and 2 mM of NH4H2PO4 as phosphate source. For 24 hours treatments (with the exception of cold treatment), plants were grown on cellophane disks (AA Package Ltd.) placed on top of untreated medium. Cellophane disks were then transferred onto untreated (control) or treated (induction) medium.
RNA extraction, cDNA synthesis and qPCR
Total RNA was extracted from ground frozen tissue with the RNeasy plant mini kit (#74904, Qiagen). On-column DNase I treatment was performed using RNase-free DNase I (#79254, Qiagen) according to the manufacturer's recommendations. Total RNA samples were quantified using a Nanodrop ND-1000 spectrophotometer. RNA integrity was checked using a 2100 BioAnalyzer (Agilent Technologies) and RNA-Nano chips (#5067-1511, Agilent Technologies). The RIN (RNA integrity number) provided by the BioAnalyzer software ranges from 1 (corresponding to a very degraded sample) to 10 (for a mostly intact sample).
cDNA was synthesized from 1 μg of total RNA. Reverse transcription was performed in a 20 μl volume with 2 μM of poly-dT17 primer and 5 U of superscript III reverse transcriptase (#18064, Fermentas) according to the manufacturer's protocol. cDNA was then diluted 10 times by adding 180 μl nuclease-free water.
Amplification experiments were carried out using a 7300 Applied Biosystem thermocycler. Reactions were performed in 10 μL volumes containing 5 μL 2X SYBR-green mastermix (#4309155, Applied Biosystem), 500 nM forward and reverse primers, and 4 μl diluted cDNA (equivalent to 20 ng of reverse transcribed total RNA). A two-step cycle composed of denaturation at 94°C for 15 seconds followed by hybridization/elongation at 60°C for 60 seconds, was repeated 40 times and then followed by a dissociation step. Three technical replicates were performed for each reaction. Genomic contamination was determined by direct amplification of 20 ng total RNA for 50 cycles.
Data analysis
Fluorescence data were first manually checked for any substantial variation between technical replicates and any outlying values were discarded. Fluorescence data were then exported and transformed using a Perl script into a format suitable for analysis using LinRegPCR v2012.0 [32]. Briefly, LinRegPCR determines mean reaction efficiency (Em) for any specific primer pair, by linearly regressing fluorescence data from several amplification reactions. Because mean efficiencies are calculated in this way, where possible, reactions were grouped in qPCR plates by amplicon rather than by treatment/growth condition. Using the mean efficiency and the window of linearity of the reaction, LinRegPCR computes a N0 value which reflects the initial amount of template in the reaction mix. The computed N0 values from the technical replicates were then averaged. Genomic contamination was taken into account by subtracting N0 values obtained from MpEF1α amplified from total RNA. Contamination was insignificant in all cases, with N0 values from genomic amplifications at least two orders of magnitude lower than N0 from cDNA amplifications.
The extent of variation in transcript levels across samples was determined using the software GenEx version 6 (http://www.biomcc.com/genex-software.html), including geNorm [16] and NormFinder [15] algorithms. N0 values from three biological replicates were first averaged and then normalized by the highest averaged N0 value for a given gene. Normalized N0 were thus rendered equivalent to relative quantification data classically obtained by the ∆Ct method and suitable to be used in GenEx (see Equation 1 adapted from Ruijeter et al. [32]).Equation1
Stability values were produced by geNorm and NormFinder algorithms (the lower the value, the more stable the gene transcript level). For a given gene, geNorm computes the standard deviation of transcript level ratios between the gene and another gene in all tested conditions. The stability value is then equal to the average of the standard deviations computed for every pairwise comparison of the gene. In contrast, NormFinder relies on a mathematical model to describe gene transcript level variations for a given tested condition and between all tested conditions. The sum of both terms produces a stability value.
Stability values were transformed into z-scores (z) using Equation 2. Equation2 with Sn the stability value of a given gene n, and δS the mean and the standard deviation of the stability values respectively.
Identification of candidate reference genes
To select putative reference genes for qPCR in M. polymorpha, we conducted a survey of genes used in other plant species [4,5,17,19–28]. 11 candidate genes were selected and orthologous sequences were identified in a M. polymorpha thallus transcriptome (Giulia Morieri, Hélène Proust & Steve Kelly, unpublished) using Arabidopsis gene sequences and reciprocal best BLAST [33] (Table 1). Proteins encoded by the selected genes play roles in genome organization and expression (EF1α, ELONGATION FACTOR 1α; ELF5, EUKARYOTIC ELONGATION FACTOR 5A-1; APT, ADENINE PHOSPHORIBOSYL TRANSFERASE; H3, HISTONE 3), cytoskeleton formation (TUB8, TUBULIN BETA CHAIN 8; ACT, ACTIN 7), protein modification and degradation (CUL, CULLIN 1; UBQ10, POLYUBIQUITIN 10; PEX, PEROXIN 4), glycolysis (GAPC1, GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE C SUBUNIT 1) and vesicle trafficking (SAND).
Quantification of transcript levels in different biological contexts
To obtain a set of reference genes that is suitable for use across a broad range of conditions, transcript levels were quantified in 22 biological contexts regrouping different tissue types and plants subjected to different treatments (Table 2). The range of developmental stages included whole plants of different ages (17 days and 24 days old) grown in sterile (Gamborg's) or soil conditions, plus gemmae (vegetative propagules), antheridiophores (male reproductive structures), and archegoniophores (female reproductive structures). Treatments included abiotic stresses such as nitrogen and phosphate starvation for 17 days or 24 hours (nitrogen only) or 24 hours cold exposure; and hormone treatments where plants were either grown on NAA and ABA-containing media or exposed to NAA, ABA and GR24 (a synthetic strigolactone) for 24 hours.
Where applicable, phenotypic differences between normal and treated plants were recorded (S1 Fig.). Plants cultivated for 17 days on ABA (S1B Fig.) showed a notable size reduction compared to untreated plants (S1A Fig.). Similarly, when cultivated on NAA (S1D Fig.), plant growth was drastically impaired compared to untreated controls (S1C Fig.). The observed ABA– and NAA–induced phenotypes are consistent with published reports [13,34]. Minimal differences were observed between growth on low (S1F Fig.) versus high (S1E Fig.) phosphate levels for 17 days, but nitrogen starvation (S1H Fig.) caused a severe reduction in growth compared to growth on high nitrate levels (S1G Fig.). Nitrogen starvation for just 24 hours did not lead to visible growth perturbations (not shown).
qPCR amplifications were carried out using RNA extracted from plants from the 22 biological contexts (Table 2) and the entire experiment was replicated three times. All RNA samples showed a RIN > 7 (S1 Table), which is sufficient integrity for qPCR experiments [35]. cDNA was synthesized from total RNA extracts to perform qPCR reactions. Primers were designed for the 11 candidate reference genes using the Primer3Plus software [36]. To minimize amplification bias, amplicons were designed to be 80 to 150 nucleotides in length and were located in the 3' part of the gene. Primer specificity was first checked by PCR and subsequent gel electrophoresis (not shown). The specificity of each qPCR reaction was then checked by performing a dissociation step. For each pair of primers, the dissociation curve showed a unique peak of fluorescence, indicating that a single DNA fragment was amplified during the PCR reaction (S2 Fig.—a few reactions are shown superimposed).
To determine transcript levels for each candidate reference gene in each of the 22 biological contexts, LinRegPCR software [32] was used to compute mean efficiencies (Em) and initial cDNA amount (N0) values for every reaction. All Em values were above 1.85 (Table 1), a value representative of an efficient amplification reaction [37]. Fig. 1 shows a box plot of the log transformed N0/N0min ratio calculated per gene across all conditions. The data reveal that MpEF1α transcript levels were the highest over all conditions tested whereas MpSAND levels were the lowest. Furthermore, MpPEX and MpAPT transcript levels varied least across the whole experiment whereas MpGAPC1 and MpUBQ10 transcript levels varied greatly depending on biological context. Importantly, however, transcript levels were not normalized by known reference genes and thus values are subject to biological and technical perturbations. Therefore, whilst this preliminary analysis showed that the amplification procedure was successful, further refinement was needed to identify reference genes.
Average N0 values per gene and condition (n = 3) were normalized on the smallest value then log transformed. Data were then represented for each gene as a box-plot.
Assessment of variation in transcript levels for each candidate reference gene
To assess variation in transcript levels across samples, qPCR data were analyzed in groups using geNorm [16] and NormFinder [15] algorithms. Groups were defined as 'all', 'development', 'abiotic stress' and 'hormone', which encompassed all biological contexts, developmental stages and organs, nutrient starvation and cold exposure, and hormone treatments respectively. Both approaches produced a ranked list of genes with the lowest values representing the most consistent transcript levels. Both geNorm and NormFinder ranked similar genes in the top five but not necessarily in the same order (S2 and S3 Tables) (see for example MpACT, MpAPT, MpCUL and MpEF1 when considering all biological contexts). However, in some cases important discrepancies existed between rankings produced by the two algorithms: for example MpELF5 is ranked 1st by geNorm but 7th by NormFinder for the abiotic stress group. In an attempt to reconcile the discrepancies observed, we averaged the values obtained from the two algorithms. Values could not be compared directly as geNorm and NormFinder do not rely on the same calculations. Consequently, we averaged z-scores derived from the data produced by each algorithm for each gene, and for each group (Fig. 2). As for rankings produced by the two algorithms, the lower the z-score for a gene, the more consistent transcript levels are across samples.
All 22 conditions tested (A, highlighted in blue), development group (B), abiotic stress group (C), and hormone group (D). Variation is represented as the average of z-scores derived from geNorm and NormFinder results. The lower the z-score, the more invariant the transcript levels between samples.
When using z-scores, a different gene ranking was revealed for each group. Transcript levels of MpGAPC1, MpUBQ10 and MpTUB8 varied most across the four groups (Fig. 2). As such, these three genes are unsuitable for use as reference genes in qPCR assays. MpEF1α and MpSAND appeared to be suitable for developmental comparisons (Fig. 2B), MpACT and MpSAND for abiotic stress (Fig. 2C), and MpCUL and MpAPT for hormone treatments (Fig. 2D). Taking into account all biological contexts, MpAPT and MpACT had the lowest z-scores. In addition, MpAPT was ranked third by geNorm and second by NormFinder, and MpACT was ranked second and fifth—in this last case with a value almost identical to the value of the genes ranked third and fourth. Consequently, MpAPT and MpACT are the most suitable reference genes for generic studies. Notably, some genes appeared to have relatively constant transcript levels in some contexts but very variable levels in others. For example, MpSAND had a very low z-score when assayed in the development and abiotic stress groups (Fig. 2B and C) but the highest score in the hormone group (Fig. 2D). In contrast, MpH3 had a low score in the hormone group but a very high score in all other cases.
Estimation of the number of reference genes required for normalization
To further improve normalization strategies, several reference genes can be used simultaneously. Vandesompele et al. [16] described a method to estimate the optimum number of genes needed that computes the variation Vn/n+1 induced by the inclusion of the (n+1)th best ranked gene in the calculation of the normalization factor. A variation greater than 0.15 was assumed to be significant [16]. We implemented the method as a Perl script (available as a supplemental file) and, for each group of biological contexts, computed the stepwise variation obtained when a new gene was added to the normalization factor, starting from the best to the worse ranked gene. When adding the second ranked gene to the normalization factor (V1/2), a variation greater than 0.15 was obtained for every group except the hormone group (Fig. 3). Using the third ranked gene in addition to the first two decreased the variation again but did not improve any of the normalization factors calculated (V2/3 < 0.15 in all cases). Adding more genes tend to make the stepwise variation rise again for three of the four groups, an observation previously reported (see for example Vandesompele et al. [16]). Since the variation when adding the third best-ranked gene was below 0.15, we concluded that using the two most highly ranked genes to compute the normalization factor was sufficient.
Estimation was done by calculating the pairwise variation (Vn/n+1) of the normalization factors NFn and NFn+1 as described in Vandesomple et al., [16].
Validation of reference genes for qPCR analysis during nitrate and phosphate starvation
In Arabidopsis, AtPHT1-1 (PHOSPHATE TRANSPORTER 1) that encodes a phosphate transporter and AtNRT2-1 (NITRATE TRANSPORTER 2) that encodes a nitrate transporter involved in the High Affinity Transport System are induced during phosphate and nitrate starvation, respectively [38,39]. We identified a similar sequence for each gene in our M. polymorpha transcriptome dataset and measured transcript levels after nutrient starvation (Fig. 4). Non-normalized qPCR data showed a ∼4-fold increase in MpPHT1 transcript levels after phosphate starvation, and a ∼27-fold or ∼14-fold increase in MpNRT2 transcript levels after long term or short term nitrate starvation (Fig. 4A and B, 'no ref'). The induction of both genes transcription is consistent with previously published results in Arabidopsis [38,39]. Given the dramatic differences detected in non-normalized qPCR between transcript levels before and after starvation, these two genes were deemed appropriate for validation of our candidate reference genes in M. polymorpha.
Histograms represent the fold-change of the average transcript levels (n = 3) compared to the long-term control condition of MpPHT1-1 (A) and MpNRT2-1 (B). Data were non-normalized ('no ref') or normalized by a factor consisting of the geometric mean of the transcript level of MpAPT and MpACT (highlighted in blue), of MpACT and MpSAND, of MpACT, MpSAND and MpELF5, and only by the transcript level of MpGAPC1. Biological replicates are depicted superimposed with crosses, diamonds and squares, respectively for replicate one, two and three. NP stands for normal phosphate, LP for low phosphate, NN for normal nitrate, and LN for low nitrate media respectively. A 's' indicates a short (24 h) treatment, a 'l' a long (17 days) treatment.
Fig. 4 illustrates different strategies of normalization. Using MpAPT and MpACT, which we showed to be the best candidate reference genes for generic studies, MpPHT1 and MpNRT2 transcript levels exhibited a profile reminiscent of non-normalized data, with fold-change values in the same range: ∼5-fold increase for MpPHT1, and ∼25-fold or ∼17-fold increase (long or short term starvation) for MpNRT2 (Fig. 4A and B, highlighted in blue). The same profile was observed when the data were normalized with the two best candidate genes for the abiotic stress group (MpACT and MpSAND), with a small change, if any, in the fold-change increase (∼5-fold, ∼25-fold and ∼14-fold). Addition of the third ranked gene of the abiotic stress group (MpELF5) to normalize the data only marginally changed the obtained fold-change increase for both MpPHT1 and MpNRT2 after starvation (Fig. 4A and B). This result confirmed that two reference genes are sufficient to normalize qPCR data in M. polymorpha. In combination, these data demonstrate that the two generic reference genes, as well as the two genes specific to the abiotic stress group can be used to reliably normalize qPCR data across abiotic stress contexts.
Data normalization was also carried out using the most variable gene, MpGAPC1. Drastically different (and obviously inaccurate) transcript levels were obtained for both MpPHT1 and MpNRT2. For example, MpPHT1 transcript levels appeared to be equivalent in high and low phosphate contexts, as opposed to being higher in low phosphate. Similarly, whereas in our experiment MpNRT2 transcript levels are higher after long-term nitrate starvation than after 24 hours (non-normalized data and data normalized with the best candidate reference genes), normalization with MpGAPC1 reversed the trend.
These results demonstrate that the methods used here for the identification of reference genes are robust, and also that improper normalization can lead to erroneous analysis of qPCR raw data.
Normalization of qPCR results is needed to correct for biological and experimentally introduced variability. A common method of normalization is to compare transcript levels of the gene of interest to those of a reference gene that, through either transcriptional or post-transcriptional mechanisms, is characterized by relatively constant transcript levels across the conditions and/or tissue types being analyzed. Selection of suitable reference genes is not a trivial task and requires careful analysis of non-normalized transcript levels. To facilitate this task, several algorithms such as geNorm and NormFinder are available [15,16]. Here we applied both algorithms, and averaged their results, to identify a set of suitable reference genes for qPCR studies in the model liverwort species M. polymorpha.
We tested eleven candidate genes in a broad range of experimental conditions. We show that genes such as GAPC1, TUB, or UBQ that are commonly used in other organisms [4,5,20] are poor reference genes in M. polymorpha. Instead, our results indicate that for generic applications, normalization using MpAPT and MpACT is optimal. Orthologs of both genes have been shown to be good reference genes in previous studies with other species [4,5,23,25].
The suitability of any particular gene as a reference for normalization can be surprisingly variable depending on biological context. For example, MpSAND transcript levels are elevated after hormone treatments, rendering the gene unsuitable for normalization in these conditions. However, MpSAND transcript levels are relatively constant under different growth conditions, after nutrient starvation and cold response and in different developmental contexts. This constancy has also been observed in other organisms such as Eucalyptus or Arabidopsis [20,25]. This example demonstrates that where possible, reference genes should be determined for each subset of conditions, as restricted as possible; a conclusion also drawn previously by others [5,18].
An important question that arises when testing for reference genes is the number of genes necessary for optimal normalization. The authors of geNorm recommend the use of at least three reference genes (and up to five in some cases) in order to compensate for individual reference gene variation in different study conditions [16]. In contrast, NormFinder authors argue that the use of several reference genes does not necessarily improve normalization accuracy [15]. The use of multiple reference genes increases the number of qPCR reactions required for any particular experiment. This can become problematic for large-scale projects or when working with material from which only small amounts of RNA can be extracted. Here we have shown that two reference genes are sufficient to produce a reliable normalization factor.
We report the identification of a reference gene set suitable for normalizing transcript level data obtained by qPCR in Marchantia polymorpha. We showed that two reference genes were necessary and sufficient to normalize qPCR data and determined the best gene pairs for different experimental conditions such as hormone treatments and abiotic stress, and across a variety of developmental stages. While different pairs were found to be the most appropriate for these biological contexts, we observed that the MpAPT and MpACT combination was the best for generic studies. Therefore, we recommend the use of MpAPT and MpACT for Marchantia polymorpha qPCR analysis.
Supporting Information
S1 Fig. Phenotype of exogenously treated plants.
17 days mock (A) and 1 μM ABA (B) treatments, mock (C) and 750 nm NAA (D) treatments, high (E) and low (F) phosphate, high (G) and low (H) nitrate. Scale bars equals to 1 cm (A, B, G, H) or 2 cm (C, D, E, F).
S2 Fig. Typical dissociation curves for each amplicon.
Pictures were taken using the qPCR instrument's software: MpAPT (A), MpACT (B), MpPEX (C), MpUBQ10 (D), MpCUL (E), MpELF5 (F), MpEF1α (G), MpTUB8 (H), MpH3 (I), MpGAPC1 (J), MpSAND (K), MpPHT1 (L), MpNRT2 (M).
S1 Table. RNA integrity number obtained for the three biological replicates of each sample.
S2 Table. Transcript level stability values calculated by geNorm algorithms for each reference gene.
S3 Table. Transcript level stability values calculated by NormFinder algorithms for each reference gene.
S1 Data. N0 data in excel file.
S2 Data. M. polymorpha gene sequences in word file.
S3 Data. Perl script implementing Vn/n+1 calculations.
We are grateful to Giulia Morieri and Steve Kelly (Department of Plant Sciences, University of Oxford) for the generation and analysis of M. polymorpha transcriptomes, Francois Didier Boyer (INRA Versailles Grignon) for providing GR24, Cécile Proust-Lima (INSERM Bordeaux) for providing advice on mathematical analysis, and Julie Bull, Mary Saxton, Helen Prescott and Lida Chen for technical assistance.
Author Contributions
Conceived and designed the experiments: DS-M HP. Performed the experiments: DS-M HP. Analyzed the data: DS-M HP. Wrote the paper: DS-M HP JAL LD.
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- 22. Le Bail A, Dittami SM, de Franco PO, Rousvoal S, Cock MJ, Tonon T, et al. Normalisation genes for expression analyses in the brown alga model Ectocarpus siliculosus. BMC Mol Biol. 2008;9: 75. pmid:18710525
- 23. Le Bail A, Scholz S, Kost B. Evaluation of reference genes for RT qPCR analyses of structure-specific and hormone regulated gene expression in Physcomitrella patens gametophytes. PLoS One. 2013;8: e70998. pmid:23951063
- 24. Manoli A, Sturaro A, Trevisan S, Quaggiotti S, Nonis A. Evaluation of candidate reference genes for qPCR in maize. J Plant Physiol. 2012;169: 807–815. pmid:22459324
- 25. Moura JC, Araujo P, Brito Mdos S, Souza UR, Viana Jde O, Mazzafera P. Validation of reference genes from Eucalyptus spp. under different stress conditions. BMC Res Notes. 2012;5: 634. pmid:23148685
- 26. Wang HL, Chen J, Tian Q, Wang S, Xia X, Yin W. Identification and validation of reference genes for Populus euphratica gene expression analysis during abiotic stresses by quantitative real-time PCR. Physiol Plant. 2014;152: 529–545. pmid:24720378
- 27. Wang Z, Chen Y, Fang H, Shi H, Chen K, Zhang Z, et al. Selection of reference genes for quantitative reverse-transcription polymerase chain reaction normalization in Brassica napus under various stress conditions. Mol Genet Genomics. 2014;289: 1023–1035. pmid:24770781
- 28. Yang H, Liu J, Huang S, Guo T, Deng L, Hua W. Selection and evaluation of novel reference genes for quantitative reverse transcription PCR (qRT-PCR) based on genome and transcriptome data in Brassica napus L. Gene. 2014;538: 113–122. pmid:24406618
- 29. Fredericq H, De Greef J. Red (R), far-red (FR) photoreversible control of growth and chlorophyll content in light-grown thalli of Marchantia polymorpha L. Naturwissenschaften. 1966;53: 337. pmid:5988692
- 30. Wann F. Some of the factors involved in the sexual reproduction of Marchantia polymorpha. Am J Bot. 1925;12: 307–318.
- 31. Johnson CM, Stout PR, Broyer TC, Carlton AB. Comparative chlorine requirements of different plant species. Plant and Soil. 1957;8: 337–353.
- 32. Ruijter JM, Ramakers C, Hoogaars WM, Karlen Y, Bakker O, van den Hoff MJ, et al. Amplification efficiency: linking baseline and bias in the analysis of quantitative PCR data. Nucleic Acids Res. 2009;37: e45. pmid:19237396
- 33. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. Basic local alignment search tool. J Mol Biol. 1990;215: 403–410. pmid:2231712
- 34. Ishizaki K, Nonomura M, Kato H, Yamato KT, Kohchi T. Visualization of auxin-mediated transcriptional activation using a common auxin-responsive reporter system in the liverwort Marchantia polymorpha. J Plant Res. 2012;125: 643–651. pmid:22311005
- 35. Schroeder A, Mueller O, Stocker S, Salowsky R, Leiber M, Gassmann M, et al. The RIN: an RNA integrity number for assigning integrity values to RNA measurements. BMC Mol Biol. 2006;7: 3. pmid:16448564
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- 38. Orsel M, Krapp A, Daniel-Vedele F. Analysis of the NRT2 nitrate transporter family in Arabidopsis. Structure and gene expression. Plant Physiol. 2002;129: 886–896. pmid:12068127
- 39. Shin H, Shin HS, Dewbre GR, Harrison MJ. Phosphate transport in Arabidopsis: Pht1;1 and Pht1;4 play a major role in phosphate acquisition from both low- and high-phosphate environments. Plant J. 2004;39: 629–642. pmid:15272879


text:
RICHMOND — Every team has a weakness and the Hofstra Pride took advantage of the Spiders' weakness from the opening tip. Like Superman reacts to kryptonite, the Spiders react to seeing a 2-3 zone. They become hesitant. They become stagnant. They miss shots.
Entering the game, Richmond ranked at the bottom of the Atlantic 10 Conference in 3-point shooting at just 13 percent from beyond the arc, making just nine through their first three contests. Unfortunately for the Spiders, Hofstra head coach Joe Mihalich read the scouting reports and made sure it stayed that way.
But Richmond found a way even after adding to their dismal start from 3-point range by shooting just 8-of-26 from beyond the arc. They regrouped and fought — the true litmus test for a team heading toward success during the season.
For the first 40:44, the Richmond Spiders could not solve the perplexing zone defense of Hofstra. Then, in the overtime period, the Spiders broke out of their malaise, scoring 11 points while finishing the game on a 19-5 run, stealing a victory from the Pride, 74-63 at The Robins Center.
"I think tonight when we had to pressure a little bit, it kind of worked in our favor," Cedrick Lindsay said after the Spiders' (3-1) overcame an 11-point deficit to defeat the Pride. "We have a bunch of scrappy guys who are kind of used to playing from behind, kind of underdogs and we've been fighting like that our whole lives."
And it showed in a game where Richmond never led until the overtime period.
Hofstra's (1-3) zone stymied any fluidity the Spiders' offense may have had, allowing them to shoot just 37 percent from the floor in the first half while the Pride also dictated the tone on the offensive end, shooting 57 percent from the field in the first half. Most of the reason for the success by the Pride comes down to the man patrolling the sidelines.
Joe Mihalich won the 2013 Metro Atlantic Athletic Conference's coach of the year award while leading Niagara to the conference's best record last season. But the 15-year veteran of Purple Eagles basketball left for a new challenge at Hofstra and in the game's opening 20 minutes, one could see the difference Mihalich can make on a game.
Like an offensive and defensive coordinator in the National Football League, Mihalich called both offensive and defensive plays from the sideline, setting up full-court pressures on inbounds and a motion-offense on the other end of the court. His play-calls worked to the benefit of Zeke Upshaw who scored a game and career-high 37 points on 9-of-19 shooting. It's the second-most points ever surrendered by a Richmond defense, trailing only Craig Sedmak's 39 points for American on Jan. 30, 1993
Richmond coach Chris Mooney could not help himself while pouring on the accolades during his opening statement.
"I got to give credit to coach Mihalich. I've known him a long time and he's a heck of a coach," Mooney said. "He inherited a team with very few players and they've pieced together a roster and I think they are playing really well over there."
Yet, Mooney's Spiders also have shown the mark of a tough team and it starts with Lindsay's play.
Entering the contest just 57 points shy of 1,000 for his career, Lindsay scored 26 points — 14 of those in the second-half and overtime periods — while shooting 10-of-19 from the field and dishing out four assists with no turnovers. In fact, with Richmond staring down a five point deficit with just 1:55 remaining, it was Lindsay nailing one of his two 3-pointers on the night, closing the gap.
Then, following a Kendall Anthony 3-pointer to give Richmond a three-point lead in the overtime period, it was Lindsay taking advantage of Hofstra turnover by driving through the lane for what would be the dagger points of overtime. Mooney had nothing but praise for the senior who is quickly becoming a luxury the Spiders need on a nightly basis.
"He's running a team that's struggling to shoot against a zone which is becoming more and more packed in and he has to turn up the heat on defense," Mooney said of Lindsay. "He picked his spots, talked guys through cuts, he was really, really good. For a point guard to have 26 points, that's a luxury that's nice to have."
But on this night Lindsay received help from his 5-foot-8 counterpart who has made a name for himself as one of the conference's most dangerous sharpshooters.
Entering the game shooting just 33 percent from the field, including 25 percent from 3-point range, Anthony stepped up when his team needing him, nailing 5-of-12 3-pointers en route to an 18-point night. Yet his biggest shot and decision of the night came after he air-balled a 3-point attempt before having the rebound fall back toward him. Without hesitating, he unloaded, giving Richmond only their second lead of the night and one they would not relinquish.
"I think it was heart and determination. We wanted to win that game," Lindsay explained of his team's rally. "We saw it slipping and coach made a good adjustment with us picking up at full-court … late in the game we were still scrambling and getting steals."
And it could be said the defensive effort saved Richmond.
After the Pride hit 12-of-21 shots in the first half, the Spiders limited them to just 5-of-24 over the second half and overtime periods, holding their fourth straight opponent to under 41 percent shooting.
With heart, determination and a strong defensive effort, Richmond pulled off grand larceny by stealing the win and also showcasing why the Spiders can never be counted out, no matter the circumstance.
Ryan Lazo is the sports editor at The Hopewell News, covering eight high schools, VCU, VSU, Richard Bland, and University of Richmond. You can view his latest articles here and can follow him on Twitter @RMLazo13.

subset: commoncrawl


text:
Refine by Subcateogry:
STEP 1: LIMIT results to these categories:
STEP 2: Return ONLY resources from:
Select multiple by holding down [control] or [command]
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posted Dec 29, 2017
This project involved resurfacing of the entire 34 miles of the Northern Rail Trail in Merrimack County.
published Feb 1, 2014
U.S. Access Board,
National Center on Accessibility
In 2007 the National Center on Accessibility (NCA) entered into an agreement with the U.S. Access Board and National Park Service to investigate natural firm and stable surface alternatives when creating accessible pedestrian trails, including crushed stones, packed soil, and other natural material.
posted May 10, 2018
On March 15, 2011, new Department of Justice rules took effect, specifying the "other power-driven mobility devices" (OPDMD) that could be used on trails by "individuals with mobility disabilities." If you manage a trail that is open to the public this rule applies to your facility.
posted Mar 30, 2018
MIG, Inc.
An important goal of this plan is to provide integrated recreational experiences for all visitors—with and without disabilities—employing the principles of universal design that are incorporated into these accessibility design guidelines.
Building an accessible trail on the Boothbay Region Land Trust's Penny Lake Preserve.
posted Jan 26, 2018
This webinar tells the story of how the Razorback Greenway was conceived, designed, funded, and implemented— a case study with multiple innovative and enabling solutions.
The Potts Memorial Park Trail Project used $28,000 in Recreational Trails Program (RTP) funds to make improvements to the trail, including making the trail more accessible to those with disabilities.
posted Apr 2, 2018
Stuart Macdonald
Questions and Answers to help trail managers respond to recent Department of Justice rule on Wheelchairs and Other Power-Driven Mobility Devices (updated February 19, 2011)
posted Nov 5, 2019
Ramps, typically used for building access, are often provided on trails.
Montgomery County used RTP funds to add a universally accessible trail in Rotary Park.
Page 4 of 6
Fort Worden State Park, Port Townsend, Washington
Routed and painted wood sign; Arches National Monument, Moab, Utah
Sign helps users find trail beyond point of interest; Arches National Monument, Moab, Utah
See more photo results
Nature Bridges
Nordic Manufacturing Ltd.
Presto Geosystems
See more business results


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6419 West Newberry Road, Gainesville, FL 32605 Directions
Since 1999
Damaged Screens, Charging Issues, Battery Replacement, Liquid Damage Treatments, Laptop Repairs, Ipad and Tablet Repairs, Advanced Board-Level Micro-Soldering, Data Recovery, Cases & Accessories
Gadget Guys has over 20 years of combined experience with technology and repairs. Their goal is to ensure a professional and customer service driven environment. Gadget Guys offers cost-effective and speedy solutions and their knowledgeable and friendly staff will properly diagnose your gadget and offer a solution. Buy, Sell & Trade gently used gadgets with warranty. They also buy and sell refurbished iPhones.
Discount: 10% Off All Repairs - If you can find a competitor, that offers a lower price than them they will match it plus $5 off your repair.
TBR® Inspection Report
4110 Southwest 34th Street, #7, Gainesville, FL 32608 Directions
Since 2014
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Orange Phone Care is a local small business owned and operated by a University of Florida alum. They provide quality iPhone and smartphone repair services at affordable prices. Orange Phone Care offers free estimates and most repairs take less than 20 minutes and backed by a lifetime warranty. They proudly provide multiple Florida cities with quality phone repair services including Daytona, Fleming Island, Gainesville, St. Petersburg and Tampa.
TBR® Inspection Report
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Repair, Buy, Sell, Trade and Accessorize All Popular Devices, Cases, Tempered Glass Screen Protectors, Standard Screen Protectors, Headphones, Chargers, USB Cables, Bluetooth Speakers, Smartphone, iPhone, Tablet and Computer Repair, Advanced Diagnostics, Charger Port Replacement, Power Supply, Audio Jack, Battery, Touch Screen & Lcd Replacement
Re-Tech provides mobile users with quality repair services, the largest selection of Certified Pre-owned Devices on the market, and a wide variety of phone and tablet accessories. The Company offers competitive prices and guarantees its exceptional repairs with Lifetime warranties. Re-Tech also gives the option to trade-in your device at any of their locations toward an upgrade, or receive cash or store credit. Every device they sell is backed with their 180-day warranty and comes with a 7-day exchange period.


text:
Warriors players react to the NBA suspending the season due to coronavirus
The NBA suspended the 2019-20 season after Utah Jazz center Rudy Gobert tested positive for the novel coronavirus, and several players across the league took to social media to share their reactions.
"Man we cancelling sporting events, school, office work, etc etc," Los Angeles Lakers forward LeBron James tweeted. "What we really need to cancel is 2020! Damn it's been a rough 3 months. God bless and stay safe."
Two members of the Golden State Warriors also tweeted their thoughts.
"2020 aint it," Stephen Curry wrote. "Don't know what to compare this situation to…just gotta buckle up and take care of yourself and those around you. Basketball will be back at some point but right now, protect yourself and stay safe out there!"
Rookie Eric Paschall also had things to say.
"This has been a wild rookie year!" he tweeted before adding, "Everyone be safe!"
Sports Illustrated's Chris Mannix reported Wednesday night the season may not resume even after the suspension.
Recommended Video:
"The people I've talked to tonight, they don't have any idea how long this is going to last, and if I had to characterize it any way, there's not a lot of optimism that this season is going to be able to be resumed," he said during a television appearance. "There's more of a sense that — and they're getting this from their own medical experts — there's a real possibility that this gets worse country-wide before it gets better.
"And if it continues to get worse, they're not going to come back and play in front of arenas. Now they've pulled the pin on the nuclear option, I do not believe based on my conversations they're going to come back and say, 'Alright, we're going to play games in empty arenas, smaller venues, just go get the TV games on the air.'"
The Warriors were scheduled to play the Brooklyn Nets Thursday night.
Sign up for 'The Daily' newsletter for the latest on coronavirus here.
- A full list of online coronavirus resources: Where to get your questions answered
- How a mild case of the novel coronavirus can quickly turn deadly
- California advises against even small social gatherings
- The two most common symptoms in coronavirus patients
- How coronavirus compares to epidemics of the past
- Coronavirus: Photos show empty scene at world's busiest attractions
- How to tell if you have a cold, the flu, or coronavirus
- These companies are asking Bay Area employees to work from home due to coronavirus
- 6 things travelers should know in the age of coronavirus
- The worst myths and misinformation about coronavirus
- Out of hand sanitizer? Here's how to make your own
- Are children at a lower risk for coronavirus complications?
- Are the elderly at a greater risk for coronavirus complications?
- Here's what BART will do if a passenger has coronavirus
Eric Ting is an SFGATE digital reporter. Email: email@example.com | Twitter:@_ericting


text:
by Matthew S. Batham
Published by Matthew S. Batham. 1st. 2006
Very good condition. A magical adventure set in a mysterious world of witches and warlocks. Pictorial cardwraps. 167 pages.
SIGNED by the author to half title-page and to a promotional card tucked inside the book. Spine faded (although it may meant to be like this!?). Lower edge of first 30 pages have a crease at the inner margin (book possibly dropped at some point?).
Stock no. 1313451
Price £15.00
Located at: Stella Books, Tintern


text:
17. French Fries
French fries taste so good, but being fried in trans fats and then heavily salted makes them a real cancer risk. Foods heated to such a high temperature also produce acrylamide, a known carcinogen found in cigarette smoke.
Alternatives – Check out some recipes for baked fries; using flavorful spices and olive oil produces a very tasty and much healthier side dish.
Continue Reading on Next Page
Continue Reading on Next Page


text:
LANSING, MI (MPRN)-- The state wants to make it easier for people to apply for public assistance.
This is all about the amount of assets a person can have and still qualify for food and cash assistance and State Emergency Relief. Starting November first, people can have up to $15,000 in assets and still be eligible for help. That's thousands of dollars more than they could previously, and now the amount will be the same for all three programs.
Currently, the caps are: $500 for State Emergency Relief; $3,000 for the Family Independence Program and other cash assistance programs; and $5,000 for food assistance.
"Getting help from the department is really hard," said Robert Gordon, Director of Michigan Department of Health and Human Services. "It is too hard for people. We ask too much of them."
Governor Gretchen Whitmer said the move is necessary because, "A family struggling to keep their head above water shouldn't have to become completely destitute to get a little help."
The department is also trying to make the application process easier for applicants and MDHHS employees. Instead of filling out an asset checklist and having department employees verify a persons' assets, the department will take people's word for what they own and how much money they have in most instances. The department will accept an applicant's statements of assets instead of requiring applicants to fill out an assets verification checklist. There are some instances where the department will verify, for example, if the person or family has more than $10,000 in assets.
Gordon said the risk of fraud is low.
"We should not visit the sins of a very few people on hundreds of thousands of Michiganders who are trying to do the right thing and trying to get by," he said.
Vehicles will also no longer be counted as an asset for food assistance.


text:
OROVILLE — Tuesday, Butte County supervisors will be asked to extend a ban on medical marijuana dispensaries for 22 months and 15 days.
On Jan. 12, the supervisors adopted an "interim urgency ordinance" that established a 45-day moratorium on new dispensaries or the operation of existing ones.
The urgency measure was enacted to give the county time to craft an ordinance that would either establish the criteria for allowing dispensaries or outlaw them.
The law allows for the extension of an urgency ordinance of this sort and in a message to the board, interim Chief Administrative Officer Scott Tandy is asking for that.
He said there are court cases in the system on issues related to the placement of dispensaries and those cases could well be settled by the end of the extension, which would clarify what the county can and cannot do.
The proposal to extend the ban will be the topic of a time-certain public hearing, scheduled for 10:30 a.m.
In another time-certain hearing, set for 2 p.m., the supervisors will almost certainly undo a decision that was originally made in August 2008.
At that time, the board, on a split vote, approved the operation of the New Era Mine, an open pit gold mine in the Dry Creek region of the county.
Opponents of the mine operation filed suit in Butte County Superior Court, and on Dec. 10, 2009, the court ruled in their favor.
In response to that decision, county staff is urging the board to rescind the 2008 resolution allowing the mine to operate, rule that mining operations at the New Era were abandoned in the 1990s and that the mining and reclamation permit was no longer valid.
County staff also recommends the supervisors advise the mine owners, North Continent, that if they want to operate the mine, they will have to seek a new permit.
The proposal also advises the supervisors to grant the appeal to block the mine that was filed by a number of area residents.
Besides saying that the mine cannot be operated, the staff proposal asks that the owners be held responsible for reclamation of the mine site at a cost of more than $267,000, and that the firm reimburse the county Department of Development Services $50,000 in costs it incurred as a result of the mine.
One item on the board"s consent agenda will have an impact on people who borrow DVDs from the Butte County Library system.
The proposal that was the topic of a cost study is to charge a $1 fee for a three-day "loan" of newly released DVDs.
By law, users" fees are not allowed to do more than cover the "estimated reasonable" cost of providing the service.
"In order to justify the fee for DVD rentals, a time study was conducted in Paradise, Oroville and Chico branches to determine the estimated county cost," states the report that is going to the supervisors.
Since the item is on the consent agenda, it won"t be discussed or debated, unless a supervisor, member of the staff or a member of the public has it "pulled." All of the items on the consent calendar are passed with a single vote of the board.
Butte County Board of Supervisors
9 a.m. Tuesday
Administration Building
25 County Center Drive, Oroville


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Anne Kingston on the Cultural Script and the Real Work in Recovery
Anne Kingston on the cultural script and the real work in recovery
Not that rehab is always required in order to recover from addictions, as Anne Fletcher points out in her acclaimed book Inside Rehab: The Surprising Truth About Addiction, published this year. The myth-busting investigation of U.S. rehab facilities … Read more on Macleans.ca
Elements Behavioral Health Enhances Its Christian Addiction Treatment …
The Recovery Place is one of Florida's premier drug and alcohol treatment centers. The Recovery Place provides individualized, client-centered addiction treatment with multiple programs to fit clients' needs, including detox, day treatment, intensive … Read more on PR Web (press release)
Throwback Thursday: 5 biggest turkeys in Suns history
Three months after the Finals and after signing a five-year, $ 9 million dollar contract, Dumas was back in rehabilitation for drugs and alcohol. He returned to the Suns … The Suns had to settle on Neal Walk, a 6-foot-10 center out of Florida. Walk … Read more on Valley of the Suns


text:
|North Carolina
|Type of Government:
|FSC Category:
|NAICS Category:
|Posted Date:
|Jan 29, 2020
|Due Date:
|May 30, 2020
|Solicitation No:
|Bid Source:
|Please Login to View Page
|Contact information:
|Please Login to View Page
|Bid Documents:
|Please Login to View Page
Delineated Area:
North: Highway 540
South: Highway 440 & Highway 64
West: Highway 540
East: Highway 440
Office Minimum Sq. Ft. (ABOA):
Office Maximum Sq. Ft. (ABOA):
Parking Spaces (Total):
Parking Spaces (Surface):
34 secured (See parking requirement details below under 'Additional Requirements')
Parking Spaces (Structured):
Parking Spaces (Reserved):
Full Term:
15 Years
Firm Term:
10 Years
Option Term:
1 Option for 5 Years
Additional Requirements:
Space shall be located in Flex or a warehouse.
•· A building equipped with at least two points of ingress/egress with a direct route to the offered space is required.
•· Offered secured parking area must be discrete.
•· The offered space must include a drive-in bay large enough to accommodate a 10 ft. long bucket truck that requires a minimum height clearance of 10 ft. or higher measured from floor to the lowest obstruction. The rollup door must be at least 10' wide.
•· This requirement will house a federal law
enforcement office where employees can or have the potential to carry weapons, and people may be temporarily held and/or questioned.
•· A minimum of 1,000 SF should be 12 feet and 0 inches or higher measured from floor to the lowest obstruction.
•· If offered space is on the 2 nd floor or higher a loading dock or loading area is required.
•· If offered space is on the 2nd floor or higher, a freight elevator is required.
Building must have a roll-up door with minimum height 10'; minimum door width is 10' to service 10' trucks.
The site must provide for 34 dedicated parking spaces.
The parking spaces may be a mix of secure and reserved spaces, depending on what the existing site allows for. As much as possible, the parking spaces should be discrete, and allow for obscure ingress and egress of the building by personnel. Parking lot layout should also allow for direct and unimpeded access to/from the street, allowing for quick vehicular ingress and egress. If necessary, the designated parking area should have privacy fencing (6 feet high minimum) if it backs to residential property.
Although the office space may be located close to major arteries or highway system, the entering and exiting of the building by personnel should be obscure as much as possible.
The ideal office space is not to be collocated with certain entities such as Probation Office, Public Defender's Office, or other Court family to help minimize mix between that group and personnel
The space must also avoid collocating with child care centers, grade schools, or other such facilities to with a high concentration of children and families.
On-site parking must meet/or exceed local code.
Reserved car parking spaces must be available adjacent to the suite.
All offered space must be contiguous within the building.
•· The Space shall be located in a modern quality Building of sound and substantial construction with a facade of stone, marble, brick, stainless steel, aluminum or other permanent materials in good condition and acceptable to the GSA Lease Contracting Officer. If not a new Building, the Space offered shall be in a Building that has undergone, or will complete by occupancy, modernization or adaptive reuse for the Space with modern conveniences.
•· A fully serviced lease is required.
•· Normal Hours of Operation: 7:00 AM - 5:00 PM M-F; offered space must be accessible 24/7.
Offered space shall not be in the 100 or 500 year flood plain.
Space must meet the Federal Government's standards for fire and life safety and be capable of being fully handicapped accessible. Building must be capable of meeting the Government's security requirements. All services, supplies, utilities, and tenant alterations are to be provided as part of the rental consideration.
Brokers or agents may represent property owner, but written authorization from the owner is required. Subleases are not acceptable. Space should not be located within the 100-year flood plain
Not all minimum requirements are reflected in this advertisement.
Other typical and special requirements as called for in the Request for Lease Proposal (RLP) package.
To submit a location for inspection, please forward information by 5:00 PM CDT, May 30, 2019.
Expression of Interest Submission Instructions/Requirements:
Please provide the following basic information and descriptions pertaining to the building you propose for consideration.
All submissions should include the following information:
•(1) (1) Name of owner;
(2) Age of building;
(3) Total existing gross square feet, and gross square feet per floor;
(4) Site plan depicting the building and parking;
(5) Floor plan and gross square footage of proposed space;
(6) Identification of on-site parking;
•(7) (7) Address or described location of building;
Location on map demonstrating the building lies within the delineated area;
(9) FEMA map evidencing that the property lies outside the 100-year floodplain;
(10) A narrative and map describing proximity of the building to the nearest bus and/or train stop, and major transportation routes;
(11) Evidence of ownership, if building submitted by owner. Non-Owners (e.g. brokers) must provide evidence of authority granted by property owner to submit the property; and
(12) Any information related to title issues, easements, or restrictions on the use of the building.
In no event shall the Offeror enter into negotiations or discussions concerning the space to be leased with representatives of any Federal Agency other than the officers and employees of General Services Administration (GSA) or their authorized representative, Studley, Inc.
All Offers of Space must be submitted in writing to: Melissa Hein
Managing Director
Savills Inc., GSA Broker Contractor
399 Park Avenue
11th floor
|May 9, 2019
|[Presolicitation] General Services Administration (GSA) seeks to lease space in Raleigh, NC
|May 10, 2019
|[Presolicitation] General Services Administration (GSA) seeks to lease space in Raleigh, NC
|Jan 29, 2020
|[Award Notice (Original)] AWARD NOTICE– THE U.S. GOVERNMENT SEEKS TO LEASE APPROXIMATELY 7,628 ANSI/BOMA OFFICE AREA SQUARE FEET OF OFFICE FLEX SPACE IN RALEIGH, NC
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Bid Number Description Date Issued Bid Opening Date Bid Opening Time Help 281-LOI
State Government of North Carolina
Bid Due: 7/17/2020
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State Government of North Carolina
Bid Due: 7/21/2020
Bid Number Description Date Issued Bid Opening Date Bid Opening Time Help 19-RFI-015124-DAD
State Government of North Carolina
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State Government of North Carolina
Bid Due: 12/15/2020


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Lexington, KY
Affordable Housing Strategy
Lexington, KY
Affordable Housing Strategy
During 2013 and 2014, czb worked with the Lexington-Fayette Urban County Government to create a framework for affordable housing strategy and development. The county's needs were stark: when czb arrived, there was a $36 million gap in affordable housing funding and a shortage of more than 6,000 affordable housing units.
"How do we create the political will to invest in fixing our affordable housing gap?"
Median Household Income
of adults have a bachelor's degree or higher
of all households live in poverty
affordable housing funding gap per year
affordable housing unit shortage
Assessment & analysis.
czb analyzed demographic factors, wages, property values, housing assistance, and rent prices in Lexington, a market of approximately 125,000 households.
A truth revealed.
czb's findings showed Lexington was meeting the needs of households with incomes of $40,000 or more but that there existed a 6,000-unit shortage of homes for the city's lowest-income households. Our analysis showed that the shortage would likely climb by 400 units each year, presenting a growing problem for the city's poor.
To fund the subsidy required to provide 6,000 affordable housing units, czb proposed the creation of an affordable housing trust fund, requiring that the local government commit to an initial investment of between $3 million and $4 million.
In 2014, Lexington's Urban County Council voted unanimously to allocate $3 million to establish a Lexington Affordable Housing Trust Fund and follow that up with $2 million annually over the next five years.
Program directors have used that fiscal commitment to leverage more than $10 million in additional funding for the construction or rehabilitation of affordable housing across the city. More than 430 affordable housing units have been built or renovated since the city began allocating taxpayer money to the fund.
2001 Mount Vernon Avenue
Alexandria, VA 22301


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With the shut down of Xively I have had to get rid of my bridge unit and revert back to the USB cable. Can anyone help me with the PVBC configuration so I can upload my consumption data to PVOutput…Thanks in advance Bill
Current Cost and PVBC USB Setup
Sunny_Boy #1
mspohr #2
This thread has a lot of information that could help
Sunny_Boy #3
Thanks for the tip I have been watching this thread however was hoping to just us the usb cable thanks.
maximo #4
I am in the same boat. Ran the bridge with c.c. for years. Before then i used to run pvbc with my pc until I had issues and the developer Dennis M-F who was originally really active went off the grid and stopped supporting it about 2014.
I use Oxley Solar app to upload my generation but want to use my envir and usb cable to upload consumption.


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Private sector lender ICICI Bank Ltd on Friday raised Rs1,635.33 crore by selling shares in its life insurance arm as part of the Rs 6,056 crore initial public offering (IPO) beginning next week, the company said in a stock exchange filing.
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In 2017, the Tax Cuts and Jobs Act eliminated the income tax deduction for investment expenses through the year 2025, leading many family offices to look for new ways of reducing costs.1
One option is for the family office to convert from a cost-basis model to a profit-based model, which might allow it to classify its operating expenses as "ordinary and necessary expenses incurred in a trade or business." This would make those expenses tax deductible.2
However, in order to take advantage of this, a family office must be properly restructured as a management company to demonstrate its status as a bona fide business. Two important Tax Court cases — Lender Management LLC v. Commissioner and Hellmann v. Commissioner — provide a clear sense of what a family office needs to do to achieve this and properly convert expenses that are currently non-deductible into fully deductible trade or business expenses.
The Internal Revenue Code does not define the terms "trade or business." Whether a particular activity falls under the umbrella of "trade or business" is thus determined on a case-by-case basis.
There are, however, some precedents to consider. For instance, it has been established that in order to be engaged in a trade or business, a taxpayer must be involved in the activity "with continuity and regularity" and that the primary purpose of the activity "must be for income or profit."3 Furthermore, investors are not considered to be engaged in a trade or business when managing and monitoring their own investments.4
The Lender and Hellmann cases offer further clarity for family offices interested in navigating these distinctions.
In 2005, the Lender family reorganized its family office, creating Lender Management LLC. In a 2017 ruling, the Tax Court held that Lender Management's activities, which involved providing investment management services to others (i.e., family members who did not have an ownership interest in the management company) for profit, were sufficient to constitute a trade or business, allowing it to fully deduct trade or business expenses.
In 2018, the Hellmann family sought similar recognition for its management company, GF Family Management, LLC (GFM). However, the Tax Court refused to issue a summary judgement, instead requesting further factual development, either through a trial or stipulated facts. Believing they would not prevail under these circumstances, the Hellmann family relented, paying the tax they owed and abandoning their effort to be recognized as a trade or business for tax purposes.
From these outcomes, we can glean several important guidelines that family offices should be mindful of when considering restructuring as a management company.
The management company must provide services beyond just investing assets, and have the personnel and expertise to do so. Primary decision-making for investments should rest with the management company.
In Lender, the Tax Court found that Lender Management did substantially more than just keeping records and collecting interest and dividends. In fact, the company provided one-on-one investment and financial planning services to family members. Furthermore, the Chief Investment Officer for Lender Management, Keith F. Lender, satisfied the expertise criteria, having an undergraduate degree in business from Cornell and an MBA from Northwestern. He headed up a team of five employees with an annual payroll of over $300,000 who made investment decisions based on the goals and needs of their family member clients.
Compensation paid to the management company must be greater than a normal investment return, and the taxpayer's ownership of the management company should not be in proportion to their interest in the investment partnership.
This was the major sticking point in Hellmann. Each family member had a 25% interest in the management company. They also each had a 25% interest in the assets being managed by the associated investment partnerships. Because these two streams of income were perfectly proportional, the compensation paid to GFM effectively replaced the investment income each family member would've derived from the investment portfolio.
This was not an issue for Lender Management. Keith Lender owned 99% of the management company, but only a minority interest in the associated investment partnerships. The 25% profits interest and the 2.5% fee on assets under management in the LLCs payable to Lender Management indicated that the management company was receiving a discrete fee for services provided to others and not simply a return on its own investment.
Ideally, no family member would have an ownership interest in the management company; if necessary, it should be limited to just a few family members.
The family should not be especially cohesive or concentrated.
Lender Management provided individualized service to a large, geographically distributed family with diverse needs. Many of the family members did not know one another, and those who did were not always in agreement. Some conflicts were so severe that the members involved refused to attend meetings together. As such, the family in Lender did not act collectively or with a singular goal in mind, and Lender Management did not make decisions for the family as a group. Rather, it treated them individually, addressing their specific needs and risk tolerances.
In Hellmann, the circumstances were almost exactly the opposite. GFM represented only four family members, all of whom lived in the Atlanta area and got along well with one another.
The family members should be free to withdraw their capital interest and hire different investment managers.
Lender Management allowed its clients, the family members, to terminate its relationship with the investment partnerships at any time, subject to liquidity requirements. This means that family members were free to seek investment management services from a different provider if they were unhappy with the management company's investment performance.
From these two cases, we can see that the Tax Court is predominantly concerned with the nature of the services provided by the management company, and the family members' relationships with the management company and among themselves, when determining whether a family office is eligible to deduct operating expenses. A management company that has a qualified staff of employees, accepts fees for individualized services and is largely independent from the familial client base it serves should be able to make the case that it is operating as a trade or business and is thus entitled to deduct its operating expenses.
1 Per Section 67(g) of the Tax Cuts and Jobs Act of 2017
2 Per Section 162 of the Internal Revenue Code
3 Commissioner v. Groetzinger, 480 U.S. 32, 35 (1987)
4 Whipple v. Commissioner, 373 U.S. 193 (1963); Higgins v. Commissioner, 312 U.S. at 218 (1941)