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Do the transcriptional mechanisms that control an individual's mitochondrial content, PGC1α (peroxisome proliferator-activated receptor γ coactivator-1α) and NRF1 (nuclear respiratory factor-1), also cause differences between species? We explored the determinants of cytochrome c oxidase (COX) activities in muscles from 12 rodents differing 1,000-fold in mass. Hindlimb muscles differed in scaling patterns from isometric (soleus, gastrocnemius) to allometric (tibialis anterior, scaling coefficient = −0.16). Consideration of myonuclear domain reduced the differences within species, but interspecies differences remained. For tibialis anterior, there was no significant scaling relationship in mRNA/g for COX4-1, PGC1α, or NRF1, yet COX4-1 mRNA/g was a good predictor of COX activity (r2 = 0.55), PGC1α and NRF1 mRNA correlated with each other (r2 = 0.42), and both could predict COX4-1 mRNA (r2 = 0.48 and 0.52) and COX activity (r2 = 0.55 and 0.49). This paradox was resolved by multivariate analysis, which explained 90% of interspecies variation, about equally partitioned between mass effects and PGC1α (or NRF1) mRNA levels, independent of mass. To explore the determinants of PGC1α mRNA, we analyzed 52 mammalian PGC1α proximal promoters and found no size dependence in regulatory element distribution. Likewise, the activity of PGC1α promoter reporter genes from 30 mammals showed no significant relationship with body mass. Collectively, these studies suggest that not all muscles scale equivalently, but for those that show allometric scaling, transcriptional regulation of the master regulators, PGC1α and NRF1, does not account for scaling patterns, though it does contribute to interspecies differences in COX activities independent of mass.
- scaling
- cytochrome c oxidase
- peroxisome proliferator-activated receptor γ coactivator-1α
- nuclear respiratory factor-1
- mitochondria
- lactate dehydrogenase
all animals have the need to alter muscle mitochondrial content during development and in response to physiological and environmental challenges. Central to this remodeling process are transcriptional master regulators, namely peroxisome proliferator-activated receptor γ coactivator-1α (PGC1α) and nuclear respiratory factor-1 (NRF1) (20, 33, 34). Mitochondrial biogenesis requires the coordination of hundreds of nuclear genes encoding mitochondrial structural and enzymatic proteins, including the proteins that control replication, transcription, and translation of mitochondrial DNA. This onerous task is mediated by the coactivator PGC1α, which regulates suites of nuclear-encoded metabolic genes through direct and indirect association with DNA-binding transcription factors. Central to control of genes in oxidative phosphorylation are NRF1, NRF2, and nuclear hormone receptors [e.g., peroxisome proliferator-activated receptor (PPAR), retinoic acid receptors, thyroid hormone receptors]. These transcription factors confer upon an individual the capacity to change muscle mitochondrial content during development (5, 13, 24, 25), and in response to exercise (1, 3, 44), dietary manipulations (33, 37), hormonal and energetic changes (2, 18, 22, 33, 45, 46). In this study, we ask if the transcriptional mechanisms that control mitochondrial content in individuals are also responsible for maintaining differences seen between species.
Interspecies differences in the muscle metabolic phenotype can arise for many reasons, such as activity levels and environment challenges. One paradigm that is perhaps most amenable to addressing interspecies differences is the variation arising in relation to body mass, or metabolic scaling. It has been known for more than 100 years that animals differing in body mass show allometric scaling of metabolic rate (29). There is no consensus on either the underlying origins or even the exact mathematical relationships (9, 16, 19, 28, 42, 43), but it is generally accepted that a plot of log mass vs. log mass-specific metabolic rate demonstrates a negative slope, or scaling coefficient (b) ranging from −0.25 to −0.33. Muscle metabolic enzymes also show allometric scale effects in fish (36) and mammals (15), although typically less than is seen in metabolic rate (29, 31). Interestingly, mitochondrial enzymes scale negatively and glycolytic enzymes scale positively. With scaling coefficients of 0.25–0.33, an animal 10 times larger than another would be expected to have about half the oxidative enzyme activity and twice the glycolytic enzyme activity. Simple stochastic models, such as size-dependent differences in turnover of mRNA or protein, cannot easily explain how muscles of larger animals maintain fewer mitochondria yet greater activities of glycolytic enzymes; the scaling patterns in oxidative and glycolytic enzymes appear to demand active regulation.
To explore the origins of interspecies differences in metabolic enzyme activities, we used rodent skeletal muscles to identify the patterns in 1) metabolic enzyme activities, both oxidative [cytochrome c oxidase (COX)] and glycolytic [lactate dehydrogenase (LDH)], 2) myonuclear content (mg DNA/g tissue), 3) enzyme transcript levels [citrate synthase (CS)], COX4-1, LDHA, and 4) transcript levels of two putative master regulators, PGC1α and NRF1. To explore the evolutionary variation in PGC1α expression, we compared the promoters of >50 mammals, constructing luciferase reporter genes from a subset to assess evolutionary variation in PGC1α proximal promoter strength.
Tissue Samples
In addition to published sequences, we used tissues from various sources to generate DNA sequence, promoter constructs, enzyme measurements, and RNA analyses (Table 1). All collections from live animals were made in accordance with an approved Queen's University Animal Care Committee protocol.
Enzyme and RNA analyses.
Animals used for tissue enzyme and RNA analyses were derived from both lab and field collections. Charles River Canada supplied lab mice (Mus musculus CD-1 males), gerbils (Meriones unguiculatus), and hamsters (Mesocricetus auratus). Rats (Rattus norvegicus Sprague-Dawley) were sentinel males from the animal care centre at Queen's University. These samples were euthanized using pentobarbital 150 mg/kg. Female guinea pigs (Cavia porcellus) were control animals used in the Reynolds and Brien lab at Queen's University and were anaesthetized and decapitated. Dwarf hamsters (Phodopus campbelli) were retired from the Wynne-Edwards colony at Queen's University and were killed by cervical dislocation. Groundhogs (Marmota monax) muscles were collected on our behalf by Cornell University staff from their colony.
Wild rodents were trapped using Sherman box traps (North American deer mice, Peromyscus maniculatus; eastern chipmunks, Tamias striatus) or Havahart traps (eastern grey squirrels, Sciurus carolinensis; American red squirrels, Tamiasciurus hudsonicus). Animals were euthanized using 150 mg/kg pentobarbital after being removed from the trap. Beaver (Castor canadensis) samples were contributed by a local trapper as part of a pest removal, killed by rifle.
DNA analysis.
Samples from many local species were obtained from buccal cell swabs of living specimens (human, Homo sapiens; domestic dog, Canis lupus familiaris; housecat, Felis catus), meat from a local butcher (domestic sheep, Ovis aries), road kill (porcupine, Erethizon dorsatum), or through hunters and trappers (moose, Alces alces; white-tailed deer, Odocoileus virginianus; black bear, Ursus americanus; least weasel, Mustela nivalis; American mink, Neovison vison; fisher, Martes pennant; raccoon, Procyon lotor; wolf, Canis lupus; coyote, Canis latrans; meadow vole, Microtus pennsylvanicus). Many samples of exotics were obtained from colleagues in Biology (mouflon, Ovis aries orientalis; chamois, Rupicapra rupicapra; muskox, Ovibos moschatus; giraffe, Giraffa camelopardalis; rhinoceros, Diceros bicornis; tapir, Tapirus bairdii; lynx, Lynx canadensis; brown hyena, Hyaena brunnea; polar bear, Ursus maritimus; gorilla, Gorilla gorilla). Others were provided by Kevin Campbell (University of Manitoba; masked shrew, Sorex cinereus; water shrew, Sorex palustris; star-nosed mole, Condylura cristata; coast mole, Scapanus orarius). The Metro Toronto Zoo provided samples of lion (Panthera leo) and capybara (Hydrochoerus hydrochaeris). In each case, DNA sequences were based upon a single individual.
Enzyme Analyses
We made measurements from whole gastrocnemius (GAST), soleus (SOL), and tibialis anterior (TA) from 12 rodents from 5 families: Muridae: lab mouse, deer mouse, gerbil, lab rat; Cricetidae: dwarf hamsters, Syrian hamsters; Sciuridae: eastern chipmunks, eastern grey squirrels, American red squirrels, groundhogs; Caviidae: guinea pigs; and Castoridae: beaver. Muscles were excised from euthanized animals and flash-frozen in liquid nitrogen. GAST, SOL, and TA were collected from each species except groundhog, for which no SOL was available, and the TA is called tibialis cranialis.
Muscles for enzyme analyses were powdered in liquid nitrogen and stored at −80°C. Between 50 and 75 mg of powdered tissue were homogenized in 20 vol of extraction buffer (20 mM Tris, 0.6 mM lauryl maltoside, pH 8.0) using a ground glass homogenizer on ice.
COX was assayed with 20 mM Tris (pH 8.0), 0.6 mM lauryl maltoside, and 100 μM cytochrome c. Cytochrome c was reduced with an excess of ascorbate, dialyzed exhaustively against 20 mM Tris (pH 8.0), and then frozen in aliquots. Each sample was assayed in a 96-well spec plate using a Molecular Devices SpectroMax Plus spectrophotometer (Sunnyvale, CA) held at a temperature of 25°C. This assay was done immediately after homogenization in quadruplicate with samples diluted 40-fold in extraction buffer and read for 90 s at a wavelength of 550 nm.
LDH was assayed in 20 mM HEPES (pH 7.0), with 1 mM pyruvate and 0.2 mM NADH. Homogenates were diluted 40-fold in assay buffer (20 mM HEPES, pH 7.0) immediately prior to the assay to obtain acceptable rates.
The enzyme homogenates were also used to measure DNA contents of rodent muscle, an indicator of myonuclear content. Aliquots of homogenates were digested in proteinase K (0.2 mg/ml) in 200 mM NaCl, 20 mM Tris, 50 mM EDTA, 0.10% SDS (pH 8.0). The digest was treated with RNase A (10 μg/ml) and then quantified using PicoGreen against standards treated in the same manner.
RNA Analyses
RNA was extracted from ∼30–50 mg of powdered rodent TA muscle using the Qiagen RNeasy kit (Mississauga, ON, Canada) following manufacturer's instructions. RNA was quantified using the 260 nm absorbance on the Molecular Devices SpectroMax Plus spectrophotometer. From this total RNA, cDNA was made from 2 μg RNA using the Qiagen QuantiTect Rev. Transcription kit (Mississauga, ON, Canada) as per the manufacturer's instructions. Real-time primers (Table 2) were constructed for COX4-1, NRF-1, PGC1α, CS, and LDHA based upon near perfect homologies in published sequences from rodents and nonrodents. To compare transcript levels between species, it was essential that the primers for a particular gene were identical for all species, with no mismatches or wobble sites. For each species, we verified that the real-time PCR reaction was linear with respect to template concentration and amplification generated a product that demonstrated a single dissociation curve. A species was excluded from the analysis when it failed to generate an appropriate dilution series or when the PCR product produced multiple dissociation peaks. Analysis was performed on each cDNA of interest and normalized to β-actin (BACT) to determine the threshold cycle (CT). The relative cDNA for a gene of interest is expressed as 2▵CT(BACT−GENE of INTEREST). The values for each gene and species were transformed into relative cDNA levels per g tissue by multiplying mg RNA per g tissue. The resulting values for multiple genes (relative mRNA per g tissue) are then arbitrarily adjusted to fit within the same values to facilitate comparisons between genes.
Real-time analysis was conducted on an ABI 7500 RT-PCR system (Foster City, CA) using 10 min at 95°C (1 cycle) followed by 40 cycles of 15 s at 95°C, 30 s at annealing temperature and 36 s at 72°C. Reactions (25 μl) contained 1× SYBR green master mix (Roche Applied Sciences, Basel, Switzerland), 100 ng cDNA, and 0.58 μM of each primer.
COX is a multimeric protein and it was not feasible to measure the expression of all subunits. COX4-1 is the muscle isoform for the subunit COX4. We developed consensus primers based upon published sequences that enabled us to amplify single products of the appropriate size in all species except American red squirrel and Syrian hamster, which yielded double dissociation peaks.
LDH is a tetrameric protein composed of subunits from five genes. In skeletal muscle, the enzyme is composed essentially from muscle subunits encoded by LDHA. We developed consensus primers based upon published sequences that enable us to amplify single products of the appropriate size in all species except beaver.
CS is a homodimeric protein with no paralogs. Its consensus primers based upon published sequences that enable us to amplify single products of the appropriate size in all species except guinea pig, which yielded a double dissociation peak.
NRF1 is a monomeric protein with no paralogs. The real-time primers were able to amplify appropriate amplicons from all species.
PGC1α is a monomeric protein with no isoforms, although 2 paralogs exist with overlapping functions. The primers were developed were specific for PGC1α and amplified a single product in all species.
Promoter Analyses
Promoter sequence analysis began with surveys of published genomic sequences. Using this information, a proximal promoter (∼700 bp) was amplified from various taxa to generate long promoter sequences. Beyond this point, there was very little homology between species. From these sequences, we designed internal primers that were able to amplify a shorter proximal promoter (∼600 bp) from a subset of species to conduct reporter gene analyses.
DNA purification.
DNA from tissues was used to provide material for promoter characterization. Tissue samples were digested in proteinase K, as described in the enzyme analyses section. The DNA was extracted in an equal volume of phenol-chloroform-isoamyl alcohol (25:24:1). After centrifugation (10 min at 1,700 g), the aqueous phase was recovered and DNA was precipitated by adding 0.1 vol of ammonium acetate (7.5 M) and 2 vol of 100% ethanol, followed by centrifugation (3 min at 1,700 g). The pellet was washed with 70% ethanol, air-dried, and resuspended in 250 μl TE buffer (10 mM Tris·HCl, 1 mM EDTA, pH 8.0). DNA purity was assessed using absorbance at 260 nm and 280 nm and then quantified based on the 260 nm reading.
Published PGC1α promoter sequences.
Promoter sequences were available for several placental mammals from GenBank or ENSEMBL databases (Table 1). In each species for which genomic data was reliable, we downloaded and aligned about 700 bp of sequence immediately proximal to the start codon of the PGC1α gene.
There was complete conservation of the arrangement of the gene between species in that there was no TATA box evident and a very short 5′-untranslated region upstream of the translation start codon. Additionally, the sequence of the gene corresponding to the NH2 terminus of the protein was very highly conserved, permitting the design of a universal reverse primer that worked for all mammals examined.
Sequencing novel promoters.
Clade-specific consensus primers were developed for about 700 bp (Table 3) of the proximal promoter region. Beyond this region, there was considerable divergence between even closely related species. This long promoter was amplified from select terrestrial mammals. Each amplification reaction contained 1× PCR buffer, 1.5 mM MgCl2, 2.5 units Taq polymerase (Qiagen, Mississauga, ON, Canada), 200 μM dNTP (Promega, Madison, WI), between 20 and 50 ng template, and water to make a final volume of 25 μl. Amplifications included 2 min at 94°C, followed by 35 cycles (1 min at 94°C, 30 s in a gradient of 57–61°C, 1 min at 72°C), and final extension at 72°C for 10 min. The resulting PCR products were visualized on 2% agarose gels with ethidium bromide. DNA bands of the appropriate size were excised and extracted from the gel, ligated into a pDrive cloning vector (Qiagen, Madison, WI), transformed into DH5α-competent cells (Invitrogen, Carlsbad, CA), and grown on agar plates containing 50 μM IPTG, 200 μM X-gal, and 130 μM ampicillin. Colonies positive for inserts were grown in Lysogeny broth with ampicillin (50 ng/μl). Plasmids were isolated with QIAprep Spin Miniprep Kit (Qiagen, Mississauga, ON, Canada) and quantified at 260 nm then sequenced (Robarts Research Institute, London, ON, Canada). The sequences obtained (see Table 1 for GenBank references) were then used to determine shorter proximal promoter sequences (∼600 bp), which were used in promoter comparisons or generation of reporter genes.
Transcription factor binding site identification.
Putative elements in the PGC1α promoter were identified, but no effort was made to assess if the elements were functional in the various species. Given the variability in consensus sequences reported in the literature, we focused on the elements most highly conserved within the promoter. Our first approach was high-stringency searches using PATCH (pattern search for transcription factor binding sites) and TRANSFAC 6.0: minimum length 6, maximum mismatch 1, penalty 100, lowest score 87.5. We excluded noncanonical sequences and or sequences that contained nucleotide mismatches. The following elements were identified in the PGC1α promoter based upon TRANSFACT and PATCH: specificity protein 1 element (GGCTGGG), cyclic AMP-response element (GACGTCA), hepatocyte nuclear factor 3 element(TGTGTGC), inverted CCAAT element (ICE)/nuclear factor Y element (ATTGG), and E1 box (CATGTG). We also noted additional conserved sequences that have been identified (35) as insulin-responsive elements T(A/G)TTTT(T/G).
Reporter gene constructs.
We used the pDrive plasmids containing the sequenced long promoter to create a series of shorter proximal promoters to be used for construction of reporter genes. The PCR primers were designed from the species-specific PGC1α promoter sequence and included the restriction sites Xho1 and Mlu1. The cloned fragment was digested with Mlu1 and Xho1 (New England Biolabs, Ipswich, MA) then ligated into a luciferase reporter plasmid (pGL2 basic, Promega). The plasmid had been prepared by digestion with Mlu1 and Xho1, treatment with calf intestinal phosphatase, and gel purification. Each reporter construct was sequenced after creation. Plasmids for transfection were transformed, grown, and purified as described in the section on sequencing novel promoters, quantified using PicoGreen and diluted to 250 ng/μl.
Cell culture and transient transfections.
Two murine muscle cell lines, Sol8 and C2C12, were used to assess promoter strength. All cells were grown in Dulbecco's modified eagle medium (DMEM; GIBCO, Burlington, ON, Canada) supplemented with either 10% (C2C12) or 20% (Sol8) fetal bovine serum (FBS, GIBCO) and containing penicillin, streptomycin, and neomycin (GIBCO). Growth conditions were maintained at 37°C with 5% CO2 and 95% humidity. When cells reached ∼50% confluence on 12-well plates, the medium was replaced with 1.5 ml per well fresh DMEM/FBS. Plasmids were prepared as follows. Renilla plasmid (4 μg) was mixed with DMEM (640 μl), and dispensed as 8 μl aliquots (50 ng per aliquot). To each aliquot, 2 μl of unique luciferase reporter plasmid (500 ng) were added, mixed, and stored at room temperature. In a separate tube, 1.5 μl FuGene (Roche Applied Science, Basel, Switzerland) was added to 40 μl DMEM (no serum), mixed by titration then incubated 5 min at room temperature. The Plasmid mix (10 μl DMEM with 500 ng luciferase reporter plasmid and 50 ng Renilla plasmid) was transferred to the FuGene mix (40 μl DMEM with 1.5 μl FuGene), then incubated at room temperature. After 30 min, the FuGene/plasmid/DMEM mixture was added drop-wise into one well of the 50% confluent 12-well plates, gently agitated, and returned to the incubator. After 24 h the medium was changed to differentiation media (DMEM, 2% horse serum, with penicillin, streptomycin, and neomycin). After 3 additional days, cells were collected for luciferase measurements.
Luciferase expression was determined using the Dual Luciferase Assay System (Promega) according to the manufacturer's instructions on a luminometer (Lmax; Molecular Devices, Sunnydale, CA) with automatic injectors. Cells were harvested in 100 μl of 1 × passive lysis buffer and frozen at −80°C. To measure firefly luciferase, 100 μl of luciferase assay reagent (LARII, Promega) were added to 10 μl of room-temperature cell lysate in a white 96-well plate. Relative light units were measured for 30 s, with a 1 s premeasurement delay. To measure Renilla luciferase, 100 μl of Stop & Glo solution (Promega) were then added to the same well, and relative light units were again measured for 30 s, with a 2 s delay.
The variation in number of animals used for each measurement complicated the analyses. We had only one beaver sample, but we conducted all analyses on this species with pseudoreplication. Regression analyses used means for each species.
The influence of body mass on enzyme activities was assessed using a simple linear regression model. To assess if transcription factor mRNA could be used to predict downstream events, we compared the 12 means of each parameter using simple linear regression and multiple regression.
The perception that mammalian muscle enzymes show an allometric scaling pattern have been based upon relatively few studies that focus on single select muscles and employ distantly related species (29). Our first goal was to assess if allometric scaling of metabolic enzymes was evident in one family of mammals (rodents).
Metabolic Enzymes in Rodents
Twelve species of lab-reared and wild-caught rodents were analyzed for hindlimb muscle nucleic acids (Table 4), enzyme activities, using the mitochondrial enzyme COX and the glycolytic enzyme LDH. COX and LDH were measured in enzyme homogenates and expressed both per g tissue and per mg DNA, to take into account differences in myonuclear domain. For COX-specific activities (Fig. 1) only TA showed a statistically significant scaling pattern whether expressed per g tissue (b = −0.16, P = 0.0089) or per mg DNA (b = −0.14, P = 0.032). Neither GAST nor SOL showed a significant mass dependence (per g tissue, GAST P = 0.36, SOL P = 0.91; per mg DNA, GAST P = 0.30, SOL P = 0.054). For LDH-specific activities (Fig. 2), only TA showed a significant scaling relationship and only when expressed per g tissue (b = +0.11, P = 0.034; per mg DNA, P = 0.068), whereas GAST and SOL showed no significant mass dependence (per g tissue, GAST P = 0.14, SOL P = 0.061; per mg DNA, GAST P = 0.19, SOL P = 0.74).
Previous studies have reported a reciprocal relationship between oxidative and glycolytic enzymes (15, 29, 36). When the muscles of different-sized rodents were compared in terms of the ratio of LDH/COX (Fig. 2), there was a strong size dependence for TA (r2 = 0.73, b = 0.29, P = 0.0004) and SOL (r2 = 0.49, b = 0.15, P = 0.02). However, the relative activities in LDH and COX showed no significant size dependence in GAST (P = 0.09). Thus, TA showed allometric scaling of COX, LDH, and LDH/COX but GAST displayed an isometric pattern for each of these enzyme patterns.
Transcript Levels in Rodents
We focused subsequent studies on the TA muscle, which showed a clear scaling pattern whether expressed per g tissue or per mg DNA. The main goal of transcript analysis was to explain the patterns in COX seen in these rodents, partitioning the variation between species to mass-dependent and mass-independent effects. In addition to analyzing the transcripts of the NRF1-PGC1α-COX4-1 pathway, we also measured transcripts for LDHA and CS. In each case, transcript levels were assessed by real-time PCR, corrected with β-actin mRNA, and multiplied by mean RNA/g (Table 4) to obtain a relative measure of transcripts per g tissue.
NRF1, PGC1α, and COX4-1 mRNA.
The first approach we employed was to express COX4-1, NRF1, and PGC1α transcripts relative to body mass using the traditional scaling axes (Fig. 3). There was no significant effect of body mass on the mRNA of these genes (COX4-1 P = 0.11, NRF1 P = 0.20, PGC1α P = 0.58).
We also performed correlational analyses of the data to determine the relationships between each mRNA and its downstream effect (Fig. 4). COX4-1 mRNA could predict 55% of the COX activity (Fig. 4A). The mRNA levels of NRF1 and PGC1α correlated with each other (r2 = 0.42). Both NRF1 (Fig. 4B, r2 = 0.52) and PGC1α (Fig. 4C, r2 = 0.48) were strong predictors of COX4-1 mRNA and COX activity (r2 = 0.55 and 0.49, respectively) (data not shown).
Next, the entire data set was analyzed using a multiple linear regression approach to examine the influences of body mass, PGC1α mRNA, NRF1 mRNA, and COX4-1 mRNA. Omission of COX4-1 mRNA from the analyses had negligible effects. As mRNA for NRF1 and PGC1α were strongly correlated, subsequent statements about PGC1α mRNA also apply to NRF1 mRNA. A model using PGC1α mRNA and body mass could explain 90% of the variation in COX activity (Fig. 4D). The standardized partial regression coefficients indicate that PGC1α (standard beta = 0.63) and mass (standard beta = −0.60) contribute about equally to explaining variation in COX (Table 5). Thus, neither PGC1α nor NRF1 mRNA predicts the effects of body mass, but either explains how individual species deviate from the value predicted from mass alone.
LDHA, LDHB, and CS.
The analysis of TA mRNA also provided the opportunity to explore other targets of interest including transcripts for another mitochondrial enzyme (CS) and a representative glycolytic enzyme (LDH).
CS mRNA (Fig. 3E) was found to have a much stronger relationship with body mass (r2 = 0.46) and display a slope (b = −0.26, P = 0.022) that was higher than COX4-1 transcripts or COX itself. Regrettably, we did not measure CS activity in this study.
When LDHA mRNA was expressed relative to body mass (Fig. 3C), no scaling emerged (b = −0.038, P = 0.84) in contrast to the positive scaling seen for the enzyme (b = +0.11). There was no correlation between LDH activity and LDHA transcript levels. LDHB mRNA (data not shown) showed a negative allometric scaling relationship (b = −0.30, P = 0.07).
PGC1α Promoter Analyses in Mammals
The proximal promoters were analyzed for a selection of laurasiatherians and euarchontoglirean mammals that were primarily terrestrial (i.e., excluding cetaceans) and nonfliers (i.e., excluding bats). We compiled sequences from the Ensembl database representing ∼800 bp upstream of the transcription start site beyond which even closely related taxa were highly divergent. Taxon-specific primers were used to amplify ∼700 bp of proximal promoter from other species. For a subset of mammals, a segment of ∼600 bp of proximal promoter was subcloned into a luciferase reporter gene to assess promoter strength.
Promoter sequence.
We did not expect that analysis of 600 bp of the PGC1α promoter would faithfully reflect taxonomic relationships, but the tree generated from the PGC1α promoter (Fig. 5) displays fairly good agreement with accepted phylogenetic relationships (38). The branch lengths within and between taxa suggest the promoter is diverging much faster in rodents than other taxa.
Proximal promoters were analyzed for the presence/absence of putative transcription factor binding sites (Fig. 5). In the proximal 600 bp of mouse sequence there are two insulin-responsive sequences (IRS) (−478 and −315 bp relative to translation start codon), E1 box (−169), an inverted MyoD site (−144), HNF3 site (−96), NF-Y site (−74), and another E1 box (−21). There were some common themes but many exceptions to the arrangement of regulatory elements in PGC1α promoters from other mammals.
The distribution on IRS elements was also highly variable, but with many taxon-specific patterns. All members of Artiodactyla and Carnivora had a single site, while Perissodactyla and Eulipotyphia had none. Within rodents, sciuromorphs had no IRS, caviomorphs possessed a single distal IRS, and murids possessed a second more proximal IRS.
A distal CRE site was present in every species except deermouse, lab mouse, meadow vole, muskrat, and capybara. A second CREB site (TGACGCCA) was seen in every species (−50), but inverted (not depicted on Fig. 5).
Elements for the myogenic factors (MyoD site and E1 box) were present in all species. Two E1 boxes were seen in every species in the same positions. A MyoD site was present in every species, though always in the (-) orientation.
An HNF3 site occurred in all species except 2 (of 7) artiodactyla and the caviomorph rodents (porcupine, guinea pig, capybara).
Sp1 sites were present in every species, though there was some variants in number and sequence. The first element GGCTGGG was present in every species (−211) except deermouse (GGTTGGG). The same Sp1 sequence was seen in the (-) orientation in each species, in each case separated by 10 nucleotides from the first (not depicted on Fig. 5). A third Sp1-like sequence (GGGGCTGGT) was found more proximal (−114) in each species except coyote and the three caviomorphs.
Some species appeared to lack an NF-Y site: bushbaby, Caviidae (guinea pig and capybara), and horse. Most species had a single NF-Y site, but a second more proximal NF-Y site was found in some groups: 6 (of 8) Artiodactylidae, 4 Eulipotyphla (moles and shrews), 2 Lagomorpha (rabbit and pika), and a single primate (gorilla).
Promoter activity.
Proximal promoters from a subset of these species were used to construct luciferase reporter genes (Table 1). The expression of firefly luciferase and Renilla luciferase were measured in two mouse myoblast lines: C2C12 and Sol8 cells. Luciferase activities were measured after 3 days of differentiation. When all species were considered, an isometric relationship between body mass and PGC1α promoter strength was seen in both Sol8 cells and C2C12 cells (Fig. 5C).
Although much has been learned about the mechanisms by which an animal changes metabolic enzyme levels, much less is known about the origins of differences seen between species. Elegant gene regulatory mechanisms coordinate gene expression to maintain the integrity of complex pathways during metabolic remodeling. An evolutionary change that generates a difference between species cannot compromise the capacity of an individual to undertake compensatory metabolic remodeling. Thus differences in metabolic enzyme activities between species may arise by mechanisms that are distinct from those involved in metabolic remodeling.
Scaling of Metabolic Enzyme Activities
Our goal was to assess the underlying mechanisms that determine why muscle metabolic enzymes differ among species, focusing on patterns related to body size. More than 50 years ago, it was first recognized that muscle mitochondrial enzymes showed negative allometric scaling (29). Likewise, it has been known for more than 30 years that glycolytic enzymes show positive allometric scaling in fish (36) and mammals (15). The magnitude and origin of the scaling of mitochondrial enzymes have received the most attention in recent years. One possible confounding influence is a difference in myonuclear content. Whether considering it as myonuclear domain (fiber volume per nucleus) or nuclear content (DNA per g tissue), the abundance of nuclei can in principle have a dramatic effect on constitutive gene expression. Each nucleus produces mRNA to provide the precursors in support of the surrounding cytoplasm (32). It follows that more nuclei per g tissue would lead to more precursors per g tissue (14, 31, 39). Thus, higher levels of nuclear gene transcripts (per g tissue) can arise even if there are no differences in gene expression per se. There is a strong relationship between myonuclear domain size and body mass in both fast twitch and slow twitch mammalian muscle fibers (26). In this study on rodents, we found that only TA showed significant negative scaling of mitochondrial enzymes (per g) and positive scaling of glycolytic enzymes (per g), with little effect when myonuclear domain was considered. However, when COX was expressed relative to DNA, differences between muscles diminished and the scaling coefficients converged. Thus, consideration of myonuclear content reduced the differences between muscles of a species, but not differences between species. Previous studies with fish reached the same conclusion: expressing mitochondrial enzyme activities relative to DNA content largely negated differences in mitochondrial enzymes in relation to size (10) and between fibers (8) but did not affect differences between species (8).
Though the main focus of this study is the impact of body size, it is also important to consider the potential impact of other factors, including lifestyle and phylogenetic relationships (23). Squirrels possessed COX activities that were higher than would be expected based on the regression of all rodents. The interspecies differences were most obvious when comparing chipmunk to gerbil, red squirrel to hamster, and eastern gray squirrel to guinea pig. The squirrels have ∼50% more COX activity than the other size-matched rodents. It is not possible from our data set to distinguish between factors such as phylogenetic signatures (sciuromorphs vs. muroids), activity levels (active vs. more sedentary), or rearing conditions (wild vs. lab bred or colony reared). Allometric patterns in metabolic rate can be influenced by diet and habitat (28) and activity levels (41). Body mass is thought to be the major factor in interspecies differences in metabolic rate, accounting for up to 96% of variation with size (28). Though the differences in metabolic enzymes seen between species may arise from complex evolutionary and physiological factors, the more proximate question is how the muscle cell determines the activities of metabolic enzymes and the extent to which the differences arise from genetics or environment.
Transcriptional Origins of Interspecies Differences in COX activity in TA Muscle
Regardless of whether the differences in COX are linked to size or other factors, the interspecies differences seen in TA muscle COX afford an opportunity to explore the underlying basis of the variation. Most of the work on transcriptional master controllers has been performed on mice, rats and humans, but in these models mitochondrial content has been shown to be under transcriptional control in response to energy challenges such as exercise and dietary stress (20, 34). The question becomes whether interspecies differences are driven by the same factors. Therefore, we asked if the "master controllers" of mitochondrial biogenesis were regulated in a way that would account for scaling patterns in mitochondrial enzymes. Transcriptional control of the genes for PGC1α and NRF1 is central to the capacity of these factors to act as master regulators of mitochondrial biogenesis (1, 6, 7, 18, 20, 22, 34, 46). Consequently, we expected their expression to drive differences seen in COX4-1 mRNA and ultimately COX activity.
COX activity in TA muscle (Fig. 1C) showed a clear scaling pattern (b = −0.16) and body mass explained about half of the interspecies differences in COX activities (r2 = 0.51). Surprisingly, there was no significant regression between body mass and mRNA for COX4-1, PGC1α, or NRF1 (Fig. 3), yet each of these mRNAs was a good predictor of COX activity (Fig. 4). When all of the parameters were evaluated in a multivariate analysis, the paradoxical patterns were more clearly resolved. As expected, PGC1α and NRF1 mRNA were good predictors of COX4-1 mRNA, which in turn was a good predictor of COX activity. This simple transcription pathway could predict about half of the differences seen in COX activity between species. However, body mass, independently of PGC1α or NRF1 transcript levels, also explained about half of the variation in COX activities among these animals. Thus, COX activity is determined in equal measure by unknown mechanisms linked to body mass (independent of PGC1α or NRF1 transcript levels) and by PGC1α and NRF1 (independent of body mass). In more general terms, the COX activity in an animal may be determined by its body mass, but PGC1α and/or NRF1 regulate the actual COX activity to be higher or lower than value predicted by mass alone.
We have little evidence of the mechanisms determining interspecies differences in COX activity acting independently of PGC1 and NRF1 transcript levels. In adaptive remodeling of individuals, posttranscriptional regulation of activity plays a role in the function of both PGC1α [e.g., SIRT1-mediated acetylation (33)] and NRF1 [serine kinase-dependent phosphorylation (17)]. Alternately, there may be a role for other transcription factors known to affect mitochondrial gene expression, such as NRF2, PPAR, thyroid hormone receptors, or retinoic acid receptors (20, 34). As a first attempt to explain regulation of interspecies differences, we further explored the mechanisms by which the differences in mRNA levels of the transcription factors arise between species, focusing on the proximal promoter of PGC1α. The NRF1 gene has a very large and variable 5′-untranslated region, with considerable variation between species, making this promoter difficult to analyze across species. Our analysis of the PGC1α gene was limited to ∼700 bp of proximal promoter. More distal regions of the promoter have considerable sequence variation that precludes cloning novel sequences through consensus primers.
PGC1α Promoter Analyses
We analyzed ∼600 bp of the PGC1α promoter from the start codon to an upstream conserved element for IRS. We assessed the location, number, and arrangement of transcription factor binding elements, and used the sequence to construct neighbor-joining trees. We were surprised to find such a short sequence provided a faithful rendering of mammalian phylogeny (38). Each gene has functional constraints that can cause them to evolve in ways that misrepresent phylogeny (1, 11). Noncoding regions, such as promoters and introns, can experience many point mutations, substitutions, insertions, and deletions with little selective penalty. However, promoters, as well as introns, possess sequences that are conserved because they are specific binding sites (11, 12, 27).
The variation seen in the promoters for mammals we studied fell into three categories: sequence variation between elements, presence or absence of specific elements, and arrangement of elements. The overall variation in sequence was sufficient to derive a robust tree (Fig. 5A) that depicted mammalian phylogeny despite the size (38). The most remarkable feature of the tree was the unexpectedly long branch lengths within rodents. The factor driving this divergence is not immediately clear, but in addition to overall sequence variation there was considerable variation in the representation of specific elements, particularly more distal IRS and NFAT sites (Fig. 5B).
Elements may act as binding sites in vitro or in engineered settings but be transcriptionally irrelevant in vivo (40). Most of the elements identified in this study have been shown to bind transcription factors and regulate transcription in some physiological context (20, 34). These include sites involved in constitutive expression and basic cellular function, such as sites for Sp1, NF-Y, and HNF3, sites that impart sensitivity to metabolism, including CRE, and IRS sites, and muscle-specific factors, such as MyoD sites and E-boxes. For most of these elements, there were instances when select species showed gain or loss but in ways that reflected no particular relationship to size pattern. There were also many potential sites for nuclear hormone receptors, and there was considerable diversity in the number and arrangement of these elements though no phylogenetic or mass-dependent pattern was apparent.
We used reporter genes to assess the activity of the PGC1α promoter in rodents and other placental mammals. There was no simple allometric relationship observed, despite sampling from species that differed in body mass by 1,000-fold. The lack of difference in promoter strength between species, but a clear difference in mRNA levels points to the potential role for more distal elements (46). There is also a potential role for hormones, such as insulin-like growth factors and thyroid hormone, which are known to exert effects on mitochondrial gene expression (6, 7, 20–22, 34).
Reciprocal Regulation of OXPHOS and Glycolytic Genes
Somero and Childress (36) were the first to note the reciprocal pattern in scaling of mitochondrial and glycolytic enzymes, the same relationship observed in our study and others (10, 15). It is unclear whether this pattern arises as a result of independent regulatory pathways or a single pathway with reciprocal effects. The current study provides evidence that these two aspects of the metabolic phenotype, mitochondrial and glycolytic enzyme levels, arise through different cellular mechanisms. In the case of LDH, the scaling coefficients for LDHA mRNA and catalytic activity were quite dissimilar. LDH activity showed positive scaling, whereas LDHA mRNA showed no mass dependence. One possible explanation invokes a role for cytoplasmic protein turnover. Larger animals have a lower metabolic rate, which is likely accompanied by decreases in protein damage and turnover. Thus, larger animals may have less need for LDH synthesis to maintain their LDH activities. We have seen in other studies that larger animals have lower than expect LDHA mRNA, given their LDH activities, which we also attributed to cytoplasmic protein turnover (4).
In summary, our data have provided important insights into the mechanisms by which allometric scaling of metabolic enzymes arise in muscle. Not all muscles show allometric scaling of metabolic enzymes, though all muscles show a reciprocal relationship between oxidative and glycolytic enzymes. In muscles that show allometric scaling of COX, the differences can be attributed in roughly equal parts to transcriptional regulation through NRF1 and PGC1α and body mass effects acting through other cellular mechanisms, such as posttranscriptional effects on NRF1 and PGC1α activities (e.g., SIRT1 acetylation, MAPK phosphorylation) or other transcriptional regulators. Remarkably, almost half of the variation in COX seen between species is consistent with a simple model of transcriptional regulation of NRF1 and PGC1α activities. How their respective promoters are controlled to achieve this difference remains unanswered but sequences differences in the proximal promoter do not appear to explain why PGC1α mRNA levels vary among species.
No conflicts of interest, financial or otherwise, are declared by the author(s).
We thank many colleagues for their assistance providing tissue samples, particularly Kevin Campbell (Univ. of Manitoba), and colleagues at Queen's University including Candace Scott, Peter de Groot, Tice Post, and Sharon Regan. Thanks to Chris Monk and Trevor Snider for assistance with PGC1α promoter characterization, Robert Montgomerie (Queen's University) for his advice on multivariate analysis, and Steve Lougheed (Queen's University) for his help in the phylogenetic analyses.
- Copyright © 2011 the American Physiological Society


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#5 Song of 2010 [VIDEO]
Just in time for the new year!
KICKS 105 is counting down the Top 5 Country Songs of 2010, according to the Billboard Charts.
Just in time for the new year!
KICKS 105 is counting down the Top 5 Country Songs of 2010, according to the Billboard Charts.
It's official (yet, not surprising) -- LeAnn Rimes and Eddie Cibrian are engaged.
The pair, whom met on the set of the movie "Northern Lights" in 2008, have had quite the publicized romance. Rimes left her then-husband Dean Sheremet while Cibrian divorced Brandi Glanville after eight years of marriage, to be together.
After many rumors about Shania Twain's engagement from countless news outlets, Shania Twain set the record straight on her personal website Tuesday morning stating "I'm in love. Frederic Nicolas Thiebaud has been a true gift to me as a compassionate, understanding friend and over time, an amazing love has blossomed from this precious friendship."
A gallery featuring country star Dierks Bentley.
A gallery featuring Australian country star Keith Urban.
A gallery featuring hot country artists wearing cowboy hats.
A gallery featuring some of country's best-looking guitars
A gallery featuring country artist Josh Turner
A look at some of the hottest women in country music today.
A gallery featuring Country Legend George Strait.
The 53rd Annual Grammy Awards Nominees have been released, with a few notable country artists on the list.
This is the official trailer for the new movie "Country Strong" featuring Gwyneth Paltrow, Tim McGraw and Leighton Meester. Coming to a theater near you on January 7th, 2011...

subset: commoncrawl


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Fast start by Golding as rivals jump the gun
WHILE her erstwhile rivals on the professional tennis circuit were battling it out at the French Open yesterday, Anna Kournikova, the Russian tennis star, was to be found off the coast of Devon, somewhat improbably, firing the start gun for one of international sailing's toughest races, the single-handed transatlantic race.
On a filthy afternoon off Plymouth Sound for the start of a race that promises long stretches of hard upwind sailing, Kournikova did the honours from on board HMS Tyne, the Royal Navy warship, firing a flare gun to get matters under way. By her side stood Ellen MacArthur, who won the monohull division in this event four years ago.
In the 15-strong Open 60 fleet so eager were the sailors to get away at…


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Qqotd for 12/13
What new venture would you like to start if time, money etc were no object?
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What is SportsDB?
Posted by Casey Trauer on 24 May 2013 04:30 PM
SportsDB was designed to be an all-purpose repository of sports data as represented by the open source XML format SportsML. Indeed, many of its tables and columns represent a 1:1 relationship with SportsML elements and attributes.
SportsDB shares SportsML's design goal of creating a "core" structure for sports data -- for example, storing in one common location a game score or event data such as game times and participants. For developers creating applications for multiple sports, this creates a certain efficiency.
Just like SportsML, however, SportsDB is an evolving sports data ecosystem. Developers at XML Team Solution continue to expand the schema, not only to adapt it to more sports but also to meet the needs of increasingly extensive sports feeds. For example, the growth of real-time, play-by-play feeds across all sports means events are being described through statistical data in greater detail than ever before.
Here are a few tips and caveats to keep in mind when starting your journey with SportsDB.


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An Aging Mother's Animated Love Letter to Her Autistic Son
"Who would look after him if I wasn't here?" and other questions this mom asks herself every day.
For the 76th summer in a row, motorcycle riders and enthusiasts from around the world recently gathered in Sturgis, a small town in the Black Hills of South Dakota. It is, without a doubt, the Mecca of Motorcycles — a pilgrimage for bikers and a place to show off the mind-bending customizations they've made to their Harleys. But this year, thanks to the presence of the Bird Cage Theatrics Company, it also happened to be the place to witness the most realistic rendition of Shakespeare this side of the West End.
Along with all of those bikers, the company's actors and actresses (including one biker in their midst) made the trek from their Wyoming base to Sturgis a few weeks ago. Their aim: to bring Macbeth to a seemingly new audience — and exactly as the Bard intended it (i.e., with a bit more violence than you're likely to see in the dinner-theater version). The show's producers admit to having a rough go of it the first couple of days when bikers who just wanted a night out drinking were assaulted by Shakespearian prose. But in the end, they managed to avoid a biker brawl and even won over a few new fans for ye olde Bard.
Check out the mini-doc above for a glimpse of Macbeth … on motorcycles.
"Who would look after him if I wasn't here?" and other questions this mom asks herself every day.
Meet a teenage actor-singer-comedian with absolutely no doubt that his tumultuous adolescence will soon give way to Hollywood fame.
Get up close and personal with the athletes of the reemerging ancient pastime of mallakamb, in Narratively's first 360 film.
Once a year, residents of this mountainous island gather at two churches on opposite ends of town and launch 100,000 handmade rockets — directly at each other.
When Dee came out as a transgender, it meant the end of her marriage to Penny. And that's when the empowering journey for both women truly began.
For thousands of years, herders have grazed their livestock on the vast Southern Mongolia grasslands. As Chinese investment turns this mineral-rich region into a cash cow, does the ancient culture have any hope of survival? A few families are willing to fight for it.
This LA artist is creating ephemeral works designed to dazzle – then disintegrate.
An intimate look at life on the kidney waiting list.
After months of subsisting in overcrowded camps, migrants turned an empty building in the Greek capital into a remarkable experiment in self-managed living.
We humans are far more complex than the news headlines and clickbait would have you believe. Let the Narratively newsletter be your guide.


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Watch: Running Man Teases Animation Featuring Members As Animals
SBSs Running Man will turned into an animation!
Previously, inside sources revealed that an animated version of the variety show is currently being worked on with the goal of airing sometime this year.
Soompi.Display.News.English.300×250.BTF Soompi.Mobile.English.300×250.ATF
The animation aims to target children. The broadcasting station will be working with mobile messenger company LINE and a company specializing in creating animations.
The animated version of Running Man will feature the members as animal characters in its story. Yoo Jae Suk will be a grasshopper, Kim Jong Kook will be a tiger, Lee Kwang Soo will be a giraffe, HaHa will be a penguin, Ji Suk Jin will be an impala, and Song Ji Hyo will be a cat.
This wont be a strange concept for the members as they previously appeared as animals in the Animal Kingdom special episode in 2014.
Following this report, SBS released a teaser confirming that Running Man has an animation in the works to be revealed in July. Check it out below!
Meanwhile, you can watch the latest episode of Running Man here!


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text:
|Quoting Isitsafenow (Reply 2):
It was in the news, I believe last February
|Quoting Dtwclipper (Reply 3):
But, I found it very moving that the NY Times put her in todays magazine issue about the best and brightest who died in 2005.
|Quoting NYT:
She pleaded with the terrorists, Let the women go. The terrorists refused. The older women, then, and the children, she insisted. Amazing what can happen if you ask: they relented. Derickson rounded up the selected hostages and coaxed them down the emergency slide, even as they resisted, too terrified to move. Then it was on to Algiers.
|Quoting NYT:
And she talked to the passengers, reminding them calmly but urgently to do as the hijackers demanded, to stay silent, to keep their heads down. She advocated for the most desperate, the ones who needed the bathroom, or some water, or some more room for a pregnant wife.
The plane landed in Algiers for refueling. The ground crew had no intention of fueling the plane without pay; the terrorists became agitated, threatening violence. Derickson, like a soccer mom on board some minivan from hell, reached into her purse, pulled out her Shell credit card and told them to charge it. They did.
|Quoting Iairallie (Reply 6):
They probably wrote the article because of the Germans releasing the Hijacker last week after only 19 years.
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To George Washington from John Moylan, 23 March 1781
Newburg 23d March 1781
In Consequence of an order on the Store wch Colonel Pickering some time ago produced from your Excellency in favour of his Departmt, a return of Clothing has been lately handed in for no less than 402 persons Employed under him in this district alone, not Including those Attached to the Army—I have thought it my Duty, Sir, to refuse complying with it, untill your return, as I did not Conceive the order to Extend to the Departmt in General, but to such partial supplies as may be occasionally wanting—I am pressed by Colonel Hughs for an Answer, shou'd therefore be glad to know your Excellency's Sentiments on the matter. In the meantime I Shall take the Liberty to Observe, that whilst our Department continues in the same Circumstances it has hitherto Laboured under, from the Scarcity of Supplies, the greatest Inconveniencys must necessarily arise to the Troops, if the Quarter Masr General is Entitled to draw for the Use of his poeple at Large, be their wants Ever so great in proportion to the numbers allready returned for.
a Similar order in favr of Mr Rensalaer D. Comissy of Ordinance Stores has been produced on the Comissary of Hides at Albany, & application in Consequence in all for 900 Nine Hundred Hides, part of which I am well Informed is to go towards the paymt of Old Debts, of more than two years Standing—your Excellency must know that the Exchanging of Hides for Shoes & Boats is, from the Want of Cash, the only ressource we now have left for our supplies of those necessary Articles—the Worst Conseqce must therefore be Apprehended, shoud Drafts of this kind be admitted from all quarters—25000 pairs of Shoes—Including the quantity on hand, & what we have to Expect by the opening of the Campaign being the [Sum illegible ] of our expectations for the remainder of this years Supply. I have the pleasure to Inform your Excellency, that the Business of the Hide Department is going on as well as the Circumstces of General Distress can possibly admit of. I am however Advised & in deed I have reason to believe that Colonel Pickering is Endeavo[uring] to get the Direction of it, & I conceal that I have wrote Philadelphia to give him Every opposition, convinced as I am that the Army can receive no additional Advantage thereby particularly when the Wants of his Own Departmt—may at any time be found to clash with the wants of the Troops. I have the honour to be Your Excellency's most obedt Hble servt
John Moylan A.C.G.
DLC: Papers of George Washington.


text: TRAINING TUESDAY PODCAST 072 (Prospecting Blocks, Father's Day) THIS WEEK: THE SHANLE FAMILY, PROSPECTING PYRAMID, GENERATION iY AND PARENTING FATHER'S DAY: An interview with SFC Raymond Shanle who was featured in GX magazine because he is not only a RRNCO but has 5 children who are honorably serving in the Army National Guard. PERPETUALLY PROSPECTING: (At our core we must be … Continue reading TRAINING TUESDAY PODCAST 072 (Prospecting Blocks, Father's Day) → You are unauthorized to view this page. Username or E-mail Password Remember Me Forgot Password


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(CHARLESTON, S.C.) – February 13, 2013 – Three credit unions have utilized Optimal Talent Solutions™ and its suite of talent management services. SAFE Federal Credit Union, Health Facilities Federal Credit Union and Greater Abbeville Federal Credit Union, all of South Carolina, were able to extend member services by engaging the Charleston-based consulting group (an arm of South Carolina Financial Solutions™).
SAFE Federal Credit Union, an $825 million credit union based in Sumter utilized Optimal Talent Solutions™ for diversity training.
Health Facilities Federal Credit Union, a $23 million credit union in Florence, utilized Optimal Talent Solutions™ for employee handbook updates and employee handbook implementation services.
Greater Abbeville Federal Credit Union, an $11 million credit union in Abbeville, utilized Optimal Talent Solutions™ for assessment services and will employ job analysis, employee development and training solution services within the near future.
Optimal Talent Solutions supports the enhancement and implementation of strategies designed to improve the process of acquiring, developing, aligning, assessing, and retaining a diverse workforce with the required skills and aptitude to meet an organization's current and future needs. The consulting group also provides employee regulations support to include, but not limited to, AAP Outsourcing, Training, EEO-1 Reporting and Diversity and Inclusion programs. For more information about the services provided, visit OptimalTalent.org.
Optimal Talent Solutions™ is part of the South Carolina Financial Solutions™, LLC family.
About South Carolina Financial Solutions, LLCSouth Carolina Financial Solutions (SCFS) is a credit union service organization that is a member of the South Carolina Federal Credit Union family of service organizations. SCFS provides credit unions of all sizes with affordable, business enhancing services. With the credit union philosophy of "people helping people" in mind, SCFS and its associated service organizations offer industry professionals with extensive experience to enhance a credit union's functionality, agility and business processes. More information about South Carolina Financial Solutions can be found at SouthCarolinaFinancialSolutions.org.
Contact: Meredith Siemens VP-Corporate Communications
South Carolina Federal Credit Union
843-569-4359 (office)


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Coplay seeks another grant
By PAUL CMIL in Local News
Coplay has hit a bonanza with grant funds over the past few months and will move forward with another application. At the workshop meeting May 2, borough Secretary Sandra Gyecsek reported on another grant category that might be available....
CWSA board hears positive results of annual audit
By PAUL CMIL in Local News
The April Coplay-Whitehall Sewer Authority board meeting is an annual review of the authority's operations. This year's review was held April 20. The players are often the same, and the results are in line with past years' excellent reports....
Board backs increased vehicle fee
By AL RECKER in Local News
Whitehall Township Board of Commissioners at its Monday workshop meeting agreed, with a bit of hesitancy, to back a $5 motor registration fee proposal that is before Lehigh County Board of Commissioners — but not before stating its support must...
Cementon/Egypt: Roast beef lunch is planned
By The Press in Columns
Catasauqua: Salem UCC plans May 6 flea market
By The Press in Columns
Armstrong seeks executive seat
By The Press in Local News


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OUr story
The idea for CARSA was sparked when some Rwanda university students from different backgrounds met and began to tell their stories. They discovered they shared a burden for healing, forgiveness and reconciliation. They felt disharmony among others at the university, the churches, and their respective neighborhoods. Beneath the veneer of everyday life, people were suffering from trauma, bitterness and distrust.
The group began growing as they spent weekends praying, worshipping God, and experiencing His healing in their hearts. Training about trauma healing, reconciliation and counseling helped them to process their personal journeys of healing. After graduating, some of these group members dedicated themselves to the work of CARSA, which was officially established in 2004 with the vision to "have a society where peace, solidarity, and unity in diversity are established."
Initially, CARSA used HWEC (Healing Wounds of Ethnic Conflict) workshops in secondary schools, universities, and churches to bring healing to the youth. In 2006 CARSA started its ongoing work in rural communities, bringing together genocide offenders and survivors. Today CARSA has extended its impact through multiple programs focused on alleviating poverty, building peace, equipping youth and researching the effects of the genocide.
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Christian Action for Reconciliation and Social Assistance (CARSA) was founded in Rwanda in 2002. CARSA received its legal status on 6/10/2004 by ministerial order nº111/11. Its registered office is in Gasabo district, Kigali city council. Through the new law governing non-government organization in Rwanda, CARSA has been registered as a Faith Based organization (FBO) through an Official Gazette n° 50 of 16/12/2013. For the past 10 years of its existence, CARSA has achieved a lot, impacting the community greatly.


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My baby, Topaze. She's a 3 year old female that I adopted from our beloved Lynn Mathers. She's a very sweet girl, loved by everyone and every dogs in the neighbourhood. She's such a sweetie that she gets invitation to special occasions - for example a baby shower.
Dog's owner and/or photo submitted by Nicole Boucher
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Question of the Week
Today is October 23, 2017
On this day in 1987:
The Southern California Kerry breeder, Ray Perry, was murdered on his way back from a dog show.


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Franz Beckenbauer
Franz Beckenbauer 'investigated by Swiss authorities over 2006 World Cup'The Swiss Attorney General's Office is reportedly investigating Franz Beckenbauer in relation to Germany's bidding process for the 2006 World Cup.
Sep 1, 12:46
Franz Beckenbauer: 'Bastian Schweinsteiger better away from Manchester United'Germany legend Franz Beckenbauer claims that Bastian Schweinsteiger is more comfortable with the national team as he is away from his "problems" at Manchester United.
Jun 7, 20:50
FIFA investigating 2006 World Cup bidding processFIFA's ethics committee opens investigations into six officials regarding the bidding process for the 2006 World Cup in Germany.
Mar 22, 13:48
Pep Guardiola 'not allowed' to sign Bayern Munich players this summerBayern Munich's honorary chairman Franz Beckenbauer says that Pep Guardiola will not be allowed to poach players to take to Manchester City.
Mar 15, 12:28
Franz Beckenbauer fined, warned by FIFA's ethics committeeFranz Beckenbauer is fined and warned by FIFA's ethics committee over the German football legend's failure to co-operate with an investigation into World Cup bids.
Feb 17, 14:12
Franz Beckenbauer: 'Juventus clash is mouth-watering'Bayern Munich ambassador Franz Beckenbauer expects a "mouth-watering" tie between his side and Juventus in the last 16 of the Champions League.
Dec 14, 12:32
Franz Beckenbauer: 'Pep Guardiola may consider joining Manchester United'Franz Beckenbauer says that Bayern Munich boss Pep Guardiola may be tempted to take over at Manchester United.
Dec 11, 18:23
Franz Beckenbauer eager for Bayern Munich to keep Pep GuardiolaFranz Beckenbauer admits that it would be a "real shame" if Bayern Munich lost Pep Guardiola at the end of the season.
Nov 28, 13:57
Franz Beckenbauer denies buying votes for 2006 World CupFranz Beckenbauer clarifies why a payment was made to FIFA following recent accusations that Germany bought votes to host the 2006 World Cup.
Oct 26, 19:29
FIFA's ethics committee investigate Franz BeckenbauerFranz Beckenbauer is among the list of 11 FIFA committee members that will be under investigation by the governing body's ethics committee.
Oct 21, 15:34
Germany denies claims that 2006 World Cup was aided by bribesFIFA will investigate "very serious" allegations that the German Football Association set up a secret slush fund to buy votes so that they could host the 2006 World Cup.
Oct 17, 10:36
Former Bayern Munich manager Dettmar Cramer dies, aged 90Dettmar Cramer, who led Bayern Munich to back-to-back European Cup triumphs in 1975 and 1976, dies at the age of 90.
Sep 18, 10:36
Franz Beckenbauer: 'Mario Gotze needs to improve'Franz Beckenbauer believes that Bayern Munich's Mario Gotze must be prepared to change his game to silence his critics.
Jul 7, 12:44
Franz Beckenbauer: 'Bastian Schweinsteiger should stay at Bayern Munich'Bayern Munich legend Franz Beckenbauer urges Bastian Schweinsteiger to ignore any interest from Manchester United this summer.
Jun 26, 03:33
Report: Bastian Schweinsteiger on verge of Manchester United moveBastian Schweinsteiger withdraws from discussions over a new deal at Bayern Munich and will instead await an offer from Manchester United, according to a report.
Jun 25, 15:11
Franz Beckenbauer tells Bastian Schweinsteiger to reject Manchester UnitedBastian Schweinsteiger should stay with Bayern Munich and reject a move to Manchester United, says Franz Beckenbauer,
Jun 19, 05:30
Franz Beckenbauer: 'Jurgen Klopp will join Napoli'Franz Beckenbauer tips Jurgen Klopp to become Napoli's next manager in the summer.
May 31, 19:57
Matthias Sammer: 'Bayern Munich will support Mario Gotze'Bayern Munich sporting director Matthias Sammer insists that the club are fully behind Mario Gotze.
May 14, 03:07
Franz Beckenbauer: 'Lionel Messi the difference'Lionel Messi is the only difference between Bayern Munich and Barcelona, according to the German club's honorary president Franz Beckenbauer.
May 12, 20:06
Franz Beckenbauer laments Bayern Munich transfer businessBayern Munich legend Franz Beckenbauer laments the lack of firepower in the side after their 3-0 defeat to Barcelona in the Champions League semi-finals.
May 10, 16:21
Franz Beckenbauer: 'Jurgen Klopp would fit in well at Bayern Munich'Bayern Munich legend Franz Beckenbauer backs Jurgen Klopp to replace Pep Guardiola should the Spaniard leave the club this summer, claiming he is "definitely" an option.
Apr 27, 10:48
Franz Beckenbauer: 'Pep Guardiola is no failure'Franz Beckenbauer says that it is too hasty to think that Pep Guardiola has failed as Bayern Munich manager.
Apr 21, 11:16
Franz Beckenbauer eager for Jurgen Klopp to stay in BundesligaBayern Munich legend Franz Beckenbauer admits that he would like to see Jurgen Klopp reject a move abroad in favour of staying in the Bundesliga.
Apr 18, 14:36
Franz Beckenbauer frustrated by "black night" for Bayern MunichFranz Beckenbauer admits that it was a "black night" for Bayern Munich as they slipped to a 3-1 loss to Porto in the first leg of the Champions League quarter-final.
Apr 16, 02:07
Franz Beckenbauer: 'Mats Hummels is at best age to move to England'Franz Beckenbauer believes that 26-year-old Manchester United target Mats Hummels is at the "best age" to leave Borussia Dortmund and move to England.
Apr 6, 08:57
Franz Beckenbauer: 'Sami Khedira is not the same player'Germany legend Franz Beckenbauer has claimed that injuries have robbed Sami Khedira of the qualities that made him a top player.
Mar 17, 10:11
Franz Beckenbauer: 'Franck Ribery, Jerome Boateng hurting Bayern Munich'Bayern Munich honorary president Franz Beckenbauer believes that Franck Ribery and Jerome Boateng must be more disciplined on the field.
Mar 16, 11:22
Arsenal, Liverpool 'vying for Xherdan Shaqiri loan deal'Arsenal and Liverpool compete for the loan signing for Bayern Munich's Xherdan Shaqiri, according to reports.
Nov 26, 18:14
Xherdan Shaqiri heading for Bayern Munich exit in JanuaryBayern Munich honorary president Franz Beckenbauer claims that winger Xherdan Shaqiri is expected to leave the club in the New Year.
Nov 26, 13:03
Video: Manchester City's Yaya Toure leaves Cristiano Ronaldo out of 'dream team'Manchester City midfielder Yaya Toure leaves Real Madrid star Cristiano Ronaldo out of his 'dream team'.
Nov 24, 12:36
Harry Redknapp defends Rio Ferdinand against criticsQueens Park Rangers manager Harry Redknapp says that he has no problem with the form of Rio Ferdinand.
Sep 21, 23:54
Franz Beckenbauer wants Bayern Munich move for Marco ReusBayern Munich's honorary president Franz Beckenbauer talks up a move for Borussia Dortmund ace Marco Reus.
Aug 21, 22:46
Franz Beckenbauer backs Bastian Schweinsteiger for captaincyGerman legend Franz Beckenbauer backs Bastian Schweinsteiger to become the next captain of the national side.
Aug 5, 05:32
Franz Beckenbauer praises Germany squadFranz Beckenbauer praises the Germany squad following their World Cup-winning exploits.
Jul 15, 05:22
Franz Beckenbauer warns of Lionel Messi "genius"Germany legend Franz Beckenbauer urges his compatriots to remember that "one stroke of genius" from Lionel Messi could hand Argentina World Cup glory on Sunday.
Jul 11, 16:46
On this day: Germany win third World CupTwenty-four years ago today, the Germans win the World Cup for the third time in their history at the expense of Argentina in Rome.
Jul 8, 10:08
Roy Hodgson: 'Luis Suarez has to perform at the World Cup'England boss Roy Hodgson believes that Luis Suarez has to produce his best form at the World Cup to be considered a world-class player.
Jun 16, 05:56
Qatar 2022 officials hit back at corruption claimsOfficials of Qatar 2022 hit back at attacks suggesting their World Cup bid was corrupt.
Jun 14, 21:59
England vs. Italy: Five key factorsAhead of England's World Cup opener against Italy this evening, Sports Mole picks out five factors that could have a telling impact on the final result.
Jun 14, 11:14
Franz Beckenbauer provisionally banned from football-related activity by FIFAFranz Beckenbauer is provisionally banned from football-related activity by FIFA for 90 days.
Jun 13, 17:30


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A report out last week shows that affinity marketing, which has grown by an average of 25% from 2002 – 2003, is expected to grow by a further 27% by mid-2005. What is driving this significant growth, what impact is it likely to have – on customer management, and how are individual market sectors affected?
Let's start by giving you some background to the report. Group 1 Software, a market-provider of software solutions for data quality, customer communications, and direct marketing applications, commissioned the report.
MarketingUK undertook the research in July 2003, with a survey population of senior marketers from the UK's top 1000 companies surveyed by telephone and email. A research objective was to find out the share of the marketing budget different industries assign to affinity or partnership marketing. Another was to check the same sectors' estimated allocation in two years, comparing this with earlier research by MarketingUK in January 2002. The summary results of the survey, by industry sector can be seen in the following graph:
The principal findings associated with the research are outlined below:
"The trend of affinity marketing is set to substantially increase as a marketing tool across all sectors over the next two years. Marketers predict an average rise of seven percentage points across all sectors by 2005. This is probably a result of the increasing need for marketers to obtain value for money and return on investment from every marketing pound.
Currently, an average of 20% of the marketing budget is devoted to affinity marketing, up from 16% in 2002. This is in line with expectations a year ago, which predicted an average of 23% by early 2004. According to Group 1 Software's new research this is set to rise to 27% by mid 2005."
It looks as if marketing budgets remain tight. The latest Bellwether report for Q2 shows that marketing budgets have been revised down for the fourth consecutive quarter. Marketing managers continue to seek ways of improving the return on investment from every pound. Affinity marketing is increasingly being viewed as one such solution, as it allows a shirt into new markets without the infrastructure or costs of adding value to the customer experience.
This general trend holds well in individual market sectors on the whole, as the following analysis of major findings of the research by market sector shows.
Financial Services
Affinity marketing varies within the financial services sector. Banks show the least interest in affinity marketing, currently allotting only 13% of the marketing budget to partnership tie-ups. Affinity spend is predicted to increase by 6 percentage points to 19% by 2005, well-below the cross-industry average of 27%.
Credit card issuers currently allot 17% of their marketing budget to partnership marketing. 2002 research predicted that by early 2004 this would have reached 23%. The recent research however, reveals a slight change in attitude, as the researchers believe the credit card sector will not allocate 23% of the budget to collaborative marketing until mid 2005. A reason for this slowdown is perhaps the sector's heavy investment into affinity credit cards during the 1990s resulting in over-saturation of the market.
Direct insurers are currently devoting 18% of the marketing budget to partnership marketing. It is predicted that this will rise by 7 percentage points to 25% in 2005.
Significantly, they are the only industry surveyed to have experienced a drop in the part of the budget allocated to affinity partnerships. The 2002 research indicated direct insurers assigned 22% of their marketing funds to partnership marketing fifteen months ago, so there has been a drop of 4 percentage points to 2003.
Retail Multiples
Retail multiples currently allot the highest proportion (31%) of their marketing budget to affinity marketing. However the industry drops to third place by 2005 with an estimated 34% of the budget being spent on affinity partnerships. Retailers traditionally have had a high level of involvement with non-conflicting third-party partners for example tie-ups between clothes multiples and washing powder brands. Retail multiples hold much consumer interest. The industry is therefore desirable as affinity partners, especially to less-exciting sectors with frequency or churn problems.
The telecoms industry, both fixed line and mobile services, spends 16% of the marketing budget on affinity marketing. However, by 2005 it is thought that this figure will rise by 7 percentage points to 23%. It is surprising the current figure is so low as the industry is becoming increasingly commoditised and deregulated, with strict price controls.
The travel industry (including hotels, travel agents and tour operators) is one of the longest established sectors at using affinity partnerships as a marketing tactic. Currently the industry allots the second highest percentage of its marketing budget to affinity marketing (28%) – just below retail multiples at 31%. By 2005 it is thought that a massive 41% of the budget will be allocated to partnership marketing – both the largest proportion and the highest increase (13 percentage points) of the industries surveyed.
As a customer facing, leisure/entertainment industry, the customer experience is paramount to the travel sector. Therefore affinity partnerships that can improve the overall holiday are an integral part of the industry's marketing strategy. For example, a travel agent's offering no longer runs to just the mode of transport and hotel, but to car rental, tours and travel insurance. Also hoteliers offer more than just bed and board. It is now common to find affinity inclusions of cut-price transport, theatre tickets and restaurant deals. However, partnerships are not only viable to hoteliers, tour operators and travel agents as a marketing tool but also strategic to increasing profits. The travel sector has changed its affinity relationships often making commission arrangements on sales to third parties. The travel sector is dependent on affinity tie-ups for growth.
The utility market currently allocates 18% of the marketing budget to affinity marketing. This is set to rise to 24% in 2005, an overall increase of 6 percentage points. Since deregulation in 1997 the utilities market has changed radically. It has been opened to fierce competition. Privatisation of the industry resulted in a plethora of new or regional companies seeking approval and national coverage. One-time monopolies no longer have a captive customer base and have to work harder to keep the loyalty of their customer base and bring in new business.
So what do we make of this move to affinity marketing and its different levels of acceptance in different industries? It seems clear the major driver in the move is to get a better return from each marketing pound (or dollar). Combining two complementary brands, and hopefully two complementary customer bases, should provide a better return from marketing spend for both the organizations. However, this doesn't necessarily do too much for the consumer.
We have long suggested that consumers are looking for solutions to their problems, and that those solutions will often need multiple suppliers to be effective. Affinity marketing at least involves companies collaborating to make an offer to the consumer, so skills are built in collaboration between companies, even if they are not yet taking a solutions-driven approach to marketing.
Perhaps what is most interesting is that in travel, perhaps the most developed sector in affinity marketing, alongside Retail, we are seeing the emergence of a solutions package. Travel, whether business or leisure, usually involves a package of needs: travel, hotel, insurance, entertainment, etc., and it is encouraging the industry is using affinity marketing to put together such solutions.
In the move from product-focused sales and marketing to customer-centric approaches we've been looking for a 'tipping point' which can help in the transition from one to the other. Could affinity marketing provide such a transition?
If you want to know more about the report on affinity marketing, contact Louisa Osmond of Lindsell Marketing at [email protected]
What do you make of this? If you have comments to make, we'd love to hear them. Make them below by clicking on the 'add your own comments to this story' or e-mail me at [email protected].
Richard Forsyth


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Finally, a meditation practice that isn't just one more thing to do in a day.
AMP (Active Meditation Practice)
The first thing people say to us when we tell them we teach meditation is, "Oh, I can't do that. I can't quiet my mind!" followed closely by, "And besides, I don't have time."
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It's not necessarily practical, or entirely reasonable, to equate meditating with quieting the mind - especially in the beginning of your practice. We like to use the analogy of training for a marathon: you don't decide to run one, and then go out for a 26.2-mile training run. You begin with a shorter run, or even a walk. You begin your training wherever you are.
Meditation is the same way.
Active meditation doesn't ask you to stop what you're doing so you can meditate. The AMP techniques fit in with all the things you're doing every day. Get a handle on how the tools work, and you can use them all day long to feel calmer, less stressed, more focused. You'll find that you have more energy to devote to the things you love, and less desire to stay bogged down in the things you don't. Making choices, making changes, feeling happier, and bringing more of your vibrant self to all the things in your life will become more natural, easier, and more fun.
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Here's what AMP users are saying:
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Using techniques from our signature meditation program, AMP (Active Meditation Practice), these guided meditation recordings will help you apply your AMP tools to specific areas of your life.
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In just 10-15 minutes, these recordings will guide you to deeper insights and greater clarity. They also give you a framework for using your AMP tools in ways that are relevant to you - once you've mastered the tools, there's no limit to the life situations they can light up!
If your picture of meditation is velvet cushions and a quiet mind, watching your thoughts drift by as you breathe incense and chant a mantra to pan flutes and the sound of waves crashing on the shore, this is *not* the meditation practice you're looking for.
Upon completing your purchase, you'll receive an email containing links to your recordings and an invitation to our closed Facebook group where you can connect with us and others who are using active meditation to transform their lives and the world. We'll see you there!
Active Meditation on Chakras
Your chakras influence the way you express yourself. Each chakra has a corresponding phrase that will clue you in to which one is dominating at any moment:
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See what all the chakra hype is about and how to give yours a gentle tune-up.
This active meditation on the chakras is designed to give you an overview of the seven main chakras as you work with a healing neutral gold vibration to connect to and find balance in your chakras.
In a nutshell, your chakras correspond with the following real-life experiences:
First: basic survival, family of origin
Second: emotions, sexuality, creativity
Third: personal power, activity
Fourth: affinity for self and others
Fifth: communication, speaking your truth
Sixth: intuition, clairvoyance
Seventh: knowingness, connection to your higher power
Healing your chakras essentially cleans the grit from the gears of your life. It's like tuning up the engine in your car, in a way - everything just runs more smoothly.
Upon completing your purchase, you'll receive an email containing links to your recordings and an invitation to our closed Facebook group where you can connect with other daring spirits and have access to the DS team for support. The group is a fantastic resource for questions, and we have a lot of fun in there!


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LayStar Home > Reading Magazine > Berkshire Directory > Searching: Reading Directory
Commercial Services - Party Services in Reading
Are you missing out? Your business or your favourite beaty spot or attraction could be listed here in the LayStar Directory.
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Tidy Homes Cleaning
Main Category: Commercial Services
Tidy Homes is a local, professional cleaning company offering domestic and office cleaning as well as commercial cleaning services
Website: Tidy Homes Cleaning
Telephone: 07724 632 132
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The LayStar online web directory is completely free and is the place to advertise attractions and services. If you own a business or if you want to sing the praise of a good local attraction or service, get it listed here for free. With the LayStar directory you can also list your telephone number and website link, along with an image such as your logo. Not many directories give you nearly as much for free. Simply register with us (that's free too), use the search form on the left to find the UK location of the attraction or service (suggest the location if it doesn't yet exist on LayStar) and then follow the link that will appear here to build your advert.
LayStar Directory Terms of Use