text:
Under normoxia, non-malignant cells rely on oxidative phosphorylation for their ATP production, whereas cancer cells rely on Glycolysis; a phenomenon known as the Warburg effect. We aimed to elucidate the mechanisms contributing to the Warburg effect in human breast cancer.
Experimental design
Lactate Dehydrogenase (LDH) isoenzymes were profiled using zymography. LDH-B subunit expression was assessed by reverse transcription PCR in cells, and by Immunohistochemistry in breast tissues. LDH-B promoter methylation was assessed by sequencing bisulfite modified DNA.
Absent or decreased expression of LDH isoenzymes 1-4, were seen in T-47D and MCF7 cells. Absence of LDH-B mRNA was seen in T-47D cells, and its expression was restored following treatment with the demethylating agent 5'Azacytadine. LDH-B promoter methylation was identified in T-47D and MCF7 cells, and in 25/ 25 cases of breast cancer tissues, but not in 5/ 5 cases of normal breast tissues. Absent immuno-expression of LDH-B protein (<10% cells stained), was seen in 23/ 26 (88%) breast cancer cases, and in 4/8 cases of adjacent ductal carcinoma in situ lesions. Exposure of breast cancer cells to hypoxia (1% O2), for 48 hours resulted in significant increases in lactate levels in both MCF7 (14.0 fold, p = 0.002), and T-47D cells (2.9 fold, p = 0.009), but not in MDA-MB-436 (-0.9 fold, p = 0.229), or MCF10AT (1.2 fold, p = 0.09) cells.
Citation: Brown NJ, Higham SE, Perunovic B, Arafa M, Balasubramanian S, Rehman I (2013) Lactate Dehydrogenase-B Is Silenced by Promoter Methylation in a High Frequency of Human Breast Cancers. PLoS ONE 8(2): e57697. https://doi.org/10.1371/journal.pone.0057697
Editor: Paul van Diest, University Medical Centre Utrecht, The Netherlands
Received: August 13, 2012; Accepted: January 28, 2013; Published: February 21, 2013
Copyright: © 2013 Brown 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.
Funding: Sheffield Breast Cancer support group funding to NJB is acknowledged (Grant code X/001592). 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.
Breast cancer is the most common cancer diagnosis and the leading cause of cancer-related deaths in women worldwide [1]. Moreover, the disease incidence has increased over the past three decades [2]. Despite advances in early detection and treatment, around 30% of patients with early-stage breast cancer will develop recurrent disease, which often presents as metastasis and a poor prognosis [3].
Normal cells in the presence of oxygen (aerobic conditions), derive most of their energy from glycolysis coupled with oxidative phosphorylation which is a highly efficient process yielding 38 molecules of ATP for each molecule of glucose consumed [4]. Glucose enters into the cell and undergoes a series of steps to generate pyruvate, which then enters into the mitochondria where it is metabolized to CO2 and water with the formation of ATP. Furthermore, this process is facilitated by the presence of oxygen which activates oxidative phosphorylation and inhibits glycolysis (known as the Pasteur effect), [5], [6]. However, when oxygen is limited (anaerobic conditions), such as following prolonged exercise, pyruvate undergoes fermentation where it is converted to lactate by a reaction catalyzed by Lactate Dehydrogenase (LDH), [7], [8].
LDH is a tetrameric enzyme, containing 2 major subunits (A and B), encoded by 2 different genes, LDH-A and LDH-B [8], [9]. The LDH-A and LDH-B subunits associate as tetramers to form 5 different isoenzymes (LDH-1 to LDH-5), which are comprised of either subunits LDH-B4 (LDH-1); LDH-B3:A1 (LDH-2); LDH-B2:A2 (LDH-3), LDH-B1:A3 (LDH-4) and LDH-A4 (LDH-5) subunits. These LDH isoenzymes catalyze the interconversion of pyruvate and lactate, which is a key step in the glycolysis pathway as it also regenerates NAD+, necessary for continued glycolysis [10]. The LDH-5 isoenzyme which is composed solely of four LDH-A subunits (LHD-A4), catalyzes the reduction of pyruvate into lactate with unparalleled efficiency, particularly under anaerobic conditions [8], [11]. However, this function of LDH gradually decreases as the number of LDH-B subunits compared to the LDH-A subunits increase. Thus, the LDH-1 isoenzyme composed solely of four LDH-B subunits, efficiently favours the backward conversion of lactate into pyruvate [8],[10].
In contrast to normal cells, cancer cells derive a major source of their energy from glycolysis even in the presence of oxygen, despite the fact that glycolysis alone is an energetically inefficient process, yielding only 2 molecules of ATP per molecule of glucose [12], [13], [14]. This increased glycolysis even in the presence of oxygen was first described by Otto Warburg in the 1920's, and is now known as the Warburg effect or aerobic glycolysis [13], [14]. Activation of aerobic glycolysis is thought to be a mechanism used by cancer cells to liberate them from a dependency on oxygen for their ATP production, particularly under hypoxic tumour microenvironments [7]. Furthermore, this altered metabolism of cancer cells is thought to confer a selective advantage for their survival and proliferation [10].
Although the precise molecular mechanisms underlying the Warburg effect are incompletely understood, environmental (nutrients, hypoxia, acidity, etc), genetic and epigenetic factors are known to play a role [5], [14], [15], [16]. A role for genetic and /or epigenetic factors has also emerged from the finding that the glycolytic phenotype persists in cancer cells isolated from tumours growing in vivo [5]. Tumour cells have been shown to undergo aerobic glycolysis following oncogene activation (myc, ras, AKT), and tumour suppressor (p53, VHL) gene inactivation, which act to upregulate Hypoxia-Inducible Factor 1 alpha (HIF-1α) levels [5], [7]. Increased HIF-1α, in turn results leads to increased transcription of genes involved in glucose transport, glycolysis, and lactate formation through up-regulation of the LDH-A subunit. Interestingly, the LDH-A subunit has been found to be frequently up-regulated in clinical tumours and is often associated with disease progression and a poor prognosis [11], [17], [18]. In addition to the above mechanisms contributing to the Warburg effect, we and others have previously reported absence or decreased expression of the LDH-B subunit in prostate and gastric cancers, due to hyper-methylation of the LDH-B promoter [19], [20]. Recently, decreased expression of LDH-B has been associated with higher histological grade and perineural invasion in bladder cancer, suggesting that LDH-B may be related to tumour progression [21].
The aim of the current study was to elucidate the mechanism(s) contributing to the Warburg effect in human breast cancer. Panels of human breast derived cell lines representing various stages of breast cancer development and progression (non-malignant, pre-malignant, pre-invasive, invasive and metastatic disease), were profiled for expression of LDH isoenzymes using zymography. Absent or decreased expression of the LDH-B subunit was seen in T-47D and MCF7 cells, and the underlying mechanisms were investigated by sequencing the LDH-B promoter region in bisulfite modified DNA from the cell lines, and human breast cancer tissues. Furthermore, functional relevance of the absent or decreased expression of the LDH-B subunit was investigated by subjecting both LDH-B deficient and proficient cells to conditions of hypoxia, and measuring lactate levels as an indicator of their dependency on glycolysis.
Materials and Methods
Cell lines and culture
Human breast cancer cell lines MDA-MB-436, MDA-MB-231, MCF7, T-47D, and the prostate cancer cell line LNCaP were obtained from the American Type Culture Collection (ATCC), (http://www.atcc.org/). The MCF10A, MCF10AT and MCF10DCIS.com breast epithelial cell lines, representing normal (immortalized) breast epithelial cells, pre-malignant and pre-invasive ductal carcinoma in situ (DCIS) disease respectively [22], were a kind gift from the Barbara Ann Karmanos Cancer Institute, Detroit, Michigan, USA. The LNCaP-LN3 prostate cancer cell line used as a positive control was a kind gift from Dr. Curtis Pettaway (MD Anderson Cancer Centre, USA), [23]. The MCF7, MDA-MB-436 and MDA-MB-231 cells were maintained in RPMI-1640 media containing Glutamax, supplemented with 10% fetal calf serum and Penicillin/ Streptomycin (Gibco-BRL, Paisley, United Kingdom). The T-47D cells were cultured in DMEM containing Glutamax, supplemented with 10% fetal calf serum and Penicillin/ Streptomycin (Gibco-BRL). The MCF10A, MCF10AT and MCF10DCIS.com cells were cultured in DMEM/F12 Advanced media supplemented with Penicillin/ Streptomycin, 5% horse serum, 10 mM HEPES, 10 µg/ml insulin, 20 ng/ml EGF, 100 ng/ml cholera toxin and 0.5 µg/ml hydrocortisone. All cells were confirmed to be free from Mycoplasma using the EZ-PCR Mycoplasma test kit (Geneflow Ltd, UK).
Short tandem repeat profiling of cells
In order to confirm the authenticity of representative breast cell lines, DNA from MCF7, T-47D and MCF10 was analyzed by Short Tandem Repeat (STR) profiling of 10 loci, at the University of Sheffield Core Genomics facility (http://genetics.group.shef.ac.uk/), using the StemEliteTM ID system (Promega, Product code: G9530), and the products run on a ABI 3730 DNA analyzer, according to the manufactures instructions. STR matches were searched using the LGC standards database (may 2012): http://www.lgcstandardsatcc.org/ATCCCulturesandProducts/CellBiology/STRProfileDatabase/tabid/986/Default.aspx. The STR profiles of the cells tested were as expected with 18/18 matching alleles, thus confirming their authenticity.
Patient tissue material
Archived paraffin-embedded tissues obtained from patients having surgery for breast cancer at the Royal Hallamshire Hospital were used. Prior local ethics committee approval (South Yorkshire Research Ethics reference: 09/H1310/54), and informed patient consent was obtained in all cases. Genomic DNA was extracted from micro-dissected tissue sections as previously described [24], and used for sequence analysis. In addition, genomic DNA extracted from 5 cases of normal human breast tissue was purchased from Ambsbio (www.ambsbio.com), with patient ages ranging from 21-83 yrs. For sequencing of the LDH-B gene promoter region, 25 cases of adenocarcinoma were used which comprised of grade 1 (n = 5); grade 2 (n = 10), and grade 3 (n = 10) tumours. For immunohistochemistry, 26 cases of adenocarcinoma were used; 18 of which were also used for DNA sequence analysis.
Zymography for lactate dehydrogenase isoenzymes
Zymography for LDH isoenzymes was performed using cellulose acetate membranes (Helena Biosciences, Titan III (94×76 mm)), with Tris-Glycine buffer according to the supplied manufacturer's instructions and as previously described [25]. Following electrophoresis, the gels were placed in 10 ml of staining solution containing 5 mM NAD, 50 mM lithium lactate, 0.1 mM Tris-HCl (pH 8.6), 0.2 mM Phenazinmethosulphate (PMS), 2.0 mM nitrobluetetrazolium (NBT), and 0.8 mM (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and the colour was allowed to develop (3- 6 min) in the dark at 37°C.
Reverse transcription PCR
Total RNA was extracted using TRI Reagent (Sigma-Aldrich, Dorset, UK), according to the manufacturer's instructions. After precipitation, the RNA pellet was washed three times with 75% ethanol. RNA was quantified spectrophotometrically. RNA (2 µg) was reverse transcribed into cDNA using the SuperScript® III First-Strand Synthesis System (Invitrogen, Paisley, UK), with 250 ng of random primers, according to the manufacturer's instructions. LDH-B mRNA expression was assessed by reverse transcription PCR (RT-PCR), using primers and conditions as previously described [19].
Sequencing of sodium bisulfite-modified DNA
DNA was extracted from breast cancer cell lines using the Gentra Puregene cell kit (Qiagen), according to the supplied manufacturer's instructions. Prior to sequencing, DNA was subject to sodium bisulfite-modification using the CpGenomeTM DNA modification kit (Invitrogen), according to the supplied manufacturer's instruction manual. For sequencing of the LDH-B promoter region, sodium bisulfite-modified DNA was amplified using previously published PCR primer sequences and conditions [19]. Only those cases showing an intense single PCR band following electrophoresis through a 2% agarose gel were excised and subject to sequence analysis. Sequencing of bisulfite modified DNA was performed at the University of Sheffield Core Genomics facility and the chromatograms visualized using the freely downloaded Chromas software (http://www.technelysium.com.au/chromas.html).
Hypoxic cell culture
For the hypoxic cell culture experiments, T-47D, MCF7, MDA-MB-436, and MCF10AT cells were initially cultured under normoxic conditions as described above. Cells were then trypsinized and plated into T25cm2 flasks, and the flasks transferred into a normoxic (21% O2) incubator, or hypoxic (1% O2) chamber, as previously described [26]. The cell culture medium used for hypoxic growth had been pre-equilibrated to the same oxygen tension as in the hypoxic chamber for 6 hours. Lactate levels in the conditioned medium were measured following 48 hours of cell growth under hypoxic or normoxic conditions.
Lactate measurements
Lactate concentrations in the conditioned medium were measured using a protocol available from the Sigma Aldrich website: http://www.sigmaaldrich.com/etc/medialib/docs/Sigma/Enzyme_Assay/llacticacidmw.Par.0001.File.tmp/llacticacidmw.pdf. Briefly, the assay depends on the reduction of NAD+ to NADH and a change in absorbance, which is proportional to the concentration of lactate in the medium. A standard curve was constructed using lactate concentrations ranging from (0–1000 µM lactate). In addition, lactate concentrations were assayed at the Department of Clinical Chemistry, Royal Hallamshire Hospital, Sheffield, using a Roche 501 analyzer, and the Lact2 lactate Gen.2 assay (Roche). Lactate levels were expressed as µM per million cells, by counting the final cell numbers using a coulter counter (Beckman Coulter model Z2), and after subtracting the values for the basal lactate levels present in the unconditioned media.
Immunohistochemistry and scoring of staining
Immunohistochemistry for expression of the LDH-B protein in human breast tissue sections was performed as previously described, using 3,3'-Diaminobenzidine (DAB) as the chromogen [19]. The anti-LDH-B antibody used has previously been shown to be LDH-B specific and non-reactive against LDH-A [27]. Immunostained sections were assessed by 2 independent histopathologists (BP and MA), and the percentage of cells stained recorded. Relevant tissue areas scoring <10% of stained cells were recorded as showing absent LDH-B expression, while tissues showing ≥10% of stained cells were recorded as showing expression. Images were taken using a Nikon Eclipse 80i microscope attached to a Nikon digital camera (DS-2mv), and the images acquired using the Nikon Nis-Elements imaging software.
5'- Azacytidine treatment
T-47D cells were cultured in T75 cm2 flasks to ∼20% confluence. A final concentration of 1 µM freshly prepared 5'-azacytidine (Sigma-Aldrich), was added to the culture medium and the drug containing medium was changed every 2 days [19]. After 5 days of treatment, total RNA was isolated using TRI reagent (Sigma) as described above. The RNA was quantified and 200 ng was used for RT-PCR as described above.
LDH isoenzymes are absent or decreased in T-47D and MCF7 cells
Following visualization of the stained zymography gel, absent or decreased expression of LDH isoenzymes 1–4 were seen in T-47D and MCF7 cells, with the isoenzyme pattern being similar to the LNCaP-LN3 prostate cells used as a control (Figure 1). In contrast, expression of LDH isoenzymes 1–4 were seen in both MDA-MB-436 and MDA-MB-231 cell lines, although these cells showed absent or reduced expression of the LDH-5 isoenzyme. Expression of LDH isoenzymes 2–5 were prominent in the MCF10AT, MCF10DCIS.com and MCF10A cells. Interestingly, in MCF10AT, MDA-MB-436, MDA-MB-231, MCF10DCIS.com and MCF10A cells, an additional band was visible just below the LDH-4 isoenzyme band (Figure 1, arrowhead). This may represent posttranslational modified forms of the LDH protein, since it has been reported that LDH may be subject to several posttranslational modifications such as phosphorylation, acetylation and methylation [28].
Note the absent or decreased expression of LDH isoenzymes 1–4 in T-47D and MCF7 cells. In contrast, expression of LDH isoenzymes 1–4, can be seen in MDA-MB-436 and MDA-MB-231 cells. Expression of LDH isoenzymes 2-5, are predominant in the MCF10AT, MCF10DCIS.com and MCF10A cells. The expression of all 5 LDH isoenzymes can be seen in LNCaP cells, while absent or decreased expression of LDH isoenzymes 1–4, can be seen in the LNCaP-LN3 cells used as controls. In MCF10AT, MDA-MB-436, MDA-MB-231, MCF10DCIS.com and MCF10A cells, a weak band (arrow head), can be seen under the LDH-4 isoenzyme band, and may represent a posttranslationally modified form of LDH [28].
LDH-B promoter is methylated in T-47D and MCF7 cells
In mammals, DNA methylation of cytosine residues located at 5' to guanine residues (CpG sites), represents a major epigenetic mechanism for regulating gene expression, including X-chromosome inactivation and genomic imprinting [29]. DNA hypermethylation has been shown to result in gene silencing with consequent loss of gene expression. To understand the mechanisms underlying the absent or decreased expression of the LDH-B subunit as seen in the T-47D and MCF7 cells, we sequenced the LDH-B promoter region in these and the other breast cell lines. Sequencing of bisulfite modified DNA showed methylation of the LDH-B promoter region in both T-47D and MCF7 cells (Figure 2a), while the LDH-B expressing cells; MDA-MB-436, MDA-MB-231, MCF10A, MCF10AT, were all seen to be unmethylated.
Methylation at CpG sites (marked by arrowheads), can be seen in (a) T-47D cells and in (b) a case of human breast cancer tissue by the presence of unmodified CpG residues (arrowheads). Identical sites can be seen to be modified and therefore unmethylated in (c) normal human breast tissue and (d) peripheral blood lymphocyte DNA from a patient with breast cancer.
LDH-B promoter methylation is frequent in breast cancer tissues
Sequencing of the LDH-B promoter region in DNA extracted from 25 cases of human breast cancer tissues of various grades (1–3), showed evidence of LDH-B promoter methylation in all 100% (25/25) cases (Figure 2b). Careful examination of all 14 of the CpG sites within the amplifed promoter region, determined that all of the sites showed evidence of methylation in tumour tissues and the methylated breast cancer cell lines. In contrast, LDH-B promoter methylation was absent in all 5 cases of normal breast tissue DNA (Figure 2c), in peripheral blood lymphocytes from matched (n = 10), or unmatched patients (n = 15) (Figure 2d), and in normal breast skin epithelium (n = 13), used as control tissues. In these cases, an absence of methylation was noted at all 14 of the CpG sites within the promoter region analysed.
LDH-B protein expression is absent in breast cancer and pre-invasive lesions
Immunoexpression of the LDH-B protein was assessed in 26 cases of breast cancer tissues, which in some cases included adjacent pre-invasive DCIS lesions and non-malignant glands. Following microscopic examination, LDH-B protein expression was seen to be localized to within the cytoplasm of non-malignant glands, in 4/ 8 cases of DCIS lesions and in 3/ 26 cases (12%), of breast cancers (Figure 3a & 3b). However, absent expression of LDH-B protein expression was seen in 23/26 (88%) cases of breast cancer, and in 4/8 (50%) cases of adjacent DCIS lesions (Figure 3c & 3d).
(a) Strong cytoplasmic expression of LDH-B protein can be seen in the glands of non-malignant epithelium (x20 magnification), and (b) in a case of pre-invasive DCIS lesion (x10). (c) A DCIS lesion (marked by asterix) showing absent expression of LDH-B, while strong expression can be seen in the adjacent and surrounding non-malignant glands (x10). (d) A breast cancer case showing absent LDH-B expression, while expression can be seen in non-malignant glands at the top left of the image (x10).
Re-expression of LDH-B mRNA by 5'-azacytidine treatment
We next tested the ability of the DNA methyltransferase inhibitor, 5'-Azacytidine [30], to restore expression of the LDH-B mRNA in T-47D cells, which were seen not to express LDH-B mRNA by RT-PCR (Figure 4). Treatment of T-47D cells with 5'-Azacytidine for 5 days showed re-expression of the LDH-B mRNA.
Untreated (U) T-47D cells showed an absence of LDH-B mRNA expression. Treatment (T) of cells with 1 µM 5'-Azacytidine for 5 days, was able to restore mRNA expression. GAPDH was used as a loading and amplification control.
Hypoxia leads to increased lactate levels in LDH-B deficient breast cancer cells
It has previously been reported that the LDH-A subunit is highly efficient at converting pyruvate to lactate particularly under hypoxic conditions [8], [16]. We therefore investigated the effects of hypoxia on both LDH-B promoter methylated (T-47D and MCF7), and unmethylated (MDA-MB-436, MCF10AT) cells, as the T-47D and MCF7 cells were seen to predominantly express the LDH-A4 (LHD-5) isoenzyme. All 4 cell lines were cultured under conditions of normoxia (21% O2), or hypoxia (1% O2) for 48 hours, and lactate levels were measured in the conditioned medium as described above. Following hypoxic growth, mean lactate levels in the conditioned medium from MCF10AT cells increased from 27.9 µM to 33.6 µM per 1×106 cells (1.2 fold change), but this difference was not statistically significant (p = 0.09, Mann Whitney test), (Figure 5). In MDA-MB-436 cells the mean lactate levels decreased slightly from 38.5 µM to 34.5 µM per 1×106 cells (-0.9 fold change), but was not statistically significant (p = 0.229). However, following hypoxic growth of MCF7 cells, mean lactate levels increased significantly (p = 0.002), from 2.9 µM to 40.7 µM per 1×106 cells (14.0 fold change). Similarly, in T-47D cells the mean lactate levels were found to increase significantly (p = 0.009), from 15.2 µM to 43.5 µM per 1×106 cells (2.9 fold change) following hypoxic growth (Figure 5).
Following hypoxic growth, mean lactate levels in the conditioned medium from MCF10AT cells increased from 27.9 µM to 33.6 µM per 1×106 cells (1.2 fold change), but this difference was not statistically significant (p = 0.09, Mann Whitney test). In MDA-MB-436 cells the mean lactate levels decreased slightly from 38.5 µM to 34.5 µM per 1×106 cells (-0.9 fold change), but was not statistically significant (p = 0.229). However, following hypoxic growth of MCF7 cells, mean lactate levels increased significantly (p = 0.002), from 2.9 µM to 40.7 µM per 1×106 cells (14.0 fold change). Similarly, in T-47D cells the mean lactate levels were found to increase significantly (p = 0.009), from 15.2 µM to 43.5 µM per 1×106 cells (2.9 fold change), following hypoxic growth. * indicate statistically significant differences (p<0.05).
In order to determine the effects of hypoxia on LHD-5 isoenzyme activity, T-47D cells were cultured under both normoxic and hypoxic conditions and subjected to zymography analysis. As expected, the LDH-5 isoenzyme activity was seen to increase following hypoxic growth (Figure 6), since hypoxia has previously been shown to elevate expression of the LDH-A subunit [11], [17].
Note the relative increased activity of LDH-5 following hypoxic growth.
It has been known since the 1920's that cancer cells derive a major source of their energy by aerobic glycolysis; a phenomenon known as the Warburg effect, in contrast to normal cells which rely mainly on oxidative phosphorylation [6], [12], [14]. Although the precise mechanism(s) for the altered metabolism in cancers is unknown, previous studies have demonstrated diverse mechanisms, including oncogene activation, tumour suppressor gene inactivation, hypoxia, up-regulation of the LDH-A subunit, and absent or decreased expression of the LDH-B subunit [5], [14], [15], [16], [19]. Our current study has shown for the first time in human breast cancer that loss of expression of the LDH-B subunit may occur at an early (DCIS) stage, and occurs in a high frequency of breast cancers, with the underlying mechanism likely to involve methylation of the LDH-B promoter. Furthermore, since the LDH-B deficient cells showed significantly increased lactate levels when exposed to hypoxic conditions, the loss or reduction in LDH-B expression may provide these cancer cells with a growth and survival advantage. In addition, the finding of absent or reduced expression of the LDH-B subunit in 2 widely used human breast cancer cell lines (T-47D and MCF7), could have consequences for data interpretation during the use of these cell lines in metabolic or other studies.
By investigating a range of breast cell lines for their LDH expression profile, we have shown that the T-47D and MCF7 cells predominantly express the LDH-5 (LDH-A4) isoenzyme, with relatively low levels of the LDH-4 (LDH-A3:B1) isoenzyme. Although our results are in stark contrast to the data reported by Hussien and Brooks (2011), who showed that MCF7 cells expressed mainly the LDH-B subunit [31], our data are in partial agreement with those reported by Burke et al., (1978), who showed that MCF7 cells expressed only the LDH-5 isoenzyme [32]. The difference between our data and those reported by Burke et al., (1978), may be related to technical factors such as differences in the sensitivity of the staining procedure, amounts of protein loaded or the possibility that the LDH-B gene promoter may have become unmethylated in a small proportion of MCF7 cells to allow re-expression of the LDH-B protein, which could explain the relatively low level expression of the LDH-4 isoenzyme seen in the MCF7 cells (Figure 1). The possibility that the MCF7 cells were contaminated with another cell line can be firmly ruled out from our STR profiling data, which authenticated these cells.
Due to the absent or decreased expression of the LDH-B subunit, T-47D and MCF7 cells were seen to express predominantly the LDH-5 (LDH-A4), and LDH-4 (LDH-A3:B1) isoenzymes, which are enriched for the LDH-A subunit. This data is in agreement with a previous study showing that LDH-A is the predominant isoform expressed in breast tissue [33]. Other studies have demonstrated a critical role for LDH-A in promoting the growth of breast and other cancer types. For instance, silencing the expression of LDH-A has been shown to decrease cellular proliferation and tumourigenic ability of breast cancer cells [34], and furthermore LDH-A has been shown to be involved in tumour progression [35].
Previous studies have reported that the glycolytic rate in cultured cells appears to correlate with tumour aggressiveness [5]. Thus, it has been reported that the non-invasive MCF7 cells have a much lower aerobic glucose consumption rate compared with the highly invasive MDA-MB-231 breast cancer cell line [5]. Although this finding is in line with our data where we found that during conditions of normoxia, lactate levels were higher in the highly invasive MDA-MB-436 cells compared with MCF7 cells, we did not expect this finding based on the LDH isoenzyme profiles observed in these cells. Since the MCF7 cells are deficient in LDH-B, even under normoxic conditions they were expected to show higher levels of lactate production due to their expression of LDH-A, which is the subunit that has the highest efficiency for the conversion of pyruvate to lactate [8], [10]. These findings suggest that there are factors other than the loss of LDH-B expression, which may underlie the Warburg effect in human breast cancer.
Tumour cells are periodically exposed to hypoxic and normoxic environments [5]. Following hypoxic growth, we measured significant changes in lactate levels in the LDH-B deficient MCF7 and T-47D cells, whereas the LDH-B proficient MCF10AT and MDA-MB-436 cells showed insignificant changes, compared to normoxic growth conditions (Figure 5). Thus, our data show that cells with absent or decreased expression of LDH-B (but express LDH-A), may be better adapted to hypoxic growth by up-regulating fermentative glycolysis. Even under normoxic conditions, we were expecting breast cancer cells with absent or decreased expression of LDH-B to show relatively higher levels of lactate production [8], [10]. However, we observed the opposite result, in that cells with absent or decreased LDH-B expression had relatively low levels of lactate production under normoxic conditions. Thus, our data suggest that under normoxic growth conditions, breast cancer cells which express both LDH-A and LDH-B subunits rely on glycolysis for their energy production, as evidenced by the relatively higher rates of lactate production, and this is maintained even under hypoxic conditions. In contrast, cells with absent or decreased expression of LDH-B (but express LDH-A), under normoxic conditions rely mainly on non-glycolytic pathways for their energy production, but under hypoxic conditions may switch to fermentative glycolysis for their energy production. This data is in keeping with other published data suggesting that certain tumours may rely more on non-glucose carbon sources, even in glucose replete conditions [36].
Previous studies have shown that lactate plays a number of key roles in cancer development, progression and metastasis [10], [37], [38]. For instance, in colorectal cancer a study has shown that lactate can be extruded from cancer cells into the extracellular matrix and subsequently taken up by tumour-associated stromal fibroblasts to be used as a fuel [39]. Additionally, lactate may constitute an alternative metabolic fuel for cancer cells, as oxidative tumour cells can use lactate in preference to glucose, thereby sparing glucose to fuel hypoxic tumour cell growth [37]. In tumours with increased glycolysis and consequently increased lactate production, the extracellular pH has been shown to be acidic [40], [41]. Furthermore, a low pH has been shown to stimulate in vitro invasion and in vivo metastasis of cancer cells [42]. Clinical based studies in patients with cervical cancer or head and neck squamous cell cancer (HNSCC), have shown lactate levels to be inversely correlated with patient survival [38]. Thus, our finding of increased lactate levels in breast cancer cells with absent or reduced expression of the LDH-B subunit when exposed to hypoxic conditions, suggests that the increased lactate levels may contribute to fuel aggressive tumour behavior.
The glycolytic phenotype has been shown to coincide with the transition from pre-malignant lesions to invasive cancer [5]. This finding is in agreement with our data from breast cancer, since by immunohistochemistry we found LDH-B protein to be expressed in 4/ 8 pre-invasive DCIS lesions, but an absence of expression in the adjacent cancer. However, LDH-B protein expression was found to be absent in the remainder 4/ 8 cases of DCIS lesions, which suggests that the loss of LDH-B protein expression may occur earlier than previously reported i.e. during the transition from non-malignant epithelium to pre-invasive DCIS lesions. In support of this, Ong and colleagues reported an increase in glucose consumption in pre-malignant intestinal polyps and cancers compared with non-malignant mucosa [43].
Despite the gaps in our knowledge concerning the glycolytic phenotype, our data showing absent or decreased expression of the LDH-B subunit, may open up a new therapeutic strategy for the treatment of breast and other cancer types. Targeting the remaining LDH-A subunit with therapeutic agents, specifically in those tumours with an absent or decreased expression of LDH-B is expected to lead to a greater cell death, as this is expected to inhibit all cellular LDH activity, and lead to a depletion of the NAD+ stock necessary for self-sufficient glycolysis to occur. In support of this idea, a recent study has shown that specifically targeting the LDH-A subunit using a small molecule inhibitor (FX11), or by siRNA knock-down, increased cell death and inhibited the progression of tumours in vivo [35]. Furthermore, MCF7 cells (with decreased expression of LDH-B), were highly sensitive to growth inhibition by FX11 [35]. Interestingly studies on naturally occurring LDH-A null patients have shown that such patients do not usually develop symptoms during normal physiological conditions, although excessive fatigue and myoglobulinuria have been noted after intense anaerobic exercise [44]. Taken together with published data, our study provides promising evidence for the possibility that the LDH-A subunit may be a safe 'drugable' target for the development of novel anti-cancer agents [45]. Future efforts should therefore be focused on developing non-toxic and highly specific small molecule inhibitors of the LDH-A subunit, in order to block glycolysis, particularly in those tumours which have absent or decreased expression of the LDH-B subunit, thus, 'cutting-off' a major pathway used by breast and other cancer cells for their energy production.
We wish to thank Martin Loxley at the Dept. Clinical Chemistry, Royal Hallamshire Hospital for lactate measurements. Receipt of the breast cell lines MCF10A, MCF10AT, MCF10DCIS.com from the Barbara Ann Karmanos Cancer Institute, Detroit, Michigan, USA is gratefully acknowledged.
Author Contributions
Conceived and designed the experiments: NJB SB IR. Performed the experiments: SEH SB IR. Analyzed the data: NJB SEH BP MA IR. Contributed reagents/materials/analysis tools: NJB SB IR. Wrote the paper: NJB SEH SB IR.
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- 9. Markert CL (1963) Lactate Dehydrogenase Isozymes: Dissociation and Recombination of Subunits. Science 140: 1329–1330.
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- 12. Bensinger SJ, Christofk HR (2012) New aspects of the Warburg effect in cancer cell biology. Semin Cell Dev Biol 23: 352–361.
- 13. Dell' Antone P (2012) Energy metabolism in cancer cells: How to explain the Warburg and Crabtree effects? Med Hypotheses 79: 388–92.
- 14. Koppenol WH, Bounds PL, Dang CV (2011) Otto Warburg's contributions to current concepts of cancer metabolism. Nat Rev Cancer 11: 325–337.
- 15. Dang CV, Semenza GL (1999) Oncogenic alterations of metabolism. Trends Biochem Sci 24: 68–72.
- 16. Koukourakis MI, Giatromanolaki A, Sivridis E (2003) Lactate dehydrogenase isoenzymes 1 and 5: differential expression by neoplastic and stromal cells in non-small cell lung cancer and other epithelial malignant tumors. Tumour Biol 24: 199–202.
- 17. Kolev Y, Uetake H, Takagi Y, Sugihara K (2008) Lactate dehydrogenase-5 (LDH-5) expression in human gastric cancer: association with hypoxia-inducible factor (HIF-1alpha) pathway, angiogenic factors production and poor prognosis. Ann Surg Oncol 15: 2336–2344.
- 18. Koukourakis MI, Kontomanolis E, Giatromanolaki A, Sivridis E, Liberis V (2009) Serum and tissue LDH levels in patients with breast/gynaecological cancer and benign diseases. Gynecol Obstet Invest 67: 162–168.
- 19. Leiblich A, Cross SS, Catto JW, Phillips JT, Leung HY, et al. (2006) Lactate dehydrogenase-B is silenced by promoter hypermethylation in human prostate cancer. Oncogene 25: 2953–2960.
- 20. Maekawa M, Taniguchi T, Ishikawa J, Sugimura H, Sugano K, et al. (2003) Promoter hypermethylation in cancer silences LDHB, eliminating lactate dehydrogenase isoenzymes 1–4. Clin Chem 49: 1518–1520.
- 21. Liao AC, Li CF, Shen KH, Chien LH, Huang HY, et al. (2011) Loss of lactate dehydrogenase B subunit expression is correlated with tumour progression and independently predicts inferior disease-specific survival in urinary bladder urothelial carcinoma. Pathology 43: 707–712.
- 22. Miller FR, Santner SJ, Tait L, Dawson PJ (2000) MCF10DCIS.com xenograft model of human comedo ductal carcinoma in situ. J Natl Cancer Inst 92: 1185–1186.
- 23. Pettaway CA, Pathak S, Greene G, Ramirez E, Wilson MR, et al. (1996) Selection of highly metastatic variants of different human prostatic carcinomas using orthotopic implantation in nude mice. Clin Cancer Res 2: 1627–1636.
- 24. Rehman I, Takata M, Wu YY, Rees JL (1996) Genetic change in actinic keratoses. Oncogene 12: 2483–2490.
- 25. Glen A, Gan CS, Hamdy FC, Eaton CL, Cross SS, et al. (2008) iTRAQ-facilitated proteomic analysis of human prostate cancer cells identifies proteins associated with progression. J Proteome Res 7: 897–907.
- 26. Elbarghati L, Murdoch C, Lewis CE (2008) Effects of hypoxia on transcription factor expression in human monocytes and macrophages. Immunobiology 213: 899–908.
- 27. Tsoi SC, Zheng J, Xu F, Kay HH (2001) Differential expression of lactate dehydrogenase isozymes (LDH) in human placenta with high expression of LDH-A(4) isozyme in the endothelial cells of pre-eclampsia villi. Placenta 22: 317–322.
- 28. Zhou W, Capello M, Fredolini C, Racanicchi L, Piemonti L, et al. (2012) MS analysis reveals O-methylation of L-lactate dehydrogenase from pancreatic ductal adenocarcinoma cells. Electrophoresis 33: 1850–1854.
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- 35. Le A, Cooper CR, Gouw AM, Dinavahi R, Maitra A, et al. (2010) Inhibition of lactate dehydrogenase A induces oxidative stress and inhibits tumor progression. Proc Natl Acad Sci U S A 107: 2037–2042.
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Biswas S, Lunec J, Bartlett K (2012) Non-glucose metabolism in cancer cells-is it all in the fat? Cancer Metastasis Rev [Epub ahead of print].
- 37. Feron O (2009) Pyruvate into lactate and back: from the Warburg effect to symbiotic energy fuel exchange in cancer cells. Radiother Oncol 92: 329–333.
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Ong ES, Zou L, Li S, Cheah PY, Eu KW, et al.. (2010) Metabolic profiling in colorectal cancer reveals signature metabolic shifts during tumorigenesis. Mol Cell Proteomics: 1–45.
- 44. Yoshikuni K, Tagami H, Yamada M, Sudo K, Kanno T (1986) Erythematosquamous skin lesions in hereditary lactate dehydrogenase M-subunit deficiency. Arch Dermatol 122: 1420–1424.
- 45. Ward RA, Brassington C, Breeze AL, Caputo A, Critchlow S, et al. Design and synthesis of novel lactate dehydrogenase a inhibitors by fragment-based lead generation. J Med Chem 55: 3285–3306.


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See All
Video Recipes
"Pachakam" means cooking in kerala and Pachakam.Com welcomes you to the magnificent world of easy and healthy Indian cooking with a probe into the fascinating Indian cooking recipes. Cooking is not just about making food but is an art of presenting your culinary skills to those who love food with a different flavor and Indian cooking, by no means is an exception.
Braised Duck in tofu,peanuts and eggs
Category :
Published On: 29 Oct 2009
English: Salt
tamil: Uppu
malayalam: Uppu
telugu: Uppu
kannada: Uppu
hindi: Namak
Bengali: Laban / Noone
Gujarati: Meethu
Konkani: Meet
Marathi: Mith
for more details, click the name of the ingredient
English: Sugar
tamil: Sakarai
malayalam: Panjasara
telugu: Chakara
kannada: Sakkare
hindi: Cheeni / Shakkar
Gujarati: Khand
Konkani: Peyndaar
Marathi: Sakhar
English: Garlic
tamil: Poondu / Vellaipoondu
malayalam: Veluthulli / Vellulli
telugu: Vellulli / Velluri / Ullipayilu / Vellipaaya / Tellapaayalu / Velluli Payalu
kannada: Bellulli
hindi: Lehsan / Lasun
Bengali: Rasoon / Rashun
Gujarati: Lasan
Konkani: Lahsun / Losan
Marathi: Lasun / Lusson
Oriya: Rasuna
Punjabi: Lassan / Lahsun
Tulu: Bollulli
Kashmiri: Ruhan
Urudu: Lassun / Leshun
English: Ginger
tamil: Inji
malayalam: Inchi
telugu: Allam / Allamu / Sonthi
kannada: Shunti / Alla / Ashi Shunti / Ardraka
hindi: Adrak / Adu / Soonth
Bengali: Ada
Gujarati: Aadu / Soonth
Konkani: Alley
Marathi: Aale
Oriya: Ada
Punjabi: Adrak
Tulu: Soonthi
Urudu: Adrak / Adhrak
English: Egg
tamil: Muttai
malayalam: Mutta
telugu: Gruddu
kannada: Motte
hindi: Anda
Gujarati: Endu
Konkani: Motto
Marathi: Ande
English: oil
tamil: Ennai
malayalam: enna
telugu: camuru
kannada: taila
hindi: tela
Punjabi: moyen
Wipe the duck (with the skin on) inside out with paper towels to dry.
Marinate the duck with soya sauce , five spice powder and salt. Leave to marinate for 11/2 hours.
Heat a large wok, (big enough to hold the duck) add sugar and 2 tbs spoons water. Let the sugar dissolve on low heat. The syrup becomes a clear liquid and turns light golden. .
Throw in galangal, chopped garlic, ginger, star anise, cloves, and cinnamon and stir to combine.
Slide in the marinated duck and coat with the caramel sauce evenly.
Fry the marinated duck over medium heat .(There is sufficient oil in the skin) .
Using a wooden spoon, flip the duck pieces constantly till they are evenly browned. (The darker the browning at this stage the better the colour when finished).
Pour 2 cups of hot water.
Add raw peanuts and cook it along with the duck.
Bring the mixture to a boil, reduce the heat to a simmer and cook for about 35 minutes till the duck pieces are tender.
Add tofu and cooked eggs and continue to simmer till the eggs and tofu are well blended in the sauce.
Use a big ladle to skim off excess fat from the duck. :- There may be plenty of fat floating on the sauce.
Season with salt and pepper and allow to stand for 10 minutes before serving.
Arrange the duck pieces on a large serving plate.
:- Braised Duck In Soya Sauce is ready!!:- Garnish the Braised Duck In Soya Sauce with cilantro or water cress.:-Braised Duck In Soya Sauce... serve hot!!
Fish lightly fried in oil...
This is a favourite among my friends at pot luck coffee mornings...
Nizana Shammas is a hosewife who has lots of interest n cooking. She is an expert in providing various traditional recipes.
Renukadevi Kumanan is a well versed person in providing varities of South Indian recipes and also some modern recipes. She used to try varities of recipes from different cuisines.
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1: I dedicate this to my brother
2: hi I am a baby star and I was born as a ball of gas then the gravity pulled the gas and dust into a sphere then I turn denser and I turn my hydrogen into helium.
4: In my child hood I started to get formed into a sphere by the gravity pulling gas and dust together to make me a sphere of helium
6: As an adult I turn into a perfect star. as a sphere. Then I expload. as I get very old.
8: recourses Text book


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Duckworld Smart Adventures
The time has come: you too can live inside Duckburg! Take a refreshing dive in Scrooge McDuck his moneybin, design your own soapbox, race down the mountain and try to figure out who sabotages the zoo. You need to stop it before it's too late!
Duckworld Smart Adventures contains two episodes, fourteen ingame storytelling cutscenes and over twenty mini games!
For more info and a demo visit the website of Duckworld Smart Adventures.
In may 2014 Sanoma asked me to join their team of about fifteen freelance game developers to create Duckworld Smart Adventures. As the lead programmer my role was to create, maintain and take responsibility for the technical design as well as to lead a team of three talented programmers. Next to these tasks I also developed a few mini games and the ingame smartphone.
Duckworld Smart Adventures has been made by Sanoma, the Donald Duck IP is owned by Disney.


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BY yveet On May 30, 2018 Template
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As the release date nears for the print edition of Jess Brallier's Tess's Tree (HarperCollins, Aug. 25), illustrated by Peter H. Reynolds, two questions remain: can a picture book with an online following succeed in print? And can lightning strike twice for a book originally "published" at FunBrain.com? (Hint: Jeff Kinney's Diary of a Wimpy Kid already showed that a series of graphic novels can successfully make the transition.)
"You can engage with a book online and on the page," maintains author Jess Brallier, the man behind the Grossology series and publisher and general manager of FunBrain.com, a part of Pearson's Family Education Network. "The Diary of a Wimpy Kid books prove one doesn't have to think of the Internet being a threat. It's reaching out to a bunch of kids whose parents may not be in a bookstore habit."
When Brallier was first casting about for children's books to "publish" at FunBrain.com that would also appeal to teachers and parents, he turned inward and asked FEN editorial director Jeff Kinney.Once Kinney's comics went live on the site, Brallier looked for more manuscripts, but found it hard. Most authors are locked into contracts, he says. So once again he looked to another FEN staffer: himself.
Tess's Tree, Brallier's 30th, was inspired by a friend's daughter, Tess, who held a funeral for a tree that had to be taken out of her yard. "It was a period of my life where the previous generation was starting to pass," says Brallier, who was excited by the idea of celebrating life, especially the life of a tree. "I think it's difficult for kids to deal with loss," he says. "It can be scary and lonely. They watch trusted adults being sad. I thought a tree was a softer way to explore that."
Illustrator Reynolds, too, was moved by the story of loss—and its eco component, remembering a tree. On his Stellar Cafe blog he calls it a "gem about dealing with grief and in these eco-aware times, the theme packs a two-fer." Reynolds not only offered to do the illustrations for Tess's Tree but created a TeleFable, his company Fablevision's signature online book format, to post the book on FunBrain.com.
Although their online picture book didn't get quite as much attention as Kinney's graphic novels, which received between 60,000 and 80,000 hits a day, it has had as many as 49,000 reads. And within a month of its being online, Brenda Bowen, then publisher of an eponymous imprint at HarperCollins, offered Brallier and Reynolds a book contract.
Although Harper didn't change a single word of the text, says Brallier—a company first—at 60 pages, the FunBrain version was too long. Reynolds condensed it to 17 pages and completely reillustrated the book.
The book has an announced first printing of 75,000 copies. Inspired by Reynolds's new art, which he says "really made Tess come alive," Brallier is working on additional story lines featuring the nine-year, two-month-old Tess.
Tess's Tree by Jess M. Brallier, illus. by Peter H. Reynolds. HarperCollins, $16.99 ISBN 978-0-06-168752-5


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The Texting Habits You Should Avoid When Talking To Guys
So you're on a date with this cute guy you met at a friend's birthday party when you feel your phone buzz in your bag. Do you check for messages or leave it be? Answer: Leave it be. According to a survey by Match.com, 70 percent of singles want their dates to keep their phones OFF the table. Unless you've been waiting for an important message, checking for phone can wait until later.
In the same survey, Match.com was able to list down some of the guys' three texting turn-offs. Are you guilty of some of these?
1 Typos and wrong grammar.
According to the report, 36% of guys don't like receiving poorly structured messages. So if you want to thank him for the time he's spent with you, make sure you do a quick spell check first.
READ: LOL of the Day: Texts From Overprotective Pinoy Parents
2 Using ALL CAPS.
READ: This Instagram Account Shows You the Text Messages of Couples Who are Bound to Call It Quits
3 Texting them while they're in school.
If you want to go out with him again, resist the urge to send your date a text during school hours. Survey says 47 percent of single guys don't like it. Besides, it's not a bad idea to make him wait a little.
Of course, all the annoying text habits in the world wouldn't make a difference if he's already majorly attracted to you. According to a study by lead author Alex Jones, Ph.D. and psychologist Robin Kramer, there are three things that will make you irresistible to men. And no, makeup doesn't have a lot to do with it.
READ: 7 Text Messages That Have Stayed In Your Drafts Folder
This story, 3 Texting Habits That Turn Guys Off, originally appeared on FemaleNetwork.com.
* Minor edits have been made by the Candymag.com editors.


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Venetta's debut children's book conveys the thrill of growing one's own food.
Lexi Williams and her brother Jason lack their mother's passion for gardening until they find out about a kids' contest. Suddenly, they both want to grow potatoes, carrots, and broccoli in order to win a prize of two tickets to the fall festival and $100. Lexi dreams of taking her best friend, Amy Atkins, to the festival, while Jason longs to spend the cash prize on new soccer shoes. The suspense builds as Lexi, Jason, and their mother plant tiny seeds, ward off caterpillars that threaten to devour their vegetables, and try to train the family's youngest child, Timmy, to pull up weeds. Venetta credibly captures the children's voices; for example, Lexi keeps a list of the many ways in which siblings are annoying. "Reason number seven hundred and thirty-two not to like brothers: They're downers," she thinks, after Jason frets that their vegetables are too small. Parents will recognize themselves, too: when Jason's mother tells him to be careful with kitchen cutlery, Venetta writes, "Jason doubted the knife his mom used to spread peanut butter on his sandwiches was going to cut off his finger, but nodded anyway." The book is also a worthy guide for parents and caregivers aiming to cultivate children's interest in gardening. The author extols the virtues of worm feces as plant food, touts peppermint to deter ants, and points out that worker bees are female. Still, readers may find it difficult to sustain their interest for the book's 260 pages, which include recipes and gardening lessons; there's just enough suspense to make readers want to read to the end, but Venetta probably could have achieved her goals in two-thirds of the space. Some of the dialogue is mundane, and the book repeats points as it alternates between the narrative and the children's journal entries. The book is also slightly prone to gender stereotypes; for instance, the girls make corn-cob dolls while Jason builds a box for a beehive with his father. However, Motz's (What Shall We Dream, 2016, etc.) vivid color illustrations re-create both the contemplative and exhilarating feelings of being in a garden. They convey the kids' expressions of wonderment and further the author's goal of interesting children in gardening.
A productive, if overlong, mix of storytelling and gardening tips.


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Having never actually read a contemporary ghost story, I wondered whether people's assertions that 'books are scarier than films' was in fact true. In a world where graphic, explicit scenes of violence in video games and films are commonplace, I thought perhaps it would be far more difficult for a book to elicit that same chill using only words.
I could not in fact, have been more wrong, although it would take me a while to discover it when reading Dark Matter. I found protagonist Jack's neuroticism fairly irritating at the start. Other reviewers have supposed that his preoccupations with class and general tendency towards depression are important as the book moves on and becomes darker. In the beginning though, these traits are not particularly well explained, nor are they rooted in concrete relationships or events. In many ways, it seems as though Michelle Paver is merely trying to tick all of the 'sad loner' boxes – no family, no friends, dead end job, depressed....
As the narrative moves on from London into the North and eventually to Gruhuken, these niggling matters melt away as they do (much later) for Jack. Paver's descriptions are just right, being only sentences here and there evoking shapes and colours as opposed to long-winded paragraphs. There is a definite monochrome palette running throughout the story, which makes phenomena such as the Northern Lights stand out, and the disappearance of the twilight is almost as unnerving for the reader as it is for Jack.
It is when the reader joins in his frequent swings between terror and rationality that Jack's psychological profile comes into its own. Paver is able to make the slow, encroaching footsteps of the ghostly trapper echo in readers' minds as much as they do in Jack's. The storyline of this ghostly presence is woven into the fabric of Jack's everyday life: much as Jack does, the reader goes through periods of believing the ghost is real and then suddenly the real world will intrude in the form of Algie or Gus on the wireless, and for a time it appears that everything is alright.
By the end of the novel, with footsteps pounding around the hut, Jack's terrified realisation that the ghost is able to enter the hut is as chilling for the reader as it must be for Jack. Another reviewer mentioned that at this point she became afraid to look out of the windows of her own house, and this particular feeling of jumpy paranoia also afflicted me as I read on to the end.
Given that the prickly, unsettled feeling this book gave me lingered for an entire weekend, I must say that this book is by far scarier than any film. I was not just a voyeur, instead I was forced into feeling and experiencing everything as Jack does, as my mind created a picture of Paver's perpetually dark Arctic winter.
Perhaps I'm just a little bit wimpish, but I dare you to try it for yourself.
Sophie Long is an online contributor and until recently was an intern at New Welsh Review.
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Home body wraps are often used for firming the skin and weight reduction. Determining the best home body wrap kits comes down to the ingredients, the desired results, ease of use, price and personal preference. Make sure the ingredients are pure and safe. Follow the instructions as outlined and don't over use them. If you have sensitive skin, you should consult a dermatologist before purchasing a kit.
Ultimate Body Wrap
The Ultimate Body Wrap is used for firming the skin and weight reduction. It includes Ingredients such as seaweed for stimulating the body, and dead sea mud and clays for holding the heat of the body wrap to encourage the seaweed to do its job. The ingredients in this product also target and eliminate cellulite patches. The product claims it can bring a loss of 5-15 inches in one use.
Anti-Cellulite Body Wrap Kit
The Anti-Cellulite Body Wrap kit contains Bentonite clay, sea kelp, seaweed and bladderwrack. The makers say the ingredients work together to eliminate the appearance of cellulite. Aside from that, its aloe vera base feeds the skin with nourishing moisture. Ella Simpson, owner of Wrap Yourself Slim, a store that specializes in body wrap kits, claims that the Anti-Cellulite Body Wrap kit will diminish the appearance of cellulite in as little as one wrap.
Aloe Vera Detox Body Wrap Kit
This kit uses a combination of herbs in a clay base. It claims to rid your body of cellulite, as well as reduce scarring or stretch marks. The clay base works by drawing out impurities through the perspiration it creates. Herbs such as Japanese green tree help stimulate the release of the toxins and Irish moss will help to increase the circulation to fuel perspiration. The makers say you will lose at least four inches in one use.


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I have read quite a few books, articles, and studies, and they all have come to a consensus as to what foods should be avoided if you have Hashimoto's. Some should be cut out of your life forever, while others should be cut out for a few weeks and then gradually brought in to see if you have reactions to them. I am following that elimination approach and hopefully I will be able to bring some of these back into my life, after healing my gut properly. Most of these no-no foods can cause gut problems and a healthy gut is key to overall health.
1. GlutenThe main reason to stay away from gluten has nothing to do with gluten! The way wheat is harvested is the problem. Common protocol for wheat harvest is to dowse it in glyphosate a week before harvest in order to make the wheat die, which makes it go to seed which yields higher crops. What is glyphosate? Glyphosate is a pesticide in Round-Up. This chemical causes digestive issues similar to celiac disease as well as keeps us from being able to absorb proper nutrients like vitamin D, causes reproductive problems, and causes imbalances in the gut, among other problems. This is one of the biggest reasons many people say they are gluten-intolerant. It is the toxic chemicals farmers use in order to save money and increase profits.
2. Dairy
The proteins that are in cow's milk are different than proteins in human milk. Those with leaky gut might see these as foreign invader which will trigger the body to fight it. That means inflammation. This is an over-simplified explanation, so check out Dr. Wentz's better explanation. Also, apparently 60% of thyroid patients feel better going dairy-free. I would love to be able to add more dairy back in, but to be honest, I don't eat much of it these days anyway. I would miss cheese, but it isn't a deal-breaker.
3. Nightshades
Nightshades like tomatoes and peppers can damage intestinal lining, and aid in leaky gut syndrome and imflammation. Leaky gut is common with people who have autoimmune disease, so it is good to eliminate while helping your gut heal. I expect to be able to add these back in without much problem.
4. Legumes
Legumes can prohibit the absorption of nutrients and aid in leaky gut. This is another one I will eliminate for now and add in later, but eat in moderation as in occasional legumes, not a daily meal.
5. Soy
Soy is also sprayed with glyphosate... nuff said, but they also mimic estrogen and no one needs *more* estrogen in their body. Why put your hormones out of whack?
6. Coffee
This one makes me so so sad. First, coffee is very acidic and can cause some pH imbalance in the gut. It also affects your insulin reaction, and Hashimoto's patients are usually insulin-sensitive. Caffein also boosts your stress hormone which puts your adrenals into crisis mode, which they are unfortunately in ALL THE TIME when you have Hashimoto's. (Adrenal fatigue is a real thing!) Coffee can also mess with your thyroid medications, not allowing it to be absorbed well.
7. Sugar and Alcohol
Balancing blood sugar levels is critical when you have Hashimoto's. Blood sugar swings weaken the adrenals and cause spikes in thyroid antibodies. There was a study that found 50% of Hashimoto patients had carbohydrate sensitivities that made them unable to control the insulin spikes that would happen after eating a carb-heavy meal. This is taxing on the adrenals. The typical SAD (standard American diet) is carb heavy and loaded with sugar. Check out your low-fat foods. They add sugar when they take out fat! Fat and protein is important to eat with carbs so that your body doesn't spike.
8. Eggs
Egg whites can permeate the gut lining and cause the immune system to attack the thyroid more. This isn't necessarily a no-no food, but I am going to limit my egg intake. I used to eat 3-4 eggs a day. For now I am not eating eggs, but I will add them back in at a reduced raite. I might eat 3-4 a week instead of a day.


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Amir Khan close to battle with Floyd
Amir Khan will be out to prove himself to Floyd Mayweather Jr when facing Devon Alexander on December 13, according to Golden Boy Promotions boss Oscar De La Hoya.
De La Hoya says a meeting is close to being confirmed but the contest will likely propel the winner into a fight against Mayweather in 2015.
Khan has constantly chased a fight with the unbeaten American, spurning the chance to face Alexander last year in order to enhance his bid to secure the career-defining bout.
"There are just little details that we are finishing but Khan and Alexander are going to fight," De La Hoya said.
"Amir Khan looked good against my boy Luis Collazo but I have a feeling he wants to make a statement against Alexander, and Alexander is coming off a good win against Soto Karass.
"They realise they have to prove themselves if they want to get a really big fight, like against Mayweather."
At 27, Khan had seemingly abandoned plans to face Mayweather, who says he will retire next year.
The Briton is likely to be sceptical regarding a future meeting with Mayweather, should he beat Alexander, having been continually overlooked as a potential opponent for the 47-0 fighter.
But Alexander says the carrot of Mayweather will work as motivation for Khan but still expects to prevail in Las Vegas.
"It looks like it will happen, and I'm excited," said Alexander. "It should be a big win for me. I have the ability to outshine him."
Belfast Telegraph


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The Boy and I were at a lovely wedding this summer. Our friends were married by the same priest that married us, in the same church. The Boy was the Best Man, and Fr. Joe took more than a few jabs at him in throughout the ceremony.
Our friend the priest did have something to say that day that has really stuck with me over the last few months (and it wasn't about how the groom should really reconsider his friends). He told the happy, crying, over-the-moon in love couple that he hoped their wedding day was the least happy day of their marriage. He said in your marriage you should love each more today than yesterday, and a little bit more tomorrow.
I can't say that I love the Boy more today than yesterday, or more than a week ago, or last month. But I do love him more than our wedding day. We are stronger today than we were then. We've been through some truly craptastic days in the last 3 years, and while I would happily trade many of them in, there is no one else I would have rather spent those days with.
We are a team. Happy as a team. Sad as a team. Successful as a team. Frustrated as a team. Elated as a team. Scared as a team. Some days he has to be the team captain, and some days it's me. Through it all what keeps me the most steady and focused and optimistic as we face our toughest opponents is the knowledge that we're in it together, and reading from the same page of the playbook.
Look at me with the sports analogy. He must be rubbing off on me.
Happy Anniversary Buddy.
I thought I loved you then.


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Francesca and Lisa are back with another collection of warm and witty stories that will strike a chord with every woman. This six book series is among the best reviewed humor books published today and has been compared to the late greats, Erma Bombeck and Nora Ephron. Delia Ephron said of the fifth book in the series, Have a Nice Guilt Trip, "Lisa and Francesca, mother and daughter, bring you the laughter of their lives once again and better than ever. You will identify with these tales of guilt and fall in love with them and fierce (grand) Mother Mary." This seventh volume will not disappoint as it hits the humorous and poignant note that fans have come to expect from the beloved mother-daughter duo.
More light, bright essays to delight fans of this mother-daughter writing team. This collection offers a gateway to their humorous, breezy style, featuring rapid-fire paragraphs and plenty of sarcasm.
– Kirkus Reviews
Family is a perennially popular subject, and Scottoline and Serritella bring wisdom and laughs to make sure it never gets boring.
– Publishers Weekly
This collection is ideal for sampling between slathering on sunscreen and dozing in a beach chair.
– Booklist


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Anyway, I've gotten inspired to do so again by catching up to episodes of the AMC series "The Killing," which returns next month for its third season. I'm currently on the first few episodes of season 2 and thankfully have managed to avoid any major spoilers. (Please don't tell me any!) I had heard people were disappointed and felt let down that season 1 did not conclude with a resolution of the murder case at the center of the story, but when you marathon-watch the episodes, that doesn't matter so much.
From the start of watching The Killing, I was immediately reminded of one of my favorites, the 1990s NBC series "Homicide," whose creators Tom Fontana and David Simon went on to do the HBO series "Oz," "The Corner," "The Wire" and "Treme" (the latter three being all Simon's), among other works. Despite the mastery of those shows, The Killing, in tone and subject matter, could be called the true successor to Homicide.
It starts with something as small as the credits' theme music -- airy flutes over percussion that sounds like wood striking wood – and the visuals of abstract close-up colors depicting sirens, rain, a murdered body and other images. The tone of this introduction then bleeds into the action and the story itself. The Killing's central detectives, Linder (Mireille Enos) and Holden (Joel Kinnaman), parallel Homicide's Pembleton (Andre Braugher) and Bayliss (Kyle Secor). Linder and Pembleton are the intense, focused ones and Holden and Bayliss are more casual and sometimes screwed-up.
A large portion of Homicide's first season also focused on the murder of a young girl, with Pembleton and Bayliss chasing several red herrings and dead ends. Some of the criticism of The Killing – admittedly I had to skim it to avoid spoilers – sprung from fan frustration at the way one possible suspect after another was eliminated as a possibility, leaving our protagonists Linder and Holden no closer to catching the killer. In the current television and pop culture universe, audiences have less patience for this type of storytelling than they might have when Homicide aired – hence the online chatter.
Just as Homicide did, The Killing points out that real life doesn't usually offer the decisive resolutions that the Law & Order franchise, for all its dramatic excellence, still does. With that, The Killing yields great characterizations even in small parts. The detectives' interrogation of Belko Royce (Brendan Sexton III) and that character's twisted relationship with his mother recalls a scene in Homicide's first season about Calpurnia Jones, who kills off husbands and family members for insurance money. The detectives are interviewing her very dim nephew, who reveals, "My wife, Aunt Calpurnia…" You can just imagine the rest.
Aside from this dark comic example, larger aspects of both shows have parallels – highlighting depressed American cities outside the media capitals of NY, LA, SF and DC as their settings – Seattle and Baltimore respectively. The police brass in both shows are mired in department politics and bureaucratic pressures. And when there's real danger, you really feel it, as when Pembleton is threatened in "The Gas Man" episode and when Linder makes a discovery about mayoral candidate Richmond toward the end of the first season, while at his apartment to speak with him.
With so many parallels, the creator of The Killing, Veena Sud, has got to be a fan of Homicide. Sud previously did extensive writing and editing work on the late, lamented CBS show Cold Case, and undoubtedly is steeped in crime fiction television as a viewer as well.
Even if The Killing doesn't go beyond a third season – which is still a possibility if the twists and turns don't continue to stay true to this crime fiction TV standard, it's already made a mark as a classic piece of work, and an inheritor of the Homicide mantle.


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Todd Riley
Magnolia High Magnolia, TX Class of 1988
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I'm six years old. As was our weekly tradition; it's a Saturday night and I'm staying at yours. My hair, which, by the way, is circa 3ft long, has been tied into one of your trademark messy, fuzzy plaits, curled gently across my shoulder as I lay there in the dark. There's a storm outside, and as it picks up and the thunder starts roaring across the black, sinister sky and the rain starts hammering on the window, you potter in with your cup of tea and your packet of TUC crackers, wrapped up in your ivory, floral nightgown, and you sit on the chair at the end of my bed, crumbs falling into your lap. You didn't say anything. You just sat, eating those crackers. I didn't stay awake for much longer, because having you there was always enough, but, even in those brief, sleepy moments of mine, I remember thinking how lucky I was to have a Grandma who would happily sit up until 3am if it meant I didn't have to. I also remember thinking you were the ultimate criminal for eating crackers after you'd brushed your teeth.
I'm eighteen years old. We're sat next to each other at Tommy's funeral. You've just lost the man you've loved and squabbled with for over fifty years. The most integral part of your life has come to its end and I know you're heartbroken. You're holding my hand as the poem I wrote for him is being read out. Your bottom lip is shaking and your eyes are laced with tears. Not a single one of them fell. I look at you and I know you're holding it together for me. You whispered how beautiful my words were and how proud you were of me. I bought you a Baileys at the wake and we said goodbye to him together. I never would have made it through that day without you.
I'm twenty one years old. You've made the spontaneous decision that you need to get a boyfriend so you've got someone around who can help you with the hoovering. We're sat at the dining table on my laptop, and we've loaded up 'Plenty of Fish.' You can barely navigate a Nokia 3310, let alone an entire website, so I wasn't too sure how many folk of your generation would be clued up on internet dating. 600+ results later; I don't think you've ever been more thrilled that I was wrong. We cry-laughed at 74 year old Londoner Terry and his explicitly written desires for a young, curvy, Asian babe, and then we cry-laughed some more at topless Peter from Gloucestershire, whose questionable camera angles had made him look like a cross between a wild yeti, and my left thumb. Once we caught our breath, you asked if we could look at the under 25s instead. You never did get your date, but it never really mattered. Topless Peter took pride of place on the cover of your birthday card that year and we laughed about it all over again.
In March 2013, 5 months after we struggled to find you a boyfriend on the Internet, you got diagnosed with bowel cancer. For the first time in my life, I experienced days where I just couldn't find a bright side or a silver lining, or some great, philosophical reasoning as to why this might be happening. Those positive things that I cling onto when things are shit were just completely non-existent; there was nothing positive about seeing you, this sharp, funny, vibrant woman laying helpless and confused in a hospital bed. There was nothing positive about the idea that my entire world was going to be cruelly and suddenly stripped of your presence. You weren't having any of it. You made it your mission to see me as a bridesmaid at my best friend's wedding that coming August. You promised me you'd be there.
You underwent major surgery to remove the cancer and ultimately to save your life. I spent evening after evening sat with you at the hospital, chinwagging about the ways of the world, mutually lusting over that Aussie anaesthetist, and generally feeling an overwhelming sense of gratitude to the universe for letting you live. In hindsight; yes, your cancer was an awful experience, but it was also one that brought us together in a way that I could never have envisaged. I learnt so much from you during that time. I learnt so much about your life and your beliefs and your innate courage and your penchant for dishy young men with kind eyes and scattered facial hair....Familiar, I know. I learnt that it's okay not to be okay, and that I can still function as a human and be there for other people even when I'm tempted to break into a Havisham-style sob every five minutes. I discovered a new sadness that makes me treasure happiness even more-so. And of course, I learnt that hospital doughnuts are surprisingly addictive. You ordered one whenever they were on the menu and sneaked it into my hands every single time.
I've spent so much of my life with you and I've adored every second of it. I can't remember a time when you haven't supported me, except perhaps for that moment I cracked open the third bottle of wine on Christmas Day. You've shared with me your silly witticisms and you've let me drag you to the pantomime every year, and you've asked for every last tiny detail when I've been on a date. 'Ooooh look, isn't he lovely?! I think he might be the one Kath!' You've bought me slippers every winter and you've been the first person on the phone whenever anything noteworthy has happened. You've believed in me when I haven't believed in myself. You've loved me so fiercely and so purely, and it is a love I'll carry with me always.
I look at you and I know that heroism isn't about having a superpower, or being utterly fearless, or having cheese-grater abs. It isn't about living an insane fictional lifestyle or getting viral media coverage (I know you don't know what that means), or possessing the expert skill of kissing whilst upside down. (Your arthritic knees would never allow it anyway.) It's not even about prancing around in a fancy costume or swishing through the street in a cape. Nope, perhaps the truest of heroes are those who walk among us: those who throw goodwill around like confetti, those whose hearts are made of the finest gold, those who are extraordinary just by existing and make us feel extraordinary with just one kind glance. Perhaps the most worthy heroes are those who can inspire us to be so much better without ever thinking it or telling us that we need to be. That's you Granny dearest. And every last pinch of love that I have within me is yours. I'll never forget your face when you saw me as a bridesmaid.
If I'm anything like you are when I'm silver-haired and wrinkly, I'll know that it's been a life well lived.
Yours always,
Kath x
Next Sunday, I'll be dressing up as my dear old Granny B and walking 10k at St Wilfrid's Hospice's first ever Hero Walk. If you've got a spare fiver and want to make a difference to this incredible charity and the community it serves, you can sponsor me right here!


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Construction work for the new Cortex MetroLink Station may result in delays for some Red Line and Blue Line MetroLink passengers May 14-20.
MetroLink will operate on a single track at the Grand MetroLink Station, and Blue Line trains will only operate between the Forest Park-DeBaliviere and Shrewsbury-Lansdowne I-44 MetroLink Stations during the following times:
During these times, westbound Blue Line passengers will need to board a Red Line train between the Fairview Heights and Central West End MetroLink Stations, and travel to the Forest Park-DeBaliviere Station to transfer to a Blue Line train and continue their westbound trips. Eastbound Blue Line passengers will need to board a Red Line train at the Forest Park-DeBaliviere Station to continue their eastbound trips.
Red Line MetroLink passengers may experience delays of 5 to 10 minutes. Passengers should allow 10 minutes when transferring between Red Line and Blue Line trains.


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2019 Twin Counties Heart & Stroke Walk
Rocky Mount, NC
Saturday, October 26, 2019
Life is why we walk.
Thanks to all our walkers, donors and volunteers who have accepted the challenge to help fight heart disease and stroke. We cannot achieve our mission without each one of you!
Date & Time
Check-In 9 a.m.
Starts 10 a.m.
Rocky Mount Mills
1147 Falls Rd
Rocky Mount, NC 27804
Get Directions
Rachel Urban
Email Rachel Urban
(336) 580-1813
By participating in the Heart Walk, you're joining a million Heart Walk Heroes from across the nation raising funds for lifesaving science. Science that can teach us all how to live longer and be Healthy For Good.
Take a few minutes to consider the lives you are going to help change for the better through your leadership and donations. We're talking more moms, dads, brothers, aunts and babies' lives saved. Help keep hearts everywhere beating. Let's take steps together to cure heart disease and stroke.


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Two years after the outbreak of what has come to be known as the Arab Spring, the bloom is off the rose. Fledgling democracies in North Africa are struggling to move forward or even maintain control, government crackdowns in the Persian Gulf and elsewhere have kept liberalization at bay, and Syria is slipping ever deeper into a vicious civil war that threatens to ignite the Middle East. Instead of widespread elation about democracy finally coming to the region, one now hears pessimism about the many obstacles in the way, fear about what will happen next, and even open nostalgia for the old authoritarian order. Last June, when the Egyptian military dismissed parliament and tried to turn back the clock by gutting the civilian presidency, The Wall Street Journal's chief foreign policy columnist cracked, "Let's hope it works." (It didn't.) And Egyptian President Mohamed Morsi's attempted power grab in November made such nostalgia commonplace.
The skepticism is as predictable as it is misguided. Every surge of democratization over the last century -- after World War I, after World War II, during the so called third wave in recent decades -- has been followed by an undertow, accompanied by widespread questioning of the viability and even desirability of democratic governance in the areas in question. As soon as political progress stalls, a conservative reaction sets in as critics lament the turbulence of the new era and look back wistfully to the supposed stability and security of its authoritarian predecessor. One would have hoped that by now people would know better -- that they would understand that this is what political development actually looks like, what it has always looked like, in the West just as much as in the Middle East, and that the only way ahead is to plunge forward rather than turn back.
The first error critics make is treating new democracies as blank slates, ignoring how much of their dynamics and fate are inherited rather than chosen. Turmoil, violence,
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