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HIV-1 replication depends on a delicate balance between cellular co-factors and antiviral restriction factors. Lens epithelium-derived growth factor (LEDGF/p75) benefits HIV, whereas apolipoprotein B mRNA-editing catalytic polypeptide-like 3G (APOBEC3G), tripartite motif 5alpha (TRIM5α), and tetherin exert anti-HIV activity. Expression levels of these proteins possibly contribute to HIV-1 resistance in HIV-1-exposed populations.
We used real-time PCR and flow cytometry to study mRNA and protein levels respectively in PBMC and PBMC subsets. We observed significantly reduced LEDGF/p75 protein levels in CD4+ lymphocytes of HIV-1-exposed seronegative subjects relative to healthy controls, whereas we found no differences in APOBEC3G, TRIM5α, or tetherin expression. Untreated HIV-1-infected patients generally expressed higher mRNA and protein levels than healthy controls. Increased tetherin levels, in particular, correlated with markers of disease progression: directly with the viral load and T cell activation and inversely with the CD4 count.
Our data suggest that reduced LEDGF/p75 levels may play a role in resistance to HIV-1 infection, while increased tetherin levels could be a marker of advanced HIV disease. Host factors that influence HIV-1 infection and disease could be important targets for new antiviral therapies.
Citation: Mous K, Jennes W, Camara M, Seydi M, Daneau G, Mboup S, et al. (2012) Expression Analysis of LEDGF/p75, APOBEC3G, TRIM5alpha, and Tetherin in a Senegalese Cohort of HIV-1-Exposed Seronegative Individuals. PLoS ONE 7(3): e33934. https://doi.org/10.1371/journal.pone.0033934
Editor: Rupert Kaul, University of Toronto, Canada
Received: October 19, 2011; Accepted: February 23, 2012; Published: March 27, 2012
Copyright: © 2012 Mous 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: This work is supported by the fund for Scientific Research (FWO) Flanders (www.fwo.be) (grant G.0660.06) and an FWO doctoral fellowship to KM. 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.
Human immunodeficiency virus type 1 (HIV-1) interacts with many cellular host proteins during its replication cycle –. Some of these proteins are required for HIV-1 replication while others exhibit antiviral activity. Lens epithelium-derived growth factor p75 (LEDGF/p75)  is a cellular co-factor, whereas apolipoprotein B mRNA-editing catalytic polypeptide-like 3G (APOBEC3G, A3G) , tripartite motif 5alpha (TRIM5α) , and tetherin (BST2, CD317, HM1.24)  are cellular factors with distinct antiviral activities. The main working mechanisms of these factors can be summarized as follows. LEDGF/p75 assists in the integration of viral cDNA into specific regions of the host's genetic material , . APOBEC3G inhibits HIV replication by inducing G-to-A hypermutation of the viral HIV-1 genome during reverse transcription , . TRIM5α structurally disorders the retroviral capsid, leading to the interruption of the natural “uncoating” process in a species-specific manner , ; and was recently described to promote innate immune signaling . Tetherin, finally, inhibits HIV release by tethering newly formed retrovirus particles to the cell membrane .
The significance of these four proteins as possible correlates of protection against HIV-1 remains to be determined. Several studies on peripheral blood mononuclear cells (PBMC) have found that APOBEC3G mRNA levels correlate positively with the CD4 count and negatively with the viral load of untreated HIV-1-infected subjects –, suggesting that APOBEC3G contributes to the control of HIV-1 infection. However, other studies did not find such correlations for APOBEC3G – or TRIM5α  mRNA. In addition, certain studies compared the expression of APOBEC3G or TRIM5α between distinct study populations like untreated HIV-1-infected subjects, healthy controls, and HIV-1-exposed seronegative individuals. Some studies observed lower mRNA expression levels of APOBEC3G , ,  or TRIM5α  in untreated HIV-1-infected subjects compared to healthy controls, although the opposite was also found for APOBEC3G . APOBEC3G mRNA and protein expression levels were also studied respectively in PBMC and PBMC-subsets (CD4+ and CD8+ lymphocytes, and CD14+ monocytes) of HIV-1-exposed seronegative subjects relative to healthy controls , , . Here too, conflicting data were obtained, showing either similar  or higher ,  levels of antiviral APOBEC3G in HIV-1-exposed seronegative subjects relative to healthy controls.
In the present study, we investigated mRNA and protein expression levels of the 4 HIV-1-related factors LEDGF/p75, APOBEC3G, TRIM5α, and tetherin in HIV-1-exposed seronegative subjects, healthy controls, untreated HIV-1-infected subjects, and antiretroviral therapy-treated HIV-1-infected subjects. HIV-1-exposed seronegative subjects are individuals who remain HIV-1 seronegative despite frequent and unprotected exposure to HIV, and are observed in cohorts of commercial sex workers, men having sex with men, intravenous drug users, and discordant couples . In this study, HIV-1-exposed seronegative subjects were enrolled from a Senegalese cohort of HIV-1 serodiscordant couples. Real-time quantitative polymerase chain reaction (qPCR) was applied to study mRNA expression in PBMC. Protein expression levels were determined simultaneously in different PBMC subsets by flow cytometry. We hypothesized that expression levels of the above-mentioned proteins may contribute to the putative in vivo HIV-1 restriction in HIV-1-exposed seronegative subjects.
Materials and Methods
The study was approved by the Internal Review Board of the Institute of Tropical Medicine (Antwerp, Belgium) and by the Ethical Committees of the Senegalese Ministry of Health (Dakar, Senegal) and the University Hospital of Antwerp (Belgium). All study subjects gave written informed consent prior to enrollment.
This study was conducted on a cohort of heterosexual couples recruited at the Department of Infectious Diseases at the Fann University Teaching Hospital, Dakar, Senegal, as previously described . Twenty three HIV-1 exposed seronegative subjects (HESN) in HIV-1 serodiscordant couples, 23 healthy controls (HC) in HIV-1-negative seroconcordant monogamous couples, and 45 HIV-1-infected patients in HIV-1 serodiscordant or HIV-1-positive seroconcordant couples were studied. Within the group of 45 HIV-1-infected patients, 9 patients were untreated HIV-1-infected subjects (HIV-UT) and 36 patients were antiretroviral therapy-treated HIV-1-infected subjects (HIV-ART). All couples had a sexual relationship of at least 7.5 years. HESN and HC were matched for gender. Blood samples and standard questionnaires with information on socio-demographics and sexual behavior were collected. Male condom provision and sexual risk reduction counseling were provided during every visit.
Sample collection and processing
Whole blood samples were collected in EDTA tubes. CD4+ T cell counts and T cell activation levels were determined in fresh whole blood using CD3, CD4, CD8, and CD38 fluorochrome-labeled antibodies and a FACSCalibur flow cytometer (BD Biosciences) like previously reported . In this article, the T cell activation status is determined by the percentage of CD38+ cells among the CD8+ T cells. Plasma and PBMC were separated from fresh whole blood, frozen down at −80°C and in liquid nitrogen, respectively, and shipped to Belgium. HIV and HSV-2 (Herpes simplex virus type 2) status were determined in plasma by ELISA. HIV-1 viral loads were quantified in plasma by the Amplicor HIV-1 Monitor assay, version 1.5 (Roche Diagnostics GmbH). PBMC were thawed, washed twice, and counted prior to mRNA and protein expression analysis.
RNA extraction, DNase treatment and reverse transcription
PBMC samples were dissolved in TRIzol® Reagent (Invitrogen) and stored at −80°C for less than three months. Total RNA was isolated according to the manufacturer's instructions (Invitrogen). Removal of genomic DNA was performed with the DNA-free Kit (Ambion) following the manufacturer's protocol. Additional wash steps, once with 200 µl isopropanol (in the presence of 0.7 µl of glycogen) and twice with 75% ethanol, were performed to remove traces of contaminants. The NanoDrop ND-1000 spectrophotometer (Thermo Scientific) was used to quantify total RNA levels. Total DNase-treated RNA was stored at −80°C for less than one month. The iScript cDNA synthesis Kit (Bio-Rad Laboratories NV-SA) was applied to 400 ng of total DNase-treated RNA following the manufacturer's instructions. In parallel, 100 ng of the same RNA was dissolved in RNase/DNase-free water (no-RT control) at the same final concentration (20 ng/10.5 µl) as the RT (reverse transcribed) samples. Both RT and no-RT samples were stored at −80°C for less than one month. For each sample, the difference in quantification cycle (Cq)-value between RT and no-RT samples was higher than 10. During optimization of the qPCR protocol, RNA quality was evaluated by RNA gel electrophoresis (1% agarose gel). The 18S and 28S fragments were visualized with GelRed (VWR) versus a 1-kb DNA ladder (Invitrogen) and were observed in the expected 1∶2 ratio on representative samples.
Messenger RNA (mRNA) expression levels were quantified by real-time PCR using the Bio-Rad CFX96 system and SYBR Green mix (Bio-Rad). Target-specific primers were designed using the Harvard website: http://pga.mgh.harvard.edu/primerbank/, except for TRIM5α and TBP, for which Primer3 software (v.0.4.0) was used. Specificity was tested using BLAST (NCBI). All primer pairs, except for TRIM5α, could be designed to span at least one intron. Name, GeneID, primer sequences, exon localization and amplicon length are listed in Table 1. The primer mixes were prepared by combining Forward (Fw) and Reverse (Re) Primers (Eurogentec), both at a concentration of 2 pmol/µl. Each optimized 25 µl reaction contained 12.5 µl of iQ SYBR Green Supermix (Bio-Rad), 2 µl of the primer mix and 5, 10 or 20 ng of the cDNA template at a volume of 10.5 µl. Each assay included, in triplicate, a standard curve, a no-template control (water), the RT samples, and, in the case of TRIM5α, the no-RT samples. The following subsequent heating steps were included as PCR cycling conditions: 50°C (10 min), 95°C (3 min), 40 cycles of 95°C (10 s) and 60°C (30 s) with fluorescence capturing following each cycle, 95°C (10 s), and melting curve acquisition with fluorescence capturing every 5 s during a temperature increase in 0.5°C increments from 65°C to 95°C. The amplicon length of the PCR product was validated by 2% agarose DNA gelelectrophoresis. PCR products were visualized with GelRed (VWR) versus a 100-bp DNA ladder (Invitrogen).
mRNA data analysis
The obtained results (Cq-values) were exported from the Bio-Rad CFX Manager version 1.5 and imported into qbasePLUS software, version 1.5, from BioGazelle (Ghent, Belgium) following baseline subtraction and manual threshold matching. The GeNorm applet selected the reference genes B2M and GAPDH out of five genes (UBC, B2M, GAPDH, RPL13A, TBP) as the most stably expressed across the different study populations (25 samples, all applied in triplicate). Both reference genes were used to normalize target gene expression in the qbasePLUS software. For the standard curve, a 2-fold serial dilution of cDNA of one Caucasian healthy control was used. Cq-values were converted into relative expression values taking into account amplification efficiencies, inter-run variations, and normalization factors. Outliers were excluded when a difference of more than 1.0 was registered in the Cq-values. Finally, CNRQ (Calibrated Normalized Relative Quantity) values were exported from the qbasePLUS software and statistically investigated.
Intracellular and surface protein staining
The protein expression levels of LEDGF/p75, APOBEC3G, TRIM5α, and tetherin were investigated in CD4+ T cell and CD14+ monocyte subsets based on simultaneous intracellular and/or cell surface staining of PBMC. The following reagents were used: 0.1% bovine serum albumin (Acros Organics) and 0.05% sodium azide (Merck) in phosphate buffered saline (PBS, Lonza) as washing buffer; Reagents A and B to fix and permeabilize cells, respectively (Leucoperm kit, AbD Serotec); primary mouse unlabeled antibody (isotype IgG1 and IgG2a) and anti-LEDGF/p75 (IgG1) from BD Biosciences, anti-APOBEC3G and anti-TRIM5α (both IgG1) from the AIDS Research and Reference Reagent Program (Division of AIDS, NIAID, NIH), and anti-tetherin (IgG2a) as kindly provided by Chugai Pharmaceutical Co., Japan; secondary FITC-labeled antibody (BD Biosciences); normal mouse serum (eBioscience); membrane antibodies anti-CD4-PerCP, anti-CD45RO-APC, anti-CD38-PE, anti-CD14-APC, and anti-CD16-PE from BD Biosciences; and 1% paraformaldehyde (Sigma-Aldrich). LEDGF/p75, APOBEC3G, TRIM5α, and tetherin were analyzed using an in-house optimized intracellular staining (ICS) protocol , . Cells were washed (630×g, 10 min, RT), fixed (10 min, RT), washed twice, permeabilized (30 min, 4°C), washed, incubated with primary antibody (24 h, 4°C), washed twice, incubated with secondary antibody supplemented with reagent B (30 min, 4°C), washed twice, incubated with normal mouse serum (10 min, 4°C), incubated with a membrane antibody cocktail (CD4/CD45RO/CD38 or CD4/CD14/CD16 to study lymphocytes or monocytes, respectively, 20 min, 4°C), washed twice, and dissolved in 1% paraformaldehyde prior to data acquisition (FACSCalibur flow cytometer, BD Biosciences). To study membrane tetherin levels, PBMC were processed according to the protocol above with a 30-min anti-tetherin incubation at 4°C, without fixation or permeabilization steps. In general, at least 25000 and 50000 events were respectively measured to study expression patterns in CD4+ lymphocytes and CD14+ monocytes. Data analysis with CellQuest Pro Software (BD Biosciences) produced median fluorescence intensity (MFI) values, reflecting the median amount of bound antibody per cell and thus, the protein's abundance. Host protein MFI-values were corrected for non-specific background staining by subtracting isotype MFI-values.
Differences in expression levels between individuals of different study groups were investigated with non-parametric Mann-Whitney U tests. Correlation analyses were performed using non-parametric Spearman's rank correlation tests. Observations were considered statistically significant when p<0.05. Statistical analyses were performed with SPSS version 17.0, and graphs were drawn with GraphPad Prism, version 5.
Twenty three HIV-1-exposed seronegative subjects (HESN), 23 healthy controls (HC), and 45 HIV-1-infected patients were studied. Nine HIV-1-infected patients were untreated (HIV-UT) and 36 were antiretroviral therapy-treated (HIV-ART). Several parameters like age, gender, duration of the sexual relationship, CD4 count, number of sexual contacts per month, condom use, and HSV-2 serostatus were compared between HESN and HC, HIV-UT and HC, and HIV-ART and HIV-UT (Table 2). The HC subjects were in a monogamous relationship for at least 7.5 years at the time of enrollment. They reported a significantly lower percentage of condom use. HIV-1-infected subjects were more often male individuals. Therapy-naïve HIV-1 infected subjects (HIV-UT) showed significantly lower CD4 count levels than HC, and a higher prevalence of HSV-2 seropositivity.
Differences in mRNA expression in PBMC
The mRNA expression levels of LEDGF/p75, APOBEC3G, TRIM5α, and tetherin were analyzed for all study subjects in PBMC by real-time qPCR (Table 3). None of the genes displayed a significant difference in mRNA expression between HESN and HC. HIV-UT showed significantly lower levels of LEDGF/p75 (p = 0.008) and higher levels of tetherin (p = 0.027) than HC. Additionally, higher expression levels of APOBEC3G (p = 0.078), TRIM5α (p = 0.016), and tetherin (p<0.001) were observed in HIV-UT relative to HIV-ART subjects.
Differences in protein expression in lymphocytes and monocytes
Next, we analyzed protein expression levels of LEDGF/p75, APOBEC3G, TRIM5α, and tetherin in CD4+ lymphocytes and CD14+ monocytes by a novel intracellular staining assay . Expression of LEDGF/p75 was found to be significantly reduced (p = 0.022) in CD4+ lymphocytes of HESN compared to HC (Table 3). APOBEC3G, TRIM5α, and tetherin protein levels were similar for HESN and HC. HIV-UT showed significantly higher tetherin levels in CD4+ lymphocytes and significantly higher levels of all four proteins in CD14+ monocytes compared to HC and HIV-ART.
Correlations between mRNA and protein levels
Subsequently, we correlated mRNA levels in PBMC with protein levels specifically in lymphocytes and monocytes within the entire study population including HESN, HC, HIV-UT, and HIV-ART subjects (Figure 1). For most HIV-related host factors, we observed a significant positive correlation between mRNA and protein level as was most prominent for total tetherin, whereas no significant correlations were observed for LEDGF/p75.
Correlation patterns were studied in all study subjects between mRNA (CNRQ-values, x-axis) and protein (MFI-values, y-axis) levels of (A) LEDGF/p75 (LGF), (B) APOBEC3G (A3G), (C) TRIM5α (T5a), (D) membrane tetherin (memTeth), and (E) total tetherin (totTeth). mRNA levels were studied in PBMC while protein levels were analyzed in lymphocytes (Ly, left), CD4+ lymphocytes (CD4Ly, middle), and CD14+ monocytes (CD14Mo, right). CNRQ-values refer to Calibrated Normalized Relative Quantity of mRNA expression levels and MFI-values refer to Median Fluorescence Intensity of protein expression levels. Spearman's Rho (Rho) and p-values were obtained by non-parametric Spearman's rank correlation tests. P-values below 0.05 are in bold.
Correlates of reduced LEDGF/p75 in HESN
To obtain insight into the possible role played by LEDGF/p75 in resistance to HIV-1 infection, we studied the reduced LEDGF/p75 expression levels in HESN considering the duration of the sexual relation, the number of sexual contacts per month, the frequency of condom use, and the HSV-2 serostatus (Table 4). We found that LEDGF/p75 expression levels were most reduced among HESN with the highest number of sexual contacts per month (p = 0.065), whereas no difference in LEDGF/p75 expression was found in the HIV-1-negative control group (Table 4).
Correlations between expression levels and T cell activation
Finally, we investigated whether the observed differences in mRNA and/or protein expression level could be correlated to T cell activation levels and/or disease status (viral load and CD4 count). Whereas no significant difference was observed in CD4+ T cell activation level (% CD38+ cells among the CD4+ T cells, p = 0.297), HESN showed a trend towards lower CD8+ T cell activation levels than HC (p = 0.095) in Figure 2A. Reduced T cell activation levels among HESN did not correlate with their reduced LEDGF/p75 protein expression levels (p = 0.970, Figure 2B). Neither did we observe differences in LEDGF/p75 expression between distinct CD4+ lymphocytes based on CD45RO (memory) or CD38 (activation) staining among HESN (data not shown). In HIV-UT, T cell activation levels correlated directly with TRIM5α (p = 0.002) and tetherin (p = 0.086) mRNA levels, and inversely with LEDGF/p75 mRNA levels (p = 0.037) (data not shown). Membrane and total expression levels of tetherin, in particular, correlated directly with T cell activation and the viral load, and inversely with the CD4 count. These correlations were most prominent for membrane tetherin as is depicted in Figure 2C–E.
(A) Difference in CD4 (left) and CD8 (right) T cell activation status (y-axis) between HESN and HC (x-axis). P-values were obtained through the Mann Whitney U test. (B) Correlation pattern in HESN subjects between LEDGF/p75 protein levels (LGF, y-axis) in CD4+ lymphocytes (CD4 Ly, left) and CD14+ monocytes (Mo, right) and T cell activation status (x-axis). (C–E) The graphs depict correlation patterns in CD4+ lymphocytes (CD4 Ly, left) and CD14+ monocytes (Mo, right) of HIV-UT subjects between membrane tetherin protein levels (memTeth, y-axis) and T cell activation status (x-axis, C), CD4 count (cells/mm3) (x-axis, D) and log viral load (x-axis, E). The subject's general T cell activation status is described by the percentage of CD38+ cells among the CD8+ T cells (B,C). Spearman's Rho (Rho) and p-values were obtained by non-parametric Spearman's rank correlation tests. P-values below 0.05 are in bold. MFI = Median Fluorescence Intensity.
In this study, we explored whether the expression of LEDGF/p75, APOBEC3G, TRIM5α, and tetherin could play a role in HIV-1 resistance. We studied mRNA levels in PBMC and protein levels in CD4+ lymphocyte and monocyte subsets. We observed significantly reduced levels of LEDGF/p75 protein expression in the CD4+ T cells of HESN compared to those of HC, whereas similar mRNA levels were found for both HESN and HC. Our findings of lower levels of LEDGF/p75 in CD4+ lymphocytes of HESN are in line with two recent HESN-studies showing reduced gene expression levels of several host genes important for HIV replication , . Lower LEDGF/p75 protein levels may cause HIV-1 integration to occur less efficiently, thus contributing to HIV-1 resistance in HESN subjects. This hypothesis is supported by an in vitro experiment that found a direct correlation between LEDGF/p75 expression levels and the efficiency of HIV-1 integration in the host genome . Other studies, however, reported that efficient knockdown or knock-out of LEDGF/p75 is not sufficient to completely abolish HIV-1 replication –, suggesting that other factors may be needed in concert with LEDGF/p75 to limit HIV integration in HESN subjects.
We observed no differences in mRNA or protein levels of APOBEC3G, TRIM5α, or tetherin between HESN and HC. These findings are at variance with previous reports showing elevated APOBEC3G expression levels in HESN compared with HC , . On the other hand, it has been suggested that reduced exposure to HIV-1 results in comparable APOBEC3G mRNA levels for HESN and HC over time . In our HESN population, most HIV-1-infected partners were treated with antiretroviral therapy, and all couples received counseling to promote condom use. Therefore, the lack of distinct expression patterns may indeed result from the relatively low levels of HIV exposure in our population. Moreover, Reddy et al  observed similar levels of APOBEC3G mRNA between pre-infection values of seroconverters and exposed persistently negative individuals, suggesting that APOBEC3G mRNA levels would not contribute to protection against HIV-1.
Untreated HIV-1-infected patients (HIV-UT) showed significantly lower LEDGF/p75 and higher tetherin mRNA levels than HC, and higher APOBEC3G, TRIM5α, and tetherin mRNA levels than HIV-ART. At protein level, HIV-UT expressed significantly enhanced levels of tetherin in the lymphocytes and of all four proteins in the monocytes. These data suggest that HIV-1-infected subjects try to curb HIV-1 replication by increasing the expression level of the antiviral restriction factors.
Interestingly, increased levels of tetherin in HIV-UT correlated directly with T cell activation and the viral load and inversely with the CD4 count. This finding suggests that tetherin is not capable of controlling viral replication; instead it appears to be a marker of advanced HIV disease in therapy-naïve HIV-1-infected patients. Recently, Coleman et al  also reported increased tetherin expression levels upon exposure to HIV-1 without it being capable of restricting cell-to-cell spread of the virus. At least two other studies support our observations by suggesting that the tetherin-induced accumulation of unreleased virions at the cell membrane could actually promote cell-to-cell spread, e.g., by enhancing fusogenicity or regulating the integrity of the viral synapse , . In turn, accumulation of virus particles at the cell membrane may trigger further increases in immune activation , . Thus, tetherin may have a dual role during HIV-1 infection and disease. As an innate mediator, tetherin may be involved in the control of early or acute virus replication but it could become ineffective as the infection progresses.
T-cell activation levels also correlated directly with mRNA levels of TRIM5α but not with those of APOBEC3G; nevertheless we expect that similar mechanisms like those observed for tetherin are involved here. Indeed, type I interferons which are induced upon viral replication are known to upregulate APOBEC3G , –, TRIM5α , , and tetherin , . High-level interferon receptor expression on monocytes  may explain why our data showed relatively higher increases of APOBEC3G, TRIM5α, and tetherin in monocytes than in lymphocytes.
Intriguingly, HIV-UT expressed reduced mRNA levels in PBMC and elevated protein levels in monocytes for LEDGF/p75, relative to HC. The mRNA levels correlated inversely (p = 0.05; Rho = −0.667) and the protein levels tended to correlate directly (p = 0.170; Rho = 0.5) with the viral load of these patients (data not shown). These correlations suggest that LEDGF/p75 expression may also account for disease progression in HIV-UT, supporting recently described observations by Madlala et al . Parallel to type I interferon induction, viral replication induces the production of several cytokines and chemokines amongst which Tumor Necrosis Factor alpha (TNF-α), which can induce LEDGF/p75 levels , . Of note, long period or high level exposure to TNF-α makes LEDGF/p75 levels decrease again , which may play a role in the difference seen between LEDGF/p75 mRNA and protein levels.
Apart from the reasoning in the previous paragraph, mRNA and protein levels may differ as mRNA is not necessarily translated into (functional) protein , and external factors may act differently upon protein and mRNA levels , . Our data e.g. also show a difference in mRNA versus protein expression for LEDGF/p75 in HESN (Figure 1A). Nevertheless, we did observe some correlations between protein levels in (CD4+) lymphocytes, and mRNA levels in PBMC which are predominantly composed of lymphocytes (Figure 1B–E). Of note, due to the limited number of CD4+ cells in HIV-1-infected subjects, we were obliged to work with PBMC for quantification of mRNA levels. On the other hand, the flow cytometry based method allowed us to investigate protein levels in PBMC subsets. As earlier studies also focused on mRNA levels in PBMC, we could easily compare our results with these results previously obtained. For example, our data supported that APOBEC3G mRNA levels are similar for HESN and HC . On the other hand, in literature, limited information is forehand regarding protein expression in particular cell-types while proteins are considered to be of greater relevance in the “direct” fight against HIV-1 infection as they are the “work horses” of the cell.
In summary, we observed significantly lower levels of LEDGF/p75 in CD4+ lymphocytes of HESN subjects, suggesting that LEDGF/p75 may play a role in the in vivo resistance to HIV-1 infection. Untreated HIV-1-infected patients showed increased levels of the antiviral proteins APOBEC3G, TRIM5α, and tetherin, with tetherin in particular as a possible marker of advanced HIV disease. Future studies of LEDGF/p75 expression in other populations of HESN subject will be required to confirm our findings. Understanding the role of HIV-1-related host factors in HIV-1 susceptibility and disease could be of great interest for the development of future antiviral therapies.
We thank the individuals who kindly participated in the study; Dr Ndeye Fatou Ngom Gueye, Dr Ibrahima Ndiaye, and Dr Khady Ba Fall for recruiting and Marianne Ndiaye for counseling and follow-up of the Senegalese participants; Dr Aïssatou Gaye Ndiaye for HIV serological testing; and Katrien Fransen for HIV-1 viral load and HSV-2 testing. We acknowledge Chugai Pharmaceutical Co. (Japan) for kindly providing the anti-tetherin antibody and the NIH AIDS Research and Reference Reagent Program (Division of AIDS, NIAID, NIH) for providing the anti-APOBEC3G and anti-TRIM5α antibodies.
Conceived and designed the experiments: WJ LK XVO. Performed the experiments: KM MC. Analyzed the data: KM WJ GD. Contributed reagents/materials/analysis tools: MC MS SM. Wrote the paper: KM WJ LK XVO.
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- 19. Reddy K, Winkler CA, Werner L, Mlisana K, Abdool Karim SS, et al. (2010) APOBEC3G expression is dysregulated in primary HIV-1 infection and polymorphic variants influence CD4+ T-cell counts and plasma viral load. AIDS 24: 195–204. 10.1097/QAD.0b013e3283353bba [doi].
- 20. Sewram S, Singh R, Kormuth E, Werner L, Mlisana K, et al. (2009) Human TRIM5alpha expression levels and reduced susceptibility to HIV-1 infection. J Infect Dis 199: 1657–1663. 10.1086/598861 [doi].
- 21. Biasin M, Piacentini L, Caputo SL, Kanari Y, Magri G, et al. (2007) Apolipoprotein B mRNA-Editing Enzyme, Catalytic Polypeptide-Like 3G: A Possible Role in the Resistance to HIV of HIV-Exposed Seronegative Individuals. J Infect Dis 195: 960–964.
- 22. Miyazawa M, Lopalco L, Mazzotta F, Lo CS, Veas F, et al. (2009) The ‘immunologic advantage’ of HIV-exposed seronegative individuals. AIDS 23: 161–175. 10.1097/QAD.0b013e3283196a80 [doi];00002030-200901140-00002 [pii].
- 23. Camara M, Dieye TN, Seydi M, Diallo AA, Fall M, et al. (2010) Low-level CD4+ T cell activation in HIV-exposed seronegative subjects: influence of gender and condom use. J Infect Dis 201: 835–842. 10.1086/651000 [doi].
- 24. Jennes W, Camara M, Dieye T, Mboup S, Kestens L (2008) Higher homologous and lower cross-reactive Gag-specific T-cell responses in human immunodeficiency virus type 2 (HIV-2) than in HIV-1 infection. J Virol 82: 8619–8628.
- 25. Mous K, Jennes W, De RA, Pintelon I, Kestens L, et al. (2011) Intracellular detection of differential APOBEC3G, TRIM5alpha, and LEDGF/p75 protein expression in peripheral blood by flow cytometry. J Immunol Methods. S0022-1759(11)00152-9 [pii];10.1016/j.jim.2011.06.028 [doi].
- 26. McLaren PJ, Ball TB, Wachihi C, Jaoko W, Kelvin DJ, et al. (2010) HIV-exposed seronegative commercial sex workers show a quiescent phenotype in the CD4+ T cell compartment and reduced expression of HIV-dependent host factors. J Infect Dis 202: Suppl 3S339–S344. 10.1086/655968 [doi].
- 27. Madlala P, Gijsbers R, Christ F, Hombrouck A, Werner L, et al. (2011) Association of Polymorphisms in the LEDGF/p75 Gene (PSIP1) with Susceptibility to HIV-1 Infection and Disease Progression. AIDS. 10.1097/QAD.0b013e328349c693 [doi].
- 28. Marshall HM, Ronen K, Berry C, Llano M, Sutherland H, et al. (2007) Role of PSIP1/LEDGF/p75 in lentiviral infectivity and integration targeting. PLoS One 2: e1340. 10.1371/journal.pone.0001340 [doi].
- 29. Shun MC, Raghavendra NK, Vandegraaff N, Daigle JE, Hughes S, et al. (2007) LEDGF/p75 functions downstream from preintegration complex formation to effect gene-specific HIV-1 integration. Genes Dev 21: 1767–1778. 21/14/1767 [pii];10.1101/gad.1565107 [doi].
- 30. Zheng Y, Ao Z, Jayappa KD, Yao X (2010) Characterization of the HIV-1 integrase chromatin- and LEDGF/p75-binding abilities by mutagenic analysis within the catalytic core domain of integrase. Virol J 7: 68. 1743-422X-7-68 [pii];10.1186/1743-422X-7-68 [doi].
- 31. Zielske SP, Stevenson M (2006) Modest but reproducible inhibition of human immunodeficiency virus type 1 infection in macrophages following LEDGFp75 silencing. J Virol 80: 7275–7280. 80/14/7275 [pii];10.1128/JVI.02470-05 [doi].
- 32. Coleman CM, Spearman P, Wu L (2011) Tetherin does not significantly restrict dendritic cell-mediated HIV-1 transmission and its expression is upregulated by newly synthesized HIV-1 Nef. Retrovirology 8: 26. 1742-4690-8-26 [pii];10.1186/1742-4690-8-26 [doi].
- 33. Jolly C, Booth NJ, Neil SJ (2010) Cell-Cell Spread of Human Immunodeficiency Virus Type 1 Overcomes Tetherin/BST-2-Mediated Restriction in T cells. J Virol 84: 12185–12199. JVI.01447-10 [pii];10.1128/JVI.01447-10 [doi].
- 34. Gummuluru S, Kinsey CM, Emerman M (2000) An in vitro rapid-turnover assay for human immunodeficiency virus type 1 replication selects for cell-to-cell spread of virus. J Virol 74: 10882–10891.
- 35. Malim MH, Emerman M (2008) HIV-1 accessory proteins–ensuring viral survival in a hostile environment. Cell Host Microbe 3: 388–398. S1931-3128(08)00126-1 [pii];10.1016/j.chom.2008.04.008 [doi].
- 36. Tokarev A, Skasko M, Fitzpatrick K, Guatelli J (2009) Antiviral activity of the interferon-induced cellular protein BST-2/tetherin. AIDS Res Hum Retroviruses 25: 1197–1210. 10.1089/aid.2009.0253 [doi].
- 37. Chen K, Huang J, Zhang C, Huang S, Nunnari G, et al. (2006) Alpha interferon potently enhances the anti-human immunodeficiency virus type 1 activity of APOBEC3G in resting primary CD4 T cells. J Virol 80: 7645–7657.
- 38. Stopak KS, Chiu YL, Kropp J, Grant RM, Greene WC (2007) Distinct patterns of cytokine regulation of APOBEC3G expression and activity in primary lymphocytes, macrophages, and dendritic cells. J Biol Chem 282: 3539–3546. M610138200 [pii];10.1074/jbc.M610138200 [doi].
- 39. Koning FA, Newman EN, Kim EY, Kunstman KJ, Wolinsky SM, et al. (2009) Defining APOBEC3 expression patterns in human tissues and hematopoietic cell subsets. J Virol 83: 9474–9485. JVI.01089-09 [pii];10.1128/JVI.01089-09 [doi].
- 40. Asaoka K, Ikeda K, Hishinuma T, Horie-Inoue K, Takeda S, et al. (2005) A retrovirus restriction factor TRIM5alpha is transcriptionally regulated by interferons. Biochem Biophys Res Commun 338: 1950–1956. S0006-291X(05)02472-1 [pii];10.1016/j.bbrc.2005.10.173 [doi].
- 41. Sakuma R, Mael AA, Ikeda Y (2007) Alpha interferon enhances TRIM5alpha-mediated antiviral activities in human and rhesus monkey cells. J Virol 81: 10201–10206. JVI.00419-07 [pii];10.1128/JVI.00419-07 [doi].
- 42. Kawai S, Azuma Y, Fujii E, Furugaki K, Ozaki S, et al. (2008) Interferon-alpha enhances CD317 expression and the antitumor activity of anti-CD317 monoclonal antibody in renal cell carcinoma xenograft models. Cancer Sci 99: 2461–2466. CAS968 [pii];10.1111/j.1349-7006.2008.00968.x [doi].
- 43. Sharma P, Singh DP, Fatma N, Chylack LT Jr, Shinohara T (2000) Activation of LEDGF gene by thermal-and oxidative-stresses. Biochem Biophys Res Commun 276: 1320–1324. 10.1006/bbrc.2000.3606 [doi];S0006-291X(00)93606-4 [pii].
- 44. Takamura Y, Fatma N, Kubo E, Singh DP (2006) Regulation of heavy subunit chain of gamma-glutamylcysteine synthetase by tumor necrosis factor-alpha in lens epithelial cells: role of LEDGF/p75. Am J Physiol Cell Physiol 290: C554–C566. 290/2/C554 [pii];10.1152/ajpcell.00398.2005 [doi].
- 45. Bustin SA, Benes V, Garson JA, Hellemans J, Huggett J, et al. (2009) The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem 55: 611–622. clinchem.2008.112797 [pii];10.1373/clinchem.2008.112797 [doi].
- 46. Chiu YL, Greene WC (2006) Multifaceted antiviral actions of APOBEC3 cytidine deaminases. Trends Immunol 27: 291–297.

domain: biology


text:
I’d heard about Kate Bingaman Burt’s work awhile back, glanced at it, but didn’t really pay much mind. Then I heard she had published a book with a decent sized independent publisher, I decided to dig deeper. Perhaps I’m behind in the game here, but I’m really intrigued by the work. It’s so simple, and really, benign in nature, but captures my attention; illustrated lists of consumable items? A whole poster filled with nothing but bad passwords? Who would think of this stuff? No one but Kate, obviously, but she’s doing a kick-ass job of it and it seems to be working. More power to her.
Of course she has an Etsy shop. You can buy pretty much all of it there except the book, which is easily found on Amazon.

domain: artistic


Results: Febvre and Prado win qualifiers in Loket
With traction at a premium and ruts at a minimum, there was some great racing during the qualifying races at Loket for round 13 of the world championship in the Czech Republic.
Jeremy Seewer led early in MXGP but Romain Febvre passed Gajser then eventually got his teammate, after putting on some serious pressure, to take a very impressive race win, with Frenchman using his hard pack skills to find grip on the slippery surface.
Gajser tipped over while third chasing the Yamahas but slipped off, however would still work his way back into third, passing Arnaud Tonus.
Coldenhoff had been running inside the top five but a pass by Arnaud Tonus left him off the track and then Searle and Paulin found their way by in the closing laps.
For Searle it was one of his best rides of the season. Finally getting a good start, he got into the pace of the leaders and held Paulin off for sixth in his first Grand Prix as a factory Kawasaki rider and also posted the second fastest lap of the race to show he still has the speed to run at the sharp end in the right surroundings.
Jorge Prado worked his way by Henri Jacobi and Darian Sanayei to take the lead and clear off at Loket but the battled raged throughout the top ten. Vialle followed Prado through the Kawasaki duo to take second,while Jacobi just held off a charging Calvin Vlaanderen, who had worked through the top ten to take third.
Dylan Walsh rode great for fifth with Sterry a strong sixth with Thomas Kjer Olsen coming from a first turn pile-up, that also claimed Jags Geerts, to take seventh. Mitchell Harrison came from 14th to ninth to show he is adapting well to the unique circuit.


Wire Rod Packing Machine
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Please send us more information about your project, we would like to give you our proposal immediately after getting your details. Sincerely welcome you to come and visit our plant.


text:
This year AltGen strapped on their boots and grabbed their hard hats as we set-out to celebrate ‘Global Wind Day’ across the African continent. With most of our staff visiting various wind farms and experiencing the magic for themselves, our consulting team attended the graduation ceremony of 18 students who successfully completed the internationally recognized Wind Turbine Service Technician Training Programme at the South African Renewable Energy Technology Centre (SARETEC).
AltGen congratulates all of the students and we look forward to tracking their professional progress.
Our team in Kenya took the scenic road to the idyllic Ngong Hills where they visited the Ngong Hills Wind Farm owned and operated by KenGen. Observations by our team was the accessibility and beauty of the farm, it is open to visitors and the local community bring their cattle to graze.
ZA Employees visited West Coast One Wind Farm in Vredenburg, where they were given an in-depth view of the SCADA operations and the important role it plays, and taken on a tour inside the gentle giants – wind turbines supplied by Vestas.

domain: geography


text:
Invader - Avatar Beat - "It was really hard to find a good song for invader."
Witness - theyaremanycolors (FLASHING LIGHTS WARNING) - "A chaotic song for a chaotic character that is completely overwhelmed by everything happening all at once."
Artisan - Vagrant Keepsake of Oblivion - "It took a while to find a good song for Artisan, but I think this one works well!"
Vestige - Desert Battle 2 - "Do I really need to explain this one?"
Visitor - Asleep Atop An Atom - "A slower song for one of the slower hosts."
Subject - 4 - "I dunno man, it just fits. Please ignore the album art."
Dreamer - Does the sheep count the sheep? - "Good song + song related to sleeping."
Parting - Diva - "dude...the vocals are easy lover and the guitar is parting...woah"
Hot Rash - 11 Lifeline - "We will become stronger."
Hellion - Agent Tex - "uhh...funny car?"
Equinox - ORDER - "Thy end is now!"
Bloxxer - Goodbye Baby - "Today is the day Bloxxerman lives."

domain: entertainment


During his Wednesday press conference, President-elect Trump attacked both BuzzFeed and CNN, refusing to answer a question from the latter.
While taking questions from the press, Trump didn’t mince words about BuzzFeed, which, on Tuesday, published a 35-page dossier of unverified intelligence memos about Russia and Trump in its entirety, uncensored. One part of that memo included a paragraph alleging that in 2013, Russian intelligence operatives surreptitiously recorded video of the President-elect paying Russian sex workers to urinate in front of him at the Ritz-Carlton hotel in Moscow.
CNN reported on the two-page synopsis of that memo — which John McCain recently admitted he gave to FBI director James Comey — which was shown to President-elect Trump during an intelligence briefing. In addition to slamming BuzzFeed as a “failing pile of garbage” that would “suffer the consequences,” Trump refused to answer a question from CNN’s Jim Acosta, calling CNN “fake news.” When Acosta insisted that Trump allow CNN to have a question answered since Trump attacked their reporting, Trump simply responded by saying, “Don’t be rude,” and “Quiet, quiet.”
— BBC News (World) (@BBCWorld) January 11, 2017
— ABC7 Eyewitness News (@ABC7) January 11, 2017
The press conference was largely rambling and disorganized, with the President-elect referring to himself in the third person twice in just 30 seconds:
— CNN Newsroom (@CNNnewsroom) January 11, 2017
Journalists and political commentators observed on Twitter that the President-elect’s treatment of critical journalists is not unlike how some of history’s most notorious dictators treated news outlets that provided oppositional coverage. Journalists also remarked that Trump blatantly disregarded the strict provisions in the Constitution that prevent presidents and elected officials from making decisions that would result in their own enrichment:
"You are fake news!" says the guy who spent 8 years birthering Obama, and said Ted Cruz's father helped kill JFK….. #TrumpPressConference
— Josh Mallon (@jmallon199) January 11, 2017
The guy who started the birther campaign against Obama just called CNN fake news. Let that sink in. #TrumpPressConference
— Caroline O. (@RVAwonk) January 11, 2017
This is the beginning. All real news is about to become fake. This is straight out of the Authoritarian playbook. #TrumpPressConference
— Laura Miers (@LauraMiers) January 11, 2017
Trump just threatened BuzzFeed. Actually this is how Nazi Germany started. #TrumpPressConference
— imfabulous (@imfabulous13) January 11, 2017
Trump just refused to answer questions from a CNN reporter. Because he doesn't like their coverage of him.#TrumpPressConference
— Caroline O. (@RVAwonk) January 11, 2017
Trump insists that the press are 'fake news' at his own press conference. This is very very bad indeed. #TrumpPressConference
— Laurie Penny (@PennyRed) January 11, 2017
— Amy Siskind (@Amy_Siskind) January 11, 2017
It isn’t hyperbolic to compare Trump’s treatment of the media to Hitler’s treatment of the press in the prelude to Germany’s Third Reich. As the Washington Post reported, the term “Lügenpresse,” which translates to “lying press,” was a slur popularized by Nazi supporters during Hitler’s reign, and encouraged by Joseph Goebbels, Hitler’s director of propaganda. Hitler’s treatment of the press resulted in physical attacks against journalists:
The verbal attacks against journalists soon turned into physical violence in Germany. At times, media members were unable to cover the Pegida-organized protest marches without private security personnel. Some reporters who risked going in without bodyguards were beaten up. It is without doubt that the word “Lügenpresse” has an extremely ugly meaning in modern-day Germany.
The term “Lügenpresse” remained in relative obscurity until Trump’s supporters began using it to describe reporters covering his rallies. The Atlantic’s Rosie Gray captured video of two Trump supporters haranguing members of the media as “Lügenpresse” outside the press pen during the campaign:
Friendly interaction outside the press pen. "Lugenpresse!" pic.twitter.com/MWUZynJ8jx
— Rosie Gray (@RosieGray) October 23, 2016
During the campaign, Trump banned Washington Post reporters from obtaining press passes to his events between June and September, citing their critical coverage of him. It remains unclear whether or not CNN or BuzzFeed will be allowed into White House press conferences during the Trump administration, particularly since Trump press secretary Sean Spicer said that the President-elect won’t follow traditional media rules during his administration.
Tom Cahill is a writer for US Uncut based in the Pacific Northwest. He specializes in coverage of political, economic, and environmental news. You can contact him via email at [email protected], or follow him on Facebook.


text:
Santa Claus has always been one of the bargain bin warriors of the cult movie world: in other words, a popular title amongst trash aficionados that has never gotten a proper release due to tricky rights situations and a number of grey-market editions. It recently got a new kick at the can via a blu-ray edition from VCI. While the results aren’t as technically pristine as fans might have hoped for, it’s a big improvement on past discs and adds a number of swell, subject-appropriate extras in the bargain.
This transfer was taken from a film source: the element mostly looks good but there is the occasional bit of damage, mainly a few scratches that run down the right side of the frame on a few occasions. A couple of brief sections seem faded in their colors in comparison to the rest of the film and there are also a handful of moments where the frame seems to briefly “flash” as if the color timing slipped for a second.
It’s also worth noting that the disc claims to have both the Mexican and K. Gordon Murray versions of the film. This is only partially true: this appears to be a Spanish language print that is edited to reflect the American version, excising one sequence in set in hell, but retaining the original Spanish language titles and signs used in the Mexican version. In fairness to VCI, they do include the excised Hell sequence and the English language titles and signs in a deleted scenes section. That material is taken from a video master that was drawn from a 16mm source.
As for the transfer itself, it’s a mixed bag: color representation is pretty strong, particularly the vivid primary reds on Santa’s and Pitch’s costumes, but the detail level wavers depending on the closeness of the shot. Close-ups fare the best while wide shots get kind of hazy in detail. The end result isn’t an ideal blu-ray transfer but it’s still a big step up from all the dupey, standard-def transfers of Santa Claus that have flooded the grey market for years.
Both English and Spanish mono soundtracks are provided: aside from the occasional pop, both sound fine. Subtitles are provided for the Spanish track but unfortunately they are printed in white lettering that blends into the background, particularly during the scenes in Santa’s castle. There is also a 5.1 remix for the English language track but it’s an artificial-sounding affair that is too boomy to make a comfortable listen.
The best element of the disc is a surprisingly generous assortment of extras. Viewers are treated to theatrical, t.v. and radio spots for Santa Claus, all built around K. Gordon Murray’s unique showmanship (the film was put out as a limited weekend-only engagement). In addition to the aforementioned deleted/alternate scenes, there are also a trio of “Santaland” shorts that Murray assembled by shooting footage at Santa-themed roadside attractions. Two of these shorts use footage from Santa Claus itself and all three use other “mascots” that Murray adopted from his other Mexican releases, albeit in cheaply-redesigned costumes. This little trilogy is a smirk-inducing trip back to when kiddie entertainment could be really cheap and weird. Elsewhere, there is an unexpectedly comprehensive animated stills gallery that features all sorts of ad materials (print ads, posters, color & black and white stills) as well as a Christmas-themed Howdy Doody short.
That said, the best bonus inclusions come from Daniel Griffith. He does a commentary track for the film that works quite well thanks to his extensive knowledge on K. Gordon Murray and the films he distributed. His comments cover the history of the personnel involved, how Murray repackaged and sold it to an American audience and, most interestingly, the fact that the Santa Claus legend wasn’t part of the usual Christmas celebrations in Mexico, which leads into discussion of how the filmmakers tried to make it palatable to their home audience via a number of eccentric additions to Kris Kringle lore.
The commentary is nicely complemented by “Santa Claus Conquers The Devil,” a new featurette that presents a concise, 14-minute introduction to the mind-melting wonders of Santa Claus. It incorporates input from cult film personalities like Larry Blamire and MTS3K‘s Kevin Murphy as it divides its time between explaining how this odd film came to be and the effect that it had on the young American minds that viewed it during its initial release. As usual with a Griffith featurette, it has nice graphics and a punchy editing style.
In short, this is a flawed disc but the uptick in visual quality and particularly the supplements make it worthwhile for anyone obsessed with this holiday oddity. If Santa Claus is your cup of yuletide madness, it definitely beats those grey market dupes.

domain: hobby


text:
Fri, Oct 30, 2009
Says Billings For Flights With A Friend Were Legitimate
The former Governor of North Carolina testified before the State
Board of Elections that invoices filed by a political ally for
flights on private aircraft had nothing to do with getting a break
on repairs for his home in Raleigh.
Mike Easley directly disputed the sworn comments made by McQueen
Campbell earlier this week. Campbell is a long-time Easley friend
whom Easley appointed to the N.C. State University board of
Television Station WRAL reports that the board is investigating
the possibility that Easley's campaign or the state Democratic
party violated campaign finance laws.
In his testimony, Campbell said Easley suggested he bill the
state for airplane flights he gave the then-Governor to pay for
repairs Campbell made on Easley's home. Campbell said two such
invoices were false, which would violate campaign laws. But Easley
denied having made such a suggestion to Campbell.
Easley, who left office in January, said he told Campbell in
2005 to pre-bill for flights he anticipated using, which is not
illegal under North Carolina law.
Easley did say he had asked Campbell to have repairs made on his
home in Raleigh, which he rented out while he occupied the
Governor's mansion. The home suffered water damage, but Easley said
he thought Campbell would bill the real estate company that was
managing the property.
The station reports that Campbell testified he had flown Easley
in his plane many times from 1999 to 2004, and that those flights
were never reported by the campaign committee. Campbell says those
flights were worth almost $88,000. Easley testified that Campbell
had been "paid in full" for those flights.
Easley has been under a cloud since leaving office in January
after 8 years as Governor. He is the first Governor to be called to
testify before the elections board.
Also: Legend Cub, Piper Orders, Postal UAVs?, IMC Club 'Brown Jacket Award', X-47B Refueling The Diamond DA62 has received its EASA Type certificate. After a sunny and warm day Wed>[...]
Also: United Bars Security Expert, Airborne Advisors, NTSB Video, Avidyne, Cessna, Airport Access Tempest Plus Marketing Group has announced support for Able Flight as a Platinum L>[...]
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Aerospace Web Aerospaceweb.org is a non-profit site operated by engineers and scientists in the aerospace field. The goal of this site is to provide educational information on a va>[...]

domain: news


text:
Illustrated Stories from Aesop (Illustrated Stories)
Only 1 in stock
This new collection of Aesop's fables is sumptuously illustrated by Giuliano Ferri and presented as a hardback gift edition to be read again and again. Each of these timeless stories conveys a message about how to behave towards others. Stories include The Fox and the Crow, The Hare and the Tortoise, The Boy who cried Wolf and The Goose that Laid the Golden Eggs.
Number Of Pages:
- ID: 9781409538875
- Saver Delivery: Yes
- 1st Class Delivery: Yes
- Courier Delivery: Yes
- Store Delivery: Yes
Prices are for internet purchases only. Prices and availability in WHSmith Stores may vary significantly
© Copyright 2013 - 2018 WHSmith and its suppliers.
WHSmith High Street Limited Greenbridge Road, Swindon, Wiltshire, United Kingdom, SN3 3LD, VAT GB238 5548 36


text:
Kotdwara (India): Weather for the next 7 days and accommodation
7-day weather forecast for Kotdwara
booked.net offers an extended weather forecast for the next 7 days in Kotdwara and in any other city around the globe.
Searching for an extended weather forecast in Kotdwara for today?
Maximum daytime temperature will be +32ºC and the minimum +23ºC.
Winds will persist through the day from the west-southwest at 2.78 km/h.
It's forecasted 61% of humidity, followed by 1008 hPa of atmospheric pressure and 0% of cloudiness.
Looking for an extended weather forecast in Kotdwara for tomorrow?
Maximum daytime temperature for October 01: +32ºC and minimum nighttime temperature: +23ºC.
It’s forecasted 61% of humidity, followed with northeast winds.
Cloud coverage will be 0% and the atmospheric pressure will be near 1007 hPa.
Frequently Asked Questions for USA travelers for Kotdwara, India:
1. What makes Kotdwara a must-visit destination?
Kotdwara is a small town in the Indian state of Uttarakhand that boasts of breathtaking natural beauty and fascinating religious significance. The town acts as a gateway to the famous Jim Corbett National Park and is also home to a number of ancient temples and vibrant cultural festivals.
2. When is the best time to visit Kotdwara?
The ideal time to visit Kotdwara is from October to June when the weather is pleasant and perfect for outdoor activities like trekking, hiking and exploring the natural beauty of the region.
3. What events should travelers attend in Kotdwara?
Travelers should attend the Kotdwar Mahotsav, an annual cultural and religious festival which is celebrated with great zeal and enthusiasm in the month of February.
4. Can you recommend some hotels in Kotdwara?
Some of the best hotels in Kotdwara are Hotel Krishna Palace, Hotel Shree Hari, and Hotel Samrat. These hotels offer a comfortable stay with modern amenities and excellent hospitality.
5. What is the high and low season for Kotdwara?
The high season for Kotdwara is from October to June while the low season is from July to September.
6. What is the temperature like throughout the year in Kotdwara?
The temperature in Kotdwara varies from 8°C to 40°C throughout the year.
7. What is the temperature by month in Kotdwara?
- January: 8°C to 22°C
- February: 10°C to 25°C
- March: 15°C to 30°C
- April: 20°C to 35°C
- May: 25°C to 40°C
- June: 28°C to 38°C
- July: 26°C to 34°C
- August: 25°C to 33°C
- September: 22°C to 32°C
- October: 18°C to 30°C
- November: 13°C to 27°C
- December: 9°C to 22°C
8. What is the number of rainy days in Kotdwara?
Kotdwara experiences an average of 140 rainy days per year.
9. What is the number of rainy days by month in Kotdwara?
- January: 4 rainy days
- February: 4 rainy days
- March: 4 rainy days
- April: 4 rainy days
- May: 17 rainy days
- June: 23 rainy days
- July: 27 rainy days
- August: 23 rainy days
- September: 14 rainy days
- October: 4 rainy days
- November: 1 rainy day
- December: 2 rainy days
10. What is the number of sunny days in Kotdwara?
Kotdwara experiences an average of 220 sunny days per year.
11. What is the number of sunny days by month in Kotdwara?
- January: 25 sunny days
- February: 28 sunny days
- March: 31 sunny days
- April: 30 sunny days
- May: 14 sunny days
- June: 7 sunny days
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#26 - Marc-André Giroux - The Road to a $70,000 E-Book Launch
Marc talks about the recent launch of his first e-book, Production Ready GraphQL, and how it netted him over $70k during the launch. He talks about the fears he had going into launch day, the challenges he faced during the writing process, and how he was able to overcome impostor syndrome to make the launch happen. Marc also goes into detail about the tools he used to sell his book, his writing process, how he built an audience over time.
Production Ready GraphQL Book
Marc on Twitter


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Drink and Draw at the Miller Taproom
Drink and Draw will be making the move back to our Miller Taproom! Every first Wednesday of the month join us as we get the creative juices flowing.
NWI, the Region, and the Chicagoland area is home to a huge creative community. We'd like to invite the creatives, and the community out for our Drink and Draw event.
18th Street's Drink and Draw is a fun way for creatives to get out of the house, show their work, meet other artists and fans, and become acquainted with possible buyers in an open and unbiased environment.
-How does it work for artists?
Do you like to draw, or paint, or decoupage? Come in and make some art while enjoying great beer (or craft cocktails, or soda or water). If you would like, hang what you have created on our display wall to show and sell during the event- there is no commission- you keep all money from the sale. It is a great way to show your work to a whole new audience, gain some new fans, and maybe make some money.
Have premade work? Bring it in a portfolio to show off. We want to keep the display wall open for art that is made at the Drink & Draw. Also make sure to bring business cards to hand out to anyone interested in your work.
If you want to just come in and enjoy a beer and sketch- then hell yeah, we'd love to have ya.
There is no sign up, no paperwork, no fees, no jury, just show up and do your thing.
-How does it work for the public?
Come in and see art being created right before your eyes. If you see something you like you can talk directly with the artist about purchasing it on the spot. Or, maybe you have been wanting a piece of art for your living room- now you have a room full of artists that you can talk to about what you would like.
Sounds great, right? It's because it is!


You are expected to contact Quanzhou YiXin Umbrella Co.,Ltd. to get instant info on Automatic Folding Umbrella production. We plan to develop a system to enable you to directly monitor the production. You will be informed about the significant progress.
Over the past years, YiXin Umbrella has been focusing on the development, design, production, and marketing of reverse folding umbrella in China. The reverse umbrella
is one of the main products of YiXin Umbrella. Good bendability is one of its highlighted selling points. The air-permeated design enables the moisture and foot sweat to remove from the shoe. The brand logo can be printed on this product. YiXin Umbrella's customer service always works on a professional level. It has a strong metal shaft and tips to prevent breaking or folding.
We are innovative: We embrace innovation, it’s at our core. We always put customers’ needs first – we go the extra mile.


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Younger Next Year Exercise Program combines the best information from Younger Next Year with the cutting-edge workouts from Thinner This Year. Here is the 10-minute warm-up critical for maintaining ankle, shoulder, and hip mobility. The five amazing things aerobic exercise will do for your body, and finding the method that...
A giftable little hardcover that is packed with full-colour photos of dogs in action and at rest paired with text revealing everything dogs can teach us about life.
A collection of recipes that are inspired by the cultures that shapped the American identity - and palate. From the familiar Northern Italian to the more unexpected North African, it includes: Mexican, French, German, Greek, Chinese, Japanese, Thai, Scandinavian and more.
By Liz Gutman
Includes recipes for: Chocolate Mint Meltaways, PB&J Cups, Chai Latte Lollipops, Cherry Cordials, Spicy Pralines, and the cult favourite, Beer and Pretzel Caramels, Plus candy bars - the Twist Bar, the Nutty Bar, the Coconut-Lime Bar, inspired by commercial favourites Snickers, Twix but taken to new heights of deliciousness.
Specifically created to cross-train the brain, this title is a mix of challenges, riddles, and brainteasers - all vetted by a neuroscientist who specializes in aging brains and designed to work in areas of cognitive function that are normal aging: long-term memory, executive functioning, and processing speed.
By Austin Kleon
When asked to talk to students at Broome Community College in upstate New York in the spring of 2011, the author wrote a simple list often things he wished he'd heard when he was their age. After giving the speech, he posted the text and slides to his popular blog,...
Blends middle school math with fantasy to create an adventure in problem-solving. This title includes word problems that are perilous, do-or-die scenarios of blood-sucking vampires, or a rowboat of 5 shipwrecked sailors with a single barrel of freshwater. It presents problems that are as fun to read as they are...
When a boy named Oscar discovers a giant - a very hungry giant holding a sign that says "Food Please" - in his backyard, he knows he can't turn his back on him. Yet it's not easy feeding a hungry giant. A whole pizza disappears in a single gulp. So...

domain: library


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Haleakala Crater lit just after sunrise.
This image was captured last month during the 3rd Annual Maui Photo Festival as part of their Haleakala Sunrise excursion. It is actually an HDR image created from 3 exposures and using Photomatix Pro. At first glance, it might not strike someone as the type of image people think of when they hear the term HDR. But here, I used multiple exposures to capture detail in the clouds as well as the crater below – which would have not been possible in a single exposure due to the range in highlights and shadows created by the intensity of the newly risen sun.
Here, I wanted to get as much detail as possible, so I shot at f/16. I could have framed the shot without the sun, but knowing that such a small aperture would give me a starburst effect (I was playing around with earlier in the trip) I waited to get enough separation between the clouds and the sun and placed it in the corner of the shot. Of course, it also gave me a bit of lens flare, but I didn’t mind.
Stick Around, It Ain’t Over
This was my 3rd Haleakala sunrise, and just like the previous two trips, right after sunrise, the summit and lookout points go from being shoulder-to-shoulder crowds right before the big moment, to a sparse group of folks right after. I don’t know what everyone’s hurry is, there’s still more to see (and shoot) than just the sunrise.
Better Next Time
I did want to fill in some of the foreground rocks, but my attempts with using a fill flash came up fairly lame. In hindsight, had I brought along my 32″ 5-in-1 reflector with me instead of leaving it in the hotel room that morning, it may have given me a better result than the flash. Something to try for next time.
One Last Note…
Hopefully this goes without saying, but never, ever stare at the sun through your camera’s viewfinder to get a shot like this, or any other sunrise or sunset image. You’re only asking for trouble.
For this and my other sunrise shots from the crater, I have a tripod setup and was using the Live View mode on my LCD to frame the image, switched back out of Live View (to activate autofocus again), and used the autofocus focal point selector to pick my focus point. Finally, I had a remote shutter release to fire off the brackted shots (remember, I was shooting for HDR).
Net result, I wasn’t staring at the sun through the viewfinder. Please don’t learn this the hard way.


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domain: medical


Armenian police have demanded that the protesters on the Baghramyan Avenue in the downtown Yerevan immediately leave the area and move towards the Freedom Square.
The police also threatened to use force, said Russian news agency Interfax June 28.
Armenian police chief, Vladimir Gasparyan gave the protesters 30 minutes to leave the Baghramyan Avenue.
The police have already seized microphones and loudspeakers from the protesters.
Police forces and special equipment have also been dispatched to the Baghramyan Avenue.


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The popular name Las Vegas comes from the Spanish term “meadows” or the grassland meadow. The city is situated in Nevada at the Western United States. It was discovered in the year 1829 by the league of Mexican merchants spearheaded by Antonio Arnijo when they arrived at the grassland meadow as they journeyed to Los Angeles.
In the year 1855, a small group of Mormon missionaries from the Salt Lake City was sent in the area to educate the native Indian settlers in cultivating the land for farming. They built a fort and created a way station in between Los Angles and Salt Lake City. This was the beginning of the non-Indian period.
As the years went by and the population had become fast increasing, Las Vegas was officially established in the year 1902 with the initial population of 800 people. As a fast growing city, it was estimated that in each month, four thousand to eight thousand are moving in. Tourism, gaming or gambling was born as the city grows popularly.
Las Vegas allures and attracts many tourists not only as the city entertainment facilities but also of enjoyment and satisfactions of all especially to those who love nightlife. If you are looking for a better place to spend most of your time, Vegas has its great accommodations for visitors especially in summer months. Yes it’s true that summer makes your day frustrating because of the blistering heat of the sun. Pools are the primary options like Hard Rock Hotel pool and many others.
The month of June, July and August it is the best summer month of Vegas. Enjoying the best book you have in the travel, sipping favorite cocktails, and sitting in the side of the pool sounds great and relaxing. If you are a person with great interest in entertainment shows, the whole year round is yours to visit Vegas. However, if you are a gambler the best month for you is opening weekends of March Madness—the Super Bowl.
If you want have a good night during that summer visit, Casinos like in Stratosphere Hotel Casino is the best choice. Or visit entertainment shows every night as much as you like in the whole night of your travel. Get lots of surprises in the fascinating and spectacular Bellagio Fountains located at the Las Vegas Boulevard in front of Bellagio Hotel Casino.
Furthermore, if you like to have a superb experience to these marvelous sightseeing creations in the city Las Vegas has it all for you.

domain: travel


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He was reported to be in the water near The New Navigation off Titford Road, in Oldbury, early on Wednesday morning.
Police, paramedics and firefighters were sent to the scene and pulled him from the canal but he could not be saved.
The death of the man is not being treated as suspicious.
A spokesman for West Midlands Police said on Wednesday: "We received a 999 call reporting a body in the canal off Titford Road, Oldbury, at around 6.40am.
"A man was rescued from the canal but he was sadly pronounced dead at the scene.
"We’re working hard to identify him so that we can pass on the sad news to his family.
"At this stage his death is not being treated as suspicious and the coroner has been informed.
"The charity Samaritans is available 24/7 should you need to talk."
A spokesman for West Midlands Ambulance Service added: "“We were called by the police at 6.46am on Wednesday to reports of a patient in the canal near the Navigation Inn, Titford Road, Oldbury.
"Two ambulances, a paramedic officer, a critical care paramedic and HART paramedics were sent to the scene.
"On arrival crews found a man in the canal.
"Crews worked quickly with fire colleagues to rescue him from the water before ambulance staff commencing advanced life support on scene.
"Sadly, despite their best efforts nothing could be done to save the man and he was confirmed dead a short time later."
If you have been affected by this article, you can call the Samaritans on 116123 or visit Samaritans.org


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Phosphates in Packaged Foods May Be Putting Your Health at Risk
Dr. Kristie Leong
There are lots of reasons not to eat a lot of processed or packaged foods and not to sip soft drinks. They're high in sugar and salt and they often contain unhealthy additives. One unhealthy additive you may not be aware of is dietary phosphates. Phosphates are added to packaged pastries, cookies, buns, energy drinks and soft drinks, and they're naturally found in some meat and dairy products, especially processed meats. Phosphates are used to thicken packaged foods and make them look and taste better. What's concerning is the effect they have on your health when you get too much.
Phosphates are the form in which phosphorus is found in the body. You need a certain amount of phosphorus for strong teeth and bones and for producing ATP, the form of energy the body uses to do work. Its also important for regulating the action of certain enzymes.
On the other hand, most people easily meet their requirement for dietary phosphate and phosphorus when they eat a healthy diet. When you get too many dietary phosphates from soft drinks and packaged foods it can increase your risk for health problems.
Why Is Too Much Dietary Phosphate a Problem?
When researchers fed groups of mice a diet containing differing amounts of phosphates, the mice that ate a high-phosphate diet had 40% more arterial inflammation and congestion than mice that ate a low-phosphate diet after 20 weeks. The mice on the high-phosphate diet also had more fat tissue and insulin resistance. These are all risk factors for heart disease.
A diet high in phosphates is also suspected to increase the risk of heart disease in humans, which is one reason why processed foods and soft drinks shouldn't be on the menu. Not only is a high phosphate diet bad for your heart, it may also trigger metabolic syndrome because of it increases insulin resistance. Not a good thing for your heart or your waistline.
How to Lower the Amount of Phosphates in Your Diet
One of the easiest ways to reduce phosphates in your diet is to eliminate most processed and packaged foods from your diet. If you read the packages of these foods you'll find phosphates in the form of disodium phosphate, calcium phosphate, tricalcium phosphate, monosodium phosphates, pyrophosphate polyphosphates or phosphoric acid. Soft drinks are also loaded with phosphates.
Some foods are naturally high in phosphorus such as yogurt, pork, milk, cheese and organ meats, beans, mushrooms, and some starchy vegetables. You can find complete lists of foods high in phosphorus and phosphates online. Some of these foods such as yogurt, milk, beans and mushrooms contain other nutrients that still make them a healthy choice in moderation. The best way to lower the amount of dietary phosphate you take in is to eliminate processed foods and soft drinks since they have little nutritional value anyway.
The Bottom Line?
Now there's another reason to stay away from soft drinks and packaged foods. They contain too much dietary phosphate and other additives and too little nutritional value. Give your heart a break and eat more whole foods.
Health Disclaimer. Copyright ©2011-2020. Dr. Kristie Leong is a family practice physician and medical writer. Published with permission.
"Dietary Phosphate Modulates Atherogenesis and Insulin Resistance in apolipoprotein E knockout mice - brief report." Arteriosclerosis, Thrombosis and Vascular Biology.
Avoid This Common Food Chemical" Men's Health.
"Low-Phosphorus Diet: Best for Kidney Disease?" Mayo Clinic.

domain: food


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Surprise, AZ (SportsNetwork.com) - The Texas Rangers announced Tuesday that staff ace Yu Darvish had successful ligament reconstruction surgery performed on his right elbow.
The procedure, performed by noted sports surgeon Dr. James Andrews in Gulf Breeze, Florida, will sideline the standout right-hander for the entire 2015 season.
Darvish left his 2015 spring training debut on March 5 after just one inning after experiencing discomfort in his elbow. An MRI taken by the Rangers' medical staff the following day revealed a sprained ulnar collateral ligament that necessitated Tommy John surgery.
The All-Star hurler missed the final seven weeks of last season due to mild inflammation in his right elbow. Darvish finished the year 10-7 with a 3.06 ERA over 22 starts and struck out 182 hitters in 144 1/3 innings.


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European plastics and rubber machinery accounts for 40 per cent or 13 billion euro of global output
Venice, 10 September 2015: The output of the nine members of EUROMAP - the European Association of Plastics and Rubber Machinery Manufacturers - amounted to 13.0 billion euro in 2014, a year-on-year increase of 1.9 per cent.
In the same period global exports from EUROMAP countries rose by 1.6 per cent to 9.7 billion euro.
EUROMAP represents almost 1,000 manufacturing companies with a combined workforce of more than 57.000 in Austria, France, Germany, Great Britain, Italy, Luxembourg, Spain, Switzerland and Turkey.
At a press briefing held at EUROMAP's General Assembly in Venice on 10 September, the focus was on the BRIC countries as customers for plastics and rubber machines from EUROMAP suppliers. EUROMAP President Luciano Anceschi says: "The performance of those markets did not meet our or our manufacturers' expectations. The shortfall mainly resulted from a slump in demand in Brazil and an even sharper drop in Russia. As for China, the plastics and rubber machinery industry's development was uneven and needs to be closely monitored. India, on the other hand, is giving cause for optimism after a couple of years of declining exports."
"Overall, global output of plastics and rubber machinery reached 32.5 billion euro in 2014 with EUROMAP accounting for 40 per cent of this total," explains EUROMAP Vice President Helmut Heinson. "Despite China's share rising sharply over the past few years, EUROMAP managed to hold its own. The same applies to exports with EUROMAP maintaining a market share of around 50 per cent over the past five years."
EUROMAP forecasts a 2 per cent increase for the current year with output rising to 13.3 billion euro.
Image source: jovannig--fotolia.com
EUROMAP Supported ExhibitionsPDF, 188.03 KB
EUROMAP Press Conference - Presentation Global ChangesPDF, 893.16 KB
EUROMAP Press Release - Economic SituationPDF, 28.91 KB

domain: finance


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Mika slipped her nightdress on over her head, and shivered. Not from the sensual feel of the grey-blue silk sliding over her bare skin, for it had been ten years since wearing silk on a regular basis had been a novelty, but simply from the chill in the elegantly minimalist bedroom with its bare polished floors and blank white walls. She supposed she could have worn a rather more practical (and warm) garment than the scrap of strap-shouldered material, but if she had ceased to wear clothes like this just because there was no one there who would notice it then she would have stopped a long time before. She wore silk because that was who she was.
It was past two o’clock of a cold night, and her usual bed-warmer was on the other side of the planet. But she refused to think too much about that, because if she did she would only start to worry. Tohma would surely be all right – she knew how strong and capable he was – but it was at times like this when her mind liked to remind her of how vulnerable he could be at times. At times related to a certain other person. And when that other person had bolted for a cold city thousands of miles away and vanished without trace –
/Don’t think about it./
She hadn’t heard from her husband in two days, since that brief phone call to tell her he had arrived safely at New York, and was about to start checking hotels. He had sounded as calm and reassuring as ever, but she couldn’t forget the fearful expression of his usually shuttered eyes when he had left. As he had reassured her that nothing was more important than Eiri to him at the moment – as if she hadn’t known that already.
But there was nothing she could do, and worrying wouldn’t help her or anyone else. If anyone could find Eiri it would be Tohma, and she had to trust him. She was a practical woman, and knew that if she didn’t get some sleep soon she would be worse than useless at work tomorrow. Nobody needed a company director who looked and thought like one of the living dead. So she brushed the few knots from her obedient hair, braided it carefully so it wouldn’t annoy her when she lay down, and got into bed.
By an effort of will she forced her tense muscles to relax – a hot bath would have been nice, but it was rather too late now – and half an hour later had almost managed to convince her mind to stop chasing itself in circles. She was just skirting the edges of sleep, when her ears caught the slight snick of the lock on the front door of the apartment, and the faintest of swishing sounds as it swung open and closed again.
For a long moment she lay there, waiting for the light in the living room to turn on, or – if it was her husband as she hoped – for him to come through into the bedroom. But minutes past with no other sound, except the gradually increasing drum of her heart against her ribs. Softly, she got up, wrapped a satin dressing gown around herself, and carefully took the small handgun from the drawer of the bedside table. Both she and Tohma hated the thing – Tohma particularly, as he had had more than enough of hand guns six years ago – but hating guns and not owning them when one was a the head of a major company and married to a millionaire music idol were two entirely separate things.
Still, she kept it at her side rather than in front of her as she crept over to the door of the room. If it was Tohma, she didn’t want to frighten him half to death by appearing fully armed.
The living room was in near darkness, broken by the strange glimmering non-light of the street-lamps outside filtering through pale curtains, but she could make out a figure sitting silent in the gloom.
Her voice was a little uncertain, though it was difficult to imagine who else could have been sitting quietly in their darkened living room at three in the morning. Even more difficult to imagine anyone else with that artful mess of pale gold hair which somehow managed to catch any light in the room and shimmer with faint silvery lines.
Mika flicked on the light switch, and winced a little as the harsh whiteness assaulted her eyes. Her husband sat in one of the deep leather chairs, his small carry-on bag by his feet, his hat held gently between his hands. Mika looked at him with a concerned frown, and set the handgun on a side-table.
“What are you doing sitting here in the dark?” she asked, perhaps slightly warily, though the yawn which accompanied her words added to the sense of careful nonchalance. “Why didn’t you call and tell me you were coming back?”
He looked up at her with his usual sweet smile. “Gomen, Mika. I didn’t want to disturb you…” Then his turquoise eyes flickered back to the dark material clasped between his hands, and Mika had the odd feeling that she had ceased to exist to him.
“Why are you back so soon?” she asked, still standing by the doorway. “Did you find Eiri?”
Another flick of eyes in her direction. “I…briefly. I’m surprised he hasn’t called.”
“Shindou-kun found him and persuaded him to come back.” His voice sounded no different from usual, but Mika’s grey-blue eyes widened, wondering how much it had cost him to say those plain words. “I think he was on his way to the airport when I saw him, so he probably beat me back.”
“Was…was he all right?”
“Better, I think. He seemed…better.”
The vagueness of his tone almost frightened her. He was normally so decisive in everything, not tolerating anything less than perfection from himself or anyone else. She took a half step towards him, then hesitated.
“I’m surprised he hasn’t called,” the fair-haired man murmured to himself. “But then, he’s probably getting reacquainted with –“
The end of the sentence was bitten off. And Mika would have asked what he meant, but she knew that if she did he would only turn to her with that bright, reassuring smile, and tell her kindly that it didn’t matter. And every time he did that it seemed to drive another icy needle into her.
“Would you like me to get some food ready for you?” she asked. “I know they never give you anything that approximates to genuine food on these long flights.” Bright, empty words, as bright and empty as his smiles.
“No thank you,” he replied with perfect politeness. He was always perfectly polite. “I’m really not hungry.”
“A drink?” She was rambling, and she hated herself for it. But she knew he was hurting, could even guess why, and knew that he would never let her help him in any way more than fetching food. She was his wife and his closest friend, but that had never meant too much. “At least let me find you some tea -”
That was the iron control slipping for a brief second, narrowed eyes snapping up to catch her own, the word that same whiplash of sound which faced his employees on a daily basis but he scarcely ever used with her. And like them she cringed back, even as she berated herself. Then the cold blue-green eyes clouded with a passing shadow of weariness, and he pushed his glasses up to pinch the bridge of his nose. He always wore glasses for long haul flights, she knew, because the air-conditioning always dried out his contact lenses and made his eyes painful. He disliked the glasses for their discomfort and appearance – such weakness was not part of Seguchi Tohma’s carefully projected image – but for Mika they were always a little welcome. They reminded her of a strange bespectacled boy she had met more than twenty-five years before.
“I…thank you. I’m just a little tired, Mika,” he continued after a moment in which his eyes flitted shut. “I’m suffering through two lots of jet lag. I…just want to sleep.”
A brief pause. “Come to bed then,” she said softly, holding out her hand in a gesture of invitation. After a second in which Tohma seemed to look at it blankly, he stood up, and walked past her into the bedroom. He didn’t take her hand.
Mika frowned again, and nervously fiddled with a few strands of brown hair which had escaped from her plait. /I wish I didn’t know what was wrong…/
The fair-haired man sloughed off his dark green trousers, before folding them neatly and placing them on a chair. The same meticulous pattern followed with the forest-green jacket and the paler shirt – though his normally agile fingers fumbled over the cuff buttons. At last the vivid purple undershirt joined the meticulous pile, and, unselfconsciously naked, he reached for night clothes from a drawer. Mika stood in the doorway, watching yet not watching – this evening ritual was too familiar to warrant anything more. Tohma had never seemed to care whether she looked or not.
Pyjamas were put on with the same neat, precise movements as always – aqua silk veiling pale gold skin by inches as he meticulously fastened tiny buttons – but Mika half fancied she could see less of the man’s characteristically feline grace this night than usual. She could have put it down to his tiredness, but she knew him better than that. And she knew better than to comment on it.
So she took off her dressing gown, draping it neatly over a chair as long years of living with him had taught her, and pushed back the crisp white sheets of the bed, sliding in without saying a word. Tohma sat down on the opposite edge with perfect control, no wearily throwing himself down between the welcoming covers, before swinging his legs up and lying down on his side, facing away from her.
Mika flicked off the bedside lamp, and lay in the half-darkness gazing at the back of the fair head a few inches from her own. The electric orange light snaked through a gap in the imperfectly closed curtains, dyeing the night a faintly lurid hue and illuminating the hunched curl of her husband’s spine above the line of sheet.
Soundlessly, Mika shifted a little closer to him – not intimately close, nor intrusively – just enough that she could feel the warmth from his skin and knew he could feel hers, and that her bare arms brushed watery silk. She knew he was not asleep, no matter how tired he might claim to be. The tensely unhappy feeling in the air told her that much, and Tohma was never one blessed with easy sleep at the best of times. She had woken more times than she could remember in the small hours of the morning to find the bed beside her empty, and heard the softest strains of the piano from within the imperfectly soundproofed music room.
She half wondered why he had not turned to the instrument the moment he got back. When he got this bad, it seemed to be the only thing that could calm him, soothing his turbulent mind as she had never quite been able. She had never before known him to have a problem that music could not help.
With infinite softness, she touched her fingers to the white-gold strands of his hair. The minutes of silence stretched out through the cool air, and he didn’t react to her touch.
“He went back to Shindou-kun, didn’t he.”
No need to specify who ‘he’ was. The presence of that ‘he’ hung heavy over her husband’s slim form as he lay there, just as he always hovered between them. She could not blame her brother – she loved him too much for that – but sometimes she half wondered if Eiri knew just how much power he had over her husband.
There was a long moment of quiet, as she continued to gently card her fingers through golden hair. Below their window the occasional car was a distant rush of sound, a brief gleam of pale light travelling across the wall and away.
“He thinks he doesn’t need me any more.”
The answer was breathed softly, in much the same tones as he used every day. No melodrama or tears over the man he had devoted his life to for perhaps more than twenty years. Just that same calm softness as always.
“He’s found that boy, and he thinks he doesn’t need me. But he has to. He…he’s always needed me.”
Mika could almost have imagined she heard the faintest of doubts in those last words – impossible, that the ever controlled, ever confident Seguchi Tohma should have doubts, but the slight tremor told otherwise. So Mika didn’t voice the thought that maybe Eiri had needed him because he had only been a child, in need of help and protection, and then a boy on the verge of adulthood in need of support and healing. And that now Eiri was grown, and had found a life away from such dependence.
Instead, she continued that gentle stroking of silken strands, allowing her forehead to rest slightly against the warm shoulder.
“He’ll always need a friend he can trust like you,” she said quietly. “You know him better than anyone else. He’ll understand that some day.”
Another lengthy pause, during which she felt the tense muscles of the man’s back begin to relax a fraction, and heard his breathing deepen a little.
“He has to need me…” came the whisper from the darkness, soft with the edge of sleep. “He has to…”
Mika stayed there, gently touching golden hair, until she felt him slip away. Only then did she wrap a protective arm around his waist, and closed her eyes.