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Frank, Ines PhD; Robbiani, Melissa PhD
From the Center for Biomedical Research, Population Council, New York, NY.
Received for publication June 23, 2010; accepted September 27, 2010.
Supported by the National Institutes of Health AI052048, R37 AI040877, and the International Partnership for Microbicides (to M.R.). M.R. is a 2002 Elizabeth Glaser Scientist. This work was also funded in part with federal funds from the National Cancer Institute, NIH, under Contract No. NO1-CO-12400 (JDL, JWB). Provision of the inhibitors by Dr Moore was achieved with the support of the National Institutes of Health grant U19 AI076982.
Correspondence to: Robbiani Melissa, PhD, Senior Scientist and Director of Biomedical HIV Research, Center for Biomedical Research, HIV and AIDS Program, Population Council, 1230 York Avenue, New York, NY 10065 (e-mail: email@example.com).
Supplemental digital content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions this article on the journal's Web site (www.jaids.com).
Dendritic cells (DCs) efficiently transfer captured (trans) or de novo-produced (cis) virus to CD4 T cells. Using monocyte-derived DCs, we evaluated entry inhibitors targeting HIV envelope (BMS-C, T-1249) or CCR5 (CMPD167) for their potency to prevent DC infection, DC-driven infection in T cells in trans and cis, and direct infection of DC-T-cell mixtures. Immature DC-T-cell cultures with distinct mechanisms of viral transfer yielded similar levels of infection and produced more proviral DNA compared with matched mature DC-T-cell cultures or infected immature DCs. Although all compounds completely blocked HIV replication, 16 times more of each inhibitor (250 vs 15.6 nM) was required to prevent low-level infection of DCs compared with the productive DC-T-cell cocultures. Across all cell systems tested, BMS-C blocked infection most potently. BMS-C was significantly more effective than CMPD167 at preventing DC infection. In fact, low doses of CMPD167 significantly enhanced DC infection. Elevated levels of CCL4 were observed when immature DCs were cultured with CMPD167. Viral entry inhibitors did not interfere with Candida albicans-specific DC cytokine/chemokine responses. These findings indicate that an envelope-binding small molecule is a promising tool for topical microbicide design to prevent the infection of early targets needed to establish and disseminate HIV infection.
HIV mucosal transmission represents the principal route of virus acquisition, particularly of R5-tropic virus.1,2 In the initial phase of infection, HIV crosses the mucosal epithelial barrier likely within hours3 to access target cells in the submucosa including CD4 T cells, dendritic cells (DCs), and macrophages.4 Small local founder populations of infected cells are generated5,6 that then undergo local expansion to produce enough virus and infected cells that can migrate to proximal lymphoid tissue to establish a systemic infection.5,7-9 More recently, sequencing viruses in heterosexual transmission pairs and in acute HIV infection provided evidence that a single virus (or infected cell) initiated productive infection in close to 80% of the individuals tested, with the remaining individuals infected by between 2 and 5 viruses.10,11 This underscores the challenges faced in preventing HIV infection and spread. Productive infection of a cell is initiated through attachment of HIV envelope to CD4. This interaction triggers conformational changes in the viral envelope that enable HIV to bind to its coreceptor CCR5 or CXCR4.12-14 Further conformational changes in the envelope lead to the formation of a fusion pore mediating infection of the target cell.15,16
The availability of CD4 and CCR5 molecules on immature DCs render them vulnerable to infection via fusion. Although immature DCs produce much less virus compared with CD4 T cells,17-19 limited amounts of de novo-produced virions are efficiently transmitted (in cis) to resting CD4 T cells,19 especially effector memory CD4 T cells abundant in mucosal tissues.20 Additionally, immature and mature DCs express C-type lectin receptors and may express undefined molecules that facilitate binding and internalization.21-25 Such captured virus can be rapidly (within hours) and efficiently transferred (in trans) to CD4 T cells upon cell-cell contact, referred to as infectious or virological synapses,26-29 a process that is independent of productive DC infection.25,27 Typically the efficiency of HIV transmission can be increased by maturation of DCs,18,27,30,31 indicating that mature DCs in the local and draining lymphoid tissues might further facilitate HIV spread to CD4 T cells. Not surprisingly, numerous studies have highlighted an important role for DC-mediated HIV transmission during mucosal HIV infection, which likely involves the multiple modalities of DC-driven infection.3,6,17,32-38 Thus, topical microbicides that contain agents targeting specific attachment and fusion events need to be extremely efficient in preventing these critical virus-cell interactions.
During each stage of the sequential entry process, HIV is vulnerable to blockade by compounds that bind to viral envelope or the cellular receptors CD4 and CCR5 or CXCR4. Recent in vitro studies showed that DC-mediated infection in trans or cis are sensitive to fusion inhibitors.7,28,39 The virological synapse is also sensitive to a CCR5 antagonist (TAK779) and several viral envelope-binding neutralizing antibodies.28,40 Other small molecule CCR5 ligands being tested in the macaque model show promise.41-44 An envelope-binding small molecule inhibitor (BMS-C) showed in vitro activity against HIV isolates from multiple genetic subtypes.45 Because of the central role of DCs in initiating HIV infection, we were interested to more extensively compare the efficiency of 3 inhibitors that antagonize distinct steps of virus entry, to prevent DC infection and DC-driven viral spread. T-1249, a gp41 peptide fusion inhibitor, is a 39-amino acid synthetic peptide and blocks viral fusion with the cell membrane by inhibiting late-stage conformational changes within gp41.46 BMS-C is a small molecule attachment inhibitor that binds to gp120 to inhibit CD4 binding and subsequent conformational changes associated with coreceptor binding,47 whereas CMPD167 is a CCR5-specific receptor antagonist.48,49 Applying these 3 viral entry inhibitors, we specifically compared distinct viral transfer mechanisms (trans vs cis) and a DC-T-cell coculture setting that involves cell-free virus, allowing both infection mechanisms to occur simultaneously along with direct infection of CD4 T cells. We demonstrate the greater potency of BMS-C in preventing DC and DC-driven infection without perturbing innate DC function.
The HIV entry inhibitors T-1249,46 BMS-C,47 and CMPD16748,49 were kindly provided by John P. Moore. For all inhibitors, working stocks at a maximum stock concentration of 0.1 mM (CMPD167, in DMSO), and 503.6 μM (T-1249, in PBS) were prepared and kept at −80°C until use. After thawing, aliquots were kept at 4°C for a maximum of 4 weeks.
Human monocyte-derived DCs were generated from highly enriched populations of CD14 monocytes and autologous CD4 T cells isolated from CD14-negative cell fractions, as previously described, with the minor modification that PBS supplemented with 1% human serum (Sigma, St. Louis, MO) and 2 mM EDTA (Sigma) was used as the buffer.28 As needed, cells were activated on day 5 for 48 hours with the cocktail of prostaglandin E2 (PGE2), IL-1, IL-6, and tumor necrosis factor α (TNF-α) as described.50 For immature DC infection, cells were used on day 5. When comparing immature and mature DCs in the cocultures, the cells were cultured in parallel and day 7 DCs used. Comparable levels of HIV infection are seen for day 5 or day 7 immature DCs. CD4 lymphocytes were sorted from CD14-negative cells by negative selection using the CD4 lymphocyte isolation kit (Miltenyi Biotech, Auburn, CA). Cells were cultured in complete RPMI-1640 (R1) as described.28 R10 contained 10% fetal bovine serum (GIBCO-BRL Life Technologies, Grand Island, NY).
Phenotypic characterization of immature and mature DCs including purity controls of generated DCs and CD4 T-cell populations (purity ≥97% for DCs and T cells) were routinely monitored by 2-color flow cytometry as previously reported.51 DCs were stained with a fluorescein isothiocyanate (FITC)-conjugated anti-HLA-DR against a panel of phycoerythrin (PE)-conjugated monoclonal antibodies (mAbs) characterizing an immature or mature DC phenotype: anti-CD25, anti-CD80, anti-CD86 (all from BD/Pharmingen, San Jose, CA), and anti-CD83 (Immunotech-Beckman-Coulter, Marseille, France). A PE-conjugated anti-CD3 mAb (BD/Pharmingen) was used to detect contaminating T cells in DC preparations. The purity of CD4 T-cell populations was evaluated by stained cells with a FITC-conjugated anti-CD3 mAb against PE-conjugated mAbs anti-CD4 and anti-CD8 (all from BD/Pharmingen). Isotype controls IgG2a-FITC, IgG1-PE, and IgG2b-PE (all from BD/Pharmingen) were included in each experiment. Cell samples were acquired using a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA) and data analysis was carried out using FlowJo software (Tree Star, Ashland, OR).
Sucrose gradient purified infectious HIV BaL was kindly provided by the AIDS and Cancer Virus Program (SAIC Frederick, National Cancer Institute at Frederick, Frederick, MD). HIV BaL (lot # P3953, P4022, P4126, P4143, P4237) was propagated (in SUPT1-CCR5 Cl.30 or Jurkat-TAT-CCR5 cells) and purified as described.52 Aliquots of virus stocks were thawed, and single use aliquots were stored at −80°C before the titer of virus was verified by titration on TZM-bl cells.53 Virus stocks were treated with 10 mg/mL DNase (Roche Applied Science, Indianapolis, IN) for 30 minutes at 37°C before use. For the trans phase of viral transfer, immature and mature DCs were pulsed with HIV (8 × 103 50% tissue culture infective dose (TCID50) per 1 × 105 DCs) for 2 hours at 37°C in a 15 ml conical tube (pretreated with R10 for 2 minutes on ice) at a concentration of 106 DCs/100 μL (with a maximum of 1 × 107 cells/tube). During the last 30 minutes of incubation staphylococcal enterotoxin B (SEB) peptide (Sigma; S4881) at a final concentration of 0.5 mg/mL was added before cells were washed 4 times with ice-cold R1, the viable cells recounted by trypan blue exclusion and cell numbers adjusted to 2.5 × 106 cells/mL. For the cis phase of viral transfer virus-pulsed immature DCs were recultured at a concentration of 1 × 106 cells/mL in a total volume of 3 mL per well in a 6-well plate (in R1 with IL-4/GM-CSF) for additional 48 hours before virus-exposed DCs were collected, incubated with SEB peptide, washed, and cell numbers adjusted as described for the trans phase. For viral replication in DC-T-cell mixtures (mix), immature and mature DCs were pretreated with SEB peptide, washed, and cell numbers adjusted (as above). T cells (3 × 105 per well) were seeded in a 96-well flat bottom plate and the inhibitors (T-1249, BMS-C, or CMPD167; 0.06 to 250 nM) added just before addition of the virus/SEB-pulsed or SEB-pulsed DCs (1 × 105 cells/well). Virus (8 × 103 TCID50) was added directly to the DC-T-cell cocultures that contained SEB-treated DCs. For immature DC infections, cells (3 × 105 per well) were seeded in a 96-well flat bottom plate and inhibitors (T-1249, BMS-C, or CMPD167; 0.06 to 250 nM) added before addition of 2.4 × 104 TCID50 virus per well. Samples were set up in duplicate. After 7 days of culture cells were harvested, washed, and lysed. Samples were stored at −80°C until quantitative PCR (qPCR) analyses.
Candida albicans (strain SC5314, obtained from the American Type Culture Collection) was cultured and maintained as previously described.54 After overnight amplification in Sabouraud dextrose broth (Sigma) at 30°C, Candida was washed 4 times in PBS before viable Candida yeasts were counted by trypan blue exclusion and resuspended in R1. Immature DCs (3 × 105/well of a 96-well flat bottom plate) were cultured in the presence and absence of 3 × 105Candida albicans yeast. Amphotericin B (5 μg/mL, Sigma) was added to all conditions to limit Candida overgrowth. Viral entry inhibitors were added at a final concentration of 250 nM/well. Cells were cultured at 37°C, and supernatants harvested 24 hrs or 7 days later. Harvested supernatants were spun and transferred to fresh plates to remove any cellular debris and immediately frozen at −80°C until further analysis. Cytokines and chemokines were detected using a Beadlyte 24-Plex Detection System as previously described.54
qPCR was performed as previously described28 with the minor modification that HIV copies were normalized on cell numbers by using qPCR for albumin gene copy number. Albumin (Alb) forward (F) and reverse (R) primer/probe sequences were AlbF: TGC ATG AGA AAA CGC CAG TAA, AlbR: ATG GTC GCC TGT TCA CCA A, and AlbP: 5′ FAM-TGA CAG AGT CAC CAA ATG CTG CAC AGA A-TAMRA 3′. Standards for quantification of viral and albumin copy numbers were set up by adding titrated quantities of the plasmid HIV AD8 NL43 DNA into a constant genomic background of SUPT1/CCR5 CL.30 cells. For albumin copies, known numbers of lysed uninfected SUPT1/CCR5 CL.30 cells were serially diluted in lysis buffer.
Data were analyzed using GraphPad Prism software version 5.03 (GraphPad InStat Inc, San Diego, CA). Results of experiments have been summarized as the arithmetic mean and standard error of the mean (SEM). When 2 groups were compared, the null hypothesis of no group difference was evaluated with the nonparametric Mann-Whitney, the Wilcoxon Signed-Ranked, or the Student t test. The 50% or 90% inhibitory concentration (IC50 and IC90) values of different compounds were modeled with nonlinear regression employing a constant slope algorithm. The conventional measure of P < 0.05 was used to determine whether experimental differences were statistically significant.
To evaluate the efficacy of viral entry inhibitors to prevent HIV infection of immature DCs and DC-driven infection of CD4 T cells, it was necessary to establish a system with high reproducibility and sensitivity to detect low levels of viral replication. We used sensitive qPCR to monitor viral HIV late gag proviral DNA as a quantitative measure of productive infection.28,55 Although qPCR was sensitive down to detecting one copy of proviral DNA, immature DCs and cocultures of immature DCs with resting CD4 T cells required a minimum of about 4000 (for DCs) or 2000 (for cocultures) TCID50 per 1 × 105 DCs for reliable detection of low copy numbers of viral DNA (data not shown). Therefore, we increased the viral inoculum to 8000 TCID50 per 1 × 105 DCs for more consistent viral amplification across different donors. To increase levels of viral replication in cocultures, DCs were pre-exposed to an SEB peptide to uniformly activate the T cells. SEB-activation of DC-T-cell cocultures has been used previously.56-58 SEB did not impact the DC phenotype in the DC or DC-T-cell cocultures (data not shown).
As previously reported,18,19,59 immature DCs alone exhibited low-level infection with R5 HIV BaL compared with the significantly greater replication seen in the presence of CD4 T cells (Fig. 1). The latter was true for both modes of DC-mediated transmission of virus to the T cells, and when virus was added directly to DC-T-cell mixtures. All immature DC-T-cell cocultures replicated virus to similar levels, independent of mode of infection (trans, cis, or cell-free). Notably, under coculture conditions, the immature DCs promoted more virus replication than their mature counterparts in trans infection systems and in the cell-free virus infected DC-T-cell mixtures (P < 0.02).
The ability of CMPD167, BMS-C, and T-1249 to block DC infection, and the different modes of DC-driven infection in DC-T cell co-cultures was measured. No toxicity was detected at any of the inhibitor doses tested (data not shown). Although each inhibitor effectively blocked HIV infection across all settings, inhibitor-dependent and cell system-dependent differences were observed. A complete block of infection in immature DCs required 250 nM of each inhibitor, which was 16-fold greater than for the various DC-T-cell cocultures (Fig. 2). Although all 3 compounds show comparable efficiency in the higher dose range (250 nM to 15.6 nM), the dose-response curve of the CCR5 inhibitor CMPD167 markedly deviated from the others at doses below 15.6 nM. In fact, DC infection was significantly enhanced in the presence of low dose of CMPD167 (0.06 nM; P < 0.005) (Fig. 2A). A similar effect was observed for concentrations lower than 0.06 nM (data not shown). Furthermore, area under the curve analyses highlight significant differences between the activity of CMPD167 and T-1249 (P < 0.04) or BMS-C (P < 0.02) (Fig. 2B). On the other hand, the inhibitors exhibited similar levels of potency against HIV infection of the various DC-T-cell environments (Fig. 2A), despite immature DCs driving more virus replication than mature DCs (Fig. 1).
Although all inhibitors completely blocked HIV infection in different cell culture settings, the inhibitory doses varied within and across cultures. The most dramatic differences between inhibitors were observed in blocking the low level infection in immature DCs, whereas in contrast, dose-response curves of all 3 inhibitors were similar for the DC-mediated transfer in cis (Fig. 2A). To examine this more closely, we calculated the IC50 and IC90 values of the inhibitors in each biological system (Fig. 3). Significantly more T-1249 (P values from 0.008 to 0.02 for 50% and P values from 0.008 to 0.04 for 90% inhibition) and CMPD167 (P values from 0.0002 to 0.07 for 50% and P values from 0.001 to 0.03 for 90% inhibition) was needed to block immature DC infection compared with all other DC-T-cell systems. In contrast, comparable amounts of BMS-C were required to block infection of immature DCs and the immature DC-T-cell cocultures. However, ∼10-fold less BMS-C was necessary to prevent trans-mediated infection of mature DC-T cocultures and mature DC-T-cell mixtures (P < 0.03; IC50 = 0.19 nM and 0.14 nM, respectively) compared with that required to inhibit immature DC infection (IC50 = 1.58 nM). Thus, although similar amounts of the inhibitors were needed to block the different modes of infection in the various DC-T-cell systems, BMS-C was more efficient in preventing immature DC infection.
As sentinels of the immune system, DCs produce cytokines and chemokines that mediate direct effector and immunoregulatory functions. Although entry antagonists have significant potential to prevent HIV entry into permissive target cells, inhibitors must not trigger the release of inflammatory factors that might exacerbate HIV infection or impede responses to other pathogens. Therefore, we investigated whether these viral entry inhibitors affected DC biology. Exposure of immature DCs to CMPD167, BMS-C, or T-1249 for up to 7 days did not alter their typical pattern of cell surface receptor expression (data not shown) including CD4, which is the main viral entry receptor that mediates HIV infection. However, when supernatants of such cultures were tested for the presence of different cytokines and chemokines, CCL4 levels were significantly increased after 7 days of culture with CMPD167 (P < 0.02) (Fig. 4A, left panel). The increased level of CCL4 after 24 hours of culture with CMPD167 was not significant. Minor increases in the levels of CCL5 after culture with CMPD167 were not significantly different from the control (Fig. 4A, right panel). No other cytokines or chemokines were elevated after culture with any of the inhibitors at these time points (data not shown for IL-1β, IL-1RA, IL-2, IL-2R, IL-5, IL-6, IL-7, IL-10, IL-12, IL-13, IL-15, IL-17, TNF-α, IFN-α, IFN-g, CCL2, CCL3, CXCL8, CXCL9, CXCL10).
To determine if the inhibitors might impair innate DC responses to common pathogens, we measured the impact of each inhibitor on the DC cytokine and chemokine production induced by Candida albicans. Immature DCs produce IL-1β, IL-1RA, IL-2R, IL-6, IL-10, IL-12, TNF-α, CCL2, CCL3, CCL4, CXCL8, and CXCL10 in response to Candida yeast (see Figure, Supplemental Digital Content 1, http://links.lww.com/QAI/A107).54 After 7 days, there was an increase in the production of CCL4 in the presence of CMPD167 (Fig. 4B, left panel), but this was not significant and a marginal increase of CCL5 in the presence of all three compounds (Fig. 4B, right panel). There was no impact on any of the other cytokine and chemokine responses induced by Candida yeast (data not shown). Thus, apart from the higher levels of CCL4 detected in the presence of CMPD167, the inhibitors had minimal impact on innate DC cytokine/chemokine responses.
Dendritic cells are believed to play a central role in the onset of HIV infection at the body surfaces through a variety of mechanisms.2,17,37 Immature and mature DCs can capture and internalize HIV that is then rapidly disseminated to CD4 T cells on DC-T-cell contact.27-29,60 Upon productive infection, immature DCs produce much smaller amounts of virus compared with CD4 T cells, but they are extremely efficient in transmitting progeny virions to CD4 T cells.32,61 Additionally, DCs readily trigger virus growth upon contacting virus-carrying CD4 T cells.62,63 It is proposed that the role of immature DCs is more restricted to local HIV replication at the mucosal surface, while mature DCs might drive trans infection in the lymph nodes.64 Activation of DCs triggered by local pathogens may also facilitate trans infection at body surfaces.
We examined the ability of 3 viral entry inhibitors that are being explored as potential microbicide candidates to prevent cell-free infection of immature DCs and DC-T-cell mixtures, and DC-mediated infection of CD4 T cells, using the blood monocyte-derived DC model. This system provides sufficient cell numbers to carry out side-by-side analyses of the 3 inhibitors. Unlike in an earlier study,61 the levels of virus replication after trans vs cis transfer by DCs were comparable. This might be due to methodology differences, especially the inclusion of the SEB peptide herein to activate the recipient T cells to allow more robust replication of virus in both settings. Similarly, the use of SEB peptide-pulsed immature DCs here likely overcame the greater efficiency in DC-T-cell communication and conjugate formation by mature DCs, facilitating relatively greater replication in the presence of immature DCs than previously observed.28,65 Augmented DC-T-cell communication in the presence of SEB peptide may also provide signals to increase replication in the immature DCs66,67 that would further amplify virus directly and provide additional new virus to spread to T cells.
The compounds used in this study represent the three main classes of HIV entry inhibitors. All three inhibitors prevented R5 HIV replication in the different DC systems, impeding the distinct stages of the sequential cascade of viral fusion. This is in agreement with previous findings by us and others, where fusion peptides28,68-70, CCR5 antagonists,69,70 or other potential entry inhibitors61 readily blocked DC-mediated infection in trans or cis. Recent reports suggest that even neutralizing Abs are more efficient at blocking HIV transfer than originally thought.67,71-74 Similarly, cell-free or cell-to-cell spread between T cells were recently shown to be equally sensitive to viral entry inhibition.40 Inhibition of DC-driven trans and cis infection further suggests that the recently described formation of nanotubes75,76 or cytoplasmic extensions by immature DCs that envelop CD4 T cells77 and are enriched with immature virus particles are freely accessible to viral entry inhibitors. Therefore, even in the presence of virological synapse formation, each step of the viral fusion cascade is vulnerable to the inherent antiviral potency of the different viral entry inhibitors. Immature and mature DCs (1) sequester entrapped virions in unique compartments, (2) exhibit differences in DC-T-cell conjugate formation, and (3) release entrapped particles and/or produce new viruses at CD4 T-cell-DC junctions.28 These features may contribute to differences in the sensitivity of immature or mature DC-driven, and the trans or cis DC-driven virus spread to inhibition.
Another main finding in this study is the enhancement of DC infection at low doses of CMPD167. This possibly relates to increased levels of CCL4 induced by CMPD167 and likely parallels what CCL5 may do in macrophages.69,70,78 An increase in CCL4 levels in DC cultures could also explain previous observations where only partial inhibition of immature DC infection was achieved using CCL5, a natural ligand for CCR5, to block infection. Additionally, CCL4 could encourage the recruitment of additional target cells to augment HIV amplification79 in vivo.
The remarkable low efficiency of CCR5 and fusion inhibitors to block DC infection compared with the various DC-T-cell cocultures is in agreement with previous findings, where immature DC infection was more resistant to complete inhibition by 20-fold or 36-fold for a CCR5 (SCH-C) vs 6 -fold or 10-fold for a fusion peptide inhibitor than PBMCs or trans-mediated infection.69,70 This effect possibly relates to the common sensitivity of CCR5 and fusion inhibitors to the level of cell surface expression of CCR5. Although the cooperation of multiple CCR5 coreceptors is required for infection by HIV,79 high or low levels of CCR5 affect the efficiency of a CCR5 antagonist and can impact the speed of fusion pore formation and, therefore, the efficiency of a fusion peptide inhibitor.80 Unlike in DC infection, HIV receptors CD4 and CCR5 (or CXCR4) on the T cell are recruited to the DC-T-cell contact sites thereby enhancing productive infection events and possibly the inhibition of such events by CCR5 or fusion inhibitors. On the other hand, direct binding to the virus, thereby preventing virus attachment to its target cell, likely explains the high potency of BMS-C to prevent cell-free and DC-driven infection to comparable levels. Although herein inhibitors were only tested against one R5-virus and variations in the potency of the 3 chosen inhibitors across several R5-viruses cannot be excluded, a recent study showed no substantive virus-specific pattern to the actions of the CMPD167, BMS-C, or a fusion peptide against a panel of R5 viruses.45 This is consistent with earlier reports on other entry inhibitors and with the subtype-independent nature of the HIV fusion process.81,82
Several approaches utilizing inhibitors of virus-cell interactions are being explored for topical microbicides,28,40,44,83 particularly CCR5 inhibitors.42,84,85 Although CMPD167 has been shown to inhibit vaginal transmission of an R5 SHIV to macaques,42,85 there might be concern that residual low levels of drug might ultimately enhance infection if animals were exposed to virus at later time points when drug would become limiting. Alternatively, small molecule inhibitors like BMS-C that directly target viral envelope may have considerable advantages compared with CCR5 or fusion peptide inhibitors due to their superior ability to efficiently prevent DC-driven HIV infection and spread coincident with their limited impact on innate immune function. Despite a preferential transmission of R5 HIV during mucosal infection, future studies are needed to evaluate the potency of entry inhibitors against X4-tropic and dual-tropic HIV to advance our understanding for the development of a potent topical prevention strategy. Overall, combination strategies using inhibitors targeting multiple stages of the infection process will likely be most effective against HIV.
The authors would like to thank Julian W. Bess Jr and Jeffrey D. Lifson for providing the HIV used in these studies and for critical review of the article. The following reagents were obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH: pNL(AD8), HIV AD8 Macrophage-Tropic R586 from Dr Eric O. Freed. We thank Daniel Gawarecki for statistics advice. The assistance and use of the Population Council's Flow Cytometry Facility is gratefully acknowledged.
CCR5 antagonist; dendritic cells; fusion inhibitor; HIV transmission; HIV envelope-binding small molecule inhibitor
© 2011 Lippincott Williams & Wilkins, Inc.
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I definitely know how hard it is when people you are around every day don't support your lifestyle and dietary choices. Luckily for me my boyfriend is on board, so we support each other, but almost everyone else that knows how I eat thinks it's incredibly strange and some sort of "fad" diet that I will eventually ditch for a standard American diet (even though it's been almost 2 years since I took the plunge into primal).
With that said, there's nothing wrong with getting support from strangers if everyone shares your values and viewpoints. I didn't join this forum until just a few months ago, but I wish I had much, much sooner. It's really nice to be able to ask questions that you know will be understood, and get relevant, meaningful answers and support, rather than "oh that silly diet you're on" kind of stuff.
If you can start leading by example by eating and living according to the primal blueprint, your family may become more interested and accepting of the lifestyle once they see how good you feel all the time. It's hard to change people's opinions, but sometimes seeing is believing. Good luck to you!

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To the Editor.—
While we agree with Levinson and Simpson1 that reported cases of neuroleptic malignant syndrome (NMS) are heterogeneous and that extrapyramidal symptoms (EPS) and medical disorders ought to be treated, several points require clarification.The authors reevaluated data on 67 patients drawn from over 300 case reports of NMS published since 1960. They suggested that NMS, as conceptualized by most investigators, is neither "unitary" nor malignant. However, this conclusion may be unwarranted in view of inconsistencies and ambiguities in their analysis. For example, concurrent treatment with lithium carbonate was used initially to eliminate two cases from analysis, while six of 14 "not ill" patients were also receiving lithium carbonate. Similarly, dehydration, which is unlikely to be neglected as a cause of fever by experienced clinicians, was used both to exclude cases and, as a relevant medical factor, to classify cases. In addition, the criteria used to define the
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Reports of animal bites increase in Erie County
Erie County saw a significant increase in the number of reported animal bites through the first two months of 2013.
Local emergency departments, physician offices and animal enforcement officials reported 101 animal bites to the Erie County Department of Health in January and February. Only 72 bites were reported during the first two months of 2012.
But an Erie County health official isn't sure more people are getting bitten.
"I think our reporters of animal bites, the ERs and doctor's offices, are on the ball," said Charlotte Berringer, R.N., director of community health for the county Health Department. "We have the right people in those places, better than we did before."
Saint Vincent Health Center's director of emergency medicine said his emergency department hasn't seen an increase in the number of patients with animal bites in 2013.
"I have talked with our staff and they said we have seen a typical number of bites so far this year," said Wayne Jones, D.O.
The county Health Department tracks mammal bites to humans because of the possibility of rabies, a viral disease animals can transmit to humans through bites or scratches. It is almost always fatal if left untreated.
Only mammals can transmit rabies, Berringer said. About half of the county's reported animal bites come from dogs, while another 26 percent are cat bites or scratches, and 19 percent are bat bites or exposures.
"If you wake up and there's a bat in your room, that's considered a bat bite because it could have bitten you and you wouldn't have felt it," Berringer said.
If you are bitten by a wild animal or a domesticated one with no proof of being vaccinated for rabies, you will need to be treated for rabies exposure.
Rabies treatment involves a dose of rabies immunoglobulin based on your weight and a series of four rabies shots over the next 14 days, Jones said.
"We like to start treatment within 24 hours of the bite," Jones said.
Spring is usually the time of year when reports of animal bites start to increase, said Brian Froess, the Pennsylvania Dog Warden for Erie County. Froess said he hasn't seen an unusual number of animal bites this year.
"I can tell when the weather warms up without going outside just by the number of calls I get," Froess said. "Everyone heads outside and the dogs are outside."
In order to reduce your risk of getting bit, Berringer offered these tips:
- Leave animals alone when they are eating or drinking.
- Never try to scare or tease animals.
- Don't mistreat or hurt an animal.
- Never pet an animal with its babies.
- Don't try to stop an animal fight.
- Leave injured, sick or wounded animals alone.
DAVID BRUCE can be reached at 870-1736 or by e-mail. Follow him on Twitter at twitter.com/ETNbruce.


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What is in this article?:
- Beginning farmer, rancher legislation re-introduced in House, Senate
- Need to provide training, tools
• The legislation reduces barriers, such as credit and land access issues, that new agriculture entrepreneurs face, and invests in successful new-farmer training programs and grants to help farmers capture more of the retail food dollar through value-added enterprises.
Senator Tom Harkin of Iowa and Representative Tim Walz of Minnesota have announced the introduction of the Beginning Farmer and Rancher Opportunity Act of 2013 in both the Senate and the House of Representatives.
The two identical bills expand opportunities and remove barriers for beginning farmers and those who wish to pursue a career in agriculture.
The bill reduces barriers, such as credit and land access issues, that new agriculture entrepreneurs face, and invests in successful new-farmer training programs and grants to help farmers capture more of the retail food dollar through value-added enterprises.
"We applaud Senator Harkin and Representative Walz and their co-sponsors for re-introducing this legislation and for championing the needs of beginning farmers as Congress heads into another Farm Bill," says Juli Obudzinski, Policy Associate with the National Sustainable Agriculture Coalition.
"The Beginning Farmer and Rancher Opportunity Act helps new farmers get started in agriculture, and invests in programs that have a proven track record of equipping farmers with the tools and skills they need to be successful in their farming career."
The Beginning Farmer and Rancher Opportunity Act is a comprehensive legislative package that invests in critical federal conservation, credit, research, and rural development programs that support opportunities for new farmers and ranchers.
The bill was originally introduced in the previous Congress, and gained the support of 37 members in the House and Senate who signed on to co-sponsor the bill.
In addition to NSAC, over 170 organizations officially endorsed the bill, including the National Farmers Union, National Association of Counties, Organic Valley, and the Farmer-Veteran Coalition.
The National Sustainable Agriculture Coalition has been closely involved in the development of this bill, and has worked in close partnership with both Congressional offices along with several farmer advocacy organizations across the country.
"With the average age of the U.S. farmer at 57, ensuring that the next generation of American farmers is able to provide the world with a safe, abundant supply of food should be a top priority," said Congressman Walz, Ranking Member of the U.S. House Agriculture Subcommittee on Conservation, Energy, and Forestry.


text: The construction, finance, management, and preservation of commercial assets is a critical aspect of economic revitalization. Commercial development and finance programs seek to encourage commercial districts to strengthen small businesses, improve the physical environment, and provide organizational support, business counseling, and loan packaging. The intent is to revitalize the business climate in impoverished and depressed neighborhoods and to provide additional goods, services, and employment for the community.


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Lighting up Windows: From websites to apps
- Date: to
- Day 2
- B33 Hood
- 3-137
- Speakers: Can Comertoglu, Justin Garrett
- 2 comments
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Great tips presented in this session. The only thing I missed here is how pinned website tiles can become live (for updates etc.)?
The guy from ClipBoard was unnecessary. I correlate him to a video advertisement that you have to watch before watching some web video like on Youtube. No one likes commercials. Aside from that, I liked this session. I'd say it was slightly better than average.
Comments have been closed since this content was published more than 30 days ago, but if you'd like to continue the conversation, please create a new thread in our Forums, or Contact Us and let us know.


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General Manager
Tensions between members of the Campbellsburg City Council and the city's attorney flared Monday night when one member questioned the city's legal expenses.
Mayor Jan Fletcher was poised to adjourn the city council's Monday night meeting, when council member Rex Morgan said he wanted to address the attorney's expenses.
As a tax paying resident of Henry County, I think it's only reasonable to expect that the road I live on receive the same care and maintenance as any other road in the county.
Preparing for the North Central Kentucky Conference championship, which was played all day Tuesday at the Henry County Country Club, the Henry County boys' golf team played in two matches last week.
The Wildcats hosted Owen County last Monday and traveled to Whitefield Academy on Thursday in the final two outings before the all-important conference tournament arrived. Afterwards, coach Willie Peyton said his team played well against Owen and struggled against Whitefield, but it was the NCKC that was his team's first real focus of the season.
Lindsey Susan Cottrell, 28, Lockport, to Stephen Ryan Davie, 28, Lockport.
Emily Gail Cross, 33, Louisville, to Samuel Scott Watson, 35, Louisville.
Andrea Acs, 31, Brooklyn, New York, to Patrick Anthony Kiefer, 28, Brooklyn, New York.
Dennis Martin, 58, Smithfield, and Cindy Hawkins, 49, Ontario, Canada.
Nicholas Anton Filacchione, 50, Edgewood, and Caroline Ann Filacchione, 50, Edgewood.
Staff writer/photographer
There's a new face patrolling the streets of Eminence.
John Wilson, 36, joined the Eminence Police Department on Sept. 9, replacing former EPD Officer Rodney Hawkins.
The dedication perseverance and volunteer efforts were apparent at the park dedication and ribbon cutting Thursday, Sept. 9.
Taxes, and utility rates, soon will go up in New Castle.
Monday night, the city's commission approved the first reading of the 2010 tax rate ordinance, which increased the property tax rate by 0.7 cents.
Landmark News Service
Nearly two years after its escape, an Owenton woman's missing kangaroo may have been spotted Thursday morning in the rural Henry County community of Bethlehem.
Jeannie Stivers was driving through the small community when something caught her eye.
Hampton's loss could be Campbellsburg's gain.
Since 1951, the small Georgia city 30 miles south of Atlanta hosted two races in the NASCAR Sprint Cup series. In August, NASCAR and the owners of Kentucky Motor Speedway in Sparta, announced that at least one of those races will come to the Kentucky track next year.
Wednesday, Sept. 15
Children's Story and Craft hour every Wednesday from 10 to 11 a.m. at Pleasureville Christian Church. Also, the community library at the church is open to the public.
Thursday, Sept. 16
Free adult scrapbooking offered at the Eminence Community Life Center from 10 a.m. until noon on Thursdays.
Copyright © 2016
The Henry County Local is your source for local news, sports, events, and information in Eminence, KY and the surrounding area.


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- Policy
The Australian Conservation Foundation has welcomed today's bipartisan motion on nuclear non-proliferation and disarmament and called for real action to match the rhetoric.
"The Prime Minister and Opposition Leader have taken an important step in acknowledging the grave threat posed by nuclear weapons and the importance of nuclear disarmament and abolition," said ACF nuclear free campaigner Dave Sweeney.
"The motion recognises that the Treaty on the Non Proliferation of Nuclear Weapons (NPT) is the essential foundation for the achievement of nuclear disarmament and the cornerstone of the nuclear non-proliferation regime.
"Yet Australia is actively undermining non-proliferation efforts and weakening the NPT by selling uranium to nuclear armed states and planning to sell to non NPT signatories.
"Australia currently sells uranium to nuclear weapons states such as France, the UK, China and the USA and is advancing plans to sell uranium to Russia and India — both of which are nuclear weapons states with secretive weapons programs.
"India is not a party to the NPT and there is clear legal advice that selling uranium to India would be in conflict with our obligations under the South Pacific Nuclear Weapons Free Zone Treaty.
"As well as joining international efforts to abolish nuclear weapons, Australia should suspend uranium sales to nuclear weapons states that don't comply with or are not signatories to the NPT.
"Australia must become much more diligent in ensuring we are not supplying uranium to countries with inadequate safety standards in their civil nuclear power programs.
"We know Australian uranium was present in the reactors at Fukushima and that rocks from South Australia and Kakadu are fuelled fallout in Japan and beyond.
"Australian uranium has already spread serious problems and nuclear insecurity around the world. While this motion is a significant step, we need to be far more rigorous in making sure we do not continue to fuel nuclear threats."


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I made green juice. I affectionately refer to it as green monster juice. I've been drinking it in the morning instead of coffee, since I gave up caffeine. Yes, I know, I'm crazy. It is day three, and just passing the coffee shop at work is enough to make me cry. I won't mention the headaches. You see, I'm doing a 21 day sugar detox I feel amazing (other than the headaches).
Breakfast has been a thickly sliced tomato topped with caramelized onions, a runny egg and 1/2 an avocado. There's crust less quiche, burgers without buns, and spaghetti squash in lieu of a big ol' pot of pasta. I'm surviving just fine thankyouverymuch. However, a glass of wine would be FAN.TAS.TIC. 21 days, won't be so bad, right?
If you find me in a corner hoovering a dozen donuts try not to judge me… okaythanks.
In all seriousness, this juice will wake you right up, I think it's the ginger/cinnamon combination. I obsess. I try to make it the night before I go to work, leaving it in a mason jar over night. That way, I can sleepily grab it and sip on it while I get ready for work. You should try it. Until then … think of me as you drink your coffee, and add a little extra creamer for me. Pretty please?
Green Monster Juice
yield: 1 SERVING
total time: 5 MINUTES
1 cup of kale leaves
1 medium apple, chopped
2 teaspoons fresh lemon juice
1 inch of fresh ginger
pinch of cinnamon
3/4 cup of water
Place all the ingredients in a blender, and blend until well combine. Press the juice through a fine mesh strainer. Serve immediately over ice.
Slightly adapted from It's All Good


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Karhulan Uimahalli (Finland)
- Overview
- Hotels
- Map
- Photos
Practical Information
Address: Karhulantie 46, Kotka
Zip code: FIN-48100
City: Kotka
Region: Kymi
Country: Finland
Phone 1: (05) 234 5415
Phone 2: 2345416
Entrance fee: €3.00.
More info: 25m x 12.5m, 1.2m - 3.8m depth, indoors, 26°C (79°F).
Hotels nearby
With its central location, Karhulan Hovi Manor Hotel is within easy reach of most tourist attractions and business addresses in Kotka.The Karhulan Hovi Manor Hotel boasts a convenient location with moRead moredern amenities in every guestroom and superb service.Room amenities include non smoking rooms, bathtub, shower, separate shower and tub.Hotel facilities offered at this Kotka accommodation include shops, laundry service/dry cleaning, meeting facilities, restaurant, disabled facilities.For a more enjoyable stay, guests can take advantage of a variety of recreational facilities, including water sports (non-motorized), garden.Modern comfort and convenience are seamlessly combined to ensure the guests' satisfaction.To proceed with your booking at the Karhulan Hovi Manor Hotel, simply choose your travel dates and fill in our secure online booking form.Hide
The Cumulus Hotel is located in Kotka making it one of the best hotels to stay at while in town.The hotel has a total of 93 well-appointed rooms designed with the guests' comfort in mind.Each guestrooRead morem is tastefully appointed with mini bar, internet access – LAN, hair dryer.The amenities and services offered at this Kotka accommodation include car park, concierge, bar/pub, elevator.To unwind, guests can enjoy the leisure facilities provided on the hotel's property, including sauna.The hotel provides a warm and welcoming service of international standard.For your reservation at the Cumulus Hotel Kotka, please choose your period of stay and fill out our secure online booking form.Hide
With its central location, Sokos Seurahuone Kotka Hotel is within easy reach of most tourist attractions and business addresses in Kotka.The Sokos Seurahuone Kotka Hotel boasts a convenient location wRead moreith modern amenities in every guestroom and superb service.The guestrooms are equipped with internet access – wireless, daily newspaper, desk.To suit guests' convenience, this Kotka accommodation offers restaurant, 24hr room service, nightclub.Leisure and sports facilities available on the hotel's property comprise gym.The hotel creates a balance of rich culture and modern convinience to ensure you a memorable stay. Make your reservation at the Sokos Seurahuone Kotka Hotel Kotka now by selecting your chosen dates of stay and submitting our secure online booking form. Hide
The Hotel Leikari is perfectly located for both business and leisure guests to Karhula.The Hotel Leikari boasts a convenient location with modern amenities in every guestroom and superb service.Room aRead moremenities include non smoking rooms, daily newspaper, desk, hair dryer, television, shower.The amenities and services offered at this Karhula accommodation include 24hr room service, meeting facilities.Hotel's guests can experience on-site latest leisure and sports facilities such as indoor pool, sauna.This hotel is characterized by a combination of modern comfort and traditional element of Karhula, making it a distinct accommodation. To book a room at the Hotel Leikari, please select your dates of travel and complete fill out our secure online booking form.Hide
Motel Road 66. 3-star hotel "Motel Road 66" is located in Kotka. This accommodation has a total of 18 rooms. The interior temperature is never uncomfortable thanks to the air conditioning. Fishing, kaRead moreraoke and mini-golf are some of the activities the hotel offers. For guests who appreciate getting in some sports practice, the hotel also offers options, such as pool and golf. This hotel hosts a pool table and a golf course. For guests' well-being, there is a sauna, a welcome relaxation for body and mind after all your busy activities of the day. Dinner can be enjoyed at the onsite restaurant which is known for its American, Russian and Asian specialties. A lounge bar is open for you to have a drink in company. Extra amenities include free of charge Internet access and an airport shuttle service.Hide
Customer reviews
Activities nearby
- Harbor and marina
- 1.9 mi Itaranta Marina:
- 2.4 mi Sunila Marina:
- 2.6 mi Metsola Marina:
- 2.4 mi Suulisniemi Marina:
- 3.1 mi Silkosaari Marina:
- 3.3 mi Pitkasaari Yacht Harbour:
- 2.6 mi Vanha Satama Harbour:
- 3 mi Keisarinsatama Marina:


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We're exploring the Black Death and its influence in the lives of composers from the Middle Ages, Renaissance, and beyond.
A live performance by Ensemble REBEL from the 2011 Indianapolis Early Music Festival…plus, recorder music from a featured release by Nina Stern.
Renaissance music by Jewish composers with the viola da gamba consort Fretwork.


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"With the Aircraft De-icing Forecast Service we have proven that winter icing delays can be reduced by 84%" Steve Crawley, bmi
bmi is part of the Lufthansa group and the second largest airline based at London Heathrow. From their Heathrow hub, bmi operates services in the UK to Europe, the Middle East, Central Asia and Africa.
During cold weather, ice can form on aircraft wings presenting a serious danger to aircraft safety. If ice is present, bmi must de-ice aircraft before take off. bmi needs accurate predictions of when to de-ice in order to improve planning, save unnecessary costs and reduce relays.
Every minute of delay costs on average £50. Costs increase considerably if long flights with connections are delayed, as bmi has to provide hotels, taxis, meals and seats on other carriers if passengers miss their connections. Additionally, the low cost operation has short turnarounds (twenty five minutes) with up to six flights a day, so it is vital the first flight departs on time.
bmi takes the Met Office's Aircraft De-icing Forecast Service which forecasts aircraft icing conditions worldwide. Our service provides timely de-icing predictive information which enables improved logistical planning.
Our accurate, tailored guides to holdover times, and proactive SMS and email alerts to ground staff enable improved de-icing operations.
bmi has 52 de-icing stations around the world to monitor, therefore it finds the help from the Met Office invaluable. By using our service to decide whether to anti-ice an aircraft and protect it during an overnight stop can save bmi in excess of £9,000. Importantly, with our Aircraft De-icing Forecast Service, bmi has reduced winter icing delays by 84%.
Every airline knows that by reducing delays you reduce costs, keep customer satisfaction, and reduce human-factor elements associated with stress and pressure.
Last updated: 20 April 2016
Aircraft De-icing Forecast case study


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Mercyhurst men's hockey preview, Feb. 28
Mercyhurst (18-12-6, 16-4-5 AHA) vs. Robert Morris (13-15-4, 13-8-4)
- When: Today and Saturday, 7:05 p.m.
- Where: Today -- Mercyhurst Ice Center; Saturday -- RMU Island Sports Center, Pittsburgh
- On the air: www.americaonesports.com (live stream), www.hurstathletics.com & www.rmucolonials.com (live audio & real-time statistics)
- What to expect: Mercyhurst has clinched the Atlantic Hockey Association's regular-season title -- the first in program history -- as well as the No. 1 seed, a first-round bye and home-ice advantage in the best-of-three quarterfinal round in the upcoming conference tournament. ... Robert Morris stands fifth in the standings with 30 points, one behind third-place Air Force and Connecticut and two behind runner-up Bentley, entering the regular season's final weekend. The Colonials can clinch one of the three remaining first-round byes, as well as finish as high as the second seed. ... The Lakers are unbeaten in their past four games (3-0-1) and 9-3-3 in conference play in the second half, while RMU has won nine of their last 13 games (9-2-2). ... The Lakers and Colonials tied 5-5 on Dec. 14 at MIC in their first meeting of the season. ... The Lakers are without junior F Matthew Zay (lower body), their leading scorer with 16 goals and 40 points, and freshman D Philippe Drouin (upper body). ... Junior G Jimmy Sarjeant, who is unbeaten in AHA play with a 14-0-4 record, will start tonight, while redshirt junior Jordan Tibbett will start Saturday. ... Junior F Cody Wydo (21-17-38) and sophomore F Zac Lynch (16-18-34) lead the Colonials' offense. ... Sophomore G Terry Shafer has a 13-13-4 record, 2.81 goals-against average and .919 save percentage this season.
-- Victor Fernandes


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Notebook: Abdelkader, Mrazek return; Nosek skates
Blashill pleased with team's performance in Bostonby Dana Wakiji @Dwakiji / DetroitRedWings.com
DETROIT - The Red Wings are starting to get their World Cup players back.
After skating on their own after the team's morning skate Wednesday, Justin Abdelkader and Petr Mrazek took part in a full practice at Joe Louis Arena Thursday.
Notebook: Mantha bigger; lineups for Boston
Abdelkader, Mrazek return; Hronek expected to play Fridayby Dana Wakiji @Dwakiji / DetroitRedWings.com
GROSSE POINTE WOODS, Mich. - Bigger is better.
At least that's the way Anthony Mantha operated this past summer.
Trending: Callahan, Howard, Coreau, Svechnikov
First preseason game is a 4-2 victory over the Penguinsby Dana Wakiji @Dwakiji / DetroitRedWings.com
1. Mitch Callahan: Callahan, 25, is not going to make the Red Wings this season but that doesn't mean he can't have a good game this preseason.
Callahan, playing on a line with center Matt Lorito and Eric Tangradi, scored the first two goals of the game, the first on the power play at 11:35 of the first, and the second even-strength going to the net at 1:45 of the second.
Red Wings release Kirwan from amateur tryout
Camp Roster Reduced to 55; Red Wings Host Penguins Tonight in Preseason Openerby Kyle Kujawa / DetroitRedWings.com
DETROIT - The Detroit Red Wings today released forward Luke Kirwan from his amateur tryout. Kirwan will return to the Ontario Hockey League's Flint Firebirds.
Notebook: Svechnikov's family nearby; lineup for Pens
Zetterberg, Kronwall, Pulkkinen still working their way back from injuriesby Dana Wakiji @Dwakiji / DetroitRedWings.com
DETROIT - If you can't be at home, sometimes home can come to you.
That's the case this season for Evgeny Svechnikov, who will be playing with the AHL's Grand Rapids Griffins this season.
Red Wings to wear commemorative patch in honor of #9
Commemorative patch to be worn for all home and road games this seasonby Alex DiFilippo / Detroit Red Wings
DETROIT - The Detroit Red Wings today announced that the team will wear a commemorative patch on their jerseys for the duration of the 2016-17 regular season in honor of "Mr. Hockey" Gordie Howe, who passed away in June at the age of 88.
The Red Wings will wear the commemorative "9" patch above the logo on the front of the jersey on the left side of the chest for all home and road games this season, starting with the Season Opener on Thursday, Oct. 13 against the Tampa Bay Lightning at Amalie Arena. The Red Wings will wear a white "9" patch on the red home jerseys and a red "9" patch on the white road jerseys.
Coreau, Larkin, Mantha shine in Red-White Game
Tatar, Nielsen and Vanek advance to World Cup Finals with Team Europeby Dana Wakiji @Dwakiji / DetroitRedWings.com
TRAVERSE CITY, Mich. - The Red-White game couldn't have gone much better for Jared Coreau.
Red Wings fans were treated to a fast-paced, high-scoring scrimmage as the team concluded training camp.
DeKeyser's Drive
Red Wings defenseman Danny DeKeyser has gone from an undrafted college signee to an elite, No. 1 blueliner in Detroit in under four yearsby Josh Berenter @JBerenter / DetroitRedWings.com
TRAVERSE CITY, Mich. -- It was a life-changing summer for Danny DeKeyser.
The Red Wings defenseman signed a six-year, $30 million contract on July 26, got married September 3, helped start his own apparel line and now, he's come into training camp ready be a No. 1 blueliner and a leader in the Wings' locker room.
Notebook: Ott hopes to contribute on and off the ice
Veteran foward wants to lead by example and make it difficult on opponentsby Dana Wakiji @Dwakiji / DetroitRedWings.com
TRAVERSE CITY, Mich. - On the ice, Steve Ott is considered highly annoying by opponents but off the ice, he's just the guy you want as your teammate.
The Red Wings signed Ott on July 1. For a guy who grew up across the river in Windsor, it's been a dream come true.
Red Wings send seven back to juniors
Camp Roster Reduced to 56; Preseason Begins Tuesday at Joe Louis Arenaby Kyle Kujawa / Detroit Red Wings
DETROIT - The Detroit Red Wings today assigned defenseman Jordan Sambrook to the Ontario Hockey League's Erie Otters and released six players from tryout agreements, who will all return to their respective major junior teams: forwards Justin Brazeau, Luke Coleman and Jeff de Wit; defenseman Dylan Coghlan; and goaltenders Stephen Dhillon and Connor Hicks.
The Red Wings' roster stands at 56 players - with another six World Cup of Hockey participants slated to rejoin the team early this week - as training camp wraps up with tomorrow's Red & White Game at 12 p.m. at Centre Ice Arena in Traverse City, Mich. Detroit's preseason schedule begins on Tuesday, Sept. 27 when the Red Wings welcome the defending Stanley Cup-champion Pittsburgh Penguins to Joe Louis Arena.


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Interest Master
|Rakesh Kumar
Followers : 1665
Date Of Birth : 1987-05-16
Occupation : Model
Sonal Singh Chauhan (born on 16 May 1987) is an Indian fashion model and actress. She has won several beauty contests and made her acting debut in the movie Jannat directed by Kunal Deshmukh. Sonal Chauhan was born in Noida, India. Her family hailed from famous Royal Chauhan Rajput of Mainpuri. Her father is a police inspector, and she has one elder brother and two sisters, one younger and one elder. She studied at Delhi Public School, Noida and then Philosophy honors at Gargi College in New Delhi. She was crowned as the Miss World Tourism 2005 at Miri, Sarawak state of Malaysia. She is the first Indian to have claimed Miss World Tourism title. She has been a popular model of Delhi, and has appeared in a number of advertisement campaigns in print and electronic media. She appeared in the ads of brands like Ponds and Nokia. She also appeared on the cover page of FHM and The Man magazine. She walked the ramp as show stopper for jewellery brand YS18 at the India International Jewelry...


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BROOKLYN, MI — The second and final race weekend at Michigan International Speedway is finally here, and there was a buzz around the track early Friday, Aug. 16.
A few hours before practice began for the Pure Michigan 400, numerous drivers expressed their excitement to be back at the fast track at MIS.
Speaking alongside Roush Fenway Racing teammate and 2012 winner of the Pure Michigan 400, Greg Biffle, Carl Edwards said he always enjoys returning back to MIS, the site of his first Sprint Cup Series start.
"I think this is one of our favorite racetracks," he said. "For me, this is the site of my first start so it is real special for that reason. It is just a fun place."
In June, Biffle was victorious in the Quicken Loans 400, holding off Kevin Harvick to deliver Ford its 1,000th NASCAR Victory. Sitting in ninth place in the NASCAR Sprint Cup Series standings, Biffle said he wouldn't mind repeating that feat Sunday.
"I really love this place," he said. "It is one of my favorite places to come and you can run two and three wide through the corner in a downforce situation where you normally can't do that at most race tracks.
"This is just a fun place for myself and our team to race."
Kyle Busch, driver of the No. 18 Interstate Batteries Toyota for Joe Gibbs Racing,
said while he has had some "good" and "not so good" days at MIS, the spring
race at MIS went pretty well. He is coming off a victory last weekend at Watkins Glen.
"(We) finished fourth, which was good for us and our team and how sometimes we run here," he said. "(I'm) excited about the opportunity to come back and try to change it up a little and maybe run a little better this time and just be able to get the Interstate Batteries Camry back to victory lane hopefully here in Michigan."
Leading the Sprint Cup standings is Jimmie Johnson with 15 top-10 finishes in 22 starts this year. Johnson was in the hunt for his first victory at MIS during the Quicken Loans 400, but blew a front left tire with three laps to go and fell to 28th.
"We're excited to be here with the way the No. 48 ran and all
of Hendrick Motorsports ran here in the spring," Johnson said. "We've all been excited to come
back and hopefully have that pace and lead the race and hopefully get one of the cars in victory lane.
"I certainly want to get the No. 48 into victory lane. We've tried for a lot of years now and have been very close here, but haven't been able to pull that off."
Follow Justin Dacey on Twitter: @JustinDacey or email him at firstname.lastname@example.com


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Amarillo artist Patsy Kisor is featured through the end of August in an exhibition at the Carson County Square House Museum in Panhandle, according to a news release.
Kisor's art career began in 1983 when she took a clay sculpture class at Amarillo Collage. After two years of sculpture classes, she taught herself how to sculpt rock and metal. She has won numerous awards in various Texas, Oklahoma and New Mexico art contests.
The exhibition will hang in the Square House Gallery. For information, call 537-3524.
© 2016. All Rights Reserved. | Contact Us


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Through the Boiler Room
Pick up the goodies hidden on both sides of the staircase, and then go through the eastern door. After a cutaway scene involving Ada and Krauser, the scene will switch back to you. Smash the barrels for some items, and then aim directly ahead and down a bit through the wooden boards to snipe off a crossbowman (you can also snipe another enemy, but he'll be wearing a helmet and hiding almost entirely behind the wall, and therefore hard to aim at). A Soldier with a stun rod will come up the stairs, kill him and then go down to kick open (there are enemies right on the other side) the door to enter the boiler room.
Kill the remaining enemies here, and then start salvaging the area. There are some nice items in the narrow side room and some ammo in the cabinet upstairs. Then go through the door.
Knife Fight
Keep heading down the path until you reach the elevator, there are no enemies along the way. Operate it to go up. Once you reach the large rectangular region, you will be confronted by Krauser and challenged to a knife fight.
This whole portion is an interactive cutscene, so always be prepared to press buttons as they show up. Leon and Krauser will be talking the entire time, try and listen in (it's interesting) without losing awareness of Krauser's attacks, which are done very suddenly after he or Leon finishes a sentence. Failure to press the buttons quick enough will result in instant death. At one point, Krauser will pin you to the ground, perform the action as fast as you can to avoid being stabbed in the neck.
Ada will eventually come to your aid. Krauser will do a supernatural fifty-foot leap to a high platform and leave.
Leon's Pro Skillz
Jump down and keep heading down the path until you come to the automatic door. As you run down the corridor, a set of lasers will appear and block your path. One touch of them and you are dead. To get past the first set of lasers, wait for the two beams to become vertically parallel, and then run through. Another set of lasers appears, wait for all three beams to become parallel (two of them will form a v-shape prior to the moment, start approaching the lasers at that time because the lasers will have already moved to the next position)and that is when you can pass through.
Another set of lasers appears, and there are so many that you can't just run through. Approach the beams, and press the onscreen buttons quickly to have Leon demonstrate his pro skillz in dodging lasers. Some black bug-like things will now cover the sides of the wall, they will project lasers when you get near. Do the same thing to flip through them all.
When you reach the door at the end, it will be locked. Check the control panel to the right, and the final laser set will be activated, moving at you from behind. Run straight at it and press the buttons to have Leon run up the wall and flip straight over the lasers. Note that these lasers won't come at you at a constant speed, they accelerate at the end to throw you off guard.
If you wait too long too long at the door when the final lasers come, a ton of lasers will immediately materialize where you are and kill you, so get running right after you check the control panel. Open the door after you have done the wallclimb-flip laser dodge.
Falling Containers of Doom
Take the treasure off the red throne. You can sit on it if you like, but it does nothing. Shoot the gem off the ceiling after you get up. After you are done resting, go behind the throne and operate the circular elevator.
Up ahead is a Merchant. Get some First Aid Sprays and upgrade your weapons if you like, because there is a boss fight coming up. Open the gate and keep going down the path. You can take some items in the small gated area if you like. A cutscene will show three large containers suspended precariously from a sky cable. After Leon finds the GPS dot that he threw on Ashley, the U3 (Saddler introduced you to It, remember?) will burst out of the wall and you will end up in the first container.
The point of all three containers is to find and press two red buttons, and then exit to the next container within 30 seconds, after which the container falls into the depths of nowhere. Each red button (except one) has a shutter blocking access to it, which you must open by shooting a green panel. All the while the U3 will be trying to kill you.
You can not kill the U3 in the containers, so don't waste ammo. If you shoot the head and deal enough damage, the U3 will hide into the ceiling for a while. However, you can always run past the U3 without much chance of getting hurt. Stay close and its attacks will miss.
For the first container, the red buttons are in the southwest and the northeast. The southwest one should be pressed first. The fight begins with the U3 right in front of you, run past. Most of its attacks can be dodged, but you will be disoriented afterwards, so check the map often to see that you are on the right track. Go around the corner, shoot the green panel to open the shutter, and press the red button. Use the map to find your way to the northeast, shoot the green panel, and press the button. You must exit in 30 seconds, but it shouldn't be a problem. Run past the U3 as it drops down right behind you and enter the red door.
The second container is a bit tougher, as the green panels aren't located exactly next to the shutters. The red buttons are in the northwest and southeast. The U3 will climb back up, run past it and shoot the green panel on the beam. Press the northwest red button and head to the southeast. The green lock is high above the shutter, shoot it and press the red button. You again have just 30 seconds to exit, but you have to go all the way around. If the U3 shows up, again, ignore it and run. Enter the red door to get to the final container.
A red button is visible in the area to your right just as you board the third container, press it and then the U3 will appear again, this time with a deadly pincer appendage (it can kill you in one hit). Run away from it and head into the narrow hall spanning the middle of the container. At the end, shoot the green lock through the bars, and then retrace your steps to the other side and press the red button. Again, 30 seconds to run back in the narrow passage and exit. The U3 will be blocking your way, quickly make it leave, take the same path you took just before to shoot the green lock, and leave.
Jump on the crane hook thing to get back onto spacey, solid, non-falling ground.
U3 (It)
That's not the end of it though. After you take all the goodies lying on the right, the U3 will still climb up the cliffside, and now you have to kill him for good. There are two explosive cans (one around the rock, one in the tunnel) for you to use. You can try to trap the U3 using the gates, but it will manage to break through after a while. Refer to Bosses and Strategies on how to defeat the U3.
When you have dealt with the hideous creature, go to the next area for some items. You can take the lift back across the abyss for a green herb, and then take the lift back. When finished, go up the ladder.
The Underground Detour
Smash the barrels around here, and head to the cliff edge. Don't jump down just yet. There are several enemies wandering about, but they take a long time to notice you. They are also stupid enough to stand right next to explosive barrels. Scan the area to find the red cans and shoot them to take out most of the Soldiers. Kill any survivors by aiming down with the rifle, and then hop down.
Search the area for free items before going on. The path is blocked by a wire fencing, so go back into the tent and climb down the hole to enter a tunnel. There are no enemies in it, so just run to the end to meet up with a Merchant. Restock your health items and ammo for another boss fight coming up. Also, if you havn't already gotten the Exclusive upgrade for the Striker, do it now. You will have to go a very long stretch without another Merchant, and if you run out of shotgun shells it could be devastating. The Merchant is also operating the last target practice gallery, so go ahead and get some bottlecaps if you feel like it. Climb up the ladder and enter the sandy double doors.
Krauser Preshowdown
Get the goodies from the barrels along the path, and then jump down and head west. Krauser will appear on a far ledge, chat a bit, and start off the fight by firing his TMP. He might also throw a grenade, which you can avoid by pressing the dodge buttons. Snipe Krauser with one handgun shot to make him go away for the moment, and go through the east door.
Take the magnum ammo and other items on the table. Quickly go up the ladder, run to a corner, and face the hole. Krauser will flip up, run straight to him and start slashing with the knife (it is his only weakness). All of Krausers attacks can be dodged by pressing the buttons, if you have fast reflexes you can get through it unscathed. Krauser will eventually use a flash grenade and escape.
Jump back to the first floor, kick down the gate, and proceed. Krauser will confront you again when you approach the ruined walls on your left. Do the same thing as before to make him flash grenade away, and then keep heading east. When you get to the large square building (don't enter, there are just a few items to grab and only means another skirmish with Krauser), go right of it. Jump down the ledge, and Krauser will fire his TMP again. Run for the tunnel, grabbing the green herb to your right along the way.
When you get to the eastmost region and take the Piece of the Holy Beast, Panther, the gates close and Krauser appears on the ledge above. He starts off with TMP and grenades, hide under the ledge and you cannot be hit. When Krauser hops down, run up and knife. When he hops back on the ledge, press the action button to respond to his taunt, and repeat the procedure until he flash grenades away. A statue will then come out of the ground, push it into the nearby floor indentation to reveal the gate levers.
Pull the lever and head west until you reach the gate (northwest of the square building). Pull the lever next to it and keep going. There will be no more Krauser mini-fights for this part, but there will be electronic droids that can deal a lot of damage. The grounded ones will scuttle near and self-destruct, the flying ones will fire a machine gun at you. One shot will blow them up. Use your map to find the way to the northern circular area. Krauser will appear and taunt you again, make sure to respond. Go up the circular staircase and climb the ladder to reach the final battleground.
Jack Krauser
First, go left and take the magnum ammo and green herb. Then go to the opposite area and take the Piece of the Holy Beast, Eagle off the scaffolding. Krauser will appear and sprout a new arm. His attacks with it include shoving it forward and stabbing you after a kick, these are both instant deaths unless you press the dodge buttons. Even worse, you only have three minutes to defeat Krauser and exit the area before the bombs go off.
Refer to Bosses and Strategies on how to defeat Krauser. Remember that he retains his weakness to knife. When he is dead, take the Piece of the Holy Beast, Serpent. Hop down the ladder and jump down the ledge. Insert all three pieces into the wall to unlock it. After you enter, the chapter ends.
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Download the Good Practice Case Study (27 Kb PDF)
Summary: The project consists of construction and operation of a SHPP on Treshtena, River Dalgi Del village, Georgy Damjanovo Municipality, Montana Region, Bulgaria. The project is financed through EBRD Bulgarian Energy Efficiency and Renewable Energy Credit Line (BEERECL) and as such received 20 % grant of the total capital costs after project completion. Project documentation was prepared and submitted by EnCon Services - the Consultant, approved by EBRD to operate and manage the facility in Bulgaria.
Aims: To produce electricity and sell it to the grid thus ensuring both economic and environmental benefits.
Summary of Results: The main result is a SHPP operating and gaining profit for the owners
Planning Time: Construction period from 01/06/2004 to 31/08/2004
Planning issues: The hydro power plant is operational
Operation Time: 25 years
To find similar reports, click on a keyword below:
T@W: Sustainable Energy Technology at Work (2006-2008) : Renewable Energy Technologies : Small Hydro Power


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Thank you for visiting our past auction result archives. If you have an item identical (or similar) to this auction lot, please call, write or contact us to discuss. We will be able to help you.
Lot # 1409 (of 1594) < Previous Lot | Next Lot >
1983-1984 North Carolina Tar Heels Team-Signed Basketball with Michael Jordan
Starting Bid - $500.00, Sold For - $5,581.25
Spalding basketball, displaying alternating panels of white and "Tar Heel" blue, signed in black Sharpie by eighteen members of the 1983-1984 North Carolina men's basketball team including Michael Jordan (directly on the ball label), Sam Perkins, Matt Doherty, Brad Daugherty, Kenny Smith, Steve Hale, Dean Smith (head coach), Roy Williams (asst. coach), and Bill Gutheridge (asst. coach). The notation "1983-84 Tar Heels" is written in black marker on one of the blue panels. The signatures are all fairly uniform in strength, grading "8/9," and the ball is in Excellent condition. The 1983-1984 season marked Jordan's final season at North Carolina, where he starred for three seasons. Jordan's college career began on a high note as he helped lead the Tar Heels to the 1982 NCAA Championship as a freshman. He closed out his career in equally spectacular fashion. Jordan was named the Naismith College Player of the Year in 1984 and was also awarded the prestigious John R. Wooden Award. Jordan-signed items dating from his college days are very scarce, and this team-signed basketball, commemorating his final college season, is a particularly rare, substantial, and historically significant early Jordan-signed item. LOA from James Spence/JSA. Reserve $500. Estimate $1,000/$2,000. SOLD FOR $5,581.25
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No ...
Old member.
yes i do remember you.
U dont remember me? i was the nab roaming the forum before the wii launched, and i am bbbbACK. Since my last post i have gotten a Ps3.
Anyone remember me?!
My name is Alan, gaming name is ZaLaN
No ...
The name rings a bell
Sadly its the bell of forgetfulness
Spoiler Alert!
TNOG.ORG - Games events in Manchester
MikeDBloke - PSN
I was into weird games before people knew what they where
@D_Bloke - My inane prattle
nope, and i doubt many people will care.
Wii Games: Legend of Zelda: Twilight Princess, Heatseeker, Excite Truck, Wii Play, NHL 2k9, Super Mario Galaxy, Super Strikers Charged, Shaun White Snowboarding, The Bigs, Guitar Hero: World Tour, Ghost Squad, Call of Duty: World at War, Wii Sports Resort, Punch-out!
VC: Wave Race 64
Homebrew Channel
I do indeed remember you.
I remember I used to complain about how awful Piczo is, whenever I saw your name.
Enoch was right.
no clue who u are.
That's because you're a new member, Krunch. >.>
To be honest, I'm not an old member either, so I obviously don't remember you (I actually joined last year, btw). But welcome back either way! It's always good to see old members returning... especially one all the way back from 2006. o_o
Yes, I do indeed.
Joined in Dec. 07, so no.


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LC-MS Gains Favor Due To the...
As Immunotherapy Revolutionizes the Cancer Treatment...
Expanding Use of NGS Has Largely...
Developers of DTC Genetic Testing Are...
A new approach to high-throughput screening has been developed by microPEP. Rather than imprint reagents onto microarrays, microPEP's approach injection-molds microwell arrays to be used with microbeads. These microarrays contain 40 million locations that are 3 µm in diameter and 3 µm deep, making them the smallest microwell arrays in the world, according to the company.
"This molded slide is meant to be an option to the 25 x 75 mm microarrays," according to Scot MacGillivray, business development manager. "We think we're the only company in the world to offer this," MacGillivray reported at the Knowledge Foundation conference on "Detection Technologies" late last year.
"This molded microarray has the potential to change the way healthcare is delivered and assays are performed," and it certainly makes high-throughput screening more cost effective. For example, traditionally microbead assays could only be performed by first etching away a silicon wafer. That's expensive, costing hundreds of dollars. Molding that substrate, however, costs less than $30, MacGillivray says. "When we get into the area of thousands of slides, the cost is under $10 per microwell slide." The microwell slides meet Class 10,000 cleanroom specifications.
The tooling is based upon standard injection technology and some proprietary technology. The next step for microPEP is to explore the submicron fabrication limits. "We're interested in trying nanofeatures," MacGillivray said. Additionally, the company has developed 50 and 100 µm well slides. The 3 µm microwell slides are available now.
While microPEP is advancing miniaturization for biodetection, other presenters discussed new types of arrays, expanded capabilities, increased speeds, new combinations of capabilities, and novel molecular methods to immobilize and label DNA.
Platypus Technologies is developing protein-detection assays based upon liquid-crystal technology to rapidly detect and report the presence of target molecules bound to specially designed surfaces.
As Richard Schifreen, Ph.D., president and CEO, explained, liquid-crystal molecules are rod-shaped and tend to align parallel with each other. That alignment changes, however, when target molecules bind to receptors that are immobilized on the surface of liquid crystal-coated substrate. The standard analogy compares the alignment to upright bowling pins, and the binding to the scattered pins after the bowling ball—the target molecule—has struck.
The dramatic difference makes direct detection of viruses captured by surface-bound antibodies possible with low- or no-power readouts based upon interrogation with polarized light. For a simple "yes/no" readout, a light image indicates presence of the target molecule and a dark image indicates absence of the target.
"The work is at the precommercialization phase," Dr. Schifreen said. "The sensor can be packaged into an instrument, a badge, a handheld device, or incorporated into an alarm system."
Platypus is currently developing a protein-detection assay for influenza exposure and water-borne pathogens, and gas sensors to detect nitric oxide and pesticides and to monitor asthma. "The advantages," he said, "are low- or no-energy requirements, the inherent flexibility of a single format for multiple analytes, and the simplicity of a solid-state system. It's very inexpensive, costing a couple of dollars at research scale," versus about $100 for an electrochemical sensor.
"Detection speed depends on the particular system," Dr. Schifreen said, but ranges from a few seconds to one or two minutes. "The assay can be either qualitative or quantitative. We're working to optimize the system so it's more specific, more reproducible, and, in some applications, reversible."
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City Filter Press
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FSA-Group, Técnicas de Filtración S.A., were established in 1974 with the idea to study and solve any project related to filtration and dehydration. In this sense, TEFSA,Técnicas de Filtración S.A. has developed specialized projects for specific situations in these last 30 years. A broad fabrication program, being one on of the main competitive advantages of the company, ...
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The company ENVITES, spol. s r.o. was established in 1990. The establishment of the company is historically related to South Moravia, which is well-known for its engineering industry associated with the production of water and wastewater treatment equipment, ecology and production of filtration systems. Since the time of its establishment the company has been offering a ...
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Filter press design, manufacture, installation and maintenance expertise in filtration technology. Belt presses, sludge driers. Welders Filtration Technology is a long-established and widely respected leader in the world of filter press design, manufacture, installation and maintenance.
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based in Tiantai County, CHINA
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