text:
Proteomic Analysis of HIV-Infected Macrophages
- First Online:
- Received:
- Accepted:
DOI: 10.1007/s11481-010-9253-4
- Cite this article as:
- Meléndez, L.M., Colon, K., Rivera, L. et al. J Neuroimmune Pharmacol (2011) 6: 89. doi:10.1007/s11481-010-9253-4
- Citations
- 932 Downloads
Mononuclear phagocytes (monocytes, macrophages, and microglia) play an important role in innate immunity against pathogens including HIV. These cells are also important viral reservoirs in the central nervous system and secrete inflammatory mediators and toxins that affect the tissue environment and function of surrounding cells. In the era of antiretroviral therapy, there are fewer of these inflammatory mediators. Proteomic approaches including surface enhancement laser desorption ionization, one- and two-dimensional difference in gel electrophoresis, and liquid chromatography tandem mass spectrometry have been used to uncover the proteins produced by in vitro HIV-infected monocytes, macrophages, and microglia. These approaches have advanced the understanding of novel mechanisms for HIV replication and neuronal damage. They have also been used in tissue macrophages that restrict HIV replication to understand the mechanisms of restriction for future therapies. In this review, we summarize the proteomic studies on HIV-infected mononuclear phagocytes and discuss other recent proteomic approaches that are starting to be applied to this field. As proteomic instruments and methods evolve to become more sensitive and quantitative, future studies are likely to identify more proteins that can be targeted for diagnosis or therapy and to uncover novel disease mechanisms.
KeywordsMonocytes Macrophages HIV SELDI-TOF 2D DIGE Tandem mass spectrometry Proteomics
Two-dimensional difference in gel electrophoresis
Acquired immunodeficiency syndrome
Blood–brain barrier
Cognitive impairment
Central nervous system
Cerebrospinal fluid
Generalized estimating equation
HIV-1 associated dementia
HIV-associated neurological disorders
Human brain microvascular endothelial cell
A macrophage tropic R5 virus isolated from blood of a patient with HIV dementia
High-performance liquid chromatography
Isotope coded affinity tags
- IL-1β
Isobaric tag for relative and absolute quantitation
Liquid chromatography coupled with electrospray ionization and a hybrid quadrupole linear ion-trap and Fourier-transform ion-cyclotron-resonance mass spectrometry
Liquid chromatography/mass spectrometry/mass spectrometry
Linear ion trap Fourier transform instrument
- M-tropic
Macrophage tropic
- M-tropism
Matrix assisted laser desorption/ionization-mass spectrometry
Matrix-assisted laser desorption ionization/time of flight
Minor cognitive motor disorder
Monocyte derived macrophage
Mass spectrometry
Normal cognition
Placental macrophages
- PTMs
Post-translational modifications
Tyrosine phosphorylation
Quadrupole time-of-flight
Sodium dodecyl sulfate polyacrylamide gel electrophoresis
Surface enhancement laser desorption/ionization
Surface-enhanced laser desorption/ionization time-of-flight
Stable isotope labeling of cells in culture
Simian immunodeficiency virus
Superoxide dismutase
Signal transducer and activator of transcription-1
A monocyte cell line
Tumor necrosis factor
Vesicular stomatitis virus
Macrophages and HIV infection: early evidence
When HIV infection was first discovered in 1981, multiple studies pointed to CD4+ T cells as the only targets of infection (Maddon et al. 1986). A few years later, new evidence revealed that different HIV isolates could productively infect other CD4+ cells including monocytes and macrophages (Cheng-Mayer et al. 1988; Gendelman et al. 1988; Collman et al. 1989). Macrophages are mononuclear phagocytes involved in both innate and adaptive immune responses. These cells act as sentinels of the immune system because of their phagocytic and inflammatory functions. Those HIV isolates that preferentially infect macrophages are termed macrophage tropic (M-tropic) or non-syncytia inducing, whereas those that productively infect CD4+ T cells are termed T cell tropic or syncytia-inducing based on the phenotype developed after infection. HIV uses the CCR5 (R5), and the CXCR4 (X4), as co-receptors for entry (Alkhatib et al. 1996; Deng et al. 1996). In addition, several groups have provided evidence of the dual usage of chemokine co-receptors by some viral isolates (Doranz et al. 1996). A classification system was developed wherein viral isolates were designated as R5, X4, or R5X4 viruses, depending on co-receptor usage (Berger et al. 1998). Shortly after identification of co-receptors for HIV entry, it was demonstrated that co-receptor use cannot be assumed to be a surrogate for tropism, owing to the presence of dual-tropic viral strains that are X4 but not R5 or vice versa (Goodenow and Collman 2006). CD4(+) T cells and macrophages can be infected by R5-using viruses, as they both bear R5 co-receptors (Duenas-Decamp et al. 2010; Hladik et al. 1999), but R5-using viruses vary in their capacity to infect macrophages (Duenas-Decamp et al. 2010; Tuttle et al. 2002). The presence of X4 viruses correlates with risk of disease progression (Raymond et al. 2010; Tuttle et al. 2002). But highly macrophage-tropic R5 viruses have been related to neurological complications (Peters et al. 2007). HIV-1 disease progression is also associated with an increased capacity of the virus to replicate in macrophages, indicating that M-tropism of HIV-1 is an important determinant (Gendelman et al. 1990; Li et al. 1999; Tuttle et al. 2002; as reviewed by Gorry et al. 2005). During initial viral transmission in vivo, preferential infection is via M-tropic, R5 viruses (Bieniasz and Cullen 1998; Bachis et al. 2010; Raymond et al. 2010). X4 viruses in primary infections are not usual (Raymond et al. 2010). Macrophages are to be among the first cells infected with HIV-1 following sexual transmission (Zhu et al. 1993) although cervical mucosa CD4(+) T cells can also be infected as they possess CXCR4 and CCR5 co-receptors (Hladik et al. 1999), and R5 viruses from a few patients have been shown to replicate in T cells and not in macrophages (Li et al. 2010).
Macrophages as HIV reservoirs
HIV persists in the host system despite antiretroviral treatment. There are essentially two theories of persistent infection: ongoing replication, which is a consequence of drug resistance, and latency, which involves the presence of HIV in reservoir cells such as resting memory CD4+ T cells, and in mononuclear phagocytes including peripheral blood monocytes, macrophages, microglia, and dendritic cells (Le Douce et al. 2010; Embretson et al. 1993; Finzi et al. 1997; Chun et al. 1997; Wong et al. 1997; Bailey et al. 2006; Keele et al. 2008; Zhu 2002). Mononuclear phagocytes are important sites of viral persistence (Popovic et al. 1988; Gendelman et al. 1988; Le Douce et al. 2010). Since monocytes and tissue macrophages live for a long time, they can act as reservoirs and vehicles for viral dissemination (Crowe et al. 2003). Infectious virus is present not only in differentiated macrophages but also in circulating monocytes (Zhu et al. 2002; Lambotte et al. 2000) affecting cellular receptors (Melendez-Guerrero et al. 1990) and antigen presentation (Melendez-Guerrero et al. 1991). It has been shown that the CD16+ subset of monocytes is more permissive to HIV infection than CD16- cells (Ellery et al. 2007). These CD16+ monocytes are recruited to sites of infection or inflammation (Crowe et al. 2003; Alexaki and Wigdahl 2008), and represent an intermediate state of activation between monocytes and macrophages (Ancuta et al. 2009). Upon subsequent differentiation, they become resting cells and viral reservoirs in different tissues including the brain (Gartner 2000; Fischer-Smith et al. 2001).
Macrophages and HIV-associated neurological disorders
HIV-associated neurological disorders (HAND) is a repertoire of several central nervous system (CNS) disease manifestations caused by HIV infection in advanced stages of the disease that range from mild forgetfulness to frank dementia. CNS disease results as a consequence of the neuronal death caused by the secretion of soluble viral and cellular neurotoxins from activated and/or infected perivascular macrophages and microglia (Gonzalez-Scarano and Martin-Garcia 2005; Kaul et al. 2001). These neurotoxins include the cytokines [tumor necrosis factor-α (TNF-α) or interleukin-1β (IL-1β)] (Fischer-Smith and Rappaport 2005), excitatory amino acids (Dreyer and Lipton 1995; Yeh et al. 2000), chemokines that induce cellular inflammatory cascades, and viral proteins (Gonzalez-Scarano and Baltuch 1999; Kaul et al. 2001). The secretion of these factors, together with a severe dysregulation of the normal functions of macrophages, can lead to neuronal dysfunction and apoptosis (Adle-Biassette et al. 1995; Shi et al. 1998), resulting in the development of severe dementia commonly called HIV-1 associated dementia (HAD; Gonzalez-Scarano and Martin-Garcia 2005; Kaul et al. 2001).
Two theories of viral entry into the CNS currently prevail: cell-free entrance and the Trojan horse model. HIV can enter the brain as cell-free virus at an early stage of the disease (Banks et al. 2001). Once in the brain, HIV establishes productive infection mostly in the mononuclear phagocytes of the CNS. These include perivascular macrophages derived from blood monocytes, meningeal macrophages, macrophages of the choroid plexus, and the microglia. The perivascular macrophages and the microglia are the major HIV-producing cells in the CNS (Fischer-Smith et al. 2001; Williams and Hickey 2002; Kim et al. 2003, 2005, 2006). Mononuclear phagocytes are less susceptible to the cytopathic effects of HIV infection compared to lymphocytes (Collman et al. 1989). In contrast to lymphocytes, viral replication is more persistent in mononuclear phagocytes, which leads to a continuous low-level virus production for the lifespan of the cells.
The Trojan horse model establishes that HIV-infected blood monocytes traffic from the periphery to the brain and hence are the primary source of virus in the brain (Narayan et al. 1982; Peluso et al. 1985; Meltzer et al. 1990; Davis et al. 1992; Fischer-Smith et al. 2001; Williams and Blakemore 1990; Kim et al. 2003, 2005, 2006). Monocyte trafficking to the CNS is part of the normal turnover and is augmented with inflammation and viral infection (Hickey and Kimura 1988). HIV-infected monocytes have been shown to cross the blood–brain barrier (BBB) more efficiently than non-infected monocytes (Persidsky et al. 1999). More importantly, at late stages of the disease, when the immune system is highly compromised and the BBB is deteriorated, there is an increased accumulation of monocyte-derived macrophages (MDM) from the circulation in the brain. Most of macrophages accumulating in the perivascular space appear to be an activated CD14+/CD16+ blood monocyte subpopulation (Fischer-Smith et al. 2001; Williams et al. 2001), which is more permissive to HIV infection (Ellery et al. 2007; Jaworski et al. 2007) and is increased in patients with HAD (Pulliam et al. 1997). The activated CD14+/CD16+ population is elevated in the blood of Hispanic women taking antiretroviral therapy (unpublished) as well as in their cerebrospinal fluid (CSF; Agasalda et al. 2010). Altogether, current data support the influence of the peripheral compartment in the development HIV-induced CNS disease, as reviewed by Fischer-Smith et al. (2008).
HIV alterations of cellular functions of macrophages: evidence before the proteomics era
Perivascular macrophages and resident microglia are the principal targets for HIV in the CNS. As described above, these cells play an important role in HIV pathogenesis and persistence in the CNS due to the secretion of toxic factors that affect neuronal function and survival (Giulian et al. 1990). Understanding how HIV infection modulates the normal physiology of these cells and contributes to neuronal injury has been of great interest. A key question is how specific proteins are modulated during HIV infection and how such modulation affects neuronal survival. The initial evidence of modulation of host cellular proteins was gathered via the study of individual proteins as small components of the whole cellular system. The most widely used methods were one dimensional (1D) gel electrophoresis, high-performance liquid chromatography (HPLC), Western blot, peptide sequencing by mass spectrometry, enzyme-linked immunosorbent assay (ELISA), flow cytometry, and electron microscopy. For example, in an early study Jiang et al. (2001) using several time-consuming and complex methods, which included supernatant fractionation, HPLC, and immunostaining, showed that glutamate is a mediator of neurotoxicity. The role of secreted arachidonic acid in neuronal injury was demonstrated by using HPLC, ELISA, and Southern blot (Genis et al. 1992). The group of Talley (1995) demonstrated the expression and activity of TNF- α, after fractionation, HPLC, and immunostaining, that this protein promotes loss of viability in neurons treated with HIV-infected MDM conditioned media. Although valuable information was found by the methods available before the 1990s, investigations had to rely on known proteins and pathways and thus were limited in a way that proteomic analysis is not.
Monocytes, macrophages, and microglia in the proteomics era
Before the proteomics era fully developed, researchers accumulated vast databases from genome sequences, but it became clear that sequences were not enough to elucidate the cell's proteome or biological functions as their information is restricted to DNA and RNA. An organism's genome is quite stable, whereas its proteome is highly dynamic. Proteins are very difficult to predict from genomic data. The existence of an open-reading frame does not necessarily indicate the presence of a functional protein; one gene can give rise to more than one functional protein. Also, protein post-translational modifications (PTMs) are not marked on the genome, and more importantly mRNA concentration does not necessarily correlate with protein concentration (Pandey and Mann 2000). Protein modifications such as PTMs, alternative RNA splicing, RNA editing, and proteolytic processing give rise to a very diverse proteome (Link and LaBaer 2009). Generally, most diseases are the consequence of the absence or dysfunction of a protein. Proteomics is a rapidly evolving field that gives a comprehensive view of the characteristics and activity of all cellular proteins expressed at a given time under specific conditions. By studying the cell proteome and secretome, we can examine PTMs, interactions, localization, conformation, stability, and trace of their functions in the cell (Morrison et al. 2002). These proteomic-based techniques have replaced in part traditional identification methods that failed to provide answers to important questions in research.
Proteomics approaches applied to studies of HIV-infected macrophages
Uses, advantages, and disadvantages
One-dimensional SDS polyacrylamide gel electrophoresis (1DE)
Standard mean for molecular weight determination of entire proteins
Ciborowski et al. 2007
All bands are cut for protein identification by LC/MS/MS
Luciano-Montalvo et al. 2008
Difficult to correlate protein expression with identification data
Garcia et al. 2009
Surface-enhanced laser desorption/ionization (SELDI)
Detect protein profiles and enzymatic activities. Increased sample throughput Does not provide protein identification.
Luo et al. 2003; Sun et al. 2004; Wojna et al. 2004; Carlson et al. 2004; Kadiu et al. 2007; Toro-Nieves et al. 2009; Wiederin et al. 2009
Matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry
Vaporization and ionization of both small and larger molecules, high accuracy, and sub-picomole sensitivity
Chertova et al. 2006
Electrospray ionization liquid chromatography tandem mass spectrometry (ESI LC/MS/MS)
Ionize samples to obtain peptide sequences, used with LC. Allows protein identification.
Ciborowski et al. 2007; Kraft-Terry et al. 2010; Wiederin et al. 2009; Ciborowski et al. 2007
Two-dimensional differential in-gel electrophoresis (2D DIGE)
Enables separating up to three samples with the same 2D gel according to isoelectric point and weight. Accurate analysis of differences in protein concentration between samples. Includes an internal standard. Difficult to detect membrane proteins and low molecular weight proteins
Kraft-Terry et al. 2010; Dukelow et al. 2007; Kadiu et al. 2009
Differentially expressed proteins in monocytes and MDM of patients with HAD
Patient sample
Proteome comparison
Differences in protein profiles
Differentially identified proteins
Related function
HIV-1-infected MDM from patients with or without CI
MDM lysate
Profiles of HIV-1-infected MDM from 22 patients: CI vs. non CI
177 peaks (2–80 m/z); 38 peaks differentially expressed between NC and CI
Luo et al. 2003
Protein fingerprints of MDM from HAD patient
MDM lysate
Profiles of HIV-infected MDM of one case study: HAD vs. non HAD
7 peaks (3–20 m/z): ↑ HAD
Wojna et al. 2004
HIV-1-infected monocytes/macrophages from NC and HAD patients
MDM supernatant
HIV-1-infected MDM: NC vs. HAD in 24 patients and 9 HIV seronegative controls
1 peak (↓ HAD)
Sun et al. 2004
HIV-1-infected monocytes from Hispanic women
Monocyte lysate
2D DIGE and LC/MS/MS
Monocytes from 11 patients with NC vs. HAD
18 spots significantly differentially regulated between NC and HAD
18; 6 selected for validation
Antioxidants, structural, regulatory, enzymes
Kraft-Terry et al. 2010
Differentially expressed proteins in HIV-1 "in vitro" infected macrophages
Proteome comparison
Differences in protein profiles
Differentially identified proteins
Related function
Proteome of HIV-1 ADA-infected MDM
Cell lysate
HIV-1 ADA-infected MDM vs. control
Up-regulation 2 peaks in HIV-1-infected MDM
Structural, regulatory, enzymes, HIV proteins
Carlson et al. 2004
HIV-1 NLAD8-infected MDM
HIV-1 NLAD8 virions produced by infected MDM
253 unique proteins
Cytoskeleton, adhesion, signaling, intracellular trafficking, metabolism, immune response
Chertova et al. 2006
Cytoskeletal transformation of HIV-1 ADA-infected MDM
HIV-1 ADA-infected MDM vs. control
21 peaks up- or downregulated
Kadiu et al. 2007
HIV-1 ADA-infected MDM in HBMEC proteins
Cell lysate
HBMEC co-cultured with control MDM vs. with HIV-1 ADA infected MDM
161 spots upregulated
Structural/cytoskeletal, regulatory, redox, enzymes, HIV proteins
Ricardo-Dukelow et al. 2007
47 spots downregulated
HIV-1 ADA-infected MDM secretome
1D electro-phoresis
HIV-1 ADA-infected MDM secretome
9 differentially expressed proteins
Cytoskeletal, enzymes, redox, immunoregulation
Ciborowski et al. 2007
Microglia astrocyte crosstalk
Cell lysate
HIV-1 ADA-infected microglia/absence of astrocytes
39 spots upregulated
Structural, regulatory, enzymes
Wang et al. 2008
24 spots downregulated
Effects of HIV-1 ADA in monocyte plasma membrane proteome
Cell lysate
HIV-1-infected monocytes vs. control
67 spots upregulated
Structural/cytoskeletal, regulatory, enzymes, oxidative stress
Kadiu et al. 2009
172 spots downregulated
MDM: uninfected vs. infected with HIV-1 SF162 and primary isolates from patients with NC or CI
Cell lysate
Control vs. NC
2 downregulated
Structural, replication, metabolism, signaling
Toro-Nieves et al. 2009
Control vs. CI
1 downregulated
Replication, chemotaxis, protein trafficking, apoptosis, redox
1 upregulated
Control vs. SF162
1 upregulated
Metabolism, replication, viral assembly, structural
NC vs. CI
1 upregulated
Common to SF162 and CI : 3
Structural, signaling
SF-162 vs. NC
3 downregulated)
Common to NC and CI : 2
Metabolism, viral proteins
Common to SF162, NC, and CI : 3
Rat microglia and astrocytes infected with VSV recombinant HIV-ADA
Cell lysate
HIV-1-infected microglia in presence of astrocytes
149 upregulated
Structural, regulatory, enzymes
Wang et al. 2008
360 downregulated
Monocyte cell line (THP-1) infected with HIV-1 HTLVIIIB
Cell lysate
Protein profile of HIV-1- infected undifferentiated monocytic THP-1 cells
26 HIV vs. uninfected; 9 downregulated and 17 up-regulated in HIV-infected THP-1 cells
Structural, regulatory, inflammation, signaling, antibiotic
Pathak et al. 2009
Matrix-assisted laser desorption ionization/time of flight (MALDI-TOF) functions similarly to SELDI-TOF as both are mass spectrometry based and are used for analysis of complex peptide mixtures (Table 1). The obtained peptide mass fingerprint is subsequently compared to virtual fingerprints of protein sequences in databases, and the top-scoring proteins are retrieved as possible candidate proteins (Gevaert and Vandekerckhove 2000). Using MALDI-TOF and liquid chromatography/mass spectrometry/mass spectrometry (LC/MS/MS), Chertova et al. (2006) characterized proteins in HIV-1 virions produced from MDM infected with HIV clone NLAD8, an R5 virus (Table 3). Their findings revealed proteins related to cell signaling, intracellular trafficking, cytoskeleton, and activation of immune response systems that contributed to understanding the viral assembly process in macrophages (Chertova et al. 2006). Other studies integrating MALDI-TOF and 2D electrophoresis have shown macrophage dysfunction related to inflammatory and infectious diseases (Dupont et al. 2004). These studies showed the first two-dimensional maps of the human macrophage and secretome with a tremendous utility for understanding the biological functions of this immune cell.
LC/MS/MS or tandem mass spectrometry is the most sensitive technique used to identify proteins present in a biological sample (Table 1). This method works by ionizing molecules and sorting and identifying them according to their mass-to-charge (m/z) ratios (Jemal and Xia 2006). The importance of protein identification lies in the fact that each protein has a unique cell function that may be altered when, for example, an obligate parasite such as HIV-1 interacts with the cell causing a proteome unbalance that may correlate with disease pathogenesis.
Current in vitro and ex vivo studies of HIV-infected macrophages were done mostly with non-quantitative or semi-quantitative proteomic methods (Tables 2 and 3). One limitation of the proteomics approaches described in studies of patient MDM has been the amount of protein required by gel-based studies (Luo et al. 2003; Wojna et al. 2004; Table 2). Another limitation has been the challenge in verification of proteins in finding antibodies that recognize the same epitopes detected by MS/MS (Kraft-Terry et al. 2010; Table 2).
Future proteomic methods for quantitative determinations of proteins and protein modifications
Stable isotope labeling of cells in culture (SILAC)
Cellular signaling and protein-protein interactions
Inexpensive. Can be used in dividing and non-dividing cells
Requires cells in culture
LC-based quantitative proteomics
Stable Isotope labeling post-culture with oxygen (18O)
LC-based quantitative proteomics
Can be applied after protein digestion. Can be done in low and high resolution mass spectrometers
Sophisticated statistical analysis
Jorge et al. 2009
Stable isotope labeling with isotope coded affinity tags (ICAT)
LC-based quantitative proteomics
Can be applied after protein digestion. Can be done in low and high resolution mass spectrometers
Missed identification of proteins with few or no cysteine residues, loss of information for post-translational modifications, and complicated interpretation of spectra due to the biotin group
Wu et al. 2006
Isobaric tags for relative and absolute quantitation (ITRAQ): 4plex and 8plex
LC-based quantitative proteomics
Can be applied after protein digestion. Can be done in low and high-resolution mass spectrometers. Most sensitive
Requires a Nano LC ESI MS MS QStar XL MS MS or LTQ-Orbitrap for analysis.
Song et al. 2008
Post-translational modifications (PTMs)
(a) Protein purification based in its affinity for metal ions
(a) More efficient for multiple phosphorylated proteins
(a) Non-specific binding of acid peptides to the matrix
Kota et al. 2009
(a) Immovilized metal affinity chromatography
(b) Phosphoprotein isotope coded solid phase Tag
(b) Abundance of phospho- proteins from digested complex mixtures using heavy or light stable isotopes
(b) Reliable quantitation of O-phosphorylations
(b) Cannot be applied to phosphotyrosine residues, bias toward pSer and pThr residues
Protein microarray
Protein abundance, interactions, affinities or activity
Accurate, rapid, low volume required
Database needed, depends on protein quality within the chip
Speer et al. 2005; Charboneau et al. 2002; Bertone and Snyder 2005
(a) Analytical
(b) Functional
(c) Reverse phase
In the SILAC method, experimental and control cells are labeled in cultures containing different types of media, one with a "heavy" arginine and lysine containing carbon (13 C) isotope and nitrogen isotope (15 N) and "light" and media containing l-lysine and l-arginine. Subsequently, cell extracts are combined and analyzed with a high-resolution MS/MS or a linear ion trap Fourier transform instrument (LTQ-FT) or LTQ-Orbitrap, recently developed by Thermo-Fisher (Table 4). Originally, the SILAC approach was recommended for dividing cells that can easily incorporate these amino acids in culture while undergoing several cell divisions (Mann 2006). However, recent studies have demonstrated that even primary can incorporate these isotopes in culture (Spellman et al. 2008). Therefore, this method could potentially be applied to obtain quantitative protein analysis of HIV-infected MDM in vitro and ex vivo.
Other stable isotope labeling methods that can be performed after protein digestion include ICAT, 18O, and ITRAQ (Table 4). ICAT is used to compare proteins that are labeled with light and heavy tags carrying a biotin moiety. After cation exchange chromatography, the mixed peptides are purified using immobilized avidin. Peaks corresponding to the same peptide are identified as doublets in the mass spectra due to mass differences in the light and heavy isotopes. The peaks' intensities correlate with the relative abundance of the proteins in experimental and control samples. The limitation of this method is missed identification of proteins with few or no cysteine residues, loss of information for post-translational modifications, and complicated interpretation of spectra due to the biotin group (Wu et al. 2006). In the 18O method, the protease catalyzes the incorporation of two 18O atoms at the carboxy terminal end of peptides and increases their mass by 4 Da. The labeled sample is combined with the non-labeled one, and both are processed and analyzed by MS as doublets (Jorge et al. 2009). Sensitive statistical methods have been developed to analyze 18O peptides with medium and high resolution mass spectrometers (Jorge et al. 2007). Large data sets have been generated by applying the 18O method from plasma exposed to lipopolysaccharides (LPS) and from burn patients (Qian et al. 2005, 2010).
ITRAQ is the most sensitive quantitative proteomics method currently available (Table 4). A comparison of ITRAQ with ICAT, 2-D DIGE, and ITRAQ revealed a larger number of proteins identified by ITRAQ (Wu et al. 2006). This technology employs a 4- and 8-plex set of amine reactive isobaric tags to label lysine side chains at the N-terminus in a digested mixture. The Q-TOF mass spectrometer and the LTQ Orbitrap that can perform higher energy collision-induced dissociation are necessary for ITRAQ analysis. The Q-TOF quantitative proteomics requires the MASCOT analysis program; Orbitrap uses Proteome Discoverer for analyses (Casado-Vela et al. 2010). Recent studies applied iTRAQ to determine the role of T regulatory (Treg) cells in the proteome of HIV-infected MDM (Huang et al. 2010). ITRAQ using the LTQ Orbitrap has also been recently applied to studies of plasma from the simian immunodeficiency (SIV) monkey model of HIV encephalitis in search of biomarkers for HAND (Pendyala et al. 2010).
Protein PTMs (hydroxylations, methylations, nitrosylations, thiolations, γ-carbonylations, glycosylations, and phosphorylations) can alter a protein's function, structure, location, and life span. In the past, irreversible protein modifications were assessed using 2D gel electrophoresis, and spots of interest were identified using immunological procedures, specifically, with antibodies against PTMs, such as nitrotyrosine (Dalle-Donne et al. 2006). Now PTMs can be analyzed using proteomic approaches, mostly MS/MS, but other techniques are also used (Table 4). Cysteine oxidation and thiolation can be directly and easily detected by MS approaches (Dalle-Donne et al. 2006). Numerous protein activities depend on phosphorylation or dephosphorylation, that can occur in serine, threonine, tyrosine, and histidine residues. Using databases, investigators can predict possible phosphosites, but not all phosphosites are physically available to be phosphorylated or they simply are not used. Some useful databases for prediction of phosphosites are PHOSIDA, a PTM database that can predict phosphorylations, acetylations, and N-glycosylations, and Phospho.ELM, a database designed exclusively for phosphosite prediction in eukaryotes. Ibarrola et al. (2003) used SILAC to quantitate the extent of phosphosites as well as to identify and quantitate novel phosphorylation sites. Other techniques such as immobilized metal affinity chromatography, which uses Ga3+ to enrich phosphopeptides from cell lysates and phosphoprotein isotope-coded solid-phase tag, which labels and enriches phosphopeptides from complex mixtures, have also been used for the proteomic analysis of phosphopeptides (Kota et al. 2009). More recently MS/MS/MS or MS3 methodologies have been applied to phophopeptide PTMs (Ulintz et al. 2009).
Another important feature of proteins is glycosylation. Proteins can be N- and/or O-glycosylated. N-gycosylation attaches to the nitrogen of asparagines or arginines; O-glycosylation attaches to the oxygen of serine, threonine, and tyrosine. Glycans can undergo changes from being very homogenous and simple to highly complex and heterogeneous structures. Because glycosylations are immunogenic, making a glycosylation profile is potentially useful for HIV vaccine development. Glycosylation of HIV proteins influences tropism, neutralization, viral infectivity, and viral processing (Graham et al. 2008). Go et al. (2009) used a combination of MALDI-MS and LC/ESI-FTICR MS, in addition to reverse-phase HPLC, to separate glycopeptides and to determine the glycosylation profile of two clade C recombinant HIV envelopes. Graham et al. (2008) assessed the glycosylation in all HIV and SIV proteins using 2D DIGE and either ESI-MS/MS or LTQ-ion trap-MS/MS.
There are three different types of protein microarrays: analytical, functional, and reverse phase. Analytical microarrays are mostly used to detect affinity and expression (Table 4). Usually antibodies are attached to a microscope slide to detect the protein or probe (Bertone and Snyder 2005). Functional microarrays, which contain full-length functional proteins or domains, are used to study activity or interactions: protein–protein, protein–RNA, protein–phospholipid, or protein–ligand (Bertone and Snyder 2005). The reverse phase microarray uses denaturated protein lysates fixed to the slide and then probed, whereas conventional microarrays immobilize the antibodies or probes. The reverse-phase technique has been used to detect either quantitative protein changes in healthy and diseased tissue (Charboneau et al. 2002) or PTMs (Speer et al. 2005).
Proteomics of monocytes, macrophages in HIV replication
HIV-1 infection alters the proteome and secretome of macrophages (Tables 2 and 3). How HIV-1 affects the intracellular and secreted proteins is incompletely understood. Several studies have shown that shortly after the viral protein gp120 interacts with the CD4 and the seven transmembrane G protein-coupled receptors, CCR5- or CXCR4-specific signaling pathways such as the intracellular calcium mobilization, PI-3K activation, phosphorylation of mitogen-activated protein kinases (MAPKs), ERK1/2, JNK/SAPK, and p38 are engaged (Lee et al. 2005; Cicala et al. 2002). This HIV-1 activation of MAPKs leads to up- and downregulation of several genes and consequently influences the transcriptional and post-transcriptional events related to the formation of important cellular and viral proteins (Wahl et al. 2006). Consequently, the gp120-mediated activation of these protein kinases leads to the formation of pro-inflammatory cytokines and chemokines such as TNF-α and MCP-1, respectively, which can influence HIV-1 replication and cell functions (Del Corno et al. 2001; Lee et al. 2003). In monocytes, one of the functional consequences of viral exposure is the facilitation of protein transformation from the cytosol to the plasma membrane (Kadiu et al. 2009). The dysregulation of macrophage function is mediated by HIV envelope gp120 protein with cellular co-receptor activation that results in the inflammatory responses observed in HAD pathogenesis (Gendelman et al. 2006). Some reports have shown the involvement of CXCR4 in the gp120 macrophage/microglia activation with the induction of pro-inflammatory cellular pathways that may contribute to AIDS dementia (Bezzi et al. 2001).
Microarray studies or MDM transcriptome have provided larger data sets for pathway analysis than proteomics (Brown et al. 2008; Giri et al. 2009; Tsang et al. 2009; Ancuta et al. 2009; Van den Bergh et al. 2010). A macrophage pro-inflammatory M1 phenotype after HIV infection was demonstrated with increased mRNAs for calcium upregulation, cell cycle, apoptosis, MAPK, and cytokines/chemokines (Brown et al. 2008). Since many of the over-expressed mRNAs may not represent the final protein product owing to delays in translation, the authors were able to validate ∼18% of these proteins including calcium related and pro-apoptotic proteins (caspase 7). They concluded that HIV primes macrophages to a pro-inflammatory M1 phenotype that is independent of toll-like receptor activation (Brown et al. 2008). However, subsequent studies on the HIV-infected MDM transcriptome had the opposite result, an anti-inflammatory phenotype (Giri et al. 2009; Tsang et al. 2009). Agreement with some aspects of these studies related to the mechanisms of evasion of innate immunity by HIV interaction with MDM has been confirmed by proteomics studies, with the activation of oxidative stress associated proteins, inflammation, and signaling cascades (Ciborowski et al. 2007; Luciano-Montalvo and Meléndez 2009; Toro-Nieves et al. 2009). Moreover, some inflammatory proteins identified from a large data set generated by quantitative proteomics from plasma exposed to LPS and from burn patients (Qian et al. 2005, 2010) are also present in monocytes from HIV-infected women (Velazquez et al. 2009; Kraft-Terry et al. 2010).
Proteomics of patient cells has spearhead research toward the diagnosis and treatment of HIV-related disease (Luo et al. 2003; Wojna et al. 2004; Kraft-Terry et al. 2010). These studies include the discovery of proteins as candidates for biomarkers for HIV associated cognitive dysfunction at different stages (Velazquez et al. 2009) as well as other HIV-associated cellular changes and pathologies (Rasheed et al. 2009). The validation of the protein candidates as biomarkers in different populations and the discovery of novel biomarkers and possible targets for therapies by more sensitive and quantitative proteomics approach remains to be studied.
Proteomics of placental macrophages and MDM: a model of HIV persistence
The HIV-1 persistence in the monocyte–macrophage lineage has been explained by two theories: the lack of proviral gene expression (latency) and continuous viral expression without cytopathic effects (ongoing replication; Le Douce et al. 2010). Several traits of monocyte–macrophage lineage make it an important source of viral persistence: the HIV-1 infection is generally non-lytic for these cells; they can harbor viruses longer than CD4+ T cells; and cells from monocyte–macrophage are more resistant to cytopathic effects. For example, microglial cells in the brain can produce viruses during their total lifespan (Williams et al. 2001). However, not all of the mononuclear phagocyte populations equally support viral growth. The placental macrophages (PM) are a target for HIV-1 infection inside the placenta, but these cells are also barriers to primary infection by HIV-1. They can be productively infected by laboratory strains and clinical isolates (Fear et al. 1998; Kesson et al. 1993, 1994; McGann et al. 1994; Meléndez-Guerrero et al. 1994; Meléndez et al. 2001; Plaud-Valentin et al. 1999), but they are less susceptible to HIV-1 than are MDM. Although PM express lower levels of HIV-1 receptor CD4 and co-receptors CCR5 and CXCR4 than do MDM, the receptor expression is not the sole determinants of HIV replication in these macrophages (Luciano-Montalvo et al. 2008; Torres et al. 2001; Melendez et al. 2001). In contrast to the consistent levels of replication exhibited by MDM, HIV replication in PM reaches a peak of viral replication around 3–7 days after infection and then decreases (Kesson et al. 1994). Furthermore, levels of replication in PM measured by production of HIV-1 viral capsid protein (p24) are ten times lower than in MDM (Plaud-Valentin et al. 1999), a finding that cannot be explained solely by co-receptor expression. The HIV-1 persistence in MDM and the low permissiveness exhibited by PM make them good candidates for the study of cellular factors involved in restriction of viral replication.
Intracellular and secreted proteins associated with HIV restriction in Placental Macrophages as compared with MDM
Protein name
Total peptides detected in sequencing
Peak intensity in SELDI-TOF compared with MDM
From whole cells lysates:
Cytoskeletal 14-like protein
Luciano-Montalvo et al. 2008
SH3 glutamic acid rich like protein 3
Luciano-Montalvo et al. 2008
Protein S-100 A8 (calgranulin A)
Luciano-Montalvo et al. 2008
10 kDa heat shock protein
Luciano-Montalvo et al. 2008
Cystatin B
Luciano-Montalvo et al. 2008
Cytochrome C
Luciano-Montalvo et al. 2008
SH3 glutamic acid rich like protein 1
Luciano-Montalvo et al. 2008
Luciano-Montalvo et al. 2008
Protein S-100 A8 (calgranulin B)
Luciano-Montalvo et al. 2008
Luciano-Montalvo et al. 2008
Luciano-Montalvo et al. 2008
Superoxide dismutase CuZn
Luciano-Montalvo et al. 2008
From macrophage secretome:
Fatty acid-binding protein 3
Garcia et al. 2009
Garcia et al. 2009
Garcia et al. 2009
Peroxiredoxin 5
Garcia et al. 2009
Proteomics platforms were also used to identify secreted factors that might be associated with HIV-1 restriction in PM and HIV persistence in MDM. The screening of the secretome using SELDI-TOF followed by LC/MS/MS confirmed that PM and MDM were secreting different proteins (Garcia et al. 2009). After sequencing and identification, several proteins were validated for differential expression in PM and MDM by Western blot analysis. This study reported that peroxiredoxin 5 is significantly more abundant in PM than in MDM supernatants. This protein is important in the cellular antioxidant mechanisms, and other members of its family have shown antiviral activity. Furthermore, peroxiredoxin 5 has been found to negatively regulate TNF-α signaling, and so it could also suppress NF-κB activity and HIV-1 replication in PM (Garcia et al. 2009). Cystatin B was also found to be significantly more abundant in MDM than in PM supernatants (Garcia et al. 2009). These data confirmed the higher levels of cystatin B in MDM cell lysates reported by Luciano-Montalvo et al. (2008). We suggested that high expression of peroxiredoxin 5 could be one of the mechanisms by which HIV-1 replicates inefficiently in placental macrophages, whereas low expression of cystatin B could impair their capacity to replicate HIV-1, but the mechanisms remain under investigation (Luciano-Montalvo et al. 2008; Luciano-Montalvo and Meléndez 2009; Garcia et al. 2009). Interestingly, peroxiredoxin 5 was one of the proteins identified as downregulated in recent proteomics studies of monocytes derived from women with HAND, a finding that suggests chronically activated monocytes act as a Trojan horse that carries HIV to the brain (Kraft-Terry et al. 2010).
Taken together, proteomics approaches have been used to elucidate the proteome and secretome of tissue macrophages in order to determine how these new players interact with proteins related to HIV-1 replication.
Proteomics of monocytes, macrophages, and microglia in HIV dementia
After HIV-1 enters the CNS, either as a cell-free virus or in infected macrophages, several signaling pathways are activated with the release of inflammatory products, chemokines, and neurotoxins (cellular and viral) that attract other cells to the site of infection, resulting in an increase of intra neuronal calcium and subsequently producing neuronal death (Gendelman et al. 1997; Li et al. 2005; Persidsky et al. 1997). This damage results in psychomotor slowing, memory impairment, and brain atrophy (Anderson et al. 2002). In fact, apoptosis of neurons and astrocytes has been detected in brain tissue autopsies from AIDS patients with HAD (Adle-Biassette et al. 1995).
The communication between the nervous and the peripheral immune systems is an important pathway for activation of macrophages during HAD (Ballabh et al. 2004). The response of the CNS to systemic immune challenge results in brain inflammation caused by infected monocytes/macrophages trafficking into the brain from the periphery, producing disruption of the BBB by oxidative proteins (Gonzalez-Scarano and Martin-Garcia 2005).
Luo et al. (2003) identified several protein peak differences between uninfected and HIV-1-infected MDM linked to the pathogenesis of HAD. We identified 177 protein peaks in lysates of HIV-1-infected MDM from Hispanic patients receiving highly active antiretroviral treatment, among which 38 peaks were related to cognitive impairment (Table 2). These data supported the hypothesis of the emergence of a monocyte subset in patients at the onset of dementia. Afterwards, our collaborators examined the proteome of HIV-infected MDM (Carlson et al. 2004). They used SELDI-TOF to detect 58 differentially expressed proteins in MDM after in vitro infection with HIV-1 ADA, a macrophage tropic R5 virus (Gendelman et al. 1988) (Table 3). Microsequencing of these proteins by LC/MS/MS permitted identification of important cellular proteins such as ß-actin, annexin A5, vimentin, l-plastin, and desmoyokin, in addition to viral proteins such as gp120 and vif (Table 3). The fact that many of these proteins are related to changes in cellular structure and functions supports the hypothesis of alterations in the cell after HIV infection (Carlson et al. 2004). Sun et al. (2004) used the same approach to compare profiles of secreted MDM proteins from HIV-seropositive individuals with normal cognition, HAD, and HIV-negative controls. This group found a decrease in secretion of lysozyme in MDM supernatants from individuals with HAD, thus demonstrating alterations in cell function as a consequence of HAD (Table 2).
The SELDI-TOF approach was used initially to profile the defining characteristics of disease progression (Luo et al. 2003; Wojna et al. 2004). However, the spectra developed with this approach were not comparable among instruments at various institutions and did not allow us to identify the proteins that were differentially expressed. Therefore, identification of the proteome was the next approach to be used, first by 1D gel electrophoresis and sequencing by LC/MS/MS. Using this approach, studies aimed at deciphering the effect of HIV variants from patients with cognitive impairment on the macrophage proteome, our group identified 20 proteins related to apoptosis, chemotaxis, inflammation, and redox metabolism (Toro-Nieves et al. 2009; Table 3). Some of the identified proteins in HIV-infected MDM, including ferritin, ubiquitin, and apoptosis-related proteins, have been identified in macrophage-induced inflammation (Xue et al. 2008). These studies showed that the macrophage's proteome can change depending on the infecting viral strain by stimulating an inflammatory and pro-apoptotic phenotype. The mechanisms by which these viruses affect the macrophage proteome are currently being investigated.
After HIV-1 infects macrophages, several signals are activated causing morphological and functional changes to the cell (Chertova et al. 2006; Ciborowski et al. 2007; Kadiu et al. 2007; Lee et al. 2003). By using SELDI-TOF, LC/MS/MS, and Western blots, culture fluids derived from in vitro HIV-1-infected MDM identified 15 differentially expressed proteins, including cytoskeletal proteins (Kadiu et al. 2007; Table 3). Their findings demonstrated HIV-1 can drive the cell to a permissive state for viral replication or can enhance phagocytosis and intracellular microbial killing. This group subsequently demonstrated how HIV-1 transforms the monocyte plasma membrane proteome by their use of cell surface labeling with fluorescent dyes followed by 2D DIGE and LC/MS/MS analysis (Kadiu et al. 2009). They found that 53% of HIV-1-induced proteins were associated with the plasma membrane, cellular activation, and oxidative stress, which are processes related to HAD neuropathogenesis (Table 3). Using a similar proteomics platform, Ricardo-Dukelow et al. (2007) established how HIV-1-infected MDM induced upregulation of 161 human brain microvascular endothelial cell proteins that are related to important cellular processes such as metabolism, transport, structural changes, and regulation. Their findings support the role of HIV-1-infected MDM in BBB dysfunction, which contribute to HAD (Table 3).
Despite all the confirmed information about the interactions between HIV-1 and macrophages, the exact pathways of macrophage activation after HIV-1 infection remain incompletely understood. Using proteomics, other studies have shown that the macrophage secretome is affected by HIV-1 infection (Ciborowski et al. 2007) where cystatins B and C, l-plastin, superoxide dismutase, and α-enolase were identified preferentially from HIV-infected cells (Table 3).
Taken together, proteomics studies clearly demonstrate the effects of HIV-1 on the macrophage activation, structure, and function. These results are not distant from microarray studies where an inflammatory M1 HIV macrophage predominates following HIV infection with activation of apoptotic signaling that is modulated after time in culture (Brown et al. 2008).
Wang et al. (2008) established a proteomic modeling for crosstalk between HIV-1-infected microglia and astrocytes. Using 2D DIGE, Western blot, and LC/MS/MS they detected changes in the microglial cytoskeleton and alterations in the microglial–astrocyte communication. These observations provided unique insights into glial crosstalk and how it is related to HAD.
In the neuroAIDS field, proteomics approaches have also been used for studying the CSF (Yuan and Desiderio 2005; Laspiur et al. 2007; Rozek et al. 2007). Currently, some of the proteins uncovered in HAND that are common to CSF and HIV-infected MDM are subject to functional studies including cystatins, cathepsins, and SOD (Kadiu et al. 2009; Luciano-Montalvo and Meléndez 2009; Rodriguez-Franco et al. 2010; Rivera et al. 2010). These proteomics studies have helped to elucidate important mechanisms in HIV infection, MDM activation, and disease.
Conclusion and future directions
This review provides an overview of the knowledge about the function of HIV-infected monocytes/macrophages that has been obtained by using various proteomics approaches. It includes studies from the pre-proteomics and post-proteomics eras with a detailed description of the proteomics approaches used (Table 1). These methods have helped to identify the differences in proteins that are resolved in a gel electrophoresis approach and, if coupled to fluorescence tagged markers as in 2D DIGE, have helped to quantify their relative abundance in experimental and control samples.
Proteomic studies applied to the discovery of potential biomarkers from ex vivo studies of macrophage proteins derived from patients with HIV-associated neurocognitive dysfunction have been cumbersome and provide few protein candidates because of the limited number of MDM available for gel-based studies. However, these studies have yielded promising candidates and spearheaded the discovery of new disease mechanisms (Table 2).
Proteomic studies applied to in vitro studies of HIV-infected macrophages have been facilitated by an abundance of macrophages from leukopheresis and elutriation provided by Dr. Gendelman's laboratory (Table 3). These studies have revealed the presence of proteins related to inflammation, oxidative stress, apoptosis, cytoskeleton, and signaling pathways. Many of these proteins are being tested in functional studies and could be potential avenues for increasing our understanding of the biology of HIV to eventually eradicate cellular reservoirs and add new targets for therapies and vaccines.
Novel system biology and proteomics approaches for quantitative proteomics have been reviewed (Table 4). These methods will likely lead to more complex proteomes of HIV-infected MDM to facilitate the discovery of increased number of biomarkers for diagnosis and therapies against HAND and other infectious and neuroinflammatory diseases, which will further the understanding of the role of macrophages in different diseases.
This work was supported in part by grants from the National Institutes of Health to Dr. Melendez (R01MH083516, U54NS4301, GM08224, G12RR03051) and to Krystal Colon and Eillen Rodriguez-Franco (GM061838) and by institutional funds. We thank Alexandra Cabán for her help in manuscript writing, sponsored by Melendez R01MH083516.
Conflict of interest disclosure
The authors report no conflicts of interest.
Open Access
This article is distributed under the terms of the Creative Commons Attribution Noncommercial License which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited.


text:
This item is temporarily out of stock. However, we can notify you as soon as this product becomes available.
Thank you
We have received your request and you will be notified as soon as this product becomes available.
In order to keep our prices as low as possible, all CreateForLess order subtotals must be at least $10. This helps us save on labor costs when packing your order so we can extend the savings to you!
Description: Authentique Paper is perfect to use for scrapbook layouts, memory albums, greeting cards, invitations and more. Reliable paper from the Durable collection is double-sided cardstock. One side has stripes in Tan, Ivory, and Light Blue. The reverse side is Light Teal with large Tan polka dots. All edges have a distressed look. Acid-free, lignin-free and laser friendly. 80 lb. 12 x 12 inch.
See more Authentique Durable Collection.
Review this product and Enter
to Win a $100 shopping spree!

subset: commoncrawl


text:
�� � � ��
Cycle Works is proud of our active community support programs. The nature of our business dictates the support path that our company must follow. We focus our support on local organizations dedicated to the following causes:
If you feel that Cycle Works' support will enhance your event, please fill out the following form and be sure to answer ALL questions in the Summary of Donation Request field.We ask that you please take the time read and answer the questions instead of just sending a form letter as this helps us better choose charities and events that align with our corporate values.
Please be aware that requests for donations will only be considered if this form is completely filled out with all questions answered and at least 30 days notice is given before the event.
Cycle Works firmly believes that any cause that enhances our community deserves our consideration and all efforts will be made to help as many as we can. Thank you for including us and we wish you the best of luck!
� � ��
Calgary, Alberta
Edmonton, Alberta
Acheson, Alberta
Okotoks, Alberta
Red Deer County, Alberta


text:
MEDICA.de spoke with adjunct professor Dr. Matthias Orth, MD, Head of Laboratory Medicine at the Marienhospital in Stuttgart, about the risks of direct-to-consumer testing.
In the past, the analytical emphasis of DTC testing was on clinical and chemical testing. What research areas do these tests cover nowadays?
What role does the Genetic Diagnostics Act play here?


text:
MMA Forum - UFC Forums - UFC Results - MMA Videos (http://www.mmaforum.com/forumindex.php)
- MMA Smacktalk (http://www.mmaforum.com/mma-smacktalk/)
- - I Think Arlovski Is Gonna Kill Himself.. (http://www.mmaforum.com/mma-smacktalk/87522-i-think-arlovski-gonna-kill-himself.html)
I Think Arlovski Is Gonna Kill Himself..
after his ko loss to brett rogers arlovski admitted he was suicidal and even played a game of russian roulette. now after 4 losses in a row, 3 of them being brutal ko's, i cant imagine his mental state. someone put this man on suicide watch
lets just hope he make the right decision.
I genuinely worry but somehow expressing that in a emoticon seems wrong.
As far as skillset and technique goes I'm generally always impressed with Andre, just like Goulet he doesn't have the chin for fighting, really is a shame like.
Hope he is okay.
Arlovski should hang it up now. For is own health if nothing else.
He has one of the scarriest KO'd faces when he's on the mat as well. Always looks bad.
This might sound corny, but I wrote him an email via Myspace...cuz I remember he broke up with his Playboy model girlfriend around the same time. Any man would have suicidal tendencies with a break up like that! That's no laughing matter. Forget about the KOs. Imagine the ass he's missing...
Any proof behind these stories?
Andrei is way stronger than you guys give him credit for. He's already stated he is not retiring and he will continue to fight. As for that roulette story, can you prove he said that?
|All times are GMT -4. The time now is 03:14 PM.
Powered by vBulletin Version 3.8.7 , Copyright ©2000 - 2017, Jelsoft Enterprises Ltd.
vBulletin Security provided by vBSecurity v2.2.2 (Pro) - vBulletin Mods & Addons Copyright © 2017 DragonByte Technologies Ltd.
User Alert System provided by Advanced User Tagging (Pro) - vBulletin Mods & Addons Copyright © 2017 DragonByte Technologies Ltd.


text:
Your child's age at the date of departure for each flight on your journey determines whether or not they are eligible for an infant or child fare.
|Age range
|Acceptance criteria
|Fare type eligibility
|8 days until 2nd birthday
One infant with one adult
Infant fare if travelling on a parent's lap
Child fare* if travelling in a separate seat or car seat*
Two infants with one adult:
If both infants are less than 12 months old, they need to be accompanied by a second adult passenger (at least 16 years of age).
|Child fare* if second infant is over 12 months old and is travelling in a separate seat or car seat*
Three infants with two adults:
If all three infants are less than 12 months old, they need to be accompanied by a third adult passenger (at least 16 years of age).
Note: Any additional infant (e.g. 4 infants and 3 adults) must be accompanied by an additional passenger (at least 16 years of age). Booking details of the accompanying passengers must be provided at the time of reservation.
Child fare* if third infant is over 12 months old and is travelling in a separate seat or car seat*
|2 to 11 years
Children between 2-5 years should be accompanied by an adult over 16 years of age when travelling.
Children between 5-12 years can travel as unaccompanied minors. Click here for details on unaccompanied minors.
Child fare*
|12 years and older
Adult fare
*Car seats may be used for infants and children aged between 6 months and 36 months only.
Infants less than 8 days old may be accepted to travel under the following circumstances:
|Fare type
|10% of the adult fare
|Varies per flight route*
*Child fares are available on select routes. For all other routes, children must travel at the adult fare.
|Checked baggage
|First Class
|Business Class
|Economy Class
|Flights to and from Argentina, Brazil, Canada, and the USA
|One piece, not to exceed 23kg (50lb) or a maximum dimension of 115cm (45in)
|Flights to and from all other destinations
|10kg (22lb) not to exceed a maximum dimension of 115 cm (45in)
One stroller, collapsible carrycot, or pushchair per infant is accepted without charge.
Children and infants travelling on a child fare are eligible for the same baggage allowances as adults.


text:
Sri Lanka's east coast, running from Trincomalee in the north to the grasslands of Yala in the south, has good reason to feel optimistic about the future
Boeing 777 was in a 'high and increasing rate of descent' in its final moments, indicates latest analysis of satellite data by Australian Transport Safety Bureau
Poor weather has, until now, prevented ships from deploying the drone. With the weather improving, a video camera-equipped vehicle will scrutinize the sonar contacts
Two men stole Buddy from a conservation facility and released him at a beach — his first time in the sea. He is likely to die there, officials say
Families of the lost plane's 239 passengers and crew were angry over the decision to stop what is already the most expensive search in aviation history
If confirmed to come from the missing aircraft, the discovery would be the first part of MH370 found east of the main search zone in the southern Indian Ocean
Seychelles is nothing less than another Galapagos, with wondrous plants and animals — species you won't find anywhere else, writes Suzanne Morphet
With about 15,000 square kilometres left to scour of a 120,000 square-kilometre search area, there's a 'decreasing possibility' of success, Martin Dolan said
Investigators looking for MH370 found a 19th century ship in December after already having found one last March. The search for the missing plane continues
Warning — Video contains graphic content. Law enforcement agencies only learned of the murders when the video was found on a cellphone left in a taxi in Fiji


text:
Overseas investors bought Indian equity derivatives worth 41.22 billion rupees ($677.4 million) on Tuesday, NSE data shows.
The amount included 28.53 billion rupees worth of index options.
"This kind of buying in Nifty options happened in March and April and is pointing towards a big move on the long side even from record highs," says a derivatives analyst at a foreign broker.
Data shows 2 million August put contracts added for NSE at 7,900 level.
Concentration of put contracts has also shifted from 7,600 to 7,700 for the August series, implying a support around 7,700 levels.
Data also shows 1.4 million call contracts added at 8,000 level and 0.9 million for 8,100 August strike.


text:
If sport is war without the shooting, tomorrow's World Cup final between Spain and the Netherlands in South Africa picks up a most venerable conflict.
These two European superpowers went at it for 80 years back in the 16th and 17th centuries. Perhaps we should place our bets on Sunday's clash in Soccer City going to extra time. But who should England get behind on the historical record? It's a tough one. England sided with the Dutch against the Spanish in the Eighty Years' War. But, not long after, Cromwell was tangling with these Protestant cousins.
Spare a thought, too, for our Deputy Prime Minister. Nick Clegg has a Spanish wife and a Dutch mother. Perhaps Howard Webb will not be the only Englishman playing referee tomorrow.
It would, of course, have been good if England had made it to Johannesburg for tomorrow's final. But we have to admit that it is rather nice to be a neutral, for a change, as old empires clash. Hup Holland! Viva España! And may the best team win.
- More about:
- Howard Webb
- Referee
- Southern Europe
- Spain


text:
Welcome to MotoHouston.com! You are currently viewing our forums as a guest which gives you limited access to the community. By joining our free community you will have access to great discounts from our sponsors, the ability to post topics, communicate privately with other members, respond to polls, upload content, free email, classifieds, and access many other special features. Registration is fast, simple and absolutely free, join our community!
If you have any problems with the registration process or your account login, please contact us.
|Like us on Facebook! Regular shirt GIVEAWAYS and more
Share This Thread:
|Subscribe to this Thread
|Thread Tools
Senior Member
Join Date: Nov 2008
Feedback Rating: (0)
Posts: 136,324
Review Revit Replica onepiece suit 79999
We test the topend suit from Revit
THIS is the top-of-the-range one-piece leather suit from Rev'it, which the firms says is the product of its experience in MotoGP with Alvaro Bautista, Danilo Petrucci and Alex Rins.
Called the Replica, it comes in three eye-catching colours, but protection is the most important thing here, and you'll find CE Level 2 (the highest) armour in the shoulders, elbows, knees and hips.
Hip protection isn't something I've ever seen in any of the leathers I've worn, and it seems like a no-brainer – who wouldn't want additional protection in this area? I don't think I've ever come off a bike without my hips hitting the ground.
The armour attaches to the inside of the suit with Velcro, so its position can be adjusted for optimum fit. The inside of the armour is well padded with foam, which makes it comfortable.
The whole suit is quite comfortable. Usually when I get a new suit it takes a little while to break in, like a pair of boots, but I found this one comfortable straight away. The Kevlar stretch panels provide unrestricted movement in all the right places and the neoprene cuffs give a comfortable fit and low profile that makes it easy for gloves to slide over.
Elasticated panels on the backs of the shoulders, above the elbows and knees make for ease of movement. There's another elasticated panel across the lower back which, unusually, continues around to the front of the hips. It might not sound like much but it really makes the suit more comfortable in a tuck.
The first time I wore this suit I was testing in Spain and the ambient temperature was 34C. As you'd expect, I was warm at a standstill, but on the move the airflow was great thanks to the perforated chest area. It felt like it was sucking in air.
The speed hump can double as a water container, but left empty it allows air through to your back, which I could also feel. It lets in so much air that you'd definitely need a warm base layer in cooler temperatures. The perforations are located away from the seams, to retain the latter's integrity.
There's a removable washable lining. If you sweat a lot, like me, that means no more excuses for stinking the house out.
The Replica is an unashamed race suit and among its more technical features are the replaceable rubber grips on the insides of the knees. I loved these as they really helped me grip the tank. I'm an a big fan of grip patches on the tank and don't usually like riding without them, so a similar effect built in to a suit is a huge bonus. My only criticism is that I'd like the suit's grips to have larger surface areas.
There are hard sliders at the elbows, shoulders and knees, which are designed to absorb some impact as well as abrasive forces in a crash.
There's also a built-in foam tailbone protector - something that sounds uncomfortable but which I never thought about while riding. You can add a built-in back protector as an option. I wear a separate heavy-duty back protector, which fitted under the suit no problem.
The Replica is very comfortable, with a high level of technical protective features and airflow in the right places. At £800, it's competitively priced, too.
Related Content
New: Aerostitch Roadcrafter one-piece suit
New: Tecnik Viper one-piece suit
New: Richa Rebel two-piece leathers
Showcase: Visordown's Top 3 Retro leathers
Used Review: Berik 3256 one piece leathers
|Similar Topics
|Thread Starter
|Last Post
|Dainese valentino rossi replica repsol suit NEW!!
|Off Topic
|My Spartan Leather custom suit review!
|Gear Talk
|Thread Tools


text:
Need an LH Test in Sonora, CA? Simply order online 24/7 and get a fast, affordable LH Test at a lab near you (Find More Locations):
What is an LH Test?
The LH Test, also known as a Luteinizing Hormone test, is a lab test that measures the LH level in the body. The LH Test is used to help guide fertility decisions for people in Sonora, CA.
How much does an LH Test cost?
Our LH Test price includes the doctor's order that you need to get tested in Sonora, CA, all lab fees and taxes, and a pdf copy of your results. For current prices, CLICK HERE.
What does an LH Test report look like?
For an example of an LH Test sample report, please CLICK HERE.
We are delighted to offer the LH Test in Sonora, CA.
LH Test in other cities near Sonora, CA:Avery | Big Oak Flat | Chinese Camp | Columbia | Douglas Flat | Hathaway Pines | Jamestown | Mi Wuk Village | Moccasin | Murphys | Soulsbyville | Standard | Tuolumne | Twain Harte | Vallecito


text:
Twelve hours later, southeast England had been laid to waste by the most powerful storm for two centuries. Winds of up to 100mph ripped in from the south coast, shearing off roofs and flattening fences and trees; power lines crashed and whole villages were cut off. Nineteen people died, the number mercifully reduced by the fact the storm struck during the early hours of the morning of 16 October 1987.
The iconic image of the storm, however, became the trees: 15m conifers, oaks and beeches bent to the hurricane-force winds and found they could not stand again. Trees which had stood through the Industrial and French revolutions, the battles of Trafalgar and Waterloo, the invention of the motorcar and two world wars and had withstood two or three centuries of floods, droughts and mini-ice ages finally crashed under the force of the Great Storm, metres-high root plates towering over the men who came to saw them up. Part of Britain lost a generation of trees, and with them ecosystems of bugs, plants and animals that loved their shady undergrowth. Most famously, the village of Sevenoaks in Kent was left with only one of its world-famous trees standing.
Perhaps the biggest-affected landowner was the National Trust - the custodian of hundreds of historic houses, gardens and woodlands - which woke to find 50 of its properties in the east and southeast of England damaged by the storm. The Trust alone estimated that it lost a third of a million trees.
As the charity's headquarters began to piece together the historic scale of the destruction, it hired a helicopter and sent the now head of gardens and parks, Mike Calnan, and photographer Mike Howarth up into the steely autumn sky to record how the storm had changed entire landscapes. This summer, 20 years later, Calnan and Howarth returned to the air to revisit the once windblasted scenes and record their regeneration.
What they found shows an almost unthinkable rejuvenation: the flourishing of a 20-year restoration programme, one which at some properties is still in the finishing stages. But the pictures show something else, too - the storm-devastated scenes, the juvenile growth of replanted properties, and the virility of places they left alone are all reminders of the fragility of man's control over nature.
'It takes less than a minute for the tree to go down, but 200 to 300 years to grow back to the size it was,' says Ray Hawes, the Trust's head of forestry. 'Until then it was almost like we felt we could control what we wanted to grow, when we felled them. The storm came along and we realised we couldn't.'
The results have led the National Trust, one of Britain's biggest landowners, to change the way it manages its land in important ways - lessons that could be invaluable if, as expected, global warming brings more such great storms. 'In the long history of land management in this country, over hundreds of years, a storm like that is a minor event,' says Calnan. 'It's about adapting to change: in the 17th century, gardens were about imposing your will on nature; now in terms of how we're making the most of - even conserving - great landscapes, it's about accepting that change is inevitable.'
Michael Fish was, he claimed, quoted 'completely out of context'. The hurricane he referred to was heading for Florida days before. The Met Office admits, though, that it got the forecast terribly wrong: forecasters had tracked the storm across the Atlantic and saw it gathering force as it spun over the Bay of Biscay. But they mistakenly thought it would clip the southeast corner of Britain, taking most of its destructive power across the Channel.
Later that night, duty forecasters at the Met Office's Bracknell HQ realised their mistake and alerted emergency services. An hour later, around midnight, the storm made landfall on the south coast and began to push a path north and east, reaching London at about 2am and finally blowing out over East Anglia just before dawn.
Just outside the storm zone in Swindon, and before the advent of 24/7 news coverage, Calnan arrived at the National Trust's head office later that morning oblivious to the events of the night before. 'The phone rang and a gardener said: "I have had a few trees down", then another call came through saying: "I have had a lot of trees down",' he recalls. 'Another gardener was almost in tears and said: "My garden's gone." Then I started to realise something major had happened and listened to the radio, and realised we had missed the great once-in-300-years event.'
The list of National Trust properties blown through by the force-12 storm includes some of the country's most famous historic homes and gardens: the Elizabethan castle at Sissinghurst; Capability Brown's landscaped gardens at Petworth House; Sir William Churchill's family home at Chartwell, and the prized plant collection at Nymans Garden. Probably the worst affected were Chartwell, the pleasure gardens at Sheffield Park, and Toys Hill woodlands high on the North Downs, where up to 98 per cent of the beech and oak woodlands was, says Calnan, 'obliterated as far as the eye can see'.
When the weather cleared enough a few weeks later to take the helicopter up, Calnan remembers their first sight of Toys Hill and the exotic neighbouring Emmetts Garden: 'There had been a continuous chatter, then we just went silent. The pilot hovered about 100ft above Emmetts; we just stared, trying to believe what we were seeing, which was almost like an atom bomb blew above ground level... This garden was completely flattened, like some giant had put their foot on it and squashed it.'
When they took stock, the Trust realised that the damage would cost millions of pounds to put right: drainage pipes to formal gardens had been crushed and ripped out of the ground, carefully landscaped pleasure grounds such as Sheffield Park were strewn, and fabulous plant collections blown about, scattering valuable genetic material and generations of history of Britain's dual obsessions with global expansion and collecting exotic species. 'They are more or less works of art,' says Calnan, 'as well as plants of great historical and botanical interest.'
More than the individual trees and shrubs, though, whole ecosystems were lost as wildlife which thrived in the dense shade and even temperatures under tree canopies was exposed to summer sun and winter frost; bluebells and azaleas withered, sparrow hawks and roe deer fled; in their place grass and heather began to grow, and butterflies and nightjars thrived. 'Before the storm, if you were walking all day you had a job to find anywhere to walk in the sun,' says Alan Bradford, the only gardener left at Sheffield Park who was working there during the storm. 'Even now you have a job to find anywhere to walk in the shade.'
Good things appeared, too: new vistas were revealed, relics of garden history uncovered, and gardeners and foresters had once-unthinkable opportunities to replace over-mature trees they had felt unable to cut down for sentimental reasons and because of the fear of public outcry.
But putting right the damage to centuries-old trees and gardens was going to take many years. 'Working for the National Trust you're dealing with growth, decay and death all the time,' says Calnan. 'You're also thinking ahead to ensure the propagation of the character of the garden and landscape. That's what we do. The Great Storm presented us with that 20 times over, so it was a massive opportunity.'
'The great ponds at Sheffield Place at the right season of the year are bordered with red, white and purple reflections, for rhododendrons are massed upon the banks and when the wind passes over the real flowers the water flowers shake and break into each other.' Virginia Woolf's Reflections at Sheffield Place shimmers with the colour and grandeur that draws nearly 200,000 visitors a year to what is now called Sheffield Park Garden, in East Sussex.
Since at least 1730 the owners of Sheffield Park and designers such as Capability Brown and Humphry Repton have planted and sculpted the grounds as pleasure gardens, with avenues of trees, lakes, waterfalls, clumps of lakeside rhododendrons, and the autumn fire of Japanese maples, nyssas and beds of gentians. What they created was less a collection of plants than a living work of art designed to be explored through sunken paths, every turn of which reveals a fresh view, says head gardener Andy Jesson.
The wind which passed over Sheffield Park in the early hours of that stormy October Friday did more than shake the reflections in the lakes. Half of all the trees and shrubs, an estimated 2,000 specimens so carefully planted over 120 acres, were plucked up at their roots or wrenched off from their stems and tossed carelessly across the once-immaculate grounds. Such was the chaos that 'when we came in we couldn't find our way around,' remembers Alan Bradford.
It took the gardeners a week just to clear a 300m path from their hut to the shop, and three years to saw and clear the trees - and replanting is only just finishing this year.
Looking back from the famous cricket pitch at the southeast end of the garden, over the lakes towards the big Gothic-style house, Sheffield Park looks a complete picture of colour and shapes again. Bradford, though, can see enduring changes, like the way silvery Scots pines no longer glisten against the dark-green backdrop of the storm-felled trees behind, and the bare columns of the palm walk are no longer heavily buttressed by white rhododendrons.
It was a clean-up operation repeated across the National Trust's properties. The first priority was restoring the morale of devastated gardeners, some of whom were 'literally in tears', says Calnan: 'They'd seen their lifetime's work obliterated in four hours.'
Then paths and roads had to be cleared, the most dangerously toppled trees removed, and the gardens reopened. Rare specimens and seeds to propagate and replenish the gardens had to be collected as quickly as possible before they were lost or died.
'It was the equivalent of your roof blowing off and the walls falling in and trying to rush in and rescue the Titians and Botticellis,' says Calnan.
After that, the long slog of clearing and replanting hundreds of acres of gardens and woodlands got under way. Within weeks the Trust raised £3m from a public appeal and made thousands more from selling valuable ancient windfall wood. In the 20 years that have passed, staff and hundreds of volunteers have put in tens of thousands of man-hours collecting and sourcing, propagating and placing half a million trees and shrubs. Among and alongside these, fallen trees have carried on growing, younger trees which survived have flourished in the space and light, and others have reseeded themselves. And with time, other plants, birds and animals that lived among them have begun to return.
Studying the pictures of this summer's flyover, Hawes sees both an almost unthinkable resurrection and renewal, and the legacy of the storm damage, still there if you know what to look for: too many trees of the same age and height, too much sunlight climbing down between the thin young growth; in the few places where they were left, old stumps and oaks have been colonised by insects and lichens or are magnificently continuing to grow along the forest floor and sending new shoots up towards the light. 'Those trees had been there for 200 or 300 years - it will take an age to get back to full height... maybe 50 years,' says Hawes. There are some things, though, that will probably never be the same again, he says, pointing to the distinctive red blobs in the canopy where grey squirrels introduced from North America have barked the young trees, especially the beech, which might now never grow to the majestic heights they once did.
A few miles north of Sheffield Park is Toys Hill, more than 400 acres of woodland. Perched 770ft above sea level, Toys Hill was hit by the full force of the storm, which literally flattened most of the south side and plateau.
If the first lesson of 16 October was how efficiently centuries of manmade creations, from homes and gardens to whole woodlands, could be wiped out by a single storm, the second lesson was how ruthlessly nature could reassert itself. 'If you don't manage land in the UK for 20 years, come back and you'll see trees growing; they'll even propagate in brick walls, they'll rip up tarmac,' says Trevor White, who took over as the North Kent head warden a few months after the storm. 'Nature doesn't like a vacuum, so something will come in.'
The importance of the natural rehabilitation goes further, for nature often turned out to be more successful than the multimillion-pound efforts of the human experts. And nowhere was - is - this more vividly evident than at Toys Hill, where 80 acres were set aside as a historic experiment to watch and compare what would happen with no outside interference or 'management' of the recovery.
The first challenge at Toys Hill was to reconsider what was 'natural' for the site. Within weeks of the storm, foresters were astonished to find heather blooming in the newly opened space as the rare lowland heathland habitat which had existed for centuries before the 1850s enclosures reasserted itself. But attempts to fence off some of the area and return it to heathland failed when local people removed the fences.
Instead the Trust decided to return the site to woodland, which, after all, would have covered most of England before settled farmers began grazing. Most of the site was cleared by machines and manpower, and replanted with prized beech and oak saplings. Two decades later, the difference between the woodland and the 'non-intervention zone' is clear: in the managed area, birch and willow quickly took root, while the oak and beech often struggled to take hold in soil which had been compacted by the heavy clearing machinery. Meanwhile, many of the old trees left for dead in the non-intervention zone carried on growing along the forest floor, sending young shoots upwards towards the light, while insects thrived in the rotting wood of dead trees, and roe deer and other animals took refuge in what are sometimes impenetrable depths behind the fallen trunks and root plates. The result, says White, is a richer variety of trees and ecology in the non-intervention zone than where the planters tried to recreate these habitats. Elsewhere there are reports of similar findings: at Sheffield Park, for example, gardeners no longer water young trees and shrubs, having discovered that they grow more strongly if they are forced to send out roots for moisture; at other properties, naturally propagated plants show more genetic variety and strength than mass plantations, and the thriving of younger surviving trees was a reminder of how important it is to keep renewing woodland, sometimes replacing older specimens with new saplings. 'Human beings do like to control everything around us,' says White. 'By deciding we're going to have a non-intervention zone, we have given over control of that land and decided to accept whatever nature gives us.'
Looking back over 20 years, National Trust staff say they have learnt several lessons. 'Perhaps we shouldn't have rushed in and cleared up in a good-husbandry and good-housekeeping way,' says Calnan. 'In hindsight we missed an opportunity to show people the power of nature and the scale of the work we had to do.' More importantly, they would avoid using so much machinery, not just at Toys Hill but other properties too - though not at Sheffield Park. 'One of the biggest lessons is that what's going on under the ground is more important than what's going on over it,' says Jesson. In future, too, more dead trees would be left for insects, birds and fungi. And, of course, the lesson of Toys Hill is that many more woodlands could be left to recover themselves.
'I would never say that what was done and all the money spent was wasted - it wasn't at the time,' says Hawes. But when the next Great Storm comes, the National Trust would do things differently, he says: 'If we can leave woods and gardens which have a bit of resilience for the next generations, we'll have done what we can.'


text:
The content on this page is provided by a Time Out partner.
After winning a prize, two brothers from a small Welsh town, David "Dai" Jones (Donald Houston) and Thomas "Twm" Jones (Meredith Edwards), make their first excursion into London to see a rugby match. Once they arrive in the big city, however, David and Thomas miss their contact, Whimple (Alec Guinness), and get separated. While David falls into the scheming hands of a con artist named Jo (Moira Lister), Thomas visits an array of pubs, making for a lively urban outing.
Release details
|0 mins
Cast and crew
|Charles Frend
|Richard Hughes, Charles Frend, Leslie Norman
|Donald Houston
Meredith Edwards
Moira Lister
Alec Guinness
Hugh Griffith
Julie Milton
Joyce Grenfell


text:
It only takes a minute to get started. Fill out our uncomplicated sign up form and login to the website.
This step is discretionary but will dramatically multiply your chances of your profile being viewed by others. Write a few sentences about yourself, your interests and upload any nice photographs of yourself.
Utilise our website's cutting-edge search to pinpoint members who fulfill your requirements. Or just browse through listings and pinpoint folks that interest you.
Identified someone who meets your requirements? Send a message to them or just a wink and see what they say. With a little luck you'll be on your way to a once in a lifetime experience.
It's really quick


text:
The content team is busily ramping up on doc plans for the next version of Operations Manager. (And I'll be very careful not to reveal anything about product changes, features, etc.) I'll be owning the operations content, so I'm working on new outlines and identifying current gaps.
As I was going through the current content, it seemed like we mention user roles in a few places but don't really integrate them in the rest of the content. I'm thinking for v.Next, I should add which user roles can do something for every task/procedure. So, if I'm a Read-Only Operator looking at the operator's guide, I can immediately see whether I can do this thing I'm looking up or not.
I don't know yet whether to include it at the beginning of each topic ("this information applies to the Operator user role"), at the end, preceding each procedure…somewhere consistent, definitely.
What do you think, would user roles be a useful addition to relevant topics? Or are user roles easy enough to understand that it isn't necessary?


text:
- Forums
- Features
- Disney News
- Disney Parks
- Events
- Other Parks
- MiceTrips
- MiceDeals
- MicePod
MiceChat / Seven Seas Lagoon
by Cory Disbrow on Aug 17, 2016 | No Comments
Hello, and welcome to Dateline Disney World! This week, we are heading to the Magic Kingdom bright and early to check out what's happening at the park. But first, a bit of news from around…
Posted in Dateline Disney World, Disney Parks, Magic Kingdom, Walt Disney World
Copyright © 2017 MiceChat. MiceAge and MiceChat, LLC are not associated in any manner whatsoever with the Walt Disney Company, its subsidiaries and/or its affiliates. Our purpose is to present completely independent editorials, reviews, and guides about theme parks and other products from Disney and similar companies. Disney's site is available via this link.


text:
Jen Hadfield
From Wikipedia, the free encyclopedia
Jen Hadfield (born 1978 in Cheshire) is an English poet.
Hadfield was educated at Withington Girls' School.
She won the 2008 T.S. Eliot Prize for poetry for her second collection, Nigh-No-Place.[1] This was a notable honour for a relative newcomer, as the award's previous recipients include such major poets as Carol Ann Duffy, Seamus Heaney, and Ted Hughes.
Hadfield also won an Eric Gregory Award in 2003.
Hadfield currently resides in Shetland, off the north of mainland Scotland.[2] She publishes a line of artbooks, using linocut, photography, woodwork, bookbinding and fly-tying, which she calls "Rogue Seeds".[citation needed]
Her third poetry collection, Byssus, was published in February 2014.
- ^ Mark Brown (2009-01-12). "Jen Hadfield wins TS Eliot prize for poetry". The Guardian.
- ^ "Jen Hadfield". United Kingdom - Poetry International Web.
External links[edit]
- Jen Hadfield's website
- Scottish Poetry Library profile
- Poetry Archive profile and poems written and audio
- British Council profile
- BBC 8 January 2009. Jen Hadfield reads her poetry". Reading "Paternoster". (audio, 2 mins)
- "TS Eliot judge Tobias Hill on the noteworthy Jen Hadfield" by Tobias Hill 14 January 2009 Guardian
- Hadfied's blog
|This article about an English writer, poet or playwright is a stub. You can help Wikipedia by expanding it.


text:
The Redneck Badge of Courage
Holy flyin' fried chicken! Comb out your best wig, whip up a batch of banana puddin' and practice your Camel Walk! SCOTS is comin' to town!
This finger-lickin' foursome, better known as Southern Culture on the Skids, has been dishing up its distinctive version of trailer-park twang since 1985. Based in Chapel Hill, North Carolina, singer/guitarist Rick Miller, bassist Mary Huff, drummer Dave Hartman and the band's newest addition, keyboardist Cousin Crispy, play music that pays tribute to their Appalachian roots. White trash is king in their Southern-fried world of halter tops, mobile homes, girl fights, demolition derbies, Naugahyde, shotgun weddings and shag carpet.
Since being signed to the major label DGC in 1995, SCOTS has recorded and released two albums. The first, Dirt Track Date, provides a taste of their diverse musical range--a hat is tipped to the likes of rockabilly, funk, garage rock, hillbilly-tinged country and California surf instrumentals. It's like Link Wray meets The Cramps. Dirt Track Date also exposes the band's collective food fetish--lowbrow Southern delicacies like fried chicken, Little Debbie Snack Cakes and sweet tea loom large throughout.
Plastic Seat Sweat, released in 1997, picks up where Dirt Track Date left off. Recorded at Reflection Sound Studios in Charlotte, North Carolina, in 1997, it remains true to the band's unique brand of white-trash gospel, but takes more chances. A veritable cornucopia of musical delights, this CD dishes up Martin Denny-esque sounds of exotica, belly-dancing instrumentals, and turbo-speed rockabilly riffs. The songs about Southern eats still abound, like "Carve That Possum," an Uncle Dave Macon classic, and "Banana Puddin'," which the band prefers to eat when it's "day old and bold."
Upcoming Events
Phoenix Mercury vs. Minnesota Lynx
TicketsFri., Jun. 30, 7:00pm
All You Can Eat Value Pack - Mercury v Lynx
TicketsFri., Jun. 30, 7:00pm
TicketsFri., Jun. 30, 8:00pm
Hot August Night
TicketsSat., Jul. 1, 7:30pm
Sure, their albums are great, but the best way to enjoy SCOTS is to see them live. The dedicated touring band has played more than 400 shows in the past two years and developed something of a cult following in the process. In fact, their live shows could easily qualify as performance art. Where else can you expect to be pelted with fried chicken and spoonfuls of banana pudding? For the fashion mavens in the crowd, bassist Mary Huff delivers the goods--the towering, stylishly coifed wigs she dons would make any truck-stop waitress envious. If the saying "the bigger the hair, the closer to God" were true, she would have been sainted by now.
So grab yourself an eight-piece box and head on over to Jackson Hole on October 30 to catch SCOTS live. It's an experience in white-trash culture that you won't soon forget. Just make sure to dodge the flyin' drumsticks.
--Kaytie Johnson
Southern Culture on the Skids is scheduled to perform on Friday, October 30, at Jackson Hole, 1 East Jackson. Showtime is 8 p.m.; Big Sandy and His Fly Rite Boys, and Neko Case share the 18-and-older bill. Tickets are $10 in advance, $12 the day of the show. 257-0990, 503-5555.
Get the Things to Do Newsletter
Sign up for our weekly guide to events in Phoenix, and never be bored again. With suggestions for every day of the week, our recommendations will keep you busy on any budget.
Upcoming Events
Chase Field Stadium Tours
TicketsTue., Jun. 27, 9:30am
Arizona Diamondbacks vs. St. Louis Cardinals
TicketsTue., Jun. 27, 6:40pm
TicketsWed., Jun. 28, 7:00pm
Arizona Diamondbacks vs. Colorado Rockies
TicketsFri., Jun. 30, 6:40pm
Sponsor Content


text:
Wheeler del Torro may call himself a "culinary designer," but we'd say his title also deserves "innovative" and "pioneer." In addition to the 5,000 vegan and boozy ice cream flavors he's created, he was far ahead of the curve with underground dinners; he held his first one 15 years ago. Be a part of his avant-garde experiences during the latest Barrio pop-up: a series of six-course meals with a guest chef each night ($110; or $130 with a signed copy of del Torro's The Vegan Scoop). The price includes money to spend at 3 Scoops, gratuities, and all other fees.
Held in the test kitchen for Wheeler's Catering & Frozen Dessert Co., these hearty dinners will fuse Wheeler's Afro-Cuban flavors with each guest chef's cooking style. While del Torro likes to keep his guest-chef lineup under wraps, we know that Strip T's Tim Maslow—a Momofuku veteran known for his love of meat—will be cooking on April 26, and Matt Drummond of Brasserie JO will be the collaborator on April 28. Past Barrio dinners have included slow-cooked yuca with citrus-garlic sauce, a ragout of three kinds of mushrooms with sweet peppers and tomatoes, and a corn cake with a guava glaze and grilled pineapple.
After taking advantage of the 3 Scoops credit and perusing your signed copy of del Torro's book, you'll have a full picture of the chef as not just a culinary designer but also an innovator and pioneer.
Barrio by Wheeler del Torro
Six-Course Culinary Adventure with Guest Chefs and $10 Credit at 3 Scoops ($110); With Signed Cookbook ($130)
- from $110


text:
The hotel is a luxury aparthotel, mixing the services of a hotel with the convenience and privacy of an apartment. It offers 24-hour reception and check-out services, and free telephone calls to UK landlines are included. Further facilities include air conditioning, a hotel safe, lift access to the upper floors as well as Internet access.
This stylish aparthotel is located in the centre of Leeds, less than 1.6 km from Leeds Rail Station.
The large, modern apartments have flat-screen TVs with satellite/cable channels and free broadband Internet access. All are en suite with a shower and hairdryer. Further in-room amenities include a double bed, direct dial telephone and a safe. There is a kitchen with a fridge, microwave and tea and coffee making facilities. Individually regulated air conditioning and heating are standard in all accommodation.
The establishment accepts MasterCard and VISA as payment methods.
Room Facilities:
Direct dial telephone
Satellite cable TV
Internet access
Tea Coffee
Double bed
Individual airconditioned
Individual heating


text:
SailNet Community (http://www.sailnet.com/forums/)
- Gear & Maintenance (http://www.sailnet.com/forums/gear-maintenance/)
- - Outboard Motor Bracket "not for use with 4 stroke engines" (http://www.sailnet.com/forums/gear-maintenance/35195-outboard-motor-bracket-not-use-4-stroke-engines.html)
Outboard Motor Bracket "not for use with 4 stroke engines"
I am looking at an adjustable outboard motor bracket. I have found several brackets which take my motor size, Honda 8 four stroke, but state they are "not for use with 4 stroke engines". Supposedly, this has something to do with torque. I also realize that 4 strokes tend to be heavier than 2 strokes but how much heavier can they be. Or is there some other characteristic of Four Strokes that require a special bracket?
I give two example below. In the first the bracket is good up to 20HP but only apparently for 2 STROKE not FOUR. The second bracket is for only a 9.9 FOUR STROKE but costs $115 more.
Are these valid claims be the manufacturer or just a way to generate more profit on four strokes?
Stainless steel springs. Maximum 20hp (not for use with 4- stroke engines). Made entirely of non-corrosive material. CAD$169.95
Extra strength stainless steel torsion springs to counter balance motor. Heavy duty U channel construction for more stability. CAD$284.95 "
You're gonna have to spend the money. The 2-stroke brackets are just not designed for the larger, heavier 4-strokes. The 4-stroke brackets are generally constructed with channel aluminum, not flat bar, they have much stronger springs, they have larger and better grade bolts, and the mounting pads are wider. I put a 9.9 4-stroke (125lbs) on an old 2-stroke mount. I used it that was for about a month, and it didn't work very well. It took two people to lift the motor, as the springs were undersized, and the bracket would sway back and forth with the movement of the boat, making me very nervous, (hence the channel construction on the 4-stroke models). Note, that the mounting plates are much wider on the 4-stroke models, and will not line up with the holes from an old 2-stroke mount. Also....make sure you get one that has enough vertical lift, they range from 9" to 15.5". I bought the Fulton (which is on the boat now) but it does not drop the motor far enough into the water, creating problems if someone is on the bow, I just bought a Garelick, with 15.5" of travel (for $350!!!) and will be swapping it out whenever it stops raining. If you want to buy my like new, 4-stroke Fulton bracket let me know...
I feel your pain. Been trying to fit a new one on my SJ23. The Fultons are way too narrow, and the Garelicks are wide and pretty pricey. The big question though: What boat are you mounting the bracket onto? The mounting bracket, that part that attachs to your transom, is a big issue. Usually the dealers only list the motor mount size. The Garelick models 71090 and 71091 are terrific mounts and have a 8Wx10H footprint. Other models get even larger. Check your backing plate or board that is probably well hidden or virually inaccessable, except for Plastic Man. Check Garelick's website for some good mounting instructions. Thier customer service was great too, sending me detailed mounting templates for the 71090.
Good Luck and fair winds,
Lake Norman, NC
Ive used a bracket rated for 30hp 4-stroke to hold a 40hp 2 stroke for the last few years now with no issues. I can say that the 4stroke brackets are alot stronger than the others, but if your motor falls under the weight limit, i dont see a reason it wouldnt work.
As tempting as it is, don't convince yourself it's all a marketing scam in order to save a bit of money. Do it right and you won't have anything to worry about.
Aloha Peter,
Looks like you've already received some very good advice... just my $0.02 worth, but I'd spend the extra money and get a bracket rated for the type and size OB I intended to use and not cut corners.
True, there are occasions where 'the biggest, most expensive' choice is a waste of money, but for a crummy $115, is it worth losing an OB?
As with lots of things in life, you generally get what you pay for; Just MHO. All the best to you what ever course you choose.
John K.
The other point is that if you're using the four-stroke outboard as the primary auxilliary engine for your boat, and you go cheap on the bracket... there is a much higher chance that the bracket will break if you're motoring in heavy weather or seas. Murphy's law says that the bracket will break in such a situation and possibly lead to you losing the entire boat. Is that really worth saving $100 or so???
Re: Outboard Motor Bracket "not for use with 4 stroke engines"
like everyone else has said , use a 4 stroke bracket for a 4 stroke motor ,
i used my 2 stroke bracket for a 4 stroke one trip and it looked like it was going to snap off . replaced it with the right mount for 4 strokes no problems
Re: Outboard Motor Bracket "not for use with 4 stroke engines"
Once again, I'm the odd man out.
I don't see why a bracket designed for a 20 hp 2 stroke wouldn't work on an 8 hp 4 stroke. The torque and weight on the 8 hp shouldn't come anywhere close to a 20 hp 2 stroke outboard. I would contact the manufacturer, but as I said, I can't see a problem.
Re: Outboard Motor Bracket "not for use with 4 stroke engines"
2 Nd on capta
|All times are GMT -4. The time now is 08:18 AM.
Powered by vBulletin® Version 3.8.7
Copyright ©2000 - 2017, vBulletin Solutions, Inc.
vBulletin Security provided by vBSecurity v2.2.2 (Pro) - vBulletin Mods & Addons Copyright © 2017 DragonByte Technologies Ltd.
User Alert System provided by Advanced User Tagging v3.1.0 (Pro) - vBulletin Mods & Addons Copyright © 2017 DragonByte Technologies Ltd.
(c) Marine.com LLC 2000-2012


text:
Win $496 worth of Philosophy products!
One lucky winner will receive a huge assortment of Philosophy's star products. From their cult-favorite skincare line to their heavenly-scented shower gels and body lotions, there's a little bit of everything to enjoy. Good luck!
Note to all BFFs: If you are logged in, please log out before filling in the form. Don't worry, we'll fix that real soon!
[contest-form id=12345]


text:
We're a quiet, clean, relaxed bunch of people. Two uni students and one full time worker. The house is a five minute walk from Armidale's CBD, and just 50 metres from a bus stop to the University and TAFE. Room's aren't huge, but they have built-ins, and there's a large living and dining area, and huge backyard.
1 empty room to rent out in an otherwise fully furnished, modern, 3 bedroom house near the centre of Armidale. On a quiet street, walking distance from the city centre shops, hospital and 5 minute drive to UNE. Students and couples welcome. Outdoor area with firepit, great to sit around for a drink on a cold winters night. Empty garage for your car if you want to use it. All bills spilt evenly.