text:
- Research
- Open Access
Overlapping effector interfaces define the multiple functions of the HIV-1 Nef polyproline helix
© Kuo et al.; licensee BioMed Central Ltd. 2012
- Received: 14 February 2012
- Accepted: 31 May 2012
- Published: 31 May 2012
HIV-1 Nef is a multifunctional protein required for full pathogenicity of the virus. As Nef has no known enzymatic activity, it necessarily functions through protein-protein interaction interfaces. A critical Nef protein interaction interface is centered on its polyproline segment (P69VRPQVPLRP78) which contains the helical SH3 domain binding protein motif, PXXPXR. We hypothesized that any Nef-SH3 domain interactions would be lost upon mutation of the prolines or arginine of PXXPXR. Further, mutation of the non-motif "X" residues, (Q73, V74, and L75) would give altered patterns of inhibition for different Nef/SH3 domain protein interactions.
We found that mutations of either of the prolines or the arginine of PXXPXR are defective for Nef-Hck binding, Nef/activated PAK2 complex formation and enhancement of virion infectivity (EVI). Mutation of the non-motif "X" residues (Q, V and L) gave similar patterns of inhibition for Nef/activated PAK2 complex formation and EVI which were distinct from the pattern for Hck binding. These results implicate an SH3 domain containing protein other than Hck for Nef/activated PAK2 complex formation and EVI. We have also mutated Nef residues at the N-and C-terminal ends of the polyproline segment to explore interactions outside of PXXPXR. We discovered a new locus GFP/F (G67, F68, P69 and F90) that is required for Nef/activated PAK2 complex formation and EVI.
MHC Class I (MHCI) downregulation was only partially inhibited by mutating the PXXPXR motif residues, but was fully inhibited by mutating the C-terminal P78. Further, we observed that MHCI downregulation strictly requires G67 and F68. Our mutational analysis confirms the recently reported structure of the complex between Nef, AP-1 μ1 and the cytoplasmic tail of MHCI, but does not support involvement of an SH3 domain protein in MHCI downregulation.
Nef has evolved to be dependent on interactions with multiple SH3 domain proteins. To the N- and C- terminal sides of the polyproline helix are multifunctional protein interaction sites. The polyproline segment is also adapted to downregulate MHCI with a non-canonical binding surface. Our results demonstrate that Nef polyproline helix is highly adapted to directly interact with multiple host cell proteins.
- Nef
- MHC class I
- p21-activated protein kinase
- Protein-protein interaction interface
- SH3 domain
HIV-1 Nef is a highly significant pathogenic factor, but its role in the progression to AIDS is not mechanistically understood. Nef is remarkable in the number and diversity of its functions, and its pathogenicity may depend on the collective impact of its highly conserved multiple activities [1, 2]. Among its numerous activities, Nef downregulates major histocompatibility complex class I (MHCI) from the cell surface, downregulates CD4, activates and forms a complex with p21-activated protein kinase 2 (PAK2) and enhances the infectivity of the HIV-1 virion [2, 3]. It is plausible that each of these four Nef functions makes contributions to HIV-1 Nef pathogenicity. Nef/activated PAK2 complex formation may enhance cellular activation and, as a result, viral replication [4]. MHCI downregulation appears to blunt the host cytotoxic T cell response to the virus [5–7]. CD4 downregulation may enhance the efficiency of viral production [8–10]. Finally, enhancement of virion infectivity (EVI) may facilitate the spread of the virus throughout the infected patient [11].
Nef downregulates or upregulates a large number of cell surface receptors, though it has recently been suggested that the mechanism of modulating the levels of many of these proteins is indirect [12, 13]. The mechanisms of these indirect effects are, in general, unknown; and ultimately Nef must directly interact with host cell proteins as it has no enzymatic activity [2]. Here, we have studied Nef activities that appear to depend on direct interactions between Nef and relevant host cell target proteins. These activities include CD4 and MHCI downregulation, as direct binding of Nef to the cytoplasmic tails of these membrane proteins has been demonstrated [14–19]. Also, direct roles for SH3 domain proteins have been suggested for several Nef activities: 1) the SH3 domain protein, Hck, is known to bind Nef with nM affinity [20]; 2) Activation of Hck and other Src family kinases has been proposed to facilitate MHCI downregulation by formation of a Nef/Src family kinase/ ZAP70/PI3K complex [21, 22]; 3) The activation of PAK2 and the formation of the Nef/activated PAK2 complex appear to require an SH3 domain protein [20]. Finally, mutational evidence suggests the involvement of an SH3 domain containing protein for EVI [2].
To begin to unravel the complex interactions between Nef and the multiple host cell proteins involved in these Nef activities, we have used fine structure mutational analysis to describe the roles of the polyproline segment, P69VRPQVPLRP78, residues. First, we mutated each residue of the core sequence, PQVPLR, that represents the Nef SH3 domain binding motif (PXXPXR) [23, 24]. Our analysis confirmed the importance of the prolines and established the significance of the arginine in the Nef PXXPXR SH3 domain binding motif [20]. The remaining three residues (X's) were expected to contribute to the specificity of binding for a particular Nef/SH3 domain pair [25–28].
We also searched for functionally significant residues near PQVPLR that could be necessary for Nef/activated PAK2 complex formation, MHCI downregulation, and EVI. Two residues to the N-terminal side P69VRPQVPLRP78, G67 and F68, were found to be critical for Nef/activated PAK2 complex formation, EVI and MHCI downregulation. Based on our mapping analysis, we have proposed a new multifunctional Nef-host cell protein interface for these activities.
To the C-terminal side of PXXPXR, we also characterized the functional significance of P78. In contrast to mutations of PXXPXR, G67 and F68, the phenotype of the P78 mutant was not multiply defective but specific for MHCI downregulation. This result suggests a unique mechanism independent of Nef interacting with an SH3 domain protein. With the current data, we and others have now characterized the dependence of MHCI downregulation on the stretch of Nef amino acids from E62 to P78 plus the D123 residue that is spatially near P78[29–33]. These results confirm in detail the recently reported structure for the tripartite complex formed by Nef, AP-1 μ1, and the cytoplasmic tail of MHCI downregulation [34]. Our new results greatly expand the understanding of the polyproline segment, and represent the first advance in integrating Nef structure and function.
SH3 domain protein involvement in three Nef activities
The identities of the putative SH3 domain proteins necessary for Nef/activated PAK2 complex formation, MHCI downregulation, and EVI are not known: therefore, the Nef-SH3 domain interactions could only be assessed by functional assays. The three assays were all performed with intact cells which make analysis of the Nef-SH3 domain interaction more difficult to interpret. However, if a direct interaction with an SH3 domain protein is mechanistically required for a given Nef activity, then all three of the point mutations of the prolines and the arginine in PXXPXR should eliminate the activity. If Nef binds multiple SH3 domain proteins, the mutation of the remaining residues PQVPLR, should distinguish between different SH3 domain proteins. Therefore, we have used the requirement that single mutations of both prolines and the arginine are fully defective before concluding that there is a Nef-SH3 domain interaction involved. Conservative mutations of the other three residues of PXXPXR then distinguish between different SH3 proteins that bind Nef [25–27].
The polyproline helix
We began our investigation of Nef effector interfaces by assaying Nef-Hck SH3 domain binding in vitro. The results of these direct binding assays can then be used to evaluate subsequent results of functional assays. The importance of the motif-designating proline residues in PXXPXR have been demonstrated for Nef-Hck SH3 domain binding with the mutations, P72A and P75A, but R77 has not been adequately tested [20]. Here, we have used mutations P72G, P75G and R77K. For the other three residues, we mutated Q73 to R, V74 to I, and L76 to V. All of the residues in PQVPLR are very highly conserved (HIV-1 subtype B data base of 1643 sequences, >97% [32]); therefore, even conservative amino acid replacements (as defined by BLOSUM62, [35]) are expected to disrupt of at least one important Nef function. Summarizing, it is expected that the one to one binding of Nef to the Hck SH3 domain will give structurally interpretable information needed to evaluate functional assays.
Nef-Hck SH3 interaction
Two additional mutations were made to further characterize Nef binding to Hck. In X-ray crystal structures, P78 is predicted to bend away from the planar hydrophobic interface of the Hck SH3; and therefore it defines the border of the core SH3 domain binding site [23, 24]. As expected, the P78G mutation has minimal impact on Nef-Hck SH3 domain binding. Nef also interacts with the RT loop of the Hck SH3 domain which is outside of the SH3 planar hydrophobic interface [24, 36, 37]. An isoleucine in the Hck RT loop is buried in a hydrophobic pocket on the surface of Nef formed by L87, F90, W113, I114, T117 and Q118[23, 24, 38]. Unexpectedly, we found no reduction in binding by the non-conservative F90A mutation (Figure 1). The simplest explanation is that the remaining residues in the pocket are sufficient to stabilize the Nef-Hck SH3 interaction. With these eight mutations, we have a Nef-Hck SH3 domain interaction signature to compare with other Nef activities.
Nef-SH3 domain interaction required for Nef/activated PAK2 complex formation
Having established a basis for comparing putative Nef SH3 interactions to Nef-Hck SH3 binding, we investigated the role of PQVPLR in Nef/activated PAK2 complex formation. In extracts from cells not expressing Nef, PAK2 autophosphorylation is not detectable by the sensitive in vitro kinase activity assay (IVKA) using anti-PAK2 antibody. IVKAs of extracts from cells expressing Nef exhibit both PAK2 autophosphorylation and phosphorylation of the added substrate myelin basic protein ( Additional file 1: Figures S1A and S1B). We have previously demonstrated that in Nef-expressing cells only a small fraction of activated PAK2 is not immunoprecipitated by anti-Nef antibody and that the activation of PAK2 by Nef is sufficient to alter the phosphorylation state of an intracellular protein [39, 40]. The components of the Nef/activated PAK2 complex have not been identified leaving open the possibility that the activation of PAK2 by Nef could be a multi-step process with activation separate from complex formation.
To demonstrate a role for an SH3 domain protein in Nef/activated PAK2 complex formation, it is necessary for single mutations of the prolines and the arginine in PXXPXR to abrogate this activity as was observed for Hck binding. It has been previously shown that Nefs with P72A or P75A mutations fail to form the Nef/activated PAK2 complex which suggests SH3 protein involvement [20, 41]. However, this suggestion would be called into question if Nef with the R77K mutation gave a functional phenotype. Mutations of the other residues in PQVPLR (Q, V, and L) should give a different mutational profile for Nef/activated PAK2 complex formation compared to Hck (Figure 1).
Role of PQVPLR, P78 and F90 in MHCI downregulation
The double mutation of P72 and P75 to alanines (AQVALR) abrogates the ability of Nef to downregulate MHCI [44, 45]. It was subsequently reported that the single mutations, P72A and P75A, are only partially defective for MHCI downregulation, but the P78A mutation abolishes this activity [30, 33]. Thus, the case can be made that the double mutation of P72A/P75A yields a synthetic phenotype that is the result of a local disruption of Nef structure. In Figure 2B, we evaluated this hypothesis. P72 and P75 mutated to glycine gave partial defects in MHCI downregulation. Nef with P78 mutated to glycine was fully defective for MHCI downregulation (Figure 2B). Significantly, we also demonstrated only a small effect of the R77K mutation on MHCI downregulation (77.3 ± 5.3% of SF2Nef activity), despite the fact that this mutation totally prevents Hck binding and Nef/activated PAK2 complex formation (Figures 1 and 2A). The L76V and F90A mutations also had partial deficits for MHCI downregulation (Figure 2B). Therefore, only the mutation of P78 fully abrogates MHCI downregulation in the polyproline region. Our results are suggestive of a relatively non-specific structural role for PQVPLR in MHCI downregulation. CD4 downregulation was also monitored as a positive control of overall Nef activity and was fully functional in all cases. Finally, it should be noted that all the mutations presented in Figure 2 were stably expressed at levels similar to the wild type protein (Figure 2AMiddle Panel).
Our results do not support the involvement of an SH3 domain protein in MHCI downregulation but point to P78 and a nearby residue D123. The Nef mutant with D123E is devoid of MHCI downregulation activity [31, 32]. The mutational phenotypes of P78 and D123 are fully consistent with recent proposals of a Nef-induced multi-protein complex that lacks an SH3 domain protein and blocks MHCI transport to the cell membrane [15, 18, 19, 46]. In the Discussion, we review a recent report of the ternary structure of Nef, the cytoplasmic tail of MHCI (ctMHCI) and AP-1 μ1 [34] that is in full accord with the mutational data presented here and previously published [18, 19, 29, 30, 32, 33].
Enhancement of virion infectivity (EVI)
The N-terminal side of the polyproline helix
The impact of the V66A, G67A and F68A mutations on EVI
Qualitatively similar phenotypes for Nef/activated PAK2 complex formation (Figures 2 and 4) and EVI (Figures 3 and 5) suggest interactions by Nef with very similar SH3 proteins, or even the same SH3 protein for these activities. It should be noted that, even if Nef uses the same SH3 protein for Nef/activated PAK2 complex formation and EVI, the overall mechanisms are nonetheless distinct, since EVI is not dependent on Nef/activated PAK2 complex formation [32, 50].
PQVPLR, GFP/F, and P78 reside in a region of overlapping, multifunctional effector domains on the surface of Nef
Each of the colors represents potential multifunctional interaction domains for Nef binding to cellular proteins. Brown residues are important for MHCI downregulation. These include the tetra-glutamate segment (residues E62-E65), G67 and F68. The quadruple mutation of the tetra-glutamate segment to four alanines has been reported by many groups to be defective for MHCI downregulation [15, 19, 29, 44, 45]. The proximity of G67 and F68 to the tetra-glutamate segment suggests concerted action by these residues in MHCI downregulation.
G67 and F68 are also required for Nef/activated PAK2 complex formation and EVI. P69 is included in this patch as tan because it contributes to Nef/activated PAK2 complex formation, but not MHCI downregulation. Gold residues have a role in Hck binding [23, 24, 38, 52]. Here, we report for the first time the importance of F90 for Nef/activated PAK2 complex formation and EVI. To reflect the overlapping functionalities of its residues we represented GFP/F in three different colors (Figure 6A).
Blue residues are Nef's multifunctional PXXPXR motif, and pink residues surround P78 and D123 (Figure 6A). D123 is multifunctional and required for MHCI downregulation, EVI and CD4 downregulation [31, 32, 53]. The conservative mutation D123E is fully defective for these three Nef activities and enhances Nef/activated PAK2 complex formation several fold [31, 32]. This latter effect may be related to the fact that mutating the nearby F121 to arginine prevents Nef/activated PAK2 complex formation [20]. D123, P78, T80, and F121 residues are part of a predicted protein-protein interaction interface [54]. The mechanism of action of D123 for different functions has remained obscure, although the recent report of the ternary complex of Nef, AP-1 μ1 and ctMHCI explains the roles of P78 and D123 in MHCI downregulation [34].
Identification of critical amino acids in Nef protein-protein interaction interfaces
We have carefully mapped by mutational analysis residues that could possibly act in concert with PQVPLR in Nef-SH3 domain protein interactions. Mutations were found that resulted in partial defects, and mutations were found that eliminated one or more Nef activities. Mutations of the latter phenotype were observed for G67, F68, P72, V74, P75, R77, P78, and F90. The mutational properties of these residues fit the expected properties of critical residues in protein-protein interfaces. Typically, critical residues in protein-protein interfaces exhibit packing densities equivalent to that found in the interiors of proteins which account for the large disruption of binding by a conservative replacement or by creating a hole in the interface with an alanine substitution (glycine excepted). Further, these critical amino acids are typically found in the central core of protein-protein interfaces [51]. For a given interface only a few of the residues account for most of the binding affinity, and identifying these amino acids localizes the interface [51, 55, 56].
The multifunctional PXXPXR motif
Summary of phenotypes for mutations in the Nef polyproline region
Hck SH3
There are overlapping effector surfaces at the N-terminal end of the polyproline helix
We have defined a new interface, GFP/F, which is necessary for Nef/activated PAK2 complex formation and full activity of EVI (Table 1). In Figure 6A Function, GFP/F is presented in three colors with G67 and F68 (brown) residing between F90 (gold) and P69 (tan). We propose that GFP/F is part of a protein–protein interaction interface for Nef/activated PAK2 complex formation based on our mutational analysis and the 99% conservation of these residues [32]. To test this model we made the conservative mutation, F68Y, and we found this mutant Nef to be fully defective for Nef/activated PAK2 complex formation ( Additional file 2: Figure S2A). The most straightforward explanation for this result is that F68 is a critical residue for Nef/activated PAK2 complex formation engaging in hydrophobic interactions which the extra hydroxyl group of tyrosine disrupts.
A recently proposed interface overlapping GFP/F is the secretion modulation region (SMR, spanning V66GFPV70 but not including F90). Bond and co-workers have shown that secretion of Nef in exosomes and the binding of Nef to mortalin depends on this motif. In silico modeling suggests the SMR motif forms a protein binding pocket [57, 58]. Therefore, there appear to be at least four Nef functions modulated by this region of the Nef surface including MHCI downregulation, Nef/activated PAK2 complex formation, mortalin binding and EVI but not Hck binding [59].
A multifunctional surface at the C-terminus of the polyproline helix
P78 and D123 Nef mutants are defective for MHCI downregulation (Table 1). D123 represents a multifunctional residue as the conservative mutation, D123E, is highly defective for MHCI downregulation, CD4 downregulation and EVI. A computer analysis of the surface of Nef (PPI-Pred) included P78 and D123 along with T80 and F121 as part of a putative protein-protein interaction site [54]. All of these residues (pink in Figure 6A) are highly conserved [32]. Further D123 and F121 are part of a proposed dimerization interface [53, 60, 61]. This region of the Nef surface appears to resemble the region at the N-terminus of the polyproline helix in its multifunctionality.
MHCI downregulation
MHCI is a common target for anti-host defenses of human viruses. HIV-1 Nef counters MHCI directed cytoxicity in a distinct mechanism from those of adenovirus, human CMV and Kaposi's sarcoma-associated herpesvirus and even simian immunodeficiency virus Nef [62, 63]. Key to HIV-1 Nef's mechanism is residue P78. P78 appears to be specifically involved in MHCI downregulation as mutating P78 to alanine has little or no effect on other Nef activities (Figures 2,3 and [20, 30, 33, 41, 58]. The recent description of a ternary structure of ctMHCI, AP-1 μ1 and Nef (http://retroconference.org/2012b/Abstracts/45450.htm) strongly implies that P78 is critical for Nef binding and rerouting MHCI away from the cell surface [34].
An extended stretch of Nef amino acids from E62 to P78 binds to both AP-1 μ1 and ctMHCI in a manner entirely different from the binding of Nef to SH3 domains [23, 24, 34]. In the model of Jia et al., the tetra-glutamate segment (E62E63E64E65) electrostatically interacts with a patch of four lysines in AP-1 μ1. This ionic interaction is consistent with our previous report that several combinations of the tetra-glutamate segment mutated to two glutamates and two alanines all remain functional for MHCI downregulation [29]. In the new structure, G67 and F68 are part of a bend in the Nef main chain that allows the polyproline helix to be positioned in the complex near the YXXL binding site of AP-1 μ1. The polyproline segment of Nef serves as a clamp over the low affinity combination of ctMHCI bound to AP-1 μ1. ctMHCI binds weakly to the YXXL site of AP-1 μ1 because the cryptic motif in ctMHCI is the defective YXXA. Down the length of the polyproline segment the interactions between Nef residues and the complex are not extensive. Therefore, the structure rationalizes the partial defects observed with single mutations of PXXPXR residues while the double mutation of AXXAXR is fully defective [15, 19, 29, 44, 45]. P78 is the exceptional residue in that it is buried in the ternary complex. Spatially near P78 is D123, which is also critical for MHCI downregulation [31, 32, 53]. D123 forms a salt bridge with R393 of AP-1 μ1 as part of a network of electrostatic interactions between all three proteins. Therefore, the model predicts the mutational defects seen in MHCI downregulation with P78G and D123E.
The ternary complex model originally proposed by Guatelli and co-workers and Collins and co-workers [15, 18, 19, 46], has been contested as others have argued for involvement of SH3 domain host cell proteins, specifically Hck, Lyn and Src or alternatively a decisive role for Nef dimerization that is dependent on residue D123[21, 44, 53, 61]. SH3 domain binding is not part of the new structure, and the role of D123 in the new structure excludes it from participating in dimerization.
The structure of Jia et al is a major advance but important questions remain unanswered [34]. Residue M20 is analogous to P78 in its significance and specificity for MHCI downregulation ( Additional file2: Figure S2B and [19, 64, 65]). Here we have found G67 and F68 to also be structurally required for MHCI downregulation. These Nef residues may be involved in binding additional factor(s) to the Nef effector complex for MHCI downregulation [46].
Analysis of the in vivo significance of Nef activities
Detailed characterization of Nef protein-protein interaction interfaces makes feasible targeted studies of the impact of Nef activities on HIV-1 replication and cytotoxic impact in vivo. BLT humanized mice represent an advanced model for reconstitution of the human immune system [66–68]. We have recently published the complex phenotype of nef(-)HIV-1 in this system of reduced viral replication and highly cytotoxic effects on T cells and thymocytes [69]. Significance for a given Nef activity can be established by mutations specific for that function that result in either reduced HIV-1 replication/cytotoxicity or rapid reversion of the mutation.
It should also be noted that a second recent report investigated the three amino acid loop, V66, G67, and F68[70]. Mutations of these amino acids resulted in defects for MHCI downregulation, Nef/activated PAK2 complex formation, EVI but not CD4 downregulation. In addition, the Nef activities of inhibition of actin remodeling following T cell receptor stimulation, hyper-phosphorylation of cofilin and CXCR4 downregulation were dependent on the loop residues.
We have undertaken detailed mutational analysis of polyproline helix and proximal surface residues that are critical for multiple Nef activities. Four of the Nef activities that have been suggested to be dependent on host cell SH3 domain proteins have been investigated. We have demonstrated that three Nef activities, Nef/activated PAK2 complex formation, Hck binding and EVI are dependent on a Nef-SH3 domain protein interaction. When subjected to detailed analysis a fourth activity, MHCI downregulation, does not exhibit the properties expected of an activity dependent on an SH3 domain protein. Particularly telling in this regard is the small impact of the R77K mutation on MHCI downregulation. Furthermore, the three Nef activities that definitely require SH3 domain proteins do not depend on the same host cell protein. Hck and at least one additional SH3 domain protein interact with Nef.
The functionally dense region of the Nef surface investigated here serves as a model for the protein's structural complexity. The stretch of amino acids from E62 to P69 plus the spatially close F90 contains at least four combinations of residues that effect separate Nef activities-Nef/activated PAK2 complex formation, EVI downregulation, MHCI downregulation, and mortalin binding. From P72 to R77, PQVPLR acts as a PXXPXR motif to bind at least two different SH3 domain proteins and contributes to MHCI downregulation. Near the C-terminal end of the polyproline helix D123 is involved in at least three different Nef activities [31, 32]. For MHCI downregulation, D123 acts in conjunction with P78 to pin the ctMHCI to AP-1 μ1. The rest of the polyproline segment plays a supporting role in ternary complex by positioning the tetra-glutamate segment to interact with a lysine patch on AP-1 μ1.
Maintenance of the structure of this critical region of Nef enforces strict conservation of the component residues and implies that the associated activities confer fitness to the virus. Extending the type of analysis presented here to the rest of the Nef surface will define which Nef activities are sufficiently important to structurally impose overlapping protein-protein interaction interfaces and the attending extreme conservation of amino acid residues. Detailed functional analysis will allow activity-specific mutations in Nef to be evaluated in BLT humanized mice. Highly conserved protein-protein interaction interfaces for significant Nef activities would then be preferred targets for developing drug-like inhibitors of Nef function [71].
Cell lines and culture conditions
Human CEM T cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (Hyclone), 50 IU of penicillin per ml, 50 μg streptomycin per ml, 2 mM L-glutamine and 1 mM sodium pyruvate. HEK 293T cells were cultured in DMEM with 10% fetal bovine serum, 50 IU of penicillin per ml, 50 μg/ml of streptomycin and 2 mM L-glutamine. HeLa-MAGI cells were cultured in the presence of 1.0 mg/ml puromycin. Cell lines were maintained at 37°C in a humidified incubator with 5% CO2 (CEM) or 10% CO2 (293T and HeLa-MAGI).
Plasmid DNA constructs
The pcDNA3.1-SF2Nef plasmid DNA expression vector was utilized for transient transfection of 293T cells. Bacteria expression vectors were pGEX2TSF2Nef which expresses full length Nef as a GST fusion protein and pET28aHckSH3 which expresses amino acids 61–117 of Homo sapiens Hck (gi:56203176) in frame with polyhistidine. The pLXSN, pLNefSNSF2 (wild type or mutated), and pEQPAM vectors were transfected into HEK 293T cells to generate amphotropic vectors for subsequent transduction of CEM T cells. The p9B18 HIV-1SF2 proviral clone was transfected into HEK 293T producer cells to generate infectious virus for HeLA-MAGI infectivity assays. Point mutations were made by site-directed mutagenesis in SF2Nef as previously described [72]. All mutations were verified by DNA sequencing and are numbered in accordance with the standard Nef length of 206 amino acids.
Transfection of HEK 293T cells
One day prior to transfection, HEK 293T cells were plated at 800,000 cells per well in six-well plates such that the cells would be 90% confluent upon transfection. The pcDNA3.1, pcDNA3.1-SF2Nef, p9B18-SF2, pLXSN and pEQPAM plasmid DNA vectors were transfected into the cells using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. Four hours post-transfection, the culture medium was replaced with fresh DMEM with 10% fetal bovine serum and antibiotics. 36–48 hours post-transfection, cell lysates and/or virus supernatants were harvested for subsequent experiments.
Transduction of human CEM T cells
pEQPAM (2 μg) and pLNefSNSF2 (2 μg) were co-transfected into HEK 293T cells to generate amphotropic virus as described above. Harvested viral supernatants were filtered through 0.45 μm filters. 24-well plates were coated with 400 μg of Retronectin (Takara) followed by 500 μl of 2% bovine serum albumin in phosphate buffered saline. Wells were then rinsed and virus was absorbed upon incubation with 500 μl of virus supernatant twice at one-hour intervals. CEM T cells (300,000 per well) were incubated overnight. The next day, a third addition of 500 μl of viral supernatant was added on top of the cells. Twenty four hours later, cells were collected, washed and re-suspended in complete RPMI containing 1.5 mg/ml G418 for selection. Cells were expanded into T-75 cm2 flasks, at which point the cells were harvested for subsequent Western blot analysis, in vitro kinase assay, and flow cytometric analysis.
Western blot analysis and antibodies
Cells were washed in phosphate-buffered saline and lysed in 1 ml of Buffer A (50 mM Tris, pH = 8.0, 100 mM NaCl, 25 mM NaF, 25 mM benzamidine, 20 mM β-glycerophosphate, 2 mM Na3VO2, 3 mM EDTA, 10% glycerol, and 0.5% IGEPAL-630) containing one Roche protease inhibitor tablet per 10 ml. Lysates were centrifuged at 13,000 X g for 30 minutes and the supernatant fraction was prepared for SDS-PAGE gel electrophoresis. Separated proteins were transferred to nitrocellulose. The transferred blots were blocked with 10% milk and subsequently incubated with primary antibodies for one-hour at room-temperature, washed, and probed with HRP-conjugated secondary antibodies. Western blots were visualized by chemiluminescence upon incubation with luminol and p-coumaric acid. Nef protein expression was determined using the sheep polyclonal anti-SF2Nef serum, as previously described [32, 72].
In vitro kinase assay
Endogenous Nef/activated PAK2 complex formation assays were performed as previously described [32, 72]. Nef-expressing 293T cells were lysed in Buffer A containing one Roche protease inhibitor tablet per 10 ml. Lysate containing 600 μg of protein was completely immunoprecipitated with 10 μl sheep anti-SF2 Nef serum and protein A agarose beads (Sigma) [39]. In one experiment ( Additional file 1: Figure S1A), 10 μl of anti-PAK2 antibody (Cell Signaling) was used. Protein A beads were washed twice in lysis buffer, once in 1M MgCl2, and twice in kinase buffer (10 mM Tris, pH = 7.5, 5 mM MgCl2 and 1% Triton X-100). The beads were then resuspended in 100 μl of kinase buffer. γ-32P]-ATP was incubated with the immunoprecipitates for 10 minutes at room temperature. The kinase reaction was terminated upon addition of EDTA. Proteins were eluted with SDS Laemmli buffer and resolved by 9% SDS-PAGE. Phosphorylated proteins were quantitated by phosphoimager analysis (Packard Cyclone). In Additional file 1: Figure S1B, the combined assay of PAK2 atuophosphorylation and myelin basic protein phosphorylation was performed in identical fashion to the autophosphorylation assay except that 15 μg of myelin basic protein were added to the 0.1 ml autophosphorylation reaction.
Flow cytometry analysis
For analysis of cell surface CD4 and MHCI levels, transduced CEM cells (500,000) were first incubated with mouse monoclonal anti-haplotype A1, A11, and A26 MHC class I antibody (One Lambda) for 20 minutes on ice and then washed. Cells were then incubated with fluorescein isothiocyanate (FITC)-labeled rabbit anti-mouse IgG for 20 minutes on ice and then washed. Cells were then incubated with 2 μg of mouse IgG for 20 minutes on ice and then washed. Cells were then incubated with phycoerythrin (PE)-conjugated IgG1 monoclonal antibody to human CD4 (Exalpha) for 20 minutes on ice and then washed. Stained cells were analyzed on a BD FACSCalibur instrument equipped with CellquestPro software. CEM cells transduced with LXSN served as the positive control. For negative controls mouse isotype antibody replaced anti-MHC class I, and PE-conjugated mouse IgG1 (Exalpha) replaced PE-conjugated anti-CD4.
HeLa-MAGI infectivity assays
As described above, HEK 293T cells were transfected with the proviral clone for SF2 (p9B18) to generate infectious virus. Virus was harvested 48 hours post-transfection, filtered through a 0.45-μm filter, and stored frozen in aliquots at −70°C. The virus produced was normalized by p24gag ELISA. HeLa-MAGI cells (80,000) were plated in 12-well plates and infected 24 hours later in triplicate with 5 ng of p24 gag of virus in 400 μl of cell culture medium containing 20 μg/ml of DEAE-dextran (Sigma) at 37°C. After a two-hour incubation, fresh culture medium was added, and the cells were incubated for an additional 36 hours. The cells were then fixed and stained with X-gal. Blue cells were counted as indicators of infected cells. Expression of Nef and p24gag in lysates of 293T producer cells was analyzed by Western blot analysis as described above.
In vitro binding assay
BL-21 pLysS bacteria were transformed with pGEX4T, pGEX2TSF2Nef, pGEX2TSF2NefP72G, pGEX2TSF2NefQ73R, pGEX2TSF2NefV74I, pGEX2TSF2NefP75G, pGEX2TSF2NefL76V, pGEX2TSF2NefR77K, pGEX2TSF2NefP78G, pGEX2TSF2NefF90A, and pET28HckSH3 (Hck amino acids 61-117). The fusion protein derived from pGEX2TSF2Nef has been previously shown to bind the SH3 domain of Hck in pulldown assays [29]. A 10 ml starter culture in Luria-Bertani medium with 34 μg/ml chloramphenicol and either 50 μg/ml of ampicillin (pGEX construct) or kanamycin (pET28 construct) was initiated from a single colony, grown at 37°C 225 RPM, and used to inoculate 250 ml of Luria-Bertani medium with antibiotics as described previously [29]. The 250 ml culture was grown at 37°C, 225 RPM until optical density at 600 nm reached 0.6. Isopropyl-ß-D-thiogalactopyranoside was added to the culture to a final concentration of 3 mM and allowed to induce for 3 hours shaking at 37°C and 225 RPM. Bacteria were pelleted and stored frozen at -20°C.
Bacteria pellets containing glutathione S-transferase (GST) fusion proteins or histidine (His) tagged protein were resuspended in 15ml of PBS containing one tablet of Roche protease inhibitor per 10 ml. The resuspended bacteria were sonicated on ice using a microtip for 1 minute at power level 3.5; 10s on and 10s off. Bacterial lysates were centrifuged at 10,500 RPM, 4°C, 20 minutes. Supernatants of GST fusion proteins were rotated with 0.5 ml packed glutathione Sepharose beads (GE Healthcare) for 30 min at room temperature on a rotator. His-tagged protein was incubated with 0.5 ml packed HisPur Cobalt Resin (Pierce) for 30 minutes at 4°C on a rotator. At 4°C, the Sepharose beads were washed with 10ml cold PBS three times, and HisPur cobalt resin was washed with 6ml HisPur wash buffer (50 mM sodium phosphate, 300 mM NaCl, and 10 mM imidazole) three times. The bound GST fusion protein was eluted with 20 mM reduced glutathione pH 8.0, and the bound His-tagged protein was eluted with HisPur elution buffer (50 mM sodium phosphate, 300 mM NaCl, 150 mM imidazole). Concentration of all proteins was determined by Bradford; purity was determined by SDS-PAGE electrophoresis; and each protein was exhaustively dialyzed in binding buffer (50 mM Tris pH7.5, 1% IGEPAL CA-630, 2 mM MgCl2, 200 mM NaCl and 5 mM ß-mercaptoethanol). Binding assays with proteins at 3 μM were performed as described previously [29] and analyzed by Western blotting with polyclonal anti-polyhistidine (Abcam).
We are especially grateful to Kalle Saksela for pGEX-4T-1-Hck-SH3 and Paul Luciw for the SF2 proviral clone, p9B18. We also thank Paul Denton for assistance in making the Figures. This work was supported by Grant AI33331 from the National Institute of Allergy and Infectious Diseases of the National Institutes of Health, USA and UNC CFAR P30 A1504410. Richard L Watkins was supported by NIH Virology Training Grant 5T32A1007419. Mingjie Liu was supported by UNC AIDS International Training and Research Program: 5 D43 TW001039-14.
Authors' Affiliations
- Fackler OT, Moris A, Tibroni N, Giese SI, Glass B, Schwartz O, Krausslich HG: Functional characterization of HIV-1 Nef mutants in the context of viral infection. Virology. 2006, 351: 322-339. 10.1016/j.virol.2006.03.044.View ArticlePubMedGoogle Scholar
- Foster JL, Garcia JV: HIV-1 Nef: at the crossroads. Retrovirology. 2008, 5: 84-10.1186/1742-4690-5-84.PubMed CentralView ArticlePubMedGoogle Scholar
- Foster JL, Garcia JV: Role of Nef in HIV-1 replication and pathogenesis. Adv Pharmacol. 2007, 55: 389-409.View ArticlePubMedGoogle Scholar
- Olivieri KC, Mukerji J, Gabuzda D: Nef-mediated enhancement of cellular activation and human immunodeficiency virus type 1 replication in primary T cells is dependent on association with p21-activated kinase 2. Retrovirology. 2011, 8: 64-10.1186/1742-4690-8-64.PubMed CentralView ArticlePubMedGoogle Scholar
- Collins KL, Chen BK, Kalams SA, Walker BD, Baltimore D: HIV-1 Nef protein protects infected primary cells against killing by cytotoxic T lymphocytes. Nature. 1998, 391: 397-401. 10.1038/34929.View ArticlePubMedGoogle Scholar
- Tomiyama H, Akari H, Adachi A, Takiguchi M: Different effects of Nef-mediated HLA class I down-regulation on human immunodeficiency virus type 1-specific CD8(+) T-cell cytolytic activity and cytokine production. J Virol. 2002, 76: 7535-7543. 10.1128/JVI.76.15.7535-7543.2002.PubMed CentralView ArticlePubMedGoogle Scholar
- Yang OO, Nguyen PT, Kalams SA, Dorfman T, Gottlinger HG, Stewart S, Chen IS, Threlkeld S, Walker BD: Nef-mediated resistance of human immunodeficiency virus type 1 to antiviral cytotoxic T lymphocytes. J Virol. 2002, 76: 1626-1631. 10.1128/JVI.76.4.1626-1631.2002.PubMed CentralView ArticlePubMedGoogle Scholar
- Cortes MJ, Wong-Staal F, Lama J: Cell surface CD4 interferes with the infectivity of HIV-1 particles released from T cells. J Biol Chem. 2002, 277: 1770-1779. 10.1074/jbc.M109807200.View ArticlePubMedGoogle Scholar
- Lundquist CA, Tobiume M, Zhou J, Unutmaz D, Aiken C: Nef-mediated downregulation of CD4 enhances human immunodeficiency virus type 1 replication in primary T lymphocytes. J Virol. 2002, 76: 4625-4633. 10.1128/JVI.76.9.4625-4633.2002.PubMed CentralView ArticlePubMedGoogle Scholar
- Ross TM, Oran AE, Cullen BR: Inhibition of HIV-1 progeny virion release by cell-surface CD4 is relieved by expression of the viral Nef protein. Curr Biol. 1999, 9: 613-621. 10.1016/S0960-9822(99)80283-8.View ArticlePubMedGoogle Scholar
- Pizzato M, Helander A, Popova E, Calistri A, Zamborlini A, Palu G, Gottlinger HG: Dynamin 2 is required for the enhancement of HIV-1 infectivity by Nef. Proc Natl Acad Sci U S A. 2007, 104: 6812-6817. 10.1073/pnas.0607622104.PubMed CentralView ArticlePubMedGoogle Scholar
- Baur AS: HIV-Nef and AIDS pathogenesis: are we barking up the wrong tree?. Trends Microbiol. 2011, 19: 435-440. 10.1016/j.tim.2011.06.002.View ArticlePubMedGoogle Scholar
- Mitchell RS, Chaudhuri R, Lindwasser OW, Tanaka KA, Lau D, Murillo R, Bonifacino JS, Guatelli JC: Competition model for upregulation of the major histocompatibility complex class II-associated invariant chain by human immunodeficiency virus type 1 Nef. J Virol. 2008, 82: 7758-7767. 10.1128/JVI.02668-07.PubMed CentralView ArticlePubMedGoogle Scholar
- Grzesiek S, Stahl SJ, Wingfield PT, Bax A: The CD4 determinant for downregulation by HIV-1 Nef directly binds to Nef. Mapping of the Nef binding surface by NMR. Biochemistry. 1996, 35: 10256-10261. 10.1021/bi9611164.View ArticlePubMedGoogle Scholar
- Noviello CM, Benichou S, Guatelli JC: Cooperative binding of the class I major histocompatibility complex cytoplasmic domain and human immunodeficiency virus type 1 Nef to the endosomal AP-1 complex via its mu subunit. J Virol. 2008, 82: 1249-1258. 10.1128/JVI.00660-07.PubMed CentralView ArticlePubMedGoogle Scholar
- Preusser A, Briese L, Baur AS, Willbold D: Direct in vitro binding of full-length human immunodeficiency virus type 1 Nef protein to CD4 cytoplasmic domain. J Virol. 2001, 75: 3960-3964. 10.1128/JVI.75.8.3960-3964.2001.PubMed CentralView ArticlePubMedGoogle Scholar
- Preusser A, Briese L, Willbold D: Presence of a helix in human CD4 cytoplasmic domain promotes binding to HIV-1 Nef protein. Biochem Biophys Res Commun. 2002, 292: 734-740. 10.1006/bbrc.2002.6700.View ArticlePubMedGoogle Scholar
- Singh RK, Lau D, Noviello CM, Ghosh P, Guatelli JC: An MHC-I cytoplasmic domain/HIV-1 Nef fusion protein binds directly to the mu subunit of the AP-1 endosomal coat complex. PLoS One. 2009, 4: e8364-10.1371/journal.pone.0008364.PubMed CentralView ArticlePubMedGoogle Scholar
- Wonderlich ER, Williams M, Collins KL: The tyrosine binding pocket in the adaptor protein 1 (AP-1) mu1 subunit is necessary for Nef to recruit AP-1 to the major histocompatibility complex class I cytoplasmic tail. J Biol Chem. 2008, 283: 3011-3022.View ArticlePubMedGoogle Scholar
- Manninen A, Hiipakka M, Vihinen M, Lu W, Mayer BJ, Saksela K: SH3-Domain binding function of HIV-1 Nef is required for association with a PAK-related kinase. Virology. 1998, 250: 273-282. 10.1006/viro.1998.9381.View ArticlePubMedGoogle Scholar
- Dikeakos JD, Atkins KM, Thomas L, Emert-Sedlak L, Byeon IJ, Jung J, Ahn J, Wortman MD, Kukull B, Saito M, et al: Small molecule inhibition of HIV-1-induced MHC-I down-regulation identifies a temporally regulated switch in Nef action. Mol Biol Cell. 2010, 21: 3279-3292. 10.1091/mbc.E10-05-0470.PubMed CentralView ArticlePubMedGoogle Scholar
- Youker RT, Shinde U, Day R, Thomas G: At the crossroads of homoeostasis and disease: roles of the PACS proteins in membrane traffic and apoptosis. Biochem J. 2009, 421: 1-15. 10.1042/BJ20081016.PubMed CentralView ArticlePubMedGoogle Scholar
- Arold S, Franken P, Strub MP, Hoh F, Benichou S, Benarous R, Dumas C: The crystal structure of HIV-1 Nef protein bound to the Fyn kinase SH3 domain suggests a role for this complex in altered T cell receptor signaling. Structure. 1997, 5: 1361-1372. 10.1016/S0969-2126(97)00286-4.View ArticlePubMedGoogle Scholar
- Lee CH, Saksela K, Mirza UA, Chait BT, Kuriyan J: Crystal structure of the conserved core of HIV-1 Nef complexed with a Src family SH3 domain. Cell. 1996, 85: 931-942. 10.1016/S0092-8674(00)81276-3.View ArticlePubMedGoogle Scholar
- Kay BK, Williamson MP, Sudol M: The importance of being proline: the interaction of proline-rich motifs in signaling proteins with their cognate domains. FASEB J. 2000, 14: 231-241.PubMedGoogle Scholar
- Li SS: Specificity and versatility of SH3 and other proline-recognition domains: structural basis and implications for cellular signal transduction. Biochem J. 2005, 390: 641-653.PubMed CentralView ArticlePubMedGoogle Scholar
- Musacchio A: How SH3 domains recognize proline. Adv Protein Chem. 2002, 61: 211-268.View ArticlePubMedGoogle Scholar
- Zarrinpar A, Bhattacharyya RP, Lim WA: The structure and function of proline recognition domains. Sci STKE. 2003, 2003: RE8-PubMedGoogle Scholar
- Baugh LL, Garcia JV, Foster JL: Functional characterization of the human immunodeficiency virus type 1 Nef acidic domain. J Virol. 2008, 82: 9657-9667. 10.1128/JVI.00107-08.PubMed CentralView ArticlePubMedGoogle Scholar
- Casartelli N, Giolo G, Neri F, Haller C, Potesta M, Rossi P, Fackler OT, Doria M: The Pro78 residue regulates the capacity of the human immunodeficiency virus type 1 Nef protein to inhibit recycling of major histocompatibility complex class I molecules in an SH3-independent manner. J Gen Virol. 2006, 87: 2291-2296. 10.1099/vir.0.81775-0.View ArticlePubMedGoogle Scholar
- Kwak YT, Raney A, Kuo LS, Denial SJ, Temple BR, Garcia JV, Foster JL: Self-association of the Lentivirus protein, Nef. Retrovirology. 2010, 7: 77-10.1186/1742-4690-7-77.PubMed CentralView ArticlePubMedGoogle Scholar
- O'Neill E, Kuo LS, Krisko JF, Tomchick DR, Garcia JV, Foster JL: Dynamic evolution of the human immunodeficiency virus type 1 pathogenic factor, Nef. J Virol. 2006, 80: 1311-1320. 10.1128/JVI.80.3.1311-1320.2006.PubMed CentralView ArticlePubMedGoogle Scholar
- Yamada T, Kaji N, Odawara T, Chiba J, Iwamoto A, Kitamura Y: Proline 78 is crucial for human immunodeficiency virus type 1 Nef to down-regulate class I human leukocyte antigen. J Virol. 2003, 77: 1589-1594. 10.1128/JVI.77.2.1589-1594.2003.PubMed CentralView ArticlePubMedGoogle Scholar
- Jia X, Singh R, Homann S, Yang H, Guatelli J, Xiong Y: Structural Basis of HIV-1 Nef mediated Down-regulation of MHC-1. 19th Conference on Retroviruses and Opportunistic Infections. 2012, Paper #40LBGoogle Scholar
- Henikoff S, Henikoff JG: Amino acid substitution matrices from protein blocks. Proc Natl Acad Sci U S A. 1992, 89: 10915-10919. 10.1073/pnas.89.22.10915.PubMed CentralView ArticlePubMedGoogle Scholar
- Arold S, O'Brien R, Franken P, Strub MP, Hoh F, Dumas C, Ladbury JE: RT loop flexibility enhances the specificity of Src family SH3 domains for HIV-1 Nef. Biochemistry. 1998, 37: 14683-14691. 10.1021/bi980989q.View ArticlePubMedGoogle Scholar
- Lee CH, Leung B, Lemmon MA, Zheng J, Cowburn D, Kuriyan J, Saksela K: A single amino acid in the SH3 domain of Hck determines its high affinity and specificity in binding to HIV-1 Nef protein. Embo J. 1995, 14: 5006-5015.PubMed CentralPubMedGoogle Scholar
- Collette Y, Arold S, Picard C, Janvier K, Benichou S, Benarous R, Olive D, Dumas C: HIV-2 and SIV nef proteins target different Src family SH3 domains than does HIV-1 Nef because of a triple amino acid substitution. J Biol Chem. 2000, 275: 4171-4176. 10.1074/jbc.275.6.4171.View ArticlePubMedGoogle Scholar
- Arora VK, Molina RP, Foster JL, Blakemore JL, Chernoff J, Fredericksen BL, Garcia JV: Lentivirus Nef specifically activates Pak2. J Virol. 2000, 74: 11081-11087. 10.1128/JVI.74.23.11081-11087.2000.PubMed CentralView ArticlePubMedGoogle Scholar
- Wei BL, Arora VK, Raney A, Kuo LS, Xiao GH, O'Neill E, Testa JR, Foster JL, Garcia JV: Activation of p21-activated kinase 2 by human immunodeficiency virus type 1 Nef induces merlin phosphorylation. J Virol. 2005, 79: 14976-14980. 10.1128/JVI.79.23.14976-14980.2005.PubMed CentralView ArticlePubMedGoogle Scholar
- Wiskerchen M, Cheng-Mayer C: HIV-1 Nef association with cellular serine kinase correlates with enhanced virion infectivity and efficient proviral DNA synthesis. Virology. 1996, 224: 292-301. 10.1006/viro.1996.0531.View ArticlePubMedGoogle Scholar
- Karkkainen S, Hiipakka M, Wang JH, Kleino I, Vaha-Jaakkola M, Renkema GH, Liss M, Wagner R, Saksela K: Identification of preferred protein interactions by phage-display of the human Src homology-3 proteome. EMBO Rep. 2006, 7: 186-191. 10.1038/sj.embor.7400596.PubMed CentralView ArticlePubMedGoogle Scholar
- Renkema GH, Manninen A, Saksela K: Human immunodeficiency virus type 1 Nef selectively associates with a catalytically active subpopulation of p21-activated kinase 2 (PAK2) independently of PAK2 binding to Nck or beta-PIX. J Virol. 2001, 75: 2154-2160. 10.1128/JVI.75.5.2154-2160.2001.PubMed CentralView ArticlePubMedGoogle Scholar
- Blagoveshchenskaya AD, Thomas L, Feliciangeli SF, Hung CH, Thomas G: HIV-1 Nef downregulates MHC-I by a PACS-1- and PI3K-regulated ARF6 endocytic pathway. Cell. 2002, 111: 853-866. 10.1016/S0092-8674(02)01162-5.View ArticlePubMedGoogle Scholar
- Greenberg ME, Iafrate AJ, Skowronski J: The SH3 domain-binding surface and an acidic motif in HIV-1 Nef regulate trafficking of class I MHC complexes. Embo J. 1998, 17: 2777-2789. 10.1093/emboj/17.10.2777.PubMed CentralView ArticlePubMedGoogle Scholar
- Wonderlich ER, Leonard JA, Kulpa DA, Leopold KE, Norman JM, Collins KL: ADP ribosylation factor 1 activity is required to recruit AP-1 to the major histocompatibility complex class I (MHC-I) cytoplasmic tail and disrupt MHC-I trafficking in HIV-1-infected primary T cells. J Virol. 2011, 85: 12216-12226. 10.1128/JVI.00056-11.PubMed CentralView ArticlePubMedGoogle Scholar
- Craig HM, Pandori MW, Riggs NL, Richman DD, Guatelli JC: Analysis of the SH3-binding region of HIV-1 nef: partial functional defects introduced by mutations in the polyproline helix and the hydrophobic pocket. Virology. 1999, 262: 55-63. 10.1006/viro.1999.9897.View ArticlePubMedGoogle Scholar
- Goldsmith MA, Warmerdam MT, Atchison RE, Miller MD, Greene WC: Dissociation of the CD4 downregulation and viral infectivity enhancement functions of human immunodeficiency virus type 1 Nef. J Virol. 1995, 69: 4112-4121.PubMed CentralPubMedGoogle Scholar
- Chutiwitoonchai N, Hiyoshi M, Mwimanzi P, Ueno T, Adachi A, Ode H, Sato H, Fackler OT, Okada S, Suzu S: The identification of a small molecule compound that reduces HIV-1 Nef-mediated viral infectivity enhancement. PLoS One. 2011, 6: e27696-10.1371/journal.pone.0027696.PubMed CentralView ArticlePubMedGoogle Scholar
- Luo T, Livingston RA, Garcia JV: Infectivity enhancement by human immunodeficiency virus type 1 Nef is independent of its association with a cellular serine/threonine kinase. J Virol. 1997, 71: 9524-9530.PubMed CentralPubMedGoogle Scholar
- Lo Conte L, Chothia C, Janin J: The atomic structure of protein-protein recognition sites. J Mol Biol. 1999, 285: 2177-2198. 10.1006/jmbi.1998.2439.View ArticlePubMedGoogle Scholar
- Choi HJ, Smithgall TE: Conserved residues in the HIV-1 Nef hydrophobic pocket are essential for recruitment and activation of the Hck tyrosine kinase. J Mol Biol. 2004, 343: 1255-1268. 10.1016/j.jmb.2004.09.015.View ArticlePubMedGoogle Scholar
- Liu LX, Heveker N, Fackler OT, Arold S, Le Gall S, Janvier K, Peterlin BM, Dumas C, Schwartz O, Benichou S, Benarous R: Mutation of a conserved residue (D123) required for oligomerization of human immunodeficiency virus type 1 Nef protein abolishes interaction with human thioesterase and results in impairment of Nef biological functions. J Virol. 2000, 74: 5310-5319. 10.1128/JVI.74.11.5310-5319.2000.PubMed CentralView ArticlePubMedGoogle Scholar
- Foster JL, Denial SJ, Temple BR, Garcia JV: Mechanisms of HIV-1 Nef function and intracellular signaling. J Neuroimmune Pharmacol. 2011, 6: 230-246. 10.1007/s11481-011-9262-y.PubMed CentralView ArticlePubMedGoogle Scholar
- Bogan AA, Thorn KS: Anatomy of hot spots in protein interfaces. J Mol Biol. 1998, 280: 1-9. 10.1006/jmbi.1998.1843.View ArticlePubMedGoogle Scholar
- Cunningham BC, Wells JA: Comparison of a structural and a functional epitope. J Mol Biol. 1993, 234: 554-563. 10.1006/jmbi.1993.1611.View ArticlePubMedGoogle Scholar
- Ali SA, Huang MB, Campbell PE, Roth WW, Campbell T, Khan M, Newman G, Villinger F, Powell MD, Bond VC: Genetic characterization of HIV type 1 Nef-induced vesicle secretion. AIDS Res Hum Retroviruses. 2010, 26: 173-192. 10.1089/aid.2009.0068.PubMed CentralView ArticlePubMedGoogle Scholar
- Shelton MN, Huang MB, Ali SA, Powell MD, Bond VC: Secretion modification region-derived peptide disrupts HIV-1 Nef's interaction with mortalin and blocks virus and Nef exosome release. J Virol. 2012, 86: 406-419. 10.1128/JVI.05720-11.PubMed CentralView ArticlePubMedGoogle Scholar
- Jung J, Byeon IJ, Ahn J, Gronenborn AM: Structure, dynamics, and Hck interaction of full-length HIV-1 Nef. Proteins. 2011, 79: 1609-1622. 10.1002/prot.22986.PubMed CentralView ArticlePubMedGoogle Scholar
- Arold S, Hoh F, Domergue S, Birck C, Delsuc MA, Jullien M, Dumas C: Characterization and molecular basis of the oligomeric structure of HIV-1 nef protein. Protein Sci. 2000, 9: 1137-1148. 10.1110/ps.9.6.1137.PubMed CentralView ArticlePubMedGoogle Scholar
- Poe JA, Smithgall TE: HIV-1 Nef dimerization is required for Nef-mediated receptor downregulation and viral replication. J Mol Biol. 2009, 394: 329-342. 10.1016/j.jmb.2009.09.047.PubMed CentralView ArticlePubMedGoogle Scholar
- Petersen JL, Morris CR, Solheim JC: Virus evasion of MHC class I molecule presentation. J Immunol. 2003, 171: 4473-4478.View ArticlePubMedGoogle Scholar
- Swigut T, Iafrate AJ, Muench J, Kirchhoff F, Skowronski J: Simian and human immunodeficiency virus Nef proteins use different surfaces to downregulate class I major histocompatibility complex antigen expression. J Virol. 2000, 74: 5691-5701. 10.1128/JVI.74.12.5691-5701.2000.PubMed CentralView ArticlePubMedGoogle Scholar
- Akari H, Arold S, Fukumori T, Okazaki T, Strebel K, Adachi A: Nef-induced major histocompatibility complex class I down-regulation is functionally dissociated from its virion incorporation, enhancement of viral infectivity, and CD4 down-regulation. J Virol. 2000, 74: 2907-2912. 10.1128/JVI.74.6.2907-2912.2000.PubMed CentralView ArticlePubMedGoogle Scholar
- Iijima S, Lee YJ, Ode H, Arold S, Kimura N, Yokoyama M, Sato H, Tanaka Y, Strebel K, Akari H: A non-canonical mu-1A-binding motif in the N-terminus of HIV-1 Nef determines its activity to down-regulate MHC-I in T lymphocytes. J Virol. 2012, 86: 3944-3951. 10.1128/JVI.06257-11.PubMed CentralView ArticlePubMedGoogle Scholar
- Denton PW, Estes JD, Sun Z, Othieno FA, Wei BL, Wege AK, Powell DA, Payne D, Haase AT, Garcia JV: Antiretroviral pre-exposure prophylaxis prevents vaginal transmission of HIV-1 in humanized BLT mice. PLoS Med. 2008, 5: e16-10.1371/journal.pmed.0050016.PubMed CentralView ArticlePubMedGoogle Scholar
- Denton PW, Krisko JF, Powell DA, Mathias M, Kwak YT, Martinez-Torres F, Zou W, Payne DA, Estes JD, Garcia JV: Systemic administration of antiretrovirals prior to exposure prevents rectal and intravenous HIV-1 transmission in humanized BLT mice. PLoS One. 2010, 5: e8829-10.1371/journal.pone.0008829.PubMed CentralView ArticlePubMedGoogle Scholar
- Melkus MW, Estes JD, Padgett-Thomas A, Gatlin J, Denton PW, Othieno FA, Wege AK, Haase AT, Garcia JV: Humanized mice mount specific adaptive and innate immune responses to EBV and TSST-1. Nat Med. 2006, 12: 1316-1322. 10.1038/nm1431.View ArticlePubMedGoogle Scholar
- Zou W, Denton PW, Watkins R, Krisko JF, Nochi T, Foster JL, Victor Garcia J: Nef functions in BLT mice to enhance HIV-1 replication and deplete CD4+CD8+ 2 thymocytes. Retrovirology. 2012, 2012: 2012-in pressGoogle Scholar
- Meuwissen PJ, Stolp B, Iannucci V, Vermeire J, Naessens E, Saksela K, Geyer M, Vanham G, Arien KK, Fackler OT, Verhasselt B: Identification of a highly conserved valine-glycine-phenylalanine amino acid triplet required for HIV-1 Nef function. Retrovirology. 2012, 9: 34-10.1186/1742-4690-9-34.PubMed CentralView ArticlePubMedGoogle Scholar
- Betzi S, Restouin A, Opi S, Arold ST, Parrot I, Guerlesquin F, Morelli X, Collette Y: Protein protein interaction inhibition (2P2I) combining high throughput and virtual screening: Application to the HIV-1 Nef protein. Proc Natl Acad Sci U S A. 2007, 104: 19256-19261. 10.1073/pnas.0707130104.PubMed CentralView ArticlePubMedGoogle Scholar
- Foster JL, Molina RP, Luo T, Arora VK, Huang Y, Ho DD, Garcia JV: Genetic and functional diversity of human immunodeficiency virus type 1 subtype B Nef primary isolates. J Virol. 2001, 75: 1672-1680. 10.1128/JVI.75.4.1672-1680.2001.PubMed CentralView ArticlePubMedGoogle Scholar
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Wind Man
film soundtracks
Yuri Poteyenko
3.99 usd + shipping
Loosely based on A Very Old Man with Enormous Wings by Gabriel Garcia Marquez, Khuat Akhmetov's Wind Man (Chelovek-veter) takes place in the steppes of Kazakhstan where one unsuspecting village is about to meet an extraordinary visitor. During the middle of a thunderstorm, a man with wings (played by Igor Yasulovich) falls onto the ground. Is he an angel? Or is he the devil? What are his intentions? It all doesn't matter, because the wing man is just a catalyst of crazy events, a serious of vignettes that unveil the corrupt nature of post-Soviet societies, who can't tolerate anything that doesn't fit into the category of normal.
After the discovery of the frail man, the villagers have an argument about what to do with him. While one Koranic scholar believes that the man is some sort of angel, the village mullah (Muslim leader) condemns the oddity a demon simply because he fails to understand Allah's language. Then it's up to the fat capitalist to make final judgment on the man. When he concludes that the "angel" will fail to turn any profit and is just a financial liability, the poor soul is officially labeled as a burden. Village officials finally decide by the banning of the anomaly, who is still struggling to fly away…
Poteyenko's haunting score gives the story an eternal, magical quality that's also a great way to represent the deliberate anachronisms within the film. The music illustrates the great variety of reactions people approach the angel with: some are friendly and curious, others are neutral while the last group is religiously charged and frantically evil. From the percussive hunt to the glorious choral beauty of resurrection, you can also enjoy the winged man's fantastic adventure through the illuminating score that also cleverly incorporates the sound of wind into its fabric.
Collectors edition of 1000 copies.
Label: KMRCD 004
Date: 2007
Tracks: 13
Time: 28"03
01 - Appearance of Madar (01:53)
02 - Fall of an Angel (Main Titles) (02:41)
03 -
"He Reminds Me of Someone"
04 - Madar - Harbinger of Death (01:58)
05 -
New Friend
06 - Angel in Anger (01:20)
07 - Flight / Broken wing (02:26)
08 - Madar is Close (00:47)
09 - Hunting Madar (03:25)
10 -
"I Will Be Flying" / Fall
11 -
Wind Man
12 -
13 -
© Copyright KeepMoving Records 2006

subset: commoncrawl


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Posts Tagged With: lime juice
Pâtes aux Saucisson Sauce Blanche ( Pasta with a Sausage Creme Sauce)
The combination of flavors in this incredible pasta dish will blow you away. The French elements of dried sausage (saucisson) and creme fraiche makes it feel like home to me. So simple and can be made easily in less than 3o minutes!! The Recipe! ~ Pasta of choice. I used Orecchiette. Bow tie pasta would … Continue reading
Categories: Fun Stuff Tags: 30 minutes, butter, creme fraiche, dried sausage, lime juice, mushrooms, pasta, rosemary, saucisson Leave a comment


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The Glass Reunion
April 30, 2013 in Technology at Home, Technology at Work, Technology for Fun, Technology on the Move | Tags: application software, class reunion, digital technology, gadget nut, Google Glass, mobile technology, mobility, social media, software, technology | 1 comment
Class reunions are events unlike any other in our life experience. What I mean by that is they offer us a chance to measure or at least notice specific net changes in our cultural milieu. Since they occur at regular ten-year intervals and because you typically only see the folks in your class during the once per decade event, they make it easy to see how much life has changed since the last get together.
For instance, if you attended your reunion in 1980, you probably didn't see a single cell phone or video cam. That's because 1980 was still the era of land-lines and Kodak Instamatics.
By 1990, there were likely a number of cell phones and a few video cameras. We would laugh at these people today because the cell phones would look like World War Two era Army field phones and the Video cameras would make you look like the crew backing up Mike Wallace on 60 Minutes. How could we think THAT was cool?
I went to a reunion in 1999 and I was amazed at the number of video recorders. They were starting to get pretty small by then. I remember looking across the banquet room and seeing about fifty of these things just rolling, taking in the ambiance of the Great American High School Reunion. Our reunion featured plentiful amounts of free booze so I'm sure the antics of my classmates, and me for that matter, were recorded for future use as blackmail fodder.
Now, we are about to enter another one of those technologically significant, yet potentially embarrassing eras. Google Glass will soon become as ubiquitous as the video cam and the smart phone. Will it be the tech equivalent of the duck-tail or mullet? Or, will it be world changing?
Personally, I'm skeptical. Imagine walking into the ball room to register. The person behind the table is smiling sweetly and looks vaguely familiar. Before she can say, "Hi, I'm …." you are mumbling to your eyepiece to activate your facial recognition software. Glass quickly whispers her name into your ear "Betty Jo Belitnikoff".
"Why Betty Jo! You haven't changed a bit. I love the blue rinse and those sensible shoes . . . very cool!"
That happens to be the last thing you say directly to any of your classmates that night. The rest of the evening, you are conversing with the Glass friend dangling in front of your eyeball. Everyone else is doing the same thing.
All you hear is low murmuring of Glass commands – Identify Face, . . .record, . . . retrieve email, . . .open Facebook, . . . open LinkedIn. . . . message to Frank.
And so it goes all night long. Everyone is prowling around identifying faces and pulling up cyber versions of each other. No direct conversation is required. Everyone who wants to interact can simply message via Glass. An eerie silence hovers over the entire room. It's like a zombie convention.
There are undeniably definite advantages. You get to avoid answering the unpleasant questions and you don't have to listen to boring stories about kids and pets. You are in control; you can ignore or dig as deeply into the life of each classmate as you might care too. If it gets too bad, you'll likely have the option to pull up something to stream from Netflix.
This is when you realize that the real world and the cyber or virtual worlds have partially merged. It is so disorienting, you are suddenly not sure what has happened or where you are.
Are you a warm blooded human visiting a virtual class reunion? Have you somehow been sucked into the virtual world where you can only interact via the technology you bring with you.
Or, has the virtual world expanded into the three dimensional universe, blurring the once distinct boundaries into a sort of cyber no man's land? Has your Second Life merged with your real life?
You will ponder this as you move about the ball room, but you will come up with more questions than answers. A whiff of familiar perfume will pull you in one direction while a Facebook posting will jerk you back into reality, or was reality found only in a perfume scented distant memory?
Which is real; the warm and funny close friend of forty years ago or the bitter, angry guy spewing venomous political pabulum on Facebook?
The great television writer, Rod Serling would have recognized this dilemma right away. He would tell you without hesitation, you have indeed entered, The Twilight Zone.


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Home backup
Portable generators
Pressure washers
Water pumps
Generac PRO
Wi-Fi enabled
Parts & accessories
All products
Business standby
Industrial generators
Mobile generators
Light towers
New Team Members
Welcome to the Team!
We are pleased to welcome the newest member of Generac's international sales team, Christina Lay. Born and raised in Peru, where she participated as a member of the Peruvian national swim team for several years, Christina moved to Wisconsin in 2009. Having worked previously for Kohl's, Northwestern Mutual and Xerox, Christina will use over seven years of bi-lingual administrative, client relations and customer service experience to help provide sales support for the Pramac and Ottomotores teams.
We are thrilled to introduce Generac's newest International Sales Representative, Adriana Sofia Llantada. Sofia moved to Wisconsin in 2006 after spending the majority of her life in Mexico City. Having already earned an Associate's degree in Information Technology and Web Design in addition to a Bachelor's degree in Marketing, Sofia is currently enrolled in a Master's program for Business Administration. With over 20 years of sales and customer service experience, Sofia has worked for several other companies including S.C. Johnson, Conley Equipment, Monsanto, Xerox and Jacques Trading Company. Sofia will use her bicultural background and bilingual skills to support Generac's international customers, business partners and internal team members.
Help us welcome Generac's newest International Account Manager, Felix Bergollo. Felix joins us from Whirlpool Corporation and brings more than 20 years of experience in B2B sales, business development, market strategies & product sales training. Felix will utilize his international retail experience as a Retail Sales Account Manager to facilitate sales initiatives for Generac's retail accounts.
We are thrilled to welcome Regional Development Manager, Antonio Nava to the Generac Team! Antonio was born in Mexico, and now resides in Miami with his wife, Lourdes, and his daughter, Pamela, who is 22 months old. He has worked previously as a sales executive and has experienced success on multiple energy projects for a number of industries around the world. His career experience includes developing distribution partners for the commercialization of power generation equipment, most notably complex solutions in the energy industry and rental market. Antonio combines a multicultural background with diverse business administration skills, Six Sigma certification and mechanical/electrical engineering experience.
In his last position as a general sales manager for Cummins, Antonio was responsible for leading a team of sales representatives, engineers and project managers of gaseous standby generators in South America. During this time, he managed to increase sales 80% and expanded Cummins' global presence from three to seven countries through new distribution partners and the negotiation of lucrative contracts. He will apply his past experience to help Generac increase sales in South America and expand the company's global footprint.
Tradeshows/Events Attended
Generac Benefits from Attending 6th Annual BNAmericas Summit
From 10-11 of August, members of the Generac Industrial Power team traveled to Santiago, Chile to attend the 6th Annual BNAmericas Latam Power Generation Summit. Regional Development Manager, Jesus Campos, joined Marketing Director, Samara Salgado, and Director of Latin American Sales, Frank Moreno, at the event to learn about new business opportunities in the commercial and industrial sector, including the new political reality in Argentina, where subsidies and rate controls are being lifted and how Mexico's landmark energy reform continues to bring about change within the industry.
Also serving as a forum for industry-related brainstorming, the event allowed different experts to share their insights regarding innovation, efficiency, navigating local bureaucracies and making long-lasting connections with the industry's project stakeholders. Using this knowledge to enhance future initiatives will allow Generac Industrial Power's sales and marketing teams to take advantage of regional opportunities and contribute to the company's global success in 2016 and beyond.
Pictured above from left to right are Frank Moreno, Jesus Campos and Samara Salgado at this year's event.
Dealer News
Bonyplus Now Protected by Generac
Bonyplus, one of the largest cosmetic companies in Brazil, purchased a gas-powered generator in early Q3 from Generac's international dealer, Powercom. After 13 years of activity selling energy solutions to local companies, Powercom looks to carry their regional success through the end of 2016 and into next year.
Generac Joins Local Distributor to Discuss Power in the Dominican Republic
In early August, Generac Industrial Power's Regional Development Manager, Del Maccio, traveled to join members from Generac Industrial Distributor, Equimax at their joint "Present and Future of Electric Power Systems in the Dominican Republic" conference in the Dominican Republic. Along with other Equimax representatives, Del was joined by Juan Carlos Perez, Equimax's General Director. Items of discussion included a diagnosis of the current state of the Dominican Republic's electricity sector, implications of Electrical Covenant in the new order of the sector, and the main challenges to solving the current state of the Dominican Republic's national electricity infrastructure.
Attending the event was an overall success, as having Generac and a local distributor on hand not only increased visibility in the region, but also opened the door for future business opportunities with other organizations in the Dominican Republic and surrounding areas.
Pictured above is Del Maccio (second from left) with members of the Equimax team.
New Product Introductions
Generac Launches New Website for Latin American Countries
The Generac website, www.Generac.com, is being launched in Latin America to foster our market share and create stronger bonds with our clients across the region. For Generac, Latin America is a strategic region with huge potential to develop our presence in the infrastructure, mining, oil and gas markets among others. The website offers insight and helpful tools to all Latin American countries except Brazil; an independent website will be managed solely for Brazil.
The website displays a contemporary design with automatic country detection features, as well as an innovative structure that facilitates simple navigation and enables users to intuitively find all information available on Generac's industrial, commercial, residential and mobile products. It is also possible to make a manual country selection to search our dealer network, find more technical information with better illustrations, review offered services and find details on the courses and certifications we provide locally.
The current site facilitating these initiatives, www.generacottomotores.com, will soon be shut down, and users who are not aware of the new site will be automatically redirected to the new Generac portal when they search for the old domain.
News from Italy & Europe
News from Africa, Asia, & Middle East


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Side note: this is not an admission that will get you very far in an interview for a librarian job. Just so you know. Customer services skills are much more relevant.
Reading rules, do you have them? This is a kind of meme coming from Bookriot, via Bookshelves of Doom and Flexnib.
The writer at Bookriot says,
"...I thought about it as just a list of my (many!) seemingly arbitrary rules for reading. Once I got started, though, I discovered that those rules actually tell you so much about me that they double as personality traits..."
So, in the interests of delving into my personality, here are my reading rules. What are yours?
1. One book at a time. I do occasionally break this rule, especially if I pick up one I must read AT ONCE and I'm in the middle of another. Or, I'll sometimes read a fiction and a non-fiction at the same time.
2. Must read before turning off the light. It's a conditioned reflex for me. Reading is relaxation and so reading before going to sleep is signalling to my brain to turn off.
3. I rarely re-read things. Only a few books will be revisited.
4. A good book is one where I emerge from it with a book hangover.
5. Don't mark the pages. I'm not sure where this one comes from since I have no issue with writing all over printed out journal articles and other documents. But, books? No. On the other hand I do highlight non-fiction if I'm reading it as an ebook.
6. I rarely use bookmarks, preferring to turn the book face down at the page I'm reading. Bookmarks are used for ebooks though.
7. I usually prefer the book to the movie. I can appreciate a movie as a separate interpretation but most of the time the book resonates more.
8. I will read the "in" book if it appeals. But some genre's don't appeal. Fifty shades? Nope. Life of Pi? Okay.
9. There are some books I won't read because I find their content too much to deal with. For example, dystopian books rarely appear on my reading lists. Harrowing books about children don't work for me right now. E.g. Sarah's Key is not something I can face reading right now. Unless there is some kind of Happy End, I can't deal with that sort of story.
10. I like Happy End. Please give it to me.


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Here are the rules which you must abide by if you are tagged.
1. Link to your tagger and post these rules.
2. Share 7 facts about yourself: some random, some weird.
3. Tag 3 people at the end of your post and list their names (linking to them).
4. Let them know they've been tagged by leaving a comment at their blogs.
Golly, gotta figure this out....
1. I have a masters in nursing and practice as a nurse practitioner in family medicine in a family practice residency program. I love my job and teaching new docs
2. I am constantly singing or whistling in the halls and stairwells...great accoustics.
3. I have tatted for thirty years..hard to believe since I am only 20.
4. My toes are REALLY weird too...gripper toes we call them
5. One of my favorite phrases is..."let me just be the devil's advocate for a second."..
6. I have three kitties and would take them over the human's any day...they don't talk back and always want to snuggle! (just kidding)
7. I have a very bizaar and quirky sense of humor that some folks just don't get...and I married a guy just like me!! It makes for some really high times
I am going to "tag" (I just figured out how! Do I have to ask/tell first? Hope not cause I didn't!! (LOL)


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How special; Sabbath lambs!ReplyDelete
The lambs are so cute. Why the four horns? I find that interesting. They sure look like good mothers. Thanks for sharing.ReplyDeleteReplies
Jacob Sheep have between 2 and 6 horns but usually they are 2 horned or 4 horned. It's one of the things that first peaked our interest in Jacobs. That and they are a heritage breed in need of conservation and of course their spots. You can read more about our flock by clicking on the S-Squared Sheep in the sidebar.
They ARE darling!ReplyDelete
how adorableReplyDelete
How exciting! Your lambs are so cute. I see that mothering, "I'm only thinking of my babies right now" look, just like our does get when they've kidded; how precious! Do they make little maa-ing/rumbling sounds to their lambs? What a blessing!ReplyDelete
Beautiful babies!ReplyDelete


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Wednesday, January 2, 2013
Jacquelynne, who blogs at The Noble Wife http://thenoblewife.blogspot.com/2013/01/happy-2013-this-is-third-year-in-row.html posted the most beautiful poem about Choices by Max Lucado yesterday. I think it is a perfect reminder of the choices I should make every day. Please take a moment to go to her blog and check it out - you won't be sorry! (She has a great blog too!!)
2 comments:
- Jacquelynne Steves said...
Thanks for the link to my blog! Happy New Year!
- January 3, 2013 at 12:08 PM
- StitchinByTheLake said...
I read that just the other day and, like you, was very touched by it. blessings, marlene
- January 15, 2013 at 11:39 AM
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About: Our Physicians
Ray Bologna, MD, MBA – The Creator of The Accidental Sisterhood
Dr. Ray Bologna has spent much of his 20+ year medical career helping women understand and treat their pelvic health problems. In 2005, he created a five-step pelvic floor exercise program (based on Kegel exercises) to improve bladder problems like urinary frequency, urgency, urge incontinence, and stress urinary incontinence. He published the program in his book, The Accidental Sisterhood, currently in its 4th edition with thousands of copies sold. Over the years, Dr. Bologna realized the one missing component from his book was a network of support – the bringing together of women who share the same issues and can learn from one another. He launched The Accidental Sisterhood website as a way to create this community.
Dr. Bologna currently serves as the chairman of the Department of Urology at Akron General Medical Center, and practices Urology and Female Pelvic Medicine and Reconstructive Surgery in Akron, Ohio at Physicians Urology. A graduate of the University of Notre Dame, he received his doctor of medicine from Northeast Ohio Medical University (NEOMED) and completed his residency in urology at NEOMED's affiliated Urology Program. He completed a fellowship in female medicine and reconstructive surgery at Graduate Hospital in Philadelphia, PA. Dr. Bologna is board certified in both urology and female pelvic medicine and reconstructive surgery.
Click here to learn more about Dr. Bologna's practice.
Mary South, MD
Dr. Mary South is a leading expert in Urogynecology, more formally described as Female Pelvic Medicine and Reconstructive Surgery. An Obstetrician Gynecologist by training, Dr. South focuses on pelvic floor disorders and the procedures and surgical management to treat the challenges that many women face.
A native Ohioan, Dr. South is a graduate of the College of Wooster ('96) and Case Western Reserve School of Medicine ('01). She completed residency and fellowship at the University of North Carolina and Duke University, and went on to start her own joint fellowship program to support the next generation of Female Pelvic Medicine and Reconstructive Surgery specialists at the University of Cincinnati. During fellowship and beyond, Dr. South has published various peer-reviewed journal articles and review articles. he has also contributed chapters to several books, including Overactive Bladder and Nocturia and Continence: Current Concepts and Treatment Strategies, and presented award-winning educational videos on surgical techniques at national medical conferences.
Dr. South is board-certified in both Obstetrics and Gynecology and Female Pelvic Medicine and Reconstructive Surgery, and currently owns a successful independent practice—Northeast Ohio Urogynecology—in Akron, Ohio.
Click here to learn more about Dr. South's practice.


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Enjoy! This is little bear
Will post more pics when I'm finished with him. Questions? email me! email@example.com
We've had a very busy week at church this week with VBS! You know what I'm talking about! Kids, Kids everywhere! But, such fun!
I haven't made anything this week, due to some small minor shoulder injury and now, I can only move my arm so much, but, here is a link to a cute pencil, pen case that would make some young school person happy! If you make it share your thoughts with the author! Enjoy!
I might give this a whirl soon!
Have a great Thursday!
Are these not just the most precious little ones? Here is our new grandbaby Everett! So cute!
This is the Lord's Day and we are now home from Worship! Just a great time of singing and praising God! We're gearing up for a week of Vacation Bible School--begins tomorrow night and runs through Friday. I'm in charge of registration and medical release forms. Yippee!!
It's a fairly easy system however, since, it's all done on the computer! My dedicated hubby built the database we use and is all ready to go for this year!
Guessing I should get busy on the Nursing Wrap today, maybe have it done by tomorrow when I'm sure we'll be over at Grace's part of the day.... then off to church in the evening.
Today's verse from Bible Gateway: "Are not five sparrows sold for two pennies? Yet not one of them is forgotten by God. Indeed, the very hairs of your head are all numbered. Don't be afraid; you are worth more than many sparrows." Luke 12: 6-7
Happy Sunday! Don't let the heat of summer drag you down!
Tim, Pam, and Bryton came over tonight and brought (yum) pizza & ice cream! Wow--fattening! Anyway, we sat and watched a movie---"Coraline" It was a strangely disturbing weird movie. I wasn't impressed with it. It made me feel sad, cause here I am a sewer and I would make a doll like that, but, it was too weird. The sewer was evil, and I didn't like the way it turned out.
I don't imagine any one who sews cute toys and dolls would find this movie too fun. But, it was good that "real" came back and they all lived happily ever after.
The last sewing project I made was a crocheted dish rag and potholder and dishtowel for Pam when she went to her Camino weekend. I also made myself a crocheted dish rag.
I recently made an over the head nursing wrap for Grace, but, she thought it wasn't enough to cover everything, so, I have to research and make another type. I have the material and stuff to work on it, but, I left it in Melanie's car and she's over at Grace's tonight, helping with the baby. I came home, and we watched the movie with Pam & Tim instead.
The new little baby is here. He's a week old and just as cute as can be! I won't post his picture here, but, suffice it to say he's adorable!
I'm also getting ready to make a bassinet covering, did you see anything on the web that costs about 850.00! Yikes! That's the kind they want me to make! I'm hoping I can pull it off! We'll see. Now, if I do end up doing that project, I will post pictures!


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Senate Majority Leader Mitch McConnell isn't willing to share the details of his bill to repeal Obamacare with the public, but he's also not willing to share any details with industry groups that represent millions of patients.
According to the LA Times, requests from over a dozen groups have been turned down.
This week, a group of more than 15 patients groups -- including the American Heart Assn., the March of Dimes, the American Lung Assn. and the American Diabetes Assn. -- asked McConnell's office to meet with them next week, proposing any time between Friday and June 22.
A representative from McConnell's office told them staff schedules were too busy, according to representatives of several of the organizations.
They're "too busy" fidgeting with legislation that will impact the healthcare industry to meet with the... healthcare industry.
These patient groups are far from alone. Vendors and providers are also being left out of the process. This isn't even a case of Republicans advancing a bill scripted for them by lobbyists; they're making strictly political calculations. Senate Republicans, like their House colleagues, are not interested in the myriad of ways in which their bill will effect health, patients and the economy.
Their only goal is to devise a legislative formula that cuts taxes for the rich at the expense of providing healthcare while mustering enough support to actually pass it with a bare minimum of votes. Expanding or improving access to healthcare isn't even given lip service at this point.
These are the calculations of unaccountable sociopaths.


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Build Tolko. Build Green.
We work to balance environmental, social and economic performance through our policies, principles and actions. This commitment to the environment, and the people we serve, ensures that the resources we manage today will contribute to future generations tomorrow.
We are committed to achieving world leading standards from forest management through the manufacturing of environmentally friendly products.
Green Building Standards
All Tolko products meet international Green Building standards, including LEED and Green Globes.
Tolko products earn the certified wood credit under these standards through Sustainable Forestry Initiative (SFI), our Sustainable Forest Management (SFM) certification. SFI fully qualifies under the LEED standard via the LEED Alternative Compliance Path process. In addition, our panel products qualify for incremental LEED credits under the low emitting materials requirements.
View Tolko's Build Green brochures in English and French
Build Green (pdf)
Bâtissez Vert (pdf)


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Teahouses or lodges are the standard accommodation on a trek in Nepal. They offer rooms with beds, communal dining rooms, bathrooms (sometimes attached) and home cooking. At MHE, we aim to secure the best lodge accommodation in every location. Please remember, that the location and altitude plays a big part in the style and services of accommodation available.
Depending on the location and your budget, they will be anything from a basic wooden/stone construction with the bare minimum facilities, to places with hot water, electrical outlets in the room, and wifi facilities. The higher you go, the more basic the accommodation is.
The local tea houses are basic but clean and they do not often have attached bathrooms or hot showers. The price is based on twin share and does not include any extra charge that lodge owners may levy for single rooms, attached bathrooms or hot showers (if these facilities are available) or for battery charging. We can't know in advance what they may charge for such things in the different lodges along the way, so we prefer to leave this cost out, rather than pass along an estimated charge to you. We cannot guarantee that if you would like a single room on trek, that you will be able to get one. We will always try our best, but supply and demand in some cases means that it just isn't possible.
All building material, from wood, to glass for windows, mattresses, blankets, cooking pots, bathroom fittings, has to be flown to Lukla and then carried by porters up the trail. Satellite dishes, gas cylinders for heating water, foodstuffs, beer, soft drinks and snacks are also carried up from the airport at Lukla by porters, donkeys or yaks. This costs time and money.
Since many lodges were damaged in the earthquake in 2015, rebuilding was a priority. The Nepal government was extremely slow in handing out cash for this, and many lodge owners sank all their savings into rebuilding as best they could, so that they would have a functioning business when tourists returned. Whatever facilities they have managed to provide from their often limited resources, is to make your trek more comfortable.
In lodges located from Lukla to Namche Bazaar, you will be surprised to find that most rooms will have an attached bathroom – though not like a hotel – which may have hot water and a shower head – but most likely a bucket – and comfortable mattresses and blankets or duvets.
Once you leave Namche Bazaar for the higher, more remote elevations, the lodges become simpler, and attached bathrooms are a rarity. The rooms will not have electrical outlets, and you will have to ask the lodgeowner to recharge your gadgets. Most of these places use solar power, so if it hasn't been sunny there will be very limited electricity available for lights in the lodge, and you may not be able to fully charge gadgets. There is, of course a separate charge for this service.
You will always get a room which has a wooden bed base, a mattress of some sort, a pillow, and a blanket or duvet. There will usually be a window, a door, and a light fitting. Perhaps even somewhere to hang your jacket. The rooms aren't large, as all you are doing is sleeping in there. We always recommend you bring a sleeping bag as we cannot guarantee the warmth or cleanliness of the blankets – it's rather hard to wash and dry them in the mountains. Everyone sits in the communal dining room for meals, tea and chats. There will usually be a heater of some sort in the communal dining room ONLY, which keeps you warm and helps dry out your socks and boots. Bedrooms are unheated.
The kitchen is usually a small affair with limited cooking points, so it is important to not order 20 different dishes and expect them to come out at the same time. This is why we usually stick to dal bhat – it's easy, nutritious and quickly made. Hot water is always available for tea, soup, etc and you will be refilling your bottles from this. The water is often carried from a nearby stream if tank storage is not available, so please do not waste the water you have been given. You will find that a daily shower is not necessary or even desirable in the higher, colder places.
No matter the facilities in your teahouse, you will always find warm Sherpa hospitality, and those fantastic mountain views. So join us in Nepal!
21/11/2017 01:24:02 am
Thanks for post this very informative piece! it's very usefull! I enjoyed reading it! keep up the great work!
31/1/2018 08:04:24 pm
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Bulls, Dwyane Wade agree to a buyout: Report
CHICAGO – It appears that the era of Dwyane Wade will come to an end after just one season.
On Sunday night, just before the Bulls are scheduled to open training camp this week, the Chicago Tribune is reporting that the Bulls have reached a buyout agreement on the final year of the guard's contract.
Wade, according to the repot from K.C. Johnson, will give up between $8-10 million of his $23.8 million dollar contract for the 2017-2018 season.
Reporter K.C. Johnson tweeted the news on Sunday night.
This brings a quick close to Wade's time in his hometown in which he spent just a single season. The 14-year veteran played in 60 games in the 2016-2017 season, scoring 18.3 points per game with 4.5 rebounds and 3.8 assists.
Check back with WGNTV.com for more on this story.


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N. Needleman wrote:Commentary from a fan who met Sabrina Sutherland at the TP Fest and learned this:
David and Mark obviously co-wrote the series... to a point. Around the time they handed things over to Showtime, things changed a bit. Mark went off to write The Secret History of Twin Peaks and David directed the whole thing, but the interesting thing to note here is that, as you all may recall, the series Showtime announced was going to be 8 or 9 episodes. There was that whole drama where David announced he quit, Showtime caved, and now we have 18 episodes. This was NOT a case of the existing material simply being stretched out. According to Sabrina, David actually continued to write. So this seems to have shifted the level of writing involvement more heavily to Lynch than Frost. EDIT: For clarity, it's not that David wrote nine more episodes. It's more like he wrote more scenes interspersed throughout the existing material.
More here, including Jan D'Arcy talking about how Eamon Farren was so upset by the assault scene in Part 10 he called his own grandmother.
I think this closely matched what Fenn said on a podcast interview that was quickly pulled down. Since that interview, I suspected Lynch wrote a significant amount of the new material himself, if not all of it.
Which does not, in my mind, in any way, shape or form diminish Frost's contribution. You can still see Frost's fingerprints all over S3 (Dr. Amp anyone?)


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Dr. Gordon L. Fung is a cardiologist specializing in the treatment of heart disease with a special interest in cardiac rehabilitation, cardiovascular pharmacology, electrocardiography, and preventive cardiology. At UCSF Medical Center at Mount Zion, he is Director of the Cardiology Consultation Service, the Cardiac Noninvasive Laboratory, and the nation's first Asian Heart & Vascular Center. At Moffitt - Long Hospitals, he directs the Electrocardiography Laboratory and is the Associate Director of the Non-Invasive Laboratory. His research explores advances in preventive cardiology and ethnic disparities in cardiovascular disease.
Fung earned a master's degree in public health in hospital economics and administration at the University of California, Berkeley and a medical degree at the University of California, San Francisco. He completed an internal medicine residency and was chief resident at Highland General Hospital in Oakland. He completed a cardiology fellowship at the Veterans Administration Medical Center in Martinez, California. He is active in the American Heart Association and has served on the association's national board of directors. He is also active in the American College of Cardiology and was a member of its Board of Governors. In 2007, Dr. Fung was awarded a Doctorate of Philosophy degree in Chinese Philosophy. Fung is a clinical professor of medicine and cardiology at UCSF School of Medicine.
In 2007, Fung was appointed by Governor Arnold Schwarzenegger to the Council of Multicultural Health which advises the State Department of Public Health and the State Department of Health Services. In 2006, he was appointed by Mayor Gavin Newsome in San Francisco to represent the San Francisco Medical Society Healthy San Francisco Advisory Oversight Committee. At the National American Heart Association, he serves on the Diversity Leadership Committee as well as the Asian Outreach Task Force of the Western States Affiliate and member of the Chinese Community Cardiac Council at the San Francisco Division. Fung also directs an Annual Symposium Cardiovascular Diseases in Asians.
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Audrina Patridge was spotted at a screening of "Waiting for Lightning" presented by DC Shoes last night (April 10).
Joined by boyfriend Corey Bohan, the former reality star looked to be having a good time as she headed inside to watch the flick, a documentary about skateboarder Danny Way.
In a recent interview, Audrina shared her personal style obsessions as of late- "Right now I'm really all about shoes. I love crazy shoes. If you have a simple dress on and amazing shoes on it just completes [the outfit]."
"I [also] love all the high-waisted shorts. I'm all about very high-waisted and cropped tops right now. It's very 80s and 90s, I'm into it."
credit - celebrity paradise, zimbio, gossipcenter.com
Click here for more posts on Audrina
~Kelli at Hills Freak
Click here for more posts on Audrina
~Kelli at Hills Freak


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Previously when I have made chicken in the crockpot, it has disintegrated and smelled like someone has gotten sick in the pot and then cooked it. However this time the chicken was tasty and delicious. BAP and SSP loved the "dragon meat."
Lemon Garlic Chicken
adapted from: http://www.number-2-pencil.com/2013/05/23/slow-cooker-lemon-garlic-chicken/
4 lemons (3 cut in half with the ends sliced off and the other one sliced thinly)
2 heads garlic, sliced in half
1T steak seasoning
1/2 t dried thyme
4.5lb chicken, cleaned out and patted dry
Place garlic and the halved lemons in the bottom of the crockpot. Place other lemon chunks inside the chicken. Sprinkle steak seasoning and dried thyme on top of chicken. Cook chicken chicken on HIGH for 4hours (until chicken reaches 165). Let chicken rest 15minutes. Serve with mashed potatoes and steamed broccoli.