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Inhibition of Cell Cycle Progression by a Synthetic Peptide Corresponding to Residues 65–79 of an HLA Class II Sequence: Functional Similarities but Mechanistic Differences with the Immunosuppressive Drug Rapamycin
Michelle L. Boytim, Shu-Chen Lyu, Ron Jung, Alan M. Krensky and Carol Clayberger
J Immunol March 1, 1998, 160 (5) 2215-2222;
Michelle L. Boytim
Shu-Chen Lyu
Ron Jung
Alan M. Krensky
Carol Clayberger
A synthetic peptide corresponding to a region of the α1 α-helix of DQA03011 (DQ 65–79) inhibits the proliferation of human PBL and T cells in an allele-nonspecific manner. It blocks proliferation stimulated by anti-CD3 mAb, PHA-P, and alloantigen, but not by PMA and ionomycin. Substitution of each amino acid with serine shows that residues 66, 68, 69, 71–73, and 75–79 are critical for function. Inhibition of proliferation is long lasting and is not reversible with exogenous IL-2. The peptide can be added 24 to 48 h after stimulation and still block proliferation. The DQ 65–79 peptide does not affect expression of IL-2 or IL-2R; however, IL-2-stimulated proliferation is inhibited. Cell cycle progression is blocked at the G1/S transition, and the activity of cdk2 (cyclin-dependent kinase 2) kinase is impaired by the continued presence of p27. Although these results suggest a mechanism similar to that of rapamycin, the peptide inhibition is not reversed with FK-506, which indicates a distinct mechanism.
Tcells are crucial for the defense of the host against infection, and the key to T cell activation is recognition of nonself. Processed peptides from both internal and external sources are bound to MHC molecules and presented to T cells by APC (1). Other regions of MHC molecules interact with T cell coreceptors CD4 or CD8 (2, 3, 4, 5, 6). Not only are MHC molecules critical for inducing immune responses, but they are also important in controlling them. Thymocytes are deleted if they cannot recognize self MHC molecules or if they strongly recognize self peptides in the context of self MHC molecules. In the periphery, recognition of self peptides presented by self MHC molecules can lead to deletion or anergy of mature T cells (7, 8). Recently, it has been demonstrated that lysis of target cells by NK cells can be inhibited by the specific recognition of MHC class I molecules on the target cell by killer cell-inhibitory receptors (KIR)3 on the NK cell (9, 10). Thus, MHC molecules are important for both initiation and regulation of normal immune responses.
In addition to their natural role in host defense and T cell education, MHC molecules play a pivotal role in the process of graft rejection. Both direct recognition of allogeneic MHC and indirect recognition through presentation of allogeneic MHC molecules as peptides in host MHC molecules can stimulate rejection (11, 12). Conversely, MHC molecules have also been implicated in the prevention of graft rejection. Circumstantial evidence comes from studies in which concurrent donor-specific transfusions prolong graft survival in humans, and studies in rodents in which injection of spleen cells or lymphocytes from the donor strain induce tolerance to the graft (13, 14). MHC molecules themselves are shown to induce unresponsiveness by injection of cells transfected with class I or class II donor MHC molecules into graft recipients (14). High doses of purified class I molecules induce tolerance in the rat allograft model; however, this is not the case for purified class II molecules (15, 16). In contrast, peptides corresponding to the β-chain of donor rat class II MHC could initiate unresponsiveness to a transplant via oral or intrathymic administration in the recipient (17, 18). Additionally, synthetic peptides derived from nonpolymorphic regions of MHC class II have inhibitory effects (19).
During the past decade, we have characterized the effects of synthetic peptides corresponding to HLA class I sequences on immune responses. A peptide corresponding to residues 222–235 of the α3 domain, the CD8 binding domain of the HLA class I molecule, blocks the differentiation of pre-CTL into mature effector cells in an allele-unrestricted manner (5). We reported that peptides corresponding to residues 98–113 or 56–69 of HLA-A2 block CTL responses in an allele-specific manner (20, 21). Finally, a peptide derived from residues 75–84 of the α1 α-helix of HLA-B2702 inhibits lysis of target cells by CTL in an allele-nonspecific manner (22). This peptide prevents graft rejection in rodent models (23, 24, 25).
We sought to determine whether synthetic peptides corresponding to conserved regions of class II molecules could have similar immunoregulatory effects. After testing a panel of peptides, we found that a peptide corresponding to residues 56–80 of DQA03011 inhibits a variety of immune responses, and this activity is localized to residues 65–79. In this study, we characterize the inhibitory effects of this peptide and describe its mechanism of action.
Peptides, Abs, and reagents
Peptides were synthesized using Fmoc chemistry on an Applied Biosystems (Foster City, CA) peptide synthesizer, and were purified to greater than 95% purity by HPLC (26), and the composition was confirmed by mass spectrometry. Stock solutions of peptide were made by dissolving peptide in DMSO (25 mg/ml). Cyclosporin A was a gift from Merck Research Laboratories (Rahway, NJ), and rapamycin was a gift from Wyeth Ayerst (Philadelphia, PA). rIL-2 was obtained from Sigma Chemical Co. (St. Louis, MO). Abs specific for cyclin E, p21, p27, and cdk2 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Goat anti-mouse horseradish peroxidase and goat anti-rabbit horseradish peroxidase were obtained from Caltag (South San Francisco, CA). Abs to CD2, CD3, CD4, CD8, CD28, CD45RO, CD69, IL-2Rα,β,γ, class I, and class II were purchased from PharMingen (San Diego, CA). Unless specified, all other reagents were obtained from Sigma Chemical Co.
PBL and T cell isolation, cell culture
PBL were obtained from volunteer donors or buffy coats from Stanford Blood Center and were isolated via Ficoll-Hypaque density centrifugation. T cells were isolated following adherence of macrophages on plastic petri dishes and removal of B cells on nylon wool columns (27). T cell purities were greater than 90% by staining with anti-CD3 mAb. Cells were cultured in RPMI 1640 supplemented with 10% FBS, 100 U/ml penicillin/streptomycin, 2 mM l-glutamine, and 1 mM HEPES. For preactivated cells, purified T cells were cultured with 0.8 μg/ml PHA-P for 72 h before stimulation with rIL-2 (100 U/ml), as described (28).
Proliferation assay
Proliferation assays were performed essentially as described (27). For each stimulation protocol/cell type, the time for the optimal proliferative response was determined empirically. For human PBL, 96-well plates were coated with 50 μl of anti-CD3 mAb (5 μg/ml) in PBS for 1 h at 37°C. PBL were added at 4 × 105 cells/well and peptide at a concentration of 40 μM. The cells were pulsed with [3H]TdR (1 μCi/well) (DuPont NEN, Boston, MA) and were harvested with a PhD cell harvester at 72 h. [3H]TdR incorporation was determined by liquid scintillation counting. Purified T cells were cultured at 2 × 105 cells/well, pulsed at 72 h, and harvested at 96 h. Where indicated, rIL-2 (100 U/ml) or anti-CD28 mAb (1 μg/ml) was added at the start of culture. In indicated assays, PHA-P (5 μg/ml) or PMA and ionomycin (10 ng/ml and 250 ng/ml, respectively) were used to stimulate cells, and with these treatments, cells were pulsed at 24 h and harvested at 48 h. Preactivated cells were stimulated with rIL-2 (100 U/ml), pulsed at 24 h, and harvested at 48 h. Cyclosporin A (100 nM) and rapamycin (100 nM) were used in indicated experiments.
Restimulation assay
PBL (4 × 106/ml) were incubated in the presence of peptide (40 μM) or rapamycin (100 nM) with or without immobilized anti-CD3 mAb (5 μg/ml) for 24 h at 37°C in 2-ml wells. The cells were then washed three times with PBS and were replated at the same density. After a 7-day incubation, the cells were washed, plated at equivalent densities in 96-well microtiter plates, and restimulated with anti-CD3 mAb (5 μg/ml). After 48 h, the cells were pulsed with [3H]TdR and were harvested at 72 h. In some experiments, 100 U/ml of rIL-2 was added during the restimulation.
Cell cycle analysis.
PBL (2 × 105/well) were cultured with PHA-P (5 μg/ml) in the presence of peptide (40 μM), cyclosporin A (100 nM), rapamycin (100 nM), or medium. At 48 h, the cells were washed in PBS and resuspended in 250 μl of a solution containing 4 mM sodium citrate, 0.05% Nonidet P-40, 0.45 mg/ml RNase, and 50 μg/ml propidium iodide. The cells were incubated on ice for 10 min, at which time 25 μl of 1.5 M sodium chloride was added. The cells were analyzed on a Becton Dickinson (San Jose, CA) FACScan at the Stanford FACS facility. Propidium iodide fluorescence was plotted on a linear scale vs orthogonal scatter.
Cell surface receptor analysis.
PBL (2 × 105 cells) were stained as previously described (29) with the indicated FITC-conjugated Abs (1 μg/106 cells). Cells were also stained with propidium iodide immediately before analysis to exclude dead cells.
Kinase assay
Assays were performed as described (30) with the following modifications. Preactivated T cells (5 × 106) were stimulated with 100 U/ml rIL-2. At 24 h, the cells were washed in PBS and lysed in lysis buffer (0.5% Triton X-100, 1 mM DTT, 10 mM β-glycerophosphate, 20 mM sodium fluoride, 0.2 mM sodium orthovanadate, 10 μg/ml leupeptin, and 5 μg/ml aprotinin). Lysates were incubated on ice for 10 min, then spun down for 3 min in a microcentrifuge. The amount of protein in each supernatant was measured using the Bio-Rad protein assay (Bio-Rad, Hercules, CA). Anti-cyclin E Ab (2 μg) was added to supernatants containing equivalent amounts of protein, and the samples were rocked at 4°C for 1 h. Protein A/G beads (15 μl) (Santa Cruz Biotechnology) were added, and the samples were rocked for 45 min at 4°C. The beads were washed twice in lysis buffer and twice in kinase buffer (20 mM HEPES, pH 7, 10 mM β-glycerophosphate, 5 mM magnesium chloride, 10 μg/ml leupeptin, and 1 mM DTT). Kinase activity was assayed by resuspending the beads in 40 μl of kinase buffer supplemented with 8 μCi [γ-32P]ATP, 4 μM ATP, and 20.6 mg/ml histone H1 type III-S. Samples were incubated at 37°C for 15 min, and the reaction was stopped by adding 40 μl of ice-cold 20 mM EDTA, pH 8. Triplicate aliquots were spotted onto phosphocellulose filters (Life Technologies, Grand Island, NY). The filters were soaked briefly in 1% H3PO4/10 mM Na4P2O7 and washed three times for 15 min in 1% H3PO4. 32P incorporation was determined by liquid scintillation counting.
Immunoprecipitations and Western blots
Preactivated cells (1–2 × 107) were treated with peptide and 100 U/ml IL-2 for 24 h, and immunoprecipitations were performed as described (28). For Western blots, 2.5 × 106 preactivated T cells were incubated with peptide (40 μM) and 100 U/ml rIL-2 and prepared as described (31). Proteins were separated on either 7.5% or 12% SDS-PAGE gels. The proteins were transferred electrophoretically to supported nitrocellulose (Amersham, Arlington Heights, IL), and the membrane was blocked with a solution of 5% nonfat dry milk in 5 mM Tris-buffered saline, pH 7.5, with 0.05% Tween-20. Blots were probed with the indicated Abs at 1:1000. This step was followed by a secondary Ab conjugated to horseradish peroxidase at 1:3000, and proteins were visualized by chemiluminescence.
Synthetic peptides corresponding to HLA class II sequences inhibit proliferation of PBL and T cells
HLA class II molecules are heterodimers composed of an α- and a β-chain. While the β-chain is highly polymorphic, the α-chain is relatively conserved (32). From the hypothetical model of class II, later confirmed by the crystal structure of HLA-DR (33, 34), we selected the α1 α-helix region for study based on our previous studies with MHC class I (20, 21, 22). Peptides corresponding to the α-helix of DRA, DQA, and DPA (32) were synthesized.
The peptide corresponding to residues 56–80 of DQA03011 blocked proliferation of PBL to anti-CD3 mAb (data not shown). Overlapping 15-amino acid peptides were synthesized from this sequence and were tested for function. A peptide corresponding to residues 65–79 (Table I⇓), designated DQ 65–79, recapitulated the effects of the full-length peptide. The DQ 65–79 peptide inhibited [3H]thymidine incorporation by PBL or purified T cells stimulated with anti-CD3 mAb (Fig. 1⇓, A and B). Furthermore, the inhibitory effect of the peptide was not reversed by the addition of anti-CD28 mAb to purified T cells stimulated by anti-CD3 mAb (Fig. 1⇓C). Inhibition was not limited to stimulation via anti-CD3 mAb, as PHA-P-stimulated (Fig. 2⇓) or alloantigen-stimulated cells (data not shown) were also blocked by peptide; however, DQ 65–79 did not block PMA- and ionomycin-stimulated proliferation (Fig. 1⇓D). Inhibition of proliferation by peptide was observed with cells obtained from 35 donors, indicating the effects were allele unrestricted. The inhibition of proliferation was not due to cell death because no significant increase in staining by either trypan blue or propidium iodide was observed over the course of the proliferation assay (data not shown). A second peptide, in which aspartic acid was substituted for asparagine at amino acid 72, did not inhibit proliferation like the DQ 65–79 peptide (Table I⇓ and Figs. 1⇓ and 2⇓). The DQ 65–79 peptide and its derivatives were used to characterize the immunoregulatory effects and mechanism of action.
The DQ 65–79 peptide inhibits PBL and T cell proliferation. PBL (A and D) or purified T cells (B and C) were stimulated with anti-CD3 mAb (A and B), anti-CD3 plus anti-CD28 mAb (C), or PMA and ionomycin (D) in the presence of 40 μM peptide. Cells were pulsed with [3H]TdR at 24 (D), 48 (A and C), or 72 (B) h and harvested 24 h later. Each point is a mean of six replicates with the SD. Results are representative of at least three experiments with PBL from different donors. DMSO indicates the media control.
Serine substitutions affect the function of the DQ 65–79 peptide. Peptide sequences are as shown in Table I⇓. The proliferation assay was conducted using PBL stimulated with PHA-P in the presence of 40 μM peptide. Cells were pulsed at 24 h and harvested 24 h later. Each point is a mean of six replicates with the SD. Results are representative of at least three experiments.
Amino acid sequences of DQA03011-derived peptides
The inhibitory effects of the DQ 65–79 peptide are sequence specific and long lasting
To determine which amino acids were necessary for peptide inhibition, peptides were synthesized in which each amino acid of the sequence was substituted individually with serine (Fig. 2⇑). Peptides with serine substitutions at residues 65 and 67 maintained activity, while serine substitutions at 70 or 74 increased activity. All remaining substituted peptides lost functional activity.
We next examined whether peptide-induced unresponsiveness was transitory or long-lived. PBL treated with peptide and/or anti-CD3 mAb were washed, rested 7 days, and restimulated with anti-CD3 mAb alone. Cells initially treated with both the DQ 65–79 peptide and anti-CD3 mAb were unresponsive to restimulation when compared with cells initially treated with anti-CD3 mAb with either DMSO or the DQ 65–79 72D peptide (Fig. 3⇓C). Addition of exogenous IL-2 during the restimulation did not reverse the unresponsiveness of the DQ 65–79 peptide-treated cells (Fig. 3⇓D). Interestingly, cells cultured with the DQ 65–79 peptide alone were not unresponsive upon restimulation (Fig. 3⇓B). These cells, after washing and resting, were indistinguishable from control cells in their response to anti-CD3 mAb. Thus, simultaneous signals provided by peptide and anti-CD3 mAb are required to induce long term unresponsiveness.
DQ 65–79 induces a long term unresponsiveness that is not reversed with IL-2. PBL were cultured with anti-CD3 mAb in the presence of the indicated agent and were pulsed with [3H]TdR at 72 h (A). In B, C, and D, PBL were preincubated for 24 h with the indicated agent, washed at 24 h, and then cultured in medium for 7 days. The cells were then restimulated with anti-CD3 mAb and pulsed at 24 h with [3H]TdR. In B PBL were preincubated with peptide alone, while in C and D PBL were preincubated with peptide plus anti-CD3 mAb. rIL-2 (100 U/ml) was added to the restimulation in D. Each point is the mean of six replicates with the SD. Results are representative of four similar experiments with PBL from different donors.
The DQ 65–79 peptide does not inhibit early activation events
We examined the effect of the DQ 65–79 peptide on two key events involved in T cell activation: early gene expression and cell surface receptor expression. In some experiments, the effects of the DQ 65–79 peptide were compared with the immunosuppressive drugs cyclosporin A or rapamycin, agents that have divergent mechanisms of action (35).
The hallmark of cyclosporin A activity is its block of IL-2 gene expression, a critical early event in T cell proliferation (35). Cyclosporin A treatment also decreases expression of other early genes such as IFN-γ and IL-2Rα (36). In contrast, rapamycin does not affect the expression of these early genes, but instead blocks signaling events downstream of the IL-2R (29). No decrease in IL-2, IL-2Rα, or IFN-γ gene expression was detected by Northern blot analysis of PHA-P-activated PBL treated with the DQ 65–79 peptide as compared with DMSO-treated cells (data not shown). In agreement with these data, both the DQ 65–79 peptide and rapamycin blocked anti-CD3- or rIL-2-induced proliferation of preactivated PBL, while cyclosporin A blocked only anti-CD3-induced proliferation (Figs. 4⇓, A and B).
The DQ 65–79 peptide blocks proliferation of preactivated T cells to both anti-CD3 mAb and IL-2. Preactivated T cells (see Materials and Methods) were stimulated with immobilized anti-CD3 mAb (A) or 100 U/ml IL-2 (B) in the presence of 40 μM peptide, 100 nM cyclosporin, or 100 nM rapamycin. Cells were pulsed with [3H]TdR at 24 h (B) or 48 h (A) and harvested 24 h later. Results are representative of at least three experiments.
Because the DQ 65–79 peptide inhibited IL-2-mediated proliferation, cell surface expression of proximal signaling molecules was examined. The levels of CD2, CD4, CD8, MHC class I, and MHC class II were identical in anti-CD3 mAb-stimulated PBL in the presence or absence of the DQ 65–79 peptide (data not shown). Additionally, there was no difference between the expression of the activation markers IL-2Rα, CD69, and CD45RO in peptide-treated or untreated cells (data not shown). Because the peptide inhibited IL-2-mediated proliferation (Fig. 4⇑B), the expression of the IL-2Rβ and IL-2Rγ chains also was examined; however, DQ 65–79 had no effect on either of these molecules (data not shown). These findings indicate that, like rapamycin, the functional effects of the DQ 65–79 peptide are most likely downstream of the IL-2R.
The DQ 65–79 peptide impedes cell cycle progression
To further examine the effect of the DQ 65–79 peptide on proliferation, cell cycle and kinetics studies were performed. Analysis of DNA content showed that the peptide, along with cyclosporin A and rapamycin, prevented DNA replication by blocking the G1 to S transition (Table II⇓). We examined the kinetics of peptide activity by adding it at various time points after activation. [3H]Thymidine incorporation of PBL stimulated with anti-CD3 mAb was inhibited when the DQ 65–79 peptide was added up to 24 h after stimulation (Fig. 5⇓). The kinetics data concur with the block at G1/S of the cell cycle. The ability of the DQ 65–79 peptide to block T cell proliferation at a relatively late time was similar to that of rapamycin, but contrasted with cyclosporin A, which lost its effect by 24 h.
The DQ 65–79 peptide blocks PBL proliferation when added 24 h after activation. PBL were stimulated with immobilized anti-CD3 mAb. Peptide (40 μM), cyclosporin (100 nM), or rapamycin (100 nM) was added at the indicated times after the start of culture. Cells were pulsed with [3H]TdR at 48 h and harvested at 72 h. Each point is the mean of six replicates with the SD. Results are representative of at least three experiments with different donors.
The DQ 65–79 peptide inhibits cell cycle progression
The DQ 65–79 peptide inhibits cdk2 kinase activity
Because the DQ 65–79 peptide functioned until relatively late after activation and blocked the G1/S cell cycle transition, we focused on events that occur during this time period. The cyclin-dependent kinases (cdks) are key regulators of cell cycle progression. Activation of the cdks is a complex process, requiring both the recruitment of positive and the release of negative regulators. These kinases are regulated positively by association with cyclin proteins and by threonine phosphorylation, and they are regulated negatively by tyrosine phosphorylation and the association with either the Ink, Cip, or Kip families of inhibitory proteins. The negative signals from these inhibitory proteins overwhelm positive signals. Thus, an inhibitory protein must be removed from the cyclin-cdk complex before the kinase can become active (37, 38).
Cdk2 is critical for the transition from G1 into S phase of the cell cycle. It must associate with cyclin E and be phosphorylated on threonine 160 for activation. Cdk2 can be regulated negatively by phosphorylation of tyrosine 15 and by the association of either p21Cip1 or p27Kip1 (28). Because the DQ 65–79 peptide inhibited cell cycle progression at G1/S, we examined its effects on cdk2 kinase activity. T cells stimulated with IL-2 in the presence or absence of the DQ 65–79 peptide were assayed for cdk2 kinase activity. Phosphorylation of the cdk2 kinase substrate histone H1 was decreased with DQ 65–79 peptide treatment (Fig. 6⇓A). This effect was not due to a change in the expression of cdk2 or cyclin E because the amount of these proteins was essentially equivalent between cells treated with the DQ 65–79 peptide and controls over a 2-day time course (Fig. 6⇓C). Nor was inhibition due to a detectable change in phosphorylation, as the phosphorylation pattern was equivalent between treatment conditions, as shown by the doublet bands (Fig. 6⇓B). Cdk2 in DQ 65–79 peptide-treated cells was able to form a complex with cyclin E, as shown by the coimmunoprecipitation of cdk2 with Abs specific for cyclin E (Fig. 6⇓B). There was no significant change in complex formation in cells treated with the DQ 65–79 peptide as compared with controls (Fig. 6⇓, B and C). However, the steady state level of the inhibitory protein p27 was higher, while the level of p21 was lower, in the cells treated with the DQ 65–79 peptide as compared with cells treated with the control 72D peptide (Fig. 6⇓C). Rapamycin retards G1/S cell cycle progression by the inhibition of the cdk2 kinase via a block in p27 degradation, and also blocks the up-regulation of p21 (Fig. 6⇓C) (30, 39, 40). Thus, DQ 65–79 affected events downstream of the IL-2R in a manner similar to rapamycin, resulting in the inhibition of cell cycle progression and subsequent proliferation.
The DQ 65–79 peptide inhibits cdk2 kinase activity and prolongs p27 expression. Incorporation of 32P into histone H1 by cdk2 kinase immunoprecipitated from IL-2-stimulated preactivated T cells treated with the indicated agents at 24 h after stimulation (A). Each point is the mean of triplicates with the SD. Lysates from IL-2-stimulated preactivated T cells treated with the indicated agents (D = medium control; Q = DQ 65–79; 72 = DQ 65–79 72D; and R = rapamycin) were immunoprecipitated with anti-cyclin E mAb, separated on a 12% SDS-PAGE gel, and probed with anti-cdk2 mAb (B). Preactivated T cells were activated for 0, 1, or 2 days with IL-2 plus the indicated agent. Lysates were separated on 7.5% SDS-PAGE gels. Western blots were probed with the indicated Abs (C). β-Tubulin was the control for loading. Analysis by densitometry from left to right: p27 (1026, 954, 617; 950, 651, 203; 1064, 877, 481); β-tubulin (904, 870, 614; 926, 1007, 1054; 774, 829, 658); cdk2 (997, 1101, 941; 1117, 1154, 1156; 877, 852, 764); cyclin E (1096, 1101, 815; 1159, 1175, 1273; 915, 1087, 860); p21 (360, 140, 104; 284, 255, 892; 251, 429, 189).
The DQ 65–79 peptide is not antagonistic with FK-506
Because of the multiple functional similarities between DQ 65–79 and rapamycin, we asked whether the DQ 65–79 peptide bound to the immunophilin FK-506-binding protein (FKBP). Rapamycin and FK-506 bind to FKBP, and this complex formation is critical for their function (41). Rapamycin and FK-506 have different effects and are mutually antagonistic. Excess FK-506 prevents rapamycin-mediated inhibition of IL-2-mediated proliferation, and likewise, an excess of rapamycin prevents FK-506-mediated inhibition of PMA- and ionomycin-stimulated proliferation (41). Since the DQ 65–79 peptide blocked IL-2-mediated proliferation, but not PMA- and ionomycin-mediated proliferation, we conducted similar experiments in which the DQ 65–79 peptide was substituted for rapamycin. The DQ 65–79 peptide had no effect on FK-506-mediated inhibition of PBL proliferation stimulated with PMA and ionomycin in a range of 10 to 10,000 molar excess (Fig. 7⇓A). Likewise, no concentration of FK-506 tested blocked the effect of the DQ 65–79 peptide on IL-2-mediated proliferation, although FK-506 did reverse the inhibition of proliferation by rapamycin (Fig. 7⇓B). Therefore, the DQ 65–79 peptide does not compete for binding to the FK-506 binding site on FKBP, and probably does not bind to FKBP at all. Thus, while DQ 65–79 is functionally similar to rapamycin, it acts via a distinct mechanism.
The DQ 65–79 peptide and FK-506 are not mutually antagonistic. PBL were stimulated with 10 ng/ml PMA and 250 ng/ml ionomycin in the presence of the indicated concentrations of FK-506 and DQ 65–79 (A). Preactivated T cells were stimulated with 25 U/ml rIL-2 in the presence of the indicated concentrations of FK-506, rapamycin, and DQ 65–79 (B).
A synthetic peptide corresponding to residues 65–79 of the α-chain of DQA03011 inhibits the proliferation of PBL and purified T cells in an allele-nonspecific manner. This peptide induces long-term unresponsiveness that is not reversible with IL-2 and is dependent upon concomitant activation through the TCR or IL-2R. The DQ 65–79 peptide does not inhibit early mRNA accumulation or cell surface receptor expression, but instead blocks the G1 to S transition of the cell cycle. Cells treated with the DQ 65–79 peptide exhibit repressed cdk2 kinase activity and prolonged expression of the p27 inhibitor.
We and others have demonstrated that either intact MHC molecules or synthetic peptides corresponding to polymorphic regions of MHC molecules inhibit target cell lysis of CTL specific for those MHC Ags (17, 21). We later demonstrated that synthetic peptides corresponding to residues 75–84 of the class I molecule HLA-B2702 block lysis of target cells by CTL in an allele-nonrestricted manner (22). Peptides derived from this region of class I, either alone or in combination with a subtherapeutic dose of cyclosporin A, prevent rejection of heterotopic heart allografts in rodents (23, 24, 25, 42). Lymphocytes isolated from tolerant animals differentiate into donor-specific CTL in vitro, consistent with the notion that the cells are functionally inactivated in vivo (42).
The inhibitory effect of the DQ 65–79 peptide is sequence specific, but not MHC restricted. Peptides synthesized with single amino acid substitutions at individual residues 66, 68, 69, 71–73, and 75–79 lost function. These residues are located throughout the length of the peptide, indicating that the overall sequence of the peptide is important for its function. This contrasts with our previous findings with the synthetic peptide corresponding to residues 75–84 of the class I HLA-B2702 sequence. This peptide blocks lysis of target cells by CTL, and this effect is completely dependent on the presence of an isoleucine at position 80 (22). Substitution of the other residues had little, if any, effect on the function of the class I peptide.
A potential mechanism for DQ 65–79 peptide function is that it interferes with Ag presentation by binding to the peptide groove of MHC molecules. Peptides derived from this conserved region of HLA DQ do bind to HLA class II molecules (43, 44). However, the DQ 65–79 peptide blocks proliferation of all T cells tested, indicating an allele-unrestricted effect. Furthermore, the DQ 65–79 peptide inhibits proliferation of purified T cells stimulated by anti-CD3 mAb or PHA-P, both of which bypass APC. These findings suggest that the peptide-mediated inhibition is independent of Ag presentation.
It has been postulated that soluble whole or fragmented MHC molecules modulate immune responses through interaction with the TCR, potentially by steric inhibition of the TCR interaction with membrane-bound MHC/Ag complexes (3, 5). It is possible that the DQ 65–79 peptide bypasses allele specificity by binding to conserved regions of the TCR. It is also possible that the DQ 65–79 peptide could interfere with costimulation, causing cells to become anergic (45). This type of unresponsiveness can often be overcome by the addition of exogenous IL-2. However, neither exogenous IL-2 nor anti-CD28 mAb reversed the inhibitory effect of the peptide. Therefore, the DQ 65–79 peptide functions through a mechanism not involving TCR/MHC interactions or the induction of classical anergy.
The DQ 65–79 peptide described in this work and the class I-derived B2702 75–84 peptide identified previously are derived from analogous regions of MHC class II and MHC class I, respectively. This region of native MHC class I is important in binding the p58 and p70 KIR of NK cells (9). There is evidence to suggest that functionally similar inhibitory receptors for MHC class II exist, although they have not yet been identified (46, 47). Although the B2702.75–84 peptide blocks NK-mediated killing, peptide function is not dependent on any defined KIR (J. E. Goldberg and C. Clayberger, unpublished observations). Various KIRs are expressed on subsets of NK cells and a small percentage of T cells (9). Unlike KIR, the allele-nonspecific effects of the DQ 65–79 peptide suggest that a potential inhibitory ligand would have to be broadly expressed.
The expression of cytokines and signaling receptors is critical for the initiation of proliferation. Unlike cyclosporin A (36), no inhibition of IL-2 or IFN-γ expression was detected in DQ 65–79 peptide-treated T cells stimulated with PHA-P. Expression of all cell surface receptors tested, including all three chains of the IL-2R, was normal in peptide-treated cells stimulated with anti-CD3 mAb or PHA-P. Instead, the peptide interfered with signals downstream of the IL-2R. Although cdk2 activity was reduced, we could not detect any differences in the phosphorylation pattern of cdk2 or its association with cyclin E in lysates from cells activated in the presence of the DQ 65–79 peptide. Instead, the peptide prolonged the presence of the inhibitor molecule p27 (39). Although it is not known how rapamycin or the DQ 65–79 peptide modulates p27 expression, it may involve the ubiquitin degradation pathway (48).
The DQ 65–79 peptide and rapamycin share functional and mechanistic activities, but they differ in several important ways. First, rapamycin, in a complex with FKBP, blocks Saccharomyces cerevisiae yeast cell division by binding to and inhibiting the activity of TOR (target of rapamycin), a phosphoinositol kinase family member required for growth (49). In contrast, the DQ 65–79 peptide did not affect division of S. cerevisiae yeast cells at any concentration tested (S.-C. Lyu and C. Clayberger, unpublished observations). Furthermore, our experiments show that DQ 65–79 peptide and rapamycin do not bind to the same site on FKBP, and it is likely that the DQ 65–79 peptide does not bind FKBP at all.
Rapamycin has pleiotropic effects on cell proliferation downstream of the IL-2R. As a complex with FKBP, rapamycin blocks the activity of mTOR, the mammalian homologue of TOR, which is critical for cap-dependent translation. and for the activation of p70 S6 kinase (50, 51). The p70 S6 kinase has also been implicated in the regulation of CREB/ATF transcription factors, which are involved in the expression of proliferating cell nuclear Ag, a protein that is required for entry into the S phase of the cell cycle (52). We are currently investigating whether the DQ 65–79 peptide affects these and other signaling events downstream of the IL-2R.
Do analogous peptides corresponding to regions of MHC molecules exist in vivo, and do they mediate similar functions? Although there are no definitive answers to these questions, several lines of evidence suggest that soluble peptides of MHC molecules are present and can mediate immunoregulatory functions in vivo. It has been recognized for some time that class I MHC molecules exist in both membrane-bound and soluble forms, the latter resulting from alternative splicing (53). Soluble class II molecules also exist, and activated lymphocytes produce large quantities of soluble MHC molecules (54, 55, 56). Soluble MHC molecules inhibit immune responses in both allele-restricted and unrestricted ways, and they are likely to be responsible for part or all of the nonspecific immunosuppression observed following blood transfusion in a number of clinical settings (57, 58).
MHC-derived peptides may also mediate some of the phenomena attributed to suppressor cells. There are many studies that document a potent suppressor effect of T lymphocytes or their products, and many of these effects map to the MHC. However, when a thorough analysis of the MHC was conducted, no suppressor genes were identified (59). Our findings suggest that peptides, derived from intact MHC molecules by either pre- or post-translational events, can mediate immunoregulatory effects. The generation of peptides from MHC molecules may occur only in restricted sites or in response to particular stimuli, and secretion and/or processing of these molecules by activated lymphocytes may be important in turning off immune responses. Characterization of such peptides in vitro should prove useful in the design of novel therapies to dampen the in vivo immune response in transplant rejection and autoimmune disease.
We thank Elizabeth Mellins and Jodi Goldberg for helpful discussions and critical reading of the manuscript.
↵1 This work was supported by National Institutes of Health Grants AI 35125, DK 35008, AI 41520, and HD 34214 and by funds from the Ralph and Marion Falk Memorial Research Trust. A.M.K. is the Shelagh Galligan Professor of Pediatrics at Stanford University and a Burroughs Wellcome Scholar in Experimental Therapeutics. M.L.B. is a Howard Hughes Medical Institute Predoctoral Fellow.
↵2 Address correspondence and reprint requests to Dr. Carol Clayberger, Department of Cardiothoracic Surgery, Stanford University, Stanford, CA 94305-5407. E-mail address: cclay{at}leland.stanford.edu
3 Abbreviations used in this paper: KIR, killer cell-inhibitory receptor; cdk, cyclin-dependent kinase; FKBP, FK-506-binding protein; PHA-P, phytohemagglutinin-P; TOR, target of rapamycin.
Received August 8, 1997.
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You are going to email the following Inhibition of Cell Cycle Progression by a Synthetic Peptide Corresponding to Residues 65–79 of an HLA Class II Sequence: Functional Similarities but Mechanistic Differences with the Immunosuppressive Drug Rapamycin
Michelle L. Boytim, Shu-Chen Lyu, Ron Jung, Alan M. Krensky, Carol Clayberger
The Journal of Immunology March 1, 1998, 160 (5) 2215-2222;


Italian architect and writer. He studied painting in Rome. Returning to Turin in 1733, he observed the late works of Filippo Juvarra under construction and, in 1737, edited the papers of Guarino Guarini, the Architettura civile.
Vittone obtained spectacular visual and structural effects in a number of small, central-plan churches that he designed in Turin and elsewhere in the Piedmont from 1737 to 1770. These churches had multiple-level interiors and used innovative vaulting techniques for their complex domes. A central dome might have two or even three successive vaults, the lower ones being pierced to allow the viewer to see through them to the ones above. This placing of structures within structures might also be illusionistically achieved or enhanced by skillful painting or by the manipulation of lighting through cleverly placed windows. A prime example is the Church of Santa Chiara at Bra (1742); it has a low vault pierced by windows through which one sees a second shell, painted with heavenly scenes and lit by windows not visible from the interior.
Vittone would frequently place smaller, subsidiary domes around a larger, lower, central dome and would open up the space for viewing by using relatively slim piers whose curving forms contribute to an impression of light, airy soaring movement in the elegantly decorated interior. Among his other masterpieces are the Chapel of the Visitation at Vallinotto (1738), and the churches of San Bernardino in Chieri (1740) and Santa Chiara in Turin (1742). His later churches, such as the Assunta at Grignasco (1750) and that of San Michele at Rivarolo Canavese, are larger, simpler, and more monumental but feature the same kinds of diminishing successions of curving, converging vaults and piers.
Considered to be the last of the three great masters of Piedmontese Baroque architecture, Vittone achieved a reconciliation of the ideas of his predecessors Guarino Guarini and Filippo Juvarra in developing an immensely creative type of architecture, especially for churches. He published two treatises, "Istruzioni elementari" (1760) and "Istruzioni diverse" (1766).


We can help your business grow and improve your sales performance by analyzing your current sales process, methodology and people and then report back on what should be changed or developed to bring forward a positive change.
We provide an objective, independent view and are able to see any issues clearly to ensure we design a bespoke plan of action based specifically around your needs and requirements.
This could be of assistance with development of effective sales and marketing propositions, providing strategies for testing new products or services, staff recruitment and mentoring, performance management and incentive planning, or generally preparing how to best manage any growth of your business.
We put the right people in the right teams, provide them with the right technology, and design the right process to manage performance within your business.
Our Consultancy services implement practical and realistic changes that can be left in place to ensure we leave your business in a much stronger position than when we arrive.
How can Insight Market Research Consultancy help and support your business?
Our highly trained specialists will analyse your business and implement strategies to help growth.
We will build a step by step plan to support you and your staff whilst positive changes are implemented.
Insight Market Research Consultancy will identify specific training needs and offer solutions to address these areas.
Insight Market Research will develop a friendly and constructive plan to support positive changes within your business.
We approached Insight Market Research Ltd regarding mystery shopping in Germany as a result of a successful project they were already running in the UK. They have since been carrying out research for us on a weekly basis throughout our entire network of German branches and the quality of the services they provide has always been first class. I would have no hesitation in recommending them to anyone requiring a cost effective and flexible mystery shopping provider.


MTV hasn't had much success with its reality programming over the last few years. Recently the spin-off show Snooki & JWoww said goodbye, closing the chapter on the network's association with the hugely popular Jersey Shore. They appeared to be building a sizable audience for their Jersey Shore meets West Virginia reality series Buckwild, but that momentum was lost completely when the show's breakout star Shain Gandee passed away tragically at the age of 21. Since then, MTV's follow-up shows like Big Tips Texas and Slednecks have failed to gain an audience and each lasted only one season.
Given these struggles, it only makes sense that the network would double down on it's only popular non-competition reality show franchise by bringing the original Teen Mom back and recently officially announcing they are casting for a brand new season of 16 and Pregnant.
MTV is casting for the thought-provoking documentary series 16 and Pregnant. This series follows the journeys of young women as they face unexpected pregnancies and navigate the difficulties of becoming parents at a young age.
Our goal is to give viewers a look inside the realities of teen pregnancy — from morning sickness and balancing school responsibilities to navigating incredibly difficult decisions and relationships with boyfriends and/or parents, ultimately to the day of the baby's arrival and beyond. MTV is looking for young women from varying backgrounds who are facing an unplanned pregnancy and would like to share their personal story about this life-changing and complicated journey.
If you would like to share your story in order to raise awareness about this issue, please email us as soon as possible at 16andPregnantCasting@mtv.com. Include the city and state you live in, your contact details, a picture and the reason why you'd like to share your story as part of 16 and Pregnant.
Teen Mom Junkies reports that there may be some changes for Season 6. For starters, the MTV cameras may be following the girls for nine or more months instead of the usual five to better document the impact of having a child so young on these teens' lives.
Teen Mom Junkies also says producers may be proactively searching out expectant teen moms in more extreme situations, and are reportedly reaching out to hospitals to help find them.
As you probably surmised given that MTV has just issued the casting call and the fact that they may be filming the new moms for a period of nine months or more, fans shouldn't expect 16 and Pregnant Season 6 to air until late 2015 or, more likely, 2016.


text:
<?php

namespace app\models\tours;

use Yii;

/**
 * This is the model class for table "tour_fields_values".
 *
 * @property integer $id
 * @property integer $tour_id
 * @property integer $field_id
 * @property string $value
 *
 * @property Value $field
 * @property Tour $tour
 */
class Value extends \yii\db\ActiveRecord
{
    /**
     * @inheritdoc
     */
    public static function tableName()
    {
        return 'tour_fields_values';
    }

    /**
     * @inheritdoc
     */
    public function rules()
    {
        return [
            [['tour_id', 'field_id'], 'integer'],
            [['value'], 'string']
        ];
    }

    /**
     * @inheritdoc
     */
    public function attributeLabels()
    {
        return [
            'id' => 'ID',
            'tour_id' => 'Tour ID',
            'field_id' => 'Field ID',
            'value' => 'Value',
        ];
    }

    /**
     * @return \yii\db\ActiveQuery
     */
    public function getField()
    {
        return $this->hasOne(Field::class, ['id' => 'field_id']);
    }

    /**
     * @return \yii\db\ActiveQuery
     * TODO: личная заметка - заменить костыли на рациональное решение.
     * очередной минус.. Tour::className() - костыль, сразу палящий отсутствие хорошего саппорта у фв.
     * В php уже давно существует саппорт Class::class
     */
    public function getTour()
    {
        return $this->hasOne(Tour::class, ['id' => 'tour_id']);
    }
}

redpajama_set_name: RedPajamaGithub


text:
A first look at Hearts in Space "Shores of Time" video from the band's new Already Gone EP
Already Gone EP"> Thorin Klosowski
| Video |
Thorin Klosowski | November 9, 2011 | 7:30am
Already Gone EP" href="javascript:;">
It looks like Audio Visual Violence Club has been busy lately. On the heels of the recent video for Snake Rattle Rattle Snake's "Like a Moth," the Violence Club just dropped a video for "Shores of Time," the lead track from Hearts in Space's new record, Already Gone, slated for release this Friday, November 11, at the Larimer Lounge. The video, which appears to have been shot in Predator-vision, is going to be familiar to anyone who has seen a bulk of AVVC's work, but this one seems slightly different than the usual cut-up, distorted shots the editors are known for.
The clip does, however, still have tons of random footage from old films, and it captures Hearts in Space's sound remarkably well. The band doesn't really sound like surf rock, but there has always been a little bit of it buried in there. So when the waves are crashing down on the surfers at the beginning of the video and the distortion is going all crazy, it works really well. Like most of AVVC's videos, there are also a bunch of possible metaphors going on, including an effect that makes it look like a house is on fire and the repeated showing of "emergency stop" sign. The whole thing has a weird, haunted-house-on-the-ocean vibe to it. Have a look.

redpajama_set_name: RedPajamaCommonCrawl


The Hauled Water Initiative is an effort by the County to determine the feasibility of using hauled water as a potable water source for new single-family residential development, where there is no other feasible source of municipal or well water available. Documents related to this proposed initiative are available under the Documents section on this page.
In response to a motion by Supervisor Antonovich, the County's Chief Executive Officer (CEO) convened the Hauled Water Task Force ("Task Force") comprised of staff from the Departments of Public Health, Public Works, Regional Planning, Fire, and County Counsel, to study the use of hauled water as a potential source of potable water in certain parts of the County. The Task Force prepared an informational report on the feasibility of using hauled water and presented the report to the Board of Supervisors in 2009.
The Task Force's recommendations were compiled into a revised report, which the CEO presented to the Board on August 17, 2012. On September 4, 2012, the Board instructed the Task Force to prepare the appropriate environmental documentation analyzing the potential environmental impacts of a hauled water policy, and to prepare an ordinance for a single-family residential hauled water use policy for new development. The Task Force determined that an environmental impact report (EIR) is required for the ordinance.
The County of Los Angeles released the Draft EIR for public review on May 31, 2016.
The Notice of Availability is available.
The Draft EIR is completed and is available under the "Documents" section of this web page. The 50-day public review period for the Draft EIR starts on Tuesday, May 31, 2016, and ends on Wednesday, July 20, 2016.


Created a proof-of-concept DApp for a peer-to-peer, blockchain-backed, off-chain oracle, over/under bet. Used Solidity to guarantee the integrity of the bet and oracle. Used Node.js for the off-chain services. Used Vue.js for the front-end. Accessed the blockchain from both Node.js and the browser via Web3. Used Truffle to execute unit tests.
Delivered Solidity smart contracts & an off-chain to on-chain bridge with unit tests for IOV's token sale. Dealt with the non-trivial requirement of accounting for Bitcoin to Ether conversions and refunds.
Used Electron with React and Redux to create a cross-platform graphical user interface (GUI) for CPU/GPU proof-of-work (PoW) cryptocurrency mining. Ran a Monero mining pool using Angular and Node.js. Administered a Cosmos proof-of-stake (PoS) validator node with sentry nodes to mitigate distributed denial of service (DDoS) attacks.
Created a full-stack framework that generates iOS/Android/web app front-ends from a single code base. That same code base provides shared code for the back-end (Google App Engine, MySQL database, etc).
Architected and built the full-stack of a scalable and distributable C++ back-end of microservices for a Google Web Toolkit (GWT) and d3 based front-end for algorithmic trading. Controlled the microservices using websockets. Used shared memory for automatic registration of services co-hosted with the registry service; used websocket registration otherwise. Tied into the Interactive Brokers API.
Quadrupled Baby Doctor, Inc.'s market size. Disobeyed a directive from the CEO that saved him from criminal indictment. Gave a presentation that won a spot in Plug & Play's healthcare start-up accelerator program. Helped raise 320k$. Designed & implemented a HIPAA compliant, GWT compatible tech infrastructure. Managed a remote team.
Managed a remote team. Spearheaded automation of middle office systems. Architected and deployed a robust, OS and database agnostic infrastructure with auditing, role based security, presentation template engine, and distributed job scheduling and execution. Client apps for the system ranged from Excel (via C# dll and/or VBA) to Perl. Used the Model-View-Presenter pattern to facilitate desktop/tablet/phone user interfaces.
Invented profitable directional intra/interday trading algorithms for bond futures. Designed and delivered a Matlab/CQG high-frequency trading system in 4 weeks. Performed ad hoc event simulations. Created event study templates. Bridged Matlab, Interactive Brokers API, Excel, and a proprietary real-time trading system with a C# dll. Manually intervened into an automated process that would have resulted in failing on delivery of a 107MM short for repo.
Consulted for Bigbox Red SL. Automated systems using NetMorpher freeing-up an entire man-month of data entry, extraction, and transformation. Created a NetMorpher based search engine optimization (SEO) tool for publishing classified ads, forum posts, etc with either a few clicks manually or entirely automatically.
Consulted for properazzi.com. Wrote Java and pig code and deployed it to the production hadoop cluster one week after starting. Performed ad hoc statistical analysis for data for press releases. Upgraded and maintained the cluster. Optimized map-reduce jobs. Wrote payment processing servlets using Google's guice for dependency injection. Refactored existing code. Wrote an automated, future-proof back-up system using protobufs and amazon.com's S3.
Consulted for Prometheus Research by pure telecommute. Delivered a 1-month-allotted, one-off web app in 3 weeks and turned it into an integral part of their flagship product. Architected/implemented their web-based data collection services for use in both online and offline modes. Wrote extensions for Firefox.
Developed a digital mash-up machine. The Firefox-based app, tested on Linux and Win32, runs in desktop or server mode and provides a browser-based drag-and-drop (Web 2.0) user interface to instruct the C++ back-end. The machine can automate, personalize, and combine data entry/extraction from disparate sinks/sources (web pages, streaming media, databases, proprietary apps, etc) and output to mobile phone(s), cell(s) in an Excel spreadsheet, specific locations in a Word doc, a browser, printer(s), database(s), etc. The design allows for massive scalability using distributed computers. Conceived a multi-faceted revenue strategy to accompany the machine allowing content providers to earn/charge micro payments for access to their data, for one.
Invented the Internet Oasis - a battery powered, portable Internet cafe. Obtained corporate sponsorship from Via. Liaised with investors, business developers, VC funds, corporations, etc.
Developed and maintained real-time, electronic market making pricing engines for US treasury and agency bonds and optimized a real-time, aggressive proprietary trading engine. Coded real-time asset swap spread and agency discount note pricing engines. Directly supported bond traders' applications. Created a bond calculator for long end US treasury manual trades. Completed the GS Fixed Income Associate Training Program.
Consulted for weather.com and discovery.com. Wrote data capture templates, presentation templates, and workflows. Liaised with clients. Configured TeamSite and OpenDeploy.
Setup an Internet cafe and designed and implemented automatic, fault-tolerant, multithreaded, distributed accounting software using Java, C++, and CORBA. Coded automatic stock market order entry programs and confirmation scripts. Installed numerous intranets with a Linux box server/gateway. Devised and coded the infrastructure for a totally customizable web portal. Designed and implemented an efficient object persistence scheme permitting multiple inheritance, including virtual base classes, and circular object references using recursion.
Joined the POLYSIM team and was christened "Programming Wizard" by project manager and team leader Dr. Rudi Stocker. Participated in the full development lifecycle. Coded the front end of the world's foremost polymer simulator including details regarding the fluid package, reaction packages, and databases. Designed the entire reaction class hierarchy for five major chemistries and their associated sub-chemistries, thermodynamic classes, estimable parameter classes, controller classes, and designed a generic collocation point container class. Coded the interface for all five reactors in the Ziegler-Natta chemistry in addition to reactors in three other chemistries. Provided guidance for several Research & Development Engineers of the eight-member team.
Created a predictive, two-dimensional, time-averaged hydrodynamic model for a circulating fluidized bed (CFB) riser operating in the fast fluidization regime with downward flow of gas and solids near the wall. Modification of the elaborate two-dimensional model allowed for its coupling with kinetic reaction equations to form a general, tractable CFB riser reactor model. The culmination of the research manifested itself in the form of a fully object-oriented, multi-threaded simulator written in C++. The simulator included an MFC graphical user interface supporting drag-and-drop process flow diagram construction, novel object persistence design, and generic flash calculations.
Substantial projects stemming from course work: design and implementation of a generic, multi-purpose chemical plant simulator, Gibbs energy minimization of a modified Claus plant reaction furnace, simulation of transpiration cooling of an insulated cylinder, steam reforming of methane, and isentropic discharge of an ideal gas from a duct.
Achieved a GPA of 3.85/4.0. Awarded John S. Poyen Scholarship & DB Robinson and Associates Travel Grant.


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text:
Microcephaly Could Be Just The Tip Of The Effects Of Zika Virus The devastating birth defect microcephaly may be just a warning sign for the range of problems babies will suffer. Rob Stein tells Linda Wertheimer about what else scientists are learning about Zika.
Microcephaly Could Be Just The Tip Of The Effects Of Zika Virus
Microcephaly Could Be Just The Tip Of The Effects Of Zika Virus 3:45
August 28, 20168:57 AM ET
Heard on Weekend Edition Sunday
The devastating birth defect microcephaly may be just a warning sign for the range of problems babies will suffer. Rob Stein tells Linda Wertheimer about what else scientists are learning about Zika.
LINDA WERTHEIMER, HOST:
This is WEEKEND EDITION from NPR News. Rachel Martin's away. I'm Linda Wertheimer. The Food and Drug Administration now says all U.S. blood donation centers must screen for the Zika virus. News about the first cases of local transmission in Florida have raised fears among women who are pregnant or want to start a family. But what are the odds of contracting the Zika virus, and what does that virus really do? Joining us now to talk about this is NPR health correspondent Rob Stein. Rob, welcome.
ROB STEIN, BYLINE: Nice to be here.
WERTHEIMER: So now we've heard a lot about how Zika causes this terrible birth defect called microcephaly. What do we know about how much of a risk there really is or does it matter how much risk there is if something as catastrophic as microcephaly might happen?
STEIN: Yeah, so the first thing I ought to say, Linda, is that with most things about Zika at this point, unfortunately there's still more questions than definitive answers. It's so new that there isn't really a lot that we know for sure. One thing we do know, though, is that it's pretty clearly been shown that Zika can cause microcephaly in babies that are born to women who get infected when they're pregnant. Now, how often that occurs, we still don't really know. But there are some estimates, and they range from anywhere from about 1 percent to about 13 percent of cases in which the woman gets infected during her first trimester. But there is also increasing evidence that the virus probably poses a risk no matter when a woman gets infected during her pregnancy. And there's probably no time when the pregnancy is completely safe.
WERTHEIMER: What kind of damage does the virus do?
STEIN: It can be really devastating. In fact, there's some new research that just came out. Some researchers published brain scan images from dozens of Brazilian babies who were born to women who got infected when they were pregnant. Whole portions of these babies and fetuses' nervous systems are just missing, like parts of their brain stem, parts of their spinal cord. And in some cases, there are babies that are born whose brains - they seem like they're OK, but after they're born, they realize that parts of their brain or the ventricles were filled up with fluid, sort of puffed up their brains so they looked normal, but, in fact, they were severely damaged.
WERTHEIMER: Now, I know you spent some time with some of these babies when you were in Brazil earlier in the year. Can you tell us anything about what these babies need, how they can be cared for, what it was like to be around them?
STEIN: Yeah, so, you know, some of these babies seem, you know, pretty much like normal babies. I mean, their heads looked a little small, but otherwise, they seemed like your typical baby. Other babies, it was more clear that they had severe problems. They were starting to have seizures. They were starting to have problems eating. Some of them seemed like they were developing OK, but then they stopped eating, started losing weight and started, like, losing ground developmentally. And one of the things that really strikes you is how these babies sound. They often cry way more than normal babies, and their cries can sound really disturbing in some ways. They're much more kind of pained and anguish sounding than a normal baby's cry, and it's much more difficult for the mothers or anyone else to soothe these babies.
WERTHEIMER: So, Rob, we've had cases of the Zika virus appear in Florida. It's at the end of the summer. We have all of next summer to look forward to. How serious is this? How frightened should we be?
STEIN: You know, it's definitely something to be concerned about and keep an eye on, but we just don't know yet how bad it will get. And public health people tell me that, look, we've had other diseases like this. We've had dengue, chikungunya. They're spread by the same mosquitoes. They're very similar viruses. They've come into this country, and we haven't had widespread outbreaks. We've had little clusters pop up that die off very quickly, and the same thing could happen with Zika. And so it could end up being not a huge problem in this country. We just don't know yet.
WERTHEIMER: NPR health correspondent Rob Stein, thank you.
STEIN: Thank you.


In order to better understand fluctuations in behaviour and experiences in the flow of daily life, EU-GEI scientists have developed the PsyMate device and application. This data collection and software tool is able to capture multiple parameters in the flow of people's everyday life. The PsyMate tool helps to empower patients in managing their own life, it helps clinicians in terms of diagnosis, and it can contribute to the evaluation of mental health care interventions. The tool received the (Dutch) Mental Health TECH award in 2015.
The PsyMate App is a platform to run ecological data-collection schemes and embedded mobile interventions that can be customized to different needs in research and basic clinical practice.
The PsyMate App implements the "PEAS 4.5 specs" that define the intended functionality of the PsyMate and the context in which it should operate. The PsyMate is protocol-driven. Protocols specify the different moments for data collection (e.g. based on time or beeper based algorithms or on events) and the type of data ((self-report) questionnaires or sensor data). Algorithms can be added that trigger additional data collection (or provide the user with instructions). These triggers can be defined on user-based reports (e.g. high anxiety) or contextual information (e.a. changes in places tracked by GPS or activation tracked by the motion sensors).
The data capture is very user-friendly: at irregular intervals during the day (i.e. randomized), the PsyMate App will beep to prompt the user to fill in basic information on mood states and current circumstances and experience (this is called the "Experience Sampling Method" or ESM). Filling in the prompted questionnaire takes around 2 minutes. Data are then stored and can be analysed after e.g. 1 week to build up what is called a "film" rather than a "snapshot" of daily life.
A so-called 'demo' App is available for free and accessible without restrictions for users and clinicians. It is actually available in 5 languages (English, French, German, Dutch, Spanish) and other translations are in preparation. Because everyone can subscribe to it, it is also used in fields outside mental health, such as somatic care (primarily stress related illnesses as IBS, post stroke rehabilitation, etc), and organisational psychology.


text:
Q: Check for duplicate arrays in buffer I am working on an embedded system in which a packet is saved if the device cannot establish a communication. I have many different scenarios where the packet can be saved and I want to avoid saving repeated packages.
The packets of the system are 16 bytes long and I store them in a buffer of 320 bytes (in order to keep 20 packages maximum).
How can I check if the packet I am about to save is repeated somewhere in the buffer?

A: Just loop through saved packets and compare them with the new one to be added
#include <stdio.h>
#include <assert.h>
#include <string.h>
#include <stdlib.h>

// represents a 16 char packet
typedef char packet_t[16];

/* pktbuf library - used to store up to 20 packet_t packets */

struct pktbuf_s {
    packet_t buffer[20];
    size_t packetcnt;
};

#define PKTBUF_INIT()  {0}

int pktbuf_save(struct pktbuf_s *t, packet_t *packet)
{
    assert(t != NULL);
    assert(packet != NULL);
    for (size_t i = 0; i < t->packetcnt; ++i) {
        if (!memcmp(&t->buffer[i], packet, sizeof(*packet))) {
            // duplicate packet found in buffer, notify user application
            return 1;
        }
    }
    if (t->packetcnt == sizeof(t->buffer)/sizeof(*t->buffer)) {
        // no place in buffer, notify user
        return -1;
    }
    // save the packet and increment packet count
    memcpy(&t->buffer[t->packetcnt], packet, sizeof(*packet));
    t->packetcnt++;
    // succcess
    return 0;
}

void pktbuf_clear(struct pktbuf_s *t)
{
    assert(t != NULL);
    t->packetcnt = 0;
}

size_t pktbuf_get(struct pktbuf_s *t, packet_t **packets)
{
    assert(t != NULL);
    *packets = t->buffer;
    return t->packetcnt;
}

/* User application example ----------------------------------- */

void packet_receive(packet_t *packet)
{
    memset(packet, 1, sizeof(*packet));
}

int main()
{
    struct pktbuf_s pktbuf = PKTBUF_INIT();

    // some user app receives packet
    packet_t packet;
    packet_receive(&packet);
    // save received packet in bnuffer
    if (pktbuf_save(&pktbuf, &packet) == -1) {
        // no place in buffer to save the packet
        // print error message? blink with the led? or just ignore?
    }
    // continue the user application, until we want to...

    // drain packet buffer
    packet_t *packets;
    const size_t packetscnt = pktbuf_get(&pktbuf, &packets);
    for (size_t i = 0; i < packetscnt; ++i) {
        // do smth with packet: packets[i]
    }
    // clear buffered packets
    pktbuf_clear(&pktbuf);

    // continue with user application

    return 0;
}

A: Packet identifiers / Sequence number
It is difficult to give relevant advice without knowing the packet structure or intended purpose. E.g., is this an RS485 network? UART communication with a single device? Something else?
However, if you have control over the packet structure, you could use a packet identifier or sequence number. If you are only communicating with one device, this alone may be sufficient to discriminate between packets. The packet structure might look something like the following. The relevant field here is seq no. (sequence number). Numbers indicate byte offsets.
0         1         2     3      15
---------------------------------------------------
| control | seq no. | len | data | checksum / crc |
---------------------------------------------------
           ^^^^^^^^^

If there are multiple devices, your packet probably includes source and address fields. In this case, a combination of the address information and the packet identifier would be adequate.
0         1         2        3        4     5      15
---------------------------------------------------------------------
| control | seq no. | source | dest   | len | data | checksum / crc |
---------------------------------------------------------------------
           ^^^^^^^^^^^^^^^^^^

The onus would be on the device generating the packet to increment the sequence number for every new packet. Since your buffer can only hold 20 packets, a 1-byte ID / sequence field would probably be adequate.
(Sequence numbers can be used for other purposes also, such as checking if a packet has been lost in transit - in which case there will be a 'gap' in the sequence numbers of received packets.)
It is hard to know whether such optimizations are necessary - or whether using a simple memcmp() to compare packets is adequate.

redpajama_set_name: RedPajamaStackExchange


For Loughton, Chigwell and Woodford the fabulous historical Epping Forest is perhaps our greatest asset. Epping Forest is one of London's largest open spaces and as a registered charity is managed by the City of London with little cost to the general public.
Many use the forest for walking, running, cycling, horse riding, football, angling even golf. Alongside these pursuits there are a whole host of other activities to get involved with. Currently there is a fantastic exhibition on called Forest Charter 800 for all. The original Forest Charter was signed by a 9 year old Henry III in 1217. The exhibition explores what Epping Forest was used for then. Its provisions protected the rights of 'free men' to enter and earn a living on Forest land for centuries afterwards.
You can discover why your dog should be very grateful that 'expeditation' of dogs is no longer required; find out more about what on earth your pigs' 'pannage' opportunities and how to avoid charges for 'chiminage'. There are some newly commissioned illustrations and activity sheets that open a window on what people spent time doing in Epping forests way back a 800 years ago.
The Exhibition is being held in the Queen Elizabeth's Hunting Lodge, Rangers Road, Chingford, E4 7QH and is on until Monday 4 Sep 2017. It is open 10am until 5pm (excluding Mondays) and entry is free and requires no booking.
Epping Forest is open 24 hours a day, 365 days a year if you've never been there its time to check it out and find out more about what life is like just beyond Walthamstow.


text:
Alien Storm on the Master System is a severly scaled-back port of the Genesis version. It's single-player only, only Slammer and Garth are playable, there are only three simultaneous enemies, and certain attacks were removed. However, it still retains the original's "triplicated fun" in the form of traditional brawler stages, auto-scrolling shooter sections, and first-person turret stages.
Shape-changing alien scum take over your city. Fry the slimy horrors and smash their bid for global conquest. Blast into action with fire blazers, electric whips and more!
This port lacks the ability to jump. You can still roll and do dash-attacks, however.
In the original and Genesis versions, the robot is named "Scooter." In this version, he's known as "Slammer."
Running, Shooting
Attack, Roll
Cartoony
Alien Storm is a decent port of Sega's overlooked arcade brawler, at least considering it's on the Master System. Of course, it's heavily scaled back, although it does have all the variety stages intact. Like most SMS games, the graphics and sound are passable but not amazing. Its challenge level and length are both moderate.
Dynamite Dux


text:
Proceedings [of The] Annual Business Meeting, Издања 41-44
Аутор: State Historical Society of Wisconsin
consin Library Association and this Society, is the directing force in all these movements, and allows none of them to suffer for lack of energetic, professional advice and assistance. It is confidently expected that at the session of the Wisconsin legislature, this winter, the commission will be granted an increased appropriation, with an expert official staff. The State will, in consequence, soon take a ieading stand, not only in the matter of free traveling libraries for rural communities, but in free public libraries for municipalities.
CONSTITUTIONAL REVISION. The Society is working under a constitution adopted in 1853. At that time, there were no models for such a document, other than those of the older State historical societies of the Atlantic slope, which are private, and more or less exclusive, corporations. Our Society, however, after the coming of Secre. tary Draper, the popularizing of the membership list, the granting of State aid, and the assumption of State trusteeship, gradually, at successive stages, took on a character quite distinct from its sister organizations in the East; its aims, its methods, have become in many respects different, — so different, in fact, that the old constitution, although frequently patched, is in many particulars unsuited to our work. During the greater part of these forty-three years of development, we have come to rear a code of usage which represents more to us, in our daily operations, than the letter of the constitution. On the eve of moving into a new and perinanent home, the first roof-tree which we can call our own, it would seem appropriate that our fundamental rules be so revised as authoritatively to recognize existing, well-tried methods, to eliminate such of the machinery of our organization as experience has found useless, and in general to improve its working. The appointment at this meeting of a committee on the revision of the constitution and by-laws, seems advisable.
PROGRESS UPON THE NEW BUILDING.
It is with great pleasure that we are able to announce satisfactory progress upon our new building.
At a special meeting of the Board of Commissioners for Erecting the State Historical Library, held January 7th and 8th, Messrs. Ferry & Clas, of Milwaukee, were chosen as the architects for the building; and they were requested to develop the two sets of plans which had been presented by them. The plans which they submitted in competition, and upon which they had won their case, provided for a cruciform building, of classical design, with the reading room and administrative offices in the center, and book-stacks as wings on either side. The architects had presented an alternative plan, being also a classical building of the Ionic order, -- but with a solid structure in front, containing reading room, seminary rooms, and offices, and two book-stacks projecting from the rear, – the museum to cover all, in the fourth story. At a meeting of the board, held March 27th, the alternative plan, last described, was adopted, and the architects instructed to prepare at once detailed plans and specifications.
Meanwhile, the Board of State University Regents, in accordance with the terms of chapter 298, laws of Wisconsin for 1895, formally transferred to the State, for the site of the building, a large plot of ground at the west end of the property between Park, Langdon, and State streets, known as the "lower campus." A loan of $60,000 was obtained from the Commissioners of Public Lands, with which to commence the work of construction.
The special committee on the selection of facing stone met May 6th, and, after thorough discussion of various samples of stone and of a special report by the architects, selected the buff-Bedford (Ind.) limestone, and the Berea and Amherst (0.) sandstones, as a basis for the bids of contractors. Their report was adopted by the executive committee, the same day. Later (May 28th), the detailed plans for the basement and first story of the building were adopted by the full Board. Sealed proposals were duly advertised for, in accordance with the terms of the creating act.
On the 23rd of May, the chairman of the Society's delegation upon the Board of Commissioners, Gen. Lucius Fairchild, had passed away. At a meeting of the executive committee of the Society, held June 25th, the Hon. William F. Vilas was chosen
to succeed him, and took his seat upon the Board at the meeting of the latter on the 29th of June. The Board at this meeting unanimously adopted the following resolution:
Resolved, that the Board has received with great sorrow, information of the death of Commissioner Fairchild. In him, his fellow commissioners recognized, by common consent, one to whom, perhaps more than to any other person, was due the passage of the act providing for the noble building in the construction of which this board is now engaged, and which will in some sense be his monument. At the several sittings of the Board, throughout the first year of its existence, his well-considered counsel has been of the greatest value in times of perplexity, and his charming manner a delight and an inspiration to all. In looking upon his vacant chair, the surviving commissioners feel that there has gone forth from their midst, one who was not only a valued co-laborer in a work upon which he had dearly set his heart, but also a most beloved friend.
Bids for constructing the basement and first story being opened, in the presence of competitors, the contract was awarded to the lowest bidder, Thomas R. Bentley, of Milwaukee, for $42,553 — buff-Bedford stone, from the Dark Hollow (Ind.) quarry, to be the facing material. The executive committee of the Board, in whose hands the supervision of the construction was placed, afterwards made a contract with the Consolidated Stone Co., of Chicago, owners of the quarry selected, by which the latter agreed to furnish stone to bidders for the remainder of the building, at the same price paid by Bentley for use in the basement and first story. T. C. McCarthy, of Madison, was appointed inspector of the works. Later, the Pittsburg Testing Laboratory, of Pittsburg and Chicago, was contracted with for mill and shop inspection of the steel work used in the construction.
At the quarterly meeting held September 28th, a report of the summer's work, and of receipts and expenses to date, was made by the executive committee, in the course of which this comment was made: "The work has been delayed, at times, by unavoidable difficulties: such as a strike in the Dark Hollow quarry; non-arrival of stone and steel, in the size immediately required; and, throughout September, by stormy weather. As a whole, however, the progress has been quite equal to the expectation of the committee, and the contractor now hopes to have the present contract completed before December 1st next. The quality of the work appears to be excellent, at every point; and the Committee feel confident that the building, when completed, will be a credit to the Commission, the Historical Society, and the State."
Continued stormy weather and aggravating non-arrival of stone have still further delayed operations, but it is expected that the Bentley contract will be completed by the first of January next.
At the September meeting of the Board, the architects were directed to complete at once the plans and specifications for the remainder of the part of the building now to be erected. Messrs. Ferry and Clas are now engaged upon this work, and it is hoped that proposals may be advertised for in February next, to the end that work can be resumed early in the spring
Provided no unforeseen event occurs to interrupt the operations of the Board, there is every reason to suppose that the Society may be enabled to move into its new quarters two years hence, or certainly not later than the spring of 1899. If the removal could take place sooner than this, it would be the better for all concerned. Our present rooms in the Capitol are already overcrowded with books, and two years more of accumulation, at our ever-increasing ratio, will find us seriously hampered for space for administrative and reading-room purposes; the State government urgently needs, in its growth, the three floors we now occupy; and already considerable uneasiness is felt by our neighbors in the south wing, at the enormous weight of our possessions. Doubtless this alarm is thus far without sufficient warrant; but it exists, and we must reckon with it. Were the legislature to enable us to spend more than the stipulated $60,000 within 1897, the work of construction could be materially hastened, and thus the interest of all parties concerned be advanced. It is worth while considering, also, whether the completion of the permanent home of the State Historical Society would not be a highly desirable achievement for the semi-centennial year (1898).
In any event, the Society and the great educational interests which it represents, have in the present outlook much to be thankful for. The near future is bright with the promise of still better days. On behalf of the Executive Committee,
REUBEN G. THWAITES,
E. – REPORT OF EXECUTOR OF DRAPER ESTATE.
Madison, Wis., November 16, 1896. Mr. R. G. Thwaites, Secretary State Historical Society
DEAR Sir: Final judgment in the matter of the estate of Lyman C. Draper was entered on the 6th day of November, 1896. By this judgment, the homestead of the late Lyman C. Draper, namely, lot 4, block 51, in the city of Madison, was confirmed in the State Historical Society, and also the title to the library and manuscripts.
The hope and expectation of Mr. Draper was, that the Society would receive a very considerable sum from his personal estate, particularly from his iron-mining property; but after the payment of the expenses of his last illness, the funeral expenses, the debts, and the costs of administration, there was no estate left for distribution to the residuary or general legatees.
Owing to the fact that some claims which were made against the estate, amounting to a considerable sum, were either successfully contested or were withdrawn, the library and manuscripts were saved to the Society. The claims so presented, and the disposition of them, were as follows: (1) A claim on the part of Mrs. Draper, widow of the deceased, for her support and maintenance; and (2) a claim on the part of George W. Hoyt, for $1,160.50, were both disallowed after contest. The claim (3) of M. H. Chynoweth and others, for $1,200 and interest, was withdrawn.
Questions having arisen in the mind of the executor as to the proper construction of the will, he petitioned the court for a construction thereof. At the hearing of such petition for construction, Mr. H. M. Lewis appeared in behalf of the State Historical Society, Mr. Burr W. Jones, guardian ad litem, appeared in behalf of the infant, and Messrs. Bashford, O'Connor, & Aylward in behalf of the adult and general legatees named in said will.
The particulars in which the executor sought information, were whether the legacy of the library and manuscripts to the State Historical Society was a specific or general legacy; whether certain of the property belonging to said deceased, which came to the hands of the executor, was liable in the first instance to the payment of debts, costs of administration, or to the payment of legacies mentioned in said will; and whether the said


Last month, activist Charmian Gooch won the 2014 TED Prize, giving her organisation, Global Witness, $1 million toward her cause: making company ownership more transparent and accountable.
This is a huge win for the fight against global corruption. Secret deals and anonymous companies deprive some of the world's poorest people of money that could go toward education or health care programmes. It's an issue that ONE's working on too – our phantom firms campaign has made real progress in calling for European leaders to crack down on these shady companies.
So many of the countries rich in natural resources like oil or diamonds or timber are home to some of the poorest and most dispossessed people on the planet. And much of this injustice is made possible by currently accepted business practices. And one of these is anonymous companies.
Want to know the worst part? It's legal.
In an era where information is so easily accessible, Gooch argues that it's unacceptable that company information is so hard to get hold of. Protecting tax evaders, corrupt government officials and criminals has become an accepted business practice. But that must change, and they must be exposed and stopped.
This isn't just a problem in some far-off tropical tax haven. The current system allows the true owners of companies around the world to remain hidden from the public, creating opportunities for scandal and abuse on your own doorstep. For example, Ukranian investigative journalists revealed that the former Ukraine presidential compound is owned by UK phantom firm.
For us to know who owns and controls companies, so that they can no longer be used anonymously against the public good. Together, let's ignite world opinion, change the law, and launch a new era of openness in business.
Like Charmian, we want to see information about who owns companies and trusts to be made public. Join us in calling for a global crackdown on phantom firms.


text:
Plumbers in St Louis Park, Minnesota
Dean's Professional Plumbing, Heating and Air has been the premier company to go to for plumbers in St Louis Park, Minnesota. Dean's plumbing and heating specialists have the experience and knowledge you need to handle all of your service needs. Using only certified and licensed technicians, Dean's crew is able to complete jobs quickly and efficiently, specializing in service and small jobs. Even though they started as a plumbing company and have grown over the years to include full service heating and A/C, Dean's Professional Plumbing, Heating and Air has remained family owned and operated.
At Dean's, you can expect clean, fast and friendly service. Customer satisfaction is their number one priority. Their licensed specialists always arrive on time, are professionally dressed and take care to leave your home cleaner than when they arrived. They offer a 24-hour emergency service to St Louis Park customers for those unexpected plumbing and heating emergencies. From service and installation, to repair and remodeling, Dean's crew of professional plumbers and heating experts can do it all.
Services Available in St Louis Park and Twin Cities Metro Include:
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Plumbers in St. Louis Park, Minnesota
St. Louis Park is a city adjacent to Minneapolis, Edina, Golden Valley, Minnetonka, Plymouth and Hopkins. Dean's plumbers in St. Louis Park offer fast, efficient plumbing services. Dean's professionals are licensed specialists who always arrive on time. You can expect plumbers professionally dressed who will deliver clean, fast and friendly service. Customer satisfaction is their number one priority. They offer a 24-hour emergency service to St. Louis Park customers for those unexpected plumbing emergencies.
Dean's Plumbers in St. Louis Park, home of U.S. Senator Al Franksen
St. Louis Park is home of United States Senator Al Franken. Dean's plumbers in St. Louis Park as well as the Twin Cities, offer fast, efficient plumbing services. Dean's plumbers in St. Louis Park have a lot of plumbing experience, knowledge and licensed professionals to be proud of. Dean's professional standards are high. They use only certified and licensed technicians. For plumbers in St. Louis Park, Minnesota, Dean's Professional Plumbing, Heating and Air is the company to turn to. Even though they started as a plumbing company and have grown over the years to include full service heating and A/C, Dean's Professional Plumbing, Heating and Air has remained family owned and operated.
Plumbing Services Available in St.Louis Park and Twin Cities Metro Include:
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I am using Unity to develop a Hololens app.
Most of the time the room mesh that I receive from the Lens is aligned with the UP direction in Unity's world (0,1,0).
However, it sometimes occurs that the room meshes suddenly losses its alignment with Unity UP vector, which results in a tilted mesh (about 40 degree off Unity's UP direction).
(Please also note that once the tilt happens the room meshes will be tilted both on the editor with remote debug, and when running the app directly on the lens itself).
Now I understand that the origin of the room is different from Unity but 40 degrees seems a bit off, that would also mean that many game properties would have to be changed accordingly (gravity direction for instance).
Could anybody confirm that this behavior is not to be expected, and that a room mesh should always more or less be aligned with Unity's UP vector?


who have exhibited leadership potential.
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