Through the actions of one or more isoforms of the enzyme 5α-reductase in many male reproductive tissues, circulating testosterone (T) undergoes metabolic conversion into 5α-dihydrotestosterone (DHT), which binds to and activates androgen receptors (AR) with greater potency than T. In birds, T is also subject to local inactivation into 5β-DHT by the enzyme 5β-reductase. Male golden-collared manakins perform an androgen-dependent and physically elaborate courtship display, and these birds express androgen receptors in skeletal muscles and spinal cord at levels far greater than those expressed in species with more limited courtship routines, including male zebra finches. To determine if local T metabolism facilitates or impedes activation of male manakin courtship, we examined expression of two isoforms of 5α-reductase, as well as 5β-reductase, in forelimb muscles and spinal cords of males and females of the two aforementioned species.We found that all enzymes were expressed in all tissues, with patterns that partially predict a functional role for 5α-reductase in these birds, especially in both muscle and spinal cord of male manakins. Moreover, we found that 5β-reductase was markedly different between species, with far lower levels in golden-collared manakins, compared to zebra finches. Thus, modification to neuromuscular deactivation of T may also play a functional role in adaptive behavioral modulation.Given that such a role for 5α-reductase in androgen-sensitive mammalian skeletal muscle is in dispute, our data suggest that, in birds, local metabolism may play a key role in providing active androgenic substrates to peripheral neuromuscular systems. Similarly, we provide the first evidence that 5β-reductase is expressed broadly through an organism and may be an important factor that regulates androgenic modulation of neuromuscular functioning. Sex steroid hormones activate diverse vertebrate reproductive behaviors by their actions on central and peripheral tissues. In reproductively active males, gonadal testosterone (T) increases aggressive behavior, as well as appetitive (courtship) and consummatory (copulatory) behavior. To achieve these effects, T acts on neural circuits and/or skeletal muscles that motivate behavioral performance and enhance motor output –3. The aIn many male reproductive tissues, T undergoes conversion into the potent androgen 5α-dihydrotestosterone (5α-DHT) by one of two main isoforms (types 1 and 2) of the enzyme 5α-reductase –6. This In other tissues of some species, especially birds, T can undergo conversion into an inactive androgen, 5β-dihydrotestosterone (5β-DHT), by the enzyme 5β-reductase . AlthougManacus vitellinus) and the zebra finch (Taenoygia guttata). Male manakins perform a complex, physically intensive courtship routine that involves elaborate dances above the forest floor and the production of mechanical wing-snaps  to meet conditions of normality. Statistical analyses were performed using SPSS v20.In both manakin and zebra finch, we successfully amplified fragments of the SRD5A1, SRD5A2, and SRD5B1 genes, which encode the type 1 5α-reductase, type 2 5α-reductase, and 5β - reductase, respectfully. Alignment analyses revealed that the gene sequences were highly similar between species, with ≈ 90 % homology for SRD5A1, ≈96 % homology for SRD5A2, and ≈ 96 % homology for SRD5B1. These data suggest that the genes that express androgenic reductase enzymes are relatively conserved between the golden-collared manakin and zebra finch. Furthermore, these data may even imply broader conservation in reductase enzymes among passerines, given that manakins and finches are relatively distantly related within their common Order.p = 0.03), with males expressing this transcript more abundantly than females (p < 0.05). However, we discovered no regional differences in type 1 5α-reductase across the spinal cord  = 0.52, p = 0.60), nor did we find a sex × spinal cord region interaction  = 0.58, p = 0.57).We first examined type 1 5α-reductase expression in the spinal cord of the golden-collared manakin  = 0.12, p = 0.89; sex × spinal cord region interaction: F  = 0.62, p = 0.55) or 5β-reductase  = 1.60, p = 0.22; spinal cord region: F  = 0.23, p = 0.80; sex × spinal cord region interaction: F  = 0.04, p = 0.96).We detected no evidence of significant variation in expression patterns of either type 2 5α-reductase  = 1.14, p = 0.72; region: F  = 0.45, p = 0.64; interaction: F  = 0.68, p = 0.52), type 1 5α-reductase  = 3.87, p = 0.06; region: F  = 0.34, p = 0.72; interaction: F  = 0.27, p = 0.77), or 5β-reductase  = 1.07, p = 0.31; region: F  = 0.29, p = 0.76; interaction: F  = 1.84, p = 0.18).We also examined enzyme expression in the spinal cords of zebra finches Fig. . Overallp = 0.03), such that males expressed more of this gene in these muscles than females (p < 0.05). There was no difference in type 1 5α-reductase across muscle  = 0.66, p = 0.53), nor was there a sex × muscle interaction  = 0.35, p = 0.71).Next, we examined the expression patterns of reductase enzymes in the main wing muscles of the golden-collared manakins Fig. . We uncop = 0.12) and muscle  = 0.70, p = 0.50). Further, there was no evidence of a significant sex × muscle interaction on the expression of this gene  = 0.53, p = 0.60).We found that muscle expression of type 2 5α-reductase was statistically indistinguishable across sex  = 2.63, p < 0.01), in that they were significantly higher in the SH compared to either the SC (p < 0.001) or PEC (p < 0.01). Yet, there was no difference in 5β-reductase mRNA expression between the SC and PEC. We found no sex difference  = 0.80, p = 0.38) or sex × muscle interaction  = 0.26, p = 0.78) in the expression of this gene.With respect to the expression of 5β-reductase, levels of this transcript varied among muscles  = 8.40, p = 0.58) or muscle  = 3.06, p = 0.06) on mRNA levels. However, we did uncover a significant sex × muscle interaction  = 5.33, p = 0.01), and further post-hoc tests showed that males maintained higher levels of type 1 5α-reductase in their SH (p < 0.05). In the other two wing muscles, expression levels of this gene were indistinguishable between the sexes (p > 0.05).We similarly compared reductase expression patterns in the wing muscles of zebra finches Fig. . For typp < 0.001), such that transcript levels were higher in the SH compared to the SC (p <0.001) and PEC (p <0.001). Otherwise, we found no difference in on type 2 5α-reductase expression between the sexes  = 0.87, p = 0.36), nor did we find evidence of a sex × muscle interaction  = 0.26, p = 0.77).There was an effect of muscle on type 2 5α-reductase expression  = 34.17, p = 0.043), with expression being greater in the SH compared to both the SC (p < 0.05) and the PEC (p < 0.05). Moreover, while there was no overall sex difference in  = 1.32, p = 0.26), we did detect a significant sex × muscle interaction  = 6.39, p < 0.01). Post-hoc analyses showed that males express more 5β-reductase in the SH than females (p < 0.05), but that such differences were not present in either the SC (p > 0.05) or the PEC (p > 0.05).For 5β-reductase, we found that expression levels varied among muscles  = 3.55, p = 0.11) or type 2 5α-reductase  = 1.96, p = 0.17). However, spinal cord expression of 5β-reductase was significantly greater in zebra finches, compared to golden-collared manakins  = 9.32, p = 0.003).In a final analysis, we compared expression patterns of reductase enzymes across species Fig. . In the p = 0.003). By contrast, expression of type 2 5α-reductase in the muscle was statistically indistinguishable between these two species  = 0.23, p = 0.63). Importantly, we also found that levels of muscular 5β-reductase were significantly greater in the zebra finch, compared to the golden-collared manakin  = 18.66, p < 0.001).We found that muscle expression of type 1 5α-reductase was greater in zebra finches than in golden-collared manakins  = 9.61, The general hypothesis motivating this research is that, in both wing muscle and spinal cord, enzymes involved in the synthesis of 5α-reduced androgens are expressed at greater levels in birds that demonstrate significant androgen-dependent motor function as part of masculine courtship displays. Thus, we predicted higher expression of 5α-reductase isoforms not only in male birds compared to females, but also in golden-collared manakins compared to zebra finches. Because 5β-reductase inactivates androgens, we predicted an inverse expression pattern for this enzyme, relative to the activating 5α-reductase isoforms.Our results provide several lines of evidence, but not all, to support the predictions outlined above. First, we detected expression of both type 1 and type 2 5α-reductase isoforms in all the tissues and in both of the species we examine. Expression of these enzymes suggests that androgen metabolism actively occurs in the spinal cord and wing muscles, effects likely conserved across both oscine and suboscine passerine species. Second, we found that male golden-collared manakins express relatively higher levels of type 1 5α-reductase in their spinal cord and wing muscles, compared to females of this species. This overall sex difference was not present in zebra finches, which instead showed a sex differences in the expression of type 1 5α-reductase only in the SH muscle. Thus, the capacity to form the active androgen 5α-DHT in the manakin spinal tissue and wing musculature may be greater in males than in females, but this sex difference is apparently absent in zebra finches.Interestingly, zebra finches expressed more type 1 5α-reductase in their muscles than golden-collared manakins, regardless of sex or wing muscle in question. This result is contrary to our prediction, as it suggests that zebra finches have a greater capacity to metabolize T into its more potent state. Androgens do impact zebra finch muscles , 28, so Given that the wing muscles constitute a large portion of a bird’s mass, our result showing abundant expression of 5β-reductase in both the manakin and zebra finch indicate that this enzyme is transcribed throughout much of the avian body. Past work in several birds shows that 5β-reductase is located in the brain, pituitary, skin and cloacal gland , 50, wheAs discussed previously, both manakins and zebra finches express AR in the spinal cord and several skeletal muscles, with expression in manakins greatly exceeding that of zebra finches and a host of other species that do not perform extravagant physical displays , 44. OthNotably, we find a variety of muscle-specific effects in enzyme transcription. For example, in zebra finch muscles, type 1 5α-reductase 1 was expressed at significantly higher levels in the male SH compared to females, with no sex difference in other muscles. These results indicate that males exceed females in the capacity to provide 5α-reduced androgenic metabolites to AR in some passerine skeletal muscles, a result predicted by our initial hypothesis. Furthermore, in contrast to our hypothesis, male zebra finches expressed more 5β-reductase in the SH than females, but there was no sex differences in the expression of this gene in the other muscles. This level of muscle specificity in enzyme expression was not present in golden-collared manakins; thus, even though both type 1 5α-reductase and 5β-reductase can be expressed in some wing muscles at greater levels in males than in females, it is clear that such effects are not consistent across species. The functional significance of these muscle-specific sex differences in androgenic reducing ability within the zebra finch muscular system is currently unclear.Across all regions of the manakin spinal cord, type 1 5α-reductase was expressed at greater levels in males compared to females. By contrast, no differences across sex were observed in zebra finches type 1 5α-reductase, and there were no sex differences for type 2 in either manakins or zebra finches. Moreover, there were no sex differences in 5β-reductase expression in the spinal cord of either species, although golden-collared manakins expressed far less of this enzyme than zebra finches. These results suggest that the type 1 5α-reductase isoform may be especially important in the male manakin spinal cord and that relatively low levels of 5α-reductase may exacerbate the capacity to metabolize androgens into 5α-DHT (see above). Interestingly, we have previously observed that after injection of radiolabeled T, radioactivity accumulates to a greater degree in the spinal cords of male manakins, compared to females . Thus, tFactors other than courtship behavior may account for some of the differences in reductase expression we uncover. For example, in theory, species and sex differences in circulating androgens may influence the expression of reductase enzyme . We can Equally important to recognize are some of the limitations of our species comparison. Golden-collared manakins and zebra finches are distantly related within the passerine family, and thus a variety of ecological, social, and neutral evolutionary processes may influence the evolution of peripheral androgenic control systems . Still, In general, expression levels of the two isoforms of 5α-reductase were similar enough to each other to suggest that neither isoform exerted the predominant or exclusive functional effect. Thus, while patterns of expression indicate that the type 1 isoform may be locally regulated to enhance formation of more potent 5α-reduced metabolites in manakin skeletal muscle and spinal cord, the type 2 isoform would appear abundant enough in both species to similar have some effect on androgenic metabolism. These differences may stem from the possibility that the two isoforms utilize different substrates with different functional endpoints across species, and this idea merits further investigation in birds.A receptors are expressed, locally produced allopregnanolone might regulate neurotransmission [Androgen-reducing enzyme may also have other physiological functions in manakins and zebra finches. For example, in addition to androgens, some progestins can be 5α-reduced producing physiologically relevant products. One of the best examples of this is allopregnanolone, which is an endogenous steroid that potently facilitates GABAergic inhibition and is produced via 5α-reduction and 3α–hydroxylation of progesterone . It is tsmission . Elevatesmission . We havesmission . Thus, iThere is also recent evidence that 5α-reductase can metabolize glucocorticoids, thus limiting their actions . TherefoIn summary, our results demonstrate that type 1 and type 2 5α-reductase, as well as 5β-reductase, are expressed in skeletal muscle and spinal cords of two passeriform species, the sub-oscine golden-collared manakin and the oscine zebra finch. Patterns of expression in these tissues and between these species are consistent with a role for metabolic regulation of androgenic hormones within the neuro-motor apparatus to influence courtship behavior, especially the golden-collared manakin’s physically demanding courtship display. Whereas there is debate as to whether 5α-reductase plays a significant role the activation of AR in mammalian skeletal muscle –32, our AR, androgen receptor; PEC, pectoralis; SC, surprcoricoideus; SH, scapulohumeralis caudalis; T, testosterone


TNF leak is reversible and independent of cell death. Leak correlates with disruption of continuous claudin-5 immunofluorescence staining, myosin light chain phosphorylation and loss of claudin-5 co-localization with cortical actin. All these responses require NF-κB signaling, shown by inhibition with Bay 11 or overexpression of IκB super-repressor, and are blocked by H-1152 or Y-27632, selective inhibitors of Rho-associated kinase that do not block other NF-κB-dependent responses. siRNA combined knockdown of Rho-associated kinase-1 and -2 also prevents myosin light chain phosphorylation, loss of claudin-5/actin co-localization, claudin-5 reorganization and reduces phase 1 leak. However, unlike H-1152 and Y-27632, combined Rho-associated kinase-1/2 siRNA knockdown does not reduce the magnitude of phase 2 leak, suggesting that H-1152 and Y-27632 have targets beyond Rho-associated kinases that regulate endothelial barrier function. We conclude that TNF disrupts TJs in HDMECs in two distinct NF-κB-dependent steps, the first involving Rho-associated kinase and the second likely to involve an as yet unidentified but structurally related protein kinase(s).Capillary leak in severe sepsis involves disruption of endothelial cell tight junctions. We modeled this process by TNF treatment of cultured human dermal microvascular endothelial cell (HDMEC) monolayers, which unlike human umbilical vein endothelial cells form claudin-5-dependent tight junctions and a high-resistance permeability barrier. Continuous monitoring with electrical cell-substrate impedance sensing revealed that TNF disrupts tight junction-dependent HDMEC barriers in discrete steps: an ~5% increase in transendothelial electrical resistance over 40 minutes; a decrease to ~10% below basal levels over 2 hours (phase 1 leak); an interphase plateau of 1 hour; and a major fall in transendothelial electrical resistance to < 70% of basal levels by 8–10 hours (phase 2 leak), with EC During acute inflammation, an increase in endothelial permeability (leak) above basal levels permits an exudate of large plasma proteins  to form a provisional matrix in tissues upon which extravasating inflammatory leukocytes can migrate. This inducible (hyper)permeability is normally confined to post-capillary venule segments of the microcirculation ,2 but inin situ  × 100. Co-localization of anti-CL5 with actin staining was calculated using the Volocity 6.1.1 software co-localization tool with automatic thresholding based on Costes et al. sulfonyl]-2-propenenitrile  or by transduction with the murine S32/36A mutant SR-IκBα that had been inserted into the pLZRS retroviral vector downstream of an CMV promoter; the corresponding negative control was cells transduced by the same vector but without any insert . RetroviHDMECs plated on gelatin-coated (Sigma) 6-well plates at 50% confluence were spin-transfected with Oligofectamine (Invitrogen) complexed to siRNA sequences that specifically targeted TNFR1, ROCK-1, ROCK-2, both ROCK-1 and -2 or MYPT1. First, siRNA complexes of Oligofectamine (Invitrogen) at 50 μg/ml and siRNA at 100 nM were prepared in Opti-MEM I Reduced Serum Medium (Invitrogen) and then diluted five-fold in Opti-MEM to yield a final siRNA concentration of 20 nM. The siRNA-Oligofectamine were then added to HDMECs cultures and centrifuged at 1200 RCF for 5 minutes, followed by incubation for 2 hours at 37°C. Fresh medium  was then added overnight and cells were re-transfected 24 hours later. After resting for 24 hours, HDMEC were then trypsinized and seeded into fibronectin-coated ECIS 8-chamber arrays  at 100,000 cells per well. Cell viability was assessed by re-plating and knockdown specificity assessed in comparison to a non-targeting siRNA control. siRNA sequences used were, as non-targeting siRNA control: 5'-UGGUUUACAUGUCGACUAA-3' (Dharmacon # D-001910-01-05); for targeting TNFR1: 5’-GUACAAGUAGGUUCCUUUGUU-3’ and 5'-UGGUUUACAUGUCGACUAA-3' (Dharmacon #D-005197-01 and #D-005197-02); for targeting MYPT: 5'-CAUCAGCUGGUGAUCGAUAtt-3’ and 5’-GCAGUACCUCAAAUCGUUUtt-3’ (Life Technologies # s9235 and # s9237); for targeting ROCK-1: 5’-CGGUUAGAACAAGAGGUAAtt-3’ and 5’-GGUUAGAACAAGAGGUAAAtt-3’ (Life Technologies # s12098 and s12097); and for ROCK-2: 5’-GGAGAUUACCUUACGGAAAtt-3’ and 5’-GAGAUUACCUUACGGAAAAtt-3’ (Life Technologies # s18161 and s18162).For immunoblot analyses, HDMECs cultured in C12 plastic wells (BD Biosciences) were washed twice in ice cold PBS, scrape-harvested into Laemmli sample buffer  subjected to freeze-thaw and boiled for 5 minutes. Lysates were fractionated on SDS-PAGE gels, transferred at 4°C onto PVDF filters (Millipore), incubated in Blocking buffer , and bound with primary antibody overnight by rocking at 4°C. Blocking buffer containing 5% BSA in place of milk was used for blots involving anti-phospho-MLC. After incubation for 1 hour at room temperature with species-specific horse radish peroxidase-conjugated secondary antibody (Jackson ImmunoResearch Laboratories) detection of bound antibodies was performed with SuperSignal West Pico or Femto chemiluminescent substrates (Thermo Fisher Scientific). Immunoblot images display bands of interest and controls from the same contiguous immunoblots with background levels minimized by adjusting all pixels equally with ImageJ 1.48 software. Molecular weights in kDa calculated from protein size markers are included in each figure.50 values for TNF were analyzed by non-linear regression (curve fit) using a least squares method. Data are expressed as a mean value ± SEM. P values of p <0.05 were considered significant.Data were analyzed with Prism 6.0e software. Significance of differences among groups was tested by one-way analysis of variance (ANOVA) followed by the Bonferroni post-test or by two-tailed t-tests that were paired or unpaired as fitting the experimental design. EC50 TNF concentration for phase 2 leak (150±41 pg/ml) was five-fold higher than phase 1 leak  respectively and did not cause cell death in two different experiments (data not shown).TNF, at a concentration of 20 ng/ml, caused an immediate but small rise in TEER that varied inversely relative to basal TEER levels as assessed by electrical cell-substrate impedance sensing ECIS; . The mectrations . Howevertrations , a targetrations , and req50 for phase 2 leak computed from ECIS measurements -induced leak in HDMECs is complex, involving two distinct phases with different EC50 values for these cytokines. Second, both phases are dependent upon activation of NF-κB. Third, the initial fall in barrier function (phase 1 leak) correlates temporally with disruption of CL5 junctional continuity and with onset of MLC phosphorylation, which also are NF-κB-dependent and as shown by siRNA knockdown, are dependent on activation of ROCK-1 and -2. Interestingly, the TNF-induced, ROCK-mediated phosphorylation of MLC appears largely independent of MYPT1. Fourth, the second phase of TNF-induced leak, while blocked by selective inhibitors of ROCK, are largely unaffected by siRNA combined knockdown of ROCK-1 and -2, implying the involvement of other structurally related but distinct kinase(s).Enhanced endothelial leak through post-capillary venules is a crucial component of inflammation that involves disruption of VE-cadherin organized AJs. There have been many studies analyzing this process as a response to inflammatory mediators, such as TNF or IL-β, de novo gene expression by activating the master transcriptional regulator NF-κB. These pathways have been studied intensively but less so with regard to the effect of TNF on EC barrier function. TNF invariably triggers two distinct phases of leak. Both phases of TNF-induced leak in HDMEC cannot be attributed to apoptosis or necroptosis based on the absence of nuclear condensation or fragmentation, respectively. Further evidence against irreversible injury is that there is a recovery to basal barrier levels at effective TNF doses within 18 hours. The NF-κB-dependent programs, which remain to be identified, must be highly regulated in order to produce the statistically uniform kinetics and leak nadirs described. The explanation we propose for how the same cytokine, acting through the same receptor (TNFR1), can initiate two processes with different kinetics and concentration dependency is that the more rapid process involves transcriptional events dependent solely upon preformed transcription factors (as seen in early induction of E-selectin) whereas the slower process requires de novo synthesis of additional transcription factors (as seen in the later induction of VCAM-1) [50 values of TNF or IL-β required for phase 1 vs. phase 2 leak may be explained by different signal strengths being required for the activation of different transcription factors [ECs are perhaps the major systemic target of TNF, which triggers  VCAM-1) ,29. Simi factors .Our results differ from a previous report describing immediate rapid changes in the HDMEC barrier by TNF and IL-β involving the small G protein Arf6 that are independent of NF-κB-activation . HoweverWe noted that CL5 was displaced but not degraded at TJs disrupted by TNF. In contrast to our findings in HDMECs, others have described rapid CL5 degradation in bovine retinal EC cultures treated with TNF  a findinOur data show ROCK is activated downstream of NF-κB activation and that both responses contribute to phase 1 TNF leak. In support of these conclusions, genetic and pharmacologic methods of inhibiting TNF-induced activation of NF-κB or ROCK-1 and -2 each suffice to inhibit MLC phosphorylation, loss of cortical actin filaments, TJ disruption and leak, and at concentrations effective at inhibiting TNF-induced MLC phosphorylation, H-1152 and Y-27632 did not decrease NF-κB-dependent induction of ICAM-1. TNF-induced ROCK-dependent MLC phosphorylation appears to be independent of myosin phosphatase, because siRNA silencing of MYPT1 expression did not inhibit leak or TNF-induced MLC phosphorylation. Corroborating the latter point, TNF-induced ROCK activity (as measured by increased MLC phosphorylation) is still inhibited by Y-27632 in MYPT1 KD HDMEC. ROCK may instead directly phosphorylate MLC via the alternative pathway described by Amano et al . In humasiRNA knockdown of both ROCK isoforms showed that the contributions of ROCK-1 and -2 activity are additive and that phase 1 but not phase 2 leak is ROCK-dependent. Although the TEER decrease until the nadir of phase 1 (circa 2 h) was largely inhibited by combined siRNA knockdown of ROCK-1 and ROCK-2 expression, the subsequent TEER decrease from the interphase plateau until the phase 2 nadir (through 8 hours TNF) was not. In contrast, two different pharmacologic reagents widely regarded and commercially distributed as ROCK specific inhibitors effectively inhibited both nadirs of leak. That knockdown of ROCK-1 and -2 suffices to block loss of CL5/actin co-localization suggests that actin changes deriving from MLC phosphorylation are mostly relevant to phase 1 and not phase 2 TNF leak. We considered if the distinct effects of Y-27632 and H-1152 on phase 2 leak could derive from the very small amount of residual ROCK expression not silenced by siRNA. This seems unlikely because no inhibition of phase 2 leak was detected following highly effective knockdown of ROCK-1 and -2 expression. We therefore conclude that the inhibition of phase 2 leak by the serine/threonine kinase inhibitors H-1152 and Y-27632 likely derives from inhibition of different TNF-activated ser/thr kinases. The concentrations used here are comparable to their previous use on EC as ROCK-specific inhibitors in publications involving Y-27632 ,36,42–44In summary, we have investigated the effect of TNF on TJ-dependent human microvascular EC barriers that are organized around CL5. We report for the first time that such a process takes place in discrete steps, and by the failure of ROCK siRNA to reduce phase 2, that prevention of capillary leak may require targeting more than one pathway . The con


Geranium wilfordii is one of the major species used as Herba Geranii (lao-guan-cao) in China, it is commonly used solely or in polyherbal formulations for treatment of joint pain resulted from rheumatoid arthritis (RA) and gout. This herb is used to validate a target-based drug screening platform called Herbochip® and evaluate anti-inflammatory effects of Geranium wilfordii ethanolic extract (GWE) using tumor necrosis factor-alpha (TNF-α) as a drug target together with subsequent in vitro and in vivo assays.in vitro model.A microarray-based drug screening platform was constructed by arraying HPLC fractions of herbal extracts onto a surface-activated polystyrene slide (Herbochip®). Using TNF-α as a molecular probe, fractions of 82 selected herbal extracts, including GWE, were then screened to identify plant extracts containing TNF-α-binding agents. Cytotoxicity of GWE and modulatory effects of GWE on TNF-α expression were evaluated by cell-based assays using TNF-α sensitive murine fibrosarcoma L929 cells as an in vivo anti-inflammatory effects of GWE were further assessed by animal models including carrageenan-induced hind paw edema in rats and xylene-induced ear edema in mice, in comparison with aspirin. The hybridization data obtained by Herbochip® analysis showed unambiguous signals which confirmed TNF-α binding activity in 46 herbal extracts including GWE. In L929 cells GWE showed significant inhibitory effect on TNF-α expression with negligible cytotoxicity. GWE also significantly inhibited formation of carrageenan-induced hind paw edema and xylene-induced ear edema in animal models, indicating that it indeed possessed anti-inflammatory activity.The We have thus validated effectiveness of the Herbochip® drug screening platform using TNF-α as a molecular target. Subsequent experiments on GWE lead us to conclude that the anti-RA activity of GWE can be attributed to inhibitory effect of GWE on the key inflammatory factor, TNF-α. Our results contribute towards validation of the traditional use of GWE in the treatment of RA and other inflammatory joint disorders. Geranium wilfordii Maxim (GW), Erodium stephanianum Willd., and other congenic plants including G. Carolinianum in the Geraniaceae family are used as Chinese medicinal plant lao-guan-cao or Herba geranii/Herba erodii. According to Pharmacopoeia of People’s Republic of China 2010 [etc., ailments that related to inflammation, swelling, pain, and other discomforts in muscles, bones, and joints. GW can be used in polyherbal formulation such as in Yunnan Baiyao or alone such as in the lao-guan-cao paste/cream (Unguentum Geranii) for topical therapy of the aforementioned ailments, particularly RA [The dried aerial parts of larly RA . Unfortui.e., interactions between genetic and environmental factors are involved [RA is a progressive joint damaging and disabling disease characterized by pain, swelling, and stiffness of the synovial joints. This autoimmune disease is resulted from dysregulations of both innate and adaptative immunity, involved -6. Cumulinvolved . Among tinvolved ,8. TNF-αinvolved . The sucinvolved . Before involved ,11.Plasmodium falciparum as the drug target of artemisinins (qinghaosu) [Artemisia annua L. (qinghao) [et al. published a microarray-based drug screening technology (Herbochip®) which allowed functional characterization of herbal compounds fractionated by HPLC using a defined protein drug target in a reverse screening fashion. The prototype of this technology was reported to be used for finding anti-TNF-α active herbal fractions as mentioned in a patent application [Sophora flavescens (SFE-herbochip) were screened using cytochrome P450 3A4 (CYP450 3A4) as a molecular target, hoping to obtain anti-CYP450 3A4 active components that might be useful in the design of regimens for the treatment of human immunodeficiency virus (HIV) infection [in vitro and in vivo assays. Our results present a platform that can be used for drug screening with known drug targets and contribute towards validation of the traditional use of GWE in the treatment of RA and other inflammatory joint disorders.A decade ago, a WHO  report snghaosu)  from theqinghao) ,17. It infection . Herein,L929  cells line was purchased from the China Center for Type Culture Collection . Aspirin was from Shandong Xinhua Pharmaceutical Co. Ltd., Shangdong, China. Actinomycin D (ActD) was from Beyotime Institute of Biotechnology, Jiangsu, China. Sodium hydrogen carbonate, dimethyl sulfoxide, Tween 20, sodium chloride, and xylene were from Sinopharm Chemical Reagent Co. Ltd., Shanghai, China. Other reagents and chemicals were from Sigma-Aldrich, USA.Geranium wilfordii Maxim were obtained from Yannan Baiyao Group Tianzihong Pharmaceutical Co. Ltd.  and identified by Yunnan Institute of Materia Medica  according to The Pharmacopoeia Commission of PR China 2010 [G. wilfordii as well as all plant materials screened were stored in our laboratory (Table G. wilfordii (GWE) was needed in subsequent experiments, the starting material was increased to 3 kg for preparation of GWE according to the same protocol. The positive control, aspirin (95% pure), was obtained from the Shandong Xinhua Pharmaceutical Co., Ltd. .For primary screenings, most of the plant materials of 82 selected Chinese herbs were either self-collected or purchased from market Table . SpecifiGeranium wilfordii (GWE), were dissolved in 50% ethanol to a concentration of 50 mg/mL. The sample was then resolved into 96 fractions by a HPLC system equipped with a C18 column, an Agilent1100 instrument, a quaternary pump, a manual injector, a UV detector and a workstation for peak identification and integration . The column used was a Hypersil ODS C-18 analytical column  and the detection wavelength was set at 254 nm. For each fractionation, 50 μL sample was loaded and the elution was carried out by a water to EtOH gradient at a constant flow rate of 0.75 mL/min at 30°C for 96 min as follows: 0-50% EtOH (90 min), 50-100% EtOH (6 min). One-minute fractions were collected and then 150 μL each fraction was transferred into a 96-well microplate . Thus each plate represented a complete HPLC run of the fractions/samples load. The plates were vacuum-dried by a Speed Vac  and stored at 4°C until use.Fractionation of the plant extract by HPLC: The plant extracts obtained above, including that made from 2O), NH4OH , and 1,4-butanediol diglycidyl ether . After treatments, the slides were washed with 0.1 M NaHCO3 (pH 8.0), dried, sealed and stored at 4°C until use (blank chips). Prior to spotting, 30 μL OptiFix I  was added to each well of the 96-well plates. The samples were then spotted onto the blank chips using an automatic arrayer  according to the format shown in Figure Fabrication of herbochips - arraying the HPLC fractions on plastic slides: The blank chips were manufactured by mode injection followed by surface-activation . Briefly3, pH 8.0), and 50 μL the BNHS solution (2 mg/mL biotinamidohexanoic acid N-hydroxysuccinimide ester in DMSO) was added. The biotinylation reaction was carried out for 1 h on ice and then stopped by adding equal volume of glycine (50 mM in coupling buffer). The mixture was then dialyzed against coupling buffer to remove unreacted BNHS and excess glycine. For hybridization, each cave on the chips was covered by a cover glass (22 × 22 mm) under which 20 μL biotinylated-TNF-α in TBST  or TBST alone was added. The slides were then incubated at 37°C for 1 h. While placed in a slide washing jar, the slides were washed in TBST (4 × 2 min), rinsed in water (4 × 2 min) on a horizontal shaker at 80 rpm at room temperature, and then dried at 37°C (30 min). The same hybridization procedure was used in the subsequent reaction with Cy5-labeled streptavidin (SA-Cy5) where 20 μL 2.5 μg/mL SA-Cy5 in TBST was used. The dried slides were scanned by a laser scanner  and the fluorescent intensity of red spots in the image was analyzed at emission wavelength of 635 nm by GenePix 5.0 Software. The fluorescence value (FV) of each hybridization reaction was then recorded.Screening TNF-α binding activities in the plant extracts by using the Herbochip® platform: Purified TNF-α was purchased from PrimeGene Technical . For preparation of biotinylated TNF-α, 1 mg protein was dissolved in 1 mL coupling buffer  for an additional 24, 48, and 72 h. Cell viability was determined by incubation with 3--2,5-diphenyl tetrazolium bromide (MTT) (0.5 mg/mL) for 1 h, solubilization in dimethyl sulfoxide (DMSO), and spectrophotometric measurement at 550 nm [ActD+test drugs/O.D.ActD. Effect of the GWE on TNF-α activity was evaluated using L929 cell bioassay in the presence of 1 μg/mL Actinomycin D (ActD) as described by Kiemer et al. [4 cells/well in 100 μL of culture media. Following incubation for 24 h, the medium in the wells was replaced with fresh medium containing ActD (2 μg/mL). After 1 h of preincubation with ActD, serial dilutions of test drugs and a final concentration of 0.2 ng/mL TNF-α was added. The plates were then incubated for an additional 24 h at 37°C and then MTT test was performed as described above. The TNF-α inhibitory effects of the test drugs were expressed as % inhibition = (O.D.ActD+TNF-α+test drugs – O.D.ActD+TNF-α)/(O.D.ActD – O.D.ActD+TNF-α).The TNF-α sensitive murine fibrosarcoma L929 cells were used for determination of cytotoxicity and inhibitory effect on TNF-α of the samples. L929 cells were cultured in RPMI-1640 medium  supplemented with 10% fetal bovine serum (FBS), 100 μg/mL streptomycin, 100 U/mL penicillin, and 0.03% L-glutamine, and maintained at 37°C in humidified air containing 5% COt 550 nm . The cytr et al. . In briead libitum.Anti-inflammatory activities of GWE were tested using rat carrageenan-induced hind paw edema and mouse xylene-induced ear edema experimental models. The animal experiments were carried out at Yunnan Institute of Materia Medica (YIMM) and approved by the Animal Experimental Ethics Committee of YIMM  2005–0008). Based on the recommended dose of the raw drug for human  [Carrageenan-induced rat hind paw edema model: Male Sprague–Dawley (SD) rats, weighting 160–210 g, were used. For each experiment, 30 SD rats were randomized into 3 groups, untreated control (water), treatments with GWE (1.69 g/kg/d) and reference drug control aspirin (0.1 mg/kg/d). Test drugs were administrated intragastrically each day for 5 consecutive days. One hour after the last drug dosing, each rat was injected with 0.1 mL freshly prepared suspension of 1% carrageenan in physiological saline into subplantar tissue of the right hind paw. Paw edema was then measured at 1, 2, 4, and 6 h after induction of inflammation.Xylene-induced mouse ear edema model: Male ICR mice weighting 18–20 g were used. For each experiment, 30 ICR mice were randomized into 3 groups, untreated control (water), treatments with GWE (1.69 g/kg/d) and reference drug control aspirin (0.1 mg/kg/d). Test drugs were administrated intragastrically each day for 5 consecutive days. One hour after the last drug dosing, 50 μL xylene was applied to both anterior and posterior surfaces of the right ear for another hour. Subsequently, discs of 8 mm diameter were removed from each ear and weighed in a balance. The swelling was estimated as the difference in weight between the punches from right and left ears.In vivo data are represented as Mean ± SE . The In vitro assay are comparison between groups was made by One-way ANOVA, The differences with p < 0.05 or p < 0.01 were considered significant or highly significant, respectively.The Herein, a refined Herbochip® screening platform was constructed and reported. Treatment of the polystyrene plastic slides with glutaraldehyde led to formation of free aldehyde groups on the surface which subsequently reacted with 1,4-butanediol diglycidyl ether. The resulted terminally located epoxyl groups would then be able to react with carboxyl-, hydroxyl-, amino-, and sulfhydryl-groups to form covalent bonds. Thus most biological molecules including nucleic acids, proteins, and small molecules containing aforementioned functional groups would be conjugated onto the surface-activated slides (blank chips) when presented. The formatting of the HPLC fractions onto the chip was presented in Figure Pueraria thromsonii extract)-, AKE (Amomum kravanh extract)-, SCE (Syzygium caryophyllaceum extract)- and GBE (Garcinia bancans extract)-herbochips, were chosen to represent different binding signal strengths from negative binding (−) to strong binding (+++). Likewise, TNF-α binding activity of GWE-herbochip was shown in Figure A total of 82 selected herbs were made into herbochips and 46 herbs were found to possess TNF-α binding activities Table . As showThe cytotoxic effects of GWE on L929 cells were assayed in cultures exposed to GWE at indicated concentrations for 24, 48, and 72 h. As shown in Figure In the carrageenan-induced hind paw edema assay, GWE inhibited swelling significantly 1 h post carrageenan injection Table . In the G. wilfordii extract (GWE), was further explored. The Herbochip® technology reported here was basically a reverse screening method. Unlike most high-throughput-screening (HTS) formats, in which large chemical libraries were screened against biological targets via the use of automation, miniaturized assays and large-scale data analysis [We have reported a microarray-based drug screening platform referred to as the Herbochip® for target-based discovery of natural compounds for functional characterization and therapeutic uses. The application of this platform was validated using TNF-α as the target for screening and one of the results, namely the anti-inflammatory fractions of the analysis ,22, the analysis . In thisetc., indicating that this novel HTS platform could be applied to a wide range of known drug targets. It is well known that the disease target of most, if not all, of herbal medicines remain unknown, the utilization of this target-based drug screening platform on developments of herbal medicine is definitely worth further explored.During the course of this study, thousands of different herbochips including the GWE-herbochip reported in details here, were fabricated. The protein drug target TNF-α was employed to screen 82 herbochips and the hit rate was as high as 56% (46/82) when herbs reported to have anti-inflammatory activity were counted. As mentioned, the platform was initially used to screen for herbal species that inhibit CYP450 3A4, an important enzyme for the metabolism of a broad range of therapeutic agents . In addiTNF-α plays a central role in most inflammatory responses and it is a validated target for drugs against a number of chronic inflammatory disorders resulting from immune dysregulations such as Crohn’s disease, ulcerative colitis, ankylosing spondylitis, rheumatoid arthritis (RA), ,24,25. UWe have thus validated effectiveness of the Herbochip® drug screening platform using TNF-α as a molecular target. Subsequent experiments on GWE lead us to conclude that the anti-RA activity of GWE can be attributed to inhibitory effect of GWE on the key inflammatory factor, TNF-α. Our results contribute towards validation of the traditional use of GWE in the treatment of RA and other inflammatory joint disorders.


His scientific career started even before his graduation with a Master’s Degree in Pharmacy at the Institute of Pharmacognosy at the University of Vienna. From the beginning, he dedicated his scientific efforts to medicinal plants. In 1965, he finished his Ph.D. thesis dealing with cardiac glycosides of Convallaria majalis.Wolfgang Kubelka was born on February 18He had the chance to gain more experience in the field of natural products chemistry during his research stays in the laboratories of outstanding experts like the professors Reichstein (Basel), Tschesche (Bonn), Beal, and Doskotch (Ohio State University) during 1965–1971.Convallaria accompanied the scientific work of Prof. Kubelka through all these years, resulting in the Habilitation for Pharmacognosy in 1973. He is author of more than 100 publications dealing, for example, with the isolation and characterization of plant constituents, development of analytical methods suitable for the proof of quality of herbal substances and herbal preparations, biosynthesis of plant constituents, chemotaxonomy, and ethnopharmacology. He always took his responsibility for the education of students of pharmacy very seriously and introduced, for example, pharmacobotanical field excursions into the curriculum.From 1983 to 2003, Prof. Kubelka headed the Institute of Pharmacognosy at the University of Vienna. His dedication to the development of sciences at the University resulted in his election as dean for the faculty of natural sciences for several years.Beside this scientific career, Prof. Kubelka always tried to link the sciences with practical implementation. His initiative was essential for the foundation of the Austrian Pharmaceutical Society (ÖPhG 1979) and the Austrian Society for Phytotherapy (ÖGPhyt 1991). Although being emeritus professor since 2003, he is still active in the training of pharmacists and physicians, in organizing congresses, and among other topics, in being chief editor of several journals. His expertise is also still acknowledged in the Quality Drafting Group of the Committee on Herbal Medicinal Products at the European Medicines Agency.th birthday with the best wishes for his future.Since 1985, Prof. Kubelka has been the editor-in-chief of Scientia Pharmaceutica. During these years, he guided this journal from the printed version to the online edition with free access to full texts. The editorial board of Scientia Pharmaceutica congratulates Prof. Kubelka on the occasion of his 80Ad multos annos! The Editors


The effect of the carbimazole on expression of tumor necrosis factor (TNF-α) in liver, was investigated in an experimental model of high fat diet (HFD) induced obesity. The HFD  induced TNF-α in liver tissue along with raised serum triglyceride (TG), cholesterol and high TSH (62%). In carbimazole (1 mg/100 gbw) treatment, the induction of TNF-α was significantly inhibited, without affecting other parameters. It also improved the liver function, which was raised due to HFD in experimental control rats. The inflammation is the primary cause of metabolic syndrome [MS] . It is aThe rats were divided into group-1 . Group −2 [received high fat diet (HFD)  1 mg/100 gbw)]. Above treatments were continued for 4 months and finally rats were sacrificed to assess lipid profile (serum triglyceride (TG) and cholesterol) and liver function test namely aminotransferase (AST), Alanine transaminase (ALT), and alkaline phosphatase (ALKP) and thyroid stimulating hormone (TSH). The expression of TNF-alpha was determined in Liver tissue by RT-PCR [0 gbw]. AEthical clearance: The protocol was approved by animal ethics committee of our Institution .Lipid profile, liver function and TSH were significantly raised in animals fed with HFD Table . There wIt is well documented that faulty diet and life style mediated physiological changes induces systemic low grade inflammation [LGI]. Our results indicate the raised serum TSH and TNF-α in high fat diet fed rats and lower in carbimazole treated rats . This could be due to inhibitory action of carbimazole on Rac1, involved in expression of TNF-α . Since, The inhibition of TNF –α expression in carbimazole treated group could be its direct anti-inflammatory effect, but it can also be through high TSH, which needs further exploration.


Peer evaluation is the cornerstone of science evaluation. In this paper, we analyze whether or not a form of peer evaluation, the pre-publication selection of the best papers in Computer Science (CS) conferences, is better than random, when considering future citations received by the papers.Considering 12 conferences , we collected the citation counts from Scopus for both the best papers and the non-best papers. For a different set of 17 conferences, we collected the data from Google Scholar. For each data set, we computed the proportion of cases whereby the best paper has more citations. We also compare this proportion for years before 2010 and after to evaluate if there is a propaganda effect. Finally, we count the proportion of best papers that are in the top 10% and 20% most cited for each conference instance.The probability that a best paper will receive more citations than a non best paper is 0.72  for the Scopus data, and 0.78  for the Scholar data. There are no significant changes in the probabilities for different years. Also, 51% of the best papers are among the top 10% most cited papers in each conference/year, and 64% of them are among the top 20% most cited.There is strong evidence that the selection of best papers in Computer Science conferences is better than a random selection, and that a significant number of the best papers are among the top cited papers in the conference. In these examples of peer evaluation of noteworthy or “best” papers, the selection happens after the paper’s publication, and thus the peers may have some information about how the paper has been accepted by readers.Peer evaluation is the cornerstone of scientific evaluation focusing on two main aspects: the evaluation of scientists and the evaluation of their scientific work as reported in papers. Evaluation of scientists by peers is a central aspect of hiring, promotion, granting awards and distinctions, and it is an important aspect of most research grant evaluation. The peer evaluation, or peer review, of papers and manuscripts, in turn, is a much more frequent task—tens to hundreds of thousands manuscripts are evaluated by peers each year as the main part of the scientific publication process. A different form of peer evaluation of papers is the selection of particularly noteworthy papers. Some journals (for example in Civil Engineering and in Finance/Economics) award a yearly best paper selected from the papers published in the journal in the previous year. The Faculty of 1000 (F1000) , or an Economics or Sociology paper may change government policies. In these cases, the practical importance of the paper may not be reflected in the number of citations received. In Computer Science, we are not aware of research that links publication of papers to changes in the practice of programmers or system designers, hence we will constrain our analysis to the “scientific importance” of a paper, and measure it by the number of citations received over time. We will evaluate how many more citations a “best” paper receives in comparison to the other papers of the same conference and year. In particular, we are interested in answering the following questions:There has been a large volume of publications that discuss peer evaluation of scientists, the correlation or non-correlation of their evaluation and standard bibliometric measures –5. Furtha posteriori correct measure of the importance of a paper, and use it to evaluate how correct the peer decision is, which was based on limited information of the impact of the paper on other readers. In this paper, we follow the latter approach.Regarding one form of paper evaluation, the selection of noteworthy published papers, one must first note that there are two approaches to research in this area. One may consider that peer evaluation is the gold standard, and use it to evaluate how “correct” a standard measure such as citation count is. Or one may consider that citation count is the Sen and Patel  study tha best paper has a significantly higher number of citations than the median paper in the same volume;in 25% of the cases, the best paper was the highest cited paper in the volume;in a small majority of cases, the best paper has a higher citation count than the runner up.Coupé  analyzesThe paper also studies the influence of other factors on receiving the award, such as length of the paper, number and gender of authors, and so on.Faculty of 1000 is a service that recommends papers in life sciences based on the evaluation of 5,000 experts. Each expert may recommend any published paper, and classify it as “Good”, “Very Good” or “Exceptional” and must further classify the paper into groups, such as “changes clinical practice” or “good for teaching” and so on. A paper can receive recommendations from more than one expert, which adds to its ranking.Wardle  comparesr2 = 0.20), which indicates that further positive evaluations by F1000 does not result in a higher number of citations. Notice that this research does not compare non-selected papers with selected papers by F1000; it only compares the correlation of “better ranking” to more citations.Bornmann and Leydesdorff  correlatWaltman and Costas  analyze Michayluk and Zurbruegg  study thhttp://arnetminer.org/conferencebestpapers. They rank the papers according to citation counts and show the top three per conference instance as well as the rank and citation count of the best papers.To our knowledge, there is no other published work that compares the impact of best papers to non-best papers at computer science conferences. The only work we found that shows the citation counts of best papers in computer science conferences and ranks their citation counts among other papers at that conference instance is We first became aware of that page in June 2014. We did our data collection and analysis independently and without knowledge of that webpage. It is worth noting, however, that our analysis is significantly different and one would likely draw different conclusions from that page vs. our statistical analysis. For example, from that page, one might draw the conclusion that best papers are generally not cited as well as non-best papers. See, for example, for AAAI 2012, the two best papers are ranked 59 & 174 in number of citations. However, our statistical analysis concludes that there is a high probability that the best paper will have more citations than a random paper in the same conference instance.Computer Science (CS) research practices place much more value on scientific communication through conference and meetings than other scientific areas –19. TherIn fact there is a perceived correlation between the prestige of a conference and lower acceptance rates, although one empirical evaluation shows that the correlation is not real . Freyne In 2008, Web of Science (WOS) added some selected conferences to its indexed sources. Bar-Ilan  showed thttp://jeffhuang.com/best_paper_awards.html with the list of best papers for the CS conferences below. The list below contains the conference name and its abbreviation. We got our list of which papers were the best from that site.Jeff Huang at Brown University maintains a website at AAAI Conference on Artificial Intelligence (AAAI)Annual Meeting of the Association for Computational Linguistics (ACL)ACM SIGCHI Conference on Human Factors in Computing Systems (CHI)ACM International Conference on Information and Knowledge Management (CIKM)IEEE Conference on Computer Vision and Pattern Recognition (CVPR)Annual Symposium on Foundations of Computer Science (FOCS)ACM Foundations of Software Engineering Conference (FSE)International Conference on Computer Vision (ICCV)International Conference on Machine Learning (ICML)International Conference on Software Engineering (ICSE)International Joint Conference on Artificial Intelligence (IJCAI)International Conference on Computer Communications (INFOCOM)ACM SIGKDD Conference on Knowledge Discovery and Data Mining (KDD)ACM International Conference on Mobile Computing and Networking (MOBICOM)USENIX Symposium on Networked System Design and Implementation (NSDI)USENIX Symposium on Operating Systems Design and Implementation (OSDI)ACM Programming Language Design and Implementation (PLDI)ACM Symposium on Principles of Database Systems (PODS)IEEE Symposium on Security and Privacy (S&P)ACM Annual Conference of the Special Interest Group on Data Communication (SIGCOMM)ACM Annual Conference of the Special Interest Group on Information Retrieval (SIGIR)ACM Annual Conference of the Special Interest Group for the Computer Systems Performance Evaluation (SIGMETRICS)ACM Annual Conference of the Special Interest Group on Management of Data (SIGMOD)ACM-SIAM Symposium on Discrete Algorithms (SODA)ACM Symposium on Operating Systems Principles (SOSP)ACM Symposium on the Theory of Computing (STOC)ACM Symposium on User Interface Software and Technology (UIST)International Conference on Very large Data Bases (VLDB)International Conference on the World Wide Web (WWW)The list includes very prestigious conferences in different areas of Computer Science. AAAI and IJCAI are in the general area of Artificial Intelligence; ACL is in Natural Language Processing; CHI and UIST are in User Interfaces; CIKM and SIGIR are in Information Retrieval; CVPR and ICCV are in Computer Vision; FOCS, STOC, and SODA are in Theory; FSE and ICSE are in Software Engineering; ICML and KDD are in Machine Learning; INFOCOM, MOBICOM, NSDI, and SIGCOMM are in Networking; OSDI and SOSP are in Operating Systems; PLDI is in Programming Languages; PODS, SIGMOD, and VLDB are in Databases; S&P is in Computer Security; SIGMETRICS is on System Performance, and WWW is in the World Wide Web.conference instance.In this paper, we will call AAAI, for example, a conference, and the AAAI that happened in 2002, which selected one best paper, a Scopus explicitly indexes the following conferences from the list above: AAAI, CIKM, FSE, ICCV, ICSE, INFOCOM, KDD, MOBICOM, PODS, SIGMOD, SODA, and STOC. We collected the Scopus citation counts received for each of the best and non-best papers in these conferences for the years 1998 to 2012 inclusive. The Scopus data were collected in March 2013.The Scopus data refer to 19,421 papers, of which 168 are best papers. For the remaining 17 conferences in the list above, we collected the data using DBLP and Google Scholar. DBLP is a website that lists all papers and authors of a large set of computer science conferences and journals . From DBLP, we collected the title of all the papers published in the remaining conference instances from 1996 to 2012. We then selected a random sample of papers listed and queried Google Scholar using the paper’s title. We made sure that we retrieved the citation counts of at least eight papers besides the best paper for each conference instance. The Google Scholar collection was performed in January 2013.Scholar data refer to 1,577 papers, 170 of which are best papers. b, nb) such that b is a best paper, nb is a non-best paper, and the number of citations of b is larger than nb, divided by the total number of pairs . Formally, the probability Pc for a conference instance c is:cit(x) is the number of citations received by a paper x, b is a best paper of conference instance c, and nb is a non-best paper in c. If we rank all papers in a conference instance c from lowest to highest citation (the higher cited has higher rank), and assign rank = 0 to the lowest cited paper, then the formula for Pc simplifies to:Nbc is the number of best papers in c and Nnbc is the number of non-best papers in c. A similar procedure to estimate probabilities was used in 11].


HIV virions assemble on the plasma membrane and bud out of infected cells using interactions with endosomal sorting complexes required for transport (ESCRTs). HIV protease activation is essential for maturation and infectivity of progeny virions, however, the precise timing of protease activation and its relationship to budding has not been well defined. We show that compromised interactions with ESCRTs result in delayed budding of virions from host cells. Specifically, we show that Gag mutants with compromised interactions with ALIX and Tsg101, two early ESCRT factors, have an average budding delay of ~75 minutes and ~10 hours, respectively. Virions with inactive proteases incorporated the full Gag-Pol and had ~60 minutes delay in budding. We demonstrate that during budding delay, activated proteases release critical HIV enzymes back to host cytosol leading to production of non-infectious progeny virions. To explain the molecular mechanism of the observed budding delay, we modulated the Pol size artificially and show that virion release delays are size-dependent and also show size-dependency in requirements for Tsg101 and ALIX. We highlight the sensitivity of HIV to budding “on-time” and suggest that budding delay is a potent mechanism for inhibition of infectious retroviral release. ESCRTs are implicated in cellular processes which require fission of budding membranes. Likely the most studied of these processes is the HIV-ESCRT interactions. The canonical view is that interference with ESCRT recruitment results in a late budding arrest of virions at the plasma membrane and this mechanistic view of ESCRTs has shaped our understanding of their function in almost all cell biology. In this manuscript, we present a full kinetic analysis of HIV virion release under all known mutations in Gag that affect HIV-ESCRT interactions. Our data show that contrary to the canonical view, a defect in ESCRT recruitment does not inhibit virion budding, however it creates a delay. We further show that during budding delay, activated proteases release critical HIV enzymes back to host cytosol, leading to budding of non-infectious progeny virions. We suggest that budding delay is a potent mechanism for inhibition of infectious retroviral release and can be the basis for developing antiviral treatments which slow the budding process and therefore disproportionally affect infectious retroviral release. We also suggest that such budding delay may be one of the mechanisms underlying cellular innate immune responses which inhibit the spread of retroviral infection. The m1 sites) ,13,14. O1 sites) . While t budding , remainsESCRTs are implicated in cellular processes which require fission of budding membranes and are shown to play a major role in multivesicular body formation , envelopMonteCarlo simulations show that the protease activation after complete assembly of HIV virions on plasma membrane can quantifiably explain the loss of Pol specific proteins to host cell cytosol before VLP release.Here we found that late budding arrest of HIV, due to mutations of its late domains, is transient. We have characterized the budding kinetics starting with Gag virus like particles (VLPs). HIV Gag protein is sufficient for assembly of Gag VLPs with the same size as HIV virions . Using GR8.2) and its parental full length HIV-1 R9 vector (HIVR9). HIVR8.2 after budding incorporates all components of the virion except ENV proteins and the genomic RNA.Humanized HIV Gag protein expressed in cells supports production of VLPs with similar size distributions as HIV virions . We initR9 and HIVR8.2 were compared to Gag VLP release 24 hours after transfection. Using Gag domain mutagenesis, we observed that while HIV release and maturation are affected by p6 late domain mutations, VLP production by Gag remains almost unaffected . Shown are the following mutations in Gag p6, HIVR9 and HIVR8.2: ΔPTAP incorporate a 7LIRL10 instead of 7PTAP10 [36SR37 instead of 36YP37 [7LIRL10 plus 36SR37). 24 hours post-transfection, cells and VLPs were collected as described in Materials and Methods and analyzed by immunoblotting using p24, ALIX and Tsg101 specific antibodies.In parallel experiments, budding of HIVf 36YP37 , and ΔPTR9 and HIVR8.2 VLPs was sensitive to late domain mutagenesis; Tsg101 was fully sensitive to ΔPTAP mutation, and ALIX was only slightly affected by ΔYP mutation . ALIX migrates as two separate bands with the upper band likely related to a post-translational modified form. We don’t know yet at this stage the nature of this ALIX modification. ALIX background level corresponds to exosome release . As commonly reported, HIV virion release (here shown HIVR9 and HIVR8.2) was detectably reduced under the ΔPTAP mutation in addition to a clear defect in Gag processing. Also, a slight change in release and maturation profiles was observed in ΔYP mutant HIVR9 and HIVR8.2.We found that incorporation of early ESCRTs in released Gag as well as HIVR9 and HIVR8.2, production of Gag VLPs was only slightly affected by mutations within p6 . Indeed, expression of Gag with alteration in late domains either as humanized, non-humanized co-expressed with Rev or within R9 with an abrogated ribosomal slippage leads to the same results . Aside from interacting with the p6 domain, ALIX also binds to the NC domain and mutations affecting NC have recently been implicated in HIV virion release [Fig 1B), however, none of the amino acid substitutions and/or truncations had a marked effect on Gag VLP release . The observations related to Gag versus HIVR8.2 VLP release were confirmed by pulse/chase 35S-labeling experiments .In contrast to HIV release . We founS3 Fig). Plasma membrane binding requirement was tested by G2A mutation [S1A Fig).The Tsg101/ALIX engagement-independent release of Gag VLPs was tested on different cell types with no major changes in the VLP release except for the apparent cell type specific NC effect  to budding Gag VLPs, we used HA-tagged forms under tight control of their expression levels along with Gag NC and/or p6 mutants . We found that Gag VLPs were released with similar yield even when recruitment of Tsg101 and ALIX were compromised due to p6 and/or NC mutations, however surprisingly these VLPs retained both CHMP4b and VPS4A independently of p6 and NC alterations that are inhibiting the early ESCRTs recruitments. Fluorescently tagged ESCRT-III components have been previously localized within budding wild type Gag VLPs, however not in the presence of p6 mutations [Given that compared to HIVutations . Even ifFig 2B, the expression of VPS4ΔE228Q had a substantial negative effect on all Gag VLP production which confirms a requirement for VPS4 in eventual Gag VLP production.We further tested the requirement for VPS4 engagement in production of VLPs with compromised interactions with early ESCRTs. To this end, we expressed a dominant negative VPS4 (ΔE228Q) during production of HIV Gag VLPs. As shown in Fig 3 is the VLP production comparing WT, ΔPTAP, ΔYP, Δp6 and control ΔG2A. U2OS cells were used for both immunoblotting and microscopy . Our analysis shows that the kinetics of VLP release is delayed by ~20 minutes (ΔYP) to ~1 hour (ΔPTAP and Δp6) which is consistent with the similar VLP release observed 24 hours post-transfection .To confirm that the Gag variants detected in VLPs using immunoprobing indeed originate from VLPs produced by cells, we visualized the released VLPs by total internal reflection microscopy (TIRF) on individual cells. Using TIRF and Gag p6 variants fused to mCherry, we followed the assembly and release of VLPs in live cells. We confirmed the similar VLP assembly on cellular plasma membrane between all Gag variants, and 12 hours post-transfection, we artificially detached the cells to visualize released VLPs as described in Materials and Methods. VLPs were indeed observed immobilized on the cell-free surface accordingly as shown in R9 or HIVR8.2 express Gag along with Gag-Pol and all other HIV co-factors aside from ENV for HIVR8.2. We chose to generate a system that only express Gag and Gag-Pol proteins for more accurate comparison with Gag to investigate whether the observed differences between Gag and HIV VLPs can be sufficiently explained by the packaging of Gag-Pol. We constructed the Gag plus Gag-Pol open reading frames in a single encoding cassette using humanized Gag and preserving the HIV ribosomal slippage ; the Gag plus Gag-Pol VLPs produced are referred to as “Gag.Pol”. We further generated variants of Gag.Pol by mutating p6 as described for Fig 1 , with either active (PRwt) or inactive protease . Gag.Pol with ΔPTAP and ΔYP mutations resulted in formation of VLPs with defects in terms of VLP yield and maturation . Interestingly, PRΔD25N VLPs showed dramatic release defect in all p6 mutants . Over-expression of ALIX substantially rescued the maturation defect due to ΔPTAP mutation , as commonly reported.Having established that the HIV Gag VLPs with abrogated interactions with Tsg101 and ALIX are delayed in their release, we set out to investigate the discrepancy in budding of HIV versus Gag VLPs. Upon transfection in cells, the HIVPRΔD25N; ). As shoFig 4A and 4B and S5 Fig). The RT loss is reversed in ΔPTAP VLPs by over-expression of ALIX as shown in Fig 4A. Interestingly, we observed that while ΔPTAP mutation induces identical RT loss in both Gag.Pol and HIV VLPs, the RT loss induced by ΔYP mutation in Gag.Pol VLPs is not occurring in HIVR9 and HIVR8.2 . These data suggest the potential engagement of an HIV effector(s) missing in the minimal Gag.Pol system, that is likely capable of supporting ALIX function in the context of ΔYP mutation.Immunoprobing for Gag and Pol domains indicates that ΔPTAP VLPs are devoid of any detectable RT, while an average of 70% RT loss is observed in ΔYP VLPs . Supporting the notion of a race between VLP neck closure and PR activation, we also found that WT Gag.Pol showed a ~25% RT loss when compared to ΔPR Gag.Pol . Based on the yields of Gag.Pol VLP production (comparing both PRwt and PRΔD25N to Gag VLPs), we suspected a longer delay in release of Gag.Pol VLPs with altered p6 compared to Gag VLPs.There is a reduction in the amount of incorporated RT within Gag.Pol p6 mutants when compared to incorporated RT in WT Gag.Pol. We hypothesized that delayed VLP release in addition to activation of PR before closure of the VLP neck would result in Pol auto-processing and subsequent diffusion of Pol products back to the host cytosol. Indeed, PR was also lost equivalently to RT in Gag.Pol p6 mutants, and follows the same profile in HIVFig 5. As expected, Gag.Pol VLPs budded out at a slower rate compared to Gag VLPs, likely due to the Pol cargo size. To this end, all delays related to p6 mutations were extended in time. Unlike Gag VLPs, which were released with a constant delay measured with respect to the cytosolic Gag concentration, the delay in Gag.Pol VLPs did not follow the same curve as the cytosolic fraction. These kinetics indicates the occurrence of parallel processes during Gag.Pol VLPs production.VLP release kinetics of Gag.Pol variants were analyzed as shown in ), we observed that the appearance of mature p24 versus p55 precursor and related products (p48 and p41) were not necessarily synchronized. Indeed, ΔYP mutation shows a delay in release of mature VLPs however their production does not continue to the same extent as for WT, instead, it saturates earlier while budding follows with VLPs enriched with Gag precursors. ΔPTAP mutation releases VLPs with mainly Gag precursors, especially Gag p48, and with a substantial delay. To test the effect of packaging full length Pol we performed kinetics on PRΔD25N  with p6 mutations. VLP production kinetics in Gag.Pol PRΔD25N with p6 mutants were all significantly affected, strongly suggesting the importance of early ESCRT engagement (both Tsg101 and ALIX) when large cargo is loaded. Importantly, in any case, no full abrogation of VLP release was observed under any p6 mutation.Interestingly, in the context of PRwt . The p6 late domain mutation directly dictates the efficiency based on the severity of p6 alterations . These observations were confirmed by pulse/chase 35S-labeling experiments .Our observations in budding kinetics of Gag.Pol VLPs demonstrated that when protease activation is inhibited and VLPs incorporate the full length Gag-Pol protein, the kinetics of VLP release is further delayed and becomes strongly dependent on early ESCRTs. These observations suggest a dependence of VLP release on cargo size. To evaluate the influence of cargo size on VLP production, we artificially fused GFPs in frame and in tandem to Gag C-terminus . We found that indeed VLP release by Gag-GFPFig 7A).There is a predominant impact for PTAP and at lower extent for YP.We further confirmed that intact Gag p6 is required for efficient VLP production with large cargo through rescue of p6 mutant Gag-3x.GFP VLP release by co-transfection of Gag with wt p6 . In both cases, VLP production is negatively affected depending on the length of cargo and nature of p6 alteration.We further modulated the cargo size using Pol truncations in the context of PRΔD25N for maintaining the integrity of Pol cargo. Experiments were performed both under physiological frame shifted expression of Gag.Pol along with Pol proteins expressed in frame with Gag which resulted in 10 fold increase of Pol incorporation in released VLPs. Under both conditions, we observed the same effect of p6 late domain mutations on VLP release . The effect of p6 alteration on VLP release by Gag.Pol full length was similar to our findings above, however, in both cases of “in frame” or “frame shift” expression of Gag-Pol, PR activation appeared to be tightly regulated by Pol C-terminus, likely the IN domain. Indeed, when IN is deleted, PR was activated before VLP budding and this activation accounted for a substantial loss in VLP yield. This premature activation of Gag.Pol PRwt ΔIN (deletion of IN domain) seems likely to occur before Gag.Pol clusters on the plasma membrane as a ΔG2A mutation of the same Gag.Pol constructs showed exactly the same profile of PR premature processing. These findings are in line with the absence of VLP release when Gag-Pol full length is expressed in frame due to drastic delay of Gag-Pol VLP production .In the context of truncated Gag.Pol with wild type protease, VLP production profile is more complex as deletions in Pol also influence timing of PR activation, as shown for Pol truncations  Late domain mutations of HIV Gag result in transient delay of virion release from the plasma membrane. ii) HIV protease is activated following full assembly of virions on the plasma membrane and delays in virion release result in loss of Pol associated enzymes to the cell cytosol and budding of non-infectious virions. iii) Size of cargo attached to the C-terminus of Gag modulates the speed and requirements for early ESCRT factors during HIV budding. While small cargo sizes rely mostly on Tsg101, larger cargo sizes are similarly dependent on both Tsg101 and ALIX for efficient VLP budding.R8.2) .We show that alteration of Gag p6 late domains do not inhibit the release of HIV VLPs but rather result in delayed release. We characterized this effect for both VLPs that package HIV Gag only and for VLPs packaging both Gag and Gag-Pol (Gag.Pol). For the Gag.Pol VLPs, the delay ranges from ~70 minutes for the ΔYP mutants that lose proper interaction with ALIX to more than 10 hours for the ΔPTAP mutants which completely lose Tsg101 recruitment. Since the assembly of VLPs takes approximately 45 minutes, a ~10 fold delay in release of the budding VLP will result in a substantial accumulation of ΔPTAP VLPs at the cell surface when analyzed 12 to 24 hours post-transfection. ΔYP mutation has a much shorter delay of ~70 minutes and therefore would result in lesser fold increase in budding VLPs at the cell surface. Importantly, these accumulation levels of VLPs are consistent with the observed phenotypes of HIV late domain mutagenesis ,15. InteR8.2 pseudotyped with VSV-G have shown that VLPs produced by HIVR8.2 ΔYP have a decreased infectivity of approximately 50% compared to wild type HIVR8.2 while HIVR8.2 ΔPTAP VLPs are non-infectious [Our results rationally explain the infectivity assays previously reported on progeny virions lacking engagement of ESCRTs. Specifically, infectivity experiments using HIVfectious . While tfectious . AnalysiR8.2 VLPs, as shown in S6 Fig. The observed reduction of VLP release due to endocytosis is in agreement with a balance between fast budding and endocytosis of delayed VLPs. Prior to our observations it was shown that HIV Gag with mutated or even deleted p6 releases VLPs from cells [HIV Gag protein alone is capable of budding from the plasma membrane. We found that Gag still efficiently buds out under severe p6 mutations but with delay at the cell surface for periods of ~20 minutes to ~1 hour. There is some minimal endocytosis of VLPs assembled under mutated Gag compared to Gag.Pol and HIVom cells –49. Thesom cells . Here, oIn vitro, direct recruitment of CHMPs onto negatively curved membranes has been recently observed [Interestingly, using a slight over-expression of CHMP4 and VPS4, we observed the incorporation of these proteins within released VLPs even in the context of severe p6 and NC mutations, and expression of VPS4DN markedly reduced the efficiency of VLP release. Engagement of Tsg101 and ALIX during the HIV budding is generally assumed to allow the recruitment of downstream ESCRT-III proteins which polymerize at the neck of the budding VLPs before release ,50–54. Bobserved . We cannobserved –60. In cobserved ,62, baseIn line with the above, we found that ESCRT engagement during VLP budding grows more critical by addition of cargo to the Gag C-terminus. We have measured the kinetics of release for Gag.Pol VLPs with inactivated protease. The Pol protein has a large protein mass (150 kDa) and, in absence of processing, the full length Pol incorporates within the VLP. We found that under these conditions, Gag.Pol VLP production is similarly sensitive to Tsg101 as well as ALIX interactions as shown with ΔPTAP and ΔYP p6 mutants. These results are surprising since typically Tsg101 is the primary interaction during HIV VLP budding, however they agree with the increased importance of ALIX when the budding neck diameter is large like during cytokinesis ,19,63. AR8.2 PRΔD25N VLP release is tested , full length Pol (large cargo) release is more sensitive to PTAP integrity than to YP. We hypothesize that there is an HIV factor that is absent in the Gag.Pol and is promoting the efficient VLP release in absence of functional PTAP/YP sites. This factor is likely acting to mimic some of the Tsg101/ALIX function in accelerating ESCRT-III recruitment and/or promoting Pol packaging before PR activation.Compared to the minimal Gag.Pol system, when HIVThe activation of HIV protease immediately post-assembly on plasma membrane is supported by some experimental evidence suggesting that increased packaging of Gag-Pol results in premature activation of PR . Rapid mFig 9), ΔPTAP and to lesser extent ΔYP VLPs are enriched mainly of Gag p48 and p41 forms, accordingly, clearly suggesting a loss of PR activity in these released VLPs.The observed kinetics of Gag precursor release from budding virions analyzed using computer simulations support activation of PR immediately post-virion assembly. Early biochemical characterization of PR cleavage sites showed that Gag and Gag-Pol SP1/NC and SP2/p6 sites are the first to get cleaved by PR . TherefoFig 4 and demonstrates that this phenotype is due to delayed release of ΔPTAP PRΔ VLPs with intact Pol domains.The first report identifying the importance of the PTAP sequence within Gag p6 used RT activity within the released HIV virions as a measure of viral fitness [All together, our observations suggest that the engagement of early ESCRTs during HIV budding is obligatory for speeding up the closure of budding virions and release of fully formed particles before the HIV protease activation occurs, which is fundamental for safeguarding the infectivity of progeny HIV virions. Other viruses and cellular processes whose cargo are not as time sensitive may forego some interactions with ESCRTs therefore possibly explaining the diverse requirements of ESCRTs in these processes ,69.Our observations show that ‘budding delay’ is a potent mechanism for inhibition of infectious retroviral release and suggest that this mechanism can be used for developing antiviral treatments that would not block ESCRT-dependent cellular processes but slow them to the point of infectious retroviral release inhibition. We also speculate that such mechanism maybe exploited by host cells to inhibit spread of infection.HIV-1ΔR8.2 (HIV-1NL4-3 R9ΔApa ) and HIVAll cell lines used were grown in complete DMEM medium under standard conditions, excepted for TIRF experiments where cells were incubated in CO2-independent medium (LifeTechnologies).Anti-ALIX , anti-TsAll cell lines used were transfected using lipofectamine 2000 (LifeTechnologies), except for 293T cells using standard CaPO4 precipitation technique. Both cells and media were collected for analysis. Cells were lysed in RIPA buffer , and after removal of residual cell debris by centrifugation, VLPs were pelleted from cell supernatants by centrifugation for 2 hours through 10% (w/v) sucrose cushion at 15,000 x g. Final VLP samples were re-suspended in PBS. VLP release yields/ratio were calculated as VLPs-associated Gag forms per cell-associated Gag forms based on either CA or MA probing, after densitometry analysis of the immunoblotting data using the Image Studio Lite software (LI-COR). HIV Gag kinetics were fit using a boltzman equation to calculate the delay times for various mutants as described in Supporting Information.Fig 3 (right panels).Live images were acquired using iMIC Digital Microscope made by TILL photonics controlled by TILL’s Live Acquisition imaging software see also . U2OS ceSimulations were setup following the Gillipsie algorithm . Processkp is the processing rate, kd is the diffusion rate of Pol from the formed VLP with open neck, kr is the rate of VLP release before processing, and The concentration shown in brackets is the number of molecules within one VLP. At time t = 0 therefore Fig 8A to fit the experimental p24 and p55 concentrations measured in Gag.Pol kinetics experiments. In these simulations, kp and kd rates are kept constant while each specific p6 mutation is simulated with a corresponding kr. The simulated internal Pol (Red) and Gag.Pol (Blue) concentrations in three VLPs are shown in Fig 8B.Simulated curves of p24 and p55  are used in S1 Fig(A) Effect of the G2A mutation. 200 ng of each Gag construct were used for transfection, and samples were collected 24 hours post-transfection. (B) Time course  of VLPs release. 200 ng of each Gag construct were used for transfection. (C) Gag dose-dependent of VLPs release yields. 50, 100, and 200 ng of each Gag construct were used for transfection, and samples were collected 24 hours post-transfection. All panels correspond to Gag immunoprobing using p24 antibody. Experiments were performed 1 time for (A) and 3 times for (B) and (C) with very similar results.(TIF)Click here for additional data file.S2 Fig(A) 200 and 250 ng of each Gag and HIVR8.2 constructs, respectively, were used for transfection. 11 hours post-transfection, cells were pulsed 1 hour then chased for 12 hours. Samples were collected and analyzed accordingly (see (B) Time course of VLPs release by Gag-nxGFP cargo. 200 ng of each Gag construct were used for transfection. Each 8 hours during the kinetic, cells were pulsed 30 min then chased the remaining time before samples collection. Both experiments in (A) and (B) were performed twice with the same outcome.ngly see . (B) Tim(TIF)Click here for additional data file.S3 FigCells and VLPs were collected 24 hours post-transfection. All panels correspond to p24 immunoprobing. This experiment was performed 3 times with similar results.(TIF)Click here for additional data file.S4 Fig(A) Comparison of the Gag-Pol vector previously used in the literature  and our Gag.Pol construct. (see (B) p6 re-introduction in the original GagPol vector (in frame) rescued VLP release, with reduced negative regulation of the integrase domain during the VLPs budding process. The original Gag-Pol vector consists of GagΔp6 fused in frame to Pol starting the TF domain till the IN (integrase) end. We fused full length Gag (p6 included) to Pol as in the original Gag-Pol vector and analyzed the VLPs release profile. 250 ng of each construct were used for transfection, and both Cells and VLPs were analyzed 24 hours post-transfection. (C) Full length Pol regulates proper PR activation during budding and release independently of intact Gag p6. Variants of our Gag.Pol p6 mutants were generated by modulating Pol length via Pol truncation as indicated. 250 ng of each construct were used for transfection, and both Cells and VLPs were analyzed 24 hours post-transfection. The vectors were expressed in 293T cells, and all panels correspond to p24 immunoprobing. All these experiments were performed at least 3 times with similar results.uct. see . 250 ng (TIF)Click here for additional data file.S5 Fig(A) Independent triplicate assessments of VLPs release by the Gag.Pol variants in 293T cells as indicated, with either active PR (PRwt) or inactive PR (PRΔD25N). (B) Comparative evaluation of p24-related Gag products and RT in VLPs released by Gag.Pol PRwt versus PRΔ in 293T cells. Final VLP samples were re-suspended in the same volume, and the indicated volume folds were analyzed by immunoblotting as shown. (C) Densitometry values from Panels (A) and (B) were processed accordingly and plotted as the corresponding relative amounts compared to the standards that equalize to 1 for p24 in Gag.Pol PRwt and 100% for RT in Gag.Pol PRΔD25N. 250 ng of each construct were used for transfection, and VLPs were analyzed 24 hours post-transfection. These experiments were performed 3 times as shown for (A) with similar results.(TIF)Click here for additional data file.S6 Fig(A) Gag (200 ng of each construct were used for transfection). (B) Gag.Pol (250 ng of each construct were used for transfection). These experiments were performed 3 times with similar results.Dynasore, a noncompetitive inhibitor of the GTPase activity of Dynamin which blocks cellular dynamin-dependent endocytosis , 73, was(TIF)Click here for additional data file.S1 Text(DOCX)Click here for additional data file.