Ten days and counting. When the curtain goes up on the G20 summit on November 30 in Buenos Aires, the spotlight will fall on the planned trade talks between United States President Donald Trump and his China counterpart Xi Jinping.
For the global community, it can not come quick enough.
So far, the shockwaves from what is starting to look like a new Cold War have left international markets wobbling and the world’s second-largest economy cooling amid heightened concerns from the global community.
But hopes of a quick fix might be premature.
“China-US divisions are structural problems, which may not be solved in the short term,” said Lou Jiwei, the former minister of finance. “However, a break-up will cause huge damage to both, as China is not the former Soviet Union, and it has become highly integrated into the current global system.”
The fallout has already descended on China’s economy. Last week, a batch of data released by the National Bureau of Statistics showed that the impact of the trade dispute is having a significant effect.
While industrial output and investment grew faster than expected in October on the back of a raft of government measures, retail sales have softened.
The property market has also suffered while new car sales have stalled as consumers rein in spending. Local government and corporate debt are other ongoing issues.
As for the markets, they have reacted wildly in the past 24 hours after the spat between Beijing and Washington spilled over at the Asia Pacific Economic Co-operation meeting at the weekend.
The Shanghai Composite Index dropped 2.1% to close at 2,645.85 while the Shenzhen Composite Index shed 2.7%. Hong Kong’s Hang Seng fell nearly 2% with the rest of Asia’s major indices in the red following an overnight sell-off on Wall Street.
Reports that Xi’s top trade negotiator, Vice-Premier Liu He, had canceled plans to visit the US added to the turmoil.
Yet this can be put down to a change of strategy from Beijing. A source told the South China Morning Post that the decision had been made after “work-level discussions” between the two sides.
“The latest plan is that the two countries’ trade negotiation teams will meet in Buenos Aires,” the person, who declined to be identified, was quoted as saying, adding that they would work behind the scenes to flesh out even a tentative agreement between Trump and Xi.
Still, China is pressing ahead with its “opening up” policy to cope with rising trade tensions, according to Liu Shijin, an adviser to the People’s Bank of China.
Although a timeline was not mentioned for these “market reforms,” he told a finance forum in Beijing that they could not be stopped.
“The Sino-US trade frictions are still ongoing, private firms’ expectations are unstable, and some people are saying they cannot see clearly and are a bit anxious,” Liu said.
“To cope with the trade frictions, China should implement a high level of opening up of its markets and economy, but it will push ahead with those reforms on its own steam and [will] not be forced by others,” he added.
His remarks come at a time when tit-for-tat tariffs have been imposed on a range of imports from both countries, including duties worth more than US$200 billion on Chinese goods and products entering the US with the threat of more to come.
“Through its strategic choice of Chinese products, the US government was not only able to minimize the negative effects on US consumers and firms, but also to create substantial net welfare gains in the US,” authors Benedikt Zoller-Rydzek and Gabriel Felbermayr wrote in a paper for EconPol Europe, a network of researchers in the European Union.
“As the trade conflict escalates, however, the US administration may not be able to restrict its selection to products with high import elasticities,” they added. “And US welfare might decrease as more of the tariff incidence falls on consumers.”
Only a deal in Buenos Aires would eliminate that scenario.


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Current therapeutic antiangiogenic biologics used for the treatment of pathological ocular angiogenesis could have serious side effects due to their interference with normal blood vessel physiology. Here, we report the generation of novel antivascular endothelial growth factor‐A (VEGF) biologics, termed VEGF “Sticky‐traps,” with unique properties that allow for local inhibition of angiogenesis without detectable systemic side effects. Using genetic and pharmacological approaches, we demonstrated that Sticky‐traps could locally inhibit angiogenesis to at least the same extent as the original VEGF‐trap that also gains whole‐body access. Sticky‐traps did not cause systemic effects, as shown by uncompromised wound healing and normal tracheal vessel density. Moreover, if injected intravitreally, recombinant Sticky‐trap remained localized to various regions of the eye, such as the inner‐limiting membrane and ciliary body, for prolonged time periods, without gaining access either to the photoreceptors/choriocapillaris area or the circulation. These unique pharmacological characteristics of Sticky‐trap could allow for safe treatment of pathological angiogenesis in patients with diabetic retinopathy and retinopathy of pre‐maturity.
A locally delivered, bifunctional recombinant “Sticky‐trap” comprising a VEGF‐trap and a heparin‐binding domain (HBD) can prevent aberrant ocular angiogenesis without affecting systemic physiological VEGF‐processes.
Bifunctional “Sticky‐trap” is comprised of VEGF‐trap and a carboxy‐terminal HBD.
Treatment with recombinant Sticky‐trap can prevent the development of abnormal ocular angiogenesis.
Sticky‐trap remains at the site of delivery; thus, it has a local antiangiogenic activity.
Sticky‐trap applied in the eye does not compromise systemic physiological VEGF‐processes, such as wound healing and kidney function.
Abnormal vessel formation is implicated in the pathophysiology of a number of diseases including diabetic retinopathy (DR), retinopathy of pre‐maturity (ROP), the wet form of age‐related macular degeneration (AMD), cancer and obesity (Carmeliet, 2003, 2005; Folkman, 2007). Inhibition of pathological angiogenesis has been shown to improve vision in patients with AMD, DR and ROP (Ferrara & Kerbel, 2005; Gasparini et al, 2005; Kerbel, 2008; Mintz‐Hittner et al, 2011), as well as help maintain metabolic homeostasis in obese individuals (Sun et al, 2012). Over the past decade, different approaches to inhibit VEGF signalling have been developed, such as recombinant trap proteins (VEGF‐trap; Holash et al, 2002), monoclonal antibodies (bevacizumab; Ellis, 2005; Ferrara et al, 2004) and small multi‐kinase inhibitors (Wilhelm et al, 2006; Chow & Eckhardt, 2007), some of which have been approved for clinical use (Grothey & Galanis, 2009; Heath & Bicknell, 2009) in treating eye diseases as well as certain types of cancer.
However, the aforementioned approaches may be accompanied by a number of severe side effects due to the disruption of proper vessel function in various organs (Eremina et al, 2003, 2006; Kamba et al, 2006; Verheul & Pinedo, 2007; Chen & Cleck, 2009; Boehm et al, 2010). As a consequence, the use of anti‐VEGF therapies may be relatively contraindicated, particularly in certain patient populations, such as pregnant women (Petrou et al, 2010), patients undergoing surgery (Cortes et al, 2012), pre‐mature infants (Hard & Hellstrom, 2011) and elderly patients with comorbidities, such as cardiovascular insufficiency (Chen & Cleck, 2009; Nazer et al, 2011).
The storage of numerous growth factors in the extracellular matrix (ECM), as well as the creation of morphogen gradients during development, is controlled by the interaction between the heparin‐binding domain (HBD; a sequence of positively charged amino acids) of the growth factor and extracellular‐matrix heparan sulphate proteoglycans (HSPGs). VEGF contains two regions with HBDs coded by exons 6 and 7. Alternative splicing of these exons results in distinct isoforms (Robinson & Stringer, 2001; Ladomery et al, 2007). VEGF121 is missing both exons and is therefore completely soluble. VEGF145 and VEGF165 contain exon 6 and 7, respectively, and are therefore partially soluble. VEGF189 has both exons and is strictly retained at the secretion site (Houck et al, 1992; Park et al, 1993).
In order to circumvent the aforementioned disadvantages of current antiangiogenic therapies, we have developed novel biologics with properties that allow for local inhibition of angiogenesis. By genetically fusing the original VEGF‐trap (Holash et al, 2002) to the HBDs of VEGF, we created novel VEGF‐traps, referred to as Sticky‐traps, engineered to possess: (i) a short systemic half‐life in it's soluble form and (ii) local antiangiogenic activity via the ability to bind to the ECM thus leading to locally retained, biologically active molecules that provide prolonged angiostatic activity at the site of pathological neovascularization.
Design and production of VEGF Sticky‐traps
The original VEGF‐trap (Holash et al, 2002) is composed of IgG‐like domains 2 and 3 of VEGF‐R1 and VEGF‐R2, respectively, fused to the constant region (Fc) of immunoglobulin IgG1 (Fig 1A and Supplementary Fig S1A). The Fc region is composed of two IgG‐like domains, CH2 and CH3, and is responsible for the long serum half‐life of VEGF‐trap, due to its ability to bind to the FcRn receptor located on endothelial, epithelial and circulating blood cells, after which it is recycled back into the circulation instead of undergoing degradation (Ghetie & Ward, 1997). In order to shorten the half‐life, we replaced the CH2 domain, which has been previously shown to be necessary for binding to FcRn (Mueller et al, 1990), with a hinge domain (H'; Glaser et al, 2005) and a poly‐glycine‐serine linker (Glaser et al, 2005; Fig 1A and Supplementary Fig S1A), and named this molecule Short‐trap. To derive Sticky‐traps, we modified the Short‐trap by adding the HBDs of the VEGF isoforms (HBDs; Munoz & Linhardt, 2004), encoded by exons 6 and 7, at the carboxy‐terminus. We also included VEGF exon 8, which is necessary for proper disulphide‐bond formation of the HBD (Krilleke et al, 2007). In total, we generated three Sticky‐traps: Sticky‐trap68, Sticky‐trap78 and Sticky‐trap678, where the numbers indicate which VEGF exons were included (Fig 1A and Supplementary Fig S1A and B). Finally, we generated a “trapless” construct coding only for the short Fc (shFc), as a negative control (Fig 1A and Supplementary Fig S1A).
We then used genetic and recombinant protein‐based approaches to characterize these new biologics. In both approaches, we combined the piggyBac transposon (Ding et al, 2005) transgene delivery system with a tetracycline‐based ON/OFF switch (Agha‐Mohammadi et al, 2004; Supplementary Fig S2A) for transgene expression in a doxycycline (dox)‐dependent manner. To test the biologic activity/property of the engineered novel biologics with genetic approaches, we derived transgenic cancer cell lines (PC‐3, A‐673 and HT‐29; Supplementary Fig S2B–D). In the absence of doxycycline (dox), none of the transgenes were expressed, while in the presence of dox, all were expressed at a high level and in comparable amounts (Supplementary Fig S2B–D). We also attached green fluorescent protein (EGFP) as a reporter for transgene expression. Flow cytometry for EGFP in the induced cells showed that on average 88% of the cells expressed the transgene (Supplementary Fig S2E and Supplementary Table S1).
To produce and purify recombinant proteins, we generated stably transfected 293 suspension cell lines using vectors allowing for expression of recombinant traps in a dox inducible manner (Li et al, 2013). Of the Sticky‐traps, Sticky‐trap678 was selected for further characterization, given its superior potency in a xenograft assay (discussed below). All of the traps formed homodimers, had the predicted molecular weight (Supplementary Fig S3A) and were glycosylated (Supplementary Fig S3B).
Molecular and biochemical characterization of Sticky‐traps
Immunostaining, performed on transgenic PC‐3 (Fig 1B) and A‐673 cells (Supplementary Fig S4A), for the Fc domain showed—as expected—that shFc, VEGF‐trap and Short‐trap did not bind to the ECM. However, Sticky‐traps were bound and retained by the ECM components, indicating that the VEGF HBDs remained functional in these molecules. Further support for the strong affinity of Sticky‐traps to the ECM was obtained from Western blot analysis, in which high levels of soluble (free) shFc, VEGF‐trap and Short‐trap were detected in the culture media, while Sticky‐traps were hardly detectable (Fig 1B and Supplementary Fig S4A). Using recombinant proteins, we performed ECM‐based and heparin column assays (Fig 1C and D). Sticky‐trap showed a dose‐dependent ability to bind to the ECM (Matrigel matrix), while VEGF‐trap, Short‐trap and IgG showed no affinity (Fig 1C). Furthermore, high NaCl concentration was required for elution of Sticky‐trap, further indicating its strong affinity for the ECM (Fig 1D).
Sticky‐traps also retained their ability to bind VEGF, as confirmed by a competitive ELISA that detects only free VEGF (Maynard et al, 2003; Tissot van Patot et al, 2005). After addition of dox to the media of transgenic cell lines, both Short‐trap and Sticky‐traps, similar to VEGF‐trap, blocked the appearance of free VEGF in the conditioned media (Supplementary Fig S4B–D). Binding was also shown to be extracellular as VEGF was still completely sequestered from the supernatant when a 1:1 mix of wild‐type and transgenic PC‐3 cells was assayed (Fig 1E).
We used equilibrium binding assays to determine the binding affinity of recombinant traps for VEGF. Various amounts of traps were incubated with VEGF165, and the free, unbound VEGF165 was measured using a sensitive immunoassay. All traps showed strong affinity for VEGF with similar KD (VEGF‐trap; 9.0 pM, Short‐trap; 12.2 pM, and Sticky‐trap 13.9 pM; Fig 1F). Finally, as a functional assay, we used human umbilical vein endothelial cell (HUVEC) to examine the ability of recombinant traps to inhibit VEGF‐induced proliferation and inhibit VEGFR‐2 Y1175 phosphorylation. Various amounts of traps were pre‐incubated with fixed amounts of VEGF and added to HUVEC cell cultures. All traps were able to efficiently block VEGF‐induced proliferation in a dose‐dependent manner (Fig 1G; percentage inhibition compared to vehicle only), as well as inhibit VEGFR2 phosphorylation at Y1175 (Supplementary Fig S3C). The enhanced ability of Sticky‐trap to inhibit HUVEC proliferation in doses higher than 10 pM is presumably attributed to the fact that both Sticky‐trap and VEGF have the same HBD. Therefore, it is possible that Sticky‐trap can affect the bioavailability of VEGF by competing for available binding sites in the ECM, as well as compromise the ability of VEGF to bind to neuropilin‐1, which is mediated through exons 7 and 8 of the HBD, and is required for VEGFR‐2 activation (Parker et al, 2012).
Pharmacokinetics and systemic distribution
In order to characterize the in vivo pharmacokinetics of Sticky‐trap, after subcutaneous injection, we used an ELISA assay to measure their concentration in the serum. As expected, high amount of VEGF‐trap was detected (Cmax = 10 μg/ml; AUC = 24 μg × days/ml), while both Short‐trap and Sticky‐trap had an AUC that was around 400‐fold lower (Cmax = 0.10 μg/ml; AUC = 0.065 μg × days/ml and Cmax = 0.10 μg/ml; AUC = 0.06 μg × days/ml, respectively; Fig 2A), and were practically undetectable 24–48 h after injection.
Previous studies have reported a spike of serum VEGF after treatment with antiangiogenic agents, such DC101 (i.e. anti‐VEGF‐R2 Ab) and sunitinib (Bocci et al, 2004; Ebos et al, 2007). Therefore, we examined whether this was also the case for traps. We observed an increase of circulating VEGF in the serum within 1 h post‐injection of VEGF‐trap, which plateaued between 24 and 48 h (Fig 2B). In the case of Short‐trap and Sticky‐trap, we did not observe any increase of serum VEGF 1, 8 h and 1 day post‐injection, indicating the absence of any systemic activity (VEGF levels were not assayed for the rest of the time points, while VEGF baseline levels in the control (vehicle only) mice were undetectable).
Finally, to exclude the possibility that Sticky‐trap due to its high affinity to the ECM is deposited in tissues, we examined the distribution in various tissues and especially those with fenestrated vessels. Two days post‐subcutaneous injection, we showed that in contrast to VEGF‐trap, Short‐trap and Sticky‐trap were undetectable in kidney, small‐intestine, liver and adrenal gland (Fig 2C–F).
Sticky‐traps inhibit angiogenesis in vivo and remain at the site of introduction
Although our intention here is to propose the use of Sticky‐traps as therapeutic agents to suppress pathological neovascularization in eye diseases, in order to initially explore and evaluate the in vivo effect of Sticky‐traps with ease, we used tumour xenograft assays (Fig 3 and [Link], [Link], [Link], [Link], [Link], [Link], [Link], [Link]). Nude mice were used as recipients for subcutaneous xenografts, and transgenic expression of traps (and shFC control) was induced with dox‐containing food (dox‐chow). Expression was visualized by EGFP. Prior to dox administration, none of the xenografts expressed EGFP, while expression was detected as early as 2 and 4 days post‐administration and reached a plateau at day 4 and 8, in the A‐673 and HT‐29 xenografts, respectively ([Link], [Link]). EGFP expression by PC‐3 cancer lines was very weak (data not shown).
Upon trap expression, the PC‐3 derived xenografts significantly decreased in size, some completely regressed (Supplementary Fig S5A) with only small foci remaining (Supplementary Fig S5B). Tumour‐stasis was observed in HT‐29 xenografts expressing Sticky‐trap678, while a small increase in tumour size was observed when VEGF‐trap was induced. Likely due to its short half‐life, Short‐trap was the least effective; tumours expressing this variant kept growing, albeit at a much slower rate compared to the shFc control (Supplementary Fig S6B). A‐673‐derived xenografts stopped growing and the average tumour volume remained constant during the treatment (Supplementary Fig S7B). The stasis of the tumour was independent of the initial volume of the A‐673 xenograft tumours (data not shown).
The results from the HT‐29 xenografts indicated that Sticky‐trap678 was the most efficient trap; thus, we produced recombinant protein corresponding to this isoform. To characterize the ability of the recombinant traps to inhibit tumour progression, we established xenografts using wild‐type HT‐29 and A‐673 cells (Fig 3A and B). HT‐29‐derived tumours were not affected by intratumourally injected Short‐trap (Fig 3A), while Sticky‐trap resulted in tumour growth delay, similar to VEGF‐trap (Fig 3A).
In our A‐673 xenograft studies, we also performed systemic delivery of recombinant VEGF‐trap via subcutaneous injections. Sticky‐trap was able to almost completely inhibit tumour progression and was significantly more efficient than equal amounts of systemically administered VEGF‐trap (VEGF‐trap 100 μg s.c., P < 0.01; Fig 3B). Intratumourally injected Sticky‐trap was also initially more efficient than a 5‐times higher dose of systemically administered VEGF‐trap, losing statistical significance between day 9 and 14 (P = 0.0048 at day 5, P = 0.0343 at day 9, and P = 0.1857 at day 14).
We then analysed the effect of trap expression on tumour microvascular density by immunostaining of xenografts with antibodies recognizing the endothelial marker CD31 (PECAM). Although all traps dramatically decreased tumour microvascular density (Fig 3C and D, and [Link], [Link], [Link], [Link]), recombinant Sticky‐trap was the most potent (Fig 3C and D). The vessel‐density decrease resulted in hypoxic regions in the HT‐29 tumours (Supplementary Fig S6D), and extensive areas of necrosis in both A‐673 and HT‐29 tumours ([Link], [Link]).
To examine the levels of the traps in the systemic circulation, we collected serum from tumour‐bearing mice treated with recombinant traps and measured the levels of free circulating traps using a functional ELISA. Circulating VEGF‐trap could be detected in both xenograft models— HT‐29 and A‐673—while Short‐trap and Sticky‐trap were undetectable (Fig 3E and F). We observed a similar pattern in the case of xenografts using transgenic cancer cell lines ([Link], [Link]). We also collected urine sample from mice bearing xenografts expressing either VEGF‐trap, Short‐trap or Sticky‐traps. By Western blotting, we detected high amount of intact VEGF‐trap in urine of mice with either A‐673 or HT‐29 xenograft (Supplementary Fig S12A and B), while only fragments of Sticky‐trap678 were detected in urine of mice with A‐673 xenografts (Supplementary Fig S12A).
Sticky‐traps have a favourable toxicity profile
As mentioned previously, systemic anti‐VEGF therapies may be associated with adverse side effects. We therefore investigated the potential systemic effects of Sticky‐traps outside the tumour environment of A‐673 xenograft‐bearing animals. At a tumour size of 1.5 cm3, transgene expression was induced, and 8 days later, full‐thickness excisional wounds were created in the neck area of the animals (Niethammer et al, 2002; Seavey et al, 2009; Fig 4A). Consistent with the known wound healing delay following systemic VEGF suppression (Bose et al, 2010), 4 days after wounding, the wound size of animals with tumours expressing the soluble VEGF‐trap was significantly larger than those expressing Sticky‐trap (Fig 4B and C; P < 0.05). Animals with tumours expressing Sticky‐traps and control non‐tumour‐bearing animals fed with dox‐chow showed the same wound size (Fig 4B and C; P > 0.05). Similarly, a longer time period was required for the scab to fall off from the wounds on animals bearing tumours expressing VEGF‐trap compared to control animals and those expressing Sticky‐traps (Fig 4B, D and E; P < 0.01). No difference was observed between the latter two (Fig 4B, D and E; P > 0.05).
In a second model, we also measured systemic VEGF suppression by examining tracheal micro‐vessels (Kamba et al, 2006) by whole‐mount immunostaining with antibodies recognizing endothelial cells (anti‐CD31) and their basement membrane (anticollagen IV). Functioning vessels are double positive, while vessel regression leads to areas positive for collagen IV and negative for CD31 (called empty sleeves; Kamba et al, 2006). Animals with tumours expressing Sticky‐trap678 and non‐tumour‐bearing controls had very few empty sleeves and the majority of the vessels were double positive (Fig 4F and G; P > 0.05). In contrast, animals with tumours expressing the VEGF‐trap showed a fivefold increase in empty sleeve formation (Fig 4F and G; P < 0.001).
In addition, we also used recombinant proteins in order to further examine the safety of Sticky‐trap. Due to the limited amount of recombinant Sticky‐trap, we decided to use pups and examine the functionality of kidney glomeruli vessels after treatment via lectin immunostaining (Fig 5). In order to model systemic administration, traps (25 mg/kg) were given subcutaneously once every 3 days, starting at P3, and the mice were euthanized after three injections at P12. We observed decreased perfusion of glomeruli vessels in pups treated with VEGF‐trap (Fig 5A and B), which caused some of the glomeruli to collapse (Fig 5C), while the glomeruli of pups treated with Sticky‐trap appeared normal. We also detected high levels of VEGF‐trap in kidney tissue lysates, while Sticky‐trap was undetectable (Fig 5D).
Taken together, these results suggest that (i) systemic VEGF antagonism has adverse effects on the maintenance of normal vessels and (ii) no such adverse events are observed with the Sticky‐trap.
Sticky‐trap binds to various anatomical areas of the eye
Given the clinical value of antiangiogenic molecules in the treatment eye diseases and the demonstrated strong retention of Sticky‐trap at the site of introduction, we characterized its distribution in the eye after intravitreal injection using immunostaining of cross‐sectioned whole eyes. Two days post‐injection, only Sticky‐trap could be detected in all eyes, while VEGF‐trap and Short‐trap were not present by then (Fig 6A). Sticky‐trap remained in the eye at least for 12‐days post‐injection (Fig 6A). In contrast to VEGF‐trap and Short‐trap, Sticky‐trap specifically localized to the lens, the inner‐limiting membrane, the ciliary body and the ciliary body zonula fibres (Fig 6B and C and [Link], [Link], [Link], [Link]). This distribution was in part overlapping with collagen IV (Supplementary Fig S13). Small amounts of Short‐trap and VEGF‐trap could be detected in the choroid of some eyes analysed 2 and 8 h post‐injection ([Link], [Link]). Since Sticky‐trap was not detected posterior to the inner‐limiting membrane, we also performed subretinal injections. Three days post‐injection, we could detect Sticky‐trap in the subretinal space, between the retinal pigment epithelium (RPE) and outer nuclear layers (Supplementary Fig S17). We also examined the circulating levels of traps after intravitreal injections. Short‐trap and Sticky‐trap were undetectable, while VEGF‐trap was present at a significant level (Fig 6D).
Sticky‐trap efficiently inhibits eye neovascularization
The ability of Sticky‐trap to specifically bind to the inner‐limiting membrane prompted us to study its efficacy in an oxygen‐induced retinopathy (OIR) mouse model for DR and ROP. We examined the ability of traps to inhibit ocular neovascularization after intravitreal injections in the eyes of mice exposed to high oxygen from P7 until P12 followed by return to normoxia; one eye was injected with trap, while the contralateral eye served as vehicle control. Pathological vascularization was quantified by immunostaining at either P17 (Fig 7A–D) or P21 (Fig 7E–H). At P17, we found a 65 and 70%, reduction of tuft formation by VEGF‐trap and Sticky‐trap, respectively (Fig 7B and C). Persisting vaso‐obliteration (i.e. avascular area) was reduced by 46% by Sticky‐trap compared to 18% when VEGF‐trap was used (Fig 7B and D). At P21, Sticky‐trap reduced tuft formation by 95%, and VEGF‐trap by 90% (Fig 7F and G). Moreover, 74% of the eyes treated with Sticky‐trap had no obliteration and/or tufts at all, compared to 39 and 26% in the case of VEGF‐trap and vehicle, respectively (Fig 7F and H). Immunostaining for traps showed that Sticky‐trap remains in the eye after injection and localizes to the inner‐limiting membrane and the ciliary body (Supplementary Fig S18A). We observed the same results when traps were injected at P7 (pre‐high oxygen exposure) and analysed at P17 (Supplementary Fig S18B). We also examined the level of circulating traps, and in contrast to VEGF‐trap, Sticky‐trap was undetectable in the serum (Supplementary Fig S18C).
Finally, we assessed vascular leakage using intravenous Evans Blue injection. Both Sticky‐trap and VEGF‐trap were able to inhibit vascular leakiness and reduce the levels of Evans Blue in the vitreous to the same level found in wild‐type eyes under normoxia (Fig 8).
Sticky‐trap is not toxic to the eye
Using genetic models, it has previously been reported that deletion of VEGF in adult RPE cells rapidly leads to the ablation of choriocapillaris, pronounced loss of the outer segments of cone photoreceptors and as a consequence to blindness (Nishijima et al, 2007; Kurihara et al, 2012). In this study, we performed three intravitreal injections of Sticky‐trap, over a 6‐week span and then examined the function of photoreceptors using electroretinography (ERG; Fig 9A). Using both scotopic and photopic ERGs, we demonstrated that the function of rod (a‐wave; scotopic ERG and flicker; photopic ERG) and cone (flash; photopic ERG) photoreceptors was not affected (Fig 9B–G). In addition, the unaltered b‐wave amplitude of scotopic ERG indicates that the function of the cells of the inner nuclear layer, such as bipolar and Muller cells, as well as the function of ganglion cells was also not affected (Fig 9D; Dong & Hare, 2000; Bui & Fortune, 2004). Similar results were also observed 1 week after a single subretinal injection of Sticky‐trap (Supplementary Fig S19).
Furthermore, using immunohistochemistry to stain for cone photopigment opsin, we showed that there was not any difference in the number of cone photoreceptor outer segments between vehicle and Sticky‐trap treated mice (Fig 9H and I). Finally, using electron micrographs, we examined the integrity of the choriocapillaris and showed that it was not affected (Fig 9J). Therefore, we could conclude that the extended presence of Sticky‐trap in the eye does not interfere with retina function.
In this study, we employed both genetic and pharmacological approaches to characterize novel anti‐VEGF biologics: VEGF Sticky‐traps. In contrast to VEGF‐trap (Holash et al, 2002) that has a broad systemic activity, Sticky‐traps are locally retained at the delivery site for an extended amount of time by binding to the ECM; this feature results in a safer toxicity profile. The ability of Sticky‐traps to remain at the site of delivery was achieved by engineering the original VEGF‐trap to contain a strong HBD and thus to be able to bind to negatively changed heparan sulphate glycosaminoglycans of various HSPGs of the ECM and cell surface. Furthermore, Sticky‐traps are at least as effective as VEGF‐trap in inhibiting pathological angiogenesis.
Currently approved angiostatic treatments of neovascular eye diseases require frequent intravitreal injections of the eye using expensive drugs (Stewart, 2012). Longer acting compounds, and the use of sustained delivery devices, would allow for less frequent administration and as a consequence could lower the costs and risks associated with repeated intraocular injections. Recent clinical trials have provided evidence that VEGF‐trap administered every other month in patients with AMD can be as effective as monthly ranibizumab (Stewart et al, 2012a); likely due to its longer predicted intravitreal half‐life (4.8 days versus 3.2 days; Stewart et al, 2012b) and its higher VEGF‐binding affinity (Papadopoulos et al, 2012). In the case of bevacizumab, various approaches have been evaluated with the goal of increasing the intravitreal half‐life (Pan et al, 2011). In this study, we showed that Sticky‐trap was more effective compared to VEGF‐trap in inhibiting tuft formation and vaso‐obliteration in a mouse model of DR and ROP. Moreover, Sticky‐trap remained in the eye up to 12 days, which is longer than the time periods reported in the literature for biologics targeting ocular abnormal angiogenesis. This extended retention strongly indicates that Sticky‐trap might allow for administration in less frequent intervals.
We provide compelling evidence that in contrast to VEGF‐trap, Sticky‐trap binds to various areas of the eye, including the inner‐limiting membrane after intravitreal injections. Thus, Sticky‐trap would be very appealing for treatment of patients with diseases such as DR and ROP, in which new vessels emerge from the retinal vascular network, penetrate the inner‐limiting membrane (ILM) and advance into the posterior hyaloid face (Antonetti et al, 2012). Previous studies have shown that both the thickness of the ILM and the amount of HSPGs increase with disease progression in the case of DR (Hanneken et al, 1991; Matsunaga et al, 2005). The later will allow for even higher retention of Sticky‐trap in patients with advanced disease, which may result in higher clinical benefit. We were also able to show that after subretinal injections, Sticky‐trap binds and remains in the subretinal space; the origin of new vessels observed in AMD (Jager et al, 2008). This retention property could allow local AMD treatment with delivery of Sticky‐trap in the RPE layer with various methods, such as slow‐release implants (Janoria et al, 2007), liposomes (Abrishami et al, 2009), viruses (Pechan et al, 2009) and RPE cells (Otani et al, 2002).
Delivery of soluble VEGF antagonists can be worrisome due to the high risk of severe side effects, especially since many of the elderly patients with DR and AMD might have coexisting risk factors (Stewart, 2012). The local activity and short systemic half‐life of Sticky‐trap may prove especially helpful in these cases. Moreover, pre‐mature infants suffering from ROP could also greatly benefit from this novel biologic. Treatment of ROP typically occurs during the neonatal stage (between 30 and 40 weeks post‐menstrual age), during organogenesis. During this period, it has been shown that VEGF is very important for the proper development of both kidneys and lungs; its inhibition can lead to dysfunction of glomerular filtration and bronchopulmonary dysplasia (Eremina et al, 2006; Thebaud, 2007). In the recent BEAT‐ROP clinical trial comparing intravitreal bevacizumab to conventional diode laser photocoagulation for ROP treatment, increased mortality was observed in the bevacizumab versus laser group (6.6 versus 2.6%). Interestingly, four of five deaths in the bevacizumab group (and one of two in the laser group) were due to pulmonary complications (Mintz‐Hittner et al, 2011). Recent reviews point out that systemic VEGF suppression in pre‐mature infants can be detrimental, and strongly suggest cautious and limited use of bevacizumab for this condition (Hard & Hellstrom, 2011; Avery, 2012; Darlow et al, 2013). Given the similar pharmacokinetic profile of VEGF‐trap, and its even higher affinity, it is reasonable to assume that similar concerns will arise. In contrast, the Sticky‐trap could provide a safer therapy for ROP since following intravitreal injections; it does not escape into the circulation.
In this study, we characterized our novel bifunctional antiangiogenic biologic, the Sticky‐trap, and demonstrated its efficacy in neovascularization models. Due to its unique property to locally inhibit abnormal angiogenesis, Sticky‐trap merits further exploration as a key component of antiangiogenic therapies.
Materials and Methods
Construction of VEGF‐traps and piggyBac expression system
Traps were generated using basic molecular biology techniques. VEGF‐trap (1479 bp; 492 a.a.; M.W. 54.8 kDa) is composed by (i) the signal peptide (NP_002010, a.a. 1–31), (ii) domain‐2 of human VEGFR‐1 (NP_002010, a.a. 131–231), (iii) domain‐3 of human VEGFR‐2 (NP_002244, a.a. 226–327) and (iv) the Fc region of human IgG1 (H domain; P01857.1, a.a. 99–113, plus CH2 domain; P01857.1, a.a. 114–223, plus CH3 domain; P01857.1, a.a. 224–330). Two epitope tags (FLAG: DYKDDDDK and His: HHHHHHHH) were added to the carboxy‐terminus with GS1 linkers (GGGS) in between. For the generation of Short‐trap (1227 bp, 408 a.a., M.W. 44.8 kDa), the CH2 domain was substituted by (i) a H' domain (17 a.a.; EPKSCDTPPPCPRCPAR; Glaser et al, 2005) and (ii) a GS2 linker (GGGSSGGGS; Glaser et al, 2005).
For the generation of the Sticky‐traps (Sticky‐trap68: 1332 bp, 443 a.a., M.W. 51.0 kDa; Sticky‐trap78: 1392 bp, 463 a.a., M.W. 51.0 kDa; and Sticky‐trap678: 1464 bp, 487 a.a., M.W. 54.0 kDa), the mouse vascular endothelial growth factor (VEGF‐A) exons 6 (NP_001020421, a.a 319–342), 7 (NP_001020421, a.a 343–386) and 8 (NP_001020421, a.a 387–392) were used, and a GS3 linker (GGGAS) was placed between the CH3 and VEGF‐A exons.
The transposons, PB‐TAG and PB‐rtTA, were generated by modifying PB‐TET (Woltjen et al, 2009) and pcDNA‐rtTA/neo (IMP, AN unpublished). PB‐TAG is composed of the second‐generation tetracycline‐regulated promoter (TRE; Agha‐Mohammadi et al, 2004) driving the expression of the transgene coupled to EGFP through an internal ribosomal entry site (IRES). PB‐rtTA is composed of the reverse‐transactivator of tetracycline (rtTA; Urlinger et al, 2000) and the neomycin resistance gene, both driven by separate constitutive promoters.
Cell culture and generation of cancer cell lines
The human prostate cancer PC‐3, human rhabdomyosarcoma A‐673 and human colon adenocarcinoma HT‐29 cell lines were purchased from ATCC and maintained in RPMI‐1640, DMEM and McCoy's 5A media, respectively, all containing 10% foetal bovine serum.
Briefly, for the generation of stable cell lines, 1 × 106 cells were plated per well (9.6 cm2/well) in a 6‐well plate and were transfected 16 h after with 0.5 μg of PB‐rtTA/neo and 2.5 μg of PB‐TAG‐GOI along with 0.5 μg PBase using ExGen500 (Fermentas, cat. # R0511) according to the manufacturer's protocol. Stable clones were derived after 2 weeks of selection using 750 μg/ml G418. To simplify the method and maintain a heterogeneous cancer cell population, all G418 resistant cells were trypsinized and pooled together. For induction of gene expression, we used 2 μg/ml of doxycycline (Sigma, cat. # D9891) in the culture media. Cell lines were cultured at 37°C, in 100% humidity and 5% CO2 containing atmosphere.
Generation of mammalian cell lines for protein expression
Stable mammalian cell lines expressing recombinant proteins were established as has been previously described (Li et al, 2013).
The harvested media containing the secreted proteins were concentrated 10‐ to 20‐fold on a Prep/Scale Spiral Wound 10‐KDa MWCO Ultrafiltration module (Millipore, cat. # SK1P003W4). Recombinant proteins were purified from the concentrated media by immobilized‐metal affinity chromatography on Ni‐NTA columns (Bio‐Rad, cat. # 156‐0135). Purified proteins were quantified using absorbance at A280 nm (NanoDrop UV‐Vis spectrophotometer; Thermo Scientific) and confirmed by Coomassie blue staining after SDS–PAGE.
For isolation of cytoplasmic and secreted protein from stably transfected cancer lines, 1 × 106 cells were plated per well (9.6 cm2/well) in a 6‐well plate and cultured with and without doxycycline for 48 h. Supernatants were collected, centrifuged and stored at −70°C. Subsequently, the cytoplasmic proteins along with ECM proteins of the cell monolayer were isolated using RIPA lysis buffer containing protease inhibitors (Roche, cat. # 11836170001). Plates containing 250 μl of lysis buffer per well were incubated for 30 min on ice. The lysis was collected and stored at −70°C.
Protein from xenografts was isolated from frozen tumour sections. Around a half gram of tissue was quickly homogenized on ice in 3 ml ice‐cold RIPA lysis buffer containing protease inhibitors mentioned above using homogenizer. Homogenates were incubated for 1 h at 4°C and centrifuged at 15,000 × g for 30 min at 4°C. The supernatant was collected and frozen at −20°C. Aliquots of supernatant were collected for protein determination by the Bradford method (Bio‐Rad protein assay).
Western blot assays
Cell culture extracts and supernatants, tumour protein extracts and plasma were resolved by 4–20% SDS–PAGE and transferred to nitrocellulose membranes. Membranes were blocked in 5% non‐fat milk in TBS‐T buffer (10 mM Tris pH 7.5, 150 mM NaCl and 0.1% Tween 20). A goat anti‐human Fc IgG1‐HRP‐conjugated antibody (1 in 5,000; Jackson Immunoresearch, cat. # 109‐035‐098) was used for detection of VEGF‐traps. Loading for cell culture and tumour extracts was assessed with rabbit antibody against human beta‐actin (1 in 10,000; Sigma, cat. # A5441) followed by anti‐rabbit IgG1‐HRP‐conjugated antibody (1 in 10,000; Bio‐Rad, cat. # 170‐6515).
VEGFR2 tyrosine phosphorylation assay
Human umbilical vein endothelial cells grown to confluency were starved in serum‐free media overnight and then treated with 1–10 μg/ml VEGF‐trap inhibitors for 2 h at 37°C prior to the tyrosine phosphorylation assay. Cells were pre‐treated with 200 μM Na3VO4 in serum‐free media for 5 min at 37°C and then exposed to 100 ng/ml VEGF, which was pre‐incubated with and without inhibitors for 30 min prior to use, in serum‐free media for 5 min 37°C. Cells were then lysed in modified RIPA (mRIPA) buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 1% NP‐40, 0.25% sodium deoxycolate, 1 mM EDTA, 1 mM sodium orthovanadate, 10 mM β‐glycerophosphate and protease inhibitors [1 mM phenylmethanesulfonyl fluoride (PMSF), 20 μg/ml leupeptin, and 20 μg/ml aprotinin]) and evaluated by immunoblot analysis using the following antibodies at a dilution of 1:1000: rabbit anti‐VEGFR2 (55B11) (Cell Signaling, Danvers, MA, USA), rabbit anit‐phospho‐VEGFR2 Y1175 (Cell Signaling) and mouse anti‐GAPDH (EMD Millipore, Billerica, MA, USA).
Flow cytometry analysis
For flow cytometry analysis, 1 × 106 cells were plated per well (9.6 cm2/well) in a 6‐well plate and cultured with or without doxycycline for 48 h, trypsinized and suspended into PBS containing 1% v/v of 7‐AAD (BD Pharmingen, cat. # 559925) for detection of apoptotic cells. The FACSAria™ cell sorter (BD Biosciences) was used for single cell analysis.
Enzyme‐linked immunosorbent assay for VEGF‐trap was developed as has been previously described (Koh et al, 2010). A 96‐well plate was coated overnight at 4°C with 200 ng of VEGF in 100 μl of PBS. The plate was washed four times with 400 μl wash buffer [PBS, 10% SBlock (Pierce, cat. # 37538), 0.05% Tween‐20] and then coated for 2 min with SBlock at RT. Fifty microlitres of trap standards, samples and controls were added along with 50 μl of incubation buffer (100% SBlock, 0.1% Tween‐20) and incubated on the bench, at RT for 2 h. After washing four times with 400 μl of wash buffer, 50 μl of HRP‐conjugated goat anti‐human IgG‐Fc (1:10,000 dilution in PBS; Jackson Immunoresearch, cat. # 109‐035‐098) along with 50 μl of incubation buffer was added in each well and the plate was incubated at RT for 2 h. The plate was washed four more times with 400 μl of wash buffer, before addition of 50 μl of 3,3′,5,5′‐tetramethylbenzidine (TMB) solution (Thermo Scientific, cat. # 34028) and incubation at RT for 30 min. The reaction was stopped with stopping solution (Sigma, cat. # S5814), and the absorbance at 450 nm was measured using an ELISA reader (Bio‐Rad).
Human VEGF was measured using a commercially available sandwich ELISA (R&D systems, cat. # DVE00) following the manufacturer's protocol. One hundred microlitres of tissue supernatant was used for each sample.
Binding affinity of traps was determined using an ELISA able to detect unbound human VEGF (R&D Systems, cat. # DVE00). Various concentrations of traps (0.15–160 pM) were incubated with 10 pM VEGF165 (R&D Systems, cat. # 293‐VE‐010) and resuspended at final concentration 0.1 μg/μl in PBS, 1 mg/ml Fraction V BSA) in incubation buffer (50 mM phosphate buffer, 150 mM NaCl, 1 mg/ml fraction V BSA, pH 7.5) overnight at room temperature. Low‐binding tubes were used for the incubation. For the ELISA assay, 200 μl of each incubation sample was added per each well containing 50 μl of assay diluent RD1W.
Matrigel‐based ECM binding assays
Matrigel‐matrix‐coated plates (BD Biosciences, cat. # 354607) were incubated with 100% SBlock (Pierce, cat. # 37538), for 10 min at room temperature. One hundred microlitres of varying amounts recombinant proteins diluted in 50% of Sblock in PBS was added and incubated at RT for 2 h. After washing four times with 400 μl of wash buffer (10% SBlock, 0.1% Tween‐20 in PBS), 100 μl of HRP‐conjugated goat anti‐human IgG‐Fc (1:10,000 dilution in 50% of SBlock in PBS; Jackson Immunoresearch, cat. # 109‐035‐098) was added in each well and the plate was incubated at RT for 2 h. The plate was washed four more times with 400 μl of wash buffer, before addition of 50 μl of TMB solution (Thermo Scientific, cat. # 34028) and incubation at RT for 30 min. The reaction was stopped with stopping solution (Sigma, cat. # S5814), and the absorbance at 450 nm was measured using an ELISA reader (Bio‐Rad).
Cell culture monolayer ECM binding assays
One million cells were plated per well (9.6 cm2/well) in a 6‐well plate, containing microscope cover slips, and cultured with or without doxycycline for 48 h. Subsequently, supernatant were collected for Western blot analysis, and cell monolayers were washed three times with PBS. After 10‐min fixation with 1% paraformaldehyde in PBS, cell monolayers were incubated with a goat anti‐human Fc IgG1‐cy3‐conjugated antibody (1 in 500 in PBS; Jackson Immunoresearch, cat. # 115‐165‐008) at 4°C overnight. Cover slips were mounted and visualized as described below.
HUVEC proliferation assay
Primary human umbilical vein endothelial cells (HUVEC‐2, BD Biosciences, cat. # 354151) were cultured in 96‐well plates (5,000 cells per well) in 100 μl of basic EBM‐2 media (Lonza, cat. # CC‐3156) per well, along with various amounts of traps (0–218 nM) and VEGF165 (1 nM; R&D Systems, cat. # 293‐VE‐010). After 72 h, 10 μl of AlamarBlue® (Invitrogen, cat. # DAL1025) was added per well and incubated for 18 h. Fluorescence was measured on a plate reader (560EX nm/590EM nm filter settings).
To test the affinity of recombinant traps to heparin, 1 μg of trap was mixed with 20 μl of immobilized heparin‐Sepharose beads (Pierce, cat. # 20207) in 400 μl of 0.05 M phosphate buffer and 0.25 M NaCl and incubated at room temperature for 2 h. Elution was performed with 500 μl of 0.05 M phosphate buffer with NaCl concentrations of 0.25, 0.5, 1.0 and 1.5 and 2.0 M. Ten microlitres from each elution fraction, along with proper controls, was resolved by SDS–PAGE gel, and Western blot was done using a goat anti‐human Fc IgG1‐HRP‐conjugated antibody.
To assess the pharmacokinetic profile, traps (4 mg/kg) were injected subcutaneously in 8‐week‐old male C57BL/6J mice (average 25 g body weight), and blood was collected 1, 2, 4, 6, 24, 48, 96 and 168 h post‐injection. The serum levels of the traps were measured using ELISA.
For the xenograft assays, we used 8‐ to 12‐week‐old male nude mice for the PC‐3 prostate and A‐673 rhabdomyosarcoma cancer lines and 12‐ to 16‐week‐old female nude mice for the HT‐29 colon cancer line. Briefly, cells were trypsinized and suspended into serum‐free media containing 33% Matrigel (BD Biosciences, cat. # 356234). Mice were anesthetized using isoflurane and injected subcutaneously with 5 × 106 transgenic cancer cells or 1 × 106 wild‐type cancer cells, per site in both dorsal flanks (150 μl per site). The mice were housed a specific‐pathogen‐free (SPF) condition, and the care of the animals was in accordance with institutional guidelines (Toronto Centre for Phenogenomics, Toronto, ON, Canada). Doxycycline was administered using doxycycline‐containing pellets (0.625 g/kg; Harlan Laboratories, cat. # TD.01306). EGFP expression was monitored using a 388‐nm wavelength (blue) LED light source, the appropriate cut‐off filters and a conventional camera. Tumour size was monitored using callipers and the volume was calculated using the formula V = (L × W × H)π/6.
Wound healing assay
Wounding was performed and healing assessed as previously described (Niethammer et al, 2002; Seavey et al, 2009). Wounds were created in the dorsal area, close to the neck, of male nude mice (8–12 weeks of age) bearing bilateral subcutaneous tumours in the flank areas. For these studies, we used the A‐673 cell lines stably transfected with traps. Once the combined size of both tumours was approximately 1.5 cm3, doxycycline was administered for 8 days in order to induce the expression of traps. At day 8, mice were anesthetized using isoflurane, and full‐thickness wounds were created using 6‐mm sterile Biopsy Punch (Miltex, cat. # MIL33‐36) at the nape area. One drop of liquid bandage was applied to the wound. Post‐operatively, liquid Metacam (meloxicam) was given subcutaneously for 3 days for pain management, and Novo‐Trimel (trimethoprim and sulfamethoxazole) was given per os for 5 days in order to avoid any bacterial infections. The wound was photographed every other day for a period of 12–14 days. Blood samples were also collected before doxycycline administration, at day 8 and at the end of the study, as described below. At the end of the study (day 12–14), the mice were euthanized and the wound area was dissected and further analysed using haematoxylin and eosin (H&E) staining.
Blood samples were collected in Microtainer plasma‐separating tubes (Becton Dickinson, cat. # 365985) from retro‐orbital sinus during the study and by cardiac puncture of mice under anaesthesia with isoflurane at the end of the study. Samples were centrifuged, aliquoted and stored at −70°C until assayed. All capillary tubing, syringes and needles used for bleeding were heparin‐coated to avoid clotting.
Tumour hypoxia was detected using a Hypoxyprobe Plus Kit (Natural Pharmacia International Inc.) following the manufacturer's instructions. Briefly, 60 mg/kg of pimonidazole hydrochloride was injected i.p. into tumour‐bearing mice 1.5 h before tissues were harvested. Pimonidazole adducts were detected as described below.
Blood vessels were detected by staining for endothelial cells using hamster anti‐CD31 (PECAM) monoclonal antibody (1:200; Millipore, cat. # MAB1398Z). Vascular basements were examined with rabbit polyclonal antibody against type‐IV collagen (1:5000; Cosmo Bio CO, cat. # LSL‐LB‐1403‐EX). Pimonidazole adducts were detected using hypoxyprobe rabbit antisera (1:50; Natural Pharmacia International Inc., cat. # PAB2627). Proliferation was assessed by phospho‐histone H3 (PH3) antibody (1:200; Upstate, cat. # 06‐570). Secondary antibodies were anti‐hamster Cy‐3 conjugated for CD31 (1:500), anti‐rabbit Cy‐5 conjugated for collagen, PH3 and pimonidazole (1:500).
Tumour samples were frozen using O.C.T. compound (Sakura Tissue‐Tek, cat. # 4583) immediately after dissection and stored at −70°C. Cryostat sections were cut into fragments of 18 μm thickness and stored at −70°C until assayed. For immunostaining, sections were fixed with cold methanol for 10 min at RT and left to dry. Tracheas were fixed with 1% paraformaldehyde in PBS for 1 h at RT. Specimens were incubated in 5% goat serum in PBS containing 0.3% Triton X‐100 (PBS‐T) for 1 h at RT in order to permeabilize them and block any non‐specific antibody binding. Primary antibodies were incubated at 4°C overnight, and secondary antibodies for 2 h at RT.
Image acquisition and quantification
Sections were visualized using Zeiss LSM510 Meta two‐photon microscope. Images were captured with a Zeiss Axiocam camera connected to the microscope using AxionVision software. Images of at least six fields were taken for each tumour section (from at least three different tumours), while images of 8–12 fields were taken for each trachea specimen. Images were analysed and quantified using ImageJ (http://rsbweb.nih.gov/ij/).
Tumour samples were fixed in 10% neutral‐buffered formalin prior to paraffin embedding and subsequent H&E staining. Digital slides were created from microscope slides using ScanScope CS, and images were acquired using ImageScope viewing software (Aperio Technologies, Inc.).
Injections were performed into 6‐ to 8‐week‐old adult mice (CD1) using a 34‐gauge beveled needle attached to the Nanofil submicrolitre injection system (World Precision Instruments, Sarasota, FL, USA) as previously described (Ballios et al, 2010). Injections were made into the vitreous fluid by injecting through the sclera 1–2 mm below the limbus. Procedures were performed using a Möller Hi‐R 900C surgical microscope (Innova Medical Ophthalmics, Inc., Toronto, ON, Canada).
Oxygen‐induced retinopathy mouse model
Oxygen‐induced retinopathy experiments were performed as previously described (Smith et al, 1994). Briefly, P7 pups were exposed to hyperoxia (75% oxygen) for 5 days in an enclosed chamber. P12 pups were then returned to room air (~20% oxygen) and injected with traps (2.5 μg) or saline, and the retinas were examined at either P17 or P21 stages. Injections were performed by injecting 0.5 μl of each reagent using a 33‐gauge Hamilton syringe. Mouse eyes were enucleated and flat mounts were performed as previously described (Dorrell et al, 2002). The eyes were immunolabelled with GS Lectin (Life Invitrogen, cat. # I32450) overnight and washed vigorously before mounting in antifade media. Images were captured using a Zeiss 700 confocal microscope, and the images were processed with ImageJ (NIH) and Adobe Photoshop software (Adobe). The effects were quantified using published protocols as previously described to quantify the volume of the neovascular tufts and vaso‐obliteration (Banin et al, 2006).
ERG measurements and electron microscopy
Electroretinography measurements and electron microscopy were performed as previously described (Dorrell et al, 2009).
Quantification of vascular permeability
Vascular permeability was determined using slightly modified published protocols (Scheppke et al, 2008). P12 OIR pups were injected intravitreally with VEGF‐traps or PBS. P17 OIR mouse pups were injected (intramuscular) with 100 μl Evans Blue dye (Sigma, cat. # E2129). The eyes were enucleated 4 h later and the retinas were isolated. Several μl of blood was extracted using cardiac punction. Evans Blue dye that accumulated in the retinas was extracted and solubilized in formamide (0.2 ml per retina) at 78°C overnight. The solutions were ultracentrifuged at 4°C at 128,000 g for 45 min to pellet cellular debris. Evans Blue dye in the supernatant was detected with a spectrophotometer at 620 nm (blue signal) and 740 nm (for background subtraction). Non‐solubilized blood samples were also spun for 15 min at 3,550 g at 25°C and diluted 1:1,000 for spectrometry. Leakage was calculated with the following equation: [Evans Blue concentration (mg/ml)]/[blood Evans Blue concentration (mg/ml) × circulation time (h)].
Results are reported as mean ± s.e.m. Statistical significance of differences was assessed by one‐way ANOVA followed by the Newman–Keul test, using PRISM, ver. 5.0 (Graph‐Pad, San Diego, CA, USA).
The paper explained
Excessive production of vascular endothelial growth factor (VEGF‐A) leads to abnormal retinal angiogenesis in patients with DR and in newborns suffering of ROP. If left untreated, both conditions often lead to blindness. Current clinical therapies aimed at inhibiting VEGF have shown some success. However, systemic VEGF inhibition has detrimental side effects, such as kidney toxicity, impaired wound healing, highly elevated blood pressure, gastrointestinal perforation, haemorrhage, thrombosis, reversible posterior leukoencephalopathy, cardiac impairment and endocrine dysfunction. Infants—in which organogenesis is still active—are even more critically affected.
In this study, we developed a novel inhibitor of VEGF, referred to as VEGF Sticky‐trap. It comprised of three components: (a) the VEGF‐trap region (b) a modified Fc region that ensures short serum half‐life and (c) a HBD that allows Sticky‐trap to remain at the place of injection. Using a mouse model of DR and ROP, we proved Sticky‐trap to be highly effective. In contrast to currently available treatments, once injected intravitreally, Sticky‐trap does not exit into the circulation. Instead, it binds the ECM and remains in the eye for a prolonged period of time. Based on these findings, we investigated and showed that local Sticky‐trap treatments do not affect distant wound healing, neither do they compromise the kidney vasculature.
This is the first report of a local acting angiogenesis inhibitor that can immediately be translated into the clinic as an effective, and safe treatment of DR and ROP. This is of particular importance for elderly patients and pre‐mature infants requiring VEGF suppression and who are especially sensitive to potential side effects of such therapies.
IPM designed the traps and the overall study; IPM, PDW, BGB, SMH, HS designed experiments; IPM, PDW, SH, ACG, TK, BGB, ZL, CMW, PZ, EA, LD, VM, TB, SMH, HS conducted experiments; IPM, PDW, SH, TK collected data; IPM, PDW, TK performed data analysis; MLIA, JMR, DvdK, MF supervised the study; AN conceived and supervised the study; IPM and AN wrote the manuscript; all authors discussed the results and commented on the manuscript.
Conflict of interest
The authors declare that they have no conflict of interest.
Supplementary Figure S1
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Supplementary Table S1
We thankfully acknowledge Kristina Nagy for her invaluable intellectual input and for critical reading of the manuscript. We would like to thank the staff of the Toronto Centre for Phenogenomics (TCP), especially Angela Walker, for excellent technical support. We also thank Dr Malcom Wood for his excellent assistance with the EM studies, as well as Stephen Bravo and Daniel Feitelberg for their help with immunostaining analysis. This work was supported by grants award to P.W. by the National Eye Institute; J.M.R. by CIHR; to D.v.d.K. by CIHR, the Foundation Fighting Blindness and the McEwen Centre for Regenerative Medicine; to M.F. by the National Eye Institute and the Lowy Medical Research Institute MacTel Program; and to A.N. by the McEwen Centre for Regenerative Medicine, Robert and Sheryl McEwen, and CCSRI.
FundingNational Eye Institute
See also: DM Favara & AL Harris (May 2014)
This is an open access article under the terms of the Creative Commons Attribution 4.0 License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.
- © 2014 The Authors. Published under the terms of the CC BY 4.0 license

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Credits: hmdPL on YouTube I did it, I post it on the video section! Yeah!!!
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Hameron dreams come true in this chỉnh sửa - the lines from Act Your Age are tweaked so that House tells Cameron he has a crush on her and he's out of his mind about her. Awesome.
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PURPOSE: To establish Articles of Cooperation with the Residence Hall Association (RHA).
WHEREAS, RHA governs through the educational and social programming and policy-making decisions for the representation of students residing in the residence halls, and
WHEREAS, RHA also facilitates communications between the residents and the Department of Residential Life, university administration, and other organizations, and
WHEREAS, MSA has a responsibility to ensure that the voice of the student body is heard in all settings and that the rights of students are protected, and
WHEREAS, it is in the best interest of both MSA and RHA to have an established relationship because it ensures MSA and RHA’s ability to meet the needs of students, and
WHEREAS, the proposed Articles of Cooperation will help both organizations achieve their goals, and
WHEREAS, the proposed Articles of Cooperation will ensure consistency in the relationship between MSA and RHA.
THEREFORE, let the proposed Articles of Cooperation between MSA and RHA be enacted and entered into MSA’s bylaws and the appropriate section of RHA’s bylaws upon passage by both MSA and RHA.
6.90 THE RESIDENCE HALL ASSOICIATION (RHA)
A. INTER-GOVERNMENT COMMUNICATION –
(1) EXECUTIVE AND LEGISLATIVE OFFICERS: To ensure communication between the Missouri Students Association (MSA) and the Residence Hall Association (RHA), the President of RHA, the President of MSA, and the Speaker of MSA shall meet at least twice during both the fall and winter semesters to discuss planned referenda by either government, position statements, policy decisions, inter-organizational workings, and the composition of internal and external committees, or any other business. In addition to these meetings, they may meet at any time deemed necessary. By-laws of each organization shall have specific language requiring their officers to attend these meetings.
(2) MINUTES AND LEGISLATION: MSA and RHA shall send copies of the minutes, programming events, and passed legislation of their senate meetings to the presiding officer of the other legislative body. MSA and RHA shall send copies of their agendas for Senate and Congress meetings respectively within a week of the meetings. The Director of the Department of Student Activities, Student Services, or Department of Student Communications shall send the President of RHA a list of events, plans, and activities they will put on monthly.
(1) The RHA President shall have a seat reserved on the MSA Senate Cabinet.
(2) MSA and RHA shall also have the ability to appoint liaisons to each respective policy making bodies as deemed appropriate by the executive and legislative officers of both respective organizations.
(3) A permanent liaison position for RHA Congress shall be in place for an MSA representative assigned by the President of MSA.
C. RATIFICATION AND CHANGE
(1) RATIFICATION AND REVIEW: Upon receiving a two-thirds vote in both the MSA Senate and RHA Congress, these Articles of Cooperation shall be adopted immediately and will be reviewed for accuracy and relevancy at least every three years or as deemed necessary by the executive and legislative officers of either respective organization.
(2) AMENDMENT PROCEDURE: To change the Articles of Cooperation, both the MSA Senate and the RHA Congress must ratify the proposed change by a two-thirds majority vote.
(3) WITHDRAWAL PROCEDURE: Either MSA or RHA may withdraw from these Articles of Cooperation at any time by a two-thirds majority vote by their respective legislative bodies.
Corey Gibson Greg Davis Jonathan Mays
Operations Chair President Speaker
MSA RHA MSA
Rachel Anderson Robb Loran Nick Trusty
President Director of the DSS Director of the DSA
MSA MSA MSA
Director of the DSC


Thursday, April 30, 2009
Where did you grow up? Is your family musical or artistic?
I grew up in Louisville. My mother is a painter and my father is a writer and a songwriter/musician.
What is your musical background / training?
I started playing violin in the third grade and played through high school. I also sang in a church choir for a few years during elementary school, but didn't write songs until I began playing guitar. I taught myself guitar. Three years ago I decided to try playing bass, as well.
Who are your musical favorites/inspirations?
I was deeply inspired by all the local bands and touring indie rock bands I saw as a young teenager in the early '90s, in particular - Rodan, Ruby Falls, Drinking Woman, Crain, Evergreen, Unwound, Sleater-Kinney, Come, Versus, etc. Some of my current favorites are Neko Case, TV on the Radio, Shannon Wright and Mirah. I also love a lot of music made by women from the past like Loretta Lynn, Patsy Cline, Kitty Wells, Janis Joplin, Billie Holiday, etc.
What bands/projects are you currently involved with? How did you get involved with them?
I play guitar and sing with Second Story Man. I've been playing music with bandmate Evan Bailey since I was sixteen years old and we were in a band together called Itch House. A couple years later, Evan introduced me to Jeremy Irvin and the three of us started Second Story Man. We've been together ever since. I also play bass and sing in Minnow. I had never played bass before and when Rob Pennington asked me to play with his wife Becca and their friend Doug Maxson, I jumped at the opportunity to work in a totally different musical context.
What do you hope to achieve with music?
I want to continue to be a part of the creation of thoughtful and exciting music - and to have my bands' music be available to others to hear. I love collaborating with others and seeing song ideas transform into something beyond where I ever expected them to go because of the input of others. Of course I love performing live, too. I guess I just want to be able to continue doing this as long as I can.
at 7:45:00 AM


text:
Whether or not our education system is relevant to preparing citizens for a rightful role and contribution sparks a perennial debate.
The world may be on different pages but there is fair agreement that education systems are not addressing what national and global societies need.
Because of examinations teaching tends to underscore one domain of learning — the cognitive domain — effectively reducing all learning to processes of drilling and cramming for a good pass.
Good students have been defined by their mastery of all manner of content and by quality of passes in tests and examinations. Teachers and parents alike celebrate children who obtain the best grades in total disregard of who the children are as social and political animals.
Parents and teachers spend extra time and resources to support additional classes; they organise highly competitive cluster mock examinations all in a bid to improve learners’ grades in percentage points.
Perfectly fine I would say. This is what the whole world is doing much worse in the countries of Asia and East Asia where education is a fierce contest. But honestly, this is not education. That’s not in keeping with the purpose of education.
Limiting teaching and the learning experience to passing one-time examinations reduces learning to a series of rote contests measured by a mechanical testing system which neither prepares the individual for real life experiences nor builds in the quest for life-long learning.
Certainly, such drill type rote learning is poison for societies like Malawi subjugated by a dangerous decline in values and yet increasingly beset with complex social, economic and political problems.
You see, passing tests and examinations is not the best predictor of success in the life of the children that parents, teachers and leaders are entrusted with. Yes, children must develop and be challenged cognitively, but there is more to life than ‘brain intelligence’
I am sure it’s perfectly fine to ‘manufacture the geniuses’ who will develop the technologies that our world needs — to manufacture the work horses of technological innovation. Yet we must also admit that this is not how the greater social-political world is designed and that’s not how the world should define people development.
We know all too well how brilliant people have failed to lead people and the world. The world knows that the best scientists have not created the most useful technologies for human kind — quite the contrary in many cases — nor have the best known political scientists created the peace the world so desperately needs.
That’s why the richest people are not necessarily the most educated nor were they the cleverest in school. Some of the best businesspeople studied no business management and the best men or women of God never studied complex theology, let alone obtained lofty degrees.
Yes, people have to learn and train their brains. But to me personal attitudes, values and principles such as perseverance, reliability, honesty or hard work are better predictors of success in later life than grades.
Simple taken-for-granted skills like reading, writing, speech or use of technology are a great asset for people advancement much more than distinctions in the most difficult subjects on a national curriculum.
Practical capacities for team work; collaboration; networking and social interaction with people are a priceless bank for individual and group performance in everyday life, by far more valuable than a string of high sounding certificates.
Defining a good child purely on the basis of academic grades has enormous pitfalls. It sets standards which create stiff competition, sometimes in areas that children may not be good at. High contest learning environments make children who ‘under-perform to the standards set’ feel stupid and useless with a high likelihood for failure in real life.
Cognitively inclined definition of ‘good learners’ prevents children learning from the mistakes they make in school because by this model of education good students excel, not make mistakes. Those that make mistakes are not good!
And this has destroyed many a life.
Millions of children who would go on to become useful citizens and leaders in so many ways within their own societies ‘have been killed prematurely’ by systems of learning which glamorise grades, worship intelligence and celebrate only the highest academic passes.
But I argue that training the brain is only a small part of educating the whole person. Good education underscores the development of social capital; that is transfer of human values such as love of one another, honesty, keeping promises, keeping commitments, reliable performance of duty, enjoyment of a shared daily life and trust of both self and others.
Have you ever wondered why everybody is complaining about the performance of today’s products of the education system?
The answer lies in lack of discipline, responsibility and accountability. There is pervasive lack of honesty, avoidance of hard work and clamour for personal attention.
This is why we have today people who want money — lots of money — but they are unready to work hard. We have graduates today who will fight with every rule, disagree with every code of conduct because they worship freedom without responsibility.
Our education produces people who make light of very serious mistakes because they have limited sense of accountability and moral judgement. It churns out dangerously impressionable people who are neither self-conscious nor self-aware.
These are people who may be engaged in a great profession but do not have a clear sense of who they want to become in life.
This sounds pretty harsh, yet so real. And it starts with how we define teaching, learning and education. You see, education — western type or traditional — is intended to make good people better, bad people good and everybody a human being. It is failing miserably for so many unsuspecting users!
As we close: it is not education if it creates competition, aggression or social degeneration. It is certainly not if the result is senseless excitement about life without the exercise of basic human values.
A vibrant writer who gives a great insight on hot topics and issues

domain: sociology


Views: 61 Author: Site Editor Publish Time: 2017-11-02 Origin: Site
Methods for controlling the moisture content of pellet feed
There are two kinds of solutions for real-time on-line moisture detection and control technology of pellet feed. One is feedback control, which is online testing of the water content in the feed after cooling. The other is feedforward control, which is on-line testing the water content in the feed before conditioning. In comparison with the pre-set value, the two methods can control the moisture content of the adjustment steam and the spray adding of the granulator. Both schemes can maintain the moisture content of the finished product in the range of the predetermined standard values, which are conducive to the stability of the moisture content of the finished product. However, the feedforward method is better in controlling the final product than the feedback type, and the real-time performance is better.
Reasonable moisture content
The diversity of sources and variety of raw materials used in production results in the variability of water content in pellet feed products. After mixing, the moisture content of the semi-finished mixture should be controlled at 9% to 14%. The moisture content should be controlled at 12.5%, and should not exceed 13% at any time. Otherwise, it will be susceptible to moldy deterioration. After the conditioning, the moisture content of the feed should be controlled at 15% ~ 16%, so that the quality of the granulated feed produced is better, smoother, the powder rate is lower, and the moisture content of the finished product can meet standard requirements.
Water content control
In actual production, the water content should be adjusted according to the water content of the powder to achieve the ideal temperature and moisture content levels. The normal moisture content of the powder needs dry steam when conditioning, water vapor will have a negative effect on the quality of the adjustment and granulation. For feed that contains lower water content and needs more moisture, this can be done by reducing steam pressure, closing all or part of the trap, adding non-saturated steam or increasing the conditioning time to increase the water content in the feed to improve the moisture. For feed that has higher moisture content you should use high-pressure supersaturated steam so that the feed after conditioning has met the requirements for temperature and the moisture content. The cooling process is to reduce the pellet feed temperature so that it does not exceed room temperature by 3 to 5 ℃ and to remove the water content in the feed so that the water content of granular feed products meet the required standards. After cooling the pellet feed, temperature and moisture must meet the requirements in order to be packaged, otherwise, the finished product in the storage process becomes moldy more easily. For the smaller pellet feed, this should be used when the cooling air volume is smaller, the cooling time is shorter; and for the wetter or larger pellet feed, you should increase the air volume and extend the cooling time. For relatively dry and small feed particles, the amount of cooling air used should be smaller, the cooling time should be shorter and the wetter, larger feed particles should have their cooling air flow increased and their cooling time prolonged.
The addition of water
Normally the automatic water adding system is used which is suitable for adding water into the mixer. The water addition system is mainly composed of a stainless steel storage tank, a rust-proof solenoid valve, a water pump, a rust-proof flow meter and an intelligent flow meter, etc. The water spray and water spray delay time can be set on the intelligent flow meter. The system uses PLC automatic control technology with a high degree of automation so that the proportion of water adding is accurate, reliable to control, easy to operate and so on.
Pellet feed should go through high-precision moisture detection and control to help control the moisture content of the pellets, so as to achieve the maximum input-output ratio and the best product quality.


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Award for lung research
23 February 2005
A UCL academic accepted the first of three annual cheques for $96,000 at an award ceremony at the Rayne Institute in February 2005.
The ceremony was introduced by Professor Geoff Laurent, Director of the CRR, who talked about the centre's research into lung cancer, asthma and other respiratory diseases. Dr Janes' award was then presented by two representatives from Johnson & Johnson's Office of Science & Technology: Dr Joni Catalano-Sherman and Mr David Bowser. Johnson & Johnson awards around £2.5 million every year in Focused Giving grants, supporting seed grants as well as many 'fund-matching' programmes, where a department or research project needs to raise 50% of the necessary funds in order to secure government funding for the other 50%.
Dr Janes' study, 'Cell Therapy To Repair Injured Lung Parachyma by Genetically Modified Bone Marrow-Derived Stem Cells', will investigate whether transplanting certain stem cells found in bone marrow into the bloodstream can repair acute lung damage caused by illness or injury. Dr Janes said: "This award will enable me to carry out vital research work that may lead to real breakthroughs in the treatment of lung disease. I'd like to thank Johnson & Johnson for supporting this project."
Image: Dr Sam Janes and Mr David Bowser.
To find out more about the centre use the link below.

domain: biology


text:
- Harbor Freight Tools plans to move ahead with Cape Girardeau store (12/5/17)2
- Feds ask judge to impose $6.5 million punishment for Cape surgeon (12/7/17)9
- Business Notebook: Yule Log Cabin gets home feel honestly (12/4/17)
- Former Wimpy's Drive-In owner Freeman Lewis dies (12/9/17)2
- Makeover at the movies: Transformation complete inside Cape theater (12/8/17)4
- Sugarfire Cape barbecue restaurant to open June 2018 (12/7/17)
- Rep. Lichtenegger proposes change to term limits (12/4/17)7
- Fire displaces family of seven (12/5/17)1
- Buffalo Wild Wings moving to new location in March (12/2/17)2
- Fruitland Army veteran spends weeks helping in ravaged Puerto Rico (12/5/17)2
Pot affects gas tank reading
SANDY, Utah -- A Utah man took his newly acquired used sport utility vehicle to a mechanic to find out why the gas gauge always read half-full.
The mechanic looked inside the gas tank and found about 35 pounds of marijuana in plastic-wrap packages. They filled about half of the tank's volume. Police estimate the pot is worth about $35,000.
The Nissan Armada has had several owners and was once a rental car. Sandy police are trying to figure out who stashed the pot but say the current owner is not a suspect.
Investigators in the town south of Salt Lake City say the drug packs could have been in the tank for months.

domain: news


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Burnsville MN, United States1 month ago on ebay.com
For sale $119
...problems, please contact us. Please Note: All included items are shown in the pictures. Accord Trombone ... Features: Great for a student! What's Included: Trombone Case Mouthpiece What's Not ...
Shingle Springs 95682, CA, United States1 month ago on ebay.com
For sale $89
...to the bottom of this page to take a better look. This nice vintage trombone from C.G. Conn dates to 1920 according to the serial number lists I‘ve ...looks to mean that it‘s a medium bore and a high pitch model. The trombone sports a beautiful bit of stenciling, and is in nice overall condition, although it ...
Jersey City 07395, NJ, United States2 weeks ago on ebay.com
For sale $87
Jack teagarden Meet Me Where They Play The Blues Period spl- ... item: Meet Me Where They Play The Blues, Jack Teagarden, 1955, on Period spl-1106, Jazz ten inch. ...Condition. Clean surface areas with vibrant color and side seam wear. grading: All grades vetted by two graders. All records graded according to the protocol found in the 2006 Goldmine ... ...
Huntington Beach CA, United States3 weeks ago on ebay.com
For sale $1,900
...and had been previously owned. The bachloyalist website database places its manufacture date at about ... according to its serial number, which jives with a copy of an instruction sheet for ...
Leland 49654, MI, United States1 week ago on ebay.com
For sale $32
...I could not find any markings. It is in nice shape. It has a trombone clasp and according to many websites that would date it to the early part of ...
Baltimore 21278, MD, United States2 weeks ago on ebay.com
For sale $15
According ebay jewelry pro this is Italian style from 1960s , unfortunately I cannot read maker stamp, pin are proxy 1,5 " and gold fill is lost, description on picture, please check my collection maybe you find something else
Chattanooga TN, United States1 month ago on ebay.com
For sale $1,199
...and very light tarnish overall. The slide moves fast and freely. The serial number (1301) according to the Bach Loyalist website dates this instrument to approx. ... ? Please scroll down and ...
Greenville, SC, United States1 week ago on ebay.com
For sale $24
...Lot in good condition, in original vintage boxes. Includes (according to my research, please verify instruments by pictures) Cello, attached to sheet of "The Messiah" music (3)Trumpet (3)Trombone (3)French Horn (3)Tuba (3)Harp (2)Each ornament length varies from 2.5 - 4.5 inches. Boxes measure just under 7 inches square. These are made of lightweight ...
Houston 77056, TX, United States2 weeks ago on ebay.com
For sale $80
For sale is a used Greg Black 5gs Large shank trombone mouthpiece. This mouthpiece is in the regular weight. The specs, according to Greg Black's website, are as follows: Cup Diameter: 25.8mm, 1.016” Cup Depth: Medium, Bore - .281” Rim Width: Medium Wide This was purchased new in 2007, and has obviously seen some wear. Despite its ...
Johnstown PA, United States4 weeks ago on ebay.com
For sale Auction
Really cool vintage Holton "Collegiate" trombone estate find. I'm a guitar guy, so my knowledge on this instrument is extremely limited. According to research the serial number 177076 indicate is was made mid/late 1947. Appears to be all original and in relatively good condition for its age. It has dents and dings, nothing that seems to impair ...
Phoenix AZ, United States3 weeks ago on spreadmyad.com
For sale $600
Accord trombone with f attachment. Classic brass, silver, and rose colors. Kept in great condition, just don't play anymore. 2 very small dings in the bell, they do ...make it just a straight mute. There is a marching band lyre in the case but it doesn't fit this trombone. Local, cash only, email with a reasonable offer. ...
Ashland 97520, OR, United States1 month ago on ebay.com
For sale $1,499
Pre - Owned Conn 88H Trombone with F attachment. Perfect playing condition and good cosmetic condition. Only one major dent (pictured) and wear and tear of areas with frequent skin ...is excellent. Purchase comes with 6.5 al mouthpiece and case. Serial number is: 5 577399. According to Conn's serial number chart this places it somewhere post 2000's ... ...
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domain: hobby


February 12, 2018
The Olympics Drone Game is Strong
In case you missed the opening ceremony of the Olympics last week (and not many of us did with 28.3 million viewers tuning in) there was an outstanding light display featuring drones.
From The Engineer: “A light show put on by a coordinated swarm of 1218 Intel Shooting Star drones formed an animated display, setting a Guinness world record for “the most unmanned aerial vehicles airborne simultaneously”. The swarm will also be in action throughout the games during medal ceremonies, which will include animations of the appropriate sports and Olympic-related logos including the familiar linked rings.”
This means we can look forward to more awe-inspiring moments brought to us by the beautiful light displays of the Olympic drones.


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When it comes to taking stock, festivals often put forward high audience attendance figures as irrefutable proof of success. That’s fine. In these times of crisis, lack of public support usually has lethal consequences. But it is also worth considering carefully other indicators of good artistic health
Hiring the undisputed stars of the classical music world such as Plácido Domingo, Juan Diego Flórez, Daniel Barenboim and Lorin Maazel is all very well; they provide glamour and almost always guarantee a full house, though bearing in mind the exorbitant fees they are paid it is never clear whether their concerts are profitable.
We are not going to discuss the rules of the market here. In Spain, hiring with an open wallet has been, and continues to be, the main impulse behind the programming at many theatres and festivals.
This is why it is so important to look at other indicators.
It would be best to put the brakes on those festivals which, with public money, openly apply a arts policy based on the cult of the big stars, always hiring the diva in vogue, the pianist who’s biggest in the media or the flashiest orchestra.
One is the production itself, which does not always depend on the artists, orchestras or directors on tour, with closed programmes which often consist of the same old worn-out works so as not to frighten away the public. Take it (if you can afford it) or leave it.
Some festivals opt for higher-risk formulas: they commission works, promote co-productions, design exportable programmes, revive musical heritage, support local musicians and seek a balance between the most innovative proposals and the better-known repertoire, which must never be ignored because it is basic to building up interest.
Though this makes it more difficult to fill the halls, the effort does not fall on deaf ears, because it helps to create artistic fabric from source
, much needed to avoid being regarded on the international circuit as the last haven of profligate concert programming.
It would be best to put the brakes on those festivals which, with public money, openly apply a arts policy based on the cult of the big stars, always hiring the diva in vogue, the pianist who’s biggest in the media or the flashiest orchestra. If the initiative is taken from private sector, we think it’s great, because famous artists are always the icing on the musical cake. But using public resources, the objectives have to be different.


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Through the years we have seen all sorts of creative designs that are made of material we often take for granted. We have seen sculptures made from ‘trash,’ we have seen other artistic approaches to other items that we discard daily. Today we get a look at something unique from Zimbabwe-based artist Zayd Menk. We get a look at Zayd Menk’s Scale Model Of Manhattan, which is made from recycled motherboards, hot glue sticks, various CPU bits and more.
The creation is seen in 0.0635:100 scale, which was done for his school art project. To be exact, the piece features 263 sticks of hot glue, 27 motherboards, 10 CRT monitor motherboards, 18 sticks of RAM, 15 batteries, 12 Nokia E-series phones, 4 watches, 3 hard drives, 2 telephones and many other pieces of recycled tech hardware. Each building, including the Bank of America tower, are seen, with the Empire State Building even lighting up with LED’s.
Check out the design in the images below. Keep it locked for more outstanding creations and artistic designs coming soon.


I recently re-enabled feature on a ASP.NET site. This feature (a set of pages) had been running OK about six months ago for an initial pilot project but was disabled until now until a decision was made to develop the feature fully. In intervening time the web site had other modification and had been rebuilt (it was targeted at .NET 3.5) without issue.
When I re-enabled the feature (renamed a .ASPX file) I got the error
Could not load file or assembly 'System.Web.Abstractions, Version=188.8.131.52, Culture=neutral, PublicKeyToken=31bf3856ad364e35' or one of its dependencies. The system cannot find the file specified.
Now this surprised me, I had not change the .NET version targeting or changed development environment. In the end I fixed it by copying up the missing DLL to the bin directory. I had tried adding the following to the web.config to no effect
<assemblyIdentity name="System.Web.Abstractions" publicKeyToken="31BF3856AD364E35"/>
<bindingRedirect oldVersion="0.0.0.0-184.108.40.206" newVersion="0.0.0.0"/>
On think a bit more I think the issue must have been caused by:
- I may have upgraded to .NET to 3.5. SP1 in the time frame (but I thought I did it earlier)
- I have installed MVC on my development PC, and most other posts that mention this issue also mention MVC, usually beta versions.
EIther way it is fixed now


Search term must have more than 2 characters.
Expanded metal is produced by no-waste technology, where the material is suitable for production cuts and stretches. For the production of expanded metal can therefore be used solely tensile materials such as steel DC04, stainless steel, aluminum and other metals. The production of expanded metal can be realised from both sheets and coils.
The most frequent is flattening of expanded metal with square and circular hole. By flattening expanded metal loses its three-dimensional shape and provide a flat and smooth surface. It's up to you and your needs to use the rolled metal. It's used, for example, like a covers, fillers and air conditioning components.


TR swimmers compete at region
By Zakary Rodriguez | Advertising Manager |
Over Friday, Jan. 31, and Saturday, Feb. 1 the Roosevelt swim team made their first regionals debut in three years at Bill Walker Natatorium. Swimming for a chance to go to the state meet, was the boys relay team along with Matthew Swanson.
As opposed to districts, at the regional meet, there would be A-Finals and B Finals, meaning the top 16 swimmers from preliminaries would move to the finals. Once in the finals, only the fastest two swimmers from every event would be selected to automatically move to the state meet. After that, the top 8 swimmers across the entire state of Texas, from their respective event, will be selected to move on.
First to swim in preliminaries was Swanson, swimming his 100 backstroke. He swam at a time of 55.48 seconds, putting him easily into the top 16 fastest swimmers in the event and into the B-Finals. The boys relay team was up next, and they swam in the last heat of the last event of the day on Friday. Unfortunately, they barely missed the finals, placing 17 with a time of 3:42.66, meaning Swanson would be the only one swimming in finals the following day.
“Going into the finals, I wanted to get a time drop,” Swanson said. “I wanted to finish first in B-finals.”
Swanson got his diving block and in the water. It was time for him to show out and swim faster than he had swam before. As Swanson started his third lap, he had a narrow lead of less than one second, and he maintained that lead into the start of his fourth lap. Finally, Swanson swam into the wall and stopped his time. “54.58” was prominently displayed next to “Swanson” and the number one. Swanson had finished in first place in the B-finals.
“I looked up and saw that one next to my name. I just started bawling,” Swanson said. “I felt like I owned the water and had to show it, show everyone that that race was mine.”
After getting out of the water, Seth Gutierrez rushed to Swanson and embraced him. Not only was Swanson the first Roosevelt swimmer to swim individually at regionals in three years, but he swam in the finals and dominated his heat to finish first. Following the B-finals, the A-finals heat took place and unfortunately, Swanson was moved to ninth place. Despite being moved to ninth place, and being stopped short of going to state, Swanson wasn’t short of accolades. He finished with the third fastest time for the 100 backstroke in the district and the sixth fastest time in the region. As for what’s next for Swanson, he has his eyes set on swimming at the collegiate level.
“Swimming has been such a big part of my life,” Swanson said. “I find a passion in it and can’t imagine not having it being a part of my life.”


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Friday, January 8, 2010
Barner Exclusive Post Cards - NY's Psychadelic Reservoir
We're now starting to carry a line of exclusive post cards. Mostly done by our own staff.
A sample follows. It's "New York's Psychadelic Reservoir." The shot was taken by David (the owner) and then edited to give it the counter culture look.
The resevoir in the picture is really the Ashokan Reservoir, which is part of New York City's water system -- located in Ulster County New York (just as we are).

domain: geography