text:
Skip to comments.Anti-French feelings run high in most Gallic US city
Posted on 05/01/2003 5:47:59 PM PDT by MadIvan
The big chill freezing relations between the US and France has reached America's most Gallic city, New Orleans, otherwise known as the Big Easy.
A statue of Joan of Arc gazes at the Mississippi and the French Quarter is still festooned with tricolours and fleur de lys flags but the malaise afflicting the two nations has even spread to the bicentennial celebration of their common past.
A lavish exhibition marking one of the most important events in their joint history, the Louisiana Purchase, is drawing large crowds but its organisers at one stage feared that bad feeling over Iraq would scupper the project.
Laura Bush, the president's wife and honorary head of the organising committee, stayed away from the opening gala and her greeting to visitors to the museum hosting the show is remarkable for its nuanced language.
The exhibition "offers great insights into the founding of our nation, the nature of leadership and the rich artistic dealings between America and France," the First Lady says, tactfully omitting any mention of France as the US's oldest ally.
"Jefferson's America and Napoleon's France" at the New Orleans Museum of Art commemorates the 200th anniversary of what is arguably the most lucrative real estate bargain in history, at least for the buyer. The infant American republic doubled its size thanks to the deal with France's then First Consul, snapping up the territory from the Mississippi to the Rocky Mountains for the knockdown price of four cents an acre
French copies of the purchase documents are some of the exhibition's highlights. But its organisers were afraid that these, and other French treasures, might not show up, victims of this spring's war of words between Washington and Paris over the Middle East.
To everyone's relief, all the promised items turned up, a victory hailed by some as proof of the two countries' enduring cultural ties overcoming petty political differences.
In fact, the show only underlines the historical contrast between the two nations, despite Marquis de Lafayette helping the colonies to win the American Revolution and the gift of the Statue of Liberty.
A sumptuous gilded throne used by Bonaparte stands beside an austere, high-backed leather armchair belonging to Jefferson, admittedly the most passionate Francophile of all US presidents. Among the ironies highlighted by the show was France's then status as the world's most powerful nation on land, one which celebrated a "cult of war" and far removed from the image of "cheese-eating surrender monkeys" coined by The Simpsons.
In another piquant parallel, Napoleon and his armies had just returned from a victorious military expedition to the Orient, to Egypt.
"After seeing all that, I understand that we have much less in common than I thought," said one visitor to the show, Cookie Sampson. "Napoleon must have been quite an egomaniac."
Another irony of the Louisiana Purchase was that the Creoles of New Orleans, the only sizeable town in the wilderness sold by the transaction, were decidedly ambivalent about the deal.
This week's 200th anniversary of the treaty's drafting was marked with a Mass in the cathedral but the real action was in the streets where the city held a traditional New Orleans funeral for the R&B guitarist, Earl King.
Despite its veneer of French influence, New Orleans seems to be part of the US mainstream in its attitude to the country itself.
Another local venue which celebrates a significant chapter in US-French history is the National D-Day Museum. New Orleans was the home of the Higgins boats which landed Allied troops on the beaches of Normandy. On the pavement outside, anti-French feeling was running predictably high.
"I have no use for them now," said Joe Thurman. "We protected their butt back then and now they fight against us tooth and toenail. And when we make a success of it in Iraq, now they want a piece of the action."
"Maybe they have something to offer with their food and wine but it's their principles - if they have any - and their leaders I can't stand."
Regards, Ivan
De Cajuns, dey no git no killed off in dey World War One un dey World War Two. Dey no get no emasculated. Dey no sip no cup of no bad communism. Dey families left France jus in time, no.
"Maybe they have something to offer with their food and wine but it's their principles - if they have any - and their leaders I can't stand."
Basically says it all.
Hardly "victorious." Nelson whipped the French fleet and Bonney barely escaped, abandoning his troops to deal with the plague and finally to surrender to the Turks.
Napoleaon's spinning of this adventure into a victory is surely one of history's notable con jobs.
What does France "owe" us? Nothing. They owe us nothing, zilch, zero, nada. Gratitude is a dog's disease from which nations never suffer in dealing with others. No nation- including this one- will ever operate it's foreign policy on past alliances or percieved debts. Any nation that does is a fool.
Is the current French policy of opposition to the current US foreign policy wrong? Perhaps. Is it based on it's own percieved interests (even if they are not the right interests)? Most likely.
But let's stop all this nonesence about the French being "ingrates" and "traitors". France is an independent nation that rightly or wrongly is acting in what she believes to be her interests. They "owe" us nothing. What if France supported us? Would it have mattered a bit in the outcome of this war? Why the hatred?
French Canadians are the principal reason that Canada has turned from Ally to Enemy.
True enough. But they're not in love with France. They just hate America a lot more (because the Anglosphere is more of a threat to them because France is too far away). If Canada was attached to France they would hate that. They just don't fit in, anywhere in the world. It is a sad story. They should just secede and make their own state and be happy there.
Matters not. Whether our liberation of France was incidental or not, the fact remains that we saved their behinds from the Nazi occupation and it took them just a few years to forget that. As to the French being mature, well, nations are judged by the actions of their leaders. The British Prime Minister was mature; the French PM was not. He pandered (that's a word indicating immaturity) to the whims of his leftist populace, while the British PM ignored a firstorm of criticism from his own leftists, and did the right thing.
"What does France "owe" us? Nothing. They owe us nothing, zilch, zero, nada. Gratitude is a dog's disease from which nations never suffer in dealing with others. No nation- including this one- will ever operate it's foreign policy on past alliances or percieved debts."
Yes, we got nada, but we deserved more. And plenty of nations have operated their foreign policy on past alliances. The British entered WWII based on an alliance with Poland, and WWI erupted when nation after nation fell in like dominoes according to their particular alliances. Britain entered this present war based on an alliance with the US, as did Australia. If you aren't buying any of this I would suggest that you go to the history books and read about the causes of any given war. Time after time wars occur because of one country's alliance with another.
"Lafayette, we are here." It was allegedly uttered by one of Pershing's subordinates when the doughboys arrived in France during WWI. The phrase alludes to the US returning a favor to France for honoring an alliance with the Colonists during the American Revolution. WE REMEMBERED WHAT WE OWED AFTER 135 YEARS HAD PASSED.
It has been 58 years since we liberated France. The French owed us. But they screwed us.
And you are correct on one point; no nation should feel indebted to another in perpetuity. However, as I pointed out, we remembered a debt we owed France for 135 years, over three lifetimes by the life-expectancy standards of that period, but they can't find it in themselves to be grateful for the WWII French liberation of 1945, less than one lifetime.
I can see where you're coming from; standards of international politics and loyalty are much different in this day, the world is a much more fast-paced place, and most importantly, modern governments are much less entrenched than the monarchies and oligarchies of old. Governments and government men come and go much more quickly than they used to. Today it's the classic case of What-Have-You-Done-For-Me-Lately? That said, I still believe France owes us and they should have supported us in this war. Because once again we are fighting to safeguard French society as well as American society. When a dirty suitcase goes off in Paris they'll be asking why someone didn't do anything to prevent it.
We ressurected Lafayette during WWI, not to compliment the French, but to persuade an Isolationist population to go into a war that really didn't involve us. Don't blame the French that we bought that hooey- they had nothing to do with it.
Nations act in their interests- period. Don't confuse propaganda with truth.
That's what I learned in school. Considering the French sold out the other French Canadians after some war, though (and sold Louisiana to America!), I doubt the people there are big fans of the French.
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Gene expression profiling in circulating endothelial cells from systemic sclerosis patients shows an altered control of apoptosis and angiogenesis that is modified by iloprost infusion
- Elisa Tinazzi†1,
- Marzia Dolcino†2,
- Antonio Puccetti2, 3Email author,
- Antonella Rigo4,
- Ruggero Beri1,
- Maria Teresa Valenti5,
- Roberto Corrocher1 and
- Claudio Lunardi1Email author
DOI: 10.1186/ar3069
© Tinazzi et al.; licensee BioMed Central Ltd. 2010
Received: 2 October 2009
Accepted: 7 July 2010
Published: 7 July 2010
Circulating endothelial cells are increased in patients affected by systemic sclerosis (SSc) and their number strongly correlates with vascular damage. The effects of iloprost in systemic sclerosis are only partially known. We aimed at studying the gene expression profile of circulating endothelial cells and the effects of iloprost infusion and gene expression in patients with systemic sclerosis.
We enrolled 50 patients affected by systemic sclerosis, 37 patients without and 13 patients with digital ulcers. Blood samples were collected from all patients before and 72 hours after either a single day or five days eight hours iloprost infusion. Blood samples were also collected from 50 sex- and age-matched healthy controls. Circulating endothelial cells and endothelial progenitors cells were detected in the peripheral blood of patients with systemic sclerosis by flow cytometry with a four-colour panel of antibodies. Statistical analysis was performed with the SPSS 16 statistical package.Circulating endothelial cells were then isolated from peripheral blood by immunomagnetic CD45 negative selection for the gene array study.
The number of both circulating endothelial cells and progenitors was significantly higher in patients affected by systemic sclerosis than in controls and among patients in those with digital ulcers than in patients without them. Circulating endothelial cells and progenitors number increased after iloprost infusion. Gene array analysis of endothelial cells showed a different transcriptional profile in patients compared to controls. Indeed, patients displayed an altered expression of genes involved in the control of apoptosis and angiogenesis. Iloprost infusion had a profound impact on endothelial cells gene expression since the treatment was able to modulate a very high number of transcripts.
We report here that circulating endothelial cells in patients with systemic sclerosis show an altered expression of genes involved in the control of apoptosis and angiogenesis. Moreover we describe that iloprost infusion has a strong effect on endothelial cells and progenitors since it is able to modulate both their number and their gene expression profile.
Systemic sclerosis (SSc) is a rare systemic autoimmune disease characterized by a preminent vascular endothelial dysfunction, by immunological abnormalities, and by excessive extracellular matrix accumulation leading to fibrosis of the skin and internal organs [1].
Endothelial cell (EC) damage defines a crucial step during the pathogenesis of vascular disorders since its injury leads to the loss of the anti-thrombotic properties of the vessels wall and rapidly enhances the number of damaged circulating endothelial cells (CECs). CECs are likely to represent those cells shed from vascular luminal endothelium as a result of insults in disease states [2]. They correlate with physiological markers of endothelial damage/dysfunction and they have been identified as a marker of vascular damage in a variety of disorders, including malignancy, cardiovascular diseases and autoimmune disorders such as systemic sclerosis (SSc) and vasculitides [3–9]. In healthy subjects, CECs are rarely detectable and probably represent the effect of natural endothelial cells turnover [10]. Therefore, complete regeneration of injured endothelium is of particular importance and may occur by migration and proliferation of surrounding mature endothelial cells.
CECs are terminally differentiated cells with a low proliferative potential and their capacity to substitute damaged endothelial cells and to create new vessels is relative limited [11]. Moreover accumulating evidence indicates that bone marrow-derived progenitor cells have the potential to differentiate into mature CECs and they have been termed endothelial progenitor cells (EPCs) [12–15]. As a consequence, EPCs can give an effective contribution to endothelization and neo-vascularization as shown by different studies in animal models and humans [10, 16–18].
Iloprost, a chemically stable prostacyclin analog [19], has been shown to induce long-term clinical improvement in various vascular conditions, including ischemic ulcers and pulmonary hypertension primary or secondary to SSc [20]. Iloprost infusion increases arteriolar distension and blood flow as a result of a vasodilating effect. The drug inhibits platelet activation and aggregation, and leukocyte activity [21]. Iloprost therapy has also a protective and reparatory effect by influencing EPCs [22]. The pharmacological effect on ECs modulates the adhesion molecules (E-selectin, ICAM-1, VCAM-1) expression and growth factors release, particularly VEGF and CTGF [23, 24].
The biological activity is mediated by a specific interaction with the I prostanoid (IP) membrane receptor [25], the same receptor as prostaglandin I2. Iloprost is a potent IP receptor agonist that activates adenylate cyclase, resulting in an acute increase in intracellular cyclic AMP. Such an increase in cAMP has profound effects on cellular function in platelets, endothelial cells, smooth muscle cells, fibroblasts, and in a number of different cell types involved in both innate and acquired immunity [23, 24, 26, 27]. We reasoned that such a strong impact on the function of different cell types and particularly of endothelial cells is the result of the modulation of several genes, an aspect that has never been looked at, in vivo.
We therefore aimed to evaluate the role played by iloprost infusion on circulant endothelial cell number and to clarify the molecular effects of the treatment in patients with SSc by studying CECs gene expression profiling before and after the treatment. Moreover, since digital ulcers are the key clinical manifestation of severe vascular damage, we considered a group of patients with skin ulcers separately, in order to evaluate whether in this subset of patients both the numbers and the gene expression of CECs is different from patients with a less severe vascular involvement.
Materials and methods
Patients and controls
We enrolled 50 patients affected by SSc: 37 without skin ulcers and 13 with digital ulcers; 18 patients were affected by the diffuse cutaneous form and 32 by the limited cutaneous form of the disease. Fifty age- and sex-matched healthy donors were enrolled as controls.
Blood samples collected in EDTA using a Vacutainer system (Becton Dickinson, NJ, USA) were drawn from patients before, and 72 hours after a single day or five days of being infused with iloprost for eight hours. In both cases the first 7 ml of blood was discarded and blood was processed within three hours after collection.
The study was approved by the local ethics committee (Comitato Etico per la Sperimentazione, Azienda Ospedaliera Universitaria di Verona) and informed written consent was obtained from all the participants to the study.
Detection of circulating endothelial cells and progenitors by flow-cytometry
CECs and EPCs were directly detected in whole peripheral blood in EDTA by lyse-no-wash method. Two hundred μL of each sample were incubated with a mixture of monoclonal antibodies for 20 minutes at room temperature after a 10-minute preincubation with a blocking serum. Fluorescein isothiocyanate (FITC)-conjugated anti-CD45, R-Phycoerythrin (PE)-conjugated anti-CD146, -CD31, -CD133 and -CD34 or isotype-matched control (IgG1), allophyco-cyanine (APC)-conjugated anti-CD3, -CD16, -CD19 and -CD33 were used. 7-Amino-actinomycin D (7-AAD) was added for dead cells exclusion. Samples were also stained with anti-CD45 FITC, anti-CD146, -CD31, -CD133, -CD34 PE, anti-CD106 or anti-VEGFR2 APC and peridin-chlorophill-protein (PerCP)-conjugated anti-CD3, -CD16, -CD19 and -CD33.
All reagents were purchased from Becton Dickinson (San Jose, CA, USA), except for anti-CD16 (Caltag, Burlingame, CA, USA), anti-CD106 (Biolegend, San Diego, CA, USA) and anti-VEGFR2-APC (R & D Systems, Minneapolis, MN, USA)
After labeling, red blood cells were lysed by incubation with 2 ml of ammonium chloride solution. The samples were analysed on a FACS Calibur cytometer (Becton Dickinson). The sensitivity of fluorescence detectors was set and monitored using Calibrite Beads (Becton Dickinson) according to the manufacturer's recommendations; 500.000 cells per sample were acquired in live gating. FlowJo 8.8.2 software (Tree Star, Ashland, OR, USA) was used to analyze data. A sequential Boolean gating strategy [28], designed to remove dead cells, platelet aggregates and debris, and to exclude CD45 + and CD3 +/CD19 +/CD16 +/CD33 + hematopoietic cells (dump channel), was used to accurately enumerate total CECs and EPCs [29]. The absolute number of CECs and EPCs was established in double platform, combining the flow-cytometrically assessed per cent cells and the white blood cells (WBC) count assessed using a haematology cell analyser [30].
Isolation of CECs and EPCs from peripheral blood
Twenty ml of blood obtained from all patients were added to 40 ml of phosphate buffered saline (PBS) solution. Mononuclear cells were isolated by density gradient centrifugation using Ficoll-Paque, washed twice with PBS and suspended in 80 μl of degassed separation buffer (PBS pH 7.2, 0.5% BSA, 2 mM EDTA) per 107 cells. Cells were incubated with 20 μl of anti-CD45 coated immunomagnetics micro-beads (Miltenyi Biotech, Auburn, CA, USA) for 15 minutes at 4°C with gentle rotation. Bead-bound cells were then separated from unbound cells by a magnetic sorting on LD columns (Miltenyi). CECs and EPCs were found in the fraction of unbound cells (CD45 low/negative). An aliquote of each fraction was analyzed by FACS using anti-CD45 FITC, anti-CD146/CD31/CD34/CD133 PE and 7-AAD to confirm the endothelial origin and quantify the possible lymphocyte contamination.
RNA extraction
We obtained CECs and EPCs from peripheral blood of 13 patients affected by SSc with digital ulcers and 37 patients without any skin ulcer before, and 72 hours after, iloprost infusion. Cells within each patient's group were counted and pooled together for RNA extraction. Each patient contributed to the pool with the same number of CECs. Control RNA was extracted from circulating endothelial cells (CECs + EPCs) obtained from 50 healthy donors.
Gene array analysis
Cell pellets of CECs and EPCs obtained from SSc patients, with and without digital ulcers, before and 72 hours after iloprost infusion both after one and five days of therapy (test samples) were used for gene array experiments. CECs and progenitors purified from healthy donors were used as control samples.
Isolation of total RNA, preparation of cRNA, hybridization, and scanning of probe arrays were performed according to the protocols of the manufacturer (Affymetrix, Santa Clara, CA, USA) by Cogentech (Consortium for Genomic Technologies c/o IFOM-IEO Campus, Milano, Italy). To ensure that a sufficient amount of cDNA was available, the RNA extracted from CECs was subjected to a two-cycle cDNA synthesis according to Affymetrix protocol. Biotinylated target cRNA was hybridized to the Human Genome U133A 2.0 GeneChip (Affymetrix). The Human Genome U133A GeneChip is a single array representing 14,500 well-characterized human genes and includes more than 22,000 probe sets and 500,000 distinct oligonucleotide features.
The different gene expression patterns were analyzed using Array Assist version 5.0 (Stratagene, La Jolla, CA, USA), which calculates background-adjusted, normalized, and log-transformed intensity values applying the PLIER algorithm [31–33].
The PLIER method uses quartile normalization and runs an optimization procedure which determines the best set of weights on the perfect match (PM) and mismatch (MM) for each probe pair. Finally, the normalized, background-corrected data were transformed to the log2 scale. A signal log2 ratio of 1.0 indicates an increase of the transcript level by two-fold change (2 F.C.) and -1.0 indicates a decrease by two-fold (-2 F.C.). A signal log2 ratio of zero would indicate no change.
Genes were selected for final consideration when their expression (F.C.) was at least two-fold different in the test sample versus the control sample. Experiments were performed in duplicates [34].
Selected genes were submitted to a functional classification according to the Gene Ontology (GO) annotations [35]. To find the GO terms overrepresented in our dataset, a GO enrichment was calculated with Array Assist that operates a statistical computation using a hypergeometric distribution [36].
Real time RT-PCR
Total RNA was extracted from endothelial cells using TRIzol reagent (Invitrogen, Carlsbad, CA, USA), following manufacturer's instructions. First-strand cDNA was generated using the SuperScript III First-Strand Synthesis System for RT-PCR Kit (Invitrogen), with random hexamers, according to the manufacturer's protocol. RT product was aliquoted in equal volumes and stored at -20°C.
PCR was performed in a total volume of 25 μl containing 1× Taqman Universal PCR Master mix, no AmpErase UNG and 2.5 μl of cDNA; pre-designed, Gene-specific primers and probe sets for each gene (BCL2 Hs99999018-m1) (ICAM1 Hs00164932-m1) (VEGFA Hs00900055-m1) were obtained from Assay-on-Demande Gene Expression Products (Applied Biosystems).Real Time PCR reactions were carried out in a two-tube system and in singleplex. The Real Time amplifications included 10 minutes at 95°C (AmpliTaq Gold activation), followed by 40 cycles at 95°C for 15 seconds and at 60°C for one minute. Thermocycling and signal detection were performed with ABI Prism 7300 Sequence Detector (Applied Biosystems). Signals were detected according to the manufacturer's instructions. This technique allows the identification of the cycling point where PCR product is detectable by means of fluorescence emission (Threshold cycle or Ct value). As previously reported, the Ct value correlates to the starting quantity of target mRNA [37]. Relative expression levels were calculated for each sample after normalization against the housekeeping gene GAPDH, using the ΔΔCt method for comparing relative fold expression differences [38]. The data are expressed as mRNA fold.
Ct values for each reaction were determined using TaqMan SDS analysis software. For each amount of RNA tested triplicate Ct values were averaged. Because Ct values vary linearly with the logarithm of the amount of RNA, this average represents a geometric mean.
Statistical analysis
Calculations were performed with the SPSS 16 statistical package. Comparison of CECs and EPCs levels between healthy controls and patients affected by SSc with and without ulcers were performed by T-test and Pearson test. Correlations between CECs and EPCs number before and after iloprost infusion were assessed with a non parametric test (Wilcoxon test).
Comparison of gene expression by Real Time RT-PCR was carried out by T-test.
CECs and EPCs in patients with SSc
CECs and EPCs are extremely rare in the peripheral blood of healthy people, representing somewhere between 0.01% and 0.0001% of mononuclear cells [11, 29]. Flow-cytometry offers the advantage of a rapid and accessible technique [29, 30], with the availability of multiple markers as well as the possibility of distinguishing CECs and EPCs using a small blood volume.
Key elements for accurate detection and enumeration of rare events in flow cytometry are the number of events acquired and the signal to noise ratio. Collection of a large number of events is mandatory to identify an adequate number of a rare event population; therefore, we stored 500,000 cells per sample in live gating. To minimize noise, we reduced non-specific binding by preincubating cells with blocking serum and doublets acquisition by an adequate flow rate. Dead cells can be a major source of non-specific staining by monoclonal antibodies. A real-time viability stain (7-AAD) was used to identify dead cells and to exclude them from analysis. We also established a dump channel (CD3, CD16, CD19, CD33) to exclude cells not of interest for the analysis. Indeed, the interest of the method reported here lies in the high intra-assay reproducibility and the high precision in the detection of both CECs and EPCs due to the gating strategy and to the presence of a dump channel [39, 40].
Finally, since no markers are entirely specific for endothelial cells, we used a multicolour approach and to maximize the signal we used the best fluorochrome (PE) for the most critical detection. CD146 and CD31 are useful as endothelial cell markers and were used in combination, since both these markers are individually expressed by other cell types, such as activated T-lymphocytes, pericytes, bone marrow fibroblasts, nerve fibers and leukocytes subsets and platelet/leukocytes aggregates respectively [41]. CD34, CD133 and VEGFR2 were used to more precisely identify EPCs.
CECs were defined as CD45 negative, CD146/CD31/CD34 positive and CD133 negative. EPCs are greater than CECs and are CD146/CD31 negative, CD34/CD133 positive, CD45 low positive and VEGFR2 positive [29].
Comparison of CECs and EPCs number between patients affected by SSc and healthy controls
SSc patients (50)
Healthy controls (50)
P value
689 ± 464
22 ± 17
< 0,0001
146 ± 92
1 ± 1
< 0,0001
Comparison of CECs and EPCs number between patients with and without skin ulcers
Skin ulcers - SSc patients (33)
Skin ulcers + SSc patients (17)
P value
600 ± 401
968 ± 553
142 ± 93
158 ± 92
Number of CECs and EPCs before and after iloprost infusion
Data before iloprost infusion
661 ± 404
152 ± 93
Data 72 h after one day iloprost therapy
745 ± 453
186 ± 104¶
Data 72 h after five days iloprost therapy
775 ± 382
206 ± 139¥
Taken together, these data indicate that the CECs and EPCs count is significantly higher in patients compared to healthy controls and that iloprost infusion induces a significant enrichment in both cell populations.
Gene array analysis of endothelial cells
We decided to use a gene array approach to analyse the transcriptional profiles of CECs in SSc patients. Since the purification procedure allows the recovery of a very limited amount of cells, our samples were prepared by mixing both EPCs and CECs, therefore, from now on and for this set of experiments, the term CECs will refer to the cell population that includes the two cell subtypes.
CECs were isolated from 37 patients without ulcers and from 13 patients with ulcers. CECs obtained from each group of subjects were then pooled for RNA extraction. Each patient contributed to the pooled sample with the same number of cells. CECs were also isolated from the blood of 50 healthy donors.
We compared the gene expression patterns of CECs obtained from SSc patients either in presence or in absence of digital ulcers with those obtained from normal healthy donors. As described in the Methods section only those genes modulated more than two-fold compared to the control sample (normal healthy donors) were considered in our analysis.
All the results of the gene array analysis have been deposited in the public repository ArrayExpress (accession number: [E-MEXP-2769]).
In CECs from patients with ulcers 6,544 genes were modulated when compared to the healthy counterpart, in particular 5,260 transcripts were down-regulated and 1,284 genes were up-regulated (Additional files 1, 2, 3).
A profound difference in gene expression was also observed in CECs obtained from patients without ulcers with 6,672 modulated genes (5,425 down-regulated genes and 1,247 up-regulated genes) (Additional files 4, 5).
These data showed that the transcriptional profiles of CECs in SSc were profoundly different from the transcriptional profiles of CECs of healthy donors, indicating that the two populations were quite heterogeneous at least at transcriptional level.
Among the genes differently expressed in these two populations, the number of down-regulated genes was significantly higher when compared to the number of the up-regulated ones.
CECs were also obtained from the same patients 72 hours after treatment with iloprost and the gene expression profiles of these cells were compared to the ones of CECs obtained from the same patients before treatment.
The treatment resulted in differential expression of 2,133 genes (1,080 up-regulated and 1,053 down-regulated) in patients with digital ulcers (Additional files 6, 7). A higher number of genes (6,643) was modulated by the iloprost infusion in patients without digital ulcers: the up-regulated were 5,081, while the down-regulated ones were 1,562 (Additional files 8, 9).
The results so far obtained showed that iloprost treatment had a strong impact on the transcriptional activity of CECs derived from SSc patients with and without digital ulcers.
Given the high number of modulated genes, we next decided to focus our attention on the effect of the treatment on the genes differently expressed in patients affected by SSc versus healthy donors. We therefore selected within the 6,544 transcripts differently expressed in patients with digital ulcers only those genes which were also modulated after iloprost treatment in the same patients. This subset of genes included 1,211 transcripts.
Functional classification of genes modulated by iloprost in SSc patients with digital ulcers
Probe set ID
Gene Title
Gene symbol
F.C. SSc ulcers/healthy
F.C. SSc ulcers post-treatment/SSc ulcers pre-treatment
Representative Public ID
Positive regulation of anti-apoptosis
RAS p21 protein activator (GTPase activating protein) 1
8.72 down
4.28 up
protein kinase, AMP-activated, alpha 1 catalytic subunit
6.49 down
6.60 up
BCL2/adenovirus E1B 19 kDa interacting protein 3
69.04 down
11.78 up
Response to stress
CD59 molecule, complement regulatory protein
18.76 down
2.72 up
12.38 down
4.28 up
mitogen-activated protein kinase 11
17.27 up
10.62 down
growth arrest and DNA-damage-inducible. beta
13.58 down
8.27 down
vascular endothelial growth factor
36.08 down
5.58 up
heat shock transcription factor 1
8.12 up
3.47 down
thymidylate synthetase
4.21 down
3.30 up
serum/glucocorticoid regulated kinase family. member 3
8.24 down
9.60 down
Response to wounding and wound healing
Tissue factor pathway inhibitor 2
9.85 down
2.62 up
lectin, mannose-binding, 1
11.53 up
9.84 down
5.13 down
3.61 down
connective tissue growth factor
595.44 down
14.43 up
Wnt receptor activity
frizzled homolog 6
39.71 down
2.18 up
Receptor complex
integrin. alpha 3
5.21 down
2.36 up
integrin. beta 3
2.17 up
2.47 down
integrin. alpha 9
3.18 down
3.58 down
cholinergic receptor. nicotinic. alpha 3
2.54 up
4.46 down
interleukin 11 receptor. alpha
15.25 down
2.90 down
intercellular adhesion molecule 1 (CD54)
28.90 down
6.61 up
deiodinase, iodothyronine, type II
7.75 up
10.10 down
fucosyltransferase 8 (alpha (1,6) fucosyltransferase)
17.44 down
17.71 up
endothelin receptor type B
10.11 up
2.75 down
solute carrier family 22, member 3
6.50 up
6.21 down
CD47 molecule
12.43 down
10.98 up
chemokine (C-X-C motif) ligand 13
3.03 down
7.56 up
chemokine (C-X-C motif) ligand 12
24.98 down
2.54 up
plasminogen activator, urokinase receptor
12.79 down
2.05 up
chemokine (C-X-C motif) ligand 3
24.57 down
3.15 down
chemokine (C-X-C motif) ligand 11
34.76 down
5.60 up
phospholipase D1, phosphatidylcholine-specific
12.37 down
3.08 up
cytochrome P450, family 26, subfamily B, polypeptide 1
17.74 down
15.25 up
DNA-dependent DNA replication
origin recognition complex, subunit 1-like
10.43 down
7.25 up
REV3-like, catalytic subunit of DNA polymerase zeta
38,90 down
2.80 down
high-mobility group box 2
5.53 down
2.41 up
RAB28, member RAS oncogene family
23.27 down
2.09 up
general transcription factor II, i
3.72 down
3.64 up
DNA-dependent DNA replication
carbonyl reductase 1
2.39 up
2.34 down
G0 to G1 transition
Mdm4, p53 binding protein
3.27 down
6.07 up
platelet-derived growth factor beta-receptor activity
platelet-derived growth factor receptor-like
5.33 up
9.50 down
actin cytoskeleton organization and biogenesis
pleckstrin homology domain containing, family C, member1
51.02 down
13.59 up
Rho-associated, coiled-coil containing protein kinase 1
8.58 down
8.52 up
diaphanous homolog 3 (Drosophila)
3.72 down
10.53 up
filamin B, beta (actin binding protein 278)
59.49 down
2.91 down
spectrin, alpha, non-erythrocytic 1 (alpha-fodrin)
13.23 down
2.62 down
FYVE, RhoGEF and PH domain containing 2
12.94 up
2.60 down
Innate immune response
toll-like receptor 2
5.94 down
3.16 up
toll-like receptor 3
55.81 down
6.59 down
CD180 molecule
23.53 down
10.73 up
toll-like receptor 5
27.49 down
2.03 up
complement factor H
561.66 down
2.36 down
pentraxin-related gene, rapidly induced by IL-1 beta
5.17 down
12.45 up
interleukin 7
3.06 down
11.97 up
complement component 9
6.43 up
9.24 down
Noteworthy is that most of these genes showed a significant change at transcription level after iloprost infusion.
Among genes related to apoptosis, for instance, anti-apoptotic genes such as RAS p21 protein activator 1 (RASA1), protein-kinases, AMP-activated alpha1 (PRKAA1) and BCL2 interacting protein 3 (BNIP3) were down-regulated in sclerodermic patients (F.C. -8.72, -6.49 and -69.05 respectively) but up-regulated after treatment (F.C. + 4.29, + 6.61, + 11.78).
Genes involved in the cellular response to stress had a similar behaviour; CD59, a complement regulatory protein, was strongly down-regulated in SSc patiens (F.C. -18.77) and up-regulated by the treatment (F.C. + 2.72). Vascular endothelial growth factor (VEGF) a well-known mitogen for vascular endothelial cells and a fundamental molecule for the EPCs recruitment from bone marrow, was greatly repressed in SSc patients (FC -36.08) but highly induced (F.C. + 5.58) after iloprost treatment.
The high increase of heat shock transcription factor 1 (HSF1) (F.C. + 8.12) was followed by a marked reduction (F.C. -3.47) after iloprost treatment.
Another cluster of modulated genes was represented by genes involved in the process of wounding and wound healing. Tissue factor pathway inhibitor-2 (TFPI2) is regulated by vascular endothelial growth factor and indeed its expression profile varied similarly to VEGF (F.C. -9.85 before and F.C. + 2.62 after iloprost). Indeed connective tissue growth factor (CTGF) showed the strongest down-regulation in SSc patients (F.C. -595.44) which was followed by a marked up-regulation (F.C. + 14.43) after treatment.
Iloprost also influenced the adhesion properties of CECs since several integrin genes were modulated in SSc patients after treatment. Expression level of intercellular adhesion molecule 1 (ICAM1) varied from a down-regulation of -28.91 F.C. to an up-regulation of + 6.61 F.C. The transcription level of endothelin receptor type B (EDNRB) gene varied from F.C. + 10.11 to F.C. -2.75.
The functional class named chemotaxis included genes encoding for chemokines, a group of molecules able to attract leukocytes and regulate angiogenesis, vascular proliferation and fibrosis. Several genes encoding for chemokines (CXCL13, CXCL12; CXCL3, CXCL11) had a significant change at the transcription level after iloprost infusion.
The CECs transcriptome modulated by iloprost treatment was also enriched in transcripts involved in the innate immune response regulation. This functional class included several toll like receptors (TLR2, 3 and 5) in particular TLR3 and TLR5 expression underwent extensive variation in SSc patients after iloprost infusion (F.C. -55.82 and -27.49 before treatment to F.C. -6.59 and + 2.03 after treatment).
A very strong reduction in expression (F.C. -561.66) of the gene encoding for complement factor H (CFH) was observed in CECs during SSc, however such reduction was less pronounced (F.C. -2.36) after iloprost infusion.
The same analysis was performed on CECs isolated from SSc patients without digital ulcers. Therefore we focused our attention on the genes significantly modulated in SSc patients, whose expression was also influenced by iloprost treatment.
Functional classification of genes modulated by iloprost in SSc patients without digital ulcers
Probe Set ID
Gene Title
Gene symbol
FC SSc/healthy
FC SSc post-treatment/SSc pre-treatment
Representative Public ID
Positive regulation of anti-apoptosis
BCL2/adenovirus E1B 19 kDa interacting protein 3
19.65 down
5.93 up
RAS p21 protein activator (GTPase activating protein) 1
5.35 down
2.02 up
protein kinase, AMP-activated, alpha 1 catalytic subunit
2.88 down
3.50 up
Response to stress
3.83 down
4.33 up
mitogen-activated protein kinase 11
3.53 up
2.15 up
growth arrest and DNA-damage-inducible, beta
11.23 down
4.25 up
vascular endothelial growth factor
7.03 down
2.38 up
thymidylate synthetase
2.76 down
5.84 up
Response to wounding and wound healing
connective tissue growth factor
1912.1 down
11.18 up
Tissue factor pathway inhibitor 2
3.36 down
8.90 down
Wnt receptor activity
frizzled homolog 6
19.85 down
3.25 up
Receptor complex
integrin, alpha 3
3.37 down
2.04 up
integrin, alpha 9
2.05 down
2.02 up
cholinergic receptor, nicotinic, alpha 3
2.10 up
12.73 down
interleukin 11 receptor, alpha
10.76 down
6.66 up
intercellular adhesion molecule 1 (CD54)
21.89 down
2.00 up
endothelin receptor type B
12.18 up
9.07 up
solute carrier family 22, member 3
14.37 up
8.31 down
CD47 molecule
6.17 down
3.51 up
chemokine (C-X-C motif) ligand 3
35.50 down
2.32 up
chemokine (C-X-C motif) ligand 11
2.98 down
2.32 down
phospholipase D1, phosphatidylcholine-specific
7.72 down
2.07 up
cytochrome P450, family 26, subfamily B, polypeptide 1
25.09 down
3.98 down
mitogen-activated protein kinase 9
9.53 down
7.02 up
DNA-dependent DNA replication
REV3-like, catalytic subunit of DNA polymerase zeta
21.36 down
8.90 up
high-mobility group box 2
2.78 down
3.18 up
RAB28, member RAS oncogene family
7.25 down
3.79 up
General transcription factor II, i
4.51 down
4.52 up
Prostaglandin-E2 9-reductase activity
transcription factor EB
6.09 down
11.09 up
G0 to G1 transition
metaxin 1
6.35 down
4.85 up
platelet-derived growth factor beta-receptor activity
platelet-derived growth factor receptor-like
5.04 up
2.72 up
actin cytoskeleton organization and biogenesis
filamin B, beta (actin binding protein 278)
92.27 down
2.75 up
spectrin, alpha, non-erythrocytic 1 (alpha-fodrin)
17.80 down
8.95 up
FYVE, RhoGEF and PH domain containing 2
15.22 down
5.67 up
Innate immune response
toll-like receptor 3
8.66 down
10.24 down
toll-like receptor 5
7.83 down
2.95 up
complement factor H
295.64 down
3.15 down
interleukin 7
10.94 down
2.96 up
complement factor I
578.23 down
16.50 up
complement component 9
11.81 down
10.01 up
Noteworthy was that most of the selected genes had a similar response to iloprost infusion when compared to the other disease subset. The results further confirm that iloprost treatment exerts a strong effect on the transcriptional profiles of CECs obtained from SSc patients.
Functional enrichment in gene categories in patients with skin ulcers compared to those without ulcers
Biological process
Number of probe sets
Immune response
Defense response
Response to wounding
Signal transducer activity
Response to stress
Regulation of cell proliferation
Positive regulation of nitric oxide biosynthesis
Positive regulation of biosynthesis
Receptor activity
Cell activation
Cell proliferation
Positive regulation of innate immune response
Regulation of immune response
Inflammatory response
Positive regulation of immune response
Cell adhesion
Cytokine activity
Interestingly, iloprost treatment modulated 59.5% of these transcripts (1,370/2,303).
These data show that there is a significant difference in the trascriptional profiles of CECs isolated from SSc patients with or without digital ulcers. The results therefore indicate that CECs are quite heterogeneous within the same disease and that these differences may be associated to the presence of a particular clinical subset.
Real Time RT-PCR validation of gene array results
We have detected and quantified CECs and EPCs in the peripheral blood of 50 SSc patients using a four-color flow-cytometry approach. The gating strategy and the presence of a dump channel allows the detection of both CECs and EPCs with high precision and a high intra-assay reproducibility. Moreover, we have followed the EULAR recommendations on endothelial precursor cells quantification [42]. Most of the reports on CECs and EPCs enumeration have used a three-color flow-cytometry [6, 43] and different markers from those recommended by EULAR explaining the controversial results obtained by different groups [6, 44]. We needed a precise enumeration of CECs and EPCs also because we had to use them for the gene array study.
In our cohort of SSc patients, the number of both CECs and EPCs was higher than in healthy donors as already reported [6]. The increased EPC levels in SSc support their mobilisation from bone marrow in the attempt of revascularization in response to vascular ischemia. Moreover the counts of CECs correlated with the clinical stage of the disease, since a higher number was detectable in patients with a more severe vascular damage (presence of digital ulcers). Patients with digital vascular lesions did not show a significant increased number of EPCs in accordance with previous data [45] and suggesting an increased homing at this stage.
We observed that iloprost infusion significantly increased the number of both cell types in all the patients treated. To our knowledge, the finding of increased levels of CECs and EPCs in patients with SSc after iloprost treatment has not been previously reported and may be of difficult interpretation since one would expect a reduction of these cells to the levels similar to those seen in healthy controls. A possible explanation for these findings is that iloprost infusion may be responsible for the in vivo recruitment of EPCs from bone marrow and for their homing into sites of angiogenesis and/or vascular damage, thus contributing to neovascularization and/or wound-healing processes. Moreover, the drug may favour the migration and proliferation of mature endothelial cells surrounding the sites of vascular damage thus leading to an increase shedding of damaged cells. However, the increase of EPCs is not confined to iloprost therapy since a statistically significant increase in EPCs has also been observed during atorvastatin treatment in patients with SSc [43].
In SSc patients, CECs were not only increased in their number but also revealed a completely different transcriptional profile when compared to that of CECs obtained from healthy donors. We decided to focus our attention on the different expression of genes strictly related to vasculogenesis, reparative processes, cell migration and homing, since a deficient vascular repair mechanism and defective vasculogenesis are the main contributors to vasculopathy in Ssc.
We found a significant decrease in expression of genes encoding for molecules involved in the negative regulation of apoptosis (RASA1, PRKAA1 and BNIP3) suggesting that the cells are prone to apoptosis. Apoptosis of endothelial cells is considered the primary pathogenic event in SSc and the downmodulation of genes encoding for antiapoptotic molecules helps in understanding the molecular basis of this event. Moreover, the downregulation of SGK3, also called cytokine-independent survival kinase (CISK), a survival kinase involved in cellular response to stress protecting cells from apoptosis [46], further indicates an impaired regulation of cellular survival pathways.
In SSc patients CECs showed also a decreased transcription of TFPI2, EREG and CTGF, molecules crucially involved in tissue-specific proliferation/differentiation homeostasis and effective reparative activity, indicating that these cells are also compromised in their wound healing capacity. CTGF, a growth factor produced as part of a growth factor cascade during vascular injury responses, can also modulate the activity of angiogenic molecules such as VEGF [47, 48] and similarly, VEGF is a potent inducer of CTGF mRNA [49, 50]. Therefore the simultaneous under-expression of both genes is not surprising.
Another function severely compromised in SSc is the adherence ability of CECs, demonstrated by the reduced transcription levels of several adhesion molecules such as ICAM1 and integrins. This aspect together with the downregulation of different chemokines may suggest a reduced cell migration and therefore a reduced andothelial cell homing. In particular, the reduced expression of CXCL12 (SDF1) in patients with digital ulcers is in accordance with previous findings on the reduced expression of this chemokine in late phase of the disease [51].
Endothelin-1 and its receptors A and B play a pivotal role both in vasoconstriction and in fibrosis. Receptors A are expressed on smooth muscle cells and mediate vasoconstriction, whereas receptors B are expressed on endothelial cells and mediates primarily vasodilatation through nitric oxide production. The upregulation of endothelin-1 receptor B gene expression by CECs suggests the attempt of endothelial cells to privilege the vasoactive effect and to increase tissue blood flow.
VEGF, a potent angiogenic mitogen playing a crucial role in angiogenesis under various pathophysiological conditions, was strongly down-regulated probably in relation to a reduced proangiogenic activity of CECs in SSc. Interestingly VEGF can mobilize EPCs from bone marrow and accelerate endothelial damage repair [52, 53]. Therefore the increase in VEGF gene expression after iloprost therapy may explain the EPCs increase in an attempt to repair the vascular damage. The increase of the VEGF gene expression after iloprost stimulation has already been described in lung fibroblasts [24]. Moreover iloprost has been reported to induce VEGF production from intestinal epithelial cells [54], from monocytes [55] and to modulate VEGF secretion from platelets isolated from SSc patients [56]. It is well known that VEGF has been found elevated in the sera of patients with SSc. However, we must consider first of all that the gene array analysis has been carried out in CECs, which can exhibit a different transcriptional profile from that expected and seen in the same cells in different conditions, that is, cells isolated from skin and coltured [57] or in a whole tissue [58]. Secondly, VEGF is produced from different cell types as mentioned above and this may account at least in part for the increased levels of circulant VEGF in patients with SSc. As for VEGF gene expression, iloprost infusion showed a strong effect on the transcriptional profiles of CECs isolated from patients with SSc both in the presence or absence of digital ulcers, since it induced a marked modulation of the differently expressed genes.
When we considered the genes over- and under-expressed in SSc patients versus healthy donors we found that the treatment induced an opposite behaviour of most of these transcripts. Thus several molecular functions repressed in CECs of scleroderma patients, were restored after iloprost infusion as shown by the over-expression of antiapoptotic genes (RASA1, PRKAA1 and BNIP3) and of the transcripts encoding for adhesion molecules (ICAM1, ITGA3, ITGA9), chemokines and wound healing process (EREG, CTGF). Also, the decrease in heat shock transcription factor 1 expression after iloprost treatment is in accordance with the protective effects of the drug on endothelial cells. The gene array data concerning the drug modulation of genes involved in the control of apoptosis, in cell adhesion and in vasculogenesis were validated by quantitative RT-PCR of selected genes (BCL2, ICAM1, VEGF) in SSc patients with and without digital ulcers.
In this study we analyzed gene expression profiles of CECs and EPCs obtained from healthy subjects and from SSc patients with and without digital ulcers before and after iloprost treatment using a gene array approach. Based on gene ontology analysis we found that CECs from patients show down-modulation of genes involved in the control of apoptosis, in cell migration and adhesion and in angiogenesis. Several impaired cellular function were reversed by iloprost treatment and the gene array modulation was validated by quantitative RT-PCR of selected genes. The different expression profile of CECs in SSc patients compared to normal subjects account for endothelial cell apoptosis and for the impaired angiogenesis in the disease. Moreover, our data give a novel insight into the vascular repairing effects of iloprost treatment.
circulating endothelial cells
connective tissue growth factor
endothelial progenitor cells
fruorescence activated cell sorting
progressive systemic sclerosis
vascular endothelial growth factor
All the authors were supported by grants from The Italian Ministry of University, Technology and Scientific Research (ex MURST 60%). CR was supported also by a grant from Cariverona foundation and CL from a grant from the Italian Association of sclerodermic patients (AILS).
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- Cipriani P, Franca Milia A, Liakouli V, Pacini A, Manetti M, Marrelli A, Toscano A, Pingiotti E, Fulminis A, Guiducci S, Perricone R, Kahaleh B, Matucci-Cerinic M, Ibba-Maneschi L, Giacomelli R: Differential expression of stromal cell-derived factor 1 and its receptor CXCR4 in the skin and endothelial cells of systemic sclerosis patients: pathogenic implications. Arthritis Rheum. 2006, 54: 3022-3033. 10.1002/art.22047.View ArticlePubMedGoogle Scholar
- Beadry P, Hida Y, Udagawa T, Alwayn IP, Greene AK, Arsenault D, Folkman J, Heymach JV, Ryeom S, Puder M: Endothelial progenitor cells contribute to accelerate liver regeneration. J Pediatr Surg. 2007, 42: 1190-1198. 10.1016/j.jpedsurg.2007.02.034.View ArticleGoogle Scholar
- Ferreira LS, Gerecht S, Shieh HF, Watson N, Rupnick MA, Dallabrida SM, Vunjak-Novakovic G, Langer R: Vascular progenitor cells isolated from human embryonic stem cells give rise to endothelial and smooth muscle-like cells and form vascular networks in vivo. Circ Res. 2007, 101: 286-294. 10.1161/CIRCRESAHA.107.150201.View ArticlePubMedGoogle Scholar
- Stratton R, Rajkumar V, Ponticos M, Nichols B, Shiwen X, Black CM, Abraham DJ, Leask A: Prostacyclin derivates prevent the fibrotic response to TGF-beta by inhibiting the Ras/MEK/ERK pathway. FASEB J. 2002, 16: 1949-1951.PubMedGoogle Scholar
- Lederle FA, Nelson DB, Joseph AM: Smokers' relative risk for aortic aneurysm compared with other smoking-related diseases: a systematic review. J Vasc Surg. 2003, 38: 329-334. 10.1016/S0741-5214(03)00136-8.View ArticlePubMedGoogle Scholar
- Solanilla A, Villenueve J, Auguste P, Hugues M, Alioum A, Lepreux S, Ducroix JP, Duhaut P, Conri C, Viallard JF, Nurden AT, Constans J, Ripoche J: The transport of high amounts of vascular endothelial growth factor by blood platelets underlines their potential contribution in systemic sclerosis angiogenesis. Rheumatology. 2009, 48: 1036-1044. 10.1093/rheumatology/kep154.View ArticlePubMedGoogle Scholar
- Giusti B, Fibbi G, Marheri F, Serrati S, Rossi L, Poggi F, Lapini I, Magi A, Del Rosso A, Cinelli M, Guiducci S, Kahaleh B, Bazzicchi L, Bombardieri S, Matucci-Cerinic M, Gensini GF, Del Rosso M, Abbate R: A model of anti-angiogenesis: differential trasncriptosome profiling of microvascular endothelial cells from diffuse systemic sclerosis patients. Arthritis Res Ther. 2006, 8: R115-10.1186/ar2002.PubMed CentralView ArticlePubMedGoogle Scholar
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This article is published under license to BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

subset: commoncrawl


text:
Studies on the diastereoselective, asymmetric total synthesis of vindoline and the clinically important antitumor drug vinblastine are detailed based on the implementation of a tandem [4+2]/[3+2] cycloaddition cascade of 1,3,4-oxadiazoles, and a recently developed single step Fe(III)-promoted biomimetic coupling and subsequent oxidation reaction of vindoline with catharanthine. Extensions of these studies to the synthesis and evaluation of vinblastine analogues containing previously inaccessible deep-seated structural changes will be pursued, new insights into the mechanism of the biomimetic Fe(III)-promoted coupling of vindoline with catharanthine will be established further expanding access to unique vinblastine analogues, two new alternatives to existing coupling methods will be examined and developed expanding the range of synthetically accessible vinblastine analogues available for examination, and key insights into the structural features of vinblastine and vincristine integral to their binding to tubulin, inhibition of microtubulin formation, and inhibition of cell mitosis and tumor cell growth will be established. Not only will a fundamental understanding of the structure-function relationships of vinblastine's antitumor properties emerge from the studies, but drugs with improved potency, selectivity, efficacy, and/or tumor resistance profiles can be expected to continue to emerge from the studies.
Fundamentally new approaches to the synthesis of vinblastine and vincristine, clinically employed antitumor drugs, will be developed, unique insights into the mechanism of a key biosynthetic (biomimetic) coupling reaction will emerge from the studies, a fundamental understanding of the interaction of vinblastine/vincristine with its biological target (tubulin) will be defined, and new drugs that further improve on the potency, selectivity, and efficacy of the clinically used natural products will be discovered including those that are active against vinblastine-resistant and multidrug-resistant (MDR) tumors.
|Turner, Travis C; Shibayama, Kotaro; Boger, Dale L (2013) Hypervalent iodine(III)-promoted intermolecular C-C coupling of vindoline with ýý-ketoesters and related substrates. Org Lett 15:1100-3
|Barker, Timothy J; Duncan, Katharine K; Otrubova, Katerina et al. (2013) Potent Vinblastine C20' Ureas Displaying Additionally Improved Activity Against a Vinblastine-Resistant Cancer Cell Line. ACS Med Chem Lett 4:
|Campbell, Erica L; Skepper, Colin K; Sankar, Kuppusamy et al. (2013) Transannular Diels-Alder/1,3-dipolar cycloaddition cascade of 1,3,4-oxadiazoles: total synthesis of a unique set of vinblastine analogues. Org Lett 15:5306-9
|Leggans, Erick K; Duncan, Katharine K; Barker, Timothy J et al. (2013) A remarkable series of vinblastine analogues displaying enhanced activity and an unprecedented tubulin binding steric tolerance: C20' urea derivatives. J Med Chem 56:628-39
|Schleicher, Kristin D; Sasaki, Yoshikazu; Tam, Annie et al. (2013) Total synthesis and evaluation of vinblastine analogues containing systematic deep-seated modifications in the vindoline subunit ring system: core redesign. J Med Chem 56:483-95
|Leggans, Erick K; Barker, Timothy J; Duncan, Katharine K et al. (2012) Iron(III)/NaBH4-mediated additions to unactivated alkenes: synthesis of novel 20'-vinblastine analogues. Org Lett 14:1428-31
|Gotoh, Hiroaki; Duncan, Katharine K; Robertson, William M et al. (2011) 10'-Fluorovinblastine and 10'-Fluorovincristine: Synthesis of a Key Series of Modified Vinca Alkaloids. ACS Med Chem Lett 2:948-952
|Va, Porino; Campbell, Erica L; Robertson, William M et al. (2010) Total synthesis and evaluation of a key series of C5-substituted vinblastine derivatives. J Am Chem Soc 132:8489-95
|Tam, Annie; Gotoh, Hiroaki; Robertson, William M et al. (2010) Catharanthine C16 substituent effects on the biomimetic coupling with vindoline: preparation and evaluation of a key series of vinblastine analogues. Bioorg Med Chem Lett 20:6408-10
|Kato, Daisuke; Sasaki, Yoshikazu; Boger, Dale L (2010) Asymmetric total synthesis of vindoline. J Am Chem Soc 132:3685-7
Showing the most recent 10 out of 15 publications


text:
A public school principal in Louisiana is under investigation for violating the federal ban on school-sponsored prayer. She allegedly handed out prayers — complete with grammar and punctuation errors — for students to recite before taking standardized tests.
Hey, shouldn't there be a federal ban on school principals who write grammatically incorrect anything? Maybe I'm just being picky.
Apparently, the principal has no faith in the students or teachers because she thinks each child needs to pray for help in passing the tests, referred to in the prayer as the "enemies." And she also seems to think the kids need to be spoon-fed a prayer, as if they aren't capable of saying their own prayers in their own way, should they wish to do so. [Which, by the way, would be the legal form of prayer in school.]
I'm surely no fan of standardized tests as the sole barometer of a school's or an individual's performance, but shouldn't the principal at least pretend she doesn't loathe the tests, just so the kids have, oh, I don't know, a positive attitude toward taking them? Or at least not a "the test is my enemy" attitude? I'll settle for a neutral attitude, okay? The "enemy" attitude can't be a good thing, particularly for kids who are struggling academically.
Do You Believe in Miracles? NOOOOOOOO!
The principal also distributed a "special announcement" to teachers last year, dealing with the school's troubled status: it's in danger of being taken over by the state for poor performance. Like the prayer, the announcement contains grammar and punctuation errors and asserts faith in God. To me, it also shows a lack of faith in the teachers and students:
We will not be taken over by the state but by faith. … We will need a miracle. And after all isn't that what he specializes in?
Perhaps miracles are God's specialty. But doesn't the specialty of school principals and teachers have something to do with educating students so that they don't need miracles to bail them out? I guess it's too much to ask for a little positive thinking and perhaps a plan of action to improve the students' skills and, as a byproduct, their test scores.
BHW Gets the Whole Scoop and Nothing But the Scoop
Thus far, news stories have published only excerpts of the prayer and announcement. But the BHW investigative team has unearthed the complete text of both documents, and it's a doozey of a read. [You should have seen the rough drafts!] Please note that I have reproduced the writings exactly from the originals, blemishes and all.
First, the announcement given to teachers:
To: All Green School Staph
From: Principal Stroller
Lord have mercy on us, we are UP the crick. Education in New Orleans, is in trouble.
Teachers, there is no way on Gods green earth that those children will improve there test scores this year, I don't now what the problem with those kids is but they still can't read and rite. Basic skills, my ass. I wish I'da nown this sooner. Then I couldof done something about it.
Well its to late for that now. Were headed for state takeover if we don't start asking for help from the man upstairs, because he's all weve got left. So start praying for a miracle, we need it.
You need to be a prayer warrior and pray for each other, each others family, and for our school. I'm sure well prevail if you can do a lot of praying. Don't bother with the books and lesson plans now. That stuff doesn't work with these kids.
The only thing left is a little divine intervention. I really believe that we will not be taken over by the state but by faith. We will need a miracle. And after all isn't that what he specializes in?
Amen it is. Now go get those prayers started and get me that miracle!
Now, the prayer given to students to recite:
Dear God,
We need your help today, we are not ready to face our enemies but we have to because the tests are mandatory. But we cant defeat them without your help because we sure cant read or rite. And forget add, subtract, or divide. And so we pray to you:
That we can prepare ourselves in heart and mind to take this test,
so we can randomly fill in circles in #2 pencil,
and with your help and a little from a freind at the testing service,
we can get a miraculous pasing grade.
We receive your help faith, knowing that through you we shall do valiantly, for you are the one who treads down our enemies.(LEAP, Iowa).
Please, God, dont let the evil state come in and improve our schools. We dont need to learn anything, we just need to pass the tests.
And then our principal can keep her job.
Is anyone dialing 1-800-state-of-louisiana-board-of-education yet?
On the up side, the principal is actually a role model for the students in her school: she's living proof that being literate doesn't matter much at all, even for an educator. In fact, she's the perfect embodiment of the failed American education system: "Don't you pay any attention to those grammatical errors, honey. You can always be a teacher. Or even a principal."Powered by Sidelines


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3D Architectural Visualization video (Interior and Exterior)
This project was successfully completed by hambaka90 for $800 USD in 14 days.Get free quotes for a project like this
Project Budget$750 - $1500 USD
Completed In14 days
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Project Description
I need to please create a 3D architectural animated video for a commercial and residential building. The time frame is 40 second exterior and 20 second interior. The exterior has to be 360, aerial view, show skyline view from the 10th floor view and have details of the landscape, parking lot, plants, street view and people (black images are fine). There will be 2 interior designs - one unit which is a commercial unit and the 2nd unit is residential. I need 2D of the floor plans for the units as well. The interior units can be a mix of rendered images for a kitchen, living room, bedroom, open office floor space and an executive office and animation.
I will be sending live videos of the building and surrounding area as soon as I have selected the right designer.
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text: Sleep-Deprived College Students: College to Open Nap Rooms Jul 1, 2014 by Suada Kolovic We've all been there: Going about our day without a care in the world when it dawns on us that (go figure) our term paper on the pros and cons of procrastination in the creative process is due tomorrow. Panicked, we consider emailing our professor an excuse about a death in the family but given we killed off Nana (who's actually alive and well back home) last semester during finals week, we decide it's best to pull an all-nighter. The next day, we're irritable, unmotivated and just plain sluggish and while the simple solution is to overcome procrastination and not leave an assignment until the last minute, the Art Center College of Design has suggested a different approach: a nap room on campus. The Pasadena, Calif.-based college opened its first nap room last year in an effort to combat exhaustion among its mostly commuter students. "Our Nap Room is available for students four weeks during each 14-week term. We are a year-round program with three terms a year," said spokesperson Teri Bond. "The room is available 24 hours on Mondays, Thursdays, Fridays, Saturdays and Sundays during those four weeks each term." And while there isn't a determined space just yet, Art Center students are hoping to follow in the footsteps of the University of Colorado-Boulder and the University of Michigan in instituting a permanent nap room on campus. (For more on this story, click here.) If you're a fan of napping between classes, do you think it's your university's responsibility to provide nap rooms for students? Let us know what you think in the comments section. And for a more detailed look into the Art Center College of Design or hundreds of other universities, check out our College Search. And don't forget, you should pay for your college education with as much free money as possible! Find as many scholarships and grants as you can before turning to student loans. Visit the Scholarships.com free college scholarship search today where you'll get matched with countless scholarships and grants for which you qualify, then apply and win! It's that easy!


text:
Makes one 9-inch pie
One 9-inch unbaked pie shell
1/4 cup shortening
1-1/2 cups flour
1 cup brown sugar
Work the above ingredients together and set aside.
3/4 tsp. baking soda
1/8 tsp. nutmeg
Pinch of ginger, cinnamon and cloves
1/4 tsp. salt
3/4 cup molasses
3/4 cup hot water
Mix all ingredients together until well blended.
Into an unbaked 9-inch pie shell, combine the crumbs and liquid in alternate layers with crumbs on bottom and top. Bake 15 minutes at 450 degrees F., then 20 minutes at 350 degrees, F. Serve after cooling, or warm with ice cream or whipped cream, if desired.
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Suits Season 4 DVD Box set is for sale now at cheap golf shop with best price and free shipping.
Suits Season 4 DVD Box set
Actors: Gabriel Macht,Patrick J. Adams,Meghan Markle,Sarah Rafferty,Gina Torres,Rick Hoffman
Creator: Aaron Korsh
Category: Comedy | Drama
Seasons: Season 4
Year of Release: 2015
Number of Discs: 7
Language: English with removable subtitles
Audio Format: Dolby Digital 5.1
Aspect Ratio: 1.78:1
Video Format: Support both NTSC & PAL
Region: 1,2,4,5
Suits Season 4 DVD Description
Fourth season of Suits follows the final of Sutis Season 3, the company that is the life's work of Walter Gillis is ripe for a takeover, and Mike has a plan to save it that is derided as insufficiently ruthless by both Harvey and Sidwell. Mike puts his representation by Pearson Specter under review, and when Harvey's other client Logan Sanders announces his plan for a hostile takeover of the company, Harvey is obligated to help him against Mike. Meanwhile, feared SEC litigator Jeff Malone tells Jessica and Harvey that Woodall, now at that agency, is about to pursue his vendetta against the firm by having Malone attack their clients. Louis has already learned of this from Katrina and hopes to be asked to save the firm with his litigation skills, but Malone plans instead to leave the SEC and join PS as a partner.
Buy Suits Season 4 DVD Box set from UK Golf Shop - uknit.co.uk, Where you can enjoy such Promotion Activities:
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text:
Annie leibovitz
Εξερευνήστε σχετικά θέματα
Kate Winslet by Annie Leibovitz - tone, pose, exposure
To Annie Leibovitz, Jessica Chastain is A Work Of Art
This is another one of my favorite Annie Leibovitz photos. Although it's a little strange to be posing a celebrity in a tub full of milk, I think it's totally something Whoopi Goldberg would do.
The 11 actresses who posed for Annie Leibovitz for 2017's Hollywood Portfolio have riveted moviegoers this year, in a stunning range of cinematic styles. James Wolcott reviews their awards-season glow.
Adele by Annie Leibovitz | GRAVERAVENS
Adele,Annie Leibovitz. Short lit. Pretty
Annie Leibovitz. portrait, Leonardo di Caprio
Jonah Hill, Paul Rudd, Seth Rogen Jason Segel by Annie Leibovitz
Angelina Jolie by Annie Leibovitz. This portrait from Leibovitz remind me one of my favourites old school fashion photographer Irving Penn. From the composition to the lighting and b&w treatment. Love it.
Annie Leibovitz More


text:
- Home
- Award Details
High Spatial Resolution Ultrafast Sum-Frequency Generation Microscope
Award Information
Department of Energy
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Phase I
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Physics Materials and Applied Mathematics Research LLC
1665 E. 18th Street, Suite 112, Tucson, AZ, 85719-6808
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Name: Kevin Kremeyer
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Research Institution
Name: University of California, Irvine (UCI)
Address: Natural Sciences II
Irvine, CA, 92697-1075
Phone: () -
Type: Nonprofit college or university
Address: Natural Sciences II
Irvine, CA, 92697-1075
Phone: () -
Type: Nonprofit college or university
AbstractVibrationally resonant sum-frequency generation (SFG) spectroscopy is a second order nonlinear optical technique that features high chemical selectivity and a set of selection rules that makes it an ideal complement to coherent Raman scattering (CRS) as an imaging tool. Attempts have been made to construct high-resolution, high speed SFG microscopes, but to date both the resolving power and image acquisition speed have been limited to unacceptable ranges due to the lack of purely refractive optical elements capable of operating without deleterious chromatic effects over the wavelengths of the two beams used to produce SFG in samples. In order to increase both the spatial resolution and the scanning speed of SFG microscopes, we will design custom achromatic optical elements for operation at wavelengths in the near- and mid-infrared regions. Additionally, we use these improvements to incorporate both CRS and second harmonic generation into the microscope. Commercial Applications and Other Benefits: The proposed technology will introduce a microscope that will greatly enhance researchers ability to image fragile and sensitive materials without the use of exogenous agents. Examples include biological tissues, works of art and ancient objects of archeological significance. In particular, a multimodal microscope incorporating SFG and CRS will provide researchers with the ability to image biological tissue structure that cannot be currently imaged without labels or destructive methods. Such a microscope could profoundly affect fields of study such as medicine and biomechanical engineering.
* information listed above is at the time of submission.
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text:
Our stretch goal is to fund production of a second CD during the next year, "Friend in the Dark," collecting experimental arrangements of our Norse music and many of Ada's earlier songs, which have been out of print while we concentrated on the Sundown project, performed by Ada and Lauren, performing as the duo "Sassafrass: Trickster & King."
We've started recording for "Friend in the Dark" and all completed tracks are availble streaming for free online. If we make our stretch goal of $8,606, we will give every backer who pledges at the $15 level or above an instant download of all the current finished tracks of "Friend in the Dark", and a digital download of the finished album when complete, and we will also pledge to complete the album within one year. If we don't make the stretch goal, we will still release the current tracks as a pre-order, but we will put finishing the CD project on the back burner.
So if you've already pledged or signal boosted the Kickstarter, thank you, and please keep spreading the word; we're very close to making the stretch goal!
Sassafrass will also be performing at Worldcon on Saturday at 2:30pm; the schedule says Trickster and King, but we've had a last-minute addition of another member, which means we can do trios!


text:
I have a probe type -- high/low -- thermometer, but I didn't check it every hour -- the low matched the spot in my garden that I believe to be the coldest. (Farthest north, exposed, and lowest in elavation), but I have to "trust" the site for the time factor!
To sign-off this list, send email to email@example.com with the
message text UNSUBSCRIBE HOSTA-OPEN


text:
Sales Calls...Cold calling for new clients
This project received 3 bids from talented freelancers with an average bid price of $194 USD.Get free quotes for a project like this
Project Budget$30 - $250 USD
Total Bids3
Project Description
Looking for someone who is very good with cold calling to create new leads, increase overall exposure to social media & web-hits. We hare selling a high end product in the travel/aviation market. Need to connect with travel managers, high wealth individuals, entertainment industry, sports figures, personal assistants, executive assistants, yacht & jet owners etc. Additional commissions offered on leads that turn into sales. We are based in the US, but our services are offered internationally. Will provide and basic script to start. Can provide basic calling list to start but need new leads as well.
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Hotel Village Club Perla Majestic
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TripAdvisor Reviews Hotel Village Club Perla Majestic Cosenza
Travel Blogs from Cosenza
A trip to Devil's bridge
Today we set off on a longer day trip to see Devil's bridge. To be fair, the trip was great, and Devil's bridge was nice to see, but it was a heck of a trip to get there !! Not helped by our satnav which decided to take us on a 10km detour only to bring us back to where we ...
Chilled weekend
... is still a big hotel at the top of the hill with amazing 180 degree views of the coast - but it is now completely derelict. We air wheeled up there to explore and it's a real shame that it is no longer used as it is in a fantastic location - and apparently it used to have a swimming pool on the roof too ! We were also lucky to have another nice sunset down on the beach so yet more sunset photies coming up ...
Here we are in Calabria
... Camerota. On the on the way back down the coast, we spotted a statue, way up on top of one of the mountains and decided to go and find it !! It was the statue Il Redentore which was literally on top of the mountain, with a platform specially built for it, and the small church next to it, complete with a hair-raising bit of track on stilts to get up there. We managed to get there just in time to enjoy the sunset too which was ...
Trains, trains and automobiles
... through and still haven't really got a feeling of her personality. It's all a bit superficial, a point that the author makes as an apology but it doesn't really help. Of course no journey is possible without its delays and this was no exception, as with that long arduous journey a week ago, we had a long delay at Salerno, sitting on the train that was to complete our journey. Once it got going, some 70 minutes later, it did move quite fast and hugged ...
Day 45 - Taormina to Castrovillari
... to find a very large ship with its mouth wide open and so we just drove up and onboard without really being sure that we were even on the correct boat. Virtually as soon as we got Travis onboard the ship started to sail
Now we weren't even certain that we had driven onto the correct ship let alone where it may be heading. We eventually consoled ourselves with the thought that at least the pointy end of the boat was facing towards mainland Italy - the ship did in ...
Other places to stay in Cosenza
Italiana Hotels CosenzaVia Panebianco 452, Cosenza | 4 star hotelfrom $69
Home Club Suite HotelViale Giacomo Mancini, Cosenza | 4 star hotelfrom $73
Hotel EuropaVia J F Kennedy, Cosenza | 4 star hotelfrom $84
Royal HotelVia delle Medaglie d'Oro, Cosenza | 4 star hotelfrom $82
Hotel Sant' AgostinoVico Fiumicello 6, Paola | 3 star hotelfrom $94
Albergo BlumentagVia Magna Grecia, 8, Paola | 3 star hotelfrom $101
Park Hotel TyrrenianVia Stromboli 227, Cosenza | 4 star hotelfrom $72
Hotel CentraleVia del Tigrai 3, Cosenza | Hotelfrom $71
Link HotelVia Raffaele Coscarella, Cosenza | 4 star hotelfrom $105
Hotel AlhambraVia della Civilta' 56, Paola | Hotelfrom $53
AldebaranMessinette 8, Cosenza | Hotelfrom $67
Hotel San FrancescoVia Della Timpa, Paola | Hotelfrom $115
Centro Turistico Marina Di SibariShimoda Onsen, Cosenza | Hotelfrom $140
Hotel Baia del SoleStrada Statale 18 Localita' Coreca, Cosenza | Hotelfrom $66
Hotel La CalavrisellaL'Antiga Esquerra de l'Eixample, Cosenza | Hotelfrom $33


text:
Clinical Trials Resource Center
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Following are the most recent results for new drug therapies currently in clinical trials worldwide. Results
are also searchable by therapeutic area beginning with the most recent updates.
ASLAN Pharmaceuticals issued results of a phase II study of varlitinib (ASLAN001) in combination with capecitabine as second-line treatment for metastatic HER2-positive breast cancer patients progressing on trastuzumab (Herceptin). The multinational, randomized, open-label study compared the effects of varlitinib combined with capecitabine versus that of lapatinib (Tykerb) combined with capecitabine on tumor shrinkage at week 12. Top-line data showed significantly greater tumor shrinkage at week 12 of the treatment cycle in patients who were on therapy for more than a month, receiving 400mg varlitinib (36.4%) twice daily compared to 1,250mg lapatinib (17.8%) once daily (p=0.075). A total of 50 patients were enrolled in the study. While the study was not powered for objective response rate (ORR), it found that patients dosed with varlitinib showed higher ORR compared to patients on lapatinib (60% versus 46%). There were no differences in progression-free survival (PFS) or overall survival (OS) in the study. Adverse events reported included nausea, vomiting and diarrhoea, and occurred at similar frequency in both arms. The incidence of grade three diarrhea was 12.5% in the varlitinib group and was clinically manageable. There were no instances of grade four diarrhea.
Merck KGaA released results of a phase III study of Cladribine Tablets for brain atrophy related to relapsing remitting multiple sclerosis (RRMS). The CLARITY study was a two-year (96-week), randomized, double-blind, placebo-controlled, international study. It randomized 1,326 patients with relapsing remitting MS according to the revised McDonald criteria. Study participants were randomized to one of three different treatment groups consisting of two different dose regimens of Cladribine Tablets or matching placebo tablets (1:1:1 ratio). The brain atrophy analysis evaluated the effect of Cladribine tablets on brain volume loss (BVL) over two years in RMS and the association of BVL with confirmed disability progression in 1,025 (77.3%) of the patients in CLARITY. The mean percentage brain volume loss per year was significantly reduced in patients treated with Cladribine Tablets 3.5mg/kg (0.56%±0.68, p=0.010, n=336) and 5.25mg/kg (0.57%±0.72, p=0.019, n=351) compared with patients treated with placebo (0.70%±0.79, n=338). The risk of disability progression was also significantly lower in patients treated with Cladribine Tablets 3.5mg/kg (HR 0.63, 95% CI 0.438, 0.894; p=0.010) and 5.25mg/kg (HR 0.58, 95% CI 0.406, 0.833; p=0.003) than in those treated with placebo. After adjusting for treatment group, percentage brain volume loss per year showed a significant correlation with the cumulative probability of disability progression in the overall study population (HR 0.67, 95% CI 0.571, 0.787; p<0.0001). Lymphopenia was the most commonly reported adverse event (AE) in patients treated with Cladribine Tablets. The incidence of infections was 48.3% with Cladribine Tablets and 42.5% with placebo, with 99.1% and 99% rated mild-to-moderate by investigators.
Theratechnologies released results of a phase III trial of ibalizumab for treatment-experienced patients infected with multidrug resistant HIV-1. TMB-301 was a single arm, 24-week study of ibalizumab plus optimized background regimen (OBR). Patients receiving their current failing antiretroviral therapy (ART), or no therapy, were monitored during a seven-day control period. Thereafter, a single loading dose of 2,000mg of intravenous (IV) ibalizumab was the only ART added to their regimen. The primary efficacy endpoint was the proportion of patients achieving a 0.5 log10 decrease in HIV-1 RNA seven days after initiating ibalizumab therapy, day 14 of the study. Ibalizumab was continued at doses of 800mg IV every two weeks through 24 weeks on study treatment. A total of 40 patients have been enrolled in the study. The new data showed that patients with multidrug resistant (MDR) HIV-1 infection experienced a mean increase in CD4+ T cell of 48 cells/μL after 24 weeks of treatment with ibalizumab plus an optimized background regimen (OBR). These data supplement previously reported findings, where 83% of patients achieved a 0.5 log10 decrease in viral load from baseline seven days after the single loading dose of 2000 mg of ibalizumab (primary endpoint) and a mean reduction in viral load of 1.6 log10 over the 24 week treatment period with more than 48% of patients experiencing a viral load reduction of more than 2.0 log10. A BLA to the FDA has been submitted.
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