Aptamer-Based Affinity Chromatography for Protein Extraction and Purification.

Aptamers are oligonucleotide molecules able to recognize very specifically proteins. Among the possible applications, aptamers have been used for affinity chromatography with effective results and advantages over most advanced protein separation technologies. This chapter first discusses the context of the affinity chromatography with aptamer ligands. With the adaptation of SELEX, the chemical modifications of aptamers to comply with the covalent coupling and the separation process are then extensively presented. A focus is then made about the most important applications for protein separation with real-life examples and the comparison with immunoaffinity chromatography. In spite of well-advanced demonstrations and the extraordinary potential developments, a significant optimization work is still due to deserve large-scale applications with all necessary validations. Graphical Abstract Aptamer-protein complexes by X-ray crystallography.

Direct measurement of the mechanism by which magnesium specifically modifies the mechanical properties of DNA.

We examine the effect of physiological cations Na+, K+, Mg2+, and Ca2+ on the mechanical properties of bundles of λ-phage DNA using silicon nanotweezers (SNTs). Integrating SNTs with a microfluidic device allows us to perform titration experiments while measuring the effect in real-time. The results show that only for Mg2+ and in particular, at the intra-nuclear concentration (100 mM), the interaction occurs.

Kinetic analysis and binding studies of a new recombinant human factor VIIa for treatment of haemophilia.

INTRODUCTION/AIM: LR769 is a new second-generation recombinant human Factor VIIa (rhFVIIa) developed for haemophilia treatment. We determined enzymatic properties of LR769 and its interaction with antithrombin, tissue factor, platelets and endothelial protein C receptor (EPCR), compared with NovoSevenRT. METHODS: Kinetic enzyme assays and active site titration were used for enzymatic studies. Surface Plasmon Resonance (SPR) was used for determination of binding constants. Cellular binding was determined for platelets and cultured human umbilical vein endothelial cells (HUVEC). RESULTS: The dissociation constant (Kd ) for activated platelet binding was in the 1 µm range for both products. At saturation, more LR769 than NovoSevenRT was bound to the platelets. Binding to HUVEC was 25-50% higher for LR769 than for NovoSevenRT. Protein C, soluble EPCR, and anti-EPCR antibody all reduced the binding, indicating specificity for EPCR. LR769 was similar to NovoSevenRT in all kinetic assays. Active site titration demonstrated 0.7 mole of active site/mole of protein. The kcat /Km values for activation of FX and FIX with purified recombinant tissue factor and phospholipids were 10.5 s-1 /0.32 µm and 3.3 s-1 /0.44 µm respectively. The apparent second-order rate constant for inactivation by human plasma AT was 5.9 ± 0.4 × 103 m-1 s-1 . The Kd values for binding of LR769 to soluble tissue factor and full-length tissue factor were 8.1 nm and 0.9 nm, respectively, and the Kd for binding to soluble EPCR was 41 nm. CONCLUSION: Overall, LR769 exhibited characteristics similar to NovoSevenRT, but bound EPCR on HUVEC with somewhat higher affinity than NovoSevenRT.

3D-shape recognition and size measurement of irregular rough particles using multi-views interferometric out-of-focus imaging.

We realize simplified-tomography experiments on irregular rough particles using interferometric out-of-focus imaging. Using two angles of view, we determine the global 3D-shape, the dimensions, and the 3D-orientation of irregular rough particles whose morphologies belong to families such as sticks, plates, and crosses.

[About a case of uterine per-partum rupture, 37months after resection of a rectovaginal endometriosis nodule]. / À propos d'un cas de rupture utérine per-partum, 37 mois après une résection d'un nodule recto-vaginal d'endométriose.

Endometriosis is a common condition in women, whose main repercussions are painful symptoms. In addition, it was shown that endometriosis was a major cause of infertility and various obstetric complications could be related to this pathology. Uterine rupture is a rare but serious complication whose incidence tends to decrease with the screening of women at risk, however, its fetal, maternal morbidity and mortality causes remains important. We were confronted with a case of posterior uterine rupture in a patient of 36 years, primipare term exceeded in immediate postpartum period. The patient's primary antecedent of uterine surgery torus was responsible for infertility endometriosis. The outcome was favorable for the mother, after a surgical treatment by laparotomy, and for the child. In the literature, two cases have been reported of uterine rupture after endometriosis surgery, which is why we found it interesting to report this rare case. Given the increase in surgical management of this disease, it seems relevant to ask whether, in the future, we should be more vigilant in monitoring pregnancy for these women.

Dual-wavelength digital holography for 3D particle image velocimetry: experimental validation.

A multi-exposure digital in-line hologram of a particle field is recorded by two successive pulses of different wavelengths. During the reconstruction step, each recording can be independently analyzed by selecting a given wavelength. This procedure enables avoiding the superimposition of particle images that may be close to each other.

Simultaneous 3D location and size measurement of bubbles and sand particles in a flow using interferometric particle imaging.

We present a system to characterize a triphasic flow in a 3D volume (air bubbles and solid irregular particles in water) using only one CCD sensor. A cylindrical interferometric out-of-focus imaging setup is used to determine simultaneously the 3D position and the size of bubbles and irregular sand particles in a flow. The 3D position of the particles is deduced from the ellipticity of their out-of-focus image. The size of bubbles is deduced from analysis of interference fringes. The characteristics of irregular sand particles are obtained from analysis of their speckle-like pattern. Experiments are confirmed by simulations.

[Prediction of spontaneous preterm birth in symptomatic patients: A review]. / Prédiction de la prématurité en cas de menace d'accouchement prématuré: revue de la littérature.

OBJECTIVE OF THE REVIEW: To identify predictors of preterm delivery in the context of threatened preterm labour. MAIN POINTS: Tobacco use and previous history of preterm delivery are the main anamnestic elements to predict preterm birth. High positive predictive value of vaginal examination is restricted to cases with strong cervical alterations like dilatation over 4 cm. In case of discrete cervical alterations, literature confirms the great interest for cervical length ultrasonographic measurement as it reduces false positive cases. Absence of fetal respiratory movements appears to be as sensitive as cervical length and could be more specific but its clinical use remains rare. Vaginal detection of fetal fibronectin is the most useful biomarker with high negative predictive value (>90%). Fibronectin quantitative test seems to enhance the positive predictive value. No other biomarker is currently used in clinical practice. Electromyography and elastography of the cervix appear to be promising approaches.

In vitro and in vivo properties differ among liquid intravenous immunoglobulin preparations.

OBJECTIVE: To compare in vitro and in vivo biological and biochemical properties of five liquid intravenous immunoglobulin (IVIg) preparations licensed for therapeutic use in Europe. METHODS: ClairYg(®) was compared in a blinded manner to four other liquid IVIg preparations licensed in Europe (Octagam(®) , Kiovig(®) , Gamunex(®) , Privigen(®) ). Three batches of each preparation were tested, except for the IgG repertoires and the animal model. RESULTS: Levels of anti-A and anti-B antibodies were lower in ClairYg(®) (0·11/0·11) relative to a positive EDQM standard and Octagam(®) (0·11/0·08) than in other preparations (0·33-0·69/0·42-0·46). IgG in ClairYg(®) recognized 365 and 416 protein spots in HEp-2 cell and Escherichia coli protein extracts vs. 230-330 and 402-842 protein spots, respectively, for IgG in other preparations. IgA content (301 vs. 165-820 ng/mg of IgG), Factor XI and Factor XII antigen (0·46 vs. 0·85-2·40 mU/mg of IgG and 7·8 vs. 20·0-46·2 lU/mg of IgG) C1q binding (0·42 vs. 0·67-1·89 arbitrary units) and C5a uptake (0·41 vs. 0·45-0·66% of activation) were lower in ClairYg(®) than in other preparations. Finally, intravenous infusion of ClairYg(®) , Gamunex(®) and Privigen(®) had no major effect on arterial blood pressure in spontaneously hypertensive rats. CONCLUSIONS: Our results evidence some differences in the biological and biochemical properties among licensed liquid IVIg preparations.

Metabolomic profiling with NMR discriminates between biphosphonate and doxorubicin effects on B16 melanoma cells.

The metabolomic profiles of B16 melanoma cells were investigated in vitro with high resolution-magic angle spinning proton magnetic resonance spectroscopy and OPLS multivariate statistical analyse. We compared the profiles for untreated melanoma B16-F10 cells and Ca(2+) chelating EGTA, doxorubicin or BP7033 bisphosphonate treated cells. The two last molecules are known to induce anti-proliferative effects by different mechanisms of action in cells. Untreated and EGTA treated cells had similar profiles and were considered together as control cells. Several spectral regions could discriminate control from doxorubicin as well as BP7033 treated cells. Doxorubicin and BP7033 displayed distinct metabolic profiles. Important changes in neutral lipids and inositol were related to doxorubicin activity whereas BP7033 affected essentially phospholipids and alanine/lactate metabolism. These results provide new putative targets for both drugs. Metabolomics by NMR is shown here to be a good tool for the investigation of the mechanisms of action of drugs in pre-clinical studies.

Pharmacological strategies and micrometastasis: what is known? What must be done?

Metastasis accounts for over 90% of cancer patient deaths. In spite of this , the majority of the currently used drugs are aimed at eradicating or controlling the primary tumor. Hardly any cancer therapies used at the moment interfere only with metastasis and there is a need for drugs acting specifically on the metastatic processes, curing the minimal residual disease and being less toxic than the current drugs. Searching for drugs that do not affect the growth of the primary tumor but inhibit the colonization and/or the outgrowth in distant sites could lead to new drugs specifically aimed at treating the minimal residual disease. The aim of this work is to search at what conditions such drugs might be discovered and if some exist already. For that, mechanisms of actions of current experimental and clinical antimetastatic drugs are described and their utility for different clinical settings (primary prevention, adjuvant setting) is analyzed. In conclusion, clues exist that specific antimetastatic drugs could exist and will be developed in a near future.

New pharmacological strategies against metastatic spread.

Although metastatic spread is the most frequent cause of death in cancer patients, there are very few drugs specifically targeting this process. Bases for a new antimetastatic drug discovery strategy are weak because a great number of unknowns characterize the complete understanding of the metastatic cascade mechanisms. Moreover, the current experimental models are too simplistic and do not account for the complexity of the phenomenon. Some targets have been identified but too few are validated. Among them, the metastasis suppressor genes seem to be the most promising. In spite of this, during recent years, a dozen of molecules, which fulfil the definition of a specific metastatic drug that inhibits the metastases without altering the growth of the primary tumour (which can be eradicated by surgery), have been identified and assessed for the proof of the concept. The continuation of this effort would benefit in terms of efficiency, if the objectives were defined more precisely. It is particularly important to distinguish molecules that prevent spread of the metastatic cells of the early-stage primary tumour from the ones which induce a regression of the established metastases or to inhibit the transition from disseminated occult tumour cells to dormant micrometastasis. This second goal is a priori more relevant in the current clinical setting where the detection of early metastatic spread is very difficult, and therefore would call for greater effort on the part of the scientific community.

[Metastatic spread: mechanisms and therapies]. / Les mécanismes de la dissémination métastatique et ses thérapies.

Although metastatic spread is the most frequent cause of deaths in cancer patients, there are very few drugs specifically targeting this process. Bases for a new antimetastatic drug discovery strategy are weak because a great number of unknowns characterizes the whole understanding of the metastatic cascade mechanisms. Moreover, the current experimental models are too simplistic and do not account for the complexity of the phenomenon. Some targets have been identified but too few are validated. Among them, metastasis suppressor genes seem to be the most promising. In spite of this, during the last years, a dozen of molecules which fulfill the definition of a specific metastatic drug, namely that inhibit metastases without altering growth of the primary tumor (which can be eradicated by surgery), have been identified and tried out to assess the proof of the concept. The continuation of this effort would be more efficient if the objectives were defined more precisely. It is particularly important to distinguish molecules aimed at preventing metastic cell spreading at the primary tumour early stage and molecules which have to induce a regression of established metastases or to inhibit the transition from disseminated occult tumour cells to dormant micrometastasis. This second goal is a priori more relevant in the current clinical setting where detection of the early metastatic spread is very difficult, and therefore it should focus a greater effort of the scientific community.

Microsatellite instability and sensitivitiy to FOLFOX treatment in metastatic colorectal cancer.

BACKGROUND: Microsatelite instability (MSI) is the consequence of the inactivation of a mismatch repair gene and is observed in approximately 15% of colon cancer cases. Patients with MSI colon cancer do not benefit from 5-fluorouracil (5-FU)-based chemotherapy. A current treatment of reference for colon cancer is a combination of 5-FU and oxaliplatin (FOLFOX). The aim of this study was to determine the efficiency of the FOLFOX treatment in patients with metastatic MSI colon cancer. PATIENTS AND METHODS: Tumour specimens were collected from patients with metastatic colon cancer treated with FOLFOX 4 modified or FOLFOX 6; these two regimens are based on 85 mg/m2 and 100 mg/m2 oxaliplatin, respectively. The MSI status was assessed by measuring the length of five monomorphic mononucleotide markers. The FOLFOX regimen was evaluated as a first-line treatment according to WHO criteria. RESULTS: Forty patients (22 men, 18 women), median age 63.5 years (27-83 years) were treated with FOLFOX 4 or 6. Nine patients had tumours exhibiting high MSI (MSI group) and 31 patients had tumours exhibiting microsatellite stability (MSS group). In the MSS group, 11 partial responses (36%) were observed, while there were only two in the MSI group (22%) (no significant difference). The two patients who were responders in the MSI group were treated with FOLFOX 6. The overall survival was not significantly different for MSI and MSS patients. CONCLUSION: No significant differences in the overall response rate or overall survival between the two groups of patients were observed. However, these results suggest that patients with MSI colon cancer are more sensitive to a higher dose of FOLFOX.

Microvessel density and VEGF expression are prognostic factors in colorectal cancer. Meta-analysis of the literature.

We performed a meta-analysis of all published studies relating intratumoural microvessel density (MVD) (45 studies) or vascular endothelial growth factor (VEGF) expression (27 studies), both reflecting angiogenesis, to relapse free (RFS) and overall survival (OS) in colorectal cancer (CRC). For each study, MVD impact was measured by risk ratio between the two survival distributions with median MVD as cutoff. Eleven studies did not mention survival data or fit inclusion criteria, six were multiple publications of same series, leaving 32 independent studies for MVD (3496 patients) and 18 for VEGF (2050 patients). Microvessel density was assessed by immunohistochemistry, using antibodies against factor VIII (16 studies), CD31 (10 studies) or CD34 (seven studies). Vascular endothelial growth factor expression was mostly assessed by immunohistochemistry. Statistics were performed for MVD in 22 studies (the others lacking survival statistics) including nine studies (n = 957) for RFS and 18 for OS (n = 2383) and for VEGF in 17 studies, including nine studies for RFS (n = 1064) and 10 for OS (n = 1301). High MVD significantly predicted poor RFS (RR = 2.32 95% CI: 1.39-3.90; P < 0.001) and OS (RR = 1.44; 95% CI: 1.08-1.92; P = 0.01). Using CD31 or CD34, MVD was inversely related to survival, whereas it was not using factor VIII. Vascular endothelial growth factor expression significantly predicted poor RFS (RR = 2.84; 95% CI: 1.95-4.16) and OS (RR=1.65; 95% CI: 1.27-2.14). To strengthen our findings, future prospective studies should explore the relation between MVD or VEGF expression and survival or response to therapy (e.g. antiangiogenic therapy). Assessment of these angiogenic markers should be better standardised in future studies.

Sudden, unexpected death due to undiagnosed frontal glioblastoma in a schizophrenic patient.

The incidence of sudden death due to undiagnosed primary intracranial tumor is low in forensic autopsy. We report a case of a 48-year-old white male, known to be a schizophrenic patient for several years, and in whom a medico-legal autopsy disclosed a large, previously undiagnosed, bilateral frontal glioblastoma infiltrating the genu of corpus callosum. We emphasize the importance of performing complete autopsy, including a thorough neuropathological examination, in all cases of sudden unexpected death, especially in those cases in which no extracerebral cause of death had been established and whose clinical history was primarily of a psychiatric nature.

Integrative analysis of gene expression patterns predicts specific modulations of defined cell functions by estrogen and tamoxifen in MCF7 breast cancer cells.

To explore the mechanisms whereby estrogen and antiestrogen (tamoxifen (TAM)) can regulate breast cancer cell growth, we investigated gene expression changes in MCF7 cells treated with 17beta-estradiol (E2) and/or with 4-OH-TAM. The patterns of differential expression were determined by the ValiGen Gene IDentification (VGID) process, a subtractive hybridization approach combined with microarray validation screening. Their possible biologic consequences were evaluated by integrative data analysis. Over 1000 cDNA inserts were isolated and subsequently cloned, sequenced and analyzed against nucleotide and protein databases (NT/NR/EST) with BLAST software. We revealed that E2 induced differential expression of 279 known and 28 unknown sequences, whereas TAM affected the expression of 286 known and 14 unknown sequences. Integrative data analysis singled out a set of 32 differentially expressed genes apparently involved in broad cellular mechanisms. The presence of E2 modulated the expression patterns of 23 genes involved in anchors and junction remodeling; extracellular matrix (ECM) degradation; cell cycle progression, including G1/S check point and S-phase regulation; and synthesis of genotoxic metabolites. In tumor cells, these four mechanisms are associated with the acquisition of a motile and invasive phenotype. TAM partly reversed the E2-induced differential expression patterns and consequently restored most of the biologic functions deregulated by E2, except the mechanisms associated with cell cycle progression. Furthermore, we found that TAM affects the expression of nine additional genes associated with cytoskeletal remodeling, DNA repair, active estrogen receptor formation and growth factor synthesis, and mitogenic pathways. These modulatory effects of E2 and TAM upon the gene expression patterns identified here could explain some of the mechanisms associated with the acquisition of a more aggressive phenotype by breast cancer cells, such as E2-independent growth and TAM resistance.

Carboxymethyl benzylamide dextran inhibits angiogenesis and growth of VEGF-overexpressing human epidermoid carcinoma xenograft in nude mice.

Vascular endothelial growth factor (VEGF) expression is elevated in a wide variety of solid tumours. Inhibition of VEGF activities is able to reduce angiogenesis and tumour growth. We have recently shown in vitro that carboxymethyl dextran benzylamide (CMDB7) prevents the binding of VEGF(165) to its cell surface receptors and thus inhibits VEGF activities on endothelial cells. In the present study, we explored the effects of CMDB7 on highly aggressive human epidermoid carcinoma A431 cells known to overexpress epidermal growth factor receptors (EGFRs) and produce a high amount of VEGF and a minor quantity of bFGF. In vitro, CMDB7 blocked the mitogenic activity of A431-conditioned medium on endothelial cells. Concerning A431 cells, CMDB7 inhibited their proliferation and the VEGF(165) binding to them. In vivo, administration of CMDB7 (10 mg kg(-1)) three times per week for 2 weeks inhibited the growth of A431 xenografts in nude mice by 73% as compared to the control group. Immunostaining of endothelial cells with mouse-specific GSL-1 lectin in tumour sections revealed that CMDB7 also inhibited the density of intratumour endothelial cells by 66%. These findings demonstrate that CMDB7 has an efficient antiangiogenic and antitumour action in vivo even when tumour cells produce a high level of VEGF and EGFRs.

Inhibition of epidermoid carcinoma A431 cell growth and angiogenesis in nude mice by early and late treatment with a novel dextran derivative.

We investigated the effect of a new dextran derivative, phenylacetate carboxymethyl benzylamide dextran (NaPaC), on epidermoid carcinoma A431 cells secreting a large quantity of angiogenic factor, vascular endothelial growth factor (VEGF). In vitro, NaPaC inhibited the proliferation of A431 cells (IC(50)=5 micro M). Also, NaPaC decreased the binding of radiolabelled VEGF(165) to endothelial cells (IC(50)=0.2 micro M). In vivo, we explored the effects of NaPaC (15 mg kg(-1)) on A431 xenograft growth starting the drug administration at the time of tumour cell inoculation (early treatment) and 1 week later, when tumours were well established (late treatment). Early treatment was more efficient on tumour inhibition (70% vs control) than late treatment (50% vs control). Early and late NaPaC-treatment increased the aponecrosis in tumour by 70 and 30%, respectively. Whatever treatment, NaPaC inhibited the intratumour endothelial cell density in the same manner. In contrast, vessel area was decreased only when NaPaC was injected early (35%). These results show that NaPaC has a potent inhibitory effect, dependent on treatment outset, on epidermoid carcinoma growth associated with an intratumour microvascular network diminution and an aponecrosis increase. As this drug is nontoxic at efficient dose, it offers interesting perspectives for the therapy of malignant lesions.