Drug-resistant epimastigotes of Trypanosoma cruzi and persistence of this phenotype after differentiation into amastigotes

Des formes épimastigotes du parasite #Trypanosoma cruzi ont été rendues résistantes au benznidazole en utilisant, in vitro, une pression médicamenteuse continue. Les parasites ont été sélectionnés en fonction de la caractérisation génétique antérieure de leurs loci isoenzymatiques. Tous les clones résistants sont capables de croître en culture à long terme en présence d'une concentration d'au moins 50 microM de benznidazole qui est la dose circulante de drogue chez un patient en cours de traitement. Le niveau le plus élevé de résistance atteint est 220 microM. Après la différenciation des formes épimastigotes en amastigotes, le niveau de résistance n'est pas altéré. La vitesse de croissance des formes résistantes d'épimastigotes et d'amastigotes est inférieure à celle des formes sauvages mais leur viabilité n'est pas affectée. Ces résultats peuvent avoir des conséquences à la fois pour entreprendre l'étude du phénotype résistant de clones du parasite #T. cruzi mais également pour suivre le devenir de ce phénotype résistant au cours du développement du cycle expérimental in vivo. (Résumé d'auteur

A focused antibody library for selecting scFvs expressed at high levels in the cytoplasm

<p>Abstract</p> <p>Background</p> <p>Intrabodies are defined as antibody molecules which are ectopically expressed inside the cell. Such intrabodies can be used to visualize or inhibit the targeted antigen in living cells. However, most antibody fragments cannot be used as intrabodies because they do not fold under the reducing conditions of the cell cytosol and nucleus.</p> <p>Results</p> <p>We describe the construction and validation of a large synthetic human single chain antibody fragment library based on a unique framework and optimized for cytoplasmic expression. Focusing the library by mimicking the natural diversity of CDR3 loops ensured that the scFvs were fully human and functional. We show that the library is highly diverse and functional since it has been possible to isolate by phage-display several strong binders against the five proteins tested in this study, the Syk and Aurora-A protein kinases, the αβ tubulin dimer, the papillomavirus E6 protein and the core histones. Some of the selected scFvs are expressed at an exceptional high level in the bacterial cytoplasm, allowing the purification of 1 mg of active scFv from only 20 ml of culture. Finally, we show that after three rounds of selection against core histones, more than half of the selected scFvs were active when expressed <it>in vivo </it>in human cells since they were essentially localized in the nucleus.</p> <p>Conclusion</p> <p>This new library is a promising tool not only for an easy and large-scale selection of functional intrabodies but also for the isolation of highly expressed scFvs that could be used in numerous biotechnological and therapeutic applications.</p

Selective BuChE Inhibitory Activity, Chemical Composition, and Enantiomeric Content of the Essential Oil from Salvia leucantha Cav. Collected in Ecuador

The essential oil (EO) of Salvia leucantha Cav. was isolated by steam distillation of the aerial parts collected in the South of Ecuador. Its physical properties were evaluated and the chemical composition of the oil was determined by GC-MS and GC-FID analyses using two chromatographic columns, DB-5ms and HP-INNOWax. Six major compounds were identified, namely, the sesquiterpenes 6.9-guaiadiene (19.14%), (E)-caryophyllene (16.80%), germacrene D (10.22%), (E)-β-farnesene (10.00%), and bicyclogermacrene (7.52%), and the monoterpenoid bornyl acetate (14.74%). Furthermore, four pairs of enantiomers were determined by enantioselective GC-MS of the essential oil. (−)-germacrene D and (+)-α-pinene showed the highest enantiomeric excess (ee%). In an in vitro assay, the essential oil demonstrated an interesting inhibitory activity of the enzyme butyrylcholinesterase (BuChE), with an IC50 = 32.60 µg/mL, which is the highest determined for a Salvia species. In contrast, the oil was weakly active against acetylcholinesterase (AChE) with an IC50 > 250 µg/m

Constituents and Selective BuChE Inhibitory Activity of the Essential Oil from Hypericum aciculare Kunth

A potential source of new inhibitors of cholinesterase enzymes are certain compounds of natural plant origin; therefore, in the study described herein we have determined the chemical composition and the acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) inhibitory activities of the essential oil (EO) steam distilled from aerial parts of Hypericum aciculare, which was collected in southern Ecuador. The oil qualitative and quantitative composition was determined by GC-FID and GC-MS using a non-polar and a polar chromatographic column. A total of fifty-three constituents were identified, that accounted for about 98% of the EO content. The hydrocarbon n-nonane (16.4–28.7%) and the aldehyde n-decanal (20.7–23.1%) were the predominant oil constituents. In addition, the EO showed significant inhibition of BuChE (IC50 = 28.3 ± 2.7 μg/mL) and moderate activity towards AChE (IC50 = 82.1 ± 12.1 µg/mL). Thus, the EO from H. aciculare aerial parts is an interesting candidate to investigate the mechanism of selective ChE inhibition by the two ChE enzymes with the aim to discover potential targets to control the progression of the Alzheimer’s disease (AD)

Overexpression of phosphoserine aminotransferase PSAT1 stimulates cell growth and increases chemoresistance of colon cancer cells

International audienceABSTRACT: BACKGROUND: Colorectal cancer (CRC) is one of the most common causes of cancer death throughout the world. In this work our aim was to study the role of the phosphoserine aminotransferase PSAT1 in colorectal cancer development. RESULTS: We first observed that PSAT1 is overexpressed in colon tumors. In addition, we showed that after drug treatment, PSAT1 expression level in hepatic metastases increased in non responder and decreased in responder patients. In experiments using human cell lines, we showed that ectopic PSAT1 overexpression in colon carcinoma SW480 cell line resulted in an increase in its growth rate and survival. In addition, SW480-PSAT1 cells presented a higher tumorigenic potential than SW480 control cells in xenografted mice. Moreover, the SW480-PSAT1 cell line was more resistant to oxaliplatin treatment than the non-transfected SW480 cell line. This resistance resulted from a decrease in the apoptotic response and in the mitotic catastrophes induced by the drug treatment. CONCLUSIONS: These results show that an enzyme playing a role in the L-serine biosynthesis could be implicated in colon cancer progression and chemoresistance and indicate that PSAT1 represents a new interesting target for CRC therapy

Improving dental epithelial junction on dental implants with bioengineered peptides

Introduction: The functionalization of titanium (Ti) and titanium alloys (Ti6Al4V) implant surfaces via material-specific peptides influence host/biomaterial interaction. The impact of using peptides as molecular linkers between cells and implant material to improve keratinocyte adhesion is reported.Results: The metal binding peptides (MBP-1, MBP-2) SVSVGMKPSPRP and WDPPTLKRPVSP were selected via phage display and combined with laminin-5 or E-cadherin epithelial cell specific peptides (CSP-1, CSP-2) to engineer four metal-cell specific peptides (MCSPs). Single-cell force spectroscopy and cell adhesion experiments were performed to select the most promising candidate. In vivo tests using the dental implant for rats showed that the selected bi functional peptide not only enabled stable cell adhesion on the trans-gingival part of the dental implant but also arrested the unwanted apical migration of epithelial cells.Conclusion: The results demonstrated the outstanding performance of the bioengineered peptide in improving epithelial adhesion to Ti based implants and pointed towards promising new opportunities for applications in clinical practice

Improving dental epithelial junction on dental implants with bioengineered peptides

Introduction: The functionalization of titanium (Ti) and titanium alloys (Ti6Al4V) implant surfaces via material-specific peptides influence host/biomaterial interaction. The impact of using peptides as molecular linkers between cells and implant material to improve keratinocyte adhesion is reported.Results: The metal binding peptides (MBP-1, MBP-2) SVSVGMKPSPRP and WDPPTLKRPVSP were selected via phage display and combined with laminin-5 or E-cadherin epithelial cell specific peptides (CSP-1, CSP-2) to engineer four metal-cell specific peptides (MCSPs). Single-cell force spectroscopy and cell adhesion experiments were performed to select the most promising candidate. In vivo tests using the dental implant for rats showed that the selected bi functional peptide not only enabled stable cell adhesion on the trans-gingival part of the dental implant but also arrested the unwanted apical migration of epithelial cells.Conclusion: The results demonstrated the outstanding performance of the bioengineered peptide in improving epithelial adhesion to Ti based implants and pointed towards promising new opportunities for applications in clinical practice

Seismotectonics of southeast France: from the Jura mountains to Corsica

The analysis of the seismicity catalog (1996 to 2019) covering the region from the Jura mountains to Corsica provides a first-order image of the distribution of earthquakes, highlighting large structures such as the Briançonnais and Piedmontais seismic arcs, the eastward deepening of the focal depths through the Western Alps, several large active faults (e.g. Belledonne, Middle Durance, Ligure). Over this period the magnitudes are moderate and the focal mechanisms of the main events display a diversity of seismic behaviors that can be explained by the complexity of the different geological domains with a more or less strong structural inheritage, by variable rheological characteristics at the scale of the crust and by the joint action of different mechanisms of deformation. The distribution of the historical events is in fairly good agreement with the instrumental seismicity, but several earthquakes of $M >6$ are highlighted since the 14th century until the beginning of the 20th

Seismotectonics of southeast France: from the Jura mountains to Corsica

The analysis of the seismicity catalog (1996 to 2019) covering the region from the Jura mountains to Corsica provides a first-order image of the distribution of earthquakes, highlighting large structures such as the Briançonnais and Piedmontais seismic arcs, the eastward deepening of the focal depths through the Western Alps, several large active faults (e.g. Belledonne, Middle Durance, Ligure). Over this period the magnitudes are moderate and the focal mechanisms of the main events display a diversity of seismic behaviors that can be explained by the complexity of the different geological domains with a more or less strong structural inheritage, by variable rheological characteristics at the scale of the crust and by the joint action of different mechanisms of deformation. The distribution of the historical events is in fairly good agreement with the instrumental seismicity, but several earthquakes of $M >6$ are highlighted since the 14th century until the beginning of the 20th