Methods for producing active scfv antibodies and libraries therefor

The present disclosure describes scFv antibody libraries, antibodies isolated from the libraries, and methods of producing and using the same.Extensions au Japon (n°5,385,796), aux USA et au Canada. Licence concédée à 2 sociétés (2012 et 2013

Association between Mycoplasma genitalium infection and HIV acquisition among female sex workers in Uganda: evidence from a nested case-control study.

OBJECTIVES: Cross-sectional studies have shown a strong association between Mycoplasma genitalium and HIV infections. We previously reported that in a cohort of female sex workers in Uganda, M genitalium infection at baseline was associated with HIV seroconversion. Here we examine the temporal association between the M genitalium infection status shortly before HIV seroconversion and HIV acquisition. METHODS: A nested case-control study was conducted within a cohort of women at high risk for HIV in Kampala. Cases were those of women acquiring HIV within 2 years of enrolment. For each of the 42 cases, 3 controls were selected from women HIV negative at the visit when the corresponding case first tested HIV seropositive. The association between HIV acquisition and M genitalium infection immediately prior to HIV testing was analysed using conditional logistic regression. RESULTS: There was weak evidence of an association between M genitalium infection and HIV acquisition overall (crude OR=1.57; 95% CI 0.67 to 3.72, aOR=2.28: 95% CI 0.81 to 6.47). However, time of M genitalium testing affected the association (p value for effect-modification=0.004). For 29 case-control sets with endocervical samples tested 3 months prior to the first HIV-positive result, M genitalium infection increased the risk of HIV acquisition (crude OR=3.09; 95% CI 1.06 to 9.05, aOR=7.19; 95% CI 1.68 to 30.77), whereas there was little evidence of an association among the 13 case-control sets with samples tested at an earlier visit (crude OR=0.30: 95% CI 0.04 to 2.51; aOR=0.34; 95% CI 0.02 to 5.94). CONCLUSIONS: Our study showed evidence of a temporal relationship between M genitalium infection and HIV acquisition that suggests that M genitalium infection may be a co-factor in the acquisition of HIV infection

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

Proton Sponge Trick for pH-Sensitive Disassembly of Polyethylenimine-Based siRNA Delivery Systems

Small interfering RNAs offer novel opportunities to inhibit gene expression in a highly selective and efficient manner but depend on cytosolic translocation with synthetic delivery systems. The polyethylenimine (PEI) is widely used for plasmid DNA transfection. However, the water-soluble PEI does not form siRNA polyplexes stable enough in extracellular media for effective delivery. We previously showed that rendering PEI insoluble in physiological media, without modifying drastically its overall cationic charge density, by simple conjugation with natural hydrophobic α-amino acids, can lead to effective siRNA delivery in mammalian cells. In here, we comprehensively investigated the mechanism behind the excellent efficacy of the leading PEIY vector. Our data revealed that the underlining proton sponge property is key to the effectiveness of the tyrosine−polyethylenimine conjugate as it may allow both endosomal rupture and siRNA liberation via an optimal pH-sensitive dissolution of the PEIY self-aggregates. Altogether, these results should facilitate the development of novel and more sophisticated siRNA delivery systems

Medieval coastal Syrian vegetation patterns in the principality of Antioch

The coastal area of Jableh, in the vicinity of the Saladin and Al-Marquab castles, is a fertile alluvial plain located on the northwestern part of Syria, in what was once the crusader Principality of Antioch. In order to detail the coastal environment during the crusader period in the Middle East, palynological analyses have been conducted on the underlying coastal-alluvial deposits. The recovered sediments represent a continuous record of the environmental history of the area spanning a c. AD 850—1850 cal. yr period, from the Muslim Era up to and including the late Ottoman times. During the local crusader period (AD 1100—1270), the area was dominated by an arborescent mattoral mixed with a xerophytic shrub-steppe. The alluvial plain was slightly waterlogged and colonized by a wetland meadow with an open vegetation of steppe-like character on bare surfaces and fresh arable soils. The riparian and open deciduous riverine forests were weakly developed. Signs of agricultural activities are mainly recorded for the High Medieval period (AD 1000—1300), with an increase of vineyards in the coastal area. Since c. AD 1250 cal. yr until the end of the crusader period, agricultural activities never reached the same intensity as during the Mameluke Sultanate and the Ottoman Empire

Amenability of groups and GG-sets

This text surveys classical and recent results in the field of amenability of groups, from a combinatorial standpoint. It has served as the support of courses at the University of G\"ottingen and the \'Ecole Normale Sup\'erieure. The goals of the text are (1) to be as self-contained as possible, so as to serve as a good introduction for newcomers to the field; (2) to stress the use of combinatorial tools, in collaboration with functional analysis, probability etc., with discrete groups in focus; (3) to consider from the beginning the more general notion of amenable actions; (4) to describe recent classes of examples, and in particular groups acting on Cantor sets and topological full groups

Live cell immunogold labelling of RNA polymerase II

Labeling nuclear proteins with electron dense probes in living cells has been a major challenge due to their inability to penetrate into nuclei. We developed a lipid-based approach for delivering antibodies coupled to 0.8 nm ultrasmall gold particles into the nucleus to label RNA polymerase II. Focussed Ion Beam slicing coupled to Scanning Electron Microscopy (FIB/SEM) enabled visualization of entire cells with probe localization accuracy in the 10 nm range

Chronic multifocal non-bacterial osteomyelitis in hypophosphatasia mimicking malignancy

BACKGROUND: Hypophosphatasia (HP) is characterized by a genetic defect in the tissue-nonspecific alkaline phosphatase (TNSALP) gene and predominantly an autosomal recessive trait. HP patients suffer from reduced bone mineralization. Biochemically, elevated concentrations of substrates of TNSALP, including pyridoxal-5'-phosphate and inorganic pyrophosphate occur in serum, tissues and urine. The latter has been associated with chronic inflammation and hyperprostaglandinism. CASE PRESENTATION: We report on 2 affected children presenting with multifocal inflammatory bone lesions mimicking malignancy: A 6 years old girl with short stature had been treated with human growth hormone since 6 months. Then she started to complain about a painful swelling of her left cheek. MRI suggested a malignant bone lesion. Bone biopsy, however, revealed chronic inflammation. A bone scan showed a second rib lesion. Since biopsy was sterile, the descriptive diagnosis of chronic non-bacterial osteomyelitis (CNO) was established. The diagnostic tests related to growth failure were repeated and subsequent analyses demonstrated a molecular defect in the TNSALP gene. The second girl (10 years old) complained about back pain after she had fallen from her bike. X rays of her spine revealed compressions of 2 thoracic vertebrae. At first these were considered trauma related, however a bone scan did show an additional lesion in the right 4(th )rib. A biopsy of this rib revealed a sterile lympho- plasmocytoid osteomyelitis suggesting multifocal CNO. Further analyses did show a decreased TNSALP in leukocytes and elevated pyridoxal phosphate in plasma, suggesting a heterozygous carrier status of HP. CONCLUSION: Chronic bone oedema in adult HP and chronic hyper-prostaglandinism in childhood HP do suggest that in some HP patients bone inflammation is present in conjunction with the metabolic defect. Sterile multifocal osteomyelitis could be demonstrated. Non-steroidal anti-inflammatory treatment achieved complete remission. These cases illustrate chronic inflammation of the bone as a new feature of HP

Synergy Between Intercellular Communication and Intracellular Ca2+ Handling in Arrhythmogenesis

Calcium is the primary signalling component of excitation-contraction coupling, the process linking electrical excitability of cardiac muscle cells to coordinated contraction of the heart. Understanding Ca2þ handling processes at the cellular level and the role of intercellular communication in the emergence of multicellular synchronization are key aspects in the study of arrhythmias. To probe these mechanisms, we have simulated cellular interactions on large scale arrays that mimic cardiac tissue, and where individual cells are represented by a mathematical model of intracellular Ca2þ dynamics. Theoretical predictions successfully reproduced experimental findings and provide novel insights on the action of two pharmacological agents (ionomycin and verapamil) that modulate Ca2þ signalling pathways via distinct mechanisms. Computational results have demonstrated how transitions between local synchronisation events and large scale wave formation are affected by these agents. Entrainment phenomena are shown to be linked to both ntracellular Ca2þ and coupling-specific dynamics in a synergistic manner. The intrinsic variability of the cellular matrix is also shown to affect emergent patterns of rhythmicity, providing insights into the origins of arrhythmogenic Ca2þ perturbations in cardiac tissue in situ