Random antral follicle count performed on any day of the menstrual cycle has the same predictive value as AMH for good ovarian response in IVF cycles.

OBJECTIVE: To determine whether the predictive value of AFC for ovarian response to stimulation for IVF depends on the day of the menstrual cycle when ultrasound is performed. METHODS: 410 women undergoing their first IVF cycle were included. All the women had AFC performed twice. The first measurement, random AFC (r-AFC), was performed during the fertility workup whatever the day of their menstrual cycle. Three groups were constituted according to the period of ultrasound performance: at early follicular phase i.e., day 1 to day 6 (eFP-AFC); at mid follicular phase i.e., day 7 to 12 (mFP-AFC) and at luteal phase i.e., day 13 or after (LP-AFC). A second AFC measurement was performed before the start of the ovarian stimulation (SD1-AFC). AMH dosing was done in the early follicular phase. RESULTS: Random AFC (r-AFC) was correlated to AMH (r = 0.69; p<0.001), SD1-AFC (r = 0.75; p<0.001) and number of oocytes retrieved (r = 0.49; p<0.001). When regarding AFC depending on the cycle day group, the correlation with AMH was 0.65, 0.66 and 0.85 for the eFP-AFC, the mFP-AFC and the LP-AFC respectively (all p were <0.001). The ROC analysis showed the same predictive value for good ovarian response (more than 6 oocytes retrieved) for the eFP-AFC, mFP-AFC and LP-AFC (AUC 0.73, 0.75 and 0.84 respectively; p = 0.28). The AUC of r-AFC (0.76) were similar to those of AMH (0.74) and SD1-AFC (0.74) (p = 0.21 and 0.92 respectively). CONCLUSION: AFC is strongly correlated with AMH and highly predictive of good ovarian response during the whole menstrual cycle.

Nascent teichoic acids insertion into the cell wall directs the localization and activity of the major pneumococcal autolysin LytA.

The bacterial cell wall is in part composed of the peptidoglycan (PG) layer that maintains the cell shape and sustains the basic cellular processes of growth and division. The cell wall of Gram-positive bacteria also carries teichoic acids (TAs). In this work, we investigated how TAs contribute to the structuration of the PG network through the modulation of PG hydrolytic enzymes in the context of the Gram-positive Streptococcus pneumoniae bacterium. Pneumococcal TAs are decorated by phosphorylcholine residues which serve as anchors for the Choline-Binding Proteins, some of them acting as PG hydrolases, like the major autolysin LytA. Their binding is non covalent and reversible, a property that allows easy manipulation of the system. In this work, we show that the release of LytA occurs independently from its amidase activity. Furthermore, LytA fused to GFP was expressed in pneumococcal cells and showed different localization patterns according to the growth phase. Importantly, we demonstrate that TAs modulate the enzymatic activity of LytA since a low level of TAs present at the cell surface triggers LytA sensitivity in growing pneumococcal cells. We previously developed a method to label nascent TAs in live cells revealing that the insertion of TAs into the cell wall occurs at the mid-cell. In conclusion, we demonstrate that nascent TAs inserted in the cell wall at the division site are the specific receptors of LytA, tuning in this way the positioning of LytA at the appropriate place at the cell surface.

Specific and spatial labeling of choline-containing teichoic acids in Streptococcus pneumoniae by click chemistry.

Propargyl-choline was efficiently incorporated into teichoic acid (TA) polymers on the surface of Streptococcus pneumoniae. The introduction of a fluorophore by click chemistry enabled sufficient labeling of the pneumococcus, as well as its specific detection when mixed with other bacterial species. The labeling is localized at the septal site, suggesting a similar location of the TA and peptidoglycan (PG) synthetic machineries. This method is a unique opportunity to improve our understanding of the spatial location of pneumococcal TA biosynthesis.

Radiological assessment of the placement of Essure® in order to reduce hysterosalpingography: Pelvic X-ray versus combined pelvic X-ray and ultrasound.

OBJECTIVE: Three months after hysteroscopic sterilisation with Essure®, a confirmation test is required to evaluate the correct location of the inserts. The test may be conducted using a pelvic ultrasound (2D or 3D) or an abdominal X-ray. Should the location not look satisfactory on these tests, a follow-up hysterosalpingography (HSG) would be required. The objective of our study is to assess whether the Essure® 3-month confirmation test using a single X-ray or a combination of X-ray and ultrasound could reduce the use of HSG. STUDY DESIGN: This retrospective study examined patients who underwent birth control Essure® procedure between 2009 and 2015 in the Gynaecological Surgery Department at the Regional University Hospital Centre (CHRU) in Lille. We divided patients into two groups based on the imaging tests performed: single X-ray (2009-2010) versus X-ray and pelvic ultrasound (2014-2015). We then compared the results of the imaging tests and the use of HSG between the two groups. RESULTS: One hundred and thirty-four patients were tested, of which 60 (44.8%) using a single X-ray and 74 (55.2%) using a combination of X-ray and ultrasound. We note that the combined X-ray/ultrasound test reduces significantly the number of HSG performed (26.7% versus 12.2%, P=0.04). CONCLUSION: Compared to a single X-ray, the combination of X-ray and ultrasound enables to significantly limit the use of HSG.

Autocatalytic association of proteins by covalent bond formation: a Bio Molecular Welding toolbox derived from a bacterial adhesin.

Unusual intramolecular cross-links present in adhesins from Gram-positive bacteria have been used to develop a generic process amenable to biotechnology applications. Based on the crystal structure of RrgA, the Streptococcus pneumoniae pilus adhesin, we provide evidence that two engineered protein fragments retain their ability to associate covalently with high specificity, in vivo and in vitro, once isolated from the parent protein. We determined the optimal conditions for the assembly of the complex and we solved its crystal structure at 2 Å. Furthermore, we demonstrate biotechnological applications related to antibody production, nanoassembly and cell-surface labeling based on this process we named Bio Molecular Welding.

[Are ultrasound measurements of the cervical length and fetal head-perineum distance predictive of delivery outcome in post-term pregnancies?]. / Les mesures échographiques de la longueur cervicale et de la hauteur de la présentation fœtale sont-elles prédictives de l'issue d'accouchement chez des patientes à terme ?

OBJECTIVE: Post-term pregnancy is frequently associated with higher fetal and maternal morbidity and mortality. Its management essentially depends on clinical cervical characteristics as evaluated by the Bishop score (BS). However, BS is poorly predictive of the delivery outcome. We sought to demonstrate that ultrasound measurement of cervical length and evaluation of fetal height could predict the outcome in post-term pregnancies. METHODS: A prospective single center study was undertaken between the 21st of January and the 1st of June 2013. Fetal height was measured using a transperineal technique and cervical length was evaluated by a vaginal ultrasound on patients consulting and their term date. C-section rates were considered to be the primary judgment criteria. RESULTS: A total of 136 patients were included. C-section rates in this population was 19%. Fetal height and cervical length were not different between the C-section group and the vaginal delivery group. CONCLUSION: Our study demonstrates that ultrasound measurement of cervical length and fetal height do not show better results than BS in predicting the outcome of post-term pregnancy. Combining these ultrasound measurements has already been suggested in other studies and promising results have been shown. More studies are necessary to further these results.

Recombinant expression of the precursor of the hemorrhagic metalloproteinase HF3 and its non-catalytic domains using a cell-free synthesis system

Snake venom metalloproteinases (SVMPs) participate in snakebite pathology such as hemorrhage, inflammation, and necrosis. They are synthesized as latent multi-domain precursors whose processing generates either catalytically active enzymes or free non-enzymatic domains. Recombinant expression of the precursor of P-III class SVMPs has failed due to the instability of the multi-domain polypeptide structure. Conversely, functional recombinant non-catalytic domains were obtained by prokaryotic expression systems. Here, we show for the first time the recombinant expression of the precursor of HF3, a highly hemorrhagic SVMP from Bothrops jararaca, and its non-catalytic domains, using an E. coli-based cell-free synthesis system. The precursor of HF3, composed of pro-, metalloproteinase-, disintegrin-like-, and cysteine-rich domains, and containing 38 Cys residues, was successfully expressed and purified. A protein composed of the disintegrin-like and cysteine-rich domains (DC protein) and the cysteine-rich domain alone (C protein) were expressed in vitro individually and purified. Both proteins were shown to be functional in assays monitoring the interaction with matrix proteins and in modulating the cleavage of fibrinogen by HF3. These data indicate that recombinant expression using prokaryotic-based cell-free synthesis emerges as an attractive alternative for the study of the structure and function of multi-domain proteins with a high content of Cys residues

Interaction of Penicillin-Binding Protein 2x and Ser/Thr protein kinase StkP, two key players in Streptococcus pneumoniae†R6 morphogenesis.

Bacterial cell growth and division require the co-ordinated action of peptidoglycan biosynthetic enzymes and cell morphogenesis proteins. However, the regulatory mechanisms that allow generating proper bacterial shape and thus preserving cell integrity remain largely uncharacterized, especially in ovococci. Recently, the conserved eukaryotic-like Ser/Thr protein kinase of Streptococcus pneumoniae (StkP) was demonstrated to play a major role in cell shape and division. Here, we investigate the molecular mechanisms underlying the regulatory function(s) of StkP and show that it involves one of the essential actors of septal peptidoglycan synthesis, Penicillin-Binding Protein 2x (PBP2x). We demonstrate that StkP and PBP2x interact directly and are present in the same membrane-associated complex in S. pneumoniae. We further show that they both display a late-division localization pattern at the division site and that the positioning of PBP2x depends on the presence of the extracellular PASTA domains of StkP. We demonstrate that StkP and PBP2x interaction is mediated by their extracellular regions and that the complex formation is inhibited in vitro in the presence of cell wall fragments. These data suggest that the role of StkP in cell division is modulated by an interaction with PBP2x.

Association of RrgA and RrgC into the Streptococcus pneumoniae pilus by sortases C-2 and C-3.

Pili are surface-exposed virulence factors involved in the adhesion of bacteria to host cells. The human pathogen Streptococcus pneumoniae expresses a pilus composed of three structural proteins, RrgA, RrgB, and RrgC, and requires the action of three transpeptidase enzymes, sortases SrtC-1, SrtC-2, and SrtC-3, to covalently associate the Rrg pilins. Using a recombinant protein expression platform, we have previously shown the requirement of SrtC-1 in RrgB fiber formation and the association of RrgB with RrgC. To gain insights into the substrate specificities of the two other sortases, which remain controversial, we have exploited the same robust strategy by testing various combinations of pilins and sortases coexpressed in Escherichia coli. We demonstrate that SrtC-2 catalyzes the formation of both RrgA-RrgB and RrgB-RrgC complexes. The deletion and swapping of the RrgA-YPRTG and RrgB-IPQTG sorting motifs indicate that SrtC-2 preferentially recognizes RrgA and attaches it to the pilin motif lysine 183 of RrgB. Finally, SrtC-2 is also able to catalyze the multimerization of RrgA through the C-terminal D4 domains. Similar experiments have been performed with SrtC-3, which catalyzes the formation of RrgB-RrgC and RrgB-RrgA complexes. Altogether, these results provide evidence of the molecular mechanisms of association of RrgA and RrgC with the RrgB fiber shaft by SrtC-2 and SrtC-3 and lead to a revised model of the pneumococcal pilus architecture accounting for the respective contribution of each sortase.

VL position 34 is a key determinant for the engineering of stable antibodies with fast dissociation rates.

Predictive engineering of antibodies exhibiting fast kinetic properties could provide reagents for biotechnological applications such as continuous monitoring of compounds or affinity chromatography. Based on covariance analysis of murine germline antibody variable domains, we selected position L34 (Kabat numbering) for mutational studies. This position is located at the VL/VH interface, at the base of the paratope but with limited antigen contacts, thus making it an attractive position for mild alterations of antigen binding properties. We introduced a serine at position L34 in two different antibodies: Fab (fragment antigen binding) 57P (Asn34Ser) and scFv (single chain fragment variable) 1F4 (Gln34Ser), that recognize peptides derived from the coat protein of tobacco mosaic virus and the oncoprotein E6, respectively. Both mutated antibodies exhibited similar properties: (i) expression levels of active fragments in Escherichia coli were markedly improved; (ii) thermostability was enhanced; and (iii) dissociation rate parameters (k(off)) were increased by 2- and at least 57-fold for scFv1F4 and Fab57P, respectively, while their association rate parameters (k(on)) remained unchanged. The L34 Ala and Thr mutants of both antibody fragments did not possess these properties. This first demontration of similar effects observed in two antibodies with different specificities may open the way to the predictive design of molecules with enhanced stability and fast dissociation rates.

BIACORE data processing: an evaluation of the global fitting procedure.

Data from real-time molecular interaction analysis using BIACORE are currently evaluated by numerical integration. We have investigated the ability of two software packages (BIAevaluation 3.0 and CLAMP99) to analyze complex interactions. Three experimental data sets of high quality obtained with BIACORE upgraded and 2000 instruments, representative of simple bimolecular, heterogeneous ligand, and mass-transport-limited interactions, were processed by the global fitting procedure. The two software, which differ mainly in the statistical assessment of the output values, were able to discriminate correctly between various interacting models and provided very close output parameters with satisfactory statistical tests.

Cysteine protease isoforms from Trypanosoma cruzi, cruzipain 2 and cruzain, present different substrate preference and susceptibility to inhibitors.

Cysteine-proteinases from parasitic protozoa have been recently characterized as factors of virulence and pathogenicity in several human and veterinary diseases. In Chagas' disease, the chronic infection caused by Trypanosoma cruzi, structure-functional studies on cysteine proteases were thus far limited to the parasite's major isoform, a cathepsin L-like lysosomal protease designated as cruzipain, cruzain or GP57/51. Encoded by a large gene family, cruzipain is efficiently targeted by synthetic inhibitors, which prevent parasite intracellular growth and differentiation. We have previously demonstrated that the multicopy cruzipain gene family includes polymorphic sequences, which could encode functionally different isoforms. We report here a comparative kinetic study between cruzain, the archetype of the cruzipain family, and an isoform, termed cruzipain 2, which is expressed preferentially by the mammalian stages of T. cruzi. Heterologous expression of the catalytic domain of cruzipain 2 in Saccharomyces cerevisae yielded an enzyme that differs markedly from cruzain with respect to pH stability, substrate specificity and sensitivity to inhibition by natural and synthetic inhibitors of cysteine proteases. We suggest that the structural-functional diversification imparted by genetic polymorphism of cruzipain genes may have contributed to T. cruzi adaptation to vertebrate hosts.

Self-assembly of the vascular endothelial cadherin ectodomain in a Ca2+-dependent hexameric structure.

Vascular endothelial cadherin (VE-cadherin) is a transmembrane protein essential for endothelial cell monolayer integrity (Gulino, D., Delachanal, E., Concord, E., Genoux, Y., Morand, B., Valiron, M. O., Sulpice, E., Scaife, R., Alemany, M., and Vernet, T. (1998) J. Biol. Chem. 273, 29786-29793). This molecule belongs to the cadherin family of cell-cell adhesion receptors, for which molecular details of homotypic interactions are still lacking. In this study, a recombinant fragment encompassing the four N-terminal modules of VE-cadherin (VE-EC1-4) was shown to associate, in solution, as a stable Ca(2+)-dependent oligomeric structure. Cross-linking experiments combined with mass spectrometry demonstrated that this oligomer is a hexamer. Gel filtration chromatography experiments and analytical ultracentrifugation analyses revealed the existence of an equilibrium between the hexameric and monomeric species of VE-EC1-4. The concentration at which 50% of VE-EC1-4 is in its hexameric form was estimated as 1 microm. The dimensions of the hexamer, measured by cryoelectron microscopy to be 233 +/- 10 x 77 +/- 7 A, are comparable to the thickness of adherens endothelial cell-cell junctions. Altogether, the results allow us to propose a novel homotypic interaction model for the class II VE-cadherin, in which six molecules of cadherin form a hexamer.

Molecular mechanisms of antibiotic resistance in gram-positive pathogens.

The widespread and uncontrolled use of antibiotics, both for human consumption and animal feed, has encouraged the development of drug resistance in a variety of pathogenic bacteria. Gram-positive species employ resistance mechanisms which include the modification of the antibiotic structure, mutagenesis of key amino acids in the macromolecular targets of specific chemotherapeutics, or drug efflux from the cell, among others. These three main mechanisms have been identified in resistance profiles for systems involved in protein biosynthesis, nucleic acid replication, and bacterial cell wall generation. This work will review how Gram-positive bacteria have manipulated all three classes of targets in the generation of resistance. Upcoming and recently approved antibacterials for human consumption will also be highlighted.

Calcium-dependent conformational stability of modules 1 and 2 of human gelsolin.

Gelsolin modulates the actin cytoskeleton in the cytoplasm and clears the circulation of stray filaments. In vitro, gelsolin cleaves, nucleates and caps actin filaments, activities that are calcium-dependent. Both cellular and secreted forms share a sequence of 730 residues comprising six homologous modules termed G1-G6. A disulphide bond is formed in secreted G2, whereas in the cytoplasm it remains reduced. A point mutation in G2 causes an amyloidosis with neurological, ophthalmological and dermatological symptoms. This mutation does not affect the cytoplasmic form, while the secreted form is proteolysed. As a first step towards understanding how gelsolin folds and functions in different cellular compartments, we have characterized at equilibrium the urea-induced unfolding of G1 and G2, with or without calcium and/or disulphide bond. G1 and G2 both exhibit two-state unfolding behaviour and are stabilized by calcium. The disulphide bond also contributes to the stability of G2. In the absence of Ca(2+) and disulphide bond, G2 adopts a non-native conformation, suggesting that folding of G2 in the cytoplasm relies on the presence of surrounding modules or other molecular partners.

Deacylation kinetics analysis of Streptococcus pneumoniae penicillin-binding protein 2x mutants resistant to beta-lactam antibiotics using electrospray ionization- mass spectrometry.

Penicillin-binding proteins (PBPs) catalyze the transpeptidase reaction involved in peptidoglycan synthesis and are covalently inhibited by the beta-lactam antibiotics. In a previous work we have focused on acylation efficiency measurements of various Streptococcus pneumoniae PBP2x* mutants to study the molecular determinants of resistance to beta-lactams. In the present paper we have developed a method to improve an accurate determination of the deacylation rate constant using electrospray ionization-mass spectrometry. This method is adaptable to the analysis of deacylation of any beta-lactam. Compared to the fluorographic technique, the ESI-MS method is insensitive to variations in the concentration of functional proteins and is therefore more reliable. We have established that the resistance of PBPs to beta-lactams is mostly due to a decrease of the acylation efficiency with only marginal effects on the deacylation rates.

Effects on interaction kinetics of mutations at the VH-VL interface of Fabs depend on the structural context.

The influence of framework residues belonging to VH and VL modules of antibody molecules on antigen binding remains poorly understood. To investigate the functional role of such residues, we have performed semi-conservative amino acid replacements at the VH-VL interface. This work was carried out with (i) variants of the same antibody and (ii) with antibodies of different specificities (Fab fragments 145P and 1F1h), in order to check if functional effects are additive and/or similar for the two antibodies. Interaction kinetics of Fab mutants with peptide and protein antigens were measured using a BIACORE instrument. The substitutions introduced at the VH-VL interface had no significant effects on k(a) but showed small, significant effects on k(d). Mutations in the VH module affected k(d) not only for the two different antibodies but also for variants of the same antibody. These effects varied both in direction and in magnitude. In the VL module, the double mutation F(L37)L-Q(L38)L, alone or in combination with other mutations, consistently decreased k(d) about two-fold in Fab 145P. Other mutations in the VL module had no effect on k(d) in 145P, but always decreased k(d) in 1F1h. Moreover, in both systems, small-magnitude non-additive effects on k(d) were observed, but affinity variations seemed to be limited by a threshold. When comparing functional effects in antibodies of different specificity, no general rules could be established. In addition, no clear relationship could be pointed out between the nature of the amino acid change and the observed functional effect. Our results show that binding kinetics are affected by alteration of framework residues remote from the binding site, although these effects are unpredictable for most of the studied changes.

Expression, purification, crystallization and preliminary X-ray analysis of the iota-carrageenase from Alteromonas fortis.

This is the first crystallization report of a glycoside hydrolase which belongs to family 82. A recombinant form of His-tagged iota-carrageenase from Alteromonas fortis was expressed, purified and crystallized. Crystals were obtained by the vapour-diffusion method using polyethylene glycol (M(W) = 6000) as a precipitant. They belong to space group P2(1), with unit-cell parameters a = 56. 75, b = 91.04, c = 125.01 A, beta = 93.41 degrees. The unit cell contains two molecules in the asymmetric unit related by a non-crystallographic twofold axis. Crystals diffracted to 2.0 A resolution on a synchrotron beamline.

Mapping of the interaction between the immunodominant loop of the ectodomain of HIV-1 gp41 and human complement protein C1q.

The human immunodeficiency virus type 1 transmembrane envelope glycoprotein gp41 has been previously shown to activate the C1 complex of human complement through direct interaction with its C1q subunit. The major interaction site has been located within the gp41 immunodominant region (residues 590-620), and a synthetic peptide overlapping residues 601-613 of gp41 (sequence GIWGCSGKLICTT) was shown to inhibit binding of gp41 to C1q in vitro (Thielens, N.M., Bally, I.M., Ebenbichler, C.F., Dierich, M.P. & Arlaud, G.J. (1993) J. Immunol. 151, 6583-6592). The ectodomain of gp41 (s-gp41) was secreted from the methylotrophic yeast Pichia pastoris and purified by immunoaffinity chromatography. Enzymatic deglycosylation of the recombinant s-gp41 was necessary to allow its in vitro interaction with C1q. A solid-phase competition assay was used to monitor the effect of mutant peptides derived from segment 601-613 of gp41 on the binding of deglycosylated s-gp41 to C1q. Whereas mutation of Ser606 had no effect, replacement of Ile602, Trp603, Lys608, Leu609 and Ile610 by Ala abolished the ability of the resulting peptides to inhibit binding of s-gp41 to C1q, suggesting that these residues participate in the interaction between gp41 and C1q. These findings are discussed in the light of a structural model of the immunodominant loop of gp41. It is proposed that the recognition of gp41 by C1q is driven by hydrophobic interactions, and that the sites of gp41 responsible for interaction with gp120 and C1q partly overlap.

Schmid's metaphyseal chondrodysplasia mutations interfere with folding of the C-terminal domain of human collagen X expressed in Escherichia coli.

Human collagen X contains a highly conserved 161-amino acid C-terminal non-triple helical domain that is homologous to the C-terminal domain of collagen VIII and to the C1q module of the human C1 enzyme. We have expressed this domain (residues 545-680) in Escherichia coli as a glutathione S-transferase fusion protein. The purified fusion protein trimerizes spontaneously in vitro, and after thrombin cleavage, the purified C-terminal domain trimer (46.2 kDa) is extremely stable and trypsin-resistant. Mutations within the C-terminal domain have been observed in patients with Schmid's metaphyseal chondrodysplasia (SMCD). Some of these mutations (Y598D, G618V, W651X, or H669X; X is the stop codon) were constructed by site-directed mutagenesis. Each mutation had identical consequences regarding the fusion protein: 1) absence of trimeric formation, 2) copurification of the approximately 60-kDa GroEL chaperone protein, and 3) sensitivity of the monomeric fusion protein to trypsin digestion. These results show that the C-terminal domain of collagen X is sufficient to produce a very stable and compact trimer in the absence of collagen Gly-X-Y repeats. Moreover, mutations causing SMCD interfere in this system with the correct folding of the C-terminal domain. The existence of a similar mechanism in chondrocytes might explain the relative homogeneity of phenotypes in SMCD despite the diversity of mutations.