Optimized Coordination of Uranyl in Engineered Calmodulin Site 1 Provides a Subnanomolar Affinity for Uranyl and a Strong Uranyl versus Calcium Selectivity.

As an alpha emitter and chemical toxicant, uranium toxicity in living organisms is driven by its molecular interactions. It is therefore essential to identify main determinants of uranium affinity for proteins. Others and we showed that introducing a phosphoryl group in the coordination sphere of uranyl confers a strong affinity of proteins for uranyl. In this work, using calmodulin site 1 as a template, we modulate the structural organization of a metal-binding loop comprising carboxylate and/or carbonyl ligands and reach affinities for uranyl comparable to that provided by introducing a strong phosphoryl ligand. Shortening the metal binding loop of calmodulin site 1 from 12 to 10 amino acids in CaMΔ increases the uranyl-binding affinity by about 2 orders of magnitude to log KpH7 = 9.55 ± 0.11 (KdpH7 = 280 ± 60 pM). Structural analysis by FTIR, XAS, and molecular dynamics simulations suggests an optimized coordination of the CaMΔ-uranyl complex involving bidentate and monodentate carboxylate groups in the uranyl equatorial plane. The main role of this coordination sphere in reaching subnanomolar dissociation constants for uranyl is supported by similar uranyl affinities obtained in a cyclic peptide reproducing CaMΔ binding loop. In addition, CaMΔ presents a uranyl/calcium selectivity of 107 that is even higher in the cyclic peptide.

Accumulation and Speciation of Cobalt in Paracentrotus lividus.

Since the first human release of radionuclides on Earth at the end of the Second World War, impact assessments have been implemented. Radionuclides are now ubiquitous, and the impact of local accidental release on human activities, although of low probability, is of tremendous social and economic consequences. Although radionuclide inventories (at various scales) are essential as input data for impact assessment, crucial information on physicochemical speciation is lacking. Among the metallic radionuclides of interest, cobalt-60 is one of the most important activation products generated in the nuclear industry. In this work, a marine model ecosystem has been defined because seawater and more generally marine ecosystems are final receptacles of metal pollution. A multistep approach from quantitative uptake to understanding of the accumulation mechanism has been implemented with the sea urchin Paracentrotus lividus. In a well-controlled aquarium, the day-by-day uptake of cobalt and its quantification in different compartments of the sea urchin were monitored with various conditions of exposure by combining ICP-OES analysis and γ spectrometry. Cobalt is mainly distributed following the rating intestinal tract ≫ gonads > shell spines. Cobalt speciation in seawater and inside the gonads and the intestinal tract was determined using extended X-ray absorption fine structure (EXAFS). The cobalt inside the gonads and the intestinal tract is mainly complexed by the toposome, the main protein in the sea urchin P. lividus. Complexation with purified toposome was characterized and a complexation site combining EXAFS and AIMD (ab initio molecular dynamics) was proposed implying monodentate carboxylates.

Uranium Uptake in Paracentrotus lividus Sea Urchin, Accumulation and Speciation.

Uranium speciation and bioaccumulation were investigated in the sea urchin Paracentrotus lividus. Through accumulation experiments in a well-controlled aquarium followed by ICP-OES analysis, the quantification of uranium in the different compartments of the sea urchin was performed. Uranium is mainly distributed in the test (skeletal components), as it is the major constituent of the sea urchin, but in terms of quantity of uranium per gram of compartment, the following rating: intestinal tract > gonads ≫ test, was obtained. Combining both extended X-ray Absorption Spectroscopy and time-resolved laser-induced fluorescence spectroscopic analysis, it was possible to identify two different forms of uranium in the sea urchin, one in the test, as a carbonato-calcium complex, and the second one in the gonads and intestinal tract, as a protein complex. Toposome is a major calcium-binding transferrin-like protein contained within the sea urchin. EXAFS data fitting of both contaminated organs in vivo and the uranium-toposome complex from protein purified out of the gonads revealed that it is suspected to complex uranium in gonads and intestinal tract. This hypothesis is also supported by the results from two imaging techniques, i.e., Transmission Electron Microscopy and Scanning Transmission X-ray Microscopy. This thorough investigation of uranium uptake in sea urchin is one of the few attempts to assess the speciation in a living marine organism in vivo.

A conductive hydrogel based on alginate and carbon nanotubes for probing microbial electroactivity.

Some bacteria can act as catalysts to oxidize (or reduce) organic or inorganic matter with the potential of generating electrical current. Despite their high value for sustainable energy, organic compound production and bioremediation, a tool to probe the natural biodiversity and to select most efficient microbes is still lacking. Compartmentalized cell culture is an ideal strategy for achieving such a goal but the appropriate compartment allowing cell growth and electron exchange must be tailored. Here, we develop a conductive composite hydrogel made of a double network of alginate and carbon nanotubes. Homogeneous mixing of carbon nanotubes within the polyelectrolyte is obtained by a surfactant assisted dispersion followed by a desorption step for triggering electrical conductivity. Dripping the mixture in a gelling bath through simple extrusion or a double one allows the formation of either plain hydrogel beads or liquid core hydrogel capsules. The process is shown to be compatible with the bacterial culture (Geobacter sulfurreducens). Bacteria can indeed colonize the outer wall of plain beads or the inner wall of the conductive capsules' shell that function as an anode from which electrons produced by the cells are collected.

Cellular capsules as a tool for multicellular spheroid production and for investigating the mechanics of tumor progression in vitro.

Deciphering the multifactorial determinants of tumor progression requires standardized high-throughput preparation of 3D in vitro cellular assays. We present a simple microfluidic method based on the encapsulation and growth of cells inside permeable, elastic, hollow microspheres. We show that this approach enables mass production of size-controlled multicellular spheroids. Due to their geometry and elasticity, these microcapsules can uniquely serve as quantitative mechanical sensors to measure the pressure exerted by the expanding spheroid. By monitoring the growth of individual encapsulated spheroids after confluence, we dissect the dynamics of pressure buildup toward a steady-state value, consistent with the concept of homeostatic pressure. In turn, these confining conditions are observed to increase the cellular density and affect the cellular organization of the spheroid. Postconfluent spheroids exhibit a necrotic core cemented by a blend of extracellular material and surrounded by a rim of proliferating hypermotile cells. By performing invasion assays in a collagen matrix, we report that peripheral cells readily escape preconfined spheroids and cell-cell cohesivity is maintained for freely growing spheroids, suggesting that mechanical cues from the surrounding microenvironment may trigger cell invasion from a growing tumor. Overall, our technology offers a unique avenue to produce in vitro cell-based assays useful for developing new anticancer therapies and to investigate the interplay between mechanics and growth in tumor evolution.

Thermodynamics of Calcium binding to the Calmodulin N-terminal domain to evaluate site-specific affinity constants and cooperativity.

Calmodulin (CaM) is an essential Ca(II)-dependent regulator of cell physiology. To understand its interaction with Ca(II) at a molecular level, it is essential to examine Ca(II) binding at each site of the protein, even if it is challenging to estimate the site-specific binding properties of the interdependent CaM-binding sites. In this study, we evaluated the site-specific Ca(II)-binding affinity of sites I and II of the N-terminal domain by combining site-directed mutagenesis and spectrofluorimetry. The mutations had very low impact on the protein structure and stability. We used these binding constants to evaluate the inter-site cooperativity energy and compared it with its lower limit value usually reported in the literature. We found that site I affinity for Ca(II) was 1.5 times that of site II and that cooperativity induced an approximately tenfold higher affinity for the second Ca(II)-binding event, as compared to the first one. We further showed that insertion of a tryptophan at position 7 of site II binding loop significantly increased site II affinity for Ca(II) and the intra-domain cooperativity. ΔH and ΔS parameters were studied by isothermal titration calorimetry for Ca(II) binding to site I, site II and to the entire N-terminal domain. They showed that calcium binding is mainly entropy driven for the first and second binding events. These findings provide molecular information on the structure-affinity relationship of the individual sites of the CaM N-terminal domain and new perspectives for the optimization of metal ion binding by mutating the EF-hand loops sequences.

Physicochemical properties of aqueous core hydrogel capsules.

Capsules having a thin alginate hydrogel membrane and an aqueous core can be obtained by a process that involves a co-extrusion step in air followed by a sol-gel transition of the shell after immersion into a gelling bath. The possibility to encapsulate cells that further grow in these biocompatible compartments, and thus offer a versatile tool for cell culture, led us to investigate the physicochemical properties of the capsules. A cut-off pore size of the semi-permeable membrane is extrapolated from the release of polymers out of the capsule. When polymers cannot diffuse through the membrane, the osmotic pressure mismatch between the core and the surrounding medium triggers an inflation of the capsule. The swelling may reach a steady state that allows the determination of the elastic features of the hydrogel shell. On the other hand, the capsule membrane may rupture and then contract. From this stress-relaxation process, a critical deformation of the hydrogel shell above which plasticity occurs can be deduced. Finally, thanks to the physical nature of the hydrogel, the core content can be released by dissolving the membrane with the help of small electrolytes. The shell life is shown to vary inversely with the ionic strength of the solution.

Thin adhesive oil films lead to anomalously stable mixtures of water in oil.

Oil and water can only be mixed by dispersing droplets of one fluid in the other. When two droplets approach one another, the thin film that separates them invariably becomes unstable, causing the droplets to coalesce. The only known way to avoid this instability is through addition of a third component, typically a surfactant, which stabilizes the thin film at its equilibrium thickness. We report the observation that a thin fluid film of oil separating two water droplets can lead to an adhesive interaction between the droplets. Moreover, this interaction prevents their coalescence over timescales of several weeks, without the use of any surfactant or solvent.

Electrochemically induced far-infrared difference spectroscopy on metalloproteins using advanced synchrotron technology.

New information on a protein's structure, intra- and intermolecular hydrogen bonds, or metal-ligand bond properties can be unraveled in the far-infrared (far-IR)-terahertz-domain (600-3 cm(-1) or 18-0.1 THz). In this study, we compare the performances of thermal sources with synchrotron far-IR to record reaction-induced Fourier transform infrared (FT-IR) difference signals with proteins in solution. Using the model protein Cu-azurin placed in a short path length electrochemical cell adapted for transmission spectroscopy in vacuum-purged optics, we show that minute spectral shifts induced by metal isotope labeling or temperature changes are detected using the far-IR beamline AILES of the synchrotron SOLEIL. On one hand, these data allow us to identify modes involving Cu-ligand vibrations and pave the way for the analysis of metal sites or metal redox states of proteins not amenable to resonance Raman spectroscopy. On another hand, small band shifts or changes in band intensity upon temperature modifications show that far-IR difference spectroscopy allows one to extract from a complex background hydrogen-bonding signatures directly relevant to the protein function. For Cu-azurin, a temperature-sensitive IR mode involving Cu(II)-His vibrations points to the role of a hydrogen bond between a Cu histidine ligand and the water solvent in tuning the Cu(II)-histidine bond properties. Furthermore, these experimental data support the possible role of a His117-water interaction in electron-transfer activity of Cu-azurin proposed by theoretical studies.

Droplet-based microfluidic platform for viscosity measurement over extended concentration range.

Rheology of concentrated protein solutions is crucial for the understanding of macromolecular crowding dynamics as well as the formulation of protein therapeutics. The cost and scarcity of most protein samples prevents wide-scale rheological studies as conventional viscosity measurement methods require large sample volume. There is a growing need for a precise and robust viscosity measurement tool that minimizes consumption and simplifies the handling of highly concentrated protein solutions. This objective is achieved by combining microfluidics and microrheology: we developed a specific microsystem to study the viscosity of aqueous solutions at high concentrations. The PDMS chip allows in situ production, storing and monitoring of water-in-oil nanoliter droplets. We perform precise viscosity measurements inside individual droplets by particle-tracking microrheology of fluorescent probes. Pervaporation of water through a PDMS membrane induces aqueous droplet shrinking, concentrating the sample up to 150 times, thus allowing viscosity measurements along an extended concentration range in just one experiment. The methodology is precisely validated by studying the viscosity of sucrose solutions. Two model proteins are also studied with sample consumption reduced to as little as 1 µL of diluted solution, showcasing the viability of our approach for the study of biopharmaceuticals.

Thoracoscopic Anterior Vertebral Body Tethering in Lenke Type-1 Right Adolescent Idiopathic Scoliosis.

Background: Vertebral body tethering (VBT) is indicated for skeletally immature patients with progressive adolescent idiopathic scoliosis (AIS) who have failed or are intolerant of bracing and who have a major coronal curve of 40° to 65°. The vertebral body must be structurally and dimensionally adequate to accommodate screw fixation, as determined radiographically. The best indication for VBT is a flexible single major thoracic curve with nonstructural compensating lumbar and proximal thoracic curves (Lenke 1A or 1B). VBT allows for progressive correction of the deformity without spinal fusion by utilizing a minimally invasive fluoroscopic technique. Description: The procedure for a right thoracic curve is performed with use of a right thoracoscopic approach with the patient in the left lateral decubitus position. The thoracoscope is introduced through a portal at the apex of the curvature in the posterior axillary line. Instrument portals are created lateral to each vertebral body in the mid-axillary line. Screws are inserted into each vertebral body under biplanar fluoroscopic control and with intraoperative neuromonitoring. An electroconductivity probing device, while not mandatory, is routinely utilized at our practice. The tether is attached to the most proximal screw of the construct, and then reduction is obtained sequentially by tensioning the tether from one vertebral screw to the next. Alternatives: Bracing is the gold-standard treatment for progressive AIS involving the immature spine. The most commonly utilized surgical treatment is posterior spinal fusion (PSF), which should be considered when the major coronal curve exceeds 45°. Rationale: PSF has proven to be a dependable technique to correct scoliotic deformities. It has a low complication rate and good long-term outcomes. However, concerns exist regarding the stiffness conferred by PSF and the long-term effects of adjacent segment disease. Thus, interest had developed in non-fusion solutions for AIS correction. VBT utilizes the Hueter-Volkmann principle to guide growth and correct deformity. Compressive forces applied to the convexity of the deformity by a polyethylene tether allow the patient's growth to realign the spine. Intraoperative correction triggers growth modulation, and most of the modulation seems to occur during the first 12 months postoperatively. The best results have been seen with a short Lenke type-1A curve in a patient with closed triradiate cartilage, a Risser 3 or lower (ideally Risser 0) iliac apophysis, and a flexible curve characterized by a 50% reduction of the major coronal curve angle on side-bending radiographs. Expected Outcomes: In 57 immature patients with a Lenke type-1A or 1B curve (i.e., a 30° to 65° preoperative Cobb angle), Samdani et al.3 found a main thoracic Cobb angle reduction from 40° ± 7° preoperatively to 19° ± 13° at 2 years after VBT. In the sagittal plane, the T5-T12 kyphosis measured 15° ± 10° preoperatively, 17° ± 10° postoperatively, and 20° ± 13° at 2 years. No major neurologic or pulmonary complications occurred. A total of 7 (12.3%) of the 57 patients underwent surgical revision, including 5 for overcorrection and 2 to span additional vertebrae. In a study of 21 skeletally mature patients, Pehlivanoglu et al.4 found that the Cobb angle was reduced from 48° preoperatively to 16° on the first-erect postoperative radiograph and finally to 10° at the latest follow-up (mean, 27.4 months). The 2 main complications of VBT reported in the literature are overcorrection and tether breakage. Both may require revision, which explains the higher rate of revision observed for VBT compared with PSF. Important Tips: Good patient selection is important. VBT is most appropriate in cases of a flexible Lenke type-1A or 1B curve in an immature child before Risser stage 3 and after triradiate cartilage closure.Always monitor and control screw positioning in both anteroposterior and lateral planes fluoroscopically.The screws should be placed parallel to the vertebral end plates or, even better, be angled inferiorly for the upper vertebrae and angled superiorly for the lower vertebrae to decrease the risk of pull-out when tensioning the device and during growth modulation. Less tension on the uppermost and lowermost instrumented vertebrae than at the apex, as controlled by the tensioning device, can also help to limit pull-out. Acronyms and Abbreviations: VBT = vertebral body tetheringAIS = adolescent idiopathic scoliosisIONM = intraoperative neuromonitoringPSF = posterior spinal fusionUIV = upper instrumented vertebraLIV = lower instrumented vertebraAP = anteroposteriorK-wire = Kirschner wire.

Long-term Outcomes of Tibial Spine Avulsion Fractures After Open Reduction With Osteosuturing Versus Arthroscopic Screw Fixation: A Multicenter Comparative Study.

Background: More information is needed regarding return to preinjury sport levels and patient-reported outcomes after tibial spine avulsion (TSA) fracture, which is most common in children aged 8 to 12 years. Purpose: To analyze return to play/sport (RTP), subjective knee-specific recovery, and quality of life in patients after TSA fracture treated with open reduction with osteosuturing versus arthroscopic reduction with internal screw fixation. Study Design: Cohort study; Level of evidence, 3. Methods: This study included 61 patients <16 years old with TSA fracture treated via open reduction with osteosuturing (n = 32) or arthroscopic reduction with screw fixation (n = 29) at 4 institutions between 2000 and 2018; all patients had at least 24 months of follow-up (mean ± SD, 87.0 ± 47.1 months; range, 24-189 months). The patients completed questionnaires regarding ability to return to preinjury-level sports, subjective knee-specific recovery, and health-related quality of life, and results were compared between treatment groups. Univariate and multivariate logistic regression analyses were conducted to determine variables associated with failure to return to preinjury level of sport. Results: The mean patient age was 11 years, with a slight male predominance (57%). Open reduction with osteosuturing was associated with a quicker RTP time than arthroscopy with screw implantation (median, 8.0 vs 21.0 weeks; P < .001). Open reduction with osteosuturing was also associated with a lower risk of failure to RTP at preinjury level (adjusted odds ratio, 6.4; 95% CI, 1.1-36.0; P = .035). Postoperative displacement >3 mm increased the risk of failure to RTP at preinjury level regardless of treatment group (adjusted odds ratio, 15.2; 95% CI, 1.2-194.9; P = .037). There was no difference in knee-specific recovery or quality of life between the treatment groups. Conclusion: Open surgery with osteosuturing was a more viable option for treating TSA fractures because it resulted in a quicker RTP time and a lower rate of failure to RTP as compared with arthroscopic screw fixation. Precise reduction contributed to improved RTP.

Isolated MPFL reconstruction with soft tissue femoral fixation technique in 54 skeletally immature patients: Clinical outcomes at 2 years follow-up. A French multicenter retrospective study.

BACKGROUND: Medial patello-femoral ligament (MPFL) reconstruction is one of the therapeutic options to treat patellofemoral instability. Classically, a à la carte treatment of skeletal and ligament abnormalities is described. This option is difficult to achieve in children because bony procedures can damage the femoral and/or tibial growth plate. The objective was to evaluate a strategy for isolated reconstruction of the MPFL in the treatment of objective patellar instabilities in children, in a large cohort. The return to sport, knee function and pain or discomfort were studied as secondary endpoints. METHODS: This French multicenter retrospective study included 54 pediatric patients with objective patellofemoral instability. Patients were included if they had presented at least 2 episodes of objective patella dislocation. A Deie-like technique with gracilis tendon graft, soft tissue femoral fixation and patellar bone tunnels for patellar fixation was used. Recurrence of dislocation was studied as the primary endpoint, and the recurrence rate was compared with the literature. A comparison of functional scores (Kujala, Lille femoro-patellar instability score or LFPI Score and Tegner activity score) and NRS between pre- and postoperative was studied as a secondary objective. RESULTS: A recurrence of femoro-patellar instability was observed for five patients within 2 years follow up (9%). A significant improvement of the Kujala, LFPI score, Tegner and NRS scores was observed (p<0.001). CONCLUSION: Isolated reconstruction of the MPFL presents a risk of recurrence of 9% at 2years follow-up. This technique significantly improves the functional scores of the knee. This modified Deie technique provides good clinical and functional results, allowing return to sports with an acceptable risk of recurrence of patellar dislocation, similar to those observed in the literature. Isolated MPFL reconstruction as a first-line treatment appears to be a reliable and effective technique in terms of recurrence of dislocation and functional scores. It allows early recovery and rehabilitation and has lower morbidity than procedures requiring bone gestures. LEVEL OF EVIDENCE: III, retrospective comparative study.

Development of an Efficient FRET-Based Ratiometric Uranium Biosensor.

The dispersion of uranium in the environment can pose a problem for the health of humans and other living organisms. It is therefore important to monitor the bioavailable and hence toxic fraction of uranium in the environment, but no efficient measurement methods exist for this. Our study aims to fill this gap by developing a genetically encoded FRET-based ratiometric uranium biosensor. This biosensor was constructed by grafting two fluorescent proteins to both ends of calmodulin, a protein that binds four calcium ions. By modifying the metal-binding sites and the fluorescent proteins, several versions of the biosensor were generated and characterized in vitro. The best combination results in a biosensor that is affine and selective for uranium compared to metals such as calcium or other environmental compounds (sodium, magnesium, chlorine). It has a good dynamic range and should be robust to environmental conditions. In addition, its detection limit is below the uranium limit concentration in drinking water defined by the World Health Organization. This genetically encoded biosensor is a promising tool to develop a uranium whole-cell biosensor. This would make it possible to monitor the bioavailable fraction of uranium in the environment, even in calcium-rich waters.

Management of anterior cruciate ligament tears in Tanner stage 1 and 2 children: a narrative review and treatment algorithm guided by ACL tear location.

The incidence of anterior cruciate ligament (ACL) tears in skeletally immature patients has acutely increased over the last 20 years, yet there is no consensus on a single "best treatment." Selection of an optimal treatment is critical and based on individual circumstances; consequently, we propose a treatment-selection algorithm based on skeletal development, ACL tear location, type, and quality, as well as parental perspective in order to facilitate the decision-making process. We combined our surgical group's extensive case histories of ACL tear management in Tanner Stage 1 and 2 patients with those in the literature to form a consolidated data base. For each case the diagnostic phase, communication with patient and parents, treatment choice(s), selected surgical techniques and rehabilitation schedule were critically analyzed and compared for patient outcomes. MRI-imaging and intraoperative tissue quality assessment were preeminent in importance for selection of the optimal treatment strategy. Considerations for selecting an optimal treatment included: associated lesions, the child/patient and parent(s)' well-informed and counseled consent, biological potential, and the potential for successful ACL preservative surgery. Complete ACL tears were evaluated according to tear-location. In type I and II ACL tears with remaining good tissue quality, we propose primary ACL repair. In type III and IV ACL tears we propose physeal-sparing reconstruction with an iliotibial band graft. Finally, in the case of a type V ACL tear, we propose that the best treatment be based on the Meyers-McKeever classification. We present a facile decision-making algorithm for ACL management in pediatric patients based on specific elements of tissue damage and status.

From stability to permeability of adhesive emulsion bilayers.

Water drops dispersed in chloroform and stabilized with phospholipids become adhesive if a bad solvent for lipids, such as silicone oil, is added to the continuous phase. In this way, two sticking drops are separated by a bilayer of phospholipids. By using microfluidic technologies, we probe the stability and properties of such membranes likewise encountered in foams or vesicles. We first establish the stability diagram of adhering drop pairs as a function of the continuous phase composition. We found two regimes of destabilization of the bilayer. The first one concerns a competition between the dynamics of adhesion and the transport of surfactants toward the interfaces that leads to a dilute surfactant coverage. The second one corresponds to a dense surface coverage where the lifetime distribution of the bilayer exponentially decreases as a signature of a nucleation process. In the stable regime, we observe the propagation of adhesion among a concentrated collection of drops. This is another remarkable illustration of the suction consequence when two close deformable objects are pulled apart. Moreover, the present experimental strategy offers a novel way to study the phase diagrams of bilayers from a single phospholipid to a mixture of phospholipids. Indeed, we detect phase transitions at a liquid-liquid interface that are ruled by the amount of bad solvent. Finally, we probe the transport of water molecules through the bilayer and show that its permeability is linked to the adhesion energy that reflects its fluidity.

Encapsulation of Cells in a Collagen Matrix Surrounded by an Alginate Hydrogel Shell for 3D Cell Culture.

Numerous techniques for mammalian cell culture have been developed to mimic the complex in vivo three-dimensional structure of tissues and organs. Among them, the sole use of proteins to create a matrix where cells are embedded already gives rise to self-organized multicellular assemblies. Loading cells in a controlled extracellular matrix along with cell culture and monitoring through a strategy that is compatible with pipetting tools would be beneficial for high throughput screening applications or simply for a standardized method. Here, we design submillimeter compartments having a thin alginate hydrogel shell and a core made of a collagen matrix where cells are embedded. The process, using a microfluidic device, is based on a high speed co-extrusion in air, leading to a compound jet whose fragmentation is controlled. The resulting core-shell liquid drops are then collected in a gelling bath that triggers a fast hardening of the shell and is followed by a slower self-assembly of collagen molecules into fibers. We show how to formulate the core solution in order to maintain cell viability at physiological conditions that otherwise induce tropocollagen molecules to self-assemble, while being able to prevent flow disturbances that are detrimental for this jetting method. Encapsulated Caco-2 cells, mainly used to model the intestinal barrier, proliferate and form a closed polarized epithelial cell monolayer where the apical membrane faces the continuous medium.

Inter-Site Cooperativity of Calmodulin N-Terminal Domain and Phosphorylation Synergistically Improve the Affinity and Selectivity for Uranyl.

Uranyl-protein interactions participate in uranyl trafficking or toxicity to cells. In addition to their qualitative identification, thermodynamic data are needed to predict predominant mechanisms that they mediate in vivo. We previously showed that uranyl can substitute calcium at the canonical EF-hand binding motif of calmodulin (CaM) site I. Here, we investigate thermodynamic properties of uranyl interaction with site II and with the whole CaM N-terminal domain by spectrofluorimetry and ITC. Site II has an affinity for uranyl about 10 times lower than site I. Uranyl binding at site I is exothermic with a large enthalpic contribution, while for site II, the enthalpic contribution to the Gibbs free energy of binding is about 10 times lower than the entropic term. For the N-terminal domain, macroscopic binding constants for uranyl are two to three orders of magnitude higher than for calcium. A positive cooperative process driven by entropy increases the second uranyl-binding event as compared with the first one, with ΔΔG = -2.0 ± 0.4 kJ mol-1, vs. ΔΔG = -6.1 ± 0.1 kJ mol-1 for calcium. Site I phosphorylation largely increases both site I and site II affinity for uranyl and uranyl-binding cooperativity. Combining site I phosphorylation and site II Thr7Trp mutation leads to picomolar dissociation constants Kd1 = 1.7 ± 0.3 pM and Kd2 = 196 ± 21 pM at pH 7. A structural model obtained by MD simulations suggests a structural role of site I phosphorylation in the affinity modulation.

Discovery and characterization of UipA, a uranium- and iron-binding PepSY protein involved in uranium tolerance by soil bacteria.

Uranium is a naturally occurring radionuclide. Its redistribution, primarily due to human activities, can have adverse effects on human and non-human biota, which poses environmental concerns. The molecular mechanisms of uranium tolerance and the cellular response induced by uranium exposure in bacteria are not yet fully understood. Here, we carried out a comparative analysis of four actinobacterial strains isolated from metal and radionuclide-rich soils that display contrasted uranium tolerance phenotypes. Comparative proteogenomics showed that uranyl exposure affects 39-47% of the total proteins, with an impact on phosphate and iron metabolisms and membrane proteins. This approach highlighted a protein of unknown function, named UipA, that is specific to the uranium-tolerant strains and that had the highest positive fold-change upon uranium exposure. UipA is a single-pass transmembrane protein and its large C-terminal soluble domain displayed a specific, nanomolar binding affinity for UO22+ and Fe3+. ATR-FTIR and XAS-spectroscopy showed that mono and bidentate carboxylate groups of the protein coordinated both metals. The crystal structure of UipA, solved in its apo state and bound to uranium, revealed a tandem of PepSY domains in a swapped dimer, with a negatively charged face where uranium is bound through a set of conserved residues. This work reveals the importance of UipA and its PepSY domains in metal binding and radionuclide tolerance.

Propagation of drop coalescence in a two-dimensional emulsion: a route towards phase inversion.

The phase inversion that undergoes an emulsion while being sheared is a sudden phenomenon that is still puzzling. In this Letter, we report an experimental investigation on propagative coalescence by using a microfluidic device where a calibrated two-dimensional emulsion is created and destabilized. The velocity of propagation and the probability of the coalescence are reported as a function of the size and the spatial distribution of the drops, respectively. We then discuss the efficiency of this novel scenario of phase inversion and suggest that inversion can be favored by the existence of a drop size distribution.