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Introduction: The aim of this study was to evaluate the difference between symptomatic discoid lateral meniscus (DLM) and healthy knees in terms of gait analysis., Evidence Acquisition: A systematic review was conducted from the electronic databases PubMed/MEDLINE, EMBASE and Scopus. The review was performed on studies that reported data on kinematics, gait analysis, biomechanics in discoid lateral meniscus, before and after surgery., Evidence Synthesis: Out of 127 identified items, six full-text articles were assessed for eligibility. Three of these studies were included in the quantitative synthesis. Those studies reported a significantly slower gait speed and shorter stride length, smaller excursion during weight acceptance and mid-stance phases and during the whole gait cycle, smaller maximum flexion angle in swing phase, lower frontal excursion, smaller axial excursion in the DLM groups compared to the control groups. One study reported a significative increase in the walking speed and in the stride length, increase both in the maximum knee flexion angle during stance phase and swing phase, and in the range of flexion-extension after arthroscopic surgical treatment for symptomatic DLM., Conclusions: Literature about gait analysis on DLM patients is still very scarce and heterogeneous. The few studies on this topic show a different gait pattern between DLM knees and healthy knees and are encouraging about the surgical outcomes of DLM in terms of gait. Larger and standardized studies should be designed to assess a more suitable number of subjects with DLM.

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.

Introduction: Leg length discrepancy (LLD) and malalignment of long bones are frequent orthopedic problems encountered in Maffucci syndrome and Ollier disease (OD). Orthopedic surgeons used historically external fixators to address the deformities. In this multicentric case series, we propose the use of motorized intramedullary nails., Methods: We retrospectively reviewed for 9 years, in four different centers, patients with OD and Maffucci's syndrome that had lengthening nails for LLD with or without associated deformities. The minimum follow-up period was 24 months. We reported complications, clinical tolerance of lengthening, lengthening rate and target, bone healing index, and EQ-5D-Y functional and visual analog scores (VAS). We also saw on X-rays the whole lengthened bone and its regenerate zone to assess the evolution of the enchondromas., Results: we used the nailing technique in 8 femurs and 2 tibias in 8 patients (mean age: 13.3 years, range: 11-16, mean follow-up time: 53.8 months, range: 26-108). The mean correction amount was 6.44 cm for the femur over 76.8 days and 3.75 cm over 44 days for the tibia with a mean VAS score of 6.63/15 and mean EQ-5D-Y of 81/100. The lengthening goal was achieved in all patients. No mechanical complications were noted. The medullary canal of the operated bones showed improvement and healing in 8 out of 10 segments., Discussion: Besides achieving the goals of surgery with good functional outcomes, lengthening nails has a therapeutic effect on enchondromas with fewer complications than traditional correction methods., (© The Authors, published by EDP Sciences, 2024.)

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., (Copyright © 2022. Published by Elsevier Masson SAS.)

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.

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., Competing Interests: Disclosure: The Disclosure of Potential Conflicts of Interest forms are provided with the online version of the article (http://links.lww.com/JBJSEST/A421)., (Copyright © 2023 by The Journal of Bone and Joint Surgery, Incorporated.)

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., Competing Interests: The authors have declared that there are no conflicts of interest in the authorship and publication of this contribution. AOSSM checks author disclosures against the Open Payments Database (OPD). AOSSM has not conducted an independent investigation on the OPD and disclaims any liability or responsibility relating thereto., (© The Author(s) 2023.)

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.

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.

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  K pH7 = 9.55 ± 0.11 ( K dpH7 = 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 10 7 that is even higher in the cyclic peptide.

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.

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 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 UO 2 2+ and Fe 3+ . 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., (© 2021. The Author(s), under exclusive licence to International Society for Microbial Ecology.)

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.

[This corrects the article DOI: 10.1063/1.5037795.].

Droplet-based microfluidics, using water-in-oil emulsion droplets as micro-reactors, is becoming a widespread method for performing assays and especially in the cell biology field. Making a simple and highly portable system for creating emulsion droplets would help to continue the popularization of such a technique. Also, the ability to emulsify all the samples would strengthen this compartimenlization technique to handle samples with limited volume. Here, we propose a strategy of droplet formation that combines a classical flow-focusing microfluidic chip, which could be commercially available, with a standard laboratory adjustable micropipette. The micropipette is used as a negative pressure generator for controlling liquid flows. In that way, emulsification does neither require any electrical power supply nor a cumbersome device and functions with small liquid volumes. Droplet formation can be easily and safely performed in places with limited space, opening a wide range of applications especially in biological laboratory environments with higher level of safety regulations, i.e., BSL-3/4. Fortunately, the present methodology that involves small fluid volumes, and thus possible time dependent flow conditions, allows to minimize dead volume while keeping drops' size homogeneous. A physical characterization of droplet production and a model that describes the emulsion features, in terms of drop size and size distribution, are proposed for rationalizing the performances of the micropipette-powered emulsification process.

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.

Chemotaxis is an important biological process involved in the development of multicellular organisms, immune response and cancer metastasis. In order to better understand how cells follow chemical cues in their native environments, we recently developed a microfluidics-based chemotaxis device that allows for observation of cells or cell aggregates in 3D networks in response to tunable chemical gradients (Aizel et al., 2017). Here, we describe the methods required for fabrication of this device as well as its use for live imaging experiments and subsequent analysis of imaging data. This device can be adapted to study a number of different cell arrangements and chemical gradients, opening new avenues of research in 3D chemotaxis., (© 2018 Elsevier Inc. All rights reserved.)

In many cell types, migration can be oriented towards a chemical stimulus. In mammals, for example, embryonic cells migrate to follow developmental cues, immune cells migrate toward sites of inflammation, and cancer cells migrate away from the primary tumour and toward blood vessels during metastasis. Understanding how cells migrate in 3D environments in response to chemical cues is thus crucial to understanding directed migration in normal and disease states. To date, chemotaxis in mammalian cells has been primarily studied using 2D migration models. However, it is becoming increasingly clear that the mechanisms by which cells migrate in 2D and 3D environments dramatically differ, and cells in their native environments are confronted with a complex chemical milieu. To address these issues, we developed a microfluidic device to monitor the behaviour of cells embedded in a 3D collagen matrix in the presence of complex concentration fields of chemoattractants. This tuneable microsystem enables the generation of (1) homogeneous, stationary gradients set by a purely diffusive mechanism, or (2) spatially evolving, stationary gradients, set by a convection-diffusion mechanism. The device allows for stable gradients over several days and is large enough to study the behaviour of large cell aggregates. We observe that primary mature dendritic cells respond uniformly to homogeneous diffusion gradients, while cell behaviour is highly position-dependent in spatially variable convection-diffusion gradients. In addition, we demonstrate a directed response of cancer cells migrating away from tumour-like aggregates in the presence of soluble chemokine gradients. Together, this microfluidic device is a powerful system to observe the response of different cells and aggregates to tuneable chemical gradients.

Liquid core capsules having a hydrogel membrane are becoming a versatile tool for three-dimensional culture of micro-organisms and mammalian cells. Making sub-millimeter capsules at a high rate, via the breakup of a compound jet in air, opens the way to high-throughput screening applications. However, control of the capsule size monodispersity, especially required for quantitative bioassays, was still lacking. Here, we report how the understanding of the underlying hydrodynamic instabilities that occur during the process can lead to calibrated core-shell bioreactors. The requirements are: i) damping the shear layer instability that develops inside the injector arising from the co-annular flow configuration of liquid phases having contrasting viscoelastic properties; ii) controlling the capillary instability of the compound jet by superposing a harmonic perturbation onto the shell flow; iii) avoiding coalescence of drops during jet fragmentation as well as during drop flight towards the gelling bath; iv) ensuring proper engulfment of the compound drops into the gelling bath for building a closed hydrogel shell. We end up with the creation of numerous identical compartments in which cells are able to form multicellular aggregates, namely spheroids. In addition, we implement an intermediate composite hydrogel layer, composed of alginate and collagen, allowing cell adhesion and thus the formation of epithelia or monolayers of cells.

Determining the phenotype and genotype of single cells is central to understand microbial evolution. DNA sequencing technologies allow the detection of mutants at high resolution, but similar approaches for phenotypic analyses are still lacking. We show that a drop-based millifluidic system enables the detection of heritable phenotypic changes in evolving bacterial populations. At time intervals, cells were sampled and individually compartmentalized in 100 nL drops. Growth through 15 generations was monitored using a fluorescent protein reporter. Amplification of heritable changes-via growth-over multiple generations yields phenotypically distinct clusters reflecting variation relevant for evolution. To demonstrate the utility of this approach, we follow the evolution of Escherichia coli populations during 30 days of starvation. Phenotypic diversity was observed to rapidly increase upon starvation with the emergence of heritable phenotypes. Mutations corresponding to each phenotypic class were identified by DNA sequencing. This scalable lineage-tracking technology opens the door to large-scale phenotyping methods with special utility for microbiology and microbial population biology.

The local functionalities of biocompatible objects can be characterized under conditions similar to the operating ones, using scanning electrochemical microscopy (SECM). In the case of alginate beads entrapping carbon nanotubes (CNTs), SECM allows evidencing of the local conductivity, organization, and communication between the CNTs. It shows that the CNT network is active enough to allow long range charge evacuation, enabling the use of alginate/CNT beads as soft 3D electrodes. Direct connection or local interrogation by a microelectrode allows visualization of their communication as a network and eventually the study of them individually at the nanoscale.

To address possible cell-to-cell heterogeneity in growth dynamics of isogenic cell populations of Chlamydomonas reinhardtii, we developed a millifluidic drop-based device that not only allows the analysis of populations grown from single cells over periods of a week, but is also able to sort and collect drops of interest, containing viable and healthy cells, which can be used for further experimentation. In this study, we used isogenic algal cells that were first synchronized in mixotrophic growth conditions. We show that these synchronized cells, when placed in droplets and kept in mixotrophic growth conditions, exhibit mostly homogeneous growth statistics, but with two distinct subpopulations: a major population with a short doubling-time (fast-growers) and a significant subpopulation of slowly dividing cells (slow-growers). These observations suggest that algal cells from an isogenic population may be present in either of two states, a state of restricted division and a state of active division. When isogenic cells were allowed to propagate for about 1000 generations on solid agar plates, they displayed an increased heterogeneity in their growth dynamics. Although we could still identify the original populations of slow- and fast-growers, drops inoculated with a single progenitor cell now displayed a wider diversity of doubling-times. Moreover, populations dividing with the same growth-rate often reached different cell numbers in stationary phase, suggesting that the progenitor cells differed in the number of cell divisions they could undertake. We discuss possible explanations for these cell-to-cell heterogeneities in growth dynamics, such as mutations, differential aging or stochastic variations in metabolites and macromolecules yielding molecular switches, in the light of single-cell heterogeneities that have been reported among isogenic populations of other eu- and prokaryotes.

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.

Better understanding of uranyl toxicity in bacteria is necessary to optimize strains for bioremediation purposes or for using bacteria as biodetectors for bioavailable uranyl. In this study, after different steps of optimization, Escherichia coli cells were exposed to uranyl at low pH to minimize uranyl precipitation and to increase its bioavailability. Bacteria were adapted to mid acidic pH before exposure to 50 or 80 µM uranyl acetate for two hours at pH≈3. To evaluate the impact of uranium, growth in these conditions were compared and the same rates of cells survival were observed in control and uranyl exposed cultures. Additionally, this impact was analyzed by two-dimensional differential gel electrophoresis proteomics to discover protein actors specifically present or accumulated in contact with uranium.Exposure to uranium resulted in differential accumulation of proteins associated with oxidative stress and in the accumulation of the NADH/quinone oxidoreductase WrbA. This FMN dependent protein performs obligate two-electron reduction of quinones, and may be involved in cells response to oxidative stress. Interestingly, this WrbA protein presents similarities with the chromate reductase from E. coli, which was shown to reduce uranyl 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.

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.

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., (© 2012 American Chemical Society)

The Escherichia coli ChrR enzyme is an obligatory two-electron quinone reductase that has many applications, such as in chromate bioremediation. Its crystal structure, solved at 2.2 Å resolution, shows that it belongs to the flavodoxin superfamily in which flavin mononucleotide (FMN) is firmly anchored to the protein. ChrR crystallized as a tetramer, and size exclusion chromatography showed that this is the oligomeric form that catalyzes chromate reduction. Within the tetramer, the dimers interact by a pair of two hydrogen bond networks, each involving Tyr128 and Glu146 of one dimer and Arg125 and Tyr85 of the other; the latter extends to one of the redox FMN cofactors. Changes in each of these amino acids enhanced chromate reductase activity of the enzyme, showing that this network is centrally involved in chromate reduction.

We present a novel millifluidic droplet analyser (MDA) for precisely monitoring the dynamics of microbial populations over multiple generations in numerous (≥10(3)) aqueous emulsion droplets (~100 nL). As a first application, we measure the growth rate of a bacterial strain and determine the minimal inhibitory concentration (MIC) for the antibiotic cefotaxime by incubating bacteria in a fine gradient of antibiotic concentrations. The detection of cell activity is based on the automated detection of an epifluorescent signal that allows the monitoring of microbial populations up to a size of ~10(6) cells. We believe that this device is helpful for the study of population dynamic consequences of microbe-environment interactions and of individual cell differences. Moreover, the fluidic machine may improve clinical tests, as it simplifies, automates and miniaturizes the screening of numerous microbial populations that grow and evolve in compartments with a finely tuned composition.

Water-in-oil emulsion drops are formed and stabilized with phospholipids which can adhere and form a bilayer. Using microfluidics, adhesive drop pairs are then trapped and submitted to an ac electric field. We observe three distinct states as a function of the adhesion energy and the electric field intensity. The pair can be either stable, though slightly deformed, or unzip and separate, or coalesce. The frontiers between the different states directly reflect vesicle detachment forces and electroporation theories. The experimental approach that we propose for probing liquid interface wetting between monolayers allows us to finely tuned the tension in the bilayer and gives access to bilayer unzipping.

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., (© 2011 American Physical Society)

Pharmacokinetic analyses of clopidogrel are hampered by the existence of multiple active metabolite isomers (H1 to H4) and their instability in blood. We sought to retest the pharmacodynamic activities of the four individual active metabolite isomers in vitro, with the ultimate aim of determining the isomers responsible for clopidogrel activity in vivo. In vitro activity was evaluated by measuring binding of [³³P]-2-methylthio-ADP on P2Y₁₂-expressing Chinese hamster ovary (CHO) cells and human platelets in platelet-rich plasma (PRP). A stereoselective method that used reverse-phase ultra high-performance liquid chromatography (UHPLC) and tandem mass spectrometry (MS) was developed to measure individual concentrations of the stable 3'-methoxyacetophenone (MP) derivatives of H1-H4. The new method was used to analyze plasma samples from clopidogrel-treated subjects enrolled in a phase I clinical trial. In vitro binding assays confirmed the previously observed biological activity of H4 (IC₅₀: CHO-P2Y₁₂: 0.12 μM; PRP: 0.97 μM) and inactivity of H3, and demonstrated that H1 was also inactive. Furthermore, H2 demonstrated approximately half of the biological activity in vitro compared with H4. Optimisation of UHPLC conditions and MS collision parameters allowed the resolution and detection of the four derivatised active metabolite isomers (MP-H1 to MP-H4). The stereoselective assay was extensively validated, and was accurate and precise over the concentration range 0.5-250 ng/ml. Only MP-H3 and MP-H4 were quantifiable in incurred clinical samples. Based on in vitro pharmacodynamic data and found concentrations, the active metabolite isomer H4 is the only diastereoisomer of clinical relevance for documenting the pharmacokinetic profile of the active metabolite of clopidogrel.