Bottom-up assembly of functional intracellular synthetic organelles by droplet-based microfluidics

Bottom-up synthetic biology has directed most efforts toward the construction of artificial compartmentalized systems that recreate living cell functions in their mechanical, morphological, or metabolic characteristics. However, bottom-up synthetic biology also offers great potential to study subcellular structures like organelles. Because of their intricate and complex structure, these key elements of eukaryotic life forms remain poorly understood. Here, the controlled assembly of lipid enclosed, organelle-like architectures is explored by droplet-based microfluidics. Three types of giant unilamellar vesicles (GUVs)-based synthetic organelles (SOs) functioning within natural living cells are procedured: (A) synthetic peroxisomes supporting cellular stress-management, mimicking an organelle innate to the host cell by using analogous enzymatic modules; (B) synthetic endoplasmic reticulum (ER) as intracellular light-responsive calcium stores involved in intercellular calcium signalling, mimicking an organelle innate to the host cell but utilizing a fundamentally different mechanism; and (C) synthetic magnetosomes providing eukaryotic cells with a magnetotactic sense, mimicking an organelle that is not natural to the host cell but transplanting its functionality from other branches of the phylogenetic tree. Microfluidic assembly of functional SOs paves the way for high-throughput generation of versatile intracellular structures implantable into living cells. This in-droplet SO design may support or expand cellular functionalities in translational nanomedicine

C-axis electronic Raman scattering in Bi_2Sr_2CaCu_2O_{8+\delta}

We report a c-axis-polarized electronic Raman scattering study of Bi_2Sr_2CaCu_2O_{8+\delta} single crystals. In the normal state, a resonant electronic continuum extends to 1.5 eV and gains significant intensity as the incoming photon energy increases. In the superconducting state, a coherence 2\Delta peak appears around 50 meV, with a suppression of the scattering intensity at frequencies below the peak position. The peak energy, which is higher than that seen with in-plane polarizations, signifies distinctly different dynamics of quasiparticle excitations created with out-of-plane polarization.Comment: 12 pages, REVTEX, 3 postscript figure

Electronic Raman scattering in YBCO and other superconducting cuprates

Superconductivity induced structures in the electronic Raman spectra of high-Tc superconductors are computed using the results of ab initio LDA-LMTO three-dimensional band structure calculations via numerical integrations of the mass fluctuations, either in the whole 3D Brillouin zone or limiting the integrations to the Fermi surface. The results of both calculations are rather similar, the Brillouin zone integration yielding additional weak structures related to the extended van Hove singularities. Similar calculations have been performed for the normal state of these high-Tc cuprates. Polarization configurations have been investigated and the results have been compared to experimental spectra. The assumption of a simple d_(x^2-y^2)-like gap function allows us to explain a number of experimental features but is hard to reconcile with the relative positions of the A1g and B1g peaks.Comment: 14 pages, LaTeX (RevTeX), 5 PostScript figures, uses multicol.sty, submitted to PR

Multiband model of high Tc superconductors

We propose an extension to other high T_{c } compounds of a model introduced earlier for YBCO. In the ''self-doped'' compounds we assume that the doping part (namely the BiO, HgO, TlO planes in BSCCO, HBCCO, TBCCO respectively) is metallic, which leads to a multiband model. This assumption is supported by band structure calculations. Taking a repulsive pairing interaction between these doping bands and the CuO_{2} bands leads to opposite signs for the order parameter on these bands and to nodes whenever the Fermi surfaces of these bands cross. We show that in BSCCO the low temperature dependence of the penetration depth is reasonably accounted for. In this case the nodes are not located near the 45^{o} direction, which makes the experimental determination of the node locations an important test for our model. The situation in HBCCO and TBCCO is rather analogous to BSCCO. We consider the indications given by NMR and find that they rather favor a metallic character for the doping bands. Finally we discuss the cases of NCCO and LSCO which are not ''self-doped'' and where our model does not give nodes.Comment: 11 pages, revtex, 1 figure

Diagnostic accuracy of non-invasive tests for advanced fibrosis in patients with NAFLD: An individual patient data meta-analysis

Objective Liver biopsy is still needed for fibrosis staging in many patients with non-alcoholic fatty liver disease. The aims of this study were to evaluate the individual diagnostic performance of liver stiffness measurement by vibration controlled transient elastography (LSM-VCTE), Fibrosis-4 Index (FIB-4) and NAFLD (non-alcoholic fatty liver disease) Fibrosis Score (NFS) and to derive diagnostic strategies that could reduce the need for liver biopsies. Design Individual patient data meta-analysis of studies evaluating LSM-VCTE against liver histology was conducted. FIB-4 and NFS were computed where possible. Sensitivity, specificity and area under the receiver operating curve (AUROC) were calculated. Biomarkers were assessed individually and in sequential combinations. Results Data were included from 37 primary studies (n=5735; 45% women; median age: 54 years; median body mass index: 30 kg/m2; 33% had type 2 diabetes; 30% had advanced fibrosis). AUROCs of individual LSM-VCTE, FIB-4 and NFS for advanced fibrosis were 0.85, 0.76 and 0.73. Sequential combination of FIB-4 cut-offs (<1.3; ≥2.67) followed by LSM-VCTE cut-offs (<8.0; ≥10.0 kPa) to rule-in or rule-out advanced fibrosis had sensitivity and specificity (95% CI) of 66% (63-68) and 86% (84-87) with 33% needing a biopsy to establish a final diagnosis. FIB-4 cut-offs (<1.3; ≥3.48) followed by LSM cut-offs (<8.0; ≥20.0 kPa) to rule out advanced fibrosis or rule in cirrhosis had a sensitivity of 38% (37-39) and specificity of 90% (89-91) with 19% needing biopsy. Conclusion Sequential combinations of markers with a lower cut-off to rule-out advanced fibrosis and a higher cut-off to rule-in cirrhosis can reduce the need for liver biopsies

Can bottom-up synthetic biology generate advanced drug-delivery systems?

Nanomedicine has demonstrated the potential of nanotechnology in treating diseases by selectively targeting pathogenic cells and releasing their cargo on site, but the complexity of molecular engineering such drug-delivery vehicles impedes their broad application and clinical translation. New methodologies to generate more advanced and intelligent systems are required. Bottom-up synthetic biology, empowered by microfluidics, allows the conception of multifunctional cell-mimicking structures – such as synthetic exosomes – that showcase its ability to create sophisticated systems. Recently, considerable progress has been made towards the assembly of complex structures that can dynamically release therapeutics, sustain protein biosynthesis, and sense and interact with the nearby environment. These functionalities will propel the creation of advanced drug-delivery platforms. Creating a magic bullet that can selectively kill cancer cells while sparing nearby healthy cells remains one of the most ambitious objectives in pharmacology. Nanomedicine, which relies on the use of nanotechnologies to fight disease, was envisaged to fulfill this coveted goal. Despite substantial progress, the structural complexity of therapeutic vehicles impedes their broad clinical application. Novel modular manufacturing approaches for engineering programmable drug carriers may be able to overcome some fundamental limitations of nanomedicine. We discuss how bottom-up synthetic biology principles, empowered by microfluidics, can palliate current drug carrier assembly limitations, and we demonstrate how such a magic bullet could be engineered from the bottom up to ultimately improve clinical outcomes for patients

Polymer‐based porous microcapsules as bacterial traps

Exposure to live bacteria and accumulation of dead bacteria during bactericidal processes can cause bacterial infectious diseases, implant failure, and antibacterial surface deterioration. Microcapsules with asymmetrically distributed, funnel‐shaped pores, which are capable of capturing, retaining, and killing bacteria are developed, offering a solution to bacterial contamination in liquids. It is found that bacterial isolation inside microcapsules is mainly driven by the bacteria's own motility and the microcapsules' geometry. After entry into the microcapsule cavity, the bacteria are stably retained inside. The microcapsules shield surrounding cells from exposure to bacterial toxins, as demonstrated by the coculture of rat embryonic fibroblast cells with microcapsules loaded with live Escherichia coli. The microcapsules can be enhanced with a bactericidal coating covering only the interior cavity. This confines the bacteria‐killing process, thereby further increasing biocompatibility. The microcapsules may offer a viable bacteria combatant approach as a potentially advantageous method to eradicate bacterial contamination

Bottom-up assembly of viral replication cycles

Bottom-up synthetic biology provides new means to understand living matter by constructing minimal life-like systems. This principle can also be applied to study infectious diseases. Here we summarize approaches and ethical considerations for the bottom-up assembly of viral replication cycles