Search

Magnetic guidance shows promise as a strategy for improving the delivery and performance of cell therapeutics. However, clinical translation of magnetically guided cell therapy requires cell functionalization protocols that provide adequate magnetic properties in balance with unaltered cell viability and biological function. Existing methodologies for characterizing cells functionalized with magnetic nanoparticles (MNP) produce aggregate results, both distorted and unable to reflect variability in either magnetic or biological properties within a preparation. In the present study, we developed an inverted-plate assay allowing determination of these characteristics using a single-platform approach, and applied this method for a comparative analysis of two loading protocols providing highly uniform vs. uneven MNP distribution across cells. MNP uptake patterns remarkably different between the two protocols were first shown by fluorimetry carried out in a well-scan mode on endothelial cells (EC) loaded with BODIPY558/568-labeled MNP. Using the inverted-plate assay we next demonstrated that, in stark contrast to unevenly loaded cells, more than 50% of uniformly functionalized EC were captured within 5 min over a broad range of MNP doses. Furthermore, magnetically captured cells exhibited unaltered viability, substrate attachment, and proliferation rates. Conducted in parallel, magnetophoretic mobility studies corroborated the markedly superior guidance capacity of uniformly functionalized cells, confirming substantially faster cell capture kinetics on a clinically relevant time scale. Taken together, these results emphasize the importance of optimizing cell preparation protocols with regard to loading uniformity as key to efficient site-specific delivery, engraftment, and expansion of the functionalized cells, essential for both improving performance and facilitating translation of targeted cell therapeutics.

Kaposiform lymphangiomatosis (KLA) is a complex lymphatic anomaly involving most commonly the mediastinum, lung, skin and bones with few effective treatments. In recent years, RAS-MAPK pathway mutations were shown to underlie the pathogenesis of several complex lymphatic anomalies. Specifically, an activating NRAS mutation (p.Q61R) was found in the majority of KLA patients. Recent reports demonstrated promising results of treatment with the MEK inhibitor, Trametinib, in patients with complex lymphatic anomalies harboring gain of function mutations in ARAF and SOS1, as well as loss of function mutation in the CBL gene, a negative regulator of the RAS-MAPK pathway. We present a 9-year-old child with a severe case of KLA harboring the typical NRAS (p.Q61R) mutation detected by plasma-derived cell free DNA, responsive to trametinib therapy.The NRAS somatic mutation was detected from plasma cfDNA using droplet digital PCR. Concurrent in-vitro studies of trametinib activity on mutant NRAS affected lymphatic endothelial cells were performed using a three-dimensional spheroid sprouting assay.Trametinib treatment lead to resolution of lifelong thrombocytopenia, improvement of pulmonary function tests and wellbeing, as well as weaning from prolonged systemic steroid treatment. Concurrent studies of mutant NRAS-expressing cells showed enhanced lymphangiogenic capacity along with over activation of the RAS-MAPK and PI3K-AKT-mTOR pathways, both reversed by trametinib.Trametinib treatment can substantially change the prognosis of patients with RAS pathway associated lymphatic anomalies.This is the first description of successful trametinib treatment of a patient with KLA harboring the most characteristic NRAS p.Q61R mutation. Treatment can significantly change the prognosis of patients with RAS pathway-associated lymphatic anomalies. We devised an in vitro model of KLA enabling a reproducible method for the continued study of disease pathogenesis. Mutated NRAS p.Q61R cells demonstrated increased lymphangiogenic capacity.

The treatment of lymphatic anomaly, a rare devastating disease spectrum of mostly unknown etiologies, depends on the patient manifestations1. Identifying the causal genes will allow for developing affordable therapies in keeping with precision medicine implementation2. Here we identified a recurrent gain-of-function ARAF mutation (c.640T>C:p.S214P) in a 12-year-old boy with advanced anomalous lymphatic disease unresponsive to conventional sirolimus therapy and in another, unrelated, adult patient. The mutation led to loss of a conserved phosphorylation site. Cells transduced with ARAF-S214P showed elevated ERK1/2 activity, enhanced lymphangiogenic capacity, and disassembly of actin skeleton and VE-cadherin junctions, which were rescued using the MEK inhibitor trametinib. The functional relevance of the mutation was also validated by recreating a lymphatic phenotype in a zebrafish model, with rescue of the anomalous phenotype using a MEK inhibitor. Subsequent therapy of the lead proband with a MEK inhibitor led to dramatic clinical improvement, with remodeling of the patient's lymphatic system with resolution of the lymphatic edema, marked improvement in his pulmonary function tests, cessation of supplemental oxygen requirements and near normalization of daily activities. Our results provide a representative demonstration of how knowledge of genetic classification and mechanistic understanding guides biologically based medical treatments, which in our instance was life-saving.

Increased susceptibility to thrombosis, neoatherosclerosis, and restenosis due to incomplete regrowth of the protective endothelial layer remains a critical limitation of the interventional strategies currently used clinically to relieve atherosclerotic obstruction. Rapid recovery of endothelium holds promise for both preventing the thrombotic events and reducing post-angioplasty restenosis, providing the rationale for developing cell delivery strategies for accelerating arterial reendothelialization. The successful translation of experimental cell therapies into clinically viable treatment modalities for restoring vascular endothelium critically depends on identifying strategies for enhancing the functionality of endothelial cells (EC) derived from high cardiovascular risk patients, the target group for the majority of angioplasty procedures. Enhancing EC-associated nitric oxide (NO) synthesis by inducing overexpression of NO synthase (NOS) has shown promise as a way of increasing paracrine activity and restoring function of EC. In the present study, we developed a direct contact co-culture approach compatible with highly labile effectors, such as NO, and applied it for determining the effect of EC functionalization via NOS gene transfer on the growth of co-cultured arterial smooth muscle cells (A10 cell line) exhibiting the defining characteristics of neointimal cells. Bovine aortic endothelial cells magnetically transduced with inducible NOS-encoding adenovirus (Ad) formulated in zinc oleate-based magnetic nanoparticles (MNP[(iNOS)Ad]) strongly suppressed growth of proliferating A10 and attenuated the stimulatory effect of a potent mitogen, platelet-derived growth factor (PDGF-BB), whereas EC functionalization with free (iNOS)Ad or MNP formulated with a different isoform of the enzyme, endothelial NOS, was associated with lower levels of NO synthesis and less pronounced antiproliferative activity toward co-cultured A10 cells. These results show feasibility of applying magnetically facilitated gene transfer to potentiate therapeutically relevant effects of EC for targeted cell therapy of restenosis. The direct contact co-culture methodology provides a sensitive and reliable tool with potential utility for a variety of biomedical applications.

It is important to synthesize materials to recapitulate critical functions of biological systems for a variety of applications such as tissue engineering and regenerative medicine. The purpose of this study was to synthesize a chimeric hydrogel as a promising extracellular matrix (ECM) mimic using gelatin, a nucleic acid aptamer and polyethylene glycol (PEG). This hydrogel had a macroporous structure that was highly permeable for fast molecular transport. Despite its high permeability, it could strongly sequester and sustainably release growth factors with high bioactivity. Notably, growth factors retained in the hydrogel could maintain ~50% bioactivity during a 14-day release test. It also provided cells with effective binding sites, which led to high efficiency of cell loading into the macroporous hydrogel matrix. When cells and growth factors were co-loaded into the chimeric hydrogel, living cells could still be observed by day 14 in a static serum-reduced culture condition. Thus, this chimeric aptamer-gelatin hydrogel constitutes a promising biomolecular ECM mimic for loading cells and growth factors.

The treatment of lymphatic anomaly, a rare devastating disease spectrum of mostly unknown etiologies, depends on the patient manifestations

Surfaces functionalized with affinity ligands have been widely studied for applications such as biological separations and cell regulation. While individual ligands can be directly conjugated onto a surface, it is often important to conjugate polyvalent ligands onto the surface to enhance ligand display. This study was aimed at exploring a method for surface functionalization via polymerization of affinity ligands, which was achieved through ligand hybridization with DNA polymers protruding from the surface. The surface with polyvalent ligands was evaluated via aptamer-mediated cell binding. The results show that this surface bound target cells more effectively than a surface directly functionalized with individual ligands in situations with either equal amounts of ligand display or equal amounts of surface reaction sites. Therefore, this study has demonstrated a new strategy for surface functionalization to enhance ligand display and cell binding. This strategy may find broad applications in settings where surface area is limited or the surface of a material does not possess sufficient reaction sites.

Biomaterials for the precise control of protein release are important to the development of new strategies for treating human diseases. This study aimed to fundamentally understand aptamer--protein dissociation triggered by complementary oligonucleotides, and to apply this understanding to develop affinity hydrogels for controlled protein release. The results showed that the oligonucleotide tails of the aptamers played a critical role in inducing intermolecular hybridization and triggering aptamer--protein dissociation. In addition, the attachment of the oligonucleotide tails to the aptamers and the increase of hybridizing length could produce a synergistic effect on the dissociation of bound proteins from their aptamers. More importantly, pegylated complementary oligonucleotides could successfully trigger protein release from the aptamer-functionalized hydrogels at multiple time points. Based on these results, it is believed that aptamer-functionalized hydrogels and complementary oligonucleotides hold great potential of controlling the release of protein drugs to treat human diseases.

Nucleic acid aptamers are an emerging class of synthetic ligands and have recently attracted significant attention in numerous fields. One is in biosensor development. In principle, nucleic acid aptamers can be discovered to recognize any molecule of interest with high affinity and specificity. In addition, unlike most ligands evolved in nature, synthetic nucleic acid aptamers are usually tolerant of harsh chemical, physical, and biological conditions. These distinguished characteristics make aptamers attractive molecular recognition ligands for biosensing applications. This review first concisely introduces methods for aptamer discovery including upstream selection and downstream truncation, then discusses aptamer-based biosensor development from the viewpoint of signal production.

Synthetic molecular libraries hold great potential to advance the biomaterial development. However, little effort is made to integrate molecules with molecular recognition abilities selected from different libraries into a single biomolecular material. The purpose of this work is to incorporate peptides and nucleic acid aptamers into a porous hydrogel to develop a dual-functional biomaterial. The data show that an anti-integrin peptide can promote the attachment and growth of endothelial cells in a 3D porous poly(ethylene glycol) hydrogel and an antivascular endothelial growth factor aptamer can sequester and release VEGF of high bioactivity. Importantly, the dual-functional porous hydrogel enhances the growth and survival of endothelial cells. This work demonstrates that molecules selected from different synthetic libraries can be integrated into one system for the development of novel biomaterials.

While the discovery of highly potent biologics has led to the development of promising therapies for various human diseases, biologics can cause severe toxicity if delivered inappropriately. Thus, great efforts have been made to synthesize polymeric systems for safe and efficient delivery of biologics. However, the application of polymeric delivery systems is often limited by problems such as harsh reaction conditions, low drug sequestration efficiency, and difficult drug release regulation. This study was aimed at developing a superporous material system with a hydrogel and an aptamer to overcome these challenges. The results have shown that the superporous hydrogel is capable of instantaneously and fully sequestering a large amount of growth factors, owing to the presence of superporous architectures and aptamers. Moreover, the sequestering and loading procedure does not involve any harsh conditions. The release kinetics of growth factors can be molecularly modulated by either changing the binding affinity of the aptamer or by using a triggering effector. Therefore, this study presents a promising superporous material for the delivery of highly potent biologics such as growth factors for clinical applications.

In this paper, we report on a passive duplexer based on reflective modulation for single-mode fibers. The component integrates a silicon micromechanical modulator and a photoreceiver in a hybrid assembly. Fabricated by the silicon micromachining technology reflective modulators allow a considerable cost reduction of single mode fiber-optic links as compared to systems operating with active light sources at both link terminals. The micromechanical modulator is characterized by a low insertion loss of less than 3 dB and polarization insensitivity. The ON-OFF ratio is typically 9 dB over a wavelength range from 1260 to 1360 nm. The operation frequency is limited by the mechanical movement of the modulator membrane to 1.3-2 MHz. We report on the characterization of fully packaged duplexer components.

The development of biomaterials has shifted from the development of simple, biocompatible materials to advanced biomaterials with functions to mimic biological systems. To develop these advanced materials, biomaterials are often functionalized with ligands for cell attachment or protein binding. However, many ligands lack specificity of recognizing their targets or their binding functions are difficult to control. In this chapter, nucleic acid aptamers are introduced and many of their uses in biomaterial functionalization are discussed. Nucleic acid aptamers are single-stranded oligonucleotides ligands that have many advantages over other ligands. It is expected that nucleic acid aptamers can be used to develop the next generation of biomaterials.

We report on vertical mirrors fabricated by deep reactive ion etching of silicon. The mirror height is 75 /spl mu/m, covering the fiber core of a single-mode fiber when the latter is placed into a groove of equal depth and etched simultaneously with the mirror. To obtain a uniform etch depth, etching is stopped on a buried oxide layer. Using the buried oxide as a sacrificial layer allows to fabricate mirrors with suspension and actuation structures as well as fiber-alignment grooves in one and the same processing step. A minimal mirror thickness of 2.3 /spl mu/m was achieved, resulting in an aspect ratio higher than 30. The verticality was better than 89.3/spl deg/. In the upper part of the mirror a surface roughness below 40 nm rms was obtained. At a wavelength of 1300 nm the reflectivity of the aluminum-coated mirrors was measured to be higher than 76%. Using a reactive ion etched mirror we have fabricated an optical fiber switch with electrostatic actuation. The coupling loss in the bar state of two packaged prototypes was between 0.6 and 1.7 dB and between 1.4 and 3.4 dB in the cross state. The switching time is below 0.2 ms.

Reversible DNA hybridization can be used as a new mechanism to control the sustained and triggered release of therapeutic oligonucleotides from hydrogels.

The ability to regulate cell-material interactions is important in various applications such as regenerative medicine and cell separation. This study successfully demonstrates that the binding states of cells on a hydrogel surface can be programmed by using hybridized aptamers and triggering complementary sequences (CSs). In the absence of the triggering CSs, the aptamers exhibit a stable, hybridized state in the hydrogel for cell-type-specific catch. In the presence of the triggering CSs, the aptamers are transformed into a new hybridized state that leads to the rapid dissociation of the aptamers from the hydrogel. As a result, the cells are released from the hydrogel. The entire procedure of cell catch and release during the transformation of the aptamers is biocompatible and does not involve any factor destructive to either the cells or the hydrogel. Thus, the programmable hydrogel is regenerable and can be applied to a new round of cell catch and release when needed.

Polymeric delivery systems have been extensively studied to achieve localized and controlled release of protein drugs. However, it is still challenging to control the release of multiple protein drugs in distinct stages according to the progress of disease or treatment. This study successfully demonstrates that multiple protein drugs can be released from aptamer-functionalized hydrogels with adjustable release rates at predetermined time points using complementary sequences (CSs) as biomolecular triggers. Because both aptamer–protein interactions and aptamer–CS hybridization are sequence-specific, aptamer-functionalized hydrogels constitute a promising polymeric delivery system for the programmable release of multiple protein drugs to treat complex human diseases.

Capteur de temperature reparti utilisant la reflectometrie optique dans le domaine temporel. Raman anti Stokes et le comptage de photons

High power pump modules are used for industrial as well as telecom applications such as for fiber lasers and cladding pumped erbium doped fiber amplifiers (EDFA) [Lichtenstein, N., et al., 2004]. Key element of a pump module is a single emitter laser diode, which normally has an aperture of around 90 mum, in order to provide best coupling into an optical multi-mode fiber with a 105 mum core diameter. Increasing the brightness of the pump module lends additional flexibility in the design of fiber lasers and amplifiers and enables higher output power. In this communication we report on two approaches to increase the brightness of our latest generation of high power pump modules. Within the first approach the electro-optical performance of the single emitter laser diode is improved by optimizing Bookham's mature chip technologies such as InGaAs/AlGaAs structures grown by MBE, the ridge-waveguide manufacturing process, the E2 facet passivation and the mounting technology. The combination of improved chip design and new coupling scheme leads to reliable broad area modules with outstanding brightness.

Newly developed wavelength stabilized 980 nm pump lasers demonstrate uncooled operation with >100 mW ex-fiber power over a record -15/spl deg/C to 120/spl deg/C. With uncooled modules using these lasers, low cost optical amplifiers with ultra low power dissipation for metro systems become possible.

It is anticipated that in the near future erbium doped fiber amplifiers (EDFA) will find their way into new optical networking concepts, ranging from WDM systems in the metro sector to loss-compensation in optical switching. In order to be viable, these applications demand a technology, which in comparison to traditional systems is more compact, less costly, and more readily integrated, yet offers comparable performance and reliability. These demands are probably most significant for the EDFA pump laser. Our contribution discusses general reliability issues in pump module packaging, with special emphasis on how they will likely be addressed in future designs. As an example of this emerging trend in pump laser packaging we will present some of the design concepts and the reliability performance of our recently introduced family of un-cooled 980 nm pump modules. For 980 nm pumps, the key target specifications defined by new applications may be summarised as follows: chip reliability < 500 FIT; target package reliability < 500 FIT; wavelength-stabilised output power 200 mW; roadmap to 300 mW; operating case temperature 0/spl deg/C to 70/spl deg/C; power consumption < 1 W; packaging technology opens path to sustainable cost reduction. We and other manufacturers have recognised that these requirements can only be addressed by a significant differentiation of conventional 980 nm module technology, notably the transition to cooler-less operation. Our design features an 8-pin ceramic (Al/sub 2/O/sub 3/) miniDIL housing and a near planar assembly technology. The rated output power of this product family is 200 mW.

We present a fully packaged duplexer implementing a reflective Fabry-Perot modulator realised by silicon surface micromachining. The device demonstrates excellent performance for bit rates up to 2.8 Mbit/s. Relaxed positioning tolerances, a fiber to fiber loss below 3 dB, an extinction ratio better than 12 dB, and the potential for low cost fabrication make this device very attractive for use in economical optical links.

We report on vertical mirrors fabricated by deep reactive ion etching of silicon. In view of their application in fiber optical switches the mirror height is 75 /spl mu/m. So the mirror covers well the fiber core of a single mode fiber, when the fiber is placed in a groove of the same depth and etched at the same time as the mirror. A minimal mirror thickness of 2.3 /spl mu/m was achieved, resulting in an aspect ratio over thirty. The verticality was better than 89.3/spl deg/. The surface roughness of the etched surface is 36 nm rms. For an increased reflectivity the silicon mirrors are coated with aluminium. Their reflectivity was measured to be 76%. To obtain an uniform etch depth, etching is stopped on a buried oxide layer. This allows to integrate at the same time mirrors with suspension and actuation structures as well as fiber-alignment grooves in one processing step. Using this technology we have fabricated a fiber optical switch with promising performance: The coupling loss in the OFF position is 2.5 dB and 4 dB in the ON position. The switching time is below 0.2 ms.

Analysis showed that a new clip design is needed meeting the following requirements: (a) suitability for automation (e.g. handling, laser welding and fine adjustment), (b) tolerance for gaps between the rod and the clip walls to obtain good weld spot quality, (c) stable pivot point for readjustment by applying plastic deformation and (d) flexibility for assuring bending of the clip rather than in the rod. These requirements have been addressed by the clip design presented in this paper. The clip is made of Kovar using high precision electro-erosion techniques. It consists of a rigid front section and a flexible back section. The fiber is fixed by laser welding to both sections. The weld in the rigid front section acts as pivot point whereas movements on the back part results in fiber tip travel with an approx. 5:1 ratio. The final mechanical fine adjustment is done by bending (plastic deformation) the flexible back part of the clip or by laser hammering. This flexibility assures that the clip bends rather than the rod itself, which could lead to reliability problems. The flexibility was achieved by narrowing and rounding off the joints between welding area and clip base as well as by the positioning of the laser welds.

Many applications display a distinctly asymmetric traffic profile. Large traffic volume, for instance video data, travels in the downstream direction but only moderate bandwidth is carried in the opposite direction (service requests, telemetry, POTS). Therefore, solutions that trade reflective modulation speed for a cost advantage can be very attractive. It has been noted that a Fabry-Perot resonator fabricated in silicon micro-technology is a promising candidate to realise this cost advantage. We discuss the design and performance of a (3 Mbit/s) micro-mechanical Fabry-Perot reflective modulator. (4 pages)

Optical coupling from single mode fibers to integrated waveguides of semiconductor optoelectronic devices with less than 0.5 dB packaging loss generally requires positioning accuracies on the order of ±0.5 to ±1.5 μm. Laser welding is widely used for fixing because it guarantees long term stability, and it is the preferred method for hermetic packaging [1-3]. Furthermore, no thermal strain on the module parts and precise positioning of the welds are attractive features of this technique. A Nd:YAG laser equipped with fiber guided energy distribution to the welding heads is generally used. However, commercial laser systems do not guarantee a stable distribution of the energy into the different arms. This leads to different volumes and temperatures of the melted material in the weld spots, preventing a simultaneous solidification and cooling process. Hence, an unequal shrinking of the welding spots occurs, resulting in a shift of a few microns of the welded parts, strongly increasing the assembly loss. However, the shrinking of welding spots can be turned into an advantage by using it for readjustment purposes.

A new generation of wavelength stabilised, uncooled 980 nm pump modules in MiniDIL housings is presented, enabling 400 mW ex-fibre power over a temperature range of 10/spl deg/C to 70/spl deg/C. At 100/spl deg/C 200 mW power is still obtained with a robust fibre coupling scheme.

The simulated annealing algorithm is applied to the design of binary holographic optical elements for the connection of single-mode structures. The system energy used with the simulated annealing algorithm is the absolute square of the overlap between the transmitted wave front and the modal characteristics of the receiving device; the system variables are the binary states of the hologram pixels. The plausibility of the obtained binary patterns is discussed for examples involving plane and spherical modal characteristics. The ability of the annealing process to avoid local minimum trapping is demonstrated.

A novel fiber-optic Doppler velocimeter is presented. By suitably controlling the polarization state of the local oscillator, illuminating beam, and signal light, balanced mixing with a noise reduction of 30 dB as compared to unbalanced mixing can be realized. The remaining noise level is predominantly due to shot noise. Allowing for losses in the receiving optics and for nonperfect mixing efficiency, the minimum detectable signal power is estimated to be 10/sup -18/ W-Hz/sup -1/. The high sensitivity of the system is demonstrated by the detection of light scattered from a distant moving object. >

A fiberoptic acoustic sensor constructed in the Mach-Zehnder arrangement for use in a resonant photoacoustic cell is reported. Tests indicate a minimum detectable concentration of 0.5 ppm NOT, in Ar for an input power of 50 mW. This extrapo-lates for 500 mW input to a minimum detectable concentration of 50 ppb.

Opto- acoustic Fiber SensorQ. Munir, H.P. Weber, and R. BattigInstitute of Applied Physics, University of Bern,SwitzerlandAbstractAn optoacoustic sensor for trace analysis in gases was constructed and experimentallytested. The crucial element in such systems, the highly sensitive electronic microphone,was replaced by a fiberoptic interferometer in the Mach -Zehnder configuration. The fiber ofthe sensing arm was wrapped onto a thin foil arranged close to the cylindrical walls of theacoustic cell. Thus the acoustical waves generated by the absorbed light were transferredto an elongation of the fiber. The cell was operated at one of its resonances. A sensitivi-ty of ~ 50 ppb was evaluated for an input power of 500 mW radiation of an Ar -laser for thedetection of NO2 in an inert buffer gas. Presently the minimum observable power is 7.10 -8W /cm I. Details on this device are described in