Upgrading the Nutritional Value of PKC Using a Bacillus subtilis Derived Monocomponent β-Mannanase

Palm kernel cake (PKC) is an abundant side stream that can only be added to non-ruminant feed in small concentrations due to its content of antinutritional factors, mainly galactomannan, which cannot be digested by non-ruminants. β-mannanases can be added to partially hydrolyze galactomannan to form mannose oligosaccharides, which are known to be prebiotic. We here investigate the action of a β-mannanase from B. subtilis on PKC by colorimetry, NMR and fluorescence microscopy. The amount of mannan oligosaccharides in solution was significantly increased by the β-mannanase and their degree of polymerization (DP) was significantly reduced. There was a dose-response behavior in that larger β-mannanase concentrations increased the amount of soluble mannose oligosaccharides while reducing their average DP. Using confocal immunofluorescence microscopy, solubilization of galactomannan in PKC was clearly visualized. Images show a clear disruption of the cellulose and galactomannan structures of the PKC cell walls. We thus show in this study that using commercial dosages of β-mannanase on PKC can lead to formation of prebiotic compounds. Thus, this study suggests that utilization of PKC in poultry feed formulation might be increased by addition of a β-mannanase and would improve the return on investment

Broadband single-chip transceivers for compact NMR probes

Nuclear magnetic resonance (NMR) is one of the most relevant spectroscopic tools in use today. However, NMR requires relatively expensive and complicated experimental settings given by the combination of high homogeneous magnetic fields and a relatively complex radio-frequency (RF) electronics. This thesis concerns the development of RF electronics hardware, specifically introducing new complementary-metal-oxide-semiconductor (CMOS) transceiver designs. This work stems from a collaboration between EPFL and Metrolab SA, and aims at pushing in two directions: first, NMR-oriented CMOS transceivers will simplify the implementation of NMR probes for both experimental and commercial applications; second, novel CMOS ultra-compact probes will deliver experimental versatility and improved sensing power at the nL and sub-nL scale. We describe broadband 1 mm^2 transceivers operating in the range from 1 MHz to 1 GHz. The microchips include a RF power amplifier, a low-noise RF preamplifier, a frequency mixer, an audio-frequency (AF) amplifier, fully integrated transmit-receive switches, IQ signal generation, and broadband quadrature detection. In this work we show multi-nuclear NMR spectroscopy in combination with excitation/detection probe-heads based on micro-solenoids, therefore validating the broadband functioning. A combination of the transceivers and Metrolab's technology is also shown to deliver state-of-art performance in prototypes of commercial probes aimed for magnetometry. We shown that custom multichannel probes employing water samples of 500 nL are capable of measurement resolutions as high as 0.06 ppb/Hz^(1/2) at 7 T, and that magnetic noise due to field fluctuations can be directly measured at this resolution level and distinguished by the electronic noise. Overall, the results of this package indicate that NMR-oriented CMOS transceivers simplify the implementation of NMR probes for both experimental and commercial applications. When CMOS transceivers are combined to external resonators the resulting NMR probe may be called "compact" in the sense that the overall probe size is dominated by the excitation/detection resonator itself. Besides the implementation of compact probes, in this thesis we introduce the concept of ultra-compact NMR probes, where a single-chip transceiver is co-integrated with a multilayer microcoil realized with the metals of the CMOS technology. We demonstrate that with a non-resonant integrated coil of about 150 µm external diameter a 1H spin sensitivity of about 1.5·10^13 spins/Hz^(1/2) is achieved at 7 T. This value of sensitivity compares well with the most sensitive inductive probes previously reported at similar volume scales, with the resulting device showing an exceptional degree of versatility. We use, for the first time, a ultra-compact CMOS probe for the NMR spectroscopy of intact, static, sub-nL single ova of 0.1 and 0.5 nL, thereby reaching the relevant volume scale where life development begins for a broad variety of organisms, humans included. Thanks to the robustness and the versatility of the probe we could deliver a first extensive study of sub-nL single ova and indicate that ultra-compact probes are promising candidates to enable NMR-based study and selection of microscopic entities at biologically relevant volume scales. Overall, the results of this study indicate that CMOS ultra-compact probes will deliver experimental versatility and improved sensing power at the nL and sub-nL scale

Acrylamides with hydrolytically labile carbonate ester side chains as versatile building blocks for well-defined block copolymer micelles via RAFT polymerization

En route towards improved delivery systems for targeted chemotherapy, we propose a straightforward approach for the hydrophobic modification of the acrylamide N-(2-Hydroxyethyl) acrylamide (HEAm). An ethyl or benzyl group was introduced via a hydrolytically sensitive carbonate ester yielding HEAm-EC and HEAm-BC, respectively. Block copolymers of HEAm, respectively PEG and HEAm-EC or HEAm-BC were successfully synthesized by reversible addition-fragmentation chain transfer (RAFT) polymerization, obtaining a library of well-defined block copolymers with different degrees of polymerization (DP). To further explore the versatility of our approach in terms of polymer synthesis, self-assembly, drug solubilization and in vitro cell interaction, polyethylene glycol (PEG) and polyHEAm as hydrophilic polymer blocks were compared. The block copolymers formed micellar nanoparticles (10-100 nm) in PBS and could efficiently solubilize hydrophobic dyes and anti-cancer drugs. Benzyl carbonate ester side chains increased micellar stability and drug loading capacity. Moreover, PEG as hydrophilic block showed in comparison to HEAm more promising results concerning both colloidal stability and drug loading capacity. Confocal microscopy showed that the micelles could efficiently deliver a hydrophobic dye inside the cells. Finally, we also demonstrated efficient formulation of the anti-cancer drug paclitaxel with an in vitro cancer cell killing performance comparable or even better than the two commercial PTX nano-formulations Abraxane and Genexol-PM at equal drug dose. In conclusion, modification of HEAm through carbonate linkages offers a versatile platform for the design of degradable polymers with potential for biomedical applications

Nanomagnetic Resonance Imaging (Nano-MRI) Gives Personalized Medicine a New Perspective

This chapter provides a brief overview of molecular imaging techniques and its present and future potential in personalized medicine, with special a focus on the magnetic resonance imaging (MRI) approach. It discusses the current techniques that allow for the in vivo visualization of molecular processes at the nanoscale resolution (nano-MRI). Nano-MRI is progressing rapidly thanks to the work of a very small but extremely brilliant community of experts. This paper is not intended to be a comprehensive review of nano-MRI written for these experts, but rather a concise description of the present achievements for a much broader audience of medical professionals. The goal is to bridge the gap between the nano- MRI community and those in the medical field that will ultimately benefit from the further development of nano-MRI targeting specific medical goals. The aim of this review is to highlight the potential of nano-MRI in the improvement of MRI sensitivity and consequently on the impact of this widely used technique for diagnosis and personalized treatment. Sensitivity improvements are based on the use of magnetic nanoprobes in conventional MRI as well as novel nanoscale imaging based on nitrogen-vacancy (NV) centers in diamonds

A selective ratiometric fluorescent probe for no‐wash detection of PVC microplastic

Microplastics (MP) are micrometric plastic particles present in drinking water, food and the environment that constitute an emerging pollutant and pose a menace to human health. Novel methods for the fast detection of these new contaminants are needed. Fluorescence‐based detection exploits the use of specific probes to label the MP particles. This method can be environmentally friendly, low‐cost, easily scalable but also very sensitive and specific. Here, we present the synthesis and application of a new probe based on perylene‐diimide (PDI), which can be prepared in a few minutes by a one‐pot reaction using a conventional microwave oven and can be used for the direct detection of MP in water without any further treatment of the sample. The green fluorescence is strongly quenched in water at neutral pH because of the formation dimers. The ability of the probe to label MP was tested for polyvinyl chloride (PVC), polyethylene (PE), polyethylene terephthalate (PET), polypropylene (PP), polystyrene (PS), poly methyl methacrylate (PMMA) and polytetrafluoroethylene (PTFE). The probe showed considerable selectivity to PVC MP, which presented an intense red emission after staining. Interestingly, the fluorescence of the MP after labeling could be detected, under excitation with a blue diode, with a conventional CMOS color camera. Good selectivity was achieved analyzing the red to green fluorescence intensity ratio. UV– Vis absorption, steady‐state and time‐resolved fluorescence spectroscopy, fluorescence anisotropy, fluorescence wide‐field and confocal laser scanning microscopy allowed elucidating the mechanism of the staining in detail

Development and application of microtechnologies in the design and fabrication of cell culture biomimetic systems

“Lab-On-a-chip” systems have proved to be a promising tool in the field of biology. Currently, cell culture is performed massively on Petri dishes, which have traditionally been used in cell culture laboratories and tissue engineering. However, having proved to be a widely used tool until now, the scientific community has largely described the lack of correlation between the results obtained in the laboratory and the clinical results. This lack of connection between what has been studied in the laboratories and what has been observed in the clinic has led to the search for more advanced alternative tools that allow results to be obtained closer to reality. Thus, the use of microtechnologies in the field of biomedical engineering, presents itself as the perfect tool as an alternative to obsolete traditional media. Thanks to the low volumes of liquid it presents for its use, it also makes it an essential technology for the testing of drugs, new compounds and materials. By being able to more accurately reproduce the biomimetic environment of cell cultures and tissues, they make this technique fundamental as an intermediate step between basic in vitro laboratory tests and preclinical animal tests, resulting from this way in the best alternative for the reduction of both the use of animal models, as in times and costs. For a biomimetic system to be as such, it also needs another series of complementary devices for its better functioning. Micro-valves, micro pumps, flow sensors, O2 sensors, pH, CO2 are fundamental for the correct functioning andsophistication of biomimetic systems. This complexity, on the other hand, is often not perceived by the user since the miniaturization of all these components makes “Lab-On-a-Chip” systems smaller every day, despite numerous control components that can be incorporated.This thesis presents some examples of different microfluidic devices designed and manufactured through the use of microtechnologies, with all applications, focused on their use in biomimetic systems.<br /

Manufacturing Methods for Magnetic Resonance Microscopy Tools with Application to Neuroscience

Magnetresonanztomographie (MR) ist ein unverzichtbares nicht-invases und hochselektives bildgebendes Verfahren in der Medizin. MR Tomographie wird kommerziell in der klinischen Diagnostik und der Forschung für Gehirnkrankheit, z.B. Epilepsie, Alzheimer und Parkinson, angewandt. In den Neurowissenschaften haben sich Kleintiere als biologische Modelle für die grundlegenden Studien zur diesen Gehirnkrankheiten etabliert. MR Methoden sind ein wertvolles Werkzeug um die Morphologie und den Metabolismus von Kleintieren zu untersuchen. Die Modelle für die Untersuchung von Gehirnkrankheiten schließen Zellen/Zellkulturen und organotypische hippocampale Schnittkulturen (OHSC) mit ein. Obwohl die MR Mikroskopie für die Untersuchung von OHSC schon angewandt wurde fehlt eine effektive Plattform für umfangreiche longitudinale Studien an OHSC wie sie in den Neurowissenschaften üblich sind. Zwei Detektorkonzepte für die MR Mikroskopie inklusive ihrer Auslegung, der Herstellung und der Charakterisierung, werden in dieser Arbeit beschrieben. Beide Konzepte basieren auf Herstellungsmethoden welche hohe Fertigungsgenauigkeiten zulassen und in ihrem Herstellungsvolumen skalierbar sind. Hohle solenoide Mikrospulen welche für hochauflösende Untersuchung von Zell und Zellanhäufungen geeignet sind werden eingeführt. Die Herstellung basiert auf dem automatisierten wickeln von Mikrospulen, eine skalierbare und hochpräzise Fertigungsmethode der Mikrotechnologie. Zudem werde induktiv gekoppelte Ober ächenspulen eingeführt. Diese Oberflächenspulen fokussieren den magnetischen Fluss und werden deshalb Lenz Linsen genannt. Die Lenz Linsen werden mit kabelgebundenen und induktiv gekoppelten Spulen verglichen. Ihre Breitband-Fähigkeit machen sie zu einem idealen Kandidaten für die Nutzung in verschiedensten MR Tomographie Systemen. Die Lenz Linsen wurden für den Einsatz in einer MR kompatiblen Inkubationsplattform ausgelegt, welche in dieser Arbeit entwickelt wurde. Der MR Inkubator erweitert die Funktionalität eines MR Tomographen um neurologische Gewebe (z.B. OHSC) über mehrere Stunden andauernde MR Messungen am Leben zu erhalten. Der MR Inkubator erlaubt longitudinale Studien an OHSC und bietet damit eine Plattform für umfangreiche Studien in den Neurowissenschaften. Die Lenz Linsen wurden zusammen mit dem MR Inkubator für MR Mikroskopie Mes- sung von akuten/ xierten hippocampalen Schnitten und OHSC genutzt. Die Resultate dieser MR Mikoskopie Messungen zeigen dass in OHSC die grobe Zytoarchitektur sicht- bar ist, ohne dass die OHSC während der Messungen sterben. Somit ist das eingeführte System bereit für longitudinale Studien an OHSC, welche bereits für die Aufklärung der Epilepsieprogression begonnen wurden