TensorFit: A torch-based tool for ultrafast metabolite fitting of large MRSI data sets.

PURPOSE: To introduce a tool (TensorFit) for ultrafast and robust metabolite fitting of MRSI data based on Torch's auto-differentiation and optimization framework. METHODS: TensorFit was implemented in Python based on Torch's auto-differentiation to fit individual metabolites in MRS spectra. The underlying time domain and/or frequency domain fitting model is based on a linear combination of metabolite spectroscopic response. The computational time efficiency and accuracy of TensorFit were tested on simulated and in vivo MRS data and compared against TDFDFit and QUEST. RESULTS: TensorFit demonstrates a significant improvement in computation speed, achieving a 165-times acceleration compared with TDFDFit and 115 times against QUEST. TensorFit showed smaller percentual errors on simulated data compared with TDFDFit and QUEST. When tested on in vivo data, it performed similarly to TDFDFit with a 2% better fit in terms of mean squared error while obtaining a 169-fold speedup. CONCLUSION: TensorFit enables fast and robust metabolite fitting in large MRSI data sets compared with conventional metabolite fitting methods. This tool could boost the clinical applicability of large 3D MRSI by enabling the fitting of large MRSI data sets within computation times acceptable in a clinical environment.

Deep learning pipeline for quality filtering of MRSI spectra.

With the rise of novel 3D magnetic resonance spectroscopy imaging (MRSI) acquisition protocols in clinical practice, which are capable of capturing a large number of spectra from a subject's brain, there is a need for an automated preprocessing pipeline that filters out bad-quality spectra and identifies contaminated but salvageable spectra prior to the metabolite quantification step. This work introduces such a pipeline based on an ensemble of deep-learning classifiers. The dataset consists of 36,338 spectra from one healthy subject and five brain tumor patients, acquired with an EPSI variant, which implemented a novel type of spectral editing named SLOtboom-Weng (SLOW) editing on a 7T MR scanner. The spectra were labeled manually by an expert into four classes of spectral quality as follows: (i) noise, (ii) spectra greatly influenced by lipid-related artifacts (deemed not to contain clinical information), (iii) spectra containing metabolic information slightly contaminated by lipid signals, and (iv) good-quality spectra. The AI model consists of three pairs of networks, each comprising a convolutional autoencoder and a multilayer perceptron network. In the classification step, the encoding half of the autoencoder is kept as a dimensionality reduction tool, while the fully connected layers are added to its output. Each of the three pairs of networks is trained on different representations of spectra (real, imaginary, or both), aiming at robust decision-making. The final class is assigned via a majority voting scheme. The F1 scores obtained on the test dataset for the four previously defined classes are 0.96, 0.93, 0.82, and 0.90, respectively. The arguably lower value of 0.82 was reached for the least represented class of spectra mildly influenced by lipids. Not only does the proposed model minimise the required user interaction, but it also greatly reduces the computation time at the metabolite quantification step (by selecting a subset of spectra worth quantifying) and enforces the display of only clinically relevant information.

Crystallization-Enhanced Emission and Room-Temperature Phosphorescence of Cyclic Triimidazole-Monohexyl Thiophene Derivatives.

The development of organic room-temperature phosphorescent (ORTP) materials represents an active field of research due to their significant advantages with respect to their organometallic counterparts. Two cyclic triimidazole (TT) derivatives bearing one and three hexyl-thiophene moieties, TT-HThio and TT-(HThio)3, have been prepared and characterized. Both compounds display enhanced quantum yields in their crystalline form with respect to those in a solution state, revealing crystallization-enhanced emissive (CEE) behavior. Importantly, while single fluorescence is observed in solution, crystalline powders also feature dual ORTP, whose respective molecular and aggregate origins have been disclosed through X-ray diffraction analysis and DFT/TDDFT calculations. The relation between the photophysical properties of TT-HThio and its crystallinity degree has been confirmed by a decrease in photoluminescent quantum yield (Φ) and loss of vibronic resolution when its crystals are ground in a mortar, revealing mechanochromic behavior and confirming CEE features.

Combined clarification and affinity capture using magnetic resin enables efficient separation of rAAV5 from cell lysate.

Recombinant adeno-associated virus (rAAV) vectors have displayed enormous potential as a platform for delivery of gene therapies. Purification of rAAV at industrial scale involves a series of elaborate, material, and time-consuming midstream steps, such as clarification by depth filtration and concentration/buffer exchange by tangential flow filtration. In this study, we developed a filter-less flow capture method for purification of rAAV serotype 5, using a high-gradient magnetic separator and magnetic Mag Sepharose beads coupled to an AVB affinity ligand. In under 2 h, we captured and eluted rAAV5 directly from ∼5 L of cell lysate with a recovery yield of 63% (±5%, n = 3). Compared to cell lysate, the eluate showed a 3-log reduction of host cell DNA and host cell proteins. The process developed eliminates the need for filtration and column chromatography in the early steps of industrial rAAV purification. This will be of high value for industrial-scale manufacturing of rAAVs by reducing time and material in the purification process, without compromising product recovery and purity.

Analysis of the robustness and dynamics of spin-locking preparations for the detection of oscillatory magnetic fields.

Extracting quantitative information of neuronal signals by non-invasive imaging is an outstanding challenge for understanding brain function and pathology. However, state-of-the-art techniques offer low sensitivity to deep electrical sources. Stimulus induced rotary saturation is a recently proposed magnetic resonance imaging sequence that detects oscillatory magnetic fields using a spin-lock preparation. Phantom experiments and simulations proved its efficiency and sensitivity, but the susceptibility of the method to field inhomogeneities is still not well understood. In this study, we simulated and analyzed the dynamic of three spin-lock preparations and their response to field inhomogeneities in the presence of a resonant oscillating field. We show that the composite spin-lock preparation is more robust against field variations within the double resonance effect. In addition, we tested the capability of the chosen composite spin-lock preparation to recover information about the spectral components of a composite signal. This study sets the bases to move one step further towards the clinical application of MR-based neuronal current imaging.

Synthetic Biology Toolbox, Including a Single-Plasmid CRISPR-Cas9 System to Biologically Engineer the Electrogenic, Metal-Resistant Bacterium Cupriavidus metallidurans CH34.

Cupriavidus metallidurans CH34 exhibits extraordinary metabolic versatility, including chemolithoautotrophic growth; degradation of BTEX (benzene, toluene, ethylbenzene, xylene); high resistance to numerous metals; biomineralization of gold, platinum, silver, and uranium; and accumulation of polyhydroxybutyrate (PHB). These qualities make it a valuable host for biotechnological applications such as bioremediation, bioprocessing, and the generation of bioelectricity in microbial fuel cells (MFCs). However, the lack of genetic tools for strain development and studying its fundamental physiology represents a bottleneck to boosting its commercial applications. In this study, inducible and constitutive promoter libraries were built and characterized, providing the first comprehensive list of biological parts that can be used to regulate protein expression and optimize the CRISPR-Cas9 genome editing tools for this host. A single-plasmid CRISPR-Cas9 system that can be delivered by both conjugation and electroporation was developed, and its efficiency was demonstrated by successfully targeting the pyrE locus. The CRISPR-Cas9 system was next used to target candidate genes encoding type IV pili, hypothesized by us to be involved in extracellular electron transfer (EET) in this organism. Single and double deletion strains (ΔpilA, ΔpilE, and ΔpilAE) were successfully generated. Additionally, the CRISPR-Cas9 tool was validated for constructing genomic insertions (ΔpilAE::gfp and ΔpilAE::λPrgfp). Finally, as type IV pili are believed to play an important role in extracellular electron transfer to solid surfaces, C. metallidurans CH34 ΔpilAE was further studied by means of cyclic voltammetry using disposable screen-printed carbon electrodes. Under these conditions, we demonstrated that C. metallidurans CH34 could generate extracellular currents; however, no difference in the intensity of the current peaks was found in the ΔpilAE double deletion strain when compared to the wild type. This finding suggests that the deleted type IV pili candidate genes are not involved in extracellular electron transfer under these conditions. Nevertheless, these experiments revealed the presence of different redox centers likely to be involved in both mediated electron transfer (MET) and direct electron transfer (DET), the first interpretation of extracellular electron transfer mechanisms in C. metallidurans CH34.

Oxygen-Tolerant RAFT Polymerization Initiated by Living Bacteria.

Living organisms can synthesize a wide range of macromolecules from a small set of natural building blocks, yet there is potential for even greater materials diversity by exploiting biochemical processes to convert unnatural feedstocks into new abiotic polymers. Ultimately, the synthesis of these polymers in situ might aid the coupling of organisms with synthetic matrices, and the generation of biohybrids or engineered living materials. The key step in biohybrid materials preparation is to harness the relevant biological pathways to produce synthetic polymers with predictable molar masses and defined architectures under ambient conditions. Accordingly, we report an aqueous, oxygen-tolerant RAFT polymerization platform based on a modified Fenton reaction, which is initiated by Cupriavidus metallidurans CH34, a bacterial species with iron-reducing capabilities. We show the synthesis of a range of water-soluble polymers under normoxic conditions, with control over the molar mass distribution, and also the production of block copolymer nanoparticles via polymerization-induced self-assembly. Finally, we highlight the benefits of using a bacterial initiation system by recycling the cells for multiple polymerizations. Overall, our method represents a highly versatile approach to producing well-defined polymeric materials within a hybrid natural-synthetic polymerization platform and in engineered living materials with properties beyond those of biotic macromolecules.

Design and characterization of a synthetic minimal promoter for heterocyst-specific expression in filamentous cyanobacteria.

Short and well defined promoters are essential for advancing cyanobacterial biotechnology. The heterocyst of Nostoc sp. is suggested as a microbial cell factory for oxygen sensitive catalysts, such as hydrogenases for hydrogen production, due to its microoxic environment. We identified and predicted promoter elements of possible significance through a consensus strategy using a pool of heterocyst-induced DIF+ promoters known from Anabaena sp. PCC 7120. To test if these conserved promoter elements were crucial for heterocyst-specific expression, promoter-yfp reporter constructs were designed. The characterization was accomplished by replacing, -35 and -10 regions and the upstream element, with well described elements from the trc promoter of Escherichia coli, which is also functional in Nostoc sp. From the in vivo spatial fluorescence of the different promoter-yfp reporters in Nostoc punctiforme ATCC 29133, we concluded that both the consensus -35 and extended -10 regions were important for heterocyst-specific expression. Further that the promoter strength could be improved by the addition of an upstream element. We designed a short synthetic promoter of 48 nucleotides, PsynDIF, including a consensus DIF1 sequence, a 17 base pair stretch of random nucleotides and an extended consensus -10 region, and thus generated the shortest promoter for heterocyst-specific expression to date.