Bit-error-rate testing of fiber optic data links for MMIC-based phased array antennas

The measured bit-error-rate (BER) performance of a fiber optic data link to be used in satellite communications systems is presented and discussed. In the testing, the link was measured for its ability to carry high burst rate, serial-minimum shift keyed (SMSK) digital data similar to those used in actual space communications systems. The fiber optic data link, as part of a dual-segment injection-locked RF fiber optic link system, offers a means to distribute these signals to the many radiating elements of a phased array antenna. Test procedures, experimental arrangements, and test results are presented

Engineering synucleinopathy‐resistant human dopaminergic neurons by CRISPR‐mediated deletion of the SNCA gene

An emerging treatment for Parkinson's disease (PD) is cell replacement therapy. Authentic midbrain dopaminergic (mDA) neuronal precursors can be differentiated from human embryonic stem cells (hESCs) and human induced pluripotent stem cells (iPSCs). These laboratory‐generated mDA cells have been demonstrated to mature into functional dopaminergic neurons upon transplantation into preclinical models of PD. However, clinical trials with human fetal mesenchephalic cells have shown that cell replacement grafts in PD are susceptible to Lewy body formation suggesting host‐to‐graft transfer of α‐synuclein pathology. Here, we have used CRISPR/Cas9n technology to delete the endogenous SNCA gene, encoding for α‐synuclein, in a clinical‐grade hESC line to generate SNCA+/− and SNCA−/− cell lines. These hESC lines were first differentiated into mDA neurons, and then challenged with recombinant α‐synuclein preformed fibrils (PFFs) to seed the formation for Lewy‐like pathology as measured by phosphorylation of serine‐129 of α‐synuclein (pS129‐αSyn). Wild‐type neurons were fully susceptible to the formation of protein aggregates positive for pS129‐αSyn, while SNCA+/− and SNCA−/− neurons exhibited significant resistance to the formation of this pathological mark. This work demonstrates that reducing or completely removing SNCA alleles by CRISPR/Cas9n‐mediated gene editing confers a measure of resistance to Lewy pathology

Discovery, characterization and engineering of bacterial thermostable cellulose- degrading enzymes

Lignocellulose is the most abundant biomass on Earth, and thus our largest organic carbon reservoir. Enzymatic depolymerization of recalcitrant polysaccharides, notably cellulose, is a major cost driver in accessing the renewable energy stored within lignocellulosic biomass. Natural biodiversities may be explored to discover microbial enzymes that have evolved to conquer this task in various environments. We are studying novel enzymes from various biodiversities for the conversion of lignocellulosic materials, using (meta)genome mining and functional screening of fosmid libraries. Targeted biodiversities include deep-sea hot vents of the Arctic mid-ocean ridge (AMOR), the microbiome of the wood-eating Arctic shipworm, thermophilic enrichment cultures from biogas reactors, the Svalbard reindeer gut microbiome, and publicly available metagenomic data from various hot environments. Bioprospecting of the different biodiversities has so far resulted in the discovery of approximately 20 novel enzymes active on lignocellulosic substrates. The significant differences in the origin of the enzymes is reflected in their properties, both beneficial and challenging, and provide us with interesting engineering targets for improved performance in industrial settings. We will present case studies, including work on a novel thermostable cellulase named mgCel6A, with good activity on sulfite-pulped Norway spruce. This enzyme consists of a glycoside hydrolase family 6 catalytic domain (GH6) connected to a family 2 carbohydrate binding module (CBM2) and both the activity profile and predicted structural similarities to known cellulases suggest that mgCel6A is an endo-acting cellulase. Comparison of the full-length enzyme with the catalytic domain showed that the CBM strongly increases substrate binding, while not affecting thermal stability. However, importantly, in reactions with higher substrate concentrations the full-length enzyme was outperformed by the catalytic domain alone, underpinning previous suggestions that CBMs may be less useful in high-consistency bioprocessing. This enzyme is currently being targeted for rational engineering in an effort to decrease the pH optimum and improve the pH stability. Other case studies include GH48 cellulases and lytic polysaccharide monooxygenases (LPMOs). One important aspect of this work concerns the possible assembly of novel enzyme cocktails for lignocellulose processing that can compete with exiting commercial cocktails, which are primarily composed of fungal enzymes. Thus, comparative studies of our most promising bacterial enzymes with their well-known fungal counterparts are also being conducted

Tectonic Controls on Gas Hydrate Distribution off SW Taiwan

The northern part of the South China Sea is characterized by widespread occurrence of bottom simulating reflectors (BSR) indicating the presence of marine gas hydrate. Because the area covers both a tectonically inactive passive margin and the termination of a subduction zone, the influence of tectonism on the dynamics of gas hydrate systems can be studied in this region. Geophysical data show that there are multiple thrust faults on the active margin while much fewer and smaller faults exist in the passive margin. This tectonic difference matches with a difference in the geophysical characteristics of the gas hydrate systems. High hydrate saturation derived from ocean bottom seismometer data and controlled source electromagnetic data and conspicuous high‐amplitude reflections in P‐Cable 3D seismic data above the BSR are found in the anticlinal ridges of the active margin. In contrast all geophysical evidence for the passive margin points to normal to low hydrate saturations. Geochemical analyses of gas samples collected at seep sites on the active margin show methane with heavy δ13C isotope composition, while gas collected at the passive margin shows light carbon isotope composition. Thus, we interpret the passive margin as a typical gas hydrate province fuelled by biogenic production of methane and the active margin gas hydrate system as a system that is fuelled not only by biogenic gas production but also by additional advection of thermogenic methane from the subduction system

Highly selective BSA imprinted polyacrylamide hydrogels facilitated by a metal-coding MIP approach

We report the fabrication of metal-coded molecularly imprinted polymers (MIPs) using hydrogel-based protein imprinting techniques. A Co(II) complex was prepared using (E)-2-((2 hydrazide-(4-vinylbenzyl) hydrazono)methyl)phenol; along with iron(III) chloroprotoporphyrin (Hemin), vinylferrocene (VFc), zinc (II) protoporphyrin (ZnPP) and protoporphyrin (PP), these complexes were introduced into the MIPs as co-monomers for metal-coding of non-metalloprotein imprints. Results indicate a 66% enhancement for bovine serum albumin (BSA) protein binding capacities (Q, mg/g) via metal-ion/ligand exchange properties within the metal-coded MIPs. Specifically, Co(II)-complex-based MIPs exhibited 92 ± 1% specific binding with Q values of 5.7 ± 0.45 mg BSA/g polymer and imprinting factors (IF) of 14.8 ± 1.9 (MIP/non-imprinted (NIP) control). The selectivity of our Co(II)-coded BSA MIPs were also tested using bovine haemoglobin (BHb), lysozyme (Lyz), and trypsin (Tryp). By evaluating imprinting factors (K), each of the latter proteins was found to have lower affinities in comparison to cognate BSA template. The hydrogels were further characterised by thermal analysis and differential scanning calorimetry (DSC) to assess optimum polymer composition

α-Synuclein-Confocal Nanoscanning (ASYN-CONA), a Bead-Based Assay for Detecting Early-Stage α-Synuclein Aggregation

α-Synuclein fibrils are considered a hallmark of Parkinson’s disease and other synucleinopathies. However, small oligomers that formed during the early stages of α-synuclein aggregation are thought to be the main toxic species causing disease. The formation of α-synuclein oligomers has proven difficult to follow, because of the heterogeneity and transient nature of the species formed. Here, a novel bead-based aggregation assay for monitoring the earliest stages of α-synuclein oligomerization, α-Synuclein–Confocal Nanoscanning (ASYN-CONA), is presented. The α-synuclein A91C single cysteine mutant is modified with a trifunctional chemical tag, which allows simultaneous fluorescent labeling with a green dye (tetramethylrhodamine, TMR) and attachment to microbeads. Beads with bound TMR-labeled α-synuclein are then incubated with a red dye (Cy5)-labeled variant of α-synuclein A91C, and EtOH (20%) to induce aggregation. Aggregation is detected by confocal scanning imaging, below the equatorial plane of the beads, which is known as the CONA technique. On-bead TMR-labeled α-synuclein and aggregated Cy5-labeled α-synuclein from the solution are quantitatively monitored in parallel by detection of fluorescent halos or “rings”. α-Synuclein on-bead oligomerization results in a linear increase of red bead ring fluorescence intensity over a period of 5 h. Total internal reflection fluorescence microscopy was performed on oligomers cleaved from the beads, and it revealed that (i) oligomers are sufficiently stable in solution to investigate their composition, consisting of 6 ± 1 monomer units, and (ii) oligomers containing a mean of 15 monomers bind Thioflavin-T. Various known inhibitors of α-synuclein aggregation were used to validate the ASYN-CONA assay for drug screening. Baicalein, curcumin, and rifampicin showed concentration-dependent inhibition of the α-synuclein aggregation and the IC₅₀ (the concentration of the compound at which the maxiumum intensity was reduced by one-half) were calculated

A rapid and sensitive assay for quantification of siRNA efficiency and specificity

RNA Interference has rapidly emerged as an efficient procedure for knocking down gene expression in model systems. However, cross-reactivity, whereby multiple genes may be simultaneously targeted by a single short interfering RNA (siRNA), can potentially jeopardize correct interpretation of gene function. As such, it is essential to test the specificity of a siRNA prior to a full phenotypic analysis. To this end, we have adapted a reporter-based assay harnessing the sensitivity of luciferase activity to provide a quantitative readout of relative RNAi efficacy and specificity. We have tested different siRNAs directed against Thymosin β4 (Tβ4); determined their effectiveness at silencing Tβ4 and have both excluded off-target silencing of the Tβ4 homologue Thymosin β10 (Tβ10) and demonstrated partial knockdown of Tβ10 despite significant (12/23; 52%) sequence mismatch. This assay system is applicable to any RNAi study where there is a risk of targeting homologous genes and to the monitoring of off-target effects at the genome level following microarray expression profiling

Protein aggregation and calcium dysregulation are hallmarks of familial Parkinson's disease in midbrain dopaminergic neurons

Mutations in the SNCA gene cause autosomal dominant Parkinson’s disease (PD), with loss of dopaminergic neurons in the substantia nigra, and aggregation of α-synuclein. The sequence of molecular events that proceed from an SNCA mutation during development, to end-stage pathology is unknown. Utilising human-induced pluripotent stem cells (hiPSCs), we resolved the temporal sequence of SNCA-induced pathophysiological events in order to discover early, and likely causative, events. Our small molecule-based protocol generates highly enriched midbrain dopaminergic (mDA) neurons: molecular identity was confirmed using single-cell RNA sequencing and proteomics, and functional identity was established through dopamine synthesis, and measures of electrophysiological activity. At the earliest stage of differentiation, prior to maturation to mDA neurons, we demonstrate the formation of small β-sheet-rich oligomeric aggregates, in SNCA-mutant cultures. Aggregation persists and progresses, ultimately resulting in the accumulation of phosphorylated α-synuclein aggregates. Impaired intracellular calcium signalling, increased basal calcium, and impairments in mitochondrial calcium handling occurred early at day 34–41 post differentiation. Once midbrain identity fully developed, at day 48–62 post differentiation, SNCA-mutant neurons exhibited mitochondrial dysfunction, oxidative stress, lysosomal swelling and increased autophagy. Ultimately these multiple cellular stresses lead to abnormal excitability, altered neuronal activity, and cell death. Our differentiation paradigm generates an efficient model for studying disease mechanisms in PD and highlights that protein misfolding to generate intraneuronal oligomers is one of the earliest critical events driving disease in human neurons, rather than a late-stage hallmark of the disease