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The exosporium of Bacillus megaterium QM B1551 spores is morphologically distinct from exosporia observed for the spores of many other species. Previous work has demonstrated that unidentified genes carried on one of the large indigenous plasmids are required for the assembly of the Bacillus megaterium exosporium. Here, we provide evidence that pBM600-encoded orthologues of the Bacillus subtilis CotW and CotX proteins, which form the crust layer in spores of that species, are structural components of the Bacillus megaterium QM B1551 spore exosporium. The introduction of plasmid-borne cotW and orthologous cotX genes to the PV361 strain, which lacks all indigenous plasmids and produces spores that are devoid of an exosporium, results in the development of spores with a rudimentary exosporium-type structure. Additionally, purified recombinant CotW protein is shown to assemble at the air-water interface to form thin sheets of material, which is consistent with the idea that this protein may form a basal layer in the Bacillus megaterium QM B1551 exosporium. [ABSTRACT FROM AUTHOR]

Focalised hypoxia is widely prevalent in diseases such as stroke, cardiac arrest, and dementia. While in some cases hypoxia improves cellular functions, it mostly induces or exacerbates pathological changes. The lack of methodologies that can simulate focal acute hypoxia, in either animal or cell culture, impedes our understanding of the cellular consequences of hypoxia. To address this gap, an electrochemical localised oxygen scavenging system (eLOS), is reported, providing an innovative platform for spatiotemporal in vitro hypoxia modulation. The electrochemical system is modelled showing O 2 flux patterns and localised O 2 scavenging and hypoxia regions, as a function of distance from the electrode and surrounding flux barriers, allowing an effective focal hypoxia tool to be designed for in vitro cell culture study. O 2 concentration is reduced in an electrochemically defined targeted area from normoxia to hypoxia in about 6 min depending on the O 2 -flux boundaries. As a result, a cell culture-well was designed, where localised O 2 scavenging could be induced. The impact of localised hypoxia was demonstrated on human neural progenitor cells (hNPCs) and it was shown that miniature focal hypoxic insults can be induced, that evoke time-dependent HIF-1α transcription factor accumulation. This transcription is "patterned" across the culture according to the electrochemically induced spatiotemporal hypoxia gradient. A basic lacunar infarct model was also developed through the application of eLOS in a purpose designed microfluidic device. Miniature focal hypoxic insults were induced in cellular processes of fully oxygenated cell bodies, such as the axons of human cortical neurons. The results demonstrate experimentally that localised axonal hypoxic stress can lead to significant increase of neuronal death, despite the neurons remaining at normoxia. This suggests that focal hypoxic insult to axons alone is sufficient to impact surrounding neurons and may provide an in vitro model to study the impact of microinfarcts occurring in the deep cerebral white matter, as well as providing a promising tool for wider understanding of acute hypoxic insults with potential to uncover its pathophysiology in multiple diseases., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Wong, Varga, Káradóttir and Hall.)

A rational design approach is proposed for a multifunctional enzyme reagent for point-of-care diagnostics. The biomaterial reduces downstream isolation steps and eliminates immobilization coupling chemicals for integration in a diagnostic platform. Fusion constructs combined the central functional assay protein (e.g. monomeric sarcosine oxidase, mSOx, horseradish peroxidase, HRP), a visualizing protein (e.g. mCherry) and an in-built immobilization peptide (e.g. R5). Monitoring protein expression in E.coli was facilitated by following the increase in mCherry fluorescence, which could be matched to a color card, indicating when good protein expression has occurred. The R5 peptide (SSKKSGSYSGSKGSKRRIL) provided inbuilt affinity for silica and an immobilization capability for a silica based diagnostic, without requiring additional chemical coupling reagents. Silica particles extracted from beach sand were used to collect protein from crude protein extract with 85-95% selective uptake. The silica immobilized R5 proteins were stable for more than 2 months at room temperature. The K m for the silica-R5 2 -mCh-mSOx-R5-6H was 16.5 ± 0.9 mM (compared with 16.5 ± 0.4 mM, 16.3 ± 0.3 mM, and 16.1 ± 0.4 mM for R5 2 -mCh-mSOx-R5-6H, mSOx-R5-6H and mSOx-6H respectively in solution). The use of the "silica-enzymes" in sarcosine and peroxide assays was shown, and a design using particle sedimentation through the sample was examined. Using shadowgraphy and particle image velocimetry the particle trajectory through the sample was mapped and an hourglass design with a narrow waist shown to give good control of particle position. The hourglass biosensor was demonstrated for sarcosine assay in the clinically useful range of 2.5-10 μM in both a dynamic and end point measurement regime., (Copyright © 2018. Published by Elsevier Ltd.)

Colloidosomes are polymer shell microcapsules. They are stable and easy to prepare and have been used to encapsulate drugs for release at specific areas in the body. Traditional polymer shell capsules cannot totally seal drugs, since they are porous, and small molecules diffuse through the polymer shell. In this paper, we report a method for encapsulating an antibiotic kanamycin using gold or silver coated colloidosomes. The colloidosomes are impermeable and can be triggered using ultrasound. To investigate the application of the capsules in a biological system, Escherichia Coli ( E. coli ) was chosen as a model organism. After triggering, the released antibiotic, as well as the metal shell fragments, kill E. coli . Both the silver and gold shells colloidosomes are toxic to this bacterial system and the gold coated colloidosomes can load a higher concentration of kanamycin.

The combination of enzymatic recognition and manometric transduction is explored, using enzymes that consume or evolve a gas with low solubility in aqueous media. A design is discussed whereby change in partial pressure of a gas in the headspace is related to the turnover of analyte by the enzyme. Headspace and sample volume dimensions are considered, demonstrating the influence of flux at the air-water interface. The relative importance of diffusion and reaction for the enzyme solution is shown. When enzyme kinetics dominate, the concentration gradient is low and the overall kinetics are determined by the total amount of active enzyme, reducing either enzyme concentration or enzyme layer thickness will reduce the diffusion limitation. A Teflon-enzyme composite is presented to allow a reuseable immobilised enzyme preparation and a disc with stirring magnet identified as an efficient configuration. A glucose oxidase system was tested in the monitoring of glucose consumption during fermentation. Application to other enzyme systems is discussed.

A new displacement hybridisation method is reported using a single strand DNA probe, labelled with an acceptor fluorophore (oregon green 488). Detection of double stranded sample target is shown, with discrimination between the probe, duplexed during the assay, and free single stranded probe DNA achieved through the FRET from a donor grove fluorophore (Hoechst 33258). A model for the kinetics of the displacement assay is presented and the course of the assay predicted according to probe/target ratios and sequence. The modelled predictions are consistent with the experimental data showing single base pair mismatch discrimination. The pattern of response according to the mismatch/perfect complement ratio in a mixed sample is also considered with an allele-discrimination ratio lying between the homozygous gene and total mismatch case, according to ratio. The assay is shown to be tolerant of different probe concentrations and ratios and through the dual wavelength recorded signals from donor and FRET acceptor, internal baseline correction is achieved with excellent noise reduction through ratiometric measurement.

Short peptides sequences were selected that showed binding selectivity towards healthy or oxidised (unhealthy) low density lipoprotein (LDL), respectively. These were investigated for application in atherosclerosis risk monitoring. Comparison was also made with the LDL receptor ligand repeat peptide (LR5). The peptides were immobilised on a gold surface plasmon resonance surface and LDL binding detected as a shift in the resonance. 3.7x10(7) (+/-5.6x10(6)) LDL/mm(2)/microg/ml solution LDL were bound on GlySerAspGlu-OH and 6.8x10(7) (+/-9.2x10(6)) LDL/mm(2)/microg/ml on GlyCystineSerAspGlu, compared with approximately 10(8) LDL/mm(2)/microg/ml on LR5. In this first group, binding of LDL decreased with oxidation level and a good correlation was found between LDL binding and residual amino groups on the apoprotein of the LDL following oxidation, or the change in relative electrophoretic mobility (REM) of LDL. The decrease in binding was 1.1x10(7) LDL particles/mm(2) per% oxidation for GlySerAspGlu-OH, 1.8x10(7) LDL particles/mm(2) per% oxidation for GlyCystineSerAspGlu and 2.4x10(7) LDL particles/mm(2) per% oxidation for LR5. A second group of three peptides were also selected showing increased binding with LDL oxidation: GlyCystineCysCys (1.5x10(7) LDL/mm(2) per microg/ml), GlyLysLysCys-SH (10(7) LDL/mm(2) per microg/ml) and GlyLysLys-OH (5.6x10(7) LDL/mm(2) per microg/ml). The latter gave a linear increase in LDL binding with oxidation level (1.2x10(7) LDL particles/mm(2) per% oxidation). LDL concentration is around 2-3 mg/ml in plasma compared with the low detection levels with this method (1-10 microg/ml), allowing a strategy to be developed requiring the minimum sample volume and diluting with physiological buffer prior to assay. By using a comparative reading between LDL adsorption on surfaces from the first and second group of peptides (e.g. GlyCystineSerAspGlu and GlyLysLys-OH, respectively), LDL oxidation could be determined without knowledge of LDL concentration. Higher binding was seen on GlyCystineSerAspGlu than GlyLysLys-OH below 30% LDL oxidation, whereas above 30% oxidation the binding on the latter surface was greater. Simple correlation of this form could provide good tests for atherosclerosis risk.