Development of spirulina for the manufacture and oral delivery of protein therapeutics.

The use of the edible photosynthetic cyanobacterium Arthrospira platensis (spirulina) as a biomanufacturing platform has been limited by a lack of genetic tools. Here we report genetic engineering methods for stable, high-level expression of bioactive proteins in spirulina, including large-scale, indoor cultivation and downstream processing methods. Following targeted integration of exogenous genes into the spirulina chromosome (chr), encoded protein biopharmaceuticals can represent as much as 15% of total biomass, require no purification before oral delivery and are stable without refrigeration and protected during gastric transit when encapsulated within dry spirulina. Oral delivery of a spirulina-expressed antibody targeting campylobacter-a major cause of infant mortality in the developing world-prevents disease in mice, and a phase 1 clinical trial demonstrated safety for human administration. Spirulina provides an advantageous system for the manufacture of orally delivered therapeutic proteins by combining the safety of a food-based production host with the accessible genetic manipulation and high productivity of microbial platforms.

Shielding of viruses such as SARS-Cov-2 from ultraviolet radiation in particles generated by sneezing or coughing: Numerical simulations of survival fractions.

SARS-CoV-2 and other microbes within aerosol particles can be partially shielded from UV radiation. The particles refract and absorb light, and thereby reduce the UV intensity at various locations within the particle. Previously, we demonstrated shielding in calculations of UV intensities within spherical approximations of SARS-CoV-2 virions within spherical particles approximating dried-to-equilibrium respiratory fluids. The purpose of this paper is to extend that work to survival fractions of virions (i.e., fractions of virions that can infect cells) within spherical particles approximating dried respiratory fluids, and to investigate the implications of these calculations for using UV light for disinfection. The particles may be on a surface or in air. Here, the survival fraction (S) of a set of individual virions illuminated with a UV fluence (F, in J/m2) is assumed described by S(kF) = exp(-kF), where k is the UV inactivation rate constant (m2/J). The average survival fraction (Sp) of the simulated virions in a group of particles is calculated using the energy absorbed by each virion in the particles. The results show that virions within particles of dried respiratory fluids can have larger Sp than do individual virions. For individual virions, and virions within 1-, 5-, and 9-µm particles illuminated (normal incidence) on a surface with 260-nm UV light, the Sp = 0.00005, 0.0155, 0.22, and 0.28, respectively, when kF = 10. The Sp decrease to <10-7, <10-7, 0.077, and 0.15, respectively, for kF = 100. Results also show that illuminating particles with UV beams from widely separated directions can strongly reduce the Sp. These results suggest that the size distributions and optical properties of the dried particles of virion-containing respiratory fluids are likely important to effectively designing and using UV germicidal irradiation systems for microbes in particles. The results suggest the use of reflective surfaces to increase the angles of illumination and decrease the Sp. The results suggest the need for measurements of the Sp of SARS-CoV-2 in particles having compositions and sizes relevant to the modes of disease transmission.

Viruses such as SARS-CoV-2 can be partially shielded from UV radiation when in particles generated by sneezing or coughing: Numerical simulations.

UV radiation can inactivate viruses such as SARS-CoV-2. However, designing effective UV germicidal irradiation (UVGI) systems can be difficult because the effects of dried respiratory droplets and other fomites on UV light intensities are poorly understood. Numerical modeling of UV intensities inside virus-containing particles on surfaces can increase understanding of these possible reductions in UV intensity. We model UV intensities within spherical approximations of virions randomly positioned within spherical particles. The model virions and dried particles have sizes and optical properties to approximate SARS-CoV-2 and dried particles formed from respiratory droplets, respectively. In 1-, 5- and 9-µm diameter particles on a surface, illuminated by 260-nm UV light from a direction perpendicular to the surface, 0%, 10% and 18% (respectively) of simulated virions are exposed to intensities less than 1/100th of intensities in individually exposed virions (i.e., they are partially shielded). Even for 302-nm light (simulating sunlight), where absorption is small, 0% and 11% of virions in 1- and 9-µm particles have exposures 1/100th those of individually exposed virions. Shielding is small to negligible in sub-micron particles. Results show that shielding of virions in a particle can be reduced by illuminating a particle either from multiple widely separated incident directions, or by illuminating a particle rotating in air for a time sufficient to rotate through enough orientations. Because highly UV-reflective paints and surfaces can increase the angular ranges of illumination and the intensities within particles, they appear likely to be useful for reducing shielding of virions embedded within particles.

Detailed investigations into the Akabori-Momotani reaction for the synthesis of amphetamine type stimulants: Part 2.

The Akabori-Momotani reaction can be used to synthesise pseudoephedrine in 50% yield from N-methylalanine and benzaldehyde. This paper investigates electronic effects of substituted benzaldehydes on the reaction to synthesise amphetamine type stimulants and identifies several new Akabori-Momotani by-products, 1-[(4-methoxybenzyl)(methyl)amino]ethanol (11c), 2-(4-methoxyphenyl)-3,4-dimethyl-1,3-oxazolidine (12c), 1,2,3,4-tetramethyl-5,6-di-(4-methoxyphenyl)piperazine (13c) and 1,2,4,5-tetramethyl-3,6-di-(4-methoxyphenyl)piperazine (14c). This paper also investigates pseudoephedrine and methamphetamine isomeric distribution from the Akabori-Momotani reaction with the aid of molecular modelling to understand why more pseudoephedrine than ephedrine is produced.

The synthesis and investigation of impurities found in Clandestine Laboratories: Baeyer-Villiger Route Part I; Synthesis of P2P from benzaldehyde and methyl ethyl ketone.

The synthesis of impurities detected in clandestinely manufactured Amphetamine Type Stimulants (ATS) has emerged as more desirable than simple "fingerprint" profiling. We have been investigating the impurities formed when phenyl-2-propanone (P2P) 5, a key ATS precursor, is synthesised in three steps; an aldol condensation of benzaldehyde and methyl ethyl ketone (MEK); a Baeyer-Villiger reaction; and ester hydrolysis. We have identified and selectively synthesised several impurities that may be used as route specific markers for this series of synthetic steps. Specifically these impurities are 3-methyl-4-phenyl-3-buten-2-one 3, 2-methyl-1,5-diphenylpenta-1,4-diene-3-one 9, 2-(methylamino)-3-methyl-4-phenyl-3-butene 16, 2-(Methylamino)-3-methyl-4-phenylbutane 17, and 1-(methylamino)-2-methyl-1,5-diphenylpenta-4-ene-3-one 22.

Probing the subcellular localization of hopanoid lipids in bacteria using NanoSIMS.

The organization of lipids within biological membranes is poorly understood. Some studies have suggested lipids group into microdomains within cells, but the evidence remains controversial due to non-native imaging techniques. A recently developed NanoSIMS technique indicated that sphingolipids group into microdomains within membranes of human fibroblast cells. We extended this NanoSIMS approach to study the localization of hopanoid lipids in bacterial cells by developing a stable isotope labeling method to directly detect subcellular localization of specific lipids in bacteria with ca. 60 nm resolution. Because of the relatively small size of bacterial cells and the relative abundance of hopanoid lipids in membranes, we employed a primary (2)H-label to maximize our limit of detection. This approach permitted the analysis of multiple stable isotope labels within the same sample, enabling visualization of subcellular lipid microdomains within different cell types using a secondary label to mark the growing end of the cell. Using this technique, we demonstrate subcellular localization of hopanoid lipids within alpha-proteobacterial and cyanobacterial cells. Further, we provide evidence of hopanoid lipid domains in between cells of the filamentous cyanobacterium Nostoc punctiforme. More broadly, our method provides a means to image lipid microdomains in a wide range of cell types and test hypotheses for their functions in membranes.

Identification and quantification of polyfunctionalized hopanoids by high temperature gas chromatography-mass spectrometry.

Hopanoids are triterpenoids produced mainly by bacteria, are ubiquitous in the environment, and have many important applications as biological markers. A wide variety of related hopanoid structures exists, many of which are polyfunctionalized. These modifications render the hopanoids too involatile for conventional gas chromatography (GC) separation, so require either laborious oxidative cleavage of the functional groups or specialized high temperature (HT) columns. Here we describe the systematic evaluation and optimization of a HT-GC method for the analysis of polyfunctionalized hopanoids and their methylated homologs. Total lipid extracts are derivatized with acetic anhydride and no further treatment or workup is required. We show that acid or base hydrolysis to remove di- and triacylglycerides leads to degradation of several BHP structures. DB-XLB type columns can elute hopanoids up to bacteriohopane-tetrol at 350 °C, with baseline separation of all 2-methyl/desmethyl homologs. DB-5HT type columns can additionally elute bacteriohopaneaminotriol and bacteriohopaneaminotetrol, but do not fully separate 2-methyl/desmethyl homologs. The method gave 2- to 7-fold higher recovery of hopanoids than oxidative cleavage and can provide accurate quantification of all analytes including 2-methyl hopanoids. By comparing data from mass spectra with those from a flame ionization detector, we show that the mass spectromet (MS) response factors for different hopanoids using either total ion counts or m/z 191 vary substantially. Similarly, 2-methyl ratios estimated from selected-ion data are lower than those from FID by 10-30% for most hopanoids, but higher by ca. 10% for bacteriohopanetetrol. Mass spectra for a broad suite of hopanoids, including 2-methyl homologs, from Rhodopseudomonas palustris are presented, together with the tentative assignment of several new hopanoid degradation products.

The RND-family transporter, HpnN, is required for hopanoid localization to the outer membrane of Rhodopseudomonas palustris TIE-1.

Rhodopseudomonas palustris TIE-1 is a gram-negative bacterium that produces structurally diverse hopanoid lipids that are similar to eukaryotic steroids. Its genome encodes several homologues to proteins involved in eukaryotic steroid trafficking. In this study, we explored the possibility that two of these proteins are involved in intracellular hopanoid transport. R. palustris has a sophisticated membrane system comprising outer, cytoplasmic, and inner cytoplasmic membranes. It also divides asymmetrically, producing a mother and swarmer cell. We deleted genes encoding two putative hopanoid transporters that belong to the resistance-nodulation-cell division superfamily. Phenotypic analyses revealed that one of these putative transporters (HpnN) is essential for the movement of hopanoids from the cytoplasmic to the outer membrane, whereas the other (Rpal_4267) plays a minor role. C(30) hopanoids, such as diploptene, are evenly distributed between mother and swarmer cells, whereas hpnN is required for the C(35) hopanoid, bacteriohopanetetrol, to remain localized to the mother cell type. Mutant cells lacking HpnN grow like the WT at 30 °C but slower at 38 °C. Following cell division at 38 °C, the ΔhpnN cells remain connected by their cell wall, forming long filaments. This phenotype may be attributed to hopanoid mislocalization because a double mutant deficient in both hopanoid biosynthesis and transport does not form filaments. However, the lack of hopanoids severely compromises cell growth at higher temperatures more generally. Because hopanoid mutants only manifest a strong phenotype under certain conditions, R. palustris is an attractive model organism in which to study their transport and function.

The continuing puzzle of the great oxidation event.

The rise of atmospheric O(2) was a milestone in the history of life. Although O(2) itself is not a climate-active gas, its appearance would have removed a methane greenhouse present on the early Earth and potentially led to dramatic cooling. Moreover, by fundamentally altering the biogeochemical cycles of C, N, S and Fe, its rise first in the atmosphere and later in the oceans would also have had important indirect effects on Earth's climate. Here, we summarize major lines of evidence from the geological literature that pertain to when and how O(2) first appeared in significant amounts in the atmosphere. On the early Earth, atmospheric O(2) would initially have been very low, probably <10(-5) of the present atmospheric level. Around 2.45 billion years ago, atmospheric O(2) rose suddenly in what is now termed the Great Oxidation Event. While the rise of oxygen has been the subject of considerable attention by Earth scientists, several important aspects of this problem remain unresolved. Our goal in this review is to provide a short summary of the current state of the field, and make the case that future progress towards solving the riddle of oxygen will benefit greatly from the involvement of molecular biologists.

Demonstration of a mobile Flux Laboratory for the Atmospheric Measurement of Emissions (FLAME) to assess emissions inventories.

The advancement of air quality science and the development of effective air quality management plans require accurate estimates of emissions. In response to the need for new approaches to quantifying emissions, we have designed a mobile Flux Lab for the Atmospheric Measurement of Emissions (FLAME) that uses eddy covariance for the direct measurement of anthropogenic emissions at the neighborhood scale. To demonstrate the FLAME's capabilities, we have deployed it in the Huntington-Ashland region at the borders of Ohio, Kentucky and West Virginia. This area routinely experiences high ozone and fine particulate matter (PM(2.5)) concentrations and is home to a significant amount of industrial activity, including coal storage and transport. Experiments focused on carbon dioxide (CO(2)), nitrogen oxides (NO(x)) and fine particulate matter (PM(2.5)). Spikes in CO(2) and NO(x) concentrations were correlated with the passage of trains and barges through the FLAME's footprint. Calculated barge emission factors ranged from 49 to 76 kg NO(x) tonne(-1) fuel and agreed well with previously published values. Fluxes measured at three sites in the town of Worthington were mainly positive. They ranged between -6.5 to 29 mg m(-2) s(-1) for CO(2) and -9.7 x 10(-5) to 9.1 x 10(-5) mg m(-2) s(-1) for PM(2.5). We illustrate how the measurements can be compared to emissions inventories on a per capita basis for greenhouse gases and countywide for other pollutants. The results show that a mobile eddy covariance system can be used successfully to measure fluxes of multiple pollutants in a variety of settings. This alternative method for estimating emissions can be a useful tool for assessing uncertainties in emissions inventories and for improving their accuracy.

Involvement of BmoR and BmoG in n-alkane metabolism in 'Pseudomonas butanovora'.

'Pseudomonas butanovora' uses an alcohol-inducible alkane monooxygenase (BMO) to grow on C(2)-C(9) n-alkanes. Five ORFs were identified flanking the BMO structural genes. Two of the ORFs, bmoR, encoding a putative sigma(54)-transcriptional regulator BmoR, and bmoG, encoding a putative GroEL chaperonin BmoG, were analysed by gene-inactivation experiments. The BmoR-deficient mutant grew at slower growth rates than the wild-type on C(2)-C(5) n-alkanes and showed little to no growth on C(6)-C(8) n-alkanes within 7 days. A BmoR-deficient mutant was constructed in the 'P. butanovora' bmoX : : lacZ reporter strain and used to test whether bmoR was involved in bmoX induction after growth on C(2)-C(8) carbon sources. In acetate- or lactate-grown cells, C(2)-C(8) n-alcohols failed to induce beta-galactosidase activity. In contrast, in propionate-, butyrate- or pentanoate-grown cells, n-butanol induced approximately 45 % of the beta-galactosidase activity observed in the control bmoX : : lacZ strain. In propionate-grown cells, C(2)-C(5) n-alcohols induced beta-galactosidase activity, whereas C(7) and C(8) n-alcohols did not. BmoR may act as a sigma(54)-transcriptional regulator of bmo that is controlled by the n-alcohol produced in the alkane oxidation. During growth on short-chain-length fatty acids, however, another BMO regulatory system seems to be activated to promote transcription of bmo by short-chain-length alcohols (i.e.

Evidence for modified mechanisms of chloroethene oxidation in Pseudomonas butanovora mutants containing single amino acid substitutions in the hydroxylase alpha-subunit of butane monooxygenase.

The properties of oxidation of dichloroethene (DCE) and trichloroethylene (TCE) by three mutant strains of Pseudomonas butanovora containing single amino acid substitutions in the alpha-subunit of butane monooxygenase hydroxylase (BMOH-alpha) were compared to the properties of the wild-type strain (Rev WT). The rates of oxidation of three chloroethenes (CEs) were reduced in mutant strain G113N and corresponded with a lower maximum rate of butane oxidation. The rate of TCE degradation was reduced by one-half in mutant strain L279F, whereas the rates of DCE oxidation were the same as those in Rev WT. Evidence was obtained that the composition of products of CE oxidation differed between Rev WT and some of the mutant strains. For example, while Rev WT released nearly all available chlorine stoichiometrically during CE oxidation, strain F321Y released about 40% of the chlorine during 1,2-cis-DCE and TCE oxidation, and strain G113N released between 14 and 25% of the available chlorine during oxidation of DCE and 56% of the available chlorine during oxidation of TCE. Whereas Rev WT, strain L279F, and strain F321Y formed stoichiometric amounts of 1,2-cis-DCE epoxide during oxidation of 1,2-cis-DCE, only about 50% of the 1,2-cis-DCE oxidized by strain G113N was detected as the epoxide. Evidence was obtained that 1,2-cis-DCE epoxide was a substrate for butane monooxygenase (BMO) that was oxidized after the parent compound was consumed. Yet all of the mutant strains released less than 40% of the available 1,2-cis-DCE chlorine, suggesting that they have altered activity towards the epoxide. In addition, strain G113N was unable to degrade the epoxide. TCE epoxide was detected during exposure of Rev WT and strain F321Y to TCE but was not detected with strains L279F and G113N. Lactate-dependent O(2) uptake rates were differentially affected by DCE degradation in the mutant strains, providing evidence that some products released by the altered BMOs reduced the impact of CE on cellular toxicity. The use of CEs as substrates in combination with P. butanovora BMOH-alpha mutants might allow insights into the catalytic mechanism of BMO to be obtained.

Product and product-independent induction of butane oxidation in Pseudomonas butanovora.

Pseudomonas butanovora grows on butane by means of an inducible soluble alkane monooxygenase (sBMO). The induction of sBMO was studied using the wild type and a sBMO reporter strain. The reporter strain has the lacZ::kan cassette inserted into bmoX, the gene that encodes the alpha-subunit of the hydroxylase of sBMO. The beta-galactosidase activity in the reporter strain was not induced by butane, but was induced by 1-butanol and butyraldehyde. P. butanovora expressed sBMO product-independent activity at 3.0+/-1 nmol ethylene oxide min(-1) mg protein(-1) in stationary phase. The sBMO product-independent activity likely primes the expression of sBMO by butane.

Comparison of a micro-multileaf collimator with a 5-mm-leaf-width collimator for intracranial stereotactic radiotherapy.

PURPOSE: To dosimetrically compare a micro-multileaf collimator (minimum leaf width of 3 mm) with the 5-mm-leaf multileaf collimator (MLC) of a standard linear accelerator for stereotactic conformal radiotherapy treatment of intracranial lesions. MATERIALS AND METHODS: Fourteen patients previously treated for a variety of irregularly shaped intracranial lesions using BrainLAB's micro-MLC were retrospectively replanned using the Varian Millennium MLC (5 mm leaf width). All planning was performed with the BrainSCAN v 5.1 software. The same fixed, noncoplanar beam arrangement was used for both plans, and identical target coverage was achieved by adjusting the MLC shape around the planning target volume (PTV). The isodose distributions and dose-volume histograms (DVH) were computed and plans were compared in terms of conformity of the prescription isodose to the PTV and dose received by surrounding normal tissue. RESULTS: Equivalent PTV coverage was achieved using the 5-mm collimator by adjusting the MLC shape around the target in every case. There was a statistically significant increase in the conformity index for the Varian MLC compared with the micro-MLC (p < 0.001), indicating a worse conformity of the prescription isodose to the PTV, but this parameter was within our (and Radiation Therapy Oncology Group) clinical criterion in all cases. There was no statistically significant difference in the maximum dose to critical structures, but DVH curves demonstrated an increased volume of normal tissue irradiated to the lower isodose levels. The mean increase in the volume of critical structure enclosed within the 50% and 70% isodose surfaces was 5.7% and 4.9%, respectively. CONCLUSIONS: The micro-MLC consistently improves both PTV conformity and surrounding tissue sparing when compared to that of a standard linear accelerator. However, when viewed quantitatively, the improvements are small enough that individual centers may question their choice of equipment when outfitting a stereotactic radiotherapy service.