Chromosome-free bacterial cells are safe and programmable platforms for synthetic biology.

A type of chromosome-free cell called SimCells (simple cells) has been generated from Escherichia coli, Pseudomonas putida, and Ralstonia eutropha. The removal of the native chromosomes of these bacteria was achieved by double-stranded breaks made by heterologous I-CeuI endonuclease and the degradation activity of endogenous nucleases. We have shown that the cellular machinery remained functional in these chromosome-free SimCells and was able to process various genetic circuits. This includes the glycolysis pathway (composed of 10 genes) and inducible genetic circuits. It was found that the glycolysis pathway significantly extended longevity of SimCells due to its ability to regenerate ATP and NADH/NADPH. The SimCells were able to continuously express synthetic genetic circuits for 10 d after chromosome removal. As a proof of principle, we demonstrated that SimCells can be used as a safe agent (as they cannot replicate) for bacterial therapy. SimCells were used to synthesize catechol (a potent anticancer drug) from salicylic acid to inhibit lung, brain, and soft-tissue cancer cells. SimCells represent a simplified synthetic biology chassis that can be programmed to manufacture and deliver products safely without interference from the host genome.

Proteorhodopsin Overproduction Enhances the Long-Term Viability of Escherichia coli.

Genes encoding the photoreactive protein proteorhodopsin (PR) have been found in a wide range of marine bacterial species, reflecting the significant contribution that PR makes to energy flux and carbon cycling in ocean ecosystems. PR can also confer advantages to enhance the ability of marine bacteria to survive periods of starvation. Here, we investigate the effect of heterologously produced PR on the viability of Escherichia coli Quantitative mass spectrometry shows that E. coli, exogenously supplied with the retinal cofactor, assembles as many as 187,000 holo-PR molecules per cell, accounting for approximately 47% of the membrane area; even cells with no retinal synthesize ∼148,000 apo-PR molecules per cell. We show that populations of E. coli cells containing PR exhibit significantly extended viability over many weeks, and we use single-cell Raman spectroscopy (SCRS) to detect holo-PR in 9-month-old cells. SCRS shows that such cells, even incubated in the dark and therefore with inactive PR, maintain cellular levels of DNA and RNA and avoid deterioration of the cytoplasmic membrane, a likely basis for extended viability. The substantial proportion of the E. coli membrane required to accommodate high levels of PR likely fosters extensive intermolecular contacts, suggested to physically stabilize the cell membrane and impart a long-term benefit manifested as extended viability in the dark. We propose that marine bacteria could benefit similarly from a high PR content, with a stabilized cell membrane extending survival when those bacteria experience periods of severe nutrient or light limitation in the oceans.IMPORTANCE Proteorhodopsin (PR) is part of a diverse, abundant, and widespread superfamily of photoreactive proteins, the microbial rhodopsins. PR, a light-driven proton pump, enhances the ability of the marine bacterium Vibrio strain AND4 to survive and recover from periods of starvation, and heterologously produced PR extends the viability of nutrient-limited Shewanella oneidensis We show that heterologously produced PR enhances the viability of E. coli cultures over long periods of several weeks and use single-cell Raman spectroscopy (SCRS) to detect PR in 9-month-old cells. We identify a densely packed and consequently stabilized cell membrane as the likely basis for extended viability. Similar considerations are suggested to apply to marine bacteria, for which high PR levels represent a significant investment in scarce metabolic resources. PR-stabilized cell membranes in marine bacteria are proposed to keep a population viable during extended periods of light or nutrient limitation, until conditions improve.

Episodic bouts of hyperaemia and shear stress improve arterial blood flow and endothelial function.

INTRODUCTION: Exercise and heat stress lead to systemic improvements in arterial endothelial function, vascular stiffness, and cardiopulmonary capacity. The improvements in endothelial function may be primarily mediated via increases in shear stress. This study examined whether improvements in arterial function may be achieved in the absence of systemic vascular adaptations. Specifically, we hypothesized that repeated bouts of brief occlusion would improve arterial endothelial function via shear stress-dependent mechanisms. METHODS: Eleven healthy males underwent a shear stress intervention (5 s brachial occlusion, 10 s rest) for 30 min, five times weekly for 6 weeks on one arm while the other acted as an untreated control. Ultrasound was used to assess brachial arterial forearm blood flow (FBF) and vascular conductance (FVC), diameter, and shear rate (SR), while endothelial function was assessed by flow-mediated dilatation (FMD). Post-occlusive reactive hyperaemia and pulse wave velocity (PWV) were also measured. RESULTS: There were no changes in any of the measures in the control arm (all d < 0.2, p > 0.05). After 3 weeks of the intervention, FMD was increased from baseline (7.6 ± 0.6 vs. 5.9 ± 0.9%; d = 1.3, p = 0.038) and further increased after 6 weeks to 9.5 ± 2.6% (d = 1.7, p < 0.001). SR was also increased following the 6-week intervention (all d ≥ 0.6, p < 0.001). Resting and peak FBF and FVC were also increased in response to the intervention (all d ≥ 0.6, p < 0.001) and PWV was reduced. CONCLUSIONS: These data demonstrate that episodic increases in shear stress elicit marked increases in arterial endothelial function and vascular reactivity.

The role of shear stress on cutaneous microvascular endothelial function in humans.

PURPOSE: Previous studies suggest that exercise and heat stress improve cutaneous endothelial function, caused by increases in shear stress. However, as vasodilatation in the skin is primarily a thermogenic phenomenon, we investigated if shear stress alone without increases in skin temperature that occur with exercise and heat stress increases endothelial function. We examined the hypothesis that repeated bouts of brief occlusion would improve cutaneous endothelial function via shear stress-dependent mechanisms. METHODS: Eleven males underwent a shear stress intervention (forearm occlusion 5 s rest 10 s) for 30 min, five times·week-1 for 6 weeks on one arm, the other was an untreated control. Skin blood flow was measured using laser-Doppler flowmetry, and endothelial function was assessed with and without NOS-inhibition with L-NAME in response to three levels of local heating (39, 42, and 44 °C), ACh administration, and reactive hyperaemia. Data are cutaneous vascular conductance (CVC, laser-Doppler/blood pressure). RESULTS: There were no changes in the control arm (all d ≤ 0.2, p > 0.05). In the experimental arm, CVC to 39 °C was increased after 3 and 6 weeks (d = 0.6; p ≤ 0.01). Nitric oxide contribution was increased after 6 weeks compared to baseline (d = 0.85, p < 0.001). Following skin heating to 42 °C and 44 °C, CVC was not different at weeks 3 or 6 (d ≤ 0.8, p > 0.05). For both 42 and 44 °C, nitric oxide contribution was increased after weeks 3 and 6 (d ≥ 0.4, p < 0.03). Peak and area-under-the-curve responses to ACh increased following 6 weeks (p < 0.001). CONCLUSIONS: Episodic increases in shear stress, without changes in skin or core temperature, elicit an increase in cutaneous microvascular reactivity and endothelial function.

Porphyrin Binding to Gun4 Protein, Facilitated by a Flexible Loop, Controls Metabolite Flow through the Chlorophyll Biosynthetic Pathway.

In oxygenic phototrophs, chlorophylls, hemes, and bilins are synthesized by a common branched pathway. Given the phototoxic nature of tetrapyrroles, this pathway must be tightly regulated, and an important regulatory role is attributed to magnesium chelatase enzyme at the branching between the heme and chlorophyll pathway. Gun4 is a porphyrin-binding protein known to stimulate in vitro the magnesium chelatase activity, but how the Gun4-porphyrin complex acts in the cell was unknown. To address this issue, we first performed simulations to determine the porphyrin-docking mechanism to the cyanobacterial Gun4 structure. After correcting crystallographic loop contacts, we determined the binding site for magnesium protoporphyrin IX. Molecular modeling revealed that the orientation of α6/α7 loop is critical for the binding, and the magnesium ion held within the porphyrin is coordinated by Asn-211 residue. We also identified the basis for stronger binding in the Gun4-1 variant and for weaker binding in the W192A mutant. The W192A-Gun4 was further characterized in magnesium chelatase assay showing that tight porphyrin binding in Gun4 facilitates its interaction with the magnesium chelatase ChlH subunit. Finally, we introduced the W192A mutation into cells and show that the Gun4-porphyrin complex is important for the accumulation of ChlH and for channeling metabolites into the chlorophyll biosynthetic pathway.

Structural and functional consequences of removing the N-terminal domain from the magnesium chelatase ChlH subunit of Thermosynechococcus elongatus.

Magnesium chelatase (MgCH) initiates chlorophyll biosynthesis by catalysing the ATP-dependent insertion of Mg2+ into protoporphyrin. This large enzyme complex comprises ChlH, I and D subunits, with I and D involved in ATP hydrolysis, and H the protein that handles the substrate and product. The 148 kDa ChlH subunit has a globular N-terminal domain attached by a narrow linker to a hollow cage-like structure. Following deletion of this ~18 kDa domain from the Thermosynechoccus elongatus ChlH, we used single particle reconstruction to show that the apo- and porphyrin-bound forms of the mutant subunit consist of a hollow globular protein with three connected lobes; superposition of the mutant and native ChlH structures shows that, despite the clear absence of the N-terminal 'head' region, the rest of the protein appears to be correctly folded. Analyses of dissociation constants shows that the ΔN159ChlH mutant retains the ability to bind protoporphyrin and the Gun4 enhancer protein, although the addition of I and D subunits yields an extremely impaired active enzyme complex. Addition of the Gun4 enhancer protein, which stimulates MgCH activity significantly especially at low Mg2+ concentrations, partially reactivates the ΔN159ChlH-I-D mutant enzyme complex, suggesting that the binding site or sites for Gun4 on H do not wholly depend on the N-terminal domain.

Conserved chloroplast open-reading frame ycf54 is required for activity of the magnesium protoporphyrin monomethylester oxidative cyclase in Synechocystis PCC 6803.

The cyclase step in chlorophyll (Chl) biosynthesis has not been characterized biochemically, although there are some plausible candidates for cyclase subunits. Two of these, Sll1214 and Sll1874 from the cyanobacterium Synechocystis 6803, were FLAG-tagged in vivo and used as bait in separate pulldown experiments. Mass spectrometry identified Ycf54 as an interaction partner in each case, and this interaction was confirmed by a reciprocal pulldown using FLAG-tagged Ycf54 as bait. Inactivation of the ycf54 gene (slr1780) in Synechocystis 6803 resulted in a strain that exhibited significantly reduced Chl levels. A detailed analysis of Chl precursors in the ycf54 mutant revealed accumulation of very high levels of Mg-protoporphyrin IX methyl ester and only traces of protochlorophyllide, the product of the cyclase, were detected. Western blotting demonstrated that levels of the cyclase component Sll1214 and the Chl biosynthesis enzymes Mg-protoporphyrin IX methyltransferase and protochlorophyllide reductase are significantly impaired in the ycf54 mutant. Ycf54 is, therefore, essential for the activity and stability of the oxidative cyclase. We discuss a possible role of Ycf54 as an auxiliary factor essential for the assembly of a cyclase complex or even a large multienzyme catalytic center.

Structure of the cyanobacterial Magnesium Chelatase H subunit determined by single particle reconstruction and small-angle X-ray scattering.

The biosynthesis of chlorophyll, an essential cofactor for photosynthesis, requires the ATP-dependent insertion of Mg(2+) into protoporphyrin IX catalyzed by the multisubunit enzyme magnesium chelatase. This enzyme complex consists of the I subunit, an ATPase that forms a complex with the D subunit, and an H subunit that binds both the protoporphyrin substrate and the magnesium protoporphyrin product. In this study we used electron microscopy and small-angle x-ray scattering to investigate the structure of the magnesium chelatase H subunit, ChlH, from the thermophilic cyanobacterium Thermosynechococcus elongatus. Single particle reconstruction of negatively stained apo-ChlH and Chl-porphyrin proteins was used to reconstitute three-dimensional structures to a resolution of ∼30 Å. ChlH is a large, 148-kDa protein of 1326 residues, forming a cage-like assembly comprising the majority of the structure, attached to a globular N-terminal domain of ∼16 kDa by a narrow linker region. This N-terminal domain is adjacent to a 5 nm-diameter opening in the structure that allows access to a cavity. Small-angle x-ray scattering analysis of ChlH, performed on soluble, catalytically active ChlH, verifies the presence of two domains and their relative sizes. Our results provide a basis for the multiple regulatory and catalytic functions of ChlH of oxygenic photosynthetic organisms and for a chaperoning function that sequesters the enzyme-bound magnesium protoporphyrin product prior to its delivery to the next enzyme in the chlorophyll biosynthetic pathway, magnesium protoporphyrin methyltransferase.

A systematic review of using electrical stimulation to improve clinical outcomes after hip fractures.

BACKGROUND: Pain and muscles weakness often delays regaining independent mobility following hip fracture surgery. Electrical stimulation may relieve pain and improve muscle strength and function. PURPOSE: To systematically review and evaluate available literature examining the effectiveness of using electrical stimulation to promote clinical outcomes after hip fractures. METHODS: Two researchers independently searched MEDLINE, CINAHL, EMBASE, Web of Science, Cochrane Reviews, Physiotherapy Evidence Database, and PsycInfo from inception to July 1, 2018, with no restrictions. The quality and fidelity of the included interventions were assessed, and expert consultation was conducted to help explain the results. RESULTS: We identified 432 records through database searching. Initial screening indicated 24 articles were appropriate for full-text review, and four articles met the inclusion criteria. In included studies, electrical stimulation (i.e. TENS) reduced pain (mean difference (MD) = 3.3 points on 10-point Visual Analogue Scale, p < .001), improved range of motion (ROM) (MD: 25.7°, p < .001), and accelerated functional recovery immediately after hip fracture (p < .001). Conflicting evidence existed when using neuromuscular electrical stimulation to improve muscle strength and other functional outcomes (e.g. mobility); however, nine experts advised that longer-term interventions might be necessary to achieve significant improvment in muscle strength. CONCLUSION: Available evidence, albeit limited, supports the early application of noninvasive electrical stimulation (e.g. TENS) for improving clinical outcomes (i.e. reducing pain, improving ROM, and accelerating functional recovery after hip fractures). We could not find conclusive evidence on the effectiveness of using electrical stimulation to improve muscle strength. This review establishes the need for future additional high-quality trials in this field.

Engineering a Rhodopsin-Based Photo-Electrosynthetic System in Bacteria for CO2 Fixation.

A key goal of synthetic biology is to engineer organisms that can use solar energy to convert CO2 to biomass, chemicals, and fuels. We engineered a light-dependent electron transfer chain by integrating rhodopsin and an electron donor to form a closed redox loop, which drives rhodopsin-dependent CO2 fixation. A light-driven proton pump comprising Gloeobacter rhodopsin (GR) and its cofactor retinal have been assembled in Ralstonia eutropha (Cupriavidus necator) H16. In the presence of light, this strain fixed inorganic carbon (or bicarbonate) leading to 20% growth enhancement, when formate was used as an electron donor. We found that an electrode from a solar panel can replace organic compounds to serve as the electron donor, mediated by the electron shuttle molecule riboflavin. In this new autotrophic and photo-electrosynthetic system, GR is augmented by an external photocell for reductive CO2 fixation. We demonstrated that this hybrid photo-electrosynthetic pathway can drive the engineered R. eutropha strain to grow using CO2 as the sole carbon source. In this system, a bioreactor with only two inputs, light and CO2, enables the R. eutropha strain to perform a rhodopsin-dependent autotrophic growth. Light energy alone, supplied by a solar panel, can drive the conversion of CO2 into biomass with a maximum electron transfer efficiency of 20%.

Revealing CO2-Fixing SAR11 Bacteria in the Ocean by Raman-Based Single-Cell Metabolic Profiling and Genomics.

The majority of marine microbes remain uncultured, which hinders the identification and mining of CO2-fixing genes, pathways, and chassis from the oceans. Here, we investigated CO2-fixing microbes in seawater from the euphotic zone of the Yellow Sea of China by detecting and tracking their 13C-bicarbonate (13C-HCO3-) intake via single-cell Raman spectra (SCRS) analysis. The target cells were then isolated by Raman-activated Gravity-driven Encapsulation (RAGE), and their genomes were amplified and sequenced at one-cell resolution. The single-cell metabolism, phenotype and genome are consistent. We identified a not-yet-cultured Pelagibacter spp., which actively assimilates 13C-HCO3-, and also possesses most of the genes encoding enzymes of the Calvin-Benson cycle for CO2 fixation, a complete gene set for a rhodopsin-based light-harvesting system, and the full genes necessary for carotenoid synthesis. The four proteorhodopsin (PR) genes identified in the Pelagibacter spp. were confirmed by heterologous expression in E. coli. These results suggest that hitherto uncultured Pelagibacter spp. uses light-powered metabolism to contribute to global carbon cycling.

Mutagenesis alters the catalytic mechanism of the light-driven enzyme protochlorophyllide oxidoreductase.

The light-activated enzyme protochlorophyllide oxidoreductase (POR) catalyzes an essential step in the synthesis of the most abundant pigment on Earth, chlorophyll. This unique reaction involves the sequential addition of a hydride and proton across the C17=C18 double bond of protochlorophyllide (Pchlide) by dynamically coupled quantum tunneling and is an important model system for studying the mechanism of hydrogen transfer reactions. In the present work, we have combined site-directed mutagenesis studies with a variety of sensitive spectroscopic and kinetic measurements to provide new insights into the mechanistic role of three universally conserved Cys residues in POR. We show that mutation of Cys-226 dramatically alters the catalytic mechanism of the enzyme. In contrast to wild-type POR, the characteristic charge-transfer intermediate, formed upon hydride transfer from NADPH to the C17 position of Pchlide, is absent in C226S variant enzymes. This suggests a concerted hydrogen transfer mechanism where proton transfer only is rate-limiting. Moreover, Pchlide reduction does not require the network of solvent-coupled conformational changes that play a key role in the proton transfer step of wild-type POR. We conclude that this globally important enzyme is finely tuned to facilitate efficient photochemistry, and the removal of a key interaction with Pchlide in the C226S variants significantly affects the local active site structure in POR, resulting in a shorter donor-acceptor distance for proton transfer.

The role of echocardiography in diagnosis, monitoring closure and post-procedural assessment of patent foramen ovale.

Patent foramen ovale (PFO) is common, with a probe-patent PFO present in 15-35% of the general population. Patent foramen ovale has been implicated in the aetiology of a number of different pathologies, including cryptogenic stroke, decompression sickness in divers, platypnea orthodeoxia, and migraine with aura. Cardiac ultrasound has a major role not only in the diagnosis of PFO but also in monitoring subsequent therapeutic intervention and in the post-procedural assessment of patients following percutaneous closure. The aim of this review was to outline the data regarding the role of echocardiography in diagnosis, during monitoring and post-procedural assessment so as to provide practical advice to minimize error and optimize patient outcomes. The review will seek to outline the limitations of the available techniques and factors that should be taken into account during percutaneous device closure.

Bryoplana xerophila n. g. n. sp., a new limnoterrestrial microturbellarian (Platyhelminthes, Typhloplanidae, Protoplanellinae) from epilithic mosses, with notes on its ecology.

Bryoplana xerophila, a new genus and species of limnoterrestrial protoplanelline platyhelminth, was found in moss and soil covering a concrete wall in northern Alabama, USA. Bryoplana xerophila is the first taxon of limnoterrestrial Protoplanellinae recorded from North America and is one of the few rhabdocoels known from dry habitats. It is unique within Protoplanellinae in lacking rhabdites, having a pharynx rosulatus in the frontal half of the body, and lacking sclerotized parts in the male system. Notes on encystment, reproduction and feeding behavior are given. An updated identification key to all known genera of Protoplanellinae is presented.

Sexual addiction 25 years on: A systematic and methodological review of empirical literature and an agenda for future research.

In 1998, Gold and Heffner authored a landmark review in Clinical Psychology Review on the topic of sexual addiction that concluded that sexual addiction, though increasingly popular in mental health settings, was largely based on speculation, with virtually no empirical basis. In the more than two decades since that review, empirical research around compulsive sexual behaviors (which subsumes prior research about sexual addiction) has flourished, ultimately culminating in the inclusion of a novel diagnosis of Compulsive Sexual Behavior Disorder in the eleventh edition of the World Health Organization's International Classification of Diseases. The present work details a systematic review of empirical research published between January 1st, 1995 and August 1st, 2020 related to compulsive sexual behaviors, with a specific focus on evaluating the methodologies of that literature. This review yielded 371 papers detailing 415 individual studies. In general, the present review finds that, although research related to compulsive sexual behaviors has proliferated, much of this work is characterized by simplistic methodological designs, a lack of theoretical integration, and an absence of quality measurement. Moreover, the present review finds a virtual absence of high-quality treatment-related research published within this time frame. Implications of these findings for both clinical practice and future research are discussed.

The active site of magnesium chelatase.

The insertion of magnesium into protoporphyrin initiates the biosynthesis of chlorophyll, the pigment that underpins photosynthesis. This reaction, catalysed by the magnesium chelatase complex, couples ATP hydrolysis by a ChlID motor complex to chelation within the ChlH subunit. We probed the structure and catalytic function of ChlH using a combination of X-ray crystallography, computational modelling, mutagenesis and enzymology. Two linked domains of ChlH in an initially open conformation of ChlH bind protoporphyrin IX, and the rearrangement of several loops envelops this substrate, forming an active site cavity. This induced fit brings an essential glutamate (E660), proposed to be the key catalytic residue for magnesium insertion, into proximity with the porphyrin. A buried solvent channel adjacent to E660 connects the exterior bulk solvent to the active site, forming a possible conduit for the delivery of magnesium or abstraction of protons.

Site-specific immobilization and micrometer and nanometer scale photopatterning of yellow fluorescent protein on glass surfaces.

A simple method is described for the site-specific attachment of yellow fluorescent protein (YFP) to glass surfaces on length scales ranging from tens of micrometers to ca. 200 nm. 3-Mercaptopropyl(triethoxy silane) is adsorbed onto a glass substrate and subsequently derivatized using a maleimide-functionalized oligomer of ethylene glycol. The resulting protein-resistant surface is patterned by exposure to UV light, causing photochemical degradation of the oligo(ethylene glycol) units to yield aldehyde groups in exposed regions. These are covalently bound to N-(5-amino-1-carboxypentyl)iminoacetic acid, yielding a nitrilotriacetic acid (NTA)-functionalized surface, which following complexation with Ni(2+), is coupled to His-tagged YFP. Using scanning near-field photolithography, in which a UV laser coupled to a scanning near-field optical microscope is utilized as the light source for photolithography, it is possible to fabricate lines of protein smaller than 200 nm, in which the biomolecules remain strongly optically active, facilitating the acquisition of diffraction-limited fluorescence images by confocal microscopy.

The xanthophyll cycle pool size controls the kinetics of non-photochemical quenching in Arabidopsis thaliana.

Arabidopsis plants overexpressing beta-carotene hydroxylase 1 accumulate over double the amount of zeaxanthin present in wild-type plants. The final amplitude of non-photochemical quenching (NPQ) was found to be the same in these plants, but the kinetics were different. The formation and relaxation of NPQ consistently correlated with the de-epoxidation state of the xanthophyll cycle pool and not the amount of zeaxanthin. These data indicate that zeaxanthin and violaxanthin antagonistically regulate the switch between the light harvesting and photoprotective modes of the light harvesting system and show that control of the xanthophyll cycle pool size is necessary to optimize the kinetics of NPQ.