The Rhodium Analogue of Coenzyme B12 as an Anti-Photoregulatory Ligand Inhibiting Bacterial CarH Photoreceptors.

Coenzyme B12 (AdoCbl; 5'-deoxy-5'-adenosylcobalamin), the quintessential biological organometallic radical catalyst, has a formerly unanticipated, yet extensive, role in photoregulation in bacteria. The light-responsive cobalt-corrin AdoCbl performs this nonenzymatic role by facilitating the assembly of CarH photoreceptors into DNA-binding tetramers in the dark, suppressing gene expression. Conversely, exposure to light triggers the decomposition of this AdoCbl-bound complex by a still elusive photochemical mechanism, activating gene expression. Here, we have examined AdoRhbl, the non-natural rhodium analogue of AdoCbl, as a photostable isostructural surrogate for AdoCbl. We show that AdoRhbl closely emulates AdoCbl in its uptake by bacterial cells and structural functionality as a regulatory ligand for CarH tetramerization, DNA binding, and repressor activity. Remarkably, we find AdoRhbl is photostable even when bound "base-off/His-on" to CarH in vitro and in vivo. Thus, AdoRhbl, an antivitamin B12, also represents an unprecedented anti-photoregulatory ligand, opening a pathway to precisely target biomimetic inhibition of AdoCbl-based photoregulation, with new possibilities for selective antibacterial applications. Computational biomolecular analysis of AdoRhbl binding to CarH yields detailed structural insights into this complex, which suggest that the adenosyl group of photoexcited AdoCbl bound to CarH may specifically undergo a concerted non-radical syn-1,2-elimination mechanism, an aspect not previously considered for this photoreceptor.

Complete Genome Sequence Assembly and Annotation for Myxococcus xanthus Strains DK1050 and DK101.

Myxococcus xanthus is a social Gram-negative soil bacterium and the best studied member of the order Myxococcales in the class Deltaproteobacteria, which was recently reclassified as the phylum Myxococcota. Here, we report complete genomes, obtained using Illumina and PacBio sequencing, of M. xanthus strains DK1050 and DK101 (GenBank accession numbers CP104804 and CP104803, respectively).

An analysis of environment effect on ethanol blends with plastic fuel and blend optimization using a full factorial design.

There is a growing amount of plastic waste that needs to be properly disposed of in order to protect the environment from the negative effects of increasing reliance on plastic products. Recent interest has focused on chemical recycling as a means of reducing plastic's negative environmental effects. Converting waste plastics into basic petrochemicals allows them to serve as hydrocarbon feedstock or fuel oil through pyrolysis operations. Scientists have taken a keen interest in the production of bioethanol from renewable feedstocks due to its potential as a source of energy and alternative fuel. Due to its beneficial effects on the environment, ethanol has emerged as a promising biofuel. In this paper, energy recovered from low-density polyethylene and high-density polyethylene waste was converted into an alternative plastic fuel and evaluated for its environmental impact with the blending of ethanol in a diesel engine. Ternary fuel blends with 20%, 30%, and 40% waste plastic fuel and 10%, 15%, and 20% ethanol with standard diesel were tested. The study found that blending 10% ethanol with 20% plastic fuel decreased fuel consumption by around 7.9% compared to base diesel. Carbon monoxide emissions are reduced by about 10.2%, and hydrocarbon emissions are reduced by about 13.43% when using the same ternary blend. The optimum values of fuel consumption and emissions were obtained by full factorial design for a ternary fuel blend of 10% ethanol and 20% plastic fuel at the full load condition.

Plasmalogens and Photooxidative Stress Signaling in Myxobacteria, and How it Unmasked CarF/TMEM189 as the Δ1'-Desaturase PEDS1 for Human Plasmalogen Biosynthesis.

Plasmalogens are glycerophospholipids with a hallmark sn-1 vinyl ether bond that endows them with unique physical-chemical properties. They have proposed biological roles in membrane organization, fluidity, signaling, and antioxidative functions, and abnormal plasmalogen levels correlate with various human pathologies, including cancer and Alzheimer's disease. The presence of plasmalogens in animals and in anaerobic bacteria, but not in plants and fungi, is well-documented. However, their occurrence in the obligately aerobic myxobacteria, exceptional among aerobic bacteria, is often overlooked. Tellingly, discovery of the key desaturase indispensable for vinyl ether bond formation, and therefore fundamental in plasmalogen biogenesis, emerged from delving into how the soil myxobacterium Myxococcus xanthus responds to light. A recent pioneering study unmasked myxobacterial CarF and its human ortholog TMEM189 as the long-sought plasmanylethanolamine desaturase (PEDS1), thus opening a crucial door to study plasmalogen biogenesis, functions, and roles in disease. The findings demonstrated the broad evolutionary sweep of the enzyme and also firmly established a specific signaling role for plasmalogens in a photooxidative stress response. Here, we will recount our take on this fascinating story and its implications, and review the current state of knowledge on plasmalogens, their biosynthesis and functions in the aerobic myxobacteria.

Vitamin B12 photoreceptors.

Photoreceptor proteins enable living organisms to sense light and transduce this signal into biochemical outputs to elicit appropriate cellular responses. Their light sensing is typically mediated by covalently or noncovalently bound molecules called chromophores, which absorb light of specific wavelengths and modulate protein structure and biological activity. Known photoreceptors have been classified into about ten families based on the chromophore and its associated photosensory domain in the protein. One widespread photoreceptor family uses coenzyme B12 or 5'-deoxyadenosylcobalamin, a biological form of vitamin B12, to sense ultraviolet, blue, or green light, and its discovery revealed both a new type of photoreceptor and a novel functional facet of this vitamin, best known as an enzyme cofactor. Large strides have been made in our understanding of how these B12-based photoreceptors function, high-resolution structural descriptions of their functional states are available, as are details of their unusual photochemistry. Additionally, they have inspired notable applications in optogenetics/optobiochemistry and synthetic biology. Here, we provide an overview of what is currently known about these B12-based photoreceptors, their discovery, distribution, molecular mechanism of action, and the structural and photochemical basis of how they orchestrate signal transduction and gene regulation, and how they have been used to engineer optogenetic control of protein activities in living cells.

Coenzyme B12 -dependent and independent photoregulation of carotenogenesis across Myxococcales.

Light-induced carotenogenesis in Myxococcus xanthus is controlled by the B12 -based CarH repressor and photoreceptor, and by a separate intricate pathway involving singlet oxygen, the B12 -independent CarH paralogue CarA and various other proteins, some eukaryotic-like. Whether other myxobacteria conserve these pathways and undergo photoregulated carotenogenesis is unknown. Here, comparative analyses across 27 Myxococcales genomes identified carotenogenic genes, albeit arranged differently, with carH often in their genomic vicinity, in all three Myxococcales suborders. However, CarA and its associated factors were found exclusively in suborder Cystobacterineae, with carA-carH invariably in tandem in a syntenic carotenogenic operon, except for Cystobacter/Melittangium, which lack CarA but retain all other factors. We experimentally show B12 -mediated photoregulated carotenogenesis in representative myxobacteria, and a remarkably plastic CarH operator design and DNA binding across Myxococcales. Unlike the two characterized CarH from other phyla, which are tetrameric, Cystobacter CarH (the first myxobacterial homologue amenable to analysis in vitro) is a dimer that combines direct CarH-like B12 -based photoregulation with CarA-like DNA binding and inhibition by an antirepressor. This study provides new molecular insights into B12 -dependent photoreceptors. It further establishes the B12 -dependent pathway for photoregulated carotenogenesis as broadly prevalent across myxobacteria and its evolution, exclusively in one suborder, into a parallel complex B12 -independent circuit.

Light-Triggered Carotenogenesis in Myxococcus xanthus: New Paradigms in Photosensory Signaling, Transduction and Gene Regulation.

Myxobacteria are Gram-negative δ-proteobacteria found predominantly in terrestrial habitats and often brightly colored due to the biosynthesis of carotenoids. Carotenoids are lipophilic isoprenoid pigments that protect cells from damage and death by quenching highly reactive and toxic oxidative species, like singlet oxygen, generated upon growth under light. The model myxobacterium Myxococcus xanthus turns from yellow in the dark to red upon exposure to light because of the photoinduction of carotenoid biosynthesis. How light is sensed and transduced to bring about regulated carotenogenesis in order to combat photooxidative stress has been extensively investigated in M. xanthus using genetic, biochemical and high-resolution structural methods. These studies have unearthed new paradigms in bacterial light sensing, signal transduction and gene regulation, and have led to the discovery of prototypical members of widely distributed protein families with novel functions. Major advances have been made over the last decade in elucidating the molecular mechanisms underlying the light-dependent signaling and regulation of the transcriptional response leading to carotenogenesis in M. xanthus. This review aims to provide an up-to-date overview of these findings and their significance.

Reagent integration and controlled release for multiplexed nucleic acid testing in disposable thermoplastic 2D microwell arrays.

The seamless integration of reagents into microfluidic devices can serve to significantly reduce assay complexity and cost for disposable diagnostics. In this work, the integration of multiplexed reagents into thermoplastic 2D microwell arrays is demonstrated using a scalable pin spotting technique. Using a simple and low-cost narrow-bore capillary spotting pin, high resolution deposition of concentrated reagents within the arrays of enclosed nanoliter-scale wells is achieved. The pin spotting method is further employed to encapsulate the deposited reagents with a chemically modified wax layer that serves to prevent disruption of the dried assay components during sample introduction through a shared microchannel, while also enabling temperature-controlled release after sample filling is complete. This approach supports the arbitrary patterning and release of different reagents within individual wells without crosstalk for multiplexed analyses. The performance of the in-well spotting technique is characterized using on-chip rolling circle amplification to evaluate its potential for nucleic acid-based diagnostics.

The Photoactive Excited State of the B12-Based Photoreceptor CarH.

We have used transient absorption spectroscopy in the UV-visible and X-ray regions to characterize the excited state of CarH, a protein photoreceptor that uses a form of B12, adenosylcobalamin (AdoCbl), to sense light. With visible excitation, a nanosecond-lifetime photoactive excited state is formed with unit quantum yield. The time-resolved X-ray absorption near edge structure difference spectrum of this state demonstrates that the excited state of AdoCbl in CarH undergoes only modest structural expansion around the central cobalt, a behavior similar to that observed for methylcobalamin rather than for AdoCbl free in solution. We propose a new mechanism for CarH photoreactivity involving formation of a triplet excited state. This allows the sensor to operate with high quantum efficiency and without formation of potentially dangerous side products. By stabilizing the excited electronic state, CarH controls reactivity of AdoCbl and enables slow reactions that yield nonreactive products and bypass bond homolysis and reactive radical species formation.

Deregulation of hepatic lipid metabolism associated with insulin resistance in rats subjected to chronic monocrotophos exposure.

In our previous study, we demonstrated the potential of monocrotophos (MCP), an organophosphorus insecticide (OPI), to induce glucose intolerance, insulin resistance (IR), and dyslipidemia with hyperinsulinemia in rats after chronic exposure. As hyperinsulinemia is likely to exert an impact on hepatic lipid metabolism, we carried out this study to establish the effect of chronic MCP exposure (0.9 and 1.8 mg/kg/day for 180 days) on hepatic lipid metabolism in rats. The state of IR induced by MCP in rats was associated with an increase in the liver lipid content (triglyceride and cholesterol) and expression levels of sterol regulatory element-binding proteins, PPARγ, acetyl-CoA carboxylase, and fatty acid synthase in the liver. Similarly, activities of key enzymes (acetyl-COA carboxylase, fatty acid synthase, lipin 1, malic enzyme, glucose-6-phosphate dehydrogenase, and glycerol-3-phosphate dehydrogenase), which regulate lipogenesis, were enhanced in livers of pesticide-treated rats. A strong correlation was observed between insulin levels, hepatic lipid content, and plasma lipid profile in treated rats. Our study suggests that long-term exposure to OPIs not only has a propensity to induce a state of hyperinsulinemic IR, but it is also associated with augmented hepatic lipogenesis, which may explain dyslipidemia induced by chronic exposure to MCP.

Enhanced sample filling and discretization in thermoplastic 2D microwell arrays using asymmetric contact angles.

Sample filling and discretization within thermoplastic 2D microwell arrays is investigated toward the development of low cost disposable microfluidics for passive sample discretization. By using a high level of contact angle asymmetry between the filling channel and microwell surfaces, a significant increase in the range of well geometries that can be successfully filled is revealed. The performance of various array designs is characterized numerically and experimentally to assess the impact of contact angle asymmetry and device geometry on sample filling and discretization, resulting in guidelines to ensure robust microwell filling and sample isolation over a wide range of well dimensions. Using the developed design rules, reliable and bubble-free sample filling and discretization is achieved in designs with critical dimensions ranging from 20 µm to 800 µm. The resulting devices are demonstrated for discretized nucleic acid amplification by performing loop-mediated isothermal amplification for the detection of the mecA gene associated with methicillin-resistant Staphylococcus aureus.

Combustion and emission analysis of hydrogenated waste polypropylene pyrolysis oil blended with diesel.

Petroleum-based plastic pyrolysis oil contains unsaturated compounds, and the presence of these compounds makes the produced fuel unsuitable for combustion in diesel engines. Hydrogenation of pyrolysis oil is performed to convert unsaturated compounds to saturated compounds. Past studies have shown that hydrogenation of petroleum-based plastic pyrolysis oil is viable; however, its combustion and emissions analysis in diesel engines has not yet been reported. In this study, we investigated the combustion, performance, and emissions of hydrogenated polypropylene pyrolysis oil (HPPO) blended with diesel. Polypropylene (PP) was converted to pyrolysis oil using ZSM-5 as the catalyst. The hydrogenation of polypropylene pyrolysis oil (PPO) was conducted at pressure of 70 bar, and the reaction temperature was maintained at 350 °C. Ni metal impregnated on the ZSM-5 base support was used as the catalyst of choice. The produced HPPO possessed physicochemical properties that match the EN590 standards(European diesel fuel standards). Gas chromatography-mass spectrometry (GC-MS) studies of PPO and HPPO showed the effectiveness of hydrogenation for the complete conversion of alkenes to alkanes, and hydrocracking resulted in cracking higher carbon number alkanes to lower values. HPPO was blended with diesel in ratios of 10 wt.%, 20 wt.%, 30 wt.%, and 40 wt.%. The diesel engine performance results for the blended fuel showed combustion, performance, and emissions on par with pure diesel fuel for blending ratios up to 20 wt.%. As is known, plastic solid waste (PSW) materials pose serious hazards to the environment. Our HPPO physicochemical properties matched the EN590 standards for diesel fuel. The combustion of HPPO in diesel engines can provide an option for environmentally cleaner disposal of PSW.

A bacterial light response reveals an orphan desaturase for human plasmalogen synthesis.

Plasmalogens are glycerophospholipids with a hallmark sn-1 vinyl ether bond. These lipids are found in animals and some bacteria and have proposed membrane organization, signaling, and antioxidant roles. We discovered the plasmanylethanolamine desaturase activity that is essential for vinyl ether bond formation in a bacterial enzyme, CarF, which is a homolog of the human enzyme TMEM189. CarF mediates light-induced carotenogenesis in Myxococcus xanthus, and plasmalogens participate in sensing photooxidative stress through singlet oxygen. TMEM189 and other animal homologs could functionally replace CarF in M. xanthus, and knockout of TMEM189 in a human cell line eliminated plasmalogens. Discovery of the human plasmanylethanolamine desaturase will spur further study of plasmalogen biogenesis, functions, and roles in disease.

Prophylactic neuroprotective propensity of Crocin, a carotenoid against rotenone induced neurotoxicity in mice: behavioural and biochemical evidence.

Previously we have demonstrated the potential neuroprotective propensity of saffron and Crocin (CR) employing a Drosophila model of Parkinsonism. Rotenone (ROT) has been extensively used as a model neurotoxin to induce Parkinson's disease (PD) like symptoms in mice. In the present study, as a proof of concept we evaluated the efficacy of CR prophylaxis (25 mg/ kg bw/d, 7d) to attenuate ROT(0.5 mg/Kg bw/d,7d) -induced neurotoxic effects in male mice focussing on neurobehavioural assessments and biochemical determinants in the striatum. CR prophylaxis significantly alleviated ROT-induced behavioural alterations such as increased anxiety, diminished exploratory behaviour, decreased motor co-ordination, and grip strength. Concomitantly, we evidenced diminution of oxidative stress markers, enhanced levels of antioxidant enzyme and mitochondrial enzyme function in the striatal region. Further, varying degree of restoration of cholinergic function, dopamine and α-synuclein levels were discernible suggesting the possible mechanism/s of action of CR in this model. Based on our earlier data in flies and in worm model, we propose its use as an adjuvant therapeutic agent in oxidative stress-mediated neurodegenerative conditions such as PD.

B12-based photoreceptors: from structure and function to applications in optogenetics and synthetic biology.

Vitamin B12-based photoreceptor proteins sense ultraviolet (UV), blue or green light using 5'-deoxyadenosylcobalamin (AdoCbl). The prototype of this widespread bacterial photoreceptor family, CarH, controls light-dependent gene expression in photoprotective cellular responses. It represses transcription in the dark by binding to operator DNA as an AdoCbl-bound tetramer, whose disruption by light relieves operator binding to allow transcription. Structures of the 'dark' (free and DNA-bound) and 'light' CarH states and studies on the unusual AdoCbl photochemistry have provided fundamental insights into these photoreceptors. We highlight these, the plasticity within a conserved mode of action among CarH homologs, their distribution, and their promising applications in optogenetics and synthetic biology.

A Scalable Random Access Micro-traps Array for Formation, Selective Retrieval and Capturing of Individual Droplets.

Formation, selective retrieval and capturing of individual droplets are key operational capabilities needed for a broad range of droplet microfluidic applications. The membrane displacement trap (MDT) element gives a robust method for uniform discretization and controllable manipulation of aqueous droplets using an enclosed micro-well covered by an elastomer membrane. This capability can be scaled up by combining the modular elements with a system design that requires a minimal number of signal inputs. Incorporation of MDT elements with a pneumatically-controllable multiplexer system can lead to a scalable random access MDT array platform for liquid discretization and selective manipulation. Herein, we report the design and development of a programmable droplet microfluidic platform for liquid sampling and selectively handling up to 32 individual droplets using 10 pneumatic signal inputs. The multiplexer system can logarithmically scale up capacity of the MDT array platform, making it possible to manipulate hundreds droplets.

Plasticity in oligomerization, operator architecture, and DNA binding in the mode of action of a bacterial B12-based photoreceptor.

Newly discovered bacterial photoreceptors called CarH sense light by using 5'-deoxyadenosylcobalamin (AdoCbl). They repress their own expression and that of genes for carotenoid synthesis by binding in the dark to operator DNA as AdoCbl-bound tetramers, whose light-induced disassembly relieves repression. High-resolution structures of Thermus thermophilus CarHTt have provided snapshots of the dark and light states and have revealed a unique DNA-binding mode whereby only three of four DNA-binding domains contact an operator comprising three tandem direct repeats. To gain further insights into CarH photoreceptors and employing biochemical, spectroscopic, mutational, and computational analyses, here we investigated CarHBm from Bacillus megaterium We found that apoCarHBm, unlike monomeric apoCarHTt, is an oligomeric molten globule that forms DNA-binding tetramers in the dark only upon AdoCbl binding, which requires a conserved W-X9-EH motif. Light relieved DNA binding by disrupting CarHBm tetramers to dimers, rather than to monomers as with CarHTt CarHBm operators resembled that of CarHTt, but were larger by one repeat and overlapped with the -35 or -10 promoter elements. This design persisted in a six-repeat, multipartite operator we discovered upstream of a gene encoding an Spx global redox-response regulator whose photoregulated expression links photooxidative and general redox responses in B. megaterium Interestingly, CarHBm recognized the smaller CarHTt operator, revealing an adaptability possibly related to the linker bridging the DNA- and AdoCbl-binding domains. Our findings highlight a remarkable plasticity in the mode of action of B12-based CarH photoreceptors, important for their biological functions and development as optogenetic tools.

Multifactorial control of the expression of a CRISPR-Cas system by an extracytoplasmic function σ/anti-σ pair and a global regulatory complex.

Expression of CRISPR-Cas systems is a prerequisite for their defensive role against invading genetic elements. Yet, much remains unknown about how this crucial step is regulated. We describe a new mechanism controlling CRISPR-cas expression, which requires an extracytoplasmic function (ECF) σ factor (DdvS), its membrane-bound anti-σ (DdvA) and a global regulatory complex (CarD-CarG). Transcriptomic analyses revealed that the DdvS/CarD/CarG-dependent regulon comprises a type III-B CRISPR-Cas system in Myxococcus xanthus. We mapped four DdvS-driven CarD/CarG-dependent promoters, with one lying immediately upstream of the cas cluster. Consistent with direct action, DdvS and CarD-CarG localize at these promoters in vivo. The cas genes are transcribed as a polycistronic mRNA that reads through the leader into the CRISPR array, a putative σA-dependent promoter in the leader having negligible activity in vivo. Consequently, expression of the entire CRISPR-Cas system and mature CRISPR-RNA (crRNA) production is DdvS/CarD/CarG-dependent. DdvA likely uses its large C-terminal domain to sense and transduce the extracytoplasmic signal triggering CRISPR-cas expression, which we show is not starvation-induced multicellular development. An ECF-σ/anti-σ pair and a global regulatory complex provide an effective mechanism to coordinate signal-sensing with production of precursor crRNA, its processing Cas6 endoribonuclease and other Cas proteins for mature crRNA biogenesis and interference.

Controlled droplet discretization and manipulation using membrane displacement traps.

An innovative platform enabling complex discretization and manipulation of aqueous droplets is described. The system uses simple membrane displacement trap elements to perform multiple functions including droplet discretization, release, metering, capture, and merging. Multi-layer PDMS devices with membrane displacement trap arrays are used to discretize sample into nanoliter scale droplet volumes, and reliably manipulate individual droplets within the arrays. Performance is characterized for varying capillary number flows, membrane actuation pressures, trap and membrane geometries, and trapped droplet volumes, with operational domains established for each platform function. The novel approach to sample digitization and droplet manipulation is demonstrated through discretization of a dilute bacteria sample, metering of individual traps to generate droplets containing single bacteria, and merging of the resulting droplets to pair the selected bacteria within a single droplet.

Genome-wide association study of alcohol consumption and genetic overlap with other health-related traits in UK Biobank (N=112 117).

Alcohol consumption has been linked to over 200 diseases and is responsible for over 5% of the global disease burden. Well-known genetic variants in alcohol metabolizing genes, for example, ALDH2 and ADH1B, are strongly associated with alcohol consumption but have limited impact in European populations where they are found at low frequency. We performed a genome-wide association study (GWAS) of self-reported alcohol consumption in 112 117 individuals in the UK Biobank (UKB) sample of white British individuals. We report significant genome-wide associations at 14 loci. These include single-nucleotide polymorphisms (SNPs) in alcohol metabolizing genes (ADH1B/ADH1C/ADH5) and two loci in KLB, a gene recently associated with alcohol consumption. We also identify SNPs at novel loci including GCKR, CADM2 and FAM69C. Gene-based analyses found significant associations with genes implicated in the neurobiology of substance use (DRD2, PDE4B). GCTA analyses found a significant SNP-based heritability of self-reported alcohol consumption of 13% (se=0.01). Sex-specific analyses found largely overlapping GWAS loci and the genetic correlation (rG) between male and female alcohol consumption was 0.90 (s.e.=0.09, P-value=7.16 × 10-23). Using LD score regression, genetic overlap was found between alcohol consumption and years of schooling (rG=0.18, s.e.=0.03), high-density lipoprotein cholesterol (rG=0.28, s.e.=0.05), smoking (rG=0.40, s.e.=0.06) and various anthropometric traits (for example, overweight, rG=-0.19, s.e.=0.05). This study replicates the association between alcohol consumption and alcohol metabolizing genes and KLB, and identifies novel gene associations that should be the focus of future studies investigating the neurobiology of alcohol consumption.