Host-derived O-glycans inhibit toxigenic conversion by a virulence-encoding phage in Vibrio cholerae.

Pandemic and endemic strains of Vibrio cholerae arise from toxigenic conversion by the CTXφ bacteriophage, a process by which CTXφ infects nontoxigenic strains of V. cholerae. CTXφ encodes the cholera toxin, an enterotoxin responsible for the watery diarrhea associated with cholera infections. Despite the critical role of CTXφ during infections, signals that affect CTXφ-driven toxigenic conversion or expression of the CTXφ-encoded cholera toxin remain poorly characterized, particularly in the context of the gut mucosa. Here, we identify mucin polymers as potent regulators of CTXφ-driven pathogenicity in V. cholerae. Our results indicate that mucin-associated O-glycans block toxigenic conversion by CTXφ and suppress the expression of CTXφ-related virulence factors, including the toxin co-regulated pilus and cholera toxin, by interfering with the TcpP/ToxR/ToxT virulence pathway. By synthesizing individual mucin glycan structures de novo, we identify the Core 2 motif as the critical structure governing this virulence attenuation. Overall, our results highlight a novel mechanism by which mucins and their associated O-glycan structures affect CTXφ-mediated evolution and pathogenicity of V. cholerae, underscoring the potential regulatory power housed within mucus.

The Rvv two-component regulatory system regulates biofilm formation and colonization in Vibrio cholerae.

The facultative human pathogen, Vibrio cholerae, employs two-component signal transduction systems (TCS) to sense and respond to environmental signals encountered during its infection cycle. TCSs consist of a sensor histidine kinase (HK) and a response regulator (RR); the V. cholerae genome encodes 43 HKs and 49 RRs, of which 25 are predicted to be cognate pairs. Using deletion mutants of each HK gene, we analyzed the transcription of vpsL, a biofilm gene required for Vibrio polysaccharide and biofilm formation. We found that a V. cholerae TCS that had not been studied before, now termed Rvv, controls biofilm gene transcription. The Rvv TCS is part of a three-gene operon that is present in 30% of Vibrionales species. The rvv operon encodes RvvA, the HK; RvvB, the cognate RR; and RvvC, a protein of unknown function. Deletion of rvvA increased transcription of biofilm genes and altered biofilm formation, while deletion of rvvB or rvvC lead to no changes in biofilm gene transcription. The phenotypes observed in ΔrvvA depend on RvvB. Mutating RvvB to mimic constitutively active and inactive versions of the RR only impacted phenotypes in the ΔrvvA genetic background. Mutating the conserved residue required for kinase activity in RvvA did not affect phenotypes, whereas mutation of the conserved residue required for phosphatase activity mimicked the phenotype of the rvvA mutant. Furthermore, ΔrvvA displayed a significant colonization defect which was dependent on RvvB and RvvB phosphorylation state, but not on VPS production. We found that RvvA's phosphatase activity regulates biofilm gene transcription, biofilm formation, and colonization phenotypes. This is the first systematic analysis of the role of V. cholerae HKs in biofilm gene transcription and resulted in the identification of a new regulator of biofilm formation and virulence, advancing our understanding of the role TCSs play in regulating these critical cellular processes in V. cholerae.

Reciprocal c-di-GMP signaling: Incomplete flagellum biogenesis triggers c-di-GMP signaling pathways that promote biofilm formation.

The assembly status of the V. cholerae flagellum regulates biofilm formation, suggesting that the bacterium senses a lack of movement to commit to a sessile lifestyle. Motility and biofilm formation are inversely regulated by the second messenger molecule cyclic dimeric guanosine monophosphate (c-di-GMP). Therefore, we sought to define the flagellum-associated c-di-GMP-mediated signaling pathways that regulate the transition from a motile to a sessile state. Here we report that elimination of the flagellum, via loss of the FlaA flagellin, results in a flagellum-dependent biofilm regulatory (FDBR) response, which elevates cellular c-di-GMP levels, increases biofilm gene expression, and enhances biofilm formation. The strength of the FDBR response is linked with status of the flagellar stator: it can be reversed by deletion of the T ring component MotX, and reduced by mutations altering either the Na+ binding ability of the stator or the Na+ motive force. Absence of the stator also results in reduction of mannose-sensitive hemagglutinin (MSHA) pilus levels on the cell surface, suggesting interconnectivity of signal transduction pathways involved in biofilm formation. Strains lacking flagellar rotor components similarly launched an FDBR response, however this was independent of the status of assembly of the flagellar stator. We found that the FDBR response requires at least three specific diguanylate cyclases that contribute to increased c-di-GMP levels, and propose that activation of biofilm formation during this response relies on c-di-GMP-dependent activation of positive regulators of biofilm production. Together our results dissect how flagellum assembly activates c-di-GMP signaling circuits, and how V. cholerae utilizes these signals to transition from a motile to a sessile state.

A safe and sustainable bacterial cellulose nanofiber separator for lithium rechargeable batteries.

Bacterial cellulose nanofiber (BCNF) with high thermal stability produced by an ecofriendly process has emerged as a promising solution to realize safe and sustainable materials in the large-scale battery. However, an understanding of the actual thermal behavior of the BCNF in the full-cell battery has been lacking, and the yield is still limited for commercialization. Here, we report the entire process of BCNF production and battery manufacture. We systematically constructed a strain with the highest yield (31.5%) by increasing metabolic flux and improved safety by introducing a Lewis base to overcome thermochemical degradation in the battery. This report will open ways of exploiting the BCNF as a "single-layer" separator, a good alternative to the existing chemical-derived one, and thus can greatly contribute to solving the environmental and safety issues.

High transmittance and deep RGB primary electrochromic color filter for high light out-coupling electro-optical devices.

We report a transmittance controllable electrochromic color filter (TCECF) by incorporating new electrochromic leuco dyes and their optimized composition. Each primary color red (R), green (G), and blue (B) electrochromic filter has an excellent transmittance of more than 84% at 650 nm, 540 nm, 450 nm, and the color coordinates are controllable from white (0.332, 0.347) to deep-red (0.621, 0.344), deep-green (0.327, 0.646), and deep-blue (0.179, 0.085), respectively. Also, each TCECF has good coloration efficiencies of 188.7 cm2 C-1 (R), 189.3 cm2 C-1 (G), and 147.8 cm2 C-1 (B) with high optical density change. A full color producible electrochromic color filter (ECF) is designed and fabricated by integrating primary RGB color filters with a refractive index matching adhesive layer. The fabricated three-stack full color producible ECF enables high transmittance of about 61% for clear white light extraction, and it can produce various colors including RGB. This TCECF technology will be very useful for high light out-coupling electro-optical applications, such as smart lighting, smart window, and display.

Genomic and metabolic analysis of Komagataeibacter xylinus DSM 2325 producing bacterial cellulose nanofiber.

Bacterial cellulose nanofiber (CNF) is a polymer with a wide range of potential industrial applications. Several Komagataeibacter species, including Komagataeibacter xylinus as a model organism, produce CNF. However, the industrial application of CNF has been hampered by inefficient CNF production, necessitating metabolic engineering for the enhanced CNF production. Here, we present complete genome sequence and a genome-scale metabolic model KxyMBEL1810 of K. xylinus DSM 2325 for metabolic engineering applications. Genome analysis of this bacterium revealed that a set of genes associated with CNF biosynthesis and regulation were present in this bacterium, which were also conserved in another six representative Komagataeibacter species having complete genome information. To better understand the metabolic characteristics of K. xylinus DSM 2325, KxyMBEL1810 was reconstructed using genome annotation data, relevant computational resources and experimental growth data generated in this study. Random sampling and correlation analysis of the KxyMBEL1810 predicted pgi and gnd genes as novel overexpression targets for the enhanced CNF production. Among engineered K. xylinus strains individually overexpressing heterologous pgi and gnd genes, either from Escherichia coli or Corynebacterium glutamicum, batch fermentation of a strain overexpressing the E. coli pgi gene produced 3.15 g/L of CNF in a complex medium containing glucose, which was the best CNF concentration achieved in this study, and 115.8% higher than that (1.46 g/L) obtained from the control strain. Genome sequence data and KxyMBEL1810 generated in this study should be useful resources for metabolic engineering of K. xylinus for the enhanced CNF production.

An accurate measurement of the dipole orientation in various organic semiconductor films using photoluminescence exciton decay analysis.

In this study, we report an accurate and more reliable approach to estimate the dipole orientation of emitters especially phosphorescence, fluorescence and even thermally activated delayed fluorescence. The dipole orientation measurements are performed by examining the variation of the photoluminescence (PL) exciton decay rate from time-resolved PL and optical analysis. Our anisotropic dipole orientation results are consistent with those of previous reports. The studied measurement approach is very reliable and accurate to estimate the dipole orientation of any organic semiconductor materials regardless of whether they are doped or neat films.

SimCAL: a flexible tool to compute biochemical reaction similarity.

BACKGROUND: Computation of reaction similarity is a pre-requisite for several bioinformatics applications including enzyme identification for specific biochemical reactions, enzyme classification and mining for specific inhibitors. Reaction similarity is often assessed at either two levels: (i) comparison across all the constituent substrates and products of a reaction, reaction level similarity, (ii) comparison at the transformation center with various degrees of neighborhood, transformation level similarity. Existing reaction similarity computation tools are designed for specific applications and use different features and similarity measures. A single system integrating these diverse features enables comparison of the impact of different molecular properties on similarity score computation. RESULTS: To address these requirements, we present SimCAL, an integrated system to calculate reaction similarity with novel features and capability to perform comparative assessment. SimCAL provides reaction similarity computation at both whole reaction level and transformation level. Novel physicochemical features such as stereochemistry, mass, volume and charge are included in computing reaction fingerprint. Users can choose from four different fingerprint types and nine molecular similarity measures. Further, a comparative assessment of these features is also enabled. The performance of SimCAL is assessed on 3,688,122 reaction pairs with Enzyme Commission (EC) number from MetaCyc and achieved an area under the curve (AUC) of > 0.9. In addition, SimCAL results showed strong correlation with state-of-the-art EC-BLAST and molecular signature based reaction similarity methods. CONCLUSIONS: SimCAL is developed in java and is available as a standalone tool, with intuitive, user-friendly graphical interface and also as a console application. With its customizable feature selection and similarity calculations, it is expected to cater a wide audience interested in studying and analyzing biochemical reactions and metabolic networks.

Next generation smart window display using transparent organic display and light blocking screen.

Transparent organic light emitting diodes (TOLED) have widespread applications in the next-generation display devices particularly in the large size transparent window and interactive displays. Herein, we report high performance and stable attractive smart window displays using facile process. Advanced smart window display is realized by integrating the high performance light blocking screen and highly transparent white OLED panel. The full smart window display reveals a maximum transmittance as high as 64.2% at the wavelength of 600 nm and extremely good along with tunable ambient contrast ratio (171.94:1) compared to that of normal TOLED (4.54:1). Furthermore, the performance decisive light blocking screen has demonstrated an excellent optical and electrical characteristics such as i) high transmittance (85.56% at 562nm) at light-penetrating state, ii) superior absorbance (2.30 at 562nm) in light interrupting mode, iii) high optical contrast (85.50 at 562 nm), iv) high optical stability for more than 25,000 cycle of driving, v) fast switching time of 1.9 sec, and vi) low driving voltage of 1.7 V. The experimental results of smart window display are also validated using optical simulation. The proposed smart window display technology allows us to adjust the intensity of daylight entering the system quickly and conveniently.

Improved Thermal Stability of Lithium-Rich Layered Oxide by Fluorine Doping.

The thermal stability of lithium-rich layered oxide with the composition Li(Li1/6 Ni1/6 Co1/6 Mn1/2 )O2-x Fx (x=0.00 and 0.05) is evaluated for use as a cathode material in lithium-ion batteries. Thermogravimetric analysis, evolved gas analysis, and differential scanning calorimetry show that, upon fluorine doping, degradation of the lithium-rich layered oxides commences at higher temperatures and the exothermic reaction is suppressed. Hot box tests also reveal that the prismatic cell with the fluorine-doped powder does not explode, whereas that with the undoped one explodes at about 135 °C with a sudden temperature increase. XRD analysis indicates that fluorine doping imparts the lithium-rich layered oxide with better thermal stability by mitigating oxygen release at elevated temperatures that cause an exothermic reaction with the electrolyte. The origin of the reduced oxygen release from the fluorinated lithium-rich layered oxide is also discussed.

ReactPRED: a tool to predict and analyze biochemical reactions.

MOTIVATION: Biochemical pathways engineering is often used to synthesize or degrade target chemicals. In silico screening of the biochemical transformation space allows predicting feasible reactions, constituting these pathways. Current enabling tools are customized to predict reactions based on pre-defined biochemical transformations or reaction rule sets. Reaction rule sets are usually curated manually and tailored to specific applications. They are not exhaustive. In addition, current systems are incapable of regulating and refining data with an aim to tune specificity and sensitivity. A robust and flexible tool that allows automated reaction rule set creation along with regulated pathway prediction and analyses is a need. ReactPRED aims to address the same. RESULTS: ReactPRED is an open source flexible and customizable tool enabling users to predict biochemical reactions and pathways. The tool allows automated reaction rule creation from a user defined reaction set. Additionally, reaction rule degree and rule tolerance features allow refinement of predicted data. It is available as a flexible graphical user interface and a console application. AVAILABILITY AND IMPLEMENTATION: ReactPRED is available at: https://sourceforge.net/projects/reactpred/ CONTACT: anirban.b@samsung.com or ty76.kim@samsung.comSupplementary information: Supplementary data are available at Bioinformatics online.

The cabABC Operon Essential for Biofilm and Rugose Colony Development in Vibrio vulnificus.

A transcriptome analysis identified Vibrio vulnificus cabABC genes which were preferentially expressed in biofilms. The cabABC genes were transcribed as a single operon. The cabA gene was induced by elevated 3',5'-cyclic diguanylic acid (c-di-GMP) and encoded a calcium-binding protein CabA. Comparison of the biofilms produced by the cabA mutant and its parent strain JN111 in microtiter plates using crystal-violet staining demonstrated that CabA contributed to biofilm formation in a calcium-dependent manner under elevated c-di-GMP conditions. Genetic and biochemical analyses revealed that CabA was secreted to the cell exterior through functional CabB and CabC, distributed throughout the biofilm matrix, and produced as the biofilm matured. These results, together with the observation that CabA also contributes to the development of rugose colony morphology, indicated that CabA is a matrix-associated protein required for maturation, rather than adhesion involved in the initial attachment, of biofilms. Microscopic comparison of the structure of biofilms produced by JN111 and the cabA mutant demonstrated that CabA is an extracellular matrix component essential for the development of the mature biofilm structures in flow cells and on oyster shells. Exogenously providing purified CabA restored the biofilm- and rugose colony-forming abilities of the cabA mutant when calcium was available. Circular dichroism and size exclusion analyses revealed that calcium binding induces CabA conformational changes which may lead to multimerization. Extracellular complementation experiments revealed that CabA can assemble a functional matrix only when exopolysaccharides coexist. Consequently, the combined results suggested that CabA is a structural protein of the extracellular matrix and multimerizes to a conformation functional in building robust biofilms, which may render V. vulnificus to survive in hostile environments and reach a concentrated infective dose.

Correction: The cabABC Operon Essential for Biofilm and Rugose Colony Development in Vibrio vulnificus.

[This corrects the article DOI: 10.1371/journal.ppat.1005192.].

Improvement of succinate production by release of end-product inhibition in Corynebacterium glutamicum.

Succinate is a renewable-based platform chemical that may be used to produce a wide range of chemicals including 1,4-butanediol, tetrahydrofurane, and γ-butyrolactone. However, industrial fermentation of organic acids is often subject to end-product inhibition, which significantly retards cell growth and limits metabolic activities and final productivity. In this study, we report the development of metabolically engineered Corynebacterium glutamicum for high production of succinate by release of end-product inhibition coupled with an increase of key metabolic flux. It was found that the rates of glucose consumption and succinate production were significantly reduced by extracellular succinate in an engineered strain, S003. To understand the mechanism underlying the inhibition by succinate, comparative transcriptome analysis was performed. Among the downregulated genes, overexpression of the NCgl0275 gene was found to suppress the inhibition of glucose consumption and succinate production, resulting in a 37.7% increase in succinate production up to 55.4g/L in fed-batch fermentation. Further improvement was achieved by increasing the metabolic flux from PEP to OAA. The final engineered strain was able to produce 152.2g/L succinate, the highest production reported to date, with a yield of 1.1g/g glucose under anaerobic condition. These results suggest that the release of end-product inhibition coupled with an increase in key metabolic flux is a promising strategy for enhancing production of succinate.

Using the Endoscopic Transconjunctival and Transcaruncular Approach to Repair Combined Orbital Floor and Medial Wall Blowout Fractures.

PURPOSE: To demonstrate the effectiveness of the endoscopic transcaruncular and transconjunctival approach in the repair of combined medial and inferior orbital wall fractures. METHODS: A retrospective chart review was conducted on 160 patients with combined medial and inferior orbital wall fractures. All patients underwent surgery via an endoscopic transcaruncular and transconjunctival approach without lateral canthotomy, performed by a single surgeon. Porous polyethylene sheets (1.0 mm in thickness) were implanted to cover the orbital defects. The minimal postoperative follow-up period was 6 months. The authors evaluated enophthalmos, diplopia, and ocular motility pre and postoperatively and report surgical complications. RESULTS: A total of 160 patients were included, comprising 121 men and 39 women. The mean patient age was 33.9 ±â€Š14.1 years, and the mean postoperative follow-up period was 12 months. The average enophthalmos was 3.20 mm preoperatively, and the mean improvement at 6 months after surgery was 2.82 mm. One patient suffered a canalicular laceration after surgery, and another retrobulbar hemorrhage; however, both of these complications resolved with appropriate management. Otherwise, there were no significant surgical complications including newly developed diplopia, decreased visual acuity, or cerebrospinal fluid leakage. CONCLUSIONS: The endoscopic transcaruncular and transconjunctival approach is a useful and promising technique to repair combined medial and inferior orbital wall fractures.

Functional elucidation of the non-coding RNAs of Kluyveromyces marxianus in the exponential growth phase.

BACKGROUND: Non-coding RNAs (ncRNAs), which perform diverse regulatory roles, have been found in organisms from all superkingdoms of life. However, there have been limited numbers of studies on the functions of ncRNAs, especially in nonmodel organisms such as Kluyveromyces marxianus that is widely used in the field of industrial biotechnology. RESULTS: In this study, we measured changes in transcriptome at three time points during the exponential growth phase of K. marxianus by using strand-specific RNA-seq. We found that approximately 60% of the transcriptome consists of ncRNAs transcribed from antisense and intergenic regions of the genome that were transcribed at lower levels than mRNA. In the transcriptome, a substantial number of long antisense ncRNAs (lancRNAs) are differentially expressed and enriched in carbohydrate and energy metabolism pathways. Furthermore, this enrichment is evolutionarily conserved, at least in yeast. Particularly, the mode of regulation of mRNA/lancRNA pairs is associated with mRNA transcription levels; the correlation between the pairs is positive at high mRNA transcriptional levels and negative at low levels. In addition, significant induction of mRNA and coverage of more than half of the mRNA sequence by a lancRNA strengthens the positive correlation between mRNA/lancRNA pairs. CONCLUSIONS: Transcriptome sequencing of K. marxianus in the exponential growth phase reveals pervasive transcription of ncRNAs with evolutionarily conserved functions. Studies of the mode of regulation of mRNA/lancRNA pairs suggest that induction of lancRNA may be associated with switch-like behavior of mRNA/lancRNA pairs and efficient regulation of the carbohydrate and energy metabolism pathways in the exponential growth phase of K. marxianus being used in industrial applications.

Stationary-phase induction of vvpS expression by three transcription factors: repression by LeuO and activation by SmcR and CRP.

An exoprotease of Vibrio vulnificus, VvpS, exhibits an autolytic function during the stationary phase. To understand how vvpS expression is controlled, the regulators involved in vvpS transcription and their regulatory mechanisms were investigated. LeuO was isolated in a ligand-fishing experiment, and experiments using a leuO-deletion mutant revealed that LeuO represses vvpS transcription. LeuO bound the extended region including LeuO-binding site (LBS)-I and LBS-II. Further screening of additional regulators revealed that SmcR and cyclic adenosine monophosphate-receptor protein (CRP) play activating roles in vvpS transcription. SmcR and CRP bound the regions overlapping LBS-I and -II, respectively. In addition, the LeuO occupancy of LBS-I and LBS-II was competitively exchanged by SmcR and CRP, respectively. To examine the mechanism of stationary-phase induction of vvpS expression, in vivo levels of three transcription factors were monitored. Cellular level of LeuO was maximal at exponential phase, while those of SmcR and CRP were maximal at stationary phase and relatively constant after the early-exponential phase, respectively. Thus, vvpS transcription was not induced during the exponential phase by high cellular content of LeuO. When entering the stationary phase, however, LeuO content was significantly reduced and repression by LeuO was relieved through simultaneous binding of SmcR and CRP to LBS-I and -II, respectively.

Enhancement in the electrochemical performance of zirconium/phosphate bi-functional coatings on LiNi0.8Co0.15Mn0.05O2 by the removal of Li residuals.

The effect of bi-functional coatings consisting of Zr and phosphate (P) on the electrochemical performance of Li1.0Ni0.8Co0.15Mn0.05O2 (NCM) has been investigated. The presence of various types of Zr and P compounds such as oxides (ZrO2 and Li2ZrO3) and phosphates (Zr2P2O9, ZrP2O7 and LiZr2(PO4)3) in the coating was confirmed by experiments as well as density functional theory (DFT) calculations. When the NCM samples were coated with the Zr/P hybrid material, the cycle retention and the amount of removed Li residuals (LiOH, Li2CO3) were enhanced by the synergistic effect from Zr and P. The NCM sample coated with a Zr/P layer with a Zr/P ratio of 1 : 1 exhibited an increase in the initial capacity (209.3 mA h g-1) compared to the pristine sample (207.4 mA h g-1) at 0.1C, owing to the formation of the coating layer. The capacity retention of the Zr/P coated sample (92.4% at the 50th cycle) was also improved compared to that of the pristine NCM sample (90.6% at the 50th cycle). Moreover, the amount of Li residuals in the Zr/P coated NCM sample was greatly reduced from 3693 ppm (pristine NCM) to 2525 ppm (Zr/P = 5 : 5).

Engineering of a butyraldehyde dehydrogenase of Clostridium saccharoperbutylacetonicum to fit an engineered 1,4-butanediol pathway in Escherichia coli.

1,4-Butanediol (1,4-BDO) is currently produced from succinate via six enzymatic reactions in an engineered Escherichia coli strain. Butyraldehyde dehydrogenase (Bld) and butanol dehydrogenase of Clostridium saccharoperbutylacetonicum were selected based on their activities of catalyzing the final two reactions in the 1,4-BDO pathway. To fit Bld into the non-natural 1,4-BDO pathway, we engineered it through random mutagenesis. Five Bld mutants were then isolated using a colorimetric Schiff's reagent-based method. Subsequent site-directed mutagenesis of Bld generated the two best Bld mutants, L273I and L273T, which produced 1,4-BDO titers fourfold greater than those of wild-type Bld. The enhanced 1,4-BDO titers obtained using L273I and L273T clearly correlated with their enhanced activities, which were caused by amino acid mutations at position 273 of Bld. The highest titer of 1,4-BDO (660 ± 40 mg/L) was obtained in a knock-out E. coli strain [ΔldhA ΔpflB ΔadhE ΔlpdA::K. lpd(E354K) Δmdh ΔarcA gltA(R164L)] coexpressing Bld273T+Bdh.

A Polyolefin Elastomer Encapsulant Modified by an Ethylene-Propylene-Diene Terpolymer for Photovoltaic Applications.

In this study, a newly designed adhesion promoter, a modified ethylene-propylene-diene terpolymer (m-EPDM), was constructed via a simple thiol-ene click reaction between the ethylene-propylene-diene terpolymer (EPDM) and 3-mercaptopropyltrimethoxysilane (MPTS) to employ polyolefin elastomer (POE) encapsulants in photovoltaic modules. The grafting reaction of MPTS on an EPDM backbone (thiol-ene click reaction) was verified using 1H NMR, 29Si NMR, and SEM/EDX. The thermal and mechanical characteristics of the POE compounds did not significantly change with an increasing m-EPDM content irrespective of the cross-linking state. Interestingly, the adhesion strength to the glass substrate increased linearly with an increasing m-EPDM content until 9 phr. Also, the POE compounds containing more than 12 phr m-EPDM showed cohesion failure of the encapsulant layer, remaining as a residue of the encapsulant layer on the glass surface after peel testing. The damp-heat test was conducted to evaluate the long-term durability of the photovoltaic module encapsulated with m-EPDM, and no significant power loss was found even after 1000 h under the test conditions.