Paucibacter sediminis sp. nov., isolated from sediment in a freshwater pond.

A Gram-stain-negative, aerobic, oxidase-positive, weakly catalase-positive, motile by means of a single polar flagellum, rod-shaped bacterium designated as strain S2-9T was isolated from sediment sampled in Wiyang pond, Republic of Korea. Growth of this strain was observed at 10-40 °C (optimum, 35 °C) and pH 5.5-9.5 (optimum, pH 7.0-8.0) and in the presence of 0-0.5 % NaCl in Reasoner's 2A broth. The major fatty acids (>10 %) of strain S2-9T were C16 : 0 and summed feature 3 (comprising a mixture of C16 : 1 ω7c and/or C16 : 1 ω6c). Ubiquinone-8 was detected as the respiratory quinone. The major polar lipids were phosphatidylethanolamine and phosphatidylglycerol. Strain S2-9T showed the highest 16S rRNA gene sequence similarity to Paucibacter oligotrophus CHU3T (98.7 %), followed by 'Paucibacter aquatile' CR182 (98.4 %), all type strains of Pelomonas species (98.1-98.3 %), Mitsuaria chitosanitabida NBRC 102408T (97.9 %), Kinneretia asaccharophila KIN192T (97.8 %), Mitsuaria chitinivorans HWN-4T (97.4 %), and Paucibacter toxinivorans 2C20T (97.4 %). Phylogenetic trees based on the 16S rRNA gene and whole-genome sequences showed that strain S2-9T formed a tight phylogenetic lineage with Paucibacter species (CHU3T, CR182, and 2C20T). Average nucleotide identity and digital DNA-DNA hybridization values between strain S2-9T and Paucibacter strains were 76.6-79.3% and 19.5-21.5 %, respectively. The genomic DNA G+C content of strain S2-9T was 68.3 mol%. Notably, genes responsible for both sulphur oxidation and reduction and denitrification were found in the genome of strain S2-9T, suggesting that strain S2-9T is involved in the nitrogen and sulphur cycles in pond ecosystems. Based on the polyphasic taxonomic results, strain S2-9T represents a novel species of the genus Paucibacter, for which the name Paucibacter sediminis sp. nov. is proposed. The type strain is S2-9T (= KACC 22267T= JCM 34541T).

Community recovery of benthic macroinvertebrates in a stream influenced by mining activity: Importance of microhabitat monitoring.

The decrease in freshwater biodiversity owing to anthropogenic disturbances such as mining activity is a global challenge; hence, there is an urgent need for systematic approaches to continuously monitor such disturbances and/or the recovery of biodiversity in freshwater habitats. The Hwangjicheon Stream is the source of South Korea's longest river and has been subjected to runoff from coal mining. We investigated changes in the diversity of the benthic macroinvertebrate community in various microhabitats, including riffle, run, and pool, to monitor the recovery of biodiversity in the stream following the improvement of a mining water treatment plant in 2019. The dataset comprised 111 samples obtained from four types of microhabitats (riffle, run, pool, and riparian) over a four-year period from 2018 to 2021. The mining-affected sites had lower macroinvertebrate community complexities according to a network analysis, and grouped into the same cluster based on self-organizing map (SOM) analysis. Moreover, 51 taxa selected as indicator species represented each cluster obtained through the SOM analysis. Among them, only Limnodrilus gotoi and Radix auricularia were included as indicator species at the mining-affected sites. However, after 2020, the benthic macroinvertebrate community complexity increased, and some of the microhabitats at the mining-affected sites were included in the same cluster as the reference sites in the SOM analysis, indicating that the recovery of benthic macroinvertebrate communities had initiated in certain microhabitats (e.g., riparian). Further analysis confirmed that the macroinvertebrate community clearly differed according to the survey year, even in different microhabitats at the same sites. This suggests that more acute microhabitat monitoring may be necessary to quickly confirm biodiversity restoration when assessing the degree of the recovery in river biodiversity from anthropogenic disturbances.

Chlorophyll a Synthesis in Rhodobacter sphaeroides by Chlorophyll Synthase of Nicotiana tabacum.

The production of phytylated chlorophyll a (Chl aP) in Rhodobacter sphaeroides, which uses phytylated bacteriochlorophyll a (BChl aP), is the first step in expanding the light absorption spectra. Unlike the chlorophyll synthase (ChlG) of the Synechocystis sp. PCC6803, ChlGs of angiosperms, including Arabidopsis thaliana, Nicotiana tabacum, Avena sativa, and Oryza sativa, showed bacteriochlorophyll synthase activity and resistance to inhibition by bacteriochlorophyllide a (BChlide a), geranylgeranylated BChl a (BChl aGG), and BChl aP, collectively called bacteriochlorins. Among the angiosperm ChlGs, N. tabacum ChlG had the highest bacteriochlorophyll synthase activity and resistance to inhibition by bacteriochlorins. Expression of N. tabacum chlG in R. sphaeroides resulted in the formation of free Chl aP in the presence of BChl aP during photoheterotrophic growth, even though reactive oxygen species were generated.

The Photoactive Photosynthetic Reaction Center of a Rhodobacter sphaeroides Mutant Lacking 3-Vinyl (Bacterio)Chlorophyllide a Hydratase Contains 3-Vinyl Bacteriochlorophyll a.

Rhodobacter sphaeroides mutant BF-lacking 3-vinyl (bacterio)chlorophyllide a hydratase (BchF)-accumulates chlorophyllide a (Chlide a) and 3-vinyl bacteriochlorophyllide a (3V-Bchlide a). BF synthesizes 3-vinyl bacteriochlorophyll a (3V-Bchl a) through prenylation of 3V-Bchlide a and assembles a novel reaction center (V-RC) using 3V-Bchl a and Mg-free 3-vinyl bacteriopheophytin a (3V-Bpheo a) at a molar ratio of 2:1. We aimed to verify whether a bchF-deleted R. sphaeroides mutant produces a photochemically active RC that facilitates photoheterotrophic growth. The mutant grew photoheterotrophically-implying a functional V-RC-as confirmed by the emergence of growth-competent suppressors of bchC-deleted mutant (BC) under irradiation. Suppressor mutations in BC were localized to bchF, which diminished BchF activity and caused 3V-Bchlide a accumulation. bchF expression carrying the suppressor mutations in trans resulted in the coproduction of V-RC and wild-type RC (WT-RC) in BF. The V-RC had a time constant (τ) for electron transfer from the primary electron donor P (a dimer of 3V-Bchl a) to the A-side containing 3V-Bpheo a (HA) similar to that of the WT-RC and a 60% higher τ for electron transfer from HA to quinone A (QA). Thus, the electron transfer from HA to QA in the V-RC should be slower than that in the WT-RC. Furthermore, the midpoint redox potential of P/P+ of the V-RC was 33 mV more positive than that of the WT-RC. R. sphaeroides, thus, synthesizes the V-RC when 3V-Bchlide a accumulates. The V-RC can support photoheterotrophic growth; however, its photochemical activity is inferior to that of the WT-RC. IMPORTANCE 3V-Bchlide a is an intermediate in the bacteriochlorophyll a (Bchl a)-specific biosynthetic branch and prenylated by bacteriochlorophyll synthase. R. sphaeroides synthesizes V-RC that absorbs light at short wavelengths. The V-RC was not previously discovered because 3V-Bchlide a does not accumulate during the growth of WT cells synthesizing Bchl a. The levels of reactive oxygen species increased with the onset of photoheterotrophic growth in BF, resulting in a long lag period. Although the inhibitor of BchF is unknown, the V-RC may act as a substitute for the WT-RC when BchF is completely inhibited. Alternatively, it may act synergistically with WT-RC at low levels of BchF activity. The V-RC may broaden the absorption spectra of R. sphaeroides and supplement its photosynthetic ability at various wavelengths of visible light to a greater extent than that by the WT-RC alone.

In vitro and in vivo phototoxicity on gastric mucosa induced by methylene blue.

Background: Methylene blue (MB) is used endoscopically to demarcate tumors and as a photosensitizer in photodynamic therapy (PDT). However, there are few in vivo studies about its toxicity in healthy stomach tissue. We performed sequential in vitro and in vivo analyses of MB-induced phototoxicity. Methods: We performed in vitro experiments using the AGS human gastric cancer cell line treated with light-emitting diode (LED) irradiation (3.6 J/cm2) and MB. Cytotoxicity was evaluated using terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay. In vivo toxicity was evaluated in the stomach of beagles using the same dose of fiber-optic LED via gastroscopy, after spraying 0.1% and 0.5% MB solutions. Stomach tissue was also evaluated using the TUNEL assay. Results: In vitro, increased concentrations of MB led to higher TUNEL scores. However, cell viability was significantly lower after MB plus LED irradiation than after treatment with MB alone (P < 0.001). In vivo, the TUNEL score was highest immediately after treatment with 0.1% or 0.5% MB plus light irradiation, and the score was significantly higher in the LED illumination plus MB group than in the control group (P < 0.05). The elevated TUNEL score was maintained for 3 days in the MB plus light irradiation group but returned to normal levels on day 10. Conclusions: : Endoscopic light application with MB 0.5% concentration to the stomach may be regarded as a safe procedure despite some DNA injuries in the early period.

Comparison of Bacterial Assemblages Associated with Harmful Cyanobacteria under Different Light Conditions.

Harmful cyanobacterial blooms in freshwater ecosystems are closely associated with changes in the composition of symbiotic microbiomes, water quality, and environmental factors. In this work, the relationship between two representative harmful cyanobacterial species (Anabaena sp. and Microcystis sp.) and their associated bacterial assemblages were investigated using a 16S rRNA-based meta-amplicon sequencing analysis during a large-scale cultivation of cyanobacteria under different light conditions with limited wavelength ranges (natural light, blue-filtered light, green-filtered light, and dark conditions). During the cultivation periods, the growth pattern of cyanobacteria and bacterial composition of the phycosphere considerably varied in relation to light restrictions. Unlike other conditions, the cyanobacterial species exhibited significant growth during the cultivation period under both the natural and the blue light conditions. Analyses of the nitrogenous substances revealed that nitrogen assimilation by nitrate reductase for the growth of cyanobacteria occurred primarily under natural light conditions, whereas nitrogenase in symbiotic bacteria could also be activated under blue light conditions. Sphingobium sp., associated with nitrogen assimilation via nitrogenase, was particularly dominant when the cell density of Microcystis sp. increased under the blue light conditions. Thus, cyanobacteria could have symbiotic relationships with ammonium-assimilating bacteria under light-limited conditions, which aids the growth of cyanobacteria.

Sustainability of in vitro light-dependent NADPH generation by the thylakoid membrane of Synechocystis sp. PCC6803.

BACKGROUND: NADPH is used as a reductant in various biosynthetic reactions. Cell-free bio-systems have gained considerable attention owing to their high energy utilization and time efficiency. Efforts have been made to continuously supply reducing power to the reaction mixture in a cyclical manner. The thylakoid membrane (TM) is a promising molecular energy generator, producing NADPH under light. Thus, TM sustainability is of major relevance for its in vitro utilization. RESULTS: Over 70% of TMs prepared from Synechocystis sp. PCC6803 existed in a sealed vesicular structure, with the F1 complex of ATP synthase facing outward (right-side-out), producing NADPH and ATP under light. The NADPH generation activity of TM increased approximately two-fold with the addition of carbonyl cyanide-p-(trifluoromethoxy) phenylhydrazone (FCCP) or removal of the F1 complex using EDTA. Thus, the uncoupling of proton translocation from the electron transport chain or proton leakage through the Fo complex resulted in greater NADPH generation. Biosilicified TM retained more than 80% of its NADPH generation activity after a week at 30°C in the dark. However, activity declined sharply to below 30% after two days in light. The introduction of engineered water-forming NADPH oxidase (Noxm) to keep the electron transport chain of TM working resulted in the improved sustainability of NADPH generation activity in a ratio (Noxm to TM)-dependent manner, which correlated with the decrease of singlet oxygen generation. Removal of reactive oxygen species (ROS) by catalase further highlighted the sustainable NADPH generation activity of up to 80% in two days under light. CONCLUSION: Reducing power generated by light energy has to be consumed for TM sustainability. Otherwise, TM can generate singlet oxygen, causing oxidative damage. Thus, TMs should be kept in the dark when not in use. Although NADPH generation activity by TM can be extended via silica encapsulation, further removal of hydrogen peroxide results in an improvement of TM sustainability. Therefore, as long as ROS formation by TM in light is properly handled, it can be used as a promising source of reducing power for in vitro biochemical reactions.

Multi-scale causality analysis between COVID-19 cases and mobility level using ensemble empirical mode decomposition and causal decomposition.

The global spread of the coronavirus disease 2019 (COVID-19) pandemic has affected the world in many ways. Due to the communicable nature of the disease, it is difficult to investigate the causal reason for the epidemic's spread sufficiently. This study comprehensively investigates the causal relationship between the spread of COVID-19 and mobility level on a multi time-scale and its influencing factors, by using ensemble empirical mode decomposition (EEMD) and the causal decomposition approach. Linear regression analysis investigates the significance and importance of the influential factors on the intrastate and interstate causal strength. The results of an EEMD analysis indicate that the mid-term and long-term domain portrays the macroscopic component of the states' mobility level and COVID-19 cases, which represents overall intrinsic characteristics. In particular, the mobility level is highly associated with the long-term variations of COVID-19 cases rather than short-term variations. Intrastate causality analysis identifies the significant effects of median age and political orientation on the causal strength at a specific time-scale, and some of them cannot be identified from the existing method. Interstate causality results show a negative association with the interstate distance and the positive one with the airline traffic in the long-term domain. Clustering analysis confirms that the states with the higher the gross domestic product and the more politically democratic tend to more adhere to social distancing. The findings of this study can provide practical implications to the policymakers that whether the social distancing policies are effectively working or not should be monitored by long-term trends of COVID-19 cases rather than short-term.

Photoautotrophic Growth Rate Enhancement of Synechocystis sp. PCC6803 by Heterologous Production of 2-Oxoglutarate:Ferredoxin Oxidoreductase from Chlorobaculum tepidum.

2-Oxoglutarate:ferredoxin oxidoreductase from Chlorobaculum tepidum (CtOGOR) is a carbon-fixing enzyme in the reductive TCA cycle that reversibly carboxylates succinyl-CoA to yield 2-oxoglutarate. CtOGOR is a heterotetramer of two large (α = 68 kDa) and two small (ß = 38 kDa) subunits. The αß protomer harbors one thiamine pyrophosphate and two 4Fe-4S clusters. Nonetheless, the enzyme has a considerable oxygen tolerance with a half-life of 143 min at 215 µM dissolved oxygen. Kinetic analyses of the purified recombinant CtOGOR revealed a lower Km for succinyl-CoA than for 2-oxoglutarate. Cellular levels of 2-oxoglutarate and glutamate­a product of glutamine oxoglutarate aminotransferase and glutamate dehydrogenase­increased more than twofold in the exponential phase compared with the control strain, leading to an approximately >30% increase in the photoautotrophic growth rate. Thus, CtOGOR was successfully produced in Synechocystis, thereby boosting carboxylation, resulting in enhanced photoautotrophic growth.

Evaluation of the Impacts of Abandoned Mining Areas: A Case Study with Benthic Macroinvertebrate Assemblages.

Mining activities are among the most long-lasting anthropogenic pressures on streams and rivers. Therefore, detecting different benthic macroinvertebrate assemblages in the areas recovered from mining activities is essential to establish conservation and management plans for improving the freshwater biodiversity in streams located near mining areas. We compared the stability of benthic macroinvertebrate communities between streams affected by mining activities (Hwangjicheon: NHJ and Cheolamcheon: NCA) and the least disturbed stream (Songjeonricheon: NSJ) using network analysis, self-organizing map, and indicator species analysis. Species richness was lowest at sites where stream sediments were reddened or whitened due to mining impacts in NHJ and NCA. Among functional feeding groups, the ratio of scrapers was lower (i.e., NHJ) or not observed (i.e., NCA) in the affected sites by mining. The networks (species interactions) were less connected in NHJ and NCA than in NSJ, indicating that community stability decreased in the area affected by mining activity. We identified five groups based on the similarity of benthic macroinvertebrate communities according to the gradients of mining impacts using a self-organizing map. the samples from the reference stream (clusters 1 and 5), sites located near the mining water inflow area (cluster 4), sites where stream sediments acid-sulfated (cluster 2), and sites that had recovered from mining impacts (cluster 3). Among the 40 taxa selected as indicators defined from the five clusters in self-organizing map, only few (Physa acuta, Tipula KUa, and Nemoura KUb) indicator species were selected in each cluster representing the mining-impacted sites. Our results highlighted that the benthic macroinvertebrate community complexity was lower in streams affected by mining activity. Furthermore, the range of disturbed areas in the streams, where conservation and management plans should be prioritized, can be quantified by examining alterations in the benthic macroinvertebrate community.

Application of naïve Bayesian approach in detecting reproducible fatal collision locations on freeway.

Detecting high-collision-concentration locations based solely on collision frequency may produce different results compared to those considering the severities of the collisions. In particular, it can lead government agencies focusing sites with a high collision frequency while neglecting those with a lower collision frequency but a higher percentage of injury and fatal collisions. This study developed systematic ways of detecting reproducible fatal collision locations (R) using the naïve Bayes approach and a continuous risk profile (CRP) that estimates the true collision risk by filtering out random noise in the data. The posterior probability of fatal collisions being reproducible at a location is estimated by the relationship between the spatial distribution of fatal-collision locations (i.e., likelihood) and the CRP (i.e., prior probability). The proposed method can be used to detect sites with the highest proxy measure of the posterior probability (PMP) of observing R. An empirical evaluation using 5-year traffic collision data from six routes in California shows that detecting R based on the PMP outperform those based on the SPF-based approaches or random selection, regardless of various conditions and parameters of the proposed method. This method only requires traffic collision and annual traffic volume data to estimate PMP that prioritize sites being R and the PMPs can be compared across multiple routes. Therefore, it helps government agencies prioritizing sites of multiple routes where the number of fatal collisions can be reduced, thus help them to save lives with limited resources of data collection.

Anti-Inflammatory Effects of a Polyphenol, Catechin-7,4'-O-Digallate, from Woodfordia uniflora by Regulating NF-κB Signaling Pathway in Mouse Macrophages.

Inflammation is a defense mechanism that protects the body from infections. However, chronic inflammation causes damage to body tissues. Thus, controlling inflammation and investigating anti-inflammatory mechanisms are keys to preventing and treating inflammatory diseases, such as sepsis and rheumatoid arthritis. In continuation with our work related to the discovery of bioactive natural products, a polyphenol, catechin-7,4'-O-digallate (CDG), was isolated from Woodfordia uniflora, which has been used as a sedative and remedy for skin infections in the Dhofar region of Oman. Thus far, no study has reported the anti-inflammatory compounds derived from W. uniflora and the mechanisms underlying their action. To investigate the effects of CDG on the regulation of inflammation, we measured the reduction in nitric oxide (NO) production following CDG treatment in immortalized mouse Kupffer cells (ImKCs). CDG treatment inhibited NO production through the downregulation of inducible nitric oxide synthase expression in lipopolysaccharide (LPS)-stimulated ImKCs. The anti-inflammatory effects of CDG were mediated via the inhibition of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) activation, an important inflammatory-response-associated signaling pathway. Moreover, CDG treatment has regulated the expression of pro-inflammatory cytokines, such as IL-6 and IL-1ß. These results suggested the anti-inflammatory action of CDG in LPS-stimulated ImKCs.

Spatiotemporal filtering method for detecting kinematic waves in a connected environment.

Backward-moving kinematic waves (KWs) (e.g., stop-and-go traffic conditions and a shock wave) cause unsafe driving conditions, decreases in the capacities of freeways, and increased travel time. In this paper, a sequential filtering method is proposed to detect KWs using data collected in a connected environment, which can aid in developing a traffic control strategy for connected vehicles to stop or dampen the propagation of these KWs. The proposed method filters out random fluctuation in the data using ensemble empirical mode decomposition that considers the spectral features of KWs. Then, the spatial movements of KWs are considered using cross-correlation to identify potential candidate KWs. Asynchronous changes in the denoised flow and speed are used to evaluate candidate KWs using logistic regression to identify the KWs from localized reductions in speed that are not propagated upstream. The findings from an empirical evaluation of the proposed method showed strong promise for detecting KWs using data in a connected environment, even at 30% of the market penetration rates. This paper also addresses how data resolution of the connected environment affects the performance in detecting KWs.

In Vitro Bactericidal Effects of Photodynamic Therapy Combined with Four Tetracyclines against Clostridioides difficile KCTC5009 in Planktonic Cultures.

Surface disinfection in health-care facilities is critical to prevent dissemination of Clostridioides difficile (C. difficile). Tetracyclines (TCs) are broad-spectrum antibiotics that are associated with a low risk of development of C. difficile infection (CDI) and are used as photosensitizers (PS) in photodynamic therapy (PDT). We evaluated whether TCs may be useful environmental cleansing agents. We compared the in vitro ability to kill C. difficile of four TCs (TC, doxycycline, minocycline, and tigecycline) combined with PDT using ultraviolet A (UVA). We included chitosan, a cationic material, as a booster to increase the photodynamic bactericidal efficacy of TCs. PDT-induced bactericidal effects were assessed by the number of viable cells and the degree of DNA damage and membrane integrity. To avoid the intrinsic antibacterial activity of TCs at high concentrations, we used low concentrations of TCs (0.05 and 0.1 mg/mL). The bactericidal effect of treatment with chitosan plus PDT was over 100 times higher than that with PDT alone for each of the four TCs. DNA damage measured by ethidium bromide monoazide and real-time quantitative polymerase chain reaction was also greater for PDT plus chitosan treatment than for PDT alone or under control conditions: the threshold cycle (Ct) values for the control, PDT, and PDT plus chitosan were 14.67 ± 0.22, 20.46 ± 0.12, and 25.54 ± 0.17, respectively. All four TCs caused similar levels of severe cell membrane damage during PDT compared with control conditions. These data suggest that PDT combined with any of the four TCs plus chitosan might be an available tool to kill efficiently planktonic form of C. difficile.

Comparison of high and low molecular weight chitosan as in-vitro boosting agent for photodynamic therapy against Helicobacter pylori using methylene blue and endoscopic light.

BACKGROUND: We reported in a previous study that photodynamic therapy (PDT) of Helicobacter pylori(H. pylori) could potentiate bactericidal effect by adding chitosan. As a next step, we compared the bactericidal effects of low molecular weight (LMW) combined with Photodynamic Therapy to high molecular weight (HMW) chitosan. METHOD: To perform PDT to kill H. pylori, we used endoscopic light as light source, methylene blue (MB) as a photosensitizer and chitosan (310-375, 50-190 kDa). We evaluated bacterial removal rate and its membrane damage by ethidium bromide monoazide PCR method (EMA q-PCR). 8-oxo-2'-dexoyguanosine by ELISA was measured for oxidative stress. RESULTS: At a chitosan concentration of ≤0.05%, the killing effect did not differ between the two molecular weights, and 100% bacterial removal rate was observed at a light energy ≥ 6.23 mJ/cm2 powers under 0.02% MB. After 15 min irradiation, LMW chitosan with high concentration of MB (0.004%) showed highest killing effects, which were consistent with the results of EMA q-PCR but not with the level of 8-OHdG. Bactericidal effects of LMW chitosan plus PDT using 0.002 and 0.004% MB for 15 min irradiation were significantly higher than those using HMW chitosan plus PDT. CONCLUSION: We found that PDT using methylene blue with LMW chitosan to kill H. pylori exerted greater bactericidal effects through bacterial membrane damage than PDT with HMW chitosan. These results suggest that it would be better to choose LMW chitosan to enhance the effect of PDT for clinical application, even at a very low concentration of PS.

The neuroplastic effect of olfactory training to the recovery of olfactory system in mouse model.

BACKGROUND: Several studies have reported the benefits of olfactory training (OT) in the olfactory nervous system of mouse models. Therefore, in this study we performed next-generation sequencing to evaluate the effects of OT on mRNA sequencing in the olfactory area. METHODS: Mice in each group were administered 300 mg of 3-methylindole per kilogram of mouse weight. The olfactory function was evaluated by a food-finding test once a week. The olfactory neuroepithelium was harvested for histologic examination and protein analysis. Subsequently, data analysis, gene ontology and pathway analysis, quantitative real-time polymerase chain reaction of mRNA, and Western blot analysis were conducted. RESULTS: Mice were divided into 4 groups according to treatment. Control, anosmia, training, and steroid group mice resumed food finding. Olfactory Maker Protein, olfr1507, ADCY3, and GNAL mRNA expression was higher in the olfactory neuroepithelium of OT than anosmia group mice. In total, 26,364 mRNAs were analyzed. Comparison of the results of OT vs anosmia revealed that ADCY8,10, GFAP, NGF, NGFR, GFAP, and BDNF mRNAs were upregulated in the gene ontology. CONCLUSION: OT improved olfactory function, as indicated by the food-finding test. OT improved the olfactory recovery time to stimulate olfactory nerve regeneration. OT may initially stimulate the olfactory receptor, followed by neurogenesis. Steroid therapy and OT operated under completely different mechanisms in the upregulated gene study. These results indicate that OT may be one of the future modalities for treating olfactory impairment.

Preparation of Radiation Cross-Linked Poly(Acrylic Acid) Hydrogel Containing Metronidazole with Enhanced Antibacterial Activity.

Metronidazole (MD) is known as a periodontitis medicine and has been widely used in antibiotics for resistance to anaerobic bacteria, periodontal disease, and other threats. To treat diseases, drug delivery carriers are needed with a high bioadhesive property and enhanced drug penetration. Poly (acrylic acid) (PAA) hydrogel films have a good bioadhesive property and are able to localize the absorption site and increase the drug residence time. In this study, we fabricated a MD loaded PAA hydrogel with different MD content (0.1, 0.25, 0.5, and 1 wt%) using varying doses (25, 50, and 75 kGy) and the radiation doses (25, 50, or 75 kGy) in a one-step gamma-ray irradiation process. The chemical and physical structure were determined through a Fourier transformed infrared spectroscopy, X-ray photoelectron spectroscopy, gel content, and compressive strength. In addition, MD loaded PAA hydrogels were performed to MD release behaviors and cytotoxicity. Finally, we conducted antibacterial activity to demonstrate the prevention of growth of bacteria as a therapeutic dressing. The basic chemical structure analysis of MD was changed greatly at radiation doses of 50 and 75 kGy due to degradation by gamma-ray irradiation. However, when the absorbed dose was 25 kGy, the chemical structure analysis of MD did not change significantly, and the gel content and compressive strength of MD/PAA hydrogel were approximately 80% and 130 kPa, respectively. The MD/PAA hydrogels exhibited no cytotoxicity and good antibacterial activity against Escherichia coli, Staphylococcus aureus, and Streptococcus mutans. These results provide good evidence that MD/PAA hydrogel prepared by gamma-ray irradiation has potential as a competitive candidate for the therapeutic dressing.

Building a Thermostable Metabolon for Facilitating Coenzyme Transport and In†Vitro Hydrogen Production at Elevated Temperature.

To facilitate coenzyme transport and in vitro enzymatic hydrogen production, a multi-enzyme metabolon comprising a miniscaffoldin containing three cohesins, a dockerin-containing mutant dehydrogenase, a dockerin-containing diaphorase, and a Histidine-tagged (His-tagged) NiFe hydrogenase was constructed. As the NiFe hydrogenase has very complicated structure and cannot be fused directly with a dockerin, a bifunctional peptide was designed. The bifunctional peptide, in which one terminus contains a modified dockerin binding the cohesin of the miniscaffoldin and the other, after chemical modification, binds the His-tag of NiFe hydrogenase, enabled His-tagged proteins to be integrated into the cohesin-dockerin-based metabolon. The metabolon exhibited an initial reaction rate 4.5 times that of the enzyme cocktail at the same enzyme loading, which indicated enhanced coenzyme transport of the metabolon. However, this metabolon was unstable owing to the degradation of the miniscaffoldin at elevated temperature. Glutaraldehyde was used to cross-link the metabolon for locking its spatial organization. The cross-linked metabolon not only exhibited 2.5 times the reaction rate of the enzyme cocktail, but also retained its stability at 70 °C. The amount of hydrogen production catalyzed by the cross-linked metabolon was nearly twice that of the metabolon without glutaraldehyde cross-linking and four times that of the enzyme cocktail at 70 °C after 22 h of reaction.

Advanced water splitting for green hydrogen gas production through complete oxidation of starch by in vitro metabolic engineering.

Starch is a natural energy storage compound and is hypothesized to be a high-energy density chemical compound or solar fuel. In contrast to industrial hydrolysis of starch to glucose, an alternative ATP-free phosphorylation of starch was designed to generate cost-effective glucose 6-phosphate by using five thermophilic enzymes (i.e., isoamylase, alpha-glucan phosphorylase, 4-α-glucanotransferase, phosphoglucomutase, and polyphosphate glucokinase). This enzymatic phosphorolysis is energetically advantageous because the energy of α-1,4-glycosidic bonds among anhydroglucose units is conserved in the form of phosphorylated glucose. Furthermore, we demonstrated an in vitro 17-thermophilic enzyme pathway that can convert all glucose units of starch, regardless of branched and linear contents, with water to hydrogen at a theoretic yield (i.e., 12 H2 per glucose), three times of the theoretical yield from dark microbial fermentation. The use of a biomimetic electron transport chain enabled to achieve a maximum volumetric productivity of 90.2mmol of H2/L/h at 20g/L starch. The complete oxidation of starch to hydrogen by this in vitro synthetic (enzymatic) biosystem suggests that starch as a natural solar fuel becomes a high-density hydrogen storage compound with a gravimetric density of more than 14% H2-based mass and an electricity density of more than 3000Wh/kg of starch.

Exceptionally High Rates of Biological Hydrogen Production by Biomimetic In Vitro Synthetic Enzymatic Pathways.

Hydrogen production by water splitting energized by biomass sugars is one of the most promising technologies for distributed green H2 production. Direct H2 generation from NADPH, catalysed by an NADPH-dependent, soluble [NiFe]-hydrogenase (SH1) is thermodynamically unfavourable, resulting in slow volumetric productivity. We designed the biomimetic electron transport chain from NADPH to H2 by the introduction of an oxygen-insensitive electron mediator benzyl viologen (BV) and an enzyme (NADPH rubredoxin oxidoreductase, NROR), catalysing electron transport between NADPH and BV. The H2 generation rates using this biomimetic chain increased by approximately five-fold compared to those catalysed only by SH1. The peak volumetric H2 productivity via the in vitro enzymatic pathway comprised of hyperthermophilic glucose 6-phosphate dehydrogenase, 6-phosphogluconolactonase, and 6-phosphogluconate dehydrogenase, NROR, and SH1 was 310 mmol H2 /L h-1 , the highest rate yet reported. The concept of biomimetic electron transport chains could be applied to both in vitro and in vivo H2 production biosystems and artificial photosynthesis.