Human ocular thelaziasis with genetic analysis in Niigata Prefecture, Japan: A case report on an emerging zoonosis.

Purpose: We report the clinical findings and molecular identification of ocular Thelazia callipaeda from Niigata Prefecture in the Hokuriku area of Japan during winter. Observations: A 77-year-old male visited an ophthalmology clinic in Niigata Prefecture in January 2022 after a 2-week-duration of a conjunctival injection in the left eye and foreign body sensation. Slit-lamp microscopy revealed 11 active nematodes in the left conjunctival sac. Morphological characteristics included longer female body length than male, buccal cavity lacking teeth and lips, and serrated striations along the body surface. The specimens were determined to be T. callipaeda. Genetic analysis of the mitochondrial cytochrome c oxidase subunit 1 (cox1) gene revealed an h9 haplotype. Conclusions and Importance: T. callipaeda infection, especially the h9 haplotype, commonly occurs in western Japan owing to its higher incidence in warmer climates, suggesting the origin of the case. Here, we report a human case of Thelaziasis diagnosed in a cold region of Japan (the Hokuriku area) during winter. This human case of T. callipaeda infection from a cold, previously unassociated region, raises concerns about the potential geographical widening of its distribution, and further investigation may be warranted to prevent its spread.

Cellulose-fueled microbial fuel cells equipped with a bipolar membrane using hydrogen phosphate as the final electron acceptor.

OBJECTIVES: A bipolar membrane microbial fuel cell (bMFC) is used to generate electricity using cellulose in phosphate buffer solution as fuel, and the mechanism of electricity generation is elucidated from five reference experiments. RESULTS: The bMFC was operated for 20 days using cellulose as fuel and Cellulomonas fimi. In the first reference experiment, no microorganism was used. In the second experiment, a cation-exchange membrane was used instead of a bipolar membrane. In the third experiment, the bipolar membrane was used in the opposite orientation as in the main experiment. In the fourth experiment, D2O was used instead of H2O in the cathode chamber. In the final experiment, the tris-maleate buffer was used instead of a phosphate buffer. Sufficient power generation did not occur in either reference experiment. CONCLUSIONS: The bMFC continuously generated electricity for 20 days, and elucidated H+ and OH- react in bipolar membrane, where the counter cation of dihydrogen phosphate served as the final electron acceptor.

Production of glycerate from glucose using engineered Escherichiacoli.

In this study, glycerate was produced from glucose using engineered Escherichia coli BW25113. Plasmid pSR3 carrying gpd1 and gpp2 encoding two isoforms of glycerol-3-phosphate dehydrogenase from Saccharomyces cerevisiae and plasmid pLB2 carrying aldO encoding alditol oxidase from Streptomyces violaceoruber were introduced into E. coli to enable the production of glycerate from glucose via glycerol. Disruptions of garK and glxK genes in the E. coli genome were performed to minimize the consumption of glycerate produced. As a result, E. coli carrying these plasmids could produce nearly three times higher concentration of glycerate (0.50 ± 0.01 g/L) from 10 g/L glucose compared to E. coli EG_2 (0.14 ± 0.02 g/L). In M9 medium, disruption of garK and glxK resulted in an impaired growth rate with low production of glycerate, while supplementation of 0.5 g/L casamino acids and 0.5 g/L manganese sulfate to the medium replenished the growth rate and elevated the glycerate titer. Further disruption of glpF, encoding a glycerol transporter, increased the glycerate production to 0.80 ± 0.00 g/L. MR2 medium improved the glycerate production titers and specific productivities of E. coli EG_4, EG_5, and EG_6. Upscale production of glycerate was carried out in a jar fermentor with MR2 medium using E. coli EG_6, resulting in an improvement in glycerate production up to 2.37 ± 0.46 g/L with specific productivity at 0.34 ± 0.11 g-glycerate/g-cells. These results indicate that E. coli is an appropriate host for glycerate production from glucose.

Production of R- and S-1,2-propanediol in engineered Lactococcus lactis.

1,2-propanediol (1,2-PDO) is a versatile chemical used in multiple manufacturing processes. To date, some engineered and non-engineered microbes, such as Escherichia coli, Lactobacillus buchneri, and Clostridium thermosaccharolyticum, have been used to produce 1,2-PDO. In this study, we demonstrated the production of R- and S-1,2-PDO using engineered Lactococcus lactis. The L- and D-lactic acid-producing L. lactis strains NZ9000 and AH1 were transformed with the plasmid pNZ8048-ppy harboring pct, pduP, and yahK genes for 1,2-PDO biosynthesis, resulting in L. lactis LL1 and LL2, respectively. These engineered L. lactis produced S- and R-1,2-PDO at concentrations of 0.69 and 0.50 g/L with 94.4 and 78.0% ee optical purities, respectively, from 1% glucose after 72 h of cultivation. Both 1% mannitol and 1% gluconate were added instead of glucose to the culture of L. lactis LL1 to supply NADH and NADPH to the 1,2-PDO production pathway, resulting in 75% enhancement of S-1,2-PDO production. Production of S-1,2-PDO from 5% mannitol and 5% gluconate was demonstrated using L. lactis LL1 with a pH-stat approach. This resulted in S-1,2-PDO production at a concentration of 1.88 g/L after 96 h of cultivation. To our knowledge, this is the first report on the production of R- and S-1,2-PDO using engineered lactic acid bacteria.

Microbial fuel cells using α-amylase-displaying Escherichia coli with starch as fuel.

Escherichia coli JM109 (pGV3-SBA) can assimilate starch by fusing the starch-digesting enzyme α-amylase from Streptococcus bovis NRIC1535 to an OprI' lipoprotein anchor on the cell membrane. This study shows microbial fuel cells (MFCs) development using this recombinant type of E. coli and starch as fuel. We observed the current generation of MFCs with E. coli JM109 (pGV3-SBA) for 120 h. During this period, it consumed 7.1 g/L of starch. A mediator in the form of anthraquinone-2,6-disulfonic acid disodium salt at 0.2, 0.4, and 0.8 mM was added to the MFCs. The highest maximum-current density (271 mA/m2) and maximum-power density (29.3 mW/m2) performances occurred in the 0.4 mM mediator solution. Coulomb yields were calculated as 3.4%, 3.0%, and 3.5% in 1.0, 5.0, and 10.0 g/L of initial starch, respectively. The concentrations of acetic acid, succinic acid, fumaric acid, and ethanol as metabolites were determined. In particular, 38.3 mM of ethanol was produced from 7.1 g/L of starch. This study suggests the use of recombinant E. coli which can assimilate starch present in starch-fueled MFCs. Moreover, it proposes the possibility of gene recombination technology for using wide variety of biomass as fuel and improving MFC's performance.

Engineering Escherichia coli for Direct Production of 1,2-Propanediol and 1,3-Propanediol from Starch.

Diols are versatile chemicals used for multiple manufacturing products. In some previous studies, Escherichia coli has been engineered to produce 1,2-propanediol (1,2-PDO) and 1,3-propanediol (1,3-PDO) from glucose. However, there are no reports on the direct production of these diols from starch instead of glucose as a substrate. In this study, we directly produced 1,2-PDO and 1,3-PDO from starch using E. coli engineered for expressing a heterologous α-amylase, along with the expression of 1,2-PDO and 1,3-PDO synthetic genes. For this, the recombinant plasmids, pVUB3-SBA harboring amyA gene for α-amylase production, pSR5 harboring pct, pduP, and yahK genes for 1,2-PDO production, and pSR8 harboring gpd1-gpp2, dhaB123, gdrAB, and dhaT genes for 1,3-PDO production, were constructed. Subsequently, E. coli BW25113 (ΔpflA) and BW25113 strains were transformed with pVUB3-SBA, pSR5, and/or pSR8. Using these transformants, direct production of 1,2-PDO and 1,3-PDO from starch was demonstrated under microaerobic condition. As a result, the maximum production titers of 1,2-PDO and 1,3-PDO from 1% glucose as a sole carbon source were 13 mg/L and 150 mg/L, respectively. The maximum production titers from 1% starch were similar levels (30 mg/L 1,2-PDO and 120 mg/L 1,3-PDO). These data indicate that starch can be an alternative carbon source for the production of 1,2-PDO and 1,3-PDO in engineered E. coli. This technology could simplify the upstream process of diol bioproduction.

Itaconic acid derivatives: structure, function, biosynthesis, and perspectives.

Itaconic acid possessing a vinylidene group, which is mainly produced by fungi, is used as a biobased platform chemical and shows distinctive bioactivities. On the other hand, some fungi and lichens produce itaconic acid derivatives possessing itaconic acid skeleton, and the number of the derivatives is currently more than seventy. Based on the molecular structures, they can be categorized into two groups, alkylitaconic acids and α-methylene-γ-butyrolactones. Interestingly, some itaconic acid derivatives show versatile functions such as antimicrobial, anti-inflammatory, antitumor, and plant growth-regulating activities. The vinylidene group of itaconic acid derivatives likely participates in these functions. It is suggested that α-methylene-γ-butyrolactones are biosynthesized from alkylitaconic acids which are first biosynthesized from acyl-CoA and oxaloacetic acid. Some modifying enzymes such as hydroxylase and dehydratase are likely involved in the further modification after biosynthesis of their precursors. This contributes to the diversity of itaconic acid derivatives. In this review, we summarize their structures, functions, and biosynthetic pathways together with a discussion of a strategy for the industrial use. KEY POINTS: • Itaconic acid derivatives can be categorized into alkylitaconic acids and α-methylene-γ-butyrolactones. • The vinylidene group of itaconic acid derivatives likely participates in their versatile function. • It is suggested that α-methylene-γ-butyrolactones are biosynthesized from alkylitaconic acids which are first synthesized from acyl-CoA and oxaloacetic acid.

One-pot chemoenzymatic synthesis of glycopolymers from unprotected sugars via glycosidase-catalysed glycosylation using triazinyl glycosides.

Glycopolymers were successfully synthesised from unprotected sugars in aqueous media via a one-pot chemoenzymatic process of three reactions; the direct synthesis of 4,6-dimethoxy-1,3,5-triazin-2-yl glycosides from unprotected sugars, a glycosidase-catalysed glycosylation using the triazinyl glycoside to afford glycomonomers and a radical polymerisation. The resulting glycopolymers exhibited specific interactions with the corresponding lectin as glycoclusters.

Correction: Sano, M., et al. Microbial Screening Based on the Mizoroki-Heck Reaction Permits Exploration of Hydroxyhexylitaconic-Acid-Producing Fungi in Soils. Microorganisms 2020, 8, 648.

The authors wish to make the following correction to this paper [...].

Biobased Poly(itaconic Acid-co-10-Hydroxyhexylitaconic Acid)s: Synthesis and Thermal Characterization.

Renewable vinyl compounds itaconic acid (IA) and its derivative 10-hydroxyhexylitaconic acid (10-HHIA) are naturally produced by fungi from biomass. This provides the opportunity to develop new biobased polyvinyls from IA and 10-HHIA monomers. In this study, we copolymerized these monomers at different ratios through free radical aqueous polymerization with potassium peroxodisulfate as an initiator, resulting in poly(IA-co-10-HHIA)s with different monomer compositions. We characterized the thermal properties of the polymers by thermogravimetric analysis (TGA) and Fourier-transform infrared spectroscopy (FT-IR). The nuclear magnetic resonance analysis and the gel permeation chromatography showed that the polymerization conversion, yield, and the molecular weights (weight-averaged Mw and number-averaged Mn) of the synthesized poly(IA-co-10-HHIA)s decreased with increasing 10-HHIA content. It is suggested that the hydroxyhexyl group of 10-HHIA inhibited the polymerization. The TGA results indicated that the poly(IA-co-10-HHIA)s continuously decomposed as temperature increased. The FT-IR analysis suggested that the formation of the hydrogen bonds between the carboxyl groups of IA and 10-HHIA in the polymer chains was promoted by heating and consequently the polymer dehydration occurred. To the best of our knowledge, this is the first time that biobased polyvinyls were synthesized using naturally occurring IA derivatives.

Microbial Screening Based on the Mizoroki-Heck Reaction Permits Exploration of Hydroxyhexylitaconic-Acid-Producing Fungi in Soils.

Recently, we developed a unique microbial screening method based on the Mizoroki-Heck reaction for itaconic acid (IA)-producing fungi. This method revealed that 37 out of 240 fungal strains isolated from soils produce vinyl compounds, including IA. In this study, we further characterized these compounds in order to verify that the screening method permits the isolation of fungi that produce other vinyl compounds, excluding IA. HPLC analysis showed that 11 out of 37 isolated strains produced IA, similar to Aspergillus terreus S12-1. Surprisingly, the other 8 isolated strains produced two vinyl compounds with HPLC retention times different from that of IA. From these strains, the vinyl compounds of Aspergillus niger S17-5 were characterized. Mass spectrometric and NMR analyses showed that they were identical to 8-hydroxyhexylitaconic acid (8-HHIA) and 9-HHIA. This finding showed that 8-HHIA- and 9-HHIA-producing fungi, as well as IA-producing fungi, are ubiquitously found in soils. Neither 8-HHIA nor 9-HHIA showed antibacterial or anti-inflammatory activities. Interestingly, 8-HHIA and 9-HHIA showed cytotoxicity against the human cervical cancer cell line (HeLa) and human diploid cell line (MRC-5), and MRC-5 only, respectively, compared to IA at the same concentration. This study indicates that the screening method could easily discover fungi producing 8-HHIA and 9-HHIA in soils.

Aqueous One-pot Synthesis of Glycopolymers by Glycosidase-catalyzed Glycomonomer Synthesis Using 4,6-Dimetoxy Triazinyl Glycoside Followed by Radical Polymerization.

Glycopolymers have attracted increased attention as functional polymeric materials, and simple methods for synthesizing glycopolymers remain needed. This paper reports the aqueous one-pot and chemoenzymatic synthesis of four types of glycopolymers via two reactions: the ß-galactosidase-catalyzed glycomonomer synthesis using 4,6-dimetoxy triazinyl ß-D-galactopyranoside and hydroxy group-containing (meth)acrylamide and (meth)acrylate derivatives as the activated glycosyl donor substrate and as the glycomonomer precursors, respectively, followed by radical copolymerization of the resulting glycomonomer and excess glycomonomer precursor without isolating the glycomonomers. The resulting glycopolymers bearing galactose moieties exhibited specific and strong interactions with the lectin peanut agglutinin as glycoclusters.

DISCOVER: A facile structure-based screening method for vinyl compound producing microbes.

Here we report a novel structure-based microbial screening method for vinyl compound discovery, DISCOVER (direct screening method based on coupling reactions for vinyl compound producers). Through a two-step screening procedure based on selective coupling reactions of terminal alkenes, the thiol-ene reaction (1st step of screening) and Mizoroki-Heck reaction, followed by iodine test (2nd step of screening), microbes producing vinyl compounds like itaconic acid (IA) can be isolated from soil samples. In the 1st step of screening, soil sources are plated on agar medium supplemented with an antimicrobial agent, α-thioglycerol (TG), and a radical initiator, VA-044 (VA). In the 2nd step of screening, vinyl compounds produced in the cultures are labelled with iodobenzene via the Mizoroki-Heck reaction, followed by an iodine test, leading to the detection and characterisation of labelled products. We evaluated the validity of DISCOVER using IA and its producer Aspergillus terreus. Experimental data supported our hypothesis that IA reacts with TG in the medium via the thiol-ene reaction and consequently, A. terreus rapidly forms colonies on the agar medium because of the loss of the antimicrobial activity of TG. Using DISCOVER, high throughput and selective isolation of A. terreus strains producing IA was possible from soils.

A high-throughput screening method based on the Mizoroki-Heck reaction for isolating itaconic acid-producing fungi from soils.

In this study, we report a novel method based on the Mizoroki-Heck reaction followed by an iodine test for the screening of itaconic acid-producing fungi from soils. This method is simple, rapid, and requires 10 µL of culture; results are obtained within 1.5 h. The detection limit of itaconic acid in the cultures was 0.13 mM. This is the first report on the direct screening of itaconic acid-producing fungi using a coupling reaction.

Engineering Lactococcus lactis for D-Lactic Acid Production from Starch.

Bioprocess development is a current requirement to enhance the global production of D-lactic acid. Herein, we report a new bioprocess for D-lactic acid production directly from starch using engineered Lactococcus lactis NZ9000. To modify L. lactis as a D-lactic acid producer, its major endogenous L-lactate dehydrogenase (L-Ldh) gene was replaced with a heterologous D-Ldh gene from Lactobacillus delbrueckii subsp. lactis JCM 1107. The resulting strain AH1 showed a somewhat slower growth rate but similar lactic acid production compared to those of the intact strain when cultivated with glucose as a carbon source. The chemical purity of D-lactic acid produced by L. lactis AH1 was 93.8%, and the enzymatic activities of D- and L-Ldh in AH1 were 1.54 U/mL and 0.05 U/mL, respectively. Next, a heterologous α-amylase gene from Streptococcus bovis NRIC 1535 cloned into an expression vector pNZ8048 was introduced into AH1. The resulting strain AH2 showed an amylolytic activity of 0.26 U/mL in the culture supernatant. Direct production of D-lactic acid from starch as the carbon source was demonstrated using L. lactis AH2, resulting in D-lactic acid production at a concentration of 15.0 g/L after 24 h cultivation. To our knowledge, this is the first report on D-lactic acid production in engineered L. lactis.

Direct electron transfer of Cellulomonas fimi and microbial fuel cells fueled by cellulose.

The strain of Cellulomonas fimi NBRC 15513 can generate electricity with cellulose as fuel without mediator using a single chamber type microbial fuel cell (MFC) which had 100 mL of chamber and 50 cm2 of the air cathode. The MFCs were operated over five days and showed the maximum current density of 10.0 ± 1.8 mA/m2, the maximum power density of 0.74 ± 0.07 mW/m2 and the ohmic resistance of 6.9 kΩ. According to the results of cyclic voltammetry, the appearance of the oxidation or reduction peak was not observed from the cell removed solution. The fact is that C. fimi does not secrete mediator-like compounds, while the oxidation peak was observed at +0.68 V from the phosphate buffer containing the washed cell. The peak appearance was caused by the electron transfer activity of which corresponds to cytochrome c, and disappeared after adding antimycin A which inhibits the electron transfer activity. The cell was alive throughout the experiment as the result of a colony forming unit on Luria-Bertani agar plates. This was thought that cytochrome c was on the membrane surface of the living cell and played a role in the direct electron transfer between the cells and anode.

Continuous production of d-lactic acid from cellobiose in cell recycle fermentation using ß-glucosidase-displaying Escherichia coli.

The present study demonstrates continuous production of d-lactic acid from cellobiose in a cell recycle fermentation with a hollow fiber membrane using recombinant Escherichia coli constructed by deleting its pyruvate formate-lyase activating enzyme gene pflA and expressing a heterologous ß-glucosidase on its cell surface. The ß-glucosidase gene bglC from Thermobifida fusca YX was cloned into a cell surface display vector pGV3, resulting in pGV3-bglC. Recombinant E. coli JM109 harboring the pGV3-bglC showed ß-glucosidase activity (18.9 ± 5.7 U/OD600), indicating the cell surface functioning of mutant ß-glucosidase. pH-stat cultivation using d-lactic acid producer E. coli BW25113 (ΔpflA) harboring pGV3-bglC in minimum medium with 10 g/L cellobiose in a jar fermentor under anaerobic condition resulted in 5.2 ± 0.1 g/L of d-lactic acid was obtained after 84 h cultivation, indicating that the engineered E. coli produced d-lactic acid directly from cellobiose. For continuous d-lactic acid production, cell recycle fermentation was conducted under anaerobic condition and the culture was continuously ultrafiltrated with a hollow fiber cartridge. The permeate was drawn to the reservoir and a minimum medium containing 10 g/L cellobiose was fed to the fermentor at the same rate (dilution rate, 0.05 h-1). Thus, this system maintained the d-lactic acid production (4.3-5.0 g/L), d-lactic acid production rate (0.22-0.25 g/L/h), and showed no residual cellobiose in the culture during 72 h operation. Interestingly, the d-lactic acid production rate in cell recycle fermentation was more than 3 times higher than that in the batch operation (0.06 ± 0.00 g/L/h).

Comparison of cellulose nanofiber properties produced from different parts of the oil palm tree.

Cellulose nanofibers (CNFs) were obtained from three types of oil palm wastes, mesocarp, empty fruit bunch (EFB), and palm kernel shell (PKS), as well as the trunk of the oil palm tree, to compare their morphological, thermal, and mechanical properties. Despite large differences in the chemical components of cell walls in the raw materials, the production of CNFs from all parts of the oil palm were achieved in this work. The morphology and mechanical properties of the CNF sheets obtained from the trunk had advantages over the CNF sheets from wastes, while the thermal degradation properties showed no advantage. Cellulose crystallinity of the CNF sheet from the mesocarp and PKS had lower crystallinity (69.1 and 71.1%), and the highest crystallinity of 77.0% was exhibited by the sheet from the trunk. The value of specific tensile strength and specific Young's modulus were highest in the CNF sheet of the trunk, and lowest mechanical properties shown in the CNF sheet from the mesocarp. These results strongly suggested that the CNF could be obtained from all parts of the plants, but their properties may vary.

Microbial fuel cells equipped with an iron-plated carbon-felt anode and Shewanella oneidensis MR-1 with corn steep liquor as a fuel.

A single chamber type microbial fuel cell (MFC) with 100 mL of chamber volume and 50 cm2 of air-cathode was developed in this study wherein a developed iron-plated carbon-felt anode and Shewanella oneidensis MR-1 were used. The performance of the iron-plated carbon-felt anode and the possibility of corn steep liquor (CSL) as a fuel, which was the byproduct of corn wet milling and contained lactic acid, was investigated here. MFCs equipped with iron-plated or non-plated carbon-felt anodes exhibited maximum current densities of 443 or 302 mA/m2 using 10 g/L of reagent-grade lactic acid, respectively. In addition, using centrifuged CSL without insoluble ingredients or non-centrifuged CSL as a fuel, the maximum current densities of the MFCs with iron-plated carbon-felt anode were 321 or 158 mA/m2, respectively. This report demonstrated the effect of iron-plated carbon-felt anode for electricity generation of MFC using S. oneidensis MR-1 and the performance of CSL as a fuel.

Synthesis and Characterization of Alkali Metal Ion-Binding Copolymers Bearing Dibenzo-24-crown-8 Ether Moieties.

Dibenzo-24-crown-8 (DB24C8)-bearing copolymers were synthesized by radical copolymerization using a DB24C8-carrying acrylamide derivative and N-isopropylacrylamide monomers. The cloud point of the resulting copolymers changed in aqueous solution in the presence of cesium ions. In addition, the ¹H NMR signals of DB24C8-bearing copolymers shifted in the presence of alkali metal. This shift was more pronounced following the addition of Cs⁺ compared to Rb⁺, K⁺, Na⁺, and Li⁺ ions due to recognition of the Cs⁺ ion by DB24C8.