Long-Term Culturing of Placozoans (Trichoplax and Hoilungia).

The phylum Placozoa remains one of the least explored among early-branching metazoan lineages. For over 130 years, this phylum had been represented by the single species Trichoplax adhaerens-an animal with the simplest known body plan (three cell layers without any organs) but complex behaviors. Recently, extensive sampling of placozoans across the globe and their subsequent genetic analysis have revealed incredible biodiversity with numerous cryptic species worldwide. However, only a few culture protocols are available to date, and all are for one species only. Here, we describe the breeding of four different species representing two placozoan genera: Trichoplax adhaerens, Trichoplax sp. H2, Hoilungia sp. H4, and Hoilungia hongkongensis originating from diverse biotopes. Our protocols allow to culture all species under comparable conditions. Next, we outlined various food sources and optimized strain-specific parameters enabling long-term culturing. These protocols can facilitate comparative analyses of placozoan biology and behaviors, which together will contribute to deciphering general principles of animal organization.

Complete genome sequence of a species of the genus Agarivorans from the Sea of Japan.

A strain of Agarivorans sp., named OAG1, predicted to be capable of degrading agar, was isolated from the Sea of Japan. Its 4.7 Mbp genome with 4,224 predicted protein-coding genes offers insight into the varied genes responsible for agar degradation, as observed in the comparative genomic study of Agarivorans species.

Investigation of treatment continuity, usefulness, and nocebo effect in switching from the original etanercept to its biosimilar in patients with rheumatoid arthritis: A JET observational study in Japanese clinical practice.

OBJECTIVES: To assess the usefulness and onset of nocebo effects after switching from the original etanercept (ETN) to a biosimilar (BS) in routine clinical practice at rheumatology clinics in Japan (13 sites). METHODS: A total of 165 patients (87.0% women, age = 57.88 ± 15.07 years, and disease duration = 10.32 ± 7.71 years), whose low disease activity was maintained with the original ETN for ≥12 weeks, and who agreed to switch treatment to its BS, were included. The end-points were disease activity score 28 (DAS28)-C-reactive protein and DAS28-erythrocyte sedimentation rate. RESULTS: No significant difference was observed between the changes in DAS28-C-reactive protein and DAS28-erythrocyte sedimentation rate >12 weeks before switching and >12 weeks after switching (P = 0.132 and 0.334, respectively). The treatment continuation rate during the 52 weeks after switching to BS was 97.3%. During this period, BS was discontinued in only four patients, and no nocebo effects were suspected in these four patients. CONCLUSION: Switching from ETN to BS was effective even in routine clinical practice at rheumatology clinics in Japan, and no nocebo effects were observed. Sufficient explanations to patients by rheumatologists and the additional payment for drug costs between patients at hospital visits effectively improved the continuation rate without any nocebo effect.

Molecular basis of the unique osmoregulatory strategy in the inshore hagfish, Eptatretus burgeri.

Hagfishes are characterized by omo- and iono-conforming nature similar to marine invertebrates. Conventionally, hagfishes had been recognized as the most primitive living vertebrate that retains plesiomorphic features. However, some of the "ancestral" features of hagfishes, such as rudimentary eyes and the lack of vertebrae, have been proven to be deceptive. Similarly, by the principle of maximum parsimony, the unique body fluid regulatory strategy of hagfishes seems to be apomorphic, since the lamprey, another cyclostome, adopts osmo- and iono-regulatory mechanisms as in jawed vertebrates. Although hagfishes are unequivocally important upon discussing the origin and evolution of the vertebrate osmoregulatory system, the molecular basis for the body fluid homeostasis in hagfishes has been poorly understood. In the present study, we explored this matter in the inshore hagfish, Eptatretus burgeri, by analyzing the transcriptomes obtained from the gill, kidney and muscle of the animals acclimated to distinct environmental salinities. Together with the measurement of parameters in the muscular fluid compartment, our data indicate that the hagfish possesses an ability to conduct free amino acids (FAAs)-based osmoregulation at a cellular level, which is in coordination with the renal and branchial FAA absorption. We also revealed that the hagfish does possess the orthologs of the known osmoregulatory genes, and that the transepithelial movement of inorganic ions in the hagfish gill and kidney is more complex than previously thought. These observations pose a challenge to the conventional view that the physiological features of hagfishes have been inherited from the last common ancestor of the extant vertebrates.

The GPR84 molecule is a mediator of a subpopulation of retinal microglia that promote TNF/IL-1α expression via the rho-ROCK pathway after optic nerve injury.

Resident microglia are important to maintain homeostasis in the central nervous system, which includes the retina. The retinal microglia become activated in numerous pathological conditions, but the molecular signatures of these changes are poorly understood. Here, using an approach based on FACS and RNA-seq, we show that microglial gene expression patterns gradually change during RGC degeneration induced by optic nerve injury. Most importantly, we found that the microglial cells strongly expressed Tnf and Il1α, both of which are known to induce neurotoxic reactive astrocytes, and were characterized by Gpr84high -expressing cells in a particular subpopulation. Moreover, ripasudil, a Rho kinase inhibitor, significantly blunted Gpr84 expression and cytokine induction in vitro and in vivo. Finally, GPR84-deficient mice prevented RGC loss in optic nerve-injured retina. These results reveal that Rho kinase-mediated GPR84 alteration strongly contribute to microglial activation and promote neurotoxicity, suggesting that Rho-ROCK and GPR84 signaling may be potential therapeutic targets to prevent the neurotoxic microglial phenotype induced by optic nerve damage, such as occurs in traumatic optic neuropathy and glaucoma.

DPP-4 Inhibitors Attenuate Fibrosis After Glaucoma Filtering Surgery by Suppressing the TGF-ß/Smad Signaling Pathway.

Purpose: This study investigated the effect of dipeptidyl peptidase-4 inhibitors (DPP-4is) on fibrosis after glaucoma filtering surgery with clinical data and an in vitro model that used transforming growth factor-ß (TGF-ß) to induce human Tenon's fibroblast (HTF) fibrosis. Methods: The medical records of 41 eyes of 35 patients with diabetes with neovascular glaucoma (NVG) who received initial trabeculectomy were retrospectively reviewed. The surgical success rate was compared between cases that received (n = 23) and did not receive (n = 18) DPP-4i treatment for diabetes. The antifibrotic effects of linagliptin (a DPP-4i) were evaluated with quantitative real-time PCR for fibrosis markers (α-smooth muscle actin, collagen Iα, and fibronectin), a scratch assay, and a collagen gel contraction assay of primary cultured HTFs treated with TGF-ß1 and linagliptin. Western blotting analysis was performed to evaluate the levels of phosphorylated Smad2 and Smad3 in the presence of linagliptin. Results: The Kaplan-Meier curve for bleb survival was higher in patients who received DPP-4is (P = 0.017, log-rank test). The in vitro experiments demonstrated that treatment with linagliptin attenuated the elevated levels of fibrosis markers induced by TGF-ß1 in HTFs. Linagliptin treatment also prevented the migration and gel contraction of HTFs. Linagliptin inhibited the phosphorylation of Smad2 and Smad3, which is the canonical pathway of TGF-ß signaling. Conclusions: The current study indicates the potential effect of DPP-4is for maintaining bleb function after glaucoma filtering surgery in patients with diabetes with NVG. Our results demonstrate that linagliptin attenuates fibrotic change in HTFs by inhibiting TGF-ß/Smad signaling.

Deep ocean water alters the cholesterol and mineral metabolism of squid Todarodes pacificus and suppresses its weight loss.

This study is the first to demonstrate that deep ocean water (DOW) has physiological significant effects on squid. After 36 h of rearing squids, those reared with DOW had significantly higher total and free cholesterol levels and lower alanine transaminase activity in hemolymph as compared with those reared with surface sea water (SSW). SSW rearing also resulted in 6.95% weight loss, while DOW rearing caused only 2.5% weight loss, which might be due to liver metabolism suppression. Furthermore, both monovalent (sodium, chloride, and potassium ions) and divalent (calcium, inorganic phosphorus, and magnesium ions) ions in hemolymph were elevated when reared with DOW compared to those when reared with SSW. A study of genes expressed in the brain revealed that five genes were specifically remarked in DOW rearing. Most altered genes were neuropeptides, including those from vasopressin superfamily. These neuropeptides are involved in cholesterol and/or mineral metabolisms and physiological significant effects on squid. This study is the first report the effects of DOW on cholesterol and mineral metabolism of squid and will contribute to squid aquaculture using DOW.

Kynurenine promotes Calcitonin secretion and reduces cortisol in the Japanese flounder Paralichthys olivaceus.

Deep ocean water (DOW) exerts positive effects on the growth of marine organisms, suggesting the presence of unknown component(s) that facilitate their aquaculture. We observed that DOW suppressed plasma cortisol (i.e., a stress marker) concentration in Japanese flounder (Paralichthys olivaceus) reared under high-density condition. RNA-sequencing analysis of flounder brains showed that when compared to surface seawater (SSW)-reared fish, DOW-reared fish had lower expression of hypothalamic (i.e., corticotropin-releasing hormone) and pituitary (i.e., proopiomelanocortin, including adrenocorticotropic hormone) hormone-encoding genes. Moreover, DOW-mediated regulation of gene expression was linked to decreased blood cortisol concentration in DOW-reared fish. Our results indicate that DOW activated osteoblasts in fish scales and facilitated the production of Calcitonin, a hypocalcemic hormone that acts as an analgesic. We then provide evidence that the Calcitonin produced is involved in the regulatory network of genes controlling cortisol secretion. In addition, the indole component kynurenine was identified as the component responsible for osteoblast activation in DOW. Furthermore, kynurenine increased plasma Calcitonin concentrations in flounders reared under high-density condition, while it decreased plasma cortisol concentration. Taken together, we propose that kynurenine in DOW exerts a cortisol-reducing effect in flounders by facilitating Calcitonin production by osteoblasts in the scales.

Incidence of Mid-Term Prognostic Events in Patients With Acute Coronary Syndrome During the Late 2010s in 2 Tertiary Hospitals in a Rural Area of Japan　- A Temporal Comparison.

Background: Data on the incidence of mid-term prognostic events in patients who developed acute coronary syndrome (ACS) in the late 2010s are scarce. Methods and Results: We retrospectively included and collected data for 889 patients with ACS (ST-elevation myocardial infarction [STEMI]/non-ST-elevation ACS [NSTE-ACS]) discharged alive from 2 tertiary hospitals in Izumo City, in rural Japan, between August 2009 and July 2018. Patients were divided into 3 time groups (T1: August 2009-July 2012; T2: August 2012-July 2015; T3: August 2015-July 2018). The cumulative incidence of major adverse cardiovascular events (MACE; comprising all-cause death, recurrent ACS, and stroke), major bleeding, and heart failure hospitalization within 2 years of discharge was compared among the 3 groups. The incidence of freedom from MACE was significantly higher in the T3 group than in the T1 and T2 groups (93 [95% confidence interval {CI} 90-96%] vs. 86% [95% CI 83-90] and 89% [95% CI 90-96], respectively; P=0.03). There was a tendency for a higher incidence of STEMI among patients in T3 (P=0.057). The incidence of NSTE-ACS was comparable among the 3 groups (P=0.31), as was the incidence of major bleeding and hospitalization for heart failure. Conclusions: The incidence of mid-term MACE in patients who developed ACS during the late 2010 s (2015-2018) was lower than that in prior periods (2009-2015).

Long-Term Surgical Outcomes and Possible Postoperative Complication with Severe Corneal Endothelial Cell Loss After Trabeculectomy for Cytomegalovirus-Associated Anterior Uveitis with Secondary Glaucoma.

PURPOSE: We assess long-term surgical outcomes after an initial trabeculectomy for cytomegalovirus-associated anterior uveitis with secondary glaucoma (CMV-SG). METHODS: We retrospectively reviewed the medical records of 16 eyes of 15 patients with CMV-SG and 157 eyes of 157 patients with primary open-angle glaucoma. The average follow-up period was approximately 3 years. Surgical success was defined as intraocular pressure (IOP) below 18 mmHg and at least 20% lower than baseline. RESULTS: Kaplan-Meier survival analysis revealed that bleb survival rates were not significantly different in the CMV-SG and POAG groups (P = 0.75). Bullous keratopathy occurred in 2 of 16 eyes with CMV-SG postoperatively but did not occur in the POAG group. The corneal endothelial cell density decreased by 34.2 ± 22.7% in the CMV-SG group during an average follow-up period of 2.7 ± 2.0 years. CONCLUSION: Trabeculectomy effectively controlled IOP in CMV-SG, but attention must be paid to corneal endothelial cell loss.

Phylogenetic and functional properties of hagfish neurohypophysial hormone receptors distinct from their jawed vertebrate counterparts.

Vertebrate neurohypophysial hormones, i.e., vasopressin- and oxytocin-family peptides, exert versatile physiological actions via distinct G protein-coupled receptors. The neurohypophysial hormone receptor (NHR) family was classically categorized into four subtypes (V1aR, V1bR, V2R and OTR), while recent studies have identified seven subtypes (V1aR, V1bR, V2aR, V2bR, V2cR, V2dR and OTR; V2aR corresponds to the conventional V2R). The vertebrate NHR family were diversified via multiple gene duplication events at different scales. Despite intensive research effort in non-osteichthyes vertebrates such as cartilaginous fish and lamprey, the molecular phylogeny of the NHR family has not been fully understood. In the present study, we focused on the inshore hagfish (Eptatretus burgeri), another group of cyclostomes, and Arctic lamprey (Lethenteron camtschaticum) for comparison. Two putative NHR homologs, which were previously identified only in silico, were cloned from the hagfish and designated as ebV1R and ebV2R. In vitro, ebV1R, as well as two out of five Arctic lamprey NHRs, increased intracellular Ca2+ in response to exogenous neurohypophysial hormones. None of the examined cyclostome NHRs altered intracellular cAMP levels. Transcripts of ebV1R were detected in multiple tissues including the brain and gill, with intense hybridization signals in the hypothalamus and adenohypophysis, while ebV2R was predominantly expressed in the systemic heart. Similarly, Arctic lamprey NHRs showed distinct expression patterns, underscoring the multifunctionality of VT in the cyclostomes as in the gnathostomes. These results and exhaustive gene synteny comparisons provide new insights into the molecular and functional evolution of the neurohypophysial hormone system in vertebrates.

Feasibility of methotrexate discontinuation following tocilizumab and methotrexate combination therapy in patients with long-standing and advanced rheumatoid arthritis: a 3-year observational cohort study.

OBJECTIVES: Methotrexate (MTX) is associated with extensive side effects, including myelosuppression, interstitial pneumonia, and infection. It is, therefore, critical to establish whether its administration is required after achieving remission with tocilizumab (TCZ) and MTX combination therapy in patients with rheumatoid arthritis (RA). Therefore, the aim of this multicenter, observational, cohort study was to evaluate the feasibility of MTX discontinuation for the safety of these patients. METHODS: Patients with RA were administered TCZ, with or without MTX, for 3 years; those who received TCZ+MTX combination therapy were selected. After remission was achieved, MTX was discontinued without flare development in one group (discontinued [DISC] group, n = 33) and continued without flare development in another group (maintain [MAIN] group, n = 37). The clinical efficacy of TCZ+MTX therapy, patient background characteristics, and adverse events were compared between groups. RESULTS: The disease activity score in 28 joints-erythrocyte sedimentation rate (DAS28-ESR) at 3, 6, and 9 months was significantly lower in the DISC group (P < .05, P < .01, and P < .01, respectively). Further, the DAS28-ESR remission rate at 6 and 9 months and Boolean remission rate at 6 months were significantly higher in the DISC group (P < .01 for all). Disease duration was significantly longer in the DISC group (P < .05). Furthermore, the number of patients with stage 4 RA was significantly higher in the DISC group (P < .01). CONCLUSIONS: Once remission was achieved, MTX was discontinued in patients who responded favorably to TCZ+MTX therapy, despite the prolonged disease duration and stage progression.

High Current Density Oxygen Evolution in Carbonate Buffered Solution Achieved by Active Site Densification and Electrolyte Engineering.

High current density reaching 1 A cm-2 for efficient oxygen evolution reaction (OER) was demonstrated by interactively optimizing electrolyte and electrode at non-extreme pH levels. Careful electrolyte assessment revealed that the state-of-the-art nickel-iron oxide electrocatalyst in alkaline solution maintained its high OER performance with a small Tafel slope in K-carbonate solution at pH 10.5 at 353 K. The OER performance was improved when Cu or Au was introduced into the FeOx -modified nanostructured Ni electrode as the third element during the preparation of electrode by electrodeposition. The resultant OER achieved 1 A cm-2 at 1.53 V vs. reversible hydrogen electrode (RHE) stably for 90 h, comparable to those in extreme alkaline conditions. Constant Tafel slopes, apparent activation energy, and the same signatures from operando X-ray absorption spectroscopy among these samples suggested that this improvement seems solely correlated with enhanced electrochemical surface area caused by adding the third element.

Understanding the intermediates and carbon dioxide adsorption of potassium chloride-incorporated graphitic carbon nitride with tailoring melamine and urea as precursors.

In this study, we demonstrated the synthesis of potassium chloride (KCl)-incorporated graphitic carbon nitride, (g-C3N4, CN) with varying amounts of N-vacancies and pyridinic-N as well as enhanced Lewis basicity, via a single-step thermal polymerization by tailoring the precursors of melamine and urea for carbon oxide (CO2) capture. Melamine, as a precursor, undergoes a phase transformation into melam and triazine-rich g-C3N4, whereas the addition of urea polymerizes the mixture to form melem and heptazine-rich g-C3N4 (CN11). Owing to the abundance of pyridinic-N and the high surface area, CN11 adsorbed higher amounts of CO2 (44.52 µmol m-2 at 25 °C and 1 bar of CO2) than those reported for other template-free carbon materials. Spectroscopic analysis revealed that the enhanced CO2 adsorption is due to the presence of pyridinic-N and Lewis basic sites on the surface. The intermediates of CO2adsorption, including carbonate and bicarbonate species, attached to the CN samples were identified using in-situ Fourier-transform infrared spectroscopy (FTIR). This work provides insights into the mechanism of CO2 adsorption by comparing the structural features of the synthesized KCl-incorporated g-C3N4 samples. CN11, with an excellent CO2 uptake capacity, is viewed as a promising candidate for CO2 capture and storage.

Ripasudil Eyedrops Ameliorated Bullous Keratopathy Complicated with Cytomegalovirus Corneal Endotheliitis: A Case Report.

PURPOSE: We describe a case of bullous keratopathy complicated with cytomegalovirus (CMV) corneal endotheliitis that was successfully treated with ripasudil eye drops. METHODS: A retrospective case report. RESULTS: A 65-year-old female patient diagnosed with CMV-associated anterior uveitis in the right eye was referred to us when anterior uveitis recurred with bullous keratopathy. Initial best-corrected visual acuity (BCVA) was 0.4 (decimal visual acuity). Her condition did not improve with anti-CMV treatment, and BCVA decreased to 0.07. At this point, intraocular pressure (IOP) was 20 mmHg, and ripasudil eye drops were started for IOP control. After 1 month, not only had IOP decreased to 14 mm Hg but the condition of the corneal edema had also improved. The central corneal thickness decreased to a normal level, and the BCVA recovered to 0.8. CONCLUSION: Ripasudil eye drops not only lower IOP in patients with CMV corneal endotheliitis but may also have the potential to treat bullous keratopathy.

Gene Recruitments and Dismissals in the Argonaut Genome Provide Insights into Pelagic Lifestyle Adaptation and Shell-like Eggcase Reacquisition.

The paper nautilus or greater argonaut, Argonauta argo, is a species of octopods which is characterized by its pelagic lifestyle and by the presence of a protective spiral-shaped shell-like eggcase in females. To reveal the genomic background of how the species adapted to the pelagic lifestyle and acquired its shell-like eggcase, we sequenced the draft genome of the species. The genome size was 1.1 Gb, which is the smallest among the cephalopods known to date, with the top 215 scaffolds (average length 5,064,479 bp) covering 81% (1.09 Gb) of the total assembly. A total of 26,433 protein-coding genes were predicted from 16,802 assembled scaffolds. From these, we identified nearly intact HOX, Parahox, Wnt clusters, and some gene clusters that could probably be related to the pelagic lifestyle, such as reflectin, tyrosinase, and opsin. The gene models also revealed several homologous genes related to calcified shell formation in Conchiferan mollusks, such as Pif-like, SOD, and TRX. Interestingly, comparative genomics analysis revealed that the homologous genes for such genes were also found in the genome of the shell-less octopus, as well as Nautilus, which has a true outer shell. Therefore, the draft genome sequence of Arg. argo presented here has helped us to gain further insights into the genetic background of the dynamic recruitment and dismissal of genes to form an important, converging extended phenotypic structure such as the shell and the shell-like eggcase. Additionally, it allows us to explore the evolution of from benthic to pelagic lifestyles in cephalopods and octopods.

A Rare Case of Fungal Keratitis Caused by Plectosphaerella cucumerina Diagnosed With Repeated Corneal Scrapings: A Case Report.

Plectosphaerella cucumerina is a filamentous fungus that infects plants and crops, but there are few previous reports of human infections. The current case was an 82-year-old woman who was referred to us for corneal infection in her left eye that did not improve with antibacterial and anti-inflammatory treatment. The best-corrected visual acuity (BCVA) of the eye at the first visit to us was hand motion. Slit-lamp examination revealed extensive white infiltration and ulceration in the anterior corneal stroma of the left eye. Intensive antibacterial and antiviral treatment for one month did not improve the condition of the cornea. Although initial culture testing and polymerase chain reaction (PCR) testing of corneal scraping samples did not reveal the causative microorganism, repeated culture testing identified P. cucumerina. The corneal infection eventually subsided after topical and systemic treatment with voriconazole (VRCZ). The final BCVA in the left eye was 1.3 logMAR. This was a rare case of fungal keratitis due to P. cucumerina. Our case suggests that it is important to perform repeated examinations with corneal scrapings, especially when the treatment response is poor.

Whole-Genome Sequencing of Pseudoalteromonas sp. Strain KAN5, an Agar-Degrading Bacterium Isolated from the Gut of an Alga-Eating Fish.

A strain of Pseudoalteromonas that degrades agar was isolated from the intestines of an alga-eating fish (Andamia tetradactyla). We named the strain KAN5 and report on the genome sequenced with the Oxford Nanopore Technologies platform. The 3.8-Mbp genome contains 3,428 protein-coding genes, and the genes involved in agar degradation were confirmed.

Ag-modified TiO2/SiO2/Fe3O4 sphere with core-shell structure for photo-assisted reduction of 4-nitrophenol.

Nitrogen-containing contaminants, such as 4-nitrophenol (4-NP), cause detrimental effects when discharged into the environment and thus should be reduced or removed from ecosystems. In this study, an Ag-loaded TiO2-SiO2-Fe3O4 (TSF) with a core-shell structure was employed for the photo-assisted reduction of 4-NP. Fe3O4, SiO2, and TiO2 in the core-shell structure served as a magnetic center, protective layer, and light absorber, respectively. To improve the reduction activity of 4-NP, Ag was loaded onto TSF under stirring, with a variation of the temperature (2-130 °C) and reaction time (1, 2, and 4 h). Under the optimized conditions, 5Ag-TSF (with 5 wt% of Ag) could promote the reduction of aqueous 4-NP solution (2 × 10-4 M, 75 mL) in the presence of NaBH4 (0.1 M, 5 mL) under irradiation by a metal halide lamp, affording over 98% reduction within 5 min and a rate constant of 0.185 min-1, demonstrating its promising activity. Moreover, due to the advantages of the core-shell structure, the magnetic properties of Fe3O4 were sufficient to enable facile recycling of the sample for further reaction; SiO2 could protect the Fe3O4 center from oxidation or reduction; TiO2 enabled Ag accommodation and absorbed light to generate electron-hole pairs. In summary, an Ag-loaded TiO2-SiO2-Fe3O4 sphere with high activity and recyclability for 4-NP reduction was prepared via a facile and simple stirring method, where the sample can be used as a promising material in environmental remediation.