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Corneal hysteresis (CH) is associated with glaucomatous structural changes. We retrospectively investigated the association between CH and the regional circumpapillary retinal nerve fibre layer thickness (cpRNFLT) in 419 eyes of 419 patients with normal-tension glaucoma (NTG) and primary open-angle glaucoma (POAG). CH was used as the explanatory variable, and cpRNFLT (total and quadrant) was used as the dependent variable. Standardized β coefficients were compared both overall and between the NTG and POAG groups. Multiple regression analysis with CH as the explanatory variable and cpRNFLT parameters as the dependent variables suggested that even after adjusting for age, sex, intraocular pressure (IOP), axial length, and central corneal thickness, all areas of the cpRNFLT were significantly associated with CH, except for the nasal quadrant. In the stratified analysis by glaucoma type, a positive relationship between CH and regional cpRNFLT tended to be maintained in the NTG group but not in the POAG group (P = 0.060 for interaction). Additionally, in the NTG group, the CH-inferior cpRNFLT relationship was particularly strong in the less-IOP fluctuation group. These results suggest that CH may be a potential predictor of anatomical vulnerability around the optic nerve, particularly enhancing the inferior regions of NTG., (© 2024. The Author(s).)

The formation of the topological surface state originates from bulk band inversion at the top of the valence band and the bottom of the conduction band. The transition between normal and topological insulators is known as a topological phase transition. Here we show spin-polarized electronic states of Pb-based ternary topological insulators Pb(Bi 1-x Sb x ) 2 Te 4 (x = 0.55, 0.70, 0.79) investigated by spin- and angle-resolved photoemission spectroscopy and first-principles calculations. We visualize a pair of spin-polarized surface resonances dispersing along the upper edge of projected bulk bands in occupied states. Interestingly, a branch of the spin-polarized surface resonances continuously connects to the topological surface state. The coexistence of the topological surface state and the spin-polarized surface resonances can be explained by considering the topological phase transition., (© 2024. The Author(s).)

The aim of this study was to investigate the effect of a ginger extract on optic nerve head blood flow (ONH BF) under endothelin-1 (ET-1) stimulation. Using laser speckle flowgraphy, we measured ONH BF in brown Norway rats. To establish the ONH BF impairment profile under ET-1 stimulation, we administered an intravitreal injection of ET-1 under anesthesia. We then gave the ginger extract sublingually to assess its effect on ONH BF in both normal and ET-1-induced ischemic conditions. Post ET-1 injection, there were no significant changes in parameters including intraocular pressure or systemic factors. ONH BF showed a dose-dependent decline after ET-1 injection, with a significant reduction after a 2.50 pmol ET-1 dose. Sublingual administration of the ginger extract significantly improved ONH BF in both normal and ET-1-stimulated rats. This suggests that our newly developed supplement for improving ONH BF has a potential role in retinal ischemic diseases, including glaucoma., (© 2023. The Author(s).)

We aimed to investigate the impact of glutathione trisulfide (GSSSG) on lipopolysaccharide (LPS)-induced inflammation in retinal glia. Inflammatory responses in mouse-derived glial cells and Wistar rat retinas were stimulated with administration of LPS. Cell survival and proinflammatory cytokine production were examined using the Calcein-AM assay, and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and enzyme-linked immunosorbent assay (ELISA), respectively. Retinal microglia were visualized with immunohistochemistry for Iba1. Administration of LPS (10 µg/mL) or GSSSG (less than 100 µM) did not affect survival of cultured primary Müller cells and established microglial cells (BV-2). RT-qPCR and ELISA indicated that GSSSG inhibited LPS-induced gene upregulation and protein secretion of proinflammatory cytokines in these glial cells and rat retinas. GSSSG inhibited LPS-induced activation of TGF-β-activated kinase 1 (TAK1), which is an upstream kinase of NF-κB, in BV-2 cells. Finally, in vivo experiments indicated that intravitreal administration of GSSSG but not its relative glutathione disulfide (GSSG) inhibited LPS (500 ng)-induced accumulation of Iba1-immunopositive microglia in rat retinas. Taken together, GSSSG has the potential to prevent pathogenesis of inflammation-associated ocular diseases by inhibiting proinflammatory cytokine expression in retinal glial cells., (© 2023. The Author(s).)

Anti-oxidative intraocular irrigating solutions (IISs) based on reactive persulfides, such as oxidized glutathione disulfide (GSSG), are commonly used worldwide. However, even with GSSG-based IISs, it has been shown that oxidative stress can occur during surgery, posing a risk to intraocular tissues. This study compared two IISs: one containing GSSG and one containing an oxidized glutathione trisulfide (GSSSG). Experimental in vivo irrigation with the IISs in rabbits showed that there was less leakage into the anterior chamber of rabbit serum albumin during perfusion with a 300-μM GSSSG IIS than with a 300-μM GSSG IIS. Experimental in vivo cataract surgery in rabbits showed that aqueous flare was suppressed 3 days after surgery with a 600-μM GSSSG IIS, but not with a 300-μM GSSSG or 300-μM GSSG IIS. Furthermore, an in vitro experiment, without any live tissue, showed that reactive oxygen species were suppressed more strongly with a 600-μM GSSSG IIS than with a 300-μM GSSG IIS. Thus, this study found that novel IISs based on GSSSG had anti-inflammatory and anti-oxidative effects during and after intraocular surgery and may decrease the rate of complications after surgery., (© 2022. The Author(s).)

Many psychrophilic microorganisms synthesize ice-binding proteins (IBPs) to survive the cold. The functions of IBPs are evaluated by the effect of the proteins on the nonequilibrium water freezing-point depression, which is called "thermal hysteresis (TH)", and the inhibitory effect of the proteins on the growth of larger ice crystals, which is called "ice recrystallization inhibition (IRI)". To obtain mechanical insight into the two activities, we developed a modified method of ice affinity purification and extracted two new IBP isoforms from Psychromyces glacialis, an Arctic glacier fungus. One isoform was found to be an approximately 25 kDa protein (PsgIBP_S), while the other is a 28 kDa larger protein (PsgIBP_L) that forms an intermolecular dimer. Their TH activities were less than 1 °C at millimolar concentrations, implying that both isoforms are moderately active but not hyperactive IBP species. It further appeared that both isoforms exhibit high IRI activity even at submicromolar concentrations. Furthermore, the isoforms can bind to the whole surface of a hemispherical single ice crystal, although such ice-binding was generally observed for hyperactive IBP species. These results suggest that the binding ability of IBPs to whole ice crystal surfaces is deficient for hyperactivity but is crucial for significant IRI activity., (© 2022. The Author(s).)

Non-proteinaceous components in membranes regulate membrane protein insertion cooperatively with proteinaceous translocons. An endogenous glycolipid in the Escherichia coli membrane called membrane protein integrase (MPIase) is one such component. Here, we focused on the Sec translocon-independent pathway and examined the mechanisms of MPIase-facilitated protein insertion using physicochemical techniques. We determined the membrane insertion efficiency of a small hydrophobic protein using solid-state nuclear magnetic resonance, which showed good agreement with that determined by the insertion assay using an in vitro translation system. The observed insertion efficiency was strongly correlated with membrane physicochemical properties measured using fluorescence techniques. Diacylglycerol, a trace component of E. coli membrane, reduced the acyl chain mobility in the core region and inhibited the insertion, whereas MPIase restored them. We observed the electrostatic intermolecular interactions between MPIase and the side chain of basic amino acids in the protein, suggesting that the negatively charged pyrophosphate of MPIase attracts the positively charged residues of a protein near the membrane surface, which triggers the insertion. Thus, this study demonstrated the ingenious approach of MPIase to support membrane insertion of proteins by using its unique molecular structure in various ways., (© 2022. The Author(s).)

AlGaN-based deep ultraviolet light-emitting diodes (LEDs) have a wide range of applications such as medical diagnostics, gas sensing, and water sterilization. Metal-organic vapor phase epitaxy (MOVPE) method is used for the growth of all-in-one structures, including doped layer and thin multilayers, using metal-organic and gas source raw materials for semiconductor devices. For AlN growth with high crystalline quality, high temperature is necessary to promote the surface migration of Al atoms and Al-free radicals. However, increase in temperature generates parasitic gas-phase prereactions such as adduct formation. In this work, AlN growth at 1500 °C by a stable vapor phase reaction has been achieved by jet stream gas flow metal-organic vapor phase epitaxy. The AlN growth rate increases with gas flow velocity and saturates at ~ 10 m/s at room temperature. Moreover, it is constant at an ammonia flow rate at a V/III ratio from 50 to 220. These results demonstrate the reduction in adduct formation, which is a typical issue with the vapor phase reaction between triethylaluminum and ammonia. The developed method provides the in-plane uniformity of AlN thickness within 5%, a low concentration of unintentionally doped impurities, smooth surface, and decrease in dislocation density because of the suppression of parasitic reactions., (© 2022. The Author(s).)

Antifreeze proteins (AFPs) inhibit ice growth by adsorbing onto specific ice planes. Microbial AFPs show diverse antifreeze activity and ice plane specificity, while sharing a common molecular scaffold. To probe the molecular mechanisms responsible for AFP activity, we here characterized the antifreeze activity and crystal structure of TisAFP7 from the snow mold fungus Typhula ishikariensis. TisAFP7 exhibited intermediate activity, with the ability to bind the basal plane, compared with a hyperactive isoform TisAFP8 and a moderately active isoform TisAFP6. Analysis of the TisAFP7 crystal structure revealed a bound-water network arranged in a zigzag pattern on the surface of the protein's ice-binding site (IBS). While the three AFP isoforms shared the water network pattern, the network on TisAFP7 IBS was not extensive, which was likely related to its intermediate activity. Analysis of the TisAFP7 crystal structure also revealed the presence of additional water molecules that form a ring-like network surrounding the hydrophobic side chain of a crucial IBS phenylalanine, which might be responsible for the increased adsorption of AFP molecule onto the basal plane. Based on these observations, we propose that the extended water network and hydrophobic hydration at IBS together determine the TisAFP activity.

Systemic antioxidative status has been implicated in glaucoma pathogenesis. Additionally, corneal hysteresis (CH) may contribute to glaucoma progression. Here, we evaluated the relationship between biological antioxidant potential (BAP) and CH. This study included 103 patients with open-angle glaucoma (OAG). We used a free radical analyzer to measure BAP, and an ocular response analyzer to measure CH and corneal resistance factor (CRF). We evaluated the relationship between systemic oxidative stress and other clinical parameters with Spearman's rank correlation test and a multi-regression analysis. BAP was not correlated to either CH or CRF in the male or female OAG patients. BAP was correlated to both CH and CRF in the female OAG patients older than 57 years (r = 0.51, P = 0.003; r = 0.49, P = 0.004), but uncorrelated in the female OAG patients younger than 57 years. Multiple regression analysis revealed that BAP independently contributed to CH (P = 0.025) and CRF (P = 0.015) in the older female OAG patients. Systemic oxidative stress may significantly affect the viscoelasticity of the cornea in older female OAG patients. Future studies are needed to confirm that low systemic antioxidative status and low corneal hysteresis contribute to glaucoma pathogenesis.

Treatments for refractory glaucoma include trabeculectomy, in which a filtering bleb is created to reduce aqueous pressure. Mitomycin C (MMC) is often used as an adjuvant to reduce post-trabeculectomy bleb scarring and consequent failure. However, scarring sometimes still occurs. Thus, we searched for more effective trabeculectomy adjuvants with high-throughput screening (HTS) of a library of 1,165 off-patent drug compounds. This revealed that amsacrine (AMSA), a DNA topoisomerase II (TOP2) inhibitor, was the top candidate. Compared to MMC, rabbits that underwent trabeculectomy with 10% AMSA had lower IOP at 42, 56, and 70 days (P < 0.01 at all measurement points) and a higher bleb score at 28, 42, 56, and 70 days (P =  < 0.01, 0.04, 0.04, and < 0.01, respectively). Compared to saline, rabbits that received 1% AMSA also had lower IOP and better bleb score at all time points, without a sharp drop in IOP just after surgery (all P < 0.01). Both effects were milder than MMC at 7 days (P = 0.02 and <0.01, respectively). Thus, this study showed that HTS may help identify new, promising uses for off-patent drugs. Furthermore, trabeculectomy with AMSA at a suitable concentration may improve the prognosis after trabeculectomy compared to MMC.

Pregnancy is a stress factor culminating into mild endoplasmic reticulum (ER) stress, which is necessary for placental development. However, excessive or chronic ER stress in pre-eclamptic placentas leads to placental dysfunction. The precise mechanisms through which excessive ER stress impacts trophoblasts are not well understood. Here, we showed that ER stress reduces the number of lysosomes, resulting in inhibition of autophagic flux in trophoblast cells. ER stress also disrupted the translocation of lysosomes to the surface of trophoblast cells, and inhibited lysosomal exocytosis, whereby the secretion of lysosomal-associated membrane protein 1 (LAMP1) into culture media was significantly attenuated. In addition, we found that serum LAMP1 and beta-galactosidase levels were significantly decreased in pre-eclampsia patients compared to normal pregnant women, potentially indicating lysosomal dysfunction through ER stress in pre-eclamptic placentas. Thus, we demonstrated that excessive ER stress essentially disrupts homeostasis in trophoblasts in conjunction with autophagy inhibition by lysosomal impairment.

Ice-binding proteins (IBPs) are capable of binding ice crystals and inhibiting their growth at freezing temperatures. IBPs are also thought to stabilize the cell membrane at non-freezing temperatures near 0 °C. These two effects have been assumed to reduce cold- and freezing-induced damage to cells and tissues. However, knowledge regarding the effects of IBP on the living animals is limited. Here, we characterized the relationship between the IBP effects and the physiological role by using the nematode Caenorhabditis elegans. The expression of fish (NfeIBPs)- and fungus-derived IBPs (AnpIBPs and TisIBP8) in C. elegans improved its survival rate during exposure to 0 and -2 °C (cold shock) and -5 °C (freezing). The observed cold tolerance of C. elegans after cold shock is attributable to the stabilization of cell-membrane lipids with IBPs, and the freezing tolerance at -5 °C can be attributed to the inhibition of ice-crystal growth by the IBPs. Significantly, the survival rate of C. elegans at -5 °C was improved by expression of wild-type AnpIBP and maximized by that of TisIBP8, whereas it was lowered when a defective AnpIBP mutant was expressed. These results suggest that the ice-binding ability of IBP has a good correlation with the survival rate of C. elegans during freezing.

Nanoparticles are widely used in commodities, and pregnant women are inevitably exposed to these particles. The placenta protects the growing fetus from foreign or toxic materials, and provides energy and oxygen. Here we report that autophagy, a cellular mechanism to maintain homeostasis, engulfs platinum nanoparticles (nPt) to reduce their cytotoxicity in trophoblasts. Autophagy was activated by nPt in extravillous trophoblast (EVT) cell lines, and EVT functions, such as invasion and vascular remodeling, and proliferation were inhibited by nPt. These inhibitory effects by nPt were augmented in autophagy-deficient cells. Regarding the dynamic state of nPt, analysis using ICP-MS demonstrated a higher accumulation of nPt in the autophagosome-rich than the cytoplasmic fraction in autophagy-normal cells. Meanwhile, there were more nPt in the nuclei of autophagy-deficient cells, resulting in greater DNA damage at a lower concentration of nPt. Thus, we found a new protective mechanism against the cytotoxicity of nPt in human trophoblasts.

Ice recrystallization is a phenomenon observed as the increase in ice crystal size within an already frozen material. Antifreeze proteins (AFPs), a class of proteins capable of arresting ice crystal growth, are known to inhibit this phenomenon even at sub milli-molar concentrations. A tremendous range in the possible applications of AFPs is hence expected in both medical and industrial fields, while a key determinant of the ice recrystallization inhibition (IRI) is hardly understood. Here, IRI efficiency and ice plane affinity were examined for the wild-type AFPI-III, a defective AFPIII isoform, and a fungal AFP isoform. To simplify the IRI analysis using the formal representation of Ostwald-ripening (r 3  = r 0  + kt), we monitored specific ice grains exhibiting only uniform growth, for which maximum Feret diameter was measured. The cube of an ice grain's radius (r 3  + kt), we monitored specific ice grains exhibiting only uniform growth, for which maximum Feret diameter was measured. The cube of an ice grain's radius (r 3 ) increased proportionately with time (t), and its slope gave the recrystallization rate (k). There was a significant difference in the IRI efficiency between the samples, and the fungal AFP possessing the activity with the smallest amount (0.27 μM) exhibited an affinity to multiple ice planes. These results suggest that the IRI efficiency is maximized when AFPs bind to a whole set of ice planes.

Ghrelin is a unique fatty acid-modified peptide hormone produced in the stomach and has important roles in energy homeostasis and gastrointestinal motility. However, the medium-chain fatty acid source for ghrelin acyl-modification is not known. We found that a fat-free diet and the removal of intestinal microbiota did not decrease acyl-ghrelin production in the stomach or plasma acyl-ghrelin levels in mice. RT-PCR analysis showed that genes involving fatty acid synthesis, metabolism, and transport were expressed in pancreas-derived ghrelinoma (PG-1) cells. Treatment with an irreversible inhibitor of carnitine palmitoyltransferase-1 (CPT-1) strongly decreased acylated ghrelin levels but did not affect ghrelin or ghrelin o-acyl transferase (GOAT) mRNA levels in PG-1 cells. Our results suggest that the medium-chain fatty acid used for the acyl-modification of ghrelin is produced in ghrelin-producing cells themselves by β-oxidation of long-chain fatty acids provided from the circulation.

Optical measurement of membrane potentials enables fast, direct and simultaneous detection of membrane potentials from a population of neurons, providing a desirable approach for functional analysis of neuronal circuits. Here, we applied recently developed genetically encoded voltage indicators, ASAP1 (Accelerated Sensor of Action Potentials 1) and QuasAr2 (Quality superior to Arch 2), to zebrafish, an ideal model system for studying neurogenesis. To achieve this, we established transgenic lines which express the voltage sensors, and showed that ASAP1 is expressed in zebrafish neurons. To examine whether neuronal activity could be detected by ASAP1, we performed whole-cerebellum imaging, showing that depolarization was detected widely in the cerebellum and optic tectum upon electrical stimulation. Spontaneous activity in the spinal cord was also detected by ASAP1 imaging at single-cell resolution as well as at the neuronal population level. These responses mostly disappeared following treatment with tetrodotoxin, indicating that ASAP1 enabled optical measurement of neuronal activity in the zebrafish brain. Combining this method with other approaches, such as optogenetics and behavioural analysis may facilitate a deeper understanding of the functional organization of brain circuitry and its development.

High-density lipoprotein (HDL) interacts with various cells, particularly macrophages, in functional cell-HDL interactions. Here, we found that HDL protein quality and lipid quality play critical roles in HDL functions. HDL fractions from healthy volunteers (HDL Healthy ) and patients with recurrent coronary atherosclerotic disease (HDL CAD ) were prepared. To analyse functional HDL-macrophage interactions, macrophages were co-incubated with each HDL, and lipid mediator production was assessed by liquid chromatography/mass spectrometry-based metabololipidomics. HDL Healthy treatment attenuated the pro-inflammatory lipid mediator production, particularly that of leukotriene (LT) B 4 , and this treatment enhanced lipoxin (LX) B 4 and resolvin (Rv) E2 production. HDL Healthy treatment enhanced the proteasome-mediated degradation of the LTB 4 -producing enzyme 5-lipoxygenase (LO) in activated macrophages; however, HDL CAD did not show these anti-inflammatory effects. HDL Healthy was engulfed by macrophages via clathrin-mediated endocytosis, which was a critical step in 5-LO/LTB 4 regulation. We also found that HDL CAD showed higher levels of the LTB 4 -producing enzymes and thus promoted LTB 4 production from HDL CAD . In addition, LTB 4 attenuated HDL endocytosis, HDL-mediated 5-LO degradation in macrophages, and HDL-derived augmentation of macrophage phagocytosis. These results indicated that local LTB 4 produced de novo from HDL CAD regulates HDL-macrophage functional interactions and plays critical roles in dysfunctional, inflammatory HDL characteristics.

Gene rearrangement is a widely-shared phenomenon in spore forming bacteria, in which prophage(-like) elements interrupting sporulation-specific genes are excised from the host genome to reconstitute the intact gene. Here, we report a novel class of gene-intervening elements, named gin, inserted in the 225 bp gerE-coding region of the B. cereus ATCC10987 genome, which generates a sporulation-specific rearrangement. gin has no phage-related genes and possesses three site-specific recombinase genes; girA, girB, and girC. We demonstrated that the gerE rearrangement occurs at the middle stage of sporulation, in which site-specific DNA recombination took place within the 9 bp consensus sequence flanking the disrupted gerE segments. Deletion analysis of gin uncovered that GirC and an additional factor, GirX, are responsible for gerE reconstitution. Involvement of GirC and GirX in DNA recombination was confirmed by an in vitro recombination assay. These results broaden the definition of the sporulation-specific gene rearrangement phenomenon: gene-intervening elements are not limited to phage DNA but may include non-viral genetic elements that carry a developmentally-regulated site-specific recombination system.

A supersoluble 40-residue type I antifreeze protein (AFP) was discovered in a righteye flounder, the barfin plaice (bp). Unlike all other AFPs characterized to date, bpAFP transitions from moderately-active to hyperactive with increasing concentration. At sub-mM concentrations, bpAFP bound to pyramidal planes of ice to shape it into a bi-pyramidal hexagonal trapezohedron, similarly to the other moderately-active AFPs. At mM concentrations, bpAFP uniquely underwent further binding to the whole ice crystal surface including the basal planes. The latter caused a bursting ice crystal growth normal to c-axis, 3 °C of high thermal hysteresis, and alteration of an ice crystal into a smaller lemon-shaped morphology, all of which are well-known properties of hyperactive AFPs. Analytical ultracentrifugation showed this activity transition is associated with oligomerization to form tetramer, which might be the forerunner of a naturally occurring four-helix-bundle AFP in other flounders.

A previous report showed that the consumption of glutathione through oxidative stress activates the glutathione synthetic pathway, which is accompanied by production of ophthalmic acid from 2-aminobutyric acid (2-AB). We conducted a comprehensive quantification of serum metabolites using gas chromatography-mass spectrometry in patients with atrial septal defect to find clues for understanding myocardial metabolic regulation, and demonstrated that circulating 2-AB levels reflect hemodynamic changes. However, the metabolism and pathophysiological role of 2-AB remains unclear. We revealed that 2-AB is generated by an amino group transfer reaction to 2-oxobutyric acid, a byproduct of cysteine biosynthesis from cystathionine. Because cysteine is a rate-limiting substrate for glutathione synthesis, we hypothesized that 2-AB reflects glutathione compensation against oxidative stress. A murine cardiomyopathy model induced by doxorubicin supported our hypothesis, i.e., increased reactive oxygen species are accompanied by 2-AB accumulation and compensatory maintenance of myocardial glutathione levels. Intriguingly, we also found that 2-AB increases intracellular glutathione levels by activating AMPK and exerts protective effects against oxidative stress. Finally, we demonstrated that oral administration of 2-AB efficiently raises both circulating and myocardial glutathione levels and protects against doxorubicin-induced cardiomyopathy in mice. This is the first study to demonstrate that 2-AB modulates glutathione homeostasis in the myocardium.

Glaucoma is a group of eye diseases characterized by alterations in the contour of the optic nerve head (ONH), with corresponding visual field defects and progressive loss of retinal ganglion cells (RGCs). This progressive RGC death is considered to originate in axonal injury caused by compression of the axon bundles in the ONH. However, the molecular pathomechanisms of axonal injury-induced RGC death are not yet well understood. Here, we used RNA sequencing (RNA-seq) to examine transcriptome changes in rat retinas 2 days after optic nerve transection (ONT), and then used computational techniques to predict the resulting alterations in the transcriptional regulatory network. RNA-seq revealed 267 differentially expressed genes after ONT, 218 of which were annotated and 49 unannotated. We also identified differentially expressed transcripts, including potentially novel isoforms. An in silico pathway analysis predicted that CREB1 was the most significant upstream regulator. Thus, this study identified genes and pathways that may be involved in the pathomechanisms of axonal injury. We believe that our data should serve as a valuable resource to understand the molecular processes that define axonal injury-driven RGC death and to discover novel therapeutic targets for glaucoma.

Glaucoma, a leading cause of blindness worldwide, is instigated by various factors, including axonal injury, which eventually leads to a progressive loss of retinal ganglion cells (RGCs). To study various pathways reportedly involved in the pathogenesis of RGC death caused by axonal injury, seven pathways were investigated. Pathway-specific fluorescent protein-coded reporters were each packaged into an adeno-associated virus (AAV). After producing axonal injury in the eye, injected with AAV to induce RGC death, the temporal activity of each stress-related pathway was monitored in vivo through the detection of fluorescent RGCs using confocal ophthalmoscopy. We identified the activation of ATF6 and MCP-1 pathways involved in endoplasmic reticulum stress and macrophage recruitment, respectively, as early markers of RGC stress that precede neuronal death. Conversely, inflammatory responses probed by NF-κB and cell-death-related pathway p53 were most prominent in the later phases, when RGC death was already ongoing. AAV-mediated delivery of stress/response reporters followed by in vivo cellular imaging is a powerful strategy to characterize the temporal aspects of complex molecular pathways involved in retinal diseases. The identification of promoter elements that are activated before the death of RGCs enables the development of pre-emptive gene therapy, exclusively targeting the early phases of diseased cells.

Aqueous humor (AH) is one of the body fluids in the eye, which is known to be related with various ocular diseases, but the complete RNAs characteristic of the AH in patients is not yet known. The aim of this study was, with a microarray analysis, to reveal the disease-related extracellular miRNAs profiles in individual patients AH. 100 μl of AH was collected by anterior chamber paracentesis from 10 glaucoma, 5 cataract, and 5 epiretinal membrane patients. The extracted total RNAs were shorter than 200 nt, and their amount was 5.27 ± 0.41 ng in average. Among 530.5 ± 44.6 miRNA types detected in each sample with a microarray detectable 2019 types of matured miRNAs, 172 miRNAs were detected in all 10 glaucoma or control patients. From the glaucoma group, 11 significantly up-regulated and 18 significantly down-regulated miRNAs (P < 0.05 for both) were found to have areas under the curve better than 0.74 in a receiver operating characteristic analysis. They also formed a cluster composed only of glaucoma patients in a hierarchal cluster analysis. AH had a possibility of becoming a source of miRNA that can serve as a biomarker and a therapeutic target.

Cryopreservation methods using liquid nitrogen (LN(2)) for gametes and embryos are prevalent in mammalian artificial reproduction. However, the pregnancy rate from frozen embryos has not improved over the past two decades because freeze-thawing causes significant damage. The strict regulation of transportation of LN(2) containers by airlines also limits exchange between breeders. In this article, we introduce a medium that enabled bovine embryos to be held for up to 7 days at 4°C. A pregnancy rate of 75% (24/32) was obtained for embryos held for 7 days in this medium and transferred to primed recipients. Its constituents were medium 199, foetal bovine serum, and HEPES for buffering. This technique will enable LN(2)-free storage and air transportation of embryos provided transplantation to recipients can be completed within 7 days.