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Abstract Microvascular decompression (MVD) has proven efficacy in trigeminal neuralgia (TN) and hemifacial spasm (HFS). This study utilized computational fluid dynamics (CFD) to investigate the impact of MVD on wall shear stress (WSS) of responsible arteries (RAs) at the neurovascular contact (NVC). A total of 21 cases (10 TN, 11 HFS) were analyzed, involving RAs at NVC validated through intraoperative photographs. Hemodynamic parameters (WSS, vessel diameter, flow rate, pressure drop) was calculated using CFD for the RAs based on 3D silent-magnetic resonance angiograms. The NVC was segmented into NVC-proximal, NVC-site, and NVC-distal portions using simulated 3D CFD images that correlated with surgical observations. WSS ratios of NVC-site to NVC-proximal (NVC-site/proximal) was calculated both before and after MVD. Prior to MVD, WSS in the RA at the NVC displayed a peaked curve with a maximum at NVC-site; however, post MVD, it presented a smooth curve without peaks. The WSS ratio exhibited a significant decrease after MVD. The impact of MVD on WSS of RAs at NVC was evaluated in both TN and HFS cases. Analyzing the hemodynamics of RAs through CFD and identifying WSS peaks at NVC portions before MVD provided a more detailed and localized understanding of the morphologically depicted NVC.

Abstract DNA hypomethylating agents (HMAs) are used for the treatment of myeloid malignancies, although their therapeutic effects have been unsatisfactory. Here we show that CRISPR-Cas9 screening reveals that knockout of topoisomerase 1-binding arginine/serine-rich protein (TOPORS), which encodes a ubiquitin/SUMO E3 ligase, augments the efficacy of HMAs on myeloid leukemic cells with little effect on normal hematopoiesis, suggesting that TOPORS is involved in resistance to HMAs. HMAs are incorporated into the DNA and trap DNA methyltransferase-1 (DNMT1) to form DNA-DNMT1 crosslinks, which undergo SUMOylation, followed by proteasomal degradation. Persistent crosslinking is cytotoxic. The TOPORS RING finger domain, which mediates ubiquitination, is responsible for HMA resistance. In TOPORS knockout cells, DNMT1 is stabilized by HMA treatment due to inefficient ubiquitination, resulting in the accumulation of unresolved SUMOylated DNMT1. This indicates that TOPORS ubiquitinates SUMOylated DNMT1, thereby promoting the resolution of DNA-DNMT1 crosslinks. Consistently, the ubiquitination inhibitor, TAK-243, and the SUMOylation inhibitor, TAK-981, show synergistic effects with HMAs through DNMT1 stabilization. Our study provides a novel HMA-based therapeutic strategy that interferes with the resolution of DNA-DNMT1 crosslinks.

Abstract Epidural spinal cord stimulation (SCS) is indicated for the treatment of intractable pain and is widely used in clinical practice. In previous basic research, the therapeutic effects of SCS have been demonstrated for epileptic seizure. However, the mechanism has not yet been elucidated. In this study, we investigated the therapeutic effect of SCS and the influence of epileptic seizure. First, SCS in the cervical spine was performed. The rats were divided into four groups: control group and treatment groups with SCS conducted at 2, 50, and 300 Hz frequency. Two days later, convulsions were induced by the intraperitoneal administration of kainic acid, followed by video monitoring to assess seizures. We also evaluated glial cells in the hippocampus by fluorescent immunostaining, electroencephalogram measurements, and inflammatory cytokines such as C–C motif chemokine ligand 2 (CCL2) by quantitative real-time polymerase chain reaction. Seizure frequency and the number of glial cells were significantly lower in the 300 Hz group than in the control group. SCS at 300 Hz decreased gene expression level of CCL2, which induces monocyte migration. SCS has anti-seizure effects by inhibiting CCL2-mediated cascades. The suppression of CCL2 and glial cells may be associated with the suppression of epileptic seizure.

Badminton is one of the most popular racket sports played by all age groups worldwide. Anyone can practice and play badminton as a leisure or competitive sport, regardless of age, experience, or skill level. It does not require physical contact among players or expensive equipment. Compared with closed-skill exercises (e.g., running and swimming), open-skill exercises (e.g., badminton and table tennis) have been reported to significantly improve cognitive function and prevent cognitive decline. This mini review aimed to investigate the findings related to badminton intervention for cognitive function, with attention to the modifiable risk factors of dementia in adults. Additionally, we outlined the characteristics of badminton’s physical demands. This narrative review encompassed studies reported from 2013 to 2023 from multiple databases, including PubMed and Google Scholar. Keywords such as “badminton”, “dementia”, “cognitive function”, “physical activity”, and “depression” were used to identify relevant articles. Various studies, including randomized controlled trials, cohort studies, and case–control studies, were selected to provide a comprehensive overview of badminton intervention. Quantitative data analysis suggests that at a cognitive level, high-intensity badminton can enhance cognitive function with at least 10 min of play once weekly or 20 min of play once weekly on average, whereas moderate-intensity badminton can be effective when played for at least 30 min once weekly or 35 min twice weekly on average. For modifiable risk factors, recreational badminton for about 3 h weekly could effectively manage blood pressure. For body fat percentage and fasting serum glucose levels, recreational badminton for about 10 h weekly exerted positive effects. For depressive states, a moderate-intensity badminton program for 100 min weekly was effective. For lipid profiles associated with dementia occurrence, prolonged badminton intervention for more than 3 months may be beneficial. The characteristics presented here indicate that intervention with regular badminton exercise could effectively improve cognitive function and prevent cognitive decline in older adults.

Abstract Antisense oligonucleotides (ASOs) are synthetic single-stranded oligonucleotides that bind to RNAs through Watson–Crick base pairings. They are actively being developed as therapeutics for various human diseases. ASOs containing unmethylated deoxycytidylyl-deoxyguanosine dinucleotide (CpG) motifs are known to trigger innate immune responses via interaction with toll-like receptor 9 (TLR9). However, the TLR9-stimulatory properties of ASOs, specifically those with lengths equal to or less than 20 nucleotides, phosphorothioate linkages, and the presence and arrangement of sugar-modified nucleotides—crucial elements for ASO therapeutics under development—have not been thoroughly investigated. In this study, we first established SY-ODN18, an 18-nucleotide phosphorothioate oligodeoxynucleotide with sufficient TLR9-stimulatory activity. We demonstrated that an unmethylated CpG motif near its 5′-end was indispensable for TLR9 activation. Moreover, by utilizing various sugar-modified nucleotides, we systematically generated model ASOs, including gapmer, mixmer, and fully modified designs, in accordance with the structures of ASO therapeutics. Our results illustrated that introducing sugar-modified nucleotides in such designs significantly reduces TLR9-stimulatory activity, even without methylation of CpG motifs. These findings would be useful for drug designs on several types of ASOs.

Due to the sporadic distribution and trace amount of environmental DNA (eDNA) in deep-sea water, in the context of biodiversity monitoring, large volumes of filtration and multiple filtration replicates are required for eDNA metabarcoding. To address issues tied to the use of multiple filtration devices and large filtration volumes (e.g., contamination, time consumption, etc.), we have developed two systems for simple, rapid, and contamination-less filtration simultaneously that allow for the processing of multiple sample replicates from large volumes of water. First, the water from a Niskin bottle was filtered directly using a solenoid pump. Second, the pumped deep-sea water, using the siphon effect, was directly filtered by a filtration device driven by water pressure. This system can process 24 replicates simultaneously without the need for expensive equipment and active driving force. Compared with conventional filtering methods, e.g., peristaltic pumps, the proposed systems reduce filtration time, minimizing contamination, and enabling the simultaneous acquisition of multiple replicates. Overall, the systems presented here provide an effective approach for eDNA metabarcoding analysis, particularly for the filtration of large volumes of water containing small amounts of eDNA, such as deep-sea water. • The present systems reduce filtration time and contamination without water having to be transferred. • Simultaneous multiple replicates improve the efficiency and reliability of biodiversity assessments.

Abstract Extracellular vesicles (EVs), including exosomes, are recognized as promising functional targets involved in disease mechanisms. However, the intravital heterogeneity of EVs remains unclear, and the general limitation for analyzing EVs is the need for a certain volume of biofluids. Here, we present cellulose nanofiber (CNF) sheets to resolve these issues. We show that CNF sheets capture and preserve EVs from ~10 μL of biofluid and enable the analysis of bioactive molecules inside EVs. By attaching CNF sheets to moistened organs, we collect EVs in trace amounts of ascites, which is sufficient to perform small RNA sequence analyses. In an ovarian cancer mouse model, we demonstrate that CNF sheets enable the detection of cancer-associated miRNAs from the very early phase when mice did not have apparent ascites, and that EVs from different locations have unique miRNA profiles. By performing CNF sheet analyses in patients, we identify further location-based differences in EV miRNA profiles, with profiles reflecting disease conditions. We conduct spatial exosome analyses using CNF sheets to reveal that ascites EVs from cancer patients exhibit location-dependent heterogeneity. This technique could provide insights into EV biology and suggests a clinical strategy contributing to cancer diagnosis, staging evaluation, and therapy planning.

Judo is a well-known Japanese martial art that also features in Olympic games. Recently, an increasing interest has been observed in the application of judo as a multicomponent exercise, with a growing body of evidence showing that this feature improves physical and cognitive performance in older adults. Therefore, this review highlights the benefits of judo training in preventing physical and cognitive decline in older adults. Specifically, first, this review outlines the basic characteristics of judo (philosophy, match, and training). Subsequently, prior research examining the impacts of judo training on the physical and cognitive aspects of elderly individuals is reviewed. Thereafter, the brain mechanisms underlying the effects of judo training in improving physical and cognitive performance are discussed. Throughout this review, judo training demonstrated some positive effects on physical (gait and balance, among others) and cognitive (memory and executive function) function in older adults. These positive effects are attributed to a variety of changes in the brain (e.g., increased neurotrophic factor expression and increased cerebral blood flow, among others) that affect different brain regions and networks both functionally and structurally. From these findings, this review concludes that judo training can be an effective way to maintain and prevent physical and cognitive decline in older adults.

Abstract Ensemble simulations of climate models are used to assess the impact of climate change on precipitation, and require downscaling at the local scale. Statistical downscaling methods have been used to estimate daily and monthly precipitation from observed and simulated data. Downscaling of short-term precipitation data is necessary for more accurate prediction of extreme precipitation events and related disasters at the regional level. In this study, we developed and investigated the performance of a downscaling method for climate model simulations of hourly precipitation. Our method was designed to recognize time-varying precipitation systems that can be represented at the same resolution as the numerical model. Downscaling improved the estimation of the spatial distribution of hourly precipitation frequency, monthly average, and 99th percentile values. The climate change in precipitation amount and frequency were shown in almost all areas by using the 50 ensemble averages of estimated precipitation, although the natural variability was too large to compare with observations. The changes in precipitation were consistent with simulations. Therefore, our downscaling method improved the evaluation of the climatic characteristics of extreme precipitation events and more comprehensively represented the influence of local factors, such as topography, which have been difficult to evaluate using previous methods.

Table tennis is an extremely popular sport throughout the world as it requires no expensive equipment, specialized amenities, or physical contact among players, and the pace of play can be adapted to allow participation by players of all skill levels, ages, and abilities. It is an aerobic-dominant sport driven primarily by the phosphagen system because rallies are relatively brief (several seconds) and separated by longer rest periods. Several studies have shown that physical interventions including table tennis can help prevent cognitive decline and dementia. Accordingly, the present paper provides an overview of the basic physical and cognitive demands of table tennis, reviews previous studies reporting improvements in physical and brain health across different non-clinical and clinical populations, and critically evaluates the usefulness of table tennis intervention for the prevention of cognitive decline and dementia. This review suggests that table tennis intervention could be a powerful strategy to prevent cognitive decline and dementia in the elderly.

Abstract 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.

Tirabrutinib is a highly selective Bruton's tyrosine kinase (BTK) inhibitor used to treat hematological malignancies. We analyzed the anti-tumor mechanism of tirabrutinib using phosphoproteomic and transcriptomic methods. It is important to check the drug's selectivity against off-target proteins to understand the anti-tumor mechanism based on the on-target drug effect. Tirabrutinib's selectivity was evaluated by biochemical kinase profiling assays, peripheral blood mononuclear cell stimulation assays, and the BioMAP system. Next, in vitro and in vivo analyses of the anti-tumor mechanisms were conducted in activated B-cell-like diffuse large B-cell lymphoma (ABC-DLBCL) cells followed by phosphoproteomic and transcriptomic analyses. In vitro kinase assays showed that, compared with ibrutinib, tirabrutinib and other second-generation BTK inhibitors demonstrated a highly selective kinase profile. Data from in vitro cellular systems showed that tirabrutinib selectively affected B-cells. Tirabrutinib inhibited the cell growth of both TMD8 and U-2932 cells in correlation with the inhibition of BTK autophosphorylation. Phosphoproteomic analysis revealed the downregulation of ERK and AKT pathways in TMD8. In the TMD8 subcutaneous xenograft model, tirabrutinib showed a dose-dependent anti-tumor effect. Transcriptomic analysis indicated that IRF4 gene expression signatures had decreased in the tirabrutinib groups. In conclusion, tirabrutinib exerted an anti-tumor effect by regulating multiple BTK downstream signaling proteins, such as NF-κB, AKT, and ERK, in ABC-DLBCL.

Deep-sea ecosystems present difficulties in surveying and continuous monitoring of the biodiversity of deep-sea ecosystems because of the logistical constraints, high cost, and limited opportunities for sampling. Environmental DNA (eDNA) metabarcoding analysis provides a useful method for estimating the biodiversity in aquatic ecosystems but has rarely been applied to the study of deep-sea fish communities. In this study, we utilized pumped deep-sea water for the continuous monitoring of deep-sea fish communities by eDNA metabarcoding. In order to develop an optimum method for continuous monitoring of deep-sea fish biodiversity by eDNA metabarcoding, we determined the appropriate amount of pumped deep-sea water to be filtered and the practical number of filtered sample replicates required for biodiversity monitoring of deep-sea fish communities. Pumped deep-sea water samples were filtered in various volumes (5–53 L) at two sites (Akazawa: pumping depth 800 m, and Yaizu: pumping depth 400 m, Shizuoka, Japan) of deep-sea water pumping facilities. Based on the result of evaluations of filtration time, efficiency of PCR amplification, and number of detected fish reads, the filtration of 20 L of pumped deep-sea water from Akazawa and filtration of 10 L from Yaizu were demonstrated to be suitable filtration volumes for the present study. Fish biodiversity obtained by the eDNA metabarcoding analyses showed a clear difference between the Akazawa and Yaizu samples. We also evaluated the effect of the number of filter replicates on the species richness detected by eDNA metabarcoding from the pumped deep-sea water. At both sites, more than 10 sample replicates were required for the detection of commonly occurring fish species. Our optimized method using pumped deep-sea water and eDNA metabarcoding can be applied to eDNA-based continuous biodiversity monitoring of deep-sea fish to better understand the effects of climate change on deep-sea ecosystems.

Gel growth induced by contact of polymer solutions with crosslinker solutions yields an emerging class of anisotropic materials with many potential applications. Here, we report the case of a study on the dynamics in forming anisotropic gels using this approach with an enzyme as a trigger of gelation and gelatin as the polymer. Unlike the previously studied cases of gelation, the isotropic gelation was followed by gel polymer orientation after a lag time. The isotropic gelation dynamics did not depend on concentrations of the polymer turning into gel and of the enzyme inducing gelation, whereas, for the anisotropic gelation, the square of the gel thickness was a linear function of the elapsed time, and the slope increased with polymer concentration. The gelation dynamics of the present system was explained by a combination of diffusion-limited gelation followed by free-energy-limited orientation of polymer molecules.

Abstract General population-based cohort studies provide solid evidence on mass Helicobacter pylori (HP) eradication effects. Self-reported questionnaires are occasionally used in such studies to ascertain the HP eradication history. However, reports on the reliability of these questionnaires are lacking. This general population-based cohort study included 899 individuals with HP infection at the baseline survey who were reported to have eradicated it at the 5-year follow-up survey. Of these, the medical records of 280 patients were available for investigation, and the HP eradication status of 93 individuals was ascertained. Their medical records were reviewed, and the reliability of the self-reported questionnaire responses was assessed. Of the 91 individuals who successfully eradicated HP based on the medical records, 90 (98.9%) answered the self-reported questionnaire correctly, with an unweighted kappa value of 0.661 (p

The Yokozuna Slickhead Narcetes shonanmaruae is a recently described deep-sea fish species and an active-swimming, relatively large top predator in Suruga Bay, Japan. Its only known habitat is the deepest part of the bay (>2,000 m); six individuals have been collected thus far (up to 138 cm in total length). During our monitoring survey of faunal diversity on seamounts within marine protected areas in Japanese waters, environmental DNA (eDNA) metabarcoding revealed the Yokozuna Slickhead 12S ribosomal RNA gene sequence on/around three seamounts belonging to the Nishi-Shichito Ridge (at depths of around 2,000 m) located 400–600 km south of the known locality. A baited camera system deployed at the foot of one of the three seamounts at a depth of 2,091 m captured a Yokozuna Slickhead individual that was over 250 cm in total length, threatening Pacific Grenadiers Coryphaenoides acrolepis around the bait and attacking the bait cage. A combination of eDNA metabarcoding and baited camera observation represents a powerful tool for the detection of rare predatory fish species and the study of their ecology even in the deep sea, thus helping to better understand vulnerable marine ecosystems and reveal the impact of the rapidly changing global ocean.

Chemical modifications have been extensively used for therapeutic oligonucleotides because they strongly enhance the stability against nucleases, binding affinity to the targets, and efficacy. We previously reported that oligonucleotides modified with an N-methylguanidine-bridged nucleic acid (GuNA[Me]) bearing the thymine (T) nucleobase show excellent biophysical properties for applications in antisense technology. In this paper, we describe the synthesis of GuNA[Me] phosphoramidites bearing other typical nucleobases including adenine (A), guanine (G), and 5-methylcytosine (mC). The phosphoramidites were successfully incorporated into oligonucleotides following the method previously developed for the GuNA[Me]-T-modified oligonucleotides. The binding affinity of the oligonucleotides modified with GuNA[Me]-A, -G, or -mC toward the complementary single-stranded DNAs or RNAs was systematically evaluated. All of the GuNA[Me]-modified oligonucleotides were found to have a strong affinity for RNAs. These data indicate that GuNA[Me] could be a useful modification for therapeutic antisense oligonucleotides.

Gelation through the liquid-liquid contact between a polymer solution and a gelator solution has been attempted with various combinations of gelator and polymer solutions. In many combinations, the gel growth dynamics is expressed as X∼t, where X is the gel thickness and t is the elapsed time, and the scaling law holds for the relationship between X and t. In the blood plasma gelation, however, the crossover of the growth behavior from X∼t in the early stage to X∼t in the late stage was observed. It was found that the crossover behavior is caused by a change in the rate-limiting process of growth from the free-energy-limited process to the diffusion-limited process. How, then, would the crossover phenomenon be described in terms of the scaling law? We found that the scaling law does not hold in the early stage owing to the characteristic length attributable to the free energy difference between the sol-gel phases, but it does in the late stage. We also discussed the analysis method for the crossover in terms of the scaling law.

Abstract Clostridioides difficile infection (CDI) is an important nosocomial infection and is the leading cause of infectious diarrhea in hospitalized patients. We aimed to assess the effect of bowel rest on the management of CDI. A single-center retrospective cohort study was conducted. The primary outcome was the composite of the all-cause mortality and CDI recurrence within 30 days. The main secondary outcome was switching from metronidazole to vancomycin. Of the 91 patients with CDI enrolled as the full cohort, 63 patients (69%) and 28 patients (31%) constituted the control group and the bowel rest group, respectively. After one-to-one propensity score matching, a total of 46 patients were included as the matched cohort. In the full cohort, the composite outcome occurred in 19.0% and 14.3% of the patients in the control and the bowel rest group, respectively (p = 0.768). In the matched cohort, it was 17.4% in each group. Although there was no statistically significant difference, the trend of switching was lower in the bowel rest group. The bowel rest may not affect the all-cause mortality and CDI recurrence within 30 days. However, in those prescribed bowel rest, switching from metronidazole to vancomycin may reduce.

Abstract The risk of developing metachronous gastric cancer (MGC) following curative endoscopic submucosal dissection (ESD) of early gastric cancer (EGC) remains even after eradicating Helicobacter pylori (HP) successfully. We screened initial EGC and adjacent non-cancerous mucosa ESD-resected specimens for somatic variants of 409 cancer-related genes, assessing their mutational burden (MB) to predict molecular markers for metachronous post-ESD development. We compared variants between ten patients diagnosed with MGC more than 3 years after ESD and ten age-matched patients who did not have MGC developments after successful HP eradication. We found no significant background differences between the two groups. In adjacent non-cancerous mucosa, the MB tended to be higher in the patients with metachronous developments than in the others. Somatic genomic alterations of RECQL4, JAK3, ARID1A, and MAGI1 genes were significantly associated with MGC development. The criteria including both the MB and their variants, which had potential significant values for predicting MGC. In conclusion, combined of assessing specific somatic variants and MB may be useful for predicting MGC development. This study included a limited number of subjects; however, our novel findings may encourage further exploration of the significance of the molecular features of EGC that predict MGC development, thereby promoting focused follow-up strategies and helping elucidate the mechanisms.

Animals that live in nutrient-poor environments, such as the deep sea, often establish intracellular symbiosis with beneficial bacteria that provide the host with nutrients that are usually inaccessible to them. The deep-sea mussel Bathymodiolus japonicus relies on nutrients from the methane-oxidizing bacteria harboured in epithelial gill cells called bacteriocytes. These symbionts are specific to the host and transmitted horizontally, being acquired from the environment by each generation. Morphological studies in mussels have reported that the host gill cells acquire the symbionts via phagocytosis, a process that facilitates the engulfment and digestion of exogenous microorganisms. However, gill cell phagocytosis has not been well studied, and whether mussels discriminate between the symbionts and other bacteria in the phagocytic process remains unknown. Herein, we aimed to investigate the phagocytic ability of gill cells involved in the acquisition of symbionts by exposing the mussel to several types of bacteria. The gill cells engulfed exogenous bacteria from the environment indiscriminately. These bacteria were preferentially eliminated through intracellular digestion using enzymes; however, most symbionts were retained in the bacteriocytes without digestion. Our findings suggest that regulation of the phagocytic process after engulfment is a key mechanism for the selection of symbionts for establishing intracellular symbiosis.

Extreme storms, such as tropical cyclones, are responsible for a significant portion of the plastic debris transported from land to sea yet little is known about the storm response of microplastics and other debris in offshore and open waters. To investigate this, we conducted floating plastic surveys in the center of Sagami Bay, Japan approximately 30 km from the coastline, before and after the passage of a typhoon. The concentrations (number of particles/km2) of micro- and mesoplastics were two orders of magnitude higher 1-day after the typhoon than the values recorded pre-typhoon and the mass (g/km2) of plastic particles (sum of micro- and mesoplastics) increased 1,300 times immediately after the storm. However, the remarkably high abundance of micro- and mesoplastics found at 1-day after the typhoon returned to the pre-typhoon levels in just 2 days. Model simulations also suggested that during an extreme storm a significant amount of micro- and mesoplastics can be rapidly swept away from coastal to open waters over a short period of time. To better estimate the annual load of plastics from land to sea it is important to consider the increase in leakages of plastic debris into the ocean associated with extreme storm events.

Some sea slugs sequester chloroplasts from algal food in their intestinal cells and photosynthesize for months. This phenomenon, kleptoplasty, poses a question of how the chloroplast retains its activity without the algal nucleus. There have been debates on the horizontal transfer of algal genes to the animal nucleus. To settle the arguments, this study reported the genome of a kleptoplastic sea slug, Plakobranchus ocellatus, and found no evidence of photosynthetic genes encoded on the nucleus. Nevertheless, it was confirmed that light illumination prolongs the life of mollusk under starvation. These data presented a paradigm that a complex adaptive trait, as typified by photosynthesis, can be transferred between eukaryotic kingdoms by a unique organelle transmission without nuclear gene transfer. Our phylogenomic analysis showed that genes for proteolysis and immunity undergo gene expansion and are up-regulated in chloroplast-enriched tissue, suggesting that these molluskan genes are involved in the phenotype acquisition without horizontal gene transfer.

Analyses of environmental DNA (eDNA) from macroorganisms in aquatic environments have greatly advanced in recent years. In particular, eDNA metabarcoding of fish using universal PCR primers has been reported in various waters. Although pumped deep-sea water was used for eDNA metabarcoding of deep-sea fish, conventional methods only resulted in small amounts of extracted eDNA and subsequent few or no PCR amplicons. To optimize eDNA metabarcoding of deep-sea fish from pumped deep-sea water, we modified conventional procedures of eDNA extraction and PCR amplification. Here, we propose a modified eDNA extraction method, in which a filter used for eDNA sampling was shredded and incubated in microtubes for efficient lysis of eDNA sources. Total eDNA yield extracted using the modified protocol was approximately six-fold higher than that extracted by the conventional protocol. The PCR enzyme Platinum SuperFi II DNA Polymerase successfully amplified a target region of fish universal primers (MiFish) from trace amounts of eDNA extracted from pumped deep-sea water and suppressed nonspecific amplifications more effectively than the enzyme used in conventional methods. Approximately 93% of the sequence reads acquired by next generation sequencing of these amplicons were derived from fish. The improved procedure presented here provided effective eDNA metabarcoding of deep-sea fish. • A modified eDNA extraction protocol, in which a filter was shredded and incubated in microtubes, increased eDNA yields extracted from pumped deep-sea water over the conventional method. • The PCR enzyme Platinum SuperFi II DNA polymerase improved the amplification efficiency of trace amounts of MiFish objectives in eDNA extracted from pumped deep-sea water with suppressing nonspecific amplifications. • The use of Platinum SuperFi II DNA polymerase for eDNA metabarcoding using MiFish primers resulted in the acquisition of abundant sequence reads of deep-sea fish through next generation sequencing.

Drought resistant plants typically have reduced growth. Here the authors show that overexpression of the BRL3 brassinosteroid receptor confers drought tolerance and accumulation of osmoprotectant metabolites without penalizing growth, demonstrating that drought response and growth can be uncoupled.

Abstract Telomere maintenance by telomerase activity supports the infinite growth of cancer cells. MST-312, a synthetic telomerase inhibitor, gradually shortens telomeres at non-acute lethal doses and eventually induces senescence and apoptosis of telomerase-positive cancer cells. Here we report that MST-312 at higher doses works as a dual inhibitor of telomerase and DNA topoisomerase II and exhibits acute anti-proliferative effects on cancer cells and xenografted tumours in vivo. Our cell-based chemical fingerprinting approach revealed that cancer cells with shorter telomeres and lower expression of lamin A, a nuclear architectural protein, exhibited higher sensitivity to the acute deleterious effects of MST-312, accompanied by formation of telomere dysfunction-induced foci and DNA double-strand breaks. Telomere elongation and lamin A overexpression attenuated telomeric and non-telomeric DNA damage, respectively, and both conferred resistance to apoptosis induced by MST-312 and other DNA damaging anticancer agents. These observations suggest that sufficient pools of telomeres and a nuclear lamina component contribute to the cellular robustness against DNA damage induced by therapeutic treatment in human cancer cells.

Abstract The radioactive materials are generally concentrated downwind of their origins when the prevailing winds blow continuously in one direction. If this principle determined the pattern of dispersion in all cases, dispersion directions could be estimated by wind patterns. However, this hypothesis has not been sufficiently verified because of the complexity of dispersion processes and weather systems. Here, we show that dispersion directions, which are divided into four ranges, can be estimated by wind patterns using a machine learning approach. The five-year average hit rates of the directions of dispersion estimated using near-surface winds exceed 0.85 in all months. The dispersion directions can be estimated up to 33 hours in advance using forecast winds. In particular, high hit rates exceeding 0.95 are achieved in January and March, when large-scale weather systems dominate. These results indicate that the dispersion directions are determined by the wind patterns that correspond to large-scale weather systems and diurnal circulation patterns in most cases. Our findings also provide more reliable information on dispersion patterns with reduced uncertainties, given that reasonable skill is achieved at a sufficient lead time for evacuation.

Abstract Hepatocellular carcinoma (HCC) is a typical hyper-vascular tumor, so the understanding the mechanisms of angiogenesis in HCC is very important for its treatment. However, the influence of the exosomes secreted from HCC cells (HCC-exosomes) on angiogenesis remains poorly understood. We herein examined the effects of the exosomes secreted from HepG2 cells (HepG2-exosomes) on the lumen formation of human umbilical vein endothelial cells (HUVECs) by the imaging of angiogenesis. The degree of lumen formation of HUVECs was dependent on the number of HepG2-exosomes. The HepG2-exosomes expressed NKG2D, an activating receptor for immune cells, and HSP70, a stress-induced heat shock protein associated with angiogenesis through the vascular endothelial growth factor (VEGF) receptor. In addition, the HepG2-exosomes contained several microRNAs (miRNAs) reported to exist in the serum of HCC patients. These results suggest that the HCC-exosomes play an important role in angiogenesis. Further studies on the function of HCC-exosomes may provide a new target for HCC treatment.

Barnyardgrass is a problematic agricultural weed. Here, via genomic analysis, Guo et al. identify genes potentially underlying its success, including a gene cluster activated in response to co-cultivation with rice that leads to synthesis of the allelochemical DIMBOA.

Abstract We previously showed that disruption of intermolecular interactions, e.g., by lowering the molecular planarity and/or introducing bent structures, improves the aqueous solubility of compounds, and based upon that work, we hypothesized that azobenzene trans-to-cis photoswitching could also be utilized to enhance the aqueous solubility of compounds. Here, we demonstrate that UV/visible light irradiation can reversibly switch the aqueous solubilization of an anti-cancer candidate drug, a low-molecular-weight kinase inhibitor bearing an azobenzene moiety. The increase of solubilization associated with UV-induced trans-to-cis conversion may have clinical relevance, because the time-scale of thermal cis-to-trans reversion at 37 °C is longer than that of oral absorption.

Abstract G-quadruplex (G4) is a higher-order nucleic acid structure that is formed by guanine-rich sequences. G4 stabilization by small-molecule compounds called G4 ligands often causes cytotoxicity, although the potential medicinal impact of this effect has not been fully established. Here we demonstrate that a synthetic G4 ligand, Y2H2-6M(4)-oxazole telomestatin derivative (6OTD), limits the growth of intractable glioblastoma (grade IV glioma) and glioma stem cells (GSCs). Experiments involving a human cancer cell line panel and mouse xenografts revealed that 6OTD exhibits antitumor activity against glioblastoma. 6OTD inhibited the growth of GSCs more potently than it did the growth of differentiated non-stem glioma cells (NSGCs). 6OTD caused DNA damage, G1 cell cycle arrest, and apoptosis in GSCs but not in NSGCs. These DNA damage foci tended to colocalize with telomeres, which contain repetitive G4-forming sequences. Compared with temozolomide, a clinical DNA-alkylating agent against glioma, 6OTD required lower concentrations to exert anti-cancer effects and preferentially affected GSCs and telomeres. 6OTD suppressed the intracranial growth of GSC-derived tumors in a mouse xenograft model. These observations indicate that 6OTD targets GSCs through G4 stabilization and promotion of DNA damage responses. Therefore, G4s are promising therapeutic targets for glioblastoma.

Plasma is regarded as a solution of precursor polymers specifically transformed to gel-forming polymers by a reaction with initiators. We developed a theory for the gel growth dynamics of plasma induced by contact with a source of gelators that are yielded by the initiation. In developing the theory, we combined the Ginzburg–Landau type dynamics with the gelator diffusion dynamics expressed by the moving boundary picture. The theory predicts the crossover of the rate-limiting process in the time course of the thickness of the gel layer X from the energy-limited process expressed by X∼t to the diffusion-limited process expressed by X∼t, where t is the time elapsed from when the plasma comes into contact with the source of gelators. A demonstration experiment was performed by placing a tissue factor coating plate as the initiator in plasma. Log–log plot of X vs. t showed a crossover as predicted by the theory, and the parameters characterizing plasma were determined.

The volume of a cylindrical polyacrylamide gel was measured when immersed in a binary mixture of isobutyric acid–water at different temperatures and weight fractions of isobutyric acid. Near the upper critical solution temperature of the binary mixture, the curve for gel volume vs. isobutyric acid weight fraction has a shoulder or a peak near the critical weight fraction. On the other hand, in a region away from the critical temperature, the gel volume decreased monotonically with increasing isobutyric acid weight fraction. The cloud point temperature of the binary mixture inside the gel was lower than that outside the gel. Thermodynamic description for the gel in the critical mixture is derived on the basis of the Ising model. By the description, the experimental results are explained consistently. The theoretical analysis shows that the shoulder and the peak appearing in the swelling behavior of the gel are respectively induced by the criticalities of the binary mixture outside and inside the gel. It also shows that the cloud point temperature lowering of the binary mixture inside the gel is attributed to the effective enhancement of the temperature of the binary mixture inside the gel induced by the presence of the gel polymer.

[This corrects the article DOI: 10.1371/journal.pone.0171274.].

Intracellular thioautotrophic symbionts of deep-sea vesicomyid clams lack some DNA repair genes and are thought to be undergoing reductive genome evolution (RGE). In this study, we addressed two questions, 1) how these symbionts lost their DNA repair genes and 2) how such losses affect RGE. For the first question, we examined genes associated with nucleotide excision repair (NER; uvrA, uvrB, uvrC, uvrD, uvrD paralog [uvrDp] and mfd) in 12 symbionts of vesicomyid clams belonging to two clades (5 clade I and 7 clade II symbionts). While uvrA, uvrDp and mfd were conserved in all symbionts, uvrB and uvrC were degraded in all clade I symbionts but were apparently intact in clade II symbionts. UvrD was disrupted in two clade II symbionts. Among the intact genes in Ca. Vesicomyosocius okutanii (clade I), expressions of uvrD and mfd were detected by reverse transcription-polymerase chain reaction (RT-PCR), but those of uvrA and uvrDp were not. In contrast, all intact genes were expressed in the symbiont of Calyptogena pacifica (clade II). To assess how gene losses affect RGE (question 2), genetic distances of the examined genes in symbionts from Bathymodiolus septemdierum were shown to be larger in clade I than clade II symbionts. In addition, these genes had lower guanine+cytosine (GC) content and higher repeat sequence densities in clade I than measured in clade II. Our results suggest that NER genes are currently being lost from the extant lineages of vesicomyid clam symbionts. The loss of NER genes and mutY in these symbionts is likely to promote increases in genetic distance and repeat sequence density as well as reduced GC content in genomic genes, and may have facilitated reductive evolution of the genome.

Lipid nanoparticles (LNPs) or liposomes are the most widely used drug carriers for nanomedicines. The size of LNPs is one of the essential factors affecting drug delivery efficiency and therapeutic efficiency. Here, we demonstrated the effect of lipid concentration and mixing performance on the LNP size using microfluidic devices with the aim of understanding the LNP formation mechanism and controlling the LNP size precisely. We fabricated microfluidic devices with different depths, 11 μm and 31 μm, of their chaotic micromixer structures. According to the LNP formation behavior results, by using a low concentration of the lipid solution and the microfluidic device equipped with the 31 μm chaotic mixer structures, we were able to produce the smallest-sized LNPs yet with a narrow particle size distribution. We also evaluated the mixing rate of the microfluidic devices using a laser scanning confocal microscopy and we estimated the critical ethanol concentration for controlling the LNP size. The critical ethanol concentration range was estimated to be 60-80% ethanol. Ten nanometer-sized tuning of LNPs was achieved for the optimum residence time at the critical concentration using the microfluidic devices with chaotic mixer structures. The residence times at the critical concentration necessary to control the LNP size were 10, 15-25, and 50 ms time-scales for 30, 40, and 50 nm-sized LNPs, respectively. Finally, we proposed the LNP formation mechanism based on the determined LNP formation behavior and the critical ethanol concentration. The precise size-controlled LNPs produced by the microfluidic devices are expected to become carriers for next generation nanomedicines and they will lead to new and effective approaches for cancer treatment.

Individuals with autism spectrum disorder (ASD) show superior performance in processing fine detail, but often exhibit impaired gestalt face perception. The ventral visual stream from the primary visual cortex (V1) to the fusiform gyrus (V4) plays an important role in form (including faces) and color perception. The aim of this study was to investigate how the ventral stream is functionally altered in ASD. Visual evoked potentials were recorded in high-functioning ASD adults (n = 14) and typically developing (TD) adults (n = 14). We used three types of visual stimuli as follows: isoluminant chromatic (red/green, RG) gratings, high-contrast achromatic (black/white, BW) gratings with high spatial frequency (HSF, 5.3 cycles/degree), and face (neutral, happy, and angry faces) stimuli. Compared with TD controls, ASD adults exhibited longer N1 latency for RG, shorter N1 latency for BW, and shorter P1 latency, but prolonged N170 latency, for face stimuli. Moreover, a greater difference in latency between P1 and N170, or between N1 for BW and N170 (i.e., the prolongation of cortico-cortical conduction time between V1 and V4) was observed in ASD adults. These findings indicate that ASD adults have enhanced fine-form (local HSF) processing, but impaired color processing at V1. In addition, they exhibit impaired gestalt face processing due to deficits in integration of multiple local HSF facial information at V4. Thus, altered ventral stream function may contribute to abnormal social processing in ASD.

Symbiont transmission is a key event for understanding the processes underlying symbiotic associations and their evolution. However, our understanding of the mechanisms of symbiont transmission remains still fragmentary. The deep-sea clam Calyptogena okutanii harbours obligate sulfur-oxidizing intracellular symbiotic bacteria in the gill epithelial cells. In this study, we determined the localization of their symbiont associating with the spawned eggs, and the population size of the symbiont transmitted via the eggs. We show that the symbionts are located on the outer surface of the egg plasma membrane at the vegetal pole, and that each egg carries approximately 400 symbiont cells, each of which contains close to 10 genomic copies. The very small population size of the symbiont transmitted via the eggs might narrow the bottleneck and increase genetic drift, while polyploidy and its transient extracellular lifestyle might slow the rate of genome reduction. Additionally, the extracellular localization of the symbiont on the egg surface may increase the chance of symbiont exchange. This new type of extracellular transovarial transmission provides insights into complex interactions between the host and symbiont, development of both host and symbiont, as well as the population dynamics underlying genetic drift and genome evolution in microorganisms.

We present a scaling model based on a moving boundary picture to describe heterogeneous gelation dynamics. The dynamics of gelation induced by different gelation mechanisms is expressed by the scaled equation for the time taken for development of the gel layer with a few kinetic coefficients characterizing the system. The physical meaning obtained by the analysis for a simple boundary condition from the standpoint of the phase transition shows that the time development of the gelation layer depends on whether the dynamics of the order parameter expressing the gelation of the polymer solution is fast or slow compared with the diffusion of the gelators in the heterogeneous gelation. The analytical method is used to understand the coagulation of blood from various animals. An experiment using systems with plasma coagulation occurring at interfaces with calcium chloride solution and with packed erythrocytes is performed to provide the data for model fitting and it is clarified that a few key kinetic coefficients in plasma coagulation can be estimated from the analysis of gelation dynamics.

ObjectiveIntra-arterial stem cell transplantation exerts neuroprotective effects for ischemic stroke. However, the optimal therapeutic time window and mechanisms have not been completely understood. In this study, we investigated the relationship between the timing of intra-arterial transplantation of allogeneic mesenchymal stem cells (MSCs) in ischemic stroke model in rats and its efficacy in acute phase.MethodsAdult male Wistar rats weighing 200 to 250 g received right middle cerebral artery occlusion (MCAO) for 90 minutes. MSCs (1 × 10(6) cells/ 1 ml PBS) were intra-arterially injected at either 1, 6, 24, or 48 hours (1, 6, 24, 48 h group) after MCAO. PBS (1 ml) was intra-arterially injected to control rats at 1 hour after MCAO. Behavioral test was performed immediately after reperfusion, and at 3, 7 days after MCAO using the Modified Neurological Severity Score (mNSS). Rats were euthanized at 7 days after MCAO for evaluation of infarct volumes and the migration of MSCs. In order to explore potential mechanisms of action, the upregulation of neurotrophic factor and chemotactic cytokine (bFGF, SDF-1α) induced by cell transplantation was examined in another cohort of rats that received intra-arterial transplantation at 24 hours after recanalization then euthanized at 7 days after MCAO for protein assays.ResultsBehavioral test at 3 and 7 days after transplantation revealed that stroke rats in 24h group displayed the most robust significant improvements in mNSS compared to stroke rats in all other groups (p'sConclusionsThese results suggest that intra-arterial allogeneic transplantation of MSCs provides post-stroke functional recovery and reduction of infarct volumes in ischemic stroke model of rats. The upregulation of bFGF and SDF-1α likely played a key mechanistic role in enabling MSC to afford functional effects in stroke. MSC transplantation at 24 hours after recanalization appears to be the optimal timing for ischemic stroke model, which should guide the design of clinical trials of cell transplantation for stroke patients.

Fibroblast-like stromal cells modulate cancer cells through secreted factors and adhesion, but those factors are not fully understood. Here, we have identified critical stromal factors that modulate cancer growth positively and negatively. Using a cell co-culture system, we found that gastric stromal cells secreted IL-6 as a growth and survival factor for gastric cancer cells. Moreover, gastric cancer cells secreted PGE2 and TNFα that stimulated IL-6 secretion by the stromal cells. Furthermore, we found that stromal cells secreted glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Extracellular GAPDH, or its N-terminal domain, inhibited gastric cancer cell growth, a finding confirmed in other cell systems. GAPDH bound to E-cadherin and downregulated the mTOR-p70S6 kinase pathway. These results demonstrate that stromal cells could regulate cancer cell growth through the balance of these secreted factors. We propose that negative regulation of cancer growth using GAPDH could be a new anti-cancer strategy.

In clinical practice, deep brain stimulation (DBS) is effective for treatment of motor symptoms in Parkinson's disease (PD). However, the mechanisms have not been understood completely. There are some reports that electrical stimulation exerts neuroprotective effects on the central nervous system diseases including cerebral ischemia, head trauma, epilepsy and PD, although there are a few reports on neuroprotective effects of spinal cord stimulation (SCS). We investigated the neuroprotective effects of high cervical SCS on PD model of rats. Adult female Sprague-Dawley rats received hour-long SCS (2, 50 or 200 Hz) with an epidural electrode at C1-2 level for 16 consecutive days. At 2 days after initial SCS, 6-hydroxydopamine (6-OHDA) was injected into the right striatum of rats. Behavioral evaluations of PD symptoms were employed, including cylinder test and amphetamine-induced rotation test performed at 1 and 2 weeks after 6-OHDA injection. Animals were subsequently euthanized for immunohistochemical investigations. In order to explore neurotrophic and growth factor upregulation induced by SCS, another cohort of rats that received 50 Hz SCS was euthanized at 1 and 2 weeks after lesion for protein assays. Behavioral tests revealed that the number of amphetamine-induced rotations decreased in SCS groups. Immunohistochemically, tyrosine hydroxylase (TH)-positive fibers in the striatum were significantly preserved in SCS groups. TH-positive neurons in the substantia nigra pars compacta were significantly preserved in 50 Hz SCS group. The level of vascular endothelial growth factor (VEGF) was upregulated by SCS at 1 week after the lesion. These results suggest that high cervical SCS exerts neuroprotection in PD model of rats, at least partially by upregulation of VEGF. SCS is supposed to suppress or delay PD progression and might become a less invasive option for PD patients, although further preclinical and clinical investigations are needed to confirm the effectiveness and safety.

We recently reported isolation of viable rat amniotic fluid-derived stem (AFS) cells [1]. Here, we tested the therapeutic benefits of AFS cells in a rodent model of ischemic stroke. Adult male Sprague-Dawley rats received a 60-minute middle cerebral artery occlusion (MCAo). Thirty-five days later, animals exhibiting significant motor deficits received intravenous transplants of rat AFS cells or vehicle. At days 60-63 post-MCAo, significant recovery of motor and cognitive function was seen in stroke animals transplanted with AFS cells compared to vehicle-infused stroke animals. Infarct volume, as revealed by hematoxylin and eosin (H&E) staining, was significantly reduced, coupled with significant increments in the cell proliferation marker, Ki67, and the neuronal marker, MAP2, in the dentate gyrus (DG) [2] and the subventricular zone (SVZ) of AFS cell-transplanted stroke animals compared to vehicle-infused stroke animals. A significantly higher number of double-labeled Ki67/MAP2-positive cells and a similar trend towards increased Ki67/MAP2 double-labeling were observed in the DG and SVZ of AFS cell-transplanted stroke animals, respectively, compared to vehicle-infused stroke animals. This study reports the therapeutic potential of AFS cell transplantation in stroke animals, possibly via enhancement of endogenous repair mechanisms.

The sea slug Plakobranchus ocellatus (Sacoglossa, Gastropoda) retains photosynthetically active chloroplasts from ingested algae (functional kleptoplasts) in the epithelial cells of its digestive gland for up to 10 months. While its feeding behavior has not been observed in natural habitats, two hypotheses have been proposed: 1) adult P. ocellatus uses kleptoplasts to obtain photosynthates and nutritionally behaves as a photoautotroph without replenishing the kleptoplasts; or 2) it behaves as a mixotroph (photoautotroph and herbivorous consumer) and replenishes kleptoplasts continually or periodically. To address the question of which hypothesis is more likely, we examined the source algae for kleptoplasts and temporal changes in kleptoplast composition and nutritional contribution. By characterizing the temporal diversity of P. ocellatus kleptoplasts using rbcL sequences, we found that P. ocellatus harvests kleptoplasts from at least 8 different siphonous green algal species, that kleptoplasts from more than one species are present in each individual sea slug, and that the kleptoplast composition differs temporally. These results suggest that wild P. ocellatus often feed on multiple species of siphonous algae from which they continually obtain fresh chloroplasts. By estimating the trophic position of wild and starved P. ocellatus using the stable nitrogen isotopic composition of amino acids, we showed that despite the abundance of kleptoplasts, their photosynthates do not contribute greatly to the nutrition of wild P. ocellatus, but that kleptoplast photosynthates form a significant source of nutrition for starved sea slugs. The herbivorous nature of wild P. ocellatus is consistent with insights from molecular analyses indicating that kleptoplasts are frequently replenished from ingested algae, leading to the conclusion that natural populations of P. ocellatus do not rely on photosynthesis but mainly on the digestion of ingested algae.

BACKGROUND:The rotenone-insensitive internal NADH-quinone oxidoreductase from yeast, Ndi1, has been shown to work as a replacement molecule for complex I in the respiratory chain of mammalian mitochondria. In the so-called transkingdom gene therapy, one major concern is the fact that the yeast protein is foreign in mammals. Long term expression of Ndi1 observed in rodents with no apparent damage to the target tissue was indicative of no action by the host's immune system. METHODOLOGY/PRINCIPAL FINDINGS:In the present study, we examined rat skeletal muscles expressing Ndi1 for possible signs of inflammatory or immune response. In parallel, we carried out delivery of the GFP gene using the same viral vector that was used for the NDI1 gene. The tissues were subjected to H&E staining and immunohistochemical analyses using antibodies specific for markers, CD11b, CD3, CD4, and CD8. The data showed no detectable signs of an immune response with the tissues expressing Ndi1. In contrast, mild but distinctive positive reactions were observed in the tissues expressing GFP. This clear difference most likely comes from the difference in the location of the expressed protein. Ndi1 was localized to the mitochondria whereas GFP was in the cytosol. CONCLUSIONS/SIGNIFICANCE:We demonstrated that Ndi1 expression did not trigger any inflammatory or immune response in rats. These results push forward the Ndi1-based molecular therapy and also expand the possibility of using foreign proteins that are directed to subcellular organelle such as mitochondria.

Leber's hereditary optic neuropathy (LHON) is a maternally inherited disorder with point mutations in mitochondrial DNA which result in loss of vision in young adults. The majority of mutations reported to date are within the genes encoding the subunits of the mitochondrial NADH-quinone oxidoreductase, complex I. Establishment of animal models of LHON should help elucidate mechanism of the disease and could be utilized for possible development of therapeutic strategies.We established a rat model which involves injection of rotenone-loaded microspheres into the optic layer of the rat superior colliculus. The animals exhibited the most common features of LHON. Visual loss was observed within 2 weeks of rotenone administration with no apparent effect on retinal ganglion cells. Death of retinal ganglion cells occurred at a later stage. Using our rat model, we investigated the effect of the yeast alternative NADH dehydrogenase, Ndi1. We were able to achieve efficient expression of the Ndi1 protein in the mitochondria of all regions of retinal ganglion cells and axons by delivering the NDI1 gene into the optical layer of the superior colliculus. Remarkably, even after the vision of the rats was severely impaired, treatment of the animals with the NDI1 gene led to a complete restoration of the vision to the normal level. Control groups that received either empty vector or the GFP gene had no effects.The present study reports successful manifestation of LHON-like symptoms in rats and demonstrates the potential of the NDI1 gene therapy on mitochondrial optic neuropathies. Our results indicate a window of opportunity for the gene therapy to be applied successfully after the onset of the disease symptoms.

It is widely recognized that mitochondrial dysfunction, most notably defects in the NADH-quinone oxidoreductase (complex I), is closely related to the etiology of sporadic Parkinson's disease (PD). In fact, rotenone, a complex I inhibitor, has been used for establishing PD models both in vitro and in vivo. A rat model with chronic rotenone exposure seems to reproduce pathophysiological conditions of PD more closely than acute mouse models as manifested by neuronal cell death in the substantia nigra and Lewy body-like cytosolic aggregations. Using the rotenone rat model, we investigated the protective effects of alternative NADH dehydrogenase (Ndi1) which we previously demonstrated to act as a replacement for complex I both in vitro and in vivo. A single, unilateral injection of recombinant adeno-associated virus carrying the NDI1 gene into the vicinity of the substantia nigra resulted in expression of the Ndi1 protein in the entire substantia nigra of that side. It was clear that the introduction of the Ndi1 protein in the substantia nigra rendered resistance to the deleterious effects caused by rotenone exposure as assessed by the levels of tyrosine hydroxylase and dopamine. The presence of the Ndi1 protein also prevented cell death and oxidative damage to DNA in dopaminergic neurons observed in rotenone-treated rats. Unilateral protection also led to uni-directional rotation of the rotenone-exposed rats in the behavioral test. The present study shows, for the first time, the powerful neuroprotective effect offered by the Ndi1 enzyme in a rotenone rat model of PD.