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Yamamoto, H.

Publications and source records attributed to Yamamoto, H..

At least 37 records · Page 2Linked to original sources

Microfluidic platforms for probing spontaneous functional recovery in hierarchically modular neuronal networks

Inherent capacity to flexibly reorganize after injury is a hallmark of brain networks, and recent studies suggest that functional consequences of focal damage are strongly influenced by the networks non-random connectivity. Although many of these insights have been derived from animal models and computational simulations, experimental platforms that enable bottom-up investigations of the structure-function relationships underlying damage and recovery processes remain limited. In this study, we used polydimethylsiloxane microfluidic devices to construct hierarchically modular neuronal networks that mimic the architectural features of the mammalian cortex. Laser microdissection was employed to selectively sever intermodular connections, enabling controlled damage to either hub or peripheral connections. Damage to hub connections led to delayed recovery, requiring more than three days for correlations to re-emerge. In contrast, peripheral damage resulted in faster recovery. Experiments that induced repeated injury to neuronal networks further demonstrated that recovery primarily occurred through the formation of alternative pathways rather than restoration of the original connections. These findings highlight how topological features of neuronal networks shape their response to injury and subsequent reorganization, providing mechanistic insights into the intrinsic self-repair capacity of biological systems.

neuroscience↗

Deep Learning-coupled Proximity Proteomics to Deconvolve Kinase Signaling In Vivo

Deconvolving the substrates of hundreds of kinases linked to phosphorylation networks driving cellular behavior is a fundamental, unresolved biological challenge, largely due to the poorly understood interplay of kinase selectivity and substrate proximity. We introduce KolossuS, a deep learning framework leveraging protein language models to decode kinase-substrate specificity. KolossuS achieves superior prediction accuracy and sensitivity across mammalian kinomes, enabling proteome-wide predictions and evolutionary insights. By integrating KolossuS with CRISPR-based proximity proteomics in vivo, we capture kinase-substrate recognition and spatial context, obviating prior limitations. We show this combined framework identifies kinase substrates associated with physiological states such as sleep, revealing both known and novel Sik3 substrates during sleep deprivation. This novel integrated computational-experimental approach promises to transform systematic investigations of kinase signaling in health and disease.

cell biology↗

Unraveling anti-inflammatory metabolic signatures of Glycyrrhiza uralensis and isoliquiritigenin through multiomics

Glycyrrhiza uralensis, known for its diverse pharmacological effects, including immunoregulation, anti-tumor, and antioxidant properties, is a widely used medicinal plant found in more than 70% of traditional herbal medicines (Kampo) in Japan. Although over 300 compounds have been discovered in G. uralensis, the molecular mechanisms underlying its bioactivity remain largely unknown due to the chemical diversity of its compounds. Here, we performed a multiomics analysis incorporating untargeted hydrophilic metabolomics, lipidomics, and phosphoproteomics to elucidate the molecular mechanisms distinguishing the effects of a single bioactive compound, isoliquiritigenin (ILG), and the extract of G. uralensis (GU). Multiomics time-course data were obtained for lipopolysaccharide (LPS)-stimulated RAW264.7 cells under four experimental conditions: control, LPS(+), LPS(+)/ILG(+), and LPS(+)/GU(+), where 182 hydrophilic metabolites, 381 lipids, and 13,211 phosphopeptides were characterized. The metabolic signatures of inflammatory macrophages, including increased levels of glycolytic intermediates, succinate, citrulline, triacylglycerols, and cholesteryl esters, were attenuated in both the GU(+) and ILG(+) groups. Using a multivariate approach based on a partial least squares algorithm with an imposed inflammation level order information, we identified upregulated phosphorylation of sirtuin 1 and 2 (SIRT1/2) along with alterations in nicotinamide adenine dinucleotide metabolism in the ILG(+) group. The inhibition of SIRT2 suppressed the anti-inflammatory effect of ILG, as indicated by a reduction in interleukin-6 (IL-6) levels. Furthermore, we discovered a substantial increase in {gamma}-aminobutyric acid (GABA) and its downstream metabolite, 4-guanidinobutyric acid, in the GU(+) group. These increases were attributed to endogenous GABA production through glutamic acid decarboxylase rather than uptake via GABA transporters. Exogenous GABA administration significantly suppressed IL-6 and IL-1{beta} expression in LPS-stimulated cells, and the simultaneous administration of GABA and ILG enhanced the anti-inflammatory effects. Consequently, this study presents an approach to elucidating the importance of traditional herbal formulations and demonstrates the utility of multiomics in uncovering that endogenous GABA production would facilitate anti-inflammatory effects with ILG in GU administration.

systems biology↗

Modular architecture confers robustness to damage and facilitates recovery in spiking neural networks modeling in vitro neurons

Impaired brain function is restored following injury through dynamic processes that involve synaptic plasticity. This restoration is supported by the brains inherent modular organization, which promotes functional separation and redundancy. However, it remains unclear how the modular structure interacts with synaptic plasticity, most notably in the form of spike-timing-dependent plasticity (STDP), to define the damage response and recovery efficiency. In this work, we numerically modeled the response and recovery to damage of a neuronal network in vitro bearing a modular structure. Consistent with the in vitro observations, the in silico numerical model effectively captured the decline and subsequent recovery of spontaneous activity following the injury. We revealed that the modular structure confers robustness to injury, minimizes the decrease in neuronal activity, and promotes recovery via STDP. Finally, using the reservoir computing framework, we show that information representation in the neuronal network improves with the recovery of synchronous activity. Our work provides an experimental-numerical platform for predicting recovery in damaged neuronal networks and may help developing effective models for brain injury.

neuroscience↗

Implication of S-1 biomarkers on aging and prognosis in elderly patients with lung cancer treated by adjuvant S-1 chemotherapy: Accompanying results of the Setouchi Lung Cancer Group Study 1201

BACKGROUNDSThe management of elderly patients presents several challenges due to age-related declines; however recent recommendations advocate for age not being the sole determinant for adjuvant treatment decisions in patients with non-small cell lung cancer (NSCLC). Aging may alter expression levels of 5-fluorouracil (5-FU) biomarkers. METHODSExpression changes with aging were explored using The Cancer Genome Atlas (TCGA) database. 5-FU-related biomarker expressions, including thymidylate synthase (TS), dihydropyrimidine dehydrogenase (DPD), orotate phosphoribosyltransferase, epidermal growth factor receptor (EGFR), and excision repair cross-complementation group-1 (ERCC1), were assessed by the quantitative reverse-transcription PCR assays in 89 NSCLCs elderly patients ([&ge;] 75 years old) receiving adjuvant S-1, an oral fluoropyrimidine agent, therapy in a SLCG1201 trial. RESULTS: TCGA database analysis (n=955) indicated decreased TS expression with aging, particularly in those 75 or older. In the SCLG1201 trial, univariate analysis revealed that high EGFR and low TS expressions correlated with favorable recurrence-free survival (RFS)(p=0.0264) and overall survival (OS)(p=0.0365), respectively. Multivariable analysis confirmed pathological stage as an independent prognostic factor for both RFS and OS (p=0.0052 and 0.0352, respectively). EGFR mutant tumors (n=27) showed significantly higher DPD (p=0.0066) and EGFR (p<0.0001) expressions, and lower TS(p=0.0125) and ERCC1(p=0.0015) expressions. CONCLUSIONDespite pathological stage being an independent prognostic factor, high EGFR and low TS expressions may predict better clinical outcomes in elderly NSCLC patients using adjuvant S-1. The age-related decrease in TS expression supports the potential benefit of 5-FU-based therapy in them compared to younger patients. Further research is warranted to validate these clinical implications.

pharmacology and toxicology↗

A simple immunohistochemical method for perinatal mammalian ovaries revealed different kinetics of oocyte apoptosis caused by DNA damage and asynapsis.

Oocytes having meiotic defects are assumed to be eliminated by apoptosis in perinatal period. However, the oocyte apoptosis caused by meiotic defects has not been well analyzed, partly due to the great technical demands for tissue sectioning of perinatal ovaries. In the present study, we applied a squash method for immunohistochemical analysis of perinatal mouse ovaries as a substitute for tissue sectioning. As a result, we could show different kinetics of apoptosis caused by DMC1- and SPO11-deficiencies, indicating that DNA damage-induced apoptosis precedes asynapsis-induced apoptosis in mouse oocytes. Double mutant analysis revealed that only asynapsis-induced apoptosis was significantly dependent on HORMAD2. The present method is simple, easy, and able to analyze a sufficient number of oocytes for detecting infrequent events in a single specimen, accelerating detailed immunohistochemical analyses of mammalian ovaries during the fetal and perinatal periods.

developmental biology↗

The triad interaction of ULK1, ATG13, and FIP200 is required for ULK complex formation and autophagy

In mammals, autophagosome formation, a central event in autophagy, is initiated by the ULK complex comprising ULK1/2, FIP200, ATG13, and ATG101. However, the structural basis and mechanism underlying the ULK complex assembly have yet to be fully clarified. Here, we predicted the core interactions organizing the ULK complex using AlphaFold, which proposed that the intrinsically disordered region of ATG13 engages the bases of the two UBL domains in the FIP200 dimer via two phenylalanines and also binds the tandem MIT domain of ULK1, thereby yielding the 1:1:2 stoichiometry of the ULK1-ATG13-FIP200 complex. We validated the predicted interactions by point mutations and demonstrated direct triad interactions among ULK1, ATG13, and FIP200 in vitro and in cells, wherein each interaction was additively important for autophagic flux. These results indicate that the ULK1-ATG13- FIP200 triadic interaction is crucial for autophagosome formation and provide a structural basis and insights into the regulation mechanism of autophagy initiation in mammals.

cell biology↗

Synergistic reinforcement learning by cooperation of the cerebellum and basal ganglia

The cerebral cortex, cerebellum and basal ganglia are essential for flexible learning in mammals. Although traditionally thought to operate under different learning rules, recent evidence suggests that both the basal ganglia and the cerebellum may employ reinforcement learning mechanisms. This raises the question of how these structures coordinate when a common reward prediction error mechanism is active. To address this issue, we first examined output signals from the basal ganglia and cerebellum following the activity of the cerebral cortex. We recorded single-neuron activity from the output regions of the cerebellum and basal ganglia - the cerebellar nuclei (CN) and substantia nigra pars reticulata (SNr) - in both male and female ChR2 transgenic rats. Neurons in the CN and SNr exhibited distinct temporal response patterns; notably, the fast excitatory response in the CN, driven by mossy fiber input, was synchronized with the inhibitory response in the SNr, mediated via the direct pathway. Using these experimental findings together with connectome data, we developed both a semi-realistic spiking network model and a reservoir-based reinforcement learning model. In the latter model, successful learning depended on synaptic plasticity in both the cerebellum and basal ganglia with a temporal precision on the order of 10 ms. Furthermore, cortical {beta}-oscillations enhanced learning and optimal reinforcement learning occurred when the output of cerebellar and basal ganglia signal phase-locked at the frequency of cortical oscillation. Taken together, our results suggest that the coordinated output of the cerebellum and basal ganglia, driven by tightly tuned cortical input, underlies brain-wide synergistic reinforcement learning. Significance StatementThe cerebral cortex, cerebellum, and basal ganglia support learning. Recent research suggests that both the basal ganglia and cerebellum use a similar learning process called reinforcement learning, which involves predicting rewards. To understand how these brain regions work together, we recorded brain activity in rats while photo-stimulating the cerebral cortex. We found that two types of responses in the cerebellum and basal ganglia were synchronized, which might help activate the cerebral cortex. A computer model showed that precise timing of signals from both the cerebellum and basal ganglia is important for learning. This timing was important only when the cerebral cortex worked in a specific frequency range. Our findings suggest that coordinated brain activity enhances learning.

neuroscience↗

A nitrogenase-like enzyme is involved in the novel anaerobic assimilation pathway of a sulfonate, isethionate, in the photosynthetic bacterium Rhodobacter capsulatus

Prokaryotes contribute to the global sulfur cycle by using diverse sulfur compounds as sulfur sources or electron acceptors. Here we report that a nitrogenase-like enzyme (NFL) and a radical SAM enzyme (RSE) are involved in the novel anaerobic assimilation pathway of a sulfonate, isethionate, in the photosynthetic bacterium Rhodobacter capsulatus. The nflHDK genes for NFL are localized at a locus containing genes for known sulfonate metabolism in the genome. A gene nflB encoding an RSE is present just upstream of nflH, forming a small gene cluster nflBHDK. Mutants lacking any nflBHDK genes lost the ability to grow with isethionate as the sole sulfur source under anaerobic photosynthetic conditions, indicating that all four NflBHDK proteins are essential for the isethionate assimilation pathway. Heterologous expression of the islAB genes encoding a known isethionate lyase that degrades isethionate to sulfite and acetaldehyde restored the isethionate-dependent growth of a mutant lacking nflDK, indicating that the enzyme encoding nflBHDK is involved in an isethionate assimilation reaction to release sulfite. Furthermore, heterologous expression of nflBHDK and ssuCAB encoding an isethionate transporter in the closely related species R. sphaeroides, which does not have nflBHDK and cannot grow with isethionate as the sole sulfur source, conferred isethionate-dependent growth ability to this species. We propose to rename nflBHDK as isrBHDK (isethionate reductase). The isrBHDK genes are widely distributed among various prokaryote phyla. Discovery of the isethionate assimilation pathway by IsrBHDK provides a missing piece for the anaerobic sulfur cycle and for understanding the evolution of ancient sulfur metabolism. ImportanceNitrogenase is an important enzyme found in prokaryotes that reduces atmospheric nitrogen to ammonia and plays a fundamental role in the global nitrogen cycle. It has been noted that nitrogenase-like enzymes (NFLs), which share an evolutionary origin with nitrogenase, have evolved to catalyze diverse reactions such as chlorophyll biosynthesis (photosynthesis), coenzyme F430 biosynthesis (methanogenesis), and methionine biosynthesis. In this study, we discovered that an NFL with unknown function in the photosynthetic bacterium Rhodobacter capsulatus is a novel isethionate reductase (Isr), which catalyzes the assimilatory degradation of isethionate, a sulfonate, releasing sulfite used as the sulfur source under anaerobic conditions. Isr is widely distributed among various bacterial phyla, including intestinal bacteria, and is presumed to play an important role in sulfur metabolism in anaerobic environments such as animal guts and microbial mats. This finding provides a clue for understanding ancient metabolism that evolved under anaerobic environments at the dawn of life.

microbiology↗

Microevolution toward loss of photosynthesis: Mutations promoting dark-heterotrophic growth and suppressing photosynthetic growth in cyanobacteria

The prevalence of parasitic plants suggests frequent evolution of photosynthetic capacity loss in the natural environment. However, no studies have observed such evolutionary events as a loss of photosynthetic capacity. Herein, we report mutations that lead to a loss or decrease in photosynthetic growth capacity of dark-adapted variants of the cyanobacterium Leptolyngbya boryana, which can grow heterotrophically even in the dark. We isolated 28 dark-adapted variants through long-term cultivation (7-49 months) under dark-heterotrophic conditions. All variants showed significantly faster dark-heterotrophic growth than the parental strains, accompanied by the loss of photosynthetic growth capacity in 15 variants. Genome resequencing of the variants revealed that 19 of the 28 variants carried various mutations in a common single gene (LBDG_21500) encoding a protein phosphatase 2C (PP2C) RsbU that is involved in the partner switching system (PSS). Phenotypic and transcriptomic analyses of a LBDG_21500-knockout mutant suggested that the PSS, including LBDG_21500, is involved in the global transcriptional regulation of various genes under both photoautotrophic and dark-heterotrophic conditions. We propose the renaming of LBDG_21500 to phsP (phototrophic-heterotrophic switching phosphatase). Our results imply that mutations in the global transcriptional regulatory system serve as the first evolutionary step leading to the loss of photosynthetic capacity. ImportancePhotosynthetic organisms that grow using minimal resources: light, water, and CO2, support most heterotrophic organisms as producers on the Earth. When photosynthetic organisms thrive over long generations under environments where organic compounds are readily available, they may lose the photosynthetic capacity because of the relief of selective pressure to maintain photosynthesis. The prevalence of parasitic plants in the natural environment supports this idea. However, there have been no actual observations of evolutionary processes leading to a loss of photosynthetic growth capacity. The significance of our research is in observing microevolution of a cyanobacterium through a long-term cultivating under dark heterotrophic conditions. In particular, the high frequency of mutations to a gene involved in the global transcriptional regulatory system suggests that such mutations in regulatory systems are regarded as an example of the initial evolutionary processes toward complete loss of photosynthesis.

evolutionary biology↗

Extracellular vesicle-mediated secretion of chlorophyll biosynthetic intermediates in the cyanobacterium Leptolyngbya boryana

Extracellular vesicles (EVs) are derived from the outer membrane (OM) in Gram-negative bacteria and have diverse physiological functions such as toxin transport, extracellular signal transduction, nutrient acquisition, and horizontal gene transfer. EV-mediated secretion of monovinyl protochlorophyllide (MV-Pchlide), the chlorophyll a (Chl) biosynthetic intermediate, was previously reported in a mutant lacking dark-operative Pchlide reductase in the cyanobacterium Leptolyngbya boryana. This study showed a detailed characterization of EVs from the wild-type (WT) of L. boryana grown under photoautotrophic and dark heterotrophic conditions, focusing on the accumulation of Chl intermediates. WT L. boryana cells produce two types of EVs, low-density EVs (L-EVs) and high-density EVs (H-EVs), both under light and dark conditions. L-EVs and H-EVs showed distinct morphological features and protein compositions. L-EVs from cells grown under both light and dark conditions commonly contained carotenoids, myxol glycoside, and zeaxanthin, as major pigments. Based on the protein compositions of EVs and other cellular membrane fractions, L-EVs and H-EVs are probably derived from low-density OM and high-density OM interacting with cell walls, respectively. Fluorescence detection of pigments was applied to EVs, and the three Chl intermediates, protoporphyrin IX, demetallated Mg-protoporphyrin IX monomethyl ester, pheophorbide, and were commonly detected both L-EVs from light- and dark-grown cells, whereas L-EVs from dark-grown cells contained additional MV-Pchlide and MV-protopheophorbide. These Chl intermediates appear to transfer from the thylakoid membrane to L-EVs via an unknown transport system. Cyanobacterial EVs may play a novel function in alleviating the accumulation of Chl intermediates in cells.

microbiology↗

Using variable data independent acquisition for capillary electrophoresis-based untargeted metabolomics

Capillary electrophoresis coupled with tandem mass spectrometry (CE-MS/MS) offers advantages in peak capacity and sensitivity for metabolic profiling, owing to the electroosmotic flow-based separation. However, the utilization of data-independent MS/MS acquisition (DIA) is restricted due to the absence of an optimal procedure for analytical chemistry and its related informatics framework. We assessed the mass spectral quality using two DIA techniques, namely, all-ion fragmentation (AIF) and variable DIA (vDIA), to isolate 60[~]800 Da precursor ions with respect to annotation rates. Our findings indicate that vDIA, coupled with the updated MS-DIAL chromatogram deconvolution algorithm, yields higher spectral matching scores and annotation rates compared to AIF. Additionally, we evaluated a linear migration time (MT) correction method using internal standards to accurately align chromatographic peaks in a dataset. After the correction, the peaks exhibited less than 0.1 min MT drifts, a difference mostly equivalent to that of conventional reverse-phase liquid chromatography techniques. Moreover, we conducted MT prediction for metabolites recorded in mass spectral libraries and metabolite structure databases containing a total of 469,870 compounds, achieving an accuracy of less than 1.5 min root mean squares. Thus, our platform provides a peak annotation platform utilizing MT information, accurate precursor m/z, and the MS/MS spectrum recommended by the metabolomics standards initiative. Applying this procedure, we investigated metabolic alterations in lipopolysaccharide (LPS)-induced macrophages, characterizing 170 metabolites. Furthermore, we assigned metabolite information to unannotated peaks using an in-silico structure elucidation tool, MS-FINDER. The results were integrated into the nodes in the molecular spectrum network based on the MS/MS similarity score. Consequently, we identified a significantly increased amount of metabolites in the LPS-administration group, glycinamide ribonucleotide, not present in any spectral libraries. Additionally, we retrieved metabolites of false-negative hits in the initial spectral annotation procedure. Overall, our study underscores the potential of CE-MS/MS with DIA and computational mass spectrometry techniques for metabolic profiling.

biochemistry↗

Transcription factor activity profiling reveals the role of REST and LEF1 in the recovery from depression

Psychophysiological disorders chronically impair brain functions, often accompanied by dysregulation of multiple genes, suggesting a multifaceted etiology behind the symptoms. To explore transcription factors (TFs) involved in such transcriptomic changes, we analyzed TF-activity profiles (TFAPs) from the brains of mice experienced chronic stress, and revealed alteration in TF-activity correlating with their pathophysiological phenotypes. We identified REST/NRSF and TCF/LEF associated with depressive phenotypes and discovered that neuropsychiatric drugs sertraline and lithium influence REST- and TCF/LEF-activity, both in vitro and in vivo, thereby affecting gene expression profiles. Pharmacological or genetic manipulation of REST- or TCF/LEF-activity in defeated mice impacts post-stress recovery from depressive phenotypes, with combined treatment further augmenting the outcomes. Our TFAP analysis enhances understanding of molecular mechanisms underpinning chronic diseases, aiding future therapeutic strategy development.

neuroscience↗

A Tetravalent Dengue Virus-like Particle Vaccine Induces High levels of Neutralizing Antibodies and Inhibits Dengue Replication in Non-Human Primates

Dengue virus (DENV) represents a significant global health burden, with 50% of the worlds population at risk of infection, and there is an urgent need for next-generation vaccines. Virus-like particle (VLP)-based vaccines, which mimic the antigenic structure of the authentic virus but lack the viral genome, are an attractive approach. Here we describe a dengue VLP (DENVLP) vaccine which generates a robust and long-lasting neutralizing antibody response against all four DENV serotypes in non-human primates. Importantly, DENVLP vaccination produced no ADE response against any of four DENV serotypes. Finally, we demonstrate in a non-human primate challenge model that DENVLP vaccination substantially reduces viral replication. We also transfer the purified IgG from the immunized monkeys into immunodeficient mice, where they protect against subsequent lethal dengue virus challenge, indicating a humoral mechanism of protection. These results indicate that a DENVLP vaccine is a safe and effective vaccine candidate. One Sentence Summary: Immunization of non-human primates with a tetravalent dengue VLP vaccine induces high levels of neutralizing antibodies and reduces the severity of infection for all four dengue serotypes.

microbiology↗

Syntaxin 17 recruitment to mature autophagosomes is temporally regulated by PI4P accumulation

During macroautophagy, cytoplasmic constituents are engulfed by autophagosomes. Lysosomes fuse with closed autophagosomes but not with unclosed intermediate structures. This is achieved in part by the late recruitment of the autophagosomal SNARE syntaxin 17 (STX17) to mature autophagosomes. However, how STX17 recognizes autophagosome maturation is not known. Here, we show that this temporally regulated recruitment of STX17 depends on the positively charged C-terminal region of STX17. Consistent with this finding, mature autophagosomes are more negatively charged compared with unclosed intermediate structures. This electrostatic maturation of autophagosomes is likely driven by the accumulation of phosphatidylinositol 4-phosphate (PI4P) in the autophagosomal membrane. Accordingly, dephosphorylation of autophagosomal PI4P prevents the association of STX17 to autophagosomes. Furthermore, molecular dynamics simulations support PI4P-dependent membrane insertion of the transmembrane helices of STX17. Based on these findings, we propose a model in which STX17 recruitment to mature autophagosomes is temporally regulated by a PI4P-driven change in the surface charge of autophagosomes.

cell biology↗

TAX1BP1 recruits ATG9 vesicles through SCAMP3 binding

Macroautophagy is a cellular process that delivers cytoplasmic material to lysosomes for degradation via autophagosomes. It often involves the selective degradation of ubiquitinated proteins. During selective macroautophagy, five ubiquitin-binding adaptors, p62, NBR1, OPTN, NDP52, and TAX1BP1, form biomolecular condensates with ubiquitinated proteins and recruit ATG9 vesicles, which serve as the initial membrane source required for autophagosome formation. However, the molecular details underlying the cargo/adaptor-dependent recruitment of ATG9 vesicles remain unclear. Here, we show that ATG9 vesicles are recruited by three cargo adaptors: TAX1BP1, NBR1, and OPTN. We also find that ATG9A itself is not the determinant for recruitment by these cargo adaptors, and that TAX1BP1-dependent ATG9 vesicle recruitment is mediated by SCAMP3, a transmembrane protein on the ATG9 vesicles, through binding to the coiled-coil 1 domain of TAX1BP1. These findings provide mechanistic insights into the cargo/adaptor-dependent assembly of ATG9 vesicles in mammals.

molecular biology↗

Recombinase-Independent AAV for Anterograde Transsynaptic Tracing

Viral transsynaptic labeling has become indispensable for investigating the functional connectivity of neural circuits. Adeno-associated virus serotype 1 (AAV1) allows for anterograde transneuronal labeling and manipulation of postsynaptic neurons. However, it is limited to delivering an AAV1 expressing a recombinase which relies on using transgenic animals or genetic access to postsynaptic neurons. We reasoned that a strong expression level could overcome this limitation. To this end, we used a self-complementary AAV1 (scAAV1) under a strong promoter (CAG). We demonstrated the anterograde transneuronal efficiency of scAAV1 by delivering a fluorescent marker in mouse retina-superior colliculus and thalamic-amygdala pathways in a recombinase-independent manner. In addition to investigating neuronal connectivity, scAAV1.CAG may be suitable for functional manipulation and imaging.

neuroscience↗

Environmental RNA as a non-invasive tool for assessing toxic effects in fish: a proof-of-concept study using Japanese medaka exposed to pyrene

Although environmental RNA (eRNA) is emerging as a non-invasive tool to assess the health status of aquatic macro-organisms, the potential of eRNA remains largely untested. In this study, we investigated the ability of eRNA to detect changes in gene expression in Japanese medaka fish (Oryzias latipes) in response to sub-lethal pyrene exposure, as a model toxic chemical. We performed standardized acute toxicity tests and collected eRNA from tank water and RNA from fish tissue after 96 h of exposure. Our results showed that over 1000 genes were detected in eRNA and the sequenced read counts of these genes correlated with those in fish tissue (r = 0.50). Moreover, eRNA detected 86 differentially expressed genes in response to pyrene, some of which were shared by fish RNA, including suppression of collagen fiber genes. These results suggest that eRNA has the potential to detect changes in gene expression in fish in response to environmental stressors without the need for sacrificing or causing pain to fish. However, we also found that the majority of sequenced reads of eRNA (> 99%) were not mapped to the reference medaka genome and they originated from bacteria and fungi, resulting in low sequencing-depth. In addition, eRNA, in particular nuclear genes, was highly degraded with a median transcript integrity number (TIN) of < 20. These limitations highlight the need for future studies to improve the analytical methods of eRNA application. TOC Art O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=92 SRC="FIGDIR/small/538202v1_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@98c437org.highwire.dtl.DTLVardef@17a1f31org.highwire.dtl.DTLVardef@95d1e7org.highwire.dtl.DTLVardef@7a253_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗