Biology subjects

Inoue, K.

Publications and source records attributed to Inoue, K..

72 records · Page 4Linked to original sources

bioRxiv2020

Exploration of natural red-shifted rhodopsins using a machine learning-based Bayesian experimental design

Microbial rhodopsins are photoreceptive membrane proteins utilized as molecular tools in optogenetics. In this paper, a machine learning (ML)-based model was constructed to approximate the relationship between amino acid sequences and absorption wavelengths using ~800 rhodopsins with known absorption wavelengths. This ML-based model was specifically designed for screening rhodopsins that are red-shifted from representative rhodopsins in the same subfamily. Among 5,558 candidate rhodopsins suggested by a protein BLAST search of several protein databases, 40 were selected by the ML-based model. The wavelengths of these 40 selected candidates were experimentally investigated, and 32 (80%) showed red-shift gains. In addition, four showed red-shift gains > 20 nm, and two were found to have desirable ion-transporting properties, indicating that they were potentially useful in optogenetics. These findings suggest that an ML-based model can reduce the cost for exploring new functional proteins.

biochemistry↗

bioRxiv2020

Chloroplast chaperonin-mediated targeting of a thylakoid membrane protein

Post-translational protein targeting requires chaperone assistance to direct insertion-competent proteins to integration pathways. Chloroplasts integrate nearly all thylakoid transmembrane proteins post-translationally, but mechanisms in the stroma that assist their insertion remain largely undefined. Here, we investigated how the chloroplast chaperonin (Cpn60) facilitated the thylakoid integration of Plastidic type I signal peptidase 1 (Plsp1) using in vitro targeting assays. Cpn60 bound Plsp1 in the stroma. In isolated chloroplasts, the membrane integration of imported Plsp1 correlated with its dissociation from Cpn60. When the Plsp1 residues that interacted with Cpn60 were removed, Plsp1 did not integrate into the membrane. These results suggested Cpn60 was an intermediate in Plsp1s thylakoid targeting. In isolated thylakoids, the integration of Plsp1 decreased if Cpn60 was present in excess of cpSecA1, the stromal motor of the cpSec1 translocon which inserts unfolded Plsp1 into the thylakoid. An excess of cpSecA1 favored integration. Introducing Cpn60s obligate substrate RbcL displaced Cpn60-bound Plsp1; then, the released Plsp1 exhibited increased accessibility to cpSec1. These in vitro targeting experiments support a model in which Cpn60 captures and then releases insertion-competent Plsp1, while cpSecA1 recognizes free Plsp1 for integration. Thylakoid transmembrane proteins transiting the stroma can interact with Cpn60 to shield from the aqueous environment. One-sentence summaryThe chloroplast chaperonin captures and releases Plastidic type I signal peptidase 1 during its targeting to the thylakoid membrane.

plant biology↗

bioRxiv2020

Lab-on-a-graphene-FET detection of key molecular events underpinning influenza virus infection and effect of antiviral drugs

Small solid-state devices are candidates for accelerating biomedical assays/drug discovery, however their potential remains unfulfilled. Here, we demonstrate that graphene-field effect transistors (FET) can be used to successfully detect the key molecular events underlying viral infections and the effect of antiviral drugs. Our device success is achieved by bio-mimicking the host-cell surface during an influenza infection at the graphene channel. In-situ AFM confirms the biological interactions at the sialic acid-functionalized graphene: viral hemagglutinin (HA) binds to sialic acid, and neuraminidase (NA) reacts with the sialic acid-HA complex. The graphene-FET detects HA binding to sialic acid, and NA cleavage of sialic acid. The inhibitory effect of the drug "zanamivir" on NA-sialic acid interactions is monitored in real-time; the reaction rate constant of NA-sialic acid reaction was successfully determined. We demonstrate that graphene-FETs are powerful platforms for measurement of biomolecular interactions and contribute to future deployment of solid-state devices in drug discovery/biosensing.

bioengineering↗

bioRxiv2019

A non-mosaic humanized mouse model of Down syndrome, trisomy of the long arm of human chromosome 21 in mouse chromosome background

Down syndrome (DS) is a complex human condition, and animal models trisomic for human chromosome 21 (HSA21) genes or orthologs provide insights into better understanding and treating DS. However, HSA21 orthologs are distributed into three mouse chromosomes, preventing us from generating mouse models trisomy of a complete set of HSA21 orthologs. The only existing humanized mouse DS model, Tc1, carries a HSA21 with over 20% of protein coding genes (PCGs) disrupted. More importantly, due to the human centromere, Tc1 is mosaic (a mix of euploid and trisomic cells), which makes every mouse unique and compromises interpretation of results. Here, we used mouse artificial chromosome (MAC) technology to "clone" the 34 MB long arm of HSA21 (HSA21q). Through multiple steps of microcell-mediated chromosome transfer we created a new humanized DS mouse model, Tc(HSA21q;MAC)1Yakaz ("TcMAC21"). Constitutive EGFP expression from the transchromosome and fluorescent in situ hybridization validate that TcMAC21, containing a hybrid chromosome of HSA21q and mouse centromere, is not mosaic. Whole genome sequencing shows that TcMAC21 contains a nearly complete copy of HSA21q with 93% of intact PCGs, while RNA-seq and additional mRNA/protein expression analyses confirm that PCGs are transcribed and regulated. A battery of tests show that TcMAC21 recapitulates many DS phenotypes including morphological anomalies in heart, craniofacial skeleton and brain, pathologies at molecular and cellular level, and impairments in learning, memory and synaptic plasticity. TcMAC21 is the most complete mouse model of DS extant and has potential for supporting a wide range of basic and preclinical research. Significance StatementIn the last 25 years, mouse models of trisomy 21 have supported research into Down syndrome, from defining the basis for developmental effects up to support for clinical trials. However, existing models have significant shortfalls, especially for preclinical studies. These deficiencies include incomplete or inappropriate representation of trisomic genes, absence of an extra chromosome, and mosaicism. Using cutting edge technologies we produced a mouse artificial chromosome containing the entire 34Mb long arm of human chromosome 21 and, with assisted reproductive technologies, established it in the germ line of mice. This trisomic mouse manifests developmental and functional features of Down syndrome, including hippocampal-based learning and memory deficits. This is the most complete model of Down syndrome produced to date.

genetics↗

bioRxiv2019

Proprioception is subject-specific and improved without performance feedback

Accumulating evidence indicates that the humans proprioception map appears subject-specific. However, whether the idiosyncratic pattern persists across time with good within-subject consistency has not been quantitatively examined. Here we measured the proprioception by a hand visual-matching task in multiple sessions over two days. We found that people improved their proprioception when tested repetitively without performance feedback. Importantly, despite the reduction of average error, the spatial pattern of proprioception errors remained idiosyncratic. Based on individuals proprioceptive performance, a standard convolutional neural network classifier could identify people with good accuracy. We also found that subjects baseline proprioceptive performance could not predict their motor performance in a visual trajectory-matching task even though both tasks require accurate mapping of hand position to visual targets in the same workspace. Using a separate experiment, we not only replicated these findings but also ruled out the possibility that performance feedback during a few familiarization trials caused the observed improvement in proprioception. We conclude that the conventional proprioception test itself, even without feedback, can improve proprioception but leave the idiosyncrasy of proprioception unchanged.

neuroscience↗

bioRxiv2019

Lipid Chaperoning of a Thylakoid Protease Whose Stability is Modified by the Protonmotive Force

Protein folding is a complex cellular process often assisted by chaperones but can also be facilitated by interactions with lipids. Disulfide bond formation is a common mechanism to stabilize a protein. This can help maintain functionality amidst changes in the biochemical milieu which are especially common across energy-transducing membranes. Plastidic Type I Signal Peptidase 1 (Plsp1) is an integral thylakoid membrane signal peptidase which requires an intramolecular disulfide bond for in vitro activity. We have investigated the interplay between disulfide bond formation, lipids, and pH in the folding and activity of Plsp1. By combining biochemical approaches with a genetic complementation assay, we provide evidence that interactions with lipids in the thylakoid membrane have chaperoning activity towards Plsp1. Further, the disulfide bridge appears to prevent an inhibitory conformational change resulting from proton motive force-mimicking pH conditions. Broader implications related to the folding of proteins in energy-transducing membranes are discussed.

biochemistry↗

bioRxiv2019

A Distinct Subset of Human Short Introns: U2AF Heterodimer Is Replaced by SPF45 (RBM17) as General Splicing Factor

Human pre-mRNA introns vary in size from under fifty to over a million nucleotides. We searched for essential factors involved in the splicing of human short introns by screening siRNAs against 154 human nuclear proteins. The splicing activity was assayed with a model HNRNPH1 pre-mRNA containing short 56-nucleotide intron. We identified a known alternative splicing regulator SPF45 (RBM17) as a constitutive splicing factor that is required to splice out this 56-nt intron. Whole-transcriptome sequencing of SPF45-deficient cells revealed that SPF45 is essential in the efficient splicing of many short introns. To initiate the spliceosome assembly on a short intron with the truncated poly-pyrimidine tract, the U2AF-homology motif (UHM) of SPF45 competes out that of U2AF65 for binding to the UHM-ligand motif (ULM) of the U2 snRNP protein SF3b155 (SF3B1). We propose that splicing in a distinct subset of human short introns depends on SPF45 but not U2AF heterodimer.

molecular biology↗

bioRxiv2019

Origination of the circadian clock system in stem cells regulates cell differentiation in Arabidopsis thaliana.

The circadian clock regulates various physiological responses. To achieve this, both animals and plants have distinct circadian clocks in each tissue that are optimized for that tissues respective functions. However, if and how the tissue-specific circadian clocks are involved in specification of cell types remains unclear. Here, by implementing a single-cell transcriptome with a new analytics pipeline, we have reconstructed an actual time-series of the cell differentiation process at single-cell resolution, and discovered that the Arabidopsis circadian clock is involved in the process of cell differentiation through transcription factor BRI1-EMS SUPPRESSOR 1 (BES1) signaling. In this pathway, direct repression of LATE ELONGATED HYPOCOTYL (LHY) expression by BES1 triggers reconstruction of the circadian clock in stem cells. The reconstructed circadian clock regulates cell differentiation through fine-tuning of key factors for epigenetic modification, cell-fate determination, and the cell cycle. Thus, the establishment of circadian systems precedes cell differentiation and specifies cell types.

plant biology↗

bioRxiv2019

A slight decrease in serum albumin level is associated with rapid progression of kidney dysfunction even within the normal range: The Yuport Health Checkup Center Cohort Study

ObjectiveA low-normal albumin level is associated with a high risk of cardiovascular disease and mortality in the general population. However, the relationship between serum albumin level and future decline of kidney function is unclear. We aimed to clarify the effect of serum albumin level on the decline of kidney function in the general population.\n\nMethodsThe data used were from 11,000 participants of a voluntary health checkup program between 1998 and 2006 conducted in Japan. The primary outcome for kidney function was a difference in estimated glomerular filtration rate ({Delta}eGFR) of [≥]3 mL/min/1.73 m2/year. The association of the risk of decreased kidney function with albumin level was determined using a logistic regression analysis. We fit separate multivariable logistic regressions for serum albumin levels (g/dL) as a continuous variable and as categorical data, classified as [≤]4.3 (n=2,530), 4.4- 4.6 (n=5,427), and [≥]4.7 (n=3,043).\n\nResultsOf 11,000 participants, 346 had a {Delta}eGFR/year of [≥]3. As compared with the participants with albumin levels of [≥]4.7 g/dL, the risk of decline in kidney function was higher not only in those with albumin levels of [≤]4.3 g/dL (adjusted OR = 2,29, 95% CI: 1.65-3.18) but also in 4.4-4.6 g/dL (adjusted OR = 1.60, 95% CI: 1.20-2.14).\n\nConclusionDecreased albumin level is an independent risk factor for rapid decline in kidney function even within the normal range.

epidemiology↗

bioRxiv2019

Multiplexed 129Xe HyperCEST MRI detection of genetically-reconstituted bacterial protein nanoparticles in human cancer cells.

Gas vesicle nanoparticles (GVs) are gas-containing protein assemblies expressed in bacteria and archaea. Recently, GVs have gained considerable attention for biotechnological applications as genetically-encodable contrast agents for MRI and ultrasonography. However, at present, the practical use of GVs is hampered by a lack of robust methodology for their induction into mammalian cells. Here, we demonstrate the genetic reconstitution of protein nanoparticles with characteristic bicone structures similar to natural GVs in a human breast cancer cell line KPL-4, and genetic control of their size and shape through expression of reduced sets of humanized gas vesicle genes cloned into Tol2 transposon vectors, referencing the natural gas vesicle gene clusters of the cyanobacteria planktothrix rubescens/agardhii. We then report the utility of these nanoparticles as multiplexed, sensitive and genetically-encoded contrast agents for hyperpolarized xenon chemical exchange saturation transfer (HyperCEST) MRI.

biophysics↗

bioRxiv2019

Genome sequence of the euryhaline Javafish medaka, Oryzias javanicus: a small aquarium fish model for studies on adaptation to salinity.

BackgroundThe genus Oryzias is constituted of 35 medaka-fish species each exhibiting various ecological, morphological and physiological peculiarities and adaptations. Beyond of being a comprehensive phylogenetic group for studying intra-genus evolution of several traits like sex determination, behaviour, morphology or adaptation through comparative genomic approaches, all medaka species share many advantages of experimental model organisms including small size and short generation time, transparent embryos and genome editing tools for reverse and forward genetic studies. The Java medaka, Oryzias javanicus, is one of the two species of medaka perfectly adapted for living in brackish/sea-waters. Being an important component of the mangrove ecosystem, O. javanicus is also used as a valuable marine test-fish for ecotoxicology studies. Here, we sequenced and assembled the whole genome of O. javanicus, and anticipate this resource will be catalytic for a wide range of comparative genomic, phylogenetic and functional studies.\n\nFindingsComplementary sequencing approaches including long-read technology and data integration with a genetic map allowed the final assembly of 908 Mbp of the O. javanicus genome. Further analyses estimate that the O. javanicus genome contains 33% of repeat sequences and has a heterozygosity of 0.96%. The achieved draft assembly contains 525 scaffolds with a total length of 809.7 Mbp, a N50 of 6.3 Mbp and a L50 of 37 scaffolds. We identified 21454 expressed transcripts for a total transcriptome size of 57, 146, 583 bps.\n\nConclusionsWe provide here a high-quality draft genome assembly of the euryhaline Javafish medaka, and give emphasis on the evolutionary adaptation to salinity.

genomics↗

bioRxiv2019

Computational Intractability Law Molds the Topology of Biological Networks

Virtually all molecular interaction networks (MINs), irrespective of organism or physiological context, have a majority of loosely-connected leaf genes interacting with at most 1-3 genes, and a minority of highly-connected hub genes interacting with at least 10 or more other genes. Previous reports proposed adaptive and non-adaptive hypotheses describing sufficient but not necessary conditions for the origin of this majority-leaves minority-hubs (mLmH) topology. We modeled the evolution of MINs as a computational optimization problem which describes the cost of conserving, deleting or mutating existing genes so as to maximize (minimize) the overall number of beneficial (damaging) interactions network-wide. The model 1) provides sufficient and, assuming [Formula], necessary conditions for the emergence of mLmH as a software adaptation to circumvent computational intractability, 2) predicts the percentage number of genes having d interacting partners, and 3) when employed as a fitness function in an evolutionary algorithm, produces mLmH-possessing synthetic networks whose degree distributions match those of equal-size MINs.\n\nSummaryOur results indicate that the topology of molecular interaction networks is a selected-for software adaptation that minimizes the evolutionary cost of re-wiring the network in response to an evolutionary pressure to conserve, delete or mutate existing genes and interactions.

systems biology↗

bioRxiv2018

The sunlit microoxic niche of the archaeal eukaryotic ancestor comes to light

Recent advances in phylogenomic analyses and increased genomic sampling of uncultured prokaryotic lineages brought compelling evidence in support of the emergence of eukaryotes from within the Archaea domain of life. The discovery of Asgardaeota archaea and their recognition as the closest extant relative of eukaryotes fuelled the revival of a decades-old debate regarding the topology of the tree of life. While it is apparent that Asgardaeota encode a plethora of eukaryotic-specific proteins (the highest number identified to date in prokaryotes), the lack of genomic information and metabolic characterization has precluded inferences about their lifestyles and the metabolic landscape that may have favoured the emergence of the hallmark eukaryotic subcellular architecture. Here, we use advanced phylogenetic analyses to infer the deep ancestry of eukaryotes and genome-scale metabolic reconstructions to shed light on the metabolic milieu of the closest archaeal eukaryotic ancestors discovered till date. In doing so, we: i) generate the largest Asgardaeota genomic dataset available so far, ii) describe a new clade of rhodopsins encoded within the recovered genomes, iii) provide unprecedented evidence for mixotrophy within Asgardaeota, iv) present first-ever proofs that the closest extant archaeal relatives to all eukaryotes (Heimdallarchaeia) have microoxic lifestyles with aerobic metabolic pathways unique among Archaea (i.e. kynurenine pathway) and v) generate the first images of Asgardaeota.

microbiology↗

bioRxiv2018

Monkey Features Location Identification Using Convolutional Neural Networks

Understanding animal behavior in its natural habitat is a challenging task. One of the primary step for analyzing animal behavior is feature detection. In this study, we propose the use of deep convolutional neural network (CNN) to locate monkey features from raw RGB images of monkey in its natural environment. We train the model to identify features such as the nose and shoulders of the monkey at about 0.01 model loss.

animal behavior and cognition↗

bioRxiv2018

Deletion of class II ARF causes essential tremors through Nav1.6 traffic impairment

ADP-ribosylation factors (ARFs) are a family of small monomeric GTPases consisting of three classes. In the present study, we generated class II ARF-deficient mice (ARF4+/-/ARF5-/-) and found that they exhibited severe movement-associated tremors. Treatment of the mice with propranolol and gabapentin, which alleviate symptoms in patients with essential tremors, similarly reduced the amplitude of the pathologic tremors. In vivo electrophysiological recordings of the ARF4+/-/ARF5-/- mice revealed that they exhibited reduced excitability of their cerebellar Purkinje cells. Immunohistochemical studies revealed that ARF4+/-/ARF5-/- mice exhibit a severe, selective reduction of Nav1.6 proteins that are important for maintaining repetitive action potential firing in the axon initial segments (AISs) of the Purkinje cells. This decrease in Nav1.6 protein expression and the consequent tremors were alleviated by Purkinje cell-specific expression of ARF5. These results indicate that class II ARF mediates the selective trafficking of Nav1.6 to the AISs in cerebellar Purkinje cells, and suggest that the essential tremors can be ascribed to the reduced intrinsic excitability of Purkinje cells, caused by the selective decrease of Nav1.6 proteins in the AISs.

neuroscience↗

bioRxiv2018

Heliorhodopsins are absent in diderm (Gram-negative) bacteria: Some thoughts and possible implications for activity

Microbial heliorhodopsins are a new type of rhodopsins with an opposite membrane topology compared to type-1 and -2 rhodopsins, currently believed to engage in light sensing. We determined their presence/absence is monoderms and diderms representatives from the Tara Oceans and freshwater 25 lakes metagenomes. Heliorhodopsins were absent in diderms, confirming our previous observations in cultured Proteobacteria. Based on these observations, we speculate on the putative role of heliorhodopsins in light-driven transport of amphiphilic molecules.

microbiology↗

bioRxiv2017

Toward Machine Learning-based Data-driven Functional Protein Studies: Understanding Colour Tuning Rules and Predicting the Absorption Wavelengths of Microbial Rhodopsins

The light-dependent ion-transport function of microbial rhodopsin has been widely used in optogenetics for optical control of neural activity. In order to increase the variety of rhodopsin proteins having a wide range of absorption wavelengths, the light absorption properties of various wild-type rhodopsins and their artificially mutated variants were investigated in the literature. Here, we demonstrate that a machine-learning-based (ML-based) data-driven approach is useful for understanding and predicting the light-absorption properties of microbial rhodopsin proteins. We constructed a database of 796 proteins consisting of microbial rhodopsin wildtypes and their variants. We then proposed an ML method that produces a statistical model describing the relationship between amino-acid sequences and absorption wavelengths and demonstrated that the fitted statistical model is useful for understanding colour tuning rules and predicting absorption wavelengths. By applying the ML method to the database, two residues that were not considered in previous studies are newly identified to be important to colour shift.

biophysics↗

bioRxiv2017

Glutamine metabolism regulates Th2 cell differentiation via the α-ketoglutarate-dependent demethylation of histone H3K27

The acquisition of T cell functions seems to be closely linked to the reprogramming of the metabolic pathway. However, the impact of metabolic changes on the differentiation of helper T cell subsets remains unclear. We found that TCR-mediated activation of glutamine metabolism regulates Th2 cell differentiation via the supplementation of -ketogulutalate (-KG) and histone H3K27 demethylation. Deprivation of glutamine or pharmacological inhibition of glutamine metabolism blocks the induction of Th2 cell differentiation without affecting Stat6 phosphorylation. The methylation status of H3K27 at the Th2 cytokine gene locus was significantly increased in Th2 cells cultured under glutamine-deprived conditions. The inhibitory effect of glutamine-deprivation was antagonized by -KG, and the -KG-dependent induction of Th2 cell differentiation was reduced in utx- and jmjd3-deficient naive CD4 T cells. These findings show that the glutamine-a-ketoglutarate axis is crucial to regulating the epigenetic status at the Th2 cytokine gene locus and subsequent Th2 cell differentiation.

immunology↗