Biology subjects

Ishida, H.

Publications and source records attributed to Ishida, H..

At least 19 recordsLinked to original sources

bioRxiv2026

Leveraging archaeological specimens to investigate failed invasions: A case study on Okhotsk pigs, northern Japan

Invasive species pose a major threat to biodiversity, yet our understanding of failed invasions/translocations, instances where alien/introduced species fail to establish, remains limited. Investigating the factors behind failed invasions is critical for improving prevention and management strategies for modern biological invasions. Here, we propose a novel framework that utilizes archaeological archives to uncover evidence of failed invasions. We estimated the biological and ecological factors contributing to the failed invasion of pigs from later prehistory to recent times (299 cal BC to 1900 AD) on Rebun Island in far northern Japan by synthesizing the evidence obtained from stable isotopes, zooarchaeology, and historical documents. Despite the anthropogenic introductions of pigs into Rebun Island, pigs did not establish a feral population and disappeared after ca. 1200 AD. We identified reduced propagule pressure, abiotic resistance due to the cold climate, and decreased resources as the three key factors that contributed to the disappearance of pigs. Pigs are one of the most widespread invasive species globally, and this study represents a novel approach to studying failed invasions using archaeological data, which aligns with the framework of conservation paleobiology.

ecology↗

bioRxiv2026

Impact of intercalators on the properties of DNA analyzed by molecular dynamics simulations

Intercalation of small molecules between DNA base pairs affects DNA conformation, disrupting essential cellular processes including replication, transcription, and repair. We investigated conformational changes in 18-mer DNA upon intercalation of doxorubicin, SYBR Gold and YOYO-1 using extensive MD simulations. Two main patterns for the intercalation were identified: RISE-type intercalation occurs between adjacent base pairs and extends the DNA helix with decreased twist angles, while OPEN-type intercalation proceeds through base-pair opening without significant DNA extension. Kinetic analysis revealed that association rates for intercalation followed the order: first YO-moiety (mono-intercalation) > SYBR Gold > doxorubicin > YOYO-1 (bis-intercalation). Free energy landscape showed that forces at DNA termini reached up to 117 pN during stretching. Notably, base pairs adjacent to intercalators were protected from strand separation, accompanied by additional helical unwinding. MM-PBSA/GBSA analysis revealed that the driving force for intercalation is the stacking energy, and the binding affinity was highest for minor groove binding. Persistence length decreased with single molecule binding but recovered with two molecules due to their electrostatic repulsion. Mechanical properties of intercalated DNA showed position-dependence, demonstrating that multiple intercalation modes coexist in solution. The heterogeneous nature of intercalation explains why experimental measurements reflect ensemble averages rather than single binding configurations.

biophysics↗

bioRxiv2025

Long-distance gene flow and contrasting population structures of reef-building corals and their algal symbionts inform adaptive potential across the Western Pacific

The genetic diversity and connectivity of reef-building coral populations are key to their survival in warming oceans. Yet our understanding of corals demographic resilience and adaptive potential is complicated by cryptic species diversity, wide geographic distributions, and complex coral-algal symbioses. To address these challenges, we investigated genetic connectivity and diversity of the broadcast-spawning coral Acropora spathulata and its associated Symbiodiniaceae across 29 reefs spanning the Great Barrier Reef, the Coral Sea, and New Caledonia, using whole-genome sequencing of 1,088 colonies. We identified four genetically distinct coral populations that diverged between 0.27 and 0.65 million years ago, likely due to geographic isolation across thousands of kilometers. These populations maintained asymmetrical gene flow along major ocean currents despite demographic isolation, and sustained large local effective population sizes ([~]2,900), supported by a high dispersal range of [~]100 km per generation. In contrast, their Symbiodiniaceae partners varied over finer spatial scales, with five distinct Cladocopium taxa distributed along latitudinal and cross-shore gradients, likely driven by local environmental conditions. These results suggest that high dispersal capacity and large local population size promote demographic resilience within reef systems, while environment-specific symbioses and long-distance gene flow across reef-systems support adaptation and evolutionary rescue.

molecular biology↗

bioRxiv2025

Structure and inhibition mechanism of the helicase-primase complex from human herpesvirus 1

Human herpesviruses (HHVs) are widespread pathogens causing severe diseases, especially in immunocompromised individuals, and are linked to neurodegenerative and autoimmune disorders. Current antiviral therapies primarily target the -subfamily, with limited options for {beta}- and {gamma}-subfamilies. The helicase-primase complex (HPC) is essential for viral DNA replication and represents a key antiviral target, but its structure and inhibition mechanisms were previously unclear. Here, cryo-EM structures of the HHV1 (-subfamily) HPC bound to single-stranded DNA and two clinical inhibitors, amenamevir and pritelivir, are reported. The HPC features flexible helicase and primase modules, with both inhibitors binding a shared allosteric pocket in the helicase module. Structural, biochemical, and molecular dynamics analyses indicate that these inhibitors lock the helicase in an open, inactive conformation. Combined molecular dynamics and fragment molecular orbital analyses explain the -subfamily selectivity and distinct antiviral spectra of these drugs. These findings provide a structural framework to guide the development of novel inhibitors targeting {beta}- and {gamma}-subfamily herpesviruses. SignificanceHerpesviruses are ubiquitous human pathogens responsible for a wide spectrum of diseases, including severe infections in immunocompromised individuals and associations with neurodegenerative and autoimmune disorders. Despite the clinical importance of these viruses, current antiviral therapies are largely limited to the -subfamily, with few effective options for {beta}- and {gamma}-subfamilies. The helicase-primase complex (HPC) is a critical enzyme for viral DNA replication and a validated antiviral target, yet its structural basis and inhibition mechanisms have remained elusive, hindering the rational development of next-generation therapeutics. This study provides the first high-resolution cryo-EM structures of the human herpesvirus 1 (HHV1) HPC bound to single-stranded DNA and two clinically relevant inhibitors, amenamevir and pritelivir. These structures reveal the modular organization of the HPC and identify a shared allosteric pocket where both inhibitors bind. Structural, biochemical, and molecular dynamics analyses demonstrate that these inhibitors lock the helicase in an open, catalytically inactive conformation, thereby blocking ATP binding and halting DNA unwinding. Fragment molecular orbital (FMO) calculations further elucidate the molecular determinants of inhibitor binding, explaining the -subfamily specificity and the distinct antiviral spectra of amenamevir and pritelivir. The integrated approach combining cryo-EM, molecular dynamics, and quantum chemical calculations not only clarifies the mechanism of action of current clinical inhibitors but also establishes a robust framework for the rational design of novel antivirals. By providing detailed insights into the structural and dynamic properties of the HPC and its inhibition, this work paves the way for the development of new drugs with tailored specificity and improved pharmacokinetic properties, potentially expanding therapeutic options to currently untreatable herpesvirus infections. These findings have broad implications for antiviral drug discovery and for understanding the molecular mechanisms underlying DNA replication in herpesviruses.

biochemistry↗

bioRxiv2023

Autophagosome development and chloroplast segmentation occur synchronously for piecemeal degradation of chloroplasts

Plants distribute many nutrients to chloroplasts during leaf development and maturation. When leaves senesce or experience sugar starvation, the autophagy machinery degrades chloroplast proteins to facilitate efficient nutrient reuse. Here, we report on the intracellular dynamics of an autophagy pathway responsible for piecemeal degradation of chloroplast components. Through live-cell monitoring of chloroplast morphology, we observed the formation of chloroplast budding structures in sugar-starved leaves. These buds were then released and incorporated into the vacuolar lumen as an autophagic cargo termed a Rubisco-containing body. The budding structures did not accumulate in mutants of core autophagy machinery, suggesting that autophagosome creation is required for forming chloroplast buds. Simultaneous tracking of chloroplast morphology and autophagosome development revealed that the isolation membranes of autophagosomes interact closely with part of the chloroplast surface before forming chloroplast buds. Chloroplasts then protrude at the site associated with the isolation membranes, which divide synchronously with autophagosome maturation. This autophagy-related division does not require DYNAMIN-RELATED PROTEIN 5B, which constitutes the division ring for chloroplast proliferation in growing leaves. An unidentified division machinery may thus fragment chloroplasts for degradation in coordination with the development of the chloroplast-associated isolation membrane.

plant biology↗

bioRxiv2023

Palaeoproteomic investigation of an ancient human skeleton with abnormal deposition of dental calculus

Detailed investigation of extremely severe pathological conditions in ancient human skeletons is important as it could shed light on the breadth of potential interactions between humans and disease etiologies in the past. Here, we applied palaeoproteomics to investigate an ancient human skeletal individual with severe oral pathology, focusing our research on bacterial pathogenic factors and host defense response. This female skeleton, from the Okhotsk period (i.e., 5th-13th century) of Northern Japan, poses relevant amounts of abnormal dental calculus deposition and exhibits oral dysfunction due to severe periodontal disease. A shotgun mass-spectrometry analysis identified 81 human proteins and 15 bacterial proteins from the calculus of the subject. We identified two pathogenic or bioinvasive proteins originating from two of the three "red complex" bacteria, the core species associated with severe periodontal disease in modern humans, as well as two additional bioinvasive proteins of periodontal-associated bacteria. Moreover, we discovered defense response system-associated human proteins, although their proportion was mostly similar to those reported in ancient and modern human individuals with lower calculus deposition. These results suggest that the bacterial etiology was similar and the host defense response was not necessarily more intense in ancient individuals with significant amounts of abnormal dental calculus deposition.

evolutionary biology↗

bioRxiv2022

Influenza A virus agnostic receptor tropism revealed using a novel biological system with terminal sialic acid-knockout cells

Avian or human influenza A viruses bind preferentially to avian- or human-type sialic acid receptors, respectively, indicating that receptor tropism is an important factor for determining the viral host range. However, there are currently no reliable methods for analyzing receptor tropism biologically under physiological conditions. Here, we established a novel system using MDCK cells with avian- or human-type sialic acid receptors and with both sialic acid receptors knocked out (KO). When we examined the replication of human and avian influenza viruses in these KO cells, we observed unique viral receptor tropism that could not be detected using a conventional solid-phase sialylglycan binding assay, which directly assesses physical binding between the virus and sialic acids. Furthermore, we serially passaged an engineered avian-derived H4N5 influenza virus, whose PB2 gene was deleted, in avian-type receptor-KO cells stably expressing PB2 to select a mutant with enhanced replication in KO cells; however, its binding to human-type sialylglycan was undetectable using the solid-phase binding assay. These data indicate that a panel of sialic acid receptor-KO cells could be a useful tool for determining the biological receptor tropism of influenza A viruses. Moreover, the PB2-KO virus experimental system could help to safely and efficiently identify the mutations required for avian influenza viruses to adapt to human cells that could trigger a new influenza pandemic. Author summaryInfluenza A virus initiates infection via hemagglutinin by binding to avian- or human-type receptors. The acquisition of mutations that allow avian virus hemagglutinins (HAs) to recognize human-type receptors is mandatory for the transmission of avian influenza viruses to humans, which could lead to a pandemic. Therefore, it is important to detect such mutation(s) in animal influenza viruses for pandemic surveillance and risk assessment. In this study, we established a novel system using a set of genetically engineered MDCK cells with knocked out sialic acid receptors to biologically evaluate the receptor tropism for influenza A viruses. Using this system, we observed unique receptor tropism in several virus strains that was undetectable using conventional solid-phase binding assays that measure physical binding between the virus and artificially synthesized sialylglycans. This study makes a significant contribution to the literature because our findings suggest the pitfall of conventional receptor binding assay and the existence of a sialic acid-independent pathway for viral infection. In addition, our system could be safely used to identify mutations that could acquire human-type receptor tropism. Thus, this system could contribute not only toward basic analyses, such as elucidating the mechanism of influenza virus host range determination, but also the surveillance of viruses of animal origin that could be capable of infecting via human-type receptors, triggering a new influenza pandemic.

microbiology↗

bioRxiv2021

Brain fatty acid binding protein exhibits non-preferential and mutation-resistant binding towards fatty acids

Members of the fatty acid binding protein (FABP) family function as intracellular transporters of long chain fatty acids and other hydrophobic molecules to different cellular compartments. Brain fatty acid binding protein (FABP7) exhibits ligand-directed differences in cellular transport behavior. For example, when FABP7 binds to docosahexaenoic acid (DHA), the complex relocates to the nucleus and influences transcriptional activity, whereas FABP7 bound with monosaturated fatty acids remain in the cytosol. We used a variety of biophysical techniques to enhance understanding of ligand-directed transport. Specifically, we examine how FABP7 binds to fatty acids, including saturated stearic acid (SA), monounsaturated oleic acid (OA), and polyunsaturated DHA. We find that at 37{degrees}C FABP7 has near equivalent binding affinities for the fatty acids, while at lower temperatures, FABP7 exhibits a preference for the unsaturated fatty acids. Therefore, nuclear localization of the FABP7-DHA complex cannot be explained by binding preferences. Using NMR spectroscopy and molecular dynamics simulations, we observe that DHA uniquely affects the portal region of FABP7, which could enhance the complexs nuclear localization. Mutations to purported critical binding residues (R126L and Y128F) have little effect on fatty acid binding, with molecular dynamics simulations revealing that the bound fatty acid can adopt binding poses that can accommodate the mutations. SignificanceThis work studies FABP7 at physiological temperature and shows that nuclear localization of FABP7 cannot be initiated by tighter ligand interactions. Through biophysical experiments and simulations, we show ligand-dependent conformational changes, instead of binding affinities, are associated with certain biological outcomes. Extensive simulations reveal redundancy in available ligand binding conformations, which permits mutant-resistant binding. This suggests that these mutations do not affect ligand binding affinities, but changes in protein conformation and dynamics may result in disease associated cellular outcomes.

biophysics↗

bioRxiv2021

Simultaneous Binding of the N- and C-terminal Cytoplasmic Domains of Aquaporin 4 to Calmodulin May Contribute to Vesicular Trafficking

Aquaporin 4 (AQP4) is a water transporting, transmembrane channel protein that has important regulatory roles in maintaining cellular water homeostasis. Several other AQP proteins exhibit calmodulin (CaM)-binding properties, and CaM has recently been implicated in the cell surface localization of AQP4 that occurs in response to osmotically-driven changes in cell swelling in the central nervous system. The objective of the present study was to assess the CaM-binding properties of AQP4 in detail. Inspection of AQP4 revealed two putative CaM-binding domains (CBDs) in the cytoplasmic N- and C-terminal regions, respectively. The Ca2+-dependent CaM-binding properties of synthetic and recombinant AQP4 CBD peptides were assessed using fluorescence spectroscopy, isothermal titration calorimetry, and two-dimensional 1H, 15N-HSQC NMR with 15N-labeled CaM. The N-terminal CBD peptide of AQP4 predominantly interacted with the N-lobe of CaM with a 1:1 binding ratio and a Kd of 3.4 M. CaM bound two C-terminal AQP4 peptides with interactions observed for both the C- and N-lobes of CaM (Kd1: 3.6 M, Kd2: 113.6 M, respectively). A recombinant AQP4 protein domain (rAQP4ct, containing the entire cytosolic C-terminal domain sequence) bound CaM in a 1:1 binding mode with a Kd of 6.1 M. A ternary bridging complex could be generated with the N- and C-lobes of CaM interacting simultaneously with the N- and C-terminal CBD peptides. These data suggest that this unique adapter protein binding mode of CaM and AQP4 may be an important regulatory mechanism for the vesicular trafficking of AQP4.

biochemistry↗

bioRxiv2021

Meclizine and metabotropic glutamate receptor agonists attenuate severe pain and primary sensory neuron Ca2+ activity in chemotherapy-induced peripheral neuropathy

Chemotherapy-induced peripheral neuropathy (CIPN) affects about 68% of patients undergoing chemotherapy and causes severe neuropathic pain which is debilitating health problem and greatly reduces quality of life. Cisplatin is a commonly used platinum-based chemotherapeutic drug known to cause CIPN, possibly by causing oxidative stress damage to primary sensory neurons. Metabotropic glutamate receptors (mGluRs) are widely hypothesized to be involved in pain processing. Meclizine is an H1 histamine receptor antagonist which is known to have neuroprotective effects including anti-oxidative effect. Here, we used a mouse model of cisplatin-induced CIPN to test agonists of mGluR8 and group II mGluR as well as meclizine as interventions to reduce cisplatin-induced pain. We performed behavioral pain tests and in vivo entire DRG neurons Ca2+ imaging using genetically-encoded Ca2+ indicator, Pirt-GCaMP3 to monitor different drug interventions on a populational ensemble level. CIPN induced increased spontaneous Ca2+ activity in DRG neurons, increased Ca2+ transient amplitudes, and hyperresponses to mechanical, thermal, and chemical stimuli. We found mGluR8 agonist, DCPG, group II mGluR agonist, LY379268, and Histamine1 receptor antagonist, meclizine all significantly attenuated mechanical and thermal pain caused by CIPN. LY379268 and meclizine, but not DCPG, attenuated DRG neuronal Ca2+ activity elevated by CIPN. Furthermore, meclizine attenuated cisplatin-induced weight loss. These results suggest group II mGluR agonist, mGluR8 agonist, and meclizine are excellent candidates to study for mechanisms and new treatment option for CIPN.

neuroscience↗

bioRxiv2021

Capsaicin pretreatment alleviates postoperative pain and reduces primary sensory neuron Ca2+ activity

After surgeries, especially thoracotomy incision, patients develop unbearable pain. Opioids are used for reducing pain but often cause serious side effects. Previously, we found that capsaicin pretreatment of the incision area alleviated spontaneous and thermal pain in a postoperative pain animal model. In the present study, we aimed to monitor primary sensory neuron Ca2+ activity in in vivo dorsal root ganglia (DRG) in a postoperative pain model using Pirt-GCaMP3 treated with capsaicin or controls. Intraplantar injection of capsaicin (0.05%) alleviated spontaneous, mechanical, and thermal postoperative pain. The Ca2+ response in in vivo DRG and in in situ spinal cord was significantly enhanced in the ipsilateral side compared to contralateral side or naive control. Primary sensory nerve fiber length was significantly decreased in the incision skin area in capsaicin-pretreated animals detected by immunohistochemistry and placental alkaline phosphatase (PLAP) staining. Thus, capsaicin pretreatment alleviates postoperative pain by suppressing Ca2+ response due to degeneration of primary sensory nerve fibers in the skin.

neuroscience↗

bioRxiv2021

Mast cell-specific receptor/corticotropin-releasing factor axis regulates alcohol withdrawal-associated headache

Rehabilitation from alcohol addiction or abuse is challenging due to alcohol withdrawal symptoms. Headache is a severe alcohol withdrawal symptom that frequently contributes to rehabilitation failure. Despite the need for treating alcohol withdrawal-induced headache, there is no appropriate therapeutic option available. Development of improved therapeutics will depend on obtaining a clearer understanding of alcohol withdrawal-induced headache pain mechanisms. Here, we show that the mast cell-specific receptor MrgprB2 controls development of alcohol withdrawal-induced headache. Withdrawing alcohol from alcohol-acclimated mice induces strong headache behaviors, including facial allodynia, facial pain expressions, and reduced walking movement, symptoms often observed in humans suffering from headache. Observed pain behaviors were abolished in MrgprB2-deficient mice. We observed in vivo spontaneous activation and hypersensitization of trigeminal ganglia neurons in alcohol withdrawal mice, but not in MrgprB2-deficient mice. Corticotropin-releasing factor (CRF) was increased in dura mater after alcohol withdrawal. Injection of CRF into dura mater resulted in activation of trigeminal ganglia neurons and vasodilation, which was accompanied by headache behavior. In cells, CRF evoked Ca2+ transients via MrgprB2 or human MrgprX2. The results indicate that alcohol withdrawal causes headache via mast cell degranulation in dura mater. The process is under control of MrgprB2/MrgprX2, which would appear to represent a potential target for treating alcohol withdrawal-related headache.

neuroscience↗

bioRxiv2021

Imaging sensory transmission and neuronal plasticity in primary sensory neurons with genetically-encoded voltage indicators

Detection of somatosensory inputs requires conversion of external stimuli into electrical signals by activation of primary sensory neurons. The mechanisms by which heterogeneous primary sensory neurons encode different somatosensory inputs remains unclear. In vivo dorsal root ganglia (DRG) imaging using genetically-encoded Ca2+ indicators (GECIs) is currently the best technique for this purpose by providing an unprecedented spatial and populational resolution. It permits the simultaneous imaging of >1800 neurons/DRG in live mice. However, this approach is not ideal given that Ca2+ is a second messenger and has inherently slow response kinetics. In contrast, genetically-encoded voltage indicators (GEVIs) have the potential to track voltage changes in multiple neurons in real time but often lack the brightness and dynamic range required for in vivo use. Here, we used soma-targeted ASAP4.4-Kv, a novel GEVI, to dissect the temporal dynamics of noxious and non-noxious neuronal signals during mechanical, thermal, or chemical stimulation in DRG of live mice. ASAP4.4-Kv is sufficiently bright and fast enough to optically characterize individual neuron coding dynamics. Notably, using ASAP4.4-Kv, we uncovered cell-to-cell electrical synchronization between adjacent DRG neurons and robust dynamic transformations in sensory coding following tissue injury. Finally, we found that a combination of GEVI and GECI imaging empowered in vivo optical studies of sensory signal processing and integration mechanisms with optimal spatiotemporal analysis. HighlightsO_LIIn vivo ultra fast and sensitive dynamic voltage imaging of peripheral primary sensory neurons by a newly generated genetically-encoded voltage indicator. C_LIO_LIIdentification of mechanical, thermal, or chemical stimuli-evoked voltage signals with superior temporal resolution. C_LIO_LISingle-cell detection of changes in sub- and suprathreshold voltage dynamics across different disease conditions. C_LIO_LICombination of voltage (by ASAP4.4-Kv) and Ca2+ (by Pirt-GCaMP3) signals to facilitate the understanding of signal processing and integration of primary sensory neurons, especially for noxious versus non-noxious sensation. C_LI

neuroscience↗

bioRxiv2020

Identification of differential gene expression pattern in lens epithelial cells derived from cataractous and non-cataractous lenses of Shumiya cataract rat

The Shumiya cataract rat (SCR) is a model for hereditary cataract. Two-third of these rats develop lens opacity within 10-11-weeks. Onset of cataract is attributed to the synergetic effect of lanosterol synthase (Lss) and farnesyl-diphosphate farnesyltransferase 1 (Fdft1) mutant alleles that lead to cholesterol deficiency in the lenses, which in turn adversely affects lens biology including the growth and differentiation of lens epithelial cells (LECs). Nevertheless, the molecular events and changes in gene expression associated with the onset of lens opacity in SCR is poorly understood. In the present study, a microarray-based approach was employed to analyze comparative gene expression changes in LECs isolated from the pre-cataractous and cataractous stages of lenses of 5-, 8- and 10-week-old SCRs. The changes in gene expression observed in microarray results in the LECs were further validated using real-time PCR and western blot analyses. Lens opacity was not observed in 5-week-old rats. However, histological analysis revealed mild dysplasia in the anterior suture and poorly differentiated fiber cells at the bow region. Expression of approximately 100 genes, including major intrinsic protein of lens fiber (MIP and Aquaporin0), deoxyribonuclease II beta (Dnase2b), heat shock protein B1 (Hspb1), and crystallin {gamma} ({gamma}Cry) B, C, and F were found to be significantly downregulated (0.07-0.5 fold) in rat LECs derived from cataract lenses compared to that in noncataractous lenses (control). Thus, our study aimed to identify the gene expression patterns during cataract formation in SCRs, which may be responsible for cataractogenesis in SCR. We proposed that mutation in lanosterol synthase was responsible for the downregulation of genes associated with lens fiber differentiation, which in turn leads to the formation of cataract in these rats. Our findings may have wider implications in understanding the effect of cholesterol deficiency and the role of cholesterol-lowering therapeutics on cataractogenesis.

cell biology↗

bioRxiv2020

Cep57 and Cep57L1 cooperatively maintain centriole engagement during interphase to ensure proper centriole duplication cycle

Centrioles duplicate in the interphase only once per cell cycle. Newly formed centrioles remain associated with their mother centrioles. The two centrioles disengage at the end of mitosis, which licenses centriole duplication in the next cell cycle. Therefore, timely centriole disengagement is critical for the proper centriole duplication cycle. However, the mechanisms underlying centriole engagement during interphase are poorly understood. Here, we show that Cep57 and Cep57L1 cooperatively maintain centriole engagement during interphase. Co-depletion of Cep57 and Cep57L1 induces precocious centriole disengagement in the interphase without compromising cell cycle progression. The disengaged daughter centrioles convert into centrosomes during interphase in a Plk1-dependent manner. Furthermore, the centrioles reduplicate and the centriole number increases, which results in chromosome segregation errors. Overall, these findings demonstrate that the maintenance of centriole engagement by Cep57 and Cep57L1 during interphase is crucial for the tight control of centriole copy number and thus for proper chromosome segregation.

cell biology↗

bioRxiv2019

Machine-driven parameter optimisation of biochemical reactions

The development of complex, multi-step methods in molecular biology is a laborious, costly, iterative and often intuition-bound process where an optimum is sought in a multidimensional parameter space through step-by-step optimisations. The difficulty of miniaturising reactions under the microliter volumes usually handled in multiwell plates by robots, plus the cost of the experiments, limit the number of parameters and the dynamic ranges that can be explored. Nevertheless, because of non-linearities of the response of biochemical systems to their reagent concentrations, broad dynamic ranges are necessary. Here we use a high-performance nanoliter handling platform and computer generation of liquid transfer programs to explore in quadruplicates more than 600 combinations of 4 parameters of a biochemical reaction, the reverse-transcription, which lead us to uncover non-linear responses, parameter interactions and novel mechanistic insights. With the increased availability of computer-driven laboratory platforms for biotechnology, our results demonstrate the feasibility and advantage of methods development based on reproducible, computer-aided exhaustive characterisation of biochemical systems.

molecular biology↗

bioRxiv2019

Establishment of a Reverse Genetics System for Influenza D Virus

Influenza D virus (IDV) was initially isolated in the USA in 2011. IDV is distributed worldwide and is one of the causative agents of bovine respiratory disease complex (BRDC), which exhibits high morbidity and mortality in feedlot cattle. Molecular mechanisms of IDV pathogenicity are still unknown. Reverse genetics systems are vital tools not only for studying the biology of viruses, but also for use in applications such as recombinant vaccine viruses. Here, we report the establishment of a plasmid-based reverse genetics system for IDV. We first verified that the 3'-terminal nucleotide of each 7-segmented genomic RNA contained uracil in contrary to the previous report, and were then able to successfully generate recombinant IDV by co-transfecting 7 plasmids containing these genomic RNAs along with 4 plasmids expressing polymerase proteins and NP into HRT-18G cells. The recombinant virus had a growth deficit compared to the wild-type virus, and we determined the reason for this growth difference by examining the genomic RNA content of the viral particles. We found that recombinant virus incorporated an unbalanced ratio of viral RNA segments into particles as compared to the wild-type virus, and thus we adjusted the amount of each plasmid used in transfection to obtain recombinant virus with the same replicative capacity as wild-type virus. Our work here in establishing a reverse genetics system for IDV will have a broad range of applications, including uses in studies focused on better understanding IDV replication and pathogenicity as well as those contributing to the development of BRDC countermeasures.\n\nIMPORTANCEBovine respiratory disease complex (BRDC) exhibits high mortality and morbidity in cattle, causing economic losses worldwide. Influenza D virus (IDV) is considered to be a causative agent of BRDC. Here, we developed a reverse genetics system that allows for the generation of IDV from cloned cDNAs, and the introduction of mutations into the IDV genome. This reverse genetics system will become a powerful tool for use in studies related to understanding the molecular mechanisms of viral replication and pathogenicity, and will also lead to the development of new countermeasures against BRDC.

microbiology↗

bioRxiv2019

Jomon genome sheds light on East Asian population history

Anatomical modern humans reached East Asia by >40,000 years ago (kya). However, key questions still remain elusive with regard to the route(s) and the number of wave(s) in the dispersal into East Eurasia. Ancient genomes at the edge of East Eurasia may shed light on the detail picture of peopling to East Eurasia. Here, we analyze the whole-genome sequence of a 2.5 kya individual (IK002) characterized with a typical Jomon culture that started in the Japanese archipelago >16 kya. The phylogenetic analyses support multiple waves of migration, with IK002 forming a lineage basal to the rest of the ancient/present-day East Eurasians examined, likely to represent some of the earliest-wave migrants who went north toward East Asia from Southeast Asia. Furthermore, IK002 has the extra genetic affinity with the indigenous Taiwan aborigines, which may support a coastal route of the Jomon-ancestry migration from Southeast Asia to the Japanese archipelago. This study highlight the power of ancient genomics with the isolated population to provide new insights into complex history in East Eurasia.

genomics↗