Biology subjects

Nakamura, S.

Publications and source records attributed to Nakamura, S..

At least 55 records · Page 3Linked to original sources

bioRxiv2022

Multiple genes evolved for fungal septal pore plugging identified via large-scale localization and functional screenings

Multicellular organisms exhibit cytoplasmic exchange using porous structures for cooperation among cells. Fungal multicellular lineages have evolved septal pores for this function. Interconnected hyphal cells possess the risk of wound-related cytoplasmic loss unless the septal pores are plugged. However, the gene evolution of regulatory mechanisms underlying fungal septal pore plugging remains poorly understood. To identify novel septal components, 776 uncharacterized proteins were identified using genomic comparisons between septal pore-bearing and -lacking ascomycete species. We then determined their subcellular localizations, and in total 62 proteins localized to the septum or septal pore. We analyzed the effects of deleting the encoding genes on septal pore plugging upon hyphal wounding. Of the 62 proteins, 23 were involved in regulating septal pore plugging. Here, using orthologous group and phylogenetic analyses, this study suggests that septal pore regulation has evolved either by co-option of preexisting genes or by Pezizomycotina-specific gene acquisition.

microbiology↗

bioRxiv2022

Collective gradient sensing by swimming bacteria without clustering

We characterize the taxis enhancement of swimming bacteria by collective migration without apparent clustering. We confine dilute Salmonella suspension in a shallow channel and evaluate the thermotaxis response to local heating and diffusion. By combining cell tracking analysis and numerical simulation based on simple modeling, we show that the alignment interaction suppresses orientation fluctuation, strengthens migration bias, and also prevents the dispersion of accumulated population. The results show a prominent example of how a collective motion of active matter implements a biological function.

biophysics↗

bioRxiv2022

Preclinical Study on SARS-CoV-2 Sublingual Vaccine with RBD Antigen and Poly(I:C) Adjuvant in Cynomolgus Macaques

Mucosal vaccine for sublingual route was prepared with recombinant SARS-CoV-2 spike protein receptor binding domain (RBD) antigen and poly(I:C) adjuvant components. The efficacy of this sublingual vaccine was examined using Cynomolgus macaques. Nine of the macaque monkeys were divided into three groups of three animals; control (just 400 g poly(I:C) per head); low dose (30 g RBD and 400 g poly(I:C) per head); and high dose (150 g RBD and 400 g poly(I:C) per head), respectively. N-acetylcysteine (NAC), a mild reducing agent losing mucin barrier, was used to enhance vaccine delivery to mucosal immune cells. RBD-specific IgA antibody secreted in pituita was detected in two of three monkeys of the high dose group and one of three animals of the low dose group. RBD-specific IgG and/or IgA antibodies in plasma were also detected in these monkeys. These indicated that the sublingual vaccine stimulated mucosal immune response to produce antigen-specific secretory IgA antibodies in pituita and/or saliva. This sublingual vaccine also affected systemic immune response to produce IgG (IgA) in plasma. Little RBD-specific IgE was detected in plasma, suggesting no allergic antigenicity of this sublingual vaccine. Thus, SARS-CoV-2 sublingual vaccine consisting of poly(I:C) adjuvant showed reasonable efficacy in a non-human primate model.

immunology↗

bioRxiv2022

Novel monoclonal antibodies showing broad neutralizing activity for SARS-CoV-2 variants including Omicrons BA.5 and BA.2.75

We identified novel neutralizing monoclonal antibodies against SARS-CoV-2 variants (including Omicron) from individuals received two doses of mRNA vaccination after they had been infected with wildtype. We named them MO1, MO2 and MO3. MO1 shows high neutralizing activity against authentic variants: D614G, Delta, BA.1, BA.1.1, BA.2, and BA.2.75 and BA.5. Our findings confirm that the wildtype-derived vaccination can induce neutralizing antibodies that recognize the epitopes conserved among the SARS-CoV-2 variants (including BA.5 and BA.2.75). The monoclonal antibodies obtained herein could serve as novel prophylaxis and therapeutics against not only current SARS-CoV-2 viruses but also future variants that may arise.

microbiology↗

bioRxiv2022

Genetic and lipidomic identification of tuberculostearic acid as a controller of mycobacterial membrane compartmentalization

Mycobacteria diverge in a basic way from other bacterial and eukaryotic cells based on their distinct membrane structures. Here we report genome-wide transposon sequencing to discover the controllers of membrane compartmentalization in Mycobacterium smegmatis. cfa, a gene that encodes a putative cyclopropane-fatty-acyl-phospholipid synthase, shows the most significant effect on recovery from a membrane destabilizer, dibucaine. Lipidomic analysis of cfa deletion mutants demonstrates an essential role of Cfa in the synthesis of specific membrane lipids containing a C19:0 monomethyl-branched stearic acid. This molecule, also known as tuberculostearic acid (TBSA), has been intensively studied for decades due to its high level and genus-specific expression in mycobacteria. The proposed Cfa-mediated conversion of an unsaturation to a methylation matched well with its proposed role in lateral membrane organization, so we used new tools to determine the non-redundant effects of Cfa and TBSA in mycobacterial cells. cfa expression regulated major classes of membrane lipids including phosphatidylinositols, phosphatidylethanolamines and phosphatidylinositol mannosides. Cfa localized within the intracellular membrane domain (IMD), where it controls both cellular growth and recovery from membrane fluidization by facilitating subpolar localization of the IMD. Overall, cfa controls lateral membrane partitioning but does not detectably alter orthogonal transmembrane permeability. More generally, these results support the proposed role of the subpolar IMD as a subcellular site of mycobacterial control of membrane function. SignificanceMycobacteria remain major causes of disease worldwide based in part on their unusual membrane structures, which interface with the host. Here we discover the long sought biosynthetic origin of tuberculostearic acid (TBSA), a major fatty acid found selectively in mycobacteria, as well as its role in mycobacterial cells. The lipid is produced by an enzyme called Cfa, whose loss causes a growth defect and slow reformation of a membrane domain near the pole of the rod-shaped cell. Thus, our study offers mechanistic insights to the intrinsic molecular factors critical for mycobacterial plasma membrane partitioning.

microbiology↗

bioRxiv2022

Phenotypic screening using waveform analysis of calcium dynamics in primary cortical cultures

At present, in vitro phenotypic screening methods are widely used for drug discovery. In the field of epilepsy research, measurements of neuronal activity have been utilized for predicting efficacy of anti-epileptic drugs. Fluorescence measurements of calcium oscillations in neurons are commonly used for measurement of neuronal activities, and some anti-epileptic drugs have been evaluated using this assay technique. However, changes in waveforms were not quantified in previous reports. Here, we have developed a high-throughput screening system containing a new analysis method for quantifying waveforms, and our method has successfully enabled simultaneous measurement of calcium oscillations in a 96-well plate. Features of waveforms were extracted automatically and allowed the characterization of some anti-epileptic drugs using principal component analysis. Moreover, we have shown that trajectories in accordance with the concentrations of compounds in principal component analysis plots were unique to the mechanism of anti-epileptic drugs. We believe that an approach that focuses on the features of calcium oscillations will lead to better understanding of the characteristics of existing anti-epileptic drugs and allow prediction of the mechanism of action of novel drug candidates.

neuroscience↗

bioRxiv2022

The cell wall polymer initiates plasma membrane partitioning in mycobacteria

Lateral partitioning of proteins and lipids shapes membrane function. In model membranes, partitioning can be influenced by interactions with other membranes and solid supports. While cellular membranes can departition in response to various perturbations, including disruption of bilayer-extrinsic structures, the mechanisms by which they partition de novo are largely unknown. The plasma membrane of Mycobacterium smegmatis can be spatially and biochemically departitioned by the fluidizing agent benzyl alcohol. By screening for mutants that are sensitive to benzyl alcohol, we show that the bifunctional cell wall synthase PonA2 promotes membrane partitioning and cell growth upon fluidizer washout. PonA2s role in membrane repartitioning and regrowth depends solely on its conserved transglycosylase domain. We find that the cell wall polymer, but not active cell wall polymerization, is critical for membrane partitioning. Our work highlights a key initiating role for bilayer-extrinsic structures in patterning cellular membranes.

molecular biology↗

bioRxiv2022

No circadian clock-gene circuit in the generation of cellular bioluminescence rhythm of CaMV35S::PtRLUC in duckweed

Physiological circadian rhythms are coordinated in the plant body in an orderly manner. The coordination of time information has been studied from the aspects of cell-cell local coupling and long-distance communication between tissues. These studies were based on the idea that the behavior of the clock gene circuit represents the physiological rhythms. Here we report the cellular circadian rhythm of a bioluminescence reporter which is not governed by the clock gene circuit in the expressing cells. Using a dual-color bioluminescence monitoring system in Lemna minor transfected with the AtCCA1::LUC+ and CaMV35S::PtRLUC reporters, cellular bioluminescence rhythms with different free-running periods (FRPs) were detected in the same cells. Co-transfection experiments with the two reporters and a clock gene overexpressing effector revealed that the circadian properties of the AtCCA1::LUC+ rhythm, but not those of the CaMV35S::PtRLUC rhythm, were altered in the cells with a dysfunctional clock gene circuit. This indicates that the AtCCA1::LUC+ rhythm is a direct output of the cellular circadian clock while the CaMV35S::PtRLUC rhythm is not. After plasmolysis, the CaMV35S::PtRLUC rhythm disappeared while the AtCCA1::LUC+ rhythm persisted. The plant circadian system consists of both cell-autonomous rhythms and non-cell-autonomous rhythms that are unaffected by the cellular clock.

plant biology↗

bioRxiv2022

Oxytocin and Emotional Tear in Dogs

In humans, tear volume increases in emotional arousal. No studies have investigated the relationship of emotional arousal and tear volume in animals. We measured tear volume in dogs before and after reunion with their owners. Tear volume increased significantly during owner, but not familiar non-owner, reunions but not during reunions with familiar non-owners. When oxytocin instillation was applied to dogs, the tear volume increased, suggesting that oxytocin can mediate the emotion-induced tear secretion in dogs. When the photos of dogs face in which artificial tear was applied to the dogs eyes, the positive impression of these photo increased. These suggest that emotional tear can facilitate human-dog emotional connection.

animal behavior and cognition↗

bioRxiv2022

The oxytocin system regulates tearing

Tears are an exocrine physiological fluid secreted onto the ocular surface from the lacrimal apparatus in all mammals. Limited research has been conducted on the functional neuronal circuitry of tear production. In particular, the neuronal mechanisms of emotional tearing, which is a physiological reaction harmonized with enhanced emotional arousal and assumed to be unique to humans, remain unclear. We identified that the oxytocin neurons in the paraventricular hypothalamus is functionally projected to the oxytocin receptor-expressing neurons in the lacrimation center of the superior salivatory nucleus. Optogenetic activation or inhibition of these neurons and/or receptors can modulate the superior salivatory nucleus dependent tear secretion mediated through oxytocin. Moreover, we identified that maternal behavior, nociceptive behavior stimulation, and aversive memory retrieval are linked to tearing in mice, and that these emotional linked tearing are suppressed by optogenetic inhibition of the corresponding oxytocin system. Thus, tearing could be regulated through functional connections between central oxytocin systems in the paraventricular hypothalamus and the superior salivatory nucleus.

animal behavior and cognition↗

bioRxiv2022

Molecular mechanisms of Plasmodium development in male and female Anopheles mosquitoes

Vector competence influences the ability of Anopheles mosquitoes to transmit Plasmodium parasites. The aim of this study was to determine the competence of male and female Anopheles mosquitoes to support the development of Plasmodium parasites. Male and female A. stephensi (STE2 strain) were infected with in vitro-cultured P. berghei (ANKA strain) ookinetes. We found that the number of oocysts produced was higher in males than in females. RNA-seq analysis of male and female mosquitoes injected with P. berghei ookinetes showed that predominantly genes of unknown function changed in expression levels in response to ookinete infection; however, further studies are required to elucidate their functions. Moreover, male mosquitoes were injected with in vitro-cultured P. falciparum (3D7 strain) gametocytes or zygotes. The development of P. falciparum in males was detected using nested polymerase chain reaction. We found the DNA content was higher on day 15 than on day 0, indicating that P. falciparum developed in the mosquito hemocoel. This study revealed promising new mechanisms underlying the interactions between Plasmodium and mosquitoes. Author SummaryAnopheles mosquitoes transmit Plasmodium parasites that cause malaria disease. Vector competence is the ability of vectors to support pathogen development. The aim of this study was to determine the competence of male and female mosquitoes for Plasmodium parasites. Oocysts were formed in both male and female mosquitoes injected with ookinetes from in vitro-cultured Plasmodium berghei, which causes malaria in mice. The number of oocysts was higher in males, indicating that the competence in males was higher than that in females. RNA-seq analysis showed that the expression of genes of unknown function was highly variable in males and females. The genes defining the competence factors need further study, but the results indicate novel genes may be discovered. Furthermore, the development of Plasmodium falciparum, which causes the most severe malaria in humans, in male mosquitoes injected with in vitro-cultured gametocytes or zygotes was detected. As male mosquitoes do not suck blood, this method may allow us to experiment with Plasmodium more safely.

microbiology↗

bioRxiv2021

Engineered ACE2 counteracts vaccine-evading SARS-CoV-2 Omicron variant

The novel SARS-CoV-2 variant, Omicron (B.1.1.529) contains an unusually high number of mutations (>30) in the spike protein, raising concerns of escape from vaccines, convalescent sera and therapeutic drugs. Here we analyze the alteration of neutralizing titer with Omicron pseudovirus. Sera obtained 3 months after double BNT162b2 vaccination exhibit approximately 18-fold lower neutralization titers against Omicron than parental virus. Convalescent sera from Alpha and Delta patients allow similar levels of breakthrough by Omicron. Domain-wise analysis using chimeric spike revealed that this efficient evasion was primarily achieved by mutations clustered in the receptor-binding domain, but that multiple mutations in the N-terminal domain contributed as well. Omicron escapes a therapeutic cocktail of imdevimab and casirivimab, whereas sotrovimab, which targets a conserved region to avoid viral mutation, remains effective. The ACE2 decoy is another virus-neutralizing drug modality that is free, at least in theory, from complete escape. Deep mutational analysis demonstrated that, indeed, engineered ACE2 prevented escape for each single-residue mutation in the receptor-binding domain, similar to immunized sera. Engineered ACE2 neutralized Omicron comparable to Wuhan and also showed a therapeutic effect against Omicron infection in hamsters and human ACE2 transgenic mice. Like previous SARS-CoV-2 variants, some sarbecoviruses showed high sensitivity against engineered ACE2, confirming the therapeutic value against diverse variants, including those that are yet to emerge. One Sentence SummaryOmicron, carrying [~]30 mutations in the spike, exhibits effective immune evasion but remains highly susceptible to blockade by engineered ACE2.

microbiology↗

bioRxiv2021

Myogenetic oligodeoxynucleotides as anti-nucleolin aptamers inhibit the growth of embryonal rhabdomyosarcoma cells

BackgroundEmbryonal rhabdomyosarcoma (ERMS) is the muscle-derived tumor retaining myogenic ability. iSN04 and AS1411, which are myogenetic oligodeoxynucleotides (myoDNs) serving as anti-nucleolin aptamers, have been reported to inhibit the proliferation and induce the differentiation of myoblasts. The present study investigated the effects of iSN04 and AS1411 on the growth of multiple ERMS1 cell lines in vitro. MethodsThree patient-derived ERMS cell lines, ERMS1, KYM1, and RD, were used. Nucleolin expression and localization in these cells was confirmed by immunostaining. The effects of iSN04 or AS1411 on the growth of ERMS cells were examined by cell counting, EdU staining, quantitative RT-PCR (qPCR), and three-dimensional culture of tumorspheres. ResultsIn all ERMS cell lines, nucleolin was abundantly expressed, and localized and concentrated in nucleoli, similar to myoblasts. Both iSN04 and AS1411 (10-30 M) significantly decreased the number of all ERMS cells; however, their optimal conditions were different among the cell lines. iSN04 (10 M) markedly reduced the ratio of EdU+ cells, indicating the inhibition of cell proliferation. qPCR demonstrated that iSN04 suppressed the cell cycle, partially promoted myogenesis, but did not induce apoptosis. Finally, both iSN04 and AS1411 (10-30 M) disrupted the formation and outgrowth of RD tumorspheres mimicking in vivo tumorigenesis. ConclusionsERMS cells expressed nucleolin, and their growth was inhibited by the anti-nucleolin aptamers, iSN04 and AS1411. The present study provides the first evidence that anti-nucleolin aptamers can be used as nucleic acid drugs for chemotherapy against ERMS.

cell biology↗

bioRxiv2021

Enterococcus spp. have higher fitness for survival, in a pH-dependent manner, in pancreatic juice among duodenal bacterial flora

ObjectivesBacterial infection is involved in the progression of many gastrointestinal diseases, including cancer; however, how and which bacteria colonize in pancreatic juice and tissue have yet to be elucidated. Recently, we reported that Enterococcus faecalis exists in the pancreatic juice and tissues of patients with chronic pancreatic disease. Here, we investigated the survival of E. faecalis in duodenal juice with different pH conditions. MethodsPancreatic juice samples from 62 patients with cancers of the duodeno-pancreato-biliary region were evaluated for the presence of E. faecalis. 16S ribosomal RNA PCR and 16S-based metagenome analyses were performed to determine the bacterial composition. The survival of E. faecalis in various pancreatic juice conditions was evaluated. ResultsOf 62 samples, 27% (17/62) were positive for Enterococcus spp., among which 71% (12/17) contained E. faecalis. Enterococcus spp. showed the highest fitness for survival in alkaline pancreatic juice among various bacterial species. The microbiome of pancreatic juice from patients with pancreatic and bile duct cancer showed diversity, but Enterococcus spp. were enriched among duodenal tumors and intraductal papillary mucinous neoplasms. ConclusionsAlkalinity is important for the selective survival of E. faecalis among microbiota. E. faecalis may induce pancreatic inflammation with changes in pancreatic juice conditions.

microbiology↗

bioRxiv2021

Light-dependent synthesis of a nucleotide second messenger controls bacterial motility

Nucleotide second messengers are universally crucial factors for the signal transduction of various organisms. In prokaryotes, cyclic nucleotide messengers are involved in the bacterial life cycle and function, such as virulence, biofilm formation, and others mainly via gene regulation. Here we show that the swimming motility of a soil bacterium is rapidly modulated by cyclic adenosine monophosphate (cAMP) synthesized upon light exposure. Analysis of a loss-of-photoresponsivity mutant obtained by transposon random mutagenesis determined the novel sensory gene, and its expression in Escherichia coli through codon optimization revealed the light-dependent synthesis of cAMP. GFP labeling showed the localization of the photoresponsive enzyme at the cell poles where flagellar motors reside. The present findings highlight the new role of cAMP that rapidly controls the flagella-dependent bacterial motility and the global distribution of the discovered photoactivated cyclase among diverse microbial species.

microbiology↗

bioRxiv2021

Adaptive diversification in the cellular circadian behavior of Arabidopsis leaf- and root-derived cells

The plant circadian system is based on self-sustained cellular oscillations and is utilized to adapt to daily and seasonal environmental changes. The cellular circadian clocks in the above- and belowground plant organs are subjected to diverse local environments. Individual cellular clocks are affected by other cells/tissues in plants, and the intrinsic properties of cellular clocks remain to be elucidated. In this study, we showed the circadian properties of leaf- and root-derived cells of a CCA1::LUC Arabidopsis transgenic plant and demonstrated that the cells in total isolation from other cells harbor a genuine circadian clock. Quantitative and statistical analyses for individual cellular bioluminescence rhythms revealed a difference in amplitude and precision of light/dark entrainment between the two cell-types, suggesting that leaf-derived cells have a clock with a stronger persistence against fluctuating environments. Circadian systems in the leaves and roots are diversified to adapt to their local environments at the cellular level.

plant biology↗

bioRxiv2021

Depression-like state induced by low-frequency repetitive transcranial magnetic stimulation to ventral medial frontal cortex in monkeys

The medial frontal cortex (MFC), especially its ventral part, has long been of great interest with respect to the pathology of mood disorders. A number of human brain imaging studies have demonstrated the abnormalities of this brain region in patients with mood disorders, however, whether it is critically involved in the pathogenesis of such disorders remains to be fully elucidated. In this study, we conducted a causal study to investigate how the suppression of neural activity in the ventral region of the MFC (vMFC) affects the behavioral and physiological states of monkeys by using repetitive transcranial magnetic stimulation (rTMS). By using low-frequency rTMS (LF-rTMS) as an inhibitory intervention, we found that LF-rTMS targeting the vMFC induced a depression-like state in monkeys, which was characterized by a reduced spontaneous behavioral activity, increased plasma cortisol level, impaired sociability, and decreased motivation level. On the other hand, no such significant changes in behavioral and physiological states were observed when targeting the other MFC regions, dorsal or posterior. We further found that the administration of an antidepressant agent, ketamine, ameliorated the abnormal behavioral and physiological states induced by the LF-rTMS intervention. These findings indicate the causal involvement of the vMFC in the regulation of mood and affect and the validity of the LF-rTMS-induced dysfunction of the vMFC as a nonhuman primate model of the depression-like state.

neuroscience↗

bioRxiv2021

Identification, crystallization and epitope determination of public TCR shared and expanded in COVID-19 patients

T cells play pivotal roles in protective immunity against SARS-CoV-2 infection. Follicular helper T (Tfh) cells mediate the production of antigen-specific antibodies; however, T cell receptor (TCR) clonotypes used by SARS-CoV-2-specific Tfh cells have not been well characterized. Here, we first identified and crystallized public TCR of Tfh clonotypes that are shared and expanded in unhospitalized COVID-19-recovered patients. These clonotypes preferentially recognized SARS-CoV-2 spike (S) protein epitopes which are conserved among emerging SARS-CoV-2 variants. These clonotypes did not react with S proteins derived from common cold human coronaviruses, but cross-reacted with symbiotic bacteria, which might confer the publicity. Among SARS-CoV-2 S epitopes, S864-882, presented by frequent HLA-DR alleles, could activate multiple public Tfh clonotypes in COVID-19-recovered patients. Furthermore, S864-882-loaded HLA tetramer preferentially bound to CD4+ T cells expressing CXCR5. In this study, we identified and crystallized public TCR for SARS-CoV-2 that may contribute to the prevention of COVID-19 aggravation.

immunology↗