Biology subjects

Yamamoto, S.

Publications and source records attributed to Yamamoto, S..

At least 55 records · Page 3Linked to original sources

bioRxiv2024

AAV delivery of GBA1 suppresses alpha-synuclein accumulation in Parkinson's disease models and restores motor dysfunction in a Gaucher's disease model

Biallelic mutations in the glucosylceramidase beta 1 (GBA1) gene are the underlying genetic cause of Gauchers disease (GD), resulting in a deficient lysosomal hydrolase and subsequent accumulation of glycosphingolipids. More recently, GBA1 mutations have been identified as the most prevalent genetic risk factor for Parkinsons disease (PD), associated with more pronounced symptoms characterized by earlier onset and accelerated cognitive decline. In these GBA-associated PD patients the -synuclein pathology is more prominent, and recent data suggest a link between -synucleinopathies and GBA1 mutations. Here, we explored the effect of GBA1 gene supplementation on the GD phenotypes and -synuclein pathology by using the adeno-associated virus (AAV) system. We have compared two AAV serotypes, AAV5 and AAV9, and two different ubiquitous promoters, and demonstrate that both promoters work efficiently albeit not the same in vitro and in vivo. GBA1 overexpression reduces the accumulation of glucosylsphingosine (GlcSph) and restores motor dysfunction in a GD mouse model. We further demonstrate that GBA1 overexpression can dissolve phospho--synuclein aggregation induced by the addition of -synuclein pre-formed fibril (PFF) in a mouse primary neuron model suggesting the direct effect of {beta}-Glucocerebrosidase (GCase) on -synuclein accumulation. In vivo, we show that GCase inhibition can induce insoluble high-molecular-weight -synuclein aggregation and that delivery of GBA1 achieves robust reduction of the -synuclein aggregates in the mouse brain. In summary, GCase expression not only reduces GlcSph, but also restores GD motor dysfunction and removes -synuclein aggregates which are the hallmark for PD and -synucleinopathies. AAV delivery of GBA1 is a powerful approach to restore glucocerebrosidase function and to resolve misfolded -synuclein protein, with applications for GD and PD.

neuroscience↗

bioRxiv2024

Multimodal mechanisms of human centriole engagement and disengagement

The DNA and the centrioles are the only cellular structures that uniquely replicate to produce identical copies, which is crucial for proper chromosome segregation in mitosis. A new centriole termed "daughter" is progressively assembled adjacent to a pre-existing, "mother" centriole. Only after the daughter centriole is structurally completed as an identical copy, it disengages from its mother to become the core of a new functional centrosome. The mechanisms preventing precocious disengagement of the immature copy have been previously unknown. Here, we identify three key centriole-associated mechanisms that maintain the mother-daughter engagement: the cartwheel, the torus, and the pericentriolar material pathways. Among these, the torus is critical for establishing the characteristic orthogonal engagement between the mother and daughter centrioles. Furthermore, we show that the engagement mediated by the cartwheel and the torus pathways is released stepwise through structural changes in the daughter centriole, known as centriole blooming and centriole distancing, respectively. Disruption at any stage of these structural transitions leads to the failure of all subsequent steps, ultimately blocking centriole disengagement and centrosome conversion at the end of mitosis. Overall, this study provides a comprehensive understanding of how the maturing daughter centriole progressively disengages from its mother through multiple maturation steps, to ensure its complete structure and conversion into an independent centrosome.

cell biology↗

bioRxiv2024

Seesaw protein: Design of a protein that adopts interconvertible alternative functional conformations.

Classical Anfinsen's dogma states that a protein folds into a single unique conformation with minimal Gibbs energy under physiological conditions. However, recent advances have revealed that single amino acid sequences can fold into two or more conformations. Here, we propose a novel approach to design a protein that adopts interconvertible alternative functional conformations, termed "seesaw" protein (SSP). An SSP was engineered by fusing GFP lacking the C-terminal {beta}-strand and DHFR lacking the N-terminal {beta}-strand with an overlapping linker, which can be competitively incorporated into either the GFP or the DHFR moiety. In vivo and biochemical analysis, including AFM imaging, demonstrated that the SSP adopts two alternative conformations, which can be biased by point mutations and ligand binding. In addition, the balance of the seesaw can be reversibly changed depending on buffer conditions. In summary, our design strategy for SSP provides a new direction for creating artificial proteins with on-off behaviors.

biochemistry↗

bioRxiv2024

Visualization of intracellular ATP dynamics in the whole kidney under pathophysiological conditions using the kidney slice culture system

ATP depletion plays a central role in the pathogenesis of kidney diseases. Recently, we reported spatiotemporal intracellular ATP dynamics during ischemia reperfusion (IR) using GO-ATeam2 mice systemically expressing an ATP biosensor. However, observation from the kidney surface did not allow visualization of deeper nephrons or accurate evaluation of ATP synthesis pathways. Here we established a novel ATP imaging system using slice culture of GO-ATeam2 mouse kidneys, evaluated ATP synthesis pathway, and analyzed intracellular ATP dynamics using ex vivo IR and cisplatin nephropathy models. We demonstrated that proximal tubules (PTs) were strongly dependent on oxidative phosphorylation (OXPHOS), whereas podocytes relied on both OXPHOS and glycolysis using ATP synthesis inhibitors. We also confirmed that the ex vivo IR model could recapitulate ATP dynamics in vivo; ATP recovery in PTs after reperfusion varied depending on ischemia length, whereas ATP in distal tubules (DTs) recovered well even after long ischemia. After cisplatin administration, ATP levels in PTs decreased first, followed by a decrease in DTs. An organic cation transporter 2 inhibitor suppressed cisplatin uptake in kidney slices, leading to better ATP recovery in PTs, but not in DTs. Finally, we confirmed that a mitochondria protection reagent delayed cisplatin-induced ATP decrease in PTs. This novel system may provide new insights into the energy dynamics and pathogenesis of kidney disease.

molecular biology↗

bioRxiv2024

Fetal Kidney Transplantation for In Utero Fetuses

Potter sequence, characterized by bilateral renal agenesis, oligohydramnios, and consequent pulmonary hypoplasia, presents a significant challenge in the management of affected neonates. Due to their prematurity and associated abdominal complications, these infants often fail to reach a stage where dialysis can be safely initiated and sustained, leading to an exceedingly high mortality rate. Therefore, there is hopeful anticipation that interventions serving as a bridge to achieve a state where dialysis can be safely performed will markedly improve life expectancy. We have developed a unique approach of "transplantation of fetal kidneys from a different species during the fetal period" as a bridge therapy until stable dialysis therapy can be implemented. This is a new concept of fetal therapy, targeting the fetus in utero and utilizing fetal kidneys of an appropriate size for transplantation. In this study, we first validated the approach using allogeneic transplantation. Fetal kidneys with bladders from GFP-expressing rats (gestational age 14.0-16.5 days) were transplanted subcutaneously into allogeneic rat fetuses in utero (gestational age 18.0-18.5 days) using a special needle transuterinally, and live pups were successfully obtained. The transplanted fetal kidneys with bladders were confirmed to have urine production capability. By periodic aspiration of the subcutaneous urinary cyst after birth, urine produced by the transplanted fetal kidney was successfully drained outside the body for an extended period (up to 150 days). Biochemical tests confirmed the solute removal capacity of the transplanted fetal kidney. Furthermore, despite allogeneic transplantation, long-term urine production was sustained without the use of immunosuppressants, confirming that organ transplantation into fetuses is associated with lower rejection compared to adult transplantation. Next, xenotransplantation was performed. When GFP-expressing mouse fetal kidneys (gestational age 13.0-13.5 days) were transplanted into rat fetuses in utero, maturation of renal tissue structures was confirmed even in the interspecies setting.

bioengineering↗

bioRxiv2024

Flow zoometry of Drosophila

Drosophila serves as a highly valuable model organism across numerous fields including genetics, immunology, neuroscience, cancer biology, and developmental biology. Central to Drosophila-based biological research is the ability to perform comprehensive genetic or chemical screens. However, this research is often limited by its dependence on laborious manual handling and analysis, making it prone to human error and difficult to discern statistically significant or rare events amid the noise of individual variations resulting from genetic and environmental factors. In this article we present flow zoometry, a whole-animal equivalent of flow cytometry for large-scale, individual-level, high-content screening of Drosophila. Our flow zoometer automatically clears the tissues of Drosophila melanogaster, captures three-dimensional (3D) multi-color fluorescence tomograms of single flies with single-cell volumetric resolution at an unprecedented throughput of over 1,000 animals within 48 hours (24 hr for clearing; 24 hr for imaging), and performs AI-enhanced data-driven analysis - a task that would traditionally take months or years with manual techniques. To demonstrate its broad applications, we employed the flow zoometer in various laborious screening assays, including those in toxicology, genotyping, and tumor screening. Flow zoometry represents a pivotal evolution in high-throughput screening technology: previously from molecules to cells, now from cells to whole animals. This advancement serves as a foundational platform for "statistical spatial biology", to improve empirical precision and enable serendipitous discoveries across various fields of biology.

bioengineering↗

bioRxiv2024

Whole organism snRNA-seq reveals systemic peripheral changes in Alzheimer's Disease fly models

Peripheral tissues become disrupted in Alzheimers Disease (AD). However, a comprehensive understanding of how the expression of AD-associated toxic proteins, A{beta}42 and Tau, in neurons impacts the periphery is lacking. Using Drosophila, a prime model organism for studying aging and neurodegeneration, we generated the Alzheimers Disease Fly Cell Atlas (AD-FCA): whole-organism single-nucleus transcriptomes of 219 cell types from adult flies neuronally expressing human A{beta}42 or Tau. In-depth analyses and functional data reveal impacts on peripheral sensory neurons by A{beta}42 and on various non-neuronal peripheral tissues by Tau, including the gut, fat body, and reproductive system. This novel AD atlas provides valuable insights into potential biomarkers and the intricate interplay between the nervous system and peripheral tissues in response to AD-associated proteins.

genetics↗

bioRxiv2024

Loss of function in RBBP5 results in a syndromic neurodevelopmental disorder associated with microcephaly

PurposeEpigenetic dysregulation has been associated with many inherited disorders. RBBP5 encodes a core member of the protein complex that methylates histone 3 lysine-4 (H3K4) and has not been implicated in human disease. MethodsWe identify five unrelated individuals with de novo heterozygous pathogenic variants in RBBP5. Three truncating and two missense variants were identified in probands with neurodevelopmental symptoms including global developmental delay, intellectual disability, microcephaly, and short stature. Here, we investigate the pathogenicity of the variants through protein structural analysis and transgenic Drosophila models. ResultsBoth missense p.T232I and p.E296D variants affect evolutionarily conserved amino acids and are expected to interfere with the interface between RBBP5 and the histones. In Drosophila, ubiquitous overexpression of human RBBP5 is lethal in the larval developmental stage. Loss of Rbbp5 leads to a reduction in brain size, and the human reference, p.T232I, or p.E296D variant transgenes fail to rescue loss of Rbbp5. Expression of either missense variant in an Rbbp5 null background results in a less severe microcephaly phenotype than the human reference, indicating both p.T232I and p.E296D variants are loss-of-function alleles. ConclusionDe novo heterozygous variants in RBBP5 are associated with a syndromic neurodevelopmental disorder. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=193 HEIGHT=200 SRC="FIGDIR/small/578086v1_ufig1.gif" ALT="Figure 1"> O_LINKSMALLFIG WIDTH=200 HEIGHT=24 SRC="FIGDIR/small/578086v1_ufig1a.gif" ALT="Figure 1"> View larger version (59K): org.highwire.dtl.DTLVardef@11337daorg.highwire.dtl.DTLVardef@b7f4d8org.highwire.dtl.DTLVardef@12bc283org.highwire.dtl.DTLVardef@1ef4e09_HPS_FORMAT_FIGEXP M_FIG C_FIG

genetics↗

bioRxiv2023

Effects of local brain temperature on somatosensory evoked potentials in rats

Although the focal brain cooling technique is widely used to examine brain function, the effects of cortical temperature at various levels on sensory information processing and neural mechanisms remain underexplored. To elucidate the mechanisms of temperature modulation in somatosensory processing, this study aimed to examine how P1 and N1 deflections of somatosensory evoked potentials (SEPs) depend on cortical temperature and how excitatory and inhibitory inputs contribute to this temperature dependency. SEPs were generated through electrical stimulation of the contralateral forepaw in anesthetized rats. The SEPs were recorded while cortical temperatures were altered between 17-38 {degrees}C either without any antagonists, with a gamma-aminobutyric acid type A (GABAA) receptor antagonist (gabazine), with aminomethylphosphonic acid (AMPA) receptor antagonist (NBQX), or with N-Methyl-D-aspartic acid (NMDA) receptor antagonist ([R]-CPP). The effects of different gabazine concentrations (0, 1, and 10 {micro}M) were examined in the 35-38 {degrees}C range. The P1/N1 amplitudes and their peak-to-peak differences plotted against cortical temperature showed an inverted U relationship with a maximum at approximately 27.5 {degrees}C when no antagonists were administered. The negative correlation between these amplitudes and temperatures of [≥]27.5 {degrees}C plateaued after gabazine administration, which occurred progressively as the gabazine concentration increased. In contrast, the correlation remained negative after the administration of NBQX and (R)-CPP. These results suggest that GABAergic inhibitory inputs contribute to the negative correlation between SEP amplitude and cortical temperature around the physiological cortical temperature. HighlightsO_LIFocal cortical cooling altered somatosensory evoked potentials (SEPs). C_LIO_LISEP amplitude was negatively correlated with cortical temperatures of 27.5-38.0 {degrees}C. C_LIO_LIGABAAR but not AMPAR nor NMDAR antagonists eliminated the negative correlation. C_LIO_LIGABAergic signaling is involved in the temperature dependency of SEPs. C_LI

neuroscience↗

bioRxiv2023

Role of the medial agranular cortex in unilateral spatial neglect

Unilateral spatial neglect (USN) results from impaired attentional networks and can affect various sensory modalities, such as visual and somatosensory. The rodent medial agranular cortex (AGm), located in the medial part of the forebrain from rostral to caudal direction, is considered a region associated with spatial attention. The AGm selectively receives multisensory input with the rostral AGm receiving somatosensory input and caudal part receiving visual input. Our previous study showed slower recovery from neglect with anterior AGm lesion using the somatosensory neglect assessment. Conversely, the functional differences in spatial attention across the entire AGm locations (anterior, intermediate, and posterior parts) are unknown. Here, we investigated the relationship between the severity of neglect and various locations across the entire AGm in a mouse stroke model using a newly developed program-based analysis method that does not require human intervention. Among the various lesion positions, acute severity was higher with the lesion in the intermediate rostrocaudal position. On the other hand, the recovery from USN-like behavior after this phase tended to be slower in cases with more rostral lesions in the AGm. Additionally, no motor paralysis was observed in any of the mice with lesions in each AGm. These results suggest that the intermediate rostrocaudal position of the AGm may significantly influence selection of the direction, regardless of the areas to which it is connected. On the contrary, recovery from USN-like behavior may be dependent on the areas to which it is connected. HighlightsLesion of the rodent medial agranular cortex (AGm) results in unilateral spatial neglect (USN). In the acute phase, the severity was higher with lesions in the intermediate AGm position. Recovery from somatosensory USN tended to be slower with rostral AGm lesions. Recovery from USN may depend on sensory modalities associated with the connected areas. Our results revealed location-dependent differences in attentional functions within the AGm.

animal behavior and cognition↗

bioRxiv2023

RNAi-based screen for cuticle pigmentation in Drosophila melanogaster reveals novel regulators of brain dopamine

The dopaminergic system has been extensively studied for its role in behavior and neurological diseases. Despite this, we still know little about how dopamine levels are regulated in vivo. To identify regulators of dopamine, we utilized Drosophila melanogaster cuticle pigmentation as a readout, where dopamine is used as a precursor to melanin. We started by measuring dopamine from known pigmentation mutants (e.g. tan, ebony, black) and then performed an RNAi-based screen to identify new regulators. We found 153 hits, which were enriched for developmental signaling pathways and mitochondria-associated proteins. From 35 prioritized candidates, 11 had an effect on head dopamine levels. Effects on brain dopamine were mild even when the rate-limiting synthesis enzyme Tyrosine hydroxylase (TH) was knocked down, suggesting changes in dopamine levels are tightly regulated in the nervous system. We pursued two of our hits that reduced brain dopamine levels, clueless and mask. Further examination suggests that mask regulates transcription of TH and affects dopamine-dependent sleep patterns. In summary, by studying genes that affect cuticle pigmentation, we were able to identify genes that affect dopamine metabolism as well as a novel regulator of behavior.

neuroscience↗

bioRxiv2023

Analysis of histone H3K4me3 modifications in bovine placenta derived from different calf-production methods

In ruminants, overgrowth of offspring produced by in vitro fertilization (IVF) is a common problem. Abnormal epigenetic modifications caused by environmental factors during the early embryonic period are suspected as an etiology of overgrowth. In this study, we investigated the genome-wide histone H3K4me3 profiles of bovine placentae that play a pivotal role in fetal development and compared their characteristics between artificial insemination (AI)- and IVF-derived samples. Cotyledons were harvested from the placentae obtained at parturition of 5 AI- and 13 IVF-derived calves, and chromatin immunoprecipitation sequencing was performed for H3K4me3. We confirmed no significant maternal tissue contamination in the samples we used. The revealed H3K4me3 profiles reflected the general characteristics of the H3K4me3 modification, which is abundantly distributed in the promoter region of active genes. By extracting common modifications from multiple samples, the genes involved in placenta-specific biological processes could be enriched. Comparison with the H3K4me3 modifications of blastocyst samples was also effective for enriching the placenta-specific features. Principal component analysis suggested the presence of differential H3K4me3 modifications in AI- and IVF-derived samples. The genes contributing to the difference were related to the developmental biological processes. Imprinted genes such as BEGAIN, ZNF215, and DLX5 were among the extracted genes. Principal component and discriminant analyses using only male samples categorized the samples into three groups based on fetal weight and calf-production methods. To our knowledge, this is the first study to profile the genome-wide histone modifications of bovine fetal placentae and reveal their differential characteristics between different calf-production methods.

genomics↗

bioRxiv2023

Comparable analysis of multiple DNA double-strand break repair pathways in CRISPR-mediated endogenous tagging

CRISPR-mediated endogenous tagging, utilizing the homology-directed repair (HDR) of DNA double-strand breaks (DSBs) with exogenously incorporated donor DNA, is a powerful tool in biological research. Inhibition of the non-homologous end joining (NHEJ) pathway has been proposed as a promising strategy for improving the low efficiency of accurate knock-in via the HDR pathway. However, the influence of alternative DSB repair pathways on gene knock-in remains to be fully explored. In this study, our long-read amplicon sequencing analysis reveals various patterns of imprecise repair in CRISPR/Cas-mediated knock-in, even under conditions where NHEJ is inhibited. Suppression of the microhomology-mediated end joining (MMEJ) or the single strand annealing (SSA) repair mechanisms leads to a reduction in distinct patterns of imprecise repair, thereby elevating the efficiency of accurate knock-in. Furthermore, a novel reporter system shows that the SSA pathway contributes to a specific pattern of imprecise repair, known as asymmetric HDR. Collectively, our study uncovers the involvement of multiple DSB repair pathways in CRISPR/Cas-mediated gene knock-in and proposes alternative approaches to enhance the efficiency of precise gene knock-in.

genomics↗

bioRxiv2023

A Zika virus protein expression screen in Drosophila to investigate targeted host pathways during development

In the past decade, Zika virus (ZIKV) emerged as a global public health concern. While adult infections are typically mild, maternal infection can lead to adverse fetal outcomes. Understanding how ZIKV proteins disrupt development can provide insights into the molecular mechanisms of symptoms caused by this virus including microcephaly. In this study, we generated a toolkit to ectopically express Zika viral proteins in vivo in Drosophila melanogaster in a tissue-specific manner using the GAL4/UAS system. We use this toolkit to identify phenotypes and host pathways targeted by the virus. Our work identified that expression of most ZIKV proteins cause scorable phenotypes, such as overall lethality, gross morphological defects, reduced brain size, and neuronal function defects. We further use this system to identify strain-dependent phenotypes that may contribute to the increased pathogenesis associated with the more recent outbreak of ZIKV in the Americas. Our work demonstrates Drosophilas use as an efficient in vivo model to rapidly decipher how pathogens cause disease and lays the groundwork for further molecular study of ZIKV pathogenesis in flies.

developmental biology↗

bioRxiv2023

Statistical mining of triple-negative breast cancer-specific nanobodies among huge libraries from immunized alpacas

Breast cancer can be classified into several types according to the expression patterns of human epidermal growth factor receptor 2 (Her2), oestrogen receptor (ER), and progesterone receptor (PgR) proteins. The prognosis of patients with tumors showing low Her2 expression and no ER and PgR expression--categorized as triple-negative breast cancer (TNBC)--is worst among these groups. Due to the lack of specific antibodies for TNBC, curative treatments for TNBC remain limited. Antibodies targeting TNBC have potential as diagnostic and therapeutic tools. Here, we generate a panel of nanobodies targeting TNBC cell lines by immunizing alpacas and subsequently panning the resulting phage libraries with TNBC cell lines. We show that several clones exclusively stain Her2-negative cells in tissues of breast cancer patients, and a few clones stain both Her2-positive and Her2-negative regions in these tissues. These clones can be applied to patient-specific therapies using drug-conjugated antibodies, radiolabelled antibodies, chimaera antigen receptor T cells, or drug delivery components, as well as to TNBC diagnosis.

cancer biology↗

bioRxiv2022

De Novo Variants in MRTFB have gain of function activity in Drosophila and are associated with a novel neurodevelopmental phenotype with dysmorphic features.

Myocardin-Related Transcription Factor B (MRTFB) is an important transcriptional regulator which promotes the activity of an estimated 300 genes during different stages of development. Here we report two pediatric probands with de novo variants in MRTFB (R104G and A91P) and mild dysmorphic features, intellectual disability, global developmental delays, speech apraxia, and impulse control issues. As the MRTFB protein is highly conserved between vertebrate and invertebrate model organisms, we generated a humanized Drosophila model expressing the human MRTFB protein in the same spatial and temporal pattern as the fly gene. Expression of the human MRTFBR104G variant using a mrtf-T2A-GAL4 line proved to be embryonic lethal. Additional phenotypes were also identified by expressing the MRTFBR104G and MRTFBA91P variant in a subset of Drosophila tissues. Notably, expression within wing tissues resulted in an expansion of intervein tissue, wing vein thickening, shortening or loss of wing veins, and blistering. The MRTFBR104G and MRTFBA91P variants also display a decreased level of actin binding within critical RPEL domains, resulting in increased transcriptional activity and changes in the organization of the Actin cytoskeleton. These changes were not observed in flies expressing two additional candidate variants, MRTFBN95Sand MRTFBR109Q, highlighting that the location of the mutation within the 2nd RPEL domain is critical to the pathogenicity of the variant. These changes suggest that the MRTFBR104G and MRTFBA91P alleles we have identified affect the regulation of the protein and that these variants in MRTFB underly a novel neurodevelopmental disorder.

genetics↗

bioRxiv2022

The molecular mechanism for carbon catabolite repression of the chitin response in Vibrio cholerae

Vibrio cholerae is a facultative pathogen that primarily occupies marine environments. In this niche, V. cholerae commonly interacts with the chitinous shells of crustacean zooplankton. As a chitinolytic microbe, V. cholerae degrades insoluble chitin into soluble oligosaccharides. Chitin oligosaccharides serve as both a nutrient source and an environmental cue that induces a strong transcriptional response in V. cholerae. Namely, these oligosaccharides induce the chitin sensor, ChiS, to activate the genes required for chitin utilization and horizontal gene transfer by natural transformation. Thus, interactions with chitin impact the survival of V. cholerae in marine environments. Chitin is a complex carbon source for V. cholerae to degrade and consume, and the presence of more energetically favorable carbon sources can inhibit chitin utilization. This phenomenon, known as carbon catabolite repression (CCR), is mediated by the glucose-specific Enzyme IIA (EIIAGlc) of the phosphoenolpyruvate-dependent phosphotransferase system (PTS). In the presence of glucose, EIIAGlc becomes dephosphorylated, which inhibits ChiS transcriptional activity by an unknown mechanism. Here, we show that dephosphorylated EIIAGlc interacts with ChiS. We also isolate ChiS suppressor mutants that evade EIIAGlc-dependent repression and demonstrate that these alleles no longer interact with EIIAGlc. These findings suggest that EIIAGlc must interact with ChiS to exert its repressive effect. Importantly, the ChiS suppressor mutations we isolated also relieve repression of chitin utilization and natural transformation by EIIAGlc, suggesting that CCR of these behaviors is primarily regulated through ChiS. Together, our results reveal how nutrient conditions impact the fitness of an important human pathogen in its environmental reservoir. AUTHOR SUMMARYVibrio cholerae is a facultative human pathogen that most commonly inhabits aquatic environments and can cause the disease cholera when ingested in the form of contaminated food or drinking water. The survival of this pathogen in the marine environment is dependent upon its ability to colonize chitin, an insoluble polysaccharide that is the main constituent of the shells of crustacean zooplankton. V. cholerae degrades chitin into soluble oligomers, which can be taken up by this microbe and used as a nutrient. Importantly, chitin oligomers also induce the activity of a chitin-sensing protein, ChiS, to activate the expression of genes required for both (1) eating chitin and (2) undergoing horizontal gene transfer by natural transformation. Previous work demonstrated that more energetically favorable carbon sources, like glucose, can inhibit chitin-induced behaviors. However, the mechanism underlying this response remained unclear. Here, we show how a dephosphorylated intermediate in the glucose uptake pathway - EIIAGlc - interacts with ChiS to repress its activity. Furthermore, we demonstrate that this repression of ChiS by EIIAGlc is responsible for inhibiting both growth on chitin and natural transformation. Together, our results reveal how nutrient conditions impact the fitness of a major human pathogen in its environmental reservoir.

microbiology↗

bioRxiv2022

Cardiac neural crest lineage diversity and underlying gene regulatory networks revealed by multimodal analysis

Neural crest cells (NCCs), a multipotent stem cell population, contribute to cardiac development as a source of the outflow septum, vascular smooth muscle and semilunar valves. However, genetic programs underlying lineage diversification of cardiac NCCs remain largely unknown. Using single-cell (sc) and spatial transcriptomics, we demonstrate multiple NCC subpopulations with distinct gene expression signatures; smooth muscle(-like), non-muscle mesenchymal, and Schwann cell progenitor/melanoblast-like cells. Integrative scRNA-seq and scATAC-seq analyses predict lineage trajectories starting from immature NCCs, which bifurcate into smooth muscle(-like) and non-muscle mesenchymal lineages in association with hierarchical transcription factor networks. Combinatory analyses with Cre-mediated genetic lineage tracing characterize intermediate NCCs at the bifurcation as Sox9+/Scx+ tendon and cartilage progenitor-like cells with genetic programs, some of which are common to skeletal tissues whereas others are unique to cardiac NCCs. These findings provide a basis for understanding the roles of NCCs in cardiac development and pathogenesis particularly associated with calcification.

developmental biology↗