Biology subjects

Ramos, C.

Publications and source records attributed to Ramos, C..

16 recordsLinked to original sources

bioRxiv2026

Twin-prime editing enables endogenous protein visualization and mutant allele tracking

Precise analysis of endogenous protein behavior in mosaic tissues requires strategies that both modify native alleles and identify successfully edited cells at single-cell resolution. Here, we establish a CRISPR-based genome-editing platform that combines endogenous protein visualization with allele-specific mutant tracking in vivo. Using the chick neural tube and {beta}-catenin as a model system, we validate cytosine base editing, prime editing, and twin-prime editing by introducing stabilizing mutations that reproduce their expected cellular and morphological phenotypes. We then develop a twin-prime editing strategy that couples installation of a defined oncogenic mutation to simultaneous insertion of minimal peptide tags, thereby making productive editing directly observable at single-cell resolution without clonal selection. Comparative analysis of ALFA, V5, and split-GFP tags identifies split-GFP as the most reliable strategy for endogenous protein visualization in vivo, and fluorescence-based cell selection strongly enriches for the intended twin-prime editing product. Finally, we extend the approach to endogenous wild-type {beta}-catenin, identifying a functionally neutral insertion site that enables visualization at physiological, non-stabilized levels while preserving normal protein behavior. Together, these results establish twin-prime editing as a versatile platform for directly linking precise endogenous genome modification to protein visualization and allele-specific mutation analysis in intact vertebrate tissues.

cell biology↗

bioRxiv2026

Proteomics to uncover new actors in the antifungal action of metformin. Impact on virulence traits, oxidative stress, and essential proteins from Candida albicans.

Metformin is one of the most widely prescribed drugs with a proven safety profile, making it an excellent candidate for drug repurposing strategies. Its anti-Candida effect and its synergistic potential with azoles have already been described against C. albicans and C. glabrata, alongside AMPK involvement and its divergent consequences on autophagy. However, metformin has additional effects on C. albicans biology that remain to be explored using differential proteomics. In the present study, C. albicans SC5314 was treated with metformin to identify the proteins involved in its anti-Candida activity. First, we demonstrated that metformin inhibits C. albicans SC5314 growth in a dose-dependent manner, an effect that intensifies under low glucose (0.2%) and filament-inducing conditions. It also reduces virulence traits such as filamentation, biofilm formation, and invasive growth. Furthermore, it induces significant oxidative stress, which is neutralized by antioxidant agents such as N-acetylcysteine and glutathione. Additionally, 50 mM metformin substantially increased fluconazole efficacy under 0.2% glucose. Our label-free proteomic study of C. albicans SC5314 exposed to 50 mM metformin allowed the identification and quantification of 1,899 proteins, with 95 and 47 proteins showing increased and decreased abundance, respectively. Notably, 26 of the down-regulated proteins were encoded by essential genes, demonstrating the drastic effect of metformin on C. albicans viability. GO Term analysis revealed that the most relevant functions affected were ATP binding, inhibition of ATPase activity (with reduced ATP levels), translation inhibition, mitochondrial function, filamentation, and responses to oxidative stress and antifungals. In conclusion, metformin significantly compromises the viability and virulence of C. albicans at high concentrations. Given that high concentrations of metformin have recently been reported in the intestinal tract, and that intestinal Candida is a known source of invasive infections in immunocompromised, under chemotherapy, and post-surgery patients, understanding metformin anti-Candida action becomes increasingly relevant.

microbiology↗

bioRxiv2025

Secreted LysM proteins are required for niche competition and full virulence in Pseudomonas savastanoi during host plant infection

Phytopathogenic bacteria secrete diverse virulence factors to manipulate host defenses and establish infection. Characterization of the type III secretion system (T3SS)- and HrpL-independent secretome (T3-IS) in Pseudomonas savastanoi pv. savastanoi (Psv), the causal agent of olive knot disease, identified five secreted LysM-containing proteins (LysM1-LysM5) associated with distinct physiological processes critical for infection. Functional predictions from network analyses suggest that LysM1, LysM2, and LysM4 may participate in type IV pilus-related functions, while LysM3 and LysM5 are likely to possess peptidoglycan hydrolase domains critical for cell division. Supporting these predictions, loss of LysM1 function resulted in impaired twitching and swimming motility, highlighting a role in pilus-mediated movement and early host colonization. In contrast, mutants lacking LysM3 or LysM5 exhibited pronounced filamentation and defective bacterial division, underscoring their essential role in septation, a process crucial for both in planta fitness and tumor formation. Structural modeling and protein stability assays demonstrate that LysM3 interacts with peptidoglycan fragments such as tetra-N-acetylglucosamine and meso-diaminopimelic acid, as well as with zinc ions, through conserved LysM and M23 domains. LysM3 also displayed selective bacteriostatic activity against co-inhabiting Gram-negative bacterial competitors, such as Pantoea agglomerans and Erwinia toletana. Our findings highlight the relevance of LysM proteins in maintaining bacterial integrity, motility, and competitive fitness, which are crucial for successful host infection. This study expands the functional repertoire of LysM-containing proteins and reveals their broader impact on bacterial virulence and adaptation to the plant-associated niche. Author SummaryPlant pathogenic bacteria secrete a variety of proteins to manipulate host responses and outcompete microbial rivals. In this study, we investigated a group of five secreted proteins with conserved LysM domains produced by the bacterium responsible for olive knot disease, Pseudomonas savastanoi. We found that these proteins fulfill distinct and critical functions during infection. Three are potentially involved in the assembly of type IV pili, structures that help bacteria move and colonize plant tissues, while the other two are related to enzymes that remodel the bacterial cell wall and are essential for proper cell division. Mutants lacking these proteins failed to divide normally and were significantly impaired in their ability to infect olive plants. One of these proteins, LysM3, also exhibited the ability to inhibit other Gram-negative bacteria that coexist with the pathogen in the plant tumor, pointing to a role in microbial competition. These findings shed light on how LysM-containing proteins contribute to both disease progression and bacterial competition and survival in the plant environment. This work paves the way for new insights into bacterial pathogenesis and offers potential strategies for controlling olive knot disease and other plant infections.

microbiology↗

bioRxiv2025

Soil Carbon Sequestration in Nothofagus obliqua Forests with Different Canopy Cover Levels under Silvopastoral Management

Agroforestry contributes to slowing deforestation, favouring ecosystem regeneration and improving land use sustainability. In this study, we evaluated the effect of agroforestry on the recovery of degraded soils in a native forest and its capacity to capture and stabilize carbon (C) and nitrogen (N) in the soil. We conducted the study in a native forest of Nothofagus obliqua (oak) located in Ranchillo Alto (37{degrees}04'52'' S, 71{degrees}39'14'' W), in the Nuble region, Chile. This site includes three silvopastoral systems with different levels of forest degradation: open (Op), semi- open (SOp) and semi-closed (SC), together with a control treatment without silvopastoral treatment (Ctr). The analysis was conducted at four soil depths (0-10, 10-20, 20-30 and 30-60 cm). To evaluate carbon and nitrogen sequestration and fixation in relation to the different forest cover treatments, we performed chemical, physical and biological soil analyses. We also included a physical fractionation by particle size to separate soil organic matter (SOM) into meaningful fractions with different characteristics and dynamics: particulate organic matter (POM) and organic matter associated with the mineral matrix (MAOM). Our results indicated that Op, despite being the most degraded condition, showed the highest values of C, N and non- oxidizable carbon (Cnox). This condition could be associated with the history of potato burning in this sector, which generates stable pyrogenic carbon in the soil. Analyses of C and N, as well as Cnox in the MAOM fraction, and C stock revealed that the three silvopastoral systems showed higher values than the control treatment without silvopastoral treatment. This suggests that the adoption of silvopastoral practices can improve soil quality and contribute to long-term carbon sequestration. These results support the application of sustainable practices to mitigate soil degradation and enhance carbon sequestration in degraded ecosystems.

ecology↗

bioRxiv2024

Meioc-Piwil1 complexes regulate rRNA transcription for differentiation of spermatogonial stem cells

Ribosome biogenesis is vital for sustaining stem cell properties, yet its regulatory mechanisms are obscure. Herein, we show unique properties of zebrafish meioc mutants in which spermatogonial stem cells (SSCs) do not differentiate or upregulate rRNAs. Meioc colocalized with Piwil1 in perinuclear germ granules, but Meioc depletion resulted in Piwil1 accumulation in nucleoli. Nucleolar Piwil1 interacted with 45S pre-rRNA. piwil1+/-spermatogonia with reduced Piwil1 upregulated rRNAs, and piwil1+/-;meioc-/- spermatogonia recovered differentiation later than those in meioc-/-. Further, Piwil1 interacted with Setdb1 and HP1, and meioc-/- spermatogonia exhibited high levels of H3K9me3 and methylated CpG in the 45S-rDNA region. These results indicate that zebrafish SSCs maintain low levels of rRNA transcription with repressive marks similar to Drosophila piRNA targets of RNA polymerase II, and that Meioc has a unique function on preventing localization of Piwil1 in nucleoli to upregulate rRNA transcripts and to promote SSC differentiation.

developmental biology↗

bioRxiv2024

Controlling interfacial protein adsorption, desorption and aggregation in biomolecular condensates

The aggregation of amyloidogenic proteins is linked to age-related diseases. The presence of interfaces can affect their aggregation mechanism, often speeding up aggregation. -Synuclein (Syn) can adsorb to biomolecular condensates, leading to heterogenous nucleation and faster aggregation. Understanding the mechanism underlying localization of amyloidogenic proteins at condensate interfaces is crucial for developing strategies to prevent or reverse their binding. We show that Syn localization to the surface of peptide-based heterotypic condensates is an adsorption process governed by the proteins condensate-amphiphilic nature. Adsorption occurs in multiple layers and levels off at micromolar concentrations. Based on these findings, we design three strategies to modulate Syn accumulation: (i) addition of biomolecules that decrease the condensate {zeta}-potential, such as NTPs and RNA, (ii) competitive adsorption of proteins targeting the condensate interface, such as G3BP1, DDX4-YFP, EGFP-NPM1, Hsp70, Hsc70, and (iii) preferential adsorption of Syn to membranes. Removing Syn from the condensate interface slows aggregation, highlighting potential cellular control over protein adsorption and implications for therapeutic strategies. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=128 SRC="FIGDIR/small/619145v1_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@159b012org.highwire.dtl.DTLVardef@1369671org.highwire.dtl.DTLVardef@ced970org.highwire.dtl.DTLVardef@2578b0_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2024

Altered cellular metabolic pathway and epithelial cell maturation induced by MYO5B defects are partially reversible by LPAR5 activation

Functional loss of the motor protein, Myosin Vb (MYO5B), induces various defects in intestinal epithelial function and causes a congenital diarrheal disorder, microvillus inclusion disease (MVID). Utilizing the MVID model mice, Vil1-CreERT2;Myo5bflox/flox (MYO5B{Delta}IEC) and Vil1-CreERT2;Myo5bflox/G519R(MYO5B(G519R)), we previously reported that functional MYO5B loss disrupts progenitor cell differentiation and enterocyte maturation that result in villus blunting and deadly malabsorption symptoms. In this study, we determined that both absence and a point mutation of MYO5B impair lipid metabolism and alter mitochondrial structure, which may underlie the progenitor cell malfunction observed in MVID intestine. Along with a decrease in fatty acid oxidation, the lipogenesis pathway was enhanced in the MYO5B{Delta}IEC small intestine. Consistent with these observations in vivo, RNA-sequencing of enteroids generated from two MVID mouse strains showed similar downregulation of energy metabolic enzymes, including mitochondrial oxidative phosphorylation genes. In our previous studies, lysophosphatidic acid (LPA) signaling ameliorates epithelial cell defects in MYO5B{Delta}IEC tissues and enteroids. The present study demonstrates that the highly soluble LPAR5-preferred agonist, Compound-1, improved sodium transporter localization and absorptive function, and tuft cell differentiation in patient-modeled MVID animals that carry independent mutations in MYO5B. Body weight loss in male MYO5B(G519R) mice was ameliorated by Compound-1. These observations suggest that Compound-1 treatment has a trophic effect on intestine with MYO5B functional loss through epithelial cell-autonomous pathways that may improve the differentiation of progenitor cells and the maturation of enterocytes. Targeting LPAR5 may represent an effective therapeutic approach for treatment of MVID symptoms induced by different point mutations in MYO5B. NEW & NOTEWOTHYThis study demonstrates the importance of MYO5B for cellular lipid metabolism and mitochondria in intestinal epithelial cells, a previously unexplored function of MYO5B. Alterations in cellular metabolism may underlie the progenitor cell malfunction observed in microvillus inclusion disease (MVID). To examine the therapeutic potential of progenitor-targeted treatments, the effects of LPAR5-preferred agonist, Compound-1, was investigated utilizing several MVID model mice and enteroids. Our observations suggests that Compound-1 may provide a therapeutic approach for treating MVID. Graphic Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=142 SRC="FIGDIR/small/610579v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@54b5borg.highwire.dtl.DTLVardef@1966a5aorg.highwire.dtl.DTLVardef@2073f1org.highwire.dtl.DTLVardef@9c12e1_HPS_FORMAT_FIGEXP M_FIG C_FIG

physiology↗

bioRxiv2024

The Dystrophin-Dystroglycan complex ensures cytokinesis efficiency in Drosophila epithelia

Cytokinesis physically separates daughter cells at the end of cell division. This step is particularly challenging for epithelial cells, which are connected to their neighbors and to the extracellular matrix by transmembrane protein complexes. To systematically evaluate the impact of the cell adhesion machinery on epithelial cytokinesis efficiency, we performed an RNAi-based modifier screen in the Drosophila follicular epithelium. Strikingly, this unveiled adhesion molecules and transmembrane receptors that facilitate cytokinesis completion. Among these is Dystroglycan, which connects the extracellular matrix to the cytoskeleton via Dystrophin. Live imaging revealed that Dystrophin and Dystroglycan become enriched in the ingressing membrane, below the cytokinetic ring, during and after ring constriction. Using multiple alleles, including Dystrophin isoform-specific mutants, we show that Dystrophin/Dystroglycan localization is linked with unanticipated roles in regulating cytokinetic ring contraction and in preventing membrane regression during the abscission period. Altogether, we provide evidence that, rather than opposing cytokinesis completion, the machinery involved in cell-cell and cell-matrix interactions has also evolved functions to ensure cytokinesis efficiency in epithelial tissues.

cell biology↗

bioRxiv2024

High-Throughput Phenotyping of Seed Quality Traits Using Imaging and Deep Learning in Dry Pea

Seed traits, such as seed color and seed size, directly impact seed quality, affecting the marketability and value of dry peas [1]. Assessing seed quality is integral to a plant breeding programs to ensure optimal seed standards. This research introduced a phenotyping tool to assess seed quality traits specifically tailored for pulse crops, which integrates image processing with cutting-edge deep learning models. The proposed method is designed for automation, seamlessly processing a sequence of images while minimizing human intervention. The pipeline standardized red-green-blue (RGB) images captured from a color light box and used deep learning models to segment and detect seed features. Our method extracted up to 86 distinct seed characteristics, ranging from basic size metrics to intricate texture details and color nuances. Compared to traditional methods, our pipeline demonstrated a 95 percent similarity in seed quality assessment and increased time efficiency (from 2 weeks to 30 minutes for processing time). Specifically, we observed an improvement in the accuracy of seed trait identification by simply using an RGB value instead of a categorical, non-standard description, which allowed for an increase in the range of detectable seed quality characteristics. By integrating conventional image processing techniques with foundational deep learning models, this approach emerges as a pivotal instrument in pulse breeding programs, guaranteeing the maintenance of superior seed quality standards.

plant biology↗

bioRxiv2024

C. elegans display antipathy behavior towards food after contemporaneous integration of nutritional needs and dietary lipid availability

Organisms utilize sophisticated neurocircuitry to select optimal food sources within their environment. Methylobacterium is a lifespan-promoting bacterial diet for C. elegans that drives faster development and longevity, however after ingestion, C. elegans consistently choose any other food option available. A screen for genetic regulators of the avoidance behavior toward Methylobacterium identified the AWB and AWC sensory neurons and the odr-1 guanylate cyclase expressed exclusively in those four ciliated neurons as mediators of the antipathy response. Metabolic profiling of the Methylobacterium diet reveals a macromolecular profile enriched in saturated fats and here we show that C. elegans sense and integrate signals related to the type of ingested lipids that subsequently cues food-related behaviors. Moreover, disruption of endogenous lipid metabolism modifies the intensity of antipathy toward Methylobacterium which suggests that the current state of lipid homeostasis influences food preference. Enhanced expression of the sphingolipid degradation enzyme Saposin/spp-9 enhances antipathy behaviors and activation of the sphingosine rheostat and more specifically modulation of the bioactive lipid mediator sphingosine-1-phosphate (S1P) acts as a signal to promote avoidance of Methylobacterium. Taken together, our work reveals that C. elegans modify food choices contemporaneously based on the availability of dietary lipids and the ability to metabolize dietary lipids. HIGHLIGHTSO_LIUncover new molecular mechanisms underlying the decision matrix an animal uses to choose what foods to eat. C_LIO_LIDefine the molecular mechanisms underlying an antipathy behavioral response toward foods after initial ingestion that contemporaneously integrates dietary needs with nutritional profile. C_LIO_LIODR-1 signaling from AWB and AWC ciliated neurons of the C. elegans nervous system mediate the antipathy response to diet. C_LIO_LIManipulation of sphingosine-1-phosphate (S1P) of the sphingosine rheostat controls the intensity of the antipathy behavioral response. C_LIO_LIModulating antipathy behaviors can impact the magnitude of the lifespan-promoting effects of longevity diets. C_LI

physiology↗

bioRxiv2023

A metagenomics approach to identification of eukaryotes in metazoan-associated microbiomes

BackgroundMicrobial eukaryotes are integral components of the microbiome where they shape community composition and ecological interactions. However, the abundance and diversity of eukaryotic species within the microbiome, the eukaryome, remains poorly defined. These deficiencies arise from unresolved technical limitations in recovering DNA from microbial eukaryotes due to their relatively low abundance in most samples and resilience to extraction. To overcome these limitations, we developed an extraction protocol that specifically targets recovery of eukaryotic microbes from microbiome samples and allows for metagenomics sequencing of eukaryotic species. MethodsMicrobes were seeded in synthetic stool prior to DNA extraction to mimic microbiome samples from the gastrointestinal tract. Assessment of cell disruption was performed using intracellular staining with the azo dye trypan blue or quantification of DNA recovery. A mock microbial community of five bacteria and five eukaryotes was built to test the effectiveness of the full protocol by seeding stool with defined numbers of cells from each species. ResultsMechanical disruption efficiently released DNA from bacterial, fungal, and protozoan species where standard microbiome DNA extraction kits did not. Optimization of the bead beating parameters lysed >95% of fungal cells within synthetic stool samples. In a mixed bacteria- eukaryote sample, eukaryotic DNA could be further enriched by targeting methylated DNA for destruction with methyl-specific restriction endonucleases. Application of this approach to a defined community of 10 different microbes, five eukaryotes and five bacteria, seeded in synthetic stool demonstrated the success of this strategy by enriching for eukaryotes approximately 72-fold and producing a eukaryote-dominated DNA pool. ConclusionsOverall, development of a microbiome sample protocol that includes DNA extraction and enrichment from eukaryotic species will facilitate exploration of the eukaryome and its impact on human health.

microbiology↗

bioRxiv2023

A dicer-related helicase opposes the age-related pathology from SKN-1 activation in ASI neurons

Coordination of cellular responses to stress are essential for health across the lifespan. The transcription factor SKN-1 is an essential homeostat that mediates survival in stress-inducing environments and cellular dysfunction, but constitutive activation of SKN-1 drives premature aging thus revealing the importance of turning off cytoprotective pathways. Here we identify how SKN-1 activation in two ciliated ASI neurons in C. elegans results in an increase in organismal transcriptional capacity that drives pleiotropic outcomes in peripheral tissues. An increase in the expression of established SKN-1 stress response and lipid metabolism gene classes of RNA in the ASI neurons, in addition to the increased expression of several classes of non-coding RNA, define a molecular signature of animals with constitutive SKN-1 activation and diminished healthspan. We reveal neddylation as a novel regulator of the SKN-1 homeostat that mediates SKN-1 abundance within intestinal cells. Moreover, RNAi-independent activity of the dicer-related DExD/H-box helicase, drh-1, in the intestine, can oppose the e2ffects of aberrant SKN-1 transcriptional activation and delays age-dependent decline in health. Taken together, our results uncover a cell non-autonomous circuit to maintain organism-level homeostasis in response to excessive SKN-1 transcriptional activity in the sensory nervous system. SIGNIFICANCE STATEMENTUnlike activation, an understudied fundamental question across biological systems is how to deactivate a pathway, process, or enzyme after it has been turned on. The irony that the activation of a transcription factor that is meant to be protective can diminish health was first documented by us at the organismal level over a decade ago, but it has long been appreciated that chronic activation of the human ortholog of SKN-1, NRF2, could lead to chemo- and radiation resistance in cancer cells. A colloquial analogy to this biological idea is a sink faucet that has an on valve without a mechanism to shut the water off, which will cause the sink to overflow. Here, we define this off valve.

genetics↗

bioRxiv2023

Dopamine D2 receptors in mossy cells reduce excitatory transmission and are essential for hippocampal function

Hilar mossy cells (MCs) are principal excitatory neurons of the dentate gyrus (DG) that play critical roles in hippocampal function and have been implicated in brain disorders such as anxiety and epilepsy. However, the mechanisms by which MCs contribute to DG function and disease are poorly understood. Expression from the dopamine D2 receptor (D2R) gene (Drd2) promoter is a defining feature of MCs, and previous work indicates a key role for dopaminergic signaling in the DG. Additionally, the involvement of D2R signaling in cognition and neuropsychiatric conditions is well-known. Surprisingly, though, the function of MC D2Rs remain largely unexplored. In this study, we show that selective and conditional removal of Drd2 from MCs of adult mice impaired spatial memory, promoted anxiety-like behavior and was proconvulsant. To determine the subcellular expression of D2Rs in MCs, we used a D2R knockin mouse which revealed that D2Rs are enriched in the inner molecular layer of the DG, where MCs establish synaptic contacts with granule cells. D2R activation by exogenous and endogenous dopamine reduced MC to dentate granule cells (GC) synaptic transmission, most likely by a presynaptic mechanism. In contrast, removing Drd2 from MCs had no significant impact on MC excitatory inputs and passive and active properties. Our findings support that MC D2Rs are essential for proper DG function by reducing MC excitatory drive onto GCs. Lastly, impairment of MC D2R signaling could promote anxiety and epilepsy, therefore highlighting a potential therapeutic target. SIGNIFICANCEGrowing evidence indicates that hilar mossy cells (MCs) of the dentate gyrus play critical but incompletely understood roles in memory and brain disorders, including anxiety and epilepsy. Dopamine D2 receptors (D2Rs), implicated in cognition and several psychiatric and neurological disorders, are considered to be characteristically expressed by MCs. Still, the subcellular localization and function of MC D2Rs are largely unknown. We report that removing the Drd2 gene specifically from MCs of adult mice impaired spatial memory and was anxiogenic and proconvulsant. We also found that D2Rs are enriched where MCs synaptically contact dentate granule cells (GC) and reduce MC-GC transmission. This work uncovered the functional significance of MC D2Rs, thus highlighting their therapeutic potential in D2R- and MC-associated pathologies.

neuroscience↗

bioRxiv2021

Single-cell transcriptomics reveals a conserved metaplasia program in pancreatic injury

BACKGROUND & AIMSAcinar to ductal metaplasia (ADM) occurs in the pancreas in response to tissue injury and is a potential precursor for adenocarcinoma. The goal of these studies was to define the populations arising from ADM, the associated transcriptional changes, and markers of disease progression. METHODSAcinar cells were lineage-traced with enhanced yellow fluorescent protein (EYFP) to follow their fate upon injury. Transcripts of over 13,000 EYFP+ cells were determined using single-cell RNA sequencing (scRNA-seq). Developmental trajectories were generated. Data were compared to gastric metaplasia, KrasG12D-induced neoplasia, and human pancreatitis. Results were confirmed by immunostaining and electron microscopy. KrasG12D was expressed in injury-induced ADM using several inducible Cre drivers. Surgical specimens of chronic pancreatitis from 15 patients were evaluated by immunostaining. RESULTSscRNA-seq of ADM revealed emergence of a mucin/ductal population resembling gastric pyloric metaplasia. Lineage trajectories suggest that some pyloric metaplasia cells can generate tuft and enteroendocrine cells (EECs). Comparison to KrasG12D-induced ADM identifies populations associated with disease progression. Activation of KrasG12D expression in HNF1B+ or POU2F3+ ADM populations leads to neoplastic transformation and formation of MUC5AC+ gastric-pit-like cells. Human pancreatitis samples also harbor pyloric metaplasia with a similar transcriptional phenotype. CONCLUSIONSUnder conditions of chronic injury, acinar cells undergo a pyloric-type metaplasia to mucinous progenitor-like populations, which seed disparate tuft cell and EEC lineages. ADM-derived EEC subtypes are diverse. KrasG12D expression is sufficient to drive neoplasia from injury-induced ADM and offers an alternative origin for tumorigenesis. This program is conserved in human pancreatitis, providing insight into early events in pancreas diseases.

cell biology↗

bioRxiv2020

Neuron-specific cilia loss alters locomotor responses to amphetamine.

The neural mechanisms that underlie responses to drugs of abuse are complex, and impacted by a number of neuromodulatory peptides. Within the past ten years it has been discovered that several of the receptors for neuromodulators are enriched in the primary cilia of neurons. Primary cilia are microtubule-based organelles that project from the surface of nearly all mammalian cells, including neurons. Despite what we know about cilia, our understanding of how cilia regulate neuronal function and behavior is still limited. The primary objective of this study was to investigate the contributions of primary cilia on specific neuronal populations to behavioral responses to amphetamine. To test the consequences of cilia loss on amphetamine-induced locomotor activity we selectively ablated cilia from dopaminergic or GAD2-GABAergic neurons in mice. Cilia loss had no effect on baseline locomotion in either mouse strain. Both female and male mice lacking cilia on dopaminergic neurons showed significantly reduced responses to acute administration of 3.0 mg/kg amphetamine compared to wildtype mice. In contrast, changes in the locomotor response to amphetamine in mice lacking cilia on GAD2-GABAergic neurons were primarily driven by reductions in locomotor activity in males. Following repeated amphetamine administration (1.0 mg/kg/day over 5 days), mice lacking cilia on GAD2-GABAergic neurons exhibited enhanced sensitization of the locomotor stimulant response to the drug, whereas mice lacking cilia on dopaminergic neurons did not differ from their wildtype controls. These results indicate that cilia play neuron-specific roles in both acute and neuroplastic responses to psychostimulant drugs of abuse.

neuroscience↗

bioRxiv2018

Integrity and stability of virulence plasmids from Pseudomonas syringae are modulated by mobile genetic elements and multiple toxin-antitoxin systems

BackgroundVirulence plasmids are critically exposed to genetic decay and loss, particularly in Pseudomonas syringae strains because of their high content of mobile genetic elements and their exploitation of environmental niches outside of the plant host. The demonstrated high plasticity and adaptability of P. syringae plasmids, involving the acquisition and loss of large DNA regions, contrasts with their usual high stability and the maintenance of key virulence genes in free living conditions. The identification of plasmid stability determinants and mechanisms will help to understand their evolution and adaptability to agroecosystems as well as to develop more efficient control measures.\n\nResultsWe show that the three virulence plasmids of P. syringae pv. savastanoi NCPPB 3335 contain diverse functional stability determinants, including three toxin-antitoxin systems (TA) in both pPsv48A and pPsv48C, whereas one of the two replicons of pPsv48C can confer stable inheritance by itself. Loss of pPsv48A increased by two orders of magnitude upon functional inactivation of its TA systems. However, inactivation of the TA systems from pPsv48C did not result in its curing but led to the recovery of diverse deletion derivatives. One type consisted in the deletion of an 8.3 kb fragment, with a frequency of 3.8 {+/-} 0.3 x 10-3, by recombination between two copies of MITEPsy2. Likewise, IS801 promoted the occurrence of deletions of variable size by one-ended transposition with a frequency of 5.5 {+/-} 2.1 x 10-4, 80 % of which resulted in the loss of virulence gene idi. These deletion derivatives were stably maintained in the population by replication mediated by repJ, which is adjacent to IS801. IS801 also promoted deletions in plasmid pPsv48A, either by recombination or one-ended transposition. In all cases, functional TA systems contributed significantly to reduce the occurrence of plasmid deletions in vivo.\n\nConclusionsVirulence plasmids from P. syringae harbour a diverse array of stability determinants with a variable contribution to plasmid persistence. Additionally, multiple TA systems favour the long-term survival and integrity of virulence plasmids, as well as the maintenance of pathogenicity genes in free-living conditions. This strategy is likely widespread amongst native plasmids of P. syringae and other bacteria.

microbiology↗