Biology subjects

Harrington, A.

Publications and source records attributed to Harrington, A..

11 recordsLinked to original sources

bioRxiv2025

Exosome Trafficking Is a Key Regulator of Adipocyte Thermogenesis

Activation of beige adipocytes enhances energy expenditure and promotes metabolic health, presenting a promising approach for combating obesity and diabetes. As part of this process, thermogenesis, fueled in part by uncoupled mitochondrial respiration, plays a central role in converting calories into thermal energy, thereby preventing their storage as fat. Here, we identify a role for exosome trafficking as an intrinsic regulator of beige adipocyte thermogenesis. Exosomes are small extracellular vesicles that mediate cell-cell and intracellular communication by transporting regulatory cargo, including microRNAs, proteins, and lipids. Using both human cells and mouse models, we show that thermogenic activation of beige adipocytes promotes the rapid release of exosomes enriched in microRNAs known to suppress thermogenic programs. Genetic or pharmacological blockade of exosome secretion attenuates thermogenesis, whereas enhancing exosome release amplifies thermogenic output. Mice deficient in the exosome secretion regulator Rab27a exhibit reduced energy expenditure in response to both cold exposure and {beta}3-adrenergic stimulation. These findings establish exosome trafficking as a key contributor to beige adipocyte thermogenic capacity, highlighting an intracellular mechanism that may be leveraged to enhance energy expenditure and treat obesity-related metabolic diseases. Significance StatementThermogenic adipocytes, including beige fat cells, help maintain energy balance by converting excess nutrients into heat, thereby reducing fat storage and supporting metabolic function. Although these cells are known to promote energy expenditure, the intracellular processes that enable their full thermogenic response are not well defined. Here, we show that exosome secretion is required for beige adipocytes to reach their full thermogenic potential. Blocking exosome release dampens this response, while boosting it amplifies thermogenic output. These findings point to exosome release as an essential part of thermogenic regulation and a potential target for improving metabolic health.

cell biology↗

bioRxiv2023

Preimplantation Embryos Amplify Sperm-Derived miRNA Levels to Mediate Transgenerational Epigenetic Inheritance

The effects of chronically stressing male mice can be transmitted across generations by stress-specific changes in their sperm miRNA content that induce stress-specific phenotypes in their offspring. But how each stress paradigm alters the levels of distinct sets of sperm miRNAs is not known. We showed previously that exposure of male mice to chronic social instability (CSI) stress results in elevated anxiety and reduced sociability specifically in their female offspring across multiple generations because it reduces miR-34c levels in sperm of stressed males and their unstressed male offspring. Here we describe evidence that astrocyte-derived exosomes (A-Exos) carrying miR-34c mediate how CSI stress has this transgenerational effect on sperm. We found that CSI stress decreases miR-34c carried by A-Exos in the prefrontal cortex and amygdala, as well as in the blood of males. Importantly, miR-34c A-Exos levels are also reduced in these tissues in their F1 male offspring, who despite not being exposed to stress exhibit reduced sperm miR-34c levels and transmit the same stress-associated traits to their male and female offspring. Furthermore, restoring A-Exos miR-34c content in the blood of CSI-stressed males by intravenous injection of miR-34c-containing A-Exos restores miR-34c levels in their sperm. These findings reveal an unexpected role for A-Exos in maintaining sperm miR-34c levels by a process that when suppressed by CSI stress mediates this example of transgenerational epigenetic inheritance.

genetics↗

bioRxiv2022

Gallocin A, an atypical two-peptide bacteriocin secreted by Streptococcus gallolyticus subsp. gallolyticus

Streptococcus gallolyticus subsp. gallolyticus (SGG) is an opportunistic gut pathogen associated with colorectal cancer. We previously showed that colonization of the murine colon by SGG in tumoral conditions was strongly enhanced by the production of gallocin A, a two-peptide bacteriocin. Here, we aimed at characterizing the mechanisms of its action and resistance. Using a genetic approach, we demonstrated that gallocin A is composed of two peptides, GllA1 and GllA2, which are inactive alone and act together to kill "target" bacteria. We showed that gallocin A can kill phylogenetically close relatives. Importantly, we demonstrated that gallocin A peptides can insert into membranes and permeabilize lipid bilayer vesicles. Next, we showed that the third gene of the gallocin A operon named GIP, is necessary and sufficient to confer immunity to gallocin A. Structural modelling of GllA1 and GllA2 mature peptides suggested that both peptides form alpha-helical hairpins stabilized by intramolecular disulfide bridges. The presence of a disulfide bond in GllA1 and GllA2 was confirmed experimentally. Addition of disulfide reducing agents abrogated gallocin A activity. Likewise, deletion of a gene encoding a surface protein with a thioredoxin-like domain impaired gallocin A ability to kill Enterococcus faecalis. Structural modelling of GIP revealed a hairpin-like structure strongly resembling that of the GllA1 and GllA2 mature peptides, suggesting a mechanism of immunity by competition with GllA1/2. Finally, identification of other class IIb bacteriocins exhibiting a similar alpha-helical hairpin fold stabilized with an intramolecular disulfide bridge suggests the existence of a new subclass of class IIb bacteriocins. IMPORTANCEStreptococcus gallolyticus subsp. gallolyticus (SGG), previously named Streptococcus bovis biotype I, is an opportunistic pathogen responsible for invasive infections (septicemia, endocarditis) in elderly people and often associated with asymptomatic colon tumors. SGG is one of the first bacteria to be associated with the occurrence of colorectal cancer in humans. Previously, we showed that tumor-associated conditions in the colon provide to SGG with the ideal environment to proliferate at the expense of phylogenetically and metabolically closely related commensal bacteria such as enterococci (Aymeric et al., 2017). SGG takes advantage of CRC-associated conditions to outcompete and substitute commensal members of the gut microbiota using a specific bacteriocin named gallocin and renamed gallocin A recently following the discovery of gallocin D in a peculiar SGG isolate. Here, we showed that gallocin A is a two-peptide bacteriocin and that both GllA1 and GllA2 peptides are required for antimicrobial activity. Gallocin A was shown to permeabilize bacterial membranes and to kill phylogenetically closely related bacteria such as most streptococci, lactococci and enterococci, probably through membrane pore formation. GllA1 and GllA2 secreted peptides are unusually long (42 and 60 amino acids long) and with very few charged amino acids compared to well-known class IIb bacteriocins. In silico modelling revealed that both GllA1 and GllA2 exhibit a similar hairpin-like conformation stabilized by an intramolecular disulfide bond. We also showed that the GIP immunity peptide also forms a hairpin like structure like GllA1/GllA2. Thus, we hypothesize that GIP blocks the formation of the GllA1/GllA2 complex by interacting with GllA1 or GllA2. Gallocin A may constitute the first class IIb bacteriocin displaying disulfide bridges important for its structure and activity and the founding member of a subtype of class IIb bacteriocins.

microbiology↗

bioRxiv2022

An international scholastic network to generate LexA enhancer-trap lines for Drosophila

Conditional gene regulation in Drosophila through binary expression systems like the LexA-LexAop system provides a superb tool for investigating gene and tissue function. To increase the availability of defined LexA enhancer trap insertions, we present molecular, genetic and tissue expression studies of 301 novel Stan-X LexA enhancer traps derived from mobilization of the index SX4 line. This includes insertions into distinct loci on the X, II and III chromosomes that were not previously associated with enhancer traps or targeted LexA constructs, an insertion into ptc, and eleven insertions into natural transposons. A subset of enhancer traps was expressed in CNS neurons known to produce and secrete insulin, an essential regulator of growth, development and metabolism. Fly lines described here were generated and characterized through studies by students and teachers in an international network of genetics classes at public, independent high schools, and universities serving a diversity of students, including those underrepresented in science. Thus, a unique partnership between secondary schools and university-based programs has produced and characterized novel resources in Drosophila, establishing instructional paradigms devoted to unscripted experimental science.

genetics↗

bioRxiv2022

Mink on the brink: Comparing survey methods for detecting a Critically Endangered carnivore, the European mink Mustela lutreola

Monitoring rare and elusive species is critical in guiding appropriate conservation management measures. Mammalian carnivores are challenging to monitor directly, due to their generally nocturnal and solitary lifestyle, and relatively large home ranges. The European mink Mustela lutreola is a Critically Endangered, small, semi-aquatic carnivore and is one of the most threatened mammal species in Europe. In northern Spain, the European mink population is monitored regionally using different methods and approaches, making assessment of national population status difficult. There is an urgent need to 1) assess the efficacy of survey methods, and 2) identify a standard monitoring methodology that can be deployed rapidly and inexpensively over large areas of the minks range. We deployed four methods - camera trapping, hair tubes, live trapping, and environmental DNA (eDNA) from water samples - to compare the probability of detecting European mink when present at 25 sampling sites within five 10x10km squares, and the economic cost and time required for each method. All four methods successfully detected European mink but the probability of detection varied by method. Camera trapping and hair tubes had the highest probability of detection; however, eDNA and live trapping detected mink in one 10x10km square where the latter two methods did not. For future European mink monitoring programmes, we recommend a combination of at least two methods, and suggest that camera traps or hair tubes are combined with live trapping or eDNA (depending on the scale and aims of the study), to gather critical information on distribution, occupancy and conservation status.

ecology↗

bioRxiv2022

Peripheral Auditory Nerve Impairment in a Mouse Model of Syndromic Autism

Dysfunction of the peripheral auditory nerve (AN) contributes to dynamic changes throughout the central auditory system, resulting in abnormal auditory processing, including hypersensitivity. Altered sound sensitivity is frequently observed in autism spectrum disorder (ASD), suggesting that AN deficits and changes in auditory information processing may contribute to ASD-associated symptoms, including social communication deficits and hyperacusis. The MEF2C transcription factor is associated with risk for several neurodevelopmental disorders, and mutations or deletions of MEF2C produce a haploinsufficiency syndrome characterized by ASD, language and cognitive deficits. A mouse model of this syndromic ASD (i.e., Mef2c+/- or Mef2c-Het) recapitulates many of the MEF2C Haploinsufficiency syndrome-linked behaviors including communication deficits. We show here that Mef2c-Het mice exhibit functional impairment of the peripheral AN and a modest reduction in hearing sensitivity. We find that MEF2C is expressed during development in multiple AN and cochlear cell types, and in Mef2c-Het mice, we observe multiple cellular and molecular alterations associated with the AN, including abnormal myelination, neuronal degeneration, neuronal mitochondria dysfunction, and increased macrophage activation and cochlear inflammation. These results reveal the importance of MEF2C function in inner ear development and function and the engagement of immune cells and other non-neuronal cells, which suggests that microglia/macrophages and other non-neuronal cells might contribute, directly or indirectly, to AN dysfunction and ASD-related phenotypes. Finally, our study establishes a comprehensive approach for characterizing AN function at the physiological, cellular, and molecular levels in mice, which can be applied to animal models with a wide range of human auditory processing impairments. Significance StatementThis is the first report of peripheral auditory nerve (AN) impairment in a mouse model of human MEF2C haploinsufficiency syndrome that has well-characterized ASD related behaviors including communication deficits, hyperactivity, repetitive behavior, and social deficits. We identify multiple underlying cellular, sub-cellular, and molecular abnormalities that may contribute to peripheral AN impairment. Our findings also highlight the important roles of immune cells (e.g., cochlear macrophages) and other non-neuronal elements (e.g., glial cells and cells in the stria vascularis) in auditory impairment in ASD. The methodological significance of the study is the establishment of a comprehensive approach for evaluating peripheral AN function and impact of peripheral AN deficits with minimal hearing loss.

neuroscience↗

bioRxiv2021

The regenerative response of cardiac interstitial cells

Understanding how certain animals are capable of regenerating their hearts will provide much needed insights into how this process can be induced in humans in order to reverse the damage caused by myocardial infarction. Currently, it is becoming increasingly evident that cardiac interstitial cells play crucial roles during cardiac regeneration. To understand how interstitial cells behave during cardiac regeneration, we performed single-cell RNA sequencing (scRNA-seq) of regenerating zebrafish hearts. Using a combination of immunohistochemistry, chemical inhibition and novel transgenic animals, we were able to investigate the role of cell type specific responses during cardiac regeneration. This approach allowed us to identify a number of important regenerative mechanisms within the interstitial cell populations. Here, we provide here a detailed insight into how interstitial cells behave during cardiac regeneration and identify a number of novel features of these cells which will serve to increase our understanding of how this process could eventually be induced in humans.

cell biology↗

bioRxiv2020

Secretion, maturation and activity of a quorum-sensing peptide (GSP) inducing bacteriocins transcription in Streptococcus gallolyticus

Streptococcus gallolyticus subsp. gallolyticus (Sgg) is an emerging opportunistic pathogen responsible for septicemia and endocarditis in the elderly. Invasive infections by Sgg are strongly linked to the occurrence of colorectal cancer (CRC). It was previously shown that increased secondary bile salts in CRC-conditions enhances the bactericidal activity of gallocin, a bacteriocin produced by Sgg, enabling it to colonize the mouse colon by outcompeting resident enterococci. In a separate study, we have shown that Sgg produces and secretes a 21-mer peptide that activates bacteriocin production. This peptide was named CSP because of its sequence similarity with competence stimulating peptides found in other streptococci. Here we demonstrate that CSP is a bona fide quorum-sensing peptide involved in activation of gallocin gene transcription. We therefore refer to CSP as GSP (gallocin stimulating peptide). GSP displays some unique features since its N-terminal amino-acid lies three residues after the double glycine leader sequence. Herein, we set out to investigate the processing and export pathway that leads to mature GSP. We also conducted the first comprehensive structure-activity relationship (SAR) of Sgg GSP to identify its key structural features. SignificanceStreptococcus gallolyticus subsp. gallolyticus (Sgg) is an opportunistic pathogen associated with colorectal cancer (CRC) and endocarditis. Sgg utilizes quorum-sensing (QS) to regulate the production of a bacteriocin (gallocin) and gain selective advantage in colonizing the colon. In this manuscript, we report 1) the first structure-activty relationship study of the Sgg QS pheromone that regulates gallocin production; 2) evidence that the active QS pheromone is processed to its mature form by a unique ABC transporter and not processed by an extracellular protease; and 3) supporting evidence of interspecies interactions between streptococci pheromones. Our results revealed the minimal pheromone scaffold needed for gallocin activation and uncovered unique interactions between two streptococci species QS signals that warrant further studies.

microbiology↗

bioRxiv2019

The PAR2 signal peptide prevents premature receptor cleavage and activation

Unlike closely related GPCRs, protease-activated receptors (PAR1, PAR2, PAR3, and PAR4) have a predicted signal peptide at their N-terminus, which is encoded by a separate exon, suggesting that the signal peptides of PARs may serve an important and unique function, specific for PARs. In this report, we show that the PAR2 signal peptide, when fused to the N-terminus of IgG-Fc, effectively induced IgG-Fc secretion into culture medium, thus behaving like a classical signal peptide. The presence of PAR2 signal peptide has a strong effect on PAR2 cell surface expression, as deletion of the signal peptide (PAR2{Delta}SP) led to dramatic reduction of the cell surface expression and decreased responses to trypsin or the synthetic peptide ligand (SLIGKV). However, further deletion of the tethered ligand region (SLIGKV) at the N-terminus rescued the cell surface receptor expression and the response to the synthetic peptide ligand, suggesting that the signal peptide of PAR2 may be involved in preventing PAR2 from intracellular protease activation before reaching the cell surface. Supporting this hypothesis, an Arg36Ala mutation on PAR2{Delta}SP, which disabled the trypsin activation site, increased the receptor cell surface expression and the response to ligand stimulation. Similar effects were observed when PAR2{Delta}SP expressing cells were treated with protease inhibitors. Our findings indicated that these is a role of the PAR2 signal peptide in preventing the premature activation of PAR2 from intracellular protease cleavage before reaching the cells surface. The same mechanism may also apply to PAR1, PAR3, and PAR4.

biochemistry↗

bioRxiv2018

Re-Evaluating One-step Generation of Mice Carrying Conditional Alleles by CRISPR-Cas9-Mediated Genome Editing Technology

CRISPR-Cas9 gene editing technology has considerably facilitated the generation of mouse knockout alleles, relieving many of the cumbersome and time-consuming steps of traditional mouse embryonic stem cell technology. However, the generation of conditional knockout alleles remains an important challenge. An earlier study reported up to 16% efficiency in generating conditional knockout alleles in mice using 2 single guide RNAs (sgRNA) and 2 single-stranded oligonucleotides (ssODN) (2sgRNA-2ssODN). We re-evaluated this method from a large data set generated from a consortium consisting of 17 transgenic core facilities or laboratories or programs across the world. The dataset constituted 17,887 microinjected or electroporated zygotes and 1,718 live born mice, of which only 15 (0.87%) mice harbored 2 correct LoxP insertions in cis configuration indicating a very low efficiency of the method. To determine the factors required to successfully generate conditional alleles using the 2sgRNA-2ssODN approach, we performed a generalized linear regression model. We show that factors such as the concentration of the sgRNA, Cas9 protein or the distance between the placement of LoxP insertions were not predictive for the success of this technique. The major predictor affecting the methods success was the probability of simultaneously inserting intact proximal and distal LoxP sequences, without the loss of the DNA segment between the two sgRNA cleavage sites. Our analysis of a large data set indicates that the 2sgRNA-2ssODN method generates a large number of undesired alleles (>99%), and a very small number of desired alleles (<1%) requiring, on average 1,192 zygotes.

genetics↗

bioRxiv2018

A cross-organism framework for supervised enhancer prediction with epigenetic pattern recognition and targeted validation

Enhancers are important noncoding elements, but they have been traditionally hard to characterize experimentally. Only a few mammalian enhancers have been validated, making it difficult to train statistical models for their identification properly. Instead, postulated patterns of genomic features have been used heuristically for identification. The development of massively parallel assays allows for the characterization of large numbers of enhancers for the first time. Here, we developed a framework that uses Drosophila STARR-seq data to create shape-matching filters based on enhancer-associated meta-profiles of epigenetic features. We combined these features with supervised machine learning algorithms (e.g., support vector machines) to predict enhancers. We demonstrated that our model could be applied to predict enhancers in mammalian species (i.e., mouse and human). We comprehensively validated the predictions using a combination of in vivo and in vitro approaches, involving transgenic assays in mouse and transduction-based reporter assays in human cell lines. Overall, the validations involved 153 enhancers in 6 mouse tissues and 4 human cell lines. The results confirmed that our model can accurately predict enhancers in different species without re-parameterization. Finally, we examined the transcription-factor binding patterns at predicted enhancers and promoters in human cell lines. We demonstrated that these patterns enable the construction of a secondary model effectively discriminating between enhancers and promoters.

bioinformatics↗