Biology subjects

Smith, C. J.

Publications and source records attributed to Smith, C. J..

49 records · Page 3Linked to original sources

bioRxiv2021

Multi-modal adaptor-clathrin contacts drive coated vesicleassembly

Clathrin-coated pits are formed by the recognition of membrane and cargo by the heterotetrameric AP2 complex and the subsequent recruitment of clathrin triskelia. A potential role for AP2 in coated-pit assembly beyond initial clathrin recruitment has not been explored. Clathrin binds the {beta}2 subunit of AP2, and several binding sites on {beta}2 and on the clathrin heavy chain have been identified, but our structural knowledge of these interactions is incomplete and their functional importance during endocytosis is unclear. Here, we analysed the cryo-EM structure of clathrin cages assembled in the presence of {beta}2 hinge and appendage ({beta}2HA) domains. We find that the {beta}2-appendage binds in at least two positions in the cage, demonstrating that multi-modal binding is a fundamental property of clathrin-AP2 interactions. In one position, {beta}2-appendage cross-links two adjacent terminal domains from different triskelia below the vertex. Functional analysis of {beta}2HA-clathrin interactions reveals that endocytosis requires two clathrin interaction sites: a clathrin-box motif on the hinge and the "sandwich site" on the appendage, with the appendage "platform site" having less importance. From these studies and the work of others, we propose that {beta}2-appendage binding to more than one clathrin triskelion is a key feature of the system and likely explains why clathrin assembly is driven by AP2.

biochemistry↗

bioRxiv2020

Temporal changes in the gut microbiota in farmed Atlantic cod (Gadus morhua) outweigh the response to diet supplementation with macroalgae.

BackgroundAquaculture successfully meets global food demands for many fish species. However, aquaculture production of Atlantic cod (Gadus morhua) is modest in comparison to market demand. For cod farming to be a viable economic venture specific challenges on how to increase growth, health and farming productivity need to be addressed. Feed ingredients play a key role here. Macroalgae (seaweeds) have been suggested as a functional feed supplement with both health and economic benefits for terrestrial farmed animals and fish. The impact of such dietary supplements to cod gut integrity and microbiota, which contribute to overall fish robustness is unknown. The objective of this study was to supplement the diet of juvenile Atlantic cod with macroalgae and determine the impacts on fish condition and growth, gut morphology and hindgut microbiota composition (16S rRNA amplicon sequencing). Fish were fed one of three diets: control (no macroalgal inclusion), 10% inclusion of either egg wrack (Ascophyllum nodosum) or sea lettuce (Ulva rigida) macroalgae in a 12-week trial. ResultsThe results demonstrated there was no significant difference in fish condition, gut morphology or hindgut microbiota between the U. rigida supplemented fish group and the control group at any time-point. This contrasts with the A. nodosum treatment. Fish within this group were further categorised as either Normal or Lower Growth. Lower Growth individuals found the diet unpalatable resulting in reduced weight and condition factor combined with an altered gut morphology and microbiome relative to the other treatments. Excluding this group, our results show that the hindgut microbiota was largely driven by temporal pressures with the microbial communities becoming more similar over time irrespective of dietary treatment. The core microbiome at the final time-point consisted of the orders Vibrionales (Vibrio and Photobacterium), Bacteroidales (Bacteroidetes and Macellibacteroides) and Clostridiales (Lachnoclostridium). ConclusionsOur study indicates that U. rigida macroalgae can be supplemented at 10% inclusion levels in the diet of juvenile farmed Atlantic cod without any impact on fish condition or hindgut microbial community structure. We also conclude that 10% dietary inclusion of A. nodosum is not a suitable feed supplement in a farmed cod diet.

microbiology↗

bioRxiv2020

Neonatal immune challenge induces female-specific changes in social behavior and somatostatin cell number, independent of microglial inflammatory signaling

Decreases in social behavior are a hallmark aspect of acute "sickness behavior" in response to infection. However, immune insults that occur during the perinatal period may have long-lasting consequences for adult social behavior by impacting the developmental organization of underlying neural circuits. Microglia, the resident immune cells of the central nervous system, are sensitive to immune stimulation and play a critical role in the developmental sculpting of neural circuits, making them likely mediators of this process. Here, we investigated the impact of a postnatal day (PND) 4 lipopolysaccharide (LPS) challenge on social behavior in adult mice. Somewhat surprisingly, neonatal LPS treatment decreased sociability in adult female, but not male mice. LPS-treated females also displayed reduced social interaction and social memory in a social discrimination task as compared to saline-treated females. Somatostatin (SST) interneurons within the anterior cingulate cortex (ACC) have recently been suggested to modulate a variety of social behaviors. Interestingly, the female-specific changes in social behavior observed here were accompanied by an increase in SST interneuron number in the ACC. Finally, these changes in social behavior and SST cell number do not appear to depend on microglial inflammatory signaling, because microglia-specific genetic knock-down of myeloid differentiation response protein 88 (MyD88; the removal of which prevents LPS from increasing proinflammatory cytokines such as TNF and IL-1{beta}) did not prevent these LPS-induced changes. This study provides novel evidence for enduring effects of neonatal immune activation on social behavior and SST interneurons in females, independent of microglial inflammatory signaling.

neuroscience↗

bioRxiv2020

Reverse transcriptase enzyme and priming strategy affect quantification and diversity of environmental transcripts

RT-Q-PCR, and RT-PCR amplicon sequencing, provide a convenient, target-specific, high-sensitivity approach for gene expression studies and are widely used in environmental microbiology. Yet, the effectiveness and reproducibility of the reverse transcription step has not been evaluated. Therefore, we tested a combination of four commercial reverse transcriptases with two priming techniques to faithfully transcribe 16S rRNA and amoA transcripts from marine sediments. Both enzyme and priming strategy greatly affected quantification of the exact same target with differences of up to 600-fold. Furthermore, the choice of RT system significantly changed the communities recovered. For 16S rRNA, both enzyme and priming had a significant effect with enzyme having a stronger impact than priming. Inversely, for amoA only the change in priming strategy resulted in significant differences between the same sample. Specifically, more OTUs and better coverage of amoA transcripts diversity were obtained with GS priming indicating this approach was better at recovering the diversity of amoA transcripts. Moreover, sequencing of RNA mock communities revealed that, even though transcript diversities (i.e. OTU counts within a sample) can be biased by the RT, the comparison of {beta} diversities (i.e. differences in OTU counts between samples) is reliable as those biases are reproducible between environments. Originality-Significance StatementIs the complementary DNA (cDNA) produced after Reverse Transcription (RT) a faithful representation of the starting RNA? This is a fundamental and important question for transcriptomic-based studies in environmental microbiology that aim to quantify and/or examine the diversity of transcripts via RT approaches. Yet little is known about the reliability and reproducibility of this step. Here, we evaluated the effect of the two main components of the RT reaction - the retro transcriptase enzyme and priming strategy (gene specific vs random priming), on the quantification and diversity of cDNA. We found that both have a significant impact. We further provide evidence to enable informed choices as to the enzyme and priming combinations to improve the performance of RT-PCR approaches. Taken together, this work will improve the reliability and reproducibility of transcript-based studies in environmental microbiology.

molecular biology↗

bioRxiv2020

Anomalous Diffusion Characterization by Fourier-Transform FRAP with Patterned Illumination

Fourier transform fluorescence recovery after photobleaching (FT-FRAP) with patterned illumination is theorized and demonstrated for quantitatively evaluating normal and anomalous diffusion. Diffusion characterization is routinely performed to assess mobility in cell biology, pharmacology, and food science. Conventional FRAP is noninvasive, has low sample volume requirements, and can rapidly measure diffusion over distances of a few micrometers. However, conventional point-bleach measurements are complicated by signal-to-noise limitations, the need for precise knowledge of the bleach beam profile, potential for bias due to sample heterogeneity, and poor compatibility with multi-photon excitation due to local heating. In FT-FRAP with patterned illumination, the time-dependent fluorescence recovery signal is concentrated to puncta in the spatial Fourier domain through patterned bleaching, with substantial improvements in signal-to-noise, mathematical simplicity, representative sampling, and multiphoton compatibility. A custom nonlinear-optical beam-scanning microscope enabled patterned illumination for photobleaching through two-photon excitation. Measurements in the spatial Fourier domain removed dependence on the bleach profile, suppressing bias from imprecise knowledge of the point spread function. For normal diffusion, the fluorescence recovery produced a simple single-exponential decay in the spatial Fourier domain, in excellent agreement with theoretical predictions. Simultaneous measurement of diffusion at multiple length scales was enabled through analysis of multiple spatial harmonics of the bleaching pattern. Anomalous diffusion was characterized by FT-FRAP through a nonlinear fit to multiple spatial harmonics of the fluorescence recovery. Constraining the fit to describe diffusion over multiple length scales resulted in higher confidence in the recovered fitting parameters. Additionally, phase analysis in FT-FRAP was shown to inform on flow/sample translation. Statement of SignificanceFourier transform fluorescence recovery after photobleaching (FT-FRAP) with patterned illumination greatly improves the accuracy of diffusion assessments and simultaneously accesses information on both normal and anomalous diffusion in a single experiment.

biophysics↗

bioRxiv2020

Instant FLIM enables 4D in vivo lifetime imaging of intact brains

Traditional fluorescence microscopy is blind to molecular microenvironment information that is present in fluorescence lifetime, which can be measured by fluorescence lifetime imaging microscopy (FLIM). However, existing FLIM techniques are typically slow to acquire and process lifetime images, difficult to implement, and expensive. Here, we present instant FLIM, an analog signal processing method that allows real-time streaming of fluorescence intensity, lifetime, and phasor imaging data through simultaneous image acquisition and instantaneous data processing. Instant FLIM can be easily implemented by upgrading an existing two-photon microscope using cost-effective components and our open-source software. We further improve the functionality, penetration depth, and resolution of instant FLIM using phasor segmentation, adaptive optics, and super-resolution techniques. We demonstrate through-skull intravital 3D FLIM of mouse brains to depths of 300 m and present the first in vivo 4D FLIM of microglial dynamics in intact and injured zebrafish and mouse brains up to 12 hours.

neuroscience↗

bioRxiv2020

CRISPR-mediated biocontainment

We have exploited the repetitive nature of transposable elements of the human genome to generate synthetic circuits. Transposable elements such as LINE-1 and Alu have successfully replicated in mammalian genomes throughout evolution to reach a copy number ranging from thousands to more than a million. Targeting these repetitive elements with programmable DNA nucleases such as CRISPR-Cas9 rapidly induce extremely high levels of cell death. We use this genotoxic feature to build synthetic biocontainment circuits: CRISPR defense system (CRISPR-DS) capable of preventing CRISPR genome editing, and we introduce the proof-of-concept of CRISPR Safety-Switch, an inducible, stringent and non-leaky kill-switch capable of clearing out cell lines resistant to DNA breaks.

synthetic biology↗

bioRxiv2019

Interleukin-1 receptor antagonist treatment in acute ischaemic stroke does not alter systemic markers of anti-microbial defence

AimBlockade of the cytokine interleukin-1 (IL-1) with IL-1 receptor antagonist (IL-1Ra) is a candidate treatment for stroke entering phase II/III trials, which acts by inhibiting harmful inflammatory responses. Infection is a common complication after stroke that significantly worsens outcome and is related to stroke-induced deficits in systemic immune function thought to be mediated by the sympathetic nervous system. Therefore, immunomodulatory treatments for stroke, such as IL-1Ra, carry a risk of aggravating stroke-associated infection. Our primary objective was to determine if factors associated with antibody-mediated antibacterial defences were further compromised in patients treated with IL-1Ra after stroke. MethodsWe assessed plasma concentrations of immunoglobulin isotypes and complement components in stroke patients treated with IL-1Ra or placebo and untreated non-stroke controls using multiplex protein assays. Activation of the SNS was determined by measuring noradrenaline, a major SNS mediator. ResultsThere were significantly lower plasma concentrations of IgM, IgA, IgG1 and IgG4 in stroke-patients compared to non-stroke controls, however there were no differences between stroke patients treated with placebo or IL-1Ra. Concentrations of complement components associated with the classical pathway were increased and those associated with the alternative pathways decreased in stroke patients, neither being affected by treatment with IL-1Ra. Noradrenaline concentrations were increased after stroke in both placebo and IL-1Ra-treated stroke patients compared to non-stroke controls. ConclusionThese data show treatment with IL-1Ra after stroke does not alter circulating immunoglobulin and complement concentrations, and is therefore unlikely to further aggravate stroke-associated infection susceptibility through reduced availability of these key anti-microbial mediators.

immunology↗

bioRxiv2019

Enabling large-scale genome editing by reducing DNA nicking

To extend the frontier of genome editing and enable the radical redesign of mammalian genomes, we developed a set of dead-Cas9 base editor (dBE) variants that allow editing at tens of thousands of loci per cell by overcoming the cell death associated with DNA double-strand breaks (DSBs) and single-strand breaks (SSBs). We used a set of gRNAs targeting repetitive elements - ranging in target copy number from about 31 to 124,000 per cell. dBEs enabled survival after large-scale base editing, allowing targeted mutations at up to ~13,200 and ~2610 loci in 293T and human induced pluripotent stem cells (hiPSCs), respectively, three orders of magnitude greater than previously recorded. These dBEs can overcome current on-target mutation and toxicity barriers that prevent cell survival after large-scale genome engineering.\n\nOne Sentence SummaryBase editing with reduced DNA nicking allows for the simultaneous editing of >10,000 loci in human cells.

synthetic biology↗

bioRxiv2018

Differential amplicons for the evaluation of RNA integrity extracted from complex environmental samples

BackgroundReliability and reproducibility of transcriptomics-based studies are highly dependent on the integrity of RNA. Microfluidics-based techniques based on ribosomal RNA such as the RNA Integrity Number (RIN) are currently the only approaches to evaluate RNA integrity. However, it is not known if ribosomal RNA reflects the integrity of the meaningful part of the sample, the mRNA. Here we test this assumption and present a new integrity index, the Ratio amplicon, Ramp, to monitor mRNA integrity based on the differential amplification of long to short RT-Q-PCR amplicons of the glutamine synthetase A (glnA) transcript.\n\nResultsWe successfully designed and tested two Ramp indexes targeting glnA transcripts. We showed in a suite of experimental degradations of RNA extracted from sediment that while the RIN in general did reflect the degradation status of the RNA well the Ramp mapped mRNA degradation better as reflected by changes in Reverse Transcriptase Quantitative PCR (RT-Q-PCR) results. Furthermore, we examined the effect of degradation on transcript community structure by amplicon sequencing of the 16S rRNA, amoA and glnA transcript which was successful even form the highly-degraded samples. While RNA degradation changed the community structure of the mRNA profiles, no changes were observed between successively degraded 16S rRNA transcripts profiles.\n\nConclusionAs demonstrated, transcripts can be quantified and sequenced even from highly degraded samples. Therefore, we strongly recommend that a quality check of RNA is conducted to ensure validity of results. For this both the RIN and Ramp are useful, with the Ramp better evaluating mRNA integrity in this study.

microbiology↗

bioRxiv2018

Optimisation of a novel method for the production of single-span membrane proteins in Escherichia coli

The large-scale production and isolation of recombinant protein is a central element of the biotechnology industry and many of the products have proved extremely beneficial for therapeutic medicine. Escherichia coli is the microorganism of choice for the expression of heterologous proteins for therapeutic application, and a range of high-value proteins have been targeted to the periplasm using the well characterised Sec protein export pathway. More recently, the ability of the second mainstream protein export system, the twin-arginine translocase, to transport fully-folded proteins into the periplasm of not only E. coli, but other Gram-negative bacteria, has captured the interest of the biotechnology industry.\n\nIn this study, we have used a novel approach to block the export of a heterologous Tat substrate in the later stages of the export process, and thereby generate a single-span membrane protein with the soluble domain positioned on the periplasmic side of the inner membrane. Biochemical and immuno-electron microscopy approaches were used to investigate the export of human growth hormone by the twin-arginine translocase, and the generation of a single span membrane-embedded variant. This is the first time that a bona-fide biotechnologically-relevant protein has been exported by this machinery and visualised directly in this manner. The data presented here demonstrate a novel method for the production of single-span membrane proteins in E. coli.\n\nHighlightsO_LIThe Tat translocase has captured the interest of the biotechnology industry\nC_LIO_LIBiochemical and immuno-EM approaches showed efficient export of hGH by Tat\nC_LIO_LIA novel approach was used to block export of hGH by Tat in E. coli\nC_LIO_LIWe demonstrate a novel method for producing single-span membrane proteins in E. coli\nC_LI

biochemistry↗

bioRxiv2017

Microglial elimination of dopamine D1 receptors defines sex-specific changes in nucleus accumbens development and social play behavior during adolescence

Adolescence is a developmental period in which the mesolimbic dopaminergic reward circuitry of the brain, including the nucleus accumbens (NAc), undergoes significant developmental plasticity and neural circuit maturation. Dopamine D1 receptors (D1rs) in the NAc have recently been demonstrated to be critical modulators of social behavior, but how these receptors are regulated in adolescence to mediate social behavior is not well understood. In this report, we used multi-plexed immunohistochemistry with volumetric reconstructions, co-immunoprecipitation, ex vivo, and in vivo stereotaxic, microglial manipulation, and social behavior assessment to demonstrate that microglia and complement-mediated phagocytic activity shapes sex-specific NAc development. Moreover, we report for the first time that microglia-mediated phagocytosis is required for natural developmental changes in behavior, specifically, adolescent male social play behavior. These data have broad implications for understanding how experience interacts with the developing reward circuity, sex-specific responses to stimuli in adolescence, and how neuropsychiatric disorders may arise in a sexually dimorphic manner.

neuroscience↗

bioRxiv2017

Single-Neuron Gene Expression Analysis Using the Maxwell 16 LEV System in the Neural Systems and Behavior Course

Gene expression analysis from single cells has become increasingly prominent across biological disciplines; thus, it is important to train students in these approaches. Here, we present an experimental and analysis pipeline that we developed for the Neural Systems & Behavior (NS&B) course at Marine Biological Laboratory. Our approach used the Maxwell(R) 16 LEV simplyRNA Tissue Kit and GoTaq(R) 2-Step RT-qPCR System for gene expression analysis from single neurons of the crustacean stomatogastric ganglion, a model system to study the generation of rhythmic motor patterns. We used double-stranded RNA to knockdown expression of a putative neuromodulator-activated sodium channel. We then examined the electrophysiological responses to known neuromodulators and confirmed that the response was reduced. Finally, we measured how mRNA levels of several ion channel genes changed in response. Our results provide new insights into the neural mechanisms underlying the generation and modulation of rhythmic motor patterns.

neuroscience↗