Biology subjects

Smith, C. J.

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

5 recordsLinked to original sources

bioRxiv2026

Adolescent blockade of complement signaling in the lateral septum increases social novelty seeking behavior in male mice

Social behaviors are critical for survival and change dramatically over the lifespan. Adolescence is a critical period of development during which social novelty seeking peaks before declining into adulthood. Adolescence is also a time of pronounced neural circuit refinement as excess synapses are eliminated. One critical mechanism supporting this maturation of neural circuits is microglial pruning of synapses through the classical complement signaling cascade. However, it remains unclear how microglial pruning of the neural circuitry supporting social novelty preference shapes the trajectory of this behavior during adolescence. To address this, we blocked microglial complement-dependent pruning during adolescence by injecting neutrophil inhibitory factor (NIF; blocks the adhesion of ligands to CD11b/C3 receptor) in the lateral septum (LS), a key node in the social circuitry supporting social novelty preference, in male mice. We found that NIF administration into the LS during adolescence increased preference for the novel social chamber over the familiar as compared to control PBS administration. LS-NIF treatment had no impact on anxiety-like behavior in the light-dark box test and no effect on sociability. LS-NIF treatment also decreased the expression of immune-related genes in the LS as compared to PBS treatment. These data support the hypothesis that complement-dependent microglial synaptic elimination in the LS is critical for the developmental progression of social novelty preference.

neuroscience↗

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↗