Biology subjects

Kennedy, R. T.

Publications and source records attributed to Kennedy, R. T..

15 recordsLinked to original sources

bioRxiv2026

Deep in vivo brain metabolomics maps vulnerability-associated and cocaine-induced neurochemical states

Innate temperament affects vulnerability to substance use disorders (SUDs). Identifying the neurochemistry that differentiates temperaments, alongside the metabolic impact of drugs of abuse, can provide new insights into pathways to SUD and treatments. We show that heritable emotional temperaments associated with SUD vulnerability are encoded by distinct extracellular neurochemical states in the living brain. We apply untargeted metabolomics to microdialysis samples to define the chemical composition of the living brain metabolome with unprecedented depth. Using two phenotypes from a rodent model of diverging temperaments linked to SUDs, dialysate samples were collected before and after acute exposure to cocaine (15 mg/kg, i.p.). Metabolomic analysis revealed previously unknown basal differences in stress-response, bioenergetics, and amino acid neurotransmitters and their related pathways between the two phenotypes. Cocaine administration also had a profound impact on the metabolome, altering amino acid, purine, and arachidonic acid pathways along with cognition-related metabolites. Despite these broad metabolomic changes, cocaine administration narrowed several pre-existing differences between phenotypes, highlighting divergent responses across genetic lines. These findings demonstrate that in vivo metabolomics not only offers novel insights into the neurochemistry underlying temperament and drug response but also serves as framework to advance a systems-level understanding of other brain-related disorders.

neuroscience↗

bioRxiv2026

Integrating mass spectrometry with Nanopore direct RNA sequencing for de novo modification profiling of bacteriophage MS2

RNA modifications contribute to the regulatory and structural complexity of RNA molecules, influencing key biological processes. Nanopore direct RNA sequencing (DRS) enables the detection of these modifications at single-molecule resolution without chemical conversion. Oxford Nanopore Technologies recently introduced RNA004 sequencing chemistry and the Dorado basecaller, which improves accuracy and enables the identification of eight RNA modifications. However, the reliability of these predictions requires careful validation using orthogonal approaches. Here, we profiled RNA modifications in the MS2 bacteriophage genome using both LC-MS/MS and Nanopore DRS. LC-MS/MS analysis revealed that {Psi} is the predominant modification, present at approximately one site per RNA molecule, while all other modifications were found at low levels (< 0.04 modifications per RNA). In contrast, Dorado-based modification calling not only confirmed {Psi} but also predicted multiple m6A, m5C, 2OMe, and inosine sites not supported by LC-MS/MS data. To refine modification calling, we used in vitro transcribed (IVT) RNA as a negative control and subtracted IVT-derived false positive rates from native RNA predictions. This adjustment reduced overcalling and improved confidence in site-specific predictions. These data demonstrate that while Dorado can detect RNA modifications de novo, its predictions require careful filtering and validation. Our studies of provide a rare benchmark dataset for assessing the ability of transcriptome-wide methods to accurately identify RNA modifications and estimate modification stoichiometry. These findings support the use of orthogonal approaches, including LC-MS/MS and IVT controls, alongside Nanopore sequencing to provide a more reliable and interpretable strategy for studying RNA modification patterns. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=138 SRC="FIGDIR/small/729303v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@637a55org.highwire.dtl.DTLVardef@f48272org.highwire.dtl.DTLVardef@a0465aorg.highwire.dtl.DTLVardef@6af045_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2026

RNase 4 improves bottom-up modification mapping of E. coli total tRNAs using HILIC-MS/MS

The structural and functional diversity of RNAs is expanded by the post-transcriptional incorporation of nucleoside variants. Emblematic of this, tRNAs contain extensive modifications that ensure their function during protein synthesis. Mass spectrometry has long been the field standard for identifying specific sites of chemical modifications on RNA. Nonetheless, mass spectrometry-based mapping approaches are not widely implemented. This is partially due to technical challenges associated with current methodologies including the limited diversity of available RNases, complexity of RNA mixtures, and conventional use ion-pairing reagents that require dedicated instrumentation. Here, we present a bottom-up liquid chromatography-tandem mass spectrometry (LC-MS/MS) workflow employing hydrophilic interaction liquid chromatography (HILIC) without ion-pairing reagents to globally map E. coli tRNA modifications. We implement orthogonal digestions using RNase 4 and a folded digestion scheme with RNase T1 to generate uniquely mappable oligonucleotides compatible with HILIC-MS/MS analysis and achieve 75-100% sequence coverage for most tRNA isoacceptors. HILIC-MS/MS matches the performance of traditional ion-pairing reverse-phased LC-MS/MS. This level of coverage allowed us to discover a new site of methylation (Gm17) in tRNAGly, and confirm the presence of an s4U8 modification predicted in tRNAArg. Furthermore, by applying this method to E. coli lacking the m5U54 methyltransferase (trmA) we confirmed the established dependence of acp3U47 insertion on m5U54 in tRNAPhe. Our findings show that RNase 4 improves bottom-up tRNA sequencing, enabling high-quality E. coli tRNA analysis without ion-pairing reagents. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=72 SRC="FIGDIR/small/727428v1_ufig1.gif" ALT="Figure 1"> View larger version (14K): org.highwire.dtl.DTLVardef@1f483a3org.highwire.dtl.DTLVardef@1ee5e01org.highwire.dtl.DTLVardef@5db88borg.highwire.dtl.DTLVardef@fee855_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2024

Neurochemical and Neurophysiological Effects of Intravenous Administration of N,N-dimethyltryptamine in Rats

N,N-dimethyltryptamine (DMT) is a serotonergic psychedelic that is being investigated clinically for the treatment of psychiatric disorders. Although the neurophysiological effects of DMT in humans are well-characterized, similar studies in animal models as well as data on the neurochemical effects of DMT are generally lacking, which are critical for mechanistic understanding. In the current study, we combined behavioral analysis, high-density (32-channel) electroencephalography, and ultra-high-performance liquid chromatography-tandem mass spectrometry to simultaneously quantify changes in behavior, cortical neural dynamics, and levels of 17 neurochemicals in medial prefrontal and somatosensory cortices before, during, and after intravenous administration of three different doses of DMT (0.75 mg/kg, 3.75 mg/kg, 7.5 mg/kg) in male and female adult rats. All three doses of DMT produced head twitch response with most twitches observed after the low dose. DMT caused dose-dependent increases in serotonin and dopamine levels in both cortical sites along with a reduction in EEG spectral power in theta (4-10 Hz) and low gamma (25-55 Hz), and increase in power in delta (1-4 Hz), medium gamma (65-115 ), and high gamma (125-155 Hz) bands. Functional connectivity decreased in the delta band and increased across the gamma bands. In addition, we provide the first measurements of endogenous DMT in these cortical sites at levels comparable to serotonin and dopamine, which together with a previous study in occipital cortex, suggests a physiological role for endogenous DMT. This study represents one of the most comprehensive characterizations of psychedelic drug action in rats and the first to be conducted with DMT. Significance StatementN,N-dimethyltryptamine (DMT) is a serotonergic psychedelic with potential as a tool for probing the neurobiology of consciousness and as a therapeutic agent for psychiatric disorders. However, the neurochemical and neurophysiological effects of DMT in rat, a preferred animal model for mechanistic studies, are unclear. We demonstrate that intravenous DMT caused a dose-dependent increase in serotonin and dopamine in medial prefrontal and somatosensory cortices, and simultaneously increased gamma functional connectivity. Similar effects have been shown for other serotonergic and atypical psychedelics, suggesting a shared mechanism of drug action. Additionally, we report DMT during normal wakefulness in two spatially and functionally distinct cortical sites -- prefrontal, somatosensory -- at levels comparable to those of serotonin and dopamine, supporting a physiological role for endogenous DMT.

neuroscience↗

bioRxiv2023

Conserved 5-methyluridine tRNA modification modulates ribosome translocation

While the centrality of post-transcriptional modifications to RNA biology has long been acknowledged, the function of the vast majority of modified sites remains to be discovered. Illustrative of this, there is not yet a discrete biological role assigned for one the most highly conserved modifications, 5-methyluridine at position 54 in tRNAs (m5U54). Here, we uncover contributions of m5U54 to both tRNA maturation and protein synthesis. Our mass spectrometry analyses demonstrate that cells lacking the enzyme that installs m5U in the T-loop (TrmA in E. coli, Trm2 in S. cerevisiae) exhibit altered tRNA modifications patterns. Furthermore, m5U54 deficient tRNAs are desensitized to small molecules that prevent translocation in vitro. This finding is consistent with our observations that, relative to wild-type cells, trm2{Delta} cell growth and transcriptome-wide gene expression are less perturbed by translocation inhibitors. Together our data suggest a model in which m5U54 acts as an important modulator of tRNA maturation and translocation of the ribosome during protein synthesis.

biochemistry↗

bioRxiv2023

Offline Two-dimensional Liquid Chromatography-Mass Spectrometry for Deep Annotation of the Fecal Metabolome

Compound identification is an essential task in the workflow of untargeted metabolomics since the interpretation of the data in a biological context depends on the correct assignment of chemical identities to the features it contains. Current techniques fall short of identifying all or even most observable features in untargeted metabolomics data, even after rigorous data cleaning approaches to remove degenerate features are applied. Hence, new strategies are required to annotate the metabolome more deeply and accurately. The human fecal metabolome, which is the focus of substantial biomedical interest, is a more complex, more variable, yet lesser-investigated sample matrix compared to widely studied sample types like human plasma. This manuscript describes a novel experimental strategy using multidimensional chromatography to facilitate compound identification in untargeted metabolomics. Pooled fecal metabolite extract samples were fractionated using offline semi-preparative liquid chromatography. The resulting fractions were analyzed by an orthogonal LC-MS/MS method, and the data were searched against commercial, public, and local spectral libraries. Multidimensional chromatography yielded more than a 3-fold improvement in identified compounds compared to the typical single-dimensional LC-MS/MS approach and successfully identified several rare and novel compounds, including atypical conjugated bile acid species. Most features identified by the new approach could be matched to features that were detectable but not identifiable in the original single-dimension LC-MS data. Overall, our approach represents a powerful strategy for deeper annotation of the metabolome that can be implemented with commercially-available instrumentation, and should apply to any dataset requiring deeper annotation of the metabolome.

systems biology↗

bioRxiv2022

Methylated guanosine and uridine modifications in S. cerevisiae mRNAs modulate translation elongation

Chemical modifications to protein encoding messenger RNA (mRNA) can modulate their localization, translation and stability within cells. Over 15 different types of mRNA modifications have been identified by sequencing and liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) technologies. While LC-MS/MS is arguably the most essential tool available for studying analogous protein post-translational modifications, the high-throughput discovery and quantitative characterization of mRNA modifications by LC-MS/MS has been hampered by the difficulty of obtaining sufficient quantities of pure mRNA and limited sensitivities for modified nucleosides. To overcome these challenges, we improved the mRNA purification and LC-MS/MS pipelines to identify new S. cerevisiae mRNA modifications and quantify 50 ribonucleosides in a single analysis. The methodologies we developed result in no detectable non-coding RNA modifications signals in our purified mRNA samples and provide the lowest limit of detection reported for ribonucleoside modification LC-MS/MS analyses. These advancements enabled the detection and quantification of 13 S. cerevisiae mRNA ribonucleoside modifications and revealed four new S. cerevisiae mRNA modifications at low to moderate levels (1-methyguanosine, N2-methylguanosine, N2, N2-dimethylguanosine, and 5-methyluridine). We identified four enzymes that incorporate these modifications into S. cerevisiae mRNAs (Trm10, Trm11, Trm1, and Trm2), though our results suggest that guanosine and uridine nucleobases are also non-enzymatically methylated at low levels. Regardless of whether they are incorporated in a programmed manner or as the result of RNA damage, we reasoned that the ribosome will encounter the modifications that we detect in cells and used a reconstituted translation system to discern the consequences of modifications on translation elongation. Our findings demonstrate that the introduction of 1-methyguanosine, N2-methylguanosine and 5-methyluridine into mRNA codons impedes amino acid addition in a position dependent manner. This work expands the repertoire of nucleoside modifications that the ribosome must decode in S. cerevisiae. Additionally, it highlights the challenge of predicting the effect of discrete modified mRNA sites on translation de novo because individual modifications influence translation differently depending on mRNA sequence context.

biochemistry↗

bioRxiv2021

Mapping enzyme catalysis with metabolomic biosensing

Enzymes are represented across a vast space of protein sequences and structural forms and have activities that far exceed the best chemical catalysts; however, engineering them to have novel or enhanced activity is limited by technologies for sensing product formation. Here, we describe a general and scalable approach for characterizing enzyme activity that uses the metabolism of the host cell as a biosensor by which to infer product formation. Since different products consume different molecules in their synthesis, they perturb host metabolism in unique ways that can be measured by mass spectrometry. This provides a general way by which to sense product formation, to discover unexpected products and map the effects of mutagenesis.

bioengineering↗

bioRxiv2021

Viperin inhibits cholesterol biosynthesis and interacts with enzymes in the cholesterol biosynthetic pathway

Many enveloped viruses bud from cholesterol-rich lipid rafts on the cell membrane. Depleting cellular cholesterol impedes this process and results in viral particles with reduced viability. Viperin (virus inhibitory protein endoplasmic reticulum-associated, interferon-induced) is an ER membrane-associated enzyme that when expressed in response to viral infections exerts broad-ranging antiviral effects, including inhibiting the budding of some enveloped viruses. Here we have investigated the effect of viperin expression on cholesterol biosynthesis. We found that viperin expression reduces cholesterol levels by 20 - 30 % in HEK293T cells. A proteomic screen of the viperin interactome identified several cholesterol biosynthetic enzymes among the top hits. The two most highly enriched proteins were lanosterol synthase and squalene monooxygenase, enzymes that catalyze key steps establishing the sterol carbon skeleton. Co-immunoprecipitation experiments established that viperin, lanosterol synthase and squalene monooxygenase form a complex at the ER membrane. Co-expression of viperin was found to significantly inhibit the specific activity of lanosterol synthase in HEK293T cell lysates. Co-expression of viperin had no effect on the specific activity of squalene monooxygenase, but reduced its expression levels in the cells by approximately 30 %. Despite these inhibitory effects, co-expression of either LS or SM failed to reverse the viperin-induced depletion of cellular cholesterol levels in HEK293T cells. Our results establish a clear link between the down-regulation of cholesterol biosynthesis and viperin, although at this point the effect cannot be unambiguously attributed interactions between viperin and a specific biosynthetic enzyme.

biochemistry↗

bioRxiv2020

Synaptotagmin-7 is a calcium sensor for facilitation at the splanchnic-chromaffin cell synapse

Disturbances that threaten homeostasis elicit activation of the sympathetic nervous system (SNS) and the adrenal medulla. The effectors discharge as a unit to drive global and immediate changes in whole-body physiology. Descending sympathetic information is conveyed to the adrenal medulla via preganglionic splanchnic fibers. These fibers pass into the gland and synapse onto chromaffin cells, which synthesize, store, and secrete catecholamines and vasoactive peptides. While the importance of the sympatho-adrenal branch of the autonomic nervous system has been appreciated for many decades, the mechanisms underlying transmission between presynaptic splanchnic neurons and postsynaptic chromaffin cells have remained obscure. In contrast to chromaffin cells, which have enjoyed sustained attention as a model system for exocytosis, even the Ca2+ sensors that are expressed within splanchnic terminals have not yet been identified. This study shows that a ubiquitous Ca2+-binding protein, synaptotagmin-7 (Syt7), is expressed within the fibers that innervate the adrenal medulla, and that its absence can alter synaptic transmission in the preganglionic terminals of chromaffin cells. The prevailing impact in presynapses that lack Syt7 is a decrease in synaptic strength and neuronal short-term plasticity. Evoked excitatory postsynaptic currents (EPSCs) in Syt7 KO preganglionic terminals are smaller in amplitude than in wild-type synapses stimulated in an identical manner. Splanchnic inputs also display robust short-term presynaptic facilitation, which is compromised in the absence of Syt7. These data reveal, for the first time, a role for any synaptotagmin at the splanchnic-chromaffin cell synapse. They also suggest that Syt7 has actions at synaptic terminals that are conserved across central and peripheral branches of the nervous system.

neuroscience↗

bioRxiv2019

Targeting Viperin to the Mitochondrion Inhibits the Thiolase Activity of the Trifunctional Enzyme Complex

Understanding the mechanisms by which viruses evade host cell immune defenses is important for developing improved antiviral therapies. In an unusual twist, human cytomegalovirus (HCMV) co-opts the antiviral radical SAM enzyme, viperin (Virus inhibitory protein, endoplasmic reticulum-associated, interferon-inducible), to enhance viral infectivity. This process involves translocation of viperin to the mitochondrion where it binds the {beta}-subunit (HADHB) of the mitochondrial trifunctional enzyme complex that catalyzes the thiolysis of {beta}-ketoacyl-CoA esters as part of fatty acid {beta}-oxidation. We have investigated how the interaction between these two enzymes alters their activities and their effect on cellular ATP levels. Studies with purified enzymes demonstrated that viperin inhibits the thiolase activity of HADHB, but, unexpectedly, HADHB activates viperin to synthesize the antiviral nucleotide 3-deoxy-3,4-didehydro-CTP. Enzyme activities were also measured in lysates prepared from transfected HEK 293T cells transiently expressing these enzymes. Mirroring the studies on purified enzymes, localizing viperin to the mitochondria decreased thiolase activity whereas co-expression of HADHB significantly increased viperin activity. Furthermore, targeting viperin to mitochondria also increased the rate at which HADHB was retro-translocated out of mitochondria and degraded, providing an additional mechanism for reducing HADHB activity. Targeting viperin to the mitochondria decreased cellular ATP levels by over 50 %, consistent with the enzyme disrupting fatty acid catabolism. These results provide biochemical insight into the mechanism by which HCMV subjugates viperin; they also provide a biochemical rational for viperins recently discovered role in regulating thermogenesis in adipose tissues.

biochemistry↗

bioRxiv2019

The Interaction of Viperin with Hepatitis C Virus Non-Structural Protein 5A Inhibits the Catalytic Activity of Viperin

The radical SAM enzyme viperin exerts a wide range of antiviral effects through both the synthesis of the antiviral nucleotide 3-deoxy-3, 4-didehydro-CTP (ddhCTP) and through its interactions with various cellular and viral proteins. Here we investigate the interaction of viperin with hepatitis C virus non-structural protein 5A (NS5A) and the host sterol regulatory protein, vesicle-associated membrane protein A (VAP-33). NS5A and VAP-33 form part of the viral replication complex that is essential for copying the RNA genome of the virus. Using transfected enzymes in HEK293T cells, we show that viperin binds to both NS5A and VAP-33 independently and that this interaction is dependent on all three proteins being localized to the ER membrane. Co-expression of viperin with VAP-33 and NS5A was found to reduce NS5A levels, most likely by increasing the rate of proteasomal degradation. However, co-expression of viperin with VAP-33 and NS5A also reduces the specific activity of viperin by ~ 3-fold. This observation suggests that NS5A may have evolved to bind viperin as a strategy to reduce ddhCTP synthesis, thereby reducing possibility of the replication complex introducing this chain-terminating nucleotide during genome synthesis.

biochemistry↗

bioRxiv2018

Incentive and dopamine sensitization produced by intermittent but not long access cocaine self-administration

Recent studies suggest that the temporal pattern of drug use (pharmacokinetics) has a profound effect on the ability of self-administered cocaine to produce addiction-like behavior in rodents, and to change the brain. To further address this issue, we compared the effects of Long Access (LgA) cocaine self-administration, which is widely used to model the transition to addiction, with Intermittent Access (IntA), which is thought to better reflect the pattern of drug use in humans, on the ability of self-administered cocaine to increase dopamine (DA) overflow in the core of the nucleus accumbens (using in vivo microdialysis), and to produce addiction-like behavior. IntA experience was more effective than LgA in producing addiction-like behavior - a drug experience-dependent increase in motivation for cocaine assessed using behavioral economic procedures, and cue-induced reinstatement - despite much less total drug consumption. There were no group differences in basal levels of DA in dialysate, but a single self-administered IV injection of cocaine increased DA in the core of the nucleus accumbens to a greater extent in rats with prior IntA experience than those with LgA or Short Access (ShA) experience, and the latter two groups did not differ. Furthermore, high motivation for cocaine was associated with a high DA response. Thus, IntA, but not LgA, produced both incentive and DA sensitization. This is consistent with the notion that a hyper-responsive dopaminergic system may contribute to the transition from casual patterns of drug use to the problematic patterns that define addiction.

neuroscience↗

bioRxiv2018

In vivo detection of optically-evoked opioid peptide release

Though the last decade has seen accelerated advances in techniques and technologies to perturb neuronal circuitry in the brain, we are still poorly equipped to adequately dissect endogenous peptide release in vivo. To this end we developed a system that combines in vivo optogenetics with microdialysis and a highly sensitive mass spectrometry-based assay to measure opioid peptide release in freely moving rodents.

neuroscience↗

bioRxiv2018

Regulation of IRAK1 Ubiquitination by the Antiviral Radical SAM Enzyme Viperin

Viperin is a radical S-adenosylmethionine (SAM) enzyme that plays a multifaceted role in the cellular antiviral response. Viperin was recently shown to catalyze the SAM-dependent formation of 3'-deoxy-3',4'-didehydro-CTP (ddhCTP), which inhibits some viral RNA polymerases. Viperin is also implicated in regulating K63-linked poly-ubiquitination of interleukin-1 receptor-associated kinase-1 (IRAK1) by the E3 ubiquitin ligase TNF Receptor-Associated Factor 6 (TRAF6) as part of the Toll-like receptor-7 and 9 (TLR7/9) innate immune signaling pathways. We show that IRAK1 and TRAF6 activate viperin to efficiently catalyze the radical-mediated dehydration of CTP to ddhCTP. Furthermore, poly-ubiquitination of IRAK1 requires the association of viperin with IRAK1 and TRAF6. Poly-ubiquitination appears dependent on structural changes induced by SAM binding to viperin but does not require catalytically active viperin. The synergistic activation of viperin and IRAK1 provides a mechanism that couples innate immune signaling with the production of the antiviral nucleotide ddhCTP.

biochemistry↗