Gut dysbiosis was inevitable, but tolerance was not: temporal responses of the murine microbiota that maintain its capacity for butyrate production correlate with sustained antinociception to chronic voluntary morphine.

The therapeutic benefits of opioids are compromised by the development of analgesic tolerance, which necessitates higher dosing for pain management thereby increasing the liability for dependence and addiction. Rodent models indicate opposing roles of the gut microbiota in tolerance: morphine-induced gut dysbiosis exacerbates tolerance, whereas probiotics ameliorate tolerance. Not all individuals develop tolerance which could be influenced by differences in microbiota, and yet no study has capitalized upon this natural variation to identify specific features linked to tolerance. We leveraged this natural variation in a murine model of voluntary oral morphine self-administration to elucidate the mechanisms by which microbiota influences tolerance. Although all mice shared similar and predictive morphine-driven microbiota changes that largely masked informative associations with variability in tolerance, our high-resolution temporal analyses revealed a divergence in the progression of dysbiosis that best explained differences in the development in tolerance. Mice that did not develop tolerance also maintained a higher abundance of taxa capable of producing the short-chain fatty acid (SCFA) butyrate, known to bolster intestinal barriers, suppress inflammation, and promote neuronal homeostasis. Furthermore, dietary butyrate supplementation significantly reduced the development of tolerance. These findings could inform immediate therapies to extend the analgesic efficacy of opioids.

Deletion of arrestin-3 does not improve compulsive drug-seeking behavior in a longitudinal paradigm of oral morphine self-administration.

Opioid drugs are potent analgesics that mimic the endogenous opioid peptides, endorphins and enkephalins, by activating the µ-opioid receptor. Opioid use is limited by side effects, including significant risk of opioid use disorder. Improvement of the effect/side effect profile of opioid medications is a key pursuit of opioid research, yet there is no consensus on how to achieve this goal. One hypothesis is that the degree of arrestin-3 recruitment to the µ-opioid receptor impacts therapeutic utility. However, it is not clear whether increased or decreased interaction of the µ-opioid receptor with arrestin-3 would reduce compulsive drug-seeking. To examine this question, we utilized three genotypes of mice with varying abilities to recruit arrestin-3 to the µ-opioid receptor in response to morphine in a novel longitudinal operant self-administration model. We demonstrate that arrestin-3 knockout and wild type mice have highly variable drug-seeking behavior with few genotype differences. In contrast, in mice where the µ-opioid receptor strongly recruits arrestin-3, drug-seeking behavior is much less varied. We created a quantitative method to define compulsivity in drug-seeking and found that mice lacking arrestin-3 were more likely to meet the criteria for compulsivity whereas mice with enhanced arrestin-3 recruitment did not develop a compulsive phenotype. Our data suggest that opioids that engage both G protein and arrestin-3, recapitulating the endogenous signaling pattern, will reduce abuse liability.

Temperature effects on RNA polymerase initiation kinetics reveal which open complex initiates and that bubble collapse is stepwise.

Transcription initiation is highly regulated by promoter sequence, transcription factors, and ligands. All known transcription inhibitors, an important class of antibiotics, act in initiation. To understand regulation and inhibition, the biophysical mechanisms of formation and stabilization of the "open" promoter complex (OC), of synthesis of a short RNA-DNA hybrid upon nucleotide addition, and of escape of RNA polymerase (RNAP) from the promoter must be understood. We previously found that RNAP forms three different OC with λPR promoter DNA. The 37 °C RNAP-λPR OC (RPO) is very stable. At lower temperatures, RPO is less stable and in equilibrium with an intermediate OC (I3). Here, we report step-by-step rapid quench-flow kinetic data for initiation and growth of the RNA-DNA hybrid at 25 and 37 °C that yield rate constants for each step of productive nucleotide addition. Analyzed together, with previously published data at 19 °C, our results reveal that I3 and not RPO is the productive initiation complex at all temperatures. From the strong variations of rate constants and activation energies and entropies for individual steps of hybrid extension, we deduce that contacts of RNAP with the bubble strands are disrupted stepwise as the hybrid grows and translocates. Stepwise disruption of RNAP-strand contacts is accompanied by stepwise bubble collapse, base stacking, and duplex formation, as the hybrid extends to a 9-mer prior to disruption of upstream DNA-RNAP contacts and escape of RNAP from the promoter.

Pharmacological and genetic manipulations at the µ-opioid receptor reveal arrestin-3 engagement limits analgesic tolerance and does not exacerbate respiratory depression in mice.

Opioid drugs are widely used analgesics that activate the G protein-coupled µ-opioid receptor, whose endogenous neuropeptide agonists, endorphins and enkephalins, are potent pain relievers. The therapeutic utility of opioid drugs is hindered by development of tolerance to the analgesic effects, requiring dose escalation for persistent pain control and leading to overdose and fatal respiratory distress. The prevailing hypothesis is that the intended analgesic effects of opioid drugs are mediated by µ-opioid receptor signaling to G protein, while the side-effects of respiratory depression and analgesic tolerance are caused by engagement of the receptor with the arrestin-3 protein. Consequently, opioid drug development has focused exclusively on identifying agonists devoid of arrestin-3 engagement. Here, we challenge the prevailing hypothesis with a panel of six clinically relevant opioid drugs and mice of three distinct genotypes with varying abilities to promote morphine-mediated arrestin-3 engagement. With this genetic and pharmacological approach, we demonstrate that arrestin-3 recruitment does not impact respiratory depression, and effective arrestin-3 engagement reduces, rather than exacerbates, the development of analgesic tolerance. These studies suggest that future development of safer opioids should focus on identifying opioid ligands that recruit both G protein and arrestin-3, thereby mimicking the signaling profile of most endogenous µ-opioid receptor agonists.

RNA Polymerase: Step-by-Step Kinetics and Mechanism of Transcription Initiation.

To determine the step-by-step kinetics and mechanism of transcription initiation and escape by E. coli RNA polymerase from the λPR promoter, we quantify the accumulation and decay of transient short RNA intermediates on the pathway to promoter escape and full-length (FL) RNA synthesis over a wide range of NTP concentrations by rapid-quench mixing and phosphorimager analysis of gel separations. Experiments are performed at 19 °C, where almost all short RNAs detected are intermediates in FL-RNA synthesis by productive complexes or end-products in nonproductive (stalled) initiation complexes and not from abortive initiation. Analysis of productive-initiation kinetic data yields composite second-order rate constants for all steps of NTP binding and hybrid extension up to the escape point (11-mer). The largest of these rate constants is for incorporation of UTP into the dinucleotide pppApU in a step which does not involve DNA opening or translocation. Subsequent steps, each of which begins with reversible translocation and DNA opening, are slower with rate constants that vary more than 10-fold, interpreted as effects of translocation stress on the translocation equilibrium constant. Rate constants for synthesis of 4- and 5-mer, 7-mer to 9-mer, and 11-mer are particularly small, indicating that RNAP-promoter interactions are disrupted in these steps. These reductions in rate constants are consistent with the previously determined ∼9 kcal cost of escape from λPR. Structural modeling and previous results indicate that the three groups of small rate constants correspond to sequential disruption of in-cleft, -10, and -35 interactions. Parallels to escape by T7 RNAP are discussed.

Mechanism of transcription initiation and promoter escape by E. coli RNA polymerase.

To investigate roles of the discriminator and open complex (OC) lifetime in transcription initiation by Escherichia coli RNA polymerase (RNAP; α2ßß'ωσ70), we compare productive and abortive initiation rates, short RNA distributions, and OC lifetime for the λPR and T7A1 promoters and variants with exchanged discriminators, all with the same transcribed region. The discriminator determines the OC lifetime of these promoters. Permanganate reactivity of thymines reveals that strand backbones in open regions of long-lived λPR-discriminator OCs are much more tightly held than for shorter-lived T7A1-discriminator OCs. Initiation from these OCs exhibits two kinetic phases and at least two subpopulations of ternary complexes. Long RNA synthesis (constrained to be single round) occurs only in the initial phase (<10 s), at similar rates for all promoters. Less than half of OCs synthesize a full-length RNA; the majority stall after synthesizing a short RNA. Most abortive cycling occurs in the slower phase (>10 s), when stalled complexes release their short RNA and make another without escaping. In both kinetic phases, significant amounts of 8-nt and 10-nt transcripts are produced by longer-lived, λPR-discriminator OCs, whereas no RNA longer than 7 nt is produced by shorter-lived T7A1-discriminator OCs. These observations and the lack of abortive RNA in initiation from short-lived ribosomal promoter OCs are well described by a quantitative model in which ∼1.0 kcal/mol of scrunching free energy is generated per translocation step of RNA synthesis to overcome OC stability and drive escape. The different length-distributions of abortive RNAs released from OCs with different lifetimes likely play regulatory roles.