Consumption of oxycodone prevents oxytocin from attenuating alcohol intake in rats.

Alcohol and opioid polysubstance use (PSU) is common and often accompanied by higher trait anxiety. Oxytocin decreases anxiety, alcohol- and opioid-seeking and -taking but has not been assessed in the context of PSU. Here we developed a rat model of sequential oxycodone and alcohol PSU to examine the relationship between anxiety, alcohol and oxycodone intake, and the efficacy of systemic oxytocin to attenuate alcohol intake. Male and female Sprague-Dawley rats were assessed for baseline anxiety-like behavior using acoustic startle and the elevated plus maze (EPM). Rats were then given 2-bottle choice access to oxycodone and/or water for 6-hr/day for 7 days, followed by 2-bottle choice access to alcohol (20% v/v) and/or water for five 24-hr sessions across 10 days. Next, monosubstance (oxycodone- or alcohol-alone) rats continued to have access to only one substance/day while PSU rats had access to oxycodone and water for 3-hr, followed by alcohol and water for 6-hr. After 12 days, rats were tested in the EPM 20 hours after alcohol access to examine withdrawal-related anxiety. Next, oxytocin (0, 0.3 or 1.0 mg/kg IP) was administered following the oxycodone/water session, 30 minutes prior to alcohol access. Rats received intragastric oxycodone (2 mg/kg) or water followed by intragastric alcohol (2 g/kg) and blood was collected to determine blood alcohol levels. Elevated baseline anxiety-like behavior was accompanied by reduced alcohol intake. Consumption of oxycodone did not alter alcohol intake but resulted in less anxiety-like behavior during withdrawal and prevented oxytocin from attenuating alcohol intake. Oxytocin (1 mg/kg) reduced alcohol intake in the alcohol-only condition, an effect that persisted for days after a single oxytocin administration. Rats that received oxycodone prior to non-contingent alcohol displayed higher blood alcohol levels than those that did not. These results support the necessity for the testing of medications for substance use in rodent models of PSU.

Evidence for elevated psychiatric distress, poor sleep, and quality of life concerns during the COVID-19 pandemic among U.S. young adults with suspected and reported psychiatric diagnoses.

We report distress levels and functional outcomes based on self-reported pre-existing mental health conditions among U.S. young adults (N=898) during the COVID-19 pandemic (April 13-May 19, 2020). Depression, anxiety, and PTSD symptoms, as well as COVID-19-related concerns, sleep problems, and quality of life were compared across the following pre-existing mental health groups: 1) no diagnosis, 2) suspected diagnosis, 3) diagnosed and untreated, and 4) diagnosed and treated. Compared to those without a diagnosis, the likelihood of scoring above the clinical threshold for those with a diagnosis - whether treated or not - was more than six-fold for depression, and four-to six-fold for anxiety and PTSD. Individuals with a suspected diagnosis were 3 times more likely to score above the clinical threshold for depression and anxiety and 2 times more as likely to score above this threshold for PTSD compared to those with no diagnosis. We also present higher levels of COVID-19-related worry and grief, poorer sleep, and poorer reported health-related quality of life among those with either a suspected or reported mental health diagnosis. Findings provide evidence of vulnerability among individuals with a mental health diagnosis or suspected mental health concerns during the initial weeks of the COVID-19 pandemic.

Structure-based design of potent, amidine-derived inhibitors of factor Xa: evaluation of selectivity, anticoagulant activity, and antithrombotic activity.

To enhance the potency of 1,2-dibenzamidobenzene-derived inhibitors of factor Xa (fXa), an amidine substituent was incorporated on one of the benzoyl side chains to interact with Asp189 in the S1 specificity pocket. Lead molecule 1 was docked into the active site of fXa to facilitate inhibitor design. Subsequently, iterative SAR studies and molecular modeling led to a 1000-fold increase in fXa affinity and a refined model of the new inhibitors in the fXa active site. Strong support for the computational model was achieved through the acquisition of an X-ray crystal structure using thrombin as a surrogate protein. The amidines in this series show high levels of selectivity for the inhibition of fXa relative to other trypsin-like serine proteases. Furthermore, the fXa affinity of compounds in this series (K(ass) = 50-500 x 10(6) L/mol) translates effectively into both anticoagulant activity in vitro and antithrombotic activity in vivo.

Adapting selected nucleic acid ligands (aptamers) to biosensors.

A flexible biosensor has been developed that utilizes immobilized nucleic acid aptamers to specifically detect free nonlabeled non-nucleic acid targets such as proteins. In a model system, an anti-thrombin DNA aptamer was fluorescently labeled and covalently attached to a glass support. Thrombin in solution was selectively detected by following changes in the evanescent-wave-induced fluorescence anisotropy of the immobilized aptamer. The new biosensor can detect as little as 0.7 amol of thrombin in a 140-pL interrogated volume, has a dynamic range of 3 orders of magnitude, has an inter-sensing-element measurement precision of better than 4% RSD over the range 0-200 nM, and requires less than 10 min for sample analysis. The aptamer-sensor format is generalizable and should allow sensitive, selective, and fast determination of a wide range of analytes.

In vitro selection methodologies to probe RNA function and structure.

In vitro selection, or SELEX, has been used both to characterize the interaction of natural nucleic acids with proteins and to generate novel nucleic acid-binding species, or aptamers. Although numerous reports have demonstrated the power of the technique, they have not expanded on the methodologies that can be used for selection. This review focuses on the considerations and problems involved in selecting protein-binding aptamers from a random-sequence RNA pool. As an illustration, we describe two approaches to selecting aptamers to a particular target, the HTLV-I Rex protein. In the first, complete randomization is used to find an artificial, high-affinity RNA binding site. In the second, the contributions of individual nucleotides and/or base pairs to the natural Rex-binding element are determined by mutating the wild-type sequence and selecting active binding variants.

The C. elegans trans-spliced leader RNA is bound to Sm and has a trimethylguanosine cap.

mRNA splicing in C. elegans is unusual: most introns are very short (approximately 50 bases), and many mRNAs receive a leader by trans-splicing. The donor in trans-splicing is a 94 nucleotide molecule, termed the leader RNA, that contributes its 5' 22 nucleotides to a variety of mRNAs. We show here that C. elegans has the usual snRNAs, which presumably catalyze the splicing reactions. As expected, they are bound to the Sm antigen and have 2,2,7-methylguanosine caps. Remarkably, the trans-spliced leader RNA is also Sm-associated and has this special cap. Hence, a molecule discovered as a substate of splicing has properties of molecules heretofore known only to facilitate splicing of other RNAs. Mature mRNAs that have received the leader evidently lack 2,2,7-methylguanosine caps, suggesting that these caps are removed or altered during processing.

A cyanogen bromide fragment of S4 that specifically rebinds 16S RNA.

Escherichia coli ribosomal protein S4 was subjected to cyanogen bromide cleavage and was found to generate a complete cleavage product capable of rebinding 16S rRNA. This fragment, consisting of residues 1-103, was found to bind with an apparent association constant of 11 microM-1. This fragment was used in place of S4 in an in vitro reconstitution experiment. Although the particles formed had a protein composition not significantly different from reconstituted 30S ribosomal subunits, their sedimentation behavior was more like that of particles reconstituted without S4. These results indicate to us that, although residues 104-203 of S4 are involved in the assembly of the 30S ribosome, they are not necessary for the binding of S4 to 16S RNA. Taken with previous results, the domain of S4 involved in specific binding of 16S RNA can be confined to residues 47-103.

A rapid and preparative method for the separation of yeast ribosomal proteins by using high-performance liquid chromatography.

Ribosomal proteins from the yeast Saccharomyces cerevisiae were separated, on a preparative scale, by ion-exchange h.p.l.c. Proteins from the small and large ribosomal subunits were resolved, respectively, into 33 and 23 peaks, and most of the proteins present in these peaks were identified by using one- and two-dimensional gel electrophoresis. Several of the peaks appeared to contain a single protein uncontaminated by other species. Ribosomal proteins were also separated by using reverse-phase h.p.l.c. Analysis of the peaks resolved indicated that the order of elution for the proteins of both ribosomal subunits is, in certain cases, different for each of the two h.p.l.c. techniques used. Thus a combination of the two chromatographic methods employed here has the potential to facilitate the rapid and preparative separation of each of the proteins present in yeast ribosomes.

Chemical and functional characterization of an altered form of ribosomal protein S4 derived from a strain of E. coli defective in auto-regulation of the alpha operon.

We have isolated a mutant form of Escherichia coli ribosomal protein S4. This mutant is temperature sensitive and apparently fails to autogenously regulate the gene products of the alpha operon, which consists of the genes for proteins S13, S11, S4, L17, and the alpha subunit of RNA polymerase (1). We have shown that this mutation results in the production of an S4 protein with a molecular weight approximately 4,000 daltons less than the wild-type protein. Our chemical analyses demonstrate that the mutant protein is missing its C-terminal section consisting of residues 170-203. However, our studies to determine the capacity of this mutant protein to bind 16S RNA show that this protein is unimpaired in RNA binding function. This observation suggests that the functional domain of protein S4 responsible for translational regulation of the S4 gene products requires more of the protein than the 16S RNA binding domain.

Application of high-performance liquid chromatography to the purification and characterization of ribosomal protein L3 from trichodermin-resistant yeast mutants.

A new h.p.l.c. cation-exchange method has been used to separate proteins from 60S ribosomal subunits prepared from strains of Saccharomyces cerevisiae sensitive or resistant to trichodermin. Ribosomal protein L3 was identified in column eluates by one-dimensional and two-dimensional gel electrophoresis and purified further by reverse-phase h.p.l.c. The protein was cleaved with CNBr and the products were analysed, again by reverse-phase h.p.l.c. A marked difference was observed in the peptide profiles between preparations from trichodermin-sensitive and trichodermin-resistant yeast strains. These results provide the first direct demonstration that, in yeast, mutationally induced resistance to trichodermin can alter the covalent structure of ribosomal protein L3. They convincingly demonstrate the potential of the experimental technique for the rapid and preparative separation of a selected yeast ribosomal protein and its subsequent characterization.