The effectiveness of a transitional pain service in patients undergoing surgery with an increased risk of developing chronic postsurgical pain (TRUSt study). A randomized clinical trial.

STUDY OBJECTIVE: Poorly controlled acute postsurgical pain is associated with delayed recovery, chronic postsurgical pain (CPSP), chronic opioid use and impaired functioning in daily activities. The aim was to determine the effectiveness of a transitional pain service (TPS) to improve quality of recovery for patients at risk of CPSP. We hypothesized that a TPS improves the quality of recovery in patients at risk of CPSP. DESIGN: Single-center, pragmatic, randomized, superiority trial. SETTING: Tertiary hospital in the Netherlands. PATIENTS: Assessed for eligibility if ≥18 years of age, undergoing elective surgery, and had an increased risk of developing CPSP. After being stratified for sex, 176 patients were included. INTERVENTION: Patients were randomized to receive TPS or standard of care (SOC). TPS was a multidisciplinary intervention providing a patient-tailored perioperative pain management plan, throughout all phases of surgery. MEASUREMENTS: The primary outcome was the difference in quality of recovery on the third postoperative day, measured by the Quality of Recovery (QoR)-15 questionnaire. Secondary outcomes include the between group differences in opioid consumption. MAIN RESULTS: The primary outcome was available in 169 (96.0%) patients. No difference between groups was found in QoR-15 on the third postoperative day (mean difference 2.0, 95% CI -5.5 to 9.4, p = 0.607). A decrease in opioid usage (compared to baseline) was observed in chronic opioid users, the median [IQR] reduction in total daily oral morphine milligram equivalents (MME) for TPS was -30 [-60, 0] at three and - 29.3 [-65.6, 0] at six months, whereas SOC had a median reduction of 0 [-56, 0] at three, and 0 [-60, 7.5] at six months. CONCLUSIONS: TPS did not significantly affect short-term quality of recovery but might improve long-term outcomes, such as the incidence of chronic pain, opioid consumption, and functioning in daily life. However, sample size in the present study was too small to provide solid evidence for this positive signal.

C-Sulfonylation of 4-Alkylpyridines: Formal Picolyl C-H Activation via Alkylidene Dihydropyridine Intermediates.

4-Picoline derivatives are converted to the corresponding aryl picolyl sulfones upon treatment with aryl sulfonyl chlorides and Et3N in the presence of catalytic DMAP. The reaction proceeds smoothly for a variety of alkyl and aryl picolines using a range of aryl sulfonyl chlorides. The reaction is believed to involve N-sulfonyl 4-alkylidene dihydropyridine intermediates and results in formal sulfonylation of unactivated picolyl C-H bonds.

Imidazolidine Hydride Donors in Palladium-Catalyzed Alkyne Hydroarylation.

Aldehyde-derived imidazolidines participate as hydride donors in intramolecular reductive Heck-type reactions. N,N'-Diphenylimidazolidines prepared from ortho-alkynyl benzaldehydes underwent regio- and stereoselective palladium-catalyzed hydroarylation followed by formal 1,5-hydride transfer and reductive elimination to afford substituted alkenes and imidazolium moieties, the latter conveniently converted in situ to ring-opened benzanilides to simplify product isolation. Internal alkynes were converted to trisubstituted alkenes via a syn hydroarylation process, while a terminal alkyne was converted to a cis alkene via a formal trans hydroarylation reaction. Benzanilide products could be converted to carboxylic acid derivatives under basic conditions, resulting in the net conversion of alkynyl aldehydes to alkenyl carboxylic acids. A styrene derivative with an attached N,N'-dimethylbenzimidazoline hydride donor was also found to undergo an analogous hydroarylation/benzimidazoline oxidation to give a diarylethane product.

Study protocol for the TRUSt trial: a pragmatic randomised controlled trial comparing the standard of care with a transitional pain service for patients at risk of chronic postsurgical pain undergoing surgery.

INTRODUCTION: Patients with either surgery-related or patient-related risk factors are at an increased risk of acute and chronic postsurgical pain (CPSP) and long-term opioid use. To improve recovery, prevent CPSP and decrease opioid use, we need to identify these patients before surgery and provide a multidisciplinary pain management strategy throughout hospital admission and follow-up in the postdischarge period. We hypothesise that a multidisciplinary transitional pain service (TPS) improves quality of recovery and reduce the incidence of CPSP and opioid consumption. METHODS AND ANALYSIS: We aim to investigate the effectiveness of implementation of a TPS for patients at risk of developing CPSP. The trial design is a pragmatic, open-label, randomised controlled trial (RCT). After stratification for sex, patients are randomly assigned to the TPS or standard of care (SOC) group. Our primary outcome is the quality of recovery, measured at the morning of the third postoperative day, employing the quality of recovery (QoR)-15 questionnaire. Secondary outcomes are the incidence of CPSP, opioid consumption and patient-reported outcome measures at 3 and 6 months postoperatively. We need to enrol 176 patients to detect a minimal clinical important difference of 8 points on the QoR-15 score. ETHICS AND DISSEMINATION: Ethics approval was obtained by the accredited medical research ethics committee of the Academic Medical Center in Amsterdam (2020_211) on 15 October 2020. Protocol version 3.2 was approved on 25 January 2020. The trial is registered with the Netherlands Trial Register, NL9115. The results will be disseminated by open access publication in a peer-reviewed journal.Trial registration number NL9115.

Uterine artery pseudoaneurysm: A case of late intra-abdominal haemorrhage after caesarean section.

Uterine artery pseudoaneurysm (UAP) is a rare acquired vascular malformation associated with vaginal bleeding or intraabdominal haemorrhage occurring after pelvic surgery. Pseudoaneurysm may present with delayed, severe haemorrhage after a seemingly uncomplicated initial postoperative period. Treatment is therefore necessary to prevent further complications. We describe here a case of a 32-year-old mother, who presented with abdominal pain and intraabdominal bleeding, 20 days after Caesarean Section. Computerised Tomography (CT) scan showed the presence of haemoperitoneum, suggestive of pseudoaneurysm at the right cervical artery which was successfully managed with emergency angiographic embolisation.

Intratumoral toremifene therapy and tissue distribution in the baboon.

The purpose of the present study was to evaluate the tissue distribution of toremifene (TOR) in baboons following intra-tissue injections and to examine the effectiveness of intratumoral TOR therapy of baboons with various spontaneous neoplasms. Five healthy baboons (Papio sp.) were used to examine the distribution of TOR following intra-tissue injections. Twenty-three different tissue specimens were collected for HPLC analysis. In addition, four baboons with various spontaneous neoplasms (myxoma, squamous cell carcinoma, lymphosarcoma and adenocarcinoma) were treated with intratumoral TOR and their responses were evaluated. Tissue TOR distribution was also examined in these animals. In the tissue distribution study, target tissue/serum TOR concentration ratios ranged from 138 to 8873 and the target tissue/other tissue ratios ranged from 1.2 to 2428. The distribution of TOR was very favorable, with the highest concentrations outside the injection sites noted in adjacent organs. A marked response was observed in the myxoma and partial responses were observed in the other three cases. Drug level analysis data from these four animals revealed tissue concentrations similar to those seen in the TOR tissue distribution study. Intratumoral administration of TOR can achieve effective local tumor and tissue concentrations, while systemic distribution via circulation to other organs is limited.

Tissue distribution of transdermal toremifene.

PURPOSE: Toremifene is an orally administered triphenylethylene derivative with antiestrogenic activity that is primarily used in the treatment of patients with metastatic breast cancer. The purpose of this study was to evaluate the therapeutic advantage of local (transdermal) administration of toremifene in several animal models. Local (subcutaneous and skin) versus systemic concentrations of toremifene were evaluated serially following transdermal application of the drug. With high local concentrations and minimal distribution to other organs via the circulation, topical toremifene may deliver maximal therapeutic effects to local tissue while avoiding the side effects seen with systemic therapy. METHODS: Three animal models (nude mice, baboons, and a horse) were used to examine topically administered toremifene for kinetic measurements. RESULTS: In nude mice implanted subcutaneously with MDA-MB-231 human breast tumors, topical toremifene (2.5 mg/day x 5 days) produced greater than 50-fold higher tumor concentrations compared with intraperitoneal (i.p.) administration (1.0 mg/day x 5 days). Systemic distribution in plasma, uterus, and liver was lower following topical than following i.p. administration. In nude mice inoculated subcutaneously with estrogen receptor-positive (ER +) MCF-7 human breast cancer cells, topical toremifene and 4-hydroxytoremifene (4-OH) prevented tumor growth in the presence of estradiol. In four nontumor-bearing baboons that were given transdermal toremifene, relatively high distribution of drug was noted in normal breast tissue and fat, compared with undetectable serum concentrations. Finally, a new topical formulation of toremifene (a gel preparation for human use, Orion-Farmos, Finland) achieved high local tumor toremifene concentrations in a horse melanoma, with minimal systemic distribution. CONCLUSIONS: Transdermal toremifene can achieve high local tissue concentrations with minimal systemic distribution.

Identification of a domain required for autoproteolytic cleavage of murine coronavirus gene A polyprotein.

The 5'-most gene of the murine coronavirus genome, gene A, is presumed to encode viral RNA-dependent RNA polymerase. It has previously been shown that the N-terminal portion of this gene product is cleaved into a protein of 28 kilodaltons (p28). To further understand the mechanism of synthesis of the p28 protein, cDNA clones representing the 5'-most 5.3 kilobases of murine coronavirus mouse hepatitis virus strain JHM were sequenced and subcloned into pT7 vectors from which RNAs were transcribed and translated in vitro. The sequence was found to encode a single long open reading frame continuing from near the 5' terminus of the genome. Although p28 is encoded from the first 1 kilobase at the 5' end of the genome, translation of in vitro-transcribed RNAs indicated that this protein was not detected unless the product of the entire 5.3-kilobase region was synthesized. Translation of RNAs of 3.9 kilobases or smaller yielded proteins which contained the p28 sequence, but p28 was not cleaved. This suggests that the sequence in the region between 3.9 and 5.3 kilobases from the 5' end of the genomic RNA is essential for proteolytic cleavage and contains autoproteolytic activity. The p28 protein could not be cleaved from the smaller primary translation products of gene A, even in the presence of the larger autocleaving protein. Cleavage of the p28 protein was inhibited by addition of the protease inhibitor ZnCl2. This study thus identified a protein domain essential for autoproteolytic cleavage of the gene A polyprotein.

Sequence analysis of nucleocapsid gene and leader RNA of human coronavirus OC43.

The nucleotide sequence of the 3'-end of the genomic RNA of human coronavirus OC43 (HCV-OC43) was determined from the cDNA clones of the intracellular virus-specific mRNAs. The nucleotide sequence and the predicted amino acid sequence of the main open reading frame (ORF), which represents the nucleocapsid (N) protein, were highly homologous to those of bovine coronavirus (BCV) Mebus strain. This ORF predicts a protein of 448 amino acids. Additional smaller ORFs are also present in a different reading frame. We have also determined the leader sequence present at the 5'-end of HCV-OC43 mRNAs by a primer extension study. This sequence is highly homologous to that of mouse hepatitis virus, particularly in the 3'-end of the leader sequence, which is postulated to be involved in the unique mechanism of leader-primed transcription. These data suggest that HCV-OC43 and BCV might have diverged from each other fairly recently and that the 3'-end of the leader sequence has significant functional roles.

Specific interaction between coronavirus leader RNA and nucleocapsid protein.

Northwestern blot analysis in the presence of competitor RNA was used to examine the interaction between the mouse hepatitis virus (MHV) nucleocapsid protein (N) and virus-specific RNAs. Our accompanying article demonstrates that anti-N monoclonal antibodies immunoprecipitated all seven MHV-specific RNAs as well as the small leader-containing RNAs from infected cells. In this article we report that a Northwestern blotting protocol using radiolabeled viral RNAs in the presence of host cell competitor RNA can be used to demonstrate a high-affinity interaction between the MHV N protein and the virus-specific RNAs. Further, RNA probes prepared by in vitro transcription were used to define the sequences that participate in such high-affinity binding. A specific interaction occurs between the N protein and sequences contained with the leader RNA which is conserved at the 5' end of all MHV RNAs. We have further defined the binding sites to the area of nucleotides 56 to 65 at the 3' end of the leader RNA and suggest that this interaction may play an important role in the discontinuous nonprocessive RNA transcriptional process unique to coronaviruses.

Primary structure and translation of a defective interfering RNA of murine coronavirus.

An intracellular defective-interfering (DI) RNA, DIssE, of mouse hepatitis virus (MHV) obtained after serial high multiplicity passage of the virus was cloned and sequenced. DIssE RNA is composed of three noncontiguous genomic regions, representing the first 864 nucleotides of the 5' end, an internal 748 nucleotides of the polymerase gene, and 601 nucleotides from the 3' end of the parental MHV genome. The DIssE sequence contains one large continuous open reading frame. Two protein products from this open reading frame were identified both by in vitro translation and in DI-infected cells. Sequence comparison of DIssE and the corresponding parts of the parental virus genome revealed that DIssE had three base substitutions within the leader sequence and also a deletion of nine nucleotides located at the junction of the leader and the remaining genomic sequence. The 5' end of DIssE RNA was heterogeneous with respect to the number of UCUAA repeats within the leader sequence. The parental MHV genomic RNA appears to have extensive and stable secondary structures at the regions where DI RNA rearrangements occurred. These data suggest that MHV DI RNA may have been generated as a result of the discontinuous and nonprocessive manner of MHV RNA synthesis.

Discontinuous transcription generates heterogeneity at the leader fusion sites of coronavirus mRNAs.

Coronavirus mRNA is synthesized by a discontinuous transcription process, which involves a free leader RNA species. As a result, each virus-specific mRNA contains an identical leader RNA derived from the 5', end of the genomic RNA. In this study, we demonstrate by primer extension studies that the leader-fusion sites on a given species of coronavirus subgenomic mRNA are heterogeneous. The heterogeneity was due to variation in the number of pentanucleotide (UCUAA) repeats present at the leader fusion site. This pentanucleotide repeat region was complementary between the free leader RNA and the transcription start sites on the template RNA. This result suggests that the discontinuous transcription of coronavirus mRNAs occurs within the complementary sequences localized in two different RNA segments and that RNA joining occurs at variable sites.

Human hepatitis delta antigen is a nuclear phosphoprotein with RNA-binding activity.

The genetic origin, structure, and biochemical properties of the delta antigen (HDAg) of a human hepatitis delta virus (HDV) were investigated. A cDNA fragment containing the open reading frame encoding the HDAg was transcribed into RNA and used for in vitro translation in rabbit reticulocyte lysates. The HDAg open reading frame was also inserted into an expression vector containing a simian virus 40 T-antigen promoter and expressed into COS 7 cells. In both systems, a protein species of 26 kilodaltons was synthesized from this open reading frame and could be specifically immunoprecipitated with antisera obtained from patients with delta hepatitis. A similar protein was also synthesized from antigenomic-sense monomeric HDV RNA in both systems, although the efficiency of translation was lower than that of the isolated open reading frame. This protein was found to be phosphorylated at the serine residues. Immunoperoxidase studies with anti-HDV sera demonstrated that the HDAg was expressed mainly in the nuclei of the transfected COS 7 cells. Moreover, the HDAg was shown to bind the genomic RNA of HDV. These studies indicate that HDAg is encoded by the antigenomic-sense RNA of HDV and is a nuclear phosphoprotein associated with an RNA-binding activity.

RNA recombination of murine coronaviruses: recombination between fusion-positive mouse hepatitis virus A59 and fusion-negative mouse hepatitis virus 2.

It has previously been shown that the murine coronavirus mouse hepatitis virus (MHV) undergoes RNA recombination at a relatively high frequency in both tissue culture and infected animals. Thus far, all of the recombination sites had been localized at the 5' half of the RNA genome. We have now performed a cross between MHV-2, a fusion-negative murine coronavirus, and a temperature-sensitive mutant of the A59 strain of MHV, which is fusion positive at the permissive temperature. By selecting fusion-positive viruses at the nonpermissive temperature, we isolated several recombinants containing multiple crossovers in a single genome. Some of the recombinants became fusion negative during the plaque purification. The fusion ability of the recombinants parallels the presence or absence of the A59 genomic sequences encoding peplomers. Several of the recombinants have crossovers within 3' end genes which encode viral structural proteins, N and E1. These recombination sites were not specifically selected with the selection markers used. This finding, together with results of previous recombination studies, indicates that RNA recombination can occur almost anywhere from the 5' end to the 3' end along the entire genome. The data also show that the replacement of A59 genetic sequences at the 5' end of gene C, which encodes the peplomer protein, with the fusion-negative MHV-2 sequences do not affect the fusion ability of the recombinant viruses. Thus, the crucial determinant for the fusion-inducing capability appears to reside in the more carboxyl portion of the peplomer protein.

Sequence and translation of the murine coronavirus 5'-end genomic RNA reveals the N-terminal structure of the putative RNA polymerase.

A 28-kilodalton protein has been suggested to be the amino-terminal protein cleavage product of the putative coronavirus RNA polymerase (gene A) (M.R. Denison and S. Perlman, Virology 157:565-568, 1987). To elucidate the structure and mechanism of synthesis of this protein, the nucleotide sequence of the 5' 2.0 kilobases of the coronavirus mouse hepatitis virus strain JHM genome was determined. This sequence contains a single, long open reading frame and predicts a highly basic amino-terminal region. Cell-free translation of RNAs transcribed in vitro from DNAs containing gene A sequences in pT7 vectors yielded proteins initiated from the 5'-most optimal initiation codon at position 215 from the 5' end of the genome. The sequence preceding this initiation codon predicts the presence of a stable hairpin loop structure. The presence of an RNA secondary structure at the 5' end of the RNA genome is supported by the observation that gene A sequences were more efficiently translated in vitro when upstream noncoding sequences were removed. By comparing the translation products of virion genomic RNA and in vitro transcribed RNAs, we established that our clones encompassing the 5'-end mouse hepatitis virus genomic RNA encode the 28-kilodalton N-terminal cleavage product of the gene A protein. Possible cleavage sites for this protein are proposed.

The 5'-end sequence of the murine coronavirus genome: implications for multiple fusion sites in leader-primed transcription.

The coronavirus leader-primed transcription model proposes that free leader RNA species derived from the 5'-end of the genomic RNA are utilized as a primer for the transcription of subgenomic mRNAs. To elucidate the precise mechanism of leader-priming, we cloned and sequenced the 5'-end of the mouse hepatitis virus genomic RNA. The 5'-terminal sequences are identical to the leader sequences present at the 5'-end of the subgenomic mRNAs. Two possible hairpin loop structures and an AU-rich region around the 3'-end of the leader sequence may provide the termination site for leader RNA synthesis. The comparison of 5'-end genomic sequences and the intergenic start sites for mRNA transcription revealed that there are homologous regions of 7-18 nucleotides at the putative leader/body junction sites. Some intergenic regions contain a mismatching nucleotide within this homologous region. We propose that free leader RNA binds to the intergenic region due to this homology and is cleaved at the mismatching nucleotide before serving as a primer. Thus, the free leader RNA species may be longer than the leader sequences in the subgenomic mRNAs and different mRNAs may have different leader/body junction sites.

Multiple recombination sites at the 5'-end of murine coronavirus RNA.

Mouse hepatitis virus (MHV), a murine coronavirus, contains a nonsegmented RNA genome. We have previously shown that MHV could undergo RNA-RNA recombination in crosses between temperature-sensitive mutants and wild-type viruses at a very high frequency (S. Makino, J.G. Keck, S.A. Stohlman, and M.M.C. Lai (1986) J. Virol. 57, 729-737). To better define the mechanism of RNA recombination, we have performed additional crosses involving different sets of MHV strains. Three or possibly four classes of recombinants were isolated. Recombinants in the first class, which are similar to the ones previously reported, contain a single crossover in either gene A or B, which are the 5'-most genes. The second class of recombinants contain double crossovers in gene A. The third class of recombinants have crossovers within the leader sequence located at the 5'-end of the genome. The crossover sites of the third class have been located between 35 and 60 nucleotides from the 5'-end of the leader RNA. One of these recombinants has double crossovers within the short region comprising the leader sequences. Finally, we describe one recombinant which may contain a triple crossover. The presence of so many recombination sites within the 5'-end of the genome of murine coronaviruses confirms that RNA recombination is a frequent event during MHV replication and is consistent with our proposed model of "copy-choice" recombination in which RNA replication occurs in a discontinuous and nonprocessive manner.