Characterization of a Truncated Metabotropic Glutamate Receptor in a Primitive Metazoan, the Parasitic Flatworm Schistosoma mansoni

A novel glutamate-binding protein was identified in Schistosoma mansoni. The protein (SmGBP) is related to metabotropic glutamate receptors from other species and has a predicted glutamate binding site located within a Venus Flytrap module but it lacks the heptahelical transmembrane segment that normally characterizes these receptors. The SmGBP cDNA was cloned, verified by 5′ and 3′ Rapid Amplification of cDNA Ends (RACE) and shown to be polyadenylated at the 3′end, suggesting the transcript is full-length. The cloned cDNA was subsequently expressed in bacteria and shown to encode a functional glutamate-binding protein. Other studies, using a specific peptide antibody, determined that SmGBP exists in two forms, a monomer of the expected size and a stable but non-covalent dimer. The monomer and dimer are both present in the membrane fraction of S. mansoni and are resistant to extraction with high-salt, alkaline pH and urea, suggesting SmGBP is either an integral membrane protein or a peripheral protein that is tightly associated with the membrane. Surface biotinylation experiments combined with western blot analyses and confocal immunolocalization revealed that SmGBP localized to the surface membranes of adult male schistosomes, especially the dorsal tubercles. In contrast, we detected little or no expression of SmGBP either in the females or larval stages. A comparative quantitative PCR analysis confirmed that the level of SmGBP expression is several-fold higher in male worms than cercariae, and it is barely detectable in adult females. Together, the results identify SmGBP as a new type of schistosome glutamate receptor that is both gender- and stage-specific. The high-level expression of this protein in the male tubercles suggests a possible role in host-parasite interaction

A Novel G Protein-Coupled Receptor of Schistosoma mansoni (SmGPR-3) Is Activated by Dopamine and Is Widely Expressed in the Nervous System

Schistosomes have a well developed nervous system that coordinates virtually every activity of the parasite and therefore is considered to be a promising target for chemotherapeutic intervention. Neurotransmitter receptors, in particular those involved in neuromuscular control, are proven drug targets in other helminths but very few of these receptors have been identified in schistosomes and little is known about their roles in the biology of the worm. Here we describe a novel Schistosoma mansoni G protein-coupled receptor (named SmGPR-3) that was cloned, expressed heterologously and shown to be activated by dopamine, a well established neurotransmitter of the schistosome nervous system. SmGPR-3 belongs to a new clade of “orphan” amine-like receptors that exist in schistosomes but not the mammalian host. Further analysis of the recombinant protein showed that SmGPR-3 can also be activated by other catecholamines, including the dopamine metabolite, epinine, and it has an unusual antagonist profile when compared to mammalian receptors. Confocal immunofluorescence experiments using a specific peptide antibody showed that SmGPR-3 is abundantly expressed in the nervous system of schistosomes, particularly in the main nerve cords and the peripheral innervation of the body wall muscles. In addition, we show that dopamine, epinine and other dopaminergic agents have strong effects on the motility of larval schistosomes in culture. Together, the results suggest that SmGPR-3 is an important neuronal receptor and is probably involved in the control of motor activity in schistosomes. We have conducted a first analysis of the structure of SmGPR-3 by means of homology modeling and virtual ligand-docking simulations. This investigation has identified potentially important differences between SmGPR-3 and host dopamine receptors that could be exploited to develop new, parasite-selective anti-schistosomal drugs

Impact of opioid-free analgesia on pain severity and patient satisfaction after discharge from surgery: multispecialty, prospective cohort study in 25 countries

Background: Balancing opioid stewardship and the need for adequate analgesia following discharge after surgery is challenging. This study aimed to compare the outcomes for patients discharged with opioid versus opioid-free analgesia after common surgical procedures.Methods: This international, multicentre, prospective cohort study collected data from patients undergoing common acute and elective general surgical, urological, gynaecological, and orthopaedic procedures. The primary outcomes were patient-reported time in severe pain measured on a numerical analogue scale from 0 to 100% and patient-reported satisfaction with pain relief during the first week following discharge. Data were collected by in-hospital chart review and patient telephone interview 1 week after discharge.Results: The study recruited 4273 patients from 144 centres in 25 countries; 1311 patients (30.7%) were prescribed opioid analgesia at discharge. Patients reported being in severe pain for 10 (i.q.r. 1-30)% of the first week after discharge and rated satisfaction with analgesia as 90 (i.q.r. 80-100) of 100. After adjustment for confounders, opioid analgesia on discharge was independently associated with increased pain severity (risk ratio 1.52, 95% c.i. 1.31 to 1.76; P < 0.001) and re-presentation to healthcare providers owing to side-effects of medication (OR 2.38, 95% c.i. 1.36 to 4.17; P = 0.004), but not with satisfaction with analgesia (beta coefficient 0.92, 95% c.i. -1.52 to 3.36; P = 0.468) compared with opioid-free analgesia. Although opioid prescribing varied greatly between high-income and low- and middle-income countries, patient-reported outcomes did not.Conclusion: Opioid analgesia prescription on surgical discharge is associated with a higher risk of re-presentation owing to side-effects of medication and increased patient-reported pain, but not with changes in patient-reported satisfaction. Opioid-free discharge analgesia should be adopted routinely

Burnout among surgeons before and during the SARS-CoV-2 pandemic: an international survey

Background: SARS-CoV-2 pandemic has had many significant impacts within the surgical realm, and surgeons have been obligated to reconsider almost every aspect of daily clinical practice. Methods: This is a cross-sectional study reported in compliance with the CHERRIES guidelines and conducted through an online platform from June 14th to July 15th, 2020. The primary outcome was the burden of burnout during the pandemic indicated by the validated Shirom-Melamed Burnout Measure. Results: Nine hundred fifty-four surgeons completed the survey. The median length of practice was 10 years; 78.2% included were male with a median age of 37 years old, 39.5% were consultants, 68.9% were general surgeons, and 55.7% were affiliated with an academic institution. Overall, there was a significant increase in the mean burnout score during the pandemic; longer years of practice and older age were significantly associated with less burnout. There were significant reductions in the median number of outpatient visits, operated cases, on-call hours, emergency visits, and research work, so, 48.2% of respondents felt that the training resources were insufficient. The majority (81.3%) of respondents reported that their hospitals were included in the management of COVID-19, 66.5% felt their roles had been minimized; 41% were asked to assist in non-surgical medical practices, and 37.6% of respondents were included in COVID-19 management. Conclusions: There was a significant burnout among trainees. Almost all aspects of clinical and research activities were affected with a significant reduction in the volume of research, outpatient clinic visits, surgical procedures, on-call hours, and emergency cases hindering the training. Trial registration: The study was registered on clicaltrials.gov "NCT04433286" on 16/06/2020

Characterization of novel glutamate and dopamine neurotransmitter receptors in the bloodfluke Schistosoma mansoni

Schistosomes are among the most complex parasites affecting humans. They are dioecious parasites with an indirect life cycle that requires two hosts, a snail intermediate host and a human definitive host. The complexity of the life cycle is made possible, in part by the parasite's nervous system, which coordinates behavior and all major physiological activities of the worm. Neuronal signaling in schistosomes is mediated by a variety of neurotransmitters and associated receptors. Among these transmitters are dopamine and the neuroactive amino acid, glutamate. Both have been implicated in the control of neuromuscular function and movement but their mode of action is largely unknown. Here we provide the first molecular evidence for the existence of dopamine and glutamate receptors in Schistosoma mansoni. Two glutamate receptors were identified: One (SmGluR) is distantly related to heptahelical (metabotropic) glutamate receptors from other species and the other (SmGBP) is an unusual, truncated receptor that resembles the extracellular binding domain of glutamate receptors but lacks the remaining heptahelical transmembrane sequence. When expressed heterologously in vitro, SmGluR and SmGBP were both responsive to glutamate but did not recognize many classical (mammalian) glutamate agonists and antagonists, suggesting these are novel receptors with different pharmacological properties. Immunolocalization studies revealed that the two glutamate receptors have different tissue distributions. SmGluR is strongly expressed in neurons of the central and peripheral nervous system, including the peripheral innervation of the body wall muscles and the acetabulum, and it is also present in the female reproductive tract. In contrast, SmGBP is male-specific and was found only in the tegument, particularly the tubercles. The third receptor described in this thesis, SmD2, is structurally related to dopaminergic type 2 receptors and it is selectively activated by dopamine in vitro. SmD2 is localized entirely in the body wall musculature of both male and female worms and larvae. Together these results suggest that SmD2 and SmGluR could play important roles in the control of schistosome movement, SmD2 by targeting the musculature directly, whereas SmGluR works indirectly via the innervation of the musculature. The results also identify novel functions for glutamate receptors in the control of the acetabulum (SmGluR), egg production in females (SmGluR) and the tubercles of the male tegument (SmGBP). The widespread distribution of these receptors, combined with their unusual structures and pharmacological profiles, make them promising targets for discovery of new anti-schistosomal drugs.Schistosoma est une des espèces les plus complexes de parasites humains. Il est dioïque et requiert un cycle de vie indirect : un hôte intermédiaire, l'escargot, et un hôte définitif, l'homme. Le système nerveux du parasite contrôle le comportement et toutes les activités physiologiques importantes et est en grande partie responsable de la complexité du cycle de vie du parasite. La signalisation neuronale des schistosomes est médiée par une variété de neurotransmetteurs et récepteurs correspondants, dont la dopamine et le glutamate. Ces transmetteurs régissent en partie les fonctions neuromusculaires et le mouvement, mais leur mode d'action demeure inconnu. Cette étude divulgue l'existence de récepteurs de la dopamine et du glutamate chez Schistosoma mansoni. Deux récepteurs de la dopamine ont été identifiés : SmGluR est indirectement associé au domaine heptahélice (métabotropique) des récepteurs du glutamate chez d'autres espèces, et SmGBP est un récepteur tronqué qui ressemble au domaine de liaison extracellulaire des récepteurs du glutamate dont la séquence transmembranaire heptahélice est absente. SmGluR et SmGBP sont tout deux sensibles au glutamate lorsqu'exprimés in vitro de façon hétérologue, mais ne reconnaissent pas plusieurs agonistes et antagonistes classiques (mammaliens) du glutamate. Cela suggère que ce sont de nouveaux récepteurs ayant des propriétés pharmacologiques différentes. Ces deux récepteurs du glutamate ont une expression tissulaire différente : SmGluR est fortement exprimé dans les neurones du système nerveux central et périphérique, et l'innervation périphérique des muscles de la paroi corporelle et de l'acétabulum, ainsi que dans le système reproductif de la femelle. Par contre, SmGBP est spécifique aux mâles et ne s'exprime que dans le tégument, particulièrement les tubercules. Le troisième récepteur identifié, SmD2, est structurellement lié aux récepteurs dopaminergiques de type 2, et sélectivement activé in vitro par la dopamine. SmD2 est uniquement retrouvé dans la musculature de la paroi corporelle chez les vers adultes et les larves des deux sexes. SmD2 et SmGluR semblerait donc important quant à la mobilité des schistosomes. SmD2 ciblerait la musculature directement, et SmGluR agirait indirectement sur l'innervation musculaire. Ces résultats définissent aussi de nouvelles fonctions des récepteurs du glutamate : le contrôle de l'acétabulum (SmGluR), la production des œufs chez les femelles (SmGluR) et des tubercules du tégument chez les mâles (SmGBP). La grande distribution de ces récepteurs combinée à leurs structures et profils pharmacologiques inhabituels en font des cibles prometteuses quant au développement de nouvelles thérapies contre la bilharziose

Immunolocalization of SmGPR-3 in larval <i>Schistosoma mansoni</i>.

<p><i>S. mansoni</i> cercaria were probed with affinity purified anti-SmGPR-3 antibody, followed by fluorescein isothiocyanate (FITC)-labelled secondary antibody. (A) Immunoreactivity (green) can be seen along the major longitudinal nerve cords (solid arrowheads) and in transverse commissures (open arrowhead), including the posterior transverse commissure near the base of the tail (open arrow). (B) No significant immunoreactivity was observed in negative controls probed with anti-SmGPR-3 antibody that was pre-adsorbed with peptide antigens or (C) controls probed with secondary antibody only. (*) non-specific labelling.</p

Sequence alignment of dopaminergic G protein-coupled receptors with <i>Schistosoma mansoni</i> SmGPR receptors.

<p>A ClustalW alignment was performed using representative examples of vertebrate dopaminergic GPCRs (D1–D5), the <i>S. mansoni</i> dopamine D2-like receptor (SmD2) and several members of the SmGPR clade. SmGPR sequences are boxed (horizontal box) and SmGPR-3 is marked by an arrow. Receptor sequences are identified by their accession numbers (brackets). The positions of the predicted seven transmembrane domains are marked by horizontal lines and the invariant residue in each TM segment <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001523#pntd.0001523-Ballesteros1" target="_blank">[37]</a> is identified by an asterisk (*) Other conserved residues of functional relevance are marked by circles (•) and conserved motifs are boxed (vertical boxes). Residues discussed in this study, R^2.64 (Arg96), D^3.32 (Asp117), S^5.42 (Ser198), T^7.39 (Thr462) and Y^7.43 (Tyr466) are identified by vertical arrows.</p

Effects of dopamine and related substances on schistosome motility.

<p>(A) <i>In vitro</i> transformed 3-day-old schistosomula were incubated with test drug, dopamine (DA) or epinine (EPN), each at (10⁻⁴ M) or vehicle (CT, control). Animals were treated for 5 min at room temperature, after which they were examined with a compound microscope equipped with a digital video camera and SimplePCI (Compix Inc.) for image acquisition. Images were recorded for 1 minute (∼3 frames/second) and an estimate of body length in µm was obtained for each animal in every frame. Each tracing shown is of an individual animal and is representative of 12–15 larvae per experiment and 3–4 independent experiments per treatment. (B) Experiments were repeated with various concentrations of test agonist in a range of 10⁻⁷ M–10⁻⁴ M, or in the absence of test substance (CT, control). Images were recorded as above and body length was measured for each frame. Motility is defined as the frequency of length changes (shortening and elongation) per minute of observation, as described in the Methods. The data are presented as the means and SEM of three separate experiments each with 12–15 animals. (C) Schistosomula were treated with test substances at a single concentration or in the absence of drug (CT, control) and motility was measured as above. Dopamine (DA), epinine (EPN), flupenthixol (FLPX), promethazine (PRMZ) were each tested at 50 µM. The remaining substances, adenaline (A), metanephrine (MTN) and haloperidol (HLRD) were tested at 500 µM. The data are the means and SEM of three separate experiments each with 12–15 animals. * Significantly different from the no drug control at P<0.05.</p

Functional expression of the <i>Schistosoma mansoni</i> SmGPR-3 receptor in yeast.

<p>(A) The full-length SmGPR-3 cDNA was expressed in <i>Saccharomyces cerevisae</i> strain YEX108 and grown in selective leu/histidine-deficient (leu⁻/his⁻) medium containing 2×10⁻⁴ M of each biogenic amine or vehicle (no drug control, ND). Yeast cells transformed with empty plasmid were used as a negative control (mock). Receptor activation was quantified from measurements of yeast growth in relative fluorescence units (RFU), using an Alamar blue fluorescence assay. The results are the means ± S.E.M. of 5–6 independent clones, each assayed in triplicate. The following biogenic amines were tested: adrenaline (A), noradrenaline (NA), dopamine (DA), epinine (EPN), serotonin (5-hydroxytryptamine, 5HT), octopamine (OA), tyramine (TA) and histamine (HA). (B) Functional assays were repeated with the same SmGPR-3-expressing yeast strain and variable concentrations of DA (△) or EPN (□). The mock control was tested with DA (•). EC₅₀ values for DA and EPN are 3.10×10⁻⁵ M and 2.85×10⁻⁵ M, respectively. The data are the means ± S.E.M. of two experiments, each in triplicate.</p