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Abstract Since its emergence, SARS-CoV-2 Omicron clade has shown a marked degree of variability and different clinical presentation compared with previous clades. Here we demonstrate that at least four Omicron lineages circulated in children since December 2021, and studied until November 2022: BA.1 (33.6%), BA.2 (40.6%), BA.5 (23.7%) and BQ.1 (2.1%). At least 70% of infections concerned children under 1 year, most of them being infected with BA.2 lineages (n = 201, 75.6%). Looking at SARS-CoV-2 genetic variability, 69 SNPs were found to be significantly associated in pairs, (phi 0.3 and p-value 0.75). One of these clusters (A23040G, A27259C, T23617G, T23620G) was also positively associated with moderate/severe COVID-19 presentation (AOR [95% CI] 2.49 [1.26–4.89] p-value: 0.008) together with comorbidities (AOR [95% CI] 2.67 [1.36–5.24] p-value: 0.004). Overall, these results highlight the extensive SARS-CoV-2 Omicron circulation in children, mostly aged

Abstract The chemokine receptor CCR5 is known to exist in cell surface subpopulations that differ in their capacity to engage ligands. One proposed explanation for this phenomenon is the presence of CCR5 species with different levels of post-translational modifications (PTMs). Tyrosine sulfation and O-glycan sialylation are PTMs that add negative charges to the extracellular domain of CCR5 and make strong contributions to chemokine binding but it is not known whether cellular mechanisms to control their levels exist. In this study we used a combination of sulfation-sensitive and sulfation-insensitive CCR5 ligands to show that the rate of turnover of CCR5 tyrosine sulfation is more rapid than the rate of turnover of the receptor itself. This suggests that the steady state level of CCR5 sulfation is maintained through the combination of tyrosine protein sulfotransferase (TPST), the trans-Golgi network (TGN)-resident ‘source enzyme, and a ‘sink’ activity that removes tyrosine sulfation from CCR5. By measuring the effects on ligand binding of knockdown and overexpression experiments, we provided evidence that non-lysosomal cellular arylsulfatases, particularly ARSG, ARSI and ARSJ, are CCR5 sulfation ‘sink’ enzymes. We also used targeted knockdown and sialylation-sensitive and insensitive chemokines to identify the sialidase NEU3 as a candidate ‘sink’ enzyme for CCR5 O-glycan sialylation. This study provides the first experimental evidence of activity of sulfatase and sialidase ‘sink’ enzymes on CCR5, providing a potential mechanism for cells to control steady-state levels of these PTMs and thereby exert dynamic control over receptor-ligand interactions at the cell surface and during receptor desensitization.

The coronavirus disease (COVID-19) pandemic has spread in the US with more than 100 million human infections and a million deaths since March 2020. A population of concern are high-risk individuals such as adults who are tobacco users, since COVID-19 is a respiratory disease that affects the lungs. Although 79% of the US population ≥ 18 years of age have completed the primary COVID-19 vaccine series; it is imperative to understand the factors associated with receiving or declining the COVID-19 vaccine among high-risk populations to improve vaccination rates. Guided by the diffusion of innovations (DOI) theory, this study identified factors associated with COVID-19 vaccination and the impact on COVID-19 vaccine uptake in adults who use tobacco. We conducted a cross-sectional study using a sample of Pennsylvanian adult tobacco users by sending a unique survey link to 4,081 email addresses in April 2022. Participants were asked about tobacco use, COVID-19 vaccination status, and reasons for receiving/declining the COVID-19 vaccine. Participants (n = 157) were 75% female, 96% White, 74% current tobacco users, and had a mean age of 50.1 (SD = 10.8) years. Nearly 78% (n = 119) received at least one dose of the COVID-19 vaccine (primary series). We categorized all vaccinated tobacco users into adopter categories of the DOI theory; innovators (10%), early adopters (14%), early majority (33%), late majority (11%), and laggards (32%). The major reason that prompted participants to get the COVID-19 vaccine was to ensure they were well protected against COVID-19 infection (77%). Additionally, the only reason for receiving the vaccine that significantly predicted early vaccine uptake (being an innovator or early adopter) was "to loosen restrictions on mask mandates and social/physical distancing" (p = 0.0180). Among the 22% that did not receive a COVID-19 vaccine, the most common major reason they declined the vaccine was because they felt politics played a big role in the vaccine development process (94%). Our findings suggest that major f actors that influenced why adult tobacco users would receive or decline the COVID-19 vaccine included infection control mandates, protection from the COVID-19 infection, and politics. Investigating these factors can help public health professionals design or develop future vaccination programs for high-risk populations in order to scale up vaccination rates.

Summary: An efficient neutrophil response is critical for fighting bacterial infections, which remain a significant global health concern; therefore, modulating neutrophil function could be an effective therapeutic approach. While we have a general understanding of how neutrophils respond to bacteria, how neutrophil function differs in response to diverse bacterial infections remains unclear. Here, we use a microfluidic infection-on-a-chip device to investigate the neutrophil response to four bacterial species: Pseudomonas aeruginosa, Salmonella enterica, Listeria monocytogenes, and Staphylococcus aureus. We find enhanced neutrophil extravasation to L. monocytogenes, a limited overall response to S. aureus, and identify IL-6 as universally important for neutrophil extravasation. Furthermore, we demonstrate a higher percentage of neutrophils generate reactive oxygen species (ROS) when combating gram-negative bacteria versus gram-positive bacteria. For all bacterial species, we found the percentage of neutrophils producing ROS increased following extravasation through an endothelium, underscoring the importance of studying neutrophil function in physiologically relevant models.

Extracellular vesicles released by the primary pathogen of periodontal disease Porphyromonas gingivalis (Pg), referred to as outer membrane vesicles (OMVs), have been associated with the pathogenesis of systemic diseases like cardiovascular disease, rheumatoid arthritis, and Alzheimer’s disease. A pathogenic role for Pg by disrupting placental homeostasis was proposed in the association between periodontal disease and adverse pregnancy outcomes. On the basis that trophoblast-derived factors modulate endothelial and immune cell profiles in normal pregnancy and the scarce presence of Pg in placenta, we hypothesized that OMVs from Pg affect trophoblast cell phenotype, impairing trophoblast–endothelium and trophoblast–neutrophil interactions. By means of in vitro designs with first-trimester human trophoblast cells, endothelial cells, and freshly isolated neutrophils, we showed that Pg OMVs are internalized by trophoblast cells and modulate the activity and expression of functional markers. Trophoblast cells primed with Pg OMVs enhanced neutrophil chemoattraction and lost their anti-inflammatory effect. In addition, reduced migration with enhanced adhesion of monocytes was found in endothelial cells upon incubation with the media from trophoblast cells pretreated with Pg OMVs. Taken together, the results support a pathogenic role of Pg OMVs at early stages of pregnancy and placentation through disruption of trophoblast contribution to vascular transformation and immune homeostasis maintenance.

Low complexity regions (LCRs) play a role in a variety of important biological processes, yet we lack a unified view of their sequences, features, relationships, and functions. Here, we use dotplots and dimensionality reduction to systematically define LCR type/copy relationships and create a map of LCR sequence space capable of integrating LCR features and functions. By defining LCR relationships across the proteome, we provide insight into how LCR type and copy number contribute to higher order assemblies, such as the importance of K-rich LCR copy number for assembly of the nucleolar protein RPA43 in vivo and in vitro. With LCR maps, we reveal the underlying structure of LCR sequence space, and relate differential occupancy in this space to the conservation and emergence of higher order assemblies, including the metazoan extracellular matrix and plant cell wall. Together, LCR relationships and maps uncover and identify scaffold-client relationships among E-rich LCR-containing proteins in the nucleolus, and revealed previously undescribed regions of LCR sequence space with signatures of higher order assemblies, including a teleost-specific T/H-rich sequence space. Thus, this unified view of LCRs enables discovery of how LCRs encode higher order assemblies of organisms.

Abstract This study aimed to characterize amino acid sensing systems in the gastrointestinal tract (GIT) of the carnivorous fish model species rainbow trout. We observed that the trout GIT expresses mRNAs encoding some amino acid receptors described in mammals [calcium-sensing receptor (CaSR), G protein-coupled receptor family C group 6 member A (GPRC6A), and taste receptors type 1 members 1 and 2 (T1r1, T1r2)], while others [taste receptor type 1 member 3 (T1r3) and metabotropic glutamate receptors 1 and 4 (mGlur1, mGlur4)] could not be found. Then, we characterized the response of such receptors, as well as that of intracellular signaling mechanisms, to the intragastric administration of l-leucine, l-valine, l-proline or l-glutamate. Results demonstrated that casr, gprc6a, tas1r1 and tas1r2 mRNAs are modulated by amino acids in the stomach and proximal intestine, with important differences with respect to mammals. Likewise, gut amino acid receptors triggered signaling pathways likely mediated, at least partly, by phospholipase C β3 and β4. Finally, the luminal presence of amino acids led to important changes in ghrelin, cholecystokinin, peptide YY and proglucagon mRNAs and/or protein levels. Present results offer the first set of evidence in favor of the existence of amino acid sensing mechanisms within the fish GIT.

Sepsis is a dysregulated host response to infection that can cause widespread effects on other organs including cardiovascular depression, hypotension and organ failure. The receptor for Nociceptin/Orphanin FQ (N/OFQ), NOP is expressed on immune cells and these cells can release the peptide. Exogenous N/OFQ can dilate blood vessels and this peptide is increased in animal and human sepsis. We hypothesise that NOP receptors are present on vascular endothelial cells and therefore provide the target for released N/OFQ to cause vasodilation and hence hypotension. Using human umbilical vein endothelial cells (HUVEC) and human vascular smooth muscle cells (HVSMC) freshly prepared from umbilical cords and up to passage 4, we assessed NOP mRNA expression by Polymerase Chain Reaction (PCR), NOP surface receptor expression using a fluorescent NOP selective probe (N/OFQATTO594) and NOP receptor function with N/OFQ stimulated ERK1/2 phosphorylation. As an in vitro sepsis mimic we variably incubated cells with 100ng/ml Lipopolysaccharide and Peptidoglycan G (LPS/PepG). HUVECs express NOP mRNA and this was reduced by ~80% (n = 49) after 24–48 hours treatment with LPS/PepG. Untreated cells do not express surface NOP receptors but when treated with LPS/PepG the reduced mRNA was translated into protein visualised by N/OFQATTO594 binding (n = 49). These NOP receptors in treated cells produced an N/OFQ (1μM) driven increase in ERK1/2 phosphorylation (n = 20). One (of 50) HUVEC lines expressed NOP mRNA and receptor protein in the absence of LPS/PepG treatment. In contrast, HVSMC expressed NOP mRNA and surface receptor protein (n = 10) independently of LPS/PepG treatment. These receptors were also coupled to ERK1/2 where N/OFQ (1μM) increased phosphorylation. Collectively these data show that an in vitro sepsis mimic (LPS/PepG) upregulates functional NOP expression in the vascular endothelium. Activation of these endothelial receptors as suggested from in vivo whole animal work may contribute to the hypotensive response seen in sepsis. Moreover, blockade of these receptors might be a useful adjunct in the treatment of sepsis.

Sepsis is a dysregulated host response to infection that can cause widespread effects on other organs including cardiovascular depression, hypotension and organ failure. The receptor for Nociceptin/Orphanin FQ (N/OFQ), NOP is expressed on immune cells and these cells can release the peptide. Exogenous N/OFQ can dilate blood vessels and this peptide is increased in animal and human sepsis. We hypothesise that NOP receptors are present on vascular endothelial cells and therefore provide the target for released N/OFQ to cause vasodilation and hence hypotension. Using human umbilical vein endothelial cells (HUVEC) and human vascular smooth muscle cells (HVSMC) freshly prepared from umbilical cords and up to passage 4, we assessed NOP mRNA expression by Polymerase Chain Reaction (PCR), NOP surface receptor expression using a fluorescent NOP selective probe (N/OFQATTO594) and NOP receptor function with N/OFQ stimulated ERK1/2 phosphorylation. As an in vitro sepsis mimic we variably incubated cells with 100ng/ml Lipopolysaccharide and Peptidoglycan G (LPS/PepG). HUVECs express NOP mRNA and this was reduced by ~80% (n = 49) after 24-48 hours treatment with LPS/PepG. Untreated cells do not express surface NOP receptors but when treated with LPS/PepG the reduced mRNA was translated into protein visualised by N/OFQATTO594 binding (n = 49). These NOP receptors in treated cells produced an N/OFQ (1μM) driven increase in ERK1/2 phosphorylation (n = 20). One (of 50) HUVEC lines expressed NOP mRNA and receptor protein in the absence of LPS/PepG treatment. In contrast, HVSMC expressed NOP mRNA and surface receptor protein (n = 10) independently of LPS/PepG treatment. These receptors were also coupled to ERK1/2 where N/OFQ (1μM) increased phosphorylation. Collectively these data show that an in vitro sepsis mimic (LPS/PepG) upregulates functional NOP expression in the vascular endothelium. Activation of these endothelial receptors as suggested from in vivo whole animal work may contribute to the hypotensive response seen in sepsis. Moreover, blockade of these receptors might be a useful adjunct in the treatment of sepsis.

Introduction Little data exists from sub-Saharan Africa describing incidence and outcomes of sepsis in emergency units and uncertainty exists surrounding optimal management of sepsis in low-income settings. There exists limited data regarding quality care metrics for non-physician clinicians trained in emergency care. The objective of this study was to describe changes in septic patients over time and evaluate associations between sepsis care and mortality. Methods Secondary analysis of a prospective cohort of all consecutive patients seen from 2010–2019 in a rural Ugandan emergency unit staffed by non-physician clinicians was performed using an electronic database based on paper charts. Sepsis was defined as suspected infection with a quick Sequential Organ Failure Assessment score (qSOFA)≥1. Multi-variable logistic regression was used to analyze three-day mortality. Results Overall, 48,653 patient visits from 2010–2019 yielded 17,490 encounters for patients age≥18 who had suspected infection, including 10,437 with sepsis. The annual proportion of patients with sepsis decreased from 45.0%% to 21.3% and the proportion with malarial sepsis decreased from 17.7% to 2.1% during the study period. Rates of septic patients receiving quality care (“both fluids and anti-infectives”) increased over time (21.2% in 2012 to 32.0% in 2019, pConclusion The largest study of sepsis management and outcomes ever published in both Uganda and sub-Saharan Africa showed sepsis and malarial sepsis decreasing from 2010 to 2019. The increasing quality of non-physician clinician care did not significantly reduce mortality and treatment with “both fluids and antibiotics” increased mortality. With causal associations between antibiotics and mortality deemed implausible, associations between sepsis mortality and interventions likely represent confounding by indication. Defining optimal sepsis care regionally will likely require randomized controlled trials.

IntroductionLittle data exists from sub-Saharan Africa describing incidence and outcomes of sepsis in emergency units and uncertainty exists surrounding optimal management of sepsis in low-income settings. There exists limited data regarding quality care metrics for non-physician clinicians trained in emergency care. The objective of this study was to describe changes in septic patients over time and evaluate associations between sepsis care and mortality.MethodsSecondary analysis of a prospective cohort of all consecutive patients seen from 2010-2019 in a rural Ugandan emergency unit staffed by non-physician clinicians was performed using an electronic database based on paper charts. Sepsis was defined as suspected infection with a quick Sequential Organ Failure Assessment score (qSOFA)≥1. Multi-variable logistic regression was used to analyze three-day mortality.ResultsOverall, 48,653 patient visits from 2010-2019 yielded 17,490 encounters for patients age≥18 who had suspected infection, including 10,437 with sepsis. The annual proportion of patients with sepsis decreased from 45.0%% to 21.3% and the proportion with malarial sepsis decreased from 17.7% to 2.1% during the study period. Rates of septic patients receiving quality care ("both fluids and anti-infectives") increased over time (21.2% in 2012 to 32.0% in 2019, pConclusionThe largest study of sepsis management and outcomes ever published in both Uganda and sub-Saharan Africa showed sepsis and malarial sepsis decreasing from 2010 to 2019. The increasing quality of non-physician clinician care did not significantly reduce mortality and treatment with "both fluids and antibiotics" increased mortality. With causal associations between antibiotics and mortality deemed implausible, associations between sepsis mortality and interventions likely represent confounding by indication. Defining optimal sepsis care regionally will likely require randomized controlled trials.

The μ-opioid peptide (MOP) receptor is a member of the opioid receptor family and an important clinical target for analgesia. Measuring MOP receptor location and tracking its turnover traditionally used radiolabels or antibodies with attendant problems of utility of radiolabels in whole cells and poor antibody selectivity. To address these issues we have synthesized and characterised a novel ATTO488 based fluorescent Dermorphin analogue; [Cys(ATTO 488)8]Dermorphin-NH2 (DermATTO488). We initially assessed the binding profile of DermATTO488 in HEK cells expressing human MOP and CHO cells expressing human MOP, δ-opioid peptide (DOP), κ-opioid peptide (KOP) and Nociceptin/Orphanin FQ peptide (NOP) receptors using radioligand binding. Functional activity of the conjugated peptide was assessed by measuring (i) the ability of the ligand to engage G-protein by measuring the ability to stimulate GTPγ[35S] binding and (ii) the ability to stimulate phosphorylation of ERK1/2. Receptor location was visualised using confocal scanning laser microscopy. Dermorphin and DermATTO488 bound to HEKMOP (pKi: 8.29 and 7.00; p0.05). Both ligands were inactive at KOP and NOP. Dermorphin and DermATTO488 stimulated the binding of GTPγ[35S] with similar pEC50 (7.84 and 7.62; p>0.05) and Emax (1.52 and 1.34fold p>0.05) values. Moreover, Dermorphin and DermATTO488 produced a monophasic stimulation of ERK1/2 phosphorylation peaking at 5mins (6.98 and 7.64-fold; p>0.05). Finally, in confocal microscopy DermATTO488 bound to recombinant MOP receptors on CHO and HEK cells in a concentration dependent manner that could be blocked by pre-incubation with unlabelled Dermorphin or Naloxone. Collectively, addition to ATTO488 to Dermorphin produced a ligand not dissimilar to Dermorphin; with ~10fold selectivity over DOP. This new ligand DermATTO488 retained functional activity and could be used to visualise MOP receptor location.

BackgroundThe early use of broad-spectrum antibiotics remains the cornerstone for the treatment of neonatal late onset sepsis (LOS). However, which antibiotics should be used is still debatable, as relevant studies were conducted more than 20 years ago, recruited in single centres or countries, evaluated antibiotics not in clinical use anymore and had variable inclusion/exclusion criteria and outcome measures. Moreover, antibiotic-resistant bacteria have become a major problem in many countries worldwide. We hypothesized that efficacy of meropenem as a broad-spectrum antibiotic is superior to standard of care regimens (SOC) in empiric treatment of LOS and aimed to compare meropenem to SOC in infants aged Methods and findingsNeoMero-1 was a randomized, open-label, phase III superiority trial conducted in 18 neonatal units in 6 countries. Infants with post-menstrual age (PMA) of ≤44 weeks with positive blood culture and one, or those with negative culture and at least with two predefined clinical and laboratory signs suggestive of LOS, or those with PMA >44 weeks meeting the Goldstein criteria of sepsis, were randomized in a 1:1 ratio to receive meropenem or one of the two SOC regimens (ampicillin+gentamicin or cefotaxime+gentamicin) chosen by each site prior to the start of the study for 8-14 days. The primary outcome was treatment success (survival, no modification of allocated therapy, resolution/improvement of clinical and laboratory markers, no need of additional antibiotics and presumed/confirmed eradication of pathogens) at test-of-cure visit (TOC) in full analysis set. Stool samples were tested at baseline and Day 28 for meropenem-resistant Gram-negative organisms (CRGNO). The primary analysis was performed in all randomised patients and in patients with culture confirmed LOS. Proportions of participants with successful outcome were compared by using a logistic regression model adjusted for the stratification factors. From September 3, 2012 to November 30th 2014, total of 136 patients (instead of planned 275) in each arm were randomized; 140 (52%) were culture positive. Successful outcome at TOC was achieved in 44/136 (32%) in the meropenem arm vs. 31/135 (23%) in the SOC arm (p = 0.087). The respective numbers in patients with positive cultures were 17/63 (27%) vs. 10/77 (13%) (p = 0.022). The main reason of failure was modification of allocated therapy. Treatment emergent adverse events occurred in 72% and serious adverse events in 17% of patients, the Day 28 mortality was 6%. Cumulative acquisition of CRGNO by Day 28 occurred in 4% of patients in the meropenem and 12% in the SOC arm (p = 0.052).ConclusionsWithin this study population, we found no evidence that meropenem was superior to SOC in terms of success at TOC, short term hearing disturbances, safety or mortality were similar in both treatment arms but the study was underpowered to detect the planned effect. Meropenem treatment did not select for colonization with CRGNOs. We suggest that meropenem as broad-spectrum antibiotic should be reserved for neonates who are more likely to have Gram-negative LOS, especially in NICUs where microorganisms producing extended spectrum- and AmpC type beta-lactamases are circulating.

Abstract A diastereoselective synthesis of the title compound as a single E diastereomer has been efficiently accomplished by assembling the featured pyrano-indole scaffold of the spiro[cyclohexane-dihydropyrano[3,4-b]-indole]-amine framework through an oxa-Pictet-Spengler reaction, promoted by a cheap and green Zeolite catalyst. Basic pharmacological experiments demonstrate that Cebranopadol acts as a mixed nociception/orphanin FQ (NOP) and mu (MOP) opioid receptor agonist useful for treatment of chronic pain.

The Nociceptin/Orphanin FQ (N/OFQ) peptide NOP receptor is coupled to pertussis toxin (PTX)-sensitive G proteins (Gi/o) whose activation leads to the inhibition of both cAMP production and calcium channel activity, and to the stimulation of potassium currents. The label free dynamic mass redistribution (DMR) approach has been demonstrated useful for investigating the pharmacological profile of G protein-coupled receptors. Herein, we employ DMR technology to systematically characterize the pharmacology of a large panel of NOP receptor ligands. These are of peptide and non-peptide nature and display varying degrees of receptor efficacy, ranging from full agonism to pure antagonism. Using Chinese hamster ovary (CHO) cells expressing the human NOP receptor we provide rank orders of potency for full and partial agonists as well as apparent affinities for selective antagonists. We find the pharmacological profile of NOP receptor ligands to be similar but not identical to values reported in the literature using canonical assays for Gi/o-coupled receptors. Our data demonstrate that holistic label-free DMR detection can be successfully used to investigate the pharmacology of the NOP receptor and to characterize the cellular effects of novel NOP receptor ligands.

The abuse of anabolic androgenic steroids (AAS) has been considered a major public health problem during decades. Supraphysiological doses of AAS may lead to a variety of neuroendocrine problems. Precisely, the hypothalamic-pituitary-gonadal (HPG) axis is one of the body systems that is mainly influenced by steroidal hormones. Fluctuations of the hormonal milieu result in alterations of reproductive function, which are made through changes in hypothalamic neurons expressing gonadotropin-releasing hormone (GnRH). In fact, previous studies have shown that AAS modulate the activity of these neurons through steroid-sensitive afferents. To increase knowledge about the cellular mechanisms induced by AAS in GnRH neurons, we performed proteomic analyses of the murine hypothalamic GT1-7 cell line after exposure to 17α-methyltestosterone (17α-meT; 1 μM). These cells represent a good model for studying regulatory processes because they exhibit the typical characteristics of GnRH neurons, and respond to compounds that modulate GnRH in vivo. Two-dimensional difference in gel electrophoresis (2D-DIGE) and mass spectrometry analyses identified a total of 17 different proteins that were significantly affected by supraphysiological levels of AAS. Furthermore, pathway analyses showed that modulated proteins were mainly associated to glucose metabolism, drug detoxification, stress response and cell cycle. Validation of many of these proteins, such as GSTM1, ERH, GAPDH, PEBP1 and PDIA6, were confirmed by western blotting. We further demonstrated that AAS exposure decreased expression of estrogen receptors and GnRH, while two important signaling pathway proteins p-ERK, and p-p38, were modulated. Our results suggest that steroids have the capacity to directly affect the neuroendocrine system by modulating key cellular processes for the control of reproductive function.

C5-substituted 2,4-diaminoquinazoline inhibitors of the decapping scavenger enzyme DcpS (DAQ-DcpSi) have been developed for the treatment of spinal muscular atrophy (SMA), which is caused by genetic deficiency in the Survival Motor Neuron (SMN) protein. These compounds are claimed to act as SMN2 transcriptional activators but data underlying that claim are equivocal. In addition it is unclear whether the claimed effects on SMN2 are a direct consequence of DcpS inhibitor or might be a consequence of lysosomotropism, which is known to be neuroprotective. DAQ-DcpSi effects were characterized in cells in vitro utilizing DcpS knockdown and 7-methyl analogues as probes for DcpS vs non-DcpS-mediated effects. We also performed analysis of Smn transcript levels, RNA-Seq analysis of the transcriptome and SMN protein in order to identify affected pathways underlying the therapeutic effect, and studied lysosomotropic and non-lysosomotropic DAQ-DCpSi effects in 2B/- SMA mice. Treatment of cells caused modest and transient SMN2 mRNA increases with either no change or a decrease in SMNΔ7 and no change in SMN1 transcripts or SMN protein. RNA-Seq analysis of DAQ-DcpSi-treated N2a cells revealed significant changes in expression (both up and down) of approximately 2,000 genes across a broad range of pathways. Treatment of 2B/- SMA mice with both lysomotropic and non-lysosomotropic DAQ-DcpSi compounds had similar effects on disease phenotype indicating that the therapeutic mechanism of action is not a consequence of lysosomotropism. In striking contrast to the findings in vitro, Smn transcripts were robustly changed in tissues but there was no increase in SMN protein levels in spinal cord. We conclude that DAQ-DcpSi have reproducible benefit in SMA mice and a broad spectrum of biological effects in vitro and in vivo, but these are complex, context specific, and not the result of simple SMN2 transcriptional activation.

Do adversarial environmental conditions create social cohesion? We provide new answers to this question by exploiting spatial and temporal variation in exposure to earthquakes across Chile. Using a variety of methods and controlling for a number of socio-economic variables, we find that exposure to earthquakes has a positive effect on several indicators of social cohesion. Social cohesion increases after a big earthquake and slowly erodes in periods where environmental conditions are less adverse. Our results contribute to the current debate on whether and how environmental conditions shape formal and informal institutions.

Nociceptin/orphanin FQ (N/OFQ) controls several biological functions by selectively activating an opioid like receptor named N/OFQ peptide receptor (NOP). Biased agonism is emerging as an important and therapeutically relevant pharmacological concept in the field of G protein coupled receptors including opioids. To evaluate the relevance of this phenomenon in the NOP receptor, we used a bioluminescence resonance energy transfer technology to measure the interactions of the NOP receptor with either G proteins or β-arrestin 2 in the absence and in presence of increasing concentration of ligands. A large panel of receptor ligands was investigated by comparing their ability to promote or block NOP/G protein and NOP/arrestin interactions. In this study we report a systematic analysis of the functional selectivity of NOP receptor ligands. NOP/G protein interactions (investigated in cell membranes) allowed a precise estimation of both ligand potency and efficacy yielding data highly consistent with the known pharmacological profile of this receptor. The same panel of ligands displayed marked differences in the ability to promote NOP/β-arrestin 2 interactions (evaluated in whole cells). In particular, full agonists displayed a general lower potency and for some ligands an inverted rank order of potency was noted. Most partial agonists behaved as pure competitive antagonists of receptor/arrestin interaction. Antagonists displayed similar values of potency for NOP/Gβ1 or NOP/β-arrestin 2 interaction. Using N/OFQ as reference ligand we computed the bias factors of NOP ligands and a number of agonists with greater efficacy at G protein coupling were identified.

Morphine and structurally related derivatives are highly effective analgesics, and the mainstay in the medical management of moderate to severe pain. Pharmacological actions of opioid analgesics are primarily mediated through agonism at the µ opioid peptide (MOP) receptor, a G protein-coupled receptor. Position 17 in morphine has been one of the most manipulated sites on the scaffold and intensive research has focused on replacements of the 17-methyl group with other substituents. Structural variations at the N-17 of the morphinan skeleton led to a diversity of molecules appraised as valuable and potential therapeutics and important research probes. Discovery of therapeutically useful morphine-like drugs has also targeted the C-6 hydroxyl group, with oxymorphone as one of the clinically relevant opioid analgesics, where a carbonyl instead of a hydroxyl group is present at position 6. Herein, we describe the effect of N-substituent variation in morphine and oxymorphone on in vitro and in vivo biological properties and the emerging structure-activity relationships. We show that the presence of a N-phenethyl group in position 17 is highly favorable in terms of improved affinity and selectivity at the MOP receptor, potent agonism and antinociceptive efficacy. The N-phenethyl derivatives of morphine and oxymorphone were very potent in stimulating G protein coupling and intracellular calcium release through the MOP receptor. In vivo, they were highly effective against acute thermal nociception in mice with marked increased antinociceptive potency compared to the lead molecules. It was also demonstrated that a carbonyl group at position 6 is preferable to a hydroxyl function in these N-phenethyl derivatives, enhancing MOP receptor affinity and agonist potency in vitro and in vivo. These results expand the understanding of the impact of different moieties at the morphinan nitrogen on ligand-receptor interaction, molecular mode of action and signaling, and may be instrumental to the development of new opioid therapeutics.

Microvascular inflammation occurs during sepsis and the endogenous opioid-like peptide nociceptin/orphanin FQ (N/OFQ) is known to regulate inflammation. This study aimed to determine the inflammatory role of N/OFQ and its receptor NOP (ORL1) within the microcirculation, along with anti-inflammatory effects of the NOP antagonist UFP-101 (University of Ferrara Peptide-101) in an animal model of sepsis (endotoxemia). Male Wistar rats (220 to 300 g) were administered lipopolysaccharide (LPS) for 24 h (-24 h, 1 mg kg(-1); -2 h, 1 mg kg(-1) i.v., tail vein). They were then either anesthetised for observation of the mesenteric microcirculation using fluorescent in vivo microscopy, or isolated arterioles (~200 µm) were studied in vitro with pressure myography. 200 nM kg(-1) fluorescently labelled N/OFQ (FITC-N/OFQ, i.a., mesenteric artery) bound to specific sites on the microvascular endothelium in vivo, indicating sparse distribution of NOP receptors. In vitro, arterioles (~200 µm) dilated to intraluminal N/OFQ (10(-5)M) (32.6 + 8.4%) and this response was exaggerated with LPS (62.0 +7.9%, p=0.031). In vivo, LPS induced macromolecular leak of FITC-BSA (0.02 g kg(-1) i.v.) (LPS: 95.3 (86.7 to 97.9)%, p=0.043) from post-capillary venules (