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Long natural antisense transcripts (NATs) have been demonstrated in significant numbers in a variety of eukaryotic organisms. They are particularly prevalent in the nervous system suggesting their importance in neural functions. However, the precise physiological roles of the overwhelming majority of long NATs remain unclear. Here we report on the characterization of a novel molluscan nitric oxide synthase (NOS)-related long non-coding NAT (Lym-NOS1AS). This NAT is spliced and polyadenylated and is transcribed from the non-template strand of the Lym-NOS1 gene. We demonstrate that the Lym-NOS1AS is co-expressed with the sense Lym-NOS1 mRNA in a key neuron of memory network. Also, we report that the Lym-NOS1AS is temporally and spatially regulated by one-trial conditioning leading to long term memory (LTM) formation. Specifically, in the cerebral, but not in the buccal ganglia, the temporal pattern of changes in Lym-NOS1AS expression after training correlates with the alteration of memory lapse and non-lapse periods. Our data suggest that the Lym-NOS1AS plays a role in the consolidation of nitric oxide-dependent LTM.

Neural circuits underlying reflex and rhythmic behaviours in molluscs can be understood at the level of single identified neurons. Using these systems, the most important advances have been made in the understanding of the cellular and molecular basis of learning and memory. Keywords: molluscs; nervous systems; neurons; behaviour; learning

ObjectivesTo investigate the intensity and longevity of SARS-CoV-2 vaccination response in patients with immune-mediated inflammatory disease (IMID) by diagnosis, treatment and adapted vaccination schedules.MethodsSARS-CoV-2 IgG antibody response after SARS-CoV-2 vaccination was measured longitudinally in a large prospective cohort of healthy controls (HC) and IMID patients between December 2020 and 2021. Demographic and disease-specific data were recorded. Humoral response was compared across treatment and disease groups, and with respect to receipt of booster vaccinations. Age and sex adjusted SARS-CoV-2 antibody response was modelled over time. Marginal mean antibody levels and marginal risks of poor response were calculated at weekly intervals starting from week-8 after the first vaccination up to week 40.ResultsAmong 5076 individuals registered, 2535 IMID patients and 1198 HC were eligible for this analysis. Mean antibody levels were higher in HC compared to IMIDs at all-time points, with peak antibody response in HC more than twice that in IMIDs (12.48 (11.52-13.52) vs. 5.71 (5.46-5.97)). Poor response to vaccination was observed in IMID patients treated with agents affecting B- and T-cell functions. Mean differences in antibody response between IMID diseases were small. After additional vaccinations, IMID patients could achieve higher antibody levels than HC vaccinated according to the two-dose schedule, even-though initial antibody levels were lower.ConclusionsIMID patients show a lower and less durable SARS-CoV-2 vaccination response and are at risk to lose humoral immune protection. Adjusted vaccination schedules with earlier boosters and/or more frequent re-doses could better protect IMID patients.

In eukaryotes, CREB-binding protein (CBP), a coactivator of CREB, functions both as a platform for recruiting other components of the transcriptional machinery and as a histone acetyltransferase (HAT) that alters chromatin structure. We previously showed that the transcriptional activity of cAMP-responsive element binding protein (CREB) plays a crucial role in neuronal plasticity in the pond snail Lymnaea stagnalis. However, there is no information on the role CBP plays in CREB-initiated plastic changes in Lymnaea. In this study, we characterized the Lymnaea CBP (LymCBP) gene and investigated the roles it plays in synaptic plasticity involved in regulating feeding behaviors. Similar to CBPs of other species, LymCBP possesses functional domains, such as KIX domain, which is essential for interaction with CREB and was shown to regulate long-term memory (LTM). In situ hybridization showed that the staining patterns of LymCBP mRNA in the central nervous system were very similar to those of Lymnaea CREB1 (LymCREB1). A particularly strong LymCBP mRNA signal was observed in the Cerebral Giant Cell (CGC), an identified extrinsic modulatory interneuron of the feeding circuit, key to both appetitive and aversive LTM for taste. Biochemical experiments using the recombinant protein of LymCBP HAT domain showed that its enzymatic activity was blocked by classical HAT inhibitors such as curcumin, anacardic acid and garcinol. Preincubation of Lymnaea CNSs with these HAT inhibitors blocked cAMP-induced long-term potentiation between the CGC and the follower B1 motoneuron. We therefore suggest that HAT activity of LymCBP in the CGCs is a key factor in synaptic plasticity contributing to LTM after classical conditioning.

Applications of key technologies in biomedical research, such as qRT-PCR or LC-MS based proteomics, are generating large biological (-omics) data sets which are useful for the identification and quantification of biomarkers involved in molecular mechanisms of any research area of interest. Genome, transcriptome and proteome databases are already available for a number of model organisms including vertebrates and invertebrates. However, there is insufficient information available for protein sequences of certain invertebrates, such as the great pond snail Lymnaea stagnalis, a model organism that has been used highly successfully in elucidating evolutionarily conserved mechanisms of learning and memory, ageing and age-related as well as amyloid-β induced memory decline. In this investigation, we used a bioinformatics approach to designing and benchmarking a comprehensive CNS proteomics database (LymCNS-PDB) for the identification of proteins from the Central Nervous System (CNS) of Lymnaea stagnalis by LC-MS based proteomics. LymCNS-PDB was created by using the Trinity TransDecoder bioinformatics tool to translate amino acid sequences from mRNA transcript assemblies obtained from an existing published Lymnaea stagnalis transcriptomics database. The blast-style MMSeq2 software was used to match all translated sequences to sequences for molluscan proteins (including Lymnaea stagnalis and other molluscs) available from UniProtKB. LymCNS-PDB, which contains 9,628 identified matched proteins, was then benchmarked by performing LC-MS based proteomics analysis with proteins isolated from the CNS of Lymnaea stagnalis. MS/MS analysis using the LymCNS-PDB database led to the identification of 3,810 proteins while only 982 proteins were identified by using a non-specific Molluscan database. LymCNS-PDB provides a valuable tool that will enable us to perform quantitative proteomics analysis to identify a plethora of protein interactomes involved in several CNS functions in Lymnaea stagnalis including learning and memory, aging-related memory decline and others.

BackgroundRA is characterized by synovial inflammation resulting in local bone loss [1]. Inhibitors of JAK/Stat pathways, such as baricitinib, demonstrated efficacy in reducing signs and symptoms of RA in clinical trials, however, little is known about their effects on synovitis and bone structure [2]. Preclinical and clinical observations suggest a positive effect JAK inhibitors on bone mass and microstructure, however no prospective, interventional clinical trial has been performed so far [3].ObjectivesThe aim of this study is to evaluate the effect of baricitinib on local inflammation (synovitis and osteitis) and bone structure (erosions) in RA patients failing on cs/bDMARD therapy using hand MRI.MethodsBAREBONE is a prospective, interventional, open label, monocentric single center study (EUDRACT 2018-001164-32 / NCT03701789) to assess the effect of baricitinib (4mg/day) on local MRI inflammation and structure in patients with active RA. Besides demographic and clinical characteristics, hand joint inflammation was assessed by magnetic resonance imaging (MRI) using a 1.5 Tesla scanner (Siemens Magnetom Aera T1w TSE cor, T2w TIRM cor, T2w TSE fat-sat trans, T1w TSE fat-sat trans + cor after KM;). at baseline, week 24 and week 48. Scans were assessed for synovitis, osteitis and bone erosions using the RAMRIS scoring system using two independent blinded readers (SK and SB). Intraclass correlation coefficients were calculated for total RAMRIS and synovitis, erosion and osteitis subscores and in a second step differences between cs and bDMARD failure were elaborated. Variables are summarized descriptively using means and 95% bootstrap confidence intervals for continuous outcomes and as number and percentages for categorical outcomes.ResultsThirty- two RA patients were screened and 30 patients were included (age: 53.4 [SD 12.6] years; sex: f/m N 24/6; disease duration: 3 [IQR 2.0 – 8.0] years; biologic naïve/bDMARD failure 16/14). 27 patients completed the trial while MRI data was available for 24 patients at week 48. Demographics and clinical characteristics can be seen in Table 1. Total RAMRIS scores slightly decreased from 20.6 (95% CI 14.4 -27.8) at baseline (BL) to 18.3 (11.5 -26.5) at week 48. The synovitis subscore mainly contributed to total RAMRIS reduction by significantly improving from 5.3 (4.0 - 6.8) at BL to 2.7 (1.5 - 4.0) at week 48 with a score change of -2.9 (-4.0 to -1.8). At week 48, 12 patients (44.4%) had no signs of synovitis compared to only 3 patients at BL. In contrast, RAMRIS osteitis subscores only marginally decreased from 4.9 (2.2 - 8.4) at BL to 4.0 (1.9 - 6.7) at week 48. RAMRIS erosion score remained stable over the 48-week observation time. A significant difference in RAMRIS synovitis change for biologic naïve -3.8 (-5.2 to -2.6) vs biologic failure -1.0 (-2.2 to 0.4 could be observed at week 48).With respect to clinical disease activity, DAS 28 score decreased from 4.8 (4.5 – 5.1) at BL to 2.9 (2.5 – 3.3) at week 48. Detailed results can be found in Table 1 and Figure 1. Intraclass coefficient (95%CI) for RAMRIS scoring was high for both readers 0.997 (0.994 to 0.998).Table 1.Demographics, DAS 28 ESR, RAMRIS total score and RAMRIS subset scores at baseline, week 24 and week 48 are shown as well as number of patients with improvement and resolution of synovitis.BaselineWeek 24Week 48N303027AgeMean [SD]53.5 (12.6)Genderfemalen [%]24 (80.0)malen [%]6 (20.0)Disease duration, yearsMedian (IQR)3.0 (2.0-8.0)DAS-28 ESRMean [95%CI]4.8 (4.5 to 5.1)3.0 (2.7 to 3.3)2.7 (2.4 to 3.0)MRI availablen [%]30 (100.0)28 (93.3)24 (88.9)RAMRIS totalMean [95%CI]20.6 (14.4 to 27.6)18.4 (12.6 to 25.4)18.3 (11.5 to 26.5)RAMRIS total changeMean [95%CI]0.0 (0.0 to 0.0)-2.1 (-4.0 to -0.4)-3.9 (-7.2 to -0.5)RAMRIS synovitisMean [95%CI]5.3 (3.9 to 6.9)3.5 (2.2 to 4.9)2.7 (1.5 to 4.0)RAMRIS synovitis changeMean [95%CI]0.0 (0.0 to 0.0)-1.8 (-2.5 to -1.0)-2.9 (-4.0 to -1.8)RAMRIS synovitis improvedpatients n [%]10 (33.3)13 (48.1)RAMRIS synovitis resolvedpatients n [%]10 (33.3)12 (44.4)RAMRIS osteitisMean [95%CI]4.9 (2.2 to 8.4)3.7 (1.5 to 6.2)4.0 (1.9 to 6.7)RAMRIS osteitis changeMean [95%CI]0.0 (0.0 to 0.0)-0.9 (-3.1 to 1.0)-1.9 (-5.7 to 1.1)RAMRIS osteitis improvedpatients n [%]2 (6.7)4 (14.8)RAMRIS erosionMean [95%CI]10.4 (7.3 to 14.6)11.2 (7.7 to 15.0)11.6 (7.5 to 16.6)RAMRIS erosion changeMean [95%CI]0.0 (0.0 to 0.0)0.6 (0.1 to 1.2)0.9 (0.0 to 2.1)RAMRIS erosion worsenedpatients n [%]2 (6.7)3 (11.1)ConclusionOur study shows that baricitinib primarily reduces MRI synovitis in RA patients that have previously failed csDMARD and bDMARD therapy and particularly in patients who are biologic naïve.References[1]McInnes, I.B. and G. Schett, The pathogenesis of rheumatoid arthritis. N Engl J Med, 2011.[2]Genovese, M.C., et al., Baricitinib in Patients with Refractory Rheumatoid Arthritis. N Engl J Med, 2016[3]Adam, S., et al., JAK inhibition increases bone mass in steady-state conditions and ameliorates pathological bone loss by stimulating osteoblast function. Sci Transl Med, 2020.AcknowledgementsLilly Deutschland GmbH funded the Barebone trialDisclosure of InterestsStephan Kemenes: None declared, Sara Bayat: None declared, David Simon Speakers bureau: Lilly Pharma Deutschland GmbH, Janssen, Consultant of: BMS, Pfizer, Sanofi, Abbvie, Janssen, Medac, Novartis,Lilly Deutschland GmbH, GileaBMS, Pfizer, Sanofi, Abbvie, Janssen, Medac, Novartis,Lilly Deutschland GmbH, Gilead, Amgend,, Grant/research support from: Novartis, Gilead, Abbvie, Lilly, Gerhard Krönke Speakers bureau: GSK, Novartis, Consultant of: GSK, Lilly, Novartis, Janssen, Grant/research support from: Lilly, Novartis, BMS, Janssen, Daniela Bohr: None declared, Larissa Valor: None declared, Fabian Hartmann: None declared, Louis Schuster: None declared, Koray Tascilar Speakers bureau: Gilead speaker, Consultant of: UCB, Lilly, Georg Schett Speakers bureau: Janssen, Abbvie, BMS, Lilly, Novartis, Roche, AMGEN, Gilead, UCB, Consultant of: Lilly, Novartis, Abbvie, Grant/research support from: Chugai, Lilly, Novartis, Arnd Kleyer Speakers bureau: Lilly, Novartis, Abbvie, Consultant of: BMS, Pfizer, Sanofi, Abbvie, Janssen, Medac, Novartis,Lilly Deutschland GmbH, Gilead, Amgen, Grant/research support from: Novartis, Lilly Deutschland GmbH, Gilead

How an animal responds to a particular sensory stimulus will to a great extent depend on prior experience associated with that stimulus. For instance, aversive associative learning may lead to a change in the predicted outcomes, which suppresses the behavioural response to an otherwise rewarding stimulus. However, the neuronal mechanisms of how aversive learning can result in the suppression of even a vitally important innate behaviour is not well understood. Here we used the model system ofLymnaea stagnalisto address the question of how an anticipated aversive outcome can alter the behavioural response to a previously effective feeding stimulus. We found that aversive classical conditioning with sucrose as the CS (conditioned stimulus) and strong touch as the aversive US (unconditioned stimulus) reverses the decision so that the same salient feeding stimulus inhibits feeding, rather than activating it. Key to the understanding of the neural mechanism underlying this switch in the behavioural response is the PlB (pleural buccal) extrinsic interneuron of the feeding network whose modulatory effects on the feeding circuit inhibit feeding. After associative aversive training, PlB is excited by sucrose to reverse its effects on the feeding response. Aversive associative learning induces a persistent change in the electrical properties of PlB that is both sufficient and necessary for the switch in the behavioural output. In addition, the strong touch used as the US during the associative training protocol can also serve as a sensitizing stimulus to lead to an enhanced defensive withdrawal response to a mild touch stimulus. This non-associative effect of the strong touch is probably based on the facilitated excitatory output of a key identified interneuron of the defensive withdrawal network, PeD12.

Interference-based forgetting occurs when new information acquired either before or after a learning event attenuates memory expression (proactive and retroactive interference, respectively). Multiple learning events often occur in rapid succession, leading to competition between consolidating memories. However, it is unknown what factors determine which memory is remembered or forgotten. Here, we challenge the snail, Lymnaea, to acquire two consecutive similar or different memories and identify learning-induced changes in neurons of its well-characterized motor circuits. We show that when new learning takes place during a stable period of the original memory, proactive interference only occurs if the two consolidating memories engage the same circuit mechanisms. If different circuits are used, both memories survive. However, any new learning during a labile period of consolidation promotes retroactive interference and the acquisition of the new memory. Therefore, the effect of interference depends both on the timing of new learning and the underlying neuronal mechanisms., Michael Crossley et al. use the snail Lymnaea to investigate the factors involved in determining which memories are remembered or forgotten when multiple learning events happen close together. They find that timing of events and whether the same neuronal mechanisms are used are key factors in determining whether interference will occur.