Cooperative Folding as a Molecular Switch in an Evolved Antibody Binder.

Designing proteins with tunable activities from easily accessible external cues remains a biotechnological challenge. Here, we set out to create a small antibody-binding domain equipped with a molecular switch inspired by the allosteric response to calcium seen in naturally derived proteins like calmodulin. We have focused on one of the three domains of Protein G that show inherent affinity to antibodies. By combining a semi-rational protein design with directed evolution, we engineered novel variants containing a calcium-binding loop rendering the inherent antibody affinity calcium-dependent. The evolved variants resulted from a designed selection strategy subjecting them to negative and positive selection pressures focused on conditional antibody-binding. Hence, these variants contained molecular "on/off" switches, controlling the target affinity towards antibody fragments simply by the presence or absence of calcium. From NMR spectroscopy we found that the molecular mechanism underlying the evolved switching behavior was a coupled calcium-binding and folding event where the target binding surface was intact and functional only in the presence of bound calcium. Notably, it was observed that the response to the employed selection pressures gave rise to the evolution of a cooperative folding mechanism. This observation illustrates why the cooperative folding reaction is an effective solution seen repeatedly in the natural evolution of fine-tuned macromolecular recognition. Engineering binding moieties to confer conditional target interaction has great potential due to the exquisite interaction control that is tunable to application requirements. Improved understanding of the molecular mechanisms behind regulated interactions is crucial to unlock how to engineer switchable proteins useful in a variety of biotechnological applications.

Clinical use of cone-beam computed tomography in Western Norway: a referral-based retrospective study.

OBJECTIVE: To elucidate cone-beam computed tomography (CBCT) referral profiles in Western Norway. MATERIALS AND METHODS: In all, 3,031 referrals to oral- and maxillofacial radiologist were reviewed. Patient data were retrieved retrospectively from electronic charts. The patient's age, gender, and perceived clinical indication were noted, as well as relevant medical and dental history and whether the referring clinician was a general dentist or held a clinical dental specialty. RESULTS: A total of 2,680 referrals fulfilled the inclusion criteria (UiB n = 1,471, and TkVestland, n = 1,209). The female:male ratio was 1,427:1,253. Mean age was 33 years - 35 years for females compared to 31 years for males (p < 0.001). The most common clinical indications were related to impacted teeth (29%), endodontic issues (17%), cleft lip palate (12%), and resorptions (10%). Less common were bone lesions, implant planning, trauma to the teeth or jaws, atypical orofacial pain, and temporo-mandibular joint (TMJ). The patient age-profiles mirrored differences in indications within the cohort. Most referrals were from specialist dentists such as orthodontists, oral surgeons, and endodontists. Interestingly, 543/2,680 (20%) referrals were from general dentists.  Conclusions: Specialist dentists such as orthodontists, oral surgeons, and endodontists refer most patients for clinical indications such as impacted teeth, endodontic issues, and resorptions.

A One Health real-time surveillance system for nowcasting Campylobacter gastrointestinal illness outbreaks, Norway, week 30 2010 to week 11 2022.

BackgroundCampylobacter is a leading cause of food and waterborne illness. Monitoring and modelling Campylobacter at chicken broiler farms, combined with weather pattern surveillance, can aid nowcasting of human gastrointestinal (GI) illness outbreaks. Near real-time sharing of data and model results with health authorities can help increase potential outbreak responsiveness.AimsTo leverage data on weather and Campylobacter on broiler farms to build a risk model for possible human Campylobacter outbreaks and to communicate risk assessments with health authorities.MethodsWe developed a spatio-temporal random effects model for weekly GI illness consultations in Norwegian municipalities with Campylobacter monitoring and weather data from week 30 2010 to 11 2022 to give 1-week nowcasts of GI illness outbreaks. The approach combined a municipality random effects baseline model for seasonally-adjusted GI illness with a second model for peak deviations from that baseline. Model results are communicated to national and local stakeholders through an interactive website: Sykdomspulsen One Health.ResultsLagged temperature and precipitation covariates, as well as 2-week-lagged positive Campylobacter sampling in broilers, were associated with higher levels of GI consultations. Significant inter-municipality variability in outbreak nowcasts were observed.ConclusionsCampylobacter surveillance in broilers can be useful in GI illness outbreak nowcasting. Surveillance of Campylobacter along potential pathways from the environment to illness such as via water system monitoring may improve nowcasting. A One Health system that communicates near real-time surveillance data and nowcast changes in risk to health professionals facilitates the prevention of Campylobacter outbreaks and reduces impact on human health.

Genetic and clinical basis for two distinct subtypes of primary Sjögren's syndrome.

OBJECTIVES: Clinical presentation of primary Sjögren's syndrome (pSS) varies considerably. A shortage of evidence-based objective markers hinders efficient drug development and most clinical trials have failed to reach primary endpoints. METHODS: We performed a multicentre study to identify patient subgroups based on clinical, immunological and genetic features. Targeted DNA sequencing of 1853 autoimmune-related loci was performed. After quality control, 918 patients with pSS, 1264 controls and 107 045 single nucleotide variants remained for analysis. Replication was performed in 177 patients with pSS and 7672 controls. RESULTS: We found strong signals of association with pSS in the HLA region. Principal component analysis of clinical data distinguished two patient subgroups defined by the presence of SSA/SSB antibodies. We observed an unprecedented high risk of pSS for an association in the HLA-DQA1 locus of odds ratio 6.10 (95% CI: 4.93, 7.54, P=2.2×10-62) in the SSA/SSB-positive subgroup, while absent in the antibody negative group. Three independent signals within the MHC were observed. The two most significant variants in MHC class I and II respectively, identified patients with a higher risk of hypergammaglobulinaemia, leukopenia, anaemia, purpura, major salivary gland swelling and lymphadenopathy. Replication confirmed the association with both MHC class I and II signals confined to SSA/SSB antibody positive pSS. CONCLUSION: Two subgroups of patients with pSS with distinct clinical manifestations can be defined by the presence or absence of SSA/SSB antibodies and genetic markers in the HLA locus. These subgroups should be considered in clinical follow-up, drug development and trial outcomes, for the benefit of both subgroups.

Assessment of major salivary gland ultrasonography in Sjögren's syndrome. A comparison between bedside and post-examination evaluations.

OBJECTIVES: Major salivary gland ultrasonography (SGUS) is a suitable diagnostic tool in Sjögren's syndrome (SS). We aimed to determine the more representative gland, projection and format most applicable for reproducible image analysis. METHODS: One investigator performed SGUS in patients with SS. Parotid and submandibular glands were examined in longitudinal and transverse planes and evaluated bedside using a simplified scoring system (0-3). Longitudinal and transverse images and videos of all glands were stored and later evaluated/graded by three investigators, at two time-points. Agreement was calculated using intraclass correlation coefficient (ICC). RESULTS: The ICC for static image and video scoring compared to bedside evaluation ranged from 0.131 to 0.882. Average ICC for longitudinal/transverse image was 0.667/0.662, and 0.683/0.510 for longitudinal/transverse video. Interobserver reliability was good to excellent (0.81-0.94). Intraobserver reliability scores ranged from fair to excellent (0.46-0.96). The correlation between image and video evaluations of all modalities and examiners was good to excellent (0.614-0.904). The best mean ICC was found for the longitudinal projection of the left parotid gland (0.861) and the lowest mean ICC was for the transverse projection of the left submandibular gland (0.66). CONCLUSIONS: Our study indicates a trend favouring longitudinal video of the parotid gland as preferred projection, gland and storage format.

Biomechanical differences in double poling between sexes and level of performance during a classical cross-country skiing competition.

Biomechanical differences in double poling (DP) between sex and performance level were investigated in female and male cross-country skiers during a classical race (10/15 km). Skiers were divided into faster and slower on basis of race performance: females faster (n=20), females slower (n=20), males faster (n=20), and males slower (n=20). Based on video analysis while DP in a flat section of the track, joint and pole angles at pole plant (PP) and pole-off, cycle characteristics and the use and coordination pattern of heel-raise (raise of heels from the ground to have a higher body position at PP) were analysed. Faster females and males had 4.3% and 7.8% higher DP velocity than their slower counterparts (both P<0.001). Faster males had 6.5% longer cycles than slower males (P<0.001). Faster skiers stopped heel-raise later than slower skiers (females: 2.0±3.4% vs. -1.0±3.5%, P<0.05; males: 3.9±2.4% vs. 0.8±3.2% of cycle time in relation to PP, P<0.001). At PP, faster skiers and male skiers had a smaller pole angle and greater ankle to hip and ankle to shoulder angle with respect to vertical, resulting in a more distinct forward body lean. However, the majority of the differences are likely due to higher DP velocity.

Dehydroevodiamine and hortiamine, alkaloids from the traditional Chinese herbal drug Evodia rutaecarpa, are IKr blockers with proarrhythmic effects in vitro and in vivo.

Evodiae fructus is a widely used herbal drug in traditional Chinese medicine. Evodia extract was found to inhibit hERG channels. The aim of the current study was to identify hERG inhibitors in Evodia extract and to investigate their potential proarrhythmic effects. Dehydroevodiamine (DHE) and hortiamine were identified as IKr (rapid delayed rectifier current) inhibitors in Evodia extract by HPLC-microfractionation and subsequent patch clamp studies on human embryonic kidney cells. DHE and hortiamine inhibited IKr with IC50s of 253.2±26.3nM and 144.8±35.1nM, respectively. In dog ventricular cardiomyocytes, DHE dose-dependently prolonged the action potential duration (APD). Early afterdepolarizations (EADs) were seen in 14, 67, 100, and 67% of cells after 0.01, 0.1, 1 and 10µM DHE, respectively. The proarrhythmic potential of DHE was evaluated in 8 anesthetized rabbits and in 8 chronic atrioventricular block (cAVB) dogs. In rabbits, DHE increased the QT interval significantly by 12±10% (0.05mg/kg/5min) and 60±26% (0.5mg/kg/5min), and induced Torsade de Pointes arrhythmias (TdP, 0.5mg/kg/5min) in 2 rabbits. In cAVB dogs, 0.33mg/kg/5min DHE increased QT duration by 48±10% (P<0.05*) and induced TdP in 2/4 dogs. A higher dose did not induce TdP. In human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), methanolic extracts of Evodia, DHE and hortiamine dose-dependently prolonged APD. At 3µM DHE and hortiamine induced EADs. hERG inhibition at submicromolar concentrations, APD prolongation and EADs in hiPSC-CMs and dose-dependent proarrhythmic effects of DHE at micromolar plasma concentrations in cAVB dogs should increase awareness regarding proarrhythmic effects of widely used Evodia extracts.

Current concepts on Sjögren's syndrome - classification criteria and biomarkers.

Sjögren's syndrome is a lymphoproliferative disease with autoimmune features characterized by mononuclear cell infiltration of exocrine glands, notably the lacrimal and salivary glands. These lymphoid infiltrations lead to dryness of the eyes (keratoconjunctivitis sicca), dryness of the mouth (xerostomia), and, frequently, dryness of other surfaces connected to exocrine glands. Sjögren's syndrome is associated with the production of autoantibodies because B-cell activation is a consistent immunoregulatory abnormality. The spectrum of the disease extends from an organ-specific autoimmune disorder to a systemic process and is also associated with an increased risk of B-cell lymphoma. Current treatments are mainly symptomatic. As a result of the diverse presentation of the syndrome, a major challenge remains to improve diagnosis and therapy. For this purpose an international set of classification criteria for primary Sjögren's syndrome has recently been developed and validated and seems well suited for enrolment in clinical trials. Salivary gland biopsies have been examined and histopathology standards have been developed, to be used in clinical trials and patient stratification. Finally, ultrasonography and saliva meet the need of non-invasive imaging and sampling methods for discovery and validation of disease biomarkers in Sjögren's syndrome.

Antibodies against carbamylated proteins are present in primary Sjögren's syndrome and are associated with disease severity.

OBJECTIVES: Herein, we investigate the presence and prognostic value of autoantibodies against carbamylated proteins (anti-CarP) in the serum of patients with primary Sjögren's syndrome (pSS). PATIENTS AND METHODS: Serum levels of anti-CarP antibodies were measured in Norwegian patients with pSS (n=78) and corresponding controls (n=74) using ELISA and analysed in relation with exocrine gland function, degree of salivary gland inflammation, signs of ectopic germinal centre (GC) formation and immunological markers. For univariate comparisons, the Mann-Whitney U test and χ(2) or Fisher's exact tests were used. Correlations were assessed with Spearman's rank testing. Multivariate regression analyses were used to assess the effect of anti-CarP positivity on clinical manifestations. RESULTS: Of the patients with pSS, 27% were positive for anti-CarP IgG antibodies. Levels of anti-CarP correlated positively with total IgG, IgM, rheumatoid factor and ß2-microglobulin. Importantly, after adjusting for confounding factors, patients positive for anti-CarP had significantly higher focus score. Furthermore, positive anti-CarP status coincided with 9.2-fold higher odds of having developed GC-like structures in the minor salivary glands. As a patient group considered having worse disease outcome, individuals with ectopic GC-like structures also presented with significantly higher levels of anti-CarP antibodies. CONCLUSIONS: Presence of anti-CarP in patients with pSS is strongly associated with increased focal lymphocytic infiltration, formation of ectopic GC-like structures in minor salivary glands, and diminished salivary gland function. Even taking into consideration our relatively small cohort we believe that anti-CarP antibodies offer new possibilities for identifying patients with more active disease and at risk of developing additional comorbidity.

Sjögren's syndrome patients with ectopic germinal centers present with a distinct salivary proteome.

OBJECTIVE: Clinical expression of SS shows considerable interpatient heterogeneity. Thus, the aim of this study was to assess whether individual salivary proteomic profiles provide a framework for identification of disease-phenotype-driven biomarker signatures. METHODS: Using a 187-plex capture antibody-based assay, proteomic biomarker profiles from unstimulated whole saliva were generated from a SS-cohort representing six clinically distinct disease phenotypes. Discriminant function analyses identified the most powerful biomarker signatures for correct recapitulation of each patient's status with respect to hyposalivation and histopathological features of salivary gland inflammation. In addition, gene ontology-based network analyses allowed systematic interpretation of the molecular patterns underlying these specific disease features. RESULTS: Presentation of hyposalivation was associated with significant alteration in 22 out of 119 reliably detectable biomarkers. Thereof, a 4-plex signature allowed accurate prediction of salivary gland function for >80% of the cases. With respect to histopathological features, the most distinct profiles were identified in conjunction with ectopic germinal centres. Selected from the 13 analytes relevant here, pregnancy-associated plasma protein A, thrombospondin 1 and peptide YY would recapitulate the presence or absence of tertiary lymphoid organization for 93.8% of the patients. Whereas functional annotation of alterations associated with hyposalivation identified the IL1 system as a dominant pro-inflammatory component, changes observed in context with ectopic lymphoid organization revealed specific shifts in chemotactic profiles and altered regulation of apoptotic processes. CONCLUSION: Multivariate analyses of a patient's salivary proteome could reliably recapitulate specific aspects of SS disease. Accessible and repetitively collectable, such biomarker signatures harbour great potential for patient subclassification and subsequent follow-up.

Is salivary gland ultrasonography a useful tool in Sjögren's syndrome? A systematic review.

OBJECTIVE: Ultrasonography (US) is a sensitive tool in the diagnosis of major salivary gland abnormalities in primary Sjögren's syndrome (pSS). The aim of this systematic review was to assess the metric properties of this technique. METHODS: PUBMED and EMBASE databases were searched. All publications between January 1988 and January 2013 were considered. Data were extracted from the articles meeting the inclusion criteria according to US definition of salivary gland scoring system and metric properties studied. The type and number of glands tested, study design and metric properties according to OMERACT filter (truth, discrimination, feasibility) were assessed. RESULTS: Of 167 publications identified initially with PUBMED and EMBASE, 31 met the inclusion criteria. The number of pSS patients varied among the studies from 16 to 140. The diagnosis of pSS was in line in most of the cases with the American-European Consensus Group (AECG) classification criteria for Sjögren's syndrome. The US examination was performed in suspected pSS only in studies in which the sensitivity ranged from 45.8 to 91.6% and specificity from 73 to 98.1%. There was heterogeneity in regard to the definition of US in B-mode and few studies used US in colour Doppler. Few studies reported reliability of US and sensitivity to change in pSS. CONCLUSION: US is a valuable tool for detecting salivary gland abnormalities in pSS. Its reliability has been poorly investigated and there is considerable variation in the definition of US abnormalities. Further studies are required to validate and standardize the US definition of salivary gland in pSS.

Single-shot photofragment imaging by structured illumination.

A laser method to suppress background interferences in pump-probe measurements is presented and demonstrated. The method is based on structured illumination, where the intensity profile of the pump beam is spatially modulated to make its induced photofragment signal distinguishable from that created solely by the probe beam. A spatial lock-in algorithm is then applied on the acquired data, extracting only those image components that are characterized by the encoded structure. The concept is demonstrated for imaging of OH photofragments in a laminar methane/air flame, where the signal from the OH photofragments produced by the pump beam is spatially overlapping with that from the naturally present OH radicals. The purpose was to perform for the first time, to the best of our knowledge, single-shot imaging of HO(2) in a flame. These results show an increase in signal-to-interference ratio of about 20 for single-shot data.

Diagnostic utility of major salivary gland ultrasonography in primary Sjögren's syndrome.

OBJECTIVES: To investigate major salivary gland ultrasonography (US) in relation to symptoms and findings of oral and ocular dryness, and autoimmune disease, for potential use in diagnosis and follow-up of patients with primary Sjögren's syndrome (pSS). METHODS: Patients with pSS were recruited from the Department of Rheumatology, Haukeland University Hospital. The parotid and submandibular salivary glands were examined by US using a simplified scoring system for glandular homogeneity and hypoechogenic areas. Scans were graded on a scale 0-3, grades 0-1 considered corresponding to normal/non-specific changes and grades 2-3 to pathological changes. Sicca symptoms of the mouth and eyes, salivary gland capacity, tear secretion, minor salivary gland inflammation, serum autoantibodies, and fatigue were also investigated. RESULTS: US was performed in 97 patients. Oral and ocular sicca symptoms correlated with US score and decreased saliva levels. Fatigue VAS correlated with oral sicca symptoms but was inversely correlated with age. Patients with normal/non-specific US findings tended to be older than patients with pathological US findings. US score correlated with unstimulated and stimulated salivary secretion and tear secretion. Minor salivary gland inflammation correlated with major salivary gland US findings, and lymphoid organisation, germinal centre (GC)-like structures, in the minor salivary gland tissue biopsies was seemingly related to US pathology. Serum autoantibodies against Ro/SSA and/or La/SSB were associated with US pathology. CONCLUSIONS: US findings in major salivary glands correlate with subjective and objective oral and ocular items as well as systemic autoimmune features of pSS. US represents a useful imaging tool for diagnostics and follow-up of pSS.

Lessons from the heart: mirroring electrophysiological characteristics during cardiac development to in vitro differentiation of stem cell derived cardiomyocytes.

The ability of human pluripotent stem cells (hPSCs) to differentiate into any cell type of the three germ layers makes them a very promising cell source for multiple purposes, including regenerative medicine, drug discovery, and as a model to study disease mechanisms and progression. One of the first specialized cell types to be generated from hPSC was cardiomyocytes (CM), and differentiation protocols have evolved over the years and now allow for robust and large-scale production of hPSC-CM. Still, scientists are struggling to achieve the same, mainly ventricular, phenotype of the hPSC-CM in vitro as their adult counterpart in vivo. In vitro generated cardiomyocytes are generally described as fetal-like rather than adult. In this review, we compare the in vivo development of cardiomyocytes to the in vitro differentiation of hPSC into CM with focus on electrophysiology, structure and contractility. Furthermore, known epigenetic changes underlying the differences between adult human CM and CM differentiated from pluripotent stem cells are described. This should provide the reader with an extensive overview of the current status of human stem cell-derived cardiomyocyte phenotype and function. Additionally, the reader will gain insight into the underlying signaling pathways and mechanisms responsible for cardiomyocyte development.

Genetic associations to germinal centre formation in primary Sjogren's syndrome.

BACKGROUND: Primary Sjögren's syndrome (pSS) is an autoimmune rheumatic disease mainly characterised by focal mononuclear cell infiltration in the salivary and lacrimal glands, and by the symptoms xerostomia and keratoconjunctivitis sicca. Germinal centre-like structures (GC) are found in the minor salivary glands of approximately 25% of patients. In this study, we aimed to assess genetic variations in pSS patients with GC-like formations (GC+) compared with patients without such formations (GC-). METHODS: Minor salivary gland biopsies from Swedish and Norwegian pSS patients (n=320) were evaluated for GC-like formations, identifying 76 GC+ and 244 GC- patients. A panel of 1536 single-nucleotide polymorphisms (SNPs) in 107 genes was genotyped. Minor allele frequencies in GC+ and GC- patients were compared using Fisher's exact test, and associations were considered significant when p<4.7×10(-4) and suggestive when p<0.01. RESULTS: In this case-only analysis, we identified two SNPs in CCL11 (eotaxin) associated with GC-like structures (p<4.7×10(-4), OR 0.45 and 0.41, respectively). A haplotype of the two minor alleles was associated with GC status with p=2.6×10(-4), OR 0.40. Suggestive associations (p<0.01) were found in SNPs in the B cell activation and/or GC-formation related genes AICDA, BANK1 and BCL2. Furthermore, SNPs in IL17A, ICA1, PKN1 and SNPs in the NF-κB pathway genes CARD8, IKBKE and TANK were found suggestively associated with GC-like structures. CONCLUSIONS: Our findings suggest that genetic variations may explain why ectopic GC-like structures are present in some pSS patients, and support the hypothesis that GC+ and GC- patients represent distinct disease phenotypes.

Transient compartment-like syndrome and normokalaemic periodic paralysis due to a Ca(v)1.1 mutation.

We studied a two-generation family presenting with conditions that included progressive permanent weakness, myopathic myopathy, exercise-induced contracture before normokalaemic periodic paralysis or, if localized to the tibial anterior muscle group, transient compartment-like syndrome (painful acute oedema with neuronal compression and drop foot). 23Na and 1H magnetic resonance imaging displayed myoplasmic sodium overload, and oedema. We identified a novel familial Ca(v)1.1 calcium channel mutation, R1242G, localized to the third positive charge of the domain IV voltage sensor. Functional expression of R1242G in the muscular dysgenesis mouse cell line GLT revealed a 28% reduced central pore inward current and a -20 mV shift of the steady-state inactivation curve. Both changes may be at least partially explained by an outward omega (gating pore) current at positive potentials. Moreover, this outward omega current of 27.5 nS/nF may cause the reduction of the overshoot by 13 mV and slowing of the upstroke of action potentials by 36% that are associated with muscle hypoexcitability (permanent weakness and myopathic myopathy). In addition to the outward omega current, we identified an inward omega pore current of 95 nS/nF at negative membrane potentials after long depolarizing pulses that shifts the R1242G residue above the omega pore constriction. A simulation reveals that the inward current might depolarize the fibre sufficiently to trigger calcium release in the absence of an action potential and therefore cause an electrically silent depolarization-induced muscle contracture. Additionally, evidence of the inward current can be found in 23Na magnetic resonance imaging-detected sodium accumulation and 1H magnetic resonance imaging-detected oedema. We hypothesize that the episodes are normokalaemic because of depolarization-induced compensatory outward potassium flux through both delayed rectifiers and omega pore. We conclude that the position of the R1242G residue before elicitation of the omega current is decisive for its conductance: if the residue is located below the gating pore as in the resting state then outward currents are observed; if the residue is above the gating pore because of depolarization, as in the inactivated state, then inward currents are observed. This study shows for the first time that functional characterization of omega pore currents is possible using a cultured cell line expressing mutant Ca(v)1.1 channels. Likewise, it is the first calcium channel mutation for complicated normokalaemic periodic paralysis.

Reliability Exercise of Ultrasound Salivary Glands in Sjögren's Disease: An International Web Training Initiative.

INTRODUCTION: Major salivary gland ultrasonography (SGUS) demonstrated its good metric properties as an outcome measure for diagnosing primary Sjögren's disease (SD). The objective was to assess SGUS reliability among sonographers with different levels of experience, using web training. METHODS: Sonographers from expert centers participated in the reliability exercise. Before exercises, training was done by videoconferencing. Reliability of the two most experienced sonographers (MES) was assessed and then compared to other sonographers. Intra-reader and inter-reader reliability of SGUS items were assessed by computing Cohen's κ coefficients. RESULTS: All sets were read twice by all 14 sonographers within a 4-month interval. Intra-reader reliability of MES was almost perfect for homogeneity, substantial for Outcome Measures in Rheumatology (OMERACT) scoring system (OMERACTss). Among LES (less experienced sonographers), reliability was moderate to almost perfect for homogeneity, fair to moderate for OMERACTss, and fair to almost perfect for binary OMERACTss. Inter-reader reliability between MES was almost perfect for homogeneity, substantial for diagnosis, moderate for OMERACTss, and substantial for binary OMERACTss. Compared to MES, reliabilities of LES were moderate to almost perfect for both homogeneity and diagnosis, only fair to moderate for OMERACTss, but increased in binary OMERACTss. CONCLUSIONS: Videoconferencing training sessions in an international reliability exercise could be an excellent tool to train experienced and less-experienced sonographers. SGUS homogeneity items is useful to distinguish normal from abnormal salivary glands parenchyma independently of diagnosis. Structural damage evaluations by OMERACT scoring system is a new comprehensive score to diagnose patients with SD and could be easily used by sonographers in a binary method.

The goal of this project was to evaluate the reliability of salivary gland ultrasonography in patients with Sjögren's disease using online training in an international study. Currently, salivary gland ultrasonography is routinely used only by European expert sonographers but few studies have studied intra-reader and inter-reader reliability, among less experienced international sonographers. Many salivary gland ultrasonography scoring systems are used today, but it is difficult to know how to put them into practice. Online training on an international level allows a significant number of practitioners to use the different scoring systems including the latest OMERACT (Outcome Measures in Rheumatology) score, which is simple and comprehensive. There were two phases to this project: A first step consisted in a training session by videoconferencing to all sonographers, the second step was an inter and intra-reader reliability exercises. The results of our study showed satisfactory results, especially for parenchyma homogeneity. Regarding the comprehensive OMERACT score, the results are quite disparate, notably for less experienced sonographers and could be explained by this new comprehensive scoring system. However, when binary OMERACT score (minor damage versus major damage of salivary gland parenchyma (OMERACT score 0­1 vs. 2­3) was employed, reliability increased and can be very useful for novice sonographers in routine practice because it does not require scoring of all the pathological features in Sjögren's disease. This study highlights the need to train non-experts interested in this field and demonstrates the potential for beginners to quickly become experts.

Association of genes in the NF-κB pathway with antibody-positive primary Sjögren's syndrome.

Primary Sjögren's syndrome (SS) is a systemic autoimmune inflammatory disease characterized by focal lymphocytic infiltrates in the lachrymal and salivary glands and autoantibodies against the SSA/Ro and SSB/La antigens. Experimental studies have shown an activation of NF-κB in primary SS. NF-κB activation results in inflammation and autoimmunity and is regulated by inhibitory and activating proteins. Genetic studies have shown an association between multiple autoimmune diseases and TNFAIP3 (A20) and TNIP1 (ABIN1), both repressors of NF-κB and of IKBKE (IKKε), which is an NF-κB activator. The aim of this study was to analyse single nucleotide polymorphisms (SNPs) in the IKBKE, NFKB1, TNIP1 and TNFAIP3 genes for association with primary SS. A total of 12 SNPs were genotyped in 1105 patients from Scandinavia (Sweden and Norway, n = 684) and the UK (n = 421) and 4460 controls (Scandinavia, n = 1662, UK, n = 2798). When patients were stratified for the presence of anti-SSA and/or anti-SSB antibodies (n = 868), case-control meta-analysis found an association between antibody-positive primary SS and two SNPs in TNIP1 (P = 3.4 × 10(-5) , OR = 1.33, 95%CI: 1.16-1.52 for rs3792783 and P = 1.3 × 10(-3) , OR = 1.21, 95%CI: 1.08-1.36 for rs7708392). A TNIP1 risk haplotype was associated with antibody-positive primary SS (P = 5.7 × 10(-3) , OR = 1.47, 95%CI: 1.12-1.92). There were no significant associations with IKBKE, NFKB1 or TNFAIP3 in the meta-analysis of the Scandinavian and UK cohorts. We conclude that polymorphisms in TNIP1 are associated with antibody-positive primary SS.

An easy-to-use high-throughput selection system for the discovery of recombinant protein binders from alternative scaffold libraries.

Selection by phage display is a popular and widely used technique for the discovery of recombinant protein binders from large protein libraries for therapeutic use. The protein library is displayed on the surface of bacteriophages which are amplified using bacteria, preferably Escherichia coli, to enrich binders in several selection rounds. Traditionally, the so-called panning procedure during which the phages are incubated with the target protein, washed and eluted is done manually, limiting the throughput. High-throughput systems with automated panning already in use often require high-priced equipment. Moreover, the bottleneck of the selection process is usually the screening and characterization. Therefore, having a high-throughput panning procedure without a scaled screening platform does not necessarily increase the discovery rate. Here, we present an easy-to-use high-throughput selection system with automated panning using cost-efficient equipment integrated into a workflow with high-throughput sequencing and a tailored screening step using biolayer-interferometry. The workflow has been developed for selections using two recombinant libraries, ADAPT (Albumin-binding domain-derived affinity proteins) and CaRA (Calcium-regulated affinity) and has been evaluated for three new targets. The newly established semi-automated system drastically reduced the hands-on time and increased robustness while the selection outcome, when compared to manual handling, was very similar in deep sequencing analysis and generated binders in the nanomolar affinity range. The developed selection system has shown to be highly versatile and has the potential to be applied to other binding domains for the discovery of new protein binders.

Variants in the DDX6-CXCR5 autoimmune disease risk locus influence the regulatory network in immune cells and salivary gland.

Fine mapping and bioinformatic analysis of the DDX6-CXCR5 genetic risk association in Sjögren's Disease (SjD) and Systemic Lupus Erythematosus (SLE) identified five common SNPs with functional evidence in immune cell types: rs4938573, rs57494551, rs4938572, rs4936443, rs7117261. Functional interrogation of nuclear protein binding affinity, enhancer/promoter regulatory activity, and chromatin-chromatin interactions in immune, salivary gland epithelial, and kidney epithelial cells revealed cell type-specific allelic effects for all five SNPs that expanded regulation beyond effects on DDX6 and CXCR5 expression. Mapping the local chromatin regulatory network revealed several additional genes of interest, including lnc-PHLDB1-1. Collectively, functional characterization implicated the risk alleles of these SNPs as modulators of promoter and/or enhancer activities that regulate cell type-specific expression of DDX6, CXCR5, and lnc-PHLDB1-1, among others. Further, these findings emphasize the importance of exploring the functional significance of SNPs in the context of complex chromatin architecture in disease-relevant cell types and tissues.