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SignificanceArtificial synthesis of spider silk has been actively pursued. However, until now, the natural mechanical properties of spider silk have been largely unreproducible. We thoroughly investigated the genomes and transcripts of four related species of orb-weaver spiders as well as the proteins in their silk threads. Then, in addition to spidroin, we found several low-molecular-weight proteins in common. Interestingly, the low-molecular-weight protein component of spider dragline silk doubled the tensile strength of artificial silk–based material. This discovery will greatly advance the industry and research on the use of protein-based materials. Dragline silk of golden orb-weaver spiders (Nephilinae) is noted for its unsurpassed toughness, combining extraordinary extensibility and tensile strength, suggesting industrial application as a sustainable biopolymer material. To pinpoint the molecular composition of dragline silk and the roles of its constituents in achieving its mechanical properties, we report a multiomics approach, combining high-quality genome sequencing and assembly, silk gland transcriptomics, and dragline silk proteomics of four Nephilinae spiders. We observed the consistent presence of the MaSp3B spidroin unique to this subfamily as well as several nonspidroin SpiCE proteins. Artificial synthesis and the combination of these components in vitro showed that the multicomponent nature of dragline silk, including MaSp3B and SpiCE, along with MaSp1 and MaSp2, is essential to realize the mechanical properties of spider dragline silk.

The complement system is essential for an adequate immune response. Much attention has been given to the role of complement in disease. However, to better understand complement in pathology, it is crucial to first analyze this system under different physiological conditions. The aim of the present study was therefore to investigate the inter-individual variation in complement activity and the influences of age and sex. Complement levels and functional activity were determined in 120 healthy volunteers, 60 women, 60 men, age range 20-69 year. Serum functional activity of the classical pathway (CP), lectin pathway activated by mannan (MBL-LP) and alternative pathway (AP) was measured in sera, using deposition of C5b-9 as readout. In addition, levels of C1q, MBL, MASP-1, MASP-2, ficolin-2, ficolin-3, C2, C4, C3, C5, C6, C7, C8, C9, factor B, factor D, properdin, C1-inhibitor and C4b-binding protein, were determined. Age- and sex-related differences were evaluated. Significantly lower AP activity was found in females compared to males. Further analysis of the AP revealed lower C3 and properdin levels in females, while factor D concentrations were higher. MBL-LP activity was not influenced by sex, but MBL and ficolin-3 levels were significantly lower in females compared to males. There were no significant differences in CP activity or CP components between females and males, nevertheless females had significantly lower levels of the terminal components. The CP and AP activity was significantly higher in the elderly, in contrast to MBL-LP activity. Moreover, C1-inhibitor, C5, C8, and C9 increased with age in contrast to a decrease of factor D and C3 levels. In-depth analysis of the functional activity assays revealed that MBL-LP activity was predominantly dependent on MBL and MASP-2 concentration, whereas CP activity relied on C2, C1-inhibitor and C5 levels. AP activity was strongly and directly associated with levels of C3, factor B and C5. This study demonstrated significant sex and age-related differences in complement levels and functionality in the healthy population. Therefore, age and sex analysis should be taken into consideration when discussing complement-related pathologies and subsequent complement-targeted therapies.

Spider silk is one of the best natural fibers and has superior mechanical properties. However, the large-scale harvesting of spider silk by rearing spiders is not feasible, due to their territorial and cannibalistic behaviors. The silkworm, Bombyx mori, has been the most well known silk producer for thousands of years and has been considered an ideal bioreactor for producing exogenous proteins, including spider silk. Previous attempts using transposon-mediated transgenic silkworms to produce spider silk could not achieve efficient yields, due to variable promoter activities and endogenous silk fibroin protein expression. Here, we report a massive spider silk production system in B. mori by using transcription activator-like effector nuclease-mediated homology-directed repair to replace the silkworm fibroin heavy chain gene (FibH) with the major ampullate spidroin-1 gene (MaSp1) in the spider Nephila clavipes. We successfully replaced the ∼16-kb endogenous FibH gene with a 1.6-kb MaSp1 gene fused with a 1.1-kb partial FibH sequence and achieved up to 35.2% chimeric MaSp1 protein amounts in transformed cocoon shells. The presence of the MaSp1 peptide significantly changed the mechanical characteristics of the silk fiber, especially the extensibility. Our study provides a native promoter-driven, highly efficient system for expressing the heterologous spider silk gene instead of the transposon-based, random insertion of the spider gene into the silkworm genome. Targeted MaSp1 integration into silkworm silk glands provides a paradigm for the large-scale production of spider silk protein with genetically modified silkworms, and this approach will shed light on developing new biomaterials.

The most commonly used markers to assess complement activation are split products that are produced through activation of all three pathways and are located downstream of C3. In contrast, C4d derives from the cleavage of C4 and indicates either classical (CP) or lectin pathway (LP) activation. Although C4d is perfectly able to distinguish between CP/LP and alternative pathway (AP) activation, no well-established markers are available to differentiate between early CP and LP activation. Active enzymes of both pathways (C1s/C1r for the CP, MASP-1/MASP-2 for the LP) are regulated by C1 esterase inhibitor (C1-INH) through the formation of covalent complexes. Aim of this study was to develop validated immunoassays detecting C1s/C1-INH and MASP-1/C1-INH complex levels. Measurement of the complexes reveals information about the involvement of the respective pathways in complement-mediated diseases. Two sandwich ELISAs detecting C1s/C1-INH and MASP-1/C1-INH complex were developed and tested thoroughly, and it was investigated whether C1s/C1-INH and MASP-1/C1-INH complexes could serve as markers for either early CP or LP activation. In addition, a reference range for these complexes in healthy adults was defined, and the assays were clinically validated utilizing samples of 414 COVID-19 patients and 96 healthy controls. The immunoassays can reliably measure C1s/C1-INH and MASP-1/C1-INH complex concentrations in EDTA plasma from healthy and diseased individuals. Both complex levels are increased in serum when activated with zymosan, making them suitable markers for early classical and early lectin pathway activation. Furthermore, measurements of C1-INH complexes in 96 healthy adults showed normally distributed C1s/C1-INH complex levels with a physiological concentration of 1846 ± 1060 ng/mL (mean ± 2SD) and right-skewed distribution of MASP-1/C1-INH complex levels with a median concentration of 36.9 (13.18 - 87.89) ng/mL (2.5-97.5 percentile range), while levels of both complexes were increased in COVID-19 patients (p<0.0001). The newly developed assays measure C1-INH complex levels in an accurate way. C1s/C1-INH and MASP-1/C1-INH complexes are suitable markers to assess early classical and lectin pathway activation. An initial reference range was set and first studies showed that these markers have added value for investigating and unraveling complement activation in human disease.

The major ampullate Spidroin 1 (MaSp1) is the main protein of the dragline spider silk. The C-terminal (CT) domain of MaSp1 is crucial for the self-assembly into fibers but the details of how it contributes to the fiber formation remain unsolved. Here we exploit the fact that the CT domain can form silk-like fibers by itself to gain knowledge about this transition. Structural investigations of fibers from recombinantly produced CT domain from E. australis MaSp1 reveal an α-helix to β-sheet transition upon fiber formation and highlight the helix N o 4 segment as most likely to initiate the structural conversion. This prediction is corroborated by the finding that a peptide corresponding to helix N o 4 has the ability of pH-induced conversion into β-sheets and self-assembly into nanofibrils. Our results provide structural information about the CT domain in fiber form and clues about its role in triggering the structural conversion of spidroins during fiber assembly. The mechanical properties of spider silk are a consequence of the structural organisation of proteins known as spidroins. Here the authors investigate the structure of the fibers formed by a C-terminal domain of a major spidroin: the study elucidates the mechanisms by which spidroins are transformed from soluble form into a fiber.

Plant growth is coordinated with environmental factors, including water availability during times of drought. Microtubules influence cell expansion; however, the mechanisms by which environmental signals impinge upon microtubule organization and whether microtubule-related factors limit growth during drought remains unclear. We found that three Clade E Growth-Regulating (EGR) Type 2C protein phosphatases act as negative growth regulators to restrain growth during drought. Quantitative phosphoproteomics indicated that EGRs target cytoskeleton and plasma membrane-associated proteins. Of these, Microtubule-Associated Stress Protein 1 (MASP1), an uncharacterized protein, increased in abundance during stress treatment and could bind, bundle, and stabilize microtubules in vitro. MASP1 overexpression enhanced growth, in vivo microtubule stability, and recovery of microtubule organization during drought acclimation. These MASP1 functions in vivo were dependent on phosphorylation of a single serine. For all EGR and MASP1 mutants and transgenic lines examined, enhanced microtubule recovery and stability were associated with increased growth during drought stress. The EGR-MASP1 system selectively regulates microtubule recovery and stability to adjust plant growth and cell expansion in response to changing environmental conditions. Modification of EGR-MASP1 signaling may be useful to circumvent negative growth regulation limiting plant productivity. EGRs are likely to regulate additional proteins involved in microtubule stability and stress signaling.

MASP-3 was discovered 15 years ago as the third mannan-binding lectin (MBL)-associated serine protease of the complement lectin pathway. Lacking any verified substrate its role remained ambiguous. MASP-3 was shown to compete with a key lectin pathway enzyme MASP-2 for MBL binding, and was therefore considered to be a negative complement regulator. Later, knock-out mice experiments suggested that MASP-1 and/or MASP-3 play important roles in complement pro-factor D (pro-FD) maturation. However, studies on a MASP-1/MASP-3-deficient human patient produced contradicting results. In normal resting blood unperturbed by ongoing coagulation or complement activation, factor D is present predominantly in its active form, suggesting that resting blood contains at least one pro-FD activating proteinase that is not a direct initiator of coagulation or complement activation. We have recently showed that all three MASPs can activate pro-FD in vitro . In resting blood, however, using our previously evolved MASP-1 and MASP-2 inhibitors we proved that neither MASP-1 nor MASP-2 activates pro-FD. Other plasma proteinases, particularly MASP-3, remained candidates for that function. For this study we evolved a specific MASP-3 inhibitor and unambiguously proved that activated MASP-3 is the exclusive pro-FD activator in resting blood, which demonstrates a fundamental link between the lectin and alternative pathways.

BackgroundThe biological processes involved in the development of structural changes associated with radiographic axial spondyloarthritis (r-axSpA) remain largely unraveled. Still, high disease activity, elevated C-reactive protein (CRP), and existing syndesmophytes are associated with radiographic progression. The complement system is an inflammation-generating part of the innate immune system. Animal models have shown inhibition of complement activation to diminish structural changes associated with axSpA [1].ObjectivesThis project aimed to investigate complement activation and serum levels of complement proteins and their correlations with radiographic spinal progression over a two years follow-up period in a longitudinal cohort of axSpA patients with active r-axSpA, and high risk of radiographic progression, recruited from the randomized controlled trial CONSUL.MethodsAll patients had active r-axSpA and risk factors for radiographic spinal progression (BASDAI ≥4, and elevated CRP and/ or ≥1 syndesmophyte(s)). Serum samples were collected at baseline (n = 96) and after 108 weeks (n = 89) of TNF-I therapy and analyzed by immunoassays for complement lectin pathway proteins (L-ficolin, M-ficolin, H-ficolin, CL-L1, MBL, MASP-1, MASP-2, MASP-3, and MAp44) and the complement activation product C3dg. X-rays were performed at baseline and after 108 weeks and read blinded for clinical data and chronology by three independent expert readers. New bone formation was defined as the growth of syndesmophyte(s) and/or new syndesmophyte(s) determined by 3-reader-agreement.ResultsPatient characteristics are shown in Table 1. In total, 19 patients developed new bone formation at week 108. Baseline serum levels of MASP-1, MASP-2, and C3dg were elevated in patients with new bone formation, whereas baseline serum levels of MASP-3 were decreased (all p<0.05). Baseline MASP-1, MASP-3, and C3dg predicted the development of new bone formation in a univariate logistic regression analysis, whereas CRP did not. Baseline MASP-1, MASP-3, and C3dg remained significant in a multivariate logistic regression analysis. L-ficolin and C3dg levels at week 108 were elevated in patients with new bone formation, and the serum levels at week 108 predicted development of new bone formation in a univariate logistic regression analysis. In a multivariate regression analysis, C3dg remained significant (p<0.05).ConclusionIn this study, complement activation measured by C3dg and serum levels of MASP-1 and MASP-3, prior to TNF-I therapy, predicted development of new bone formation at week 108. Furthermore, elevated levels of C3dg and L-ficolin at week 108 were associated with new bone formation. These findings support the involvement of complement activation in new bone formation in r-axSpA.Reference[1]Yang C et al. Sci Rep. 2016.Table 1.Baseline demographics of the investigated patient population from the CONSUL RCT (n = 96)Age, median (IQR)37 (31-45)Male, n (%)70 (73)HLA-B27 positive, n (%)80 (83)Previous bDMARD treatment, n (%)21 (22)Symptom duration in years, median (IQR)12 (6.0-20)Smoking, n (%)36 (38)mSASSS, median (IQR)5.0 (0.3-18)Syndesmophytes, median (IQR)1.8 (0-6.3)Presence of ≥1 syndesmophyte(s)δ, n (%)47 (49)CRP, median (IQR)9.2 (3.2-19)Elevated CRP (>5 mg/L), n (%)64 (67)ASDAS-CRP, median (IQR)3.6 (3.1-4.1)BASDAI, median (IQR)6.2 (5.2-6.8)δDetermined by 3 expert readers blinded for clinical data.Figure 1.a) Baseline MASP-1, MASP-2, MASP-3, and C3dg levels according to development of new bone formation at week 108. b) L-ficolin and C3dg levels at week 108 according to development of new bone formation at week 108. New bone formation: growth of syndesmophytes and/or new syndesmophytes determined by 3-reader-agreement. p-values indicate comparisons between groups by t-test. Bars indicate median and IQR (MASP-1, MASP-2, MASP-3, C3dg), and mean and sd (L-ficolin).Acknowledgements:NIL.Disclosure of InterestsClara Elbæk Mistegaard: None declared, Anne Troldborg: None declared, Anne Gitte Loft Speakers bureau: AbbVie, MSD, Novartis, Pfizer, UCB, Consultant of: AbbVie, MSD, Novartis, Pfizer, UCB, Grant/research support from: Novartis, Steffen Thiel: None declared, Burkhard Muche: None declared, Valeria Rios Rodriguez: None declared, Murat Torgutalp: None declared, Mikhail Protopopov: None declared, Joachim Listing: None declared, Joachim Sieper: None declared, Denis Poddubnyy: None declared, Fabian Proft Speakers bureau: AMGEN, AbbVie, BMS, Celgene, Janssen, MSD, Novartis, Pfizer, Roche, UCB, Consultant of: Novartis, Grant/research support from: Novartis, Lilly, UCB.

BackgroundThe pathogenesis of axial spondyloarthritis (axSpA) remains largely unexplained, but recent evidence supports an involvement of the innate immune system. The lectin pathway (LP) of complement activation plays an essential role in innate immunity. Previous studies have shown that elevated levels of specific complement proteins are associated with disease activity in axSpA. However, little is known about alterations in LP proteins and complement activation in relation to treatment with TNF-inhibitors (TNF-I).ObjectivesTo determine changes in LP protein levels and complement activation in a longitudinal multi-center cohort of axSpA-patients with high disease activity from the RCT CONSUL before and after 12 weeks of treatment with a TNF-I. Furthermore, to investigate the correlations between LP protein levels, and disease activity, and treatment response.MethodsSerum samples at baseline and after 12 weeks of treatment with the TNF-I golimumab were obtained from 108 radiographic axSpA-patients with high disease activity (BASDAI ≥4) and high risk for radiographic progression (elevated CRP or ≥1 syndesmophyte present at inclusion) recruited from the CONSUL cohort. Serum levels of the LP proteins MBL, CL-L1, H-ficolin, L-ficolin, M-ficolin, MASP-1, MASP-2, MASP-3, and MAp44, and complement activation product C3dg were measured by immunoassays.ResultsSerum levels of L-ficolin, M-ficolin, MBL, MAp44, and C3dg decreased significantly after 12 weeks of treatment with TNF-I, whereas serum levels of MASP-3 and CL-L1 increased significantly (Figure 1). After adjustment for CRP, significant changes were observed for L-ficolin, M-ficolin, MASP-1, MASP-2, MASP-3, and MAp44. Baseline serum levels of L-ficolin, M-ficolin, and C3dg correlated positively with baseline ASDAS-CRP (Spearman’s rho 0.290, 0.190, and 0.328, respectively, all p<0.05), whereas baseline MASP-1 and MAp44 correlated negatively with baseline ASDAS-CRP (Spearman’s rho -0.204, and -0.218, respectively, both p<0.05). In a univariate logistic regression analysis, baseline serum levels of MASP-1 predicted achievement of clinical important improvement (ΔASDAS≥1.1) at week 12, and baseline serum levels of C3dg predicted inactive disease (ASDAS<1.3) at week 12. In corresponding multivariate logistic regression analyses, only C3dg remained significantly associated with inactive disease (ASDAS<1.3) (p = 0.025).ConclusionBaseline serum levels of L-ficolin, M-ficolin and C3dg correlated positively with baseline ASDAS-CRP, and the protein serum levels decreased significantly after TNF-I therapy. Baseline levels of complement activation measured by C3dg predicted achievement of inactive disease (ASDAS<1.3) at week 12 in both a univariate and a multivariate logistic regression analysis. Our findings suggest an involvement of the complement system in the underlying inflammatory processes of axSpA and prompt further investigations.Table 1.Baseline demographics of the investigated patient population from the CONSUL RCT (n = 108)Age, median (IQR)38 (31-49)Male, n (%)77 (71)HLA-B27 positive, n (%)89 (82)Previous treatment with bDMARD, n (%)26 (24)Time since diagnosis in years, median (IQR)3.0 (0.0-9.0)Years since onset of symptoms, median (IQR)12 (6.0-21.0)CRP, mg/L (IQR)9.1 (4.1-20)Elevated CRP (> 5mg/L), n (%)75 (69)mSASSS, median (IQR)4.3 (0.3-18)#Syndemophytes present, median (IQR)1.6 (0.0-6.3)#Presence of ≥1 syndesmophyte(s) §, n (%)43 (48) #ASDAS-CRP, median (IQR)3.6 (3.1-4.1)BASDAI, median (IQR)6.2 (5.2-7.0)BASFI, median (IQR)5.2 (4.0-6.4)§ Determined by 3 calibrated readers blinded for clinical data. # data available on n = 89Figure 1.Differences in lectin pathway protein serum levels were compared by paired t-tests. Bars indicate median and IQR (MASP-3, MAp44, and C3dg) or mean and sd (L-ficolin, M-ficolin, MBL, and CL-L1).REFERENCES:NIL.Acknowledgements:NIL.Disclosure of InterestsClara Elbæk Mistegaard: None declared, Anne Troldborg: None declared, Anne Gitte Loft Speakers bureau: AbbVie, MSD, Novartis, Pfizer, and UCB, Consultant of: AbbVie, MSD, Novartis, Pfizer, and UCB, Grant/research support from: Novartis, Steffen Thiel: None declared, Burkhard Muche: None declared, Valeria Rios Rodriguez: None declared, Murat Torgutalp: None declared, Mikhail Protopopov: None declared, Joachim Listing: None declared, Joachim Sieper: None declared, Denis Poddubnyy: None declared, Fabian Proft Speakers bureau: AbbVie, AMGEN, BMS, Celgene, Hexal, Janssen, MSD, Novartis, Pfizer, Roche und UCB, Consultant of: AbbVie, AMGEN, BMS, Celgene, Hexal, Janssen, MSD, Novartis, Pfizer, Roche und UCB, Grant/research support from: Novartis, Eli Lilly and UCB.

BackgroundAxial spondyloarthritis (axSpA) is a chronic inflammatory rheumatic disease without specific diagnostic biomarkers. However, recent evidence suggests an involvement of the innate immune system in the pathogenesis of axSpA [1]. The lectin pathway of complement activation serves essential functions in the innate immune system, regulates homeostasis and development. We have previously shown the lectin pathway proteins (LPPs) L-ficolin and M-ficolin to be elevated in axSpA-patients compared with clinically relevant controls with unspecific low back pain (uLBP).ObjectivesTo investigate the diagnostic potential of LPP levels in patients recruited from a well-defined cross-sectional prospective cohort (OptiRef), including newly diagnosed axSpA-patients and uLBP-patients.MethodsSerum samples were obtained from 515 individuals from the OptiRef cohort; 151 newly diagnosed axSpA-patients and 364 uLBP-patients [2]. All patients were assessed according to a standardized protocol, incl. clinical information as demographics, SpA features, symptom duration, and disease activity. Routine lab tests (HLA-B27 and CRP) were performed. Imaging (x-ray and MRI) was performed if deemed clinically relevant. Serum levels of all 10 LPPs (MBL, CL-L1, M-ficolin, H-ficolin, L-ficolin, MASP-1, MASP-2, MASP-3, MAp19, and MAp44) and the complement activation product C3dg were measured by immunoassays.ResultsPatient characteristics are shown in Table 1. L-ficolin, MASP-2, and C3dg serum levels were increased in axSpA-patients compared with uLBP-patients, whereas CL-L1 and MASP-3 serum levels were decreased (Figure 1). After adjustments for CRP, C3dg serum levels remained significantly increased in axSpA-patients, whereas M-ficolin and MASP-3 serum levels were decreased in axSpA-patients. The diagnostic potential of combining either L-ficolin, MASP-3, and C3dg with HLA-B27 increased specificity to 93-95% compared with HLA-B27 alone (77%) but decreased sensitivity to 29-33% compared with HLA-B27 alone (83%). In a univariate logistic regression analysis, CL-L1, MASP-2, MASP-3, and C3dg were associated with an axSpA-diagnosis, and C3dg and MASP-3 remained significant in a multivariate logistic regression analysis.ConclusionIn this study, serum levels of C3dg, MASP-3, and CL-L1 differed significantly between axSpA-patients and uLBP-patients after adjustment for CRP. Although combining HLA-B27 with measurements of L-ficolin, MASP-3, and C3dg increased diagnostic specificity for axSpA, it seems unjustified due to the concomitant loss of sensitivity. However, both C3dg and MASP-3 were associated with the axSpA-diagnosis in multivariate logistic regression, indicating the complement system’s involvement in axSpA-pathogenesis. This should be further investigated.References[1]Ambarus, C., et al. Curr Opin Rheumatol. 2012[2]Poddubnyy, D., et al. Rheumatology (Oxford). 2021Table 1.Patient characteristics of the included patients from the OptiRef cohortTotal cohortn = 515axSpAn = 151uLBPn = 364p-value*Median age, years (IQR)36 (29-45)33 (26-40)38 (30-46)<0.001aMales, n (%)240 (47)83 (55)157 (43)0.008bHLA-B27 positive, n (%)203 (41)121 (83)θ82 (23)θθ<0.001bSmoking, n (%)138 (30)47 (31)91 (25)0.154bBMI, median (IQR)24 (22-27)24 (22-26)ϵ24 (22-28)ϵϵ0.464aSymptom duration, years median (IQR)6 (2-12)5 (2-10)7 (2-14)0.027aInflammatory back pain, n (%)210 (41)130 (86)175 (48)<0.001bGood response of back pain to NSAID, n (%)274 (73)102 (84)σ172 (68)σσ0.002bElevated CRP (> 5 mg/L), n (%)91 (18)53 (35)Δ38 (10)ΔΔ<0.001bRadiographic axSpA, n (%)77 (59)ASDAS-CRP, median (IQR)2.7 (1.8-3.3)§BASDAI, median (IQR)4.6 (2.9-5.7)ω* axSpA vs. uLBP. a Mann Whitney U test. b Chi2 test. Notations in the table indicate available data. If no marks, data were available on all patients. θ n=146. θθ n=353 ϵ n=141. ϵϵ n=340. σ n=122. σσ n=252. Δ n=150.ΔΔ n=358. § n=142. ω n=148.Figure 1.Serum levels of significantly altered complement LPPs in the two patient groups.Acknowledgements:NIL.Disclosure of InterestsClara Elbæk Mistegaard: None declared, Anne Troldborg: None declared, Anne Gitte Loft: None declared, Steffen Thiel: None declared, Laura Spiller: None declared, Valeria Rios Rodriguez: None declared, Burkhard Muche: None declared, Judith Rademacher: None declared, Anne-Katrin Weber: None declared, Susanne Lueders: None declared, Joachim Sieper: None declared, Denis Poddubnyy: None declared, Fabian Proft Speakers bureau: AMGEN, AbbVie, BMS, Celgene, Janssen, MSD, Novartis, Pfizer, Roche, UCB, Consultant of: Novartis, Grant/research support from: Novartis, Lilly, UCB.