The Ser52Pro variant of transthyretin (TTR) produces aggressive, highly penetrant, autosomal-dominant systemic amyloidosis in persons heterozygous for the causative mutation. Together with a minor ...quantity of full-length wild-type and variant TTR, the main component of the ex vivo fibrils was the residue 49-127 fragment of the TTR variant, the portion of the TTR sequence that previously has been reported to be the principal constituent of type A, cardiac amyloid fibrils formed from wild-type TTR and other TTR variants Bergstrom J, et al. (2005) J Pathol 206(2):224–232. This specific truncation of Ser52Pro TTR was generated readily in vitro by limited proteolysis. In physiological conditions and under agitation the residue 49-127 proteolytic fragment rapidly and completely self-aggregates into typical amyloid fibrils. The remarkable susceptibility to such cleavage is likely caused by localized destabilization of the β-turn linking strands C and D caused by loss of the wild-type hydrogen-bonding network between the side chains of residues Ser52, Glu54, Ser50, and a water molecule, as revealed by the high-resolution crystallographic structure of Ser52Pro TTR. We thus provide a structural basis for the recently hypothesized, crucial pathogenic role of proteolytic cleavage in TTR amyloid fibrillogenesis. Binding of the natural ligands thyroxine or retinol-binding protein (RBP) by Ser52Pro variant TTR stabilizes the native tetrameric assembly, but neither protected the variant from proteolysis. However, binding of RBP, but not thyroxine, inhibited subsequent fibrillogenesis.
This study looked at using modified camelina oil to develop sustainable coatings that could replace those derived from petroleum-based materials for use in packaging and other industrial sectors. ...Solvent-free synthesis of maleic anhydride grafted camelina oil (MCO) was carried out at two different temperatures (200 and 230 °C) to obtain sustainable hydrophobic coating materials for paper substrates. Maleic anhydride grafting of camelina oil was confirmed with attenuated total reflectance-Fourier transform infrared and NMR spectroscopic techniques, and up to 16% grafting of maleic anhydride was achieved, as determined by the titration method. MCO, obtained at different reaction temperatures, was coated onto cellulosic paper and evaluated for its hydrophobicity, mechanical, oxygen, and water vapor barrier properties. Scanning electron microscopy indicated the homogeneous dispersion of coating material onto the paper substrate. MCO-coated papers (MCO-200C paper and MCO-230C paper) provided a water contact angle of above 90° which indicates that the modified oil was working as a hydrophobic coating. Water vapor permeability (WVP) testing of coated papers revealed a reduction in WVP of up to 94% in comparison to the uncoated paper. Moreover, an improved oxygen barrier property was also observed for paper coated with both types of MCO. Analysis of the mechanical properties showed a greater than 70% retention of tensile strength and up to a five-fold increase in elongation at break of coated versus uncoated papers. Overall, the results show that camelina oil, a renewable resource, can be modified to produce environmentally friendly hydrophobic coating materials with improved mechanical and water vapor barrier properties that can serve as a potential coating material in the packaging industry. The results of this research could find applications in the huge paper packaging industries, specially in food packaging.
Calcium Binds to Transthyretin with Low Affinity Cantarutti, Cristina; Mimmi, Maria Chiara; Verona, Guglielmo ...
Biomolecules (Basel, Switzerland),
08/2022, Letnik:
12, Številka:
8
Journal Article
Recenzirano
Odprti dostop
The plasma protein transthyretin (TTR), a transporter for thyroid hormones and retinol in plasma and cerebrospinal fluid, is responsible for the second most common type of systemic (ATTR) amyloidosis ...either in its wild type form or as a result of destabilizing genetic mutations that increase its aggregation propensity. The association between free calcium ions (Ca2+) and TTR is still debated, although recent work seems to suggest that calcium induces structural destabilization of TTR and promotes its aggregation at non-physiological low pH in vitro. We apply high-resolution NMR spectroscopy to investigate calcium binding to TTR showing the formation of labile interactions, which leave the native structure of TTR substantially unaltered. The effect of calcium binding on TTR-enhanced aggregation is also assessed at physiological pH through the mechano-enzymatic mechanism. Our results indicate that, even if the binding is weak, about 7% of TTR is likely to be Ca2+-bound in vivo and therefore more aggregation prone as we have shown that this interaction is able to increase the protein susceptibility to the proteolytic cleavage that leads to aggregation at physiological pH. These events, even if involving a minority of circulating TTR, may be relevant for ATTR, a pathology that takes several decades to develop.
Abstract
Systemic amyloidosis is caused by misfolding and aggregation of globular proteins
in vivo
for which effective treatments are urgently needed. Inhibition of protein self-aggregation ...represents an attractive therapeutic strategy. Studies on the amyloidogenic variant of β
2
-microglobulin, D76N, causing hereditary systemic amyloidosis, have become particularly relevant since fibrils are formed
in vitro
in physiologically relevant conditions. Here we compare the potency of two previously described inhibitors of wild type β
2
-microglobulin fibrillogenesis, doxycycline and single domain antibodies (nanobodies). The β
2
-microglobulin -binding nanobody, Nb24, more potently inhibits D76N β
2
-microglobulin fibrillogenesis than doxycycline with complete abrogation of fibril formation. In β
2
-microglobulin knock out mice, the D76N β
2
-microglobulin/ Nb24 pre-formed complex, is cleared from the circulation at the same rate as the uncomplexed protein; however, the analysis of tissue distribution reveals that the interaction with the antibody reduces the concentration of the variant protein in the heart but does not modify the tissue distribution of wild type β
2
-microglobulin. These findings strongly support the potential therapeutic use of this antibody in the treatment of systemic amyloidosis.
The tissue diagnosis of amyloidosis and confirmation of fibril protein type, which are crucial for clinical management, have traditionally relied on Congo red (CR) staining followed by ...immunohistochemistry (IHC) using fibril protein specific antibodies. However, amyloid IHC is qualitative, non‐standardised, requires operator expertise, and not infrequently fails to produce definitive results. More recently, laser dissection mass spectrometry (LDMS) has been developed as an alternative method to characterise amyloid in tissue sections. We sought to compare these techniques in a real world setting. During 2017, we performed LDMS on 640 formalin‐fixed biopsies containing amyloid (CR+ve) comprising all 320 cases that could not be typed by IHC (IHC−ve) and 320 randomly selected CR+ve samples that had been typed (IHC+ve). In addition, we studied 60 biopsies from patients in whom there was a strong suspicion of amyloidosis, but in whom histology was non‐diagnostic (CR–ve). Comprehensive clinical assessments were conducted in 532 (76%) of cases. Among the 640 CR+ve samples, 602 (94%) contained ≥2 of 3 amyloid signature proteins (ASPs) on LDMS (ASP+ve) supporting the presence of amyloid. A total of 49 of the 60 CR‐ve samples were ASP–ve; 7 of 11 that were ASP+ve were glomerular. The amyloid fibril protein was identified by LDMS in 255 of 320 (80%) of the IHC–ve samples and in a total of 545 of 640 (85%) cases overall. The LDMS and IHC techniques yielded discordant results in only 7 of 320 (2%) cases. CR histology and LDMS are corroborative for diagnosis of amyloid, but LDMS is superior to IHC for confirming amyloid type.
Systemic amyloidosis is a serious disease which is caused when normal circulating proteins misfold and aggregate extracellularly as insoluble fibrillary deposits throughout the body. This commonly ...results in cardiac, renal and neurological damage. The tissue target, progression and outcome of the disease depends on the type of protein forming the fibril deposit, and its correct identification is central to determining therapy. Proteomics is now used routinely in our centre to type amyloid; over the past 7 years we have examined over 2000 clinical samples. Proteomics results are linked directly to our patient database using a simple algorithm to automatically highlight the most likely amyloidogenic protein. Whilst the approach has proved very successful, we have encountered a number of challenges, including poor sample recovery, limited enzymatic digestion, the presence of multiple amyloidogenic proteins and the identification of pathogenic variants. Our proteomics procedures and approaches to resolving difficult issues are outlined.
Lethal necrotizing fasciitis caused by Streptococcus pyogenes is characterized by a paucity of neutrophils at the site of infection. Interleukin (IL)–8, which is important for neutrophil ...transmigration and activation, can be degraded by S. pyogenes. Blood isolates of S. pyogenes were better able to degrade human IL-8 than throat isolates. Degradation of IL-8 was the result of a single specific cleavage between 59glutamine and 60arginine within the IL-8 C-terminal α helix. Cleaved IL-8 reduced neutrophil activation and migration. IL-8–cleaving activity was found in partially purified supernatant of a necrotizing fasciitis isolate, and this activity was associated with an ∼150-kDa fraction containing S. pyogenes cell envelope proteinase (SpyCEP). IL-8–cleaving activity corresponded with the presence of SpyCEP in the supernatant. Cleavage of IL-8 by S. pyogenes represents an unprecedented mechanism of immune evasion, effectively preventing IL-8 C-terminus–mediated endothelial translocation and subsequent recruitment of neutrophils
The Galaxy and Mass Assembly (GAMA) survey has been operating since 2008 February on the 3.9-m Anglo-Australian Telescope using the AAOmega fibre-fed spectrograph facility to acquire spectra with a ...resolution of R≈ 1300 for 120 862 Sloan Digital Sky Survey selected galaxies. The target catalogue constitutes three contiguous equatorial regions centred at 9h (G09), 12h (G12) and 14.5h (G15) each of 12 × 4 deg2 to limiting fluxes of r
pet < 19.4, r
pet < 19.8 and r
pet < 19.4 mag, respectively (and additional limits at other wavelengths). Spectra and reliable redshifts have been acquired for over 98 per cent of the galaxies within these limits. Here we present the survey footprint, progression, data reduction, redshifting, re-redshifting, an assessment of data quality after 3 yr, additional image analysis products (including ugrizYJHK photometry, Sérsic profiles and photometric redshifts), observing mask and construction of our core survey catalogue (GamaCore). From this we create three science-ready catalogues: GamaCoreDR1 for public release, which includes data acquired during year 1 of operations within specified magnitude limits (2008 February to April); GamaCoreMainSurvey containing all data above our survey limits for use by the GAMA Team and collaborators; and GamaCoreAtlasSV containing year 1, 2 and 3 data matched to Herschel-ATLAS science demonstration data. These catalogues along with the associated spectra, stamps and profiles can be accessed via the GAMA website: http://www.gama-survey.org/
A critical challenge of training deep learning models in the Digital Pathology (DP) domain is the high annotation cost by medical experts. One way to tackle this issue is via transfer learning from ...the natural image domain (NI), where the annotation cost is considerably cheaper. Cross-domain transfer learning from NI to DP is shown to be successful via class labels 1. One potential weakness of relying on class labels is the lack of spatial information, which can be obtained from spatial labels such as full pixel-wise segmentation labels and scribble labels. We demonstrate that scribble labels from NI domain can boost the performance of DP models on two cancer classification datasets (Patch Camelyon Breast Cancer and Colorectal Cancer dataset). Furthermore, we show that models trained with scribble labels yield the same performance boost as full pixel-wise segmentation labels despite being significantly easier and faster to collect.
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