High Molecular Weight Aggregates Research Articles

Indigestible proteins and peptide-bound AGEs in ultra-processed foods: Dulce de leche as a model of severely transformed protein food systems

Caramel milk confectioneries, such as dulce de leche, are produced by simmering whole or reconstituted milk with sugar and alkali at temperatures ranging from 115 to 130 °C for several hours. This process triggers extensive non-enzymatic browning through the Maillard reaction, leading to significant modifications in milk proteins. Our research focused on the effects of such severe processing conditions on the structural integrity and digestibility of milk proteins, using dulce de leche as a model for severely transformed food protein systems. Due to structural modifications, covalent interchain cross-links, and sugar-derived bridging moieties, milk proteins in dulce de leche form high molecular weight aggregates that are not resolvable by SDS-PAGE. Proteomic analysis revealed that all major milk proteins are involved in the formation of these covalent molecular complexes. When subjected to static gastro-duodenal digestion using both adult and infant models, these macroaggregates showed resistance to pepsin and marginal susceptibility to duodenal proteases. High pressure liquid chromatography-tandem mass spectrometry (HPLC-MS/MS)-based peptidomic analysis of dulce de leche digests allowed identifying variously sized polypeptides bearing the hallmarks of the Maillard reaction. However, many MS signals could not be assigned due to unknown modifications. The prevalence of free and peptide-bound advanced glycated end products (AGEs) in caramel milk products and other ultra-processed foods poses significant health concerns, stressing the need for a comprehensive dedicated evaluation.

Leveraging High-Throughput Analytics and Automation to Rapidly Develop High-Concentration mAb Formulations: Integrated Excipient Compatibility and Viscosity Screening

Abstract Background Formulation screening is essential to experimentally balance stability and viscosity in high-concentration mAb formulations. We developed a high-throughput approach with automated sample preparation and analytical workflows to enable the integrated assessment of excipient compatibility and viscosity of mAb formulations. Methods Ninety-six formulations of a trastuzumab biosimilar were screened by combining 8 types of excipient modifiers with 4 types of buffers across a pH range of 4.5 to 7.5. Key stability risks, including high molecular weight (HMW) aggregation and fragmentation, were thoroughly assessed along with viscosity at high concentrations. Additionally, several biophysical parameters were evaluated for their ability to predict stability or viscosity outcomes. Multiple linear regression was applied to fit the data and identify key factors. Results The optimal pH range for the trastuzumab biosimilar was found to be 5.0 to 6.5, based on opposing pH dependencies for stability and viscosity. Buffer type had a minor effect on viscosity and fragmentation but played a significant role in influencing HMW aggregates, with Na-acetate and histidine-HCl being the best candidates. The impact of excipient modifiers on viscosity, HMW, and fragmentation depended on both pH and buffer type, showing strong interactions among factors. Arginine-HCl and lysine-HCl effectively lowered viscosity of the trastuzumab biosimilar at pH levels above 6.0, while glycine formulations were more effective at reducing viscosity below pH 6.0. Histidine-HCl, arginine-HCl, and lysine-HCl lowered the risk of HMW aggregation, whereas formulations containing Na-phosphate or NaCl showed higher HMW aggregation. Formulations with arginine-HCl, lysine-HCl, and NaCl demonstrated a rapid increase in fragmentation at pH levels below 5.0, while Na-aspartate formulations showed increased fragmentation at pH levels above 6.5. Conclusion Hence, it is important to optimize the levels of each chosen excipient in the formulation study to balance their benefits against potential incompatibilities. This study serves as a foundation for identifying high-concentration antibody formulations using a high-throughput approach, where minimal materials are required, and optimized formulation design spaces can be quickly identified.

Analysis of aggregation profile of glucagon using SEC-HPLC and FFF-MALS methods.

Recently, the first generic glucagon for injection was approved for the treatment of severe hypoglycemia. Unlike its brand name recombinant glucagon, the generic glucagon is synthetic. Since glucagon has a high propensity to form aggregates in solution, it is essential to assess the aggregation profile of the synthetic glucagon compared to the recombinant glucagon. In this study, two robust separation methods, size-exclusion chromatography (SEC-HPLC) and field-flow fractionation coupled with a multi-angle light scattering detector (FFF-MALS), were employed to characterize generic and brand glucagon aggregation in six lots (three newly released, three expired). The presence of aggregation in samples was determined from the generated chromatograms and analyzed. The study showed that both products have comparable aggregation profiles. The SEC-HPLC demonstrated that in both glucagon versions, the expired lots had a higher percentage of dimers than the newly released lots, but even at expiration, the amount was negligible (∼0.1%). The FFF-MALS method did not detect any dimers or higher molecular weight aggregates. Further evaluation of the detection limit found that FFF-MALS was unable to detect aggregates at amounts lower than 0.5% of total glucagon. The negligible amounts of dimer detected in the generic and brand glucagon indicate that both versions are physically stable and are not prone to aggregation under clinically relevant conditions.

The new missense G376V-TDP-43 variant induces late-onset distal myopathy but not amyotrophic lateral sclerosis.

TAR DNA binding protein of 43 kDa (TDP-43)-positive inclusions in neurons are a hallmark of several neurodegenerative diseases including familial amyotrophic lateral sclerosis (fALS) caused by pathogenic TARDBP variants as well as more common non-Mendelian sporadic ALS (sALS). Here we report a G376V-TDP-43 missense variant in the C-terminal prion-like domain of the protein in two French families affected by an autosomal dominant myopathy but not fulfilling diagnostic criteria for ALS. Patients from both families presented with progressive weakness and atrophy of distal muscles, starting in their fifth to seventh decade. Muscle biopsies revealed a degenerative myopathy characterized by accumulation of rimmed (autophagic) vacuoles, disruption of sarcomere integrity and severe myofibrillar disorganization. The G376V variant altered a highly conserved amino acid residue and was absent in databases on human genome variation. Variant pathogenicity was supported by in silico analyses and functional studies. The G376V mutant increased the formation of cytoplasmic TDP-43 condensates in cell culture models, promoted assembly into high molecular weight oligomers and aggregates in vitro, and altered morphology of TDP-43 condensates arising from phase separation. Moreover, the variant led to the formation of cytoplasmic TDP-43 condensates in patient-derived myoblasts and induced abnormal mRNA splicing in patient muscle tissue. The identification of individuals with TDP-43-related myopathy, but not ALS, implies that TARDBP missense variants may have more pleiotropic effects than previously anticipated and support a primary role for TDP-43 in skeletal muscle pathophysiology. We propose to include TARDBP screening in the genetic work-up of patients with late-onset distal myopathy. Further research is warranted to examine the precise pathogenic mechanisms of TARDBP variants causing either a neurodegenerative or myopathic phenotype.

Physiological and pathological 3R and 4R tau expression in marmoset brain

AbstractBackgroundCommon marmosets (Callithrix jacchus) have recently emerged as an important model for studying aging and neurodegenerative disorders, including Alzheimer’s disease (AD). Amyloid‐β deposits in the cerebral cortex of marmosets present naturally with aging, which has now been widely reported. However, the role of tau in the marmoset brain has been understudied. The purpose of the present studies was to comprehensively examine 3R and 4R tau isoform expression in the marmoset brain.MethodsThe mRNA expression in the marmoset brain was confirmed by RT‐PCR. The total mRNA was isolated from frozen tissue of various age groups and reverse transcribed to cDNA, which was used for RT‐PCR. The sequence of each fragment was confirmed by the Sanger sequencing method. The tau protein expression in Sarkosyl soluble and insoluble fractions was examined by western blot using tau‐specific antibodies, and expression levels of 3R/4R were quantified by Dot blot. Synaptic tau expression was analyzed from crude synaptosome extractions of the frontal cortex. Pathological tau aggregates in cryo‐slices of a 10‐year‐old marmoset brain were visualized by immunohistochemistry.Results3R and 4R tau were expressed in the marmoset brain, with 3R tau predominately expressed in neonatal brains and 4R tau in adult/aged brains. 3R and 4R tau were also localized in synaptic regions of the frontal cortex. The Sarkosyl soluble tau was phosphorylated on T181, T217, and T231 sites with evidence of oligomers. Insoluble tau was phosphorylated on S396/S404 and S202/S205 sites and contained high molecular weight aggregates comprising 3R and 4R tau. Moreover, a 10‐year‐old marmoset brain case study demonstrated a paired helical filament structure in the hippocampus and intracellular 3R and 4R tau accumulation.ConclusionsOur results reveal physiological and pathological 3R/4R tau distributions in the frontal and temporal cortex of the marmoset brain, confirming the presence of 3R tau and AD‐like pathological tau in aging marmosets. These data support the utility of common marmosets as appropriate models for studying AD and neurodegenerative disorders.

Properties and structural features of native and modified proteins of concentrates from white and brown rice

The physicochemical properties and structural features of proteins of concentrates (PC) from white and brown rice are described. Differences in a degree of proteolysis and the relationship between the functional and technological properties of proteins and their structure were established. Proteins from white rice had lower molecular weights (MW) than proteins from brown rice: 1.3–199.5 kDa versus 1.5–251.1 kDa. Most proteins were grouped in three low-molecular-weight fractions (МW 11.7–27.5 kDa) in PC from white rice and in one fraction with the high molecular weight (182–251.1 kDa) in PC from brown rice. In the process of hydrolysis, the MW of proteins (high-molecular-weight) of PC from white rice decreased from 199.5 to 120.2 kDa with generation of peptides with a molecular weight of <1.3 kDa, while the MW remained unchanged upon hydrolysis of proteins of PC from brown rice. In the composition of PC from white rice, flavonoids interacted with protein fractions with molecular weights of 131, 10, and 4.0 kDa; while in PC from brown rice, they interacted with only one fraction with a molecular weight of 216 kDa. The quantity of flavonoids in PC from white rice was 2.3 times less than that in PC from brown rice. Elements of protein secondary structure were established for PC: α–helix, 310 — helix, β-structure, β-bends, and irregular shape. Proteolysis of proteins was accompanied by a decrease in the number of α-helices, increase in the proportion of β-structures and irregular regions, weakening of the hydrophobic properties of proteins and an increase in the number of S–S bonds; PC from brown rice was characterized by a higher content of –СН2 groups from the flavonoid family, as well as by a higher degree of unsaturation of groups of benzene nuclei, ketone and ester groups. A negative correlation was found between the foaming ability of PC and the upper boundaries of molecular weights (r = — 0.95), quantity of high-molecular weight proteins (r = — 0.80) and aggregation constants, and a positive correlation (r = + 0.8) with the number of S–S bonds. The high foaming ability is interrelated with proteins with a molecular weight of no more than 120 kDa. The results are intended to regulate the functional properties of protein products based on the characteristics of the physicochemical properties of native and modified proteins.

Effects of microwave on the structural and emulsifying properties and interfacial properties of oxidized soybean protein aggregates.

This research explored microwave treatment impact on the structuro-functional aspects of oxidized soy protein aggregates (OSPI). Data showed that oxidative treatment promoted the formation of high molecular weight aggregates through hydrophobic interactions, thereby disrupting the structure of natural soy protein isolates (SPI). Microwave treatment for an appropriate time (≤30s) caused the molecular structure of OSPI to open up and reduction in molecular weight and disulfide bond content, while absolute zeta potential increased. These modifications increased emulsifying capacity of OSPI, as well as the interfacial adsorption of protein. Longer microwave treatment times (>30s) caused OSPI to exhibit a tendency to aggregate in TEM and CLSM images. It indicated the appropriate microwave electromagnetic field effect and microwave heating effect could coordinatively regulate soy protein functional properties by modifying their aggregation behavior. The results provided new ideas for reducing resource waste, and further expanding soy protein application in the food industry.

Cloning, expression, purification and characterization of the recombinant nucleocapsid protein from SARS-CoV-2 and its combination with a CpG ODN-39M

Several studies have reported the ability of the nucleocapsid (N) protein from the SARS-CoV-2 virus to interact with RNA in vitro. Nevertheless, its capacity to interact with deoxyribonucleic acid has yet to be widely described. In the present work, the nucleocapsid protein from the SARS-CoV-2 Delta variant was obtained in E. coli. The recombinant protein was purified, immune identified and incubated with different quantities of the ODN-39M, a CpG ODN with adjuvant properties. As a result, a curve of precipitation was obtained. The analysis by agarose gel electrophoresis of the mixtures revealed that ODN-39M retarded its mobility, whereas the protein increased its migration. When a sample of N+ODN-39M was crosslinked and analyzed by SDS-PAGE, high molecular weight aggregates were detected. Accordingly, upon analysis by Transmission Electron Microscopy, particles of approximately 20 nm were visualized in both samples of N protein, with and without ODN-39M. However, aggregates of particles were abundant in the mixture. Notably, such structures kept the ability of N protein to be recognized by human sera of COVID-19 convalescent donors. We can assert that nucleocapsid protein can bind to deoxyribonucleic acid. Such interaction changed the protein conformation, contributing to the previously reported immunogenicity in mice of N+ODN-39M complexes. Keywords: Nucleocapsid, SARS-CoV-2, ODN-39M, Particles

Refolding Increases the Chaperone-like Activity of αH-Crystallin and Reduces Its Hydrodynamic Diameter to That of α-Crystallin.

αH-Crystallin, a high molecular weight form of α-crystallin, is one of the major proteins in the lens nucleus. This high molecular weight aggregate (HMWA) plays an important role in the pathogenesis of cataracts. We have shown that the chaperone-like activity of HMWA is 40% of that of α-crystallin from the lens cortex. Refolding with urea significantly increased-up to 260%-the chaperone-like activity of α-crystallin and slightly reduced its hydrodynamic diameter (Dh). HMWA refolding resulted in an increase in chaperone-like activity up to 120% and a significant reduction of Dh of protein particles compared with that of α-crystallin. It was shown that the chaperone-like activity of HMWA, α-crystallin, and refolded α-crystallin but not refolded HMWA was strongly correlated with the denaturation enthalpy measured with differential scanning calorimetry (DSC). The DSC data demonstrated a significant increase in the native protein portion of refolded α-crystallin in comparison with authentic α-crystallin; however, the denaturation enthalpy of refolded HMWA was significantly decreased in comparison with authentic HMWA. The authors suggested that the increase in the chaperone-like activity of both α-crystallin and HMWA could be the result of the correction of misfolded proteins during renaturation and the rearrangement of protein supramolecular structures.

Combination and precipitation mechanism of soy protein and tea polyphenols

Non-covalent interactions of plant proteins and polyphenols is often occurred in food production and processing. However, the combination of the two may form a precipitate to reduce the aesthetic value and taste. In this study, we prepared different proportions of soy protein isolate (SPI) and catechin/epigallocatechin-3-gallate (EGCG) non-covalent complexes. The structural and conformational changes of the complex in the non-covalent interaction system were explored by multi-spectral technology, and the transition mechanism from the solution-state to the precipitation-state was analyzed. As a “bridge”, polyphenols made more SPI cross-linked and aggregate, and reduce hydrophobicity. The enhancement of resonance Rayleigh scattering further confirmed the formation of macromolecular particles. SPI mainly combined with catechin/EGCG through hydrogen bonds, and more benzene rings and phenol hydroxyl structure of EGCG were conducive to the formation of a more stable complex with protein. In addition, the increase of polyphenol concentration promoted the conjugation effect (hydrophobic interaction and electrostatic interaction) with protein, resulting in insoluble high molecular weight aggregate precipitation.

Hypochlorous acid inactivates myeloperoxidase inside phagocytosing neutrophils

When neutrophils phagocytose bacteria, they release myeloperoxidase (MPO) into phagosomes to catalyse the conversion of superoxide to the potent antimicrobial oxidant hypochlorous acid (HOCl). Here we show that within neutrophils, MPO is inactivated by HOCl. In this study, we aimed to identify the effects of HOCl on the structure and function of MPO, and determine the enzyme's susceptibility to oxidative inactivation during phagocytosis. When hydrogen peroxide was added to a neutrophil granule extract containing chloride, MPO activity was rapidly lost in a HOCl-dependent reaction. With high concentrations of hydrogen peroxide, western blotting demonstrated that MPO was both fragmented and converted to high molecular weight aggregates. Using the purified enzyme, we showed that HOCl generated by MPO inactivated the enzyme by destroying its prosthetic heme groups and releasing iron. MPO protein was additionally modified by forming high molecular weight aggregates. Before inactivation occurred, MPO chlorinated itself to convert most of its amine groups to dichloramines. When human neutrophils phagocytosed Staphylococcus aureus, they released MPO that was largely inactivated in a process that required production of superoxide. Enzyme inactivation occurred inside neutrophils because it was not blocked when extracellular HOCl was scavenged with methionine. The inactivated enzyme contained a chlorinated tyrosine residue, establishing that it had reacted with HOCl. Our results demonstrate that MPO will substantially inactivate itself during phagocytosis, which may limit oxidant production inside phagosomes. Other neutrophil proteins are also likely to be inactivated. The chloramines formed on neutrophil proteins may contribute to the bactericidal milieu of the phagosome.

506-P: Regulation of ANGPTL3 by SEL1L-HRD1 ER-Associated Degradation

Angiopoietin-like 3 (ANGPTL3) is a liver-derived circulating factor critical for systemic lipid metabolism via the inhibition of lipoprotein lipase (LPL). Loss-of-function mutations in ANGPTL3 in humans and mice reduce circulating levels of triglycerides. Here, we report the discovery of a novel mechanism regulating the biosynthesis and maturation of nascent ANGPTL3 in the endoplasmic reticulum (ER). Mice with acute or chronic liver-specific deletion of SEL1L-HRD1, the most conserved branch of mammalian endoplasmic reticulum (ER)-associated degradation (ERAD) machinery, exhibit significantly lower levels of triglyceride and cholesterol in the serum with reduced weights of both epididymal and inguinal white adipose tissues (WAT). Various proteomics screenings have identified ANGPTL3 as a possible link between SEL1L-HRD1 ERAD and hypolipidemia. Indeed, ANGPTL3 is a bona fide endogenous substrate of SEL1L-HRD1 ERAD in the liver and in the absence of ERAD, ANGPTL3 protein is retained in the ER, forming high-molecular weight aggregates. This abnormal ER retention of ANGPTL3 accounts for a reduced circulating level of ANGPTL3, therefore contributing to hypolipidemia in ERAD-deficient mice. Together, this study not only uncovers a novel regulator for ANGPTL3 and novel targets in the treatment of hyperlipidemia in obesity and diabetes. Disclosure L.Zhou: None. L.Lin: None. S.Sun: None. L.Qi: None.

P203 PLASMA TRANSTHYRETIN FORMS IN PATIENTS WITH WILD–TYPE TRANSTHYRETIN CARDIAC AMYLOIDOSIS: INFLUENCED OF TAFAMIDIS TREATMENT

Abstract Introduction Transthyretin (TTR) is a plasma protein that transports thyroxin and retinol complexed to retinol–binding protein (RBP). TTR misfolding and aggregation can lead to the extracellular deposition of amyloid in the myocardium, causing TTR cardiac amyloidosis (ATTR–CA). Aim of this study is to develop a native electrophoretic method to characterize circulating TTR forms in plasma samples of ATTR–CA patients, before and after administration of tafamidis, a TTR stabilizer. Material & Methods Plasma samples from patients affected by wild–type ATTR amyloidosis (ATTRwt, n=10) were collected before (T0) and during tafamidis treatment at 14, 30 and 90 days. Plasma samples of sex– and age–matched healthy controls (n=6) were also collected. All samples were separated on a native 4–20% Tris–Gly polyacrylamide gel. Western blot analysis was performed with anti–TTR or anti–RBP antibodies. Proteins were detected by Clarity ECL substrate. ImageLab software was used for image acquisition and densitometric analysis of the blots. Total TTR (mg/dl) and RBP (mg/dl) were quantified by nephelometry. Results In both groups, the most represented forms were: TTR dimers or trimers (37–50kDa), TTR tetramers complexed with RBP protein in 1:1 (75kDa,) or 1:2 ratio (100kDa), and high molecular weight aggregates (>150kDa). RBP protein was detectable in association with TTR tetramers and some of the TTR aggregates (250kDa, 480kDa). Based on TTR electrophoretic pattern at T0, patients could be divided in 2 groups: ATTR–1, characterised by the presence of TTR tetramers (55kDa), and ATTR–2, characterised by its absence. The ATTR–1 group showed also higher levels of total TTR in comparison with ATTR–2 (mean±SD 25.5±3.8, 16.6±4.2 respectively, p=0.004), while plasma RBP levels were similar (5.1±0.7, 5.1±1.4 respectively). The electrophoretic patterns of ATTR–1 at T0, as well as TTR and RBP levels were comparable to those of controls (TTR 22.6±3.8, RBP 4.4±0.9). Tafamidis treatment was associated with a progressive increase of total TTR levels both in ATTR–1 and –2 (T90 31.4±5.8, 28.7±6.8) but the 2 groups continued to be distinguishable by the electrophoretic pattern. Conclusions The native electrophoresis allowed us to detect diverse circulating TTR forms and to classify patients in 2 groups according to their electrophoretic pattern. TTR quali/quantitative evaluation by electrophoresis could represent a valid tool to assess the individual pharmacological response to tafamidis.

Next generation multimodal chromatography resins via an iterative mapping approach: Chemical diversity, high-throughput screening, and chromatographic modelling

Multimodal chromatography resins are becoming a key tool in the purification of biomolecules. The main objective of this research was the establishment of an iterative framework for the rapid development of new multimodal resins to provide novel selectivity for the future purification challenges. A large chemically diverse virtual library of 100 multimodal Capto™ MMC ligand analogues was created, and a broad array of chemical descriptors were calculated for each ligand in silico. Principal component analysis (PCA) was used to map the chemical diversity and guide selection of ligands for synthesis and coupling to the Capto ImpRes agarose base matrix. Twelve new ligands were prepared in two groups: ‘group one’ consist of L00-L07 and ‘group two’ consist of L08-L12. These ligands are diverse in the influence of varied secondary interactions such as hydrophobic interactions, H-bonding, etc. Additional resin prototypes were also prepared to look at the chromatographic impact of ligand density variation. High-throughput plate-based studies were performed for parallel resin screening for batch-binding of six model proteins at different chromatographic binding pH and sodium chloride concentration conditions. Principal component analysis of the binding data provided a chromatographic diversity map leading to the identification of ligands with improved binding. Further, the new ligands have improved separation resolution between a monoclonal antibody (mAb1) and product related impurities, a Fab fragment and high molecular weight (HMW) aggregates, using linear salt gradient elutions. To quantify the importance of secondary interactions, analysis of the retention factor of mAb1 on the ligands at various isocratic conditions lead to estimations of (a) the total number of water molecules and counter salt ions released during adsorption, and (b) hydrophobic contact area (HCA). The iterative mapping approach of chemical and chromatography diversity maps described in the paper proves to be a promising method for identifying new chromatography ligands for biopharmaceutical purification challenges.

Effect of the Ultraviolet Radiation on the Lens.

The lens is a transparent, biconvex anatomical structure of the eyes responsible for light transmission and fine focusing on the retina. It is fundamentally constituted by water-soluble proteins called crystallins which are responsible for lens transparency due to their stable and highly organized disposition in the lens fiber cells. Some conformational changes and the subsequent aggregation of crystallins lead to loss of transparency in the lens and are the beginning of cataracts, which is the most frequent cause of reversible blindness in the world. Ultraviolet radiation is considered one of the risk factors for cataract development. The lens is exposed to radiation between 295 and 400 nm. This UV radiation may induce several processes that destroy the crystallins; the most significant is the oxidative stress due to increased free radicals formation. The oxidative stress is directly involved in modifications of the crystallin proteins leading to the formation of high molecular weight aggregates and then the subsequent opacification of the lens, known as cataracts. This review aims to summarize current knowledge about the damage of the lens proteins caused by ultraviolet radiation and its role in developing cataracts.

Aggrecan and Hyaluronan: The Infamous Cartilage Polyelectrolytes - Then and Now.

Cartilages are unique in the family of connective tissues in that they contain a high concentration of the glycosaminoglycans, chondroitin sulfate and keratan sulfate attached to the core protein of the proteoglycan, aggrecan. Multiple aggrecan molecules are organized in the extracellular matrix via a domain-specific molecular interaction with hyaluronan and a link protein, and these high molecular weight aggregates are immobilized within the collagen and glycoprotein network. The high negative charge density of glycosaminoglycans provides hydrophilicity, high osmotic swelling pressure and conformational flexibility, which together function to absorb fluctuations in biomechanical stresses on cartilageduring movement of an articular joint. We have summarized information on the history and current knowledge obtained by biochemical and genetic approaches, on cell-mediated regulation of aggrecan metabolism and its role in skeletal development, growth as well as duringthe development of joint disease. In addition, we describe the pathways for hyaluronan metabolism, with particular focus on the role as a "metabolic rheostat" during chondrocyte responses in cartilage remodeling in growth and disease.Future advances in effective therapeutic targeting of cartilage loss during osteoarthriticdiseases of the joint as an organ as well as in cartilage tissue engineering would benefit from 'big data' approaches and bioinformatics, to uncover novel feed-forward and feed-back mechanisms for regulating transcription and translation of genes and their integration into cell-specific pathways.

1107 PLASMA CIRCULATING TRANSTHYRETIN FORMS IN PATIENTS WITH WILD TYPE TRANSTHYRETIN CARDIAC AMYLOIDOSIS

Abstract Background and aims Wild type transthyretin (TTR) cardiac amyloidosis (ATTRwt-CA) is caused by the misfolding, aggregation and tissue deposition of native TTR, leading to cardiac structural remodeling. In the last decade, the validation of non-invasive diagnostic approaches and the development of novel therapeutic options targeting the amyloidogenic cascade, such as the TTR stabilizer tafamidis, have dramatically changed the epidemiology and the prognosis of the disease. Nonetheless, the basic pathophysiological mechanisms underlying TTR misfolding and aggregation are still poorly understood. We aimed to characterize circulating TTR forms in plasma samples of ATTRwt-CA patients before and after treatment with tafamidis through a native electrophoretic method. Methods Plasma samples from 6 male patients with ATTRwt amyloidosis (median age 82 years, IQR 80-83), collected before (T0) and during tafamidis treatment, and from 6 healthy controls were collected. Plasma samples were separated on a native 4–20% Tris-Gly polyacrylamide gel. Western blot analysis was performed with anti-TTR or anti-retinol-binding protein (RBP) antibodies. Proteins were detected by Clarity ECL substrate. Results Circulating TTR forms were qualitatively similar between ATTRwt-CA patients at T0 and controls. In both groups, the most represented forms were TTR dimers or trimers (∼37-50 kDa), TTR tetramers complexed with RBP in 1:1 ratio (∼80 kDa) or 1:2 ratio (∼100 kDa), and high molecular weight (MW) aggregates (>150 kDa). Neither TTR monomers nor tetramers could be detected. RBP protein was detectable in association with TTR tetramers and some of the higher MW fractions (∼150 kDa, >250 kDa). Following tafamidis treatment, all ATTRwt-CA patients displayed a progressive increase of the intensity of the band corresponding to TTR-RBP complexes, in agreement with the a stabilizing action on TTR tetramers. Interestingly, dimers and trimers, detectable at T0, were progressively lost during tafamidis treatment. Conclusions TTR tetramer exists only complexed with RBP and in equilibrium with low and high MW forms, without apparent qualitative differences between ATTR-CA and healthy controls. Tafamidis increases circulating TTR tetramers complexed with RBP. Evaluation of TTR isoform may prove useful as a circulating biomarker for the assessment of response to treatment in patients with ATTRwt-CA.

276 CIRCULATING FORMS OF PLASMA TRANSTHYRETIN IN PATIENTS WITH WILD-TYPE TRANSTHYRETIN AMYLOIDOSIS AND EFFECTS OF TREATMENT WITH TAFAMIDIS

Abstract Introduction Transthyretin (TTR) is a homotetrameric 55-KDa plasma protein that transports thyroxin and retinol complexed to retinol-binding protein (RBP). TTR misfolding and aggregation can lead to the extracellular deposition of amyloid (ATTR) representing one of the most frequent forms of amyloidosis in elderly. Aim of this study is to develop a native electrophoretic method to characterize circulating TTR in plasma samples of ATTR patients before and during treatment with tafamidis, a TTR stabilizer. Methods Plasma from ATTR patients (n=6), collected before (T0) and during tafamidis treatment, and plasma of healthy controls (n=6) were obtained from Fondazione Toscana G. Monasterio (Pisa, Italy). Plasma samples were separated on a native 4–20% Tris-Gly polyacrylamide gel. Western blot analysis was performed with anti-TTR (DAKO) or anti-RBP (Siemens Healthineer) antibodies. Proteins were detected by Clarity ECL substrate (BioRad). Results Circulating forms of TTR were qualitatively similar between ATTRwt patients at T0 and controls. In both groups, the most represented forms were: TTR dimers or trimers (∼37-50 kDa), TTR tetramers complexed with RBP protein in 1:1 ratio (∼80 kDa,) or 1:2 ratio (∼100 kDa), and high molecular weight (MW) aggregates (>150 kDa). Neither TTR monomers nor the tetramers were visible. RBP protein was detectable in association with TTR tetramers and some of the higher MW fractions (∼150 kDa, >250 kDa). Following tafamidis treatment, all ATTRwt patients displayed a progressive increase of the intensity of the band corresponding to TTR-RBP complexes, in agreement with the drug stabilizing action on TTR tetramers. Interestingly dimers and trimers, detectable at T0, were progressively lost during tafamidis treatment. Conclusions The native electrophoretic allowed us to detect several circulating TTR fractions. Data suggest a similar pattern of circulating TTR both in patients and healthy control: TTR tetramer exists only complexed with RBP and in equilibrium with low and high MW forms. Furthermore, the method allowed to appreciate the stabilizing effect of tafamidis on circulating TTR tetramers complexed with RBP. The study of circulating TTR fractions can expand our knowledge on mechanisms triggering its destabilization even when not mutated.

SEL1L-HRD1 ER-associated degradation suppresses hepatocyte hyperproliferation and liver cancer

SummaryEndoplasmic reticulum (ER) homeostasis has been implicated in the pathogenesis of various forms of cancer; however, our understanding of the role of ER quality control mechanisms in tumorigenesis remains incomplete. Here, we show that the SEL1L-HRD1 complex of ER-associated degradation (ERAD) suppresses hepatocyte proliferation and tumorigenesis in mice. Hepatocyte-specific deletion of Sel1L or Hrd1 predisposed mice to diet/chemical-induced tumors. Proteomics screen from SEL1L-deficient livers revealed WNT5A, a tumor suppressor, as an ERAD substrate. Indeed, nascent WNT5A was misfolding prone and degraded by SEL1L-HRD1 ERAD in a quality control capacity. In the absence of ERAD, WNT5A misfolds is largely retained in the ER and forms high-molecular weight aggregates, thereby depicting a loss-of-function effect and attenuating WNT5A-mediated suppression of hepatocyte proliferation. In humans, SEL1L-HRD1 ERAD expression correlated positively with survival time for patients with liver cancer. Overall, our data reveal a key role of SEL1L-HRD1 ERAD in suppressing hepatocyte proliferation and liver cancer.

Self-aggregating properties of inulin in a dilute solution

The creation of functional food products based on inulin-containing vegetable raw materials can provide the population with functional diabetic nutrition. In this regard, investigation of the technological parameters of obtaining inulin from Jerusalem artichoke tubers (Helianthus tuberosus L.) and determination of its quantitative characteristics seem highly relevant. This study aims to determine the qualitative characteristics of inulin obtained from Jerusalem artichoke tubers by both flash extraction and conventional methods. Jerusalem artichoke inulin samples were obtained by the flash extraction method at a high temperature of 105 °C during both shorter and longer periods of time and by the conventional method at a temperature of 75 °C in a neutral medium. The hydrodynamic properties and molecular weight of the samples demonstrated the self-aggregating properties of this biopolymer. Inulin obtained by the flash extraction method consists of two fractions: low-molecular weight inulin and high-molecular weight aggregate represented by a polysaccharide complex. These aggregates can be formed both by inter- and intramolecular interactions of various inulin fractions in the solution. As expected, their isolation using conventional methods appeared impossible: the method of concentration yielded a number of subfractions on the UV membrane and a large amount of aggregated water-insoluble microgel. At the same time, inulin obtained by the conventional method consists of one fraction, although having a high degree of polydispersity. In order to obtain high-quality inulin intended for nutritional and prophylactic purposes, it is preferable to use the flash extraction method over short periods of time.