Heat of supersaturation-limited amyloid burst directly monitored by isothermal titration calorimetry

Amyloid fibrils form in supersaturated solutions via a nucleation and growth mechanism. Although the structural features of amyloid fibrils have become increasingly clearer, knowledge on the thermodynamics of fibrillation is limited. Furthermore, protein aggregation is not a target of calorimetry, one of the most powerful approaches used to study proteins. Here, with β₂-microglobulin, a protein responsible for dialysis-related amyloidosis, we show direct heat measurements of the formation of amyloid fibrils using isothermal titration calorimetry (ITC). The spontaneous fibrillation after a lag phase was accompanied by exothermic heat. The thermodynamic parameters of fibrillation obtained under various protein concentrations and temperatures were consistent with the main-chain dominated structural model of fibrils, in which overall packing was less than that of the native structures. We also characterized the thermodynamics of amorphous aggregation, enabling the comparison of protein folding, amyloid fibrillation, and amorphous aggregation. These results indicate that ITC will become a promising approach for clarifying comprehensively the thermodynamics of protein folding and misfolding.Aggregation has often been an obstacle to studying the structure, function, and physical properties of proteins. However, a large number of aggregates associated with serious diseases, including Alzheimer’s, Parkinson, and prion diseases (1, 2) promoted the challenge of studying protein misfolding and aggregation. Researchers succeeded in distinguishing amyloid fibrils and oligomers from other amorphous aggregates and characterized the ordered structures present in amyloid fibrils or oligomers, which led to the development of the field of amyloid structural biology (3–8). These advances have been attributed to various methodologies that are also useful for studying the structural properties of globular proteins. Even X-ray crystallography has become a powerful approach for studying amyloid microcrystals (5) or oligomers (9). The atomic details of amyloid fibrils are becoming increasingly clearer, and a cross-β structure was shown to be the main structural component of fibrils (5, 6, 8). Although tightly packed core regions of amyloid fibrils have been reported, the overall structures were shown to be dominated by common cross-β structures, which supported the argument for the main-chain dominated architecture in contrast to the side-chain dominated architecture of globular native states (10–12).These structural studies have been complemented by a series of efforts to clarify the mechanism for the formation of amyloid fibrils (i.e., amyloid fibrillation). The presence of a long lag time in spontaneous fibrillation and rapid fibrillation by the addition of preformed fibrils represent a similarity with the supersaturation-limited crystallization of substances (13–18). We have revisited “supersaturation” and argued its critical role for amyloid fibrillation (17–19). The role of supersaturation in neurodegenerative diseases at the proteome level has been reported recently (20).However, calorimetry, one of the most powerful methods used to study the thermodynamic properties of globular proteins (21–24), has not played a significant role in understanding protein aggregation. The aggregation of proteins following heat denaturation as monitored by differential scanning calorimetry is an infamous example demonstrating how aggregation can prevent exact analyses (25, 26). To date, few studies have investigated protein aggregation including amyloid fibrils with calorimetry (27–32). Our previous study on the exothermic heat effects accompanying fibril growth was achieved by monitoring the seed-dependent elongation of fibrils formed by β₂-microglobulin (β2m), a protein responsible for dialysis-related amyloidosis, using isothermal titration calorimetry (ITC) (28).In the present study using β2m, we succeeded in characterizing the total heat of spontaneous fibrillation and amorphous aggregation. An analysis of the heat burst associated with fibrillation or amorphous aggregation under various temperatures clarified their thermodynamic properties. The results obtained enabled the calorimetric characterization of amyloid fibrils and amorphous aggregates relative to that of the native globular structures, which opens a new field for the calorimetric study of protein aggregates.     

Intestinal necrosis related to administration of cation exchange resin without sorbitol: A retrospective analysis of 61 patients with end‐stage renal diseases

Intestinal necrosis associated with cation exchange resin (CER) is considered related to sorbitol, but it has been reported even in patients receiving CER alone. This study was performed to identify the risk factors of CER‐related intestinal necrosis. The pathological database of 61 end‐stage renal disease patients with surgical intervention for intestinal perforation was reviewed. The correlations between CER treatment and clinicopathological factors were studied among three groups: (i) patients administered CER and with CER at the perforation site (n = 23), (ii) patients administered CER with undetected CER at the perforation site (n = 12) and (iii) patients not administered CER (n = 26). The majority of the perforation site in group 1 was in the sigmoid colon (82.6%) with significantly higher average age and more frequent CER adhesion rates to the mucosa around the perforation site than group 2. The laxative administration rate in group 1 was significantly higher than group 3 and tended to be higher than group 2. The incidence of CER‐related intestinal necrosis was estimated at 0.57%. CER should be used with extreme caution in elderly patients with passage disturbance.     

Glucocorticoid receptor and serum‐ and glucocorticoid‐induced kinase‐1 in esophageal adenocarcinoma and adjacent Barrett's esophagus

Barrett's esophagus (BE) is a consequence of gastroesophageal reflux disease and is predisposed to esophageal adenocarcinoma (EAC). EAC is an exemplar model of inflammation‐associated cancer. Glucocorticoids suppress inflammation through glucocorticoid receptor (GR) and serum‐ and glucocorticoid‐induced kinase‐1 (Sgk1) expressions. Therefore, we immunolocalized GR and Sgk1 in EAC and the adjacent BE tissues and studied their association with clinical disease course in 87 patients with EAC who underwent surgical resection (N = 58) or endoscopic submucosal dissection (N = 29). Low GR and Sgk1 expressions in adjacent BE tissues were associated with adverse clinical outcomes (P = 0.0008 and 0.034, respectively). Patients with low Sgk1 expression in EAC cells exhibited worse overall survival (P = 0.0018). In multivariate Cox regression analysis, low GR expression in the adjacent nonmalignant BE tissues was significantly associated with worse overall survival (P = 0.023). The present study indicated that evaluation of GR and Sgk1 expressions in both the EAC cells and adjacent nonmalignant BE tissues could help to predict clinical outcomes following endoscopic and surgical treatments. In particular, the GR status in BE tissues adjacent to EAC was an independent prognostic factor.     

Engineering of cell membranes with a bisphosphonate-containing polymer using ATRP synthesis for bone targeting

The field of polymer-based membrane engineering has expanded since we first demonstrated the reaction of N-hydroxysuccinimide ester-terminated polymers with cells and tissues almost two decades ago. One remaining obstacle, especially for conjugation of polymers to cells, has been that exquisite control over polymer structure and functionality has not been used to influence the behavior of cells. Herein, we describe a multifunctional atom transfer radical polymerization initiator and its use to synthesize water-soluble polymers that are modified with bisphosphonate side chains and then covalently bound to the surface of live cells. The polymers contained between 1.7 and 3.1 bisphosphonates per chain and were shown to bind to hydroxyapatite crystals with kinetics similar to free bisphosphonate binding. We engineered the membranes of both HL-60 cells and mesenchymal stem cells in order to impart polymer-guided bone adhesion properties on the cells. Covalent coupling of the polymer to the non-adherent HL-60 cell line or mesenchymal stem cells was non-toxic by proliferation assays and enhanced the binding of these cells to bone.     

Frank's sign with acute aortic dissection

A 59‐year‐old man with a long smoking history presented with sudden back pain. Frank''s sign was noticed in his bilateral ears, and computed tomography revealed Stanford type A acute aortic dissection. If young patients have Frank''s sign, attention should be paid to aortic disease in addition to coronary artery disease.     

Wound,pressure ulcer and burn guidelines – 4: Guidelines for the management of connective tissue disease/vasculitis-associated skin ulcers

The Japanese Dermatological Association prepared guidelines focused on the treatment of skin ulcers associated with connective tissue disease/vasculitis practical in clinical settings of dermatological care. Skin ulcers associated with connective tissue diseases or vasculitis occur on the background of a wide variety of diseases including, typically, systemic sclerosis but also systemic lupus erythematosus (SLE), dermatomyositis, rheumatoid arthritis (RA), various vasculitides and antiphospholipid antibody syndrome (APS). Therefore, in preparing the present guidelines, we considered diagnostic/therapeutic approaches appropriate for each of these disorders to be necessary and developed algorithms and clinical questions for systemic sclerosis, SLE, dermatomyositis, RA, vasculitis and APS.