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.     

Features and prognostic impact of distant metastasis in patients with stage IV lung adenocarcinoma harboring EGFR mutations: importance of bone metastasis

Mutated epidermal growth factor receptor (EGFR) and signaling pathways were associated with multiple brain and intra-pulmonary metastases, oncogenic progression and metastasis. However, features of metastasis to other organs and the independent prognostic influence of metastatic lesions were not elucidated in patients with lung cancer harboring EGFR mutations. Between January 2007 and April 2012, we treated 277 patients diagnosed with stage IV lung adenocarcinoma. Studied were 246 patients with available tumor EGFR mutation data who also underwent radiographic evaluation of lung, abdominal, brain, and bone metastases. The EGFR mutated group (N = 98) had significantly more metastatic lesions in the brain and bone than the wild-type group (N = 148): brain, 3 (1–93) versus 2 (1–32) median (range), P = 0.023; bone, 3 (1–43) versus 2 (1–27), P = 0.035, respectively. In addition, EGFR mutations were significantly more frequent in patients with multiple than non-multiple lung metastases (24/40 vs. 12/42, P = 0.004). Multivariate analysis showed that bone metastasis was a significant independent negative predictive factor of overall survival (OS) in patients with mutated [hazard ratio (HR) 2.04; 95 % confidence interval (CI) 1.17–3.64; P = 0.011] and wild-type EGFR (HR 2.09; 95 % CI 1.37–3.20; P < 0.001). In conclusion, patients with mutated EGFR had more lung, brain, and bone metastases, and bone metastasis was an independent negative predictor of OS.     

Correction to: Quick and effective improvement of leucine enriched dietary supplement on malnutrition in acute stroke patients receiving enteral tube feeding

Emergency Department (ED) crowding occurs when demand for care exceeds the available resources. Crowding has been associated with decreased quality of care and increased mortality, but the prevalence on a national level is unknown in most countries. We performed a national, cross-sectional study on staffing levels, staff workload, occupancy rate and patients waiting for an in-hospital bed (boarding) at five time points during 24 h in Swedish EDs. Complete data were collected from 37 (51% of all) EDs in Sweden. High occupancy rate indicated crowding at 12 hospitals (37.5%) at 31 out of 170 (18.2%) time points. Mean workload (measured on a scale from 1, no workload to 6, very high workload) was moderate at 2.65 (±1.25). Boarding was more prevalent in academic EDs than rural EDs (median 3 vs 0). There were an average of 2.6, 4.6 and 3.2 patients per registered nurse, enrolled nurse and physician, respectively. ED crowding based on occupancy rate was prevalent on a national level in Sweden and comparable with international data. Staff workload, boarding and patient to staff ratios were generally lower than previously described.     

Exercise-induced changes of functional mitral regurgitation in asymptomatic or mildly symptomatic patients with idiopathic dilated cardiomyopathy

It has remained unclear why functional mitral regurgitation (MR), even if it is of a mild degree, has prognostic importance in patients with idiopathic dilated cardiomyopathy (IDC). Exercise-induced changes in functional MR, which might be a clue to this question, have not been fully clarified. Thus, in this study, semisupine exercise echocardiography was performed on 32 asymptomatic or mildly symptomatic patients with IDC (29 men, mean age 45 +/- 14 years). The mean ejection fraction was 28 +/- 10% (range 13% to 45%). The effective regurgitant orifice (ERO) area of MR was measured, as well as echocardiographic parameters including mitral valve geometry. ERO at rest was associated best with systolic mitral tenting area (r(S) = 0.85, p <0.001). Functional MR did not newly appear during exercise in 9 subjects without MR at rest. In the remaining 23 subjects with functional MR at rest, all showed exacerbations of MR, with a median ERO of 10.5 mm(2) (interquartile range 6.3 to 16.5) to 18.7 mm(2) (interquartile range 9.5 to 29.3) (p <0.001). An increase in ERO was correlated best with the enlargement of tenting area (r(S) = 0.90, p <0.001) and was the strongest independent determinant of exercise duration (beta = -0.55, p = 0.002, multiple R(2) = 0.46). In conclusion, functional MR complicated with IDC was significantly exacerbated during exercise, with mitral valve deformation, which was strongly related to exercise intolerance; thus, the clinical impact of functional MR in patients with IDC could be more serious than can be expected by its degree at rest.     

Hepatocellular carcinoma presenting with obstructive jaundice: a clinicopathological study of eight cases

BACKGROUND/AIMS: The prognosis of icteric type hepatocellular carcinoma is extremely poor, not only because of obstructive jaundice, but also because of difficulties for early diagnosis. The aim of this study is to evaluate characteristics of icteric hepatocellular carcinoma for early diagnosis. METHODOLOGY: Eight patients with icteric hepatocellular carcinoma among 326 patients with hepatocellular carcinoma in our hospitals were retrospectively examined by laboratory data, image studies and pathology studies. RESULTS: Most cases were already advanced, with a portal tumor thrombus at the time of diagnosis. Imaging studies fail to reveal tumors because this type of hepatocellular carcinoma has an irregular faint margin and has lost the characteristic pattern of hepatocellular carcinoma, such as capsular formation or early enhancement. Pathology observations demonstrated poorly or moderately differentiated hepatocellular carcinoma in all our cases. CONCLUSIONS: This type of hepatocellular carcinoma should be considered in cirrhotic patients with obstructive jaundice or in patients with high tumor marker levels even if image studies fail to reveal tumors. For better prognosis, combination therapies such as biliary drainage, support for portal flow as well as treatment for the hepatocellular carcinoma, are necessary.     

Resistance of Helicobacter pylori to quinolones and clarithromycin assessed by genetic testing in Japan

Background and Aim: As bacterial resistance to clarithromycin limits the efficacy of clarithromycin‐based regimens for Helicobacter pylori infection, attention has turned to quinolone‐based rescue therapies. Resistance of H. pylori to both clarithromycin and quinolone can be predicted by genetic testing. Here, we used this approach to evaluate the prevalence of clarithromycin‐ and quinolone‐resistant strains of H. pylori in Japan. Methods: DNA was extracted from gastric tissue samples obtained from 153 patients infected with H. pylori (103 naive for eradication therapy and 50 with previous eradication failure following triple proton pump inhibitor/amoxicillin/clarithromycin therapy). Mutations in H. pylori 23S rRNA and gyrA genes associated with resistance to clarithromycin and quinolones, respectively, were determined. Results: Of 153 patients, 85 (55.6%) were infected with clarithromycin‐resistant strains. The prevalence of clarithromycin‐resistant strains in patients with previous eradication failure (90.0%, 45/50) was significantly higher than that (38.8%, 40/103) of those naive for eradication therapy (P < 0.001). Fifty‐nine patients (38.6%) were infected with strains resistant to quinolones. The incidence of quinolone‐resistant strains also appeared higher in patients with eradication failure (48.0%, 24/50) than in those who had not undergone therapy (34.0%, 35/103); however, the difference was not statistically significant (P = 0.112). The incidence of quinolone‐resistance in clarithromycin‐resistant strains (44/85, 51.8%) was significantly higher than that in clarithromycin‐sensitive strains (15/68, 22.1%) (P < 0.001). Conclusions: A high incidence of quinolone‐resistance was found in clarithromycin‐resistant strains of H. pylori, particularly in patients with previous eradication failure. Our results suggest that testing for susceptibility of H. pylori to quinolones is useful for determining the optimal rescue eradication regimen.