Pan-cancer methylome analysis for cancer diagnosis and classification of cancer cell of origin.

The accurate and early diagnosis and classification of cancer origin from either tissue or liquid biopsy is crucial for selecting the appropriate treatment and reducing cancer-related mortality. Here, we established the CAncer Cell-of-Origin (CACO) methylation panel using the methylation data of the 28 types of cancer in The Cancer Genome Atlas (7950 patients and 707 normal controls) as well as healthy whole blood samples (95 subjects). We showed that the CACO methylation panel had high diagnostic potential with high sensitivity and specificity in the discovery (maximum AUC = 0.998) and validation (maximum AUC = 1.000) cohorts. Moreover, we confirmed that the CACO methylation panel could identify the cancer cell type of origin using the methylation profile from liquid as well as tissue biopsy, including primary, metastatic, and multiregional cancer samples and cancer of unknown primary, independent of the methylation analysis platform and specimen preparation method. Together, the CACO methylation panel can be a powerful tool for the classification and diagnosis of cancer.

Single-cell temperature mapping with fluorescent thermometer nanosheets.

Recent studies using intracellular thermometers have shown that the temperature inside cultured single cells varies heterogeneously on the order of 1°C. However, the reliability of intracellular thermometry has been challenged both experimentally and theoretically because it is, in principle, exceedingly difficult to exclude the effects of nonthermal factors on the thermometers. To accurately measure cellular temperatures from outside of cells, we developed novel thermometry with fluorescent thermometer nanosheets, allowing for noninvasive global temperature mapping of cultured single cells. Various types of cells, i.e., HeLa/HEK293 cells, brown adipocytes, cardiomyocytes, and neurons, were cultured on nanosheets containing the temperature-sensitive fluorescent dye europium (III) thenoyltrifluoroacetonate trihydrate. First, we found that the difference in temperature on the nanosheet between nonexcitable HeLa/HEK293 cells and the culture medium was less than 0.2°C. The expression of mutated type 1 ryanodine receptors (R164C or Y523S) in HEK293 cells that cause Ca2+ leak from the endoplasmic reticulum did not change the cellular temperature greater than 0.1°C. Yet intracellular thermometry detected an increase in temperature of greater than ∼2°C at the endoplasmic reticulum in HeLa cells upon ionomycin-induced intracellular Ca2+ burst; global cellular temperature remained nearly constant within ±0.2°C. When rat neonatal cardiomyocytes or brown adipocytes were stimulated by a mitochondrial uncoupling reagent, the temperature was nearly unchanged within ±0.1°C. In cardiomyocytes, the temperature was stable within ±0.01°C during contractions when electrically stimulated at 2 Hz. Similarly, when rat hippocampal neurons were electrically stimulated at 0.25 Hz, the temperature was stable within ±0.03°C. The present findings with nonexcitable and excitable cells demonstrate that heat produced upon activation in single cells does not uniformly increase cellular temperature on a global basis, but merely forms a local temperature gradient on the order of ∼1°C just proximal to a heat source, such as the endoplasmic/sarcoplasmic reticulum ATPase.

Sarcomeric Auto-Oscillations in Single Myofibrils From the Heart of Patients With Dilated Cardiomyopathy.

BACKGROUND: Left ventricular wall motion is depressed in patients with dilated cardiomyopathy (DCM). However, whether or not the depressed left ventricular wall motion is caused by impairment of sarcomere dynamics remains to be fully clarified. METHODS AND RESULTS: We analyzed the mechanical properties of single sarcomere dynamics during sarcomeric auto-oscillations (calcium spontaneous oscillatory contractions [Ca-SPOC]) that occurred at partial activation under the isometric condition in myofibrils from donor hearts and from patients with severe DCM (New York Heart Association classification III-IV). Ca-SPOC reproducibly occurred in the presence of 1 µmol/L free Ca2+ in both nonfailing and DCM myofibrils, and sarcomeres exhibited a saw-tooth waveform along single myofibrils composed of quick lengthening and slow shortening. The period of Ca-SPOC was longer in DCM myofibrils than in nonfailing myofibrils, in association with prolonged shortening time. Lengthening time was similar in both groups. Then, we performed Tn (troponin) exchange in myofibrils with a DCM-causing homozygous mutation (K36Q) in cTnI (cardiac TnI). On exchange with the Tn complex from healthy porcine ventricles, period, shortening time, and shortening velocity in cTnI-K36Q myofibrils became similar to those in Tn-reconstituted nonfailing myofibrils. Protein kinase A abbreviated period in both Tn-reconstituted nonfailing and cTnI-K36Q myofibrils, demonstrating acceleration of cross-bridge kinetics. CONCLUSIONS: Sarcomere dynamics was found to be depressed under loaded conditions in DCM myofibrils because of impairment of thick-thin filament sliding. Thus, microscopic analysis of Ca-SPOC in human cardiac myofibrils is beneficial to systematically unveil the kinetic properties of single sarcomeres in various types of heart disease.

In vivo cardiac nano-imaging: A new technology for high-precision analyses of sarcomere dynamics in the heart.

The cardiac pump function is a result of a rise in intracellular Ca2+ and the ensuing sarcomeric contractions [i.e., excitation-contraction (EC) coupling] in myocytes in various locations of the heart. In order to elucidate the heart's mechanical properties under various settings, cardiac imaging is widely performed in today's clinical as well as experimental cardiology by using echocardiogram, magnetic resonance imaging and computed tomography. However, because these common techniques detect local myocardial movements at a spatial resolution of ∼100 µm, our knowledge on the sub-cellular mechanisms of the physiology and pathophysiology of the heart in vivo is limited. This is because (1) EC coupling occurs in the µm partition in a myocyte and (2) cardiac sarcomeres generate active force upon a length change of ∼100 nm on a beat-to-beat basis. Recent advances in optical technologies have enabled measurements of intracellular Ca2+ dynamics and sarcomere length displacements at high spatial and temporal resolution in the beating heart of living rodents. Future studies with these technologies are warranted to open a new era in cardiac research.

Simultaneous imaging of local calcium and single sarcomere length in rat neonatal cardiomyocytes using yellow Cameleon-Nano140.

In cardiac muscle, contraction is triggered by sarcolemmal depolarization, resulting in an intracellular Ca(2+) transient, binding of Ca(2+) to troponin, and subsequent cross-bridge formation (excitation-contraction [EC] coupling). Here, we develop a novel experimental system for simultaneous nano-imaging of intracellular Ca(2+) dynamics and single sarcomere length (SL) in rat neonatal cardiomyocytes. We achieve this by expressing a fluorescence resonance energy transfer (FRET)-based Ca(2+) sensor yellow Cameleon-Nano (YC-Nano) fused to α-actinin in order to localize to the Z disks. We find that, among four different YC-Nanos, α-actinin-YC-Nano140 is best suited for high-precision analysis of EC coupling and α-actinin-YC-Nano140 enables quantitative analyses of intracellular calcium transients and sarcomere dynamics at low and high temperatures, during spontaneous beating and with electrical stimulation. We use this tool to show that calcium transients are synchronized along the length of a myofibril. However, the averaging of SL along myofibrils causes a marked underestimate (∼50%) of the magnitude of displacement because of the different timing of individual SL changes, regardless of the absence or presence of positive inotropy (via ß-adrenergic stimulation or enhanced actomyosin interaction). Finally, we find that ß-adrenergic stimulation with 50 nM isoproterenol accelerated Ca(2+) dynamics, in association with an approximately twofold increase in sarcomere lengthening velocity. We conclude that our experimental system has a broad range of potential applications for the unveiling molecular mechanisms of EC coupling in cardiomyocytes at the single sarcomere level.

Relationship between chain collapse and secondary structure formation in a partially folded protein.

Chain collapse and secondary structure formation are frequently observed during the early stages of protein folding. Is the chain collapse brought about by interactions between secondary structure units or is it due to polymer behavior in a poor solvent (coil-globule transition)? To answer this question, we measured small-angle X-ray scattering for a series of ß-lactoglobulin mutants under conditions in which they assume a partially folded state analogous to the folding intermediates. Mutants that were designed to disrupt the secondary structure units showed the gyration radii similar to that of the wild type protein, indicating that chain collapse is due to coil-globule transitions.

Effect of non-native helix destabilization on the folding of equine ß-lactoglobulin.

ß-lactoglobulin forms a non-native α-helix during an early stage of folding. To address the role of the non-native structure in the folding process, we designed several mutants of equine ß-lactoglobulin with reduced helical propensity in the non-native helix region. One of them, A123T, showed a similar structure to that of the wild-type protein; its folding kinetics was investigated by stopped-flow circular dichroism (CD) and fluorescence. Although A123T showed a reduced burst-phase CD intensity, its folding rate was similar to that of the wild-type protein, which indicated that the formation of the non-native helix does not accelerate or decelerate the folding reaction.

Delineation of solution burst-phase protein folding events by encapsulating the proteins in silica gels.

Many studies have shown that during the early stages of the folding of a protein, chain collapse and secondary structure formation lead to a partially folded intermediate. Thus, direct observation of these early folding events is crucial if we are to understand protein-folding mechanisms. Notably, these events usually manifest as the initial unresolvable signals, denoted the burst phase, when monitored during conventional mixing experiments. However, folding events can be substantially slowed by first trapping a protein within a silica gel with a large water content, in which the trapped native state retains its solution conformation. In this study, we monitored the early folding events involving secondary structure formation of five globular proteins, horse heart cytochrome c, equine ß-lactoglobulin, human tear lipocalin, bovine α-lactalbumin, and hen egg lysozyme, in silica gels containing 80% (w/w) water by CD spectroscopy. The folding rates decreased for each of the proteins, which allowed for direct observation of the initial folding transitions, equivalent to the solution burst phase. The formation of each initial intermediate state exhibited single exponential kinetics and Arrhenius activation energies of 14-31 kJ/mol.

Fatty acids bound to recombinant tear lipocalin and their role in structural stabilization.

A variant of human tear lipocalin was expressed in Escherichia coli, and the bound fatty acids were analysed by gas chromatography, mass spectroscopy and nuclear magnetic resonance spectroscopy. Five major fatty acids were identified as hexadecanoic acid (palmitic acid, PA), cis-9-hexadecenoic acid (palmitoleic acid), 9,10-methylenehexadecanoic acid, cis-11-octadecenoic acid (vaccenic acid) and 11,12-methyleneoctadecanoic acid (lactobacillic acid). The composition of the bound fatty acids was similar to the fatty acid composition of E. coli extract, suggesting that the binding affinities are similar for these fatty acids. The urea-induced and thermal-unfolding transitions of the holoprotein (nondelipidated), apoprotein (delipidated) and PA-bound protein were observed by circular dichroism. Holoproteins and PA-bound proteins showed the same stability against urea and heat, and were more stable than apoprotein. These results show that each bound fatty acid stabilizes recombinant tear lipocalin to a similar extent.

Non-native alpha-helix formation is not necessary for folding of lipocalin: comparison of burst-phase folding between tear lipocalin and beta-lactoglobulin.

Tear lipocalin and beta-lactoglobulin are members of the lipocalin superfamily. They have similar tertiary structures but unusually low overall sequence similarity. Non-native helical structures are formed during the early stage of beta-lactoglobulin folding. To address whether the non-native helix formation is found in the folding of other lipocalin superfamily proteins, the folding kinetics of a tear lipocalin variant were investigated by stopped-flow methods measuring the time-dependent changes in circular dichroism (CD) spectrum and small-angle X-ray scattering (SAXS). CD spectrum showed that extensive secondary structures are not formed during a burst-phase (within a measurement dead time). The SAXS data showed that the radius of gyration becomes much smaller than in the unfolded state during the burst-phase, indicating that the molecule is collapsed during an early stage of folding. Therefore, non-native helix formation is not general for folding of all lipocalin family members. The non-native helix content in the burst-phase folding appears to depend on helical propensities of the amino acid sequence.