Myosin and tropomyosin-troponin complementarily regulate thermal activation of muscles.

Contraction of striated muscles is initiated by an increase in cytosolic Ca2+ concentration, which is regulated by tropomyosin and troponin acting on actin filaments at the sarcomere level. Namely, Ca2+-binding to troponin C shifts the "on-off" equilibrium of the thin filament state toward the "on" state, promoting actomyosin interaction; likewise, an increase in temperature to within the body temperature range shifts the equilibrium to the on state, even in the absence of Ca2+. Here, we investigated the temperature dependence of sarcomere shortening along isolated fast skeletal myofibrils using optical heating microscopy. Rapid heating (25 to 41.5°C) within 2 s induced reversible sarcomere shortening in relaxing solution. Further, we investigated the temperature-dependence of the sliding velocity of reconstituted fast skeletal or cardiac thin filaments on fast skeletal or ß-cardiac myosin in an in vitro motility assay within the body temperature range. We found that (a) with fast skeletal thin filaments on fast skeletal myosin, the temperature dependence was comparable to that obtained for sarcomere shortening in fast skeletal myofibrils (Q10 ∼8), (b) both types of thin filaments started to slide at lower temperatures on fast skeletal myosin than on ß-cardiac myosin, and (c) cardiac thin filaments slid at lower temperatures compared with fast skeletal thin filaments on either type of myosin. Therefore, the mammalian striated muscle may be fine-tuned to contract efficiently via complementary regulation of myosin and tropomyosin-troponin within the body temperature range, depending on the physiological demands of various circumstances.

Trans-scale thermal signaling in biological systems.

Biochemical reactions in cells serve as the endogenous source of heat, maintaining a constant body temperature. This process requires proper control; otherwise, serious consequences can arise due to the unwanted but unavoidable responses of biological systems to heat. This review aims to present a range of responses to heat in biological systems across various spatial scales. We begin by examining the impaired thermogenesis of malignant hyperthermia in model mice and skeletal muscle cells, demonstrating that the progression of this disease is caused by a positive feedback loop between thermally driven Ca2+ signaling and thermogenesis at the subcellular scale. After we explore thermally driven force generation in both muscle and non-muscle cells, we illustrate how in vitro assays using purified proteins can reveal the heat-responsive properties of proteins and protein assemblies. Building on these experimental findings, we propose the concept of 'trans-scale thermal signaling'.

Simulation toolkits at the molecular scale for trans-scale thermal signaling.

Thermogenesis is a physiological activity of releasing heat that originates from intracellular biochemical reactions. Recent experimental studies discovered that externally applied heat changes intracellular signaling locally, resulting in global changes in cell morphology and signaling. Therefore, we hypothesize an inevitable contribution of thermogenesis in modulating biological system functions throughout the spatial scales from molecules to individual organisms. One key issue examining the hypothesis, namely, the "trans-scale thermal signaling," resides at the molecular scale on the amount of heat released via individual reactions and by which mechanism the heat is employed for cellular function operations. This review introduces atomistic simulation tool kits for studying the mechanisms of thermal signaling processes at the molecular scale that even state-of-the-art experimental methodologies of today are hardly accessible. We consider biological processes and biomolecules as potential heat sources in cells, such as ATP/GTP hydrolysis and multiple biopolymer complex formation and disassembly. Microscopic heat release could be related to mesoscopic processes via thermal conductivity and thermal conductance. Additionally, theoretical simulations to estimate these thermal properties in biological membranes and proteins are introduced. Finally, we envisage the future direction of this research field.

The Kuchi-kara Taberu index as a predictive marker of oral intake recovery in patients with aspiration pneumonia.

AIM: In recent years, the Kuchi-kara Taberu (KT) index has been used as a new tool for the evaluation of dysphagia; however, its predictive ability remains unknown. This study was conducted to examine the validity of oral intake recovery prediction using the KT index in patients with aspiration pneumonia. METHODS: The subjects were older inpatients admitted to an acute-care hospital for the treatment of aspiration pneumonia. A logistic regression analysis was conducted to clarify factors significantly associated with oral intake recovery. In addition, cut-off values of the predictors were calculated using a receiver operating characteristic curve analysis with the area under the curve (P < 0.05). RESULTS: A total of 124 patients (mean age 84.3 ± 7.9 years old, 70 males, 54 females) were enrolled in this study and divided into two groups at discharge: the oral intake group (62.1%) and the non-oral intake group (37.9%). The significant predictors of the recovery of oral intake were the pre-treatment period, treatment period, and the total score of the KT index; the respective odds ratios were 1.082 (95% confidence interval [CI] 1.013-1.156), 1.018 (1.003-1.033) and 0.850 (0.780-0.927), and the respective areas under the curve were 0.407 (95% CI 0.300-0.515), 0.304 (0.208-0.399), and 0.732 (0.640-0.824). The cut-off value of the total score of the KT index was 30. The model showed 85.7% sensitivity and 57.4% specificity. CONCLUSION: The KT index is suggested to be a valid variable for predicting whether or not a patient with aspiration pneumonia can recover their oral intake ability during acute-care hospitalization. Geriatr Gerontol Int 2023; 23: 221-226.

Mice with R2509C-RYR1 mutation exhibit dysfunctional Ca2+ dynamics in primary skeletal myocytes.

Type 1 ryanodine receptor (RYR1) is a Ca2+ release channel in the sarcoplasmic reticulum (SR) of the skeletal muscle and plays a critical role in excitation-contraction coupling. Mutations in RYR1 cause severe muscle diseases, such as malignant hyperthermia, a disorder of Ca2+-induced Ca2+ release (CICR) through RYR1 from the SR. We recently reported that volatile anesthetics induce malignant hyperthermia (MH)-like episodes through enhanced CICR in heterozygous R2509C-RYR1 mice. However, the characterization of Ca2+ dynamics has yet to be investigated in skeletal muscle cells from homozygous mice because these animals die in utero. In the present study, we generated primary cultured skeletal myocytes from R2509C-RYR1 mice. No differences in cellular morphology were detected between wild type (WT) and mutant myocytes. Spontaneous Ca2+ transients and cellular contractions occurred in WT and heterozygous myocytes, but not in homozygous myocytes. Electron microscopic observation revealed that the sarcomere length was shortened to ∼1.7 µm in homozygous myocytes, as compared to ∼2.2 and ∼2.3 µm in WT and heterozygous myocytes, respectively. Consistently, the resting intracellular Ca2+ concentration was higher in homozygous myocytes than in WT or heterozygous myocytes, which may be coupled with a reduced Ca2+ concentration in the SR. Finally, using infrared laser-based microheating, we found that heterozygous myocytes showed larger heat-induced Ca2+ transients than WT myocytes. Our findings suggest that the R2509C mutation in RYR1 causes dysfunctional Ca2+ dynamics in a mutant-gene dose-dependent manner in the skeletal muscles, in turn provoking MH-like episodes and embryonic lethality in heterozygous and homozygous mice, respectively.

Heat-hypersensitive mutants of ryanodine receptor type 1 revealed by microscopic heating.

Thermoregulation is an important aspect of human homeostasis, and high temperatures pose serious stresses for the body. Malignant hyperthermia (MH) is a life-threatening disorder in which body temperature can rise to a lethal level. Here we employ an optically controlled local heat-pulse method to manipulate the temperature in cells with a precision of less than 1 °C and find that the mutants of ryanodine receptor type 1 (RyR1), a key Ca2+ release channel underlying MH, are heat hypersensitive compared with the wild type (WT). We show that the local heat pulses induce an intracellular Ca2+ burst in human embryonic kidney 293 cells overexpressing WT RyR1 and some RyR1 mutants related to MH. Fluorescence Ca2+ imaging using the endoplasmic reticulum-targeted fluorescent probes demonstrates that the Ca2+ burst originates from heat-induced Ca2+ release (HICR) through RyR1-mutant channels because of the channels' heat hypersensitivity. Furthermore, the variation in the heat hypersensitivity of four RyR1 mutants highlights the complexity of MH. HICR likewise occurs in skeletal muscles of MH model mice. We propose that HICR contributes an additional positive feedback to accelerate thermogenesis in patients with MH.

Whole picture of human stratum corneum ceramides, including the chain-length diversity of long-chain bases.

Ceramides are essential lipids for skin permeability barrier function, and a wide variety of ceramide species exist in the stratum corneum (SC). Although ceramides with long-chain bases (LCBs) of various lengths have been identified in the human SC, a quantitative analysis that distinguishes ceramide species with different LCB chain lengths has not been yet published. Therefore, the whole picture of human SC ceramides remains unclear. Here, we conducted LC/MS/MS analyses to detect individual ceramide species differing in both the LCB and FA chain lengths and quantified 1,327 unbound ceramides and 254 protein-bound ceramides: the largest number of ceramide species reported to date. Ceramides containing an LCB whose chain length was C16-26 were present in the human SC. Of these, C18 (28.6%) was the most abundant, followed by C20 (24.8%) and C22 (12.8%). Each ceramide class had a characteristic distribution of LCB chain lengths and was divided into five groups according to this distribution. There was almost no difference in FA composition between the ceramide species containing LCBs of different chain lengths. Furthermore, we demonstrated that one of the serine palmitoyltransferase (SPT) complexes, SPTLC1/SPTLC3/SPTSSB, was able to produce C16-24 LCBs. The expression levels of all subunits constituting the SPT complexes increased during keratinocyte differentiation, resulting in the observed chain-length diversity of LCBs in the human SC. This study provides a molecular basis for elucidating human SC ceramide diversity and the pathogenesis of skin disorders.

Modulation of Local Cellular Activities using a Photothermal Dye-Based Subcellular-Sized Heat Spot.

Thermal engineering at the microscale, such as the regulation and precise evaluation of the temperature within cellular environments, is a major challenge for basic biological research and biomaterials development. We engineered a polymeric nanoparticle having a fluorescent temperature sensory dye and a photothermal dye embedded in the polymer matrix, named nanoheater-thermometer (nanoHT). When nanoHT is illuminated with a near-infrared laser at 808 nm, a subcellular-sized heat spot is generated in a live cell. Fluorescence thermometry allows the temperature increment to be read out concurrently at individual heat spots. Within a few seconds of an increase in temperature by approximately 11.4 °C from the base temperature (37 °C), we observed the death of HeLa cells. The cell death was observed to be triggered from the exact local heat spot at the subcellular level under the fluorescence microscope. Furthermore, we demonstrate the application of nanoHT for the induction of muscle contraction in C2C12 myotubes by heat release. We successfully showed heat-induced contraction to occur in a limited area of a single myotube based on the alteration of protein-protein interactions related to the contraction event. These results demonstrate that even a single heat spot provided by a photothermal material can be extremely effective in altering cellular functions.

Opto-thermal technologies for microscopic analysis of cellular temperature-sensing systems.

Could enzymatic activities and their cooperative functions act as cellular temperature-sensing systems? This review introduces recent opto-thermal technologies for microscopic analyses of various types of cellular temperature-sensing system. Optical microheating technologies have been developed for local and rapid temperature manipulations at the cellular level. Advanced luminescent thermometers visualize the dynamics of cellular local temperature in space and time during microheating. An optical heater and thermometer can be combined into one smart nanomaterial that demonstrates hybrid function. These technologies have revealed a variety of cellular responses to spatial and temporal changes in temperature. Spatial temperature gradients cause asymmetric deformations during mitosis and neurite outgrowth. Rapid changes in temperature causes imbalance of intracellular Ca2+ homeostasis and membrane potential. Among those responses, heat-induced muscle contractions are highlighted. It is also demonstrated that the short-term heating hyperactivates molecular motors to exceed their maximal activities at optimal temperatures. We discuss future prospects for opto-thermal manipulation of cellular functions and contributions to obtain a deeper understanding of the mechanisms of cellular temperature-sensing systems.

Microscopic Temperature Control Reveals Cooperative Regulation of Actin-Myosin Interaction by Drebrin E.

Drebrin E is a regulatory protein of intracellular force produced by actomyosin complexes, that is, myosin molecular motors interacting with actin filaments. The expression level of drebrin E in nerve cells decreases as the animal grows, suggesting its pivotal but unclarified role in neuronal development. Here, by applying the microscopic heat pulse method to actomyosin motility assay, the regulatory mechanism is examined from the room temperature up to 37 °C without a thermal denaturing of proteins. We show that the inhibition of actomyosin motility by drebrin E is eliminated immediately and reversibly during heating and depends on drebrin E concentration. The direct observation of quantum dot-labeled drebrin E implies its stable binding to actin filaments during the heat-induced sliding. Our results suggest that drebrin E allosterically modifies the actin filament structure to regulate cooperatively the actomyosin activity at the maintained in vivo body temperature.

In situ measurements of intracellular thermal conductivity using heater-thermometer hybrid diamond nanosensors.

Understanding heat dissipation processes at nanoscale during cellular thermogenesis is essential to clarify the relationships between the heat and biological processes in cells and organisms. A key parameter determining the heat flux inside a cell is the local thermal conductivity, a factor poorly investigated both experimentally and theoretically. Here, using a nanoheater/nanothermometer hybrid made of a polydopamine encapsulating a fluorescent nanodiamond, we measured the intracellular thermal conductivities of HeLa and MCF-7 cells with a spatial resolution of about 200 nm. The mean values determined in these two cell lines are both 0.11 ± 0.04 W m-1 K-1, which is significantly smaller than that of water. Bayesian analysis of the data suggests there is a variation of the thermal conductivity within a cell. These results make the biological impact of transient temperature spikes in a cell much more feasible, and suggest that cells may use heat flux for short-distance thermal signaling.

DICOM segmentation and STL creation for 3D printing: a process and software package comparison for osseous anatomy.

BACKGROUND: Extracting and three-dimensional (3D) printing an organ in a region of interest in DICOM images typically calls for segmentation as a first step in support of 3D printing. The DICOM images are not exported to STL data immediately, but segmentation masks are exported to STL models. After primary and secondary processing, including noise removal and hole correction, the STL data can be 3D printed. The quality of the 3D model is directly related to the quality of the STL data. This study focuses and reports on the DICOM to STL segmentation performance for nine software packages. METHODS: Multidetector row CT scanning was performed on a dry human mandible with two 10-mm-diameter bearing balls as a phantom. The DICOM image file was then segmented and exported to an STL file using nine different commercial/open-source software packages. Once the STL models were created, the data (file) properties and the size and volume of each file were measured, and differences across the software packages were noted. Additionally, to evaluate differences between the shapes of the STL models by software package, each pair of STL models was superimposed, with the observed differences between their shapes characterized as the shape error. RESULTS: The data (file) size of the STL file and the number of triangles that constitute each STL model were different across all software packages, but no statistically significant differences were found across software packages. The created ball STL model expanded in the X-, Y-, and Z-axis directions, with the length in the Z-axis direction (body axis direction) being slightly longer than that in the other directions. The mean shape error between software packages of the mandibular STL model was 0.11 mm, but there was no statistically significant difference between them. CONCLUSIONS: Our results revealed that there are some differences between the software packages that perform the segmentation and STL creation of the DICOM image data. In particular, the features of each software package appeared in the fine and thin areas of the osseous structures. When using these software packages, it is necessary to understand the characteristics of each.

The challenge of intracellular temperature.

This short review begins with a brief introductory summary of luminescence nanothermometry. Current applications of luminescence nanothermometry are introduced in biological contexts. Then, theoretical bases of the "temperature" that luminescence nanothermometry determines are discussed. This argument is followed by the 105 gap issue between simple calculation and the measurements reported in literatures. The gap issue is challenged by recent literatures reporting single-cell thermometry using non-luminescent probes, as well as a report that determines the thermal conductivity of a single lipid bilayer using luminescence nanothermometry. In the end, we argue if we can be optimistic about the solution of the 105 gap issue.

Multifunctional temozolomide-loaded lipid superparamagnetic nanovectors: dual targeting and disintegration of glioblastoma spheroids by synergic chemotherapy and hyperthermia treatment.

Aiming at finding new solutions for fighting glioblastoma multiforme, one of the most aggressive and lethal human cancer, here an in vitro validation of multifunctional nanovectors for drug delivery and hyperthermia therapy is proposed. Hybrid magnetic lipid nanoparticles have been fully characterized and tested on a multi-cellular complex model resembling the tumor microenvironment. Investigations of cancer therapy based on a physical approach (namely hyperthermia) and on a pharmaceutical approach (by exploiting the chemotherapeutic drug temozolomide) have been extensively carried out, by evaluating its antiproliferative and pro-apoptotic effects on 3D models of glioblastoma multiforme. A systematic study of transcytosis and endocytosis mechanisms has been moreover performed with multiple complimentary investigations, besides a detailed description of local temperature increments following hyperthermia application. Finally, an in-depth proteomic analysis corroborated the obtained findings, which can be summarized in the preparation of a versatile, multifunctional, and effective nanoplatform able to overcome the blood-brain barrier and to induce powerful anti-cancer effects on in vitro complex models.

Microscopic heat pulses activate cardiac thin filaments.

During the excitation-contraction coupling of the heart, sarcomeres are activated via thin filament structural changes (i.e., from the "off" state to the "on" state) in response to a release of Ca2+ from the sarcoplasmic reticulum. This process involves chemical reactions that are highly dependent on ambient temperature; for example, catalytic activity of the actomyosin ATPase rises with increasing temperature. Here, we investigate the effects of rapid heating by focused infrared (IR) laser irradiation on the sliding of thin filaments reconstituted with human α-tropomyosin and bovine ventricular troponin in an in vitro motility assay. We perform high-precision analyses measuring temperature by the fluorescence intensity of rhodamine-phalloidin-labeled F-actin coupled with a fluorescent thermosensor sheet containing the temperature-sensitive dye Europium (III) thenoyltrifluoroacetonate trihydrate. This approach enables a shift in temperature from 25°C to ∼46°C within 0.2 s. We find that in the absence of Ca2+ and presence of ATP, IR laser irradiation elicits sliding movements of reconstituted thin filaments with a sliding velocity that increases as a function of temperature. The heating-induced acceleration of thin filament sliding likewise occurs in the presence of Ca2+ and ATP; however, the temperature dependence is more than twofold less pronounced. These findings could indicate that in the mammalian heart, the on-off equilibrium of the cardiac thin filament state is partially shifted toward the on state in diastole at physiological body temperature, enabling rapid and efficient myocardial dynamics in systole.

Functional significance of HCM mutants of tropomyosin, V95A and D175N, studied with in vitro motility assays.

The majority of hypertrophic cardiomyopathy (HCM) is caused by mutations in sarcomere proteins. We examined tropomyosin (Tpm)'s HCM mutants in humans, V95A and D175N, with in vitro motility assay using optical tweezers to evaluate the effects of the Tpm mutations on the actomyosin interaction at the single molecular level. Thin filaments were reconstituted using these Tpm mutants, and their sliding velocity and force were measured at varying Ca2+ concentrations. Our results indicate that the sliding velocity at pCa ≥8.0 was significantly increased in mutants, which is expected to cause a diastolic problem. The velocity that can be activated by Ca2+ decreased significantly in mutants causing a systolic problem. With sliding force, Ca2+ activatable force decreased in V95A and increased in D175N, which may cause a systolic problem. Our results further demonstrate that the duty ratio determined at the steady state of force generation in saturating [Ca2+] decreased in V95A and increased in D175N. The Ca2+ sensitivity and cooperativity were not significantly affected by the mutations. These results suggest that the two mutants modulate molecular processes of the actomyosin interaction differently, but to result in the same pathology known as HCM.