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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.
The skeletal muscle contracts when depolarization is transmitted to the dihydropyridine receptor in the T-tubule, and Ca2+ is released through type 1 ryanodine receptor (RYR1) from the sarcoplasmic reticulum (SR; Meissner, 1994; Iino, 1999; Rios, 2018; Woll and Van Petegem, 2022). RYR1 is a monomeric protein molecule consisting of ∼5,000 amino acids that form a tetramer; this protein functions as a giant Ca2+ release channel with Ca2+-induced Ca2+ release (CICR) properties (Schneider, 1994; Endo, 2009; Rios, 2018). Mutations in RYR1 have been reported to induce severe muscle diseases, such as malignant hyperthermia (MH; MIM accession no. 145600) and central core disease (CCD; MIM accession no. 117000), the latter of which is characterized by muscle weakness (Treves et al., 2008; Allard, 2018; Jungbluth et al., 2018).
MH is induced by inhalational anesthetics, such as halothane and isoflurane, as well as by depolarizing muscle relaxants. Typical symptoms of MH are elevated body temperature (up to ∼42°C) and enhanced skeletal muscle contraction. MH-related mutations in RYR1 are thought to cause massive Ca2+ release from the SR due to acceleration of CICR. MH is a relatively rare disease, occurring in only one in tens of thousands of cases of anesthesia; however, once it occurs, it can be fatal unless treated promptly (Sinha et al., 2017). It has also been reported that MH mutations in RYR1 are involved in severe heat stroke (Wappler et al., 2001; Davis et al., 2002). Analyses of the RYR1 gene in patients with MH and related muscle diseases have identified >300 different mutations located throughout the molecule, including in the N-terminal, central, and C-terminal regions (Lanner et al., 2010; Pancaroglu and Van Petegem, 2018).
The x-ray crystal structure of the RYR1 N-terminal region was resolved in 2010 (Tung et al., 2010). In 2015, three research groups (Efremov et al., 2015; Yan et al., 2015; Zalk et al., 2015) reported its protein structure at the near-atomic level using cryo-electron microscopy. In the following year, the structure of the channel in its open state was reported (des Georges et al., 2016), improving the understanding of its 3-D structure. To understand the detailed pathogenesis of MH, the structure and function of the mutants must also be comprehensively analyzed.
A heterologous expression system in HEK293 cells has been used for functional analysis of MH-related mutations in RYR1 (Tong et al., 1997; Tong et al., 1999). We previously performed functional analysis using HEK293 cells overexpressing a disease mutant of RYR1 that causes MH syndrome in humans (Murayama et al., 2015; Murayama et al., 2016; Yamazawa et al., 2020). We reported that the Y523S mutation in the N-terminal region (Murayama et al., 2015; Yamazawa et al., 2020) and R2508C mutation in the central region (Murayama et al., 2016) lead to extremely severe phenotypes that reduce the stability of the closed state of the channel, thereby causing Ca2+ leakage from the SR.
To fully understand the mechanisms by which MH occurs in humans, valid animal models are needed. Since the turn of the century, studies using knock-in/knockout mouse models with RYR1 mutations, corresponding to human MH mutations, have been reported (Chelu et al., 2006; Yang et al., 2006; Yuen et al., 2012; Lopez et al., 2018). We previously generated an MH mouse model (R2509C-RYR1 mice) carrying a p.R2509C mutation in RYR1 using the CRISPR/Cas9 system (Yamazawa et al., 2021). In R2509C-RYR1 heterozygous (R2509C-Het) mice, MH and MH-like episodes were induced by volatile anesthetics as well as by an increase in environmental temperature (Yamazawa et al., 2021). As in humans with MH, MH responses in R2509C-Het mice were characterized by sustained skeletal muscle contractions, resulting in hyperthermia, hyperventilation, rhabdomyolysis, and, ultimately, death. Additionally, R2509C-RYR1 homozygous (R2509C-Hom) mice died in utero (Yamazawa et al., 2021), as did knock-in homozygous mice with R163C (Yang et al., 2006) and Y524S (Chelu et al., 2006) RYR1 mutations. Therefore, no experiments on intracellular Ca2+ responses have been performed using skeletal muscle cells from R2509C-Hom mice. In the present study, we generated primary cultured skeletal myocytes to investigate Ca2+ dynamics in R2509C-Hom mice. We found that Ca2+ homeostasis was markedly disrupted in myocytes from R2509C-Hom mice, accompanied by sarcomere shortening.
Materials and methods
All animal experiments were performed in accordance with the regulations and guidelines of the Institutional Animal Care and approved by the Ethics Committees of The Jikei University School of Medicine. Mice were housed in isolator cages, fed with food and water ad libitum, and kept in a controlled environment with 12-h light/12-h dark cycles, 23–25°C temperature, and 50–60% relative humidity under specific pathogen-free conditions in the animal facilities of The Jikei University of Medicine. Homozygous mutant neonates were obtained by mating heterozygotes, and their littermates were used as controls. The genotypes of all neonates and embryos used in the experiments were determined using PCR analysis.
Preparation of skeletal muscle primary culture
Primary cultured myocytes at embryonic days 17–18 were prepared using a previously described procedure with some modifications (Rando and Blau, 1994; Yamazawa et al., 1996; Yamazawa et al., 1997). Briefly, the forelimbs and hindlimbs were removed from mice embryo, and the bones were dissected. The muscle was cut into small fragments and enzymatically dissociated with 2 mg/ml collagenase (Worthington Biochemical) and 2 mg/ml dispase (FUJIFILM Wako Pure Chemical Corporation) at 37°C for 40–50 min. The fragments were passed through a 40-μm cell strainer, and the suspension was subjected to low-speed centrifugation. The pellet was resuspended in Ham’s Nutrient Mixture (F10), supplemented with 20% (v/v) FBS, penicillin (100 U/ml), streptomycin (100 U/ml), and 5 ng/ml recombinant human fibroblast growth factor-basic (Nacalai Tesque Inc.). Myoblasts were differentiated into myotubes/myocytes using Dulbecco’s modified Eagle’s medium (Nacalai Tesque Inc.) containing 2% horse serum. After 2–5 d of culture in differentiation medium, the myocytes were used in experiments. In optical heating experiments, 100 µM N-benzyl-N-nitroso-p-toluenesulfonamide (FUJIFILM Wako Pure Chemical Corporation) was added to HEPES-Krebs solution (140 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 11 mM glucose, and 5 mM HEPES, pH 7.4) to suppress the movement of skeletal myocytes upon activation.
Preparation of single flexor digitorum brevis cells
Skeletal muscle cells isolated from mice were prepared based on a previously described procedure, with some modifications (Yamazawa et al., 2021). Briefly, WT and R2509C-Het mice (8–20 wk old) were anesthetized through intraperitoneal injection of an anesthetic mixture (0.75 mg/kg medetomidine, 4 mg/kg midazolam, and 5 mg/kg butorphanol) before euthanasia. Flexor digitorum brevis (FDB) muscles were dissected and incubated with 2 mg/ml collagenase (Worthington Biochemical) in the HEPES-Krebs solution containing 2 mg/ml BSA (A8806; Merck) for 2 h at 37°C. Following incubation, single cells were separated by gentle trituration in HEPES-Krebs solution. Isolated single FDB fibers were seeded onto a laminin (Sigma-Aldrich)-coated or iMatrix-511 (T311; Takara Bio)-coated cover slip of a 35-mm glass-bottom dish. Next, 100 µM N-benzyl-N-nitroso-p-toluenesulfonamide, in HEPES-Krebs solution, was applied to suppress the movement of FDB fibers upon activation.
Cultured primary myocytes were incubated with 4 µM Cal-520-AM (AAT Bioquest) or fura-2-AM (Thermo Fisher Scientific) dye in HEPES-Krebs solution, containing 1 mg/ml BSA, for 30 min at room temperature (RT; 23–26°C). The cells were then washed thrice with HEPES-Krebs solution to remove excess dye; after 30 min of de-esterification, fluorescence images were obtained with a 20× objective lens (NA 0.75, UPlanSApo; Olympus) of an inverted microscope (DMI6000B; Leica) equipped with a complementary metal-oxide-semiconductor camera (ORCA-Flash4.0; Hamamatsu Photonics), at the rate of one frame every 0.5–2 s, using a MetaFluor v7 imaging system (Molecular Devices). The fluorescence wavelength was switched using a fluorescence filter exchanger (LAMBDA 10-3; Sutter Instrument). For Cal-520, it was excited at 480 ± 9 nm, and fluorescence was measured at 520 ± 14 nm. For fura-2, it was excited at alternating wavelengths of 340 (340 ± 13) nm and 380 (387 ± 6) nm, and the fluorescence was measured at 520 ± 14 nm for both wavelengths. Regions of interest corresponding to individual cells were selected and the mean fluorescence intensity (F) of each region of interest minus the background intensity was calculated for each frame.
We used the F340/F380 ratio (R; the value of F at an excitation wavelength of 340 nm divided by the value at an excitation wavelength of 380 nm) to estimate intracellular Ca2+ concentration ([Ca2+]cyt), as described previously (Murayama et al., 2016; Yamazawa et al., 2020).
To measure the cell temperature, we used ERthermAC, a photostable fluorescent molecular thermometer that selectively targets the sarco-endoplasmic reticulum (SR/ER). Cultured primary myocytes were loaded with 4 µM Cal-520-AM and 200 nM ERthermAC in HEPES-Krebs solution, containing 1 mg/ml BSA, for 30 min at RT. ERthermAC was excited at 556 ± 10 nm, and fluorescence was measured at 617 ± 37 nm. Excitation light and emission filters for Cal-520 and ERthermAC were exchanged alternately.
Drugs were applied to the myocytes or FDB fibers using a homemade 6-channel puffing pipette (Yamazawa et al., 2020), which changed the solution surrounding the cells within ∼1 s.
Microscopy system for optical heating
The microscopy system used for optical heating has been described previously (Oyama et al., 2022). Briefly, confocal images were obtained using an inverted microscope (IX73; Olympus) equipped with a Nipkow confocal scanner unit (CSU-X1; Yokogawa Electric), a dichroic mirror (Di01-T405/488/561; Semrock), a 20× objective lens (UPlanSApo, NA = 0.75; Olympus), and an EM-CCD camera (iXon Ultra; Andor Technology). For imaging of Cal-520, a 488-nm laser light (Vortran Laser Technology) and an emission filter (FF01-520/35; Semrock) were used. The local temperature was increased using an infrared laser (KPS-STD-BT-RFL-1455-05-CO; Keopsys) with an IR-LEGO mini (SIGMAKOKI). The change in local temperature was calculated through thermal quenching of 10 µg/ml Alexa Fluor 555 conjugated to 10-kD dextran (D34679; Thermo Fisher Scientific) in HEPES-Krebs solution, as previously described (Oyama et al., 2015). The dye was excited using a 561-nm laser light (Cobolt), and the fluorescence was captured through an emission filter (FF01-617/73; Semrock). Microscopic experiments were performed at 24°C. To inhibit the activity of sarco- and endoplasmic reticulum Ca2+-ATPase (SERCA), the myocytes were incubated in HEPES-Krebs solution containing 1 mg/ml BSA, 4 µM Cal-520AM, and 20 µM cyclopiazonic acid (CPA), for 30 min at 24–26°C, and then observed 60 min later in HEPES-Krebs solution containing 20 µM CPA.
Analyses for local heating
Microscopic images were analyzed using the ImageJ software (National Institutes of Health). The fluorescence intensity of Cal-520 was measured in an area of 139 µm2. To minimize artifacts caused by movement and drifting focus due to contracture during heating, the area at the center of the cell was selected. The maximum changes in the relative intensity of Cal-520 (ΔFmax/F0) were calculated from the maximum and minimum F/F0 for 5 s during heating, where F is the fluorescence intensity at an arbitrary time and F0 is the averaged intensity for 1.2 s after the initiation of recording (i.e., 10 s before beginning the heating). If the maximum F/F0 – 1 > 1 – minimum F/F0, then ΔFmax/F0 = maximum F/F0 – 1. If the maximum F/F0 – 1 < 1 – minimum F/F0, then ΔFmax/F0 = minimum F/F0 – 1. The background intensity without excitation was subtracted.
To measure the change in local temperature, relative changes in Alexa Fluor 555 intensity were calculated as ΔF/F0 = (Fheating − Iback)/(Fafter − Iback), where Fheating is the averaged intensity for 1 s at the end of the heating period, Fafter is the averaged intensity for 1 s at ∼20 s after the end of heating, and Iback is the background intensity when the excitation light is turned off. The ΔF/F0 of Alexa Fluor 555 was converted to ΔT using the relationship between ΔT and ΔF/F0 (Oyama et al., 2015).
Primary cultured myocytes were fixed with 4% paraformaldehyde in PBS (T900; Takara Bio) and permeabilized with 0.3% Triton X-100 (X100; Sigma-Aldrich) in PBS. After rinsing with PBS, the cells were incubated with 2% BSA (01281-84; Nacalai Tesque Inc.) in PBS for 1 h at RT. For immunofluorescence staining, the cells were incubated overnight at 4°C with mouse–anti-myosin heavy chain antibody (MF20, 1:200; 14-6503-82; Thermo Fisher Scientific). After washing, the cells were incubated with Alexa Fluor 546 rabbit anti-mouse IgG (H+L; 1:1,000; A-11060; Thermo Fisher Scientific) and 4′,6-diamidino-2-phenylindole dye (1:1,000; 340-07971; Dojindo Laboratories) for 1 h at RT. Images of the primary cultured cells were captured using an LSM 880 confocal laser-scanning microscope (Carl Zeiss) equipped with a 20× objective (α Plan-Apochromat, NA 0.8; Carl Zeiss).
Myoblasts and myotubes were lysed with Pro-Prep protein extraction solution (iNtRON Biotechnology). The extracted proteins were separated on 3–15% polyacrylamide gels and transferred to PVDF membranes. Membranes were probed with the primary antibodies against myosin heavy chain (MHC; MF20; Thermo Fisher Scientific), RYR1 (F1; Santa Cruz Biotechnology), and GAPDH (6C5; Santa Cruz Biotechnology), followed by HRP-labeled anti-mouse IgG (04-18-18; Kirkegaard & Perry Laboratories). Positive bands were detected by chemiluminescence using ImmunoStar LD (FUJIFILM Wako Pure Chemical Corporation) as a substrate.
Transmission electron microscopy
Transmission electron microscopy (TEM) experiments were performed based on a previously described procedure (Yamazawa et al., 2016), with slight modifications. Briefly, primary skeletal muscle culture cells were fixed with 2% glutaraldehyde (Nacalai Tesque Inc.) in 0.1 M phosphate buffer for 1 h at 4°C and post-fixed with 1% osmium tetroxide (Nisshin EM Co., Ltd.) in the same buffer at 0°C for 30 min. The tissues were first dehydrated with a graded series of ethanol, further with 100% ethanol for 10 min three times, and embedded in Epok 812 (Oken). Sections of 60-nm thickness were cut with a diamond knife and mounted on grids. After staining with uranyl acetate and lead citrate, sections were examined using an H-7500 electron microscope (Hitachi) at 80 kV.
The unpaired Student’s t test was used for comparisons between two groups. A one-way ANOVA followed by Tukey’s test was performed to compare three or more groups. Multiple groups were compared using the Steel test. Mann–Whitney U test was used for comparisons of two independent samples. These tests were performed using Prism v7 and v9 (GraphPad Software, Inc.).
Online supplemental material
Fig. S1 shows the morphology of R2509C-Hom embryos. Fig. S2 shows the distribution of the lengths of A-band in WT and R2509C mutant primary myocytes. Fig. S3 shows spontaneous Ca2+ transients in WT and R2509C mutant primary myocytes. Fig. S4 shows the time-course of heat-induced Ca2+ release in WT and R2509C primary myocytes. Fig. S5 shows temperature measurement of the SR in WT and R2509C-Het FDB-isolated fibers. Fig. S6 shows ERthermAC staining in primary myocytes. Video 1 shows spontaneous contraction in WT myocytes. Video 2 shows spontaneous contraction in R2509C-Het myocytes. Video 3 shows spontaneous contraction in R2509C-Hom myocytes. Video S4 shows spontaneous Ca2+ transients in WT myocytes. Video 5 shows spontaneous Ca2+ transients in R2509C-Het myocytes. Video 6 shows spontaneous Ca2+ transients in R2509C-Hom myocytes.
To characterize skeletal muscle cells from R2509C-Hom mice, primary cultures were prepared from WT and R2509C mouse skeletal muscle at embryonic days 17−18 (E17.5). We initially attempted to prepare primary-cultured skeletal muscle cells from neonatal R2509C mice; however, R2509C-Hom fetuses showed abnormal deaths between E17 and E19, with subcutaneous hemorrhage, subcutaneous effusion, and translucent skeletal muscles (see Fig. S1). These phenotypes were distinct from those in homozygous RYR1 knockout mice that have been reported to die in the neonatal period (dyspedic mice; see Takeshima et al., 1994). Indeed, we successfully prepared cultured skeletal muscle cells from neonatal dyspedic mice at E19.5 (Yamazawa et al., 1996).
Morphology of primary myocytes
Under the present culture conditions, primary myotubes/myocytes from WT and R2509C (R2509C-Het and R2509C-Hom) mice developed multiple nuclei within 3 d of differentiation induction (Fig. 1 A). The morphology of the cells was similar. Interestingly, primary myocytes from WT (Video 1) and R2509C-Het (Video 2) mice showed spontaneous contractions, whereas those from R2509C-Hom mice (Video 3) did not. Fluorescence immunostaining with anti-myosin heavy chain antibodies revealed a similar level of expression of MHC in WT, R2509C-Het, and R2509C-Hom myocytes (Fig. 1 B). Also, Fig. 1 C presents representative Western blots of MHC in WT, R2509C-Het, and R2509C-Hom myocytes up to 4 d after differentiation. There was no significant difference in average MHC expression levels between WT and R2509C myocytes from day 0–4 (P > 0.05). Therefore, it is unlikely that the absence of spontaneous contractions in R2509C-Hom myocytes occurred because of loss of myosin molecules.
The morphology of primary myocytes was further investigated after 3 d of differentiation using TEM (Figs. 2 and S2). Sarcomere structures were preserved with clear Z-lines in all three types of primary myocytes (Fig. 2 A); however, the I-bands were not detected in R2509C-Hom myocytes, indicating the occurrence of sarcomere shortening. Indeed, the sarcomere length (SL) in R2509C-Hom myocytes (1.7 ± 0.3 µm) was significantly shorter than that in the WT (2.2 ± 0.3 µm) or R2509C-Het (2.3 ± 0.4 µm) myocytes (P < 0.0001 and 0.0001, respectively; Fig. 2 C). To confirm the contribution of RYR1 mutants to the marked shortening of SL in R2509C-Hom myocytes, we tested the effects of a novel RYR1 inhibitor, 6,7-(methylenedioxy)-1-octyl-4-quinolone-3-carboxylic acid (Cpd1; Mori et al., 2019; Yamazawa et al., 2021). Cpd1 (2 μM) was continuously applied to the culture media from the onset of differentiation. The SL in Cpd1-treated R2509C-Hom myocytes (2.0 ± 0.3 µm) was significantly longer than that in the R2509C-Hom myocytes (1.7 ± 0.3 µm; Fig. 2, B and C). To further investigate the distribution of SL, we have analyzed the histograms, as shown in Fig. 2 D. It is reasonable to assume that the distribution shows a single peak in WT, because myocytes are in the resting state. Indeed, the Gaussian fitting provided the mean ± SD as 2.2 ± 0.3 µm. The data in R2509C-Het were similarly fitted by a single Gaussian, providing the value as 2.3 ± 0.3 µm. In contrast, R2509C-Hom and R2509C-Hom+Cpd1 were not fitted well by a single Gaussian (Fig. 2 D). Therefore, these data sets were analyzed using two Gaussians (discussed below in Discussion); one of the peak values was fixed at 2.2 µm in both groups, and the other peak values were derived as 1.6 ± 0.2 and 1.8 ± 0.2 µm in R2509C-Hom and R2509C-Hom+Cpd1, respectively.
Resting intracellular Ca2+ concentrations in R2509C primary myocytes
It was previously shown in MH mouse models that dyshomeostasis of [Ca2+]cyt in skeletal myocytes is among the most common features of MH (Chelu et al., 2006; Yang et al., 2006). Therefore, we investigated whether and how Ca2+ transients are altered in R2509C-Hom myocytes. Fluorescence Ca2+ imaging showed that spontaneous Ca2+ transients occurred similarly as in WT (Fig. S3 A and Video 4) and R2509C-Het (Fig. S3 B and Video 5) myocytes but not in R2509C-Hom (Fig. S3 C and Video 6) myocytes (Fig. S3 D). We measured the resting intracellular Ca2+ concentration ([Ca2+]cyt) using fura-2, a ratiometric fluorescent Ca2+ indicator, in myocytes differentiated from primary myoblasts from WT and R2509C-RYR1 mice (at day 2−4 after the onset of differentiation). The average resting [Ca2+]cyt in WT myocytes (103.7 ± 40.7 nM) was close to the reported value of ∼100 nM (see Harkins et al., 1993; Royer et al., 2008). Resting [Ca2+]cyt was significantly higher in R2509C-Het myocytes (124.5 ± 42.0 nM), and highest in R2509C-Hom myocytes (186.4 ± 71.4 nM; Fig. 3 A and Table 1). Therefore, the data for R2509C-Hom myocytes were further analyzed by varying the days of differentiation (Fig. 3 B). In addition, the large distributions of values in R2509C-Hom myocytes may be related to the variance of individual cellular functions involved in Ca2+ homeostasis. We found that [Ca2+]cyt was elevated in R2509C-Hom myocytes at day 2 (176.6 ± 54.3 nM) after differentiation, as compared to those in WT (103.7 ± 40.7 nM) and R2509C-Het (124.5 ± 42.0 nM) myocytes (P < 0.0001 and 0.0001, respectively). Resting [Ca2+]cyt increased with the days of differentiation, and the mean value was significantly high at day 4 of differentiation (222.5 ± 100.3 nM). To confirm the contribution of RYR1 mutants to the marked increase in [Ca2+]cyt, we tested the effects of Cpd1. Cpd1 (2 μM) was continuously applied to the culture media from the onset of differentiation. In R2509C-Hom myocytes, a significant decrease in resting [Ca2+]cyt by Cpd1 was observed at day 3 (179.5 ± 63.2 and 140.6 ± 52.7 nM for myocytes in the absence and presence of Cpd1, respectively) and day 4 (222.5 ± 100.3 and 151.4 ± 73.5 nM for control and Cpd1, respectively; Fig. 3 B and Table 1). In addition, Western blot analysis of RYR1 was performed to examine the relationship between RYR1 expression level and increase in resting [Ca2+]cyt in R2509C-Hom myocytes. As shown in Fig. 3 C, the expression level of RYR1 tended to increase depending on the differentiation days.
Excitation–contraction coupling and caffeine sensitivity
We next examined how excitation–contraction coupling is altered in mutant myocytes. Electrical stimulation induced significant Ca2+ transients (responses >10% of the baseline fluorescence value, i.e., F0) in WT, R2509C-Het, and R2509C-Hom myocytes without apparent differences in the response rate (>90%). However, the peak amplitude of Ca2+ transients (Fmax/F0) was reduced in the order of WT ≥ R2509C-Het > R2509C-Hom (Fig. 4 A, Table 2), although the amplitude for R2509C-Hom may be somewhat underestimated due to an increase in F0 in R2509C-Hom myocytes (see Fig. 3 A). Similarly, in KCl-induced depolarization, the peak amplitude of Ca2+ transients (Fmax/F0) at KCl concentrations above 20 mM was significantly reduced in the order of WT R2509C-Het > R2509C-Hom (Fig. 4 B and Table 2). To further characterize the functional properties of depolarization-induced Ca2+ transients, we plotted KCl dose-response curves. In each curve, the amplitude of Ca2+ transients was normalized by the maximum response to obtain the EC50 value (Fig. 4 C). The order of the sensitivity to KCl stimulation was R2509C-Hom > R2509C-Het > WT (Fig. 4 C and Table 2). Subsequently, we characterized caffeine-induced Ca2+ transients as an index for CICR activity (Fig. 4, D and E, and Table 2). Significant Ca2+ transients were observed at and above 3 mM in WT myocytes, whereas the corresponding caffeine concentrations for R2509C-Het and R2509C-Hom were 1 and 0.3 mM, respectively. As followed for the response to electrical stimulation, there were no significant differences between groups in the reaction rate (>90%) in the presence of 10 or 20 mM caffeine. Although the absolute peak amplitude (Fmax/F0) varied in the order of WT ≥ R2509C-Het > R2509C-Hom, the caffeine sensitivity was found to vary in the order of R2509C-Hom > R2509C-Het > WT (Fig. 4, D and E, and Table 2).
Heat-induced Ca2+ release in R2509C primary myocytes
We previously reported that Ca2+ transients are induced by infrared laser-based micro-heating in HEK293 cells expressing various RYR1 mutants as well as in isolated FDB fibers prepared from WT and R2509C-Het mice (Oyama et al., 2022). We found that mild heating (which did not affect [Ca2+]cyt in WT muscle cells) significantly increased [Ca2+]cyt in R2509C-Het muscle cells, demonstrating the heat-hypersensitivity of RYR1. Based on these results, we proposed a heat-mediated Ca2+ release mechanism through RYR1 from the SR, i.e., heat-induced Ca2+ release (HICR) that is likely to play a crucial role in the progression of MH at the cellular level (Oyama et al., 2022). Therefore, we next applied a heat pulse (ΔT = 11 ± 2°C from the base temperature T0 = 24 ± 1°C; central value ± range) to primary myocytes from WT, R2509C-Het, and R2509C-Hom mice after loading with the fluorescent Ca2+ indicator Cal-520 (Fig. 5, A–F). An infrared laser beam focused with an objective lens formed a concentric temperature gradient around cells (Oyama et al., 2022). The temperature gradient obtained depends on the laser power used. In the present study, the maximum temperature rise was 13°C at the focal point, with a minimum of 10°C or less at the end of the field of view. The duration of heating was controlled by a mechanical shutter. The temperature gradient was formed within 0.1 s of the opening of the shutter, and then was eliminated within 0.1 s by heat diffusion when the shutter was closed (Itoh et al., 2014; Oyama et al., 2020). The change in temperature was calculated with temperature-sensitive fluorescent dextran (Oyama et al., 2015).
Then, we performed time-dependent analyses in WT, R2509C-Het, and R2509C-Hom myocytes. No significant differences were observed in Ca2+ level among the before (F0), during (14–15 s), and after (19–20 s) heating in WT (Fig. 5 G). In R2509C-Het, [Ca2+]cyt significantly increased during heating (Fig. 5 H), while in R2509C-Hom, it decreased during heating and increased after heating (Fig. 5 I). The fluorescence intensity of Cal-520 before heating was increased in the order of WT ≤ R2509C-Het < R2509C-Hom (Fig. 5 J), which is consistent with the order for the resting [Ca2+]cyt (Fig. 3 A). Although little or no change in [Ca2+]cyt was detected during heating in WT myocytes (Fig. 5, A and B; see thick black line), a substantial increase in [Ca2+]cyt was detected in R2509C-Het myocytes (Fig. 5, C and D; see thick red line). The maximum changes in the relative fluorescence intensity of Cal-520 during heating (ΔFmax/F0) were greater in R2509C-Het myocytes than in WT or R2509C-Hom myocytes (Fig. 5 K and Fig. S4). This finding is consistent with our previous result obtained in isolated R2509C-Het FDB fibers (Oyama et al., 2022).
Why did R2509-Het cause a heat-induced Ca2+ release, whereas R2509C-Hom did not, and rather caused a decrease in [Ca2+]cyt? We recently reported that the ER/SR is a major Ca2+ source of the heat-induced Ca2+ bursts in HEK293 cells expressing WT and mutant RYR1 (Oyama et al., 2022), and that ER/SR Ca2+ is substantially depleted in cells expressing severe RYR1 mutation, such as R2509C. Subsequently, we tested whether the decrease in [Ca2+]cyt during heating in R2509C-Hom myocytes is due to heat activation of SERCA. In R2509C-Hom myocytes, inhibition of SERCA by 20 μM CPA significantly suppressed the decrease in Ca2+ levels during heating (Fig. 6, A–D). The results suggest that ER/SR Ca2+ in R2509C-Hom myocytes were severely depleted, and heating caused Ca2+ uptake rather than Ca2+ release.
Simultaneous measurement of [Ca2+]cyt and cellular temperature in isolated skeletal cells
Finally, we investigated whether an increase in the cellular temperature is associated with the increase in [Ca2+]cyt upon application of anesthesia. In a previous study, we reported that inhalation of isoflurane increases the rectal temperature in R2509C-Het mice, and that the exposure to isoflurane increases the [Ca2+]cyt in isolated FDB fibers (Yamazawa et al., 2021). At the cellular level, Meizoso-Huesca et al. (2022) demonstrated that heat production is closely related to Ca2+ leakage through RYR1 in the skeletal muscles, using a fluorescent molecular thermometer (Meizoso-Huesca et al., 2022). In the present study, we used ERthermAC, a small-molecule fluorescent dye (Kriszt et al., 2017), to perform the SR-targeted thermometry in living cells (Oyama et al., 2022). This dye is highly photostable and can be used with Cal-520 for intracellular Ca2+ imaging (e.g., Hou et al., 2017; Oyama et al., 2022). Striation patterns following ERthermAC treatment represent the morphology of the SR along myocytes, indicating successful staining by the dye (Fig. 7 A). We found that application of 1% isoflurane using a puffer pipette to R2509C-Het FDB fibers increased the fluorescence intensity of Cal-520 (Fig. 7 B, right top) and that of ERthermAC decreased (Fig. 7 B, right bottom). Note that the fluorescence intensity of ERthermAC decreased in response to an increase in temperature (as in Krisz et al., 2017; Hou et al., 2017; Oyama et al., 2022). WT FDB fibers showed no apparent decrease in the fluorescence intensity of Cal-520 or ERthermAC (Fig. 7 B, left), except for a gradual time-dependent reduction because of the photobleaching effect (Fig. S5). The results suggest that the prominent reduction in the fluorescence intensity of ERthermAC in R2509C-Het FDB fibers (Fig. 7 C) is not an artifact caused by application of isoflurane, but rather, at least partially, is due to the isoflurane-induced temperature increase in the cells.
We compared several aspects of Ca2+ dynamics (resting Ca2+, KCl, caffeine) in primary cultured myocytes from recently generated MH model mice (R2509C mice). R2509C-Hom myocytes exhibited markedly increased resting [Ca2+]cyt, reduced depolarization evoked and caffeine-induced Ca2+ release, and lacked spontaneous Ca2+ transients. R2509C-Hom myocytes also exhibited significantly shortened sarcomeres, consistent with elevated resting [Ca2+]cyt. In addition, MH is induced by inhaled anesthetics to increase body temperature; however, the actual relationship between heat and Ca2+ transients remains unclear. Consequently, we measured [Ca2+]cyt and cellular temperature simultaneously in the present study.
There was no significant difference in the SL between WT and R2509C-Het myocytes, and the value was within the range reported for resting skeletal myocytes (i.e., 2.0–2.5 µm; Goldspink, 1968). In contrast, the SL of R2509C-Hom myocytes was abnormally shortened compared to those for WT or R2509C-Het myocytes. These findings are consistent with the notion that sarcomeres in R2509C-Hom myocytes are contracted even under resting conditions. Excessive shortening of SL has been reported around the core in the leg muscles of aged Y522S heterozygous mice (Boncompagni et al., 2009), which is consistent with our results.
We also analyzed the mechanisms by which SL was shortened to ∼1.7 µm in R2509C-Hom myocytes (Fig. 2). Samples for TEM imaging were chemically treated with glutaraldehyde, followed by dehydration (see Materials and methods). This protocol has been reported to reduce the size of cells by ∼10% (Wyffels, 2001). If this is the case, SL in living R2509C-Hom myocytes can be calculated within the ∼1.3–1.7 μm range, which is the physiologically relevant A-band length in vertebrate striated muscles (cf. Suzuki et al., 2005; Udaka et al., 2008; Shimomura et al., 2016). The short SL in R2509C-Hom myocytes indicates that abnormal contractions occur in these cells, even in the resting state. Such abnormal contractions can likely be induced by enhanced Ca2+ leakage from the SR, resulting in a sustained increase in [Ca2+]cyt (Fig. 3 and Tables 1 and 2). Extensively elevated [Ca2+]cyt may cause the disorganization of myofibrillar structures in the skeletal muscles (Fig. S1), resulting in early death of R2509C-Hom embryos (see above).
The higher Ca2+ levels in R2509C-Het and R2509C-Hom myocytes than in WT (Fig. 3 A and Table 1) suggest that SL is equal to that in WT, or shortened due to tension generation according to the pCa-tension curve. R2509C-Het was well fit by a Gaussian, showing the peak and the SD as 2.3 µm (Fig. 2 D), which is quite similar to the case in WT, and their distributions are not significantly different (Fig. 2 C). Therefore, we can conclude that Ca2+ level distribution in R2509C-Het myocytes was in the region of relaxation. However, R2509C-Hom and R2509C-Hom+Cpd1 were not fit well by a single Gaussian. Therefore, we switch to the other possibility that the distribution of Ca2+ level is centered at the overlapping region between the relaxed and the partially activated state; hence, there are two respective distributions. These findings are consistent with those of previous studies (Balnave and Allen, 1996; Glass et al., 2018); i.e., in mouse FDB fibers, active force through actomyosin interaction is generated from 150 nM [Ca2+]cyt, and [Ca2+]cyt for half-maximal activation is 314 nM (Balnave and Allen, 1996; Glass et al., 2018). The SL of a myocyte in the group in the relaxed state should be similar to that in the WT that is also in the relaxed state. Therefore, the distributions of R2509C-Hom and R2509C-Hom+Cpd1 were analyzed based on two Gaussians, where one of the peaks was fixed at 2.2 μm. According to this analysis, the shorter peak is at 1.6 μm in R2509C-Hom, and it is recovered slightly to 1.8 μm in R2509C-Hom+Cpd1 (Fig. 2 D).The remarkable differences in the SL distributions of R2509C-Het and R2509C-Hom are consistent with the less significant difference of the resting [Ca2+]cyt between WT and R2509C-Het myocytes when compared with that between R2509C-Het and R2509C-Hom (Fig. 3 A). It is also noticeable that the population at 2.2 μm that corresponds to the sarcomere in the relaxed state is higher in R2509C-Hom+Cpd1 than in R2509C-Hom. The recovery of SL to the longer distributions strongly suggests that the Ca2+ level, and in turn the tension level, was lowered in the presence of Cpd1.
It has been reported that the expression of RYR1 mRNA, which is not expressed in pre-differentiated myoblasts, is induced at ∼24 h after the onset of differentiation, with the expression level reaching the maximum at day 4 (Aley et al., 2010). Given this previous finding, the abrupt increase in the resting [Ca2+]cyt in R2509C-Hom myocytes at day 4 is likely coupled with an increase in RYR1 expression and Ca2+ leakage from the SR. This prediction is supported by our previous result on R2508C RYR1-mediated Ca2+ leakage from the ER in HEK293 cells expressing mutant RYR1 (Murayama et al., 2016). This indicated that the increase in resting [Ca2+]cyt in R2509C-Het and R2509C-Hom myocytes is caused by enhanced Ca2+ leakage through RYR1 from the SR.
Notably, Fmax/F0 in R2509C-Hom myocytes were unexpectedly low relative to those in WT or R2509C-Het myocytes, although it is underestimated because F0 is increased. The results suggest that Ca2+ concentration in the SR lumen of R2509C-Hom myocytes was markedly reduced, as observed in the ER lumen with HEK293 cells overexpressing R2508C mutants (Murayama et al., 2016). In addition, the Fmax/F0 induced by 10 or 20 mM caffeine reportedly represents the maximal response of Ca2+ released from the SR/ER (Murayama et al., 2015; Murayama et al., 2016; Yamazawa et al., 2020). Therefore, we calculated the Fmax/F0 values under electrical stimulation and under maximal caffeine. As summarized in Table 2, the ratio values of WT (0.51: 3.59/7.01) and R2509C-Het (0.34: 2.42/7.04) myocytes were comparable, whereas the ratio values (0.70: 1.53/2.19) in R2509C-Hom myocytes were markedly greater than the others; therefore, the sensitivity to depolarization was likely increased in these cells. The increased sensitivity to depolarization in R2509C-Hom myocytes was also indicated by the EC50 values determined from the dose–response curve for KCl (Fig. 4, B and C; and Table 2). These findings are consistent with those of a previous study showing increased sensitivity to K+-induced depolarization in myotubes from homozygous R163C mice (Yang et al., 2006).
Heat-induced Ca2+ release and cell temperature
In R2509C-Hom myocytes, fluorescence intensity was lower during heating and higher after heating than before heating (Fig. 5, E and F; and Fig. S4), coupled presumably with the heat-induced Ca2+ release mechanism, as demonstrated in our previous study using R2508C HEK293 cells (Oyama et al., 2022). CPA, a SERCA inhibitor, inhibited the decrease in [Ca2+]cyt during heating in R2509C-Hom myocytes (Fig. 6 D). This result may have been caused by thermal activation of the SERCA, as demonstrated in our previous studies (Tseeb et al., 2009; Itoh et al., 2014; Oyama et al., 2022). This is because [Ca2+]cyt is increased in R2509C-Hom myocytes coupled with decreased [Ca2+] in the SR lumen; therefore, [Ca2+]cyt is lowered via heating-induced activation of SERCA. Hence, the differential Ca2+ response in R2509C-Het versus R2509C-Hom myocytes may be attributable to a difference in Ca2+ concentration between the cytoplasm and SR lumen.
The fluorescence intensity of ERthermAC started to decrease with a delay after onset of the isoflurane-induced Ca2+ increase (Fig. 7 B). A similar type of delay was observed in our previous studies using HeLa cells (Suzuki et al., 2007; Takei et al., 2014). The delay of a temperature increase following an increase in [Ca2+]cyt may reflect the time for SERCA to be activated for its thermogenesis, as efficient heat release by the Ca2+-ATPase should occur only after [Ca2+]SR is lowered to a certain value (de Meis et al., 2005). In the present study, this hypothesis was directly tested in R2509C-Hom myocytes, in which Ca2+ concentration in the SR was significantly reduced compared to that in WT and R2509C-Het myocytes (Fig. 3 and Table 1). We consider that in R2509C-Hom myocytes, the thermogenic activity of SERCA is immediately increased after onset of the isoflurane-induced Ca2+ increase and, therefore, the temperature increases with a shorter delay.
However, this experimentation could not be performed in primary myocytes because of the pre-differentiated myoblasts (MF20-negative cells) present at high density, which caused a strong background signal overlapping that of primary myocytes (Fig. 1 B and Fig. S6). Further studies are required to resolve this technical issue by developing (1) a method of obtaining differentiated myoblasts from primary myocytes with a sufficiently homogeneous quality and (2) a fluorescent probe that specifically targets the SR in living skeletal muscles.
In summary, we analyzed morphological and functional changes in myocytes from R2509C-RYR1 mice. In R2509C-Hom myocytes, Ca2+ release by depolarization or caffeine was decreased and resting [Ca2+]cyt was increased, indicating a Ca2+ leak and a reduced SR Ca2+ content. In R2509C-Hom myocytes, sarcomeres were remarkably shortened to the A-band length, even in the resting condition, which was associated with abnormal Ca2+ homeostasis, particularly an increase in the resting [Ca2+]cyt. In R2509C-Het myocytes, heat sensitivity for intracellular Ca2+ release via the heat-induced Ca2+ release mechanism was increased, and the cellular temperature was elevated in the presence of isoflurane. These findings suggest that (1) the abnormal morphological and functional changes underlie the embryonic lethality of homozygotes, and (2) environmental heat stress or exposure to an inhalation anesthetic, or both, accelerates the progression of MH in heterozygotes.
Eduardo Ríos served as editor.
We would like to thank Dr. Togo Shimozawa (The University of Tokyo) for critical reading of the manuscript, and Ms. Ikue Hiraga (Juntendo University) for her technical assistance. We thank Dr. Young-Tae Chang (Pohang University of Science and Technology, Gyeongsangbuk, Korea) for provision of ERthermAC.
This work was supported by JSPS KAKENHI Grant Numbers 19K07306, 20H04511, 21K09755 22H05055 (to T. Yamazawa), 22H05053 (to M. Suzuki), 22H05054 (to K. Oyama), 21K06789 (to F. Kobirumaki-Shimozawa), 20H03421, 21K19929 (to N. Fukuda), 19H03404, 22H02805 (to T. Murayama), 19K07105 (to N. Kurebayashi), 19H03198 (to K. Oyama, T. Yamazawa, and M. Suzuki), and 22H03198 (to N. Fukuda, F. Kobirumaki-Shimozawa, and T. Yamazawa), and the Platform Project for Supporting Drug Discovery and Life Science Research (BINDS; Basis for Supporting Innovative Drug Discovery and Life Science Research) from AMED under Grant Number JP20am0101080 (to T. Murayama and N. Kurebayashi, support No. 2533), Vehicle Racing Commemorative Foundation (#6237, 6303 to T. Murayama), and The Naito Foundation (to F. Kobirumaki-Shimozawa), and an Intramural Research Grant for Neurological and Psychiatric Disorders from the National Center of Neurology and Psychiatry (2-5 to T. Murayama).
The authors declare no competing financial interests.
Author contributions: T. Yamazawa, K. Oyama, N. Fukuda, and M. Suzuki designed the research study; Y. Tsuboi, T. Yamazawa, F. Kobirumaki-Shimozawa, T. Murayama, N. Kurebayashi, Y. Manome, E. Kikuchi, and T. Tachibana performed the experiments; S. Noguchi, T. Inoue, Y.U. Inoue, S. Mori, R. Ishida, H. Kagechika, and I. Nishino contributed new reagents/analytic tools; Y. Tsuboi, K. Oyama, M. Suzuki, N. Fukuda, and T. Yamazawa analyzed the data. All authors were involved in drafting this work.
This work is part of a special issue on excitation–contraction coupling.
Y. Tsuboi and K. Oyama contributed equally to this paper.

domain: literature


text:
Heres' an other GEAT DEAL from DontCrack : BUY MORE / PAY LESS...
- Buy a second product and get 5% EXTRA DISCOUNT OFF both products
- Buy a third product and get 10% EXTRA DISCOUNT OFF all 3 products
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- Buy five products or more and get 20% EXTRA DISCOUNT off all products!!!
In some cases, buying more products will make your total cart even cheaper than buying a single product ;-)

domain: economics


text:
SKETCH BOOK includes sketches of interior designers and architects, showing their own ideas and philosophy. Sketch acts as a tool to develop a design concept and give inspiration to designers. This book helps the readers to understand their view and design process clearly.
Language Korean/English Size 150 X 220 Page 416
List Price Korea Won 28,000, USD 28

domain: library


A North American chlorine shortage is straining prices, and the effects are already being felt here in New Brunswick. The cause is an unfortunate double whammy, with a production disaster and the pandemic each playing a role.
In August 2020, a massive fire at a Louisiana factory took North America’s largest pool and spa care provider offline.
The BioLab plant produces about 70 percent of the chlorine treatments in the North American market, according to Dion Rodrigues, director of marketing for Pool Supplies Canada, based in Burlington, Ont.
The plant should not resume production until spring 2022.
“So this situation certainly has an impact on the Canadian swimming pool industry,” said Rodrigues.
But the demand for chlorine was increasing even before the fire, he said, as Canadians shut down by the pandemic plunged into pool and spa purchases in record numbers.
“Basically they took their unused travel budget and said, ‘We’re going to put the hotel pool in the backyard.’ “
Prices already up by almost 20%
Shelley Melvin, of Emmerson Pools in Rothesay, said she has certainly seen this play out here in New Brunswick.
Melvin said Emmerson’s pool and spa facilities “have at least doubled already”, with more to come this summer.
This has not only caused a shortage of chemicals, but also liners and other pool-related items as staff at many manufacturing plants are at half capacity due to the pandemic, Melvin said.
People panic a bit and buy more than they need. I think if people didn’t do that it would definitely help a lot.– Paul Crowdis, owner of the pool and hot tub
Kelly Hare, who co-owns Premium Pools and Spas with her husband Tim Hickey, said prices have already gone up 10-15%, and in some cases 20%, on everything from chlorine washers to spas, and there are “huge safeguards for the supply chain”.
Plus, she said, manufacturers have limited the amount of spas and other items that can be ordered.
“They’ve capped what you can get, so anything we have to sell sells out fast,” Hare said.
Concerns about panic buying relapses
Some predict that the shortage will trigger another pandemic phenomenon: hoarding.
This is certainly a concern in the United States, where several media outlets warn of tight supplies and locals lament chlorine droughts on Facebook.
“Dude. Who accumulates the chlorine tablets ??? All the surrounding stores… none! Someone posted Thursday on the public page of the Above Ground Pools group, which lists Canadian and US members.
“I bought a tub two weeks ago (5 pounds) for $ 29 and Amazon is trying to sell for $ 160. If you accumulate, STOP! … We all want to enjoy our pools here. “
Rothesay resident Paul Crowdis said he wouldn’t be surprised to see the same happen here.
It’s one thing to stock up on what you need for the season; it’s another to pick up enough for five seasons, leaving none for anyone else, he said.
“When the pandemic started, you couldn’t buy a roll of toilet paper in this town,” Crowdis said.
“It’s the same with chlorine, people panic a bit and buy more than they need. I think if people didn’t do that it would definitely help a lot. “
Crowdis, which has both a pool and a hot tub, said it stocked up on chlorine pucks last season when prices were cut.
“But when I went to buy some pellet [chlorine] I noticed the price had gone up… about 20 percent. And the price of the washers has also gone up, by about 15 percent. ”
As an example, he said, a large container of granulated chlorine that cost $ 79 last year will now cost $ 99.
Finding alternatives to chlorine
Crowdis said the price has yet to reach a point where it would consider closing its pool or hot tub, but is already considering alternatives, such as a saltwater system.
Hare said Premium Pools had several inquiries about alternative systems, such as ionizers and saltwater systems.
” More [the salt-water systems] are also out of stock, and there’s no salt, “Hare said.” Canadian Tire, Walmart, they’re all sold out.
Ionizers are popular, she said, but at $ 800 they’re not a cheap solution and the price will rise due to lower demand and lower supply.
“It’s just one more thing to add to the list of rising prices. “
If you’re looking for a bright spot, there are pools that won’t be affected by the price hike: public recreational pools.
Ernie Breau, director of maintenance at the Canada Games Aquatic Center, said the chlorine shortage will not affect the city’s pool or splash pads as they use chlorine washers not stabilized.
“As I understand it, the shortage affects stabilized washers and calcium hypochloride chlorine powder, used for swimming pools and home spas,” said Breau.
However, he noted, “from what I hear people are already stocking up on unstabilized pucks.”


Ahsoka is a sequel to the popular animated TV series Star Wars Rebels written by Dave Filoni. This American TV series is interconnected to some of the events of Return Of Jedi (1983). Here is the Ahsoka TV series: Poster, Cast, Release date and trailer.
The cast of the action series Ahsoka:
1. Rosario Dawson:
Rosario Dawson played the lead character Ahsoka Tano in this TV series. American actress and film producer Rosario Dawson started her acting career in 1995. Dawson also known as a voice artist gave her voice to characters in movies such as Wonder Woman, Batgirl/Barbara Gorden and Nyx. Dawson performed in famous movies and TV series such as The Mandalorian, Men in Black 2, Trance, Alexander and Daredevil. Rosari is active in politics and she endorsed many political campaigns.
2. Mary Elizabeth Winstead:
Mary Elizabeth Winstead started her acting career as a child artist. American actress and singer got her first noticeable role as Jessica Bennet on the NBC soap opera Passions. She appeared in horror movies like Final Destination 3 and Death Proof. Mary boosted her career through movies like Live Free or Die Hard and Scott Pilgrim vs. the World. Mary Elizabeth Winstead has joined the Marvel Cinematic Universe (MCU) and will be seen in upcoming Marvel movies.
3. Natasha Liu Bordizzo:
Natasha Liu Bordizzo, an Australian actress, has a Chinese mother and an Italian father. Natasha can speak Chinese and she is trained in taekwondo and karate. Natasha will be seen as a Mandalorian warrior in Ahsoka.
4. Ray Stevenson:
Northern Irish actor Late Ray Stevenson had popular roles in the movie King Arthur and the HBO television series Rome (2005-2007). He also appeared in the Marvel movies Thor: Ragnarok and Thor: Love and Thunder. Stevenson died on May 22, 2023, just four days before his birthday. The whole team of Ahsoka paid tribute to the late Ray Stevenson in the premiere event.
5. Ivanna Sakhno:
Ivanna Sakhno started her acting career through a TV series in 2005. Her first movie was a biopic Ivan the Powerful. Ivanna’s first major role in Hollywood was thriller movie ‘The Body Tree’. Sakhno gives credit to the movies Pacific Rim: Upsprong, The Spy Who Dumped Me and Let It Snow for her successful career.
6. Diana Lee Inosanto:
Versatile American actress Diana Lee Inosanto is also a director, stuntwoman, martial artist and writer. In 2020 she wrote a children’s book ‘The Curious Mind Of Sebastian’. In this series Ahsoka, she is playing the role of Magistrate. She appeared as a guest star in ‘The Mandalorian’ in 2020.
7. David Tennant:
Scottish actor David Tennant is famous for his role in the BBC sci-fi series Doctor Who (2005-2010; 2013). He has also appeared in the fantasy movie Harry Potter and Goblet of Fire (2005). David adopted his professional name ‘Tennant’ inspired by singer Neil Tennant. David performed as a voice-over artist for the character Huyang in Ahsoka.
8. Lars Mikkelsen:
Danish legendary actor Lars Mikkelsen is famous for his previous performances in The Killing, House of Cards, Sherlock and Star Wars Rebels. In the Star Wars series Ahsoka, Lars is playing the role of a ruthless leader, Thrawn, who is willing to destroy the New Republic.
Release date of Ahsoka:
The action, adventure and drama TV series Star Wars: Ahsoka released on Disney+ on August 22. This series, Ahsoka (season 1) consists of eight episodes and will be released one episode every week on Disney+ Hotstar.


text:
The $42 billion New Zealand Super Fund is investing into a $300 million hotel portfolio established by local construction and property organization the Russell Group and private investment company Lockwood Property Group, creating a platform for further investment in New Zealand’s tourism sector.
The phased investment, which is subject to normal closing conditions, includes the Four Points by Sheraton and Adina Auckland Britomart in Auckland as well as the BreakFree Hotel in Christchurch. The fund also plans to acquire and develop additional sites.
Will Goodwin, head of direct investments for the fund, said in a statement the fund had been considering opportunities to invest in New Zealand’s tourism sector. “This partnership will give the NZ Super Fund exposure to New Zealand’s fast-growing tourism sector, diversify our investment portfolio and help support the industry’s strategic objectives,” he said. “New Zealand needs additional hotel accommodation to support both growing domestic tourism and international arrivals. There are clear capacity constraints in this sector and we look forward to working with our partners to identify opportunities for future growth.”
Despite recent visitor growth, New Zealand is poised to have a significant shortfall in hotel rooms, with more than 4,500 extra beds needed by 2025. Auckland faces the biggest constraint with a requirement of up to 4,300 new hotel rooms, but only 2,500 are projected to be built, according to Colliers International.
Lockwood Property Group’s Steve Lockwood said the significant opportunity for future growth in the sector was a major motivation for the deal. “The partnership provides a unique set of skills which will enable us to capitalize on the opportunities which become available in this sector throughout metropolitan and provincial New Zealand.”
Under the arrangement, two joint ventures have been created, one to own properties operating as hotels and the other to manage properties and identify future opportunities.

domain: news


text:
With news buzzing around successful music producer and CEO of the Last Two Music Group on an upcoming musical concert aimed at celebrating him, his contribution to the Ghanaian music industry at large and his artists Sarkodie, Kwaw Kese, Obrafour, Tinny and Edem in particular, Hammer has in an interview with NY DJ on Kapital Radio explained reasons behind the success of these mainstream artists and himself.
“You can’t fall because if you do, there might be something wrong and we don’t believe in that. Even if we try to make a bad song, we can’t. We don’t know how to make bad songs. That mentality is what we work with and has gotten us here”.
Hammer, whose name cannot be left out in the success story of so many Ghanaian musicians has expressed appreciation to his five mainstream artists and the Ghanaian community for the support since the announcement of the said musical concert.

domain: celebrity


text:
Pokeno 2 (Blue Box) | Non- Casino Games | Gamblers General Store >
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domain: game


text:
Cornelius Gross, Miki Ebisuya, and Nassos Typas join the prestigious life sciences organisation
The European Molecular Biology Organisation (EMBO) awards membership to exceptional scientists every year. EMBO’s major goals are to support talented researchers at all stages of their careers, stimulate the exchange of scientific information, and help build a European research environment where scientists can achieve their best work.
For 2022, Miki Ebisuya, Group Leader at EMBL Barcelona, Cornelius Gross, Interim Head of EMBL Rome and Nassos Typas, Group Leader and Senior Scientist at EMBL Heidelberg, have been recognised by their peers and awarded membership to the organisation. EMBL alumni Friedrich Frischknecht (University of Heidelberg), Edward Lemke (Institute of Molecular Biology), Marina Mapelli (European Institute of Oncology), and Giuseppe Testa (Università degli Studi Milano), have also been elected as members. In total, 146 EMBL alumni are currently EMBO members.
EMBO funds research fellowships, courses, workshops, conferences, and science policy initiatives, and publishes several prestigious journals. “It’s an honour to be elected as an EMBO member and to join this select group of accomplished life scientists,” said Nassos Typas, who highlighted the importance of the organisation’s support in his career. “I have been a beneficiary of EMBO’s actions to empower young scientists in several occasions, and now I very much look forward to giving back to the community by helping EMBO accomplish its missions.”
Inter-species differences, human behaviour, and bacterial interactions
The three EMBL scientists elected to EMBO membership work in very diverse areas: from comparing early development across species, to studying human behaviour, to analysing bacterial interactions with the environment.
Miki Ebisuya’s group is interested in comparing the differences and similarities in development among species. Human development is, in general, slower than mouse development. To understand such timing differences, the Ebisuya group has focused on studying the segmentation clock, which is an oscillatory pattern of gene expression that regulates the speed of somite formation during the very early stages of development.
“I knew about EMBO even when I was in Japan – it’s a nice way of networking and organising for scientists,” said Ebisuya. “I’m honoured to be a new EMBO member and grateful for the lab members and colleagues.”
The Gross group studies instinctive fear-related behaviour. Fear is a mental state caused by exposure to threats or cues that signal those threats. It promotes defensive behaviours, driven by decision-making capacities that help us avoid harm and that are critical to survival. However, fear in a pathological form can trigger a wide variety of mental disorders. The goal of the Gross group is to understand, at a molecular and circuit level, the neural control of fear-related behaviour.
“EMBO has been at the forefront of shaping global scientific best practice and policy in the life sciences for decades. I am extremely proud to be able to contribute to this group to push the boundaries of how we do science,” said Gross.
The Typas group works to understand bacteria and their interactions with the environment, other bacteria, the host, and phages. Their biological focus is on the bacterial cell envelope – which is a complex multi-layered structure that serves to sense and protect these organisms from their fluctuating environments. To do so, they are developing high-throughput, multi-readout, automated approaches to quantitatively assess gene-gene, gene-drug, and drug-drug interactions in many different bacteria and the communities they live in.
Gross, Ebisuya and Typas join an organisation of more than 1,900 international scientists, including 90 Nobel Laureates who hold, or have held, EMBO membership.

domain: biology


The Commission Way awards is named for the second Executive Director of the Commission on Human Relations, Ernest Hemingway. Mr. Hemingway held the position for over 19 years between 1972 and 1991. The Commission has awarded this recognition since 1997 to honor those persons, employers, or volunteer groups that have contributed substantially to promoting diverse social, cultural, and ethnic perspectives that enrich and expand understanding among all citizens in Chemung County.
Categories for Awards
Ernest Hemingway Award- Seeks to recognize people, employers, or volunteer groups that have substantially contributed to promoting diverse social, cultural, and ethnic perspectives that enrich and expand understanding among all residents of Chemung County.
WAY Organization Award- This award recognizes a group of people who have significantly impacted the community through their organization, programs, and events.
WAY Youth Award- Two community youths that exemplify excellence, leadership, character, and community involvement.
Racial Justice Award- Given to honor a person, business, or organization from the private or public sector who works toward eliminating racism and discrimination based on race, gender, or creed in the workplace or community.


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Sallie Mae will host the inaugural Sallie Mae 5K beginning at 8:30 a.m. July 16 at the Wilmington Riverfront at 801 Shipyard Drive to raise money for the Bridging the Dream Scholarship Fund.
Sallie Mae asked 20,000 high school counselors across the country to nominate high school juniors or seniors who exemplify excellence inside and outside the classroom, have overcome obstacles and have a strong desire to attend college in 2016. Sallie Mae selected four recipients and awarded each a $25,000 scholarship.
The Bridging the Dream Scholarship Program also motivated Sallie Mae employees to take the step of organizing and participating in fundraising events to support it. Employees raised more than $50,000, which the company matched to reach the program’s $100,000 goal.
This year, Sallie Mae is expanding the program by encouraging leaders of community organizations and school counselors to nominate students, increasing the number of scholarships awarded and holding the inaugural Sallie Mae 5K. The goal is to raise at least $125,000 through a combination of company support, employee fundraising activities and contributions from the community.
Registration is $35 per person. For information or to register, visit bit.ly/2tRnTRW.


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Grow Strong, Little Ones!
Build a strong foundation for children by providing them with all the nourishment they need!
✔ Magnesium – promotes calcium retention
✔ Vitamin D3 – develop child’s motor skills
✔ Fish Collagen – to bind calcium which helps in bone binding
✔ Vitamin C – enhance absorption of iron from food
✔ Vitamin E – protect fat in body tissues from oxidation
LAC FullCal Junior is recommended to be taken daily at any time by children 3 – 12 years old.
• Age 3 : 2 sticks daily
• Age 4 – 12 : 4 sticks daily
|Serving Size||1.8g (1 stick)|
|Amount||Per 100g||Per Serving|
|Carbohydrate 76.8 g 1.4 g||76.8g||1.4g|
|Protein 2.3 g 0 g||2.3g||0g|
Ingredients: Sucrose, Calcium Citrate, Erythritol, Lemon Juice Powder, Lemon Juice, Trehalose, Magnesium Oxide, Fructooligosaccharides, Arabic Gum, Silicon Dioxide, Collagen (Fish), Vitamin C, Lemon Flavour, Citric Acid, Vitamin E, Vitamin D3, Peppermint Flavour.
Contains thickener, anticaking agent and acidity regulator as permitted food conditioner and contains permitted flavouring substances.
Excessive use can have laxative effect.
Storage: Store in a cool, dry place below 25°C. Avoid direct sunlight.

domain: health


Kennedy ruled out of clash against Tigers
West Coast forward Josh Kennedy will miss Sunday's AFL clash with Richmond to ensure he doesn't suffer any complications from his recent eye injury.
Kennedy, who suffered bleeding to his eye after being accidentally hit with a tennis ball thrown by teammate Dean Cox last Thursday, was released from hospital on Sunday after spending four days immobilised to limit the damage.
The 23-year-old is expected to make a full recovery but has been told to restrict his activity this week to avoid risking long-term damage.
"He's laying low for the week. He's going to rest up and make sure there's no exertion or possible re-bleeding around the eye," Kennedy's manager Wayne Loxley said yesterday.
"He went home yesterday so it's obviously settled down.
"But the concern is any undue exertion for the next several days can have some complications further down the track.
"The eye's good at the moment. He's just got to take it easy.
"When I spoke to him on Friday, the plan was that he wouldn't be playing this week."
Kennedy's absence will be offset by the expected return of ruckman Nic Naitanui, who missed last week's win over the Bulldogs due to soreness.
Defender Sam Butler could also return from a quad injury, but the Eagles will closely monitor the fitness of vice-captain Beau Waters, who suffered a minor knock to his left elbow at training last week, before landing awkwardly on his right elbow against the Bulldogs.
Waters, who had previously suffered serious injuries to both elbows, was subbed out of the game at three-quarter time.
But in a promising sign, the 25-year-old wasn't included on West Coast's injury list yesterday.
However, Daniel Kerr remains in doubt after missing the past three games due to a series of sore spots relating to the hamstring he injured last year.
The Eagles sit in fifth place on the ladder but are just two points adrift of fourth-placed Carlton, who have played an extra game.
West Coast will use the home game against Richmond to celebrate their 25th year in the AFL, with the club's inaugural Hall of Fame to be launched on Friday night.


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What is your name and how would you briefly describe what you do?
My name is Per Axell. I am a gardener, garden designer, artist and illustrator and I usualy take on comissions with motifs from those genres.
What are you working with right now?
I am working with illustrations for information signs for nature reserves in the municipallity of Uppsala.
What does your work table tell us about you?To the extent that I work indoors there are pencils, paper, colors that apply. The work usually swells over so I can take floor, chairs and all available surfaces. But most of the time, I'm out in nature, drawing directly, at least during periods of more free Creation.

domain: artistic


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Lebanon, Ind. – Employers are looking to fill thousands of jobs at the Boone County Job Fair, presented by the Boone County Economic Development Corporation (Boone EDC), on Thursday, September 20 from 4-7 p.m. at Lebanon High School.
”More than 30 Boone County employers are looking to hire employees in a variety of positions throughout the county,” said Boone EDC Executive Director Molly Whitehead.
The job fair will include some of the county’s top employers covering industries such as manufacturing, business services, logistics and distribution, education, healthcare and retail. Companies are looking for full-time, part-time, temporary and seasonal employees.
The job fair is free and open to all. No registration is required for job seekers. Candidates of all industries and experience levels are encouraged to come dressed professionally and bring copies of their resume. A Help Desk will be available to provide job seekers with resume and interview tips.
Companies planning to attend include:
- Accurate Personnel
- Allied Universal Security Services
- American Ultraviolet Company
- Arc of Greater Boone County
- Camping World / Gander Outdoors
- CNH Industrial
- Control Tech Heating & Air Conditioning
- Cracker Barrel
- Daimler Trucks North America
- Diverse Staffing
- Elwood Staffing
- Facility Concepts, Inc
- FedEx Ground
- Fukai Toyotetsu Indiana Corporation
- General Cable
- GetGo Cafe’ + Market
- Hachette Book Group
- Hoosier Village Retirement Community
- Integrity Staffing Solutions
- Lebanon Community School Corporation
- K. Hurst Co, Inc.
- Panera Bread
- REGO-FIX Tool Corp.
- Staffing Resources, LLC
- Stalcop, LLC
- Suburban Home Health
- The Waters of Lebanon
- United States Cold Storage
- Witham Health Services
- XPO Logistics
- Zionsville Community Schools
More information is available at www.betterinboone.org/jobfair.
About the Boone County Economic Development Corporation
A strong community is a united community, one that achieves balance between its residents and businesses. At the Boone County Economic Development Corporation, we seek the right economic opportunities to support strong communities and enhance the quality of life for all Boone County residents. We aim to be a catalyst for smart growth strategies. We forge mutually beneficial partnerships with organizations capable of understanding and further enriching the unique assets that the residents of Boone County have built. For more information, visit www.betterinboone.org.


Journalist Eva Simpson and broadcaster Iyare Ighion look back at the week's news, strange names of 2013, selfies and staying young at heart.
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Humans like oranges because they contain Citrus and are high in vitamins and minerals that everybody needs. Hedgehogs, on the other hand, do not follow human reasoning. Hedgehogs are made uniquely. The digestive and immune systems of hedgehogs vary from one another.
Hedgehogs’ digestive systems are extremely delicate, and only some meals containing specific foods that are good for them can be delivered. You can feed hedgehogs those foods at any time, and they are considered part of a balanced, organic diet. Then several foods are classified as junk foods that are rarely offered to hedgehogs as a treat.
But then there is the question of whether or not my hedgehog will eat grapes, and if so, which group it belongs in. Given the hedgehogs’ complex digestive systems, the questions tend to be difficult. But don’t be concerned! We’re here to answer your questions and make your hedgehog’s dietary journey a little easier!
Grapes and Hedgehogs:
While citrus fruits are beneficial to most digestive systems, they are not recommended for hedgehogs. Hedgehogs of all varieties, whether wild or domesticated, have been classified as hazards. Hedgehogs are known as hazards due to their small stomachs being very sensitive.
Citrus fruits, as their name indicates, are particularly acidic. They are foods that can cause stomach upset and lead to gastric diseases. Since such diseases can be extremely painful for hedgehogs, you should keep citrus fruits out of their reach. The hedgehog’s digestive system can become weakened as a result of gastric illness, and You may see the hedgehog lose its diet.
Hedgehogs’ health may deteriorate due to a lack of diet, and they can become underweight. Maintaining a hedgehog’s weight is already a challenging and perplexing task, but adding declining health to the mix will make it even more difficult. It can be a big headache for you and a source of discomfort for your hedgehog.
Can Hedgehogs Eat Grapes?
The answer is NO! Hedgehogs cannot eat grapes! Hedgehog owners now understand that grapes are toxic for their pets. Hedgehogs commonly dislike sugar, and even if they adapt to the taste and liking it, it is harmful to them to consume this. Sugars can cause obesity in hedgehogs as well as diabetic conditions, which are hard to cure.
What’s more worrying is that when hedgehogs are ill, they refuse to eat something. They refuse to feed, and as a result, their energy levels plummet. Hedgehogs’ weight is extremely significant. The majority of the characteristics of hedgehogs and the majority of their behaviors are attributed to their weights.
If your hedgehog is underweight, it has a poor digestive system and consumes unhealthy foods, which decreases its activity levels and can even cause exhaustion. Underweight hedgehogs will refuse to eat anything and will avoid feeding. Hedgehogs lose more weight as they avoid food, which can influence their age.
Hedgehogs with excess weight face a similar but distinct problem. They normally have diseases like cholesterol and diabetes and are addicted to the sweets placed in front of them. The difference is that if they are not given their preferred treats, they will refuse to eat something.
But Some Hedgehogs Like Citrus?
In some rare cases, hedgehogs have been observed to like citrus fruits. The liking is observed because they adapt to the taste well, and they seem to be enjoying the sugary food presented to them as treats. Some hedgehog owners say that feeding their hedgehogs oranges has no impact on their wellbeing.
Some people said that their hedgehogs liked oranges and that they ate them happily, asking for more. There were no adverse effects, and their mouths and digestive tracts displayed no odd or disturbing behavior.
The comments that follow imply that consumption patterns are reciprocal. Some owners say that their hedgehog died due to a poor digestive system caused by citrus fruit consumption. Others say that their hedgies developed a sore mouth after eating citrus fruits and were unable to eat for days, despite receiving renowned physicians’ care, and eventually passed away due to weak immunity. Weak immunity means that the risk of disease is higher and that the disease’s impact will also be much worse.
Consequently, we can argue that some say grapes are good for hedgehogs, and others say they’re bad for hedgehogs. In one form or the other, Citrus has been scientifically proven to be toxic to hedgehogs and should be avoided at all costs. Is it worth it to take such a big gamble to see if your hedgehog and its digestive system can handle Citrus?
Hence, Citrus fruits, especially grapes, should be kept away from hedgehogs.

domain: food


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Lincoln Campaign Print, Hand-colored Lithograph
This product might require additional shipping/packaging charges. Should this be the case, we contact you before shipping your order.
Call (312) 944-3085 or email us here.
This Lincoln Campaign Print features a medium shot of Abraham Lincoln. It is based on Lincoln’s Cooper Union pose, photographed by Mathew Brady, O-17. The blue coloring is especially vibrant, the red is very bright as well. This print, in less of a condition as this one recently sold for $875.00 at auction, with extensive mildew at the bottom and other condition issues. Ours has no mildew, and features the original glass and frame.
(Abraham Lincoln) 1860 Campaign Print. Hand-colored lithograph: “ABRAHAM LINCOLN, / REPUBLICAN CANDIDATE FOR / SIXTEENTH PRESIDENT OF THE UNITED STATES.” Hartford & New York: E. B. & E. C. Kellogg, 1860. 8-1/2″ x 12-1/2″ (sight); 11-3/4″ x 15-3/4″ (overall).

domain: hobby


text:
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He went on to score 35 points in another outstanding show
The movie is in the works for Disney+ in June
The NBA star said a Pennsylvania-based artist sold knock off products with his brand name on them
"Baby Freak coming soon!"
"The Warriors have always been the big threat to go after Giannis"
A team spokesman confirmed to AFP that the Greeks were writing to governing body FIBA seeking a ban for the three referees in charge
Hard work is the most important thing for success
The head coach left the Greek Freak out for precautionary reasons
For Greece. For Milwaukee. For the family
The Greek Freak said he was glad he had his two brothers on the Greek team


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The temperature in the sea varies dramatically between the hot tropics and the frozen poles. But do the ocean’s most famous predators only live in the warm parts, or do sharks like cold water?
The simple answer is that it depends on the species of shark. Some sharks are highly susceptible to the cold and can only survive in tropical temperatures.
In contrast, others live happily in much cooler water. There are even species that live in the freezing Arctic ocean (although not the Antarctic, which is too cold even for the hardiest sharks).
We’re going to look at the different sharks that prefer cold water, those that love warm water, and those that have adapted to exist in between.
We’ll see that in every case, there’s a temperature range that a particular shark species have evolved to thrive in and that they could ultimately die if the water was too cold or too warm.
What Water Temperature Do Sharks Prefer?
Although it’s broadly possible to say that there are probably more sharks living in tropical and temperate waters, the question “Do Sharks Like Warm or Cold Water?” can only be truly answered by talking about a specific species.
There are over 500 different types of sharks swimming in our oceans today, and they have evolved to exist in the temperatures found in almost every part of the sea.
The ocean covers 70.8% of the surface of Earth, so there’s plenty of saltwater for these different sharks to live in. In fact, the ability to spread themselves around the world has been essential for the survival of all the various shark species.
Imagine if sharks could only live in warm water and all 500 species needed to eat there. Amongst other problems, the food supply would run out very quickly!
So, through the millennia, different sharks have evolved to fill an ecological niche to live in various habitats, including those with warm, intermediate, and cold temperatures.
So, to answer “What Water Temperature Do Sharks Prefer?” we need to look at a specific shark or maybe a group of sharks.
But before we do that, it’s helpful to define the different zones of temperature that the world’s oceans are typically divided into. Specific shark species often have an optimal temperature range that crosses between these zones. However, they give us a guide to work with.
Ocean Temperature Zones
Generally, the oceans are divided into three zones that make it easier to understand the expected water temperatures you’ll find there.
Understanding that these ocean regions refer to the expected sea surface temperatures is essential. The deep ocean will be far cooler, even in temperate or tropical zones, and will usually stay the same cold temperature year-round.
Tropical – Where the water temperature is above at least 20°C / 68 °F for the entire year. Typically with a maximum of around 30°C / 86°F.
Temperate – Temperatures range between about 10°C / 50 °F and 20C / 68°F throughout the year.
Polar – Where temperatures are below 10°C / 50 °F year round.
Throughout the year, the location and size of each surface temperature region will change with the seasons. For example, tropical zones, and accordingly, the area (northerly or southerly) that the associated sharks can live in, will expand further from the equator towards the relevant pole as the weather gets warmer there in its summer months.
Shark migration patterns are often closely related to water temperature. Sharks will use expanding summer temperature zones to access food-rich areas that would be too cool for them to visit in the winter months.
Are Sharks Sensitive to Cold?
Yes, all sharks are sensitive to the cold and have a minimum temperature at which they can live.
The majority of sharks are cold-blooded animals (poikilothermic), so their internal body temperature matches their surroundings.
A few shark species, notably the shortfin mako, some thresher sharks, and the great white shark, are homeothermic with higher internal temperatures than the water around them. However, these sharks are still sensitive to the cold and will also have a lower limit at which they can survive.
As well as being sensitive to the cold, sharks are as susceptible, or even more so, to heat. Exposure to excessively warm water will cause the shark’s essential body systems to shut down.
The shark will be unable to obtain the oxygen it needs from the water and will quickly die.
Every shark has a temperature range that it has adapted to. So, water that is too cold for one species may be just right or even too warm for another.
Water temperature can also significantly affect the availability of food for the shark. Sharks, including the blue shark, have been observed to tolerate lower temperatures than usual for short periods due to the abundance of food in cold waters.
Blue sharks have even been tracked diving to depths of 400 meters / 1,312 feet where the water is only 8°C / 46°F to catch their favorite food before returning quickly to the surface to bask in much warmer water.
Sharks That Like Cold Water
Do sharks like cold water? Yes, several shark species have evolved to live successfully in cold water.
If we take cold water as the polar zones below 10°C / 50 °F year round, we’re looking at some unique sharks that have evolved specifically to live in such a harsh environment.
Most of these sharks are endotherms that have developed biological systems to raise their body temperature above that of the surrounding water.
Cold-water sharks may also include the rare deepwater sharks, which live in low temperatures all year round.
For example, the bottom temperature of the Mariana Trench is 1 to 4°C / 34 to 39°F. Although the very bottom might be a little cool, the frilled shark is known to live within the trench’s depths, preferring temperatures below 10°C / 50°F
Other deepwater and, accordingly, coldwater sharks would include the goblin shark, bluntnose sixgill, Pacific sleeper, and the Bahamas sawshark.
Unlike the other deepwater sharks that generate their own heat, the Pacific sleeper survives in the freezing depths by having an anti-freeze system inside its liver.
Instead of the usual squalene, which may solidify at low temperatures, the Pacific sleeper has diacylglycerol ethers and triacylglycerol, which maintain their fluidity in the cold.
These species have all adapted to living in cold water and thrive where others would quickly freeze to death.
For example, in common with other endotherms, the porbeagle traps and reuses the heat generated in its muscles and other organs using specialized blood vessels called retia mirabilia.
These function like an organic heat exchanger and significantly raise the shark’s body temperature to around 8 to 10°C / 14 to 18°F above the surrounding water.
The porbeagle even uses these blood vessels to keep its eyes and brain warm and keep them functioning optimally even when rapidly changing depth when the water can get a lot cooler very quickly.
This incredible system helps almost to eliminate heat loss from the body to the water, allowing these particular sharks to thrive in cold waters.
Are There Sharks in Really Cold Waters?
Yes, there are even sharks that can live in the extreme cold of the Arctic Circle.
However, the Greenland shark is undoubtedly the most famous for having an incredible tolerance to low temperatures.
Greenland sharks have an extremely slow metabolism and are estimated to live between 250 and 500 years, making them the longest-living vertebrate species.
Unlike most sharks in freezing water, these slow-moving sharks are cold-blooded. The ancient shark uses a natural antifreeze cocktail of high concentrations of urea and trimethylamine N-oxide (TMAO) in its body to prevent its proteins from freezing as it swims underneath the ice.
What About Antarctica? Do Sharks Live There?
Antarctica is too cold for even the specially adapted sharks that can live in the Arctic.
There were sharks around 40 million years ago when the waters were much warmer, and with global warming dramatically raising the Antarctic temperature, sharks may be able to return to the waters in years to come.
This would be expected to have a dramatic effect on the ecology of the area.
Sharks That Like Warm Water
Warm water sharks are some of the most well-known, and the broadest range of species lives in these zones.
These sharks take advantage of the warm temperatures and abundant food supply in tropical areas to have higher metabolisms than their cold water relations allowing them to swim faster.
Nurse sharks are exclusively found in tropical regions and are known to have a high tolerance for unusually warm water.
Tiger sharks appear to prefer water at a reasonably precise 22°C / 71.6F and quickly suffer in temperatures higher than about 28C / 82.4F, which is among the reasons that they are not good aquarium inhabitants.
The Inbetweeners – Shark Species That Like Temperate Waters
While many sharks stick to cold or warm water, some inhabit the intermediate temperate zone and migrate back and forth to reproduce or follow food.
Temperate sharks are often larger sharks, including the great white and shortfin mako, who use their relative bulk to thrive in cooler conditions that smaller tropical sharks couldn’t survive in. They may also swim in tropical waters but go deeper where the water is still chilly.
Rather than having the biological “heating systems” that some endothermic sharks have, these sharks usually have a thicker layer of body fat to help keep themselves warm.
Great white sharks are unusual for their ability to live happily in a wide range of temperatures, and the most fearsome of all ocean predators can be found in waters ranging between 12 and 24°C / 54 and 75°F.
Like the cold water shark species, the great white uses specialized heat-exchanging blood vessels to maintain its core body temperature warmer than the surrounding water when necessary.
Incredibly to conserve energy, the shark can regulate this temperature control, and when high levels of activity aren’t required, it can cool to match the surroundings.
The blue shark also has a wide oceanic distribution and lives mainly where the water is between 12 and 20°C / 54 and 68°F, although, as we’ve seen, they can tolerate much cooler water.
Blue sharks are one of the most well-traveled in the ocean and will travel huge distances each year following their favorite foods, even to the Arctic.
Will Sharks Die if the Water Is Too Cold or Too Warm?
Yes, all sharks are sensitive to temperature and will die if the surrounding water is too cold or too hot for their particular biology.
Do Great White Sharks Like Cold Water?
Great white sharks prefer water between 12 and 24°C / 54 and 75°F, which gives them one of the broadest distribution ranges of any shark.
The main reason why great white sharks may be thought to prefer cold water is that their favorite foods typically live there.
Large, fat-rich animals like sea lions, seals, and walruses all tend to live in colder regions meaning great whites will congregate there.
Do Sharks Attack in Cold Water?
Sharks do attack in cold water. However, attacks involving humans are considerably rarer than in warmer water.
The main reason is simply that there are fewer people there. Activities like surfing and swimming all take place in much higher amounts in warm waters, so there is a much greater chance of an incident.
Also, shark concentrations tend to be higher in temperate and tropical waters.
However, sharks are no more or less interested in humans in warm versus cold water, and you should always be cautious of your behavior if you’re in any area known to have sharks.
For example, if you were to swim in a cold water area with high numbers of seals known to attract great whites, you could end up being an accidental meal!
Were There Sharks When the Titanic Sank?
It is unlikely that sharks were present at the site of the Titanic when it sank, as the water temperature (about −2°C / 28 °F) would have been too cold for almost all sharks.
You can check out our full investigation into the subject here for more information.
Do sharks like cold water? Yes, some do!
Sharks, including the Greenland shark, have evolved to swim in some of the very coldest parts of the ocean, including the Arctic.
All shark species have a preferred temperature range specific to the niche that they exist in.
Many shark species we are most familiar with live in tropical and temperate waters. However, others have developed the incredible ability to keep their bodies warmer than their surroundings. In doing so, they can take advantage of the food they find in the colder seas.
British-born Dan has been a scuba instructor and guide in Egypt’s Red Sea since 2010.
Dan loves inspiring safe, fun, and environmentally responsible diving and particularly enjoys the opportunity to dive with sharks or investigate local shipwrecks.
When not spending time underwater, Dan can usually be found biking and hiking in Sharm’s desert surroundings.

domain: geography


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- Legends (10), for piano, 4 hands, B. 117 (Op. 59)
- Violin Concerto in A minor, B. 96/B. 108 (Op. 53)
In his first recording as a soloist for BIS, violinist Richard Tognetti turns in a splendid, sprightly, and deeply felt performance of Dvorák's "Violin Concerto in A minor." With his clean but sweet one, ample but focused vibrato, and impeccable technique, Tognetti delivers a performance near the top of its class, easily surpassing Perlman's overly opulent version and challenging even Josef Suk's all but definitive version. Joined by trombonist turned conductor Christian Lindberg -- alert and engaging -- and the Nordic Chamber Orchestra -- tight but relaxed -- Tognetti's performance may introduce a new generation to this delightful if underrated work. The coupling, however, is less successful. Dvorák's "Legends for orchestra," 10 short works in the style though not in the form of the same composer's "Slavonic Dances," are not quite in the same league as the dances in terms of immediate appeal, and these well-played but not especially sensitive interpretations do not display them to best advantage. For that, try earlier performances by Kubelik or Mackerras. As always with BIS, the digital sound is crisp, clear, and deep.


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UK Small Business Directory
AJ Pets and HoMe3 THE PARADE, HIGH STREET
Tel: Click Here for Phone Number
Pets Foods, Treats and Accessories for your pets
Pet Shops Swanscombe, Pet Shops KentTo add your free business listings see our Free Advertising page.
AJ Pets and HoMe are listed in; Pet Shops Directory : Pet Shops in Kent : Pet Shops in Swanscombe : Kent Business Directory : Swanscombe Business Directory : Pet Shops Listings : AJ Pets and HoMe
Search for Pet Shops by Town - search for Pet Shops by County
Business Category Search, UK County Search and UK Post Town Search

domain: pet


text:
Merchants--North Carolina--History--18th century
Subject Source: Local sources
Found in 2 Collections and/or Records:
Identifier: Mss. 65 B63
Scope and Contents Papers, 1770-1875, of the Blow family of "Tower Hill," Sussex County, Va. and of the Waller family. Prominent correspondents in the collection include Philip Barraud, John Hartwell Cocke, Henry Lee and Edmund Ruffin. Most of these accessions were integrated as the collection was partially processed, but some are boxed separately. Includes a Blow family scrapbook containing many manuscript items. Collection includes: correspondence of Richard Blow (1746-1833), merchant, who had...
Identifier: Mss. 65 B625
Scope and Contents Papers, 1772-1872, of the Blow family. Chiefly business papers, 1772-1820, of Richard Blow of Portsmouth and "Tower Hill," Sussex County, Va. and his son George Blow, Sr. and George Blow, Jr. of 'Tower Hill' and Williamsburg, Va. Richard Blow was a partner in stores in Virginia and North Carolina (trading as Richard Blow and Co., Briggs & Blow, Baker & Blow [South Quay, Nansemond County, Va.], Blow & Barksdale [Petersburg, Va.], and Blow and Scammel). He was also a plantation...