Screening for Novel Type 2 Ryanodine Receptor Inhibitors by Endoplasmic Reticulum Ca2+ Monitoring.

Type 2 ryanodine receptor (RyR2) is a Ca2+ release channel on the endoplasmic (ER)/sarcoplasmic reticulum that plays a central role in the excitation-contraction coupling in the heart. Hyperactivity of RyR2 has been linked to ventricular arrhythmias in patients with catecholaminergic polymorphic ventricular tachycardia and heart failure, where spontaneous Ca2+ release via hyperactivated RyR2 depolarizes diastolic membrane potential to induce triggered activity. In such cases, drugs that suppress RyR2 activity are expected to prevent the arrhythmias, but there is no clinically available RyR2 inhibitors at present. In this study, we searched for RyR2 inhibitors from a well-characterized compound library using a recently developed ER Ca2+-based assay, where the inhibition of RyR2 activity was detected by the increase in ER Ca2+ signals from R-CEPIA1er, a genetically encoded ER Ca2+ indicator, in RyR2-expressing HEK293 cells. By screening 1535 compounds in the library, we identified three compounds (chloroxylenol, methyl orsellinate, and riluzole) that greatly increased the ER Ca2+ signal. All of the three compounds suppressed spontaneous Ca2+ oscillations in RyR2-expressing HEK293 cells and correspondingly reduced the Ca2+-dependent [3H]ryanodine binding activity. In cardiomyocytes from RyR2-mutant mice, the three compounds effectively suppressed abnormal Ca2+ waves without substantial effects on the action-potential-induced Ca2+ transients. These results confirm that ER Ca2+-based screening is useful for identifying modulators of ER Ca2+ release channels and suggest that RyR2 inhibitors have potential to be developed as a new category of antiarrhythmic drugs. SIGNIFICANCE STATEMENT: We successfully identified three compounds having RyR2 inhibitory action from a well-characterized compound library using an endoplasmic reticulum Ca2+-based assay, and demonstrated that these compounds suppressed arrhythmogenic Ca2+ wave generation without substantially affecting physiological action-potential induced Ca2+ transients in cardiomyocytes. This study will facilitate the development of RyR2-specific inhibitors as a potential new class of drugs for life-threatening arrhythmias induced by hyperactivation of RyR2.

Treatment planning comparison of high-dose-rate brachytherapy vs. robotic and conventional stereotactic body radiotherapy for ultrahypofractionated treatment of prostate cancer.

Background and purpose: Ultrahypofractionated radiation therapy is increasingly used in the treatment of prostate cancer. High-dose-rate brachytherapy (HDR-BT) and stereotactic body radiotherapy (SBRT) are representative methods of ultrahypofractionation. This study was performed to compare clinically applied treatment plans for patients who had been treated using HDR-BT vs. conventional or robotic SBRT. Materials and methods: Calculated dose-volume indices between HDR-BT without a perirectal spacer (n = 20), robotic SBRT without a spacer (n = 40), and conventional (non-robotic) SBRT with a spacer (n = 40) were compared. Percentages against the prescription dose regarding the planning target volume (PTV), bladder, rectum, and urethra were statistically compared. Results: The D50% of the PTV with HDR-BT (140.5% ± 4.9%) was significantly higher than that with robotic or conventional SBRT (116.2% ± 1.6%, 101.0% ± 0.4%, p < 0.01). The D2cm3 of the bladder with HDR-BT (65.6% ± 6.4%) was significantly lower than those with SBRT (105.3% ± 2.9%, 98.0% ± 1.3%, p < 0.01). The D2cm3 of the rectum with HDR-BT (60.6% ± 6.2%) was also significantly lower than those with SBRT (85.1% ± 8.8%, 70.4% ± 9.6%, p < 0.01). By contrast, the D0.1cm3 of the urethra with HDR-BT (117.1% ± 3.6%) was significantly higher than those with SBRT (100.2% ± 0.7%, 104.5% ± 0.6%, p < 0.01). Conclusions: HDR-BT could administer a higher dose to the PTV and a lower dose to the bladder and rectum, at the cost of a slightly higher dose to the urethra compared with SBRT.

Stereotactic body radiation therapy for prostate cancer: a study comparing 3-year genitourinary toxicity between CyberKnife and volumetric-modulated arc therapy by propensity score analysis.

BACKGROUND: To investigate whether the rate of stereotactic body radiation therapy-related (SBRT-related) genitourinary (GU) toxicity is lower in patients with prostate cancer treated with CyberKnife. METHODS: We retrospectively reviewed the medical records of patients with nonmetastatic prostate cancer at two institutions between 2017 and 2020. We analyzed 70 patients who were extracted by propensity score matching based on age, pre-treatment International Prostate Symptom Score (IPSS), and prostate volume. The patients were treated with SBRT, with a total dose of 36.25 Gy in five fractions over five consecutive weekdays, using CyberKnife or volumetric-modulated arc therapy (VMAT). RESULTS: The low-, medium-, and high-risk patients were 2, 19, and 14, respectively, in the CyberKnife group and 4, 17, and 14, respectively, in the VMAT group. The median follow-up time in both groups was 3 years. One patient with CyberKnife died of unrelated causes. No biochemical or clinical recurrence, distant metastases, or death from prostate cancer was observed. The peak values of IPSS in the acute phase (< 3 months) were significantly lower in the CyberKnife than in the VMAT group (CyberKnife:16.2 vs VMAT:20.2, p = 0.025). In multiple regression analyses, the treatment modality (p = 0.03), age (p = 0.01), bladder medication pre-irradiation (p = 0.03), and neoadjuvant androgen deprivation therapy (p = 0.04) contributed to the peak value of the acute-phase IPSS. The incidence of treatment-related grade 2 acute GU toxicity tended to be lower in the CyberKnife than the VMAT group (CyberKnife: 22.9% vs. VMAT: 45.7%, p = 0.077). No difference was noted between the groups with regard to late IPSS or GU toxicity and gastrointestinal toxicity in all phases. Toxicities of grade ≥ 3 have not been observed to date. CONCLUSIONS: Regardless of treatment modality, SBRT is effective in treating prostate cancer without serious toxicity. However, CyberKnife has an advantage over VMAT in terms of acute prostate symptoms.

The effect of vibration generated by demolition work on irradiation position accuracy of stereotactic radiotherapy system.

PURPOSE: One existing building, adjacent to the radiotherapy building, where stereotactic radiation was done, was to be demolished to give space for the construction of a new building. However, we were concerned that the vibrations generated by this demolition work, which occurred within 2 m from the radiotherapy building, would affect the radiation position accuracy of the radiotherapy machine. METHODS: To determine whether radiotherapy could be performed safely during the demolition period, we performed simulation tests involving the vibrations generated during demolition, measured these vibrations, and verified their effect on the irradiation position accuracy of the stereotactic radiotherapy system. For effective evaluations, tests were conducted assuming the maximum vibrations that could occur during actual demolition work. RESULTS: The maximum displacement of the vibrations generated by the simulated demolition work was 3.30 µm on the floor of the treatment room and 4.68 µm at the ceiling. CONCLUSIONS: The results of the vibration measurements exceeded the limits of the criteria applicable to the electron beam system. However, the accuracy of the irradiation position of the stereotactic radiotherapy system remained unchanged during these vibrations. Therefore, the vibrations had no impact on radiotherapy safety, and radiotherapy was continued during the demolition work while coordinating with the demolition workers as necessary.

Deficiency of the splicing factor RBM10 limits EGFR inhibitor response in EGFR-mutant lung cancer.

Molecularly targeted cancer therapy has improved outcomes for patients with cancer with targetable oncoproteins, such as mutant EGFR in lung cancer. Yet, the long-term survival of these patients remains limited, because treatment responses are typically incomplete. One potential explanation for the lack of complete and durable responses is that oncogene-driven cancers with activating mutations of EGFR often harbor additional co-occurring genetic alterations. This hypothesis remains untested for most genetic alterations that co-occur with mutant EGFR. Here, we report the functional impact of inactivating genetic alterations of the mRNA splicing factor RNA-binding motif 10 (RBM10) that co-occur with mutant EGFR. RBM10 deficiency decreased EGFR inhibitor efficacy in patient-derived EGFR-mutant tumor models. RBM10 modulated mRNA alternative splicing of the mitochondrial apoptotic regulator Bcl-x to regulate tumor cell apoptosis during treatment. Genetic inactivation of RBM10 diminished EGFR inhibitor-mediated apoptosis by decreasing the ratio of (proapoptotic) Bcl-xS to (antiapoptotic) Bcl-xL isoforms of Bcl-x. RBM10 deficiency was a biomarker of poor response to EGFR inhibitor treatment in clinical samples. Coinhibition of Bcl-xL and mutant EGFR overcame the resistance induced by RBM10 deficiency. This study sheds light on the role of co-occurring genetic alterations and on the effect of splicing factor deficiency on the modulation of sensitivity to targeted kinase inhibitor cancer therapy.

Mitochondrial uncouplers induce proton leak by activating AAC and UCP1.

Mitochondria generate heat due to H+ leak (IH) across their inner membrane1. IH results from the action of long-chain fatty acids on uncoupling protein 1 (UCP1) in brown fat2-6 and ADP/ATP carrier (AAC) in other tissues1,7-9, but the underlying mechanism is poorly understood. As evidence of pharmacological activators of IH through UCP1 and AAC is lacking, IH is induced by protonophores such as 2,4-dinitrophenol (DNP) and cyanide-4-(trifluoromethoxy) phenylhydrazone (FCCP)10,11. Although protonophores show potential in combating obesity, diabetes and fatty liver in animal models12-14, their clinical potential for treating human disease is limited due to indiscriminately increasing H+ conductance across all biological membranes10,11 and adverse side effects15. Here we report the direct measurement of IH induced by DNP, FCCP and other common protonophores and find that it is dependent on AAC and UCP1. Using molecular structures of AAC, we perform a computational analysis to determine the binding sites for protonophores and long-chain fatty acids, and find that they overlap with the putative ADP/ATP-binding site. We also develop a mathematical model that proposes a mechanism of uncoupler-dependent IH through AAC. Thus, common protonophoric uncouplers are synthetic activators of IH through AAC and UCP1, paving the way for the development of new and more specific activators of these two central mediators of mitochondrial bioenergetics.

Cytosolic Ca2+-dependent Ca2+ release activity primarily determines the ER Ca2+ level in cells expressing the CPVT-linked mutant RYR2.

Type 2 ryanodine receptor (RYR2) is a cardiac Ca2+ release channel in the ER. Mutations in RYR2 are linked to catecholaminergic polymorphic ventricular tachycardia (CPVT). CPVT is associated with enhanced spontaneous Ca2+ release, which tends to occur when [Ca2+]ER reaches a threshold. Mutations lower the threshold [Ca2+]ER by increasing luminal Ca2+ sensitivity or enhancing cytosolic [Ca2+] ([Ca2+]cyt)-dependent activity. Here, to establish the mechanism relating the change in [Ca2+]cyt-dependent activity of RYR2 and the threshold [Ca2+]ER, we carried out cell-based experiments and in silico simulations. We expressed WT and CPVT-linked mutant RYR2s in HEK293 cells and measured [Ca2+]cyt and [Ca2+]ER using fluorescent Ca2+ indicators. CPVT RYR2 cells showed higher oscillation frequency and lower threshold [Ca2+]ER than WT cells. The [Ca2+]cyt-dependent activity at resting [Ca2+]cyt, Arest, was greater in CPVT mutants than in WT, and we found an inverse correlation between threshold [Ca2+]ER and Arest. In addition, lowering RYR2 expression increased the threshold [Ca2+]ER and a product of Arest, and the relative expression level for each mutant correlated with threshold [Ca2+]ER, suggesting that the threshold [Ca2+]ER depends on the net Ca2+ release rate via RYR2. Modeling reproduced Ca2+ oscillations with [Ca2+]cyt and [Ca2+]ER changes in WT and CPVT cells. Interestingly, the [Ca2+]cyt-dependent activity of specific mutations correlated with the age of disease onset in patients carrying them. Our data suggest that the reduction in threshold [Ca2+]ER for spontaneous Ca2+ release by CPVT mutation is explained by enhanced [Ca2+]cyt-dependent activity without requiring modulation of the [Ca2+]ER sensitivity of RYR2.

The mechanism of MICU-dependent gating of the mitochondrial Ca2+uniporter.

Ca2+ entry into mitochondria is through the mitochondrial calcium uniporter complex (MCUcx), a Ca2+-selective channel composed of five subunit types. Two MCUcx subunits (MCU and EMRE) span the inner mitochondrial membrane, while three Ca2+-regulatory subunits (MICU1, MICU2, and MICU3) reside in the intermembrane space. Here, we provide rigorous analysis of Ca2+ and Na+ fluxes via MCUcx in intact isolated mitochondria to understand the function of MICU subunits. We also perform direct patch clamp recordings of macroscopic and single MCUcx currents to gain further mechanistic insights. This comprehensive analysis shows that the MCUcx pore, composed of the EMRE and MCU subunits, is not occluded nor plugged by MICUs during the absence or presence of extramitochondrial Ca2+ as has been widely reported. Instead, MICUs potentiate activity of MCUcx as extramitochondrial Ca2+ is elevated. MICUs achieve this by modifying the gating properties of MCUcx allowing it to spend more time in the open state.

Dosimetric impacts of beam-hardening filter removal for the CyberKnife system.

PURPOSE: Equipment refurbishment was performed to remove the beam-hardening filter (BHF) from the CyberKnife system (CK). This study aimed to confirm the change in the beam characteristics between the conventional CK (present-BHF CK) and CK after the BHF was removed (absent-BHF CK) and evaluate the impact of BHF removal on the beam quality correction factors kQ. METHODS: The experimental measurements of the beam characteristics of the present- and absent-BHF CKs were compared. The CKs were modeled using Monte Carlo simulations (MCs). The energy fluence spectra were calculated using MCs. Finally, kQ were estimated by combining the MC results and analytic calculations based on the TRS-398 and TRS-483 approaches. RESULTS: All gamma values for percent depth doses and beam profiles between each CK were less than 0.5 following the 3%/1 mm criteria. The percentage differences for tissue-phantom ratios at depths of 20 and 10 cm and percentage depth doses at 10 cm between each CK were -1.20% and -0.97%, respectively. The MC results demonstrated that the photon energy fluence spectrum of the absent-BHF CK was softer than that of the present-BHF CK. The kQ values for the absent-BHF CK were in agreement within 0.02% with those for the present-BHF CK. CONCLUSIONS: The photon energy fluence spectrum was softened by the removal of BHF. However, no remarkable impact was observed for the measured beam characteristics and kQ. Therefore, the previous findings of the kQ values for the present-BHF CK can be directly used for the absent-BHF CK.

Stereotactic body radiation therapy for Japanese patients with localized prostate cancer: 2-year results and predictive factors for acute genitourinary toxicities.

OBJECTIVE: We aimed to report the 2-year results of stereotactic body radiation therapy for prostate cancer and identify the clinical and dosimetric factors that predict acute genitourinary toxicities. METHODS: We retrospectively reviewed the medical records of patients with non-metastatic prostate cancer treated at Toyota Memorial Hospital between 2017 and 2020. The patients were treated with stereotactic body radiation therapy with a total dose of 36.25 Gy in five fractions on consecutive weekdays. While low-risk patients received radiotherapy alone, intermediate- to high-risk patients also received androgen deprivation therapy. RESULTS: We analysed a total of 104 patients, including 10, 60 and 34 low-, intermediate- and high-risk patients, respectively. The median follow-up duration was 2 years. We did not observe biochemical/clinical recurrence, distant metastasis or death from prostate cancer. One patient died of another cause. Grade 2 acute genitourinary toxicity was observed in 40 (38%) patients. Age (P = 0.021), genitourinary toxicity of grade ≥1 at baseline (P = 0.023) and bladder mean dose (P = 0.047) were significantly associated with the incidence of grade 2 acute genitourinary toxicity. The cut-off value of 65 years for age and 10.3 Gy for the bladder mean dose were considered the most appropriate. Grade 2 acute gastrointestinal toxicity was observed in five (5%) patients. None of the patients experienced grade ≥3 acute or late toxicity. CONCLUSIONS: Stereotactic body radiation therapy is feasible for Japanese patients with prostate cancer, with acceptable acute toxicity. Age, genitourinary toxicity at baseline and bladder mean dose predict grade 2 acute genitourinary toxicity.

Exact Real-Time Longitudinal Correlation Functions of the Massive XXZ Chain.

We apply a recently developed thermal form factor expansion method to evaluate the real-time longitudinal spin-spin correlation functions of the spin-1/2 XXZ chain in the antiferromagnetically ordered regime at zero temperature. An analytical result incorporating all types of excitations in the model is obtained, without any approximations. This allows for the accurate calculation of the real-time correlations in this strongly interacting quantum system for arbitrary distances and times.

Comparison of Physician-recorded Toxicities and Patient-reported Outcomes of Five Different Radiotherapy Methods for Prostate Cancer.

BACKGROUND/AIM: To compare five radiotherapy methods for prostate cancer. PATIENTS AND METHODS: During 2005-2018, the data of patients with non-metastatic prostate cancer were retrospectively analysed. Patients were treated with high-dose-rate brachytherapy (HDR-BT); low-dose-rate brachytherapy (LDR-BT); or external-beam radiotherapy (EBRT), including conventionally fractionated radiotherapy (CFRT), moderate-hypofractionated radiotherapy (MHRT), and ultra-hypofractionated radiotherapy (UHRT). RESULTS: In total, 496 patients (149, HDR-BT; 100, LDR-BT; 100, CFRT; 97, MHRT, and 50, UHRT) with a median follow-up of 4.3 years were enrolled. The incidence of grade ≥2 acute genitourinary toxicities was significantly lower with HDR-BT (p<0.001) than with any other radiotherapy. The cumulative incidence of late grade ≥2 genitourinary toxicities was the highest with UHRT and significantly higher (p=0.005) with UHRT than with HDR-BT. Higher symptom score peaks were noted 4 weeks after therapy for LDR-BT than for EBRT. CONCLUSION: Physician-recorded toxicities were slightly lower with HDR-BT and patient-reported outcomes tended to be worse with LDR-BT.

Red fluorescent CEPIA indicators for visualization of Ca2+ dynamics in mitochondria.

Mitochondrial Ca2+ dynamics are involved in the regulation of multifarious cellular processes, including intracellular Ca2+ signalling, cell metabolism and cell death. Use of mitochondria-targeted genetically encoded Ca2+ indicators has revealed intercellular and subcellular heterogeneity of mitochondrial Ca2+ dynamics, which are assumed to be determined by distinct thresholds of Ca2+ increases at each subcellular mitochondrial domain. The balance between Ca2+ influx through the mitochondrial calcium uniporter and extrusion by cation exchangers across the inner mitochondrial membrane may define the threshold; however, the precise mechanisms remain to be further explored. We here report the new red fluorescent genetically encoded Ca2+ indicators, R-CEPIA3mt and R-CEPIA4mt, which are targeted to mitochondria and their Ca2+ affinities are engineered to match the intramitochondrial Ca2+ concentrations. They enable visualization of mitochondrial Ca2+ dynamics with high spatiotemporal resolution in parallel with the use of green fluorescent probes and optogenetic tools. Thus, R-CEPIA3mt and R-CEPIA4mt are expected to be a useful tool for elucidating the mechanisms of the complex mitochondrial Ca2+ dynamics and their functions.

Insights into channel modulation mechanism of RYR1 mutants using Ca2+ imaging and molecular dynamics.

Type 1 ryanodine receptor (RYR1) is a Ca2+ release channel in the sarcoplasmic reticulum in skeletal muscle and plays an important role in excitation-contraction coupling. Mutations in the RYR1 gene cause severe muscle diseases such as malignant hyperthermia (MH), which is a disorder of CICR via RYR1. Thus far, >300 mutations in RYR1 have been reported in patients with MH. However, owing to a lack of comprehensive analysis of the structure-function relationship of mutant RYR1, the mechanism remains largely unknown. Here, we combined functional studies and molecular dynamics (MD) simulations of RYR1 bearing disease-associated mutations at the N-terminal region. When expressed in HEK293 cells, the mutant RYR1 caused abnormalities in Ca2+ homeostasis. MD simulations of WT and mutant RYR1s were performed using crystal structure of the N-terminal domain (NTD) monomer, consisting of A, B, and C domains. We found that the mutations located around the interdomain region differentially affected hydrogen bonds/salt bridges. Particularly, mutations at R402, which increase the open probability of the channel, cause clockwise rotation of BC domains with respect to the A domain by alteration of the interdomain interactions. Similar results were also obtained with artificial mutations that mimic alteration of the interactions. Our results reveal the importance of interdomain interactions within the NTD in the regulation of the RYR1 channel and provide insights into the mechanism of MH caused by the mutations at the NTD.

Synthetic Essentiality of Metabolic Regulator PDHK1 in PTEN-Deficient Cells and Cancers.

Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is a tumor suppressor and bi-functional lipid and protein phosphatase. We report that the metabolic regulator pyruvate dehydrogenase kinase1 (PDHK1) is a synthetic-essential gene in PTEN-deficient cancer and normal cells. The PTEN protein phosphatase dephosphorylates nuclear factor κB (NF-κB)-activating protein (NKAP) and limits NFκB activation to suppress expression of PDHK1, a NF-κB target gene. Loss of the PTEN protein phosphatase upregulates PDHK1 to induce aerobic glycolysis and PDHK1 cellular dependence. PTEN-deficient human tumors harbor increased PDHK1, a biomarker of decreased patient survival. This study uncovers a PTEN-regulated signaling pathway and reveals PDHK1 as a potential target in PTEN-deficient cancers.

Inositol 1,4,5-trisphosphate receptor type 2-independent Ca2+ release from the endoplasmic reticulum in astrocytes.

Accumulating evidence indicates that astrocytes are actively involved in the physiological and pathophysiological functions of the brain. Intracellular Ca2+ signaling, especially Ca2+ release from the endoplasmic reticulum (ER), is considered to be crucial for the regulation of astrocytic functions. Mice with genetic deletion of inositol 1,4,5-trisphosphate receptor type 2 (IP3 R2) are reportedly devoid of astrocytic Ca2+ signaling, and thus widely used to explore the roles of Ca2+ signaling in astrocytic functions. While functional deficits in IP3 R2-knockout (KO) mice have been found in some reports, no functional deficit was observed in others. Thus, there remains a controversy regarding the functional significance of astrocytic Ca2+ signaling. To address this controversy, we re-evaluated the assumption that Ca2+ release from the ER is abolished in IP3 R2-KO astrocytes using a highly sensitive imaging technique. We expressed the ER luminal Ca2+ indicator G-CEPIA1er in cortical and hippocampal astrocytes to directly visualize spontaneous and stimulus-induced Ca2+ release from the ER. We found attenuated but significant Ca2+ release in response to application of norepinephrine to IP3 R2-KO astrocytes. This IP3 R2-independent Ca2+ release induced only minimal cytosolic Ca2+ transients but induced robust Ca2+ increases in mitochondria that are frequently in close contact with the ER. These results indicate that ER Ca2+ release is retained and is sufficient to increase the Ca2+ concentration in close proximity to the ER in IP3 R2-KO astrocytes.

Efficient High-Throughput Screening by Endoplasmic Reticulum Ca2+ Measurement to Identify Inhibitors of Ryanodine Receptor Ca2+-Release Channels.

Genetic mutations in ryanodine receptors (RyRs), Ca2+-release channels in the sarcoplasmic reticulum essential for muscle contractions, cause various skeletal muscle and cardiac diseases. Because the main underlying mechanism of the pathogenesis is overactive Ca2+ release by gain-of-function of the RyR channel, inhibition of RyRs is expected to be a promising treatment of these diseases. Here, to identify inhibitors specific to skeletal muscle type 1 RyR (RyR1), we developed a novel high-throughput screening (HTS) platform using time-lapse fluorescence measurement of Ca2+ concentrations in the endoplasmic reticulum (ER) ([Ca2+]ER). Because expression of RyR1 carrying disease-associated mutation reduces [Ca2+]ER in HEK293 cells through Ca2+ leakage from RyR1 channels, specific drugs that inhibit RyR1 will increase [Ca2+]ER by preventing such Ca2+ leakage. RyR1 carrying the R2163C mutation and R-CEPIA1er, a genetically encoded ER Ca2+ indicator, were stably expressed in HEK293 cells, and time-lapse fluorescence was measured using a fluorometer. False positives were effectively excluded by using cells expressing wild-type (WT) RyR1. By screening 1535 compounds in a library of well characterized drugs, we successfully identified four compounds that significantly increased [Ca2+]ER They include dantrolene, a known RyR1 inhibitor, and three structurally different compounds: oxolinic acid, 9-aminoacridine, and alexidine. All the hit compounds, except for oxolinic acid, inhibited [3H]ryanodine binding of WT and mutant RyR1. Interestingly, they showed different dose dependencies and isoform specificities. The highly quantitative nature and good correlation with the channel activity validated this HTS platform by [Ca2+]ER measurement to explore drugs for RyR-related diseases.

Clinical usefulness of MLCs in robotic radiosurgery systems for prostate SBRT.

Stereotactic body radiation therapy (SBRT) using recently introduced multileaf collimators (MLC) is preferred over circular collimators in the treatment of localized prostate cancer. The objective of this study was to assess the clinical usefulness of MLCs in prostate SBRT by comparing the effectiveness of treatment plans using fixed collimators, variable collimators, and MLCs and by ensuring delivery quality assurance (DQA) for each. For each patient who underwent conventional radiation therapy for localized prostate cancer, mock SBRT plans were created using a fixed collimator, a variable collimator, and an MLC. The total MUs, treatment times, and dose-volume histograms of the planning target volumes and organs at risk for each treatment plan were compared. For DQA, a phantom with a radiochromic film or an ionization chamber was irradiated in each plan. We performed gamma-index analysis to evaluate the consistency between the measured and calculated doses. The MLC-based plans had an ~27% lower average total MU than the plans involving other collimators. Moreover, the average estimated treatment time for the MLC plan was 31% and 20% shorter than that for the fixed and variable collimator plans respectively. The gamma-index passing rate in the DQA using film measurements was slightly lower for the MLC than for the other collimators. The DQA results acquired using the ionization chamber showed that the discrepancies between the measured and calculated doses were within 3% in all cases. The results reinforce the usefulness of MLCs in robotic radiosurgery for prostrate SBRT treatment planning; most notably, the total MU and treatment time were both reduced compared to the cases using other types of collimators. Moreover, although the DQA results based on film dosimetry yielded a slightly lower gamma-index passing rate for the MLC than for the other collimators, the MLC accuracy was determined to be sufficient for clinical use.

Genotype-Phenotype Correlations of Malignant Hyperthermia and Central Core Disease Mutations in the Central Region of the RYR1 Channel.

Type 1 ryanodine receptor (RYR1) is a Ca2+ release channel in the sarcoplasmic reticulum of skeletal muscle and is mutated in some muscle diseases, including malignant hyperthermia (MH) and central core disease (CCD). Over 200 mutations associated with these diseases have been identified, and most mutations accelerate Ca2+ -induced Ca2+ release (CICR), resulting in abnormal Ca2+ homeostasis in skeletal muscle. However, it remains largely unknown how specific mutations cause different phenotypes. In this study, we investigated the CICR activity of 14 mutations at 10 different positions in the central region of RYR1 (10 MH and four MH/CCD mutations) using a heterologous expression system in HEK293 cells. In live-cell Ca2+ imaging, the mutant channels exhibited an enhanced sensitivity to caffeine, a reduced endoplasmic reticulum Ca2+ content, and an increased resting cytoplasmic Ca2+ level. The three parameters for CICR (Ca2+ sensitivity for activation, Ca2+ sensitivity for inactivation, and attainable maximum activity, i.e., gain) were obtained by [3 H]ryanodine binding and fitting analysis. The mutant channels showed increased gain and Ca2+ sensitivity for activation in a site-specific manner. Genotype-phenotype correlations were explained well by the near-atomic structure of RYR1. Our data suggest that divergent CICR activity may cause various disease phenotypes by specific mutations.

Genetically Encoded Fluorescent Indicators for Organellar Calcium Imaging.

Optical Ca(2+) indicators are powerful tools for investigating intracellular Ca(2+) signals in living cells. Although a variety of Ca(2+) indicators have been developed, deciphering the physiological functions and spatiotemporal dynamics of Ca(2+) in intracellular organelles remains challenging. Genetically encoded Ca(2+) indicators (GECIs) using fluorescent proteins are promising tools for organellar Ca(2+) imaging, and much effort has been devoted to their development. In this review, we first discuss the key points of organellar Ca(2+) imaging and summarize the requirements for optimal organellar Ca(2+) indicators. Then, we highlight some of the recent advances in the engineering of fluorescent GECIs targeted to specific organelles. Finally, we discuss the limitations of currently available GECIs and the requirements for advancing the research on intraorganellar Ca(2+) signaling.