The dynamic relationship between inorganic polyphosphate and adenosine triphosphate in human non-small cell lung cancer H1299 cells.

Inorganic polyphosphate (polyP) plays a vital role in cellular energy metabolism and signaling, owing to its structure and high-energy phosphate bonds. Intracellular ATP functions both as a cellular energy source and a key factor in cell death, and ATP dynamics in tumor cells are crucial for advancing cancer therapy. In this study, we explored the interplay between polyP and ATP in cellular energy metabolism. Treatment with polyP did not affect cell proliferation of human non-small cell lung cancer H1299 and human glioblastoma T98G cell lines as compared to their respective control cells until 72 h post-treatment. The mitochondrial membrane potential (MMP) in polyP-treated cells was low, contrasting with the time-dependent increase observed in control cells. While the ATP content increased over time in untreated and Na-phosphate-treated control cells, it remained unchanged in polyP-treated cells. Furthermore, the addition of cyclosporine A, a mitochondrial permeability transition pore (mPTP) inhibitor, failed to restore ATP levels in polyP-treated cells. We performed lactate assays and western blot analysis to evaluate the effect of polyP on glucose metabolism and found no significant differences in lactate secretion or glucose-6-phosphate dehydrogenase (G6PD) activity between polyP-treated and control cells. Additional pyruvate restored MMP but had no effect on the cellular ATP content in polyP-treated cells. We observed no correlation between the Warburg effect and glucose metabolism during ATP depletion in polyP-treated cells. Further investigation is warranted to explore the roles of polyP and ATP in cancer cell energy metabolism, which might offer potential avenues for therapeutic interventions.

Evaluation of Image Classification for Quantifying Mitochondrial Morphology Using Deep Learning.

BACKGROUND: Mitochondrial morphology reversibly changes between fission and fusion. As these changes (mitochondrial dynamics) reflect the cellular condition, they are one of the simplest indicators of cell state and predictors of cell fate. However, it is currently difficult to classify them using a simple and objective method. OBJECTIVE: The present study aimed to evaluate mitochondrial morphology using Deep Learning (DL) technique. METHODS: Mitochondrial images stained by MitoTracker were acquired from HeLa and MC3T3-E1 cells using fluorescent microscopy and visually classified into four groups based on fission or fusion. The intra- and inter-rater reliabilities for visual classification were excellent [(ICC(1,3), 0.961 for rater 1; and 0.981 for rater 2) and ICC(1,3), respectively]. The images were divided into test and train images, and a 50-layer ResNet CNN architecture (ResNet-50) using MATLAB software was used to train the images. The datasets were trained five times based on five-fold cross-validation. RESULT: The mean of the overall accuracy for classifying mitochondrial morphology was 0.73±0.10 in HeLa. For the classification of mixed images containing two types of cell lines, the overall accuracy using mixed images of both cell lines for training was higher (0.74±0.01) than that using different cell lines for training. CONCLUSION: We developed a classifier to categorize mitochondrial morphology using DL.

Feasibility of an Ultrasound-Based Method for Measuring Talar Displacement during the Anterior Drawer Stress Test Using a Telos Device: A Preliminary Study.

This study was conducted to measured talar displacement using ultrasound during an anterior drawer test (ADT) with a Telos device. Five adults (3 men and 2 women; 8 ankles; mean age: 23.2 y) with a history of ankle sprain and eight adults (5 men and 3 women; 16 ankles; mean age: 22.1 y) without a history of ankle sprain were recruited into a history of ankle sprain (HAS) and a control group, respectively. Talar displacement was observed in response to load forces applied by a Telos device during the ultrasound stress imaging test. The ultrasound probe was placed 5 mm inside from the center of the Achilles tendon on the posterior ankle along the direction of the major axis. The inter-rater reliability for the present method was classified as good and excellent (ICC(2,2) = 0.858 and 0.957 at 120 N and 150 N, respectively) in the control group and excellent (ICC(2,2) = 0.940 and 0.905 at 120 N and 150 N, respectively) in the HAS group, according to specific intraclass correlation coefficient values. We found that talar displacement during the ADT was lower in the HAS group than in the control group. Analysis of the receiver operating characteristic curve revealed that the quantitative ultrasound-based ADT using a Telos device was superior to the X-ray-based test in detecting reduced ankle joint mobility during the ADT (area under the curve of 0.905 and 0.726 at a force of 150 N using ultrasound-based and X-ray-based tests, respectively). Further investigation is needed; nevertheless, this preliminary study suggests that the ultrasound-based quantitative ADT using a Telos device might detect talar displacement more sensitively than the conventional stress X-ray.

Contribution of Neuropilin-1 in Radiation-Survived Subclones of NSCLC Cell Line H1299.

Non-small cell lung cancer (NSCLC) is an aggressive lung cancer accounting for approximately 85% of all lung cancer patients. For the patients with Stages IIIA, IIIB, and IIIC, the 5-year survival is low though with the combination with radiotherapy and chemotherapy. In addition, the occurrence of tumor cells (repopulated tumors) that survive irradiation remains a challenge. In our previous report, we subcloned the radiation-surviving tumor cells (IR cells) using the human NSCLC cell line, H1299, and found that the expression of neuropilin-1 (NRP-1) was upregulated in IR cells by the microarray analysis. Here, we investigated the contribution of neuropilin-1 to changes in the characteristics of IR cells. Although there were no differences in angiogenic activity in the tube formation assay between parental and IR cells, the cell motility was increased in IR cells compared to parental cells in the cell migration assay. This enhanced cell motility was suppressed by pretreatment with anti-NRP-1 antibody. Although further studies are necessary to identify other molecules associated with NRP-1, the increase in cellular motility in IR cells might be due to the contribution of NRP-1. Inhibition of NRP-1 would help control tumor malignancy in radiation-surviving NSCLC.

Chromosomal Location of Genes Differentially Expressed in Tumor Cells Surviving High-Dose X-ray Irradiation: A Preliminary Study on Radio-Fragile Sites.

Altered gene expression is a common feature of tumor cells after irradiation. Our previous study showed that this phenomenon is not only an acute response to cytotoxic stress, instead, it was persistently detected in tumor cells that survived 10 Gy irradiation (IR cells). The current understanding is that DNA double-strand breaks (DSBs) are recognized by the phosphorylation of histone H2AX (H2AX) and triggers the ataxia-telangiectasia mutated (ATM) protein or the ATM- and Rad3-related (ATR) pathway, which activate or inactivate the DNA repair or apoptotic or senescence related molecules and causes the expression of genes in many instances. However, because changes in gene expression persist after passaging in IR cells, it may be due to the different pathways from these transient intracellular signaling pathways caused by DSBs. We performed microarray analysis of 30,000 genes in radiation-surviving cells (H1299-IR and MCF7-IR) and found an interesting relation between altered genes and their chromosomal loci. These loci formed a cluster on the chromosome, especially on 1q21 and 6p21-p22 in both irradiated cell lines. These chromosome sites might be regarded as "radio-fragile" sites.

Association between Physical Activity Levels and Body Composition among Healthy Older Japanese Adults during a Snowy Winter: A Cross-Sectional Study.

BACKGROUND: Despite a long average lifespan, increased life expectancy does not guarantee higher quality of life. METHODS: To contribute in understanding some determinants of healthy life expectancies in older Japanese individuals in a snowy winter region, we investigated the indicators of health. Local residents (n = 124) in the city of Iwamizawa volunteered for health examinations from January 2016 to March 2016. We recorded activity via daily steps for 2-week periods. In addition, we measured body composition, grip strength, and assessed nutritional status. RESULTS: Analysis of body composition and daily activity indicated that women who walked more than 4000 steps had lower fat mass and increased muscle mass. Men with >3.0 metabolic equivalents (METs) when walking had lower body fat. CONCLUSION: For healthy older Japanese individuals in this snowy winter region, walking >4000 steps daily for women and exercise of >3.0 METs for men may indicate health-promoting activities.

Cellular calcification induced by inorganic polyphosphate involves ATP depletion and opening of the mitochondrial permeability transition pore (mPTP).

Inorganic polyphosphate (polyP) is a linear polymer containing tens to hundreds of orthophosphate residues linked by high-energy phosphoanhydride bonds. PolyP promotes osteocalcification and bone mineralization in both mouse and human osteoblastic cells. In the present study, we examined the molecular mechanism by which polyP affects mitochondrial metabolism to promote cellular calcification in MC3T3-E1 osteoblastic cells. The cellular content of adenosine triphosphate (ATP) was diminished one day after polyP treatment, and this was accompanied by increased conversion to adenosine diphosphate. Furthermore, mitochondrial membrane potential was significantly decreased in polyP-treated cells. These results suggest that the depletion of intracellular ATP and the decrease in mitochondrial membrane potential induced by polyP treatment may be a trigger to promote cell calcification.

A Peptide Derived from Phosphoinositide 3-kinase Inhibits Endocytosis and Influenza Virus Infection.

Endocytosis mediates the internalization and ingestion of a variety of endogenous or exogenous substances, including virus particles, under the control of intracellular signaling pathways. We have previously reported that the complex formed between the small GTPase Ras and phosphoinositide 3-kinase (PI3K) translocates from the plasma membrane to endosomes, signaling from which thereby regulates clathrin-independent endocytosis, endosome maturation, influenza virus internalization, and infection. However, the molecular mechanism by which the Ras-PI3K complex is recruited to endosomes remains unclear. Here, we have identified the amino acid sequence responsible for endosomal localization of the Ras-PI3K complex. PI3K lacking this sequence failed to translocate to endosomes, and expression of the peptide comprising this PI3K-derived sequence inhibited clathrin-independent endocytosis, influenza virus internalization, and infection. Moreover, treatment of cells with this peptide in an arginine-rich, cell-penetrating form successfully suppressed influenza virus infection in vitro and ex vivo, making this peptide a potential therapeutic agent against influenza virus infection.Key words: signal transduction, endocytosis, endosome, imaging, influenza virus.

Forefoot and hindfoot kinematics in subjects with medial tibial stress syndrome during walking and running.

Excessive foot pronation during static standing, walking and running has been reported as a contributing factor for the development of medial tibial stress syndrome (MTSS). The motion of foot pronation consists of hindfoot and forefoot motion. However, no previous studies have investigated forefoot and hindfoot kinematics during walking and running in subjects with MTSS. The current study sought to compare hindfoot and forefoot kinematics between subjects with and without MTSS while walking and running. Eleven subjects with MTSS and 11 healthy controls (each group containing 10 males and one female) participated in the current study. Segment angles of the hindfoot and forefoot during walking and running barefoot on a treadmill were recorded using three-dimensional kinematic analysis. An independent t-test was used to compare kinematic data between groups. Subjects with MTSS exhibited significantly greater hindfoot eversion and abduction (p < 0.05) during walking and running than subjects without MTSS, significantly greater forefoot eversion and abduction (p < 0.05) during walking, and significantly greater forefoot abduction during running (p < 0.05). Hindfoot and forefoot kinematics during walking and running were significantly different between subjects with and without MTSS. For prevention and rehabilitation of MTSS, it may be important to focus on not only hindfoot but also forefoot kinematics during both running and walking. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res.

Factors Affecting Bone Mineral Density Among Snowy Region Residents in Japan: Analysis Using Multiple Linear Regression and Bayesian Network Model.

BACKGROUND: As the onset of osteoporosis leads to reduced activities of daily living and may result in patients being bedridden, efforts to prevent decreased bone density are necessary. Various studies on the relationship between sex, age, nutrients, and exercise habits and bone mineral density have been conducted to date. However, for snowy region residents, the magnitude of influence of various factors affecting bone mineral density and the influence level have not been clarified. OBJECTIVE: This study aimed to clarify the degree of influence and factors influencing bone mineral density based on survey results on health conditions and lifestyle habits in heavy snow areas. METHODS: A total of 354 citizens who visited a drugstore in the target area were included in a study that included using the brief-type self-administered diet history questionnaire on lifestyle and exercise habits. Height, weight, body composition, and bone densitometer values were analyzed using multiple regression to calculate their association with bone mineral density. In addition, a Bayesian network model was used to determine the influence level of each factor as a conditional probability. RESULTS: Multiple regression analysis revealed that age, sex, fracture, and calcium intake significantly influenced bone mineral density. In addition, the result of Bayesian network analysis suggested that age and sex affected bone mineral density, whereas nutrients and exercise habits might not have a direct impact. However, calcium intake and the T-score were significant factors affecting the presence or absence of fracture experiences, suggesting that adequate calcium intake is essential for preventing fractures. CONCLUSIONS: In the multiple regression analysis, age, sex, fracture, and calcium intake were selected as factors; however, in the Bayesian analysis, only age and sex affected bone mineral density while nutrients did not. In addition, the fact that calcium intake and the T-score were shown to affect bone fracture history suggests that calcium intake is an important measure that can prevent bone fractures. Overall, these results suggest that measures such as ensuring a bone fracture-free environment and providing nutritional advice for calcium intake can be effective in preventing bone loss.

Investigation of dose-rate effects and cell-cycle distribution under protracted exposure to ionizing radiation for various dose-rates.

During exposure to ionizing radiation, sub-lethal damage repair (SLDR) competes with DNA damage induction in cultured cells. By virtue of SLDR, cell survival increases with decrease of dose-rate, so-called dose-rate effects (DREs). Here, we focused on a wide dose-rate range and investigated the change of cell-cycle distribution during X-ray protracted exposure and dose-response curves via hybrid analysis with a combination of in vitro experiments and mathematical modelling. In the course of flow-cytometric cell-cycle analysis and clonogenic assays, we found the following responses in CHO-K1 cells: (1) The fraction of cells in S phase gradually increases during 6 h exposure at 3.0 Gy/h, which leads to radio-resistance. (2) Slight cell accumulation in S and G2/M phases is observed after exposure at 6.0 Gy/h for more than 10 hours. This suggests that an increase of SLDR rate for cells in S phase during irradiation may be a reproducible factor to describe changes in the dose-response curve at dose-rates of 3.0 and 6.0 Gy/h. By re-evaluating cell survival for various dose-rates of 0.186-60.0 Gy/h considering experimental-based DNA content and SLDR, it is suggested that the change of S phase fraction during irradiation modulates the dose-response curve and is possibly responsible for some inverse DREs.

Inorganic polyphosphate enhances radio-sensitivity in a human non-small cell lung cancer cell line, H1299.

Inorganic polyphosphate is a linear polymer containing tens to hundreds of orthophosphate residues linked by high-energy phosphoanhydride bonds. Polyphosphate has been recognized as a potent anti-metastasis reagent. However, the molecular mechanism underlying polyphosphate action on cancer cells is poorly understood. In this study, we investigated the involvement of polyphosphate in radio-sensitivity using a human non-small cell lung cancer cell line, H1299. We found that polyphosphate treatment decreases cellular adenosine triphosphate levels, suggesting a disruption of energy metabolism. We also found that the induction of DNA double-strand breaks was enhanced in polyphosphate-treated cells after X-ray irradiation and colony formation assay revealed that cell survival decreased compared with that of the control groups. These findings suggest that polyphosphate is a promising radio-sensitizer for cancer cells. Therefore, we hypothesized that polyphosphate treatment disrupts adenosine triphosphate-mediated energy transfer for cellular survival and DNA repair, thereby reducing the cellular capability to resist X-ray irradiation.

Nutritional Status and Body Composition of Independently Living Older Adults in a Snowy Region of Japan.

Lifestyle diseases, which are associated with nutrition, account for 30% of elderly requiring long-term care. To increase health expectancy among Japan's rapidly aging population, we investigated the nutritional status and body composition of elderly adults living in a region subject to heavy snowfall, to identify pertinent health indicators. The dietary habits of 288 local residents aged ≥50 years were analyzed using body composition and a brief-type self-administered diet history questionnaire. Body mass index of all residents was normal. Basal metabolic rate (BMR) and muscle mass were reduced in the older group. Dietary habits did not differ with age among men, but older women had significantly higher dietary intake. BMR and muscle mass declined with age, even when dietary intake was sustained. Despite sufficient dietary intake, independently living older adults demonstrate less efficient use of food with age. Interventions to reduce excessive sodium and protein intake are required.

Modeling cell survival and change in amount of DNA during protracted irradiation.

Hyper-radiosensitivity (HRS) is a well-known bioresponse under low-dose or low-dose-rate exposures. Although disorder of the DNA repair function, non-targeted effects and accumulation of cells in G2 have been experimentally observed, the mechanism for inducing HRS by long-term irradiation is still unclear. On the basis of biological experiments and a theoretical study, we have shown that change in the amount of DNA associated with accumulation of cells in G2 enhances radiosensitivity. To demonstrate continuous irradiation with 250 kVp X-rays, we adopted a fractionated regimen of 0.186 or 1.00 Gy per fraction at intervals of 1 h (i.e. 0.186 Gy/h, 1.00 Gy/h on average) to Chinese Hamster Ovary (CHO)-K1 cells. The change in the amount of DNA during irradiation was quantified by flow cytometric analysis with propidium iodide (PI). Concurrently, we attempted a theoretical evaluation of the DNA damage by using a microdosimetric-kinetic (MK) model that was modified to incorporate the change in the amount of DNA. Our experimental results showed that the fraction of the cells in G2/M phase increased by 6.7% with 0.186 Gy/h and by 22.1% with 1.00 Gy/h after the 12th irradiation. The MK model considering the change in amount of DNA during the irradiation exhibited a higher radiosensitivity at a high dose range, which could account for the experimental clonogenic survival. The theoretical results suggest that HRS in the high dose range is associated with an increase in the total amount of DNA during irradiation.

Tomosynthesis can facilitate accurate measurement of joint space width under the condition of the oblique incidence of X-rays in patients with rheumatoid arthritis.

OBJECTIVE: Accurate evaluation of joint space width (JSW) is important in the assessment of rheumatoid arthritis (RA). In clinical radiography of bilateral hands, the oblique incidence of X-rays is unavoidable, which may cause perceptional or measurement error of JSW. The objective of this study was to examine whether tomosynthesis, a recently developed modality, can facilitate a more accurate evaluation of JSW than radiography under the condition of oblique incidence of X-rays. METHODS: We investigated quantitative errors derived from the oblique incidence of X-rays by imaging phantoms simulating various finger joint spaces using radiographs and tomosynthesis images. We then compared the qualitative results of the modified total Sharp score of a total of 320 joints from 20 patients with RA between these modalities. RESULTS: A quantitative error was prominent when the location of the phantom was shifted along the JSW direction. Modified total Sharp scores of tomosynthesis images were significantly higher than those of radiography, that is to say JSW was regarded as narrower in tomosynthesis than in radiography when finger joints were located where the oblique incidence of X-rays is expected in the JSW direction. CONCLUSION: Tomosynthesis can facilitate accurate evaluation of JSW in finger joints of patients with RA, even with oblique incidence of X-rays. ADVANCES IN KNOWLEDGE: Accurate evaluation of JSW is necessary for the management of patients with RA. Through phantom and clinical studies, we demonstrate that tomosynthesis may achieve more accurate evaluation of JSW.

Detection and measurement of rheumatoid bone and joint lesions of fingers by tomosynthesis: a phantom study for reconstruction filter setting optimization.

Rheumatoid arthritis (RA) is a systemic disease that is caused by autoimmunity. RA causes synovial proliferation, which may result in bone erosion and joint space narrowing in the affected joint. Tomosynthesis is a promising modality which may detect early bone lesions such as small bone erosion and slight joint space narrowing. Nevertheless, so far, the optimal reconstruction filter for detection of early bone lesions of fingers on tomosynthesis has not yet been known. Our purpose in this study was to determine an optimal reconstruction filter setting by using a bone phantom. We obtained images of a cylindrical phantom with holes simulating bone erosions (diameters of 0.6, 0.8, 1.0, 1.2, and 1.4 mm) and joint spaces by aligning two phantoms (space widths from 0.5 to 5.0 mm with 0.5 mm intervals), examining six reconstruction filters by using tomosynthesis. We carried out an accuracy test of the bone erosion size and joint space width, done by one radiological technologist, and a test to assess the visibility of bone erosion, done by five radiological technologists. No statistically significant difference was observed in the measured bone erosion size and joint space width among all of the reconstruction filters. In the visibility assessment test, reconstruction filters of Thickness+- and Thickness-- were among the best statistically in all characteristics except the signal-to-noise ratio. The Thickness+- and Thickness-- reconstruction filter may be optimal for evaluation of RA bone lesions of small joints in tomosynthesis.

Evaluation of the cell survival curve under radiation exposure based on the kinetics of lesions in relation to dose-delivery time.

We have investigated the dose rate effects on cell damage caused by photon-beam irradiation. During a relatively long dose-delivery time with a low dose rate, lesions created in cells may undergo some reactions, such as DNA repair. In order to investigate these reactions quantitatively, we adopted the microdosimetric-kinetic (MK) model and deduced a cell surviving fraction (SF) formula for continuous irradiation. This model enabled us to estimate the SF from dose and dose rate. The parameters in the MK model were determined so as to generate the SF, and we attempted to evaluate the dose rate effects on the SF. To deduce the cell-specific parameters in the SF formula, including the dose rate, we performed a split-dose experiment and a single-dose experiment with a constant dose-delivery time (10 min) (to retain the condition for equivalent behavior of cell lesions) by means of a clonogenic assay. Then, using the MK model parameters, the SFs were reproduced for a variety of dose rates (1.0, 0.31, 0.18, 0.025 and 0.0031 Gy/min) and were compared with reported experimental data. The SF curves predicted by the MK model agreed well with the experimental data, suggesting that the dose rate effects appear in the kinetics of cell lesions during the dose-delivery time. From fitting the analysis of the model formula to the experimental data, it was shown that the MK model could illustrate the characteristics of log-SF in a rectilinear form at a high dose range with a relatively low dose rate.

Quantitative estimation of DNA damage by photon irradiation based on the microdosimetric-kinetic model.

The microdosimetric-kinetic (MK) model is one of the models that can describe the fraction of cells surviving after exposure to ionizing radiation. In the MK model, there are specific parameters, k and yD, where k is an inherent parameter to represent the number of potentially lethal lesions (PLLs) and yD indicates the dose-mean lineal energy in keV/µm. Assuming the PLLs to be DNA double-strand breaks (DSBs), the rate equations are derived for evaluating the DSB number in the cell nucleus. In this study, we estimated the ratio of DSBs for two types of photon irradiation (6 MV and 200 kVp X-rays) in Chinese hamster ovary (CHO-K1) cells and human non-small cell lung cancer (H1299) cells by observing the surviving fraction. The estimated ratio was then compared with the ratio of γ-H2AX foci using immunofluorescent staining. For making a comparison of the number of DSBs among a variety of radiation energy cases, we next utilized the survival data in the literature for both cells exposed to other photon types, such as (60)Co γ-rays, (137)Cs γ-rays and 100 kVp X-rays. The ratio of DSBs based on the MK model with conventional data was consistent with the ratio of γ-H2AX foci numbers, confirming that the γ-H2AX focus is indicative of DSBs. It was also shown that the larger yD is, the larger the DSB number is. These results suggest that k and yD represent the characteristics of the surviving fraction and the biological effects for photon irradiation.

Morphogenetic study on the maturation of osteoblastic cell as induced by inorganic polyphosphate.

Since inorganic polyphosphates [poly(P)] have an activity to induce bone differenciation in vitro and in vivo, we examined an effect of poly(P) on organelle by light microscopy and electron microscopy in Murine MC3T3-E1 osteoblastic cells. The MC3T3-E1 cells were ultrastructurally observed to possess morphological characteristics of osteoblasts. Cells cultured with poly(P) were strongly stained with an anti-collagen type I antibody but not in those cultured without poly(P). Ultrastructural analysis of cells cultured with poly(P) revealed a well-developed Golgi apparatus, swollen and elongated rough endoplasmic reticulum, large mitochondria and many coated pits. Since MC3T3-E1 cells can be transformed from a resting phase to an active blastic cell phase after supplementation with poly(P), it implies that poly(P) can be an effective material for bone regeneration.

A simulation study of the radiation-induced bystander effect: modeling with stochastically defined signal reemission.

The radiation-induced bystander effect (RIBE) has been experimentally observed for different types of radiation, cell types, and cell culture conditions. However, the behavior of signal transmission between unirradiated and irradiated cells is not well known. In this study, we have developed a new model for RIBE based on the diffusion of soluble factors in cell cultures using a Monte Carlo technique. The model involves the signal emission probability from bystander cells following Poisson statistics. Simulations with this model show that the spatial configuration of the bystander cells agrees well with that of corresponding experiments, where the optimal emission probability is estimated through a large number of simulation runs. It was suggested that the most likely probability falls within 0.63-0.92 for mean number of the emission signals ranging from 1.0 to 2.5.