Lumbar spondylosis is more prevalent among the middle-aged and elderly, but few population-based studies have been conducted, especially in Japan. The purpose of this study was to explore the prevalence of lumbar spondylosis and its associations with low back pain among community-dwelling Japanese women.Lateral radiographs of the lumbar spine were obtained from 490 Japanese women ≥ 40 years old, and scored for lumbar spondylosis using the Kellgren-Lawrence (KL) grade at lumbar intervertebral level from L1/2 to L5/S1. Height and weight were measured, and body mass index (BMI) was calculated. Low back pain in subjects was assessed using a self-administered questionnaire. Stiffness index (bone mass) was measured at the calcaneal bone using quantitative ultrasound.Prevalence of radiographic lumbar spondylosis for KL ≥ 2, KL ≥ 3 and low back pain were 76.7%, 38.8% and 20.0%, respectively. Age was positively associated with radiographic lumbar spondylosis  and low back pain. Greater BMI was associated with lumbar spondylosis with KL = 2, but not with KL ≥ 3. Stiffness index was associated with neither radiographic lumbar spondylosis nor low back pain. Multiple logistic regression analysis identified radiographic lumbar spondylosis (KL ≥ 3) at L3/4, L4/5 and L5/S1 was associated with low back pain, independent of age, BMI and stiffness index.Severe lumbar spondylosis at the middle or lower level may contribute to low back pain. Lumbar spondylosis is characterized by disc degeneration and osteophytosis, and is more prevalent among the middle-aged and elderly –9. SincePrevious studies have shown that the prevalence of lumbar spondylosis ranges from 38% to 85% , 12–16. The prevalence of lumbar spondylosis and associations with low back pain have yet to be fully elucidated. The purpose of this study was to explore the prevalence of lumbar spondylosis and its associations with low back pain among community-dwelling Japanese women.This study was conducted as part of a survey of age-related health status in a Japanese community . All women ≥ 40 years old in Oshima, a town in Nagasaki Prefecture in Japan, were invited to participate. Oshima has a population of approximately 5,800 , including approximately 2,000 women ≥ 40 years. Despite having a shipyard in the town, Oshima is primarily a rural area. The examination of each subject was performed at the Oshima Health Center between 2001 and 2003. A total of 573 women (approximately 30% of eligible women) participated in the study. All participants were non-institutionalized, lived independently, and provided written informed consent before examinations. All study protocols were approved by the Ohshima local ethics committee.2. Subjects were asked if they had low back pain on most days during the previous one month using a self-administered questionnaire (yes/no). Information on current smoking and alcohol drinking was also obtained (yes/no). Stiffness index (bone mass) was also measured at the calcaneal bone using quantitative ultrasound (QUS). Broadband ultrasound attenuation (BUA: dB/MHz) and the speed of sound (SOS: m/s) were measured with an Achilles ultrasound bone densitometer . Stiffness index, a function of BUA and SOS, was automatically calculated by using the scanner software [Height (in meters) and weight (in kilograms) were measured with the subject in light clothing and without shoes, and body mass index (BMI) was calculated as weight/heightsoftware .Lateral radiographs of the lumbar spine were obtained with the subject lying on their side with knees bent. Radiographs were scored by a single experienced orthopedic surgeon (RT) for lumbar spondylosis using the Kellgren-Lawrence (KL) grade as follows: KL0, normal; KL1, slight osteophytes; KL2, definite osteophytes; KL3, disc space narrowing with osteophytes; KL4, bone sclerosis, disc space narrowing, and large osteophytes  Seventy-seven women for whom radiographs showed poor technical quality and seven women with missing data on low back pain were excluded, leaving 490 women for analysis. The Cochran-Armitage trend test was used to evaluate differences in the prevalence of radiographic lumbar spondylosis or low back pain among age groups. Age-specific means of stiffness index were determined using a general linear modelling method. Logistic regression analysis was used to explore the associations of age, BMI and stiffness index with radiographic lumbar spondylosis (based on the worst level of spondylosis) or low back pain, and the associations of KL grade at each intervertebral level with low back pain, adjusting for age, BMI and stiffness index. Furthermore, we assigned scores as follows: 0, KL = 0–1; 1, KL = 2; and 2, KL ≥ 3. Scores for each level were totaled for each individual . We conducted logistic regression analysis in order to examine the association between total score for spondylosis at each level and low back pain, adjusting for age, BMI and stiffness index. Results are presented as odds ratios (ORs) with 95% confidence intervals (CIs). Data were analyzed using Statistical Analysis System software package version 9.2 .Intra- and inter-observer variability in the KL grading of lumbar radiographs were found to be sufficient, with “substantial” kappa scores of 0.78 (95%CI: 0.62-0.94) and 0.63 (95%CI: 0.44-0.83), respectively.2, and 68.8 (16.1), respectively.Characteristics of subjects are presented in Table p < 0.001), KL ≥ 3 (p < 0.001) and low back pain (p = 0.006) increased with age. Stiffness index decreased with age (p < 0.001). Numbers (%) of subjects with radiographic lumbar spondylosis at each intervertebral level are shown in Table Overall prevalence of radiographic lumbar spondylosis for KL ≥ 2, KL ≥ 3 and low back pain were 76.7%, 38.8% and 20.0%, respectively (Table 2) increase in BMI was 1.59 in KL = 2. BMI was not associated with KL = 3, relative to KL = 0–2 (data not shown). Stiffness index was associated with neither radiographic lumbar spondylosis nor low back pain.Age was positively associated with radiographic lumbar spondylosis  and low back pain Table . The OR Through multiple logistic regression analysis, Table We showed that the overall prevalence of radiographic lumbar spondylosis of KL ≥ 2 or KL ≥ 3 and low back pain was 76.7%, 38.8% and 20.0%, respectively, among community-dwelling Japanese women ≥ 40 years old. Previous studies have reported the prevalence as between 38 and 85% , 12–16. Muraki et al.  showed tSeveral studies have shown older age to be associated with lumbar spondylosis , 12, 16 In our study, greater BMI was associated with lumbar spondylosis with KL = 2, but not with lumbar spondylosis with KL ≥ 3, which suggests that greater BMI may be associated with moderate lumbar spondylosis. Some studies have reported that obesity is related to lumbar spondylosis , 7, 14, In our study, stiffness index from QUS was not associated with lumbar spondylosis. Mariconda et al.  studied We showed that a higher prevalence of radiographic lumbar spondylosis (KL ≥ 2) was seen at L3/4 and L2/3, and that a higher prevalence of radiographic lumbar spondylosis (KL ≥ 3) was seen at L5/S1 and L4/5. O’Neill et al.  reportedThe association between lumbar spondylosis and low back pain is controversial. Some studies have reported an association , 7, 9–11This study has several limitations that must be considered. First, since this study used a cross-sectional design, our results do not necessarily show a causal relationship. Second, data on spinal canal stenosis, degeneration at facet joints, or other spinal disorders or psychological distress causing low back pain were not available in our study. Third, we did not collect data on pain severity. Further study is needed to clarify the severity of low back pain using a visual analogue or numeric pain rating scale to assess the relationship between severity of degenerative changes and severity of low back pain. The KL grade does not discriminate according to the degree of disc space narrowing. Caution is thus needed when interpreting the results. Fourth, a relatively high proportion of women (13.4%: 77/573) was excluded from analysis because of the poor technical quality of radiographs, which may have led to selection bias. Fifth, because this study only included women, our findings may not be generalizable to men.Lumbar spondylosis (KL ≥ 3) at L3/4, L4/5 or L5/S1 was associated with low back pain among community-dwelling Japanese women. Severe lumbar spondylosis at the middle or lower level may contribute to low back pain.


We compiled all available data on soil organic carbon (OC) storage in Australia’s tidal marshes (323 cores). OC stocks in the surface 1 m averaged 165.41 (SE 6.96) Mg OC ha−1 (range 14–963 Mg OC ha−1). The mean OC accumulation rate was 0.55 ± 0.02 Mg OC ha−1 yr−1. Geomorphology was the most important predictor of OC stocks, with fluvial sites having twice the stock of OC as seaward sites. Australia’s 1.4 million hectares of tidal marshes contain an estimated 212 million tonnes of OC in the surface 1 m, with a potential CO2-equivalent value of $USD7.19 billion. Annual sequestration is 0.75 Tg OC yr−1, with a CO2-equivalent value of $USD28.02 million per annum. This study provides the most comprehensive estimates of tidal marsh blue carbon in Australia, and illustrates their importance in climate change mitigation and adaptation, acting as CO2 sinks and buffering the impacts of rising sea level. We outline potential further development of carbon offset schemes to restore the sequestration capacity and other ecosystem services provided by Australia tidal marshes.Australia’s tidal marshes have suffered significant losses but their recently recognised importance in CO Whereas technological approaches to remove atmospheric CO2 are being sought, biosequestration of CO2 is already a proven process that can be conserved, enhanced and incorporated into emission accounting and reduction frameworks. Vegetated coastal habitats – tidal marshes, seagrasses and mangroves – known as ‘blue carbon ecosystems’, rank among the most efficient biosequestration systems on the planetincrease understanding of, and accelerate action on the important role of coastal blue carbon ecosystems in climate change action”. It will also be among the first countries to include blue carbon in its national greenhouse gas inventory. For these aspirations to be fulfilled, Australia needs to: 1) determine the stocks and distribution of blue carbon; 2) determine the factors controlling the accumulation of blue carbon around its coastline; and 3) identify and capitalise on opportunities for implementing management actions capable of enhancing carbon stocks or avoiding greenhouse gas emissions above the ‘business as usual’ scenario. Here we contribute to these goals, with a focus on tidal marshes .Removal of atmospheric carbon dioxide Information on Australian tidal marsh carbon stocks and accumulation rates has increased considerably over the past decade through localised efforts. Carbon stocks and accumulation rates for Australian tidal marshes appear to be comparable with global average values16171819174The primary goal of this study was to provide new estimates of blue carbon stocks and accumulation rates for Australian tidal marshes. A large new (previously unpublished) dataset comprising of sediment cores taken from the temperate coasts of Australia has been complied, along with all available literature data . This reSarcocornia quinqueflora (117 cores), Juncus kraussii (84 cores), Sporobolus virginicus (40 cores), Suaeda australis (14 cores), Tecticornia sp. (13 cores), Baumea juncea (6 cores) and Paspalum vaginatum (3 cores). Another 43 cores had a mix of species, and 3 cores did not have vegetation data.A total of 323 samples were analysed from the states of Victoria , New South Wales , temperate Queensland , Western Australia , South Australia  and Northern Territory . A total of 11 observations of tidal marsh carbon stocks were sourced from the literature. All data are summarised in −1 in the top 1 m, with a mean of 165.41 Mg OC ha−1 (SE 6.96). Carbon stocks at 30 cm depth varied from 8.89 to 603.67 Mg OC ha−1, with a mean of 77.92 Mg OC ha−1 (SE 3.35). Australian tidal marsh sediment vertical accretion rates ranged from 0.33 to 5.93 mm yr−1, with an overall mean of 2.09 ± 0.32 mm yr−1, while surface elevation tables (SET’s) recorded a mean elevation change of 1.44 mm yr−1   at a weighted average price of $AUD12.10 per Mg CO2-e .Carbon stocks varied from 14 to 962 Mg OC haSE 0.44, . We calc3) and carbon stock  = 84.262, P = 0.000, F = 2.094, P = 0.15, Juncus kraussii, Sarcocornia quenqueflora and mixed vegetation , Sporobolus virginicus dominated sites were found to be significantly lower  = −2.757, P = 0.006). A linear mixed-effects model detected no significant relationship between mean maximum temperature (°C) and carbon stocks (t(243) = −1.515, P = 0.13).Geomorphic setting was found to be a strong predictor of OC stocks with fluvially-influenced tidal marsh sites having approximately double the OC than of sites with greater marine influence  of OC in the top 1 m, based on an area of 1,376,500 ha .et al.17−2 yr−1, equating to 3.78 Tg C yr−1 based on a tidal marsh area of 13,765 km2 . Our estimates are lower than this; we find that Australian tidal marshes sequester 54.52 g OC m2 yr−1, which equates to 0.75 Tg OC yr−1.In this study we collected data on OC stocks from 84 tidal marsh sites (323 soil cores) around Australia – the most comprehensive Australian tidal marsh OC database to date – providing a two fold increase in estimates since global data published by Chmura et al.Juncus tidal marsh, although they included it in their summary table. After accounting for these corrections, Ouyang and Lee’s−2 yr−1. Nevertheless, our estimates remain below current global means for tidal marsh OC sequestration reported by Ouyang and Lee et al. et al. We note, however, that the two studies used by Ouyang and Lee3, DBD) were generally in the same range and, therefore, we suggest that variance in sediment accretion (mm yr−1) between previous global estimates is the likely driver of the comparatively low OC sequestration rates observed here. In this study the mean sediment accretion rate was 2.09 mm yr−1 which was based on 20 observations . Likewise, Duarte et al.−1, three-fold higher than the mean value we report for Australian marshes. Much of this data was from North America and Europe where tidal marshes are dominated by Spartina alterniflora, which occurs lower in the mid-intertidal zone, approximately at mean sea level142829Spartina sp. may be associated with differences in the position of tidal marshes in the intertidal. Elevation differences could also be limiting our comparison of carbon stocks among Australian marsh species, with Juncus often  occurring at higher elevations within the same marsh than Sarcocornia. Obtaining more data on the position of Australian saltmarshes within the tidal frame and assessing the impact of this upon sediment accumulation rates and ecogeomorphic responses is clearly a research priority for reducing uncertainty in carbon accumulation rates for the region. The collection of data from severely under-represented large, macrotidal catchments  will also present an opportunity to investigate the role of tidal range and catchment characteristics on surface and carbon accumulation in Australian marshes.Compared with Ouyang and Leervations , whereasSporobolus virginicus  indicate that like seagrasses, plant community composition in tidal marshes may be a major driver of sediment carbon concentrationsSarconornia and Juncus sp. were found to have similarly high OC stocks, and are found in both the low and high tidal zones, respectively, and Sporobolus virginicus with comparatively lower OC stocks is situated in the mid tidal zone, we can infer that tidal inundation was not the driver of OC stock variation in this study. We therefore recommend that further research into the effect of tidal marsh vegetation on soil OC focus on morphological and recalcitrant tissue variations among plant community typesThe significantly lower OC stocks found beneath tidal marsh vegetation species et al.et al.et al.et al.The significant relationship between geomorphology and OC stocks, with fluvial tidal marsh sites (landward) having twice the amount of OC as marine (seaward) sites subject to little or no fluvial influence, is consistent with findings by Saintilan −1, 1 m depth) to be $AUD9.37 billion ($USD7.19 billion). Of course, this stock can only be monetized under scenarios where the stock is vulnerable and payments can be received for avoided emissions (e.g. REDD+). In addition, we found that Australian tidal marshes accumulate OC annually at a rate of 54.52 ± 2.34 g OC m2 yr−1, which represents a financial value of $AUD33.32 ± 1.57 million ($USD28.02 million). Together, these values highlight the significant role Australian tidal marshes have played in biosequestration historically (i.e. the long-term development of the current OC stocks) and may continue to play into the future . Conversely, the size of these estimates also gives some indication of: 1) the magnitude of OC which may have been lost through past habitat destruction - with up to 70% of the pre-European extent of coastal wetlands lost in parts of eastern Australia38We estimate the potential value of the carbon stock locked in Australian tidal marshes Sea level rise is likely to have particular implications for the distribution of Australian tidal marshes, and may influence OC stocks and the capacity of marshes to accumulate OC in the future. Our assessment has indicated that Australian tidal marshes accumulate sediments at a rate of 2.09 ± 0.32 mm yrWhere there is suitable topography to enable upslope migration of marsh , new areas of tidal marsh establishment may offer an opportunity to increase OC accumulation and storage under rising sea levels. Moreover, this value compounds with that derived from other ecosystem services of tidal marshes, such as their nursery role for many marine and bird species, and for their value in coastal protection and nutrient cycling. Indeed, the loss of tidal marshes around Australia  also off2 and a global area of 41,657 km242432 emissions. Australia’s recent decision to incorporate wetlands into the National Greenhouse Gas Inventory  signifies an important step towards accounting for CO2 emissions and reductions in their carbon sequestration capacity associated with losses of tidal marsh and also for identifying opportunities for restoration of OC sequestration. This study represents a major advance in our understanding of carbon sequestration by Australian tidal marshes, with data from 323 sites around Australia, 45% of which are new data not published thus far. We stress that a large proportion of our OC stock data came from 30 cm cores, whereas international methodologies are generally based on 1 m depths For future research we recommend the following:As the world tries to transition to a low-carbon economy, there is growing opportunity to finance the conservation and protection of tidal marshes using the carbon offset market. The Verified Carbon Standard (VCS) Methodology for Tidal Wetland and Seagrass Restoration could attract carbon credits for tidal marsh restoration, but to our knowledge there have been no attempts by carbon offset providers to use the VCS scheme in Australia. At the national level, the Australian Government’s Emission Reduction Fund (ERF) is the primary policy relevant to carbon management. Considering the magnitude of OC storage and accumulation in Australian tidal marshes, we recommend their inclusion within this policy framework. This study demonstrates the value of tidal marshes as blue carbon sinks in Australia and the potential for further development of carbon offset schemes to restore the carbon sequestration capacity and other ecosystem services provided by these unique ecosystems.http://www.csiro.au/en/Research/OandA/Areas/Coastal-management/Coastal-Carbon-Cluster), with the exception of Queensland and NSW data which were derived from previously published work1519Data on OC and other sediment properties were compiled from the states of Western Australia (WA), South Australia (SA), Victoria (VIC), New South Wales (NSW), and temperate Queensland (QLD). The locations of sites are shown in Sediment cores (ranging from 10 to 100 cm long) were sampled by means of percussion and rotation, or vibrocoring. The core barrels consisted of PVC or aluminium pipes (50 to 90 mm inside diameter) with sharpened ends to cut fibrous material and minimise compression during coring. All cores were sealed at both ends, transported to the laboratory and stored at 4 °C until processing. Cores in NSW were sampled at 0–20 cm, 20–50 cm, and 50–100 cm intervals, while cores in QLD were sampled at 0–30 cm, and in VIC soils were sampled at 0–2 cm, 14–16 cm, and 28–30 cm increments. WA and SA soils were sampled in increments ranging from 1 cm to 10 cm. Samples were weighed before and after oven drying to constant weight at 60 °C (DW) to determine bulk density. Soil increments were homogenised and ground into a fine powder using a ball mill. We focussed on bulk soil OC, which included belowground living plant biomass (e.g. roots and rhizomes).The ‘Champagne test’−1) were obtained from previously published research using marker horizons2530542 yr−1) could not be statistically analysed in this study due to the use of an Australian tidal marsh mean sediment vertical accretion value (obtained from the literature) used universally in the below equation:Information on sediment accretion rates (mm yr2 yr−1), −1) obtained from the literature  to grams of OC per m2 in the top 1 mm soil layer, and n represents the number of individual cores. The value of Australia’s total OC stock was calculated through the following equation:where terature ,si repre−1, 1 m depth), and αi represents the total area of tidal marsh within each corresponding Australian state obtained from the literature  extrapolation of regressions were only conducted to 30 cm (n = 5). Predictions of 1 m deep OC stocks were calculated as follows; Logarithmic regressions were applied to sediment cores with a depth between 50 < 100 cm allowing for both a higher lied Eqs :Ai represents the actual OC stock to 1 m depth of an individual core (Mg OC ha−1), Li represents the linearly predicted OC stock value to 1 m depth of the same core , however heterogeneous variance was found between dominant vegetation types (P < 0.05). A restricted maximum likelihood (REML) estimate of the parameters in a linear mixed-effects model (“lme” function), with location included as a random factor, was used to assess the contribution of mean maximum temperature on tidal marsh OC stocks to a depth of 30 cm. A separate REML mixed effects model with the inclusion of fixed factor heterogeneity using the “varIdent” function, again with location assessed as a random factor, was used to investigate the effects of dominant vegetation type on OC stocks to 30 cm depth. All assumptions of linear mixed-effects analysis were confirmed through regression diagnostics. All OC stock data were square root transformed, and two high outliers (Amity North and Amity South) were removed, to facilitate normality of OC stock values prior to analysis.To test the effects of geomorphic setting  only locations that retained clear delineation between geomorphic settings were used. For New South Wales (NSW), 77 paired comparisons of fluvial and marine settings within the same estuarine systems were used (e.g. upper and lower river sampling sites)How to cite this article: Macreadie, P. I. et al. Carbon sequestration by Australian tidal marshes. Sci. Rep.7, 44071; doi: 10.1038/srep44071 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Background and aims: Hilar cholangiocarcinoma is a devastating malignancy with incidence varying by geography and other risk factors. Rapid progression of disease and delays in diagnosis restrict the number of patients eligible for curative therapy. The objective of this study was to determine prognostic factors of overall survival in all patients presenting with hilar cholangiocarcinoma.Methods: All adult patients with histologically confirmed hilar cholangiocarcinoma from 2003 to 2013 were evaluated for predictors of survival using demographic factors, laboratory data, symptoms and radiological characteristics at presentation.Results: A total of 116 patients were identified to have pathological diagnosis of hilar cholangiocarcinoma and were included in the analysis. Patients with a serum albumin level >3.0 g/dL (P < 0.01), cancer antigen 19‐9 ≤200 U/mL (P = 0.03), carcinoembryonic antigen ≤10 ìg/L (P < 0.01) or patients without a history of cirrhosis (P < 0.01) or diabetes (P = 0.02) were associated with a greater length of overall survival. A serum albumin level >3.0 g/dL was identified as an independent predictor of overall survival  with a survival benefit of 44 weeks.Conclusion: This study was the largest analysis to date of prognostic factors in patients with hilar cholangiocarcinoma. A serum albumin level >3.0 g/dL conferred an independent survival advantage with a significantly greater length of survival. Its incidence in Asia is as high as 71 per 100 000 men and 31 per 100 000 women. Patients are affected in the sixth or seventh decade of life, with presentation before the age of 40 being a rarity , therapeutic interventions and overall survival. The presence of cirrhosis was determined by laboratory and radiographical evidence when biopsy evidence was not available. All laboratory data were collected at the time of initial presentation.All adult patients with hilar cholangiocarcinoma, pathologically confirmed between September 2003 and September 2013 at the Cleveland Clinic Foundation, were retrospectively identified and included in the analysis. Diagnosis was confirmed histologically by bile duct brushings or biopsies. Patients included those referred to the Cleveland Clinic Foundation for further evaluation or those initially evaluated at our institution. The following data were collected for all patients: demographic data , risk factors for cholangiocarcinoma , presenting laboratory data  were evaluated. Patients with incomplete data were excluded (those with less than 20% of variables defined) or if current status  could not be verified. Categorical data were compared with Fisher’s exact tests and quantitative variables were represented as mean with standard error. Overall survival was described using Kaplan-Meier estimates, with overall survival determined by survival curves. Multivariate analysis was performed using the Cox proportional hazards model. Overall survival was defined as time from start of symptoms to time of death with patients alive at the end of the study censored and was represented as median with 95% confidence interval (CI). Sub-group analysis of patients who underwent surgical resection was completed. A P-value <0.05 was considered significant. Data were analysed using JMP version 9.0 .Univariate and multivariate analysis of demographic data  0.31; 95% CI 0.14–0.70).On univariate analysis of variables at patient presentation, the median survival period was 467 days (95% CI 257–572), which is significantly longer than 155 days (95% CI 122–194) in patients with an albumin ≤3.0 g/dL , compared with 305.5 days (95% CI 221–469) in patients with CEA ≤ 10 μg/L (P < 0.01). Patients with CA19‐9 ≤ 200 U/mL had a median survival period of 444.5 days (95% CI 210–532), compared with 203 days (95% CI 142–238) for those with a value greater than 200 U/mL (P = 0.03). In patients with a history or cirrhosis, overall survival was significantly shorter . Lastly, patients with diabetes experienced a shorter overall survival [225 days (95% CI 122–326)] than those without diabetes . Elevated tumor markers have been used to aid in the diagnosis of hilar cholangiocarcinoma; the average CEA and CA 19‐9 concentrations at presentation were 12.0 ± 2.7 μg/L and 3236.4 ± 1189.7 U/mL, respectively (n = 27).Patients were treated with surgical resection . Sub-group analysis assessed the impact of cirrhosis on lower albumin levels. Patients with underlying cirrhosis represented 6% of all patients in our data set and were associated with lower overall survival on univariate analysis; but the absence of cirrhosis was not found to be an independent predictor of survival; thus the relationship between low albumin and prognosis in hilar cholangiocarcinoma cannot be explained by cirrhosis alone. Cancer cachexia may provide an alternate explanation. Hypoalbuminemia is present in the setting of a systemic inflammatory response and is associated with weight loss [, cachexia and the inflammatory response may contribute to progressive loss of vital protein in patients with hilar cholangiocarcinoma; further, the long half-life of serum albumin may provide a longitudinal assessment of the patients’ wellbeing. As stated previously, studies have demonstrated that pre-operative albumin concentration is an independent predictor of survival after surgical resection—a finding that is now extended to the overall survival of all patients presenting with hilar cholangiocarcinoma, regardless of therapeutic management. Serum albumin is inexpensive and reproducible, and nearly all patients with biliary pathology or suspected hilar cholangiocarcinoma will have a hepatic panel measured.In early epidemiological studiessurvival . In our ght loss . Weight ,, a CEA > 10 μg/L and a CA19‐9 > 200 U/mL were associated with diminished median overall survival by 17 weeks and 35 weeks, respectively. Although not independent predictors of survival, the data suggests that patients with higher CA19‐9 and CEA serum concentrations may have diminished survival.Tumor markers have been evaluated as prognostics factors in patients undergoing surgical resection for cholangiocarcinoma—specifically CEA and CA 19‐9 ,33. In ,, one should be cautious when interpreting survival in patients with PSC and hilar cholangiocarcinoma from our data set.The prevalence of PSC in cholangiocarcinoma ranges from 5–36% ,35. Six, the retrospective nature of the study limited data collection and analysis; further, during the decade-long study period, changes in therapeutic options may have occurred. Despite these limitations, this study has provided a comprehensive assessment of prognostic factors for overall survival in patients with hilar cholangiocarcinoma.There are limitations to our study. Although the sample set was fairly large , this study was the largest retrospective analysis of prognostic factors in all patients diagnosed with hilar cholangiocarcinoma at a single institution. Overall, hilar cholangiocarcinoma is associated with a poor prognosis and the data demonstrated that an albumin level >3.0 g/dL at presentation is an independent predictor of overall survival.In summaryAbhijeet Waghray: study concept and design; acquisition of data; analysis and interpretation of data; drafting of the manuscript; critical revision of the manuscriptAnastasia Sobotka: acquisition of data; drafting of the manuscriptCarlos Romero Marrero: critical revision of the manuscriptBassam Estfan - critical revision of the manuscriptFederico Aucejo: critical revision of the manuscriptKV Narayanan Menon: study concept and design; analysis and interpretation of data; critical revision of the manuscript; study supervisionConflict of interest statement:  none declared.


Five elements Fe, Mn, Zn, Cu, and Se were determined by using inductively coupled plasma mass spectrometry (ICP-MS). Fe levels were the highest and Se levels were the lowest in all plants. The range levels of all elements in all plants were as follows: Fe 193.4–1757.9, Mn 23.6–143.7, Zn 15.4–32.7, Se 0.13–0.92, and Cu 11.3–21.8 µg/g. Intakes of essential elements from the medical plants in infusion were calculated: Fe 4.6–13.4, Mn 6.7–123.2, Zn 7.0–42.7, Se 0.14–1.5, and Cu 1.5–5.0 µg/dose. The calculated intakes of essential elements for all plants did not exceed the daily intake set by the World Health Organization (WHO) and European Food Safety Authority (EFSA). These medicinal plants may be useful sources of essential elements, which are vital for health.The objective of this study was to investigate the content of essential elements in medicinal plants in the Kingdom of Saudi Arabia (KSA). Five different medical plants (mahareeb  Medicinal plants are used by millions of people around the world. The usage of medicinal plants was mainly in primary healthcare by most of the users . This stCopper is found in various human transcription factors and enzymes and is considered as an important essential trace element. However, excessive intake of copper can lead to liver damage and Wilson's disease. Wilson's disease results from accumulation of free copper in the liver, brain, and kidney . DeficieIron is well established as an essential element and is a component of haemoglobin and is present in several human enzymes. Iron is present in high levels in red blood cells and muscle tissue. High doses of iron can cause hepatotoxicity. Acute liver failure has been reported after an overdose of ferrous sulphate in a young adult . Iron deManganese is an important element and is a constituent of metalloenzymes that oxidize cholesterol and fatty acids . A deficSelenium is an antioxidant and is involved in the biosynthesis of thyroid hormones. Selenium plays an important role in the immune system and can help suppress the progression of HIV to AIDS . Intake Zinc is an essential element, and cellular zinc promotes homeostatic control to avoid accumulation of surplus zinc. Exposure to excessive zinc results in copper deficiency and cell apoptosis. In addition, zinc deficiency has been linked to a suppressed immune response . Zinc anSyzygium caryophyllatum and Syzygium densiflorum were Fe , Zn , Cu  and Mn , respectively. The study concluded that these two plants were rich in some trace elements which are necessary for human health.Subramanian et al.  determinDifferent essential elements with levels (mg/kg), including Fe (54.2–231), Zn (4.7–19), Cu (3.2–10.5), and Mn (8.8–490), were previously determined in herbs and spices from Saudi Arabia , and no Zizyphus jujube, Eugenia jambolana, Coccinia indica, Citrus acida, Ocimum sanctum, and Trigonella foenumgraecum) from India. The concentrations (mg/kg) were in the following ranges: Fe 123–760, Zn 20.4–620, Mn 17.5–426, and Cu 0.4–35.5. The study concluded that these plants were rich in one or more of the investigated elements [Emblica officinalis, Catharanthus roseus, Azadirachta indica, Solanum anguivi, Artemisia nilargirica, and Elsholtzia communis) from India. The levels (mg/kg) of the elements in the plants were in the following ranges: Mn 31.3–183.7, Fe 311.1–820.4, Cu 3.7–30.1, Zn 23.3–62.8, and Se 0.0–0.5. They stated that trace elements in medicinal plants play an important role in treatment of disease [Essential elements including Fe, Zn, Mn, and Cu were determined in six medicinal plants , sheeh (Artemisia vulgaris), harjal (Cynanchum argel delile), nabipoot (Equisetum arvense), and cafmariam (Vitex agnus-castus). Therefore, all subsections related to sample collection and preparation, sample digestion, elemental measurements by using ICP-MS, chemicals reagents, and analytical method were the same as the previous study [Toxic elements  were already determined in the five medicinal plants mahareeb us study .3 10 times. Limits of detection and quantification were calculated  by the following formulas:  LOD = 3 × SD and LOQ = 10 × SD.The LODs were calculated after measuring 1% HNOμg/L of a mixed standard of all measured elements after each set of five samples. A sample was spiked with mixed standards (20 μg/L) of analysed elements, and the recovery was calculated for each element.For quality control, continuing calibration verification (CCV) analysis was used for each run. The CCV was performed by testing 20 t-test in Excel (paired-samples t-test). Differences were determined to be statistically significant if P < 0.05 with 95% confidence level, among measured elements, and between washed and unwashed plants. SPSS version 22 was used, comparing means using one-way ANOVA, to investigate differences among the plant species for the individual elements with significance level of 0.05.Statistical analysis was performed on all plant samples using the two-sample μg/L) were as follows: Fe 0.34, Cu 0.03, Se 0.07, Zn 0.17, and Mn 0.04. The LOQs (μg/L) were as follows: Fe 1.12, Cu 0.08, Se 0.24, Zn 0.56, and Mn 0.12.The LODs  of the five measured elements in unwashed and washed plant samples are shown in n=3) for each sample were carried out. In general, washed plants showed lower concentrations than unwashed plants for all elements measured. This indicates that washing removed essential elements from the plants. In some cases, the removal was as much as 80% of the original concentration in the unwashed sample. The reason for washing the plants was to investigate the impact of washing on the level of original concentrations of the essential elements. We can derive from these results that rigorous washing will remove high percentages of essential elements in plants in the filtrate. Therefore, the users who drink the filtrate can ingested the removed percentage, which can be source of the essential elements in such plants. The level of essential elements in filtrate can be calculated as a difference between unwashed and washed plants (The concentrations (d plants . It is n(Cymbopogon) showed higher concentrations of the measured elements compared with the other plants. The levels of elements for all medicinal plants were as follows: Fe > Mn > Zn > Cu > Se. The increasing order of the plants based on the concentrations of each individual element as follows:  Fe: mahareeb > nabipoot > cafmariam > harjal > sheeh  Cu: mahareeb > nabipoot > harjal > sheeh > cafmariam  Zn: sheeh > harjal > mahareeb > nabipoot > cafmariam  Se: nabipoot > harjal > mahareeb > sheeh > cafmariam  Mn: harjal > mahareeb > nabipoot > sheeh > cafmariamMahareeb Thus, mahareeb has a tendency to the highest contents of elements, whereas cafmariam has a tendency to the lowest in most of the cases.P < 0.05) from each other. Mahareeb reported the highest value, and sheeh had the lowest level. From the statistical analysis for Cu levels among the five plants, mahareeb and nabipoot showed no significant difference (P > 0.05), while the other three plants showed significant difference (P < 0.05) among each other. Mahareeb reported the highest value, and cafmariam had the lowest level.Based on comparison among the five plants, Fe contents in all plants showed significant difference (P < 0.05) among all plants. Harjal reported the highest value for Mn, and cafmariam had the lowest level. Moreover, the statistical analysis regarding Zn levels in the five plants showed no significant difference (P > 0.05) between mahareeb and nabipoot. However, the other three plants showed significant difference among each other. Sheeh reported the highest value, and cafmariam had the lowest level. The statistical analysis related to Se levels among the five plants showed significant difference (P < 0.05) between mahareeb and nabipoot. No significant difference (P > 0.05) was shown among the rest. Nabipoot had the highest value, and cafmariam had the lowest level. A significant difference (P < 0.05) was observed in the levels of most measured elements between the washed and unwashed plants.Based on the outcome of the statistical analysis among the five plants regarding the Mn content, there were significant differences . High percentages of all elements in the range 19%–80% were removed from mahareeb by washing, compared with all other plants. Nabipoot showed the lowest percentage of elements removed by washing (0%–51%), compared with all other plants.D (g) × Celm (μg/g), where D = amount of one dose in grams (bunch) and Celm = measured concentration of each element in the plant in μg/g. The calculated quantities for the elements in the unwashed and washed plants are shown in µg/g. The calculated dosages of exposure (μg/dose) are included in Based on personal communications with consumers and retailers, the quantity of each plant used in one dose is as follows: harjal 7.39 g, nabipoot 3.59 g, mahareeb 3.33 g, sheeh 4.49 g, and cafmariam 4.33 g. The quantity of each plant was calculated as an average of few small bunches. We calculated the quantities of each element contained in each dose of the medical plants in this study. The doses shown above for each plant were multiplied by the concentration of each measured element , using tThe investigated medicinal plants in this study are a source of the essential elements Fe, Cu, Zn, Se, and Mn. Essential elements are important for human health . In geneμg/g) and Cu (2.2–22.6 μg/g) levels in this study were within the range found in our study; Se (0.2–3.4 μg/g) had a larger range than observed in our study; Mn (2.6–102.9 μg/g) had a similar range to our study. Iron was reported in high levels in all tested medicinal plants in this study as well as in ours. These results showed that all medicinal plants, regardless of their origins, are a source of similar high concentrations of micronutrients, such as iron.Previous studies have been conducted to determine the levels of essential elements in medicinal plants. In Saudi Arabia, the element contents in various medicinal plants (different from this study) were already determined . These aμg/g) was present in the highest concentration in all investigated herbs. These results demonstrate that medicinal plants with different origins can show similar trends in the levels of essential elements, which is the trend of many terrestrial plants. It was reported that trace element uptake by plants due to their concentrations in nutrient solutions, which reflect the similar soil availability [The levels of trace elements, including Cu, Fe, Zn, Mn, and Se, have been determined in Chinese herbal medicines used for relieving heat . The reslability . The abslability , 25. MorAzadirachta indica and Ocimum sanctum) from India is consistent with the trends observed in our study. Additionally, Zn (200.8–254.5 μg/g) > Cu (63.5–129.2 μg/g) followed the same trend as observed for all plant samples in the present study. This similar trend could indicate that different herbal plants in different geographic areas uptake elements in similar ratios.The results from a recent study  investigμg/g) > Mn (0.321–19.63 μg/g) > Zn (0.086–0.146 μg/g) > Cu (0.075–0.091 μg/g), which was the same trend observed for all the medicinal plants in our study. Again, it can be seen that similar proportions of elements are present in plants regardless of the geographic area of origin.Another recent study  measuredμg/g) > Zn (12.65–146.67 μg/g) > Cu (1.44–156.24 μg/g). Again, this trend matches what was found in the present study.A study  in the UFrom our previous study , surprisWe conclude from this study that medicinal plants are an important source of essential elements, such as Fe, Mn, Zn, Cu, and Se. Fe and Mn were the elements in the highest concentrations in all plants, with Se having the lowest concentration in all plants. In general, mahareeb contributed to higher levels of the essential elements compared with all other plants, while cafmariam reported the lowest levels of the essential elements. The washing process removed a percentage of the essential elements from all investigated plants. Infusion of all plants was measured, and calculated intake levels of essential elements per dose were below the recommended daily intake levels set by the WHO and EFSA.


To define more precisely the molecular mechanisms of EnMSC therapy, in the present study, we assessed in parallel the paracrine and therapeutic properties of MSCs derived from endometrium, bone marrow, and adipose tissues in a rat model of myocardial infarction (MI). EnMSCs, BMMSCs, and adipose‐derived MSCs (AdMSCs) were characterized by fluorescence‐activated cell sorting (FACS). Paracrine and cytoprotective actions were assessed in vitro by coculture with neonatal cardiomyocytes and human umbilical vein endothelial cells. A rat MI model was used to compare cell therapy by intramyocardial injection of BMMSCs, AdMSCs, and EnMSCs. We found that EnMSCs conferred superior cardioprotection relative to BMMSCs or AdMSCs and supported enhanced microvessel density. Inhibitor studies indicated that the enhanced paracrine actions of EnMSCs were mediated by secreted exosomes. Analyses of exosomal microRNAs (miRs) by miR array and quantitative polymerase chain reaction revealed that miR‐21 expression was selectively enhanced in exosomes derived from EnMSCs. Selective antagonism of miR‐21 by anti‐miR treatment abolished the antiapoptotic and angiogenic effects of EnMSCs with parallel effects on phosphatase and tensin homolog (PTEN), a miR‐21 target and downstream Akt. The results of the present study confirm the superior cardioprotection by EnMSCs relative to BMMSCs or AdMSCs and implicates miR‐21 as a potential mediator of EnMSC therapy by enhancing cell survival through the PTEN/Akt pathway. The endometrium might be a preferential source of MSCs for cardiovascular cell therapy. S The present report describes the functional and mechanistic comparisons of mesenchymal stem cells (MSCs) from three different sources  as therapeutic agents for the treatment of cardiovascular disease. MSCs from the endometrium have markedly superior therapeutic potential relative to the other cell types, properties that can be explained by the paracrine actions mediated by exosomal microRNAs, in particular microRNA‐21, with downstream effects on cell survival and angiogenesis. The findings are important from a clinical perspective because the endometrium could be a preferential source of MSCs for cardiovascular cell therapy.Stem cell infusion and intramyocardial injection remain promising approaches for the treatment of coronary artery disease, myocardial infarction (MI), and heart failure Exosomes are cell‐derived microvesicles that are released from the plasma membrane into the extracellular space, where they play key roles in intercellular signaling In the present study, we compared paracrine functions, in vivo therapy, and exosomal profiles of human EnMSCs, BMMSCs, and adipose‐derived MSCs (AdMSCs). We report the superior actions of EnMSCs in a rat MI model relative to BMMSCs and AdMSCs that correlated with enhanced paracrine actions and higher expression of exosomal miR‐21. The results are consistent with a role for miR‐21 by downregulation of phosphatase and tensin homolog (PTEN) and enhancement of Akt survival kinase activity in the superior actions of EnMSCs in this model.http://www.sebio.net.cn). Human AdMSCs were purchased from Cyagen Bioscience . Human umbilical vein endothelial cells (HUVECs) were provided by AllCells . The cells were maintained in Dulbecco's modified Eagle's medium  with low glucose, supplemented with 10% fetal bovine serum  at 37°C in a humidified atmosphere with 5% CO2.Human BMMSCs and EnMSCs were obtained from S‐Evans Biosciences . In brief, 1 × 106 cells suspended in 100 μl of phosphate‐buffered saline (PBS) with 1% FBS were stained with a panel of antibodies: fluorescein isothiocyanate (FITC)‐CD45, phycoerythrin (PE)‐CD29, allophycocyanin‐CD90, PE‐CD34, PE‐CD105, PE‐CD166, PE‐CD117, and isotype‐matched control for 1 hour. The cells were washed twice with PBS, and at least 10,000 cells per tube were acquired for each test. Surface marker expression was quantified using FACSCount II software (BD Biosciences).MSCs at passages 6–8 were characterized for surface markers using a FACS Canto II Flow Cytometer  for 90 minutes at 37°C with 5% CO2. Nonadherent cells were collected and seeded onto 24‐well plates (2.5 × 105 cells per well) or 6‐well plates . The culture medium was replaced 48 hours after seeding. CMs were ready for experimentation after 72 hours of incubation.Neonatal rat cardiomyocytes (CMs) were isolated and cultured as described previously http://www.roche_applied_science.com). In brief, the cells or samples were fixed in 4% paraformaldehyde and permeabilized with 0.2% Triton X‐100 for 10 minutes. Nucleotide and TdT enzyme mixture was added to the cells or samples for 1 hour of incubation at 37°C. The nuclei were stained with 4′,6‐diamidino‐2‐phenylindole (DAPI). The percentage of apoptotic nuclei per total CM nuclei was calculated as the index of apoptosis. Images were taken at five randomly selected fields in the border zone of the ischemic myocardium in each sample or per well.Terminal deoxynucleotidyl transferase dUTP nick‐end labeling (TUNEL) assays were performed in accordance with the manufacturer's protocol . Sections or cells were washed with PBS‐0.05% Tween (PBST) followed by incubation of secondary antibody . After 1 hour of incubation at 37°C, cell nuclei were stained with DAPI. Images were obtained by fluorescence microscope  at five randomly selected fields for each sample or well and the mean positive/negative numbers calculated.Cells and heart samples were fixed with 4% paraformaldehyde for 10 minutes and permeabilized with 0.2% Triton. After 1 hour of blocking by 10% bovine serum albumin (BSA), the cells or tissue samples were incubated with primary antibodies as follows: anti‐troponin I .For tube formation assays, 96‐well plates were precoated with 50 μl of growth factor‐reduced Matrigel (BD Biosciences) for 30 minutes at 37°C. HUVECs were seeded at a density of 20,000 cells per well and incubated with conditioned medium at 37°C. After 4–6 hours, capillary network structures were imaged by phase‐contrast microscopy (Leica Microsystems) and the total number of interconnecting branches in each group counted using Image‐Pro software . The mixture was centrifuged at 10,000http://www.hitachi.com), and micrographs were taken to quantify the size of the exosomes.To investigate exosome morphology, 3 μl of exosome pellets were layered onto 200‐mesh copper electron microscopy grids for 5 minutes at room temperature and stained with uranyl acetate g for 10 minutes. The supernatant was discarded, and the cell pellets were resuspended in 10 ml of PBS and centrifuged at 400g for 5 minutes. The cell pellets were washed twice with 10 ml of complete medium to remove any unbound dye.To label the exosomes, 4 µl of PKH‐26, (Sigma‐Aldrich) was added to 1 ml of diluent containing 30 μg of MSC‐derived exosomes and incubated at room temperature for 15 minutes. Then, 1 ml of 5% BSA was added to stop the reaction, and the suspension was centrifuged at 400EnMSCs were cultured to 90% confluence in complete medium. The media were replaced with DMEM containing 2% exosome‐depleted FBS, and the cells were conditioned with or without 20 μM GW4869 (Sigma‐Aldrich) for an additional 48 hours. Conditioned media were collected for in vitro studies, and EnMSCs were prepared for in vivo studies.http://www.genomics.agilent.com). RNA samples of 100 ng were used for miR microarrays. RNA was labeled using a miRCURY Hy3/Hy5 Power labeling kit , hybridized on a miRCURY LNA Array, version 18.0 (Exiqon), and scanned using an Axon GenePix 4000B microarray scanner . Expression data were analyzed using GenePix Pro 6.0 software .Total RNA was extracted from exosomes using a total exosome RNA isolation kit (Thermo Fisher Scientific), according to the manufacturer's instructions. RNA pellets were dissolved in RNase‐free water, and RNA content was determined using an Agilent 2100 Bioanalyzer . For viral infection of EnMSCs, CMs, and HUVECs, the cells were seeded at a density of 1 × 105 cells per well in a six‐well plate and infected with lentiviral vectors in the presence of 10 μg/ml polybrene . After 12 hours of infection, the medium was replaced with growth medium. At 72 hours after infection, the efficiency of the infection was assessed as green fluorescent protein expression by fluorescence microcopy and precisely quantified by real‐time polymerase chain reaction (RT‐PCR).Recombinant lentivirus was constructed by Genechem , and mRNAs were reverse transcribed using a PrimeScript Stand cDNA Synthesis Kit . The mRNA primers were selected using Primer3 Input online software (available at: http://primer3.ut.ee/) as shown in supplemental online Table 1. The primers of miRs were designed using miRBase online software (available at: http://www.mirbase.org/) as shown in supplemental online Table 2. RT‐PCRs were conducted using SYBR green reaction mixture in the Step One Plus Real‐Time PCR System . The PCR conditions were 95°C for 10 minutes and 40 cycles at 95°C for 30 seconds, 60°C for 30 seconds, and 72°C for 1 minute. We used β‐actin as the internal control for normalization and calculated the relative expression of mRNA using a (ΔCt) value method. Relative expression of miRs were also evaluated by a ΔCt method and normalized to U6.Total RNAs and miRs were extracted, miRs were reverse transcribed using a miDETECT A Track quantitative RT (qRT)‐PCR Kit  containing Protease Inhibitor Cocktail Set III, EDTA‐Free  on ice for 30 minutes. Then, cell lysates were collected and centrifuged at 12,000 rpm for 30 minutes at 4°C to acquire extracts. Protein concentrations were assessed using a BCA Protein Assay Kit (Thermo Fisher Scientific). Next, 30 μg of protein was analyzed by SDS‐polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes (EMD Millipore). After 1 hour of blocking with PBST containing 5% BSA at room temperature, primary antibodies, including anti‐Bcl‐2, Bcl‐2‐associated X protein (Bax), caspase‐3, phosphorylated‐Akt/total‐Akt, PTEN, vascular endothelial growth factor (VEGF), CD63, and β‐actin (Abcam) were diluted to 1:1,000 and incubated with the membrane overnight at 4°C. The membranes were washed with PBST and incubated with 1:3,000 secondary conjuncted with horseradish peroxidase (HRP) antibody for 1 hour at room temperature. The membranes were immersed in enhanced chemiluminescence solution and images acquired by digital development with β‐actin as control for normalization.In brief, cells and exosomes were rinsed with PBS and lysed in RIPA buffer . The control groups received the same volume of PBS.Rats  were fed a standard laboratory diet. All animal research protocols met the Guide for the Care and Use of Laboratory Animals  and were approved by the Animal Care and Use Committee of Zhejiang Province Medical Institute. The MI model was established as described previously 2 and placed in the supine position. Two‐dimensional and M‐model echocardiographic images  were obtained at the level of the papillary muscles in an investigator‐blinded manner. The left ventricular end‐systolic diameter (LVESD) and left ventricular end‐diastolic diameter (LVEDD) were measured for at least three separate cardiac cycles. The left ventricular end‐systolic volume (LVESV) and left ventricular end‐diastolic volume (LVEDV) were calculated as 7.0 × LVESD3/(2.4 + LVESD) and 7.0 × LVEDD3/(2.4 + LVEDD), respectively. The ejection fraction (EF) and fractional shortening (FS) were calculated as [(LVEDV − LVESV)/LVEDV] × 100 and [(LVEDD − LVESD)/LVEDD] × 100, respectively.Echocardiography was implemented at baseline and 3, 28, and 56 days after surgery. The rats were anesthetized with 2% isoflurane in 100% Ohttp://www.sakura-finetek.com) and snap frozen on dry ice. Samples were cut at 7 μm thickness and preserved at −80°C.The rats were sacrificed at 3, 28, and 56 days after MI. The hearts were excised and cut into transverse slices at the ventricle midline between the atrioventricular groove and the apex. The hearts were dehydrated in 30% sucrose solution for 12–24 hours at 4°C, embedded in Tissue‐Tek O.C.T. compound  and anti‐CD31 antibody (Abcam) at 4°C overnight. After several washes with PBST, the samples were reacted with HRP‐conjugated secondary antibody for 1 hour at room temperature. Immunopositive reactions were developed using a polymer 3,3′‐diaminobenzidine and sections mounted on gelatin‐coated slides. Images were taken at four to five randomly selected high‐magnification fields in the border zone of the ischemic myocardium, and the average number of microvessels per visual field was calculated.http://www.maxim.com.cn). The infarct area was calculated by the sum of the endocardial and epicardial length of the infarct zone divided by the total length of the endocardial and epicardial left ventricle using Image‐Pro software (Media Cybernetics) and expressed as a percentage of infarct size.At 28 and 56 days after MI, Masson trichrome staining was performed for infarct zone evaluation. In brief, sections from different groups were fixed with 4% paraformaldehyde, washed with PBS, and stained using a Masson trichrome kit . One‐way analysis of variance tests were used to compare the differences between groups. Data are presented as the mean ± SD. Differences were considered statistically significant at p < .05.Statistical analyses were calculated using SPSS, version 16.0, software . Cell surface marker expression of MSCs from each source was determined by flow cytometry. All samples coexpressed equivalent mesenchymal surface markers, including CD29, CD90, CD105, CD166, and were negative for hematopoietic surface markers CD34 and CD45. The expression of CD117 was very low in all MSCs .Paracrine actions can contribute significantly to the cardioprotective effects of MSC‐based therapy The role of paracrine action as a mechanism for MSC‐mediated therapy for myocardial infarction is well recognized. Therefore, the paracrine effects of each MSC treatment group were compared by quantifying apoptosis, neovascularization, infarct size, and cardiac function in parallel in a rat model of MI.TUNEL staining of cardiac cells in the border zones after MI revealed that hearts in the EnMSC group had the lowest apoptotic index compared with that of the other groups , programmed cell death 4 (PDCD‐4), S‐phase kinase‐associated protein 2 (Skp‐2), Yes‐associated protein 1 (Yap‐1), POC1 centriolar protein A (Pix‐2), YOD1 deubiquitinase (YOD‐1), signal transducer and activator of transcription 3 (Stat3), phosphatase and tensin homolog (PTEN), toll‐like receptor 4 (TLR‐4), sprouty homolog 1 (SPRY‐1), and Bcl‐2 interacting mitochondrial ribosomal protein 2 (BMRP‐2). Angiogenesis target genes included polycystin 2, transient receptor potential cation channel (PKD‐2), reversion‐inducing cysteine‐rich protein with Kazal motif (RECK), Kruppel‐like factor 6 (KLF‐6), fibroblast growth factor 18 (FGF‐18), Stat3, PTEN, TLR‐4, SPRY‐1, and BMRP‐2. Target gene primers were selected using Primer3 Input online software (available at: http://primer3.ut.ee/), as listed in supplemental online Table 1. To identify possible apoptotic target genes, CMs were cocultured with exosome fractions under hypoxia , and RNA and protein were extracted after 48 hours. For angiogenesis, HUVECs were incubated with exosomes and RNA and protein extracted after 24 hours. Putative target genes were identified by qRT‐PCR. Both assays revealed that PTEN mRNA was uniquely decreased by treatment with EnMSC exosomes (Fig. Target genes were predicted by gen ontology analysis, and genes were classified into three groups: molecular function, biological process, and cellular component. Apoptosis‐ and angiogenesis‐related miR‐21 targets were selected for further identification. Putative miR‐21 apoptotic target genes included HS1‐associated protein X‐1 (mes Fig. –5C. Westmes Fig. , 5E.miR‐21‐KD, EnMSCNull, EnMSCs, BMMSCs, and AdMSCs were incubated with CMs or HUVECs under hypoxic or normoxic conditions, followed by apoptosis or angiogenesis assays, respectively. For in vivo validation, EnMSCmiR‐21‐KD and EnMSCNull, respectively, were delivered into infarcted rat hearts. PTEN and its downstream targets were simultaneously evaluated by Western blotting. In parallel experiments, CMs and HUVECs were transfected directly with miR‐21 or a negative control and subjected to hypoxic or normoxic incubations. As shown in Figure miR‐21‐KD than CMs cocultured with EnMSCNull and EnMSC‐derived exosomes, and HUVECs incubated with exosomes of EnMSCmiR‐21‐KD had a lower capillary density (Fig. miR‐21‐KD treatment group, including the loss of antiapoptosis and cardioprotective properties (Fig. To validate a role for miR‐21 in EnMSC‐specific paracrine and cardioprotective effects, EnMSCs were transfected with a miR‐21 antagonist miR or negative control as described in Materials and Methods. For in vitro study, exosomes derived from EnMSCity Fig. , 6D. Mories Fig. , 6I, losies Fig. , 6J, 6K,ies Fig. , 6L, andOur results suggest that MSCs derived from the endometrium have greater therapeutic potential compared with cells derived from bone marrow or adipose tissue, the two most common sources of therapeutic MSCs Clinical trials of MSCs derived from bone marrow or adipose tissue have demonstrated improved cardiac function that is probably mediated primarily by paracrine actions of secreted cytokines rather than by cardiac regeneration or myogenic differentiation Exosomal mRNA and miRs could be key components for biological information exchange and cell‐to‐cell communication It was recently reported that exosomal miR‐21 derived from induced pluripotent stem cells protected the ischemic myocardium from apoptosis Our study has important clinical implications. MSCs are potential off‐the‐shelf reagents that are immune‐privileged, without autologous restrictions, and with wide application. The safety and feasibility of MSC therapy for ischemic myocardial disease has been well documented in both preclinical and early‐phase clinical trials, but improvements in myocardial function have been modest We have described the superior properties of EnMSCs relative to AdMSCs or BMMSCs for the treatment of myocardial infarction. EnMSCs conferred enhanced myocardium salvage and microvessel regeneration that was associated with significant improvement of cardiac function after MI. The possible mechanism for enhanced paracrine activity and therapy by EnMSC treatment might involve elevated expression and cellular delivery of exosomal miR‐21, with corresponding downstream effects on PTEN and Akt. Further investigations and clinical trials are essential to confirm EnMSCs as the preferred MSC source for myocardial regenerative medicine. Our study was limited to only three sources of MSCs. Thus, it is possible that MSCs from other early‐stage tissue sources, including placenta, fetus, and embryo, might have similar or superior properties to EnMSCs.K.W.: conception and design, collection and/or assembly of data, data analysis and interpretation, manuscript writing; Z.J.: collection and/or assembly of data, data analysis and interpretation; K.A.W.: conception and design, data analysis and interpretation; J.C.: data analysis and interpretation, manuscript writing; H.H., Y.Z., L.W., Y.W., C.N., and Q.L.: collection and/or assembly of data; J.Z.: collection and/or assembly of data, data analysis and interpretation; Z.Z.: data analysis and interpretation, manuscript writing; C.X: provision of study material or patients; L.Z.: data analysis and interpretation, manuscript writing; R.W., W.Z., and H.Y.: data analysis and interpretation, manuscript writing; X.H. and J.W.: conception and design, data analysis and interpretation, provision of study material or patients, financial support, final approval of manuscript.K.A.W. is founder, president, and CEO of Integene International LLC, a gene therapy company based in Miami, FL. The other authors indicated no potential conflicts of interest.Supporting InformationClick here for additional data file.


There is growing need to develop efficient methods for early-stage drug discovery, continuous manufacturing of drug delivery vehicles, and ultra-precise dosing of high potency drugs. Here we demonstrate the use of solvent-free organic vapor jet printing to deposit nanostructured films of small molecular pharmaceutical ingredients, including caffeine, paracetamol, ibuprofen, tamoxifen, BAY 11-7082 and fluorescein, with accuracy on the scale of micrograms per square centimeter, onto glass, Tegaderm, Listerine tabs, and stainless steel microneedles. The printed films exhibit similar crystallographic order and chemistry as the original powders; controlled, order-of-magnitude enhancements of dissolution rate are observed relative to powder-form particles. In vitro treatment of breast and ovarian cancer cell cultures in aqueous media by tamoxifen and BAY 11-7082 films shows similar behavior to drugs pre-dissolved in dimethyl sulfoxide. The demonstrated precise printing of medicines as films, without the use of solvents, can accelerate drug screening and enable continuous manufacturing, while enhancing dosage accuracy. Traditional approaches used in the pharmaceutical industry are not precise or versatile enough for customized medicine formulation and manufacture. Here the authors produce a method to form coatings, with accurate dosages, as well as a means of closely controlling dissolution kinetics. However, traditional approaches are not precise or versatile enough for customized medicine formulation and manufacture4. Severe trade-offs exist between drug discovery rate, dose customizability, and manufacturing scalability. Here we describe an approach that produces coatings with accurate, customizable dosages, as well as a means of closely controlling dissolution kinetics of active pharmaceutical ingredients (APIs). As proof-of-principle, we demonstrate the printing of widely used yet poorly soluble cancer drugs (BCS II type)5, among other APIs, achieving enhanced bioavailability. The combination of improved process control for dose accuracy and consistency, scalable yet customizable process, and controlled dissolution kinetics suggests a pathway for accelerating drug discovery and manufacture.Much of drug research and development now focuses on high potency and personalized medicines, and on alternative delivery vehicles7. Micronization and nanonization8 techniques have been developed to enhance the bioavailability by accelerating the dissolution of an API, commonly made in powder form to increase the surface area to volume ratio9 and, hence, dissolution rate. However, mechanical methods  for producing small particles are energy- and time-consuming, while the resulting nanoparticles may lack stability during storage and controlled release8. Formulating medicines with nanoparticles is also challenging, since homogeneity and stability are difficult to achieve due to particle agglomeration and changes in crystallinity8, compromising batch-to-batch consistency and safety, particularly for high-potency APIs (HPAPIs). Furthermore, prior to approval for general use, drugs undergoing initial testing are typically dissolved in organic solvents, dimethyl sulfoxide (DMSO) being one of the most common, leading to erroneous estimation of drug efficacy and bioavailability, as compared to direct application of the API. The lack of rapid polymorph phase screening methods combined with limited drug amounts leads to higher candidate attrition rates during drug discovery10. Finally, traditional process requirements to achieve high-throughput manufacturing compete with those required for personalization of dosage12. In contrast, film-form drug delivery vehicles have been demonstrated14 and have the potential to enable high throughput, continuous manufacturing, and personalized dosage simultaneously. For topical applications, thin film pharmaceutical coatings are used in transdermal drug delivery systems, such as patches and microneedles15. For localized drug delivery, thin films of APIs are applied in the form of coatings onto various delivery vehicles 1. Film-form drug systems are also used for transmucosal and oral drug delivery, providing the means for rapid drug dissolution and transport to the systemic circulation17. Manufacturing of film-form drugs includes dispersion of API particles in a polymer matrix by mixing, dipping, or spraying, followed by polymer casting or extrusion18. These approaches, however, suffer from limited particle dispersion, stability, and drug loading, especially when working with nanoparticles14.There are several challenges on the road from drug discovery, to formulation, to screening, and to manufacturing. For example, low solubility and dissolution rates pose severe limitations for new drug discovery19. In VTE, the source material is heated in vacuum, where the molecular mean free path is long, resulting in line-of-sight deposition by physisorption on a cooled surface20. Drug films obtained by VTE can exhibit enhanced dissolution due to increased surface area, while high dosing accuracy is in principle possible. Although VTE is a widely used technique for inorganic and organic material deposition, its scalability for the pharmaceutical industry is limited due to relatively low, controlled deposition rates (on the order of Å per s) and low material yield. Additionally, due to the deposition mechanism, some morphologies are difficult to access23, while significant cross-contamination risk of the common evaporation chamber and parasitic deposits makes it poorly suited for drug discovery. To overcome the challenges outlined above, we adapt a process originally developed to achieve continuous, solvent-free, large-scale, high-throughput, yet ultra-precise printing of small-molecular organic semiconductors: organic vapor jet printing (OVJP)22. It proceeds by thermally evaporating the substance  into a stream of inert carrier gas , followed by jetting onto a substrate, where the API forms a film  and large area deposits, while also enabling additive patterning with high material utilization efficiency (>70%). When film thickness exceeds 200 nm, the films can evolve into unique three-dimentional nano and microstructures21, such as shown in Fig. 21. Unlike other vapor-based deposition techniques , OVJP can be performed at atmospheric pressure and in some cases in ambient atmosphere25. These characteristics and the fact that the OVJP apparatus itself is very simple suggest that OVJP is a perfect candidate for the multiple uses outlined above.More recently, vacuum thermal evaporation (VTE) was used to create micro- and nano-structured thin films of drugsilm Fig. . Figure 25 is unlike traditional ink-jet printing in that it operates without liquid solvents, eschewing the need to pre-dissolve the API27, in principle making it attractive for application to printing APIs. However, typical API films deposited onto 1 × 1 cm2 surface area should in some cases be thicker than the organic semiconductor films used in optoelectronic devices, e.g., on the order of 10–1000 nm vs. 10–100 nm. How does the  evaporation process affect the chemistry, structure, and functionality of a drug compound? Will advantageous film and particle morphologies be obtained when the API is printed at useful thicknesses and onto biocompatible substrates? Here we address these questions and demonstrate how films of small molecular medicines can be made controllably by OVJP, obtaining simultaneously: crystalline deposits, enhanced dissolution kinetics and bioavailability, and precise dosages.OVJP29 —were characterized by thermogravimetric analysis (TGA)Examples of the obtained film microstructures are shown in Fig. We attribute the latter morphology to the fact that paracetamol and ibuprofen were deposited at temperatures of 190 and 75 °C, respectively, which were 20 and 5 °C above their melting point, suggesting that adsorption took place via a transient liquid phase, followed by dewetting and crystallization. In this case, the deposit morphology can be tuned by changing the source temperature, carrier gas flow rate, and adjustments to the deposition flux.To test the chemical stability of deposited substances, drug films were dissolved in methanol and analyzed by ultra-performance liquid chromatography (UPLC), for comparison to as-received API powder. Similar retention times and peak structures were obtained for deposited films and the original powder samples, Fig.  suggesti32. Hence, the crystal structure of the API films was studied using synchrotron X-ray diffraction and compared to the structure of the original powder and reference experimental data. The diffraction patterns of films were in very good agreement with both bulk powder used in the evaporation source and reference data available on stable materials polymorphs, indicating that OVJP did not change the crystal structure/polymorph composition of the API  is solute concentration as a function of time, t is time, D is diffusion coefficient in the solvent, A is solute-liquid contact area, V is solvent volume, δ is boundary layer thickness, Cs is the solubility limit in a given solvent. While A can sometimes be measured, boundary layer behavior is often a confounding and poorly controlled variable. OVJP enables the small molecular API to be formed as a continuous film. Although the surface profile may exhibit nanoscale roughness, this fine-scale roughness helps to quickly establish a virtual surface—i.e., a saturated boundary layer, which remains at a relatively constant thickness throughout the dissolution process, and therefore opens a simplified way of controlling and studying the dissolution process of a drug. Hence, for OVJP-deposited API films, Eq. , the dissolution rate becomes essentially constant, precisely controlled by the film’s projected area.For the sink condition  buffer (pH = 1.2) solution45, and tamoxifen in aqueous acetate buffer solution46 (pH = 4.9).  First, the solubility of these compounds in corresponding solvents was measured at 20 ± 1 °C. The saturation limit for fluorescein in deionized water was measured to be 10 ± 0.5 μg ml−1, for ibuprofen in HCl 22.5 ± 0.5 μg ml−1, and for tamoxifen in acetate 23.6 ± 0.5 μg ml−1. To measure dissolution kinetics, a rotating disk dissolution apparatus max is the maximum slope in the dissolution curve that plots the amount of dissolved material vs. time, typically evaluated at the start of dissolution process, where the concentration driving force is highest. IDR is an intrinsic property of a dissolving material, which depends for a given solvent on the material’s degree of crystallinity, crystalline and chemical structures. In all cases, intrinsic dissolution of films was comparable to one of compressed pellets )). In the case of printed films, IDR was essentially unaltered, indicating that the material's chemical and structural form was not altered either, corroborating XRD and UPLC results.The dissolution kinetics of three materials having poor aqueous solubility were studied: fluorescein in deionized waterFigure 35 over time, clearly present, for example, in the dissolution of ibuprofen powder  for tamoxifen (4.5 µg per film) and 500 nM (0.1 µg ml−1) for BAY 11-7082 (0.25 µg per film).To determine in a controlled manner whether enhanced API dissolution rates translate effectively to the API’s action on live cells, two different cancer cell lines  in growth medium were exposed to tamoxifen films and BAY 11-7082 films printed on glass slides. The cells were plated onto 12-well tissue culture dishes, and allowed to adhere and grow for 24 h in 2.5 ml of 10% fetal bovine serum-supplemented growth medium. Tamoxifen and films were inverted and placed onto the surface of the growth media  exhibit enhanced cancer cell-killing properties in vitro, without having to pre-dissolve them in organic solvents, such as DMSO. While amorphous dispersions have been used to increase the APIs’ solubility, their stability remains questionable49, eliminating the need for mixing and powder preparation.More generally, the ability to deposit small molecular drugs from their pure form, as films and without the use of solvents as shown here, opens an alternative approach to drug screening and manufacturing, where accurate dosage, chemical and structural stability, and processing flexibility are needed without hindering drug functionality. We therefore anticipate benefits for: early-stage drug discovery—eliminating the need for organic solvents for poorly soluble drugs during screening processes, development of drug delivery vehicles—e.g., films for transdermal drug delivery, directly coated patches, microneedles, encapsulated dissolvable films or implants, manufacturing and administration of HPAPIs with accurate, individualized dosing and nanogram-level accuracy during manufacturing. The technique demonstrated here also potentially enables continuous manufacturing, Caffeine (C0750) >99% was purchased from Sigma-Aldrich. Tamoxifen (ICN15673883) >99.24% and paracetamol (5074863) >99.5% were purchased from Fisher Scientific. BAY 11-7082 (19542-67-7) >98% and ibuprofen (401003) >99% were purchased from EMD Millipore, fluorescein (32615) >99.8% was purchased from Fluka; all were used as received. Borosilicate glass slides, 12 mm diameter, and 0.2 mm thick, were used as substrates for film deposition. Substrates were cleaned by ultrasonication in detergent solution and deionized water, followed by acetone and isopropanol rinses, for 10 min each. Substrates were then placed in boiling isopropanol for 5 min and dried in pure nitrogen gas prior to film deposition.OVJP nozzles used in this study were made from quartz tubes having 12.5 mm outer diameter and a nozzle tip of 500 μm internal diameter, with an ~15° inside wall taper from the nozzle axis. The inert carrier gas was 99.99% pure nitrogen.−2. The heating tape leads were connected to a temperature controller , and a 1/16 inch diameter K type thermocouple was used to monitor and maintain the temperature of the source. The source consisted of ~0.15 g of powder embedded in a porous SiC ceramic foam of 100 dots per inch (DPI) and placed in the heated source section of the tube. The gas flow rates were maintained using mass flow controllers .The nozzles were cleaned with acetone and isopropanol solvents, dried and wrapped with 36″ gauge heavy insulated tape heater  with a power density of 8.6 W·in The process parameters that were kept constant are: nozzle-substrate separation distance 1.5 mm) and substrate temperature (20 °C). The process was performed in glove box purged with 99.99% pure nitrogen gas. Detailed processing conditions are given in Table .5 mm andDeposits were formed by rastering the nozzle over the substrate to form adjacent, overlapping lines at 0.2 mm center-to-center spacing, allowing for homogeneous thickness of deposit for a nozzle of 0.5 mm inner diameter positioned 1.5 mm from substrate surface. Fluorescein films on microneedles were deposited through a flexible mask. The same process can be performed without a mask when using nozzle with appropriate printing resolution.Surface X-ray diffraction measurements of the films were performed at Sector 13-BM-C at the Advanced Photon Source, Argonne National Laboratory using a Newport 6-circle kappa diffractometer. Figure An FEI Nova 200 Nanolab scanning electron microscope with accelerating voltage of 5–10 kV and current 0.1–0.5 nA was used to obtain the surface morphology images. Optical microscopy analysis was performed with Zeiss microscope .To determine evaporation temperature of the powders, and subsequently source temperature in the system, TGA was used. All measurements were performed using a TA Instruments Thermogravimetric Analyzer Q500 at 0.01% accuracy, with nitrogen sample purge flow rate 60 ml per min and balance purge flow rate of 40 ml per min. Heating rate was 5 °C per min.−1 for duration of 20 min–12 h . λmax for absorbance testing was: 490 nm for fluorescein, 224 nm for ibuprophen, and 240 nm for tamoxifen.Drug dissolution was monitored by in situ UV–VIS Ocean Optics USB + 2000 spectrometer with T300-UV–VIS transmission dip probe, with a 10 mm optical path. Solution absorbance was recorded with frequency of 2–60 s2O + 0.1% formic acid and B: methanol + 0.1% formic acid. The detection wavelength was set to 280 nm. Caffeine and paracetemol were run isocratic at 100% B, while Tamoxifen and BAY had a gradient elution starting at A/B: 75/25, to 25/75 at 0.5 min, steady through 5 min, then returned to 75/25.To determine the concentration of analyte, and to screen for degradation or impurities, drug powders and films were dissolved in methanol and run on a Waters Acquity H Class UPLC equipped with a Waters Acquity UPLC C18 column (2.1 × 100 mm). Drug powders were used as standards in all cases, and were diluted such that film samples fell within the range of standards. For all molecules, the mobile phase was pumped at 0.3 ml per min, and consisted of A: ddHFor dissolution of the film, the glass substrate with the printed film was attached to a rotating rod with a substrate holder. In the case of compressed powder pellet, a 1.57 mm diameter pellet was compressed at 50 ± 2 psi for ~10 s into a holder having same size as disk substrate holder. In the case of loose powder, the weighed powder was dissolved into solution and stirred with blank substrate holder at 100 r.p.m. The detailed dissolution setup is shown in Supplementary Figure −1 of solid) were shaken at ~1000 r.p.m. for 48 h, followed by solution filtration with a 0.4 μm filter after 24 h. Filtered solution concentrations were then measured using UV–VIS spectrometry, calibrated with known solution concentrations. Five different concentrations for calibration were used in each test. The concentrations used were as follows: for fluorescein in dionized water −1, 2, 3, 4, 5 μg ml−1, ibuprofen in HCl pH 1.2 buffer: 2, 4, 6, 8, 10 μg ml−1, tamoxifen in acetate buffer pH 4.9: 1, 2, 4, 6, 10 μg ml−1.Solubility tests were performed at ambient temperature (20 ± 1 °C). Solutions with excess concentration (at least 100 μg ml50 was estimated to be ~40 μm. Since the boundary layer thickness in the rotating disk setup does not depend on compacted powder diameter, D was extracted for each compound based on compacted powder measurement and used for theoretical prediction of loose powders and printed films in plots for Figs. Theoretical predictions were calculated according to Noyes–Whitney theory (Eqs. The data that support the findings of this study are available from the corresponding author on request.Supplementary Information


In the present study, we have shown that ImPACT, when modified for cardiac applications, can assess both absorbed and effective doses. The results of our dose comparison indicate that modified ImPACT dose assessment is a promising and practical method for evaluating coronary CTA.The primary study objective was to assess radiation doses using a modified form of the Imaging Performance Assessment of Computed Tomography (CT) scanner (ImPACT) patient dosimetry for cardiac applications on an Aquilion ONE ViSION Edition scanner, including the Ca score, target computed tomography angiography (CTA), prospective CTA, continuous CTA/cardiac function analysis (CFA), and CTA/CFA modulation. Accordingly, we clarified the CT dose index (CTDI) to determine the relationship between heart rate (HR) and X‐ray exposure. As a secondary objective, we compared radiation doses using modified ImPACT, a whole‐body dosimetry phantom study, and the k‐factor method to verify the validity of the dose results obtained with modified ImPACT. The effective dose determined for the reference person (4.66 mSv at 60 beats per minute (bpm) and 33.43 mSv at 90 bpm) were approximately 10% less than those determined for the phantom study (5.28 mSv and 36.68 mSv). The effective doses according to the k‐factor (PACS number(s): 87.57.Q‐, 87.59.Dj, 87.57.uq According to a 2008 report by the United Nations Scientific Committee on the Effects of Atomic Radiation (UNSCEAR), effective doses of CCTA range from 3 to 27.5 mSv.0.9−7.9 mSv; chest, 2.212.9 mSv; abdomen, 3.1−16.1 mSv. In addition, the effective dose of CCTA is higher than that of other CT examinations; therefore, appropriate dose management is needed. These effective doses were determined using a number of estimation methods, including a whole‐body dosimetry phantom study with a thermoluminescent dosimeter (TLD)The phantom study represents a sophisticated clinical method. However, the absorbed and effective doses are obtained easily with the ImpactDose application, as the software can select cardiac scan protocols from a “User‐specified” or “SOMATOM Definition Flash scanner” during CCTA examinations.European Commission,0.014 mSv·mGy−1·cm−1) for estimating the effective dose was introduced in the 2004 European Commission guidelines.0.017 mSv·mGy−1·cm−1 reported in the 2000 guidelines0.027−0.034mSv·mGy−1·cm−1), which is important because the adult chest k‐factor tended to underestimate the effective dose for CCTA. However, the k‐factor is limited by its lack of assessment of organ‐ and/or tissue‐absorbed doses.Coronary computed tomography angiography (CCTA) is considered a reliable practical diagnostic method for coronary artery disease. Although an estimated 410,000 examinations are performed annually at 1,535 cardiology and/or cardiovascular surgery hospitals in Japan,To date, little information has been reported regarding electrocardiogram (ECG)‐gated scanning‐based dose assessment methods.The primary objective of this study was to assess radiation doses using modified ImPACT for cardiac applications , and CTA/CFA modulation), as described by Kobayashi et al.,A multidetector row CT (MDCT) scanner with 320 rows of detector elements , which is capable of data acquisition at a slice thickness of 0.5 mm and coverage of 160 mm, was used in this study. The applicable CTDI for each of the scan protocols was obtained from the CT console display, as summarized in CTDIwithout  ECG is the standard CTDI with one rotation scan, as defined by a number of international organizations,CTDIwith  ECG is the CTDI normalized with a single‐rotation ECG‐gated scanning (not one rotation) of the cardiac applications .where 70−120 bpm.40−120 bpm in 1 bpm increments was used to estimate CTDIwith  ECG, except for modulation, for which an HR gradient of 40–80 in 1 bpm increments was used. Because of the modulation did not work in high HR. All CTDIwith  ECG values were obtained from the CT console display.The 320‐MDCT has cardiac applications of the prospective ECG‐triggering scans  and retrospective ECG reconstruction scans (continuous CTA/CFA and CTA/CFA modulation) as are explained in sections B.1 to B.5 below. Completely different scan techniques are used in accordance with the patient's HR and the purposes to reduce their radiation dose. It is common knowledge that these scan timings of scan techniques are closely related to HR. According to previous publications, the optimal cardiac phase is generally 75% of the R‐R interval (late diastole) for HR 40–60 beats per minute (bpm) or 40% (end systole) for B.1CTDIwith  ECG is obtained during the prespecified phases of late diastole (40−70 bpm) and end systole (60−120 bpm).The Ca score (a Framingham Risk Score) is an effective index for determining the appropriate program to treat the amount of Ca in coronary arteries. This is a low‐dose scanning technique involving the exposure time and a single cardiac phase. Therefore, B.2CTDIwith  ECG for this method is obtained during the same as phase as the Ca score according to the acquisition time.Target CTA is a low‐dose scanning technique in which the exposure time and a single cardiac phase are manually preset before scanning to ensure that the patient receives consistent exposure. The actual exposure time depends on the acquisition time . Therefore, this scan mode cannot correspond to arrhythmia. B.3CTDIwith  ECG is obtained during the prespecified cardiac phase range of 1%–99% in 1% increments .Prospective CTA is a low‐dose scanning technique in which exposure occurs only during the prespecified range of the cardiac phase.B.4CTDIwith  ECG is obtained throughout the cardiac cycle . In general, only data acquired during the cardiac phase with the least amount of motion are used for image reconstruction.Continuous CTA/CFA is a scanning technique in which exposure occurs throughout the R‐R interval over one or more heartbeats. Therefore, a functional analysis can be performed using the obtained data. Similar to prospective CTA, multisegmental reconstruction is available for patients with high HRs in whom multiple beats are scanned. In this scan mode, B.5CTDIwith  ECG is obtained as follows: the tube current decreases automatically, except for the prespecified dose increase phase of 1%–99%. In this case, the 100% phase was not estimated because the tube current was fixed at that point. The dose reduction rate ranges from 5% to 80%. The C.F. is subsequently calculated using CTA/CFA modulation is a scanning technique in which exposure occurs throughout the R‐R interval and over one or more heartbeats. Exposure is also available to reduce the mA during portions of the R‐R interval that do not require high‐resolution imaging. Therefore, CTDIair) and CTDI in the center  with either a standard head or standard body polymethylmethacrylate (PMMA) phantom. The Medical International Radiation Dose Radiation dose‐five (MIRD‐5) mathematical phantom used in ImPACT was divided from head to mid‐thigh into 208 axial slabs of 5 mm thick.ImPACT patient dosimetry spreadsheet software, version 1.0.4 for Excel , was developed by the ImPACT group to provide a convenient user interface for determining organ‐ and tissue‐absorbed doses according to the National Radiological Protection Board SR250 Monte Carlo dose data sets (NRPB‐SR250) .22, 2General usage for assessing the radiation dose  was determined according to the following parameters: CT scanner, tube voltage, tube current, rotation time, spiral pitch, collimation, scan range, scan region (head or body), and organ weighting scheme, as described in ICRP 60CTDIwithout  ECG of cardiac applications in the ImPACT ScanCalculation worksheet . The bolus tracking time was calculated as the total time of the intermittent scans and continuous scan. However, 320‐MDCT is incapable of stopping the continuous scan automatically within 10 s for mechanical restriction. Therefore, we performed four intermittent scans  to provide stable reproducibility of the bolus tracking scan time, as a continuous scan with a manual stop is not stably reproducible. The scan areas were then determined, as shown in The organ‐ and tissue‐absorbed doses and effective dose were estimated for the CCTA scanning protocols  and 3 toThe effective dose was defined in ICRP Publication 103,The reference phantom C.F. includes 14 CT examination categories . During CCTA examination, we considered the chest C.F. to be appropriate.6 cm3  and a dosimeter  that was annually calibrated by a standard dosimetry laboratory. TLDs were then calibrated at an air kerma of 10 mGy from an effective energy of 54.6 keV  of aluminum (99.9 %); 7.88 mmAl) using the above‐described ionization chamber and diagnostic X‐ray equipment  with an X‐ray tube aluminum filter , collimation‐filter , and additional 6.0 mmAl filter (purity: 99.9%).TLDs were calibrated using an ionization chamber with a volume of The phantom study used a human body phantom in which molded polymer shapes, intended to simulate bone, were embedded in a material equivalent to soft tissue ; 233 TLD elements  were additionally inserted into this phantom. The RANDO phantom was placed in the supine position on a 320‐MDCT table and irradiated to evaluate the radiation doses from CCTA  and 3. ADair was calculated from the fluorescence (f), as indicated in Kyokko, Hiroshima, Japan). The air‐absorbed dose Dair and the ratio of the air and organ mass energy‐absorption coefficient (μen/ρ) at an effective energy of 54 keV, as follows:The sex‐averaged organ‐ and tissue‐absorbed dose D (Gy) was calculated using HT (Sv) was then calculated using D and a radiation weighting factor (1.0) specified in the ICRP Publication 103:The sex‐averaged equivalent dose HT and the age‐ and sex‐averaged weighting factor WT for each organ or tissue:The effective dose E (Sv) was calculated using Regarding the remaining tissues and organs, the adrenal gland, gallbladder, heart, kidney, pancreas, prostate gland , small intestine, spleen, and uterus  were evaluated. The endosteal bone surface dose enhancement factors, referenced by Nishizawa et al.0.014 mSv·mGy−1·cm−1) from the 2004 European Commission Guidelines:In contrast, the effective dose was estimated according to the following equation for comparison with the k‐factor study; specifically, the effective dose was calculated from the DLP displayed on the CT console and the k‐factor for adult chest  according to CTDIwith  ECG Accordingly, the cardiac phase limitation was characterized by HR . To reduce the patient dose, the cardiac phase range should be kept as narrow as possible. If the prespecified narrow cardiac phase range exceeds these values, the patient dose will not be reduced. 0.014 mSv·mGy−1·cm−1; 2.57 mSv at 60 bpm and 17.10 mSv at 90 bpm) were significantly lower than those obtained with the other two types of measurements; for example, the differences from ImPACT were 2.09 mSv (45%) and 16.33 mSv (50%), respectively. In contrast, the effective doses obtained with the C.F. values reported by Einstein et al.0.017 mSv·mGy−1·cm−1, respectively; 5.65 mSv at 60 bpm and 37.81 mSv at 90 bpm) were more similar to those of the phantom study.CTDIwith  ECG). These results suggest that dose assessment with modified ImPACT is a practical method for CCTA evaluation.In this study, we have shown that modified ImPACT, when used to evaluate CCTA with cardiac applications , can assess radiation doses . We have additionally demonstrated that the CTDI can be characterized by cardiac applications (0.014 mSv·mGy−1·cm−1) is used.0.028 mSv·mGy−1·cm−1, respectively. Although these adult coronary k‐factors can assess the effective dose, they cannot assess the organ‐ and tissue‐absorbed doses. Therefore, the effective dose for CCTA is possibly underestimated when the adult chest k‐factor is used. In contrast, the absorbed doses are assessed easily with modified ImPACT.In many dose estimations involving CCTA cases, the k‐factor  is independent of the cardiac phase and HR. Target CTA features a similar X‐ray irradiation activation pattern, and the resulting dose increases linearly as the acquisition time increases. During prospective CTA, X‐ray irradiation is active for one or more X‐ray tube rotations  because the prespecified cardiac phase time exceeds one rotation time, and the resulting dose increases at low HRs with a wide prespecified cardiac phase range because the absolute time increases. However, in this study, we clarified a limitation of the prospective CTA dose when determining the cardiac phase range required for dose reduction. Therefore, narrow cardiac phase specification may not lead to dose reduction. During continuous CTA/CFA, X‐ray irradiation remains active throughout the cardiac cycle, resulting in a much larger dose than that in other ECG‐gated scans. The CTA/CFA modulation dose over a wide cardiac phase range of 70%–90% is similar to the continuous CTA/CFA dose because the dose reduction phase time is too short to modulate the tube current. We confirm that the results of the present study are useful for determining scanning protocols in ECG‐based examinations. Therefore, these results were subsequently used for the C.F. (An earlier study by Kobayashi et al.the C.F. .One limitation of modified ImPACT is that it reduces radiosensitive organs to simple geometric shapes. Therefore, the doses absorbed by the lung, stomach, and liver differed from those measured in the RANDO phantom study. According to ImPACT, the doses absorbed by the bone surface were twofold higher than those obtained in the phantom study. Although the tissue‐absorbed doses in the phantom study were estimated using the method reported by Nishizawa and colleagues,Modified ImPACT yields a more accurate effective dose when compared with the k‐factor method. In addition, the effective dose for the reference male phantom was approximately 25% less than those for the reference female phantom. We confirm that this difference is due to differences in the dose absorbed by the breasts. In contrast, the effective doses for the reference phantom were approximately 10% less than obtained in the phantom study. These results occurred because of differences in factors such as phantoms, organ locations, TLD sensitivity, TLD insert positions, and estimated tissue‐absorbed doses. For example, the effective dose was approximately 7.5‐fold higher at 90 bpm than at 60 bpm. However, the continuous CTA/CFA three‐beat dose at 90 bpm was 8.4  because Finally, we consider the credibility of the C.F. values reported by Einstein et al.The present study demonstrated that modified ImPACT could perform ECG‐gated, scanning‐based radiation dose assessments and may thus be useful in future applications.We especially thank Dr. Alan Britten for permission to use of the ImPACT.This work is licensed under a Creative Commons Attribution 3.0 Unported License.Supplementary MaterialClick here for additional data file.