The weightless environment during spaceflight induces site-specific bone loss. The 30-day Bion-M1 mission offered a unique opportunity to characterize the skeletal changes after spaceflight and an 8-day recovery period in mature male C57/BL6 mice. In the femur metaphysis, spaceflight decreased the trabecular bone volume (−64% vs. Habitat Control), dramatically increased the bone resorption (+140% vs. Habitat Control) and induced marrow adiposity invasion. At the diaphysis, cortical thinning associated with periosteal resorption was observed. In the Flight animal group, the osteocyte lacunae displayed a reduced volume and a more spherical shape , and empty lacunae were highly increased (+344% vs. Habitat Control). Tissue-level mechanical cortical properties  were locally decreased by spaceflight, whereas the mineral characteristics and collagen maturity were unaffected. In the vertebrae, spaceflight decreased the overall bone volume and altered the modulus in the periphery of the trabecular struts. Despite normalized osteoclastic activity and an increased osteoblast number, bone recovery was not observed 8 days after landing. In conclusion, spaceflight induces osteocyte death, which may trigger bone resorption and result in bone mass and microstructural deterioration. Moreover, osteocyte cell death, lacunae mineralization and fatty marrow, which are hallmarks of ageing, may impede tissue maintenance and repair. Under the weightless spaceflight environment, astronauts are subject to factors such as radiation exposure, biorhythm changes, and vestibular alterations, and spaceflight induces site-specific bone loss in both humans and rodents6. These experiments showed either deterioration in trabecular and cortical bone parameters11 or no changes16 depending on the animal’s age, strain levels, habitat, flight duration and the delay between landing and sample collection. Nevertheless, in the context of growing bone, most of the results provide evidence for the spaceflight-induced inhibition of bone formation as well as impairment in periosteal expansion and longitudinal growth11, whereas bone resorption is either unchanged18 or increased2. Impaired mechanical properties have also been reported in the humerus and vertebrae19, although these changes were mostly caused by geometrical characteristics rather than differences in material properties19. Studies have also provided evidence of alterations in the bone matrix, mineral maturation and degree of mineralization (DMB) in the cortical compartment, suggesting that spaceflight also affects bone cortical-quality properties in growing rats23.Previous in-flight animal research conducted on young growing rats during relatively short-term missions (from 4 to 19 days) has considerably advanced our understanding of skeletal adaptations to spaceflight, including the mechanisms of bone loss and skeletal development in microgravity24. In brief, investigations of the tibia, femur and caudal vertebrae26 or the ischium1 revealed bone loss with increased bone resorption26 and decreased bone formation26.Because of the relationship between launch weight and cost as well as the constraints of launch vehicles, lighter experiments that use mice are now more common. The availability of multiple genetically modified mice strains also offers additional research opportunities. Excluding the topic of this paper (the Bion M1 flight), four previous mice missions have provided data on in-flight skeletal changes. These missions included short 12–15 day shuttle missions  with female C57BL6/J mice (2 to 4.5 months old) and a 91-day mission aboard the International Space Station (ISS) with 2-month-old male mice. Unfortunately, in the latter experiment, only 1 wild-type and 2 pleiotrophin-transgenic mice survived the flight3, highlighting the usefulness of prolonged mission studies. In addition, bone recovery after spaceflight is a concern because studies on spacemen27 and young rats28 have suggested that bone mass recovery after reambulation takes longer than the mission duration.Taken together, data from rats and mice emphasize an imbalance between bone resorption and formation, which results in deep alterations of the bone structure under space conditions. Furthermore, the bone loss appears to increase with time as also observed in space crewmembers30 after a one-month mission. In addition, a subset of flight mice was allowed to recover over the 8 days after landing to study their behavioural and locomotor functions31. Overall, studying the early dynamic events during bone re-adaptation to normal gravity is important, particularly for the development and prescription of post-flight countermeasures.Thus, our knowledge of skeletal changes during and after spaceflight remains incomplete. The BionM1 mission provided additional invaluable evidence to help further our understanding of the cellular, microstructural and qualitative bone changes that occur in skeletally mature miceTherefore, we analysed the microstructure of trabecular and cortical bone in the peripheral and axial skeleton of mature male mice and hypothesized that greater bone loss would be observed in the weight-bearing femur compared with the less weight-bearing vertebra. We also performed a cellular analysis of osteoblasts, osteoclasts, osteocytes and adipocytes at the different sites. Finally, the bone sub-microstructure and nanostructure were investigated to assess bone fragility, bone matrix collagen and mineralization properties as well as the 3D osteocyte-lacuna morphometric shape and distribution.Four groups of C57BL/6N mature male mice (23 weeks old) were compared. Two groups were exposed to a one-month spaceflight: (i) a Flight group, which included mice killed within the day after landing; and (ii) a Flight + Rec recovery group, which included mice that were allowed to recover over 8 days. Two ground control groups were also included: a Habitat Control group, which was kept under spacecraft housing conditions; and a Control group, which was kept under standard housing conditions .The body mass did not differ between the groups before the flight and at landing Fig. . During A comparison between the two control groups (Control and Habitat Control) was performed to estimate the impact of spacecraft housing, which was found to negatively affect the femur and lumbar vertebrae (but not the thoracic vertebrae). Indeed, the L3 vertebrae and femur trabecular bone volume fraction (BV/TV) and connectivity density were decreased in the Habitat Control group vs. the Control group Figs . After sThe trabecular BV/TV of the L3 vertebrae analysed by microtomography decreased in the Flight group vs. both the Habitat Control and Control groups  because of decreases in the trabecular number (Tb.N) and thickness (Tb.Th) Fig. . These pTo evaluate whether spaceflight and recovery also altered the bone material properties, nanoindentation analyses of the L1 interstitial (older) and peripheral trabecular bone (younger bone) were performed  groups and between the Flight + Rec and Flight groups (p = 0.032), and it was significantly decreased between the Flight and Control groups and between the Flight + Rec and Habitat Control groups  were performed  was greater in the Flight and Flight + Rec groups vs. the Habitat Control and Control groups and the fraction of larger lacunae (more than 200 µm3) was smaller in the Flight and Flight + Rec groups vs. the control groups  containing more than 1500 lacunae each were assessed. Figure ups Fig. .To determine whether osteocyte survival was compromised by spaceflight, we assessed the number of empty lacunae via a histological analysis Fig. . All lac5. We further revealed a number of unidentified spaceflight-induced effects. The nanomechanical characterization showed that the intrinsic mechanical properties were locally affected in both the axial and appendicular skeletal sites. We also demonstrated that spaceflight compromised the cortical osteocyte survival and decreased the lacunar volume. Furthermore, 8 days after landing, these bone adaptations did not recover despite normalized bone resorption and increased bone formation.This study provides multilevel analyses of weight-bearing (femur) and less weight-bearing (vertebrae) bones of the mature mouse skeleton after a one-month residency in space without any countermeasures and with an 8-day recovery period in male mice. We first confirmed the site-specific cortical and trabecular alteration induced by spaceflight, the weight bearing femur being more severe affected than vertebraAlthough the small sample size of the mice on board Bion-M1 (n = 5 for the Flight and Flight + Rec groups) and the inter-individual variability limited the statistical power of our analyses, a nonparametric test with a p-value < 0.017, which was considered significant (Bonferroni’s adjustment), provided robustness by limiting the risk of false interpretation.31 and body mass changes were not observed among the Control, Habitat Control and Flight animals. The effects of a JAXA habitat mouse cage (different from the Bion-M1) have been tested in young mice32, and contrary to our results, femur cortical thinning without alteration of the trabecular compartment was observed. These observations highlight the importance of dissociating the effects of specific spacecraft housing conditions from spaceflight effects when interpreting results. Noteworthy, however, the mice experienced different spatial environments in the spacecraft housing on Earth (2D surface) and in flight .Before specifying the spaceflight effects, we acknowledge that spacecraft housing, per se, as observed in the Habitat Control group induced alterations compared with the standard Control group. Indeed, deteriorated trabecular bone was observed in the femur and vertebrae, although the changes were less severe than those in the Flight group. Compared with the Flight group, the cortical bone structure, material properties and osteocyte parameters in the Habitat Control group were preserved, even when endosteal bone resorption was specifically increased in this group. The spacecraft housing  consisted of a cylindrical module, and it primarily differed from the standard housing (Control) by the space volume. Such confinement and the resulting reduction in physical activity likely induced the observed bone alterations. Furthermore, a special paste-like diet and distribution (6 times daily) may have contributed to the changes, although the nutrient and energy intake was generally consistent between groups26 or longer24 missions with mice. Moreover, spaceflight-induced fat marrow invasion at the femur metaphysis based on a significant appearance of lipid droplets that remained 8 days post flight. This in-flight fat increase was previously reported in the humeral bone marrow of young growing rats17 as well as in human vertebrae after 60 days of bed rest33. At this stage, linking adipogenesis with a potential decrease in osteoblastogenesis is still speculative, although these lineages originate from the same precursor cells. The osteoblastic activity (posterior to osteoblastogenesis) was assessed by quantifying the osterix-positive osteoblast surfaces (vertebra and femur) and osteoid seams (vertebra), and changes were not observed in our flight mice. These results are inconsistent with reports indicating that bone formation was inhibited in rapidly growing rats10, which likely occurred because of bone growth inhibition. In astronauts, regardless of their level of physical exercise, an increase in serum or urine bone resorption markers is a consistent feature, whereas the level of bone formation markers are decreased35 or unchanged37. Furthermore, after one week of Earth recovery, we observed that active osteoclastic surfaces returned to the control value and that osteoblastic parameters increased in the trabecular compartments of both bone sites. Such bone formation rebound over the preflight values has also been observed in astronauts38 and may reflect the differentiation-related lag time of precursor cells in the bone response to mechanical 1 g reloading39. Overall, these data show that mature mice are a more appropriate human model than growing rats when investigating bone cellular alterations during and after spaceflight.In mice of the spaceflight groups, bone resorption was dramatically stimulated in both the trabecular femur and vertebrae, which is consistent with reports in shorteret al. also found a decrease in the BV/TV compared with that of the standard controls after the Bion-M1 mission25.One-month spaceflight affects the vertebral column in a site-dependent manner. Micro-CT analyses revealed bone loss at the lumbar vertebral body, whereas the thoracic vertebral body was likely spared because of mechanical respiratory movements. Moreover, the BV/TV was initially higher in the thoracic vertebrae than the lumbar vertebrae (31 ± 1.5% vs. 17.9 ± 1.2%) and may have decreased the likelihood of spaceflight-induced bone loss in the thoracic vertebrae. In the caudal vertebrae, a site with no weight-bearing function, Berg-Johansen 26. In our study and concomitant with the in-flight bone structural alterations, we found that the hardness, or elastic modulus, and the maximal load measured by nanoindentation were deteriorated in the Flight animals at the periosteal side and the inner portion  of the lateral, anterior and posterior quadrants of the femoral cortex. At 8 days after re-entry, the femur showed no sign of trabecular recovery, a worsening of cortical thickness, and partial recovery of the nanomechanical parameters. Despite the decreased bone resorption and increased formation activities, no sign of bone structure rescue was observed.In the femur, the Flight animals suffered from more severe trabecular bone loss relative to the Habitat Control animals. This loss in the Flight group was associated with strut disappearance and disconnection, and thinning of the remaining struts. The Flight animals also displayed a thinner cortex compared with the Control animals as well as periosteal bone resorption and unaffected endosteal resorption compared with the Habitat Control group. Such cortical thinning has also been found in growing female mice after a shorter-duration spaceflight, and a further decrease in femur biomechanical properties  was reported40, should be further investigated, and the expression of enzymes involved in collagen crosslink formation should be assessed because an in vitro study showed that the conversion rate of immature crosslinks to mature compounds is decreased in microgravity41. Notably, recent investigations have shown that the nanomorphology and tissue mechanics are altered in mice subjected to osteocyte apoptosis induced by truncating connexin 43, which is the most abundant connexin protein in osteocyte gap junctions42.To further understand the alteration of intrinsic local mechanical properties in-flight mice, we evaluated the bone mineral characteristics . No changes were evident, showing that the remaining bone preserved its mineral quality properties during spaceflight. We then evaluated the collagen quality because collagen is the main organic component of the bone matrix, and changes were not observed. Thus, to better comprehend the local impairment of mechanical properties, additional studies are still necessary. In particular, the orientation of the collagen fibers, which is known to be influenced by the loading direction43. This finding suggested that osteocyte lacunae in weightless environments adapt their shape from elongated to more spherical, which is also observed in non-weight-bearing bones. A histological analysis of several hundred osteocytes per sample revealed that the fraction of empty lacunae was more than doubled in the Flight and Flight + Rec groups vs. the other groups, suggesting massive osteocyte death. More numerous empty osteocyte lacunae were also observed in an iliac crest biopsy of a Rhesus monkey flown on board the BION-11 flight44 and in neurectomised rat tibia by synchrotron imaging45. However, after a 14-day spaceflight, the empty lacunae ratio was unaffected and the 2D lacunae area increased in the pelvic bone of female mice1. It thus seems that different stages in osteocyte adaptation to spaceflight likely occur, and they are characterized by osteocyte osteolysis 1 or osteocyte death as well as bone resorption stimulation. Osteocyte death also occurs after immobilization, microdamage and ageing46. Recently, osteocytic death during ageing has been considered to precede lacunae occlusion by minerals, which is called “micropetrosis”48. Therefore, in the Flight group, dead osteocytes may have left uninhabited lacunae that would progressively mineralize and shrink.By analysing more than 7000 osteocyte lacunae per femur cortex using SR imaging, we demonstrated that long-term spaceflight compromised the 3D lacunae shape descriptors. The most significant changes were a decrease in lacunar volume , with the lacunae becoming more spherical in the posterior zone, and the same trends were observed in the anterior, medial and lateral zones. Interestingly, the osteocyte lacunae in the calvaria (non-weight-bearing bone) have been shown to be more spherical that those observed in the fibula (a weight-bearing bone) in mature miceIn conclusion, we provide evidence that a one-month spaceflight affects both axial and appendicular skeletal sites and leads to trabecular disconnection and cortical thinning, which are associated with increased osteoclastic resorption at the trabecular and periosteal levels, and fatty bone marrow. Although not exhaustive, multi-scale analyses of the bone matrix revealed new insights. At the tissue level, microradiography or FTIR measurements revealed that the bone mineral and collagen properties were preserved in the animals exposed to flight conditions. However, at the nanoscale level, the material properties were reduced at specific sites, such as at the trabeculae edges in the vertebrae or at the femur periosteal and mid-cortex. In the animals exposed to spaceflight conditions, osteocyte death was evidenced along with a smaller and more spherical shape of the osteocyte lacunae because of lacunar mineralization. Because osteocytes are long-lived key regulators of bone remodelling, their compromised survival may jeopardize bone deterioration reversibility, primarily in aging process. Further experiments should consider much longer post-flight periods to assess this hazard.31.The experimental design was approved by the IACUC of the MSU Institute of Mitoengineering  and the Biomedical Ethics Commission of the IBMP . This study in compliance with the European Convention for the Protection of Vertebrate Animals used for Experimental and Other Scientific Purposes3). Each habitat contained three mice. All groups were exposed to 12-h light/dark cycles with mean (±S.D.) temperatures of 23.0 ± 1.5 °C, 21.1 ± 0.4 °C and 21.3 ± 0.8 °C for the Habitat Control, Flight and Control groups, respectively. The Flight and Habitat Control animals consumed a paste-like diet containing 74.6% H2O and a casein gelling agent, whereas the Control mice consumed a standard pellet diet. The femurs and the vertebral columns were dissected and the soft tissue removed before being dehydrated for transport.Two groups of five C57BL/6N male mice exposed to one-month spaceflight on board the Russian Bion-M1 satellite were compared. One group was euthanized between 13–25 h after landing , and the other group was euthanized 8 days later . To evaluate the impact of spacecraft housing, two groups of ground control mice were investigated. A ground habitat control group  was placed in the same module under the same climate and food conditions as in the spacecraft, and a standard control group was placed in standard housing conditions (Control). For the microarchitecture, nanoindentation and FTIRM analyses, fifteen control mice were investigated. For the other investigations, 6 random control mice  were investigated. Three days prior to launch, all mice (except the Control group) were placed in the cylindrical animal module habitats , trabecular thickness , trabecular number , trabecular separation , structure model index (SMI), connectivity density (Conn. D.), and degree of anisotropy (DA). The cortical thickness (mm), cortical bone area (mm2), and cortical tissue mineral density  were calculated by integrating the value on each transverse section. The cortical bone marrow area (mm2) was calculated as follows:All left femurs were fixed in formalin and dehydrated in ethanol. All L3 and T12 vertebrae (n = 5/6) were scanned with a high-resolution μCT  at the 12.5 μm cubic resolution and analysed as described in David For the protocol details, see the SI text.Five and six left femurs and L3 vertebrae from the Flight and Flight + Rec and the Control and Habitat Control groups, respectively, were embedded in paraffin, cut and stained for osteoclast, osteoblast, osteocyte, marrow adiposity and growth plate assessments. For the protocol details, see the SI text.−1). Twenty scans per spectrum were acquired at a 4 cm−1 resolution. The FTIRM analysis of the vertebrae was performed in the transmission mode on 2-μm thick sections with a Perkin-Elmer GXII Auto-image Microscope . Ten measurements per vertebrae were performed at a spatial resolution of 30 × 40 μm. Each spectrum was collected at a 4 cm−1 resolution, and 50 scans were performed per spectrum. For the protocol details, see the SI text. The following parameters were determined: mineral crystallinity index, which is inversely proportional to the full width at half maximum (FWMH) of the 604 cm−1 peak (apatitic phosphate environment) and corresponds to the crystal size and perfection (organization of the apatite lattice)50; the mineralization index, which is the area ratio of the bands of mineral matrix (ν1ν3PO4) over the organic matrix (Amide I) (1184–910 cm−1/1712–1592 cm−1), reflects the relative mineral content in the organic matrix; mineral maturity ), corresponds to the progressive transformation of immature surface-hydrated domains into a mature and more stable apatite lattice and reflects the mineral age50; the collagen maturity, which is calculated as the ratio of the organic matrix bands in Amide I vibration (1660/1690 cm−1 area ratio) and reflects changes in the secondary structure of collagen in relation to the mineralization process52; and the carbonation, which is calculated as the area ratio of the ν2CO3 and ν1ν3PO4 bands and reflects the incorporation of CO3 ions in the crystal .To assess the bone matrix characteristics, a different procedure was performed on femurs and vertebrae because a 2-µm thick section of femur could not be cut with a microtome . Thus, a macro-analysis (FTIR) was performed on the femur, and a microanalysis (FTIRM) was performed on the vertebrae. The FTIR analysis was performed on bone femur powder diluted in potassium bromide (KBr). Bone pellets were analysed in the transmission mode with a spectrum 100 spectrometer  equipped with a wideband detector (mercury–cadmium–telluride)  from the Flight and Flight + Rec and the Control and Habitat Control groups, respectively, were cut from the blocks embedded in methylmethacrylate with a precision diamond wire saw . For the protocol details, see the SI text. For the cortical section, the following variables were measured using MATLAB software: mean DMB and heterogeneity index . For the vertebrae, the HI measurements were not considered because of trabecular thickness heterogeneity. For the cortical analyses, three layers  inside three anatomical regions  were analysed independently because of possible mineralization heterogeneity between zones. The anterior and posterior zone analyses were pooled because the anterior and posterior zones could not be precisely differentiated in the femoral mid-diaphysis. The results are expressed in grams of mineral/cmAll mid-diaphyses of the left femur and L1 vertebrae were embedded in polymethylmethacrylate and cut transversely to assess the bone material-level properties in the femur cortex and the vertebrae trabeculae. Because of potential differences in the bone material-level properties between the cortical zones, three anatomic cortical zones  were analysed, thus allowing for measurements in areas with or without muscle fixation and with different mechanical loads. Within these anatomic zones, three zones of interest  were analysed independently. Similarly, the interstitial trabecular vertebral bone (older formed bone) was analysed separately from the peripheral remodelled bone (younger formed bone). The following parameters were determined: maximal force (mN); hardness (mPa), which was interpreted as the mean pressure that the material can resist and calculated as the ratio of maximum force to contact area; modulus (gPa), which was defined by the initial slope of the unloading section of the curve; and dissipated energy (pJ), which was calculated as the area under the load-displacement curve. For the protocol details, see the SI text.3 field of view was scanned. The acquisitions were processed using a 3D-filtered back-projection algorithm to obtain 3D (2024)3 reconstructed images. To decrease the computational burden and independently analyse the anatomic specificity zones, four volumes of interest (VOIs)  were determined within each volume at a size of 700 × 700 × 500 voxels, which corresponds to a physical size of 490 × 490 × 350 µm. The 3D images included several thousands of osteocyte lacunae per VOI. For the protocol details, see the SI text.Five cortical femur sections per group were imaged using SR µCT on beamline ID19 at the ESRF  with a 0.7 µm isotopic voxel size. For each sample, 2000 projection images were recorded over a total angle of 360° at a fixed energy of 26 keV. An approximately 2.7 × 2.7 × 1.5 mm3) was obtained directly by summing the individual volume of each lacuna. The lacunar number density (N.Lc/TV) and the lacunar volume density Lc.V/TV (%) were also assessed. Because the shape of the osteocyte lacunae is generally assumed to be ellipsoidal, second-order moments can efficiently be used to determine the lengths of the main axis of the best fitting ellipsoid. This method allowed for the extraction of the three-dimensional descriptor of lacunae: length (Lc.L1), width (Lc.L2) and depth (Lc.L3). The shape of the lacunae was evaluated by the ratios of the axis lengths Lc.L1/Lc.L2 and Lc.L1/Lc.L3. The canal volume fraction  was calculated as (TV − (BV + Lc.V))/TV.The total lacunar volume  was performed between groups to test the effects of the spacecraft housing conditions, flight and recovery. The data are presented as clusters of individual points (bars = median); p < 0.017 was considered significant.Supplementary Info


Although the effect of weather and air pollution on the occurrence of subarachnoid hemorrhage (SAH) has been investigated, results have remained inconsistent. The present study aimed to determine the seasonality of aneurysmal subarachnoid hemorrhage occurrence and mortality.We used the National Inpatient Sample database to evaluate the effect of meteorological factors and air pollutants on patients with subarachnoid hemorrhage in Korea between 2011 and 2014. Monthly variations in SAH occurrence and mortality were analyzed using locally weighted scatter plot smoothing curves. Multivariate Poisson generalized linear regression models were used to evaluate potential independent meteorological and pollutant variables associated with SAH occurrence and mortality.In total, 21,407 patients who underwent clip or coil treatment owing to aneurysmal SAH in Korea from January 1, 2011, to December 31, 2014, were included. The crude incidence rate of SAH in Korea was 10.5 per 100,000 people per year. An approximately 0.5% lower risk of SAH was observed per 1°C increase in mean monthly temperature , while an approximately 2.3% higher risk of SAH was observed per 1°C increase in mean monthly diurnal temperature.We showed distinct patterns of seasonal and monthly variation in the occurrence and mortality of SAH. Our findings suggest that meteorological factors may play an important role in monthly variations in the occurrence of aneurysmal SAH. The influence of weather and air pollution on the occurrence of aneurysmal subarachnoid hemorrhage (SAH) has been widely investigated numerous countries; however, their results remain inconsistent. Although several researchers have reported that the incidence of SAH is lower in the summer,,2 additiWe aimed to determine the seasonality of aneurysmal SAH occurrence and mortality. In addition, we used the National Inpatient Sample (NIS) database to evaluate the effect of meteorological factors and air pollutants on SAH occurrence and mortality in Korea from 2011 to 2014.This study was approved by the institutional review board of Gachon University (IRB No. 1044396-201606-HR-043-01). Owing to the retrospective nature of the study, the need for informed consent was waived.http://opendata.hira.or.kr) in Korea each year. Detailed information regarding the NIS and HIRA has been described elsewhere.[We collected NIS data on patients with SAH from January 1, 2011, to December 31, 2014, available by the Health Insurance Review and Assessment Service . These data were collected and analyzed from 73 observation stations located throughout South Korea.[Meteorological data including monthly measures of mean temperature, average diurnal temperature range, and insolation from 2011 to 2014 in Korea were obtained from the Meteorological Administration of South Korea (th Korea.10), nitrogen dioxide (NO2), and sulfur dioxide (SO2) in Korea for the duration of the study period. These data were collected from Air Korea, which was established in 2002 by the Ministry of Environment of South Korea (www.airkorea.or.kr/eng/). The comprehensive air pollution data were collected and analyzed from 251 sites in 79 cities.[Air pollutant data included average monthly measures of particulate matter with an aerodynamic diameter < 10 μm (PMSeasons were defined as winter (December through February), spring (March through May), summer (June through August), and autumn (September through November).The crude SAH incidence rate was estimated as the average number of SAHs occurring in the study period per 100,000 people per year. Discrete variables are expressed as percentages; continuous variables, the mean ± standard deviation or median with interquartile range (IQR).Descriptive statistics were used to determine the median and IQR of monthly meteorological and air pollution measures. The relationships among meteorological and air pollution factors were evaluated using the Pearson correlation test.t-test was used to identify seasonal variations in SAH occurrence and mortality. Linear regression lines for the LOWESS curve were used to identify the association among temperature, diurnal temperature range, and SAH occurrence. SAH occurrence was natural log-transformed to normalize distributions.We used a locally weighted scatter plot smoothing (LOWESS) curve with a 95% confidence interval (CI) to graphically represent the total monthly variation in the occurrence/mortality of aneurysmal SAH, which was then stratified in accordance with each year. Student’s http://rcps.egov.go.kr).Univariate and multivariate Poisson generalized linear regression models were used to evaluate potential independent meteorological and pollutant variables associated with SAH occurrence and mortality, using a log-linkage function offset by the log of the population in each year from 2011 to 2014. The population of Korea between 2011 and 2014 was investigated based on the Korean population census , y = Total, group = 1))+ stat_smooth +geom_point  +… and ggplot )+ stat_smooth , se = FALSE)+stat_smooth , se = FALSE)+…). All statistical analyses were performed using R version 3.3.2.We used the ggplot function in R to visualize the map of South Korea using “ggplot2” and “Kormaps” packages ) +geom_polygon (colour = "black")+…). We created a map of South Korea in Excel to display geographic distribution of average temperature, diurnal temperature range, and PMDuring the study period, the average population of Korea was 51,037,983.8 .10 in South Korea between 2011 and 2014 based on summer and winter seasons. The average temperature varied by about 3°C ~ 8°C and 4°C ~ 6°C variation of diurnal temperature range throughout nation from 2011 to 2014 in summer and winter. PM10 ranged from approximately 13 μg/m3 to 27 μg/m3. We also present detailed regional measures of the meteorological factors and air pollutants in South Korea each year classified by summer and winter seasons  and lowest in Jeonnam (1.4) . One pos seasons .The average age of patients at SAH occurrence was 56.3 years, and 64.1% of patients were women. Surgical clipping was performed in 11,485 (53.7%) patients. The median length of admission was 20 days, and the mortality rate was 9.0%. Patient characteristics are further detailed in SAH occurrence was highest in March  and lowest in July  . The oveSAH occurrence was lower in the summer than in the winter each year . This paMortality was highest in March (n = 199) and lowest in December (n = 124) . Variati2 = 0.396). A continuous downward slope was also noted for temperatures above approximately 10–15°C . An upward slope was also observed between 8–11°C in the LOWESS curve , after adjusting for all meteorological factors and pollutants .In the present study, we found that both mean monthly and diurnal temperature range affect SAH occurrence. Lower mean temperatures were significantly correlated with higher aneurysmal SAH occurrence. In addition, higher diurnal temperature range was positively associated with SAH, this association being significantly more prominent in men than in women. In-hospital mortality was highest in March and tended to decrease at the end of the year, despite an increase in the incidence of SAH at this time. Mortality exhibited no association with either meteorological factors and pollutants in the multivariate analysis.Previous studies have supported our finding that the incidence of SAH is lower in the summer than in other seasons.–28 A recCold temperatures are known to increase blood pressure.–33 BloodSeveral studies have reported a possible correlation between SAH occurrence and atmospheric pressure, humidity, or insolation.,13,38,39No significant association was observed between in-hospital SAH mortality and any average monthly meteorological factors or pollutant. Our findings indicate that mortality was highest in March, gradually decreasing from March to December with some fluctuations, in accordance with the pattern of SAH occurrence. Several studies have documented the “July effect”: a transient increase in adverse SAH outcomes during July,,41 probaWith consideration of all findings and hypotheses of the study, we think that exposure to abrupt or chronic cold temperature may affect risk of aneurysm rupture in patients with unruptured intracranial aneurysms. Therefore, precaution may be helpful for prevention of SAH in patients with unruptured aneurysms. In addition, previous studies indicated that higher body temperatures may have a larger benefit for treatment in patients with stroke.,43 Our fOur study has the following limitations. First, we could not include all patients with SAH owing to the inherent limitations of using the NIS database. However, random selection of patients with SAH who had undergone clipping or coiling procedures did not influence monthly variations in SAH. In addition, we think that this inevitable selection due to using NIS database naturally excludes milder cases and non-aneurysmal spontaneous SAH which account for between 10 and 20% of all spontaneous SAH. Second, Despite these limitations, we observed a trend toward lower SAH occurrence in the summer and higher SAH occurrence in January and during the change of seasons. These trends were significantly associated with the mean monthly temperature and diurnal temperature range. However, we observed no correlation between SAH mortality and meteorological/air pollution parameters. Our findings suggest that meteorological factors may play an important role in monthly variations in the occurrence of aneurysmal SAH, although aneurysm rupture itself is likely to be multifactorial.S1 Data(XLSX)Click here for additional data file.S2 Data(XLSX)Click here for additional data file.S1 Fig(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S5 Table(DOCX)Click here for additional data file.


Manihot esculenta Crantz.) varieties developed by the Crops Research Institute of Ghana (CRIG) was studied in paracetamol tablet formulations. The yield of the starches ranged from 8.0 to 26.7%. The starches were basic (pH: 8.1–9.9), with satisfactory moisture content (≤15%), swelling capacity (≥20%), ash values (<1%), flow properties, and negligible toxic metal ion content, and compatible with the drug. The tensile strength (Ts), crushing strength (Cs), and friability (Ft) of tablets containing 5–10% w/w of the cassava starches were similar (p > 0.05) to those containing maize starch BP. The disintegration times of the tablets decreased with increase in concentration of the cassava starches. The tablets passed the disintegration test (DT ≤ 15 min) and exhibited faster disintegration times (p > 0.05) than those containing maize starch BP. The disintegration efficiency ratio (DER) and the disintegration parameter DERc of the tablets showed that cassava starches V20, V40, and V50 had better disintegrant activity than maize starch BP. The tablets passed the dissolution test for immediate release tablets (≥70% release in 45 min) with dissolution rates similar to those containing maize starch BP. The disintegrant potential of native starches of five new cassava ( Starch and its derivatives  are principally used as disintegrants in pharmaceutical tablet formulations. Starch and its derivatives are also used as diluents, binding agents, glidants, and thickeners. Disintegrants are pharmaceutical excipients that are included in tablet formulations with the aim of facilitating the break-up of the compressed tablets into small fragments in aqueous media. The enhanced splitting of the tablets in aqueous media enhances the dissolution, absorption, and bioavailability of orally administered drugs. Other substances employed as disintegrants in pharmaceutical formulations are cellulose and its derivatives , resin and its derivatives, and crospovidone .Native starches derived from botanical sources are commonly employed as disintegrants in pharmaceutical tablet formulations usually in a concentration range of 2–10% w/w . Other fVarious mechanisms of disintegration have been proposed for disintegrants. These mechanisms which have variable application to different types of disintegrants are swelling, capillary action or wicking, strain recovery, heat of interaction, and interruption of particle-particle bonds , 5. SwelThe first disintegrating agents to be used in tablet formulations were native starches of maize, potato, and wheat . In geneThe Crop Research Institute of Ghana (CRIG), Fumesua, is using genetic techniques to develop new cassava varieties with high starch, fiber, nutrient content, and other functional properties. The aim of the current study was to evaluate the disintegrant properties of native starches of five improved cassava varieties developed by CRIG, Fumesua, in paracetamol tablet formulations. Paracetamol was used as a model drug because it has poor flow and compression properties and high capping and lamination tendencies and lacks any inherent disintegrant activity. The effects of the cassava starch disintegrants on the physicomechanical and drug release properties of the formulated paracetamol tablets were evaluated.Manihot esculenta Crantz.), namely, Sika Bankye, Ampong, AW/10/008, 12/0245, and 12/0197 , were obtained from CRIG, Fumesua, Ghana. The cassava varieties were planted in March 2014 and harvested in October 2015, and starch extractions were undertaken within two days of harvesting. Maize starch BP  was used as the reference disintegrant (coded V60). Paracetamol BP , magnesium stearate , talc and lactose , polyvinylpyrrolidone (PVP) , sulphuric acid, nitric acid, hydrochloric acid and sodium hydroxide pellets , ethanol 96% , and potassium dihydrogen orthophosphate  were used.Five varieties of matured fresh root tubers of cassava . The total ash, acid insoluble ash, and water soluble ash of the cassava starches were determined using official methods .Vd). The starch powder was dispersed in 70 ml of distilled water and made up to 100 ml with water. After 18 hours of standing the volume of the sediment (Vw) was determined. The swelling index (%) was calculated using the equation: The tapped volume occupied by 10 g of the powdered cassava starch in a 100 ml measuring cylinder was recorded  at 25°C. Five hundred milligrams of the starch powder was added to 50 ml of solvent and allowed to stand overnight. Twenty-five milliliters of the supernatant was placed in preweighed Petri dishes and evaporated to dryness over a water bath and further dried to constant weight in an oven at 100°C. The mass of the residues was determined with an analytical balance  and expressed as the percent solubility of the cassava starch in the respective solvents.M1) of cassava starch powder was weighed into a 100 ml measuring cylinder and the volume occupied (V1) noted. The measuring cylinder was then tapped on a hard surface to consolidate the powder to a constant volume (V2). The fluff density, tapped density, Hausner ratio, and Carr's index were calculated as follows: Thirty grams (h) and the mean diameter (D) of the base of the starch powder cone were determined. The angle of repose (θ) was calculated using the equation A funnel was clamped with its tip 2 cm above a hard horizontal surface. The starch powder was allowed to flow through the funnel until the apex of the powder formed just touched the funnel's tip. The height , arsenic (As), lead (Pb), and mercury (Hg) in the cassava starches was determined with an atomic absorption spectrophotometer (Buck Scientific Model 210V GP) as previously reported , 12.−1 using the FTIR spectrometer by placing the sample into a diffuse reflectance sampler. The spectra of the three samples were superimposed to assess whether or not the principal absorption bands present in the drug and starches are present in the physical mixtures.A Fourier transform infrared spectrometer (FTIR)  was used to determine the compatibility between paracetamol and the cassava starches. The spectra of the individual starches, paracetamol, and the physical mixtures of the drug and starch were recorded by scanning in the wavelength region 4000–400 cmμm) and dried in a hot air oven at 50°C for 2 h. The dry granules were manually screened using a number 16 mesh sieve (1190 μm) and mixed with talc for 5 min in the V-blender. Finally, magnesium stearate was added and blended for a further 5 min and discharged. The different batches of the paracetamol granules in the size range of 595–1190 μm were compressed into tablets (~600 mg) at pressures of 45–50 KN using a Cadmach CTX 26 tableting machine . The tablets were stored for 24 h after compression to allow for elastic recovery and hardening before evaluation.Paracetamol granules (420 g) comprising of paracetamol (83.3%) as active pharmaceutical ingredient, lactose  as diluent, polyvinylpyrrolidone, PVP (4.5%), as binder, talc (1.2%) as glidant, magnesium stearate (0.2%) as lubricant, and cassava starch or maize starch BP  as disintegrant were prepared by the wet granulation method. The required amount of paracetamol was dry-mixed with lactose and starch and moistened with PVP solution and massed for 10 min in a V-blender . The damp mass was manually screened through a number 12 mesh sieve .λ-max of 275 nm using the regression data of the calibration curve  of paracetamol (0.25–1.5 mg/100 ml) in 0.1 M sodium hydroxide.The paracetamol content of the tablets produced was determined using the procedure described in the British Pharmacopoeia . The amoCs) of ten randomly selected tablets from each batch was determined with an Electrolab hardness tester  as described in the British Pharmacopoeia [Ts) was determined using the equation D is the diameter and t is the thickness of the tablet [The crushing strength . Ten tablets weighing ~6.0 g from each batch were placed in the friabilator and it was operated for 4 min at 100 rpm. The tablets were dedusted and reweighed and the difference in tablet weight was determined. The friability was calculated as follows: The disintegration time of the tablets of each batch was determined using an Erweka disintegration apparatus  as described in the British Pharmacopoeia .Cs, Ft, and DT are the crushing strength, friability, and disintegration time, respectively. The disintegration parameter DERc was determined using the formula test is the DER of tablets containing the test disintegrant , while DERreference is the DER of tablets containing the reference disintegrant (V60). When DERc > 1, the DER of the test cassava starch disintegrant is considered to possess better disintegrant action than the reference disintegrant and vice versa [The disintegration efficiency ratio (DER) was determined using the relationship ce versa , 15.y = 624.16x + 0.0283, R2 = 0.9975). Triplicate determinations were performed for each batch. Graphs of percentage drug released were plotted against time to establish the dissolution profiles of paracetamol.In vitro tablet dissolution tests were undertaken with an Erweka dissolution apparatus  under sink conditions. Dissolution was determined in 900 ml phosphate buffer pH 5.8 at a paddle speed of 50 rpm and temperature of 37 ± 0.5°C. A tablet was carefully placed into each vessel to exclude air bubbles from its surface. At times 5, 10, 15, 30, 45, and 60 min, 20 ml of the dissolution medium was withdrawn and filtered and replaced with an equal volume of medium. The filtrates were diluted to 50 ml with 0.1 M NaOH and the absorbance of each solution was measured at 257 nm with a UV spectrophotometer . The amount of paracetamol dissolved was determined using the regression data equation  followed by Tukey's post hoc multiple comparison test with GraphPad Prism version 5.00 for Windows . At 95% confidence interval, Manihot esculenta Crantz.) after extraction produced starch yields ranging from 8.0% to 26.8% with the ranking of V20 > V40 > V10 > V30 > V50. Cassava starch yield was affected by the cassava variety used. Other factors which are known to affect cassava starch yield include processing factors [All the five varieties of cassava  and 1561.11 cm−1, 1504.98 cm−1 (amide II band) which are characteristic of the drug. The cassava starches showed intense functional groups at 3274.71 cm−1 (free hydroxyl group) and 1336.55 cm−1 and 997.37 cm−1  which characterized them as starches. The physical mixtures contained all the principal bands of the two constituents and showed no shifting of the peaks, indicating the stability and compatibility of the starches with the drug.n = 20) [p > 0.05) between the crushing strength of tablets containing maize starch and the cassava starches.Paracetamol tablets containing different concentrations of the cassava starches as disintegrants were produced by the wet granulation technique. The starch disintegrants were added to the tablet formulations intragranularly. n = 20) , except n = 20) , and alln = 20) . There wp > 0.05) between the tensile strength of tablets containing the cassava starches and that of maize starch BP, even though the tensile strength of the latter was higher. Friability test is used to evaluate the capacity of tablets to withstand abrasion and chipping associated with handling, packaging, and shipping. Apart from tablets which contained 10% w/w of cassava starch V10, all the tablets passed the friability test (tablet loss ≤ 1%). An increase in cassava starch disintegrant concentration did not produce any specific effect on tablet friability and there were no significant differences (p > 0.05) between the friability of tablets which contained cassava starch and those containing maize starch BP. The disintegration of a tablet is affected by the temperature of the disintegration medium, the nature and amount of binder used, force of compression, and the nature and amount of disintegrant used [p > 0.05) were observed between the disintegration times of tablets containing cassava starches and those containing maize starch BP.Tablets should have strong tensile strength to withstand pressure due to handling, film-coating, and packaging but must be weak enough to allow drug release after administration. In general, the tensile strength of the tablets reduced with increase in cassava starch concentration. There were no significant differences (ant used . An incrant used . No signCt/Ft) is a better measure of the mechanical strength of tablets than crushing strength as it removes the weakness related to the friability of a tablet [Ct/Ft values have strong mechanical strength [c values of tablets containing the cassava starches V40, V50, and V20, which were generally >1. This provides an indication that these experimental or test cassava starches had better DER values than that of the reference disintegrant. On the other hand, tablets containing cassava starches V10 and V30 generally had DERc values <1 which shows that the two cassava starches had inferior DER values than that of the reference disintegrant.The crushing strength-friability ratio  and crushing strength (Cs) of the tablets decreased with increase in starch concentration. All tablets containing the cassava starches disintegrated in aqueous medium in less than 15 minutes and the disintegration time decreased with increase in starch concentration. The friability of the tablets was generally unaffected by an increase in starch concentration. The disintegration efficiency ratio (DER) of tablets containing the cassava starches generally increased with increase in starch concentration. Tablets containing different concentrations of the cassava starches released more than 70% of the drug in phosphate buffer pH 5.8 at 45 minutes and were comparable to tablets containing maize starch BP. Even though starch from cassava variety V40 had a rather low starch yield of 11.2%, it appeared to be the preferred cassava starch disintegrant as the different concentrations of the starch generally produced tablets with superior physicomechanical and in vitro drug dissolution characteristics.The results showed that the five cassava varieties have low starch yields. The starches contained negligible amounts of toxic metal ions and generally exhibited good physicochemical properties required for use as pharmaceutical excipients. The five cassava starches employed as intragranular disintegrants in concentrations of 5–10% w/w produced paracetamol tablets with satisfactory physical properties comparable with maize starch BP. There were no interactions between the starches and the active pharmaceutical ingredient and excipients used in producing the tablets. Generally, the tensile strength (


Spermophilus beldingi) are routinely shot in California, Nevada, and Oregon across habitats that overlap with breeding avian scavengers. Ground squirrels shot with lead (Pb)-based bullets may pose a risk to avian scavengers if they consume carcasses containing Pb fragments. To assess the potential risk to breeding avian scavengers we developed a model to estimate the number, mass, and distribution of Pb fragments in shot ground squirrels using radiographic images. Eighty percent of shot carcasses contained detectible Pb fragments with an average of 38.6 mg of Pb fragments. Seven percent of all carcasses contained Pb fragment masses exceeding a lethal dose for a model raptor nestling . Bullet type did not influence the number of fragments in shot ground squirrels, but did influence the mass of fragments retained. Belding’s ground squirrels shot with .17 Super Mag and unknown ammunition types contained over 28 and 17 times more mass of Pb fragments than those shot with .22 solid and .22 hollow point bullets, respectively. Ground squirrel body mass was positively correlated with both the number and mass of Pb fragments in carcasses, increasing on average by 76% and 56% respectively across the range of carcass masses. Although the mass of Pb retained in ground squirrel carcasses was small relative to the original bullet mass, avian scavenger nestlings that frequently consume shot ground squirrels may be at risk for Pb-induced effects . Using modeling efforts we found that if nestling golden eagles (Aquila chrysaetos), red-tailed hawks (Buteo jamaicensis), and Swainson’s hawks (B. swainsoni) consumed shot ground squirrels proportionately to the nestling’s mass, energy needs, and diet, 100% of the nestling period would exceed a 50% reduction in delta-aminolevulinic acid dehydratase production threshold, the last 13–27% of the nestling stage would exceed a reduced growth rate threshold, but no nestlings would be expected to exceed a level of Pb ingestion that would be lethal.Recreational ground squirrel shooting is a popular activity throughout the western United States and serves as a tool for managing ground squirrel populations in agricultural regions. Belding’s ground squirrels ( Lead (Pb) exposure is a serious conservation threat to birds  because Urocitellus richardsonii; [Cynomys ludovicianus; [Medicago sativa) across the central and western United States from mammalian pests can result in substantial financial loss, thus shooting may be a valuable and effective management tool [-1; [Haliaeetus leucocephalus), common ravens (Corvus corax), ferruginous hawks , golden eagles (Aquila chrysaetos), red-tailed hawks (Buteo jamaicensis), and Swainson's hawks (B. swainsoni) [Recreational varmint shooting may provide a substantial delivery vehicle for Pb exposure to avian scavengers –21, esperdsonii; , black-tent tool ,23. Grouool [-1; ), make iool [-1; . The totool [-1; . Moreoveainsoni) –27.Little is quantitatively known about the extent to which shot ground squirrels may act as a vector for Pb to avian scavengers. More specifically, an understanding of the number of bullet fragments, their mass, and distribution within shot ground squirrels carcasses is critical for understanding risk to avian scavengers, particularly during their breeding season when ground squirrels may comprise a substantial portion of nestling diets; see –30. The U. beldingi) from California and Oregon, and quantified the number, mass, and distribution of fragments in carcasses relative to ammunition types using radiography. We validated radiography results by extracting bullet fragments from a subset of shot ground squirrel carcasses. Additionally, using estimates of Pb fragments from shot ground squirrels, we modeled potential Pb exposure in nestling raptors relative to published toxicity benchmarks  while accounting for nestling age, diet, food mass requirements, and Pb content of shot ground squirrels. We discuss these results in the context of potential exposure to breeding avian scavengers that routinely scavenge at shooting fields and bring ground squirrels to their nestlings.Our objectives in this study were to quantify the potential for shot grounds squirrels to act as a vector for Pb exposure in breeding avian scavengers and determine if specific types of commonly used ammunition differed with respect to numbers, mass, or distribution of fragments in carcasses. To do so, we examined shot Belding’s ground squirrels  are major producers of alfalfa in the US, with approximately 40% of the US production and nearly 3 million ha of alfalfa acreage coming from just 11 western states . Within -1 . We condDuring 2014 and 2015, we collected carcasses of shot ground squirrels from these three areas. Ground squirrel shooting routinely occurs throughout all three areas, both as a recreational activity and as a tool to manage ground squirrel populations. These activities mostly occur between March, when ground squirrels emerge from hibernation, and August, when ground squirrels often begin hibernation.n = 6 fields), and 2) squirrels that were shot with known ammunition types (n = 5 fields). In the case ground squirrels shot with known ammunition types, details on ammunition type were provided by land owners or private individuals that shot ground squirrels. The first approach to ground squirrel carcass collections allowed us to assess the number of fragments, distribution, and mass of fragments from carcasses that were readily available on the landscape. Secondly, the collection of carcasses shot with known ammunition type allowed us to determine if specific types of ammunition resulted in different numbers, distribution, or mass of Pb fragments in shot carcasses. All ground squirrels were shot and killed under natural field conditions at distances ranging from approximately 20–150 m. In order to ensure that Pb fragments in salvaged ground squirrels were representative of common conditions, ground squirrels were shot and retrieved by private citizens engaged in this activity, or by private land-owners that shot, retrieved, and then donated carcasses. No ground squirrels were shot specifically for the purposes of this study and U.S. Geological Survey staff did not shoot any of the ground squirrels in the this study. Specimens were placed in polyethylene storage bags, labeled with ammunition type, placed on wet ice in the field, and then stored frozen at -20°C until processing and analysis.We salvaged shot ground squirrels immediately after shooting events on alfalfa fields under two different scenarios; 1) squirrels that were shot with mixed, unknown ammunition types , removed the dermal tissue (skin and fur) from each specimen, and stored them frozen in plastic bags until radiographic analysis. The dermal tissue was removed from each carcass to facilitate improved digestion and extraction of bullet fragments from carcasses. We assumed that the dermal layer retained a limited amount of Pb and is not a significant source of Pb to avian scavengers because it is typically regurgitated upon consumption ,37. Howe2) by calibrating the digital image using a scale of known size that was next to each carcass during radiograph acquisition , and couisition . We measuisition . We thenn = 30) using a digestion procedure. We used a three-step process to separate bullet fragments from each carcass. (1) We conducted a preliminary tissue decomposition step in a pressure cooker  with 1 liter of deionized water for 9 hours at a maximum pressure of 90 kpa and temperature of 120°C. (2) We then further digested the squirrels in a 2.5% solution of WME-2717, an industrial enzyme blend, , used to treat drainage systems at slaughterhouses by rapidly digesting protein. We digested each squirrel in solution at 50°C for 7–10 days. To stop the enzyme digestion process, we added a 10% bleach solution. We then stored the resulting solution in a refrigerator at 4C until further processing. (3) To extract bullet fragments from the digest, we first filtered the solution through 500μm and 210μm sieves to remove bones and large, undigested tissue particles. Sieves were examined under a dissecting microscope to ensure no visible bullet fragments remained. We then used a gold prospecting sluice box  to recover bullet fragments from the remaining carcass solution. We positioned the sluice box at an angle of 12° with running water flowing at variable rates, and then gradually fed material from the digest into the sluice to ensure maximum capture of bullet particles. All material that passed through the sluice box was collected in a container at the end of the sluice box. Bullet fragment material caught in the sluice box riffles were extracted with forceps or by using a 5 mL bulb pipette and placed in labeled 25 mL glass scintillation vials. All carcass material was processed twice through the sluice box. Material collected from the sluice box riffles was then oven dried at 50°C in uncapped scintillation vials. Once dry, vials were capped and stored at room temperature. We visually inspected all fragments to determine if they were Pb core material, jacket material, or combined Pb/jacket fragment material under a stereomicroscope , separating the three fragment types. Recovered fragments were individually weighed using a micro balance . Fragments that were too small to be weighed on their own because they could not be handled without breaking were counted and weighed together as an aggregate.In order to validate the accuracy of radiograph images for estimating bullet fragment numbers, distribution, and mass in ground squirrels, we extracted bullet fragments from a subset of carcasses (n = 78) to the same procedures, and then measured fragment masses pre and post tissue decomposition. Pb fragments subjected to this procedure lost an average 1.6% (± 0.2) of their initial mass indicating there was a minimal effect from this procedure. We also tested whether the enzyme solution would change the mass of Pb fragments by placing 15 Pb fragments of varying mass (3–28.6 mg) in the same solution and following the identical procedures as above. As with the initial decomposition step, the enzyme solution did not have an appreciable influence on the mass of Pb fragments (mean mass loss = 0.42% ± 0.18). Finally, we tested the efficacy of fragment capture through the sluice box by adding 10 Pb bullet fragments of varying masses (6–35 mg) each to the digests of two separate ground squirrel carcasses that had not been shot but were salvaged after being hit by a vehicle on an adjacent highway to the study area. The sluice box operator was blind to the number of fragments. In both trials, 100% of the fragments were recovered using the gold sluice box procedure.In order to test whether the digest and extraction procedure resulted in any appreciable digestion and loss of solid Pb particles, we subjected Pb fragments spanning a range of sizes . This approach allowed us to estimate bullet fragment numbers in carcasses that were only radiographed, and not digested. We excluded data from one ground squirrel carcass from all analyses because the bullet fragments in the radiograph were so highly clustered in the head region of the ground squirrel that they could not be counted or measured accurately by the Image J software. In this case, the Image J software identified 94 fragments in the carcass, but the extracted carcass actually contained 398 bullet fragments. Removing this data point decreased the value of the beta coefficient estimate, thus our estimates derived from this model were lower than if we had left this outlier in the model. We then tested whether the number of estimated fragments in carcasses differed by type of ammunition  using an analysis of covariance (ANCOVA) model. The estimated number of fragments was included as the response variable, with type of ammunition as the independent variable. We included ground squirrel mass as a covariate to control for variation in squirrel size differences because individuals ranged in mass from 54–521 g during shooting activities. We also included an interaction term for ammunition type × ground squirrel mass in order to test for slope differences among bullet types across the mass of shot ground squirrels. We tested for differences in the distribution of bullet fragments throughout shot ground squirrel carcasses by the type of ammunition  using an ANCOVA model. We included the percent distribution of fragments as the response variable, bullet type as the independent variable, carcass mass as a covariate to control for size differences among shot ground squirrel carcasses, and an interaction term for ammunition type × ground squirrel mass.2; 21). Using a subset of the carcasses where we had both fragment area (from radiographs) and bullet fragment mass (from digests), our linear regression model included fragment mass as the response variable and fragment area as the independent variable. We used the parameter estimates from this linear regression model to estimate the total mass of fragments across all ground squirrel carcasses . We then tested whether the estimated mass of Pb fragments in carcasses differed by ammunition type  using an ANCOVA model where the estimated mass of fragments was the response variable, and ammunition type was the independent variable. We included mass of ground squirrel carcasses as a covariate to control for carcass size differences and an interaction term for ammunition type × ground squirrel mass. Lastly, to determine if there was a relationship between the number of bullet fragments and mass of bullet fragments in shot ground squirrel carcasses we ran a correlation analysis where the log estimated fragment mass was the response variable and log estimated number of bullet fragments was the independent variable. We dropped interaction terms from all final models if they were non-significant (P>0.10) and natural log-transformed data to improve normality of residuals where appropriate.Similar to estimating the number of bullet fragments in shot ground squirrel carcasses above, we used a two-step statistical procedure to estimate the total mass (mg) of bullet fragments in shot ground squirrel carcasses. We first developed a linear regression model to estimate the total mass of bullet fragments in shot carcasses using the radiograph estimate of fragment area . We calculated estimates of daily ground squirrel consumption rates (g/day) for each species based upon nestling growth, food requirements, and diet selection. We then applied our estimates of Pb availability in shot ground squirrels (mg Pb/g ground squirrel) to model potential daily Pb exposure associated with the ground squirrel consumption for each species. Finally we applied those estimates to thresholds for toxicity effects  based on published data.Nestling growth rates (mass) were adjusted for each day in the nest based upon growth models derived from nest observations for each species –42. The Falco sparverius). Growth rates were impaired by Pb at a dosing rate of 125 mg Pb/kg body mass, resulting in lower growth rates than controls after 4 days of exposure and a cumulative consumption of approximately 6 mg of Pb or 306 mg/kg body mass [d is a day in a nest, mf = mass of food/day required in g, gsd = proportion of diet derived from ground squirrels, Pb = mass of Pb (mg)/g of ground squirrel, and mn = nestling mass on each day in the nest in kg. We defined the numbers of days in the nest post hatch as 70, 32, and 32 for golden eagles, red-tailed hawks, and Swainson’s hawks respectively.A threshold for physiological effects was estimated from a Pb dosing study on red-tailed hawks. Red-tailed hawk adults dosed with 0.82 mg Pb/kg body weight suffered a 55% reduction in delta-aminolevulinic acid dehydratase activity after seven days exposure; cumulative consumption was approximately 6.34 mg of Pb or 5.74 mg/kg body mass over that time period . Lead caody mass . A surviody mass . Forty pody mass ) or 2294ody mass ,32. PrevOur field conditions model did not allow for the generation of confidence intervals around the daily Pb exposure estimate because it was developed using individual data for each day . To provide a credible interval (95% CI) around our estimates of daily cumulative Pb exposure, we used a Monte Carlo simulation  to generGiven the large amount of variation in both the mass of Pb fragments in shot ground squirrels and the proportion of raptor nestling diet that is derived from ground squirrels we developed three additional Pb exposure models to provide insight into the varying effects of diet and the amount of Pb in carcasses on Pb exposure and risk to nestling raptors. (1) We estimated the daily cumulative Pb exposure associated with the maximum proportion diet of derived from ground squirrels  for golden eagles, red-tailed hawks, and Swainson’s hawks respectively ,29,43 whn = 81) and mixed unknown (n = 46) ammunition types. Known ammunition consisted of Hornady.17 HMR V-Max , Hornady .17 Mach II V-Max (n = 13), Hornady .17 Win Super Mag (n = 6), Remington Thunderbolt .22 solid , and CCI .22 hollow point  rounds  rounds . Among tF1,28 = 26.39, P < 0.0001, R2 = 0.96; F5,87 = 1.97, P = 0.09; F1,87 = 8.63, P = 0.004). The number of fragments increased by 76%, on average . However, after controlling for the overall alpha level in our Tukey’s pairwise tests we did not detect a significant difference in the distribution of bullet fragments between any of the bullet types (P >0.05). However, there was a general trend of the distribution of bullet fragments being greater in carcasses shot with .17 Super Mag bullets and random unknown bullet types . Because we did not digest dermal material, estimates of bullet fragment numbers and mass in dermal material are derived strictly from the radiographic methods; across all carcasses we observed fragments in dermal tissues in 57% (17 of 30) of the carcasses, but the dermal tissue contained only 3.8% of the total number of fragments and 6.1% of mass of fragments associated with a ground squirrel carcass.Radiograph-derived estimates of bullet fragment numbers were highly correlated with fragment number recovered from digested ground squirrels  and the largest range within an individual squirrel was 0.008 to 96.252 mg. Most fragments recovered were relatively small; 76% of the fragments weighed less than 12.5 mg and had a mean surface area of 6.05 mm2 ± 2.61 SE . After accounting for mass of ground squirrel carcasses, we found that bullet type influenced the log mass of bullet fragments retained in carcasses . Post hoc Tukey’s HSD tests found that ground squirrels shot with unknown and Super Mag .17 bullets retained more bullet fragment mass than .22 hollow point (P = 0.04) and .22 solid ammunition (P = 0.009) but similar amounts to .17 HMR and .17 Mach II  and .22 solid ammunition (P = 0.004) but similar amounts to .17 HMR, .17 Mach II, and .17 Super Mag bullets . The total mass of Pb fragments increased on average by 56% , increasing the potential for fragmentation and increased retention of fragments [The influence of target mass and bullet type has important implications for potential avian exposure to Pb fragments in ground squirrels and other related taxa. Similar to our findings, 87% of black-tailed prairie dog carcasses shot in Wyoming prairie dog colonies contained bullet fragments . Howeverragments  in the lragments  relativeβ = 1.77), indicating that radiographic methods fail to detect a proportionally great number of fragments as the number of fragments in a carcass increases  after six days of dosing [Across bullet types, less than 1% of the original bullet mass remained in ground squirrel carcasses as fragments. This differs considerably other estimates where over 7% of the original bullet mass remained in shot ground squirrel carcasses . As discf dosing . Thus, wAlectoris rufa) have shown that even low one-time doses of 109 mg resulted in substantial suppression of delta-aminolevulinic acid dehydratase production, and activity levels were only 34% of normal after 21 days [Estimating potential risk to nestlings from this form of Pb exposure is complicated by a number of ecological and toxicological factors such as diet, changing energetic requirements of growing nestlings, Pb assimilation rates, and differences in Pb toxicity among species. We used literature-derived estimates of effects of Pb exposure on nestling physiology, growth and survival to model potential cumulative Pb exposure throughout the nestling stage for golden eagles, red-tailed hawks, and Swainson’s hawks. Under all models, we found that all species exceeded the 50% reduction in delta-aminolevulinic acid dehydratase production threshold for the entire nestling stage. It is unclear what, if any, potential long term effects could occur from such a substantial reduction in delta-aminolevulinic acid dehydratase production; however, nestlings have been able to recover after reduction to less than 5% of normal without severe clinical hematological effects . However 21 days . Regardl 21 days . To our Sturnus vulgaris; [Anas platyrhynchos; [Reduced growth rates of nestlings can also occur in association with Pb exposure, although generally only at high doses ,58. Undeulgaris; ), and mahynchos; ) has beehynchos; ,62.Canis latrans) and a variety of rabbit species (Leporidae) are also shot as pests during the nesting season; these carcasses also could contain Pb fragments and would increase the overall Pb burden in nestling raptors [Alectoris chukar) are common in the diets of golden eagles and recent research has shown that nearly 10% of chukars’ gizzards contained Pb-based pellets [Experimental Pb exposure effects on raptor nestling survival have only been found to occur under relatively high Pb doses . Corres raptors ,64. Addi pellets . As an a pellets . In othe pellets ). If BelDespite these uncertainties, these findings illustrate the importance of more accurate estimates of raptor dietary reliance (and other avian scavengers) on shot carcasses, and strengthening these estimates would improve our knowledge of the risk of Pb-laced ground squirrels to nestling physiology, growth, and survival. For some species that rely heavily on already dead prey and in particular prey that have been shot , we Differences in the bullet fragment mass retained in carcasses among ammunition types may be a valuable factor for supporting management decisions. We found that ground squirrels shot with .17 Super Mag bullets retained over 28 times as much bullet fragment mass than either .22 solid or .22 hollow point bullets. Outreach and education efforts that communicate how bullet type influences potential scavenger exposure may be a useful approach for resource management. Reducing the number of carcasses on the landscape that contain bullet fragments may be another useful tool for reducing Pb exposure in avian scavengers ,64,71. H


Harvest of orchid tubers for salep production is widespread in southwestern Asia and the Balkans and constitutes a major conservation risk for wild orchid populations. Synanthropic habitats, such as graveyards, are important refuges for orchids and other organisms and could offer protection from salep harvesting because of their special cultural role. However, little is known about the occurrence and factors influencing harvesting of salep in graveyards. During field surveys of 474 graveyards throughout Turkey, we observed 333 graveyards with orchids, 311 graveyards with tuberous orchids, and salep harvest in 14 graveyards. Altogether, 530 individuals of 17 orchid species were collected, representing 9% of the individuals recorded. Harvesting intensity was relatively low, and populations were usually not wholly destroyed. However, some species were clearly more affected than others. Salep harvesting risk of orchid species was significantly associated with flowering time, with early‐flowering species being more affected. A marginally significant positive relationship between harvesting risk and species‐specific tuber size was also detected. Our data suggest that graveyards might offer some protection against salep harvesting in Turkey, but they also show that some orchid taxa are much more affected than others. Overall, our observations add more weight to the conservation value of these special habitats. We hypothesized that specific traits of tuberous orchids, such as conspicuousness, tuber size, and flowering phenology, might predict salep harvesting. Conspicuousness of different orchids can be very different as a consequence of variation in height of flowering shoot. Mean tuber size is also highly variable between species. Specific variability of tuber size causes substantial differences in average weight of dried tubers of salep originated in different regions of Turkey Sezik, , charact22.1Himantoglossum Spreng. s.l. (incl. Barlia Parl. and Comperia K. Koch), where we followed the nomenclature of Sramkó, Molnár V., Hawkins, and Bateman  regardless of their spatial dimension, position within settlements, or presence of religious facilities in 2 years: 300 graveyards have been evaluated in 2014  L. C. M. Rich. lasts from beginning of April (10) to mid‐July (20); hence, average flowering time of this species is 15.Average flowering time of orchids was obtained from flowering intervals published in Kreutz and Çolak . These dAnacamptis and Ophrys in vegetative stage which were unidentifiable at the species level we excluded from the analyses.Excavated specimens of 2.3To understand which species characteristics affect salep harvesting, we used data from 14 graveyards in which salep harvesting was observed. For these graveyards, the number of harvested and unharvested orchids (treated separately for each species) was used as a bivariate response in a binomial Generalized Linear Mixed Model (GLMM). Plant height, volume of new tubers, and the average flowering time were included as explanatory variables. We also included genus as a random factor in these models to take into account the fact that closely related species are more similar to each other than expected by chance . Explanatory variables were Box‐Cox transformed and standardized to optimize model fit. We removed nonsignificant predictors from the complete model in a stepwise manner  in order to get a minimal model which contained only significant predictors. All models were built in the R Statistical Environment . We observed salep harvesting activity only in 14 graveyards (4.5%) of 311 graveyards hosting tuberous orchids . The highest frequency of salep collection in graveyards (in eight cases) was observed in Muğla province. The number of excavated individuals in a graveyard varied from 6 to 172. Himantoglossum robertianum (Lois.) P. Delforge  and Anacamptis pyramidalis (152) were collected in the highest individual number, while Himantoglossum robertianum (six graveyards), Himantoglossum jankae Somlyay, Kreutz, and Óvári (3), Anacamptis pyramidalis (3), and Ophrys holoserica subsp. heterochila Renz and Taubenheim (3) were harvested in most graveyards. The number of harvested species in a graveyard varied from 1 to 5. In those graveyards where salep harvesting occurred, mean ± SD = 37.0 ± 20.8% of species were excavated. In two graveyards, each visible individual of H. robertianum was excavated. This species was collected in both studied years in the settlements of Kemer and Meşelik (Muğla); in 2014, 50% of the individuals were removed, while in 2015, 100% in Meşelik, and 94% in Kemer was removed (Table Ophrys subfusca subsp. blithopertha (Paulus & Gack) Kreutz as a new taxon for salep harvesting from the graveyard of Bayır .We found orchids in 208 of 300 visited graveyards in 2014  suggest that harvesting intensity might be relatively low in graveyards, thereby providing some degree of protection to orchids inhabiting these seminatural anthropogenic habitats.First, although salep harvesting is widespread in Turkey, we detected excavation of orchid tubers in only 14 graveyards (4.5%) of a sample of 311 graveyards hosting tuberous orchids. As graveyards are places that are relatively highly frequented by the local inhabitants, this low intensity likely indicates lower preference for harvesting at these sites, rather than reduced detection ability. Second, in graveyards where salep collection was observed, the proportion of harvested individuals was nearly always smaller than the full population size, indicating that not all individuals were harvested . Third, compared to the average salep harvesting activity required for an economically sufficient amount of profit . The high frequency of harvested Himantoglossum individuals (39.5%), and the low frequency of Orchis (0%) within our sample of harvested individuals, contrasts with a previous study from Iran . In a wider outlook, enhancing long‐established burial practices and traditional management of Turkish graveyards (including minimization of human intervention) may allow survival of natural vegetation.Due intensification of agriculture and rapidly changing land use, the role and significance of graveyards in conservation of living natural heritage of Turkey will probably grow. Therefore, following disposes promoting long‐term maintenance of viable and valuable populations of orchids  are essentially required: (1) supporting awareness of sociocultural and conservational importance of graveyards among Turkish public; (2) In subsidizing of long‐term survival of orchids  it is especially important to keep on at least on recent low level of salep harvesting in graveyards or even reducing its intensity; (3) our dataset may help in designation of most important graveyards in orchid conservation. National official protection or at least local council protection of graveyards hosting more than five orchid species can be recommended; (4) Development of field and/or tissue culture cultivation of orchids is highly recommended to satisfy of increasing (partly foreign) commercial salep demand in favor and saving wild orchid populations. Based on our results, the highest yield is expected from the cultivation of the largest tuberous orchids (especially The authors declare that they have no conflict of interest.AMV conceived and designed the study; AMV, TN, VL, KS, and AT collected the data; TN and JT performed the analyses; JT, TN, and AMV led the writing with contributions from all authors. Click here for additional data file.


Breast cancer is the second leading cause of cancer related deaths among women aged 40–55 in the United States and currently affects more than one in ten women worldwide. It is also one of the most diagnosed cancers in women both in wealthy and poor countries. Fortunately, the mortality rate from breast cancer has decreased in recent years due to increased emphasis on early detection and more effective treatments in White population. Although the mortality rates have declined in some ethnic populations, the overall cancer incidence among African American and Hispanic populations has continued to grow. The goal of the present review article was to highlight similarities and differences in breast cancer morbidity and mortality rates primarily among African American women compared to White women in the United States. To reach our goal, we conducted a search of articles in journals with a primary focus on minority health, and authors who had published articles on racial/ethnic disparity related to breast cancer patients. A systematic search of original research was conducted using MEDLINE, PUBMED and Google Scholar databases. We found that racial/ethnic disparities in breast cancer may be attributed to a large number of clinical and non-clinical risk factors including lack of medical coverage, barriers to early detection and screening, more advanced stage of disease at diagnosis among minorities, and unequal access to improvements in cancer treatment. Many African American women have frequent unknown or unstaged breast cancers than White women. These risk factors may explain the differences in breast cancer treatment and survival rate between African American women and White women. New strategies and approaches are needed to promote breast cancer prevention, improve survival rate, reduce breast cancer mortality, and ultimately improve the health outcomes of racial/ethnic minorities. Breast cancer is the second leading cause of cancer related deaths of women aged 40–55 in the United States and currently affects more than one in ten women worldwide . In the https://scholar.google.com/). Protocols and initiatives by government programs and other non-governmental agencies were extensively studied, and archives of media coverage on breast cancer. Additionally MEDLINE, and PUBMED (https://www.ncbi.nlm.nih.gov/pubmed) searches were conducted. We mostly examined populations of White women and African American women in the United States. An attempt was made to include all relevant references examining the nature and magnitude as well as the major risk factors associated with breast cancer disparity.A review of relevant scientific publications addressing breast cancer disparity and mortality rate primarily among African American women compared to White women in the United State was done using Google Scholar  and for the most common cancers, including cancers of the prostate, lung, colorectum, kidney, and pancreas. In contrast, Black females have a 6% lower overall incidence rate for all cancers combined and for many cancers, including lung, breast (only 3% lower), and uterine cancers . Cancer The specific causes of breast cancer still remain unknown, but we do know that a woman’s chances of developing breast cancer are related to a wide range of risk factors including woman’s age, genetic factors, family history, personal health history, and diet. Lifestyle and environmental factors are also known to be responsible for a considerable portion of cancer incidence worldwide .Age and sex are the most important risk factors to be considered in breast cancer incidence rates. Breast cancer incidence rates are higher among Blacks than Whites for women under age 45. The median age of diagnosis is 58 years for Black women compared with 62 years for White women . The oveFamily history has long been known to be a risk factor for breast cancer. For example, if members of a patient's family have had particular types of cancer, the patient will have an increased risk of developing breast cancer ,35.BRCA1 and BRCA2 are human genes that produce tumor suppressor proteins. These proteins help repair damaged DNA and, therefore, play a role in ensuring the stability of the cell’s genetic material. When either of these genes is mutated, altered, or does not function correctly, DNA damage may not be repaired properly. As a result, cells are more likely to develop additional genetic alterations that can lead to cancer development. The mutations of these two genes (BRCA1 and BRCA2) are thought to account for between 5% and 10% of all breast cancer cases [BRCA1 gene have an increased risk of developing breast cancer at an early age [er cases  and apprer cases . Female arly age .BRCA1 or BRCA2 mutation can be inherited from a person’s mother or father. Each child of a parent who carries a mutation in one of these genes has a 50% chance to inherit the mutation. Specific inherited mutations in BRCA1 and BRCA2 increase the risk of female breast and ovarian cancers, and they have been associated with increased risks in many cancer types. Together, BRCA1 and BRCA2 mutations account for about 20 to 25% of hereditary breast cancers [p53 at chromosome 17q13 [BRCA-1 at 17q21 [BRCA-2 at 13q12-13 [BRCA1 and BRCA2 genes, mutations of ATM, CDH1, CHEK2, PALB2, PTEN, STK11, TP53 (p53) genes can also increase the risk of breast cancer [A harmful  cancers  and abou cancers . In huma cancers . The Losme 17q13 ,42, BRCAat 17q21  and BRCA13q12-13 . The dif13q12-13 . Finding13q12-13 . In addit cancer . PossiblDespite most of the breast cancers are not of hereditary origin, lifestyle and environmental factors such as diet, obesity, smoking, alcohol consumption, infection diseases, and radiation have a profound influence on cancer development . AlthougBreast cancer has been one of the most extensively studied cancers in relation to physical activity, with more than 60 studies that have reported this association published in Asia, and Australia, Europe, and United State. Many studies in the United States and around the world have consistently found that physically active women have a lower risk of developing breast cancer compared to inactive women. However, the percentage of risk reduction achieved through physical exercise varies between 20% and 80% with the greatest risk reduction seen among those who are most active ,49. SimiA systematic review conducted by Albuquerque and his collaborators found that Mediterranean dietary pattern and diets rich of vegetables, fruit, fish, and soy are associated with a decreased risk of breast cancer . Other s2. A BMI of greater than 35 kg/m2 is associated with a doubling of the risk of breast cancer [A recent report has linked obesity to an increased risk of developing breast cancer . Other rt cancer . How diet cancer . In addiAlcohol consumption plays an important role in the development of breast cancer. For example, previous studies have suggested that increased breast cancer risk is associated with both the amount and the duration of alcohol consumption ,61. NagaA recent report suggests that half of breast cancer cases may be prevented if chemoprevention is applied in appropriate at-risk populations and the major modifiable risk factors such as achieving and maintaining a healthy weight, regular physical activity, and minimal alcohol intake are instituted . HoweverNational Cancer Institute (NCI)-supported research indicated that aggressive breast tumors are more common in younger African American and Hispanic women living in low socioeconomic status (SES) areas. This more aggressive form of breast cancer is less responsive to standard cancer treatments and is associated with lower survival rate . A 2000 A 2014 study by the Carolina Breast Cancer found racial differences in physical activity among breast cancer survivors revealed that African American, compared to Caucasian women, are significantly less likely to meet national physical activity guidelines after diagnosis . The lacRASSF1A modulates multiple apoptotic and cell cycle checkpoint pathways, restricting exit from G1 [RASSF1A in breast cancer among women aged <50 years was found to be methylated in 76% of cases of African American women versus 29% of cases of Caucasian women (p < 0.0001) [RARβ  and HIN-1 (p < 0.0001) are more often methylated in the tumors of African American women [Apoptosis resistance in breast cancer is the major cause for female cancer mortality in the wealthy countries. Understanding apoptosis in disease conditions is very important as it not only gives insights into the pathogenesis of a disease, but may also give clues on how the disease can be treated. In cancer, there is a loss of balance between cell division and cell death and cells that should have died, but did not receive the signals to do so. The problem can arise in any one step along the way of apoptosis. Several tumor suppresser genes have been identified, and mutations in these genes have been associated with a variety of neoplastic conditions. One example is the downregulation of p53, a tumor suppressor gene, which results in reduced apoptosis and enhanced tumor growth and development . Inactiv from G1 . RASSF1A 0.0001) . RARβ  = 1.25, p = 1.1 × 10−9), which was present at greater frequency in African Americans than in women of European ancestry, and was significantly associated with triple negative breast cancer in younger women  [Previous genome-wide association studies have identified a common risk variant at the  × 10−9) . A serie × 10−9) . This is × 10−9) . Other r × 10−9) . These f × 10−9) . Recent  × 10−9) .Collectively, this review paper provides considerable evidence that breast cancer mortality rate is significantly high among minority African American women population compared to White women in the United States ,94,95,96