The most important carbon sequestration factors included returning of crop straw (41–90%), chemical nitrogen fertiliser application (10–59%) and no-till farming practices (0–10%). Different factors dominated in different crop systems in different regions. To identity site-specific key factors and take countermeasures could significantly lower carbon footprint, e.g., ban straw burning in northeast and south China, stopping continuous flooding irrigation in wheat and rice production system.Due to the increasing environmental impact of food production, carbon footprint as an indicator can guide farmland management. This study established a method and estimated the carbon footprint of grain production in China based on life cycle analysis (LCA). The results showed that grain production has a high carbon footprint in 2013, i.e., 4052 kg ce/ha or 0.48 kg ce/kg for maize, 5455 kg ce/ha or 0.75 kg ce/kg for wheat and 11881 kg ce/ha or 1.60 kg ce/kg for rice. These footprints are higher than that of other countries, such as the United States, Canada and India. The most important factors governing carbon emissions were the application of nitrogen fertiliser (8–49%), straw burning (0–70%), energy consumption by machinery (6–40%), energy consumption for irrigation (0–44%) and CH So identifying the carbon footprint of a crop is an important component of sustainable agriculture2. A carbon footprint of crop is defined as the carbon emission or carbon consequences induced by production practices in growing season5. Advances in carbon footprint research have supported carbon trade in 14 developed countries including the United States, Japan and various countries in the European Union6. For instance, carbon labelling is used to inform consumers of the greenhouse gas emissions generated throughout the entire life cycle of a product or service , which can help consumers select low-carbon products and guide producers towards low-carbon production strategies7. With the increase in carbon emissions and the need for social-economic transformation in China, a carbon market and associated management techniques are urgently needed9, but there is insufficient research related to quantifying carbon footprints to support such a management system.The carbon balance can be an indicator of agricultural production efficiency  has been developed as a tool to estimate carbon emissions internationallyThe establishment of a new method to calculate the carbon footprint of different crops, regions and production techniques is necessary to promote coordinated economic and environmental development. This study focused on matching local factors to production practices in the current farming system.The survey results showed significant differences in the farming practices used in the eight major maize production zones in China as well as among farmers in the same zone Table . In geneThe production practices used in the four wheat-producing areas of China differed markedly from one another as well as between households in the same area. The rate of nitrogen fertiliser application generally exceeded 200 kg N/ha, but that in IV was far greater than in other regions. In contrast, manure inputs were rare in most regions, except Gansu and Shaanxi , while fuel consumption to operate machinery was generally higher than 600 kg/ha due to mechanisation throughout the entire production process. The proportion of straw returned to the field was especially high in region IV due to the high degree of mechanisation, moderate temperatures and the use of advanced technologies. In contrast, straw burning was significantly higher in Anhui and Jiangsu of II (double-cropping rice area in South China) because the wheat season is directly followed by the rice season. Therefore, straw is not able to decay before the next planting and thus would negatively affect rice production. Electricity consumption for wheat irrigation was generally higher in IV, primarily due to the dominance of well irrigation and the depth of the groundwater Table .Production practices also varied greatly across the 16 primary rice-producing areas of China as well as among farmers. The nitrogen fertiliser application rate exceeded 300 kg N/ha for single-crop rice fields in IV, and it exceeded 200 kg N/ha for single-crop rice in III. Manure application was rare in most regions with the exception of I and double-cropping rice areas. Fuel consumption for machinery operation was highest in single-crop rice fields in VII (Northeast China) because of the high degree of mechanisation. No-till techniques were not employed by any of the surveyed farmers . In VII, 70% of the straw was burned in the field due to the low temperature, but this proportion decreased to 45% in II and to 18% in I (triple-cropping area in South China) because the very tight rotation schedule resulted in the non-decomposed straw becoming hazardous for the subsequent rice crop. Significantly higher electricity consumption was found for single-crop rice fields in VII, which was likely due to the use of groundwater in this region while surface water or rainfall were the dominant water sources for rice production in the other regions.We developed a carbon footprint inventory for maize, wheat and rice in different production regions based on the site-specific farming practices Figs  and A4.FIn calculating the carbon footprint (emission minus soil carbon sequestration), we found that the three crops emitted more carbon than they sequestered. Of the three main crops in 2013, maize had the lowest carbon footprint, i.e., 4052 kg ce/ha of carbon per unit area or 0.48 kg ce/kg per unit yield. The carbon footprint of wheat was 5455 kg ce/ha per unit area or 0.75 kg ce/kg per unit yield, while rice had the highest carbon footprint, i.e., 11881 kg ce/ha per unit area or 1.60 kg ce/kg per unit yield.4 derived from paddy fields, 21% from straw burning, 14% from nitrogen fertiliser, 13% from fossil fuels for agricultural machinery and 4% from electricity consumption for irrigation. Wheat exhibited a high carbon emission value of 9119 kg ce/ha, of which 37% came from electricity consumption for irrigation, 28% from nitrogen fertilisers, 25% from fuel consumption by agricultural machinery and 6% from straw burning. Maize emitted 7900 kg ce/ha with 39% coming from nitrogen fertiliser, 20% from fuel consumption by agricultural machinery, 18% from electricity consumption for irrigation and 18% from straw burning.The factors contributing to these emissions varied markedly between crops. Rice yielded the greatest emissions (maximum: 15679 kg ce/ha) with 45% consisting of CHThere were also marked differences in the soil carbon sequestration under the three crops. In terms of carbon sequestration per unit area, maize ranked highest (3848 kg ce/ha) followed by rice (3798 kg ce/ha) and wheat (3664 kg ce/ha). For all three crops, carbon sequestration was primarily determined by straw treatment; for example, straw returning accounted for 96% of the carbon sequestration in rice paddies, while the remaining 4% was associated with the application of nitrogen fertiliser. Likewise, 80% of the carbon sequestration in maize fields resulted from straw returning while 15% was associated with the application of nitrogen fertiliser, and 5% stemmed from no-till practices. For wheat, 88% of the carbon sequestration was attributed to straw returning with the remaining 12% stemming from the application of nitrogen fertiliser.The carbon footprint of maize production varied substantially among the eight regions in China, ranging from 1192 kg ce/ha to 9282 kg ce/ha Figs  and A4 ) so thatThe second group included summer maize in VI (arid area in Northwest China), where the carbon per unit area reached 6276 kg ce/ha; this area also had the lowest carbon sequestration values (2762 kg ce/ha) and relatively high carbon emissions per unit area (9038 kg ce/ha). The carbon footprint of summer maize in the second group was similar to that in the first group and explained by similar factors, namely, high nitrogen fertiliser inputs, high fossil fuel consumption by agricultural machinery and low soil carbon sequestration efficiency. The primary difference between these two groups was that VI was rain-fed, so emissions from energy consumption for irrigation were zero.17, resulting in carbon emissions of 2557 kg ce/ha and 1575 kg ce/ha for the two crop types, respectively. However, the high carbon emissions were also the result of a high degree of mechanisation  that resulted in significant emissions from fossil fuel consumption.The third group included spring maize in VI, spring maize in VII, spring and autumn maize in I and summer maize in IV. Despite the high carbon sequestration values recorded in these areas, the carbon emissions per unit area were relatively high, which was partly due to straw burning. For example, grain yield and straw production were high for spring maize in VII, while straw decomposition was slow due to low temperatures. Meanwhile, there was a lack of industrial demand for straw, which resulted in straw burning-induced carbon emissions of 4331 kg ce/ha. In region I, straw burning was relatively popular because straw returning negatively affected rice cultivationThe lowest maize carbon footprint was found for summer maize in III (1192 kg ce/ha), where there were low carbon emissions per unit area (6796 kg ce/ha) and a relatively high degree of carbon sequestration per unit area (5604 kg ce/ha). In this area, straw burning was not prominent; emissions from energy consumption for irrigation were not high; and the carbon sequestration efficiency was relatively high.The grain yields per unit area were not linearly correlated with the carbon footprint per unit area, so the carbon footprints per unit grain yield showed slightly different trends Fig. . For exaThe total carbon emissions differed markedly across the four wheat production areas of China. The carbon footprint per unit area ranged from 502 kg ce/ha to 7513 kg ce/ha Figs  and A4 ) and wasWinter wheat in IV ranked second (5619 kg ce/ha), with carbon emissions per unit area of 10042 kg ce/ha and carbon sequestration of 4423 kg ce/ha, for the following reasons. (1) The reliance on deep groundwater for irrigation resulted in the attribution of 44% of the total emissions to electricity consumption. (2) Nitrogen fertiliser inputs were high, i.e., 254 kg N/ha, so carbon emissions accounted for 27% of the total emissions. (3) Mechanisation was also high, and the related fossil fuel consumption accounted for 24% of the total emissions.Winter wheat in VI ranked third (3999 kg ce/ha), and this area exhibited the lowest carbon sequestration levels per unit area (2651 kg ce/ha). However, the carbon emissions per unit area were not high (6650 kg ce/ha) because low temperatures and drought conditions reduced the efficiency of soil organic carbon transformation after straw returning compared to the other areas .Winter wheat in III ranked last in terms of the carbon footprint per unit area (502 kg ce/ha) due to the high soil carbon sequestration efficiency  as well as the low carbon emissions per unit area 5781 kg ce/ha). Carbon emissions were lower because straw burning was uncommon . The ni4 output that accounted for 61–73% of total emissions. Among the households, 23% and 20% were found to apply manure and continuous irrigation, respectively, which resulted in CH4 emission factors of up to 32.2 mg CH4/(m2 h) and 22 mg CH4/(m2 h), respectively. These values were markedly higher than those found for the other areas. In addition, straw burning was a problem to be solved for late rice in II, accounting for 20% of emissions.Regions in the first group exhibited the highest carbon footprint per unit area (>15000 kg ce/ha) primarily due to high emissions, i.e., 17568 kg ce/ha and 16542 kg ce/ha for late rice in II and I, respectively, which were attributed to high CH4 outputs was relatively high (32–63%). (2) Emissions from straw burning constituted 38% of the total emissions for single-crop rice in VII. (3) Emissions from nitrogen fertiliser reached 27% for single-crop rice in the paddy-upland rotation area. (4) High emissions from the operation of machinery and energy consumption for irrigation accounted for 20% and 17%, respectively, of the total carbon emissions from single-crop rice in VII.The high carbon emissions in the second group were due to the following reasons. (1) Except for single-cropping rice in VII, the proportion of emissions stemming from CH4 emission factor of rice paddies in the north was lower than that in the south due to climate and soil factors. On the other hand, the ratio of households who used intermittent irrigation without manure application was relatively high in this area (maximum: 46%). (2) These groups exhibited the highest soil carbon sequestration from straw returning and nitrogen fertiliser application (high mechanisation with 99% returned straw); carbon sequestration through the application of nitrogen fertiliser was linearly and positively correlated with the application rate. Although the carbon sequestration efficiency of nitrogen fertiliser in this area was low (0.53 kg C/kg N) compared to VI (1.74 kg C/kg N) and the southern areas  (1.53 kg C/kg N), nitrogen fertiliser was applied at much higher rates (304 kg N/ha) compared to the south (196 kg N/ha).The third group exhibited the lowest carbon emissions per unit area (11807 kg ce/ha) and the highest carbon sequestration per unit area (6305 kg ce/ha) for the following reasons. (1) The CHThe yield levels per unit area were not linearly correlated with the carbon emission levels of the study areas.et al.18 and 3000 kg ce/ha in Yan et al.19 (Table The carbon footprints (per unit area and per unit grain yield) obtained in this study is different than those of other studies. For example, the carbon footprint of wheat in our research is 5455 kg ce/ha, which is higher than 794 kg ce/ha in Cheng 19 Table . That maet al.19 used farmer survey data, it was limited by sample size and thus do not adequately represent potential regional variations . In this study, 3240 farmer survey data across seven grain production areas was used, which can clearly reflect the farmers real practices and regional variations. For example, the carbon footprint of winter wheat in Yan’s study is 3000 kg ce/ha, which is similar to our study in Henan province (3792 kg ce/ha) in region IV, because his study region for winter wheat is Henan. In fact, regions IV comprise 9 provinces  and the variation of carbon emission in region IV is very high (2397 kg ce/ha-30825 kg ce/ha), because farmers’ practices (i.e. fertiliser and irrigation) is differed markedly from one another.Although Yan 19 had typically adopted “half” life cycle analysis approaches and ignored emissions from manure processing and application, straw burning, and plastic film. These ignored emissions account for 2842 kg ce/ha, 1226 kg ce/ha and 3873 kg ce/ha of maize, wheat and rice respectively.In present study, the carbon footprint included emissions from field emissions and upstream inputs , thus accounting for the complete grain production life cycle. The emissions from upstream inputs account for 63%, 86% and 50% total emissions of maize, wheat and rice respectively. Yan’s studyet al.18). The statistical data supply general agriculture inputs, but cannot reflect critical regional farming practices, such as straw treatment and manure processing and application, which are critical factors for carbon footprints. For example, the straw burning was responsible for an average of 18% (as high as 70% for wheat in II), and manure processing and application was responsible for an average of 15% (as high as 70% for autumn maize in I). Therefore, the ignored emissions from manure and straw burning .When compared to other countries, the carbon footprint per unit yield of maize, wheat and rice in China were noticeably higher than those in the United States, Canada and India Table . They ad4 emissions were also enormous for rice, but intermittent irrigation can be enhanced as a form of mitigation, especially for late rice in II and I, where CH4 emission factors are high.Therefore, our results implied that the crop production practices in China have big potential to be optimized. For example, excess nitrogen fertiliser is currently being applied on each crop in national scale. The best nutrient management practices  could lower carbon footprint significantly. Moreover, the proportion of emissions from electricity consumption for irrigation was higher for wheat, which due to the use of flood irrigation. Water-saving irrigation techniques could be applied to minimise these carbon emissions. The proportion of emissions from straw burning was tremendous for rice and maize, especially in the northeast, so optimising the energy associated with straw regeneration and straw decomposition technologies is critical for reducing carbon emissions in this region. Additionally, CH2O emission factor in the present study. (1) The N2O emission factor is only able to simulate the rate of nitrogen fertiliser application (or nitrogen surplus); it does not reflect the influence of climate and soil factors26 and does not fully capture regional differences. (2) Interactions between different factors may affect the results; for example, the condition of soil water has a major impact on the emission of N2O from rice paddies. Additionally, there is a reciprocal relationship between the carbon emissions from CH4 and N2O28, while straw returning affects N2O emissions to a certain extent by altering nitrification and denitrification processes32. To date, research has failed to find consistent solutions to these issues33. (3) Manure-induced carbon emissions have a great impact on the overall carbon footprint, but there are large differences in the types and treatment of manure, which substantially alter the parameters of interest. In this study, our parameters were based on data from research on pig dung, which accounts for 48.2% of the total livestock excrement in China34. However, the application of cow dung is common in many areas, particularly in the north and northwest34. Furthermore, production processes associated with animal faeces and urine affect the nitrogen content of manure to a certain extent, thus influencing the emission of N2O and CH435. These examples indicate that the current parameters used to estimate carbon footprints are insufficiently detailed.Establishing a complete life cycle approach for carbon footprint estimation requires ever-evolving systematic research; in particular, more detailed site-specific parameters are required to understand the interactions among parameters. For example, we found the following uncertainties in the N2O and CH4 emissions but resulted in considerable consumption of energy from fossil fuels. Straw burning further increased the carbon emissions of these grain crops. It is recommended that intermittent irrigation be optimised to reduce the emission of CH4 from rice paddies and that the irrigation of wheat, nitrogen fertiliser management for maize and straw returning for rice and maize be optimised to reduce the carbon footprint of these crops.When the complete life cycles of the three primary grain crops produced in China were considered, the carbon emissions from the production of these crops were found to be much higher than the associated soil carbon sequestration, suggesting that they are net emission systems. High water and fertiliser inputs as well as the operation of machinery for agricultural purposes not only induced soil NThe carbon emissions from grain crop production consist of the following four components: (1) emissions from the upstream production and transport of agricultural inputs, (2) direct or indirect emissions induced by various agricultural management processes, (3) emissions from the burning of crop residues and (4) the soil carbon sequestration associated with various production methods  implemented during crop growth.According to LCA theory, the field production carbon footprint includes carbon emissions and carbon sequestration at two points in the production life cycle: the upstream links are associated with the creation of inputs used in agricultural production and processing  and the in-field emissions and sequestration are associated with field cultivation  Fig. .2, N2O and CH4), and sequestered carbon refers to the organic carbon in the soil , SOCSR is the carbon sequestered per unit area by grain crop production (kg ce/ha). More details are available in the supplementary (Appendix B).36, three types of nitrogen sources yield direct N2O emissions from crop cultivation: chemical nitrogen fertiliser applied in-field, manure applied in-field and crop straw inputs returned directly to the field, which retain residual nitrogen in the root stubble. In addition, three primary nitrogen sources result in indirect N2O emissions from crop cultivation: atmospheric nitrogen deposition due to ammonia volatilisation and nitrogen oxide emission from fertilised fields, nitrogen discharged into water bodies through leaching or runoff from manure-treated fields and atmospheric nitrogen deposition from nitrogen-containing active substances released from crop straw during field burning .According to the “Study on the Greenhouse Gas Inventories in China”2O emissions model described in Chen et al.9 was selected to represent the N2O emission factor for the application of chemical nitrogen fertiliser. This model is based on studies of nitrogen loss, including ammonia volatilisation, nitrate leaching and N2O emissions in the primary grain-producing areas of China before June 2012; it was published in Nature.In this study, the N2O emission factor for manure application was synthesised from the literature on field manure management in China and overseas prior to the beginning of 2014A total of 62 samples were obtained from 10 studies of direct N2O emissions46, and 211 samples were derived from 13 studies related to NH3 volatilisation-induced indirect N2O emissions56. More details are available in the supplementary (Appendix C1).The N4 emissions from rice paddies were considered in this study, and there were large differences due to a series of factors including soil type, climate conditions, rice variety, irrigation method and field management. To reflect these differences, we used the CH4-related carbon emission model created by Yan et al.57 for rice paddies in China based on 204 records collected from 23 sampling points. The independent variables included four categorical variables: rice-growing area (five rice-producing areas), manure application (with or without), watering method (intermittent irrigation or continuous flooding) and rice type .Only CHet al.58. The database linked to this model contains data from long-term, fixed-point experiments performed in the primary agricultural production areas of China; the large sample selected for this study was collected over more than three years and included results from 93 studies and 220 data pairs. More details are available in the supplementary. The soil carbon sequestration was calculated based on the specific farmers practices , and the SOC conversion factors .The carbon emission factor for straw burning used in this study was derived from the results discussed in Zhang According to LCA theory, carbon emissions produced during the upstream development of agricultural inputs required for grain crop production should be considered when determining the carbon footprint of a system. These production goods primarily include nitrogen fertiliser, phosphate fertiliser, potassium fertiliser, pesticides, agricultural film, agricultural machinery and manure composting. Moreover, modern agricultural production processes consume a large amount of energy in the form of the diesel fuel and electricity required to operate machinery and facilitate irrigation.et al.59 quantified greenhouse gas emissions from the production and transport of nitrogen fertiliser as well as the production processes, energy types and energy consumption efficiency of 230 nitrogen fertiliser manufacturers nationwide. In that study, the emission of greenhouse gases in the production of nitrogen fertiliser reached 6.9 tons of CO2 per ton of fertiliser; this factor was published in PNAS. Additionally, Chen et al.60 analysed data from China and used the LCA method to calculate carbon emission factors at the general manufacturing level for phosphate and potassium fertilisers.Based on the LCA method, Zhang 4 and N2O emissions from composting) were 177.75 and 189.14 kgce t−1 DS61, respectively. Based on nitrogen content values of 2.18% and 1.36%, the average carbon emission factor of manure  was estimated to be 0.223 kg ce/kg dry matter62.In the present study, the model assumes that all manure inputs are pig dung, and the carbon emission factors for sludge composting and pig dung composting  were selected following the method in Audsley.In this study, we divided the main grain crop-producing areas of China by climatic zone and cropping system. First, China was divided into the following three main areas by the annual cumulative temperature: a tropical/subtropical zone, a warm temperate zone and a middle temperate zone. These areas were further divided into the following seven subareas: I (triple-cropping area in South China), II (double-cropping rice area in South China), III (lower basin of the Yangtze River (paddy–upland rotation area)), IV (North China Plain), V (irrigated area in Northwest China), VI (arid area in Northwest China) and VII (single crop area in Northeast China). More details are available in the supplementary (Appendix D). A rural household survey of the typical crops in each area was carried out in 2014, and the following three main grain crops were determined: wheat, maize and rice. The survey samples included 3240 households from 42 counties in 11 provinces (regions), including Heilongjiang (including the reclamation area), Jilin, Hebei, Henan, Shandong, Shaanxi, Gansu, Anhui, Jiangsu, Hunan and Guangxi.The indicators assess in the survey questionnaire included the maximum plot yield, maximum plot area and growth period of crops; the rate of application of chemical fertiliser, manure, pesticides and film; the fuel consumed in the operation of machinery  and the electricity consumed for irrigation; the tillage (ploughing and minimum or no-till) and rice irrigation (intermittent irrigation or long-term flooding) practices; and the amount of straw burning and straw returning. More details are available in the supplementary (Appendix E).Supplementary information


Hamsters, infected subcutaneously with 104 bioluminescent leptospires, were analyzed by in vivo imaging, organ culture, and microscopy. The results showed that the luminescence from the leptospires spread through each hamster’s body sequentially. The luminescence was first detected at the injection site only, and finally spread to the central abdomen, in the liver area. Additionally, the luminescence observed in the adipose tissue was the earliest detectable compared with the other organs, indicating that the leptospires colonized the adipose tissue at the early stage of leptospirosis. Adipose tissue cultures of the leptospires became positive earlier than the blood cultures. Microscopic analysis revealed that the leptospires colonized the inner walls of the blood vessels in the adipose tissue. In conclusion, this is the first study to report that adipose tissue is an important colonization site for leptospires, as demonstrated by microscopy and culture analyses of adipose tissue in the hamster model of Weil’s disease.Leptospirosis is one of the most widespread zoonoses in the world, and its most severe form in humans, “Weil’s disease,” may lead to jaundice, hemorrhage, renal failure, pulmonary hemorrhage syndrome, and sometimes,fatal multiple organ failure. Although the mechanisms underlying jaundice in leptospirosis have been gradually unraveled, the pathophysiology and distribution of leptospires during the early stage of infection are not well understood. Therefore, we investigated the hamster leptospirosis model, which is the accepted animal model of human Weil’s disease, by using an  Leptospira interrogans, is one of the most widespread and potentially fatal zoonoses in the world [Rattus norvegicus) is the most important source of human infections [L. interrogans has more than 250 serovars [Leptospirosis, which is mainly caused by the pathogenic spirochete he world . Many wifections . Rat urifections . The earfections –6. BecauLeptospira culture from infected organs or quantitative polymerase chain reaction analysis of leptospiral DNA have been performed to detect Leptospira in the target organs of experimentally infected animals [in vivo imaging system (IVIS) to observe the distribution of leptospires in the whole bodies of infected animals.Leptospires infect humans percutaneously or across mucous membranes by penetrating abraded skin and/or mucous membrane , move im animals , 12. It Leptospira spp. were developed [Leptospira line (strain M1307) [SC189, which was modified to incorporate the luxCDABE cassette from Photorhabdus luminescens [flgB promoter from Borrelia burgdorferi [L. interrogans [Leptospira strains expressing either fluorescent GFP or mRFP proteins, or firefly luciferase [in vivo imaging studies were performed using zebrafish and mouse models of infection [L. interrogans mouse infection model provided a powerful way to study both acute and chronic leptospirosis and, notably, revealed that leptospires could escape antibody attack by residing in the kidneys [Nowadays, IVIS is frequently used for following stem cell and bacterial dynamics in live animals . To analeveloped , and ourn M1307) . Transpoinescens  and the gdorferi , was useerrogans . After tciferase , 22. To nfection . These snfection . It was Golden Syrian hamsters are highly susceptible to leptospiral infection and are considered to be good animal models for leptospirosis , 25. TheAnimal experiments were reviewed and approved by the Ethics Committee on Animal Experiments at the Faculty of Medical Sciences, Kyushu University (Permit Number: A26-057). The experiments were carried out under the conditions indicated in the Regulations for Animal Experiments of Kyushu University and law 105 and notification 6 of the Government of Japan.L. interrogans serovar Manilae strain M1307, derived from the parent strain L495 by insertion of the luxCDABE cassette from plasmid pSB406 obtained from Dr. S. Swift, University of Nottingham, UK [flgB constitutive promoter from Borrelia burgdorferi [In this study, we used gham, UK , 17. Thegdorferi , 19. Thegdorferi  with kan4) in 100 μl of Korthof’s medium or 100 μl of Korthof’s medium without leptospires was injected subcutaneously into the right inguinal region of hamsters from the experimental (n = 8) or uninfected control (n = 2) groups, respectively. Hamsters were observed for 14 days, and moribund animals were euthanized by cervical dislocation under anesthesia in accordance with the animal ethics guidelines. Excised organs from the infected hamsters were homogenized with disposable syringes and then cultured in Korthof’s medium containing a combination of five antimicrobials  [Leptospira growth in these cultures was monitored by dark-field microscopy every day for 7 days.For the infection experiments, 8-week-old female Golden Syrian hamsters were purchased from Japan SLC Inc. . M1307 (10ouracil)  at 30°C.4L. interrogans M1307 or injected with Korthof’s medium only. The infected hamsters were anesthetized with isoflurane by inhalation and placed in an acrylic plastic box. Bioluminescent images of the hamsters were then acquired using an IVIS . Data acquisition and analysis were performed using Living Image 4.4 software . Quantification was performed using a region of interest (ROI) defined manually , and the results were expressed as photons (P) per second (s). All the acquired images and defined ROIs are shown in Bioluminescent imaging by IVIS was conducted as described previously . For whoex vivo analyses were performed to obtain more precise bioluminescent images. A total of 21 hamsters were infected with 104L. interrogans M1307. The hamsters (n = 3) were imaged with the IVIS  and washed with PBS. The cut skin was immersed in 10–20% sucrose overnight, placed in isopentane, and frozen on a metal plate chilled with liquid nitrogen. Each piece of skin was then sliced to a thickness of 60 μm using a cryotome  and then mounted on a microscopic slide. The slices were blocked with 3% bovine serum albumin in PBS (blocking buffer), washed with PBS, and incubated with a rabbit anti-serovar Manilae antiserum (1:200) at 4°C overnight . After wAfter perfusion and fixation of the whole hamster bodies with a mixture of 2.5% paraformaldehyde and 2% glutaraldehyde in PBS, the adipose tissues were cut into 2×2×10 mm pieces and kept in 2% glutaraldehyde at 4°C. The pieces were fixed with 4% osmium tetroxide for 45 min and then stained with uranyl acetate for 1 h, after which they were embedded in Epon and sliced with an ultramicrotome. The samples were observed with an HT7700 transmission electron microscope .t test  was used to compare two groups at the same time point. Values are expressed as mean ± standard error of the mean (SEM). A p value <0.05 was considered significant. p values: *p<0.05Statistical analysis was performed using JMP Pro 11.0.0 (SAS Institute Inc.). The unpaired 4L. interrogans luminescent strain M1307. Seven of the eight infected hamsters (87.5%) required euthanasia by day 9 post-infection ; Phase 2, luminescence was detected at the injection site only (days 4 to 5); Phase 3, luminescence was detected in the injection area and the center of the abdomen (day 6 to 7); and Phase 4, luminescence was detected in the greater part of the body (day 7 to 8).ex vivo examinations to identify which organs were colonized by Leptospira cells. Therefore, we acquired five types of images during each phase: i) subcutaneous tissue after skin incision; ii) organs after laparotomy; and iii) to v) organs ex vivo   . There wLeptospira. We therefore cultured adipose tissue from the infected animals as well as from their blood, livers, and kidneys  of the leptospires in the adipose tissue, confocal laser scanning microscopy was performed. These images show that Leptospira colonized along the inner walls of the blood vessels in the adipose tissues .To confirm the presence of e tissue . In the r dermis . Interes tissues . To conf vessels  and coexWe performed this study to determine where in the body leptospires colonize and grow at the early phase of an infection. Although the clinical manifestations of leptospirosis are relatively mild, they may eventually worsen and become lethal if appropriate therapy is not initiated. It is already known that when pathogenic leptospires infect percutaneously, they immediately move into the bloodstream and persist there during the leptospiremic phase of the illness , 25. OurIVIS is a useful tool for long-term observation of the whole bodies of living animals infected with pathogens . In our luxCDABE is molecular oxygen [We found that the luminescence faded immediately after the hamsters were euthanized though the luminescence could be detected immediately before death. Animals still alive while undergoing IVIS will of course take in oxygen with the anesthetic gas. In the experiment for r oxygen , this woIn this study, we combined IVIS Figs  and 2 anL. interrogans, a pathogenic species, shows chemotactic behavior towards 100 mM palmitic acid 16 times stronger than that recorded for L. biflexa, a nonpathogenic species. We also tested whether leptospires display chemotaxis towards oleic acid using a microscopic agar-drop assay [Our findings are consistent with the fact that the essential carbon source for leptospiral growth is long-chain fatty acids . Some stop assay , but no op assay .p = 0.0502 and 0.1052 respectively, p = 0.0760, 0.0896 and 0.1052 respectively, In the animal experiments we have described, only one infected hamster survived the infection . In thisIntriguingly, in microscopic observations of the adipose tissue blood vessels in the infected hamsters, leptospires were detected only in a hazy structure with the red blood cells . This haLeptospira cells into the bloodstream immediately after infection, this study shows a component of the pathological mechanisms of leptospiral infection during the early step whereby, in the hamster infection model, leptospires colonize the blood vessel inner walls of the adipose tissue. This is the first demonstration of the significant involvement played by the adipose tissues during the early stage of leptospiral infection.Besides adding to conventional knowledge about the transition of S1 FigCircles represent ROIs in the injection site (blue) or the abdominal center (red).(TIF)Click here for additional data file.S2 Figex vivo analyses each day.The 3 hamsters were performed skin incision, laparotomy and subsequent (TIF)Click here for additional data file.S3 Fig(TIF)Click here for additional data file.


Microbial life can leave various traces (or biosignatures) in rocks, including biotic alteration textures, biominerals, enrichments of certain elements, organic molecules, or remnants of DNA. In basalt glass from the ocean floor, microbial alteration textures as well as chemical and isotopic biosignatures have been used to trace microbial activity. However, little is known about the relationship between the physical and chemical nature of the habitat and the prevalent types of biosignatures. Here, we report and compare strongly variable biosignatures from two different oceanic study sites. We analyzed rock samples for their textural biosignatures and associated organic molecules. The biosignatures from the 8 Ma North Pond Region, which represents young, well-oxygenated, and hydrologically active crust, are characterized by little textural diversity. The organic matter associated with those textures shows evidence for the occurrence of remnants of complex biomolecules like proteins. Comparably the biosignatures from the older Louisville Seamount Trail (~70 Ma) are more texturally diverse, but associated with organic molecules that are more degraded. The Louisville Seamount has less fresh glass left and decreased permeability, which metabolic pathways may dominate that only leave molecular biosignatures without textural evidence of glass alteration. We propose that diverse biosignatures in oceanic crust may form during different stages of crustal evolution. Subseafloor basalts are one of the largest and most intriguing habitats on Earth. Oceans cover two-thirds of Earth’s surface and the upper 500 m of the underlying igneous crust represents Earth’s largest aquifer .2+ and Mn2+ from the glass for chemolithoautotrophic energy gain with electron acceptors like O2, sulfates, or CO2 from seawater [Evidence for microbial life in the basaltic ocean crust has been found in a number of different areas and settings , which are two distinctly different sites in terms of their crustal evolution. Age of ocean crust plays a crucial role, as it dictates factors such as the crust hydrological state, the extent of fluid-rock interactions, the abundance and type of secondary minerals, and the magnitude of geochemical change. These different factors influence the microbial communities and their potential in altering ocean crust along with the associated biosignatures.We sampled basalt glass from drill cores with a focus on areas of secondary mineral formation . Furthermore, we thoroughly documented the types of tubular alteration textures in the volcanic glass and assessed their spatial relationship to alteration fronts. We then used Scanning Electron Microscopy (SEM) and Fourier Transform InfraRed (FTIR) microscopy to locate and characterize the organic matter associated with basalt glass alteration.We analyzed glassy rock samples from drill cores U1372A and U1376A from Integrated Ocean Drilling Program (IODP) Expedition 330  to the LThe Louisville Seamount trail  is a 75 The North Pond region is a sediment pond of 8×15 km in size, located on the western flank of the Mid-Atlantic Ridge in 8 Ma basement. The sediment pond of up to ~200 m thickness is surrounded by steep rift mountains that act as recharge and discharge zones of circulating seawater. Heat flow data indicate rapid SE-NW directed movement of large amounts of seawater at temperatures of 2–15°C . This opDrilling derived contamination was assessed with the use of fluorescent microspheres of 0.52 μm diameter were added to the drill fluid as a particulate tracers mimicing microbial cells at both sites. While microsphere were present in the drill cores of both IODP expeditions ;32, we dTwo types of samples were analyzed for the work presented here:A set of 18 thin sections of 30 μm thickness (11 from Louisville and 7 from North Pond), each one of which has > 100 individual tubular alteration textures. These thin sections were not used in the search for organic molecules due to contamination issues in the thin section preparation involving organic resin and glue.Pieces of the same samples were prepared for the identification of organic molecules. Samples were cleared from external parts and then cut into thin slices, so that all surfaces were fresh cuts. We used a saw blade cleaned with 5% sodium hypochlorite and rinsed with sterilized ultrapure water. Afterwards the slices were polished by hand on silicon carbon abrasive discs with absolute ethanol to a thickness of approximately 100 μm and analyzed via scanning electron microscopy and Fourier Transformation Infrared microscopy. At all stages the rocks were only handled with clean sterile nitrile gloves. This procedure should limit contamination with organic and post sampling colonization.For 18 of 27 total thin sections of 30μm thickness, we characterized 100 randomly chosen individual microtunnels under a transmission light microscope according to Fisk & McLoughlin’s Atlas of alteration textures in volcanic glass [We used a Zeiss Supra 55 VP Field Emission® SEM coupled to energy dispersive X-ray spectrometry (EDS) at the Université Pierre et Marie Curie . Images were collected with a beam acceleration voltage of 15 kV using a backscattered electron (BSE) detector (AsB). EDS analyses and chemical mapping were carried out with a Bruker Xflash Quad® spectrometer using the microanalysis system Quantax. All the samples analyzed by SEM were prepared free of epoxy resin to avoid organic contamination (see “Sample preparation” section) and Au-coated. The samples were treated in an ultrasonic bath to remove particulates from all surfaces.-1 by mapping over areas of several mm2 with individual spot sizes of 30×30 μm and steps of 30 μm. Each spectrum corresponded to 128 summed scans collected with a spectral resolution of 4 cm-1. The raw data was converted into–ASCII files with the Omnic Atlus® imaging software by Thermo ScientificTM and afterwards normalized and background-reduced using the ChemoSpec package for the software R [FTIR measurements were carried out at the Institut de Physique du Globe de Paris, France with a Thermo Scientific iN10 MX® (Ever-Glo infrared source) equipped with a ×15 objective (NA = 0.7) and a liquid nitrogen cooled MCT-A detector in transmission and reflection mode. We acquired spectra in the range 4000–710 cmftware R .The vibrational bands summarized in 3/2 value was first introduced by Lin and Ritz [3/CH2 absorbance ratio of the respective asymmetric stretching band heights. The R3/2 value hence reflects the degree of branching and chain length of side groups associated with aliphatic chains. Low R3/2 values suggest that mainly long or less-branched aliphatic carbon chains as one would expect for bacterial or eukaryotic membrane phospholipids. Conversely, archaeal lipids lack true fatty acid side chains and hence yield higher R3/2 values. Igisu et al. [3/2 values . Individual bacterial cell components however, plot significantly higher (proteins) or slightly (membranes) to significantly (lipids) lower compared to whole cell R3/2 values [3/2 value to compare the fossilized organic matter found within the glassy basalts from the North Pond region and the Louisville Seamounts and unravel their connection to tubular alteration textures.The Rand Ritz  and Marsand Ritz  in orderu et al.  recently2 values . We usedThe tubular alteration textures as identified by transmitted light microscopy  were claThe tubules  in the North Pond samples were almost exclusively < 2 μm in width (98%) and there were more short tubes (59%), than long tubes (41%). Curvature was wide spread, with 62% of the tubes being curvilinear, 12% being kinked, and 26% convoluted. There were no knotted tubes identified in the North Pond samples. Variation in width was very low, the majority being uniform in width (82%); only 18% of the tubes were tapered, and there was no variation at mid-section and terminus, nor any branching or tunnel contents.Conversely, the Louisville tubular alteration textures (N = 1100) on the other hand were much more diverse in every respect. Short and thin tubules were the most abundant ones (47%), similar to North Pond, however long and thin (19%), short and wide (18%), and long and wide tubes (16%) were commonly found as well throughout all the samples. The variation in curvature was less pronounced, but all types were present, with 84% being curvilinear, 13% kinked, 1% knotted, and 2% convoluted. Variations in width were very common. Although most of the tubes were uniform in width, similarly to the North Pond textures , there was some variations at mid-section and terminus, with 4% engorged, 1% bumpy, 5% annulated, and 3% mushroom textured. Unlike in the North Pond samples, branching occurred in the Louisville tubes, although 88% of the tubes were not branched. 8% showed simple branching, whereas complex branching was less prominent . Furthermore only 73% of the tubules were empty, 14% were filled with dark contents, 6% with ovoid bodies, 6% showed septae, and 1% were filled with filaments. Ovoid bodies and septae were exclusively found in long and wide tubules.We found the presence organic compounds both in samples from the North Pond area and the Louisville Seamounts. However, they were distinctly different in their location and composition.-1, 2920cm-1, 2850 cm-1, and 1460–1470 cm-1 representing the asymmetrical stretch of CH3, the asymmetrical stretch of CH2, the symmetrical stretch of CH2, along with the asymmetrical bend of CH2/CH3, and the scissoring of CH2, respectively). Furthermore, the spectra showed evidence for the presence of esters/fatty acids and/or polysaccharides (absorption band at 1743 cm-1 representing the C = O stretching mode) and proteins . Associated R3/2 values ranged from 0.45 to 1.20.The North Pond samples contained only little carbon-rich phases, which appeared as tiny aggregates exclusively restricted to the palagonite/glass interface . SEM-EDS-1, 2920cm-1, and 2850 cm-1 representing the asymmetrical stretch of CH3 and CH2 as well as the symmetrical stretch of CH2, respectively). These long chain hydrocarbons were widely spread in the samples and occurred both in palagonites and vein filling zeolites or SiO2  area of ridge flank, which has undergone and is still undergoing oxidative alteration with temperatures at or below 20°C. Here, tubular alteration textures are rare, thin, and show little morphological diversity, with only small variations in width, curvature, and branching . The lowmponents . As FTIR3/2 values in the range of the ones predicted from archaeal lipids [2 (The Louisville samples are much older (~70 Ma ) and havl lipids ; Fig 8) ipids [2 . This in2) seal fractures and fluid flow ceases. When basalt ages, the organic matter is found within the palagonites and close to the sealing SiO2, which indicated to us that it is not directly associated with the formation of tubular alteration textures. It could either be added to the rocks from circulating seawater, which may drop dissolved organic carbon in the basement upon water-rock interactions  near the palagonite/fresh glass interface in young basalts where the glass likely serves as solid energetic substrate (North Pond) and (ii) within palagonites close to fractures in older basalts (Louisville) where hydrogen might be produced by water radiolysis.The relationship between alteration  and biosignatures is further illustrated in ns (e.g. ), perhapns , we suggest that the biosignatures observed within the subseafloor basalts have not been formed by the present day microbial community and have rather been produced earlier in the crustal evolution. However, our data does not suggest any apparent relation between rock alteration and the presence of organic matter. The two sets of observations may be reconciled either if the rocks presently merely provide a shelter for microbes that derive their metabolic energy from percolating seawater, as proposed by Templeton et al. [As the Rn et al.  for alteIn any case, this is an interesting observation as it implies that the community structure of the basalt hosted subseafloor biosphere changes with changing conditions during crustal evolution.As the geochemical environment changes significantly during crustal evolution (e.g. oxic in during early stages to suboxic when buried under sediments), basalts might host different communities during different stages of crustal aging.2.Our findings support the idea that microbial life in the young basaltic aquifer at North Pond, which alters oxidatively, is energetically driven by glass oxidation processes. The reduced form of iron is found at the glass interface, where the organic matter in the North Pond samples is also preserved. In the much older basaltic crust of Louisville that has undergone a considerably more complex alteration history, the textural biosignatures textures are also more complex and diverse. In the latest stages, life seems to be hosted in voids within the rock, such that basalt acts as a solid support to grow on. The organic matter is then preserved when these voids are filled by secondary phases like calcite, zeolite, or SiO


Based on the Beijing Eye Study 2011, a detailed ophthalmic examination was performed including spectral-domain optical coherence tomography (SD-OCT) with enhanced depth imaging for measurement of subfoveal choroidal thickness (SFCT) and relative height of posterior scleral staphyloma. OCT images were obtained in 103 highly myopic eyes (≤−6.00 diopters) and 227 normal eyes. The mean SFCT in highly myopic eyes was 110.6 ± 85.2 μm . The SFCT of high myopia without posterior scleral staphyloma(55 eyes) was 157.79 ± 85.18 μm, which was significantly greater than that (54.94 ± 49.96 μm) of high myopia with posterior scleral staphyloma (48 eyes) (P < 0.001). In multivariate analysis, posterior scleral staphyloma was the most important factor of choroidal thinning in high myopia , then age , axial length  and gender . The SFCT in highly myopic eyes is very thin and undergoes further thinning with increasing age and axial length (refractive error). Posterior staphyloma formation was a key factor in choroidal thinning in highly myopic eyes and to be a good indicator for risk management of choroidal thinning. Abnormalities of the choroid may play a role in the pathogenesis of myopic degeneration. Generally, high myopia is related to axial extension of the eyeball, which can cause a variety of abnormalities in fundus, such as lacquer cracks in the Bruch membrane, posterior staphyloma, choroidal neovascularization (CNV), and chorioretinal atrophy6. Based on these findings, we defined the pathologic myopia as myopic refractive error equal or more than 6 diopters (D); an axial length more than 26.5 mm; or posterior pole abnormalities with high refractive errors9. Histologic studies suggested that choroid was thinner in high myopia, for choriocapillaris lost in some areas10. The choroid circulation receives about 95% of blood from the ophthalmic artery, and provides oxygen and nutrition to the outer retinal layers. Therefore, the choroidal circulation may play an important role in the retinal dysfunction and vision loss. Posterior staphyloma formation is significantly associated with macular hole, retinal detachment and myopic foveoschisis, so it is recognized as a hallmark of high myopia.In Asia, Europe, and some races in the United States, a major cause of visual impairment is high myopiain vivo11. Using this method, several studies have reported the SFCT in normal subjects and patients with abnormalities in fundus14, including high myopia16. However, these studies were hospital-based studies which probably may not avoid a selection bias. Therefore, based on a population-based study, we attempted to study the SFCT in highly myopic eyes with EDI SD-OCT and to identify the risk factors for development of choroidal thinning with high myopia.Enhanced depth imaging spectral-domain optical coherence tomography (EDI SD-OCT) has supported a way to detect choroidal thickness 18. According to the Declaration of Helsinki, The Medical Ethics Committee of the Beijing Tongren Hospital had approved the study protocol and all participants had given informed consent. The Beijing Eye Study included 4439 subjects out of 5324 subjects invited to participate, corresponding to a response rate of 83.4% at the first time of the study in 2001. Participants were invited for the second five-year follow-up examination in 2011, at which time the SD-OCT was first performed on the participants. Of the 4403 subjects who agreed to participate at baseline, 3468[1963 (56.6%) women] agreed to continue to participate in 2011, corresponding to a response rate of 78.8%. The mean age of all participants was 64.6 ± 9.8 years .The Beijing Eye Study is a population-based prospective cohort study with an age of 50 + years in Northern China. The study has been described in detailBased on the data of Beijing Eye Study 2011, we conducted a cross-sectional study of highly myopic patients . Out of the total 3468 subjects, 135 (1.95%) eyes  of 93 (2.68%) subjects  were regarded as highly myopic eyes. 103 eyes of 78 (83.9%) participants (50 (64.1%) women) have sufficient quality OCT images for examination. For 15 (16.1%) people, OCT images were not taken or images could not be assessed owing to the serious opacity of refractive media. Compared with the highly myopic subjects with OCT images, the group without images was significantly older  but did not vary significantly in gender (P = 0.85) and refractive error (P = 0.67).As control, 227 apparently normal eyes of 227 people were randomly selected from the base, with age and gender matched in proportion to the population. The exclusion criteria included amblyopia, glaucoma, uveitis, diabetic retinopathy, retinal vascular abnormalities, laser treatment, drusen, severe chorioretinal atrophy, macular scarring, refractive surgery, intraocular surgery (such as cataract extraction and vitrectomy), which were found by fundus photographs and questionnaire. Besides, patients with severe cataract, moderate to severe chorioretinal atrophy and pathologic structures were excluded resulting from SD-OCT.20. The measurements were performed by two examiners  independently and the interobserver reproducibility was proved high . The relative heights of scleral staphyloma at the posterior pole were measured, and the method is as follows: 31 sections were scanned to cover 8 mm * 8mm areas of macular fovea, which means almost all of the posterior pole was covered, and each section was the average image of 20 times scanning images. Vertical line was made 2.5 mm away from macular fovea to intersect with the horizontal line, and the vertical distance was measured as the relative height of scleral staphyloma. The relative heights of scleral staphyloma was measured in nasal, temporal, superior and inferior quadrants, and the four points 2.5 mm away from the fovea of every quadrants were measured  using a spectral domain optical coherence tomography  with enhanced depth imaging (EDI) modality. SFCT was the vertical distance from the hyperreflective line of the Bruch’s membrane to the hyperreflective line of the inner surface of the sclera . SFCT was described by the mean values (presented as mean ± SD). Categorical variables were assessed individually with the chi-square test, and when the expectancy of less than 5, the Fisher’s exact test was used. Continued data were analyzed using an independent sampled t-test. Simple linear regression was calculated for variations in SFCT relative to systemic and ocular risk factors in highly myopic eyes. Multiple linear regression was used to evaluate the explanatory variables with regard to the dependent variable. Only the right eye of the participant was assessed in linear regression analysis. 95% Confidence intervals (CI) were presented. All P-values were 2-sided and were considered statistically significant when < 0.05.et al.Highly myopic group contained 103 eyes of 78 patients and control group contained 227 eyes of 227 participates. Table Stepwise multiple linear regression analysis confirmed that posterior scleral staphyloma was the most important factor of SFCT in high myopia , then age , axial length  and gender . Table In patients with high myopia, the relative heights of posterior scleral staphyloma 250 μm away from the macular foveola in four quadrants were measured. Analysis of variance and intergroup comparison showed that the average height of the superior (277.8 ± 232.3 μm) and temporal (246.6 ± 156.8 μm) posterior scleral staphyloma was larger (P = 0.52), followed by the nasal  and the inferior quadrant   by using multivariate regression analysis. Then, the statistical comparison of the systemic and ocular features of highly myopia with and without staphyloma showed that the group without staphyloma had obviously better BCVA, less myopia, shorter axial length, thicker subfoveal retina, and even taller and fatter than those in group with staphyloma. For the SFCT, the group without staphyloma(157.79 ± 85.18) was nearly 2 times more than staphyloma group (54.94 ± 49.96 μm) (P < 0.001). Therefore, the posterior scleral staphyloma can serve as a hallmark of high myopia. Previous researches pointed out that the formation of the posterior scleral staphyloma was closely related to the occurrence of some posterior retinopathy, including macular hole, retinal detachment and myopic retinoschisis19. Thus, we concluded that the formation and severity of posterior scleral staphyloma might be affected by forces generated by axial elongation and vitreous liquefaction and detachment in highly myopic eyes.In this study, the relative heights of posterior scleral staphyloma 250 μm from the macular foveola in four quadrants of patients with high myopia were further measured. Final results showed that the average height of superior and temporal posterior scleral staphyloma was bigger, followed by the nasal and the lowest was inferior. The reason for the obvious differences of the relative height of posterior scleral staphyloma among different quadrants is unclear, which may relate to axial elongation, vitreous traction as well as the effect and the direction of gravity. In addition, this study found that the distribution law of the relative height of posterior scleral staphyloma was the same with that of the height of incomplete posterior vitreous detachment in each quadrant. Another study of our research team showed that, for incomplete posterior vitreous detachment, the highest quadrant was superior and temporal, followed by the nasal and the lowest was inferior19. When nasal posterior scleral staphyloma in high myopia forms, the traction of the choroid and bilateral retina in the areas of macular fovea and optic disc concentrates and the resultant force is relatively big to subsequently cause abnormal choroidal structure and function and even atrophy due to its twisting. When posterior scleral staphyloma in other quadrants formed, the impact of nasal posterior scleral staphyloma on SFCT does not appear due to the absence of close vitreous adhesion to the optic disc, which causes relatively small disturbance to the corresponding choroidal areas.Univariate regression analysis of the relative heights of posterior scleral staphyloma revealed that the relative height of nasal posterior scleral staphyloma negatively correlated with SFCT, nasal choroidal thickness and diopter, and it marginally, positively correlated with the axial length. Further multivariate regression analysis found that the relative height of nasal posterior scleral staphyloma still significantly negatively correlated with diopter and SFCT, but it had no significant correlation with the nasal choroidal thickness and axial length. The results showed that the degree of posterior scleral staphyloma, especially posterior scleral staphyloma in macular nasal quadrant may be the most important factors for macular choroidal thinning in high myopia. However, univariate regression analysis indicated that the relative height of temporal, superior and inferior posterior scleral staphyloma had no significant correlation with SFCT and choroidal thickness of corresponding quadrants. Only the reason for the significantly negative correlation of the relative height of nasal posterior scleral staphyloma with SFCT is still uncertain. Possible explanation for this was that area of 250 μm away from macular nasal side  lies between the fovea and the optic disc and the two regions are closely adhesive with vitreous body24. However, the axial length of patients with pathological myopia was also in the normal range, for example 24.5 mm, and the axial length of emmetropia can increase to 27.3 mm9. The threshold of axial length causing myopic maculopathy is still unclear. Eye elongation may induce uneven choroidal thinning, which is thinner in the foveal area with lower perfusion. We proposed that in place of age and axial length, the posterior scleral staphyloma may become to the most important factor of subfoveal choroidal thickness in high myopia  was markedly thinner than normal eyes (263.0 ± 92.9 μm). It was most associated with posterior staphyloma, followed by age, axial length and gender. These observations may reveal that posterior staphyloma was an independent factor of choroidal thinning in highly myopic eyes, and abnormalities of the choroid may play a role in the pathogenesis of myopic degeneration.


Based on these data, this trial assessed the efficacy and safety of the combination of paclitaxel and valproic acid for the treatment of ATC. This was a randomized, controlled phase II/III trial, performed on 25 ATC patients across 5 centers in northwest Italy. The experimental arm received the combination of paclitaxel (80 mg/m2/weekly) and valproic acid ; the control arm received paclitaxel alone. Overall survival and disease progression, evaluated in terms of progression-free survival, were the primary outcomes. The secondary outcome was the pharmacokinetics of paclitaxel. The coadministration of valproic acid did not influence the pharmacokinetics of paclitaxel. Neither median survival nor median time to progression was statistically different in the two arms. Median survival of operated-on patients was significantly better than that of patients who were not operated on. The present trial demonstrates that the addition of valproic acid to paclitaxel has no effect on overall survival and disease progression of ATC patients. This trial is registered with EudraCT 2008-005221-11. Anaplastic thyroid cancer (ATC) has a median survival less than 5 months and, to date, no effective therapy exists. Taxanes have recently been stated as the main drug treatment for ATC, and the histone deacetylase inhibitor valproic acid efficiently potentiates the effects of paclitaxel Although rare , ATC accounts for 14–50% of total mortality for thyroid cancer, with survival rates of 10–20% at one year and less than 2% at ten years . ATC is raplatin , but unfraplatin . Belinosraplatin . In bothactivity  and induactivity . Very reactivity . We prevactivity  and paclin vitro . These fThe study was performed across 5 centers in northwest Italy between 2009 and 2012, on patients of both genders affected by ATC and eligible for treatment with TAX. The diagnosis was obtained by cytological examination of specimens following fine-needle aspiration and was confirmed by histologic examination in all patients who underwent surgery. Examination was performed by two different pathologists with proven experience in thyroid cancer. Inclusion criteria were (a) patients with radiologically or clinically confirmed disease progression during or after multimodal therapy ); (b) patients not eligible for surgery or EBR because of tumor local invasion or severe comorbidities. Patients had to have an Eastern Cooperative Oncology Group (ECOG) Performance Score <3. Liver function had to be adequate as evidenced by serum total bilirubin <2x the upper limit of normal (ULN) (<3x ULN in patients with liver metastases); AST (aspartate aminotransferase)/ALT  <3x the ULN for the local reference laboratory (<5x the ULN for patients with liver metastases). Exclusion criteria were liver and/or kidney failure; thrombocytopenia and/or thrombocytopathy; treatment with aspirin or antiplatelet agents; treatment with antipsychotic drugs; impossibility of obtaining an informed consent due to impaired consciousness; pregnant or breastfeeding women; fertile women not taking contraceptive pills. The trial was approved by the local ethic committee for each study site. Patients or their legal representatives needed to be able to read, understand, and provide written informed consent to participate in the trial, according to the Declaration of Helsinki.2) and VPA ; the control arm received TAX alone (80 mg/m2). TAX was administered weekly intravenously, dose reduction being allowed in case of hematological or gastrointestinal toxicity; VPA was administered daily p.o. in two divided doses of 500 mg. In the absence of disease progression, patients of both groups were treated up to 18 chemotherapy cycles. Primary outcomes were (1) overall survival and (2) disease progression, evaluated in terms of progression-free survival (PFS). The extent of thyroid neoplasia and secondary lesions were assessed at the beginning of treatment by computer tomography (CT) scans of head, neck, chest, and abdomen. Local recurrence or lymph node metastases were evaluated also by high definition neck ultrasonography (US). CT and US were repeated every 6–8 weeks, to assess the response to treatment according to the modified World Health Organization (WHO) criteria: (a) complete response (CR), disappearance of all known lesions and no new lesions; (b) partial response (PR), ≥50% decrease in the sum of the products of diameters of lesions; (c) disease stabilization (SD), absence of CR or PR, without any evidence of disease progression; (d) progressive disease (PD), ≥25% increase in the size of one or more measurable lesions or the appearance of new lesions. Modifications of WHO criteria refer to the persistence of response for 4 weeks after evaluation; this last criterion cannot be applied to ATC, whose clinical doubling time is very rapid, up to three days. Clinical follow-up was performed every two weeks and will last up to 12 months. (3) Quality of life is evaluated using the EORTC QLQC30 v3 ranking every six weeks.Patients were openly randomized into two arms: the experimental arm received the combination of TAX . A temporary suspension of TAX treatment, followed by a 25% reduction at the restart, was foreseen in the presence of mucositis (Grade 3); hematologic toxicity (Grade 2); gastrointestinal toxicity (Grade 3); neurotoxicity (Grade 3); cardiac toxicity (Grade 3); muscle and skeletal toxicity (Grade 3). In the presence of Grade 4 toxicity or in case of severe allergic reaction, treatment was definitively stopped.Cmax, AUC, Kel, t1/2, clearance, and Vss) were evaluated in 20 patients  by a noncompartmental pharmacokinetic analysis performed by Kinetica 2000 4.1.1 software . Blood samples were collected in heparinized tubes, from a large vein in the arm opposite that receiving the drug infusion, at the following times: time 0 (immediately before TAX infusion), 1.5 hours , 3 hours (end of infusion), 4 hours, 5 hours, and 6 hours. Blood samples were centrifuged immediately for 10 min at 1,000 ×g at room temperature and the resulting plasma was frozen and stored at −80°C until analysis.The secondary outcome of the study was the pharmacokinetics of TAX. During the first chemotherapy cycle, the pharmacokinetic parameters of TAX  and a Symmetry C18 guard column supplied by Waters . Chromatographic analysis was performed at room temperature (20 ± 2°C) by isocratic elution with a mobile phase consisting in a mixture of acetonitrile—0.1% phosphoric acid in deionized water   at a flow-rate of 1.0 mL/min. UV detection wavelength was 227 nm and TAX retention time was about 10 min. TAX concentrations were determined from the peak area ratios versus a standard curve obtained with the same procedure. The limit of quantitation for this method was 5 ng/mL.TAX plasma concentrations were measured according to a procedure described elsewhere . BrieflySerum levels of VPA were evaluated in 11 patients at 2 and 4 weeks after they started VPA treatment. Drug measurement was performed using the Dimension Vista System . The therapeutic range was 50–100 g/mL.Median and range were reported for demographic and clinical variables descriptions. Probabilities of overall survival (OS) and PFS associated with combination treatment (VPA + TAX) versus TAX treatment alone were estimated from the time of enrollment using Kaplan-Meier method; log-rank test compared distributions of the two groups. Assuming that the predicted 1 year overall survival of patients treated with TAX alone is 0.1% and that the addition of VPA may lead to an overall survival of 20%, 22 patients could permit us to demonstrate a significant difference in the primary outcomes, accepting an error of 0.05 and 80% power. The occurrence of death within 365 days from enrollment was considered the event for estimated OS. Patients who were alive after 365 days were censored for the survival analysis. PFS was defined as the time to disease progression as evaluated by CT. Patients with clinical events unrelated to treatments or disease progression were censored.The Mann–Whitney nonparametric test was used to compare pharmacokinetic values of the two different groups of patients; statistical evaluation was performed using Instat 3.05 software .Patient characteristics are reported in P = 0.9) (P = 0.6) (P = 0.03) (P = 0.6)  . Median P = 0.6) . Median  = 0.03) . Median P = 0.6) .The oral administration of 1,000 mg valproate given once daily induced the raise of valproate blood levels to values within the proper therapeutic range for epilepsy treatment after two weeks of treatment . No increase in serum values of liver enzymes was observed.For 24/25 patients enrolled in the study, the scheduled dose of TAX was maintained. In one patient, TAX was reduced to 75%, as a consequence of allergic reaction during the first TAX infusion. No severe adverse effect was observed. The median duration of treatment was not statistically different between the group treated with VPA + TAX  and the group treated with TAX alone . For all the patients enrolled, therapy was administered as planned by study design, until clinical progression or death.2/weekly as the most effective single therapeutic agent. Both guidelines conclude that current systemic treatments lead to poor outcome and fail in improving survival or PFS. Several novel therapies have been evaluated in clinical trials, such as vascular disrupting agents  and multitargeted therapies . None of these agents showed any relevant effect on survival or PFS, being similar to those obtained with the traditional multimodal approach. These discouraging results were obtained mainly from observational studies performed in small series of patients. The present study is a rare example of an interventional randomized trial comparing one established treatment (TAX) with a combination one (TAX plus VPA).An effective treatment for ATC, one of the most lethal cancers, has not been established yet , 22. Theα-tubulin is also a substrate of histone deacetylases [ in vitro data clearly showed that VPA induced tubulin acetylation and maximally potentiated the acetylating activity of TAX. Beyond our previous in vitro data, several groups have investigated the possibility of combining VPA with different chemotherapeutic agents in patients with leukaemia and solid tumors. VPA has been shown to potentiate the activity of doxorubicin in patients with breast cancer [The decision to combine VPA with TAX in the present clinical trial stems from data obtained (by our group) with VPA as histone deacetylase inhibitor. The advantage of using VPA, a potent anticonvulsant widely used to treat epilepsy and mood disorders that acts as DCI at therapeutic concentrations , 26, is etylases . In lineetylases . The effetylases : rapid dt cancer ; moreovet cancer . Finallyt cancer . On the  in vitro on the enhancing action of VPA on TAX were not confirmed in the clinical setting of our ATC patients. In fact, mean overall survival and PFS were comparable in the 2 groups of patients, and they did not differ from those reported in other previous series of patients treated with traditional multimodal treatment. This observation is not new, since, for several therapeutic agents such as fosbretabulin [Unfortunately, the encouraging results obtainedetabulin , 33 and etabulin  and, moretabulin  and pazoetabulin , the pro in vitro and in vivo experiments demonstrated that VPA is able to slightly inhibit CYP3A4 (Ki = 7975 μM) and to induce the overexpression of P-gp, therefore a pharmacokinetic interaction between the two drugs could occur in the patients of the experimental arm [Patients treated with TAX plus VPA did not experience more side effects than those treated only with TAX. Drug interaction between TAX and antiepileptic drugs has been previously described . TAX is ntal arm , 38. OurIn conclusion, regardless the promising preclinical results, the present trial demonstrated that the combination of VPA and TAX was not superior to TAX alone. Nevertheless, our results did not differ from those observed in other studies in patients with advanced ATC.


Activation timings of postural muscles of lower legs and prediction of postural disturbance were investigated in young and older adults during bilateral arm flexion in a self-timing task and an oddball task with different probabilities of target presentation. Arm flexion was started from a standing posture with hands suspended 10 cm below the horizontal level in front of the body, in which postural control focused on the ankles is important.Fourteen young and 14 older adults raised the arms in response to the target sound signal. Three task conditions were used: 15 and 45% probabilities of the target in the oddball task and self-timing. Analysis items were activation timing of postural muscles  with respect to the anterior deltoid (AD), and latency and amplitude of the P300 component of event-related brain potential.For young adults, all postural muscles were activated significantly earlier than AD under each condition, and time of preceding gastrocnemius activation was significantly longer in the order of the self-timing, 45 and 15% conditions. P300 latency was significantly shorter, and P300 amplitude was significantly smaller under the 45% condition than under the 15% condition. For older adults, although all postural muscles, including gastrocnemius, were activated significantly earlier than AD in the self-timing condition, only activation timing of gastrocnemius was not significantly earlier than that of AD in oddball tasks, regardless of target probability. No significant differences were found between 15 and 45% conditions in onset times of all postural muscles, and latency and amplitude of P300.These results suggest that during arm movement, young adults can achieve sufficient postural preparation in proportion to the probability of target presentation in the oddball task. Older adults can achieve postural control using ankle joints in the self-timing task. However, in the oddball task, older adults experience difficulty predicting the timing of target presentation, which could be related to deteriorated cognitive function, resulting in reduced use of the ankle joints for postural control. Deterioration of equilibrium function is considered as a primary cause of falls among older individuals . In seveWhen arm movements are rapidly executed while standing, the postural muscles of the legs and trunk are automatically activated before the focal muscles of the arms, to moderate any disturbances caused by the arm movements . In oldeWhen arm flexion was performed from a position with the hands suspended in front of the body, preceding activation of the triceps surae has been clearly observed –18. In tIn an oddball task, where cognitive function is strongly concerned with task execution, a target or non-target stimulus appears repeatedly in random order at a fixed inter-stimulus interval and the participant is required to perform a specific response to the target stimulus –23. In tIn contrast, changes in P300 amplitude with increasing target probability (from about 20% to about 80%) were reportedly smaller for older adults than for young adults , 28. ThiThe purpose of this study was to investigate the postural control pattern in older adults focused on the ankle joints during arm movement. For that, older adults performed bilateral arm flexion from a suspended arm position in the self-timing task. Furthermore, to investigate the relationship between anticipatory postural control and cognitive function, this arm movement was carried out in oddball tasks with different probabilities of target presentation.Our working hypotheses were as follows. First, for young adults, as the probability of target presentation increases, the preceding time of the triceps surae activation with respect to the anterior deltoid (AD) activation would be longer and become closer to that in the self-timing task, with shorter latency and smaller amplitude of P300. Second, for older adults, ES activation would be earlier than AD activation, but the triceps surae activation would not be earlier than AD activation even in the self-timing task. Onset time of postural muscle activation, and latency and amplitude of P300 would be unaffected by target probability in the oddball task, and onsets of the triceps surae would be markedly later than in the self-timing task.Subjects comprised 14 young adults  and 14 older adults . Mean values (standard deviation (SD)) for age, height, weight, and foot length were 22.4 (1.9) years, 163.7 (5.5) cm, 57.0 (8.1) kg, and 24.4 (1.2) cm for young adults, and 70.3 (5.7) years, 156.3 (9.0) cm, 56.2 (7.3) kg and 23.6 (1.4) cm for older adults. Subject health status was assessed from a questionnaire. All subjects reported no history of neurological or orthopedic impairment and had normal hearing. Older subjects were community-dwelling individuals who could walk independently and perform activities of daily living without assistance. In accordance with the Declaration of Helsinki, all subjects provided informed consent after receiving an explanation of the experimental protocol, which was approved by the ethics committee at Kanazawa University.To measure the center of pressure in the anteroposterior direction (CoPap), a force platform  was used Fig. . CoPap eSubjects held wooden grips attached beneath a wooden board , with hands fixed to the board by acrylic belts   were used in bipolar derivation to record EMG activity of the following muscles: AD as a focal muscle for arm flexion; and ES at the level of the iliac crest, long head of biceps femoris (BF) at the midpoint between the ischial tuberosity and head of the fibula, and medial head of gastrocnemius (GcM) as postural muscles. Electrode location for both AD and GcM was the midportion of the muscle belly. Since the direction of arm movement  was forward only and preceding activation of postural muscles was observed mainly in dorsal muscles , only doAg-AgCl cup electrodes  for recording EEG were affixed to the scalp at Fz, Cz, and Pz in accordance with the international 10–20 system and referred to linked earlobes. A ground electrode was placed at Fpz. An electrooculogram (EOG) was recorded from a pair of electrodes placed immediately above and below the right eye.Electrode input impedance was reduced to < 5 kΩ. Signals from electrodes were amplified  and band-pass filtered  using an amplifier . All electrical signals were sent to the computer for analysis via A/D converters (ADA16-32/2(CB) F; Contec, Japan) with a sampling rate of 1000 Hz and 16-bit resolution.All measurements were performed on the force platform while standing barefoot with feet 10 cm apart and parallel, hands suspended 10 cm below the shoulder joints, and elbows flexed 60°  during the period from 200 ms before to 800 ms after target stimulus onset were also excluded. The order of conditions was randomized for each subject. Subjects were given 3 min of seated rest between conditions.All data were analyzed while blinded to subject condition, using BIMUTAS II signal analysis software .EMGs were analyzed as described below with reference to previous studies –18 Fig.. To exclThe P300 component is elicited by target stimuli in a discrimination task  and showt test was used to assess whether burst onset of the postural muscles differed significantly from that of AD. The following analyses were separately used for subject groups. After Levene’s tests confirmed that the variances of onset times of each postural muscle were equal in every condition, two-way mixed factorial analysis of variance was used to assess the effects of condition  and muscle  on the onset time of postural muscle activation, with repeated measures on the condition factor. Greenhouse-Geisser correction was applied when Mauchly’s test of sphericity was not met. When a significant interaction between these effects or a main effect of condition or muscle was shown, post hoc multiple-comparison analyses using Tukey’s honestly significant difference were performed separately to assess differences among conditions and muscles. A paired t test was used to assess differences in latency and amplitude of P300 between conditions in oddball tasks (15 and 45%). Pearson correlations were used to evaluate the magnitude of correlation in oddball tasks between latency or amplitude of P300 and onset time of each postural muscle activation, and between latency and amplitude of P300. For each onset time of postural muscles under each condition, Student’s t test was used to investigate differences between young and older adults, after an F test was used to confirm whether variance was equal. Welch’s correction was applied when equal variance was rejected. Alpha level was set at p < 0.05. All statistical analyses were performed using IBM SPSS Statistics version 19 .The Shapiro-Wilk tests confirmed that all data satisfied the assumptions of a normal distribution. A one-sample t13 > 5.4, p < 0.001). A significant interaction between condition and muscle was found for the onset time of postural muscles in young adults . For the significant differences in onset time among conditions, ES showed no differences, BF was earlier in the self-timing condition than in 15 and 45% conditions (p < 0.01), and GcM was earlier in the order of self-timing, 45 and 15% conditions (p < 0.05). In the self-timing condition, onset time tended to be earlier in GcM than in ES (p = 0.09). In 15 and 45% conditions, no significant differences were found in onset time among muscles. In older adults, activation significantly preceding AD was observed in all muscles under the self-timing condition , and in ES and BF under the 15 and 45% conditions . However, preceding activation was not found in GcM under the 15 and 45% conditions. Only a significant main effect of condition was found for onset time of postural muscle activation in older adults . Onset time of all muscle activations was significantly earlier under the self-timing condition than under 15 and 45% conditions (p < 0.001), and no significant difference was found between 15 and 45% conditions. Only for the onset time of GcM in the self-timing task, variance was significantly larger in older adults than in young adults . Onset times of BF and GcM in the 15% condition and ES and GcM in the 45% condition were significantly later in older adults than in young adults . No significant differences were found between young and older adults in onset time of ES in 15% condition, BF in 45% condition, and all postural muscles in the self-timing condition.Figure t13 = 2.7, amplitude; t13 = 5.6, p < 0.05). In older adults, no significant differences in latency or amplitude of P300 were found between conditions.Figure In both young and older adults, no significant correlations were found between latency or amplitude of P300 and onset time of each postural muscle activation, or between latency and amplitude of P300.In the self-timing task for young adults, all postural muscles activated significantly earlier than AD, and the preceding time tended to be longer in GcM than in ES Fig. . Older aIn the oddball task for young adults, all postural muscles activated earlier than AD in both 15 and 45% conditions, and no significant differences in preceding time were seen among muscles (Fig. For older adults, preceding activation was found in ES and BF, but not in GcM in the oddball task, regardless of the probability. No significant differences in onset time of all postural muscles were seen between 15 and 45% conditions (Fig. The present study could not demonstrate which age-related deficits of multiple cognitive aspects, including memory, attention, information processing, and executive function , would sDuring arm movement, young adults can achieve sufficient postural preparation in proportion to the probability of target presentation in the oddball task. Older adults can achieve postural control using ankle joints in the self-timing task. However, in the oddball task, older adults experience difficulty predicting the timing of target presentation, which could be related to deteriorated cognitive function, resulting in reduced use of the ankle joints for postural control.