The dry subtropics are subject to a rapid expansion of crops and pastures over vast areas of natural woodlands and savannas. In this paper, we explored the effect of this transformation on vegetation productivity  along aridity gradients which span from semiarid to subhumid conditions, considering exclusively those areas with summer rains (>66%). Vegetation productivity was characterized with the proxy metric “Enhanced Vegetation Index” (EVI) (2000 to 2012 period), on 6186 natural and cultivated sampling points on five continents, and combined with a global climatology database by means of additive models for quantile regressions. Globally and regionally, cultivation amplified the seasonal and inter-annual variability of EVI without affecting its magnitude. Natural and cultivated systems maintained a similar and continuous increase of EVI with increasing water availability, yet achieved through contrasting ways. In natural systems, the productivity peak and the growing season length displayed concurrent steady increases with water availability, while in cultivated systems the productivity peak increased from semiarid to dry-subhumid conditions, and stabilized thereafter giving place to an increase in the growing season length towards wetter conditions. Our results help to understand and predict the ecological impacts of deforestation on vegetation productivity, a key ecosystem process linked to a broad range of services. Although the dry subtropics have been historically subject to a diverse array of human interventions, including logging, grazing, and cropping , in the Cultivation of crops and pastures often leads to shifts in ecosystem functioning through the modification of the composition and structure of vegetation and consequently, of resource acquisition strategies and potential growth rates ,6. In thIn most of the classical and more recent empirical models of vegetation productivity drivers, climate interacts with vegetation structure dictating the temporal and spatial productivity patterns of natural ecosystems ,9. In paOur understanding of the link between productivity and water availability has grown steadily in the last decades, focused initially on the water-use efficiencies  and lateThe aim of our study is to compare the vegetation productivity patterns of implanted crops and pastures  with the natural–predominantly woody–vegetation that they replace , across climatic water availability gradients in the dry subtropics (only those with summer rains). We focus our analysis on the magnitude, seasonality and inter-annual variability of vegetation productivity. Our guiding questions are: (1) How do key vegetation productivity attributes respond to cultivation?, and (2) How does this response vary along water availability gradients? The analyses are conducted at global and regional levels by means of the “Enhanced Vegetation Index” (EVI) and additive models for quantile regressions.We focused on the dry subtropics receiving summer rains, as defined by climatic and topographic features: warm temperatures , dry winters/wet summers , semiarid to subhumid conditions defined by the ratio of mean annual precipitation to potential evapotranspiration , gentle slopes (<0.7%), and low elevation (<1200 m). Resulting regions were named as Chaco, India-Pakistan, Mesquite, North-eastern Australia, and Zambezi-Kalahari . These ahttp://www.google.com/earth/index.html) and online photographic archives . Cultivated sampling points represented areas where natural vegetation was removed in order to implant artificial communities. We restricted all analyses to points with a homogeneous and constant land use/cover at the measurement scale and throughout the study period. We confirmed the homogeneity through the visual inspection of Google Earth images on 1.5 km-radius windows, while we resolved the constancy by restricting analyses to natural points characterized as such in 2012, and to cultivated points characterized as such in 2000 or earlier. These conditions were evaluated by visual inspection of circa 2000 imagery from the “GeoCover” Orthorectified Landsat ETM+ Mosaics project [Across the study area, we generated a regular grid of sampling points distributed within 35 transects (20 km-wide and 125 to 250 km away from each other) that covered major PPT:PETgradients. These points, that maintained an approximate distance of 7 km with each other, were classified into two possible land use systems, natural or cultivated, based on a visual inspection of high resolution satellite images  time series, produced by the Terra “Moderate Resolution Imaging Spectroradiometer” (MODIS) instrument . By appl-2*y-1 in Mesquite or India & Pakistan to 3,800 Mj*m-2*y-1 in NE Australia or Zambezi-Kalahari, Following Xiao et al. "Vegetation Photosynthesis Model"  and othewww.daac.ornl.gov). We only considered EVI values with the highest quality (flagged as category VI)–representing 79% of the entire data set–, eliminating the potential noise from clouds and aerosols. We used the software TIMESAT v.3.1 to reconstruct the EVI time series [For the 6,186 sampling points, we downloaded EVI data from 2000 to 2012  from the ORNL “MODIS Global Subsets: Data Subsetting and Visualization” tool  for quantile regression [50) in order to provide a description of the effect of X on the central tendency of Y, and 0.9 and 0.1 quantiles  to describe the behavior of the Y variable when X is the dominant constraining variable [90 to represent the healthy or permissive state of unmeasured factors in variables like the magnitude of productivity, which are maximized by water availability. The opposite occurs for the variability of productivity, which is likely minimized by water availability and thus τ10 is the logical option. We selected for all regressions a smoothing term λ = 0.5, which empirically implied a good compromise between the goodness of fit and model simplicity.To obtain a first graphic description of the productivity/water availability relationship, we represented seasonal dynamics of EVI for four PPT:PET equal intervals (0.2 to 0.4 up to 0.8 to 1.0). At each sampling point, we averaged the reconstructed EVI values of the 23 dates per year provided by MOD13Q1 for the temporal series of 13 years. We then explored the relationship by regressing functional metrics against PPT:PET by means of additive models . All information is available at the For the global level approach, we balanced sampling size differences among land use/cover systems and regions  by applyg method . This img method  tube app50), natural and cultivated systems did not differ in terms of mean EVI . Initially, along the gradient of increasing water availability, maximum productivity grew in parallel on both systems; however, at PPT:PET > 0.6 a major functional change occurred with cultivation, since the maximum productivity of the implanted systems stabilized, diverging from the natural vegetation–which continued increasing up to the humid end of the gradient– than by water availability, showing little variation along the PPT:PET gradient  resulting from a relatively low productivity of cultivated systems. At last, contrary to the global behavior (τ50 and τ10 models), we found that the intra-annual CV markedly decreased towards humid conditions in Chaco and NE Australia.Regional results generally sustained the global patterns in terms of average contrasts between systems , yet loo6 km2, five continents, 13 years of data), revealed that deforestation and subsequent agricultural expansion in the dry subtropics with summer rains appears to have a nil effect on the productivity of vegetation–given by the mean EVI–, independently from the water availability conditions . This amCultivation also increased significantly the variability of production from year to year  but lessFrom a morphological/ecophysiological perspective, the differences in inter-annual variability between systems could also be ascribed to human-selected vs. nature-selected plant adaptations. Native species display hydraulic redistribution, have larger rooting depths, slow-growth strategies, and particular structural tissues that confer individuals a high water use efficiency and stress tolerance, allowing for a stable production in dry and wet years ,56. OppoUndoubtedly, the conversion of natural vegetation increased the appropriation of vegetation productivity for human consumption ,17. Neve90 and τ10), we revealed at the global level that cultivated systems can display the highest long-term stability under the driest conditions  or net primary productivity (NPP) remains a major challenge. Regarding annual GPP, uncertainties are related to the apparent need of site-specific empirical scaled EVI-fAPAR functions , and theOur remote sensing approach provides a new quantitative insight on the relative productive differences between original natural woody systems and novel cultivated systems in dry summer-rains subtropics. Cultivation increased the seasonality and the inter-annual variability of vegetation productivity without affecting its magnitude, which responded mainly to water availability. Climatic water availability is important determining productivity in natural systems; however, it loses its strength in cultivated systems, which seem to saturate their productivity at dry subhumid conditions (PPT:PET around 0.5). In the last decades, many researchers have explored the physical constraints of vegetation productivity in order to predict its response to global climate change. Using similar data and methodologies, we assess a different dimension of change, driving the functional debate towards the effects of the ubiquitous and accelerated land use/cover shifts. We highlight the considerable changes in seasonal vegetation activity and long-term variability  and reveal that the implications of the land transformations depend both on the physical and human contexts (accounted here by the water availability gradients and by the regions).S1 Dataset(XLSX)Click here for additional data file.S1 Fig-2) from the Climatic Research Unit–UEA “Ten Minute Climatology” data base [We calculated precipitation and PAR Click here for additional data file.S2 Fig50 and τ90 or τ10) in relation to PPT:PET. Numbers within panels indicate average values about the differences between land use/cover systems according to the τ50 and τ90or τ10 models. Data came from the global additive median models  for the seven EVI-based functional metrics.Each panel represents the difference between cultivated and natural additive models for the 0.5 and 0.9 or 0.1 quantiles (τ(EPS)Click here for additional data file.S3 Fig90 or τ10) after a resampling approach (500 points). The thick line represents the averaging (with a median) of these individual models.Each panel represents the behavior of an EVI-based functional metric in relation to the PPT:PET. The thin lines represent the individual additive models for the 0.9 or 0.1 quantiles (τ(EPS)Click here for additional data file.S4 Fig50) model. Note that not all regions cover the entire water availability gradient. Gray bands indicate 95% confidence intervals according to the Hotelling [Each panel represents the behavior of an EVI-based functional metric and region in relation to the PPT:PET. Each dot represents a sampling point and each line an additive 0.5 quantile (τotelling  tube app(EPS)Click here for additional data file.S5 Fig90 or τ10) model. Note that not all regions cover the entire water availability gradient. Gray bands indicate 95% confidence intervals according to the Hotelling [Each panel represents the behavior of an EVI-based functional metric and region in relation to the PPT:PET. Each dot represents a sampling point and each line an additive 0.9 or 0.1 quantiles (τotelling  tube app(EPS)Click here for additional data file.S1 Table(DOC)Click here for additional data file.S2 Table90) and 0.9quantile (τ10) additive median models of natural and cultivated systems Click here for additional data file.S3 Table(DOC)Click here for additional data file.


Sickness absence (SA) among healthcare workers is associated with occupational and non-occupational risk factors and impacts employee health, healthcare delivery and patient health. At the same time, healthcare is one of the employment sectors with the highest rates of work-related ill health in the UK. Musculoskeletal (MSK) and mental health (MH) issues are leading causes of SA, but there is a lack of research on how certain MSK/MH conditions impact on SA duration. The study aim is to determine differences in SA duration by MH and MSK disorders in healthcare employees.Survival analyses were used to estimate SA duration due to MSK and MH problems over 6 years, and Cox’s proportional hazards models to determine the HRs of returning to work, using a bespoke Scottish health board database with over 53 000 SA events. SA duration and time to return-to-work (RTW) were estimated for employees by age, gender, job and health conditions.50: 7 days), whereas employees absent due to depression took the longest (P50: 54 days). The most influential sociodemographic variables affecting RTW were age, gender and job category.MSK and MH conditions accounted for 27% and 6% of all SA events and 23.7% and 19.5% of all days lost, respectively. Average SA duration was 43.5 days for MSK and 53.9 days for MH conditions. For MSK conditions, employees with low back or neck pain had the fastest RTW (median PUsing a unique and rich database, we found significant differences in SA duration by presenting condition in healthcare workers. MH conditions, and depression specifically, accounted for the most working days’ absence. Significant variations in duration were also observed for MSK conditions. Our findings can inform public health practitioners and healthcare managers of the most significant factors impacting MSK-related and MH-related SA to develop and implement tailored and targeted workplace interventions. Healthcare is one of the employment sectors with significantly higher rates of sickness absence (SA), impacting on employee health, healthcare delivery and patient health. This study uses a unique, data-rich and bespoke database that allows for detailed assessment of the impact of musculoskeletal (MSK) and mental health (MH) conditions on absence duration and return-to-work (RTW) for healthcare employees of a Scottish health board.Cox’s proportional hazards models applying time-varying coefficients were used to estimate SA duration due to MSK and MH problems over 6 years (n=48 007 unique SA events).The size and granularity of the database, giving six full years of SA and RTW data, and the range of variables collected–including demographic, job, SA start and end dates and self-reported conditions–allowed for the investigation of SA duration and time to RTW for healthcare employees by age, gender, job and health conditions.SA data are from a single large health board (approximately 12 000 employees) which limits the degree of generalisability. Also as SA absence is multicausal and it is necessary to consider an individual’s work and workplace environment, there are a number of variables that are not collected as part of the service  which could potentially be important risk factors for SA and RTW in this population.Sickness absence (SA) is a significant public health burden on government, employees, employers and public resources.8–12events, makes up approximately 75% of absence costs, with musculoskeletal (MSK) and mental health (MH) disorders being two of the leading causes of LTSA.2 3 14 17 18Multiple factors need to be considered to effectively manage SA through policy and practice.The healthcare sector is a diverse entity, which presents a unique set of challenges in relation to SA duration.31Certain health conditions result in longer periods of SA.18Health Response UK reports MSK injury as the leading cause of absenteeism and related cost to UK industry, with back pain as one of the most common causes of MSK-related SA.Neck problems are also a common MSK condition resulting in lost working days.37A number of systematic reviews highlight the importance of recognising MH issues in the workplace to assist in the reduction of associated SA.Depression is a leading cause of MH-related absence.40–42For those experiencing anxiety that results in SA, previous anxiety episodes, older age, lower education levels and long durations of untreated and undiagnosed symptoms contribute to longer absences.42Detailed RTW figures following LTSA are scarce. Estimates suggest that as few as 13% of people who have experienced depression and anxiety are in employment, compared with 33% of people experiencing other chronic illnesses.43Several studies have investigated the associations between SA and an employee’s sociodemographic and socioeconomic characteristics.49 50Despite a great deal of research recognising the prevalence of MSK and MH issues and their impact on SA ratesThe study population includes all participants in the EASY (Early Access to Support for You) SA management service of a Scottish health board.SA duration was calculated from the difference between the first date of absence and the RTW date. Cause of absence was grouped into seven categories: respiratory; MSK; MH; GI; ear, nose and throat; CCI and all others. Conditions assigned to the MSK category were grouped into eight subgroups according to anatomical site: hip, knee, low back, lower limb, neck, shoulder, upper limb and all others. MH conditions were allocated to eight subgroups according to diagnostic group: bipolar disorder, anxiety, depression, schizophrenia, panic attacks, self-harm, stress and all other psychiatric disorders.After missing data were eliminated (<0.05%), four main exclusion criteria were applied: (1) if there was no FDA (n=196), (2) if the FDA was a Saturday or Sunday (n=4881), as there was no SA service on those days and absences would not be recorded on FDA, (3) if the ‘date opened’  was before the FDA (n=828) and (4) if the ‘date opened’ was equal to or after the RTW date (n=3465). Due to overlaps among the missing data and exclusion criteria, a total of 13 286 absences were excluded, giving a total of 53 193 unique absence events; hence, analysis was based on these 53 193 unique absence events.Descriptive statistics and crosstabs were produced for the entire EASY population, which included all causes of SA (n=53 193). Descriptive statistics and crosstabs were then produced for two specific causes, MH (n=3093) and MSK (n=6969) conditions.Mean absence duration was calculated for the entire EASY population in the study, the entire EASY population without MSK and MH (n=43 131), MSK only, MH only, MSK subcategories and MH subcategories for each year from May 2008 to December 2015. The data were divided into 7 years as follows: May 2008–April 2009 (Year 1), May 2009–April 2010 (Year 2), May 2010–April 2011 (Year 3), May 2011–April 2012 (Year 4), May 2012–April 2013 (Year 5), May 2013–April 2014 (Year 6) and May 2014–December 2015 (Year 7). Absences commencing in Year 7 were not included as the year was incomplete and in numerous cases, absence could still be ongoing; hence, further analyses presented were based on 48 007 unique absence events.Absence duration was analysed using Kaplan-Meier survival curves and Cox’s proportional hazards models to determine the HRs of absentees returning to work. We tested the assumption of proportional hazards using Schoenfeld residuals and where violated, we corrected for this by adding the relevant time-varying covariate online . The mod10.1136/bmjopen-2017-018085.supp1Supplementary file 1All statistical analyses were conducted using STATA V.12.0 and R V.3.1.1.Our sample contains 11 694 individual people and 48 007 absences (May 2008–April 2014). Descriptive statistics for our sample  are reported in Fewer of those with MSK-related absences (n=6530) were in the two youngest age groups, than in the total population, and the proportion of those with MSK absences who were in the nursing/midwifery (n=3428) job category (52.5%) was even higher than seen in the total population (45.3%). In the MH population, 91.8% are women, compared with 87.9% of the total population .events, 28% and 18%, respectively. However, impact, in number of days absent, these causes have on total number of working days lost is much less (11.8% for GI and 6.3% for CCI). The health conditions with the highest impact on total number of working days lost are MSK (24%) and MH (20%) conditions. Mean absence duration ranged from 5.6 days for CCI-related to 53.3 days for MH-related absences. MSK absences had an overall mean duration of 28.9 days.GI and CCI problems account for the largest number of SA The three most common types of MSK problems in this population are low back pain , lower limb (9%) and upper limb problems (9%). The three most common types of MH problems among SA episodes in the EASY population are stress (64.8%), anxiety (15.9%) and depression (13.7%). The job categories with the highest cases of MH-related absences are nursing/midwifery (55.3%) and administrative services (18.8%). Mean absence duration in Years 2, 3, 4, 5 and 6 of the EASY service was compared with Year 1 using linear regression for all causes of SA to examine potential significant changes over time and then for each of the nine conditions online . Compare50) between 10 and 14 days. MH-related absences are much longer , the risk of RTW was 52%  and 77%  longer for absences due to MSK and MH conditions, respectively. No real differences were seen after adjusting for gender, age, job family, job type, cause of absence, day of absence, season of absence and year of absence.For MSK-related absences, staff absent due to neck problems were at risk of being off 17% longer  compared with those absent because of lower back problems, whereas employees who were absent because of all other MSK conditions  had quicker RTW.The risk of returning to work after being absent due to anxiety, stress and all other MH-related absences was significantly higher, when compared with depression, and was 64% , 65%  and 78%  of longer duration, respectively .The analysis by gender demonstrated that for all absences (minus MSK and MH) and for MSK absences, women exhibited longer RTW times than men . No signIn terms of job category, for all conditions (minus MSK and MH), nurses had the longest SA duration prior to RTW followed by staff in ‘support services’ . EmployeThe analysis by year of absence using Year 1 as the reference category showed that staff took significantly longer to RTW in Years 3–6 for all conditions (minus MSK and MH). Among MSK absences, in Years 2–4, staff time to RTW was significantly shorter than for Year 1. There were no significant differences in time to RTW by year for staff absences due to an MH condition. Additionally, when examining time to RTW by day of absence start, absences starting on a Tuesday and Friday took significantly longer to RTW online . No signWe found significant differences in SA duration by presenting condition in a population of healthcare workers. MH conditions, and depression specifically, accounted for the most working days’ absence. We also observed significant variations in duration for different MSK conditions. Upper limb disorders resulted in the longest SA durations among these healthcare workers, with 50% of staff returning to work by day 25. Lower back and neck problems resulted in the shortest MSK-related absences (50% RTW by 7 days), whereas knee, lower limb, shoulder and other MSK conditions resulted in SA durations that were comparable to each other (50% RTW between 10 and 14 days).Employees within the nursing and midwifery job category accounted for almost half (45.3%) of all SA events recorded in the 6-year period and over half of the MSK-related (52.5%) and MH-related (55.3%) absence events. Nurses and midwives also had longer times to RTW than every other job category. In terms of gender, no significant differences were detected in RTW between men and women who were absent from work because of MH conditions, but for all other absence events, women took longer to RTW than men.et al (2016) also demonstrated that occupation and health condition were significant factors, among others, in the rehabilitation process of people affected by MSK conditions.44The results of our study are in agreement with previous published work on the impact of MSK and MH conditions on SA and RTW.The latest HSE report lists healthcare staff as one of the categories with the highest SA rates and MH and MSK as leading causes.While the availability of such rich data on routine SA is a major strength of this study, there are also some limitations to the data. This SA management service is only available from Monday to Friday, and, therefore, not all absences may have been recorded. To avoid any confounding, we removed absences starting on a weekend. The cause of absence is self-reported by the employee when they call the service and not based on a clinical diagnosis, and comorbidities–which may also impact on absence duration–are not collected.53SA is multicausal, and it is necessary to consider an individual’s work and workplace environment. However, the lack of granularity in SA data recorded is often the barrier into investigating the risk factors and causes impacting on SA duration. Linking bespoke databases, such as the one in this study, to personnel data or having uniformity in the variables collected across health boards would allow investigation of the impact of operational  and organisational (region and structures) risk factors on SA rates and duration.As this health board has a unique SA management service, the results may not be representative of all healthcare workers. However, a national standard requires all health boards in Scotland to work towards a 4% or less SA rate.60Due to the financial and morale repercussions SA among healthcare employees has not only on healthcare staff themselves but also on their patients and employers,13 61The results of this study further establish the need for occupational health, organisational and management interventions to address recognised individual and workplace stressors that can impact on SA duration. Our results suggest that employees with upper limb problems and depressive symptoms in particular could benefit from more tailored interventions to assist them in the RTW process following an SA event. A great burden of work loss due to both MSK and MH conditions was observed for nurses and midwives.This research is important in terms of improving the health and well-being of NHS staff but may also improve the quality of patient care and subsequently public health. SA has far-ranging economic consequences for both employers and employees, as it simultaneously impacts on NHS resources/service delivery and on people’s earnings if the SA is prolonged. These findings give a deeper insight into the link between health, organisational, operational and sociodemographic factors influencing SA. Understanding these relationships allows health providers to be better placed to plan the allocation of resources, build better models of SA management and inform the development of tailored SA interventions for NHS staff.


Patients undergoing endovascular repair of thoracic aortic aneurysm should be carefully monitored for the development of postimplantation syndrome. This clinical condition is relatively common after the endovascular repair of aortic aneurysm but is rarely observed after endovascular repair of thoracic aortic aneurysms. The different known therapeutical approaches are still empiric, with reported beneficial effects with the use of NSAID, corticosteroids, and danazol.After Endovascular repair of thoracic aortic aneurysm, a systemic inflammatory response, named postimplantation syndrome, can develop. This syndrome is characterized by fever, leukocytosis, and elevated CRP plasma levels and its pathogenetic mechanisms are still unknown. Although this syndrome generally resolves within few days, some patients develop a persisting severe inflammatory reaction leading to mild or severe complications. Here we describe the case of a male patient who developed postimplantation inflammatory syndrome and severe thrombocytopenia after endovascular repair of thoracic aortic aneurysm. Treatment with prednisone (50 mg/bid) for two weeks did not improve the clinical and laboratory findings. We utilized danazol, a weak androgen that has been shown to be effective in the treatment of immune and idiopathic thrombocytopenic purpura, and after 12 days of treatment with danazol (200 mg/bid), the patient improved progressively and platelet number increased up to 53,000/ Aortic aneurysms are a relatively common disease that can lead to potentially fatal consequences , 2. EndoWe describe the case of a male patient who developed postimplantation inflammatory syndrome and severe thrombocytopenia after endovascular repair of thoracic aortic aneurysm.μL, neutrophils 9,340/μL, and monocytes 1,390/μL), and an increase in inflammatory markers (CRP 251 mg/L). The chest X-ray was compatible with left pleural effusion. The patient was initially treated with oxygen therapy, intravenous corticosteroid, and antibiotic treatment with partial improvement of the dyspnea and increase in oxygen saturation. Eleven days after the admission, the patient reported worsening of the dyspnea and a second chest X-ray showed complete opacity of the left lung field , and arterial hypertension. After admission, the patient reported epigastric pain radiating to the back many weeks previously. He showed normal body temperature (36.6°C) and heart rate (90 beats/min), with elevated arterial pressure (170/100 mmHg) and a pulse oximetry of 90%. On physical examination, he presented an area of dullness at the lower half of the left hemithorax with abolished vesicular murmur in the same area. Peripheral arterial pulses were symmetric. Routine laboratory data showed normocytic-normochromic anemia (Hb 105 g/L) with increased reticulocyte percentage (2.40%), neutrophilic and monocytic leukocytosis  [μL) with WBCs up to 18,000/μL during the second postoperative day; furthermore, there was an increase in CRP values. The patient presented fever with a peak of 38°C despite negative blood, urine, and bronchial aspirate cultures. Plasma concentrations of TNF-α and IL-6 were elevated, respectively  and 37.9 ng/L ). As empiric therapeutic approach to thrombocytopenia, we first started corticosteroid therapy (prednisone 50 mg/bid) for two weeks without significant improvement in platelet count. After this unsuccessful therapy, we used danazol, a weak androgen that has been shown to be effective in the treatment of immune and idiopathic thrombocytopenic purpura and in thrombocytopenia in patients with myelodysplastic syndrome [μL. The patient was discharged and admitted to a long-term care facility.Contrast chest CT-scan revealed a descending thoracic and proximal abdominal aortic aneurysm, with maximal axial diameter of 7 cm, presenting a thick parietal plaque (17 mm) with ulcerative aspects and hyperdensity on the left anterolateral side compatible with intramural hematoma . The patbin-III) . Laboratsyndrome , 11. Aftα, IL-1 α, IL-1β, and IL-6 [Endovascular repair of thoracic aortic aneurysm is a minimally invasive surgery presenting many advantages over the open repair , 4. Afteand IL-6 –7. IL-6 To this regard, the impact of PIS on patient outcome is still unknown but the systemic inflammatory response could be responsible for an increased cardiovascular morbidity, since the intensity of inflammation seems to be correlated with the presence of cardiovascular or any other adverse event during the first month after the procedure . FurtherThen patients with PIS may require closer surveillance during the first year after TEVAR , althougα and IL-6 plasma levels in our patient were increased, possibly confirming this hypothesis. The increased plasma levels of laboratory inflammatory parameters observed in our patient are similar to those reported in previous series of patients who developed PIS after EVAR for AAA [A specific pharmacological treatment for PIS has not been suggested yet, although, as for SIRS, the use of nonsteroidal anti-inflammatory drugs in mild forms and of corticosteroids in severe forms has been suggested, together with intensive care assistance , 4, 6. I for AAA , confirm for AAA , 4, 12.  for AAA . Less cl for AAA . AlthougμL→53,000/μL). The mechanism of action of danazol in MDS and in ITP remains unclear. It has been hypothesized that danazol, similar to the stimulatory effects of androgens on erythropoietin, could increase thrombopoietin plasma levels or reduce the level of antiplatelet antibodies as observed in ITP [The pharmacological treatment of thrombocytopenia in the present case was empirically based on corticosteroids but we observed poor effects on platelet count. Interestingly, in a recent paper by de la Motte et al., it has been reported that preoperative treatment of patients undergoing EVAR with methylprednisolone attenuates the inflammatory response with a faster recovery after EVAR for AAA . Our patd in ITP , 15.In conclusion, patients undergoing TEVAR should be monitored for the development of PIS and we recommend careful monitoring of postoperative blood inflammatory markers and blood cell count above all leukocytes and platelets. The role of the different therapeutical approaches of PIS is still empiric, with reported beneficial effects with the use of NSAIDs, corticosteroids, and danazol. The possible beneficial effects of eltrombopag, a novel agonist of the thrombopoietin receptor which promotes megakaryopoiesis similar to endogenous human thrombopoietin , have no


We examined the spatial and temporal variability of ciliate community in a subtropical estuary by rRNA and rDNA-based high throughput sequencing of 97 samples collected along the entire salinity gradient at two-month intervals in 2014. Community divided statistically into three groups: freshwater , oligohaline and mesohaline , and polyhaline and euhaline . Across all three groups, salinity explained most of the community variability. Within each group, seasonal shifts in community formed cool (spring and winter) and warm (summer and autumn) subgroups, indicating that spatial variability overrode seasonal changes in determining community composition. Cool and warm groups showed opposite associations with temperature and prey proxies, suggesting distinct seasonal niche separation. The community reassembly of cool and warm groups was essentially due to transitions between intermediate (with relative abundance of 0.01–1%) and abundant (with relative abundance > 1%) OTUs. Further analyses demonstrated that the intermediate group not only encompassed comparable OTU richness to that of the total community and maintained high metabolic activity but also had the highest proportion in transition, either to abundance or rarity, thus offering a first view on how it varies across space and time and revealing the essential role it played in maintaining stability and functionality within the community. However, our knowledge of the spatial and temporal distribution patterns of microzooplankton across a wide variety of environments and their shaping factors is still limited. Ciliates are single-celled eukaryotes (protists) that dominate many microzooplankton communities in terms of both species diversity and abundance3. By virtue of their short generation times, ciliates can quickly respond to environmental fluctuations4. Therefore, understanding the community dynamics of ciliates is increasingly important in an era of rapid environmental change, particularly in habitats where such changes are likely to be sufficiently pronounced to affect ecosystem function. Estuaries are highly dynamic ecosystems that undergo constant change due to both natural, e.g. daily  and seasonal variations, and anthropogenic factors. These variations in space and time make estuaries ideal ecosystems to perform investigations targeting the community dynamics of ciliates.Microzooplankton are heterotrophic organisms with body size between 20 and 200 µm. They can feed on pico- and nanoplankton unavailable to most meso- and macrozooplankton. They therefore occupy a key position in aquatic foodwebs as their grazing significantly affects primary producers and bacteria5 or protists6, or alongside studies of various metazoas groups, e.g. copepods, fishes, molluscs and crustaceans7. Other studies have focused on specific groups of ciliates e.g. tintinnids8 or aloricate ciliates9. Studies on the full spectrum of ciliates along entire salinity gradients are limited, and those dealing with both temporal and spatial variations are even rarer11. Furthermore, most of these studies were done with classical methods, e.g. fixing water samples with Lugol’s or Bouin’s and directly identifying and/or counting species under the microscope. There is increasing evidence, however, that convergent or plastic morphologies can mask major molecular, physiological and ecological differences, so spatial and temporal patterns based morphospecies data may be confounded by cryptic and polymorphic species13.Estuarine ciliates have been included as part of investigations on wider taxonomic scales, e.g. protozoa18. Compared with other habitats, only three investigations using molecular methods, i.e. clone library, DGGE and high throughput sequencing (HTS), have been performed on estuarine ciliates21. Although these revealed higher taxonomic richness than microscopic surveys, they did not test19 or failed to find21 relationships between ciliate community composition and environmental factors. Also, the studies were conducted at a handful of locations in the same sampling area, and did not consider the effect of temporal changes. HTS holds the potential to detect species even when present in low abundance and for sufficient representative taxa of the targeted area to be obtained, thus avoiding the bias associated with morphological and classic molecular methods. Furthermore, recent studies on prokaryotes have shown the importance of differentiating the active from the total communities24. This concept, however, has rarely been applied to ciliate communities25. Therefore, exploration of spatial and temporal patterns of distribution using rRNA-based HTS appears to be a promising approach for revealing the structure and stability of ciliate communities and for investigating relationships between community composition and environmental factors.In recent years, culture-independent molecular techniques have been applied to study ciliates in a wide variety of marine environments30. Several investigations have revealed that eukaryotic assemblages can rapidly and regularly reconstruct themselves and rare taxa may contribute to such shifts in community structure, becoming dominant when environmental conditions are favorable33. In a recent study, Logares et al. found that up to 30% of the sequences per sample belonged to neither rare nor abundant groups, but rather to an intermediate group35. Despite its considerable relative abundance, this intermediate group is understudied compared with abundant and rare groups. In a study of short-term dynamics of lacustrine small eukaryotes, Mangot et al. found that freshwater microeukaryotes were largely composed of rare OTUs that never became abundant, and a core group that involved about 20 abundant OTUs and a handful of intermediate OTUs which sporadically transitioned into dominant taxa during the study period36. This study was conducted at a permanent station in Lake Geneva for one summer with relative stable environmental conditions36. Whether this rule can be applied to a more dynamic environment on a wider spatial and temporal scales is not known. Also, the metabolic activity/inactivity of the intermediate biosphere and the role it might play in the overall community remains largely unknown.Abundant members of the biosphere attracted much attention in early ecological and biodiversity studies because of their ease of collection and likely influence on biogeochemical cycles. Recently, HTS techniques have broadened the scope of biodiversity, with the discovery of the “Rare Biosphere”. The rare biosphere, a term coined by ref. Here, we investigated the stability of community composition and structure of ciliates across the full salinity gradient in the Jiulong River Estuary near the southwestern Taiwan Strait in southeast China. A large dataset of 18 S rRNA and rDNA fragments were produced from 97 samples collected from one to three water depths at two-month intervals in 2014. The fine-scale spatial and temporal coverage, encompassing the full length of the estuary with replicate samples collected throughout the year, combined with ultra-deep sequencing for both 18 S rRNA and rDNA, enabled shifts in ciliate community composition and structure to be determined at high resolution across space and time. The present study aims to address the following questions: (i) Is the ciliate community composition and structure stable on a seasonal time-scales across the full salinity gradient? (ii) Are changes in community composition correlated with environmental factors? (iii) Considering their likely considerable relative abundance, are communities of intermediate forms stable or do they transit between being abundant and rare across space and time?In rarefaction analysis, OTU richness approached saturation in most local communities (32–186 OTUs) and the total ciliate community  corresponding with salinity zones in Jiulong River Estuary (JRE): freshwater (F), oligohaline-mesohaline (OM), and polyhaline-euhaline (PE) groups. The F group included samples with salinity less than 0.5‰, the PE group consisted of samples with salinity range of 18–40‰, and the OM group from samples with salinity between the F and PE groups (0.5–18‰). Similarities among samples were higher within groups than between groups Table . The nonity Fig. . In oligoup Fig. . In polyoup Fig. . Contraroup Fig.  and 2. Ioup Fig. .Figure 1a, and violaxanthin explained most of the community variability across the entire community dataset, with a rank coefficient of ρ = 0.669  of 0.859, which accounted for 40% of the freshwater group  based on morphological characters10. This study demonstrated that the ciliate communities were delineated into three main groups that were composed of taxa mainly appearing in freshwater, mid-estuary, and seaward zones, respectively.Previous studies on the dynamics of estuarine ciliate communities were restricted to one or two dimensions, being based either on horizontal surveys across salinity gradients, time series at one to two fixed locations, or spatial and temporal variations of a single assemblage of ciliateset al. also found that ciliate communities in river and plume surface waters resembled that in the deep river water in Long Island Sound and suggested physically vertical mixing might be one of the reasons for this21.In contrast to the horizontal variation, ciliate community variability did not show a correlation with depth Tables  and S4. 39. However, the degree to which water masses are mixed/separated largely depends on the magnitude of mixing caused by river flow and tides. This mixing of waters from surface to bottom layers and from euhaline to freshwaters can lead to the formation of ciliate communities in zones that comprise populations from multiple water mass sources. Our results support this hypothesis in that oligohaline-mesohaline group (OM) included OTUs from the freshwater (F) and polyhaline-euhaline groups (PE), sharing 43.8% and 56.6% of OTUs with F and PE, respectively  revealed that species distribution was strongly affected by temperature, which was the most important environmental factor determining their seasonality40. Mironova et al. identified a similar pattern among 30 surface water samples collected from two sites in Neva Estuary  in which two distinct groups succeeded each other during different seasons11. In the present study, community composition in the warm assemblages in the mesohaline and polyhaline zones were indistinguishable  of transitions among the three groups  largely ignoredups Fig. . Indeed,et al. was performed at a single location in Lake Geneva representing a relatively stable environment36. Nevertheless, both this and the present study showed that the intermediate groups make a contribution to community variability. Compared with the previous report, however, the present study revealed a higher contribution to the community variability, suggesting the important role of the intermediate group in maintaining community stability in a more dynamic environment. Analysis of the DNA:RNA ratio indicated that the intermediate forms are metabolically active because the regression line deviated from 1:1 line toward RNA axis . A total of 97 samples were collected from eight sampling sites across the full salinity gradient from freshwater to euhaline  is located in a subtropical monsoon zone of southeastern China, near southwestern end of the Taiwan Strait Fig. . The estine Fig. . Site A545. For chlorophyll a and pigment analyses, 200 ml of water was immediately filtered through 47 mm GF/F filter, stored in liquid nitrogen, and later extracted in acetone. Extracts were analyzed for both chlorophyll a and pigment composition by Trilogy fluorometer and Agilent series 1100 HPLC, respectively46. Water samples were also analyzed for nutrients including nitrate (NO3-N), nitrite (NO2-N), ammonia (NH4-N), dissolved total nitrogen (DTN) and dissolved reactive phosphorus (DRP) as described previously47. Information of environmental characteristics of the sampling sites is included in Table Surface waters were collected with Niskin Bottles. For freshwater site A5 and coastal site JY2, water was collected at three depths . For each sample, 2 L of water was pre-filtered with a 200 µm nylon mesh (Sefar Nitex) to remove mesozooplankton and then directly filtered onto a polycarbonate filter, 3 µm pore size  which was stored in RNAlater  at −20 °C. Water temperature, salinity, and depth were determined using a SeaBird CTD profiler. 1.8 ml of Water for bacterial analysis were pre-filtered with 20 µm mesh, fixed with ice-cool glutaraldehyde at a final concentration of 1%, stored in liquid nitrogen and later analyzed using a flow cytometer 48. Each sample was amplified in triplicate, pooled, and purified using the MiniElute Gel Extraction Kit . All purified hypervariable V4 region amplicons were sent to Majorbio sequencing company  for sequencing using Illumina MiSeq platform. Short reads were deposited in NCBI Sequence Read Archive (accession code SRP115671).High throughput sequencing of 97 samples were performed, with 71 samples from the whole year for RNA sequencing and 26 samples from both dry (February) and wet (June) seasons for DNA sequencing. All spatial and temporal distribution patterns were inferred from rRNA dataset. The rDNA dataset was used for revealing the metabolic activity of the intermediate group only. Extraction of DNA and RNA, reverse-transcription and PCR amplification followed49 and Flash software50. Chimeras were identified using UCHIME as implemented in USEARCH51. Operational Taxonomic Units (OTUs) clustering was done using Uparse at the 97% similarity cutoff 52. Generation of OTU tables and taxonomy assignment of each OTU were done with QIIME53 using the reference taxonomic database of ciliates from the Protist Ribosomal Reference database (PR2)54. Sequences were normalized by randomly resampling 14,825 reads per sample (the lowest number of sequences recovered for 97 samples), which minimized bias associated with sequencing depth and allowed for comparison of results for all samples.Quality filtering, demultiplexing and assembly of raw data were performed with Trimmomatic29. Between these two is the intermediate group36. To test whether the intermediate group was stable or shifted between the abundant and rare groups across space and time, the proportion of the intermediate group in transition across all samples was calculated. In order to reveal metabolic activity of the intermediate group, linear regression of 18 S rDNA/rRNA ratio of samples collected in dry and wet seasons was performed in R.In addition to the total community, separate analyses of abundant, intermediate, and rare assemblages were performed. OTUs were considered to be abundant or rare when they comprised more than 1% or less than 0.01% of the reads in a sample, respectively55. Ordination of samples was done by Non-Metric Multidimensional Scaling (nMDS) using Bray-Curtis dissimilarity matrices. OTU network was constructed in QIIME based on the normalized OTU table and visualized in Cytoscape56. Global and pairwise differences among groupings of samples were tested by ANOSIM within PRIMER V.6.0 package. To identify the specific OTUs that characterized each of the salinity zones, we used Indicator Species Analysis run in R using the package Indicspecies57. Only OTUs with indicator values (IV) > 0.3 and P < 0.05 were considered good indicators. Environmental data were compiled and variables not normally distributed were transformed as close to normality as possible. Analyses were completed with a reduced set of 65 samples due to missing environmental data in some samples. Spearman rank correlation coefficient (ρ) was used in BV-STEP and Bio-ENV to determine the degree of association between similarity matrices of 18 S rRNA amplicons and environmental data. Prior to performing the analyses, highly correlated environmental variables (ρ > 0.90) were removed to ensure more interpretable results. Partial Mantel tests were conducted in R with Vegan package to assess individual effects of salinity/temperature on β-diversity after controlling for salinity or temperature55.To assess similarities between ciliate communities, pairwise similarities among samples were calculated by Bray-Curtis similarity coefficientSupplementary file


Observations and model experiments highlight the importance of ocean heat in forcing ice sheet retreat during the present and geological past, but past ocean temperature data are virtually missing in ice sheet proximal locations. Here we document paleoceanographic conditions and the (in)stability of the Wilkes Land subglacial basin (East Antarctica) during the mid-Miocene (~17–13.4 million years ago) by studying sediment cores from offshore Adélie Coast. Inland retreat of the ice sheet, temperate vegetation, and warm oligotrophic waters characterise the mid-Miocene Climatic Optimum . After the MCO, expansion of a marine-based ice sheet occurs, but remains sensitive to melting upon episodic warm water incursions. Our results suggest that the mid-Miocene latitudinal temperature gradient across the Southern Ocean never resembled that of the present day. We demonstrate that a strong coupling of oceanic climate and Antarctic continental conditions existed and that the East Antarctic subglacial basins were highly sensitive to ocean warming. Ocean heat is important in forcing ice sheet retreat, yet past ocean temperature data from proximal ice sheet locations are sparse. Here, the authors present temperature reconstructions from the Wilkes Land subglacial basin during the mid-Miocene, and show that warm waters sustained ice sheet retreat 17–14.8 Ma. Satellite monitoring demonstrates that the rate of basal melting (warm ocean melting the marine-terminated ice sheet margins from below) has exceeded that of surface melting (due to radiative forcing on the ice sheet surface)2. About 74% of the glaciers covering the Wilkes Land sector of East Antarctica have progressively retreated since 2000 ad due to ingression of warm waters, likely as a consequence of a reduction in sea–ice production and changes in ocean stratification4. East Antarctica marine-based subglacial basins have the potential of ~14 m sea level rise and are vulnerable to marine ice sheet instability5. Cryosphere–-ocean interactions, therefore, play a critical role in the current ice sheet mass imbalance. Observational data7 rarely cover a period of time longer than few decades6, and are therefore insufficient to provide a record of cryosphere (in)stability at adequately varying CO2 concentrations and temperatures as those predicted for the near future. Studying ice sheet instability and ice–ocean interactions during past warm geologic episodes, when atmospheric CO2 was analogous to present day or higher, can shed light on the long-term stability of continental cryosphere for our future.Assessment of the stability and dynamics of the Antarctic ice sheet in a changing climate is fundamental given its role in the climate system including global sea-level change. Observational data and modelling studies suggest consistently that influx of warm waters onto the Antarctic continental shelf invigorates ice retreat11. The Miocene Climatic Optimum  represents one of the warmest intervals since the inception of Antarctic glaciation12, with atmospheric CO2 concentrations as high as 500–600 parts per million by volume (ppmv)17, analogous to those expected for the end of the century given unabated carbon emissions. Global surface-ocean and deep-sea temperatures were ~3–6 °C and 5–6 °C above present-day values, respectively18. The termination of the MCO, widely referred to as mid-Miocene Climatic Transition , was characterised by progressive cooling and an expansion of global ice volume10; it coincided with a CO2 decline to close to pre-industrial values (200–300 ppmv17). To date, the variability in Antarctic ice sheet volume during the Miocene has mostly been inferred from far-field deep-sea oxygen isotope data11 and numerical modelling18, and attributed to the combined forcing of atmospheric greenhouse-gas concentrations and orbital variations. However, far-field sedimentary records leave ambiguity as to how much ice volume change is involved in deep-sea oxygen isotope variability, while numerical modelling have difficulties replicating the warm polar climates as derived from proxy data. The few available ice-proximal records, such as from the Ross Sea Antarctic Geological Drilling project (ANDRILL)21, have provided first evidence for a dynamic ice sheet during the Miocene and suggest extensive melting during peak MCO warmth21. Modelling experiments22 infer substantial ice-mass loss at boundary conditions of atmospheric ~500 CO2 ppmv, astronomical configuration favourable for deglaciation and 2 °C of surface-ocean warming. Ice sheet advances onto the continental shelves, with grounded ice extending into the Ross Sea, are simulated at 280 CO2 ppmv and a cold orbit. Hence, both the available field data and modelling efforts indicate that the mid-Miocene Antarctic ice sheets were highly sensitive to relatively small changes in atmospheric CO2 concentrations22. Such sensitivity can be triggered by changes in the ocean dynamics, which are complex, include several feedbacks, and have not yet been considered in most model simulations23.For the mid-Miocene epoch, geological records show major variations in Antarctic ice sheet volume, global sea level, ocean temperatures, and marine fauna and flora24 , subantarctic (SAF) and subtropical (STF) Fronts25  retrieved sediments from Site U1356  at the continental rise/abyssal plain boundary, at ~4000 m water depth, ~350 km offshore the Adélie Coast along the Wilkes Land margin, East Antarctica27, the Wilkes Land Site U1356, and the Ocean Drilling Program (ODP) Site 1171 south of Tasmania 9 contain mid-Miocene sediments and are ideally situated along a north–south latitudinal gradient 28 records 24 in the context of available Miocene data from the Ross Sea AND-2A record21 and ODP Site 11719. We demonstrate that marine-based ice sheets are absent or extremely reduced compared to present day during the MCO at the Wilkes Land. Surface waters similar to those found today close to the subtropical front bath this area and sustain ice sheet melting. A much greater ocean temperature gradient between the Ross Sea and the Wilkes Land sites exists compared to the present day, suggesting a different oceanographic structure. Continental conditions at the margin sustain growth of temperate vegetation and soil formation. After the MCO, sea ice occurs and ocean temperatures generally cool at the margin. However, episodic reoccurrence of warm waters destabilises the marine-based ice sheet and the continental cryosphere along the margin is substantially reduced compared to the present day even at pre-industrial atmospheric CO2 values. A larger-than-today ocean temperature gradient between the Ross Sea and the Wilkes Land site still exists.Here we document the environmental dynamics characterising both the circum-Antarctic ocean and the Antarctic continent during the early to mid-Miocene based on new palynological , organic geochemical, and sedimentological data from Wilkes Land Site U135624. The analysed sediment cores are well dated29. We herein use novel techniques of constrained optimisation (CONOP)30 to further improve the age model  found in the recovered part of the record  are dominated by comparatively higher concentrations of clay and dispersed gravel. Clast-poor diatom-rich beds locally preserve an interlaminated structure of clay-rich diatom ooze and diatom-rich silty clay. Clast-rich mudstones are interbedded with laminated silty claystones. Laminae are predominately sub-mm in thickness, and have sharp upper and lower contacts, suggesting localised reworking of the turbidite levee deposits by bottom currents32  in the Pacific sector of the Southern Ocean25, a setting consistent with the available tectonic reconstructions and core lithology24 for the site during the Miocene. A common to abundant component of the assemblages is the genus Operculodinium, which today rarely occurs in assemblages south of the Subantarctic Front33 , which occurs in the Ross Sea AND-2A record exclusively in the two warmest pulses of the MCO21, is here present throughout the record  oceanic environments occur. I. pallidum is the only species presently found in Antarctic environments in the vicinity of the polar front, where sea ice occurs, salinity can be seasonally reduced due to ice melting, and seasonality is high33. However, this is a species for which a clear tolerance to high temperatures has been demonstrated35, and its value as sea ice indicator can hence be questioned. Protoperidinioid heterotrophic dinocysts are present, but never abundant except at 15.5 Ma when a single peak (13.5%) of Selenopemphix antarctica  sea ice. Moreover, the absence or low percentages of reworked dinocysts encountered in this interval  and conifers (Podocarpidites spp.), both of which indicate woody subantarctic or sub-alpine vegetation , shrubs of the family Haloragaceae , grasses (Graminidites spp.), Caryophyllaceae (Colobanthus-type), and Sphagnum-moss (Stereisporites sp.) are abundant, particularly after the MCO (Supplementary Data Myricipites (nearest living relative (NLR): Casuarinaceae) as well as regularly occurring tree ferns (NLR: Cyathea) suggest that even after the MCT a cold-temperate woody vegetation with shrubs and trees still existed in sheltered areas of the coastal lowlands. Temperature reconstructions derived from the fossil pollen assemblages suggest mean annual temperatures (MATs) between 5.8 and 13 °C, and summer temperatures >10 °C  of the Wilkes Land sector of East Antarcticaion Fig. . Element41. We are also confident that most of the pollen, as well as any terrestrial organic matter (including soil), must have originated from close to the depositional site, as most pollen grains are well preserved and Antarctica was already isolated from surrounding landmasses at the Miocene time.Comparison of Miocene pollen assemblages with Eocene and Oligocene species from the same Site U1356 reveals a different taxonomic composition in all samples. We therefore exclude the possibility of reworked Palaeogene pollen being re-deposited into these sediments42) suggest temperatures of 11.2–16.7 °C   were fundamentally different from that of today. Deposition occurred in an open-water environment where only occasional erosion or reworking of older marine sediments and transport to the core location took place. Open-marine surface waters were warm temperate, lacked a strong sea ice component, and were relatively low in nutrients compared to today. Specifically, dinocyst assemblages resemble those found today in the Pacific sector of the Southern Ocean at around or north of the Subtropical Fronts, where sea-surface temperatures vary between 8 °C and 17 °C7–14.8 Ma46 during the MCO, i.e., 4° more to the north than today , humid, locally ice-free coastal zone along the Wilkes Land margin covered by woody vegetation dominated by southern beech and Podocarpaceae conifers. Plant communities similar to that reconstructed from the Wilkes Land record have also been derived from Miocene pollen records near McMurdo Sound/Ross Sea49, McMurdo Dry Valleys50, and the Antarctic Peninsula51. However, in contrast to previously published records, the pollen assemblages here contain exceptionally high percentages of Podocarpaceae conifers and a greater diversity of woody taxa, suggesting warmer conditions at the Adélie Coast than at other Antarctic locations, with maximum temperatures around the MCO. Temperature based on branched tetraether lipids, likely reflecting mean annual temperature or mean temperature of growing degree days above freezing52, are 10–12 °C . An additional palaeotemperature proxy based on inorganic chemical weathering indices53 indicates comparable, albeit lower mean annual temperatures of 6–8 °C to its terrestrial margin at peak warmth and ocean conditions warmer than those of today.46, Fig. Selenopemphix antarctica ) at 14–13.8 and 10.8 Ma suggest that nearby glaciers were terminating into the marine system, much in contrast to the MCO for which such evidence is entirely lacking. Few icebergs reach the latitude of Site U1356 today, despite abundant marine-based ice sheets nearby due to deflection by the strong Polar Current. The episodic presence of extensive IRD at Site U1356 after the MCO thus requires the presence of marine-based ice sheets and an ocean circulation different from present. The location of Site U1356 at ~13 Ma was at 60°S, close to the present-day position of the Antarctic Divergence. If the ocean structure were similar to today, high surface water productivity would be expected, and heterotrophic protoperidinioid dinocysts would dominate the assemblages. However, episodic high abundance of gonyaulacoids temperature cysts and N. labyrinthus, which is at present associated to northern oceanic fronts, suggest a different oceanographic structure. A weakened circulation compared to that of present day is indicated allowing episodic southward transport of warm waters at Wilkes Land Site and icebergs to escape northward out of the Polar Current.Our reconstructions for the post-MCO interval show a dynamic ice sheet and a fundamentally different environment compared to that of MCO. Wilkes Land Site U1356  in these sediments is generally low compared to those of the MCO, which is consistent with a colder, more productive ocean and less soil formation due to continental ice cover compared to the MCO. However, the pollen flora remains broadly unchanged, although higher abundance of the southern beech (Nothofagus spp.) and mosses  and sedimentological proxy data indicate erosion of older sediments from the shelf, likely during ice advance and stronger current activity. A hiatus between 13.4 and 11 Ma and seismic profiles suggesting the migration of the channel levee systems depocentres in the region point to ice sheet expansion on the continental shelf46) with existing Miocene reconstructions from the Ross Sea AND-2A record21  south of Tasmania  Circumpolar Deep Waters55 (CDW), if circulation was similar to that of today, or south displacement of warm surface waters. A weak (proto-) Antarctic polar front could possibly explain the movement of warmer waters from the north. The Southern Ocean circulation, the Antarctic Circumpolar Current (ACC), the front developments, and the upwelling of warm CDW depend on the wind patterns and topography, conditions for which there are poor constraints for the Miocene.To further investigate the importance of the Southern Pacific Ocean surface oceanographic structure and temperature patterns in relation to the continental cryosphere dynamics during and after the MCO, we integrated our Wilkes Land record , the latitudinal temperature gradient between both sites appears strongly reduced, with the Wilkes Land site recording exceptionally warm surface seawater. A much-reduced temperature gradient between the two sites could exist if the ACC and its oceanic fronts were weaker compared to today and/or the ACC was displaced southwards.A comparison between the Wilkes Land and the Southern Tasmanian ODP Site 1171 records shows comparable surface seawater temperatures  during the MCO Fig.  that are59, although younger ages have been proposed60. As the modern Southern Ocean circulation was possibly developed during the MCO, oceanic fronts were in place. The ACC and associated fronts are very sensitive to seafloor topography61, and a different topography may have had consequences for latitudinal heat transport. During the MCO the Tasmanian Gateway was narrower than at present . In conclusion, our data infer a fundamentally different oceanographic and environmental setting across the Pacific sector of the Southern Ocean during the MCO compared to the modern day.The exact timing of a modern-strength fully developed ACC is still uncertain, but usually indicated between 41 and <23 Ma28) show mean annual values of 16.5–20 °C  were derived using Constrained Optimisation methodology as presented in Crampton et al.30 for diatom turnover events in the Southern Ocean. We apply the same methodology but use both radiolarian and diatom data. CONOP generates a parsimonious, best-fit composite sequence of biostratigraphic events of radiolarian and diatom derived from 36 cores in the Southern Ocean, to which observed biostratigraphic events in U1356 could be placed. This paper uses the “hybrid range” model, which was shown by Cody et al.66 to be most suitable for sites close to the Antarctic continental margin. We use two versions of this model, a “strict” and “relaxed” version, which prohibits or allows for FAD and LAD contraction to move out of its observed range as a correlation is made between U1356 observations and the composite sequence. Full details on this methodology are discussed in Cody et al.66 and Crampton et al.30. To represent uncertainties, we apply the spread of the ages from all CONOP-placed FAD and LAD data in these two models at every depth where there is an observed biostratigraphic event. This is in contrast to the approach used by Tauxe et al.29, which assigned absolute values for FADs and LADs as were presented in the CONOP Average age range model of Cody et al.67. However, Cody et al.66 showed that the Hybrid age model converged upon a robust age model earlier than the Average age range model and thus is more appropriate to use. The magnetostratigraphy presented in Tauxe et al.29 is revised on the basis of these new CONOP constraints. We also include palynology-based FADs presented in Tauxe et al.29, and note there is now an improved fit between the various chronological datasets.We developed the age model to U1356, using an integrated biostratigraphic and magnetostratigraphic methodology similar to that presented in Tauxe et al.Lycopodium clavatum tablet with a known amount of marker spores  was added for quantification of palynomorph abundances68.Sample processing was performed at Utrecht University, following standard techniques of the Laboratory of Palaeobotany and Palynology. Samples were oven-dried and weighed (~15 g dry weight sediment each), and one 69 and informally and formally described species in the literature70. Of the 31 palynological samples analysed for dinocysts, 11 were either totally or almost barren (yielding only 15–25 dinocysts). These samples are still considered in our dataset; however, because of the low dinocyst yield, careful interpretation is required for these samples. Dinocyst percentages were calculated based on the total in situ dinocysts counted, excluding reworked specimens  dinocysts mostly include Impagidinium spp., Operculodinium spp., Batiacasphaera spp., Nematosphaeropsis labyrinthus, and Spiniferites/Achomosphaera spp. Protoperidinioid cyst percentages were calculated to identify productivity trends, as P dinocysts are likely produced by heterotrophic dinoflagellates71, while G are generally produced by phototrophic dinoflagellates. Reworked dinocysts include Eocene and Oligocene taxa . Reworked dinocyst percentages, calculated based on a sum comprising both in situ and reworked dinocysts, vary between 0% and 34%. In situ dinocyst absolute abundance  and cold 38% HF (Hydrofluoric acid), and sieved over a 10 µm mesh with occasional mild ultrasonic treatment. To avoid any potential processing-related preservation bias, no oxidation was carried out. The processed residue was transferred to microscope slides using glycerine jelly as a mounting medium, and 2–3 slides were analysed per sample at 400× magnification. Slides were used for both marine  and terrestrial (pollen and spores) palynological analyses. Dinocysts were identified based on a taxonomical index73. Percentages of reworked pollen and spores were calculated based on the sum of total pollen and spores. For calculation of in situ palynomorph percentages, reworked pollen and spore counts were excluded from the total sum. Reworked pollen and spores have been identified using visual colour and fluorescence microscopy41. Permian to Palaeogene reworked pollen and spores have a dark, yellowish colour indicating geothermal maturity, and were clearly distinguishable from well-preserved in situ palynomorphs.Of the 31 samples analysed for pollen and spores, 15 were productive, and total counts range between 80 and 210 pollen and spores 33. Prebble et al.32 augmented the information available from the Southern Ocean by increasing the number of samples analysed to 311. We use their data to derive present-day dinocyst assemblages across the Southwest Pacific fronts and produced the data presented in Fig. 32 analysed the relationships between assemblages and sea-surface temperature, water column depth, and productivity by means of ordination techniques and identified seven clusters. One of these clusters (Cluster 7) groups the South Atlantic samples only, north of the STF and is not considered here. Data from the other six clusters are presented in Supplementary Selenopemphix antarctica (SA), other protoperidinioids (oP), Impagidinium spp. (I), Nematosphaeropsis labyrinthus (Nl), Operculodinium spp. (O), and other Gonyaulacoids (oG) and are presented in percent relative to the total assemblage. These six groups are chosen either based on specific ecological preferences or because of their cosmopolitan occurrence33. Importantly, dinocysts in our Miocene record can also be categorised in the same six groups. In this way, we aimed to minimise the uncertainties in the environmental reconstructions potentially caused by extinct Miocene species, although we cannot discard the possibility that some taxa may have partly changed their preferred ecological niche through time35. However, we defer from quantitatively interpreting the changes observed and use the modern dinocyst distribution only as a qualitative tool. The percentages presented in Fig. Present-day ecological preference of dinocysts is based on the analyses of more than 2400 globally distributed surface sediment samples2O3 column chromatography using hexane/DCM (9:1), 100% DCM, and DCM/methanol (1:1) to yield the apolar, ketone, and polar fractions, respectively. The ketone fractions were checked for alkenones, but none of the samples were found to contain them. The polar fractions were analysed for tetraether lipids and were used to calculate the 75 the branched vs. isoprenoid tetraether BIT43, and the relative distribution of branched tetraether lipids used to estimate mean annual temperature45. The TEX86 ratio is based on isoprenoidal–glycerol dialkyl glycerol tetraether (GDGTs)76, which are assumed to derive mainly from Thaumarchaeota, formerly known as Crenarchaeota group 177, which is an abundant group of marine Archaea. The polar fractions were dissolved in a 99:1 hexane/propanol solvent, and were filtered using a 0.45 μm, 4 mm diameter polytetrafluoroethylene filter, before being analysed using a high-performance liquid chromatography/atmospheric pressure positive ion chemical ionisation mass spectrometry (HPLC/APCI-MS) as described by Schouten et al.78. Extractions were performed at Utrecht University, and HPLC/MS analyses were carried out at the NIOZ .Twenty-nine powdered and freeze-dried sediments (~15 g dry weight) were extracted with dichloromethane (DCM)/methanol (9:1) using the Dionex accelerated solvent extraction (ASE) technique. The extracts were separated by Al42 for water depth of 0–200 m. The analytical error is 0.3 °C, while the uncertainty introduced by the calibration error for 42  values based 42 and for the Bayesian calibration of TEX8681. These two calibrations produce similar values compared to each other, but are up to 8.5 °C degrees higher than the 82 due to changes in the GDGT 2/3 ratio. However, similar trends between The uncertainty introduced by the calibration error is in this case ±4 °C. We calculated the SST based on the 43 is used as indicator for continental organic matter input. A large input of organic matter from the continent also carries isoprenoid GDGTs, which can mask the autochthonous TEX86 signal83. We discarded 44.BIT indexConsequently, 10 of the 29 samples measured in the Wilkes Land record were excluded from the 86 can be further biased by an input of GDGTs derived from methane-utilising Archaea85. Furthermore, methane-generating Archaea, which mainly produce GDGT-0 but also minor amounts of GDGTs 1–3, can alter the TEX86 signal. The “Methane Index” can be calculated86, which evaluates the contribution of methanotrophic archaea to the total GDGT pool:TEXValues of 0.3–0.5 of MI mark the boundary between “normal marine sediments” and methane-impacted sediments. Calculations of this MI index for the studied Wilkes Land record vary between 0.05 and 0.23, in the samples for which temperatures are calculated and used, and between 0.03 and 0.06 for the AND-2A record , annual MAT can be estimated in samples where high BIT index values (>0.3). For this, the calibration of Peterse et al.52. For this, samples with high BIT indices were rerun using improved chromatography of brGDGTs89. Temperature were estimated using the calibration of De Jonge et al.45:However, newer methods and calibrations have recently become availableThe uncertainty introduced by the calibration error is ±4.6 °C . Original raw data GDGTs concentrations and chromatograms) are available from the corresponding author upon request.Supplementary InformationDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Supplementary Data 3Supplementary Data 4Supplementary Data 5


In vivo matured oocytes were utilized for canine somatic cell nuclear transfer (SCNT), and serum progesterone levels were assessed to accurately determine ovulation and oocyte maturation. Canine serum progesterone concentrations during both proestrus and estrus were analyzed by RIA and ECLI to determine the ovulation day. Although both methods detected similar progesterone levels before ovulation, the mean progesterone concentration determined using ECLI was significantly higher than of RIA three days before ovulation. Following ovulation, oocytes were collected by surgery, and a lower percentage of mature oocytes were observed using ECLI (39%) as compared to RIA (67%) if 4-8ng/ml of progesterone were used for determination of ovulation. A high percentage of mature oocytes was observed using ECLI when 6–15 ng/mL of progesterone was used for ovulation determination. To determine whether ECLI could be used for canine cloning, six canines were selected as oocyte donors, and two puppies were obtained after SCNT and embryo transfer. In conclusion, compared to the traditional RIA method, the ECLI method is a safe and reliable method for canine cloning.Radioactive immunoassay (RIA) is a traditional serum hormone assay method, but the application of the method in reproductive studies is limited by the associated radioactivity. The aim of present study was to evaluate the reliability of RIA and to compare its canine serum progesterone concentration determination accuracy to that of the electric chemiluminescence immunoassay (ECLI).  Several animals have been produced by nuclear transfer since the first successful cloning of an animal by somatic cell nuclear transfer (SCNT) was performed in 1996 , sheep , pigs 44, mice , rats 6, camels in vivo should be developed to obtain high quality oocytes for canine cloning.Unlike other mammalian oocytes, dog oocytes mature after ovulation in the oviduct . After oFor over 40 years, serum progesterone (P4) has been used to detect ovarian characteristics, estrous detection, and pregnancy in humans , cattle Recently, the non-radioactive electro chemiluminescence immunoassay (ECLI) was developed for antigen detection ,26. The Unless otherwise indicated, all reagents were purchased from Sigma Chemical Co. . Chemicals used for P4 detection were ordered from Roche Diagnostics .ad libitum. In all of the animal experiments, dogs were initially anesthetized using 6 mg/kg ketamine and xylazine, and anesthesia was maintained with 2% isoflurane. During surgery, the condition of each dog was continuously monitored using a pulse oximeter. After surgery, antibiotics (penicillin G and dihydrostreptomycin sulfate) and 2 mg/10 kg meloxicam were administered to prevent inflammation. The dogs were subsequently transferred to individual cages containing fresh water. The dogs were then monitored by the veterinarian in charge, and care was provided if the animals required specific treatments.In this study, 25–30-kg mixed origin, large-breed bitches, 2–5 years of age, were obtained from the canine research center of the National Institute of Animal Science (NIAS). All animal care methods and experiments were conducted in accordance with the Guide for the Care and Use of Laboratory Animals established by the Institutional Animal Care and Use Committee (IACUC) of NIAS . Dogs were raised indoors in separate cages in a temperature-controlled room, and natural dark-light cycles were utilized. Dogs were a fed commercial diet twice daily, and were provided with water Blood was collected from dogs in proestrus or estrus each morning. Serum samples were separated into two parts, and P4 concentrations were detected using RIA and ECLI methods. Regarding the RIA method, serum was analyzed using a 1470 Wizard r-counter at the Seegene Medical Foundation (South Korea). A “cobas e” automatic analyzer and an ECLI kit were employed for ECLI methods, according to the user manual. Before analysis, the analyzer was calibrated using the calibrated reagents supplied in the kit. The date when serum progesterone levels reached special levels based on the experimental design was considered the day of ovulation.in vivo cumulus oocyte complexs (COCs) obtained from oviducts were denuded and stained with 10 μg/mL Hoechst 33342, and oocytes with the 1st polar body (PBI) were designated as mature. However, oocytes with the PBI and a perivitelline space >25 μm and oocytes without the PBI were designated as ageing and immature, respectively.After 70–72 h of hypothetical ovulation, 2 and 95% air. A fibroblast monolayer was established after being cultured for 7 d, and it was then passaged, cryopreserved in 10% DMSO, and stored in liquid nitrogen. Cells from passages 3–5 were used as donor cells for SCNT.Ear skin fibroblast cells were surgically collected from dogs, and were then transported to the lab within 4 h in ice-cold D-PBS. The tissue was washed three times using D-PBS, minced, and cultured in ADMEM supplemented with 10% fetal bovine serum at 38°C in a humidified atmosphere composed of 5% COin vivo matured oocytes were recovered via aseptic surgical procedures 70–76 h after the day of ovulation. Oocytes surrounded by cumulus cell layers were denuded and stained with 10 μg/mL Hoechst 33342 for 5 min. The first polar body and metaphase II spindle of denuded oocytes were removed using micromanipulation with fluorescence. One donor somatic cell was injected into the perivitelline space of each enucleated oocyte, and was then fused using electric stimulation with two pulses of DC at 3 kV/cm for 15 μs. The fused couplets were activated by calcium ionophore treatment for 5 min, and were then cultured in mSOF medium supplemented with 1.9 mmol/L 6-DMAP for 4 h.For nuclear transfer, canine After activation, SCNT couplets were immediately surgically transferred using a 3.5-Fr Tom Cat Catheter into the ampullary portion of the oviducts of naturally synchronous recipients. Recipients were prepared by predicting the ovulation time based on serum progesterone concentrations. Pregnancy diagnoses were assessed using ultrasound detection approximately 31 days after the embryo transfer.t-test. The significance level was established as P<0.05.Statistical analyses were performed with SPASS , and statistical significance of the P4 concentrations were analyzed using the To compare the differences in serum P4 levels determined using RIA and ECLI methods, serum samples collected from 100 dogs were analyzed. Serum samples were separated into 10 groups based on the concentration determined by the RIA method. Colocalization and averages of the values determined using the two methods were analyzed.To detect the effects of RIA and ECLI methods on the determination of the oocyte stage, 90 dogs (30 dogs for each group) were collected as oocyte donors. Serum P4 levels reached 4.0–8.0 ng/mL, which was selected as the ovulation level when RIA and ECLI methods were used. Then, a new 6.0–15.0 ng/mL level was set for ovulation determination based on ECLI methods, and oocytes stages were determined at this level.For confirmation of whether ECLI methods could be used for dog cloning, the ovulation of oocyte donors and the estrus determination of recipient dogs were determined using RIA and ECLI, and SCNT and embryo transfer were subsequently conducted.2 = 0.9417) at low P4 concentrations (<1 ng/mL), but they sharply decreased at high concentrations  using chemiluminescence immunoassays (CLIA) has been broadly applied to clinical diagnoses and environmental analyses . In the In the present study, P4 data obtained by ECLI methods displayed a higher and broader range than that obtained using RIA methods. These results could explain the high number of immature oocytes and the low number of mature oocytes recovered by ECLI methods. Prior to experimentation, all machines were calibrated using commercial calibration solutions. However, according to the manual of the analyzer, only one commercial calibration solution was employed for quality control for ECLI analyses. Therefore, the difference between RIA and ECLI might have resulted from different calibration methods. To obtain further accurate data by using ECLI in future studies, different P4 standard solutions should be employed to determine a standard curve. Based on the data from the present study, manual calibration techniques should be introduced into animal production.Although data obtained using the ECLI method was higher than that acquired using RIA methods, more than 53% of the oocytes reached the MII stage. SCNT was performed to evaluate the feasibility of ECLI in canine cloning, and the production of a cloned canine was successful. Therefore, the results of this study indicated that ECLI methods are suitable for canine cloning.According to our data, the P4 concentration used for ovulation judgment should be increased 1–2 units if ECLI methods are used for canine reproduction. In conclusion, ECLI is an environmentally friendly and relative accurate method for canine cloning.S1 FilePercentage of oocyte donors at each stage detected by RIA (4-8ng/mL) or ECLI methods using P4 concentration of 4–8 ng/mL or 6–15 ng/mL as ovulation standards.(XLSX)Click here for additional data file.


In the newly diagnosed tuberculosis group with no history of MDR, mutations in rpoB531 were seen in 10 of the samples (11.6%). Collectively, all mutations in the RRDR region studied were observed in 80 (61.5%) of known MDR cases and in 14 (16.3%) of the newly diagnosed cases. Using the RRDR as a surrogate marker for MDR, sequences for the newly diagnosed (presumed sensitive) group indicate much higher levels of MDR than the 3.9% WHO 2015 global estimate and suggests that molecular screening directly from sputum is urgently required to effectively address the detection and treatment gaps to combat MDR in this high burden country.Molecular screening of new patients suspected for TB could help in the effective control of TB in Pakistan as it is a high TB burden country. It will be informative to understand the prevalence of multi drug resistance for a better drug regimen management in this geographical area. The Rifampicin resistance determining region (RRDR) sequencing was used to identify mutations associated with drug resistance in DNA extracts from 130 known multidrug resistant (MDR) cultured strains and compared with mutations observed in DNA extracts directly from 86 sputum samples from consecutive newly diagnosed cases in Lahore, Pakistan. These newly diagnosed samples were positive for smear microscopy, chest X-ray and presumed sensitive to first line drugs. In the known MDR group the most frequent mutations conferring resistance were found in  M. tuberculosis drug resistance detection using conventional methods is by culture of bacilli on a medium containing antibiotic and can require several weeks. However, with the development of rapid molecular methods it is possible to detect mutations in genes associated with resistance in a much shorter time [rpoB, particularly mutations at codons 516, 526 and 531. MDR-TB is defined as resistance to rifampicin and isoniazid, the two most effective anti-TB drugs. In December 2010, WHO recommended the use of the GeneXpert MTB/RIF to detect and infer resistance to rifampicin directly from sputum [Pakistan is among the top 20 countries with a high TB and MDR-TB burden . Chest Xter time . Resistater time . Moleculter time  and alleter time . Severalter time , 7, 8. Mter time . PCR tecter time . Whole Gter time  but requter time . Resistam sputum . In May This work was carried out in Pakistan to characterize mutations associated with rifampicin resistance directly from sputum samples from newly diagnosed (ND) patients with no history of drug resistance and identify their key risk factors in this setting. Identifying resistance in presumed resistant samples and inferring resistance profiles directly from sputum may enable a better tailored drug regimen where possible. It will also inform patient management more rapidly and consequently reduce the rate of onward transmission of MDR tuberculosis in high burden countries such as Pakistan.Tuberculosis patients attending the Ghulab Devi Chest Hospital, Lahore, Pakistan in collaboration with University of Health Sciences, Lahore were enrolled in this study over 18 months between 2013 and 2015, based on the following inclusion criteria. For the MDR group, patients diagnosed previously with TB, and with a history of resistance to first line anti-tuberculosis drugs, were included. Sputum samples were taken from this group and culture and Drug Susceptibility Testing (DST) were performed. The second group of patients included freshly diagnosed consecutive cases, presumed drug susceptible with clinical symptoms of TB, positive in sputum smear microscopy AFB, chest X-ray positive and no history of resistance to any first line tuberculosis drugs. Sputum samples were taken from this group and culture was not performed. Not all individuals were included; those with clinical complications in addition to tuberculosis were not included in this study.The patient’s history was collected using a proforma and included age, gender, area, economic status (earning less than 300 US dollars per month), information of previous anti-tuberculosis therapy, chest x-ray, AFB test and family history of TB. The environmental parameters studied were animal contact, source of drinking water, un-boiled milk use and smoking or drug use.GeneXpert testing was carried out only for six samples of the MDR group only due to lack of global funding, according to the manufacturer’s instructions. Newly diagnosed presumed susceptible samples were not tested due to the reason that these samples have no history of drug resistance. This test was performed in order to confirm the samples of MDR group before carrying out DST.M. tuberculosis culture under strict aseptic conditions. The colonies appeared on the LJ medium after 4–6 weeks of culturing at 37°C.For the MDR group, sputum suspension of each patient was made by mixing 0.5ml sputum in equal volume of autoclaved deionized water under aseptic conditions. 0.1ml of sputum sample was spread on LJ medium for M. tuberculosis colonies from the MDR group was carried out on LJ medium containing the different first line anti-tuberculosis drugs. The concentrations of drugs added were: rifampicin (40 μg/ml); isoniazid (0.2 μg/ml); ethambutol (2 μg/ml); pyrazinamide (50 μg/ml) andstreptomycin (4 μg/ml). Following a sterility check by incubating the culture bottles at 37°C for one week, the bottles were inoculated with an M. tuberculosis suspension of the previous culture. After 4–6 weeks incubation at 37°C in incubator, growth on a drug-free control medium was compared with growth on culture media containing each concentration of anti-tuberculosis drug. Any growth of M. tuberculosis colonies on drug containing LJ media were designated drug resistant while samples where no growth was observed on drug containing LJ media, were declared to be drug susceptible Mycobacterium tuberculosis.Sub culturing of Sputum samples from the presumed susceptible group of patients were collected and then DNA extraction was carried out. Both cultures (described above) and sputum samples were extracted using the column based TIANamp genomic DNA isolation kit  method. Quantity and quality of the isolated genomic DNA was determined by NanoDrop  using 1μL sample of purified DNA.rpoB gene of all strains were sequenced using published primers [2, 0.2mM dNTPs , 20μM each of both rpoB-RRDR forward (5’- CGATCACACCGCAGACGTTGA) and reverse primers (5’-GGCACGCTCACGTGACAGACC) and 5U recombinant Taq polymerase . The following PCR conditions were carried out using a Veriti thermocycler : 94°C for 2 min followed by 35 cycles of 94°C for 30 sec, 60°C for 30 sec and 72°C for 1 min. Finally, an extension of 72°C for 10 min was performed before cleaning the products using AmpureXP magnetic beads  according to the manufacturer’s protocol and forward and reverse sequencing performed.The fragment containing 81bp Rifampicin Resistant Determining Region (RRDR) of the  primers  and analThe present research work was approved by the ethical committee of University of the Punjab, Lahore, Pakistan in accordance with the ethical standards of the responsible committee on human experimentation and with the latest (2008) version of Helsinki Declaration of 1975 . The purGeneXpert testing was positive for six of the samples of MDR group and further verified by DST.Of the 130 MDR cultures tested for resistance to isoniazid (I), rifampicin (R), ethambutol (E), pyrazinamide (P) and streptomycin (S), 96 were resistant to IREPS, 26 were resistant to IREP, three were resistant to IR, two were resistant to IRES, one was resistant to IRP and two resistant to IRPS. Streptomycin was added so that to kill any other bacterial contamination in the culture in addition to drug sensitive mycobacteria.rpoB mutations seen in both groups is shown in An overview of the For the ND group, 14/86 of the extracted DNAs (16.3%) carried mutations associated with MDR status; the remainder showed wild type RRDR sequence. Of the 14, 8 DNAs carried a single mutation at position 531, TCG>TTG/Ser>Leu (57.1%); 2 DNAs carried a single mutation at position 531, TCG>TGG/Ser>Trp (14.3%); 2 DNAs carried a single mutation at position 516, GAC>GTC/Asp>Val (14.3%) and a further 2 DNAs carried a single mutation at position 516, GAC>TAC/Asp>Tyr (14.3%).M. tuberculosis–containing sputum samples for drug resistance, although recommended by the WHO, is expensive and inaccessible to many high incidence areas such as Pakistan. To gain further information about the prevalence of MDR in newly diagnosed patients in this area of Lahore, Pakistan, the RRDR region of the rpoB gene from two groups was sequenced; the first group comprised extracted DNA from130 MDR strains from patients diagnosed previously with TB, and with a history of resistance to first line anti-tuberculosis drugs; the second group comprised 86, DNA extracts directly from sputum samples from consecutive newly diagnosed patients, with clinical symptoms of TB, positive in sputum smear microscopy AFB, chest X-ray positive and presumed drug susceptible with no history of resistance to any first line tuberculosis drugs. The most common mutation found in both groups was in line with other studies, i.e. at position 531, TCG>TTG/Ser>Leu, and at 61.3% and 57.1% for MDR and ND groups respectively, at a similar prevalence.Molecular screening of rpoB gene so sequencing the RRDR does not correlate with rifampicin resistance as successfully. The reasons for this may be that resistance to rifampicin is conferred by mutations in other parts of the gene or genome or that the DST testing was sub optimal in some way and indicated resistance when none was present, although all patients from this group had a history of resistance to first line anti-tuberculosis drugs. Antimicrobial resistance testing (AST) or Drug resistance testing was established in the 1960s [Studies indicate that 96.1% of the rifampicin resistant strains worldwide will have rpoB mutations (so a surrogate marker for MDR) studies , 16. Comhe 1960s  and therThe main finding of this study however is the high incidence of rifampicin resistance associated mutations, which is often used as a surrogate marker for MDR. At 16.3%, it is considerably higher than the WHO estimate of 3.9% of new cases of multi drug resistant tuberculosis [S1 Fig(DOC)Click here for additional data file.