Here we review current knowledge specifically on layer 5 (L5) inhibitory microcircuits, which play a critical role in controlling cortical output. We focus on recent work from the well-studied rodent barrel cortex, but also draw on evidence from studies in primary visual cortex and other cortical areas. The diversity of both deep inhibitory neurons and their pyramidal cell targets make this a challenging but essential area of study in cortical computation and sensory processing.Inhibitory neurons play a fundamental role in cortical computation and behavior. Recent technological advances, such as two photon imaging, targeted  Before surveying the existent literature on layer 5 (L5) inhibitory neurons, we briefly review current knowledge on the connectivity and physiological properties of L5 pyramidal cells (PCs), as it provides important context for understanding L5 inhibitory circuits. One feature that distinguishes L5 from other cortical layers is the diversity of its PCs, which send a myriad of long-range projections to other cortical and sub-cortical structures  the pyramidal tract (PT) neurons, which project to subcortical regions, are located mainly in layer 5B, and display impressive “thick-tufted” apical dendritic morphologies; and (2) intratelencephalic (IT) neurons, which project mainly to other cortical regions and striatum, are found mainly in layer 5A, and have smaller and less complex dendrites  cells in L5, and especially L5B, encode a high degree of information about the temporal features of a sensory stimulus and may play an important role in initiating precisely timed sequences of spikes during sensory responses  between L5 PCs. FDDI is generated when an L5 PC fires a high-frequency burst of action potentials and excites a postsynaptic L5 Martinotti cell  suggest that inhibition onto L5 PCs most strongly originates from interneurons within L5 , is a nearly ubiquitous motif in cortical microcircuits where afferent excitatory input simultaneously recruits local inhibitory neurons that provide inhibition to the same cells receiving excitation. Circuits for FFI have been well studied at the projections from thalamus to L4  can recruit both L5 PV and SOM cells driving various forms of FFI. Interestingly, as described for FFI circuits in hippocampal CA1  can also lead to unintended problems. First, these lines are not completely non-overlapping. Up to ~10% of Cre expressing cells in the widely used SOM-IRES-Cre line may actually be PV expressing, fast-spiking interneurons (Hu et al., However, there are potential solutions to these problems. The first would be the development and adoption of new transgenic lines that target highly specific inhibitory cell types (Taniguchi et al., in vivo has mostly focused on superficial interneurons due to the technical difficulty of imaging deeper cells in the cortex due to light scattering, and the practical challenges of maneuvering recording pipettes into deeper layers under visual guidance. A number of recent technical advantages should overcome these challenges. These include adaptive optics for deeper in vivo imaging (Wang et al., As we alluded to above, the recent explosion of functional information on inhibitory subtypes New optical methods will also open new avenues for manipulation of interneuron activity. Novel techniques for spatial light modulation are poised to provide unprecedented optogenetic precision, enabling experimenters to arbitrarily target manipulations to single cells or even specific ensembles of neurons (Bovetti and Fellin, AN and HA researched, wrote, and edited the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


New data are presented using inhibitors of specific ROS producing enzymes to curb nodularin/MC-induced liver cell (hepatocyte) death. They indicate that enzymes of the arachidonic acid pathway, notably phospholipase A2, 5-lipoxygenase, and cyclooxygenases, may be required for nodularin/MC-induced (and presumably OA-induced) cell death, suggesting new ways to overcome at least some aspects of OA and MC toxicity.Okadaic acid (OA) and microcystin (MC) as well as several other microbial toxins like nodularin and calyculinA are known as tumor promoters as well as inducers of apoptotic cell death. Their intracellular targets are the major serine/threonine protein phosphatases. This review summarizes mechanisms believed to be responsible for the death induction and tumor promotion with focus on the interdependent production of reactive oxygen species (ROS) and activation of Ca The dinoflagellate toxin okadaic acid (OA) and the toxic cyanobacterial microcystins , pose a health hazard to both humans and livestock . Althougd-Ala1-Xaa2-d-MeAsp3-Yaa4-Adda5-d-Glu6-Mdha7) where the amino acids in the two and four position are variable. The most common structure is MC-LeuArg (MC-LR), but other variants such as TyrArg, LeuTrp, and LeuAla are also common. MC contains several non-proteinogenic amino acids, such as Adda , d-MeAsp (d-erythro-β-methylaspartic acid), and Mdha . The related PP inhibiting cyanobacterial cyclic pentapeptide nodularin (Nod) shares several non-proteinogenic amino acids with MC, such as Adda and d-MeAsp, but instead of Mdha, the dehydro-amino acid Mdhb (2-(methylamino)-2-dehydrobutyric acid) [OA and MC are relatively complex structures A,B, withN-methyldehydroalanine by N-methyldehydrobutyrine prevents the formation of a covalent linkage with Cys273.The striking similar action of these distinct compounds, which differ in primary structure, becomes understandable upon inspection of their in solution structure, and their docking into the PP active site A,B. The 50 values). MC-LR has high potency (IC50 ≤ 1 nM) against PP1, PP2A, PP3, PP4 and PP5, and may therefore be the inhibitor of choice to obtain a more generalized PP inhibition  to PP2A (50 = 0.015 nM) and that tautomycin, often considered a preferential PP1 inhibitor [50 < 0.6 nM for PP2A  to achieve significant dephosphorylation. This may lead to under-estimation of the ICxin  is a late phenomenon in MC or nodularin exposed hepatocytes . In contet al. [The ingestion of MC or Nod producing cyanobacteria can cause lethal acute liver damage. The death occurs too rapidly to be explained by hepatocyte metabolic failure alone, and is probably due to disintegration of the liver micro-architecture, accompanied by lethal bleeding ,44. As fet al. . The actet al. . Both shLoosening of cell-cell and cell-substratum contacts has been observed in hepatocyte monolayers . In factIt is questionable if mitochondrial are early targets in PP-inhibitor induced cell death. Thus, OA or CalA failed to synergize with cAMP to induce cell death in AML cells , in whicOA became early known as a promoter of skin tumors , and MC 2+/calmodulin dependent protein kinase II (CaM-KII) was found essential for OA-, MC- and nodularin- induced apoptosis and early phosphorylation events in a number of cell types, as well as for the OA-induced inhibition of hepatocyte DNA replication [Phosphatase inhibition can increase phosphorylation of a protein only if the relevant protein kinase(s) is active. The ubiquitously expressed Calication ,57,62. Slication  is reverlication , it is elication . More sulication ,62, whiclication ,62. Anotlication .Protein phosphatases are inhibited by ROS that target specific redox sensitive amino acids. The reversible inhibitory oxidation of protein tyrosine phosphatases by hydrogen peroxide was discovered in the mid-1990s, whereas redox regulation of Ser/Thr phosphatases is a more recent discovery . The PP2N-acetyl-cysteine and redox active proteins like peroxyredoxins and methionine sulfoxide reductases can facilitate the normalization of ROS modified proteins. PP inhibitors can therefore enhance ROS accumulation also by inhibiting the action of ROS scavengers or neutralizers.Several cellular activities can produce ROS A and are2O2 by mitochondrial superoxide dismutase. Mitochondria may not be the major primary target of CaM-KII mediated PP-inhibitor induced death as little impairment of nodularin-induced apoptosis was observed in hepatocytes pretreated (5 min) with inhibitors against mitochondrial complex I and III (rotenone and azide). Increased mitochondrial ROS has been reported in neurons undergoing OA-induced death [N-terminal Kinase and p38-MAPKinase apoptotic pathways [The oxidative metabolism in mitochondria forms superoxide  that caned death . Mitochopathways ,71,72,73The plasma membrane NADPH-oxidase generates superoxide, normally through stimulation by Rac , but als2O2 when the oxidation of substrate is uncoupled [The activity of oxygenases like xanthine oxidase (XO) and monoamine oxidases (MAO) can produce both peroxide and superoxide ,77. The ncoupled ,79, as wncoupled . It is oncoupled , suggestCytochrome P450 monooxygenases (CYP) can also generate ROS during conditions of poor coupling. The NADPH-CYP reductase and cytochrome b5 is a major source of ER-generated ROS . Other kInhibitors of cyclooxygenases COX1 and 2 (indomethacine) and against LOXs in general (nordihydroguaiaretic acid) were potent antagonists of nodularin-induced apoptosis. Interestingly, the 5-lipoxygenase (5-LOX) inhibitor AA861 protected against nodularin while the 12-lipoxygenase (12-LOX) inhibitor baicalein did not B. AlthouIt should be noted that okadaic acid enhances cellular CaM-KII catalyzed phosphorylation and activation of PLA2, which subsequently translocates from cytoplasm to perinuclear membranes , where iGimap5, also known as Ian5 and IROD, is located in the perinuclear centrosomal/Golgi/ER compartment and its coiled-coil domain is essential for protection against both OA and acute ionizing radiation . It was In addition to their obvious health consequences, the marine protein phosphatase targeting toxins like okadaic acid give insight into cell death mechanisms. It is particularly intriguing that major actions of the protein phosphatase inhibiting toxins are mediated by CaM-KII in conjunction with increase of cellular ROS, possibly primarily in the perinuclear region of the target cell.


Background: The El Niño/Southern Oscillation (ENSO) is a global climate phenomenon that impacts human infectious disease risk worldwide through droughts, floods, and other climate extremes. Throughout summer and fall 2014 and winter 2015, El Niño Watch, issued by the US National Oceanic and Atmospheric Administration, assessed likely El Niño development during the Northern Hemisphere fall and winter, persisting into spring 2015.Methods: We identified geographic regions where environmental conditions may increase infectious disease transmission if the predicted El Niño occurs using El Niño indicators  and literature review of El Niño-infectious disease associations.Results: SSTs in the equatorial Pacific and western Indian Oceans were anomalously elevated during August-October 2014, consistent with a developing weak El Niño event. Teleconnections with local climate is evident in global precipitation patterns, with positive OLR anomalies (drier than average conditions) across Indonesia and coastal southeast Asia, and negative anomalies across northern China, the western Indian Ocean, central Asia, north-central and northeast Africa, Mexico/Central America, the southwestern United States, and the northeastern and southwestern tropical Pacific. Persistence of these conditions could produce environmental settings conducive to increased transmission of cholera, dengue, malaria, Rift Valley fever, and other infectious diseases in regional hotspots as during previous El Niño events.Discussion and Conclusions: The current development of weak El Niño conditions may have significant potential implications for global public health in winter 2014-spring 2015. Enhanced surveillance and other preparedness measures in predicted infectious disease hotspots could mitigate health impacts. El Niño and La Niña, the warm and cold phases, respectively, of the ENSO cycle, impact human health across much of the world through increased risk of natural disasters, such as droughts, floods, and tropical cyclones. These in turn affect agricultural yields, cause air pollution due to landscape fires, and enable transmission of various infectious diseasesDuring the summer and fall of 2014 and winter of 2015, the US National Oceanic and Atmospheric Administration (NOAA) Climate Prediction Center (CPC) issued an El Niño Watch. The most recent estimate of January 8, 2015 assessed an approximately 50-60% chance of El Niño developing during the next 2 months (Northern Hemisphere winter) and persisting into spring ,,,ad hoc updates of ENSO assessments and possible ENSO-associated infectious disease risks. Here we draw on recent climatological data, and historical El Niño-health associations, to assess the likelihood of El Niño in coming months. We identify geographic regions where infectious disease transmission may increase if the predicted El Niño occurs.Following a strong El Niño in 1997-1998, US Government organizations and international partners collaborated to initiate a climatological monitoring program as part of a global surveillance network for emerging infectious diseasesDetailed descriptions of the El Niño indicators and other climatological data used in our program are available elsewhereSST in the NINO3.4 region , often used as an indicator of the phase and amplitude of ENSO events .SST anomalies in the Western Indian Ocean (WIO); warmer NINO3.4 and WIO SSTs indicate a higher likelihood of El Niño.Global SSTs (1981-present).Outgoing Longwave Radiation , an indicator of cloudiness .Precipitation .To identify regions at risk for El Niño-associated infectious disease activity, we used the literature review of Kovats et al.SSTs in the NINO3.4 and WIO regions reached higher-than-normal temperatures in October 2014  after cooling during the summer.The tongue-appearing pattern of the SST anomaly off the Peruvian coast, with SST anomalies ≈ +1.5°C along the equator, is typical of a developing El Niño event .WIO warming  off the east African coast also is consistent with previous El Niño events. Anomalous positive SST in the eastern Pacific Ocean drives tropical and extra-tropical convective activity influencing global rainfall and temperature patterns.The SST anomalies during August-October 2014 are comparable to those preceding the 2006-2007 El Niño, with more extreme elevations in the WIO and less extreme but positive and rising NINO3.4 elevations (Table 1).2]) in OLR across Indonesia and coastal southeast Asia indicate drier than average conditions, while large negative departures (<-40 W/m2) across northern China, the western Indian Ocean, central Asia, north-central and northeast Africa, Mexico/Central America, the southwestern United States, and the northeastern and southwestern tropical Pacific suggest wetter than normal conditions .Some impacts from the current SST anomaly patterns can be observed in the pattern of global convective activity illustrated by the OLR anomaly patterns. During August through October 2014, large positive departures .Currently, several regions show extreme departures in rainfall, including southeast Asia, northeast Africa, Mexico/Central America, and the southwestern United States .Considering these typical El Niño effects on regional weather and the recent El Niño forecast, there is a high likelihood for drought conditions to prevail in southeast Asia, west-central and southern Africa, northeast Brazil, Ecuador, and Mexico during the next 5 to 8 months; as well as elevated rainfall and flooding in southwest Asia, eastern Africa, coastal Peru and Ecuador, much of the southern portion of South America, and the US southwest, including southern California.Vibrio parahaemolyticusThe development of these conditions has important implications for global public health. For each of several diseases, including ones of high global health significance, multiple epidemiological studies have linked El Niño events with increased incidence in human populations (Table 2).Aedes aegypti and Aedes albopictus mosquitoes) and often circulates in the same areas. Wet conditions linked to El Niño have been associated with increased dengue incidence on the Mexico-United States (Texas) borderEl Niño may impact dynamics of other diseases related to ones with demonstrated El Niño associations. For example, chikungunya virus shares vectors with dengue virus .Based on current global climate anomaly conditions and forecasts, El Niño is likely to develop during late 2014 and persist into early 2015. The expected effects on regional weather patterns include persistent high temperatures and drought in some areas, and heavy rainfall and flooding in others. This may enhance populations of particular vectors and the transmission of various infectious diseases in human and animal populations.Although local weather conditions mediate part of ENSO’s influence on infectious disease transmission (“teleconnections”), incorporating ENSO indicators into disease risk predictions offers advantages. ENSO forecasts typically can anticipate local weather effects several months before they manifest, providing lead-time for public health risk communication, enhancement of disease and vector surveillance programs, provisioning of clinical resources , and other preparedness measures. Also, a large-scale climate phenomenon such as ENSO may predict ecological processes better than weather-based models by integrating the effects of multiple weather variables that interact in complex waysOur El Niño-based infectious disease risk assessment for coming months requires some caveats. The development of El Niño events cannot be predicted with complete certainty. However, we expect predictability to be higher for this near-term forecast . Predictability even at longer lead-times has been favorable without a high false-alarm rate during 1857-2003,A limitation to ENSO-based infectious disease predictions is the complex relationship between ENSO the and dynamics of certain diseases. The relationship has been strong and consistent for some diseases, such as RVF in East AfricaOur observations do not attempt to predict disease outbreaks or even elevated transmission risk, but instead highlight the occurrence of current and future ENSO conditions that might influence environmental conditions that influence the dynamics of certain diseases . The linkages we describe between climate and disease transmission are based on many years of observations of teleconnections between specific persistent weather/climate anomalies and specific vector-borne and water-borne disease outbreaks in specific regions.We encourage public health organizations to take steps to mitigate El Niño impacts, such as increased incidence of infectious disease, in areas likely to be affected. While some measures are specific to certain diseases, preparations should include risk communication to the public and relevant sectors; vector and public health surveillance to detect increased disease activity; and planning for surge capacity to respond to increased disease activity. In particular, we emphasize the need for public health monitoring as El Niño develops to improve understanding of regional health effects and facilitate development of early warning systems.The authors have declared that no competing interests exist.


Pyramidal cells have two different action potential initiation sites; one at the soma, which receives inputs from synapses contacting the basal dendrites or the soma, and one at the distal apical dendrite and tuft . At the We investigated whether these two firing patterns can be synchronized by oscillatory inhibition in order to transmit top-down or combined top-down and bottom-up information to different brain areas. We hypothesize this mechanism as an explanation for the synchronization of burst spiking to distant local field potentials at beta frequencies (12-20 Hz) that was recorded in the prefrontal cortex and the anterior cingulate cortex during attentional control .We simulated the behavior of pyramidal cells stimulated by oscillatory inhibition at the tuft and noisy excitatory inputs at the basal dendrite and tuft. First, we investigated the occurrence and phase-locking of FAC and BAC firing in one cell as function of the ratio of excitatory basal and tuft inputs. Second, we examined the synchronization of a population of pyramidal cells downstream to oscillatory inhibition with frequencies in the beta range. The pyramidal cells were modeled using four compartments and including calcium dynamics in the apical dendrite and tuft .For single pyramidal cells FACs predominantly occur when the tuft receives stronger excitation than the basal dendrites, while BACs occur predominantly when the tuft and basal dendrites are excited equally strong. Both mechanisms have in common that they dominate the behavior of the pyramidal cell only when oscillatory inhibition is in the lower frequency range (2-20 Hz). For a 1:2 ratio between excitatory basal and tuft input FACs accounted for up to 85% of all spike events, whereas for a ratio of 1:1 BACs constituted up to 68%. Bursts activated by FACs and BACs were strongly phase-locked to the inhibitory drive, while bursts activated by other mechanisms had a lower coherence. The synchrony between pairs of pyramidal cells was quantified using the Schreiber measure [


The dentate gyrus (DG) of mammals harbors neural stem cells that generate new dentate granule cells (DGCs) throughout life. Behavioral studies using the contextual fear discrimination paradigm have found that selectively augmenting or blocking adult hippocampal neurogenesis enhances or impairs discrimination under conditions of high, but not low, interference suggestive of a role in pattern separation. Although contextual discrimination engages population-based coding mechanisms underlying pattern separation such as global remapping in the DG and CA3, how adult hippocampal neurogenesis modulates pattern separation in the DG is poorly understood. Here, we propose a role for adult-born DGCs in re-activation coupled modulation of sparseness through feed-back inhibition to govern global remapping in the DG. The dentate gyrus (DG) of all mammals harbors neural stem cells that generate new dentate granule cells (DGCs) throughout life  engendered a reduction in spread of VSD signal and strength of neuronal activation in the DG. Conversely, ablating 14 weeks of age and younger adult-born DGCs produced a trend towards increased excitability of the DG. In these experiments the change in the VSD signal was seen throughout the GCL suggesting a non-cell autonomous role for adult-born DGCs in modulating the activation of mature DGCs. Mossy fibers of DGCs synapse onto hilar interneurons and mossy cells, both of which can modulate the activity of the DG. Activation of hilar interneurons and mossy cells is thought to promote local inhibition and long-range activation and inhibition of DGCs, respectively  interneurons in vivo. Whether activation of 4 weeks old adult-born DGCs activate other populations of hilar interneurons that mediate feed-back inhibition onto the DG was not addressed. These observations suggest that 4 weeks old adult-born DGCs may exert modest feed-back inhibition through a potentially different mechanism, at least ex vivo. As with our study, the use of ex vivo slices precluded the possibility of addressing the potentially important contribution of mossy cells to adult-born DGC dependent recruitment of feed-back inhibition in vivo. Nevertheless, the findings of this study unequivocally demonstrate that 8 weeks old adult-born DGCs exert significantly greater feed-back inhibition onto the DG than 4 weeks old adult-born DGCs ex vivo  are encoded by 4–6 weeks old adult-born DGCs (time point t1). Maturation of this population of 4–6 weeks old adult-born DGCs generates a population of mature adult-born DGCs (>4–6 weeks of age) with high input specificity for features of context A. In addition, feed-back inhibition recruited by 4–6 weeks old adult-born DGCs ensures sparseness of activation in the DG Figure . DecreasUpon exposure to a similar context B that shares features with context A Figure . Greaterin vivo? Second, do adult-born DGCs at distinct stages of maturation or developmentally generated DGCs differentially modulate sparseness and recruit different levels of feed-back inhibition in vivo? Third, how are mossy cells and specific populations of interneurons recruited by DGCs to modulate sparseness and global remapping? The complexity of addressing this undertaking is underscored by the fact that engagement of hilar interneurons and mossy cells may be biased by different neuromodulatory systems that innervate these cell types and firing patterns of dentate granule neurons , or place cells recordings in vivo with concomitant assessment of identity will unequivocally enable direct assessment of DGC re-activation. Together, these approaches will permit resolution of how ablating or expanding populations of adult-born DGCs of different ages influences global remapping in the DG.The predictions of this model are readily testable and will necessitate addressing the following questions. First, how does enhancing or silencing populations of adult-born DGCs at distinct stages of maturation or developmentally generated DGCs, impact global remapping in the contextual discrimination paradigm The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Reduced estimated glomerular filtration rate (eGFR) using the cystatin-C derived equations might be a better predictor of cardiovascular disease (CVD) mortality compared with the creatinine-derived equations, but this association remains unclear in elderly individuals.The aims of this study were to compare the predictive values of the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI)-creatinine, CKD-EPI-cystatin C and CKD-EPI-creatinine-cystatin C eGFR equations for all-cause mortality and CVD events .Prospective cohort study of 1165 elderly women aged>70 years. Associations between eGFR and outcomes were examined using Cox regression analysis. Test accuracy of eGFR equations for predicting outcomes was examined using Receiver Operating Characteristic (ROC) analysis and net reclassification improvement (NRI).Risk of all-cause mortality for every incremental reduction in eGFR determined using CKD-EPI-creatinine, CKD-EPI-cystatin C and the CKD-EPI-creatinine-cystatic C equations was similar. Areas under the ROC curves of CKD-EPI-creatinine, CKD-EPI-cystatin C and CKD-EPI-creatinine-cystatin C equations for all-cause mortality were 0.604 (95%CI 0.561–0.647), 0.606  and 0.606  respectively. For all-cause mortality, there was no improvement in the reclassification of eGFR categories using the CKD-EPI-cystatin C  and CKD-EPI-creatinine-cystatin C  compared with CKD-EPI-creatinine equation. Similar findings were observed for CVD events.eGFR derived from CKD-EPI cystatin C and CKD-EPI creatinine-cystatin C equations did not improve the accuracy or predictive ability for clinical events compared to CKD-EPI-creatinine equation in this cohort of elderly women. However, epidemiological studies have also shown that eGFR between 60–74.9 mL/min/1.73 m2 is associated with a higher risk of CVD-related death compared to eGFR of ≥75 mL/min/1.73 m2 in patients following myocardial infarction suggesting that the risk of adverse clinical events is not confined to those with eGFR of less than 60 mL/min/1.73 m2Chronic kidney disease (CKD) is a major public health burden worldwide. Patients with CKD, especially those on dialysis, suffer from reduced life expectancy and quality of life Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) equation 2.Several newly-derived eGFR equations such as the CKD-EPI cystatin C and CKD-EPI creatinine-cystatin C equations have shown improvement in the precision and accuracy of determining GFR compared to CKD-EPI creatinine equation, but uncertainties remain as to the clinical significance and cost-effectiveness of using cystatin C-derived eGFR estimations over creatinine-derived eGFR estimations in the general population, particularly in elderly individuals. A recent meta-analysis of sixteen population cohorts reported both CKD-EPI cystatin C and combined CKD-EPI creatinine-cystatin C equations improved the accuracy in predicting all-cause and CVD mortality compared to CKD-EPI creatinine equation, but the majority of the included population cohorts were younger individuals of mixed gender with dissimilar proportion of muscle mass One thousand five hundred women were recruited in 1998 to a five-year prospective, randomized, controlled trial of oral calcium supplements  to prevent osteoporotic fractures, the Calcium Intake Fracture Outcome study  Baseline medical history including the presence of diabetes, hypertension, smoking history (current/former smokers or non-smokers) and medications were obtained from all participants. Blood pressure was measured on the right arm with a mercury column manometer using an adult cuff after the participants have been seated in an upright position and had rested for 5 minutes. An average of three blood pressure readings was recorded.Inker et al and these are presented in Fasting blood samples were collected at baseline (i.e. at time of randomisation in 1998) with sera stored in −70°C freezer until analysis. Creatinine and cystatin C measurements were performed using stored sera after 2008 and results were available in 1165 women (77%). Serum creatinine was analysed using an isotope dilution mass spectrometry (IDMS) traceable Jaffe kinetic assay for creatinine on a Hitachi 917 analyser . Serum cystatin C was measured on the Siemens Dade Behring Nephelometer, traceable to the International Federation of Clinical Chemistry Working Group for Standardization of Serum cystatin C and the Institute for Reference Materials and Measurements certified reference materials. eGFR was estimated by three equations derived by Participants' general practitioners verified their medical histories and medications where possible, and were coded using the International Classification of Primary Care–Plus (ICPC-Plus) method th Revision, Australian Modification (ICD-10-AM), I00-I99 The primary outcomes of the study were all-cause mortality and CVD hospitalizations and/or mortality retrieved from the Western Australian Data Linkage System (WADLS) for each of the study participants from 1998 until 10 years following their initial study visit. CVD hospitalizations and mortality were defined using primary diagnosis codes from ICD-9-CM, 390-459 Baseline characteristics were expressed as mean and standard deviation (SD) for continuous variables or as number and proportion for categorical variables. Association between eGFR and all-cause mortality and CVD hospitalization and/or mortality was examined using Cox proportional hazard regression model and results were expressed as hazard ratio (HR) with 95% confidence interval (CI) for every incremental reduction in eGFR to allow comparison between equations. The covariates included in the Cox regression models were age, smoking history, body mass index (BMI), diabetes, antihypertensive medications, systolic blood pressure, treatment code, prevalent renal and CVD.2), and then reclassified into new eGFR categories with CKD-EPI cystatin C equation and CKD-EPI creatinine-cystatin C equation as compared with CKD-EPI creatinine equation. P-values of less than 0.05 in two tailed testing were considered statistically significant. The data was analysed using SPSS  and STATA .To assess performance of the different equations for estimating eGFR, we assessed the discrimination of the three different models using the Area Under Curve (AUC). Discrimination refers to how well the model distinguishes individuals with and without the outcomes of interests. To assess discrimination, we calculated the area under the receiver operating characteristic (ROC) curve (AUC). An area of 1 implies perfect discrimination, whereas an area of 0.5 represents random discrimination. The sidak option provides adjusted p-values comparing the ROC areas between eGFR equations, assuming a “gold standard” being the CKD-EPI creatinine equation. For net reclassification improvement (NRI), participants were classified into three eGFR categories for all-cause and CVD hospitalization and/or mortality , which was similar for CKD-EPI cystatin C and CKD-EPI creatinine-cystatin C equations.There was at least over 30% increase in CVD events between participants with eGFR of <60 mL/min/1.73 mquations . Howeverquations . For CKDFor prediction of all-cause mortality, the AUCs varied between 0.604 , 0.606  and 0.606  respectively using the CKD-EPI creatinine, CKD-EPI cystatin C and CKD-EPI creatinine-cystatin C equations adjusted for age, BMI, hypertension, diabetes, systolic blood pressure, prevalent renal disease and CVD, smoking history and treatment group . The corFor the prediction of CVD hospitalization and/or mortality, the AUCs varied between 0.660 , 0.659  and 0.660  respectively using the CKD-EPI creatinine, CKD-EPI cystatin C and CKD-EPI creatinine-cystatin C equations adjusted for age, BMI, hypertension, diabetes, systolic blood pressure, prevalent renal disease and CVD, smoking history and treatment group . The corThe reclassification of eGFR categories in predicting all-cause mortality and CVD hospitalization and/or mortality between CKD-EPI cystatin C and CKD-EPI creatinine-cystatin C equations compared with CKD-EPI creatinine equation is shown in In elderly individuals, the accurate evaluation of eGFR for CKD staging is critical to determine correct drug dosing and risk stratification for major clinical events including CVD and all-cause mortality. Our study findings suggest that the association between reduced GFR and clinical outcomes is similar for eGFR equations with and without cystatin C. In addition, the combined CKD-EPI creatinine-cystatin C eGFR or CKD-EPI cystatin C prediction equations were not superior in predicting or reclassifying CVD hospitalization and/or mortality or all-cause mortality over the CKD-EPI creatinine eGFR equation in a cohort of elderly women.Eriksen et al. has shown that cystatin C was not superior in estimating measured GFR compared to creatinine in the general population Cystatin C appears to be a superior GFR marker compared to creatinine 2. The use of the combined CKD-EPI creatinine-cystatin C equation was able to improve reclassification of individuals with creatinine-derived eGFR of 45–74 ml/min/1.73 m2 , and also 17% of individuals with creatinine-based eGFR of 45–59 ml/min/1.73 m2 to ≥60 ml/min/1.73 m2. In a recent meta-analysis of 11 general population studies comprising of 90,750 participants, there was a more consistent linear association between reduced eGFR derived from CKD-EPI cystatin C and CKD-EPI creatinine-cystatin C equations and increased risks of all-cause and CVD mortality for all eGFR values below 85 mL/min/1.73 m2 compared with CKD-EPI creatinine equation, well above the threshold of 60 mL/min/1.73 m2 for the detection of CKD with CKD-EPI creatinine-based eGFR Two recently developed CKD-EPI creatinine-cystatin C and CKD-EPI cystatin C equations were shown to perform better in predicting measured radionuclide GFR compared to CKD-EPI creatinine equation 2) and therefore the applicability of our study findings to males, other ethnic minorities or racial groups and those with poor nutrition and low muscle mass remains unclear.The strengths of this study include the use of a large prospective cohort of subjects with complete and accurate data collection over a 10-year period. We were able to accurately examine the association between estimates of GFR using the newly developed cystatin C equations and clinical outcomes in a population with a low prevalence of CVD and renal diseases, which further strengthens this association. However, the strengths of the study must be balanced against the limitations, which include a lack of radionuclide GFR measurements and availability of single time-point measurements of creatinine and cystatin C to estimate baseline GFR. In addition, our study cohort only included white female participants with presumed adequate nutrition and muscle mass Click here for additional data file.


Andropogon gerardii), widely used in restorations, and resulting in local adaptation. We seeded three regional ecotypes of A. gerardii in reciprocal transplant garden communities across 1150 km precipitation gradient. We measured ecological responses over several timescales  in response to climate and biotic factors and tested if ecotypes could expand range. The ecotype from the driest region exhibited greatest cover under low rainfall, suggesting local adaptation under abiotic stress. Unexpectedly, no evidence for cover differences between ecotypes exists at mesic sites where establishment and cover of all ecotypes were low, perhaps due to strong biotic pressures. Expression of adaptive differences is strongly environment specific. Given observed adaptive variation, the most conservative restoration strategy would be to plant the local ecotype, especially in drier locations. With superior performance of the most xeric ecotype under dry conditions and predicted drought, this ecotype may migrate eastward, naturally or with assistance in restorations.Identifying suitable genetic stock for restoration often employs a ‘best guess’ approach. Without adaptive variation studies, restoration may be misguided. We test the extent to which climate in central US grasslands exerts selection pressure on a foundation grass big bluestem ( Worldwide, temperate grasslands are one of the most threatened ecosystems , these restoration efforts have often used genetic cultivars , a dominant C4, long-lived, perennial outcrossing grass (Weaver and Fitzpatrick A. gerardii) across the 1150 km longitudinal precipitation gradient of the Great Plains of the United States (latitude remains approximately constant). Andropogon gerardii has a wide natural distribution across the eastern United States (http://plants.usda.gov), with greatest dominance in the Great Plains  gradient in our study plots . Yet, the degree of adaptive variation within species across precipitation gradients is poorly characterized for most plants. Grassland covers one-third of continental North America  to test the extent to which ecotypes might respond to increasingly dry conditions  What is the relative role of genotype and environment in controlling these differences? (iii) How will different ecotypes of A. gerardii respond under different climatic conditions, especially precipitation, in native and reciprocally transplanted environments? (iv) How can we use this information to make predictions regarding restoration of A. gerardii in current and changing climates? To answer these questions, we measured responses of A. gerardii over several timescales, ranging from instantaneous gas exchange to medium-term responses (chlorophyll absorbance) to establishment and cover over 2 years. This combination of short-, medium-, and long-duration responses should provide a comprehensive understanding of A. gerardii ecotype responses to climate across the Great Plains.This study addressed the following questions: (i) Are gs, and higher photosynthesis, A), higher chlorophyll absorbance, greater seedling establishment, and increased vegetative cover, (ii) the central KS (CKS) ecotype, collected from xeric source populations in the west, would maintain higher gs, A, and chlorophyll absorbance, and have greater plant cover compared to ecotypes from more mesic source populations in the east  when all were grown in xeric planting sites, (iii) mesic sites  were expected to have greater plant cover, irrespective of ecotype, and plants growing there should have higher A and gs, and (iv) if local adaptation is strongly enforced by climatic selection for drought ecotypes in arid areas, then the CKS ecotype should outperform other ecotypes when planted in Colby, KS, the most arid end of the gradient. Here, we present results from the first 2 years of vegetative performance as an indication of fitness potential in the ongoing reciprocal garden restoration experiment.Specifically, we hypothesized that (i) regional ecotypes  would perform the best relative to nonresidents in their home environment as measured by gas exchange ; Hays, KS (580 mm); Manhattan, KS (871 mm); and Carbondale, IL (1167 mm)  Moench ssp. drummondii (Nees ex Steud.) de Wet & Harlan) and sorghum (S. bicolor (L.) Moench ssp. bicolor) were most recent in Hays. Soybeans (Glycine max (L.) Merr.) were planted in Manhattan for 5 years prior to the study. The Carbondale site was cultivated to corn (Zea mays L.) the year prior to seeding. Soils were all classified as loams; soil at the eastern three sites was classified as silt loam, and Colby as silt clay loam. Other chemical properties varied somewhat from site to site  were cultivated for >30, 70, and 74 years prior to seeding, respectively. The easternmost site, Carbondale, IL, had a more variable land-use history and was cultivated since 2004. In Colby, wheat and sorghum were the most recent crops, whereas sudangrass  for which resident seeds were collected and planted at each site, were purchased from a commercial supplier . Seeds sold by this supplier were sourced from across the Great Plains and grown for production in the Harpers Ferry nurseries. Species composition of the planted seed as well as seeding rate is typical for prairie restorations. While we would have preferred collecting local seed of all species used in the experiment, this was intractable due to the plot size and overall size of the experiment . Furthermore, broad-scale restorations typically use commercial seed making our experiment relevant for restorations. Additionally, plants of volunteer species from regional propagule sources also established on their own in the garden sites, making the composition of the community at each garden site a mix of volunteer and planted species  and sown to each regional ecotype in June 2009, using a 70:30 ratio of live Cies Goad . Each plWe monitored soil moisture at each site and compiled precipitation data for each region. Soil moisture from 0 to 20 cm depth was monitored using four EC-20 soil moisture probes  that were arranged diagonally across the plots at each site, with one probe per block. Probes were positioned at the beginning of the growing season (early May) and measured every 3–4 days in all sites with the exception of Colby, KS  to account for phenology differences across the 1150 km gradient. GDD was later used as a covariate in statistical analyses. In this manner, we could explore differences among sites for a given GDD, thus accounting for percent of the elapsed growing season. We developed GDD accumulations for big bluestem, with modifications from maize  were randomly positioned in four locations within each plot to count A. gerardii seedlings. The purpose of these measurements was to quantify establishment in the first growing season following seeding in June 2009. No other species were counted. Establishment was evaluated using a one-time measurement at each site at the end of the growing season in mid-October. The majority of the measurements were carried out the following year to minimize maternal effects.Establishment and cover are intended to represent long-term measures of plant performance. Establishment of 2 quadrat was used, the quadrant consisted of one intersection every 10 cm for a total of 81 intersections  of each species in the plots  was harvested at the end of each growing season, weighed, and analyzed separately to provide an estimate of community-level responses. These data are reported separately  3–4 times during summer 2011. SPAD represents a medium-term (weeks) response variable. Chlorophyll absorbance , stomatal conductance , transpiration , internal CO2 , and intrinsic water-use efficiency (WUE = A/gs) from six plants randomly selected from each plot using an LI-6400 . Gas exchange was measured on a young, fully expanded leaf with CO2 levels at 385 ppm, humidity and temperature at ambient levels, and photosynthetically active radiation (PAR) at 1500 μmol photons m−2 s−1. Measurements were made on sunny days between 10:00 and 15:00 h to minimize adjustment time to leaf chamber conditions. Measurements were made when photosynthesis and stomatal conductance had stabilized (<3% coefficient of variation). Gas exchange was measured in the plants three times during the 2010 growing season: early in the growing season, mid-season, and late in the growing season. Different instruments were used to measure gas exchange at each site. We tested each instrument against a gas standard, and values were accurate within 2.8% of actual values.Gas exchange measures are intended to represent instantaneous measures of plant performance. We measured photosynthesis , ecotype , and their two-way interaction. Random effects of block were nested within site and crossed with ecotype to recognize the appropriate experimental unit for each fixed effect factor. Parameter estimation was conducted using residual pseudo-likelihood with Newton Raphson with ridging as the optimization technique. Kenward-Roger’s procedure was used to estimate degrees of freedom and to make the corresponding adjustments in estimation of standard errors.Vegetative cover, SPAD, and gas exchange were measured multiple times through the season at the four sites. In all cases, a generalized linear mixed model was fit to the response variable. The statistical model included the fixed effects of planting site , ecotype , and their two-way interaction. Proportion GDD in linear and quadratic polynomial form  was fit to the model as a covariate, along with all two- and three-way interactions with the fixed effects of site and ecotype. Due to significant high-order interactions involving GDD for many of the response variables, comparisons between sites and ecotypes were conducted at selected values of GDD that lay within the range of the groups intended for comparison. More specifically, for cover, comparisons were conducted at 0.35 and 0.65 GDD of the growing season elapsed, as this was the range of GDD for which cover was observed in all sites. Chlorophyll absorbance was compared between sites at 0.50, 0.60, and 0.70 GDD of the growing season elapsed, as  was the range of GDD for which chlorophyll absorbance was observed in all sites. In turn, for gas exchange measures, comparisons were conducted between 0.40 and 0.70 GDD, as this was the range of GDD across sites for data collection on gas exchange.Ci and WUE, the random effect of block nested within site yielded a variance estimate that converged to zero and was thus removed from the model. Furthermore, for Ci and WUE, the Bayesian information criterion indicated improved model fit when heterogeneous residual variances were specified for each site. Variance components were estimated using residual maximum likelihood. Kenward-Roger’s procedure was used to estimate degrees of freedom and make the corresponding adjustments in estimated standard errors. Model assumptions were evaluated using externally studentized residuals and were considered to be appropriately met.Random effects included block nested within site and also crossed with ecotype, to recognize their appropriate experimental units. For the models fit to sas . Least square mean estimates, as well as estimated standard errors or 95% confidence intervals, are reported for each response analyzed. Comparisons of interest were adjusted using Tukey–Kramer  or Bonferroni (simple effect differences) to avoid inflation of Type I error rate due to multiple comparisons.Statistical analyses described thus far were implemented using the GLIMMIX procedure of anova method to compute a coefficient of determination R2 for each ecotype. Model assumptions were evaluated using studentized residuals and were considered to be reasonably met. The statistical models for each ecotype were fitted using the GLM procedure of sas . Parameter estimates and corresponding standard errors are reported.For each ecotype, linear regression models were fitted to the response percent cover for big bluestem (average of measurements early and late in the season for a given block) as a function of historic mean annual precipitation (MAP1961). For each ecotype, the statistical model evaluated linear, quadratic, and cubic effects of MAP1961 on percent cover. Variance components were estimated using the P = 0.832). However, a main effect of ecotypes was identified on seedling counts , with the CKS ecotype having disproportionately greater cover at the drier Hays and Colby planting sites, relative to the other ecotypes  and at 0.65 GDD (P < 0.001). These values of GDD were selected for inference as they were the most extreme within the observed range of GDD for all planting sites. In Hays and Colby, the CKS ecotype had significantly greater cover than the SIL or EKS ecotypes, regardless of GDD . SIL and EKS ecotypes had cover that was not significantly different from each other at any of the sites throughout the growing season (P > 0.30). In general, total plant cover  at all sites was high >80%, suggesting good establishment of focal grass species A. gerardii, as well as plants from the eight seeded species and plants of volunteer species arriving from the regional propagule pool , with greatest cover observed in the drier planting locations. For EKS and SIL ecotypes, the functional relationship between percent cover and historic MAP was characterized as quadratic in shape (P < 0.0001 in each case). This implied that for the more mesic EKS and SIL ecotypes, greatest cover occurred at planting sites with intermediate precipitation levels. The coefficient of determination R2 obtained from the fitted linear regressions indicated historic MAP accounted for 82.1%, 69.1%, and 74.1% of the variability in percent cover of big bluestem for CKS, EKS, and SIL ecotypes, respectively.To further investigate the differential performance of the ecotypes across the climatic gradient, we plotted vegetative cover of each ecotype as a function of the historical mean annual rainfall at each site Fig.. The natP < 0.001, Fig.There was no evidence for any three-way interaction between planting site, ecotype, and GDD. However, a two-way interaction was apparent between planting site and the quadratic term for proportion GDD on the SPAD chlorophyll absorbance (P ≤ 0.04) than other ecotypes at the Colby planting site. At the Hays planting site, the CKS and SIL ecotypes were not significantly different from each other, yet had consistently greater SPAD values than the EKS ecotype (P ≤ 0.04) in the earlier part of the growing season. Similarly, at the Manhattan planting sites, the CKS and SIL ecotypes were not significantly different from each other and had consistently greater SPAD values compared to the EKS ecotype (P < 0.002). No evidence for ecotypes differences in SPAD values was apparent at the Carbondale planting site at any point during the growing season (P ≥ 0.89).Pairwise comparisons between ecotypes at each site indicated that, for most of the growing season, the CKS ecotype generally had greater SPAD values  was mostly influenced by site and stage of the growing season, as a two-way interaction was apparent between planting site and GDD in quadratic form . By 0.7 GDD, A was significantly lower at the driest site in Colby, KS relative to the other sites (P ≤ 0.045).Leaf-level photosynthesis (P = 0.032). At 0.6 GDD in Colby, photosynthesis was marginally greater for the CKS ecotype (9.9 ± 1.5) compared to the EKS ecotype .Pairwise differences between ecotypes were identified at drier planting sites, where the CKS ecotype differed in photosynthetic rates from the more mesic ecotypes from EKS and SIL. More specifically, at 0.3 GDD, photosynthesis was higher in the CKS ecotype (20.7 ± 1.5) compared to the SIL ecotype (15.1 ± 1.5) at the Hays site  showed patterns similar to those observed in A and differed mostly by planting site and growing season . Regardless of ecotype, differences in stomatal conductance between sites varied dynamically through the growing season. During the early growing season, gs was greater at the wettest site in Carbondale compared to all other sites (P ≤ 0.003). As the growing season progressed, gs decreased quadratically at Carbondale, Colby, and Manhattan planting sites. In the Hays planting site, gs showed a slight decrease followed by an increase so that by late in the growing season, gs in Hays was significantly greater than in all other sites (P < 0.001). Pairwise differences between ecotypes were identified only at the Hays site for 0.3 GDD, with the CKS ecotype showing greater gs stomatal conductance (0.2 ± 0.01 mol H2O m−2 s−1) compared to the more mesic ecotypes from EKS (0.15 ± 0.01) and SIL (0.15 ± 0.01) in these conditions (P ≤ 0.026).Stomatal conductance to water vapor  showed a site-specific quadratic pattern over the growing season . Early in the growing season (GDD = 0.4), Ci in Manhattan was greater than in Carbondale (P = 0.019), whereas the remaining sites were intermediate and not significantly different from either. As the growing season progressed, Ci in Colby and Hays increased whereas Ci in Carbondale decreased, all in a quadratic manner , and Ci in Colby was greater than at the Manhattan and Carbondale sites (P ≤ 0.011). No differences in Ci among ecotypes were detected at any of the sites throughout the growing season (P ≥ 0.387).Intercellular COason Fig.C, regardnner Fig.C. Later E, mmol H2O m−2 s−1) showed patterns similar to those reported for other gas exchange measures, where a site-specific quadratic pattern was apparent for E over the growing season, regardless of ecotype (P = 0.002). For all ecotypes, differences in E between sites varied dynamically through the growing season. Early in the growing season (GDD = 0.4), Hays had greater E compared to the Manhattan and Carbondale planting sites (P < 0.02). From the middle of the growing season through the end of measures (GDD ≥ 0.5), the Hays site had significantly greater E compared to all other sites (P < 0.001), which were not significantly different from each other (P > 0.32).Leaf-level transpiration  than the SIL ecotype , in both Hays and Colby sites (P < 0.06). Also at the Hays site, the CKS ecotype had greater E than the EKS ecotypes . For most of the remaining growing season, there was no evidence for ecotypes differences at any of the sites. Nevertheless, at 0.8 GDD in Manhattan, KS, the CKS ecotype (4.8 ± 0.46) outperformed the SIL ecotype (3.2 ± 0.46) and showed greater E than the SIL ecotype (P < 0.01).Pairwise comparisons indicated differences in A/gs, had a site-specific quadratic pattern over the growing season . Early in the growing season (GDD = 0.4), WUE in Hays and Carbondale was greater than in Manhattan (P ≤ 0.006), but none was significantly different from the Colby planting site, which showed an intermediate WUE. As the growing season progressed, the estimated WUE decreased quadratically in the drier sites [i.e. Hays and Colby (KS), Fig.P ≤ 0.016), that is, with higher WUE at wetter sites. Ecotype differences in WUE were apparent only at the Hays site early in the growing season (0.3 GDD), whereby the EKS ecotype (119 ± 3.7) had significantly greater WUE compared to the CKS (104.5 ± 3.7) and SIL (106 ± 3.7) ecotypes .Intrinsic WUE, calculated as A. gerardii across the climatic gradient of the US Great Plains. Within this variation, we found evidence of local adaptation in A. gerardii performance (establishment and vegetative cover) at xeric locations along the climatic gradient, suggesting prevailing local adaptation under abiotic stress. This suggests that in spite of gene flow, large population sizes, and an outbreeding mating system, we find clear evidence of local adaptation in some ecotypes. Thus, historical and current climate selection pressures appear strong enough to overcome homogenizing effects of gene flow , with ecotypes from local populations outperforming nonlocal transplants in subenvironments such as different climates  will detect if this same pattern persists over time or changes dynamically.In our study, we characterized this phenotypic variation and further detected evidence for local ecotypic adaptation for the CKS ecotype in the form of the longest timescale measurements of establishment and cover. At a shorter timescale, subtle ecotype differences were also detected in leaf chlorophyll absorbance, where the CKS ecotype exhibited higher SPAD values across sites relative to the other ecotypes of Additional insights can be gleamed about the extent to which big bluestem can extend its range into even drier environments. Using our Colby site as a surrogate for harsher abiotic conditions, we can make predictions if there are certain ecotypes that might survive and maintain populations in the arid part of the Great Plains or under future drier climates. Although there were few differences in short-term measures of gas exchange, meaningful ecotypic differences were detected for the long-term measures of establishment and cover. In Colby, the CKS ecotype far outperformed (by a factor of 2–3 times) the SIL and EKS ecotypes in terms of establishment and cover. Interestingly, when planted with competition in Manhattan and Carbondale, there was no difference in establishment or cover between the CKS ecotype and the SIL and EKS counterparts, thereby suggesting local adaptation of the CKS ecotype to the more xeric region of the Great Plains. Moreover, greater success in vegetative performance of the CKS ecotype in Hays and Colby occurred despite no evidence for ecotypic differences in short-term gas exchange measures. This suggests the range of the CKS ecotype could potentially expand eastward with increased predicted drought.A. gerardii. This suggests a role for climatic selection of ecotypes and their adaptive variation to abiotic stress tolerance at the extreme of the climatic range. Similar results on the role of adaptive variation to extreme ranges have also been found by Barnes , neither the EKS nor the SIL ecotypes showed any evidence for ‘home site advantage’. In contrast, both SIL and EKS ecotypes showed a quadratic functional relationship between cover and precipitation Fig., such thIn contrast to the strong evidence for ecotypic differences on establishment, cover, and SPAD, planting site factors (not ecotypes) appeared to control gas exchange almost exclusively. Few ecotype differences in gas exchange were detected throughout the growing season, and any differences were of small magnitude. Instead, any anticipated home site advantage of the CKS ecotype was overshadowed by strong site effects on various components of short-term water use and gas exchange Fig., Table 4One advantage of our study is we carried out our reciprocal garden experiment in a plant community (polyculture) with focal big bluestem and nonfocal species all planted in the field as seed, thus allowing us to test for local adaptation to biotic stress under more realistic ecological conditions. In contrast, many reciprocal garden experiments are carried out with weeded or herbicided, spaced plants in monoculture McMillan . Thus, owww/aqueduct.wri.org/atlas). Investigation of ecotypic variation of an ecologically dominant grass in the current and changing climate of the Great Plains is essential to make predictions about grassland response to climate change. The climate system of the Great Plains is dynamic, with increasing frequency and severity of current and predicted droughts . Indeed, A. gerardii is a foundation species used in these conservation plantings on marginal lands throughout the US grasslands. Furthermore, only 4% of historical tallgrass prairie remained in the United States. In the eastern Great Plains, where prairie conversion to row crop agriculture has been most intense, mere hundreds of hectares remain  ecotype work of Casler et al. (A. gerardii ecotype, and cautiously emphasize planting across precipitation zones.Our study provides evidence for different s Bailey  of Greats Bailey . Early sr et al. , 2007a,br et al.  recognizA. gerardii, given the high within-population genetic diversity reported in these populations (Price et al. The potential to use local ecotypes for restoration practices (Hufford and Mazer


Mutants of lamin A cause diseases including the Hutchinson-Gilford progeria syndrome (HGPS) characterized by premature aging. Lamin A undergoes a series of processing reactions, including farnesylation and proteolytic cleavage of the farnesylated C-terminal domain. The role of cleavage is unknown but mutations that affect this reaction lead to progeria. Here we show that interphase serine 22 phosphorylation of endogenous mutant lamin A (progerin) is defective in cells from HGPS patients. This defect can be mimicked by expressing progerin in human cells and prevented by inhibition of farnesylation. Furthermore, serine 22 phosphorylation of non-farnesylated progerin was enhanced by a mutation that disrupts lamin A head to tail interactions. The phosphorylation of lamin A or non-farnesylated progerin was associated to the formation of spherical intranuclear lamin A droplets that accumulate protein kinases of the CDK family capable of phosphorylating lamin A at serine 22. CDK inhibitors compromised the turnover of progerin, accelerated senescence of HGPS cells and reversed the effects of FTI on progerin levels. We discuss a model of progeria where faulty serine 22 phosphorylation compromises phase separation of lamin A polymers, leading to accumulation of functionally impaired lamin A structures. The nuclear lamina is a fibrous arrangement underneath the inner nuclear membrane that plays an important structural role determining the mechanical properties of the nucleus ,2. One oLamin A is expressed as prelamin A and undergoes post-translational modifications, including farnesylation of C-terminal CaaX motif, endoproteolytic cleavage of the last three amino acids, methylation of C-terminal cysteine, and a second C-terminal endoproteolysis that removes the farnesyl group . FarnesyA major question in the understanding of laminopathies is how the molecular defect in the lamin A gene translates into disease symptoms. Laminopathic mutations interfere with the functions of the nuclear lamina leading to delayed or aberrant mitosis  and defeHere, we investigated the role of serine 22 phosphorylation in both lamin A and progerin functions. We found that progerin is defective for serine 22 phosphorylation in interphase but the defect can be corrected by farnesyl transferase inhibitors or mutations that prevent farnesylation. Further serine 22 phosphorylation of progerin can be stimulated by a mutation that prevents head to tail interactions in lamin A. Intriguingly, progerin mutants that undergo serine 22 phosphorylation as well as a phosphomimetic S22D lamin A mutant form intranuclear lamin droplets. CDK inhibitors inhibit serine 22 phosphorylation of lamin A, increasing the levels of this protein accelerating the entry in senescence of fibroblasts from progeria patients. We present a new model of progeria where permanent farnesylation leads to defects in serine 22 phosphorylation. We propose that lack of phosphory-lation at serine 22 compromises phase separation of solid lamin A polymers, leading to accumulation of a fibrous lamin A structure that alters the functions of both the nuclear lamina and nucleoplasmic lamin A.To study the biology and turnover of progerin, the lamin A mutant protein responsible for Hutchinson-Gilford progeria syndrome, we used retroviral vectors that express the wild type prelamin A, mature lamin A (mimicking the protein obtained after cleavage by ZMPSTE24) and mutant derivatives Figure . ExpressWe were not able to detect endogenous lamin A in the nuclear lamina in cells with foci of Progerin CS probably because non-farnesylated progerin transits through the nuclear lamina, interacts with lamin A and carries it to the foci. To confirm the idea that Progerin CS can interact with wild type lamin A and recruit it to foci, we cotransfected U-2 OS cells with GFP-pre-lamin A and RFP-progerin CS and verified their localization using fluorescence microscopy. We found that non-farnesylated progerin and lamin A-GFP colocalized in foci Figure .We next studied the phosphorylation of progerin and progerin CS at serines 22 and 392 in U-2 OS cells using phospho-specific antibodies. In non-synchronized cells, little progerin is phosphorylated at serine 22 but phosphorylation at serine 392 was readily detectable Figure . ProgeriSince FTI-277 can correct the farnesylation defect of progerin and improve its phosphorylation at serine 22, we next investigated its effects on cell growth. Consistent with previous report , FTI-277Inhibition of lamin A farnesylation does not completely abolish its incorporation into the nuclear lamina ,25. In aStaining cells expressing these constructs with the anti-phosphoserine 22 antibody revealed a nucleoplasmic signal. Of note, none of the lamin A at the nuclear envelope was positive for serine 22 phosphorylation except the few foci at the nuclear envelope formed by R50P lamin A . On immuNext we performed a similar mutational analysis in progerin. Progerin R50P localized mainly in the nuclear envelope similar to lamin A R50P. In contrast, Progerin CS with the R50P mutation formed intranuclear foci Figure  similar Of note, lamin A contains several regions of low complexity according to DisEMBL , a methoNext we evaluated the effects of R50P and CS mutations on the ability of progerin to inhibit cell growth in the tumor cell line U-2 OS. The CS mutation, but not the R50P mutation, prevented growth inhibition by progerin Figure . This isThe phosphorylation of serine 22 of lamin A is a function of CDK1 during mitosis . HoweverTo investigate whether inhibition of phosphorylation affects lamin A levels, we expressed mature lamin A . Using this construct we can now visualize that both flavopiridol and the CDK4/6 inhibitor PD0332991 increased mature lamin A levels Figure  suggestiNext we studied whether preventing farnesylation could increase the phosphorylation of endogenous progerin in cells obtained from patients with HGPS. Treatment with FTI-277 decreased the levels of endogenous progerin so we normalized the signal of serine 22 phosphorylation on the levels of progerin in cells. After this normalization, it is clear that FTI-277 increased serine 22 phosphorylation of endogenous progerin Figures . We concTo determine whether inhibition of farnesylation affected the localization of endogenous progerin, we used immunofluorescence in normal and HGPS fibroblasts treated with FTI-277. Both wild type lamin A and progerin localized to intranuclear foci after treatment with FTI-277 Figure  and thesIt is known that older cells express higher levels of CDK inhibitors such as p16INK4a  and p21 We propose a dynamic model of lamin A functions, localization and turnover controlled by defarnesylation and serine 22 phosphorylation Figure . The firHowever, inhibition of farnesylation cannot correct all defects associated to progerin expression. First, treatment with FTI does not prevent the ability of progerin to inhibit cell growth (this study and ). SecondMature lamin A remains in the nuclear lamina after defarnesylation by head to tail contacts between lamin filaments  and by iCDK1 phosphorylates lamin A at serine 22 in the nuclear envelope as part of the mitotic nuclear envelope disassembly pathway . CDK4/6 In cells taken from HGPS patients, progerin accumulates in older cell cultures in association with nuclear abnormalities and mitotic defects ,39. A neIn conclusion, we show that defects in defarnesylation of prelamin A impair serine 22 phosphorylation. Hence, mutations in ZMPSTE24 leading to accumulation of farnesylated prelamin A and the progerin mutation have a common molecular defect that could explain why they are both associated to cellular senescence and aging ,42. The http://www.selleckchem.com/). PD0332991 and FTI-277 were purchased from Sigma-Aldrich.Human osteosarcoma U-2 OS cells , BJ fibroblasts (a gift from S. W Lowe) and fibroblasts from progeria patient  were cultured in Dulbecco's modified Eagle's medium  supplemented with 10% (for U-2 OS and BJ) or 15% (for HGADFN003) fetal bovine serum (FBS) (Wisent). Flavopiridol was from , lysed in sodium dodecyl sulfate (SDS) sample buffer , and boiled for 5 min. For Western blotting, 25 μg of total protein were separated on SDS-polyacrylamide gel electrophoresis gels and transferred to Immobilon-P membranes . We used the following primary antibodies: anti-LaminA/C , anti-phospho-Ser22LaminA , anti-phospho-Ser392LaminA , anti-progerin , anti-p38 , anti-CDK6 , anti-CDK2 , anti-MCM6 , anti-Tubulin . Signals were revealed after incubation with anti-mouse or anti-rabbit secondary antibodies coupled to peroxidase (Biorad) by using enhanced chemiluminescence (Amersham Pharmacia).Cells were plated on coverslips at least 24h prior to fixation with 4% paraformaldehyde in PBS for 15 minutes at room temperature. After washing with PBS, cells were permeabilized using 0.2% Triton X-100 in PBS–3% bovine serum albumin (BSA) solution for 5 min. The cells were then washed three times with PBS–3% BSA and incubated for 1-2 h at room temperature with the primary antibody: anti-laminA/C (636) , anti-phospho-Ser22LaminA , anti-CDK4 , anti-CDK2 , anti-CDK6 , anti-p38 , anti-phospho-MSK1 , anti-phospho-RSK , anti-MEK1/2 , anti-phospho-ERK1/2  diluted in PBS/BSA. Next, cells were washed three times in PBS/BSA and incubated with the appropriate secondary antibody  for 1h at room temperature. Finally, cells were rinsed three times with PBS alone and once with PBS containing 300 nM DAPI for 10 minutes. Images from independent fields were captured with a fluorescence microscope and processed with Metamorph.For superresolution structured illumination microscopy, cells were fixed as described above and stained with anti-lamin A/C antibody (as above) or anti-fibrillarin . After washing they were stained with Alexa Fluor 568 goat anti-rabbit IgG (H+L)  and Alexa Fluor 568 goat anti-rabbit IgG (H+L) . Images were obtained with Super Resolution microscope Axio Observer Z1 ZEISS Elyra PS.1, SR-SIM, 3D PALM, STORM from Zeiss. We picked up images sets of 5 subsets each captured after rotating the grid by 5 degrees. A high resolution image was extracted from the raw data using the Structured Illumination Microscopy and the Maximum Intensity Projection algorithms at a lateral resolution (XY) of 120 nm and an axial resolution (Z) of 300 nm.


Immunological investigations of β-lg have reported several IgE-binding regions of the protein that were linked with allergic reaction. Most IgE epitopes are specific for surface features of the protein. However, no clear association of structural feature or function with allergenicity has been established.Heat treatment has been used as a means to influence the functional properties  of β-lg and it has been proposed as a means to reduce milk protein allergenicity. Although pressure -and temperature- induced denaturation of β-lg has also been used for modification of the functional properties of the protein, there is a lack of information about the antigenicity as a marker for the allergenic status of β-lg. Then, the objective of this study is to determine the modification in IgE binding to β-lg when applying High Pressure (HP), High Temperature (HT) and the combination of both technologies, HPHT.o C. Concurrently, same samples were treated only by heat treatment at 75 and 95o C for same times. Then, an in vitro test by indirect competitive enzyme-linked immunosorbent assay (ELISA) with recombinant monoclonal IgE was performed in order to determine the alterations produced in the specific antigen-antibody immunoreactivity.Samples of β-lg from bovine milk in several media at different pH were pressurized at 600 MPa in a range time of 0 to 6 minutes at 25, 75 and 95o C clearly affect the antigenity of β-lg. In addition, beyond pressure and holding time temperature is an important parameter to modulate the antigenity of this allergen.The experiments showed little changes in the allergenicity of the β-lg when heat treatments or HP at room temperature were applied. Nevertheless, results of combined HPHT treatments at 600 MPa and temperatures thereupon 75HPHT treatment should be considered as an alternative processing technology for reduction of allergenicity especially in the field of milk and milk products with high allergy risks that can even improve the nutritional and physic-chemical properties.


Miniaturisation of somatic cells in animals is limited, for reasons ranging from the accommodation of organelles to surface-to-volume ratio. Consequently, muscle and nerve cells vary in diameters by about two orders of magnitude, in animals covering 12 orders of magnitude in body mass. Small animals thus have to control their behaviour with few muscle fibres and neurons. Hexapod leg muscles, for instance, may consist of a single to a few 100 fibres, and they are controlled by one to, rarely, 19 motoneurons. A typical mammal has thousands of fibres per muscle supplied by hundreds of motoneurons for comparable behavioural performances. Arthopods—crustaceans, hexapods, spiders, and their kin—are on average much smaller than vertebrates, and they possess inhibitory motoneurons for a motor control strategy that allows a broad performance spectrum despite necessarily small cell numbers. This arthropod motor control strategy is reviewed from functional and evolutionary perspectives and its components are described with a focus on inhibitory motoneurons. Inhibitory motoneurons are particularly interesting for a number of reasons: evolutionary and phylogenetic comparison of functional specialisations, evolutionary and developmental origin and diversification, and muscle fibre recruitment strategies. The central nervous systems of animals supply the muscles of body and limbs via motor nerves. The motor neurons in these nerves activate the muscles to produce contractions and ultimately control behaviour. Muscle relaxation occurs spontaneously after excitatory input has ceased, affording no particular control of muscle inactivation. Excitatory motoneurons are all that is needed, and they are the only motoneuron type present in vertebrates. This makes the existence of inhibitory motoneurons in arthropods appear enigmatic, and inhibitory motoneurons actually remain unknown even to many contemporary physiologists. By the same token, the function of inhibitory motoneurons has evaded proper understanding , and in spiders and their kin, the chelicerates. The ultimate reason for the evolution of inhibitory motoneurons probably lies in the small size of most arthropods, compared to vertebrates, resulting in a roughly proportionately small number of muscle and (motor) neurons. Small cell numbers cause problems for the smooth control of movement and for a broad spectrum of muscle performance, from tonic posture control to fast evasive movements or predatory strikes. In conjunction with a set of further features, inhibitory motoneurons allow the sophisticated control of muscle contraction with very few motoneurons. In this way, arthropods achieve motor performances that are quite comparable to those of higher vertebrates, a surprising feat considering their small bodies, small cell numbers, and other features, such as lack of axon myelination  supplies the majority of leg muscles in the orthopteran hexapods examined to date. This has been demonstrated in varying detail in locusts  and correspondingly reduced muscle supply, and it is not used for postural maintenance and locomotion but for chewing  motoneuron should supply most or all muscles of a leg Wiersma  Fig. 1)1), thus The cellular characteristics of muscle fibres are notably similar throughout the animal kingdom. This is particularly striking when comparing the well-studied skeletal muscles of vertebrates and arthropods, and it indicates that specialised muscle cells already existed before these major animal groups diverged  cells in arthropods probably for this very reason. Smaller cells provide more capacity for the particular cellular function, here storage capacity through modification of synaptic contacts. As outlined above, this option does not exist for muscle cells due to the necessary accommodation of contractile machinery, and it is restricted for motoneurons due to the reduced conduction velocity with small axon diameters, among other reasons.The muscles of mammals contain between a few hundred and a few million muscle fibres, depending on animal and muscle size   inhibitory innervation. This is true for the scaphognathite, an appendage that is used in malacostracans to ventilate the respiratory cavity below the carapace  or chloride (Cl−) ions  inhibitory input is seemingly paradox, which has made an understanding of presynaptic inhibition more difficult initially.In some neurons, the equilibrium potential for ClLike in the case of postsynaptic inhibition, the structure of the synaptic contact may contribute significantly to the inhibitory effect . A backfill of the main leg nerve marks the motoneurons supplying the leg muscles, and subsequent immunohistochemistry directed against GABA, the transmitter of inhibitory motoneurons, marks candidate inhibitory moto- and interneurons. Any neuron bearing a double label after this procedure is a good candidate for an inhibitory motoneuron. This is because there are no other neurons known that are motoneurons in the sense that they have an axon in a motor nerve, and that are also immunoreactive to the inhibitory transmitter GABA. This is how the candidate inhibitors in scorpions and chilopods have been identified Fig. c, in theInhibitory motoneurons in scorpions and chilopods are not just present but appear in an intriguing pattern reminiscent of the situation in malacostracans and hexapods. Most notably, though, there are not just three inhibitory neurons in scorpions but three groups of neurons  in crabs and crayfish The inhibitors in the hexapods that are presumably homologous to the malacostracan specific inhibitors are common inhibitors CI2 and CI3 (above); they supply four distal leg muscles and serve common inhibitory motor control as outlined above.(ii)The last leg article in hexapods, is considered to be the claw for the present purpose; this article has only a flexor muscle (Radnikow and Bässler (iii)The extensor muscle in the hexapod leg that is closest to the claw is the levator of the tarsus, a muscle that is supplied by a single excitatory motoneuron.As outlined above, the two specific inhibitory motoneurons supplying the malacostracan leg are necessary to uncouple the stretcher and opener muscles innervated by a single common excitatory motoneuron. The existence of a common excitatory motoneuron is an extremely unusual situation by any standards. When considering the available data, the following tempting but still preliminary interpretation emerges on how this innervation pattern evolved  for a brief interval. This agent labels newly synthesised genetic material in dividing cells, including neurons. In this way, immunocytochemical staining for BrdU after incubation for selected time intervals in the developing embryo allowed identification of the progeny of the different neuronal stem cells, or neuroblasts. Additional injection of the fluorescent marker Lucifer yellow into the primordial neurons was able to identify inhibitory motoneurons CI1 and CI3 as the first progeny of neuroblast 5.5. Primordial neurons stay dye-coupled for some time after they have been produced by their neuroblast, facilitating identification of groups of progeny sharing the same origin. It turned out that CI1 and CI3 are produced by a neuroblast that does not generate any other motoneurons. Instead, neuroblast 5.5 gives rise to inhibitory interneurons, probably exclusively so. That is, according to their developmental origin CI1 and CI3 are closely related to inhibitory interneurons rather than to “normal” excitatory motoneurons. Excitatory motoneurons are produced by a set of neuroblasts located at some distance from and not related to 5.5. A similar line of argument as for CI1 probably holds for CI2, although this inhibitor is produced by a different neuroblast, located in the immediate vicinity of neuroblast 5.5. Development of CI2 has not been studied in any detail.A possible scenario for the evolutionary origin of inhibitory neurons is suggested by this developmental origin of CIs. Inhibitory motoneurons might have originated from “misdirected” inhibitory interneurons that sent their axonal growth cones into the periphery rather than to targets in the central nervous system, due to a change in the expression of cell adhesion and receptor molecules in their cell membranes, for example. This is as yet pure speculation, however, and detailed genetic and developmental analyses would be necessary to lend more substance to this idea. It is, however, intriguing under this perspective that inhibitory motoneurons in malacostracans possess interneuronal properties in the sense that they make synaptic output connections in the central ganglion. One example are the mutual synapses between the dactylopodite closer excitors and the opener inhibitor, as mentioned above (Wiens and Atwood Inhibitory motoneurons are not just an integral part of arthropod motor control strategy but warrant further study with regard to several aspects, ranging from cellular and molecular mechanisms of inhibition to developmental and evolutionary questions. The latter should profit from modern “evo-devo” approaches, employing inhibitory motoneurons as character sets for evolutionary and developmental analysis. Of particular interest in this respect are the possible presence and the detailed structure of inhibitory motoneurons in basal, apterygote hexapod groups and in advanced, holometabolous hexapod groups. By the same token as yet little studied arthropods such as diplopods and araneids merit investigation, as well as phylogenetically more distantly related animals such as onychophorans, annelids and molluscs. Study of these topics would also help to resolve evolutionary important questions such as the origin of the comparatively large neuron numbers in the chelicerates, whether it is ancestral or derived. Although the function of common and specific inhibitors in motor control now appears to be well understood, evolution has always been good for surprises. Analysis of CI function in the hexapod antenna and particularly in hexapod body wall muscles is badly needed under this perspective. The same is true for possible interactions of peripheral inhibition and neuromodulation, both often impinging on the same muscles.