Over the recent years, it has been found that microglia pseudopodia contact synapses, detect sick ones and prune them, even in adult animals. Myelinated nerves also carry out plasticity in which microglia remove myelin debris by phagocytosis. However, it remains unknown whether microglia explore structures on nerve fibers, such as Ranvier’s node (RN) or myelin sheath, before they become debris. By double or triple staining RNs or myelin sheathes and microglia in healthy rat corpus callosum, this study unveiled direct contacts of microglia pseudopodia with RNs and with para- and inter-nodal myelin sheathes, which was then verified by electron microscopic observations. Our data indicated that microglia also explore unmyelinated nerve fibers. Furthermore, we used the animals with matured white matter; therefore, microglia may be actively involved in plasticity of matured white matter tracts as it does for synapse pruning, instead of only passively phagocytize myelin debris.  However, in recent decades, microglia are found to be more than passive responders. They show high levels of activity with their pseudopodia exploring the surrounding environment, which is known as “dynamic surveillance of brain parenchyma”[3–. One of the newly observed physiological functions of microglia is their involvement in synaptic stripping or pruning[4–. Under a two-photon live-image microscope, fine microglia pseudopodia are viewed to extend to pre- and post-synaptic structures at a certain rate. Further, direct contacts of labeled microglia processes onto synapses and/or engulfed post-synaptic protein in microglia cytoplasm have been envisioned under the electron microscope[4–. During development, microglia are responsible for removing the less-used synapses[5,. This was verified by comparing microglia activity in neonatal visual cortex with normal visual experience to that activity in the visual cortex with monocular deprived visual experience. In addition, possible participation of microglia in shaping white matter tracts in visual callosum has been reported in a similarly designed study.Microglia, the brain resident cells that can sense pathological tissue alterations, are believed to be quiescent in physiological circumstances[10–. It is rational to contemplate that microglia may also surveil and maintain the integrity of white matter tracts. Myelinated nerve fibers among white matter tracts play a pivotal role in the brain communication between neurons or nuclei in a manner of saltatory conduction. The Ranvier’s node (RN) and the internodal myelin sheath are key structures for the generation of saltatory conduction because they concentrate voltage gated sodium (Na+v) channels on the node and insulate internode axons with myelin sheath[13–. The node area is protected by microvilli of Schwan cells in myelinated peripheral nerve fibers as this area is extremely important for the rapid transmission of neuronal signals[15–. In addition, microvilli may be involved in stabilizing Na+v channels at the node through trans interaction of microvilli dystroglycan with nodal axolemmal molecules, since selective knockout of Schwan cell’s dystroglycan can result in nodal Na+v channel density reduction and nerve conduction slowdown.Like synapses in the neuropil, nerve fibers, especially the myelinated nerve fibers, also undergo plasticity in both developing and matured white matter tracts[17–. A function proposed by some scholars is that astrocyte nodal processes buffer fluctuation of ions around the node, but the molecular details and the mechanism are still unknown[17–. Moreover, electron microscopy studies on contacts of labeled astrocyte processes with RNs in the corpus callosum (CC) and optic nerve have revealed that a considerable number of RNs therein are still uncovered or only partially covered[17,, leaving a broad room on nodal axolemma for other cellular components to reach. In light of the recent findings aforementioned, we hypothesized that microglia pseudopodium might extend to both CNS RN and myelin sheath, during developing myelination or post white matter tract maturation. Of particular interest was the role of pseudopodia in adult white matter tracts, as that might be related to some white matter injuries in adulthood. The concentration of Na+v channels at the nodal area allowed us to apply double and triple immunofluorescent staining of the node and microglia. In combination of labeling para- or inter-nodal domains by pertained markers, we showed in this work the potential contacts of microglia pseudopodia with the RNs and with para- and inter-nodal myelin sheathes in the rat CC. Finally, electron microscopy was conducted to verify contacts between microglia pseudopodium and RN. All experiments were performed using young adult rats of 45–55 days, when major developing myelination finished and white matter had developed into a matured stage[20–.However, which structure in the brain white matter tracts can play the same role as microvilli does in peripheral nerves is still not clear. Fine process of astrocyte has been known to contact the RNs in the central nerve system (CNS), but the functional profile of these contacts are still equivocalGuide for the Care ofLaboratory Animals in Research and approved by the Institutional Animal Care and Use Committee of the University of Nebraska Medical Center. All efforts were made to minimize animal suffering and the number of animals used in this study.Thirteen adult Sprague–Dawley rats , purchased from Charles River Laboratories , were used in this study. Samples from ten rats  underwent double and triple labeling, and then were observed under confocal microscopy. Samples from three animals  were used for electron microscopy. All experimental protocols and animal care were carried out in accordance with the National Institutes of Health plus-coated slides. The sections with the CC were incubated with either rabbit anti-myelin basic protein  plus goat anti- Ionized calcium binding adaptor-1 (Iba-1) or rat anti-MBP  with rabbit anti-Iba-1  overnight at room temperature (RT). Alex Flour conjugated secondary antibodies  against primary antibodies were applied to display fluorescent labeling. Control staining for different antigens was carried out without primary antibody.Animals were euthanized with isoflurane and transcardially perfused with saline followed by 4% paraformaldehyde in phosphate buffer . The brain containing the CC was cut into 10-μm coronal or sagittal frozen sections and directly mounted on +v; 1:200, Alomone, Jerusalem, Israel) with goat anti-Iba-1 , or mouse anti-Na+v subunit 1.6  with rabbit anti-Iba-1  for revealing RN and microglia with ramified pseudopodia. Alex Flour conjugated secondary antibodies  against primary antibodies were applied to display fluorescent labeling.Brain tissues were processed in the same way as aforementioned. The coronal sections were incubated with either rabbit anti-pan Voltage-Gated sodium channels  and goat anti-Iba-1 . Then, Alexa Flour 488 conjugated donkey anti-rabbit, Alexa Flour 568 attached donkey anti-mouse and ByLight 405 linked donkey anti goat  were applied to visualize the structures labeled by primary antibodies.Brain tissues were processed in the same way as aforementioned except that 7.5% saturated picric acid (75%) was added to the fixative. The CC sections were immunostained with a cocktail of rabbit anti-MBP , mouse anti-Na. Thus, antibody against Caspr1 was added in triple labeling of RNs, paranodal domains and microglia. Then, the sections were incubated in cockatiel of rabbit anti-pan Na+v , mouse anti-Caspr1  and goat anti-Iba-1  overnight at RT. Next, Alexa Flour 568 conjugated donkey anti-rabbit, fluorescein conjugated donkey anti-mouse and ByLight 405 conjugated donkey anti goat (provided by UNL) were applied to reveal the node, paranodal domain and microglia.Brain tissues were processed in the same way as aforementioned. As has been known, a characteristic of the RN in myelinated nerve fiber is that the node is always nested in-between two paranodal domains that are contactin-associated protein 1 (Caspr1) positive.Animals were euthanized by isoflurane and transcardially perfused with saline followed by PB, as mentioned above, containing 2% paraformaldehyde and 0.5% glutaraldehyde with 7.5% saturated picric acid (75%). The front brain containing the CC was extracted and cut into 50-μm coronal sections by a vibratome and saved in PB. The sections were immunohistochemically stained as previously described. The labeled microglia in the embedded sections were identified under light microscope , and then trimmed off for ultrathin sectioning. Ultrathin sections were cut with Leica EM UC7 Ultracut microtome, stained with lead citrate and observed with Hitachi H7500 TEM . And the work continued afterward at Computer Imaging and Microscopy Core Facility, Affiliated Hospital of Academy of Military Medicine Sciences, Beijing, China. Ultrathin sections were cut with LKB ultra-microtome, stained with lead citrate and observed with FEI Tecnai G2 Spirit transmission electron microscope.The sections then underwent osmification and plate embedding as described previously2 Spirit electron microscope.Digital images of fluorescent labeling were collected using Nikon A1 confocal system (Japan) equipped with Nikon 90i upright fluorescence microscope and BioRad Laser Sharp 2000 imaging program . Alexa Fluor 568 labeling was viewed through 561 excitation laser line with 10 nm resolution of spectra. Alexa Fluor 488 and ByLight 405 were viewed through 488 and 405 excitation laser line, also through 10 nm resolution spectra. Confocal images were captured with ×20 and ×40 objective at iris of 2.0–2.5 in box size of 1,024×1,024. All Z-scan was set up at 1 μm layer of laser scan step and saved as “avi” video files and the clearest double or triple labeling image was selected and converted to Photoshop 7.0.1  through Bio-Rad Plug-In software and stored as “tiff” file at 1,024×1,024 pixels. The electron microscopy microphotographs presented here were taken with FEI Tecnai GFig. 1A–C) and sagittal sections  that microglia pseudopodia (arrowheads) explore the inter-nodal myelin sheath (opened arrowheads) frequently . All control staining without primary antibody resulted no labeling.Using MBP to label myelin sheath and Iba-1 to mark microglia, we observed in both coronal (+v 1.6 labeled RN (opened arrows) that was touched by a microglia process marked by Iba-1 . Fig. 1H showed that a single microglia with its pseudopodia (arrowhead) appeared to explore a RN and its nearby paranodal (a pair of opened arrows and arrowhead pointing up), and a continued internodal (a pair of arrowheads and opened arrowhead pointing down) myelin sheath simultaneously.Unlike Caspr1, which is expressed only on paranodal domains, MBP can mark both paranodal and internodal myelin sheath. We encountered a paranode – RN – microglia complex, characterized by MBP positive paranodal apparatus immediate abut against a Na+v channel . However, in Fig. 2A–C, a nerve fiber lining and connecting with the labeled node (opened arrow) was also stained by anti- Na+v antibody, inferring this nerve fiber was an unmyelinated fiber, since the Na+v channels appears to evenly distribute along the unmyelinated nerve fibers in addition to aggregating in the node-like area. Besides, clusters of Na+v channels on unmyelinated nerve fiber is suggested by a recent finding that Na+v channels piled fragmentally upon lipid rafts on axolemma of node like area in unmyelinated fibers.Under laser scan of 1-µm layer of the CC section, it was explicitly observed that microglia pseudopodia (arrowheads) immediately contact the possible RNs or clusters of Na, we attempted to show the node and paranodal domain simultaneously to clarify whether microglia pseudopodia indeed explore the RN in myelinated nerve fibers. Then, by means of triple labeling, we unveiled that microglia pseudopodia (arrowheads) stretched to contact RNs labeled by Na+v channels antibody (opened arrows) and/or paranodal domains marked by Caspr 1 . In some cases, the Iba-1 positive microglia pseudopodia are closely apposite upon both RN and paranodal domains simultaneously , although we did not know whether these contacts were instant or constant.As the RN in myelinated nerve fiber is always situated in-between of paranodal domains that express Caspr1Fig. 4A and B, arrowheads). The tissue block containing labeled microglia  was marked and extracted from plate-embedded sections under anatomical microscope for further preparation of ultrathin section. Ultrathin sections were cut along with longitudinal axis of nerve fibers in the CC as shown in Fig. 4C and D. Ends of microglia pseudopodia (opened arrowheads in C and D) were seen to contact axolemma on RNs. These contacts appeared between the naked axolemma on the node and the ends of pseudopodia without any gap (C and D). The contact between paranodal myelin sheath and microglia pseudopodia was also observed . That paranodal domain was termed as axoglial apparatus ultrastructurally characterized by spiral loops formed by paranodal myelin sheath . There appeared to be a tiny gap (about 5–10 nm) between plasm membrane of microglia and paranodal myelin sheath; however, the other end of pseudopodia seemed to have direct contact with the RN and another two myelin sheathes of myelinated fibers .With plate-embedding, labeled microglia and their pseudopodia could be visualized under conventional light microscope  concentration can elicit microglia activation. A few of studies uncovered that a reduction of extracellular Ca2+ concentration around neuronal somata or dendrites evoked the convergence of microglia pseudopodia which then moved toward dendrites[32–. This finding suggests microglia may be able to sense Ca2+ surrounding the RNs. Commonly, Ca2+ around RN is stable and Ca2+ influx occurs only when axolemma or paranodal apparatus are disrupted[34–. However, physiological Ca2+ influx through the RN axolemma was found recently in cases where the axons discharged at a remarkably high frequency[36–. Hence, monitoring the K+ and/or Ca2+ concentration around RNs might be one of the duties for those microglia to have close apposition or contact upon the RNs.Furthermore, the RN is a key structure to accomplish saltatory conduction as aforementioned. It is rational to presume if the nerve fibers malfunction, activities of the RNs will be altered and per se would induce white matter tract function changes, especially declination of axon compound action potentials. The length and thickness of microglia pseudopodia dramatically changed from delicate soma with thin ramified pseudopodia to amoeboid cells with short hyper-ramified morphology when microglia were activated. The number of contacts between microglia and RNs or myelin sheath might change in this circumstance. More interestingly, the extent of declination of compound action potentials is correlated with the states of microglia activation. However, the functional significance of those contacts of microglia pseudopodia with RNs and para- or inter- nodal myelin sheathes is still an enigma. Nonetheless, this finding will open a new avenue for exploration into the effect of microglia, in addition to overt phagocytosis and/or invisible cytokines secretion, on white matter tract integrity.If microglia monitor and maintain the function of RNs by means of close apposition of their pseudopodia upon RNs, it is intriguing for us to know what will happen when those processes are shortened or shrunken upon microglia activation. Our recent study showed that the activation of microglia


The representation of carbon-nitrogen (N) interactions in global models of the natural or managed land surface remains an important knowledge gap. To improve global process-based models we require a better understanding of how N limitation affects photosynthesis and plant growth. Here we present the findings of a meta-analysis to quantitatively assess the impact of N limitation on source (photosynthate production) versus sink (photosynthate use) activity, based on 77 highly controlled experimental N availability studies on 11 crop species. Using meta-regressions, we find that it can be insufficient to represent N limitation in models merely as inhibiting carbon assimilation, because in crops complete N limitation more strongly influences leaf area expansion (-50%) than photosynthesis (-34%), while leaf starch is accumulating (+83%). Our analysis thus offers support for the hypothesis of sink limitation of photosynthesis and encourages the exploration of more sink-driven crop modelling approaches. We also show that leaf N concentration changes with N availability and that the allocation of N to Rubisco is reduced more strongly compared to other photosynthetic proteins at low N availability. Furthermore, our results suggest that different crop species show generally similar response patterns to N limitation, with the exception of leguminous crops, which respond differently. Our meta-analysis offers lessons for the improved depiction of N limitation in global terrestrial ecosystem models, as well as highlights knowledge gaps that need to be filled by future experimental studies on crop N limitation response. We therefore had to use very general search terms in the literature search and the keywords used for searching literature databases yielded 169,236 records , we only screened the first 100 records (sorted by relevance); for crop species with > = 2000–10,000 records identified  we screened the first 140 records; and for crop species with >10,000 records identified  we screened the first 240 records. To decide how many search records to include in the screening, we chose a cut-off point that appeared feasible in terms of number of records to be screened, and then checked whether we could find additional suitable studies after our cut-off point for 3 crop species . As no additional suitable studies could be found for these 3 crop species after our cut-off point, our study sample would most likely not have looked very different if we had screened more studies. This resulted in a total of 2740 records being screened   and by searching the reference lists of published articles. Many studies suitable for our analysis did not have the impact of N limitation on crop photosynthesis as their primary focus, but N availability was either varied only as a secondary experimental factor (e.g. in studies interested in the effects of elevated  treatments.The freeware program DataThief III  was usedN limitation rate was thus expressed relative to the control rate, ranging from 0% N limitation  to 100% .Nutrient limitation is defined as the limitation of the productivity of a plant due to inadequate supply of the nutrient in the soil . To examFor some response variables—namely chlorophyll, Rubisco, leaf starch, and leaf sugar- we did not find enough data expressed per unit leaf area and per unit leaf mass to allow separate analyses for each unit . For Rubrr). The loge of the response ratio was used to linearize and improve normality of errors [e rr is calculated as (SDE2 ⁄ nEE2) + (SDC2 ⁄ncC2) [e rr)-1] x 100) under N limitation, unless response ratios are compared to each other.For the investigated response variables, each experiment was standardized prior to analyses by expressing the effect of N limitation  relative to the control , i.e., the response ratio (f errors . As a tr2 ⁄ncC2) , where n2 level) influence the effect of N limitation on our response variables. We therefore used a meta-regression, which is an extension of a classic meta-analysis and allows a mix of continuous (here N limitation) and categorical explanatory variables  treatments or N supplies sharing the same control group, and ii) study dependence: same study includes many experiment with separate control groups but still share the same experimental setup (e.g. multiple cultivars). Here we accounted for these dependencies by employing mixed linear models  on photosynthesis also showed that the CO2 fertilization effect on photosynthesis was reduced under limiting N supply [2] compared to the overall N limitation effect. Nonetheless, our results confirm the increasing importance of N limitation under elevated [CO2], as the negative effect of N limitation on photosynthesis is greater under elevated [CO2] relative to ambient [CO2] , as only three studies out of 50 measured Rubisco content at elevated [CO2]. Similarly, while we did not detect an interaction of [CO2] level with N limitation on leaf sugar or leaf starch contents, this is probably caused by the few studies in our database that were conducted at elevated [CO2] (N = 6/25 of leaf starch and N = 14/57 of leaf sugar), which reduces our power to detect differences.Under elevated   ,90.Our analysis leads to five key conclusions on crop physiological processes under N limitation that are relevant for the depiction of C-N processes in TEMs:N limitation impacts leaf growth more strongly than leaf photosynthesis.Photosynthesis is sink-limited under N limitation.L changes in response to N supply.NL invested in Rubisco changes with N supply.The fraction of NLeguminous crops respond differently to N limitation than other crops.In the following we will discuss each of these conclusions in turn.First, our analysis shows that crops respond to N limitation primarily through a reduction in leaf area rather than in photosynthesis and that the reduction in leaf area is not necessarily proportional to the reduction in photosynthesis see . This isL, and NL in turn is influenced by N uptake and availability . ,91. L, aty  concentrations a lower CO2 resistance increases the carboxylation efficiency of Rubisco. The correct depiction of CO2 mesophyll diffusion has been suggested as a general shortcoming in TEMs )Note that our analysis focused on crops and it is not clear whether the observed relationships also apply to natural vegetation. But given that the crops included represent a range of different plant traits found in natural vegetation , across The clear relationships observed between leaf-level variables and the level of N limitation–where the magnitude of change increased with increasing N limitation —shows thN experiments have been criticised for not providing sufficiently controlled conditions that allow a constant and defined N supply to plants ,54. As p2] conditions. More N limitation experiments should examine these under-studied crops and under-studied variables.We focused our study on the 15 globally most important crop species. However, due to a substantial crop and biome bias in experimental studies  we were S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S3 Table(PDF)Click here for additional data file.S4 Table(PDF)Click here for additional data file.S5 Table(PDF)Click here for additional data file.S6 Table(PDF)Click here for additional data file.S1 Fig(PDF)Click here for additional data file.S1 Text(PDF)Click here for additional data file.


Perioperative bleeding during cardiac surgery are known to make patients susceptible to adverse outcomes and several bleeding classifications have been developed to stratify the severity of bleeding events. Further validation of different classifications was needed. The aim of present study was to validate and explore the prognostic value of different bleeding classifications in patients undergoing off-pump coronary artery bypass grafting (OPCAB).Data on baseline and operative characteristics of 3988 patients who underwent OPCAB in Beijing Anzhen Hospital from February 2008 to December 2014 were available. The primary endpoint was a composite of in-hospital death and nonfatal postoperative myocardial infarction (MI). The secondary endpoint was postoperative acute kidney injury (AKI). We explored the association of major bleeding defined by the European registry of Coronary Artery Bypass Grafting (E-CABG), Universal Definition of Perioperative Bleeding (UDPB), Bleeding Academic Research Consortium (BARC) classification and Study of Platelet Inhibition and Patient Outcomes (PLATO) with primary endpoints by multivariable logistic regression analysis and investigated their significance of adverse event prediction using goodness-of-fit tests of − 2 log likelihood.n = 49) and postoperative MI was observed in 4.76% (n = 190) of patients, AKI in 24.69% (n = 985). The incidence of the primary outcome was 5.99% (n = 239). Multivariable logistic regression analysis showed that BARC type 4 , UDPB class 4  and E-CABG class 2–3  bleeding but not PLATO bleeding were associated with an increased risk of in-hospital death and postoperative MI. Major bleeding defined by all the four classifications mentioned above was an independent risk factor of AKI after surgery. Inclusion of major bleeding defined by these four classifications improved the predictive performance of the multivariable model with baseline characteristics.In-hospital mortality was 1.23% (Bleeding assessed by BARC, E-CABG and UDPB classifications were significantly associated with poorer immediate outcomes. These classifications seemed to be valuable tool in the assessment of prognostic effect of perioperative bleeding. Perioperative bleeding is a common concern in patients undergoing cardiac surgery , 2. BleeA variety of definitions of bleeding were proposed to stratify the severity of bleeding which was as an important endpoint to assess the efficacy and safety of procedure . HoweverOur retrospective, single-center, cohort study enrolled a total of 3988 consecutive patients aged > 18 years who had undergone isolated OPCAB between February 2008 to December 2014 in Beijing Anzhen Hospital , including emergency and elective operations performed in off-pump setting. The major exclusion criteria were: 1) preoperative exposure to oral anticoagulants including warfarin and direct oral anticoagulants, 2) preoperative severe renal insufficient, 3) undergoing any other major cardiac surgery procedure including valvular repair or replacement, 4) and with insufficient records to specify bleeding events. The detailed study flowchart is depicted in Fig. All data used int the present study was collected from the electronic medical records by two trained medical staffs. The important data information was monitored and revised by a third medical staff to ensure the accuracy. The database of the electronic medical system contained the baseline and operative data and immediate postoperative adverse events, which provided available complete pre-, intra-, and postoperative data for all included patients.The primary endpoint of this study was a composite of in-hospital death and postoperative myocardial infarction (MI). In-hospital death included all-cause death before discharge and post operation. The postoperative MI was defined as nonfatal elevation of cardiac troponin (cTn) values > 10 times the 99th percentile upper reference limit (URL) in patients with normal baseline cTn values . The secMajor bleeding events were assessed and adjudicated according to the classification from European registry of Coronary Artery Bypass Grafting (E-CABG),  the UnivBaseline continuous variables were presented as mean value ± standard deviation if normally distributed and otherwise as median (interquartile range). Categorical variables are expressed as percentages.Multivariable logistic regression analysis was used to investigate the association between perioperative major bleeding and endpoints adjusting for potential confounding factors which had been reported in previous studies, using forward stepwise selection to identify significant confounding variables. Potential confounders reported in previous studies as important determinants of perioperative outcomes and variables suggested to be associated with clinical outcomes by univariate logistic analysis would be offered to the logistic regression models. Power of the association between risk factors and outcomes was expressed as odds ratio (OR) and 95% confidence intervals (CI). Goodness-of-fit tests of − 2 log likelihood were used to evaluate the improved model performance comparing models with and without bleeding.p value < 0.05.Statistical analysis was performed with the SPSS version 24.0 statistical software . All statistical tests were 2-sided and results were considered to be statistically significant at a Baseline demographic characteristics of the overall 3988 patients included in the present study were reported in Table n = 49) and postoperative MI occurred in 5.01% (n = 200) of patients. And 24.70% (n = 985) of patients suffered from AKI after procedure.In the 3988 OPCAB patients analyzed, in-hospital mortality was 1.23% (n = 166) and 9.85% (n = 393). And the incidence of major bleeding according to PLATO criteria was 36.28% (n = 1447). All major bleeding events defined as E-CABG class 2–3 were concomitantly captured in both BARC type 4 and UDPB class 3–4 bleeding (98.8% in BARC type 4 and 42.2% in UDPB class 3–4). The incidence rates of adverse outcomes including composite endpoints, in-hospital death, MI and AKI among patients with minor or major bleeding classified by different bleeding classifications were summarized in Table p < 0.001).BARC CABG-related bleeding (type 4) occurred in 168 4.21%) patients. The incidence of major bleeding defined by E-CABG class 2–3 and UDPB class 3–4 were 4.16% (1% patienP < 0.001). Major bleedings defined as E-CABG class 2 and 3  or UDPB class 4  were significantly associated with poorer perioperative clinical outcomes.The results of the multivariable logistic models to evaluate the association between bleeding and adverse outcomes were shown in Table We created a multivariable model using baseline characteristics such as age, gender, BMI, diabetes, hypertension, prior MI, renal insufficiency, decreased left ventricular ejection fraction (LVEF), systolic blood pressure (SBP) values and preoperative haematocrit (Hct) values and different bleeding classifications were added to this multivariable model to evaluate whether the inclusion of bleeding events could improve the predictive value of this model. The inclusion of bleeding stratified by BARC, E-CABG and UDPB improved the prediction of in-hospital death and postoperative MI Table . BARC tyOur results indicated that BARC, E-CABG and UDPB but not PLATO classifications can effectively stratify the severity of postoperative bleeding. Moreover, bleedings classified as BARC type 4, E-CABG class 2 and 3 and UDPB class 4 were independently associated with an increased risk of in-hospital death and postoperative MI.Bleeding has been recognized as important determinants of outcomes after cardiac surgery according previous studies –18. QuanIn accordance with our results, the UDPD classification was suggested to have an important effect on both short- and long-term survival after CABG by several studies , 23. KinBARC type 4 bleeding is the only one specific CABG-related bleeding definition . BleedinThe present study provided validation of four different bleeding classification in a large cohort of patients undergoing CABG. We observed that bleeding defined as BARC type 4, E-CABG class 2 and 3 and UDPB class 4 carried a higher risk of in-hospital death and postoperative MI as well as AKI. Such results of our study confirmed that BARC, E-CABG and UDPB were promising research tools for stratification of bleeding risk. Appropriate stratification of bleeding events can provide prognostic information and be useful for estimating the risk of adverse outcomes.There are some limitations in our study that must be acknowledged. Firstly, the retrospective nature of the present study was an important limitation and prospective data collection might yielded better source documentation to classify bleeding complication. However, the data regarding hemoglobin, transfused blood products and outcomes in hospital were retrieved from medical records system which was considerably reliable. Secondly, the lack of data on follow-up after surgery prevented us to evaluate the long-term effect of major bleeding on the clinical outcomes. But majority of bleeding events and adverse events resulting from bleeding occurred during perioperative period. Thirdly, the present study only enrolled patients undergoing OPCABG and further studies are needed to investigate the prognostic significance of bleeding classifications in patients undergoing other cardiac surgery.Our present study confirmed that bleeding assessed by BARC, E-CABG and UDPB classifications were significantly associated with poorer immediate outcomes but not PLATO classifications. These classifications seemed to be valuable tool in the assessment of prognostic effect of perioperative bleeding events on clinical outcomes. And the significance of these bleeding classifications needs to be validated in a larger population undergoing other cardiac surgery.


Friendreich's ataxia skin cells are highly sensitive to ultraviolet A due to their high levels of mitochondrial redox-active iron. via ATP depletion. Mitochondria iron overload is a key feature of the neurodegenerative disease Friedreich's ataxia (FRDA). Here we show that cultured primary skin fibroblasts from FRDA patients are 4 to 10-fold more sensitive to UVA-induced death than their healthy counterparts. We demonstrate that FRDA cells display higher levels of mitochondrial LI  and show higher increase in mitochondrial reactive oxygen species (ROS) generation after UVA irradiation (up to 2-fold on average), consistent with their differential sensitivity to UVA. Pre-treatment of the FRDA cells with a bespoke mitochondrial iron chelator fully abrogates the UVA-mediated cell death and reduces UVA-induced damage to mitochondrial membrane and the resulting ATP depletion by a factor of 2. Our results reveal a link between FRDA as a disease of mitochondrial iron overload and sensitivity to UVA of skin fibroblasts. Our findings suggest that the high levels of mitochondrial LI in FRDA cells which contribute to high levels of mitochondrial ROS production after UVA irradiation are likely to play a crucial role in the marked sensitivity of these cells to UVA-induced oxidative damage. This study may have implications not only for FRDA but also for other diseases of mitochondrial iron overload, with the view to develop topical mitochondria-targeted iron chelators as skin photoprotective agents.Mitochondrial labile iron (LI) is a major contributor to the susceptibility of skin fibroblasts to ultraviolet A (UVA)-induced oxidative damage leading to necrotic cell death  Significance to metallomicsIron is a vital element for cell processes such as mitochondrial energy production. In Friedreich's ataxia (FRDA), dysregulation of iron metabolism causes mitochondrial iron overload, leading to excess reactive oxygen species production and cytotoxicity. We report that high levels of mitochondrial iron in skin cells from FRDA patients render them extremely sensitive to oxidative stress induced by solar ultraviolet A (UVA) compared to their healthy counterparts. Moreover, a bespoke mitochondrial iron-trapping molecule protects the FRDA-affected skin cells from UVA-induced damage. This study highlights the sensitivity to UVA as a novel connection to FRDA and possibly other mitochondrial iron overload disorders.via Fenton chemistry.10Iron is an indispensable element for life as it participates in several crucial cellular functions.2O2.2O2-induced cytotoxicity can be partially reversed when cells are pre-treated with iron chelators, implying an important contribution from mitochondrial LI in oxidative injury. Treatment of a mouse model of FRDA with highly lipophilic iron chelators mitigated the symptoms of the disease on the heart, further supporting the benefits of this approach.i.e. mitochondria) and the consequent risks including disruption of normal homeostasis and depletion of patient's iron levels, resulting in anaemia. Hence there is a clear need to design iron chelators with improved properties, such as targeting them to their site of action, the mitochondria.In view of the central role of mitochondria in cells, it is not surprising that deregulation of mitochondrial iron metabolism, such as that which occurs in Friedreich's ataxia (FRDA), has profound consequences on cell integrity and function. FRDA is a disease caused by deficient levels of frataxin (FXN), a mitochondrial proteinOne approach to reduce/avoid unwanted effects and improve the effectiveness of the chelator consists in targeting the chelating unit to the mitochondria by linking it to a mitochondria-specific “SS peptide”. Such peptides, based on alternate hydrophobic and basic amino acid side chains readily permeate cell plasma membranes and selectively accumulate in the mitochondria.10Because mitochondrial iron overload has been recognised as a key feature of FRDA,2 (“cpd 2”) and compound 13 (“cpd 13”), the chelator–peptides used in this study have been documented elsewhere.The design, synthesis and characterization of compound All reagents used for cell cultivation were obtained from Life Technologies . Phosphate buffered saline (PBS) was from Oxoid Ltd . The mitochondrial markers tetramethylrhodamine methyl ester (TMRM) and the mitochondrial ROS indicator mitoSOX™ red were purchased from Invitrogen . Annexin V-FLUOS was from Roche . All other reagents were from Sigma-Aldrich .The human primary skin fibroblasts GM04078 , GM03816  and GM03665  from clinically affected Friedreich's ataxia patients and fibroblasts AG09429  from an apparently healthy (control) donor were obtained from the Coriell Institute for Medical Research . These lines have been characterized elsewhere notably with respect to their relative levels of FXN.422 atmosphere, as described previously.The human primary fibroblast cells were routinely grown in Earle's MEM supplemented with 15% FCS, glutamine and antibiotics, at 37 °C in a 5% CO2 and cpd 13) were prepared as 100 mM stock solutions in DMSO , diluted to the final desired concentration of 50 μM in conditioned medium and added to cells overnight as described previously.The iron chelator–peptides . Immediately following treatment, cells were incubated in serum-free and phenol red-free EMEM containing 1.5 μM mitoSOX™ red for 30 min at 37 °C. After two washes with PBS, cells were collected by trypsinization and resuspended in PBS containing 0.1% BSA. Cells were then counted and triplicates of each condition, containing equal numbers of cells (0.1–0.2 × 106 cells) were analysed using a spectrofluorimeter Clariostar  using PBS–0.1% BSA as blank. Red fluorescence was detected using 451–522 nm bandwidth for excitation and 550–650 nm bandwidth for emission, for broad detection of mitochondrial ROS.44Cells were grown in 6 cm plates in duplicates for three days to reach 70–80% confluency. On day 2 of the seeding, cpd ca. 0.2 kJ m–2 s–1. UV doses were measured using an IL1700 radiometer . Control cells were treated in the same manner to irradiated ones, except that they were not irradiated (i.e. sham-irradiated).Irradiations were performed in PBS at approximately 25 °C using a broad spectrum Sellas 4 kW UVA lamp , as described previously.–1 MTT -2,5-diphenyl-tetrazolium bromide). MTT solution was then removed and DMSO was added to the cells in order to dissolve the formazan formed. Absorbance was read at 550 nm using a spectrophotometer Fluostar Optima . The percentage of viable cells was calculated by comparing the absorbance of treated versus non-treated control cells.Cells grown in 35 mm dishes were treated as indicated in the figure legends and grown for a further 24 h in conditioned medium, after which the latter was removed and the cells were incubated for 3 h at 37 °C in serum-free medium containing 0.5 mg mLAll flow cytometry protocols are described in Supplementary Materials and methods ESI.ATP production by cells following various treatments was monitored with a luminometer (Turner Designs Model TD-20/20) 2 h after UVA, using the ViaLight™ plus kit  as described previously.46via a Moticam 580 digital camera and a Plan 10× objective.Bright-field images used to document morphological changes following treatments were captured on a Motic inverted microscope (model AE2000)  P < 0.05) were determined by either paired or unpaired t-test after one-way analysis of variance.Results are expressed as the mean ± standard deviation (SD). Significant differences  all three FRDA cell lines displayed an increasingly higher sensitivity than their healthy counterparts, with GM04078 and GM03665 being ca. 3-fold more sensitive. At the highest dose of 500 kJ m–2 , the sensitivity of the FRDA lines rose up to 10-fold over that of cells from healthy donors. Our data show that FRDA skin cells are more sensitive than their healthy counterparts to physiologically relevant doses of UVA. Microscopic images of control (AG09429) and FRDA (GM04078) cells irradiated at 250 kJ m–2 UVA capture the morphological changes 24 h post-treatment  with the compound as indicated in Materials and methods, then irradiated with a UVA dose of 500 kJ m–2 and live and dead/dying cells were scored 24 h following irradiation using flow cytometry and dual labelling of cells with fluorescently-labelled Annexin V and propidium iodide (PI). This technique allows to determine whether cells are viable, apoptotic or necrotic via differences in plasma membrane integrity and permeability. Percentages of live cells obtained are plotted as bar charts in –2), the population of live AG09429 cells decreased by ca. 10%. In stark contrast, irradiated FRDA GM03816 and GM04078 cells incurred a significant drop of 50–60% of viable cells compared to non-irradiated sample. However, pre-treatment with cpd 2 prior to irradiation effectively restored viability of control AG09429 cells, but also of the two FRDA fibroblasts to the level of untreated sample. Visual inspection of microscopy images of these experiments  to UVA compared to human fibroblasts (see –2 (approximately equitoxic to 500 kJ m–2 in human FRDA fibroblasts) to carry out our investigation and cells were pre-treated (or not) with cpd 2 as in ca. 40% live cells. Pre-treatment of cells with cpd 2 prior to irradiation restored the percentage live cells to its value in untreated sample in Y47R and to ca. 85% of its value in untreated sample in YG8sR. Representative dot plots from these experiments are depicted in 2. Taken together, these data show that cpd 2 is capable of effectively protecting highly UVA-sensitive FRDA cells, from both patients and a mouse model of FRDA against UVA-induced death.We then sought to interrogate the responsiveness of the mouse cell lines Y47R and YG8sR to cpd asts see , we chos13  using flow cytometry and the fluorescent cationic dye tetramethylrhodamine methyl ester (TMRM) which accumulates only in intact mitochondria. Mitochondrial membrane depolarization translates into a decrease in TMRM fluorescence. Human healthy and FRDA fibroblasts were irradiated with a UVA dose of 250 kJ m–2 and fluorescence was monitored 2 h post-irradiation, a time-point at which we have previously shown in healthy fibroblasts that both the level of labile iron in the cell and damage to membranes are at their highest.–2 was chosen so that the resulting membrane damage would not lead to significant dye leakage, which would invalidate the assay. The results, reported as a bar chart in i.e. control). In healthy AG09429 cells, irradiation caused a non-significant drop in TMRM fluorescence of ca. 10% compared to control cells. In contrast, in both FRDA cell lines GM03816 and GM04078, a significant decrease of respectively 70 and 80% in TMRM fluorescence could be observed, indicative of the exacerbated sensitivity of mitochondria. Pre-treatment of FRDA cells with cpd 2 prior to irradiation afforded a significant 2-fold and 3-fold protection to GM03816 and GM04078 cells, respectively while healthy AG09429 cells fully recovered their mitochondria integrity. We then interrogated the mouse fibroblasts similarly to the human fibroblasts above but using the UVA dose of 150 kJ m–2 (approximately equitoxic to 250 kJ m–2 in human FRDA fibroblasts). The FRDA YG8sR cells showed much higher sensitivity to UVA irradiation when compared to their healthy counterparts Y47R, with a ca. 50% drop in TMRM fluorescence compared to 10% in Y47R  and decreased ATP production. Furthermore, we show that both mitochondria integrity and functionality can be protected by pre-treatment of cells with a mitochondria-targeted iron chelator.We have previously shown that the measurement of cellular ATP is a good indicator of mitochondria integrity following exposure to biologically relevant doses of UVA radiation.eriments . While A2 against UVA-induced cell death supports the crucial role of mitochondrial LI in this process. We suggest that the level of mitochondrial LI could be used a prognostic indicator for skin photosensitivity, with particular relevance for FRDA patients but also, for patients with other disease of mitochondrial iron overload.Our findings reveal an important link between FRDA as a disease of mitochondrial iron overload and skin fibroblast sensitivity to UVA. Taken together, these results provide a link between the higher levels of mitochondrial LIP in FRDA cells and the higher oxidative stress observed in these organelles. These results are also in line with the observed much higher sensitivity of FRDA fibroblasts to UVA-induced damage compared to healthy counterparts. A high concentration of resident redox-active mitochondrial iron provides a rational explanation to the high oxidative burden in FRDA mitochondria described by others as a result of defective expression of frataxin.Furthermore, we show that both mitochondria integrity and functionality can be protected, at least in part by pre-treatment of cells with a mitochondria-targeted iron chelator. Exposure of cells to cumulative biologically relevant doses of UVA has been shown to induce damage to mitochondrial DNAvia the increase in matrix metalloproteinases activity, resulting in remodelling of the extracellular matrix through degradation of collagens and elastin which are hallmarks of photoaged skin.2 as part of a sunscreen formulation could be envisioned as a valuable approach for skin photoprotection of FRDA patients.The high sensitivity of FRDA fibroblasts to the oxidative solar UVA waveband has practical implications. Despite lying deep in the skin, fibroblasts are a major target for skin photodamage by long-wave UVA since up to 50% of this UV range penetrates into the dermis.Finally, the results obtained in the mouse fibroblast model of FRDA recapitulate the trend in differential sensitivities observed in human fibroblasts between healthy and FRDA cells, albeit to a lesser degree of intensity, thereby broadening the validity of this study model for the field of FRDA research.We have shown that skin fibroblasts from FRDA patients are highly sensitive to exposure to UVA. Our results altogether tie in with the concept that mitochondrial redox active iron overload is a crucial component of this sensitivity due to its ability to participate in Fenton chemistry, generating highly damaging ROS in UVA-irradiated FRDA fibroblasts.The authors state no conflict of interest.Supplementary informationClick here for additional data file.


DYRK1A, which has been implicated in DS cognitive dysfunction) and altered theta dynamics ; Dp10Yey mice show impaired alternation performance and reduced theta modulation of hippocampal low gamma; and Dp17Yey mice are not significantly different from the wild type. These results link specific hippocampal and mPFC circuit dysfunctions to cognitive deficits in DS models and, importantly, map them to discrete regions of Hsa21.Altered neural dynamics in the medial prefrontal cortex (mPFC) and hippocampus may contribute to cognitive impairments in the complex chromosomal disorder Down syndrome (DS). Here, we demonstrate non-overlapping behavioral differences associated with distinct abnormalities in hippocampal and mPFC electrophysiology during a canonical spatial working memory task in three partially trisomic mouse models of DS  that together cover all regions of homology with human chromosome 21 (Hsa21). Dp1Tyb mice show slower decision-making •These mutant mice show non-overlapping differences in spatial working memory function•Behavioral changes segregate with distinct EEG abnormalities in the hippocampus and mPFC•This links cognitive deficits to specific changes in hippocampal and mPFC circuit dynamics Chang et al. examine three partially trisomic mouse models of Down syndrome that together cover all regions of homology with human chromosome 21. They identify non-overlapping differences in spatial working memory function associated with distinct abnormalities in hippocampal and medial prefrontal electrophysiology. Dyrk1A, have been proposed to account for many of the alterations in neural development and abnormal phenotypes associated with DS and are thus targets for therapy development (Down syndrome (DS) is a complex cognitive disorder arising from the trisomy of human chromosome 21 (Hsa21), with an incidence of ∼1 in 800 live births worldwide . The curelopment .Activity in the hippocampus and mPFC can be characterized by oscillations in the theta and gamma bands. Hippocampal theta oscillations are associated with translational movement  and mnemTo further elucidate the neural mechanisms underlying cognitive deficits associated with DS, we studied three chromosome-engineered mouse models that each exhibit trisomy for one region of orthology with human chromosome Hsa21: the Dp1Tyb, Dp10Yey, and Dp17Yey strains  littermate control cohorts using a canonical spatial alternation task  makes a critical contribution to neurological and behavioral abnormalities by shifting the excitation/inhibition balance toward inhibition, for example  phenotype  in this region of Hsa21 homology.Finally, previous studies of transgenic mouse models of DS have led to the proposal that the overexpression of  example . The Dyrhenotype . Thus, tSuccessful memory encoding and retrieval are associated with increased theta power in both the hippocampus  and mPFCAs expected, the average power spectra from the mPFC A–2C and To establish whether the reduction in theta frequency observed in Dp1Tyb mice arose simply from the increased time that those animals spent immobile, we subsequently restricted our analysis to periods of movement only see . ConsistNext, we looked for differences in theta power and frequency between the first and second (sample and choice) runs, which are associated with memory encoding and retrieval, respectively. We identified a significant increase in hippocampal and medial prefrontal theta power during the second run in both the Dp10Yey and WT and the Dp17Yey and WT cohorts  and a significant interaction between run and genotype on hippocampal theta power in the Dp1Tyb and WT cohorts . SubsequCoherence between the phase of theta oscillations and the amplitude of concurrent gamma band oscillations is prevalent in the rodent hippocampus  and acroInterestingly, subsequent analyses indicated that the magnitude of hippocampal PAC in each pair of frequency bands also differed between specific DS models and WT controls. First, we found that theta-HG PAC was significantly increased in the Dp1Tyb group—which exhibited slowed decision-making—relative to WT controls, but not in any other mouse strain A. SecondTo confirm that the increased theta-HG PAC observed in Dp1Tyb mice did not arise from differences in movement statistics, we subsequently removed any effect of average time spent immobile on average theta-HG PAC values across animals in both mutant and control groups by linear regression and then compared the residual values between groups. This analysis confirmed that the increased theta-HG PAC in the hippocampus exhibited by Dp1Tyb mice was independent of differences in movement statistics . In sum,Planning, decision-making, memory encoding, and retrieval processes are each associated with increased functional connectivity between the hippocampus and mPFC in both rodents  and humaFirst, we found that theta coherence between the hippocampus and mPFC was significantly greater in Dp1Tyb mice compared to WT littermates A, while To further characterize potential changes in functional connectivity across mouse lines, we extracted the theta phase lag between the hippocampus and mPFC in order to assess the direction of communication between these regions D–4F. In Finally, we asked whether the behavioral and LFP abnormalities observed in Dp1Tyb and Dp10Yey mice persisted throughout life or were specific to the adolescent period during which they were initially tested . To thisFirst, we found that trial latency was significantly greater in Dp1Tyb mice compared to their WT control group across all three time points A, and thSecond, we found that the impaired alternation rate observed in young Dp10Yey mice persisted with age F. In conThe present study reveals distinct cognitive deficits and electrophysiological differences in three mouse models of DS, which, combined, carry duplications covering all Hsa21-orthologous regions. By taking an unbiased approach, we aimed to discover if cognitive deficits resulting from the triplication of genes/DNA elements in Hsa21 could be linked to individual regions with different sequence contents. As a measure of cognitive function, we used a canonical test of spatial working memory: spontaneous alternation in a T-maze . This beUsing this behavioral paradigm, we have shown that alternation deficits and hippocampal/mPFC neural dysfunction segregate with different regions of homology in the DS models. First, we found that spatial alternation, a putative index of mnemonic function, was impaired in Dp10Yey mice. In contrast, trial latency, which provides an independent measure of cognitive processing that includes decision-making, planning, goal-directed behavior, and attention , was proADAR2, S100B, CSTB, PRMT2, and TRPM2—have been shown to play a role in brain development and function, such that aberrant gene dosage may be related to intellectual disability in DS  responsible for the cognitive and electrophysiological phenotypes observed in these mice has the translational potential to reveal important routes toward phenotype modifying therapies, for example, by antisense oligomers, but our results indicate that targeting a single gene is unlikely to be sufficient.In summary, our study elucidates an important link among different regions of Hsa21, cognitive deficits, and both local and long-range neural circuit dysfunction. Importantly, our results imply that specific cognitive deficits in DS may result from different underlying genetic, functional, and regional abnormalities. This has important implications for understanding such cognitive deficits and indicates that therapies in DS will likely need to target multiple processes.m.walker@ucl.ac.uk). This study did not generate new unique reagents.Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Matthew Walker (cf./Nimr (hereafter referred to as Dp1Tyb); B6;129S7-Dp(10Prmt2-Pdxk)2Yey/J (hereafter referred to as Dp10Yey); B6.129S7 Dp(17Abcg1-Rrp1b)1Yey (hereafter referred to as Dp17Yey) and B6.129P2-Dyrk1atm1Mla. Dp1Tyb, Dp10Yey, Dp17Yey animals were maintained within a facility at University College London, whereas mice for the Dp1Tyb x Dyrk1atm1Mla/+ intercross were bred at the Francis Crick Institute, to generate Dp1Tyb∗Dyrk1aKO mice in which both alleles were on the same chromosome following a genetic crossover. All strains were maintained in separate colonies as hemizygous mutants backcrossed for over ten generations to C57BL/6J, with age- matched WT littermates used as controls. All experiments were undertaken using male animals, blind to genotype, which was decoded after experimental analysis and reconfirmed using an independent DNA sample isolated from post-mortem tail.We examined four mouse strains with the following alleles, previously described in All experiments were performed in accordance with the United Kingdom Animal (Scientific Procedures) Act 1986. Reporting is based on the ARRIVE Guidelines for Reporting Animal Research developed by the National Centre for Replacement, Refinement and Reduction of Animals in Research, London, United Kingdom. Mice were housed in controlled conditions in accordance with guidance issued by the Medical Research Council in Responsibility in the Use of Animals for Medical Research (1993) and all experiments were carried out under License from the UK Home Office and with Local Ethical Review panel approval. Mice were housed in individually ventilated cages (IVC) of 2-5 age-matched animals under controlled environmental conditions  with free access to food and water.Mice were anaesthetised with 2.5%–3% isoflurane  in 100% oxygen (flow rate of 1-1.5 l/min) via gas anesthesia mask  from a recently calibrated vaporizer . Body temperature was maintained with a heat blanket during surgery. A transmitter  immediately prior to the whole body completely entering the goal arm  were cut using a microtome  and stained with cresyl violet to allow histological location of the electrode track. This procedure allowed us to verify recording electrode locations, and LFP data were only included in the study if electrode tips were located in mPFC and dorsal hippocampus. In total, LFP data from just one animal was excluded because the recording site was outside the target region .For our initial analyses, continuous LFP recordings from each region were segmented into 10 s epochs that lasted from 5 s before to 5 s after animals reached the decision point on each run . Each epoch was visually inspected for artifacts prior to further analysis using custom written MATLAB  code . Any epochs that exhibited artifacts during visual inspection or for which video data (and therefore movement statistics) was either incomplete or unavailable were excluded from subsequent analysis.After de-trending and de-meaning the LFP signal from each trial, time-frequency decomposition was performed using a five cycle complex Morlet wavelet transform, with 1 s of data from the beginning and end of each epoch subsequently discarded to avoid edge effects. Time-frequency representations were then averaged across this time window to provide a power spectrum for each epoch, and each power spectrum was then normalized by its integral to facilitate comparisons between animals. Finally, these normalized power values were averaged across the 6-12Hz theta band to provide an index of theta power in each epoch for statistical comparison.th order finite impulse response (FIR) filter, discarded 1 s of data from the beginning and end of the signal to avoid edge effects, extracted the analytic signal using the Hilbert transform, and then computed dynamic power and frequency. Running speed data was up-sampled to match the power and frequency time series, allowing us to compute average theta power during movement periods only and to estimate the intercept and slope of the running speed v theta frequency relationship in each animal using linear regression.In addition, to characterize the relationship between theta power and movement, we zero-phase filtered each LFP signal in the 6-12Hz theta band using a 400To assay phase–amplitude coupling in the hippocampal LFP signal, we first computed cross-frequency coherence across a range of phase and amplitude frequencies following th order FIR filter, extracting the analytic signal in each band using the Hilbert transform, and then computing the mean amplitude of the higher frequency oscillations in each of 30 evenly distributed theta phase bins. The resulting vector length of each mean amplitude distribution, computed using the circular statistics toolbox for MATLAB .To compute an index of theta phase coherence between LFP recordings from the hippocampus and mPFC in each epoch, we first generated coherence spectra for each epoch using a window size of 1 s and an overlap between subsequent windows of 0.5 s and then averaged coherence values across the 6-12Hz theta range. In addition, to estimate the theta phase lag between concurrent oscillations in these regions, we zero-phase filtered each LFP signal in the 6-12Hz theta band using a 400Where significant differences in movement statistics between groups existed, we attempted to eliminate any potential confound on concomitant differences in theta coherence and theta-gamma PAC by linear regression. Specifically, we extracted the residual coherence or PAC values after regressing the amount of time spent immobile against those parameters across all animals (mutant and WT), and then assessed the difference in residual values between groups.Detailed statistical analysis was performed using SPSS 24 . All data are presented as mean ± SEM. Comparisons of means were performed using two-tailed Student’s t test and one way ANOVA with Tukey post hoc test if the data were normally distributed; Wilcoxon Signed test, Friedman’s test, or Mann-Whitney U-test if the data were not normally distributed . Generalized linear model (GLM) Type III tests followed by Bonferroni post hoc tests were used for analysis of repeated-measures longitudinal data. For circular  data, the Watson-Williams test was used to assess differences between groups . DiffereAll software used in this study is available and listed in the


Control over the quantization of electrons in quantum wells is at the heart of the functioning of modern advanced electronics; high electron mobility transistors, semiconductor and Capasso terahertz lasers, and many others. However, this avenue has not been explored in the case of 2D materials. Here we apply this concept to van der Waals heterostructures using the thickness of exfoliated crystals to control the quantum well dimensions in few-layer semiconductor InSe. This approach realizes precise control over the energy of the subbands and their uniformity guarantees extremely high quality electronic transport in these systems. Using tunnelling and light emitting devices, we reveal the full subband structure by studying resonance features in the tunnelling current, photoabsorption and light emission spectra. In the future, these systems could enable development of elementary blocks for atomically thin infrared and THz light sources based on intersubband optical transitions in few-layer van der Waals materials. A plethora of solid-state nanodevices rely on engineering the quantization of electrons in quantum wells. Here, the authors leverage the thickness of exfoliated 2D crystals to control the quantum well dimensions in few-layer semiconductor InSe and investigate the resonance features in the tunnelling current, photoabsorption and light emission spectra. A broad variety of electronic and optoelectronic applications have been described and proof-of-concept devices have been reported5. Along with high quantum yield3, compact design, and mechanical flexibility5, one of the attractive features of two dimensional semiconductors (2DS) is their band-gap tunability. Due to the change in quantum confinement with thickness, the primary optical transition can be tuned over a broad energy range (>1 eV8), and further fine adjustment is available through changes in dielectric environment7 and transverse electric fields9.Van der Waals crystals provide a platform for layer-by-layer material design which has expanded into a large multidisciplinary field during the last decade11. Nonetheless, experimental studies of higher energy subbands remained elusive until recently, when the first experimental observation of intersubband transitions in few-layer 12 using near-field THz absorption spectroscopy. While THz spectroscopy gives access to direct measurements of some intersubband transitions, it only works in a small energy window and requires specific doping of the materials studied. At the same time, angle-resolved photoemission spectroscopy (ARPES) only shows filled  subbands and has diminished energy resolution when applied to small (micrometer-size) crystals13.While the majority of 2DS demonstrate primary optical transitions in visible and near-infrared (IR) ranges, their intersubband transitions hold a largely unexplored potential to expand their optical activity further into the IR and THz. Depending upon the choice of material, number of layers and doping (n- or p-type), these subband transitions densely populate a region from 14.Here, we present an experimental approach that enables a comprehensive experimental study of the full subband structure of atomically thin 2DS, namely resonant tunneling spectroscopy combined with photoluminescence excitation (PLE) measurements. When applied to InSe, this method enables us to map the subbands on both conduction and valence band sides of the spectrum in agreement with theoretical band structure modeling and available ARPES data, as well as to trace the analogy between exfoliated atomically thin crystals and quantum wells in conventional semiconductors16 and features a crossover18 from weakly indirect  to direct character . Its high crystal quality has recently been demonstrated, resulting in exceptionally high electron mobility6 making it a promising avenue for developing atomically thin nanoelectronics19. For resonant tunneling measurements we build van der Waals heterostructures, where exfoliated few-layer 20 to provide atomically clean and sharp interfaces, and to avoid InSe degradation22. The assembled stacks were deposited on an oxidized silicon wafer and contacts were defined using electron beam lithography  on either side: e.g. the thresholds of EL for 4L InSe LED are Using bias values at the onset of EL, entclass1pt{minimaentclass1pt{minima24. At the same time, subbands in the middle of the spectrum  energy shown in Fig. 25. We further illustrate the quantum well behavior of the subbands in Fig. To illustrate the behavior of the subbands in the context of few-layer InSe as a quantum well, we show in Fig. 18. To provide both in- and out-of-plane polarization of photons for the excitation, we have extracted a cross-sectional block (lamella), with an hBN/InSe/hBN area of N = 2 to N = 7 see Supplementary Fig. Finally, we probed the subband states of InSe films using PLE, where the energy of the excitation light was continuously varied up to 2.7 eV while the photoluminescence intensity of the lowest energy exciton 18). Due to the p-doping, To interpret the PLE data we note that the material studied was lightly p-doped, so that 26, we estimate the layer-dependent exciton binding energies as shown in Fig. 27, while for thick flakes  silicon wafers and crystals displaying large uniform terraces were identified using optical microscopy. These flakes were picked up with graphene/hBN stack carried on a PMMA membrane20 followed by a second thin hBN crystal and a graphene monolayer. Finally, the assembled stack was released onto a thick  followed by Cr/Au (1 nm/80 nm) to prevent GaFor the measurements of PL and PLE, devices without graphene have been made to prevent the emission quenchingAll optical measurements have been performed at 4 K using an AttoDry100 cryostat and a AttoCFM I inset from Attocube. The illumination power on the sample was kept ~0.4 mW and the spot size was The tunneling measurements were carried out at 4.2 K by applying a DC bias and measuring the current between the source and drain graphene electrodes using a Keithley 2614B source-meter.Details concerning the spin-orbit coupling, the calculation of the density of states and the absorption spectra can be found in the Supplementary Fig. For high resolution scanning transmission electron microscope (STEM) imaging the FIB cross section received further thinning with a Helios Dual beam FIB-SEM, using sequentially lower milling currents of 30 kV, 16 kV, 5 kV, and 2 kV for ion beam milling and polishing. A probe side aberration corrected FEI Titan G2 80-200 kV was used with a probe convergence angle of 21 mrad, a HAADF inner angle of 48 mrad and a probe current of Supplementary Information


Adhesions are a very common complication in the abdominal surgery. Animal studies and human trials have evaluated strategies designed to reduce and prevent postsurgical adhesions but few have an evidence base that justifies routine use. A strategy to prevent adhesions effectively remains an urgent need. We studied a reproducible model of intra-peritoneal adhesion formation in rats using laparotomy with several peritoneal sutures to produce the adhesions. Here we show that entraining endogenous stem cells into injury sites using the combined effect of AMD3100 and low-dose FK-506 (AF) can reduce the adhesion score significantly and abolish peritoneal adhesions in 45% of animals in a rat model of severe postsurgical intra-abdominal adhesions, compared with saline controls. Searching for mechanisms, we found AF treatment dramatically increased SDF-1 expressing cells, HGF expressing Ym1+ M2 macrophages and CD133+ stem cells in the injury sites of peritoneal surface at day 5 post-operation. Our results demonstrate that medically induced recruitment of autologous stem cells using AF significantly reduced postsurgical intra-abdominal adhesions. These findings suggest a novel effective therapeutic approach to preventing adhesions in patients. These adhesions commonly cause problems and require additional procedures5. Up to 20% of these patients will experience symptoms ranging from pain to intestinal obstruction2. Approximately 10% of bowel obstructions caused by adhesions require reoperation known as adhesiolysis1, and the presence of severe adhesions at reoperation leads to increasing surgical difficulties and longer surgical times4. Pelvic peritoneal adhesions are responsible for approximately 10 percent of cases of female infertility6. Although attempts at amelioration have been adopted, the number of adhesiolysis procedures has been growing over the past decade5. The need for this is not only unfortunate for patients but increases the cost of health care.Formation of peritoneal adhesions which are a normal response to injury of the peritoneal surfaces aimed at repairing the damage following surgery that violates the peritoneum has become frequent11. Nevertheless, these have been only marginally helpful and a strategy to prevent adhesions effectively remains an urgent need.Peritoneal injury produces reaction aimed at healing the damaged peritoneal surfaces which results in the formation of adhesions (fibrotic scars) between two damaged peritoneal surfaces. In the last few decades attempts to stop this have included improved surgical techniques, optimized laparoscopy conditions, anti-inflammatory pharmacotherapies targeted at the inflammatory response and/or fibrin deposition, and creating a material interposition for prevention of peritoneal apposition. Sodium hyaluronate/carboxymethylcellulose (Seprafilm), oxidized regenerated celluose (Intercreed) and 4% icodextrin solution (Adept) are approved and generally viewed as best practice to prevent adhesions12. Further, treatment for a week every month for three months provided the same protection for rat and swine renal allografts14. This protection against rejection involved the presence of recipient stem cells found in the graft  causing local immunosuppression. We also showed that this medical mobilization of undifferentiated bone marrow cells produced a 25% reduction in healing time of skin wounds15. Importantly, this allowed reduced scars and provided for hair follicle growth not seen in the damaged area in controls. In the organ transplantation models, we incidentally observed that there were fewer abdominal peritoneal adhesions in small animals and no adhesion formation in large animals treated with AF combination drugs, even 3–4 years after pig kidney transplantation, however animals treated with saline or single drug formed severe intra-abdominal adhesions. In experiments with 85% partial hepatectomy it was noted that adhesions were minimized in the test group receiving combination therapy16. Based on these observations, we hypothesized that medical conscription and recruitment of undifferentiated bone marrow cells by this drug combination may promote regeneration of damaged peritoneal surfaces following surgery, and therefore prevents the formation of peritoneal adhesions. Here we test this hypothesis objectively by testing it in severe peritoneal scarring in rats. We show that this treatment was able to reduce the adhesion score significantly and abolish peritoneal adhesions in 45% of animals.We here report a new solution. This is from a novel entrainment of bone marrow precursor cells using a combination of AMD3100 (plerixafor) and FK506 (tacrolimus) that was found by chance to enable permanent liver allograft protection from rejection and survival in rats with just one week of treatmentAll animals that underwent surgery survived, no wound complications or infections occurred and all animals completed the study protocol. The surgical procedure reliably produced abdominal adhesions in control animals 14 days post-operation. Based on the adhesion grade and assessment criteria  that received saline control treatment subcutaneously developed severe intra-abdominal adhesions (adhesion scores: >4), while 16.7% (2/12) showed moderate intra-abdominal adhesions (adhesion scores: =3–4). Animals that received AMD3100 sc displayed severe  or moderate  intra-abdominal adhesions. Similarly, seven out of eight animals (87.5%) that received sc FK506 developed severe intra-abdominal adhesions, only one animal showed mild intra-abdominal adhesion (adhesion scores: =2–3). In contrast, animals that received a combination of AMD3100 and FK506 (AF) therapy sc displayed significantly less intra-abdominal adhesion formation Fig. : about 416. To find whether this treatment recruits undifferentiated bone marrow cells into the injury sites of the peritoneal surface after creating suture knots on the parietal peritoneum, immunohistochemistry staining for CD133, SDF-1 and HGF was done. Figure We have reported a synergy between AMD3100 and low-dose FK506 for entrainment of bone marrow-derived CD133 stem cells into damaged tissue18 by its attraction to the CXCR4 receptor on mobilized stem cells. Few SDF-1 positive cells were recognized histologically in animals treated with saline or single drug on day 5 after surgery. However, SDF-1 positive cells were strongly increased in treated animals  can entrain undifferentiated stem cells into injured tissuesals Fig. . Importa19, probably through stimulating the regeneration of peritoneal mesothelial cells20, inhibition of collagen deposition and its fibrinolytic capacity21. No HGF expression was found in adhesions from control animals treated with saline or AMD3100, but a few HGF positive cells were present in animals receiving low-dose FK506 alone on postoperative day 5. The number of HGF positive cells was dramatically increased in animals treated with AF combination therapy on postoperative day 5. Thus, entrainment of CD133 cells in the injury sites on the peritoneal surface was associated with increased HGF expression. The increased HGF expression may be important in preventing adhesions.HGF is crucial in regeneration, cellular growth and motility, and tissue formation. It has been reported that HGF prevents postoperative peritoneal adhesions22 of infiltrating macrophages in the injury sites of the peritoneal surface, immunofluorescent staining for the known M2 macrophage marker Ym1/Chi3l3 was performed. Few Ym1 positive cells were recognized in intra-abdominal adhesion tissues from saline control or AMD3100 treated animals, while Ym1 positive cells were slightly increased in low-dose FK506 treated animals on day 5 following surgery. In contrast, Ym1 positive cells were dramatically increased in adhesion tissues from animals with AF combination therapy  developed severe intra-abdominal adhesions on postoperative day 14  showed promising results for preventing postsurgical intra-abdominal adhesions33. This provides a precedent for prevention of abdominal adhesions by other adult stem cells. Because the in vitro preparation of autogenous stem/progenitor cells takes time and raises questions of quality, quantity and effectiveness, this stem cell therapy has limited practical application in the treatment of postsurgical abdominal adhesions. For this reason, the mobilization and recruitment of autologous stem cells that we have described is a new paradigm for a simple way to provide stem cells to injured sites of the peritoneum.Because adhesions occur at the injured surface lining of the peritoneum, a rapid rate of remesothelialization and restriction of inflammation likely are important factors that limit postoperative adhesion development. The healing time of small and large peritoneal wounds is the same16. The mechanism of action of the combination treatment is that it mobilizes bone marrow stem cells into the circulation via blocking the CXCR4 binding with SDF-1, but also promotes accumulation of the stem cells into the injured organ/tissues as the AMD3100 CXCR4 blocking effect wanes  which inhibit production of pro-inflammatory cytokines . It was found that increased M2 macrophages in sites of polyglycolic acid injury correlated with fewer peritoneal adhesions35. By using immunofluorescence staining for Ym1 (Chi3l3), a marker of M2 macrophages, we found that AF combination therapy dramatically increased Ym1 positive M2 macrophages in the injured peritoneum. Interestingly, a majority of Ym1 positive cells co-stained with hepatocyte growth factor (HGF) which is important in the action of M2 macrophages in intestinal healing through the secretion of HGF36. HGF decreases adhesion formation by inhibiting IFN-γ and PAI-137 and induces mesothelial cell proliferation38. Thus, HGF-producing M2 macrophages induced by AF combination therapy may reduce adhesion formation through regulating the inflammatory response and promoting wound repair/regeneration.Postsurgical adhesion formation is due to the peritoneal injury plus the associated inflammation. The inflammatory cells are predominantly macrophages at 24 hours following injuryet al.39. FK506 at low dose activates the bone morphogenetic protein (BMP) signaling pathway40 through FKBP12 ligands41 and BMP4 expression induces macrophage polarization towards M242. In addition, MSCs can induce M2-like macrophages in vitro that can suppress T cell and NK cell immune response and induce regulatory T cells44. CD133 is also a marker of circulating MSCs45. A high concentration of CD133 stem cells in the injured peritoneal sites may create an environment conducive to M2 macrophages.In our experiments, low-dose FK506 increased Ym1 positive M2 macrophages in the injured peritoneum. Interestingly, low-dose FK506 had a synergistic effect with AMD3100 in increasing M2 macrophages in the injured sites, similar to the report from Bai 46. Although mobilized bone marrow stem cells may generate mesothelial cells and promote remesothelialization, we chose to direct our focus to attenuation of intra-abdominal adhesion formation. Unlike reports of the effect of delivering processed adult stem cells into the peritoneal cavity in ameliorating intra-abdominal adhesion, this study expands on that to demonstrate that pharmacological mobilization and entrainment of the stem cells can reduce/attenuate postsurgical abdominal adhesion formation by stem cells accumulated directly from the circulation through release from the bone marrow. Our AF combination therapy may prevent/attenuate postsurgical intra-abdominal adhesion formation via targeting two important factors – remesothelialization and inflammation through recruiting CD133 stem cells and HGF producing M2 macrophages.Evidence from bone marrow transplant recipients suggests that the bone marrow progenitor cells might regenerate mesotheliumet al. unpublished data). In both cases this combination treatment optimized the ability of the stem cells to do the same thing: return the damaged tissue to normal. That required normal mesothelium for the peritoneal surface and normal bowel in the case of the experiments with intestinal anastomoses (and no adhesions to the suture line). This predisposition to return inflamed or damaged tissue to normal is likely a generally applicable feature of this combination therapy.This effect of warding off adhesive peritoneal healing is not incompatible with the increase in speed and completeness of healing of all three intestinal layers in colonic anastomoses that we also found with this treatment  and used at 12 to 15 weeks of ages with body weight between 250 to 350 grams. Animals were maintained in a pathogen-free facility of Johns Hopkins University School of Medicine and all animal experiments were performed in accordance with the United States National Institutes of Health (NIH) guidance13. A 3 cm vertical midline incision was made to access the abdominal cavity. Abdominal adhesions were induced by creating four 1–0 Prolene  suture knots on the anterior parietal peritoneum in a linear distribution. Each suture encapsulated approximately 2 cm of the parietal peritoneum. The diameter of the tied suture was approximately 5 mm. After the creation of the sutures, the peritoneum was closed in two layers by interrupted 3–0 silk sutures. All of the surgical procedures were performed by the same surgeon.Animals were anesthetized with the inhalation of Isoflurane  and the abdominal skin was prepared and disinfected by povidone-iodine before the procedureFifty-five animals with surgical procedures were allocated into four experimental groups: (1) Control group (n = 16): animals received the same volume of saline  immediately after surgery and every other day for 10 days; (2) AMD3100 treatment group (n = 12): animals received AMD3100  immediately after surgery and every other day for 10 days; (3) FK506 treatment group (n = 12): animals received FK506  immediately after surgery and every other day for 10 days; and (4) AF treatment group (n = 15): animals received AF combination therapy  immediately after surgery and every other day for 10 days. Sixteen animals (n = 4/group) were sacrificed for collecting tissue samples on postoperative day 5, and the other animals were sacrificed on postoperative day 14 for evaluation of intra-abdominal peritoneal adhesions.et al.47 was modified by including evaluation of the difficulty to lyse the adhesion which is described in the classification of Mazuji et al.48 . Antigen retrieval of paraffin section was achieved by a microwave, using the antigen retrieval solution . Non-specific serum blocking was performed by incubation with 5% goat serum for 30 min. Tissue sections were then incubated with primary antibodies at 4 °C overnight, followed by incubation with secondary antibodies at room temperature for 45 minutes. Tissue sections were then incubated with AB complex  for 30 min according to the manufacture instruction to amplify signals, and reacted with DAB . Counterstaining was performed by using Haematoxylin for 20 seconds. The following antibodies were used: biotin conjugated anti-CD133 antibody , anti-HGF antibody , anti-SDF-1 antibody , and biotinylated goat anti-rabbit IgG . Double staining of HGF and Ym1 was also performed by immunofluorescent stains using frozen sections. HGF staining was carried out using anti-rabbit HGF antibody  and FITC-conjugated donkey anti-rabbit IgG antibody . After HGF staining, the sections were incubated with phycoerythrin (PE) conjugated anti-Ym1 antibody  for 1 hour at room temperature. Cell nuclei were stained blue with DAPI. Tissue sections were analyzed by fluorescent microscopy.The parietal peritoneum and surrounding adhesion tissues were explanted and fixed in 2% PFA. Cut sections of 6 µm were prepared from PFA-fixed paraffin-embedded tissues for CD133, SDF-1 or HGF staining or frozen tissue for double staining of HGF and Ym1, a marker of M2 macrophages. Each representative section was stained with hematoxylin-eosin, and immunohistochemical stains were performed with the avidin-biotin-peroxidase complex methodQuantitative data are expressed as mean ± standard deviation (SD). Adhesion scores were assessed using the one-way ANOVA test followed by Bonferroni-Holm posthoc tests for comparisons between four groups. Statistical analysis was performed using SPSS 13.0 software. P < 0.05 was considered significant.