The neuroprotective effects of these drugs were mediated by modulation of targets in GABA/glycinergic and glutamatergic pathways. In vivo, the combination alleviated cognitive deficits in the acute Aβ25–35 peptide injection model and in the mouse mutant APP transgenic model. Several patterns altered in AD were also synergistically normalised. Our results open up the possibility for a promising therapeutic approach for AD by combining repurposed drugs.Alzheimer disease (AD) represents a major medical problem where mono-therapeutic interventions demonstrated only a limited efficacy so far. We explored the possibility of developing a combinational therapy that might prevent the degradation of neuronal and endothelial structures in this disease. We argued that the distorted balance between excitatory (glutamate) and inhibitory (GABA/glycine) systems constitutes a therapeutic target for such intervention. We found that a combination of two approved drugs – acamprosate and baclofen – synergistically protected neurons and endothelial structures  Alzheimer's disease (AD) is a lethal neurodegenerative disorder of older persons, affecting nearly 40 million patients worldwide. Clinically it is manifested by rapid progressive deterioration leading to the loss of independence. Mental capacities involving memory, language and everyday functioning are affected and this loss is often accompanied by psychosis. With the aging of world population and emerging of new effective treatments for other mortal diseases the importance of AD as an economic burden and limiting factor of life duration is progressively becoming more and more important. To date, all treatment options are marginally effectiveTo affect the different elements that appear to contribute to this complex disorder, we are proposing here a new combinational approach. The systematic analysis of available genetic and functional data in the literature has drawn our particular attention to the disturbance in the balance between glutamate, GABA, and glycine systems for the development of pathological features prominent in AD that could play a role in degeneration of brain structures. Glutamate system in the brain is not only important for signal transmission and plasticity but also for the regulation of survival or induction of apoptosis of brain cells. This system is counter-balanced by inhibitory GABA and glycine signalling with normal brain functions assured by the subtle equilibrium between these inhibitory and excitatory (E/I) activities.Deregulation of glutamate excitatory neuronal signalling has been proposed as a neurophysiological mechanism responsible for cognitive impairment in ADWe argued that the chronic presence of toxic Aβ oligomers could modify this balance, thereby impairing cognitive functions and enhancing the pro-apoptotic activity both in neuronal and endothelial cells. Thus, we have selected several candidate drugs, each potentially acting on multiple targets in these pathways. Since our ultimate goal has been to progress rapidly toward the AD treatment development, we focused on drugs that met two principal criteria: 1) used for a long time to treat other disordersThe first chosen drug is acamprosate calcium (ACP), an anti-craving agent that is thought to influence glutamatergic transmission and to affect the E/I balance distorted in ethanol addiction. ACP is a safe drug being used at exceptionally high doses. ACP decreases glutamate levels induced in human brain during ethanol withdrawal and reverses the resulting hyperexcitabilityB receptors. BCL has rather favourable safety profile even when used at high doses and is the only agonist of GABAB receptors that is authorised for human use. It should also be noticed that glutamate, GABA and glycine receptors targeted by ACP and BCL are also important for the function of the endothelial BBB1718Furthermore, we thought that to be effective and to resist the possible feedback changes during chronic treatment, we should use a drug combination that acts also on the brain major inhibitory system: GABA. We therefore selected a second drug, (RS)-baclofen (BCL), used for the treatment of spasticity for several decades and which activates inhibitory metabotropic GABAAt this stage we decided to test ACP or BCL alone and in combination (ABC) in a battery of cellular and animal paradigms. No satisfactory model exists for AD1–42 peptides, we used two models: a primary culture of rat cortical neurons1–42 peptides described by us previously1–42 oligomers as assessed by Western blot analyses using the 6E10 antibody. We verified that only preparations containing Aβ1–42 oligomers were able to induce toxicity in neuronal cultures, while initial peptide preparation that did not contain oligomers was not toxic1–42 toxicity  test, which explores hippocampal-dependent spatial memorym memory , as wellm memory . We also objects . ABC was objects . Furthers . This factor is thought to undergo a compensatory increase under conditions of Aβ-associated neurotoxicity25–35-injured mice, but it was maintained at normal levels by ABC treatment , was first approved in 1989 and since then has been used to treat millions of alcohol-dependent patients worldwideOur analysis of regulators acting on interconnected signalling pathways led to two well-known drugs with rather favourable safety profile: BCL and ACP. Some of their targets are well documented, while the action on others could be indirect and dose specific, especially in the case of ACP usually used at extremely high doses. Metabotropic GABAet al. demonstrated that a positive allosteric modulator of group II mGluR2 aggravated Aβ-induced neurotoxicity1–42 in cortical synaptoneurosomes in a mouse model of AD, while stimulation of group II mGluRs triggers production and release of both Aβ1–42 and Aβ1–40Calcium ACP was shown to be a weak modulator of NMDA receptors53A receptors but mostly are found in extrasynaptic sites. In our experiments, we demonstrated that neuroprotection by ACP is also mediated by its agonistic action on GlyR channels. We hypothesise that activation of GlyRs by ACP might be an important factor for the attenuating excessive/aberrant neuron excitability, such as deleterious over-activation of extrasynaptic NMDA receptors by Aβ1–42 toxicity, its antagonistic signalling through both Group I and group II metabotropic glutamate receptors as well as agonistic activation of GlyR channels is required, while BCL action is mediated by activation of GABAB receptors. Yet, our data is not conclusive at this stage but we suppose that neuroprotective effects of ABC could be mediated by a serial signalling cascade involving all these receptors. Future work is needed to further characterise the mode of action of ABC in this regard as well as its specificity on our studied targets.In the ethanol addiction model, ACP has been shown to act in the nucleus accumbens through agonism on glycine receptors (GlyRs)In vitro, ACP and BCL proved to be cytoprotective both for neurons as well as for vascular cells with strong synergistic effect and at relatively low doses. The exact mechanisms explaining such action are only emerging, but we found that neuroprotection could be due to the down-regulation of glutamate release, tau phosphorylation and diminished oxidative stress, and that it is mediated by an antagonistic action on metabotropic receptors of classes I/II and an agonistic action on GlyR in the case of ACP, and activation of GABABR in the case of BCL.While no comprehensive model for Alzheimer's disease exists1–42, oxidative stress, inflammation) of AD were significantly diminished by the drug combination. The effect was not due to a simple memory enhancement since in animals injected with nonspecific scrambled Aβ peptide as well as in normal animals cognition was not affected by ABC. In addition, ABC proved to be effective when tested in the transgenic mouse model at a stage when cognitive functions were already altered. Together, all these results open the possibility that ABC could exert a disease modifying effect. Already, we demonstrated that several patterns altered in AD, such as glutamate accumulation, tau hyperphosphorylation, endogenous Aβ oligomers and synaptic loss were synergistically normalised by ABC.The acute model of intracerebroventricular injection of Aβ peptide into healthy rodents mimics main features of AD-type dementia including neuronal degeneration, cognitive deficits accompanied by hippocampal dysfunctionin vitro studies to animal models. Over 200 compounds have been shown to affect favourably AD-like pathology in experimental modelsin vitro and in vivo improvement of the number of diverse biological AD related features as our combination. It protected both neuronal and vascular brain cells from Aβ insults, preserved synapses and reduced excessive tau protein phosphorylation, while at the same time reduced soluble endogenously produced Aβ peptides, diminished the signs of neuroinflammation and gliosis and alleviated cognitive deficits in different memory paradigms in AD animal models. We consider that the integrity of our data suggest that ABC might be a valuable disease-modifying treatment for AD, acting through a new mechanism implicating multiple molecular targets. If confirmed by powered clinical testing, low-dose ABC could serve a basis for a new conceptual approach for Alzheimer's disease therapy and also might help to understand the underlying mechanisms of this devastating dementia.Our results demonstrate the value of 1) the combinational approach and 2) using for this purpose previously existing drugs in new combinations25–35 intoxications. For transgenic mice experiments, male mice overexpressing the 751 amino acid form of human APP (hAPP) with London (V717I) and Swedish (KM670/671NL) mutations (hAPPSL) under the control of the murine Thy-1 promoterin vitro experiments, female Wistar rats (Janvier) on gestational day 15 were killed by cervical dislocation and foetuses were removed from the uterus.All animal procedures were carried out in strict adherence to the European Community Council Directive of September 22, 2010 (2010/63/UE). All experiments and protocols were authorised and approved by the French Ministry of Research, as well as by Animal Welfare Committees of Amylgen, Biotrial, QPS and Neuronexperts. All efforts were made to minimise the number of animals used. Male Swiss mice  aged 4–6 weeks and weighing 25–32 ± 2 g, and male Sprague Dawley rats (Janvier) aged 7–8 weeks weighing 220–300 g were used for Aβ1–42 peptide  was prepared in serum-free culture medium (0.1% DMSO) at 40 μM and was slowly shaken at +37°C for 3 days in the dark. After 3 days, Aβ1–42 oligomers were used on primary cortical neurons at specified concentrations and for indicated incubation times depending on the assay  to check for reproducibility between studies. In addition, we note that our Aβ1–42 oligomeric preparation was performed in media containing phenol red at concentrations (i.e. 44 μM) that did not affect oligomeric formation as assessed by Western blot analyses attesting for the presence of these toxic oligomers42 oligomerisationAβhe assay . It is w25–35) and scrambled control Aβ25–35 (Sc.Aβ) peptide were purchased from Genepep  for mice and from Bachem (Germany) for rat experiments. Peptides were solubilised in distilled water at a concentration of 3 mg mL−1 (mice) or 2 mg mL−1 (rats) and stored at −20°C until use. Before injection, peptides were incubated at +37°C for 4 days, allowing Aβ25–35 to form oligomers. A quality control analysis for Aβ25–35 aggregation was systematically performed by light scattering analysis before each injection of Aβ25–35 peptides. 9 nmol per mouse or 15 nmol per rat of Aβ25–35 peptide or Sc.Aβ were administered once icv in a final volume of 3 μL per mouse or 8 μL per ratThe amyloid-β[25–35] peptide , Strychnine , (S)-3,5-dihydroxyphenylglycine  and -amino-2,4-pyrrolidinedicarboxylic acid  were dissolved in 0.1% DMSO (except for DHPG dissolved in water) and added 2 h  before ACP (8 nM) or BCL (400 nM) to rat primary neuronal cells.(RS)-Baclofen and acamprosate calcium were provided by Sigma Aldrich. Acamprosate sodium was synthesised by NovAlix . Drugs were solubilised in distilled water and prepared before each administration. For xication . For mecper os twice daily at 8:00 am and 6:00 pm in a volume of 5 mL Kg−1 (mice) or 10 mL Kg−1 (rats). Aβ25–35 or Sc.Aβ was injected once for each experiment and treatment started 24 h before Aβ25–35 or Sc.Aβ or injection and lasted until the end of behavioural tests . Rat primary cortical and hippocampal neurons were cultured as described previously−1 of brain-derived neurotrophic factor . Cells were seeded in 96 well-plates pre-coated with poly-L-lysine  and were cultured at +37°C in a humidified air (95%)/CO2 (5%) atmosphere. After 11–13 days of culture of cortical neurons, cells were pre-treated for 1 h with ACP, BCL or combination (ABC) and then intoxicated with the appropriate concentration of Aβ1–42 during variable incubation times depending on the experiment , 2% of PS (Pan Biotech) and were seeded at the density of 20,000 cells per well in 96 well-plates  in a final volume of 100 μL  were rapidly thawed at +37°C and immediately suspended in 9 mL Dulbecco's modified Eagle's medium  containing 10% of foetal calf serum . Cell suspensions were centrifuged at 180 f 100 μL . 3 diffe1–42 intoxication.Before performing experiments with our candidate drugs, we validated experimental conditions with BDNF (50 ng/mL) for neuronal cultures or VEGF (10 nM) for endothelial cultures as positive controls to check and confirm the restoration and the validity of each endpoint after Aβ1–42 intoxication  and computed for each condition. Results were expressed as the number of overlapping stained cells per field and reported as a percentage of vehicle control.After Aβxication , cells wxication . Nuclei 1–42 intoxication  laboratories  for rats. Transgenic mice studies were performed and validated at QPS facilities (Austria). Sample sizes used for behavioural analyses were also determined in each of these companies on the basis of previous experience on assay variability. Dead mice and mice that did not succeed preliminary control tests (i.e. very weak locomotor activity) were excluded from whole analyses.Animals were tested in a random and blind manner. Treatment groups were equally represented in each cage. Mice were habituated 30 min to 2 h before each experiment. Acute Aβeriments  and read25–35 injection, with 20 min between each swim. For hAPPSL mice, training consisted in 4 swims per day for 4 days. The latency, expressed as mean ± s.e.m., was calculated for each training day. A probe test was performed 24 h after the last swim, on day 13 after Aβ25–35 injection (retention phase). The platform was removed and each animal was allowed a free 60 s swim. The time spent in each quadrant was then determined. From Day 14 to 16 after Aβ25–35 injection, or after the training test performed in hAPPSL mice, animals were tested for spatial working memory. Working memory was specifically assayed by changing the platform location everyday (four swims per day during 3 days) and by using a training inter-trial time of 2 min. The swimming times to find platform of first (swim1), second (swim2), third (swim3) and fourth trial (swim4) of every day were calculated and averaged in Aβ25–35 mice experiments. In hAPPSL mice experiments, only data of day 2 trial were used.Briefly, swimming was recorded using a Videotrack software , with trajectories being analysed as latencies and distances. The software divides the pool into four quadrants. Training consisted in three swims per day for 6 days, performed between day 7 and 12 after Aββ25–35 or Sc.Aβ peptide injection, mice were placed individually in a squared open-field and their locomotor activity was captured through an IR-sensitive camera and analysed using the Videotrack® software (Viewpoint) in terms of total distance travelled (cm). 24 h later (7 days after Aβ25–35 injection), two identical objects were placed at defined positions. Each mouse was placed in the open-field and the exploratory activity recorded during 10 min. The duration of contact with objects was analysed using the Nosetrack® protocol (Viewpoint). 24 h later (8 days after Aβ25–35 injection), the object in position #1 was replaced by a novel one differing in colour, shape and texture from the familiar object. Each mouse was placed again in the open-field and the exploratory activity recorded during 10 min. The preferential exploration index was calculated as the ratio of the duration of contacts with the object in position #1 over the total duration of contacts with both objects. For rats, 16 days after Aβ injection, rats were placed individually in a squared open-field and two trials spaced by 2 h interval were performed. During the first trial , rats were placed in the arena containing 2 identical objects and time required by each animal to complete 15 s of object exploration were determined. For the second trial , one of the objects presented in the first trial was replaced by an unknown object (novel object). Rats were placed back in the arena and exploration of each object was determined. For both trials, locomotor activity of rats was scored. Only animals having a minimal level of object exploration of 5 s during the testing trial were included in the study. The time spent in active exploration of the novel object and the familiar object (Delta Novel-Familiar) was measured.Briefly, 6 days after A25–35 or Sc.Aβ peptide injection, each mouse was placed at the end of one arm in a grey polyvinylchloride Y-maze. The series of arm entries, including possible returns into the same arm, was recorded visually. An alternation was defined as entries into all three arms on consecutive occasions. The number of the maximum alternations was the total number of arm entries minus two and the percentage of alternation was calculated as actual alternations/maximum alternations × 100. Measured parameters included the percentage of alternation (memory index) and total number of arm entries (exploration index).7 days after Aβ25–35 or Sc.Aβ peptide injection, each mouse was placed into the white compartment. After 5 s, the door was raised. When the mouse entered the darkened compartment and placed all its paws on the grid floor, the door was closed and the foot shock (0.3 mA) delivered for 3 s. The latency spent to enter the dark compartment and the number of vocalisations were recorded. The number of vocalisations did not differ among groups, indicating that shock sensitivity was unaffected by the treatments (data not shown). The retention test was carried out 24 h after training (9 days after Aβ25–35 or Sc.Aβ peptide injection). Each mouse was placed again into the white compartment. After 5 s, the door was raised and the step-through latency (latency to enter the dark compartment) was recorded up to 300 s.Briefly, the apparatus consisted of two compartments with one illuminated with white polyvinylchloride walls and the other darkened with black polyvinylchloride walls and a grid floor for electrical shocks. A guillotine door separated each compartment. 8 days after Aβ25–35 or Sc.Aβ injection, each mouse was anesthetised by intramuscular injection of 80 mg Kg−1 ketamine and 10 mg Kg−1 xylazine, and quickly perfused transcardially with 100 mL of saline solution followed by 100 mL of paraformaldehyde 4%. Brains were removed and kept for 24 h in the fixative solution at +4°C, then cut in coronal sections (20 μm) using a vibratome . Sections were stained with 0.2% cresyl violet reagent (Sigma), dehydrated with graded ethanol, treated with toluene and mounted with Mountex medium . Examination of the CA1 area was performed using a light microscope , slices being digitalised through a CCD camera  with the NIH Image version 1.63 software. CA1 measurement and pyramidal cell count have been processed by ImageJ (NIH). Data were calculated as average of 8 slices and expressed as number of CA1 pyramidal cells per millimetre for each group.Eight days after Aβ25–35 or Sc.Aβ peptide injection, Evans Blue (EB) dye  was injected i.p. 3 h prior to the transcardiac perfusion. Mice were anesthetised i.p. with 200 μL of pre-mix 80 mg Kg−1 ketamine and 10 mg Kg−1 xylazine, then perfused transcardially with 250 mL of saline solution. After decapitation, the brain was removed, dissected and weighed for quantitative measurement of EB extravasation. Samples were homogenized in PBS and mixed by vortexing after the addition of 60% trichloroacetic acid to precipitate the protein. Samples were cooled at +4°C, and then centrifuged 30 min at 10,000 g at +4°C. The supernatant was measured at 610 nm for absorbance of EB using a spectrophotometer. EB quantification was expressed as pg per mg of brain tissue.Seven days after Aβ1–42, cPARP, BDNF, IL-1β, TNFα) days after Aβ25–35 or Sc.Aβ injection, hippocampi were dissected out and rinsed in ice-cold PBS to remove excess blood, and weighed before nitrogen freezing and −80°C storage. Tissues were cut into small pieces, homogenised, sonicated and centrifuged. The supernatants were assayed immediately by ELISA for GFAP, synaptophysin, IL-1β , TNFα , BDNF , cPARP  and Aβ1–42  following manufacturer's recommendations. All samples were assayed in duplicate and only samples for which the CV was <25% between replicates were included for analyses. Results were expressed in pg protein per mg of brain tissue, or as % of Sc. control. Protein concentration was determined in brain homogenates with the BCA protein assay kit .Seven (synaptophysin), 8 (GFAP), or 9 , and 10 μL of 1 mM cumene hydroperoxide (CHP) was added to the sample and incubated for 30 min at room temperature to determine the maximal oxidation level. Absorbance was measured at 580 nm (A5802). The level of lipid peroxidation was determined as CHP equivalents according to: CHP equivalent = A5801/A5802 × (CHP (nmol)) × dilution, and expressed as CHP equivalents per mg tissue.Briefly, mice were killed by decapitation and hippocampi were rapidly isolated, weighed, and kept in liquid nitrogen until assayed. After thawing, brains were homogenised in cold methanol , centrifuged at 1,000 t-test was also used as a supportive technique. For repeated measures data (MWM acquisition and working memory tests in http://www.graphpad.com/scientific-software/prism).Statistical tests were two-tailed, and conducted at a 5% significance level. Sample sizes used for behavioural analyses were defined on the basis on previous experience on assay variability. Data distribution and within-group variation were preliminary assessed in order to guide our methodological choices. We applied an Analysis of Variance (ANOVA) with Dunnett's test for comparison of more than one experimental group against a reference. A R (http://cran.r-project.org). Briefly, CI compares dosages for a given combination to those expected to obtain the same combination effect under a simple additive assumption, and offers a quantitative definition for additive effect (CI = 1), synergism (CI < 1), and antagonism (CI > 1) in drug combinations. Isobologram is a popular graph introduced by LoeweDrug combination analyses with calculation of Combination Indexes (CI) and isobologramsSupplementary Information


As no ferromagnetic component is present in the system, the writing magnetic field does not need to be pulsed for readout, allowing permanent magnets to be used. Based on our prototypes, we construct a comprehensive model of the magnetoelectric selection mechanisms in thin films of magnetoelectric antiferromagnets, revealing misfit induced ferrimagnetism as an important factor. Beyond memory applications, the AF-MERAM concept introduces a general all-electric interface for antiferromagnets and should find wide applicability in antiferromagnetic spintronics.Magnetic random access memory schemes employing magnetoelectric coupling to write binary information promise outstanding energy efficiency. We propose and demonstrate a purely antiferromagnetic magnetoelectric random access memory (AF-MERAM) that offers a remarkable 50-fold reduction of the writing threshold compared with ferromagnet-based counterparts, is robust against magnetic disturbances and exhibits no ferromagnetic hysteresis losses. Using the magnetoelectric antiferromagnet Cr 2O3, reporting 50-fold reduction of writing threshold compared to ferromagnetic counterparts.Magnetoelectric coupling allows switching of magnetic states via gate voltage pulses. Here the authors propose and demonstrate a purely antiferromagnetic magnetoelectric random access memory based on Cr Therefore, the readout signal of MERAM cells is conventionally acquired from a ferromagnet that is coupled with the magnetoelectric antiferromagnet by exchange bias4567In the effort to develop low-power data processing and storage devices, nonvolatile random access memory schemes have received considerable attention22O3 as an AF element, we demonstrate a complete working AF-MERAM cell, proving that this concept yields substantial improvements in terms of magnetoelectric performance over comparable MERAM realizations with ferromagnets. In particular, by removing the ferromagnetic component from MERAM, we reduce the writing threshold by a factor of about 50. These characteristics render AF-MERAM a promising new member to the emerging field of purely antiferromagnetic spintronics3Here we put forth the concept of purely antiferromagnetic MERAM (AF-MERAM) , which a2O3(200 nm)/Pt(2.5 nm) that is prepared on Al2O3(0001) substrates. Similar stacks with α-Cr2O3 have been extensively studied in the scope of traditional MERAM elements with ferromagnetic Co layers413142O3(0001), which is rigidly coupled to the AF bulk and creates proximity magnetization in the Pt film16H≈+0.5 MA m−1 along the film normal. The test sequence mimics random access operations comprising the three essential elements of any memory cell: writing, storage and reading. One of the key technological advantages is that the memory cell operates in static magnetic fields and writing operations are triggered by the application of a voltage. No energy input is necessary during the storage times. The reproducibility of this process is demonstrated over 300 write–store–read cycles in To realize the memory cell, we use an epitaxial layer stack of Pt(20 nm)/α-Cr nm)/Pt2. nm that Hcool and electric cooling field Ecool=Vcool/t (t denotes the AF film thickness). For large EH fields, the order parameter selection is consistent with that expected in α-Cr2O3 C and the coercive gate voltage VC. For integration in microelectronics, the latter two are of particular relevance as the circuit voltage rating depends on them.45692O3, this leads to strongly increased magnetoelectric coercivities, especially for thin films of Cr2O3 . We find that the gate bias voltage is a material characteristic in thin films of magnetoelectric antiferromagnets, and in the following we reveal its physical origin and derive a means to control its value.The gate bias voltage of   presents2O3 thin-film systems457913151722232425262728When combining the large body of data on Crα reported to be about 1 ps m−1 in Cr2O3 . Its non-zero value in our system can be used to estimate the approximate ferrimagnetic moment density at the ordering temperature of about 21 °C, yielding a value of The gate bias voltage 2O3 antiferromagnetic film, as all magnetic moments, including boundary moments10162O3 thin films, by preparing three distinct systems with epitaxial underlayers of Al2O3(0001), Pt(111) or V2O3(0001).The presence of ferrimagnetism cannot be accounted for by any intrinsic effect within the Cr2O3 thin films are grown on a V2O3 underlayer, ferrimagnetism is almost entirely eliminated. 2O3-buffered system exhibiting virtually perfect EH symmetry. As highlighted by the indicated line profiles . While the magnetic moment of atoms near dislocations might be different from atoms in the relaxed lattice, this picture serves to illustrate that misfit dislocations do indeed unbalance the atomic populations in each of the two sublattices.Such dislocations arise due to the heteroepitaxial film growth  as the dtroscopy . As sket2O3-buffered sample emerges naturally from the previously introduced picture. Tensile misfit results in atoms being skipped from the bottom boundary sublattice instead of atoms being added. Therefore, tensile misfit results in the top boundary magnetization being aligned along the cooling field, while compressive misfit results in the top boundary magnetization being aligned opposite to the magnetic cooling field.Remarkably, the lattice misfit not only correlates with the magnitude of the emergent magnetization, but also with its sign with respect to the antiferromagnetic order parameter. This sign change of the ferrimagnetic behavior in the case of tensile misfit for the V2O3 buffered films are clearly more susceptible to the magnetic field than the V2O3-buffered films, which is in line with the substantially larger lattice misfit of the former over the latter. Moreover, a magnetic field of the same sign selects opposite antiferromagnetic states in the two systems, which corresponds to the opposite sign of the lattice misfit.To quantify ferrimagnetism, we investigate the magnetic field-induced antiferromagnetic order parameter selection with no electric field applied , which i2O3, whereas such functionality has previously been feasible only in multiferroic antiferromagnets such as BiFeO3  heated to 700 °C initially and to 500 °C after the first few monolayers. The background gas used was molecular oxygen at a partial pressure of 10−5 mbar. Chromium was evaporated from a Knudsen cell, vanadium was evaporated from a block target using an electron-beam and platinum was sputtered from a d.c. magnetron source. Deposition of the oxides was carried out using rates of about 0.4 Å s−1 and was monitored in situ by reflection high-energy electron diffraction. Oxide layers were subjected to a vacuum annealing process at 750 °C and residual pressure of 10−7 mbar directly after growth. The thin Pt top layers were deposited at lower temperatures of ≈100 °C using a higher rate of 1.0 Å s−1 to maintain layer continuity. Hall crosses were patterned from the top Pt layers, by SF6 reactive ion etching around a photoresist mask.Oxide films were grown by reactive evaporation of the base metal in high vacuum onto Transport was measured using zero-offset Hall2O3 films  to induce Zeeman splitting of the NV electronic ground-state spin. A microwave driving field was then locked to the spin transition at ≈2.864 GHz to track the additional Zeeman shift due to the stray field of the magnetic film surfaceScanning NV magnetometry was performed with a tip fabricated from single-crystal, <100>-oriented diamond that was implanted with The data underlying the present work are available upon request from the corresponding authors.How to cite this article: Kosub, T. et al. Purely antiferromagnetic magnetoelectric random access memory. Nat. Commun.8, 13985 doi: 10.1038/ncomms13985 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Supplementary Figures, Supplementary Table, Supplementary Notes and Supplementary References


These layer decoupling dynamics are a direct consequence of strongly anisotropic screening established within attoseconds, and demonstrate that important two-dimensional characteristics are also present in bulk crystals of van der Waals-layered materials, at least on ultrafast timescales.Strong quantum confinement effects lead to striking new physics in two-dimensional materials such as graphene or transition metal dichalcogenides. While spectroscopic fingerprints of such quantum confinement have been demonstrated widely, the consequences for carrier dynamics are at present less clear, particularly on ultrafast timescales. This is important for tailoring, probing, and understanding spin and electron dynamics in layered and two-dimensional materials even in cases where the desired bandgap engineering has been achieved. Here we show by means of core–hole clock spectroscopy that SnS 2, a prototypical van der Waals layered crystal.Owing to their layered nature, transition metal dichalcogenides possess an anisotropic electronic structure whose impact on carrier dynamics is not fully known. Here, the authors use X-ray spectroscopy to unveil the electronic coupling and attosecond dynamics in SnS Consequently, TMDs show promise as highly efficient field-effect transistors10, photovoltaics11 and spintronic devices12, and offer new avenues toward quantum computing. This promise hinges on a detailed understanding of the fundamental physics at play in TMDs, with particular emphasis on the spatially highly anisotropic electronic properties and the resulting consequences of quantum confinement in 2D. Indeed, exquisite band structure measurements using state-of-the-art angle-resolved photoemission (ARPES) have elucidated the electronic structure of TMDs15 and opened avenues toward tailoring bandgap and electronic properties, e.g., with different interlayer twist angles16. Despite this emerging body of ARPES work and while the electronic structure and excitations in TMDs have been investigated widely18, the extent to which the anisotropic electronic structure of layered materials confers 2D character already in the bulk crystals is not yet fully understood. If bulk crystals already exhibit essential aspects of 2D materials, simplification of device fabrication protocols may be anticipated, broadening their use in novel electronic devices. A number of studies have indeed suggested that layers in bulk TMDs, such as ReS219, WSe220, MoS221, or in graphite22, are sufficiently electronically decoupled to present as 2D materials, e.g., in terms of spin polarization. The observation of anisotropic screening and carrier dynamics in the time-domain would constitute a direct probe of layer decoupling in bulk crystals, but such studies are at present missing due to the extremely short timescales involved and the difficulty of spectroscopically resolving the anisotropic dynamical processes.In recent years, the family of two-dimensional (2D)-layered transition metal dichalcogenides (TMDs) has gained significant interest due to their unique layer-dependent electronic properties, including layer-dependent band structures225. Using core–hole clock spectroscopy26, we observe spin-dependent anisotropic charge transfer on attosecond timescales in quasi-2D bulk SnS2, and show that already in bulk crystals individual layers are indeed strongly decoupled and essentially 2D. Past ultrafast spectroscopic studies have primarily focused on excitonic carrier-relaxation dynamics in bulk TMDs28 with timescales of <100 fs in WSe229, MoS230, and SnS231. These measurements lack the atomic-scale specificity and time resolution to fully probe the anisotropic-correlated electron dynamics. Our study fills this need by investigating the carrier delocalization dynamics on attosecond timescales32: We show for the first time that van der Waals layering leads to decoupling of individual 2D layers observed dynamically on sub-fs to few-fs timescales. We demonstrate that such dynamic layer decoupling is the origin of the predominantly 2D nature and strongly anisotropic electronic properties in bulk SnS2, and discuss the broader applicability to spin and electron dynamics in layered and 2D materials.Here we address this open question by investigating the ultrafast carrier dynamics in the layered semiconductor SnS2 conduction band. In this study, we excite from Sn 3d levels (M-edge) to the SnS2 conduction band so as to access different orbital and spin characters in the conduction band. Figure 2, separated into Sn 3d3/2 (Sn M4) and Sn 3d5/2 (Sn M5) components as a result of spin–orbit coupling. Each spin–orbit component is further split into at least two distinct features, labeled A and B. Comparison to high-level electronic structure calculations18 allows us to assign these features to excitations of different orbital components of the conduction band  levels. Since dipole selection rules dictate l), the transition into Sn 5s is formally forbidden. However, we still observe a weak transition due to hybridization of Sn 5s with S 3px,y, resulting in orbitals/bands with dipole-allowed p-character. This transition represents excitation of primarily in-plane orbitals of both Sn and S character within a 2D SnS2 sheet. In contrast, the strong XA features, B1 and B2, constitute dipole-allowed excitations to deeper-lying levels in the conduction band, and they are composed primarily of weakly hybridized Sn 5px,y, Sn 5pz, S 3pz, and S 3px,y levels. Some of these excitations access therefore out-of-plane orbitals ultimately responsible for coupling between adjacent 2D SnS2 sheets. The selective excitation in the XA process into these two groups of electronic levels provides the basis for probing the anisotropy of carrier dynamics in the van der Waals-layered material SnS2. Note that surface defects do not contribute to the observed spectral features in Fig. As will be discussed in more detail below, our experimental approach is based on resonant photoemission to probe the evolution of excited states on the timescale of a core–hole decay with sub-fs time resolution. In order to properly interpret the resonant photoemission results with respect to charge-carrier dynamics, we first need to understand the character of the excited electronic states reached in the initial X-ray absorption (XA) process, in particular the excitation of electrons into the SnS2 by monitoring competing decay channels following absorption of an X-ray photon from the Sn 3d levels into the conduction band region. Core-excited SnS2 decays on ultrafast timescales by Auger-like processes, which are observable with resonant photoemission spectroscopy 33. Figure 2.We access the site- and element-specific ultrafast carrier dynamics in SnSThe resonant photoemission contour plot of Auger emission intensity as a function of photon excitation energy and photoelectron kinetic energy is shown in Fig. 4,5N4,5N4,5 Auger features when the X-ray photon energy is scanned across the Sn M5 and M4 edges . Whereas the total enhancement of all decay channels is proportional to the combined absorption cross section, the distribution between the different channels is directly related to the electronic lifetimes in the conduction band of SnS2 as discussed above. We are thus able to obtain the electron dynamics from comparing the relative amplitudes of the normal and spectator Auger contributions to the Sn M4,5N4,5N4,5 features in Fig. 4,5N4,5N4,5 transitions correspond to a final state with d8 atomic configuration . We fit transitions to each of these final states by incorporating state-dependent Coulombic repulsion and a screening term in the two-hole final states, giving excellent agreement with theory34 (see Supplementary Table 2 in resonant Auger spectroscopy.The intensity map shows a strong resonant enhancement of the Sn M5 resonance) and 498 eV (Sn M4 resonance). At hν = 490 eV, the Auger spectrum is dominated by a resonantly shifted spectator Sn M5N4,5N4,5 Auger contribution (spectator shift of 1.1 eV), with a minor normal Sn M5N4,5N4,5 Auger component. There is also a small but visible excitation of normal Sn M4N4,5N4,5. Notably, the intensity of the spectator Auger feature is approximately four times larger than that of the normal Auger feature, indicating a long-lived excited electron that screens the core–hole decay. Similarly, at hν = 498 eV, the Sn M4N4,5N4,5 feature also has a dominant resonant Auger feature (spectator shift of 0.9 eV), but with a more significant contribution from normal Auger processes. This spectral decomposition analysis is repeated across the complete resonant photoemission map and yields the evolution of the integrated spectator and normal Auger feature intensities, as shown in Fig. 2.Two representative decompositions of the Auger spectra are shown in Fig. 33. The natural lifetime of both Sn 3d3/2 and Sn 3d5/2 core–holes is identical, as predicted by theory, and verified experimentally to be τc − h = 1/kc − h = 1.69 fs (refs. 37). The influence of Coster–Kronig decay channels associated with spin–orbit component-dependent lifetimes effects is not present in Sn on this edge. The experimentally accessible charge delocalization timescale is determined from the accuracy with which RPES intensities can be determined, requiring that intensities be no more than one order of magnitude apart to still enable determination of delocalization times, and hence 0.1τc − h ≤ τdeloc ≤ 10τc − h. As a result, we expect to observe competing processes on a timescale of about 170 as to 17 fs for both spin–orbit components. Under the assumption that core holes are strongly localized to an atomic site, charge transfer or delocalization of an excited electron away from the excited core diminishes the resonant contributions to the Auger spectrum in the spectator channel, as detailed above and in Fig. kdeloc = 1/τdeloc) of the core-excited electron can thus be calculated relative to the competing intrinsic core–hole decay rate (kc − h), assuming first-order rate laws for the Auger decay26:ISA and INA are the intensities of the spectator and normal Auger components, respectively. The ISA/INA fraction varies over the investigated photon energy window of 487–499 eV, encompassing both Sn M4,5 absorption edges and corresponding to a photon energy-dependent kdeloc. A small ISA fraction reflects fast-charge delocalization (high kdeloc), while a high fraction of ISA represents localization of the conduction band electron near the core-excited atomic site . Remarkably, the energy-resolved electron dynamics vary greatly across the conduction band and even among the spin–orbit components, providing an opportunity to probe the anisotropic and spin-dependent carrier dynamics in this layered van der Waals material.We investigate the ultrafast charge-carrier dynamics using the core–hole lifetime as an internal clock of the excited state dynamics2 is an indirect bandgap semiconductor with a flat valence band and a maximum between the high-symmetry points Γ and M, whereas the conduction band minima are at Γ and M  shows that such estimates  are compatible with our experimentally determined delocalization times. Note, however, that hopping times and delocalization times are not expected to be identical due to the presence of the core hole in the RPES measurements and possible difficulties of DFT calculations to capture bandwidths correctly.A comparison of these delocalization times with a simple estimate of hopping times obtained from the density functional theory (DFT) band structure calculations . Carrier lifetimes differ by approximately a factor two, offering an opportunity for dynamical spin filtering on ultrafast timescales in SnS242. Layer decoupling observed via dynamics on ultrafast timescales may therefore be useful not only for controlling electron dynamics, but also more generally for spin and quasiparticle currents in quasi-2D-layered materials.Interestingly, our resonant photoemission data also point to the existence of spin-dependent dynamics: The two excitation edges Sn M2 single crystals of 4H polytype were grown using the Bridgman method and n-doped with chlorine to a carrier concentration of 2.3(1) × 1017 cm−343. The SnS2 substrate was cleaved via mechanical exfoliation before introducing the sample to the ultrahigh vacuum preparation chamber. This was followed by pump down, baking for 12 h, and annealing at ~200 °C for 12 h. XPS measurements of Sn 3d3/2 and 3d5/2 features revealed no reduction of Sn4+ to Sn2+.As reported elsewhere, the SnS2 single crystals were mounted on a Mo foil using tungsten wires and annealed using a UHV button heater, followed by XPS analysis before starting the RPES measurements. The RPE spectra were acquired using a double-pass cylindrical mirror analyzer, mounted in the plane of the surface normal perpendicular to the incoming radiation. The incoming synchrotron radiation is linearly polarized with the electric field vector perpendicular to surface normal. XA measurements were performed in both TEY mode, using the drain current as well as channeltron detector current, and total fluorescence yield mode using a Si photodiode (IRD AXUV100) to differentiate between surface and bulk features, respectively. Multiple X-ray incidence angles  were measured for each absorption edge to establish angular dependence and ensure surface and bulk integrity. All spectra were calibrated against the incident X-ray photon flux measured on a gold grid installed upstream of the analysis chamber. RPES was collected at a pass energy of 25 eV in photemission mode, corresponding to 0.4 eV resolution, and the slits of the beamline spherical grating monochromator were set to approximately match this resolution for a total resolution of about 0.5 eV. The base vacuum was kept below 1 × 10‒8 Torr during the measurements, and sample integrity was checked before and after each RPES run using XPS. Photon energy calibration was achieved by fixing S 2p1/2 and S 2p3/2 at 162.5 and 163.6 eV binding energies, respectively, and by XA scans conducted before and after each RPES data set.All spectra were obtained at the SLAC National Accelerator Laboratory facility Stanford Synchrotron Radiation Lightsource (SSRL) on beamline 10–1. The SnSI0) of the incoming synchrotron radiation calibrated on a gold grid upstream from the sample. Reference scans of S 2p1/2 and S 2p3/2 were measured after each RPES and XA scan for energy calibration and global vacuum-level corrections.A linear background subtraction followed by an integrated background subtraction was performed on all XP and XA spectra. All spectral intensities were normalized with respect to X-ray photon flux  functional and a hybrid functional (HSE)47. For the HSE calculations, Hartree–Fock screening and exchange parameters, with a PBE correlation, were set to 0.2 Å−1 and 25%, respectively, known as the HSE06 flavor of the available hybrid functionals within the VASP package. A zone-centered Monkhorst–Pack scheme48 was adopted to integrate over the Brillouin zone with a k-mesh of 8 × 8 × 4 for the SnS2 structure, and a plane-wave basis kinetic energy cutoff of 300 eV was used. To represent the crystalline solid, periodic boundary conditions in all three spatial dimensions were applied. The total energies and Hellmann–Feynman forces on the atoms were converged to 10−6 eV and 50 meV Å−1, respectively. Space and momentum-projected DOS calculations and band structure calculations were performed by integrating over the Brillouin zone with a 16 × 16 × 8 k-mesh using the tetrahedron method with Bloch corrections44. Spin–orbit coupling was included during density of states (DOS) and band structure calculations.First-principle DFT was performed using the projector-augmented wave methodThe data that support the findings of this study are available from the corresponding author on request.Supplementary InformationPeer Review File


The electron beam is used to initiate the crack; during in situ observation of crack propagation the electron beam effect is minimized. The observed high-frequency emission of dislocations is beyond previous understanding of the fracture of brittle MoS2. Strain analysis reveals dislocation emission to be closely associated with the crack propagation path in nanoscale. The critical crack tip plastic zone size of nearly perfect 2D MoS2 is between 2 and 5 nm, although it can grow to 10 nm under corrosive conditions such as ultraviolet light exposure, showing enhanced dislocation activity via defect generation.Whether and how fracture mechanics needs to be modified for small length scales and in systems of reduced dimensionality remains an open debate. Here, employing  in situ electron microscopy to study fracture in MoS2 monolayers and report dislocation emission rates greater than expected accompanying crack propagation.How well the linear elastic fracture picture holds at small length scales and systems with reduced dimensionality remains an active area of inquiry. Here authors use  Fracture is a process relevant to many kinds of applications of materials. Competition between two modes, namely, the crystal plane cleavage and dislocation nucleation at the crack tip, is the main factor governing the ductile or brittle behaviour of crystals142O3 show perfect fringe lattices at the vicinity of the crack tips, and confirmed the absence of dislocations2, to differentiate from the tetragonal phase) is purely brittle at room temperatureIC equals to 2γ, where γ is the surface energy per unit area42  near the crack tip in vacuum and extends to 5–10 nm under corrosive environments. Our results also suggest that the specific 2D geometry improves ductility, in combination with the reported ultrahigh strength of 2D graphene2  for taking images with a short exposure time . The occurrence of the beam damage on MoS2 requires much higher beam intensity  on the sample exceeds the critical stress intensity factor . We confirmed that crack propagation is preferably initiated at some pores  TEM images in According to the criterion of continuum fracture mechanics, a crack starts to grow when the maximum stress intensity  near the crack tip. After 10 s, the dislocation climbs along the cracking direction by 1 nm. The geometric phase analysis (GPA)22 at the crack tip zone in 12 in 2 here should be a mixed mode  cracking2 crystals on the two sides of the dislocations can have some angle difference that can be locally considered as 2 nm-sized grains.in situ TEM observations. For example, in 22) direction is towards the newly emitted dislocation (11) for the original cracking direction becomes lower, whereas tensile strain (ɛ22) is higher and the direction from crack tip to the dislocation has the maximum ɛ22, and therefore the crack turns direction in area 2. Except for the dependence of crack propagation paths on the dislocation emissions as we observed here, other factors including external stress and surface adsorbents can also play a role to determine the crack path.The dislocation dynamics near the crack tip was investigated by more location . After 9location . By idenlocation . Figure rack tip  is differack tip . The modin situ observations. We performed in situ cracking experiments in the pristine sample, as well as the sample after low-intensity beam shower without beam damage  decreases with less amount of absorbents  at the crack tip, can be estimated by models or simulationsσY is the yield stress. For monolayer MoS2, the observed rc is equal to 2–5 nm. Linear fracture mechanics can be applied if the cracking is brittle and rc<<a, where a is the characteristic dimension of intrinsic defects in the object. However, near the cracks our monolayer MoS2 sample has only sulfur vacancy defects, implying that a approaches 0 nm. This implies a higher probability of generating plasticity.According to our observations, the blunting of the crack tip with a radius of up to 2 nm in MoScracking  in line 2, the so-called stress corrosion crack (SCC). For SCC, the KIC usually becomes smaller because of a chemical reaction assisted by release of the decohesion energy. Meanwhile, the yield stress also decreases. The as-grown monolayer MoS2 sample on a sapphire substrate was exposed to ultraviolet (UV) light with controlled humidity inside the chamber (see Methods)3 and oxygen-related radicals, can be created under UV that can quickly react with MoS2. This method was first applied to visualize the grain boundaries of 2D materialsTMD materials have been known to be susceptible to environment182 sample after 2 min of UV exposure is shown in 2 monolayer is subject to a biaxial in-plane stress mainly caused by the robust adhesion between MoS2 and the sapphire substrate2 single crystals can first be determined by the rather straight lines  semi-infinite crack propagation in linear elasticity theoryThe scanning electron microscopy image of a MoSht lines . A zoom-ht lines  clearly irection , as deteirection  that is TEM grid . Insteadrc from the crack tip, we can simulate the crack path between two cracks that approach to each other  can turn the direction of cracking easily when affected by other elastic fields  extends to a distance ther see . The shang paths  are loca2 sample is rechecked by high-resolution scanning transmission electron microscope (STEM) and electron energy loss spectra (EELS) . However, this is not contradictory to the usual plasticity enhancement of ceramic materials under corrosive environmentOn the other hand, the post SCC cracking MoSa (EELS) . The pre  brought the S and MoO3 vapours to the target sapphire substrate that was located at the downstream side. The centre heating zone was heated to 635 °C at a ramping rate of 15 °C per min. After reaching 635 °C, the heating zone was maintained there for 30 min and the furnace was then naturally cooled down to room temperature.MoS2  as a supporter and a protective layer with no post-annealing step. The MoS2 and PMMA support were then detached from the sapphire substrate by floating the PMMA/MoS2/sapphire, with the PMMA side up, in a hot 2 M NaOH solution for 10 min. The PMMA/MoS2 was then transferred into deionized water (4 times) for removing any remaining NaOH in the sample. Finally, the PMMA/MoS2 was scooped out in pieces and placed on a Quantifoil TEM grid with a gold-supported thin film . The grids were kept in an ambient oven at 80 °C for 1 min to increase adhesion between the PMMA/MoS2 and the grid. PMMA was ultimately removed by acetone vapour. Before each TEM measurement, the grids were annealed at 180 °C in a vacuum chamber (∼7.5 × 10−5 Torr) for at least 12 h.PMMA  was spin-coated onto the as-grown MoS2 (90 kV)−2 in order to obtain the HRTEM images. The beam intensity at the sample in TEM mode is measured by the screen current multiplied by the square of magnifications. Because of contamination problems, it was difficult to obtain high-resolution STEM images during or after cracking. Annular dark-field STEM imaging was conducted using a probe aberration-corrected JEM ARM200F operated at 80 kV. Annular dark-field images were acquired at a 20 mrad convergence angle, with beam current 120–150 pA, beam size ∼1 Å, acquisition times of 40 μs per pixel and collection angle from 50 to 180 mrad. Because the energy of the low-voltage electron beam was below the damage threshold energy, the pristine MoS2 remains stable after regular scanning.TEM observations were conducted using a JEM ARM 200F operated at 80 kV that is lower than the knock-on damage threshold for monolayer MoS2 grown on sapphire was placed in a chamber equipped with a low-pressure Hg lamp , with the majority of emitted light at a wavelength of 254 nm and ∼10% of emitted light at a wavelength of 185 nm. Humidity was introduced into the chamber by connecting it to a humidifier. The humidity level in the chamber was monitored using a hydro-thermometer (accuracy of ±3%). The humidifier was disconnected from the chamber after reaching 65% humidity level. The MoS2 on sapphire was then irradiated under UV light for 2 min. Radicals are generated under UV and humidity conditions as follows: O2→O3; O3+H2O→O2+H2O2; 2O3+H2O2→2OH*+3O2; H2O→H*+OH*.MoS2/Si substrate was examined by field-emission SEM . An accelerating voltage of 15 kV was utilized to obtain a high contrast at different magnifications.The surface morphology of the TMDs on the SiOThe data that support the findings of this study are available from the corresponding author on reasonable request.How to cite this article: Ly, T. H. et al. Dynamical observations on the crack tip zone and stress corrosion of two-dimensional MoS2. Nat. Commun.8, 14116 doi: 10.1038/ncomms14116 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Supplementary Figures, Supplementary Tables, Supplementary Notes and Supplementary References


Evolution of C4 photosynthesis at low ca led to decreased stomatal conductance (gs), which provided an advantage over C3 plants that may be reduced by rising ca. Using controlled environments, we determined how increasing ca affects C4 water use relative to C3 plants. Leaf gas exchange and mass per area (LMA) were measured for four C3 and four C4 annual, crop-related grasses at glacial (200 µmol mol−1), ambient (400 µmol mol−1), and super-ambient (640 µmol mol−1) ca. C4 plants had lower gs, which resulted in a water use efficiency advantage at all ca and was broadly consistent with slower stomatal responses to shade, indicating less pressure on leaf water status. At glacial ca, net CO2 assimilation and LMA were lower for C3 than for C4 leaves, and C3 and C4 grasses decreased leaf hydraulic conductance (Kleaf) similarly, but only C4 leaves decreased osmotic potential at turgor loss. Greater carbon availability in C4 leaves at glacial ca generated a different hydraulic adjustment relative to C3 plants. At current and future ca, C4 grasses have advantages over C3 grasses due to lower gs, lower stomatal sensitivity, and higher absolute water use efficiency.Atmospheric CO They therefore proposed that in addition to biochemical advantages supporting higher A at low ci, protection of hydraulic function was an important advantage to C4 grasses at low ca.Photosynthesis in Cycle PEP . The ini greater . Importa greater . A centrlability . Recent r groups . Osborne species , might h3 and C4 species at glacial ca did not predict potential adjustments to co-ordination of leaf gas exchange and hydraulic function at low ca. Although evidence suggests that in non-woody species, decreased gs at elevated ca is associated with less negative leaf water potentials, lower hydraulic conductivity, and greater resistance to embolism, little is known about the influence of ca on co-ordination between photosynthetic capacity and hydraulic function (Kleaf) and photosynthetic capacity of woody C3 plants, optimizing leaf hydraulic function  by producing a better match between rapid photosynthetic responses and the slower stomata, which may improve overall water use efficiency, resulting in greater conservation of soil water and thereby decreased hydraulic stress  would decrease in plants with carbon limitation at low ca, and that decreases in iWUE and the extent of carbon limitation imposed by low ca would be greater for C3 than for C4 species (ca would be larger in C3 than in C4 grasses. Plants were grown in CO2 concentrations that represented: some of the lowest ca conditions (~200 µmol mol−1) that occurred in the glacial period during which C4 grass lineages diversified (ca (400 µmol mol−1); and super-ambient ca (640 µmol mol−1).Our goal was to determine whether growth  species . We ther2, and CO2 scrubber equipment. Three ca treatments were imposed: glacial (cGLA), 204 ± 27 μmol mol−1; ambient (cAMB), 408 ± 11 μmol mol−1; and super-ambient (cSUP) 640 ± 2 μmol mol−1 . The cAMB and cSUP treatments were rotated between cabinets 1 week prior to the first measurements, during the fourth week after sowing. The cGLA treatment was maintained in a single cabinet throughout the experiment because of the technical demands of obtaining a stable CO2 concentration at glacial ca. Growing conditions were set to a night-time temperature of 18 °C and a daytime temperature of 26 °C, resulting in a daily mean temperature of 22 °C . Temperatures and light levels were ramped daily in two even steps between 06.00 h and 08.00 h and between 18.00 h and 20.00 h (14 h light:10 h dark). Light was supplied by HID lamps, which provided photosynthetic photon flux density (PPFD) at the top of the canopy that varied within each cabinet between 300 μmol m−2 s−1 and 650 μmol m−2 s−1; daily quantum inputs were ~21 mol m−2 d−1 . Mean daily values for relative humidity ranged from 60% to 83% , providing VPDs of ~0.74 kPa under daytime conditions, and ~0.46 kPa during the night.Plants were grown in walk-in climate-controlled growth chambers  equipped with additive CO3 and four C4, used as food crops or close relatives of species used as food crops. In Poacaeae, all C4 grasses belong to a clade referred to as PACMAD (4 crop species have been domesticated from wild relatives in the Chloridoideae (teff and finger millet), and several from the Panicoideae subfamily , Setaria italica (foxtail millet), and Digitaria exilis (fonio millet) represent independent evolutionary origins of the NADP-malic enzyme (NADP-ME) subtype of C4 photosynthesis (Panicum miliaceum (proso millet) represents the NAD-ME C4 subtype (3 species were Panicum bisulcatum and Steinchisma laxa (two wild relatives from Panicoideae), Triticum turgidum , and Oryza sativa ssp. japonica  every 2–3 weeks during the course of the experiment.Plants were grown from seed in Osmocote Professional Seed Raising & Cutting Mix  in 0.55 litre plastic square tubes . Seeds were sown directly into six pots and germinated under the different CO2 mixers (LI-6400-01) and 2 × 3 cm red-blue LED light sources (LI-6400-02B). Pairs of LI-6400XT machines were randomly allocated to the three ca treatments and were rotated every 2 d: each pair of machines was used to measure two of the six blocks in every cabinet over the course of the experiment. Measurements were made under the growth conditions. To minimize disruption of ca treatments, the cuvette and integrated gas analysers of the LI-6400XT were placed inside the growth chambers and consoles outside the growth chambers (growth chambers were opened briefly before and after switching leaves). Measurements were conducted on the mid-section of individual, recently expanded leaves inserted parallel to the long axis of the 2 × 3 cm chamber, and leaf areas were calculated as cuvette length×average leaf width, measured to the nearest 0.5 mm with a ruler. Leaves were allowed to come to steady state [showing no systematic trends with a coefficient of variation (CV) <0.1 over a 5 min period] at a PPFD of 500 μmol m−2 s−1 (growth light levels) and cuvette CO2 concentrations matched to ca at the time of measurement: . Relative humidity was maintained at ~70% and block temperature at 26 °C, resulting in leaf VPDs of 1 ± 0.07 kPa . An auto-program (logging every 10 s) was used to record initial steady-state values for gas exchange , followed by the response of gsw to a step-change decrease in light availability from 500 μmol m−2 s−1 to 100 μmol m−2 s−1 PPFD. The rate of stomatal response to PPFD (Δgsw/Δt) was characterized based on the magnitude (Δgsw) and duration (Δt) of the initial decrease in gsw.We measured gas exchange using six LI-6400XT photosynthesis systems  equipped with CO3 and C4 species differed in the response of A to ca between cGLA and cAMB, we determined Kleaf in those treatments using the evaporative flux method (−2 s−1). Every second, output from the balance was logged to a computer and plotted to determine when transpiration  obtained a steady state for 5 min. At steady state, the temperature of the abaxial surface of the leaf (Tleaf) and air  were established using two type-K thermocouples and a Pico TC-08, then the leaf was immediately sheathed in plastic and cut at its base. After 15 min equilibration, a Scholander pressure bomb  was used to determine water potential, which estimated the leaf hydrostatic gradient (ΔΨ). Leaf area for these leaves (and an extra set collected from the cSUP treatment) was determined using a Canon LiDE 510 flatbed scanner and ImageJ software (Kleaf as E/(area×ΔΨ).Because C−2) were calculated using dry masses determined after a minimum of 48 h drying at 65 °C.LMAs (g mS) for entire leaves as (FM−DM)/(TM−DM), and leaf dry matter content (LDMC=DM/TM).Pressure–volume (P–V) relationships were determined using bench-drying. On the morning of measurement, attached flag leaves were sealed into plastic bags containing exhaled breath and were allowed to equilibrate for a minimum of 40 min to quench transpiration and ensure high turgor. Leaves sheathed in this manner were subsequently excised at the base of the lamina and moved to the laboratory. Initially, leaves remained sealed in plastic between measurements of fresh mass  and water potential . As water potential declined, leaves were occasionally removed from the plastic for short periods to increase the rate of drying. A minimum of 20 min equilibration was ensured between pressure bomb measurements. At the conclusion of FM and Ψ measurements, leaves were dried for a minimum of 48 h at 65 °C to determine dry mass . The turgid mass  was estimated by extrapolation of the initial linear FM–Ψ relationship  and used0, MPa) obtained below  and above  turgor loss, comparing all possible combinations that could be fit for each leaf within our data set. First, below-turgor loss fits for 1/Ψ=a(1−RWC)+π0,1  were obtained from all sequences representing at least three of the smallest RWC values and excluding two or more of the highest RWC values. Next, the x-intercept of the below-turgor loss relationship  was used to establish the RWC of the symplasm [RWCS=(RWC−af)/(1−af)]. Then the osmotic potential, π=1/ (MPa) and turgor pressure, ΨP=Ψ−π (MPa) were derived for the complementary above-turgor loss data. Finally, the bulk modulus of elasticity  and π0,2 were obtained from linear regression of ΨP= −ε(1–RWCS)−π0,2. Turgor loss point characteristics were calculated for the pair of linear relationships where  was smallest, and 0<af<1. Using π0,1 to estimate π0, the RWC at turgor loss (RWCTLP) was established by determining the RWCS at which ΨP=0, and was used to predict osmotic potential at turgor loss  from the equation for π.We optimized parameter selection for P–V relationships of individual leaves by minimizing the absolute difference between estimates of osmotic potential at full turgor (πhttps://www.R-project.org/). We fit linear models (lm) of species×ca responses, which we minimized using the Akaike information criterion (AIC). Where species effects were significant , we used linear contrasts to compare species means by photosynthetic type. Tests of photosynthetic type effects are approximate because the C3/C4 comparisons were not phylogenetically independent, and only a small number of species were included in our experiment. To adjust for heteroskedasticity, loge transformation was applied to values for gsw, iWUE, and Kleaf. Heteroskedasticity in Δgsw/Δt and P–V parameters could not be eliminated using transformation. For these parameters, we applied non-parametric Kruskal–Wallis tests, and present median values.Statistics were calculated using R Language and Environment  than at cAMB, while iWUE at cSUP was 4–56% higher (mean 30%) than at cAMB. Differences in iWUE were significant for comparisons between cGLA and cSUP in every species , and between cGLA and cAMB in every species except T. turgidum ; differences between cAMB and cSUP were not significant (P≥0.077).iWUE was more responsive to the difference between and cSUP . At cGLA4 species showed much larger absolute decreases in iWUE than C3 species . At every ca, iWUE was always higher in C4 species (range 51–232 μmol mol−1) compared with C3 species  than any of the four C4 grasses , A was not higher in C4 species compared with C3 species at cAMB and cSUP  compared with C3 grasses (36–49%); over the same range, relative increases in gsw were comparable for C3 and C4 species  was generally greater for C3 than C4 grasses , associated with significant photosynthetic-type effects on the average response between cGLA and cAMB , and between cGLA and cSUP . All C4 species increased Δgsw/Δt with decreasing ca, but only one C3 species (P. bisulcatum) showed a similar trend , S. bicolor , and P. miliaceum . Within their respective photosynthetic types, these three species showed the greatest values for iWUE and lowest values for gsw at cSUP, and the greatest decreases in iWUE from cSUP to cGLA . None of the C4 species exhibited significant changes in LMA in response to ca . In the C3 species, LMA was similar across the three ca treatments for S. laxa, but T. turgidum, P. bisulcatum, and O. sativa all showed significant reductions in LMA from either cSUP to cGLA  or from cAMB to cGLA , was therefore broadly associated with less sensitivity of LMA to ca among the C4 species. Conservation of LMA across CO2 treatments in most C4 species was linked with proportionate decreases in mass and area of the flag leaves as ca was reduced. Among C3 species, decreases in LMA arose because flag leaf mass decreased with ca from cSUP to cGLA, and flag leaf area decreased from cSUP to cAMB, but leaf areas were often similar at cGLA and cAMB ; however, on average, Kleaf was lower in plants grown at cGLA . The exception was D. exilis, a C4 species with small leaves, for which measurement errors were large . This difference was eliminated at cGLA because only C4 grasses decreased πTLP to more negative values (C4, −0.79 MPa to −1.27 MPa; C3, −0.91 MPa to −1.21 MPa; In the P–V analysis, the response of LDMC to 4 leaves . LDMC de species . Despitetic type . At cAMB3 and C4 grasses to atmospheric CO2 concentrations ranging from levels that occurred during the last 30 million years, when C4 lineages evolved and diversified, to those that could be experienced in the coming centuries. Across the range of ca, we expected that C4 species would maintain an iWUE advantage and show smaller physiological adjustments. Our results broadly support this expectation: the absolute response of gsw to ca was greater among C3 than among C4 grasses; and, as ca decreased from cAMB to cGLA, A, LDMC, and LMA declined more among C3 than among C4 species. Investigation of leaf hydraulic function at cAMB and cGLA showed that at cGLA, Kleaf decreased in both C3 and C4 species; and πTLP of C4 leaves became more negative, hence more similar to πTLP of C3 leaves, which did not adjust. Assaying the stomatal response to shade showed that higher steady-state gsw of C3 species was linked with more rapid adjustment of gsw to match A. Rates of stomatal closure were slightly more similar for C3 and C4 species at low ca, driven by strong responses of species that achieved high iWUE at elevated ca. These new findings are consistent with the hypothesis that carbon limitation is an important factor influencing leaf hydraulic function at different atmospheric [CO2]. Although there was substantial variation among species, photosynthetic type affected how leaf dry matter was deployed and how leaf turgor characteristics responded to cGLA.We exposed Cgs usually increased as ca decreased (gs of C3 grasses was associated with greater gs responses to ca; and low gsw of C4 leaves resulted in higher iWUE at all levels of ca. Importantly, A declined for C3 but not C4 grasses at cGLA. Greater A among some C3 species compared with C4 species at cAMB and cSUP suggested that C4 photosynthetic performance may have been limited by PPFD, so the iWUE advantage to C4 species may underestimate advantages to C4 species that could arise at higher irradiances (Steady-state gas exchange measurements provided the expected outcomes: 3 grass leaves generally closed their stomata more quickly than C4 leaves in response to shade. The higher steady-state gsw of C3 leaves may partially explain this difference between the photosynthetic types, but closer inspection of the data shows that Δgsw/Δt did not parallel the steady-state gsw for species within each photosynthetic type. Interestingly, among C4 species, the rate of gsw responses to light was slightly, but consistently, greater at cGLA compared with cAMB and cSUP. This decreased the difference in Δgsw/Δt between C3 and C4 species. However, a more striking trend, that probably underpinned the subtle difference in relative performance based on photosynthetic type, was that species with higher iWUEs showed greater changes in Δgsw/Δt in response to decreasing ca. At cSUP, species with high iWUE showed some of the slowest stomatal responses to shade. Because faster stomatal responses are consistent with improved water use efficiency (ca, supporting the overarching hypothesis that increasing ca minimizes the costs associated with hydraulic stress (ca-like scenarios that increase transpiration. For example, high iWUE is likely to be facilitated by high rates of internal diffusion, which are linked with decreases in cell wall dry matter (Cficiency , this su3 species in our study, only that with the highest iWUE, P. bisulcatum, showed a clear negative association between ca and the stomatal response to shade. At the other extreme, T. turgidum showed exceptionally high steady-state gsw and slow stomatal responses to shade in all three ca treatments, suggesting high transpiration irrespective of leaf water status, a strategy that can maximize CO2 uptake at a cost to hydraulic conductance (T. turgidum compared with other C3 species is important to note because iWUE for this species did not decrease at cGLA, contradicting the otherwise consistent trend towards greater decreases in iWUE among C3 compared with C4 species.Among-species variation was an important feature of our gas exchange results. This is consistent with previous studies, which have indicated that the degree to which grass stomata protect against decreases in hydraulic conductance varies even among genotypes a. Among ductance b. The apcGLA among C3 but not C4 grasses. This finding is consistent with observed differences in A, results from a meta-analysis addressing variation in LMA (ca treatments similar to those chosen for our experiment (4 and two of the wild C3 species included in our experiment were drawn from one subfamily of the Poaceae: Panicoideae, a broadly mesic-adapted clade (4 lineages are adapted to drier environments than those favoured by the Panicoideae (ca (cGLA was linked with greater carbon limitation in C3 grasses compared with their C4 relatives. This is important because differences in carbon supply affecting plant size and allocation at the whole-plant level have previously been highlighted as central to functional contrasts between C3 and C4 plants (LMA decreased at icoideae . In addi4 plants .3 and C4 grasses showed similar Kleaf, and Kleaf decreased at cGLA. The finding that there was no clear difference in Kleaf between photosynthetic types is consistent with a previous comparison using the high pressure flow meter technique, applied to predominantly perennial, North American prairie grasses (3 and C4 grass lineages are in the ratio of bundle sheath to mesophyll (ca and/or water availability (4 leaf anatomy or function counteract positive effects of increased bundle sheath ratios on Kleaf in C4 grasses. It is also important to note that C3 and C4 grasses often show similar average mesophyll cell sizes at ambient CO2 (CKleaf at cGLA was surprising, because xylem conductivity generally increases with declining ca to support increased gsw (in situ measurements of transpiration and leaf water potential in sunflower plants grown at ca similar to cGLA and cAMB showed the expected result: that Kleaf measured at ambient CO2 increased for plants grown at cGLA, minimizing the impact of increased gsw on ΔΨ (Kleaf for leaves grown at cGLA. However, this is not consistent with the decrease in LMA that we observed and, since transpiration was driven using moderate levels of light, we expect that the primary source of hydraulic resistance was exterior to the xylem (The evidence we found for decreased sw on ΔΨ , might cKleaf were low compared with other recently published estimates for similar species ; as in the study of Kleaf responses to ca that used sunflower (Kleaf at ambient [CO2].The values of ca on P–V characteristics also provide motivation for further investigation of photosynthetic type×ca responses. As leaf size decreased at cGLA, C4 grasses maintained LDMC and C3 grasses did not. In parallel, πTLP of C4 grasses became more negative at cGLA, while πTLP of C3 grasses did not change. This is an important result because πTLP is a powerful indicator of physiological responses that is expected to integrate smaller changes in, for example, π0 and ε (3 leaves grown at cGLA is consistent with both lower A and LMA, and previous evidence that C3 leaves decrease mesophyll cell volume and total non-structural carbohydrates as ca declines (TLP in C4 grasses therefore might be linked with solute accumulation at cGLA. Presumably, decreases in πTLP of C4 leaves at low ca would support maintenance of turgor in the presence of larger ΔΨ induced by higher gsw (TLP by C3 grasses grown at cGLA might be associated with maintenance of leaf water status if root and stem xylem hydraulic conductivity increased or xylem solute concentrations decreased.Effects of ca in C3 compared with C4 grass leaves, especially in terms of the positive relationship between iWUE and ca. We also predicted that low iWUE at cGLA would be linked with changes in leaf hydraulic properties. We found that while the iWUE advantage of some C4 grass leaves increased in absolute terms at cSUP, co-ordination among leaf traits was more strongly affected by cGLA than by cSUP. These experimental results broadly support predicted smaller impacts of cGLA on performance of C4 grasses (4 species will continue to be important in future. A finding with potential importance for crop improvement programmes is that as ca increases, pressure on plants to improve iWUE through rapid stomatal responses to shade may be reduced, particularly for species capable of achieving high iWUE. These results highlight the need for continued efforts to establish how hydraulics and photosynthetic performance are co-ordinated, both within leaves and at the scale of whole plants. The mechanistic basis of these responses still needs to be better understood to predict the physiological implications of C4 photosynthesis, both under past glacial climates and as they will affect performance in a future high CO2 world.We predicted that gas exchange would show greater absolute responses to  grasses , and sug


Ring sizing for mitral valve annuloplasty is conventionally done intraoperatively using specific ‘sizer’ instruments, which are placed onto the valve tissue. This approach is barely reproducible since different sizing strategies have been established among surgeons. The goal of this study is to virtually apply different sizing methods on the basis of pre-repair echocardiography to find out basic differences between sizing strategies.In three-dimensional echocardiographs of 43 patients, the mitral annulus and the contour of the anterior mitral leaflet were segmented using MITK Mitralyzer software. Similarly, three-dimensional virtual models of Carpentier-Edwards Physio II annuloplasty rings and their corresponding sizers were interactively generated from computer tomography images. For each patient, the matching annuloplasty ring was selected repeatedly according to popular sizing strategies, such as the height of anterior mitral leaflet, the intercommissural distance and the surface area of anterior mitral leaflet. The areas of the selected rings were considered as the neo-surface area of the mitral annulus after implantation.p ≤ 0.01). In contrary to intercommissural distance, height and surface area of the anterior mitral leaflet exhibited significant variations between the patients (p ≤ 0.01). The sizing according to the height of anterior mitral leaflet led to the maximal reduction of the mitral annulus surface area followed by the sizing according to the surface area of anterior mitral leaflet and finally by the intercommissural distance.The sizing of the mitral valve according to the height of anterior mitral leaflet (mean ring size = 29.9 ± 3.90), intercommissural distance (mean ring size = 37.5 ± 1.92) or surface area of anterior mitral leaflet (mean ring size = 32.7 ± 3.3) led to significantly different measurements  contains supplementary material, which is available to authorized users. Mitral valve repair is a common procedure in the field of cardiac surgery. Despite the huge experience in this field, there is no standard approach to repair the mitral valve. Most techniques were developed empirically including the implantation of annuloplasty rings in order to reshape, stabilize or downsize the valvular annulus such that a sufficient amount of leaflet tissue is involved in coaptation. As the usage of prosthetic implants of suboptimal size or repair without ring insertion has been shown to adversely affect surgical results , 2, chooTo date, one of the most commonly used prosthetic ring is the commercially available Carpentier-Edwards Physio II mitral ring (CE ring), which comes in 9 different sizes (size 28–40) . Over thThe advent of real-time three-dimensional transesophageal echocardiography (3D TEE) has allowed impressive volume renderings of the mitral valve anatomy and dynamics, especially the annulus, the leaflets and the papillary muscles, thereby significantly improving diagnostic capabilities and therapy planning. Additionally, the valve can be segmented from these images such that patient-specific virtual models are created. A specific software plugin called “Mitralyzer” of the freely available Medical Imaging Interaction Toolkit (MITK), which is able to process medical images , 23, wasOur goal was to study the effect of different sizing methods commonly used to implant a CE ring with regard to ring size selection, i.e. to find out if various strategies lead to the same results.Intraoperative real-time three-dimensional transesophageal echocardiographs of 43 patients, who underwent mitral valve surgery due to insufficiency, were extracted using Live-3D mode of the Philips ultrasound system iE33 xMatrix and a Philips X7-2 t matrix array transducer . The images were taken intraoperatively on the beating heart and before initiation of the cardiopulmonary bypass. Only patients with good quality images that involve the whole mitral annulus and the mitral leaflets with the subvalvular apparatus were involved in this study. The images were transferred to a workstation running Philips QLab Software and exported in DICOM Cartesian file format to enable further processing. Using MITK Mitralyzer software with mitral valve images at early systolic phase of the cardiac cycle allowed the interactive generation of mitral annulus models and the contour models of the AML. Carpentier-Edwards Physio II annuloplasty ring sizers and Carpentier-Edwards Physio II annuloplasty rings of size 26–38 were digitized using computer tomography . The images were exported to DICOM format and a three-dimensional model representing each planar sizer (sizer model) and its corresponding annuloplasty ring (ring model) were made using MITK Mitralyzer. By the help of the software, automatic quantifications could be derived from the models, such as surface area of AML model, height of the AML model, intercommissural distance (same for both models) for each patient see Fig. . From thFor each patient’s mitral annulus and AML model, we virtually selected the matching sizer model according to the following five different sizing methods: 1) corresponding height of AML (true sizing); 2) corresponding intercommissural distance (true sizing); 3) intercommissural distance with one size smaller (downsizing); 4) intercommissural distance with two sizes smaller (downsizing) and finally 5) corresponding AML surface area (true sizing). The anatomical references for the “true sizing” strategies are illustrated in Fig. Subsequently, the conformity between the results of the five strategies, i.e. the selected ring size, was compared to each other for each patient separately. The surface area of the chosen rings was considered as the surface area of the corresponding mitral annuli after annuloplasty.t-test. Results are presented as mean ± standard deviation in mm for measurements and without standard unit for ring sizes. A p-value less than 0.05 was considered significant.The collected data was analyzed using analysis of variance (ANOVA) and The mean age of the 43 patients at the time of the operation was 63.7 ± 11.8 years. About 77% of the patients were male. Thirty-four patients received mitral valve repair (81%) using Physio II annuloplasty ring. Eight patients (24%) of them needed an additional implantation of artificial polytetrafluoroethylene neochordae during mitral valve repair while sixteen patients (47%) received leaflet repair procedures and seven patients (21%) received both neochordae and leaflet repair. Nine patients underwent mitral valve replacement. More detailed characteristics of the patient cohort are summarized in Table p ≤ 0.01) according to the five applied strategies: height of AML (mean = 29.9 ± 3.90), intercommissural distance with true sizing (mean = 37.5 ± 1.92), intercommissural distance with one size smaller (mean = 35.5 ± 1.65) or intercommissural distance with two sizes smaller (mean = 33.6 ± 1.38) and AML surface area sizing (mean = 32.7 ± 3.29). Fig. Considering the whole patient collective, the sizing of the mitral valve led to significantly different average measurements (p ≤ 0.01). Intercommissural distance measurements in our patients cohort (mean = 38. 9 ± 6. 15 mm) shows no significant variation (p = 0. 31). The mean AML surface area was 711.13 ± 175.56 mm2 with significant variation between the patients (p ≤ 0. 01).Measurements of mitral valve apparatus landmarks are provided in Table 2 (16.4% reduction in AML surface area) while the reduction in AML when using AML surface area sizing was 19.43 ± 132.37 mm2 (1.2% reduction in AML surface area).The most sizing methods typically rely on measurements of the AML due to its tissue surface contribution to mitral valve closure, such that we did not consider the posterior mitral leaflet. Therefore, we compared the complete surface area of virtually implanted annuloplasty ring with the surface area of the AML. If the implanted ring was smaller than the AML area, we assumed that the implantation of such a ring will lead to a reduction of the visible AML surface area that participates in the mitral valve orifice during systole. The mean reduction in AML surface area when using height of AML for sizing was 116.7 ± 137.05 mm2, 125.46 ± 163.06 mm2 and 30.24 ± 163.93 mm2 bigger than the AML surface area respectively.Using the intercommissural distance with true sizing (strategy 2), intercommissural distance with one size smaller (strategy 3), or intercommissural distance with two sizes smaller (strategy 4) led to no reduction in AML surface area as the implanted rings were 241.1 ± 164.86 mm2. The reduction in annulus area when using height of AML for sizing was 818.665 ± 296.3 mm2 (strategy 1) while the reduction in annulus area when using AML area sizing (strategy 5), intercommissural distance with true sizing (strategy 2), intercommissural distance with one size smaller (strategy 3) and intercommissural distance with two sizes smaller (strategy 4) were 721.39 ± 249.45 mm2, 460.818 ± 311.75 mm2, 576.493 ± 315.29 mm2 and 671.715 ± 318.64 mm2 respectively.Mean mitral annulus area was 1413.09 ± 338.31 mmThe sizing results according to intercommissural distance (strategy 2) and AML surface area (strategy 5) led to the same sizing results in 14 patients 33%). For AML height (strategy 1) und AML surface area (strategy 5), ring sizing was equal in 10 patients (23%). In 4 patients (9%), the sizing results were equal according to AML height (strategy 1) and intercommissural distance (strategy 2). Note that in only 2 of the 43 patients (5%), the ring sizing in all methods was equal  has its closest relationships to the actual implanted ring size.Mitral valve reconstruction remains one of the most challenging fields in cardiac surgery with continuously changing concepts and new emerging ideas. The purpose of the current computer-based study was to find out if some of the currently used intraoperative methods for sizing the mitral valve annulus, as part of mitral valve repair using the CE physio II annuloplasty ring, result in similar ring size choices. The use of echocardiographic measurements to figure out the suitable annuloplasty ring is gaining more importance especially if three-dimensional measurements are performed . The mosIn our study, the use of the height of the AML (strategy 1) as reference to implant the annuloplasty ring was associated with the highest reduction in mitral valve orifice area and reduces the visible AML surface occupation area of the orifice; this theoretically implies the highest increase in coaptation area between AML and posterior mitral leaflet (PML). Such an increase in the coaptation area may result in a better competence of the mitral valve but may also lead to displacement of the AML causing systolic anterior movement (SAM) and left ventricular outflow tract obstruction with mitral insufficiency especially in the presence of a prominent PML. It may also cause iatrogenic mitral stenosis due to abundant valve tissue in the mitral valve orifice. Such postulations need further studies and finite element-based patient-specific annuloplasty simulations methods might be viable approach for this, such as shown by Schoch et al. . HoweverUsing the AML surface area (strategy 5) as a reference for sizing respects the AML and leads to less reduction in the mitral valve opening area which seems to be the best method to preserve the AML function but at the same time compromises the size of coaptation area , which cOur study showed that sizing according to intercommissural distance results in the implantation of bigger annuloplasty rings see Fig. , even ifTo further complicate sizing, it has been already shown by Bothe et al. , who terNote that the valve geometry under physiological loading conditions considerably differs from the cardioplegic valve morphology. Intraoperatively, when sizing is usually conducted, the valve is flaccid and collapsed. The actual difference in size and shape between these two states could be virtually visualized and quantified by our group for the first time , using nWithin the scope of this paper, we showed the potential effect of different isolated sizing strategies on the mitral valve geometry. Using the height of the AML to find the optimal size of the annuloplasty ring led to the maximal reduction in mitral valve orifice area and hypothetically to the maximal increase in the coaptation area between AML and PML when compared to the other sizing methods. In contrast, the use of the AML surface area resulted in a smaller reduction in the mitral valve orifice area and a smaller increase in the coaptation area between the leaflets in theory. Sizing the mitral valve annuloplasty ring according to the intercommissural distance led to the smallest reduction in mitral valve orifice. To sum up, our results suggest that the concordance between the strategies is very limited and that they should be rather used in a combined way to make an adequate decision for the patient.


Klebsiella pneumoniae liver abscess syndrome (KLAS) is characterized by bacteraemia, liver abscesses and metastatic infection caused by a hypervirulent strain of Klebsiella pneumoniae, usually belonging to the capsular serotype K1 or K2. Initially, KLAS was described in Eastern Asia; recently isolated cases have been reported from different parts of the world.Klebsiella pneumoniae was possible by culture from one of the cutaneous emboli and subsequently confirmed by blood cultures. The patient fully recovered after abscess drainage and prolonged antibiotic treatment.We describe the case of a woman with KLAS including meningeal, ocular and cutaneous metastatic infection and organ dysfunctions . The identification of a hypermucoviscous strain of We have pointed out about the importance of sampling each septic focus in order to identify the aetiology of a disseminate infection. Klebsiella pneumoniae liver abscess syndrome (KLAS) is a clinical syndrome characterized by bacteraemia, liver abscesses and metastatic infection caused by a hypervirulent strain of Klebsiella pneumoniae, usually belonging to the capsular serotype K1 or K2 [Klebsiella pneumoniae liver abscess described in Romania was in 2014 at the National Institute of Infectious Diseases ‘Professor Dr Matei Bals’ Bucharest [K1 or K2 . ExtraheK1 or K2 . PreviouK1 or K2 . The firucharest .A 53-year-old woman was admitted to the infectious diseases department presenting with a 24 h history of high fever, chills, vomiting, headache, confusion and altered general condition. The patient had a cholecystectomy several years ago and in the last month was evaluated for a possible diabetes mellitus. The patient had no history of travelling in foreign countries in the past five years. At admission the clinical examination revealed a partially conscious, delirious patient with nuchal rigidity, oliguria and haematuria. The skin was pale, with several bilateral ecchymoses on the legs and arms and papular exanthema on the calf . The oph−3) with neutrophilia (18 700 cells mm−3) severe thrombocytopenia (7000 cells mm−3), high d-dimers (15 505 ng ml−1), inflammation , creatinine (406.7 µmol l−1), urea (244.5 mg dl−1), hyponatraemia (131 mmol Na l−1), hypokalaemia (3.3 mmol K l−1), cytolysis: aspartate aminotransferase 1.1× upper limit of normal (ULN), alanine aminotransferase 1.5× ULN, increased gamma-glutamyl transferase level (3× ULN), hypoalbuminaemia (2.5 g dl−1) and hyperglycaemia 14.2 (mmol l−1).Laboratory test showed leukocytosis  examination was performed, showing diffuse oedema and a minor lenticular lesion, possible an infarction. The echocardiography did not retrieve endocardial lesions. Abdominal echography revealed a liver mass and MRI was performed that showed an abscess with dimensions of 9.0×6.0 cm in the VIIth liver segment. Considering the probable involvement of a Gram-negative rod, the antibiotic treatment was changed to meropenem (2 g every 8 h).Klebsiella pneumoniae through MALDI–TOF MS. The isolated strain had a positive string test, indicating a possible hypermucoviscosity phenotype. The antimicrobial susceptibility test yielded an isolate susceptible to cephalosporins, aminoglycosides and fluoroquinolones and we changed the antibiotic therapy to ceftriaxone (2 g every 12 h). On the third day of hospitalisation the blood cultures were also positive for Klebsiella pneumoniae with similar antimicrobial susceptibility.On the second day of hospitalization the cultures performed from the skin lesions yielded Gram-negative lactose-fermenting rods, subsequently identified as −3 and a lumbar puncture was performed, sampling a xanthochromic cerebrospinal fluid (CSF), with pleiocytosis, 15 leukocytes mm−3, elevated lactic acid (5.4 mmol l−1) and elevated proteins (2.54 g l−1). After 15 days of ceftriaxone (4 g per day) the patient underwent percutaneous drainage of the hepatic abscess. Combined therapy, both antibiotic and surgery, leaded to important improvement of the patient. The antibiotherapy was switched to oral amoxicillin–clavulanate (1 g three times a day) and the patient was discharged with close follow up until total remission of the hepatic abscess was confirmed through abdominal echography. Three months later she had good glycaemic control with oral medication and almost normal visual acuity.On day 7, platelets count were 84 000 mmKlebsiella pneumoniae has become the most common pathogen causing pyogenic liver abscesses in Singapore and Taiwan [Klebsiella pneumoniae isolates from KLAS patients belong to the capsular serotype K1 or K2, two hypervirulent types [The literature records that in the last 20 years d Taiwan . The Klent types Recently, KLAS cases have been reported in Europe and Americas in patients without a history of travelling in Eastern Asia; hence, patients outside the initial reporting area are not protected . In RomaEscherichia coli and Klebsiella pneumoniae are the two most frequently isolated pathogens [Usually, in the liver abscesses aetiology, polymicrobial involvement is common, but athogens . In our athogens .Klebsiella pneumoniae serotypes retained sensitivity to antibiotics [KLAS is an aggressive, complex pathology with a great possibility of generating a wide range of complications and sequelae. It is however important to note that the hypervirulent ibiotics . The strConsidering the high risk of pairing the hypervirulence with antimicrobial resistance of pathogens, it is very important to maintain as long as possible the ‘wild-type’ antimicrobial susceptibility for these K1 and K2 strains. Recently, a few case reports have described K1 isolates with multi-drug resistance to antibiotics . The ant