Saccharomyces cerevisiae). Our theoretical results indicate that the mechanical feedback provided by the Cell Wall Integrity pathway, with cell wall stress sensors Wsc1 and Mid2 increasingly activating membrane-localized cell wall synthases Fks1/2 upon faster cell wall expansion, stabilizes mating projection growth without affecting cell shape. Experimental perturbation of the osmotic pressure and cell wall mechanics, as well as compromising the mechanical feedback through genetic deletion of the stress sensors, leads to cellular phenotypes that support the theoretical predictions. Our results indicate that while the existence of mechanical feedback is essential to stabilize mating projection growth, the shape and size of the cell are insensitive to the feedback.The shaping of individual cells requires a tight coordination of cell mechanics and growth. However, it is unclear how information about the mechanical state of the wall is relayed to the molecular processes building it, thereby enabling the coordination of cell wall expansion and assembly during morphogenesis. Combining theoretical and experimental approaches, we show that a mechanical feedback coordinating cell wall assembly and expansion is essential to sustain mating projection growth in budding yeast ( All morphogenesis processes, whether at the cell scale or tissue level, require the coordination of growth and mechanics to properly shape functional structures. However, the mechanisms that coordinate these two processes in the sculpting of individual cells, and especially in walled cells, remain unknown. Using yeast mating projection growth as a model system, we show that a genetically-encoded mechanical feedback relays information about the mechanical state of the cell wall to the intracellular processes assembling it, thereby coordinating cell wall expansion and growth during cell morphogenesis. We find that mechanical feedback is essential to stabilize cell growth, but the shape and size of the cell are insensitive to the feedback and independently controlled. From cell division to polarization and growth, cells constantly change their shapes to perform specific tasks –3. TheseCell shape changes are ultimately governed by the mechanical state of the cell wall –7. Studiα- and a-factors for mating types a and α, respectively) that elicits the growth a tubular mating projection from the partner of the opposite type [In addition to well-known model systems for tip-growth, such as pollen tubes in plants and hyphal growth in higher fungi –9, buddiite type , 21  pathway is known to help the cell prevent loss of cell wall mechanical integrity in a variety of situations –30, fromUsing mating projection growth in budding yeast as a model system, and combining experiments and theory, we show that coordination between cell wall mechanics and assembly through direct Fks1/2 activation in the CWI pathway  stabilizes mating projection growth without affecting its geometry. In what follows, the term ‘mechanical feedback’ refers to the nature of the input signal that is sensed and relayed by stress sensors in the CWI pathway. We first derive a theoretical description that connects the cell wall mechanics to the intracellular processes building the wall (Fks1/2 activation dynamics) via the CWI pathway, and show that stable projection growth can only persist in the presence of mechanical feedback. In the absence of coordination between cell wall assembly and mechanics, cell wall expansion is always unstable, leading to either progressive thickening or thinning of the cell wall depending on conditions. Our experimental results indicate the compromising the mechanical feedback through genetic deletions of the wall stress sensors Mid2 and Wsc1, and also through perturbations of cell wall mechanics and increased turgor pressure, all lead to defects in mating projection growth and cell viability. Our experimental observations are in agreement with the theoretical predictions, suggesting that the mechanical feedback provided by the CWI pathway via direct activation of Fks1/2 synthases can stabilize projection growth without altering cell geometry. In addition, by directly measuring the size of the exocytosis region in wild-type (WT) and mutants with compromised mechanical feedback, we show that the size of the mating projection is controlled by the size of the exocytosis region, but is independent of the strength of the mechanical feedback, as predicted theoretically. Altogether, our results show that a mechanical feedback between cell wall mechanics and assembly is essential for stability of cell wall expansion and projection growth, but that its geometry and size are insensitive to the mechanical feedback.r, h, ρA, and ρI were described by equations evolving in time, and u, θ, κs by differential equations in s. The latter equations were solved by the method of lines; s was discretized and the s-derivatives were written as a differential matrix using fourth order central difference and one sided finite differences at the boundary. The resulting system becomes a differential algebraic system (DAE), which was solved using Sundials, a suite of nonlinear and DAE solvers. Steady state solutions were obtained by ensuring that all time derivatives of scaled variables were below 10−3.The system of Eqs bar1Δ mutation that prevents α-factor degradation by deletion of the Bar1 protease. Genetic techniques were performed per standard methods [wsc1Δmid2Δ strain was grown in YPD media with 1M sorbitol to increase viability. Gene deletions and GFP-tagging were constructed by genomic integration using vectors amplified and targeted by PCR primers [All yeast strains were derivatives of W303-1A and contained the  methods . Yeast s primers .α-factor (1 μM) for 2 hours. To observe the viability of cells after altering the osmotic pressure, we diluted the YPD media with distilled water upon addition of propidium iodide. The cells were imaged on slides after being exposed to propidium iodide for 10 minutes. Brightfield and fluorescent (RFP filter set) images were acquired using an inverted Nikon Eclipse TE300 microscope with a 60x objective (NA = 1.4). Image analysis was manually performed using ImageJ. Data from 3 samples for each condition was averaged and, for each sample, 150 cells or more were analyzed.Cell lysis was determined by propidium iodide (Molecular Probes) staining. Propidium iodide (PI) was prepared in DMSO at a concentration of 20 mM and then diluted 1:1000 for use. Propidium iodide was added to cells after being exposed to β-1,3 glucanase, to hydrolyze the glucan linkages that strengthen the wall. Zymolyase  per 100 μl of cells) was added to cells exposed to alpha-factor for 1.5 hours. Cells were treated additionally with concanavalin A to immobilize them during the imaging process. The cells were imaged on slides for 7 minutes after being exposed to zymolyase for 3 minutes. DIC images were acquired every 3 seconds. Data from 5 samples for each condition was averaged and, for each sample, 15 cells or more were analyzed. Image analysis was manually performed using ImageJ.To decrease the viscosity of the cell wall, we utilized zymolyase, which contains μM α-factor for 1 hour and 30 minutes. For spa2Δ cells, the 30 images were taken at 13 second intervals to average over a longer time period to average out the stronger fluctuations in polarization in this mutant. Images were acquired with a laser-scanning confocal microscope (Zeiss LSM 710), using a 100x objective (NA = 1.4). The cells were immobilized to a glass-bottom dish coated with concanavalin A. To horizontally orient the mating projections, we layered a YPD (supplemented with 1 μM α-factor) agarose pad on top of the cells. Image analysis was manually performed using ImageJ.The length-scale of exocytosis was measured in strains that contained Sec3 fused to GFP. Calcofluor White Stain (Sigma-Aldrich) was added to cells 10 minutes prior to imaging  to distinguish the cell wall during image analysis. To properly visualize the length-scale and reduce imaging noise, we averaged 30 consecutive confocal images, taken at 2 second time intervals, for each cell, after incubation in 1 P. Such high pressure is mechanically sustained by the cell wall, which provides mechanical integrity to the cell at all stages, including during mating projection growth. Similarly to other organisms [μm [s from the projection apex and azimuthal angle ϕ , and the principal curvatures κs = ∂θ/∂s and κϕ = sinθ/r, respectively, where θ is the angle between the local outward normal and the axis of growth   is muchisms [μm ). Since  angle ϕ . The shaf growth . The coor, ϕ, z)  are stanectively , 46  and σϕϕ are the tensions along s and ϕ in the wall (μ(s), minimal at the apex and increasing away from it  of a cell wall with constant density ρw, or its strain (expansion) rates Building on previous work combining cell wall mechanics and growth in tip-growing cells , as wellthe wall . The exp from it . The loc wall by , 46σss=β glucan [β glucan synthases Fks1/2, which localize at the apical, growing region of the mating projection [β glucan chains into the extracellular space, thereby assembling new cell wall onto the preexisting wall [h is the cell wall thickness Fig 1C). ng (∼ 1s ) and ver01 μm2/s ), the di from it . These locytosis , we assukon and koff, respectively. Inactivation of active, membrane-localized Fks1/2 synthases has been largely unexplored and assumed here to occur at a constant rate. The activation of inactive Fks1/2 is mediated by the GTPase Rho1 through the CWI pathway [kon /(12μ0mwρ0kp), which corresponds to the ratio λX/λm of the exocytosis length scale λX and a length scale λm ≡ 12μ0mwρ0kp/Pρw set by the expansion mechanics of the cell wall. The parameters D/λX have either been measured or estimated and we use below their known values [PρwλX)/(12μ0mwρ0kp) are unknown and we explore below the dynamical regimes of the system when these parameters are varied.Combining Eqs n values , 57  and only active Fks1/2 see . This inPρwλX)/(12μ0mwρ0kp) there exists a critical value of the feedback strength Γ below which mating projection growth is unstable. Similarly, for every value of the feedback strength Γ, there is a maximal value of (PρwλX)/(12μ0mwρ0kp) above which mating projection growth becomes unstable. This instability is caused by the progressive thinning of the apical cell wall, eventually causing the piercing of the cell and leading to cell lysis. The bifurcation between stable and unstable states characterizes the transition between stably growing mating projections and a situation in which this stable growth cannot be sustained because of the progressive thinning of the cell wall and its eventual piercing. This instability threshold (bifurcation) is equivalent to the existence of a maximal turgor pressure , above (below) which the cell wall progressively thins and eventually pierces at the tip of the projection, leading to cell lysis. The predicted increase of the maximal turgor pressure or decrease in the minimal wall viscosity for increasing feedback strength Γ indicates that cells with compromised mechanical feedback should be more sensitive to both an increase in turgor pressure or a decrease in wall viscosity than WT cells.In the presence of mechanical feedback (Γ > 0), numerical integration of Eqs P and cell wall viscosity μ0. In contrast to previous works, here we examine all three perturbations in the context of the stability of pheromone-induced projection growth. We first varied the feedback strength Γ by compromising the ability of the cell to sense the mechanical state of the wall. To this end, we genetically deleted the two primary cell wall stress sensors present during mating projection growth, namely Wsc1 and Mid2 [α-factor and mating projection growth, did the deletion of either of the two sensors  lead to increased levels of cell lysis compared to WT /(12μ0mwρ0kp) affected cell viability /(12μ0mwρ0kp) P and celed to WT , as preded to WT , indicatα-factor . These oiability , we indel medium . We obseα-factor , consistactor  , we adde0mwρ0kp) . Zymolyaojection . Since zntration , as theontration . Overallntration  and are PρwλX)/(12μ0mwρ0kp)  and decreases away from it (PρwλX)/(12μ0mwρ0kp) equivalently) and with decreasing mechanical feedback strength  . The sizrength Γ . Beyond  from it , eventuastrength . In contstrength , decreasall Figs , suggestρA + ρI, and the surface density of only active Fks1/2 synthases, ρA, are maximal at the apex and decrease away from it until they vanish , is also maximal at the apex and decreases away from it . Our theoretical predictions are in good agreement with the experimental results and indicate that the existence of mechanical feedback is essential to guarantee stability during cell wall remodeling and cell morphogenesis.This theoretical description of mating projection growth connects the mechanics of the cell wall to the molecular events in charge of sensing its mechanical state and controlling its assembly via well-established signaling pathways (CWI pathway), thus providing specific predictions on how mutations can affect cell morphogenesis. Various previous models accounted for both the mechanics and assembly (remodeling) of the cell wall –19, as wWe theoretically find that in the absence of any mechanical feedback relaying information about the mechanical state of the cell wall to the intracellular processes building it, cell wall expansion is unstable, leading to cell lysis. Previous works have shown that the cell wall is prone to piercing in cell if the CWI is compromised , and ourBeyond budding yeast, many other organisms, including other fungi, plants and bacteria, have walled cells that are constantly remodeled , 61, 62.While essential to ensuring stability during cell wall expansion, our results show that the strength of mechanical feedback does not affect mating projection shape or size Figs  and 5. TMore generally, the need to coordinate growth processes and mechanics during morphogenesis is important for individual cells, but also for tissues and organs. Identifying the molecular mechanisms enabling this coordination at different scales and in different organisms will substantially contribute to our understanding of morphogenetic processes.S1 File(PDF)Click here for additional data file.S1 VideoΔ cell growing a mating projection is shown after the addition of zymolyase. The cell pierces at the tip as theoretically predicted. From left to right, DIC image, PI staining, and merge.A mid2(AVI)Click here for additional data file.


The effect of such isoforms on pulmonary vascular permeability is unknown. We hypothesised that VEGF165a and VEGF165b on Human Pulmonary Microvascular Endothelial Cell (HPMEC) permeability using three different methods: trans-endothelial electrical resistance (TEER), Electric cell-substrate impedance sensing (ECIS) and FITC-BSA passage. In addition, potential downstream signalling pathways of the VEGF isoforms were investigated by Western blotting and the use of specific signalling inhibitors.To test this hypothesis we investigated the physiological effect of VEGF165a increased HPMEC permeability using all three methods (paracellular and transcellular) and led to associated VE-cadherin and actin stress fibre changes. In contrast, VEGF165b decreased paracellular permeability and did not induce changes in VE-cadherin cell distribution. Furthermore, VEGF165a and VEGF165b had differing effects on both the phosphorylation of VEGF receptors and downstream signalling proteins pMEK, p42/44MAPK, p38 MAPK, pAKT and peNOS. Interestingly specific inhibition of the pMEK, p38 MAPK, PI3 kinase and eNOS pathways blocked the effects of both VEGF165a and VEGF165b on paracellular permeability and the effect of VEGF165a on proliferation/migration, suggesting that this difference in cellular response is mediated by an as yet unidentified signalling pathway(s).VEGF165a and VEGF165b induce differing effects on permeability in pulmonary microvascular endothelial cells.This study demonstrates that the novel isoform VEGF We anntribute  but do nn CDKPRR . The recisoforms . Two spemembrane . These d165a and VEGF165b activate different signalling pathways mediating cell permeability, a potential explanation for the conflicting observations on effects in the vascular space. To explore this theory, we used three methods of assessing vascular barrier function and found contrasting effects with VEGF165a increasing permeability and VEGF165b decreasing permeability. We then explored the relationship of downstream pathways to these functional differences. We compared the effects of specific signalling pathway inhibitors of MEK/p38MAPK/PI3K and eNOS on permeability, cell migration and proliferation to identify a mechanism by which increased permeability could be resolved whilst maintaining beneficial cell proliferation and migration.We hypothesised that VEGFA detailed description of materials and methods is given in the online data supplement.Human Pulmonary microvascular endothelial cell (HPMEC) cryopreserved from passage 2  were cultured in endothelial cell basal medium MV2  complemented with supplement pack  according to manufacturer’s instructions.165a and VEGF165b  [For all experiments cells were grown to 80% confluence, quiesced (MV2 media only) and stimulated with combinations of VEGF plasma) , 6 in th2  as previously described by Bevan and al [Measurement of trans-endothelial electrical resistance (TEER) of the cell monolayer was performed using an Endohm 12 electrode chamber and an endothelial volt/ohm meter EVOMn and al .Cells were plated at 20000 cells/cm2 into 8-well arrays . Data was automatically and continuously collected every 2 min and recorded by computer. Experiments were performed after cells reached confluence with basal TEER values > 1500 Hz.Transendothelial permeability to macromolecules was assessed by the passage of FITC-conjugated BSA  across cell monolayers in tissue culture inserts as previously described .5 cells/ml) in an Ibidi culture-chamber . Cells were pre-incubated with or without inhibitor for 1 hour before removal of the chamber. Cells were then incubated in MV2 medium alone or MV2 medium with 20 ng/ml of recombinant protein VEGF165a or VEGF165b. Images were captured and analysed at 0 and 24 h.Cells were seeded with 100 μl of cell suspension (5 × 10Cell lysates were separated on sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotted. Blots were blocked with 5% bovine serum albumin (BSA)  and incubated overnight at 4 °C with primary antibodies165a, VEGF165b, VEGF165a + b or without any stimulation (control) for 10 min. They were then fixed, permeabilised and immunostained for VE-cadherin  and Alexa Fluor® 568 Phalloidin  for staining actin structures.HPMEC were stimulated with 100 ng/ml of VEGF165a significantly reduced the cell resistance (increased permeability) **p < 0.01, from 45 min onwards, in contrast, VEGF165b significantly increased resistance (decreased permeability) **p < 0.01 between 15 to 45 min compared with unstimulated control cells  in contrast to VEGF165b which induced a significant decrease (*p < 0.05) in the cell monolayer permeability  time-dependant increase in BSA permeability was detected after 45 min and persisted for at least 90 min, suggesting an increase in the cell monolayer permeability.The last experimental technique used to assess permeability was FITC-coupled BSA passage. The permeability of the HPMEC monolayer for FITC-BSA was monitored every 15 min up to 90 min by measuring the fluorescence intensity of the medium in the lower compartment. For unstimulated control cells, the level of BSA in the lower compartment increased slowly over time in a comparable manner to those treated with VEGFMEC Fig. . When th165a treated human umbilical vascular endothelial cells (HUVEC) was demonstrated using 100 ng/ml stimulation [165a  in HPMEC  with VEGF165a and subsequently decreased as represented in Fig. 165b also induced significant phosphorylation of tyr1214 site at 5 min (p < 0.05) in HPMEC, in contrast to tyr1175. These differences in receptor phosphorylation support the potential for differential binding of other adaptor proteins in addition to the previously reported changes in co-receptor binding that has been shown to contribute to VEGF-induced vascular permeability.VEGF-R2 phosphorylation at the tyrosine (tyr) 1175 site, was significantly induced by VEGFMEC Fig.  and retuHaving identified these phosphorylation differences we wanted to identify specific pathways leading to cell permeability which is less well characterised than those of other VEGF functional effects . FurtherThe MEK and mitogen activated protein kinase (MAPK) pathways are among the most widely studied in VEGF biology and considered to have critical roles in cell proliferation and cell growth and differentiation , 28. We 165a for 5 and 10 min that subsequently returned towards baseline at 60 min  for the cells stimulated with VEGF165a compared to control. The maximum response was observed at 10 min for both proteins. However, VEGF165b induced a significant increase of pMEK1/2 and p42/44MAPK phosphorylation at 10 min only. At 60 min the level of both phospho-proteins in the stimulated cells returns toward the control levels.HPMEC showed a similar activation pattern of pMEK1/2 and p42/44MAPK proteins with an increase in the phosphorylation when treated with VEGF165a and VEGF165b on HPMEC  in comparison to control . Cells stimulated with VEGF165b showed a significant increase in p38MAPK phosphorylation (p = 0.04) compared to the untreated cells  only with VEGF165a stimulation and this effect was inhibited by SB203580 , we then looked at the effect of VEGF isoforms on AKT also known as protein kinase B (PKB) protein. AKT has been shown to promote the proposed cell survival pathway mediated by the activation of PI3-kinase protein kinase [Having identified significant differences in the activation kinetics of MEK, p42/44MAPK and p38MAPK by VEGFn kinase  but it hn kinase .165b induces a rapid and robust phosphorylation of pAKT at 5 min and 10 min (p < 0.01) in HPMEC with maximal AKT phosphorylation detected at 5 min  and 10 min (p < 0.01) and returned to control levels at 60 min. LY294002 compounds is an inhibitor of phosphatidylinositol 3 kinase (PI3K) and inactivation of PI3K have been reported to lead to dephosphorylation of Akt that subsequently stop G1 cycle in cell growth and ultimately lead to cell apoptosis [165a and VEGF165b on HPMEC  may have a crucial role in causing hyperpermeability in response to pro-inflammatory agents such as VEGF , 37. The165a induced a significant increase in the phosphorylation of eNOS at 5 (p < 0.05) and 10 min (p < 0.01) compared to untreated control cells  is an analogue of L-arginine required for the nitric oxide synthesis by the vascular endothelium [165a or VEGF165b did not have any significant change in resistance compared to control  barrier function integrity, which is so essential for maintaining this critical function of the lung.Vascular endothelial growth factor (VEGF) was originally described as both an angiogenic and a permeability factor  and its Among all the pathological processes involved in. ARDS increases in lung vessel permeability are critical and non-redundant . The meaTwo types of inter-endothelial junction are present in the endothelium, adherens and tight junctions, the former being dominant in most vascular beds. The integrity of the adherens junction is particularly critical for regulating paracellular permeability via homophilic adhesions between VE-cadherin molecules , 20. Dis165a and VEGF165b induce differing effects on the permeability of pulmonary microvascular endothelial cells. Specifically, VEGF165a induced an increase and VEGF165b a decrease in permeability. The receptor binding and dimerisation domains are intact in the VEGFxxxb family of VEGF isoforms. However, in porcine aortic endothelial cells, VEGF165b has been shown to stimulate a unique pattern of VEGF-R2 tyrosine residue phosphorylation, contrasting with those activated by conventional isoforms suggesting activation of differing downstream signalling pathways in addition to partial agonist activity and changes in neuroplin-1 binding [165a and VEGF165b using what we considered to be physiologically relevant concentrations of VEGF.We have demonstrated for the first time that VEGF binding , 47, 48.165a and VEGF165b on the vascular permeability in addition to those we have previously shown and published on proliferation in HPMEC  and human alveolar epithelial cells offer a potential paradigm to explain the apparent compartmentalisation of VEGF between the alveolar and vascular space and the apparent disparity of data relating to the role of VEGF in ARDS [The differential effects of VEGF in ARDS .165a and VEGF165b lead to differential functional outcomes with VEGF165a increasing cell permeability in methods suggested to reflect both para and transcellular permeability and VEGF165b reducing paracellular permeability only. This suggested that the differences were due to divergence of signalling pathways and therefore potential targets for amelioration of outcome e.g. reducing permeability whilst preserving a pneumotropic effect. To verify this hypothesis, different protein inhibitors have been used to look at their effect on the change in resistance reflecting the paracellular permeability pathway of pulmonary microvascular ECs.We identified that VEGF165a signalling pathways have been studied extensively in HUVEC although studies in HPMEC are limited [The VEGF limited . We chos165a and VEGF165b. Being part of the same signalling pathway these results suggest that VEGF cell paracellular permeability involves the phosphoinositide 3-kinase–AKT pathway, which then further phosphorylates and activates endothelial nitric oxide synthase (eNOS) [165a and VEGF165b. Further studies of other pathways are required in order to unravel the molecules responsible for the differential permeability effects of VEGF isoforms such as Src kinase pathway and its role in the regulation of endothelial-barrier integrity as demonstrated recently by Gao et al. [The use of L-NAME (eNOS inhibitor) and LY294002 (PI3K inhibitor) on HPMEC inhibited the effect of both VEGFe (eNOS) . Also, to et al. .165a significantly increased endothelial cell monolayer permeability which would lead to failure of the ACM barrier function, of particular significance in the lung environment. On the other hand, depending on the isoforms present, VEGF may also be a protective agent, the data presented showing a decrease in permeability with VEGF165b exposure. Further work will need to be undertaken to clearly identify the divergence in the permeability signalling pathway induced by both isoforms and the potential protective proprieties of VEGF165b.HPMEC have specific signalling characteristics that are probably adaptations to the unique pulmonary environment. In the context of ARDS, VEGF  may be a triggering component leading to further endothelial dysfunction and failure of the ACM. The data presented has shown that exposure of HPMEC to exogenous VEGF


Four time periods (T) were defined: T1 birth to arrival at resuscitation table, T2 arrival to application of NIRS sensor, T3 application to first displayed cTOI or crSO2 value, and T4 from arrival at resuscitation table to first displayed values. Additionally, we compared first displayed values of cTOI and crSO2. Thirty neonates were included. Twenty-four were term and six late-preterm neonates. Fifteen neonates measured with NIRO were compared to 15 measured with INVOS. T1 was 49 (6–163) s with NIRO versus 59 (15–87) s with INVOS, T2 14 (4–20) s versus 12 (15–18) s, T3 33 (13–138) s versus 17 (6–290) s and T4 46 (20–153) s and 34 (14–300) s. The first displayed value tended to be higher for cTOI [54% (18–80)] compared to crSO2 [35% (15–87)]. There were no significant differences between devices in time periods and first values displayed. Cerebral tissue oxygenation can be measured within 1 min after arriving at the resuscitation table in term and preterm neonates after birth without difference between devices.Feasibility of cerebral tissue oxygenation measurements immediately after birth has been published starting with first values 2 min after birth. Aim of this study was to evaluate, the time periods from birth and from arrival at the resuscitation table to obtain the first cerebral tissue oxygenation values with two different near infrared spectroscopy (NIRS) devices. The present study is an analysis of exploratory parameters of two prospective observational studies. Cerebral tissue oxygen saturation was measured by the NIRO 200NX measuring “cerebral-tissue-oxygenation-index” (cTOI) or the INVOS5100C measuring “cerebral-regional-oxygen-saturation” (crSO Unfortunately, these methods do not provide information about potentially compromised oxygen delivery to the brain. Therefore, there is a growing interest, to monitor the cerebral regional tissue oxygenation by using near-infrared spectroscopy (NIRS) during immediate postnatal transition [2). With both NIRS devices centiles of cerebral tissue oxygenation immediately after birth starting with first values 2 min after birth have been already published [2 values with pulse oximetry immediately after birth [2 values in less than 1 min [2 values after the arrival of a newborn infant at the resuscitation table. Therefore, the aim of this study was to evaluate, the time periods from birth and from arrival at the resuscitation table to obtain the first cerebral tissue oxygenation values with two different devices. We hypothesized that there is no difference between these two devices.During immediate transition after birth beside the clinical assessment of newly born infants, routine monitoring with pulse oximetry/ECG is recommended. These two non-invasive continuous monitoring methods enable measurement of the arterial oxygen saturation . Parental informed consent was obtained from all individual participants included in the study. Videos of term and preterm infants during immediate transition after birth were analysed, which were recorded during these prospective studies. The video started when the whole body was delivered. Immediately after birth, the neonates were brought by the midwife from the operating theatre and placed on the resuscitation table under an overhead heater. The forehead skin area was cleaned and dried.2). Neonates were excluded when the arrival-time or application of sensor was not clearly visible on video recordings or if both NIRS sensors were applied simultaneously in one neonate. For analyses neonates, in whom INVOS 5100C was applied, were matched for gestational age (±1 week) to neonates in whom NIRO 200 NX was applied. Neonates with application of both sensors simultaneously were excluded. The sensor was fixed in both groups on the forehead with a double adhesive plaster and/or gauze bandage by a research team member, who was not involved into resuscitation. Four time periods (T) were defined: T1 from birth to arrival of the neonate on the resuscitation table, T2 from arrival to application of the NIRS sensor, T3 from application to first cTOI or crSO2 values on the monitor, and T4 from arrival to first displayed NIRS values. Additionally, we analysed first displayed values of cTOI and crSO2.In neonates either the NIRS sensor of the NIRO 200 NX  was positioned on the right forehead to evaluate the “cerebral tissue oxygenation index” (cTOI) or the neonatal sensor of INVOS 5100C  on the left forehead for evaluating the “cerebral regional oxygen saturation” (crSOThe data are presented as mean ± standard-deviation (±SD) for normally distributed continuous data and median (range) when the distribution was skewed. Data of NIRO group and INVOS group were compared using paired t-test and Wilcoxon-rank-sum-test for parametric and non-parametric continuous variables, respectively, and chi square or Fisher’s exact test for categorical variables. A p value of 0.05 was considered significant.2, also without reaching significance.Out of the 150 neonates with NIRS measurements during the first 15 min after birth, 15 neonates measured with INVOS 5100C were included, in whom time of arrival and application of the sensors were clearly visible. Main exclusion criterion was hindered visibility caused by attending staff (Fig. 2 using the Nellcor OxiMax it has been demonstrated that it took 22–32 s [2 and NIRS monitoring require careful technical and practical preparations to obtain the initial values within 1 min [The present study demonstrates that after the arrival of the neonate on the resuscitation table non-invasive cerebral NIRS monitoring is feasible within 1 min. The application of the NIRS sensor on the neonate’s forehead can be performed quickly independently of which device was used enabling first values even within 20 s after arrival of the neonate at the resuscitation table. However, most studies present data of cerebral regional tissue oxygenation starting 2 min after birth –5. The m 22–32 s , using t 22–32 s  and usin 22–32 s . However 22–32 s , 9. Bothin 1 min . In the in 1 min  and publin 1 min , 5.Cerebral tissue oxygenation can be measured within 1 min after arriving at the resuscitation table in term and preterm neonates after birth without difference between devices. Therefore, NIRS has the potential to become an additional routinely used tool for cerebral monitoring during immediate transition and resuscitation after birth.


Some of these states are at about 140 meV above the valence band maximum while others are mid-gap states at about 2.3 eV. No magnetic impurity could be detected in LaAlO3 by Proton-Induced X-ray Emission Spectroscopy. We, therefore, attribute the angular momentum-like states in LaAlO3 to cationic/anionic vacancies or anti-site defects. Comparison with the other rare earth perovskites leads to the empirical rule that the magnetic-field-sensitive transitions require planes of heavy elements (e.g. lanthanum) and oxygen without any other light cations in the same plane. These magnetic degrees of freedom in rare earth perovskites with useful dielectric properties may be tunable by appropriate defect engineering for magneto-optic applications.Here, we report the presence of defect-related states with magnetic degrees of freedom in crystals of LaAlO Magnetically and optically these oxides are of little interest in of themselves, unless appropriate cations are incorporated to tune their electronic band structure as well as their magnetic and optical properties3 and non-polar SrTiO3 raises the fundamental question of what makes the interface conduct, that has led to numerous experiments exploring oxide interfaces235673 and SrTiO3, revealing a variety of novel interface phenomena. Very recently, it has been demonstrated that oxide-heterostructures with angular momentum states in rare earth perovskites lead to long-range oscillatory magnetic interaction across oxide interfaces via orbital coupling3 and a variety of polar oxide substrates.Transition-metal oxides are important and interesting as they show a rich variety of properties such as high-k dielectric, ferroelectric, multiferroic, magnetic, colossal magneto-resistive and superconducting properties. Among them, wide band gap insulators with perovskite-based structures including LaAlO3, a rare-earth based perovskite, has a dielectric constant of ~25 and a band gap of ~5.6 eV. This material is a promising gate insulator for advanced Metal-Oxide-Semiconductor (MOS) devices10111314151617183 undergoes a structural phase transition from cubic to rhombohedral below 813 K (540 °C). The phase transition is associated with a rotation of the AlO6 octahedra that leads to lattice strain and the formation of twins and twin-boundaries in LaAlO3. Raman spectroscopy is a useful probe of the structural phase transitions in various ABO3 compounds, especially, LaAlO3. One of the early reports on Raman-active phonons of LaAlO3 at room temperature was by Scott−1. Recent reports20−1 suggesting broken-symmetry in LaAlO3. On a similar note, resonant ultrasound spectroscopic study of LaAlO3 proposes freezing of atomic motions of La/Al at off-site positions away from their high symmetry positions3 is an interesting compound that inherently hosts a variety of defects, which may promise new functionalities with appropriate defect engineering.LaAlO3 and related rare earth perovskites at low temperatures to understand the lattice phonons and the possible excitations involving defect states. In addition to the Raman modes, we have been able to observe several transitions at low temperatures that disappear at room temperature. The additional transitions are found to be magnetic field-sensitive showing Zeeman splitting or a magnetic field-dependent shift suggesting an unexpected magnetic degree of freedom in LaAlO3. We believe that inherent defects in LaAlO3 may lead to changes in the cationic/anionic oxidation states and the crystal electric field thus giving rise to spin/orbital degrees of freedom.Motivated by such possibilities, we have performed magnetic field-dependent Raman spectroscopic studies of LaAlO3  oriented along (100), (110), and (111) have been used to perform the experiments. We have not observed any gross difference in the quality of the substrates from these suppliers. We have also performed experiments on other rare-earth-based oxides, namely NdAlO3, NdGaO3, DyScO3, LSAT [(La0.18Sr0.82)(Al0.56Ta0.41)O3], LSAO (LaSrAlO4), and SrTiO3. In order to identify and quantify the contaminants in these single crystal substrates, we have conducted Particle Induced X-ray Emission (PIXE) measurements by using 2 MeV alpha particles and detecting the emitted X-rays using a Si(Li) detector that reveals the presence of no impurity above the instrumental resolution limit (~10 ppm).Single crystalline substrates of LaAlO3 (and other oxides) were recorded using k//B configuration and both being perpendicular to the substrate surface.Raman spectroscopic measurements at low temperatures as a function of magnetic field were performed using a WiTec Raman spectrometer coupled to an attoCube superconducting magnet. Further, temperature-dependent Raman spectra were recorded using a liquid nitrogen cooled Linkam stage. A 532 nm laser line was used as an excitation source while an 1800 g/mm grating was used to record the spectra. The spectra were also recorded using 488, 514.5, and 633 nm laser excitation in order to distinguish the Raman active and photoluminescent (PL) transitions. Raman spectra of LaAlOA1g + 4Eg) Raman active modes for 3. Experimentally these modes have been reportedly observed at 32 (Eg), 123 (A1g), 152 (Eg), 464 (Eg), and 487 (Eg) cm−1 while additional modes at 180 and 203 cm−1 were assigned to ghost modesE + 3A1) symmetries. A comprehensive mode-assignment of the various optical phonons may be found in ref. Group theory predicts five  upon matching with the excitation source at 532 nm. Notably these modes becomes weaker or invisible for the other excitation wavelengths. 3 at low temperatures indicates the presence of some sort of magnetic moment. Splitting of a peak in to three components implies an angular momentum quantum number of ‘1’. One can estimate the Lande-g factor using the relation, ΔE = 2gμBB, where ΔE stands for peak-peak splitting for a given applied magnetic-field B and μB is the Bohr magneton. For the PL peaks at 271 and 290 cm−1, g ≈ 3.0 ± 0.2, whereas for the Raman active peaks at 1094 and 1108 cm−1 and their higher orders, g ≈ 1.3 ± 0.2. However, it should be noted that the line-shifts are nonlinear in the applied field. It is intriguing to find the magnetic states in LaAlO3 where La3+, Al3+ and O2− all have nonmagnetic, closed shell ground states. We believe they arise from defects, as discussed below.The interaction between an external magnetic field and the magnetic dipole moment associated with the total angular momentum of an ion or defect will lead to Zeeman splitting of the energy levels. While Zeeman splitting will start in most systems right above zero field several of the transitions observed show a field threshold above which the splitting begins which we do not understand at this point. Magnetic splitting of the peaks in the spectrum of LaAlO−1 show a continuous drop in intensity as the temperature increases and practically disappearing above 70 K, as also can be seen in −1 and their higher orders (see −1 involve transitions between the ground state and excited states (~2.3 eV) whereas the Raman active peaks near 1094 and 1108 cm−1 involve transitions between an intermediate state  and excited states (situated near ~140 meV = 1120 cm−1). On looking at the temperature dependence, the PL transitions decay with increasing temperature as expected due to enhanced non-radiative transitions from phonon excitations. On the other hand, the Raman-active peaks show a bell shaped behavior as initially the transition increases with population of electrons in the intermediate level but as this population level increases further with increasing temperature it starts inhibiting the transition from the upper level.3 have shown the presence of various types of defects via sharp or broad peaks below the bandgap. Kawabe et al.et al.3 Schottky-like defects that maintain charge neutrality are dominant under all conditions, besides oxygen vacancies and anti-site defectset al. reportedet al.S = 3/2 and another level within 10 meV difference having spin S = 1/2 while aluminium vacancies with 0 or −3 charges give rise to a level at 690 meV above VBM and another at a separation of 20 meV with spin S = 3/2 and S = 1/2, respectively. Further, anti-site defects, such as La at Al-site (LaAl) and Al at La-site (AlLa) also give rise to additional levels: AlLa with 0/+1 charge introduces a level at 300 meV above VBM whereas LaAl with 0/+1 charge gives a level at 170 meV and +1/+2 charge at 40 meV. In other words, it has been proposed theoretically that various defects may introduce mid-gap states, some of which have spin character.Our experiments suggest the presence of defects with possible spin and/or orbital magnetic moments. Several recent reports of PL spectra in LaAlO3 as shown by the Zeeman splitting of peaks, which most likely arise from inherent defects. The data show a fair match with the energy levels estimated in refs 3+ that gives rise to the PL peaks while the cationic vacancies/anti-sites form another defect complex leading to the Raman active transitions. In presence of magnetic field, these defect complexes undergo structural transition at 2 and 4 T, respectively, thus giving rise to angular momentum states and the peak-splitting. In summary, our data demonstrate the idea of a magnetic excitation coupling to Raman active and photoluminescent transitions through defect formation where the spin-zero La3+ interacts with an oxygen vacancy/interstitial (or anti-site defect) to produce a defect complex with finite angular momentum. We think that in these rare-earth perovskites, in addition to such defects, the existence of an atomic plane with a high Z rare-earth ion and oxygen is a prerequisite to produce such angular momentum states. In the case of LaAlO3 it is possible that hybridization of the empty lanthanum ‘d’ or ‘f’ orbitals with the defect is somehow responsible for the effect.Our data provide direct evidence for the presence of angular momentum states in LaAlO3, NdGaO3, and DyScO3 are shown in 3+ and Dy3+ based compounds, we observe the angular momentum state transitions29303132J = 9/2 and J = 15/2 multiplets of Nd3+ and Dy3+, respectively, which exhibit Zeeman splitting under magnetic field 0.3(Sr2AlTaO6)0.7] and LSAO [LaSrAlO4], and their spectra shown in This idea is consistent with our investigation of the magnetic-field dependence of the Raman spectra in other rare earth-based perovskite oxides. Data for NdAlOpendence . Now wha3+ and Fe3+ in the Al-based compounds143 as well. PIXE measurements showed that no impurities could be detected within the resolution limit (~10 ppm) in LaAlO3 . The fact that despite the presence of higher amounts of such magnetic impurities in STO, we do not see any magnetic-field sensitive excitation thus it suggests that native impurities are not responsible for these angular momentum states.We rule out a role of any magnetic impurity element such as CrAlO3 see  and any −1  in LaAlO3 could be assigned to neutral oxygen interstitials or vacancies while the Raman active peaks near 1094 and 1108 cm−1 (~140 meV) may involve angular momentum states arising from anti-sites, especially La at Al-site (LaAl) defects, and/or cationic vacancies. It should be noted that none of these magnetic-field-sensitive peaks which indicate the presence of magnetic degrees of freedom in LaAlO3 have ever been reported before2021223 at ~900 °C in vacuum (PO2 = 1 × 10−2 mTorr) or oxygen rich environment (100 mTorr) for about 30 minutes does not have any significant effect on the magnetic-field sensitive peaks in the Raman spectrum (recorded using 532 nm laser line), demonstrating the stability and robustness of the defects present and suggesting possibilities for applications as optical magnetic-field sensor and light emitters. The key to the observation of the magnetic field sensitive states is the presence of a plane with a heavy element like the rare earth and oxygen. In SrTiO3 the absence of a plane of heavy elements means that we do not see any angular momentum state. Such angular momentum states in rare earth perovskites may be utilized to tailor the magnetic interactions across interfaces of oxide heterostructures for new emergent phenomena and device applications.Therefore, the PL angular momentum state transitions observed near 271 and 290 cm3 crystals by performing magnetic-field-dependent Raman spectroscopy. In addition to the Raman active phonons, we have observed at low temperatures (below 100 K) several hitherto unknown peaks appearing due to Raman active transitions near 1094 and 1108 cm−1 (~140 meV) as well as a doublet luminescent transition near 2.3 eV . Under magnetic field, these additional peaks show Zeeman splitting or blue-shift thus indicating an association with orbital angular momentum states. We believe that these additional peaks arise from defects, possibly cationic/oxygen vacancies that trap electrons directly or populate states below the conduction band edge.Here, we have presented evidence for the presence of magnetic degrees of freedom in LaAlO3 establish the presence of magnetic moments associated with defects in a purely diamagnetic material, which are inherent but robust. Engineering these defects and hence the angular momentum states may be a pathway to discover new phenomenaOur low temperature- and magnetic field-dependent Raman studies of LaAlOHow to cite this article: Saha, S. et al. Magnetic Modes in Rare Earth Perovskites: A Magnetic-Field-Dependent Inelastic Light Scattering Study. Sci. Rep.6, 36859; doi: 10.1038/srep36859 (2016).Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


A receptors (δGABAARs) mediate tonic inhibition and regulate network activity. However, the influence of δGABAARs on respiratory rhythm and motor outputs is unknown. We manipulated extra-synaptic GABAA receptor function in the preBötzinger Complex (preBötC), a site central to the generation of inspiratory motor activity in mammals. Activation of preBötC δGABAARs in anesthetized rats and wild-type mice decreased breathing rate. In δGABAAR knockout (Gabrd−/−) mice, however, δGABAARs activation had no effect on breathing rate. We then found that during active wakefulness associated with behaviors and movements, diaphragm activation was higher in the Gabrd−/− compared to wild-type mice, but not in other states. These findings identify that δGABAARs modulate the respiratory network, which is critical to understand how δGABAARs change breathing in pathological conditions affecting extra-synaptic GABAA receptor function such as exposure to anesthetics and neurosteroids.Persistent and stable respiratory activity across behavioral states is key to homeostasis. Extrasynaptic δ-subunit containing GABA The respiratory network produces an intricate respiratory pattern with three phases: inspiration, post-inspiration, and late expiration. Inspiratory motor output is driven by a population of neurons that forms the preBötzinger Complex (preBötC)4, whereas expiratory activity is regulated by the Bötzinger Complex5, a structure with direct projections from and to the preBötC, and the retrotrapezoid nucleus6. In the preBötC, excitatory neurons drive inspiratory output2, whereas inhibitory neurons coordinate the generation of inspiratory and expiratory phases7. Optogenetically targeted excitation of glycinergic neurons in the preBötC shape respiratory pattern by inducing prolonged apnea, but rhythm persists even after substantial inhibition of glycinergic cells8. The functional role of GABAergic neurons in the preBötC is, however, not clear. Expression of the GABA-synthesizing enzyme glutamate decarboxylase shows that GABAergic neurons constitute a large part of inhibitory preBötC neurons9. In brainstem sections in vitro, blocking GABAergic neurotransmission increased rhythm variability, but did not otherwise change the frequency of rhythmic bursts10. In vivo, application of the GABAA receptor agonist muscimol at the preBötC abolished rhythmic activity5, whereas the GABAA receptor antagonist bicuculline did not change rhythmic breathing, but suppressed the Breuer-Hering inspiratory inhibitory reflex11. In rabbits, GABAA receptor blockade in the preBötC decreased respiratory rate, but did not completely blocked it12. These studies suggest that the endogenous role of GABAA receptors in the preBötC is to shape respiratory motor output, for instance, during voluntary control of breathing11, but this hypothesis has not yet been tested.Rhythmic breathing is generated by a complex neural network in the brainstemA receptors mediate fast synaptic inhibition, whereas GABA can also activate highly-sensitive extra-synaptic GABAA receptors and produce tonic inhibition13. Extrasynaptic δ-subunit containing GABAA receptors (δGABAARs) mediate tonic inhibition and regulate network excitability14 and outputs15. δGABAARs also function to dampen network excitability and prevent excessive excitation16. In the context of endogenously active rhythmic networks, such as the respiratory network, these observations suggest that extra-synaptic δGABAARs may also play a significant role in modulating respiratory activity. However, the role of extra-synaptic δGABAARs in modulating respiratory rhythm has never been tested. Here, we propose that δGABAARs provide a level of tonic inhibition in the respiratory network to modulate rhythmic breathing. To test these concepts, we pharmacologically manipulated the preBötC of rats and mice, the latter including wild-type mice and mice lacking δGABAARs17. Our data identify for the first time that δGABAARs function to modulate respiratory network activity.GABAARs (Gabrd−/−) plus nineteen wild-type littermate controls were used for physiological recordings (body weight: 30.4–37.0 g). The generation of Gabrd−/− mice has been described previously18. Mice and rats were housed with free access to food and water under a 12-hour light:dark cycle (lights on at 7 am).All procedures were performed in accordance with the recommendations of the Canadian Council on Animal Care, and were approved by the University of Toronto Animal Care Committee. Twelve adult male Wistar rats , and nineteen adult mice lacking δGABA20. Briefly, we recorded activities of the diaphragm and tongue muscles in isoflurane-anesthetized (2–2.5% inspired), tracheotomised and spontaneously breathing  rats. Diaphragm muscle activity was recorded using stainless steel bipolar electrodes positioned and sutured onto the right crural diaphragm. Tongue muscle activity was recorded using two stainless steel needles inserted via a per-oral approach. The electromyogram signals were amplified , band-pass filtered (100–1000 Hz), integrated, and digitized at a sampling rate of 1000 Hz using an acquisition system and Spike software version 6 . Rats were kept warm with a heating pad during the experiments. Using a dorsal approach, a microdialysis probe  of 200 µm diameter and 1 mm diffusing membrane length was inserted into the preBötC using a stereotaxic frame and micromanipulator . The probe was placed 12.2 mm posterior, 2.0 mm lateral, and 10.5 mm ventral to bregma according to a standard rat brain atlas21 and our previous experience with this model20. Baseline (aCSF) data for breathing rate and diaphragm amplitude were averaged over a 30-min period preceding the start of THIP infusion. THIP data were averaged for a 30-min period starting 30 min after THIP infusion was initiated. Only experiments where the probe was located within 2 mm from the center of the preBötC were analysed.In anesthetized rats, we used reverse-microdialysis to unilaterally microperfuse selected agents into the preBötC. The experimental procedures were as described previously20: (i) When the probe was inserted into the medulla, tongue muscle activity decreased ~30% as it reached the vicinity of the preBötC. (ii) Microperfusion of the µ-opioid receptor agonist DAMGO (5 µM) decreased breathing rate by approximately 50%19. (iii) Post-mortem histology confirmed the probe location in the preBötC using anatomical markers such as the semi-compact division of nucleus ambiguus and the caudal part of the facial nucleus. We used the caudal part of the facial nucleus as a reference to identify the brain section located 11.6 mm posterior to bregma according to Paxinos atlas21. Once the coronal section where the probe was located was identified, we used the nucleus ambiguus to then determine the medial-lateral and dorsal ventral coordinates. Once the location of the probe site was determined, we determined 3-dimensional coordinates  using bregma as reference. We used these three anatomical and functional criteria, and experience from our previous studies, to confirm that the probes were positioned in the region of the preBötC. On rare occasions (~1/20 experiments), the probes damaged the preBötC and respiratory rhythm was irregular and unstable. In such an event, we discontinued the experiment.As described previously we used three criteria to target the preBötC, to position the probe, and to confirm its location2 into aCSF. A microdialysis probe was perfused with aCSF and baseline levels of the physiological variables were recorded for at least 30 min followed by 120 min of recordings during microperfusion of the selected agents. We applied the δGABAAR-preferring agonist 4,5,6,7- tetrahydroisoxazolopyridin-3-ol hydrochloride (THIP), the GABAA receptor antagonist bicuculline and the µ-opioid receptor antagonist DAMGO to the preBötC. All drugs were obtained from Tocris .Fresh artificial cerebrospinal fluid (aCSF) was made for each experiment with the following composition (in mM): NaCl (125), KCl (3), KH2PO4 (1), CaCl2 (2), MgSO4 (1), NaHCO3 (25), and glucose (30); pH was adjusted to 7.4 by bubbling CO19. The rationale for these correlations is that for a locus of effect of THIP at the preBötC, the latency for the drug to diffuse through the tissue and to progressively change respiratory activity will vary as a function of the distance of the probe from the preBötC. We first determined the locations of the intervention (perfusion) sites using the criteria described above. For each animal, we also calculated the distances from the corresponding perfusion site to the center of the preBötC . We then calculated the latencies to a 10% change in respiratory rate in response to THIP, and correlated these distances with the latencies or the magnitudes of THIP effects on respiratory rate.We calculated the correlation to relate the location of the intervention sites with the resultant effect on respiratory activity as previously describedGabrd−/− mice, we also used reverse microdialysis to microperfuse agents into the preBötC. We recorded diaphragm muscle activity using a similar approach to the rats and as previously described for mice22. The mice were also kept warm with a heating pad. We inserted the microdialysis probe into the medulla 6.7 mm posterior, 1.2 mm lateral, and 5.7 mm ventral to bregma according to the standard brain atlas23 and prior experience24. For wild-type or Gabrd−/− mice, baseline levels were recorded for at least 30 min followed by 120 min of recordings during microperfusion of the selected agents. Baseline (aCSF) mean data were calculated for the 30-min preceding the start of THIP infusion. THIP mean data were calculated between 30–60 min after the start of THIP infusion. The anatomical and functional criteria defined in rats were also used for the experiments in mice. We used the caudal part of the facial nucleus to identify the brain section 6.5 mm posterior to bregma as shown in the mouse Paxinos atlas23. All experiments with probe sites located within 1 mm were used to calculate mean data.In anesthetized , spontaneously breathing  wild-type and ARs in regulating respiratory and motor activities in freely behaving wild-type and Gabrd−/− mice, we recorded electroencephalogram (EEG) and postural (neck) muscle electrodes to identify sleep-wake states, and diaphragm muscle electrodes for respiratory muscle recordings. One week prior to the behavioural experiments, sterile surgery was performed under isoflurane anaesthesia to implant the mice with the EEG, neck, and diaphragm muscle electrodes. To record diaphragm activity, two wires were sutured onto the costal diaphragm via an abdominal approach. The mouse was placed in the prone position in the stereotaxic apparatus (Model SAS-4100) with blunt ear bars, and three holes were drilled into the skull for the placement of the EEG electrodes. Two stainless steel screws  were placed approximately 1 mm to the right and 1 mm anterior to bregma, and 1 mm to the left and 2 mm posterior to bregma for EEG activity; the third electrode used as a common reference was placed 2 mm to the left and 2 mm anterior to bregma. Insulated multi-stranded stainless steel wires were also sutured on the dorsal neck muscles to record the electromyogram. Post-surgical care consisted of a subcutaneous injection of an anti-inflammatory drug  and an analgesic .To identify the role of δGABA19.Mice recovered for one week prior to the experiments. On the first and second experimental days, mice were connected to the recording apparatus and placed in a large open-topped Plexiglas bowl filled with fresh bedding, food and water. Mice were left in the chamber for about 6 hours each day to habituate the animal to its environment. On the third day, experiments were performed to record electrophysiological signals while the mice were awake and asleep, and able to move freely. The Plexiglas bowl was placed on a rotating turntable  that automatically adjusts its position when the mouse physically moves; this response of the turntable avoids entanglements of the recording cable. The movement of the turntable were also recorded as a DC voltage. EEG, neck, and diaphragm muscle activities were recorded for a period of 3 hours from 10am to 1 pm. Data were amplified, filtered, moving-time averaged, sampled and analyzed as described previously25. EEG frequencies were also calculated in the following frequency bands: δ (1–4 Hz), θ (4–7.5 Hz), α (7.5–13.5 Hz), β1 (13.5–20 Hz), β2 (20–30 Hz). AW was characterized by low δ frequencies, high neck muscle activity and was often accompanied by body movements. QW was characterized by low δ frequencies, and low neck muscle activity without body movements. NREM sleep was characterized by high δ frequencies, high EEG amplitude and low neck muscle activity. REM sleep was characterized by low δ frequencies, high θ frequencies and low neck muscle activity.For every 10-second epoch, sleep-wake states were visually identified as active wakefulness (AW), quiet wakefulness (QW), non-rapid-eye-movement (NREM), and rapid-eye-movement (REM) sleep. Prevailing sleep/wake states were identified according to standard criteriaTo determine whether the animal was moving in its environment, we used a combination of video analysis, activation of the rotating table, and activation of postural neck muscle. Specifically, we separated periods of active wakefulness into two distinct periods for subsequent comparison with each other and with quiet wakefulness: (i) Periods of active wakefulness with body movements (i.e. MOV). These periods were recorded during the automatic adjustments of the turntable under the recording chamber as the mouse physically moved, and were verified by video recordings. (ii) Active wakefulness without such movements (i.e. NO-MOV). Such periods of NO-MOV occurred during specific behaviors while stationary, such as sniffing or grooming, and were also identified from the video recordings. (iii) Periods of quiet wakefulness as identified by low δ EEG frequencies, low neck muscle activity and no body movements.xcorr MATLAB, R12, Mathworks). This function is a measure of similarity of two waveforms. A value of 1 means that the two signals are identical, whereas a value of 0 means that they are completely different. Cross-correlation function was calculated for each 10 s epoch and associated with a specific state. Breathing rates, diaphragm amplitude and neck muscle activities were also measured for each 10-second epoch.Breathing rate was defined as the number of diaphragmatic breaths per minute. Diaphragm amplitude was defined as the amplitude of integrated diaphragm of a breath by subtracting the lower diaphragm signal to the peak diaphragm signal. To determine synchronization of postural neck muscle and diaphragm muscle activities, we computed the cross-correlation function using MATLAB Signal Processing Toolbox (function Gabrd−/−) and the second repeated factor being drug treatment (i.e. aCSF or drug of choice). If the ANOVA was statistically significant, an all pairwise multiple comparison procedure (Holm-Sidak tests) was then used to determine significant differences between conditions . In studies with small groups of animals (n = 6), the non-parametric Mann Whitney rank sum test was used to test significance. All statistical tests were two-tailed with the level of significance set at P < 0.05. All tests were performed with SigmaPlot version 11 .In the figures, group data are illustrated as the mean ± standard error of the mean (S.E.M.). Before analysis of variance tests are performed, all data are first tested for normality with the Shapiro-Wilk test and for equality of variances with the Brown-Forsythe test. For the studies in rats, we used 1-way ANOVAs with the repeated factor being the treatment with aCSF or drug of choice. For the studies in mice, we tested for group differences using 2-way ANOVAs, with one factor being genotype  into the preBötC  the magnitudes of the reductions in breathing rate  the latencies of the breathing rate reductions . Post-hoc analyses identified a decrease in breathing rate of 23.2 ± 2.6% in the wild-type mice  into the preBötC region19 of Gabrd−/− mice. DAMGO activates µ-opioid receptors and decreased breathing rate by 41.9 ± 3.6% . Post-hoc analyses identified a decrease in breathing rate of 19.9 ± 2.7% in the wild-type mice , but not in Gabrd−/− mice (P = 0.485). The data also showed that there was no effect of genotype on the response of diaphragm amplitude to systemic injection of THIP . These results further support the idea that THIP reduces rhythmic breathing without affecting motor amplitudes by specifically activating δGABAARs.To determine whether general activation of δGABAity Fig. . Exampleity Fig.  and grouity Fig.  identifi29. To determine whether the frequency of diaphragm muscle activations is influenced by δGABAARs in freely-behaving animals, we measured respiratory muscle activities in non-anesthetized wild-type and Gabrd−/− mice . During active wakefulness, Gabrd−/− mice exhibited an increased frequency of diaphragm muscle activations compared to wild-type mice  and there was no effect of genotype that depended upon sleep-wake state , and α activity was significantly lower in the Gabrd−/− mice (P = 0.038). There was no effect of genotype on EEG activity in any frequency band that depended upon the prevailing sleep-wake state . Overall, these data show that, in the Gabrd−/− mice, the increased frequency of diaphragm muscle activations in active wakefulness . Post-hoc analysis identified that during MOV, the frequency of diaphragm activations  was significantly higher in the Gabrd−/− mice compared to the wild-type mice  or quiet wakefulness (P = 0.147). In contrast, there was no effect of genotype on diaphragm amplitude that depended upon the type of behavior exhibited during wakefulness (P = 106). We also observed that neck muscle activity was lower in the Gabrd−/− mice compared to wild-type mice. These additional analyses, however, identified that this genotype effect on neck muscle activity was confined to periods of MOV , and not NO-MOV or quiet wakefulness (both P > 0.147). These data further suggest that a δGABAAR-mediated mechanism modulates the rhythmic components of respiratory network activity rather than the magnitude of the oscillatory motor output.The above data showed that reduced δGABAity Fig. . We thenity Fig. . The effAR-mediated modulation of respiratory network activity. Accordingly, using cross correlation, we quantified the degree to which the diaphragm and postural (neck) muscles are synchronized across MOV, NO-MOV and quiet wakefulness in the wild-type and Gabrd−/− mice. In the Gabrd−/− mice, the diaphragm and neck muscle activities showed distinct activations with multiple periods of coordination and synchronization during periods of MOV compared to the wild-type mice, . Post-hoc analyses showed that diaphragm and neck muscle activities were highly synchronized during MOV in the Gabrd−/− mice but were not in the wild-type mice  motor activities during periods of active wakefulness associated with movements.To identify whether the increased in diaphragm activation observed above is due to behavioural control of respiratory circuits, we tested the degree of coupling between diaphragm muscle and postural motor activities, two motor systems controlled by different neural circuits, across the behavioural states, and tested for an influence of δGABAce, Fig. . The groA receptor-mediated inhibition, is important to modulate rhythm in many neural circuits. Synaptic GABAA receptors are implicated in respiratory control and are present throughout the respiratory network30, but the role of extra-synaptic GABAA receptors is unknown. Here, we studied whether extrasynaptic δGABAARs provide a level of tonic inhibition in the respiratory network to modulate diaphragm rhythmic activity and to dampen rhythmicity during active motor behaviours. Using a combination of pharmacological and genetic approaches, we found that δGABAARs in the preBötC region function to decrease respiratory network activity. We then identified that the absence of δGABAARs in the Gabrd−/− mice increases respiratory network activity and motor activities during wakefulness when compared to wild-type mice. In Gabrd−/− mice, diaphragm activity is also synchronized to locomotor activity, an effect less pronounced in wild-type mice. The presence of an abnormal number of behavioral components in diaphragm muscle activity observed in Gabrd−/− mice suggests that δGABAARs may dampen the influence of non-respiratory behaviors on respiratory network activity to maintain breathing.Post-synaptic inhibition, such as GABAet al., 2001). In the preBötzinger Complex, GABAA receptors are present in inspiratory neurons and non-respiratory neurons7, suggesting that GABA in the preBötC may modulate inspiratory activity but also non-respiratory activity. GABAA receptor stimulation at the preBötC can depress breathing whereas antagonism has little or no effect10, suggestive of a GABAA receptor-mediated inhibition that shapes respiratory motor outputs rather than generate respiratory rhythm per se11. Although GABAA receptors mainly exert their effects by inducing fast post-synaptic hyperpolarization, GABA also activates extra-synaptic GABAA receptors located outside the synapse and generates tonic inhibition to reduce network excitability13. Our data indicate that the δGABAAR-preferring agonist THIP perfused in the ventrolateral medulla decreased breathing rate, at least in great part, by acting at the preBötC level. To confirm the site of action of THIP and to demonstrate that THIP modulates neurons in the vicinity of the preBötC, we showed that when THIP was perfused close to the preBötC, it induced a faster and more pronounced decrease in breathing rate than when it is perfused away from it. These relationships were supported by correlations between the latencies or magnitudes of THIP effect and distances from perfusion site to the preBötC as previously described and validated24. Although THIP is a δGABAAR-preferring agonist27, we further verified that THIP selectively targets δGABAARs by using Gabrd−/− mice that lack these receptors. The absence of response to THIP in Gabrd−/− mice further identified that selective activation of δGABAARs underlie inhibition of respiratory network activity by THIP. The combination of these two approaches support the idea that δGABAARs in the region of the preBötC have the capacity to modulate respiratory network activity.In the ventrolateral medulla of adult rats, glutamic acid decarboxylase immunoreactivity, a marker of GABAergic cells, has been identified in post-inspiratory and inspiratory neurons  channels22. Inhibition through GIRK channels likely involves a network level modulation32. Such large scale effect of neurotransmitters is known as volume transmission and δGABAARs reduce network excitability through this mechanism33. Volume transmission, as opposed to wiring transmission, modulate network excitability via neurotransmitters diffusing into the extracellular space and then by activating extra-synaptic receptors14. Although this type of neurotransmission is slower than wiring transmission, it is sufficiently fast to modulate the respiratory network and ongoing motor systems27. Volume transmission and GABAergic inhibition through δGABAARs may therefore be a potential mechanism modulating network excitability. Here, we identify, for the first time, the functional role of δGABAARs, which are uniquely expressed in the extrasynaptic space, in the preBötC region and the capacity of these receptors to modulate respiratory network activity during active behavior.The respiratory network, and at its core the preBötC, form a dynamic network endogenously generating rhythmic motor activity. To spontaneously generate respiratory motor rhythm, the network needs a sufficient level of excitabilityARs at the preBötC significantly reduced respiratory network activity. Although these data demonstrate that δGABAARs in the preBötC region have the capacity to modulate respiratory network activity, the role of δGABAARs in response to endogenous GABA release cannot be assessed due to the lack of a selective δGABAAR antagonist. To identify the endogenous role of δGABAARs in modulating respiratory network activity, we compared diaphragm muscle activity (a surrogate of respiratory network activity) between wild-type and Gabrd−/− mice at various states of arousal and during behaviours. Although δGABAARs did not play a significant role in modulating respiratory network activity during sleep and quiet wakefulness, rhythmic activity of the diaphragm muscle was higher in Gabrd−/− than in wild-type mice in active wakefulness, especially during movements. Gabrd−/− mice also showed synchronized diaphragm and neck muscle activities during movements, pointing out that, without δGABAARs, non-respiratory behaviours greatly influenced respiratory network activity. Although the mechanisms underlying the role of δGABAARs in inhibiting respiratory network activity only during movements have not been yet identified, our data suggest that δGABAARs play an endogenous role in the modulation of respiratory network activity, especially during active non-respiratory behaviors.In states of reduced arousal, i.e. anesthesia, when behaviour is suppressed and the respiratory network is autonomous, activation of δGABAARs play a significant role in modulating respiratory activity during conscious behaviors in vivo when the levels of excitation independent of breathing per se is high29. Excitatory inputs from descending motor pathways may be inhibited by δGABAARs to maintain the essential autonomic function of breathing while non-respiratory behaviours are performed. The notion of tonic inhibition regulating network excitability has been demonstrated in thalamo-cortical networks26, in the hippocampus34, and in the cerebellum35. Our findings identify that δGABAARs modulate rhythmic breathing, and dampen behavioral influences on rhythmic motor activity during active behaviors in vivo. These results identify a previously unrecognized role of δGABAARs in modulating the endogenously active respiratory network in vivo, the persistence and stability of which is critical to homeostasis. Identification of the role of extrasynaptic GABAA receptors provides a better understanding of how breathing is affected by changes in extrasynaptic GABAA receptor expression and function under pathological conditions such inflammation36 and exposure to anesthetics and neurosteroids18.Extra-synaptic δGABA


The diverse properties of DNA intercalators, varying in affinity and kinetics over several orders of magnitude, provide a wide range of applications for DNA-ligand assemblies. Unconventional intercalation mechanisms may exhibit high affinity and slow kinetics, properties desired for potential therapeutics. We used single-molecule force spectroscopy to probe the free energy landscape for an unconventional intercalator that binds DNA through a novel two-step mechanism in which the intermediate and final states bind DNA through the same mono-intercalating moiety. During this process, DNA undergoes significant structural rearrangements, first lengthening before relaxing to a shorter DNA-ligand complex in the intermediate state to form a molecular lock. To reach the final bound state, the molecular length must increase again as the ligand threads between disrupted DNA base pairs. This unusual binding mechanism results in an unprecedented optimized combination of high DNA binding affinity and slow kinetics, suggesting a new paradigm for rational design of DNA intercalators. DNA intercalation represents an invasive, yet reversible, mode of DNA-ligand binding. These essential features of DNA intercalation allow a wide range of precisely modulated therapeutic and biotechnological applications. Conventional intercalators, such as acridine235689571112134Ru2]4+ (Pi) shown in Fig. 1B152ip2+ in Pi are linked by two single bonds via a benzene ring (orange), providing additional rotational degrees of freedom. Previous bulk measurements reported by Chao et al. showed that Pi elongates DNAet al. found that the DNA-Pi complex does not preserve the free-DNA orientation, in contrast to conventional intercalatorsIn this work we introduce a new mechanism of DNA intercalation, where the intercalating moiety is converted from a fast assembling conventional intercalative state to a slow assembling final intercalative state. This intercalative conversion is characterized for the rotationally flexible binuclear ruthenium complex [μ-bipb(phen)Strong DNA condensation by cationic ligands with high affinity to DNA poses a great challenge in bulk experiments15We examined the kinetics of Pi interacting with single λ-DNA molecules as a function of constant applied forces of 10 to 50 pN and ligand concentrations of 0.15 to 40 nM. 5115In order to test our hypothesis of rapid formation of an intermediate state followed by slow formation of a final intercalated state, we probed the occupancy of the intermediate state by stopping the intercalation process at intermediate times and washing off the ligand . We obse511kf) and slow (ks) association rates obtained from the double exponential fits of DNA-Pi complex time-dependent extension measured at constant force. In this model, an initial fast, bimolecular binding event is followed by a slower, unimolecular binding event, as demonstrated above. Fits to this model reveal the fundamental reaction rates for both steps of this process, k1c, k1−, k2 and k2−, where k1c and k−1 represent association and dissociation to and from the intermediate intercalated state (I‡), while k2 and k2− represent association and dissociation to and from the final (I) intercalated threaded state  and ks (C) at two constant forces, while dissociation experiments that directly determine k2− show no concentration dependence, as expected for a unimolecular dissociation rate. The elementary rates k1, k1−, k2 and k2− are then fit to an exponential force dependence  values presented in 5252627Kd at each applied force, plotted in Kd(F) dependence as obtained in this equilibrium analysis confirms the Kd(F) values determined from the fitted elementary reaction rates from our kinetics measurements. The zero-force equilibrium dissociation constant Kd(0) = 11 ± 2 nM obtained from this equilibrium analysis is consistent with its value determined from kinetics measurements. In addition, we determine the equilibrium DNA elongation upon complete Pi threading to be Δxeq = 0.27 ± 0.03 nm.The force-extension curve obtained for the saturated DNA-Pi complex, xon = x+1 + x+2 = 0.35 nm, the DNA- Pi complex is relaxed to Δxeq = 0.27 nm in the final equilibrium state. These findings confirm a previous prediction by Bahira et al.11135The DNA intercalation affinity found here for the two-step threading of Pi is ~5-fold higher than that of its closely related parent molecule P, which exhibits one-step intercalation in single molecule studies572+ moiety through the DNA base pair, requiring major duplex disruption. However, threading of this pre-intercalated DNA duplex is ~10-fold faster than the threading intercalation of the P ligand (compare k2 for Pi and k1∙Kd = k−1 for P), and overall ~5-fold more driven.The results reveal a model for DNA-Pi assembly, illustrated in These novel findings for Pi not only overcome the limitation of bulk measurements in resolving the binding mode of the DNA-Δ,Δ-Pi interaction, but also present a convincing illustration of the utilization of single-molecule studies to provide important insights for the rational design of DNA-targeting ligands. The specific mechanism dissected here represents the first measurement of a two-step intercalator combined with a molecular lock mechanism. This mechanism results in the highest affinity measured for such an unconventional intercalator, as well as one of the slowest binding mechanisms observed. Using this information, DNA-ligand structural dynamics may be optimized for effective antitumor treatment in the presence of other non-intercalating DNA-targeting agents.All experiments were conducted using dual-beam optical tweezers (laser wavelength 830 nm). A single bacteriophage λ-DNA, labeled on opposite strands with biotin, was attached between two streptavidin-coated polystyrene beads (~5.6 μm); one bead is held in the optical trap and the other bead is held by a glass micropipette. A piezoelectric positioner displaces the micropipette (±10 nm) to maintain a fixed stretching force (±1 pN) on the DNA molecule. After the attachment between the two beads, the DNA-only stretching curve is obtained at a pulling rate of ~200 nm/s. Then, the DNA is stretched rapidly (~2 s) to reach the assigned constant force and the elongation from the DNA-only equilibrium extension due to the threading intercalation by Pi is traced. After the DNA-Pi complex reaches equilibrium elongation, the ligand is rinsed out by flowing ligand-free buffer solution. As the ligand dissociates, the DNA-Pi complex elongation is traced back to the free DNA extension. The force feedback reacts to any sudden force change as fast as 50 ms by displacing the micropipette to maintain the assigned force. The experiments were carried out in a 100 μl flow cell chamber volume and a constant ligand flow rate of ~2 μl/s. Constant force measurements were obtained on at least three DNA molecules for each averaged data point. All measurements were obtained at 21 °C and under buffer conditions of 10 mM Tris, 100 mM NaCl and pH 8. Pi was synthesized and purified as described elsewhereThe time-dependent DNA elongations are fit to a double-exponential dependence on fast and slow rates.The observed fast and slow rates are related to the elementary rates of two-step DNA intercalation1131k1 · C + k1− ≫ k2 + k2−), in which the non-intercalative state (NI) and the intermediate intercalative state (I‡) states rapidly equilibrate before the second transition to the final intercalative state (I), we can then fit the measured fast and slow rates in terms of the elementary rates.For the conditions  to the complex lengthening observed at saturated ligand binding, The fractional lengthening directly corresponds to the fractional ligand binding, We fit our measured fraction of ligand bound to McGhee-von Hippel (M-H) binding isotherm525262728Leq as a function of concentration at each constant force. Matching these calculated and measured Leq values allows us to determine the equilibrium dissociation constant Kd(F) and the intercalative occluded site size n.Eqs  and 9)  9) can bKd(F) values are then fit to the exponential force dependence as given by The force-dependent How to cite this article: Almaqwashi, A. A. et al. DNA intercalation optimized by two-step molecular lock mechanism. Sci. Rep.6, 37993; doi: 10.1038/srep37993 (2016).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


We read with interest the recent paper by Biglari et al. , which rThe authors pointedly reference the recent work by Zura et al. , statingBiglari et al. also noted the results of a randomized controlled trial by Schofer et al. comparing LIPUS with sham-device treatment of chronic nonunion patients . AlthougWe note several additional issues with the study by Biglari et al.: 1) compliance with device usage was raised as an important consideration, but compliance rates were not reported; (2) osteitis, which would likely impair healing substantially, was seen in 23 % 14 of 61) of cases, and was significantly more prevalent in the “unsuccessful treatment” group; (3) chronic nonunion was defined as non-healing for >90 days after surgery, which is inconsistent with most other definitions of nonunion [1 of case complian