Here, we spatially resolve and chemically characterize distinct TiOx phases in localized regions of a TiOx–based memristive device by combining full-field transmission X-ray microscopy with soft X-ray spectroscopic analysis that is performed on lamella samples. We particularly show that electrically pre-switched devices in low-resistive states comprise reduced disordered phases with O/Ti ratios around 1.37 that aggregate in a ~100 nm highly localized region electrically conducting the top and bottom electrodes of the devices. We have also identified crystalline rutile and orthorhombic-like TiO2 phases in the region adjacent to the main reduced area, suggesting that the temperature increases locally up to 1000 K, validating the role of Joule heating in resistive switching. Contrary to previous studies, our approach enables to simultaneously investigate morphological and chemical changes in a quantitative manner without incurring difficulties imposed by interpretation of electron diffraction patterns acquired via conventional electron microscopy techniques.Reduction in metal-oxide thin films has been suggested as the key mechanism responsible for forming conductive phases within solid-state memory devices, enabling their resistive switching capacity. The quantitative spatial identification of such conductive regions is a daunting task, particularly for metal-oxides capable of exhibiting multiple phases as in the case of TiO On the other hand, TiOx, one of the most celebrated metal oxides, provides wider opportunities for multi-state memory capacity due to the intrinsic variety of possible chemical phases. This complexity however makes its control more difficult, also prohibiting the rigorous study of the unrelying physical mechanisms. Whilst the electrical behaviour of TiOx based memristors has been widely investigated, much less is known about the atomic-scale changes that occur in the film as a consequence of switchingNanoscale resistive random-access memory devices (RRAM) based on metal-oxides, also known as memristors, have great prospect in becoming a mainstream memory technology, due to their infinitesimal dimensions, fast switching and capacity to store multiple bits of information per element12345et al.4O7 as the structure of conductive filaments in TiO2 based-devices by interpretation of electron diffraction patterns. However, HRTEM requires the use of high energy electron beam (200–300 kV) which could induce sample crystallization and effectively alter the physical-chemical state of the film. Furthermore, electron diffraction requires the crystals to be aligned in specific orientations with respect to the beam for unambiguously capturing distinct diffraction patterns2-based133-basedx film through the capping top electrode. When compared to electron energy loss spectroscopy (EELS), TXM-NEXAFS provides more analytically useful information per unit radiation damage2 phases and/or metallic electrodes when employing electron diffraction.Previous work by Kwon x phases that macroscopically assemble in a conductive localized area within a RRAM prototype that has been switched to a LRS. Presumably, this conductive spot can be regarded as a conductive filament. However, as reversibility of switching in this region cannot be proved after cutting the lamella for the experiment, dielectric breakdown or Joule heating could also be responsible for the formation of the reduced area. We prove that the conductive path is located right underneath a protrusion of the top electrode and it is composed by a reduced TiOx phase with a O/Ti ratio close to 1.37. Crystalline TiO2 rutile phase and an additional orthorhombic-like TiO2 phase regions were also identified in a localized area between the TiOx reduced phase and the top electrode, suggesting that the temperature due to Joule heating increases up to 1000 K in this areax reduced phase was also identified in both the top and bottom Pt electrodesThis technique has allowed us to spatially resolve distinct TiOx(50 nm)/Pt(30 nm) stacks (2 (x film was determined to be slightly reduced by X-ray photoelectron spectroscopy (The studied prototypes are Cr(3 nm)/Pt(30 nm)/TiO) stacks , fabricatacks  and O 1 s (525–555 eV) energy range. Each stack, consisting of 1053 images over the Ti 2p energy range and 351 images over the O 1 s energy range, was carefully aligned using a cross-correlation iteration process until the image shift was less than ±0.6 pixels (±3 nm) across the entire energy range (see Methods). After conversion of all stacks into optical density (absorbance), a detailed analysis involving extraction of the X-ray absorption spectra of specific regions of interest and conversion to component maps using spectral fitting or multivariate statistical analysis procedures was performed19For each of the two cases, PRI and LRS, two sequences (stacks) of X-ray images were acquired at closely spaced photon energies using a transmission X-ray microscope, in the Ti 2p edge) and 465 eV (on the strongest Ti 2p absorption peak) are presented in i.e. Si wafer, SiO2, Pt bottom electrode (BE), TiOx active film and Pt top electrode (TE) can be clearly distinguished in x active layer appears dark as Ti does not absorb at this energy. At 465 eV, the TiOx layer appears bright due to the strong absorption of Ti (p and at the O 1 s were then extracted from the TiOx film area. Two representative spectra are reported in p spectrum is complex but at a coarse level can be considered to consist of four main peaks (two doublets). The first doublet (2p3/2) occurs within 457–462 eV while the second doublet (2p1/2) appears in the range 462–468 eV p spectrum exhibits distinct features for the different polymorphs phase of crystalline TiO2 and for amorphous TiO22 are quite broad due to structural disorder caused by a range of bond angles and lengths, in contrast to spectra of crystalline samples which are characterized by sharp peaks. The Ti 2p spectrum shown in x22x thin film prepared via reactive sputtering and indicates damage/crystallisation was not caused by the sample preparation process. In agreement with our observations at the Ti 2p, the O 1 s spectrum shown in x film is amorphous all along the cross-section.X-ray images of the lamellae for the PRI case at 450 eV  and appear unaffected. In the defect region, the BE remains unaffected whereas the TE is discontinuous and delaminated from the TiOx film. At 450 eV, the Ti containing regions cannot be distinguished from void zones as both appear dark. By examining the Ti 2p image sequence at different energies, we can spatially resolve the location of regions containing Ti species as the contrast of the image changes in correspondence with changes in the Ti absorption peak intensities. The image at 458 eV  Ti containing regions, . These observations are consistent with the X-ray coloured image shown in The X-ray cross-section image of the LRS case recorded in the region underneath the TE protrusion at the energy of 450 eV is reported in t 458 eV  shows hix phase. 6 regions of interest (ROIs) were identified, indicated in p and O 1 s X-ray absorption spectra extracted from each of these localized regions are reported in p spectral shapes clearly indicating significant changes in the chemical structure of TiOx. Peak splitting and changes in relative intensities of the Ti 2p peaks can be attributed to changes in symmetry around Ti. In particular, the  orbitals at the Ti 2p point directly towards the 2p orbitals of the surrounding O and are therefore very sensitive to the titanium local environment  peak compared to that of the  peakp3/2, eg) peak around 460 eV; all these features indicate a high structural disorder as expected for amorphous TiOx222. A notable finding is the splitting of the  peak, which indicates distortion of the TiO6 octahedra2 (see Spectra extracted from undamaged areas of the film far away (ROI_1) and close (ROI_2) to the identified defect, are similar to each other. They show broad and smooth spectral features, higher intensity of the  profile has been reported for orthorhombic-like phases such as TiO2-II2-II phase reported inp3/2, eg) peak are different, being higher for the peak at higher energy. This is typical of TiO2 rutile phase which has tetragonal distortion of the TiO6 octahedra2231p1/2 peak is 2.4 eVp3/2, eg) peak is opposite to that in rutile, being the intensity higher for the peak at lower energy. This profile is typical of anatase. Moreover, the crystal-field splitting of the 2p1/2 peak is 1.9 eV, lower than the one observed in rutile, and typical of the anatase phase302-IId electrons and the 2p core holed partial density of statesIn the Ti 2p3/2, t2g) peak in the Ti 2p spectrum extracted from ROI_5 is lower than that in the spectrum from ROI_3, ROI_4 and ROI_6, indicating lower local symmetry at point 5. The low energy shoulder around 456 eV is consistent with the presence of a partially reduced phase containing Ti3+13p3/2 and 2p1/2 peak to valley ratio  show broad and smooth spectral features typical of amorphous TiOxs, t2g) and  peaks is shallow compared to the spectra of regions from ROI_3 to ROI_6 and their energy difference, which is often used to evaluate crystal field splitting, is smaller and typical of disordered materials (2.0 eV)g and t2g peaks appear more defined and intense. The energy gap between eg and t2g in ROI_4 and ROI_6 is ~3.0 eV, a value reported for both anatase and rutile phasesThe corresponding O 1 x phases across the LRS case, the aligned Ti 2p and O 1 s image sequences on an optical density scale were converted to chemical components maps by means of singular value decomposition (SVD) proceduresp  and in the two thinner filamentary regions connecting TE and BE on the left side of the protrusion (A and B).In order to better visualise the chemical distribution of the main TiOceduresp  and O 1 uresp  and in the regions A and B. As reduced TiOx has increased conductivity compared to the amorphous state, this is a direct observation of the area responsible for the LRS state of the device. Our observations are further validated by appending the Ti 2p and O 1 s stacks of the same ROI area  cross-section was obtained by adding a third component in addition to amorphous and reduced TiOx  were performed before analysis  and O 1 s (525–555 eV) X-ray absorption spectra. Our results demonstrate that the conductive localized region is composed by a reduced TiOx phase with a O/Ti ratio close to 1.37 and that it is located right underneath a protrusion of the top electrode. Crystalline TiO2 rutile phase and an additional orthorhombic-like TiO2 phase regions were identified for the first time, in a localized area between the TiOx reduced phase and the top electrode of the device. This provides the first experimental indication that the temperature in the core of the conductive region increases up to 1000 K. A Ti-containing phase similar to the TiOx reduced phase was also identified in both the top and bottom Pt electrodes, giving evidence of Ti diffusion into the Pt contact electrodes due to the switching. This technique can be adapted for other RRAM materials, such as NiO2O2By recording a sequence of X-ray images of the cross-section of the TiOi 2p 450–5 eV and x/Pt based memristor devices were fabricated on an oxidised (200 nm SiO2) 6-inch Si wafer. The bottom electrode (BE) and top electrode (TE) were fabricated using conventional optical lithography, electron-beam evaporation followed by a lift-off process, with BE and TE composed of Cr/Pt (3 nm/30 nm) and Pt (30 nm) successively. The Cr film in the BE served as adhesive layer for Pt. A 50 nm TiOx layer was then deposited using reactive sputtering from a Ti metal target with the following settings: 8 sccm O2, 35 sccm Ar, 2 kW at the cathode, and 15 sccm O2 and 2 kW at the additional plasma source.The Cr/Pt/TiOad hoc basis. All sweeping was performed using a Keithley 4200 electrical characterisation instrument.Electrical biasing of the devices has been carried out through voltage sweeping in order to assess or modify their resistive states as required (2 V maximum). Passive resistive state assessment of the Device Under Test (DUT) was carried out via low voltage, non-invasive voltage sweeps whilst resistive switching was induced through the application of more aggressive, high voltage sweeps. All voltage biasing was carried out under current compliance protection. The compliance current level was determined on A dual-beam focused ion beam/scanning electron microscope system (Zeiss NVision 40 FIB/FEGSEM) equipped with a gas injection system (GIS) was used to record SEM images and for cutting FIB cross-sections. SEM images were recorded at an accelerating voltage of 5 kV. Prior to performing FIB cross-sections, an electron beam-induced tungsten protective layer was deposited on the top of the electrodes in order to minimize damage caused by the gallium ions in the subsequent ion beam-induced tungsten deposition step. After extraction, the thickness of the lamella is further decreased to allow X-ray transmission by low energy ion polishing at a low incident angle until a thickness of 40–70 nm is achieved.The TXM-NEXAFS study was performed at the undulator beamline U41-FSGM at the BESSY II electron storage ring operated by the Helmholtz-Zentrum Berlin, Germany. The beamline itself and also the environment of the sample is kept under high vacuum conditions. The optical design of TXM has a spatial resolution of 25 nm and spectral resolution E/ΔE = 10000p images were acquired from 450 to 485 eV in 0.1 eV steps of photon energy. In order to increase the signal to noise ratio of Ti 2p spectra, three separate images were acquired for each photon energy giving a total of 1053 images. The O 1 s images were acquired from 525 to 540 eV in 0.1 eV steps and from 540 to 555 eV in 0.2 eV steps giving a total of 351 images. Images size is 1340 × 1300 pixels size corresponding to 5 nm per pixel. Each image was taken with the sample at the proper focus position to optimize spatial resolutiono for each stack was obtained from an internal region of each stack (off the FIB section) and used to convert the aligned stack from transmission to optical density (OD = −log[I(E)/Io(E)]). Ti 2p and O 1 s spectra were then extracted from the stack. Spectra can be obtained from regions as small as the spatial resolution of the microscope (25 nm). Data analysis was performed using software aXis2000Samples alignment was checked using a test specimen with defined distances between structures employing the same sample holder used for the actual experiment. The alignment along the beam direction was done by having the correct focus for each photon energy. The Ti 2AFM maps were acquired with a MultiMode Nanoscope V AFM (Veeco Metrology Group) in contact mode using Pt/Ir coated Si tips with a cantilever spring constant of 0.2 N/m and nominal radius of 12 nm .How to cite this article: Carta, D. et al. Spatially resolved TiOx phases in switched RRAM devices using soft X-ray spectromicroscopy. Sci. Rep.6, 21525; doi: 10.1038/srep21525 (2016).


Thereby we show how the contact of the nm-sized scanning probe tip results in a two-dimensional strain field with μm dimensions in the suspended membrane. Our combined AFM/Raman approach thus adds to the critically required instrumental toolbox towards nanoscale strain engineering of two-dimensional materials.We demonstrate the use of combined simultaneous atomic force microscopy (AFM) and laterally resolved Raman spectroscopy to study the strain distribution around highly localised deformations in suspended two-dimensional materials. Using the AFM tip as a nanoindentation probe, we induce localised strain in suspended few-layer graphene, which we adopt as a two-dimensional membrane model system. Concurrently, we visualise the strain distribution under and around the AFM tip  We use the AFM tip as a nanoindentation probe to controllably induce localised strain in the suspended FLG in the elastic regime, which we visualise using hyperspectral Raman mapping 2 covered Si wafers and then use N-Methyl-2-pyrrolidone (NMP) dissolvable plastic transmission electron microscopy (TEM) grids (Quantifoil) as intermediate carriers to deterministically transfer appropriately sized FLG flakes onto the holey SiN/Si chips. See ref. 2/Si substrate474714in front of the cantilever at an angle of ~128° A NT-MDT NTEGRA Spectra coupled AFM-Raman spectrometer is used in our experiments, where the AFM uses a fixed AFM probe  combined with a moveable piezo tube scanner onto which the sample is loaded. The piezo tube allows movement of the sample laterally and vertically under the AFM tip. The laser spot for Raman spectroscopy is independently moveable laterally on the sample surface via a mirror mounted onto a second piezo tube in the optical beam path of the confocal Raman spectrometer , the force applied on the FLG was systematically altered via controlling the cantilever deflection set-point  of the AFM feedback loop (where Raman maps of unstrained suspended FLG were measured with the AFM probe removed). To quantify the force applied on the membrane, measurements of cantilever deflection versus vertical sample piezo movement were obtained on the FLG membrane, as well as on the rigid SiN/Si chips, and recalculated to force-indentation curves 5354. The5314141657ωG) for four different forces applied by the AFM tip  are shown. In ωD2) are presented. The plotted Raman frequencies were obtained by fitting the G and 2D regions of the hyperspectral Raman maps’ individual spectra (examples in −1) and 2D (maximum downshift of −24 cm−1) evolves right under the position of the AFM tip (c.f. the Si intensity map in −1 and 2D to −79 cm−1) and also in spatial extent to a diameter of ~7 μm  is also well reflected in the full Raman spectra at selected positions across the sample, as shown in 00 nN is −1 for the non-loaded case to a maximum of ~90 cm−1 under ~6300 nN. After removal of the load, the 2D width profiles revert to their initial values. Such widening of the 2D width upon the application of stress has been reported previously for monolayer graphene35ωG) and 2D bands (∂ωD2) and the change in strain (∂ε) is reported3132ωG/∂ε [cm−1/% strain] and ∂ωD2/∂ε [cm−1/% strain]34ωD2/∂ε relations vary between −27 cm−1/% to −83 cm−1/% for uniaxial strain3132−1/% to −203 cm−1/% for biaxial strain3234−1/% for a radial geometryωG/∂ε for monolayer graphene, where additionally peak splitting into G− and G+ has to be considered, which is absent for FLG32ωD2/∂ε of −22 cm−1/% and ∂ωG/∂ε cm−1/% of −12 cm−1/% were reportedωD2/∂ε of −154 cm−1/% for biaxial strain was indicatedestimation of strain a ∂ωD2/∂ε of −140 cm−1/% from ref. −1/%) from ref. relative spatial extent of the localised strain distribution which is unaffected by the selection of the proportionality constants.Beyond qualitative observations, the magnitude of the here measured G and 2D downshifts in frequency can be used to obtain a quantitative estimation of the magnitude of strain in our measurements. Commonly a linear relation between the frequency shifts of the G . r(−2/3)-dependence . ExtractedωD2/∂ε) and thus validates that our combined AFM-Raman approach can also be used to measure membrane strain in a quantitative manner.To assess the validity of our strain distribution measurements, we compare our experimental findings with the results from an analytical model for the radial and circumferential strain distribution in a membrane from a point deformation. As discussed in ref. −1 would be expected from thermal effects−1. Another possibility requiring consideration is laser absorption in the AFM tip resulting in laser induced localised heating of the AFM tip and thus indirect heating of the FLG when in contact with the AFM tip. We note however that the frequencies of the first order Si Raman signal at the location of the AFM tip compared to at the location of the macroscopic SiN/Si support show negligible variation. Given that macroscopic supports under our Raman conditions show negligible heatingOne possible complication to our Raman spectroscopy-AFM based methodology needing consideration is laser induced heating of the FLG during our measurements626264Another possible complication to our analysis of AFM tip load induced strain distribution via Raman peak shifts arises from the dependence of graphene Raman peak shifts also on charge transfer dopingupshift of the G band45−1 was reporteddownshifted G band also in central area of tip contact  as intermediate carriers to deterministically transfer the FLG flakes onto the holey SiN/Si chips2/Si substrate47x,y,z-moveable piezo tube scanner under the fixed AFM probe, which has the tip emanating in front of the cantilever at an angle of ~128° to allow visual access to the sample-tip contact . Independent of the AFM control, the Raman laser spot (473 nm) is laterally x,y-moveable on the sample surface via a mirror mounted onto a second piezo tube in the laser beam path. This allows to perform AFM measurements (such as nanoindentation) on an area of interest while simultaneously and independently rastering the Raman laser spot over the same area of interest to obtain hyperspectral Raman maps in situ during the AFM measurements.Suspended FLG is prepared over single 12 μm × 12 μm holes in holey SiN-covered Si chips (SiMPore) by mechanical exfoliation from HOPG. HOPG is first exfoliated onto SiOHow to cite this article: Elibol, K. et al. Visualising the strain distribution in suspended two-dimensional materials under local deformation. Sci. Rep.6, 28485; doi: 10.1038/srep28485 (2016).


However, postmortem studies have historically demonstrated the fallibility of relying on clinical phenotype and in differentiating IPD from ‘Parkinson’s plus’ conditions such as multiple system atrophy (MSA). An in vivo accessible peripheral biomarker for IPD and MSA would, therefore, be of great value to direct therapy and more accurate patient counselling.Parkinson’s disease is one of the most common neurodegenerative disorders with a worldwide distribution. It has variable and complex phenotype with motor and non-motor manifestations, causes considerable psychosocial morbidity and consumes significant health care resources. Changing population demographics, and in particular, an ageing population in western societies is likely to result in gradually increasing prevalence. Despite significant advances in the understanding of disease pathology, the diagnosis of idiopathic Parkinson’s disease (IPD) remains largely clinical, with discriminatory investigations such as Ioflupane  which has recently been identified within dermal nerve fibres in IPD. In the first paper, the authors examine the utility of p-α-Syn as a biomarker for diagnosis of IPD, the second looks at p-α-Syn as a biomarker for MSA and the third at using p-α-Syn to differentiate IPD from MSA.123I]-metaiodobenzylguanidine), 20 patients with parkinsonism (PAR) of different pathogenesis assumed not to have p-α-Syn deposits  and 30 age-matched healthy controls were recruited for the study.This paper focuses on two hypotheses: whether p-α-Syn deposits in skin nerve fibres represent a useful biomarker for IPD and whether there is a link between these deposits and the underlying pathogenesis of peripheral neuropathy associated with IPD. The latter is based on experimental data showing that an accumulation of p-α-Syn deposits leads to neuron death. Twenty-one patients with well-characterised IPD  and distal sites . A second biopsy was also taken to assess variability of p-α-Syn expression from all areas in IPD patients, but only at the cervical area in the two other groups. No p-α-Syn deposits were identified in skin samples taken from the control or PAR groups. In contrast, when both skin samples were considered from the cervical area in the IPD group all patients were positive for p-α-Syn deposits. However, samples from thigh and distal leg sites in IPD patients were positive in only 52 and 24 %, respectively.In the IPD group, p-α-Syn deposits showed a significant indirect correlation with leg epidermal innervation. Deposits were not correlated with age, disease duration, or Unified Parkinson’s Disease Rating Scale (UPDRS) score. The authors conclude that p-α-Syn deposits in proximal skin nerve fibres are a sensitive biomarker for the diagnosis of IPD and can help differentiate between other forms of parkinsonism. As a result of variable local expression, the highest sensitivity (100 %) was achieved by analysing two cervical skin samples. The authors also concluded that neuritic synuclein inclusions were correlated with a small fibre neuropathy, suggesting a possible direct role of p-α-Syn in peripheral nerve damage.Comment. The use of skin biopsy in the diagnosis of IPD is an intriguing prospect. It is easily performed and minimally invasive with few adverse effects. This study provides some compelling evidence for use of skin biopsy in the diagnosis of IPD, although clearly larger studies would be of value in validating its use in clinical practice. The mean disease duration in the IPD group was 13 years, but it may be interesting to explore this biomarker in early or preclinical disease which may have significant implications for guiding earlier therapeutic interventions. However, the insights provided into pathophysiology remain limited since p-α-Syn deposits did not appear to correlate with disease duration or sub-phenotype.Donadio V et al. (2014) Neurology 82:1362–1369.MSA also remains a largely clinical diagnosis with support from autonomic function tests and neuroradiology. Pathologically MSA is characterised by detection of α-Syn deposits in oligodendrocytes and neurons. In this study, Doppler et al. explore whether p-α-Syn deposits in dermal nerve fibres can be used as a biomarker in MSA and whether this could differentiate MSA from tauopathies and IPD. Twelve patients with probable MSA according to the Gilman criteria (6 where MSA was supported by neuroimaging and a further 6 without MRI support), 15 patients with presumed tauopathies , 30 patients with IPD (based on the UK Brain Bank criteria) and 39 healthy controls were examined.Five-millimetre punch skin biopsies were taken from the back (Th12) and distal and proximal leg. Serial sections were undertaken to determine sensitivity and one in ten sections were double stained. p-α-Syn within dermal nerve fibres was detected in 8/12 patients with a clinical diagnosis of MSA. No p-α-Syn immunoreactive nerve fibres were identified in patients with presumed tauopathies or normal controls (p < 0.05). The sensitivity of skin biopsies stained positively for p-α-Syn in MSA was 67 % and specificity for a diagnosis of MSA versus tauopathies/controls was 100 %. In the IPD group, p-α-Syn was detected in 20 out of 30 patients, resulting in a sensitivity of 67 %. Double-staining serial sections of all p-α-Syn-negative patients identified one further patient with MSA and two more patients with IPD, increasing sensitivity to 75 and 73 %, respectively.In MSA patients, p-α-Syn was predominantly deposited in somatosensory fibres of the subepidermal plexus, in contrast to IPD where autonomic fibres were predominantly affected. In the IPD group, proximal biopsy sites showed the highest detection rate, raising the possibility of antidromic spreading of p-α-Syn from ganglia to sensory nerve findings. The authors conclude that detection of p-α-Syn in cutaneous nerve fibres may support the diagnosis of a ‘synucleinopathy’ in contrast to tauopathies. They also suggest that p-α-Syn in somatosensory fibres is associated with MSA-parkinsonism, whereas autonomic fibres are involved in IPD.Comment. This study advocates the use of skin biopsy in distinguishing IPD from MSA and other forms of parkinsonism. No attempt was made to correlate p-α-Syn deposition to disease severity or duration. Once again, a larger study would be of value in validating these results and developing wider datasets before its use can be supported in clinical practice. Mean disease duration varied considerably between clinical groups  and future studies may consider matching for both age and diseased duration.Doppler MD et al. (2015) Mov Disord 30(12):1688–1692.Zange et al. addressed the hypothesis that p-α-Syn can be detected in skin sympathetic nerve fibres in Parkinson’s disease, but not MSA. This study included 10 patients with IPD (according to the UK Parkinson’s Disease Society Brain Bank criteria) and 10 patients with MSA (according to Gilman criteria). Clinical classification was further supported by nuclear imaging and autonomic reflex screening. Six patients with an essential tremor served as controls.Three-millimetre punch biopsies were taken from the forearm of the clinically more affected side. p-α-Syn deposits were detected in nerve fibres innervating skin autonomic structures in α-Syn-dependent neurodegenerative disorders and p-α-Syn deposition was limited to Parkinson’s disease. However, dermal nerve fibres of MSA and control patients were devoid of p-α-Syn. The authors conclude that as their study did not show involvement of postganglionic sympathetic skin nerve fibres in MSA , testing sympathetic skin nerve fibres for p-α-Syn deposits is of value in differentiating Parkinson’s disease from MSA. However, they suggest that further studies of a variety of disease durations are necessary to verify this. Their data support the finding of dermal denervation in Parkinson’s disease, occurring independently of age and correlating with the extent of intra-axonal p-α-Syn deposition.Comment. This study supports the use of skin biopsy to differentiate IPD from MSA, specifically looking at autonomic skin nerve fibres. However, perhaps the most relevant conclusion that can be drawn is that autonomic skin fibre biopsies can be used to differentiate IPD from ‘not IPD’. The Parkinson’s disease patients in this study had a mean disease duration of 5.85 years, compared to 4.10 years for the MSA patients which are relatively well matched, although mean age of the cohorts differed by almost 10 years. As in the previous two papers, this study also supports the relationship of dermal denervation to p-α-Syn deposition. It would clearly be of value to obtain longer term follow-up data on these cohorts including pathology if available, to verify diagnosis and pathological conclusions.Zange et al. (2015) Brain 138:2310–2321.


Respiratory rhythmogenesis is controlled by neural networks located in the brainstem. One area considered to be essential for generating the inspiratory phase of the respiratory rhythm is the preBötzinger complex (preBötC). Rhythmogenesis emerges from this network through the interplay between the activation of intrinsic cellular properties (pacemaker properties) and intercellular synaptic connections. Respiratory activity continuously changes under the impact of numerous modulatory substances depending on organismal needs and environmental conditions. The preBötC network has been shown to become active during the last third of gestation. But only little is known regarding the modulation of inspiratory rhythmicity at embryonic stages and even less on a possible role of pacemaker neurons in this functional flexibility during the prenatal period. By combining electrophysiology and calcium imaging performed on embryonic brainstem slice preparations, we provide evidence showing that embryonic inspiratory pacemaker neurons are already intrinsically sensitive to neuromodulation and external conditions  affecting respiratory network activity, suggesting a potential role of pacemaker neurons in mediating rhythm adaptation to modulatory stimuli in the embryo.Breathing is a rhythmic behavior that requires organized contractions of respiratory effector muscles. This behavior must adapt to constantly changing conditions in order to ensure homeostasis, proper body oxygenation, and CO Breathing is a rhythmic behavior that must be continuously expressed in order to ensure life. However, this vital function must also be constantly adaptable to changing external and internal conditions to satisfy the organism's complex metabolic needs. Thus, the central command for respiration is under the influence of multiple neuromodulatory systems arising from different parts of the central nervous system, which participate in maintaining the efficacy and adaptability of respiratory motor output.μ opioid peptide receptors μOR, has been demonstrated [Respiratory function is controlled by neuronal network assemblages located in the brainstem. Several studies have shown that the neural network of the preBötzinger complex (preBötC) that generates inspiration plays a critical role in respiratory rhythm generation –5. This nstrated .μ opioid receptor agonist) [The developmental emergence of the rhythmogenic neural circuits responsible for respiration occurs during the last third of gestation. Typical anatomical and functional characteristics of the postnatal preBötC network are detectable from embryonic day (E) 15.5 and E17.5 in mouse and rat, respectively –21. At tagonist) , 21. Howagonist) , 22, andIn the present study, therefore, we aimed to investigate whether respiratory pacemaker neurons could be involved in the adaptability of the mouse embryonic respiratory network activity to the influence of modulatory signals. We present results on several such processes affecting the respiratory network activity at prenatal stages. Specifically, we show that the embryonic respiratory rhythm can be modulated by exposure to changes in temperature and also by the peptides SP and DAMGO. These two substances were chosen because they are well known respiration neuromodulators (see above) and also because they exert opposing effects on the postnatal respiratory network, thus allowing the investigation of a large range of activity changes. We find that some inspiratory network neurons identified as pacemaker cells are intrinsically sensitive to both of these modulatory peptides as well as to temperature. The effect on respiratory rhythm cycle frequency observed at the network level under these different extrinsic influences might therefore be mediated, at least in part, through changes specific to pacemaker neuron activity within the embryonic preBötC network.All experiments were conducted in accordance with guidelines issued by the European and French National legislations on animal experimentation and have also been approved by the Bordeaux University ethics committee. Pregnant OF1 mice were obtained from our local animal facility, with the presence of a vaginal plug the day after mating being considered as embryonic day (E) 0.5 in gestation. Pregnant females were isolated in a separate cage until their experimental use.2/5% CO2) in artificial cerebrospinal fluid (aCSF) with the following composition (in mM): 120 NaCl, 8 KCl, 1.26 CaCl2, 1.5 MgCl2, 21 NaHCO3, 0.58 NaH2PO4, and 30 glucose, pH 7.4. The first step of the dissection consisted of isolating the hindbrain by making a rostral transverse section at the anterior limit of the rhombencephalon and a caudal section at the level of the rostral part of the spinal cord. After isolation, hindbrains were embedded in a low melting point agar block and serially sectioned plane in the transverse in a rostral to caudal direction using a vibratome (Leica). Transverse sections were cut until the axial level of the facial motor nucleus was reached. Past its posterior limit, an additional 250/300 μm thick slice was removed in order to reach the anterior axial level of the 450 μm slice in which the preBötC network was isolated. Slice preparations were then placed in a recording chamber and continuously superfused with oxygenated aCSF maintained at 30°C. The slices were positioned rostral side up and maintained in place by a grid to avoid any movement during optical and electrophysiological recording sessions. We systematically waited for 30 min before starting any recording procedures. The temperature of the aCSF superfusant in the chamber was set using a PTC-10 temperature controller  and a HPT-2 heated perfusion tube .Brainstem transverse slices isolating the preBötC network were obtained from mouse embryos harvested during the developmental period between E16.5 and E18.5. The methods used for preparing slices have been previously described in detail , 20. BriExtracellular recordings of local population activity were made using an aCSF-filled glass macroelectrode fabricated from filamented borosilicate glass tubing  and positioned at the surface of the slice in the preBötC area on one side. This electrode was connected through a silver wire to a high-gain amplifier . The signals were filtered (bandwidth 3 Hz–3 kHz), integrated with a time constant of 100 ms using an electronic filter , recorded, and stored on a computer via a Digidata 1322 interface  and analyzed offline using PClamp10 software (Molecular Devices).μM; ThermoFisher, France). After this incubation period, preparations were placed in the recording chamber and an additional 30 min period was followed in order to let the preparation recover and stabilize in oxygenated aCSF at 30°C. Fluorescent images were captured from the rostral surface of the slice through a FN1 upright microscope (Nikon) equipped with a 40x objective, a standard epifluorescent illumination system, and a fluorescein filter, coupled to either an Exiblue camera  or an Andor camera  operating with an exposure time of 100 ms in overlapping exposure and readout mode. Images were acquired over periods lasting 120 s and analyzed using customized software developed by Dr. Mellen and Tuong [F/F) compared to silent periods.Slice preparations were incubated in the dark at room temperature for 40 min in oxygenated aCSF containing the cell-permeable calcium indicator dye Calcium Green-1 AM , 10 μM DL-2-amino-5-phosphonovaleric acid (AP5), 1 μM strychnine, and 10 μM bicuculline .The pharmacological cocktail used to synaptically isolate pacemaker neurons contained 20 μ opioid receptor agonist D-Ala2-N-Me-Phe4-Glycol5-enkephalin  were dissolved in aCSF and bath-applied at their final concentration of 0.5 μM and 0.25 μM, respectively. A single modulatory drug was applied per preparation in order to avoid cross effects. Preparations were exposed to a drug for a 15 min period and its effects on respiratory rhythm cycle frequency were analyzed at the end of this period using Clampfit software (Molecular Devices). The reversal of any observed effects was tested for each drug after a 30 min period wash-out during which fresh aCSF was superfused in the recording chamber.Substance P  and the t tests with a Mann-Whitney Rank Sum test or ANOVA tests were used to assess differences (taken to be significant at P < 0.05) between values in the presence of the drugs and controls.Values are given as mean ± SEM. Student's μm using a cryostat (Leica). Sections were incubated for 1 h30 in 1% BSA and 0.3% Triton X-100, followed by affinity-purified primary antibodies rabbit anti-NK1R  and goat anti-ChaT  overnight at room temperature. After multiple rinsing in PBS, sections were exposed for 2 h to the secondary antibodies Alexa Fluor 488-conjugated donkey anti-goat IgG  and Alexa Fluor 568-conjugated donkey anti-rabbit IgG . After rinsing with PBS, sections were cover-slipped, mounted in Vectashield, and observed on a Leica microscope. Control experiments in which the primary antibodies were replaced by normal serum were also conducted to ensure lack of labeling and antibody specificity.Isolated brainstem preparations were fixed for 3 h in 4% paraformaldehyde, cryoprotected in 20% PBS-sucrose overnight, and then cryosectioned at 30 μ opioid peptide agonist DAMGO known to depress newborn respiration [μM SP induced a significant cycle frequency increase from 10 ± 0.9 bursts/min in control conditions to 17.8 ± 1.5 bursts/min in the presence of SP (n = 20). This effect was partially reversible since the mean cycle frequency returned to 12.6 ± 1.7 bursts/min after 30 min wash-out with normal aCSF. Moreover, since SP is known to bind to neurokinin 1 receptors (NK1Rs), we verified that NK1Rs were significantly expressed in the preBötC region of our embryonic preparations. Immunostaining for NK1R revealed that the respiratory network, located ventral to the nucleus ambiguus which is itself identifiable by its choline acetyltransferase (ChaT) and NK1R coimmunoreactivity, was indeed positive for NK1R  , the opioid agonist even caused a complete suppression of the spontaneous respiratory rhythm  of the preBötC network . TherefoμM SP on the frequency of somatic fluorescent variations  in the additional presence of SP (With a test population of 24 cells (from 18 preparations) that were coactive with the preBötC network in control conditions and that remained rhythmogenic after synaptic isolation, we investigated their sensitivity to SP by assessing the effects of bath-applied 0.5 riations . As illuce of SP . We alsoμM). Among them, 20 showed a significant decrease in calcium transient frequency in the presence of the μ opioid receptor agonist  under DAMGO  in this experimental series were rhythmically active in phase with the whole network in control conditions and at a temperature of 28°C. Also, when synaptically isolated at this temperature, most of these pacemaker cells remained rhythmically active at their own inherent frequency (n = 19/23), while 4 of them ceased to generate any detectable activity (but see below). Subsequently, increasing the temperature from 28° to 32°C induced four different responses. First, rhythmic activity frequency increased for 14 of the pacemaker neurons from 3.7 ± 0.7 bursts/min at 28°C to 9.3 ± 1.3 bursts/min  the neuromodulatory processes targeting the respiratory central command are already functional at prenatal stages and (2) that respiratory pacemaker neurons play an important role in mediating these effects. Perturbations in the prenatal development of such modulatory actions might therefore be responsible for inappropriate postnatal breathing behavior, possibly leading to respiratory deficits with potential dramatic consequences as found in several human diseases. Therefore, a better understanding of these regulating processes might have clinical relevance, such as for understanding the Rett and Prader-Willy syndromes or Sudden Infant Death Syndrome, where abnormalities in respiratory neuromodulation mechanisms and temperature control are suspected –46.


Pagodroma nivea) from Adélie Land, Antarctica. We predicted that 1) blood Hg would increase with advancing age as a consequence of bio-accumulation; and that 2) increasing blood Hg would be related to decreased concentrations of LH in the most Hg-contaminated individuals. Hg concentrations were higher in females than in males (p<0.001), and contrary to our prediction, decreased with advancing age in males (p = 0.009) and tended to do so in females (p = 0.06). The analysis of stable isotopes (δ13C and δ15N) suggested that this unexpected pattern could originate from age and sex-related variations in trophic niche, and hence Hg exposure. Regarding LH, our prediction was only supported in young birds (≤23 years) where baseline LH was inversely correlated with Hg concentrations (p = 0.04). Hg burden did not predict baseline LH or GnRH-induced LH in birds that were more than 23 years old. These results show that age and contaminants may interfere with major endocrine mechanisms and, together with other recent studies, support the view that Hg could be connected to LH secretion and could then impair the fitness of long-lived birds.Seabirds, as long-lived top predators, accumulate contaminants such as mercury (Hg), an established endocrine disruptor. In long lived species hormonal secretion varies with age; therefore, Hg-induced endocrine disruption may be exacerbated in some age classes. Here we investigated relationships between blood total Hg and luteinizing hormone , in pre-laying known-age (11–45 years old) snow petrels ( Pagodroma nivea, a small (≈400 g) but very long-lived Antarctic seabird (≈50 years) 13C and δ15N values indicating the birds’ foraging habitats and trophic position, respectively Mercury (Hg) is a ubiquitous contaminant, present in aquatic biota with elevated concentrations in fish, some mammals and seabirds Rissa tridactyla, a recent study has reported that Hg could interfere with the secretion of luteinizing hormone (LH) Taking age into consideration is crucial when considering the consequences of Hg contamination. Indeed, several studies on vertebrates have reported breeding impairment from Hg including suppression of reproduction, reduced clutch size, poor breeding performances or unusual courtship behavior Animals were cared for in accordance with the guidelines of the ethics committee of the Institut Polaire Français Paul Emile Victor (IPEV) that specifically approved this study .−1 body mass in 1 ml of 0.9% saline solution, as validated in this species t = 10 − LHt = 0)/LHt = 0] and LH changes from 10 to 30 min post-GnRH injection [(LHt = 30 − LHt = 10)/LHt = 10] were used as indicators of different individuals’ abilities to release LH. Blood samples were centrifuged and plasma (for LH assay) and red blood cells  were stored at −20°C. Adults were weighted to the nearest 2 g using a spring balance and their skull length (head+bill) was measured to the nearest 0.5 mm using a caliper. In snow petrels, skull length appears to be a reliable measurement of the overall size of a bird The study was conducted on Adélie land , Antarctica. Many snow petrels were of known age, because chicks have been banded each year since 1964 −1). Hg concentrations are expressed in µg⋅g−1 dry weight (dw).Total Hg was measured as described in 13C values of seabirds indicating their foraging habitats 15N values increasing with trophic level 2 for δ13C and δ15N, respectively. Replicate measurements of internal laboratory standards (acetanilide) indicate measurement errors <0.10 ‰ for both δ13C and δ15N values. The C:N mass ratios of the samples were calculated as the ratio between the mass percentages in carbon and nitrogen. The consistently low C:N values of blood cells verified that low lipid content did not necessitate lipid extraction The isotopic niche of snow petrels was used as a proxy of their trophic niche during the pre-breeding period, as blood cells is a metabolically active tissue that integrates periods of weeks before sampling −1 and the intra-assay coefficient of variation was 6.2% (three duplicates). In 4 birds plasma levels were too low to measure LH.Plasma LH concentrations were assayed by radioimmunoassay as validated for snow petrels 13C and δ15N, scaled mass index, and interactions with sex, (ii) blood δ13C and δ15N values in relation to age, age2, scaled mass index and interactions with sex (linear and/or quadratic). To address the interaction between LH, Hg and age, the dataset was split into two groups using a recently developed Bayesian approach  and birds that were more than 23 (N = 14) hereafter ‘≤23 y.o.’ and ‘>23 y.o.’, respectively), blood Hg concentrations were significantly higher in ‘≤23 y.o’ birds than in the ‘>23 y.o.’ ones . Finally (iii) absolute plasma baseline LH concentrations, LH changes from 0 to 10 minutes, and changes from 10 to 30 minutes were tested as functions of blood Hg concentration (linear) in relation to age classes (‘≤23 y.o.’ and ‘>23 y.o.’). We performed all our model selection starting from the most parameterized model that included all the variables/factors and their interactions and we removed step by step the non-significant interactions, variables or factors. Selected models were checked for assumptions and values were log-transformed when necessary, we used GLM with normal error and identity link function to explain LH concentrations in relation with Hg and interaction with sex. Values are means ± SD.All analyses were performed using R v. 2.13.1. We used GLM with normal error and identity link function to explain: (i) blood Hg concentration in relation to age, blood δ−1 dw During the pre-laying period, blood Hg concentrations were not related to sampling date  and were higher in females than in males .In snow petrels dw blood was equivalent to 35.5±0.3% of ww blood and Hg concentration averaged 2.7±1.1  µg⋅gin males Table 1.−1) significantly increased (10 min: 11.5±0.8 ng⋅ml−1), then decreased , time as factor: F2,61 = 12.57, p<0.001) these LH variations were not different between sexes or age classes (≤23 y.o. versus >23 y.o.) . Absolute plasma LH concentrations  were significantly higher in males compared to females  and were not related to sampling date  or to scaled mass index . In birds that were ≤23 y.o., plasma baseline LH significantly decreased with increasing Hg concentration in blood  was positively related to blood δ13C values especially in females. According to the latitudinal δ13C gradient in oceanic waters of the Southern Ocean 13C values suggest the use of a high Antarctic habitat 13C values close to −23 ‰ depict warmer waters at more northern latitudes and the δ13C values close to −26 ‰ colder waters at more southern latitudes, but still within the Antarctic zone 13C values and Hg concentrations means that Hg might be more bioavailable for snow petrels at more northern latitudes .In pre-laying snow petrels, blood Hg concentrations were in the range of concentrations found in other species from the Southern ocean 15N values, was positively related to blood Hg concentration in females only. The enrichment of Hg with increasing trophic level has been described in food-webs and species communities 15N values was found in chicks but not in adults of great skuas Catharacta skua15N values suggests that self-feeding breeding adults fed from a krill- to a fish-based diet, depending on individuals. The higher blood δ15N values of males indicate they fed more on fish than females. However and unexpectedly despite their higher trophic level, males are less Hg contaminated than females, suggesting once more that females may feed in areas where Hg is more bioavailable. This is in agreement with the δ13C values-Hg concentration relationship. Alternatively, detoxification processes could be more efficient in males. Noticeably, their stable isotope values indicate that the most contaminated males fed on fish at more northern latitudes than the others, suggesting again a latitudinal effect on Hg availability Trophic position, as depicted by blood δ13C values) and age. This suggests that, compared to middle-aged birds, young and old individuals foraged at higher latitudes which may be more Hg contaminated. Isotopic signature of blood represents the isotopic niche during the last month preceding sampling Gavia immer) blood Hg concentrations of 2–3 µg⋅g−1 (ww) are considered at moderate risk to effects of Hg contamination −1 and 1.9 µg⋅g−1, for the most contaminated male and female respectively; these concentrations are close to the threshold level for moderate risk in common loons and snow petrels could maybe be more sensitive to lower Hg concentrations. However, in free-living birds, Hg has so far never been reported as affecting survival probabilities, even in highly contaminated areas Contrary to our prediction blood Hg concentration decreased with increasing age in pre-laying snow petrels, especially in males. The relationship between Hg and age in seabirds is poorly documented and often contradictory. For example, in organs involved in storage and detoxification as the liver, Hg concentration may decrease, increase, with advancing age or may not be related to age −1 against 2.0±0.5 µg⋅g−1, respectively) and LH secretion post-GnRH injection was less pronounced in snow petrels compared to kittiwakes . It is always difficult to compare different species as hazardous effects of Hg could be highly species-specific −1 dw, respectively) however south polar skuas suffered from higher Hg-induced breeding failure than their closely relative In the present study, plasma baseline LH decreased with increasing blood Hg in individuals until 23 y.o., which were those that bore the higher concentrations of blood Hg. This negative relationship between plasma baseline LH and blood Hg could either originate from a Hg-related dysfunction of the pituitary (i.e. an inability to produce enough LH) and/or of the hypothalamus (i.e. a lack of endogenous GnRH production). Since LH changes (0–10 min and 10–30 min) following an exogenous input of GnRH were not related to Hg, this means that Hg does not disrupt the capability of the pituitary to release LH in birds until 23 y.o.: their pituitary is thus functional. This rather suggests, as found in black-legged kittiwakes, of a problem originating from the hypothalamus, which may not release enough GnRH in response to high Hg exposure This study shows the importance of considering age in eco-toxicological studies. We reported an unexpected negative relationship between age and blood Hg concentrations in adult snow petrels and the use of stable isotopes suggests that this pattern could originate from an age and sex-related variation in late wintering areas, and hence Hg bioavailability. It is now possible to map migration and winter distribution of relatively small birds such as snow petrels via miniature loggers (geolocation sensors, GLS, e.g.


Plasmodium falciparum-infected erythrocytes in the brain and 2) coma with other underlying causes. Parasite sequestration in the brain is mediated by PfEMP1; a diverse parasite antigen that is inserted into the surface of infected erythrocytes and adheres to various host receptors. PfEMP1 sub-groups called “DC8” and “DC13” have been proposed to cause brain pathology through interactions with endothelial protein C receptor. To test this we profiled PfEMP1 gene expression in parasites from children with clinically defined cerebral malaria, who either had or did not have accompanying retinopathy. We found no evidence for an elevation of DC8 or DC13 PfEMP1 expression in children with retinopathy. However, the proportional expression of a broad subgroup of PfEMP1 called “group A” was elevated in retinopathy patients suggesting that these variants may play a role in the pathology of cerebral malaria. Interventions targeting group A PfEMP1 may be effective at reducing brain pathology.Retinopathy provides a window into the underlying pathology of life-threatening malarial coma , allowing differentiation between 1) coma caused by sequestration of  In children living in sub-Saharan Africa, severe malaria presents in three overlapping syndromes 23P. falciparum adhering to the endothelia of the brain microvasculature23578Autopsy studies of fatal malaria show large numbers of erythrocytes containing mature stages of P. falciparum erythrocyte membrane protein one (PfEMP1), is a diverse family of parasite proteins that are inserted into the surface of the IE. These molecules play a central role in cytoadhesion of IE to the endothelia of the microcirculationvar genes per parasite genomevar genes modifies the antigenic and binding properties of IEs17var genes have been proposed to express PfEMP1 with specificity to endothelial protein C receptor (EPCR)12var subsets and malaria retinopathy as a way of dissecting the relationship between parasite sequestration in the brain and disease pathology.Sequestration occurs as a result of interaction between IE surface ligands and receptors on the endothelial cells that make up the inner wall of the microvasculature  in 25/80(31.25%), and negative (CM-R–) in 55/80 (68.75%), which is similar to the original studyThe clinical features of the children included in this study were described in var expression, of which 52 (69.33%) were CM-R– and 23 (30.66%) were CM-R+. - group, though this was not statistically significant  but not Pfsir2b  was higher in the CM-R–group . This ra with RD  suggestivar transcript quantity used in the above analysis was calculated relative to the average expression of two reference metabolic genes, and does not provide information on the overall composition of the population of parasites causing infection. We therefore calculated the expression of each of the var gene subsets as proportion of total measured var transcript and explored the associations of these “proportional expression” values with retinopathy. Of the var subsets analyzed, proportional expression of group A var genes was positively associated with CM-R+ , we excluded transcript contributed by DC8, DC13 and gpA2 primers from the analysis. With this approach group A (gpA1) proportional expression remains positively associated with CM-R+ 1212th ang-2 . Insteadth ang-2 . Ang-2 wth ang-2 . When bovar genes, Pfsir2a, and Pfsir2b, and axillary temperature. PfHRP2, sICAM-1, ang-2, and 1/hematocrit was positively associated with factor 3, which explains 14% of the variation  in the brain4var expression profile of parasites from children with cerebral malaria with and without retinopathy (CM-R+ and CM-R–).Malarial retinopathy has been shown to be an important clinical marker of Parasites selected for binding on human brain endothelial cells were recently found to express predominantly a subset of group B and group A PfEMP1 containing domain cassettes 8 and 13 respectively17171812var transcript quantity that would support enrichment in DC8 or DC13 PfEMP1 in children with retinopathy. Overall there was either no difference between the two groups or, higher levels in the retinopathy negative (CM-R-) group  group . The posOverall, our result suggests an involvement of group A PfEMP1 in retinopathy. This PfEMP1 subset could potentially support parasite growth through strong cytoadhesive properties consistent with autopsy studies showing that cerebral malaria is associated with parasite sequestration in multiple organsvar genes and plasma ang-2 were independently associated with severe malaria suggesting that group A PfEMP1 do not play a direct role in widespread endothelial activationvar genes including DC8 and DC13  severe malaria and non-severe malaria192819Unlike previous studies, the comparison made in this study is between two groups of children with severe malaria who cannot be distinguished through their clinical symptoms. In previous studies var genes but overall lower var gene transcript quantity. We have suggested that, under the condition of reduced movement of blood in the microcirculation and congestion, parasites may be able to sequester without making the normal level of investment in the expression of PfEMP1.In summary our data suggests that retinopathy is associated with higher proportional expression of group A var expression analysis. Plasma PfHRP2 concentration was determined as described in Kariuki et al.This study is nested in a published study whose objective was to understand the value of malarial retinopathy in cerebral malariaP. falciparum malaria on a Giemsa stained blood-slide without presence of another cause of coma such as hypoglycemia or meningitisCerebral malaria was defined as admission with coma (Blantyre coma score≤2) and presence of var genes in qPCR19var genes (gpA1 and gpA2) were used to quantify group A var gene expression  here referred to as “transcript quantity” using the formula (Tus = 2(5-∆∆ct)) modified from lavstsen et al.var subset within each sample comparison, we used the formula (Tus = 2(5-∆ct))19. We assigned a zero Tu value if a reaction did not result in detectable amplification after 40 cycles of amplification, i.e. the Ct value was undetermined. Samples were excluded from the analysis if amplification of either of the two reference genes, i.e. seryl tRNA synthetase and fructose biphosphate aldolase could not be achieved.Patient samples were processed as described previously28pression . In addianalysed . Two houvar genes were measured with more than one primer, we used the median transcript quantity ) obtained with the various primers targeting group A or DC8 var genes to represent group A and DC8 var gene expressions, denoted as groupA_median and DC8_median respectively. That is groupA_median = ((gpA1 + gpA2)/2) and DC8_median = -max)/2. Since the primer dc8-3 is less specificvar subset, we summed the transcript quantity ) of all the var subsets analysed, i.e. sum var transcript = (groupA_median + dc13 + DC8_median + b1 + c2) or (gpA1 + dc13 + DC8_median_2 + b1 + c2). We then calculated the proportional contribution by each of the subsets. Mann-Whitney U test was used to assess the difference in var expression between retinopathy positive and negative groups.As group A and DC8 To test whether the relationship between proportional expression of group A and retinopathy is independent of anemia, we used three logistic regression models each predicting retinopathy using either 1) group A proportional transcript, or 2) admission hemoglobin, or 3) both as explanatory variables. Malaria retinopathy was a binary variable and was the outcome or dependent variable in all the three logistic regression models. Both group A proportional transcript and admission hemoglobin were normally distributed, and were entered into the logistic regression models as explanatory or independent variables.Was generated using Spearman’s rank correlation coefficient test. The exact p value was determined using the command di ‘r(p)’ in Stata.In this analysis factors with an Eigenvalue >1 was considered for further analysis. To optimize the factor loadings, we used promax rotation. Loadings >0.3 or < -0.3 were considered significant. We generated predicted factor scores for each individual (using the command “predict” in Stata) and used these generated factor scores as independent variables in a logistic regression analysis with retinopathy as the dependent/outcome variable.s values) data normally distributed before use in factor analysis we added 0.1 to all values (to eliminate zeros) and then log transformed all the resultant Tus values.To have the transcript quantity  Ethical Review Committee (under SSC 1131 and 1249), and written informed consent was obtained from parents/guardians of the study participants. The study methods were carried out in accordance with the approved guidelines.How to cite this article: Abdi, A. I. et al. Differential Plasmodium falciparum surface antigen expression among children with Malarial Retinopathy. Sci. Rep.5, 18034; doi: 10.1038/srep18034 (2015).


Vacancy-mediated climb models cannot account for the fast, direct coalescence of dislocation loops seen experimentally. An alternative mechanism, self climb, allows prismatic dislocation loops to move away from their glide surface via pipe diffusion around the loop perimeter, independent of any vacancy atmosphere. Despite the known importance of self climb, theoretical models require a typically unknown activation energy, hindering implementation in materials modeling. Here, extensive molecular statics calculations of pipe diffusion processes around irregular prismatic loops are used to map the energy landscape for self climb in iron and tungsten, finding a simple, material independent energy model after normalizing by the vacancy migration barrier. Kinetic Monte Carlo simulations yield a self climb activation energy of 2 (2.5) times the vacancy migration barrier for 1/2〈111〉 (〈100〉) dislocation loops. Dislocation dynamics simulations allowing self climb and glide show quantitative agreement with transmission electron microscopy observations of climbing prismatic loops in iron and tungsten, confirming that this novel form of vacancy-free climb is many orders of magnitude faster than what is predicted by traditional climb models. Self climb significantly influences the coarsening rate of defect networks, with important implications for post-irradiation annealing. Dislocation glide dominates plastic flow at low homologous temperatures, but confinement to a glide surface significantly restricts the evolution of a defect network25678The most widely studied atomic mechanism for climb transports mass through a pre-existing vacancy atmosphere, here referred to as vacancy mediated climb (VMC), with dislocations acting as perfect sources and sinks for vacancies23However, it has long been recognized567959510121351213R is the loop radius, a is the lattice parameter, β = 1/kBT, ν is an attempt frequency and ESC is a characteristic activation energy. As no vacancy atmosphere is required, self climb is suspected to be much faster than VMC as a factor ESC, the critical, rate controlling parameter in (1). In this paper, we use the results of over a hundred molecular statics barrier climbing calculations and kinetic Monte Carlo (KMC) simulations to explore the energy landscape for pipe diffusion around SIA loops. The pipe diffusion process in self climb effectively enables non-glide structural fluctuations of the loop, which enables thermally activated diffusive climb motion, with a characteristic activation energy ESC. Our main result is that the activation energy for self climb is given bywhere vacancy migration barrier and ν is taken as the attempt frequency for vacancy diffusion16ESC that find ESC ~ (0.4–0.7) Ef+Em over a wide range of materials51213where in situ transmission electron microscopy (TEM) observations of SIA loops in iron at 500 °C, where isolated SIA loops are seen to execute unbiased self climb diffusion perpendicular to their Burgers vector, whilst adjacent SIA loops directly coalesce through self climb, driven by elastic interaction forces. We also analyze recently published observations of SIA loop coalescence in tungstenThe fully parametrized mobility law is tested against direct a/2〈111〉 jumps for 1/2〈111〉 loops, and a/2〈111〉 or a〈100〉 jumps for 〈100〉 loops. As shown in E and migration barrier Em for a jumping atom can be accurately modeled simply by counting the number c of nearest SIA neighbors along the path, i.e. for each point on the path, x) is the Heaviside step function. For SIAs in the interior of an 1/2〈111〉 loops c = 6, whilst for 〈100〉 loops c = 4; classifying the SIAs on a loop perimeter in this manner allowed us to produce the distribution of migration pathways shown in As self climb is driven by pipe diffusion around the perimeter of SIA loops, we have simulated a large number (>100) of possible diffusion pathways for 1/2〈111〉 and 〈100〉 SIA loops in iron and tungsten (see methods). It was found that all relaxed pathways between metastable states could be approximated as c is the change in c between the initial and final configuration, σ = 1 for jumps along a/2〈111〉 , which typically overestimates Em, meaning (3) will give an lower bound on the self climb mobility.where Δ11〉 c.f.  and σ = 00〉 c.f. . The remOh) to vary the elastic environment of the jumping atom. For loop separations of more than ~5a we found variations in the energy barrier of less than 5%. As such close proximity was needed to produce observable changes we expect the self-climb energy landscape to be essentially unaffected by any surrounding defect network, even line dislocations which are not elastically neutral. The goal of the NEB calculations is to parametrize KMC simulations; an alternative approach to the simple model (3) used here would be to correlate all observed migration pathways for a given material with some detailed structural signature for the jumping atom involving second and third nearest neighbors, which could then be used to construct a large table of transition rates for use in KMCAs self climb is known to play an important role in prismatic loop coalescence, we also determined the effect of a nearby loop’s elastic field on the observed energy barriers by performing the same NEB calculations in the presence of another prismatic loop, rotating/inverting the loop of interest by a bcc symmetry operation , under the assumption of uncorrelated perimeter atoms we obtainAs the presence of a realistic external stress field (due to a nearby dislocation loop) was found to have only a weak influence over the measured pipe diffusion migration barriers, we can calculate the self climb velocity ter c.f. . The larMSC = βDSC gives DSC ∝ R−3 ~ N−3/2 is in agreement with our simulations (15which with the Einstein relation ulations  and simiα-Fe (RRR: 7900) and high-purity α-Fe , respectively. An isolated b = a[100] SIA loop of radius and perpendicular to its Burgers vector of [100]. We identify the perpendicular motion along [010] as a one dimensional projection of self climb in (100) planes. Through analysis of the mean squared displacement along [010]  and diffusivity (4), transmission electron microscopy (TEM) was used to observe the dynamics of both isolated and interacting SIA loops in ultra-high purity ng [010] , inset sR(0)〉 = 2.5 nm. An ensemble of 150 simulations were performed until less than 4 loops remained in the supercell, though the ensemble average behavior could be considered converged after approximately 50 simulations. In N(t) and average loop radius 〈R(t)〉, along with the predictions of the analytical VMC model, to give a measure of the expected coalescence times if VMC was the only active climb mechanism. Using the full-field elastic interaction energy given by Mura’s formulaESC is comparable to the inferred binding energy between dislocation loops and common impurities such as carbon or nitrogen , meaning that simulations which include impurity pinning effects should also account for self climb, as the timescales for the two processes are expected to be comparable. It is also possible that self climb could play a role in defect depinning process, a topic we leave for future study.We have derived and calculated a quantitative mobility law for self climb, by mapping out the energy landscape for pipe diffusion around a large number of randomly shaped prismatic dislocation loops in two different bcc metals which can then be explored in kinetic Monte Carlo simulations. Our results show that the activation energy for self climb is 2–2.5 times the vacancy migration barrier, which showed quantitative agreement with experimental observations of the same processes. Dislocation dynamics simulations, which account for self climb, show significantly accelerated prismatic loop coalescence rates at intermediate homologous temperatures, demonstrating that self climb is an essential component in models of microstructural evolution, even in regimes where vacancy-mediated climb can be neglected. Finally, we note that self climb activation energy et al.et al.a/2〈111〉, a〈100〉 or The pipe diffusion barrier calculations used the LAMMPS molecular simulation packagelibWe used the lattice KMC code KMC−6 Pa (ultra-high purity α-Fe) and at 1073 K for 2 hours under a hydrogen atmosphere at 1 atm (high purity α-Fe) to reduce dislocation density and remove any residual stress before electro-polishing. In the TEM-visible regions, the residual-dislocation density was less than 109 m−2, allowing the observation of individual loops with negligible effect from the stress field of residual dislocations. Loops were introduced through high-energy electron irradiation, using an ultra-high voltage TEM H-3000 by Hitachi, operated at an acceleration voltage of 2000 kV. The irradiation was performed with a beam dose of 1 × 1025 e/m2 at 290 K (ultra-high purity α-Fe) and 1 × 1025 e/m2 at 150 K (high purity α-Fe), producing SIAs that agglomerated to form loops. Behavior of SIA loops in response to specimen heating were observed using a diffraction-contrast technique at an acceleration voltage of 200 kV to avoid additional knock-on displacement, recording at a frame rate of 30 s−1 with a Gatan 676 camera.Thin foils with thickness less than 0.08 mm were pre-annealed at 773 K for 0.5 hours under a vacuum of 3 × 10b and climb in two dimensions perpendicular to their Burgers vector. The shape of the dislocation loop was fixed to be constant throughout, allowing the total elastic energy of the system Eelastic, to be a function purely of the N loop positions . In all our simulations the loops were assumed to have identical Burgers vectors bi = b. The equation of motion for an individual loop i therefore readIn our DD simulations, prismatic dislocation loops were allowed to glide along the direction of their Burgers vector Mgl is a glide mobility suitable for prismatic dislocation loops at high temperature3132Mscl is the self climb mobility given in Mscl replaced by the VMC law (5). As the glide mobility Mgl is much greater than the climb mobility Mscl we split the integration of (6), first allowing glide-only motion until a local steady state had been reached, defined as when the total magnitude of the glide force, δr = 1 Å in the direction of the climb force, and the simulation time was updated by where b; the loops under study were either circular or pill shaped , and were chosen to approximate as closely as possible the shape and position of the coalescing loops seen under the TEM, though the effect of the free surface and the surrounding defect network is neglected.For the binary loop coalescence simulations the elastic interaction energy was given by Mura’s formula  for two R1, R2 and positions x1, x2For the simulations treating the coarsening of a distribution of dislocation loops, all loops were taken to be collinear and circular, with coalescence occurring instantaneously on contact, whereupon the larger loop was expanded to accommodate the area of the smaller loop. To speed up the simulations we used an analytical far field solution for the loop-loop interactiond = x1 − x2, where How to cite this article: Swinburne, T. D. et al. Fast, vacancy-free climb of prismatic dislocation loops in bcc metals. Sci. Rep.6, 30596; doi: 10.1038/srep30596 (2016).


Turbulence intensity, or hydromechanical stress, is a parameter that influences a broad range of processes in the fields of chemical engineering and biotechnology. Fermentation processes are often characterized by high agitation and aeration intensity resulting in high gas void fractions of up to 20% in large scale reactors. Very little experimental data on hydromechanical stress for such operating conditions exists because of the problems associated with measuring hydromechanical stress under aeration and intense agitation.3 and 40 m3 volume with a broad range of operating conditions and impeller geometries (Rushton turbines). Results for impellers within each scale for the ratio of maximum to specific energy dissipation rate ϕ based on measured values of maximum stable drop size for aerated operating conditions are qualitatively in agreement with results from literature correlations for unaerated operating conditions. Comparison of data in the different scales shows that there is a scale effect that results in higher values for ϕ in larger reactors. This behavior is not covered by the classic theory of turbulent drop dispersion but is in good agreement with the theory of turbulence intermittency. The data for all impeller configurations and all aeration rates for the three scales can be correlated within ±20% when calculated values for ϕ based on the measured values for dmax are used to calculate the maximum local energy dissipation rate. A correlation of the data for all scales and all impeller configurations in the form ϕ = 2.3∙(ϕunaerated)0.34∙(DR)0.543 is suggested that successfully models the influence of scale and impeller geometry on ϕ for aerated operating conditions.An indirect method to quantify hydromechanical stress for aerated operating conditions by the measurement of maximum stable drop size in a break-up controlled dispersion was applied to characterize hydromechanical stress in reactor scales of 50 L, 3 mThe results show that besides the impeller geometry, also aeration and scale strongly influence hydromechanical stress. Incorporating these effects is beneficial for a successful scale up or scale down of this parameter. This can be done by applying the suggested correlation or by measuring hydromechanical stress with the experimental method used in this study. The development of these large scale processes is conducted in lab or pilot scale fermenters. This poses the question how to scale down the conditions prevalent in large production scale to the development scale and vice versa. Typical parameters of interest may be mass transfer coefficient kLa, volumetric power input P/VL, impeller tip speed utip, turbulence intensity in the form of maximum local energy dissipation rate ϵmax, circulation frequency tc, or a combination of these parameters like the energy dissipation to circulation function EDCF  can be estimated that resemble the respective values of these different parameters in large scale. Testing these operating conditions in small scale may reveal which of these parameters  is a suitable proxy for scaling up or scaling down a particular process. The work of Jüsten 3 reactore et al. , e.g., fMaximum stable drop sizes calculated from these distributions are indicators for hydromechanical stress. To perform a scale-up or scale-down of hydromechanical stress it is necessary to correlate the maximum stable drop size with operating conditions. This is the focus of the following paragraphs.3 scale are in good agreement with each other. This shows that a correlation of hydromechanical stress with impeller tip speed ut = π∙n∙d gives reasonable results as long as the scale is not changed. However, a comparison of the results for the different scales shows that a scale-up with constant impeller tip speed will not result in comparable values of dmax. Therefore, the levels of hydromechanical stress in the different scales will be different if ut is kept constant. This is in accordance with the results of Jüsten et al. C: Bottom clearance of first impeller [m]ΔC: Impeller spacing [m]d: Impeller diameter [m]max: Maximum stable drop size [μm]dR: Reactor diameter [m]D32: Sauter mean diameterdFl: Impeller flow number [-]Fr: Impeller Froude number [-]h: Impeller blade height [m]H: Unaerated liquid height [m]1: Constant in Eq. 2 [-]K2: Constant in Eq. 2 [-]Kn: Agitation rate [1/s]bl: Number of impeller blades [-]nP: (Aerated) power input [W]0: Unaerated power input [W]PPo: Power number [-]t: Impeller tip speed [m/s]uL: Total liquid volume [m3]Vw: Impeller blade width [m]Ø: Volume averaged energy dissipation rate [W/kg]ϵmax: Maximum local energy dissipation rate [W/kg]ϵc: Continuous phase density [kg/m3]ρmax/ϵØ [-]ϕ: ϵσ: Interfacial tension between dispersed and continuous phase [N/m]D: Dynamic viscosity of dispersed phase [Pa∙s]ηThe authors declare that they have no competing interests.AD developed the method, performed experiments and prepared the manucript. SD, MB and MM performed experiments and contributed to experimental planning and analysis of data. GS and JB initiated the project. GS gave important guidance for the experimental work. JB contributed to method development, data analysis and manuscript preparation. All authors approved the final manuscript.


In vitro studies show that estrogens acutely modulate synaptic function in both sexes. These acute effects may be mediated in vivo by estrogens synthesized within the brain, which could fluctuate more rapidly than circulating estrogens. For this to be the case, brain regions that respond acutely to estrogens should be capable of synthesizing them. To investigate this question, we used quantitative real-time PCR to measure expression of mRNA for the estrogen-synthesizing enzyme, aromatase, in different brain regions of male and female rats. Importantly, because brain aromatase exists in two forms, a long form with aromatase activity and a short form with unknown function, we targeted a sequence found exclusively in long-form aromatase. With this approach, we found highest expression of aromatase mRNA in the amygdala followed closely by the bed nucleus of the stria terminalis (BNST) and preoptic area (POA); we found moderate levels of aromatase mRNA in the dorsal hippocampus and cingulate cortex; and aromatase mRNA was detectable in brainstem and cerebellum, but levels were very low. In the amygdala, gonadal/hormonal status regulated aromatase expression in both sexes; in the BNST and POA, castration of males down-regulated aromatase, whereas there was no effect of estradiol in ovariectomized females. In the dorsal hippocampus and cingulate cortex, there were no differences in aromatase levels between males and females or effects of gonadal/hormonal status. These findings demonstrate that long-form aromatase is expressed in brain regions that respond acutely to estrogens, such as the dorsal hippocampus, and that gonadal/hormonal regulation of aromatase differs among different brain regions. Cytochrome P450 aromatase, the product of the CYP19 gene, catalyzes the biosynthesis of estrogens from androgens. Multiple peripheral tissues including ovaries and testes, placenta, adipose tissue, bone, and muscle express aromatase and hence could synthesize estrogens  must be capable of synthesizing estrogens. Studies using a ribonuclease protection assay demonstrated that there are two forms of aromatase mRNA in the rat brain: a long form and a short form, and suggested that the long form generates the active enzyme To investigate the distribution of aromatase in the rat brain, and avoid caveats of antibody specificity, we used quantitative real-time PCR (qPCR) to measure relative mRNA levels specifically of long-form aromatase in the amygdala, BNST, POA, dorsal hippocampus, cingulate cortex, brainstem and cerebellum in both male and female rats. We also evaluated possible hormonal regulation of long-form aromatase in each sex. We were particularly interested to determine whether the female rat hippocampus expresses long-form aromatase, as this has not been shown previously, yet is predicted by the observation that E2 acutely modulates synaptic transmission in the hippocampus of females.Guide for the Care and Use of Laboratory Animals and were approved by Northwestern University Animal Care and Use Committee (protocol numbers: 2011-2521-5 and 2011-1400-7). All animals were young adult Sprague Dawley rats . Six gonadally intact females were used to assess fluctuation in ovarian aromatase expression across the estrous cycle. In these intact females, estrous cycle stages were monitored for at least 5 consecutive days and ovaries were harvested from 2 rats each in proestrus, estrus, and diestrus. The remaining 9 males and 9 females were divided into the following groups: castrated males (n = 4); gonadally intact (sham castrated) males (n = 5); ovariectomized (ovx) females (n = 4); and ovariectomized/estradiol (E2)-treated (ovx+E2) females (n = 5). All gonadectomies were carried out under general anesthesia  using aseptic surgical protocol. For ovx, ovaries were removed through bilateral incisions on the dorsal flank after blood vessels were ligated. Ovariectomy of females was performed in diestrus and ovaries were collected during the surgery, frozen immediately on dry ice and stored at −80°C for later analysis. For castration, testes were removed through a single incision along the midline in the scrotum after blood vessels were ligated. For sham castration, the incision was made and closed without removal of testes. Animals were given one week to recover after surgery. Seventy-two and 48 hours prior to sacrifice, female rats in the E2-treated group were given two s.c. injections of 10 µg 17β-estradiol benzoate in 100 µl sesame oil vehicle. This dose of estradiol benzoate produces supraphysiological estradiol levels shortly after injection, which are metabolized to within the physiological range by 24 hours later All animal procedures were performed in accordance with the National Institutes of Health All tissue collection was carried out under RNase-free conditions. At the time of sacrifice, rats were deeply anesthetized with sodium pentobarbital , and then tissue from peripheral organs , the cerebellum, and brainstem (BS) was collected from each animal, pooled within group into a 1.5 ml RNase-free tube, and snap frozen on dry ice. The rest of the brain from each animal was frozen on dry ice and stored at −80°C. Frozen brains were then individually mounted in a cryostat, warmed to −4°C and 500 µm coronal sections were cut. Tissue samples from the preoptic area (POA), amygdala (Amyg), bed nucleus of the stria terminalis (BNST), dorsal hippocampus (dHipp), and cingulate cortex (CC) were punch dissected from these coronal sections using either 12 ga  or 15 ga  punches, and the tissue from the same region within each group was pooled into 1.5 ml RNase-free tubes. The tubes first were kept on dry ice to prevent thawing of the tissue and subsequently were stored at −80°C for later analysis.Tissue samples were thawed in 1 ml TRIzol Reagant (Ambion) and homogenized with a tissue sonicator (Qsonica) using three 3 sec pulses. Samples were centrifuged at 12,000 x g for 10 min at 4°C and then the supernatant was carefully removed and transferred to a new tube. After 5 min at room temperature, 0.2 ml chloroform was added; tissue was mixed vigorously, allowed to stand at room temperature for 3 min before centrifugation at 12,000 x g for 15 min at 4°C. The aqueous phase was transferred to a new tube, and total RNA was isolated and purified using Purelink RNA Mini Kit (Ambion) according to the manufacturer's instructions. RNA was eluted with 30 µl RNase-free water. RNA concentration was measured using a spectrophotometer  at a wavelength of 260 nm and RNA purity was assessed based on an A260/A280 ratio of 1.8–2.0.15 Primer, 1 µl random primers and nuclease-free water for a total volume of 5 µl. Samples were incubated in a heat block at 70°C for 5 min followed by immediate chilling on ice for 5 min. The reverse transcription master mix for each sample consisted of 4 µl GoScript 5x reaction buffer, 3 µl MgCl2, 1 µl PCR nucleotide mix, 0.5 µl recombinant RNasin (ribonuclease inhibitor), 1 µl GoScript reverse transcriptase, and 5.5 µl nuclease-free water. The master mix was added to each sample for a total reaction volume of 20 µL. Samples were incubated at 25°C for 5 min , 42°C for 1 hr (extending), and 70°C for 15 min (reverse transcriptase inactivation). After the reaction was completed, samples were spun down in a microfuge for 5 sec and stored at −20°C.Reverse transcription reactions were performed using the GoScript Reverse Transcription System (Promega). RNA (2 µg) was mixed with 1 µl Oligo(dT)5′-CTCCTCCTGATTCGGAATTGT-3′ and (R) 5′-TCTGCCATGGGAAATGAGAG-3′; GAPDH (F) 5′-GGGTGTGAACCACGAGAAATA-3′ and (R) 5′-AGTTGTCATGGATGACCTTGG-3′; HPRT (F) 5′-GGCCAGACTTTGTTGGATTTG-3′ and (R) 5′-CTTTCGCTGATGACACAAACAT-3′, and SDHA (F) 5′-ATGGAAAATGGGGAGTGCCG-3′ and (R) 5′-GCTGAAGTAGGTTCGCCCAT-3′. The primer pair efficiencies were determined based on the slope of standard curve that was generated after plotting the results of the titration of the target cDNA , GAPDH –99.8% (slope: −3.3), HPRT –100% (slope: −3.39) and SDHA –97.8% (slope: −3.376). The primer pair for aromatase targeted a region that contains parts of exons 2 and 3, which is present only in long-form aromatase. The PCR product was a 90 bp fragment , hypoxanthine phosphoribosyltransferase (HPRT) and succinate dehydrogenase complex, subunit A (SDHA). Target sequences were amplified using the following primer pairs: aromatase (F) shown in  within t–ΔΔ(CT) method (CT = cycle threshold), where ΔΔCT  =  ΔCT (sample) – ΔCTThe PCR reactions were prepared in triplicate in a 384-well plate by mixing 1.2 µl cDNA (10 µg) and 1.2 µl commercially available GoTaq qPCR master mix (Promega) using a Mosquito LCP automated liquid handler (TTP Labtech) and then adding 10 nl primer mix  with an Echo Liquid Handler (Labcyte). qPCR was carried out using the following program: 95°C for 5 min, 40 cycles of 95°C for 15 sec and 60°C for 1 min, and a melt curve analysis . The melt curve was used to confirm the specificity of each PCR reaction. Relative gene expression was determined using the 2t-tests with Bonferroni's correction for post hoc analysis when applicable. p<0.05 was considered statistically significant.Gene expression data were obtained from 4 qPCR runs per group using cDNA products of 2 independent reverse transcription reactions for each group. Each qPCR run was treated as an independent experiment. Mean aromatase mRNA levels were normalized to the levels of the housekeeping genes as described above, and used for statistical analysis (n = 4). Results were analyzed statistically using either a one-way ANOVA (estrous cycle stages) followed by Tukey's multiple comparison, or two-way ANOVA (sex x gonad/hormone condition), followed by independent-sample T values among different brain regions in qPCR analysis, the cycle number for each region was calculated using the average CT value for that region and adding 6 cycles, which was the earliest stage of amplification in which DNA amplification products were reliably detectable on a gel. We used the same aromatase and GAPDH primer pairs for RT-PCR as in the qPCR analysis. The PCR reaction (in duplicates) consisted of 1 µl cDNA, 1.25 µl forward primer (10 µM), 1.25 µl reverse primer (10 µM), 12.5 µl GoTaq colorless master mix (Promega) and 9 µl nuclease-free water for a total reaction volume of 25 µl. The optimal hot-start PCR program was as follows: 95°C for 15 sec, 60°C for 1 min and 72°C for 4 min, with a final hold at 4°C. A 2% agarose gel was prepared using TAE buffer, and ethidium bromide was added before gel casting. DNA loading dye was mixed with the PCR products and loaded on the gel. The gel was run at 120V for 1 h and viewed with a gel imager (Kodak).RT-PCR and gel electrophoresis was used to visualize the amplified aromatase cDNA fragment for each brain region and thereby corroborate qPCR results. Based on the differences in CBecause aromatase is expressed broadly, we first sought to identify reliable positive and negative controls. Among the peripheral tissues that we tested , liver showed no detectible aromatase mRNA and hence was used as a negative control. Ovary was chosen as a positive control for its high aromatase expression T = 22) was followed by BNST  and POA . Cortical structures exhibited intermediate aromatase mRNA expression; higher in dorsal hippocampus  than in cingulate cortex . The two other brain areas examined, brainstem  and cerebellum , showed much lower aromatase expression .Next, we carried out qPCR analyses of aromatase expression in different brain regions of rats from 4 groups: castrated males, gonadally intact males, ovx females and ovx+E2 females. Because the range of aromatase expression between brain areas was much larger than group differences within an area, we first plotted the mean aromatase expression for all 4 groups in each region . Of all To determine the contributions of sex and hormonal/gonadal status on aromatase expression, aromatase mRNA levels within each brain region were compared between castrated males, gonadally intact males, ovx females, and ovx+E2 females. This showed 3 distinct patterns of regulation; 1) both sex and gonadal hormones affect aromatase expression , 2) gonadal hormones up-regulate aromatase mRNA expression only in males (BNST and POA), or 3) no differences in aromatase expression regardless of sex or hormonal/gonadal status . Aromatase mRNA levels were too low in the brainstem and cerebellum for meaningful comparisons between groups. For quantitative comparisons between groups, the mean aromatase mRNA expression value of ovx females was set as 1.0 and data for the other groups were expressed relative to ovx females.In the amygdala, which showed the highest aromatase expression among the brain areas we examined, aromatase mRNA levels were higher in males by 52% compared to females [sex: F  = 10.98, p  = 0.006, In the BNST  and the In contrast to the amygdala, BNST, and POA, aromatase expression in the dorsal hippocampus  and cingin vivo in both sexes.The present study investigated relative expression of mRNA for the long form of aromatase in distinct regions of the male and female rat brain and the effect of gonadal/hormonal status on aromatase expression in both sexes. We focused on the long form of aromatase based on previous evidence that this is the catalytically active enzyme. Using qPCR, we found highest aromatase expression in the amygdala followed closely by the BNST and the POA, then the dorsal hippocampus and cingulate cortex, and much lower expression in the brainstem and cerebellum. This pattern was true for all groups regardless of sex or gonadal/hormonal status. The amygdala was the only brain region in which gonadal/hormonal upregulation of aromatase expression occurred in both sexes, though these effects were moderate in each sex. In the BNST and POA, castration had a strong effect in males to reduce aromatase expression to a level similar to that in females, whereas there was no evidence for estrogenic regulation in females. In the hippocampus and cingulate cortex, there were no significant differences in aromatase expression between sexes or gonadal/hormonal conditions. That the long form of aromatase is expressed in the dorsal hippocampus, including in females, supports the idea that estrogens synthesized within the hippocampus could acutely modulate synaptic transmission Multiple previous studies in rodents have investigated aromatase levels in the brain, however few have taken into account evidence that aromatase exists in two forms of which the long form is the active one The aromatase gene contains both androgen and estrogen response elements Regulation of brain aromatase by gonadal hormones in rodents has been demonstrated previously, although most of these studies included only one sex and/or focused on brain regions involved in reproductive function. Our findings that expression of long-form aromatase in the BNST and POA of males is upregulated by the testis are consistent with previous studies in which castration of adult male rats decreased aromatase activity in hypothalamus-preoptic area and administration of testosterone or dihydrotestosterone prevented this decrease In contrast to the role of androgens, less is known about how estrogens influence brain aromatase activity or gene expression. Several groups have reported estrogenic upregulation of aromatase expression in the POA, medial basal hypothalamus or amygdala in castrated or intact males While a majority of studies in the brain have focused on hormonal modulation, the structure of the aromatase gene indicates multiple points of regulation, and aromatase is regulated by non-hormonal factors in peripheral tissues. For example, tissue-specific expression of aromatase is regulated through different promoter regions and alternative splicing. Cloning and structural characterization of the aromatase gene showed that the coding region spans 9 exons beginning with exon 2 We found that levels of long-form aromatase in non-reproductive brain regions such as the hippocampus were similar between males and females and not affected by gonadal/hormonal status. This is useful in the context of acute E2 modulation of synaptic transmission in the hippocampus, as it eliminates sex and gonadal/hormonal status as factors that could influence the supply of locally synthesized estrogens through differential aromatase expression. Future studies will need to investigate non-hormonal factors that regulate aromatase expression and/or activity in the hippocampus and that could therefore alter the capacity of the hippocampus to synthesize estrogens. Identifying non-hormonal factors that regulate aromatase in non-reproductive brain regions such as the hippocampus will be critical to understanding the physiological role of acute E2 modulation of synaptic physiology for non-reproductive brain functions such as affective behaviors and learning and memory.