Yet, no systematic studies of fear acquisition/extinction or their underlying neural mechanisms have been conducted in this preclinical genetic SERT+/− model. Here we sought to determine if SERT+/− or SERT−/−, compared to wildtype, rats would show exacerbated panic responses and/or persistent conditioned fear responses that may be associated with PTSD or phobia vulnerability. Results: Only SERT−/− rats showed increased baseline anxiety-like behaviors with heightened panic respiratory responses. However SERT+/−  rats showed enhanced acquisition of fear and delayed extinction of fear that was associated with changes in serotonergic-related genes  and disrupted inhibition within the basolateral amygdala (BLA). Furthermore, the disrupted fear responses in SERT+/− rats were normalized with 5HT1A antagonist infusions into the BLA. Enhanced acquisition and failure to extinguish fear memories displayed by both SERT−/− and SERT+/− rats are cardinal symptoms of disabling anxiety disorders such as phobias and PTSD. The data here support the hypothesis that reduced SERT function is a genetic risk that disrupts select gene expression and network properties in the amygdala that could result in vulnerability to these syndromes.Genetic variation in serotonin transporter (SERT) that reduces transcriptional efficiency is associated with higher anxiety and fear traits and a greater incidence of post traumatic stress disorder (PTSD). Although previous studies have shown that rats with no expression of SERT (SERT Severe and or repeated psychological trauma can result in PTSD, which is associated with symptoms such as persistent fear associated memories  that can trigger panic attacks (PA)2 and lead to avoidance of activities, places and stimuli that produce these flashbacks. In regards to PD, the cardinal symptoms are unexpected and recurrent (uPA)’s that occur in the absence of a clear external trigger and are estimated to account for ~40% of PA’s3. Recurrent unexpected PAs can produce a conditioned avoidance response that occurs when people with PD are traumatized by the uPA’s and begin to fear situations that are associated with the uPA’s. This can then induce expected PA’s3 and agoraphobia in ~50% of humans with PD4. Although unexpected, PA’s can be reliably induced in humans with PD with viscerosensory associated stimuli such as 5–7% hypercapnic gas exposure7, which do not induce PAs in healthy controls at these concentrations. The relevance is that subtle increases in CO2 in the blood caused by hypoventilation or holding one’s breath result in acidosis in peripheral and central brain structures that initially results in an increase in respiration activity to help “blow off” excess CO2 [see review8]. However, a sense of suffocation occurs if CO2 levels continue to increase, which also produces adaptive behavioral and autonomic responses that recapitulate symptoms of PA’s. For example, higher concentrations of 20% CO2 will induce symptoms consistent with PA’s in healthy humans9 and also induce concentration dependent increases in fear in healthy humans that is greater (more severe) in humans with PD10. Thus there is evidence that the initial PD pathology is associated with an alteration in central neural pathways which renders them susceptible to uPA’s when exposed to otherwise non panic inducing viscerosensory stimuli11.Post traumatic stress disorder (PTSD) and panic disorder (PD) represent some of the most severe and disabling trauma/anxiety related disorders12, yet PTSD occurs in only about 7% of the population13 [see also review14]. Therefore the risk of being traumatized is much higher than the prevalence of PTSD. This suggests that the majority of people have some resiliency to traumatic events, but others may be more vulnerable . In regards to PD, the prevalence in the general population is ~2–5%15 with a strong heritability in first degree relatives (~11%) and monozygotic twins (30–40%)17, suggesting that in this case there are clear genetic contributions. Consistent with this hypothesis is that in humans with PD a meta-analysis of candidate genes identified several replicable candidate genes18 such as serotonin transporter (SERT) polymorphisms which is associated with severity of PAs19. SERT reuptake inhibitors (SSRIs) are also the gold standard for treating severe anxiety/trauma disorder such as PD and PTSD21. In 1996 Lesch and colleagues assessed anxiety traits in humans carrying a specific polymorphism in the promoter region of the SERT gene, where the dominant short (s) allelic variant reduces transcriptional efficiency of the SERT22, which is evidenced by reduced brain SERT mRNA levels23, and reduced binding24. It has been determined that humans with this SERT polymorphism have increased anxiety-associated traits22 and enhanced fear conditioned responses26. This specific SERT ss-allele polymorphism is also associated with a higher risk of PTSD in humans exposed to high trauma27, and although it is not associated with a higher risk for PD27 it is associated with more severe PA’s29.In the US, there is evidence that the risk of being exposed to a severe trauma could be as high as 75%34. The BLA also plays a critical role in fear conditioned responses [see reviews36]. Humans with the SERT polymorphism have enhanced baseline amygdala activity and reactivity to fearful stimuli that is likely a contributing factor to increased anxiety and fear-associated traits38. However humans with atrophy of the amygdala region, whom have disrupted fear conditioned responses, display similar if not worse panic responses to a 20%CO2 challenge39, and in one case study an intact amygdala was not necessary for a subject to experience PA’s40. Consistent with this finding, Schruers and colleagues determined that humans with the SERT s-allele polymorphism displayed less fear to a panicogenic CO2 challenge41. Taken together this suggests that the SERT polymorphism may not be associated with initial unexpected PAs that are the initial phase of PD, but may predispose them to the later development of amygdala regulated conditioned responses such as agoraphobia.Among anxiety circuits, the basolateral amygdala (BLA) is a critical site where serotonin plays an important role in modulating anxiety and fear responses and is highly responsive to stressful stimuli+/−) is also associated with reduced transcriptional efficiency in rats42. Rats with complete loss of SERT (SERT−/−) have high baseline anxiety associated behaviors, but the SERT+/− rats have normal anxiety behaviors, which suggests that other risk factors such as acute or chronic stress may be needed to observe anxiety vulnerability44. In light of the SERT gene being a strong candidate gene associated with the severity of PA’s, but not being associated with increase panic symptoms post a CO2 challenge, we assessed baseline anxiety; innate panic responses to a CO2 challenge ; and fear conditioned responses in SERT+/− and SERT−/−.Similar to human SERT polymorphism, a heterozygous null mutation of the SERT gene  and were cross bred with wildtype SERT+/+ Wistar rats to produce SERT+/− heterozygous Wistar Rats42. The behavior and gene array experiments were conducted on male 300–325 g rats, and the electrophysiological experiments male 150–200 g rats. All rats were housed individually in plastic cages under standard environmental conditions  for 7–10 days prior to the surgical manipulations. Food and water were provided ad libitum. All experiments were conducted in accordance with the Guide for the Care and Use of Laboratory Animals, Eighth Edition  and the guidelines of the IUPUI Institutional Animal Care and Use Committee.SERT°C. In a RNase free environment, frozen brains were sliced coronally at 300 µm  using a cryostat, and sections were placed onto slides. The dorsal raphe nucleus (DRn) and median raphe nucleus (MRn) were dissected out of 3 adjacent sections and the BLA from 2 adjacent sections using a 1.0 mm Harris Micro-punch (Electron Microscopy Sciences) and RNA processing and RT-PCR analysis of custom TLDA were performed as previously described45. For DRn/MRn, comparative evaluation of quantitative real-time PCR was performed with validated Taqman probes  using the Taqman gene expression master mix and 7900HT real-time PCR system (Applied Biosystems). Each sample was analyzed in triplicate, and beta actin was used as an internal standard. The BLA tissue was processed for serotonergic-related genes using the custom-designed TaqMan Low Density Array (TLDA), see supplemental table 45.Rats were rapidly anesthetized via IsoFlurane, decapitated, and brains were removed and frozen in isopentane chilled with dry ice, then stored at −80 Details are provided in The elevated plus maze consisted of 2 long arms with no walls, an intersection, and 2 closed walls. On the test day, the animals were placed in the center area of the maze and allowed to explore the open and closed arms for 5 min. The total amount of time spent in each arm was recorded directly to the computer from the infrared beam breaks.2, 21% O2, 79% N2) for baseline measurements, then (2) either the control gas or experimental normoxic, hypercarbic gas  for 5 min, and finally, (3) 5 min infusion of atmospheric gas. A pressure amplifier  was connected to a Powerlab data 8/35 acquisition system  and respiration rate was assessed by calculated using LabChart software (ADInstruments), and sighing was also visualized using Labchart software. A sigh was defined as an initial respiration that immediately precedes an additional second deeper inspiration. The sigh is followed by an expiration that is larger than usual, and is often accompanied by a post sigh apnea46. Supplemental Fig. 2 and O2 that occur prior to, during and at offset of gas infusion.All rats were handled 3 times a week for 3–5 min by experimenter prior to the day of gas infusions. Rats were placed in a clear Plexiglass® cylindrical plethysmograph chamber with atmospheric air infused at a flow rate of 2.8 l/min using a Vetamac flowmeter  until a steady baseline respiration rate was noted. All rats had infusions of the following: 1) 5 min infusion of atmospheric gas , followed by PBS containing 4% paraformaldehyde (PFA) and 3% sucrose and the brains were removed and processed for immunohistochemistry as described in detail previously2O2 in PBS for 20 min. Immunostaining for SERT protein and c-Fos protein was accomplished with sequential immunohistochemical procedures using (1) primary antibodies directed against SERT  and c-Fos  which were incubated on orbital shaker 12–14 h then washed in PBS prior to and in between 2 h incubations in: (1) biotinylated secondary antibodies; (2) avidin biotin complex kit; and (3) chromogen kit as described previously47.Free-floating sections were washed in 0.05 M PBS for 10 min × 3 between initial incubation in 1% H48. Photomicrographs of the SERT immunostaining in the DRn were taken at ×400 magnification using a DMLB Leica binocular brightfield microscope at 3 Bregma levels  for each rat. Densitometry analyses were done on grayscale inverted photographs using Adobe Photoshop version 16 for each Bregma level for each rat and combined prior to analyses. The WT mean represented 100% which was compared to SERT+/− and SERT−/− values.Selection of anatomical levels for analysis of SERT immunostaining in the DRn and c-Fos-immunostaining in anxiety circuits was conducted with reference to illustrations from a rat brain stereotaxic atlasThe numbers of c-Fos-ir cells were counted in the entire field of view at ×400 magnification in brain regions implicated in innate panic responses  and conditioned fear responses . All cell counts were done by an observer that was blind to the experimental treatment of each animal.Photomicrographs were obtained the Leica DMLB microscope connected to a Leica DFC 300 digital camera and Leica Applications Suite 4.1 for Windows.All rats were handled 3 times a week for 3–5 min by experimenter prior to the day of behavioral assays. The fear-conditioning chamber has a grid floor composed of 6 stainless steel rods connect to a shock generator . The fear conditioning protocol was 4 days long. On day 1, rats were placed in the conditioning chamber and allowed to habituate for 10 min. On day 2, test day 1, the rats were placed back in the conditioning chamber and was exposed to 5 tone + shock pairings, with a 120 s inter-trial interval with a tone conditioned stimuli  co-terminating with a single shock unconditioned stimuli . On day 4, test day 2, rats underwent an extinction paradigm where they were presented with 20 tone CS only with a 120 s inter-trial interval. All sessions were video-recorded and the total time spent freezing during the tones on all 2 test days was scored blind by the investigator Stephanie Fitz.+/− rats (n = 9) were anaesthetized by placing them in a closed Plexiglas® box that was connected to an isoflurane system  and then with a nose cone connected to the same system during the stereotaxic surgery. Rats were placed into an ultra-precise stereotaxic instrument  with the incisor bar set at –3.3 mm and nose cone connected to the same system during the surgery. Two stainless steel guide cannulae  were situated into guide cannulae holders fixed onto the stereotaxic arms. The 33 gauge injector was lowered into position of the BLA using coordinates  according to a standard stereotaxic atlas of the adult rat brain48. The guide cannulae were secured into place using three 2.4 mm screws anchored into the skull along with cranioplastic cement. Following placement of dummy cannulae into the guide cannulae, rats were removed from the stereotaxic apparatus and allowed to recover for 72 h prior to experimental testing. SERT+/− rats received bilateral 200 nL injections of either artificial cerebrospinal fluid  or 20 mM 8-Hydroxy-2-(dipropylamino)tetralin hydrobromide  20 min prior to acquisition and recall/extinction at a rate of 100 nL/min using 10-μl Hamilton syringes attached to an infusion pump  and subsequently connected to the injection cannulae via polyethylene (PE 50) tubing . Once the injection cannulae were securely placed into the rat, the infusion pump was turned on and set to automatically deliver 100 nL/site over 2 min. Following the injection, the cannulae remained in place for an additional min before being removed. Smooth flow of the solutions via the tip of the injection cannulae was verified before and after each injection to ensure proper drug delivery.All rats were handled 3 times a week for 3–5 min by experimenter prior to the day of behavioral assays. SERT49. Briefly, slices were incubated in an oxygenated (95% O2/5% CO2 mixture) artificial cerebrospinal fluid (ACSF) of the following composition (in mM): 130 NaCl, 3.5 KCl, 1.1 KH2PO4, 1.3 MgCl2, 2.5 CaCl2, 10 glucose, 30 NaHCO3 at room temperature for ~1 h before the recording. Slices containing the BLA were then transferred to a recording chamber mounted on the stage of the upright Nikon E600FN microscope  and perfused at rate of 2–3 ml/min with ACSF heated to 30 °C. Whole-cell patch clamp recordings were obtained using standard techniques with borosilicate glass electrodes  filled with a potassium gluconate-based solution of the subsequent composition (in mM): 140 potassium gluconate, 2 KCl, 3 MgCl2, 10 HEPES, 5 phosphocreatine, 2 potassium-ATP, 0.2 sodium-GTP) adjusted to pH 7.3 with KOH, and having an osmolarity of 285–295 mOsm. Whole-cell access resistance varied between 5 to 20 MΩ and was examined during each experiment; a change of less than 15% was acceptable. The data were obtained only from pyramidal neurons located in BLA. Whole-cell recordings were made with a Multiclamp 700B amplifier  using the pClamp 10.3 software and a Digidata 1322 A interface .Once anesthetized, brains were quickly dissected, and 350-μm coronal slices were obtained as described earliergenotype as independent variable and time as the repeated measures. In the presence of significant effects, between and within subjects post-hoc tests were conducted using a Fisher’s LSD or Dunnett’s post-hoc test, respectively. Statistical significance was accepted with p ≤ 0.05. All statistical analyses were done with SPSS 25 and Graphpad Prizm 7.0; and all graphs were generated using Graphpad Prizm 7.0 for Windows. Figureplates were made with CorelDraw X for Windows.The following dependent variables were analyzed using an ANOVA with genotype as the main independent variable . Respiratory responses over time and fear conditioned freezing behavior was analyzed using a one way ANOVA with repeated measures with +/− and SERT−/− rats, we measured SERT mRNA in micropunched tissue containing the DRn/MRn region, which revealed that, compared to wildtype SERT+/+ controls (n = 3), SERT−/+ rats (n = 5) had approximately a 40% reduction SERT mRNA and SERT mRNA levels were at the limit of detection for SERT−/− rats  = 83.1, p < 0.001, Fig. n = 6), SERT+/− (n = 6) and SERT−/− (n = 6) rats had ~57% and ~88% reduction in SERT immunostaining, respectively . This was also below the 0.8 mA shock used for fear conditioning experiment.In this experiment the mean shock needed to induce a startle response was ~0.4 mA with no difference noted between genotypes [Suppl. Fig. —Bar graph represents the mean intensity of shock needed to induce a startle response between wildtype (n = 6), SERT+/− (n = 5) and SERT−/− (n = 5) rats. The arrow indicates the shock intensity used for fear conditioning experiment in Fig. Supplemental Fig. 4—List of the 21 serotonin related genes assessed in the BLA using a custom TaqMan RT-PCR array microfluidic card.Supplementary Table 1Supplemental Figure 1Supplemental Figure 2Supplemental Figure 3Supplemental Figure 4Supplemental Table 1


These devices do not require any post-fabrication treatments such as filament creation by electroforming that would impede circuit scalability. Instead these devices operate due to Poole-Frenkel defect controlled transport where the high defect density is inherent to the Nb2O5−x deposition rather than post-fabrication treatments. Temperature dependent measurements reveal that the dominant trap energy is 0.22 eV suggesting it results from the oxygen deficiencies in the amorphous Nb2O5−x. Rectification occurs due to a transition from thermionic emission to tunneling current and is present even in thick devices (>100 nm) due to charge trapping which controls the tunneling distance. The turn-on voltage is linearly proportional to the Schottky barrier height and, in contrast to traditional metal-insulator-metal diodes, is logarithmically proportional to the device thickness. Hysteresis in the I–V curve occurs due to the current limited filling of traps.Metal-Nb In 2013, a NbO2 memristor was demonstrated that replicates the voltage triggered switching behavior observed in biological ion channels enabling a physical neuristor circuit to be built2. These NbO2 memristors successfully replicate the rectification observed in voltage-gated biological ion channels3 and the hysteresis necessary for the proposed neuristor circuit to operate2. The NbO2 memristors are manufactured by electroforming an insulating layer of Nb2O5 into the semiconductor NbO2, a process that introduces challenges for scaling to dense circuits. Additionally, previous work has not reported a capacitance in NbO2 memristors requiring an additional parallel capacitance for the neuristor circuit to operate2. Herein, an Nb2O5−x based metal-insulator-metal (MIM) device is reported that does not require electroforming, possesses a dispersive capacitance advantageous for neuromorphic circuits, and exhibits the rectification and hysteresis necessary to replicate ion channel functionality for the neuristor circuit.In 1962, a neuristor circuit was theorized for propagating electrical signals without attenuation as is observed between biological neurons2O5−x devices function in the same way as the previously published voltage threshold switches consisting of electroformed NbOx filaments within Nb2O52. When used in a neuristor circuit as described in Pickett et. al.2 the switching device, whether it is a memdiode or the NbO2 device, operates as a voltage threshold triggered switch. The neuristor oscillates when the circuit is excited by a current. Initially, a capacitor is charged in parallel with one switch, and that switch turns on when a certain threshold voltage is reached. This switch will also turn off at a voltage lower than the turn-on voltage. When this first switch turns on, the voltage increases on a second memdiode/capacitor pair which causes the second memdiode to switch. This time delayed switching raises then lowers the voltage, creating an oscillatory pulse. The memdiodes presented herein act in the same way as the prior NbO2 threshold switches (both act as voltage threshold switches) with the added benefits of integrated capacitance and as-deposited switching. In this way two such memdiodes can form the basis of a neuristor by incorporating both the switching element and the capacitance into one device that does not require electroforming. At the envisioned scale for a neuromorphic system, 104 synapses per neuron and 106 neurons/cm2 4, electroforming each individual NbO2 memristor becomes unfeasible, and with the constraint upon device size capacitor/switch integration becomes highly desirable.The Nb2O5) has been studied as an insulating layer in MIM diodes since at least the 1960’s5. These early studies on Nb – Anodized Nb2O5 – Au devices found rectification5 and a Poole-Frenkel conduction mechanism6. The defect state enabling Poole-Frenkel conduction was found to lie between 0.22–1.2 eV6 below the conduction band edge and was attributed to oxygen vacancies6. Additionally, it was found that upon dielectric breakdown these devices exhibited bistable switching in small conducting regions, similar to the NbO2 devices used in the 2013 neuristor demonstration7. The rectification observed in these NbO2 memristors was originally attributed the to the metal-insulator transition (MIT) occurring at 1081 K. However, there is debate in the literature regarding the operating mechanism of the electroformed NbO2 devices with both a Mott MIT2 and a Joule heat driven Poole-Frenkel mechanism9 suggested. Other studies of traditional transition metal oxide memristors show multiple conduction mechanisms for different resistance states, transitioning from metallic conduction at LRS to electron hopping at intermediate states and Schottky emission at HRS11. Simulations suggest that these NbO2 devices rectify through a modest temperature increase of 380 K–400 K which significantly increases the contribution of the highly non-linear Poole-Frenkel current transport9. Most recently two negative differential resistance regions have been demonstrated in current-controlled NbO2 devices, indicating the presence of a lower temperature Poole-Frenkel based transition and a higher temperature MIT.Niobium Pentoxide  and high resistance state (HRS) are not as relevant as the turn-on and turn-off voltages.The r is 37.1 ± 1.0 at 1 MHz. As shown in Fig. r > 180) than the capacitance at 1 MHz. This native capacitance allows the memdiode to be scaled to nanometer sizes without large external capacitors for application in the neuristor circuit. The device shown in Fig. In addition to rectification and hysteresis, it is desirable for the memristors in a neuristor circuit to exhibit capacitance. This eliminates the need for an external parallel capacitor, enabling higher density circuits. Capacitance measurements of various sized memdiodes indicate the relative dielectric constant εAs shown in Fig. 13, as shown in Fig. 2O5−x Schottky barrier height. Memdiodes with a minimum transition voltage of 0.128 V were obtained with Cu electrodes, a promising result for low voltage and low power operation. In addition, the electron affinity of amorphous Nb2O5−x is estimated to be 4.5 eV ± 0.07 eV by extrapolating the ideal work function to zero transition voltage.Devices of equal thickness (25 nm) were fabricated with Cu, Au, Ni, and Pt contacts in order to investigate the effects of the metal work function on device performance. As shown in Fig. 2O5−x thicknesses ranging from 10 nm–300 nm. As shown in Fig. Devices were also fabricated with a range of Nb14. However, this interpretation should lead to a turn-on voltage which is linearly dependent on the thickness and weakly dependent on the metal-insulator Schottky barrier height. The devices discussed herein exhibit rectification at lower voltages more consistent with the expected Schottky barrier heights for the metal-Nb2O5−x junction and possess turn-on voltages logarithmically dependent on thickness. This can be explained by considering charge transport via the defect states of the Nb2O5−x layer and by considering two separately charged regions within the Nb2O5−x layer. As shown in Fig. Rectification in MIM diodes is typically associated with a transition in current mechanism from thermionic emission in the off state to Fowler-Nordheim tunneling in the on stateAfter sufficient voltage is applied to reach the device turn-on, shown in Fig. 2O5−x-metal memdiodes predicts that for voltages below turn-on the conduction will be controlled by the Schottky barrier height and the associated thermionic current. Above the turn-on voltage, current will be limited by the Poole-Frenkel current described by Eq.  for the thermionic current at high temperatures and ln(I) for the Poole-Frenkel current, which controls the tunneling current as previously shown, at low temperatures, the Schottky barrier height and the Poole-Frenkel trap depth can be determined.To determine the trap energy in Eq. 2O5 of 1.0 eV, but slightly lower than the measured turn-on voltage for this device of 1.12 V. The higher experimental turn-on voltage is likely due to an additional voltage drop over Region 2 in thicker devices as shown in Fig. 2O56. Emission from such a deep defect would be extremely slow, resulting in a negligible contribution to current. It is therefore assumed that the observed temperature rectification is due to a transition from a shallow defect Poole-Frenkel current at low temperatures to thermionic emission over the Schottky barrier at high temperatures.The Schottky barrier height was determined for the thickest device in Fig. 2O56.As shown in Eq. 15. It was experimentally observed that the time scale to reach steady state is on the order of 10’s to 100’s of seconds indicating any reasonable neuromorphic frequency, f > 0.1 Hz, will exhibit hysteresis. The fixed charge in Region 1 determines the Region 1 width and thus the current through the device. Therefore, the rate of filling and emptying of traps at the boundary between Region 1 and Region 2 sets the time scale to reach a steady state current. Such slow trap dynamics could be caused by either slow emission of electrons from the trap states or a current limited filling and emptying of traps. The capture (ce) and emission (ee) rates for the trap states can be calculated according to Eqs 14.th is the thermal velocity, σn is the electron capture cross section, n is the density of available electrons, Nt is the density of traps, NC is the effective density of states in the conduction band, and (EC − Et) is the depth of the trap below the conduction band edge. While the exact values for some of these variables are unknown for amorphous Nb2O5, typical ranges for these variables are: νth ≈ 107 cm/s, σn ϵ  cm2, and NC ϵ  cm−3. Based on these values, the timescale for an emission limited process (Nt/ee) is nanoseconds to milliseconds, significantly faster than the experimentally observed current transients. However, for the experimentally observed range of currents, the timescale for a current supply limited capture process (Nt/ce) is milliseconds to kiloseconds, matching the experimental timescale for the current transients. Therefore, the memdiode hysteresis is likely caused by a current limited electron capture process which enables a wide range of frequencies for which the memdiode will exhibit the hysteresis necessary for neuromorphic circuitry.The observed hysteresis, a necessary property for neuristor circuit operation, is caused by non-steady state current transients when operating at sufficiently low frequencies. Low frequency operation, less than 1 kHz, is the primary region of interest for neurological processes2O5−x memdiodes, devices were fabricated at a multiple oxygen stoichiometries for chemical and electrical characterization. Film composition and the formation of oxygen deficiency related subgap states was further investigated using both hard and soft x-ray photoelectron spectroscopy (HAXPES and XPS). The oxygen content was varied by manipulating the Nb target applied power while holding the oxygen flow constant. Three deposition conditions were investigated: 50 W , 40 W, and 30 W Nb target power.To corroborate the role of an oxygen deficient active layer in the Nb2O5−x films as determined from XPS core-level measurements of a large sample set (refer to SI for more details). To circumvent issues associated with preferential sputtering during the etching, XPS measurements were performed on films transferred from growth under inert conditions. Multiple growth sets and sample locations were measured to account for chemical variations between films, similar to previous XPS studies of amorphous a-IGZO16. While the determined compositions are oxygen rich when including all measured oxygen in the compositional analysis, excluding the oxygen species attributed to surface over-oxidation and contamination from the compositional analysis results in a clear positive trend between oxygen deficiency and applied power during the growth (refer to SI for more details). We report oxygen deficient Nb:O ratios of 0.481 ± 0.027, 0.482 ± 0.021, 0.497 ± 0.018 for 30, 40, and 50 W Nb target powers respectively when excluding surface oxygen from the analysis.Figure 2 = 0.90 for the ratios including surface oxygen, and a slope = 0.0008 ± 0.0004, y-intercept = 0.46 ± 0.02, and R2 = 0.80 for the ratios excluding surface oxygen. Regardless of whether the surface over-oxidation is accounted for in the analysis, the same positive trend is observed with nearly identical slopes, confirming the bulk film indeed becomes more oxygen deficient at higher Nb deposition powers.Shown in Fig. 3/2 peak. In addition, after scaling both valence band spectra to the background, states within the gap above the main valence band edge are found to possess considerably more weight in the 50 W film. These states exist within the ~3.15 eV band gap, indicating they are stoichiometry-related defect states or split-off states rather than simple conduction band filling, and are primarily of Nb 4d orbital character. As the Nb:O ratio is increased (with increasing power) the Nb 4d state increases in intensity and shifts towards the expected bulk conduction band minimum (CBM) of pure Nb2O5. As a result, the 50 W film has more accessible states at 0.22 eV below the CBM than the 40 W film, consistent with the extracted Poole-Frenkel activation energy in Fig. Hard x-ray photoelectron spectroscopy (HAXPES), which provides a much deeper probing depth than traditional XPS, was performed at beamline I09 at the Diamond Light Source (DLS), Ltd. Although precise compositions cannot be determined from these measurements, the Nb 3d and O 1 s core level spectra, shown in Fig. I–V data from films with higher oxygen content show an order of magnitude decrease in current at 2.25 V, well above the turn-on voltage. The higher oxygen content devices also show a less defined turn-on, consistent with lower curvature in the band bending across a wider space-charge region due to a lower hole doping profile. This electrical analysis is consistent with the transport model presented above, and when considered with the XPS and HAXPES measurements corroborates the hypothesis that current rectification in metal-Nb2O5−x-metal memdiodes is controlled by Poole-Frenkel defect transport through oxygen deficiencies in the as-deposited material.The current-voltage characteristics of these devices were tested in the same manner shown in Fig. 2O5−x-metal device that exhibits the rectification, hysteresis, and capacitance necessary for high density neuristor circuitry without the need to electroform a conducting filament. These devices operate by defect controlled transport. Fixed charge accumulates in oxygen deficiency related defects near the grounded electrode supporting large portions of the applied voltage and creating two regions with differing charge occupations. One region has empty charged defects while the second region has filled neutral defects. Rectification occurs when the voltage drop across the charged region causes the current to transition from thermionic emission in the off state to a Poole-Frenkel current in the on state, which in turn controls tunneling through the charged defect region. The high density of defects causes the turn-on voltage to be dominated by the Schottky barrier height rather than the device thickness in contrast to traditional MIM diodes.We have demonstrated a metal-Nb2O5−x and the inverse correlation between oxygen concentration and additional subgap states. I–V curves corroborate the correlation between oxygen concentration and rectification. Temperature dependent measurements of the Poole-Frenkel activation energy reveal that the defect energy is approximately 0.22 eV ± 0.01 eV in agreement with the presented HAXPES measurements and past measurements of 0.22 eV for oxygen vacancies in amorphous Nb2O5. Calculating the expected rates at which the current controlling defects can capture and emit electrons reveals that the observed current transients are likely caused by capture rates limited by the supplying current of electrons. This slow trap process limits the speed at which the device can respond creating hysteresis at neurological frequencies.XPS and HAXPES confirm both the deposition condition dependence of oxygen concentration in sputter deposited NbMIM memdiodes were produced on sapphire substrates in three steps. First, a ground plane consisting of a 50 nm titanium adhesion layer and 300 nm of metal  was deposited by electron beam evaporation across the entire substrate. Next, a layer of niobium oxide was deposited through a shadow mask by room temperature reactive sputtering. Finally, 300 nm thick top metal contacts were deposited through a second shadow mask using electron beam evaporation, matching the underlying metal to form a symmetric device. The top metal contact defines the circular geometry of the devices which range in diameter from 100 μm to 1000 μm.2 environment using gas flows of 35 sccm and 15 sccm respectively. The chamber pressure was maintained at 10 mTorr. The niobium target was energized with 2.88 WDC/cm2–4.80 WDC/cm2 (30 W–50 W applied power) producing a maximum deposition rate of 5 angstroms per minute. It was previously shown that even low temperature annealing impacts the structural and electrical properties of Nb2O517. Thus, the low power deposition allowed precise control of the oxide thickness while minimizing unintentional heating of the devices.The niobium oxide insulating layer was sputter deposited at room temperature using a reactive deposition process. A metallic niobium target was sputtered in a mixed Ar/O2O5−x thin films were characterized by x-ray diffraction (XRD), x-ray reflectivity (XRR), optical transmission spectroscopy, x-ray photoelectron spectroscopy (XPS), and hard x-ray photoelectron spectroscopy (HAXPES). Symmetric XRD measurements revealed no peaks except for the sapphire substrate indicating the deposited niobium oxide is amorphous. The density of the deposited niobium oxide was determined by XRR to be 4.1 g/cm3 ± 0.1 g/cm3, significantly lower than the ideal density of 4.55 g/cm3 for Nb2O5 consistent with oxygen deficient material17. Applying the Tauc method to the optical transmission spectra revealed an optical band gap of 3.15 eV ± 0.004 eV, slightly lower than the reported range of band gaps from 3.2 eV–4 eV18. This matches previous reports of a lower band gap for low density Nb2O517. Additional material characterization details are available in the supplementary information including optßical transmission spectra, XRD, XRR, and XPS. Fabricated memdiodes were electrically tested to determine their steady state current-voltage relation, frequency dependent impedance, and capacitance both with and without bias. Steady state measurements were performed using a Keithley 6517A Electrometer. Frequency dependent measurements were performed using an Agilent 4294A Precision Impedance Analyzer with a frequency range from 40 Hz–10 MHz.Nb7/2 peak of gold foil references. To reduce surface contamination and oxidation from atmospheric exposure, samples were vacuum sealed after growth before being shipped to Binghamton University. Once at Binghamton University, the samples were opened and mounted for measurement under an inert Ar atmosphere in a glovebox and then transferred to the XPS chamber via a sealed vacuum suitcase.XPS measurements were performed using a laboratory-based monochromated Al Kα x-ray source with a hemispherical analyzer located at the Analytical and Diagnostics Laboratory at Binghamton University. Measurements were performed with a pass energy of 23.5 eV, corresponding to an instrumental resolution of 0.51 eV, determined from analyzing the Fermi edge and Au 4fHAXPES measurements were performed using a photon energy of hν ≈ 5935 eV at beamline I09 of the Diamond Light Source, ltd. synchrotron at the Harwell Science and Innovation Campus in Oxfordshire, UK. The photon beam was monochromated using a channel cut Si (004) crystal, providing an energy resolution of <0.5 eV. Binding energy calibration was performed using Au foil reference spectra. Due to the reduced surface sensitivity from the much deeper probing depth of HAXPES versus XPS, the samples were not mounted in a glovebox before performing HAXPES measurements, however, they were still vacuum sealed during shipping and stored in a vacuum desiccator when not being mounted or measured.Supplementary Information


The properties of van der Waals (vdW) materials often vary dramatically with the atomic stacking order between layers, but this order can be difficult to control. Trilayer graphene (TLG) stacks in either a semimetallic ABA or a semiconducting ABC configuration with a gate-tunable band gap, but the latter has only been produced by exfoliation. Here we present a chemical vapor deposition approach to TLG growth that yields greatly enhanced fraction and size of ABC domains. The key insight is that substrate curvature can stabilize ABC domains. Controllable ABC yields ~59% were achieved by tailoring substrate curvature levels. ABC fractions remained high after transfer to device substrates, as confirmed by transport measurements revealing the expected tunable ABC band gap. Substrate topography engineering provides a path to large-scale synthesis of epitaxial ABC-TLG and other vdW materials. The semiconducting ABC configuration of trilayer graphene is more challenging to grow on large scales than its semimetallic ABA counterpart. Here, an approach to trilayer growth via chemical vapor deposition is presented that utilizes substrate curvature to yield enhanced fraction and size of ABC domains. One example is trilayer graphene (TLG)8, which has two stacking configurations in its natural form. The ABA or Bernal configuration is a semi-metal with tunable band overlap10, while the ABC or rhombohedral configuration is a semiconductor with a gate-tunable band gap12. In fact, few-layer graphene with ABC stacking is an interesting system for all thicknesses, as it is predicted to have topologically-protected surface states15 and high-temperature surface superconductivity17. Although ABA appears to be the ground state and is observed more often in exfoliated trilayer graphene, the thermodynamic preference over ABC is small, and a mixture is usually found. There are indications that the choice of substrate or the number of layers can affect this delicate ordering balance19.Van der Waals (vdW) materials composed of individual atomic layers held together by vdW interactions have attracted considerable attention due to their unique physical properties that can be tuned by manipulating interlayer twisting angles22, which would catalyze a wide range of studies of the unexplored physics and applications of this unique material system. To answer this need, we develop a high-yield chemical vapor deposition (CVD) process to grow epitaxial TLG on Ni–Cu gradient alloy substrates. Angle resolved photoemission spectroscopy (ARPES) is used to confirm the epitaxial growth process and the characteristic band structures of ABC-TLG and ABA-TLG. Transmission electron microscopy is used to assess the atomic structure of the material, and it reveals the existence of quasi-lamellar patterns of ABA-TLG and ABC-TLG. Based on these observations, we develop a model for how substrate topography and, more specifically, the curvature of surface corrugations, plays a principal role in stabilizing ABC-TLG during growth. This curvature-based stacking selection (CBSS) mechanism is confirmed by an experiment correlating CVD-grown TLG material with the original growth substrate. We then demonstrate that the substrate curvature can be engineered through CVD growth time for controllable ABC-TLG yields of ~59%. We further find that transfer of TLG from the corrugated growth substrate to flat SiO2 leads to annihilation of ABA-ABC domain walls and increased ABC and ABA domain size, as revealed by infrared scanning near-field optical microscopy (IR-SNOM). Electron transport measurements demonstrate that the band gap of the CVD-grown ABC-TLG could be tuned by a perpendicular electric field. Our finding that substrate topography can be used to stabilize the ABC stacking structure may enable development of related CBSS synthesis strategies for other vdW epitaxies with specific interlayer stacking configurations and study concerning their physical properties and applications.To date, electronic transport studies of ABC-TLG have relied heavily on exfoliated samples, a process that is not scalable and limits the range of experimental investigations. A critical experimental challenge is to develop large-area synthesis of high-quality ABC-TLG23. Catalytic substrates were prepared by depositing a 100-nm thick Ni film on one side of a flat Cu foil. The Ni–Cu foils were then annealed at 1050 °C for 5 min to create a Ni–Cu gradient alloy substrate with a Ni-rich side and a Ni-poor side. During CVD growth, the top TLG layer forms first over the Ni-rich side of the substrate, and then the middle and bottom TLG layers grow beneath the top layer via carbon back-diffusion from the Ni-poor side23. Additional information about the growth process is provided in the Methods section. As shown in Fig. As shown schematically in Fig. E vs. 24. The orientation, size, and distribution of graphene grains, as well as their registry with the substrate were investigated using micro-ARPES. Figure d bands from Cu and/or Cu–Ni alloy are evident with binding energies of 2–4 eV. Distinctive d and sp substrate bands in the 3D ARPES plots indicate a high degree of crystallinity in the metallic substrate foils after CVD growth.We performed systematic micro-ARPES and nano-ARPES measurements of the band dispersions of the material on the growth substrate, with 140 µm and 120 nm spot size respectively, to evaluate the electronic properties of the as-grown material. We directly probed the binding energy vs. momentum  plane. Three well-defined Dirac cones are observed at −1, corresponding to the π graphene bands that cross the Fermi level. In contrast to earlier reports on few-layer graphene grown by CVD24, no arc-like shape states around the cones are observed, indicating that our graphene flakes are oriented along the ΓΚgraphene [25. These results demonstrate that our process leads to epitaxial growth of TLG on the Ni–Cu gradient alloy substrate.The graphene-substrate registry and the orientation of the graphene flakes are revealed by the multi-grain Fermi surface shown in Fig. E vs. momentum) was recorded for individual graphene grains using high energy and momentum resolution nano-ARPES26. The most intense flakes in Fig. 28. TLG flakes containing a mixture of both stacking types were also identified  calculations . Figure 33, which with ABC stacking completely destructively interferes and with ABA stacking only partially interferes. The 34 and larger than usual levels in graphite. We observed flakes with up to 100% ABC content  was used to probe the stacking morphology and crystallinity of the as-grown TLG. This method can provide layer number contrast for oriented grapheneage Fig.  of the s34. Importantly, this near equivalence between ABA and ABC energies suggests that the abundance of the two states can be tuned in finite-sized flakes by modulating other factors that slightly bias their relative energetics.The large-area fraction of ABC phase and the quasi-lamellar ABA/ABC domain morphology Fig.  suggest d is the distance between graphene layers  becomes energetically preferable to maintaining atomic registry between layers at all points. For weakly bound atomic layers of the type studied here36, κc is κ0 > κc will result in arrays of interlayer dislocation lines, several types of which induce stacking shifts between ABA and ABC  less prevalent ABC phase. Our growth substrates had a quasi-periodic pattern of surface corrugations (discussed further below) with typical curvatures of ima Fig. . When aders Fig. . Graphenentclass1pt{minima37 formation and domain size, we formulated a first principles-informed continuum mechanics description of the structure of vdW materials on non-flat surfaces (see Methods section for details). The dislocation character of a domain wall is denoted z = zigzag or a = armchair), X and Y specify the stacking configuration  between the upper two and lower two graphene layers, respectively  to be, see Fig. The results demonstrated that curvature stabilizes interlayer dislocations and that certain types of stable dislocations in turn imply the presence of ABC domains. Specifically, our model indicates that the equilibrium state changes with increasing curvature from perfect ABA to states containing interlayer dislocations Fig. . A substPES Fig.  and DFTEentclass1pt{minimaentclass1pt{minima38) and/or structural domains with only simple periodic stacking sequences  are not likely representative of actual states.Our description of CBSS indicates that rich internal stacking structures will be commonplace in CVD multilayer graphene systems  and only 7% ABC coverage over the other two grains which have significantly lower curvatures (~1–2 × 106 m−1). Further details of the IR-SNOM technique are provided below.To directly test the CBSS mechanism, we marked a CVD-grown sample such that DFTEM images of domain wall morphologies could be mapped back to AFM images of the substrate topography at the precise location where growth occurred  that the primary driving force for this phenomenon is the reduction of the total step/curvature-induced interlayer disregistry/dislocation energy . Accordingly, increasing growth time from 1 to 6 h led to a monotonic increase in the final average peak corrugation curvatures (measured on our Cu (111) substrates) from ~7.0 × 10m−1 Fig. . ABC yiem−1 Fig. . The inc2 substrate and imaged with infrared scanning near-field optical microscopy , greatly superior to the ~1 µm typically achieved by Raman spectroscopy34.We performed additional experiments to confirm that curvature-stabilized ABC domains were preserved and exhibited appropriate electronic properties after transfer and conformation to flat dielectric substrates commonly used for device fabrication. TLG flakes were transferred to a SiO2, and three striking phenomena were identified. (1) Quasi-lamellar stacking domains, similar to the DFTEM image of Fig. 2 (5.6 µm2)  curves of an ABC-TLG channel. The existence of a tunable energy band gap in the material is evidenced by the increase in the on/off ratio as the strength of the out-of-plane electric field is increased. The same measurement showed that a sizeable ABC band gap was also achieved at room temperature ; the use of substrate curvature on the nanoscale to generate interlayer dislocations and locally stabilize ABC domains. The quasi-periodic corrugated topography of our growth substrate (commonly observed) typically generates alternating lamellar regions of ABC and ABA, as revealed by DFTEM and IR-SNOM measurements. An optimal range of curvatures, tunable through growth time, leads to maximized ABC yield, consistent with our continuum CBSS model predictions. The coarsening of substrate topography beneath growing graphene overlayers is attributed to the reduction of interlayer disregistry/dislocation energy achieved by increasing corrugation scale. ARPES results on as-grown TLG confirmed the epitaxial nature and electronic hallmark of ABC-TLG and ABA-TLG, while the existence of a sizable, gate-tunable band gap for ABC-TLG regions was confirmed by electron transport measurements. Significant increases in ABC and ABA domain size were observed following transfer from growth substrates to flat SiO3 for 40 s and two DI water baths for 2 min each. A nickel film of 100 nm thickness was sputtered onto one side of the Cu foil (Lesker PVD75 DC/RF Sputter System). Ni–Cu foils were cut into 1 cm × 4 cm pieces and then loaded into the CVD furnace. The furnace was ramped to 1050 °C at a rate of 60 °C/min in a flow of 500 sccm Ar without H2. The growth substrate was annealed for 5 min at 1050 °C in a flow of 500 sccm Ar + 30 sccm H2. TLG was grown using 1.8 sccm CH4 (1% in Ar) + 30 sccm H2 for 3 h. The reactor was then rapidly cooled to room temperature in a flow of 10 sccm H2 and 1000 sccm Ar.The TLG growth was carried out in an atmospheric pressure CVD system with a 1-inch furnace . Cu foils of 25 µm thickness  were cleaned with 5.4% HNO2/Si substrate with prefabricated gold makers. After air drying and baking at 150 °C for 3 min, PMMA was removed by soaking the sample in acetone overnight and cleaning with IPA. TLG on SiO2/Si substrates were annealed in H2/Ar forming gas at 400 °C for 1 h before IR-SNOM characterization.PMMA  was spun over the surface of the TLG as grown on Ni–Cu foils and then baked at 100 °C for 2 min The substrate was removed by an etcher solution . The PMMA supported TLG was cleaned in 10% HCl solution and two water baths, and was transferred onto a 250 nm SiO2 laser beam with wavelength 10.6 µm and the enhanced local field under the tip interacts strongly with the sample underneath it. The elastically scattered light containing the local optical properties of the sample substrate is collected by a MCT (HgCdTe) detector in the far field. Near-field optical images with spatial resolution of sub 20 nm can be achieved simultaneously with topography by recording the scattered light while scanning the sample. ABA/ABC graphene have different optical conductivities at 10.6 µm due to their different band structures and thus can be differentiated in the near-field images. In bilayer graphene, the contrast of domain walls in near-field images stems from surface plasmon reflection at the domain walls and feature a single or double bright line depending on the domain wall type47. Operation in tapping mode rather than contact mode40 prevents imaging-induced domain wall motion, and no such motion was observed in these experiments.Scattering-type scanning near-field optical microscopy is based on tapping mode atomic force microscopy (AFM). A metallic AFM tip is illuminated with a focused COAn atomic force microscope  equipped with a probe with a tip radius of 10 nm  was used to characterize the topography of the growth substrate, in a different facility from that in which IR-SNOM measurements were performed.2 for 1 h to reduce the PMMA residuals on TLG channels. The devices were spin-coated with a HSQ buffer layer , followed by atomic layer deposition (Cambridge Nanotech S200) of 40 nm Al2O3 as the top-gate dielectric. Lastly, e-beam lithography was carried out to deposit 5 nm Ti/40 nm Au top-gates on the Al2O3 dielectric to form the dual-gate FET devices.ABA-TLG and ABC-TLG regions were first identified by IR-SNOM. ABA-TLG and ABA-TLG channels were then defined by e-beam lithography and oxygen plasma etching . The remaining e-beam resist  was removed using acetone and isopropanol (IPA). The top contact metallization for the FET device (5 nm Cr/40 nm Au) was deposited by e-beam lithography followed by thermal evaporation. After liftoff process, the device was annealed at 225 °C in the forming gas of 1000 sccm Ar + 250 sccm HR–VTG curve was measured with a fixed back-gate voltage (VBG) in steps of 20 V, and VTG was swept continuously.Low temperature electrical transport measurements were performed in a Quantum Design Physical Property Measurement System at an ambient He pressure of ~5 Torr. Transport measurements were recorded using the drain as a common ground for the source and gates. Voltages for top and back gates were independently controlled by Keithley 2410 and Keithley 237 voltage sources, respectively. Gate leakage was verified to be <0.5 nA. Source-drain were biased for constant voltage using a second Keithley 237, with maximum power <0.5 nW across all measurements. After verifying source-drain linearity, source-drain current-voltage data with mV scale source bias was used to determine the channel resistance for each gate configuration. Each 33. The 32. This can be seen in Fig. TLG was transferred onto a Cu grid with an amorphous carbon support. An aperture was placed in the diffraction plane of an electron microscope to create a real space image from electrons scattered only at a specified angle. With oriented stacked graphene, the The ARPES and nano-ARPES experiments were carried out at the ANTARES beamline of synchrotron SOLEIL. It is equipped with a Fresnel zone plate (FZP) to focalize the beam and order selection aperture (OSA) to eliminate higher diffraction orders. The sample was mounted on a nano-positioning stage which was placed at the coincident focus point of the electron analyzer and the FZP. Nano-ARPES can be operated in two ways: (1) the point mode where a spectrum is collected from a specific point of the sample and (2) the imaging mode, where photoelectron intensity from a specific electron energy range is mapped over the sample to create a two dimensional image of the electronic states of interest. All photoemission measurements were performed at a temperature of ∼60 K. The photoelectron spectra were obtained using a hemispherical MBS analyzer. The instrumental energy resolution is ∼12 meV and practical momentum resolution is ∼0.02 1/Å for an emission angle ∼20°48 package using the Perdew-Burke-Ernzerhof (PBE) generalized-gradient approximation49 supplemented by the TS method50 to account for dispersive interactions. We use the suggested tight setup and accompanied basis functions throughout. A Cu (111) surface was generated from an optimized copper slab using six layers, where the bottom three layers were kept fixed to their bulk positions. A vacuum layer of 30 Å was introduced to separate periodic images along z, and a finite-size correction for the slab was employed. The Brillouin zone was sampled by a 11 × 11 × 1 Monkhorst-Pack k-point grid51, and k-point convergence was improved by filling the electronic states with a Gaussian broadening of 0.1 eV. Models including nickel doping of 3.7% were calculated in 3 × 3 surface supercells of the above-described copper surface, reducing the number of layers to three and replacing one copper atom with nickel. Binding energies were defined throughout as E0(Cu), and n-graphene sheet system, the defined Cu(111) surface, and n-graphene sheets, respectively. Transition states were searched with the climbing-image nudged-elastic-band method using five images.52All-electron density-functional theory (DFT) calculations were carried out with the Fritz-Haber-Institute-Ab initio-Molecular-Simulationsx such that the disregistry energy over a region of size LCu (per unit length y) is53 is the 2D Young’s modulus of graphene, i is the interlayer index , Ni is the integer dislocation density between x = 0 and x = LCu, ε,i0 is the in-plane misfit strain between flat layers in i, and di = 0.335 nm54 is the interlayer separation. We separate geometric strains from elastic strains in εκ.i is that introduced via curvature under the condition of epitaxial registry between layers  or the graphene layer below it (i = 1 or 2). This form also invokes the assumption that dislocations relieve strain uniformly over each local peak or valley. The only non-material-parameter input into the model, the substrate curvature profile κ(x), was determined by fitting AFM-measured substrate topographies to a Fourier sine series and parameterizing the resulting curvatures at each corrugation extremum as i = 3 (the substrate). Bending energy differences between states can also be neglected . The total dislocation line energy  was expressed asa = 0.14 nm is the nearest neighbor distance in graphene, Eedge = 0.318 × 10−10 J/m and Escrew = 1.091 × 10−10 J/m are the core energy coefficients, B = 22.08 × 10−20 J is the bending modulus of graphene. The first term in the square brackets is the core energy and the second term is the contribution from long-range interactions between interlayer dislocations. The bulk energy of the trilayer is Ei is the bulk energy of ABA or ABC per unit volume, and The total energy of an elastic multilayer system (weakly bonded sheets) was divided into contributions from in-plane disregistry strain, interlayer dislocations, and stacking phase bulk energies; 2σ2 Fig. . We furt32). The energy of each state was determined as a function of maximum local curvature κ0 , as shown in Fig. We considered ten possible TLG states; perfect ABA, perfect ABC, and eight configurations with periodic dislocation arrays Fig. . There aSupplementary Information


Undisturbed flows are critical in these systems, and air bubbles entering microfluidic channels can lead to device delamination or cell damage. To prevent bubble entry into microfluidic channels, we report a low-cost, Rapidly Integrated Debubbler (RID) module that is simple to fabricate, inexpensive, and easily combined with existing experimental systems. We demonstrate successful removal of air bubbles spanning three orders of magnitude with a maximum removal rate (dV/dt) In the 1990s microfluidic systems gained popularity in analytical “lab-on-a-chip” platforms owing to unique microscale capabilities including robust sample routing, rapid multiplexed analysis, and laboratory portability . Over thmax ranging from 0.5 μL min−1 to > 2 mL min−1.Bubble removal can be divided into two general approaches, (i) bubble traps and (ii) debubblers. Traps focus on guiding bubbles to a containment reservoir before they enter the cell culture region; traps leverage the buoyancy of air bubbles and can be vented with an external vacuum source as the reservoir capacity is reached ,13,14. DAlthough myriad debubblers have been reported in the literature, they are often complex and highly application-specific; thus, integration into more general microfluidic systems represents a significant implementation barrier. To address hurdles related to both complex fabrication and integration of debubblers, we introduce a simple workflow to create a rapidly integrated debubbler (RID) module that can be easily combined with existing microfluidic systems. Key practical features of the RID module include (i) an accessible fabrication process with rapid assembly (<2 min), (ii) low device cost (<$0.50 at lab prototype level), and (iii) press-to-fit tubing connections to simplify component integration. Controlled shear stimulation of cultured cells is a hallmark capability in microfluidic systems that enables quantitative correlation between applied fluid-induced wall shear stresses (WSS) and cellular responses including endothelial cell alignment , calcium2 laser  with inlet and outlet ports in L1 designed to house #003 rubber O-rings (outer diameter (OD) = 3/16”, inner diameter (ID) = 1/16”, Durometer 70A, McMaster-Carr). Pressure sensitive adhesive films  were rolled onto the top surfaces of L1–L2 prior to laser cutting using a cold roll laminator. A polytetrafluoroethylene (PTFE) membrane  was cut with a razor blade and placed in contact with L3. L3 (PMMA) was used to protect the PTFE membrane from damage due to improper insertion of the tubing. The 2 mm wide by 12 mm region cut into L5 (polyester film with PSA on bottom surface) defined the degassing region of the device, and the PSA lamination produced liquid-tight sealing between layers. Once all layers were cut in a batch process, individual devices could be assembled in less than 2 min. The packed O-ring assembly allowed simple press-to-fit connection with 1/16” OD tubing, and the addition of barbed end fittings  allowed attachment to flexible tubing. The overall footprint of the RID module was 10 mm × 18.5 mm (W × L). As shown in total minimization as the vapor (v) phase  and liquid (l) phase interact with the solid components of the PTFE membrane. The change in total surface energy, ΔEtotal, can be expressed as the change in interfacial area, ΔA, during the attachment of vapor to the membrane, and the difference in surface energy (γ) between the solid-vapor (sv) and solid-liquid (sl) interfaces [The operating principle of the RID module is based on total surface energy Eterfaces : (1)ΔEtosl to the interfacial energies: The Young equation relates the solid-liquid contact angle θsl > 90°) the right-hand side of Equation (2) is negative implying that γsv < γsl; from Equation (1), there is a corresponding energetically favorable decrease in total surface energy. Conversely, for a hydrophilic material (θsl < 90°), γsv > γsl with unfavorable ΔEtotal > 0. Thus, the hydrophobic PTFE membrane used in the RID module energetically favors the formation of vapor-solid interfaces when compared with a hydrophilic material.With a hydrophobic material,  or by decreasing the pore size (Equation (3)). At P > Pb, and (iii) Vb:VT ratio . A test condition failed when a bubble was visualized in the fluid exiting the device via image analysis. We conservatively report the failure threshold as the lower bound of the 95% confidence interval determined from failure testing of four independent RID devices. The equation WSS = 6 μQw–1 h–2  [channel = 1 μL). To characterize RID bubble removal, individual streams of air and liquid  were combined to create segmented flow streams and directed toward the input port of the RID. The test liquid was complete cell culture media Dulbecco’s Modified Eagle Medium /F-12  with food color added to increase contrast during imaging. Flow streams entering and exiting the RID module were simultaneously recorded  and analyzed with a custom MATLAB image processing script see . Black a h << w)  was usedopen. Results reported as mean ± standard deviation (SD) from four independent devices.A 5 mL fluid reservoir was connected vertically to the input of the debubbler. With the debubbler dry, cell culture media was added to the reservoir to generate a hydrostatic pressure head ΔP = ρgΔh with Δh denoting the difference in height between the media level and the outlet port. Using video analysis, the height difference required to initiate flow through the debubbler was determined and converted to the opening pressure Pcritical at which fluid was forced through the PTFE membrane. Results reported as mean ± SD from four independent devices. A syringe pump was used to introduce cell culture medium to the RID, while the outlet port was connected to dead-end tubing. Sensors on either side of the debubbler  measured the pressure across the module as a function of time. Video analysis was used to determine the critical liquid pressure PA glass capillary viscometer was used to measure the viscosity of DMEM/F12 cell culture media with 10% FBS. Fluid density was determined by measuring mass of a known media volume using an analytical balance  at 37 °C. A goniometer  was used to measure the interfacial tension of cell culture media in air, and the contact angle between the culture medium and PTFE membrane using the hanging drop and sessile drop methods respectively. Results reported as mean ± SD from four independent devices. –2 on Geltrex coated tissue culture flasks. Media was replaced every 24–48 h. For routine culture, human umbilical vein endothelial cells (HUVEC) were cultured in DMEM/F12 with 10% v/v FBS using the manufacturer’s protocols. Briefly, HUVECs were enzymatically disassociated using Trypsin/EDTA  and sub-cultured when they reached 70% confluence. Cells were centrifuged at 250 g, resuspended in medium, and counted. Cells were plated at 5000–10,000 cells cm−1) at 37 °C for 1 h to improve cell adhesion. Excess Geltrex solution was aspirated, and a PDMS microfluidic network was magnetically sealed against the glass substrate. The microfluidic channel network was fabricated using a soft lithography process described previously [−2 and allowed to attach overnight. Cells were then treated with Calcein AM  and the cell permeable Hoechst 33342 stain  for 15 min to aid visualization of HUVEC cytoplasm and nuclei respectively. HUVECs in culture were imaged after exposure to a segmented air-liquid stream with flow rate corresponding to WSS of 11.5 dyne cm−2 with and without a RID module upstream of the culture device. The RID modules were sterilized with ethanol followed by 3× wash with PBS to remove residual ethanol and allowed to dry before use. For bubble exposure studies, a microfluidic channel network was sealed against a glass slide using our previously reported reversibly Sealed, Easy Access, Modular (SEAM) culture platform ,25. Glaseviously . HUVECs −2) [−2) [channel = 1 μL. The range of tested bubble volumes was selected to evaluate bubble removal capabilities under common flow disruption scenarios: (i) Vb < Vchannel, (ii) Vb ≥ Vchannel, and (iii) Vb >> Vchannel . A custom image processing algorithm was used to measure Vb, Vb:VT, and Q. Results from the RID bubble removal testing are summarized in To ensure that RID modules could be used for shear stimulation studies relevant to human (1–50 dyne cm−2)  and rode−2) [−2) , segmentb ≤ 100 μL) from segmented flows (0.07 ≤ Vb:VT ≤ 0.7) including the challenging high Q (high WSS), small Vb, and high Vb:VT conditions where dV/dt must be sufficiently high to remove bubbles before they are displaced downstream by the incoming flow. The average WSS at failure = 289 ± 48 dyne cm−2 with 95% confidence interval, 213 < WSS < 352 dyne cm−2; the dashed line in −2 or Q = 2.6 mL min−1), and represents a conservative limit of the operational range. Although the 95% confidence interval is relatively large, the RID reproducibly removed large and small volume bubbles at flow rates corresponding to WSS that span the physiological ranges of both human and rodent cells commonly used to study shear stimulation in vitro. RID (dV/dt)max = 1.5 mL min−1, enables rapid removal of bubbles, and is competitive with more complex and specialized debubblers. Traditionally, debubblers are designed for a particular bubble removal operation range; to the best of our knowledge, this is the first demonstration of a rapid debubbler spanning at least three orders of magnitude in removed Vb with a single device. It should be noted that Vb = 100 μL was the maximum bubble volume tested due to experimental limitations and was not the operational failure limit of the RID. Although bubbles with Vb < 200 nL could not be reproducibly generated using our experimental setup, we anticipate that smaller volume bubbles would be removed either at (i) the entrance of the RID module via contact with the PTFE membrane due to the perpendicular orientation of the input tubing or (ii) in the gap between the membrane and PMMA where the bubble interactions with the membrane are energetically favorable. The RID successfully removed bubbles across three orders of magnitude  at Q = 1.1 mL min−1 (liquid WSS = 90 dyne cm−2); flow direction is left to right. Bubbles entering the RID module were removed with dV/dt = 440 μL min−1 and single-phase flow exited the device  was maintained for both experimental conditions. −1 h−2. Under the test conditions, we estimate the amplification factor Φ comparing WSS with and without bubbles  = 55 , we measured (i) the minimum pressure, Popen, required to deflect the PTFE membrane from the PMMA layer and (ii) the maximum pressure, Pcritical, above which cell culture media was forced through the hydrophobic PTFE membrane pores. The operating pressure range spanned three orders of magnitude with Popen = 7.4 ± 0.4 Pa and Pcritical = 9.7 ± 1.7 kPa. The small opening pressure can be attributed to the limited adhesion between the hydrophobic PTFE and the PMMA surface that must be overcome by the flow stream pressure. Pcritical is a function of membrane pore diameter; the manufacturer’s reported PTFE pore diameter of 10 μm was sufficient for our applications. However, a membrane with a smaller pore size can be used if a higher Pcritical is desired (see Equation (3)). It should be noted that the PTFE membranes do not contain well-defined circular pores but are comprised of a fibrous mesh that contain voids between fibers; the relatively large 95% CI for WSS at failure can be attributed in part to inhomogeneities between membranes. To determine the operating pressure range for the RID . The interconnection problem can be a source of frustration because each component can have different tubing requirements and corresponding mating mechanisms . A goal of this work was to increase accessibility by providing an integration approach to simplify connections. Here, we implemented a simple connection mechanism via embedded O-rings in the top housing layer of the RID module that form a compression seal against inserted rigid tubing. The press-to-fit O-ring connectors See  were ablTo facilitate customization and improve debubbler accessibility for the microfluidics community, the RID fabrication workflow uses equipment commonly found in a community makerspace or general research laboratory. Our approach incorporates commercially available materials , and modules can be assembled in less than 2 min when layers  are cut We have demonstrated a simple fabrication workflow to create a debubbler that can rapidly remove bubble volumes spanning three orders of magnitude from segmented flows at flow rates compatible with those required for microfluidic shear stimulation studies. The fabrication process can be carried out in a general makerspace or research laboratory and the footprint and fluidic interconnections can be customized as needed to fit existing experimental setups. We anticipate that the combination of simple fabrication, integration, and functional capabilities will enable RIDs to be easily implemented into microfluidic applications where the entry of bubbles is undesired.


N = 136) of zebrafish egg clutches, each clutch being split to monitor developmental success and perform transcriptome analysis in parallel. We aimed at drawing a molecular portrait of the egg in order to characterize the relation between egg transcriptome and developmental success and to subsequently identify new candidate genes involved in fertility.Egg quality can be defined as the egg ability to be fertilized and subsequently develop into a normal embryo. Previous research has shed light on factors that can influence egg quality. Large gaps however remain including a comprehensive view of what makes a bad egg. Initial development of the embryo relies on maternally-inherited molecules, such as transcripts, deposited in the egg during its formation. Bad egg quality is therefore susceptible to be associated with alteration or dysregulation of maternally-inherited transcripts. We performed transcriptome analysis on a large number . Statistical modeling using partial least squares regression and genetics algorithm demonstrated that gene signatures from transcriptomic data can be used to predict developmental success. The identity and function of differentially expressed genes indicate a major dysregulation of genes of the translational machinery in poor quality eggs. Two genes, ootulina and slc29a1a, playing an important role for fertilization. Together, our observations highlight the diversity of the possible causes of egg quality defects and reveal mechanisms of maternal origin behind the lack of fertilization and early embryonic failures that can occur under normal reproduction conditions.Here we show that egg transcriptome contains molecular signatures, which can be used to predict developmental success. Our results also indicate that poor egg quality in zebrafish is associated with a dysregulation of (i) the translational machinery genes and (ii) novel fertility genes, The online version of this article (10.1186/s12864-019-5930-8) contains supplementary material, which is available to authorized users. Good quality or developmentally competent fish eggs are defined as those that are successfully fertilized and develop normally as viable, non-malformed embryos . Howevernpm2a and npm2b) genes were recently found to be crucial for egg quality; suppression of npm2b resulted in embryonic arrest before zygotic genome activation (ZGA) in mouse and zebrafish, and npm2a deficiency in zebrafish led to a complete lack of embryonic development , ribosomal protein S27 (isoform 2) [rps27.2], and U1 spliceosomal RNA [U1] with fold changes of 7.81, 1.90, and − 2.33/− 2.35, respectively) are associated with ‘translation’ and ‘ribosomes’.We submitted the 66 DEGs to Gene Ontology functional annotation analysis using the online program, DAVID  with thep-value and fold change in the microarray analysis are shown in Additional file rpf2 , spon1b [spondin 1b] , tspan7b [tetraspanin 7b] , rps27.2 , stra13 [stimulated by retinoic acid 13 homolog/centromere protein X] , and rtn4ip1 [reticulon 4 interacting protein 1]  was increased in bad quality eggs as compared to good quality eggs, while that of U1  and slc29a1a  were increased in bad relative to good quality eggs  in good quality eggs, using RNA-seq data stored in the PhyloFish [slc29a1a and otulina were predominantly expressed in ovary, egg, and/or embryo, thus, functional knockouts of these genes would mostly affect the female reproduction and the ensuing embryogenesis with minimal effect on non-reproductive organs. qPCR analysis for otulina  were identified by fin clip genotyping and subsequently mated with wild-type (WT) or Dr_vasa:eGFP C3  males, and embryonic development was recorded. While mutations could be detected in F0 females, these mutations were not transmitted to the next generation thus making it impossible to generate non-mosaic homozygote mutant fish for further analysis. Since the mutagenesis efficiency of the CRISPR/Cas9 system was very high [otulina and slc29a1a genes were however sufficiently knocked-out in the transgenic mosaic F0 females to study the phenotype associated with the partial knock out of target genes, as previously shown for other maternal genes in zebrafish [otulina and slc29a1a in the eggs of these mutant mosaic females as compared to those from control WT pairings  and slc29a1a (n = 10) mutants could be observed even in the F0 generation. All of our observations were therefore obtained from the F0 generation using eggs from mosaic mutant females in which target genes were partially knocked out and corresponding expression significantly reduced in eggs.In order to validate the in vivo significance of some of the DEGs, we performed functional analysis by genome editing using the CRISPR/Cas9 system. We first examined the tissue localization of our validated differentially abundant transcripts, hyloFish  online dery high , 18, theebrafish . This waotulina and slc29a1a mutant-derived eggs had a significantly lower developmental success, defined as the proportion of surviving embryos at 24 hpf to the total number of spawned eggs  in comparison to controls (74.61 ± 7.9%) , partially cellularized , and normal development, as shown in Table slc29a1a mutants displayed some heterogeneity in their offspring; while a proportion of the spawn did not develop and did not undergo cell division as observed previously, a number of cells underwent abnormal development characterized by asymmetrical cell division and the appearance of a cell mound on top of an enlarged cytoplasm, which occurred until approximately 4–5 hpf  Fig. d. Eggs fotulina, we assessed the spawned eggs from otulina-mutant females crossed with WT males for the transcript levels of components of the wnt  and tnf/nf-κb  pathways as otulina plays a role in these pathways in mammalian models [wnt3a, tcf7, lef1, and dvl2, but not tcf3, transcript levels were significantly decreased in the otulina mutant-derived eggs . In addition, we compared the distribution of selection frequencies of each gene in the final populations using the actual and randomized data. We did not observe significant peaks (genes with high selection frequency) in the randomized data . We ran the PLS-GA procedure with 70 populations of 500 potential solutions, i.e. subsets of 1 to 20 randomly selected genes. We thus obtained 35,000 final potential solutions, which we evaluated by 10 runs of 2-fold cross validation (2-FCV); we defined the average cross-validated Rrpf2 [rps27.2 [spond1b [rtn4ip1 [tspan7b [stra13 [slc29a1a [In this study, we identified 66 DEGs between good and bad quality eggs at the one-cell stage, which is a relatively low number as compared to other studies. In fact, it must be reiterated that all of the couples that were mated and produced clutches were wildtype without any particular treatment in contrast to most existing studies on fish egg quality. Thus, there may have been multiple natural causes behind the decline in quality of the eggs from the different mothers, such as nutrition, density, age of parents, delay from last spawn, and genetics just to name a few , 23, 24.rpf2  is a rib[rps27.2  is a str[rtn4ip1  is a mit[tspan7b  is a cel [stra13  has roleslc29a1a  is transrpf2 and rps27.2 were found by microarray and confirmed by qPCR to be increased in bad quality eggs ; it was revealed that rpl24 transcript, which encodes a structural component of the large ribosome subunit, was more abundant in the latter which had higher mortality [With regard to the functional characteristics of the DEGs in the study using all samples, overrepresentation analyses of the GO terms by the DAVID gene ontology program found that genes that function in ribosome and translation were predominantly enriched. A recent study demonstrated that ribosome was one of the top over-represented KEGG pathways in the expressed transcriptome of unfertilized zebrafish eggs, which suggests that the ribosome/translation process plays a major role in fertilization and subsequent embryonic development . Furtherortality . Thus, iObtaining a gene signature to predict the survival rate is valuable and of practical interest as the identification of a set of genes that correlates with the rate of survival can open up avenues for understanding the biological phenomena to explain egg quality and for future applications in aquaculture . Existinotulina and sc29a1a due to their ovarian-predominant expression and dysregulation in bad quality eggs . This ubiquitination-deubiquitination system is a key regulator of important signaling pathways, including Wnt, TNF-α, and NF-κb. The otulin gene, the mammalian ortholog, has been previously shown to be involved in early development in mice since a functionally-disruptive gene mutation results in embryonic lethality due to perturbed Wnt signaling and angiogenesis [otulin has also been shown to be a key factor in regulating inflammation and immunity through its modulatory role in the TNF-α and NF-κb pathways [wnt components including wnt3a, tcf7, lef1, and dvl2  pathways [slc29a1 in fish is still unknown since these species usually undergo external fertilization and embryonic growth. Further investigations into their physiological functions are warranted.On the other hand, clampsia –47. In apathways . Howeverotulina and slc29a1a, that play an important role to allow the production of fertilizable eggs.In this report, we show that statistically robust gene signatures exist in the maternally-inherited transcriptome that could be used to predict development competence. We also report that poor zebrafish egg quality is associated with a dysregulation of the translational machinery genes. Finally, we identified novel fertility-related genes, Danio rerio) of the AB strain were maintained at 25 °C in a central filtration recirculating system with a 12 h light/dark cycle in the INRA LPGP fish facility . Breeding pairs were kept in the same tank overnight separated by a partition, and in the morning, the divider was removed after which the female released her eggs to be fertilized by the male. One hundred and thirty-six clutches of fertilized zebrafish eggs at the one-cell stage were harvested and divided into two parts. One part was flash-frozen in TRI reagent  and stored at -80 °C for molecular biology analyses. The other part was cultured in modified Yamamoto’s embryo solution  and monitored for up to 48 h, and the number of survivors was counted at 8, 24, and 48 hpf. Good quality eggs were defined as embryos that had a very high survival rate (> 93%) at 48 hpf and bad quality eggs were those that suffered a very low survival rate (< 38%) at 48 hpf. Those percentages were selected to obtain two groups of contrasted phenotype  with sufficient statistical power.Wildtype zebrafish . All samples were confirmed to have a RIN (RNA integrity number) of 9–10 which are generally accepted as reflecting very good quality RNA.Zebrafish gene expression profiling was conducted using an Agilent 8x60K high-density oligonucleotide microarray. Labeling and hybridization steps were performed following the Agilent “One-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp labeling)” protocol. Briefly, for each sample, 150 ng of total RNA was amplified and labeled using Cy3-CTP. Yield (> 825 ng cRNA) and specific activity (> 6 pmol of Cy3 per μg of cRNA) of Cy3-cRNA produced were checked with the NanoDrop 2000 spectrophotometer . 600 ng of Cy3-cRNA was fragmented and hybridized on a sub-array. Hybridization was carried out for 17 h at 65 °C in a rotating hybridization oven prior to washing and scanning with an Agilent Scanner  using the standard parameters for a gene expression 8x60K oligoarray (3 μm and 20 bits). Data were then obtained with the Agilent Feature Extraction software (10.7.3.1) according to the appropriate GE protocol (GE1_107_Sep09) and imported into GeneSpring GX software (Agilent Technologies) for analysis. The data were first normalized by median centering, log-transformed, and and filtered considering expressed genes those which have a signal above background in at least 80% of the samples in at least one of the two conditions. Then differential expressed genes (DEGs) were determined by performing a T-Test with a Benjamini-Hochberg (BH) corrected pval < 0.05. Genes and biological samples were finally classified according to their gene expression profiles using a hierarchical clustering method (Cluster software) and the results were visualized with Treeview .https://david.ncifcrf.gov/) [p < 0.05).The differentially expressed genes (DEGs) obtained from the microarray analysis were subjected to overrepresentation analyses using the DAVID version 6.7 (rf.gov/)  online phttp://primer3.ut.ee) and extended across an intron when possible to eliminate the contribution from genomic DNA. qPCR was performed in triplicate using the GoTaq qPCR Mastermix kit , which utilizes carboxy-X-rhodamine (CXR) as the reference fluorochrome, using the following cycling condition: 95 °C for 10 s and 60 °C for 30 s for 40 cycles. The data were collected with the StepOnePlus apparatus  and quantitation of the samples was conducted using standard curves. LSM couples member 14B (lsm14b), prefoldin subunit 2 (pfdn2), and ring finger protein 8 (rnf8) had the most stable expression in the microarray dataset and were thus used as internal controls for qPCR. Further, 18S rRNA, beta-actin (bact), and elongation factor 1 alpha (EF1α) were also used as internal controls for qPCR [One μg of RNA was used as template for synthesis of cDNA using the Maxima First Strand cDNA Synthesis Kit (Thermo Fisher Scientific) as per the manufacturer’s protocol. The cDNA samples were then diluted 20-fold and subjected to qPCR using the primers listed in Additional file for qPCR . The geootulina are: 5′ GGAGACGCATGAGGATGAAC 3′; 5′ GGAAACAAACAGCATATTCT 3′; and 5′ GGGTCAGGTATCAAATAACT 3′. The gRNA sequences for slc29a1a are: 5′ GGGAGCCGCGTTATCCTT3’; 5′ GGGGCTTGTCAGAAACTA 3′; and 5′ TAGGAACCATATACAAAAAA 3′. Nucleotide sequences containing the gRNA were ordered, annealed together, and cloned into the DR274 plasmid. In vitro transcription of the gRNA from the T7 initiation site was performed using the Maxiscript T7 kit (Applied Biosystems), and their purity and integrity were assessed using the Agilent RNA 6000 Nano Assay kit and 2100 Bioanalyzer (Agilent Technologies). Zebrafish embryos at the one-cell stage were micro-injected with approximately 30–40 pg of each CRISPR/Cas9 guide along with 8–9 nM of purified Cas9 protein . The embryos were allowed to grow to adulthood, and genotyped using fin clip and PCR that detected the deleted regions. The PCR bands of the mutants were then sent for sequencing to verify the deletion. Once confirmed, the mutant females were mated with wildtype males to produce F1 embryos, whose phenotypes were subsequently recorded. Images shown on Fig. CRISPR/Cas9 guide RNA (gRNA) were designed using the ZiFiT Targeter online software version 4.2) , 52 and .2 , 52 avasa:eGFP males. These samples were lysed with 5% chelex containing 100 μg of proteinase K at 55 °C for 2 h and then 99 °C for 10 min. The extracted DNA was subjected to PCR using the AccuPrime system (Promega) for slc29a1a, Advantage 2 system for nucleoplasmin 2b (npm2b), and Jumpstart Taq polymerase (Sigma-Aldrich) for otulina and vasa:eGFP. The primers are listed in Additional file Genotyping of F0 and F1 animals was performed by harvesting fin clips from adult mosaic females as well as F1 animals under anesthesia (0.1% phenoxyethanol) and F1 eggs (1–2 cell stage) from mosaic females crossed with p-value < 0.05 was considered as statistically significant.Statistical analysis of the difference in the expression of each gene between bad and good quality embryos was performed using a Student’s t-test whenever possible after determination of normality of distribution using the Anderson-Darling test. When a t-test could not be used, a Mann-Whitney’s U-test was used. All statistical determinations were conducted using Prism version 7 . Data are presented as mean ± standard error (SEM). A p-values > 0.1 were discarded. We then computed the Pearson correlation coefficient for the expression levels of each pair of genes. We considered genes with pairwise correlations higher than 0.95 as redundant and kept only the gene with the highest correlation to the survival rates in each subset of correlated genes.We first filtered uninformative genes by performing a correlation test between each gene expression levels and survival rate; genes with p genes (1 ≤ p ≤ 20) randomly among the 5410 filtered genes [2, between the actual survival rates and the estimates obtained by the 2-FCV procedure. We ranked the 500 subsets according to the 2-FCV R2 values. We selected the best individuals by associating each individual with a selection probability that was proportional to its rank [2 obtained for each individual as the final criterion to quantify the quality of each final subset of genes.We implemented a GA to select optimal individuals, i.e. subsets of genes whose expression levels predict survival rate. We generated the initial population of potential solutions by selecting 500 subsets of ed genes . We applits rank . We alte2 of the final individuals obtained on the actual and randomized data were also compared. The relationship between gene expression and survival rate was considered significant when the actual R2 values were significantly higher than the R2 values of the randomized dataset. To compare their distributions, a Mann-Whitney test was used.To assess the relevance of the procedure, we applied the same GA to randomized datasets obtained by randomly permuting the survival rate of the different observations. Genes involved in egg survival are expected to appear often in the final subsets. The distribution of the selection frequency of each gene in the randomized datasets was computed. Genes with selection frequency in the actual data higher than the 95th or 99th percentile of the selection frequencies in the randomized data were considered as relevant. The distributions of the average 2-FCV RAdditional file 1:p-value, and fold change. Analysis was performed with the GeneSpring GX program. (XLSX 13 kb)The complete list of the 66 DEGs including the gene description, Ensembl annotation, corrected Additional file 2:Sequences of the primer pairs that were used in this study. (XLSX 13 kb)Additional file 3:List of differentially expressed genes (DEGs) in bad quality eggs as compared to good quality eggs that were the most modified among the 66 DEGs found by microarray analysis. (XLSX 10 kb)Additional file 4:rpf2, (b) spond1b, (c) tspan7b, (d) rps27.2, (e) stra13, (f) rtn4ip1, (g) slc29a1a, and (h) otulina transcripts by RNA-seq retrieved from the Phylofish online database. (PDF 606 kb)Tissue localization of (a) Additional file 5:wnt, tnf, and nf-kb pathways in otulina mutant-derived eggs. Transcript levels of (a) tcf3, (b) tnfa, (c) ikkaa, (d), nf-kb2, (e) rel, and (f) rela were investigated by qPCR (PDF 76 kb)Evaluation by qPCR for transcripts of


These three polyphenols can be used proficiently as biochemical markers for distinguishing the two apples when pomological traits cannot. Conversely, ITS1 and ITS2 polymorphism did not allow us to distinguish ‘Casciana’ from ‘Rotella’ fruit. Overall, the use of polyphenolic fingerprint might represent a valid tool to ensure the traceability of products with a high economic value.Ancient apple cultivars usually have higher nutraceutical value than commercial ones, but in most cases their variability in pomological traits does not allow us to discriminate among them. Fruit of two Tuscany ancient apple cultivars, ‘Casciana’ and ‘Rotella’, picked from eight different orchards (four for each cultivar) were analyzed for their pomological traits, organoleptic qualities, polyphenolic profile and antiradical activity. The effectiveness of a polyphenol-based cluster analysis was compared to molecular markers  to unequivocally discern the two apples. ‘Casciana’ and ‘Rotella’ fruit had a higher nutraceutical value than some commercial cultivars, in terms of phenolic abundance, profile and total antiradical activity. Although pedo-climatic conditions of different orchards influenced the phenolic profile of both apples, the polyphenolic discriminant analysis clearly separated the two cultivars, principally due to higher amounts of procyanidin B2, procyanidin B3 and  In Italy, the landscape complexity from ancient times has influenced the selection of numerous local apple cultivars. As long as the communities were isolated and the exchanges were limited, these cultivars had a strictly local value, but with the opening of the communities and the intensification of the exchanges, these local cultivars began to spread, mix and sometimes get confused. ‘Rotella’ and ‘Casciana’ are two ancient apple varieties typically found in Tuscany and both are listed in the germplasm bank of the Tuscany region  . The mixture was recovered and transferred to 14 mL tubes and incubated for 20 min at 60 °C then extracted twice with isoamyl alcohol chloroform. After adding isopropyl alcohol to the upper phase, the DNA was precipitated at 4 °C for 1 h. The pellet obtained after centrifugation was washed with ethanol at 70% (v/v) and dissolved in DNase free water. The concentration of each DNA sample was measured using a WPA biowave DNA spectrophotometer , and their integrity was evaluated by agarose gel electrophoresis. The DNA was stored at −20 °C until further analysis.DNA was extracted from leave of apple trees using a modified CTAB extraction method of Gawel & Jarret . One hunThe nuclear rDNA region, comprising the first internal transcribed spacer (ITS1), the 5.8S rRNA gene and the second internal transcribed spacer (ITS2), was amplified by the polymerase chain reaction (PCR) by using two primers, respectively complementary to the 18S and 25S rDNA near the ITS1 and ITS2 borders, M15 (5’-AAGTCGTAACAAGGTTTCCGTAGG-3’) and M17 (5’-CTTTTCCTCCGCTTATTGATATG-3’) .Amplification was carried out with conventional PCR in 20 μL reactions containing 1× 10X DreamTaq Buffer and 0.5 μM of each primer, 1U of DreamTaq and 20 ng of template DNA. PCR was run in a PCR system 2700 : Thermocycling consisted of an initial denaturation step at 95 °C (5 min), which was followed of cycles for: M15/M17 , with final extension step at 72 °C (10 min).All reactions were checked for amplification by gel electrophoresis. Amplified DNA sequences were directly cloned in pGEM-T Easy Vector System . Colony PCR screening was performed on individual white colonies using as primers M13 Forward and M13 Reverse. The clones that shown inserts with different weight were sequenced by automated sequencing . The sequences were analyzed using BLASTN, for their identification in GeneBank.Malus species already present in the database allowed us to construct a phylogenetic tree using the MEGA7 program [Platanus acerifolia was used as outgroup. Asterisks represent a bootstrap higher than 70%.The sequences are multi-aligned using CLUSTALW program . The mul program . The evoData are expressed as mean ± standard deviation and are subjected to one-way ANOVA test and statistical differences among the eight groups of two apple cultivars were calculated by least significant difference (LSD) test at 95% confidence with GraphPad Software . Linear correlation between phenolic compounds and total antiradical activity was carried out with the same software. Hierarchical clusters were carried out using Ward’s method on normalized data matrix , in order to see similarities between cultivars by using their phenolic content. Cluster analysis (CA) was conducted using IBM SPSS Statistics 24 . Heatmap was elaborated using GraphPad Software on the normalized polyphenol data matrix .p-coumaroylquinic acid) that were higher in ‘Casciana’ than in ‘Rotella’ accessions, independently of the orchard of origin and, therefore, of different pedo-climatic and agronomic factors. The three polyphenols mentioned above can be used proficiently as biochemical markers and their simultaneous presence can be considered as a sensitive, rapid, cheap and reliable methodology for discrimination and traceability of ‘Casciana’ and ‘Rotella’ fruit. Conversely, the molecular marker used in the present experiment, ITS1 and ITS2, did not enable us to distinguish ‘Casciana’ from ‘Rotella’ fruits.The current study reports new information about the nutraceutical properties of two ancient apple cultivars, ‘Rotella’ and ‘Casciana’. The polyphenolic content and the total antiradical activity found in these two ancient apple cultivars were higher than values reported for some commercial cultivars, drawing attention to the need to rediscover and re-evaluate old varieties in the attempt to find new “nutrafood” sources. When polyphenolic fingerprint was used for a cluster analyses, it allowed us to clearly separate the two cultivars and individuated three polyphenols (procyanidin B2, procyanidin B3 and Although the importance of these results might seem to be circumscribed at the local level, the use of chemometric parameters based on polyphenol fingerprint and the identification of valid biochemical markers is certainly of broader interest and can allow us to ensure the traceability of products with high economic value and to contrast the fraudulence phenomena.