Background: Significant progress toward the recovery of useful vision in blind patients with severe degenerative retinal diseases caused by photoreceptor death has been achieved with the development of visual prostheses that stimulate the retina electrically. However, currently used prostheses do not provide feedback about the retinal activity before and upon stimulation and do not adjust to changes during the remodeling processes in the retina. Both features are desirable to improve the efficiency of the electrical stimulation (ES) therapy offered by these devices. Accordingly, devices that not only enable ES but at the same time provide information about the retinal activity are beneficial. Given the above, a bidirectional communication strategy, in which inner retinal cells are stimulated and the output neurons of the retina, the ganglion cells, are recorded using penetrating microelectrode arrays (MEAs) is proposed.Methods: Custom-made penetrating MEAs with four silicon-based shanks, each one with three or four iridium oxide electrodes specifically designed to match retinal dimensions were used to record the activity of light-adapted wildtype mice retinas and degenerated retinas from rd10 mice in vitro. In addition, responses to high potassium concentration and to light stimulation in wildtype retinas were examined. Furthermore, voltage-controlled ES was performed.Results: The spiking activity of retinal ganglion cells (RGCs) was recorded at different depths of penetration inside the retina. Physiological responses during an increase of the extracellular potassium concentration and phasic and tonic responses during light stimulation were captured. Moreover, pathologic rhythmic activity was recorded from degenerated retinas. Finally, ES of the inner retina and simultaneous recording of the activity of RGCs was accomplished.Conclusion: The access to different layers of the retina with penetrating electrodes while recording at the same time the spiking activity of RGCs broadens the use and the field of action of multi-shank and multi-site penetrating MEAs for retinal applications. It enables a bidirectional strategy to stimulate inner retinal cells electrically and to record from the spiking RGCs simultaneously (BiMEA). This opens the possibility of a feedback loop system to acknowledge the success of ES carried out by retinal prostheses. The retina harbors not only the photoreceptors but also a neuronal network (“inner retina”) that serves for information processing and provides the retinal output neurons, the retinal ganglion cells (RGCs). Degenerative retinal diseases caused by photoreceptor death, such as age-related macular degeneration (AMD) and retinitis pigmentosa (RP), are the third leading cause of blindness in the world . NowadayIn order to restore the lost function of photoreceptors, commercially available retinal prostheses comprise primarily of a light-sensing device and a microelectrode array (MEA). To capture visual information, a camera together with a signal processor unit can be used to detect and process light stimuli. Likewise, a light-sensitive device such as an array of photodiodes might be used. Following, visual information is transduced into electrical signals and conducted into the retinal tissue by a pulse generator or induced by the same array of photodiodes through a MEA. Thereby, retinal implants activate the remaining circuitry of the visual pathway due to the ES of bipolar and/or RGCs, depending on whether the corresponding MEA is placed between the sclera and the choroid , between the choroid and the remaining retinal cells , or between the neural layer of the retina and the vitreous body  .While current retinal prostheses allow the restoration of useful visual percepts to blind patients with RP, they do not automatically adjust to the remodeling processes of the retina that lead to an increase of the stimulation threshold and a reduction of the efficiency of the ES therapy of these devices . FurtherWith the aim to facilitate the improvement and adjustment of ES parameters in retinal implants and to provide means to examine in real-time the electrical activity within the retina, a bidirectional communication strategy between retinal cells and a prosthetic device using a penetrating MEA has been proposed . In thisin vitro in retinas of normal mice and mice showing a retinal degeneration. Furthermore, the potential field of action of penetrating MEAs in retinal applications is shown. As they allow access to the different retinal layers, a follow-up of a group of neurons corresponding to a same neuronal column is possible.In this work, a proof of concept confirming the feasibility of using penetrating MEAs as a dual purpose device that stimulates the inner retina and records local field potentials and spiking activity of retinal ganglion cells (RGCs) is unveiled see . Upon phInstitut für Werkstoffe der Elektrotechnik (IWE-1), RWTH Aachen University (Germany) after evaluating the design by Custom-made penetrating MEAs, here after called BiMEAs, were designed and fabricated at the x) electrodes. In total, each penetrating structure contains 12 or 16 electrodes, named 12-BiMEA or 16-BiMEA, respectively. The 12-BiMEAs belong to the first generation of such probes, which have 10–20 μm thick shanks with a width of 100 μm, an inter-shank distance of 150 μm, a total shank length of 1 mm, and rectangular electrodes with a surface area of either 800 μm2 (12-BiMEA-A) or 1600 μm2 (12-BiMEA-B) and a vertical inter-electrode distance of 20 μm (2 (16-BiMEA-A) or three rectangular electrodes and a bottom trapezoid electrode with the same surface area (16-BiMEA-B)  . In ordeBiMEA-B) .x was chosen as the electrode material, as it fulfills all of the aforementioned requirements , Titanium/Gold (Ti/Au) feedlines 30/300 nm thick, a first passivation layer of silicon nitride (SiN) 1 μm thick with an opening filled with Au. On top, a stack layer with 30 nm of Ti, 250 nm of platinum (Pt), and 500 nm of IrOx form the surface area of the electrodes. Afterward, a coating of 3 μm of parylene-C forms a second passivation layer with the corresponding openings to expose the surface area of the IrOx electrodes  corresponds to the bottom electrode of the right-most shank and E1.4 to the top electrode, E4.4 is the top electrode of the left-most shank.To keep track of the recordings corresponding to a same vertical column, the shanks of the BiMEA probes were numbered from 1 to 4 from right to left, and the electrodes were numbered from 1 to 4 from the bottom to the top. Thus, while electrode 1.1  via cyclic voltammetry (CV) with 500 cycles, a scan rate of 100 mV/s, and activation potentials from -0.85 to 0.85 V versus a Silver/Silver Chloride (Ag/AgCl) reference electrode in 0.9% phosphate buffered saline solution (PBS). In addition, the charge delivery capacity (Qdc) was calculated at the last cycle of the CV curve integrating the current density along the electrode potential versus the Ag/AgCl electrode as suggested by dc between 239.3 and 552.5 mC/cm2.The IrOx electrodes showed a low impedance, which decreased with respect to the increasing electrode surface area (ESA) of the BiMEA electrodes, especially in the frequency range of interest where neuronal spikes are captured (102–103 Hz). Electrochemical properties showed by the BiMEA probes are summarized in Supplementary Figure 1.The impedance of the electrodes was evaluated by electrochemical impedance spectroscopy (EIS) using a potentiostat  and a three-electrode cell setup prior to the first usage. Each IrOx electrode served as a working electrode and a Ag/AgCl electrode and a platinum (Pt) wire were used as reference and counter electrodes, respectively. The EIS measurements were carried out in 10xPBS applying a 10 mV sinus wave in a range of frequencies between 1 Hz and 100 kHz. The IrOrd10 mice were bred locally from breeding pairs obtained from Jackson (strain name: B6CXB1-Pde6brd10/J). In this line the rd10 mutation was backcrossed onto the C57BL/6J background for five generations before intercrossing to homozygosity. All animals were kept on a 12 h light/dark cycle with food and water ad libitum. All experiments were performed in accordance with the German Law for the Protection of Animals and after approval was obtained by the regulatory authorities, the Forschungszentrum Jülich and the Landesamt für Natur, Umwelt, und Verbraucherschutz of the German federal state of North-Rhine Westfalia.Wildtype animals of the strain C57BL/6 were obtained from Charles River and rd10 mice were prepared under ambient light. The animals were deeply anesthetized with isoflurane  and killed by decapitation, followed by the enucleation of the eyeballs, which were immediately transferred into oxygenated Ames’ medium  at room temperature. The physiological solution was bubbled with carbogen gas  containing 95% O2 and 5% CO2 at a pH of 7.4. In order to keep both eyes vital and ensure perfusion during preparation, the eyes were first opened along the ora serrata, allowing the removal of the cornea and the lens. Hereafter the procedure that is explained was effected for each eye as the retinal tissue was used for the experiments.Light-adapted retinas from wildtype and The lens and the vitreous body were carefully removed using fine forceps. Then, the retina was separated from the eyecup and cut into halves. One half was stored in oxygenated Ames’ medium until it was used, and the other piece was mounted with the ganglion cell layer (GCL) facing downwards onto a circular piece of nitrocellulose filter paper . The filter served as a carrier for the tissue and had a precut central hole with a diameter of 1.5 mm. Afterward, the filter/retina sandwich was transferred onto a polydimethylsiloxane (PDMS) pillow with the GCL facing up, and the filter paper was fixed to the PDMS using insect pins. Finally, the tissue was covered with fresh oxygenated medium.The experimental setup was based on s) of either 10 or 20 kHz, and a Ag/AgCl reference electrode were used for the electrical recordings.Electrical recordings were performed using the BioMAS , an in-hA Faraday cage was used to shield the measurement setup, including a support for holding the perfusion chamber, the headstage of the BioMAS system, whose front-end facilitated the handling of the BiMEAs, a LED circuit, and a micromanipulator system , which enabled the movement of the probes along three different axes . Furthermore, the perfusion chamber comprised a reservoir with a PDMS pillow to support the retinal tissue and allowed the inflow and outflow of oxygenated Ames’ medium at room temperature through a perfusion system with a flow rate between 3 and 4 ml/min, keeping the retina vital during the experiments see .0. Then, the insertion was carried out stepwise inside the tissue, at intervals of approximately 20–25 μm. In this way, further depths (Zx) inside the tissue were referenced to the electrode that recorded the first electrical activity.Once the perfusion system was set to run, the BiMEA shanks were driven down slowly with the micromanipulator system onto the epiretinal surface of the tissue until the first peaks or spikes were captured, setting this first position as Z+) was increased during the perfusion of the tissue to initiate depolarization and increase spiking activity of RGCs. To this effect, potassium bicarbonate (KHCO3) was added to the regular Ames’s medium solution to achieve a 20 mM K+ concentration. The tissue was superfused for 2 min with the high K+ solution followed by washout with the regular physiological solution.The extracellular concentration of potassium .In order to assess the efficiency of the ES, an electrical stimulation efficiency ratio (ESE) was calculated by dividing the firing rate in a window of 400 ms after the ES artifact by the firing rate averaged in 8 s before the stimulation pulse as proposed by del), which enabled also the calculation of the injected charge (Qinj) after the time integration of Idel, and the calculation of the charge density (Qd) considering the ESA of the stimulating electrode, which was 576 μm2, as only 16-BiMEA probes were used during ES experiments. Idel, Qinj, and Qd were then calculated for both cathodic and anodic phases.Additionally, the current measurement circuit inside the headstage allowed the measurement of the delivered current during ES  programs.As suggested by To determine whether or not a peak was an action potential (spike), an algorithm based on the search of spikes whose inter-spike intervals were equal or greater than 3 ms , whose ard10 retina.The feasibility of using the penetrating BiMEAs for recording retinal activity is shown for wildtype and degenerated x) of the retina. The insertion of the shanks was carried out stepwise: first the shanks were moved close to a position nearby the surface of the retina, then the insertion was continued until the top electrodes of the recording shank had captured spikes. The penetration was performed without the assistance of an optical system, but was assessed by observing the tip of the shanks through the glass ring with naked eyes and the electrical activity recorded by the electrodes. The insertion of the shanks was further confirmed in a dummy experiment , indicating that the bottom electrode of the shank was nearby the retinal surface. In this way, further depths at Z1 (21.6 μm), Z2 (39.7 μm), Z3 (61 μm), Z4 (80.8 μm), and Z5 (100.7 μm) corresponded to the position of the bottom electrode (E1.1) inside the retina with respect to Z0 (0 μm).In all experiments, the retina was penetrated from the nerve fiber layer/ganglion cell layer (NFL/GCL). In this part of the retina, action potentials are generated in somas and axons of RGC, while neurons located in deeper retinal layers do not fire action potentials. While penetrating the tissue, fast voltage deflections in the form of spikes were first recorded at the lowest of the electrodes. Spikes were observed at progressively higher electrodes when shanks were moved deeper into the retina . In this1 spikes were detected in all four electrodes, however, the spike amplitude was higher at the bottom electrodes E1.1 and E1.2, with peak heights of 28.52 ± 5.08 μV and 31.83 ± 3.19 μV accordingly (see 3 E1.3 and E1.4 recorded the spikes with the highest amplitudes (see 1.3 and E1.4) were in between the GCL and the NFL, that E1.2 was reaching the IPL, and that the bottom electrode (E1.1) was at the end of the IPL and reaching the inner nuclear layer (INL) of the retina  and the INL. In contrast, the upper electrodes E1.3 and E1.4, which had moved further into the IPL and GCL were still recording spikes. Finally, at Z5, only low amplitude peaks (≤16 μV) were detected by the upper electrodes, meaning they were already beyond the RGCs.At Zngly see , therebyudes see , and takudes see , it was 2 (42.8 μm) is shown. While the spiking activity of the vertical column in shank one is barely captured by the bottom electrode (E1.1) but detected by the upper electrodes , the bottom electrodes in shank two (E2.1 and E2.2) are the ones recording the action potentials of RGCs. The latter reveals that even though both shanks were inside the tissue, they were actually at different depths. Thus, when the Z positions were set, these corresponded to the shank whose electrodes captured the first spikes of the recording. Hence, the Z positions for the recordings displayed in It is important to notice that in cases when the explanted tissue was not completely flat on the PDMS pillow, the shanks did not contact the tissue at the same depth. The latter can be seen in Furthermore, having shanks with multiple recording sites allowed us to follow the activity of a group of cells within a same vertical column inside the tissue. As exhibited in + concentration, allowing us to observe a physiological retinal response to changes in the extracellular ionic concentration. A recording of one electrode site displaying the spiking activity and the firing rate along a complete experiment is shown in + were distinguished. First, regular SA with a firing rate of ∼17 Hz was detected , Z2 (42.5 μm), and Z3 (63 μm). At Z1, the reaction to light was evident at the two bottom electrodes, indicating their proximity to the NFL and GCL. At Z1, the upper recording sites did not capture any action potential but displayed the electrical artifact induced by the stimuli (see red arrows). At Z2 the spiking activity became more visible for E3.2. Finally, at Z3 the action potentials were diminished in amplitude at E3.1, became notorious with higher amplitude peaks at E3.2 and E3.3, and started to be captured by E3.4. Considering the responses to light captured at the upper electrodes, the reduced spikes captured by E3.1, and that the latter was at a depth of ∼63 μm inside the retinal tissue, it was an indication that the bottom electrode was entering the next retinal layer, the IPL, and that the retina was still vital. In this way, the BiMEA probes were placed in such a way that the bottom electrode would be located deep inside the retina, where spikes were barely captured or not captured at all, while the upper electrodes were still capable of recording the ongoing activity, so that further experiments, such as ES inside the retina, could be performed.Responses to light were also used in the experiments to assess the vitality of wildtype retinas while penetrating the tissue, ratifying that the same group of cells at different retinal depths were being recorded. rd10 mice, a mouse line that is considered a suitable model for the human disease RP. When positioning the shanks inside the degenerated tissue, a rhythmic activity was recorded  and 81.4 μm (E3.1 from Z2) inside the retina, it was expected that E4.1 was reaching the end of the tissue at the outer margin of the INL. While no spikes are generated at this depth, the bottom electrodes captured the same spikes as the upper electrodes, albeit at lower amplitudes of 30 ± 6.81 μV (E4.1) and 44.43 ± 9.18 μV (E3.1). Finally, the average maximum spike amplitude in wildtype retina was ∼39 μV, while in rd10 it was ∼100 μV  of the selected shank as the stimulating electrode, and the rest as recording electrodes. In this way, the shank carrying the stimulating electrode would be referred as the stimulated shank, and the others as non-stimulated shanks. Different sets of stimulation parameters termed ES-1 to ES-6 were chosen and the stimulation efficiency ESE was determined.After validating the feasibility of using the BiMEA probes for recording retinal activity and accessing the different retinal layers for both wildtype and p < 0.05) with ESEs higher than one were revealed for the three recording electrodes  of the stimulated shank  and ES-4 (in E2.4 and E2.3) in recording electrodes of non-stimulated shanks, exposing therewith an inhibition effect after ES , however, the mean ESEs of the electrodes was lower than in wildtype, between 2.2 and 2.7. Significant stimulations were also obtained using ES-3 and ES-6 on this retinal sample, yet the ESEs were lower than for ES-2, ranging between 1.4 and 2.2  and the NFL (E3.1 and E2.1) in the rd10 sample  is being captured, since the ratio would calculate an ESE higher than 1 even when one spike is detected after ES, and would rise extremely if the activity before ES is slightly higher than 0 Hz. Nevertheless, consistent and significant electrical responses generating firing rate increases were observed in shank 2 during the three different ES parameters tested on the sample  in deeper retinal layers, while at least one of the upper electrodes came in close proximity to the GCL, to continuously record the spiking activity of the retina. Recordings of spontaneous electrical activity, as well as meaningful physiological responses to optical stimulation and changes in the extracellular ionic concentrations proved that the recorded spikes originated from RGCs, showing in turn the vitality of the tissue during the intraretinal recordings. Moreover, the penetrating MEAs were also capable of crossing different retinal depths in degenerated d10 mice . After p2) when using small electrode sizes (∼706 μm2) on rd10 retinas, as recently reported by In addition, current measurements during ES exposed injected currents and charge densities within the range of subretinal ES thresholds  lead to the optimization of the shank width, which was reduced from 100 to 60 μm.On one side, the design of such probes must consider the anatomy and microstructure of the retina. The first generation of BiMEA probes (12-BiMEAs) exhibited in this work, had a shank length of 1000 μm, which was reduced to 312 μm in a newer design (16-BiMEAs), coming closer to the total retinal thickness of approximately 200–220 μm in wildtype mice and 100–120 in rd10 . Considex, PEDOT, or nanostructured Pt  helped to reduce the distance from the tip to the bottom electrode, thereby avoiding to pierce completely the retina during positioning of the electrodes. As smaller electrodes could reduce the dimensions of the penetrating shanks and increase spatial resolution, appropriate electrode materials that yield low impedances and high charge delivery capacities, such as IrOtured Pt , should tured Pt .Furthermore, new materials that attenuate the mechanical and biological mismatch between the retinal tissue and the implant should be considered. Current planar retinal implants are based on flexible substrate materials such a polyimide, parylene-C, and silicone rubber , howeverThis work unveiled the feasibility of using multi-shank and multi-site penetrating MEAs for retinal applications. In this way, different layers of the retina were accessed, offering at the same time the possibility to stimulate the inner retina and to follow-up the electrical activity along the same neuronal column by simultaneous recording of RGCs. Thus, the use of such systems could enable a bidirectional communication that provides feedback about the success of ES, captures the presence of abnormal retinal activity, and in principle could perform an autonomous calibration of stimulating parameters. While this proof of concept opens the door to potential intraretinal implants, it must be taken into account that it is an invasive technique and that the biological impact on the retina has not been established yet. Therefore, future bidirectional penetrating implants must focus on the assessment and reduction of potential damages to the retina, as well as on the development of flexible and complaint penetrating probes.All experiments were performed in accordance with the German Law for the Protection of Animals and after approval was obtained by the regulatory authorities, the Forschungszentrum Jülich and the Landesamt für Natur, Umwelt und Verbraucherschutz of the German federal state North-Rhine Westfalia.WM, PW, FM, and AO conceived the idea and designed the experiments. SL fabricated the devices. VRM and JG performed electrical and biological experiments. VRM and SL performed the characterization of the electrodes. The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Brassica napus is of substantial economic value for vegetable oil, biofuel, and animal fodder production. The breeding of yellow-seeded B. napus to improve seed quality with higher oil content, improved oil and meal quality with fewer antinutrients merits attention. Screening the genes related to this phenotype is valuable for future rapeseed breeding.B. napus, and yellow rapeseed was shown to be an introgression line between black-seeded B. napus and yellow-seeded Sinapis alba. A total of 15,251 differentially expressed genes (DEGs) were identified among all the libraries, and 563 and 397 common DEGs were identified throughout black and yellow seed development, including 80 upregulated and 151 downregulated genes related to seed development and fatty acid accumulation. In addition, 11 up-DEGs and 31 down-DEGs were identified in all developmental stages of yellow rapeseed compared with black seed. Enrichment analysis revealed that many DEGs were involved in biosynthetic processes, pigment metabolism, and oxidation-reduction processes, such as flavonoid and phenylpropanoid biosynthesis, phenylalanine metabolism, flavone and flavonol biosynthesis, and fatty acid biosynthesis and metabolism. We found that more than 77 DEGs were related to flavonoid and lignin biosynthesis, including 4CL, C4H, and PAL, which participated in phenylalanine metabolism, and BAN, CHI/TT5, DFR, F3H, FLS, LDOX, PAP, CHS/TT4, TT5, bHLH/TT8, WD40, MYB, TCP, and CYP, which were involved in flavonoid biosynthesis. Most of these DEGs were downregulated in yellow rapeseed and were consistent with the decreased flavonoid and lignin contents. Both up- and down-DEGs related to fatty acid biosynthesis and metabolism were also analyzed, which could help to explain the improved oil content of yellow rapeseed.A total of 85,407 genes, including 4317 novel genes, were identified in the developing seeds of yellow- and black-seeded B. napus with a unique genetic background, and all the DEGs in comparison with the black-seeded counterpart could help to explain seed quality differences, such as lower pigmentation and lignin contents, and higher oil content.This research provided comprehensive transcriptome data for yellow-seeded The online version of this article (10.1186/s12870-019-1821-z) contains supplementary material, which is available to authorized users. Brassica napus, as the third leading oilseed crop grown worldwide, greatly contributes to providing edible oils, biofuels, and animal fodder [B. napus was improved with better seed oil and meal quality due to fewer pigments and polyphenols; thus, breeding yellow rapeseed has been preferred for decades, and studies on the molecular mechanism of this phenotype have been reported as well [B. napus has been selected from interspecific hybridization of Brassica species and intergeneric hybridization [Arabidopsis thaliana, mutations in transparent testa (TT) genes are responsible for seed coat color variations, including early (EBGs) and late (LBGs) biosynthesis genes [TT4/CHS, TT5/CHI, TT6/F3H, and TT7/F3’H, and LBGs include TT3/DFR, TT18/LDOX/ANS, BAN/ANR, TT12, TT19/GSTF12/GST26, and AHA10. In addition, the MYB-bHLH-WD40 (MBW) complex has been reported to have important regulatory functions in anthocyanin accumulation. Four MBW have been reported, including TT2-TT8-TTG1, MYB5-TT8-TTG1, TT2-EGL3-TTG1, and TT2-GL3-TTG1 [TTs related to seed coat variation in Brassicaceae have been delineated, including 95 copies of 21 TTs in B. napus [F3’H, TT2, PAL, BAN, TTG1, TT10, and TT1 were cloned and shown to have functions in flavonoid biosynthesis [Brassica species, including the homologous TTs cloned in Brassicas [B. rapa [B. juncea [B. napus. Liu et al. (2012) reported a lignin biosynthesis gene, BnCCR1, associated with yellow seed character, indicating a strong correlation between seed color and lignin content [BnAHA10 had effects on seed color and lignin content [BnTT10 and BnTT1 revealed that they are involved in PA metabolism, lignin synthesis, seed coat pigmentation, and fatty acid (FA) biosynthesis [B. napus and the sensitivity of yellow seed color to environmental influences , the molecular mechanisms of this phenotype remained unclear until now [l fodder . Rapeseel fodder . Yellow- as well . Fewer a as well –6. Hithedization , 8. As ris genes . The EBGGL3-TTG1 . Homolograssicas . Besides[B. rapa , B. junc. juncea , 25 and  content . Wang et content . Functiontil now , 30.B. napus after interspecific hybridization between B. rapa and B. oleracea and the artificial selection during rapeseed breeding have greatly narrowed the genetic background [B. napus, and most of the yellow-seeded B. napus were created by crossing between Brassica species, such as hybrids between B. napus and B. juncea, B. napus and B. carinata, B. juncea and B. oleracea [Sinapis alba, S. arvensis, Camelina sativa, Crambe abyssinica, and Descurainia sophia, exhibit many desirable characteristics, such as yellow seed coat, high erucic acid, pod shattering resistance, high unsaturated FA contents, and resistance to various diseases and abiotic stresses [B. napus, accompanying desirable traits. Zhang et al. (2009) obtained yellow-seeded rapeseed from intergeneric hybrids between B. napus and D. sophia [B. napus and S. alba [B. napus from hybrid progenies with improved oil content and decreased antinutrients [B. napus, revealing the expression patterns of genes involved in various biological pathways related to seed coat pigmentation, FA biosynthesis and metabolism.The short domestication history of ckground . No yelloleracea , 33. Wilstresses –36. Intr. sophia . Previou S. alba  and seleutrients , 39, 40.B. napus to investigate the transcriptome difference that might be related to the quality variation between two rapeseeds. After quality control, ~ 30.3 to ~ 37.6 million reads from the libraries were uniquely mapped to the B. napus genome (Table RNA-Seq was performed at three weeks after flowering (3 WAF), 4 WAF, 5 WAF, 6 WAF, and mature stage of yellow- and black-seeded me Table . A totalB. napus lines, which might be related to seed development and FA accumulation  value , and the DEGs in all the clusters showed similar patterns between two rapeseed lines, except for subcluster_1_1, which includes DEGs with significant differences between Y4 and B4  were identified among all the libraries. We found 563 common DEGs in B4 (4 WAF of black seed), B5 (5 WAF of black seed), B6 (6 WAF of black seed), and BM (mature stage of black seed) compared with B3 (3 WAF of black seed), including 141 upregulated and 331 downregulated genes at each developmental stage of the black seed. A total of 397 common DEGs were identified in Y4 (4 WAF of yellow seed), Y5 (5 WAF of yellow seed), Y6 (6 WAF of yellow seed), and YM (mature stage of yellow seed) compared with Y3 (3 WAF of yellow seed), including 92 upregulated and 220 downregulated genes throughout yellow seed development  values between yellow and black rapeseed , we assigned all the DEGs to all the metabolism pathways using the Arabidopsis TAIR9 version as mapping reference. We also found many DEGs were enriched in metabolism  and lipids . These genes play important roles in lignin biosynthesis, and the expression changes should be related to the reduced lignin in yellow seed. Genes involved in the flavonoid pathway were also downregulated in yellow seed compared to black seed, including BAN, CHI/TT5, DFR, F3H, FLS, LDOX, PAP, CHS/TT4, TT5, bHLH/TT8, WD40, and MYB, which participate in the biosynthesis of chalcones, flavanones, flavonols, anthocyanins, and PAs. However, we found a MATE (BnaC01g40630D) and UGT84A1 (BnaA01g18540D) were upregulated at the early developmental stages of yellow seed compared with black seed. Comparing with other two down-regulated MATEs in yellow-seeded B. napus, BnaA01g24940D (a homologous of AT3G03620) and BnaA07g18120D (a homologous of AtTT12), we found BnaC01g40630D with lower expression throughout rapeseed development than that of BnaA01g24940D and BnaA07g18120D. Expressional differences of TCPs and CYPs were also identified, which might be related to flavonoid biosynthesis, since TCP could interact with R2R3-MYB [As mentioned above, yellow seed color is associated with flavonoid and lignin biosynthesis. We found 77 DEGs related to phenylpropanoid and flavonoid biosynthesis and the phenylalanine metabolic pathway. As shown in Fig. R2R3-MYB , and CYPL BnaC05g780D. TheB. napus.We found that genes involved in FA biosynthesis, metabolism, and degradation were also changed Fig. . For insB. napus -binding Rossmann-fold superfamily protein/short-chain dehydrogenase/reductase (SDR), BnaA03g17570D encoding acetyl CoA carboxylase carboxyltransferase alpha subunit (CAC3), BnaC03g04350D encoding 3-hydroxyacyl-CoA dehydratase PASTICCINO 2 (PAS2), 4CL (BnaC03g42000D), C4H , LBD41 (BnaC03g33010D), PAL (BnaC04g08190D), BAN BnaA01g36200D), CHI (BnaC09g50050D), LDOX (BnaC01g14310D) and CHS (BnaA10g19670D). The results showed that the qPCR data of the DEGs were in accordance with the sequencing results throughout seed development of yellow- and black-seeded 200D, CHIB. napus from the backcrossing progenies of B. napus-S. alba hybrids, including S. alba specific fragments in the 38 chromosomes [B. napus. As reported by Qu et al. (2013), accumulation of polymeric phenolic compounds were similar in yellow and black rapeseeds, but the flavonoids were more accumulated in black seed since 3 WAF [B. napus. We found a total of 15,251 DEGs among all the libraries, including 80 upregulated and 151 downregulated genes identified throughout yellow and black seed development, which might be related to seed development and FA accumulation  were downregulated at two to four consecutive developmental stages of yellow rapeseed compared with black rapeseed. Two homologs of BnDFR (BnaA09g15710D and BnaC09g17150D) were also expressed at lower levels in yellow seed, which encodes an enzyme catalyzing dihydroflavanones to leucoanthocyanidins that was then converted to anthocyanidin [BnLDOX  was downregulated in yellow seed, which encodes leucoanthocyanidin reductase and catalyzes the formation of anthocyanidins. Genes encoding chalcone synthase  were also downregulated throughout yellow seed development, which catalyzes the first step of flavonoid biosynthesis. BnTT5 (BnaC08g26020D) was also downregulated in yellow seed compared with black seed at specific stages, encoding chalcone isomerase that is redundant and responsive to lights in Brassicaceae [BnTT8 (BnaC09g24870D and BnaA09g22810D) was also downregulated in yellow seed compared with black seed at specific stages, encoding the transcription factor bHLH as an important regulator throughout flavonoid biosynthesis. Another two bHLHs (BnaA01g17420D and BnaA01g24020D) were also downregulated, which have been proven to have functions in regulating jasmonate responses [Arabidopsis [CYPs were differentially expressed between yellow and black rapeseeds, although they were not shown to have functions in the secondary metabolism of B. napus. Previously, CYP71/75/93/81 have been reported to have functions in the biosynthesis of anthocyanin pigments and condensed tannins [Arabidopsis and O. sativa, which functions in generating 3′-hydroxylated flavonoids and flavone aglycones [CYPs, including members of CYP71/72/77/78/81/85/86/90/93, were differentially expressed in Dendrobium catenatum from different locations and with different outlooks, which might be related to flavonoid biosynthesis [CYP86 has been reported to be involved in suberin monomer biosynthesis in Arabidopsis [TCPs were also identified, which might be related to flavonoid biosynthesis since TCP3 could interact with R2R3-MYB [TCP1 directly activates DWARF4 and promotes brassinosteroid biosynthesis [Flavonoid biosynthesis has been considered to be the main pathway related to plant pigmentation, and u et al. , as welllba Fig. . Four hocyanidin . BnLDOX sicaceae . BnTT8  provides most of the pyruvate for plastidic FA synthesis, and the mutation of PKP severely impairs seed storage lipid synthesis [A. thaliana has been confirmed to be a subunit of heteromeric acetyl-CoA carboxylase (ACCase), which catalyzes the carboxyltransferase (CT) reaction. ACCase is responsible for the first step of FA synthesis [UDP-glucose pyrophosphorylase (UGP) (BnaA05g32480D), a sucrose-regulated protein, was upregulated in yellow seed, which is required for fumonisin B1-induced cell death [beta glucosidase 25 (BnaC01g40610D) was also upregulated in yellow seed, which is involved in the carbohydrate metabolic process [cytochrome B5 reductase 1 (CB5R1) (BnaC02g02110D) in yellow seed might also be related to the FA variation, since CB5R is a microsomal membrane-bound protein that functions as part of the microsomal electron transfer system in FA desaturation [Since the oil content of yellow seed was higher than that of black seed, we found 12 DEGs related to FA biosynthesis, metabolism, elongation and degradation Fig. . In addiynthesis . BnaA03gynthesis . Additioll death . A homol process . Upregulturation . CB5R caturation . This miB. napus derived from hybrids of B. napus-S. alba, and black-seeded B. napus. We identified the DEGs with seed development, which might be related to the development and biosynthetic process. In addition, DEGs related to the quality difference between yellow and black rapeseed have been identified, which mainly participate in flavonoid biosynthesis, phenylpropanoid biosynthesis, phenylalanine metabolism, flavone and flavonol biosynthesis, fatty acid biosynthesis and metabolism. These down-regulated genes are helpful to explain the less pigmentation  and lignin , and higher oil content in yellow rapeseed compared with black seed  was preserved in our lab and was selected from back-crossing progenies of somatic hybrids of B. napus-S. alba. The black-seeded rapeseed (B. napus cv. ‘Yangyou 6’) was obtained from the Jiangsu Lixiahe Region Agricultural Research Institute, China [B. napus. The oil content of W82 was 6% higher than that of Yangyou 6. Higher protein and sucrose contents, less dietary fiber and crude fiber, and fewer glucosinolates accumulated in the seed meal of yellow rapeseed compared with black rapeseed . Each library was pooled by mixing equal quantities of five RNA samples. The RNA quality was validated using agarose gel electrophoresis, Nanodrop, Qubit, and Agilent 2100 to confirm the purity, concentration, and integrity, respectively. mRNA was purified using beads with Oligo (dT), and cDNA was then synthesized with random hexamers after fragmentation of mRNA. After purification of cDNA with AMPure XP beads, unique adaptors and indexes were ligated. Certain fragments were selected with beads and enriched using PCR amplification. Finally, ten cDNA libraries for five developmental stages  of yellow- and black-seeded B. napus cv. Darmor-bzh genome (version 5) using TopHat2 [10(RPKM+ 1) and log2(Fold Change) value. Gene ontology (GO) enrichment of the DEGs was performed using GOseq with a corrected p-value < 0.05 [p-value < 0.05 [After removing adaptor sequences and low-quality sequences, clean reads were mapped to the  TopHat2 , 72. Bas TopHat2 , 74. Nov TopHat2 . The abu TopHat2 , 77, ande < 0.05 , and KEGe < 0.05 . An overe < 0.05 .B. napus β-actin (NCBI AF111812) was used as an endogenous control to generate the △Ct for three technological replicates.Subsamples of RNA-Seq were reverse transcribed into cDNA for real-time qPCR validation using the Revert Aid First Strand cDNA Synthesis Kit and SYBR Green Real-Time PCR Master Mixes . qRT-PCR was performed on a fluorescence quantitative system Mx3005P . Genes, primers and size of the amplicon are listed in Additional file Additional file 1:Table S1. Summary of read mapping in RNA-Seq analysis of yellow and black rapeseeds. B3, B4, B5, B6 and BM indicate 3 ~ 6 WAF and mature black seed. Y3, Y4, Y5, Y6 and YM indicate 3 ~ 6 WAF and mature yellow seed. (XLSX 34577 kb)Additional file 2:Table S2. DEGs identified with black or yellow seed development and between same developmental stages of two rapeseeds. (XLSX 11030 kb)Additional file 3:Table S3. DEGs related to seed development. (XLSX 94 kb)Additional file 4:Table S4. Up- and downregulated genes among all the developmental stages of yellow- and black-seeded B. napus. (XLSX 25 kb)Additional file 5Figure S1. The SOM clusters of DEGs from five developmental stages of two rapeseed lines. (XLSX 173 kb)Additional file 6:Table S5. All the up- and down-DEGs with GO annotation. (XLSX 63 kb)Additional file 7:Figure S2. KEGG enrichment of DEGs (Y3 vs. B3) in flavonoid and phenylpropanoid biosynthesis. (XLSX 24 kb)Additional file 8:Figure S3. KEGG enrichment of up- and down-DEGs between yellow- and black-seeded B. napus. (XLS 31 kb)Additional file 9:Table S6. KEGG pathways of DEGs between yellow- and black-seeded B. napus. (PDF 519 kb)Additional file 10:Figure S4. Overview of pathways related to the DEGs between yellow- and black-seeded B. napus. (PDF 981 kb)Additional file 11:Figur S5. Heatmap of DEGs involved in secondary metabolism. (JPG 6164 kb)Additional file 12:Table S7. Quality differences between yellow- and black-seeded B. napus. (JPG 1896 kb)Additional file 13:Table S8. Primers for qPCR validation of DEGs (JPG 3234 kb)


TECTA) have been reported to cause non-syndromic hearing loss (NSHL) in either a dominant or recessive inheritance pattern. Among the identified TECTA mutations, H1400Y has been associated with NSHL in two independent studies. However, its exact role in contributing to genetic hearing loss remains elusive.Many mutations in the α-tectorin gene  guidelines, we conclude that H1400Y should be disqualified as a cause of genetic hearing loss. True pathogenic variants causing genetic hearing loss should be more deliberately reported in accordance with ACMG guidelines. TECTA gene encodes α-tectorin, a protein of 2156 amino acid residues, that is a major component of the non-collagenous glycoproteins comprising the tectorial membrane [TECTA is known as a causative gene of both autosomal dominant (DFNA8/A12) and autosomal recessive (DFNB21) non-syndromic sensorineural hearing loss (NSHL) [TECTA result in a broad spectrum of hearing loss depending on the domain in which the mutation occurs [TECTA cause mid-frequency hearing loss with a cookie-bite or U-shape audiometric configuration [TECTA mutations with hereditary hearing loss, the precise molecular mechanism remains unclear. Furthermore, considering that TECTA mutations account for 4% of all cases of autosomal dominant NSHL (ADNSHL), TECTA-related ADNSHL should be regarded as one of the most frequent subtypes of ADNSHL [The tectorial membrane is an extracellular matrix overlying the organ of Corti. By making contact with the stereocillia of specialized sensory hair cells, the tectorial membrane plays an important role in inducing the movement of these hair cells via intracochlear sound transmission . The TECs (NSHL) , 4. α-Teguration , 8. Analguration . Despitef ADNSHL .TECTA. Based on familial segregation analyses, we concluded that R1890C is a causative mutation for hereditary hearing loss, whereas H1400Y is not.Here, we report a 4-year-old Korean girl with non-progressive, prelingual, mid-frequency, and mild-to-moderate hearing loss, who had two missense mutations in the ZP and ZA domains of With approval from the Institutional Review Board of the Severance Hospital, Yonsei University Health System , a three-generation pedigree was obtained from the family by genetic counseling. The family comprised 11 individuals, four of whom participated in the study. The four members from three generations of the family were evaluated at Yonsei University Severance Hospital. For phenotype evaluation, we reviewed the medical and developmental history, and conducted physical examinations, pure tone audiometry, and a speech evaluation.The proband III-1) was a 4-year-old female of family YUHL-165 who failed to pass the neonate hearing screening at birth and was thus referred to our department for diagnostic auditory brain stem response (ABR) testing. There was no family history related to hearing loss . Since the two variants were in trans, the de novo R1890C variant was confirmed to be located on the paternal allele. Therefore, both the H1400Y and R1890C variants were located on the paternal allele, suggesting a cis status of the two variants  was performed. Among the 2368 variants in 182 known hearing loss genes detected in our pipeline, 11 were manually reviewed for pathogenicity determination since their allele frequencies in the population database were under 0.01 (1%). Among these 11 variants in 10 hearing loss genes, two heterozygous missense variants in the The minor allele frequencies of each variant were determined in presumptively normal hearing population databases, including ESP6500, 1000 Genomes Project, and the Korean database  Table . An exteTECTA gene, H1400Y was revealed to be inherited from the proband’s father in the trio study (Fig. TECTA mutations are generally associated with stable and post-lingual progressive hearing loss, whereas recessive TECTA mutations are generally associated with pre-lingual stable hearing loss. Therefore, phenotype-based genotype interpretation was further applied for identification of true variants.Among the two candidate variants in the udy Fig. . Howeverudy Fig. , it alsoTECTA mutation is inherited in an autosomal dominant or recessive mode. However, application of the updated hearing loss-specified ACMG guideline maximized the evidential weight for each component, thereby allowing us to designate R1890C as “pathogenic” rather than “likely pathogenic”, and H1400Y as “benign” rather than “variant of uncertain significance” (Table According to the original ACMG guideline interpretation, the variants R1890C and H1400Y could be classified as “likely pathogenic” and “variant of uncertain significance”, respectively Table . These re” Table .TECTA is one of the most common genes responsible for ADNSHL and has been associated with various types of hearing loss in terms of progression and affected frequency range [TECTA mutation. Currently established genotype-phenotype correlations in TECTA indicate that mutations in the ZP and ZA domains are generally related to mid- and high-frequency hearing loss, respectively [ADNSHL has both genetically and clinically heterogeneous characteristics, and 24 associated genes have been identified to date . TECTA icy range , 4. The ectively , 11. Howectively  challengectively .TECTA missense mutations, R1890C and H1400Y, were detected in the proband of the present trio study. The variant H1400Y is located in exon 12 corresponding to the ZA domain of α-tectorin, whereas R1890C is located in the ZP domain. R1890C was previously reported as a pathogenic mutation with a high level of evidence [TECTA. Four of the five affected family members were evaluated, all of whom demonstrated bilateral, mild to moderate symmetric sensorineural hearing loss, affecting the mid frequencies. Interestingly, the H1400Y mutation was present in the cis-form with the other deleterious mutation T1866M. The authors suggested that T1866M was likely the causative mutation of the hearing impairment in this family, although the effect of H1400Y could not be completely ruled out at that time [Two evidence . Howeverevidence , 14. Onehat time . In 2006hat time  first reTECTA associated with ADNSHL identified to date are missense variants, while nonsense mutations are associated with an autosomal recessive pattern of inheritance. As mentioned above, the specific pattern of hearing loss caused by a TECTA mutation depends on the affected domains. Missense mutations in the ZP and ZA domains are generally related to mid- and high-frequency hearing loss, respectively. However, the proband in this study showed a flat-type audiogram, which seems to affect the high frequency. There are several possibilities to explain this finding. First, the reliability of the proband’s pure tone audiometry could be relatively poor due to her young age. Second, the synergistic effect of the two mutations cannot be completely excluded. Third, the post-lingual hearing loss may be due to a genetic cause inherited from her father. Therefore, accurate follow-up hearing testing will be needed for this family.Interestingly, all of the mutations of TECTA mutations affect hearing loss. In this case, other available lines of evidence such as those derived from segregation studies should be incorporated to pinpoint true genetic causes. The combination of genetic and segregation analyses can help to provide several counseling strategies for determining the progression, prediction, and therapeutic application for this young patient.Unfortunately, there has been no in vitro functional assay to evaluate the mechanism by which TECTA is one of the major contributing causative genes of hearing loss, accurate application of hearing loss-specified ACMG rules is expected to enhance classification of all of the variants identified to date.Recent improvements of variant interpretation guidelines, including the hearing loss-specified ACMG guideline established by the ClinGen Working Group, advocated our findings. Compared to the original ACMG guideline, which provided inconclusive results even when conducting a familial segregation study, the optimized ACMG guideline maximized the pathogenicity evaluation results and pinpointed the true pathogenic mutation. As TECTA gene as a genetic cause of hearing loss, whereas the H1400Y variant was determined to be an innocent bystander. To the best of our knowledge, this is the first study to provide clear evidence as to whether or not H1400Y is responsible for hearing loss, including results from a familial segregation study and hearing loss-specified ACMG guideline application. Therefore, this study is expected to contribute to gaining a better understanding of the TECTA gene mutation spectrum.In conclusion, we identified the R1890C mutation in the


Thermoregulatory grooming, a behavioral defense against heat, is known to be driven by skin-temperature signals. Because at least some thermal cutaneous signals that drive heat defenses are likely to be generated by transient receptor potential (TRP) channels, we hypothesized that warmth-sensitive TRPs drive thermoregulatory grooming. Adult male Wistar rats were used. We showed that camphor, a nonselective agonist of several TRP channels, including vanilloid (V) 3, when applied epidermally to the back (500 mg/kg), caused a pronounced self-grooming response, including paw-licking and snout- and chest-“washing”. By the percentage of time spent grooming, the response was similar to the thermoregulatory grooming observed during exposure to ambient warmth (32 °C). Ruthenium red , when administered intravenously at a dose of 0.1 mg/kg, attenuated the self-grooming behavior induced by either ambient warmth or epidermal camphor. Furthermore, the intravenous administration of AMG8432 (40 mg/kg), a relatively selective TRPV3 antagonist, also attenuated the self-grooming response to epidermal camphor. We conclude that camphor causes the self-grooming behavior by acting on TRP channels in the skin. We propose that cutaneous warmth signals mediated by TRP channels, possibly including TRPV3, drive thermoregulatory self-grooming in rats. Studies like this shed light on the organization of thermoregulatory behaviors and pave the way to what we have termed “thermopharmacology”—the approach of modulating thermoeffector activities and behaviors by drugs that mimic thermal stimulation . a) in the range of 18–29 °C resulted in no Tb differences between the groups . At the same Ta, rats exhibited an increased Tsk (p < 0.001 vs. 26 °C). Some basic level of self-grooming (2.7–3.4% of time), presumably hygienic in nature, was observed at 18–29 °C, with no statistical differences between the groups studied at different Tas . Presumably, this was a warmth-defense response, because exposure to the same Ta resulted in a Tb increase and tail-skin vasodilation.First, we studied the self-grooming response to ambient warmth. Exposure to an ambient temperature  did not cause any Tb changes (sk (p = 0.017). Importantly, during camphor-induced self-grooming, rats “washed” their fir on the snout, neck, and chest, i.e., the areas that had no exposure to camphor.Next, we studied whether self-grooming can be induced by stimulating TRP channels. In these experiments, we used camphor, a nonselective agonist of several TRP channels, mostly warmth-sensitive, viz., TRPV1  and TRPVed above C. At thi changes A or Tsk nges . These data suggest that the self-grooming response to camphor was not induced by the odor, but rather was due to a topical, cutaneous action of this compound. We conclude that the nonselective TRP agonist camphor activates the thermoregulatory-like self-grooming behavior by acting on the skin.Another possibility that had to be addressed was that camphor, a compound with a strong odor, caused the self-grooming behavior not by acting on thermoreceptors in the skin , but by irritating the nasal mucosa (distantly). To test this possibility, we studied the self-grooming response to camphor (or PPG), when the substance was applied not to the skin, but to a cotton ball. The ball was placed in the cage and separated from the rat by mesh, thus, remaining out of the animal’s reach. Under these conditions, only the low, hygienic levels of self-grooming were observed, and no difference occurred between rats exposed to camphor or PPG . Pretreatment with ruthenium red attenuated the grooming responses to both stimuli  or in response to PPG (p = 0.079). These results suggest that the grooming behavior occurring in response to ambient warmth or camphor is mediated by TRP channels. We then investigated whether the self-grooming responses to warmth and camphor are mediated by TRP channels. First, we studied whether ruthenium red, a nonselective TRP antagonist with marked potency against several warmth-sensitive rat channels , woua of 32 °C) was evaluated. Furthermore, TRPV4 is activated by somewhat lower temperatures (<30 °C) [One potential development of this study, as pointed out by the reviewers of this manuscript, would be to assess the involvement of TRPV4 channels. However, in our earlier study , the sel(<30 °C)  than TRPp = 0.026; Next, we used AMG8432, a novel, relatively potent TRPV3 antagonist with limited selectivity , and tested whether it would attenuate camphor-induced self-grooming. Rats pretreated with AMG8432 showed a significant reduction (by ~7.4%) in camphor-induced self-grooming, as compared to vehicle (Cinnamomum camphora). Camphor activates several rat TRP channels, most notably warmth-sensitive, viz., TRPV1 and TRPV3 [In the present study, we used camphor, a bicyclic monoterpene ketone typically isolated from the wood of the camphor laurel tree , applied to the skin, causes a self-grooming response, which is similar to thermoregulatory grooming. We have further shown that ruthenium red  attenuates the grooming response to either ambient warmth or camphor, and that AMG8432 (a relatively nonselective TRPV3 antagonist) attenuates the camphor-induced self-grooming behavior. Together, these data suggest that, in rats, TRP channels, possibly including TRPV3, mediate those cutaneous warmth signals that drive thermoregulatory self-grooming.a of 22–24 °C. Standard laboratory rodent chow and tap water were available ad libitum (except during the experiments). All experiments were performed between 9 AM and 2 PM. All procedures were approved by the ethics committee of the Federal University of ABC (protocols 001/2010 and 003/2013).Male Wistar rats  were obtained from the Institute of Biomedical Sciences, University of São Paulo . All rats were housed in standard cages kept in ventilated cabinets  under a 12:12 h light-dark cycle (lights on at 7 AM) and a controlled TFor all surgical procedures, rats were anesthetized with an intraperitoneal administration of ketamine-xylazine cocktail  and treated with an antibiotic  and a nonsteroidal anti-inflammatory drug . Experiments were performed 5–7 days after surgery. At the end of the experiments, rats were euthanized with urethane 30% .Via midline laparotomy, each rat was implanted with a datalogger  in the peritoneal cavity for recording abdominal temperature, a measure of deep Tb. The abdominal wall was sutured in layers. After the experiments, the rat was euthanized, the datalogger was removed, and the Tb data were uploaded to a computer for processing. Each rat assigned to experiments involving i.v. injections was also implanted with a jugular catheter. A small incision was made on the ventral surface of the neck, to the right of and parallel to the trachea. The right jugular vein was exposed, freed from its surrounding connective tissue, and ligated. A silicone catheter , filled with heparinized (10 U/mL) saline, was passed into the superior vena cava through the jugular vein and secured in place with ligatures. The free end of the catheter was knotted, tunneled under the skin to the nape, and exteriorized. The wound was sutured. The catheter was flushed with heparinized saline the day after surgery and every other day thereafter, up to the day of the experiment. After catheterization, rats were housed individually. Camphor, a nonselective TRP agonist ,19, was a of 18, 22, 26, 29, or 32 °C. The midpoint of the thermoneutral zone for rats in this setup is ~26 °C [An acrylic arena , lined with wood bedding, was set inside a climatic chamber , at a Ts ~26 °C ,39. DuriObservations in an elevated plus maze are often used to assess anxiety-like behaviors in rats . We usedb  was recorded with implanted dataloggers every 5 min. Tail Tsk, a measure of the tail vasomotor tone, was determined based on the images taken with an A300 infrared camera . The values of Tsk were obtained at the border of the proximal and middle thirds of the tail. Large  increases in Tsk typically signify tail-skin vasodilation [as (18–32 °C), the recording started after 2 h of exposure. In experiments with camphor (or PPG) administration, the recording started at 5 min after the administration. The self-grooming response was measured as a percentage of time a rat spent engaging in this behavior. In the elevated plus maze, 5 min after the camphor (or PPG) treatment, rats were individually placed in the apparatus and the following indices of anxiety were studied: an increased time spent in and number of entries to the closed arms; a decreased time spent in and number of entries to the open arms; an increased number of events of “risk-assessment behavior” ; head dipping (when a rat stretches its body beyond the edge of an open arm and lowers the head below the surface of the arm); and rearing, as well as increased numbers of fecal boli and urine spots.Deep Tdilation ,34. The t-test was used to compare single-measurement values between the two groups. All analyses were performed using Sigma Plot 13.0 (Systat Software). Differences were considered significant at p < 0.05. Data are reported as Mean ± SE.One-way ANOVA and two-way ANOVA were used, followed by the Holm–Sidak post-hoc test, as appropriate. Student’s


MYO15A variants are responsible for human non-syndromic autosomal recessive deafness (DFNB3). The majority of MYO15A variants are associated with a congenital severe-to-profound hearing loss phenotype, except for MYO15A variants in exon 2, which cause a milder auditory phenotype, suggesting a genotype-phenotype correlation of MYO15A. However, MYO15A variants not in exon 2 related to a milder phenotype have also been reported, indicating that the genotype-phenotype correlation of MYO15A is complicated. This study aimed to provide more cases of MYO15A variation with diverse phenotypes to analyse this complex correlation.MYO15A variants  from 14 unrelated families, identified by targeted gene capture of 127 known candidate deafness genes, were recruited. Additionally, we conducted a review of the literature to further analyses all reported MYO15A genotype-phenotype relationships worldwide.Fifteen Chinese autosomal recessive non-syndromic hearing loss (ARNSHL) individuals with MYO15A variants in 15 patients, two of which presented with a milder phenotype. Interestingly, one of these cases carried two reported pathogenic variants in exon 2, while the other carried two novel variants not in exon 2. Based on our literature review, MYO15A genotype-phenotype correlation analysis showed that almost all domains were reported to be correlated with a milder phenotype. However, variants in the N-terminal domain were more likely to cause a milder phenotype. Using next-generation sequencing (NGS), we also found that the number of known MYO15A variants with milder phenotypes in Southeast Asia has increased in recent years.We identified 16 novel variants and 12 reported pathogenic MYO15A variant spectrum, enriched our knowledge of auditory phenotypes, and tried to explore the genotype-phenotype correlation in different populations in order to investigate the cause of the complex MYO15A genotype-phenotype correlation.Our work extended the The online version of this article (10.1186/s12881-019-0790-2) contains supplementary material, which is available to authorized users. More than 50% of congenital hearing impairment cases are attributable to genetic factors, with more than 100 syndromic and non-syndromic genes known to date. Non-syndromic hearing loss (NSHL) is further categorized as autosomal dominant, autosomal recessive, X-linked, Y-linked or mitochondrial hearing loss. Autosomal recessive non-syndromic hearing loss (ARNSHL) accounts for up to 70% of NSHL. As of Jan 2019, more than 70 genes have been reported to be responsible for ARNSHL (http://hereditaryhearingloss.org/). Variants of GJB2, SLC26A4, MYO15A, OTOF, CDH23, MYO7A, TMC1, HGF and CIB are worldwide major contributors to ARNSHL [MYO15A, MIM: 602666) gene variants are the third or fourth most frequent causes of ARNSHL [MYO15A variants was 3~9.9% (19/557) among Turkish ARNSHL patients, 5.7% among Pakistani patients [Sensorineural hearing loss (SNHL) is one of the most common sensory defects, affecting approximately 466 million people worldwide, 34 million of whom are children  in humans, whereas variants of homologous Myo15 genes lead to deafness and vestibular dysfunction in mice (shaker-2) [Encoded by function . As a rehaker-2) .MYO15A variants were documented in patients with severe to profound congenital sensorineural deafness phenotypes from consanguineous families in the Middle East and Southeast Asia. However, with the recent development of next-generation sequencing (NGS), the reported number of MYO15A variants has sharply increased in other countries and regions, such as East Asia and Europe. Contrary to previous reported severe auditory phenotypes, partial deafness with residual hearing at low frequencies was reported, always associated with pathologic variants in exon 2 affecting the encoding of the N-terminal domain. Interestingly, recent studies have reported that some variants of MYO15A affecting the function of other domains, which were expected to bring about congenital severe to profound hearing loss, also were observed to result in various milder auditory phenotypes [Initially, the majority of enotypes , 10.MYO15A variant cases from 14 unrelated ARNSHL families and identified 16 novel variants and 12 MYO15A variants in total by targeted gene capture and high-throughput sequencing. Additionally, we analysed all reported MYO15A genotype-phenotype relationships worldwide through a review of the literature. Our work extended the MYO15A variant spectrum, enriched our knowledge of auditory phenotypes, and tried to explore the genotype-phenotype correlation in different populations in order to investigate the cause of the complex MYO15A genotype-phenotype correlation.In the present study, we reported 15 Chinese MYO15A variants  from 14 unrelated families, were recruited by the Department of Otolaryngology, Head and Neck Surgery of the Chinese PLA General Hospital. 127 known candidate deafness genes identifications were performed with targeted gene capture.A total of 15 Chinese ARNSHL individuals, ethnic Han, with For auditory assessment in all subjects, otoscopy, tympanometry, acoustic reflex thresholds, distortion product otoacoustic emission (DPOAE), and pure-tone audiometry following standard protocols were included.  Auditory brainstem response (ABR) and auditory steady state response (ASSR) were applied if the above subjective audiometric assessments could not be performed in infants. Family history and clinical questionnaires were available from all subjects or relatives.The definition for severity of hearing impairment, according to pure-tone audiometry (PTA) of the better ear, was made based on the average hearing threshold level at four frequencies  of air conduction. 26–40 dB HL were considered to be mild hearing loss; 41–55 dB HL, moderate hearing loss; 56–70 dB, moderately severe hearing loss; 71–90 dB HL, severe hearing loss; > 90 dB HL, profound hearing loss. The occurrence of hearing loss was categorized as prelingual (≤6 years) or post-lingual (> 6 years).http://bio-bwa.sourceforge.net/) was applied to align reads to the NCBI37/hg19 assembly. SNPs, inserts and deletions were detected by Genome Analysis Toolkit (GATK) software and SAM tools (http://samtools.sourceforge.net/).Blood DNA kit  was used to extract Genomic DNA of all participants from peripheral blood. Targeted gene capture and high-throughput sequencing were then applied as reported thoroughly in a previous study . Brieflyhttp://sift.jcvi.org), PolyPhen-2 (http://genetics.bwh.harvard.edu/pph2/) and Mutation Taster analyses (http://www.mutationtaster.org), while, GERP++ software (http://mendel.stanford.edu/sidowlab/downloads/gerp/index.html) and PhyloP (http: //compgen.bscb.cornell.edu/phast/) were performed to estimate the evolutionary conservation of the amino acid sequence. Genotyping for candidate pathogenic variants of all family members were underwent with Sanger sequencing. A PE9700 thermocycler  was obtained for PCR, and then sequence analysis was carried out using an ABI 3730 Genetic Analyzer on affected cases and normal controls to determine if potential pathogenic mutations in the gene were co-segregating with the disease phenotype of these families. Further, aligning sequences were performed with the NCBI Reference Sequence and DNAStar software  to identify variants and polymorphisms. Finally, we selected 200 blood DNA samples from Chinese Han individuals with normal hearing as ethnically matched controls.Potentially pathogenic variants including missense, nonsense, splice-site or frameshift variants with an allele frequency less than 0.01 were identified using the Genome Aggregation Database, the Exome Aggregation Consortium and 1000 Genomes databases. Simultaneous detection of point mutations, micro-indels, and duplications (< 20 bp) could be achieved. Pathogenicity of each missense variant was predicted using SIFT  original articles and 2) reports of MYO15A variants with ARNSHL in humans.Relevant researches were searched through PubMed using the MeSH terms “MYO15A in 15 NSHL patients from 14 families, only one of which was homozygous and 14 were compound heterozygotes , 3 splice site variants , 3 frameshift variants  and one nonsense variant (p.Arg1898*). Coincidentally, two families  in our study shared one identical novel variant, c.7396-1G > A. The remaining 12 variants were previously reported: p.Pro286Serfs*15 [MYO15A variants  had severe-to-profound hearing loss. The locus was subsequently further refined to chromosome 17p11.2 [MYO15A were initially identified in three multigenerational ARNSHL families from Indonesia and India [MYO15A gene. In the present study, we identified 16 novel possible pathogenic variants, including one nonsense variant, two ±1 or 2 splice site variants, three frameshift variants with evidence of pathogenicity and 9 missense variants .In 1995, the  17p11.2 , 71. SinMYO15A gene contains 67 exons and encodes several alternatively spliced transcripts, and the longest mRNA transcript contains 3530 amino acids. It includes a long N-terminal extension encoded by giant exon 2, an ATPase motor domain, two light chain binding IQ motifs, and a tail region containing two myosin-tail homology 4 (MyTH4) domains, two band 4.1 superfamily  (FERM), a Src-homology-3 (SH3) domain and a C terminal class I PDZ-ligand domain. Additionally, based on the presence or lack of giant exon 2, there are two alternatively spliced transcripts, isoform classes II and I. Myosin XVa is a member of the unconventional myosin superfamily, which is indispensable in the graded elongation of stereocilia of cochlear and vestibular hair cells. Whirlin is a scaffolding protein that is essential for maintaining normal human hearing  and sight . EPS8 is an actin capping protein and is similarly essential for human hearing . Myosin XVa can transport whirlin and Eps8 to the tip of stereocilia and interacts with them to form a stereocilia tip complex, which can facilitate the extremely important transformation of microvilli into mature stereocilia [Shaker-2 (Myo15sh2) mice exhibit profound hearing loss and abnormal vestibular function caused by short stereocilia and by losing the normal staircase structure of stereocilia in hair cells [The reocilia . Shaker-MYO15A variants affecting the motor domain is the largest of all domains  had recently been identified in a non-consanguineous Chinese family. The audiograms of two affected siblings also showed moderate residual hearing at identical low frequencies, which seems to provide additional evidence of an association between the mutant allele p.Ser1176Valfs*14 and a less severe auditory phenotype, especially in Chinese families [In our literature review, the number of ins Fig. c. The mohenotype . Earlierhenotype . Howeverhenotype . In 2007henotype . More pahenotype . Furtherfamilies . In addiMYO15A variants should predict hearing loss tendency according to affected domain, which will inform the choice of optimal hearing rehabilitation methods for individuals [MYO15A was much more complicated. First, recent studies showed that some pathologic variants affecting domains  that would have been expected to cause a severe hearing phenotype, actually resulted in a milder auditory phenotype. The above relationship was further confirmed by our finding that case (1507382) showed residual low-frequency hearing and compound heterozygosity for MYO15A , affecting the motor and MyTH4 1 domains. Conversely, some variants associated with dysfunction of the N-terminal domain cause congenital severe to profound hearing loss instead of a milder phenotype. In addition, we reviewed 12 MYO15A variants associated with both severe and milder phenotypes (Table MYO15A variants [MYO15A variant (p.Tyr289*) affecting the N-terminal domain. The audiograms of three affected siblings revealed congenital severe to profound sensorineural hearing loss at all frequencies, while their mother showed progressive severe sensorineural hearing loss of a milder form, which suggested the presence of a modifier. To our knowledge, in general, autosomal dominant non-syndromic hearing loss (ADNSHL) were associated with post-lingual hearing impairment, whereas ARNSHL were associated with congenital or pre-lingual hearing impairment. However, according to the results of our literature review, there were 8 MYO15A variant cases from Japan and Korea with unconventional post-lingual hearing impairment Additional file 2:Figure S1. Pedigrees of the families carried MYO15A variants. (PDF 420 kb)Additional file 3:Table S2. Variants of MYO15A detected in this study. (DOCX 25 kb)


Ctenocephalides felis is an ectoparasitic flea species commonly found on dogs and cats. The current study verified the in vitro virulence of conidia of the entomopathogenic fungus Beauveria bassiana produced under different color LED light  to adults of C. felis. The fungal isolates were cultivated on malt extract agar (MEA). Bioassay treatments used aerial conidia in test tubes. Adult fleas were obtained from a house cat in Chiang Mai province, Thailand. The experiments were composed of one control and eleven treatment groups. All of the treatments with B. bassiana conidia caused adult mortality after an exposure of 12 h. Among the conditions used in this study, B. bassiana cultured under red LED and fluorescent light were the most effective in causing mortality (100 %) in adult fleas after 36 h. The experimental results indicate that these aerial conidia of B. bassiana have promising potential for use in control of C. felis adult stages. Ctenocephalides canis (Curtis), and Ctenocephalides felis felis (Bouché) are small, laterally flattened, wingless, and highly specialized insects. They are very important obligate hematophagous ectoparasites and have worldwide distribution  in Pitsanulok, Thailand. Stock cultures were maintained in test tubes on slants of MEA medium  at 25±1°C with a photoperiod of 12:12h (dark  :  light). B. bassiana was screened for stimulation of conidiation with six colors of LED light  and under fluorescent light and darkness. After 1 h exposure to the light treatments, cultures were kept in the dark at 27 ± 1°C and relative humidity (RH) ≥ 80% for 15 d. This study used aerial conidia produced by transferring one plug (5 mm. diam.) from the growing edge of a colony to each test tube. Conidia were suspended in a 0.01% aqueous solution of Tween 80 and quantified by haemocytometer. B. bassiana in each test tube, incubated at 27±1°C, and mortality was checked every 12 h. The experiment had two control groups: pure agar and Metarhizium anisopliae. Mean mortality time and LC50 were calculated and statistical analysis used one-way ANOVA and significance between treatments was tested with the LSD method (P < 0.05).Adult fleas used in this study were the laboratory strain that has been maintained at the Insect Pathology Laboratory, Department of Entomology and Plant Pathology, Faculty of Agriculture, Chiang Mai University, Thailand. This colony originated from a wild strain taken from a house cat in Chiang Mai area and has been maintained on cats under laboratory conditions. Fleas were removed from cats via combining and counted in a test tube for every treatment (11 tr.) which was done for 5 replications. The strain has not been pressured with any insecticides since then. Bioassay treatments using 20 live fleas per one test tube were kept at room temperature for 1-2 h and were treated with one plug (0.5 mm. diam.) of B. bassiana conidial production with a high virulence to pests. In the current study, the mortality of Ctenocephalides sp. observed in treatments 1-11 was time-dependent ( B. bassiana + , B. bassiana + (fluorescent (0.09×108 conidia/ml), dark (0.09×108 conidia/ml)), and two isolates of M. anisopliae (4849 (0.04×107 conidia/ml), 2MG (0.13×108 conidia/ml)) had significantly different mortalities at 12 h after treatment. The results revealed that the cumulative mortalities of adults at 12 h ranged between 6 and 27 percent. After 24 h, all of fungal treatments showed a cumulative flea mortality of more than 75 percent. During the same period, B. bassiana +  and two isolates of M. anisopliae  showed the highest cumulative mortalities of 93, 89, 92, and 92%, respectively, and were not significantly different, while 100% mortality was reached in the treatments B. bassiana +  and M. anisopliae (4849) after 36 h (Previous studies have shown that different color LED light has great potential to stimulateependent . Adult fter 36 h . All fleter 36 h . Ctenocephalides sp. was also only undertaken by De-Melo et al. [ B. bassiana has shown pathogenicity as a biological control agent for different arthropod species and it is commercially available as various mycoinsecticides [ B. bassiana produced under LED light start killing fleas within 12 h after treatment administration [ B. bassiana had a cumulative flea mortality of 100% in 36-48 h as short time similar some synthetic chemicals [ M. anisopliae had different effects on insect pests. So, identification, application and screening of different fungal isolates in bioassays may provide promising biopesticides. Our study screened both B. bassiana and M. anisopliae with activity against adult fleas. The current research showed that growth of B. bassiana under LED lights enhanced production of conidia that resulted in 100% overall flea mortality. Future research will examine the ovicidal and larvicidal capacity of this entomopathogenic fungus. In the future such entomopathogenic fungi could be developed as bioinsecticides for practical use in pet care products like Chaingard® pets powder. The evidence presented here indicates that both B. bassiana and M. anisopliae have a high level of adulticidal activity, and potential for inclusion in flea management systems.To the best of our knowledge, the work of seeking efficient entomopathogenic fungi againsto et al. , 21, 24.cticides , 21. In hemicals , 14, 23.hemicals , 25. Simhemicals  it was rhemicals . Previouhemicals  showed t B. bassiana produced under difference colors of LED light was relatively high ranging from 78 to 100 percent and rapidly occurred from 24 to 36 h after exposure. Comparisons of conidia of B. bassiana produced under different colors of LED light to enhance their virulence and efficacy were obtained from the experiments. Conidia of B. bassiana produced under different colors of LED light caused infection which involves integument penetration at 12 h and total colonization of the insect at 48 h after exposure. Therefore, these results indicate that B. bassiana culture under LED light has great potential as a candidate for biological control of Ctenocephalides sp. adult stages. The potential of this entomopathogenic fungus for controlling fleas needs continued study, and the research should be expanded to include other ectoparasites of dogs and cats.A previous study showed the efficacy of flea control by using aerial conidia of


This study directly compared the performance of a contralateral routing of signal (CROS)/bilateral routing of signal (BiCROS) and a soft-band bone-anchored hearing aid (BAHA) in patients with unilateral sensorineural hearing loss (SNHL) and assessed the relationship between hearing aid benefits and personal factors. Participants with unilateral SNHL were prospectively enrolled in the study and were tested under the following three conditions: unaided, with CROS/BiCROS, and with soft-band BAHA. Sound localization, consonant, hearing in noise, and psychoacoustic tests were performed. Pseudobinaural benefits  were obtained in both the CROS/BiCROS and soft-band BAHA conditions and compared to the unaided condition. Sound localization ability was not improved in either the CROS/BiCROS condition or soft-band BAHA condition. Rather, sound localization ability was significantly decreased in the CROS/BiCROS setting. A CROS/BiCROS hearing aid and a soft-band BAHA provided additional benefit for speech-in-noise perception when target speech was directed to the impaired ear side. The CROS/BiCROS hearing aid was superior to the soft-band BAHA one in decreasing the head shadow effect, but it appeared to have a negative effect when the noise was delivered to the better ear. The positive and negative effects of CROS/BiCROS for localization and speech perception were significantly correlated with personal factors such as age, hearing threshold in the better ear, and unaided psychoacoustic performances. Despite the lack of device acclimatization, we believe that this study provides counseling information for hearing aid clinics to use in the context of patients with unilateral SNHL. Individuals with severe to profound unilateral sensorineural hearing loss (SNHL) report difficulty localizing sound within a horizontal plane and with listening to sounds coming from the side of the impaired ear . DespiteThere are two main nonsurgical options currently used worldwide to help alleviate the head shadow effect. One option is a rerouting of the signal to the contralateral normal hearing ear through air conduction. This can be accomplished by use of the contralateral routing of signal (CROS) system. CROS/bilateral routing of signal (BiCROS) air conduction hearing aids use a microphone in the impaired ear that delivers sound to the functioning ear either via a wire or wirelessly. BiCROS hearing aids also provide appropriate amplification for the functioning ear when the better ear has hearing loss. The second option is the use of a nonsurgical bone conduction system . Bone-anchored hearing aids (BAHAs) route sound to the better ear by transcranial bone conduction from a microphone/processor located on the skull of the impaired ear side. A bone conduction audio processor coupled to the head with a soft band has been the most common nonsurgical option available over the past 15 years or so. Thus, wearing a CROS hearing aid or a soft-band BAHA on the impaired side limits the head shadow effect. Although both devices impair speech perception when the noise emanates from the impaired ear side –9 and doRecently, more individuals with severe to profound unilateral SNHL consider their hearing loss to be a hindrance in social interactions, which affects their emotional and psychological well-being –20. PatiThe present study compared the performances of localization ability in a horizontal plane, pseudobinaural benefits of speech perception in noisy conditions, and psychoacoustic performance between wireless CROS/BiCROS hearing aids with technologies such as digital noise reduction and directional microphone and soft-band BAHAs with similar technologies. To provide a customized rehabilitation strategy, we also assessed the relationship between hearing aid benefit and personal factors.All participants were recruited and tested in the Hearing Laboratory at Samsung Medical Center in Seoul, South Korea. Every participant provided written informed consent prior to participating in this study. Written informed consent was additionally obtained from the next of kin, caretakers, or guardians of any minors/children enrolled in this survey. The study protocol was approved by the Samsung Medical Center Institutional Review Board (2016-06-136). The study was carried out in accordance with approved guidelines.A total of 21 subjects  with unilateral SNHL were prospectively enrolled. All subjects had severe to profound hearing loss  in one ear and normal hearing or mild hearing loss  in the contralateral ear. None of the subjects had air-bone gaps of 15 dB or more at 500, 1, 2, or 4 kHz in both ears. Demographics and pure-tone thresholds for each subject are shown in The CROS/BiCROS system employed herein consisted of a wireless Phonak CROS transmitter on the poorer ear and a Phonak Audéo Q70-312T open-fit hearing aid  on the better ear. If there was some degree of hearing loss in the better ear, a BiCROS hearing system was adapted. Devices were fitted by experienced audiologists using Phonak’s fitting algorithm  as recommended by the manufacturer. The Phonak CROS/BiCROS program included (1) SoundFlow, a multi-base automatic program that actively adapts and integrates the correct parameters to ensure optimal everyday listening; (2) Rear Ear Sound, an adaptive directional microphone to improve sound localization and improve speech in noisy situations; and (3) Sound Recover, a frequency adjustment protocol that acts according to the threshold of the better ear. Detailed CROS/BiCROS hearing aid fitting information is shown in The BAHA sound processor was worn on a BAHA Softband. The tested sound processor was the programmable Cochlear Baha 3 Power (BP110). The BAHA sound processor  was fitted by experienced audiologists using the Baha Fitting Software. The Client, Indication, and Connection types were configured using the BC Select step. This enabled a quick setup of the sound processor and incorporated the latest research data on corrections for transcranial attenuation, cross-hearing, and transmission loss through the skin . The finParticipant performance was compared under the following three different conditions: unaided, with the wireless CROS/BiCROS, and with the soft-band BAHA. Participants were randomly assigned to the CROS/BiCROS hearing aid or BAHA. The order in which hearing devices were worn varied across subjects. There was no acclimatization period for the devices. All subjects participated in audiogram, localization, speech perception, and psychoacoustic tests in a double-walled and sound-attenuating booth. In addition to objective measures, subjective performance was also assessed using a questionnaire. Each participant performed all tests on the same day. Sound localization and speech-in-noise perception tests were conducted without masking the better ear, whereas for audiogram, consonant test, and psychoacoustic tests, an ear plug was inserted in the better ear only in the BAHA condition.Each participant was seated one meter from the center of a 13-loudspeaker array. The loudspeakers were placed at 15° intervals from a −90° azimuth to a +90° azimuth. Study participants sat in chairs at the center of the arc with their ears at a height equal to the loudspeaker array. Participants were informed of the number corresponding to each speaker. They were instructed that the target word would be randomly presented from one of the 13 speakers. They were asked to remain seated, facing the loudspeaker at the 0° azimuth and to hold their head still during the testing. Each participant was instructed to say the number of the loudspeaker from which they thought the target sound was coming. The localization stimulus was a Korean bisyllabic word “ja-yeon.” The stimulus was 865 ms in duration. It was recorded from a male speaker using a sampling rate of 44.1 kHz . Each stLocalization accuracy was quantified in two ways, as follows: root-mean square error (RMSE) and hemifield identification score. First, we calculated the error in degrees between the target speaker and the response speaker for each trial. Errors were squared before they were averaged. Then, the square root was taken after averaging . We alsoSpeech perception testing included the consonant test and the Korean Hearing in Noise Test (K-HINT) . For theThe participants were also subjected to K-HINT, which was modified for this experiment to measure the pseudobinaural benefit of speech perception in a noisy condition. During this trial, the study participant sat on a chair in the center of the sound field facing two loudspeakers that were located approximately one meter away at the 0° azimuth. Two additional speakers were located at the same distance from the listener at the ± 90° azimuths. Target sentences and white noise were presented from the front speaker (0°) or from one of the two side speakers (± 90°). The presentation level of the noise was fixed at 65 dBA, and the level of target sentences varied according to the adaptive procedure of the original HINT . One lisSFN); (2) for measuring the squelch effect, speech was presented from one of the two side speakers (± 90°) toward the better ear (NH), while noise was presented from one of the two side speakers (± 90°) toward the poorer ear (HL) (NHSHLN); and (3) for measuring the head shadow effect, speech was presented from one of the two side speakers (± 90°) toward the poorer ear, while noise was presented from one of the two side speakers (± 90°) toward the better ear (HLSNLN). Study participants were instructed not to move their heads. Prior to the experiment, four to five practice tests were administered. No feedback was provided during the practice or test runs.The K-HINT trial was conducted in three different conditions, as follows: (1) for measuring the summation effect, both speech and noise were presented from the front  graphical user interface was used to present acoustic stimuli to subjects for psychoacoustic tests. Stimuli were presented through a loudspeaker  in the sound field at an average level of 65 dBA. Study participants sat at a distance of one meter from the loudspeaker and were asked to face the speaker during the course of the experiment.et al. (2011) . Post-hoc tests using the Bonferroni correction revealed that mean RMSE was significantly increased when tested in the CROS/BiCROS condition as compared with in the unaided condition (p = 0.003). However, no significant difference in mean RMSE was observed when performance in the BAHA condition was compared to that in the unaided condition (p = 0.225). Separately, the mean ± SD values of hemifield identification score in the three listening conditions are presented according to the loudspeaker location  = 0.335, p = 0.717]. A paired t-test was run for each listening condition to determine whether or not there was a statistically significant mean difference between the poorer ear side and the better ear side. Participants with unaided and BAHA conditions performed significantly better at differentiating the sound location side when the sound came from the better ear side versus when the sound came from the poorer ear side . There was no significant difference in mean hemifield identification score in the CROS/BiCROS condition, regardless of where the sound originated from .The results of localization testing for participants with unilateral SNHL in each listening condition are shown in location . Loudspeχ2(2) = 31.976–38.456, all p < 0.001 for all frequencies). Post-hoc analysis with Wilcoxon signed-rank test was conducted with a Bonferroni correction applied, resulting in a significance level set at p < 0.017. At 250 Hz, BAHA performed significantly better than did CROS/BiCROS . At higher frequencies , CROS/BiCROS performed significantly better than BAHA . Speech recognition threshold (SRT) and word recognition score (WRS) values were similar between the two aided conditions.The mean ± SD of pure-tone thresholds between listening conditions are shown in χ2(2) = 18.175, p < 0.001 in a quiet condition; χ2(2) = 16.231, p < 0.001 in a steady noise condition; and χ2(2) = 23.792, p < 0.001 in a modulated noise condition). Post-hoc analysis with Wilcoxon signed-rank test (a Bonferroni correction was applied to set a significance level of p < 0.017) determined that consonant perception in a quiet condition was significantly increased when tested in the CROS/BiCROS condition versus the unaided  and BAHA  conditions. However, consonant perception in either noise condition was significantly decreased when tested in the BAHA condition as compared with in the unaided condition  and CROS/BiCROS condition .SFN), −6.90 dB ± 3.48 dB when speech was directed at the better ear and noise was directed at the poorer ear (NHSHLN), and 4.80 dB ± 2.10 dB when speech was directed to the poorer ear and noise was directed at the better ear (HLSNHN). Clearly, speech perception in the unaided condition was best in the NHSHLN condition and worst in the HLSNHN condition . Speech perception with BAHA was of a similar pattern to in the unaided condition in the three different noise conditions . The mean SNR threshold ± SD with BAHA was −1.53 dB ± 1.49 dB in the FSFN condition, −7.27 dB ± 3.23 dB in the NHSHLN condition, and 2.62 dB ± 2.52 dB in the HLSNHN condition. However, speech perception with CROS/BiCROS was not different among any of the noise conditions . The mean SNR threshold ± SD with CROS was −1.52 dB ± 1.14 dB in the FSFN condition, −3.19 dB ± 2.97 dB in the NHSHLN condition, and −1.67 dB ± 4.91 dB in the HLSNHN condition.When comparing the pseudobinaural benefits of speech perception in noise, SNR thresholds of K-HINT in the three different noise conditions are presented in p = 0.046 for the FSFN condition; F = 13.421, p < 0.001 for the NHSHLN condition; and F = 32.683, p < 0.001 for the FSFN condition]. For the FSFN condition (summation), post-hoc tests using the Bonferroni correction revealed that speech perception in the CROS/BiCROS condition was significantly improved as compared with in the unaided condition (p = 0.028). Furthermore, speech perception in the BAHA condition elicited a slight improvement versus in the unaided and CROS/BiCROS conditions, though not in a statistically significant manner . For the NHSHLN condition (Squelch), post-hoc tests using the Bonferroni correction revealed that speech perception in the CROS/BiCROS condition was significantly decreased as compared with in the unaided (p = 0.006) and BAHA (p < 0.001) conditions. In other words, CROS/BiCROS delivered noise from the poorer ear to the better ear and could not suppress the delivered noise . However, speech perception in BAHA showed a slight improvement as compared with in the unaided condition, although not in a statistically significant way (p = 1.000). For the HLSNHN condition (head shadow), post-hoc tests using the Bonferroni correction showed that both CROS/BiCROS and BAHA led to significantly better speech perception than did the unaided condition . Additionally, CROS/BiCROS performed significantly better in reducing the head shadow effect than did BAHA (p < 0.001).A repeated-measures ANOVA with a Greenhouse–Geisser correction determined that speech perceptions in the three different noise conditions differed significantly between listening conditions [F = 3.729, χ2(2) = 3.349, p = 0.187). TMD thresholds at 10 Hz are shown in χ2(2) = 14.095, p = 0.001). Post-hoc analysis with Wilcoxon signed-rank tests (a Bonferroni correction was applied to establish a significance level of p < 0.017) revealed that temporal resolution in the BAHA condition was significantly decreased as compared with in the unaided condition  and the CROS/BiCROS condition .Psychoacoustic performance for each listening condition is shown in χ2(2) = 0.353, p = 0.838), while speech quality significantly differed depending on listening condition (χ2(2) = 8.291, p = 0.016). However, there were no significant differences between listening conditions in post-hoc analysis with Wilcoxon signed-rank tests .Hearing aid preference and subjective assessments are shown in r| = 0.441–0.518, all p < 0.05). On the contrary, that in the BAHA condition were significantly increased in participants who had a worse hearing threshold in the better ear, impaired temporal resolution, and impaired spectral resolution . The benefit of a device that improves speech perception was significantly correlated with personal factors only in the CROS/BiCROS condition. The benefit of CROS/BiCROS for consonant perception in a quiet environment was significantly increased in participants who had worse hearing thresholds in the better ear, impaired spectral resolution, and impaired temporal resolution . The benefit of CROS/BiCROS for consonant perception in a steady noise was also significantly increased in participants who had impaired temporal resolution . There was no significant correlation between personal factors and benefit of a device that improved speech-in-noise perception by summation effect . The benefit of CROS/BiCROS for speech-in-noise perception was significantly increased with worse spectral resolution  when speech came from the direction of the better ear and noise came from the direction of the poorer ear . However, it was significantly increased with younger age  and better spectral resolution  when speech and noise came directionally from the poorer ear .To provide a customized rehabilitation strategy, a Pearson’s correlation was run to assess the relationship between device benefits (CROS/BiCROS or BAHA) and personal factors such as age, hearing threshold of the better ear, unaided TMD performance, and unaided SRD performance . Hearingt(11) = −1.239, p = 0.241).This study provides possible counseling information to be considered when choosing a noninvasive rehabilitative option in individuals with severe to profound unilateral SNHL. Wireless CROS/BiCROS hearing aids and soft-band BAHAs had no benefit for improving horizontal localization ability . In factA CROS/BiCROS hearing aid significantly enhanced consonant perception in a quiet situation as compared with in the unaided and BAHA conditions , and thiWhen we compared the subjective discomfort in the noisy background and sound quality, participants rated both hearing devices similarly to the unaided condition . The preThis study has several limitations. First, this study had no trial period for participants to adjust to the hearing devices. Psychoacoustic results showed that temporal resolution decreased when sound was delivered from the poorer ear to the better ear through a BAHA . In otheIn summary, sound localization ability in the horizontal plane was significantly decreased in the CROS/BiCROS condition because it diminished the monaural level and spectral cues for monaural listeners. A CROS/BiCROS hearing aid provided a slight but significant additional benefit for speech perception in a quiet and speech-in-noise perception when speech and noise were presented from the front. Both the CROS/BiCROS hearing aid and BAHA helped reduce the head shadow effect, and they significantly enhanced speech-in-noise perception when the target speech was presented to the poorer ear. A CROS/BiCROS hearing aid was superior to a BAHA in overcoming the head shadow effect, but it appeared to have a rather negative effect when the noise was delivered to the better ear. It should be noted that the performance of the CROS/BiCROS hearing aid was significantly influenced by age, hearing thresholds in the better ear, and unaided psychoacoustic performances and these factors should be considered when counseling patients in hearing aid clinics. Despite the lack of device acclimatization, we believe that this study provides potential counseling information for use in hearing aid clinics for the management of patients with severe to profound unilateral SNHL.S1 Tablep* for unaided and CROS/BiCROS conditions, p¶ for unaided and BAHA conditions, p† for CROS/BiCROS and BAHA conditions.Normal hearing on the contralateral side is defined as pure-tone average air-conduction hearing threshold  of better than or equal to 20 dB HL. (DOCX)Click here for additional data file.