In the adult auditory organ, mechanoelectrical transducer (MET) channels are essential for transducing acoustic stimuli into electrical signals. In the absence of incoming sound, a fraction of the MET channels on top of the sensory hair cells are open, resulting in a sustained depolarizing current. By genetically manipulating the in vivo expression of molecular components of the MET apparatus, we show that during pre-hearing stages the MET current is essential for establishing the electrophysiological properties of mature inner hair cells (IHCs). If the MET current is abolished in adult IHCs, they revert into cells showing electrical and morphological features characteristic of pre-hearing IHCs, including the re-establishment of cholinergic efferent innervation. The MET current is thus critical for the maintenance of the functional properties of adult IHCs, implying a degree of plasticity in the mature auditory system in response to the absence of normal transduction of acoustic signals. Mechanoelectrical transducer (MET) channels on the tips of inner hair cells are essential for transducing auditory sensory information. Here, the authors show that disrupting MET channel function also prevents the preservation of normal inner hair cell identity in adult mice. While tip links are formed by cadherin 23 (CDH23) and protocadherin 15 (PCDH15)2, several other proteins are involved in tensioning and anchoring these links3. In the absence of sound, a fraction of the MET channels are normally open, allowing a standing depolarizing current into the hair cells that is mainly carried by K+ and Ca2+. Calcium entry through the MET channels causes adaptation and reduces their open probability4. However, the endolymph that surrounds the hair bundle has a low Ca2+ concentration in both pre-hearing 5 and adult (~ 20–40 µM)6 cochleae, resulting in a larger in vivo resting open probability of the MET channel. The driving force for the MET current is provided by an electrical gradient between the endocochlear potential 7 and the hair-cell resting potential (~ −60 mV)8. Although the resting MET current contributes to setting the membrane potential of pre-hearing and adult inner hair cells (IHCs)8, its functional implication for IHC development is currently unknown.The sense of hearing relies on sound evoking the displacement of stereociliary bundles projecting from the upper surface of hair cells. Hair bundle displacement causes the opening of mechanoelectrical transducer (MET) channels located at the tips of the shorter and middle rows of stereocilia, and the transduction of acoustic information into a receptor potential inside the hair cells. Stereocilia are interconnected by several extracellular linkages2+ action potentials (APs)10, which play a crucial role in promoting neuronal survival11 and the refinement of the immature auditory system13. Functionally mature IHCs respond to sound stimulation with graded receptor potentials. This change in the receptor potential requires a completely new complement of basolateral ion channels that appear just before the onset of hearing14. We hypothesize that, in the immature cochlea, the functional MET channels together with the maturing endocochlear potential7 and endolymphatic Ca2+ concentration5, trigger the developmental switch from spiking IHCs to high-frequency signal transducers.Before the onset of hearing, IHCs fire spontaneous CaUsing transgenic mice to manipulate the expression of molecular components of the MET apparatus in vivo, we demonstrate that the MET current is required for their developmental switch from immature to functional sensory receptors. Moreover, we found that the resting MET current in adult IHCs is required for maintaining their mature biophysical identity. The loss of the MET current in adult IHCs causes them to revert to an immature, pre-hearing phenotype and they re-acquire efferent innervation.Pcdh15 lead to hair-bundle disruption and loss of the MET current from early postnatal days15. Therefore, we investigated whether the absence of the MET current in PCDH15 deficient mice affected hair cell maturation. IHCs acquire most of their mature biophysical and morphological characteristics around the onset of hearing (~ P12). This includes the expression of a rapidly activating large conductance Ca2+-activated K+ current 9 and a K+ current 14 carried by KCNQ4 channels. We found that IK,f was present in adult control IHCs  but absent in Pcdh15av3J/av3J mutant mice . The size of IK,n was significantly reduced in Pcdh15av3J/av3J IHCs  compared to that of Pcdh15+/av3J control cells . Pcdh15av3J/av3J IHCs retained an immature phenotype by expressing the inward (IK1) and slowly-activating outward (IK) K+ currents16. The size of IK, calculated as the difference between the steady-state and early  current was 2.4 ± 0.5 nA (Pcdh15+/av3J) and 1.6 ± 0.2 nA (Pcdh15av3J/av3J) . The physiological consequence of the immature current profile in adult Pcdh15av3J/av3J IHCs was that, instead of generating rapid and graded voltage responses characteristic of adult IHCs  (Deafness mouse)17, which is necessary for mechanoelectrical transduction18, and the unconventional Myo6 19, which is required for the development of the MET apparatus20. The above findings indicate the existence of a link between a functional MET apparatus and IHC maturation. Mutations in or absence of stereociliary proteins early in development normally causes severe morphological defects of the stereociliary bundle . We used targeted knockouts obtained by crossing floxed mice (Ush1cfl/fl and Myo7afl/fl) with Myo15-cre+/- mice24. Because the temporal expression of the cre driven by the hair cell specific Myo15 promoter occurs postnatally 24, the stereociliary bundle develops normally, allowing us to study the functional implications of the missing protein.Protocadherin 15 (PCDH15) is localized to the lower part of the tip link where it contacts the top of the shorter rows of stereocilia. Mutations in ice Fig. . The absHCs Fig. , they reHCs Fig. , a chara25. In Ush1c (harmonin) mutant mice the MET channel lacks the resting current and by P7 the MET current is almost completely absent23. Although the harmonin-a isoform has been implicated in Ca2+ channel regulation, this role is restricted to post-hearing IHCs26. Therefore, we recorded MET currents from Ush1cfl/fl and Ush1cfl/flMyo15-cre+/- immature IHCs 24. However, at P8  compared to control cells , and it deteriorated rapidly thereafter such that by P10 it was almost absent in Ush1cfl/flMyo15-cre+/− IHCs . The fraction of the MET channel open at rest in P10-P12 IHCs was negligible in Ush1cfl/flMyo15-cre+/- mice  and significantly reduced compared to that of control cells . Despite the abnormal MET current, the hair bundles of Ush1cfl/flMyo15-cre+/- IHCs acquired the characteristic staircase structure and appeared qualitatively similar to littermate controls at P15  were significantly reduced compared to control cells  compared to control cells . Finally, s2 in IHCs from Ush1cfl/flMyo15-cre+/- mice tended to show rounded tips rather than the characteristic prolate-shaped stereocilia tips of controls , which has to be close to the activation threshold of the Ca2+ current. In IHCs Vm is determined by the balance between an inward, depolarizing resting MET current and outward IK5. Therefore, we investigated whether the substantially reduced resting MET current in immature IHCs from Ush1cfl/flMyo15-cre+/− mice affected IHC physiology. Since the fraction of MET channels open at rest is determined by Ca2+-dependent adaptation27, the Vm of IHCs in vitro strongly depends upon the extracellular Ca2+ concentration. Using current clamp, we measured the depolarizing current required to elicit APs in IHCs at 35–37 °C using 1.3 mM Ca2+, which is the Ca2+ concentration present in the perilymphatic solution bathing the IHC basolateral membrane. Starting from the resting Vm , a similar amount of current was required to trigger APs in both control Ush1cfl/fl  and Ush1cfl/flMyo15-cre+/−  mice , and even more so when values were adjusted for the temperature difference  is the resting membrane potential  and adult (P29) IHCs from control Ush1cfl/fl mice express both IK,f and IK,n 31 and the K+ current profile of adult IHCs retains pre-hearing characteristics  and resting open probability  of the MET current in IHCs of pre-hearing Myo7afl/flMyo15-cre+/− mice was similar to that measured in control Myo7afl/fl IHCs   is another hair cell protein that binds to harmoninice Fig. , the siz10) Fig. . The norHCs Fig. , but notMyo7afl/flMyo15-cre+/- mice meant that superfusion of the estimated endolymphatic Ca2+ concentration (0.3 mM Ca2+)5, caused them to depolarize to near the expected in vivo resting Vm and elicited repetitive APs  acquired current and voltage responses similar to control Myo7afl/fl cells .The aforementioned experiments Fig.  show thaAPs Fig. . The incnce Fig. , confirmration 0. mM Ca2+5Myo7afl/flMyo15-cre+/− mice, the MET current became progressively affected from P14 compared to control Myo7afl/fl IHCs  were significantly larger than those from Myo7afl/flMyo15-cre+/− cells at P14  and P16  (one-way ANOVA). The smaller MET current in Myo7afl/flMyo15-cre+/- IHCs after the onset of hearing occurred despite the normal appearance of the stereociliary bundles and the presence of tip links as well as prolate-shaped stereocilia tips , such that by P59 Myo7afl/flMyo15-cre+/- IHCs exhibited an immature current profile  and an inward rectifier (IK1) K+ current16. IHCs from control mice  remained relatively constant as a function of age . Compared to IHCs recorded from acutely dissected gerbil cochleae 4. Therefore, in vitro, only a very small or no depolarizing inward MET current is likely to be present8. By keeping IHCs depolarized using high K+ in the culture medium (40 mM), as a proxy for the in vivo receptor potential caused by the resting MET current, IK,f was retained for longer periods , which substantially increases the resting MET current27. In 40 μM Ca2+  or when 40 mM K+ was used . When cochlear cultures were incubated with 40 μM Ca2+ together with 50 μM of the MET channel blocker d-tubocurarine34 , but produced similar results to when the open probability of the MET channel was very low . Blocking the MET channel caused IHCs not only to rapidly lose IK,f but also to express the inward rectifier K+ current IK1, which is normally present in pre-hearing cells16 . Although IK1 was also seen in IHCs with partially closed MET channels  it was significantly smaller than that measured in the presence of 40 μM Ca2+ and d-tubocurarine .To confirm the hypothesis that the resting MET current is required for IHCs to maintain their mature biophysical characteristics, we set up organotypic cultures from the adult cochlea. For these experiments we used gerbils (P18-P24), instead of mice, because their adult IHCs are more robust and can survive in vitro for the long periods required to test the hypothesis. Initially, cochleae were cultured for 1 to 8 days in the DMEM/F12 medium supplemented with CaClHCs Fig. . By the ent Fig. . The maiods Fig. . The dira2+ Fig. , the size34 Fig. , which hK,f Fig.  compareds16 Fig. . IK1 was2+-efficiency12. Presynaptic activity was estimated by measuring the size of the Ca2+ current (ICa) and the induced increase in cell membrane capacitance (ΔCm) following depolarizing voltage steps12. ICa in IHCs from both young adult  and adult  Myo7afl/flMyo15-cre+/− mice was not significantly different  to those measured in control Myo7afl/fl IHCs   and control Myo7afl/fl IHCs . The apparent larger inactivation of ICa in Myo7afl/flMyo15-cre+/− IHCs  Fig. . In addiHCs Fig.  was due HCs Fig. , which i37. IHCs are directly innervated by cholinergic efferent endings only during early stages of development, but no longer respond to acetylcholine (ACh) from about P18 onwards38. Adult IHCs from P22 Myo7afl/fl and Myo7afl/flMyo15-cre+/− mice did not respond to extracellular ACh  and amplitude  not significantly different from those previously reported in pre-hearing IHCs 39. Reversible block of the ACh-mediated currents by 1 μM strychnine  indicated that while IHCs from Myo7afl/fl mice showed the characteristic axo-dendritic contacts between efferent and afferent fibers , promoting their functional differentiation into mature graded sensory receptors able to encode sound stimuli. We also found that adult IHCs require functional MET channels to maintain their adult profile. The absence of the MET current during adult stages caused IHCs to lose their identity, regressing into an immature-like biophysical and morphological profile. Adult IHCs without functional MET channels became re-innervated by inhibitory cholinergic efferent neurons, which normally make transient axo-somatic contacts only with pre-hearing IHCs.Here we show that the MET current is essential to establish and maintain the key biophysical and morphological properties characteristic of adult IHCs. During the second post-natal week in mice, which is just before the onset of hearing at P12, open MET channels keep the IHC membrane potential at the threshold for Ca2+-dependent APs14 that can be modulated by the inhibitory efferent neurotransmitter ACh38 and by excitatory spontaneous ATP-induced Ca2+-waves in non-sensory cells, which synchronize the activity of several IHCs41. Indeed, patterned bursts of APs have been recorded from auditory afferent neurons prior to the onset of hearing in mice43, and both their survival11 and the refinement of tonotopic maps in the auditory brainstem44 rely on the firing activity of pre-hearing IHCs. However, the refinement of the auditory pathway mainly occurs during the first postnatal week45, a finding supported by evidence that surgical removal of the cochlea causes a substantial loss of cochlear nucleus neurons mainly during this period47. Despite the crucial role of APs in developing IHCs, spiking activity is not suitable to detect sound because it would modulate and degrade auditory stimuli in the mature cochlea. Therefore, just before hearing onset immature IHCs undergo major changes involving the acquisition and/or elimination of different ion channels and other membrane proteins14. Intracellular Ca2+ transients associated with a precise frequency and/or pattern of AP activity can activate transcription factors controlling the expression of proteins linked to development49. In cochlear IHCs, repetitive Ca2+ APs during the second postnatal week are driven by a standing depolarizing MET current5, resulting from the progressive reduction in endolymphatic Ca2+ concentration5 that, by reducing adaptation, increases the MET channel open probability. Therefore, AP activity driven by the greater standing MET current during the few days preceding hearing onset could provide the cue or trigger for the last step of IHC development by influencing gene expression. We demonstrate that genetic manipulations that abolish the contribution of the MET current in vivo during the second postnatal week prevented IHCs from becoming mature auditory receptors. We propose that, during pre-hearing stages of development, the primary function of IHC AP activity could switch from refining the auditory circuitry13 to promoting the maturation of the sensory receptor itself.Pre-hearing IHCs of altricial rodents exhibit spontaneous CaMyo7afl/flMyo15-cre+/- mice meant that their auditory system was able, at least initially, to mature fully as judged by the presence of normal ABRs (P15), absence of axo-somatic efferent terminals on P20-P22 IHCs, and a normal IHC basolateral current profile at P20. However, the loss of IHC MET current after the onset of hearing caused a progressive down-regulation of the adult-type basolateral ion channels carrying IK,f and IK,n. By two months of age, IHCs from Myo7afl/flMyo15-cre+/- mice have the same size and basolateral membrane currents as pre-hearing cells, including the re-expression of IK116 and ISK236. A similar progressive loss of IK,f was also observed in cultured cochlear IHCs, in which cells are not stimulated by sound and the resting open probability of the MET current is either reduced or blocked. In vitro experimental manipulations that increased the MET channel open probability (endolymphatic-like Ca2+ concentration) or mimicked the receptor potential due to sound stimulation (IHC depolarization) were able to maintain the adult current profile of cultured IHCs for longer. Calcium entering via the MET channel has been shown to be required for maintaining the morphological stability of transducing stereocilia in early postnatal hair cells50. Although in the presence of mature endolymphatic-like solution Ca2+ entering through the MET channel seems to be cleared from the stereocilia by the plasma membrane Ca2+ ATPase type 2 (PMCA2), some of it could diffuse into the cell body51 and, together with the depolarization by the MET current, potentially regulate gene expression and as such the maintenance of the adult IHC current profile. The only adult-like function that was not affected by the loss of the MET channel was that associated with the synaptic machinery, indicating that once it has matured, a process that is controlled by a critical period of pre-hearing APs12, it becomes independent of IHC activity. Loss of ribbon synapses in the adult cochlea occurs following acoustic trauma or ototoxic antibiotics52, conditions that cause either a temporary or permanent IHC damage. IHCs from aging mice have also been shown to gradually lose ribbon synapses53. The fact that exocytosis in IHCs from Myo7afl/flMyo15-cre+/- mice was normal at 7 weeks, despite IHCs starting to become smaller, may be due to ribbon loss being a slow process53.The normal AP activity in IHCs from pre-hearing 55. After the onset of hearing, efferent terminals only form axo-dendritic synapses with the auditory afferent fibers contacting IHCs56 and expression of the postsynaptic α9nAChRs and SK2 channels is rapidly down-regulated54. Therefore, adult IHCs lose the ability to respond to ACh54. However, it has been shown that efferent endings can re-innervate IHCs following cochlear damage57 or in aging mice58. We found that the re-emergence of axo-somatic efferent innervation with IHCs can also occur in mature mice with no functional MET current.While auditory ribbon synapses appeared to be resilient to post-maturational changes in MET function, the efferent fibers were more plastic. The efferent endings make transient cholinergic axo-somatic contacts with immature IHCs60, aging61 or in forms of progressive hearing loss, such as Usher syndrome type III62.The above findings, including the reduced size of the IHCs, the direct re-innervation by efferent cholinergic fibers, the complete down-regulation of the adult-like basolateral membrane ion channels and the ability to fire APs, indicate that functional MET channels are required for mature IHCs to maintain their biophysical and morphological identity. Without functional MET channels, IHCs revert to an earlier, pre-hearing stage of development. It would be interesting in the future to determine whether the re-establishment of the immature-like biophysical and morphological profile in IHCs without working MET channels is the first stage of an intrinsic attempt to repair the faulty cochlea by “recapitulating” early development. In any case, this functional regression shows a surprising plasticity in the mature cochlea in response to events that abolish normal MET currents, which could occur due to either noise63. At the end of the experiments, which lasted up to 40 minutes, mice were usually culled by a schedule 1 method. In a few animals, ABR measurements were followed by the recovery of the mice from anesthesia with intraperitoneal of atipamezole mix63.All animal studies were performed in the UK and licensed by the Home Office under the Animals (Scientific Procedures) Act 1986 and were approved by the University of Sheffield and University of Sussex Ethical Review Committees. ABR measurements (see below) were conducted under anesthesia using ketamine  and xylazine , which were administered with intraperitoneal injection as previously describedtm1a allele for both Myo7a (Myo7atm1a(EUCOMM)Wtsi allele ID: 4431921) and Ush1C (Ush1ctm1a(KOMP)Wtsi allele ID: 4363497) were generated by the Mouse Genetics Programme at the Wellcome Trust Sanger Institute . For Myo7a the critical exons 10 and 11 were floxed, while for Ush1c exons 5–8 were floxed. The tm1c allele was obtained by crossing the tm1a mouse to a FLPeR carrying mouse (Rosa26Fki). The tm1d alleles, which were used for the experiments, were obtained by crossing the tm1c mouse (Myo7afl/fl and Ush1cfl/fl) with the Myo15-cre mice and genotyped as previously described24. The genotyping protocols for mutant mice were also previously described: Myo7a (Myo7ash6J/sh6J)31 and Pcdh15 (Pcdh15av3J)15. For the conditional knockout mice  genomic DNA was amplified under standard reaction conditions using Promega Taq DNA polymerase . The reaction mix was incubated at 94 °C for 5 min followed by 40 cycles of 94 °C for 30 s, 58 °C for 45 s and 72 °C for 90 s, followed by 72 °C for 5 min. Primer pairs and product size are as follows: Myo7a: forward, 5′-GGGAGAGAAAGCGGGTGTG- 3′ and reverse, 5′- AAGCTGGACTCTCTGGTGGC-3′, producing a 360-bp product for the WT and a 460-bp product for the tm1c homozygous mutant; Ush1C : forward, 5′-ACAGAGCCGTGGGTTCATTC- 3′ and reverse, 5′-GTAATGGAGCTGAGGCAGGG-3′, producing a 326-bp product for the WT and a 430-bp product for the tm1c homozygous mutant.For the conditional knockout mice, the targeted 64. Gerbil IHCs were positioned at a frequency range of 250–420 Hz65. Animals of either sex were killed by cervical dislocation and the organ of Corti dissected in extracellular solution composed of (in mM): 135 NaCl, 5.8 KCl, 1.3 CaCl2, 0.9 MgCl2, 0.7 NaH2PO4, 5.6 d-glucose, 10 Hepes-NaOH. Sodium pyruvate (2 mM), amino acids and vitamins were added from concentrates (Fisher). The pH was adjusted to 7.5 . The dissected apical coil of the organ of Corti was transferred to a microscope chamber, immobilized using a nylon mesh fixed to a stainless steel ring and viewed using an upright microscope . Hair cells were observed with Nomarski differential interface contrast optics (×63 water immersion objectives). To expose the basolateral surface of the cells, a small tear was made in the epithelium with a suction pipette (tip diameter 3–4 µm) filled with extracellular solution.Inner hair cells (IHCs) were studied in acutely dissected organs of Corti from postnatal day 7 (P7) to P218 mice and P21-P28 gerbils, where the day of birth is P0. In mice, experiments were performed on IHCs positioned in the apical coil of the cochlea (around 12 kHz)2, 1 EGTA-KOH, 5 Na2ATP, 5 Hepes-KOH, 10 Na-phosphocreatine . When investigating MET currents or membrane capacitance, a Cs-glutamate intracellular solution was used composed of (mM): 106 Cs-glutamate, 20 CsCl, 3 MgCl2, 1 EGTA-CsOH, 5 Na2ATP, 0.3 Na2GTP, 5 Hepes-CsOH, 10 Na2-phosphocreatine . Data acquisition was performed using a Digidata 1440 A with pClamp software 10.0  or a CED Power 1401 with Signal Software . Data were filtered at 2.5 kHz (8-pole Bessel) and sampled at ≥ 5 kHz. Origin software  was used to perform offline data analysis. Membrane potentials were corrected for the residual series resistance Rs after compensation  and liquid junction potential (LJP), which for Cs-glutamate and KCl intracellular solutions were −11 mV and −4 mV, respectively.Membrane currents and voltage responses were investigated under whole cell voltage or current clamp, respectively, either at room temperature (20–25 °C) or body temperature (34–37 °C), using Optopatch amplifiers . Patch pipettes, with resistances of 2–3 MΩ, were pulled from soda glass capillaries . The shank of the electrode was coated with surf wax  to reduce the electrode capacitative transient. When investigating the basolateral properties of IHCs, the intracellular solution was composed of (mm): 131 KCl, 3 MgClIK,f was measured near –25 mV and at either 0.7 ms or 1.0 ms after the start of the voltage step, while IK,n was measured as the difference between the peak and steady state of the deactivating inward current at −124 mV. Steady-state total currents were measured at 160 ms, at a potential of 0 mV (extrapolated from the current-voltage curves). The holding potentials used for these recordings were set at either −84 mV or −64 mV .When investigating basolateral membrane properties, the size of + in order to depolarize the efferent fibers. Event analysis was performed using the Mini Analysis Program . Note that the number of events used to calculate the frequency of inhibitory postsynaptic currents (715 events) was larger than that used for the amplitude  were performed at body temperature as previously described12. Briefly, a 4 kHz sine wave of 13 mV RMS was applied to IHCs from −81 mV, unless otherwise stated, and was interrupted for the duration of the voltage step. The capacitance signal from the Optopatch was filtered at 250 Hz and sampled at 5 kHz. ΔCm was measured by averaging the Cm trace over a 200 ms period following the voltage step and subtracting the pre-pulse baseline. Data were acquired using pClamp software and a Digidata 1440 A (Molecular Devices). ΔCm experiments were performed in the presence of 30 mM TEA and 15 mM 4-AP (Fluka) to block the BK current 9 and delayed rectifier K+ currents , and linopirdine (80 μM: Tocris) to block IK,n14.Real-time changes in membrane capacitance  with a driver voltage (VPiezo) to the fluid jet of ± 20 V, with positive deflections moving the stereocilia in the excitatory direction. For some experiments a gravity fed local perfusion system was used to extracellularly apply the estimated endolymph-like solution (300 µM Ca2+)5 either alone or with 200 µM dihydrostreptomycin (DHS). The resting MET current becomes evident when the MET channels shut off in the inhibitory phase of the stimulus: the holding current minus the current present during inhibitory bundle deflection4.For mechanoelectrical transducer (MET) current recordings, a fluid jet driven by a piezoelectric disc was used to elicit bundle displacement as previously describedUnless otherwise stated, electrophysiological data was analyzed with PCLAMP software (Molecular Devices) and ORIGIN .63. Briefly, stimuli were delivered to the ear by calibrated loudspeakers  placed 10 cm from the animal’s pinna. Sound pressure was calibrated with a low-noise microphone probe system . Experiments were performed using BioSigRZ software driving an RZ6 auditory processor (Tucker-Davis Technologies). Response thresholds were estimated from the resulting ABR waveform and defined as the lowest sound level where any recognizable feature of the waveform was visible. Responses were measured for click and pure tone stimuli of frequencies between 3.0 and 24 kHz. Stimulus sound pressure levels were typically 0–95 dB SPL, presented in steps of 5 dB. The brainstem response signal was averaged over 256 repetitions. Tone bursts were 5 ms in duration with a 1 ms on/off ramp time, which was presented at a rate of 42.6/sec.Auditory brainstem responses (ABRs) were recorded from either male or female knockout mice and their littermate controls between P15 and P62. Recordings were performed in a soundproof chamber  as previously described66. Ca2+ signals were recorded using a two-photon laser-scanning microscope  based on a mode-locked laser system operating at 800 nm, 80-MHz pulse repetition rate, < 100-fs pulse width . Images were captured with a 60x objective  using a GaAsp PMT (Hamamatsu) coupled with a 525/40 bandbass filter . Images were analyzed off-line using custom built software routines written in Python  and ImageJ (NIH). Ca2+ signals were measured as relative changes of fluorescence emission intensity (∆F/F0).For calcium dye loading, acutely dissected cochleae were incubated for 40 min at 37 °C in DMEM/F12, supplemented with fluo-4 AM  as recently described1. Briefly, cochleae were dissected in HEPES buffered  Hanks’ balanced salt solution (HBHBSS), placed onto collagen-coated glass coverslips, fed with 100–150 μl of medium containing 98% or 93% standard Dulbecco’s modified Eagle’s DMEM/F12 with additional 10 mM HEPES buffer, 2% or 7% foetal bovine serum  and 10 μg/ml ampicillin (Sigma) and maintained at 37 °C for up to 11 days. The standard DMEM/F12 solution containing 4.18 mM KCl and 1.09 mM CaCl2  was used, additionally supplemented with KCl or CaCl2 depending on the experiments 67 the above medium was buffered with 2.2 mM HEDTA. For the recordings aimed at testing the effect of high extracellular K+, the above medium was supplemented with 1 M KCl to reach the final desired concentrations (15 mM or 40 mM KCl). In order to block the MET channels in the presence of the endolymphatic-like solution (40 μM Ca2+: see above), we used 50 μM of the MET channel blocker d-tubocurarine .Cochlear cultures from P18-P24 gerbils were prepared as described previouslyMyo7afl/fl and Myo7afl/flMyo15-cre+/- mice  were perfused with 4% paraformaldehyde in phosphate-buffered saline (PBS) for 45 min at 4 °C. Cochleas were microdissected, rinsed three times for 10 min, and incubated for 1 h at room temperature in PBS supplemented with 20% normal horse serum and 0.3% Triton X-100. The samples were then incubated overnight with the primary antibody: rabbit anti-myosin7a68 (1/500) in PBS supplemented with 1% horse serum. The samples were rinsed three times for 10 min in PBS, and then incubated for 1 h with ATTO-488-conjugated goat anti-rabbit IgG antibody . Actin was labeled with ATTO-647N–conjugated phalloidin . Samples were then mounted in Fluorsave . The z-stack images were captured with a Zeiss LSM-700 confocal microscope equipped with a Plan Apochromat 63X/NA 1.4 oil immersion lens .Dissected inner ears from 4, before processing through the thiocarbohydrazide-Os-repeated procedure69, followed by dehydration in an ethanol series and critical point drying. The organ of Corti samples were mounted on SEM support stubs with silver paint and sputter coated with a thin (ca 5 nm) layer of platinum before examination in a JEOL 6700 cold field-emission SEM operating at 3 or 5 kV. Images are a sample from 3 or more mice for each genotype.The auditory bullae were isolated from the heads of the mice and opened to expose the cochleae which were fixed, by intralabyrinthine perfusion using a fine hypodermic needle through the round window. The fixative was 2.5% glutaraldehyde in 0.1 M cacodylate buffer containing 3 mM calcium chloride (pH 7.3). Following perfusion the intact cochleae were immersed in the fixative for 2 h and then decalcified by immersion in 4.0% EDTA (pH 7.3) for 2 days at 4 °C. Subsequently the cochleae were dissected under cacodylate buffer to isolate approximately half turn segments of the organ of Corti from apex to base. These organ of Corti segments were post-fixed (2 h) in cacodylate buffered 1% OsO70. For ultrastructural evaluation of the nerve terminal region of IHCs, ultrathin (80–90 nm) sections were cut in radial planes from the apical coil, mounted on 200 mesh thin bar copper grids  and stained in 2% uranyl acetate in 70% ethanol for 20 min, followed by 0.4% lead citrate dissolved at high pH in distilled water for 5 min. They were examined in a JEOL 1230 TEM operated at 100 kV accelerating voltage. Digital images were acquired in using a Megaview III . The height and width of stereocilia were measured as previously described71. Semi-thin sections (250–270 nm) of the organ of Corti were obtained in a radial plane and examined unstained in a JEOL 1230 TEM operated at 100 kV. The height and width was measured using imageJ (NIH). The heights of first row IHC stereocilia (s1) were measured only when 90–100% of their length from the distal tip lay within the section . The full length of second row (s2) stereocilia was readily captured in these sections and was measured to the maximum distal tip including the bevelled portion where present. The width was measured at half maximum height of each stereocilium. For TEM, ≥ 3 mice were processed for each set of experiments and for each genotypes. Images were taken from the same region (around 12 kHz) used for the electrophysiological recordings.For TEM cochleae were fixed as for SEM but postfixed by immersion for 1 h in 1% osmium tetroxide in 0.1 M cacodylate buffer, dehydrated and embedded in Spurr resint test or for multiple comparisons, analysis of variance (one-way and two-way ANOVA followed by Bonferroni’s test) were applied. P < 0.05 was selected as the criterion for statistical significance. Mean values are quoted in text and figures as means ± SEM. apart those referring to ABR and TEM measurements, which are reported as means ± SD. Only mean values with a similar variance between groups were compared. Electrophysiological recordings in which the IHC leak conductance, normally calculated between −84 and −94 mV, was significantly larger than that previous reported for both immature and adult cells14 or with a residual series resistance (Rs) after compensation > 5 MOhm were excluded from the analysis. Animals of either sex were randomly assigned to the different experimental groups. No statistical methods were used to define sample size, which was defined based on previous published similar work from our laboratory. The majority of the experiments were performed blindly to animal genotyping.Statistical comparisons of means were made by Student’s two-tailed Supplementary InformationPeer Review File


In this study, LiInO2 and LiInO2–LiI were introduced as new cathode coating materials to suppress such undesirable reactions. The LiInO2–LiI composite coating layer reduced the undesirable interfacial reactions and prevented the diffusion of S and P ions from the sulfide electrolyte to the oxide cathode. Moreover, the electrochemical properties of all-solid-state cells were improved by the cathode coating. The LiInO2–LiI-coated electrode presented better rate capability and lower impedance than the pristine and LiInO2-coated electrodes. Hence, the LiInO2–LiI composite coating was successful at improving the cathode stability while providing superior electrochemical properties.All-solid-state batteries with inorganic solid electrolytes are ideal to overcome the safety issues related to the flammable organic electrolyte in lithium ion batteries. Sulfide materials are promising inorganic electrolytes due to their high ionic conductivity and good elasticity. Nevertheless, their application is limited by their high reactivity and instability at the cathode/electrolyte (Li[Ni However, they suffer from safety issues arising from the flammability of the organic liquid electrolyte used. Moreover, the consequences become increasingly serious for larger LIBs. Although it is not yet widely known to the public, there have been several fires in electric vehicles and energy storage systems involving LIBs. This could prove fatal for the current market expansion of LIBs. Ultimately, the flammable liquid electrolyte needs to be replaced with a non-flammable solid electrolyte11. Many superionic conductors have been explored as candidate materials17. Among them, sulfide superionic conductors have attracted a lot of attention due to their high ionic conductivity20. Several state-of-the art sulfide-based solid electrolytes such as Li10GeP2S12 (LGPS) and Li9.54Si1.74P1.44S11.7Cl0.3 (LSiPSCl) have even demonstrated conductivities far greater than those of organic liquid electrolytes13. Furthermore, sulfide materials can form interfacial connections with the cathode through mechanical pathways without the need for high temperature sintering because of their ductility and elasticity. Nevertheless, the commercialization of sulfide-based all-solid-state batteries (ASSBs) has been limited by the high reactivity of sulfides, that causes instability at the sulfide electrolyte/electrode interface. In particular, the interface reactions that occur with oxide cathodes form an undesirable interfacial layer23 which deteriorates the capacity, rate capability, and cyclic performance of ASSBs.Nowadays, lithium ion batteries (LIBs) are used to power everything from small portable devices to electric vehicles and large scale machinery such as energy storage systems324, Li4Ti5O1225, Lix3Lax2/3−TiO326, and Li2O–SiO227. However, the research on surface modification to enhance interfacial stability in sulfide-based ASSBs is in its infancy compared to the broad literature on reducing the unwanted reactions that arise from organic liquid electrolytes in LIBs. Moreover, the role of the coating layer in ASSBs is quite different from that in commercial LIBs, since the cathode degradation mechanism is different for sulfide solid electrolytes and organic liquid electrolytes. Thus, it is necessary to study cathode coating materials from a new perspective for ASSBs. Furthermore, most coating research for ASSBs has focused on the modification of LiCoO2 cathodes. In light of the recent drive to reduce the cobalt content of large-scale battery systems owing to its prohibitive cost, surface coatings are needed that are suitable for cathodes with low Co content.It has been demonstrated that such interfacial instability can be suppressed by surface modification of the cathode by coating it with a stable oxide, for example LiNbO0.8Co0.15Al0.05]O2 (NCA), a high Ni cathode material suitable for large scale LIBs, and applied a new coating material to its surface in an attempt to enhance its interfacial stability with a sulfide solid electrolyte (75Li2S–22P2S5–3Li2SO4) in an all-solid-state cell. The influence of the surface coating on the electrolyte/cathode interface is highly influenced by the coating material32. Although they have not yet been applied as cathode coating materials to our knowledge, we expect indium oxides to be promising coating materials as they are highly stable33. Since a lithium depletion layer is formed between cathode/sulfide electrolyte interface, oxides containing lithium is preferable as coating materials for sulfide based ASSBs. Fortunately, LiInO2 with α-NaFeO2 type structure has been reported as stable oxide35. Hence, we selected LiInO2 as a coating material for the NCA cathode.In this work, we selected LiO2). With the pristine cathode, a considerably thick (~38 nm) interfacial reaction layer was observed at the cathode/electrolyte interface  and LiInO2–LiI-coated cathode. The coating layer seems to have been preserved at the cathode/electrolyte interface. To observe the composition of the coating layer in more detail, the composite electrode was studied by EDS. As shown in Fig. 2–LiI composite layer.Figure 2-coated, and 0.5 wt.% LiInO2–LiI-coated electrodes in all-solid-state cells at current densities of 8.5–170 mA·g−1. The initial capacity of the LiInO2–LiI-coated electrode was slightly smaller than that of the LiInO2-coated electrode. However, as the current density increased, the LiInO2–LiI-coated electrode exhibited a superior capacity to the LiInO2-coated electrode. This means that adding LiI to the coating layer can successfully enhance the rate capability of all-solid-state cells with coated cathodes. Figure 2-coated, and LiInO2–LiI-coated electrodes in all-solid-state cells. All the Nyquist plots were composed of 2–3 overlapped semicircles. The typical Nyquist plots of lithium ion cells (using liquid electrolyte) consist of two semicircles, attributed to charge transfer resistance and the solid electrolyte interface. However, for cells using a sulfide solid electrolyte, the impedance is expected to be highly dependent upon resistance related to the interface layer between the oxide cathode and the sulfide electrolyte. The Nyquist plot of the pristine electrode shows a large semicircle followed by a small semicircle at low frequency region. In contrast, the Nyquist plots of the coated samples presented a distorted semicircle, which may consist of overlapped 2–3 semicircles. It is notable that the semicircles of the coated electrode were decreased compared to that of the pristine electrode. In particular, the Nyquist plot of the LiInO2–LiI-coated electrode showed a significantly smaller semicircle than not only the pristine electrode but also the LiInO2-coated electrodes. This results indirectly proves that the addition of I ions is efficient to reduce the impedance of the cells and enhance the lithium ion transfer, which leads to the fast rate capability of the LiInO2–LiI-coated electrode shown in Fig. Figure 2–LiI-coated electrodes that were collected from the composite electrode of all-solid-state cells after 30 charge–discharge cycles. In the pristine sample, S and P ions were detected in the cathode region, as shown in Fig. 2–LiI coating layer. This clearly confirms that the LiInO2–LiI coating layer successfully blocks the diffusion of the S and P ions from the sulfide electrolyte to the cathode.Figure 2-coated, and 0.5 wt.% LiInO2–LiI-coated cathodes were compared using XPS analysis  before the cathode was added. As shown in Fig. 1/2 and 2p3/2 components of non-bridging sulfur (S−) in the sulfide electrolyte43. The small peaks marked in red almost correspond to the peaks of binding sulfur (S0) in P2S74− units. However, we cannot exclude the possibility of these peaks arising from the products of secondary reactions such as the oxidation of sulfide. The peaks marked in blue are likely to be associated with the side reaction as well.To determine the effect of the LiI addition to the coating layer in detail, composite electrodes employing pristine, 0.5 wt.% LiInO2 coating was applied, as shown in Fig. −) (marked in orange), were somewhat higher due to the LiInO2 coating effect. This result coincides with the previous STEM-EDS analysis  presented more complicated peaks. As shown in Fig. 2 coating layer  and oxide cathode (NCA) in all-solid-state cells, LiInO2 and LiInO2–LiI were employed as new cathode coating materials. The coating layer on the surface of the cathode powder comprised homogeneously distributed nano-sized LiInO2 particles. The stability of the coated cathodes was evaluated by fabricating sulfide-based all-solid-state cells. The composite electrode employing LiInO2-coated NCA showed superior capacity and rate capability to the composite electrode employing pristine cathode, and that employing LiInO2–LiI-coated NCA showed better rate capability and lower impedance compared to both other types of electrode. Cross-sectional STEM images and EDS line profiles of the sulfide electrolyte/cathode interfaces demonstrated that the LiInO2 coating layer suppressed the diffusion of S and P ions into the cathode, indicating that the coating reduced the undesirable interfacial reactions. This phenomenon was more pronounced with the LiInO2–LiI coating material. In the STEM-EELS analysis, it was observed that the LiInO2–LiI coating layer effectively prevented the diffusion of S and P ions into the cathode region. XPS analyses confirmed that the LiInO2–LiI coating layer was better at suppressing the secondary reactions at the sulfide electrolyte/cathode interface than the LiInO2 coating. Hence, surface modification by coating with LiInO2–LiI is a promising method of reducing interfacial instability and enhancing the electrochemical properties of sulfide-based all-solid-state cells. Figure 2 and LiInO2–LiI coatings on the surface of the NCA cathode.To increase the interfacial stability between the sulfide electrolyte (75Li0.8Co0.15Al0.05]O2 (NCA) powder was used as a pristine cathode. To prepare the LiInO2 coating solution, lithium nitrate  and indium nitrate hydrate (In(NO3)3∙xH2O) were dissolved in anhydrous ethanol  at 70 °C. The amount of LiInO2 was adjusted to 0.5 wt.%, 1.0 wt.%, and 2.0 wt.% of the cathode powder. For the LiInO2–LiI coating solution, ammonium iodide and lithium nitrate were also dissolved in the solution of LiInO2 in anhydrous ethanol, at 0.25 mol% or 1 mol% relative to the molar ratio of LiInO2. Pristine cathode powder was added to the coating solution and stirred at 70 °C until the solvent was completely evaporated. The dried materials were heat treated at 650 °C (ramping rate = 2 °C/min.) for 5 h under air atmosphere to obtain LiInO2-coated NCA powder. As samples for comparison, the LiInO2–LiI coating solutions were dried at 70 °C and heat treated at 650 °C for 5 h, which is same condition for the coating process, to obtain the LiInO2–LiI composite powders.A commercial Li[Ni2-coated NCA powders as well as LiInO2 were obtained using a Rigaku MiniFlex II X-ray diffractometer over the 2θ range of 10–90° with monochromatized Cu Kα radiation (λ = 1.5406 Å). The surface morphology of the pristine and coated powder was observed using field-emission scanning electron microscopy  and high-resolution transmission electron microscopy .X-ray diffraction (XRD) patterns of synthesized samples of pristine and LiInO2S–22P2S5–3Li2SO4). The sulfide electrolyte was prepared using mechanical milling process and subsequent heat-treatment, as reported in the previous literature44. The cathode mixture for the composite electrode was prepared by hand mixing the cathode (pristine or coated NCA), sulfide solid electrolyte, and Super P carbon black at a weight ratio of 68.6: 29.4: 2.0. To form a solid electrolyte layer as a separator, 0.2 g of the sulfide electrolyte was compressed under 30 MPa pressure in a mould. Thereafter, the composite electrode (cathode) layer was formed on one side of the solid electrolyte using 0.02 g of the cathode mixture and carbon-nanotube paper . The anode electrode layer was formed on the opposite side with 0.05 g of Li–In powder and nickel foil. Each compression process was performed at 30 MPa. The cathode/electrolyte/anode assembly was placed inside a 2032 coin-type cell. The structure of this assembly is shown in Fig. For electrochemical testing, all-solid-state cells were fabricated using sulfide electrolyte , energy dispersive X-ray spectroscopy (EDS), and electron energy loss spectroscopy (EELS) analyses were employed to investigate the element diffusion between the cathode and the solid electrolyte. The cross-sectional interface was prepared using focused ion beam (FIB) milling (Quanta 3D FEG) prior to STEM analyses. A vacuum transfer system was utilized to avoid exposure to air during the FIB milling process and transfer to the STEM.The cells were subjected to galvanostatic cycling (WonATech voltammetry system) over a voltage range of 3.88–1.88 V at various charge–discharge rates. X-ray photoelectron spectroscopy  was employed to analyse the reaction products on the composite electrodes containing pristine, LiInOSupporting Information


This article presents flame retardant compounds for silicone rubber (SR) in the form of polyhedral oligomeric silsequioxanes (POSS), containing both isobutyl groups and amino-propyl (AM-POSS) or chloro-propyl group (HA-POSS) or vinyl groups (OL-POSS). Silsequioxanes were incorporated into the silicone rubber matrix in a quantity of 3 and 6 parts by wt by the method of reactive stirring with the use of a laboratory mixing mill. Based on the analyses performed by TG (Thermogravimetry) FTIR (Fourier Transform Infrared Spectroscopy), conical calorimeter, and SEM-EDX (Scanning Electron Microscopy and Energy Dispersive X-ray Spectroscopy) methods, the thermal degradation mechanism of non-cross-linked and cross-linked silicone rubber has been elucidated. The effects of POSS, and POSS in a synergic system with melamine polyphosphate (MPP), on the thermal properties and flammability of silicone rubber composites were presented. Based on the test results obtained, a mechanism of flame retardant action POSS and POSS-MPP has been proposed. It has been shown that POSS, especially with MPP, considerably increases the thermal stability and decreases the flammability of the SR rubber composites under investigation. Silicone rubbers (SR) are characterized by excellent compatibility, and chemical, thermal, and UV resistances, and also showing very good dielectric properties. Unfortunately, silicone rubbers are flammable polymers; when ignited they are totally burnt, releasing great amounts of heat within a short period of time. The flammability of silicone rubbers considerably reduces their use in the medical, automotive, power, and cable industries ,2.A commonly used method of improving the resistance of polymeric materials, including silicone rubbers, against the action of fire consists of incorporating additive flame-resistant compounds into their matrix. On account of environmental restrictions concerning the use of halogen flame retardants, at present halogen-free organophosphorus flame-retardant compounds, such as melamine polyphosphate (MPP) or ammonium polyphosphate (APP), have the greatest application potential .The action of organophosphorus compounds consists in forming an expanded, intumescent carbon layer on the polymer surface during its thermal decomposition. This layer acts as a barrier preventing heat transfer from its source to the polymer surface and reducing the fuel transfer to the flame .To obtain a high efficiency of flame retardant compounds, one has to incorporate them into the polymeric matrix in the presence of carbon-rich compounds.During the thermal decomposition of a polymeric composite, the organophosphorus compounds contained in it, e.g., MPP, undergo degradation to water, ammonia, and PP acid that when reacting with carbon compounds, forms thermally unstable esters of phosphoric acid. Their decomposition, mainly by dehydration processes, results in the formation of a carbon layer cutting off the flow of oxygen, fuel, and heat between the polymer and flame. Water vapor and nitrogen compounds formed during the MPP decomposition diffusing to the flame, foam the liquid destruction products of the composite and increase the insulating power of the boundary layer .The incorporation of MPP into the poor in carbon silicone rubber to make it flame retardant must be combined with a carbonizing additive, such as pentaerythritol (PER) .From the investigations performed so far, it distinctly follows that the satisfactory effectiveness of the MPP/PER system, especially in relation to silicone rubber, can be obtained when its content is over 30 parts by wt. Unfortunately, such a high quantity of this flame retardant negatively affects the processing, functional properties, and price of the silicone rubber composites obtained .New, alternative of halogen and halogen-free flame-retardant compounds are polyhedral oligomeric silsequioxanes (POSS) ,9. POSS POSS can be incorporated as reactive components during polymerization or combined with macromolecules by their direct plasticization with the polymeric matrix.From the investigations performed so far, it follows that POSS incorporated into silicone rubbers considerably improves their mechanical and thermal properties and reduces their flammability . Liu et The use of POSS as effective compounds for reducing the flammability of polymers is presented in a review by Zhang and co-workers . From thCarosio et al.  studied Researchers indicated that the thermal degradation in an inert atmosphere, as well as the thermo-oxidation in air of the acrylic fibers, has been significantly modified by the coatings due to their char-former character .Despite the investigations performed, the effect of POSS, especially the POSS-phosphorus compounds system, on the flammability of polymeric materials has not been totally explained.The present paper describes pioneering studies on the effect of three POSS compounds containing amino groups (AM-POSS), chlorine groups (HA-POSS), and vinyl groups (OL-POSS) on the thermal properties and flammability of high-temperature vulcanized silicone rubber (SR), also taking into account the decomposition kinetics. The effect of the synergism of POSS-MPP action on the flammability of silicone rubber was also examined. Based on the analyses performed by TG, FTIR, conical calorimeter and SEM-EDX methods, the mechanism of the flame-retardant action of POSS-MPP was explained.4 (kg/mol) and vinyl group content of 0.05–0.09%. The rubber was cross-linked with the use of bis peroxide from “Silikony Polskie” in a quantity of 0.7 part by wt/100 parts by wt. of the rubber .The object of study was methylvinylsilicone rubber (SR), made by “Silikony Polskie” , with a molecular weight of 60–70 × 103H6N6 (H3PO4)n, and polyhedral oligomeric silsesquioxanes POSS from Hybridplastics . The chemical formula of POSS is (RSiO1.5)n where R is a hydrogen atom or an organic group  , chemicaic group . Please D = 140 mm and L = 300 mm. The average temperature of the rolls was 35 °C. The rotational speed of the front roll was Vp = 20 rpm, friction 1:1.Rubber blends were prepared using a laboratory two-roll mill, with roll dimensions of The composition of the rubber mixtures are shown in Kinetic parameters of vulcanization of the elastomer blends were measured by means of MonTech DRPA 300 Rheometer , at 160 °C, according to standard PN-ISO 3417:1994, directly after the preparation of the rubber blends, and for comparison, after a week of conditioning at the ambient temperature. The blends were vulcanized in steel molds placed between electrically heated press shelves.−1 in transmittance mode with a resolution of 8 cm−1 and the speed of scanning equal to 0.6329 cm·s−1.Fourier transform infrared (FTIR) spectra were recorded on a FTIR Spectrum Two spectrophotometer from PerkinElmer . Attenuated total reflection (ATR) accessory equipped with a single reflection diamond ATR crystal on ZeSe plate was used for all of the analysis. Measurement parameters were 32 scans from 500–4000 cmEDS analysis of residue after burning were obtained with the use of scanning electron microscope (SEM), model TM3000 from Hitachi .The thermal properties of the rubber composites were tested under an air atmosphere at a temperature ranging from 25 to 700 °C with the use of a thermal analyser . Weighed portions amounted to about 5–10 mg. Investigated samples were analyzed at a heating rate of 10 °C/min.Determination of activation energy of destruction studied silicone rubber compositions.In order to determine the activation energy of destruction, compositions of silicone rubber were tested under a nitrogen atmosphere at temperatures ranging from 25 to 700 °C with the use of a thermal analyser . The samples were heated with the rate 5, 10, 15, and 20 °C/min.X is degree of decomposition; tW, W0, fW are the actual, initial, and final mass of the sample, respectively. A typical model for the kinetic process can be expressed as:dx/dt is the decomposition rate, f(X), the function of X, which depends on the particular decomposition mechanism, and k is the decomposition rate constant, which can be expressed by the Arrhenius equation:A is the pre-exponential factor (s−1), E is the activation energy (J/mol), R is the gas constant (8.314 J·mol−1·K−1), and T is the temperature (K). Substituting Equation (3) into Equation (2), givesIn general, the thermal degradation reaction of a solid polymer can be simply shown as: Bsolid → Csolid + Dgas, where Bsolid is the starting material, and Dgas and Csolid are the different products during the degradation of Bsolid. In thermogravimetric measurements, the degree of decomposition (conversion) can be calculated as follows :(1)X=W0−β (β = dT/dt), the variation of the degree of decomposition can be analyzed as a function of temperature. Therefore, the reaction rate gives:If the temperature of a sample is changed by a constant value of Equations (4) and (5) are the basic equations for the kinetic calculation.α, the degree of conversion; T, the absolute temperature to reach the conversion, and is the integral conversion functionThis iso-conversional integral method, suggested independently by Flynn and Wall and Ozawa ,22, usesα = const.), the plot log β versus (1/T), obtained from a series experiments performed at several heating rates, should be a straight line whose slope allows for the evaluation of the activation energy:Thus, at a constant conversion , sample weight during testing, total heat released (THR), effective combustion heat (EHC), average weight loss rate (MLR), heat release rate (HRR), and final sample weight.The SR composites were examined using a cone calorimeter from Fire Testing Technology Ltd. . Elastomer samples with dimensions of (100 × 100 ± 1) mm and thickness of (2 ± 0.5) mm were tested in a horizontal position with a heat radiant flux density of 35 kW·mThe thermal degradation of polysiloxanes (silicone rubbers) can proceed according to three reaction mechanisms: unzipping mechanism, random scission of Si–O bond in the main chain of polymer, and external catalytic mechanism.Polydimethylsiloxanes (PDMS), containing silanol (Si–OH) or hydroxyl-alkyl-silanol (Si–R–OH) terminal groups, undergo thermal degradation according to the unzipping mechanism  20,23,2,223,24.In the first stage of heating, the molecular weight of the polymer violently increases as a result of intermolecular condensation processes proceeding between terminal silanol groups and inter-chain Si–O groups. Further increase in temperature causes a linear decrease in the polymer molecular weight as a result of the formation of cyclic siloxanes followed by their tearing off and passing to the gas phase of cyclic siloxanes, mainly trimers and tetramers. Presumably, the cyclic siloxane that is stable at the polysiloxane degradation temperature can catalyze the resultant degradation processes of polysiloxane.The weakest bond in the structure of polydimethylsiloxane (PDMS) is the C–Si bond, whose energy amounts to 326 kJ/mol. Nevertheless, cyclic siloxanes are formed by the decomposition of a Si–O bond, whose energy value is 451 kJ/mol, which in turn suggests that the thermal decomposition of PDMS should be considered from the point of view of both kinetic parameters and molecular structure and not only the bond energy.Polysiloxanes containing terminal groups, such as a trimethylsilyl or vinyl group, as well as accidently occurring vinyl groups in the main chain, undergo thermal degradation by inter- or intra-molecular reactions proceeding between accidental bonds of the main polymer chains  23,25].,25.23,25Unlike the unzipping mechanism, the thermal degradation of polysiloxanes according to the random scission mechanism requires decisively higher temperatures, which causes a violent loss of the sample mass, which is observed already in the early stage of the thermal decomposition of polysiloxane, while the scission of the Si–O bond resulting in the formation of oligomeric final products generally occurs in the middle part of polysiloxane chain.Polysiloxanes containing ionic polar additives or impurities undergo thermal degradation according to a catalytic mechanism. Contrary to the unzipping and random scission mechanisms, the catalytic mechanism consists in hydrolytic decomposing the Si–O bond in the main polymer chain. Grassie and Macfarlane have shown that even trace quantities of KOH dramatically deteriorate the thermal stability of polysiloxanes, even within the range of moderate temperatures .Radhakrishnan , investiT = 350 °C, while the maximal rate of its decomposition at T = 396 °C amounted to 3.80%/min, the second stage began at T = 410 °C, while its maximal rate at T = 465 °C amounted to 16.90%/min (third stage).The methylvinylsilicone rubber tested in the atmosphere of a nitrogen gas underwent thermal decomposition in a single stage. The maximal rate of this process at a temperature of 483 °C amounted to 22.85%/min . Under aThe three-stage decomposition of silicone rubber under air can result from the catalytic action of air in the depolymerization reactions of polysiloxane to volatile cyclic low-molecular compounds, as confirmed by a decisively lower temperature of the beginning of thermal decomposition, TR, of SR rubber under air in relation to the value of TR under neutral gas. The cyclic compounds, formed as a result of the decomposition of SR rubber according to the random scission mechanism, are then thermally decomposed under both air and nitrogen at a temperature above 450 °C.T between 60 and 395 °C is connected with the presence of plasticizer in the form of low-molecular hydroxydimethylsiloxane, incorporated into the elastomer blend  underwent a two-stage thermal decomposition. The first principal stage of thermal decomposition with a weight loss at ΔT1 = 265 °C, T2 = 320 °C, and T3 = 375 °C amounted to 0.79, 1.80, and 0.36%/min, respectively, were connected with the degradation of unsaturated hydrocarbon chains and the detachment of hydrocarbon destruction products. The fourth stage of thermal decomposition was recorded at ΔT = 450–650 °C as in the case of HA-POSS, which was connected with the thermal degradation and decomposition of the cage structure. A very high residue after the thermal decomposition of OL-POSS, amounting to 86.5% at T = 600 °C, resulted from the susceptibility of this compound to cross-linking and thermal cyclization [Vinyl POSS (OL-POSS) underwent a four-stage thermal decomposition . The firlization .−1, which were connected with stretching vibrations of the –CH group. A peak at wavenumber of 1091 cm−1 was connected with stretching vibrations of OH group, whereas a signal at wavenumber 1459 cm−1 corresponded to asymmetric stretching vibration of the –CH3 group. The peak at 2952 cm−1 was attributed to physically connected water at the POSS surface.FTIR spectra of all studied POSS showed signals at wavenumbers of 745 and 837 cm−1, respectively. The first one was connected with valence vibrations, whereas the second was due to deformation vibrations of the –NH group.Additionally, the FTIR spectrum of POSS/AM showed signals at 1229 and 1570 cm−1 that is connected with valence vibration of the C–Cl group. In the IR spectrum of the POSS/OL, a sharp peak at 548 cm−1 was observed, which was attributed to vibration of the CH=CH2 group, whereas the signal at 1407 cm−1 corresponded to deformation vibrations of the –CH group in –C=C–H , the thermal decomposition rate of the composite (dm/dt), the maximal temperature of its thermal decomposition (TRMAX), and the residue after its thermal decomposition (Pw) and residue at T = 650 °C (P650)  .T5. It should, however, be underlined that the highest increase in the value of this parameter was recorded for the composites containing OL-POSS. In the case of samples SR/OL3 and SR/OL6, parameter T5 was increased by 200 °C (sample SR/OL3) and by 194 °C (sample SR/OL6) in relation to the cross-linked SR rubber (From the data listed in R rubber . These idm/dt). The reduction in the destruction rate of polymeric composites exerts a positive influence on decreasing their flammability. This results from the formation of lower amounts of volatile and flammable products of pyrolysis passing to flame, which inhibits the free radical reactions proceeding in the combustion zone. Among all the samples tested, the lowest value of parameter dm/dt was shown by SR/OL3 and SR/OL6. These samples also showed the highest values of residue after thermal decomposition and at T = 650 °C (parameter Pw and P650), which distinctly indicated that vinyl groups of POSS/OL, non-active in the process of reactive mixing and rubber vulcanization carried out at T = 130 °C  of SR/AM6 composite was decreased by 28.87% in relation to the cross-linked SR rubber, while it decreased in the case of HA-POSS by 14.65%, and in the case of OL-POSS by 47% , of POSS/SR composite, in relation to the unfilled, cross-linked SR rubber, were connected with the formation of a ceramic coating on the sample surface under combustion , that in the case of the composite tested, was lower by more than 43% in relation to the cross-linked SR rubber .The effect on the SR/HA composite flammability is also exerted by the chlorine atom present in the HA-POSS structure. Halogens, passing to the gaseous phase of combustion, effectively interrupt the highly energetic radical reactions in both the pre- and flame-zones, consequently decreasing the efficiency of degradation and destruction processes of elastomeric composite ,31.MAX parameters. The increase in the flammability of cross-linked silicone rubber in the presence of MPP resulted from two effects. First, the acidic products of the thermal decomposition of melamine polyphosphate incorporated into the silicone rubber matrix initiated the decomposition of the Si–O bond in the polymer main chain. Second, MPP, increasing the distances between polymeric chains, considerably reduced the thermal crosslinking process, especially the cyclization of SR rubber as confirmed by both the violent increase in the value of parameter MLR  to the silicone rubber matrix caused an increase in its flammability expressed by THR, HRR, and HRReter MLR  and the eter T50 . It shouFrom the data listed in MAX by 60.83, 40.72, and 66.97%, respectively, in relation to the SR/MPP composite. Also, the parameters of mass loss rate (MLR) of SR/AM6/MPP 14, SR/HA6/MPP14, and SR/OL6/MPP14 underwent reduction by 17.5, 29.16, and 50.8%, respectively, in relation to that of the SR/MPP composite. The flame retardant action of the synergetic POSS/MPP system expressed by parameters HRRMAX, MLR, and THR  [The acidic destruction products of melamine polyphosphate initiate the degradation and thermal destruction processes of silicone rubber, which results in the formation of very high quantities of solid carbon products that pass to the flame zone, constituting an additional source of fuel (Parameter HRRmposite) . The cerThis paper presented flame retardant compounds for silicone rubber in the form of polyhedral oligomeric silsequioxanes (POSS), containing both isobutyl groups and amino-propyl (AM-POSS) or chloro-propyl group (HA-POSS) or vinyl groups (OL-POSS). Silsequioxanes were incorporated into the silicone rubber matrix in a quantity of 3 and 6 parts by wt. by the method of reactive stirring with the use of a laboratory mixing mill.T = 130 °C, took part in the reactions of cross-linking and cyclization proceeding during the thermal decomposition and combustion of SR rubber composites.Based on the obtained results, it was found that the studied POSS, non-active in the process of reactive mixing and rubber vulcanization carried out at Vinyl POSS, among all the polyhedral oligomeric silsequioxanes tested, catalyzed the processes of cross-linking and thermal cyclization of silicone rubber.Based on the SEM-EDX analysis, it was found that the decrease of flammability of silicone rubber in the presence of POSS was connected with the formation, during thermal decomposition of POSS/SR composites, of a ceramic coating that limited flow mass and energy between sample and flame.The increase in the flammability of cross-linked silicone rubber in the presence of MPP resulted from two effects. First, the acidic products of the thermal decomposition of melamine polyphosphate incorporated into the silicone rubber matrix initiated the decomposition of the Si–O bond in the polymer main chain. Second, MPP, increasing the distances between polymeric chains, considerably reduced the thermal crosslinking process, especially the cyclization of SR rubber.A lower flammability of SR/POSS/MPP composites in relation to that of SR/POSS resulted from the synergetic action of MPP and POSS during the formation of an insulating boundary layer during the thermal decomposition and combustion of SR/POSS/MPP composites.The acidic destruction products of melamine polyphosphate initiated the degradation and thermal destruction processes of silicone rubber, which resulted in the formation of very high quantities of solid carbon products passing to the flame zone, which constituted an additional source of fuel. The ceramic structure formed in the presence of POSS, making it impossible to transfer carbon destruction products to flame, contributed to their cross-linking and cyclization, and consequently to increasing the insulation capacity of the boundary layer formed during the decomposition and combustion of SR/POSS/MPP composites.


The proto-oncoprotein MET is a receptor tyrosine kinase that plays a key role in cancer cell growth and invasion. We have used fluorescence-tagged antibodies to activate MET in live serum-starved glioblastoma cells and monitor the fate of antibody-bound MET receptor in single cell-based assays. We found that the antibodies induced rapid and transient formation of highly polarized MET clusters on the plasma membrane and promoted the activation of MET, resembling the initial effects of binding to its ligand, HGF. However, the antibody-induced clustering and activation of MET led to the rapid removal of the receptor from cell surface and altered its intracellular processing, resulted in rapid degradation of the receptor. Consequently, while cells pre-treated with HGF remain competent to respond to further HGF stimulation, cells pre-treated with antibodies are refractory to further HGF stimulation due to antibody-mediated MET depletion. Removal of MET by sustained treatment of antibodies blocked cancer cell migration and invasion. Our studies reveal a novel mechanism to alter the recycling process of MET in glioblastoma cancer cells by promoting the receptor degradation through a proteasome-sensitive and lysosome-dependent pathway through the ligand-independent activation of MET using anti-MET antibodies. Met oncogene was originally identified as a chromosomal translocation fusion gene, which encode the oncogenic TPR-MET fusion protein in a chemically transformed human osteosarcoma-derived cell line1. The TPR-MET fusion oncogene expresses a constitutively active MET receptor tyrosine kinase (RTK) activity due to the dimerization of the leucine-zipper domain in the TPR (Translocated Promoter Region) moiety of the fusion protein2. The MET  RTK is normally expressed in various cells of epithelial origins or fibroblasts, and is essential for embryonic development, mitogenesis and morphogenesis of various tissues such as skeletal muscle, limb, and neural crest development4. The MET RTK is activated by the binding of its cognate ligand, hepatocyte growth factor (HGF), which induces the phosphorylaton of two tyrosine residues, tyrosine-1234 and tyrosine-1235 (Y1234/Y1235) of the catalytic loop of the kinase domain5. MET activation mobilizes the coordinated invasive cell growth program by promoting cell proliferation, survival, migration, and morphogenesis4.The Met gene is identified in medulloblastoma, gastric and esophageal carcinomas, and non-small-cell lung (NSCL) carcinoma with acquired resistance to epidermal growth factor receptor (EGFR) inhibitor, whereas activating mutations of MET are associated with sporadic papillary renal cancer, childhood hepatocellular carcinoma and gastric carcinoma6. The expression of MET is also aberrantly up-regulated in many human malignancies including glioblastoma multiforme (GBM)7, the most aggressive and therapeutically difficult brain tumor8. In normal cells, HGF-induced MET activation is a tightly regulated process9. After ligand binding, MET is internalized via endocytosis and the tyrosine-phosphorylated receptor is recognized by CBL ubiquitin E3 ligase to target MET to multivescular bodies for subsequent degradation in lysosomes9. Notably, certain mutations in the kinase domain of MET, originally identified in human renal papillomas, allow the receptor to constitutively recycle back to the cell surface, and these mutations lead to stronger signaling activities10. Abnormal activation of MET is responsible for resistance to targeted therapies against VEGFR  in GBM12 and inhibitors of the EGFR in lung cancers14. Over-expression or ligand-mediated activation of the MET signaling pathway is an established mechanism of resistance towards the targeted therapies against members of EGFR subfamily of RTKs6.Altered expression of MET is associated with various malignancies. Amplification of the 16. An one-armed monovalent 5D5 antibody has been developed19 that binds to the monomeric MET protein on the cell surface and blocks the binding of HGF to the receptor without induction of the down-regulation of the MET receptors. A non-activating monoclonal antibody, LY2875358, was recently reported20. This antibody can prevent the MET receptor to interact with HGF, as well as to trigger receptor downregulation20. Another bivalent antibody, SAIT301, which does not activate the RTK activity of MET, was also shown to cause the downregulation of the MET protein after an extended treatment21. It appears that LY2875358 and SAIT301 employ different cellular processes to down-regulate MET receptors, although a direct comparison of these two antibodies is lacking. These studies suggest that the MET receptor, with its unique structural or conformational determinants, may be manipulated through binding with antibodies to target the receptors to degradation.Since the high level expression of MET is correlated with poor prognosis of various cancers, MET serves as an excellent target for cancer therapy. Various approaches, such as the development of small molecular chemical inhibitors or specific monoclonal antibodies, have been explored to inhibit the RTK activity of MET or to block the interaction between the MET receptor and the ligand, HGF, in a wide array of cancers22. HGF stimulation induces an enhanced formation of the MET-AXL complex to promote the MET-AXL co-clustering on the plasma membrane22. Our findings reveal that HGF-dependent clustering of the MET-AXL complex is required for the MET-mediated cell signaling. In addition, we have recently shown that the cell surface delivery of MET from Golgi is a highly regulated process which is very sensitive to the lipid modification enzyme ASM (acid sphingomyelinase) in GBM cancer cells, suggesting that the total level of cell surface MET is closely regulated by both newly synthesized MET from Golgi and recycling pool of MET23. In this report, we have examined the cellular response to the ligand-independent activation of MET receptor on the plasma membrane by antibody-mediated MET receptor clustering.We have recently found that the MET receptor is often complexed with AXL, another important RTK, in glioblastoma and breast cancer cells23 and HGF promotes the MET-AXL RTK complex formation on the plasma membrane22. To examine how MET is activated on the plasma membrane, we examined the distribution of the MET protein in serum-starved human U373-MG glioblastoma cells, which express relatively high levels of MET protein23. To monitor the distribution of MET on the plasma membrane, we exposed live serum-starved cells to mouse monoclonal anti-MET antibodies that were pre-conjugated with Allophycocyanin (APC), an intensely bright phycobiliprotein that exhibits far-red fluorescence with high quantum yields. The advantage of using the APC-conjugated MET antibody to immunostain live cells is that it only detected the plasma membrane-associated MET with high signal-to-noise ratio  of the receptor complexes on the T cell membraneTo test whether antibody-mediated cross-link of the receptor is sufficient to activate MET, we used an independent bivalent goat anti-MET antibody that was pre-conjugated to biotin. The conjugation of antibodies to biotin allows the subsequent detection of the receptors by Streptavidin-labeled Brilliant Violet 421. Since the excitation of Brilliant Violet 421 by 405 nm laser induces a fluorescence maximum emission of 421 nm, it is possible to use three spectrum channels to simultaneously monitor and compare the distributions of biotinylated antibodies (purple), the activated MET protein using a rabbit anti-phosphorylated Y1234/1235 antibody followed by a secondary antibody conjugated with the Alexa Fluor 647 fluorophore (red), and subsequent detection of the total MET proteins using an anti-MET antibody pre-conjugated to Alexa Fluor 488 (green). Our studies showed that exposure of live serum-starved cells to the biotinylated goat anti-MET antibody, but not a control biotinylated normal goat IgG, was sufficient to induce the clustering and patch formation of the MET proteins on the plasma membrane Figs , S1A. ThIn addition, the biotin-conjugated goat anti-MET antibodies also induced the cluster formation of the phosphorylated MET receptor on the plasma membrane in other human glioblastoma cells such as T98G cells Fig. , suggest22. We tested the effects of HGF at 50 ng/ml, the lowest concentration of HGF needed to maximally activate MET in U373-MG cells22, and the concentration is also in the range of HGF known to be present in the tumor microenvironment in the melanoma samples carrying BRAF mutations25. In serum-starved cells, there was no immunostaining signal when stained with the anti-phosphorylated Y1234/Y1235 antibodies on the plasma membrane, suggest that MET is not activated , as revealed by Western blotting analysis. Addition of HGF to the serum-starved cells promoted the phosphorylation of Y1234/Y1235 on MET within 7 minutes Fig. . In addi26, a relative specific tyrosine kinase inhibitor (TKI) of MET. Our studies showed that inhibition of the MET RTK activity by crizotinib indeed effectively blocked the ligand-independent MET activation and MET signaling to AKT by the biotin-conjugated or APC-conjugated anti-MET antibodies, in a manner similar to the crizotinib’s effect on the HGF-dependent activation process , which induced the MET activation and receptor clustering on the plasma membrane  to the cells that have been received the 1st HGF treatment, and examined the response of these cells to form the MET clusters  if the cells had received the prior or 1st treatment of the biotin-conjugated anti-MET antibodies  and removed the unbound antibodies by extensive washing. The antibody-pretreated cells were next incubated in the serum-starvation medium for 30 minutes (recovery time), which greatly eliminated the activated MET protein on the cell surface  and the cluster formation of the activated and phosphorylated MET protein was monitored. We found that pre-treatment of cells with the biotin-conjugated anti-MET antibodies greatly dampened the responsiveness of the cells to form clusters when exposed to HGF  to activate MET phosphorylation, as revealed by Western blot analysis , and found that the antibody-marked MET proteins were accumulated in the lysosomes following a 30-minute chase , washed and then incubated in the fresh serum-starvation medium for various time (chase) Fig. . In cell31. MVBs can further mature into late endosomes, which then fuse with lysosomes, leading to degradation of the mono-ubiquitinated receptors in the lysosomes32. Interestingly, sorting of MET RTK in MVBs is also sensitive to proteasome inhibitors, although the exact mechanism remains unclear31. To examine the cause of MET protein disappearance in serum-starved cells treated with the biotin-conjugated anti-MET antibodies, we examined if the MET protein degradation could be prevented by MG-132, an inhibitor of the 26S proteosome that targets poly-ubiquitinated proteins for degradation33, or by bafilomycin A1 (BAF)34, a specific inhibitor of vacuolar-type H(+)-ATPase that inhibits the acidification of lysosomes and consequently blocks protein degradation in lysosomes. Serum-starved cells were treated with biotin-conjugated anti-MET antibodies or treated with HGF as control. After 7 minutes, BAF or MG-132 were added to the medium and incubated for further 2 hours urs Fig. . We founurs Fig. . As conturs Fig. . Similarurs Fig. . Similarurs Fig. . The antBecause the anti-MET antibodies tested above trigger an efficient MET protein degradation, we wondered if exposure of cells to these antibodies can efficiently suppress the HGF-mediated biological events. Since the MET receptor plays a key role in cell migration and invasive growth of various cancer cells, we examined the potential effects of anti-MET antibodies on cell migration. In actively growing U373-MG cells, treatment of cells with the biotin-conjugated anti-MET antibodies for 15 hours potently suppressed the cell migration in a wound-healing assay, as compared to that of the biotin-conjugated control IgG Fig. . WesternGlioblastoma cells are known to be highly invasive. To determine whether anti-MET antibodies affect such invasiveness, we further examined the effects of these anti-MET antibodies on gliobastoma cell invasion, using Transwells pre-coated with Matrigel Fig. . We firsIn this report, we describe single cell-based assays to analyze the activation and subsequent processing of the MET receptor in response to the binding of antibodies in glioblastoma cells. By using fluorescently tagged anti-Met antibodies, we found that binding of anti-MET antibodies to the MET receptor induced the transient formation of a highly polarized receptor cluster on the plasma membrane, in a manner similar to the events when the receptors were bound by their cognate ligand, HGF Fig. . However22. In the current study, we have tested two commercially available anti-MET antibodies, a goat polyclonal antibody (biotin-conjugated) and a mouse monoclonal antibody (APC-conjugated), which are likely to recognize different epitopes in the MET extracellular domain. However, both antibodies can efficiently induce the rapid MET-AXL co-clustering and the phosphorylation and activation of both MET and AXL . Recombinant human HGF was from Invitrogen/Thermo Fisher. Antibodies against MET, including goat anti-MET antibody , and mouse monoclonal anti-MET antibody , and control goat IgG (biotin-conjugates) and control mouse IgG (APC-conjugate), antibodies against MET , phosphorylated AXL  were from R&D Systems. Rabbit antibodies against AXL, MET, Y1234/Y1235-phosphorylated MET, phosphorylated AKT , and anti-MET rabbit monoclonal antibody  were obtained from Cell Signaling Technology. Chemicals, MG-132 and bafilomycin, were from Sigma-Aldrich. Secondary antibodies, including the Alexa Fluor 488-conjugated goat anti-mouse IgG, Alexa Fluor 647-conjugated goat anti-mouse IgG and Alexa Fluor 647-conjugated goat anti-rabbit IgG antibodies were from Jackson Immunologicals. Brilliant Violet 421 or 605-conjugated streptavidin was from BioLegend. The CellTiter 96 AQueous One Solution Kit was from Promega and the Alexa Fluor 488 Annexin V/Dead Cell Apoptosis Kit was from Invitrogen/Thermo Fisher. For tyrosine kinase inhibitors of MET, crizotinib was obtained from Cell Signaling Technology; JNJ-38877605 and SGX-523 were obtained from Selleck Chemicals. Boyden chambers with 8 μm pores were from Costar , and the growth factor-reduced Matrigel was from BD Biosciences. Differential Quik Stain Kit was obtained from Electron Microscopy Sciences.36.Cells were lysed in lysis buffer containing 50 mMTris (pH 7.4), 150 mM NaCl, 1% Triton-X-100, 0.1% SDS, supplemented with 5 mM NaF, 1 mM Na3VO4, and 1X complete protease inhibitor . Western blotting was conducted as previously described23. Briefly, cells were fixed and permeabilized with Fixation and Permeabilization Solution (BD Biosciences), blocked with goat serum, incubated with primary antibodies and then secondary antibodies in phosphate-buffered saline (PBS) containing 0.05% Tween 20 and 7% goat serum. Images were acquired using a Nikon A1R confocal laser scanning microscopy system equipped with the NIS-Elements Advance Research software with 20x objective (NA 0.8). For double or triple immunostaining, fluorescence were obtained after excitation with laser light of 405 nm, 488 nm and 640 nm to capture images for Brilliant Violet 421 (laser 405), Alexa Fluor 488 and Alexa Fluor 647, respectively, and these fluorophores have minimal fluorescence bleed-throughs. Exposure settings were kept constant for all conditions within each set of experiments. The captured images were further analyzed and quantified by using the NIS Elements Viewer software.U373-MG cells were serum-starved for 3 hours, and the starved cells were then stimulated with or without HGF (50 ng/ml), APC-conjugated mouse monoclonal anti-MET antibody (1 μg/ml) and biotin-conjugated anti-MET antibody (2 μg/ml) for 7 minutes before fixation. Immunofluorescence experiments were conducted as described37. For each condition, cells were counted from 6 independent fields and integrated. All experiments were done in duplicates.U373-MG cells were seeded on the plate and cultured for 16 hours. A sterile 20 μl tip was used to scrape the cells to make a wound. The cells were washed twice with warm PBS to remove floating cells. The biotin-conjugated anti-MET antibody or Biotin-conjugated IgG (each at 2 μg/ml) were added to the cells for additional 15 hours or longer, depending on the specific cell lines. For cells to be treated with HGF or anti-MET-biotin antibody stimulated assay, cells were starved for 9 hours, then stimulated for 24 hours with HGF (50 ng/ml) or anti-MET antibodies (2 μg/ml) or the corresponding controls. Cells were photographed using the EVOS XL Core microscope system with 20x objective and the cells migrated into the wound area were quantified using the Image J softwareCell invasion assays were performed using Boyden chambers with 8 μm pores, coated with the growth factor-reduced Matrigel diluted in Dulbecco’s modified Eagle’s medium. Cells were detached by trypsin and washed once with starvation medium . Twenty thousand cells were seeded in the upper chamber for each condition in starvation medium, and cells were allowed to invade for 24 hours toward the lower chamber containing starvation medium supplemented with HGF (50 ng/ml) or PBS or IgG or anti-MET antibodies , or in combinations of HGF + anti-MET antibodies or corresponding controls. For cell invasion assay in the complete medium, cells were seed on the upper chamber, and cells were allowed to invade for 24 hours toward the lower chamber containing complete medium supplemented with IgG or anti-MET antibodies. Cells were then fixed with 4% paraformaldehyde. Cells in the upper chambers were mechanically removed using cotton swabs. Cells invaded into the lower chambers were then stained with the Differential Quik Stain Kit . Cells were imaged using an EVOS XL Core system with 20x objective and quantified using the ImageJ software. For each condition, cells were counted from 6 independent fields and integrated. All experiments were done in duplicates.38. Different data sets were considered to be statistically significant when the p-value was 0.001 (***), 0.01 (**) or <0.05 (*).Quantitative data are expressed as the mean ± S.D. , normalized to the control condition, and plots were prepared using the GraphPad Prism 5 software. Statistically significant differences between means were determined using a two-tailed equal-variance Student’s t-testSupplementary Information


This first-in-human study in Saudi orthodontic patients has evaluated the role of low-level laser therapy (LLLT) in pain perception (PP). The outcome of single application of LLLT with 4 different treatment modalities (TM) on PP are evaluated following orthodontic bracket bonding on maxilla.  A prospective clinical intervention with implementation of parallel technique in each group, 32 orthodontic patients with ectopic canine requiring fixed orthodontic appliance were enrolled and randomly allocated to the 4 groups: LLLT + self-ligating (SL) bracket, LLLT + conventional (Conv.) bracket, non-LLLT + SL bracket, and non-LLLT + Conv. bracket. Orthodontic bracket bonding from 1st molar to 1st molar and superelastic 0.012 inch NiTi were applied for the maxilla. For each patient, maxillary 1st molar to molar received a single application of LLLT using a 940 nm Ga-Al-As laser device on 5 different points labially/buccally and palatally. Main outcome measure was the degree of PP score during the 1st week of orthodontic tooth movement (OTM) after 4 hours, 24 hours, 3 days, and 7 days of both LLLT and non-LLLT treatment applications. A questionnaire with an 11-point numeric rating scale (NRS) was used for PP.  Mean ± SD of PP in the LLLT + SL group was 3.33 ± 1.4, 3.58 ± 1.06, 2.31 ± 0.67, and 1.89 ± 0.54 in 4 hours, 24 hours, 3 days, and 7 days, respectively. Compared to all 4 TM groups, LLLT groups showed better PP. More statistically significant differences were found in LLLT groups. No harms were encountered.  The intervention provider and the patient were not blinded to the intervention.  The LLLT + SL group revealed significantly promising benefits on PP during OTM. LLLT has promising benefits on OTM. It has been revealed that 940 nm diode LLLT significantly increase osteoblast cells during their proliferation and differentiation stages. Thus, it induces bone remodelling by stimulating osteogenesis , 2. QualIn a systematic review of animal studies on acceleration of OTM, using noninvasive techniques suggested for further research studies to establish protocols to use them clinically with conviction . Based oResearch in relation to pain and its control in clinical situations did not progress/advance much as one of the major impeding factors for such a delay was our incapability in measuring pain effectively. A spectrum of studies were carried out considering pain as signals of tissue injury at one end and as a completely subjective phenomenon.The effect of a single dose of LLLT on spontaneous and chewing pain after the placement of elastomeric separators on OTM , PP withStudies have revealed the usefulness of LLLT in the reduction of PP immediately following the placement of separators  and initAfter obtaining the approval from the Ethical Committee of Jouf University (LCBE#4-22-2/40), which complies with the Declaration of Helsinki, a written informed consent was obtained from all the subjects . This study was designed and conducted according to the guidelines of Strengthening the Reporting of Observational Studies in Epidemiology (STROBE).Thirty-two healthy orthodontic patients of Saudi ethnic background with ages between 14 and 25 years were selected for the study . Based oH test, all 4 TM groups in relation to PP outcome with OTM treatment time.  PP after 4 hours was LLLT + SL < LLLT + Conv.<non-LLLT + SL < non-LLLT + Conv.  PP after 24 hours was LLLT + SL < LLLT + Conv.<non-LLLT + Conv. < non-LLLT + SL.  PP after 3 days was LLLT + SL < non-LLLT + SL < LLLT + Conv. < non-LLLT + Conv.  PP after 7 days was LLLT + SL < non-LLLT + SL < LLLT + Conv. < non-LLLT + Conv.Tables U test to explore the exact differences between groups. p=0.011), 3 days (p=0.013), and 7 days (p=0.001). p=0.021) and 7 days (p=0.002). p=0.040), 24 hours (p=0.006), 3 days (p=0.002), and 7 days (p=0.001). p=0.024). p=0.013). p=0.031).Based on extensive literature search, this is first-in-human study that evaluated 4 different TM outcomes on Saudi ethnic orthodontic patient. In this study, prospective evaluation of the consequences of a single application of LLLT on pain consecutively after 4 hours, 24 hours, 3 days, and 7 days after orthodontic treatment was conducted in parallel study design. Parallel study design is efficient and has uniformity in allocation segment and has no carry-across effects or contamination which is the main disadvantage for split-mouth studies , 28, 29.An infrared radiation with a wavelength of 940 nm –8 and a Present study and 25 different global studies' results of 14 different countries are highlighted in Various studies have investigated the role of LLLT on a variety of biological processes –5, and iThe present study reports in favour of SL bracket. These results coincide with results reported by Qamruddin et al. . The advGender disparities were not considered in this report due to inequality in subject distribution between male and female. Female patients were more than the male. Qamruddin et al.  found noThis clinical prospective intervention revealed LLLT has promising benefit among all 4 treatment modalities, and LLLT + SL results the best and LLLT + Conv. as the 2nd best in lessened pain perception during the 1st week of OTM. Lastly, comparing the efficacy among 2 bracket systems, SL lessened pain perception better compared to Conv.