AUSTRAL observing program was started in 2011, performing geodetic and astrometric very long baseline interferometry (VLBI) sessions using the new Australian AuScope VLBI antennas at Hobart, Katherine, and Yarragadee, with contribution from the Warkworth  12 m and Hartebeesthoek (South Africa) 15 m antennas to make a southern hemisphere array of telescopes with similar design and capability. Designed in the style of the next-generation VLBI system, these small and fast antennas allow for a new way of observing, comprising higher data rates and more observations than the standard observing sessions coordinated by the International VLBI Service for Geodesy and Astrometry (IVS). In this contribution, the continuous development of the AUSTRAL sessions is described, leading to an improvement of the results in terms of baseline length repeatabilities by a factor of two since the start of this program. The focus is on the scheduling strategy and increased number of observations, aspects of automated operation, and data logistics, as well as results of the 151 AUSTRAL sessions performed so far. The high number of the AUSTRAL sessions makes them an important contributor to VLBI end-products, such as the terrestrial and celestial reference frames and Earth orientation parameters. We compare AUSTRAL results with other IVS sessions and discuss their suitability for the determination of baselines, station coordinates, source coordinates, and Earth orientation parameters.The  VLBI performance for geodesy and astrometry relies on a global network of antennas that are compatible in sensitivity, frequency coverage, and slew speed. The volume of the spanned network correlates with the expected accuracy of measured Earth orientation parameters Malkin , and uneBetween 1989 and 2011, the only radio telescope observing regularly for geodesy and astrometry in Australia was the 26 m antenna in Hobart (Ho), while the 34 m DSS45 and the 64 m Parkes antennas participated on an occasional basis. Titov  showed tThe IVS is currently in transition to the VLBI Global Observing System  results, a dense time series, or sessions with astronomical purposes. The AUSTRAL program comprises 24 h single sessions, 15 day CONT-like campaignsaust02) was carried out in August 2011. It was scheduled at UTAS and correlated at the Washington correlator. Then, beginning in March 2012 (aust04), the recording mode was changed from the standard IVS mode of 256 Mbps (mega bits per second) to 1 Gbps (giga bits per second). In July 2013 (aust10), a collaboration was arranged with the Technische Universität Wien to deliver the schedules for the AUSTRAL experiments using the Vienna VLBI Software  was observed  and June (A15-2) and up to In this paper, we describe the sessions up to June 2015. We note that in beginning of 2016, the AUSTRAL program could be resumed, under collaboration with the Shanghai correlator. Figure Up to now, there have been 151 AUSTRAL sessions which have produced useful data. More than half of them (81) involved the full five-station network; 42 had four stations, which are usually the three AuScope antennas and either Ww or Ht, and 22 sessions were done with only the three AuScope antennas. In the remaining six sessions, there were good data from only two stations (two sessions) or additional stations were included (four sessions). Table In total, the AUSTRAL sessions comprise about 770,000 observations. One observation means one measured time delay between two antennas  channels in X-band and six channels in S-band. To increase the sensitivity  Mark5B+ units for recording. In total, the AuScope array owns and uses more than 100 Mark5 modules, each with capacities of up to 32 TB. All AUSTRAL experiments correlated at Curtin were sent to the correlator via e-transfer. In the case of Ke and Yg, the modules were first physically shipped to Hobart, where the data were transferred from the modules to the local RAID system before it was e-transferred to Curtin. For this, the Hobart observatory is linked to the university campus at 10 Gbps and with 10 Gbps to the Australian mainland.moprinFor observing, a redundant server system was installed which can access the local field system machines, the DBBCs, the Mark5s, and the drive PC. For the observations, we use the eRemoteControl software . Therefore, we need to log as much information as possible, e.g., concerning the module allocations or data transfers. The pivot of the AUSTRAL operations is the publicly accessible AuScope operations wiki,Correlation at UTAS was performed on a six PC cluster with attached multi-TB arrays or, for bigger jobs, the UTAS HPC cluster. In terms of latency, the delay between observation and the preparation of a correlation report was usually several months.Scheduling is the process of determining which telescopes observe which source at what time. A schedule is a complex optimisation process which is usually created using a special software. For the AUSTRAL sessions, the newly developed scheduling tool Vie_sched of VieVS , or simply as the number of scans per hour for an individual station.The reality is somewhat more complicated. The largest error in VLBI at present is due to unpredictable fluctuations in the troposphere. To account for this, the general goal is to get as many observations in different directions and at different elevations for each station as possible. This is referred to as F) and the sensitivity . With a target SNR of 15 or 20, the scan duration is then determined by the brightness of the source  measured in Jansky (Jy). According to Lovell et al. , the AuSaust50 the S-band SEFD at Hb was increased to 5300 Jy to account for a large amount of S-band radio frequency interference (RFI).In the beginning, rather conservative values of about 5000 Jy were used. These values were determined with the help of colleagues from the Goddard Space Flight Centre based on the analysis of recent observations. For Yg, a bit higher values were used, accounting for hardware problems leading to failures in various channels. In October 2014, the AUSTRAL program started to maintain its own catalog files and then beginning with a1501 onwards. At the moment, Yg shows the best performance, with a sensitivity of 3600 Jy in X-band. This is close to the theoretical value of 3500. The expectations for Ww were significantly improved after a major service of the DBBC. On the other hand, the values for Ht were found to be a little too optimistic and increased to 2200 and 1400 Jy in X- and S-bands, respectively.The last change in antenna SEFD followed a thorough analysis of the A14 observations, with new SEFDs from It should be noted here that determining the antenna-specific SEFD, a posteriori of observations within a network, is not straightforward. A recent study by Gruber  revealedTo understand the effects of changing the antenna SEFDs in the scheduling, in Fig. Using the AUSTRAL observing mode (data rate of 1 Gbps) for a relatively strong source of 1.0 Jy, the scheduled scan duration changes from 24 s for two antennas with SEFD of 3500 to 31 s when both antennas have SEFDs of 4000, and up to 48 s for SEFDs of 5000. For weaker sources of 0.8 Jy flux density, the scan duration is 37 and 75 s for SEFDs of 3500 and 5000 Jy, respectively, and for a source of flux densities of 0.5 Jy, the scan duration varies between 94 and 192 s for the same respective SEFDs.we used completely new software which was originally designed for simulations and was not fully verified for real observations. When applying VieVS for the scheduling of AUSTRAL sessions, a number of new features and scheduling options were added to the software;the schedulers themselves were quite inexperienced in the beginning and they are still learning and improving their understanding of the scheduling process;the AUSTRAL sessions allow for fast slewing and short on-source times (in the style of the future VGOS), demanding for new scheduling that differs from that for the legacy experiments.In Fig. aust11, a schedule in the style of the legacy observations. On average, six scans per station per hour are done. An antenna spends 54 % of time on observations and 7 % on slewing. For 37 % of the session an antenna is idle, meaning that it is not used and presumably waiting for other antennas. This is mainly due to the inclusion of Ht. While the three AuScope antennas and Ww are practically identical and have similar capabilities, Ht is much more sensitive but also slower in slewing. In addition, the mutual visibility between Ht and Ww is limited, causing some difficulties in scheduling. For aust11, an additional 2 % of the time was spent for calibration, which are fixed-time intervals added at the beginning and the end of a scan. For the first sessions, data of up to 8.4 TB were recorded per station.Scheduling of AUSTRALs evolved for three reasons:aust30, restricting the source list to sources stronger than 0.5 Jy. Later on (starting with a1501), using simulations  and run for 24 h. This means that the last scan of a session starts within this 24 h and often finishes a couple of minutes after 17:00UT the next day. This would cause a problem when scheduling two consecutive sessions. For the AUSTRAL weekend sessions starting at 00:00UT on a Saturday and Sunday, the first session was, therefore, scheduled to be 5 min short of 24 h. In addition, the schedule file (.snp) of the first session was edited to start the following session, allowing for an automatic session changeover without additional interaction by the operator.a1501-a1515) in February, and A15-2 (a1516-a1530) in June 2015. We use the term quasi-continuous as the 15 24 h sessions were placed around other observing commitments, such as the IVS R-sessions which occur twice a week. This, on the other hand, allows for a nice comparison of measured parameters between the different types of sessions . The reasons for this exceptionally good behaviour could not be identified so far.We find a median session fit of 44 ps over all sessions. This is considerably worse than the IVS R1/R4 observations with a median value of 34 ps. The reason for this is not completely understood yet. Studying Fig. aug020, shows an improved session fit of 26 ps.One possible reason for the generally worse residuals is that due to strong RFI in two S-band channels at Hb, these data are usually omitted from correlation. Consequently, the broadband resolution function is wider, leading to higher group delay errors in S-band. Although the S-band is only used to calibrate the ionosphere and errors are usually heavily reduced  and most recent (aug) sessions quickly rise for the long baselines with Ht, the curves are much flatter for the other two sets of data. Given the fact that the reason for the curvature seen in baseline repeatabilities is a point of discussion  have been improved from about 9 mm to between 4–7 mm in baseline length repeatability. For the very long baselines, we find improvements from 2–3 to 1.5 cm for the most recent sessions. Studying Fig. 500 km haon Titov , this miThe individual values of baseline wrms are summarised in Table aug sessions). Despite these impressive improvements over the two years, results of the IVS Cont14 series are better still, especially on the long baselines . However, it has been shown Malkin  that bote Malkin .In Table y-pole with a formal uncertainty of 300 We find that the AUSTRAL sessions are not very well suited for determining polar motion. While the results from the R-sessions deviate about 100–200 Despite the poor statistics for the AUSTRAL sessions in Table y-pole and to some extent also x-pole and dUT1 for the AUST-CONT A15-1 series in February 2015, while the following sessions show large, almost systematic offsets. This also partly applies to the April sessions, which show particularly good session fits  comparison with other space geodetic techniques, including the global navigation satellite systems (GNSS).


L of the present structure, we find that the nanoring-strip graphene arrays have a wide range of resonances (resonance wavelength increases from 17.73 μm to 28.15 μm). When changing the sensing medium refractive index medn, the sensitivity of mode A and B can reach 2.97 μm/RIU and 5.20 μm/RIU. By changing the doping level gn, we notice that the transmission characteristics can be tuned flexibly. Finally, the proposed sensor also shows good angle tolerance for both transverse magnetic (TM) and transverse electric (TE) polarizations. The proposed nanoring-strip graphene arrays along with the numerical results could open a new avenue to realize various tunable plasmon devices and have a great application prospect in biosensing, detection, and imaging. In the present study, we design a tunable plasmonic refractive index sensor with nanoring-strip graphene arrays. The calculations prove that the nanoring-strip have two transmission dips. By changing the strip length  Surface plasmon resonance (SPR) on the metal/dielectric interface at the sub-wavelength scale provides an excellent platform for a variety of optoelectronic applications ,2. SPR pGraphene, a monolayer of carbon atoms, arranged in a plane with a honeycomb lattice, due to its unique optical and electronic properties, it has stimulated research interest in photonics and optoelectronics . More inL of the present structure, we find that the nanoring-strip graphene arrays have a wide range of resonances (resonance wavelength increases from 17.73 μm to 28.15 μm). When changing the sensing medium refractive index medn, the sensitivity of mode A and B can reach 2.97 μm/RIU and 5.20 μm/RIU. Such a high sensitivity will have great prospects in terms of biosensing and detection. We finally investigate the oblique incidence of incident electromagnetic waves and the results show that the resonance is angle-insensitive. The resonance is confined to the electromagnetic field at the edge of the nanoring-strip. In addition, the nanoring-strip with different doping level for electrically tunable spectral imaging provides interesting applications. The proposed sensor structure can be used in biosensing, detection, and imaging.In the present study, we design a tunable plasmonic refractive index sensor with nanoring-strip graphene arrays. The transmission response relying on geometrical sizes of the nanostructure and the sensing medium refractive index are extensively studied by the finite-difference time-domain (FDTD) method. By changing the strip length subn) and a sensing medium (medn). We assume that the substrate is semi-infinite. P = 300 nm is the fixed period of the arrays. The width of the nanoring is 1W = 30 nm and the thickness of graphene is t = 1 nm. The strip width is 2W = 30 nm and the length is L = 180 nm. In T) is set to 300 K and considering the doping level of graphene and the condition FE >> BTK can be satisfied. In general, in accordance with the Pauli Exclusion Principle conductive of the graphene surface can be approximated as an intraband Drude model expression:e is the charge of electron, the ћ = h/2π is the reduced Planck constant and the τ is the relaxation time. Where, the τ and FE can be written asgn, Fν = 106 m/s and µ ≈ 10,000 cm2/(V·s) are the graphene doping level, the velocity of graphene Fermi and the measured dc mobility, respectively  for both TM and TE polarizations. This result shows that for TM and TE polarization, the resonance dip wavelength is insensitive to the angle of incidence. There are two main reasons. Firstly, from gn = 3 × 1013 cm−2 for TM and TE polarizations, as shown in For TM and TE polarizations, the transmission spectra of the proposed nanostructure at different L of the present structure, we find that the nanoring-strip graphene arrays have a wide range of resonances (resonance wavelength increases from 17.73 to 28.15 μm). When changing the sensing medium refractive index medn, the sensitivity of mode A and B can reach 2.97 μm/RIU and 5.20 μm/RIU. Such a high sensitivity will have great prospects in terms of biosensing and detection. In addition, the transmission dip resonance wavelength of the proposed nanostructure can be tuned flexibly by changing the doping level gn. The results also show that the graphene nanoring-strip arrays is insensitive to different polarization modes (TM or TE). From the above results, we conclude that the nanoring-strip graphene arrays have good tunability and high sensitivity, we believe that this sensor has broad application prospects in biosensing, detection, and imaging. In conclusion, a tunable plasmonic refractive index sensor with nanoring-strip graphene arrays has been proposed and theoretically demonstrated. By FDTD method simulation calculation, it is found that the graphene nanoring-strip structure has a high sensitivity to the sensing medium refractive index changes. By changing the strip length


TBX5 is a T-box family transcription factor that regulates heart and forelimb development in vertebrates and functional deficiencies in this protein result in Holt-Oram syndrome. Recently, we have shown that acetylation of TBX5 potentiates its activity and is important for heart and limb development. Here we report that class II histone deacetylases HDAC4 and HDAC5 associate with TBX5 and repress its role in cardiac gene transcription. Both HDAC4 and HDAC5 deacetylate TBX5, which promotes its relocation to the cytoplasm and HDAC4 antagonizes the physical association and functional cooperation between TBX5 and MEF2C. We also show that protein kinase D1 (PRKD1) relieves the HDAC4/5-mediated repression of TBX5. Thus, this study reveals a novel interaction of HDAC4/5 and PRKD1 in the regulation of TBX5 transcriptional activity. Based on their structure and function they are classified into four different groups: class I , class II , class III  and class IV (HDAC11)3.Histone deacetylases (HDACs) are histone modifying enzymes that remove the acetyl group from histone and non-histone proteins and control vital cellular processes such as cell proliferation, differentiation and development4. HDAC1 and HDAC2 share 85% similarity and play a redundant role in cardiac morphogenesis5. Deletion of Hdac1 in mice produces embryonic lethality due to proliferation defects6. Hdac2 null mice were unable to reactivate the fetal gene program or failed to exhibit normal hypertrophic responses. In addition, transgenic overexpression of Hdac2 in the heart induces cardiac hypertrophy7. HDAC3 is predominantly located in the nucleus and it shows less similarity to HDAC1 and 2 than they do to each other. Cardiac specific deletion of Hdac3 in mice produces massive cardiac hypertrophy and upregulation of genes associated with fatty acid metabolism8. In addition, mice overexpressing Hdac3 in the heart show a distinct proliferation of postnatal cardiac myocytes, but without hypertrophy9.The class I HDACs are highly similar to yeast RPD 3 (reduced potassium dependency 3) and are ubiquitously expressed10. They are abundantly expressed in the heart, brain and skeletal muscles11 and are signal responsive repressors of cardiac hypertrophy10. Calcium/calmodulin-dependent protein kinase II (CaMKII) phosphorylates HDAC4, promoting its nuclear export and de-repression of myocyte enhancer factor-2 (MEF2) target genes in cardiomyocytes13. Protein kinase CaMKII specifically transmits signals via a unique docking site in HDAC4 that is absent from other class IIa HDACs12. HDAC5 acquires CaMKII responsiveness by interacting with HDAC414. Protein kinase C and D mediate agonist-dependent cardiac hypertrophy through nuclear export of HDAC515 and in H9C2 cells, CaMKIV and protein kinase D regulate nucleocytoplasmic localization of HDAC516.Class II HDACs are highly similar to yeast HDA1 (histone deacetylase-A 1) and suppress heart growth17 and histone deacetylases are key players in heart development and cardiac hypertrophy10. Here we investigated the possible link between histone deacetylases and TBX5. We report that TBX5 associates with both class I and class II HDACs and the association of class II HDACs with TBX5 results in suppression of cardiac gene transcription. HDAC4 competes with and disrupts the functional cooperation between TBX5 and MEF2C, which plays a key role in early heart development. HDAC4/5-mediated gene repression can be partially rescued by Protein kinase D1 (PRKD1). These studies suggest the TBX5-mediated gene regulatory pathway is linked to a signal-mediated protein kinase via PRKD1.Acetylation potentiates the transcriptional activity of TBX517. Histone deacetylases (HDACs) are transcriptional repressors and anti-hypertrophic. Given the counteracting role of HDACs in gene regulation, we investigated whether HDACs interact with TBX5 and modulate transcription. To analyse their physical interaction, we co-transfected Cos7 cells with plasmids encoding MYC-TBX5 along with each of six plasmids encoding FLAG-HDACs (1–6). Pull-down and Western blot analyses were carried out on cell extracts to examine their physical interaction. Figure Histone acetyltransferases KAT2A/2B acetylate TBX5MYH6, a luciferase reporter gene construct under the control of the TBX5 target promoter MYH6. Reporter assays were performed in cardiomyoblast cells H9c2. As shown in Fig. MYH6, whereas HDAC2 and HDAC3 did not. We then tested the effect of HDACs on TBX5-mediated transcription and found that HDAC1 led to a 75% reduction of the TBX5-mediated activation on this promoter (P < 0.02). HDAC2 did not show any effect, whereas HDAC3 increased the TBX5-mediated activation, although it was not statistically significant (P > 0.05).In light of this interaction we sought to examine the effect of both class I and class II HDACs on TBX5-mediated gene transcription. We performed reporter assays on pGL3-MYH6 by almost 50%, whereas HDAC6 had no effect   Fig. . When HD-T) Fig. . This fiThe TBX5 and MEF2C functional interaction plays a major role in cardiac gene transcription. The interaction of HDACs with TBX5 prompted us to analyse the effect of HDAC4 and 5 on TBX5-MEF2C-mediated transcription. As shown in Fig. Thus, these experiments suggest that HDAC4 and HDAC5 strongly inhibit TBX5-MEF2C complex formation on their promoter target DNA, inhibiting their functional cooperativity.19. Similar to MEF2, HDAC4 and 5 also associate with TBX5 and repress TBX5-mediated transcription. To investigate whether CaMKII and IV play any roles in TBX5-dependent gene transcription we performed reporter assays. As shown in Fig. HDAC4 and HDAC5 are signal-responsive repressors of cardiac hypertrophy. Signal-mediated kinases CaMKII and IV and serine-threonine protein kinase D1 (PRKD1) phosphorylate class IIa HDACs. This post-translational modification promotes their nuclear export, relieving repression of MEF-dependent gene transcriptionin vitro phosphorylation activity on HDAC5. HDAC5 is phosphorylated at S259 and S498 by these kinases. We generated two GST-fusion proteins corresponding to the motif 1 (GST-MT1) and motif 2 (GST-MT2) of HDAC5 containing the Ser259 and Ser498 respectively. These two motifs are highly conserved among class II HDACs. PRKD1-FLAG and CaMKII/IV-HA fusion proteins were generated in Cos7 cells . TBX5 activity can also be modulated at a post-translational level, as our recent studies suggest that acetylation of TBX5 enhances its transcriptional potential17, whereas others have shown the importance of sumoylation24.Building the heart is a complex process governed by a network of molecules such as transcription factors, cofactors, signalling molecules and structural proteins25. Our pull-down assays suggest that TBX5 associates with both class I  and class II HDACs (HDAC4 and 5). Association of Tbx5 and Hdac2 in the Nucleosome Remodeling and Deacetylase (NuRD) complex has been reported previously but Hdac4 and 5 were not detected in that complex20. The association of TBX5 with HDAC4/5 is particularly interesting, not only because they are abundantly expressed in the heart11, but also due to their roles in cardiac hypertrophy10. The association of TBX5 with HDAC4/5 resulted in repression of TBX5-mediated transcription of cardiac gene MYH6. In addition, HDAC4/5 deacetylate TBX5 leading to its partial redistribution into the cytoplasm. Interestingly, the non-acetyl version TBX5-K339R also retained HDAC5 binding activity and was repressed by both HDAC4/5 in reporter assays suggesting that a chromatin restructuring mechanism does play a significant role in the transcriptional regulation of TBX5. Taking all these facts into account, we suggest that the repression of TBX5 activity by HDAC4/5 could be attributed to two factors; 1) deacetylation of TBX5 that promotes its nuclear export which effectively reduce the nuclear pool and 2) alter chromatin structure due to TBX5-mediated promoter recruitment of HDAC4/5.HDACs are modulators of heart development and growth, and their association with MEF2 transcription factors are central to the control of myogenesis and cardiac hypertrophy18. In pull-down assays we found that HDAC4 associates with both TBX5 and MEF2C, however the association between HDAC4 and MEF2C was stronger. In competition assays, HDAC4 displaced TBX5 from the TBX5-MEF2C complex, whereas TBX5 was unable to compete with HDAC4 for MEC2C binding. In addition, HDAC4 inhibited TBX5-MEF2C complex formation on the target DNA and supressed their functional cooperation on a MYH6 promoter. TBX5 interacts with the MADS-box region (1–60 Aa) of MEF2C18 and HDAC4 interacts overlapping MADS-MEF2 region (39–72 Aa)25. Since the MEF2C interacting region for TBX5 and HDAC4 overlap significantly, it is likely they compete for MEF2C binding. In addition to cytoplasmic re-localization and alter chromatin structure, our results also suggest that HDAC4/5 disrupt the association and functional cooperation between TBX5 and MEF2C, thus ensuring maximum TBX5 repression.Functional cooperation between TBX5 and MEF2C is essential for early heart development10. Signal-mediated protein kinases such as CaMKII/IV and protein kinase A/D (PRKA/D) phosphorylate specific serine residues in HDAC4/5 that guide them into the cytoplasm and relieve repression of MEF2-mediated gene transcription26. Our studies suggest that it is PRKD1, and not CaMKII/IV, that partially relieves HDAC4/5-mediated repression of TBX5. The PRKC/PRKD signalling pathway promotes agonist-dependent cardiac hypertrophy through phosphorylation of HDAC5 and nuclear export15. PRKD-dependent phosphorylation of HDAC5 occurs at Ser 259 and Ser 498 and is critical for nuclear export27. Our studies suggest that PRKD1 strongly phosphorylates Ser259 and Ser498 on HDAC5, and that could be linked to de-repression of TBX5 target genes. It is also possible that PRKD1 could directly phosphorylate TBX5 and potentiates its transcriptional activity. CaMKII and CaMKIV, which do not de-repress TBX5, showed very weak phosphorylation activity in our assay conditions.Class II histone deacetylases are signal responsive repressors of cardiac hypertrophyTBX5 plays a key role in heart development and disorder. Many key components that interact and modulate the function of TBX5 have been identified, thus helping us to understand the molecular mechanisms of heart development and disease. In this study we revealed a novel TBX5-HDAC4/5-PRKD1 gene regulatory pathway, which might be important for several aspects of heart development, and which warrants further investigation.PRKD1 cDNA into the EcoR1 and XhoI sites in pcDNA3.1. The original clone was obtained from Source Biosciences . We also cloned two DNA fragments into pGEX4T-1 encoding the phosphorylation motifs of HDAC5: MT1 and MT2 (corresponding to the amino acid residues 254 to 264 and 493 to 503 respectively) to generate fusion proteins GST-MT1 and GST-MT2.The plasmid pcDNA-HDAC1-FLAG was a generous gift from Marian A. Martinez-Balbas . Plasmids pME18S-FLAG-Hdac2 and pcEP4F-FLAG-HDAC3 were a gift from Dr Y. E. Chin . Hdac2-FLAG was subcloned into the EcoRI and XhoI sites of pcDNA3.1. The plasmid pcDNA-HDAC4-FLAG was a generous gift from Dr S.L Schreiber . Plasmids pcDNA-HDAC5-FLAG and pcDNA-HDAC6-FLAG were generated from IMAGE clones 6043491 and 2984860 respectively (Source Biosciences). Plasmids pcDNA-HA-CaMKIIδ and pcDNA-HA-CaMKIV were made from IMAGE clones BC032784 and BC025687 respectively (Source Biosciences). The plasmid pcDNA-PRKD1 was generated by cloning 18. Rat cardiomyoblast cell line H9c2 and Cos7 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum. For reporter assays, all transfections were performed in 6 wells plates using Polyfect reagent (Qiagen) according to the manufacturer’s protocol. Cells in each well received 1.5 μg of reporter plasmid, 1.0 μg of expression plasmids pcDNA-TBX5 and 0.5 µg of pcDNA-HDAC1, pcDNA-Hdac2, pcDNA-HDAC3, pcDNA-HDAC4, pcDNA-HDAC5 or pcDNA-HDAC6. For TBX5 and MEF2C experiments we also added 0.25 μg pcDNA-MEF2C. For CaMKII and CaMKIV experiments we co-transfected 0.5 µg of pcDNA-CaMKII or pcDNA-CaMKIV. To normalize the variation in transfection efficiency between the plates 4 ng of plasmid pRL-TK was used. The total amount of plasmid DNA in each well was adjusted to 3.0 or 3.5 μg with empty vector pcDNA3.1 as appropriate. After 24 h of transfection cells were harvested in lysis buffer and luciferase activity was measured using a Dual Luciferase assay kit (Promega). Each transfection experiment was carried out in duplicate and repeated for at least three times. Values shown in the histograms are means ± SD. Significance of analysis was performed by student t-test.This was conducted as described previouslyCos7 cells were grown in 100 mm petri dishes in DMEM media. Cells were transfected with 6.0 µg of pcDNA-Myc-TBX5 and 4.0 µg of pcDNA-FLAG-HDAC plasmid DNAs using transfection reagent polyfect (Qiagen). In some experiments, we also included 4.0 µg of pcDNA-HA-CaMKII or pcDNA-HA-CaMKIV. Transfected cells were harvested after 48 h and lysed in lysis buffer (20 mM Tris (pH-7.6). 150 mM NaCl, 0.5% NP40, I mM PMSF, 1 mM aprotinin, 1 mM pepstatin and 1 mM leupeptin). Cells were left in lysis buffer for 1 h under ice, followed by brief sonication. The extracts were centrifuged at 12,000 g for 10 mins at 4 °C and stored at −80 °C until used.17. Cell extracts were incubated with 20–30 µl of washed Anti-FLAG M2-Affinity gel (Sigma) at 4 °C. For interaction studies, cell lysates were added to the affinity gel and incubated for 2 h or overnight in a shaker at 4 °C. To remove non-specific proteins, beads were washed 6 times in wash buffer (20 mM Tris (pH7.6), 150 mM NaCl, 0.5% NP40 and 0.5 mM EDTA). Bound proteins were specifically eluted from the beads with wash buffer (15.0 µl) containing 3X FLAG peptide (Sigma). Eluted proteins were fractionated on 10% Bis-Tris gels followed by Western blotting. FLAG-tagged, Myc-tagged and HA-tagged proteins were detected using monoclonal Anti-FLAG M2 antibody (Sigma), Anti-Myc antibody (Millipore) and Anti-HA antibody (Sigma) respectively.Pull-down assays were carried out as described previouslyMouse heart extract was generated in 8 M urea buffer  from E13.5 embryonic hearts. Protein estimation was carried out using Quick start Bradford protein assay reagents (Bio-Rad). Western detection was carried out on 10 µg of extract with anti-TBX5 (Abnova), anti-HDAC5 (abcam), anti-HDAC4 (Santa Cruz), anti-PRKD1 (Sigma) and anti-GAPDH (Santa Cruz).17. Cos7 cells were grown on coverslips in 6-well plates overnight and transfected with TBX5-V5 and HDAC4-FLAG/HDAC5-FLAG constructs using polyfect (Qiagen). Twenty-four hours post-transfection cells were washed in phosphate buffer saline , fixed with 4% paraformaldehyde for 10 mins and washed in PBS three times, 5 minutes each. Cells were then permeabilized with 0.25% Triton X-100 in PBS for 5 minutes washed in PBS three times and incubated in 10% BSA in PBS for 1 hour at room temperature to supress non-specific binding of IgG. After blocking the cells were washed in PBS and incubated with mouse monoclonal antibody against V5  or with rabbit monoclonal antibody against FLAG  in PBS containing 3% BSA for 2 hours at room temperature. Cells were washed three times in PBS each for 5 minutes and incubated in 3% BSA in PBS containing secondary antibody Alexa Fluor 488 goat anti-rabbit IgG and/or Alexa Fluor 594 goat anti-mouse  for 45 minutes at room temperature. Then cells were washed three times in PBS each for 5 minutes and the washed coverslips were mounted on glass slides containing a small drop of mounting media containing DAPI (Vectashield). Laser scanning confocal microscopy was performed on LSM 880 F  using a 63 × 1.4 oil objective.This was performed as described previouslyIn vitro synthesis of full-length and truncated TBX5 and MEF2C proteins was performed using coupled reticulocyte lysate (Promega) according to the manufacturer’s protocol and was described previously18. Briefly, 1.0–2.0μg of template DNA was incubated at 30 °C for 90 minutes in a 50μl reaction mixture containing 35S-methionine (Amersham). Following incubation protease inhibitors PMSF (1 mM), leupeptin (1μg/ml), pepstatin (1μg/ml) and aprotinin (1μg/ml) were added to protect samples from proteolysis. Proteins were visualized using SDS-PAGE followed by autoradiography.FLAG-PRKD1, HA-CaMKII and HA-CaMKIV proteins were overexpressed in Cos7 cells by transfecting the cells with plasmid pcDNA-PRKD1 or pcDNA-CaMKII or CaMKIV and ployfect reagent (Qiagen). Cell extracts were generated from 48 h post transfected cells. Both FLAG and HA-tagged proteins were partially purified on anti-FLAG-Agarose and anti-HA-Agarose affinity gels (Sigma) respectively, aliquoted into tubes and stored at 4 °C until use. Glutathione S-transferase (GST), GST-MT1 and GST-MT2 proteins were purified on a GST-purification kit . Protein concentration was determined by Quick start Bradford protein assay kit (Bio-Rad).2, 1 µl of cold ATP (5 mM), 1 µl Ƴ32P ATP and 4 µl of GST proteins (4 µg)). For CaMKII/IV we included 1 mM CaCl2, 0.6 mM calmodulin in the kinase buffer. The final volume of the reaction mix was kept to 25 µl. Tubes were incubated at 30 °C for 45 mins. In each tube 6 µl of 4X LDS sample buffer (Invitrogen) and 1 µl of reducing agent (Invitrogen) were added and heated at 95 °C for 8 mins. Tubes were spun down and 20 µl of each sample was loaded on 10% Bis-Tris gel (Invitrogen) and electrophoresed for 1 h at constant voltage. The gel was washed in dH2O and stained with Simply blue stain (Invitrogen). The gel was then de-stained with dH2O, dried and subjected to autoradiography.Kinase reactions were assembled in Eppendorf tubes containing beads immobilized with FLAG-PRKD1 or HA-CaMKII/IV proteins. In each tube we added 5 µl of 5X kinase buffer (150 mM Tris-HCl buffer (pH-7.5), 100 mM MgCl18. Full-length or truncated versions of TBX5 proteins were generated as MBP-fusions in bacteria following IPTG induction. These fusion proteins were partially purified from bacterial lysates on amylose resin. Their integrity was checked on PAGE by Coomassie blue staining of the gel. MBP-TBX5 fusion proteins were immobilized on amylose resin and GST-MEF2C was immobilized on glutathione sepharose beads for interaction studies. Equivalent amounts (2–5 μg) of immobilized MBP or GST fusion proteins were incubated with 35S-labelled full-length MEF2C or TBX5 (wild type or mutant) in binding buffer  for 2 hours at 4 °C. Beads were washed four times with binding buffer without BSA before the bound proteins were released from the beads by boiling with SDS sample buffer. Eluted proteins were subsequently resolved on SDS-PAGE followed by scanning on a Storm PhosphorImager (Amersham Bioscience), and band intensity was quantified using Image Quant software (Molecular Dynamics).This was performed as described previously28. A DNA fragment corresponding to bases –1491 to –1530 of the MYH6 promoter containing two TBX5 sites flanking a (A/T)-rich site (T-M-T) was used as a probe. The probe was prepared by annealing the complementary oligonucleotide corresponding to oligonucleotide CAC CTC CAC ACC CTG GAG CTA TAT TGA GAG GTG ACA GTA AAC and end-labelling. All the reactions were carried out in 15–20 μl volume and incubated for 0.5 h at room temperature.We performed electrophoretic mobility shift assays (EMSAs) as described previouslyCos7 cells were transfected with 6.0 µg pcDNA-FLAG-TBX5, 2.0 µg pcDNA-HA-KAT2B and either with 2.0 µg pcDNA-FLAG-HDAC4 or pcDNA-FLAG-HDAC5. Cell extracts were generated in lysis buffer as stated above from 48 h post-transfected cells. FLAG-TBX5 protein was partially purified from the extract and fractionated on 10% bis-Tris gel (ThermoFisher Scientific) followed by Western blotting. The acetylation status of TBX5 was monitored with anti-Acetylated- Lysine antibody .All the experiments were performed in accordance with relevant guidelines and regulations of the Animal Procedures Act under license number P375A76FE (Nottingham), issued 2018-08-22, and granted by The Home Office. All methods were carried out in accordance with relevant guidelines and regulationsAll the data shown here are means ± standard deviation (SD). The significance of differences between the groups was checked by student’s t test. The P value less than 0.05 were considered as significant.Supplementary information


Therefore, based on current knowledge and latest progress, this article aimed to present the recent achievements and provide a comprehensive literature review on potential applications of graphene that could be translated into clinical reality in dentistry.Graphene family nanomaterials, with superior mechanical, chemical, and biological properties, have grabbed appreciable attention on the path of researches seeking new materials for future biomedical applications. Although potential applications of graphene had been highly reviewed in other fields of medicine, especially for their antibacterial properties and tissue regenerative capacities, Oral cavity is an extremely demanding setting. Dental materials when placed within oral cavity are fully contacted with saliva, gingival crevicular fluid, and water. At the same time, it is exposed to high temperature, masticatory forces, and variety of abrasion causing mechanical failures and overtime requiring restoration replacement with extra cost. Furthermore, most dental materials are in intimate contact with oral tissue for a long time; they must be noncytotoxic and biocompatible for them to have a harmonious interaction with host while performing desired functions. Therefore, there is always a huge interest and strong trend in continuous development of dental materials with improving properties.2 hybridized carbon atoms with hexagonal packed configuration  include ultrathin graphite, few-layer graphene (FLG), graphene oxide , reduced graphene oxide (rGO), and graphene nanosheets (GNS) . They diAlthough the developments and researches of graphene-based biomaterials related to dentistry are still at infancy, their unique properties and their abilities to functionalize alone or combined with biomaterials offer several opportunities in possible clinical applications. In this review, we intended to provide readers with an overview of the potential applications of graphene correlated to dentistry. Their biocompatibility aspect and antibiotic properties were briefly discussed. Perspectives related to graphene-based technologies aimed at oral care are presented and organized by different fields of dentistry. in vivo and in vitro [The first aspect to consider in the introduction of a new biomedical material is its biocompatibility. For a safer development of graphene-based nanomaterial, it is necessary to understand the interaction of graphene and their derivatives with living systems and their toxicityin vitro .2+ and Fe2+, which are highly mutagenic to cells leading to high levels of cytotoxicity and random scission of DNA [2+ and Fe2+ and consequently significantly lowered the cytotoxicity of GFNs [Accumulating evidences have suggested that cytotoxicity of GFNs can not be generalized as it depends on various factors including their morphology , surface charge, surface functionalization, dispensability, state of aggregation, number of layers, purity, and methods of synthesis . This isn of DNA . Greener of GFNs . Therefo2O2. The formation of OH and the cytochrome c/H2O2 electron-transfer system could enhance oxidative and thermal stress to impair the mitochondrial respiration system and eventually impose stronger oxidative damage on normal cells displaying stronger toxicity [The exact mechanism underlying the toxicity of GFNs remains obscure; several possible toxicity mechanisms of GFNs were proposed. Oxidative stress is suggested to be the main cause as the elevated ROS level may oxidize various molecules including DNA, lipids, and proteins inducing apoptosis or necrosis . GFNs matoxicity .Studies focused on the cytotoxicity of GFNs in oral setting are very limited. A study conducted by Olteanu et al. assessed the cytotoxic potential of GO, thermally reduced graphene oxide (TRGO) and Nitrogen doped graphene (N–Gr), on human dental follicle stem cells. The result showed that GFNs, especially GO, increased the intracellular ROS generation in a concentration and time-dependent manner. At high concentration (40 ug /mL), cells viability was reduced and mitochondria membrane potential was altered. While, at low concentrations (4 ug/mL), they exhibited a good safety profile providing high antioxidant defense. Their authors concluded that, among these three investigated graphenes, GO exhibited the lowest levels of cytotoxicity and induced least amount of damage to human dental follicle stem cells . in vivo studies as concentration and variations of GFNs tested can significantly change the toxicity outcome, not a conclusive answer can suit them all [Nevertheless, lack of consensus is reflected inthem all . Thus, bInterestingly, because of GNFs' versatility, their usage as potential antimicrobial agents has gained substantial interest in the field of nanomedicine –26. Howe Escherichia coli and Streptococcus mutans on gold and copper substrate in contact with CVD graphene and the result showed that the facile transfer of electrons from microbial membranes to graphene played no role on bacterial viability, denying the influence of metal substrates' conductive character on the antibacterial activity of CVD graphene [A thorough understanding of the antimicrobial mechanism is still in its infancy, but, with an increasing number of investigations on the antimicrobial activities of GRNs, three predominate mechanisms were proposed. Physical damage is induced by blade like graphene materials piercing through the microbial cellular membrane causing leakage of intracellular substance leading to cell death. Wrapping and photothermal ablation mechanism could also provoke bacterial cell damage by enclosing the bacterial cells, providing an unique flexible barrier to isolate bacteria growth medium, inhibiting bacteria proliferation, and decreasing microbial metabolic activity and cell viability. Chemical effect is primary oxidative stress mediated with production of ROS as excessive intracellular ROS accumulation could cause intracellular protein inactivation, lipid peroxidation, and dysfunction of the mitochondria, which lead to gradual disintegration of cell membrane and eventual cell death . There hgraphene . Escherichia coli, Streptococcus aureus, Klebsiella sp., and Pseudomonas aeruginosa, only few studies focused on oral pathogens. Streptococcus mutans is the primary gram-positive facultative anaerobic bacteria involved in caries formation while Porphyromonas gingivalis and Fusobacterium nucleatum are Gram-negative anaerobic bacteria associated with periodontitis and root canal infection.Oral cavity is a complex ecosystem, forming structurally and functionally organized dental biofilm embedded in a matrix of polymers of host and bacterial origins. Of clinical relevance is the fact that imbalanced microbial homeostasis in dental biofilms is associated with etiology of dental diseases; for this reason, research efforts have been made to target putative pathogens by antimicrobial or antiadhesive strategies to prevent disease initiation and progression . Althougμg/mL, bacterial growth of P. gingivalis and F. nucleatum was inhibited, while, at concentration 80 μg/mL, GO absolutely killed all S. mutans [A study conducted by He et al. evaluated the antibacterial activity of GO nanosheets against these three common types of bacteria and found that GO nanosheets were highly effective in inhibiting the growth of dental pathogens. At GO concentration 40. mutans . This co S. mutans. Scanning electron microscopy analysis revealed that there is a strong mechanical interaction between cells and GNPs, firstly involving cell trapping and consequent shrinking and secondly involving piercing through soft cell wall with sharp edges of GNP flakes which eventually killed the planktonic form of S. mutans [ S. mutans and E. faecalis, which infer to be due to the surface properties not electron diffusion of graphene material [ S. mutans and, consequently, caries, further proving the potential graphene hold for biomedical applicationsAnother graphene derivative, graphene nanoplatelet, produced via thermal exfoliation of graphite intercalation compound, was also investigated for their antibacterial properties against. mutans . CVA-gromaterial . These r S. mutans [Graphene, when combined with other compounds, showed improved synergistic antimicrobial, antibiofilm, and antiadherence activities against oral pathogens. Zinc oxide nanoparticles have been widely used in biomedical applications for its superior bactericidal effect, but its aggregation properties producing toxicity to mammalian cells have been hindering its use. When graphene material is synthesized with zinc oxide producing graphene/zinc oxide (GZNC) nanocomposite, it not only causes a much lower toxicity, but also forms a unique nanointerface to interact with microbes as compared to ZnO alone. It was observed that GZNC could decrease the synthesis of EPS, one of the key virulence factors of cardiogenicity, and reduce the amount of insoluble glucans, which influences biofilm formation by disturbing its physical integrity and stability, significantly reducing the biofilm and cariogenic properties. mutans .These antibacterial properties of GFNs could be very beneficial when integrating into biomaterials for potential clinical application. For instance, surgical sutures may be one of the most widely used medical adjunct nowadays and a good suture material should not only possess good mechanical properties, but also the antimicrobial ability to prevent breeding of bacteria. It was recently suggested that compared with conventional polyvinyl alcohol (PVA) fiber which has no antimicrobial property, PVA matrix dispersed with mechanically exfoliated graphene (MEG) showed high antibacterial effect toward gram-positive bacteria, thereby efficaciously accelerated the healing of wound, making PVA/MEG nanocomposite fiber a promising new candidate for surgical suture . in vivo bactericidal applications are still at initial stage; whether graphene has long-term and broad-spectrum antibacterial effects stay debatable. A recent study suggested that GO is not an intrinsic antimicrobial material but a general growth enhancer that can act as a biofilm that allows bacterial attachment and proliferation [Studies on graphene-based materials forferation . Neverthferation . AdditioTissue loss due to trauma, disease, or congenital abnormality is a major healthcare concern worldwide. The ongoing challenge of dental treatment is to restore missing teeth with their periodontal structure . There hScaffolds play a significant role in tissue engineering. Either in the absence or presence of chemical inducers and growth factors, it should be optimally designed to provide a biocompatible three-dimensional environment that can not only mechanically ‘support' and ‘guide' bone regeneration, but also ‘stimulate' proliferation and differentiation of stem cells into their specific tissue lineage . So far,β-catenin protein expression and activate catenin/Wnt signaling pathway, markedly increasing the degree of proliferation and differentiation of cultured cells, and led to acceleration of bone formation [Among graphene derivatives, GO, with many functional groups, has outstanding surface activities which can exert adsorptive capability to drugs, growth factors, and other biomolecules. Several in vitro experiments have demonstrated that pristine GO may upregulate ormation , 46. A sormation .β-tricalcium phosphate scaffold modified with GO significantly stimulated the proliferation and osteogenic differentiation of human bone marrow stromal osteoprogenitor cells and, importantly, accelerated new bone formation in vivo [Besides being used in their pristine form, they can be combined with different biomaterials. The resultant graphene modified scaffold presented enhanced bioactivity. Addition of GO to chitosan 3D scaffold's composition stimulated the interconnected pore structure, improved mechanical properties, and enhanced the bioactivity of the scaffold materials for osteogenesis . β-trica in vivo . GO appl in vivo .Dental pulp stem cells (DPSC) are self-renewing and multipotent cells which contain mesenchymal stem cells that can relatively easily obtained from the extracted teeth without esthetic damage . They maAnother stem cell from dental tissue that can be influenced by graphene scaffold is periodontal ligament stem cells (PDLSCs). They could be obtained from dental tissue and have shown multiple differentiation capacity and immunomodulation, capable of differentiating into both cementoblasts and collagen forming cells . A studyResearch has also been done to analyze the performance of Silk fibroin (SF) and GO in cell proliferation and mesenchymal phenotype expression of PDLSCs . AlthougOnly part of recent studies was included in this section; nevertheless, it could be speculated that graphene is one of the most promising biocompatible scaffolds for MSCs adhesion, proliferation, and differentiation, particularly toward the osteogenic lineage. Graphene alone with optimal concentration may spontaneously drive stem cells' osteogenesis, but this effect could be further promoted with presence of growth factors. For future engineering of graphene-based substrates for targeted biomedical applications, a deeper understanding of the ability of graphene to improve the biological properties of different scaffold materials will be essential for future biomedical applications.After dental implantation, fibroosseous integration took place between host biological system and dental implant. At the hard tissue interface, osteogenic properties of implant material are essential for osseointegration while at the soft tissue interface, to ensure a tight epithelial seal preventing bacterial invasion is obligatory . Leak ofEven though the alternative materials for dental implants have gained increasing interests, titanium or titanium alloy still remained popular to be the material of choice . Recent  S. sanguinis and S. mutans. Additionally, compared to titanium alone, graphene possesses osteogenic property enhancing the expression of osteogenic related genes RUNX2, COL-I, and ALP, boosting osteocalcin gene and protein expression, and consequently increasing the deposition of mineralized matrix [When graphene is coated on titanium substrate, the hydrophobic character of graphene film exerted self-cleaning effect on its surfaces decreasing the adhesion of microorganism includingd matrix . Attemptd matrix . These f S. mutans and P. gingivalis [Multiphase nanocomposite could also be a promising biomedical material to prevent implant-associated infection. As discussed in the previous session, the antibacterial properties of GO had been contradicting; hence a study conducted by Jin et al. added an antimicrobial nanomaterial, silver nanoparticle, to the GO-Ti composite. GO, with many carboxyl, hydroxyl, and carbonyl on its surfaces, are negatively charged, which could readily combine with positively charged Ag ions in the aqueous solution. Loading GO thin film and silver nanoparticles onto titanium exhibited excellent antiadherence and antimicrobial ability, especially towardngivalis . in vivo and possessed superior osseointegration in vitro. Furthermore, bonding strength between composite coating and titanium substrate of GO/CS/HA composite coating were enhanced compared with HA, GO/HA, and CS/HA coatings [Hydroxyapatite (HA) coating is widely applied as an osteoinductive modification of titanium implant; however its slow biological interaction mechanism and low mechanical strength restricted its application . Graphencoatings .β1 and initial adhesion of MSCs, indicating that graphene-functionalized NiTi allows better MSCs cytoskeleton development and spontaneous osteogenic differentiation [The direct fabrication of graphene on NiTi based dental implant using chemical vapor deposition technique upregulated the expression level of osteogenic related genes  and promoted expression of integrin ntiation .Aside from that, GO can be used as a carrier for BMP-2, an osteoinductive, and Substance P, a MSC recruitment agent. Application of GO on titanium allowed dual delivery of SP and BMP-2, showing the greatest new bone formation on Ti implant in the mouse calvaria . HoweverDespite all the excitement, developing a universally accepted transfer route of graphene to titanium implants remains to be a challenge as the deposition technique should ensure transfer effectiveness without compromising the surface properties graphene possess. Several transferring methods have been developed in the market such as wet method, electrochemical delimitation, thermal release tape, hot press transfer, and roll to roll; these methods may allow large scale transfer of graphene onto planar substrate, but are poorly suited for transferring onto 3D objects such as dental implant. Recently, Morin et al. described a vacuum-assisted dry transfer method which is to coat graphene onto the target object and maintained by a mound to sustain conformation. Using a pressure differential provided via partial vacuum, uniform force is applied to the surface facilitating successful coating of graphene onto the object without changing graphene characteristics .Graphene and its derivatives when coated on titanium implant have remarkable abilities to improve properties of titanium, enabling binding of biomolecules, and induce osseointegration. These characteristics place them under the spotlight for improvement and modification of implant materials. E. faecalis and S. mutans, but also improved the stability and bioavailability of ICG, preventing its degradation and aggregation. [The purpose of root canal treatment is to biomechanical clean an infected root canal system to destroy intracanal pathogens, decontaminate residually infected tissue, and avoid further intraoperative/postoperative infection. The main cause of endodontic failure is persisting infection in the root canal . Recentlegation. .For many years, sodium hypochlorite has been used as the most common intracanal irrigants for its strong antibacterial and tissue-dissolving abilities. However, sodium hypochlorite extrusion during root canal treatment causes acute immediate symptoms and serious potential sequelae including rapid hemolysis and ulceration of surrounding tissues, destruction of endothelial, and fibroblast cells . IncorpoBioactive cements have been widely used in endodontics for management of perforation, retrograde root filling, and pulp capping. Among them, Biodentine (BIO) and Endocem-Zr (ECZ) are considered as the safest cements that exhibits the least discoloration and calcification of tooth, but with shortcomings such as high pull-out bond strength, long setting time, and modest mechanical properties. With 3 wt % addition of graphene nanosheets, the setting time of both cements significantly decreased, which could be explained by the role of carbon based materials to act as a matrix for the development of C–S–H and calcium hydroxide, thereby reducing the induction period and accelerating the hydration process . HoweverAnother cement, calcium silicate (CS), well known as mineral trioxide aggregate (MTA), has become popular in endodontic treatment in recent years. It is a hydration product of Portland cement, which has the capacity to upregulate differentiation of odontoblasts and promote calcium phosphate deposition . HoweverIncorporating GO into Portland cement also exhibited positive influence on the workability of the cement. It enhanced the degree of hydration by increasing the nonevaporable water content and calcium hydroxide hydroxide content, contributing to the refinement of pore structures, and subsequently increasing the compressive strengths and suppressing crack propagation in the matrix at nanoscale. Introduction of 0.03% by weight GO into cement paste can increase their compressive strength and tensile strength by more than 40% .In conclusion, addition of graphene into dental cements leads to refinement of pore structure which not only strengthens the cement material but also blocks the entry for possible bacterial invasion. It could be promising reinforcing cementitious material for future dental applications.Glass ionomer (GI), with favorable coefficient of linear thermal expansion, ability to chemically bond to sound tooth structure, and dynamic fluoride release, had been utilized in a wide range of clinical application. However, its poor physiomechanical properties remained to be a concern despite the developments in GI constituents with addition of various filler types including fibers, metallic powders, and hydroxyapatite powders . In rece S. mutans without interfering its original mechanical performances and without producing a surplus of ROS [Due to the presence of microcavities between the healthy tissue and dental restoration causing bacteria invasion, there has been an increasing interest in development of antibiofilm adhesives. One concern with dental adhesive monomers is its excessive ROS production which not only cause oxidative stress associated toxicity toward fibroblasts and pulp cell, but also affect saliva redox equilibrium and decreasing natural oral immune system defenses . Due to s of ROS .μg/mL) on the viability and metabolic activity of dental pulp mesenchymal cells. The result showed that GO coating at the higher concentration induces PGE2 secretion, controls inflammation, and promotes DPSCs differentiation which is probably due to GO's large reactive surface area providing idea platform for biofunctionalization and concentrating chemical, proteins, and growth factors for faster differentiation [In treatment for periodontal bone defects using guided tissue regeneration (GTR) and guided bone regeneration (GBR), barrier membranes have been a crucial biomaterials, which is to create a secluded space between soft connective tissue and regenerating bone, for formation of unimpeded bone promoting faster differentiation of mesenchymal cells into odontoblast/osteoblast . Attemptntiation . An animntiation . in vitro and in vivo. Then, we presented a comprehensive summary of the latest research progress related to the potential applications of GFNs in dentistry (see Researches toward graphene in dental materials mainly focused on two ways: one is to prepare new dental materials as GFNs alone, and the other is to modify the common dental materials by transferring appropriate GFNs onto different substrates. As clearly highlighted in this paper, in either way, GFNs improved the physical, chemical, and mechanical properties of biomaterials, holding enormous potential in new therapeutic strategies in dental field. In this review, we firstly discussed the toxicity and antibacterial properties of GFNs on cellsstry see . in vitro and in vivo, but no studies have been done on human subjects and in-depth studies are still scarce in oral settings. GFNs is a rather fascinating material worthy of in-depth investigation; further exploration on their underlying mechanism of interaction toward oral tissues in terms of cell-signaling, metabolic pathway, osteogenesis, and antibacterial effects is needed. We hope that this review article could provide some valuable elicitation for the future scientific and technological innovations of graphene in dentistry.Safety and potential risks of GFNs should be emphasized and research efforts should be made to ensure harmless use of graphene in oral environment. Additionally, some very promising properties of GFNs have been extensively investigated in other organs of human body


Perovskite solar cells (PSCs) have raised research interest in scientific community because their power conversion efficiency is comparable to that of traditional commercial solar cells . Apart from that, PSCs are lightweight, are flexible, and have low production costs. Recently, graphene has been used as a novel material for PSC applications due to its excellent optical, electrical, and mechanical properties. The hydrophobic nature of graphene surface can provide protection against air moisture from the surrounding medium, which can improve the lifetime of devices. Herein, we review recent developments in the use of graphene for PSC applications as a conductive electrode, carrier transporting material, and stabilizer material. By exploring the application of graphene in PSCs, a new class of strategies can be developed to improve the device performance and stability before it can be commercialized in the photovoltaic market in the near future. Introduction of graphene improves photovoltaic properties of perovskite solar cells (PSCs).Graphene can be used as a conductive electrode, carrier transporting material, or stabilizer material.Graphene enhances the electrical properties and stability of PSCs.2 [Global climate change  and rapi2 . However2 . On the 2 , 6.2 [To overcome these problems, researchers need to explore new materials for next-generation photovoltaics. At present, perovskite solar cells (PSCs) have generated broad interest because of their rapid PCE improvement from 3.8% in 2009 to 22.1% in 2016 , as show2 . Thus, PTo date, several strategies such as electrode modification –21, metasp2 hybridized carbon atoms tightly packed into a 2D honeycomb crystal lattice. It can be stacked to form three-dimensional (3D) graphite, rolled to form one-dimensional (1D) nanotubes, and wrapped to form zero-dimensional (0D) fullerenes. Figure 3 W m−1 K−1) at room temperature [5 cm2 V−1 S−1) [2 g−1) [It is well known that graphene is a name given to a two-dimensional (2D) sheet of ensional D nanotubperature , high chV−1 S−1) , high opV−1 S−1) , large s [2 g−1) , superio [2 g−1) , and hig [2 g−1) , catalys [2 g−1) , optical [2 g−1) , surface [2 g−1) , and opt [2 g−1) , 52.Here, we present the successful application of graphene for high-performance and stable PSCs. Firstly, we introduce the crystal structure and tolerance factor of organic–inorganic perovskite materials. Secondly, we briefly introduce the working principle of PSCs. Thirdly, we highlight the progress of applying graphene for high-performance and stable PSCs. Finally, we provide the outlook and conclusion on the application of graphene for PSCs.3 [3 crystal structure, where A represents an organic and/or inorganic cation such as methylammonium (CH3NH3+ or MA+), formamidinium (NH(CH3)2+ or FA+), or cesium (Cs+), B represents a divalent metal cation (Pb2+ or Sn2+), and X represents a halide anion  [3 type are shown in Fig. 3 perovskite structure, the tolerance factor, t = (RA + RX)/{√2(RB + RX)} of perovskite materials should be close to 1 (corresponding to a perfectly packed perovskite structure), where RA, RB, and RX are the effective ionic radii for A, B, and X ions, respectively.The term “perovskite” is used when referring to a large compound group that has the same crystal structure as mineral perovskite CaTiO3 . For per, or I−) , 54. Thet < 0.8, cubic structure if 0.8 < t < 1.0, and hexagonal structure if t > 1 [2+, Sn2+, Cl−, Br−, and I− are 1.19, 1.18, 1.84, 1.96, and 2.20 Å [t < 1.0  and halide anions . As a result, the working principle of PSCs has raised a number of questions because the optical absorption of the perovskite layer cannot be distinguished. It should be noted that at the beginning of its discovery, PSCs were TiO2-sensitized solar cells, using MAPbI3 and MAPbBr3 as sensitizers [2 inorganic semiconductor, followed by electron extraction to the transparent conductive oxide (TCO). The electrons would flow through the external circuit to the platinum (Pt) anode and then into the iodide electrolyte. The electrolyte would regenerate and transport the electrons back to the dye molecules. Therefore, it has been proposed that dye-sensitized solar cells (DSSCs) can be used as a model to explain the working mechanism of PSCs. Because of the dissolution issues of halides in a liquid electrolyte that could influence the stability of PSCs [In general, the perovskite layer is composed of organic materials such as organic cations  and inorganic materials such as metal cations  semiconductor. Owing to the low binding energy of perovskite materials , th−1) of single-layer CVD-produced graphene films, a thin layer (~ 20 nm) of poly-:poly(styrenesulfonate) (PEDOT:PSSS) solution doped with fluorosurfactant Zonyl-FS300 together with D-sorbitol was spin-coated on top of the graphene surface to (1) reduce the graphene sheet resistance (2) act as an adhesion layer during the lamination process, and (3) induce more hole doping to the graphene electrode, since the Fermi level of PEDOT:PSS is higher than the graphene Dirac point. After optimizing the processing condition, the device performance with double-layer graphene films could achieve up to 12.37% efficiency, which is relatively high compared to that of the reported semitransparent TCO-free PSCs [Jsc of the champion device originated from the low sheet resistance  hole transporting material (HTM) on top of the graphene electrode surface, the device performance could be improved to achieve efficiencies of up to 17.1%. The dramatic increase in the device performance was attributed to (1) the use of MoO3 HTM, which provided hydrophilicity to the graphene surface see Fig. d, e.Fig./MoO3/PEDOT:PSS/MAPbI3/C60/BCP/LiF/Al) and evaluated its operational stability against repeated bending. Under strain-free conditions, the device with a graphene conductive electrode exhibited a PCE of 16.8%, which was kept within ~ 90% of the initial value after it had been bent 1000 times at R = 6, 4, and 2 mm , the graphene-MoO3/PEN substrate showed a higher sheet resistance of 552.0 ± 24.2 Ω sq−1, which could reduce the charge collection efficiency of the device, leading to a high series resistance, low shunt resistance, and low the fill factor (FF). However, both devices showed similar Jsc values; this can be attributed to the use of the graphene-based substrate exhibiting higher transmission (~ 97% transmittance) compared to the PEN/ITO-based substrate (~ 89% transmittance) over the visible wavelength region.Later, Choi et al.  used the3 (≈ − 5.4 eV) than that of conventional HTMs such as PEDOT:PSS (≈ − 5.0 eV), which can facilitate hole transfer to the anode. Furthermore, it can also enhance the device stability owing to the hydrophobic nature of P3HT that does not absorb moisture in air [R = 0.670, 0.365, 0.175, and 0.130 cm) with 14% degradation in device performance at the bending radius of 0.175 cm after 500 cycles [3)-doped graphene anode surface could extend the diffusion length of holes to the graphene electrode from ~ 210 nm (control device) to ~ 370 nm (champion device), where the champion device performance was enhanced by 42.1% compared to the control device.On the other hand, Liu et al.  demonstre in air , 74. Aft0 cycles . Later, 0 cycles  demonstr2 nanocomposite as the ESCL and demonstrated the enhancement of the device performance (PCE = 15.6%) by 56.0% compared to the device with pure TiO2 thin films (PCE = 10.0%).Graphene has also been used as a carrier transporting material for PSC applications. For example, Chandrasekhar et al.  demonstr2 layers, the authors performed impedance spectroscopy characterization under working cell conditions and observed that the addition of graphene into TiO2 thin films could effectively reduce the series resistance of the device , Wang et al. [2/graphene-SrTiO3/MAPbI3/Spiro-OMeTAD/Ag. The authors showed that graphene could enhance the electron transfer rate. As a result, the optimized device exhibited a PCE of 10.49%, which was enhanced by 53.1% compared to that of a control device with a PCE of 6.85%.To evaluate the role of graphene addition into TiOsee Fig. e and chasee Fig. f. In addg et al.  demonstr2/graphene-doped mesoporous TiO2/MAPbI3/graphene oxide/Spiro-OMeTad/gold. The high efficiency of the device was mainly attributed to the improved charge carrier injection/extraction and the device stability [To date, the highest efficiency of graphene-based PSCs has been 18.19%, which was reported by Agresti et al. , with antability .In addition, it is well known that perovskite thin films can be easily hydrolyzed and decomposed from dark brown into yellowish thin films under humid air environment see Fig. a, b. TheIn order to solve the problem, deposition of an additional layer on top of the perovskite layer was proposed, using either a graphene layer  or dopedJsc, Voc, FF, and PCE of the devices with (without) graphene on top of the perovskite/Spiro-OMeTAD thin films are 21.1 (21.6) mA cm−2, 1.09 (1.07) V, 0.682 (0.718), and 15.7 (16.6) %, respectively. Bi et al. [For example, Hu et al.  reportedi et al.  reportedIn conclusion, graphene has been used as a conductive electrode, carrier transporting material, and stabilizer material for PSC applications Table . So far,In comparison with conventional TCO-based electrodes such as FTO and indium-doped tin oxide (ITO), graphene electrodes show high flexibility and good physical, chemical, and thermal stability. Therefore, the PCE and lifetime of the devices based on graphene electrodes are much better than those of the devices based on TCO electrodes. Apart from that, graphene can enhance the electrical properties of the devices and crystallinity of the perovskite films if graphene is n-doped into metal oxide layers to act as a carrier transporting material.To further improve the stability of PSCs, it is suggested to add a layer of graphene on top of the perovskite/hole transporting layer thin film. We believe that graphene will play an important role in PSCs in the near future. Therefore, development of novel layered graphene in different layer designs of PSCs with a controlled mechanism will be needed.We propose to utilize oxidized or reduced graphene, in addition to pristine graphene, for PSC applications. We believe that, by optimizing the deposition process in terms of time, temperature, solvent, precursor selection, etc., new designs of PSCs with good performance and stability can be developed. Once graphene-based materials have proved to be useful in photovoltaic technologies, low-cost PSCs can be realized and commercialized in the market. Further, other optoelectronic devices will be designed follow this trend, initiating the era of low-cost optoelectronic devices.


In the field of nanomechanics, parametric excitations are of interest since they can greatly enhance sensing capabilities and eliminate cross-talk. Above a certain threshold of the parametric pump, the mechanical resonator can be brought into parametric resonance. Here we demonstrate parametric resonance of suspended single-layer graphene membranes by an efficient opto-thermal drive that modulates the intrinsic spring constant. With a large amplitude of the optical drive, a record number of 14 mechanical modes can be brought into parametric resonance by modulating a single parameter: the pre-tension. A detailed analysis of the parametric resonance allows us to study nonlinear dynamics and the loss tangent of graphene resonators. It is found that nonlinear damping, of the van der Pol type, is essential to describe the high amplitude parametric resonance response in atomically thin membranes. A mechanical system can be parametrically excited when its stiffness is modulated at a frequency of 2ω0/n, where ω0 is the system’s resonance frequency and n an integer2. Above a certain modulation amplitude, the system becomes unstable and exhibits parametric resonance. The advent of micro and nano engineering brought to life new ideas for exploiting parametric excitation for enhancing force and mass sensitivity8, effective quality factor9, and signal to noise ratio3 of tiny resonators. To date, many sensors, including gyroscopes12, mass sensors8 and even mechanical memories16 employ parametric excitation for improved performance.The history of parametric oscillations dates back to the 19th century and the observation of surface waves in the famous singing wineglass experiment of Michael Faraday20. They are promising candidates for various sensing applications26 due to their ultra high surface to mass ratio, combined with their high strength27. However, the quality factor of resonance is relatively low compared to other nano-electromechanical systems28, limiting their accuracy as a resonant sensing element. It is thus of interest to apply parametric amplification schemes to raise their effective quality factor and improve their performance. Several works have successfully demonstrated such an amplification scheme by applying an electrostatic spring force to the membrane and modulating its strength30. It is well known that above a certain critical force of this parametric pump, the device will become unstable and exhibit parametric resonance32. Although such behavior has been previously observed, the nonlinear dynamics involved in parametric resonance has received less attention. We demonstrate that parametric resonance holds important information about the nonlinear damping of graphene that has been a subject of strong debate in the community36.Resonators employing two-dimensional materials such as graphene or molybdenum disulfide have attracted considerable interest in the scientific community14. The origin of the effectivity of graphene for parametric resonance is the large tension modulation that can be achieved by opto-thermal means22, which is related to the large Young’s modulus of graphene. Understanding parametric resonance is of fundamental interest, but also provides an interesting alternative to direct excitation in future applications, that could reduce noise and facilitate large amplitude driving in resonators and oscillators. The parametrically excited nonlinear mechanical response is analyzed and a model is proposed that can simulate both parametric and directly driven responses. This nonlinear Duffing response, caused by the direct drive, has previously been studied to obtain the stiffness properties of graphene devices38. In this work, we focus the analysis on dissipation mechanisms in graphene. Period doubling bifurcations are almost fully governed by the linear dissipation terms, while the saddle node bifurcation of the parametric resonance is fully governed by nonlinear dissipation terms32. From this analysis, we can conclude that nonlinear damping in graphene can be accurately described by a dissipation term of the van der Pol type. Comparing this to the cubic stiffness term allows us to extract the mechanical loss tangent of graphene which is orders of magnitude larger than expected. Tracking the period doubling bifurcations shows that the region of instability is asymmetric. This unexpected deviation from the theoretical response suggests that unconventional dynamic phenomena are governing the linewidth of graphene resonators.In this work it is demonstrated that opto-thermal tension modulated single layer graphene is an ideal system to study parametric resonance. Despite the relatively low Q-factor of the graphene resonances (<1000), it is shown that a record number of 14 modes can be brought into parametric resonanceμm and a cavity depth of 300 nm. The drums have venting channels to the environment to prevent the trapping of gas in the cavity  graphene drum resonators with a diameter of 5 ity Fig. , see Met39. The ratio of the AC voltage amplitude generated by the photodetector and the AC driving voltage of the blue laser Vac,out/Vac,in is determined by a vector network analyzer (VNA). Homodyne and heterodyne detection can be performed in this setup, such that both direct and parametric resonances can be analyzed.A red helium-neon laser is used to read out the motion by the optical interference between the graphene membrane and the fixed substratef, while detecting the photodiode signal at f. When increasing the blue laser driving voltage Vac,in a remarkable effect is observed. One-by-one, the parametric resonances of graphene appear, up to 7 different modes. Each mode reaches resonance at a different threshold driving amplitude Vac,in, due to differences in quality factor and the frequency dependence of the parametric driving parameter δ40. The experiment is repeated on a different drum in Fig. Vac,in = 382.7 mV (RMS) between the second and third resonance. Moreover, in some cases transitions between the high-amplitude and low-amplitude solutions are observed, e.g. at Vac,in = 489.6 mV (RMS) between the same 2 modes. This random process is attributed to a strong dependence of the basin of attractions of the parametric high-amplitude and low-amplitude solutions on the initial conditions41. Hence, the amplitude can fall into two stable solutions: either the high amplitude solution of the third mode or the zero amplitude solution of the third mode which is also observed at higher driving amplitudes ).In Fig. 14.Due to the overlap of parametric resonances in this drum, some resonances are skipped and not all resonances are found by sweeping from low to high frequency. Instead, when sweeping the frequency backward as shown in Fig. Vac,out is detected at half of the driving frequency Vac,in. Similar to the directly driven case, a hysteresis occurs between the forward , μ is the damping coefficient, ν the nonlinear damping coefficient, β the linear stiffness coefficient, γ the nonlinear stiffness coefficient, δ cos ωt the parametric driving and F cos ωt the direct driving term. By setting γ = 0 and ν = 0 one can fit the direct response at low drive level  and van-der-Pol terms (ν) in our resonators. As shown in the Supplementary Information δl at the resonance frequency can be determined from the ratio of these terms, tan δl = ν/γ. From the values of the fits we obtain tan δl = 0.34 for drum 2 and tan δl = 0.15 for drum 3. The values of these loss tangents are in the same range as found by Jinkins et al.45. The obtained values for the loss tangent are relatively high for a bulk crystalline material, therefore the observed loss tangent is likely dominated by effects related to the atomic thickness of graphene, such as thermodynamic fluctuations46, sidewall adhesion47 or unzipping of wrinkles48.The fit to the nonlinear response of the membrane allows us to extract a number for the Duffing (n0(t) is given by Δn0 = αE2DΔT/(1 − ν), where α is the thermal expansion coefficient, E2D the 2D Young’s modulus, ν the Poisson ratio and ΔT the temperature modulation. Using approximate values from literature49, one finds that Δn0(t) ≈ 0.003ΔT Nm−1 K−1, which means that a temperature modulation of 1 K already results in a tension modulation of the order of the intrinsic pre-tension n0 (estimated to be between 0.003 N/m and 0.03 N/m40) of the graphene membranes studied here. One can define the relative shift of the resonance frequency per unit of temperature as a figure of merit for the efficiency of the opto-thermal parametric drive: −1. This estimated value for graphene is 500–5000 times larger than in other optically excited oscillators50. From the fits in Fig. T lies between 0.13 K and 1.5 K. These moderate temperature modulations illustrate that the parametric driving scheme for graphene membranes is a very efficient method for reaching parametric resonance.Here we discuss the efficiency of the tension modulation for parametric excitation of the graphene membrane. The tension modulation Δ51. (2) Inertial imaging52, where accurate tracking of multiple resonances allows one to determine the mass, location and shape of a particle on top of a resonator, which has applications in biotechnology53. (3) Parametric oscillators can also be used to build a binary information and computation system14, where information is stored in the phase of the resonator. Multi-mode resonators have the potential of enabling parallel processing and data storage. The high resonance frequencies and relatively low Q of the graphene membranes can increase computation speed.Multi-mode parametric oscillators are interesting for applications where accurate frequency tracking of multiple modes is necessary. We list here three potential applications. (1) Radio receivers in the MHz range where multiple radio channels need to be monitored and received simultaneously to maximize data rates, or to allow seamless switching between channels without having to tune the channel2.A unique feature of the demonstrated graphene system is that all of these modes can be simultaneously parametrically amplified via tension modulation. The use of parametric amplification therefore has the advantage that no feedback loops or special filters or actuation schemes are needed to select the desired resonance mode. Moreover, parametric amplification effectively results in an amplitude dependent gain, which can be used to generate higher output signals than with a constant gain. Moreover, since the driving frequency is double the readout frequency, parametric driving is less sensitive to cross-talk that is often hampering resolving signal detection in directly driven resonatorsIn conclusion, we report on multi-mode parametric resonance and amplification in single layer graphene resonators by an opto-thermal tension modulation technique. It is demonstrated that the tension-dominated restoring force results in parametric excitation of multiple resonance modes in the system when the system is opto-thermally driven. The parametrically and directly driven resonances are compared to a single degree-of-freedom model based on the Duffing, van der Pol and Matthieu equations, with good agreement at low driving levels. This allows simultaneous determination of nonlinear stiffness and damping coefficients and results in a high-frequency determination of graphene’s mechanical loss tangent. Graphene resonators are thus an interesting platform to study parametric excitations and their utilization for sensors with improved performance.2 layer on a silicon wafer. The etching did not fully stop at the silicon layer, resulting in cavities that are 300 nm thick. Circular membranes are formed by transfer of single layer CVD graphene  has a wavelength of 405 nm and is biased with a 32 mA current, resulting in 0.76 mW of incident power measured before the objective. The red laser illuminates the sample with 1.2 mW of incident power (measured before the objective lens). The vector network analyzer is of type Rohde & Schwarz ZNB4 with the frequency conversion option (k4) installed. The frequency conversion option of the VNA enables both homodyne and heterodyne detection, such that both direct and parametric resonances can be detected.All measurements are performed at room temperature in a high vacuum environment with a pressure less than 2 × 10The authors declare that all the data in this manuscript are available upon request.Supplementary Information: Opto-thermally excited multimode parametric resonance in graphene membranes


Tooth germs undergo a series of dynamic morphologic changes through bud, cap, and bell stages, in which odontogenic epithelium continuously extends into the underlying mesenchyme. During the transition from the bud stage to the cap stage, the base of the bud flattens and then bends into a cap shape whose edges are referred to as “cervical loops.” Although genetic mechanisms for cap formation have been well described, little is understood about the morphogenetic mechanisms. Computer modeling and cell trajectory tracking have suggested that the epithelial bending is driven purely by differential cell proliferation and adhesion in different parts of the tooth germ. Here, we show that, unexpectedly, inhibition of cell proliferation did not prevent bud-to-cap morphogenesis. We quantified cell shapes and actin and myosin distributions in different parts of the tooth epithelium at the critical stages and found that these are consistent with basal relaxation in the forming cervical loops and basal constriction around enamel knot at the center of the cap. Inhibition of focal adhesion kinase, which is required for basal constriction in other systems, arrested the molar explant morphogenesis at the bud stage. Together, these results show that the bud-to-cap transition is largely proliferation independent, and we propose that it is driven by classic actomyosin-driven cell shape–dependent mechanisms. We discuss how these results can be reconciled with the previous models and data. Tooth formation is a well-established model for epithelial-mesenchymal signaling interactions and for ectodermal organ formation and organogenesis in general . HoweverThe next step of tooth germ morphogenesis is the transition from the bud stage to the cap stage. The base of the bud flattens, forming epithelial bends known as cervical loops on either side . AlthougDuring the bud-to-cap transition, the surrounding mesenchyme condenses, forming a capsule. This led to ideas that epithelial proliferation within the constraining mesenchymal capsule drives the epithelium to buckle to form the cervical loop and cusps . SupportAlthough epithelial buckling due to proliferation within a constraining structure is known in other contexts Luo) mice (Jackson Laboratories 007576) were euthanized by cervical dislocation.Animals were handled under UK Home Office licensing and King’s College London Ethics Committee approval. Pregnant wild-type CD1 mice and mT/mG (Gt(ROSA)26Sor2 humidified atmosphere. Explants were treated with aphidicolin ; 10μM BrdU was added after 3 or 23 h; and explants were fixed at 5 or 25 h in 4% paraformaldehyde (PFA) for 4 h at room temperature. Explants were photographed under a stereo zoom dissection microscope with brightfield optics at the 3- and 23-h time points . For FAK inhibition, explants were treated with 1µM PF-573228  in place of aphidicolin.Frontal slices containing tooth germs of E13.5, E14.0, and E14.5 mandibular molars were obtained as described . BrieflyFor mT/mG cellular/nuclear morphometric analyses or actin/myosin staining, whole embryos were fixed in 4% PFA for 4 h at room temperature. After a phosphate-buffered saline (PBS) wash, heads were embedded in 0.49 g of gelatin , 30 g of albumin, 20 g of sucrose, and 3.5 mL of glutaraldehyde in 100 mL of PBS. Gelatin blocks were refixed in 4% PFA at 4 °C overnight and frontally sliced on a vibratome  at 90-µm thickness.For BrdU staining, antigen retrieval (DNA denaturation) was performed with 10mM sodium citrate (pH6) at 95 °C for 20 min, permeabilized in 0.1% Triton X-100  in PBS (PBST) for 3 × 10 min, then blocked with 20% goat serum  in PBST for 30 to 60 min at room temperature. For P-cadherin staining only, 20% donkey serum  was used instead of goat. Specimens were incubated with primary antibodies at 4 °C overnight. Primary antibodies were as follows: anti-RFP , goat anti-P-cadherin , rabbit anti-non-muscle myosin IIB , and rat anti-BrdU . After six 1- to 2-h PBST washes, specimens were incubated at 4 °C overnight with Alexa Fluor–conjugated secondaries (Life Technology). Nuclei and F-actin were counterstained with DAPI  and Alexa Fluor 488/635 Phalloidin , respectively. Specimens were washed 6 times with PBST (1 to 2 h per wash) and then mounted on glass slides with 50% glycerol  in PBS. Z-stacks were acquired by a confocal microscope  with oil immersion 40× and 63× objectives.Appendix Fig. 1). Landmark coordinates were entered into the geometric morphometrics package MorphoJ . Sample numbers are in the Appendix Table.Cellular/nuclear measurements used 40× z-stack images (0.21-μm steps) of tooth germs from E13.5 to E15.5 mT/mG mice. Only basal cells not undergoing mitosis  were measured. In E13.5, the corners in the bottom one-third of the tooth bud were considered cervical loops. Cells were measured in the optical slice containing their maximum cross-sectional area to avoid grazing artifacts. Morphologic features were measured with Fiji tools. Nuclear position was defined as follows: (distance between centroid and cell base midpoint) / cell height. Basal width was defined as the width of attachment to the basal membrane, and apical width was measured perpendicularly to the cell axis at a site 20% below the cell apex  was measured in Fiji.t test. Comparison of multiple embryonic stages in cellular/nuclear measurements was analyzed by analysis of variance only (no post hoc tests). For cell division orientation, Mardia-Watson-Wheeler tests  were performed with R 3.5.0 . Comparison of tissue dimensions in the proliferation inhibition experiment was by Student’s  R 3.5.0  and the  R 3.5.0 . P < 0.0Appendix Fig. 3). Unexpectedly, despite the size difference, the epithelial shape changes were remarkably normal: nonproliferating tooth germs underwent the transition from smoothly rounded buds at E13.5 to more or less triangular shapes by E13.5 + 23 h  between control and proliferation-inhibited shape. This strongly suggests that the models for bud-to-cap morphogenesis that depend exclusively on differential proliferation do not reflect events in vivo. This finding raised the following question: if not proliferation, what does drive the bud-to-cap transition?To directly test the proposition that tooth bud-to-cap morphogenesis is caused by differential proliferation alone, we applied a proliferation inhibitor, aphidicolin, to mouse molar tooth explants. Such explants faithfully recapitulate normal morphogenesis in culture . Prelimi5 + 23 h , B and f5 + 23 h . Shape c5 + 23 h . Qualita5 + 23 h  to compaMost epithelial bending mechanisms involve cell shape changes from columnar to wedge shaped . To findAppendix Fig. 4A, B). At E14.0, there was a slight enrichment of myosin throughout the flattening bottom of the tooth germ (the prospective IDE and enamel knot), smooth basally and punctate, and co-localized with actin apically . Taken together, these results show 1) basal actin and myosin enrichment coincident with basal contraction, indicating basal constriction in the IDE cells, and 2) slight basal depletion and apical elevation of myosin in the outer cervical loops, suggesting some basal relaxation and apical constriction there.To determine whether the observed cell shape changes were driven by active actomyosin mechanisms, we used fluorescent phalloidin to detect filamentous actin and immunofluorescence against myosin light chain, followed by confocal imaging, noting that mechanical tension is well correlated with total myosin levels in other systems . OccasioAppendix Fig. 3B vs. 3A), but they did incorporate BrdU  by differential cell proliferation, which has been proposed in previous models. Although there are caveats with explant experiments and the use of inhibitors, the persistence of morphogenesis during well-controlled inhibition of proliferation is hard to explain away as an artifact of explantation or off-target inhibitor effects. Meanwhile, the proliferation inhibition was well controlled: initiated before any shape change, persisting to the end of the experiment, and resulting in complete growth arrest. Although a previously published proliferation-driven model  matched Drosophila, tension is quantitatively associated with total myosin (We investigated alternative mechanisms that are based on cell shape change. Such changes were invisible to previous computational models because they treated the tissue as a continuous material or as being made of cells in the form of undeformable particles. Myosin was enriched where the cell domains got smaller, and it was depleted where they expanded, indicating active cell-autonomous mechanisms, especially during basal contraction in IDE adjacent to the enamel knot. In l myosin , and we l myosin  and suggCan our results be reconciled with proliferation-based models and experiments? Mechanosensing can regulate proliferation, so the latter could easily be downstream of the cell-autonomous shape changes that we describe. Proliferation would thus facilitate normal morphogenesis even if not required for it. Additionally, while our proliferation-inhibited explants showed largely normal morphogenesis, subtle changes in the cell sizes and lineage distributions may have compensated for the lack of differential proliferation. Furthermore, we analyzed a narrow stage range, and it is possible that differential proliferation becomes increasingly important in later cap, bell, and cusp formation.We focused on epithelially autonomous mechanisms, but experiments in which removal of the condensing mesenchyme caused the cervical loops to splay outward at mid- to late cap stages have shown clearly that physical constraint by the surrounding mesenchymal condensation is important for correct morphogenesis. This is perfectly compatible with our interpretations, since those experiments were conducted at slightly later stages and mesenchyme removal reoriented the cervical loops but did not relax them or the epithelial bends adjacent to the enamel knot . A combiThe tooth germ is an outstanding model for developmental organogenesis and has played a major role in understanding gene action and epithelial-mesenchymal signaling. Its morphogenesis is no less interesting, and our identification of basal relaxation and constriction in the cervical loops and IDE, respectively, is a first step toward integrating genetic and signaling aspects with the physical cell behaviors that make this organ.S. Yamada, contributed to design, data acquisition, analysis, and interpretation, drafted and critically revised the manuscript; R. Lav, contributed to design, data acquisition, analysis, and interpretation, critically revised the manuscript; J. Li, contributed to conception, design, data acquisition, critically revised the manuscript; A.S. Tucker, contributed to design and data interpretation, critically revised the manuscript; J.B.A. Green, contributed to conception, design, data analysis and interpretation, drafted and critically revised the manuscript. All authors gave final approval and agree to be accountable for all aspects of the work.Click here for additional data file.Supplemental material, DS_10.1177_0022034519869307 for Molar Bud-to-Cap Transition Is Proliferation Independent by S. Yamada, R. Lav, J. Li, A.S. Tucker and J.B.A. Green in Journal of Dental Research