O(10 km), associated with bands of near-inertial wave/diurnal internal tide shear of high vertical wavenumber in the upstream Kuroshio. The turbulent kinetic energy dissipation rates within these turbulent layers are >O(10−7 W kg−1), and estimated vertical eddy diffusivity shows >O(10−4 m2 s−1) on average. These results suggest that the high vertical wavenumber near-inertial waves propagating in the upstream Kuroshio could have large impacts on the watermass modifications, momentum mixing, nutrient supply, and associated biogeochemical responses in its downstream.The upstream Kuroshio flows through Okinawa Trough and the Tokara island chain, the region near the continental shelf of the East China Sea and shallow seamounts, where the Kuroshio can induce strong mixing over the shallow topography. Also, tidal currents over the rough topography may produce internal tides, and associated turbulence. The previous observations show energetic high vertical wavenumber near-inertial wave shear in the Kuroshio thermocline, which implies strong turbulent mixing. However, direct turbulence measurements in this region are very scarce. Using high lateral resolution (1–2 km) direct turbulence measurements, we show here, for the first time, that strong turbulent layers form spatially coherent banded structures with lateral scales of > Because the Kuroshio is inherently an ocean dynamic front, where the associated jet flows along the interface between seawaters of different water properties2, mixing processes in the Kuroshio Front play very important roles in watermass formation, transformation, and subduction5. In addition to this physical or hydrographical important role, the mixing in the Kuroshio has profound implications for biogeochemistry because the Kuroshio carries waters of relatively high nutrient concentrations from the tropics through its subsurface layers, as known as the nutrient stream7 similar to the Gulf Stream11. Earlier studies in the Gulf Stream9 and relatively recent studies in the Kuroshio13 have reported that elevated nutrient concentrations are found along these western boundary currents in their subsurface layers compared to that in the ambient waters of the same density. Because along-isopycnal stirring by mesoscale eddies tends to homogenize any tracer anomaly on the density surface in the ocean interior14, the elevated nutrient concentrations at the subsurface of the Kuroshio are most likely formed by across-density/diapycnal fluxes by irreversible diabatic turbulent diffusion. A previous study9 speculated that the turbulent diffusive nutrient flux by geostrophic vertical shear contributes to the concentration of elevated nutrients below the Gulf Stream. However, a limited number of direct turbulence observations at that time did not provide the supporting evidence for this hypothesis17.The Kuroshio, the western boundary current of the subtropical gyer in the North Pacific, has been known to transport large amounts of heat and salt from the tropical ocean20. While a number of previous studies have reported that the beams of the M2 internal tide emanated from the rough topography induced strong turbulence in close proximity to canyons and seamounts22, recent shipboard and lowered Acoustic Doppler Current Profiler (ADCP) measurements in the upstream Kuroshio showed widespread large amplitude near-inertial internal wave shear of high vertical wavenumber in the Kuroshio and regions between continental margins and the Kuroshio23. A very recent field study in Tokara Strait reported near-inertial waves of even shorter vertical wavelengths ~50 m accompanied by pronounced turbulent dissipation rates of O(10−7–10−6 W kg−1)20. One of the most likely mechanisms to form the high vertical wavenumber shear is the near-inertial internal waves generated by the Kuroshio over shallow topography in Okinawa Trough and Tokara Strait19. Because the Kuroshio is forced to turn its direction from northeast to southeast when it approaches Kyushu island, high vertical wavenumber near-inertial internal waves could also be generated spontaneously by the meandering Kuroshio28. Furthermore, the upstream Kuroshio passes the so-called critical latitude, 28.9°N, where M2 internal tide energy can be efficiently converted to that of high vertical wavenumber near-inertial waves through parametric subharmonic instability (hereinafter PSI)30. Near the critical latitude, K1 or O1 diurnal internal tides generated over the rough topography are also near-inertial internal waves. In a strong baroclinic front, where geostrophic shear allows anomalously low frequency internal waves, a recent theoretical study31 showed that PSI of wind-driven near-inertial waves could generate high wavenumber internal waves with a frequency of half the local Coriolis frequency f/2. Regardless of their generation mechanism, these propagating near-inertial internal waves can be trapped on the anticyclonic side of the front where the lowest internal wave frequency is decreased by geostrophic lateral and vertical shears34. The trapped near-inertial waves can break into three-dimensional turbulence and enhance mixing of momentum and tracers37. However, direct turbulence measurements in the upstream Kuroshio are very scarce. Also, the lateral resolutions of previous turbulence measurements are not sufficient to resolve the coherent spatial patterns associated with high wavenumber near-inertial internal wave shear38. As a result, earlier studies did not find elevated turbulence in the thermocline of the Gulf Stream17, and concluded that turbulence in the ocean interior is caused by patchy random internal wave breaking, even in the Gulf Stream. In contrast, a number of recent studies have reported enhanced turbulent mixing in the thermocline of the Kuroshio42 and the Gulf Stream43 caused by propagating internal waves. However, the lateral resolutions of the turbulence data were still insufficient to provide direct evidence that the turbulence is caused coherently by banded internal-wave shear in the thermocline of the western boundary currents. Although a recent extensive microstructure time-series survey at low latitude45 reported that the strong turbulence coincided with the relatively large strain caused by wind-induced near-inertial waves of anomalously low frequency in the anticyclonic vorticity field33, it is not known if near-inertial internal waves form the spatially coherent strong turbulent layers in ocean fronts. In this study, a new turbulence and microstructure observation scheme is developed and utilized to measure turbulence directly with a high lateral resolution of 1–2 km in the upstream Kuroshio near Tokara Strait, where a large amount of energy from the Kuroshio can be dissipated. For the first time, the observations in this study suggest that propagating high vertical wavenumber near-inertial internal waves form spatially coherent banded layers of strong turbulence in the upstream Kuroshio, with O(100 m) and >O(10 km) vertical and lateral scales, respectively.On the other hand, the Kuroshio flows through shallow topography in its upstream along Okinawa Trough and near Tokara Strait, where the Kuroshio over the topography can induce strong turbulence and diapycnal mixingin-situ survey was conducted using the R/T/V Kagoshima-maru during November 12–20, 2016, near Tokara Strait where the upstream Kuroshio flows over the shallow seamounts and through the narrow straits near the Tokara island chain off Kyushu island  in vertical and O(10 km) in lateral direction, similar to Rainville and Pinkel’s study in this region23. With λh and λv, horizontal and vertical wavelengths (m), respectively, −1) (where b = −gρ/ρo is buoyancy (m s−2) with gravitational acceleration g (m s−2), seawater density ρ (kg m−3) and reference density ρo = 1025 (kg m−3), and vertical coordinate z(m)), and f Coriolis frequency (rad s−1), the internal-wave dispersion relation, then implies that the frequency of these waves N~O(10−2 rad s−1), and local f~O(10−4 rad s−1). The internal-wave ray paths computed, assuming a quiescent condition show that the angles of the internal wave rays with near-inertial frequencies are more consistent with the observed shear bands than those with the M2 tidal frequency  as a function of longitude suggests that large shear variance found at 129.3–129.575°E in Leg A is associated with the downward energy propagating internal waves, and that found at 129.4–129.55°E in Leg B is caused by upward energy propagating internal waves . These estimates are comparable to the rate of wind energy input to inertial motions computed by a slab model50  local PSI with anomalously lower minimum internal-wave frequency than f due to mean velocity shears, (3) K1 internal tides generated over rough topography, (4) near-inertial internal waves generated by the Kuroshio over shallow topography in Okinawa Trough and Tokara Strait, (5) spontaneously generated near inertial internal waves by the Kuroshio, and (6) PSI of wind-induced inertial waves to generate high wavenumber anomalously low frequency internal waves of half the Coriolis frequency, f/2 along isopycnal in a strong baroclinic front31.These results indicate that the high vertical wavenumber shear is caused by near-inertial internal waves propagating in the upward and downward directions in the Kuroshio near Tokara Strait. While the wind energy flux to the inertial currents is found to be sufficient to generate an observed amplitude of near-inertial waves, it is not clear how the observed high vertical wavenumber near-inertial internal waves are formed. We speculate that the possible mechanisms are (1) PSI of the M23. Here, we show, for the first time, that these large amplitude near-inertial internal wave shears generate strong turbulence of spatially coherent banded structures, employing a newly developed high-resolution (1–2 km) tow-yo microstructure profiling technique, using 1.5 mm dyneema rope. The thin rope allows the microstructure profiler (VMP-250) to sink relatively smoothly with small drag down to 300 m depth. The measured turbulent shear spectra by the tow-yo profiling show overall good agreement with the Nasmyth empirical shear spectrum51 , because of turbulence within the surface boundary layer, which is forced by momentum and heat fluxes at the surface , and reach O(10−6 W kg−1) at some locations  within the same orders of magnitude  and O(10 km) in the vertical and lateral directions, respectively, are coherently formed along the near-inertial internal-wave shear bands  within the surface boundary layer, which is not surprising considering continuous forcing at the surface with weak stratification in the surface boundary layer  on average, and >O(10−3 m2 s−1) at many locations below 100 m depth , as well as M2 semi-diurnal tidal frequencies. However, these M2 tidal, near-inertial and subinertial peaks in shear spectra are not clear, especially in upper layers, probably because of the Doppler smearing by the strong Kuroshio flow27  is about the half of that estimated for the semi-diurnal component (D2 = M2 + S2 + N2)  (see Methods section) is continuously dissipated by the observed bands of the turbulent dissipation rates, O(10−8–10−7 W kg−1), with the reference seawater density ρo = 1025 kg m−3, the internal waves could be dissipated completely after 1–10 days. Therefore, it is possible that the PSI-induced near-inertial waves can survive ~4 days after the 94-h advection. Even without the tide or PSI, near-inertial internal waves of high vertical wavenumber could be generated by the Kuroshio over the topography19. The globally estimated energy generation rate from geostrophic flow to lee wave by the study of Nikurashin and Ferrari19 in Okinawa Trough, the region near the observation sites, is O(0.1–1 mW m−2), consistent with the vertical energy flux of observed near-inertial internal waves.The observed strong turbulence associated with high vertical wavenumber internal wave shear implies active occurrence of PSI near the critical latitude 28.9°N. The moored ADCP observations during our shipboard experiment suggest that the velocity field is dominantly modulated at Mpth Fig. . Assumin1 diurnal internal tide is the most plausible and likely candidate for the mechanism generating high vertical wavenumber near-inertial internal waves observed near Tokara Strait. The same harmonic analysis for the diurnal and semi-diurnal tidal elevations during the shipboard observations in November 2016 at Tanegashima tidal station  in the Okinawa Trough to be 6.8–21.5 × 10−4 m2 s−1 using a steady-state salinity balance with historical current data and salinity data obtained by the profiling floats. Although the direct turbulence data obtained in this study cover only the upper 300 m depth, and did not reach the layer of σθ = 26.8 kg m−3, strong turbulence just above salinity minimum layer of NPIW, which is equivalent to >O(10−4 m2 s−1) of average eddy diffusivity, is consistent with a previous estimate57. This implies that the observed large amplitude high vertical wavenumber internal waves play very important roles in watermass mixing and modification in the study region.The induced energy dissipations and vertical mixing could have important implications for watermass modifications. A previous study−1 in the subsurface layer, σθ = 24–27 kg m−3 and transports it downstream7, similar to the nutrient stream of the Gulf Stream11. In addition, nitrate concentrations below the Gulf Stream are found to be higher than in the ambient water of the same density9. This along-isopycnal anomaly of nutrients is most likely formed and maintained by diapycnal diffusion, as opposed to homogenizing mesoscale along-isopycnal stirring. Therefore, large average turbulent diffusivity of O(10−4 m2 s−1) in the subsurface layer at 100–300 m depth, σθ = 24–26 kg m−3, reported by this study, indicates that the observed subsurface turbulence induced by the near-inertial internal waves could effectively diffuse up the subsurface nutrient stream of σθ = 24–27 kg m−3 to the shallower layer, generate the elevated nutrient concentrations along the Kuroshio, and increase primary production in its downstream.Not only is the observed pronounced turbulent diapycnal mixing important for the watermass modifications of NPIW, it is also very important for nutrient supply from the lower layer to the surface euphotic layer. Recent studies of the nutrient transport in the upstream Kuroshio showed that despite its low-nutrient oligotrophic surface water, the Kuroshio transports a large amount of nitrate at an average rate of 170.8 kmol s33 catalyzes the breaking and dissipation of the near-inertial/diurnal internal waves, and promoting watermass modification and nutrient supply. Although the contributions of PSI to form observed high vertical wavenumber near-inertial waves were found to be small compared to the diurnal internal tide in our observations, other mechanisms to generate observed inertial waves are not discussed in detail in this study. Okinawa Trough and Tokara Strait are very peculiar regions because of their strong baroclinic current, the Kuroshio , flowing through the shallow seamounts and islands, and through the critical latitude for PSI and the boundary where the diurnal period is identical to the inertial period. More comprehensive field and numerical studies are, therefore, necessary to fully understand the generation, propagation, and dissipation mechanisms of these near-inertial internal waves/diurnal internal tides propagating in the upstream Kuroshio.In this study, for the first time, the spatially coherent banded layers of strong turbulence associated with high vertical wavenumber near-inertial internal waves were directly observed using a new tow-yo microstructure profiler. Our analyses suggest that the high vertical wavenumber near-inertial waves are most likely generated by the diurnal tide over the rough topography in Tokara Strait. Because the observation site is around 30°N latitude, where the inertial period is 24 h, the generated diurnal internal tides are inherently near-inertial waves with slow group velocities. The ADCP and microstructure data suggest that these near-inertial internal waves are trapped and broken into turbulence, preferentially on the anticyclonic side of the upstream Kuroshio, suggesting that the trapping of the near-inertial waves by the Kuroshio due to anticyclonic vorticityZ(t0) = uz(t0) + ivz(t0)  referenced to the time t0, is estimated from the observations Z(t) = uz(t) + ivz(t) at the arbitrary time t asuz(t0) and meridional shear vz(t0) are obtained by taking the real and imaginary parts of (4), respectively.Near-inertial wave velocity and shear rotate clockwise in time in the Northern Hemisphere. When the observation period exceeds or is equivalent to the inertial time scale, this rotation needs to be removed to avoid temporal aliasing of lateral velocity and shear structures. The back-rotated shear −1. The descending speeds of the UVMP, gradually decreasing with depth, were between 0.3–0.8 m s−1. Taking 7 minutes to reach 300 m depth and 5 minutes for recovery, the lateral resolution of the tow-yo survey was 1–2 km, depending on the absolute current velocity. The data were internally recorded and recovered after each transect observation. However, because the both FP07 thermistors mounted initially on the VMP-250 in Leg A malfunctioned, the expendable bathythermographs (XBT-T7s) were deployed six times to obtain temperature profiles. The XBT temperature data were interpolated by objective mapping with decorrelation lengths of 15 km and 40 m along ship-track and vertical directions, respectively, which were then used to estimate the background potential density ρθ and σθ and stratification for Leg A (see below). For Leg B, two functional FP07 thermistors were installed on the VMP-250, so that the potential density ρθ and σθ could be estimated, based on high-resolution FP07 thermistor data. To cross-calibrate the FP07 thermistors, the calibrated UCTD sensor and VMP-250 were clamped together and lowered to 200 m depth with the BT-winch of the Kagoshima-maru.We used a new tow-yo Underway Vertical Microstructure Profiler (Underway-VMP: UVMP) to measure high-resolution spatial structures of microscale turbulence at the Kuroshio Front. The UVMP consists of a vertical microstructure profiler, VMP-250  and a winch for the Underway-CTD . The VMP-250 carries two shear probes, two FP07 thermistors, a pressure sensor, and a vibration sensor to measure microscale velocity shear, microscale temperature gradient, pressure and accelerations, respectively, at a frequency of 512 Hz. The UVMP was tow-yoed at a ship speed relative to water, at 1.5–2.1 m sε, the turbulent shear spectra were computed using the microscale shear data measured by the airfoil shear probe over 8 seconds, at approximately every 5–6 m depth, with an overlap of 4 s (2.5–3 m). The obtained shear spectra were integrated over the wavenumber ranges, where spectra agree with the Nasmyth empirical shear spectrum51, avoiding the integrations of electronic noise at high wavenumber ranges and low frequency instrument vibration noise at low wavenumber ranges. For the wavenumber ranges where the observed and the empirical spectra did not match, we integrated the fitted Nasmyth empirical shear spectrum51  were estimated using the close relationship between potential temperature and potential density found at the observation sites  for σθ > 25 (deeper than 150 m depth), 0.04–0.1 (kg m−3) for 24 < σθ < 25 (100–150 m depth), and ~0.12 (kg m−3) for σθ < 24 ; the estimated density had better accuracy for the denser and deeper layers.Because the UVMP does not carry a conductivity sensor to measure salinity, seawater potential density Cgz, assuming that the near-inertial waves had low potential energy. Using the dispersion relation of the internal waves, the vertical group velocity for the low frequency internal wave isω is the internal wave frequency (rad s−1), and m the internal-wave vertical wavenumber (rad m−1). The horizontal kinetic energy of the internal waves isuz and vz are ADCP zonal and meridional shear (s−1), respectively, and  represents the spatiotemporal average between 100–250 m depth and over the entire transect for Leg A and Leg B. The internal-wave frequency ω was estimated as ω = 1.01 f (rad s−1), based on the internal ray path and the measured velocity shear band  and two components for diurnal (K1 and O1) tidal elevation can explain 99% of the total variance, with the M2 tidal component accounting for 67%, S2 for 13%, N2 for 3%, K1 for 10%, and O1 for 6%. To obtain the time series of subinertial diurnal and semi-diurnal tidal elevation by the harmonic analysis, the computed diurnal D1 = K1 + O1 and semi-diurnal D2 = M2 + S2 + N2 tidal elevation amplitudes were smoothed with a 30-h lowpass filter and compared with the 30-h lowpass near-inertial shear  were analyzed from August 31, 2000 through August 14, 2001. Harmonic analysis with 59 tidal constituents was conducted for the tidal elevation record to compare with the 30-h lowpass near-inertial shear amplitude (see below). The results of the harmonic analysis showed that the variance of the dominant three components for semi-diurnal  was used. The near-inertial amplitude was computed using near-inertial zonal and meridional shear as To compare the obtained 30-h lowpass semi-diurnal and diurnal tidal amplitudes with the near-inertial shear amplitude, the relatively long-term moored ADCP data were analyzed. The moored ADCP  measured the horizontal current at 14 layers with 32 m vertical bins from 480.5 to 64.5 m depth from September 30, 2000 through July 1, 2001, at Stn. TK1 Fig.  in the vThe datasets generated and/or analyzed during the current study are available from the corresponding author upon request.Supplementary Figures


Long intergenic non-coding RNA p21 (lincRNA-p21), known as the direct transcriptional target of p53, was found down-regulated in several human solid tumors. However, little is known about the role of lincRNA-p21 in gastric cancer. The expression levels of lincRNA-p21 in tissue samples and cell lines were detected by qRT-PCR. MGC-803 and MKN-45 cells were transfected with siRNAs targeting lincRNA-p21 or control siRNAs to determine the effect of reduced lincRNA-p21 expression on tumorigenesis. We also overexpressed lincRNA-p21 in MGC-803 cells. Cell proliferation was measured by CCK-8 and Ethynyl-2-deoxyuridine (EdU) incorporation assays. Migration and invasion abilities of cells were measured by wound healing and transwell assay. We demonstrated that lincRNA-p21 was significantly reduced in gastric cancer tissues (p<0.001) compared with that in normal tissues and this lower level of lincRNA-p21 was significantly correlated with higher invasion depth grade (p=0.024), more distant metastasis (p=0.009) and advanced TNM stage (p=0.011). Further study revealed that knock down of lincRNA-p21 could promote malignant behavior of gastric cancer cells and induce epithelial to mesenchymal transition (EMT). Overexpressing lincRNA-p21 showed opposite effects. Moreover, knocking down lincRNA-p21 could elevate the expression of Yes associated protein (YAP), the core effector of Hippo signaling, by elevating mRNA levels and increasing its nucleus translocation instead of the canonical Hippo pathway. Overexpression experiments verified the regulation role of lincRNA-p21 in YAP expression. Collectively, these data suggest that lincRNA-p21 could serve as a potential biomarker and a vital therapeutic target in gastric cancer. Gastric cancer (GC) is the most common gastrointestinal cancer in East Asia and ranks as the third leading type of cancer-related death worldwide , 2. The Long noncoding RNA (lncRNA), which is defined as transcripts composed of more than 200 nucleotides in length, have attracted broad attention recently –7. Long The Hippo pathway governs the organ size and cell growth rate . Yes-assin vitro experiments.Our study first confirmed the expression pattern of the lincRNA-p21 expression in GC tissues and cell lines. We then investigated the relationship between lincRNA-p21 levels and the malignant behavior of gastric cancer in two GC cell lines. The potential lincRNA-p21-dependent mechanisms correlated with EMT and YAP expression were further explored using p<0.05)   , distant metastasis (p=0.009) and more advanced TNM stage (p=0.011)  and high  expression groups according to the median ratio Figure . Our sta) Figure . Further) Figure . However) Figure . The com1) Table .p<0.05, Figure p<0.01, Figure In order to study the biological function of lincRNA-p21 in gastric cancers, we performed knockdown and overexpression experiments. The expression level of lincRNA-p21 was significantly down-regulated in the si-lincRNA-p21-transfected cells  and a normal gastric mucosal cell line (GES-1) were obtained from the cell depository of our laboratory. These cell lines were cultured in Dulbecco's modified Eagle (DMEM)  supplemented with 10% fetal bovine serum (FBS)  in a humid atmosphere of 5% CO2 at 37%.−ΔΔCT values normalized to GAPDH.The total RNA of patients’ tissue specimen and cells were both extracted using the NucleoZOL reagent  in accordance to the protocol. The concentration and quality of RNA was detected by NanoDrop. The extracted RNA was converted to cDNA by using PrimeScript™ RT reagent Kit with gDNA Eraser  following the manufacturer's instruction. The cDNA template was mixed with gene specific primers as well as SYBR Green 2X mixture . Quantitative real time-polymerase chain reaction (qRT-PCR) was carried out on Bio-Rad CFX96 Touch . The primer sequences were listed in 5 cells/per well. Then either si-lincRNA-p21 or si-NC was transfected using Lipofectamine 2000  under the guidance of the protocol. The newly constructed plasmid was extracted by EndoFree Plasmid Mini Kit  follow the instruction. We used pcDNA 3.1 as the internal control which was directly obtained from the stock of the lab. 48h later, the transfected cells were harvested for further analysis.The small interfering (si) RNA targeted linc-RNA-p21 was designed as same as the known sequence in a former report . Both thCell proliferation was tested by Cell Counting Kit-8 (CCK-8)  following the manufacturer's instructions. MGC-803 and MKN-45 were seeded in a 96-well plate 24h before the transfection of si-lincRNA-p21 or si-NC. 100μl of serum free DMEM contains 10% CCK-8 reagent was added into each well at 0h and 48h post transfection respectively and then cultured for 1h. The absorbance was assessed at a wavelength of 450nm by a microplate reader . Experiments were repeated at least three times.in vitro kit  according to instructions. MGC-803 and MKN-45 were seeded in a 96-well plate 24h before the transfection of either si-lincRNA-p21 or si-NC. At the 48h time point, 100 μl of 50 μM EdU was added in each well and incubated for 2 h at 37%. After that, the cells were fixed in 100ul of 4% paraformaldehyde (prepared in PBS) for 30 minutes at room temperature. The cells were incubated in 50 μl of 2 mg/ml glycine for 5 minutes followed by PBS washing. Afterwards, the cells were permeabilized with 1% TritonX and reacted with 1X Apollo solution for 30 minutes at the room temperature in dark subsequently. Finally, 40μl of Hoechst solution was added in each well and incubated for 15 minutes at room temperature in dark with PBS washing followed. In the end, the cells were visualized and photographed under a fluorescence microscopy. Experiments were repeated at least three times.Cell proliferation ability was also tested by EdU Apollo DNA MGC-803 and MKN-45 were seeded in a 6-well plate 24h before the transfection of either si-lincRNA-p21 or si-NC. At the 48h time point, the transfected cells were harvested and starved in serum free DMEM and then placed in the upper chambers with or without matrigel coated in duplicate. The lower chambers were filled with DMEM containing 20% FBS. After wiping off the cells on the upper surface by wet cotton swab, the cells migrated to the lower surface of the chamber were fixed in 4% paraformaldehyde and stained with 0.5% crystal violet solution and then photographed under the microscope. Experiments were repeated at least three times.MGC-803 and MKN-45 were seeded in a 6-well plate 24h before the transfection of either si-lincRNA-p21 or si-NC. Right after the transfection, a scratch was made through the center of each well using the 200ul sterile pipette tip. The scratch was observed and pictured at 0h, 24h, 48h post-transfection. Experiments were repeated at least three times.Cellular protein extracts were obtained from cultured cells with RIPA lysis buffer . The Nuclear and Cytoplasmic Extraction Kit  was used to prepare cytoplasmic and nuclear extracts separately according to the manufacture's instruction. The protein extracts were separated by 8% SDS-polyacrylamide gels and transferred electrophoretically onto a PVDF membrane using the Bio-Rad semidry transfer system. Membranes were blocked with 5% milk overnight and incubated with primary antibodies (diluted in 1:1000) for 2 hours at room temperature. Second antibodies (diluted in 1:100) were added after washing and incubated for 1 hour in dark. After washing again, the membranes were exposed in a dark room using machine. All of the primary antibodies and secondary antibodies were purchased from the Abcam campany.MGC-803 and MKN-45 cells were transfected by either si-lincRNA-p21 or si-NC 48hrs before plating into 24-well dishes containing 12 mm glass coverslips. After 24hrs, cells were fixed in 4% paraformaldehyde and then incubated 1 h in blocking buffer (0.5% triton X-100 and 1% BSA in PBS) prior to primary antibody staining. Both primary and secondary antibodies were diluted in blocking buffer. Coverslips were incubated with primary antibodies for 1 h at room temperature followed by 1h second antibodies incubation. In the end, diluted DAPI was added on the coverslips. 10 minutes later, coverslips were mounted on the glass slide. Images were captured under Nikon Eclipse 80i  microscope with NIS-Elements software (version 4.30.01).p values were two-sided and p values less than 0.05 were considered to be statistically significant.The analysis between clinicopathological characteristics and lincRNA-p21 expression was performed using SPSS 22.0 software . The rest data analysis was performed using Graphpad Prism 5.0. Student t test was used for the comparison between two groups and Chi-square test or Fisher's exact test for analyzing the correlation between lincRNA-p21 expression level and patient's clinicopathological characteristics. Quantitative analysis from pictures was performed by ImageJ v1.04 software . All of the


Distorted negative self-images and impressions appear to play a key role in maintaining Social Anxiety Disorder (SAD). In previous research, McManus et al. (2009) found that video feedback can help people undergoing cognitive therapy for SAD (CT-SAD) to develop a more realistic impression of how they appear to others, and this was associated with significant improvement in their social anxiety. In this paper we first present new data from 47 patients that confirms the value of video feedback. Ninety-eighty percent of the patients indicated that they came across more favorably than they had predicted after viewing a video of their social interactions. Significant reductions in social anxiety were observed during the following week and these reductions were larger than those observed after control periods. Comparison with our earlier data  suggests we may have improved the effectiveness of video feedback by refining and developing our procedures over time. The second part of the paper outlines our current strategies for maximizing the impact of video feedback. The strategies have evolved in order to help patients with SAD overcome a range of processing biases that could otherwise make it difficult for them to spot discrepancies between their negative self-imagery and the way they appear on video. •Video feedback is an effective component of cognitive therapy for social anxiety disorder.•Video feedback helps correct distorted negative self-images.•Video feedback can also provide patients with insight into how their safety behaviors look to others.•Social anxiety related processing biases can undermine the effects of video feedback if not addressed.•Ways of circumventing the processing biases are described. A recent network meta-analysis  has estareexperiencing feelings when watching the video. The processing biases that can interfere with video feedback fall into five broad categories. First, selectively searching for behaviors that could be interpreted negatively. Patients with SAD have a general belief that they come across badly in social interactions. This can lead them to selectively search a video for any behavior that could conceivably be interpreted in a negative fashion. This can happen even if they were not particularly concerned about those behaviors during the interaction and they were not prominent in the negative self-image. To solve this problem, it is necessary to ask patients to make clear predictions in advance of watching the video about any negative features that they believe were evident during the interaction.Second, discounting the accuracy of the video image. If an anticipated negative feature  is not evident in the video, some patients may discount the accuracy of the video image, claiming that the camera is at fault . Therefore, it is necessary to carefully set up the recording so that patients are confident that features that they are concerned about will be visible if they occur. We call this procedure calibrating the video.Third, mistaking safety behaviors for social deficits.Fourth, reactivating habitual patterns of self-criticism. During and after social interactions, patients with SAD are highly self-critical. When subsequently watching a video of the interaction, the self-critical commentary that accompanied or followed the interaction may be reactivated, making it difficult for patients to judge themselves objectively on the video. To address this, a number of techniques have been developed to reduce the reactivation of past memories and self-critical commentaries while viewing the video.Fifth, In the present paper, we first present data on the effectiveness of our current version of video feedback and compare it with the earlier version, whose effects were reported by Video feedback can be used in a variety of different ways during a course of CT-SAD. The first time that it is used is in Session 3. In the preceding session, patients engage in an experiential exercise in which they have a social interaction under two conditions: first, while focusing their attention on themselves, evaluating their performance, and engaging in habitual safety behaviors; second, while focusing externally, getting lost in the interaction  and dropping their safety behaviors. Typically, they discover that they feel less anxious and think they come across better to other people in the second condition, a discovery that is built on in subsequent therapy. In Session 3, video feedback of both social interactions is used to help patients compare their predictions of how they think they came across with how they actually came across. Since the McManus study, improved availability and functionality of camera and smartphone technology has increased the options for using video feedback. Technology now makes it easy to capture as a still shot key moments that disconfirm patients’ negative beliefs, and we have found this to be a very useful technique for consolidating learning. Further experience in using video feedback has also made us more aware of some of the processing biases (outlined above) that can make it difficult for patients to fully benefit from viewing themselves on video and clinical strategies for overcoming these biases have been developed. In order to assess the impact of our current way of using video feedback, we present video feedback data from our most recent RCT  and compare it with that reported in the Diagnostic and Statistical Manual for Mental Disorders . Forty-nine percent (23) were female.Participants were 47 patients who met  DSM-IV;  criteriaHere we report the data from Session 3 of CT-SAD. In this session, participants viewed the video recordings of the two social interactions they had engaged in during Session 2. In most instances, the Session 2 interactions involved having a conversation with a stranger. However, if patients felt this would not elicit a significant degree of anxiety, an alternative individualized task was chosen, such as having a conversation with a small group of people. Prior to watching the videos of both interactions in Session 3, patients were asked to form a mental image of the way they thought they would appear and to make clear-cut predictions about how they would come across. The videos were then watched and discussed before patients rerated how they thought they appeared. Details of how predictions were generated with patients and how the video was viewed and discussed are given below in the clinical guidelines section.not anxious at all) to 100 (the most anxious you have ever felt)?; (b) the extent to which idiosyncratic feared outcomes might be evident ; and (c) How would you rate your performance overall (0–100)? These ratings were repeated after each video had been viewed and discussed.Before watching each video, patients were asked to rate: (a) How anxious do you think you will look, on a scale ranging from 0 . This interval provides an estimate of what happens with no intervention. The second interval was that between Session 1 and Session 2. In Session 1 therapists develop a personal version of the 2(5) = 28.45, p < .001, therefore degrees of freedom were adjusted using the Greenhouse–Geisser correction. The results show that the LSAS score differed between the sessions, F = 15.37, p < .001. Pairwise comparisons between sessions 3 and 4 showed that there was a significant reduction in LSAS scores (from 73.4 to 65.5) in the week following the video feedback session (p = .003). By contrast, there were no significant changes in LSAS in the weeks following either the initial assessment (p = 1.00) or developing the individualized cognitive model .Our present findings confirm and extend those reported by Our results suggest that video feedback is an excellent technique for helping patients to correct negative self-images and to also gain insight into the way in which their safety behaviors appear to others. There are many opportunities to use it during a course of cognitive therapy, both in the therapy office and also outside of the office when conducting behavioral experiments in the real world. Typically, the first time it is used is in Session 3 when therapist and patient have the opportunity to observe the two social interactions that form part of the self-focused attention and safety behaviors experiment in Session 2. The initial messages that patients learn from this experiment are that self-focused attention and safety behaviors make the problem worse, not better. In particular, they tend to increase anxiety, make it more difficult to focus on the interaction, and give patients enhanced access to internal information (negative images and feelings) that lead them to think that they are coming across to other people more poorly than they really are. The additional messages that patients often get from viewing the videos include: (a) that they come across more favorably than they think in both conditions; (b) some of the aspects of their behavior that they do not like are the unintended, observable consequences of their safety behaviors rather than an intrinsic feature of themselves. When used at other times in therapy, video feedback has a similar function. It also is a very good way of helping patients discover that they are less the subject of other people’s critical attention than they think.When video feedback is used, it is necessary to pay attention to the way in which the video recording is set up, how the patient is prepared in advance of viewing the recording, and how the video is subsequently viewed and discussed. With suitable attention to each of these aspects, it is often possible to overcome the substantial processing biases that have prevented patients from overcoming their negative self-perceptions before they entered therapy.As patients with social anxiety can become quite self-conscious about being recorded, it is best to make video recording a routine aspect of therapy, rather than something that is just introduced on an occasional basis for video feedback. We routinely record all therapy sessions using a small domestic video camera that is unobtrusively placed on a bookshelf at right angles to the therapist and patient’s eye line, so it is not in the normal field of view. In the initial assessment interview we explain to patients that we find it helpful to view sessions afterwards in order to reflect on progress and plan future interventions. We request written permission to make the recordings for this purpose. We also encourage patients to take audio recordings of the sessions for them to review afterwards, as we find this is a very good way of maximizing learning. Once permission for the recordings has been obtained, the videos can subsequently also be used for video feedback when therapist and patient together think this might be useful.As well as making the video less intrusive, routinely recording all sessions allows one to capitalize on unplanned therapy events that can be immensely informative. For example, when talking about a topic in the session patients may spontaneously mention that they feel they blushed a lot, had a panic, stuttered, or talked nonsense. In each case they can be asked to specify how they think they looked or sounded, before comparing their prediction with what was captured on the video. A similar process can be applied to the therapist’s behavior. For example, the patient may feel that one must always be perfectly fluent in one’s speech in order to be accepted. Being aware of this belief, the therapist may pause for a while in mid-sentence before carrying on or may start one sentence and then move onto another without completing the first sentence. Chances are that the patient is unlikely to have noticed this and will be surprised to discover that it happened. Reviewing the video with the therapist afterwards helps the patient see that the dysfluency had no real significance, even though the patient would have felt it was a serious social mistake if she had done it herself.One of the main aims of video feedback is to allow patients to see their behavior in context. For this reason, when videotaping an interaction it is best to show both people in the interaction, rather than zooming in on the patient. The latter tends to make it difficult for the patient to avoid feeling self-conscious when subsequently watching the video, and also prevents an appreciation of the true significance of behaviors. For example, patients who are concerned about fidgeting might notice their hands or feet moving in a zoomed-in shot and think this indicates that they are very fidgety. However, in a zoomed-out shot they are likely to see that the other person is also moving to a similar extent.It is important to elicit patients’ main concerns before setting up the video as knowledge about these concerns may have implications for the way in which the video is set up. For example, with patients who are concerned about blushing, it is important to have a color chart or other objects in the field of view that show different shades of red. This is not usually explained to the patient in advance (as it would make them excessively self-conscious). However, if they feel that they do blush during the recording they can subsequently be asked to point to the shade of red that they think matched the blush. Invariably they point to a much darker shade, which is a wonderfully graphic way of helping them discover that their blush is less severe than they feel.During a course of CT-SAD patients are likely to have multiple interactions with other people in therapy sessions . As well as viewing the video of such interactions with their therapist, patients can also benefit from written feedback from the other participants (“confederates”) in the interaction. In order for this feedback to meaningfully reflect everyday life, it is important that the confederates are not informed about the patient’s personal fears  as this would not be information that other people would have in a routine conversation. The confederate is encouraged to treat the patient like anyone else they would meet in life outside of the therapy session rather than someone they are trying to scrutinize.Behavioral experiments conducted outside of the office but in a public space can easily be recorded on a smartphone, tablet, or other domestic camera so that video feedback can be used to enhance the value of these exercises. The principles for setting up and viewing such out-of-the-office videos are essentially the same as those that apply to in-office videos.Before viewing a video, it is important to identify patients’ predictions of what they think they will see and to get them to visualize what these things look like. This provides them with the maximum opportunity to see the difference between their self-perceptions and reality.How anxious do you think you looked?” “How boring?” “To what extent to you think you sweated?” etc.) and to indicate how they think that will look. They should be as specific as possible. For example, if somebody says “I will look in a state—just awful,” we would want to elicit a more specific description of how they think they will look on video so that this can be compared to the actual video image. For situations like shaking, blushing, underarm sweating, and dysfluent speech, it is helpful to ask the person to demonstrate on video how they think it looked—for example, by intentionally shaking one’s hand or lips, pointing to the relevant shade of red in a color chart, indicating the size of a sweat patch, or re-creating a pause—so that this can be compared with how it actually looked in the original video recording.Patients are asked to rate  the extent to which they thought their feared catastrophes occurred  while watching the video. These feelings may also distort their perception of the video. If the therapist and patient have identified this problem, patients can be asked to specifically look for things in the video that are inconsistent with the past social trauma to help them clearly distinguish between then and now .THERAPIST: How do you listen when your friend Alex is talking? Do you just go with the flow of what he says or do you zoom in on how he says every word and ask yourself, “How boring does Alex sound?” “How weird does he look?” [use patient’s own beliefs]PATIENT: (laughs) No! I just go with the flow.THERAPIST: Ok, we would like you to watch the people on the video in a similar way.Some people find that it is very difficult not to turn on their habitual self-critical commentary when watching themselves. After discussing how this can take them away from what actually happens on the video, it can be useful to ask them to watch the video from a more compassionate stance, perhaps as they would if they were watching a close friend or somebody they like and respect. They can be asked to recall the last time they had a conversation with this friend and to consider how they would view their friend:Some people find that when they hear the sound of their own voice this automatically triggers the similar sounding self-critical commentary that they normally have in social situations. If the person is highly self-critical, to prevent this mode being activated as soon as the video starts, it can be helpful to watch the first 30 seconds or so with the sound off. This will help them to see that they look as normal as anybody else on the video. When the sound is then turned on, they are in a more appropriate cognitive set. A similar maneuver can be used for people who are highly critical of their physical appearance and find it difficult to look at anything else in the video. For these people, the therapist may initially cover up the patient’s image and let them focus on how other people are responding to them. After a minute or so, they can also be revealed.Imagine you walk into a coffee shop and see a conversation happening, look at the whole group, not just one person.”There is a risk that people may selectively zoom in on themselves looking for any imperfection, rather than watching the interaction in context. To avoid this, the therapist may say something like: “Are you watching that person (therapist points to the patient in the video) as you would anyone else, or are you watching as your worst critic? Are you looking at other people and how they respond, or just focusing onthat person?”Once the patient has clearly articulated their negative self-image and they have been carefully prepared for viewing the video, therapist and patient watch it together. Sometimes the whole interaction is initially viewed without pausing. For highly self-critical patients it can be helpful to pause early in the viewing to check the following: “Does the other person seem to have noticed?” “Are they reacting as if they have seen a big mistake?” “Do the people onscreen look markedly different from each other?” “Is the other person also moving their legs and fidgeting?” “If an alien was looking at this would they think one of these people looked really odd?” As the therapist will be aware of the patient’s own self-images and has also seen how they actually behaved, the exact choice of questions will be determined by the therapist’s goal of helping the patient see which aspects of their self-image are distorted.Once the video has been watched all the way through, it can be very helpful to rewind the video to look at particular moments that have significance to the patient. For example, rewinding to the moment when the person thought they had a panic attack, when they thought they paused for a long time, looked particularly anxious, looked fidgety or felt that they sweated. They can then be asked to compare how they looked at that moment with their expectation. Other helpful questions may include: “Patients are often unaware of the way that their safety behaviors appear to other people. Video feedback provides an ideal opportunity for them to gain insight, which in turn can help motivate them to drop the safety behaviors. For example, a patient who was concerned that his colleagues might see his hand shaking while drinking a beer often turned his back to his colleagues before taking a sip. This made him feel less self-conscious and so seemed a good way of coping with the situation until he saw what it looked like on the video. He then realized it will have appeared odd and may have conveyed to his friends that he was not really interested in them, when the exact opposite was the truth. As turning his back was an intentional strategy, he was able to choose not to do it in the future. Similarly, a patient who was concerned that other people might think she was stupid tended to run through a preprepared list of topics during conversations and to be distracted from the conversation by mentally monitoring how she thought she was coming across. On viewing the video, she realized that she was conveying the impression that she was not interested in other people and was just giving them a lecture. This was the opposite of the impression that she wanted to convey, so she experimented with just saying what came into her head and responding spontaneously to what people said. When she watched this on the video she realized that dropping her safety behaviors allowed her to come across to others in the open and friendly manner that she wished.THERAPIST: What do you notice when we compare these two sets of ratings?PATIENT: I look so much better than I thought I was going to. I look OK, not really that anxious, even though I felt it.THERAPIST: What does that tell you about how visible your anxiety is?PATIENT: Maybe it isn’t that visible to others. Maybe I come across OK.THERAPIST: So if your feelings aren’t that visible, are they a reliable judge of how you come across?PATIENT: No, I guess not.THERAPIST: So next time you are in a social interaction and feel you are coming across badly, you may want to bring to mind the image of how you actually looked on the video.A key aspect of video feedback involves comparing patients’ ratings of how they thought they would appear with how they actually appeared once the video has been viewed. This comparison usually involves looking at all the specific predictions that the patient made. The initial 0–100 ratings that patients made in advance of viewing the video are compared with their ratings of the same concerns  after they have watched and discussed the video. To facilitate comparison a two-column table is constructed. Once the discrepancies have been tabulated, the patient is asked:both conditions, confederates are asked to think back to each interaction in turn and to provide feedback. We find a two-sided form, handed to the confederate as they leave the therapy room, helpful. The first side is largely blank. Confederates are asked to write a few brief notes about their general impressions. What tends to happen is that confederates mention specific points that make it clear they were interested in the patient and noticed various things about them. But what they noticed was mainly what was talked about, not the specific fears that the patient had (such as “my lip was shaking”). The second side covers the patient’s specific predictions (such as “I’ll sound boring”) and the confederate is asked to rate on 0–100 scales the extent to which this was true. Usually the confederates’ ratings are similar to the patients’ ratings after they have viewed the video, but sometimes the confederate is even more positive. When this happens it can be useful to discuss with the patient why the confederate may have been more positive: “Is it possible that your ratings are still partly influenced by your private feelings? This may be information that nobody else could have.”It can be very helpful to supplement video viewing with feedback to the patient from other people who might have been involved in an interaction. We routinely do this for the self-focused attention and safety behaviors experiment. After they have participated in Therapists should use their discretion in deciding whether it is helpful or necessary to supplement video feedback with feedback from others. Most often we present feedback from others after patients have had a chance to view and discuss their video and it is essentially used as a way of further confirming the conclusions they have already reached. However, if the feedback is very positive and the therapist thinks that the patient’s self-criticism may make it difficult for them to view the video objectively, it can be useful to show the other person feedback first. This helps to establish a different cognitive set for viewing the video.Our data (see above) indicate that almost all (98%) patients view themselves more positively after viewing the video if the experience is set up and discussed in the manner presented here. In rare instances where that does not happen, it can be useful to focus the patient’s attention on how others in the video are reacting to them. This can help them see that features that remain prominent in their own mind have less significance to others. Feedback from the confederate is also helpful in such instances.The aim of video feedback is to help patients see that they come across to others much better than they think. There are some moments in a video that illustrate this point more clearly than others. As video is a moving image, these moments can pass in and out of consciousness quite quickly. An excellent way of overcoming this problem is to capture the moment of disconfirmation as a still image. This can be done either by taking a still from the video or by taking a separate photograph. The latter is particularly useful in out-of-the-office behavioral experiments. For example, a patient reported feeling self-conscious when walking in the street even when not interacting with other people. She felt that people were likely to be hostile and predicted that if she asked someone for directions they would respond in an irritated manner, at the very least. She agreed with her therapist that she would test this out by stopping passersby and asking them the directions for the nearby rail station. The therapist accompanied and discretely took photographs on her mobile phone. The image reproduced in Still images involving other people, like the photograph in My feelings are not as visible as I think.”Still images can also be a wonderful way to capture moments when patients realize they come across much better than their internal feelings and self-perceptions tell them they do . For example, a patient reported feeling she blushed 80% red while giving a presentation to a small audience during her therapy. Prior to viewing the video, she was asked to select the shade of red she felt she blushed at this moment using a color chart. When a still image was captured from the video at the moment she felt she blushed 80%, the color she selected from the color chart was held next to this image, providing a clear disconfirmation of her belief. A second image was then created: this contained the still taken from the video at the worst moment for the patient (when she felt she blushed red) side-by-side with the block of color she predicted she blushed from the color chart. The image reproduced in The contrast between two images that depict how patients feel and how they actually looked on video . Adding some of the key learning points in the patient’s own words, either handwritten on a printed copy or typed onto an electronic image that can be saved on a smartphone or tablet, can act as a powerful flashcard that the patient can use as a reminder the next time they enter a stressful situation.In order to abstract key principles fitting the cognitive conceptualization of the patient’s social anxiety, and to consolidate and generalize learning, the patient and therapist may add some informative text to still images captured from the video  Identifying a patient’s predictions in advance of viewing a video; (2) Preparing the patient to view the video; (3) Discussing the video and rewinding to capture key moments; (4) Comparing ratings before and after viewing the video; and (5) Freezing the moment of disconfirmation and consolidating learning.Using the link below, readers can access a 7-minute video clip (Video 1) that illustrates Video feedback is a helpful technique that can be used throughout a course of cognitive therapy to help patients correct their negative self-perceptions and gain insight into the effects of their safety behaviors. Unfortunately, social-anxiety-related processing biases can make it difficult for people with SAD to see the difference between their negative self-image and what is actually shown on the screen. To reliably overcome these biases, particular attention needs to be paid to setting up the video recording; preparing patients to view the video; and subsequently watching and discussing the footage. This clinical guideline has provided details of many of the procedures we have found helpful to maximize the beneficial effects of video feedback. In selecting the most appropriate technique, therapists need to draw on their knowledge of the discrepancy between the patients’ negative self-images and how the patients actually come across. This in turn requires an ability to explore patients’ self-perceptions sensitively and in detail.


As few patient-friendly resources about pharmacogenetics are currently available, we aimed to create and assess a patient educational video on pharmacogenetic testing. A primary literature and resources review was conducted to inform the content and the format of the video. The educational video was then created using a commercially available animation program and pilot tested in focus groups of the general public and by an online survey of pharmacists. Emerging themes from the focus groups and survey indicate a desire for appropriate risk contextualization and specific examples when pharmacogenetic testing may be beneficial. Focus group participants also expressed a preference for a video with live action, and more text to reinforce concepts. Pharmacists generally felt that the video was understandable for patients and relevant for decision-making regarding testing. Using this initial feedback and the identification of important concepts to include in pharmacogenetics educational tools, we plan to revise the video, perform additional evaluations, and publish the video for public use in the future.  Pharmacogenetic (PGt) testing analyzes genetic variants that affect drug targets, transport, and metabolism, contributing to the effectiveness of medication and risk of adverse drug reactions. The use of PGt testing has diffused across several clinical specialties and is a pillar of personalized and precision medicine . There aMany of the currently available pharmacogenetic resources have been developed and published by PGt testing laboratories and may provide incomplete or biased information, or are specific to the laboratory’s unique testing services. Both primary care providers and pharmacists have expressed an interest in patient education materials to facilitate communication about testing ,5.p53 (cancer gene) lowered decisional conflict, increased knowledge, and decreased cancer worry  so you can see the seriousness of what could happen if you take the wrong thing and you don’t have any testing. You know like the blood thinner, you could get a blood clot. You know, some kind of example like that would really push the seriousness of it.”“It seems like [reasons for testing] may be pretty broad, but …if there are certain conditions, like … heart problems or digestive problems, …then this [testing] might be more targeted for you.”“Theme 2: Four participants expressed concerns about segments of the video that discuss risks of testing such as genetic discrimination and felt that it would be more helpful to present statistics about cases of genetic discrimination, particularly in regard to PGt testing. I do kind of like worry about everything all the time, so if I heard somebody like say, like this is something that somebody could steal your identity with, that would be pretty big cause for concern, but also like there are a lot of things that could happen, so I don’t know like if there is like a way that you can kind of add in an assurance, like this is extremely unlikely or like it’s probably not going to happen, something like that.”“Those participants felt that the risks of PGt testing as portrayed in the video (with the cartoon character engaging in risky behavior—skydiving) were exaggerated when put in relative context of risks that they had experienced previously in their lives. In this frame, they did not perceive PGt testing as seriously risky.That kind of led to believe there might be something seriously risky. I mean, out of all the things, somebody turning my information over is not the most riskiest thing I can think of in my life.”“Theme 3: All seven participants expressed a desire for a video with real actors as opposed animation as they felt that the animation format minimized the seriousness of the subject matter. They felt that a serious topic like PGt testing ought to be portrayed with an equally serious presentation format. This is a serious subject for me so I’d rather, in one sense I’d rather have real people doing it…I don’t have to be entertained.”“For it to be kind of a little bit cheesy like that and kind of, you know, it takes away from I think the seriousness of the actual discussion.”“Theme 4: Three participants indicated that including more text in the video would be helpful to reinforce or emphasize important concepts and would help maximize understanding.I would’ve liked to see more text in the video. …especially like important key points or key phrases.”“Yeah maybe, even like when somebody’s talking you can have the text under or something like that...just something to kind of drive the point home.”“Other suggestions included incorporating information about panel testing for genes involved in drug response , creating a shorter video, giving patients print materials in addition to the video, using a more authoritative-sounding narrator, depicting individual differences in drug response with people holding various scales , reducing the volume of background music, and including information about family history of adverse drug response.www.rxpgx.com) were emailed an invitation to participate in the research survey and 10 respondents (15%) completed the survey in its entirety. Due to the small sample size, the demographic information collected was limited in order to protect participants’ identity. Reported characteristics of pharmacist respondents are summarized in n = 5) and a majority had taken a course on pharmacogenetics (n = 7).Sixty-six members of the Community Pharmacists Pharmacogenetics Network  of respondents strongly agreed or agreed. A majority of respondents (n = 7) also agreed that the information was relevant for a patient to make an informed decision about PGt testing. The majority of respondents (n = 7) felt that the information would likely be understandable for patients. Most respondents (n = 7) agreed that the tone of the video was appropriate for patients, and half of the respondents (n = 5) agreed that the video held their interest and attention. Statistical analysis to determine significance was not performed on this data due to the low number of responses.When asked if the information in the video was comprehensive and complete, a majority  for audiovisual materials were scored individually and are summarized in Survey respondents were asked two open-ended response questions about what additional information, if any, should be added to the video and what other improvements could be made. Analysis of the open responses yielded two major themes: inclusion of specific examples of conditions/medications and contextualizing risks of testing.Theme 1: Pharmacist respondents felt that it would be helpful to include examples of medications for which there is currently informative PGt testing to emphasize the utility of testing.Seeing some real-life examples of medication situations helped by pharmacogenetics would be more impactful. The cartoon is nice and keeps things very basic so most patients will understand, but might not fully grasp the power of pharmacogenetics without a little more reality.”“Theme 2: Some pharmacists felt that the risks of PGt testing were portrayed as overly negative and neglected to present the protections in place against genetic discrimination, even going so far as to indicate that information on the risk of genetic discrimination may be unnecessary to include as cases of genetic discrimination based on PGt testing are rarely observed. The ‘risk’ should be reoriented to focus on the protections versus the risks. Stress the positive, don't scare patients off.”“Remove the part about possibly having the test used against the patient...unless companies are doing this already. I have done over 1000 tests and not encountered this yet. As the language in the video sounds now I highly doubt many patients would choose to have the test done which negates so many benefits to a possible life-saving test.”“Pharmacist respondents had a number of other suggestions to improve the video including adding information about the influence of drug transporters as well as drug metabolizing enzymes. Respondents also suggested including more information about benefits for patients undergoing PGt testing, selecting a different background music or lower volume relative to the narration, and creating an abbreviated version of the video to generate interest for a longer version.As PGt testing will likely be offered by providers with variable knowledge about pharmacogenetics, limited time with patients, and/or access to genetically-trained providers, additional educational approaches are needed to provide patients with the necessary information to make an informed decision and to understand how the test result may impact treatment decisions. With widespread access to digital media, short videos can be used to supplement patient-provider communication and enhance patients’ knowledge of this still relatively novel test. A comprehensive literature and resource review indicated a lack of patient educational materials about PGt, and few educational videos for commercial PGt tests. Though the available PGt videos provide some general background information, they are specific to a particular test offered by a commercial testing lab. Thus, we sought to create a universal educational video to introduce pharmacogenetics and PGt testing to patients. Feedback from a small group of the general public and pharmacists suggested that an educational video about PGt testing could be a useful tool to promote patient understanding and facilitate discussions about testing, though some revisions could help improve the initial version developed and shared with participants. Although animated videos have been effectively used to deliver health information , generalBoth general public participants and pharmacists made two similar suggestions. First, both groups suggested the addition of specific examples of PGt testing to enable patients to better understand how a test would be used for a patient in need of a certain medication. Inclusion of concrete examples of situations or conditions in which PGt testing would be used may help viewers relate to the personal benefits of testing. Pharmacists also suggested that inclusion of examples of medications for which there is informative PGt testing and information on the likelihood of new applications of PGt in the future may also be helpful to emphasize the utility of PGt testing. However, this finding is contradictory to another evaluation of an educational video about genetic testing and counseling which indicated that an educational video should provide broad information that may be applicable to a wider selection of patients and clinical indications .The second suggestion made by both groups was to better balance the presentations of risks and benefits of testing. Some of the focus group participants felt the risk of testing was exaggerated and preferred for the risks to be presented relative to other types of risks of health interventions, such as risk of a serious adverse response to a medication or other physical harm. Pharmacists also suggested that information that would mitigate risk should also be presented, such as federal protections against genetic discrimination. As risk perception is very subjective, the appropriate framing of risk in a limited amount of time can be challenging. Overall, however, pharmacists felt that the information presented in the video was comprehensive, understandable, relevant for decision-making, and appropriate for patients.Some limitations of the study should be acknowledged. The content of the video was developed by the synthesis of minimal existing literature on patient communication about PGt testing along with consensus by the research team. Therefore, the content of the educational video may not accurately reflect the informational needs of a given patient population. Our study is also limited by a small sample size and therefore our findings may not accurately represent the view of a larger population of patients or pharmacists. Furthermore, there may have been some ascertainment bias in the recruitment of participants in either the general public or pharmacist populations, particularly as several of the pharmacists were familiar with PGt testing.We aimed to develop and conduct an initial qualitative assessment of an educational video about PGt testing to inform patients about the purpose, benefits, and limitations of testing. The initial version of the video was assessed by pharmacists and members of the general population through an online survey and focus groups, respectively. Information gathered from these initial assessments may be used to improve the video for future use in clinical and research settings.The purpose of the educational video is to provide general information for patients about pharmacogenetics, the process of PGt testing, risks and benefits of testing, and other relevant factors to consider before deciding whether or not to undergo testing. The script for the video was developed based on a literature review on pharmacogenetics, accessible online resources, and text included in a patient brochure that had been previously developed and assessed by patients for a clinical trial . The conThe educational video was assessed via two focus groups of the general public; focus groups enable investigation of the perceptions and feelings of the general public regarding specific issues or services . Focus gA moderator guide was used to ensure consistency between the two group discussions; targeted questions were asked to direct feedback if necessary. The guide included questions and talking points focused on format , understandability of content, and perceived utility. We showed the video in its entirety initially and replayed certain sections upon request to promote discussion, such as the sections on the risks of PGt testing, the effects of individual pharmacogenes on multiple drugs, and the importance of sharing PGt results with other prescribing providers.The education video was also assessed using an online survey of pharmacists. Pharmacists who are members of CPPN were invited to view the video and provide feedback. A link to the video and a web-based survey was sent via email for ease of access as members of CPPN are located throughout the state of North Carolina. Reminder emails were sent one month after initial contact to increase response rate. The survey included measures assessing demographic and practice variables of the respondents. Questions regarding the content and format of the educational video included open-ended responses, and the PEMAT for audiovisual materials . The PEMFocus group discussions were digitally recorded; recordings were transcribed and coded by themes by one author (M.E.). A second author (R.M.) coded transcripts independently and differences in coding were reviewed and discussed between the two until consensus was reached. Illustrative quotes were abstracted for each theme identified.Summary statistics were calculated for each question. Responses to the PEMAT questions were scored according to the published guide to assess the understandability and actionability of the educational video . One poiThe Institutional Review Boards of the Duke University Health System (Pro00067835) and the University of North Carolina at Greensboro (15-0452) approved the study.Findings from the literature review as well as the focus groups and pharmacist surveys indicate that there is a need for PGt educational resources that focus on the risks and benefits of PGt testing. Therefore, providers offering PGt testing or supporting PGt services should consider the resources available and seek to provide additional information to supplement patient-provider discussions about testing. We envision the use of an introductory PGt video to supplement provider-patient discussion and potentially be accessible to patients outside of the clinic setting. A corresponding video reporting the results and the implications for treatment could also be developed to enhance comprehension of PGt test results relevant to the current as well as future treatments. Similar to what has been accomplished for at least one type of disease (inherited cancer ), a suitIn summary, there is currently a lack of available resources for patients to learn about pharmacogenetics and PGt testing in general. We developed an educational video that pharmacist respondents believed to be understandable and that would guide patients in making an informed decision about testing. Both pharmacist and general public participants believed the video could be improved by including specific examples of drugs and PGt tests, and balancing the risks and benefits of testing. The feedback gathered will serve to inform the next iteration of the video in preparation for its implementation and evaluation in clinical settings.


Lymphatic vessels function as transport channels for tumor cells to metastasize from the primary site into the lymph nodes. In this experiment we evaluated the effect of Sulfatase-1 (Sulf-1) on metastasis by upregulating it in murine hepatocarcinoma cell line Hca-F with high lymph node metastatic rate of >75%. The study in vitro showed that upregulation of Sulf-1 in Hca-F cells significantly reduced cell proliferation, migration and invasion (p<0.05). Also, the forced expression of Sulf-1 downregulated Mesothelin (Msln) at both the protein and mRNA levels. The experiment in vivo further showed that up-regulation of Sulf-1 with the attendant downregulation of mesothelin delayed tumor growth and decreased lymph node metastasis. In conclusion, our findings show that Sulf-1 is an important tumor suppressor gene in hepatocellular carcinoma (HCC), and its overexpression downregulates Msln and results in a decrease in HCC cell proliferation, migration, invasion, and lymphatic metastasis. This functional relationship between Sulf-1 and Msln could be exploited for the development of a novel liver cancer therapy. Hepatocellular carcinoma (HCC) is one of the cancers topping the list of malignancies that are of major global health concern because of its high incidence and case fatality rate. In most cases HCC is diagnosed at an advanced stage, which limits the application of curative treatment .Metastasis is the most important process in cancer progression and it accounts for about 90% of cancer-associated mortalities. To date, the pathogenesis and the genes associated with the cancer metastasis remain poorly understood . The mecIn the past few years, our laboratory conducted a study on mouse Hca-F and Hca-P cell lines in order to screen out lymphatic metastasis associated genes. Hca-F cells originated from mouse hepatocellular carcinoma which has been shown to have high lymphatic metastatic rate of >75% , 7 compaSulfatase-1 (Sulf-1) is a heparin degrading endosulfatase which is known to inhibit tumorigenesis by desulfating cell surface proteoglycans (HSGAGs) and prevents the binding of several ligands including growth factors, interleukins and morphogens such as VEGF, FGF, HGF, EGF and Wnt . Sulf-1 Mesothelin (Msln) is a 40kDa cell membrane glycoprotein and is known to act as a tumor promoter gene in various malignancies . A studyOur results show for the first time that, upregulation of Sulf-1 in Hca-F cells affects the expression of Msln both at the protein and mRNA levels. We have also demonstrated that, upregulation of Sulf-1 in Hca-F cells reduced cell proliferation, migration, invasion and lymph nodes metastasis of hepatocellular carcinoma, both in vitro and in vivo. These results suggest that the ability of Sulf-1 to downregulate Msln may provide a potential therapeutic target in hepatocellular carcinoma.Western blot was performed in order to check the expression level of both Sulf-1 and Msln in Hca-F, Sulf-1-Hca-F, and Nc-Hca-F cells. As shown in Figure *P<0.05). On the other hand, the Δct values for sulf-1 were 1.2 and 1.6 in the in vitro samples and 1.8 and 2.0 in the in vivo samples for Hca-F and Nc-Hca-F cells, respectively. Also the Δct values for Msln were 0.15 and 0.2 in vitro and 0.25 and 0.2 in vivo for Hca-F and Nc-Hca-F cells respectively. This indicate that the mRNA expression level of Sulf-1 in Sulf-1-Hca-F group was higher with a concurrently lower expression of Msln mRNA compared to the two controls, confirming that Sulf-1 upregulation led to Msln downregulation both at the Protein and mRNA levels, Figure The levels of mRNA of both Sulf-1 and Msln were also measured in vitro and in vivo by qRT-PCR and normalized to Gapdh, and Δct was used to calculate the values. In the Sulf-1-Hca-F cells the result showed that the Δct values for Sulf-1 in vitro and in vivo were 0.32 and 0.4 while the Δct value for Msln was 0.6 and 0.8, respectively, (P< 0.05) in the Sulf-1-Hca-F cells compared to the Hca-F and Nc-Hca-F cells as shown in Figure CCK-8 cell proliferation assay was used to evaluate the role of Sulf-1 on proliferation of Hca-F, Sulf-1-Hca-F and Nc-Hca-F cells. The results showed that, there was a significant inhibitory effect on cell proliferation by 60% (*P< 0.05) as shown in Table The cell cycle analysis was carried out by PI staining followed by flow cytometry. The results showed that in the Sulf-1-Hca-F cells there was 31% increase in cells accumulation in G0-G1 phase with a concomitant reduction by 32% in the number of cells in S-phase compared to the two controls (*P> 0.05).We performed transwell assay to examine the mobility and invasion ability of the stably up regulated Sulf-1-Hca-F and the two controls (Hca-F and Nc-Hca-F cell lines). The Sulf-1-Hca-F cells were shown to have approximately 61% reduction in mobility and 55% reduction in invasion ability. No significant differences were observed in migration and invasion abilities of the Hca-F and Nc-Hca-F groups as shown in Figure After inoculation of the mice with the tumor cells we observed that the group inoculated with the Sulf-1-Hca-F cells had a slower rate of tumor growth and developed smaller tumors compared to the groups that were inoculated with the Hca-F and Nc-Hca-F, Figure P<0.05). This result showed that, upregulation of Sulf-1 reduced lymph node metastasis in Hca-F cell lines.Also, we observed reduced lymphatic metastatic rate in the Sulf-1-Hca-F compared to the Hca-F and the Nc-Hca-F as shown in Table et al reported that, Sulf-1 is localized on the cell surface /2. Four weeks later, all the mice were humanely sacrificed to dissect and weight the tumor and also to dissect the reginal lymph nodes  for lymph node metastatic rate analysis. Some of the tumor tissues were fixed in 10% formalin for subsequent histological analysis whilst other tumor tissues were refrigerated for immunohistochemistry, western blot and qRT-PCR analysis.Tumor growth was monitored and measured on days 7, 14, 21 and 28 and the values were used to calculate the tumor volume according to the formula [(length (mm) × width (mm)2O2 and then incubated with the blocking solution (Horse serum) for 30 min at room temperature. The incubation with primary antibodies  was carried out overnight at 4°C. it was then Incubated with the secondary antibody for 1hr and color was developed with diaminobenzidine (DAB) . The positive reaction manifested as a brown stain. The section was counterstained in Mayor's haematoxylin. Dehydration process started from 80%, 95% and 100% of ethanol each for 2 min and then washed with xylene 2times for 2 min each. Mounting and cover slipping were done and we proceeded to slide reading. Other slides were stained by routine Haematoxylin and Eosin staining method and the rate of lymph node metastasis was determined.The resected tumors tissues were cut into 5-μm sections and fixed in paraformaldehyde. IHC stain was employed to detect the expression of Sulfatase-1 and Mesothelin. The tissues were fixed in 4% buffered formalin for 15 min at 4°C and rinsed in TBS. paraffin sections were dewaxed in xylene and rehydrated in a series of ethanol solutions after which antigen retrieval was done with Tris buffer at pH 9.0 in a microwave. Endogenous peroxidase activity was blocked by 20 min pre-incubation with 3% HP < 0.05. The data were expressed as the mean ± standard deviation (S.D).Each assay was performed three times. SPSS 17 software was used for all statistical analysis. One-way Anova was used to determine the significant differences among the groups at