Following publication of the original article , we haveIn this correction article, all members of the SRLF Trial group are now correctly shown in the collaborator list and all members are listed in Additional file The published apologizes for any inconvenience caused by this error.Additional file 1: Table S1. List of contributors; Table S2. Patients’ characteristics according to the presence of hypoxemia; Table S3. ARDS exclusion criteria among patients with bilateral infiltrates; Table S4. ICU mortality according to ventilator support and hypoxemia class; Table S5. Univariate analysis of variables associated with mortality in hypoxemic patients; Table S6. aHRs of PaO2/FiO2 banding in different sensitivity analyses; Figure S1. Evolution of oxygen support after the day of study.


After the Expression of Concern was posted on this article , 2, the This resolves the concern about data unavailability that motivated the editorial decision to post an Expression of Concern . The corS1 FileReport of statistical analysis.(PDF)Click here for additional data file.S2 FilePRISMA 5.0 reports.(RAR)Click here for additional data file.S3 FileGlobal data.(XLSX)Click here for additional data file.


Transcription factors GATA3 and STAT4 mediate the cytokine-induced development of naïve T cells into Th1 or Th2 types. In the present study, genetic analyses of GATA3 SNP rs3824662 and STAT4 SNP rs10181656 were performed to investigate the association of allelic and genotypic variations with the risk of T2D in the Bangladeshi population. A total of 297 unrelated Bangladeshi patients with type 2 diabetes and 247 healthy individuals were included in the study. The allelic and genotypic frequencies of rs10181656 located in the STAT4 gene were not found to be associated with risk of type 2 diabetes. The GATA3 rs3824662 T allele and mutant TT genotype had a significant association with the risk of T2D . Thus, the present study postulates that the genetic variation of the transcription factor GATA3, not STAT4, is associated with the risk of type 2 diabetes in the Bangladeshi population.Type 2 diabetes mellitus is a multifactorial metabolic disorder caused by environmental factors and has a strong association with hereditary issues. These hereditary issues result in an imbalance in CD4 Diabetes is a multifaceted metabolic disorder caused by impaired glucose metabolism characterized by hyperglycemia and is mainly classified as type 1 mellitus and type 2 diabetes mellitus. Both environmental and hereditary components play pivotal roles in the onset of diabetes  Impaired+T cells and decreased levels of naïve CD4+T cells, which in turn play an important role in the pathogenesis of type 2 diabetes. GATA 3 binding protein (GATA3) and Signal Transducer and Activator of Transcription-4 (STAT4) transcription factors mediate the cytokine-induced development of naïve T cells into Th1 or Th2 type. GATA3 consists of six exons and encodes a transcription factor with two transactivation domains and two zinc finger domains , while the over-dominant model had no effect. Even when either one allele or both alleles were mutated, the study participants were at risk of type 2 diabetes, which is reflected in the dominant model (comparing GG vs GT+TT)  or recessive model (comparing GG+TT vs TT)   independently had a significant association with the risk of disease  . When st662 GT in= 0.011] . 3DSNP c= 0.011] . Thus, dAlthough diabetes mellitus has been broadly classified as type 1 and type 2, increasing evidence has shed light on the fact that these two diseases overlap in many aspects. For example, classical immunological parameters for type 1 diabetes, such as anti-islet cell antibodies, elevated circulating cytokines and chemokines, are also present in many patients with type 2 diabetes ,47. In aThe STAT4 gene polymorphism is associated with increased risk for the development of early onset type 1 diabetes  but not Our findings demonstrate that genetic variation of the transcription factor GATA3, not STAT4, is associated with the risk of type 2 diabetes in the Bangladeshi population. The mechanism behind these roles needs to be investigated further. Moreover, studies with a large number of study participants from different ethnic populations are warranted to confirm the findings of this study.S1 Table(DOC)Click here for additional data file.S2 Table(DOC)Click here for additional data file.S3 Table(DOC)Click here for additional data file.S4 Table(DOC)Click here for additional data file.S1 DataThis excel file contains the raw data for this study.(XLS)Click here for additional data file.


The connections were directed by pendant phenyl groups to be between proximal ligand ends on the same faces of the grid. The 623link was separated from the topoisomeric granny knot by recycling size‐exclusion chromatography. The identity of each topoisomer was determined by tandem mass spectrometry and the structure of the 623link confirmed by X‐ray crystallography, which revealed two 82‐membered macrocycles, each in figure‐of‐eight conformations, linked through both pairs of loops.A molecular  Also formed is a 31#31Links  are mechanically connected closed loops (macrocycles).623link621link) but with a different crossing pattern. The topology can be depicted as two figure‐of‐eight shapes linked through both pairs of loops  or a granny knot  thiazolothiazole scaffold previously used to assemble a Solomon link  direct the pendant alkene chains above or below the grid plane, the biphenyl linkage significantly restricting the space accessible by the chain ends. The chain length was chosen to avoid over‐reaching, permitting only the required strand connections. Ethyl groups were used to increase the solubility of the benzimidazole units. These are smaller than the isopentyl groups used in the Solomon link synthesisTo achieve the required connectivity of the ligand strands, we designed bis‐tridentate ligand k Figure .6m The m414](BF4)8 was synthesized in analogous fashion, using ligand 1 and a small excess of iron(II) tetrafluoroborate . The 1H NMR spectrum of [Fe414](BF4)8 showed significant downfield shifts of the pyridine protons compared to the parent ligand (1), and a significant upfield shift of the benzimidazole proton Hs , which should face the pyridine ring of the perpendicular ligand strand in [Fe414](BF4)8 and therefore is indicative of the anticipated grid structure.To confirm that the modifications of the ligand would not hamper grid formation, we prepared a model ligand with methyl groups in place of the alkene chains . Complexation of the ligand with an equimolar amount of iron(II) or zinc(II) tetrafluoroborate proceeded smoothly in a mixture of dichloromethane and acetonitrile at room temperature to quantitatively afford the corresponding 2×2 interwoven grids, which were characterised by NMR spectroscopy and X‐ray crystallography . The alkene‐bearing 2×2 interwoven grid [Fe414](BF4)8 was subjected to ring‐closing olefin metathesis (RCM) as a dilute solution (1 mm) in dichloromethane/nitromethane (3:1), employing the Hoveyda–Grubbs second generation catalyst1H NMR indicated complete consumption of the starting material, and electrospray ionization mass spectrometry (ESI‐MS) showed multiply charged peaks corresponding to the successive loss of tetrafluoroborate counteranions from the fully‐closed grid . The crude product was demetallated  to afford a mixture of the metal‐free knot and link and other reaction byproducts, which were separated by preparative recycling size‐exclusion chromatography (GPC) .The 2×2 interwoven grid (BF4)8 or [Fe43](BF4)8 respectively. The 1H NMR spectrum of each complex was significantly sharper than that of the mixture originally obtained from the RCM reaction .The individual topoisomers were each remetallated by treatment with excess iron(II) tetrafluoroborate in a chloroform/acetonitrile mixture under heating 8 obtained by remetallation of a mixture of the organic topoisomers that had been separated from other species by GPC . However, slow diffusion of toluene into a solution of [Fe42/3](BF4)8 in acetone yielded single crystals of, to our surprise, the demetallated623link. The coordination of thiazolothiazole ligands to FeII is rather weak2 causes it to crystallize from solution as it reversibly forms at very low concentration. The X‐ray crystal structure of 2 42/3] should be addressed to the authors.SupplementaryClick here for additional data file.SupplementaryClick here for additional data file.


Dear Editor,DNA methylation at the 5-position of cytosine (5mC) is a crucial epigenetic mark in regulating biological processes including gene silencing, gene imprinting, and X chromosome inactivation  in MDA-MB-231 cells, which is even higher than that between H3K36me3 and 5mC . Importantly, further genomic distribution analyses revealed that the similarity between 5mC and H3K36me3 is restricted to genic regions as reported previously , which leads to massive overexpression of NSD2 and genome-wide gain of H3K36me2 . As revealed by other studies . We also found that the upregulated genes in KMS11 showed higher upstream DNA 5mC which tended to be lost in KMS11TKO, compared to the downregulated genes . While the 5mC levels of the CpG islands at the transcription start sites (TSSs) of the up- and down- regulated genes were generally low and similar, although statistically significant . This observation raises an intriguing possibility of the upstream hyper 5mC in KMS11 being functionally involved in the downstream gene transcription, which certainly needs future investigation.To further explore the functional importance on gene expression by the H3K36me2 and DNA 5mC axis, we carried out RNA-seq analyses in KMS11 and KMS11TKO, indicating that the global hypermethylation of 5mC is required for cancerous growth driven by NSD2 overexpression in KMS11.As NSD2 overexpression was demonstrated as the driver mutation for multiple myeloma bearing T translocation Below is the link to the electronic supplementary material.


The highest concentrations originate from urban and agricultural areas, are driven by diurnal winds, and peak in the early morning. These enhanced atmospheric levels can be detected across a ~100km wide nearshore area and represent a significant addition to total oceanic CO2 uptake. A global estimate puts the added sea-air flux of CO2 from these greatly enhanced atmospheric CO2 levels at 25 million tonnes, roughly 1% of the ocean’s annual CO2 uptake. The increased uptake over the 100 km coastal swath is of order 20%, indicating a potentially large impact on ocean acidification in productive coastal waters.Greatly enhanced atmospheric carbon dioxide (CO Agricultural practices can also impact local atmospheric CO2 on a diurnal cycle with large nighttime increases due to respiration and daytime decreases associated with photosynthesis [2 might impact marine air via atmospheric circulation and therefore increase the flux of CO2 into nearshore waters enhancing ocean acidification. Here we present novel observations from nearshore moorings and an autonomous sea surface vehicle that show the magnitude of urban CO2 pollution and allow us to calculate the contribution to sea-air fluxes from this previously unquantified source.The increase in atmospheric carbon dioxide  timeseries from multiple autonomous ocean based sensor platforms (moorings and surface vehicles) to provide a detailed assessment of the impacts of these high frequency variations in atmospheric CO2 concentration on sea-air CO2 fluxes in the nearshore environment.As indicated above, traditional estimates of sea-air CO2 instruments which were developed to measure CO2 concentrations in the air and water [2 instrument and CTD. Measurements were averaged hourly in the final analysis. Wave glider measurements were taken within 5km of the Monterey Bay Time Series (MBTS) Line, a transect extending from Moss Landing out 50km along the Monterey canyon  in the Monterey Bay region. Of these, three were moorings  Reference Network [2 = 0.71), as well as CarbonTracker [2 = 0.56), showed good agreement. We used the calculated baseline pCO2 values since these sources only run until December 2016 while our timeseries extends through early 2018.In this paper “atmospheric COnTracker  modeled 2 flux was estimated using the equation:2 in seawater [pCO2 is the difference between pCO2 water and pCO2 air. Gas transfer velocity is parameterized as a function of wind speed squared, while solubility is a function of water temperature and salinity. The common convention is used whereby a negative flux indicates CO2 transfer into the ocean (a sink); while a positive flux indicates release of CO2 into the atmosphere (a source).The net sea-air CO velocity, S is thseawater  and ΔpCO2 sources, anomalies were calculated for each wave glider data point. At ten minute intervals the wind speed and direction were then used to calculate the source position of the wind; this process was repeated additively over the six hours prior to any given air CO2 measurement to construct a probable path for the measured air parcel. This method ignores local variation in wind direction and speed, mixing, and in particular excludes the effects of topography on offshore winds. In order to reduce these effects, all pixels within a five degree cone around the calculated path were assigned the value of the calculated anomaly, and added to the developing composite image containing all previous tracks. When complete the image was then divided by the number of tracks intersecting each pixel, producing an average map of probable atmospheric CO2 anomaly sources and their strengths. This method should not be seen as providing exact locations or intensities of anomaly sources but it is straightforward and provides a general picture of the origin of strong atmospheric CO2 anomalies.In order to construct a high resolution map of enhanced atmospheric CO2 anomalies to sea-air fluxes, two flux calculations were made. The first used the well-mixed oceanic air baseline CO2 concentrations calculated as described above. This dataset represents atmospheric CO2 concentrations used in studies of sea-air flux that rely on modeled or well-mixed atmospheric CO2 values. This timeseries of fluxes was subtracted from a second timeseries calculated using the observed pCO2 air concentrations. The difference between these two timeseries gives a measurement of flux due to our calculated atmospheric anomalies in air pCO2. This method preserves the convention of negative values indicating increased transport of CO2 into the ocean.To estimate the contributions of the atmospheric CO2 are detected on all platforms during periods of offshore winds. A time series of atmospheric CO2 from the OA1 mooring over 2014 and 2015 illustrates the extent of these anomalies  [2 at both these sites relative to well-mixed marine air.Significant positive anomalies in atmospheric COnomalies . In Fig t Creek)  and clea2 anomalies detected along the MBTS line originate from the Salinas valley and Hecker Pass  and the Salinas Valley . OA1 is also impacted by CO2 anomalies during periods of easterly winds from these topographic features. However, its largest anomalies are registered when winds blow from ~150 degrees. OA1 is situated only a few hundred meters north of the city of Monterey and the monthly averages at this buoy were much better correlated with the urban CO2 measurements from Walnut Creek (r2 = 0.78), than with CarbonTracker modeled atmospheric CO2 (r2 = 0.47)  . This suley area .2 anomalies with distance from shore. The wave glider provides the perfect platform to further explore this relationship. Frequent wave glider measurements extend 50km out to the end of the MBTS Line, and average anomalies of 6-10ppm are detected at the end of this line during periods of offshore winds. This shows that during offshore wind events the plume of high CO2 air extends at least 50km from shore, although its CO2 content is reduced on average from the 15 ppm anomalies seen nearshore Click here for additional data file.


The effects of exogenous application of Zn2+ and of chelation of endogenous Zn2+ were examined on long-term potentiation (LTP) of stimulus-evoked synaptic transmission at Schaffer collateral (SCH) synapses in field CA1 of mouse hippocampal slices using whole-cell patch clamp and field recordings. Low micromolar concentrations of exogenous Zn2+ enhanced the induction of LTP, and this effect required activation of NMDA receptors containing NR2B subunits. Zn2+ elicited a selective increase in NMDA/NR2B fEPSPs, and removal of endogenous Zn2+ with high-affinity Zn2+ chelators robustly reduced the magnitude of stimulus-evoked LTP. Taken together, our data show that Zn2+ at physiological concentrations enhances activation of NMDA receptors containing NR2B subunits, and that this effect enhances the magnitude of LTP.The role of zinc (Zn Under normal physiological conditions, Zn2+ is released from presynaptic vesicles by low frequency synaptic transmission and interacts with multiple receptors that are involved in the induction of LTP, including NMDAR pyrimidin-4-amine . Slices were then transferred to microcentrifuge tubes and flash frozen with liquid nitrogen and stored in a -80°C freezer.Western blotting was performed on mouse hemi-sectioned brain slices (300 μm thick), which included not only the hippocampus but overlying cortex as well. Slices were treated in pairs as control , control + ZnFrozen tissue samples were boiled for 5 minutes and homogenized with high speed vortexing in lysis buffer . The samples were centrifuged afterwards for 5 minutes and the protein concentration was determined using a protein assay kit (Bio-Rad). 15 μg of protein was separated on 4–15% SDS-PAGE gradient precast gel (Bio-Rad). A separate gel was run for each animal. For each gel, equal aliquots were run in duplicate for each slice in adjacent lanes. This pattern was duplicated on the same gel so that all samples from the same animal were transferred to the PVDF membrane at the same time under the same conditions. After electroblotting, the membrane was cut in half and one half was developed with anti NMDAR2B antibody to visualize the total amount of receptor present in the sample and the other half was developed with anti PY (TYR1472) NMDAR2B antibody. Membranes were blocked with casein then incubated with primary antibodies at room temperature overnight followed by 1hr washing in TBST  at RT. Primary antibodies were NMDAR/NR2B and phospho-NR2B Y1472 primary antibodies  and anti-β-actin to normalize band intensities. Secondary antibodies conjugated with horseradish peroxidase (Jackson ImmunoResearch) were visualized using chemiluminescence ECL-Plus reagent .The blots were densitometrically analyzed using Image-J. Quantitative comparisons were performed by normalizing each blot to a control lane and the relative value of the PY signal was divided by the total NMDAR2B signal. Thus a ratio of one is the value of control standard- the PY/NMDA for control is set to 1.P<0.05. Data points averaged for statistics are marked with a dashed bracket over the graphs. All data points analyzed were a 5 minute average per slice from the time points indicated by the brackets and are taken from within the last ten minutes of recording. Means ± SEM were calculated for each group and then compared to other experimental groups using 1-way ANOVA and Dunnett’s multiple comparison test. The number of slices used for analysis included comparison of slices from within the same mice and between slices of different mice.Electrophysiological data were analyzed initially with Clampfit (v9) . Analyzed data were further processed and presented with Origin 6.1  and CorelDraw 10.0  programs. Statistical analysis were performed with SPSS (v11). Statistical data are presented as mean ± SEM unless indicated otherwise. Significance level was preset to 2+ in regulating synaptic plasticity, we commenced by bath applying exogenous Zn2+ to mouse hippocampal slices at a concentration of 1μM to evaluate stimulus-evoked LTP at SCH synapses in field CA1. To test the potential role of Zn2 (1μM) did not significantly alter fEPSP slope during the 15 minutes of baseline recording prior to high frequency stimulation (TBS). However 1μM ZnCl2 significantly increased the magnitude of LTP after TBS. Bath application of ZnCl2 at concentrations of 0.01μM and 10μM, did not significantly enhance the magnitude of LTP after TBS stimulation , it remained to be determined whether endogenous Zn2+ released by SCH terminals can influence LTP in a similar manner. The next set of experiments were designed to determine the effects of chelating endogenous Zn2+ on LTP, using the membrane impermeable Zn2+ chelator CaEDTA  was unable to enhance LTP in the presence of Ro25-6981, confirming a requirement of NR2B containing NMDA receptor activation for the enhancement of SCH-CA1 LTP by Zn2+. Thus, Zn2+ enhancement of LTP may be mediated by the up-regulation of NR2B-containing NMDARs to contribute to induction of LTP in adult rat hippocampus.The next step in our experiments was to see if NR2B-containing NMDARs subunits were required to enhance TBS induced LTP at CA1 synapses. Ro25-6981 did not significantly alter the magnitude of LTP when bath applied alone, which suggests that NR2B-containing NMDAR do not contribute substantially to control LTP elicited by TBS stimulation. This is consistent with literature suggesting that hippocampal NR2B subunits play a more predominate role in the induction of LTP in younger mice, particularly during development . However2+ crossing the membrane through other channels and activating intracellular signaling molecules [2+ mediated NMDA fEPSP. It appears that the timing of PP2 pretreatment was important; if ZnCl2 and PP2 were co-applied with no initial pre-treatment of PP2, NMDA fEPSPs were still enhanced, while pre-treatment with PP2 blocked the actions of Zn2+. This could be explained by more rapid transmembrane penetration of the charged Zn2+ compared to the larger PP2. Nevertheless, our data do suggest that Zn2+ acts via NR2B SFK phosphorylation to enhance NMDA fEPSP activity.Zinc modulation of NMDA receptors could be the result of Znolecules , 45. A lolecules ,13. In t2, supporting a requirement for SFK activation in modulation of LTP by Zn2+.During induction of stimulus-evoked LTP, there are crucial phosphorylation cascades that are activated, resulting in phosphorylation at multiple sites  on many proteins, and multiple levels of direct and indirect modulation of AMPAR and NMDAR transmission. In our experiments, PP2 did not alter TBS-induced LTP at SCH-CA1 synapses, suggesting that SFK activity is not necessary for the induction and maintenance of this form of LTP. However, PP2 did specifically block the increase in LTP elicited by 1μM ZnCl2+ enhancement of LTP, we examined the role of phosphorylation at the Y1472. Y1472 has been implicated as a key phosphorylation site of recombinant NR2B-containing NMDAR [2+ could alter the phosphorylation of the NR2B receptor subunit at baseline, without any stimulation to the tissue. Thus allowing us to examine whether or not the NR2B subunit was “primed” by phosphorylation at Y1472 before application of high frequency stimulus. Western blot analysis revealed that 1μM ZnCl2 did not increase tyrosine phosphorylation of the NR2B subunit at Y1472 prior to TBS. The addition of the inhibitor PP2 had no influence on the basal state phosphorylation at the Y1472 site. Our findings suggest that exposure to Zn2+ without stimulation does not result in an increase in phosphorylation at the site Y1472. However, this does not rule out a role for Y1472 phosphorylation in the enhancement of LTP, which could occur with the combination of Zn2+ and high-frequency synaptic activation. There could be other sites of phosphorylation that do enhance at baseline or after stimulation which should be explored in this context.To further elucidate mechanisms by which PP2 inhibited Znng NMDAR . Phosphong NMDAR , 46,47. 2+ enhancement of LTP via activation of the SFK pathway is Ca2+-dependent, or enhanced due to a direct activation of the signaling pathway that bypasses Ca2+. Although NR2A containing NMDARs have been shown to predominantly contribute to LTP in adult mice, NR2B-containing NMDARs in Field CA1 do contribute to calcium transients, suggesting a potential regulatory role in LTP formation that could be up-regulated by Zn2+ [2+ may act to release RACK1 from NR2B thus increasing NMDAR activation.The question remains as to whether Zn by Zn2+ . Althoug by Zn2+ . A possi2+ enhancement of LTP are triheteromeric NMDA receptors. Trihetermoric NMDARs contain two NR1 subunits and both NR2A and NR2B subunits in combination, which, as some studies suggest, may comprise more than 50% of the NMDARs in the synapse in the hippocampus [2+ binding as well as to possible differences in pharmacological interaction could provide an alternative site of interaction and modulation. Further examination of the kinetics and functions of triheteromeric NMDA receptors at CA1 synapses in the presence of Zn2+ is warranted [2+ may cause a change in the interaction between NR2A and NR2B that favors one subunit over the other in the NMDA receptor complex, changing the mix of NMDARs in the synapse.Another possible contributor to Znarranted . As a co2+ as a modulator of LTP, not related to a generalized properties of heavy metals in the brain [2+ and exogenously applied Zn2+, bath application of exogenous ZnCl2 has the potential to interact with an extrasynaptic receptor population with a higher percentage of NR2B-containing NMDAR that is activated by glutamate spillover during high-frequency stimulation. In conclusion, the data presented here suggests that zinc influences activity-dependent synaptic plasticity within a relatively narrow range, the balance of which may be critical for regulation of LTP of synaptic strength underlying learning and memory formation. Dysregulation of zinc homeostasis that results in altered concentrations of Zn2+ release may be a key target for therapeutic intervention in a multitude of brain pathologies.The data presented here support the conclusion that low micromolar zinc enhances TBS-induced LTP of SCH-CA1 synapses through phosphorylation of NR2B subunits of NMDA receptors. Our findings suggests a specific endogenous role for Znhe brain . Given tS1 Fig2 . Neither 0.01μM nor 10μM ZnCl2 significantly altered LTP magnitude compared to controls .The time course of LTP in untreated control slices (circles) compared to slices treated with 10μM  or 0.01μM ZnCl(TIFF)Click here for additional data file.S2 Fig2+-free ACSF plus 25μM DNQX. ZnCl2 was bath applied after 15 minutes of stable baseline. 100nM ZnCl2  and 10μM ZnCl2  transiently enhanced NMDAR fEPSPs compared to untreated control slices , an effect that had reversed by one hour of application. (Each point mean ± SEM of n recordings). (B) Waveform of NMDA fEPSPs in presence of magnesium at 0’ without zinc and after 60’ zinc exposure demonstrating no effect in the presence of magnesium.(A)Time course of NMDAR fEPSPs (amplitude) enhanced and isolated by application of Mg(TIFF)Click here for additional data file.S3 Fig2  and NMDA evoked currents for control . Baseline was recorded for 10 minutes before application of ZnCl2. Bath application of ZnCl2 increased the area compared to baseline. .This is the time course of NMDA evoked currents for 1μM ZnCl(TIFF)Click here for additional data file.S4 Fig2 . (B) Histogram demonstrating how ifenprodil inhibited NMDA fEPSPS in the presence of 1μM ZnCl2. (C) Time course of NMDA fEPSP amplitudes in control slices  treated with NVP AAM077 alone, versus slices treated with NVP AAM077 + 1μM ZnCl2 .(A) Time course of NMDA fEPSP amplitudes in control slices  in the presence of 10μM ifenprodil alone, versus slices treated with ifenprodil + 1μM ZnCl(TIFF)Click here for additional data file.S5 FigP<0.05; Student’s t-test). (B) Summary of the normalized slope at 50 minutes after TBS stimulation. Mean ± SEM of fEPSP slopes 50 minutes post-TBS in slices treated with 1μM ZnCl2 + PP2 (n = 4) versus slices treated with 1μM ZnCl2 + PP3 (n = 3). The two groups were significantly different, with PP2, but not PP3, completely blocking the Zn2+ enhancement of LTP .(A) The time course of LTP in PP2 treated slices (circles) compared to slices treated with PP3 (squares). PP3 did not inhibit LTP magnitude compared to PP2 ((TIFF)Click here for additional data file.S6 FigA representative western blot. All samples in this figure are from the same animal. The lanes above each other are duplicate aliquots of the same slice. Upper panels are developed with anti NMDAR2B antibody and the lower panels developed with anti PY NMDAR2B antibody. Quantitation of the ECL developed image was performed using Image J software. Quantitative comparisons were performed by normalizing each blot to a control lane and the relative value of the PY signal was divided by the total NMDAR2B signal. Thus a ratio of one is the value of control standard- the PY/NMDA for control is set to 1. The quantization from this study is reported in (TIFF)Click here for additional data file.


We found that TBX3 promotes differentiation only in the presence of early growth response factor 1 (EGR1), which is differentially expressed in RMS and is also a target of the PRC2 complex. The potent regulation axis revealed in this work provides novel insight into the effects of the PRC2 complex in normal cells and RMS and further supports the therapeutic value of targeting of PRC2 in RMS.TBX2 and TBX3 function as repressors and are frequently implicated in oncogenesis. We have shown that TBX2 represses p21, p14/19, and PTEN in rhabdomyosarcoma (RMS) and skeletal muscle but the function and regulation of TBX3 were unclear. We show that TBX3 directly represses TBX2 in RMS and skeletal muscle. TBX3 overexpression impairs cell growth and migration and we show that TBX3 is directly repressed by the polycomb repressive complex 2 (PRC2), which methylates histone H3 lysine 27 (H3K27 The more common form of the disease is the embryonal subtype (ERMS), characterized by loss of heterozygosity at the 11p15 locus, a region which harbors insulin-like growth factor 2. Alveolar RMS (ARMS) is the more aggressive form of RMS that is characterized by t or t translocations. The translocations result in chimeric transcripts that fuse the 5′ portion of the paired box proteins 3 or 7 (PAX3 or PAX7), including an intact DNA-binding domain, to the transactivation domain of a forkhead transcription factor (FKHR), creating novel PAX3-FKHR ) or PAX7-FKHR ) fusion proteins3. RMS is diagnosed by observation of unique skeletal muscle cell morphology phenotypes and the presence of myogenic markers such as myogenic regulatory factors (MRFs)4, yet these factors appear to be inactive in RMS5.Rhabdomyosarcoma (RMS) is the most common soft tissue pediatric sarcoma, which is thought to largely arise from the skeletal muscle lineage6. The T-box motif binds to the core sequence GGTGTGA known as the T-element7. Distinct from most members of the T-box family, TBX2 is known as a potent transcriptional repressor that functions in both embryonic development, and if deregulated, tumorigenesis8. The oncogenic potential of TBX2 was first identified by its ability to bypass cellular senescence in a Bmi−/− mouse embryo fibroblast model9. TBX2 has been shown to promote cell proliferation and block cellular senescence by repressing CDKN1A (p21), CDKN2A (p14/19ARF)10, and PTEN11.The T-box family of transcription factors are highly conserved and related throughout all metazoan lineages. They share a common DNA-binding domain known as the T-box motif and participate in diverse types of organogenesis and developmental regulation12. TBX2 and TBX3 are highly related and thought to have arisen from a gene duplication event13. In the case of deregulation, TBX3 also acquires the potential to become an oncogene in melanoma cells14. TBX3 has been found to be overexpressed in melanoma15 and several sarcomas16. TBX3 has been shown to promote tumor metastasis and the migration of breast cancer cells17. In MCF-12A breast epithelial cells and B16 mouse melanoma cells, TBX3 has been shown to directly repress TBX2 under the control of TGF-β118. Our lab has shown that TBX3 is expressed throughout skeletal muscle differentiation, whereas the expression of TBX3 in RMS cell lines is almost completely abrogated11.Another member of the T-box family, TBX3, is also characterized as a transcriptional repressor and developmental regulator19. PRC1 and PRC2 work synergistically and indispensably together for transcriptional repression of target gene by ubiquitylation of histone H2A lysine 119 residue and methylation of histone 3 lysine 27 (H3K27) residues, respectively20. The catalytic subunit of PRC2 is enhancer of zeste 2 (Ezh2)21.The polycomb repressive complexes 1 and 2 (PRC1/2) are critical in the precise and accurate regulation of development in many physiological systems including skeletal muscle22. In RMS, pharmacological inhibition of EZH2 in ERMS resulted in reduced proliferation and pharmacological inhibition or depletion of EZH2-induced myogenic differentiation in these cells23. EZH2 depletion in ARMS has also been shown to inhibit proliferation and initiate apoptosis24. JARID2, a founding member of Jumonji family of proteins, is known to be a substoichiometric component of PRC2 that appears to function in targeting PRC2 activity25. JARID2 is highly expressed in ARMS and contributes to the inhibition of differentiation in ARMS cells26.Many studies have revealed that EZH2 is overexpressed in a large number of cancersEGR1 is correlated with the induction of differentiation and repressed by PRC2 in ARMS. Discovery of this novel PRC2-TBX3-TBX2 genetic axis has important implications for understanding the mechanisms that drive proliferation and differentiation in RMS and skeletal muscle.In this work, we show that TBX3 represses TBX2 under the control of the PRC2 complex in RMS and skeletal muscle. RMS cells and proliferating skeletal muscle cells contain high levels of EZH2 that represses TBX3 and allows TBX2 expression. Depletion of EZH2 upregulates TBX3 which then down regulates TBX2. We also show that the early growth response gene 27. In skeletal muscle, TBX2 is expressed in proliferating myoblasts, but sharply downregulated upon differentiation while TBX3 is expressed throughout myogenesis and highly expressed during differentiation27. To understand the potential role of TBX3 in RMS, we transiently transfected RMS cell lines representing both ERMS (RD and RD2) and ARMS (RH30 and RH28) with an expression plasmid for TBX311. As anticipated, we observed that TBX3 was upregulated  database using GDAC Firehose. Two cohorts of the top one-third TBX3 and TBX2 expressing samples were combined and duplicates samples were removed. A correlation analysis was performed and a scatter plot was generated for TBX3 and TBX2 expression  = −0.5173, p < 0.0001, N = 113), which was also evident from two distinct clusters on the heatmap with opposite expression (N = 263)  Fig. .TBX3 mRNA expression  assays were performed using antibodies against TBX3 and primers against the ter Fig. . Intriguter Fig. . Given t27. TBX3 has been implicated in oncogenesis in breast cancer and melanoma17. To determine if TBX3 inhibited oncogenesis by repressing TBX2 or instead had a TBX2 independent role in promoting oncogenesis in RMS cells, we assayed for the growth properties of RH30 cells expressing exogenous TBX3. We found that proliferation was strongly inhibited by TBX3 and closely correlated with the degree of TBX3 expression in each clonal isolate  by immunofluorescence. RH30 cells expressing exogenous TBX3 did not show the presence of MyHC positive cells  and MYOGENIN (MYOG) in cells under normal growth conditions and under differentiation conditions. We found no upregulation of these markers under either condition  in RH30 cells and stable cell lines expressing shEZH2 were selected. Two clones were further characterized and we noted that in both cell lines, EZH2 was modestly depleted at the level of mRNA  on the Tbx3 promoter and we found that H3K27me3 was enriched on this promoter  and western blot , identified a group of developmental regulator genes that are repressed by PRC2 to maintain pluripotency and thus poised for activation during ES cell differentiation and TBX3 was included in this group of developmental regulators34. These results are consistent with what we observe in this study.The repression of 18. However, unlike what has been observed in those previous studies which showed that TBX3 has oncogenic potential, we found no evidence that TBX3 promotes oncogenesis in RMS. Instead, we found that TBX3 acts as a tumor suppressor in RMS, largely due to its repression of TBX2.TBX3 had previously been shown to represses TBX2 expression by directly associating with the TBX2 promoter in a breast cancer cell line, MCF-12A35. JARID2, a noncatalytic, substoichiometric component of the PRC2 complex, has been shown to be upregulated in RMS, where it promotes cell viability and inhibits differentiation26. The catalytic subunit of EZH2 has been shown to be highly expressed in ERMS and siRNA-mediated depletion or pharmacological inhibition of EZH2 inhibits proliferation and induce differentiation of ERMS23. EZH2 was also shown to be required for viability of ARMS by repressing FBX03224. Treatment of RH30 or RD cells with siEZH2 leads to robust transient depletion of EZH2 after 24 h and induced apoptosis but did not induce MYOGENIN or MYOD1. Transient inhibition of EZH2 has been shown to induce MYOGENIN in skeletal muscle cells38. Our lab has recently shown that transient depletion of EZH2 or JARID2 does result in transient upregulation of MYOGENIN and MYOD1 as others have seen, but sustained depletion of either factor leads to a block in Wnt signaling, which inhibits MYOD1 and blocks differentiation32. RMS cells are deregulated in Wnt signaling39, so a similar mechanism is not anticipated in RMS. Here, we found that RH30 cells with a modest sustained depletion of EZH2 generated by stable selection of a shRNA construct against EZH2 led to a decrease in proliferation and an induction of differentiation, including the upregulation of MYOD1 and MYOG.Upregulation of the PRC2 complex has been shown be correlated with many cancer typesTBX3, which allows the expression of the potent oncogene TBX2. This genetic axis also acts in skeletal muscle and provides a molecular explanation of previous results from our lab. We have shown that TBX2 is expressed in proliferating myoblasts and is sharply downregulated upon differentiation, while TBX3 is expressed throughout myogenesis and upregulated during differentiation11. Here, we establish that the down regulation of EZH2 upon skeletal muscle differentiation leads to the upregulation of TBX3 and the subsequent downregulation of TBX2.We show here that EZH2 represses 28. We found that EGR1 is a target of EZH2 repression. EGR1 has also been shown to be a target of the PAX-FOXO1 fusion that characterizes ARMS40. Thus, EGR1 is both repressed by EZH2 and destabilized by PAX3-FOXO1 in ARMS. It remains to be determined if ERG1 is a target of PRC2 in ERMS cells, but it may be that the lack of PAX3-FOXO1 in these cells is sufficient to allow EGR1 expression. Consistent with this hypothesis, we observed that the rescue of EGR1 expression by EZH2 by inhibition or depletion was relatively modest when compared to the level of EGR1 present in ERMS cells.Our results also showed that TBX3 is not sufficient to induce myogenic differentiation in ARMS, while depletion of EZH2 is sufficient to promote differentiation. These results were correlated with the expression of EGR1, which we have shown acts as a tumor suppressor in RMS that can induce differentiation in ARMS upon ectopic expressionThe PRC2-TBX3-TBX2 axis discovered in this work has significant implications for both RMS and skeletal muscle. The PRC2 complex serves as a central mediator between proliferation and differentiation and understanding the functions of its many downstream targets offers new insight into how skeletal muscle grows and repairs. TBX2 serves as a potent oncogene in RMS and many other cancers when overexpressed and thus, understanding the deregulation of TBX2 through the PRC2-TBX3-TBX2 axis offers insight in new approaches to inhibit tumor growth of RMS and additional cancers. This work also provides a mechanistic understanding of the effect of PRC2 in RMS and offers additional support to the therapeutic value of PRC2 inhibition in RMS.RD and RH30 cells (ATCC) were grown in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS)  according to standard protocols. RD2 and RH28 were obtained from Denis Guttridge, Medical University of South Carolina, and grown as described above. All cell lines were authenticated by Bio-Synthesis  using STR analysis on September 14, 2011. Cell lines are routinely screened for mycoplasma infection using the LookOut Mycoplasma quantitative polymerase chain reaction (qPCR) Detection Kit . All cell lines in this study were found to be mycoplasma negative prior to the study and following the study. C2C12 myoblasts  were grown in DMEM supplemented with 10% FBS. To induce differentiation, cells were grown to 90% confluence and the media switched to DMEM supplemented with 2% horse serum . Cells were grown in differentiation medium for the number of days indicated in each experiment.2O at 10 mg/ml and used at 10 μg/ml in RH30 cells and 2 μg/ml in RD cells. Puromyocin  was dissolved in ddH2O at 10 mg/ml and used at 2 μg/ml in all cell lines. Drugs were diluted in DMEM for use at the indicated concentrations.GSK126  was dissolved in DMSO at a concentration of 28.5 mM and 0.1% DMSO was used as the vehicle control. Blasticidin  was dissolved in ddH11. pEF was used as the vector control and contains the gene for blasticidin resistance. shRNA against TBX2 and scr control were previously described27. shRNA constructs are in the pLKO.1 vector, which contains the gene for puromycin resistance.TBX3 was cloned into the pEF TOPO  vector as previously describedCells were transfected with the indicated plasmids using calcium phosphate according to standard protocols or Turbofect transfection reagent (Fisher Scientific) according to manufacturer’s protocol. Transient transfections were harvested at 48 h post transfection or as indicated. Stable cell lines were made by transfecting cells with linearized plasmids and selecting for drug resistant colonies. Glass rings were used to isolate individual clones. Cells were recovered by trypsinization and transferred to single wells for propagation.41 with the following modifications: A total of 1 × 107 cells were used for each immunoprecipitation and protein A agarose beads  were used to immunoprecipitate the antibody–antigen complexes. Primers are described in Supplemental Table ChIP assays were performed and quantified as described previouslyCells were grown on cover slips, fixed with paraformaldehyde, blocked with 10% goat serum, 1.0 % NP-40 in phosphate buffered saline (PBS) for 1 h and washed with PBS. Primary antibodies against MyHC were incubated overnight at 4 °C, washed with PBS, and detected by Alexa Fluor-488 goat anti-mouse antibody (Life Technologies). Cell nuclei were stained by incubating with 1 μM DAPI (Life Technologies) for 5 min.4 cells per well were seeded in six-well plates and on the indicated days, cells were counted under a light microscope using a hemocytometer. Cell viability was determined by trypan blue staining. Cell counting was performed in duplicate for four blinded biological replicates.A total of 2 × 10HPRT and/or 18s rRNA.Trizol (Life Technologies) was used for RNA extraction from cells. Two micrograms of total RNA was treated with DNase  and reverse transcribed with MultiScribe MuLV reverse transcriptase (Life Technologies). Forty nanograms cDNA was used for qPCR amplification (Life Technologies) with SYBR green PCR master mix (Life Technologies). Negative controls included no RT samples where no reverse transcriptase was added for each RNA sample. All quantitative RT-PCR (qRT-PCR) was performed in triplicate and three independent RNA samples were assayed for each time point. qRT-PCR data were calculated using the comparative Ct method (Life Technologies). Standard deviations from the mean of the [Δ] Ct values were calculated from three independent RNA samples. Primers used are listed in Supplemental Table 2 incubator at 37 °C for the indicated hours. Percentage migration rates were calculated using following formula:0” is the area of the scratch immediately after the scratch was made, and “At” is the area of the scratch after indicated time. Scratch area was measured using ImageJ software (NIH). All scratch assays shown are representative of four independent experiments.Cell mobility was assayed by scraping a straight line with a 20 µl pipet tip on a monolayer of cells grown to 100% confluency. Cell debris was removed by washing. To obtain the same field during the image acquisition, marks were created near the scratch line. The plate was placed in a CO5) were plated in triplicate. One milliliter of culture medium was added to the top of each plate every 5 days and cells were grown at 37 °C in a CO2 incubator for 30 days. The plates were stained with 1 ml of 0.05% Crystal Violet (Fisher Scientific) for >1 h and colonies were counted using a dissecting microscope. All assays shown are representative of four independent experiments.Soft agar assays were carried out in 60 mm culture dishes in which 2 ml of 0.7% Noble agar (Fisher Scientific) in 1× DMEM with 10% FBS was overlaid with 2 ml of 0.35% agar in 1× DMEM with 10% FBS containing the cells. Cells of each clone  and clear lysates obtained by centrifugation. Protein concentration was determined by the Bradford’s assay  and 50 µg of protein was loaded for each well of sodium dodecyl sulfate polyacrylamide gel electrophoresis. Resolved proteins were then transferred onto a PVDF membrane using a tank blotter (Bio-Rad). Membranes were blocked with 5% milk in 1× Tris-buffered saline plus Tween 20 (TBST) and followed by incubation with primary antibody overnight at 4 °C. After washing membranes with 1× TBST, membranes were incubated with the corresponding secondary antibody, washed with 1× TBST and incubated with chemiluminescent substrate according to the manufacture’s protocol  and visualized by autoradiography and/or an iBright FL1000 Imager . Blots were quantitated using ImageJ software (NIH) or iBright Analysis software. Four biological replicates were performed for each western blot assay.Tbx3 gene were chosen using an online CRISPR Design Tool (http://tools.genome-engineering.org). Guide containing DNA oligonucleotides was designed and cloned into pSpCas9(BB)-2A-Puro (PX459) V2.0 as described42. pSpCas9(BB)-2A-Puro (PX459) V2.0 was a gift from Feng Zhang . Following selection of individual puromycin resistant clones, genomic DNA was isolated using Trizol  and PCR amplified targeted regions were sequenced.Guide sequences for the www.cbioportal.org), mRNA expression data for TBX3 and TBX2 in sarcoma patients  were visualized on a heatmap and clustered. The mRNA expression Z-score (RNAseq V2 RSEM) was set to default (±2). For further analysis, mRNA expression (RNA Seq V2 RSEM) data of TBX3 and TBX2 for sarcoma patients were downloaded from TCGA (https://cancergenome.nih.gov/) database using GDAC Firehose. To better understand the correlation between the expression of TBX3 and TBX2 in these patients, two cohorts of the top one-third TBX3 and TBX2 expressing samples were combined and duplicates samples were removed (N = 113). A correlation analysis was performed, a Spearman correlation coefficient (rs) was calculated and a scatter plot was generated for TBX3 and TBX2 expression using GraphPad Prism 8.0.Using cBioPortal (t-tests. Spearman correlation coefficient (rs) was calculated for TBX3 and TBX2 mRNA expression in TCGA samples using GraphPad Prism 8.0. Statistical significance are denoted by asterisks as indicated in figures with “***”, “**”, and “*” for p-value ≤ 0.001, 0.01, and 0.05, respectively.Data are presented as means ± standard errors. Statistical comparisons were performed using unpaired two-tailed Student’s Supplemental Table 1Supplemental Table 2Supplemental File


This article contains quality of life data experienced by individuals before and after implantation of a press-fit or screw-type osseointegrated fixation when fitted with conventional socket-suspended and bone-anchored limb prosthesis, respectively. This specifically-designed survey was developed and administered by Queensland Artificial Limb Services (QALS), an Australian State government organization. It was an integrated part of QALS′ continuous quality improvement procedure for assessing the provision of bone-anchored prosthesis. A total of 12 out of the 65 consumers completed to the survey, giving a return rate of 18%. This benchmark information can contribute to inform the design of (A) other patients' experience surveys including those built-in governmental continuous quality improvement procedure as well as (B) clinical trials looking at the overall effects of surgical implantation of ossoeintegrated fixation on patients' quality of life. Online repository contains the files:  This cohort represented circa 16% and 7% of existing population estimated at 400 in Australia and 950 worldwide, respectively 6.1.2This specifically-designed survey data on the quality of life was administered by Queensland Artificial Limb Services (QALS), an Australian State government organization, as an integrated part of its continuous quality improvement procedure for assessing the provision of bone-anchored prosthesis •7 (28%) questions about “Osseointegration Surgery Details” ,•5 (20%) questions about “Pre-Osseointegration Surgery” ,•13 (52%) questions about “Post-Surgery Osseointegration” .First, participants were required to indicate their name, address, date of birth and email. Then, participants answered 25 questions organized around the three following sections:The 65 eligible consumers were asked to participate in this study over the phone by a QALS' agent. Consumers could choose if they preferred receiving the survey by email or by post with pre-paid return envelope.6.1.3•○4 (16%) questions focusing on efficacy, particularly the level of function ,○1 (4%) question focusing on experience, particularly the level of satisfaction ,○3 (12%) questions focusing on knowledge of the osseointegration treatment , particularly the motivation for considering the procedure and the sources of information considered where promotional information included TV and social media.7 (28%) questions providing baseline outcomes that related to the quality of life of QALS′ consumers fitted with socket-suspended prosthesis before implantation of osseointegrated fixation including:•○2 (8%) questions focusing on surgery , particularly the time of the surgery and the level of satisfaction with the procedure,○2 (8%) questions focusing on safety or harms , particularly the occurrence of infection 17 (68%) questions assessing the quality of life of QALS′ consumers fitted with bone-anchored prosthesis after implantation of osseointegrated fixation including:•○4 (16%) questions focusing on efficacy, particularly the level of function ,○1 (4%) question focusing on experience, particularly the level of satisfaction ,○3 (12%) questions focusing on knowledge of the osseointegration treatment , particularly the motivation for considering the procedure and the sources of information considered where promotional information included TV and social media.7 (28%) questions providing baseline outcomes that related to the quality of life of QALS′ consumers fitted with socket-suspended prosthesis before implantation of osseointegrated fixation including:•○2 (8%) questions focusing on surgery , particularly the time of the surgery and the level of satisfaction with the procedure,○2 (8%) questions focusing on safety or harms , particularly the occurrence of infection ○6 (24%) questions focusing on efficacy or benefits , particularly the level of function ○6 (24%) questions focusing on overall experience , particularly the limitations and level of satisfaction.17 (68%) questions assessing the quality of life of QALS′ consumers fitted with bone-anchored prosthesis after implantation of osseointegrated fixation including:•1 (4%) general comments provided by consumers focusing on limitation of their observation time as well recommendations, benefits and shortcomings of the treatment .Analysis of the survey data consisted in extracting information for:Answers to the 10 (40%) dichotomous questions  were expressed in percentage of individual responses .Answers to the 3 (12%) Likert-type scale questions were expressed as percentage of participants in each of the 10 levels between 0 for “not satisfied” and 10 for “very satisfied” .○6 (24%) questions focusing on overall experience , particularly the limitations and level of satisfaction.•1 (4%) general comments provided by consumers focusing on limitation of their observation time as well recommendations, benefits and shortcomings of the treatment .Answers to the 12 (48%) open-ended questions were coded accordingly to the recurrence of themes in the replies .Answers to the 10 (40%) dichotomous questions  were expressed in percentage of individual responses .Answers to the 3 (12%) Likert-type scale questions were expressed as percentage of participants in each of the 10 levels between 0 for “not satisfied” and 10 for “very satisfied” .Answers to the 12 (48%) open-ended questions were coded accordingly to the recurrence of themes in the replies .7Only aggregated data was presented in this study. Exploration of more detailed analysis revealed that proportionate and disproportionate stratification sampling were unattainable given the diversity of case-mix and the small number of respondents.


Rotavirus A (RVA) strains bearing the DS-1-like constellation of the non-G, non-P genes (hereafter referred to as the genotype 2 backbone) requires better understanding of their evolutionary relationship. However, within a genotype, there is lack of a consensus lineage designation framework and a set of common sequences that can serve as references. Phylogenetic analyses were carried out on over 8,500 RVA genotype 2 genes systematically retrieved from the rotavirus database within the NCBI Virus Variation Resource. In line with previous designations, using pairwise comparison of cogent nucleotide sequences and stringent bootstrap support, reference lineages were defined. This study proposes a lineage framework and provides a dataset ranging from 34 to 145 sequences for each genotype 2 gene for orderly lineage designation of global genotype 2 genes of RVAs detected in human and animals. The framework identified five to 31 lineages depending on the gene. The least number of lineages (five to seven) were observed in genotypes A2 (NSP1), T2 (NSP3) and H2 (NSP5) which are limited to human RVA whereas the most number of lineages (31) was observed in genotype E2 (NSP4). Sharing of the same lineage constellations of the genotype 2 backbone genes between recently-emerging, unusual G1P[8], G3P[8], G8P[8] and G9P[4] reassortants and many contemporary G2P[4] strains provided strong support to the hypothesis that unusual genotype 2 strains originated primarily from reassortment events in the recent past involving contemporary G2P[4] strains as one parent and ordinary genotype 1 strains or animal RVA strains as the other. The lineage framework with selected reference sequences will help researchers to identify the lineage to which a given genotype 2 strain belongs, and trace the evolutionary history of common and unusual genotype 2 strains in circulation.Recent increase in the detection of unusual G1P[8], G3P[8], G8P[8], and G9P[4]  Rotaviruses A (RVA) within the genus Rotavirus of the Reoviridae family, are a major cause of severe acute gastroenteritis in children and the young of various mammals and birds. RVAs possess a triple-layered capsid that contains a genome of 11 segments of double-stranded RNA encoding six structural viral proteins  and five to six non-structural proteins (NSP1-NSP5/NSP6) ,,40], andThe genotype 2 genes were found in RVA of human as well as a diverse range of animal host species origin. Examination of over 8,500 RVA genotype 2 genes retrieved from the NCBI Viral Genomes Resource  revealedOut of 724–1,326 sequences per gene of both human and animal rotaviruses , retrievThe maximum likelihood phylogenetic trees representing the framework for the genotype 2 backbone genes are shown in Figs A few exceptions need to be noted in which bootstrap support was lower than 70%, yet lineages previously identified by Doan et al.  were maiAs to the number of lineages, it was noted that lineages were more diverse in genes whose genotype was shared between human and animal RVA strains  among 27 emergent reassortant strains . There were four constellations in G1P reassortSecondly, no G2P strains Thirdly, unlike other emergent reassortant strains, there was no corresponding G2P strain wExamining clinical and surveillance specimens of RVA infecting humans at the level of the whole genotype constellation has provided a grand view of Wa-like, genotype 1 RVA originating from porcine RVA and DS-1-like, genotype 2 RVA originating from bovine RVA in the evolutionary perspective   8] and GS1 FigSequences were retrieved using the virus variation resource based on the search criteria described in the results. Frequency of occurrence of each host species within each genotype 2 gene was tallied and plotted against the genome segments. The legend on the right side indicates the Latin names of the host species together with the corresponding colour in the histogram.(PPTX)Click here for additional data file.S1 File(ZIP)Click here for additional data file.


Clear cell sarcoma of the kidney (CCSK) is a rare malignant pediatric renal neoplasm with a heterogeneous histological appearance which often results in misdiagnosis. There are no specific immunohistochemical markers which can help in differentiating CCSK from other pediatric renal neoplasms. Recently Cyclin D1 has been investigated as a possible marker in this regard. In this study, we aim to determine the usefulness of Cyclin D1 in differentiating between CCSK and other pediatric renal neoplasms and to compare our results with those of recently published studies.A total of 48 cases of CCSK, Wilms tumor (WT), renal rhabdoid tumor, mesoblastic nephroma, renal Ewing sarcoma and neuroblastoma were included in the study. All cases were stained with cyclin D1. Extent of Cyclin D1 staining was graded according to percentage of positive tumor cells as diffuse (> 70%), focal (5 to 70%), and negative (< 5%). Intensity of Cyclin D1 staining was graded as strong or 3+, moderate or 2+ and weak or 1 + .Most or all cases of CCSK, neuroblastoma and renal Ewing sarcoma demonstrated diffuse and strong positivity for Cyclin D1. Most cases of Wilms tumor  also demonstrated diffuse and often strong positivity for Cyclin D1. In most cases of WT, blastemal component was negative.Cyclin D1 is a sensitive but not specific immunohistochemical marker for CCSK and many other pediatric renal malignant neoplasms as well as for neuroblastoma. Hence, careful examination of histological features is important in reaching an accurate diagnosis in CCSKs. However, Cyclin D1 is very helpful in distinguishing between blastema-rich WT and CCSK. Clear cell sarcoma of kidney (CCSK) is an uncommon mesenchymal renal tumor of uncertain histogenesis which comprises approximately 3 to 4% of malignant pediatric renal neoplasms. It occurs in young children and mean age at diagnosis is approximately 36 months. It is more common in males , is centered in the renal medulla and is almost always unifocal. It is usually circumscribed but unencapsulated with a tan, soft cut surface and most tumors are quite large in size. On histological examination, classic CCSK is characterized by nests or cords of epithelioid cells with round to oval nuclei. Myxoid pools and thin and regularly branching fibrovascular septa separate the cords of tumor cells [YWHAE-FAM22- NUTM2B/E gene fusion which is also seen in high grade endometrial stromal sarcoma. Recently, CCSK has also been shown to consistently demonstrate BCOR gene abnormalities including exon 15 internal tandem duplications and BCOR-CCNB3 gene fusion which distinguish it from other pediatric renal tumors. Metastases can occur as late as ten years after initial diagnosis. Owing to the essential role of doxorubicin in the therapy of CCSK, it is imperative that pathologists identify it accurately. Failure to do so can prevent a child from getting optimal chemotherapy [However, CCSK is very heterogeneous and diverse in histological appearance and a number of variant histological patterns may be seen. It has a propensity to metastasize to bone, and “bone metastasizing renal tumor of childhood” is one of its synonyms . In the otherapy –18.BCOR gene abnormalities are not available especially in developing countries such as ours. Recently, a number of studies have suggested that immunohistochemical stain Cyclin D1 is useful in distinguishing CCSK from some pediatric renal neoplasms [Owing to its marked histological heterogeneity, CCSK can be mimicked by a number of malignant pediatric renal and extrarenal neoplasms including blastema-rich Wilms Tumor (WT), mesoblastic nephroma, neuroblastoma etc. It is often difficult to diagnose CCSK from these tumors on morphology alone , 5. Untieoplasms , 5, 19. eoplasms , 15.The aim of the present study was to determine the usefulness of Cyclin D1 in distinguishing CCSK from other pediatric renal neoplasms and to see whether our findings match those in other recently published studies.The surgical pathology files of the Section of Histopathology, Department of Pathology and Laboratory Medicine, Aga Khan University Hospital and cases were submitted from French Medical Institute for Mothers and Children were searched for cases of CCSK, WT (nephroblastoma), renal rhabdoid tumor (RT), congenital mesoblastic nephroma, Ewing sarcoma of kidney as well as retroperitoneal neuroblastoma reported over a ten year period i.e. January 1, 2008 and December 31, 2017. A total of 48 cases were included in the study. These included 19 cases of CCSK, 9 cases of WT and 4 cases each of renal RT, Mesoblastic nephroma and Ewing sarcoma. In addition, 8 cases of neuroblastoma were also included. Although neuroblastomas are not renal tumors but are retroperitoneal like the former and occur in the same age group as pediatric renal tumors. Due to these features they need to be distinguished from pediatric renal tumor. Hence, they were included in the study.All cases were reviewed by the two principal authors (NU and ZA). A number of immunohistochemical markers including Vimentin , Antismooth muscle actin , S100 protein , CD99 , WT1  protein, clone 6F-H2–7, ready to use, DAKO, Glostrup, Denmark), Neurofilament , CD56 , Synaptophysin , Desmin , Cytokeratin AE1/AE3 , Epithelial membrane antigen  were performed in different cases at the time of initial reporting and were reviewed by the authors. Cyclin D1  was retrospectively performed in all 48 cases. The staining for Cyclin D1 was performed on DAKO automated immunostainer. The antibody was optimized using Ventana DAB detection kit  and standard quality control procedures were performed. Cyclin D1 staining was performed following heat induced antigen retrieval using ER2 antigen retrieval buffer. Cases of mantle cell lymphoma were used as positive control. Negative controls were also used. For the purpose of study, extent of Cyclin D1 staining was graded according to percentage of positive tumor cells as diffuse (> 70%), focal (> 5 to 70%) or negative (< 5%); intensity of staining was graded as strong or 3+ (nuclear intensity similar to that of mantle cell lymphoma control), moderate or 2+ (definite nuclear staining weaker than 3+ but easily identifiable at × 40 magnification) and weak or 1+ (nuclear staining identifiable only at high power magnification). This scale for extent and grading was based on that used in two studies published in 2015 [A total of 48 cases were included in the study. These included 19 cases of CCSK, 9 cases of WT and 4 cases each of renal RT, Mesoblastic nephroma and Ewing sarcoma. In addition, 8 cases of neuroblastoma were also included..Patients with CCSK ranged in ages from 1 to 29 years with means and median age of 5.5 and 3 years respectively. Of the 19 patients, 13 (68.4%) were males while 6 (31.6%) were females. Tumor was located in left and right kidney in 8 cases each. In 3 cases, laterality was not known. Tumor size ranged from 8 cm to 14.5 cm with mean tumor size of 11 cm in greatest dimensionAll 19 cases of CCSK demonstrated reactivity for Cyclin D1. In 13 cases (68.4%), intensity of staining was 3+, in 4 cases (21%), intensity was 2+, while in 2 cases (10.5%), it was 1+. Extent of staining ranged from 5 to 90% with average extent of staining being 54.5%  were females, while 3 (33.3%) were males. Ages ranged from 2.5 to 7 years. Mean and median ages were 4.5 and 4 years respectively. In 6 patients (66.7%), tumor was located in the left kidney while in 3 (33.3%), tumor was located in the right kidney. Tumor size was available in 8 out 9 cases and ranged from 7.5 to 17.4 cm in largest dimension with mean tumor size of 11.5 cm.Out of 9 cases of renal WT, 2 did not show any epithelial component and tumor was composed entirely of blastema and stromal components (biphasic). In these 2 cases, Cyclin D1 was negative in both components in 1 case, and showed weak positivity in blastemal component in the other case. The remaining 7 cases showed strong (3+) positivity in the epithelial component (tubules), while blastemal component was negative in all cases. Extent of staining ranged from 5 to 70%. Average extent of staining was 37.5%  were males while 1 (25%) was female. Tumor was located in right kidney in 3 cases (75%) and left kidney in 1 case (25%). Tumor size ranged from 7 to 9 cm with mean size of 8 cm in largest dimension.Out of 4 cases of congenital mesoblastic nephroma, 3 were cellular type and 1 was of mixed type and all demonstrated reactivity for Cyclin D1. Intensity of staining was 3+ in all 4 cases. Extent of staining ranged from 10 to 50% and average extent of Cyclin D1 staining was 30% Fig. C, D. AntPatients with renal RT ranged from 1 year to 14 years in age with mean and median age of 4 and 1.5 years respectively. Of the 4 patients, 3 (75%) were males and 1 (25%) was female. Tumor was located in left kidney in 2 cases (66.7%) and in right kidney in 1 case (33.3%). Tumor laterality was not known in 3 cases. Tumor size ranged from 7 cm to 9 cm in maximum dimension. Mean tumor size was 7.5 cm.Out of 4 cases of renal RT, 3 (75%) demonstrated reactivity for Cyclin D1, intensity of staining was 2+ in all 3 cases while extent of staining ranged from 10 to 30%. Average extent of staining was 20%. Vimentin was positive in all 4 cases. EMA was performed in 3 cases and was positive in all 3. All 4 cases showed loss of INI 1.The ages of 4 patients with renal Ewing sarcoma ranged from 2 to 36 years with mean and median age of 20 and 35 years respectively. Of the 4 patients, 2(50%) were males and 2 (50%) were females. Of the 4 cases, 3 were located in the right kidney (75%) while 1 (25%) was located in the left kidney (25%). Tumor size ranged from 2.5 to 17 cm in largest dimension. Mean tumor size was 11.7 cm.Out of the 4 cases of renal Ewing sarcoma, 3 (75%) demonstrated staining for Cyclin D1. Of these 3 cases, intensity of staining was 3+ in 2 cases (66.7%) and 2+ in 1 case (33.3%). Extent of staining ranged from 10 to 30%. Average extent of Cyclin D1 staining was 16.7%  were females while 3 (37.5%) were males.Out of 8 cases of neuroblastoma in our series, 8(88.9%) demonstrated intense 3+ reactivity for Cyclin D1. Extent of Cyclin D1 staining ranged from 5 to 70%. Average extent of staining was 37.5%  in over 68% of cases and moderately intense (2+) in 21%. Thus over 89% cases showed moderate to marked intensity. Average extent of staining was almost 55%. These findings were indicative of diffuse and strong nuclear positivity for Cyclin D1 in the large majority of CCSK and were similar to the findings in other recent studies by Aw et al. (3) and Mirkovic et al. [All 4 cases of congenital mesoblastic nephroma (CMN) in our study showed markedly intense 3+) focal to diffuse staining for Cyclin D1. All 11 cases of CMN in Mirkovic et al’s study showed focal (4 cases) to diffuse (17 cases) moderate to severely intense Cyclin D1 staining . This sh+ focal tSimilarly, 7 out of 9 cases of WT in our study demonstrated intense 3+ staining for Cyclin D1 in their epithelial component with average extent of staining of almost 40%. All 4 cases of NB in Mirkovic et al.’s study also demonstrated diffuse moderate to strong nuclear staining for Cyclin D1 . Thus, COf the 4 cases of renal Ewing sarcoma in our series, 3 75%) demonstrated focal to diffuse staining for Cyclin D1 which was very intense in 2 cases. All 5 cases of renal Ewing sarcoma in the study by Mirkovic et al. % demonst.Of the 4 cases of renal RT in our study, 3 75%) showed moderately intense staining for Cyclin D1 which ranged in extent from 10 to 50% with average extent of staining of 30%. This finding is in contrast to the results in the study by Mirkovic et al. % showed . All 4 cAll 8 cases of neuroblastoma in our study demonstrated intense 3+ staining for Cyclin D1 which ranged in extent from as low as 5% to as high as 90%. In fact, 6 out of 8 cases (75%) showed diffuse 3+ positivity which ranged in extent between 70 and 90%. This finding is similar to the findings by Mirkovic et al. [As discussed by both Mirkovic et al.  and Aw eCyclin D1 is a sensitive but not specific immunohistochemical marker for CCSK and many other pediatric renal malignant neoplasms as well as for neuroblastoma. Hence, careful examination of histological features is important in reaching an accurate diagnosis in CCSKs. Cyclin D1 however is very helpful in distinguishing between CCSK and blastema-rich WT which is arguably the most difficult morphologic distinction in the differential diagnosis of CCSK. Our results were similar to two other recently published studies , 5. Howe


The use of postoperative radiation therapy after breast-conserving surgery was longstanding standard practice. The treatment protocol used a standard fractionation of 50 Gy in 25 fractions plus a boost. Recently, the hypofractionation approach has gained support based on Canadian and English studies that claimed equal tumor control and similar toxicity to the standard protocol.We conducted a review of the literature of hypofractionation studies and compared the reported toxicity with the general literature. We placed special emphasis on breast fibrosis after hypofractionation versus standard fractionation. We found a striking difference in the breast toxicity reported by the hypofractionation literature regarding breast fibrosis as compared to standard fractionation. Breast fibrosis should be explored further via additional studies and discussed with potential breast-conserving surgery patients. Recently, the American Society of Radiation Oncology released a task force guideline recommending hypofractionated radiotherapy for all women of any age whether they had received chemotherapy or not . Their eAccording to Hall, “laboratory data proved that fewer and large dose fractions result in more severe late reactions, even though the early reactions are matched by an appropriate adjustment in total dose” . Hall alWe calculated the biological effective dose (BED) for both protocols using α/β of 3 Gy for late tissues and α/β of 10 Gy for early tissues.In regards to the standard fractionation (50 Gy in 2-Gy fractions), the BED for late tissues was 83.3 Gy and 60 Gy for early tissues where:BED = nd (1 + n/ (α/β) )Comparatively, for hypofractionation (41.6 Gy in 3.2 Gy per fraction), the BED for late tissues was 86 Gy and 55 Gy for early tissues.Table The UK Standardization Breast Radiotherapy START Trial A and B found that breast appearance and breast hardness were the most common changes among the two groups but the results were similar (42.6% for the 50 Gy in two fractions and 44.6% for the 41.6 Gy in 3.2 Gy per fraction or 39 Gy in 3 Gy per fraction) [START Trial A showed no significant difference between the breast hardness caused by 50 Gy in 2-Gy fractions and 40 Gy in 15 fractions. The trial reported 38.2% of the breasts were “Harder” with hypofractionation versus 42.3% for standard fractionation.Leong et al. found no added swelling to the arm using hypofractionation. The hypofractionation daily dose was 2.25 Gy to 2.5 Gy to a total of 45 Gy to 40 Gy, respectively. The conventional fractionation ranged between 1.8 Gy and 2 Gy to a total dose of 45 Gy to 50.4 Gy, respectively. Of 1,759 patients, 708 were evaluated (40%). The two groups were not equal, 406 patients received hypofractionation, and 302 received conventional fractionation [Whelan et al. reported no difference in toxicity between 50.0 Gy in 25 fractions over a period of 35 days and a dose of 42.5 Gy in 16 fractions. Grade two subcutaneous toxicity had tripled in 10 years as compared to the 30% increase for standard fractionation [Yarnold et al. found that the probability of moderate/marked breast induration after 50 Gy in 2-Gy fractions was between 39 Gy in 13 fractions and 42.9 Gy in 13 fractions with fewer side effects for the 3.3-Gy fraction size .Reddy et al. compared hypofractionation versus standard fractionation for 287 women treated from 2011 to 2017 (42.56 Gy in 16 fractions plus 10 Gy to 12.5 Gy in four to five fractions boost or 50 Gy in 25 fractions plus 10 Gy to 14 Gy in five to seven fractions). The cosmetic assessment was dependent on photographs and the patient’s own assessment. There was no report of breast fibrosis. They reported similar or improved cosmetic outcomes with hypofractionation as compared to standard fractionation .These studies showed a high rate of breast fibrosis with hypofractionation and a similarly high rate of breast fibrosis with standard fractionation.Haviland reported more side effects with a fractionation schedule of 13 sessions at 3.3 Gy versus 50 sessions at 2 Gy or 39 sessions at 3 Gy. There was a statistically significantly increased rate of physician-assessed shoulder stiffness in the 42.9-Gy schedule compared to the 50-Gy treatment in the START pilot . There was no such effect reported for the hypofractionated schedules in START-A and START-B . Table 2Xiaofeng et al. found that 50% to 60% of the patients had developed mild to moderate late breast fibrosis one year after hypofractionation radiation therapy (40 Gy plus simultaneous cavity boost to 48 Gy in 15 fractions) . Yu et aDe La Rochefordiere et al. found that using a dose of more than 2 Gy per fraction is associated with worse cosmesis . Clark eThere is some discrepancy between hypofractionation studies and many other literature reports about the incidence of breast fibrosis reported for standard fractionation. Breast fibrosis can be a potential side effect of hypofractionated radiotherapy that needs to be further studied.


After publication of this article , the autSoon before the Correction of Parental TW02, 500 and TW02/CD24-, 500Parental TW02, 1000 and TW02/CD24-, 1000Parental TW04, 100 and TW04/CD24-, 100TW04/CD24+, 100 and TW04/CD24-, 500The authors provided an updated version of this figure in which the TW02 CD24-, TW04 parental, and TW04 CD24- panels are replaced with different images. The authors also provided images of mice from the original experiment see , but somWhen the authors contacted the journal to correct Please see the corrected Figs PLOS ONE Editors have concerns about the strength of evidence supporting the in vivo claims made in the article and, therefore, issue this Expression of Concern.We regret that the journal did not correct Figs S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file.S3 File(XLSX)Click here for additional data file.


Tai Ji Quan vs Multimodal and Stretching Exercise Interventions for Reducing Injurious Falls in Older Adults at High Risk of Falling: Follow-up Analysis of a Randomized Clinical Trial,”1 published February 15, 2019, a data error occurred in the Intervention Adherence subsection of the Results section. The third sentence should have read, “A total of 158 participants in the TJQMBB group (70.5%), 160 in the multimodal exercise group (71.7%), and 159 in the stretching exercise control group (71.3%) attended 37 or more sessions .” This article has been corrected.1In the Original Investigation titled “Effectiveness of


Proton pump inhibitors (PPIs) are a mainstay treatment for gastroesophageal reflux disease (GERD) and are mainly metabolized by CYP2C19 in the liver. However, several polymorphisms of CYP2C19 exist that affect the metabolism of PPIs. Due to the large variability of PPI pharmacokinetics among the polymorphisms, this has implications in the management of patients with refractory GERD who may be potentially undertreated. Herein, we discuss the role of CYP2C19 and its relation to PPI therapy, particularly in those with GERD. Heartburn is a symptom commonly experienced by many, but it may develop into gastroesophageal reflux disease (GERD) when heartburn occurs twice weekly or more. GERD is a chronic condition where the reflux of gastric contents into the esophagus causes symptoms (e.g. heartburn) or complications. GERD affects approximately 10–20% of those in Western countries and is becoming progressively prevalent in developing countries [CYP2C19*17 allele is an ultra-rapid metabolizer (UM) of CYP2C19 substrates and may be prone to inadequate treatment of GERD due to the PPI being metabolized much faster than usual. The Dutch Pharmacogenetics Working Group (DPWG) developed specific dosing recommendations for PPIs for UMs with insufficient responses: increase by 50–100% with esomeprazole, increase by 200% with lansoprazole, increase by 100–200% with omeprazole, and increase by 400% with pantoprazole [CYP2C19*2 or *3 loss-of-function alleles have shown to have higher cure rates of GERD due to decreased clearance of the PPI. The DPWG has no recommendations for dose adjustments of PPIs in the PM population.Polymorphisms in CYP2C19 may increase or decrease metabolism of PPIs, which ultimately affect the treatment of patients with GERD who may have these polymorphisms. A patient who homozygously expresses the oprazole . RabepraCYP2C19 allele and anti-reflux surgery (ARS) in those with medically refractory GERD [CYP2C19*1/*17 and *17/*17 genotypes were a significant predictor of ARS compared to the non-ARS controls [A recent study investigating CYP2C19 extensive metabolizer (EM) and UM phenotypes in children found an association with the presence of the ory GERD . Pediatrcontrols .With pharmacogenetic testing, determining a patient's CYP2C19 phenotype would aid in finding potential sources of treatment failure of GERD earlier on, leading to fewer complications and increased quality of life . Althoug


Xenopus neural plate, PCP is marked by the enrichment of the conserved proteins Prickle3 and Vangl2 at anterior cell boundaries. Here we show that the apical determinant Par3 is also planar polarized in the neuroepithelium, suggesting a role for Par3 in PCP. Consistent with this hypothesis, interference with Par3 activity inhibited asymmetric distribution of PCP junctional complexes and caused neural tube defects. Importantly, Par3 physically associated with Prickle3 and promoted its apical localization, whereas overexpression of a Prickle3-binding Par3 fragment disrupted PCP in the neural plate. We also adapted proximity biotinylation assay for use in Xenopus embryos and show that Par3 functions by enhancing the formation of the anterior apical PCP complex. These findings describe a mechanistic link between the apical localization of PCP components and morphogenetic movements underlying neurulation.Vertebrate neural tube formation depends on the coordinated orientation of cells in the tissue known as planar cell polarity (PCP). In the  Drosophila genetic studies. In Drosophila epithelial tissues, PCP is manifested by the distribution of the Frizzled/Dishevelled and Prickle/Van Gogh membrane complexes to opposite domains inside each cell (Planar cell polarity (PCP) is a common phenomenon that refers to the orientation of cells in the plane of the tissue. PCP requires the functions of several conserved core proteins, Prickle, Van Gogh/Strabismus, Dishevelled, Frizzled and Flamingo/Stan, originally identified in ach cell . In addiach cell . Disruptach cell . The exiach cell . Howeverapical cell corners  and recruits it to the apical cell membrane. Demonstrating the importance of this interaction, a specific Pk3-binding domain of Par3 interfered with the polarization of neuroepithelial cells. To further study PCP mechanisms, we developed an efficient in vivo proximity biotinylation approach using Pk3 fused to a bacterial biotin ligase. Using this assay, we demonstrate a novel role of Par3 in promoting the interaction of Pk3 and Vangl2 in neuroepithelial cells. These findings link the subcellular localization of two core PCP components to morphogenetic events underlying vertebrate neural tube closure.To address this issue, we examined the localization and function of Par3 in the Xenopus neural plate, using characterized anti-Par3 antibodies  with different sequences and confirmed their efficacy. . UnilatePrickle, Espinas and Testin) domain  domain , consist) domain . These iXenopus embryos using conventional pull-downs (data not shown). We then decided to examine this interaction using proximity biotinylation, a highly sensitive approach  that disrupts Par3 localization and function in Xenopus embryos and mammalian cultured cells , 5’-GCTTCAGCTAGTGACACATGCAT-3’; control MO2 (CO MO2), 5’- AGCGTTTCAGGCCGATCTCTCAGTC-3’. Vangl2 MO 5’-GAGTACCGGCTTTTGTGGCGATCCA-3’ . For depletion studies, the following MOs were purchased from Gene Tools : Par3MOATCCA-3’ .Xenopus laevis PRID:NXR_0.0095 and cultured in 0.1x Marc’s Modified Ringer’s solution (MMR)  and injected with 5–10 nl of a solution containing RNAs and/or MOs. For mosaic expression of PCP complexes, embryos were injected into two dorsal blastomeres of 16–32 cell embryos. Amounts of injected mRNAs per embryo have been optimized in preliminary dose-response experiments (data not shown) and are indicated in figure legends.In vitro fertilized eggs were obtained from on (MMR)  as descron (MMR) . Stagingon (MMR) . For micFor phenotype analysis, frequencies of neural tube defects were calculated as means ± s. d. In unilaterally injected embryos, neural tube was scored as defective when the distance between the neural fold and the midline (white line) at the injected side was at least 1.5 times of that at the uninjected side. Body axis extension was estimated by measuring the length of stage 26 embryos. Blastopore defects were scored at stage 12, the defects were considered mild if the blastopore diameter was more than twice of that of uninjected embryos and severe when the blastopore groove was not visible.HEK293T cells (ATCC) were maintained in Dulbecco’s modified Eagles medium (Corning) with 10% fetal bovine serum (Sigma) and penicillin/streptomycin (Sigma). This cell line was tested and found negative for mycoplasma contamination. Cells growing at 50–70% confluence were transiently transfected using linear polyethylenimine  as described . Each 60ζ. Secondary antibodies were against mouse or rabbit IgG conjugated to Alexa Fluor 488, Alexa Fluor 555  or Cy3 . Cryosections and explants were mounted for observation with the Vectashield mounting medium (Vector). Standard specificity controls were performed to confirm lack of cross-reactivity and no staining without primary antibodies. Images that are representative of at least 10 different fields were captured using a Zeiss AxioImager microscope with the Apotome attachment . The data shown are from two to five independent experiments with 5–15 embryos per group. Quantification for Par3 and ZO1 distribution in the neuroepithelium has been carried out using ImageJ as described  for 40 min, washed in PBS and dissected. Cryosectioning and immunostaining were performed as described . Embryosescribed . Signifi3VO4, 10 mM NaF), containing cOmplete Mini EDTA-free protease inhibitor cocktail (Roche). After centrifugation at 16,000 g, the supernatant was incubated with anti-FLAG agarose beads (Sigma) at 4°C for 2 hr or with anti-Myc antibodies (9E10) for 2 hr and Protein A Sepharose  at 4°C for 2 hr. Myc-trap beads (Chromotek) were used to pull-down Par3-containing protein complexes. The beads were washed three times in lysis buffer, and subjected to SDS-PAGE and immunoblotting using standard protocols , or HA-Vangl2 (50 pg). Embryos were collected at stages 13–14 and protein biotinylation was assessed in embryo lysates and pulldowns with mouse anti-HA (12A5) and anti-Myc (9E10) hybridoma supernatants, rabbit anti-Par3  antibodies. Proteins were detected by immunoblotting with goat anti-biotin-HRP antibodies , rabbit anti-Par3 and anti-HA , and mouse anti-Myc, anti-FLAG (M2 Sigma) and anti-GFP  antibodies. In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.eLife. Your article has been reviewed by three peer reviewers, including Yukiko M Yamashita as the Reviewing Editor and Reviewer #1, and the evaluation has been overseen by Marianne Bronner as the Senior Editor.Thank you for submitting your article "Par3 interacts with Prickle3 to generate apical planar cell polarity complexes in the vertebrate neural plate" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Xenopus neural plate. Morpholino-based Par3 knockdown revealed defects in polarization of planar cell polarity proteins Vangl2 and Pk3. The authors used co-IP and proximity biotinylation assays to show binding of Par3 with Pk3 and identify the critical domains in Par3 for the binding, and reveal an important role of Par3 in localization of Pk3 to the apical membrane domain. They show that the overexpression of a Prickle3-binding Par3 fragment disrupted PCP in the neural plate. The authors finally demonstrate that Par3 is necessary to promote Pk3-Vangl2 interaction.The authors of this manuscript reveal the role of Par3 apical-basal polarity protein in regulation of planar cell polarity. They found planar polarization of Par3 in the This is an exciting study that reveals novel role for the vertebrate Par3 protein and discovers a novel physical and functional connection between apical-basal and planar cell polarity pathways. The experiments are well done and the interpretations are appropriate. Conclusions are well supported by the evidence. The reviewers raised the following major concerns to be addressed prior to publication.Essential revisions:1) The authors assume that Par3 is upstream of Pk3/Vangl2. What if Par3 and Pk3/Vangl2 reinforce each other localization and function? Vangl2 is not showing proper planar polarization without Par3 . Is Par3 showing planar polarization without Vangl2?2) The authors assume that expression of Par3[274-544] generated phenotypes because it disrupts interaction between endogenous Par3 and Pk3. This disruption of Par3 and Pk3 interaction should be confirmed directly using conventional co-IP approach in cell line in culture.3) How Par3 promotes Pk3-Vangl2 interaction? What may be the molecular mechanism responsible? Is Par3 promoting interaction between Pk3 and Vangl2 in cell line overexpression assays?4) Why GFP-Pk3 is not localizing to the apical domain at all without Par3 overexpression ? The endogenous Par3 should be at the apical membrane domain and it should direct at least some GFP-Pk3 to the apical membrane domain.Xenopus ectoderm? At higher doses, does Par3 MO interfere with apicobasal polarity?5) The authors show that Par3 depletion does not affect apicobasal polarity. Is this due to low doses of the MO used, or is it because of a distinct function of Par3 in 6) In the presence of Par3 MO, Vangl2 is no longer seen at the apical surface. Is the protein now localized to the basolateral membrane or is it internalized into membrane vesicles?7) The effect of Par3 MO on Vangl2 distribution seems to differ from that of Par3 domains or deletion mutants on Pk3 distribution. To conclude that Par3 interacts with Pk3/Vangl2 complex, the experiments should be performed under similar conditions. For example, what happens to Pk3 with Par3 MO, or what is the distribution pattern of Vangl2 when Par3[272-544] construct is used?8) Does Par3 compete with Vangl2 for Pk3, or they form a ternary complex?9) The effect of Par3-N and Par3[272-544] on distribution of wild type Par3 should be shown, so that the mislocalization of Pk3 and the localization of endogenous Par3 can be compared and correlated. Essential revisions:1) The authors assume that Par3 is upstream of Pk3/Vangl2. What if Par3 and Pk3/Vangl2 reinforce each other localization and function? Vangl2 is not showing proper planar polarization without Par3 . Is Par3 showing planar polarization without Vangl2?We agree that Par3 and Vangl2 may potentially exhibit feedback regulation during the establishment of neural plate PCP. We performed a complementary experiment suggested by reviewers and analyzed Par3 localization in neural plates of Vangl2-depleted embryos. Indeed, Par3 planar polarity has been compromised in neuroepithelial cells depleted of Vangl2, indicating that planar polarization of Par3 requires core PCP proteins .2) The authors assume that expression of Par3[274-544] generated phenotypes because it disrupts interaction between endogenous Par3 and Pk3. This disruption of Par3 and Pk3 interaction should be confirmed directly using conventional co-IP approach in cell line in culture.Xenopus ectoderm. The interaction was measured in a proximity biotinylation assay, as the degree of biotinylation of Par3 in the presence of Biotin Ligase-tagged Prickle3. Par3[272-544] had a negative effect on this protein interaction ,supporting the view that it competes with Par3 for Prickle3 binding. Moreover, Par3[272-544] was itself biotinylated, confirming its association with Prickle3. This new experiment is described in the first paragraph of the subsection “Functional significance of the Par3-Pk3 interaction for PCP”.We found that, in HEK293T cells, the levels of Pk3 and Pk3∆PET were upregulated by Par3[272-544], an effect that we did not observe in vivo. We, therefore, examined how Par3[272-544] influences the Prickle3-Par3 association directly in 3) How Par3 promotes Pk3-Vangl2 interaction? What may be the molecular mechanism responsible? Is Par3 promoting interaction between Pk3 and Vangl2 in cell line overexpression assays?We have carried out several experiments addressing the mechanism of Par3 effects on PCP. We did not see the competition of Par3 with Vangl2 for Pk3 binding, on the contrary, we show that Vangl2 binds to Par3 in the presence of Prickle3 . Vangl2 was not detected in pulldowns with Par3 mutant lacking Pk3 binding domains, Par3∆∆, indicating that Pk3 is necessary for the Par3-Vangl2 association . The simplest interpretation of this result is the formation of the ternary complex between Par3, Prickle3 and Vangl2. Since the promoting effect of Par3 on the total amount of the Pk3-Vangl2 complex was not apparent , we propose that Par3 stimulates the interaction of Prickle3 and Vangl3 within the ternary complex.We describe this experiment in the first paragraph of the subsection “The association of Par3 with the Pk3/Vangl2 complex and the identification of Pk3-interacting domains” and in the fourth paragraph of the Discussion.4) Why GFP-Pk3 is not localizing to the apical domain at all without Par3 overexpression ? The endogenous Par3 should be at the apical membrane domain and it should direct at least some GFP-Pk3 to the apical membrane domain.We suspect that endogenous Par3 is fully engaged in preexisting binding complexes, including those containing different Prickle family members. We suspect that it may be simply unavailable for the recruitment of exogenous Prickle3.5) The authors show that Par3 depletion does not affect apicobasal polarity. Is this due to low doses of the MO used, or is it because of a distinct function of Par3 in Xenopus ectoderm? At higher doses, does Par3 MO interfere with apicobasal polarity?We have examined several apical and basal polarity markers at different doses of the MO and at additional developmental stages. These new experiments confirm our initial conclusions. Although the knockdown of Par3 is efficient and a strong effect on Vangl2 localization has been observed , we saw no obvious changes in ZO1, aPKC or β-catenin .Drosophila follicular epithelial cells in Bazooka/Par3 mutants  and in mouse retinal endothelial cells with conditionally knocked out par3 . Together, these findings suggest that the requirement for Par3 in apicobasal polarity may depend on the cell type and developmental context.Similarly, no defects in apicobasal polarity were reported in 6) In the presence of Par3 MO, Vangl2 is no longer seen at the apical surface. Is the protein now localized to the basolateral membrane or is it internalized into membrane vesicles?Cryosections revealed that Vangl2 is enriched basolaterally rather than apically after Par3 depletion .This was described in the subsection “Par3 plays an essential role in neural plate PCP”.7) The effect of Par3 MO on Vangl2 distribution seems to differ from that of Par3 domains or deletion mutants on Pk3 distribution. To conclude that Par3 interacts with Pk3/Vangl2 complex, the experiments should be performed under similar conditions. For example, what happens to Pk3 with Par3 MO, or what is the distribution pattern of Vangl2 when Par3[272-544] construct is used?We found that the staining with our Vangl2 antibodies produces a strong background in cells expressing Par3[272-544] construct. However, a new experiment with Par3 MO demonstrated that Pk3/Vangl2 patches are not anteriorly polarized in Par3-depleted cells . This result is added to the second paragraph of the subsection “Par3 is required for the formation of the apical PCP complex in the neural plate”.8) Does Par3 compete with Vangl2 for Pk3, or they form a ternary complex?Please see the response to point 3.9) The effect of Par3-N and Par3[272-544] on distribution of wild type Par3 should be shown, so that the mislocalization of Pk3 and the localization of endogenous Par3 can be compared and correlated.Prompted by the reviewers, we examined the localization of myc-Par3 in cells expressing Par3N and Par3[272-544]. Par3[272-544] did not affect apical localization of Par3  as described in the first paragraph of the subsection “Functional significance of the Par3-Pk3 interaction for PCP”, consistent with our assumption that the primary effect of this construct is on Pk3 binding. Par3N caused a pronounced decrease in apical Par3, as shown previously in cultured cells . This result is shown in Figure 7—figure supplement 1 and mentioned in the first paragraph of the subsection “Par3 is required for the formation of the apical PCP complex in the neural plate”.