In contrast to the pure P2W17La films, the nanocomposite exhibits enhanced electrochromic and electrochemical performance owing to the 3D sea-cucumber-like microstructure. An EESD using the NW–P2W17La film as the cathode exhibited outstanding electrochromic and energy storage properties, with high optical modulation (48.6% at 605 nm), high switching speeds , and high area capacitance (5.72 mF cm−2 at 0.15 mA cm−2). The device can reversibly switch between transparent and dark blue during the charge/discharge process, indicating that electrochromic contrast can be used as a quantitative indicator of the energy storage status.The key challenge in the practical application of electrochromic energy storage devices (EESDs) is the fabrication of high-performance electrode materials. Herein, we deposited K Amidst increasing environmental pollution and the gradual exhaustion of non-renewable energy resources, the development of controllable devices for energy storage, conversion, and protection, such as smart windows ,2,3, mic3  (abbreviated as P2W17La) nanoparticles onto TiO2 nanowires (TiO2 NW) to obtain an electrochromic energy storage bifunctional nanocomposite (NW–P2W17La) via a combined hydrothermal and layer-by-layer (LbL) self-assembly method. The 3D sea-cucumber-like microstructure of NW–P2W17La enhanced the coverage of the surface, exposed more reactive sites, and shortened the electron/ion diffusion pathways, resulting in rapid reaction kinetics and enhanced electrochemical performance. The optical and electrochemical properties of the NW–P2W17La nanocomposite were investigated and compared to those of a densely packed structure. An asymmetric EESD was fabricated using an NW–P2W17La film as the cathode and a TiO2 nanowire film as the anode.There is currently a dearth of research on POM-based EESDs. Recently, our research group investigated several high-performance electrochromic energy storage bifunctional composites containing saturated , lacunar2W17La nanocomposite film. UV/Vis spectroscopy was used to track the growth of the composite film. The deposition of each layer of P2W17La was accompanied by a linear increase in absorbance, confirming that the growth of the multilayer film obtained via electrostatic self-assembly was uniform and orderly, and that the convenient LbL method can achieve controlled thin-film growth (m growth .2W17La). Scanning electron microscopy (SEM) images of the FTO–P2W17La film and NW–P2W17La film are shown in 2W17La film exhibits a tightly packed microstructure; the FTO surface is densely covered with aggregates of P2W17La and polyetherimide (PEI), forming a cobblestone-like morphology. 2 nanowires obtained via hydrothermal synthesis. Compared with the pure TiO2 NW film, the nanowires in the NW–P2W17La film are thicker and denser because of the encapsulation of P2W17La nanoparticles and PEI, forming a 3D sea-cucumber-like microstructure. The TiO2 nanowires act as a skeleton on which the POM nanoparticles are deposited, thereby avoiding aggregation and exposing more reactive sites. The cross-sectional image shows that the NW–P2W17La composite film can reach a thickness of 620 nm, whereas the FTO–P2W17La film is approximately 110 nm thick. This demonstrated that the 3D sea-cucumber-like microstructure of NW–P2W17La enhanced the coverage of the surface and provided more electron/ion diffusion pathways. Additionally, as shown in 2W17La composite, confirming that the P2W17La nanoparticles are uniformly attached to the TiO2 nanowires.For comparison, a pure POM film was fabricated on a fluorine-doped tin oxide (FTO) substrate using the same method , respectively. The 3D sea-cucumber-like microstructure increases the roughness of the material, thereby increasing the active reaction surface area. The surface topographic features and roughness of the films were examined using atomic force microscopy (AFM) . The thi2 nanowires scraped from the FTO substrate  of unmodified TiOubstrate a clearlyarticles b. The TE2W17La film were further studied using high-resolution X-ray photoelectron spectroscopy (XPS)  d–f. In tubstrate ,36. As substrate . The WVI P2W17La f.2W17La film were examined using an electrochemical workstation in conjunction with a UV/Vis spectrometer, and contrasted with those of the FTO–P2W17La film. 2W17La composite film in the bleached and colored states at 0 V and −1.3 V, respectively. Digital photographs of the electrodes show that, when the applied potential is 0 V, the NW–P2W17La film appears transparent. After applying the −1.3 V negative potential, the film exhibits a deep blue color owing to the reduction of P2W17La and TiO2  and (2), respectively . W. W2W17La process . To revecharging . When b 2W17La, FTO–P2W17La, and TiO2 NW films obtained at the same scan rate in 1 Mol L−1 LiClO4/propylene carbonate (PC). The NW–P2W17La film shows the largest peak current value, demonstrating its high conductivity and low internal resistance characteristics. According to the results presented in −1, the surface coverage of NW–P2W17La was determined to be 1.74 × 10−6 mol cm−2, which is higher than that of the tightly packed FTO–P2W17La film (3.61 × 10−7 mol cm−2)  (P2W17La) was prepared according to the literature [All chemicals were of analytical grade. Fluorine-doped tin oxide (FTO)-coated glass substrates (<10 ohm/sq) were purchased from Pilkington . (3-Aminopropyl)trimethoxysilane (APS) and polyetherimide (PEI) were purchased from Aladdin Bio-Chem Technology Co., Ltd. and used without further treatment. Kterature ,49,50. T2 nanowire substrate was synthesized via a hydrothermal synthesis technique according to our previously reported procedure [2W17La solution (5 × 10−3 mol L−1) for 7 min, washed with distilled water, and dried under a nitrogen stream. The FTO glass was then immersed in PEI  for 7 min. The POMs and PEI have opposite charges and are alternately deposited on the substrate via electrostatic attraction. This procedure was repeated 30 times to obtain the NW–P2W17La film comprising 30 layers of P2W17La. For comparison, a pure P2W17La film (denoted as FTO–P2W17La), which comprised 30 layers of P2W17La, was fabricated on unmodified FTO glass using the same method.The TiOrocedure . The mul2W17La film as the cathode, a TiO2 NW film as the anode, and 0.5 M LiI in PC as the liquid electrolyte. First, the two electrodes were sandwiched together with 1 mm double-sided tape. The electrolyte was then injected into the space with a syringe to fabricate the prototype electrochromic energy storage device. The effective area of the device was 1 cm2.An asymmetric bifunctional EESD was assembled using an NW–P−1. SEM images were measured on FEI Verious 460 L scanning electron microscope . AFM images were investigated by Icon Bruker microscope . TEM images were measured on a FEI Tecnai G2F20 S-TWIN microscope equipped with an energy-dispersive spectrometer (EDS) . The TEM sample was prepared by scraping a small amount of the film (0.005 mg) off the FTO substate and dispersing the powder in 1 mL of an ethanol solution by ultrasonication for 40 min. XPS analysis were measured on a Thermo ESCALAB 250 spectrometer .FTIR spectroscopy was conducted using a GangDong FTIR-650 spectrometer  between 400 and 4000 cm4/PC (1 Mol L−1) as the electrolyte. The electrochromic and capacitive properties were studied using in situ TU-T9S PERSEE UV/Vis spectrometry  with an CHI660B Chenhua electrochemical workstation . The EESD was characterized using a two-electrode system.The UV/Vis spectra were obtaining using a PERSEE TU-1901 spectrometer . The CV and galvanostatic charge/discharge (GCD) measurements were performed using an CHI660B Chenhua electrochemical workstation  in a three-electrode configuration, where the as-prepared film served as the working electrode, Ag/AgCl (3 M KCl) as the counter electrode, Pt wire/Pt plate as the reference electrode, and LiClO2 nanowires and constructed an EESD by assembling the film with a TiO2 nanowire-film cathode. Compared with a densely packed structure, the loose 3D sea-cucumber-like microstructure provides a larger active surface area and shortens the electron/ion diffusion pathways, leading to uniform and fast redox reaction kinetics and simultaneously enhancing the EC and electrochemical performance of the composite. The designed EESD exhibits large optical contrast (48.6% at 605 nm), high switching speeds , and high areal capacitance (5.72 mF cm−2 at 0.15 mA cm−2). The developed device can power an LED bulb and function as a smart energy storage device where the level of stored energy can be visually and quantitatively monitored using rapid and reversible color variation. This work provides a novel paradigm for POM-based EESDs for optical-electrochemical energy applications.We synthesized a bifunctional nanocomposite film comprising POMs and TiO


Autophagy plays a pivotal role in physiology and pathophysiology, including cancer. Mechanisms of autophagy dysregulation in cancer remain elusive. Loss of function of TRIM28, a multifunction protein, is seen in familial kidney malignancy, but the mechanism by which TRIM28 contributes to the etiology of kidney malignancy is unclear. In this study, we show TRIM28 retards kidney cancer cell proliferation through inhibiting autophagy. Mechanistically, we find TRIM28 promotes ubiquitination and proteasome-mediated degradation of transcription factor TFE3, which is critical for autophagic gene expression. Genetic activation of TFE3 due to gene fusion is known to cause human kidney malignancy, but whether and how transcription activation by TFE3 involves chromatin changes is unclear. Here, we find another mode of TFE3 activation in human renal carcinoma. We find that TFE3 is constitutively localized to the cell nucleus in human and mouse kidney cancer, where it increases autophagic gene expression and promotes cell autophagy as well as proliferation. We further uncover that TFE3 interacts with and recruits histone H3K27 demethylase KDM6A for autophagic gene upregulation. We reveal that KDM6A contributes to expression of TFE3 target genes through increasing H3K4me3 rather than demethylating H3K27. Collectively, in this study, we identify a functional TRIM28–TFE3–KDM6A signal axis, which plays a critical role in kidney cancer cell autophagy and proliferation. Kidney cancer is one of the major cancer types worldwide with over 400,000 new cases each year, with renal cell carcinoma (RCC) being the most common type, which includes clear cell carcinoma, papillary cell carcinoma, and chromophobe cell carcinoma , 2. GeneVia these pleiotropic activities, TRIM28 plays important roles in physiology and pathophysiology. Genetic evidence shows loss of TRIM28 gives rise to familiar Wilms tumor, a renal blastoma affecting children, which strongly indicates TRIM28 has a distinct role in kidney malignancy . Next, we set out to examine the effect of TRIM28 on RCC cell proliferation with both cell counting and clonogenesis assay. As Western blot showed all three RCC cell lines that we examined expressed lower TRIM28 than 293T cells (C), we then introduced exogenous TRIM28 into Caki-1 and ACHN cells, which expressed even lower TRIM28 than 786-O. The result showed overexpressing TRIM28 significantly inhibited cell proliferation (A and B). To determine whether TRIM28 had a similar effect on RCC cell proliferation in a mouse model, we then inoculated control and TRIM28-Overexpression (OE) Caki-1 cells to immunodeficient mice. The result showed TRIM28 overexpression reduced tumor growth (C). Collectively, these data showed TRIM28 represses RCC proliferation.We first examined the relation between TRIM28 and RCC patient survival with kidney cancer datasets from the Clinical Proteomic Tumor Analysis Consortium (CPTAC) and The Cancer Genome Atlas (TCGA) projects. Both datasets indicated higher TRIM28 protein (CPTAC) and mRNA (TCGA) levels correlate with better overall survival in patients with clear cell renal carcinoma , A and B3T cells C, we theferation , A and Br growth C. CollecA and S2A). On the contrary, knocking down TRIM28 gave the opposite effect (B). We then treated cells with lysosomal inhibitor hydroxychloroquine (HCQ) and found it increased LC3 and P62 accumulation in TRIM28-Knockdown (KD) cells, indicating TRIM28-KD did not block the autophagic flow (C). As the autophagosome harbors concentrated lipidated LC3 on its membrane, LC3 immunostaining signal presents as puncta visible under microscope. As a result, LC3 puncta are widely used as a surrogate for the autophagosome. To further confirm that TRIM28 regulates autophagy, we examined its effect on the number of LC3 puncta. We first established Caki-1 and ACHN cell lines stably expressing GFP-LC3 under the control of an exogenous promoter. Afterward, we overexpressed TRIM28 in these cells and found the number of LC3B puncta was significantly decreased (D). In addition, TRIM28-OE also decreased degradation of exogenous GFP-LC3 while TRIM28-KD gave the opposite result, further supporting TRIM28 inhibited autophagic degradation (E and F and S2B). While LC3 puncta serve as a marker for the autophagosome, transmission electron microscopy (TEM) renders the opportunity to directly observe the autophagosome. With TEM, we confirmed TRIM28-OE indeed decreased the number of autophagosome/autolysosome (G). In addition, TRIM28-OE cells displayed enlarged lysosome filled with undigested cargoes, which also indicated defect in lysosomal function (C) (D). The above data showed that TRIM28 repressed RCC cell proliferation as well as the autophagy-lysosome pathway. We next investigated whether these two effects of TRIM28 were connected. We blocked the autophagy pathway through HCQ treatment or knocking out ATG3, a key protein for LC3 lipidation and autophagy (H and I). Mechanistically, autophagy was shown to maintain activity of mTOR pathway, which is a major signaling hub promoting cell growth (J and K and S2E). In summary, these data showed TRIM28 suppressed RCC cell proliferation through inhibiting autophagy.When culturing TRIM28-OE RCC cells, we serendipitously noticed that they were more sensitive to starvation than control cells, which indicated that TRIM28 might regulate autophagy in RCC cells. LC3, the mammalian homologue of yeast ATG8, undergoes lipidation during autophagy and integrates into the autophagosome membrane . AnotherFigs. 2e effect B. We thegic flow C. As theecreased D. In addlysosome G. In addfunction C (28). Ttion (C) D. The abutophagy . The resutophagy , H and Il growth . To examA and B). On the other hand, we detected increased mRNA level of multiple autophagic genes upon TRIM28-KD (A and S3C). Interestingly, TFE3 was identified as a potential interaction partner of TRIM28 in a large-scale proteomic study (D and E). To explore whether TRIM28 might affect TFE3, we first examined whether TRIM28 regulated TFE3 protein level. Western blot showed TRIM28-KD increased the protein level of TFE3 but not TFEB or MITF (B and S3F). The effect of TRIM28-KD on the TFE3 protein level could be reversed by introducing exogenous TRIM28, confirming the effect was specific rather than due to off-target effect (C). Consistently, TRIM28-OE decreased the TFE3 protein level (D). As Caki-1 and ACHN cells showed not only lower TRIM28 level but also higher ratio between TFE3 and TFEB/MITF (G), we performed mechanistic study mainly with these two cell lines hereafter. In light of the significant role of TFE3 in autophagy regulation, we next examined whether TFE3 mediated the effect of TRIM28 on autophagy in RCC cells. We performed chromatin immunoprecipitation (ChIP) analysis and found TRIM28 overexpression decreased enrichment of TFE3 at autophagic gene promoters, indicating TFE3 in RCC cells was active in binding target genes (E and S3H). Importantly, after TFE3 was knocked down, TRIM28 overexpression exhibited little further effect on autophagic gene expression (F and S3I). Consistently, our gene set enrichment analysis (GSEA) confirmed TRIM28 level negatively correlated with autophagy gene expression in those TCGA clear cell RCC samples where the TFE3 level was relatively higher than MITF and TFEB (J). These results suggested TRIM28 regulated autophagic gene expression through TFE3. Since we showed earlier autophagy underlay repressive effect of TRIM28 on RCC cell proliferation, we checked whether TFE3 also mediated effect of TRIM28 on RCC cell proliferation. We found, in TFE3-KD cells, TRIM28 overexpression rendered little further effect on cell proliferation (G). Collectively, these data showed TRIM28 regulated RCC cell autophagic gene expression and cell proliferation through TFE3.Next, we explored the mechanism how TRIM28 regulated autophagy in RCC. AMPKα, a known regulator of autophagy, was previously reported to be ubiquitinated by TRIM28 in certain cell types in a MAGE-dependent manner . HoweverRIM28-KD A and S3Cic study . We confic study , D and E or MITF B and S3Ft effect C. Consisin level D. As CakFEB/MITF G, we peret genes E and S3Hpression F and S3Iand TFEB J. These feration G. CollecA). Instead, TRIM28-KD increased TFE3 protein stability after we treated cells with the protein synthesis inhibitor Cycloheximide (A and S4B). Lysosome and proteasome are two major routes for protein degradation. We found proteasome inhibitor MG132 but not lysosome inhibitor HCQ significantly increased TFE3 protein level, indicating the proteasome pathway played a major role in TFE3 protein stability control (B and S4C). After blocking proteasome with MG132, we found TRIM28-KD failed to further increase the TFE3 protein level (C and S4D), indicating it was the proteasome pathway that mediated regulation of TFE3 by TRIM28. As TFE3 activity was previously shown to be regulated by sumoylation (E). Instead, we found TRIM28 increased the TFE3 ubiquitination level when TRIM28 was coexpressed with TFE3 (D). Consistently, knocking down TRIM28 decreased ubiquitination of endogenous TFE3 (E). The RING finger domain is a prototypical E3 ligase domain (F). Consistently, this mutant showed diminished activity to decrease the TFE3 protein level and RCC cell proliferation (G–I and S4F). These data showed TRIM28 promoted ubiquitin-proteasome-mediated degradation of TFE3 through its RING finger domain.We next set out to determine how TRIM28 regulated TFE3. RT-PCR showed TRIM28-KD did not cause significant change in TFE3 mRNA A. Insteaheximide A and S4B control B and S4Cin level C and S4Doylation  and TRIMoylation , 40, 41,oylation E. Insteaith TFE3 D. Consisous TFE3 E. The RIe domain . To detee domain F. ConsisA and B and S5A). For immunofluorescence, diminished TRIM28 signal in TRIM28-KD cells indicated the specificity of the staining. Immunostaining on RCC samples in The Human Protein Atlas also supported TRIM28's major nuclear localization (B). Unexpectedly, fractionation and immunostaining experiments showed endogenous TFE3 also mainly localized to cell nucleus in RCC cells (B and C and S5C). For immunofluorescence, diminished TFE3 signal in TFE3-KD cells indicated the specificity of the staining. Similarly, exogenous TFE3 also mainly localized to nuclei (D). Such localization pattern was also seen in cells treated with Earle’s balanced salt solution or TORIN1, which served as positive control for the immunostaining. Earle’s balanced salt solution rendered starvation while TORIN1 inhibited mTOR, both of which were known to promote nuclear localization of MITF family members (D). We next further examined whether such localization pattern of TFE3 also occurred in kidney tumor samples. We first performed immunostaining on RENCA xenograft tumor sections generated from control or TFE3-KD RENCA cells. The result not only showed TFE3 mainly localized to the nucleus but also indicated the staining procedure was specific for TFE3 in tissue samples (E). With the same staining protocol, we then analyzed TFE3 localization in kidney, liver, and lung of normal BALB/c mice for comparison. We found TFE3 was generally evenly distributed in the kidney but preferentially distributed to the cytoplasm in liver and lung (E). For human RCC tissue, we analyzed the immunohistochemistry data of RCC patient samples from The Human Protein Atlas database. The result showed TFE3 had significant distribution to the nucleus in 11 of 12 samples, which was significantly higher than lung cancer tissues (F) and at least comparable with pancreatic cancer from the same database. For comparison, we performed immunostaining for TFE3 in kidney sections from five noncancer human donors who went through kidney needle aspirate examination. The result showed TFE3 mainly localized to the cytoplasm (F). In summary, these results showed TFE3 localized to the nucleus in RCC cells constitutively, unlike the noncancer kidney tissues.Conventionally, TRIM28 is considered a nuclear protein while TFE3 mainly localizes to cytoplasm under normal conditions, so the significant effect of TRIM28 on TFE3 protein level seemed intriguing to us. We confirmed that both endogenous and exogenous TRIM28 localized to the nucleus with immunofluorescence and subcellular fractionation (lization B. Unexpeo nuclei D. Such l members . Immunos members D. We nex samples E. With tand lung E. For hu tissues F and at ytoplasm F. In sumin vitro (A and B and A and B). To study whether a similar effect occurred in a mouse model, we inoculated Caki-1 cells to immunodeficient mice and found TFE3-KD inhibited tumor growth (C). In RENCA syngenic mouse tumor model, Tfe3-KD similarly decreased tumor growth (D). Consistently, a higher TFE3 protein level correlated with worse patient survival in patients with RCC from the CPTAC dataset (C). Next, we examined whether the effect of TFE3 on RCC cell proliferation was related to cell autophagy. We first examined the effect of TFE3 on previously described autophagy markers. TFE3-KD decreased lipidation of endogenous LC3 (E). In addition, TFE3-KD also decreased GFP-LC3 puncta number and inhibited GFP-LC3 degradation in RCC cell stably expressing GFP-LC3 (F and G and S6D). To directly examine the effect of TFE3 on autophagosome and lysosomes, we then subjected TFE3-KD cells to TEM. The result confirmed TFE3-KD decreased the amount of autophagosome/autolysosome (H) and identified enlarged lysosomes filled with undigested cargos (E). Consistently, when we immunostained lysosome marker LAMP1, we confirmed TFE3-KD cells had increased lysosome size (F). These results all showed TFE3 was necessary for RCC cell autophagy. Consistently, our GSEA analysis also confirmed that TFE3 level positively correlated with autophagic gene expression in patients with RCC (G). Then we examined whether TFE3 regulates cell proliferation through autophagy. As we found earlier the effect of TRIM28 on mTOR activity, we analyzed TFE3-KD cells and found TFE3-KD decreased p-S6K, a marker of mTOR activity (I). To directly examine whether autophagy was necessary for TFE3-mediated cell proliferation, we then performed TFE3-KD in ATG3-KO cells. We found TFE3-KD exhibited little effect on cell proliferation in ATG3-KO cells (J). All these results from TFE3-KD were reminiscent of TRIM28-OE. Collectively, these data showed TFE3 promoted RCC cell proliferation and autophagy.We showed above TRIM28 could regulate autophagy and proliferation in RCC cells, so we next investigated the potential effect of TFE3 on these processes. TFE3-KD significantly decreased cell proliferation r growth C. In RENr growth D. Consis dataset C. Next, nous LC3 E. In addlysosome H and ided cargos E. Consisome size F. These with RCC G. Then wactivity I. To dirKO cells J. All thA). Interestingly, KDM6A localized to many of the known TFE3 targets, with concordant H3K4me3, a marker for active promoters (B). We performed ChIP experiment and detected KDM6A enrichment on autophagic gene promoters (A). KDM6A ChIP signals could be decreased by KDM6A knockdown, supporting the specificity of ChIP signal (A). To examine the potential connection between TFE3 and KDM6A, we performed immunoprecipitation and Western blot and found both exogenous and endogenous TFE3 could interact with KDM6A (B and C). We performed ChIP-reChIP and further confirmed colocalization of TFE3 and KDM6A to WIPI2 gene promoter (D). To examine whether TFE3 was required for KDM6A recruitment to autophagic gene promoters, we compared KDM6A ChIP signals in control and TFE3-KD cells. The result showed TFE3-KD decreased KDM6A enrichment without changing the KDM6A protein level (E). As TRIM28 decreased TFE3 level, we reasoned it might decrease KDM6A enrichment on autophagic genes as well. ChIP analysis confirmed TRIM28 overexpression indeed decreased KDM6A enrichment on WIPI2 gene promoter (F). Collectively, these results showed KDM6A could be recruited by TFE3 to autophagic gene promoters.Although change in chromatin status is widely considered a prerequisite for eukaryotic transcription regulation, little is known about how TFE3 affects chromatin status. We analyzed published ChIP-Seq data in human kidney cells and noticed TFE3 and KDM6A both localized to the promoter of target genes A. Intereromoters B. We perromoters A. KDM6A P signal A. To exath KDM6A , B and Cpromoter D. To exain level E. As TRIpromoter F. CollecA). Consistently, KDM6A-KD decreased GFP-LC3 puncta number and inhibited GFP-LC3 degradation (B and C and S8A) in RCC cells stably expressing GFP-LC3. These results showed KDM6A-KD was required for RCC cell autophagy. As our earlier data showed autophagy was critical for RCC cell proliferation, we next determined whether KDM6A also contributed to RCC cell proliferation. We found KDM6A knockdown indeed decreased RCC cell proliferation (B). We next examined whether KDM6A also regulated autophagic gene expression like TFE3. Indeed, KDM6A-KD decreased the expression of autophagic genes (D and S8C). Consistently, our GSEA analysis confirmed KDM6A positively correlated with autophagic gene expression among patients with RCC whose TFE3 level was relatively higher than TFEB and MITF (D). KDM6A has intrinsic H3K27me3 demethylase activity. To determine whether this activity was required for regulation of autophagic gene expression, we rescue-expressed KDM6A-WT or inactive H1146A mutant (DN) in KDM6A-KD cells. The result showed not only KDM6A-WT but also KDM6A-DN significantly restored expression of autophagic genes, indicating KDM6A demethylase activity was not necessary (E). In addition to demethylating H3K27me3, KDM6A was known to promote gene activation together with MLL3/4 through H3K4me3 (F). Consistently, TFE3-KD also decreased H3K4me3 but not H3K27me3 on autophagic gene promoters (G and S8E). Analysis of published ChIP-Seq result on RCC patients samples also confirmed that H3K4me3 but not H3K27me3 was enriched at autophagic gene loci (F). Consistent with the decrease in H3K4me3, KDM6A-KD as well as TFE3-KD decreased the enrichment of MLL3 on the autophagic gene promoters (H and I and S8G). In summary, these data showed KDM6A promoted autophagic gene expression through H3K4 methylation.We showed above KDM6A was recruited to autophagic gene promoters by TFE3. We next examined whether KDM6A contributed to RCC cell autophagy. Western blot showed KDM6A-KD decreased endogenous LC3 lipidation A. Consisferation B. We nexic genes D and S8Cand MITF D. KDM6A ecessary E. In add H3K4me3 , 32, 33. H3K4me3 F. Consisromoters G and S8Eene loci F. ConsisTRIM28 has multiple biochemical activities. While TRIM28 loss of function was seen in Wilms kidney blastoma, little is known about the function and the underlying mechanism of TRIM28 in etiology of kidney malignancy. On the other hand, as a pivotal transcription factor for autophagy-lysosome genes, TFE3 activation due to gene fusion also notably causes kidney malignancy. However, it is unclear whether and how TRIM28 and TFE3 are related in RCC etiology. In this study, we find higher TRIM28 level correlates with better RCC patient survival and TRIM28 suppresses RCC cell proliferation and tumor growth. Mechanistically, we show TRIM28 promotes ubiquitination and proteasome-mediated degradation of TFE3. Interestingly, we uncover TFE3 is constitutively localized to cell nucleus in RCC, which promotes RCC proliferation. Our study reveals a hitherto unappreciated link between TRIM28 and TFE3, two genes pivotal in etiology of kidney malignancy. In addition, we uncover a mechanism by which TFE3 regulates chromatin status at target genes. We find TFE3 functionally interacts with and recruits KDM6A to autophagic genes, which promotes the local H3K4me3 level. In summary, our study establishes a TRIM28–TFE3–KDM6A signal axis pivotal for RCC cell proliferation .Figure 9In tissues other than kidney, the impact of TRIM28 on autophagy was reported previously and attributed to different mechanisms. TRIM28 was reported to inhibit autophagy through promoting degradation of AMPK-α1 in lung, breast, and colon cancer cells , which dInterestingly, in this study, we find an increased portion of TFE3 is localized to the RCC cell nucleus in contrast to normal tissue. Consistent with its localization, we find TFE3 is necessary for autophagy and cell proliferation in RCC. As MITF family proteins play critical roles in autophagy regulation, extensive study has been dedicated to regulation of TFEB and showed TFEB is subject to ubiquitination , 45. YetIt is widely held that eukaryotic transcription has to engage chromatin change, yet it remains elusive how TFE3 affects local chromatin modification . In thisCaki-1 cells were cultured in McCoy's 5A (Procell #PM150710). ACHN cells were cultured in minimum essential medium supplemented with nonessential amino acids (Procell #PM150410). 786-O cells were cultured in RPMI-1640 (Procell #PM150110). HEK-293T cells were cultured in Dulbecco’s modified Eagle’s medium (Procell #PM150210). All media were supplemented with 10% fetal bovine serum. RENCA cells were cultured in RPMI-1640-based complete medium (Procell #CM-0568). All cells were cultured in 5% CO2 incubator. Cycloheximide , MG132 , and TORIN1  were added to cell culture at a final concentration of 25 mg/ml, 25 μM, and 1 μM, respectively, where indicated.Caki-1 cells were transduced with lentivirus expressing GFP-LC3B (human). After selection with 10 μg/ml blasticidin for 1 week, single cells were seeded into 96-well plate, further propagated, and validated.Cell proliferation assay was performed as we reported . BrieflyColony formation assay was performed as we reported . Brieflyad libitum. Mice were randomly assigned to experimental groups. A total of 5∗10∧6 Caki-1 cells were resuspended in a 1:1 mixture of PBS and Matrigel (BD #354248) and then inoculated subcutaneously into flanks of five 6-week-old male NOG mice (Charles River Beijing). A total of 2∗10∧6 RENCA control or TFE3-KD cells were inoculated subcutaneously into flanks of seven 6-week-old female BALB/c mice (Charles River Beijing). Tumor growth was measured with caliper every 3 to 7 days. Tumor volume was estimated by the formula 0.5∗L∗W∗W . Mice were sacrificed before the estimated volume of any tumor reaches 1 cm3.All animal experiments were performed following the institute guidelines and approved by Ethics committee of Tongji Medical College. The mice were acclimated to the new environment for at least 1 week. Mice were housed in ventilated cages in a temperature-controlled room (21 °C ± 1 deg. C) with a 12 h light/12 h darkness cycle. Food and water were available Western blot was performed exactly as we described in detail . BrieflyUse of leftover frozen sections produced from fine-needle aspirate of noncancer donors was approved by the Ethics Committee of Tongji Medical College and abides by the Declaration of Helsinki principles. Patients went through fine-needle aspiration for pathology diagnosis of nephrotic syndrome , lupus nephritis , Henoch–Schonlein purpura . For immunofluorescence on human tissue, frozen sections generated from needle aspirate of noncancer donors were first fixed with 4% paraformaldehyde for 15 min. Slides were then washed and permeabilized with 0.5% Triton-X100 in cold PBS. Slides were then blocked with 1% bovine serum albumin for 1 h, incubated with primary antibodies for 1 h, incubated with secondary antibodies for 1 h. Nuclei were counterstained with 1.5 μM DAPI  for 5 min. Slides were then washed three times with PBST (PBS + 0.2% Tween-20) and mounted onto antifading mounting medium (Abcam AB104135). Immunofluorescence for mouse tissue was performed similarly except mice kidneys or xenograft tumors were first fixed with 4% paraformaldehyde overnight. Immunofluorescence for adherent cell culture was performed with the same procedure as for frozen section. Antibodies used for immunostaining are TFE3 (CST #14779), TRIM28 (ZenBio #200280), and LAMP1 (CST #15665).The cell culture medium was discarded and cells were fixed with 2.5 glutaraldehyde in PBS (Servicebio #G1102) at 4 °C for 2 to 4 h. Cell were then scraped and centrifuged to a pellet. Further sample processing was done at Servicebio Inc. Briefly, cell pellets were embedded in 1% agarose (water), washed three times with PBS (pH 7.4) for 15 min each time, and then fixed with 1% osmium tetroxide. Samples were then dehydrated in increasing concentrations of ethanol and acetone. Afterward, samples were embedded in SPI 812. Finally, 60- to 80-nm ultrathin sections were cut with Leica EM UC7 and stained with uranyl acetate and lead citrate. Ultrathin sections were examined with a transmission electron microscope (Hitachi HT7800) at 8000-fold magnification.Sybgreen1-based real-time PCR was performed exactly as we described in detail on a Bio-Rad CFX connect PCR machine . PrimersChIP was performed exactly as we described . BrieflyChIP-reChIP was performed as we described . TFE3 anShRNA-based gene knockdown was performed exactly as we described . SynthesGene knockout was performed with the CRISPR-Cas9 system exactly as we described . SynthesLentivirus-mediated gene expression was performed as we described . ExpressIn vivo ubiquitination assay was performed exactly as we described . Exogenous or endogenous TFE3 was then immunoprecipitated with FLAG antibody (Sigma #A2220) or TFE3 antibody (Proteintech #14480-1-AP), respectively. The captured material was washed with IPE150 three times and analyzed with Western blot.escribed . Cells wN-ethylmaleimide . After sonication and centrifugation, the cell lysate was incubated with anti-FLAG M2-conjugated agarose beads for 4 h. After washing with IPE1000  three times for 5 min each time, beads-captured material was analyzed with Western blot.Equal amounts of FLAG-TFE3, HA-SUMO, and Myc-TRIM28 plasmids were cotransfected into 293T cells. Forty-eight hours later, cells were lysed in IPE150 buffer  containing 10 mM Coimmunoprecipitation was performed as we described . BrieflyDensitometry analysis for Western blot results was performed with FIJI as we described . WesternInput and HA-TFE3 ChIP-Seq data for HK-2 cells were from GSE135490. KDM6A and H3K4me3 ChIP-Seq data for HEK293 were from GSE172141. H3K4me3 and H3K27me3 ChIP-Seq data of patients with kidney cancer were from GSE75597. Raw reads were first filtered with Trim-Galore and then aligned with bowtie2  to humanGene expression data and survival information of TCGA patients were downloaded from Genomic Data Commons data portal. Protein expression data for CPTAC patients with kidney cancer were downloaded from the CPTAC data portal. Patients were divided into two groups according to expression level of corresponding genes. Survival analysis and Kaplan–Meier plot were performed with R “survival” package as we described .2, circularity 0 to 1. LAMP1 particles were called with “analyze particle” function in FIJI with parameters: size 0.066 to 6.50 μm2, circularity 0 to 1. Peculiar dots outside of cells were manually filtered out. Student's t test was used to statistically analyze the difference between particle sizes in different groups.Photos were taken with QImaging Retiga R6 Monochrome camera connected to Zeiss-A1 Axiovert A1 fluorescence microscopy equipped with 63× oil object lens. Exposure time for photos from the same batch of experiment was set at the same value for comparison. Photos were then processed and analyzed with FIJI software package. Briefly, photos were background-subtracted, made into binary with same threshold, processed with “watershed” tool in FIJI. GFP-LC3 particles were called with “analyze particle” function in FIJI with parameters: size 0.20 to 6.50 μmGSEA analysis with preranked genes was performed with R package “fgsea,” and the result was validated with the GSEA application (version 4.2.1). Plot for GSEA was generated with R package “gggsea.” Genes were first ranked according to the Pearson correlation coefficient between log2 expression values of themselves and TFE3, TRIM28, or KDM6A where indicated. Autophagy gene set was adopted from previous publication  less than 0.05 were deemed significant. All error bars denote standard deviation.For the xenograft tumor growth curve, The data that support the findings of this study are available from the corresponding authors upon reasonable request.This article contains The authors declare that they have no conflicts of interest with the contents of this article.


VRK3 knockdown emphasized the inhibition of genes involved in G1/S transition of the cell cycle resulting in growth arrest in G1. Additionally, a massive modulation of genes involved in chromosome segregation was observed, concomitantly with a reduction in the level of phosphorylation of serine 10 and serine 28 of histone H3 supporting the regulation of chromatin condensation during cell division. This last effect could be partly due to a concomitant decrease of the chromatin kinase VRK1 in DMG following VRK3 knockdown. Furthermore, a metabolic switch specific to VRK3 function was observed towards increased oxidative phosphorylation without change in mitochondria content, that we hypothesized would represent a cell rescue mechanism. This study further explored the vulnerability of DMG-H3K27M cells to VRK3 depletion suggesting potential therapeutic combinations, e.g. with the mitochondrial ClpP protease activator ONC201.We previously identified VRK3 as a specific vulnerability in DMG-H3K27M cells in a synthetic lethality screen targeting the whole kinome. The aim of the present study was to elucidate the mechanisms by which VRK3 depletion impact DMG-H3K27M cell fitness. Gene expression studies after  H3-3A, encoding H3.3 histone variant or in H3C1, encoding H3.1 variant (VRK3 (Vaccinia related kinase 3) as an essential gene that has not yet been associated with DMG oncogenesis (Diffuse midline glioma (DMG) H3K27 altered represents the most severe pediatric brain tumor. Almost all the affected children die within 2 years following diagnosis , 2 witho variant  and is cVRK3 belongs to the Vaccinia Related Kinase (VRK) family containing 3 distinct members in mammals: VRK1, VRK2 and VRK3 serine/threonine kinases, which are mainly located in the nucleus. VRKs are known to regulate several aspects in cell such as cell cycle progression, nuclear envelop dynamic during mitosis and apoptosis , 9. ThesVRK1 overexpression has been associated with poor prognosis in many solid tumors including high-grade glioma  and 2 H3.1-K27M  cellular models were grown in Neurocult™ medium supplemented with heparin , EGF/FGF  at 37°C and 5% CO2 using laminin-coating. Normal stem cell  derived as previously published (® (ThermoFischer Sicentific). EdU was incorporated into cellular DNA during replication to tag cells in S phase. Total DNA was stained with FxCycleTM Violet stain (Thermofisher Scientific). Samples were acquired using an LSR Fortessa flow cytometer (BD Biosciences), and analysis was performed with Flowjow software (Flowjow 10.8.1).Cells were exposed during 2 hours to 10 μmol/L of EdU supplied with Click-iT Flow Cytometry Assay Kit (ThermoScientific) and then detached using accutase® pLKO.1-puro non-mammalian shRNA Control Plasmid DNA, SHC002, Sigma-Aldrich).Lentiviral particles were produced in HEK293T cells using psPax2 and pMD2.g second-generation packaging plasmids  with jetPRIME Polyplus transfection reagent, and lentiviral titers were then determined by fluorescence assay. GSCs were transduced at a multiplicity of infection (M.O.I.) of 3 with shVRK3-1, shVRK3-4, shCtrl-1 (negative control vector containing a nonhairpin insert Addgene #1864) and shCtrl-2  were transduced . Total RThe RNA integrity (RNA Integrity Score≥7.0) was checked on the Agilent 2100 Bioanalyzer and quantity was determined using Qubit (Invitrogen). SureSelect Automated Strand Specific RNA Library Preparation Kit was used according to manufacturer’s instructions with the Bravo Platform. Briefly, 50 to 200ng of total RNA per sample was used for poly-A mRNA selection using oligo(dT) beads and subjected to thermal mRNA fragmentation. The fragmented mRNA samples were subjected to cDNA synthesis and were further converted into double stranded DNA using the reagents supplied in the kit, and the resulting dsDNA was used for library preparation. The final libraries were bar-coded, purified, pooled together in equal concentrations and subjected to paired-end sequencing on Novaseq-6000 sequencer (Illumina). Raw data quality checking was performed with FastQC and pseudo-mapping with Salmon (v0.9.1) using Gencode annotation (v27) based on hg38 reference genome (GRCh38); Genes with less than 10 reads in the resulting raw count matrix were filtered. After variance-stabilizing transformation and removal of sample model bias with limma removeBatchEffect, DESeq2 was used with default parameters to select differentially expressed genes (DEGs) with a pvalue threshold of 0.001 after Benjamini and Hochberg correction. Sample hierarchical clustering was performed using complexHeatmap function with Euclidian distance and complete linkage as aggregation method. For functional annotation and enrichment analysis, we used GeneTonic package, ClusterProfiler package (GO from org.Hs.eg.db GOSOURCEDATA = 2021-02-01) and Gene Set Enrichment Analysis (GSEA) to identify gene sets from the MSigDB v7.4 database (C5 (GO : BP), C2 (KEGG) and HALLMARK catalogue) overrepresented among log2FC ranked genelists, using Benjamini and Hochberg procedure as multiple testing correction. Enrichmap was obtained by computing Jaccard distance between top 100 GOenrich genesets. The communities in Enrichmap were identified by Louvain clustering. Hierarchical clustering of selected gene subsets was conducted using z-score, Pearson correlation and ward D2 as agglomeration criteria.VRK2 gene, where generated with the function getBeta from the minfi package.DNA methylation data of the samples of Capper et al.  and CastVRK3 silencing and two-and-a-half million cells were scrapped . Proteins were then extracted and purified using the kit Antibody Array Assay Kit (KAS02) according to manufacturer recommendations (Full moon BioSystem). Fifty μg of proteins were biotinylated, deposited onto the Phospho Explorer Antibody Array containing 1,318 antibodies, hybridized and washed according to manufacturer recommendations. The slides were scanned on a G2505C Agilent scanner at an excitation wavelength of 550nm at a resolution of 10 μm, and images analyzed with Agilent Feature Extraction 12.0 . The fluorescence signal of each antibody  was corrected by subtracting of the local background. Fold change of corrected signal intensity were analyzed in shVRK3-4-transduced cells versus corrected signal intensity in shCtrl2 -transduced cells.Two-hundreds microliter of lysis buffer are added to cells after For classical protein extraction, cells were lysed as described previously  in RIPA For immunofluorescence, cells were plated the day before fixation on coverslip coated with 0,1 mg/mL Matrigel (Corning). Fixation was performed for 5 minutes with PBS1X/4%paraformaldehyde, permeabilization for 5 minutes with 0,5% Triton X-100, and blocking for 30 minutes in 1X PBS/5% normal goat serum (ThermoFisher). Cells were incubated overnight at room temperature (RT) with anti-VRK3  and anti-Phalloidine  and 45 minutes with secondary antibodies  and Hoechst 33342  before washes. Slides were mounted with fluoromount-G (SouthernBiotech). Images were acquired with Leica SP8 confocal microscope. For immunofluorescence in suspension, cells were plated the day before transduction and fixed 80 hours post-transduction according to ThermoFisher’s protocol. Cells were incubated with antibodies anti-VRK3 , anti-H3S10P , anti-H3S28P (1/1000 #07-145 Millipore) overnight at 4°C and with secondary antibodies  during 1h at RT. Samples were acquired using an LSR Fortessa flow cytometer (BD Biosciences), and analysis was performed with Flowjow software (Flowjow 10.8.1).TBP (TATA-binding protein) was used as an internal loading control and 2-ΔΔCT method was used for relative expression computation. Primer sequences are summarized in Total RNAs were extracted and purified with Direct-zolTM RNA MicroPrep (Zymo Research) according to manufacturer’s instructions under RNase free conditions. Five hundred ng of total RNA was reverse-transcribed using Revertaid first strand cDNA synthesis kit (Thermo Fisher Scientific). Real time quantification was performed in triplicate with ViiA Real-Time PCR system (ThermoFisher Scientific). Each sample was analyzed in triplicates. Expression of Cells were detached with accutase and washed two times with PBS 1X. Cells were then exposed to Green mitotracker™  during 30 minutes at 37°C and washed two times with PBS 1X. Samples were acquired using an LSR Fortessa flow cytometer (BD Biosciences), and analysis was performed with Flowjow software.Mitochondrial function assays were performed using MitoPlate S-1. The substrates on MitoPlate S-1 were first dissolved by incubating the plate with 30 µl of Assay Mix, consisting of 2x BMAS, 6x Redox Dye MC and saponin (30 µg/ml) necessary for cell permeabilization in a 5% CO2 incubator at 37°C for 1 h before inoculating 400,000 cells per well in a volume of 30 µl. Color changes are read kinetically during 24h and are performed with OmniLog instrument . Signal intensity was measured at 24h and corrected by background subtraction with the negative control condition without substrate. The relative intensity was computed by dividing each intensity by the global mean of a particular sample.GSC and NSC cells were plated in duplicates at 10.000 - 15.000 cells/cm2 in 96-well plates in 100 μl of complete GSC medium. Twenty-four hours after seeding, Ro31220 , SB747651A  or vehicule DMSO (Sigma-Aldrich) were added using the D300E digital dispenser (TECAN). ONC201  was added with a range of 0.25-8µM. Cell proliferation was assessed during 7 days after treatment by videomicroscopy with an Incucyte Zoom or S3 (Sartorius). Cell confluence was determined by CellPlayer Analysis software (Essen Bioscience) and normalized against the initial time point. Inhibitory Concentration (IC50) values were obtained by plotting logarithmic concentrations of the drug against the area under the curve.VRK3 in order to evaluate the early impact of VRK3 knockdown (KD) in four independent in vitro models of DMG. VRK3 expression presented the highest reduction in GSC2 and GSC3 , with greater KD with shVRK3-1 with the same threshold of adjusted pvalue ≤ 0.001  in shVRK3 cells between the 2 timepoints showed an enrichment of genes involved in histone acetylation with an overexpression of HDAC5, HDAC8, TRERF1 and downregulation of RBM14; and cholesterol biosynthetic process with a downregulation of DHCR7, NSDHL, FASN, ACAT2 and NFYA. Gene ontology enrichment analyses of DEGs after VRK3 KD pinpointed a network of the top 100 genesets, from which four communities of highly connected genesets were identified by Louvain clustering: 1) Regulation of cell cycle transition, 2) Telomere organization, 3) Kinetochore and chromatin compaction and 4) Protein localization in mitochondrion  and an overexpression of ARPC3 . For R0318220, NSC4 appeared more sensitive than H3.3- mutated cells which was not concordant with the results of the lethality screen  plays a main role in chromatin dynamics and is also tightly associated with cell cycle progression. More precisely, during prophase and metaphase, H3S10P nucleosome occupancy increase strongly along the chromosomal arms . Our resVRK3 KD  and NPM1 exhibiting several functions including H3, H4 and H2B core histone chaperoning function. Interestingly, chromatin condensation was reported to be enhanced via phosphorylation of histone H3 on Ser10 by VRK1 (data not shown). Also, VRK2 protein was shown to display a similar cytoplasmic localization, indicating a binding to endoplasmic reticulum and to a lesser extend to mitochondria  and CDK2 in both H3.1 and H3.3 cells. Conversely, CDKN1B encoding P27 was upregulated (CCND1) and CDK2-cyclin E1 (CCNE1) protein complexes. S phase entry is mainly under the control of cyclin D/CDK4/6 activation in early G1 leading to activation of CDK2/Cyclin E in late G1  , confirming the observation of Shen and coll. This observation is in accordance with a higher mitochondrial RNA content in non-tumoral pontine tissue samples in comparison with DMG-H3K27M primary tumors (VRK3 repression was identified by QPCR even 120h after transduction (data not shown). Around 10% of OXPHOS genes (13/132 in KEGG oxidative phosphorylation pathway) are encoded by the mitochondrial genome. Looking independently at the modulation of the two subsets of OXPHOS related genes, an upregulation restricted to mtOXPHOS genes in DMG versus normal NSC was identified  was one of the most significantly upregulated genes in shVRK3 cells  5A, B. ARL cells . Additio samples . This ist cancer , and is t cancer . Indeed,t cancer .VRK3 KD . This significant efficiency appeared independent of EGFR expression level in the corresponding primary tumors despite the fact that its efficiency was reported to be inversely proportional to EGFR expression in GBM , was shown to inhibit cell proliferation and induce cell death in many cancers including GBM  and one n in GBM .i.e. H3.1- and H3.3-K27M DMG, despite that the H3.3 mutated GSC models used were TP53-mutated and that VRK3 was shown to interact with TP53 protein. A massive gene downregulation affected genes involved in chromatin segregation.Our gene expression study gave some clues to explain the profound impact of VRK3 deprivation on DMG cell fitness. There were only few differences between the two main subgroups of DMG-H3K27 altered, versus NSC. VRK3 inhibition in DMG led to a significant decrease of H3S10P-positive cells. However VRK3 protein appeared unbound to chromatin in DMG-K27M in contrast to VRK1, as also reported in fibroblasts , show important indicators of reduced OXPHOS system activity compared with that of the electron transport system. Indeed, glioma cells predominantly rely on glycolysis instead of oxidative phosphorylation to regenerate ATP  can be found below: The studies involving humans were approved by informed consent for the Translational Research Program was obtained from parent or guardian according to the IRB approved protocol (CNIL 1176643). The studies were conducted in accordance with the local legislation and institutional requirements. The human samples used in this study were acquired from primarily isolated as part of our previous study for which ethical approval was obtained.VM, DC, JG, and M-AD contributed to conception and design of the study. VM, TS, LA, CS, ML performed experiments. TK and YA performed the bioinformatic analysis. VM and M-AD wrote the manuscript. All authors contributed to the article and approved the submitted version.


BCHE, CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP3A5, F2, F5, IFNL3, SLCO1B1, TPMT, UGT1A1, VKORC1), associated to different drug metabolizing rates, were characterized by genotyping using a genome wide commercial array. Widely used drugs such as anti-coagulant warfarin and lipid lowering agent atorvastatin were associated to an alarmingly high percentage of users with intermediate/poor metabolism for them. Significant differences in the frequency of normal/intermediate/poor/ultrarapid/rapid metabolizers were observed for CYPD26 (p<0.001), CYP2C19 (p<0.001) and UGT1A1 (p<0.001) between the Czech and the Finnish study populations. Our study demonstrated that administration of some popular drugs to a Czech random sample population is associated with different drug metabolizing rates and therefore exposing to risk for ADRs. We also highlight interethnic differentiation of some common pharmacogenetics variants between Central (Czech) and North European (Finnish) population studies, suggesting the utility of PGx-informed prescription based on variant genotyping.The genetic basis of variability in drug response is at the core of pharmacogenomics (PGx) studies, aiming at reducing adverse drug reaction (ADR), which have interethnic variability. This study used the Kardiovize Brno 2030 random urban Czech sample population to analyze polymorphisms in a wide spectrum of genes coding for liver enzymes involved in drug metabolism. We aimed at correlating real life drug consumption with pharmacogenomic profile, and at comparing these data with the SUPER-Finland Finnish PGx database. A total of 250 individuals representative of the Kardiovize Brno 2030 cohort were included in an observational study. Blood DNA was extracted and 59 single nucleotide polymorphisms within 13 genes ( Evidence shows the genetic components of inter-individual variability in drug response, which has been linked to ethnicity . HoweverThe Kardiovize study is a prospective epidemiological survey of 1% n = 2160) of the urban population of Brno, the second city of the Czech Republic, a Central European country . The Kar0 of the The Kardiovize study has been previously described . RecruitBCHE, CYP1A2, CYP2C19, CYP2D6, CYP3A5, F2, F5, IFNL3, SLCO1B1, TPMT, UGT1A1, VKORC1). All DNAs were quantified with Nanodrop Lite before any futher processing. For SNP genotyping with OpenArray plate in total 60 ng of sample DNA was loaded to each OpenArray sub-array . 2x TaqMan™ OpenArray™ Real-Time PCR Master Mix was used according to manufacturer protocol . OpenArray plate preparation was done using AccuFill system according to manufacturer protocol . SNP calling was done using TaqMan Genotyper Software . Auto-calling function was used and each assay was manually checked. For CNV determination two detection assays were used: CYP2D6 exon9 in combination with RNAseP and CYP2D6 intron2 in combination with RNAseP . All reactions were carried our according to manufacturer protocol on 384-well plate, each reaction in quadruplicate. In each reaction 10 ng of DNA was used. Real-time PCR run was done in QuantStudio 12K Flex Real-Time PCR System . Data analysis was done using CopyCaller® Software . For each run a reference sample with known CNV count was used to determine sample calls.DNA was extracted from participants’ blood samples using the DNeasy Blood & Tissue Kit from QIAGEN (Germany), according to manufacturer’s instructions. We selected 59 SNPs in 13 loci identified by genome-wide association studies as being associated with different drug metabolizing rates (https://www.generx.fi/) has been developed by Abomics (www.abomics.fi) and includes information about genotypes that are associated with clinically relevant variation in >200 drugs, either considering the responsiveness to the drugs or drug-induced adverse effects. The database is a collection of recommendations for the most clinically relevant and actionable pharmacogenetic drug-gene pairs. The contents of the database reflect published expert opinion pharmacogenetic recommendations and contents of commercial pharmacogenetic test panels. The database is regularly updated based on newly published literature, which is reviewed for each gene-drug pair, and recommendations are changed when needed. Recommendations are mostly based on published expert opinions and pharmacogenetic recommendation articles, e.g. by the Clinical Pharmacogenetics Implementation Consortium (CPIC). The FDA’s list of pharmacogenetic biomarkers in drug labels is also followed for update process.The GeneRx database (SLCO1B1), the predicted phenotype classes are increased, normal, decreased, and poor function. For blood coagulation factors F2 and F5, the phenotype stratification considers risk classification for venous thromboembolism (increased or significantly increased risk of venous thromboembolism). IFNL3 phenotypes are associated with the response to antiviral hepatitis C treatment (favorable or unfavorable response genotypes). For VKORC1, the phenotypes represent the expression levels of the enzyme , which links it to warfarin sensitivity.In the database, predicted phenotypes based on genotypes, considers the four metabolizer types for drug metabolizing enzymes: 1) Poor Metabolizer (PM). Medication is broken down very slowly. May experience side effects at standard doses; 2) Intermediate Metabolizer (IM). Slow rate of metabolism. May be exposed to excessive drug plasma concentration at standard doses, potentially causing side effects; 3) Normal Metabolizer (NM). Normal rate of metabolism. Expected normal efficacy at standard doses; 4) Ultrarapid Metabolizer (UM). Medication is rapidly broken down, potentially leading to lack of efficacy. These descriptions of changes in metabolism are opposite in the case of prodrugs for which the risk of over-exposure and adverse effects is more pronounced in UMs, as lack of efficacy is expected in IMs and PMs. For transporter protein genes , and tested for differences with independent-sample ge for R  . The prevalence of hypertension, hyperlipidaemia, and diabetes mellitus in this sub-cohort was 38.9% and 71.5%, and 4.4% respectively, similar to the overall cohort. The prevalence of the combinations with either diabetes mellitus and hypertension; or diabetes mellitus and hyperlipidaemia; or hypertension with hyperlipidaemia were respectively 3.6%, 3.2%, and 36.9%.A total of randomly selected 250 participants, aged 25 to 64 years  were included for PGx profiling. No statistically significant indications for deviation from Hardy-Weinberg equilibrium were detected, suggesting a genetically balanced, random sample Table. TCYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP3AS ; F2, F5 (coagulation factors); IFNL3 (interferon Lambda 3); SLCO1B1 (a solute carrier organic anion transporter family member); TPMT (thiopurine methyltransferase); UGT1A1 (a uridine diphosphate glucuronosyltransferase); and VKORC1 (a subunit of vitamin K epoxide reductase complex).The list of the 59 SNPs that we detected in 13 drug-metabolizing genes, which have been previously identified by numerous genome-wide association studies as being associated with different drug metabolizing rates , is summSubsequently, we focused our attention on the most commonly used medications used by our participants. In our study cohort 8.5% individuals used the anticoagulant warfarin. Warfarin dosage required to reach therapeutic drug levels is heavily affected by two enzymes, CYP2C9 and VKORC1 Tables .VKORC1 gene (tested in the gene panel) is associated with increased sensitivity to the drug. Only 42.9% of individuals using warfarin exhibited normal expression of VKORC1, with 38.1% exhibiting reduced expression and 19% exhibiting markedly reduced expression. Among the warfarin users, 61.9% were normal metabolizers and 38.1% were intermediate metabolizers for CYP2C9 (CYP2C9 enzyme metabolizes warfarin while VKORC1 (an enzyme involved in vitamin K recycling) is the target of warfarin. A common variant in the r CYP2C9 . Geneticr CYP2C9 . Finallyr CYP2C9 .As a comparative ethnically different study population, we considered a Finnish reference population, consisting of 9262 non-related individuals participating in the SUPER-Finland study, previously described . We usedCYP2D6, CYP2C19 and UGT1A1 between the Czech and the Finnish populations (CYP2D6 the Czech population contained less ultra-rapid metabolizers (UM) (p<0.001); for CYP2C19 the Czech population contained more normal metabolizers (NM) (~45% versus 40%) and more intermediate metabolizers (IM) (~29% versus ~26%) (p<0.001); for UGT1A1 the Czech population contained ~37% NM, while Finns included 32% (p<0.001)  .CYP2C19 is a key marker to rationalize clopidogrel treatment, CYP2D6 is responsible for metabolizing anti-depressants and anti-psychotics, while UGT1A1 metabolises many endogenous substances and clinical drugs, such as steroids, bilirubin and irinotecan.In a Czech random population sample, around 20–30% of patients showed a poor PGx metabolism to warfarin and atorvastatin, exposing them to risk for ADRs. While these data may reflect inter-individual variability rates in the genetic responses to warfarin, atorvastatin and metoprolol that has been previously reported in several European countries , at the Recently PGx has acquired importance to define individual differences in drug efficacy and toxicity, by taking into account the underlying genetic profile . CloselyCYP2C9 [ABCB1, VDR) resembling those present in Europeans and Finnish [CYP2D6, CYP2C19 and UGT1A1) between Czech and Finnish population studies, suggesting the utility of PGx-informed prescription based on variant genotyping. For CYP2C19, our data are consistent with the differences observed in the prevalence of high-risk genotypes in a large study assessing the pharmacogenomic biomarkers allelic spectrum in 18 European populations by analyzing 1,931 pharmacogenomics biomarkers in 231 genes [CYP2D6  compared to Finnish individuals. In this respect, according to the SUPER-Finland study, based on the imputation of 9262 individuals, there is a higher frequency of CYP2D6 UM in Finland compared with non-Finnish Europeans [CYP2D6 UM phenotypes compared with the ancestral European population [CYP2D6 genotype had a substantial clinical effect on anti-psychotics exposure and on their therapeutic failure [CYP2D6 genotyping would be valuable for personalizing anti-depressant and anti-psychotics dosing and treatment.The clinical relevance of our findings showing a high prevalence of impaired metabolism in association with warfarin treatment is consistent with prior work highlighting an increased risk of bleeding in patients carrying variants for CYP2C9 . AccordiCYP2C9 . In addiCYP2C9 , which iCYP2C9 , 23. Che Finnish . Our Kar Finnish , CYP2C19 Finnish  and UGT1 Finnish . An addi31 genes . The lat31 genes . Other r31 genes –30. Our uropeans . The Finpulation . CYP2D6  failure . Pre-empUGT1A1 decreased function variant rs4148323 (UGT1A1*6) in Finland compared with non-Finnish Europeans from the GnomAD v2.1.1 database [UGT1A1 metabolizes chemotherapeutic drugs such as irinotecan, used against colorectal cancer. Our data show decreased NM in Finns compared to Czechs; the SUPER-Finland showed a 22-fold enrichment of the database , 34. Intdatabase . The Wordatabase .In terms of CVD, the first cause of death worldwide, the Central and Easter European (CEE) countries have the highest CVD mortality in the EU, which also occur at younger ages . The EurOur PGx study on the Kardiovize Brno 2030 database shows heterogeneity in the metabolic profile of the population for the most common widely used medications. Of note by crossing with validated medicine we showed that in a relevant proportion the some widely used medications, i.e. warfarin and atorvastatin, are administered to patients with high-risk genotypes exposing them at risk for ADR. Finally comparing our findings in the Czech population with a Finnish reference population we showed interethnic differences in some common pharmacogenetics variants among ethnically different populations worldwide. Our results may contribute to facilitate European integration of PGx and to support pre-emptive PGx testing.S1 Table(XLSX)Click here for additional data file.


During embryogenesis, multiple intricate and intertwined cellular signaling pathways coordinate cell behavior. Their slightest alterations can have dramatic consequences for the cells and the organs they form. The transcriptional repressor Bcl6 was recently found as important for brain development. However, its regulation and integration with other signals is unknown. Using in vivo functional approaches combined with molecular mechanistic analysis, we identified a reciprocal regulatory loop between B cell lymphoma 6 (Bcl6) and the RhoA-regulated transcriptional complex megakaryoblastic leukemia/serum response factor (MKL/SRF). We show that Bcl6 physically interacts with MKL/SRF, resulting in a down-regulation of the transcriptional activity of both Bcl6 and MKL/SRF. This molecular cross-talk is essential for the control of proliferation, neurogenesis, and spatial positioning of neural progenitors. Overall, our data highlight a regulatory mechanism that controls neuronal production and neocortical development and reveal an MKL/SRF and Bcl6 interaction that may have broader implications in other physiological functions and in diseases. Crossregulation between Bcl6 and MKL/SRF regulates neurogenesis and progenitor migration to coordinate neocortical development. The mammalian cerebral cortex plays key roles in cognition, emotions, and sensory and motor functions. These tasks rely on adequate activities of neural circuits. This connectivity depends on the ability of proliferative cells of the neuroepithelium to produce an array of neurons and glial cells that carry out specialized functions in mature neural networks . RhoA expression within the embryonic neocortex is higher at the VZ and sVZ, while RhoB is mainly expressed in the cortical plate, and RhoC is barely detectable  is extensively studied for its importance in B cell development and oncogenesis but has been recently identified as a proneurogenic factor during the embryonic development of the neocortex  or a dominant negative form of RhoA (RhoADN) or RhoA knockdown using specific small interfering RNA (siRNA)] in a subset of RGCs at embryonic day 15.5 (E15.5) in a murine model (+Sox2+ and increased the proportion of GFP+Tbr2+ and GFP+Satb2+ (+Sox2+ and a decrease in GFP+Tbr2+ and GFP+Satb2+ (+Sox2+Tbr2−), newly born committed BPs migrating away from the VZ (GFP+Sox2+Tbr2+), differentiated BPs (GFP+Tbr2+Sox2−), and newly born migrating neurons (GFP+Tbr2−Sox2− or GFP+Satb2+). We found that inhibiting RhoA reduced the proportion of RGCs and committed BPs and increased differentiated BPs and neurons (2-M phases (+ cells), which was negligible at this time of development (fig. S1A). Additional staining for the proliferation marker Ki67 showed an increased cell cycle exit induced by RhoA inhibition and reduced by RhoA GoF . Together, these data suggest that RhoA inhibition increases direct and indirect neurogenesis along with RGC depletion, whereas RhoA GoF reduces neurogenesis and maintains the RGC pool size.We considered that RhoA may have more subtle effects on the fate choice of RGCs that are not evident when RhoA is conditionally deleted throughout the VZ. Therefore, we used in utero electroporation (IUE) to manipulate RhoA activity  containing the consensus Bcl6 binding site sequence in front of the promoter as used in . Moreover, Bcl6 overexpression partially restored cell position, cell cycle exit, and differentiation disrupted by RhoA, MKL, or SRF GoF . In agreement with these results, overexpression of the Bcl6 mutant had similar effects to the wild-type Bcl6 on cell cycle exit  versus asymmetrical (neurogenic) division in early NSCs  after the surgery are the ones in M phase at the time of electroporation  . We also brought to light a reciprocal regulatory loop between the Bcl6 transcription repressor and MKL/SRF in vivo and in vitro. We show that Bcl6 and SRF have a direct molecular interaction and, together, form a complex with MKL1. Dimerization is crucial for MKL, SRF, or Bcl6 to associate with some of their partners and regulate gene expression  fusion. pEBG was a gift from D. Baltimore . RhoADN contains the T19N mutation. pCAGIG:Bcl6 was a gift from P. Vanderhaeghen. The Bcl6 form unable to interact with SRF contains the mutations C121F in the first SRF-binding site and P421L in the second SRF-binding site. Myc or HA tag sequences were inserted in one of the primers used for the polymerase chain reaction (PCR) amplifications before subcloning into pCAG vector. For the TriFC, the sfGFP1-9 complementary fragment, the β strand 10 (S10) and S11 of sfGFP were subcloned into the pCAG vector by PCR from UBQ10:sXVE:(MCS)-S11-DI-GFP1–9  and UBQ10-sXVE-S10-(MCS)-3xHA . MKL1, MKL2, SRF, and Bcl6 were subcloned into pCAG:S10 and pCAG:S11 by PCR. Dimerization-deficient MKL1 mutant (MKL1ΔLZ) was deleted of the LZ domain by PCR using junction primers that inserted Xho I sites six residues between the codons 552 and 559 and subcloned into pCAG:GFP(S10) and pCAG:GFP(S11). pLKO.1-MKL1/2 shRNA was a gift from R. Prywes . ShMKL1 and shMKL2 were constructed by insertion of the sequence GGGTAGCAGACAGTTCCTCCTTCAAGAGAGGAGGAACTGTCTGCTACCTTTTTTGGAA (shMKL1) or GGCCATCCCAAGAATCCAAATTCAAGAGATTTGGATTCTTGGGATGGCTTTTTTGGAA (shMKL2) using a primer annealing procedure into pSCV2 vector. pSCV2:shBcl6 was a gift from P. Vanderhaeghen (pCAG:GFP (Addgene) and pCAG:Nuclear Localization Signal (NLS)-GFP  were used for co-electroporation. RhoA, MKL1, MKL2, and SRF sequences were amplified from cDNA and inserted into the pCAG:GFP vector or the pEBG vector for GFP or glutathione HEK293T, HeLa, and Neuro-2a cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum, 1% glutamine, and 1% penicillin-streptomycin. All cells were transfected using the PolyJet (Tebu-bio) complex according to the manufacturer’s protocol.HeLa or HEK293T cells were spread with a density of 650,000 cells per well in a six-well plate containing three round coverslips. Twenty-four hours after transfection, the cells were gently washed three times in phosphate-buffered saline (PBS) and fixed with 3.7% paraformaldehyde  in PBS for 10 min at room temperature (RT), washed three times with PBS, permeabilized for 20 min in PBS 0.2% Triton X-100 (w/v) at RT, gently washed three times with PBS, and blocked with 1 ml of 4% normal goat serum (NGS) in PBS for 1 hour at RT. The cells were then incubated with primary antibodies diluted in blocking solution for 1 hour at RT and then washed three times with PBS. The Alexa Fluor secondary antibodies in blocking solution were added for 1 hour at RT. Last, cells were stained with 4′,6-diamidino-2-phenylindole (DAPI), washed in PBS, mounted on a slide with 20 μl of fluorescence mounting medium (DAKO), and stored at 4°C.g at 4°C.HEK293T cells were seeded at 650,000 cells per 35-mm well and transfected 16 hours later with plasmid DNA using the PolyJet reagent. Twenty-four hours after transfection, the cells were gently washed three times and lysed with 300 μl of Triton X-100 lysis buffer  protease and phosphatase inhibitor cocktail (Roche) and vigorously shaken on ice for 2 hours at 4°C. Lysates were clarified by centrifugation 10 min at 17,000For the coimmunoprecipitation, protein lysates were incubated with the antibody overnight at 4°C on a rotating wheel. Dynabeads protein A or G magnetic beads (Invitrogen) or glutathione resin (GenScript) agarose beads were washed three times in PBS, blocked in 500 μl of 1% bovine serum albumin/PBS for 2 hours at 4°C, washed twice with PBS, once in Triton X-100 lysis buffer, and added into the protein lysate mixture for 2 hours at 4°C on a rotating wheel. The beads were washed three times with Triton X-100 lysis buffer and boiled for 5 min in SDS loading buffer to elute the proteins. Proteins are then separated by SDS–polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane  by Western blot. Membranes were blocked in 5% milk and 0.1% Tween 20 in PBS for 1 hour at RT, incubated with first antibodies overnight at 4°C, washed three times in 0.1% Tween 20 in PBS for 10 min, and incubated with horseradish peroxidase (HRP)–labeled antibodies for 1 hour at RT. Last, membranes were washed two times in 0.1% Tween 20 in PBS for 10 min at RT and once in sterile demineralized water. Chemiluminescence revelation was performed using the SuperSignal West Pico chemiluminescent substrate (Pierce) and exposed to Hyperfilm ECL (Amersham Biosciences).Renilla activity. Cells were spread on the well with a density of 200,000 cells per well in a 24-well plate and were transfected using the PolyJet reagent. Brains were electroporated at E15.5. All conditions were made in triplicate. Cells or cortices were collected 24 h after transfection or electroporation and lysed and dissociated in 100 μl of 1× Passive Lysis Buffer at RT for 10 min. Lysates were collected and clarified by centrifugation 5 min at 12,000 rpm and 4°C. Gene expression was measured using the Luminometer Glomax 20/20 (Promega) and the protocol “Dual-Glo” according to the manufacturer instructions.For the luciferase assay, we used the Dual-Glo Luciferase Assay System (Promega), in vitro on HEK293T lysate cells or in vivo on E16.5 cortex lysate. Luciferase activity was calculated as the Firefly activity normalized by the In utero microinjection and electroporation were performed at E13 or E15.5, as previously described (Whole brains were collected 23 hours after electroporation at E15.5 or at E13. GFP-positive brains were fixed for 3 hours in 3.7% PFA (w/v) in PBS solution, washed in PBS for 1 hour, and cryoprotected in a 30% sucrose in PBS solution overnight at 4°C. The brains were frozen in optimal cutting temperature compound before 6- to 10-μm-thick brain cross sections were obtained with a cryostat and placed on slides. For selected antibodies, sections were antigen-retrieved by immersion of the slides in 0.01 M sodium citrate buffer (pH 6.0) at 98°C for 20 s, cooled for 7 min at RT, and rinsed in PBS. The sections were permeabilized by being washed for 5 min in 0.2% Triton X-100 (w/v) in PBS and blocked in 4% NGS (v/v) and 0.2% Triton X-100 in PBS for 30 min at RT. Primary antibodies were incubated on slides overnight at 4°C and washed three times for 5 min in 0.2% Triton X-100 (w/v) in PBS. Alexa Fluor secondary antibodies were added for 1 hour at RT. Last, the slides were stained with DAPI and washed two times as before. The slides were coverslipped with Dako mounting medium and stored at 4°C.4, 2 μM sulfo-cyanine 3 azide , and 100 mM sodium ascorbate. The slides were then rinsed three more times in PBS for 5 min at RT before continuing with the immunostaining protocol as described previously.Following IUE, the operated mouse received an intraperitoneal injection with 5 μM EdU at either 8, 10, 12, 18, or 23 hours. The embryos were dissected 30 min after injection. The collected brains were fixed and sectioned as previously described. Sections were washed three times for 5 min in PBS, permeabilized in 0.2% Triton X-100 (w/v) in PBS for 30 min, and washed three times. Sections were then incubated 30 min in a working solution containing 100 mM tris-HCl, 4 mM CuSOImages for cell and cryosection staining were acquired with an Olympus FV1000 confocal microscope or a Zeiss Axio Observer A1 epifluorescence microscope.2 and 5% CO2. The medium was flowed into the chamber at about 5 ml/hour. Repetitive acquisitions were performed in laterodorsal regions of the cortex in which 25 successive z optical planes spanning 120 mm were acquired. Z-stacks were selected and combined in Zeiss LSM Image Browser. Slight drifts of the slices were corrected using the ImageJ registration tool Turboreg .Three hundred–millimeter embryonic brain slices were prepared using a vibratome (World Precision Instruments) as previously described , mouse anti-GST (Sigma-Aldrich), mouse anti-HA.11 clone 16B12 (Covance), rabbit anti-HA.11 clone 16B12 (Eurogentec), mouse anti-Myc (Eurogentec), rabbit anti-Myc , rabbit anti–α-catenin (Sigma-Aldrich), mouse anti-Ki67 (BD Biosciences), rabbit anti–phospho-Histone H3 , rabbit anti-Sox2 (Millipore), mouse anti-Sox2 , rabbit anti-Trb2 (Abcam), mouse anti-SatB2 (Abcam), mouse anti-Nestin (Millipore), and rabbit anti-ZO.1 (Invitrogen). Goat secondary antibodies labeled with Alexa 488, 568, and 647 (Invitrogen) were used for immunofluorescence. Goat anti-mouse or anti-rabbit HRP-conjugated secondary antibodies  were used for biochemistry.For E16.5 brain, the height of the whole cerebral wall was divided into 40 bins, but only the first 20 bins were included in the analysis since no cells were observed above them in any of our experiments. For E14 brains, the height of the whole cerebral wall was divided into 20 bins, and all of the bins were included in the analysis. Counting was performed by densitometry using ImageJ. To give similar importance to low-fluorescence and high-fluorescence cells, the pictures were first modified into binary images to equalize the brightness of each positive cell. Then, the area covered by the pixels was quantified in ImageJ for each bin.+ cells. Each picture includes about 300 cells. Using ImageJ, we quantified the pixels positive for the GFP fluorescence in each picture. The GFP fluorescence was then divided by the number of cells measured per picture.Five pictures per coverslip were taken for each condition to cover the whole area of transfected cells. Using QuPath, we measured the total number of DAPIt tests for two-population comparisons or one-way analysis of variance (ANOVA) followed by Bonferroni’s post hoc test for multiple comparisons across N samples, where N is the number of embryos or experiments as defined in the figure legends. Percentage values were modified with arcsin transformations before applying statistical tests.Statistical significance was determined using two-tailed, unpaired Student’s CD1 mice were bred in standard conditions and animal procedures were carried out in accordance with European guidelines and approved by the animal ethics committee of the Université catholique de Louvain. Approval number is 2021/UCL/MD/029.Key resources are provided in table S1.


KoPIP2;1, was cloned from the mangrove plant Kandelia obovata by RACE technology. The KoPIP2;1 gene was 1404 bp in length with an open reading frame (ORF) of 852 bp, encoded with 283 amino acids. Database comparisons revealed that KoPIP2;1 protein shared the highest identity (91.26%) with the aquaporin HbPIP2;2, which was isolated from Hevea brasiliensis. Gene expression analysis revealed that the KoPIP2;1 gene was induced higher in leaves than in stems and roots of K. obovata under cold stress. Transient expression of KoPIP2;1 in Nicotiana benthamiana epidermal cells revealed that the KoPIP2;1 protein was localized to the plasma membrane. Overexpressing KoPIP2;1 in Arabidopsis significantly enhanced the lateral root number of the transgenic lines. KoPIP2;1 transgenic Arabidopsis demonstrated better growth, elevated proline content, increased superoxide dismutase (SOD) and peroxidase (POD) activities, and reduced malondialdehyde (MDA) content compared with the wild-type Arabidopsis when exposed to cold stress. The findings suggest that overexpression of KoPIP2;1 probably conferred cold tolerance of transgenic Arabidopsis by enhancing osmoregulation and antioxidant capacity. This present data presents a valuable gene resource that contributes to the advancement of our understanding of aquaporins and their potential application in enhancing plant stress tolerance.Aquaporins (AQPs) are essential channel proteins that play central roles in maintaining water homeostasis. Here, a novel aquaporin gene, named  The process of water movement plays a crucial role in facilitating optimal growth and reproductive functions in plants. Aquaporins (AQPs), a group of water channel proteins, can effectively enhance the permeability of water . In addiKandelia obovata exhibits the highest level of cold resistance among mangrove plants [K. obovata may play positive roles in response to cold stress, and/or be useful genes for studying the stress response of mangroves. However, there is currently little literature about AQPs in K. obovata or other mangrove species [Studies have shown that some AQP genes can benefit plants under favorable and/or abiotic stress conditions ,14, while plants . AQPs fue plants  and playe plants . Therefo species ,25,26,27K. obovata under cold stress, which exhibited homology with other AQPs [KoPIP2;1. Furthermore, overexpression of KoPIP2;1 in Arabidopsis thaliana was performed to test its function under cold stress. This study will provide valuable clues for the function of AQP genes in K. obovata under cold stress, and also help to improve our understanding of the mechanism of stress resistance in mangrove plants.In our previous study, we isolated an expressed sequence tag (EST) (Ko4002) from her AQPs . In ordeK. obovata were collected from Guangdong Mangrove Ecological Technology Co. LTD  and sown in clean sand at room temperature. The 3 months seedlings were transferred into growth chamber with normal condition . After 7 days, these seedlings were treated under cold treatment  for 0 h, 6 h, 12 h, 1 d, 2 d, 4 d, 7 d, 15 d and 20 d. The control group consisted of seedlings treated at 5 °C for 0 d. The leaves, stems and roots of treated seedlings were individually collected and rapidly frozen in liquid nitrogen before being stored at −80 °C until further use.The seeds of K. obovata leaves using the Tiangen RNA plant Plus Reagent  following a modified version of the described method [TM reverse transcription Kit  for synthesizing the first strand cDNA in accordance with the manufacturer’s instructions. The resulting cDNA samples were utilized to clone the full-length of the KoPIP2;1 gene and to conduct the RT-qPCR analysis.Total RNA was extracted from d method . Before KoPIP2;1 gene (GenBank accession number: JZ585815.1) has been mentioned in our previous study [KoPIP2;1 gene. Nested PCR reactions were performed using nested primers  to obtain DNA fragments and RACE products which were purified by agarose gel electrophoresis and confirmed by sequencing. The 3′- and 5′- nucleotide sequences obtained were assembled using DNAMAN 6.0 software to obtain the full-length sequence of KoPIP2;1 with overlapping regions. Subsequently, primers were designed based on the assembled sequence for cloning the complete length of KoPIP2;1. The newly obtained KoPIP2;1 sequence was then submitted to the company  for sequencing, confirming the full-length cDNA sequence of KoPIP2;1, which was subsequently submitted to GenBank and assigned accession number KP267759.1.The partial sequence of the us study . This ESKoPIP2;1 (GenBank accession number: KP267759.1) was predicted using the ORF Finder tool provided by the National Center for Biotechnology Information (NCBI) . Sequence comparisons with known sequences were conducted utilizing the NCBI BLAST tools . The molecular weight and theoretical pI were predicted using the Compute pI/MW tool , while TMHMM-2.0  was utilized to predict the transmembrane domain. Motif Scan  was employed to detect motif sequences, and subcellular localization was predicted by combining PSORT  and Softberry ProComp v. 9.0 . The ClustalX 1.83 software was utilized to perform multiple sequence alignments. A total of 40 homologues of AQPs from the NCBI database were employed to investigate the evolutionary relationship of the deduced KoPIP2;1 protein. The phylogenetic tree was constructed using the Neighbor-Joining method in MEGA 5.0 software. The three-dimensional (3D) structure of KoPIP2;1 was generated using the SWISS-MODEL tool .The deduced amino acid sequence of KoPIP2;1 gene under cold stress were determined using a real-time quantitative PCR (RT-qPCR) method. Seedlings were subjected to 5 °C for different durations , with seedlings treated at 0 day serving as control. The leaves, stems and roots were harvested separately under different conditions. Total RNA was extracted from samples and cDNA synthesis was performed following the methods according to the above K. obova’s 18S rRNA was used as reference [KoPIP2;1  were designed for RT-qPCR analysis. The SYBR Premix Ex TaqTM II  reagents were used in the RT-qPCR experiment and analyzed by iCycler iQ5 real-time PCR detection system . The cycling parameters included an initial denaturation at 95 °C for 30 s, followed by 40 cycles of denaturation at 95 °C for 5 s, annealing at 55 °C for 30 s and extension at 72 °C for 30 s. Each RT-qPCR reaction was performed in triplicate. Transcripts were quantified using 2−∆∆CT method [t-test, and graphical representation was generated with GraphPad Prism 7.0 .The transcriptional levels of the eference ,31. EachT method . The datKoPIP2;1 gene was cloned into the pFGC5941-35S-GFP vector [® Plus One Step Cloning Kit  following the manufacturer’s instructions. Subsequently, the resulting fluorescence-tagged 35S-KoPIP2;1-GFP vector was introduced into Nicotiana benthamiana epidermal cells via agrobacterium-mediated transformation. The preparation of N. benthamiana leaves and transformation procedures were carried out as previously described [KoPIP2;1-GFP was transiently introduced into the leaf epidermis of N. benthamiana via Agrobacterium tumefaciens EHA105-mediated transformation, while the vector containing only 35S-GFP served as a control. The fluorescence signal in leaves was visualized using Zeiss LSM710 laser scanning confocal microscopy (×63), with excitation and emission wavelengths of 489 nm and 510 nm for GFP signal detection, respectively. Fluorescence intensity was measured at 493 to 542 nm for GFP.The P vector  with N-tescribed . BrieflyKoPIP2;1 gene was cloned into the binary vector pGW505-1 using Invitrogen Company’s Gateway technology, following the manufacturer’s instructions, to investigate its function in a heterologous expression system. The primers TOPO-F (5′-GGGGACAAGTTTGTACAAAAAAGCAGGCTCGATGGCAAAGGACGTTGAAGTTCAAG-3′) and TOPO-R (5′-GGGGACCACTTTGTACAAGAAAGCTGGGTCAGCATTGCTCCTGAAGGATCCGA-3′) were designed for directional cloning in accordance with the specified requirements. Subsequently, the amplified PCR products were cloned into an entry vector using the pENTR™/D-TOPO™ cloning Kit  and subjected to sequencing analysis. Confirmed recombinant entry vectors were subsequently transferred into the plant binary vector pGWB505 through LR Clonase™ II Enzyme Mix  for construction of plant Gateway® expression vector and sequenced. The pGWB505 vector containing KoPIP2;1 recombinant clones was introduced into A. tumefaciens strain EHA105 using the freeze–thaw method for transformation. Positive colonies were then subjected to floral dip-mediated transformation in Arabidopsis plants [A. thaliana were subsequently obtained and selected by culturing on MS medium agar plates supplemented with 50 mg/L kanamycin. The integration of transgenes in these plants was confirmed through RT-PCR (reverse transcription-PCR) analysis using gene-specific primers F2 (5′-ATGGCAAAGGACGTTGAAGTTCAAG-3′) and R2 (5′-TTAAGCATTGCTCCTGAAGGATCCG-3′). The transgenic lines were advanced through self-pollination until T3 transgenic plants were obtained. Finally, the T3 or T4 homozygous lines were utilized for functional analysis.The s plants . PositivA. thaliana and transgenic Arabidopsis lines overexpressing KoPIP2;1  were subjected to cold treatments. Cold stress was applied to 12-day-old seedlings by incubating them at 5 °C for 10 days. Seedlings grown under normal conditions at 22 °C were used as controls. The fresh weight and number of lateral roots in these seedlings were quantified. The levels of proline and malondialdehyde (MDA), as well as the activities of superoxide dismutase (SOD) and peroxidase (POD), were quantified using the corresponding assay kits  following the manufacturer’s instructions. All experiments were conducted in triplicate. Statistical analysis was performed using GraphPad Prism 7.0 software . One-way ANOVA followed by Duncan’s test was employed to determine significant differences using SPSS statistics 25.Wild-type (WT) KoPIP2;1 (GenBank ID: KP267759.1). The KoPIP2;1 sequence comprises a 136 bp 5′-untranslated region (UTR), a 416 bp 3′-UTR, and an 852 bp complete open reading frame (ORF) that encodes a protein of 283 amino acids with a calculated molecular weight (MW) of 30.41 kDa and an isoelectric point (pI) of 6.05. Bioinformatics analysis revealed that the KoPIP2;1 protein contains 24 positively charged residues (Arg and Lys) and 21 negatively charged residues (Asp and Glu) and is predicted to be localized in the plasma membrane. Similar to other PIPs, KoPIP2;1 contains six transmembrane domains (TM1–TM6), five loops (Loop A–Loop E) and conserved dual NPA motifs (NPA Ⅰ and NPA Ⅱ)  within its central region, as well as a distinctive conserved sequence (P/K)/DYX(E/D)PP(P/R)X3-4(E/D)XXELXXWSF(Y/W)R at its N-terminal, all of which are typical features of the PIP subfamily [KoPIP2;1 exhibited the highest sequence identity (91.26%) with Hevea brasiliensis aquaporin HbPIP2;2 (GenBank accession number: XP_021662216.1). Sequence alignment also demonstrated that KoPIP2;1 displayed greater similarity to PIP2s than PIP1s in both the N-terminus and C-terminus regions  was generated and named d NPA Ⅱ) , which aubfamily . Further regions . These fKoPIP2;1  [KoPIP2;1 was acceptable. Homology modeling demonstrated that the 3D model structure of KoPIP2;1 consisted of a homo-tetramer, with each monomer containing eleven α-helixes, two β-strands, some random coils, three octyl-β-glucanpyranoside molecules, seven cadmium ions and four mercury ions. It is suggested that the presence of cadmium and mercury ions may impact the gating mechanism of AQPs [The SWISS-MODEL online tool was utilized to construct the 3D model of KoPIP2;1 . A sequejc6.2.A)  revealed of AQPs .Arabidopsis aquaporins, as well as some aquaporins that exhibit high homology with the KoPIP2;1 sequence through NCBI blast analysis. According to phylogenetic tree  and its 3D modeling template . According to these data, we named the aquaqporin KoPIP2;1 in this study.We have downloaded representative amino acid sequences of different types of tic tree , KoPIP2;KoPIP2;1 is significantly induced in various tissues compared with normal conditions   in K. obovata under cold stress. Notably, significant wilting and dehydration of K. obovata leaves were observed after 15 days under cold stress in our previous study [KoPIP2;1 is an early responsive gene and may play crucial roles in the response to cold stress in K. obovata.Under cold stress, the expression of nditions . The res79-fold) A, stems 79-fold) B, and ro79-fold) C. It canus study . These rKoPIP2;1-GFP vector, labeled with fluorescence, was introduced into the epidermal cells of N. benthamiana and examined using confocal laser-scanning microscopy. As depicted in KoPIP2;1-GFP was predominantly accumulated in the plasma membrane, whereas the control GFP protein was distributed throughout the entire cell, including the nucleus. These findings indicate that KoPIP2;1 is a plasma membrane protein and are consistent with bioinformatics predictions.The 35S-Arabidopsis and three KoPIP2;1 transgenic Arabidopsis lines  showed no significant difference in fresh weight under normal growth conditions (CK) and cold stress, respectively  and in transgenic A. thaliana overexpressing PgTIP1 (Panax ginseng TIP) [KoPIP2;1, leading to morphological changes.AQPs have been reported to mediate transcellular (across membranes) root water transport and hydraulic conductance . Many PIeng TIP) ,47. ThisKoPIP2;1 exhibit superior growth compared with WT plants when subjected to cold stress. This suggests that KoPIP2;1 confers tolerance to cold stress in transgenic Arabidopsis plants. Similarly, the overexpression of SiPIP1;5A (Saussurea involucrata PIP) enhances the cold tolerance of tomato by regulating cell water balance [AtPIP1;4 and AtPIP2;5 in plants has been seen to result in accelerated water loss during drought stress [AtPIP1;b in tobacco has led to rapid wilting under drought stress [Furthermore, our findings indicate that plants overexpressing  balance . Converst stress . Overexpt stress . TherefoKoPIP2;1 overexpressing plants exhibited higher proline level compared with WT plants, indicating that KoPIP2;1 may regulate osmotic imbalance induced by cold stress. This finding is consistent with a previous study demonstrating that overexpression of TsPIP1;1  improved cellular proline accumulation in rice under salt stress [Abiotic stresses always disturb osmotic balance in plants. In general, proline is widely distributed in many organisms, and the accumulation of this substance can improve cellular protection and reduce the cellular osmotic potential . In thist stress .KoPIP2;1 can mitigate membrane damage in overexpressing plants. This finding is consistent with previous studies, which have demonstrated that overexpression of TsPIP1;1 and MaPIP1;1 also reduces cellular MDA levels [KoPIP2;1 in plants has resulted in increased levels of both SOD and POD activities compared with WT plants. Taken together, these findings suggest that the overexpression of KoPIP2;1 can enhance plant osmotic regulation and antioxidant capacity, which in turn reduces membrane injury. Certainly, this is only the start in terms of the study of the function of KoPIP2;1, further investigations, including detailed analysis in transgenic plants, are necessary to fully elucidate its role.Abiotic stress induces the rapid accumulation of reactive oxygen species (ROS), which can cause severe damage to cell membranes by oxidizing DNA, proteins, and lipids . MDA is A levels ,52. SOD A levels . The oveKoPIP2;1 from the mangrove plant K. obovata. Overexpression of KoPIP2;1 improved the cold tolerance of transgenic A. thaliana by regulating solute accumulation and antioxidant capacity, suggesting an important role for KoPIP2;1 in K. obovata under cold stress. Future investigations will focus on identifying upstream regulators of KoPIP2;1 and evaluating the growth performance of these transgenic lines exposed to diverse stresses.In summary, we successfully cloned and characterized the aquaporin gene


A community cluster of influenza A(H3N2) caused by viruses with an E199G substitution in PA was detected in Nara, Japan, between February and March 2023. The three patients with these mutant viruses had not received antiviral treatment before specimen collection but patients in the same hospital had. The sequences of the mutant viruses were closely related, suggesting clonal spread in Nara. They showed reduced susceptibility to baloxavir in vitro; however, the clinical significance of the PA E199G substitution remains unclear. Baloxavir marboxil, a cap-dependent endonuclease inhibitor, was approved in Japan in 2018 for the treatment and prophylaxis of influenza A and B virus infections in patients 12 years and older and children younger than 12 years weighing at least 10 kg. Since the 2017/18 influenza season, we have been monitoring the susceptibility of circulating influenza viruses to baloxavir and the neuraminidase (NA) inhibitors oseltamivir, peramivir, zanamivir, and laninamivir. Approximately 10-15% of total isolates are randomly selected and analysed. During our nationwide monitoring, we detected sporadic cases of influenza A(H1N1)pdm09 and AH3N2) viruses with reduced susceptibility to baloxavir in the 2018/19 and 2019/20 seasons [N2 viruseHere, we report a community cluster of influenza A(H3N2) virus with reduced susceptibility to baloxavir due to a PA E199G substitution.In Japan, influenza activity was low throughout the COVID-19 pandemic ; howeverhttps://nextstrain.org/flu/seasonal/h3n2/ha/2y). In Japan, 159 (48%) of the 329 influenza A(H3N2) viruses tested in this season were found to belong to clade 2b, followed by 76 (23%) that belonged to clade 2a.3a. We found that three of the 12 influenza A(H3N2) viruses possessed an E199G substitution in their PA protein, which is associated with reduced susceptibility to baloxavir [During the season 2022/23, we isolated 12 influenza A(H3N2) viruses in Nara, a town located near Osaka and Kyoto, as part of our nationwide monitoring . In NaraDeep sequencing analysis revealed that the PA E199G substitution was present in the specimens. No amino acid substitutions associated with reduced susceptibility to NA inhibitors were detected in the PA E199G mutant viruses.https://gisaid.org) (To assess the frequency of the PA E199G substitution in A(H3N2) viruses, we examined 81,806 PA sequences from influenza A(H3N2) viruses between 1968 and August 2023 deposited in the GISAID EpiFlu database (aid.org) . The PA Although the frequency of the PA E199G mutant A(H3N2) viruses is low, we found that three of 12 influenza A(H3N2) viruses isolated in Nara possessed the PA E199G substitution. No epidemiological link among the patients infected with the PA E199G mutant viruses could be identified, and none of them had received baloxavir before specimen collection. The whole genome sequences of these mutant viruses were identical except for an R723L substitution in polymerase basic protein 1 (PB1) of A/Nara/12/2023, suggesting clonal spread in Nara. These results indicate a community cluster of the PA E199G mutant A(H3N2) viruses in Nara.50) values. To interpret the antiviral susceptibility, we applied the World Health Organization criteria, which are based on fold-changes in IC50 values compared with the median value for viruses from the same type/subtype/lineage [We determined the susceptibilities of the three PA E199G mutant A(H3N2) viruses to baloxavir and NA inhibitors  by using a focus reduction assay and a fluorescence-based NA inhibition assay, respectively, as previously described . Our res50 values to baloxavir compared with the median IC50 values of wild-type A(H3N2) viruses isolated in the 2022/23 season in Japan. In a previous study, similar to our results, the PA E199G mutant A(H3N2) virus showed 4.5-fold higher IC50 values to baloxavir [As shown in aloxavir . These rThe PA E199G mutant influenza A(H3N2) virus was previously detected during treatment-emergent variant monitoring in a paediatric study of baloxavir and showed reduced susceptibility to baloxavir . In the An influenza outbreak occurred in the school attended by the patient infected with A/Nara/3/2023. We previously reported that influenza viruses with reduced susceptibility to baloxavir were detected 3–6 days after baloxavir administration [Of the substitutions that emerged after baloxavir treatment in the past, PA I38 substitutions were the most frequent and had the greatest impact on baloxavir susceptibility . The freAlthough patients infected with the PA I38 mutant viruses exhibited prolonged virus shedding, a rebound in virus titres after treatment and delayed symptom alleviation ,12-15, tThe PA E199G mutant influenza A(H3N2) viruses showed reduced susceptibility to baloxavir in vitro; however, the clinical significance of the PA E199G substitution remains unclear. Therefore, baloxavir may be effective in patients infected with these mutant viruses.