Actinomycetales isolates are presented. Both genomes are G+C rich and consist of a single chromosome containing homologs of known arsenite resistance genes.The complete genome sequences of two highly arsenite-resistant  Actinomycetales isolates are presented. Both genomes are G+C rich and consist of a single chromosome containing homologs of known arsenite resistance genes.The complete genome sequences of two highly arsenite-resistant  Arsenic is a toxic metalloid and is ubiquitously found in the environment. The amount of arsenic is increasing in our environment through two processes. Naturally, weathering of rocks, erosion of land, volcanism, etc., result in a release of arsenite. Anthropogenic activities, like combustion of fuels, the use of arsenic-containing pesticides, wood preservation, etc., lead to an increased release of arsenic into the environment. It is commonly found in the two forms arsenite and arsenate, with arsenite being 100 times more toxic because it can react with thiol groups of proteins. Microorganisms have developed mechanisms  for arsenic detoxification. By studying arsenic-resistant microorganisms, we can learn how to detoxify arsenic in the environment.Brevibacterium sp. strain CS2 and Micrococcus luteus strain AS2 were isolated from industrial wastewater collected from Ittehad Chemicals in Kala Shah Kaku, Punjab, Pakistan. These bacterial strains were isolated by plating wastewater on lysogeny broth (LB) agar plates supplemented with 10 mM arsenite. The MICs for arsenite and arsenate were 40 mM and 280 mM for strain CS2 and 50 mM and 275 mM for strain AS2, respectively (ectively . The opthttps://github.com/rrwick/Porechop) was used for trimming of adapters and barcodes. Nanopore coverages were 387× and 325× for CS2 and AS2, respectively.AS2 and CS2 were inoculated in LB broth and incubated at 37°C for extraction of DNA using the MasterPure complete DNA and RNA purification kit . Illumina sequencing libraries were prepared using the Nextera XT sample preparation kit  and were sequenced on an Illumina NextSeq 550 platform with a high-output kit v2.5 yielding 2 × 151-bp paired-end sequences. All programs were run with default parameters. Sequencing adapters and barcodes were trimmed with Cutadapt (v1.18) (Micrococcus luteus strain NCTC 2665 (NCBI RefSeq accession number NR_075062) and Brevibacterium senegalense strain JC43 (NCBI RefSeq accession number NR_118221). Both strains have genes related to arsenical resistance, including arsenate-mycothiol transferases and ACR3 family arsenite efflux pumps.Long-read-only assemblies were performed with Flye v2.4-release)  and subs-release Brevibacterium sp. CS2 and Micrococcus luteus AS2 have been deposited in GenBank under accession numbers CP040020 and CP040019. Nanopore and Illumina raw read data have been uploaded to the SRA under BioProject numbers PRJNA540762 and PRJNA540761 for strains CS2 and AS2, respectively.The complete genome sequences of


The aims of the study were to investigate the PK characteristics of MMF dispersible tablet by detecting the active metabolite of MPA and to establish an accuracy and precision equation for calculating MPA-AUC0-12h by limited sampling strategy (LSS) in Chinese kidney transplant patients. A total of 60 postoperative kidney transplant recipients were given a multiple-dose of MMF dispersible tablet twice daily combination with tacrolimus (Tac) and steroids. On the 5th day post-transplantation, blood specimens were collected before drug administration and up to 12 h after MMF dispersible tablet administration. Non-compartmental PK analysis was used to determine the data obtained from individual patients. Multivariate stepwise regression analysis was used to develop models for predicting MPA-AUC0-12h. The 3- and 4-point sampling models using 2 h, 4 h, 8 h and 1 h, 2 h, 4 h and 8 h, respectively, allowed accurate estimation of MPA-AUC0-12h. PK parameters of MMF dispersible tablet were obtained and the 4-point LSS is the best model for accurate and precise estimation of MPA-AUC0-12h.The mycophenolate mofetil (MMF) dose management for optimization of post-transplant treatment especially the early postoperative phase has been well recognized. MMF is a pro-drug of mycophenolic acid (MPA) and is widely used in Chinese renal transplant patients. Until now, the pharmacokinetic (PK) characteristics and model for the area under the concentration–time curve for the 12-h (h) of exposure (AUC Figure 1. Dispersible tablet a new formulation of MMF is widely used in renal transplant patients in China. As MPA has a narrow therapeutic window and large inter-individual variability (0-12h) of MPA (MPA-AUC0-12h) is essential to improve clinical outcomes  of MPA showed a poor correlation with the MPA-AUC0-12h (0-12h monitoring is laborious. The limited sampling strategy (LSS), using a limited number of blood samples to estimate the MPA-AUC0-12h , an ester prodrug of mycophenolic acid (MPA), is the first-line immunosuppressant drug used with other drugs in prevention and treatment of graft rejection in solid organ transplantation. Chemical structures of MMF and MPA are shown in iability , therapeoutcomes . Adequatoutcomes . The MPAUC0-12h . Owing tUC0-12h , solves UC0-12h . Due to 0-12h calculation in large number of samples in such patients.The aims of the study were to investigate the PK characteristics of MPA in Chinese kidney transplant patients taking MMF dispersible tablet in association with steroids and tacrolimus (Tac) and to develop an accuracy and precision LSS for MPA-AUCConsecutive, prospective 60 patients were included in the study. All the patients were over 18 years and underwent the first kidney transplant in this single-center study. Patients were recruited after passing a physical examination and laboratory tests, which included blood biochemistry, hematology, and urine analysis. We excluded patients with cancer, patients with hematologic abnormality, hepatic abnormality, gastrointestinal disorder, pregnancy, systemic infection, or any acute disease, and patients who allergy to MMF dispersible tablet, MPA, steroids or Tac. All patients underwent the same therapeutic scheme of MMF dispersible tablet with Tac and steroids as concomitant immunosuppressive therapy. This research was conducted according to the principles of the Declaration of Helsinki (1964) and its later amendments. The clinical protocol and the informed-consent form were approved by the Ethics Committees of The First Affiliated Hospital of Zhengzhou University. All participants were informed of the details and procedures of the research before they signed a written informed consent.The study was carried out in two steps. We enrolled 40 patients in the first step as the model-building group. The model-building group was used to establish optimal LSS for predicting AUC of MPA.The 20 patients who were enrolled later, were used as the validation group. The validation group was used to assess the accurate and practical applicability of the optimal LSS. The study was carried out in the Clinical Nephrotransplantation Center of the First Affiliated Hospital of Zhengzhou University.C0, C0.5, C1, C1.5, C2, C3, C4, C6, C8, C10, and C12) after the oral morning dose of MMF dispersible tablet on the 5th day after transplantation. The samples were sent to lab on ice. The blood samples were centrifuged at 3000 r/min and plasma samples were separated. MPA concentrations were analyzed using the validated ultra-performance liquid chromatography ultraviolet (UPLC-UV) method  twice daily with water. The starting Tac dose was 0.1–0.15 mg/kg/day. Targeted Tac whole-blood trough concentration was 8–15 ng/mL and the Tac concentration was assayed by enzyme-multiplied immunotechnique. All patients received two intraoperative corticosteroid of 500 mg of methylprednisolone. Maintenance methylprednisolone dose was tapered to 500 mg on day 2, followed by a stepwise reduction to 375 mg on day 3, 250 mg on day 4, and 120 mg on day 5. Then methylprednisolone (16 mg/d) was administered. Patients fasted overnight before dosing and given standardized meals until 1.5 h after MMF dispersible tablets administration, serial blood samples (2 mL each) were drawn in vials containing EDTA at 0, 0.5, 1, 1.5, 2, 3, 4, 6, 8, 10, and 12 h  method .0-t) calculated by linear trapezoid method. Predose concentration (C0), post-dose minimum and peak plasma concentration (Cmin and Cmax), time to reach Cmax (Tmax) were obtained directly from the concentration vs. time profiles. Total body clearance (CL) was calculated by dose/AUC0-τ . Given the difference in doses, we normalized the AUC0-12h of 500–750 mg. Dose-normalized estimate of AUC0-12h,norm was derived by the corresponding value dividing the result that 750 mg divided 500 mg. Inter-individual variability was assessed using coefficient of variation (CV).The PK analysis was performed using WinNonlin version 7.0 . Non-compartmental PK analysis was used to determine the data obtained from individual patients. The area under the concentration-time curve 0 to time  and a model validation group (n = 20). All results were presented as mean ± SD. The data were performed using SPSS version 18.0 . Step 1: the fAUC0-12h was calculated by linear trapezoid method. Prediction equation models were derived by multiple regression analysis, using MPA concentrations at different time points. The fAUC0-12h as a dependent variable and the concentration of MPA at each time point were used as independent variable. The correlation coefficient (R2) was used to evaluate the regression level of the equation. The fAUC0-12h was compared with eAUC0-12h calculated using the selected equation of Step 1 in the validation group. The prediction bias were quantified as the percentage of mean prediction error (MPE%) and mean absolute prediction error (MAPE%). The two error parameters were calculated by the following equations:The study had sufficient sampling points to capture the full characteristics of MPA’s PK. Since, to date, model for estimation of MPA-AUC0-12h and eAUC0-12h in the validation group. Bias and precision were visualized by plotting the average AUC0-12h that results from the estimated and full profile. The eAUC0-12h was calculated using the selected equation. The correlation between fAUC0-12h and eAUC0-12h was expressed by scatter diagram. The best model was selected based on account the values of r2, predictive bias, and the Bland–Altman analysis.The Bland–Altman test  was perfThe investigators including clinicians and clinical pharmacists who monitored the patients for adverse events (AEs). The primary outcomes were 1-week incidence of acute rejection, graft function and other AEs during hospitalization. Delayed graft function was defined as dialysis requirement in the 1st week post-transplantation. Secondary outcomes included 6-month incidence of acute rejection, graft survival and other related AEs. Those events were also determined using patient interview  regarding the potential presence of AEs, such as diarrhea, nausea and so on, at any time during hospitalization and by comparing the results of laboratory tests , during the study and 6 months after completion of the study (follow-up visit).0-12h) were evaluated by Spearman test. All statistical analyses were performed with SPSS version 18.0. P < 0.05 was considered statistically significant.Differences in efficacy and safety end points were evaluated by the Chi-square or Fisher’s exact test. The correlations between AEs and covariates  in AUC0-12h, Cmax, Cmin, C0, CL, and Tmax, respectively, were 15.19, 27.97, 89.72, 66.53, 23.56, and 80.78%. Due to the enterohepatic recirculation (EHR) most of the patients revealed a second small peak in the 4–12 h after taking MMF dispersible tablets.A total of 60 patients participated in the PK study. The mean plasma concentration–time curves of MPA after administration of MMF dispersible tablets in all patients, model-building group and validation group are shown in 0-12h are shown in Table 3. The equation containing only the trough level showed a weak correlation (R2 = 0.501) with fAUC0-12h.The results of regression equations obtained in the study group between each sampling time point and the fAUC0-12h were obtained by multiple stepwise regression analysis. As seen in Table 4, with the number of sampling time points increasing, the linear correlations between the eAUC0-12h and fAUC0-12h also improved. Using the three selected equations obtained in the model building group, we compared the differences between eAUCs and fAUCs in the model validation group. The predictive results are shown in Figures 3, 4. All the three models were found to have good mean residuals and symmetries.Equations for two-, three-, and four-point estimations of the MPA-AUCThe differences between the eAUCs obtained with 2, 3, or 4 points and the fAUCs in the 20 validation patients were 3.78 ± 6.17, 3.41 ± 5.51, and 2.11 ± 3.50 μ⋅h/mL, respectively; 95% confidence intervals were (-8.31 to 15.88), (-7.39 to 14.22), and (-4.74 to 8.97), respectively.Figure 3A).For the two-point equation, the MPE% and MAPE% values were 10.28 and 12.99%, respectively. The mean values of fAUC and eAUC were not significantly different . The agreements between fAUCs and eAUCs were tested by Bland–Altman Analysis, 10% (2/20) of patients were outside the 95% confidence interval .For the three-point model, the MPE% and MAPE% values were 8.64 and 12.39%, respectively. The mean values of fAUC and eAUC were not significantly different . The Bland–Altman Analysis confirmed the agreement between eAUCs and fAUCs, and 5% (1/20) of patients were outside the 95% confidence interval . The abilities of the three models to predict the MPA–AUC0-12h are depicted in Figure 4. Based on the above analysis, the 4-point method  was the best LSS.The four-point model, the MPE% and MAPE% values were 5.26 and 8.35%, respectively. The mean values of fAUC and eAUC were not significantly different . The Bland–Altman Analysis showed the best agreements between the eAUCs and fAUCs, and 5% (1/20) of patients were outside the 95% confidence interval . The incidences of AEs in different MPA-AUC0-12h ranges were listed in Table 5. There were no significant differences in the incidences of AEs between the patients with MPA-AUC0-12h levels no more than 60 μg⋅h/mL and more than 60 μg⋅h/mL at the 5 days after transplantation. This may related to the small sample size. Though 60 μg⋅h/mL was not a useful threshold, a relationship was still seen between AUC and adverse effects. Mean MMF daily doses at discharge were similar with hospitalization. Two patients suffered constipation at 2 months post-transplantation. All these AEs were mild. The formulation was well tolerated in transplant patients in the study.No serious AEs occurred in the study. There were no patients who suffered from acute rejection, suspected acute rejection, delayed graft function or systemic infection during hospitalization and 6 months post-transplantation. Results of vital signs, and physical examinations were within normal limits for all participants during hospitalization. The Spearman correlations between AEs and age, body weight or MPA-AUC0-12h were lacking for the new formulation of MMF in Chinese renal transplant patients. The present study developed LSSs of MMF dispersible tablet in renal transplant patients for the first time.The MMF dose management for optimization of post-transplant treatment especially the early postoperative phase has been well recognized. PK studies in the renal transplant population are essential to determine the correct dosage strategies for optimal efficacy. There is an increasing interest in use of MMF dispersible tablet in Chinese renal transplant patients. To date, most studies investigating the PK of MPA in this population applied a sparse sampling protocol, which may have limited the overall precision of the final model. In the present study, the full PK character was available for all the patients and can be used to provide limited sampling strategies information for the early transplant phase patients. The PK characteristics of MMF and equation for estimation of MPA-AUCtmax occurred at 0.5 to 6 h after MMF dispersible tablet dosing (mean peak time point: 1.5 h post-dose) and Cmax ranged from 4.26 to 38.4 μg/mL. The interindividual variability in AUC0-12h, Cmax, Cmin, C0, CL, and Tmax were large. Previous studies . The best correlations between the single-point concentration and fAUC occurred at 3 h (R2 = 0.538), 8 h (R2 = 0.682), and 10 h (R2 = 0.548) treatment with MMF dispersible tablet during the 1st week after kidney transplantation. These results are different with that found by the previous study (R2 < 0.4 at any single-point concentration) in Chinese patients treated with MMF capsule in combination with Tac and steroids , three time points  and four time points  models were developed in this study. The frequency of sampling times was acceptable in all models.The MPA PK profile is affected by the associated calcineurin inhibitors . The cyc0-12h . The presteroids . Then, wcipients . As a re0-12h was C2 and C8 with acceptable MPE% and MAPE%. This is the best practicability model with the least blood samples based on the dynamic characters of MMF dispersible tablet in this study. However, in the literature, there were no two-sample models to predict the MPA-AUC0-12h in early kidney transplant recipients were recommended for clinical practice.In this study, the two-sample equation displayed a good agreement with the fAUC. The significant correlation for predicting MPA-AUCC0/C1/C4, C2/C6/C10, C2/C4/C9, C0/C1/C2, and C0/C0.5/C2) (C2/C6/C10)  (C2/C4/C8) possibly were the patient ethnicity, number of patients, pathological conditions and formulation of MMF.Compared with the two-sample model the 3-time point equation was better correlate with the fAUC. Our equation was different with the published three-point combinations (C0.5/C2) . In the /C6/C10)  the authC0.5/C2)  the patiC8 + 1.34 ×C2 + × 1.66 ×C4 + 0.76 ×C1 (R2 = 0.948). This equation was based on four time points provided the most reliable estimation of the MPA-AUC0-12h. The model included absorption phase, distribution phase and elimination phase after the MMF dispersible tablet administration.In this study, the best model to assess the fAUC as tested by Bland–Altman analysis was 8.36 + 7.49 ×r2, predictive bias, and the Bland–Altman analysis. The 4-point method  was the best LSS. This model can be recommended for predicting MPA-AUC0-12h in early renal transplant recipients in clinical practice.Considering the MPA kinetic characters that investigated in the present research, three model equations were suggested with strong correlations between the fAUCs and eAUCs. The long time span may restrict the models application for the outpatients, but it was a useful tool to guide the individualization prescription of MMF dispersible tablet dose for the inpatients, especially for early post-transplant phase. The best model was selected by taking into account the values of 0-12h had been validated in early Chinese kidney transplant populations. The finding can be recommended in early Chinese adult renal transplant recipients to individualize the dosage regimen in clinical application.In this study, the PK characteristics of MMF dispersible tablet was studied in the early renal transplant patients. The result revealed new insights into the PK characters of MPA after multiple dose of MMF dispersible tablet. Large interindividual variability in MPA PK was demonstrated. The accurate and clinically feasible models for evaluating MPA exposure level in renal recipients after administration of MMF dispersible tablet, Tac and steroids were developed. This information can help to apply TDM of MPA more efficient. The 4-time point model was the best LSS. The applicability of this model in predicting MPA-AUCJZ, ZS, YL, and XZ designed the research. ZZ, JK, and GF performed the experiments. JY, JZ, LZ, and LhZ analyzed the results. All authors approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer DM and handling Editor declared their shared affiliation.


The chemotherapy-resistance of triple-negative breast cancer (TNBC) remains a major challenge. The Nek2B kinase and β-catenin serve as crucial regulators of mitotic processes. The aim of this study was to test the correlation between Nek2B and TNBC chemotherapy sensitivity, and to determine the regulation of Nek2B on β-catenin and wnt/β-catenin signal pathway.Gene Expression Omnibus(GEO) databases were used to gather gene exprsssion data of TNBC patients who undergoing chemotherapy. The co-expression of Nek2B and β-catenin in TNBC surgical sections and cells were analysed by immunohistochemistry, Q-RT-PCR, Western-blot and immunofluorescent staining. The impact of the expression of Nek2B and β-catenin in prognosis was also assessed using the Kaplan-Meier curves. CCK8 assay was used to detect the IC50 value of TNBC cell line. The endogenous binding capacity of Nek2B and β-catenin and phosphorylation of β-catenin by Nek2B were detected using co-immunoprecipitation (CO-IP). Chromatin immune-precipitation (ChIP) analysis and Luciferase Assays were used to evaluate the binding ability of the Nek2B, β-catenin and TCF4 complex with LEF-1 promoter. Nek2B-siRNA and Nek2B plasmid were injected into nude mice, and tumorigenesis was monitored.We found that overexpression of Nek2B and β-catenin in TNBC samples, was associated with patients poor prognosis. Patients with positive Nek2B expression were less sensitive to paclitaxel-containing neoadjuvant chemotherapy. Interestingly, in a panel of established TNBC cell line, Nek2B and β-catenin were highly expressed in cells exhibiting paclitaxel resistance. Our data also suggest that β-catenin binded to and was phosphorylated by Nek2B, and was in a complex with TCF4. Nek2B mainly regulates the expression of β-catenin in TNBC nucleus. Nek2B, β-catenin and TCF4 can be binded with the WRE functional area of LEF-1 promoter. Nek2B can activite wnt signaling pathway and wnt downstream target genes. The tumors treated by Nek2B siRNA associated with paclitaxel were the smallest in nude mouse, and Nek2B can regulate the expression of β-catenin and wnt downstream target genes in vivo.Our study suggested that Nek2B can bind to β-catenin and the co-expression correlated with TNBC patients poor prognosis. It appears that Nek2B and β-catenin might synergize to promote chemotherapy resistance. Approximately 15% of breast cancers are classified as triple-negative breast cancer (TNBC), lacking expression of the estrogen receptor and the progesterone receptor, and which is characterized by absence or low expression or no amplification on the gene level, of the human epidermal growth factor receptor 2 .Because the centrosome cycle is regulated by protein phosphorylation and given the importance of mitotic and centrosomal kinases, they are attractive targets for anti-mitotic anticancer drugs . NIMA-reThe wnt signaling pathway is an important extracellular pathway comprises two different intracellular signaling pathways: the β-catenin-dependent pathway  and the β-catenin-independent pathway . Compared with other signaling pathways, the canonical Wnt/β-catenin pathway is highly evolutionary conserved and mainly involves β-catenin . β-catenOur goal in this study is to identify a reliable, clinically useful prognostic gene-expression signature that offers novel clinical opportunities for predicting drug resistance and developing novel treatments in TNBC. We determined the effects of Nek2B-overexpression on drug resistance and explored the binding abilitity of Nek2B and β-catenin in TNBC cells.One hundred and seventy-four TNBC breast cancer samples were obtained from 1114 breast cancer patients at the Second Clinical Medical College, Shanxi Medical University, China, during January 1, 2007 and December 31, 2012. Patients who met the following criteria were eligible for this study: (1) patients with invasive TNBC, (2) patients who have completed follow-up date during the study period.Immunohistochemical analyses were performed on formalin-fixed, paraffin-embedded sections of surgical specimen as described previously . The pri2 at 37 °C.Human TNBC cell lines Hs578T, BT20, MDA-MB-231 and MDA-MB-468 were chosen. Cells were cultured in DMEM medium (Gibco) or RPMI 1640(Gibco), 10% fetal bovine serum (FBS)(Gibco) and 1% penicillin/streptomycin (Life Technologies Inc), under a humidified atmosphere without COCell proliferation viability was measured with a Cell Counting Kit-8  following the manufacturer’s instructions. The absorbance was measured at 450 nm on a microplate reader. The OD value was checked every 24 h. Each experiment was performed in triplicate.Total RNA was isolated from cells, and qPCR was performed as described previously . Target Actin-Fwd:5′-AGCGAGCATCCCCCAAAGTT-3′Actin-Rev:5′-GGGCACGAAGGCTCATCATT-3′.Nek2B-Fwd:5′-CTAGCTAGCTAGCCGTCACGGGTCGAG-3′.Nek2B-Rev:5′-CGGGATCCTTAGAATTTGCTCCATTCATTCC-3′.Β-catenin-Fwd:5′-ACTTGCCACACGTGCAATTC-3′.Β-catenin-Rev:5′-ATGGTGCGTACAATGGCAGA-3′.The total protein was extracted from each sample using RIPA buffer containing protease inhibitors. Total cell lysates containing 10 μg of protein were electrophoresed on a SDS-PAGE and then transferred to PVDF membranes . The sample was blocked with 5% fat-free milk for 1 h, and incubated with primary antibodies for 1 h and with secondary antibodies for 1 h. The primary antibodies, including anti-Nek2B  at 1:2000 and anti-β-catenin  at 1:5000 were used in the present study. The blots were visualized using ECL assay.Immunofluorescence was performed as described previously . The twoNuclear and cytoplasmic fractions were isolated using NE-PER Nuclear and Cytoplasmic Extraction Reagents  according to the instructions provided by the manufacturer.Cell lysates were incubated for 4 h at 4 °C with the indicated antibodies and protein A/G beads  or with anti-FLAG  or anti-HA  agarose gels for FLAG-OR HA-tagged protein pull-down. After incubation, beads were washed with low salt lysis buffer, boiled with 4× SDS loading buffer. IB analysis was developed using the Western HRP Chemiluminescence Substrates  .ChIP assay was performed according to instruction (Millipore). ChIP assays were performed using primers spanning binding sites in the LEF-1 promoter. 20 μl magnetic AG-beads (Invitrogen) were used for each ChIP. DNA was purified using DNA Purification Buffer plus DNA purification magnetic beads, as recommended (Invitrogen).TNBC cells were transfected with pGL4-LEF-1-prom-Luc reporters along with plasmids using the Lipofectamie 2000 as indicated in the figures. After 48 h of transfection, cell lysates were subjected to luciferase assays according to the Dual-Luciferase Reporter Assay protocol . Each experiment was repeated in triplicates.Transwell migration assays were performed using a transwell membrane  in a 24-well plate according to the manufacturer’s instructions. The cells were placed in incubators at 37 °C for different time periods according to preliminary experiments. The upper surface cells were removed using cotton swabs, those on the lower surface were fixed with 4% paraformaldehyde in PBS, stained with 0.1% crystal violet and counted.Female BALB/c nude mice of 4–6 weeks old and body weight of 20 ± 2 g were housed in specific pathogenfree (SPF) conditions. All the rats were administered as described previously . All theThe primary endpoints were disease-free survival (DFS) and overall survival (OS). DFS was calculated as time from surgery to locoregional recurrence, distant metastasis, or death. OS was determined as the time from surgery until the date of death or was censored at the date of last follow-up.p-value of< 0.05 was considered significant. All in vitro experiments were performed in triplicate. Data are represented as mean ± S.E. All analyses were performed using SPSS 22.0.The statistical significance between Nek2B and β-catenin expression and patients’ clinicopathological status was tested using chi square (χ2) test. The correlation of Nek2B and β-catenin expression with patient prognosis was determined by Kaplan-Meier method. A two-sided P < 0.05)  and chemotherapy-resistance group . Thirteen differentially expressed genes related to the chemotherapy drug resistance were found using GEO2R analysis tools Fig. a, of whi05) Fig. b. Gene O05) Fig. c.Fig. 1, LNs metastasis group (P = 0.003), and high Ki67 index group (P<0.0001). Nek2B tended to be higher in β-catenin positive cases (72.0%). The number of patients with positive β-catenin expression was significantly higher in the high Ki67 index group (P = 0.003). As shown in Fig. P = 0.031).As shown in Table  P = 0.00, and higP = 0.003). It can be seen that patients with positive Nek2B expression developed drug resistance to paclitaxel-containing neoadjuvant chemotherapy.To explore the relationship between Nek2B expression and paclitaxel resistance, we selected 43 patients diagnosed with TNBC by needle core biopsy, all of whom received 2–3 cycles of neoadjuvant chemotherapy. pCR  was achieved in 1 patient, PR  was achieved in 14 patients, SD (stable condition) was achieved in 10 patients, and PD (disease progression) was achieved in 18 patients. Therefore, our research group detected the expression of Nek2B mRNA in 42 postoperative cancer tissues. As shown in Table P < 0.05). Thus, Hs578T and BT20 cell lines were relatively resistant, while MDA-MB-231 and 468 cell lines were relatively sensitive. Further induction in chemotherapy-sensitive cells MDA-MB-231 with different concentrations of paclitaxel  showed a concentration-dependent increase in Nek2B and β-catenin mRNA levels   experiment showed that endogenous Nek2B and endogenous β-catenin exists in the form of compounds in Hs578T cells Fig. a. At theWe want to test whether the complex formed by Nek2B and β-catenin participate in the β-catenin phosphorylation and degradation using CO-IP in Hs578T cell. Nek2B antibody is used to immunoprecipitate β-catenin, TCF4, Axin l and GSK3β protein. As shown in Fig. Nek2B-siRNA and Nek2B overexpression plasmids were transfected into Nek2B high expression cells and low expression cells, respectively. Data showed that the expression levels of both Nek2B and β-catenin mRNA were reduced in Hs578T and paclitaxel group (P < 0.05), respectively. With the overexpression of β-catenin, the tumor volume were increased. The data demonstrated that combination treatment using Nek2B siRNA and paclitaxel may suppress TNBC tumor growth rate more efficiently than paclitaxel alone or Nek2B siRNA alone, indicating the potential role of Nek2B and wnt/β-catenin pathways in the progression of drug resistance. Total RNA extracts from the tumors were examined for expression levels of Nek2B, β-catenin and downstream target genes of wnt pathway. The results indicated that Nek2B can regulate the expression of β-catenin and wnt downstream target genes in vivo (Fig. To further detect the effect of Nek2B and β-catenin in TNBC chemotherapy resiatance, we performed a combination treatment using Nek2B siRNA and paclitaxel which disrupted the therapeutic targets of spindle poisons in nude mouse model. As shown in Fig. ivo Fig. b, c.Fig.Nek2B plays an established role in centrosome separation, and a number of centrosomal substrates have been identified , 18. NekChemotherapy remains the mainstay for the treatment of TNBC due to lack of targeted therapies . HoweverAs the literatures reported by Zhang et al.  and SeunLimitations of the current study’s findings must be made. There is no validated approach for the assessment of Nek2B or β-catenin by immunohistochemistry, and we elected to use methods by Lee et al. . On the In conclusion, our study showed, for the first time, that Nek2B can bind to β-catenin and the co-expression correlated with TNBC patients poor prognosis. It appears that Nek2B and β-catenin might synergize to promote chemotherapy resistance. Further studies should include TNBC specimens and cells to determine if Nek2 and β-catenin might synergize to promote chemotherapy resistance.


Arsenite, a trivalent form of arsenic, is an element that occurs naturally in the environment. Humans are exposed to high dose of arsenite through consuming arsenite-contaminated drinking water and food, and the arsenite can accumulate in the human tissues. Arsenite induces oxidative stress, which is linked to metabolic disorders such as obesity and diabetes. Brown adipocytes dissipating energy as heat have emerging roles for obesity treatment and prevention. Therefore, understanding the pathophysiological role of brown adipocytes can provide effective strategies delineating the link between arsenite exposure and metabolic disorders. Our study revealed that arsenite significantly reduced differentiation of murine brown adipocytes and mitochondrial biogenesis and respiration, leading to attenuated thermogenesis via decreasing UCP1 expression. Oral administration of arsenite in mice resulted in heavy accumulation in brown adipose tissue and suppression of lipogenesis, mitochondrial biogenesis and thermogenesis. Mechanistically, arsenite exposure significantly inhibited autophagy necessary for homeostasis of brown adipose tissue through suppression of Sestrin2 and ULK1. These results clearly confirm the emerging mechanisms underlying the implications of arsenite exposure in metabolic disorders. White adipose tissue (WAT) and brown adipose tissue (BAT) are the main types of adipose tissue in humans5. In contrast to WAT’s functions for energy storage, BAT is specialized to produce ATP through enriched intracellular mitochondria and heat through non-shivering thermogenesis via the uncoupling protein 1 (UCP1)9. UCP1 proteins are localized in the inner membrane of brown adipocyte mitochondria to uncouple ATP synthesis from respiration13. While BAT quickly regresses following birth14, new evidence has revealed symmetrical fat depots in adults that have classic BAT features18. Therefore, BAT has become a novel target for obesity treatment and prevention.Obesity is a significant risk factor for several prevalent diseases, such as diabetes, hypertension, cardiovascular diseases, and cancers19. Arsenite is accumulated in various tissues and organs including adipose tissues, lung, heart, kidney, brain, eye, liver, hair, bone, and spleen22. Thus, arsenite contaminated drinking water has been considered as a contributing factor for numerous health concerns in humans such as diabetes, lung and skin diseases, and cancer25. Recent evidence suggests that exposure to arsenite may lead to adipose tissue dysfunction and lipodystrophy as well as inhibition of adipogenesis27. Arsenite inhibits adipogenesis and adipocyte function in human mesenchymal stem cells28, 3T3-L1 preadipocytes29, and C3H 10T1/2 preadipocytes30. The mechanisms underlying adipose tissue dysfunction and inhibition of adipogenesis by arsenite have been the focus of intense research. Recent studies suggest that arsenite-induced lipolysis is mediated through transcriptional factors, including peroxisome proliferator-activated receptor-gamma (PPARγ) and CCAAT-enhancer binding protein alpha (C/EBPα)29, as well as β-adrenergic receptor signaling31.Arsenite, a most potent trivalent form of arsenic, is presented in water, soil, and foods due to its abundance in earth crust and the use of arsenite-contaminated pesticides and insecticides32. Mechanistically known so far, AMPK positively regulates autophagy induction through phosphorylating and activating ULK1, mammalian homologue of autophagy kinase Atg1, at Ser317, Ser555, and Ser777 whereas mTORC1 inhibits autophagy induction through phosphorylating and inhibiting ULK1 at Ser75736. ULK1 regulates downstream autophagosome receptors and autophagosome maturation proteins such as p6237 and LC3B, a mammalian homologue of autophagy-related gene (Atg) 838. Recent studies have shown that autophagy plays an important role in 3T3-L1 adipogenesis and lipid metabolism. Autophagy deficient 3T3-L1 preadipocytes block differentiation into mature adipocytes39. Moreover, Atg5 or Atg7 deficient primary mouse embryonic fibroblasts (MEF) impair adipogenesis, and autophagy inhibitor, such as bafilomycin A1 and chloroquine, blocks primary MEF differentiation41.Autophagy is a delicate cellular recycling process in response to environmental and genotoxic stresses, which digests damaged proteins and organelles as a defense mechanismDespite arsenite-induced defect in adipogenesis and fat accumulation in WAT, the roles of arsenite in regulating the functions of BAT remain to be elucidated. Here, we report the impacts of arsenite exposure on the functions and activities of brown adipocytes and BAT. In cultured brown adipocytes, arsenite treatment reduced adipogenesis, mitochondrial biogenesis, respiration and thermogenesis. Arsenite exposure in live mice resulted in heavy arsenic accumulation in BAT but not in WAT. Accumulated arsenite suppressed lipogenesis, mitochondrial biogenesis and thermogenesis in BAT. Autophagy activity was significantly reduced by arsenite exposure through inhibition of Sestrin2 and ULK1. Our data provides the novel mechanisms underlying the effects of arsenite exposure on the physiological functions of BAT and reveals the significance of autophagy in protection against arsenite-induced damage in BAT.P < 0.01) was significantly decreased by arsenite treatment  or the vehicle control. Arsenite dose-dependently suppressed brown adipocyte lipid accumulation, as revealed by Oil Red O stained multiocular, lipid-filled brown adipocytes Fig. . 10 μM oent Fig. . Attenuaent Fig.  was consAnalysis of mRNA expression showed that arsenite also suppressed mRNA levels of nucleus-encoded mitochondrial genes, cytochrome c oxidase subunit IV a (Cox4a) and cytochrome b-c1 complex subunit 6 (Uqcrh), and transcription factors regulating mitochondrial gene expression, transcription factor A (Tfam) and nuclear respiratory factor 1 (Nrf-1) Fig. . Consist42. Autophagosome marker, microtubule-associated protein 1 light chain 3-II  and autophagosome substrate p62 (aka sequestosome 1) were both degraded by active autophagolysosome upto 50% by 10 µM arsenite treatment, phosphorylation of unc-51 like autophagy activating kinase 1 (ULK1) at Ser 555, which is AMPK target site, was increased and phosphorylation of ribosomal protein S6 kinase (S6K) at Thr 389, which is mTORC1 target site, was decreased upon brown adipocyte differentiation process  in mice that were treated with 5 mg/kg and 10 mg/kg treatment groups, respectively. Although previous report identified that arsenic can majorly be accumulated in liver, kidney, and lung25, we never expected such a high and differential accumulation between BAT and WAT in mice. Given that the accumulated concentration of arsenic in BAT but not in WAT was increased in dose-dependent manner, we anticipated that some of physiological parameters of mouse would be affected by arsenite accumulation in BAT. However, we could not observe any significant differences in body weight  and respiratory exchange ratio  when normalized by mouse body weight . The mRNA expression of nucleus-encoded mitochondrial biogenesis genes, Cox4a, Uqcrh, Tfam, and Nrf-1, were also measured on mice BAT . These data suggest that arsenite accumulated in 10 mg/kg group effectively suppresses brown adipocyte function and mitochondrial biogenesis. A well-known function of BAT is non-shivering thermogenesis that has been spotlighted in the regulation of obesity pandemic44. Since UCP1 expression is augmented when exposed to cold in BAT of mice45, we hypothesized that UCP1 inhibition by arsenite would be more dramatic and significant because UCP1 is further induced under by cold exposure. To investigate the effects of arsenite on UCP1 expression in non-shivering thermogenesis in mouse BAT, we challenged the mice in cold condition (4 °C) for 24 hrs at the last day of arsenite administration  and PPARγ (P < 0.05) gene expression levels while there was no significant difference in PGC1 and PRDM16 gene expression levels  whereas Cox4a and Uqcrh had no significant difference , stress-inducible protein Sestrin2, and autophagy receptor p62 under our experimental conditions. Sestrin2, ULK1, and p62 were significantly downregulated by arsenite (10 mg/kg BW of mice) , there was no significant difference in both phosphorylation of S6K (a mTORC1 target) at Thr 389 and phosphorylation of ACC (an AMPK target) at Ser 79  level and induces autophagy as a defense mechanism in human uro-epithelial cellsce) Fig.  suggesti 79 Fig. . These d 79 Fig. .Figure 649. United States environmental protection agency (EPA) classified arsenic as a Group 1A carcinogen. Over decades, majority of studies have focused on arsenic carcinogenesis in skin, lung, liver, and urinary bladder and the increased level of oxidative stress, mitochondria DNA damage, mitochondrial biogenesis, and mutation in arsenic sensitive oncogenes were believed to be major contributing factors in human50. Unlike arsenic carcinogenesis studies, the studies on the effect of arsenic on the adipose tissue metabolism, mitochondrial function in obesity were very limited albeit the positive correlation between arsenite exposure and diabetes mellitus in Michigan residing human studies25, and population studies in high arsenic areas (≥150 µg/L drinking water) of Taiwan and Bangladesh51, and the molecular mechanism underlying the arsenic-induced metabolic disorders is still remained unclear.Increased consumption of high carbohydrate and fat containing food and westernized diet would have been a serious concern correlated with the increased rate of morbidity due to metabolic diseases such as obesity, diabetes, cardiovascular diseases, and cancer. Particularly combined with the exposure to harmful environmental pollutants and genotoxic agents in the food, water and air, it would have been major attention in combating against metabolic diseases in the modern society52. However, low dose of arsenic (<500 nM) upto 24 hrs treatment decreases ROS by inducing antioxidant genes53. Furthermore, low dose of arsenic promotes cell proliferation by increasing mitochondrial biogenesis in skin cancer cells54. It is still debatable but generally ROS-mediated cell damage and apoptosis would be considered as mechanism underlying arsenic-induced toxicity. Our data suggest that upto 10 μM of arsenite treatment did not affect the cell survival without apoptosis induction, so we investigated the effect of arsenite treatment during the course of brown adipocyte differentiation. Arsenite treatment suppressed differentiation and inhibited mitochondrial biogenesis of brown adipocytes in a dose dependent manner. This data was consistent with the inhibition of white adipocyte differentiation in previous studies30. However, we observed 7.9 times more accumulation BAT compared to eWAT in 10 mg/kg administration of arsenite in mice. This data would suggest that arsenite-associated obesity is not only affected by inhibition of WAT function and is further aggravated by the malfunction of BAT in vivo. It needs further investigation why the arsenic is much heavily accumulated in BAT among adipose tissues in mice. It is possible that arsenic directly bind to the promoters of genes involved in the mitochondrial biogenesis or blocking mitochondrial turnover through inhibiting autophagy gene expression or autophagy protein activity by direct binding. Our group and others would investigate the individual arsenic specific target genes and its inhibition mechanism as further studies. High level of accumulation of arsenic prevent normal cell differentiation of adipose tissues and cause the imbalance in fat metabolism and mitochondrial functions, which can contribute to the development of metabolic disorders such as obesity and diabetes. ACC gene expression essential for lipid biogenesis was also suppressed in arsenite-administered mice group. Consistently, we have observed decrease in the lipid droplet content in BAT upon arsenite administration. Another notable result is that arsenite treatment suppresses UCP1 gene expression in the mitochondria, which are essential for thermogenic function of brown fat to dissipate energy as a heat55. In live mice, we observed similar result while total food and water intake, oxygen consumption and RER were not changed. It is possible that there would be compensation mechanism from other tissues protecting mice since oxygen consumption and RER are vital signs essential for survival. Transgenic overexpression of mitochondrial uncoupling protein UCP1-3 in skin of mice showed strong resistance to chemically induced carcinogenesis, suggesting that inhibition of mitochondrial respiration through enhancing mitochondrial uncoupling would be a therapeutic target against arsenic-induced cancer56. In the same context, UCP1 overexpression or UCP1 enhancing drugs would be a potential treatment to protect adipose tissues from arsenic-induced metabolic disorders. It is plausible that proper dose of arsenic chelating agents such as BAL, DMPS, and DMSA57 or overexpressing arsenic-sequestrating proteins such as Hexokinase-2 (HK2)58 in BAT would be beneficial in improving lipid metabolism, mitochondrial function and thermogenesis, which contribute on the prevention or treatment of arsenic-induced metabolic disorders.Other arsenic research groups reported that acute high dose of arsenic (>500 nM) increases ROS and subsequently upregulates antioxidant genes such as heme oxygenase and Nrf2 to protect cells from mitochondria damage and oxidative DNA damage61. Arsenite-induced ROS in short term treatment have been suggested as a main cause to induced autophagy activity62. However, in recent studies using NIH3T3 fibroblasts, 4 hrs of short term treatment of arsenite activates Nrf2 pathway through autophagy inhibition63. Indeed, in our autophagy studies using HIB1B brown adipocytes, we observed increased level of LC3B-II accumulation along with the accumulation of p62 by arsenite, which support the inhibition of autophagy pathway. Treatment of autophagy blocker, bafilomycin A1 also completely suppress the genes of brown adipogenesis, which also support the inhibition of brown adipocyte differentiation via autophagy inhibition by arsenite. It has been reported by several independent groups that a loss of essential autophagy genes such as Atg5 and Atg7 leads to the formation of p62-positive aggregates and accumulation of ubiquitinated protein inclusion body and Mallory bodies in the liver66. Autophagy has two LC3B or Atg8 conjugations systems, Atg5-Atg1267 and Atg4-Atg7-Atg369. However, our data showed that arsenite mediated autophagy inhibition in BAT is independent of Atg5 and Atg7 (P < 0.06). Rather, Sestrin2, ULK1 and p62 inhibition by arsenite accumulation is the underlying mechanism of autophagy inhibition in BAT. Recently, Gao et al. reported that arsenite treatment upto 10 μM for short term 24 hrs blocks autophagy flux through LncRNA UCA1 mediated activation of mTORC1/S6K pathway in human hepatocytes70. However, in our studies, arsenite treatment through oral gavage over longer term 9 days in mice suppressed Sestrin2 and ULK1 gene expressions but not significantly inhibiting AMPK and activating mTORC1. Ro et al. previously reported that Sestrin2 can directly bind with ULK1 and activate autophagy induction possibly independently from mTORC1 and AMPK71. Longer arsenite exposure would directly regulates gene expression levels of Sestrin2 and ULK1 to inhibit autophagy. Differently from cell culture model, there might be a compensation mechanism that maintains mTORC1 or AMPK activity under arsenite-induced toxicity in mammals. Our TEM image data also suggested that inhibition of autophagy activity in arsenite treatment groups may contribute on the accumulation of damaged mitochondria in BAT. Autophagy inhibition by arsenite exposure would lead to irreversible apoptotic cell death which we observed in the reduction of total BAT mass including both mitochondria and lipid mass by arsenite. In this regard, autophagy enhancing drugs such as metformin, torins and resveratrol72 or the overexpression of autophagy induction proteins such as Sestrin2 and ULK1 would be protective mechanism against deregulation or loss of BAT functions affected by arsenic-induced toxicity. While our group is preparing the manuscript, two recent papers came out on publication. Consistent with our data suggesting the autophagy inhibition by arsenite, Dodson et al. from Dr. Zhang’s group treated As (III) concentrations ranging 0.5 to 5 µM [37.5 to 375 ppb] in NIH 3T3 fibroblasts upto 48 hrs and observed that arsenic inhibits SNARE complex formation by enhancing O-GlcNAcylation of SNAP29 and drives autophagy dysfunction73. However, in another arsenic research group suggested that arsenic at 50 ppm  in drinking water administration in NMRI mice under 20 weeks of HFD actually increased both macroautophagy and Chaperon-mediated autophagy (CMA) in liver tissues74. Since the macroautophagy response is more rapid and the CMA is activated when macroautophagy is inhibited or defective under prolonged nutritional stress77, the upregulation of gene expression levels of both macroautophagy and CMA genes would be a compensation effect of serious macroautophagy or CMA defect and possible non-apoptotic cell death in liver. From these recent publications, we speculate that the arsenic effect on autophagy function is highly debatable depending on arsenic concentrations, time, tissue types, and mice species. Therefore, our study has proposed a unique mechanism and is an independent research from liver studies regarding the long term arsenic effect on BAT function and autophagy.Short term treatment upto 48 hrs or acute exposure of arsenite induces autophagy evidenced by increasing lipidated form of LC3B (LC3B-II) and decreasing level of p62 autophagy receptor in cardiac cells, neuronal cells, and cancer cells25. We believe that our study provides new clues on the connections between arsenite exposure and metabolic disorders by highlighting the major metabolic functions of BAT, which seemingly has higher accumulation of arsenic in our body. Balancing the BAT differentiation, mitochondrial integrity, thermogenesis, and autophagy activity would be novel protective mechanisms against arsenic-induced toxicity and metabolic disorders.In conclusion, we propose a model where arsenite exposure consistently suppresses the brown adipocyte differentiation, mitochondrial biogenesis and function, thermogenesis, and autophagy function, which would have harmful effect on metabolic balance in human body and leads to metabolic disorders. Our study supports the previous human studies performed in US and other developing countries in the world that have higher concentration of arsenite contamination in water and soil would develop higher chances of development of metabolic disorders such as obesity and diabetes2O).Dulbecco’s modified Eagle’s medium (DMEM) was purchased from Invitrogen . Fetal bovine serum (FBS), 3-isobutyl-L-methylxanthine, T3, dexamethasone, insulin, indomethacin, and Oil Red O were purchased from Sigma-Aldrich . MTT cell growth assay kit was purchased from Sigma-Aldrich . Primary antibodies against PPARγ, phospho-AMPK (T172), phospho-ULK1 (Ser555), ULK1, phospho-ACC (Ser79), ACC, and S6K were purchased from Cell Signaling Technology . Tubulin and Actin antibodies was purchased from Developmental Studies Hybridoma Bank . PGC1α, phospho-S6K (Thr389), and AMPK antibodies were purchased from Santa Cruz Biotechnology  and UCP1 antibody was purchased from Alpha Diagnostic . Sodium arsenite (NaAsO2) was obtained from Sigma-Aldrich  and a stock solution was prepared at a final concentration of 100 mM and was diluted for a working concentration in water . The cells were then changed into DMEM supplemented with 20% FBS, 1 nM T3, 20 nM insulin, 0.125 mM indomethacin, 5 μM dexamethasone, and 0.5 mM 3-isobutyl-L-methylxanthine for 24 hrs, followed by DMEM supplemented with 20% FBS, 1 nM T3, and 20 nM insulin, replenished every two days until the cells were fully differentiated at Day 6 (D6). Sodium arsenite was added to the media at D0 and replaced with each change of the media during the process. All experiments were performed in room temperature (23 °C) unless it is specifically indicated as in cold temperature (4 °C).Immortalized HIB1B murine brown preadipocyte cell line, generated from the interscapular brown fat of C57BL/6 mice, is a gift from Dr. Johannes Klein Fully differentiated brown adipocytes were fixed with 10% formalin overnight, and the lipid droplets were stained with Oil Red O and quantified by absorbance at 500 nm in a spectrophotometer.3VO4, 1% Triton-X-100) containing protease inhibitor cocktail (Roche) and phosphatase inhibitor (Sigma) and protein concentrations were determined by BCA assay kit . Thirty micrograms of total cell lysates were subjected to 4–20% SDS-PAGE and transferred to polyvinylidene difluoride membrane. The membrane was blocked in 20 mM Tris-HCl, 137 mM NaCl, and 0.2% Tween 20 (pH 7.6) containing 5% nonfat milk. The membrane was immunoblotted with primary antibodies at 4 °C overnight followed by secondary antibody conjugated with horseradish peroxidase  for 1 hr. The membrane was exposed on X-ray film using ECL Western blot detection reagents  or digitally captured using Odyssey blot imager . Raw uncropped images with molecular size marker were displayed in the supplementary section  or cell lysis buffer . Total RNA abundance was quantified using a NanoDrop Spectrophotometer . Reverse transcription was carried out using High Capacity cDNA Reverse Transcription kit  according to the manufacturer’s instructions. mRNA expression of target genes and housekeeping gene Table  were meaMitochondria were labeled using the mitochondria-specific dye MitoTracker Red  according to manufacturer’s protocol. Briefly, the differentiated brown adipocytes were washed and incubated with 100 nM MitoTracker Red for 30 min at 37 °C. Image was taken with Leica DMI4000B fluorescence microscope . The fluorescence intensity was measured with Synergy H1 Hybrid Multi-Mode Reader . Relative fluorescence intensity is the fold of the mean fluorescence intensity of the controls.2 incubator at 37 °C for 1 hr. The cells subjected to real-time measurements of oxygen consumption rate (OCR) using XF24 Extracellular Flux Analyzer  as previously reported55. For mitochondrial stress tests, mitochondrial complex inhibitors were injected to all of the following treatments sequentially in the following order: oligomycine (1 µM), carbonyl cyanide-ptrifluoromethoxyphenylhydrazone , antimycine A/rotenone (2.5 µM each), and 3 readings were taken after each injection. OCR was automatically recorded by XF24 software v2.3 provided by the manufacturer. Calculations of proton leak, coupling efficiency, maximal respiration, and ATP production were performed according to the manufacturer’s instructions.Five thousand of brown preadipocytes were seeded in gelatin-coated 24-well XF assay plates in the 10% FBS containing DMEM and differentiated in the presence or absence of arsenite for 6 days. Differentiated brown adipocytes were rinsed 3 times, changed to 500 µL of XF assay buffer, and equilibrated at non-CO82. They were maintained for a week to acclimate to their new environment before performing the experiments. Animals were housed in a group of three or four mice per cage  with Tek-Fresh bedding  under a 12-h light/dark cycle from 06:00 to 18:00. They were fed the standard chow diet  contains16% protein and 4% fat to cover 12% of total kcal and sterilized drinking water ad libitum. Mice were euthanized according to IACUC protocol after the completion of experiments.Housing and use of mice in the study were performed in compliance with the National Institute of Health (NIH) Principles for the Use of Animals, the American Veterinary Medical Association (AVMA) Guidelines, and the Public Health Service Policy on Humane Care and Use of Laboratory Animals awarded by University of Nebraska-Lincoln . Animal care and experimental procedures were approved by the institutional Animal Care and Use Committee . Seven-week-old male C57BL/6J mice  were purchased from Jackson Laboratory n = 6 per group). A control group was given regular water at a dosing volume of 5 mL/kg which was comparable to the volume of arsenite administered groups84. The arsenite was administered to mice once a day for 9 days (Day 1 to 9)88. In the case of in vivo, we wanted to quantify the accumulated amount of arsenic in mice adipose tissues. The major source of arsenic exposure is drinking water and food. The exposure ordinarily occurs chronically. To ensure all mice ingested equal arsenic, we decided to administer arsenite to mice orally by gavage rather than including it in drinking water and food. The arsenic dose and treatment period were determined by tissue distribution and toxicity in consideration of the previous reports88. To check the levels of mRNA in BAT and to compare the change of them under different temperature, mice were divided into room temperature  or cold temperature (4 °C). The mice under cold condition were transferred to cold room (4 °C) on Day 9 for 24 hrs. The body weight of mice was measured from Day 0 to 10. The epididymal white adipose tissue (eWAT), inguinal white adipose tissue (ingWAT), and interscapular brown adipose tissue (BAT) of all mice were collected on Day 10 under anesthesia with isoflurane and CO2. All samples were measured individually and stored at −80 °C until use. All experiments were performed in RT unless it is specifically indicated as in cold temperature.Sodium arsenite stock solution at 200 mg/mL was prepared by dissolving in distilled regular water. The arsenite stock solution was aliquoted and stored in −80 °C until use. The arsenite was orally administered to mice at doses of 5 and 10 mg/kg after diluting the arsenite stock solution with distilled water  7500cx series  and an ESI SC-4 high-throughput auto sampler . All mice adipose tissue  samples were homogenized in PBS containing Complete Protease Inhibitor Cocktail . To normalize the lysate samples, the amount of protein in each lysate was measured using the Bradford Protein Assay . The adipose tissue samples were suspended in 200 µL of nitric acid with 50 µg/L of gallium as an internal standard. For complete digestion of fat, WAT samples were incubated overnight with 200 µL of nitric acid and internal standard followed by adding 200 µL of HBrown adipocyte tissue was extracted from mice and reduced to three 1-mm pieces. Samples were fixed with 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) for 1 hr at RT and then overnight at 4 °C. Samples were then post-fixed with 1% osmium tetroxide for 1 hr at RT. Fixed tissues were dehydrated through a graduated ethanol series and embedded In Spurr . Ultrathin sections (~80 nm) were stained with uranyl acetate and lead citrate and observed under a transmission electron microscope (Hitachi H7500-I). A series of ultrastructural images were collected with a bottom-mount digital camera.n = 4 per group). The metabolic profiles were investigated by a computer controlled monitoring systems of metabolic cages as previously reported45. Mice were housed in groups before they were in the metabolic cages (Day 0 to 2) and then acclimated in metabolic cages, one mouse per cage, for a day (Day 3). They also were in the metabolic cages alone while the food and water intakes and respiratory rate were measured for 5 days (Day 4 to 9).The food and water intakes and respiratory rate of mice were measured using metabolic cages  during arsenite treatment period . The prepared sections (4 microns) were stained with haematoxylin and eosin . Tissue slides were evaluated by light microscopy at 400X magnification. Six microphotographs per mouse were obtained. The lipid-droplet diameter of BAT tissues was calculated from the mean of ten values per microphotograph.P < 0.05 or **P < 0.01.All data are presented as mean ± SEM. Measurements were performed at least in triplicates. Statistical analysis was performed using SPSS 25 . One-way ANOVA was performed by Tukey’s multiple comparison test to determine the differences of group mean among control and two treatment groups. The level of significance was set at *Supplementary Info


Introduction: Vestibular dysfunction is a common disorder that results in debilitating symptoms. Even after full compensation, the vestibulo-ocular reflex (VOR) could be further improved by using rehabilitation exercises and visual-vestibular adaptation. We hypothesized that in patients with asymmetric vestibular function, the system could be rebalanced by unidirectional rotations toward the weaker side .Methods: Sixteen subjects  with chronic vestibular dysfunction that was non-responsive to other types of medical treatment were recruited for the study . Subjects had VOR asymmetry quantified by an abnormal directional preponderance (DP) with rotation test and no previous history of central vestibular problems or fluctuating peripheral vestibular disorders. They participated either in the short-term study (one session) or the long-term study (7 visits over 5 weeks). Rehabilitation consisted of five trapezoid unidirectional rotations (peak velocity of 320°/s) toward the weaker side. Care was taken to slowly stop the rotation in order to avoid stimulation in the opposite direction during deceleration. To study the short-term effect, VOR responses were measured before and 10, 40, and 70 min after a single unidirectional rotational rehabilitation session. For long-term effects, the VOR gain was measured before and 70min after rehabilitation in each session.Results: We observed a significant decrease in VOR asymmetry even 10 min after one rehabilitation session (short-term study). With consecutive rehabilitation sessions in the long-term study, DP further decreased to reach normal values during the first 2 sessions and only one subjects required further rehabilitation after week 4. This change in DP was due to an increase in responses during rotations toward the weaker side and a decrease in VOR responses during rotations in the other direction.Conclusion: Our results show that unidirectional rotation can reduce the VOR imbalance and asymmetry in patients with previously compensated vestibular dysfunction and could be used as an effective supervised method for vestibular rehabilitation even in patients with longstanding vestibular dysfunction. Normal vestibular function is essential for proper balance control and gaze stabilization during head movements during natural activities. Vestibular dysfunction results in imbalance between inputs from the two sides, leading to symptoms such as vertigo. Vestibular disorders have a prevalence of ~35% in Americans above 40 years of age . The dysEvidence from previous studies suggest that natural vestibular compensation strategies do not use the full capacity of the system. Training programs that use visual-vestibular training in the form of bidirectional  or unidiIn order to improve compensation in patients with chronic vestibular symptoms, the multisensory nature of the vestibular compensation can be exploited through sets of rehabilitation exercises –15. OrigHere, we describe a new rehabilitation method that solely targets the vestibular pathway through a specific vestibular stimulation. The rehabilitation consists of unidirectional rotations in the dark in the direction of the less responsive (LR) side. The hypothesis behind this original idea was formalized by one of the authors (NR) and tested by pilot (unpublished) studies about 20 years ago. Basically, this hypothesis was based on changes in commissural pathways and vestibular nuclei during compensation and suggested that unidirectional rotation toward the side with lower VOR responses results in excitation of that side and simultaneous inhibition of the other side . This could result in an adaptive change, leading to an increase in responses of the weaker side and a new balance between the two sides. This effect could be due to changes in the vestibular nuclei and commissural pathways or at the peripheral level, or both. A confounding and counterproductive effect most likely also exists due to the habituation of responses resulting from repeated rotations, as shown by previous studies in normal animals and humans –30. We pR2 = 0.69). The research protocol was then explained to patients and those who agreed to participate in the study, gave written informed consent in accordance with the Declaration of Helsinki. Subjects were free to drop out of the study at any time. All tests were performed in the Audiology Center of Day General Hospital, Tehran, Iran and each patient's primary care physician or otolaryngologist was informed of their participation in this research. This study  was carried out in accordance with the recommendations of the Ethics Committee of Shahid Beheshti University of Medical Sciences, Tehran, Iran and the protocol was approved by the Institutional Review Board of the University.This study was performed as a sequential double blinded clinical trial on 16 patients . There was no sex or age limitation for selecting the patients. Regarding the etiology of the vertigo, during our initial assessment, we only asked questions to rule out any known central etiology, such as tumor or surgery (since it would interfere with compensation process) or any history of fluctuating disorders such as Meniere's or BPPV with asymptomatic periods (which would be inappropriate for studying the rehabilitation effects). Typically, subjects' symptoms were not alleviated by previous medical treatment and none of the subjects used any medication during the study. Subjects had a proven and documented history of vestibular dysfunction for 1–8 years and an abnormal asymmetric VOR response during rotation test, as evidenced by a directional preponderance (DP) >10% during rotation (see below). In the initial session, a complete vestibular examination was performed, which included saccadic, smooth pursuit, optokinetic, gaze holding, rotation, and caloric tests. We used caloric DPs as supplementary evidence of asymmetry (DP < 20%) initially. However, caloric DP was not required to be abnormal for inclusion in the study. Caloric DPs were positively correlated with rotation DPs (Eye movements were measured by electronystagmography during rotation (Nicolet Spirit). Rotations were performed at peak velocity of 40°/s and 0.2 Hz. Patients were in complete darkness with eyes open during the test. The head was positioned 30° nose down, so that the horizontal canal was in its maximum plane of activation. All recordings were done while the subjects performed mental arithmetic to increase their state of alertness.As a measure of vestibular compensation , 31, 32,HR and VLR represent peak eye velocities during rotations toward the side with higher responses (HR) and lower responses (LR), respectively. The HR and LR sides were determined on the first test for each subject and were not changed during the course of the study. In this way, a change in the direction of DP would be represented by negative values. The normal range of DP for the test as performed by our equipment was <8% as measured in 52 normal subjects. Patients with initial DP values of >10% were included in the study.where VWe used DP of responses to whole-body sinusoidal rotations as a measure of asymmetry to quantify the effect of our intervention. Comparison of rotation DP to caloric test has shown that it is a reliable measure of diagnosis of vestibular imbalance in routine vestibular clinical practice and follow up , 34. It 2 over 4 s to reach a maximum velocity of 320°/s and then slowly decelerate at 10°/s2 to stop over approximately 30 s. The slow deceleration was particularly important in order to have a smooth end of rotation since a sudden stop could function as a stimulation in the opposite direction. Each session comprised of 5 such rotations, with 1 min intervals in between. The whole session was completed in ~7 min. Rotations were performed in the dark with the subjects' eyes open and heads positioned 30 degrees nose down.The unidirectional rotation comprised of a velocity trapezoid, with acceleration of 80°/sIn each session of the study, subjects first underwent an initial DP assessment by rotation test. After 3–4 min, the unidirectional rotational rehabilitation was performed as described above. Eight subjects participated in a short-term study, for which the subjects were kept in the rotation chair and DP was assessed by sinusoidal rotation test 10, 40, and 70 min after the end of rehabilitation. VOR asymmetry was originally evaluated by rotation and caloric tests, but for further evaluations we only used rotational testing since it was less bothersome for patients and more practical for serial evaluations. Another 8 subjects participated in a long-term study. In this case, subjects were asked to rest for 1 h in a calm place in the hospital without using stimulating beverages  and post rehabilitation DP was measured only 70 min after the unidirectional rotation. The rehabilitation was performed two times a week for the first 2 weeks and once a week for the second 2 weeks, providing a total of 6 sessions in 4 weeks. One week after the last session, a sinusoidal rotation test was performed for a final DP measurement. During the course of the study, if the DP measured at the beginning of any session was in the normal range or reversed, the patient was not subjected to any additional unidirectional rotations and would be instructed to return for follow up in the next session. We did this as an ethical issue since the unidirectional rotation in a subject with normal DP was not necessary and could theoretically result in an imbalance in the opposite direction.To document symptoms of all patients before the beginning of the study in the first session, they were evaluated by one of the researchers (NGS) using a questionnaire. In particular, they were asked to specify when their vestibular symptoms  have started, whether they had a sensation of rotation  or imbalance, the frequency and duration of symptoms, any accompanying auditory problems, and any precipitating factors. Subjects that participated in the long-term study were also asked to fill in a form in order to report any occasions of vestibular symptoms and their specificities  during the days between the rehabilitation sessions.n = 8 subjects, 3 female, 5 male), data was collected at 10, 40, and 70 min after rehabilitation. For the long-term study , data was collected over 6 sessions (4 weeks), before and 70 min after rehabilitation in each session (see Methods for details). None of the patients had jobs or participated in activities that resulted in intense head movements in between sessions and none had performed rehabilitative physical exercises.We tested the effect of unidirectional rotational rehabilitation on 16 patients  with confirmed chronic vestibular dysfunction for 1–8 years (3.5 ± 2). All patients had a history of some level of auditory problem, with some degree of hearing loss. Mean age of subjects was 43.2 ± 17.0 (range: 25–64) years old. For the short-term study . Based on the initial rotation test , the two sides were labeled as “low response” (LR) and “high response” (HR). The unidirectional rotational rehabilitation was then performed with rotations toward LR as described in the Methods. Figure For each of the 8 subjects that participated in the short-term study, eye velocities were measured during sinusoidal rotations and VOR responses were evaluated for half cycles to the right and left and a DP was calculated. DPs calculated for rotation and caloric tests were linearly related to each other (slope = 0.7) and on average, were not different  and decreased at 40 min and 70 min for both directions of rotation. While the increase in eye velocity for LR rotations could be attributed to the unidirectional rotation , the increase for HR half cycles at 10 min was unexpected and could be a rebound phenomenon after the inhibition due to the fast unidirectional rotation. The general trend of these changes was in a way that the asymmetry between the two sides decreased over time as calculated by the DP value  and continued up to 70 min (p = 0.003). Seventy minutes after rehabilitation, DP was normalized in half of the patients, while the other half showed a decrease in DP. In 2 subjects with DP (post-rotation) to within the normal range, the direction of DP changed  at 40 min and in one of them remained so even at 70 min.Average eye velocities for all patients in the short-term study showed a similar trend Figure . While Ve Figure . This efTogether, these results suggest an effective improvement in VOR asymmetry up to 70 min after one session of the unidirectional rotational rehabilitation. We next investigated whether this effect could be preserved for longer periods.Eight patients  participated in the long-term study, which required 7 visits over a period of 5 weeks. Note that these subjects were different from those in the short-term study and have not had any previous experience with the unidirectional rotation. Of these, 2 female subjects only participated in the first session and dropped out of the study for personal reasons. For the other 6 subjects, we measured VOR responses at the beginning of each session and 70 min after the rehabilitation in that session.t-test, p = 0.15) or LR . All of the long-term patients also showed a decrease in their DP values 70 min after rehabilitation in the first session and the average DP decreased from 21.2 ± 4.1% initially to 1.4 ± 4.2% . When data of all 16 subjects were pooled together, the decrease at 70 min became more pronounced .In the first session, similar to that observed for subjects in the short-term study, VOR responses showed a decreasing trend for HR peak eye velocity and an increasing trend for LR peak eye velocity Figure , the chat-test, p = 0.003). At 70 min after rehabilitation, the pooled data showed no significant difference between the two sides (31.0 ± 2.3°/s vs. 27.15 ± 2.8°/s). The decreasing (non-significant) trend for HR rotations and the increasing (non-significant) trend for responses to LR rotations were opposite to that expected from simple habituation to a unidirectional rotation observed in normal subjects, which resulted in a decrease in the responses of the side ipsilateral to rotation and no change in the opposite side . This change is comparable to that observed for the short-term study . Again, there was a non-significant decreasing trend over time in responses to rotations toward HR and a non-significant increasing trend for responses to LR rotations, which were enough for a significant decrease in asymmetry and DP over time. Also, notice that all 4 subjects who returned for the last final DP measurement (with no rehabilitation rotation) had symmetric VOR responses with minimal DP values. For 3 of these subjects, the symmetry was accompanied by near normal responses  for rotations in both directions. Although a significant clinical finding, this should be considered with caution since only 4 subjects participated in the last session and 3 of them had LR peak eye velocities close to normal at this point  from 14.8 ± 3.8 to −2.2 ± 4.4. Notably, the rehabilitation had no effect on 2 subjects with near normal initial DP values , suggesting that the effect of rehabilitation was retained and did not substantially diminish between sessions. The seemingly larger differences between DPs (and larger variabilities) after session 4 are probably due to the longer times between sessions .To investigate whether the effect of the rehabilitation was preserved between sessions, we compared the 70 min post-rehabilitation DP of each session with the initial DP of the next session Figure . As mentSix of the eight subjects that participated for more than 1 session in the long-term study reported on subjective changes in their symptoms over time. At the beginning of this study, two of the patients experienced only mild imbalance, while others had true vertigo or severe imbalance. However, these 2 patients showed other signs of vestibular dysfunction, such as falling toward one side in the dark. In all subjects, the sense of imbalance was aggravated by rapid head movements. All subjects also experienced associated autonomic symptoms during vertigo. Interestingly, the 2 subjects with the least response to the unidirectional rotation Figure  were theThe results of the present study show that training by a purely unilateral vestibular stimulation could decrease the asymmetry of the VOR response in patients with chronic vestibular dysfunction. We used a unidirectional rotational stimulation in the dark  and showed that this could be an effective rehabilitation method for decreasing the DP of patients with chronic vestibular dysfunction. In most cases, the vestibular imbalance decreased by ~10 min after the rehabilitation was applied and the effect lasted for weeks. Although chronically elevated DPs are harder to change , we founRepeated unidirectional rotations have previously been shown to result in habituation of vestibular responses and a decrease in VOR gain and time constant in different animals as well as humans , 38, 40.It has been shown that the naturally occurring compensation could be improved further by specific goal-directed training exercises. Such rehabilitation exercises typically use the multisensory nature of vestibular compensation to further improve balance and gaze stability in patients. Animal studies have shown compensatory changes in the vestibular nuclei (VN) neuron responses, changes in extravestibular inputs (such as neck proprioception and efferent copy of neck motor command) to the VN –8 as welVisual inputs play a major role in vestibular compensation so that when animals were kept in darkness for 4 days after unilateral lesion, they did not show improvement in spontaneous nystagmus, which was recovered once they were moved to a lighted area . StudiesThe goal of rehabilitation exercises is to use visual and other extravestibular inputs as well as other balance cues to further compensate for the lack of vestibular inputs. Previous studies have shown a 70–80% improvement in patients using different rehabilitation protocols. One study found that 28% of patients showed complete resolution of symptoms within 1 year and 54% showed some degree of improvement . The uniMean values of DP during the 6 sessions show reductions and even reversals, demonstrating the effect of the rehabilitation to reduce the vestibular imbalance. Most of the changes were observed in the first session with about 80% decrease in DP measured 70 min after the rehabilitation. This is similar to the results of Ushio et al. , where a2) in order to avoid stimulation in the opposite direction when stopping the rotation. This is very different from the 1,000°/s2 acceleration/deceleration used by the previous study. It was suggested that the training provided by the previous study most likely affected the irregular/phasic pathway  on standardized tests are required to further evaluate the subjective effect of this rehabilitation and a comparison to other methods such as the vestibular-visual training.In general, one of the shortcomings of subjective measures is that their value is reduced due to their intrinsic variability. The observed discrepancy between the subjective and objective improvement could be related to psychological factors  and personal characteristics  , 70. It NR conceptualized the original idea and hypothesis behind the unidirectional rotational rehabilitation. NR and SGS designed the study, analyzed the data, and wrote the manuscript. NGS designed the study, performed the experiments, analyzed the data, and wrote the manuscript. BS designed the study and performed the experiments.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


In the present study, to investigate the role of AR in liver tumor development, we specifically knocked out ar gene in the liver of zebrafish via the CRISPR/Cas9 system and the knockout zebrafish was named L-ARKO for liver-specific ar knockout. We observed that liver-specific knockout of ar attenuated liver tumor development in kras transgenic zebrafish at the early stage (one week of tumor induction). However, at the late stage (two weeks of tumor induction), essentially all kras transgenic fish continue to develop HCC irrespective of the absence or presence of ar gene, indicating an overwhelming role of the driver oncogene kras over ar knockout. Consistently, cell proliferation was reduced at the early stage, but not the late stage, of liver tumor induction in the kras/L-ARKO fish, indicating that the attenuant effect of ar knockout was at least in part via cell proliferation. Furthermore, androgen treatment showed acceleration of HCC progression in kras fish but not in kras/L-ARKO fish, further indicating the abolishment of ar signalling. Therefore, we have established a tissue-specific ar knockout zebrafish and it should be a valuable tool to investigate AR signalling in the liver in future.Hepatocellular carcinoma (HCC) is one of the most severe cancer types and many genetic and environmental factors contribute to the development of HCC. Androgen receptor (AR) signaling is increasingly recognized as one of the important factors associated with HCC. Previously, we have developed an inducible HCC model in  HCC is also a sex-biased disease as men are more prone to develop HCC than women2. The typical ratio of HCC between men and women is about 2:1 to 3:13. In previous animal experiments, androgen has been documented to be an important factor to cause the higher incidence of HCC in males. After exposure to androgens, mice have increased incidence of liver tumor4. Castration or use of anti-androgen protects male rodents from liver tumor development5 while female rodents receiving testosterone have an increased susceptibility to hepatocarcinogenesis6. Moreover, clinical practice also suggests that application of androgens is associated with an increased risk of developing liver neoplasm including HCC8. For example, in male Fanconi’s anemia and aplastic anemia patients, substitute androgen intakes raised the susceptibility to develop liver neoplasm such as adenoma or HCC9. All these studies have suggested that androgen should be a target to study the gender disparity of HCC.Hepatocellular carcinoma (HCC) is the most common type of malignant liver cancer and causes millions of deaths annually. Several risk factors have been identified to associate with HCC, such as alcohol, hepatic toxins and hepatitis virus10. In addition, the expression and activation of ARs are also significantly increased in the liver tissue of rodent model during chemical-induced liver carcinogenesis11. AR has also been linked to HBV-induced hepatocarcinogenesis12. Large cohort studies have also indicated an existing synergistic effect between male gender and HBV on HCC progression13. Intriguingly, a study using hepatocyte-specific AR knockout mice has resulted in later and less HCC developed compared with their wildtype littermates in both HBV- and carcinogen-induced HCC14. In HCC cell lines, hepatic AR enhances RNA transcription of HBV via direct binding to the androgen response element on HBV genome15. Overexpression of functional AR in human HCC cells also resulted in the promotion of cell growth14. Thus, AR has played an important role in HCC, but AR-dependent sex disparity in HCC has not been well studied.Androgens exert their effects through the activation of ARs, which are present in normal liver tissue and in HCCar gene in the zebrafish genome16. ar mRNA is presented as maternal RNA from oocytes and starts to increase from 50% epiboly stage, suggesting its important role in late embryonic and early larval development16. In both male and female zebrafish, ar is widely expressed in various tissues including the brain, eyes, skin, kidney, gall bladder, gills, testis, ovary, gut, heart, muscle, spleen and liver18. Similar to the mammalian system, the most active ligand for zebrafish AR is also 11-ketotestosterone (KT11), which has similar potency to testosterone as an androgen19. To investigate the role of ar in HCC development, in the present study, we successfully developed a liver-specific ar knockout zebrafish by liver-specific expression of Cas9 nuclease using the CRISPR system20. By combination with our previously established zebrafish HCC model which has inducible expression of oncogenic kras21, we found that ar may help promote HCC progression at an early stage, but not at a late stage, in our kras-induced HCC model in zebrafish.In zebrafish, there is only a single functional kras transgenic zebrafish22, we found that ar transcripts were presented at a moderately abundant levels (1–7 transcripts per million transcript) in all samples including 6 dpf larvae and male and female adult fish -specific knockout of ar in zebrafish was designed as illustrated in Fig. kras zebrafish for the liver tumor study. Now the ARKO zebrafish have been maintained for five generations and there was no observable difference compared to wildtype zebrafish in development, growth, morphology and reproduction.With the cas9 expression in the liver, in situ hybridization was performed on 4-dpf F1 L-ARKO fish. The probe for ceruloplasmin23 was used as a positive control. As shown in Fig. ceruloplasmin mRNA was specifically expressed in the liver. Similarly cas9 expression, as assessed by a cas9 antisense probe, was also specifically expressed in the liver region  and HCC , uniformed cell shape and size, and distinct cell boundary. Hyperplasia maintains hepatic plate arrangement but shows increased prominent nuclei (arrowheads). HCA shows unclear hepatic plates but still have clear cell boundary and relatively uniformed cell shape. HCC is characterized by the loss of cell boundaries and hepatic plate structure, as well as increase of mitotic cells and appearance of multiple nucleoli (yellow arrows).At 1 wpi, there were no significant change of liver phenotype in WT and LARKO fish and normal liver histology with typical two-hepatocyte plate was observed. In contrast, majority of male HCC Fig. , consistkras fish has HCC phenotype and the percentage was reduced to 30% in kras/L-ARKO fish. However, at 2 wpi, essentially all the kras and kras/L-ARKO fish had HCC phenotype while the WT and L-ARKO control fish remained normal liver morphology and histology  and 4-hydroxy-2-noneal (for lipid peroxidation), two well established markers for oxidative stress34 (data not shown).Previously in a mouse ar knockout on liver tumor progression, KT11 was used to treat kras and kras/L-ARKO zebrafish. At 1 wpi, HCC incidence increased after KT11 treatment in the kras zebrafish compared with the kras zebrafish with Dox alone. About 60% of the kras zebrafish developed HCC after KT11 treatment, while the ratio was 40% in the kras zebrafish with Dox alone -specific fabp10a promoter to drive cas9 expression. In situ hybridization confirmed the liver-specific expression of cas9 . However, the effect of liver-specific ar knockout was overwhelmed by sustaining expression of the kras driver oncogene by 2 wpi, where essentially all kras fish developed HCC phenotype irrespective of the absence of ar gene; thus, Ar may play an important role in liver tumorigenesis only in the early stage. Consistently, cell proliferation was decreased in L-ARKO zebrafish during HCC progression at 1 wpi but not at 2 wpi, indicating that the decrease of cell proliferation might contribute to the inhibitory effect of liver-specific ar knockout.In this study, to further investigate the contribution of Ar in liver tumorigenesis and provide a valuable experimental tool, we generated a liver-specific Ar knockout mouse has also been established by a Cre-loxP mediated approach14. Similarly, hepatic knockout of Ar in mice caused later and less HCC. By further mechanistic studies, it was found that the Ar knockout mice had increased cellular apoptosis and Tp53, consistent with our findings in this study. Furthermore, there was also a decrease of oxidative stress in Ar knockout mice, but this was not observed in our zebrafish study.Previously, a liver-specific kras zebrafish39. In the present study, androgen treatment in kras/L-ARKO zebrafish showed no significant effects on liver tumor progression; thus the loss of hepatic ar has largely blocked the effects of androgen on HCC progression. Moreover, liver cell proliferation in the kras/L-ARKO zebrafish after the co-treatment of androgen and Dox showed no significant change, in contrast to the dramatic stimulation of liver cell proliferation in the androgen/Dox co-treated kras zebrafish, further demonstrating that the liver-specific ar knockout blocked the effects of androgen treatment on cell proliferation during HCC progression.Previously we have shown that liver tumorigenesis could be accelerated by androgen treatment in ar knockout transgenic zebrafish, we have clearly shown that ar contributes to liver tumor progression. The loss of hepatic ar attenuated liver tumor progression only at the early stage through reduction of cell proliferation. However, the attenuant effect was overwhelmed by the long-term overexpression of kras oncogene. To determine whether the attenuation of liver tumors at the early stage by ar knockout is only specific to kras-induced liver tumors, it will be interesting to investigate if ARKO could also suppress liver tumors induced by other oncogenes. Nevertheless, our current findings are consistent with current literature in clinical studies and other experimental models that AR plays a marked role in early stages of hepatocarcinogenesis41. Furthermore, in a similar Ar knockout mouse model, it was also observed that there was a delayed liver tumorigenesis induced by diethylnitrosamine, a chemical carcinogen14. These studies together imply that the attenuation of early liver tumor by the lack of AR is likely a broad, if not universal, phenomenon.In summary, by generation of hepatocyte-specific ar knockout (L-ARKO) transgenic zebrafish Tg were generated in the present study (see the next section). krasV12 transgenic zebrafish Tg, known as kras in the following text, were previously generated21. kras/L-ARKO double transgenic zebrafish were generated by crossing the L-ARKO and kras zebrafish. Treatments of adult zebrafish (4 months postfertilization) were conducted in 6-liter tanks and water with or without treatment chemicals was changed every other day. 20 mg/l doxycycline was employed to treat adult kras, kras/L-ARKO, L-ARKO and WT zebrafish siblings for 2 weeks. For hormone treatments, 5 μg/l 11-ketotestosterone (KT11)  was applied in conjunction with 20 mg/l doxycycline for one week.All experiments were carried out in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health and the protocol was approved by the Institutional Animal Care and Use Committee (IACUC) of the National University of Singapore (Protocol Number: 096/12). Liver-specific https://chopchop.rc.fas.harvard.edu/). Six ar-specific gRNAs were designed and one of them was selected for its high rate of germline transmission. It targeted the first exon of ar gene after the initiation ATG codon . The destination vector pDestTol2CG2-U6:gRNA36, 5′ entry vector p5E-mcs and the middle entry vector pME-Cas9 for Gateway reaction were obtained from Addgene (https://www.addgene.org). The 3′ entry vector p3E-polyA (vector #302) was requested from Tol2kit (http://tol2kit.genetics.utah.edu/index.php/P3E-polyA). A 2.8 kb promoter of the fabp10a gene43 was first cloned into pGEM T vector and then subcloned into the p5E-mcs vector. The pDestTol2CG2-U6:gRNA vector was digested with BseRI enzyme and annealed ar-specific oligos were inserted into the BseRI-digested destination vector. The multisite Gateway cloning44 was performed with destination vector containing gRNA, the 5′ entry vector containing the fabp10a promoter, the middle entry vector containing zebrafish codon-optimized Cas9 sequence and the 3′ entry vector with stop poly A signal. The resulted construct is shown in Fig. cas9 was controlled by the fabp10a promoter, and ar-specific gRNA was under the control of the ubiquitous U6 promoter. The GFP controlled by the heart-specific cmlc2 promoter serving as screen marker for positive transgenic fish.Guide RNA sequences were designed using a web tool CHOPCHOP 2, 1.5 mM HEPES buffer) at 28 °C.WT zebrafish embryos were collected for microinjection. 20 pg of DNA constructs and 20 pg of Tol2 mRNA were injected into zebrafish embryos at one-cell stage. After microinjection, embryos were raised in 0.5 × Danieau’s solution . The sense and anti-sense probes were synthesized by in vitro transcription using mMESSAGE mMACHINE T7 Kit (Thermo Fisher Scientific). In situ hybridization was performed following established protocols45.46. Two primers were designed and synthesized based on the locations upstream and downstream of the gRNA target site: TTTTTCACGGACCTTACCAAAGC and TACTCTCGGTCTTTCCTTCCTG. For DNA sequencing, PCR products were cloned into pGEM T Easy vector (Promega) and sequenced using the ABIPRISM™ BigDye™ Terminator Cycle Sequencing Ready Reaction Kit .T7E1 assay was performed following the published methodRNA extraction was carried out using TRIzol® Reagent (Thermo Fisher Scientific). The RNA samples were then treated with DNase I  to remove possible DNA contamination and cleaned RNA was reverse-transcribed into cDNA using Transcriptor First Strand cDNA Synthesis Kit (Roche). Primers for qPCR were designed using NCBI Primer-BLAST web tool. The real-time qPCR was carried out in LightCycler® 480 Real-Time PCR System (Roche). All primers used are listed in Supplementary Table 47. Through FACS by a cell sorter (BD Aria), hepatocytes were isolated based on DsRed expression from LiPan fish48. Over 10,000 cells were collected for DNA/RNA analysis. The purity of FACS isolated cells was above 90%.Zebrafish livers were freshly dissected and dissociated as previously describedThe gross appearance of the liver was examined and imaged under the bright field of Olympus MVX10 stereomicroscope and photographed with an Olympus DP72 digital camera with the Olympus cellSens dimension imaging software (Olympus).33. For PCNA (Proliferating cell nuclear antigen) staining, sections were incubated with rabbit anti-PCNA primary antibody at 1:500 dilution, followed by incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies and color development using the DAKO Real Envision Detection System.Fish abdominal regions were dissected and fixed in formalin solution (Sigma-Aldrich), followed by dehydration and embedding into paraffin. 5 μm sections were made for hematoxylin and eosin staining. Stained slides were mounted in Micromount (Leica) and imaged using a light microscope . Classification of tumor types were based on established criteria as previously reportedStatistical significance between two groups was evaluated by two-tailed unpaired Student t test (GraphPad). Statistical data were presented as mean value ± standard error of mean (SEM). P < 0.05 was chosen to be statistically significant.Supplementary Table 1 and Supplementary Figures 1–3