In contrast, [18F]tetrafluoroborate (TFB) is a novel radiolabeled PET substrate of hNIS, results in PET images with high-quality and low radiation doses, and should therefore be suited for imaging of DTC. The aim of the present study was to compare the diagnostic performance of [18F]TFB-PET to the clinical reference standard [131I]iodine scintigraphy in patients with recurrent DTC.Efficient therapy of recurrent differentiated thyroid cancer (DTC) is dependent on precise molecular imaging techniques targeting the human sodium iodide symporter (hNIS), which is a marker both of thyroid and DTC cells. Various iodine isotopes have been utilized for detecting DTC; however, these come with unfavorable radiation exposure and image quality  including SPECT-CT was present for all patients; correlative [18F]FDG-PET-CT was present for 21 patients. Histological verification of [18F]TFB positive findings was available in 4 patients.Twenty-five patients with recurrent DTC were included in this retrospective analysis. All patients underwent [18F]TFB-PET detected local recurrence or metastases of DTC in significantly more patients than conventional [131I]iodine dxWBS and SPECT-CT . The diagnosis of 6 patients with cervical lymph node metastases that showed mildly increased FDG metabolism but negative [131I]iodine scintigraphy was changed: [18F]TFB-PET revealed hNIS expression in the metastases, which were therefore reclassified as only partly de-differentiated . Highest sensitivity for detecting recurrent DTC had the combination of [18F]TFB-PET-CT/MRI with [18F]FDG-PET-CT (64%).[18F]TFB-PET shows higher sensitivity and accuracy than [131I]iodine WBS and SPECT-CT in detecting recurrent DTC. The combination of [18F]TFB-PET with [18F]FDG-PET-CT seems a reasonable strategy to characterize DTC tumor manifestations with respect to their differentiation and thereby also individually plan and monitor treatment. Future prospective studies evaluating the potential of [18F]TFB-PET in recurrent DTC are warranted.In the present cohort, [ Yet both iodine dxWBS and SPECT-CT achieve only unsatisfactory image quality, especially when compared to positron emission tomography (PET) imaging approaches [131I]iodine causes significant radiation exposure, and the administration might require an in-patient stay due to radiation protection legislation fluoride labeled tetrafluoroborate (TFB) has been employed in the primary staging of patients with DTC [18F]TFB is likewise a substrate of hNIS and therefore an analogue to [124I]iodide or [131I]iodide [18F]TFB can easily be synthesized by cyclotrons equipped with PET radiochemistry and offers a favorable half-life, dosage and biodistribution, as well as superior PET image quality [18F]TFB-PET-CT is superior to conventional [131I]iodine dxWBS/SPECT-CT in post-radioiodine ablation (RAI) patients suffering from recurrent DTC.Recently, [with DTC . [18F]TFlogue to 4Iiodide odide or 1Iiodide  quality . However18F]TFB-PET in patients with newly discovered recurrent DTC. To this end, the clinical reference standard [131I]iodine dxWBS/SPECT-CT was compared on an individual basis to [18F]TFB-PET.Therefore, the aim of the present study was to evaluate the potential of [18F]TFB-PET acquisitions combined with either CT or MRI of patients suffering from DTC  were included in this retrospective study (inclusion period: July 2018 to June 2019). Patients were referred to [18F]TFB-PET due to newly discovered increasing levels of thyroglobulin (TG), autoantibodies against thyroglobulin (antiTG), suspicious cervical lymph nodes, or combinations of the latter. Decision for [18F]TFB-PET examination was done on a case-by-case basis due to aforementioned clinical indication. Detailed patient characteristics are given by Table 18F]TFB-PET was 5.1 [6.3] years. The median cumulated therapeutic dosage of [131I]iodine that was employed in the cohort until [18F]TFB-PET was 10 [10.4] GBq. All patients were treated with Levothyroxine to suppress thyroid-stimulating hormone (TSH) levels [A consecutive number of 25 8FTFB-PET18F]TFB was prepared according to a literature procedure described previously by isotopic exchange of BF4− with [18F]fluoride in hot hydrochloric acid and purified using an alumina column [18F]fluoride, tR = 4.7 min; [18F]TFB, tR = 11.2 min). Thin-layer chromatography (TLC) was performed on alumina TLC strips  with methanol as mobile phase. Strips were scanned using a radio TLC scanner . Rf([18F]fluoride) = 0.0; Rf([18F]TFB) = 0.8.[a column . The pro18F]TFB and [131I]iodine. A median activity of 317.0 [63.0] MBq [18F]TFB was administered intravenously. The PET acquisition was initiated 40 min after tracer injection . Low-dose CT or MRI was acquired for attenuation correction of PET data. A median activity of 315.0 [516.8] MBq [131I]iodine was administered immediately after completion of the PET acquisition . WBS and SPECT-CT acquisitions were done in accordance with guidelines  [Thyrotropin alfa was administered 48 and 24 h before the administration of [131I]iodine WBS acquisition.Blood levels of TSH, free triiodothyronine (fT3), free thyroxine (fT4), TG, and antiTG were measured on the day of the first thyrotropin alfa administration . Stimulated TG levels were measured before [0 was rejected if p < 0.05. Values are presented as median with standard deviation (SD) in squared brackets.SPSS statistics 24  was used for descriptive metrics and statistical testing. The McNemar and Wilcoxon method were used to test for statistically significant differences with the “exact” option of SPSS. H18F]TFB-PET, combined with either CT (n = 24) or MRI . In the following, PET-CT is used as phrase to refer to both PET-CT and PET-MRI acquisitions. Detailed patient characteristics are given by Tables n = 19), elevated antiTG levels (n = 7), sonographically suspicious cervical lymph nodes (n = 10), or combinations of these findings. Therefore, all enrolled patients were regarded as suffering from recurrent DTC. Correlative [131I]iodine imaging was present for 25 patients. The median interval between [18F]TFB-PET-CT and [131I]iodine dxWBS/SPECT-CT was 3.0 [7.3] days (range: 2–39 days). Correlative [18F]FDG-PET-CTs were present for 21 patients. The median interval between [18F]TFB-PET-CT and [18F]FDG-PET-CT was 6.9 [10.8] months. No local therapy was performed between [18F]TFB-PET-CT and [18F]FDG-PET-CT.A total number of 25 patients underwent 8FTFB-PET18F]TFB-PET-CT was 52.0% (n = 13). Details are given in Fig. 18F]TFB-PET-CT. All lesions were delineated due to increased [18F]TFB-PET accumulation. Metastases or local recurrence were discovered in 10 additional patients compared to [131I]iodine dxWBS/SPECT-CT . A detailed list of findings is given in Table 18F]TFB-PET-CT-positive patients (n = 13) was surgery together with histological examination (in 4 patients), a corresponding [131I]iodine accumulation (in 3 patients), [18F]FDG accumulation (in 5 patients), or morphological finding on CT . Therefore, the sensitivity of [18F]TFB-PET-CT was 52% (accuracy: 52%). The median [18F]TFB uptake of delineated lesions was 4.2 SUVmax [8.4].The detection rate of 8FTFB-PET.0% n = 1. Details18F]TFB-PET-CT and [18F]FDG-PET-CT, the detection rate of [18F]TFB-PET-CT was higher (51.9%) compared to [18F]FDG-PET-CT (47.6%), yet this difference was not statistically significant. In three patients, [18F]FDG-PET-CT discovered metabolically active metastases (median SUVmax: 11.9) with no increased [18F]TFB accumulation. In contrast, [18F]TFB-PET-CT discovered lesions in six patients , with no [18F]FDG accumulation. Combining [18F]FDG-PET-CT and [18F]TFB-PET-CT for staging of suspected recurrent DTC resulted in the highest sensitivity .Looking at patients who underwent both [18F]FDG uptake (median SUVmax: 4.5). Likewise, [18F]TFB-PET-CT revealed suspicious uptake of theses lymph nodes (median SUVmax: 4.2). [18F]TFB-PET-CT-guided surgery together with histological analysis was performed in two patients. In both cases, lymph node metastases could be confirmed.In 6 patients, cervical lymph node metastases were suspected due to moderately increased [131I]iodine accumulating metastases were discovered by dxWBS and SPECT-CT . In total, 11 lesions were detected by [131I]iodine imaging, which were significantly fewer compared to [18F]TFB-PET-CT (p = 0.001). All lesions were also detected by [18F]TFB-PET-CT, which additionally provided superior image quality .In three patients, [18F]TFB-positive findings were 2.46 [1566.9] ng/ml and 98.7 [144.7] IU/ml, respectively. For comparison, [18F]TFB-negative patients had median TG levels of 1.87 [4.4] ng/ml. Only one [18F]TFB-negative patient had elevated antiTG (3267 IU/ml). In six patients, no morphological or molecular findings were detected. Median TG level of these patients was 0.83 [1.2] ng/ml (no patient with elevated antiTG levels).Median TSH suppressed TG and antiTG levels for patients with [18F]TFB-PET-CT in the diagnostic workup of patients with suspected recurrence of DTC was evaluated in the present study. To this end, 25 patients with sonographically suspicious cervical lymph nodes or elevated blood tumor markers (TG or antiTG) were included in this retrospective analysis. [18F]TFB-PET-CT detected local recurrence or metastases of DTC in significantly more patients compared to [131I]iodine dxWBS/SPECT-CT. Moreover, the accuracy of [18F]TFB-PET-CT was greater compared to [131I]iodine dxWBS/SPECT-CT.The performance of [131I]iodine WBS/SPECT-CT is inferior to TG levels in detecting patients that develop metastases or local recurrence [131I]iodine dxWBS image acquisitions [131I]iodine dxWBS is not ideal for detecting recurrent DTC. However, evidence of recurrent DTC by elevated TG levels does often not enable a localized therapy. Therefore, molecular imaging strategies are needed to localize recurrent DTC more sensitively than [131I]iodine dxWBS.It was shown previously that [currence . Gonzaleisitions . This in124I]iodine-PET is superior to [131I]iodine dxWBS in detecting recurrent DTC [124I]iodine is not available in many departments of nuclear medicine [124I]iodine requires a tracer administration days before image acquisition, which causes patient management issues. Finally, the image quality of [124I]iodine-PET is not preferable due to low signal-to-noise ratios. In contrast, [18F]TFB-PET-CT can easily be produced by a cyclotron equipped radiochemistry department and delivers excellent image to noise ratios and the image acquisition can be initiated minutes after tracer administration. Interestingly, patients 1 and 10 showed similar blood parameters ; however, only patient 10 showed suspicious [18F]TFB accumulation, which was histologically confirmed to be a metastasis. Therefore, [18F]TFB-PET-CT might be advisable in patients with minimally elevated tumor marker levels.It has been shown previously that 12Iiodine-P18F]FDG-PET-CT imaging in DTC has been proposed to overcome the limitations of diagnostic [131I]iodine WBS [18F]FDG-PET has a higher sensitivity than [131I]iodine dxWBS [18F]FDG metabolism in turn [18F]FDG positiveness together with negative [131I]iodine dxWBS does not prove complete dedifferentiation. It has been shown that [124I]iodine PET and therapeutic [131I]iodine WBS have a greater sensitivity than [131I]iodine dxWBS [18F]FDG uptake of metastases might at least partly be caused by tumor infiltrating immune cells [131I]iodine dxWBS together with [18F]FDG-PET-CT. Therefore, [18F]FDG-PET and hNIS targeting molecular imaging are not equivalent but complementary.FDG-PET-CT with high-quality hNIS targeting molecular imaging is corroborated by the present study. Cervical lymph node metastases with moderate [18F]FDG accumulation but negative diagnostic [131I]iodine WBS/SPECT-CT were discovered in six patients. Thus, TENIS was suspected in these cases. However, [18F]TFB-PET-CT was positive thus suggested only partly dedifferentiation. Moreover, [18F]TFB-PET enabled a clear delineation of the metastases that lead to successful surgery in two cases, which enabled the histological confirmation of DTC. Two different patients of this study showed strong [18F]FDG accumulation of DTC metastases without evidence of functional relevant hNIS expression (negative [131I]iodine WBS and [18F]TFB-PET). Therefore, dedifferentiation and TENIS were assumed. Taken together, the combination of [18F]FDG-PET and [18F]TFB-PET seems reasonable due to their complementary nature. The combination of both imaging modalities achieved the highest sensitivity and accuracy (both: 62%) by sensitively detecting both differentiated and dedifferentiated recurrence of DTC.The advantage of combining [131I]iodine dxWBS/SPECT-CT imaging several times with inconclusive findings. Therefore, the pretest probability of positive imaging findings might be low. This could affect the transferability of the results to the clinical routine and underestimate the diagnostic performance of [18F]TFB-PET-CT. Moreover, only a relatively small patient collective was included due to the limited application of [18F]TFB-PET-CT in the clinical routine.The present retrospective study faces some limitations. First, the enrollment of patients was done retrospectively and is therefore prone to selection biases. For instance, many enrolled patients had only minimally elevated TG levels or had undergone [18F]TFB shows increased retention in the blood, which caused difficulties in the reading of head and neck acquisitions, especially when combined with low dose CT (note the blood pool in Fig. 18F]TFB and image acquisition. An increased interval between administration and image acquisition might result in lesser presence of [18F]TFB in the blood. Additionally, it might be warranted to combine [18F]TFB-PET with contrast enhanced morphology imaging to clearly delineate cervical blood vessels. This could facilitate the characterization of cervical [18F]TFB accumulating foci as either unspecific blood retention or cervical metastases.Finally, [18F]TFB-PET-CT detected local recurrence or metastases of DTC in significantly more patients than [131I]iodine dxWBS/SPECT-CT. The combination of [18F]FDG and [18F]TFB PET-CT imaging achieved the highest diagnostic performance. Future studies evaluating [18F]TFB-PET-CT in recurrent DTC seem warranted.[


In this retrospective study, we evaluated the performance of [18F]-JK-PSMA-7 under ADT.PSMA imaging is frequently used for monitoring of androgen deprivation therapy (ADT) in prostate cancer. In a previous study, [18F]-JK-PSMA-7 in 70 patients (first cohort) with increasing or detectable PSA values under ADT (PSA < 2 ng/ml for 21/70 patients). We further analyzed 58 independent patients with PSA levels < 2 ng/ml under ADT, who were imaged with [68Ga]PSMA-11 or [18F]DCFPyL (second cohort). Finally, we compared detection rates between [18F]-JK-PSMA-7, [68Ga]PSMA-11, and [18F]DCFPyL.We examined the performance of [18F]-JK-PSMA-7-positive lesions in 63/70 patients. In patients with PSA levels ≥ 2 ng/ml, the detection rate was 100 % (49/49). In patients with PSA < 2 ng/ml, the detection rate was significantly lower  and dropped from 85.7 %  to 28.6 % (2/7) for PSA levels < 0.3 ng/ml (p = 1.73 × 10−2). In the second cohort (PSA < 2 ng/ml), the detection rate was 79.3 % (46/58) for [68Ga]PSMA-11 or [18F]DCFPyL. Again, the detection rate was significantly higher (p = 1.1 × 10−2) for patients with PSA levels between 0.3 and 2.0 ng/ml  relative to those with PSA levels < 0.3 ng/ml . No significant difference was found between [18F]-JK-PSMA-7 and [68Ga]PSMA-11 or [18F]DCFPyL in patients with PSA levels < 2 ng/ml (p = 0.4295).In the first cohort, we detected [18F]-JK-PSMA-7 PET showed a high detection rate in patients with PSA levels ≥ 0.3 ng/ml under ADT. The lower PSA threshold of 0.3 ng/ml for high detection rates was consistent across the three PSMA ligands. Thus, PSMA imaging is suitable for clinical follow-up of patients with increasing PSA levels under ADT.[ Recentl = Köln) –7. Like 68Ga]PSMA-11 (n = 195) while some were examined with [18F]DCFPyL (n = 5). When administering enzalutamide or abiraterone in addition to an LHRH analog in CRPC, expression levels of PSMA increase in metastases, as recently shown in a cohort of 7 patients PSMA-11.In contrast to castrate-resistant patients, the impact of ADT on PSMA expression in hormone-sensitive prostate cancer (HSPC) remains unclear. A comparison of PET scans before and after therapy with ADT revealed a heterogeneous pattern of change in the PSMA expression of metastases in small cohorts of 10, 7, and 5 patients, respectively . In the Data on the probability of PSMA-positive lesions in patients with low PSA levels under continuous ADT are sparse. There is no consensus as to which PSA value for PMSA PET imaging is a sensitive tool under ADT. The definition of castration-resistant prostate cancer in the guidelines of the European Association of Urology includes a castrate serum testosterone < 50 ng/dl plus three consecutive rises in PSA, resulting in two 50 % increases over the nadir, and a PSA level > 2 ng/ml . Hence, 18F]-JK-PSMA-7 PET/CT from the first year of clinical implementation in patients under ADT who were referred for PET imaging by their local urologists with a broad spectrum of PSA levels. The observed detection rate in patients with PSA levels < 2 ng/ml was re-examined in a second cohort investigated with [68Ga]PSMA-11 or [18F]DCFPyL.Here, we report the results of the [18F]-JK-PSMA-7 in 2017 as a routine clinical procedure (cohort 1). As stated in the “Results” section, hypothesis 1 was confirmed. Based on this finding, we next hypothesized that high detection rates could be achieved for patients with PSA levels < 2 ng/ml under ADT (hypothesis 2). To test this hypothesis, we retrospectively analyzed an independent cohort that consisted exclusively of patients with PSA levels < 2 ng/ml under ADT. These patients were imaged with [68Ga]PSMA-11 or [18F]DCFPyL as a routine clinical procedure in 2015 and 2016 (cohort 2). The advantage of cohort 2 was that more patients could be included in the group used to calculate the lower PSA threshold for beneficial imaging with PSMA agents. To determine the lower PSA threshold, we continuously adjusted the PSA threshold and divided the number of PSMA-positive patients with PSA levels above this threshold by the total number of patients with PSMA-positive lesions. Finally, we compared the detection rates for PSMA-positive lesions in patients with PSA < 2 ng/ml between cohorts 1 and 2, assuming the non-inferiority of the PSMA ligands [18F]-JK-PSMA-7, [68Ga]PSMA-11, and [18F]DCFPyL to one another.We hypothesized that the detection rate of PSMA-positive metastases under continuous ADT was higher in patients with PSA levels ≥ 2 ng/ml relative to patients with PSA levels < 2 ng/ml (hypothesis 1). Our first cohort was imaged with -JK-PSMA-7 tracer was completed in 2016 [18F]-JK-PSMA-7 in comparison with [68Ga]PSMA-11 was demonstrated in a pilot study and then for clinical use in BCR patients within the first year of [18F]-JK-PSMA-7 application [18F]-JK-PSMA-7 for different indications, including staging, BCR, and therapy monitoring. Our study group comprised of 70 patients (aged 70.1 ± 5.5 years) who had been referred in 2017 for PET imaging with [18F]-JK-PSMA-7 (348 ± 55 MBq) under continuous ADT with LHRH analogs and/or bicalutamide. Nine patients also received enzalutamide or abiraterone, and 11 patients had been treated with docetaxel. As a PSA level > 2 ng/ml is one of several criteria to determine therapy decisions in advanced prostate cancer under ADT -JK-PSMA-7) was compared with the detection rate in cohort 2 ([68Ga]PSMA-11 or [18F]DCFPyL) by a chi-square test.The detection rate for PSMA-positive lesions in cohort 1, the subgroup with PSA < 2 ng/ml  were acquired in a low-dose technique with kV and mA modulation and adapted to the size of the patient. PET/CT scans were interpreted according to published criteria for standardization of PSMA PET/CT interpretation -JK-PSMA-7 using the “minimalist light” protocol was described previously [The detailed procedure for the radiosynthesis of [eviously . Two bateviously –7.18F]-JK-PSMA-7 (Table 18F]-JK-PSMA-7-positive lesions in 63/70 patients (90.0 %)  higher in patients with PSA levels ≥ 2 ng/ml  compared with PSA levels < 2 ng/ml . However, individual patients with very low PSA levels still showed PSMA-positive lesions  Figs. , and 3. 18F]-JK-PSMA-7 positive will undergo PET imaging. Based on our subcohort of 21 patients with PSA levels < 2 ng/ml, 14 of these 21 patients showed PSMA-positive lesions, most of the PSMA-positive patients had PSA levels ≥ 0.3 ng/ml. For patients with PSA levels between 0.3 and 2 ng/ml under ADT, the detection rate with [18F]-JK-PSMA-7 was 85.7 % (12/14). This detection rate dropped significantly to 28.6 % (2/7) for PSA levels < 0.3 ng/ml   Fig. .Fig. 4.a18F]-JK-PSMA-7-positive lesions on the PET/CT scan. The majority (57/63) of these patients had a suspicious correlate on the CT scans. In 6 patients, PET scans were interpreted as [18F]-JK-PSMA-7 positive without any suspicious correlates on the corresponding low-dose CT scans. In these 6 patients, verification was based on an MRI scan , on decreasing PSA levels after salvage radiotherapy of the prostate fossa or the pelvic lymph nodes (n = 3), or a follow-up PSMA PET/CT scan exhibiting tumor progression (n = 2).Sixty-three patients exhibited [18F]-JK-PSMA-7, we hypothesized that many patients with PSA levels < 2.0 ng/ml under ADT exhibited robust expression of PSMA and that high detection rates could be achieved for patients with PSA levels < 2 ng/ml. To test this hypothesis, we examined the second cohort of 58 patients with PSA levels < 2 ng/ml under ADT who had undergone PET/CT imaging with [68Ga]PSMA-11 PET/CT  or [18F]DCFPyL PET/CT . In parallel to our previous analysis, we searched for a lower PSA threshold that included ≥ 85 % of the patients with [68Ga]PSMA-11-positive or [18F]DCFPyL-positive lesions  between patients with PSA levels ≥ 0.3 ng/ml  and < 0.3 ng/ml   had suspicious correlates on the corresponding CT scans. In 8 patients, PET scans were interpreted as PSMA positive without any suspicious correlates on the corresponding low-dose CT scans. In these 8 patients, verification was based on decreasing PSA levels after salvage radiotherapy of the prostate fossa or the pelvic lymph nodes (n = 3) or a follow-up PSMA PET/CT exhibiting tumor progression (n = 5).Forty-six patients showed [18F]-JK-PSMA-7 from cohort 1 and [68Ga]PSMA-11 or [18F]DCFPyL from cohort 2 . Furthermore, no significant difference was found between [18F]-JK-PSMA-7 and [68Ga]PSMA-11 or [18F]DCFPyL, when the 3 PSMA ligands were analyzed separately .The detection rates did not differ significantly between [18F]-JK-PSMA-7, PET/CT detection rates were statistically higher in prostate patients under ADT with PSA levels ≥ 2 ng/ml compared with patients with PSA level < 2 ng/ml.Using -JK-PSMA-7, [68Ga]PSMA-11, and [18F]DCFPyL in patients with PSA levels between 0.3 and 2 ng/ml. The lower PSA threshold of 0.3 ng/ml for high detection rates was consistent across the two cohorts: cohort 1 (using [18F]-JK-PSMA-7) and cohort 2 (using [68Ga]PSMA-11 or [18F]DCFPyL).Detection rates did not differ significantly between [This study revealed the following three findings on the PSMA imaging of prostate cancer under ADT:18F]-JK-PSMA-7 ligand for tumor localization after biochemical recurrence and its robust synthesis -JK-PSMA-7 PET has a high detection rate for patients under ADT with PSA levels as low as 0.3 ng/ml. This PSA threshold is as low as the thresholds derived from studies examining the performance of PSMA imaging based on biochemical recurrence after a curatively intended prostatectomy. Potential explanations of this observation include the possibility that tumor cells might increase their PSMA expression in response to long-term ADT.Consistent with a recent study by Fendler and colleagues , we foun18F]-JK-PSMA-7, [68Ga]PSMA-11, and [18F]DCFPyL. However, ADT seems to have a negative impact on PSMA expression once ADT has started in hormone-sensitive patients, and a positive impact when enzalutamide or abiraterone has been added to long-term ADT in castrate-resistant patients. The pathophysiological model is that ADT upregulates the folate hydrolase 1 gene and thereby increases PSMA expression -JK-PSMA-7 PET can detect PSMA expression in metastases and relapses under continuous ADT. Some patients were heavily pretreated. However, we have no information on castrate serum testosterone or on consecutive rises in PSA resulting in two 50 % increases over the nadir, which are defining criteria for hormone-sensitive or castration-resistant prostate cancer [Our work demonstrates that 1F-JK-PSMAe cancer . In the 18F]-JK-PSMA-7 PET cohort with the independent [68Ga]PSMA-11 or [18F]DCFPyL PET cohort was hindered by the fact that we did not have details of the Gleason score in each patient and could not therefore carry out a matched-pair analysis.Comparison of our [18F]-JK-PSMA-7 PET imaging robustly detected PSMA expression when PSA levels exceed 0.3 ng/ml under continuous ADT. This result was reproducible for [68Ga]PSMA-11 and [18F]DCFPyL. Thus, PSMA PET is a suitable imaging tool even under ADT once the PSA level starts to rise.[


Clarithromycin (CAM), a semisynthetic macrolide antibiotic, is a widely used antibacterial drug. Recently, the efficacy of CAM as an add-on drug for treating multiple myeloma (MM) has been noted. Its effect on treating MM has been confirmed in combination chemotherapies that include CAM. However, a single treatment of CAM has no efficacy for treating MM. Many myeloma growth factors (MGFs) including interleukin (IL)-6 are known to be closely involved in the development of MM. CAM has been shown to suppress many MGFs, particularly IL-6. The possible mechanisms of action of CAM in treating MM have been suggested to include its immunomodulatory effect, autophagy inhibition, reversibility of drug resistance, steroid-sparing/enhancing effect and suppression of MGFs. In addition, MM is characterised by uncontrolled cell growth of monoclonal immunoglobulin (Ig)-producing neoplastic plasma cells. Large quantities of unfolded or misfolded Ig production may trigger considerable endoplasmic reticulum stress. Thus, MM is originally a fragile neoplasm particularly susceptible to autophagy-, proteasome- and histone deacetylase 6-inhibitors. Taken together, CAM plays an important role in MM treatments through its synergistic mechanisms.In addition, CAM with its pleiotropic effects on cytokines including IL-6 and indirect antiviral effects might be worth a try for treating COVID-19. The antibacterial effect of CAM is related to its capacity to inhibit protein synthesis in bacteria by binding to subunit 50S of the bacterial ribosome  and a proliferation-inducing ligand [APRIL]), transforming growth factor-β(TGF-β), granulocyte colony-stimulating factor (G-CSF), granulocyte macrophage colony-stimulating factor (GM-CSF), epidermal growth factor (EGF) family members , basic fibroblast growth factor (bFGF), Wnt family members (Wnt5A/10B/16), Jagged family (Jag1/2) and VEGF/PDGF family members. The important MGFs and other factors which are positively or negatively influenced by CAM are shown in ng et al , Klein ang et al , Klein . In particular, CAM is also known to prolong QT interval. Rarely, it can be fatal, leading to fatal ventricular arrhythmias, including ventricular tachycardia and torsades de pointes [The frequent and common adverse effects of CAM are diarrhoea, nausea, abnormal taste, dyspepsia, abdominal pain/discomfort, headache, insomnia, tooth discolouration, smell loss and taste loss. These adverse reactions are usually mild in intensity and resolve after the discontinuation of treatment. All other adverse reactions are uncommon or rare . However pointes . Thus, CRecently, the World Health Organisation (WHO) has declared the new coronavirus (SARS-CoV-2) outbreak a pandemic. Several repurposing drugs  possibly effective for treating the disease (COVID-19) caused by SARS-CoV-2 have been tested. However, no definite drugs for treating COVID-19 have yet been established after the declaration by the WHO of the pandemic. Recently, the FDA of the United States of America authorised the emergency use of remdesivir for treating COVID-19.et al [In general, antibiotics are known to be ineffective against virus infection. Shinahara et al  treated et al [et al [https://www.clinicaltrialsregister.eu/ctr-search/trial/2020-001882-36/GR).It is interesting to note that hydroxychloroquine treatment combined with azithromycin, 15-membered macrolide, reinforced viral load reduction/disappearance in COVID-19 patients . Just reet al  first reet al . In addiet al . Cai et l [et al  demonstrWe reviewed the current preclinical, clinical and experimental evidence for supporting the efficacy of CAM for treating MM, especially when it is combined with IMiDs, steroids or proteasome inhibitors. IMiDs, steroids, proteasome inhibitors and CAM might synergistically play an important role in inducing apoptosis of MM cells. In addition, the reversibility of drug resistance by CAM to chemotherapeutic drugs including steroids seems to be more important than steroid-sparing/enhancing effect thus far reported in MM treatments. Since multiple myeloma is characterised by the uncontrolled cell growth of monoclonal Ig-producing neoplastic plasma cells, the large quantities of unfolded or misfolded immunoglobulin production itself may trigger ER stress. Thus, MM is a specific neoplasm particularly susceptible to proteasome, autophagy and HDAC6 inhibitors. The development of CAM analogues specific for more potent immunomodulatory effects may provide a novel approach to MM treatment in the future.In addition, considering the pleiotropic effects of CAM on cytokines including IL-6 and its indirect antiviral effects, CAM might be worth a try for treating COVID-19.AP-1activator proteinAPRILa proliferation-inducing ligandAREGamphiregulinBAFFB-cell activating factorbFGFbasic fibroblast growth factorEGFepidermal growth factorBCL-xLB-cell lymphoma-extralargeBipchaperone protein GRP-78BiRdLen and low-dose Dex combined with CAMBDNFbrain-derived neurotrophic factorBMSCsbone marrow stromal cellsBLTdlow-dose thalidomide and low-dose DE combined with CAMBOOPbronchiolitis obliterans organising pneumoniaBoorbortezomibCAMclarithromycinCCLF1cardiotrophin-like cytokine factor 1CHOPC/EBP homologous proteinClapdPom and low-dose Dex combined with CAMCNTFciliary neurotrophic factorCRcomplete responseDexdexamethasoneERendoplasmic reticulumG-CSFgranulocyte colony-stimulating factorGDF15growth/differentiation factor15GM-CSFgranulocyte macrophage colony-stimulating factorgp130glycoprotein 130HB-EGFheparin-binding EGF-like growth factorHDAC6histone deacetylase 6HGFhepatocyte growth factorIgimmunoglobulinIGF-1insulinlike growth factor-1ICAM-1intercellular adhesion molecule-1ILinterleukinIL-6RIL-6-receptorIMiDsimmunomodulatory drugsIFN-γinterferon-γJAK/STATJanus kinases/signal transducer-activator of transcriptionKRd®, Len and low-dose DexKyprolisLenlenalidomideLFA-1lymphocyte function-associated antigen-1LIFleukaemia inhibitory factorLTDLen, low-dose thalidomide and DexMCL-1myeloid cell leukaemia sequence-1MGFmyeloma growth factorMIPαmacrophage inflammatory protein αMMmultiple myelomaMMPmatrix metalloproteinaseMRminor responseMUC-1muchin-1NF-IL-6nuclear factor for IL-6NF-κBnuclear factor-κBNRG1~4neureglin1~4NKnatural killerNKTnatural killer TORRoverall response rateOSMoncostatin MPFSprogression-free survivalPI3K/AKTphosphatidylinositol-3 kinase/AKT kinasePDGFplatelet-derived growth factorPompomalidomidePRpartial responsePSLprednisolonePTNpleiotrophinRas/MAPKGTPase/mitogen-activated protein kinaseRdLen combined with low-dose dexRRMMrelapsed or refractory multiple myelomaSDF-1stromal cell-derived factor 1S-IgAsecretory immunoglobulin ATGF-βtransforming growth factor-βTNF-αtumour necrosis factor-αUPRunfolded protein responseVEGFvascular endothelial growth factorVCAM-1vascular cell adhesion molecule-1VGPRvery good partial responseVLAvery late activating antigenWnt5A/10B/16Wnt family membersAll of the authors declare that they have no conflicts of interest.This study was not supported by any grant.


Using smFISH we determined the localization of tbg-1 transcripts (red). tbg-1transcripts are detected within distinct foci throughout the cytoplasm during both the first (A) and second (B) mitosis. tbg-1 transcripts are not enriched at centrosomes or either blastomere. In contrast, GFP tagged TBG-1proteins  localize at centrosomes, as previously shown . Shown are projections from selected focal planes. Bar=10μm.New Findings: The first observation of tbg-1 mRNA localization in early C. elegans embryos.tbg-1 mRNAs  were designed using Stellaris Probe Designer (Biosearch Technologies). smFISH was performed as described previously . For hybridization, embryos were incubated with tbg-1 RNA probes (Quasar 670) at 39°C for four hours in the dark. Following hybridization, the embryos were washed and mounted with DAPI containing (blue) medium. To visualize centrosome-associated TBG-1 protein, we used transgenic strain that expresses GFP::TBG-1 , green).RNA probes targeting


Drosophila octopamine (OA) neurons are also glutamatergic and identify the individual contributions of each neurotransmitter on sex-specific behaviors. Males without OA display low levels of aggression and high levels of inter-male courtship. Males deficient for dVGLUT solely in OA-glutamate neurons (OGNs) also exhibit a reduction in aggression, but without a concurrent increase in inter-male courtship. Within OGNs, a portion of VMAT and dVGLUT puncta differ in localization suggesting spatial differences in OA signaling. Our findings establish a previously undetermined role for dVGLUT in OA neurons and suggests that glutamate uncouples aggression from OA-dependent courtship-related behavior. These results indicate that dual neurotransmission can increase the efficacy of individual neurotransmitters while maintaining unique functions within a multi-functional social behavior neuronal network.Neuromodulators such as monoamines are often expressed in neurons that also release at least one fast-acting neurotransmitter. The release of a combination of transmitters provides both “classical” and “modulatory” signals that could produce diverse and/or complementary effects in associated circuits. Here, we establish that the majority of  Drosophila adult CNS. By manipulating the release of glutamate in OA-glutamate neurons, we demonstrated glutamate has both separable actions and complementary actions with OA on aggression and reproductive behaviors respectively. Aggression is a behavior that is highly conserved between organisms and present in many human disease states, including depression and Alzheimer’s disease. Our results show that aggressive behavior requires the release of both neurotransmitters in dual-transmitting neurons and suggests within this set of neurons, glutamate may provide a new therapeutic target to modulate aggression in pathological conditions.Neurons communicate with each other via electrical events and the release of chemical signals. An emerging challenge in understanding neuron communication is the realization that many neurons release more than one type of chemical signal or neurotransmitter. Here we ask how does the release of more than one neurotransmitter from a single neuron impact circuits that control behavior? We determined the monoamine octopamine and the classical transmitter glutamate are co-expressed in the  The classical view of information transfer for many decades was that each neuron released a single neurotransmitter, leading to the ‘one neuron, one transmitter’ hypothesis , formaliDrosophila provide the ability to genetically dissect the signaling properties of dual transmission on behavioral networks in general and upon the circuits that control aggression in particular. Aggression is a hardwired behavior that has evolved in the framework of defending or obtaining resources , 64DrosoEAAT1-gal4;UAS-EAAT1). While a loss of EAAT1 impairs larval movement [EAAT1-gal4;UAS-EAAT1) was altered in two parameters: the latency to initiate lunging increased and lunge number decreased  a STOP cassette between VMAT coding exons 5 and 6 and, 2) six in-frame tandem copies of a V5 epitope tag within exon 8 which is common to both VMAT-A and VMAT-B isoforms  which drives expression in OGNs :HA) and re-examined V5-VMAT and dVGLUT expression in the AL and SEZ  the release of GLU and DA from different synaptic vesicles in midbrain dopamine neurons, 77 and Drosophila, recent work has identified a small population of male-specific neurons that express the alpha-like adrenergic receptor, OAMB, as aggression-promoting circuit-level neuronal targets of OA modulation independent of any effect on arousal[Rdl GABAa receptor in a specific doublesex+ population stimulated male aggression [A second mechanism by which co-transmission may generate unique functional properties relies on activating distinct postsynaptic receptors. In n arousal and sepagression . Future gression ).Deciphering the signaling complexity that allows neural networks to integrate external stimuli with internal states to generate context-appropriate social behavior is a challenging endeavor. Neuromodulators including monoamines are released to signal changes in an animal’s environment and positively or negatively reinforce network output. In invertebrates, a role for OA in responding to external chemosensory cues as well as promoting aggression has been well-established , 80–83. Finally, monoamine-expressing neurons play key roles in human behavior including aggression and illnesses that have an aggressive component such as depression, addiction, anxiety, and Alzheimer’s , 87. WhiAll flies were reared on standard cornmeal-based fly food. Unless noted otherwise, during developmental and post-eclosion, flies were raised at 25°C, ~50% humidity and a 12:12hr light-dark cycle (1400±200 lx white fluorescent light) in humidity and temperature-controlled incubators. A list of stocks can be found in 2 anesthesia for identification purposes. Flies were returned to their respective isolation tubes for a period of at least 24 hours to allow recovery. For aggression testing, pairs of 3–5 day old, socially naïve adult males were placed in 12-well polystyrene plates (VWR #82050–930) as described previously [Male pupae were isolated and aged individually in 16 x 100mm borosilicate glass tubes containing 1.5ml of standard food medium as previously described . A dab oeviously . All assAll aggression was assayed within first two hours of lights ON time (Zeitgeber hours 0–2) and scored manually using iMovie version 8.0.6. Total number of lunges, wing threats, and unilateral wing extensions were scored for a period of 30 minutes after the first lunge according to the criteria established previously , 88. TheActivity levels were measured by tracking the flies in each assay using the OpenCV module in the Python programming language to analyze the video and then output XY-coordinate and distance data. The distance traveled was calculated for each fly by determining the starting location followed by the second location after a 250-ms time interval and then taking the sum of the distance traveled in each interval. To calculate pixels moved per second, the distance data was divided by the total time spent tracking.Adult male dissected brains were fixed in 4% paraformaldehyde (Electron Microscopy Sciences) for 25 minutes and labeled using a modification of protocols previously described . The folribosomal protein 49 (Rp49) was used as the reference control to normalize expression between genotypes. Expression levels were determined using the ΔΔCT method and results from control (UAS-dVGlut-RNAi/+) and experimental  groups were normalized relative to a transgenic control . The following primers were used: Rp49 Forward: 50-CATCCGCCCAGCATACAG-3’ Rp49 Reverse: 5’-CCATTTGTGCGACAGCTTAG-3’ dVGlut Forward: 5’-GCACGGTCATGTGGTGATTTG-3’ dVGlut Reverse: 5’-CCAGAAACGCCAGATACCATGG-3’. Primer designs for all Rp49 and dVGlut primers used have been described previously [Total RNA from ~40 heads using Direct-zol RNA Miniprep Pluskit (Zymo Research)and treated with DNase I per the manufacturer’s protocol. RNA concentrations were measured with a ND-1000 nanodrop spectrometer. Reverse transcription was accomplished using iScript cDNA Synthesis kit (Bio-Rad Laboratories). RT-PCR was performed using 300 ng cDNA added to iTaq Universal SYBR Green Supermix (Bio-Rad Laboratories) and primers in a 20 μL reaction volume. All samples were run in triplicate using a Stratagene Mx3005P qPCR System(Agilent Technologies). Expression of eviously .20XUAS-His2A-GFP, 13XLexAop2-His2B-mCherry, and 20XUAS-R expression clones were assembled using Gateway MultiSite LR reactions as previously described[L1-20XUAS-DSCP-L4 and L1-13XLexAop2-DSCP-L4 entry clones contain 20 copies of UAS and 13 copies of LexAop2 upstream of the Drosophila synthetic core promoter (DSCP) [R4-His2A-R3 and R4-His2B-R3 entry clones were generated as previously described [L3-GFP-L2 entry clone was generated from template pJFRC165[L3-GFP-L2 and L3-mCherry-HA-L2 entry clones were previously described [L1-20XUAS-DSCP-R5 entry clone was previously described [pDESTp10aw destination vector was previously described[The described and as ir (DSCP) , respectescribed  using ge pJFRC165 except tescribed . The L1-escribed . The pDEdescribed. InjectiUAS-B3 was performed by Genetivision into landing site VK31.B3 recombinase derived from pJFRC157  was PCR B3RT-dVGlut-LexA chromosome was generated via CRISPR/Cas9 genome editing. Both guide RNAs were incorporated into pCFD4 using previously described methods [pCFD4-vGlut1. The donor plasmid B3RT-dVGlut-LexA used the pHSG298 backbone (Takara Bio) and was generated using NEBuilder HiFi (New England Biolabs). The complete annotated sequence of B3RT-dVGlut-LexA is shown in Supplementary Information. pCFD4-vGlut1/B3RT-dVGlut-LexA injections were performed by Bestgene, Inc.The  methods  to produdVGlut on the B3RT-dVGlut chromosome pre- and post-excision, the B3RT-dVGlut chromosome was crossed with the homozygous lethal dVGlut null allele, dVGlutSS1 in the presence and absence of the pan-neuronal driver n-syb-Gal4. In the absence of a Gal4 driver, dVGlutSS1/B3RT-dVGlut progeny are fully viable and no LexAop-driven reporter gene expression is detected (UAS-B3) is expressed in the nervous system by n-syb-Gal4, dVGlutSS1/B3RT-dVGlut;UAS-B3/n-syb-Gal4 progeny are inviable, therefore after excision, as expected with removal of the entire dVGlut protein-coding sequence, a dVGlut null allele results.To assess the functionality of detected . When B3dVGlutSS1 allele was generated by CRISPR/Cas9 genome editing with the same guide RNAs used to generate B3RT-dVGlut LexA. dVGlutSS1 was identified based on failed complementation with the existing dVGlut2 allele[dVGlutSS1 allele are indicated with the deleted region in bold: GGACCAGGCGGCGGCCACGC. . . . . .AACCTCCGGCCGAGGAGCAA.The t2 allele. SequencRSRT-STOP-RSRT-6XV5-vMAT was generated via CRISPR/Cas9 genome editing. Both upstream guide RNAs were incorporated into pCFD4-vMAT1 and both downstream guide RNAs were incorporated into pCFD4-vMAT2 as previously described [RSRT-STOP-RSRT-6XV5-vMAT donor plasmid used the pHSG298 backbone (Takara Bio) and was generated using NEBuilder HiFi (New England Biolabs). The complete annotated sequence of RSRT-STOP-RSRT-6XV5-vMAT is shown in Supplementary Information. pCFD4-vMAT1/pCFD4-vMAT2/RSRT-STOP-RSRT-6XV5-vMAT injections into the nos-Cas9 strain TH_attP2[escribed . The RSRB3RT-vGlut-LexA and RSRT-STOP-RSRT-6XV5-vMAT, the recombinases catalyze excision of the intervening DNA and leave behind a single recombinase target site.The R and B3 recombinases from yeast recognize sequence-specific recombination target sites, RSRTs and B3RTs, respectively . These rDrosophila anti-dVGLUT mouse monoclonal antibodies (10D6G) were generated (Life Technologies Europe) using the C-terminal peptide sequence TQGQMPSYDPQGYQQQ of dVGLUT coupled to KLH.S1 Fig(A) dVGLUT expression detected by mAb dVGLUT in a heterozygous yw, dVGlutSS1/+ late stage embryo. (B) dVGLUT expression is not detectable by mAb dVGLUT in a homozygous yw, dVGlutSS1/ dVGlutSS1 late stage embryo.(TIF)Click here for additional data file.S2 Fig(A-B) Although the Tβh shows weaker immunoreactivity than the anti-Tdc2 antibody, Tβh is mainly detected in dVGlut>dsRed neurons at dorsal and ventral positions . Scale bar = 20 μm.(TIF)Click here for additional data file.S3 Fig(A-A’) Schematic showing the regions (boxes) of the VNS imaged in panels B and C. (B-C) A male dVGlut>dsRed adult VNS labeled with anti-Tdc2. The majority of dVGLUT+ neurons within the thoracic VNS (B) and abdominal VNS (C) express Tdc2 with a few exceptions (arrows). Scale bar = 10 μm.(TIF)Click here for additional data file.S4 Fig(A) Schematic showing the regions imaged in panels B and C (colored boxes). (B-C) The majority of OA neurons within the PENP (B) and SEZ (C) regions co-express dVGLUT as visualized in a male tdc2>dsRed adult brain labeled with anti-dVGLUT. Scale bar = 10 μm.(TIF)Click here for additional data file.S5 Fig(A)dVGlut transcript levels were decreased in n-syb-gal4>dVGLUT-RNAi males as compared to the n-syb-gal4 control . (B-C) Representative images of ventral sections of the SEZ from a tdc2-gal4>dVGLUT-RNAi;UAS-dsRed male brain labeled with anti-Tdc2. OGN differentiation as measured by Tdc2 expression is not altered by a reduction of dVGLUT. Scale bar = 10 μm. (D-E) Dorsal sections of the SEZ, PENP and protocerebral bridge region from the same brain as in B. There are no obvious changes in ventral OGN survival and differentiation as measured by Tdc2 expression. Scale bar = 20 μm.(TIF)Click here for additional data file.S6 Fig(A) Verification that each tdc2>GFP neuron in the brain and VNS is Tdc2+. The stack for panel A contains 30 optical sections at 1.0 μm. Scale bar = 20 μm. (B) The stack for panel B contains 34 optical sections at 1.0 μm. Scale bar = 20 μm. (C-E) Verification that each tdc2-dVGlut-split>GFP neuron is Tdc2+. The stack for panels C-E contains 56 optical sections at 0.5 μm. Scale bar = 20 μm. (F) Schematic showing the locations of Tdc+ clusters in C-E.(TIF)Click here for additional data file.S7 Fig(A) The activity levels of controls and tdc2>dVGlut-RNAi males did not differ during the aggression assay as measured by pixels moved/second. (B) Total behavioral events  per minute was calculated. The average number of behavioral events per minute exhibited by experimental males  was slightly higher than controls (**p<0.01)(TIF)Click here for additional data file.S8 Fig(A) The VNS of a tdc2>mtd:HA male, note the Tdc2+ cell bodies. (B) The addition of tsh>Gal80 blocked the Gal4-mediated expression of mtd:HA in the majority of Tdc2+ VNS neurons . Axonal projections from brain Tdc2+ neurons are visualized in the VNS. (C) Significantly less Tdc2+ VNS neurons are detected in tdc2/tsh>Gal80;dsRed vs. tdc2>dsRed males. . (D) The addition of tsh>Gal80 does not alter brain tdc2-gal4 reporter driven expression.(TIF)Click here for additional data file.S9 Fig(A-D) Representative images of dorsal (A-B) and ventral (C-D) optical sections of the SEZ region from tdc2-gal4;B3RT-dVGlut/dVGLUTSS1;UAS-B3 lexAop-His2B-mCherry UAS-His2A-GFP males. OGNs are visualized by the mCherry reporter and white co-colocalization in the merged channel. Scale bar = 20 μm.Neuron survival or distribution is not altered by the complete loss of dVGLUT in OGNs (TIF)Click here for additional data file.S10 Fig(A-A’) In the presence of a Gal4 driver  to drive R recombinase (UAS-R) expression, the stop cassette of RSRT>stop>6XV5-VMAT is excised and V5-VMAT (magenta) is expressed and visualized by anti-V5. dVGLUT (green) is visualized by mAb dVGLUT. (B-B’) Without the presence of a Gal4 driver, dVGLUT expression is apparent while expression from RSRT>stop>6XV5-VMAT is not detected by anti-V5. Scale bar = 30 μm.(TIF)Click here for additional data file.S11 Fig(A) Higher magnification of the SEZ region showing V5-VMAT expression in OGNs after excision by tdc2-dVGlut-gal4 driven R recombinase. The brain is labeled with anti-V5 (magenta) and mAb dVGLUT (green). Scale bar = 15 μm. (B-B”) Higher magnification of the SEZ region of the region in the dashed box in panel B. Arrowheads indicate puncta with dVGLUT and V5-VMAT colocalization. Arrows indicate puncta with only V5-VMAT (arrows). (C) Schematic indicating the location of the SEZ region. (D) SEZ region of a representative brain with a synaptic marker incorporated . The brain is labeled with anti-HA (blue), anti-V5 (magenta), and mAb dVGLUT (green). Scale bar = 20 μm. (E) Higher magnification of the SEZ region in D. Scale bar = 10 μm. (F-H) Regions of interest from E showing puncta with dVGLUT, V5-VMAT and Syt:HA. The stack for panel B contains two optical sections at 0.45 μm. Six optical sections at 0.45 μm were stacked in panels E-H.(TIF)Click here for additional data file.S12 Fig(A-C) Brains from tdc2-dVGlut-split-gal4/UAS>stop>CD8:GFP;fru-flp males demonstrate OA-FruM+ neurons are also dVGlut+. (D) No OGNs in the VNS are FruM+ although as expected the OGN-FruM+ neurons project into the VNS. Scale bar = 20 μm. (E-G) OGN-FruM+ neurons (arrow) were also identified in dVGlut-gal4/UAS>stop>CD8:GFP;fru-flp male brains labeled with anti-Tdc2 (magenta). Scale bar = 20 μm.(TIF)Click here for additional data file.S1 Table(TIF)Click here for additional data file.S2 Table(TIF)Click here for additional data file.S1 Data(TIF)Click here for additional data file.


OsSSI2-kd) of the rice OsSSI2 gene, encoding a stearoyl acyl carrier protein FA desaturase (SACPD), remarkably enhanced resistance to blast fungus Magnaporthe oryzae and the leaf-blight bacterium Xanthomonas oryzae pv. oryzae (Xoo). Transcriptomic analysis identified six AAA-ATPase family genes (hereafter OsAAA-ATPase1–6) upregulated in the OsSSI2-kd plants, in addition to other well-known defense-related genes. Here, we report the functional analysis of OsAAA-ATPase1 in rice’s defense response to M. oryzae. Recombinant OsAAA-ATPase1 synthesized in Escherichia coli showed ATPase activity. OsAAA-ATPase1 transcription was induced by exogenous treatment with a functional analogue of salicylic acid (SA), benzothiadiazole (BTH), but not by other plant hormones tested. The transcription of OsAAA-ATPase1 was also highly induced in response to M. oryzae infection in an SA-dependent manner, as gene induction was significantly attenuated in a transgenic rice line expressing a bacterial gene (nahG) encoding salicylate hydroxylase. Overexpression of OsAAA-ATPase1 significantly enhanced pathogenesis-related gene expression and the resistance to M. oryzae; conversely, RNAi-mediated suppression of this gene compromised this resistance. These results suggest that OsAAA-APTase1 plays an important role in SA-mediated defense responses against blast fungus M. oryzae.Fatty acids (FAs) have been implicated in signaling roles in plant defense responses. We previously reported that mutation or RNAi-knockdown ( Arabidopsis, NPR1 (non-pathogenesis related 1) has been demonstrated to play a master role in SA-mediated defense activation [Salicylic acid (SA) plays an important signaling role in plant defense activation against pathogens. In response to pathogen attack, SA activates a battery of defense-related genes, including pathogenesis-related (PR) genes, throughout the plant, resulting in both local and systemic resistance to the pathogen . In Arabtivation ,3. A lostivation . In ricetivation ,6.Arabidopsis, genetic screening for mutations that can suppress npr1 phenotypes (based on their ability to restore SA-induced PR expression to npr1-5 plants) resulted in isolation of several npr1 suppressor mutants (ssi: suppressor of SA insensitivity), which exhibit constitutive defense activation [ssi mutants (ssi2) revealed that the corresponding gene (SSI2) encodes a stearoyl-ACP desaturase, which desaturates stearoyl (18:0)-ACP into oleoyl-ACP, and finally, into oleic acid (18:1) [ssi2 results in a ten-fold increase in 18:0 fatty acid (FA) content, indicating involvement of FAs in plant defense reactions [ssi2 mutant plants accumulate high levels of SA and display constitutive PR gene expression and enhanced resistance to Peronospora parasitica, Pseudomonas syringae [Cucumber mosaic virus [In tivation . Map-basd 18:1) . Disrupteactions . The ssi:1 . Disric virus ,9,10.SSI2 have also been identified in soybean (GmSACPD-A/-B) [OsSSI2) [TaSSI2) [Arabidopsis ssi2, suppression of these ortholog genes enhanced resistance to multiple pathogens: Pseudomonas syringae pv. glycinea and Phytophthora sojae in soybean [Magnaporthe oryzae and leaf-blight bacteria Xanthomonas oryzae pv. oryzae (Xoo) in rice [Blumeria graminis f. sp. tritici and Fusarium head blight fungus Fusarium graminearum in wheat [SSI2 and its orthologs in defense activation in diverse plant species.The orthologs of PD-A/-B) , rice (O[OsSSI2) , and whe[TaSSI2) ,14. Simi soybean ; blast f in rice ; and powin wheat ,14. ThesSSI2 family genes participate in defense reactions in plants remain to be fully elucidated. In rice, a DNA microarray analysis revealed several hundred genes differentially expressed between the wild-type and OsSSI2-suppressed transgenic (OsSSI2-kd) plants [OsSSI2-kd plants, in addition to the well-known defense-related genes, such as WRKY45, PR1b, and PBZ1, and a thaumatin-like gene [The molecular mechanisms whereby the ) plants . Among tike gene . These rNicotiana tabacum (NtAAA1) [Arabidopsis (AtOM66) [lesion mimic resembling (lmr) mutant/lesion resembling disease (lrd6-6) mutant revealed that the corresponding gene  encodes an AAA-ATPase, and is negatively involved in HR and disease resistance [The AAA-ATPase family occurs in all life forms, including eukaryotes, prokaryotes, and archaebacteria, and is implicated in a variety of cellular activities, including proteolysis, protein folding, membrane trafficking, cytoskeletal regulation, organelle biogenesis, DNA replication, and immune responses ,16,17. S(NtAAA1) ,19 and A(AtOM66)  are, ressistance ,22.OsAAA-ATPase1, one of the six AAA-ATPase genes upregulated in OsSSI2-kd rice plants [OsAAA-ATPase1 is transcriptionally regulated by SA, and positively involved in resistance to blast fugus M. oryzae.In this study, we conducted a functional analysis of e plants . We showOsAAA-ATPase1–6;   a.OsAAA-ATPase1–6 are related within the AAA-ATPase gene family, we performed a phylogenic comparison of the proteins predicted to be encoded by OsAAA-ATPase1–6 and several known rice AAA-ATPase proteins, including LMR/LRD6-6 [To assess how R/LRD6-6 ,22, OsCDR/LRD6-6 , RuvBL1aR/LRD6-6 , RLS3 [2R/LRD6-6 , OsSKD1 R/LRD6-6 , OsFtsH5R/LRD6-6 , and RFCR/LRD6-6 , togetheR/LRD6-6 ,19 and AR/LRD6-6 . OsAAA-AOsAAA-ATPase1–6 to the plant hormones abscisic acid (ABA), ACC (an ethylene precursor), BTH , kinetin , auxin (IAA), jasmonic acid (JA), and gibberellic acid (GA). Plant hormones have been demonstrated to play important roles in interactions between plants and pathogens. Hence, we examined the transcriptional responses of OsAAA-ATPase1 (OsAAA-ATPase3 (OsAAA-ATPase2 (OsAAA-ATPase4 (OsAAA-ATPase5 (OsAAA-ATPase6 (Os07g0517600) had no detectable transcription. -ATPase1 a and OsA-ATPase3 c were in-ATPase2 b was ind-ATPase4 d and OsA-ATPase5 e were nonahG gene (nahG-rice), at the four-leaf stage, were subjected to blast inoculation. At 2–6 days post inoculation (dpi) of the blast, the fourth leaves were sampled to examine the expression of OsAAA-ATPase1–5. Rice seedlings of non-transformant Nipponbare rice (NB) and of NB expressing the OsAAA-ATPase genes clearly showed transcriptional induction in response to blast inoculation  was used, so as to cause blast disease moderately in NB, but more severely in OsAAA-ATPase1 plants.To gain some insight into the role of OsAAA-ATPase1-ox plants (lines #27 and #29) exhibited significantly higher resistance to blast disease, as evidenced by the fact that few susceptible blast lesions appeared on their leaf blades , f blades b, and thf blades c. The enand PBZ1 d,e.OsAAA-ATPase1-kd plants (lines #25 and #32): they had ca. 2-fold more fungal growth than the NB control plants   .To determine the subcellular localization of OsAAA-ATPase1 in rice cells, the EGFP-OsAAA-ATPase1 fusion protein in the rice protoplast was examined under a confocal microscope. As shown in OsAAA-ATPase1. The deduced amino acid sequence of OsAAA-ATPase1 contains consensus motifs that are typical of the AAA-ATPase family; these include the Walker A, Walker B, and SRH motifs  is a stress-induced gene; overexpression of this gene increased SA content, accelerated cell death rates, and enhanced resistance to the biotrophic pathogen Pseudomonas syringae [LMR and LRD6-6 were map-based cloned from lesion mimic mutants lmr and lrd6-6, respectively, and were found to be the same gene (Os06g0130000). LMR/LRD6-6 was shown to be localized in the multivesicular bodies (MVBs) and was negatively involved in rice immunity and cell death [lmr and lrd6-6) resulted in constitutive expression of PR1 and PBZ1, and enhanced resistance to rice blast and bacterial blight diseases; however, no difference in SA content was determined [In relation to plant immune responses, several studies have shown important roles for AAA-ATPase genes.  tabacum ; was fousistance ,19. In csyringae . Recentlll death ,22. Mutatermined ,22. By cArabidopsis ssi2 mutant, disruption of SSI2, which encodes an FA desaturase, results in an increase in the 18:0 FA content, which in turn remarkably increases SA content, PR gene expression, and resistance against multiple pathogens [SSI2-orthologs in soybean (GmSACPD-A/-B) [OsSSI2) [TaSSI2) [SSI2 and its orthologs serve as valuable susceptibility gene (S gene) resources for the development of crop cultivars with resistance to multiple pathogens, by employing targeted mutation and genome editing technologies [Arabidopsis, a mutation in the GTPase nitric oxide associated 1 (NOA1) gene partially restored the ssi2 phenotype, whereas double mutations in NOA1 and either one of the two nitrate reductase isoforms (NIA1 and NIA2) completely restored the ssi2 phenotypes; this indicates that nitric oxide (NO) is required for constitutive defense in the ssi2 mutant [OsSSI2-kd rice plants. We previously identified a group of six AAA-ATPase genes (OsAAA-ATPase1–6) that were upregulated in OsSSI2-kd rice plants [OsAAA-ATPase1–5 each exhibited a distinct induction pattern in response to different plant hormone treatments  , rice (O[OsSSI2) , and whe[TaSSI2) ,14. Thesnologies ,44,45. I2 mutant ,47. Nevee plants . In thiseatments ; OsAAA-Ae plants . One posOryza sativa L.) was grown in commercial nursery soil  in a greenhouse at 28 °C (day)/23 ° C (night) with ca. 50% relative humidity.Japonica type rice cultivar Nipponbare . To construct a plasmid for constitutive expression of OsAAA-ATPase1 under the maize ubiquitin promoter, a DNA fragment containing a 91 bp upstream sequence followed by the full coding sequence of OsAAA-ATPase1 (nucleotides 2–1655) was amplified by PCR and cloned into the pUCAP/Ubi-NT vector, as previously described [OsAAA-ATPase1 RNAi (OsAAA-ATPase1-kd), part of the 3′-UTR (nucleotides 1543–1845) of OsAAA-ATPase1 cDNA was amplified by PCR and cloned into the pANDA vector, as previously described [The cDNA clone for escribed ,49.Agrobacterium tumefaciens (strain EHA105) mediated technique, as described earlier [nahG from Pseudomonas putida under the control of a double 35S promoter (nahG-rice) were generated using the plant expression construct previously described in Yang et al. [Nipponbare rice plants were transformed by an  earlier . Transgeg et al. .3; Wako, Osaka, Japan), abscisic acid ((±)-cis-trans, ABA; Sigma), and methyl jasmonate  were dissolved in ethanol. Kinetin (Sigma) and benzothiadiazole S-methyl ester  were dissolved in dimethyl sulfoxide (DMSO); and 1-aminocyclopropane-1-carboxylic acid  and sodium salicylate  were dissolved in H2O. The solvents did not exceed a final concentration of 0.1% in the solutions used for plant treatments, and had no effect on the expression of the rice genes examined in this study.All stock solutions were prepared at a concentration of 100 mmol/L. Indole-3-acetic acid , gibberellin A3  were transferred to a container containing each of the plant hormone solutions at 50 μM. The rice seedlings were further grown for 1 day, and fourth leaf blades were stored in liquid nitrogen for RNA preparation.OsAAA-ATPase1 sequence was amplified by PCR and cloned into the sites between BglI and HidIIIp of pET32a (Novagen) as a His-tag fusion protein; then, that was transfected into the Escherichia coli Origami strain BL21(Lys). The set of primers used was as follows: OsAAA1BglII 5′-GTAGATCTCTTGAGACAAATGGAGGCGACG-3′; OsAAA1HindIII 5′-GCTAAGCTTCTACTTATCCTTCCCGACCAC-3′. Expression of the protein was induced for 4 h at 25 °C with 0.5 mmol/L isopropyl β-d-1-thiogalactopyranoside. Escherichia coli cells were pelleted by centrifugation, resuspended in lysis buffer , and sonicated. After the cell debris was removed by centrifugation , the supernatant was loaded onto a High Affinity Ni-Charged Resin , washed with washing buffer , and eluted with elution buffer . ATPase activity was measured by the malachite green-based colorimetric method using the QuantiChromTM ATPase/GTPase activity assay kit . The elution buffer was used as the negative control, and an ATPase from potatoes  was used as the positive control. One unit is defined as the amount of enzyme that catalyzes the production of 1 μM of free phosphate per minute under the assay conditions.The For subcellular localization of OsAAA-ATPase1, the plasmid pSAT6-AFP-C1-OsAAA1 was transformed into protoplasts prepared from etiolated seedlings as previously described . As a coM. oryzae (compatible race 007.0) was conducted essentially as previously described [5/mL, and were sprayed onto rice plants at the four-leaf stage. After incubation in a dew chamber at 24 °C for 24 h, the rice plants were moved back to the greenhouse. Culture and inoculation of the blast fungus escribed , with slM. oryzae genomic 28S rDNA [Disease development was evaluated by determining the 28S rDNA  by qRT-P28S rDNA ,6, 6–7 dTotal RNA was isolated from leaf blades of the 4th leaves of rice seedlings using the Trizol reagent . Quantitative RT-PCR (qRT-PCR) was performed on a Thermal Cycler Dice TP800 system  using SYBR premix Ex Taq mixture (Takara Bio) as previously described . The priThe protein sequences were retrieved from the rice annotation project database (rap-db) and aligned using Clustal-X software, and the tree was constructed using iTOL software .OsAAA-ATPase1, one of the six AAA-ATPase genes upregulated in OsSSI2-kd rice plants [OsAAA-ATPase1 is transcriptionally regulated by SA, and plays a positive role in the SA-mediated disease resistance in rice plants. Our findings provide novel insights into SA-regulated defense activation in rice, and the molecular basis of defense activation in OsSSI2-kd rice plants.In this study, we present a novel AAA-ATPase member gene, e plants . Functio


SKN-1, the C. elegans ortholog of mammalian Nrf protein, is a well-known longevity factor, and its activation is observed in several long-lived models. SKN-1 also plays essential roles in xenobiotic and oxidative stress responses. Here, we report deleterious functions of SKN-1 in somatic stress resistance that may impair lifespan. Constitutive SKN-1 activation impairs animal resistance to several stresses, including heat, ER stress and mitochondrial stress, which result from the suppression of DAF-16, another master regulator of longevity. SKN-1 activation abrogates DAF-16 nuclear import and downregulates DAF-16 target genes under stress conditions, while SKN-1 inhibition promotes the expression of DAF-16 targets, even in long-lived mutants. Further, SKN-1 activation induces the expression of vitellogenin proteins, which are required for SKN-1-mediated suppression of DAF-16 and stress resistance. Together, these findings identify detrimental roles for SKN-1 activation in animal health, and more importantly, inspire the rethinking of the complex roles for SKN-1 in aging regulation.The transcription factor  SKN-1, the C. elegans ortholog of mammalian Nrf proteins, is one of these longevity transcription factors. The activation of SKN-1 is observed in several long-lived mutants and is required for lifespan extension  alleles unexpectedly exhibit age-dependent somatic depletion of fat (Asdf)  seeded with E. coli OP50-1 (skn-1(lax120)], SPC227[skn-1(lax188)], CF1553[sod-3p::gfp], TJ356[daf-16p::daf-16a/b::gfp], CF1038 [daf-16(mu86)], VC1772[skn-1(ok2315)/nT1 (qIs51)], DR1572 [daf-2(e1368)], RT130[vit-2::gfp], RB2365 [vit-2(ok3211)], RB2202 [vit-4(ok2982)], RB2382[vit-5(ok3239)], CB4037[glp-1(e2141)], CL2166[gst-4p::gfp], LD1[skn-1b/c::gfp]. The strain SSP171[mtl-1::gfp] was generated by cloning the full length of mtl-1 genomic DNA into pPD95.79 plasmid that was injected into gonad by standard techniques. Extrachromosomal arrays were integrated into genome by UV irradiation. Double mutants were generated by standard genetic techniques.i OP50-1 . The folFor heat shock resistance assay, day 1 adult worms were cultured at 35° for survival analysis unless otherwise indicated. For TBHP (Sigma) resistance, day 1 adult worms were transferred to NGM plates supplemented with 10mM TBHP and incubated at 20° for survival analysis. For DTT resistance, day 1 adult worms were transferred to NGM plates supplemented with 7.5mM DTT and incubated at 20° for survival analysis. For MA (sigma) resistance, day 1 adult worms were transferred to NGM plates supplemented with 34mM MA and incubated at 20° for survival analysis.sod-3p::GFP, MTL-1::GFP or gst-4p::GFP expression, worms were paralyzed with 1mM levamisole and fluorescent microscopic images were taken after mounted on slides. To study the DAF-16 or SKN-1 nuclear localization, DAF-16::GFP or SKN-1::GFP worms were mounted on slides. The levels of GFP nuclear localization were scored. Briefly, no nuclear GFP, GFP signal in the nucleus of anterior or posterior intestine cells and nuclear GFP in all intestinal cells are categorized as low, medium and high expression, respectively.For fluorescent analysis, VIT-2::GFP worms were examined at 20° and other GFP worms were exposed to 31° heat stress for 8-12 hr and then collected for imaging. Microscopic imaging was performed as previously described . To analHT115 bacteria containing specific double-stranded RNA (dsRNA)-expression plasmids were seeded onto NGM plates containing 5mM isopropyl b-D-1-thiogalactopyranoside (IPTG). RNAi was induced at 25° for 24 hr. L1 worms were added to those plates to knockdown indicated genes.skn-1 gof mutant worms were collected, washed in M9 buffer and then homogenized in Trizol reagent (Life Technologies). RNA was extracted according to manufacturer’s protocol. DNA contamination was digested with DNase I (Thermo Fisher Scientific) and subsequently RNA was reverse-transcribed to cDNA by using the RevertAid First Strand cDNA synthesis Kit (Thermo Fisher Scientific). Quantitative PCR was performed using SYBR Green (Bio-Rad). The expression of snb-1 was used to normalize samples.qRT-PCR was performed as previously described  was used to determine the retention time. The Shimadzu GCMS-TQ8040 Gas Chromatograph Mass Spectrometer equipped with SH-Rxi-5sil MS column was used.Fatty acid contents were measured as previously described  tests. The levels of fluorescent micrographs were analyzed by using Chi-square and Fisher’s exact tests. The qPCR and GC-MS data were analyzed by using paired student https://doi.org/10.25387/g3.11919495.Strains are available upon request. The authors affirm that all data necessary for confirming the conclusions of the article are present within the article, figures, and tables. Supplemental Figure S1, S2, S3 and Table S1, S2 have been uploaded to figshare at SKN-1 activation on multiple stress resistance in early adulthood (day 1 adult) skn-1 gof mutants. The skn-1 gof animals exhibited improved resistance to oxidative stress , which was consistent with the dominant role of SKN-1 in xenobiotic response  stress and mitochondrial stress  , a principle negative regulator of skn-1 , while the mRNA levels of daf-16 were not affected , suggesting that SKN-1 does not directly regulate daf-16 transcription. We also compared the published genome-wide expression data (P < 0.001) were the genes that are downregulated when DAF-16 is activated, confirming that a number of genes are oppositely regulated by SKN-1 and DAF-16. As a transcription factor, DAF-16 translocates to the nucleus upon activation. We found that the skn-1 gof mutation significantly suppressed DAF-16 nuclear accumulation under heat stress , a well-established SKN-1 reporter . Germline-deficient worms exhibited strong nuclear signals of intestinal SKN-1::GFP, facilitating the observation of SKN-1 nuclear occupancy. We also observed that SKN-1 nuclear accumulation in germline-deficient worms was abolished by heat stress , indicating the silencing of SKN-1 during heat stress. SKN-1 expression can be activated by DAF-16 overexpression . These data suggest that SKN-1 is suppressed during heat stress.We next examined SKN-1 could promote DAF-16 activity and heat stress resistance. As expected, SKN-1 inhibition, either through a loss-of-function mutation or a RNAi-mediated inhibition, significantly improved animal heat stress resistance  are neutralized and its beneficial effects (promotion of xenobiotic response) become dominant.We next asked whether the inhibition of sistance  and S3A. mutants . RNAi ofl-1::GFP  and S3B. mutants  and S3C  mutants  and S3D,DAF-16 suppression in response to SKN-1 activation? SKN-1 is an important regulator of lipid metabolism . Notably, GC-MS/MS analysis revealed reductions in many fatty acid species, mostly unsaturated ones . Using a vit-2::GFP strain, we found that SKN-1 activation increased the protein levels of VIT-2  activated the DAF-16 reporter sod-3p::GFP  and that the constitutive activation of SKN-1 results in the continual mobilization of OA, which likely accounts for DAF-16 inhibition and compromised stress resistance.What is the possible mechanism for ted ones , indicatof VIT-2 , and theresponse , which padditive . Consist-3p::GFP  and S4B,sistance . Therefoty acids , we suppty acids , and exativation  in wild-mutation ; this miSKN-1 in animal health through the interaction with DAF-16, supporting dual roles for SKN-1 in aging regulation. We propose that the lifespan extension effects of SKN-1 can only be manifested under specific conditions, such as in the context of DAF-16 activation, suggesting that different transcription factors must be coordinately regulated to ensure an optimal outcome for longevity, as in the insulin/IGF mutant and germline-deficient models.In summary, our study reports deleterious functions of


Mimicking ischemia-reperfusion injury, oxygen and glucose deprivation (OGD)-re-oxygenation (OGDR) applied to endometrial cells produces significant oxidative stress and programmed necrosis, which can be inhibited by nuclear-factor-E2-related factor 2 (Nrf2) signaling. MicroRNA (miRNA)-induced repression of Keap1, a Nrf2 suppressor protein that facilitates Nrf2 degradation, is novel strategy to activate Nrf2 cascade.MicroRNA-941 (miR-941) was exogenously expressed in HESC and primary human endometrial cells, and the Nrf2 pathway examined by Western blotting and real-time quantitative PCR analysis. The endometrial cells were treated with OGDR, cell programmed necrosis and apoptosis were tested.MiR-941 is a novel Keap1-targeting miRNA that regulates Nrf2 activity. In T-HESC cells and primary human endometrial cells, ectopic overexpression of miR-941 suppressed Keap1 3′-UTR (untranslated region) expression and downregulated its mRNA/protein expression, leading to activation of the Nrf2 cascade. Conversely, inhibition of miR-941 elevated Keap1 expression and activity in endometrial cells, resulting in suppression of Nrf2 activation. MiR-941 overexpression in endometrial cells attenuated OGDR-induced oxidative stress and programmed necrosis, whereas miR-941 inhibition enhanced oxidative stress and programmed necrosis. MiR-941 overexpression and inhibition were completely ineffective in Keap1−/Nrf2-KO T-HESC cells (using CRISPR/Cas9 strategy). Restoring Keap1 expression, using an UTR-depleted Keap1 construct, abolished miR-941-induced anti-OGDR activity in T-HESC cells. Thus Keap1-Nrf2 cascade activation is required for miR-941-induced endometrial cell protection.Targeting Keap1 by miR-941 activates Nrf2 cascade to protect human endometrial cells from OGDR-induced oxidative stress and programmed necrosis.Video Abstract Postpartum hemorrhage is one of the most common complications in obstetric practice –3, resulIt has been recently shown that cell necrosis can occur via a programmed cell death process –16, callheme oxygenase-1 (HO-1), NAD(P)H:quinone oxidoreductase 1 (NQO1), γ-glutamyl cysteine ligase catalytic subunit (GCLC) [The transcription factor nuclear-factor-E2-related factor 2 (Nrf2) promotes the expression of multiple key antioxidant genes and detoxifying enzymes, protecting cells against oxidative injury –25. Nrf2t (GCLC) , exert pt (GCLC) , 30.Studies show that forced Nrf2 activation in endometrial cells enhances antioxidant activity and mitigates OGDR-induced oxidative injury. We have previously reported that KGF-induced Nrf2 activation efficiently protected endometrial cells from OGDR . MicroRNPuromycin and ploybrene were purchased from Sigma-Aldrich Chemicals Co. . The fluorescent dye JC-1 was provided by Invitrogen Thermo-Fisher . All cell culture reagents and qPCR reagents were obtained from Gibco BRL Co. . The antibodies of the present study were provided by Santa Cruz Biotechnology  and Cell Signaling Tech . The primers, sequences, constructs and virus were designed and provided by Shanghai Genechem Co. , unless otherwise motioned.As described previously , 5, the The fresh human endometrial tissues, acquired from a written-informed consent uterine-bleeding patient  were digested with 0.15% trypsin-EDTA (Sigma) and Collagenase I (Sigma) for 1 h, and then transferred to DMEM/Hams F-12 nutrient medium plus 15% FBS. Tissues were dissolved. Blood vessel cells, immune cells and other non-endometrial cells were abandoned through gravity sedimentation. The remaining human endometrial cells were resuspended and cultured in complete DMEM medium [The pre-miR-941 nucleotide sequence and its anti-sense sequence were synthesized and sequence-verified by Shanghai Genechem Co. Each of the two was ligated to the GV248 construct (Shanghai Genechem Co.). The construct, along with the lentivirus-packing helper plasmids (psPAX2 and pMD2.G ), were cGlyceraldehyde-3-phosphatedehydrogenase (GAPDH) was always examined as the reference gene and the internal control, and the 2Ct−∆∆ method utilized for the quantification of targeted mRNAs. The following mRNA primers were utilized: NQO1 (NM_000903) forward, 5′-CATTCTGAAAGGCTGGTTTG and reverse, 5′-GGCTGCTTGGAGCAAAATAC; HO1 (NM_002133) forward, 5′-GCTACCTGGGTGACCTGTCT and reverse, 5′-GGGCAGAATCTTGCACTTTG; Nrf2 (NM_006164) forward, 5′-TGAGCATGCTTCCCATGAT and reverse, 5′-CTTCTCTAGCCGCTCTGTGG; GAPDH (NM_002046) forward, 5′-CGGAGTCAACGGATTTGGTCGTAT and reverse, 5′-AGCCTTCTCCATGGTGGTGAAGAC. Keap1 (NM_203500) forward: 5′-TACGATGTGGAAACAGAGACGTGGA and reverse 5′-TCAACAGGTACAGTTCTGGTCAATCT. The primers cover exon junction/s, and the amplicons around 90–200 bp. miR-941 was normalized to U6. miR-941 and U6 primers were obtained from OriGene .The human endometrial cells, with the applied treatments, were harvest and the total cellular RNA extracted using TRIzol protocol . We utilKeap1 3′-UTR reporter plasmid  was generated using the same protocol described previously , which w5 cells in each well). Lipofectamine 2000 was utilized for the transfection of 500 nM of the wild-type (“WT”) or the mutant (“Mut”) miR-941 mimics (synthesized by Shanghai Genechem Co.). After 48 h, miR-941 levels were determined by qPCR.Human endometrial cells were seeded into the six-well tissue culture plates . Following the applied treatments, a cell counting kit-8 (CCK-8) kit  was utilized to examine cell viability , with CC5 cells in each well). LDH release to the cell medium, the quantitative marker of cell necrosis [The human endometrial cells were seeded into the 12-well tissue culture plates  for 4 h, mimicking OGD. Thereafter, human endometrial cells were returned back to the complete medium and re-oxygenated. Cells were further cultured for applied time periods.As described previously , 5, huma5 cells in each well). Following the applied treatments, cellular lysates were achieved and quantified [Human endometrial cells were seeded into the six-well tissue culture plates . In OGDR-stimulated cells with mitochondrial depolarization (“∆Ψ”) the JC-1 fluorescent dye shall aggregate, forming green monomers [The human endometrial cells were seeded into the 12-well tissue culture plates (at 0.5 × 10monomers . The det3 cells of each well). Following the applied OGDR stimulation, the superoxide colorimetric assay kit  was applied to examine the cellular superoxide contents. In brief, the superoxide detection reagent (50 μL/well) was added for 15 min under the dark, with the superoxide’s absorbance tested at the test-wavelength of 450 nm [Endometrial cells with the applied genetic treatments were initially seeded onto 96-well tissue-culturing plates (at 3 × 10f 450 nm .5 cells per well). Following the applied OGDR stimulation, the lipid peroxidation assay kit  was utilized to examine cellular lipid peroxidation levels, via the malondialdehyde method. The lipid peroxidation levels, determined by the thiobarbituric acid reactive concentration, were tested and quantified using the previously-described protocol [As reported  endometrprotocol , 49.4 per well of a 96-well plate), with applied genetic treatments, were cultured for 24 h. The NQO1 enzyme activity was tested and quantified in cell lysates using menadione as the substrate.The detailed protocol for testing the relative NQO1 activity in human endometrial cells has been described elsewhere . In brieThe lentiCRISPR-Nrf2-KO-puro construct and the lentiCRISPR-Keap1-KO-puro construct were described early , each waThe Keap1 (with no 3′-UTR region) expression GV248 construct was designed and provided by Shanghai Genechem, transduced to T-HESC cells with miR-941 overexpression. Cells were then selected by puromycin for 10 days, with Keap1 re-expression verified by qPCR and Western blotting assays.P < 0.05 was considered statistically significant.Data were presented as mean ± standard deviation (SD). The repeated-measures analysis of variance (RMANOVA) with Dunnett’s post hoc test for multiple comparisons  were utilized to evaluate statistical significance. The two-tailed unpaired T test (Excel 2013) was carried out to examine significance between two specific treatment groups. http://targetscan.org/) [http://www.mirbase.org/) and miRDB (http://www.mirdb.org/miRDB/policy.html). The prediction algorithms identified miR-941 as a promising candidate that targets the 3′-UTR of Keap1 (at position of 276–283), with context++ score: − 0.59 and the score percentage of 99%   databasecan V7.2 , Fig. 1acan V7.2 , miR-941HO1 and NQO1 [Testing the potential effect of miR-941 on Nrf2 signaling, we found that although Nrf2 mRNA levels  exerted no significant effect on Keap1 expression and Nrf2 signaling in T-HESC cells , infection with lv-pre-miR-941 led to an over 10 fold increase in the expression of mature miR-941  was transduced into T-HESC endometrial cells, and two stable cell lines, “L1” and “L2”, established. As compared to control cells transduced with miRNA antisense control lentivirus (“antaC”), mature miR-941 levels decreased by over 90% in the antagomiR-941-expressing T-HESC cells Fig. a. SignifRNA Fig. c and proRNA Fig. d levels.els Fig. e, but diels Fig. d. Furtheels Fig. f and proels Fig. d expressels Fig. g were siNrf2 mRNA)  Fig. j and k, NA) Fig. j and l, NA) Fig. m. TherefAs miR-941 inhibition suppresses Nrf2 signaling in human endometrial cells, it could intensify OGDR-induced cytotoxicity. To verify this hypothesis, antagomiR-941-expressing T-HESC cells Fig.  were subWe tested whether Nrf2 activation is essential for miR-941-induced endometrial cell protection against OGDR. Nrf2 knockout (Nrf-KO) T-HESC stable cells were established using a CRISPR/Cas9-Nrf2-KO construct , and NrfIf targeting and silencing Keap1 is the primary mechanism of miR-941-induced endometrial cell protection against OGDR, Keap1 depletion should mimic miR-941 actions. To test this hypothesis, the CRISPR-Cas9 method was applied to knockout Keap1 in T-HESC cells (using a previously described protocol ). OGDR-iKeap1 mRNA  tissues. Functionally, miR-941 negatively regulated KDM6B (lysine (K)-specific demethylase 6B) to inhibit HCC cell epithelial-mesenchymal transition (EMT) and cell migration/invasion [, reported that miR-941 levels are significantly higher in acute coronary syndrome patients than those in healthy controls [The expression and potential function of miR-941 in human endometrial cells is unknown. The study by Hu et al.an cells . It has an cells . Zhang einvasion . Bai et controls ., has shown that Keap1-targeting miR-626 activated Nrf2 signaling and protected RPEs from oxidative injury [Recent studies demonstrate that miRNA-induced silencing of Keap1, the Nrf2 suppressor protein, is a novel strategy to regulate Nrf2activation in human cells. In breast cancer cells miR-200a targeted Keap1 to activate Nrf2 signaling cascade . In SH-Se injury . TherefoThe results of this study confirm that miR-941 is a direct and specific Keap1-targeting miRNA, that regulates the Keap1-Nrf2 cascade in human endometrial cells. In T-HESC cells and primary human endometrial cells forced miR-941 overexpression inhibited Keap1 3′-UTR reporter luciferase activity and downregulated Keap1 mRNA/protein expression, subsequently leading to Nrf2 activation. Conversely, Keap1 3′-UTR reporter luciferase activity and expression were elevated in endometrial cells with miR-941 inhibition, whereas Nrf2 activation was inhibited. RNA-Pull down experiments confirmed that miR-941 directly binds Keap1 mRNA in T-HESC cells. Functional studies confirmed that miR-941 exerted significant endometrial cytoprotection against OGDR. Ectopic miR-941 overexpression in endometrial cells largely inhibited OGDR-induced ROS production and programmed necrosis. Thus, miR-941 promotes Nrf2 cascade activation by targeting and silencing Keap1. Therefore, miR-941 expression provides a novel strategy to protect human endometrial cells from OGDR and possible ischemia-reperfusion injuries.Our results support that activation of the Keap1-Nrf2 cascade is absolutely required for miR-941-induced endometrial cell protection. Nrf2 KO, using the CRISPR/Cas9 method, abolished miR-941-induced endometrial cytoprotection against OGDR. Furthermore, CRISPR/Cas9-induced Keap1 KO mimicked miR-941 actions and largely attenuated OGDR-induced cytotoxicity in T-HESC cells. Ectopic miR-941 overexpression and miR-941 inhibition were both ineffective against OGDR in the Nrf2-KO and Keap1-KO T-HESC cells. Significantly, restoring Keap1 expression, using an UTR-depleted Keap1 construct, abolished miR-941-induced endometrial cytoprotection against OGDR. Furthermore, transfection of the miR-941 mimics, with mutations at the binding sites to Keap1’s 3′-UTR, failed to protect endometrial cells from OGDR. These results demonstrate that in endometrial cells miR-941-induced activation of the Keap1-Nrf2 cascade is responsible for protection against OGDR.MiR-941 negatively regulates Keap1 to activate Nrf2 signaling cascade, thus protecting human endometrial cells from OGDR-induced oxidative stress and programmed necrosis.