Hypoxia is a distinguished hallmark of the tumor microenvironment. Hypoxic signaling affects multiple gene expressions, resulting in tumor invasion and metastasis. Quantification of hypoxic status although challenging, can be useful for monitoring tumor development and aggressiveness. However, hypoxia-independent factors such as nonspecific binding and heterogenous probe delivery considerably influence the probe signal thereby disenabling reliable quantitative imaging in vivo. In this study, we designed a self-calibrated activatable nanoprobe Cy7-1/PG5-Cy5@LWHA that specifically detects nitroreductase activity upregulated in hypoxic tumor cells. Dual fluorescence emission of the nanoprobe enables ratiometric calibration and eliminates the target-independent interference. In orthotopic and metastatic breast cancer mouse models, Cy7-1/PG5-Cy5@LWHA demonstrated remarkable hypoxia sensing capability in vivo. Moreover, ratiometric processing provided quantitative hypoxia assessment at different tumor developmental stages and facilitated tumor burden assessment in the metastatic lymph nodes. Therefore, our study demonstrates that ratiometric imaging of Cy7-1/PG5-Cy5@LWHA can be a prospective noninvasive tool to quantitatively monitor tumor hypoxia, which would be beneficial for investigating the fundamental role of hypoxia in tumor progression and for evaluating response to novel anti-hypoxia therapeutics. Furthermore, successful detection of metastatic lymph nodes with the proposed imaging approach illustrates its potential clinical application in assessing lymph node status during surgery.The online version contains supplementary material available at 10.1186/s12951-022-01341-9. Hypoxia, resulting from the rapid proliferation of cancer cells and poor tumor vasculature, is one of the most pervasive features of solid tumors . In ordeOver the past decades, nitroreductase (NTR) has been identified as a biomarker for hypoxia –10. It hth generation, PG5), and the nanoconjugate was modified with low molecular weight hyaluronic acid  to enable cell internalization through CD44. Our results demonstrate that ratiometric imaging of Cy7-1/PG5-Cy5@LWHA enabled quantitative assessment of hypoxia in vivo in orthotopic and metastatic breast cancer models. In vivo imaging results agree with the findings of ex vivo semi-quantitative techniques including immunofluorescence and qPCR. The present study delivers a noninvasive tool to quantify the extent of tumor hypoxia, which would facilitate the study of tumor biology and the development of anti-hypoxia strategies. The successful detection of metastatic lymph node (LN) using this approach further suggests its potential use in LN assessment during surgery.Herein, we designed a self-calibrated NTR activatable nanoprobe Cy7-1/PG5-Cy5@LWHA that enables accurate hypoxia imaging. Activatable fluorescent reporter Cy7-1 was used to sense the NTR activity , and an Hypoxia-activatable NIR dye Cy7-1, an “always-on” fluorophore Cy5, and LWHA were incorporated together using PG5 to form a self-calibrated activatable nanoprobe Cy7-1/PG5-Cy5@LWHA. PG5 was selected as the nanocarrier because of its consistent nanometer size, globular shape, and polyfunctional dendrimer surface . SynthesThe fluorescence emission spectra of Cy7-1/PG5-Cy5@LWHA are presented in Fig. 800) increased with NTR concentration (top to bottom), the nanoprobe concentration also affected the signal intensity (left to right). This mimics the in vivo situation where the nanoprobe concentration in the tumor region is heterogeneous due to the differences in vasculature, interstitial pressure, and other physiological factors that may affect probe delivery. The signal enhancement of I800 is a combined result of NTR activity and probe accumulation. Thus, I800 alone cannot reliably reflect the NTR level. As shown in Fig. 680) was unaffected by NTR (top to bottom), directly reflecting the nanoprobe concentration. After ratiometric calibration, the signal ratio (I800/I680) showed a good linear relationship with the NTR concentration, and was independent of the nanoprobe concentration  for 4 h and fluorescence was subsequently observed with a confocal microscope. To investigate the effect of LWHA modification, cells were also incubated with Cy7-1/PG5-Cy5 (0.05 mg/ml) and imaged under the same conditions. As shown in Fig. Previous studies have shown that elevated NTR activity is a distinguished feature of hypoxic tumor microenvironment, and is found inside hypoxic cancer cells . Thus, t+ breast cancer cells through CD44-mediated endocytosis, and later localized to the endolysosomal compartments in the cytoplasm.To further confirm the internalization mechanism of Cy7-1/PG5-Cy5@LWHA, 4T1 cells were incubated with free LWHA to block CD44 and then incubated with Cy7-1/PG5-Cy5@LWHA. As presented in Fig. 2) was used to induce hypoxia in 4T1 cells [Cy7-1/ICy5 ratio in the cytoplasm of CoCl2-treated cells, demonstrating the sensitivity of the ratiometric approach for detecting hypoxia. Quantitative analysis of three independent batches of cells confirmed that the ICy7-1/ICy5 ratio was significantly higher in hypoxic cells than in normoxic cells (p < 0.0001).To test the performance of Cy7-1/PG5-Cy5@LWHA with respect to imaging hypoxia in living-cell assays, cobalt chloride (CoClT1 cells . HypoxicT1 cells . Cy7-1 w800), Cy5 (I680), and ratio images (I800/I680) of a representative case are shown in Fig. 800 and I680 started to increase at 12 h, reached a plateau at 24 h, and remained stable up to 36 h, before diminishing at 48 h post injection  histopathology and immunofluorescence (IF) analysis of HIF-1α were performed to examine the tissue composition and hypoxia distribution. Representative pathology images of the tumor section outlined in Fig. 800/I680 ratio (solid arrows) correlated with the HIF-1α-positive area in the IF image  was administered intravenously and mice were imaged at 4, 8, 12, 24, 28, 32, 36, and 48 h post injection. Fluorescence images of Cy7-1  were mixed with the nanoprobe  and injected into the left and right flanks of BALB/c mice. In vivo images were acquired 4 h after injection. As shown in Fig. + and hypoxia− tumors was not evident in either Cy7-1 (I800) or Cy5 (I680) fluorescence images. However, in the I800/I680 ratio image, hypoxia+ tumor on the left exhibited a higher signal than the hypoxia− control. Quantitative analysis of five mice demonstrated that the average I800/I680 ratio of hypoxia+ tumors was ~ 11% higher than that of the hypoxia− tumors  and Cy7-1 (I800) could be observed in the tumor area, indicating the accumulation of the nanoprobe regardless of the tumor size. In the example shown in Fig. 680), while the larger tumor  showed notably higher Cy7-1 fluorescence (I800) than the smaller tumor . In the I800/I680 ratio image, the signal from the 7.5 mm tumor appeared to be significantly higher than that from the 4 mm tumor, indicating a higher degree of hypoxia. The hypoxic status of the tumors was further confirmed by IF staining against HIF-1α. As shown in Fig. 800/I680 ratio was compared with the ex vivo IF result. As presented in Fig. 800/I680 ratio from in vivo imaging, with R2 = 0.7717 and p < 0.005.To further validate the imaging strategy in a more clinically relevant setting, we established subcutaneous 4T1 tumors of different sizes on the bilateral flanks of BALB/c mice, representing a serial stage of naturally developed hypoxic tumors. We assumed that tumors with larger sizes would exhibit more severe hypoxia. For in vivo imaging, mice were intravenously injected with the nanoprobe and imaged with the IVIS system 24 h post injection. Similar to the results presented in Fig. 680 fluorescence image. The tumor-associated pLN exhibited higher I680 signal intensity as compared to the control pLN, indicating more probe accumulation in the tumor-associated pLN. As a result, I800 or I680 image alone could not reveal the metastatic status of the node, since signal enhancement could either result from probe accumulation or elevated NTR activity. On the other hand, I800/I680 image rectified the concentration effect, highlighting the tumor-associated pLN. After imaging, LNs were excised and imaged under the same condition. Ex vivo imaging results were comparable to those of in vivo imaging, as shown in Fig. 800/I680 ratio of Cy7-1/PG5-Cy5@LWHA could reflect the amount of tumor cells in the LNs. For each mouse, tumor burden was determined using the equation 800/I680 showed a good linear correlation with the tumor burden in the metastatic nodes, with R2 = 0.6365 and p = 0.0572. To this end, we demonstrated the feasibility of using ratiometric imaging of Cy7-1/PG5-Cy5@LWHA to identify metastatic LNs in vivo. Quantitative imaging also allowed for tumor burden estimation with accuracy comparable to that of qPCR.The microenvironment in the primary tumor can be very different from that in the metastatic site. Nevertheless, it has been discovered that the hypoxic profile of the primary tumor could be preserved in metastatic lesions . TherefoActivatable molecular probes have gained significant interest in recent years because of their superior sensitivity and specificity  . Small-mThis problem is especially prominent in hypoxia imaging, as regions with severe hypoxia are often affected by higher levels of vascular abnormalities and limited probe delivery . In thisIn addition, our results showed that hypoxia imaging successfully identified the metastatic LNs. Previous studies have shown that the hypoxia signatures of the primary tumor may be preserved at the metastatic site . Our resOverall, this study developed a novel imaging strategy combining a self-calibrated activatable nanoprobe with ratiometric image analysis, for the quantitative assessment of tumor hypoxia in vivo. Using this strategy, dynamic changes of hypoxia during tumor growth could be monitored in vivo in real time. The successful detection of metastatic LNs presented in this work further validated the potential clinical translation of this imaging strategy.2O for two days. Purified PG5-Cy5 was retrieved by freeze-drying. Cy7-1 was synthesized in accordance with a previously reported protocol [2O (8 ml). After stirring on ice for 2 h, the mixture was transferred to a 10 kDa ultrafiltration tube  and centrifuged at 5500 rpm for 15 min at 4 °C. Subsequently, Cy7-1/PG5-Cy5 was functionalized with LWHA . Cy7-1/PG5-Cy5 was mixed with LWHA at a mass ratio of 1:1 in ddH2O. The mixture was vortexed for 30 s and incubated at 4 °C for 1 h. Finally, the reaction product was dialyzed against ddH2O overnight to obtain purified Cy7-1/PG5-Cy5@LWHA. To confirm the successful synthesis of the nanoprobe, absorption and fluorescence spectra were measured after each synthesis step. Absorption spectra were measured on a spectrophotometer , and fluorescence spectra were measured using a fluorometer . The hydrodynamic size and zeta potential were measured using a Dynamic Light Scattering Zetasizer . The size and morphology of the nanoprobes were observed by TEM . The composition of the nanoprobe was characterized by Fourier transform infrared spectroscopy .Cy5-NHS  was first labeled with PG5  to form PG5-Cy5. Briefly, 300 mg of PG5 was dissolved in 5 ml of borate buffer (pH 9.0). To this solution, Cy5-NHS (3 mg) prepared in DMF was added and stirred for 24 h at room temperature in the dark. The reaction product was then transferred into a 5 K MWCO dialysis bag  and dialyzed against ddHprotocol . To enca800) and Cy5 (I680) were generated for each ROI in ImageJ. The I800/I680 ratio was calculated and the I800/I680 vs. NTR concentration curve was plotted for each probe concentration.To verify the probe’s response to NTR, 0.25 mg/ml Cy7-1/PG5-Cy5@LWHA in Tris buffer was mixed with 500 2 at 37 ºC. To induce hypoxia in cell culture, 4T1-GFP cells were incubated with 70 µM CoCl2  in serum-free RPMI medium overnight. For confocal imaging, cells were seeded at a density of 50,000 cells in 35 mm glass-bottomed imaging dishes  and cultured overnight. Prior to imaging, cells were incubated with the nanoprobe (0.05 mg/ml) for 4 h and washed three times with PBS . Cells were then incubated with 2 μg/ml DAPI  for 20 min to label the nuclei. To block CD44, cells were first incubated with 0.05 mg/ml LWHA for 2 h. In vitro imaging was performed using a confocal microscope  with a 40 Mouse breast cancer cell lines 4T1 and 4T1 transfected with Green fluorescence protein (4T1-GFP) genes were grown in RPMI-1640 medium  supplemented with 10% fetal bovine serum . Cells were cultured in a humidified atmosphere containing 5% CO2 at 37 ºC. After monolayer formation, different concentrations of the nanoprobe .Cytotoxicity was evaluated using the MTT cell proliferation and cytotoxicity assay kit . 4T1 cells were seeded in 96-well plates and cultured overnight in a humidified atmosphere containing 5% CO6 cells in 100 µ th inguinal mammary fat pad of female BALB/c mice . Tumor growth was monitored for 3–4 weeks post inoculation. Once the tumor size reached 5–10 mm, 200 μl Cy7-1/PG5-Cy5@LWHA nanoprobe (1 mg/ml in PBS) was injected into the mice via the tail vein. Mice were imaged at 4, 8, 12, 24, 28, 32, 36, and 48 h post injection using the IVIS imaging system. The image settings for Cy5 and Cy7-1 were the same as those described above. At 48 h post injection, the mice were sacrificed, and the tumor and major organs were collected and imaged using an IVIS imaging system.Following ethical approval No. IA-202105, all animal experiments were performed in compliance with the guidelines set by the Animal Care Committee at the Institute of Automation, Chinese Academy of Science (license #SYXK(京) 2020–0049). To establish the orthotopic breast tumor model, 4T1 cells  and imaged with the IVIS imaging system 24 h post injection.To investigate the ability of the nanoprobe to quantify tumor hypoxic status, subcutaneous 4T1 tumors of different sizes were established on the flanks of female BALB/c mice. In each mouse, two tumors were grown on the bilateral flanks. The first tumor was implanted on the left flank by subcutaneous injection of 1 5 4T1-GFP cells resuspended in 30 To establish LN metastasis of 4T1 breast cancer cells, 1 Pixel-based ratio images were generated in ImageJ 1.53a according to the following equation:www.abcam.com/protocols). H&E and fluorescence images were acquired using a Pannoramic MIDI scanner (3DHISTECH).Excised tissue was fixed in 10% formalin and embedded in paraffin; 4 μm-thick sections were sectioned with a microtome and stained with H&E. Immunofluorescence was used to evaluate HIF-1α expression. The antibodies and staining reagents used were anti-HIF-1α , 488-conjugated goat anti-rabbit IgG , and DAPI . Immunofluorescence was performed according to an established protocol . Total RNA was isolated according to the protocol provided in www.abcam.com/protocols. To assess gene expression, cDNA was prepared using the Servicebio ®RT First Strand cDNA Synthesis Kit . Fifteen microliter reaction was set up using 7.5 2O. qPCR was performed using the CFX96 Touch Real-Time PCR Detection System . Each sample was tested in triplicate. GAPDH was used as an endogenous control. The relative GFP gene expression level was determined using t-test with Welch's correction was used to determine the statistical difference. For in vivo imaging experiments, the Wilcoxon matched-pairs signed rank test was used to determine the differences between different hypoxia groups. Simple linear regression was used to compare ex vivo HIF-1 800/I680 at 24 h, and to compare tumor burden determined from 800/I680 in tumor-draining LNs. All data are presented as mean ± s.d. Statistical significance was set at p < 0.05.Data analysis was performed using GraphPad Prism ver. 9. For in vitro imaging experiments, an unpaired Additional file 1: Figure S1. FTIR spectrum of Cy7/PG5-Cy5@LWHA.Additional file 2: Figure S2. NTR response of free Cy7-1.Additional file 3: Figure S3. Cytotoxicity of (a) Cy7-1/PG5-Cy5@LWHA and (b) Cy7-1/PG5-Cy5.


Manganese (Mn) as well as iron (Fe) are essential trace elements (TE) important for the maintenance of physiological functions including fetal development. However, in the case of Mn, evidence suggests that excess levels of intrauterine Mn are associated with adverse pregnancy outcomes. Although Mn is known to cross the placenta, the fundamentals of Mn transfer kinetics and mechanisms are largely unknown. Moreover, exposure to combinations of TEs should be considered in mechanistic transfer studies, in particular for TEs expected to share similar transfer pathways. Here, we performed a mechanistic in vitro study on the placental transfer of Mn across a BeWo b30 trophoblast layer. Our data revealed distinct differences in the placental transfer of Mn and Fe. While placental permeability to Fe showed a clear inverse dose-dependency, Mn transfer was largely independent of the applied doses. Concurrent exposure of Mn and Fe revealed transfer interactions of Fe and Mn, indicating that they share common transfer mechanisms. In general, mRNA and protein expression of discussed transporters like DMT1, TfR, or FPN were only marginally altered in BeWo cells despite the different exposure scenarios highlighting that Mn transfer across the trophoblast layer likely involves a combination of active and passive transport processes. Although trace elements (TE) like manganese (Mn) and iron (Fe) are essential to sustain physiological processes, many studies have shown that excessive metal uptake could lead to health issues such as neurodegenerative diseases with metal-induced oxidative stress as a potential underlying pathway ,2. Mn anMn is involved in several processes of fetal development like growth, bone formation, immune function, or neurodevelopment ,12. SincTo identify possible placental TE transfer mechanisms, Duck et al. reviewed Fe transfer across different physiological barriers including the placenta . DiffereTherefore, we investigated placental Mn transfer and cytotoxicity at the villous trophoblast barrier using an in vitro BeWo b30 transfer model. In particular, we focused on elucidating Mn transfer kinetics and underlying translocation pathways like the involvement of relevant transporters  and regulatory proteins , FTH, FTL). As previously described for other tissues, Mn and Fe might share the same transport mechanisms because they both occur in two physiological relevant species ((+II) and (+III)) ,26. Henc2 and FeCl2 was assessed in confluent BeWo b30 cells to determine non-toxic concentration ranges for transfer experiments. Indirect determination of the cell number via Hoechst assay after 24 h showed no effect for concentrations up to 1000 µM of either MnCl2 or FeCl2  [BeWo barrier integrity and tightness was verified by measuring the transepithelial electrical resistance (TEER), sodium fluorescein exclusion, and immunocytochemical staining of adherence junction (γ-catenin) and microtubule proteins (tubulin) as described in Aengenheister et al. . TEER ret shown) .2, 24 h) to 4 ± 2%  with increasing FeCl2 incubation concentration. Concentrations higher than 100 µM lead to a plateau with only 4 ± 1% of applied FeCl2, indicating a different transfer mechanism compared to Mn.Applying Mn to the apical side and quantifying the basolateral Mn amount revealed a time- and concentration-dependent Mn transfer across the BeWo b30 cell layer A,C. Inte2 for 24 h on the apical side resulted in a total crossover of 21.4 ± 4.6% to the basolateral side with a defined permeability of 1.2 × 10−6 ± 3.8 × 10−7 cm/s. Since percental Mn crossover was concentration-independent  and lower at higher FeCl2 concentrations like 500 µM FeCl2 (3.0 × 10−7 ± 1.1 × 10−7)  B.2 was set to 100 µM while various FeCl2 concentrations ranging from 10 µM–500 µM were applied. Simultaneous incubation with 100 µM MnCl2 and 10 µM FeCl2 for 24 h significantly decreased Mn transfer. While without Fe, the basolateral Mn amount was 21.4 ± 4.6% normalized to the applied dose; only 14.2 ± 1.9% were transferred to the basolateral side in combination with 10 µM FeCl2  and 11.1 ± 7.2% vs. 3.7 ± 0.8% (Fe only), respectively  and translational level (10 µM and 100 µM FeCl2)  and protein level (10 µM FeCl2 + 100 µM MnCl2) as well as in DMT1 mRNA expression (100 µM FeCl2 + 100 µM MnCl2) (2 + 100 µM MnCl2)  A,B and DµM FeCl2 C. RelatiµM FeCl2 G and FPNexposure I. As oppreatment E,F. ConcM MnCl2) A–C. DMT1M MnCl2) D. AdditiM MnCl2) H was sigreatment G. FPN1 mpression I was altµM MnCl2 B. The sareatment G. While Mn is known to be essential for fetal development, concerns are rising about consequences for the developing fetus in the case of Mn overexposure in utero. Although it is known that Mn is able to cross the placenta and excess intrauterine Mn may lead to adverse pregnancy outcomes, it is surprising that underlying mechanisms of Mn transfer in placental barrier building cells have not been established yet. This key gap was addressed in this study using human BeWo b30 cells. BeWo b30 cells cultivated on microporous inserts are widely used as a model for the placental barrier because they form a confluent polarized monolayer resembling the structure and function of trophoblast cells in vivo ,34. AddiFe is transported through TfR-mediated endocytosis, which is only unidirectional from mother to child. Once in the endosome, Fe is released from the Tf-Fe complex and subsequently transported into the cytosol from DMT1. If not stored in FTH or FTL, it can be transported to the fetus through FPN . For pla2 and FeCl2 treatment for up to 1000 µM since TEER values as well as capacitance values were not affected by TE treatment. In contrast, Bornhorst et al. showed that barrier building cells of the blood-CSF barrier, for example, are more susceptible to MnCl2 treatment, with 200 µM MnCl2 being sufficient to cause barrier leakage [2 or FeCl2 treatment, which hints that BeWo b30 trophoblasts may have adapted to relatively high levels of metal exposures. Since the placenta possesses lots of mitochondria, ROS accumulation is a constant condition which increases during gestation. Therefore, the placenta is provided with a high antioxidant capacity which allows efficient retention of metal-induced ROS [2 applied in this study are not obtained under physiological relevant conditions in maternal serum or umbilical cord blood but in placental tissue with Mn amounts ranging from 62 ng/g to 89 ng/g dry weight [Firstly, data presented in this work showed that the barrier integrity of the BeWo b30 cell layer was not perturbed by MnCl leakage . Similar leakage . Barrieruced ROS ,45. The y weight ,48,49,502 is one of the used species for supplementation [In food but also in food supplements, the most important oxidation state of Mn is the divalent form. According to the Directive 2002/46/EC of the European Parliament relating to food supplements, MnClentation . Since pTfR1, DMT1, and FPN1 was only slightly altered by Mn treatment. This is also underlining the hypothesis that Mn transfer is not only transporter mediated but likely involves a combination of active, transcellular, and paracellular transfer mechanisms, which needs to be further elucidated in future studies. Transfer experiments conducted in this study pointed out distinct crossover kinetics for Mn or Fe, respectively . ExpressThis can also be confirmed focusing on Mn bioavailability. Cellular Mn concentration does not increase over time, indicating that Mn homeostasis is maintained through effective import and export mechanisms within the cells. However, while the BeWo b30 trophoblast transfer model is recapitulating the most relevant barrier layer for nutrient transfer to the fetus, further studies should be performed using a co-culture model of trophoblasts and endothelial cells to understand the contribution of the endothelial barrier to maternal-fetal Mn transfer ,27,57.2. The 100 µM FeCl2 might be a critical concentration above which the Fe transfer is more tightly regulated to maintain Fe homeostasis and avoid toxicity from excess metal exposure. Additionally, cellular Fe uptake was affected by inhibitor treatment, leading to a significant decreased Fe uptake after inhibition of TfR1 and DMT1 by Ferristatin II and also a trend for a slight increase in cellular Fe in combination of 100 µM FeCl2 with hepcidin, which is involved in FPN regulation . To understand if shared use of transporters is one possible mechanism of transfer interaction, we determined mRNA and protein expression of different transporters, transport proteins, and storage-associated proteins involved in TE transfer and homeostasis. In general, incubation of either Mn or Fe or the combination of both affected mRNA and protein expression only marginally except for protein expression of FTH and FTL. TfR protein expression was significantly downregulated by all tested single Fe conditions and adding 10 µM FeCl2 to 100 µM MnCl2 led to a decreased TfR downregulation compared to the single TE treatments. The same trend was also seen for DMT1 mRNA expression, which was decreased in all conditions except 500 µM MnCl2 and the combination of 10 µM FeCl2 + 100 µM MnCl2. Together, these results indicate that BeWo cells responded to the increased Fe and Mn exposure in order to prevent intracellular accumulation of excess TE. However, quantifying the cellular amount, the cellular Fe concentration following 100 µM FeCl2 was significantly increased in combination with 100 µM MnCl2  and FeCl2  were prepared freshly before the experiment using sterile purified water (18 MΩ). The abbreviations Mn and Fe used in this study refer to both metals in the divalent form (Mn(II) and Fe(II)). For cytotoxicity testing via Hoechst Assay, 31,000 cells/cm2 were seeded in 96-well plates in order to reach confluency after three days [2 and FeCl2 ranging from 100–1000 µM for up to 24 h. In short, Hoechst assay was carried out by fixing cells using 4% formaldehyde followed by a permeabilization step with Triton™ X-100  to allow Hoechst staining  to interact with the DNA of living cells [Stock solutions of MnClree days . One houng cells . 4 cells were seeded in endothelial growth medium MV (EC) supplemented with 1 vial SupplementMix according to the manufacture’s manual  and 1% penicillin/streptomycin on the apical side of microporous inserts . Before cell seeding, inserts were coated using a 50 µg/mL solution of human placental collagen for 1 h at 37 °C . Three days after seeding, the medium was changed 2 h before TE treatment. Barrier tightness was monitored right before and during an experiment by measuring transepithelial electrical resistance (TEER) using the cellZscope® device . Additionally, barrier integrity was proven by sodium fluorescein translocation and staining of the tight junction and microtubule proteins γ-catenin and tubulin as described elsewhere [2 (100–1000 µM) and FeCl2 (10–500 µM) on the apical side. Samples were taken from the apical and basolateral compartment 6, 24, and 48 h after treatment. Permeability coefficients were calculated as followed: P; cm s−1) determined as the quotient of the basolateral amount  and the product of the area of the microporous insert , the apical concentration at the start of the incubation  and the respective time point For transfer experiments, 8.5 × 10lsewhere . For tra3 were measured using an inductively coupled plasma-optical emission spectrometer  or Agilent ICP-MS/MS Triple Quad system . Measurement parameters can be found in the ®-274  and SRM®-1640a . The cellular TE concentration was normalized to the protein amount determined by BCA-assay. To quantify the TE content in cells grown on inserts, the membrane was cut out with a scalpel and washed in ice-cold PBS to remove remaining medium. Cells were stored at −20 °C until lysis. For cell lysis, cells were digested using a lysis buffer consisting of 1 mM TRIS , 0.1 M NaCl , 1 mM EDTA disodium salt , and 0.1% Triton™ X-100. Lysates diluted in 2% HNO2 or FeCl2 on the apical side of the microporous insert. After 6 and 24 h, samples were taken from the apical as well as basolateral compartment and quantified using ICP-OES. Cellular Mn and Fe uptake was also assessed after cell lysis. Transporter inhibition was determined according to single or combined TE incubation as described in 5.3. Furthermore, Hepcidin/LEAP-1 (Human)  was incubated 1 h before and Ferristatin II  simultaneously in combination with MnCl® extraction kit  and RNA yield was determined with Nano Drop One Spectrometer . RNA with absorption ratios A260/A280 and A260/A230 between 1.8–2.2 were considered as pure. For cDNA synthesis, 1 µg RNA was transcribed with a High-Capacity cDNA Reverse Transcription Kit  according to the manufacturer’s protocol. Prior to RT-qPCR analysis with iQTM SYBR® Green Supermix  as fluorescence probe, primer efficiency was validated by a cDNA concentration curve and primer concentrations ranging from 0.2–0.6 µM and product purity was verified by gel electrophoresis. Only primers with an efficiency between 90–120% were used , 10% SDS (w/v), 50% Glycerol (v/v), 0.2 M Tris (pH 6.8), 0.625% Bromphenol blue) were applied for SDS-PAGE. Protein transfer on a nitrocellulose blotting membrane  via tank blotting  was verified using 0.2% Ponceau staining. After blocking membranes in 3% (w/v), non-fat dry milk in 1x Tris buffered saline containing 0.1% (v/v) Tween® 20 (T-TBS) for 1 h at RT, primary antibodies diluted in blocking solution were incubated at 4 °C over night. Recombinant anti-β-actin , anti-Metallothionein , anti-DMT1 , anti-TFRC , anti-FTH1 , and anti-FTL  were used as primary antibodies. Horseradish peroxidase (HRP)-conjugated goat anti-mouse or goat anti-rabbit antibodies  were incubated as secondary antibodies for 1 h at RT. Since the primary antibodies against TfR, FTH, and FTL are very potent and belong to the same species as the first antibody against β-actin, the secondary HRP goat anti-rabbit antibody was diluted 1:2500. Chemiluminescence detected with Amersham Imager 600  was achieved by incubation of immunoblots with Clarity™ Western ECL Substrate . Protein bands were quantified using ImageJ software and normalized to β-actin as loading control. For Western Blot analysis cell pellets, pelletized from culture dishes, were lysed in ice-cold RIPA buffer in combination with sonification using an ultrasonic probe . Protein amounts from 10–30 µg denaturated with 5x Laemmli buffer . Unless otherwise stated, data are shown as mean ± SD and significance values are depicted as *


S-adenosyl-l-methionine enzyme reactions in all domains of life. 5dAdo is also an inhibitor of the radical S-adenosyl-l-methionine enzymes themselves, making it necessary for cells to construct pathways to recycle or dispose of this toxic metabolite. However, the specific pathways involved have long remained unexplored. Recent research demonstrated a growth advantage in certain organisms by using 5dAdo or intermediates as a sole carbon source and elucidated the corresponding salvage pathway. We now provide evidence using supernatant analysis by GC–MS for another 5dAdo recycling route. Specifically, in the unicellular cyanobacterium Synechococcus elongatus PCC 7942 (S. elongatus), the activity of promiscuous enzymes leads to the synthesis and excretion first of 5-deoxyribose and subsequently of 7-deoxysedoheptulose. 7-Deoxysedoheptulose is an unusual deoxy-sugar, which acts as an antimetabolite of the shikimate pathway, thereby exhibiting antimicrobial and herbicidal activity. This strategy enables organisms with small genomes and lacking canonical gene clusters for the synthesis of secondary metabolites, like S. elongatus, to produce antimicrobial compounds from primary metabolism and enzymatic promiscuity. Our findings challenge the view of bioactive molecules as sole products of secondary metabolite gene clusters and expand the range of compounds that microorganisms can deploy to compete for their ecological niche.5-Deoxyadenosine (5dAdo) is the byproduct of many radical  S-Adenosyl-l-methionine , which is formed by ATP and the amino acid methionine, is an essential cofactor of various enzymatic reactions in all domains of life. SAM can serve as a methyl group donor for the methylation of DNA, RNA, and proteins in reactions that release S-adenosylhomocysteine (SAH) as a byproduct . Furthermore, SAM is a source of the 5-deoxyadenosylradical (5dAdo•), which is formed by the activity of radical SAM enzymes , which is released as a byproduct  is also well characterized (B). In the classical and aerobic MSP, MTA is either processed by a two-step reaction by the MTA nucleosidase (MtnN), followed by a phosphorylation by the methylthioribose (MTR) kinase or by the MTA phosphorylase (MtnP). The subsequent reactions consist of a dehydration , enolization and phosphorylation , deoxygenation (MtnD: acireductone dioxygenase), and a final transamination step (MtnE: aminotransferase).yproduct . SAM can enzymes , 3, 4, 5yproduct . Radicalyproduct , 7. Theyyproduct . Promineyproduct , 11, 12.ne cycle . MTA salcterized , 15  , where methylthioribose 1-phosphate isomerase uses MTR-1P, 5dR-1P, and 5dR as substrates .Despite the high abundance of radical SAM enzymes and thereby of 5dAdo, less is known about 5dAdo salvage. se (5dR) , 10. Theous MtnP . Therefoous MtnP . Recentl cluster . The aut salvage . In agrebstrates . Only reP shunt”  —from the supernatant of the unicellular cyanobacterium Synechococcus elongatus PCC 7942 (S. elongatus) (Anabaena variabilis American Type Culture Collection (ATCC) 29413 (A. variabilis), Saccharomyces, and Arabidopsis. It blocks the shikimate pathway presumably by inhibiting the enzyme dehydroquinate synthase  . This cosynthase . Becauseynthesis , 22, thetonensis , 23, butproducts . As from of 7dSh . In vitr of 7dSh .A). Therefore, S. elongatus can synthesize a bioactive compound from the products of the primary metabolism simply by using promiscuous enzymes.In this work, we identified the pathway for 7dSh biosynthesis, which involves a new salvage route for 5dAdo resulting in the release of 5dR and 7dSh into the culture medium A. TherefS. elongatus cultures via a bioassay based on the size of the inhibition zone of A. variabilis exposed to the supernatant of S. elongatus , 5dR gradually accumulated during growth (A and B), whereas 7dSh accumulation only occurred during a later growth phase (C). After 30 days of cultivation, the supernatant contained four times more 5dR than 7dSh. Under ambient air conditions (gray squares), small amounts of 5dR were formed, whereas 7dSh could not be detected (B and C), despite that the optical density of the air-supplied cultures in the final stage of the experiment reached values similar to those of the CO2-supplemented cultures, where 7dSh accumulation could be detected (A). This suggests that the formation of the deoxy-sugars is not only growth phase dependent but also related to a specific metabolic state.Previously, we estimated the content of 7dSh in the supernatant of longatus . To quang growth , A and Bth phase C. After detected , B and Cdetected A. This sS. elongatus as both molecules showed growth inhibition toward the producer strain at elevated concentrations  was synthesized and added at a final concentration of 20 μM to S. elongatus cultures at the beginning of the cultivation. The concentration of labeled (B and C), unlabeled, (D and E), and the total amount of 5dR and 7dSh (F and G) was determined by GC–MS at different time points over a period of 30 days.In our previous work, we reported the it donor . To deteerformed . To unam labeled , B and Clabeled, , D and Eand 7dSh , F and GS. elongatus nor the excretion of unlabeled and intracellular synthesized 5dR and 7dSh was affected by the addition of exogenous 13C5-5dR . We found that 13C5-5dR is taken up by the cultures as its concentration in the supernatant continuously decreased . Already within 2 days, 13C5-7dSh could be detected in the supernatant of these cultures , clearly proving that 13C5-7dSh was formed from the precursor molecule 13C5-5dR. However, only a small amount of exogenously added 13C5-5dR was converted into 7dSh. At the end of the experiment, 10% of the initially applied 13C5-5dR (20 μM) was converted into 13C5-7dSh (∼2 μM). Around 30% of 13C5-5dR remained in the supernatant (6.5 μM). The residual amount is assumed to be metabolized via (an)other pathway(s). Because unlabeled 5dR was excreted at the same time as 13C5-5dR was taken up (B and D), 5dR must be imported and exported in parallel.Neither the growth of C). To prove that 7dSh is formed from 5dAdo salvage in S. elongatus, 5dAdo-feeding experiments were performed, and the supernatants were analyzed by GC–MS . After 4 days, almost all 5dAdo was taken up. A control experiment showed that the rapid decline in the amount of 5dAdo in the supernatant was not caused by the instability of 5dAdo in the medium. Feeding of the cells with 5dAdo immediately led to an enhanced accumulation of 5dR in the culture supernatant (C). After 14 days, 7dSh levels in 5dAdo-supplemented cultures were clearly enhanced compared with control cultures (D), supporting our hypothesis that 5dAdo is a precursor molecule of 7dSh. However, only about half of the supplemented 5dAdo  was converted into 5dR and 7dSh: at the end of the experiment, the 5dR concentration in the supplemented cultures was increased by around 10 μM and that of 7dSh by 2 μM, suggesting additional pathway(s) for 5dAdo salvage.Next, we asked the question where 5dR is derived from. This drew our attention to 5dAdo, a byproduct of radical SAM enzymes . The comFig. 1by GC–MS . Notably quickly , A and Bernatant C. After cultures D, supporS. elongatus, no homologous gene for a MtnN was found, but gene Synpcc7942_0923 is annotated as a MtnP. Therefore, an insertion mutant was generated via the replacement of the gene by an antibiotic resistance cassette (S. elongatus mtnP::specR). Polar effects because of the insertion of the antibiotic resistance cassette were excluded by monitoring the expression of the genes with a semiquantitative RT-PCR (A). A GC–MS analysis of the culture supernatant revealed that the mutant neither excreted 5dR nor 7dSh (C and D). Instead, while undetectable in the supernatant of the wildtype strain, 5dAdo strongly accumulated in the supernatant of S. elongatus mtnP::specR cultures (B). This confirmed that 5dR/7dSh are derived from 5dAdo in a MtnP-dependent manner. Because of the detoxification via excretion, the mtnP::specR mutant escapes the toxic effect of 5dAdo and does not show any growth disadvantage (A). It has previously been reported that a mtnP knockout mutant in Saccharomyces cerevisiae as well as MtnP-deficient mammalian tumor cells excreted MTA (mtnP::specR mutant excretes MTA as well as 5dAdo (E). Since 5dR/7dSh formation strongly depends on elevated CO2 conditions, we measured the amount of 5dAdo and MTA in cultures of the mtnP::specR mutant supplied with ambient air or with air enriched with 2% CO2. However, the amounts of excreted 5dAdo and MTA  were almost identical under both conditions (E). This clearly indicates that 5dAdo salvage via 5dR/7dSh formation and excretion at high CO2 conditions is not triggered by an increased synthesis of the precursor molecule 5dAdo compared with ambient CO2 concentrations. Rather, it appears that 5dAdo is actively metabolized into 5dR/7dSh under elevated CO2 conditions, whereas 5dAdo salvage under ambient CO2 conditions is conducted by (an)other pathway(s). Since the MTA formation is also unaltered (E), we conclude that 5dAdo salvage via 5dR/7dSh formation is not triggered by an enhanced demand of MTA salvage via the MSP pathway.5dAdo is known to be cleaved by either the MtnN or the MtnP , 10, 16.e RT-PCR . Under ce RT-PCR A. A GC–Mnor 7dSh , C and Dcultures B. This cdvantage A. It haseted MTA , 26. Botas 5dAdo E. Since nditions E. This cnaltered E, we convia GC–MS . Only in three of five Synechococcus strains, the deoxy-sugars 5dR and 7dSh were detectable. All the other strains accumulated neither 5dR nor 7dSh. In the freshwater strain Synechococcus sp. PCC 6301, the amounts of 7dSh and 5dR were in a similar concentration range to those in S. elongatus. This is not surprising since the genome of Synechococcus sp. PCC 6301 is nearly identical to that of S. elongatus PCC 7942  and only occurred in wildtype cell extracts but not in extracts of mtnP::specR mutant cells. Therefore, 5dAdo cleavage in S. elongatus is strictly dependent on the presence of the MtnP. Other enzymes, for example, purine nucleosidase phosphorylases  accumulated in the crude extract –like hydrolase superfamily subfamily IA . Polar effects because of the insertion of the antibiotic resistance cassette were excluded by monitoring the expression of the genes with a semiquantitative RT-PCR (A). The wildtype excreted 5dR and 7dSh, whereas the mutant only excreted trace amounts of 5dR and not 7dSh (C and D). Instead, the mutant excreted 5dAdo, which was never detected in the supernatant of the wildtype (B). This clearly shows that the gene product of Synpcc7942_1005 is the major enzyme for the dephosphorylation of 5dR-1P. However, since in the mutant, small quantities of 5dR were detectable, other phosphatases may also contribute to minor 5dR-1P dephosphorylation. In agreement with this, Synechoccoccus sp. PCC 7002, which does not possess a homolog of Synpcc7942_1005, also excreted minor amounts of 5dR and 7dSh.As 5dR-1P is metabolically stable, we assumed that for 5dR formation, a specific phosphatase must be involved. To identify this phosphatase, we analyzed the genome of drolases . Synpcc7amily IA , 31, seee sugars , 33. To e RT-PCR . Under 5e RT-PCR A. The winot 7dSh , C and Dwildtype B. This cmtnP and mtnA), whereas the producer strains possess annotated genes for the whole MSP pathway , can also act as a promiscuous aldolase, when no specific class II aldolase, as DrdA (B. thuringiensis) or Ald2 (R. rubrum), is present . We show that 5dAdo salvage can be achieved by the excretion of 5-deoxyribose and 7-deoxysedoheptulose. 5dR as a product of 5dAdo cleavage was postulated . Thlication  and hereS. elongatus by the activity of promiscuous enzymes leading to the synthesis of the bioactive deoxy-sugars 5dR and 7dSh (A). In brief, 5dAdo is processed by the promiscuous MtnP into 5dR-1P. Under elevated CO2 conditions, this molecule is dephosphorylated to 5dR by the presumably promiscuous phosphatase, the Synpcc7942_1005 gene product, to 5dR, part of which is excreted and further metabolized by the activity of a promiscuous transketolase to 7dSh, which is also excreted to avoid the inhibition of the shikimate pathway , which excretes 5dAdo instead of 5dR/7dSh, demonstrates that 5dR and 7dSh are products of 5dAdo salvage  by a specific phosphoesterase (FdrA) is also conducted by Streptomyces sp. MA37 during the production of a specific secondary fluorometabolite (Although most bacteria possess the enzymes for a two-step reaction of 5dAdo cleavage (MtnN and MTR kinase) , 37, all salvage . The 5dA the MSP , 17, 18.942_1005 . Synpcc7erfamily  and is aerfamily . As theserfamily , 40, we tabolite  . In our previous work, we showed that the affinity of S. elongatus transketolase for 5dR is 100-fold lower than for the natural substrate d-ribose-5-phosphate (13C5-5dR is converted into 13C5-7dSh. 7dSh formation from 5dR is therefore an impressive example how a more potent “derivative” (7dSh) is formed by promiscuous enzyme activity. Interestingly, a promiscuous transketolase reaction was also suggested in later steps of anaerobic 5dAdo salvage in M. jannaschii, in which 5dRu-1P is cleaved into lactaldehyde and methylglyoxal (S. setonensis (not yet sequenced) accumulates much higher concentrations of 7dSh in the supernatant than S. elongatus but no 5dR at all. If S. setonensis synthesizes 7dSh via the same pathway than S. elongatus, the complete conversion of 5dR could be due to a more specific transketolase.In later growth phases, part of 5dR is transformed into 7dSh, which is then also immediately excreted into the supernatant , specific transport systems have to be present, which are probably essential for the survival of the producer strain.In high concentrations, 5dR exhibited toxicity toward the producer strain . 5dR toxngiensis , but theexcreted , B and Dvia 5dR and 7dSh excretion was only observed when cultures were aerated with air supplemented with 2% CO2 (B and C). Since equal amounts of 5dAdo were formed under ambient CO2 as under high CO2 conditions (E), we assumed that under ambient conditions, 5dAdo salvage is conducted via (an)other pathway(s). The occurrence of (an) additional 5dAdo salvage pathway(s) in S. elongatus is underlined by the fact that 5dAdo is not completely metabolized into 5dR/7dSh even under high CO2 conditions  as suggested for organisms that do not possess a specific gene cluster for 5dAdo salvage (E). This indicates that 5dAdo salvage via 5dR/7dSh excretion under high CO2 conditions is not triggered by an increased demand of MTA salvage. It is known that intracellular CO2/HCO3− (Ci) exhibits regulatory functions at the metabolic and transcriptomic levels . ProbabA. variabilis than for the producer strain. A bactericidal effect for A. variabilis was observed at concentrations of 13 μM 7dSh . Main cultures were cultivated in 500 to 700 ml BG11 at 27 °C in flasks that were either aerated with air or air supplemented with 2% CO2. For this purpose, cultures were inoculated with an optical density at 750 nm of 0.2 to 0.5 and then cultivated for the first 3 days at 10 μE . Later, the light intensity was set to around 30 μE. Growth was determined by measuring the absorbance at 750 nm . For feeding experiments, the cultures were supplemented at the beginning of the cultivation with 5dR, [U-13C5]-5dR, or 5dAdo (Carbosynth Ltd) at the respective concentrations  were cultivated as described previously. Synechococcus sp. PCC 7002 was cultivated in a 1:1 mixture of BG11 and ASN III + vitamin B12 (10 μg/ml) (1 medium  supplemeions see  section.0 μg/ml) .S. setonensis SF666 was cultivated for 7 days as described in our previous work (ous work .13C5]-5dR 5 were synthesized in a four-step synthesis based on the literature (13C-labeled compounds are presented in the d-ribose (Sigma) or [U-13C5]-d-ribose 1  in a 4:1 mixture of acetone:methanol with SnCl2×2 H2O (1 eq) and catalytic amounts of concentrated H2SO4 at 45 °C for 20 h. After cooling to room temperature, the mixture was filtered, neutralized with NaHCO3 solution, once again filtered, and the organic solvent was evaporated. The remaining aqueous solution was extracted with ethylacetate, dried over Na2SO4, and evaporated in vacuo to yield the acetonide-protected ribose 2 as a colorless oil .5dR and -5-deoxy-d-ribofuranose 5 as a colorless oil .Deprotecting to the target 13C5]-7dSh was synthesized in a transketolase-based reaction with 5dR or [U-13C5]-5dR as substrate as described in our previous publication . The reSynpcc7942_0932) and glucose-1-phosphatase  in S. elongatus PCC 7942, a spectinomycin resistance cassette was introduced inside the respective gene. An integrative plasmid was constructed in E. coli and then transformed into S. elongatus. For this purpose, flanking regions on both sides of the respective gene were amplified from S. elongatus colonies with primers adding an overlapping fragment . Precultures of these strains, in the following named as S. elongatus mtnP::specR or S. elongatus 1005::specR were cultivated in the presence of 20 μg/ml spectinomycin, main cultures without antibiotic.To create an insertion mutant of the 5-methylthioadenosine phosphorylase . About 200 μl of the supernatant was immediately frozen on liquid nitrogen and stored at −80 °C. Before extraction, the supernatant was lyophilized. For intracellular measurements, the cell pellets were also frozen in liquid nitrogen. Samples were extracted as described in the literature (3/MeOH/H2O in a ratio of 1/2.5/0.5 v/v/v) were either added to 200 μl of the lyophilized supernatant or to cell pellets. Samples were homogenized by vortexing, ultrasonic bath  treatment (10 min), and shaking . After that, the samples were cooled on ice for 5 min and then centrifuged . The supernatant was transferred into a new reaction tube. The pellet was again extracted with 300 μl extraction solvent as described before. The supernatants were pooled, and 300 μl ice-cold water was added for phase separation. The samples were vortexed, incubated on ice (5 min), and then centrifuged . About 900 μl of the upper polar phase was transferred into a new 2 ml reaction tube and dried in a vacuum concentrator  for approximately 4.5 h. The samples were immediately closed and then derivatized as described in the literature (N-methyl-N-(trimethylsilyl)trifluoroacetamide (Macherey-Nagel) was added, and the samples were incubated at 37 °C for 30 min (1.200 rpm). The samples were centrifuged , and 120 μl was transferred into a glass vial with microinsert. The samples were stored at room temperature for 2 h before GC–MS measurement.Culture supernatant was collected by centrifugation of 1.5 ml culture . The recovery efficiency of exogenously added standards (13C5-5dR and 13C5-7dSh) during the extraction of the cell pellets, as well as during extraction of the supernatant, is shown in the GC–MS measurements were performed on a Shimadzu GC–MS TQ 8040  with an SH-Rxi-5Sil-MS column . For GC measurement, the initial oven temperature was set to 60 °C for 3 min. After that, the temperature was increased by 10 °C/min up to 320 °C, which was then held for 10 min. The GC–MS interface temperature was set to 280 °C, and the ion source was heated to 200 °C. The carrier gas flow (helium) was 1.28 ml/min. The injection was performed in split mode 1:10. The mass spectrometer was operated in exposure index mode. Metabolites were detected in multiple reaction monitoring mode. Quantification of the metabolites was performed with a calibration curve of the respective substances , 25 μl of culture supernatant was mixed with 75 μl aqueous solution of 20% MeOH (v/v) + 0.1% (v/v) formic acid. Samples were analyzed on an LC–HR-MS system (Dionex Ultimate 3000 HPLC system coupled to maXis 4G ESI-QTOF mass spectrometer). 5dAdo and MTA were separated on a C18 column with an MeOH/H2O gradient (10%–100% in 20 min). The concentration was calculated from peak areas of extracted ion chromatograms of a calibration curve of the respective standards .For the quantification of MTA and 5dAdo E, 25 μl 2) by centrifugation . The supernatant was discarded, and the pellet was washed with 10 ml fresh medium. The pellet was resuspended in 2.5 ml lysis buffer . Cell disruption was performed in a FastPrep-24 instrument  by adding glass beads (⌀ = 0.1–0.11 mm). Cell debris was removed by centrifugation . About 200 μl of the extract was either used alone or supplemented with 5dAdo  or in combination with potassium phosphate buffer pH 7.5 . The extracts were incubated at 28 °C for 7 h, frozen in liquid nitrogen, and lyophilized. About 100 μl MeOH was added, and the samples were homogenized and centrifuged. About 50 μl was applied on a TLC plate . For the mobile phase, CHCl3/MeOH in a ratio of 9:5 (v/v) with 1% (v) formic acid was used. Visualization was performed at 254 nm (Crude extract assays were performed by harvesting 10 ml of the cultures after 14 days of cultivation (air supplemented with 2% COt 254 nm  or sprayt 254 nm .R. rubrum protein sequence (rru:Rru_A0359) was used as a query sequence, and an e value <10e-20 was used for positive results.Annotations of the different genes were obtained from the Kyoto Encyclopedia of Genes and Genomes database . Also, rkarl.forchhammer@uni-tuebingen.de).All data are presented in the article, in the supporting information, or are available upon request (please contact: Karl Forchhammer, This article contains The authors declare that they have no conflicts of interest with the contents of this article.


Pyrococcus furiosus as a template with high iron atom loading of 9517 under a designated temperature of 90 °C. Compared with the magnetoferritins synthesized at 45 and 65 °C, the one synthesized at 90 °C displays a larger average magnetite and/or maghemite core size of 10.3 nm. This yields an increased saturation magnetization of up to 49.6 emu g−1 and an enhanced specific absorption rate (SAR) of 805.3 W g−1 in an alternating magnetic field of 485.7 kHz and 49 kA m−1. The maximum intrinsic loss power (ILP) value is 1.36 nHm2 kg−1. These results provide new insights into the biomimetic synthesis of magnetoferritins with enhanced hyperthermic efficiency and demonstrate the potential application of magnetoferritin in the magnetic hyperthermia of tumors.Iron oxide nanoparticles have attracted a great deal of research interest in recent years for magnetic hyperthermia therapy owing to their biocompatibility and superior thermal conversion efficiency. Magnetoferritin is a type of biomimetic superparamagnetic iron oxide nanoparticle in a ferritin cage with good monodispersity, biocompatibility, and natural hydrophilicity. However, the magnetic hyperthermic efficiency of this kind of nanoparticle is limited by the small size of the mineral core as well as its low synthesis temperature. Here, we synthesized a novel magnetoferritin particle by using a recombinant ferritin from the hyperthermophilic archaeon  Magnetic nanoparticles (MNPs) have been applied widely in biomedical fields, including diagnosis and therapy, for their unique superparamagnetism and safety within the body . In part3O4, γ-Fe2O3) in a ferritin cage to form a composite, called magnetoferritin. Magnetoferritin has been widely used in targeted drug delivery, magnetic resonance imaging contrast agents, and MHT for its excellent biocompatibility, monodispersity, and chemical/genetic modifiability [f = 190 kHz and H = 5.9–12.0 kA m−1) [f = 805.5 kHz, H = 19.5 kA m−1) was 51.3 W g−1, and the intrinsic loss power (ILP) was 0.17 nHm2 kg−1 [−1, 183 kHz) was only 2.81 W g−1 [Ferritin is an iron-storage protein that is present in most living organisms and plays a significant role in iron management and detoxification for cellular iron homeostasis . The striability ,11,12,13 kA m−1) , but the kA m−1) . To imprHm2 kg−1 . Fantech81 W g−1 . HoweverPyrococcus furiosus by adding theoretical loading factors of 10,000 Fe/cage at temperatures of 45 °C, 65 °C, 90 °C, and 95 °C, named MPfFn-45, MPfFn-65, MPfFn-90, and MPfFn-95, respectively. PfFn is considered to be the most thermostable ferritin so far, with a melting temperature (Tm) of >120 °C or 116.8 °C measured under different conditions [m = 77 °C) [In this work, we synthesized magnetoferritin within a ferritin cage (PfFn) from the hyperthermophilic archaeon nditions . Compare= 77 °C) , PfFn ha= 77 °C) ,24. Vari= 77 °C) ,26. We f2+ and H2O2 (mole ratio = 3:1) were simultaneously and stepwisely added into the PfFn solution under strictly controlled anaerobic conditions (3O4) and/or maghemite (γ-Fe2O3). It is worth noting that further characterizations to distinguish between the two minerals are not provided in this study. We think the magnetite/maghemite ratio of the magnetoferritin core has an extremely slight effect on the hyperthermic efficiency because of the similarity in the crystal structure and magnetic properties of the two minerals [Pyrococcus furiosus, which grows between 70 °C and 103 °C and at pH values between 5 and 9, with optimal growth conditions of 100 °C and pH 7, corresponding to the shortest doubling time of 37 min [The negative stained TEM image shows that PfFn is composed of a homogeneous spherical nanocage approximately 12 nm in diameter a. When tnditions b, the synditions . As shownditions c, the irnditions e. The hinditions d demonstminerals . As seenminerals ,19,23,28f 37 min . In our f 37 min . Howeverf 37 min ,30 resulTo quantify the real loading factors of iron in the ferritin cage of the MPfFn-45, MPfFn-65, and MPfFn-90, the ferritin cage and iron contents were determined by thermogravimetric analysis (TGA) and the Ferrozine method , respect2, -COO-, -OH) on the protein cage can be used for chemical modification for diagnostics and therapeutics [−1 and 1544 cm−1 in the PfFn and MPfFn samples are specific signals of the peptide bond and correspond to amides 1 and 2, respectively [−1) of -COO stretching vibrations of amino acid side chains [−1) of -NH2 and -OH stretching vibrations [As the average core size of MPfFn-90 exceeds the inner diameter of the PfFn cage (~8 nm), it is crucial to ascertain whether the protein cage is perturbed for the reason that the functional groups  (−1) for direct comparison. The initial sample temperature of the experiment was kept at about 22 °C to minimize the influence of the coil temperature on the sample. MPfFn-90 demonstrated an excellent hyperthermia effect, with a temperature rise of about 16.8 °C for 5 min  and 4.7 times higher than that of MPfFn-65 (170.4 W g−1). Furthermore, the iron concentration-dependent heating effect of MPfFn-90 was also investigated. As shown in −1, respectively.To investigate the magnetically induced heating capability of the MPfFn samples, we measured the hyperthermia performance by recording the temperature kinetics of their colloidal dispersions under exposure to a designated AMF ( kA m−1) a. Sampleor 5 min b. In conf = 485.65 kHz and varying intensity is shown in H and followed a square trend  is not only much higher than that of reported magnetoferritins [erritins ,15,24, bcH), confirming that all samples are typical superparamagnetic nanoparticles. The saturation magnetization (sM) of MPfFn-90 (42.3 emu g−1) is much higher than that of MPfFn-45 (29.4 emu g−1) and MPfFn-65 (32.4 emu g−1). Conversely, at a low temperature (5 K) all samples display open hysteretic loops. The cH values of MPfFn samples are in accordance with that of magnetite or maghemite within 200–300 Oe [sM value among these samples at 5 K. A high value of sM can make MNPs convert more electromagnetic energy into heat energy under an applied AMF [B) of MPfFn-90 (261.5 K) is much higher than that of MPfFn-45 (82.9 K) and MPfFn-65 (90.1 K), indicating that MPfFn-90 hasa larger core size. This is in good agreement with the TEM results. Based on the Wohlfarth–Cisowski test for randomly oriented noninteracting single-domain particles, the isothermal remanent magnetization (IRM) acquisition curve and direct current demagnetization (DCD) curve intersect at R = 0.5 [sM value at both 5 K and 300 K, which might be due to the higher synthesis temperature. However, no TB value is observed, indicating a larger core caused by particle aggregation.To better understand the hyperthermic mechanisms of MPfFn samples, the magnetic properties of the samples were investigated, and the results are shown in 0–300 Oe . The MPflied AMF , which c R = 0.5 . In thisB value, the anisotropy constant KA of MNPs with no magnetic interactions can be calculated by [0 is the timescale of the measurement (100 s), τ0 is the microscopic jump time (10−9 s), kB is the Boltzmann constant, and V is the volume of MNPs, which can be estimated using the average particle diameter determined via TEM measurements . The KA values obtained for MPfFn-45, MPfFn-65, and MPfFn-90 are 1.55 × 105, 1.37 × 105, and 1.60 × 105 J m −3, respectively. However, we noticed that the magnetic interactions among the MPfFn samples are nonnegligible, and such interactions influence the real value of TB [B is the anisotropy energy barrier for magnetization reversal, EB = Keff V, and Keff is the effective anisotropy constant. τ0 is the attempt time. The plot of ln (τ) = ln (1/2πν) versus of 1/TMax (the blocking temperatures obtained from the χ″ maxima for different observation times) is a straight line  [eff with size in previous work [eff in our work decreased when the size of MPfFn increased from 7.1 to 7.6 nm but increased drastically when the size increased from 7.6 to 10.3 nm. The shape anisotropy can be ignored because of the similar shape of magnetoferritins. Besides the surface anisotropy affected by the size, crystalline anisotropy is also an important factor that influences the Keff of magnetoferritins. The elevated temperature of 90 °C largely increased the crystalline anisotropy, resulting in a higher Keff.Magnetic anisotropy is also a significant parameter that influences the hyperthermic efficiency of MNPs . With thlated by .(2)KA =ue of TB . Therefoue of TB .(3)τT=τght line a, consisectively b. It is  J m −3) ,51. It i J m −3) . The magous work ,51, and 4)2Fe(SO4)2·6H2O) was purchased from Aladdin . Sodium chloride, Tris, and sodium hydroxide were obtained from Sangon Biotech . All the water used in experiments was supplied by a Milli-Q system . 3.2. Expression and Purification of PfFnAmmonium ferrous sulfate ((NHPfFn gene was transformed into Escherichia coli BL21 (DE3). The E. coli cells were cultured at 37 °C to an OD600 of 0.6 in ampicillin-containing liquid Luria–Bertani (LB) medium and induced expression with 0.5 mM isopropyl-b-d-thiogalactoside (IPTG) overnight at 30 °C. The cells were harvested by centrifugation at 8000 rpm for 8 min, and the pellet was washed once and resuspended in Tris–HCl buffer . The cells were then incubated in lysis buffer  for 2 h at 37 °C. The purification process of PfFn was conducted by heating the lysate at 100 °C for 25 min with constant stirring. The purified proteins were obtained by collecting the supernatant after centrifugation at 20,000 g for 40 min. Finally, the purified PfFn was desalted with 0.1 M NaCl buffer for further synthesis of MPfFn particles. Protein concentrations were determined by a Pierce™ BCA protein assay kit.Recombinant PfFn was prepared as previously described . Briefly−1) in 0.1 M NaCl in reaction vessels were degassed and transferred to an anaerobic chamber. A total of 50 mM (NH4)2Fe(SO4)2·6H2O as iron source and 16.67 mM H2O2 as oxidant were dissolved in degassed water (200 mL) respectively. The Fe3O4 formation reaction can be expressed as,4)2Fe(SO4)2·6H2O solution and 41.46 mL of H2O2 solution were simultaneously added into the prepared PfFn solution at a rate of 50 Fe/(protein min) using a dosing device (800 Dosino). The reaction temperature was maintained at 45 °C, and the pH was stabilized at 8.5 by 100 mM NaOH solution with a pH stat titrator. After 200 min of titration, the reaction was finished. 1 mL of 0.3 M sodium citrate was added to chelate any free iron species. Finally, the MPfFn-45 solution was obtained after centrifugation  for 10 min. Similarly, MPfFfn-65, MPfFn-90, and MPfFn-95 were synthesized with the same procedure under 65 °C, 90 °C, and 95 °C, respectively.Four batches of prepared solution of purified PfFn  were embedded in a Plasma Cleaner HPDC32G treated copper grid and stained with 1% uranyl acetate for 1 min then imaged with a JEM-1400 100-kV TEM . The hydrodynamic sizes of PfFn and MPfFn were determined by DLS  at 25 °C with a scattering angle of 90°. TGA was used to obtain the proportion of ferritin cage of entire magnetoferritin nanoparticles by using a thermogravimetric analyzer (TGA/DSC 3 STARe Mettler Toledo). Samples were heated from 30 °C to 800 °C at 5 °C min−1 under N2 flow at 50 mL min−1. N2 flow is used to prevent the further oxidation of Fe3O4. The iron concentration of the solutions was determined by a ferrozine method [−1.The morphology and crystallography of MPfFn were analyzed by TEM  with an accelerating voltage of 200 kV. The size distribution of the magnetoferritins was measured over 300 particles, and crystallographic orientation of the core was examined by high-resolution TEM (HR-TEM). For negative staining TEM observation, apoferritin and magnetoferritin samples  was determined from the maximum of the ZFC curves. Hysteresis loops were measured in the field range of ±3 T at 5 K and 300 K. The AC susceptibility was measured at frequencies of 1, 10, 100, and 1000 Hz within a temperature range of 5 K to 300 K in a weak field of 0.4 mT.The desalted MPfFn nanoparticles were freeze dried, and magnetic measurements of the dried samples were conducted with a magnetic property measurement system . The IRM acquisition and DCD curves were measured at 5 K within 0–1 T to calculate the magnetostatic interactions. The ZFC and FC curves were measured in a 1.5 mT field from 5 K to 300 K, and the blocking temperature , V is the sample volume (1 mL), and m (Fe) is the total mass of Fe in the sample. dT/dt is the initial slope of the ΔT curve as a function of time, which was determined by fitting the curves of field application time vs. temperature with the Box–Lucas equation, T(t) = A(1−e−Bt) [f·H2).The hyperthermic efficiency of MPfFn samples was measured with a commercial system D5 series device . Each MPfFn aqueous sample (1 mL) with a Fe concentration of 0.5 mg mL(1−e−Bt) . The ILP−1 with a maximum ILP value of 1.36 nH m2 kg−1; the values are significantly higher than those of other magnetoferritins and some MNPs with the same size range. The MPfFn-90 with enhanced hyperthermic efficiency might show good application prospects in magnetic hyperthermia treatment and heat-triggered drug release.Four kinds of magnetoferritins were fabricated by using PfFn as a template under different temperatures , named MPfFn-45, MPfFn-65, MPfFn-90, and MPfFn-95, respectively. The TEM images show that 90 °C is the highest temperature for synthesis of monodispersed magnetoferritin particles through our method. The core size, magnetic properties, and magnetic hyperthermic efficiency of magnetoferritin show an increasing trend with the synthesis temperature increase. The MPfFn-90 shows a highest SAR value of 805.3 W g


The resulting hematite was investigated as adsorbent and photocatalyst for removal of ibuprofen as pharmaceutical waste. Hexagonal flake-like hematite was obtained following calcination at 500 °C with the average size was measured at 1–3 µm. Increasing the calcination temperature to 700 °C transformed the uniform hexagonal structure into cubic shape morphology. Hematite also showed high thermal stability with increasing the calcination temperatures; however, the surface area was reduced from 47 m2/g to 9 m2/g. FTIR analysis further confirmed the formation Fe-O-Fe bonds, and the main constituent elements of Fe and O were observed in EDX analysis for all samples. α-Fe2O3 samples have an average adsorption capacity of 55–25.5 mg/g at 12–22% of removal efficiency when used as adsorbent for ibuprofen. The adsorption capacity was reduced as the calcination temperatures increased due to the reduction of available surface area of the hexagonal flakes after transforming into cubes. Photocatalytic degradation of ibuprofen using hematite flakes achieved 50% removal efficiency; meanwhile, combination of adsorption and photocatalytic degradation further removed 80% of ibuprofen in water/hexane mixtures.Hematite (α-Fe For the synthesis of hematite flake, Pluronic F127 powder was added into HCl solution and stirred in room temperature for 24 h. The gelatin powder was then added into the mixture followed by ferric chloride. The mixture was stirred until a homogeneous solution was formed. The weight ratio of the synthesis materials was used as follows: 1 Pluronic F127: 0.05 gelatin: 5 Ferric chloride: 0.015 HCl. The resulting homogeneous mixture was then poured into an autoclave with Teflon liner and heated at 100 °C for 24 h. The solid was filtered and calcined at 500 °C for 5 h. Finally, the black powder was washed, filtered, and dried overnight at 100 °C. The temperature of calcination was increased to 600 °C and 700 °C to obtain hematite samples which were labeled as Fe2O3-G-xC, where ‘x’ is the calcination temperature. Ferric chloride . XRD pattern was obtained by Philips X’pert XRD  instrument with Cu Ka radiation with a step size of 0.04° and counting time of 10 s. The data were recorded in the 2θ between 5–80°. The crystallinity of iron oxide was calculated based on the ratio of the area of the crystalline peaks to the total area under all peaks as shown by Equation (1) .(1)Crys© 1994–2010, Quantachrome Instruments version 11.0-Semarang, Indonesia. The BJH and SF method were used to determine the size of mesopores and micropores, respectively. The pore size distribution curve was derived from the desorption branch using the Barrett–Joyner–Halenda (BJH) model. The functional group of the materials were characterized through Fourier transform infrared spectroscopy .The morphology of the samples and elemental composition were analyzed using scanning electron microscopy (SEM) ZEISS EVO MA  and coated by Pd/Au and energy dispersive X-ray spectroscopy (EDS) and alsoTransmission Electron Microscope . Surface area of the sample was measured using nitrogen as adsorbate at P/P0–0.99 by the BET method  is the adsorption capacity in equilibrium, whileTo investigate the adsorption profiles of hematite, ibuprofen solution was prepared in water–hexane (1:1 v/v) at 100 mg Lresearch  as shown2 plotted t vs. The linearity value of R2. Photocatalytic degradation was carried out by adding 5 mL of ibuprofen solution (5 mg/L) and 2 mg of hematite into a quartz tube and irradiated with UV light at different time intervals such as 20, 30, 40, 50, 70, 90, and 120 min under continuous stirring. Three separate experiments were carried out: in dark, under immediate light irradiation, and 20 min dark adsorption prior to UV irradiation. The percentage of ibuprofen degradation was calculated asCo and tC represent ibuprofen concentration in the solution before and after the UV irradiation respectively.Photocatalytic activity of iron oxide in the degradation of ibuprofen was determined using 125 W UV light at wavelength of 265 nm in reactor setup shown in 2O3-G) after calcination at 500 °C, 600 °C, and 700 °C. The main peaks for α-Fe2O3 (JCPDS-850897) were observed at 24.08°, 33.10°, 35.56°, 40.96°, 49.52°, 54.24°, and 57.82° with the respective miller field of (012), (104), (311), (113), (024), (116), and (018). The XRD of the synthesized hematite is also in accordance with the literature [2O3 showed a higher intensity as the calcination temperatures increased to 700 °C. The result suggests the conversion of γ-Fe2O3 to α-Fe2O3. At 500 °C, the XRD data also showed a broad peak at 20–30° due to the formation amorphous structure in hematite [, (104), 1, (113),hematite ,33. The 2O3 morphology. Although α-Fe2O3 was calcined at 500 °C in order to remove the template during synthesis, the flake-like structures were retained and stable up to 600 °C. The ability to direct the morphology strongly relied on the presence of F127 and gelatin to form a stable micellar structure [2+-gelatin complex formation stabilized iron from dissolution [2O3.Characterization results obtained from XRD, SEM, and TEM analysis provided insight into the stability of α-Fetructure . The OH tructure . Gelatintructure . Fe2+-gesolution . However2 isotherm showed a typical type-III isotherm with the H1 hysteresis loop without capillary condensation which is the characteristic of a non-porous material. The specific surface area of α-Fe2O3 oxides after calcination of 500 °C was 49 m2/g. The surface area was significantly reduced to 16 m2/g following calcination at 600 °C and further reduced to 7 m2/g when calcined at 700 °C. The pore volume analyzed using BJH method indicated the reduction from 0.161 cc/g to 0.030 cc/g when α-Fe2O3 was calcined from 500 °C to 700 °C. The surface area and the pore volume summarized in 2 adsorption–desorption isotherm plot as well as the pore size distribution plot.Although the synthesized hematite showed the typical non-porous isotherm, the presence of hysteresis loop at high pressures indicated the formation of macropores originated from the interparticle interaction. The plot of pore size distribution in 2O3-G500 occurs on the expose site consisting of dangling Fe bond which forms an interaction with the OH group in ibuprofen [2O3 at 6–15 mg/g) [buprofen . The adsbuprofen , natural15 mg/g) , and com15 mg/g) . Further15 mg/g) , pine wo15 mg/g) , and gra15 mg/g) .t/tq versus t plot for the pseudo-second-order model (2). The pseudo first order plot showed the R2 values for all the sample were within 0.5–0.7. Whereas, for the pseudo second order plot, the R2 value of each calcined samples were closed to 1 thus indicating the adsorption of ibuprofen on Fe2O3-G followed the pseudo-second kinetic model.er model a and t/qer model b were ca−1 and small band at 1624 cm−1 which indicated the stretching vibration of hydroxyl group or physisorbed water. The strong adsorption on hematite samples at 431 cm−1 and 577 cm−1 corresponded to the Fe-O bond vibrations [−1 which was associated with the stretching of the C=O carbonyl group of ibuprofen [2O3-G500, the new adsorption bands appeared at 2929 cm−1 assigned to C-H vibration of ibuprofen, 1634 cm−1 due to C=O vibration, 1527 cm−1 corresponded to C=C vibration and 1064 cm−1 originated from C-O bond in the alcohol functional group in ibuprofen [−1 of ibuprofen to 1634 cm−1 on adsorbed Fe2O3 implied a weakened C=O bond due to interaction with iron oxide.FTIR analysis was carried out to determine the adsorption of ibuprofen on hematite. buprofen . The shi−1 and 577 cm−1. The ratio between the intensity of these peak was calculated to represent the changes on crystallinity of iron oxide following calcination at high temperatures. The results in 431/I577 ratios increased from 1.030 to 1.18 when calcined at 500 °C and 700 °C, respectively , the removal of ibuprofen only reached 28%. However, the combination of adsorption followed by UV irradiation increased the ibuprofen removal up to 80%.Optimization of adsorption condition only achieved 22% of ibuprofen removal on α-Feo 600 nm , its actsorption . It is a2O3 surface to the adsorbed substrate. In photocatalytic degradation without prior adsorption, the photogenerated electron and hole formed mobile OH• radical in the solution via superoxide radicals intermediate (Equations (10)–(13)) [• radical diffuse to oxidize ibuprofen to presumably CO2 and H2O (Equation (15)). There is a possibility of OH• radical to recombine and released heat.The activity of iron oxide as photocatalyst significantly improved the removal of ibuprofen via photodegradation. Preliminary adsorption prior to irradiation has optimized the interaction between ibuprofen and iron oxide. The photodegradation process can take place by direct reaction of hydroxyl radical with the adsorbed ibuprofen on the surface of photocatalysts (Equations (6)–(9)). This process occurs faster due to efficient charge transfer from Fe0)–(13)) . The OH•Adsorption of ibuprofenElectron excitationElectron trapping by surface hydroxyl groupDirect decomposition of adsorbed ibuprofenElectron trapping by dissolved oxygen to form superoxide radicalsWater dissociationGeneration of hydroxyl radicalHole trappingIbuprofen degradation2O3-G) with uniform hexagonal flake morphology has been successfully synthesized using a combination of gelatin as a natural template and F127 as a synthetic template via sol–gel method. SEM analysis showed the formation of uniform hexagonal flake-like structure that was stable after calcination at 500 °C. The transition from hexagonal to cubic structure was observed after calcination at 700 °C. Gelatin as naturally formed polymer showed a potential as structure directing agent for the formation of a highly stable iron oxide with uniform structures. The adsorption capacity of Fe2O3-G as adsorbent for ibuprofen was determined at 55 mg/g when using the hexagonal flake-like iron oxide. Although the calcination at 700 °C produced a high purity iron oxide, the adsorption capacity and removal efficiency were significantly reduced due to the formation of low surface area cubic crystallites. The efficiency of the adsorption was strongly dependent upon the surface area of iron oxide. The combination of photocatalytic degradation and adsorption for the removal of ibuprofen using iron oxide resulted in the increase of removal efficiency to 80% under UV light irradiation.Iron oxide (Fe


However, how low NAD+ condition in cells, which mimics aged or pathophysiological conditions, affects the circadian clock is largely unknown. Here, we show that low NAD+ in cultured cells promotes PER2 to be retained in the cytoplasm through the NAD+/SIRT1 axis, which leads to the attenuated amplitude of Bmal1 promoter-driven luciferase oscillation. We found that, among the core clock proteins, PER2 is mainly affected in its subcellular localization by NAD+ amount, and a higher cytoplasmic PER2 localization was observed under low NAD+ condition. We further found that NAD+-dependent deacetylase SIRT1 is the regulator of PER2 subcellular localization. Thus, we anticipate that the altered PER2 subcellular localization by low NAD+ is one of the complex changes that occurs in the aged circadian clock.The circadian clock possesses robust systems to maintain the rhythm approximately 24 h, from cellular to organismal levels, whereas aging is known to be one of the risk factors linked to the alternation of circadian physiology and behavior. The amount of many metabolites in the cells/body is altered with the aging process, and the most prominent metabolite among them is the oxidized form of nicotinamide adenine dinucleotide (NAD NAD+ acts as a coenzyme for enzymes which are involved in energy metabolism and homeostasis pathways such as glycolysis, TCA cycle, and oxidative phosphorylation  and poly(ADP-ribose) polymerases (PARPs) to regulate a wide array of cellular processes such as survival/cell death, circadian clock, and aging  is the rate-limiting enzyme and converts nicotinamide into nicotinamide mononucleotide (NMN), the NAD+ precursor  can ameliorate many age-associated pathologies, thereby leading to healthy longevity ates CLOCK  was provided by the Mitsubishi Corporation Life Sciences Limited, Japan. Anti-Per2 (PM083), anti-Lamin B1 (PM064), anti-Myc-tag (M047-3), and anti-DDDDK-tag (M185-3) were purchased from the MBL, Japan. Anti-α-tubulin (T6074) and the secondary anti-mouse antibody Alexa Fluor Plus 488 (A32723) were purchased from the Sigma-Aldrich and Thermo Fisher Scientific, respectively. PAR/pADPr antibody was purchased from the R&D Systems. Anti-Bmal1 (ab93806) and anti-acetyl Bmal1 (Lys538) were purchased from the Abcam and Merck, respectively. Secondary anti-mouse (NA931V) or rabbit (NA934) antibodies conjugated with HRP were purchased from the GE Healthcare. Myc-mClock/pcDNA3 was subcloned from Myc-mClock/pSG5 as described . 5xMyc-m2 in a humidified incubator. Cells were treated with FK866/GMX1778 with or without 1 mM nicotinamide mononucleotide for 24 h and harvested for measuring NAD+ amount, performing qPCR, immunofluorescence, and western blotting. For experiments analyzing circadian oscillations of clock genes, luciferase, and PER2 protein, cells were pretreated with FK866 for 24 h before Dex synchronization and also treated with FK866 after synchronization.NIH3T3 cells were cultured in DMEM–4.5 g/L glucose  supplemented with 10% FBS (Sigma) and antibiotics  at 37°C and 5% CO+ was measured by an HPLC system, an Agilent 1260 Infinity Binary LC System with guard  and analytical  columns, following the protocol reported previously  and cultured for 48 h. The cells after washing twice with phosphate buffered saline (PBS) were fixed with 1 ml of 4% paraformaldehyde for 15 min with shaking at room temperature. Then, cells were washed three times with PBS and permeabilized with 1 ml of 0.5% Triton X-100 for 10 min with shaking at room temperature. After blocking the cells with 1% bovine serum albumin (BSA) for 30 min at room temperature, the cells were washed three times with PBS and incubated with Myc antibody diluted with 1% BSA/PBS for 1 h at room temperature. Then, cells were incubated with secondary antibody conjugated with Alexa Fluor Plus 488 diluted with 1% BSA/PBS for 1 h at room temperature. Nuclei were also stained with 1 μg/ml Hoechst 33342  to justify subcellular localizations of clock proteins. Fluorescent images were acquired with a confocal laser scanning microscope , and subcellular localizations of clock proteins were counted at least 100 cells. We interpreted the result for nuclear localization of PER2/CRY1 (N) as positive when fluorescence intensity of PER2/CRY1 signal that overlapped with Hoechst 33342 signal was higher than the fluorescence intensity of PER2/CRY1 in the cytoplasm  placed on a six-well plate so as to have 4 × 10ytoplasm . Meanwhig for 5 min at 4°C, and resuspended in 50 μl lysis buffer . Cells were vortexed for 10 s and allowed to stand on ice for 10 min, and the same step was repeated again to extract cell components other than the nuclear fraction. The nuclear fraction was washed three times with lysis buffer, added with 50 μl RIPA buffer , vortexed for 30 s, and then allowed to stand on ice for 20 min. The nuclear fraction was centrifuged at 15,000 × g for 5 min at 4°C, and the supernatant was collected as a nuclear extract. Total cell extract was obtained by extracting cells with RIPA buffer.NIH3T3 cells treated with or without 7.5 nM FK866 for 24 h in a 10-cm dish were synchronized with 100 μM dexamethasone for 1 h. Synchronized cells after 24–44 h were collected every 4 h. Cells were washed twice with PBS and harvested by cell scrapers, centrifuged at 300 × Per1 firefly reporter, 50 ng of EF1a Renilla reporter, and indicated expression vectors using FuGENE HD (Roche). Samples were prepared using a Dual-Luciferase promoter assay kit (Promega) according to their instructions and analyzed by a multiwell luminescence measurement plate reader . Firefly luciferase activity of the reporter was normalized by the activity of Renilla luciferase under control of EF1α promoter.NIH3T3 cells seeded in a 24-well plate were co-transfected with 500 ng of Bmal1-luc/NIH3T3  and 34.5 ± 9.2% , respectively. Furthermore, GMX1778 treatment also changed the ratio of PER2 subcellular localization from 22.2 ± 0.5 to 26.3 ± 0.1% in the cytoplasm (p = 1.0 × 10–4) and from 62.0 ± 1.9 to 50.0 ± 0.6% in the nucleus (p = 2.0 × 10–4)  and 67.1 ± 8.5% , respectively (p = 4.8 × 10–3) and nuclear localizations were 71.8 ± 2.2 and 81.7 ± 1.3% (p = 8.6 × 10–3), respectively  . Importa × 10–4) . These rectively , indicatectively . Cytoplaectively . The altp = 0.011) when PER2-NLS was co-expressed, whereas the ratio of nuclear CRY1 was decreased  when PER2-NES was co-expressed , C-terminal-tagged nuclear localization signal (-NLS), or C-terminal-tagged nuclear export signal (-NES)  was co-expressed . These rxpressed . Even whxpressed . The res+ amount was next investigated  or 5 μM EX-527 (p = 4.0 × 10–4) and 33.5 ± 2.1% (p = 1.9 × 10–4), respectively, and decreased nuclear PER2 from 42.7 ± 5.5 to 28.7 ± 3.8% (p = 3.0 × 10–4) and 30.5 ± 4.2% (p = 1.8 × 10–4), respectively (p = 0.56) and 54.6 ± 3.3% (p = 0.10), respectively] . Moreover, acetyl mimetic PER2K680Q had no effect on PER2 subcellular localization. These mutant PER2 experiments suggest that K680 of PER2 is not the responsible lysine to regulate subcellular localization by SIRT1, although we cannot deny the possibility that acetyl mimetic mutant of PER2K680Q does not completely substitute the functions of acetyl-K680 of PER2.To further investigate whether the acetylation status of lysine 680 residue of PER2, where PER2 is acetylated and deacetylated by SIRT1 , affects+, which was induced by the pharmacological inhibition of NAMPT, promoted PER2 subcellular localization from the nucleus to the cytoplasm and increased and decreased the expressions of E-box- and RORE-regulated circadian genes, respectively, leading to the attenuated Bmal1-luc circadian oscillation. Furthermore, we demonstrated that SIRT1 was responsible for NAD+-dependent PER2 subcellular regulation, and suggested that lysine 680 residue on PER2 is not responsible for SIRT1-regulated PER2 subcellular localization.In this study, we found that low intracellular NAD+ is a hallmark of senescence/aging  because of differences of three-dimensional structures triggered by the acetylated form of lysine residue and substituted glutamine residue. Further experiments including structural biological experiments will be needed to elucidate which lysine residue is responsible for subcellular localization of PER2 regulated by SIRT1.In this study, we investigated whether acetylation of lysine 680 residue on PER2 is responsible for PER2 subcellular localization; however, neither acetyl mimetic mutant (K680Q) nor non-acetyl mimetic mutant (K680R) of PER2 changed subcellular localization such as low NADus sites . This su+/SIRT1 axis. Intriguingly, we have reported that SIRT1 deacetylates lysine 9/14 residues of histone H3 on the promoters of E-box-regulated genes to repress their expressions epigenetically (+/SIRT1 axis coordinates the transcriptional repression of E-box-regulated genes by different ways. In addition to the coordinated mechanisms for E-box-regulated genes, RORE-regulated genes might also be coordinated by the NAD+/SIRT1 axis. It has been reported that NAD+ activates Bmal1 transcription by SIRT1 through the deacetylation of PGC1α (+ condition increased Reb-erbα/β which in turn might repress Bmal1 gene expression. These coordinated Bmal1 gene regulations by the NAD+/SIRT1 axis presumably regulate the amplitude of Bmal1 circadian oscillation, namely the low NAD+ condition or decreased SIRT1 activity attenuates the amplitude of Bmal1 circadian oscillation. Therefore, these coordinated transcriptional regulations of E-box- and RORE-regulated circadian genes by the NAD+/SIRT1 axis might interlock circadian negative feedback loops.Our current results suggest that PER/CRY-dependent repression against E-box-regulated genes might be dependent on the NADetically . These rof PGC1α , while w+ retains PER2 in the cytoplasm, some studies have demonstrated that the NAD+/SIRT1 axis regulates PER2 properties; PER2 in –/–Sirt1 cells is more stable (In addition to our finding that low NADe stable  and locae stable , while Pe stable . Our phaThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.AA performed and analyzed the experiments. YN designed the research and wrote the manuscript. TS, YuF, HY, and YoF provided the reagents. KS assisted with the experiments and reviewed the manuscript. TM and YB designed the research and reviewed the manuscript. All authors contributed to the article and approved the submitted version.TS and YF were employed by the Mitsubishi Corporation Life Sciences Limited. The remaining 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.