Gramicidin A (gA) forms several convertible conformations in different environments. In this study, we investigated the effect of calcium halides on the molecular state and antimicrobial activity of gramicidin A. The molecular state of gramicidin A is highly affected by the concentration of calcium salt and the type of halide anion. Gramicidin A can exist in two states that can be characterized by circular dichroism (CD), mass, nuclear magnetic resonance (NMR) and fluorescence spectroscopy. In State 1, the main molecular state of gramicidin A is as a dimer, and the addition of calcium salt can convert a mixture of four species into a single species, which is possibly a left-handed parallel double helix. In State 2, the addition of calcium halides drives gramicidin A dissociation and denaturation from a structured dimer into a rapid equilibrium of structured/unstructured monomer. We found that the abilities of dissociation and denaturation were highly dependent on the type of halide anion. The dissociation ability of calcium halides may play a vital role in the antimicrobial activity, as the structured monomeric form had the highest antimicrobial activity. Herein, our study demonstrated that the molecular state was correlated with the antimicrobial activity. Bacillus brevis during sporulation [+, Na+ and K+ across membranes [Gramicidin is an antimicrobial peptide that is a byproduct of rulation . Gramicirulation ,3. Gramirulation ,5,6,7. Irulation ,6,8. Thirulation . These drulation ,10,11,12rulation ,11,12. Tembranes ,12,13,14Gramicidin’s double-stranded helical dimer can interact with monovalent or divalent cations ,19,20,21Conversion between conformers has been studied in lipid environments ,23,24,25+ synthesis [Gramicidin A is a short peptide antibiotic effective against bacteria and fungi . The antynthesis . Recentlynthesis . HoweverIn the present study, we revisited and characterized the conformational and molecular states of gA in the presence of different calcium and halide ions using circular dichroism (CD), nuclear magnetic resonance (NMR) and state-of-art mass spectroscopy, and found that gramicidin A exists in two molecular states in the presence of calcium halides. In State 1, the main effect of calcium halides on gramicidin A is to convert the conformers into one single conformation. In State 2, calcium halides can induce a dissociation of the gramicidin A dimer into monomers and the denaturation of the gramicidin A monomer. A new finding revealed that the different halide anions may play a role in the molecular state and conformation of gramicidin A. The molecular state and conformation induced by calcium halide can further influence the antimicrobial activity of gramicidin A.Gramicidin A adopts several conformers in methanol ,6,7,8. W2 and CaBr2. For CaBr2, the CD spectra below 220 nm became noisy at the concentration ≥ 0.2 mM due to the strong adsorption from the Br− anion. Therefore, we were unable to collect CD spectra from 200 to 220 nm. A similar result was also obtained when we titrated gramicidin A with CaI2. An even stronger absorption arising from the I− anion prevented us from collecting any reasonable CD spectra. Hence, we were unable to study the effect of CaI2 on the gramicidin A conformation using CD spectroscopy.2 and CaBr2. The intensity of the two negative peaks increases with an increase in the salt concentration and reaches its maximum at around 10 mM. At salt concentrations > 10 mM, these two negative peaks gradually converted into a single broadened positive peak located at ~225 nm. The change in intensity was different between the Cl− and Br− anions. For CaCl2, the two negative peaks converted into a positive peak at a salt concentration higher than 300 mM, whereas the two negative peaks did not convert into a positive peak at 400 mM for CaBr2. 2 and CaBr2. The CD results suggest that gramicidin A may adopt different conformations in these two states for CaCl2 and CaBr2.At salt concentrations ≤ 10 mM, the CD spectra showed similar spectral patterns, with the two negative peaks located at 208 and 228 nm for both CaCl2, CaBr2 and CaI2 are shown in The conformation of gramicidin A in the presence of calcium halide salts was examined using 1D proton NMR spectroscopy. The 1D proton NMR spectra of gramicidin A in the presence of CaCl2, CaBr2 and CaI2, indicating that a mixture of conformers is present under this condition. Unlike the resonance pattern of the formyl group at a concentration of 0.2 mM, the resonance of the formyl proton became a single peak at a concentration of 10 mM for CaCl2, CaBr2 and CaI2, indicating that a single conformation was present. The dispersion of these NMR spectra was well resolved at 5.0~10.0 ppm compared that of to the spectra obtained at a concentration of 0.2 mM. The well-resolved peaks at 10–10.5 ppm were not well separated compared to those at 0.2 mM. According to a previous study [At a low salt concentration, i.e., 0.2 mM, the proton signal of the formyl group was multiple resonance for CaClus study , gramici2, the individual peaks in the NMR spectrum were less dispersed compared to those in the NMR spectrum of gramicidin A at 10 mM CaCl2. A previous study has hypothesized that gramicidin A may possibly exist in a mixture of unstructured and structured monomers under this condition [2 was even more separated, and peaks for a specific type of amino acid even coincided, indicating that gramicidin A may be further denatured rather than forming a structured/unstructured monomer in CaBr2 and CaI2. Clearly, the types of halide anions have different impacts on the conformation of gramicidin A at high salt concentrations.At a high salt concentration, i.e., 200 mM, the NMR spectra of gramicidin A were very different compared to those obtained at 10 mM or 0.2 mM and dependent on the different types of halide anion as well. At a concentration of 200 mM CaClondition . The NMR2 and CaBr2 at various concentrations, respectively. The mass spectra of gramicidin A treated with CaI2 are delineated in To further characterize the molecular state of gramicidin A at the different concentrations of calcium salt and with the different types of halide anion, we applied MALDI-TOF mass spectrometry to study the molecular state of gramicidin A. m/z 1902–1905 were assigned as (gA dimer/Ca)2+, while the peaks at m/z 1920–1921 were assigned as (gA monomer/Ca/H)+. In the case of gA/CaCl2, a new peak at m/z 1936 was assigned as (gA dimer/Ca+2Cl)2+ or (gA dimer/Ca+4H2O)2+, while this peak was not observed in the cases of CaBr2 (2 (2 at concentrations higher than 100 mM (The peaks at of CaBr2  and CaI2CaBr2 (2 . Due to n 100 mM .2)/1920. The molar ratios of the dimer/monomer are summarized in 2 and CaBr2, the majority of the gramicidin A at a low salt concentration, i.e., 0.2 mM, was in the dimeric state. The molar ratios of the dimer/monomer were 6.0 and 2.0 for CaCl2 and CaBr2, respectively. At medium salt concentrations, i.e., 10 and 40 mM, the molar ratios of the dimer/monomer for gA in the presence of CaCl2 were two- to four-fold higher than those for gA in the presence of CaBr2. At a higher salt concentration, i.e., 100 mM, gramicidin A was in the monomeric state for both CaCl2 and CaBr2. In general, CaBr2 induced the formation of the monomer more efficiently than CaCl2 did. This effect was even more profound for CaI2, as most gramicidin A species were monomeric, even at very low CaI2 concentrations. The value of the dimer/monomer ratio might be underestimated. As a α-cyano-4-OH cinnamic acid (CHCA) matrix dissolved in 50% acetonitrile/50% distilled H2O was mixed with the sample in methanol, the acetonitrile/water solvent system favored monomer formation. This may shift the equilibrium toward the monomer and underestimate the dimer/monomer ratio. However, this factor does not affect the conclusion.The molar ratios of the dimer/monomer of gramicidin A in the presence of different salt concentrations and halides were calculated using the intensity of 1905 at all growth phases. In the lag phase, the growth of S. aureus was significantly inhibited by gramicidin A with or without CaCl2. In the exponential phase, the antimicrobial activity of gramicidin A was significantly inhibited in the pretreatment with 100 mM CaCl2 or in the absence of CaCl2 compared to that with the pretreatment of 0.2 and 10 mM CaCl2. In the late-exponential-to-stationary phase, the antimicrobial activity of gramicidin A in the pretreatment with 100 mM CaCl2 was more effective than that in the pretreatments with other CaCl2 concentrations. The antimicrobial activity of gramicidin A was similar in CaBr2. The most effective concentration regarding antimicrobial activity for CaBr2 was 10 mM rather than 100 mM  and CD spectroscopic techniques ,19,20,332 by Wallace and colleagues [2, CaBr2 and CaI2, indicating that gramicidin A may exist in two molecular states, State 1 at salt concentrations ≤ 10 mM and State 2 at salt concentration > 10 mM. The observations obtained with CD and NMR are consistent with the fluorescence spectroscopic studies. In the fluorescence spectra, the intensity increased with an increase in the salt concentration, demonstrated a redshift in wavelength at concentrations ≤ 10 mM, and decreased with an increase in concentration with a blueshift in wavelength at concentrations > 10 mM. These results reinforce that there are two states for gramicidin A upon the titration of calcium halides.Our results obtained from CD and NMR spectroscopies are similar to those from the studies in CaCllleagues ,19. Acco2+ cations. The 1D NMR spectra of gramicidin A at 10 mM showed a similar resonance pattern at the amide proton region for CaCl2, CaBr2 and CaI2. These NMR spectra are similar to those in a previous NMR study [From the CD and NMR spectra, we demonstrated that the main effect of calcium salt is to drive a mixture of conformers into a single species in State 1. This is consistent with the previous study by Chen and Wallace . In addiMR study , suggest2 and CaBr2 but not for CaI2. The dimer content decreased with an increase in the salt concentration, indicating that the addition of the calcium salt drove a dissociation of gramicidin A dimers into monomers. The dissociation of the gramicidin A dimer was more effective with CaI2 or CaBr2 than with CaCl2, suggesting that the dissociation is highly dependent on the type of halide anion. The order by dissociation ability is I− > Br− > Cl−.The most interesting findings in the present study are from the mass spectroscopy analyses. The analyses of the mass spectra for all calcium halides suggest that the molecular state of gramicidin A is a mixture of the dimer and monomer in State 1. The major form of gramicidin A exists in a dimeric form for both CaClm/z 1936 assigned as (gA dimer/Ca++2Cl)2+ only appeared in the mass spectrum of CaCl2 but not in the cases of CaBr2 and CaI2. A similar observation was reported by Zhou and colleagues [−, possibly inside the pore, but not with Br− or I−. As the ionic radii of Br− and I− are larger than that of Cl−, both the Br− and I− anions are too large to accommodate them inside the dimer pore of gramicidin A. The interaction of Cl− ions with gramicidin A may stabilize the dimer and reduce the dimer-to-monomer conversion rate. Therefore, the molar ratio of dimer/monomer in CaCl2 is much higher than the molar ratios in CaBr2 and CaI2. However, the exact reason needs to be further verified with a detailed structure.The reason for this phenomenon is not apparent. A possible reason may be the ionic radii of the halide anion. A peak at lleagues . These r2, indicating that gramicidin A may adopt a different conformation. Chen and Wallace proposed that gramicidin A may be a mixture of unstructured and structured monomers in this condition. Analyses of the mass spectra indicate that the molar ratios of dimer/monomer were all less than 1 for most calcium halides except for 40 mM CaCl2. Our results support the augment that the molecular state of gramicidin A should be a monomer in State 2. Similarly, the formation of monomers is also anion-type dependent.In State 2, our CD spectra are also similar to those in a study by Chen and Wallace . The two2. This is consistent with a previous study by Chen and Wallace [2 was more unstructured compared to that in 200 mM CaCl2. The unstructured effect was even more profound with the addition of 200 mM CaI2, indicating that Br− and I− anions may induce denaturation in gramicidin A. The denaturing effect of halide anions on gramicidin A is well correlated with the trend of the dimer-to-monomer dissociation of gramicidin A: I− > Br− > Cl−.We examined the structural state of gramicidin A using NMR spectroscopy. The results showed that the effect of halide anions on the gramicidin A’s conformation was very different. Gramicidin A is a mixture of unstructured and structured monomers in the presence of 200 mM CaCl Wallace . The con− and I−, may play a role in the denaturation of gramicidin A. The denaturation ability of halide anions, particularly Br− and I−, could be taken to account for the nature of the dissociation of gramicidin A.The denaturation of gramicidin A induced by halide was further confirmed by the fluorescence spectroscopy. During protein unfolding, the fluorescence spectrum usually undergoes a blueshift in the maximum wavelength and a decrease in the intensity ,32,35. I2. At 400 mM CaCl2, the antimicrobial activity of gramicidin A was significantly inhibited. The antimicrobial activity of gramicidin A is in the order 100 mM CaCl2 >> 0 mM CaCl2 > 10 mM CaCl2 ≈ 0.2 mM CaCl2 >> 400 mM CaCl2. The molecular states of gramicidin A/CaCl2 complexes in methanol may be changed when they are added to culture medium, as the addition of the gramicidin A in methanol into culture medium may drive the conversion of dimers into monomers. Therefore, the antimicrobial activity may have been overestimated in the present study. However, this does not affect the present conclusion.The antimicrobial activity of gramicidin A increased with an increase in the calcium concentration but not for 400 mM CaCl2 concentration suggest that the monomeric gramicidin A had the most effective inhibitory ability against bacterial growth. A previous study by Jadhay et al. suggested that gramicidin A in channel form mediated the most effective antimicrobial activity against Gram-positive bacteria [2, the main form of gramicidin A is monomeric. Gramicidin A in the presence of 100 mM CaCl2 can readily form ion channels in bacterial cell walls and, hence, shows the most effective antimicrobial activity. On the other hand, the major form of gramicidin A is dimeric in 0, 0.2 and 10 mM CaCl2. When bacteria are treated with gramicidin A in these conditions, it takes time to convert the dimers into monomers. Therefore, gramicidin A in these conditions was less effective in terms of antimicrobial activity when compared to the case at 100 mM CaCl2.The results obtained for the antimicrobial activity vs. CaClbacteria . The forbacteria ,37. In 12 was different from that at 100 mM CaCl2. Gramicidin A may exist largely in the denatured state in 400 mM CaCl2. This may cause the loss of the antimicrobial function. However, we could not exclude the possibility that the decrease in antimicrobial activity may have been due to the blocking of the gramicidin A channel by the high concentration of calcium cations, as calcium cations have been shown to interact with gramicidin A channels at the mouth sites and block the conduction of monovalent cations across the cell membrane [The case of gramicidin A in 400 mM CaClmembrane ,33,38.In conclusion, we demonstrate that the molecular state of gramicidin A is highly influenced by the concentration of calcium salt and the types of halide anion. Gramicidin A exists in two molecular states. In State 1 (concentrations ≤ 10 mM), the majority of gramicidin A forms a dimer. The addition of calcium salt converts the four conformers into a single species that forms a left-handed parallel double helical structure. In State 2, the addition of calcium salt induces a dissociation of dimers into monomers and a further denaturation of the structured monomers into the unstructured monomers. The abilities of dissociation and denaturation are highly dependent on the type of halide anion. Regarding antimicrobial activity, gramicidin A in the structured monomeric state showed the most effective antimicrobial activity. As the molecular state of gramicidin A is highly dependent on the dissociation ability of the halide anions, the dissociation rate can determine the molecular state and antimicrobial activity. Taken together, our study suggests that the molecular state may play a vital role in the antimicrobial activity.Gramicidin A was purchased from Merck . Spectrograde methanol and methyl-d3-alcohol were purchased from Sigma-Aldrich . Calcium chloride, calcium bromide and calcium iodide were purchased from Sigma-Aldrich . All chemicals were reagent grade and used without further purification.Circular dichroism spectra were recorded using a J-815 CD spectrometer  or a Chirascan-plus qCD spectrometer . All measurements were performed in a quartz cell with a pathlength of 0.1 mm. The CD spectra of 100 μM gramicidin A in the presence of calcium salts with a designated concentration were collected from 200 to 250 nm with a 0.5 nm interval at 25 °C. The reported circular dichroism spectra were corrected with baseline using methanol containing the same concentration of salt and smoothed using a Savitsky–Golay function in Origin 6.0.2, CaBr2 or CaI2 at ten different concentrations  dissolved in either D2O or methanol. These samples were analyzed using MALDI-TOF mass spectrometry.Gramicidin A samples (100 µM each) were mixed with CaCl2O and 0.1% trifluoroacetic acid (TFA). The gramicidin A samples were then mixed with the CHCA matrix by adding 1 µL of the sample with 1 µL of the CHCA matrix. After mixing the sample and matrix well, 1 µL of the resulting mixtures were then spotted on to the MALDI plate for analysis. The MALDI spectra in the reflection mode were recorded with a Bruker New ultrafleXtremeTM MALDI-TOF/TOF mass spectrometer from Bruker Daltonik, Bremen, Germany, at the Academia Sinica Institute of Chemistry Mass Spectrometry Center.The matrix used for the analysis was α-cyano-4-OH cinnamic acid (CHCA). A 10 mg amount of CHCA was weighed and dissolved in 1.0 mL of 50% acetonitrile, 50% distilled H2, CaBr2 or CaI2  dissolved in 90% d4-methanol/10% methanol using solution NMR. The NMR spectra were obtained using a Bruker DRX-500 NMR spectrometer equipped with a TXI z-gradient  probe at 300 K . In these spectra, the OH signal of methanol was suppressed using the presaturation method. All the NMR spectra were phased and baseline-corrected using the Topspin software  and referenced to the chemical shift of methanol at 3.3 ppm.We analyzed gramicidin A (100 μM) with CaClAll steady-state fluorescence measurements were performed with a Jasco FP-6500 fluorescence spectrometer  equipped with a water circulator and stirring accessory. The emission spectra of 100 μM gramicidin A in the presence of calcium halide salts at designed concentrations were recorded from 310 to 500 nm with an excitation wavelength of 290 nm. The bandwidths for excitation and emission were 3 and 5 nm, respectively. All measurements were performed in a quartz cell with a path length of 1 cm. The background spectra of the salts alone were taken first for later subtraction. The reported spectra were the averages obtained from at least three individual samples and three repeated measurements of each sample. All measurements were carried out at 25.0 ± 0.5 °C.Staphylococcus aureus (S. aureus) (ATCC-25923) was kindly provided by Dr. J.W. Liou in Tzu Chi University. The S. aureus was grown in Luria–Bertani (LB) broth medium in a 250 mL flask at 37 °C overnight. This overnight S. aureus culture was then diluted to OD600 = 0.1 with LB medium. This S. aureus was then grown to lag phase (OD600 = 0.2), exponential phase (OD600 = 0.6) and late-exponential-to-stationary phase (OD600 = 1.5). At the designated growth phase, 5 mL of S. aureus culture was treated at 5% (V/V) with a stock solution containing gramicidin A  and CaCl2. The stock solution was prepared in the same way as used for the spectroscopic studies, in which a final concentration of 100 μM gramicidin A was dissolved in methanol containing 0, 0.2, 10, 100 and 400 mM of CaCl2. The S. aureus samples treated with or without gramicidin A/CaCl2 were incubated at 37 °C. The optical density at a wavelength of 600 nm, (OD600), was used to determine the growth curve of S. aureus using a microplate reader  every 30 min.


Intellectual abilities and their clinical presentations are extremely heterogeneous in autism spectrum disorder (ASD). The main causes of ASD remain unclear. ASD is frequently associated with sleep disorders. Biologic rhythms are complex systems interacting with the environment and controlling several physiological pathways, including brain development and behavioral processes. Recent findings have shown that the deregulation of the core clock neurodevelopmental signaling is correlated with ASD clinical presentation. One of the main pathways involved in developmental cognitive disorders is the canonical WNT/β-catenin pathway. Circadian clocks have a main role in some tissues by driving circadian expression of genes involved in physiologic and metabolic functions. In ASD, the increase of the canonical WNT/β-catenin pathway is enhancing by the dysregulation of circadian rhythms. ASD progression is associated with a major metabolic reprogramming, initiated by aberrant WNT/β-catenin pathway, the aerobic glycolysis. This review focuses on the interest of circadian rhythms dysregulation in metabolic reprogramming in ASD through the aberrant upregulation of the canonical WNT/β-catenin pathway. Early diagnosis is major issue for better prognosis and therapeutic care3.Intellectual abilities and their clinical presentations are extremely heterogeneous in autism spectrum disorder (ASD). ASD includes Asperger, autism and pervasive developmental disorder-not otherwise specified (PDD-NOS). ASD is characterized by numerous and complex etiologies, including inflammation, metabolic, environmental or genetic determinants. Nevertheless, the main causes of ASD remain unclear. ASD is diagnosed within the first 3 years of life. Social interaction impairment, repetitive and restrictive behaviors or stereotypic patterns of behaviors characterize the different symptoms of ASD4. Brain structures and functions development are considered as dynamic processes, dependent on genetic and social/physical environmental cues, involving homeostasis processes at each time, and maturation of new functions in time. Biologic rhythms are complex systems interacting with the environment and controlling several physiological pathways, including brain development5 and behavioral processes6.ASD is frequently associated with sleep disorders7. Several genetic mutations observed in ASD are linked with the dysregulation of the WNT/β-catenin pathway through interactions between chromodomain helicase DNA binding protein 8 (CHD8) and CTNNB1 (β-catenin)8. In many ASD findings, the WNT/β-catenin pathway is increased11.Recent studies have shown that the deregulation of the core neurodevelopmental signaling is correlated with ASD clinical presentation. One of the main pathways implicated in developmental cognitive disorders is the canonical WNT/β-catenin pathway12. One of the key integrator of these complex mechanisms is the canonical WNT/β-catenin pathway14. ASD progression is associated with a major metabolic reprogramming, initiated by aberrant WNT/β-catenin pathway, the aerobic glycolysis11. In parallel, the dysregulation of circadian rhythms (CRs) upregulates the WNT/β-catenin pathway15, which in turn participates to the ASD initiation. This review focuses on the interest of CRs dysregulation in metabolic reprogramming in ASD through the aberrant upregulation of the canonical WNT/β-catenin pathway.Circadian clocks have a main role in some tissues by driving circadian expression of genes involved in physiologic and metabolic functions16. The control of the circadian clock is based on an intracellular temporal tracking system that allows anterior organisms to change direction and thus adapt their behavior and the physiology of their life span17. It is well known that in many animals species, circadian clock is formed by a specific set of transcription factors which constitutes its molecular architecture. These determinants are both used in positive and negative feedback, which are modulated through a cell-autonomous manner18.CRs are major biological phenomena found in all universal processes. Their endogenous characteristic is an innate oscillation associated with a period of over 1 day. All the studied organisms show this oscillatory process. Numerous cell functions present temporal variations driven by these oscillatory and circadian ways, including gene expression, metabolic reprogramming, and molecular and cellular pathways. Different integration levels allow the study of the CRs, as endocrinal, physiological, neuronal cell behaviors. Although the coordination and the modulation of CRs are organized by specific pacemaker structures, the primary circadian oscillations are controlled at the cell level. These oscillations are determined by numerous clock genes19. A complex loop operates with clock gene transcriptional activators and in turn clock genes with a negative feedback role inhibiting their expression by disrupting the activity of their activators20. Several input pathways involve environmental information which interacts with the different compounds of the oscillators. The oscillators are synchronized with the 24 h solar day. The input pathways generate a day-time to transpose it by the oscillators to the output pathways. These output pathways control and regulate the expression of circadian clock genes to generate the rhythmicity. Moreover, the output pathways are predicted to be rhythmic and then controlled by the clock gene transcription factors. These compounds, in turn, regulate downstream the circadian clock genes in a time-of-day-specific manner21. This system can synchronize with its environmental time by its internal clock. To respect the environment, the input pathways are vital to maintain this timing for oscillators. A the process named entrainment, the input pathways can reset the activity of the oscillators to stay in a conform 24-h period of the environment21. Environmental cues can be detected by input pathways which in turn can modulate many mechanisms to control the activity or level of compounds of oscillators to keep a correct time-of-day expression. This phenomenon is observed in several environmental cues, including nutrition, social interactions and temperature23. Furthermore, the clock allows a strategy named gating to restrict responses to environmental cues at some day times. Diurnal mammals are insensitive to a light pulse during the day. Nevertheless, during the night, a light pulse can advance or delay the clock to synchronize diurnal mammals with the environment18. Environmental signals can interact with molecular oscillators in some cells in complex multicellular organisms. In unicellular organisms, each cell is modulated by oscillators in response to light24. However, in multicellular organisms, only a part of the cells has sensory capabilities leading to clock oscillators. The oscillators, and thus the overall rhythmicity of organisms, are concentrated into compounds including a master pacemaker and peripheral oscillators25. Face to these sensory inputs, the organism presents some nervous systems which possess environmental cue abilities as a central oscillators or pacemakers rather than to individual cells. In humans, the sensory clock inputs are localized in the brain, where signals from the master pacemaker leads to oscillators in some tissues of the organism.Endogenous oscillations generate a freewheeling period, which is close to 24 h, to maintain for the organism constant ambient conditions. These oscillators, at the molecular level, are based on the products of clock regulator genes organized in a transcriptional feedback loop. Circadian oscillations are the product of these post-transcriptional modifications of proteins6. Melatonin operates as a major synchronizer in humans and provides temporal feedback to oscillators within the nervous system to control the circadian phase and the stability of the rhythm26. In humans, as in other mammals, melatonin is considered as the main influencer of CRs through its action on receptors in the nervous system27.Nonvisual retinal ganglion cells receive and perceive the light, and transmit this information to the master pacemaker  through neural connections. The central pacemaker synchronizes oscillators to the other tissues by using circadian input pathways from the nervous system to peripheral cell systems. Moreover, to maintain the entrainment of these peripheral oscillators by the environment, this central system ensures that cellular oscillations within tissues are properly in phase to provide resonance between individual cellular rhythms28. Through a feedback, sleep–wake pattern can also control the CRs. For many studies, this pattern can be defined as an interface between environmental information  and CRs29. Moreover, these two patters are influenced by melatonin4. Melatonin level is modulated by the light–dark exposure. Melatonin is associated with sleep initiation. Mutations in Clock, Bmal1, Cry1, Cry2 genes lead to the initiation of some alterations in sleep time and sleep fragmentation31.The sleep–wake pattern is controlled by both CRs and homeostasis. Sleep pressure has been enhanced during the phase of waking and then decreases during the phase of sleeping. The sleep–wake pattern is controlled by the cycle of light–darkness33. They are modulated by positive and negative self-loop-regulation mediated by CRs34. Clock and Bmal1 heterodimerize and lead to the transcription of Per1, Per2, Cry1 and Cry2 (ref. 35). The Per/Cry heterodimer inhibits its activation by a negative feedback. It translocates back to the nucleus to directly downregulate the Clock/Bmal1 complex and then inhibits its own transcription35. The Clock/Bmal1 heterodimer activates the transcription of retinoic acid-related orphan nuclear receptors, Rev-Erbs and retinoid-related orphan receptors (RORs). Through a positive feedback loop, RORs activate the transcription of Bmal1, whereas through a negative feedback loop, Rev-Erbs downregulate their transcription35.Some biological mechanisms in humans are controlled by the circadian “clock” (circadian locomotors output cycles kaput) Fig. . The cir36. Patients suffering from ASD are more correlated with falling asleep anxiety, sleep disorders and CRs disturbance36. Moreover, the prevalence of sleep disturbance is associated with cognitive impairments38. In parallel, melatonin levels have been dysregulated in ASD patients4. Melatonin diurnal levels are decreased as well as melatonin nocturnal levels, showing a global decrease in melatonin production in ASD children and a dysregulation of day–night rhythm42. Deregulation in the production of melatonin in ASD patients has been observed due to HIOMT (hydroxiindole O-methyltransferase) deficiency43. Recent studies have shown a potential interest in melatonin therapy in the treatment of sleep onset disorder for ASD patients44. Significant allelic correlation has been observed for clock genes, including Per1, and other nucleotids47. Genes involved in ASD pathogenesis are part of pathway networks enhanced in synapse formation, including Neuroxin-Neuroligin genes and in the alteration of the balance excitation-inhibition48. Parvalbumin expressing interneurons play a main role in ASD initiation. The knockout of Parvalbumin is associated with core symptoms of ASD patients49. CRs dysregulation may downregulate the maturation of Parvalbumin cells and then the timing of critical period of plasticity50. The dysregulation of CRs could impact the temporal organization of brain maturation and could have a cascade effect on several brain functions. Negative environmental conditions  could impact the CRs and thus redox homeostasis and transcriptional control of Parvalbumin genes involved in synapse formation and maturation of brain functions.Sleep disorders are frequently associated with ASD Fig. 36. Patie51. Disturbances in sleep–wake rhythm in ASD could be due to the dysregulation of melatonin52. Sleep latency and nocturnal and early awakenings have been reported in ASD41. In ASD, the nocturnal secretion of melatonin is low53. Intellectual disabilities are associated with melatonin abnormalities54. However, in the Down syndrome, the production of melatonin is normal whereas melatonin production is increased in the Fragile X patients55. Several therapeutic studies have focused on the interest of melatonin strategy in ASD56. Melatonin use in ASD children is associated with improvement of communication57, stereotyped behaviors58, anxiety59 and social withdrawal58. Nevertheless, melatonin is influenced by age and pubertal stage60, and in these studies the autistic behavioral impairment was generally enough described57.Melatonin plays a main role in neurodevelopment2 at the end. This process occurs in an aerobic or anaerobic manner, depending on whether oxygen is available. Glycolysis is the first step in glucose metabolism signaling and occurs in the cytosol of all cells. The presence of O2 is important because of oxidation of glucose under aerobic conditions results in ~32 molecules of ATP per molecule of glucose. Under anaerobic conditions, only two molecules of ATP can be produced. Aerobic glycolysis can occur in these two stages. The first occurs in the cytosol and involves the conversion of glucose to pyruvate resulting in NADH production and generating two molecules of ATP. In normal conditions, when oxygen is available, the energy contained in NADH is further released via re-oxidization of the mitochondrial chain and leads to the release of 30 molecules of ATP per molecule of glucose. Pyruvate is reduced to lactate, instead of re-oxidized, under aerobic glycolysis61. Thus, glucose is metabolized in order to produce ATP, by a cytosolic glycolysis and oxygen-dependent mitochondrial physiological mechanism. Glucose entry into the tricarboxylic acid (TCA) cycle is modulated by pyruvate dehydrogenase complex (PDH)62. Pyruvate is oxidized to acetyl-coA in mitochondria by the PDH. Acetyl-coA translocates to the TCA cycle for oxidation. Under aerobic glycolysis, pyruvate is converted into lactate in the cytosol by lactate dehydrogenase A (LDH-A). Moreover, aerobic glycolysis is caused by the involvement of hexokinase 2 (HK2) instead of HK1 and pyruvate kinase M2 (PKM2) instead of PKM1 (ref. 63). This phenomenon is called aerobic glycolysis or the Warburg effect.In mammalian cells, glucose is the main source of energy. All tissues need ATP to function normally. Cells produce ATP by a careful drop in oxidation state from energy-rich molecules like glucose, through the cell respiration process, down to product CO11. However, numerous findings have highlighted increased lactate levels in ASD patients71.Few studies have investigated the potential impact of aerobic glycolysis in ASD, and thus the expression of the glycolytic enzymes Fig. 11. Howev67, but with an increased ratio of lactate-to-pyruvate68 showing a high glucose metabolism and LDH-A activity in ASD patients. Moreover, LDH-A expression has been increased in ASD patients71. A recent study has presented a decrease level of pH correlated with high levels of lactate in ASD patients72. These observations could suggest an increase of aerobic glycolysis in ASD since the dysregulation of this balance has been proposed as a candidate cause of ASD73.Pyruvate production is stimulated74. In the same way, melatonin expression and modulation by CRs in cancers is associated with the disruption of the aerobic glycolysis77. Thermodynamic and energy reprogramming highlight this relation in fibrosis78, neurodegenerative diseases80 and cancers81. The importance of 24-h fluctuations in the aerobic glycolysis and the availability of nicotinamide adenine dinucleotide phosphate hydrogen (NADPH) in cancer has been shown through the consideration of the redox influence on NADPH82.Few studies have focused on the relationship between CRs and aerobic glycolysis Fig. . Neverth83. Several nervous molecular mechanisms are modulated by the WNT/β-catenin pathway, including development of synapses in the central nervous system85, synaptogenesis87 and control of synaptic formation88. Numerous pathophysiologic signalings are mediated by the dysregulation of this pathway, including cancers90, fibrosis91, neurodegenerative diseases80 and angiogenesis92. Stimulation of β-catenin signaling needs the presence of the complex LRP5 /LRP693. LRP5 has a main role while LRP6 presents a minor role in the retinal vascularization95. Disheveled (Dsh) forms a complex with Axin, and this prevents the phosphorylation of β-catenin by glycogen synthase kinase-3β (GSK-3β). Then, β-catenin accumulation in the cytosol is observed and translocates to the nucleus to bind T-cell factor/lymphoid enhancer factor (TCF/LEF) co-transcription factors. This nuclear binding allows the transcription of WNT-responsive genes, such as cyclin D1, c-Myc, PDK1, MCT-1 (refs. 97).The Wingless/Int (WNT) pathway is a family of secreted lipid-modified glycoproteins Fig. 83. SeverWNT ligands’ absence is associated with cytosolic β-catenin phosphorylation by GSK-3β.98.A destruction complex is composed by tumor suppressor adenomatous polyposis coli (APC), Axin, GSK-3β and β-catenin. Then, phosphorylated β-catenin is destroyed in the proteasome. WNT inhibitors, including DKKs and SFRPs, control the WNT/β-catenin pathway by preventing its ligand–receptor interactions99. GSK-3β regulates numerous pathophysiological pathways 102. GSK-3β downregulates β-catenin cytosolic accumulation and then its nuclear translocation100. GSK-3β diminishes β-catenin, mTOR pathway, HIF-1α and VEGF expression103.GSK-3β, a neuron-specific intracellular serin-threonin kinase, is the major inhibitor of the WNT pathway106. Furthermore, many gene compounds are associated with the development of ASD, including WNT2 ligand107, the WNT target MET (hepatocyte growth factor receptor)109, CHD8 (chromo-helicase domain protein 8) and DYRK1 (refs. 112). Other components of the WNT pathway have been involved in ASD pathogenesis, including WNT1 (ref. 113), WNT2 (ref. 107), WNT3 (ref. 114), WNT7A115, APC118, β-catenin119, TCF4 (refs. 121) and TCF7 (ref. 122). The CTNNB1 gene, a β-catenin encoder, is a major controller of the WNT pathway. Its expression is associated with sporadic ASD. Alterations of CTNNB1 correspond to intellectual disability and thus, ASD8. In mice, the expression of CTNNB1 expression is correlated with brain development123. Knockout of CTNNB1 in the Parvalbumin interneurons leads to the impairment of social interactions and enhancement of repetitive behaviors.Some studies have observed the potential role of over-increased WNT/β-catenin pathway in ASD patients126. In Purkinje cells, PTEN inhibition impairs social relation, behavior and deficits in motor learning128. PTEN and β-catenin control each other to normal brain growth and development129.Phosphatase and tensin homolog protein (PTEN) inhibition is associated with the increase of the WNT pathway and high risk of ASD development130. By this interaction, circadian genes can modulate the cell cycle progression131. A Bmal1 knockdown can downregulate the WNT/β-catenin pathway132. In wild-type mice, the levels of WNT-related genes are higher than those observed in Bmal1 knockdown mice134. The proliferation and progression of cell cycle are controlled by Bmal1 by activating the WNT/β-catenin pathway135. Bmal1 involves the β-catenin transcription, diminishes the β-catenin degradation and then inhibits GSK-3β activity136. In the intestinal mucosa of ApcMin/+ mice, the degradation of Per2 leads to β-catenin increase by circadian disruption137.RORs are upstream effectors of the WNT/β-catenin pathway34. Per1 and Per2 maintain cell CRs and modulate cell-related gene activity, such as c-Myc, so as to sustain the physiologic cell cycle139.In normal conditions, the core circadian genes operate in accurate feedback loops and keep the molecular clockworks in the SCN. They allow the control of peripheral clocks142. The PI3K/Akt pathway stimulates the glucose metabolism to enhance protein and lipid synthesis143. Moreover, PI3K/Akt pathway increases the glucose metabolism to protect cells against reactive oxygen species (ROS) stress induced by activated HIF-1α and decreasing the glucose entry into the TCA cycle144. HIF-1α stimulates pyruvate dehydrogenase kinase (PDK) to phosphorylate PDH and inactivates it, leading to cytosolic pyruvate being shunted into lactate by LDH-A145. HIF-1α is transcriptionally activated by PI3K/Akt/mTOR pathway through 4E-BP1 and STAT3 (refs. 151).Some reports have highlighted that the WNT/β-catenin pathway is closely associated and a main effector of the aerobic glycolysis Fig. 11,78,140152. WNT/β-catenin pathway, by activating both the PI3K/Akt/mTOR pathway and HIF-1α, can lead to aerobic glycolysis153. PI3K/Akt pathway can also control the β-catenin accumulation and then the expression of the downstream genes154. c-Myc directly stimulates the HIF-1α155, PDK and lactate transporter (MCT-1) expressions152. The stimulation of HIF-1α leads to the overexpression of glucose transporters (Glut), hexokinase (HK), pyruvate kinase (PK), PDK1 and LDH-A159.Numerous studies have observed that the WNT/β-catenin pathway can downregulate the pyruvate oxidation in the TCA cycleChanges in energy metabolism are modulated by abnormal CRs in ASD patients. In ASD, the canonical WNT/β-catenin pathway is increased. Energy behaviors of metabolic enzymes in ASD are modified by this upregulation of the WNT/β-catenin pathway leading to the enhancement of aerobic glycolysis and thus the production of lactate. This explains the glucose hyper-metabolism observed in ASD. WNT pathway is driven by the CRs and operates under a circadian regime evolving to changes in energy metabolism. CRs directly contribute to the regulation of the molecular pathways WNT/β-catenin pathway involved in the reprogramming of cellular energy metabolism enabling ASD.


Herein, we reported a biomimetic enzyme cascade delivery nanosystem, tumor-targeted erythrocyte membrane (EM)-cloaked iron-mineralized glucose oxidases (GOx-Fe0@EM-A) for enhancing anticancer efficacy by self-activated in vivo cascade to generate sufficient high toxic •OH at tumor site.Biomineralization of enzymes for Methods: An ultra-small Fe0 nanoparticle (Fe0NP) was anchored in the inner cavity of glucose oxidase (GOx) to form iron-mineralized glucose oxidase (GOx-Fe0) as a potential tumor therapeutic nanocatalyst. Moreover, erythrocyte membrane cloaking delivery of GOx-Fe0in vivo was designed to effectively accumulate ultra-small GOx-Fe0 at tumor site.Results: GOx-Fe0@EM-A had satisfactory biocompatibility and light-trigged release efficiency. Erythrocyte membrane cloaking of GOx-Fe0@EM-A not only prolongs blood circulation but also protects in vivo enzyme activity of GOx-Fe0; Tumor targeting of GOx-Fe0@EM-A endowed preferential accumulation at tumor site. After NIR light irradiation at tumor site, erythrocyte membrane of GOx-Fe0@EM-A was ruptured to achieve light-driven release and tumor deep penetration of ultra-small nanosize GOx-Fe0 by the photothermal effect of ICG. Then, GOx-Fe0 occurred self-activated in vivo cascade to effectively eradicate tumor by producing the highly cumulative and deeply penetrating •OH at tumor site.Conclusion: Tumor-targeted erythrocyte membrane-cloaked iron-mineralized glucose oxidase (GOx-Fe0@EM-A) exhibits a promising strategy for striking antitumor efficacy by light-driven tumor deep penetration and self-activated therapeutic cascade. Firstly, glucose oxidase can catalyze glucoses into gluconic acid and H2O2 under the tumor acidic conditions. In the same time, harnessing gluconic acid to decrease pH around GOx-Fe0 in real time, the Fe0NPs can rapidly ionize into Fe2+ to catalyze H2O2 into highly toxic hydroxyl radical (•OH), causing irreversible oxidative damage to cellular macromolecules at near sites from side-effect damage in vivo cascade of GOx-Fe0.Due to the relatively low potential of the Fe0) at tumor site remains a vital problem, due to renal clearance to ultra-small nanoparticles in vivo0in vivo. More importantly, erythrocyte membrane as a natural vesicle guarantees in vivo circulation stability of GOx-Fe0in vivo in vivo. In this study, DSPE-PEG-Angiopep-2 targeting peptide was inserted on the erythrocyte membrane. Angiopep-2 peptide is a tumor targeting moiety that specifically binds to low-density lipoprotein receptor-related proteins (LRP) receptor that are overexpressed on the surface of glioma cells However, how to effectively accumulate iron-mineralized glucose oxidases (GOx-Fe0@EM-A) by self-activated in vivo cascade at tumor site to generate sufficient high toxic •OH for enhancing anticancer efficacy. In order to achieve light-driven release and deep penetration of GOx-Fe0 at tumor site, indocyanine green (ICG) was encapsulated in erythrocyte membrane, utilizing photothermal effect to rupture the enclosed erythrocyte membrane. Due to the short half-life and lipid insolubility, the diffusion of hydroxyl radical (•OH) is limited thereby 0 would produce •OH in the tumor deep region. At present, GOx-Fe0@EM-A exhibited four unique characteristics (Scheme 1), as summarized in the previous reports 0. Secondly, GOx-Fe0@EM-A effectively accumulates at tumor site. Thirdly, light-driven release and tumor deep penetration of ultra-small nanosize GOx-Fe0 after NIR light irradiation. Finally, the released GOx-Fe0 undergoes self-activated in vivo cascade at tumor site.Herein, we reported a biomimetic enzyme delivery nanosystem, tumor-targeted light-driven erythrocyte membrane-cloaked iron-mineralized glucose oxidases (GOx-Fe0 was synthesized according to the literature 4 to prepare GOx-Fe0. Transmission electron microscopy (TEM) images . Moreover, the circular dichroism (CD) spectra result revealed that the secondary structure of GOx-Fe0 was almost the same as that of GOx, indicating that GOx-Fe0 remained the enzymatic activity of GOx  of 0.27 , GOx-Fe0@EM-A exhibited similar photothermal properties to ICG  was oxidized, it showed strong absorbance at 652 nm. Hence, TMB was used to detect •OH for studying the disproportionation of H2O2 by cascade catalysis of GOx-Fe0 under acidic condition. As shown in Figure 0@EM-A was added to H2O2 of various concentrations and the absorbance curve was plotted to obtain an initial reaction rates. According to the Lineweaver-Burk equation  could be oxidized by •OH to form strong fluorescent isomeric hydroxybenzoic acids (OHBA) 4 Figure H and I, 4 Figure . Further4 Figure . In addiin vitro cascade of GOx-Fe0@EM-A on murine glioma cells (C6) was evaluated by cell cytotoxicity using a Cell Counting Kit-8 (CCK-8) assay. Upon 808 nm laser light irradiation, tumor-targeted ICG-functionalized erythrocyte membrane vesicle (ICG@EM-A) group, as a control light group for GOx-Fe0@EM-A, exhibited negligible cytotoxicity toward C6 cells after 24 hours of incubation at tested concentrations and different pH  and GOx@EM-A showed no substantial cytotoxicity against C6 cells with light irradiation  was used as a ROS fluorescent probe to further detect •OH  were constructed to mimic the morphology and microenvironment of solid tumor. As shown in Figure 0@EM-A and GOx-Fe0@EM for 12 hours, and ultra-small size GOx-Fe0 released from GOx-Fe0@EM-A upon light irradiation showed stronger fluorescence in the whole C6 3D tumor spheroids. However, in the absence of light irradiation, GOx-Fe0@EM-A (100 nm) has limited penetration ability due to larger sizes than that of GOx-Fe0 (12 nm). The above results indicated that light-driven released GOx-Fe0 from GOx-Fe0@EM-A exhibited enhanced penetration ability. Subsequently, we investigated the blood circulation time and tumor-targeting capability of GOx-Fe0@EM-A in vivo. First, after the intravenous (i.v.) injection of different groups, blood samples were collected at various time points, and the blood of the uninjected mice was used as a blank for ICP-AES measurement of Fe content. As shown in Figure -1 and 12.67% ID g-1 of GOx-Fe0@EM-A and GOx-Fe0@EM remained in the blood circulation after 48 h injection, compared to GOx-Fe0 (4.11% ID g-1). GOx-Fe0@EM-A and GOx-Fe0@EM showed a significant increase in blood retention, which attributed to the camouflage of erythrocyte membrane.To evaluate the capability of tumor deep penetration of GOx-Fe0@EM-A was injected into C6 tumor-bearing mice through tail intravenous, and fluorescent images of mice were obtained by an in vivo fluorescence imaging system. As shown in Figure 0@EM-A and GOx-Fe0@EM, the tumor site showed apparent fluorescent signal, and the mice treated with GOx-Fe0@EM-A exhibited higher tumor accumulation than the GOx-Fe0@EM group due to the active tumor-targeting. However, due to its small nanosize, Cy5.5-labeled GOx-Fe0 was rapidly eliminated from the blood, and the mice tumor showed a relatively weak fluorescent signal after 24 h. The tumor-bearing mice were sacrificed after 24 h postinjection, and the main organs and tumors were collected for ex vivo fluorescence imaging. As shown in Figure 0 was mainly distributed in the liver and lung. Importantly, GOx-Fe0@EM-A group showed the strongest fluorescence at the tumor tissue compared to Cy5.5-labeled GOx-Fe0 and GOx-Fe0@EM groups. The further semiquantitative results showed that the tumor fluorescence intensity of the mice treated with GOx-Fe0@EM-A group was 3.38-fold and 1.64-fold than that of Cy5.5-labeled GOx-Fe0 and GOx-Fe0@EM groups, respectively  and tumor hypoxic region (B) was evaluated by the immunofluorescence staining study. GOx-Fe0 or GOx-Fe0@EM-A groups showed a weak fluorescent signal at tumor blood vessel site. The weak signal of GOx-Fe0 was due to the low tumor accumulation from tumor blood vessels, whereas the GOx-Fe0@EM-A could not easily penetrate into the deep tumor space, due to the large nanosize (~100 nm). However, after membrane rupture driven by light irradiation, GOx-Fe0@EM-A and GOx-Fe0@EM released small nanosize GOx-Fe0 (~12 nm) previously encapsulated. Meantime, both of the two groups showed a stronger fluorescence signal at tumor site than that without light irradiation, and the GOx-Fe0@EM-A group showed higher tumor accumulation than the GOx-Fe0@EM group, exhibiting uniform perfusion in the tumor stroma. In addition, after light irradiation, the fluorescence intensity of GOx-Fe0 released from GOx-Fe0@EM-A significantly increased at a greater distance from the CD34-labeled tumor blood vessels  showed no obvious organ damage and inflammatory changes compared to the PBS group (0 group showed reduced plasma glucose (GLU) compared to the PBS group, probably due to free alive GOx-Fe0 promoting catalytic decomposition of glucose in the blood. However, the GOx-Fe0@EM-A and GOx-Fe0@EM group had no noticeable changes in biochemical parameters of aspartate aminotransferase (AST), albumin globulin (ALB), urea nitrogen (BUN), uric acid (UA), and GLU, demonstrating no damage to liver, kidney and blood glucose function. These results indicate that tumor-targeted GOx-Fe0@EM-A exhibited sufficient tumor accumulation, and then through light-driven enzyme biohybrid' release and tumor acidic condition to complete cascade sequential catalysis to generate large amount of highly toxic •OH and achieve efficient tumor growth inhibition, showing minimal toxicity to normal major organs at the test dose.To further confirm the above therapeutic results, we constructed another C6 tumor xenograft fors Figure . The antBS group A. Blood BS group B that th0@EM-A) by self-activated in vivo cascade of GOx-Fe0 at tumor deep site to generate sufficient high toxic •OH for enhancing anticancer efficacy. Due to the biomimetic surface functionalization of erythrocyte membrane, GOx-Fe0@EM-A showed prolonged blood circulation and enhanced tumor accumulation. Moreover, at tumor site light-driven erythrocyte memebrane rupture by photothermal effect of ICG enhanced the pinpoint release and tumor tissue penetration of small nanosize GOx-Fe0. More importantly, the released GOx-Fe0 undergoes self-activated in vivo cascade to effectively produce the highly cumulative and deeply penetrating •OH to effectively eradicate tumor. In vitro and in vivo studies indicated that GOx-Fe0@EM-A exhibited dual-enzyme-like activity of glucose oxidase and peroxidase, and showed highly effective anticancer inhibition by enzyme cascade catalysis at the tumor deep site, with negligible side effects. In short, our rationally designed biomimetic enzyme cascade delivery nanosystem exhibits a new strategy of chemodynamic therapy for effective tumor eradication and provides valuable insights in nanomedicine to develop safe and highly efficient biomimetic nanocatalytic systems for various malignant tumor treatments.In summary, we developed a biomimetic enzyme cascade delivery nanosystem, tumor-targeted light-driven erythrocyte membrane-cloaked iron-mineralized glucose oxidases (GOx-FeTransmission electron microscope (TEM) images were obtained with Hitachi H-7650 operated under 80 kV. Dynamic light scattering (DLS) and zeta potential were conducted on a Zetasizer Nano ZS particle analyzer . UV-Vis absorption spectra were performed on a Shimadzu UV-2550 UV-vis spectrophotometer. The fluorescence spectra were measured using a 970CRT spectrophotometer. The CD spectra were carried out using a chirascan-plus spectropolarimeter .0@EM-A solution (with Fe concentration at 1 mg mL-1) was sealed into a dialysis bag (molecular weight cutoff = 3000 Da) against 10 ml of the PBS buffer solution  with or without 808 nm laser (1 W/cm2) irradiation, and gently shaken at 37 °C with shaker at 100 rpm. At predetermined time intervals, 0.2 mL of solution was withdrawn and displaced with fresh solution. The released Fe concentration was analyzed by ICP-MS.1 mL of GOx-Fe2O2  was mixed with TMB (1 mM) and GOx-Fe0@EM-A (100 μg/mL) in NaAc buffer solution (20 mM pH = 5.2). The chromogenic reaction (λ = 650 nm) was monitored with UV-vis spectrometer. The Michaelis-Menten kinetic curve can then be obtained by converting the absorbance changes to the initial velocity through Beer-Lambert law and plotting against the H2O2 concentration. The Michaelis-Menten constant (KM) and the maximum velocity Vmax were calculated by a linear double reciprocal plot (Lineweaver-Burk fitting).For a typical peroxidation reaction, various concentrations of H0@EM-A  was added into a mixture solution (3 mL) containing glucose (2 mM) and different pH of BA (2 mM). After 808 nm (1 W/cm2) light irradiation, the fluorescence spectrum of BA was monitored for 60 minutes and the emission intensity of OHBA at 410 nm was plotted against time.Benzoic acid (BA) was dissolved in PBS at different pH . In a typical process, GOx-Fe2.C6 cancer cells were obtained from American Type Culture Collection (ATCC) and cultured in Dulbecco modified eagle medium (DMEM) containing 4500 mg/L glucose, 10% fetal bovine serum and 1% penicillin/streptomycin. The cells were maintained at 37 °C in humidified atmosphere with 5% CO2) for 5 min. After another 20 h incubation, the cells were mixed with 10% CCK-8 containing DMEM high-glucose medium. CCK-8 could measure by a microplate reader  at 450 nm.C6 cells were seeded in a 96-well plate with a cell density of 5000 cells per well for 12 h. Then cell culture medium were replaced with different products under different GOx or ICG concentrations in DMEM high-glucose medium (pH was adjusted to 7.4 and 6.5 by the addition of HCl) for 24 h. After 4 h incubation, cells were illuminated by 808 nm light irradiation (1 W/cm0@EM (3 μg/mL of GOx), GOx-Fe0@EM-A (3 μg/mL of GOx), or GOx-Fe0@EM-A (6 μg/mL of GOx), and then with or without irradiated by 808 nm laser (1 W/cm2). The cell culture medium was removed. After staining with Calcein AM/PI for 20 min, the cells were washed and monitored by inverted fluorescent microscope .In order to further studied the viable cells and dead cells after cytotoxicity measurements, the cells were incubated with GOx-Fe5 cells/well) for 12 h. Subsequently, the cells were incubated with RI (4 μg/mL of ICG), GOx-Fe0@EM (15 μg/mL of GOx), or GOx-Fe0@EM-A (15 μg/mL of GOx), and then with or without irradiated by 808 nm laser (1 W/cm2). After that, the cells were washed with PBS and treated with DCFH-DA (10 μM) for 30 min. Finally, the cells were detected with confocal fluorescence microscope by an excitation of 488 nm.C6 cells were seeded in 2 cm confocal microscopy dish  for 5 min. After another 8 h incubation, the spheroids were washed three times and observed with CLSM from the top to the middle of spheroid.C6 cells were seeded on an ultralow adsorption 6-well plate at a density of 2 × 100, GOx-Fe0@EM, or GOx-Fe0@EM-A at a dose of 2 mg Fe kg-1. At different time points after the injection, venous blood was collected from the tail veins. The Fe ions contents in blood were determined by ICP-MS.Female BALB/c mice bearing C6 tumor xenografts (n=3) received intravenous injected with of GOx-Fein vivo imaging studies. The mice were intravenously injected with Cy5.5-labeled GOx-Fe0@EM or GOx-Fe0@EM-A , and then the fluorescent images were observed using VIS-FL in vivo imaging system  at different time intervals. After the 24 h post-injection, the mice were sacrificed and the tumors as well as normal tissues were collected for ex vivo imaging. The region-of-interests were analyzed by using Living Image software.The female Balb/c mice with C6 tumors were performed Supplementary experimental section and figures.Click here for additional data file.


Meanwhile, pPt induces remarkable content of intracellular reactive oxygen species (ROS) under an alternating electric field, leading to cellular oxidative stress injury and promotes apoptosis following the mechanism of electrodynamic therapy (EDT). In consequence, the PtG nanocomposite exhibits significant anticancer effect both in vitro and in vivo. This study has therefore demonstrated a fascinating therapeutic platform enabling oxygen‐inductive starvation/EDT synergistic strategy for effective tumor treatment.Glucose‐oxidase (GOx)‐mediated starvation by consuming intracellular glucose has aroused extensive exploration as an advanced approach for tumor treatment. However, this reaction of catalytic oxidation by GOx is highly dependent on the on‐site oxygen content, and thus starvation therapy often suffers unexpected anticancer outcomes due to the intrinsic tumorous hypoxia. Herein, porous platinum nanospheres (pPts), incorporated with GOx molecules (PtGs), are synthesized to enable synergistic cancer therapy. In this system, GOx can effectively catalyze the oxidation of glucose to generate H 2 supply to facilitate GOx‐mediated starvation by decomposing H2O2. Meanwhile, PtG induces reactive oxygen species (ROS) under an electric field following an electrodynamic mechanism. Considerable in vitro and in vivo tumor inhibition is consequently achieved.Porous platinum nanospheres (pPts) incorporated with glucose oxidase (GOx) molecules (PtGs) are synthesized for synergistic electrodynamic/starvation therapy. Despite its feasible loading for GOx, pPt enables sufficient O The cutting‐off of nutrient supply to induce starvation may effectively inhibit tumor growth. At present, there are mainly two mechanisms involved in this strategy. One is direct cutting‐off of blood supply at the tumor regime by blocking/destroying the blood vessels or inhibiting angiogenesis. Another is to deplete essential nutrients in tumor. As a type of oxidoreductase, glucose oxidase (GOx) presents intrinsic properties in converting glucose, oxygen, and water into gluconic acid, and simultaneously hydrogen peroxide is generated. Given the crucial role of glucose metabolism in tumor growth, it is logic that the tumor would starve to death with direct glucose deprivation by GOx.In recent years, starvation therapy has aroused extensive attention as a burgeoning cancer treatment modality.1 It i Great effort has been devoted to tackling this challenge. Representative tumor reoxygenation strategies fall into three main branches: one is to directly deliver oxygen reservoirs to the tumor site using perfluorocarbon (PFC), for instance. The second is to utilize peroxidase or peroxidase‐mimicking nanoenzymes to break down H2O2 and produce oxygen in situ, such as MnO2, Prussian Blue, and catalase (CAT). Another is to generate oxygen by the decomposition of certain oxide or peroxide compounds . To combine the GOx‐mediated starvation with oxygen‐inductive compounds is therefore expected to promote the therapeutic efficacy by a large magnitude. Alternatively, the combination of GOx‐mediated starvation approach with other therapeutic modalities, including chemotherapy, phototherapy, gas therapy, chemodynamic therapy, and immunotherapy, has also been extensively explored aiming at the promoted antitumor outcomes.Nonetheless, the catalytic reaction triggered by GOx relies considerably on the presence of oxygen, and thus its enzymatic activity suffers a low efficacy from the hypoxia condition which is a characteristic hallmark of malignant solid tumors.6 Grea Its main mechanism is the on‐demand generation of reactive oxygen species (ROS) by platinum (Pt) nanoparticles (Pt NPs) under an alternating electric field. More interestingly, in the presence of chloride ions, the electric field triggers the decomposition of water molecules at the surface of Pt NPs, resulting in ROS production that could induce increased intracellular oxidative stress and initiate cell apoptosis. Owing to its strong ablation capability for tumors with relatively larger dimension, EDT has been recognized as a highly potential approach for the effective treatment of malignant solid tumor. In addition, unlike other “dynamic therapies,” the ROS production in EDT does not depend on the on‐site oxygen or H2O2 content in the tumor microenvironment. Meanwhile, it is worth noting that platinum nanoparticles, as a type of peroxidase‐like nanocatalyst, can effectively catalyze the decomposition of H2O2 to water and oxygen. Compared with MnO2 and CaO2 compounds for O2 generation, Pt possesses remarkably lower cytotoxicity and strong stability in a physiological environment. It is thus expected that the combination of Pt nanoparticles and GOx may potentially serve as an alternative therapeutic platform for EDT/starvation synergistic treatment with capability of modulating hypoxic microenvironment.Electrodynamic therapy (EDT) is an emerging external‐stimuli‐activated therapeutic modality.18 ItsScheme 2O2, while pPt replenishes oxygen by triggering the decomposition of both endogenous and exogenous H2O2. The O2 supplied in turn facilitates the glucose consumption by GOx to promote tumor starvation. Meanwhile, pPts induce a considerable ROS production under an alternating electric field, leading to oxidative stress injury of cancer cells following an EDT mechanism. In consequence, significant in vitro and in vivo anticancer effect is achieved by PtG nanoparticles. This study, for the first time, offers a highly valuable therapeutic platform that may enable oxygen‐inductive starvation and EDT approaches in a synergistic manner for effective tumor ablation.Here in this study, uniform platinum nanospheres with a porous microstructure (pPt) are synthesized and incorporated with GOx molecules (PtG) to enable synergistic EDT/starvation therapy with hypoxia modulation. As demonstrated in 22.12PtCl6 with ascorbic acid in the presence of a Pluronic F127 surfactant and KBr. After surface functionalization using methoxy polyethylene glycol sulfhydryl (mPEG‐SH), GOx molecules were loaded on pPts owing to their porous microstructure, as illustrated in Figure The pPts were synthesized following a facile one‐step method by reducing H and KBr.20 Aft2 adsorption–desorption test . It is clear that both Brunauer, Emmett and Teller (BET) surface area and Barrett, Joyner, and Halenda (BJH) pore volume decreased after the loading of GOx. The element mapping exhibits homogeneous distributions of Pt, S, and N in PtG, which indicates the successful grafting of mPEG‐SH and loading of GOx molecules , and RMPI‐1640 solutions, paving the way to the further assessments .To improve the stability of pPts in aqueous solution, mPEG‐SH was used for surface modification by virtue of the affinity of platinum to thiol group. After surface modification, a narrow size distribution of pPts, with an average hydrodynamic diameter of 115 nm, presents according to the dynamic light scattering (DLS) analysis Figure . Subsequ2.2 As shown in Figure  It is noteworthy that the loading of GOx molecules in PtG hardly induces clear adverse effects on its electrocatalytic activity .In order to investigate the electrocatalytic activity, methylene blue (MB) was used to evaluate the ROS production by pPts in the presence of a square‐wave electric field, following the procedure reported previously.18 As 2.32O2 production for PtG, while considerable content of H2O2 is generated during the glucose consumption by free GOx , and the O2 induced in turn facilitates the glucose consumption by GOx.The pH variation of PtG in PBS is examined in the presence and the absence of glucose, respectively. It is clear that, upon the addition of glucose, the pH of the PtG solution shows a significant decline Figure , implyin2O2. The H2O2 produced is decomposed by pPt particles, inducing water and oxygen. It is worth noting that, in addition to the exogenous H2O2 induced during the reaction of glucose depletion, the endogenous H2O2, with an elevated magnitude in tumor tissue, can also be catalyzed by pPt to provide a certain content of oxygen. Owing to the crucial role of oxygen played in the GOx catalytic reaction, the oxygen compensation ability of pPt can considerably promote the glucose consumption by GOx under a hypoxia condition. The reaction loop, enabled by PtG, implements the self‐boosting glucose depletion process, paving the way to its unique performance in inducing tumor starvation.Overall, the mechanism of glucose depletion by PtG becomes clear. As demonstrated in Figure r tissue,21 can2.4Figure −1, while the PtG group exhibited a remarkable inhibition effect to cellular viability. With the increased glucose concentration in the culture medium, the inhibition effect of PtG was enhanced, reflecting that its killing effect is mainly attributed to the cellular starvation induced by GOx . Subsequently, to examine the EDT effect of pPts, 4T1 cells were exposed to a square‐wave electric field, set at 5 mA and 10 mHz for 5 min, after being incubated with pPt for 4 h. The cell viabilities declined continuously with increasing concentration of pPt. In addition, when the treatment duration was extended to 10 min, the decrease in cell viabilities accelerated accordingly, as expected  assay. As shown in d Figure . It was d Figure . The cold Figure . The mosd Figure . ApproxiFigure 3]Cl2 (RDPP), was used for intracellular oxygen examination, bright red fluorescence was observed in PtG and PtG+E groups comparing to other sample groups, owing to the considerable oxygen depletion occurred .To uncover the in vitro antitumor mechanism of PtG under the square‐wave electric field, 2,7‐dichlorofluorescein diacetate (DCFH‐DA) probe was used to detect the presence of intracellular ROS Figure 4a. Cellsd Figure . It is w2O2 (100 × 10−6m) was added during cell culture to simulate the tumorous microenvironment  the hypoxia atmosphere. Strong fluorescence was observed in the control group, whereas only a faint glow was observed when cells were incubated with pPt and H2O2 : 1) untreated, 2) pPt, 3) only square‐wave electric field (E), 4) free GOx, 5) PtG, 6) pPt plus square‐wave electric field (pPt+E), and 7) PtG plus square‐wave electric field (PtG+E). After intratumoral injection, the square‐wave electric stimulation was applied at 5 mA for 10 min , and the variation of tumors in mice was examined in the following 14 days. In addition, the in vivo biodistribution was evaluated at 24 h postinjection . The distributed amount of Pt within tumor reached ≈40.9 µg g−1, while negligible presence of Pt was observed in other major organs.The in vivo antitumor activity of PtGs under square‐wave electrical stimulation was evaluated in a mouse model, following the treatment process demonstrated in While no clear changes were observed in body weight for all the seven groups , the tumor growth differed significantly in all seven mice groups Figure . The resAdditionally, hematoxylin and eosin (H&E) staining, Ki‐67 staining, and immunofluorescence terminal deoxynucleotidyl transferase‐mediated dUTP‐biotin nick end labeling (TUNEL) were conducted for histological analysis after mice scarification Figure . In the Overall, it is worth noting that, compared to the current electrochemical therapy (EChT) which has been used clinically, the electrode configuration has been significantly simplified in EDT due to its distinctive functioning mechanisms. However, the operation procedure of EDT may still remain inconvenient in the actual clinical practice. To develop EDT‐based therapeutic protocol with less invasive electrodes following its fundamental principle is therefore anticipated to tackle this challenge in the follow‐on investigations.32O2. Apart from serving as a delivery cargo, pPt catalyzes both endogenous and exogenous H2O2 to produce O2, which in turn magnifies the GOx‐mediated starvation. Meanwhile, under an alternating electric field pPt can also induce a considerable content of intracellular ROS via electrodynamic catalytic reaction to promote the cellular apoptosis. In consequence, the PtG nanocomposite exhibits remarkable synergistic curative effect toward malignant solid tumor both in vitro and in vivo. This study has therefore demonstrated a highly potential therapeutic strategy and its feasible platform for effective tumor treatment.Here for the first time, this study proposed a feasible therapeutic platform enabling synergistic multimodal approach, electrodynamic therapy combined with self‐boosting tumor starvation. For this purpose, pPts with fine microstructure were synthesized and incorporated with GOx molecules (PtG). GOx can effectively consume glucose via catalytic oxidation and generate HThe authors declare no conflict of interest.Supporting InformationClick here for additional data file.


Epoxy resin was directly grafted onto the surface of mica powder by high-temperature mechanical ball milling. This method was used to achieve a chemical reaction between the epoxy resin and mica that cannot be carried out under conventional circumstances. The results show that an epoxy resin layer with a thickness of approximately 10 nm formed on the surface of the mica. This modified mica filler exhibited a significant change in its hydrophilic properties. The dispersion of mica and its compatibility with organic coatings also significantly improved. In addition, the modified mica filler was added to the epoxy coating. The improvement of the coating’s compactness and toughness is the reason for its anti-corrosion performance enhancement. Epoxy resin has excellent adhesion; low shrinkage; and good permeability resistance to water, acids, alkalis, and other corrosive media. Thus, it has been commonly used as an anti-corrosion coating in both industrial and daily-life applications ,4,5,6,7.2 adsorption on mica in an acidic environment, which improved the whiteness and sun protection factor of mica. Hu et al. [Various physical and chemical methods are often used for the surface modification of fillers to solve the problem of interface compatibility between fillers and resins ,20,21,22u et al.  used a cu et al. ,26,27. TThe mechanochemical method is widely utilized to increase the reactivity of reactants by accumulating mechanical energy through grinding, compression, and other actions, causing changes in the physical and chemical properties and even the structures of materials ,29,30,31To answer this question, traditional mechanical ball milling was first used to try to graft epoxy resin onto the surface of mica, but this could not be successfully achieved. To this end, high-temperature mechanochemistry was tested in the hope of further increasing the energy input by providing thermal energy so that the mica and epoxy resin organic molecules would bond to complete the modification of the surface of the mica. In this article, the feasibility of the high-temperature mechanochemical modification of inorganic fillers was first studied, and organic epoxy resin was successfully grafted onto the surface of mica. High-temperature mechanochemical modification simplifies the modification process, increases the interface compatibility between the modified mica and the epoxy coating matrix, and thus improves the corrosion resistance of the coating. The main process is to open the epoxy-functional group in the epoxy resin through high temperatures and mechanochemistry to make it react with the hydroxyl group on the surface of the mica, thereby achieving the surface modification of the mica. The methods used in this paper—Fourier transform infrared spectrometry (FTIR), thermogravimetric analysis (TGA), and transmission electron microscopy (TEM)—indicated that the graft modification of organic epoxy molecules on the surface of mica was successfully achieved. At the same time, a corrosion test showed that the corrosion resistance of the modified mica epoxy coating was greatly improved, and the mechanism of the modified mica’s effect on the coating performance improvement was discussed.Commercially available mica powder of 25 μm size (the average particle size) was used . An epoxy resin-based coating was used in this study and consisted of an E44 epoxy resin  as the binder, polyamide  as the curing agent, and xylene as the solvent, with a mass ratio of 1:0.8:0.4 for a stoichiometric reaction.The epoxy resin and mica were weighed and mixed according to a mass ratio of 1:1, and the mixture was added into the grinding tank of the 500 mL high-temperature ball mill for the grinding test. Meanwhile, agate balls with the material ratio of 5:1 were used as the grinding medium. The grinding time was 6 h, the rotation speed was 600 r/min, and the experimental temperatures were set to 25 °C and 200 °C. At the end of the experiment, the reacted mixture was repeatedly filtered and washed at least three times with a mixed solvent (xylene and n-butanol in a ratio of 7:3) to remove the unreacted epoxy resin from the mica surface. The modified product was then dried in a 60 °C oven for 24 h.−1). A contact angle tester  was used to test the wettability of the mica surface; the droplet nature in the measurements was a solution formed by dissolving epoxy resin in a mixed solvent (xylene and n-butanol in a ratio of 7:3).The modified mica powder was dispersed in a mixed solvent and sonicated for 10 min to make a suspension, and the particle size distribution of the modified mica was measured with an ultrasonic particle size analyzer (Master sizer 2000). A sedimentation bottle was used for the sedimentation experiment. A thermogravimetric analyzer  was used to analyze the degradation process of the organic resin on the surface of the modified mica during the heating process with a nitrogen atmosphere. Transmission electron microscopy  was used to observe the microstructure of the mica powder surface. A Fourier transform infrared spectrometer  was used to observe the epoxy functional groups on the mica surface  was used to observe the microscopic morphology of the cross-section of the coating sample. A salt spray test chamber  was used to test the salt spray resistance of the coating; five parallel samples were set for each experiment, and the temperature was set at 35 °C.The coating samples were immersed in 3.5 wt.% NaCl solution to carry out electrochemical impedance spectroscopy  Co., Ltd., Shanghai, China) tests. The working area was 4 cmIn order to study the feasibility, experiments on unmodified mica, mechanochemically modified mica at room temperature, and mechanochemically modified mica at 200 °C were designed. The particle size distributions of the three groups are shown in −1, 2920 cm−1, and 2810 cm−1 caused by –CH3 and –CH2 stretching vibrations, and those at 1620 cm−1 and 1510 cm−1 are benzene ring stretching vibrations [−1 and 3420 cm−1 (–OH stretching vibration), 1020 cm−1 (Si–O–Si stretching) and 770 cm−1 (Si–O stretching vibration) indicates the presence of hydroxide groups and silico–oxygen bonds [−1 were caused by –CH3 and –CH2 stretching vibrations, and those at 1620 and 1510 cm−1 indicate benzene ring stretching vibrations. The new peak position is close to the absorption peak position of the group in the epoxy resin molecule, which shows that high-temperature mechanochemistry has completed the graft modification of the mica surface. The above results show that high-temperature mechanochemistry can trigger a reaction to complete the surface modification of mica.brations , and theen bonds . CompareFor the FTIR curve of mica after high-temperature mechanochemical modification, the corresponding methyl or methylene stretching vibration of the epoxy resin and the benzene ring stretching vibration peaks appeared, which proves the presence of the organic epoxy resin on the surface of the mica. However, the modified epoxy resin did not show the original vibration peak of the epoxy group. This result indicates that there exists a bonding reaction between the epoxy resin and the hydroxyl group on the surface of the mica during the modification process. Organic resin is successfully grafted onto the surface of the mica. The specific reaction process is schematically represented in On the other hand, the TGA curve of the modified mica b represePhotographs of the sedimentation for different samples are presented in 0.01Hz) is used to characterize the corrosion protection property of the coating [0.01Hz was 5.11 × 1010 Ω cm2  until 1200 h of immersion. It can be seen that the |Z|0.01Hz value of the modified mica coating was one and two orders of magnitude higher than that of the unmodified mica coating. The modified coatings provided better corrosion protection than the unmodified coatings with the same content.11 Ω cm2 a. After  coating d, the in−1 to 105 Hz, as shown in 3 and 105. By contrast, the frequency range of the modified coating phase angles close to 90° was wider than that of the unmodified mica coating, which was 100 to 105. In general, the larger the semicircle diameter in the Nyquist graph, the better the corrosion resistance of the coating [At the same time, in the initial stage of immersion, the frequency range of the two coating phase angles close to 90° was 10 coating . It can The effect of unmodified mica and modified mica particles on the adhesion behavior of the epoxy coatings was assessed by pull-off and representative dry and wet adhesion strength, respectively. The pull-off test results, along with the detaching status images, are presented in In this work, a high-temperature mechanochemical method was used to graft organic epoxy resin onto the surface of mica. The mechanochemical method inputting higher energy was used to modify the mica. Finally, the hydroxyl group on the surface of the mica reacted with the epoxy group in the epoxy coating, thus achieving the modification of the mica surface.The modified mica surface formed an organic resin layer with a thickness of about 10 nm. The properties of this layer were lipophilic and hydrophobic, causing an increase in the contact angle of the mica surface from 26 to 108°, which greatly changed the mica surface properties.The presence of the organic resin layer on the surface of the mica greatly improves the interface compatibility between the filler and the epoxy coating. The agglomeration of the filler in the coating is significantly reduced. The improvement of the coating’s compactness and toughness is the reason for its anti-corrosion performance enhancement.


The pre-implantation embryo in a competent status and post-implantation fully differentiation of the inner cell mass (ICM) and trophectoderm (TE) are prerequisites of successful implantation. Type I embryonic epithelial-mesenchymal transition (EMT) involves in these processes. A high level of the mir-let-7 family was found in the dormant mouse embryo of implantation failure in our previous study. Besides, its natural inhibitor lin28a was found to function in maintained stem cell pluripotency and involved in early embryo nucleolus construction. Until now, few studies got involved in the exact molecular mechanism that affects embryo implantation potential. In this study, the possible function of Wnt/β-catenin-lin28a/let-7 pathway in mouse embryo implantation was studied.ICR mouse, Lin28a/Let-7 g transgenic mice (Lin28a-TG/Let-7 g-TG), and implanting dormant mice models were used for the study.Wnt/β-catenin signaling is essential in embryo implantation, which promotes embryo implantation through directly trigger lin28a expression, thus represses the mir-let-7 family. Lin28a and mir-let-7 both participate in implantation via an inverse function. Lin28a and mir-let-7 participate in embryo implantation through embryonic EMT.Wnt/β-catenin signaling promotes embryo implantation and accompanying embryonic EMT, which is mediated by directly activate lin28a/let-7 axis.Video abstract Development of the blastocyst to the competent status and orchestrated embryonic EMT that accompany and underlie implantation and embryonic morphogenesis are essential for a successful pregnancy.The pre-implantation mouse blastocyst is a differentiated, fluid-filled ball that comprises two distinct cell populations, the ICM and surrounded TE . With thThe signaling pathway driven by Wnt/β-catenin seems to be one of the required pathways to achieve blastocyst competency for implantation. Silencing of Wnt/β-catenin signaling in mouse embryo reduced Cdx2, a key transcription factor involved in TE lineage specification, negatively affected the implantation competency of blastocyst . In the MicroRNAs constitute a large family of approximately 21-nucleotide-long, noncoding RNAs. They occupy 5% of the human genome but work as key post-transcriptional regulators of more than 70% of genes through mRNA decay and/or translational inhibition . MicroRNLin28a, a natural inhibitor of mir-let-7 family, is a member of a RNA-binding protein (RBP) family conserved in animals , 17. LinIn this study, the regulation of mir-let-7 by Wnt/β-catenin-lin28a signaling was investigated, and their function in mouse embryo implantation competency and embryonic EMT were firstly studied.The use of animals was approved by the Committee on Use of Live Animals in Teaching and Research, the University of Hong Kong . Imprinted-coding region (ICR) mice were purchased from Harlan UK Ltd., Bicester, Oxon, UK. We generated a chimeric let-7 g species let-7S21L  in which let-7 g can be induced with doxycycline under the control of the Rosa26 locus . Lin28a transgenic (Lin28a-TG) mice was a kind gift from Children’s Hospital Boston and Dana Farber Cancer Institute, Boston, which also can be induced expression by doxycycline. Lin28a-TG and Let-7 g-TG mice were housed with food and water under controlled temperature (24 ± 1 °C), humidity (55 ± 2%), and lighting conditions . The morning of finding a vaginal plug was designated as Day1 of pregnancy. Embryos at different developmental stages were recovered through flushing the oviduct on the morning of Day 1, 2, or 3 of gestation  or uterus on Day 4 (blastocyst). Implantation sites were detected on Day 5, Day 6 or Day 7 evening by Chicago blue  via caudal vein injection (20 μl/mice). Transgenic mice were fed with doxycycline hyclate  in water from the mating day; for the control group, no doxycycline added. For embryo Wnt activation and inhibition, Wnt activator CHIR99021  at 10 μM and Wnt inhibitor mouse DKK (sc-5897-DK) at 1 μg/ml were used.The whole surgical procedure was completed before 8:00 am. The Day 4 pregnant female mice were anesthetized by phenobarbitone. The operation area was near the abdominal cavity on two sides of the back. After removing body hair, skin, and muscle incision were made to expose the para-ovarian fatty tissue, which, together with the ovary, were cut between the oviduct and the ovary. The muscles layer and the body skin were sewed up separately. The surgical site was disinfected with 70% alcohol after surgery. Meloxicam  was fed in water to release their pain. Progesterone  was administered on Day 5 and Day 6 of pregnancy by subcutaneous injection to maintain the delayed implantation status . EstradiEmbryos were fixed in 4% paraformaldehyde at room temperature for 30 min. Then washed three times in wash buffer (0.01% triton and 0.01% NP40 in PBS), each for 5 min. Permeabilization in 0.4% triton and 0.4% NP40 in PBS for 15 min at room temperature. Incubation with 1% BSA  for 1 h at room temperature. Embryos were then incubated with primary antibodies against active-β-catenin , lin28a , total β-catenin  at 4 °C overnight. After 4 rounds of wash in wash buffer, each for 10 min, embryos were incubated with anti-rabbit AF488 , anti-rabbit AF568 , or anti-mouse AF488  secondary antibody at room temperature for 1 h, washed and incubated with DAPI  for 5 min. After washing, fluorescence signals were examined under a Zeiss LSM 700 confocal laser microscope .2O2 solution was applied to sections for 30 min to block endogenous peroxidase activity. To unmask the antigenic epitope, slides were boiled in antigen retrieval buffer . Cooled down the slides at room temperature, then rinsed with PBS. Added 1% fetal bovine serum to block the sample at room temperature for 1 h. Applied primary antibody and incubated at 4 °C overnight. Slides were washed with PBS for 4 times. Applied (1:300) biotinylated secondary antibody to the sections and incubated at room temperature for 1 h. Washed with PBS again. Applied ABC buffer  to the sections and incubated at room temperature for 30 min. Washed slides with PBS. Applied DAB substrate solution  to the sections for color development, Washed slides with PBS. Counterstained slides by Hematoxylin. Dehydrated the tissue slides again through 4 times of alcohol , 5 min each. Cleared the tissue slides in three times of xylene and mounting. Antibodies used in this assay were active-β-catenin , lin28a , E-cadherin , N-cadherin .Day 5, Day 6, and Day 7 implantation sites were cut, fixed in 4% PFA, embedded in paraffin, and cut from the transverse surface onto glass slides. Deparaffinized slides in xylene for 2 times. Rehydration the slides in gradient alcohol. 3% HThe High Capacity cDNA RT Kit  was used to produce cDNA of target miRNAs according to the manufacturer’s instructions. The template of miRNAs was obtained by heating the embryos in 6.2 μl 5% NP40  at 95 °C for 10 min. 10 embryos were pooled in each sample. The miRNA reverse transcription primers and probes for mature let-7a-5p , let-7 g-5p , U6snRNA  were used. The reverse transcription condition was 16 °C for 30 min, 42 °C for 30 min, 4 °C for 5 min. The procedure of qPCR was 50 °C for 2 min, 95 °C for 10 min, 95 °C for 10 s, and 60 °C for 1 min.Pregnant Day 4 mouse uteri were cut from mouse abdominal cavity, removed fat tissues, and washed in PBS. Cut longitudinally and digested in 0.5% trypsin  and DNase I  at 4 °C for 90 min and then at 37 °C for 30 min. Tissues were then transferred to complete DMEM/F-12  and shaken gently for 30 s, three times. The dispersed luminal epithelium and some glandular epithelium in the upper cell suspension were collected and centrifuged at 1500 rpm for 5 min, then washed by centrifugation in complete DMEM-F12 for another 2 times. The recovered epithelial cells were seeded or collected.The procedure was according to the manufacturer . The PCR primers used were as follows: Site 1 forward: GGGGTTGGGTCATTGTCTTT, Reverse: CTCGGTACCAAGCTTGCG; Site 2 forward: GCACTTTCTCTTTTCTAACTGGG, Reverse: GGAGTTCTGGACACTGGGAA, Site3 forward: GCTTACCTGGGGCTAACTTT, Reverse: ATCTGAATGTCTGCCTCCCA; Site 4 forward: GAGCGCATTTTAGGGTGGTT, Reverse: TTCCTCCCTCCAAACCTGTG; Site5 forward: CAGTCCTGTTCCTCCACCAG, Reverse: CATCCACTGCTGAGAGAGGG; Published site 1 forward: GTGCGGGGGAAGATGTAGC, Reverse: CAAACTGCTGGTTGGACACG; Published site 2 forward: GTGGTTTGTGTTTCTGATTGGC, Reverse: GAGTACCCCGGAATTTGAGATC. The PCR procedure was according to Taq 2X Master Mix , that was 95 °C for 30 s, 95 °C for 30 s, 45–68 °C for 1 min, 68 °C 1 min per kb, 68 °C for 5 min, 35 cycles from the second step to the fourth step.2 265 g mg, MgCl2 0.0469 g, KCl 0.2 g, NaCl 8 g, NaH2PO4 0.05 g to 1 l and adjust pH to 2). Embryos were then put on to Day 4 ME monolayer. To determine attachment, the plate was shaken at a rate of one rotation/second on an orbital shaker for 20 s after 24 h culture. Blastocysts that remained at the same location were designated as attached blastocysts. At 48 h, outgrowth was identified as the area covered by trophoblastic monolayer around the embryo.Embryos were retrieved from pregnant Day 4 mouse uteri. The zona pellucida of blastocysts was removed by acid tyrode , lin28a siRNA , Scramble control siRNA  were electro-transfected into blastocysts in a flat electrode (1 mm gap between electrodes) in 20 μl of Hepes-buffered saline  by two sets of 3 electric pulses of 1 ms at 30 V using a 830 Electro Square Porator . Following electro-transfection, embryos were cultured in fresh M16 medium , and waited for recovery.p < 0.05 using one way ANOVA.All the images of confocal microscopy were representatives of at least three independent experiments. qPCR assays were performed in triplicate, and each experiment was repeated for at least three times. Attachment/outgrowth assay has been done at least four times. Data shown were presented as means ± sd. Differences were considered statistically significant at Previous researches revealed that Wnt/β-catenin signaling is necessary for embryo implantation competency . Mouse eTo further demonstrate Wnt/β-catenin is necessary for embryo implantation. p-GMT-TCF/LEF-GFP vector, which produces GFP protein upon activation of the canonical Wnt/β-catenin pathway, was electro-transfected into mouse blastocysts. GFP fluorescence was seen automatically expressed in the whole embryo during outgrowth at 48 h, confirming that the pathway was stimulated within this period Fig. b.The function of Wnt in embryo implantation competency was further studied. Blastocysts were cultured on fibronectin to exclude the effect of the uterine epithelium. Embryos were cultured with Wnt activator CHIR99021 or inhibitor DKK containing M16 medium for 24 h to count attachment and 48 h to observe outgrowth. Wnt stimulation increased embryo attachment (Fig. Several observations suggest that miRNAs are involved in early pre-implantation embryo development and implantation process , 27. OurPathway analysis suggested that Wnt signaling was potentially associated with the differential miRNAs expression between activated and dormant embryos . Wnt actOur lab occasionally owns let-7 g-TG mice. As let-7 family members contain the same core sequence, function similarly but location in different chromosomes. Let-7 g-TG mice model was used to study the function of let-7 family in embryo implantation. After 4 days of induction, the let-7 g level in blastocysts was elevated by more than 15 times relative to the control blastocysts without induction Fig. a. When lFor the in vivo implantation model, 10–15 Let-7 g-TG blastocysts that have been fed with doxycycline for 4 days and control blastocysts that have a normal level of let-7 g were transferred into each pseudo-pregnant mice on Day 4. The number of implantation sites was determined on Day 6 of pregnancy Fig. f, g, h. In summary, these results demonstrate that a high level of let-7 family members inhibits embryo implantation competency.Result in Fig. At the mRNA level, lin28a mRNA increased upon activation and decreased after inhibition of Wnt Fig. b. These To verify whether the regulation of Wnt/β-catenin on let-7 was through lin28a in mouse embryos. Blastocysts were electro-transfected with scrambled control siRNA and lin28a siRNA respectively and cultured for 24 h in M16 medium. The efficiency of lin28a knockdown was confirmed by immunofluorescence staining and qPCR, respectively Fig. a, b. TheIt was further hypothesized that Wnt signaling facilitates embryo implantation via lin28a/let-7 axis in the process of implantation. Mouse blastocysts were electro-transfected with let-7a inhibitor or scramble siRNA in the presence or absence of Wnt inhibitor DKK. The treated embryos were cultured on the fibronectin-coated plate in M16 medium for 24 h. DKK alone decreased embryo attachment on fibronectin, while let-7a inhibitor alone increased embryo attachment. Co-treatment of the embryo with DKK and let-7a inhibitor abolished the suppressive effect of Wnt inhibition on embryo attachment Fig. d, suggesEmbryonic EMT participates in implantation, placentation, and organ development, which is essential for a successful pregnancy , 31, 32.Active-β-catenin has been reported expressed in the nucleus of mouse embryos from 1 cell to blastocyst . HoweverThough active-β-catenin mediated canonical Wnt signaling is not required for pre-implantation embryo development, silencing of Wnt/β-catenin signaling in pre-implantation embryo reduces Cdx2, a key transcription factor involves in TE lineage specification, and considerably reduced implantation rate upon transfer to pseudo-pregnant recipients, and the Wnt downstream target c-Myc, which has been shown to be crucial for pre-implantation embryo development, is also down-regulated in blastocyst TE . SimilarEmbryonic EMT during implantation is designed as type I EMT . In our In the present study, lin28a locates in both cytoplasm and nucleus, especially in the nucleolus of mouse embryos Fig. . Lin28a In breast cancer, Wnt/β-catenin inhibits breast cancer stem cell expansion through direct binding to lin28a promoter and inhibits let-7a . BesidesLet-7a/g were decreased suddenly in the pre-implantation embryo. Overexpressed let-7 g in let-7 g-TG mice embryo decreased embryo implantation ability. Integrin-3β has been proved as a down-stream target of mir-let-7 in our previous study . IntegriThere is only one publication mentioned that the promoter activity of mir-let-7a2 in the breast cancer cell line could be downregulated by Wnt activator lithium chloride in luciferase assay . It meanWnt signaling and the mesenchymal marker were decreased in let-7 g overexpressed embryonic cells. It means let-7 g inhibit EMT process in the embryo. It has been reported overexpressed let-7b activating epithelial marker E-cadherin and attenuating mesenchymal marker Vimentin, EMT transcriptional factor Twist as well as β-catenin in malignant pleural mesothelioma . In thisIn this study, current understanding is Wnt/β-catenin is essential for proper embryo implantation, and EMT, which function via directly binding to lin28a promoter to activate its transcription, thus represses mir-let-7 biogenesis. Lin28a and mir-let-7 family inversely function in embryo implantation competency and EMT under the regulation of Wnt signaling. In conclusion, our studies first shed light on Wnt/β-catenin-lin28a/let-7a pathway in embryo implantation and accompanying EMT.Additional file 1.Additional file 2.Additional file 3.