Antagonism of transient receptor potential vanniloid-1 (TRPV1) and desensitization of transient receptor potential ankyrin-1 (TRPA1) nociceptors alleviate inflammatory bowel diseases (IBD)-associated chronic pain. However, there is limited literature available about their role in regulating the mucosal layer, its interaction with host physiology, and luminal microbial community. The present study focuses on the effects’ intra rectal administration of capsazepine (modulator of TRPA1/TRPV1 expressing peptidergic sensory neurons) on colonic mucus production and gut health. We performed histological analysis, gut permeability alteration, gene expression changes, metabolite profiling, and gut microbial abundance in the ileum, colon, and cecum content of these animals. Intra rectal administration of capsazepine modulates TRPA1/TRPV1-positive nociceptors  and resulted in damaged mucosal lining, increased gut permeability, and altered transcriptional profile of genes for goblet cell markers, mucus regulation, immune response, and tight junction proteins. The damage to mucosal lining prevented its role in enterosyne (short chain fatty acids) actions. These results suggest that caution must be exercised before employing TRPA1/TRPV1 modulation as a therapeutic option to alleviate pain caused due to IBD. The relhe colon . Moreovehe colon  and are he colon ,5 or colhe colon . In the he colon . In the he colon . Evidenche colon ,8. Similhe colon ,10. Givehe colon ,12,13, tCapsazepine (CPZ) is a selective inhibitor for TRPV1 channel and have been employed as a treatment to reduce severity of colitis in pre-clinical models ,10,13. A3− secretion in colon, which is essential for mucus release [The intestinal mucus layer protects against mechanical, chemical, and biological attacks and contributes to the maintenance of intestinal homeostasis  as an in release . Recentl release . ConsideShort-chain fatty acids (SCFAs), the enterosyne released from anaerobic bacterial fermentation of dietary substrates (mainly prebiotic containing indigestible carbohydrates) ,20,21, pSensing the significance of TRPA1 and TRPV1 co-expression ,31, theiSpp1, Trpa1, Th, and Calca genes  and proline, one of the core amino acids of PTS domain in mucin for its structural integrity . In addi Control .+ve bacteria—Roseburia, Lactobacillus, Akkermansia, Fecalibacterium, Eubacteria, Ruminococci, Butyricicoccus pullicaecorum, Clostridium coccoides, Anaerostipes butyraticus, Lachnospiraceae, and Firmicutes significantly  from goblet cells (GC) and crypts (C) of colon, but a minimal effect was observed upon their co-administration with CPZ. In addition, SCFA co-administration was unable to reverse the CPZ-induced morphological alteration in mucosal architecture  owing to their involvement in neurogenic inflammation ,32. TherMuc3 and Muc2 genes were significantly decreased in CPZ treated animals. In studies on Muc2 knockout animals, increased intestinal permeability has been shown to trigger a cascade of events leading to secondary organ inflammation and translocation of bacterial metabolites and bacteria per se leading to sepsis [Cdx2, Dll1, Foxa2, Tff3, Dll4, Vamp8, Prkd1, and Foxa3) and Fcgbp were significantly decreased in the CPZ group. A decrease in Muc3  points to compromised cell protection  and altered signal transduction pathways associated with the microbe: host interactions [Muc15 was significantly higher in the colon of CPZ treated mice and has a role in carcinogenesis and protein metabolism [Nlrp3, Nod2, Tnfα) [Nlrp3 and Tnfα are very well known for their role in the disruption of intestinal permeability [Nod2 gene has been reported to be associated with improvement in colonic mucus production, intestinal permeability, and inflammation [Nod2 in our findings might suggest a compensatory reflex mechanism to reverse the colonic alterations developed due to CPZ treatment.o sepsis ,42. Diffractions . In conttabolism . Alterat2, Tnfα) ,46. Nlrpeability ,47,48,49ammation ,51. The Lactobacillus, Fecalibacterium, and Akkermensia, etc.) as well as SCFA producing bacteria—Roseburia, Lactobacillus, Fecalibacterium, Eubacteria, Ruminococci, Butyricicoccus pullicaecorum, Clostridium coccoides, Anaerostipes butyraticus, etc. [n = 5–6). In our future studies with IBD model (colitis), we will address this.The mucin layer is a source of nutrition and provider of attachment sites for gut microbes . In the us, etc.  in CPZ tn = 24), procured from IMTech Center for Animal Resources and Experimentation , Chandigarh, India, and housed  in Animal Experimentation Facility (2039/GO/RBi/S/18/CPCSEA), National Agri-Food Biotechnology Institute (NABI), Mohali, India. Animals were provided with water and a normal pellet diet  ad libitum. The study was conducted according to the guidelines of the Declaration of Helsinki, and study protocol was approved by the Institutional Animal Ethics Committee (IAEC) of NABI . Committee for the Purpose of Control and Supervision on Experiments on Animals (CPCSEA) guidelines on the use and care of experimental animals were followed throughout the experiment.Six–eight weeks-old male C57BL/6J mice (25–30 g) (n = 6 each housed in one cage). Intrarectal administration of CPZ was done as per previously described method with slight modification [w/v capsaicin solution  and tail-flick test (in water kept at 42 °C) were employed for the confirmation of loss of TRPV1 activity. Replicates for each animal were taken using both eyes separately. The plan of experiment is explained in After 1 week of acclimatization, animals were divided into 4 groups—Control, Capsazepine (CPZ), SCFA (Short-chain fatty acids), and CPZ + SCFA  was dissolved in 10 mL of distilled water at the same temperature. The aqueous phase was incorporated drop-by-drop into the oil phase over constant stirring of 1500 rpm and kept for 1 h, followed by sonication using a probe sonicator for 10 min (5 s each on and off-cycle at 40 Hz frequency). FITC-nanoparticles were lyophilized and dispersed in milli-Q water before administration.The fluorescent nanoparticle was prepared according to Ahmad et al. . BrieflyDetermination of colon permeability was assessed as a previously reported method  with som−ΔΔCt method [Total RNA was extracted from dorsal root ganglion (DRGs) using the TRIzol RNA extraction reagent-based method. Isolated RNA was transcribed into cDNA using the Revert Aid cDNA synthesis kit . The synthesized cDNA was then used to perform gene expression of specific markers of peptidergic sensory neurons  by qRT-PCdx2, Dll1, Dll4, Foxa1, Foxa2, Foxa3, Klf1, Prkd1, Tff3, Vamp8)/tight junction protein (TJPs) /mucin production /glycosylation enzyme /immune response /4 housekeeping genes  was used. Furthermore, 50 ng of extracted RNA sample were hybridized with unique probes using the NanoString nCounter prep station and placed into the cartridge . A NanoString nCounter digital analyzer  was employed for the detection and counting of hybridized probes. The data were analyzed using nSolver 4.0 software .For transcriptional studies in colons, a customized Nanostring nCounter GX CodeSet gene expression panel , comprising genes of goblet cell differentiation  and stored in the same until processed. Fixed tissues were serially dehydrated, cleared, and embedded using gradient ethanol, xylene, and molten paraffin, respectively. In addition, 5-µm-thick sections were prepared, deparaffinized in xylene, and rehydrated in gradient ethanol. For staining, Hematoxylin-Eosin (H&E)  and Alcian blue (AB)  were used for 30 s, 15 s, and 15 min, respectively. Slides were mounted using DPX mounting medium . Images were captured at 10× on a bright-field microscope , and the thickness of the mucus layer was measured using Imagescope  manually. For high iron diamine (HID) staining for sulfomucins, rehydrated tissue sections were stained with HID solution for 18 h at room temperature in the dark followed by washing with distilled water 3 times. The sections were then stained with alcian blue for 30 min, washed 3 times with distilled water, and mounted with DPX mounting medium. Images were captured at 20× on the bright-field microscope .Zo1)- , Occludin (Occ) , Claudin-1 (Cldn-1)  for overnight at 4 °C. Finally, the sections were incubated with Alexa fluor 488  secondary antibody for 2 h at room temperature. DAPI stain was used to stain nuclei. Images were taken under a confocal microscope  and all the images were captured at 40x magnification. Images were analyzed using ImageJ software .For immunohistochemistry, 5-µm rehydrated sections were treated for antigen retrieval using citrate buffer (pH 6.0), blocked in 5% goat serum for 1 h followed by incubation in primary antibodies, Zona occludens-1 (For Scanning Electron Microscopic (SEM) analysis, small fragments of distal colon were fixed in 2.5% glutaraldehyde solution  in PBS (pH 7.4) for 6 h at 4 °C. The tissues were washed with PBS, post-fixed in 1% osmium tetroxide  in PBS (pH 7.4) for 2 h at 4 °C. Tissues were washed with PBS and dehydrated through the serial application of increasing concentration of acetone  viz. 30%, 50%, 70%, 90%, 95%, 100% to remove water at 4 °C for a 30 min period. Samples were air-dried and spur-coated with gold (10 nm). Images were acquired using Apreo S SEM .−ΔΔCt method [DNA was isolated from 100 mg cecal content, using Nucleospin DNA Stool Genomic DNA Purification Kit , as per the manufacturer’s instruction. DNA was quantified on a spectrophotometer  and run on 0.8% agarose gel electrophoresis for integrity. qRT-PCR was performed as per the protocol detailed in t method . Ct valum/z, the gain factor was set at 10, and thermal auxiliary temperatures 1 and 2 were set at 150 and 280 °C, respectively. Mass fragmentation of each peak of the MS spectrum was matched with the NIST-MS library (version 2.0) for identifying metabolites and were reported in the form of percent change.Gas chromatography-mass spectrophotometry (GC-MS) was employed for metabolite analysis. Briefly, 80 mg of cecal content was treated with methanol, chloroform, and water (in a 2.5:1:1 ratio) at 4 °C, overnight with shaking. Samples were centrifuged (3000 rpm) and the supernatant was lyophilized. For derivatization, lyophilized samples were treated with 90 µL N, O-bis(trimethylsilyl)tri-fluoroacetamide (BSTFA), 1% trimethylchlorosilane (TMCS) , and 30 μL of pyridine  and incubated for 4 h at 70 °C. The samples were diluted up to 500 µL with dichloromethane (DCM) , and 5 µL were injected into GC-MS through an automatic sampler. An Agilent 7870A chromatograph  equipped with an Agilent triple-quadrupole 7000 mass spectrometer  was employed for performing analysis. A DB-5 column  was used for the separation of derivatized molecules before reaching the MS chamber for their efficient identification. The column oven temperature was initially set at 80 °C for 2 min, then increased to 140 °C at 10 °C/min, and then increased from 140 to 250 °C at 5 °C/min. The helium carrier gas flow was set at 1.5 mL/min in spitless mode. For MS, the source temperature was set at 230 °C, electron ionization was set at −70 eV, the scan range was kept at 30–500 w/v) was prepared in 1X PBS containing cysteine with homogenization. Fecal batch fermentation was done with 531 µM CPZ and glucose (1% w/v)  for 48 h. Basal growth media of the following composition were used (composition (g/L) peptone water 2 g/L, yeast extract 2 g/L, NaCl 0.1 g/L, K2HPO4 0.04 g/L, KH2PO4 0.04 g/L, MgSO4·7H2O 0.01 g/L, CaCl2·6H2O 0.01 g/L, NaHCO3 2 g/L, Tween 80 2 mL, hemin 0.02 g/L, vitamin k1 10 mL, cysteine HCl 0.5 g/L, pH 7.0) . Initial pH was maintained at 7. Bacterial DNA extraction was done after 48 h of fermentation using a Nucleospin DNA Stool Genomic DNA Purification Kit , and the relative abundance of selected gut bacteria was determined by qRT-PCR similar to Slurry from freshly collected fecal samples  package in R [Data were analyzed using Graphpad Prism 8 software . All the data are presented as mean ± SEM. Student’s unpaired age in R , while tIn conclusion, the present study suggests the significance of CPZ treatment for pain and its unwanted effects (possibly via TRPA1/TRPV1 modulation) on mucosal health . However


Awareness toward the risks of blue light (BL) exposure is rising due to increased use of BL-enriched LEDs in displays. Short-wave BL (400–500 nm) has a high photochemical energy, leading to the enhanced production of reactive oxygen species (ROS). BL potentially plays a role in causing dry eye, cataracts, and age-related macular degeneration (AMD). The effect of BL on retinal pigment epithelium cells (RPEs) or photoreceptors has been extensively investigated. In contrast, only a few studies have investigated the effects of BL exposure on Müller cells (MCs). This is mainly due to their lack of photosensitive elements and the common assumption that their reaction to stress is only secondary in disease development. However, MCs perform important supportive, secretory, and immune functions in the retina, making them essential for retinal survival. Increased oxidative stress is a key player in many retinal diseases such as AMD or glaucoma. We hypothesize that increased oxidative stress can also affect MCs. Thus, we simulated oxidative stress levels by exposing primary porcine MCs and human MIO-M1 cells to BL. To confirm the wavelength-specificity, the cells were further exposed to red (RL), purple (PL), and white light (WL). BL and WL exposure increased ROS levels, but only BL exposure led to apoptosis in primary MCs. Thus, BL accounted for the harmful part of WL exposure. When cells were simultaneously exposed to BL and RL , cell damage due to BL could be partly prevented, as could the inhibition of p53, demonstrating the protective effect of RL and p53 dependency. In contrast, BL hardly induced apoptosis in MIO-M1 cells, which is likely due to the immortalization of the cells. Therefore, enhanced oxidative stress levels can significantly harm MC function, probably leading to decreased retinal survival and, thus, further enhancing the progression of retinal diseases. Preventing the cell death of these essential retinal cells represents a promising therapy option to enhance retinal survival. The length of time spent indoors and in front of screens has increased drastically over the last few years, mainly due to enhanced remote working since the SARS-CoV-2 pandemic, resulting in longer and more intense exposure to artificial light . EspeciaAs multiday exposure to BL could increase load on cellular repair mechanisms, it is, furthermore, likely that the accumulation of subthreshold damage to cellular processes produces long-term sub-apoptotic cell stress that finally leads to apoptosis, associated with progressive diseases such as age-related macular degeneration (AMD) . In this2−), hydroxyl radical (•OH), hydrogen peroxide (H2O2), nitric oxide (NO), and singlet oxygen (O2). Hydroxyl radicals, the most reactive oxygen form, can form out of H2O2 [BL damage is characterized by the excessive production and accumulation of free oxygen radicals  in the mitochondria , further of H2O2 . ROS pro of H2O2 , and fur of H2O2 . This, a of H2O2 , and end of H2O2 , likely In ophthalmology, oxidative stress is suspected to be involved in the development of multiple eye diseases such as senile cataract , AMD 2727, uveitIn general, MCs are the only cells spanning the entire retinal thickness, generating numerous contacts to neighboring retinal cells. For guinea pigs, it is known that they act as wavelength-dependent optical fibers and leak blue–violet light toward the surrounding tissue . Therefo2O2) measurements    . The sig ± 0.25)  and MIO- ± 0.25)  strongly2O2 levels on these cells. To this end, caspase-mediated apoptosis, metabolic activity, cell viability, and cell density were measured in both primary MCs and the human MIO-M1 cell line over 24 h  (p < 0.0001) after exposure was detected (p < 0.001) and did not decrease before 24 h , further pointing to the highly energy-consuming death process, apoptosis, as the primary cell death pathway  A. In adddetected A. In acc pathway A. This h pathway A. During pathway C, again  pathway B. The ob pathway B. Likewi pathway B. Morpho pathway C and als pathway C. Taken 2) cells with 6 and 12 h further cultivation , which resembled a combination of BL and red light  in our exposure set up, for 1 or 1.5 h (2) exposure was performed to evaluate the non-toxic effect of this wavelength on MCs , only 1.5 h of PL exposure was able to slightly induce caspase 3/7 activity in MCs (p < 0.001). All other conditions had no effect on caspase 3/7 activation. This finding further underlines the assumption that only BL can significantly and strongly induce apoptosis in MCs, while parallel exposure to RL significantly (p < 0.001) decreased caspase 3/7 activity (p < 0.0001) decrease in cell viability 24 h after exposure , but RL exposure did not (p < 0.0001) , but to a lesser degree than BL exposure  D. PL expexposure D. Furtheexposure D. To furexposure . BL and exposure . Due to exposure .p < 0.05, p < 0.01; In the next step, the effect of PL and RL exposure on human MIO-M1 cells was investigated to determine differences between primary cells and the human cell line. Again, BL induced oxidative stress in MIO-M1 cells , whereas PL exposure only slightly induced oxidative stress levels, and RL exposure did not alter stress levels at all A. PL expTaken together, BL increased oxidative stress levels in MIO-M1 cells, but PL exposure only slightly induced oxidative stress levels and was, therefore, not able to alter caspase 3/7 activity, cell viability, or cell density . RL expop-nitro), a small, selective posttranscriptional inhibitor of p53, and pifithrin µ, a mitochondrial inhibitor of p53, was evaluated   (Only Pifithrin α treatment right after BL exposure (1.5 h) significantly decreased BL-induced oxidative stress levels after 6 h  A, demons < 0.01) ; therefo 0.0001) B, provin 0.0001) C.Treatment with pifithrin α increased the cell viability of BL-exposed MCs back to control levels, further increasing 12 and 24 h after exposure D, and reTo determine if pifithrin µ treatment is as effective as pifithrin α concerning cell viability and ATP levels, these parameters were also examined 24 h after BL exposure G,H. PifiMorphologically, more rounded cells  were observed in BL-exposed MCs A,C, furtIn summary, these data strongly suggest a role for p53 in BL-induced apoptotic processes in primary MCs.2O2 [2O2 generation [2O2  . There a2O2  or cobal2O2 ,46. A mo2O2 ,48. Thus2O2 . Further2O2 . It is f2O2 . Therefo2O2 . Mitocho2O2 , whereasneration ,54. Sincion [2O2 , it can ion [2O2 . Since aion [2O2 . This isotic MCs . BL expootic MCs ). Nevertotic MCs . AccordiFurthermore, p53 activity can be induced by oxidative stress ; hence, 2O2-treated MIO-M1 cells [2O2-exposed MIO-M1 cells [In contrast to the data generated in MCs, MIO-M1 cells were only slightly affected by BL exposure and enhanced oxidative stress levels . They deM1 cells , suggestM1 cells . In summary, BL-induced apoptosis in MCs seems to be p53-dependent, as inhibition of p53 leads to significant improvement and can prevent the cell death of MCs. Therefore, p53 represents a therapeutic target to prevent the death of MCs due to increased oxidative stress levels, thus also preventing the progression of oxidative stress-based diseases such as AMD. Furthermore, it is of great interest to investigate the influence of BL or oxidative stress on other retinal cells with respect to p53 in more detail.Porcine eyes were obtained from a local abattoir, which euthanized the pigs by electrocution. The eyes were immediately transported at 4 °C to the laboratory to ensure a maximum time of 3 h after death of the animals. The explants were prepared from 6-month-old pigs weighing 100 kg on average. Within 1 h after arrival of the eyes at the lab, retinal explants or primary cells were obtained. As a first step, eyes were cleaned and disinfected in 70% ethanol for 5 min. After two washing steps, they were opened using a scalpel under a laminar flow hood. The cornea, lens, and vitreous humor were removed.2 at 37 °C in an incubator. No passages higher than five were used.Müller glia cells were isolated by digestion of the porcine retinas with 1% papain  and DNase I  for 15 min at 37 °C with gentle shaking (900 rpm) in a thermo shaker  until homogeneous. After centrifugation  the cell pellet was resuspended in MC culture medium  supplemented with 5% fetal bovine serum  and 1% penicillin/streptomycin , and the entire cell suspension was transferred into 0.1% gelatin-coated culture vessels . By replacing the medium until cells were confluent and passaging them into freshly-coated dishes, a pure culture of MCs was obtained after three passages (identified by morphology and immunostaining against GFAP and vimentin) . Cells w2 at 37 °C in an incubator. The MIO-M1 culture medium contained high-glucose DMEM  supplemented with 5% FBS and 1% P/S.The human Müller cell line Moorfields/Institute of Ophthalmology–Müller 1 (MIO-M1) was obtained from the UCL Institute of Ophthalmology, London, UK . MIO-M1 2 and a peak at 455–465 nm wavelength , applied from below. During the exposure, the temperature was monitored. Furthermore, low-intensity BL exposure of retinal cells (15 mW/cm2) was applied from below  GmbH, Frankfurt, Germany). Purple light (PL) exposure was a combination of blue and red light (peaks at 450/630 nm) with an intensity of 15 mW/cm2 (3813.6 lx), applied from below  GmbH, Frankfurt, Germany). Retinal cells were exposed to WL with an intensity of 30 mW/cm2 , applied from below. Red light (RL) exposure (630 nm) with an intensity of 3 mW/cm2 was applied from below  GmbH, Frankfurt, Germany). The irradiation and subsequent cultivation time varied according to cell type and experiment, as indicated in each experiment’s description. Wavelengths were measured using a MAVOSPEC BASE analyzer .MCs and MIO-M1 cells were exposed to BL with an intensity of 30 mW/cm® Aqueous One Solution Cell Proliferation Assay  were used according to the manufacturer’s protocol. Absorbance was measured (490/690 nm ratio) using a Tecan Reader (NanoQuant infinite M200) [2O2 assay and white 96-well plates with a clear bottom  were used to determine the level of hydrogen peroxide in the retinal cell cultures. The assay was performed according to the manufacturer’s instructions (lytic version). Luminescence was measured using a luminometer (Tecan reader SPARK 10M). Activity of caspase 3/7 was determined using a corresponding Caspase Glo Assay from Promega, according to the manufacturer’s protocols [All assays were performed in 96-well plates. A total of 15,000 MIO-M1 cells or 20,000 MCs per well were seeded the day before the experiment (MCs on 0.1% gelatin-coated well plates), resulting in 80–90% confluency. To evaluate cell viability, transparent 96-well plates  and a CellTiter 96te M200) . In thisrotocols . The amop-nitro, ab1460 Abcam, Cambdrige, UK) or µ  at the indicated concentrations (1–10 µM) in a culture medium containing MCs for a certain amount of time. Stock solutions (100 µM) in low-glucose DMEM were aliquoted and frozen at −20 °C. Aliquots were used only once and further diluted in culture medium.p53 was inhibited using pifithrin α  was performed according to the manufacturer’s protocol. Briefly, 2 mg/mL cell lysate was used, which was obtained using the Cell Lysate Buffer + Protease Inhibitor Cocktail of the kit (according to the manufacturer’s instructions). Membranes were incubated with the lysate for 5 h at RT after blocking. Treatment with the primary antibody occurred overnight. After washing steps, incubation with the Biotinylated Antibody Cocktail was performed for 1.5 h at RT. After washing again, the membranes were treated with HRP–streptavidin for 2 h at RT, washed again, and incubated with detection solution; then, the chemiluminescence signal was recorded . The analysis was performed according to the manufacturer’s instructions, followed by statistical processing with GraphPad Prism 9 .2O stock solution, diluted 1:2000 in DPBS, Merck, Darmstadt, Germany) for 5 min, washed, and imaged using a fluorescence microscope with FITC (Annexin V) and AF555 (PI) filter sets [For annexin V/propidium iodide (PI) staining, 80,000 MCs were seeded in a 24-well plate 24 h before exposing them to BL or WL. After exposure (BL or WL for 1.5 h), MCs were washed with 1× Dulbecco’s phosphate-buffered saline  and stained with annexin V/FITC , Thermo Fisher Scientific, Taufkirchen, Germany) and propidium iodide ) for 30 min in the dark. After another washing step, cells were counterstained with Hoechst  for 15 min in the dark at RT. After washing with 1× DPBS, cells were imaged in serum-free DMEM using a fluorescence microscope with AF555 (PI) and DAPI (Hoechst) filter sets. Five pictures per well were generated (see previous section). PI-positive cells were counted using the “Analyze particles” function of ImageJ  after adjusting brightness/contrast and threshold (for a converted 16-bit image). Hoechst staining was evaluated analogously .After BL exposure (and treatment with 1 µM pifithrin α) in a 96-well plate , MCs were stained with PI  and Hoechst . Round cells were manually counted using ImageJ . Only cot-tests were used for comparison, as indicated in the figure legends. Differences were considered significant at p < 0.05. Statistical differences were indicated as follows: * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 compared to the control. In some cases, for better visibility, the control was set to 100%; therefore, bar graphs represent the mean values as percentages, with control values considered as the baseline.All results were presented as the mean ± SEM. Statistical analysis was performed using GraphPad PRISM 9. Welch’s ANOVA, Kruskal–Wallis, or Welch’s www.biorender.com (accessed on 12 October 2022).Figures were generated using Retinal glia cells undergo morphological and functional changes with aging, and these impairments affect the status of all cell types in the retina. Moreover, during AMD progress, MCs begin to migrate through the retina and are activated, likely enhancing inflammation and neuronal cell death. Consequently, damaged and activated MCs can contribute significantly to the progress and development of oxidative stress-based diseases such as AMD. In this context, we were able to prove that BL enhances oxidative stress levels and induces p53-mediated apoptosis. In the case of an insufficient clearance of apoptotic MCs in vivo, this could also lead to secondary necrosis and, thus, further inflammation in the retina. During AMD, MCs could even reach RPE cells, which are not typically in direct contact. Therefore, damaged MCs could also impair RPE function and further enhance AMD progression. Importantly, BL-induced apoptosis can be reduced by p53 inhibition, highlighting a promising therapeutic option. Using ex vivo organ cultures, the effect of BL-damaged MCs could be studied in a more complex context. In particular, the influence on other retinal cell types is an exciting topic of research, as is the question of whether retinal cell damage by increased oxidative stress can be mediated by inhibiting p53.


In addition to primary and secondary alkyl halides, sterically hindered tertiary chlorides also work very well, affording the target deuterodehalogenated products with excellent efficiency and deuterium incorporation. More than 60 examples are provided, including late-stage dehalogenative deuteration of natural products, pharmaceuticals, and their derivatives, all with excellent deuterium incorporation (up to 99% D), demonstrating the potential utility of the developed method in organic synthesis. Furthermore, the method does not require external catalysts and tolerates high current, showing possible use in industrial applications.Herein, a facile and general electroreductive deuteration of unactivated alkyl halides  or pseudo-halides (X = OMs) using D Deuterium-labelled compounds are useful for mechanistic studies and pharmaceutical science, but syntheses are not trivial. Here, the authors report deuteration of unactivated alkyl (pseudo)halides driven by electrochemical force, using deuterium oxide as the source of the isotope. Dehalogenative deuteration from organic halides is a straightforward way to obtain deuterated target compounds. However, major studies focused on dehalogenative deuteration of aryl halides and activated alkyl halides20. In contrast, corresponding reports on unactivated alkyl halides, particularly on unactivated alkyl chlorides, have not been well elucidated. Several realizations have been reported but suffer from a number of problems, such as limiting to alkyl iodides using radical initiators22, the usage of a stoichiometric metal reductant23, long reaction time, and low reactivity of tertiary alkyl halides using photocatalysis24, etc. Therefore, the development of simple, efficient, and environmentally-friendly approaches to access deuterated compounds from unactivated alkyl halides with high D-incorporation is highly desirable.Compounds with D-labeling play an important role in the realm of chemistry, mechanistic studies, and pharmaceutical science44. Organic halides are important players in traditional organic chemistry as well as promising electrochemistry, owing to their versatile reactivity and readily available properties. Thus, impressive electrochemical progress of organic halides has been made, such as reductive bifunctionalization of alkenes48, reductive coupling with halides56, and others68. Hence, electrochemical deuteration of organic halides could be a straightforward and promising route for valuable deuterated compounds, which has gained increasing attention71. However, current research and success in electrochemical deuteration have been focused on aryl halides. In sharp contrast, electrochemically driven dehalogenative deuteration of unactivated halides has proven elusive  external catalysts-free and the use of electricity as an environmentally-friendly reductant, (d) catalytic amounts of electrolyte and no sacrificial anode, (e) tolerance with high current (up to 500 mA), making it potentially applicable in industrial production, (f) late-stage deuteration of complex natural products and drug derivatives.Herein, we report our effort in developing this dehalogenative deuteration of unactivated alkyl halides, making it a general, economically and environmentally approach through electrochemistry Fig. . Notable2O as the economical deuterium source, unactivated alkyl bromide 1 as the substrate, and DMF as the solvent. After careful optimization, the corresponding product 2 was finally obtained in excellent yield and D-incorporation under 30 mA constant current with carbon felt (CF) and Pb as electrodes, in the presence of DIPEA as well as the catalytic amount of TBAI as the electrolyte in an undivided cell at room temperature for 10 hours . Finally, different electrodes were tested but none of the results surpassed entry 1 (entries 10-13).Initially, we started our investigation using D2-7). Substituents with electron-withdrawing or donating properties were compatible and showed little effect on the reaction (8-13). Besides, other aromatic rings, including naphthalene, indole, and dihydrobenzofuran (15-17), were all well tolerated. The success of this procedure could be confirmed by the excellent compatibility of a wide range of functionalities, such as terminal ether (18), ester (19), and thioether (20), unprotected lactam (21-23), Boc-protected amine (24), terminal olefin (25), tetrahydropyran (26), as well as internal ester (27). Interestingly, for substrates containing both Br and Cl, it was found that the C-Br bond was easier to cleave (28). Besides, alkyl iodides also worked smoothly in this protocol, affording the corresponding products with excellent deuterium incorporation . We were pleased to find that OMs could be a suitable leaving group to give the target product in 64% yield and 99% D-incorporation (32).With the optimized reaction conditions in hand, we probed the generality of this electroreductive deuteration reaction. Firstly, various alkyl bromides were screened Fig. . Alkyl bion Fig. , 29-31. 33-45). Furthermore, other alkyl chlorides, especially tertiary substrates that did not work well in previous studies24, yielded the desired products unquestionably in satisfactory D-incorporation using this method (46-49). To further demonstrate the generality, and environmentally-friendly nature of the developed method, as well as its application in bioactive molecules and drug discovery, the late-stage deuteration was conducted using a series of natural products, pharmaceuticals, and their derivatives , Pregnenolone (52), DL-Menthol (53), Phytol (57), and Triclosan (58) was successfully achieved in 77–83% yield with 97-98% D-incorporation. Pharmaceutical intermediates ethynyl estradiol (51) and dihydro cuminyl alcohol (54) containing unsaturated bonds can also be used in this protocol. Ibuprofen (55) and naproxen (56), which are commonly used for the ease of pain, afforded the corresponding deuterated compounds in good yields  with excellent deuterium incorporation (both 98% D). Furthermore, amino acid and glucose derivatives that widely exist in organisms can also be deuterated smoothly with high levels of D-incorporation . In addition, the tolerance of the reaction system to tertiary chlorides can be extended to complex substrates .Next, we turned our attention to a more challenging target, unactivated alkyl chlorides Fig. . To our ves Fig. . The deh2, 16, 23, and 26) as well as complex alkyl bromides derived from ibuprofen (55), L-phenylalanine (59), Diacetonefructose (61), and D-mannofuranose (63) were all amenable. level of D-incorporation has not changed at all . Simple alkyl bromides with a variety of functional groups 2, 16, 2, and 26 ent Fig. .Fig. 3Fa2O, the TEMPO adduct was obtained in 64% yield (67), together with hydrodehalogenated product 2-H in 28% yield  was observed . This might explain the improved yield once external DIPEA was added by inhibiting the undesired oxidation or decomposition of the starting material  experiments. A reduction peak of alkyl bromides ved Fig. . At the al Table .Fig. 5Cy48, a plausible mechanism is proposed halides  driven by electrochemical force with high reactivity, selectivity, and excellent D-incorporation. D2O  and DIPEA  were added via a syringe. The electrocatalysis was performed at room temperature with a constant current of 30 mA maintained for 10 h. The carbon felt anode was washed with EtOAc (3 × 5 mL) in an ultrasonic bath. H2O (20 mL) was added to the system, and the resulting mixture was extracted with EtOAc (3 ×20 mL). The combined organic phase was dried with anhydrous Na2SO4, filtered, and concentrated in vacuo. The crude product was purified by column chromatography to furnish the desired product.The electrocatalysis was carried out in an undivided cell with a carbon felt anode (10 mm × 15 mm × 5 mm) and a lead cathode (10 mm × 15 mm × 0.3 mm). To a 15 mL pre‐dried undivided electrochemical cell (15 mL) equipped with a magnetic bar were added alkyl bromide, iodide or pseudo-halides , TBAI , and DMF (5.0 mL). Then D2O  and DIPEA  were added via a syringe. The electrocatalysis was performed at room temperature with a constant current of 50 mA maintained for 10 h. The carbon felt anode was washed with EtOAc (3 × 5 mL) in an ultrasonic bath. H2O (20 mL) was added to the system, and the resulting mixture was extracted with EtOAc (3 × 20 mL). The combined organic phase was dried with anhydrous Na2SO4, filtered, and concentrated in vacuo. The crude product was purified by column chromatography to furnish the desired product.The electrocatalysis was carried out in an undivided cell with a graphite felt anode (10 mm × 15 mm × 5 mm) and a lead cathode (10 mm × 15 mm × 0.3 mm). To a 15 mL pre‐dried undivided electrochemical cell (15 mL) equipped with a magnetic bar were added alkyl chloride , TBAI  and DMF (5.0 mL). Then D2O  and DIPEA  were added via a syringe. The electrocatalysis was performed at room temperature with a constant current of 500 mA maintained for 15 min (reaction system exothermic). The carbon felt anode was washed with EtOAc (3 × 5 mL) in an ultrasonic bath. H2O (20 mL) was added to the system, and the resulting mixture was extracted with EtOAc (3 × 20 mL). The combined organic phase was dried with anhydrous Na2SO4, filtered, and concentrated in vacuo. The crude product was purified by column chromatography to furnish the desired product.The electrocatalysis was carried out in an undivided cell with a carbon felt anode (or graphite felt) (10 mm × 15 mm × 5 mm) and a lead cathode (10 mm × 15 mm × 0.3 mm). To a 15 mL pre‐dried undivided electrochemical cell (15 mL) equipped with a magnetic bar were added alkyl bromide or chloride , TBAI  and DMF (5.0 mL). Then D2O  and DIPEA . The electrocatalysis was performed at room temperature with a constant current of 500 mA maintained for 4 h (the reaction system was exothermic). The carbon felt anode was washed with EtOAc (3 × 20 mL) in an ultrasonic bath. H2O (200 mL) was added to the system, and the resulting mixture was extracted with EtOAc (3 × 200 mL). The combined organic phase was dried with anhydrous Na2SO4, filtered, and concentrated in vacuo. The crude product was purified by column chromatography to furnish the desired product.To an undivided reaction flask  equipped with a teflon-coated magnetic stirring bar and teflon cap, a carbon felt anode (25 mm × 50 mm × 5 mm), and a lead cathode (25 mm × 50 mm × 0.3 mm) were added (4-bromobutoxy)benzene , TBAI , DMF (100 mL), DSupplementary InformationPeer Review File


PPARγ and COL1A1 gene expression under the influence of different glucose doses. In particular, we observed that isoflavone and 17β-estradiol regulate blood glycemia. Renal injury was inhibited by isoflavone, observed by a reduction in KIM-1, along with glycogen accumulation. These benefits of isoflavone may be associated with PPARγ overexpression in the kidneys and endometrium of diabetic ovariectomized rats. Diabetes associated with post-menopause is related to a worse condition of kidney disease. Taking into consideration that this disorder may be regulated by estrogenic mediators, we evaluated the renal protective effect of isoflavone. We investigated the role of the PPARγ in the pathogenesis of the disease. For this study, we used diabetic female rats in a postmenopausal model through ovariectomy. The animals were treated with isoflavone or 17β-estradiol. A dosage was administered to bring on blood glycemia, and through immunohistochemistry, we evaluated the immunoreactivity of PPARγ in the endometrium and renal tissue. We analyzed the immunoreactivity of renal injury molecule KIM-1 and the collagen and glycogen densities in the kidney. Through bioinformatics analysis, we observed  Type 1 diabetes (T1D) has high rates of morbidity and mortality and an increasing incidence. From the moment of diagnosis, the management of diabetes mellitus (DM) places an enormous burden on the patient, with severe limitations, changes in lifestyle, and organ dysfunction ,3,4, as Estrogen prevents the development and progression of kidney disease and improves postmenopausal symptoms, especially in diabetes, the main risk factor for chronic kidney disease. In addition, women with lower endogenous exposure to estrogen have a higher risk of kidney disease . IsoflavPeroxisome proliferator-activated receptor gamma (PPARγ) has been closely associated with diabetes, menopause, and kidney disease. Due to its role in glucose regulation, PPARγ agonists are suggested to ameliorate the problems of diabetes . Our groOur data revealed that isoflavone protects the kidney against gonadal failure associated with DM1 damage. Increased protein and gene expression of PPAR-γ was evidenced under the influence of isoflavone. In addition, we demonstrate for the first time that PPAR-γ reduction occurs dose-dependent on glucose. On the other hand, we have not clearly demonstrated the consequences of ovariectomy in the renal injury development, and other findings are necessary to confirm this hypothesis.Our study sample consisted of 60 adult, virgin female rats (Rattus norvegicus Albinus), three months of age, weighing between 180 and 210 g. The in vivo experimental protocol was approved by the Ethics Committee (Research Ethics Committee 001676/09) of the Federal University of São Paulo. The animals were donated by the Center for the Development of Experimental Models of the Federal University of São Paulo (CEDEME). All animals were kept in plastic cages with controlled light (12/12 h light/dark cycles) and temperature (22–24 °C), received water and food ad libitum, and were fed a specific proportion of soy-free food , developed by Agribrands-Purina, São Paulo, Brazil.®). Only animals with mean plasma glucose levels > 250 mg/dL were accepted as having diabetes mellitus [After a one-week adaptation period, the rats were anesthetized with an intraperitoneal injection of 10% ketamine  and 2% xylazine . They then underwent bilateral ovariectomy immediately after surgery. One day after bilateral ovariectomy, type 1 diabetes in animals was induced by a single-dose intraperitoneal injection of freshly prepared 60 mg/kg streptozotocin  dissolved in a 0.1 mol/L citrate buffer (pH = 4.8). Hyperglycemia was confirmed 48 h after the STZ injection by measuring the tail vein blood glucose levels using a blood glucose monitoring kit (Accu Check ACTIVE Rochemellitus .β-estradiol (Sigma-Aldrich). Administration of soy isoflavones or 17β-estradiol was initiated 30 d after the ovariectomy. Soy isoflavones (150 mg/kg) were administered by gavage, and 17β-estradiol (10 mg/kg) was administered subcutaneously. Treatment with soy isoflavone and 17β-estradiol was performed for 30 days after T1D induction. Was used the concentrated extract of soy isoflavones NovasoyVR  containing 40% of total isoflavones in the ratio of 1.3:1:0.3 genistein: daidzein: glycitein, respectively, 7–12% protein, 4% ash, and 6% moisture, and the remaining 41% consists of the other soy phytocomponents. In addition, we used 17After 30 days of experimentation, all animals were anesthetized with xylazine  15 mg/kg associated with ketamine  30 mg/kg, intraperitoneally. A portion of the endometrium and kidney was immediately immersed in liquid nitrogen e and then stored in the −80 °C freezer for qPCR assay. Another portion of the endometrium and kidney were immersed in paraformaldehyde 4% for histological processing.Paraffin sections were subjected to alcohol and xylene gradient solutions, antigen retrieval, protein block, and incubation with primary antibodies against PPAR-γ polyclonal antibody  and kidney injury molecule 1 (KIM-1)  overnight at 4 °C. After this time, the sections were incubated with streptavidin-peroxidase for 30 min . Periodic acid–Schiff staining was carried out according to the manufacturer’s instructions. The obtained microscope images were calculated using Leica DFC 310 FX image analysis software  and are expressed as the percentage/stained area.To evaluate the birefringence pattern of collagen fibers in the kidney, two sections per animal (100 mm distance between sections) were selected and studied using the picrosirius red method ,22. PS-s−ΔΔCT method. All reactions were performed in triplicate.According to the manufacturer’s instructions, total RNA from the endometrium and kidney was isolated using TRIzol Reagent . Total RNA was treated with DNase  to prevent genomic deoxyribonucleic acid contamination, and the RNA pellet was resuspended in RNase-free water. For mRNA expression analysis, the total RNA (1 μg) of the endometrium and kidney samples was reverse transcribed using SuperScript III RT  according to the manufacturer’s protocol. The PCR product was amplified from cDNA using QuantiFast SYBR Green PCR kit (Qiagen) and specific primers for rats ; β-actinhttps://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE168072, accessed on 3 May 2022). Primary human renal tubular epithelial (hRTE) cells. The cells were grown using serum-free conditions. The growth formulation consisted of a 1:1 mixture of Dulbecco’s modified Eagles’ medium and Ham’s F-12 growth medium supplemented with selenium (5 ng/mL), insulin (5 μg/mL), transferrin (5 μg/mL), hydrocortisone (36 ng/mL), triiodothyronine (4 pg/mL), and epidermal growth factor (10 ng/mL). The cells were fed a fresh growth medium every 3 days, and at confluence, the cells were sub-cultured using trypsin–ethylenediaminetetraacetic acid . For use in experimental protocols, cells were subcultured at a 1:2 ratio, allowed to reach confluence (7 days following subculture), and fed with 5.5, 7.5, 11, or 16 mM glucose for 7 days, followed by sub-culturing the cells for 3 passages (P3) in media containing 5.5, 7.5, 11, or 16 mM glucose. Typically, the cells were passaged, 1:2 for three passages before the initiation of the experiment [http://www.ncbi.nlm.nih.gov/geo/geo2r/, accessed 3 May 2022), which allows users to compare different groups of samples in a GEO series to examine differentially expressed genes according to experimental conditions. GEO2R was applied to detect PPAR-γ and Collagen type I alpha 1 chain (COL1A1) genes among different experimental conditions. The p-values of gene expression after Log2 transformation were used to calculate the Z-score .A study containing publicly available transcriptome data was selected from the Gene Expression Omnibus (GEOR) repository:  software. The Kolmogorov–Smirnov or Shapiro–Wilk test was used to determine the normality of the data. The data of the experimental groups were compared using a one-way analysis of variance (ANOVA), followed by the application of the Kruskal–Wallis test for nonparametric samples. p < 0.0001) and only OVX (** p < 0.01), respectively, showed hyperglycemia. Relevant to this work, we observed isoflavone-mediated glucose modulation; we demonstrated that isoflavone but not 17β-estradiol can reduce glycemia in DM1 and DM1 + OVX groups (* p < 0.05).Firstly, we confirmed our diabetes model . We obsep < 0.05), OVX (*** p < 0.001), and untreated DM1 + OVX (**** p < 0.0001) groups. In addition, a higher immunoreactivity of PPAR-γ was observed in the DM1 + OVX group treated with 17β-estradiol compared to the untreated DM1 + OVX group (*** p < 0.001). However, in the evaluation of PPAR-γ gene expression, we observed that only the DM1 + OVX group treated with isoflavone could positively modulate PPAR-γ mRNA compared to the untreated DM1 + OVX group (* p < 0.05)  h.p < 0.05), untreated DM1 + OVX group (* p < 0.05), and DM1 + OVX group treated with 17β-estradiol (* p < 0.05) (p < 0.0001) (p *** < 0.001), there is a reduction in PPAR-γ expression; this decline occurs in a concentration-dependent manner at 11 mM (p *** < 0.001) and 16 mM (**** p < 0.0001) glucose concentrations compared to the control  g. Gene e 0.0001) h. In add control i.p < 0.0001) and even when compared to the OVX group (** p < 0.01) (p < 0.01)  g. We dem < 0.01) g.p < 0.0001) and OVX alone (*** p < 0.001) (p < 0.05)  . Treatme< 0.001) . To corr < 0.05) h.p < 0.05 and **** p < 0.0001, respectively). In addition, increased glycogen storage was found in the untreated OVX group when compared with the untreated DM1 + OVX group (*** p < 0.001)  g. HoweveIn this work, in a model of diabetes followed by ovariectomy, we demonstrated the role of isoflavone in protecting from renal injury via PPAR-γ. In our model, isoflavone-treated animals were protected from renal injury, observed by a reduction in the renal injury marker KIM-1 in the renal tissue. We postulate that this isoflavone-mediated protection of kidney injury occurs via PPAR-γ, where we observed that—mainly in the model of diabetes concomitant to ovariectomy—the endometrium as much as the kidney presents a reduction in PPAR-γ, observed at the proteinic and gene levels. Isoflavone treatment for this condition can positively modulate PPAR-γ at the protein and gene levels, demonstrating this possible relationship. The relationship of isoflavone with diabetes and menopause has been reviewed by numerous authors ,26,27. IY in renal tissue of diabetic animals than estrogen, and this promoted lower expression of KIM-1 in this condition. Thus, our data suggest that isoflavone was more effective in inhibiting the renal injury induced by hyperglycemia than the estrogen itself in our model. Therefore, these findings will be better explored in future studies.Isoflavone and 17β-estradiol negatively regulate blood glucose . This si−/− demonstrates cross-links of glucose and lipid metabolism between adipose tissue, muscle, and liver, suggesting that PPARγ is important for maintaining normal insulin sensitivity and whole-body glucose and lipid homeostasis [In animals, PPAR-γeostasis . Isoflaveostasis . We haveeostasis . In this work, we demonstrated that protein loss and gene expression of PPAR-γ in endometrium but not in the kidney of ovariectomized diabetic animals may be related to greater kidney injury due to a greater susceptibility to glucose. Furthermore, we demonstrated through transcriptome analysis in human proximal renal tubule cells that the reduction in PPAR-γ expression may be glucose-concentration-dependent. Higher concentrations of glucose  cause an expressive decline in PPAR-γ expression. We observed overexpression of KIM-1 in diabetic and ovariectomized diabetic animals, but isoflavone could reduce this marker of renal damage significantly only in the ovariectomized diabetic group. COL1A1 gene. In our in vivo model, treatment with isoflavone or 17β-estradiol could maintain the collagen profile, which was unchanged when compared to the control. Calycosin, a type of isoflavone, could also reduce pro-fibrotic factors in a high-fat diet/STZ-induced T2DM model [The fibrosis present in human diabetic nephropathy reduces renal function dramatically . In diabDM model , an effeDM model . Diabetes induced by SZT and a high-fat diet increases PAS accumulation ,39 This Through our observations, we can conclude that isoflavone protects against kidney damage induced by gonadal failure and DM1 through PPAR-γ expression. In addition, we can conclude that glucose is a potent negative regulator of PPAR-γ. Isoflavone stimulates PPAR-γ protein and gene expression. Thus, we postulate that the benefits observed by isoflavone treatment may occur via PPAR-γ. Further investigations of the downstream pathway of this receptor may allow new perspectives to be developed on therapeutic approaches for diabetic nephropathy, especially for postmenopausal women, which will benefit individuals affected by diabetes.


PAX7, the major stem cell marker of satellite cells, whereas the atrophy marker MURF1 was increased. We also isolated satellite cells from muscle biopsies and cultured them in vitro. We found that a pharmacological activation of HIF-1α and its target genes caused a reduction in skeletal muscle atrophy and activation of PAX7 gene expression. In conclusion, in this work we found that HIF-1α plays a role in sarcopenia and is involved in satellite cell homeostasis. These results support further studies to test whether pharmacological reactivation of HIF-1α could prevent and counteract sarcopenia.Sarcopenia, an age-related decline in muscle mass and strength, is associated with metabolic disease and increased risk of cardiovascular morbidity and mortality. It is associated with decreased tissue vascularization and muscle atrophy. In this work, we investigated the role of the hypoxia inducible factor HIF-1α in sarcopenia. To this end, we obtained skeletal muscle biopsies from elderly sarcopenic patients and compared them with those from young individuals. We found a decrease in the expression of HIF-1α and its target genes in sarcopenia, as well as of  The global increase in life expectancy is leading to a lasting change in the age structure of the population. The proportion of the world’s population that is 65 years or older is projected to increase from 9.3 percent in 2020 to 16.0 percent in 2050 . R. R19]. RAs hypoxia is known to trigger neo-angiogenesis , changesTo assess whether the impairment of HIF-1α signaling was associated with sarcopenia, the correlation between its expression (and that of its target genes) with the onset of the atrophic phenotype was examined. The results revealed a statistically negative correlation between HIF-1α and all its target genes , with the exception of its inhibitor PHD2 A–E.VEGF, PHD2, and GAPDH, respectively, confirming the activation of the HIF-1α pathway  and 15 sarcopenic (aged 74–90 years) patients were enrolled from IRCCS Orthopaedic Galeazzi Hospital from those who were scheduled for orthopaedic surgery . Written informed consent was obtained from all participants for participation in this study (protocol “Role of the Hypoxia Inducible Factor 1α (HIF-1α) in muscle aging”; Ethics Approval N°: 94/INT/2017 13/07/2017; Clinicaltrial.gov N°: NCT03371134). The study protocol was approved by the San Raffaele Hospital Ethical Review Board. Before surgery, anthropometric examinations, and serological and metabolic analyses were performed in all patients. In addition, the skeletal muscle mass of all sarcopenic patients was quantified by dual-energy X-ray absorptiometry (DXA).2 (m2).Standing height and weight were measured to the nearest 0.1 cm and 0.5 kg using a wall-mounted stadiometer and an automated balance. The body mass index (BMI) was calculated using the formula weight (kg)/heightVenous blood was drawn from all patients before surgery and analyzed by the central laboratory of IRCCS Orthopaedic Galeazzi Hospital, which performed preoperative routine blood tests, such as glucose test, total cholesterol, calcium, phosphorus, creatinine. Moreover, HCV, HIV, HBV, and TPHA serological tests were also tested. In case of serological positivity, samples were automatically excluded from the study and discarded.2). According to EWGSOP2 sarcopenia cut-off points, reduced muscle mass was defined when ASMI was <7.0 kg/m2 for men and <5.5 kg/m2 for women [2) in order to obtain the Fat Mass Index (FMI), which was expressed as kg/m2. According to Kelly et al., normal values for FMI were considered in the range between 3 and 6 for men, and between 5 and 9 for women [The quantitative assessments of regional lean mass, fat mass and bone mass were conducted using dual energy X-ray absorptiometry (DXA) with an Hologic QDR-Discovery W densitometer  at the IRCCS Istituto Ortopedico Galeazzi in Milano, Italy. Skeletal muscle mass was reported by using the appendicular skeletal muscle index (ASMI) according to the revised European consensus on definition and diagnosis of sarcopenia from the European Working Group on Sarcopenia in Older People (EWGSOP2) . ASMI waor women . Additioor women .Fragments of tensor fascia latae were harvested from elderly patients during hip arthroplasty, whereas fragments of gracilis muscle and/or semitendinosus muscle were collected from young patients during the tendon harvesting and neoligament preparation phase (standard procedure in anterior cruciate ligament reconstruction surgeries). Tissue samples from young patients were used as a control group. From each patient, 3 sections of skeletal muscle biopsies were taken: one for muscle cell isolation, one for RNA/proteins extraction, and one for immunohistochemistry.Skeletal muscle biopsies for isolation muscle cells were collected in sterile phosphate-buffered saline  and then dissected in a new Petri dish containing PBS. All subsequent manipulations were performed in a culture hood. Briefly, all visible fat and nerve deposits were removed with forceps. The muscle was placed on a plastic plate and the tissue was minced into a paste with small surgical scissors until no visible muscle deposits remained. The paste was transferred to a tube containing 2% type II collagenase  dissolved in DMEM High Glucose  with 1% penicillin-streptomycin . Approximately 3 mL of collagenase solution was prepared for 1 g of muscle tissue. The mixture was incubated at 37 °C for 60 min on a shaker. After incubation, the homogenate was centrifuged at 300× g for 5 min and the supernatant discarded. The pellet was resuspended in 2 mL of 0.5% trypsin (Thermo Fisher Scientific) and mechanically dissociated using a 5 mL serological pipette. After dissociation, the pellet was allowed to settle by gravity for 5 min, while the supernatant was collected in another tube containing DMEM High Glucose + 10% Horse Serum . Trypsin dissociation was repeated twice and finally the collected supernatant was filtered in a cell strainer to obtain single cells. A pre-plating phase was then performed to reduce cellular contamination. Specifically, cells were seeded in DMED containing high glucose + 10% horse serum for 2 h. After pre-plating, cells were harvested, centrifuged at 500× g for 10 min, resuspended in growth medium (GM) containing Nutrient Mixture F-10 Ham (Merck), 0.5 mg/mL Bovine Serum Albumin , 0.5 mg/mL Fetuin (Merck), 0.39 μg/mL Dexamethasone (Merck), 10 ng/mL Epidermal Growth Factor , 0.05 mg/mL Insulin (Merck), 3 mg/mL Glucose (Merck), 15% siero Fetal Bovine Serum , 1% Penicillin/Streptomycin , and incubated for the following 2 days without changing the medium .g for 5 min at 4 °C, and the clear supernatant was transferred to a new tube. Next, 0.2 mL of chloroform (Merck) was added to the suspension, which was then shaken for 15 s. The mixture was centrifuged at 14,000× g for 15 min at 4 °C after incubation at room temperature for 5 min to allow phase separation. The top layer was transferred to a new 2 mL tube and used for RNA extraction, while the bottom layer of phenol was used for protein precipitation. Briefly, RNA was isolated by adding 0.5 mL isopropanol (Merck) to the aqueous phase, incubating at 4 °C, and centrifuging at 12,000× g for 10 min at 4 °C. Then, the RNA was washed in 1 mL of 75% ethanol and centrifuged at 7500× g for 5 min at 4 °C. Finally, the supernatant was discarded and the RNA pellet was air dried for 15 min, and then resuspended in 50 µL RNase-free water (Thermo Fisher Scientific) containing 0.1 mM EDTA (Merck), and stored at −80 °C. Prior to use, RNA was purified using the ReliaPrepTM RNA Miniprep System (Promega) and transcribed into cDNA using the iScript cDNA Synthesis Kit (BioRad) according to the manufacturer’s instructions. Skeletal muscle biopsies for RNA/Protein extraction were collected in RNA Later (Thermo Fisher Scientific). TRIzol reagent (Thermo Fisher Scientific) was added to 50 mg of skeletal muscle tissue, which was minced and homogenized using a tissue lyser (Thermo Fisher Scientific). The samples were shaken with tungsten carbide beads  at 900 rpm for 2 min. Then the samples were centrifuged at 12,000× g at 4 °C. The protein pellet was then washed with f 0.3 M guanidine hydrochloride (Merck) in 95% ethanol and air dried. Finally, 1% sodium dodecyl sulfate  was added to dissolve the protein pellet, and the solution was stored at −20 °C. Total protein content was determined using the BCA Protein Assay Kit (Pierce) according to the manufacturer’s instructions.To extract the proteins, 0.3 mL of 100% ethanol was added to the interphase/phenol phase, mixed by inverting the tube several times, and incubated for 3 min. After centrifugation, the pellet containing DNA was discarded, while the phenol-ethanol supernatant was transferred to a new tube. To pellet the proteins, 1.5 mL of isopropanol was added to the phenol-ethanol supernatant, incubated for 10 min, and centrifuged for 10 min at 12,000× ® qPCR Master Mix (Promega) in 20 μL final volume using a StepOnePlus® Real-Time PCR System . The following primers were used: MURF1 forward 5′-CATCAAAAGCATTGTGGAAGC-3′ and reverse 5′-ATGTTCTCAAAGCCCTGC-3′; VEGF forward 5′-TGCCCGCTGCTGTCTAAT-3′ and reverse 5′-CCTCGGTCACTCATCTTCAC-3′; PHD2 forward 5′-TGGGAGTTGCTGTTGAAGTCG-3′ and reverse 5′-CGTGCCGCCTGGAGAAAC-3′; GAPDH forward 5′-GAGTCCACTGGCGTCTTCAC-3′ and reverse 5′-GTTCACACCCATGACGAACA-3′; PAX7 forward 5′-GAAAACCCAGGCATGTTCAG-3′ and reverse 5′-GCGGCTAATCGAACTCACTAA-3′; S14 forward 5′-GTGTGACTGGTGGGATGAAGG-3′ and reverse 5′-TTGATGTGTAGGGCGGTGATAC-3′. The amplification program consisted of an initial denaturation at 95 °C for three minutes, followed by 40 cycles of 5 s each at 95 °C and 30 s at 57 °C. Relative quantification of target genes was performed in triplicate and calculated using Equation 2−ΔΔCt.Quantitative PCR was performed on 10 ng of cDNA template, 0.2 μM primers, and GoTaqw/v) nonfat dry milk or 5% (w/v) BSA (Merck) in Tris buffer saline with 0.1% Tween 20 (TBS-T) for 1 h. Blots were incubated for 2 h at room temperature with the following primary antibodies: mouse monoclonal anti-MURF1, 1:11,000 , rabbit monoclonal anti-HIF-1α, 1:1000 dilution , rabbit polyclonal anti-WNT7a, 1:1000 dilution (abcam), anti-PHD2, 1:1000 dilution , anti-GAPDH, 1:11,000 , mouse monoclonal anti-VEGF, 1;1000 (Santa Cruz Biotechnology). Total transferred proteins were used to normalize human proteins using the REVERT Total Protein Stain Kit  according to the manufacturer’s instructions. Membranes were washed three times for 10 min with TBS-T and then incubated with the appropriate anti-mouse, anti-rabbit, or anti-goat HRP-conjugated secondary antibodies  at a dilution of 1:2000 for 1 h at room temperature. After washing three times for 10 min with TBS-T, immunoreactive bands were visualized with Enhanced Chemiluminescence Detection Kit reagents  according to the manufacturer’s instructions. Optical density was measured with Image Studio TM Lite software (LI-COR Biotechnology).Proteins were boiled for 10 min in sample buffer  and loaded into a 10% SDS-PAGE gel, then transferred to a nitrocellulose membrane  by electroblotting. The nitrocellulose membranes were incubated with a blocking solution containing 5% (Human muscle tissue was harvested in formalin, fixed, paraffin embedded and sections were prepared as previously described . Formalip values of less than 0.05 were considered to be significant. All error bars represent the standard deviation of the mean. Pearson’s correlation was used to evaluate significant associations between MurF1, PAX7 and CD31 expression with sarcopenic condition.Data were described as means ± SD. The nonparametric Mann-Whitney test was used to determine statistical significance using GraphPad Prism 9 software.


However, most polymer blends exhibit phase separation after blending. In this study, polymethylmethacrylate/polyethylene glycol (PMMA/PEG) polymer blends (70/30 and 30/70 w/w) were prepared by solution casting with and without ZnO nanoparticles (NPs) loading. The effect of loading ZnO nanoparticles on blend morphology, UV blocking, glass transition, melting, and crystallization were investigated. Without loading ZnO NP, the PMMA/PEG blends showed phase separation, especially the PEG-rich blend. Loading PMMA/PEG blend with ZnO NPs increased the miscibility of the blend and most of the ZnO NPs dispersed in the PEG phase. The interaction of the ZnO NPs with the blend polymers slightly decreased the intensity of infrared absorption of the functional groups. The UV-blocking properties of the blends increased by 15% and 20%, and the band gap energy values were 4.1 eV and 3.8 eV for the blends loaded with ZnO NPs with a PMMA/PEG ratio of 70/30 and 30/70, respectively. In addition, the glass transition temperature (T Howdustries ,13,14. Cdustries ,16.4 NPs and various amounts of multi-walled carbon nanotubes as nanofillers. The direct current conductivity increased, whereas the glass transition temperature (Tg) and crystallization degree decreased with the nanofiller amount increase in the polymer blend [Polymethyl methacrylate/polyethylene glycol (PMMA/PEG) blends have been used in many applications because they are cheap and can be easily processed ,18. For er blend .Large interfacial tensions of immiscible PMMA/PEG polymer blends lead to phase separation, and the phase-separated polymer may coalesce; this leads to larger particle size and, consequently, a deterioration of the mechanical properties ,26,27,282H5OH, 96.96%, GPR RECTAPUR, VWR, Radnor, PA, USA) with constant mixing at 50 °C for 2 h followed by sonication for 1 h to ensure ZnO NPs’ complete dispersion. PMMA/PEG/ZnO films were produced using solution casting. Two PMMA/PEG polymer blends (dry weight ratio 70/30 and 30/70) were prepared and loaded with ZnO NPs . For solution casting, first 2 g PMMA  was dissolved in 50 mL dichloromethane  under constant stirring at 50 °C for 15 min. Then, 2 g PEG  was dissolved in 50 mL dichloromethane  under constant stirring at 50 °C for 15 min. When the PMMA and PEG polymers were completely dissolved and the solutions became viscous, ZnO NPs were added. For 7PMMA/3PEG/ZnO, ZnO NPs were first added to PEG and stirred at room temperature for 20 min, followed by PMMA addition. For the PEG-rich mixture (3PMMA/7PEG/ZnO), ZnO NPs were first included in the PMMA solution that was then added to PEG. Then, the PMMA/PEG/ZnO mixtures were poured on a glass substrate (10 × 10 cm) and dried at room temperature for 48 h. About 1 g of ZnO NPs  was dispersed in 80 mL of ethanol . The Debye–Scherrer equation (D = kλ/βcosθ) was used to calculate ZnO NP crystallite size. In this equation, λ represents the X-ray wavelength (1.54056 Å), θ the Bragg diffraction angle and β the full width at half maximum . FTIR , it was important to monitor their spherulitic and crystalline morphology. PEG crystallization into spherulites was studied by SEM-EDX. The 7PMMA/3PEG blend showed PEG dispersion in the PMMA matrix like sea island, with some PEG aggregates a. The poAddition of ZnO NPs to the 7PMMA/3PEG blend led to a needle-like aggregate morphology of ZnO NPs with a diameter between 100 and 250 nm and an aspect ratio (l/d) of 5–10 c. When PThe XRD diagram of pure ZnO NPs showed the characteristic peaks at 31.9°, 34.5°, 36.4° and 47.5°, which corresponded to the crystal planes (100), (002), (101) and (102) and to the hexagonal wurtzite phase (JCPDS map number 36–1451)  Figure a. Starti−1 that corresponded to asymmetric and symmetric -CH3 stretching [−1 corresponded to the C=O stretching of PMMA. The IR bands at ~1445, 1436 and 1430 cm−1 corresponded to the C-H deformation vibrations. The stretching C-O bands were at 1132, 1092 and 1080 cm−1. The bands at 970 cm−1, 840 cm−1 and 740 cm−1 correspond to the C-C stretching vibration, the CH2 vibration and the out-of-plane bending of C=O, respectively [−1 in the 7PMMA/3PEG/ZnO and 3PMMA/7PEG/ZnO blends was attributed to the Zn–O bond [FTIR spectroscopy is a powerful tool to study possible interactions between PMMA/PEG polymer blends and ZnO NPs. The FTIR spectra of the 7PMMA/3PEG and 3PMMA/7PEG polymer blends and the 7PMMA/3PEG/ZnO and 3PMMA/7PEG/ZnO nanocomposites contained IR absorption bands at 2872, 2886 and 2867 cmretching   in the function of (αhυ)2 allowed for the determination of the Tauc diagram of the PMMA/PEG and PMMA/PEG/ZnO samples g, crystallization temperature (Tc), and Tm from DSC thermograms. Comparison of the DSC thermograms of the different samples (g of the 7PMMA/3PEG blend decreased by ~35° (from 102 °C to 67 °C), possibly due to PEG’s presence, which acts as a plasticizer and enhances the blend-free volume and flexibility [g by ~14° (from 67 to 81 °C) compared with the 7PMMA/3PEG blend  compared with neat PEG. The Tc of 3PMMA/7PEG/ZnO was increased by 14° (from 16.6 °C to 30.5 °C) compared with the 3PMMA/7PEG blend, but remained lower than that of neat PEG. Sari et al. [c and Tm decrease by ~10° and 3°, respectively, when the amorphous phase (PMMA) concentration reaches 30% in the PEG/PMMA blend. The Tm of the 3PMMA/7PEG and 3PMMA/7PEG/ZnO blends was less affected (increased by 2 °C) compared with pure PEG (c) and melting enthalpy (∆Hm) of 3PMMA/7PEG were strongly increased from 34 to 118 J/g and from 91 to 119 J/g, respectively, as previously shown by Ahmed et al. [The thermograms of PEG-rich polymer blends (3PMMA/7PEG and 3PMMA/7PEG/ZnO) lacked the PMMA transition temperature. The Tpure PEG . The crypure PEG b. ZnO NPd et al. .10%, T50% and Tmax) of the samples  and from 361 °C to 386 °C (3PMMA/7PEG/ZnO blend). This effect may be explained by the fact that the added ZnO NPs act as radical scavengers that hinder the chain cleavage and radical formation, and, thus, thermal degradation starts at higher temperatures [Then, thermal stability was investigated by thermogravimetry and derivative thermogravimetry. The thermogravimetry curves were recorded under nitrogen atmosphere a. Polyme samples  showed t results . Compari results b showed eratures .g) increased by 14 °C, the crystallinity rate increased by 15%, the melting (Tm) and crystallization temperatures (Tc) increased by 2 °C and 14 °C, respectively, and the thermal stability increased by 25 °C compared to the PMMA/PEG blends without ZnO NP loading.One of the biggest challenges is to produce high-performance immiscible polymer blends at low cost. For industrial applications, immiscible polymers require the addition of a nanofiller to reduce production costs and improve some of their properties. However, the use of nanofiller to compatibilize immiscible polymer blends remains a challenge, requiring a high degree of control over nanofiller dispersion and localization. The composition of the polymer blends and the method by which the filler is incorporated during nanocomposite fabrication have a major impact on the quality of the nanocomposite. In this study, PMMA/PEG polymer blends with different weight ratios of PMMA and PEG (70/30 and 30/70) were prepared and ZnO NPs were added as nanofiller. The PMMA/PEG phase separation was observed in polymer blends without ZnO NPs, especially in the PEG-rich blend. Scanning electron microscopy (SEM) showed that loading ZnO NPs improved the blend miscibility, but most ZnO NPs dispersed in the PEG phase. ZnO NPs can accumulate at the polymer–polymer interface and are particularly effective in refining the microstructure of the immiscible PMMA/PEG polymer blends. The underlying mechanisms remain controversial, but two main arguments are most commonly made: compatibilizing effect and effects on interfacial rheology. UV-vis spectroscopy showed that adjusting the PMMA/PEG ratio and loading ZnO NPs improved the UV-blocking properties by 15–20%, and the band gap energy was adjusted between 4.18 eV and 3.76 eV (for 3PMMA/7PEG/ZnO). In addition, the glass transition temperature (T


Human Enterovirus A species, which is a common pathogen causing hand, foot, and mouth disease in children. Currently, specific vaccines and drugs against CA16 are unavailable, and there is an unmet need to further understand the virus and invent effective treatment. Constructing a CA16 infectious clone with a reporter gene will greatly facilitate its virological studies. Here, we first reported the construction of a CA16 infectious clone (rCA16) whose progeny is highly replicative and virulent in suckling mice. On the basis of rCA16, we further inserted a NanoLuc (Nluc) reporter gene and made the rCA16-Nluc clone. We found that the Nluc gene in rCA16-Nluc is stable during continuous growing in Vero cells and thus allowed detection of a steady luciferase signal in rCA16-Nluc-infected Vero cells over 10 passages. Its application in antivirals characterization and high-throughput screening is exemplified by measuring IC50, CC50, and selection index of guanidine hydrochloride, ribavirin, chloroquine, and ammonium chloride against CA16. Finally, we showed that rCA16-Nluc based assay greatly simplified the CA16 neutralizing antibody tests. Thus, these two CA16 infectious clones will be robust tools for future enterovirus studies and antivirals development.Coxsackievirus A16 (CA16) belongs to the  Enterovirus A, genus Enterovirus, family Picornaviridae (Coxsackievirus A16 (CA16), is a member of species aviridae . As a siaviridae . Its ORFaviridae .Hand, foot, and mouth disease (HFMD) mainly infect infants and young children. Generally, HFMD is mild and self-limited. However, severe diseases may include paralysis, myocarditis, and even death . CA16 isA reporter virus is valuable for virological studies and antivirals development . Althougin vitro. Intraperitoneal inoculation of both pCA16 and rCA16 in one-week-old lactating mice led to severe multiple-organ/tissue damage and was 100% lethal within 6 days, indicating high pathogenicity of this CA16 strain. We also found that the Nluc gene in rCA16-Nluc is genetically stable over ten passages in cultured cells without losing the reporter gene. Moreover, by measuring fluorescence intensity, we have shown that the application of rCA16-Nluc has simplified testing for antivirals and neutralizing antibodies, and is fully compatible with high-throughput screening platforms, suggesting a promising application potential.Here, we successfully constructed two infectious clones of a clinical CA16 isolate (Genebank: OP293089) and characterized the rCA16 and rCA16-Nluc virus, respectively. Our data indicated that both recombinant viruses showed comparable growth characteristics as the parental CA16 (pCV16) virus 2. The CA16 strain OP293089 was isolated in the Biosafety Laboratory of Shanghai Public Health Clinical Center.African green monkey kidney (Vero) cells used in this study were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FBS, 100 U/mL of penicillin, and 100 μg/mL of streptomycin at 37°C in presence of 5% COThe CA16 viral RNA was extracted with QIAamp Viral RNA Mini Kit  and reversed transcribed with oligo dT and HiScript II 1st Strand cDNA Synthesis Kit . Using the viral cDNA as the template, three viral fragments: P1, P2, and P3 were amplified. Then, the P1 fragment was cloned in the pcDNA vector (pcDNA-P1), and P2 and P3 fragments were cloned in the pUC57 vector (pUC57-P2+P3). Next, P1 and P2+P3 two fragments were ligated with the pSVA vector to form the infectious DNA clone of CA16 (rCA16). To get the CA16 infectious clone with a Nluc reporter gene (rCA16-Nluc), the Nluc sequence was inserted between 5′UTR and VP4. The genetic stability of Nluc gene was examined by PCR amplification and sanger sequencing, the PCR parameters and primers for Nluc PCR/sequencing were described in the in vitro transcription using T7 Transcription Kit  according to the manufacturer’s instruction. Next, the purified viral mRNA was transfected to Vero cells by Lipo3000  and incubated for 6 h at 37°C before the medium was changed to fresh complete medium.To generate viral mRNA, the vectors of rCA16 and rCA16-Nluc infectious clone was first linearized by Sal I restrictive enzyme , then purified as the template for Cells for WB detection were harvested and lysed in 1 × SDS loading buffer  and heated for 10 min at 100°C. Next, cell lysates were loaded and separated by 10% SDS-PAGE gel and transferred to PVDF membrane. Then, the PVDF membrane was blocked with 5% milk, and stained with the viral 2C monoclonal antibody .Vero cells were seeded on coverslips overnight and infected with the virus at MOI = 1, for another 8 h. Then, the infected cells were washed with cold PBS twice and fixed with 4% paraformaldehyde  for 15 min. After permeabilization with 0.05% Triton X-100 in 2% FBS, the cells were stained with the 2C or 3D antibody for 1 h. Next, cells were washed with PBS twice and incubated with secondary antibodies AlexoFluo594 for 30 min, followed by washing three times. Finally, cells were stained with DAPI and analyzed using EVOSThe virus was 10-fold serially diluted and added to Vero cells in a 12-wells plate, incubated for 2 h at 37°C, then washed with PBS twice, and loaded with 2 × DMEM (4% FBS) and an equal volume of Avicel . Afterwards, the 12-wells plate was incubated at 37°C for another 3 days. Then, the plate was fixed with 4% paraformaldehyde for 2 h and stained with 1% crystal violet for another 2 h.4 Vero cells were seeded in 96-well plates and incubated overnight, before infection with rCA16-Nluc virus at MOI = 0.1. Two hours post-infection, culture medium was replaced with fresh DMEM medium containing different concentrations of GuHCl or ribavirin for 24 h. Next, luciferase activity was measured according to the manufacturer’s instructions.For testing virus replication inhibitors: 2.5 × 104 Vero cells were seeded in 96-well plates and incubated overnight, then the culure medium were replaced with DMEM medium containing different concentrations of chloroquine or NH4Cl for 2 h at 37°C. Afterwards, the cells were infected with rCA16-Nluc virus at MOI = 0.1 for 2 h; then the culture medium was replaced with fresh maintaining DMEM medium for 24 h before measuring the luciferase activity.For testing virus entry inhibitors: 2.5 × 104 Vero cells were seeded in 96-well plates and cultured overnight. Then various concentrations of GuHCl, ribavirin, NH4Cl, and chloroquine in 2%FBS DMEM were added as triplicates per condition. The next day, the CCK-8 solution was added to each well and incubated at 37°C for 1 h, after which the absorbance at 460 nm was measured .After being treated with various concentrations of chemical inhibitors, cell viability was analyzed by Cell Counting Kit-8 . Specifically, 3 * 10n = 5), two rCA16 infection groups (n = 5), and one PBS control group (n = 5). Mice of the infection group were inoculated with 105 TCID50 pCA16 and rCA16 viruses via intraperitoneal routes, while the control mice were given the same volume of PBS. The body weight of mice was followed each day post-infection. For histology and viral characterization, one group of control, pCA16 and rCA16 mice were sacrificed at 4 days after infection. The brain, thigh muscle, lung, and heart tissues were harvested for viral RNA measurement by q-PCR or Hematoxylin and Eosin (HE) staining for tissue damage evaluation.Twenty-five 7-day-old C57/B6 suckling mice were randomly divided into five groups, including two pCA16 infection groups  with 103 TCID50 rCA16-Nluc at 37°C for 2 h. Uninfected cells as a negative control, and sera from the PBS group as the positive control. The virus-sera mixtures were added to the 96-wells plate where 2.5 × 104 Vero cells/well were seeded overnight, for a 24 h-incubation at 37°C. Afterwards, the luciferase activities were measured. The nAb titers are derived from the highest dilution index to achieve a 50% reduction of the relative luciferase activity. In addition, all the immunized sera were also confirmed by the CPE-reduction observation.To inactivate complement, the collected sera were heated at 56°C for 30 min, and then were 2-fold serially diluted  in DMEM and incubated with an equal volume of 10t-test. Differences were considered to be not significant (ns), p > 0.05; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.All the viral RNA copies, luciferase assay data were obtained from at least three repeated experiments. The data were analyzed using Prism 7.0 software  and are presented as the means ± SEM. Statistical significance between the two groups was determined by unpaired two-tailed Student’s in vitro transcription, and found that the recombinant CA16 viruses (rCA16) could effectively infect Vero cells and cause significant cytopathogenic effect (CPE) at 24 h post-infection  by transfection in Vero cells. Vero cells infected with rCA16 and rCA16-Nluc showed significant CPE 24 h after infection . Nluc was inserted following 5′UTR and fused with the viral 2A cleavage site (AITTLG) before the VP4 protein . Then thnfection , althougnfection , in linenfection ,E. Furthnfection . More imnfection . Finally imaging . TogetheThe genomic stability of genetically engineered enteroviruses is challenging and unpredictable. To ensure the stability of rCA16-Nluc, the rescued virus was continuingly passaged and monitored in Vero cell cultures, as indicated in 4Cl  to measure neutralizing antibody titers based on the rCA16-Nluc reporter virus. To this end, the anti-CA16 serum was produced by immunizing the C57/B6 mice twice with CA16 as shown in 5 TCID50 viruses i.p.) in one-week-old lactating mice is 100% lethal and highly pathogenic causing serious damage of multiple tissues, including the brain, hearts, lungs and muscles. In parallel with the fact that majority of enterovirus-infected HFMD cases are under 5 years of age . The virological features of rCA16 is comparable to its parental pCA16, for both plaque morphology and replication kinetics. Besides, rCA16 infection .RY: methodology, conducting experiment, data collection, and writing–original draft. MW, LL, and JY: providing key reagent. JF, XL, and MK: data analysis. JX and XZ: conceptualization, supervision, and funding acquisition. SZ: conceptualization, supervision, project administration, writing–review and editing, and funding acquisition. All authors contributed to the article and approved the submitted version.This project was supported by the National Natural Science Foundation of China (grant nos. 82172250 and 82002135) and Shanghai Public Health Clinical Center (grant nos. KY-GW-2020-10).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Spermidine (SPD), a polyamine naturally present in living organisms, is known to prolong the lifespan of animals. In this study, the role of SPD in melanogenesis was investigated, showing potential as a pigmenting agent. SPD treatment increased melanin production in melanocytes in a dose dependent manner. Computational analysis with RNA-sequencing data revealed the alteration of protein degradation by SPD treatment without changes in the expressions of melanogenesis-related genes. Indeed, SPD treatment significantly increased the stabilities of tyrosinase-related protein (TRP)-1 and -2 while inhibiting ubiquitination, which was confirmed by treatment of proteasome inhibitor MG132. Inhibition of protein synthesis by cycloheximide (CHX) showed that SPD treatment increased the resistance of TRP-1 and TRP-2 to protein degradation. To identify the proteins involved in SPD transportation in melanocytes, the expression of several solute carrier (SLC) membrane transporters was assessed and, among 27 transporter genes, SLC3A2, SLC7A1, SLC18B1, and SLC22A18 were highly expressed, implying they are putative SPD transporters in melanocytes. Furthermore, SLC7A1 and SLC22A18 were downregulated by SPD treatment, indicating their active involvement in polyamine homeostasis. Finally, we applied SPD to a human skin equivalent and observed elevated melanin production. Our results identify SPD as a potential natural product to alleviate hypopigmentation. Hypopigmentation may be diffused or localized, acquired or congenital, and is associated with a specific distribution pattern2. Acquired disorders include vitiligo, pityriasis alba, tinea versicolor and postinflamatory hypopigmentation, while congenital disorders include albinism, piebaldism, tuberous sclerosis and hypomelanosis of Ito. In addition, premature hair graying is a condition characterized by the early appearance of gray hair, generally before the age of 20 years in Caucasians and before 30 years in African Americans3. Though benign, these conditions can cause discomfort and affect the psychological state of the individual, and also increase UV sensitivity. In the cosmeceutical sector, melanogenesis modulators derived from natural sources are typically more appealing to customers. Several treatments for hypopigmentation include steroids, oral drugs, phototherapy and transplants, raising concerns for the safety and commodity of these methods4. For example, topical corticosteroids used to treat vitiligo can cause skin atrophy6. Selenium sulfide 2.5%, used to treat pityriasis versicolor can cause contact dermatitis8. Also, imidazoles and triazoles can commonly cause gastrointestinal disturbances, such as nausea and vomiting, when treated orally10. Therefore, it is important to develop new hypopigmentation treatments that are more natural and human friendly.Hypopigmentation disorders result in the appearance of white macules in the skin. These anomalies may occur due to a decrease in the number of melanocytes in the body, their inability to produce melanin, or abnormal transfer of mature melanosomes to neighboring keratinocytes11. These molecules are formed by the reaction of two or more amino groups, and can easily bind with various negatively charged macromolecules, including DNA, RNA, proteins, and acidic phospholipids13. As a result, they play essential roles in cell growth, proliferation, differentiation, gene regulation, immunity, as well as in the synthesis of proteins and nucleic acids. Spermidine (SPD) is one of the most prevalent natural mammalian polyamines. This molecule is synthesized from putrescine (PUT) or by oxidative degradation of spermine (SPM). In the polyamine pathway, the precursor ornithine initially undergoes decarboxylation by ornithine decarboxylase to generate the diamine PUT. Afterwards, the triamine SPD and the tetraamine SPM are formed by spermidine synthase and spermine synthase reactions, respectively. Moreover, intracellular SPD levels are dependent on polyamine uptake from the extracellular space, endogenous biosynthesis, catabolism, and excretion14. SPD plays important roles in cellular biochemical functions, including nucleic acid and protein synthesis, structure maintenance, and stability. It is estimated that approximately 13% of SPD is bound to DNA, and approximately 57% is bound to RNA to stimulate and improve protein synthesis15. Despite the capacity of an organism to produce SPD by de novo biosynthesis, external supplementation is essential to maintain a balanced polyamine pool16. Plant-derived foods, such as whole grains, vegetables, and legumes contain high levels of SPD, as do aged cheese and raw animal tissues19. Moreover, tissue SPD concentrations typically diminish with aging20. As a result, SPD supplementation exerts anti-aging effects and increases the overall lifespan in several animal models, including nematodes, flies, rodents, and humans21. More specifically, SPD can reduce cancer-associated mortality, enhance autophagy, ameliorate mitochondrial biogenesis, induce intestinal maturation, and improve function of the cardiovascular system, skeletal muscle, the immune system, liver, kidneys, and nervous system.Polyamines are ubiquitous positively charged amines found in all living organisms23. TYR is a bifunctional enzyme that catalyzes the hydroxylation of l-tyrosine to l-3,4-dihydroxyphenylalanine (l-DOPA), followed by the oxidation of l-DOPA to l-dopaquinone. Moreover, TRP-2 functions as a dopachrome tautomerase that converts dopachrome to its carboxylate derivative, 5,6-dihydroxyindole-2-carboxylic acid (DHICA), whereas TRP-1 functions as a DHICA oxidase that converts DHICA to a carboxylated indole-quinone that forms eumelanin (a brown-black pigment)25. Following synthesis and subsequent processing in the endoplasmic reticulum and Golgi apparatus, melanogenesis proteins are trafficked to melanosomes for pigment formation26. Subsequently, tyrosinase degradation occurs spontaneously, which is dependent on the ubiquitin proteasome and on the endosomal/lysosomal systems27. While practically all proteins are affected by such mechanisms, diminished stability and function of tyrosinase has severe implications on pigmentation. Therefore, improving the stability of tyrosinase and its associated proteins constitutes a promising strategy to overcome pigmentation defects. Since SPD has suggested to interact with proteins and improve their stability30, we aimed to find whether this polyamine could be a potential natural compound to target hypopigmentation.Melanogenesis is a complex process that regulates pigmentation. Melanin biosynthesis is tightly regulated by melanocyte-specific enzymes: TYR, tyrosinase-related protein 1 (TRP-1) and TRP-2In this study, we analyzed the potential of SPD for hypopigmentation treatment through a systematic exploration approach which revealed changes in protein degradation. Subsequently, we analyzed the role of SPD in protein stability and, as polyamine transporter expression can vary according to cell type, we also aimed to identify the main SPD transporter present in melanocytes. Lastly, we investigated the potential of SPD application in vivo through a human skin equivalent model.7H19N3) that exerts various metabolic functions in living organisms   membrane transporters was assessed since they are reportedly involved in polyamine transportation38. Firstly, transcriptome data , confirming its potential application for treatment of hypopigmentation disorders  was dissolved in distilled water, and new stocks were prepared monthly. CHX and MG132 were purchased in Sigma-Aldrich and kept at − 30 °C until use.Moderately pigmented normal human primary melanocytes  were cultured in M-254 medium (Cascade Biologics) containing human melanocyte growth supplement  and antibiotics . MNT-1, a malignant melanoma cell line, was kindly provided by Dr. Ai-Young Lee at Dongguk University, who originally received it as a gift from Dr. Vincent J. Hearing at the National Institutes of Health . B16F10 cells were obtained from the Korean Cell Line Bank . MNT-1 and B16F10 cells were cultured in minimum essential medium (Thermo Fisher Inc.) containing 20% fetal bovine serum , antibiotics , 20 mM 4-(2-hydroxyethyl)-1-piperazineëthanesulfonic acid, and 10% Dulbecco's modified Eagle's medium/high glucose . All cells were maintained at 37 °C and 5% CO4 cells/well in a 96-well plate . After overnight incubation, cells were treated with 0, 2, 5, 10, 20, 50, and 100 μM of SPD for 24 h. Subsequently, 100 μL of 3--2,5-diphenyltetrazolium bromide reagent was added to the medium in each well and incubated for 3 h at 37 °C. The reduced formazan crystals were solubilized in 50 μL of dimethyl sulfoxide (Sigma-Aldrich) and absorbances were measured at 570 nm in a microplate reader .Cells were seeded at a density of 1 × 105 cells/dish. After overnight incubation, cells were treated with SPD for 7 days. Cells were lysed in 0.5 M Tris–HCl (pH 7.5) containing 1% NP-40, 0.15 M NaCl, and 5 mM MgCl2, as previously described56. Cell lysates were centrifuged at 15,000 rpm and 4 °C for 20 min. The protein content of each supernatant was measured using a bicinchoninic acid (BCA) assay, and 100 μL of NaOH solution was added to dissolve each pellet and heated at 80 °C for 1 h. Melanin levels in the cells were determined by measuring absorbance at 450 nm. Absorbances were divided by the amount of protein used to determine each total melanin content.Melanocytes were seeded at a density of 1 × 102. Cell lysates were centrifuged at 15,000 rpm and 4 °C for 20 min, and protein contents were quantified using a BCA assay. Aliquots of 20 μg of protein per well were separated in a sodium dodecyl sulfate–polyacrylamide gel, and electro-transferred from the gel to a polyvinylidene fluoride membrane (Sigma-Aldrich). After blocking with 2.5% bovine serum albumin for 1 h, membranes were incubated with primary antibodies targeting TYR , ubiquitin , TRP-1 (1:4000), TRP-2 (1:4000), and GAPDH (1:4000) .Cells were lysed in a solution of 0.05 M Tris–HCl, pH 7.5, 0.15 M NaCl, and 0.01 M MgCl56. Cells were stained with TA99 (Thermo Fisher Inc.) and 4′,6-diamidino-2-phenylindole . Secondary antibody staining was performed using Alexa Fluor 488–conjugated F(ab′)2 fragment of goat anti-mouse IgG antibody . Images were obtained using an LSM 800 confocal microscope .Cells were cultured on Lab-Tek chamber slides , as described previously57. The remaining reads were then aligned to the Homo sapiens reference genome (GRCh38) using TopHat2 software (version 2.1.1) with default parameters58. After the alignment, we counted the numbers of reads mapped to the gene features (GTF file of GRCh38.89) using HTSeq59. Read counts for the samples in each condition were then normalized using TMM  normalization of the edgeR package60.Total RNA was isolated from saline- and SPD-treated melanocytes using RNeasy Mini Kit columns , according to the manufacturer’s instructions. RNA quality was assessed using an Agilent 2100 bioanalyzer with an RNA 6000 Nano Chip , and RNA quantity was determined using an ND-1000 Spectrophotometer . The RNA integrity number (RIN) values for all of the samples were larger than 9. Poly(A) mRNA isolation from the total RNA and subsequent fragmentation were performed the TruSeq Stranded mRNA LT Sample Prep Kit , according to the manufacturer’s instructions. The adaptor ligated libraries were sequenced using an Illumina NovaSeq 6000 . mRNA sequencing was performed for three biological replicates of each condition. From the resulting read sequences for each sample, adapter sequences  were removed using the cutadapt software (version 2.7)57. For each gene, a T-statistic value was calculated using the Student’s t-test for comparison of saline- and SPD-treated melanocytes. An empirical distribution of the T-statistic value for null hypothesis was estimated by performing all possible combinations of random permutations of the samples. Adjusted P-values were obtained for each gene using a two-tailed Student’s t-test with an empirical null distribution. The DEGs were identified as genes with adjusted P-values ≤ 0.05, and absolute log2-fold changes > 0.428 .To identify DEGs, a statistical hypothesis test was performedMelanocytes treated with 1 μM SPD for 7 days were subjected to RNA extraction using a RNeasy Plus Mini Kit . RNA concentrations were measured using a Nano-400A Micro Spectrophotometer . cDNA synthesis was performed using the AccuPower RT PreMix . cDNA, primer (F/R), and CYBR Q GreenBlue qPCR Master Mix  were dispensed into MicroAmp Fast Reaction Tubes , and mRNA expression levels were determined using an AriaMx real-time PCR system .A human skin equivalent model composed of primary human keratinocytes and melanocytes  was used. The basal layer of this human skin equivalent model is composed of dendritic cells that produce melanin granules. These cells produced pigmentation and became fully differentiated when cultured in an air–liquid interface culture for 2 weeks. Cells were then treated with 2 and 4 µM SPD for 2 weeks.Two-tailed Student’s t-test was used to analyze the differences between two groups.Supplementary Figure S1.Supplementary Figure S2.