The expression of CD200R was mainly detected on myeloid cells such as granulocytes, monocytes/macrophages, T cells in human peripheral blood, and human and rat skin. Functional immunoassays demonstrated specific binding of skin-derived CD200+ HDMEC to myeloid CD200R+ cells in vitro. Importantly, we confirmed enhanced CD200–CD200R interaction in vascDESS in vivo. We concluded that the CD200–CD200R axis plays a crucial role in regulating tissue inflammation during skin wound healing.CD200 is a cell membrane glycoprotein that interacts with its structurally related receptor (CD200R) expressed on immune cells. We characterized CD200–CD200R interactions in human adult/juvenile (j/a) and fetal (f) skin and in in vivo prevascularized skin substitutes (vascDESS) prepared by co-culturing human dermal microvascular endothelial cells (HDMEC), containing both blood (BEC) and lymphatic (LEC) EC. We detected the highest expression of CD200 on lymphatic capillaries in j/a and f skin as well as in vascDESS in vivo, whereas it was only weakly expressed on blood capillaries. Notably, the highest CD200 levels were detected on LEC with enhanced Podoplanin expression, while reduced expression was observed on Podoplanin-low LEC. Further, qRT-PCR analysis revealed upregulated expression of some chemokines, including CC-chemokine ligand 21 (CCL21) in j/aCD200 Loss of skin due to congenital diseases or traumatic tissue defects  result in concomitant loss of its barrier function. Although some superficial skin defects with intact dermal elements may heal without autografting, deep partial-thickness and full-thickness burn wounds require autografting of healthy skin from an uninjured site (the donor site) and its placement on the primary wound site . AlthougBioengineered human dermo-epidermal full-thickness skin substitute (DESS) ,5,6,7,8 In this study, we were particularly interested in CD200 (OX-2) expression in HDMEC lining blood and lymphatic capillaries of vascDESS in vitro and in vivo. CD200 has a wide but specific tissue distribution that includes certain vascular endothelia, kidney glomeruli, placental cells, and neurons ,12. Its The presence of CD200 molecules on the cell surface is essential to interact with CD200R to keep tissue homeostasis and avoid exaggerated immune responses leading to tissue damage ,16. Acco+ macrophages to preserve tissue integrity, whereas reduced expression of CD200 attenuates macrophage-mediated tissue damage [+ macrophages and granulocytes [Furthermore, the immunosuppressive capacity of CD200 was demonstrated to regulate also tissue homeostasis in the central and peripheral nervous system , vasculae damage . Hence, ulocytes ,22. CD20ulocytes . Furtherulocytes , in metaulocytes , in chroulocytes , and acuulocytes , melanomulocytes , and mululocytes .The CD200–CD200R link was described to play a crucial role in regulating the immune response, also during skin wound healing . We aimeHere, we report for the first time the characterization of human CD200 and its cognate receptor in human juvenile/adult and fetal skin as well as in bioengineered vascDESS. We demonstrate that CD200 is expressed on both blood and lymphatic capillaries both in vitro and in vivo, whereas CD200R is detected on peripheral blood-derived monocytes, granulocytes, NK, and T-cells. Moreover, we provide functional assays confirming the importance of CD200–CD200R interaction for immune responses and for skin wound healing.www.zhbsd.ch (accessed on 18 March 2022)) according to the guidelines of the local ethics committee.All experiments were performed according to the Declaration of Helsinki Principles and after permission from the Ethics Commission of the Canton Zurich. Human skin-derived dermal microvascular endothelial cells (HDMECs), dermal fibroblasts (HDF), and keratinocytes (KC) were isolated and expanded from fetal skin obtained from Spina Bifida operations between 24 and 26 weeks of gestational age , juvenile/adult skin including foreskin, scalp, skin from the hands, abdomen, legs, and arms from children ≤18 years ; adult skin donors (18–65 years old) from various body areas such as breast and abdomen  as previously described ,33. HUVE5) were incubated for 30 min at 4 °C in with primary antibodies and live/dead Zombie Aqua  and then analyzed by flow cytometry on a FACS ARIA III 4L .Immunohistochemistry: Immunofluorescence staining on cryosections was performed as described before ,8 Table.For double immunofluorescence, some of the primary antibodies were pre-labeled with Alexa 488, 647, or 555-conjugated polyclonal goat F(ab′)2 fragments, according to the manufacturer’s instructions .+ LECs, or CD200− LECs (passage 1–2), isolated and sorted from fetal and juvenile/adult skin samples, were seeded in 24-well plates at the density of 20,000 cells/well. Triplicates were prepared for each cell type. Subsequently, cells were treated with diluted CKK-8 solution for 1 h at 37 °C, and then, the absorbance was measured at a wavelength of 450 nm with a microplate reader . The procedure was repeated every second day for 11 days. OD values reflect the number of viable cells. Mean absorbance values and SDs were calculated at each time point. The proliferation ability of cultured HDMEC was determined using colorimetric Cell Counting Kit-8 kit (CKK-8) according to manufacturer’s instructions . Briefly, BECs, CD200For the viability assay on j/aHDMECs (mixtures of BEC:LEC), cells were concomitantly stained with FdA , PI , and Hoechst 33342  was conducted. For Ki67/CD31 stainings, non-treated cells were fixed with acetone/methanol (1:1) at −20 °C for 5 min and subsequently stained as described above (section “Immunohistochemistry”). Both the viability assay as well as Ki67/CD31 staining were performed every second day for 11 days.+/− BEC and LEC were seeded onto 24 well plates and cultured until confluency. Once confluency was achieved, peripheral blood mononuclear cells (PBMCs) were isolated from fresh whole human blood. For this, buffy coats  were diluted with PBS (1:1) and gently layered over an equal volume of Ficoll-Paque PLUS  and then centrifuged for 30 min at 400 g without brake. After centrifugation, the PBMC fraction was removed, transferred into a new Falcon tube, and subsequently washed twice with PBS. Following the isolation, PBMCs were immediately sorted  into the different lymphocyte subtypes that have been shown to express the CD200 Receptor (CD200R). Next, adherent endothelial cells were stained with CellTracker Deep Red Dye , and sorted lymphocytes were stained with CellTracker Red CMTPX Dye  according to the manufacturer’s instructions. Stained cells were then co-cultured in an incubator at 37 °C for 30 min. The lymphocytes were then added on top of the endothelial cells and left for another 30 min at 37 °C. In the last step, all cells were stained with Hoechst 33342 , fixed in PFA (4%), and analyzed on the confocal microscope (Leica SP8 inverse CLSM). The quantification was performed using Fiji  by counting the number of immune cells to endothelial cells (Immune cells/EC ratio).Sorted CD200− and CD200+ LEC to form colonies in vitro, we performed a clonogenic assay. Sorted CD200− and CD200+ LEC  were seeded into gelatin-coated 6-wells in EGM-2MV medium . After seeding, the cells were left to proliferate for 14 days with medium change every 2 days. On day 14, the medium was aspirated and the cells were washed once with DPBS . Next, 3 mL of a mixture of 6% glutaraldehyde and 0.5% crystal violet solution  was added to each well for 30 min and removed carefully. Cells were washed with water and left to dry. Cell colonies were counted by eye. To assess the potential of CD200260/A280 ~2.0–2.1 and A260/A230 ~2.1–2.3 were used for qRT-PCR. RNA was stored at −80 °C until further use. qRT-PCR was performed according to published protocols [n = 3) using SYBR green chemistry . Each 10 μL reaction contained 5 ng of cDNA, and amplification steps were carried out on a QuantStudio 7 Pro Real-Time PCR System . All data were normalized to GAPDH and quantification was performed using the 2−∆∆CT method with efficiency correction (Pfaffl method) [Total RNA was isolated from cultured cells either grown on culture dishes or directly following FACS sorting according to the manufacturer’s protocol , including a DNAse treatment to remove genomic DNA. Three biological replicates were collected per condition. RNA purity was assessed using an Epoch spectrophotometer . Only pure RNA with absorption ratios Arotocols ,35,36,37 method)  and CD20 method)  (Table 35 of cells (EC and fibroblasts 1:1) were resuspended in 1 mL of collagen gel. The gels were placed in 6-well cell culture inserts with membranes of 3.0 μm pore-size  and kept for 30 min at 37 °C in a humidified incubator containing 5% CO2. After the polymerization period, EGM-2MV  was added to the upper and lower chambers of the well/insert and hydrogels were incubated for two weeks. Then, hydrogels were covered by keratinocytes (7.5 × 104/gel), cultured for an additional week, and transplanted onto immuno-incompetent rats [Collagen type I hydrogels were prepared as previously described . In totaThe surgical protocol was approved by the local Committee for Experimental Animal Research . Immuno-incompetent female nu/nu rats, eight to ten weeks old , were anesthetized by inhalation of 5% Isoflurane  and maintained by inhalation of 2.5% Isoflurane via mask. The dermo-epidermal skin substitutes were transplanted on full-thickness skin wounds created on the back of the rats. n = 2 rats per condition  were transplanted onto full-thickness skin defects prepared on the backs of the rats. To prevent wound closure from the surrounding rat skin, custom-made steel rings (diameter 2.6 cm) were sutured into full-thickness skin defects using non-absorbable polyester sutures . The transplants were then covered with a silicone foil , a polyurethane sponge , a cohesive conforming bandage , and tape as a wound dressing. Dressing changes and photographic documentation were performed once per week. Animals were euthanized using carbon dioxide and the transplanted skins analogs were harvested after 7 and 21 days by in toto excision and processed for immunohistochemical analysis.vascDESS and non-vascDESS . First, blood (CD31+Podo−) and lymphatic (CD31+Podo+) capillaries were quantified in normal j/a and f human skin. Then, the CD200 expression was quantified on those blood and lymphatic capillaries in normal j/a and f human skin.Capillary profiles of immunofluorescently stained normal j/a and f human skin, non-vasc/vascDESS analogs, and rat tissue (lymph nodes) were quantified on 6–8 μm thick cryo-sections using Fiji image analysis software . The entire view field regions at 10· magnification were counted . Comparison between two groups was performed using the two-tailed unpaired Student’s p-value 0.01 to 0.05 (significant) * indicates p-value 0.001 to 0.01 (very significant)** indicates p < 0.001 (extremely significant)*** indicates p < 0.0001 (extremely significant)**** indicates p > 0.05)ns = not significant  skin as well as in fetal (f) skin . To distThe stainings of podoplanin/CD31-positive lymphatic capillaries in human j/a and f skin were further confirmed by immunofluorescence analysis for PROX1, which is a nuclear lymphatic lineage marker . Therefo+Podo−) as compared to the number of lymphatic capillaries (CD31+Podo+) (p < 0.0001). In fetal skin, blood capillaries represented 73.0 ± 19.0%, whereas lymphatic capillaries counted for 27.0 ± 13.0% of all capillaries (p < 0.001).Whereas blood capillaries of both j/a and f skin samples were only CD31-positive (empty arrows), lymphatic capillaries showed the expression of podoplanin, a lymphatic endothelium-specific marker, and a faint CD31 co-expression (white arrows). The overall quantification of capillaries located in the dermis of both skin types revealed a significantly higher number of blood capillaries (CD311+Podo+) c. Specif+Podo− blood capillaries and CD31+Podo+ lymphatic capillaries in j/a (p = 0.0004) (p = ns).Further, we detected CD200 on CD31s in j/a a’’’ as ws in j/a b’’’. Qua 0.0004) d. By conFreshly isolated and in vitro cultured (P1) HDMEC derived from j/a and f skin were analyzed with regard to CD200 expression by flow cytometry . Human C+Podo− blood endothelial cells (BEC) in the freshly isolated j/aHDMEC, which constitute 64.13 ± 11% of all HDMEC  in the freshly isolated HDMEC fraction   d.+Podo+ lymphatic endothelial cells (LEC) in the freshly isolated j/aHDMEC, which were characterized by the different expression levels of podoplanin, namely LEC PodoHigh (25.4 ± 7.2%) as well as LEC PodoLow (10.5 ± 2.4%)  (Low displayed a significantly reduced expression of CD200 (36.9 ± 5.1%)  a. Furthe± 15.6%) b,e, wher ± 5.1%) c,e. p < 0.0001)  cells. Accordingly, we identified approximately 66.7 ± 16.2% of BEC and 33.3 ± 7.5% of LEC in cultured j/a P1 HDMEC. Further, we identified only a weak expression of CD200 on BEC (11.3 ± 2%), whereas the expression of CD200 on LEC was significantly enhanced and accounted for 48.7 ± 17.2% ( 0.0001) i. The LEp < 0.0001)  constituted approximately 58.9 ± 12.5% of all endothelial cells and that only 9.06 ± 2.0% of fBEC showed the expression of CD200 j–m. By c 0.0001) m.High and LEC PodoLow, were almost undetectable on cultured (in vitro expanded) j/a and fHDMEC at P1 in vitro, and, therefore, not further analyzed.The presence of the two subpopulations, namely LEC Podo+ LECs and CD200− LECs as well as donor-matched BEC derived and cultured from j/a and f skin were separated by fluorescence activated cell sorting (FACS) and used for colorimetric cell counting assay in vitro ; d3: p < 0.05) and BEC ; d5: p < 0.01). Interestingly, from day 7–11 during the proliferation assay, the population of j/aCD200+ LEC demonstrated a lower cycling rate as compared to j/aCD200− LEC and j/aBEC. Two-way ANOVA comparison demonstrated the following significance: d7–d11: p < 0.001 (j/aCD200+ vs. j/aCD200− LEC) and d7–d11: p < 0.001 (j/aBEC vs. j/aCD200+ LEC).CD200in vitro a. Prolif+ LEC (violet line) as compared to fCD200− LEC (green line), whereas fCD200+ LEC proliferated faster than fBEC in days 1–5 of the assay  and to fBEC (d5: p < 0.001). Thus, fBEC showed a moderate proliferation rate with cycling values ranging between fCD200+ LEC and fCD200− LEC within days 5–11 of the assay. The proliferation assay of HDMEC isolated from fetal skin revealed a slightly slower proliferation rate of fCD200he assay b. In conCell viability stainings performed on primary j/aHDMECs containing both BEC and LEC populations showed high cell viability throughout the 11-day culturing period c. In add+ as one of the markers of mouse tissue-resident vascular endothelial stem cells with enhanced clonal expansion potential [+ and CD200− LEC used in this study, we performed colony-forming assays .Further, Wakabayashi et al. identified CD200otential . To inveg assays d. The qu−/CD200+ LEC and CD200− BEC fractions, and used for qRT-PCR analysis. Gene expression levels were normalized to GAPDH and expression levels of each sample were calculated relative to the CD200-BEC population. We did not use fibroblasts at all for any kind of normalization or in general in this analysis  as compared to j/aCD200− LEC  and j/aCD200− BEC  (+ LEC (4.44 ± 0.48) as compared to j/aCD200− LEC  and j/aCD200− BEC  (+ LEC (0.22 ± 0.09), whereas it was enhanced in j/aCD200− LEC  and normalized to j/aCD200− BEC   a. Anothe 0.0003) b. Howeve 0.0186) c. The ex+ LEC  (p < 0.0001) and podoplanin  mRNA was significantly reduced in j/aCD200− LEC, and the lowest expression was detected in j/aCD200− BEC . Further, assessment of mRNA expression of PROX1 and podoplanin revealed upregulation of both markers in j/aCD200ctively) d,e. In c+ LEC (19.86 ± 0.41), reduced mRNA levels were observed in j/aCD200− LEC  and j/aCD200− BEC  (+ LEC (2.32 ± 0.11), whereas CCL27 gene expression was upregulated in j/aCD200− LEC  relative to j/aCD200−BEC 1 ± 0.33 (p = 0.0018)  f. In con 0.0018) g. As CD200R is involved in immunosuppressive activity during skin inflammation and was shown to improve graft take and survival , we soug+CD15+CD200+ granulocytes [+ expression (n = 3 blood donors)  was first verified by FACS, and then respective cells were sorted . Accordiulocytes . This gr donors) .+ T-cells in human blood PBMC [+CD56+.Further, using low forward/side scatter and CD3 marker present at all stages of T-cell development, we identified a population of CD3ood PBMC   (+CD16+ monocytes (70% ± 15.5).In addition, a human monocyte subset gated also on low forward/side scatter and on CD14 marker was identified in human blood PBMC . In deta ± 19.2)  and on nFurther, the distribution of CD200R was assessed on immune cells in rat lymph nodes. We detected the CD200R expression on rat myeloid cells using rat-specific pan-myeloid antibody . Moreove+ and CD200− LECs and HUVEC were co-cultured with human blood-derived granulocytes and/or T cells and visualized using confocal microscopy. We confirmed that HUVEC were CD31+ but completely lacked the expression of podoplanin  adhered to HUVEC in the co-culture assay (p = 0.99) and T-cells  adhered to the j/aCD200− LEC (p = 0.0005) and T-cells  adhered specifically to j/aCD200+ LEC  and co-cultured with human blood-derived granulocytes or T-cells labeled with a green cell tracker and co-cultured a–c. The re assay a. Simila200− LEC b as comp200+ LEC c as compInvolvement of endothelial CD200 in the cell–cell interactions between immune cells (blood-derived T-cells) and the endothelium was further investigated by pre-incubation of primary HDMEC with anti-CD200 antibody to block the CD200 epitopes . IgG1 tr+PROX1−) and lymphatic (CD31+PROX1+) capillaries  were positive for CD200, whereas only a few blood capillaries (CD31+LYVE1−) expressed CD200.These findings were conformed using another lymphatic marker, namely LYVE1 b,d. A trAs demonstrated on microvasculature in human skin, CD200 is specifically expressed on lymphatic rather than on blood capillaries . Since pp < 0.0001)  c–d,f. Th+ immune cells has not been investigated so far in human skin. Involvement of human endothelial CD200 in the cell–cell interactions between the endothelium and CD200R+ cells were of myeloid origin, thus co-expressing myeloid, granulocytes, and monocyte/macrophage-specific markers in rat tissue , and vascularized DESS (vascDESS) at one and three weeks after transplantation . Furthert tissue .Whereas only a few CD200R-positive cells were detected in normal human j/a skin 9.65 ± 3.56 a,a’,f, m+ cells within the human dermal compartment at one week  and at three weeks  after transplantation  DESS also showed only a scarce number of CD200Rantation b–d. p < 0.0001 vs. non-vascDESS 1w, p < 0.0001 vs. human skin) and three weeks  in vivo . This difference disappeared and there was a similar number of CD200R+ T cells was found associated (13.3 ± 2.0) and non-associated with CD200-positive endothelial cells .In contrast, human vascDESS containing human blood and lymphatic capillaries demonstrated enhanced density of rat CD200R-positive cells at 1 week  CD200 was differently expressed in blood and lymphatic capillaries, (ii) CD200R was primarily expressed by myeloid and T-cells, (iii) CD200R on myeloid cells interacts with its cognate CD200 ligand expressed on endothelial in vitro and in vivo. Overall, our study demonstrated that the active CD200–CD200R axis plays a crucial role during wound healing of skin substitutes in vivo. Certain aspects of the study deserve more careful consideration.We found that CD200 was abundantly expressed on small capillaries in normal human skin and bioengineered human vascDESS in vivo, in particular on lymphatic rather than blood capillaries of human skin and adipose tissue. This is in contrast to the study of Ko et al. that examined the distribution of endothelial CD200 in different types of vessels, including small capillaries and large-diameter vessels in different rat organs except for skin . WhereasMoreover, Ko et al.  reported+ ECs. Interestingly, CD157 was characterized in their study as a marker of tissue-resident vascular endothelial stem cells (VESC) with higher proliferative potential and clonal expansion than CD157−CD200− ECs [Another investigation performed by Wakabayashi et al. in mice detected the CD200 expression in ECs of most organs, except in the liver . Moreove200− ECs . The mou200− ECs . + EC. However, we could not observe enhanced homeostatic and regenerative stem cell properties using CD200+ as compared to CD200- EC suggesting another possible function of CD200+ cells in human tissues. Therefore, further investigations are needed to validate the potential role of CD200+ human microvascular ECs as VESC in human skin.Although the authors did not investigate CD200 expression on human ECs, nor did they distinguish between mouse blood and lymphatic endothelial cells , their sIt has been demonstrated that the specific expression of surface molecules is responsible for the trafficking of T-cells, monocytes/macrophages, and other leukocytes on the endothelium during tissue inflammation ,45. FurtHigh lymphatic endothelial cells (LEC), whereas PodoLow LEC barely expressed this marker. Interestingly, PodoHigh LEC are restricted to lymphatic precollector vessels, whereas PodoLow LEC are exclusively lining lymphatic capillaries. The heterogeneous distribution of endothelial CD200 among different sites of the lymphatic vascular bed indicates a possible function of CD200+ PodoHigh lymphatic capillaries in the formation and maintaining of HEV. Indeed, previous studies demonstrated the crucial role of podoplanin expression in HEV integrity in an immune cell trafficking into the lymph nodes [Additionally, we demonstrated the highest expression of CD200 on Podoph nodes ,48.+ ECs and CD200R-expressing immune cells in HEV. Thus, the data obtained in this study indicate that CD200–CD200R interaction is critical for the proper function of the innate immune system by providing communication between CD200+ myeloid cells, monocytes/macrophages, and dendritic cells repress their pro-inflammatory activation and interleukin production in vivo [Further, ECs lining human skin microvasculature are the first contact with most blood-borne cells, such as immune cells within the skin. In the present study, we examined the expression and distribution of the CD200R, which is a cognate receptor of CD200. We found CD200R on monocytes/macrophages, granulocytes, and T-cells in both rat and human skin as well as in tissue-engineered DESS. As previously reported CD200 binding to CD200R expressed mainly on CD11b in vivo ,49,50,51 in vivo . Further in vivo . Those results suggest that CD200–CD200R physical binding may have an important role and interactions in cell adhesion between immune cells and endothelium . We conf+ interacted with CD200R. These data are in line with the observations of Ko et al. that showed that CD200–CD200R signaling mediates cell to cell interactions of immune cells with ECs in mouse tissues in vivo [Further, we detected a specific CD200–CD200R interaction in vascDESS in vivo. In particular, mainly lymphatic endothelial CD200 in vivo . The sam in vivo . Moreove in vivo . Further in vivo , autoimm in vivo , collage in vivo , and inf in vivo . Importantly, the interaction of CD200 with CD200R has been associated with a reduced risk of graft rejection as well as prolonged transplantation survival in mice . CD200 w+ EC into prevascularized skin substitutes could prevent excessive inflammation by its immune-suppressive function, thus reducing the risk of graft rejection [Therefore, the interaction of CD200 with its CD200R is of pivotal importance for skin tissue engineering. As such, the incorporation of CD200ejection . Accordiejection ,8,59,60.ejection ,8,59,60 ejection , and thaejection . High lymphatic pre-collectors, suggesting its specific role in the regulation of immune cells trafficking. Importantly, CD200–CD200R is also highly active during wound healing of transplanted skin grafts. It appears likely that a similar interaction between CD200 and CD200R will take place when autologous tissue-engineered skin analogs are transplanted onto human patients. Further, these results implicate that the prevascularisation approach of skin substitutes allows proper cell–cell interactions in the wound bed leading to improved graft take and skin regeneration. Further investigation should explore the function of CD200R+ myeloid cells and their interaction with CD200 in human skin transplants in more detail.In summary and conclusion, this appears to be the first report demonstrating the physical interaction of CD200–CD200R in human skin and transplanted skin grafts. The CD200 was present mainly on Podo


Furthermore, resistance exercise tended to increase the CB1  and CB2  expression in muscle of older adults. Interestingly, increases in the expression of CB2 following resistance exercise positively correlated with changes in key mechanisms of muscle homeostasis, such as catabolism (FOXO3a) and regenerative capacity . This study for the first time shows that CB1 is differentially expressed with aging and that changes in CB2 expression upon resistance exercise training correlate with changes in mediators that play a central role in muscle plasticity. These data confirm earlier work in cells and mice showing that the cannabinoid system might orchestrate muscle growth, which is an incentive to further explore CB-based strategies that might counteract sarcopenia.Aged skeletal muscle undergoes metabolic and structural alterations eventually resulting in a loss of muscle strength and mass, i.e. age-related sarcopenia. Therefore, novel targets for muscle growth purposes in elderly are needed. Here, we explored the role of the cannabinoid system in muscle plasticity through the expression of muscle cannabinoid receptors (CBs) in young and old humans. The CB This condition decreases the quality of life and health through an increased risk for immobility, social isolation and metabolic conditions such as type 2 diabetes.Ageing is associated with changes that eventually affect health (span). Skeletal muscle is not only key for mobility, but also fulfills central metabolic functions, such as substrate storage and oxidation. With advancing age, there is a gradual decline in muscle strength and muscle mass, also referred to as sarcopenia2, omega-34 or vitamin D5 supplementation, is considered the primary intervention to attenuate age-related muscle deterioration. However, muscular adaptations to RE are less pronounced in old compared to young adults7. Hence, it remains important to better understand which molecular networks underlie muscular degeneration with advancing age to eventually develop novel (adjuvant) therapies that can attenuate a loss in muscle strength and muscle mass.Currently, resistance exercise (RE), whether or not combined with protein9, malnutrition10, metabolic dysregulations11 and physical inactivity12. Recently, there is a growing interest in the role of the cannabinoid system in controlling plasticity in metabolic tissues, such as adipose tissue and skeletal muscle15. For example, increased endocannabinoid activity (e.g. high tissue levels of the endocannabinoids) has been associated with metabolic dysregulation such as obesity17. The cannabinoid system is composed of endocannabinoids, e.g. anandamide and 2-arachidonoylglycerol (2-AG), which are lipid mediators binding to the cannabinoid receptor type 1 (CB1) and CB2. These CBs are expressed in many tissues, and most ubiquitously in the nervous and immune system, which might explain the central role of the cannabinoid system in neural processes and in the inflammatory signaling.Multiple factors contribute to age-related muscle wasting, e.g. inflammation18. In neural cells, CB activation induces MAPK signaling which regulates many cellular processes including proliferation, differentiation, apoptosis and energy metabolism. Whereas the signaling responses upon CB binding are well described in neural tissues, it is not understood how CB1/2 affect (MAPK-mediated) downstream signaling in skeletal muscle tissue. Nevertheless, recent developments in mice indicate that CB signaling interferes with muscle metabolism19, muscle maintenance15 and regenerative processes13.CBs, as G-coupled protein receptors, induce intracellular signaling through G proteins which in turn induce or inhibit second messengers and intermediates, including cyclic adenosine monophosphate, phosphatidylinositide-3-kinase (PI3K) and phospholipase A and C1 antagonism in mice reversed age-related metabolic dysregulations20 and improved muscle regeneration in a murine model of Duchenne muscular dystrophy14. Moreover, CB1 knock-out in skeletal muscle protected old mice from insulin resistance and upregulated intermediates of the mTORC1 pathway, responsible for muscle protein synthesis (MPS)15. In vitro data confirmed that CB1 inhibition stimulated mTORC1 and MPS in human myotubes21. Besides anabolic signaling, also muscle catabolism plays a crucial role in muscle maintenance. Unfortunately, there are no studies that investigated whether modifications in the cannabinoid tone affect catabolic mechanisms, such as the ubiquitin–proteasome system (e.g. FOXO and MuRF1) or autophagy (e.g. LC3b). Unpublished data from our lab showed that CB2 was downregulated during muscle atrophying conditions such as unloading and CTX-induced injury22 in mice , indicating an exercise-induced improvement in metabolic health and an improved myogenicity, respectively. Finally, we investigate whether responses in CB expression are related to anabolic, catabolic and regenerative muscle markers, and whether they occur in a fiber-specific way.Despite these promising data indicating that CBs are interesting candidates to target (age-related) muscle wasting, little is known about the expression and modulation of CBs in human skeletal muscle tissue. In anticipation of CB (ant)agonists approval for human application, the present study explores for the first time muscle CB1 expression was higher in healthy, old adults (65–84 years) compared to young adults , whereas CB2 was not differently expressed between age groups.Skeletal muscle CBSer2448, but not phospho/total mTORSer2448 (p = 0.337), tended to be less expressed in skeletal muscle of old vs. young adults . Expression of the catabolic marker phospho/total FOXO3aThr32 was higher in old vs. young skeletal muscle , whereas its downstream mediator, the atrogene MuRF1, was not different between both age groups (p = 0.752). The autophagy markers LC3b-II  and LC3b-II/I  were ~ 2-fold higher in older adults compared to younger adults. Finally, myogenic markers Pax7 and MyoD were not differentially expressed in the muscle of both age groups.Phospho-mTOR1 and CB2, and the expression of anabolic, catabolic and myogenic markers in the pooled dataset of young and old adults revealed no significant correlations.The correlation between the expression of both CB3; p = 0.838)  increased the 1 repetition maximal (1RM) leg press strength from 175.8 ± 16.3 to 222.1 ± 19.1 kg  and muscle density from 49.9 ± 0.6 to 51.1 ± 0.5 HU  but not upper leg muscle volume  and CB2 expression  tended to increase upon RE in old adults , but not total FOXO3a, and decreased p-FOXO1Thr24  and total FOXO1 . RE also decreased the muscle expression of autophagy markers LC3b-I  and LC3b-II  with ~ 10%. Yet, the LC3b-II/I ratio was unaffected due to RE. Finally, the expression of myogenicity markers MyoD and Pax7 was upregulated after RE , whereas phospho/total mTORSer2448 remained unaffected by RE in old adults.Skeletal muscle CB2 was associated with the change in expression in these central markers of muscle maintenance  and with both myogenic markers MyoD  and Pax7 , whereas CB1 was not related to any of the markers of muscle maintenance.The change in expression (Post minus Pre RE) of the CBnce Fig. . Pearson1 and CB2 were highly expressed in the sarcolemma. The expression of CB1 in the m. Vastus Lateralis of older adults was more pronounced in type I vs. type II fibers , whereas CB2 was more expressed in type II vs. type I fibers   Fig. .Figure 415. Unfortunately, CB modulation is not yet feasible in humans. Hence, the present explorative study for the first time investigates whether CB expression is different in old vs. young skeletal muscle tissue, and whether CB expression is responsive to anabolic stimuli such as RE.Recently, it was shown in mice that CBs are key players in the regulation of myogenicity and muscle metabolism, both features that are affected with advancing age, and that contribute to sarcopenia. Consequently, it has been suggested that CB (ant)agonism is a promising strategy to counteract muscle degeneration, and more specifically age-related sarcopenia1 is differently expressed in skeletal muscle of young (20–27 years) vs. old adults (65–84 years), and that both CB1 and CB2 expression tend to increase upon chronic RE in old adults. Interestingly, the RE-induced change in CB2 expression was associated with changes in the catabolic marker FOXO3a and with changes in markers of myogenicity, i.e. MyoD and Pax7. More interventional work in animals and humans is needed to unravel the mechanisms via which CBs might affect muscle plasticity in response to age and exercise.The findings of the present study demonstrate that CB26, and that increased CB1 expression related to a decreased metabolic health, as some28, but not all29, researchers observed higher CB1 expression levels in the adipose tissue of overweight/obese adults when compared to lean. Furthermore, the cannabinoid tone positively correlated with (markers of) adiposity and insulin resistance29 and CB1 expression in visceral adipose tissue positively correlated with BMI27. Therefore, in the present study, it is conceivable that the increased CB1 expression in the muscle of older vs. young adults reflects an increased cannabinoid tone and decreased metabolic health in the older adults. Although the older participants did not suffer from explicit metabolic conditions, it is very likely that their metabolic health is generally lower when compared to physically active, young adults . Also, half of the older adults used statins to lower increased cholesterol levels.It was reported that the cannabinoid tone (e.g. circulating AEA and 2-AG levels) was typically higher in metabolic conditions, such as obesity1 expression has previously been studied in mice, be it on the gene and not on the protein expression level. Evidence in mice was equivocal. CB1 expression was lower in the m. Gastrocnemius of 1 year vs. 2 weeks old mice30, whereas others reported a higher CB1 expression in the m. Gastrocnemius of 16-month old vs. 4-months old mice19. This latter observation is in accordance with our observation that CB1 expression was higher in old vs. young adults. Besides metabolic health, it remains unclear which behavioral  and/or biological (e.g. hormone levels) changes contribute to the effect of ageing on muscle CB1 expression.The effect of age on CB1 expression would decrease in response to a 12-weeks RE program, reflecting an exercise-induced improvement in metabolic health. Surprisingly, CB1 expression increased following RE. This observation is very unlikely to reflect a deterioration in metabolic health, which is why alternative mechanisms might be at work. One hypothesis is that the increased CB expression can be explained by a change in the muscle fiber type composition since CB1 and CB2 expression was more pronounced in type I and type II muscle fibers, respectively. However, muscle MyHC type I and II expression did not alter due to RE, and if any changes were to be expected following RE, it would rather be an increase in type II fibers, which would have resulted in a relative decrease in CB1 expression rather than an increase. Alternatively, it is possible that an RE-induced increase in mitochondrial density31 explains the increases in CB1, as the receptor is highly expressed in mitochondria of human muscle tissue32. However, in contrast to endurance training, it remains debatable whether high-load RE increases the mitochondrial content33. Finally, RE-induced changes in the relative contribution and/or signaling of muscle-resident non-myogenic cell types35 such as immune cells, vascular cells and neural cells might also explain part of the increase in CB1 expression.Whereas acute exercise upregulates circulating endocannabinoid levels (particularly AEA), the effect of chronic exercise on the endocannabinoid tone and on muscle CB expression remains poorly understood. We hypothesized that muscle CB1, also CB2 expression tended to be increased upon RT. Pharmaceutical CB2 agonism increased the expression of satellite cell growth markers such as MyoD and myogenin23, while CB2 knock-out decreased MyoD and myogenin expression during murine muscle regeneration13. Therefore, the increased CB2 expression in the present study might reflect an improved myogenic capacity, e.g. satellite cell content, in response to RE. Indeed, MyoD and Pax7 expression significantly increased upon RE, and the increase of both markers positively correlated with the increase in CB2 expression. Together, these data indicate that there might be regulatory networks that relate CB2 biology to satellite cell myogenicity. However, there is need for more evidence to explore a potential association between muscle progenitor cells and CBs, especially in humans.Similar to CB30 and frogs36 showed that CB1 gene expression was higher in predominantly fast  vs. slow (e.g. Soleus) muscles, our data show that CB1 expression was more highly abundant in type I than in type II fibers of the m. Vastus Lateralis of older adults . From a metabolic perspective, it seems indeed more logical that CB1, which is an important regulatory player in peripheral oxidative capacity32, is more abundant in type I fibers, which exhibit a higher oxidative capacity than type II fibers.Whereas evidence in mice1 is differentially expressed in muscle of old (i.e. higher expression) vs. young (i.e. lower expression) adults, and that both CB1 and CB2 expression increases upon RE, the most important anabolic intervention for muscle growth purposes. Interestingly, RE-induced increases in CB2 correlated with markers of myogenicity (MyoD and Pax7). These data imply that CB modulation might be a promising tool to combat muscle degeneration. As a next step, human intervention studies should be performed to confirm whether CBs are promising targets to improve the muscle phenotype in age-related sarcopenia and/or muscle devastating conditions, such as cancer cachexia.In conclusion, cell culture and murine experiments suggested that CBs can be a promising target to treat cachexia and sarcopenia through modulation of the metabolism and muscle regenerative capacity. Yet, the role of CB modulation as a window of opportunity to treat muscle devastating conditions in (old) humans remains unstudied. The present explorative study for the first time shows that CB−2) and 14 community-dwelling, non-sarcopenic (cfr. EWGSOP21) Caucasian old males  (age: 70.8 (65–78) years; height: 1.74 (1.70–1.80) m; body mass: 82 (71–99) kg; BMI: 27.2 (24.3–31.1) kg·m−2). All participants were recreationally active, but did not engage in a consistent training program or any sport at a competitive level. Muscle tissues were collected in previously executed studies at our laboratory  and have been stored at − 80 °C immediately after completion of the study. The only inclusion criteria for selection were age  and sufficiently muscle tissue available (≥ 15 mg). Exclusion criteria were BMI < 20 or > 35 kg·m−2, unstable body weight (i.e. 2-kg change during the past 6 months), smoking, disease , smoking or supplementation of cannabis or cannabis-related products (e.g. CBD oil), regular protein supplementation and structural RE or endurance training. None of the young participants took any medication. The medicine intake of the older participants reflected the real-life situation, e.g. a large proportion of the older participants took blood thinners , statins , beta blockers , antacids  and medication to treat osteoporosis  and to treat benign prostatic hyperplasia . All muscle biopsies were sampled by the same MD, and all samples have been prepared simultaneously (August 2020) for analyses by the same researcher. This study was approved by the Ethics Committee Research UZ/KU Leuven  and conformed to the Declaration of Helsinki. All participants gave their written informed consents.Study participants were 18 Caucasian young males  (age: 24 (20–27) years; height: 1.83 (1.72–1.91) m; body mass: 77 (71–86) kg; BMI: 23.8 (22.1–26.4) kg·m−2) with the same exclusion criteria as reported in experiment 1. The medicine intake of the older participants reflected the real-life situation, e.g. a large proportion of the older participants took blood thinners , statins , beta blockers , antacids , antidepressants , sleep medication , and medication against migraine , to treat osteoporosis  and to treat benign prostatic hyperplasia . Muscle biopsies were sampled before (Pre) and after (Post) 12-weeks RE program. All biopsies were taken at least 72 h after RE to exclude interference of acute exercise on muscle molecular signaling37. Three supervised training sessions per week started with a 10-min warm-up on a cycle ergometer, followed by lower extremity exercises . In the first 6 weeks, 2 sets of 12–15 repetitions at 70% of the 1-repetition maximum (1RM) were performed, and in the last 6 weeks, participants completed 3 sets of 10–12 repetitions at 80% of the 1RM. This study was approved by the Ethics Committee Research UZ/KU Leuven (S61809) and conformed to the Declaration of Helsinki. All participants gave their written informed consents.Study participants were 19 physically active, non-sarcopenic, Caucasian old adults  (age: 70.3 (65–78) years; height: 1.68 (1.49–1.80) m; body mass: 76 (62–99) kg; BMI: 26.8 (23.3–31.1) kg·mm. vastus lateralis was performed under local anaesthesia  with a 5-mm Bergström-type needle at the Pre and Post experimental session. The muscle sample was immediately frozen in liquid nitrogen and stored at − 80 °C for later biochemical analyses.Prior to muscle biopsies, fasted participants reported at the lab and rested for 45 min. A needle biopsy (~ 150 mg) of the 38, frozen skeletal muscle tissue (15–20 mg) was homogenized (4 × 20 s) in ice-cold lysis buffer  using the FastPrep . The obtained homogenates were centrifuged at 10,000g (20 min at 4 °C) and the supernatant was stored at − 80 °C. Protein concentrations were assessed with the DC protein assay kit using a BSA protein standard , and protein concentrations were equalized by adding lysis buffer. Eventually, laemmli was added  to obtain muscle lysates. Proteins (20–35 µg) were separated by SDS-PAGE (8–12% gels) and transferred to polyvinylidene difluoride membranes. Then, membranes were blocked in TBS-T  + 5% BSA for 1 h. Overnight, membranes were incubated at 4 °C in TBS-T + 5% BSA with the following antibodies: p-mammalian target of rapamycin , total mTOR , CB1 , CB2 , Vinculin , Pax-7 , MyoD , muscle RING finger-1 , microtubule-associated protein 1A/1B-light chain 3 , glyceraldehyde 3-phosphate dehydrogenase , p-forkhead box O (FOXO) 1Thr24/3aThr32  and FOXO3a . Hereafter, membranes were incubated at room temperature for 45 min with horseradish peroxidase-conjugated secondary antibodies . Membranes were scanned and quantified with Genesnap and Genetools Softwares . Protein targets were normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH), vinculin or the total form. Staining with ponceau red staining confirm homogenous sample loading. Ponceau red staining of the representative lanes of the western blot analyses presented in Figs. As previously describede tissue 5–20 mg we tissue 5–20 mg wogenized  × 20 s i1  or CB2 (1/200) dissolved in PBS. Hereafter, the conjugated secondary antibodies were applied for 1 h at 37 °C in a humid chamber: goat anti-mouse Alexa-488 IgG2  and goat anti-rabbit Alexa-594 IgG . Next, cryosections were washed with PBS and for 15 min incubated with Hoechst (1 µg·mL−1) at room temperature. Sections were mounted with Dako fluorescence mounting medium  and visualized by fluorescence microscopy . The epifluorescence signal was recorded with FITC (MyHC I), DAPI (Hoechst) and Texas Red (CB1 and CB2) excitation filters. CB1 and CB2 expression was quantified in 16 muscle cryosections. Six distinct regions within each cryosection were analysed for CB1/2 expression, i.e. 3 regions with type I muscle fibers (MyHC I+ fibers) and 3 regions with type II muscle fibers (MyHC I− fibers). For both receptors, regions with type I and type II muscle fibers were selected in the muscle cryosections with ImageJ 1.52a . Next, the blue (cell nuclei) and green (MyHC I) signal was removed, and the red signal (CB1 and CB2 expression) was quantified in a fiber-type specific way.Muscle tissues were embedded in tissue freezing medium  and frozen in liquid nitrogen-cooled isopentane. Serial cryosections (7 µm thick) were cut with a cryostat  at − 20 °C. Prior to histological analyses, cryosections were thawed at room temperature (RT), washed with PBS and fixed with acetone at − 20 °C. Next, cryosections were blocked for 1 h in PBS containing 2% bovine serum albumin (BSA) and incubated overnight at 4 °C in a humid chamber with the following primary antibodies: BA-F8  I, DSHB, Iowa), CB39. Shortly, participants started with a warm-up set of 8 repetitions at ~ 50% of the estimated 1-RM, followed by a set of 3 repetitions at ~ 70% of the estimated 1-RM. Subsequent lifts were single repetitions with progressively heavier resistances until failure. A 2-min recovery period was provided between each attempt. The heaviest successful lift was determined as 1-RM. Participants were familiarized with the 1-RM leg press procedure. The 1-RM was assessed at baseline (PRE), after 6 weeks of RE and after 12 weeks of RE (POST).The 1-RM was measured on the leg press device as previously described3, a computed tomography scan  was used to measure the muscle volume of the left upper leg. Four thick axial images of 5 mm were obtained at the midpoint of the distance between the medial edge of the trochanter major and the intercondyloid fossa of the femur. Standard Hounsfield units (HU) ranges for muscle (0–100) were used to segment muscle area, and were corrected for bone marrow. The 4 slices were put together as one slice of 20 mm. Total muscle volume was determined by an expert radiologist, with a software program developed at the University Hospital.As previously reportedt Test or Mann–Whitney U Test (experiment 1), or a two-tailed Pair-Sampled t Test or Wilcoxon signed-rank test (experiment 2) . Where relevant, Cohen’s D (d) was calculated as an index of effect size . Correlations were assessed by the Pearson  or Spearman  correlation coefficient analyses . Significance (*) was accepted at p < 0.05 and trends (†) were set at p = 0.05–0.10.Data are presented as mean ± SEM or mean (range), and were tested for normality with a Kolmogorov–Smirnov test. Conditions were compared with a two-tailed Independent-Sample Supplementary Figures.


The kinetics of SMZ adsorption on MSN and MSN-TETA followed a pseudo-second-order model. The adsorption isotherm is described better by a Langmuir model rather than a Temkin or Freundlich model. Release kinetics showed a burst release of SMZ from bare MSN samples (k1 = 136 h−1) in contrast to a slower release found with MSN-TETA (k1 = 3.04 h−1), suggesting attractive intermolecular interactions slow down SMZ release from MSN-TETA. In summary, the MSN surface area did not influence SMZ adsorption and release. On the contrary, the design of an effective drug delivery system must consider the intermolecular interactions between the adsorbent and the adsorbate.Mesoporous silica nanoparticles (MSN) were synthesised and functionalised with triethylenetetramine (MSN-TETA). The samples were fully characterised  and used as carriers for the adsorption of the antimicrobial drug sulphamethizole (SMZ). SMZ loading, quantified by UV–Vis spectroscopy, was higher on MSN-TETA (345.8 mg g To. To54]. As described in SAXS patterns of MSN, MSN-Cl and MSN-TETA samples showed a strong peak due to the (100) plane and other two weak peaks due to the (110) and (200) planes A. SAXS a2 adsorption/desorption isotherms of MSN, MSN-Cl and MSN-TETA samples. All curves show a sharp increase of N2 volume adsorption at a relative pressure P/P0 = 0.2–0.3, due to small-sized pores filled by relatively small volumes of N2. MSN had a BET surface area of 942 m2 g−1 that decreased after functionalisation, becoming 744 m2 g−1 and 671 m2 g−1 for MSN-Cl and MSN-TETA, respectively. The pore volume decreased consistently with TETA functionalisation , and O=S=O bending (568 a 487 cm−1), confirm the loading of the drug on both MSN and MSN-TETA samples, respectively. A mass loss at T > 200 °C is observed for MSN-SMZ (12.3%) and MSN-TETA-SMZ (33.7%). Mass loss is entirely assigned to SMZ for MSN, while in the case of MSN-TETA, the loss of 33.7% can be only in part due to SMZ, since the decomposition of propyl-TETA moiety (17.3%) occurs in the same temperature range. These mass loss data, considered the contribution of the TETA moiety, correspond to a SMZ loading of 163 mg g−1 and 123 mg g−1 for MSN-TETA and MSN, respectively.SMZ drug was then adsorbed on MSN and MSN-TETA samples. SMZ presence on MSN is qualitatively confirmed by FTIR spectra A. The batic -NH2 , while tqt, mg g−1) of SMZ drug as a function of the contact time is shown in tq increase within the first phase of adsorption reaching equilibrium after approximately 5 h. Data were analysed with pseudo-first-order . This is also confirmed by the adsorbed amounts at equilibrium (qeq) of the experimental data compared against the data calculated by the pseudo-second order model. Indeed, calculated qeq were 216.2 and 332.2 mg g−1 for MSN and MSN-TETA, respectively, with the experimental values being 232.9 and 389.1 mg g−1 for MSN and MSN-TETA, respectively  vs. its equilibrium concentration in the adsorbing solution . The highest SMZ loadings were obtained with the functionalised MSN-TETA, corroborating the figures reported above from TGA . Langmuir   than for MSN (442.0 mg g−1). In our experimental conditions, the highest adsorbed amount of SMZ is 345.8 mg g−1 for MSN-TETA and 215.4 mg g−1 for MSN samples  [Temkin A and FreLangmuir C model sctively) . This su samples . Interes3 ± 0.2) . It may 3 ± 0.2) , sulphasSN-TETA 4.3 mg g−13 ± 0.2) .max%) of SMZ was 69.8% and 42.7% for MSN and MSN-TETA, respectively , thus indicating that TETA functionalisation slows down drug release  and is also used in urinary tract infections as bacteriostatic in combination with trimethoprim [−1 [In vitro release of SMZ from MSN and MSN-TETA carriers was then studied. The maximal released amount (Aectively . The kin release . The samethoprim . After 8prim [−1 . The actprim [−1 . These v2PO4 (99%), Na2HPO4 (99%), NaCl, NaOH, 3-chloropropyltrimethoxysilane CPTMS (≥97%), toluene, ammonium nitrate NH4NO3, sulphamethizole (99%) and dimethylformamide (DMF ≥ 99.9%), were purchased from Sigma Aldrich . Ethanol (99.8%) was provided by Honeywell.Hexadecyltrimethylammonium bromide CTAB (>99%), tetraethoxysilane TEOS (98%), anhydrous toluene (99.8%), triethylenetetramine TETA (≥97%), NaH2O with 1 g of CTAB avoiding bubbles formation. The mixture was immersed in an oil bath at 80 °C with mild stirring. Before slowly adding 5 mL of TEOS with a syringe, stirring was increased up to 400 rpm. The reaction mixture was then kept at 80 °C for 2 h. The product was then collected, filtered and washed with water and ethanol. CTAB extraction was carried out by suspending 3 g of product in 1 L of a EtOH/H2O (95:5 v/v) solution containing NH4NO3 10 g L−1, kept for 2 h at 80 °C. TETA functionalisation was conducted by a method similar to that reported in the literature for other silica materials [MSN samples were synthesised as described elsewhere . Brieflyaterials . Briefly2 adsorption–desorption isotherms, Fourier-transform infrared spectroscopy (FTIR), thermogravimetric analysis (TGA) and electrophoretic light scattering . TEM images were obtained on a JEOL 100S microscope. Small-angle X-ray scattering (SAXS) patterns were recorded with a S3-MICRO SWAXS camera system . The scattering patterns were recorded for 1 h. Textural analysis was carried out on an ASAP 2020 instrument, by determining the N2 adsorption/desorption isotherms at 77 K. Before analysis, MSN samples were heated at 250 °C at a rate of 1 °C/min under vacuum for 12 h. MSN-Cl, MSN-TETA and MSN-TETA-SMZ were heated at 25 °C at the rate of 1 °C/min under vacuum for 12 h. The Brunauer–Emmett–Teller (BET) [2 isotherm). FTIR studies were conducted with a Brüker Tensor 27 FTIR spectrometer equipped with a diamond-ATR accessory in the 4000–400 cm−1 range with a number of 128 scans at a resolution of 2 cm−1. TGA measurements were carried out in the T range 25–850 °C (heating rate = 10 °C/min), and under oxygen flow by means of a Perkin Elmer TGA7/DSC7.MSN samples were characterised through transmission electron microscopy (TEM), small-angle X-rays scattering (SAXS), Ner (BET)  and Barrer (BET)  methods −1) in a mixture of EtOH/H2O 60:40, and left under rotation for 60, 100, 300, 360, 1320 and 1440 min at 25 °C. The supernatant was then assayed in a quartz cuvette taking 3 µL of solution and diluting it in 3 mL of a EtOH/H2O 60:40 solution. At different times, the supernatant of each sample was analysed through an Agilent Cary 60 UV–Vis spectrophotometer at 280 nm. The concentration of SMZ in the adsorption solution was calculated through a calibration curve (concentration range 0.5–20 mg mL−1). The residual SMZ concentration evaluated by UV–Vis and the corresponding adsorbed amount tq (mg g−1) was plotted vs. time. Kinetics measurements were analysed by different kinetic models named as:Kinetics of SMZ adsorption on MSN and MSN-TETA was determined  by suspePseudo-first-order  (1)dqtdteqq is the adsorbed quantity of adsorbate at the equilibrium and tq the adsorbed quantity (mg g−1) at time t (min), k′ is the pseudo first order kinetic constant expressed in min−1.k″ is the pseudo-second-order kinetic constant (g mg−1 min−1).Alternatively, the pseudo-second-order model was used :(3)dqtdt2O 60:40 mixture at different concentrations (from 1.1 to 16.7 mg mL−1) under constant rotation for 24 h at 25 °C. The SMZ equilibrium concentration in the supernatant, obtained by centrifugation for 2 min at 1500 rpm, was then assayed by UV–Vis spectroscopy. A volume of 3 µL of the supernatant was diluted in 3 mL of a solution EtOH/H2O 60:40 in a quartz cuvette. The absorbance of SMZ solutions was measured through an Agilent Cary 60 UV–Vis spectrophotometer at 280 nm, and the concentration of SMZ in the adsorption solution was calculated with the appropriate calibration curve (concentration range 0.5–20 mg mL−1). Adsorption data were fitted through Freundlich, Temkin, and Langmuir models.Adsorption isotherm studies were carried out by suspending 10 mg of MSN or MSN-TETA in 1 mL of a solution of SMZ dissolved in a EtOH/HFreundlich model describes the adsorption isotherm as :(5)qeq=Kqeq is the adsorbed amount (mg of adsorbate/g of adsorbent), eqC (mg mL−1) is the equilibrium concentration, FK  is Freundlich constant and n (heterogeneity factor) is a dimensionless parameter that describes the heterogeneous adsorbent surface. Both empirical parameters are specific for adsorbent–adsorbate interactions at a specific temperature. With heterogeneous adsorption sites, a formation of multilayer is to be expected.In which, eqq is the adsorbed amount at the equilibrium (mg g−1), maxq is the maximum cover capacity (mg g−1), LK is Langmuir constant (L mg−1) and eqC (mg mL−1) is the equilibrium concentration. Langmuir model assumes that the adsorbent is homogeneous, and all the sites are equivalent, and only a monolayer of molecules is permitted.The Langmuir model :(6)qeq=qTb is the Temkin constant (J mol−1), R is ideal gas constant (J mol−1 K−1), T is the absolute temperature (K), TA is the isotherm equilibrium binding constant (mL mg−1) and eqC (mg mL−1) is the equilibrium concentration. According to Temkin model, the indirect effect of adsorbent–adsorbate interactions matters. It assumes that the adsorption enthalpy of adsorbed drug is due to an increase in surface covering.Temkin model :(7)qeq=RtM (mg) is drug mass at time of the withdrawal t, M0 (mg) is the mass of drug loaded, maxA is the maximum drug release and k1 (h−1) is the release constant rate.SMZ release kinetic studies were carried out suspending 50 mg of MSN-SMZ or MSN-TETA-SMZ samples in different vessels containing 50 mL of 100 mM phosphate buffer pH 7.4 NaCl 150 mM (PBS) at 37 °C. To analyse the SMZ release, other withdrawals were made at different times within 25 h. Each withdrawal was made of 2 mL, and the same amount of liquid was reconstituted with PBS buffer in each vessel to maintain “sink conditions” . Results2-isotherms of MSN showed a surface area of 942 m2 g−1 that, as expected, and decreased because of material functionalisation. TGA and FTIR techniques were used to characterise functionalised and post-adsorption samples, highlighting a higher drug loading with MSN-TETA (up to 345 mg g−1), despite its lower surface area value (671 m2 g−1), with respect to that of MSN (up to 215.4 mg g−1). SMZ adsorption kinetics on both MSN and MSN-TETA was consistent with a pseudo-second-order model, whereas the adsorption isotherms were consistent with the Langmuir model. The SMZ release of both materials was studied achieving up to 69.8% (171 µg mL−1) for MSN and 42.7% for MSN-TETA (102 µg mL−1). The latter concentration is very similar to the concentration of similar drugs measured in the blood. Interestingly, SMZ release was slower from MSN-TETA (k1 = 3.04 h−1) compared with MSN (k1 = 136 h−1). These findings highlight the importance of the establishment of suitable drug–carrier interactions. Indeed, as observed here, the positive charged functionalisation with TETA moieties may favour the interactions with the negatively charged SMZ rather than the negatively charged MSN surface. The attractive electrostatic interaction between SMZ and MSN-TETA surface is responsible for a higher loading and a slower release compared with the SMZ-MSN pair. The present DDS, based on SMZ loaded on MSN-TETA obtained here, could be potentially used against Gram negative bacteria such as E. coli to treat urinary tract infections. The therapeutic antibacterial activity of the promising DDS will deserve to be tested in a future work.Herein, a new DDS based on SMZ loaded on MSN and MSN-TETA is reported. TEM and SAXS structural characterisations confirmed the successful synthesis of MSN having the typical hexagonal structure. N


Mechanistically, TXNIP ubiquitination through MIB1 E3 ubiquitin ligase regulates NLRP3 inflammasome activation in neuroblasts. In contrast, MPP+ activates the NLPR3 inflammasome through miR590-3p upregulation and direct interference with MIB1-dependent TXNIP ubiquitination. LncZFAS overexpression inhibits this entire pathway through direct interference with miR590-3p, exposing a novel research idea regarding the mechanism of Parkinson’s disease.Parkinson’s disease remains one of the leading neurodegenerative diseases in developed countries. Despite well-defined symptomology and pathology, the complexity of Parkinson’s disease prevents a full understanding of its etiological mechanism. Mechanistically, α-synuclein misfolding and aggregation appear to be central for disease progression, but mitochondrial dysfunction, dysfunctional protein clearance and ubiquitin/proteasome systems, and neuroinflammation have also been associated with Parkinson’s disease. Particularly, neuroinflammation, which was initially thought to be a side effect of Parkinson’s disease pathogenesis, has now been recognized as driver of Parkinson’s disease exacerbation. Next-generation sequencing has been used to identify a plethora of long noncoding RNAs (lncRNA) with important transcriptional regulatory functions. Moreover, a myriad of lncRNAs are known to be regulators of inflammatory signaling and neurodegenerative diseases, including IL-1β secretion and Parkinson’s disease. Here, LncZFAS1 was identified as a regulator of inflammasome activation, and pyroptosis in human neuroblast SH-SY5Y cells following MPPThe online version contains supplementary material available at 10.1007/s12035-021-02619-z. Parkinson’s disease is one of the leading neurodegenerative diseases in developed countries, affecting 1–2% of the elderly population (above 65 years old). Mechani+) is used to induce Parkinson’s disease-like cellular disease. MPP+ is a dopaminergic neurotoxin and the active metabolite of MPT , which is known to cause human parkinsonism after injection[Without a well-defined etiological model, the development and identification of novel therapeutic targets must rely on high throughput screening in immortalized or primary neural cell lines. SH-SY5Yinjection.+ ion imbalance or oxidative stress, through as yet unclear mechanisms[+ efflux, intracellular oxidative stress, or extracellular elevated ATP levels[Neuroinflammation was initially thought to be a side effect of Parkinson’s disease pathogenesis. However, this concept has now been revised, and microglial inflammatory signals are known to exacerbate and significantly contribute to Parkinson’s disease progression–11. Inflechanisms. Perhapsechanisms, 16. Furechanisms. NLRP3 iechanisms. In homeechanisms. In the TP levels; any of TP levels. AutocatTP levels–23. NLRPTP levels.The next-generation sequencing revolution identified a plethora of long noncoding RNAs (lncRNAs), which were previously assumed to be biologically irrelevant, but are now known to have important transcriptional regulatory functions. Recentl+ treatment. This work unveils a potential beneficial role of LncZFAS1 during Parkinson’s disease progression, which could be explored as a new research idea to elucidate the mechanism of Parkinson’s disease.ZNFX1 antisense RNA 1 (ZFAS1) was recently identified as a novel lncRNA transcribed from the antisense orientation of zinc finger NFX1-type containing 1 (ZNFX1) located on chromosome 20q13.13. Over th4 to 106 cells seeded in 10-cm tissue culture dishes. The medium was changed every other day. SH-SY5Y cells were differentiated into dopaminergic neuron-like cells with (10 μM) retinoic acid in DMEM (1%FBS) for 5 days. On differentiation, cell adherence was confirmed under the microscope, and the remaining suspension cells were removed by gentle washing.SH-SY5Y cells were acquired from ATCC and maintained in Dulbecco-modified Eagle medium (DMEM)/F12 (1:1) with penicillin (100 U/mL), streptomycin (100 μg/mL), and 10% fetal bovine serum (FBS). At 90% confluency, the monolayer was washed twice with phosphate buffered saline (PBS), and the cells were chemically detached with 0.05% trypsin for 2 min. After quenching with complete media, the cells were pelleted by centrifugation at 800 rpm for 5 min, washed twice with PBS, resuspended in complete medium and 3 × 106 cells/well for 24 h and then treated with increasing MPP+ concentrations for 24 h. Cells were gently washed with PBS and chemically detached with 0.05% trypsin/EDTA. After detachment, trypsin was quenched with complete media, cells were pelleted by centrifugation, and the caspase activity was detected with the FAM-FLICA caspase-1 assay kit  according to the manufacturer’s recommendations. Briefly, cells were stained and incubated with FLICA (1:30) for 1 h at 37 °C protected from light. Cells were then by washed with 5 volumes of 1× wash buffer, pelleted by centrifugation, resuspended in 1× apoptosis wash buffer with 5 µL of propidium iodide (PI), and incubated for 10 min at 37 °C protected from light. Data were acquired with a Beckman DxFlex flow cytometer  and analyzed with CytExpert . Single stain controls were used to calculate the “compensation,” which refers to the process of correcting fluorescence spillover, that is, removing the signal of any given fluorochrome from all detectors except the one devoted to measuring that dye. PI-positive cells were detected according to the operating procedure of PI-staining kit SH-SY5Y cells were seeded in 6-well plates at 106 cells/well and treated with 1 mM MPP+. When designated, cells were also treated with miRr590-3p mimic or inhibitor and corresponding negative controls. At designated timepoints, cells were washed with pre-cooled PBS and lysed with 1 mL of RIPA buffer supplemented with phenylmethyl sulfonyl fluoride (PMSF) for each 100 µL of sample. After full lysis, samples were centrifuged at 12,000 g, 4 ℃, for 5 min, the supernatant was immediately transferred to a clean pre-cooled tube and stored at −80 ℃ for later analysis. The total protein content was quantified by BCA method, in which 25 µg protein was added to 5×loading buffer and boiled for 10 min in a water bath for denaturing. Samples were stored at −20 ℃ until required for western blotting.Cells were seeded in 6-well plates at 10Protein extracts were prepared as described above. Protein A/G-agarose microspheres were washed twice with PBS and adjusted to a 50% agarose microsphere concentration in PBS. In detail, 100 μL of 50% of protein A/G-agarose microspheres was added to 1 mL of sample and incubated in a horizontal shaker at 4 °C for 10 min to remove unspecific binding. Samples were then centrifuged at 14,000 g for 15 min at 4 °C, and the supernatant was transferred to a clean centrifuge tube. Total protein was estimated by the BCA method and adjusted to 3 μg/μL with PBS. Immunoprecipitation microspheres were prepared in 500 μL with pre-titrated target antibody and 1 volume of bead mixed with 7 volumes of sample. Samples were incubated overnight at 4 ℃ with gentle shaking, followed by centrifugation at 14,000 g for 5 s. The precipitate was collected and washed three times with pre-cooled washing buffer (800 μL per wash). The pellet was resuspended in a suitable volume of loading buffer, and the supernatant was collected for further downstream SDS-PAGE western-blot analysis.Denatured samples were loaded in 10% or 12% resolving and 5% stacking SDS-PAGE gels prepared in house. The samples were separated by electrophoresis in the MINI-PTET  system at 120 V for 5 min (stacking) and 80 V (resolving) for approximately 30 min with pre-chilled 1× electrophoresis buffer. Five microliters of 3-color pre-stained protein ladder  was used as the standard for protein size estimation. For transfer, the PVDF membrane was pre-activated in methanol for 1 min and then immersed in the transfer buffer for 15 min. The samples were transferred with a semi-dry blot apparatus . The transfer efficiency was confirmed by Ponceau S staining. For specific protein expression, the membranes were incubated with primary antibody, at pre-titrated concentrations , and resuspended in 20 µL of RNase-free water. The RNA content, purity, and quality were estimated with Nanodrop 2000  (2 µL of sample).SH-SY5Y cells were treated with MPPMiRNA was extracted with a tissue/cell miRNA extraction kit  according to the manufacturer’s recommendations. In brief, 300 μL of miRNA ReagentA was added, and the sample was mixed by inversion and incubated at room temperature for 5 min. After lysis, 250 μL of miRNA ReagentB was added, again mixed by inversion and centrifuged at 13,000 rpm for 5 min. The supernatant was transferred to a clean 1.5-mL tube, 200 μL of absolute ethanol was added and mixed vigorously, and the sample was incubated at room temperature for 5 min with shaking. After centrifugation at 13,000 rpm for 10 min, isopropanol was added (3:7 volume ratio), and the sample was inverted several times and loaded into the miRNA adsorption column. The column was washed twice with 75% absolute ethanol, dried for 10 min, and eluted with 30 μL RnaseFree TE buffer to a new tube by centrifugation at 13,000 rpm for 2 min.−ΔΔCt method.First-strand cDNA reverse transcription from mRNA was performed using the iScript cDNA Synthesis Kit  in 20-μL reactions with 2 μL of total RNA, according to the manufacturer’s recommendations. The reaction was performed at 25 °C for 5 min, 42 °C for 30 min, and 85 °C for 5 min, and cDNA was stored at −70 °C. qRT-PCR was performed using the Sofast EvaGreen Supermix system  in 20-μL reactions with a 1-μL cDNA template according to the manufacturer’s recommendations in an ABI 7500 real-time quantitative PCR instrument . First-strand cDNA reverse transcription from miR and lncRNA was performed using the one-step miR RT kit  in 20-μL reactions with 4 μL of RNA template according to the manufacturer’s recommendations. The reaction was performed for 60 min at 37 °C, followed by 5 min at 95 °C. qRT-PCR was performed using the cDNA SYBR Green miRNA fluorescence quantitative PCR kit  in 20-μL reactions with 2.5 μL template cDNA. qRT-PCR was run in an ABI 7500 real-time quantitative PCR instrument . qRT-PCR protocols are shown in Table Escherichia coli DH5α cells in house , in a 1 mL total reaction volume, and incubated at room temperature for 15 min. The transfection mixture was slowly added to the 293T cell culture (in serum-free medium) with gentle rocking and incubated at 37 °C, 5% CO2 for 6 h. The cells were gently washed with warm PBS, and 20 mL of complete medium (with 10% FBS) was added and incubated at 37 °C and 5% CO2 for 48 h. Supernatants were collected 48 h after transfection, centrifuged at 4000 g for 10 min at 4 °C to remove cell debris, and transferred to 40-mL ultracentrifuge tubes through a 0.45-μm filter. Lentiviral particles were pelleted by ultracentrifugation at 25,000 rpm, 4 °C, for 2 h, the media supernatant was discarded, and lentiviral particles were resuspended in the residual volume and transferred to a clean tube. Samples were centrifuged again at 10,000 rpm for 5 min, and the supernatant containing lentiviral particles was transferred to a clean tube. The lentivirus titer was detected in 293 T adherent cells. Cells were seeded in 96-well plates at 4 × 104 cells/well, in a 100 μL medium and incubated for 24 h. Lentiviral preps were serial diluted (1/10) in serum free media (10 to 10−10), added to T293 cells, and incubated at 37 °C and 5% CO2 for 24 h. Following incubation, 100 μL of complete medium was added and the lentiviral titer measured after 4 days by fluorescence expression. The lentiviral titer was determined as transducing units (TU/mL) calculated as follows: RNAi sequences were cloned into the GV493 plasmid and expanded in use Fig. . The Lncuse Fig. . Lentivi5 cells/well and incubated at 37 °C, 5% CO2 overnight. Before transfection, cells were washed with PBS, and 500 μL of 0.8 μg/mL polybrene in a serum-free medium with 20 μL lentivirus at a pre-titrated MOI of 5 was added. Cells were incubated overnight at 37 °C and 5% CO2, the medium was removed and replaced with 1 mL of complete medium, and further incubated at 37 °C and 5% CO2. Transfected cells were expanded and subcultured at a 1:3 ratio. Forty-eight hours after subculture, cells were seeded in Petri dishes at 200 μg/mL puromycin; selection medium was changed every 3/4 days until clonal cell clusters appeared. Single cell clones were digested, transferred to 6-well plates, expanded, frozen, and stored in liquid nitrogen. Transfection efficiency was confirmed by western blot and qRT-PCR . Primer 5  was used to design primers  as described above. The site directed mutagenesis was performed with a QuickMutagenesis Kit  according to the manufacturer’s recommendations. SH-SY5Y cells at 50% confluency were then transfected in 96-well plates with Lipofectamine 3000 system  according to the manufacturer’s recommendations. Luciferase double reporter gene expression was detected with the dual luciferase reporter gene detection kit  according to the manufacturer’s recommendations. Briefly, after 36–48 h of plasmid co-transfection, the medium was discarded, and the cells washed with 100 μL 1× PBS. Next, 50 μL of 1× phosphate-buffered saline (PLB) was added to each well, and the plate was incubated for 20–30 min with shaking to ensure lysis. Subsequently, 10 μL of the supernatant was added to a 96-well white opaque microtiter plate , followed by 100 μL of pre-mixed Luciferase Assay Reagent II. Plates were read in a dark chamber with a Berthold LB941 microplate multi-functional microplate reader  2 s after starting the reaction to detect the luciferase activity (RLU1). Finally, 100 μL of pre-mixed Stop&Glo Reagent was then added to each well to detect the intensity of the luciferase reaction in the internal reference control (RLU2). The gene-specific luciferase activity was calculated as RLU1/RLU2.The 3′UTR full length of the pre-selected target gene MIB1 was identified based on the TargetScan Human 7.2 website , mimic NC (sequence: UUGUACUACACAAAAGUACUG), hsa-miR-590-3p inhibitor (sequence: ACUAGCUUAUACAUAAAAUUA), and inhibitor-NC (sequence: CAGUACUUUUGUGUAGUACAA) particles were purchased from Nanjing Darn Pharmaceutical Technology Co., Ltd . SH-SY5Y cells at 50% confluency were transfected into 6-well plates with Lipofectamine RNAiMAX . In detail, Lipofectamine RNAiMAX (6 μL) and 20 pmol RNAi were mixed in 200 μL OPTI-Minimal Essential Medium  and incubated at room temperature for 20 min before transfection. SH-SY5Y cells were gently washed with 1 × PBS three times, 2 mL of OPTI-MEM medium was added to each well, and acclimated to 37 ℃ in 5% CO5 SH-SY5Y cells were seeded onto 24-well microscopy slides . After treatment, cells were washed with pre-cooled PBS and fixed with 4% RNase-free paraformaldehyde at room temperature for 15 min. After washing with PBS three times for 5 min, cells were permeabilized with 0.2–0.5% Triton X-100 for 5 min at room temperature. Cells were washed again with PBS (3 × 5 min) and dehydrated . The hybridization solution was prepared in house with formamide mixed with 2 × saline-sodium citrate (SSC) at a 1:1 volume at room temperature for 10 min. Cells were washed again with PBS, and 25 µL of hybridization solution was added to each well and incubated in the hybridization furnace at 50 ℃ for 4–8 h. Following incubation, 60 µL of denaturing hybridization solution containing probe was added to each well and incubated overnight in the hybridization furnace at 50 ℃. Following incubation, the cells were washed with 0.1 × SSC with 0.1% SDS and 50% formamide for 30 min. The samples were then blocked with 90 µL 20% sheep serum at room temperature for 1 h. Hybridization was detected with 60 µL anti-digoxin antibody, prepared 1/2500 in 10% sheep serum, and incubated overnight at 4 °C. After washing with PBS, samples were counterstained with 10 µL of 200 mg/mL DAPI at room temperature for 10 min. Cells were washed once with PBS, mounted with 10 µL anti-quenching mounting agent, sealed with rubber cement, and imaged using a Zeiss LSM laser confocal microscope .The FISH staining kit was purchased from RiboBio . Digoxin labeled probes (TACTTCCAACACCCGCATTCATC) were acquired from RiboBio . Briefly, 2 × 10p values < 0.05 were considered to indicate significant differences. Data were analyzed with GraphPad Prism 6.0 .All data are expressed as the mean ± SD. One-way ANOVA and non-parametric Kruskal–Wallis test were used to examine the statistical differences between groups. + can also induce inflammasome activation and pyroptosis in human neuroblast cells, SH-SY5Y cells were treated with MPP+ in increasing concentrations, and the frequency of pyroptotic cells was quantified by PI internalization and caspase-1 activation. As expected, MPP+ treatment with increasing MTPT concentrations of 250 nM to 1 mM significantly increased the frequency of pyroptotic SH-SY5Y cells 24 h after treatment , although there was no impact on the overall pre-cleaved peptide levels   Fig. . MPP+-trels Fig. . Additioent Fig. . Finallysis Fig. .Fig. 3Ln+ induces intracellular oxidative stress in SH-SY5Y cells, intracellular ROS was measured using a DCFH-DA ROS-sensing fluorescent probe and analyzed by flow cytometry. As expected, MPP+ significantly induced intracellular ROS in SH-SY5Y human neuroblasts 24 h after treatment  system  is a key antioxidant system that protects cells from oxidative stress. Trx1 is a 12-kDa ubiquitous protein with disulfide-reducing activity, which is mainly localized in the cytoplasm. TXNIP a+ SH-SY5Y cells), which, compared to microglia, are rarely used in this field. The results showed that MPTP-induced NLRP3 inflammasome activation in microglia played a central role in dopaminergic neurodegeneration and Parkinson’s disease[+ inhibits complex I of the electron transport chain, increasing reactive ROS production, and redistributing DA to the cytosol to oxidize DA and generate more ROS[In this study, the Parkinson’s disease-associated neuroinflammatory response was investigated using MPTP-induced cells High Resolution Image (TIF 370 KB)Supplementary Fig. 2pcDNA3.1-GFP vector used for lncZFAS1 overexpression in SH-SY5 cells (PNG 18054 KB)High Resolution Image (TIF 1337 KB)Supplementary Fig. 3LncRNA ZFAS transcriptional levels in empty vector control and LncRNA ZFAS transfected SH-SY5Y cells, measured by qRT-PCR (PNG 5337 KB)High Resolution Image (TIF 500 KB)Supplementary Fig. 4MIB1 sequence cloned in house into the pmiR-report plasmid (PNG 20740 KB)High Resolution Image (TIF 4570 KB)Below is the link to the electronic supplementary material.


Saccharomyces cerevisiae. Among the 257 proteins that significantly changed RNA associations, we observed the coordinated remodeling of RNA-binding enzymes — particularly of the central carbon metabolism — that complemented known metabolic responses. Furthermore, we recognized the propensity for paralogous specific alterations of enzyme-RNA interactions. Our results suggest coordinated cross talk between RNA-enzyme interactions and intermediary metabolism to maintain the physiological and molecular balance upon oxidative stress, perhaps through specialization of paralogous during evolution.RNA-binding proteins (RBPs) are key post-transcriptional regulators that play a substantial role during stress adaptation. Recent proteome-wide surveys have uncovered a large number of new and “unconventional” RBPs such as metabolic enzymes, yet little is known about the reconfiguration of the RNA-binding proteome (RBPome) and RNA-enzyme interactions in response to cellular stress. Here, we applied RNA-interactome capture to monitor the dynamics of the mRBPome upon mild oxidative stress in the yeast   •Oxidative stress induces the rearrangement of 257 proteins on polyadenylated RNAs•Coordinated response of RNA-enzyme interactions and metabolism•Yeast RNA-binding enzymes are paralog specific•Integration of three different mass spectrometry analysis tools Molecular network; Microbial metabolism; Proteomics Caenorhabditis elegans induced changes in approximately 16% of the mRNA binding proteome (mRBPome), including translation initiation and elongation factors , acidic stress induced remodeling of 10% of RNA-associated proteins involved in ribosome biogenesis, translation and the formation of P-bodies and stress granules (RNA-binding proteins (RBPs) play essential roles in regulating the transcription, processing, transport, translation, and turnover of RNAs . Recent  factors , and ars factors . In the granules , while hgranules .Oxidative stress occurs as a consequence of aerobic respiration. Reactive oxygen species (ROS) are produced primarily through leakage of electrons from the mitochondrial electron transport chain and thus cells must uphold an antioxidant system to maintain their redox balance . OxidatiOxidative stress leads to activation of evolutionary conserved signaling pathways, that can either activate transcription factors that in turn promote stress-specific transcriptional responses, or modulate protein synthesis and RNA stability through modification of translation factors or the activity of specific RBPs . FurtherSaccharomyces cerevisiae. Surprisingly, we detected a strong rewiring of metabolic enzyme-RNA associations, a substantial fraction of them relating to enzymes acting in carbon and amino acids metabolism. Additionally, we observed that oxidative stress induced re-arrangements of enzyme-RNA interactions are highly paralogous-specific, suggesting evolutionary specification. These results suggest the coordinated response between enzyme-RNA interactions and metabolic activity for adaptation to cellular stress.Here we applied RNA-interactome capture (RIC) to profile oxidative stress induced changes of the mRBPome in the yeast S. cerevisiae cells were grown to mid-log phase in rich glucose-containing medium (YPD), and oxidative stress was induced by addition of 0.5 mM hydrogen peroxide (H2O2) for 15 min. We recorded increased expression of HSP30 and GRE2 mRNAs, both known to be induced by sub-lethal doses of H2O2 -containing RNAs with bound proteins were purified from cell lysates using oligo[dT]25 beads. We wish to note that the isolation of poly(A) RNA is thought to mainly recover mRNA-interacting proteins, although other types of RNA bearing a poly(A) tail could also be recovered. As a negative control, competition experiments with exogenously added polyadenylic acids were performed for each experiment  (r = 0.57–0.73)  and respective control samples to demarcate non-specific binders were subjected to liquid chromatography-tandem mass spectrometry (LC-MS/MS). To enable robust data interpretation and minimize method specific detection bias, we applied three label-free MS quantification approaches, namely TOP3  which we.7–0.85) A and fai57–0.73) B, the fo2O2 stress with the TOP3, iBAQ, and LFQ analysis approach, respectively , Tkl1, Tps1, and Pfk2. As negative controls, we included Act1, Sod2 and Tkl2, which have not been identified by MS and are therefore not expected to bind RNA , the translational rate (r = 0.11–0.12), or protein levels (r = −0.08)  B. Thus, 2O2 for 20 min  RNA associations of RBPs upon oxidative stress could relate to the different levels of corresponding mRNA targets. To address this, we searched for published targets of the 257 proteins that changed RNA-associations upon oxidative stress. RNA targets for 13 proteins, as previously determined by RBPs immunoprecipitation microarray analysis (RIP-Chip) were identified, namely Aco1, Gre3, Mdh1, Meu1, Nsr1, Scp160, Sti1, Pin4, Puf3, Puf5, She2, Slf1, and Ssd1 . As a cor 20 min . Interesr 20 min C. Thus, r 20 min , are sigr 20 min  and alonr 20 min . Conversr 20 min  are signr 20 min , which g−14), which includes the stress-dependent RBPs Puf3 and Slf1. More specifically, the 150 proteins increasingly associated with poly(A) RNA upon oxidative stress have assigned functions in RNA localization, ribosome biogenesis, RNA processing, and RNA degradation. Interestingly, the latter includes one of the two TRAMP complexes , an evolutionary conserved complex involved in RNA surveillance and quality control (−4), which aligns with the commonly observed ceasing of translation initiation upon stress  terms and pathways A and S2B control . Possibl control . Conversn stress  . Most striking, 12 enzymes acting in “principle pathways of carbon metabolism” were enriched among the 107 proteins with decreased RNA association upon oxidative stress (TOP3 = 5.06), Tkl1) commonly exhibited significantly lowered RNA associations in H2O2 treated cells  and therefore slightly overrepresented among the 257 proteins changing RNA associations significantly upon He stress . This ate stress . Glycolye stress , the glye stress ; and towe stress . Analogoed cells B. Likewied cells C. Convere stress  , fatty acids (e.g. Frm2) and amino acid metabolism. Regarding the latter, 20 enzymes associated with the “biosynthesis of amino acids” (KEGG) were found among the set of proteins with significantly decreased mRNA association upon oxidative stress. Likewise, GSEA across the entire mRBPome revealed significantly reduced RNA associations for proteins allocated to the “biosynthesis of amino acids” . This includes proteins required for the synthesis of aspartate (Aat2), asparagine , glutamate , serine/lysine , methionine (Met6), cysteine (Sah1), threonine (Thr4), isoleucine , and others. The relevance of the herein observed changes in RNA-enzyme interactions is not known but may suggest the existence of an RNA-imposed regulatory layer for amino acids synthesis during stress.We next asked whether functional specification toward RNA-binding enzymes could have been implemented through specialization of paralogous genes after early genome duplication in yeast. We therefore considered a list of 1,094 proteins, representing 547 paralogous pairs . One hun2O2 treatment stress, we found only two paralogous pairs  but 44 proteins that had a non-RNA binding paralog. In this regard, immunoblot analysis with tagged strains confirmed that Tkl1 but not Tkl2, and cytosolic superoxide dismutase 1 (Sod1) but not its mitochondrial paralog Sod2 interacts with poly(A) RNA, respectively (−5) and carbon metabolism  are overrepresented among those 44 unique paralogues as compared with all annotated paralogous proteins in yeast. Thus, oxidative stress may preferentially alter mRNA associations of selected paralogues acting in metabolic processes. If so, we speculate that environmental stress could have been a driver for specialization of RNA-binding paralogues during evolution.Interestingly although, among the 257 proteins that changed RNA associations significantly upon Hectively E. Furthe2O2. By performing RIC and integrating three complementary MS analysis methods, we defined a list of 257 proteins — corresponding to 25% of the yeast mRBPome — that changed association with poly(A) during oxidative stress (FDR <5%). This included stress-regulated RBPs with known roles in translation and RNA degradation, reminiscent of previous observations made with arsenite-treated human cells, which promotes a strong oxidative stress response . More surprising though, we observed profound changes in RNA-enzyme interactions, many of them acting in carbon and amino acids metabolisms that commensurate with altered metabolisms. Thereby, the imposed reconfiguration of enzymes-RNA interaction appeared to be paralogue-specific, proposing stress adaptation as a driver for the specialization of RNA-binding enzymes during evolution.We investigated the plasticity of the yeast mRBPome upon exposure of yeast cells to short mild oxidative stress caused by exposure to Hresponse . Likewisresponse . Importa2O2 stressed yeast cells  to characterize the mRBPome upon oxidative stress was based on capturing poly(A) RNAs from cells, a major component of it comprising mRNAs; likely disregarding changes for RBPs that preferentially interact with other RNA species, such as rRNAs and tRNAs. We have also analyzed changes of the mRBPome under one mild stress conditions. Therefore, we do not know whether the observed coherent changes in RNA-enzyme interactions are specific to the chosen stress conditions, and in how far they are dependent on the concentration and duration of stress application with Ha.gerber@surrey.ac.uk).Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, André P. Gerber .All S. cerevisiae strains used in this study were derived from the BY4741 background strain  was added to the culture once cells reached mid-log phase (OD600 ∼ 0.6) and incubated for 15 min at 30°C. Cells were collected by centrifugation and washed in PBS. For UV-crosslinking, cells were resuspended in 25 mL of PBS and exposed to 1,200 mJ/cm2 of 254 nm UV light in a Stratalinker 1800 (Stratagene) with two 2-min breaks on ice and gentle mixing  at 30°C with constant shaking at 220 r.p.m. To induce the oxidative stress response 0.5 mM He mixing . Cells w−1 DNase I , 100 U ml−1 RNasin , complete EDTA-free protease-inhibitor cocktail ) and mechanically broken with glass beads in a Tissue Lyser  for 10 min at 30 Hz at 4°C. The cooled lysate was cleared by three sequential centrifugations at 4°C at 3,000 g for 3 min, and 5,000 g and 10,000 g for 5 min each. A negative control was introduced at this stage: the extract was supplemented with 20 mg of poly(A) for competition experiments. To capture polyadenylated RNAs for mRBPome analysis, one milligram of oligo[dT]25 Dynabeads  was equilibrated in lysis buffer, mixed with the extracts (∼5 mg) and incubated on a shaker for 10 min at room temperature (RT). The beads were collected with a magnet and the supernatant recovered for repeat incubations (see below). The beads were washed once with 500 μL of wash buffer A  and twice with 500 μL wash buffer B . The poly(A) RNA was eluted from beads in 60 μL of 10 mM Tris-HCl, pH 7.5 at 80°C for 2 min and collected. The entire procedure was repeated twice by reapplying the supernatant to the oligo[dT]25 beads, which were recovered after elution and washed three times with lysis buffer prior to reapplication. The three sequential eluates were combined and concentrated to 70 μL in a 0.5 mL Microcon-10 kD Centrifugal Filter Unit with Ultracel-10 membrane . In total, we subjected 12 samples to MS analysis: three independent experiments  of the mRBPome in control conditions as well as three poly(A) competition control experiments; and three independent experiments for profiling the mRBPome under oxidative stress conditions along with three poly(A) competition experiments.RIC was essentially performed as described previously . Pellet HSP30 and GRE2 upon oxidative stress, reverse transcription (RT) was performed with 500 ng of total RNA combined with a mixture of with oligo[dT]18 and random hexamer primers, and the Transcriptor High Fidelity cDNA Synthesis Kit according to the manufactures' instructions . PCR was conducted with 1 μL (5%) of cDNA with the following primer pairs: HSP30_Fwd, 5′-CTAGAGGGTTCAATGCACTTAT-3′; HSP30_Rev 5′-CTCACCGTCTGGTTGAATAC-3′; GRE2_Fw, 5′-CCGGAACTATTTGGTGGATAC-3′; GRE2_Rev, 5-CCTCCGATACGATTAGTCTTTG-3′; ACT1_Fw, 5′-GTCTGGATTGGTGGTTCTATC-3′; and ACT1_Rev, 5′-GGACCACTTTCGTCGTATTC-3′. PCR was performed for 5 min at 94°C, 30 cycles at 94°C for 30 s, 57°C for 30 s, 72°C for 40 s, and 8 min at 72°C. PCR products were electrophoresed on a 2% agarose gel and visualized with peqGreen DNA/RNA dye .Total RNA was isolated from 50 μL of extracts with the ZR RNA MiniPrep kit  with in column DNA digestion. To monitor induction of −1. Solvent A was 0.1% formic acid and Solvent B was aqueous 80% acetonitrile in 0.1% formic acid. Peptides were ionized by nano-electrospray ionization at 2.1 kV using a stainless steel emitter with an internal diameter of 30 μm (Thermo Scientific) and a capillary temperature of 250°C. Tandem mass spectra were acquired using an LTQ- Orbitrap Velos mass spectrometer controlled by Xcalibur 2.1 software (Thermo Scientific) and operated in data-dependent acquisition mode. The Orbitrap was set to analyze the survey scans at 60,000 resolution (at m/z 400) in the mass range m/z 300 to 2,000 and the top twenty multiply charged ions in each duty cycle selected for MS/MS in the LTQ linear ion trap. Charge state filtering, where unassigned precursor ions were not selected for fragmentation and dynamic exclusion , was used. Fragmentation conditions in the LTQ were as follows: normalized collision energy, 40%; activation q, 0.25; activation time 10 ms; and minimum ion selection intensity, 500 counts.MS analysis was performed at the Proteomics Facility, University of Bristol. 50 μL of the samples (∼8–10 μg of protein) were run on a 4–12% NuPAGE Novex acrylamide gel (Life Sciences). The gel lane was cut into one slice and subjected to in-gel tryptic digestion using a ProGest automated digestion unit (Digilab UK). The resulting peptides were fractionated using a Dionex Ultimate 3000 nanoHPLC system in line with an LTQ-Orbitrap Velos mass spectrometer (Thermo Scientific). In brief, peptides in 1% (vol/vol) formic acid were injected onto an Acclaim PepMap C18 nano-trap column (Dionex). After washing with 0.5% (vol/vol) acetonitrile 0.1% (vol/vol) formic acid peptides were resolved on a 250 mm × 75 μm Acclaim PepMap C18 reverse phase analytical column (Dionex) over a 150 min organic gradient, using 7 gradient segments  with a flow rate of 300 nL min0.05% of the yeast input extract (∼5 μg of protein) and 10%–50% of the eluates were resolved on 4–15% SDS polyacrylamide gels and transferred to polyvinylidene difluoride (PVDF) membranes (Thermo Scientific Pierce). Membranes were blocked in PBS-0.1% Tween 20 containing 5% low fat milk, probed with designated antibodies and horseradish peroxidase (HRP)-coupled secondary antibodies, and developed with the Immobilon Western Chemiluminescent HRP Substrate (Millipore). Blots were recorded with a FluorChem (Alpha Innotech) and quantified with ImageJ. The following antibodies were used: rabbit anti-Scp160  1:10,00 ; 1,070 proteins with TOP3, and 1,310 proteins for both MaxQuant modes were retained. The filtered raw peak area data was normalized such that the total peak area within each biological sample was equal to 106. A pseudocount of 1 was added to all normalized values and log2 transformed. Only proteins with an average (across replicates) raw biological peak area ≥ 3-fold greater than the corresponding average raw control peak area in both conditions were retained, leaving 935, 986 and 1,282 proteins in TOP3, LFQ and iBAQ analysis modes, respectively. Subsequently, proteins not identified in at least two of the three biological samples for at least one of the conditions were removed, leaving a final count of 812, 933 and 1,276 proteins in Top 3, LFQ and iBAQ analysis, respectively. These sets of proteins were further analyzed to estimate an FDR for declaring differentially abundant proteins between conditions. Since samples were not paired an average pairwise fold-change between replicate samples was calculated to identify proteins that change in abundance/presence between conditions. The probability of observing such a fold-change by chance (i.e. FDR) , was based on the distribution of average fold-changes generated from 10,000 random experiments. Random experiments were generated by shuffling the experimentally obtained fold-changes of replicates. From this randomized dataset, the average log2 fold-changes were calculated to assess, for each possible log2 H2O2/WT threshold of −10 to 10 in 0.5 increments, the percentage of the 10,000 randomly generated fold-changes that were above that threshold i.e. the FDR. From this simulated data a linear model of the form FDR ∼ log2 H2O2/WT fold-change was created and subsequently used to produce FDRs for all experimentally obtained (non-random) fold changes. Proteins having an FDR ≤5% were considered to be differentially abundant  were filtered to contain only the proteins identified with at least 2 peptides in at least one biological sample  were identified with the GO Term Finder at the Saccharomyces Genome Database (http://www.yeastgenome.org/) (complete analysis is given in Significantly enriched GO terms in Database  (SGD; htOverlap significance of Venn diagrams was calculated using hypergeometric distributions.