Rationale: The migration of mesenchymal osteoprogenitor cells (OPCs) to bone formation surface is the initial step of osteoblastogenesis before they undergo osteoblast differentiation and maturation for governing bone formation. However, whether the migration capacity of OPCs is compromised during aging and how it contributes to the aging-related bone formation reduction remain unexplored. In the present study, we identified a migration inhibitory factor  and examined whether targeting lnc-PMIF could facilitate osteoprogenitor cells migrating to bone formation surface to promote bone formation during aging.Methods: Primary OPCs from young (6-momth-old) and aged (18-momth-old) C57BL/6 mice and stable lnc-PMIF knockdown/overexpression cell lines were used for in vitro and in vivo cell migration assay . RNA pulldown-MS/WB and RIP-qPCR were performed to identify the RNA binding proteins (RBPs) of lnc-PMIF. Truncations of lnc-PMIF and the identified RBP were engaged to determine the interaction motif between them by RNA pulldown-WB and EMSA. By cell-based therapy approach and by pharmacological approach, small interfering RNA (siRNA)-mediated lnc-PMIF knockdown were used in aged mice. The cell migration ability was evaluated by transwell assay and cell intratibial injection assay. The bone formation was evaluated by microCT analysis and bone morphometry analysis.Results: We reported that the decreased bone formation was accompanied by the reduced migration capacity of the bone marrow mesenchymal stem cells  in aged mice. We further identified that the long non-coding RNA PMIF (postulated migration inhibitory factor)  was highly expressed in BMSCs from aged mice and responsible for the reduced migration capacity of aged OPCs to bone formation surface. Mechanistically, we found that lnc-PMIF could bind to human antigen R (HuR) for interrupting the HuR-β-actin mRNA interaction, therefore inhibit the expression of β-actin for suppressing the migration of aged OPCs. We also authenticated a functionally conserved human lncRNA ortholog of the murine lnc-PMIF. By cell-based therapy approach, we demonstrated that replenishing the aged BMSCs with small interfering RNA (siRNA)-mediated lnc-PMIF knockdown could promote bone formation in aged mice. By pharmacological approach, we showed that targeted delivery of lnc-PMIF siRNA approaching the OPCs around the bone formation surface could also promote bone formation in aged mice.Conclusion: Toward translational medicine, this study hints that targeting lnc-PMIF to facilitate aged OPCs migrating to bone formation surface could be a brand-new anabolic strategy for aging-related osteoporosis. We showed that the decreased bone formation was accompanied by the upregulated expression of lnc-PMIF within BMSCs and the reduced migration capacity of BMSCs in aged mice. We found that the lnc-PMIF could bind to human antigen R (HuR) for interrupting the HuR-β-actin mRNA interaction, therefore inhibit the expression of β-actin for suppressing the migration of aged OPCs to bone formation surface. We also authenticated a functionally conserved human lncRNA ortholog of murine lnc-PMIF. We further showed that silencing lnc-PMIF could facilitate the aged OPC migrating to bone formation surface for promoting bone formation in aged mice.The migration of OPCs to bone formation surface is the initial step of osteoblastogenesis before they undergo osteoblast differentiation and maturation for governing bone formation  and further induced for osteogenic differentiation in vitro. It was found that a series of gene encoding the migration-related proteins, e.g. the MMP family , CD44, β-actin, Tgf-β and Pdgf-BB, were all downregulated in aged BMSCs as compared to young BMSCs in both genders . As revealed by the Alkaline phosphatase (ALP) staining and Alizarin Red S staining analysis, the ALP activity after 7 days of osteogenic induction and calcium mineral deposition after 14 days of osteogenic induction were comparable between the BMSCs from aged and young mice  and intratibial injection into another batch of young mice (1×106 cells per mouse) Figure C. The tibias were then harvested three days later for undecalcified cryosection followed by fluorescent immunostaining of the osteogenic marker Runx2. As revealed by Immunofluorescent analysis, we surprisingly found that the average number of Dil-labeled cells approaching the calcein-labelled bone formation surface was significantly lower in mice treated by aged BMSCs as compared to those treated by young BMSCs, suggesting that the migration capacity of OPCs to the bone formation surface was reduced during aging  and bone formation rate (BFR/BS) were both significant lower in aged (18-month-old) C57BL/6 mice as compared to the young (6-month-old) control mice  Macf1 was significantly downregulated in aged BMSCs as compared to young BMSCs Figure H. Interestingly, we identified a long non-coding RNA PMIF , which is transcribed from the Macf1 gene locus as another transcript Figure I. Subsequently, we performed QPCR analysis to compare the expression of lnc-PMIF and another three lncRNAs previously reported to be involved in regulating cell migration, i.e., Hotair . We then examined the subcellular localization of lnc-PMIF by QPCR and fluorescence in situ hybridization (FISH) analysis, by which we found that the lnc-PMIF transcript was detected in both nuclei and cytoplasm of MC3T3-E1 cells B-C. To determine the expression level of lnc-PMIF in osteoblast differentiation, MC3T3-E1 cells were cultured in osteogenic medium for 35 days. It was found that lnc-PMIF increased in the early stage and then decreased after mineralization initiation during osteoblast differentiation , suggesting that lnc-PMIF might play a role in the early stage of osteogenic differentiation. To investigate the effect of lnc-PMIF on Macf1, we conducted cell transfection assay by using expression plasmids and siRNA of lnc-PMIF, respectively. It was found that overexpression of lnc-PMIF had no significant effect on Macf1 expression . Importantly, knockdown of lnc-PMIF could upregulate the Macf1 expression in both RNA level and protein level . In addition, neither overexpression nor knockdown of lnc-PMIF had notable effect on the coding and noncoding transcripts that transcribed from/nearby the lnc-PMIF locus . To conduct the lnc-PMIF gain- and loss-of-function study, we transfected the MC3T3-E1 cells with the lnc-PMIF shRNA-expressing plasmid or nonsense RNA control (NC) shRNA-expressing plasmid to generate the osteogenic progenitor cell line with stable lnc-PMIF knockdown (KD) and the relative control (KD-NC), respectively A. We also transfected the MC3T3-E1 cells with lnc-PMIF-expressing plasmid or NC-expressing plasmid to generate the osteogenic progenitor cell line with stable lnc-PMIF overexpression (OE) and the relative control (OE-NC), respectively B. Interestingly, we found that the cell numbers, cell viability and the proportions of cell cycle phases were all comparable between the KD and KD-NC group and between the OE and OE-NC group, respectively C-H. Moreover, there were no significant differences in either the mRNA and protein expression of osteoblast-specific markers , ALP activity or calcium mineral deposition between the KD and KD-NC groups as well as between the OE and OE-NC groups after osteogenic induction I-N. These findings indicated that lnc-PMIF was not involved in regulating the proliferation and osteogenic differentiation of OPCs.Next, we employed the MC3T3-E1 cell line, which is a common murine osteogenic progenitor cell line, to investigate the role of lnc-PMIF in OPCs. First, we obtained the full-length of lnc-PMIF by 3' Rapid Amplification of cDNA Ends (3' RACE) technique and confirm the sequences containing 1455 bp by Sanger sequencing Figure A-B. In contrast, they were both significantly lower in OE group as compared to the OE-NC group Figure C-D. Thereafter, we harvested the KD, KD-NC, OE and OE-NC cells, which were labelled by Dil and injected into the bone marrow cavity of tibias in young C57BL/6 mice (1×106 cells per mouse) as afore described. The tibias were harvested three days later for the subsequent immunofluorescent analysis as aforementioned. We found that the average number of Dil-labeled cells approaching the calcein-labelled bone formation surface was significantly higher in the mice treated by KD cells as compared to those treated by KD-NC cells, whereas it was significantly lower in the mice treated by OE cells as compared to those treated by OE-NC cells Figure E-F. We then performed immunohistochemical (IHC) staining and found that the lnc-PMIF-related signaling molecules were higher in the mice treated by KD cells as compared to those treated by KD-NC cells . Taken together, these findings suggested that lnc-PMIF could suppress the migration of OPCs to bone formation surfaces without disturbing their proliferation and osteogenic potential.On the other hand, as revealed by Transwell migration assay and Wound healing assay, the cell migration number and the migration distance were both significantly higher in the KD group as compared to the KD-NC group Figure A. Subsequently, the bands containing the aforementioned pull-down fractions were cut and subjected for Mass Spectrometry (MS), by which we identified that the human antigen R (HuR), an RNA-binding protein previously found to promote cell migration and invasion . We then performed western blot analysis to confirm that HuR were enriched within the aforementioned pull-down fractions from the cell lysis of biotin-labeled lnc-PMIF-expressing cells Figure B. In turn, we conducted the RNA immunoprecipitation (RIP) assay in combination with QPCR analysis in the MC3T3-E1 cells, by which we detected a significantly higher level of lnc-PMIF bound to the immunoprecipitated HuR proteins as compared to that bound to the immunoprecipitated IgG control proteins Figure C. Interestingly, we also detected a significantly higher mRNA level of β-actin, one of the essential components of cell migration Figure C, whereas the mRNA levels of the other three migration-related genes, Macf1, Dynein and Mmp2, bound to the immunoprecipitated HuR and IgG proteins were alike Figure C. All these findings confirmed that lnc-PMIF could directly bind to HuR.Thereafter, we sought to understand the lnc-PMIF-mediated regulatory mechanism on OPC migration. First, we search for the interacting protein partners of lnc-PMIF by conducting RNA pulldown assay on the cell lysis of MC3T3-E1 cells transfected with biotin-labeled lnc-PMIF-expressing plasmid. The eluted pull-down fractions were resolved and separated by SDS-PAGE and visualized by silver staining, which showed that several proteins  were specifically pull-downed together with the biotin-labeled lnc-PMIF from the cell lysis of biotin-labeled lnc-PMIF-expressing cells as compared to the control cells . Accordingly, we then investigate whether lnc-PMIF is involved in the HuR-β-actin-mediated regulation on OPC migration. We found that both the mRNA and protein expression of β-actin were significantly downregulated in the aforementioned MC3T3-E1 cells with stable lnc-PMIF gene overexpression (OE), but significantly upregulated in the aforementioned MC3T3-E1 cells with stable lnc-PMIF gene knockdown (KD) Figure D-E, indicating that lnc-PMIF could inhibit the β-actin expression in OPCs. Interestingly, we found that neither gene overexpression nor knockdown of lnc-PMIF could alter the protein expression of HuR Figure E. In addition, the confocal laser scanning microscopy of the β-actin fluorescent immunostaining showed that the OPC microfilaments (MF), which are composed of polymers of actin and have revealed their pivotal role in cell migration Figure F. These findings, in combination with the aforementioned findings of Transwell migration and Wound healing assay of the KD, KD-NC, OE and OE-NC cells Figure A-D, suggested that lnc-PMIF could inhibit the expression of β-actin to suppress OPC migration without affecting the expression of HuR. To investigate whether HuR could mediate the regulation of lnc-PMIF on β-actin and OPC migration, we performed RNAi-mediated HuR gene knockdown in the aforementioned KD cells and KD-NC control cells, respectively. Interestingly, we found that the elevated mRNA and protein expression of β-actin in KD cells were remarkably downregulated when the HuR gene was simultaneously knockdown Figure G-H. Consistently, the increased cell migration ability of KD cells was also compromised when the HuR gene was simultaneously knockdown Figure I-J. Collectively, all these data suggested that lnc-PMIF could interact with HuR for inhibiting the expression of β-actin to suppress OPC migration. Meanwhile, we also identified a human lnc-PMIF ortholog , which was transcribed from the human Macf1 gene locus as another transcript . In line with the function of murine lnc-PMIF, we found that silencing of the human lnc-PMIF could directly inhibit the migration activity of the human osteoblastic precursor hFOB1.19 cells in vitroD-E, indicating that the lnc-PMIF-mediated regulatory mechanism on OPC migration were functionally conserved between murine and human.Because HuR could bind to and stabilize the β-actin mRNA for promoting the migration and invasion of tumor cells Figure A, and synthesized the biotinylated full-length lnc-PMIF  and the truncated lnc-PMIF mutants, i.e. mutant A1 (sense 590-1455 bp without the 5' stem-loop), mutant A2 (sense 860-1455 bp with the center stem-loop and 3' stem-loop) and mutant A3 , respectively. We then transfected the MC3T3-E1 cells with the aforementioned WT or mutant sequences and conducted tagged-RNA streptavidin pull-down assay. The subsequent western blot analysis showed that HuR was present in the pull-down fractions from the cells transfected with either WT lnc-PMIF, truncated lnc-PMIF mutant A1 or truncated lnc-PMIF mutant A2, but absent in the pull-down fractions from the cells transfected with truncated lnc-PMIF mutant A3 Figure B. These data indicated that the center stem-loop of lnc-PMIF were sufficient for enabling the interaction between lnc-PMIF and HuR.To elucidate how lnc-PMIF interact with HuR to regulate β-actin expression and OPC migration, we first predicted the secondary structure of lnc-PMIF by bioinformatic analysis Figure C. Meanwhile, we designed and synthesized the biotin-labeled or unlabeled probe specifically targeting the center-stem loop of lnc-PMIF and the biotin-labeled or unlabeled probe specifically targeting the sequences of β-actin mRNA previously reported for interacting with HuR protein, respectively. By RNA electrophoretic mobility shift assay (EMSA), we found that the lnc-PMIF-HuR complex could only be detected when the biotin-labeled lnc-PMIF probe was incubated with either WT HuR or truncated mutant B3 Figure D, suggesting that the RRM3 of HuR could mediate the interaction between HuR and lnc-PMIF. Intriguingly, the amount of lnc-PMIF -HuR complex detected was markedly reduced when the biotin-labeled lnc-PMIF probe was incubated either with WT HuR and unlabeled β-actin probe or with truncated mutant B3 and β-actin probe Figure D. Moreover, the β-actin mRNA-HuR complex could be detected when the biotin-labeled β-actin probe was incubated with WT HuR, whereas their amount detected was decreased after the concurrently addition of unlabeled lnc-PMIF probe in a dose-dependent manner Figure E. These data suggest that the lnc-PMIF could compete with β-actin mRNA to interact with HuR. Furthermore, we constructed the HuR-RRM3 expressing plasmid, which was transfected into the aforementioned OE MC3T3-E1 cells with stable lnc-PMIF gene overexpression. We found that the decreased mRNA and protein expression of β-actin in OE cells were remarkably upregulated after the overexpression of HuR-RRM3 Figure F-G. Consistently, the reduced cell migration activity in OE cells was notably enhanced after the overexpression of HuR-RRM3 Figure H. Further, we identified a cross-species functionally conserved HuR sequence  peptide on HuR-RRM3) binding lnc-PMIF  and the truncated mutants of HuR proteins  1 of HuR alone, mutant B2 with HuR RRM2 alone and mutant B3 with HuR RRM3 alone, respectively) in vitro as aforementioned and transfected with lnc-PMIF small interference RNA (si-PMIF) for silencing lnc-PMIF and NC siRNA (si-NC) as control, respectively Figure A. QPCR analysis showed that the expression of lnc-PMIF was markedly downregulated in the si-PMIF-treated BMSCs as compared to the si-NC-treated BMSCs Figure B, which suggested the efficient knockdown of lnc-PMIF in the aged OPCs. Flow cytometry analysis showed no significant difference in the proportions of cell cycle phases between the si-PMIF-treated aged BMSCs and si-NC-treated aged BMSCs, indicating that knockdown of lnc-PMIF did not affect the proliferation of aged BMCSs Figure C. ALP activity staining revealed no significant differences in ALP activity between the si-PMIF-treated aged BMSCs and si-NC-treated aged BMSCs after osteogenic induction, suggesting that knockdown of lnc-PMIF did not alter the osteogenic capacity of aged OPCs in vitroFigure D. However, Transwell migration assay showed that the si-PMIF-treated aged BMSCs had a significantly higher cell migration number than that of si-NC-treated aged BMSCs Figure E. These data suggested that silencing lnc-PMIF could enhance the migration capacity of aged OPCs without disturbing the proliferation and osteogenic differentiation of aged OPCs in vitro. Thereafter, we performed intratibial injection with the Dil-labelled si-PMIF-treated aged BMSCs or Dil-labelled si-NC-treated aged BMSCs on the aged male C57BL/6 mice (1×106 cells per mouse), which were sacrificed three days later to collect the tibias for the subsequent Immunofluorescent analysis as aforementioned Figure A. We found that the average number of Dil+ cells approaching the calcein-labelled bone formation surface was significantly higher in the mice receiving si-PMIF-treated aged BMSCs as compared to the mice receiving si-NC-treated aged BMSCs Figure F-G, suggesting that silencing lnc-PMIF could facilitate the aged OPCs migrating to bone formation surface in aged mice in vivo. More importantly, Runx2 expression was detected in most of the migrated Dil+ cells in the mice receiving the si-PMIF-treated aged BMSCs, indicating that the si-PMIF-treated aged BMSCs migrated to the bone formation surface could undergo normal osteogenic differentiation, which would contribute to bone formation in the aged mice. In addition, the average integral optical density (IOD) of TGF-β positive cell on bone surface was significantly higher, while TRAP positive area had no significant difference, in the mice receiving si-PMIF-treated aged BMSCs as compared to the mice receiving si-NC-treated aged BMSCs , indicating that si-PMIF-treated aged BMSCs might affect cell recruitment (for bone formation) rather than bone resorption. To evaluate the effect of replenishing the aged BMSCs with lnc-PMIF knockdown as the cell-based therapy on aging-related osteoporosis, we performed intratibial injection with the aforementioned si-PMIF-treated aged BMSCs or si-NC-treated aged BMSCs on another batch of aged male C57BL/6 mice (1×106 cells per mouse) Figure A. As shown by microCT analysis, we found the higher bone mass and superior microarchitecture of tibia metaphysis together with the higher BMD and BV/TV in the mice receiving si-PMIF-treated aged BMSCs as compared to the mice receiving si-NC-treated aged BMSCs on 4 weeks after the BMSC injection Figure H. Additionally, as revealed by dynamic bone histomorphometry analysis, we found the higher MAR and BFR/BS in the mice receiving si-PMIF-treated aged BMSCs as compared to the mice receiving si-NC-treated aged BMSCs on 4 weeks after the BMSC injection Figure I. These results demonstrated that replenishing the aged BMSCs with lnc-PMIF knockdown could promote bone formation in aged mice. Collectively, it also suggested that the aberrantly upregulated lnc-PMIF could be responsible for the reduced migration of aged OPCs to the bone formation surface, which could contribute to the aging-related bone formation reduction.Considering the compromised migration capacity of aged OPCs to bone formation surface and the aberrantly upregulated lnc-PMIF in aged OPCs as aforementioned, we next sought to investigate whether silencing lnc-PMIF could facilitate the aged OPCs migrating to bone formation surface. The aged BMSCs were harvested and proliferated i.e., si-PMIF) approaching the OPCs around the bone formation surface, the si-PMIF or si-NC was encapsulated within our previously developed bone formation surface-targeting delivery system -liposome) which could facilitate the encapsulated siRNA approaching the low crystallized bone formation surface where OPCs are recruited for osteogenic differentiation 6)-liposome-si-PMIF (10 mg/kg), (DSS6)-liposome-si-NC or (DSS6)-liposome alone (vehicle) at a 2 weeks interval Figure A. To examine the knockdown efficiency of si-PMIF, the Gli1+ mesenchymal OPCs previously shown to be responsible for cancellous bone formation , suggesting the efficient gene knockdown of lnc-PMIF within OPCs around bone formation surface. At 23 months of age, all the mice were sacrificed to collect bone specimens for subsequent microCT and dynamic bone histomorphometry. As shown by microCT analysis, we found the higher bone mass and superior microarchitecture of tibia metaphysis together with the higher BMD and BV/TV in the mice with both genders from si-PMIF-treated group as compared to the si-NC-treated and vehicle-treated groups Figure B-C. As revealed by dynamic bone histomorphometry analysis, we found the higher MAR and BFR/BS in the mice with both genders from si-PMIF-treated group as compared to the si-NC-treated and vehicle-treated groups Figure D-E. In addition, after the treatment, neither the TRAP-positive area nor TGF-β positive area had a significant difference among the three groups . Collectively, these findings suggested that targeted silencing lnc-PMIF in OPCs around bone formation surface could promote bone formation in mice with aging-related osteoporosis.Given that the aging-related osteoporosis is a systemic bone disease, we next sought to evaluate the effect of systemically targeted silencing of lnc-PMIF in aged OPCs around the bone formation surface by pharmacological approach on aging-related osteoporosis. To facilitate the lnc-PMIF siRNA  that was consistently upregulated in the aged BMSCs. Surprisingly, the gain-/loss-of-function studies demonstrated that the lnc-PMIF could not only inhibit the migration capacity of OPCs in vitro but also suppress their migration to bone formation surface in vivo. However, neither overexpression nor silencing of lnc-PMIF could influence the proliferation and osteogenic differentiation of OPCs. All these results demonstrate that the aberrantly high expression of lnc-PMIF in aged OPCs could be responsible for the compromised migration capacity of aged OPCs to bone formation surface, thus contributing to the bone formation reduction during aging A.During the physiological process of osteoblastogenesis, the OPCs are recruited to the bone remodeling site, where they proliferate, migrate to bone formation surface and undergo osteogenic differentiation toward the functional mature osteoblasts in vivo, and there were very variable results obtained in different laboratories Although it was controversial whether BMSCs undergo a decrease of proliferation and differentiation potential Bmncr as a key regulator of BMSC fate during skeletal aging by serving as a scaffold to facilitate the interaction of TAZ and ABL, and thus facilitate the assembly of the TAZ and RUNX2/PPARG transcriptional complex for promoting osteogenesis and inhibiting adipogenesis of BMSCs B. It is widely recognized that MACF1, crosslinked by F-actin and microtube, was a positive regulator in cell migration in vitro, suggesting that this lncRNA-mediated regulatory mechanism is evolutionarily conserved.The emerging roles of lncRNA in regulating the proliferation, differentiation and senescence of OPCs have been documented in previous researches Given the important role of BMSCs as the unique source of OPCs for osteoblastogenesis The C57BL/6 mice were obtained from the Chinese University of Hong Kong and the Laboratory Animal Center of Air Force Medical University of China and maintained under standard animal housing conditions  in Hong Kong Baptist University and in Northwestern Polytechnical University. All the experimental procedures were approved by the Committees of Animal Ethics and Experimental Safety of Hong Kong Baptist University (Ref No. (19-120) in DH│HT&A│8│2│6 Pt.1) and by the Institutional Experimental Animal Committee of Northwestern Polytechnical University (No.2017015 & No.2019030).6) in 10 μL of α-MEM were injected into the bone cavity of recipient mice through the hole in the femur using a micro-syringe. The recipient mice were sacrificed on 3 days after the intratibial injection. The protocol was modified according to the published reference 6-month-old C57BL/6J mice male mice will be used as recipients. The mice were anesthetized by injection of pentobarbital sodium, and the left distal femur were gently drilled with a 26 G needle through the patellar tendon. Donor cells were fluorescence labelled by Dil . The Dil-labeled cells  containing 10% FBS (Gibco) and 1% penicillin and streptomycin (Gibco). The hFOB1.19 cell line was maintained in DMEM (Gibco) with 10% FBS and 1% penicillin and streptomycin. The primary BMSCs were prepared according to the previously published protocol 6) in 10 μL of α-MEM were injected into the bone cavity of recipient mice through the hole in the femur using a micro-syringe. The recipient mice were sacrificed on 3 days after the intratibial injection. The protocol was modified according to the published reference 6-month-old C57BL/6J mice male mice will be used as recipients. The mice were anesthetized by injection of pentobarbital sodium, and the left distal femur were gently drilled with a 26 G needle through the patellar tendon. Donor cells were fluorescence labelled by Dil . The Dil-labeled cells .Cells were seed at a concentration of 1X10Transient transfection of cells with siRNA or DNA plasmids was performed in 24-well plates using Lipofectamine 3000 reagent (Invitrogen). HuR expression plasmid (500 ng per well), empty plasmid (500 ng per well), siRNA control (30 nM) and siRNA (si-HuR/si-PMIF) (30 nM) were transfected into cells in culture medium and then harvested for further detection.TM/U6 Entry vector. Then the pLenti6/ BLOCK-iTTM expression construct was then generated by recombination with pLenti puro/H1TO/BLOCK-iT/DEST vector. The sequences for shRNA against lnc-PMIF were shown as follows: shRNA targeted to 718-736 of the lnc-PMIF (CCTTAGGTGCCTTTAGAAA). (2) As for lnc-PMIF overexpression, the lnc-PMIF sequences were cloned into pENTR/D-TOPO MCS vector. Then the engineered plasmid containing lnc-PMIF sequence was used for recombined with pLenti puro/TO/V5/DEST vector and packaged by ViraPowerTM Packaging Mix, for lentivirus-mediated overexpression of interest genes. The resulting plasmid was used for lnc-PMIF overexpression in cells.(1) As for lnc-PMIF knockdown, BLOCK-iT Lentiviral RNAi Expression System (Invitrogen) was used to generate the lentiviral lnc-PMIF shRNA. Briefly, the shRNA sequences were cloned to the pENTR4 in the upper compartment of each chamber and after 16 h of serum deprivation, the serum was added in the bottom chamber. After 6 h, cells were removed from the upper membrane surface by a cotton swab and washed with PBS. Migration values were determined by counting three fields per chamber after fixing the membrane in 4% paraformaldehyde and staining with Hematoxylin Staining Solution .(1) Wound healing assay: Cell monolayers were wounded using a sterile 10 μL pipette tip. Phase contrast images were taken immediately after wounding and at 18 h post-stimulation using a microscope  connected to a video camera . To measure migration, wound area was quantified using image analysis software  and expressed as normalized width of wound closure. (2) Transwell assay: Cell migration was determined using a 6.5 mm transwell chamber with an 8 μm pore size . Cells were seeded at a density of 3×10The Vybrant® MTT Cell Proliferation Assay Kit was used. Firstly, a 12 mM MTT stock solution was prepared by adding 1 mL of sterile PBS to one 5 mg vial of MTT (Component A). Then, 10 mL of 0.01 M HCl was added to one tube containing 1 gm of SDS (Component B). The isolated osteogenic cells were washed with 100 µL of fresh medium, added with 10 µL of the 12 mM MTT stock solution and incubated at 37 °C for 4 h. Then, 100 µL of the SDS-HCl solution was added to each well, mixed thoroughly using the pipette and incubated at 37 °C for 4 h in a humidified chamber. Then, each sample was mixed again using a pipette and the absorbance was read at 570 nm.Total RNA extracted from mouse and human cells were separated by PAGE (7M urea) on 6% polyacrylamide gels and transferred to a plus positively charged nylon membrane (Invitrogen). Labelled Lnc-PMIF probes were synthesized in RiboBio Company . RNA blots were hybridized in UV Hybridization Incubator  at 68 °C overnight, washed twice (5 min) with 2X saline sodium citrate (SSC) / 0.1% SDS wash buffer at 68 °C, followed by stringent washes (2X30 min) with 0.1×SSC/0.1% SDS wash buffer at 68 °C. Blots were visualized using Bio-Rad ChemiDoc XRS+ (Bio-Rad).-ΔΔCT method Total RNA was extracted from frozen tissue with a polytron homogenizer  or directly from cells by using TRIzol reagent . Total RNA was used as a template for double-stranded cDNA synthesis . The SYBR® Premix Ex Taq™ II  was applied for the quantitative RT-PCR. GAPDH were used as endogenous controls for normalization. All the primer sequences were listed in Supplemental The transcription start/end site of lnc-PMIF were determined by using 3' and 5'RACE kits based on the manufacturer's instructions (TaKaRa). All RACE primers are listed in Fluorescence-conjugated lnc-PMIF probes (5′-ACTCAGCACAGCAACACTAGCT-3′) were synthesized in RiboBio Company  and the lnc-PMIF was detected by FISH Kit  according to the manufacturer's instructions. Images were obtained with a Nikon 80i Fluorescence Microscope.in vitro , labeled with biotin RNA labeling mix (Roche), and then incubated with nuclear extracts separated from MC3T3-E1 cells. Pulldown components were separated by SDS-PAGE and silver staining. Finally, differential bands were analyzed by western blotting or mass spectrometry (MS) as previous describe The RNA pull-down assay was conducted as described 2, 10 mM of HEPES [pH 7.0], 0.5% NP-40, and 1 mM of dithiothreitol), supplemented with complete protease inhibitors  and Recombinant RNase Inhibitor  for a 30 min ice bath and centrifugalization . Supernatants were precleared with Protein A/G beads and incubated with rabbit anti-HuR antibody  or rabbit IgG  at 4 °C overnight. Protein A/G beads were then added and incubated at 4 °C for 4 h. Finally, binding RNA was isolated by phenol-chloroform-isoamyl alcohol for RT-qPCR.Isolation of MC3T3-E1 cell was lysed in polysome lysis buffer , and labeled with biotin RNA labeling mix (Roche) following the manufacturer's instructions. The plasmid of wildtype HuR and its fragment deletion mutations were synthesized from pcDNA3.1-HuR plasmid . RNA EMSA experiments were conducted according to the manufacturer's protocol with LightShift™ Chemiluminescent RNA EMSA Kit (Thermo Fisher Scientific). HuR (WT/mutant) was incubated with or without unlabeled probe for competitive reaction and anti-Flag for supershift at 25 °C for 20 min in a reaction buffer. Then biotin-labeled probe was added into the reaction system and incubated for 20 min at 25 °C. Unlabeled probes at indicated concentrations were used for competition experiments. The reactions were then loaded onto a 1% 0.5× TBE-agarose gel and transferred to a positively charged nylon membrane (Invitrogen). The membrane was then cross-linked by exposure to UV , incubated with HRP-conjugated streptavidin and visualized with ECL reagents. The dissociation constant (Kd) was calculated as the concentration of unlabeled probe when half of the labeled probe was dissociated from the complex with HuR.The high-throughput RNA-Seq experiments were performed by Annoroad Gene Technology . Briefly, total RNAs was extracted from cells by TRIzol Reagent (Life Technologies). Then the RNA samples were evaluated for degradation and impurities by 1% agarose electrophoresis. The RNA purity was determined using a NanoPhotometer . An Agilent 2100 Bioanalyzer and Agilent RNA 6000 Nano Kit were used to assess RNA concentration and integrity . The samples were sequenced and samples with RNA Integrity Number (RIN) scores > 9.5 were used in subsequent analyses. The mouse genome was selected as the reference genome in this study. High -throughput sequencing was performed by Annoroad technologies . The lncRNA library was constructed from total RNA (2 μg) using different index tags with the next Ultra directional RNA library prep Kit for Illumina  according to the manufacturer's instructions. Data quality control involved removal of contaminated reads, low quality reads, reads containing N ratios >5%, and reads that matched the ribosomal RNA. The RNA-seq data filtered for each sample was compared with the genome by HiSAT2. FPKM was used to estimate gene expression levels quantitatively.Tissues or cells were lysed in in a buffer containing 50 mM Tris (pH 7.5), 150 mM NaCl, 0.5% NP40 , and protease inhibitors . Proteins in lysates were resolved by SDS-PAGE and then transferred to polyvinylidene difluoride membranes. The membranes were blocked with 5% BSA for 1 h at room temperature and incubated with primary antibody recognizing MACF1 , Runx2 , Osteocalcin , CollagenⅠ  , HuR , β-Actin , CBF-A  , Lamin A/C  or Gapdh  at 4 °C. Incubation with secondary horseradish peroxidase-labelled antibody was carried out for 1 h at room temperature.2O. Cells were stained with 40 mM Alizarin Red S (Sigma), pH 4.0, for 15 min with gentle agitation. Cells were rinsed five times with double-distilled H2O and then rinsed for 15 min with 1× PBS while gently agitating.Cells were fixed in 70% ice-cold ethanol for 1 h and rinsed with double-distilled HAlkaline phosphatase staining was monitored using an Alkaline Phosphatase Assay Kit . Cells were fixed by immersion 4% paraformaldehyde (PFA) solution for 10 min and rinsed in PBS for 5 min 3 times. The samples were then placed in an alkaline phosphatase staining solution (BCIP/NBT solution) for 30 min. The whole procedure was protected from light. After discarding the solution, cells were rinsed in deionized water 2 min 2 times.Flow cytometry was performed with a Becton Dickinson flow cytometer to quantify the cell cycle. In brief, cells were washed twice with phosphate-buffered saline (PBS), trypsinated, and resuspended in PBS-2% FCS-20 mM EDTA. Analysis was performed in a Becton Dickinson flow cytometer with appropriate filter settings. The data were quantified by using the CellQuest software package.The FACS samples were prepared in a RNase-free environment. Cells were harvested from the cancellous bone region of long bones as follows. The epiphysis was removed to dissect the metaphyseal bone region extending about 5 mm from the growth plate, which was crushed with mortar and pestle in FACS Buffer, followed by digestion with 0.25% collagenase type I  in PBS for 30 min at 37 °C with gentle shaking. The cells were then centrifuged and re-suspended with FACS Buffer and filtered with a 70-μm cell strainer. The cells were fixed in 0.01% formaldehyde for 10 min, washed with PBS, and incubated with Alexa Fluor 594-conjugated Mouse monoclonal anti-Gli1 antibody  diluted in cell permeabilization medium  for 1 h at room temperature. Thereafter, the cells were washed in PBS, re-suspended in FACS buffer and subjected for cell sorting by the BD FACSAria™ III cell sorter. The Gli1+ cells were collected for RNA isolation and the subsequent reverse transcription PCR and QPCR analysis.The proximal tibia from the mice were fixed with 4% PFA before embedding in optimum cutting temperature . Then, the series-frozen sections (5 μm) were prepared in a cryostat (Thermo Scientific™ CryoStar™ NX70) at -24 ºC. Undecalcified sections were generated using the Kawamoto's film method.+ cell number per bone perimeter (Dil+.N/B.Pm) The immunostained section were made using professional image analysis software  under fluorescence microscope .For immunostaining, sections were incubated with blocking buffer containing 10% donkey serum and 0.15% Triton X-100 (Abcam # ab7475) for 1 h followed by incubating with primary antibodies Rabbit monoclonal anti-Runx2 antibody  at 4 °C overnight. Thereafter, the sections were washed and incubated with Alexa Fluor 647-conjugated fluorescent secondary antibodies  for 1 h. After three wash in PBS, the sections were mounted by cover slides with Fluoroshield Mounting Medium with DAPI . DilFor immunohistochemical (IHC) staining, a Pierce™ Peroxidase IHC Detection Kit  was used. Sections were incubated with blocking buffer containing 10% donkey serum and 0.3% Triton X-100 for 1 h followed by incubating with primary antibodies Rabbit monoclonal antibody (Rb mAb) of MACF1 , Rb mAb of Dynein  and Rb mAb of TGF-beta3  at 4 °C overnight. Thereafter, the sections were washed and incubated with HRP-conjugated secondary antibodies  for 1 h. Following three washes in PBS, the sections were developed for color reaction using DAB/Metal solution and hematoxylin counterstain. The immunostained section were made using professional image analysis software  under microscope .For immunofluorescent staining, cells were fixed with either 4% PFA for 20 min at room temperature followed by permeabilization with 0.2% Triton X-100 for 2 min. Next, samples were blocked with 1% BSA diluted in 1× PBS supplemented with 0.05% Tween-20 for 45 min and sequentially incubated with primary anti-β-Actin antibody  at 4 ºC overnight and fluorescently labeled secondary antibodies for 1 h. Finally, samples were washed, dried and mounted in DAPI solution (Beyotime).The LCM samples were prepared in a RNase-free environment. Briefly, the tibias fixed by 4% PFA before embedding in OCT. Then, the series frozen sections (5 µm) were prepared using the Kawamoto's film method in a cryostat (Thermo Scientific™ CryoStar™ NX70) at -24 ºC. The sections were mounted on LMD films (SECTION-LAB Co. Ltd.). The cryosections were then incubated with Alexa Fluor 594-conjugated Mouse monoclonal anti-Gli1 antibody  for 1h at room temperature. After brief rinsing in RNase-free water, the sections were air-dried. Gil1-positive cells around the calcein labeled bone surface were isolated by microdissection with an upgraded laser pressure catapulting microdissection system (P.A.L.M.) using a pulsed 355 nm diode laser in the Leica LMD 7000 Laser Microdissection System. About 150~200 identified cells were collected in reaction tube containing 5 µL lysis buffer for total RNA extraction and the subsequent reverse transcription PCR and QPCR analysis.The distal femur was dehydrated in graded concentrations of ethanol and embedded without decalcification in the modified methyl methacrylate using our previously established protocol 3). Using the Scanco evaluation software, regions of interest (ROIs) were defined for both cortical and trabecular parameters. For the left distal femoral metaphysis, the entire femora were reoriented with the mid-diaphysis parallel to the z-axis, and bone length were measured as the distance between the most proximal and distal transverse plans containing the femur. Beginning from the most proximal aspect of the growth plate, the trabeculae region on 100 consecutive slices were selected. The trabeculae were analyzed by manually contouring excluding the cortical bone for three-dimensional reconstruction  to calculate the following trabeculae parameters: Tb.BV/TV , Tb.vBMD , Tb.Th (trabecular thickness) and Tb.Sp (trabecular separation).The femur was scanned by the microCT system  and the left distal femoral metaphysis was analyzed according to the previously published protocols Supplementary figures and tables.Click here for additional data file.


Within a research project aimed at developing novel CAAs selective for certain isoforms, three series of indole-based derivatives were investigated. Enzyme activation assay on human CA I, II, VA, and VII isoforms revealed several effective micromolar activators, with promising selectivity profiles towards the brain-associated cytosolic isoform hCA VII. Molecular modelling studies suggested a theoretical model of the complex between hCA VII and the new activators and provide a possible explanation for their modulating as well as selectivity properties. Preliminary biological evaluations demonstrated that one of the most potent CAA  While inhibitors have been widely investigated as antitumor, antiglaucoma, diuretic, anticonvulsant, and antiobesity agents, the development of activators remains a poorly exploited research field until recently−/HCO3− potentially associated with hippocampal GABAergic postsynaptic depolarisation, that in turn correlates with the synchronised neuronal activity, called theta rhythmA receptor, and to impair learning and memory processes in ratsRecent reports highlighted that CA activation in the brain has implications for Alzheimer's disease and dementia2 into HCO3−2+ ion to the reaction medium, with the generation of the active zinc hydroxide species. This is the rate-determining step of the catalytic cycle, physiologically mediated by a His residue (His64 in many isoforms); CA activators (CAAs) function as additive proton shuttle molecules, shifting the equilibrium to the active form of the enzyme,,CA inhibitors classically feature a primary sulfonamide group that interacts with the zinc and displaces the catalytic Zn-bound-hydroxide/water necessary for the catalytic conversion of CO,,Activators bind to variable sites different from the inhibitors’ binding site and far away from the metal ion; the interested region does not necessarily overlap with that occupied by His64 situated in the middle part and extending towards the edge of the active site, but in the opposite part with respect to the active site or in an outer region,A, B, C, Typically, CAAs’ design was based on histidine , the natA series arises from our longstanding experience in the synthesis of indolglyoxylamides endowed with promising in vivo or in vitro biological activity 1–4  revealed a similar activation and selectivity profile for all the compounds. In line with these results, and in consideration of the synthetic feasibility, we decided to expand the SARs on these indole-based CAAs with the synthesis of compounds of series B and C (5–7 feature a 3-carboxamide moiety decorated with a hydroxyethyl (5 and 6) or lipophilic benzyl (7) group, while a simple ethyl ester moiety characterised derivatives 8 and 9  in DMSO-d6, MeOD or CDCl3. Chemical shifts are expressed in δ (ppm) and coupling constants (J) in Hertz. Magnesium sulphate was used as the drying agent. Evaporations were made in vacuo (rotating evaporator). Analytical TLCs have been carried out on Merck 0.2 mm precoated silica gel aluminium sheets (60 F-254). Silica gel 60 (230–400 mesh) was used for column chromatography. Purity of the target compounds 1–9 was determined using a Shimadzu LC-20AD SP liquid chromatograph equipped with a DDA Detector (λ = 254 nm) using a column C18 ; the mobile phase, delivered at isocratic flow, consisted of 70% of acetonitrile and 30% H2O (0.1% NH3); flow rate 1 ml/min. All the compounds showed percent purity ≥ 95%. Reagents, starting materials, and solvents were purchased from commercial suppliers and used as received. The intermediate N-benzyl-5-methoxy-1H-indole 10 was prepared according to the reported procedureThe uncorrected melting points were determined using a Reichert Kofler hot-stage apparatus. 16 or 17 (0.31 mmol) was dissolved in 1,4-dioxane (7 ml) and cooled at 0 °C. Then a solution of NaOH 1 M (1.25 ml) was added, and the mixture was stirred at room temperature for 5 h. The reaction mixture was concentrated in vacuum, the residue dissolved in ethyl acetate and washed with saturated aqueous NaHCO3 solution, water and brine. The organic phase was dried over MgSO4 and concentrated under reduced pressure. The crude products were then purified by flash chromatography (DCM/MeOH in ratio 9:1 as eluting system).A solution of the appropriate derivative 1H NMR  δ 9.05 , 8.24 , 7.98 , 7.36–7.31 , 7.20–7.18 , 6.93 , 5.35 , 4.21 , 4.00 , 3.50–3.45 , 2.50 , 2.34 , 1.85–1.82 . 13 C NMR  δ 180.23 (C=O), 162.81 (C=O), 156.04 (C-Ar) 141.59 (C-Ar), 135.39 (C-Ar), 131.53 (C-Ar), 129.04 (2 C-Ar), 128.91 (C-Ar), 128.28 (C-Ar) 126.96 (2 C-Ar), 114.19 (C-Ar), 112.16 (C-Ar), 111.51 (C-Ar), 105.63 (C-Ar), 69.90 (CH2O), 61.55 (CH2OH), 57.58 (CH2N), 51.43 (CH2Ph), 45.12 (2CH3), 38.11 (CH2NH), 26.44 (CH2CH2CH2).Yield: 69%; oil; 1H NMR  δ 8.95 , 8.54 , 7.77 , 7.48 , 7.37–7.28 , 6.90 , 5.55 , 4.00 , 3.74 , 3.30–3.23 , 2.57–2.51 , 0.97 . 13 C NMR  δ 181.90 (C=O), 163.60 (C=O), 156.20 (C-Ar), 141.71 (C-Ar), 137.18 (C-Ar), 131.56 (C-Ar), 129.20 (2 C-Ar), 128.44 (C-Ar), 128.28 (C-Ar), 127.80 (2 C-Ar), 113.77 (C-Ar), 112.84 (C-Ar), 111.68 (C-Ar), 105.21 (C-Ar), 70.44 (CH2O), 60.12 (CH2OH), 51.48 (CH2N), 50.43 (CH2Ph), 47.10 (2CH2CH3), 37.19 (CH2NH), 12.37 (2CH2CH3).Yield: 81%; oil; 2CO3  was added to a solution of the appropriate derivative 20 or 21 or 29 (0.15 mmol) in anhydrous DMF (5 ml). In another flask, Cs2CO3  was added to a suspension of the appropriate amine  in 3 ml of the same solvent. The mixtures were stirred for about 30 min at room temperature and then put together and the resulting mixture was heated at 60 °C for 5 h. The solvent was removed under reduced pressure, then ice was added to the reaction mixture and the solid precipitate formed was collected by vacuum filtration. The crude products were finally purified by flash chromatography (DCM/MeOH in ratio 9:1 as eluting system).In a flask, Cs1H NMR  δ 8.91 , 8.68 , 7.76 , 7.47 , 7.37–7.27 , 6.89 , 5.55 , 4.53 , 4.02 , 3.49–3.45 , 3.29–3.26 , 2.47 , 2.23 , 1.92–1.88 , 1.69–1.66 . 13 C NMR  δ 182.20 (C=O), 163.87 (C=O), 156.11 (C-Ar), 141.57 (C-Ar), 137.19 (C-Ar), 131.54 (C-Ar), 129.21 (2 C-Ar), 128.41 (C-Ar), 128.27 (C-Ar), 127.80 (2 C-Ar), 113.76 (C-Ar), 112.85 (C-Ar), 111.69 (C-Ar), 105.04 (C-Ar), 66.58 (CH2O), 59.19 (CH2OH), 56.01 (CH2N), 50.41 (CH2Ph), 45.31 (2CH3), 36.68 (CH2NH), 32.42 (NHCH2CH2CH2OH), 27.07 (NCH2CH2CH2O).Yield: 56%; mp = 111–113 °C; 1H NMR  δ 9.00 , 7.97 , 7.94 , 7.34–7.31 , 7.19–7.17 , 6.90 , 5.34 , 4.32 , 3.85 , 3.60–3.56 , 3.16 , 2.92 , 1.25 . 13 C NMR  δ 179.72 (C=O), 163.48 (C=O), 156.62 (C-Ar), 141.64 (C-Ar), 135.27 (C-Ar), 131.59 (C-Ar), 129.07 (2 C-Ar) 128.88 (C-Ar), 128.33 (C-Ar), 126.97 (2 C-Ar), 113.98 (C-Ar), 112.02 (C-Ar), 111.59 (C-Ar), 105.65 (C-Ar), 61.91 (CH2O), 51.45 (CH2OH), 51.10 (CH2N), 47.60 (CH2Ph), 42.23 (2CH2CH3), 29.69 (CH2NH), 10.51 (2CH2CH3).Yield: 57%; mp = 116–118 °C; 1H NMR  δ 7.81 , 7.69 , 7.36–7.31 , 7.19–7.14 , 6.88 , 5.31 , 4.39 , 4.12 , 2.69 , 2.42 , 2.14–2.07 , 1.43 . 13 C NMR  δ 165.11 (C=O), 155.04 (C-Ar), 136.01 (C-Ar), 134.60 (C-Ar), 131.88 (C-Ar), 128.97 (2 C-Ar), 128.10 (C-Ar), 127.76 (C-Ar), 126.95 (2 C-Ar), 113.55 (C-Ar), 111.14 (C-Ar), 107.26 (C-Ar), 104.33 (C-Ar), 66.45 (OCH2CH3), 59.62 (OCH2CH2), 56.40 (CH2N), 50.92 (CH2Ph), 44.89 (2CH3), 26.96 (CH2CH2CH2), 14.62 (OCH2CH3).Yield: 88%; mp = 58–60 °C; 1H NMR  δ 7.82 , 7.71 , 7.36–7.31 , 7.19–7.14 , 6.93 , 5.31 , 4.39 , 4.21 , 2.85 , 2.42 , 1.42 . 13 C NMR  δ 165.15 (C=O), 154.97 (C-Ar), 135.99 (C-Ar), 134.59 (C-Ar), 131.89 (C-Ar), 128.97 (2 C-Ar), 128.11 (C-Ar), 127.67 (C-Ar), 126.97 (2 C-Ar), 113.85 (C-Ar), 111.16 (C-Ar), 107.27 (C-Ar), 104.14 (C-Ar), 66.10 (OCH2CH3), 59.63 (OCH2CH2), 58.27 (CH2N), 50.91 (CH2Ph), 45.65 (2CH3), 14.64 (OCH2CH3).Yield: 30%; mp = 69–71 °C; 1H-NMR  δ 7.81 , 7.71 , 7.35–7.29 , 7.18–7.13 , 6.89 , 5.29 , 4.39 , 4.20 , 2.99 , 2.74 , 1.42 , 1.14 .Yield: 42%; mp = 48–50 °C; 23 or 24 or 25 (0.25 mmol) in 5 ml of anhydrous DMF, TBTU  and DIPEA  were added and the mixture was stirred for 30 min. Then, the appropriate amine  was added and the resulting mixture was stirred at room temperature overnight. DMF was removed under reduced pressure and ice was added to the flask. The mixture was extracted with DCM, dried over MgSO4, and concentrated under reduced pressure. The crude products were purified by flash chromatography (DCM/MeOH in ratio 9:1 as eluting mixture).To an ice-cooled solution of the appropriate derivative 1H NMR  δ 7.88 , 7.69 , 7.31–7.24 , 7.17–7.15 , 6.84 , 5.31 , 4.04 , 3.74 , 3.53 , 2.55–2.51 , 2.28 , 1.99–1.93 . 13 C NMR  δ 166.98 (C=O), 154.08 (C-Ar), 137.00 (C-Ar), 132.05 (C-Ar), 131.29 (C-Ar), 128.43 (2 C-Ar), 127.50 (C-Ar), 126.78 (2 C-Ar), 112.87 (C-Ar), 111.14 (C-Ar), 109.57 (C-Ar), 103.92 (C-Ar), 60.71 (CH2O), 55.84 (CH2OH), 52.26 (CH2N), 50.05 (CH2Ph), 43.01 (2CH3), 41.52 (CH2NH), 23.01 (CH2CH2CH2).Yield: 23%; oil; 1H NMR  δ 7.90 , 7.72 , 7.32–7.26 , 7.19–7.17 , 6.89 , 5.35 , 4.16 , 3.74 , 3.53 , 2.83 , 2.38 . 13 C NMR  δ 166.86 (C=O), 153.10 (C-Ar), 136.96 (C-Ar), 132.41 (C-Ar), 131.55 (C-Ar), 128.44 (2 C-Ar), 127.52 (C-Ar), 126.78 (2 C-Ar), 112.67 (C-Ar), 111.41 (C-Ar), 109.67 (C-Ar), 104.21 (C-Ar), 62.32 (CH2O), 60.70 (CH2OH), 53.37 (CH2N), 50.10 (CH2Ph), 42.71 (2CH3), 41.54 (CH2NH).Yield: 27%; oil; 1H NMR  δ 7.99 , 7.79 , 7.41–7.30 , 7.29–7.22 , 7.22–7.20 , 6.94 , 5.42 , 4.60 , 4.35 , 3.52 , 3.26 , 1.34 . 13 C NMR  δ 166.48 (C=O), 153.51 (C-Ar), 139.40 (C-Ar), 136.92 (C-Ar), 132.42 (C-Ar), 131.37 (C-Ar), 128.44 (2 C-Ar), 128.09 (2 C-Ar), 127.66 (C-Ar), 127.51 (C-Ar), 127.05 (2 C-Ar), 126.77 (2 C-Ar), 126.63 (C-Ar), 112.73 (C-Ar), 111.26 (C-Ar), 109.60 (C-Ar), 104.26 (C-Ar), 63.02 (CH2O), 51.14 (CH2N), 50.14 (2CH2Ph), 42.48 (2CH2CH3), 8.04 (2CH2CH3).Yield:16%; mp= 57–59 °C; N-benzyl-5-methoxy-1H-indole 10  in freshly distilled diethyl ether (10 ml). The mixture was maintained at room temperature for 2 h. The generated precipitate was collected by vacuum filtration to give the acyl chloride 11 that was directly used in the subsequent reaction. A solution of the appropriate amine  in dry toluene (5 ml) was added dropwise, under a nitrogen atmosphere, to a stirred suspension, cooled at 0 °C, of 2–(1-benzyl-5-methoxy-1H-indol-3-yl)glyoxylyl chloride 11  in 5 ml of the same solvent, followed by the addition of NEt3 . The reaction mixture was left under stirring at room temperature overnight. The solvent was removed under reduced pressure, and the residue was dissolved with DCM. The organic solution was washed with a 5% solution of NaHCO3, 10% solution of HCl, H2O, dried over MgSO4 and evaporated to dryness, yielding the desired compounds 12, 13 that were used in the subsequent step without further purification.Oxalyl chloride  was added dropwise at 0 °C to a solution of 1H NMR  δ 8.93 , 8.89 , 7.78 , 7.51 , 7.38–7.28 , 6.92 , 5.57 , 3.80 , 3.31–3.28 , 3.07–3.03 , 2.73 , 1.94–1.90 .Yield: 48%; mp = 186–188 °C; 1H NMR  δ 8.97 , 8.66 , 7.77 , 7.50 , 7.34–7.29 , 6.91 , 5.56 , 3.79 , 3.37–3.34 , 2.76–2.63 , 1.03 .Yield: 77%; mp = 124–126 °C; 12, 13, 18, 19 or 28 (0.30 mmol) in anhydrous DCM, cooled at −10 °C. The mixture was left under stirring for 30 min at −10 °C and subsequently at room temperature overnight. The solvent was removed under reduced pressure, and the residue was washed twice with methanol to hydrolyse the excess of BBr3. The residues obtained were purified by flash chromatography .Boron tribromide  was added dropwise, under a nitrogen atmosphere, to a solution of the appropriate derivative 1H NMR  δ 9.25 , 8.87–8.85 , 7.67 , 7.39–7.27 , 6.75 , 5.51 , 3.30–3.25 , 3.07–3.03 , 2.74 , 1.91–1.87 .Yield: 42%; oil; 1H NMR  δ 9.25 , 8.91 , 8.71 , 7.68 , 7.40–7.28 , 6.75 , 5.52 , 3.40–3.37 , 2.78–2.77 , 1.05 .Yield: 70%; mp = 176–178 °C; 1H NMR  δ 9.24 , 8.85 , 8.67 , 7.66 , 7.38–7.28 , 6.73 , 5.50 , 4.51 , 3.49–3.44 , 3.30–3.26 , 1.69–1.65 .Yield: 30%; mp = 169–171 °C; 1H NMR  δ 9.25 , 8.88 , 8.58 , 7.67 , 7.38–7.28 , 6.73 , 5.51 , 4.79 , 3.53–3.49 , 3.31–3.26 .Yield: 67%; mp = 217–219 °C; 1H NMR  δ 9.08 , 8.16 , 7.39 , 7.35–7.25 , 6.68 , 5.42 , 4.26 , 1.33 .Yield: 54%; mp = 175–177 °C; 14 or 15 (0.26 mmol) in acetone (12 ml), Cs2CO3  and 2-bromoethyl acetate  were added. The mixture was stirred at 60 °C overnight, concentrated in vacuum and the residue suspended in ethyl acetate. The suspension was washed with H2O, saturated aqueous NaHCO3 solution and brine. The organic phase was dried over MgSO4 and concentrated under reduced pressure. The crude products were then purified by flash chromatography (DCM/MeOH in ratio 9:1 as eluting system).To a solution of the appropriate derivative 1H NMR  δ 9.03 , 8.18 , 7.97 , 7.37–7.29 , 7.21–7.18 , 6.94 , 5.36 , 4.47 , 4.29 , 3.52–3.48 , 2.64 , 2.45 , 2.13 , 1.96–1.91 .Yield: 42%; mp = 97–99 °C; 1H NMR  δ 9.04 , 8.02 , 7.98 , 7.34–7.29 , 7.19–7.16 , 6.92 , 5.35 , 4.45 , 4.28 , 3.48–3.44 , 2.69 , 2.62 , 2.11 , 1.09 .Yield: 60%; oil; 11  in 1,4-dioxane (3 ml), the appropriate amine  was added dropwise at 0 °C and the mixture was stirred for 3 h at room temperature. The dioxane was removed under reduced pressure and water was added to the mixture; the generated precipitate was collected by vacuum filtration. The crude products were finally purified by flash chromatography (EtOAc/petroleum ether 40–60 °C in ratio 7:3 as eluting system).To a solution of compound 1H NMR  δ 9.05 , 7.95 , 7.85 , 7.37–7.32 , 7.20–7.18 , 6.91 , 5.37 , 3.91 , 3.72 , 3.61–3.55 , 1.86–1.81 .Yield: 71%; mp = 125–127 °C; 1H NMR  δ 8.94 , 8.61 , 7.77 , 7.49 , 7.37–7.28 , 6.91 , 5.56 , 4.79 , 3.79 , 3.52–3.51 , 3.32–3.29 .Yield: 35%; mp = 154–156 °C; 2O  was added to an ice-cooled solution of the appropriate derivative 8, 9, or 22 (0.81 mmol) in 8 ml of MeOH/H2O mixture (3:1). The resulting mixture was refluxed overnight. The suspension was filtered off, and the filtrate was acidified with a 10% aqueous solution of HCl until pH = 1. The mixture was extracted with DCM and, after drying with MgSO4, the solvent was evaporated to dryness to yield the crude products 23–25, which did not need any further purification.LiOH·H1H NMR  δ 8.00 , 7.64 , 7.32–7.29 , 7.27–7.19 , 6.87 , 5.41 , 4.16 , 3.38 , 2.94 , 2.25–2.21 .Yield: 97%, oil, 1H NMR  δ 8.06 , 7.73 , 7.37–7.27 , 7.23–7.21 , 6.97 , 5.45 , 4.41–4.40 , 3.63–3.61 , 3.01 .Yield: 81%; mp = 130–132 °C; 1H NMR  δ 8.21 , 7.57 ; 7.47 ; 7.35–7.29 ; 7.27–7.25 ; 6.90 ; 5.48 ; 4.35 ; 3.52–3.50 ; 3.23–3.21 ; 1.25 .Yield: 90%; mp = 94–96 °C; 26  in absolute ethanol (52 ml) a catalytic amount of concentrated sulphuric acid (0.52 ml) was added, and the resulting mixture was allowed to reflux for 20 h. After completion of the reaction, ethanol was removed, and the residue was dissolved with ethyl acetate and washed with saturated NaHCO3 solution. The organic layer was dried over MgSO4, filtered, and evaporated to give the corresponding ester. The crude product was then purified by flash chromatography (EtOAc/petroleum ether 40–60 °C in ratio 5:5 as eluting system). Yield: 31%; mp = 123–125 °C; 1H NMR  δ 8.56 , 7.89 , 7.70 , 7.32 , 6.93 , 4.41 , 3.91 , 1.45 .To a solution of compound 27  in 5 ml of DMF and the mixture was stirred for about 30 min at 0 °C. Once hydrogen evolution ceased, benzyl bromide  was added dropwise, and the reaction was maintained under stirring for 5 h at room temperature. After completion of the reaction, DMF was evaporated under reduced pressure, then ice was added to the flask and the solid precipitate formed was collected by vacuum filtration. The crude product was then purified by flash chromatography (EtOAc – petroleum ether 40–60 °C in ratio 3:7 as eluting system). Yield: 83%; mp = 89–91 °C; 1H NMR  δ 8.24 , 7.50 , 7.44 , 7.35–7.25 , 6.84 , 5.47 , 4.28 , 3.78 , 1.34 .Sodium hydride  was added portionwise, under nitrogen atmosphere, to an ice-cold solution of compound 2 hydration activity with Phenol red as indicator, working at the absorbance maximum of 557 nm, following the initial rates of the CA-catalysed CO2 hydration reaction for 10–100 s. For each activator, at least six traces of the initial 5–10% of the reaction have been used for determining the initial velocity. The uncatalyzed rates were determined in the same manner and subtracted from the total observed rates. Stock solutions of activator (0.1 mM) were prepared in distilled-deionized water and dilutions up to 0.1 nM were done thereafter with the assay buffer. The activation constant (KA), defined similarly with the inhibition constant (KI), was obtained by considering the classical Michaelis–Menten equation (f is the free concentration of activator.A stopped-flow methodM ([S]≪KM), and considering that [A]f can be represented in the form of the total concentration of the enzyme ([E]t) and activator ([A]t), the obtained competitive steady-state equation for determining the activation constant is given by 0 represents the initial velocity of the enzyme-catalysed reaction in the absence of an activatorWorking at substrate concentrations considerably lower than K,2O bound to Zn2+ converted to OH-. The most active compound 7 was built into its protonated and neutral form using the 2 D sketcher of Maestro and the optimised geometry of the ligand was obtained using the “minimize ligand” option of the same Maestro suite. Via Maestro, the X-ray crystal structures of hCA I (PDB ID-6EVR)2+ ion,AD4). Using the calculated ΔGAD4 and docking cluster population size, the best binding poses of 7, both before and after the proton shuttling step, were chosen among the 100 independent conformations/configuration states resulting from docking experiments.To perform the molecular modelling studies on the newly discovered CAAs, the latest version of docking software i.e. AutodockGPU2 and 5% CO2. Viable cells (identified by counting trypan-blue-excluding cellular elements) were plated at a density of 100,000 cells each well in a 24-well plate. To evaluate the ability of target compounds to modulate the cellular viability and brain-derived neurotrophic factor (BDNF) release, C20 cells were challenged with CAAs.All materials for cell culturing were obtained by Corning, New York, USA, and all used reagents were purchased by Sigma-Aldrich . The human microglial cell line C20, originally generated by David Alvarez-Carbonell et al.7, 8, and 9 for 24 h (1 and 10 µM). The cell viability was determined using the  (MTS) assay according to the manufacturer’s instructions . This tetrazolium dye can be reduced by the metabolic reducing agents NADH and NADPH to a water-soluble formazan salt. The amount of formazan produced is considered to be a marker of cell viability. The MTS reagent was added to treated C20 cells and the colorimetric conversion of formazane was quantified after 1 h by measuring the absorbance at 490 nm .C20 cells were seeded in 96-well microplates at a density of 5000 cells/well and maintained in complete culture media. The day after the seed, cells were treated with CAAs 7 for 2 h in serum-free medium and then maintained in fresh serum-free medium for 22 h. At the end of the 24 h, the levels of BDNF were measured in the C20 conditioned medium using an enzyme-linked immunosorbent assay (ELISA) Kit , accordingly to manufacture instructions. Briefly, the cell culture supernatants were centrifuged for 20 min at 1000 × g, and used in the ELISA assay. The microplate provided in the kit has been pre-coated with an antibody specific for BDNF. Standards or samples were added to the microplate wells and incubated for 1 h at 37 °C. Next, a biotin-conjugated antibody specific to BDNF was added to the well for 1 h. After washing, avidin conjugated to Horseradish Peroxidase was added to each microplate well and following incubation and washing, TMB substrate solution was added. The enzyme-substrate reaction was terminated by the addition of sulphuric acid solution and the colour change was measured spectrophotometrically at a wavelength of 450 nm ± 10 nm. The concentration of BDNF in the samples was then determined by comparing the O.D. of the samples to the standard curve. Levels of BDNF were normalised on the number of cells counted in each well by crystal violet staining, and BDNF levels were reported as pg/mL normalised per total cells.C20 cells were seeded in 24-well microplates at a density of 100,000 cells/well and maintained in complete culture media. The day after the seed, cells were challenged with compound t-test for independent means or one way ANOVA following by Bonferroni’s post-test were used to define statistical differences between absolute values, which were considered significant at p < 0.05.Values are presented as mean ± standard error of the mean (SEM) of 3 experiments. Either a Student’s 1 and 2 were prepared according to the experimental procedure outlined in N-benzyl-5-methoxyindole 10 with oxalyl chloride, in anhydrous diethyl ether, at room temperature, yielded the corresponding indolylglyoxylyl chloride 11, which was allowed to react with the appropriate amine , in the presence of triethylamine, in dry toluene solution, at room temperature, to give compounds 12 and 13. Compounds 14 and 15 were obtained by demethylation of 12 and 13, using boron tribromide in anhydrous DCM at −10 °C for 30 min and then at room temperature overnight. Following treatment with 2-bromoethylacetate and Cs2CO3 in acetone at reflux overnight, led to compounds 16 and 17, which were finally hydrolysed with 1 M NaOH solution in 1,4-dioxane at room temperature for 5 h, to achieve compounds 1 and 2.Target compounds 3 and 4 is outlined in 11 with the appropriate amine  in 1,4-dioxane, at room temperature, yielded the amides 18 and 19, which were then demethylated by treatment with boron tribromide in dry DCM. Subsequent reaction of 20 and 21 with the appropriate chloride  and Cs2CO3 in DMF for 5 h at 60 °C furnished the target compounds 3 and 4, finally purified by flash chromatography.The general synthetic pathway for the target compounds 5–7 was carried out following the procedure reported in 8, 9, 22, prepared as outlined in 2O in a solution of MeOH/H2O (3:1), to obtain 23–25, which were then treated with 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU) as condensing agent, N,N-diisopropylethylamine (DIPEA) and the appropriate amine  in anhydrous DMF, at room temperature overnight, to yield compounds 5–7, finally purified by flash chromatography.The synthesis of target compounds 8, 9, 22. The ethyl ester derivative 27 was obtained by refluxing the commercially available 5-methoxyindole-3-carboxylic acid 26 in absolute ethanol for 20 h, in the presence of a catalytic amount of concentrated sulphuric acid. Benzylation of 27 by treatment with benzyl bromide in dry DMF in the presence of NaH, and subsequent demethylation with boron tribromide in anhydrous DCM yielded 29, which was finally reacted with the appropriate chloride  in the presence of Cs2CO3 in dry DMF at 60 °C for 5 h, yielding 8, 9, 22, finally purified by flash chromatography.1–9 were assayed for their ability to activate four catalytically active and physiologically relevant hCA isoforms expressed in the human brain, namely the cytosolic hCA I, II, and VII and the mitochondrial hCA VA.Compounds The cytosolic and ubiquitous CA I is expressed in the motor neurons in the human spinal cord1–9 are listed in CA activation data of the indole-based derivatives 2 and 8 which showed KA values of 88.9 and 69.1 µM for hCA I, respectively.the cytosolic isoforms hCA I and II were not significantly activated by these derivatives up to concentrations as high as 100 µM, with the exception of compounds 1, 2, 4–7, 9, whereas 3 and 8 showed a poor activity.The mitochondrial isoform hCA VA was moderately activated by all derivatives with activation constants ranging between 24.4 and 59.8 µM; the best activators were A values in the range of 7.2–10.8 µM, independently from the decoration pattern of the central indole nucleus, suggesting that this good activity might be ascribed to the presence of the protonatable moiety rather than to its position.The brain associated cytosolic CA isoform (hCA VII) was the most sensitive one to activation by all compounds, with K7, 8, and 9 as the most interesting ones in terms of KA values  and selectivity profile towards isoform VII, as they show selectivity ratios of >13.3–9.6 against hCAI, >13.9–>12.2 against hCAII, and 3.2–8.3 against hCA VA.Overall, these results pointed out compounds 7 that was found to be comparatively the most potent and selective hCA VII activator of the whole set. Docking calculations were performed using the AutoDockGPU software (see molecular modelling methods). Results of the docking studies  calculated into the hCA VII binding site were rigidly translated into the binding sites of hCA I and hCA II , indicates that 7 should have high brain penetration properties. Also, this compound should be orally bioavailable considering its high calculated QPPCaco, which predicts the apparent Caco-2 permeability as a model of the gut-blood barrier.Given the possible implication of the CA activation for the treatment of CNS diseases, we were further asked if the most interesting compound New York . From th,To explore the neuroprotective potential of the newly synthesised indole-based CAAs, their capacity to promote brain-derived neurotrophic factor (BDNF) release was preliminarily tested on the C20 human microglial cell line. Microglial cells protect physiological brain function by regulating neurogenesis, neuronal survival, synaptic plasticity, and responding to alterations of the extracellular environment7, 8, and 9, those with the highest activation towards the brain-related hCA VII isoform, were first tested for their cytotoxicity on microglial cells. To this end, human C20 cells were treated with the target compounds for 24 h with concentrations in the micromolar range, in line with KA values, and then cell viability was evaluated by MTS assay.In this study, compounds 7 did not exhibit cytotoxic activity on C20 cells at any of the tested concentrations and even a little increase in cell viability was evidenced at 10 µM. On the contrary, compounds 8 and 9 caused a slight, although not statistically significant, reduction in cell viability at the highest tested concentration. These results prompted us to select compound 7 for further determination of its ability to promote the release of BDNF from human microglial cells. Human microglial cells were pre-treated for 2 h with compound 7, then the medium was replaced and collected after 24 h for the evaluation of BDNF levels. Interestingly, in the medium derived from cells treated with compound 7 a significant increase in BDNF levels was evidenced, at amounts comparable to those reported in the literature for microglia  in microglial cells, combined with a lack of cytotoxicity. All in all these data highlighted 7 as a promising lead compound for the development of novel agents with potential application in the treatment of CNS-related diseases.In the present study three small series of indole-based derivatives were investigated for their activation profile on physiologically relevant human CA I, II, VA, and VII isoforms. Several compounds resulted as effective micromolar activators, with promising selectivity profiles towards the brain-associated cytosolic isoform hCA VII. Docking calculations provided a theoretical model explaining the CA activation profile of compound


We show that the inert Ar atom forms relatively strong bonds with Aun+. The strength of the bond strongly varies with the cluster size and is governed by a fine interplay between geometry and electronic structure. The chemical bond between Aun+ and Ar involves electron transfer from Ar to Au, and a stronger interaction is found when the Au adsorption site has a higher positive partial charge, which depends on the cluster geometry. Au15+ is a peculiar cluster size, which stands out for its much stronger interaction with Ar than its neighbors, signaled by a higher abundance in mass spectra and a larger Ar adsorption energy. This is shown to be a consequence of a low-coordinated Au adsorption site in Au15+, which possesses a large positive partial charge. The interaction of Au At the n forces . While tn forces ,9,10, itn forces ,11.+ dimer and therefore the formation of a relatively strong bond [The properties of gold clusters differ significantly from those of copper and silver coinage metal clusters ,13,14,15ong bond . 3+ trimer as a model system, the interaction between Au and Ar was investigated by Fielicke and collaborators, using a combination of infrared spectroscopy and density functional theory (DFT) calculations [3Arm+ complexes, something that was attributed to a relativistically enhanced covalency of the Au-Ar interaction. This conclusion was supported by performing experiments on Agn3−Aun+ clusters, where the Ar bonding decreased with increasing silver concentration. Subsequent work by the same authors demonstrated similar characteristics of the interaction of Ar with Au4+ and Au5+, namely, electron transfer from Ar to the cationic gold clusters and the formation of a strong bond of partial covalent character [Using the Auulations . Ar was haracter .n+ clusters in the n = 13–20 size range, Xe was employed as the tagging element instead of Ar, as the interaction of the latter noble gas element was seen to decrease with cluster size and therefore, it was easier to form the complexes with Xe [n+, in a broad size range, has not been explained. For instance, Au15+ has been noticed to adsorb a larger amount of Ar than the closest size neighbors [15+ possesses a closed electronic configuration, and therefore, might be expected to be less reactive than the open-shell neighboring sizes.The attachment of Ar (and other noble gas elements) to larger cationic gold clusters has been reported. For example, Ar has been used as a tagging/spectator atom when investigating the geometries and the optical absorption spectra of pure and doped gold clusters ,26,27,28 with Xe . Neverthn+ clusters with Ar, by performing density functional theory calculations in the extended n ≤ 20 size range, in combination with mass spectrometric experiments. We demonstrate the formation of a  chemical bond between Ar and the clusters, which is a consequence of an electron charge donation from Ar to Au. The strength of the cluster−argon interaction is strongly size dependent, and is well correlated with the geometry of the Aun+ clusters. In this work, we address the interaction between Aum/m ≈ 1000). The laser power density and the time delay between the gas injection and the ablation were optimized to produce Aun+ clusters in the n ≤ 20 size range. Neutral and anionic species were also formed by this technique, however, they were not detectable by the mass spectrometer in the current settings. In order to make argon complexes, the He gas was mixed with 2% of Ar and the source was cooled to 250 K, resulting in AunArm+ complexes with up to m = 5, depending on cluster size. If a lower source temperature is used, the clusters are covered with even more Ar, whereas the amount of Ar attachment was minor for the larger clusters at higher source temperatures.The gold clusters were produced with the laser ablation technique , in a senArm+ clusters with n = 3–20 and m ≤ 5. All calculations were conducted employing the ORCA 4.2.1 software package [+-Arm (m ≤ 4) [n+ (n = 3–20) clusters were adopted from previous works. Infrared multiple photon dissociation spectroscopy was used in conjunction with DFT calculations to determine the geometries of the n = 3–9 sizes in Reference [n = 20 in Reference [n = 9, and those from Reference [n = 10–20 size range. We note that in Reference [nArm+ clusters. To locate the preferred Ar adsorption sites, Ar was then placed at all possible atop coordination positions, followed by geometry optimization. Starting geometries with other Ar coordination sites were tested as well. Nevertheless, atop adsorption sites always resulted in lower total energies. Density functional theory calculations were performed to complement the mass spectrometric findings, covering the entire range of Au package , by usin package , in conj package . The Stu package . Scalar  package . This le (m ≤ 4) . The geoeference , whereaseference . Here, weference  up to n eference  in the neference , the samadsE) of the mth attached Ar atom in the AunArm+ complex was calculated by Equation (1):E is the (absolute) total energy of the cluster in parenthesis. The reported adsorption energies have not been corrected for zero point energy. Moreover, Wiberg bond indexes and electron localization functions (ELF) have been calculated using the Multiwfn software package [The adsorption energy ( package .nArm+ cluster is presented. These fractions are defined by Equation (2), with I the area of the peak corresponding to the complex within parenthesis in the mass spectrum.The mass spectrometric results are summarized in n = 7, the intensity of bare Aun+ is very small,  as is the fraction of the complexes with a single Ar atom, m = 1. (ii) Second, for these smaller clusters (n ≤ 7), the nF(m) fractions with m = 2–5 are strongly size-dependent. Au3+ and Au6+ mainly adsorb three Ar atoms, while Au4+ and Au5+ primarily attach four. For Au7+, the F7(m) fractions with m = 2, 3, 4, 5 and 6 have about the same magnitude. (iii) Au11+ adsorbs more Ar atoms than its neighboring sizes and there is a gradual decrease in Ar fraction from n = 11 onwards. (iv) From n ≥ 14 onwards, only a single Ar atom is adsorbed on the clusters. (v) Last, the cluster n = 15 stands for its very high fraction of a single Ar atom.There are several interesting observations from nArm+  clusters. The geometries of the complexes that have a single Ar atom adsorbed are depicted in n+ clusters [4Ar+ and Au6Ar+ are presented in the supporting information. The relative energies of these isomers are high enough to assume that they will not be present in large amounts in the molecular beam, however this cannot be excluded for other sizes. The results provided in the following support our analysis only considering the lowest energy configurations. In order to rationalize those mass spectrometric results, DFT calculations were performed on the Auclusters ,36. The clusters . As an en = 7 the Au framework is planar; for all larger sizes the metal frameworks are three-dimensional. Therefore, a first correspondence between the Aun+ cluster geometries and the experimental Ar fractions is already identified, namely the amount of adsorbed Ar decreases sharply at the transition size from planar to three-dimensional clusters, i.e., after Au7+. A similar effect was seen in anionic Aun− species, where the size-dependent attachment of Ar was found to decrease abruptly once the clusters were three-dimensional [15+ cluster may also provide a hint for the higher F15(1) fraction. It is formed by a double layer of atoms, with a Au atom located on one layer [15+ is the adsorption site of the first Ar. For the single Ar lowest energy isomers up to ensional . The parne layer . As shown+ clusters may help in understanding the size-dependent Ar fractions in the n ≤ 7 size range. Indeed the triangular shape of the Au3+ cluster with three equivalent atop adsorption sites is consistent with the large F3(3) fraction. Similarly, the rhombic geometry of Au4+ has four possible atop sites for Ar adsorption, and the higher fraction for this size is F4(4). Au5+ has a bowtie-like geometry with four equivalent low-coordinated sites, consistent with the observed high F5(4) fraction. The triangular shape of Au6+ has three corners on which atop Ar adsorption is possible, and F6(3) is the largest fraction . Some examples of geometries for Aun+ clusters with multiple attached Ar atoms are presented in m = 0–5 structures are available as n+ cluster geometries is illustrative, but that further analysis of energetics as well as electronic structures are required to better understand the features observed experimentally.The observation that Ar adsorbs on atop coordination sites in the Aueference  from whiadsE) of the mth adsorbed Ar atom in AunArm+ were calculated. For this, the optimized geometries of the AunArm+ clusters were employed, which are shown in m = 1  For n = 3, there is a sudden decrease in adsE at m = 4, while for n = 4 and 5, the values remain high up to m = 4. For n = 6, adsE is low for m = 4, while for n = 7, the energies are relatively high up to m = 6 . (c) There are local maxima in adsE for m = 1 at n = 11 and n = 15. (d) The adsorption energies slightly increase around n = 17, decreasing again at n = 19. Hence, the DFT calculations reproduce outstandingly well the mass spectrometric observations. Using Equation (1), the adsorption energies (n+ and that of Ar in AunAr+ are presented in panel (a) of 3+ and shows an overall decrease with size n, which is not surprising since with increasing size the total +1 charge of the cluster is distributed over more atoms. The decrease, however, is not homogeneous. In particular, the higher partial charge of the Ar adsorption site of Au15+ stands out as well as the pronounced minimum of Au16+. Clearly, there is a strong correlation between the charge of the gold adsorption site before Ar adsorption with that of Ar after adsorption: the adsorbed Ar is more positively charged if the adsorption site had a larger positive charge. The correlation between the Hirshfeld partial charges and adsE  is also clear, with an overall decrease with size and local maxima for Au5+, Au11+ and particularly Au15+. Therefore, the interaction between Aun+ and Ar involves the transfer of electrons from Ar to Au, a more favorable process if the adsorption site has a higher positive charge. The Hirshfeld partial charge at the Au adsorption site in bare Aun+ and Ar is further analyzed by calculating the Wiberg index (W) of the Aun+-Ar bond . Interestingly, the Wiberg index has local maxima at Au11+ and Au15+ and a pronounced minimum at Au16+. The correlation between the partial positive charge of Ar and the Wiberg indices, together with the W values themselves being relatively large, in particular for n ≤ 5, reflects the formation of a chemical bond between Aun+ and Ar with a partial covalent character. A higher W index correlates perfectly with a smaller bond distance between Au and Ar in AunAr+, as shown in The bonding between Au3Ar+, Au15Ar+ and Au16Ar+. In each case, the DOS was projected onto the states of the Au adsorption site and of Ar. For Au3Ar+, shown in the upper panel, there were three occupied states of major Ar character, which were highly hybridized with the states of the Au adsorption site . Such overlap between the Ar and Au states clearly indicates the formation of a formal chemical bond between the atoms, in line with the high W index (0.62) for this cluster. Similar states were found in the PDOS of Au15Ar+ (middle panel), however in this case, the hybridization was smaller, as highlighted by the smaller W index (0.44). In contrast, the hybridization between Au and Ar is minor in Au16Ar+, in correspondence with the close to zero W index in this case, being only 0.24. Such a small W index is a consequence of the very limited electron charge transfer from Ar to Au in Au16Ar+, as seen from The density of states (DOS) of three selected clusters are presented in KLD) divergence. The KLD divergence is a measure of how one distribution (p(x)) differs from another (reference) distribution (q(x)), and is defined asThe level of hybridization between the states of Au and Ar in the PDOS of 3Ar+, Au15Ar+ and Au16Ar+, respectively. Therefore, the overlap between Au and Ar is larger in Au3Ar+, as expected, given its very high Ar adsorption energy, and the larger abundance in mass spectra. The overlap is smaller in Au15Ar+, but is still larger than in Au16Ar+. The Kullback–Leibler divergence is zero when both distributions are identical, and increases the more they diverge from each other . Using tn+ and Ar. Such a bond, of significant covalent character, is strong for n ≤ 5, and presents an overall decrease with size. Au11Ar+ and particularly Au15Ar+ stand out in this trend. Au15+ adopts a geometry with a low-coordinated Au atom that possesses a high partial positive charge, favoring electron donation from Ar, thus enhancing the Au15+-Ar bonding. To the right of 3Ar+ , Au15Ar+  and Au16Ar+  are plotted. These orbitals clearly show the strong involvement of Ar in the wavefunctions of the Au3Ar+ and Au15Ar+ clusters, an atom that therefore cannot be considered inert when interacting with these Aun+ clusters. The involvement of Ar in the wavefunction of Au16Ar+ is much smaller. The binding analysis is complemented by calculations of the electron localization function (ELF), which corresponds to the probability of finding two electrons of the same spin in a determined region of space, and is therefore a useful quantity to differentiate between chemical and physical bonds. Calculations have shown ELF values above 0.1 in between atoms interacting covalently [3Ar+, Au15Ar+ and Au16Ar+, and reveals a relatively high ELF value between Au3+ and Ar, of 0.115. The ELF values decrease with size, being 0.086 and 0.056 for Au15Ar+ and Au16Ar+, respectively. Hence, our analysis clearly shows the formation of a chemical bond between Auvalently . The anan+ clusters was investigated in the large n ≤ 20 size range, using a combination of mass spectrometry and density functional theory calculations. The abundances in mass spectra reveal a high propensity of the smaller clusters to form stable AunArm+ complexes. In particular, large Ar fractions are seen in the n ≤ 7 range. With increasing cluster size, the fraction of adsorbed Ar decreases, and above n = 11, only complexes with a single Ar atom are formed. In this size range, Au15+ stands out with a very high fraction of Ar. The calculations reproduce outstandingly well the mass spectrometric observations. The formation of a chemical bond between Au and Ar is proven for the investigated complexes, and is shown to be a consequence of both the geometry adopted by Aun+ and their electronic structure. In this work, the interaction of Ar with Au


Coronavirus disease 2019 (COVID-19), currently one of the immense burdens for global healthcare, is often characterized by rapid progression and the occurrence of symptoms particularly affecting the respiratory system. Continuous refinement of treatment protocols improves prognosis; however, COVID-19 survivors are often left with the symptomatic burden of dyspnea and fatigue. Therefore, it is necessary to continue comprehensive treatment including pulmonary rehabilitation. This study aimed to review the available literature on pulmonary rehabilitation in patients diagnosed with COVID-19. The pulmonary rehabilitation programs implemented various forms, i.e., aerobic exercise, breathing exercises, effective cough exercises, diaphragmatic breathing, and respiratory muscle training. Based on the literature review, it was found that pulmonary rehabilitation programs result in an improvement of respiratory function, reduction of fatigue and dyspnea, and improvement in exercise endurance and quality of life after completing both short-term and long-term programs, but depression and anxiety problems did not improve. Pulmonary rehabilitation combined with psychological therapy is crucial for COVID-19 survivors and plays a substantial role in patients’ recovery. In late 2019, health professionals from China reported a new coronavirus, SARS-CoV-2, which occupied mainly the respiratory system ,4. ConseMany health professionals and researchers reported rehabilitation protocols, consensus statements, and therapeutic interventions regarding pulmonary physiotherapy . Some ofThe aim of this paper was to perform a literature review on the role of pulmonary rehabilitation in mild-to-severe COVID-19 patients and summarize the impact of applied programs on respiratory function, exercise capacity, mobility, quality of life, activities of daily living, anxiety, and depression.The pulmonary rehabilitation programs presented by Pancera et al. and Sakai et al. were inpatient with an inpatient remote program implemented additionally by Sakai et al. ,9. Liu eFor seven patients, early rehabilitation began at the intensive care unit (ICU) and was continued in COVID wards. Eighteen patients underwent remote physiotherapy in the COVID unit, 32 were treated in rehabilitation units, 36 participated in ambulatory rehabilitation, and for 63 individuals, physiotherapy was applied through tele-rehabilitation. During the described interventions, all patients were alive and there were no reports of death after completing the protocols.The duration of all the presented rehabilitation programs varies from one to six weeks. Two manuscripts presented the effectiveness of six-week physiotherapy programs ,10 and oAll programs implemented pulmonary rehabilitation in various forms: aerobic exercise ,9,10, brAdditionally, three programs included limb muscle strengthening exercises ,9,10 andThree investigators reported the impact on respiratory function. Liu et al. presented spirometry and diffusion capacity results: FEV1, FVC, FEV1/FVC ratio, and DLCO . All spiZha et al. assessed the impact of pulmonary rehabilitation based on self-reported respiratory symptoms such as dry or productive cough, difficulty in expectoration, and dyspnea. The symptoms reporting rate decreased three to four times after completing a modified rehabilitation program based on Chinese martial arts .Wootton et al. evaluated oxygen saturation and heart rate at rest and during exercises, and to assess fatigue and dyspnea, they used the Fatigue Severity Scale (FSS) and modified Medical Research Council (mMRC) scales. No major improvement was found after completing the rehabilitation program but in contrast with other programs, no pulmonary exercises such as diaphragmatic breathing, respiratory muscle training, or cough exercises were applied .Exercise capacity was assessed in one study by a 6 min walking distance, showing statistically significant improvement after a six-week physiotherapy program focused on breathing exercises , Short PIn addition, Pancera et al. showed a quadriceps girth increase after completing both short-term (1 week) and long-term (3 weeks) rehabilitation programs .Quality of life (QoL) was assessed in two studies using the Short Form-36 (SF-36) health survey . The impThree publications reported the impact of implemented rehabilitation programs on activities of daily living (ADL) using the Barthel Index total score ,9 and FuThe study by Liu et al. additionally presented the impact of pulmonary rehabilitation on mental health using the Self-Rating Anxiety and Depression Scales  . AnxietyThe study design, aim, investigated population, rehabilitation protocols, outcome measurements, and results of reviewed studies are summarized in COVID-19 rehabilitation protocols, consensus statements, suggestions, and perspectives have been developed in many countries since the beginning of the pandemic. All physiotherapy suggestions are built on methods previously used in chronic respiratory conditions and on experience gained through the rapidly growing number of COVID-19 patients . CurrentThe majority of the pulmonary physiotherapy programs implemented in the analyzed studies had positive effects on various health aspects affected by COVID-19. Nevertheless, all these programs need further discussion considering the impact on lung function, exercise capacity, and QoL is inconclusive.Lung function, reported by Liu et al., was significantly improved by pulmonary physiotherapy and this is clearly visible in an almost two-fold increase in FVC values . However, post-rehabilitation results of the FEV1/FVC ratio, indicating obstruction level, still remain below normal values  . The freOnly one study analyzed the impact of pulmonary physiotherapy on exercise capacity with a 6-min walk distance and the improvement in distance walked after the 6-week program was 49 m. The minimally significant difference for COVID-19 patients has not been established. However, for the moderate and severe chronic obstructive pulmonary disease (COPD) patients, it is expected to be between 25–35 m and thus we can suspect the resultant difference is clinically important ,17,18. RMobility was assessed in three studies with three instruments: the BIM score, SPPB, and STS test ,9,10. ThThe QoL was appraised using two instruments: the SF-36 and EQ-5D-3L questionnaire . In the These findings suggest that pulmonary rehabilitation improves different QoL aspects and reduces anxiety associated with coronavirus but might be insufficient to address COVID-19-related depression. Further support and strategies to minimize the psychosocial consequences of COVID-19 after discharge should be considered.Most of the reviewed pulmonary rehabilitation programs had a positive effect on lung function, exercise endurance, and QoL of patients with mild, moderate, and severe COVID-19. Nevertheless, the usefulness of these programs in everyday clinical practice requires further investigation with robust study designs and additional follow-ups.