Rhinolophus bats harbor diverse clade 3 sarbecoviruses, the structural determinants of receptor tropism along with the antigenicity of their spike (S) glycoproteins remain uncharacterized. Here, we show that the African Rinolophus bat clade 3 sarbecovirus PRD-0038 S has a broad ACE2 usage and that RBD mutations further expand receptor promiscuity and enable human ACE2 utilization. We determined a cryoEM structure of the PRD-0038 RBD bound to R. alcyone ACE2, explaining receptor tropism and highlighting differences with SARS-CoV-1 and SARS-CoV-2. Characterization of PRD-0038 S using cryoEM and monoclonal antibody reactivity revealed its distinct antigenicity relative to SARS-CoV-2 and identified PRD-0038 cross-neutralizing antibodies for pandemic preparedness. PRD-0038 S vaccination elicited greater titers of antibodies cross-reacting with vaccine-mismatched clade 2 and clade 1a sarbecoviruses compared to SARS-CoV-2 S due to broader antigenic targeting, motivating the inclusion of clade 3 antigens in next-generation vaccines for enhanced resilience to viral evolution.Although  SARS-CoV-2 emerged at the end of 2019 and led to the devastating COVID-19 pandemic which claimed millions of lives worldwide4. Both viruses enter human cells via spike (S)-mediated fusion of the viral and host membranes upon binding to the angiotensin-converting enzyme 2 (ACE2) receptor8.Two sarbecoviruses have crossed the species barrier and spilled over to humans in the past two decades. SARS-CoV-1 emerged in 2002 and spread worldwide through air travel routes, leading to an epidemic with 8,098 cases and 774 deaths10 along with detection of numerous sarbecoviruses in bats and other wild animals16 underscore the recurrent zoonotic threat to public health posed by these viruses. The S glycoprotein of some of these sarbecoviruses harbor a receptor-binding domain (RBD) that utilize the human ACE2 receptor to enter host cells, indicating that they could possibly cross the species barrier to infect humans20.Reports of additional sarbecovirus spillovers to humans22. Clade 3 sarbecoviruses have been identified in bats in Europe and Africa28, such as BtKY72 and PRD-0038 for which sequences were found in Kenya and Rwanda, respectively. We recently showed that the S glycoprotein of one of them (BtKY72) could utilize two Rhinolophus affinis ACE2 alleles to promote entry into cells29. Furthermore, two amino acid residue substitutions in the BtKY72 RBD enabled S-mediated entry into human ACE2-expressing cells, broadening the range of sarbecoviruses with spillover potential29. The importance of this observation was underscored by the recent discovery of the clade 3 Khosta-2 virus28, which independently acquired the ability to bind29 and enter cells30 using the human ACE2 receptor. Studying the structure and functional properties of clade 3 sarbecovirus spike (S) glycoproteins is therefore crucial to understand spillover risk and assist in pandemic preparedness.Phylogenetic classification of sarbecoviruses based on their RBD sequences led to the definition of at least four clades: clade 1a (e.g. SARS-CoV-1), clade 1b (e.g. SARS-CoV-2), clade 2 (e.g. RmYN02) and clade 3 (e.g. BtKY72) Rhinolophus bat species and human ACE2. We determined structures of the PRD-0038 RBD bound to R. alcyone ACE2 and of the PRD-0038 S trimer, explaining receptor tropism and the distinct antigenicity of clade 3 sarbecoviruses relative to SARS-CoV-2 and SARS-CoV-1. Evaluation of a panel of monoclonal antibodies enabled identification of PRD-0038 cross-neutralizing antibodies that could be deployed for outbreak response. Vaccination of mice with PRD-0038 S elicited greater titers of antibodies cross-reacting with vaccine-mismatched clade 2 and clade 1a sarbecoviruses, relative to SARS-CoV-2 S immunization, indicating that addition of clade 3 antigens in vaccine formulations could enhance the resilience of antibody responses to viral evolution. Our findings highlight a molecular pathway for possible zoonotic spillover of a clade 3 sarbecovirus and the necessity of developing pan-sarbecovirus vaccines and countermeasures.Here, we report that the S glycoprotein of the clade 3 sarbecovirus PRD-0038, which is a member of the largely uncharacterized African bat-borne sarbecoviruses, has a broad ACE2 usage and that PRD-0038 RBD mutations further expand entry receptor tropism to additional Rhinolophus bat ACE2 orthologs harboring a C-terminal human Fc fusion to the immobilized PRD-0038 RBD using biolayer interferometry (BLI) (29 (BtKY72 and PDF-2370) and the high sequence similarity of their S glycoproteins. Our ACE2 panel comprised eight distinct R. sinicus alleles and two distinct R. affinis alleles, which were defined based on polymorphisms within the region recognized by sarbecovirus RBDs31, as well as R. alcyone and R. landeri orthologues. R. sinicus Asian bats are probable reservoir hosts for SARS-CoV-114, R. affinis bats have been shown to host closely related viruses to SARS-CoV-23 whereas R. alcyone and R. landeri bats are found in sub-saharan Africa, overlapping with the regions of sampling of several clade 3 sarbecoviruses (the exact Rhinolophus species from which PRD-0038 and BtKY72 have been sampled is unknown)25   and 1B. known)25 . We obseur panel . The PRDeri ACE2 . Finallyvaluated .R. alcyone enabled robust entry of PRD-0038 S VSV  particles with PRD-0038 S and assessed entry into HEK293T cells transiently transfected with the corresponding set of full-length, membrane-anchored ACE2s . Concurr38 S VSV , in lined by BLI . CollectR. alcyone ACE2 (RaACE2) ectodomain using single-particle cryo-electron microscopy (cryoEM) (2 is buried at the ACE2/RBD interface as compared to ~840 Å2 for the complexes of human ACE2 (hACE2) bound to the SARS-CoV-2 RBD33 or to the SARS-CoV-1 RBD34. The relative orientation of the binding partners are similar for these three structures, likely due to conservation of several key ACE2-interacting residues including L444PRD-0038/L455SARS-CoV-2 and F445PRD-0038/F456SARS-CoV-2, L475PRD-0038/L472SARS-CoV-1, Y478PRD-0038/Y489SARS-CoV-2, G485PRD-0038/G496SARS-CoV-2, G491PRD-0038/G502SARS-CoV-2 and Y494PRD-0038/Y505SARS-CoV-2  and Y41hACE2 is replaced by tenuous contacts between the topologically equivalent residues T489/V490PRD-0038 and H41RaACE2  the isolation of a mouse-adapted SARS-CoV-2 isolate harboring the Q493K substitution35 that promotes favorable interactions with mouse ACE2, the latter ortholog also possessing residues N31/E3537; and (iii) the emergence of R493SARS-CoV-2 in Omicron BA.1 and BA.237, which was subsequently reverted to the more favorable Q493SARS-CoV-2 in subsequent variants, likely due to relieving electrostatic repulsion with K31hACE238. Overall, most of the PRD-0038 RBD binding interface with R. alcyone ACE2 is remodeled as compared to human ACE2 bound to SARS-CoV-2 or SARS-CoV-1, thereby explaining the shift in receptor species tropism.To reveal the structural determinants of ACE2 recognition by clade 3 sarbecoviruses, we determined a cryoEM reconstruction of the PRD-0038 RBD bound to the natively dimeric (cryoEM) . SymmetrRS-CoV-2 –1H. HoweRS-CoV-1 , and 1J.PRD-0038 , and S2.41RaACE2 , and 1K.nterface , and 1L.29, in line with their ability to enable BtKY72 S-mediated utilization of human ACE229. We also found that human ACE2-Fc interacted with the T487W RBD, albeit weakly -bound form of SARS-CoV-2 S previously described44  and apo SARS-CoV-1 S (PDB 5X5843), suggesting that this rotameric configuration is accessible to SARS-CoV-2 Y365 and likely changes to allow linoleic acid binding44.The overall architecture of the PRD-0038 S trimer is similar to that of SARS-CoV-1 and SARS-CoV-2 and a PRD-0038 S protomer can be superimposed with r.m.s.d. values of 0.9 and 0.8 Å to SARS-CoV-1 S and SARS-CoV-2 S with which it shares 72% and 75% amino acid sequence identity, respectively . Glycosycribed44  and S6. cribed44  or that ed above . No linoed above . The PRD50 and α- and β-coronaviruses55 to prefusion-stabilized PRD-0038 S harboring the HexaPro mutations56 using an enzyme-linked immunosorbent assay (ELISA) (22 and S2X3549 (antigenic site II) as well as S2H97 (antigenic site V) cross-reacted with PRD-0038 S whereas S30957 (antigenic site IV) bound very weakly , which neutralizes a broad panel of SARS-CoV-2 variant and resembles LY-COV140458, recognizes the SARS-CoV-2 437–448 loop which is remodeled in the PRD-0038 RBD, explaining the lack of binding to PRD-0038 S (50 antibody (antigenic site Ia), which contacts an epitope sharing several residues within the ACE2 binding site, bound very weakly to wildtype PRD-0038 S whereas S2M1159 (antigenic site Ia) did not bind at all, as a result of extensive RBM (receptor-binding motif) mutations  . We founy weakly . The mar-1 RBD21 . The S2XD-0038 S . The S2Kutations . Neverth53 antibody and the RAY5351 antibody (recognizing the fusion machinery apex) cross-reacted as efficiently with PRD-0038 S as they did with SARS-CoV-2 S whereas the fusion peptide-directed 76E152 antibody did not, possibly as a result of the F823SARS-CoV-2Y806PRD-0038 epitope mutation (which is shared with other clade 3 sarbecoviruses) along with the presence of the F800PPRD-0038 HexaPro stabilizing mutation55 , clade 1b  or clade 3 (PRD-0038 and Khosta1) S glycoproteins. SARS-CoV-2 S-immunized mice had potent serum neutralizing activity against SARS-CoV-2/G614 VSV S (vaccine-matched), which was reduced against BA.2 S VSV and even more so against BA.5 S VSV  and two mice with weakest  serum neutralizing activity from the group immunized with SARS-CoV-2 HexaPro S as well as two mice with the greatest SARS-CoV-1 cross-neutralization  from the group immunized with PRD-0038 HexaPro S . In linelization , and S9.67. These residues are highly variable among sarbecoviruses, consistent with the weaker cross-reactive breadth seen in these sera. In contrast, PRD-0038 S-elicited polyclonal serum antibodies showed no dominant targeting of specific PRD-0038 RBD antigenic sites, which could indicate a more balanced binding antibody response that would be less susceptible to single amino acid mutations. Epitope mapping to vaccine-mismatched RBDs revealed that PRD-0038 and SARS-CoV-2 S-elicited sera contained subdominant antibodies targeting antigenic sites II, IV and V49, which are typically recognized by broadly reactive and neutralizing antibodies69. However, DMS using vaccine-mismatched RBD strains also reveals the presence of antibodies in SARS-CoV-2 S-elicited sera that target suboptimal sites for sarbecovirus breadth. For example, we observed strong antibody responses to the N370 glycan hole (resulting from mutations of this glycosylation sequon in our DMS experiments) for two of the four SARS-CoV-2-immunized mice analyzed. Antibody responses targeting this site are elicited as a result of the absence of this oligosaccharide in the SARS-CoV-2 RBD immunogen but its presence in all other sarbecovirus RBDs. Restoration of the N370 glycan in SARS-CoV-2 vaccines could therefore possibly limit these off-target responses by reducing elicitation of antibodies to this strain-specific epitope. In conclusion, the DMS data indicate that PRD-0038 S-elicited serum antibodies analyzed here target a broader spectrum of antigenic sites present on vaccine-matched and mismatched RBDs than SARS-CoV-2 S-elicited antibodies, providing a molecular basis for enhanced cross-reactivity , introduction of a single amino acid RBD mutation (T487W) enabled binding and S-mediated entry into cells expressing human ACE2. Moreover, this point mutation broadened host receptor tropism by enabling utilization of the geographically-relevant R. landeri and R. ferrumequinum ACE2 receptors without compromising binding to R. alcyone, R. sinicus and R. affinis ACE2 alleles. Although the T487W RBD mutation requires 3 nucleotide substitutions, these findings point to a possible spillover pathway in which a single amino acid change expands host receptor tropism markedly. Indeed, acquisition of R. landeri, R. ferrumequinum  and human ACE2 tropism would allow PRD-0038 (and likely BtKY72 and related clade 3 viruses) to expand the geographic range of host reservoirs that can be infected and in turn the likelihood of zoonotic transmission. Broadly neutralizing antibodies with activity against sarbecovirus (RBD-directed) and beyond (fusion machinery-directed) inhibited PRD-0038 S-mediated entry into cells and could be stockpiled as possible countermeasures for pandemic preparedness.Spillover is a complex process involving multiple factors such as receptor recognition, proteolytic S activation, immune antagonism and contact opportunity. To examine potential spillover pathways of clade 3 sarbecoviruses, we evaluated binding of PRD-0038 RBD mutants to a panel of 82. Compared to SARS-CoV-2 S, we found that PRD-0038 S-elicited polyclonal serum antibodies were more broadly reactive with vaccine-mismatched antigens, including clades 1a and 2 RBDs, which likely account for the weak but detectable cross-neutralization of SARS-CoV-1 with sera from mice immunized with PRD-0038 S but not with SARS-CoV-2 S. As non- or weakly neutralizing monoclonal and polyclonal antibodies have been shown to participate in protection against SARS-CoV-2 challenge in small animal models through Fc-mediated effector functions64, our findings motivate the inclusion of clade 3 and other divergent RBD antigens in updated sarbecovirus vaccine formulations86. This would allow elicitation of potent clade 3 neutralizing antibodies and cross-clades binding  antibodies with maximal breadth to achieve optimal protection against continuously evolving SARS-CoV-2 variants and sarbecoviruses found in wildlife.We observed that three immunizations with a clade 1b or with a clade 3 sarbecovirus S trimer predominantly elicited vaccine-matched serum neutralizing antibody responses. We note that our neutralization data of clade 1b pseudoviruses underscore the distinction between antigenic and genetic distance: although RaTG13 harbors a greater number of RBD mutations than Omicron BA.2 or BA.5, neutralizing activity was higher against RaTG13 than against these SARS-CoV-2 variants which accumulated mutations to erode neutralizing antibody titersThere is currently no known animal challenge model for clade 3 sarbecoviruses and we were therefore not able to evaluate the contribution of the broadly reactive antibody responses elicited upon PRD-0038 S vaccination to in vivo protection.87. Cells were cultured in 10% FBS, 1% penicillin-streptomycin DMEM at 37°C, 5% CO2. None of the cell lines were authenticated or tested for mycoplasma contamination.Cell lines used in this study were obtained from HEK293T  and Expi293F  except for the HEK293T cells with stable human ACE2 expression which was kindly provided by Jesse BloomMT726045). Both wildtype and mutant PRD-0038 RBDs (residues 318–520) were synthesized by GenScript with an N-terminal mu-phosphatase signal peptide and C-terminal 8x His tag, a short linker (GGSS) followed by an Avi tag in a pCMVR plasmid. Expi293F cells were grown at 37°C with 8% CO2 and DNA transfections were conducted with the ExpiFectamine 293 Transfection Kit (Thermo Fisher Scientific). Cell culture supernatants were harvested three days post transfection. RBDs were purified using nickel based affinity chromatography using HisTrap™ High Performance column (Cytiva). Proteins were first washed with 10 column volumes of a buffer containing 25mM sodium phosphate (pH8.0) and 300 mM NaCl, before elution with 8 column volumes of a buffer containing 25 mM sodium phosphate (pH8.0), 300 mM NaCl, 500mM imidazole. Eluted proteins were buffer exchanged into 1x PBS  using Amicon Ultra-15 Centrifugal Filter Unit (10 kDa) (Millipore). Overnight biotinylation reactions were performed using the BirA Biotin-Protein Ligase Kit (Avidity) at 4°C in 1x BiomixA, BiomixB. Biotinylated proteins were once again affinity purified using the HisTrap column as previously described to get rid of BirA. Once purified, buffer exchanged into PBS, and concentrated, proteins were flash-frozen and stored at −80°C until use.The PRD-0038 S glycoprotein sequence was obtained from Genbank . Cell culture supernatants were harvested four days after transfection and proteins were purified using HiTrap Protein A HP (Cytiva) or HisTrap™ High Performance column (Cytiva). ACE2-Fc proteins were first washed with 10 column volumes of 20 mM sodium phosphate (pH8.0) then eluted with 0.1 M citric acid (pH 3.0) directly into tubes containing 1M Tris-HCl (pH 9.0). Purified proteins were buffer exchanged into PBS (pH 8.0), concentrated using Spin-X® UF 20 mL Centrifugal Concentrator, 100,000 MWCO Membrane (PES) (Corning), and flash-frozen. ACE2-His proteins were washed with 10 column volumes of a buffer containing 25 mM sodium phosphate (pH 8.0), 300 mM NaCl, then eluted with 8 column volumes of a buffer containing 25 mM sodium phosphate (pH 8.0), 300 mM NaCl and 500 mM imidazole. Eluted proteins were buffer exchanged into 1x PBS (pH8.0) using Amicon Ultra-15 Centrifugal Filter Unit (10 kDa) (Millipore) and flash-frozen.Genbank accession numbers for all ACE2s can be found in MT726045). Recombinant PRD-0038 S glycoprotein ectodomain (residues 16–1194) constructs with pre-fusion stabilizing mutations  were synthesized by GenScript with an N-terminal mu-phosphatase signal peptide and C-terminal short linker (SG), TEV protease site (RENLYFQ), a short linker (GGGGSG), Foldon, 8x His tag, a short linker (GGSS) followed by an Avi tag in a pCMVR plasmid. The SARS-CoV-2 S glycoprotein ectodomain construct comprises residues 1–1208 with the native signal peptide, the HexaPro prefusion stabilizing mutations56 , abrogation of the furin cleavage site  and a C-terminal short linker (GSG), followed by a foldon, HRV 3C site (LEVLFQGP), a short linker (GSG), an avi tag, a short linker (GSG), an 8x his tag in a pcDNA3.1(−) plasmid. Expi293F cells were grown at 37°C with 8% CO2 and DNA transfections were conducted with the ExpiFectamine 293 Transfection Kit (Thermo Fisher Scientific). Cell culture supernatants were harvested four days post-transfection and proteins were purified using HisTrap™ High Performance column (Cytiva). Proteins were first washed with 10–15 column volumes of a buffer containing 25 mM sodium phosphate, 300 mM NaCl, 20 mM imidazole, pH 8.0, followed by elution with 10–15 column volumes using 300 mM imidazole, pH 8.0. Eluted proteins were concentrated and buffer exchanged into 1x PBS or 1x TBS  using Amicon Ultra-15 Centrifugal Filter Unit (100 kDa) (Millipore). Purified proteins were snap frozen and stored at −80°C. Purified proteins were checked for endotoxin level using Charles River Limulus Amebocyte Lysate (LAL) cartridges PTS201F. Endotoxin-free SARS-CoV-2 HexaPro S and PRD-0038 HexaPro S were flash-frozen and stored at −80°C until the day of immunization.The PRD-0038 S glycoprotein sequence was obtained from Genbank  equilibrated in 50 mM Tris-HCl, 150 mM NaCl. Complex formation was confirmed by SDS-PAGE, and the PRD-0038 RBD- R. alcyone ACE2 complex was snap frozen and stored at −80°C until day of grid preparation.D values, RBD-loaded tips were dipped into a concentration series of ACE2-His  for 600 s followed by 600 s of dissociation in 10x kinetics buffer. Global fits were used to calculate KD values using a 1:1 binding fit model. Data were plotted using GraphPad Prism. Assays were replicated with three biological replicates (recombinant RBD proteins generated on different days) and representative graphs are shown.BLI binding assays were performed on an Octet Red (Sartorius) instrument operated at 30°C with shaking (1000 rpm). For biotinylated RBD and ACE2-Fc or ACE2-His binding assays, streptavidin biosensors were hydrated in water for 10 min prior to the experiment. Biosensors were incubated in 10x kinetics buffer for 60s followed by the loading of biotinylated RBDs to the tip, all to a final level of 1 nm. Loaded biosensors were equilibrated in 10x kinetics buffer (Sartorius) for 120s which served as our baseline. For avidity binding assays, association with 1 µM ACE2-Fc (dimeric form) was performed for 300 s followed by 300 s of dissociation in 10x kinetics buffer. For affinity binding assays to determine K2, pseudoviruses were collected and cell debris removed by centrifugation at 3,000xg for 10 min. Pseudoviruses were further filtered using a 0.45 µm syringe filter and concentrated 25–50x prior to storage at −80°C. Mock VSV pseudoviruses were prepared as above but without S transfection.Wildtype and mutant PRD-0038 S constructs consisting of residues 1–1235 and containing a 21 residue C-terminal deletion (del21) followed by a 3x FLAG tag were synthesized by GenScript and placed into an HDM plasmid. VSV pseudoviruses were produced using HEK293T cells seeded on BioCoat Cell Culture Dish : poly-D-Lysine 100 mm (Corning). Cells were transfected with PRD-0038 S-Flag constructs using Lipofectamine 2000 (Life Technologies) in Opti-MEM transfection medium. After 5h of incubation at 37 °C with 5% CO2, cells were supplemented with DMEM containing 10% of FBS. On the next day, cells were infected with VSV (G*ΔG-luciferase) for 2h, followed by five time wash with DMEM medium before addition of anti-VSV G antibody  and medium. After 18–24 h of incubation at 37 °C with 5% CORhinolophus ACE2 orthologs using Lipofectamine 2000 (Life Technologies) in Opti-MEM five hours prior to plating into 96-well plates [3610] (Corning) coated with poly-lysine [P4707] (Sigma) and incubated 18–24 h before infection with VSV pseudoviruses. For human ACE2 entry assays, HEK293T cells with stable hACE2 expression were plated into poly-lysine-coated 96-well plates and incubated for 18–24h before infection with VSV pseudoviruses. The amount of pseudovirus used for infection was adjusted using Western Blot based on S incorporation across different mutants to use a constant input of S. Detection of VSV backbone was performed with 1:1,000 Anti-VSV-M [23H12] Antibody (Kerafast). Detection of 3x-FLAG tagged S was performed with 1:400 monoclonal ANTI-FLAG® M2 antibody [F3165] produced in mouse (Sigma). 1:50,000 Alexa Fluor® 680 AffiniPure Goat Anti-Mouse IgG [115-625-174] (Jackson ImmunoResearch) was used as the secondary antibody. A representative Western Blot is shown in HEK293T cells were transfected with full length R. affinis, R. sinicus, R. landeri, and R. alcyone alleles full-length ACE2 alleles (2 incubator. Cells were plated 18–24 hours prior to transfection into 96-well plates [3610] (Corning) coated with poly-lysine [P4707] (Sigma). All transfections were performed using full-length Rhinolophus ACE2 placed into a HDM plasmid (synthesized by GenScript). Transfection of ACE2 alleles into HEK293T cells was performed using 0.2 µg DNA and 0.15 µL Lipofectamine 2000 (Life Technologies) per well in Opti-MEM. After a 5 h incubation at 37 °C in a humidified 8% CO2 incubator, DMEM was added to obtain a final concentration of 10% FBS and 1% penicillin–streptomycin. Cells were incubated at 37 °C in a humidified 8% CO2 incubator for 36–48 h prior to infection. For each infection test, 2–3 technical replicates were performed, and the assays were repeated on a second day, for a total of 4–6 technical replicates. Three biological replicates (pseudovirus generated on different days) were used for cell entry and each point shown represents the mean fold change for each biological replicate. Results were plotted using Graphpad Prism  were applied and blotted twice as previously described88, onto freshly glow discharged R 2/2 UltrAuFoil grids prior to plunge freezing using a vitrobot MarkIV (ThermoFisher Scientific) with a blot force of 0 and 5 sec blot time at 100 % humidity and 22°C. Data were acquired using an FEI Titan Krios transmission electron microscope operated at 300 kV and equipped with a Gatan K3 direct detector and Gatan Quantum GIF energy filter, operated in zero-loss mode with a slit width of 20 eV. Automated data collection was carried out using Leginon89 at a nominal magnification of 105,000x with a pixel size of 0.843 Å. The dose rate was adjusted to 15 counts/pixel/s, and each movie was acquired in counting mode fractionated in 75 frames of 40 ms. A total of 2,482 and 11,743 micrographs were collected for the PRD-0038 S and PRD-0038 RBD-ACE2 datasets, respectively.Cryo-EM grids of PRD-0038 PentaPro S were prepared using two separate methods and data were combined during data processing. The first dataset was collected from the grids prepared using a reverse grid-blotting method. 3 µL of sample was added to the carbon side of a glow discharged C-flat R2/2 copper grid and 1µL was added to the back side before addition of 1 µL of CHEMS lipid dissolved in chloroform on the front side. The sample was allowed to sit on the grid for 1 minute and then manually blotted from the back side using a strip of Whatman #1 filter paper and plunged into liquid ethane. The second dataset was collected from a lacey carbon grid with a thin home-made continuous carbon layer. 3 µL of 0.15 mg/mL PRD-0038 Pentapro S was loaded onto the glow discharged (6s at 20mA) grid followed by plunge freezing using a vitrobot MarkIV (ThermoFisher Scientific). The grid was blotted with a blot force of −1, 3 second blot time, and 10 second wait time before the plunge freeze at 100% humidity and 25 °C. For the PRD-0038 RBD-ACE2 complex, grids were prepared by applying 3 µL of 4 mg/ml PRD-0038 RBD bound to the 90 for each data set. For the PRD-0038 S structure, particle images were extracted with a box size of 260 pixels with a pixel size of 1.686Å. After two rounds of 2D classification using cryoSPARC91, well-defined particles were selected and particles from each dataset were combined and binned to a box size of 130 pixels with a pixel size of 3.372Å for subsequent 3D classification using Relion93 with 50 iterations . 103,347 particles were selected and re-extracted with a box size of 260 and pixel size of 1.686Å for cryoSPARC non-uniform refinement94 with C3 symmetry and further subjected to Bayesian polishing95 in Relion. Finally, another round of non-uniform refinement with C3 symmetry and optimized per-particle defocus was carried out to the polished particles. To improve the density of the NTD, we used symmetry expansion and local refinement using cryoSPARC. For the PRD-0038 RBD - R. alcyone ACE2 structure, two rounds of reference-free 2D classification were performed using cryoSPARC to select well-defined particle images. These selected particles were subjected to two rounds of 3D classification with 50 iterations each  using Relion with an initial model generated with ab-initio reconstruction in cryoSPARC. 3D refinements were carried out using non-uniform refinement along with per-particle defocus refinement in CryoSPARC. To improve the density of the RBD-ACE2 dimer, the particles were subjected to cryoSPARC heterogeneous refinement. Particles belonging to classes with the best resolved RBD-ACE2 density were selected and subjected to the Bayesian polishing procedure implemented in Relion before performing another round of non-uniform refinement in cryoSPARC followed by per-particle defocus refinement and again non-uniform refinement. To further improve the density of the RBD-ACE2 domains, the particles were symmetry expanded and subjected to focus 3D classification without refining angles and shifts using a soft mask encompassing the RBD and monomer ACE2 using a tau value of 40 in Relion. Particles belonging to classes with the best resolved RBD-ACE2 density were selected and then subjected to local refinement using CryoSPARC. Local resolution estimation, filtering, and sharpening were carried out using CryoSPARC. Reported resolutions are based on the gold-standard Fourier shell correlation (FSC) of 0.143 criterion and Fourier shell correlation curves were corrected for the effects of soft masking by high-resolution noise substitution97. UCSF Chimera and Coot were used to fit atomic models into the cryoEM maps. S and RBD-ACE2 models were refined and relaxed using Rosetta99 using sharpened and unsharpened maps and validated using Phenix100, Molprobity101 and Privateer102.Motion correction, contrast-transfer function (CTF) parameter estimation, automatic particle picking, and extraction were performed using Warp50 values from curve fits.For PRD-0038 HexaPro S and SARS-CoV-2 HexaPro S ELISAs, 30 μl of the proteins at 3 μg/mL were plated onto 384-well Nunc Maxisorp plate  in 1x TBS and incubated 1h at 37°C followed by slap drying and blocking with 80 μL of Casein for 1 h at 37°C. After incubation, plates were slap dried and 1:4 serial dilutions of the corresponding mAbs starting from 0.1 mg/ml were made in 30 μl TBST, added to the plate and incubated at 37°C for 1 h. Plates were washed 4x in TBST and 30 μl of 1:5,000 Goat anti-Human IgG Fc Secondary Antibody, HRP  or Goat anti-Syrian Hamster IgG (H+L) Secondary Antibody, HRP  were added to each well and incubated at 37°C. After 1 h, plates were washed 4x in TBST and 30 μl of TMB (SeraCare) was added to every well for 2 min at room temperature. Reactions were quenched with the addition of 30 μl of 1N HCl. Plates were immediately read at 450 nm on a BioTek Neo2 plate reader and data plotted and fit in Prism 9 (GraphPad) using nonlinear regression sigmoidal, 4PL, X is the concentration to determine EChttps://github.com/tstarrlab/SARSr-CoV-RBD_DMS/blob/main/results/summary/summary.md. Deep mutational scanning libraries for sarbecovirus strains including PRD-0038 and SARS-Cov-1 Urbani were constructed as previously described76. Briefly, site-saturation mutagenesis libraries spanning all RBD positions were produced by Twist Bioscience (or NNS mutagenesis for positions that failed Twist mutagenesis), tagged with an N16 barcode, and cloned into a yeast display vector backbone via Gibson Assembly. Libraries were electroporated into E. coli and plated at a target bottleneck of 40,000 unique barcodes per library to overrepresent the ~4,000 possible amino acid mutations. Colonies were scraped from each transformation plate, library plasmid purified, and transformed into the AWY101 S. cerevisiae strain103 for yeast surface-display experiments. Library plasmids were sequenced using a PacBio Sequel IIe to generate long sequence reads spanning the N16 barcodes and RBD coding sequence. Raw CCS reads are available on the NCBI Sequence Read Archive, BioProject PRJNA962117, BioSample SAMN34384156. Reads were processed using alignparse (version 0.2.4)104 to generate a table linking each N16 barcode to its unique RBD mutant, available at: https://github.com/tstarrlab/SARSr-CoV-BD_DMS/blob/main/results/variants/codon_variant_table_PRD0038.csv and https://github.com/tstarrlab/SARSr-CoV-RBD_DMS/blob/main/results/variants/codon_variant_table_SARS1.csv.The complete deep mutational scanning pipeline can be found at: 76. ACE2-binding titrations were performed by incubating induced yeast-display libraries with a concentration series of dimeric ACE2 ligands from 10−6 to 10−13 M at 1-log intervals, plus a 0 M ACE2 sample, with samples equilibrated overnight at room temperature with mixing. Yeast were washed with PBS-BSA (0.2 mg/L), labeled with 1:100 FITC-conjugated chicken anti-Myc (Immunology Consultants CMYC-45F) to detect yeast-displayed RBD and 1:200 PE-conjugated streptavidin (Thermo Fisher S866) or goat anti-human-IgG (Jackson ImmunoResearch 109-115-098) to detect binding of biotinylated  or Fc-tagged  ACE2. For each titration sample, RBD+ yeast were fractionated into four bins of PE fluorescence (ACE2 binding), grown overnight, plasmid isolated, N16 barcode amplified, and barcodes counted via high-throughput sequencing on an Illumina NextSeq. RBD expression was measured by sorting cells into four bins on the basis of Myc-FITC labeling, followed by outgrowth, plasmid isolation, N16 barcode amplification, and sequencing. Sequencing reads are available on the NCBI Sequence Read Archive, BioProject PRJNA962117, BioSample SAMN34384823.The RBD expression level and ACE2-binding avidity of each RBD mutant was determined via high-throughput FACS-seq assays as previously describedhttps://github.com/tstarrlab/SARSr-CoV-RBD_DMS/blob/main/results/counts/variant_counts.csv.gz. Reads were downweighted by the ratio of total sequence reads from a bin to the number of cells sorted into that bin. For each barcode, we inferred the apparent dissociation constant for avid binding  by fitting the standard non-cooperative Hill equation to the mean FACS bin of a barcode variant as a function of ACE2 concentration. For each barcode, expression was determined via a maximum likelihood estimator of log-MFI based on the distribution of barcode counts across FACS bins and the known fluorescence boundaries of those bins. The computational pipelines for computing per-barcode binding constants and expression phenotypes are available at: https://github.com/tstarrlab/SARSr-CoV-RBD_DMS/blob/main/results/summary/compute_binding_Kd_huACE2.md and https://github.com/tstarrlab/SARSr-CoV-RBD_DMS/blob/main/results/summary/compute_expression_meanF.md. Because most mutants in the library were independently associated with more than one N16 barcode, we derived the final mutant phenotype as the average of per-barcode measurements, as computed at: https://github.com/tstarrlab/SARSr-CoV-RBD_DMS/blob/main/results/summary/collapse_barcodes_lib40_41.md. The final per-mutant deep mutational scanning phenotypes are available at: https://github.com/tstarrlab/SARSr-CoV-RBD_DMS/blob/main/results/final_variant_scores/final_variant_scores_lib40_41.csv.Demultiplexed Illumina barcode reads were aligned to library barcodes using dms_variants (version 0.8.9), yielding a table of counts of each barcode in each FACS bin which is available at Female BALB/c mice were purchased from Envigo (order code 047) at 7 weeks of age and were maintained in a specific pathogen-free facility within the Department of Comparative Medicine at the University of Washington, Seattle, accredited by the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC). Prior to each immunization, immunogens (endotoxin-free SARS-CoV-2 HexaPro S or PRD-0038 HexaPro S) were diluted to 20 µg/mL in 1x TBS  and mixed with 1:1 vol/vol AddaVax (InvivoGen vac-adx-10) to reach a final dose of 1 µg of immunogen per injection. At 8 weeks of age, 6 mice per group were anesthetized and injected intramuscularly in the quadriceps with 50µL of immunogen per leg, 100µL total at weeks 0, 3, and 6. Mice were bled via the submental route at weeks 0, 2, 5, and 8. Blood was collected in serum separator tubes (BD # 365967) and rested for 30 min at room temperature for coagulation. Serum tubes were then centrifuged for 10 min at 2,000 x g and serum was collected and stored at −80°C until use. Animal experiments were conducted in accordance with the University of Washington’s Institutional Animal Care and Use Committee.6 RLU. The mixture was incubated at room temperature for 45–60 minutes. Media was removed from the cells and 40 μL from each well of the half-area 96-well plate containing mAb and pseudovirus were transferred to the 96-well plate seeded with cells and incubated at 37°C for 1h. After 1h, an additional 40 μL of DMEM supplemented with 20% FBS and 2% PenStrep was added to the cells. After 18–20h, 40 μL of One-Glo-EX substrate (Promega) was added to each well and incubated on a plate shaker in the dark for 5 min before reading the relative luciferase units using a BioTek Neo2 plate reader. Relative luciferase units were plotted and normalized in Prism (GraphPad): 100% neutralization being cells lacking pseudovirus and 0% neutralizing being cells containing virus but lacking mAb. Prism (GraphPad) nonlinear regression with “[inhibitor] versus normalized response with a variable slope” was used to determine IC50 values from curve fits with 3 technical repeats. 3 biological replicates were carried out for each mAb.For mAb neutralization against SARS-CoV-2 S VSV and PRD-0038 S K482Y/T487W VSV, HEK293T cells with stable human ACE2 expression in DMEM supplemented with 10% FBS and 1% PenStrep were seeded at 40,000 cells/well into 96-well plates [3610] (Corning) coated with poly-lysine [P4707] (Sigma) and incubated overnight at 37°C. After 16–20h of incubation, a half-area 96-well plate (Greiner) was prepared with 1:5 serial dilutions of S2X259 and S2X35 starting from 0.1 mg/ml in DMEM, and 1:3 serial dilutions of 76E1 starting from 0.15 mg/ml in DMEM, for a total of 22 µL per well. An equal volume of DMEM with diluted pseudoviruses was added to each well. All pseudoviruses were diluted between 1:3–1:27 to reach a target entry of ~10R. alcyone ACE2 using Lipofectamine 2000 (Life Technologies) in Opti-MEM five hours prior to plating into 96-well plates [3610] (Corning) coated with poly-lysine [P4707] (Sigma) and incubated overnight at 37°C. The following day, a half-area 96-well plate (Greiner) was prepared with 3-fold serial sera dilutions . An equal volume of DMEM with diluted pseudoviruses was added to each well. All pseudoviruses were diluted between 1:3–1:27 to reach a target entry of ~106 RLU. The mixture was incubated at room temperature for 45–60 minutes. Media was removed from the cells and 40 μL from each well of the half-area 96-well plate containing sera and pseudovirus were transferred to the 96-well plate seeded with cells and incubated at 37°C for 1h. After 1h, an additional 40 μL of DMEM supplemented with 20% FBS and 2% PenStrep was added to the cells. After 18–20h, 40 μL of One-Glo-EX substrate (Promega) was added to each well and incubated on a plate shaker in the dark for 5 min before reading the relative luciferase units using a BioTek Neo2 plate reader. Relative luciferase units were plotted and normalized in Prism (GraphPad): 100% neutralization being cells lacking pseudovirus and 0% neutralizing being cells containing virus but lacking sera. Prism (GraphPad) nonlinear regression with “log[inhibitor] versus normalized response with a variable slope” was used to determine ID50 values from curve fits with 3 technical repeats. 3 biological replicates were carried out for each sample-pseudovirus pair.For SARS-CoV-2 D614G S VSV, BA.2 S VSV, BA.5 S VSV, RaTG13 S VSV, and SARS-CoV-1 S VSV neutralization, HEK293T cells with stable human ACE2 expression in DMEM supplemented with 10% FBS and 1% PenStrep were seeded at 40,000 cells/well into 96-well plates [3610] (Corning) coated with poly-lysine [P4707] (Sigma) and incubated overnight at 37°C. For PRD-0038 S VSV and Khosta1 S VSV neutralization, HEK293T cells were transfected with full length https://github.com/tstarrlab/SARSr-CoV_MAP_PRD0038-vaccine/blob/main/results/summary/summary.md. Binding of serum was evaluated against deep mutational scanning pools for PRD-0038 and SARS-CoV-1 whose construction is described above, previously published deep mutational scanning pools for SARS-CoV-2 Wuhan-Hu-176 and Omicron BA.238, and a previously published pan-sarbecovirus panel29 that was supplemented with additional newly described sarbecovirus and SARS-CoV-2 variants. Serum was first depleted of non-specific yeast-reactive antibodies as previously described105. Yeast-display RBD libraries were pooled, induced for yeast surface expression, and labeled with serum at 1:100, 1:1000, 1:10,000, and 1:100,000 dilutions for one hour at room temperature. Yeast were washed with PBS-BSA and labeled with secondary Myc-FITC antibody and APC-conjugated goat anti-mouse-IgG (Jackson ImmunoResearch 115-605-008). As with ACE2-binding titrations, libraries were then partitioned into four bins of serum binding on a BD FACSAria, collecting a minimum of 6 million RBD+ cells per sample concentration across the four bins. Cells were grown post-sort, plasmid purified, N16 barcode amplified, and sequenced on an Illumina NextSeq. Raw Illumina sequencing data is available from the NCBI Sequence Read Archive, BioProject PRJNA714677, BioSample SAMN36715819. Barcode reads were mapped to library barcodes, with raw counts found at: https://github.com/tstarrlab/SARSr-CoV_MAP_PRD0038-vaccine/blob/main/results/counts/variant_counts.csv.The complete serum deep mutational scanning pipeline is described at: https://github.com/tstarrlab/SARSr-CoV_MAP_PRD0038-vaccine/blob/main/results/summary/compute_AUC.md, and per-barcode AUC metrics are available at: https://github.com/tstarrlab/SARSr-CoV_MAP_PRD0038-vaccine/blob/main/results/bc_sera_binding/bc_sera_binding.csv. We then computed the per-variant AUC as the robust mean of replicate barcodes linked with the identical RBD variant, by taking the mean per-barcode AUC after trimming tails of the top and bottom 2.5% of AUC values among the replicate barcodes. Because mutations that disrupt RBD expression artifactually decrease serum binding, we applied two final filters: first, we censored the AUC measurement for any mutant with a measured impact on RBD expression of greater than one log-MFI unit (RBD expression < −1) from DMS measurements described above; and second, we derived a normalization constant from the slope of the linear model relating serum AUC and expression globally across all library variants for variants with expression >-1, and normalized our raw AUC measurements by this constant. The final variant derivation can be found at: https://github.com/tstarrlab/SARSr-CoV_MAP_PRD0038-vaccine/blob/main/results/summary/collapse_barcodes_SARSr-DMS.md, and final per-variant serum-binding values are available at: https://github.com/tstarrlab/SARSr-CoV_MAP_PRD0038-vaccine/blob/main/results/final_variant_scores/final_variant_scores_wts.csv and https://github.com/tstarrlab/SARSr-CoV_MAP_PRD0038-vaccine/blob/main/results/final_variant_scores/final_variant_scores_dms.csv.For each library barcode, an area under the curve (AUC) metric was derived from its distribution of sequence reads across sort bins. First, the strength of serum binding to each barcode at each serum dilution was determined as the simple mean bin from cell counts across integer-weighted bins, and subtracted by background mean bin determined from a sort from yeast libraries not incubated with sera. Any barcode with less than 3 cell counts at any sample concentration was eliminated from analysis. An AUC metric was then calculated from the relationship between mean bin and serum dilution. AUC calculation can be found at: Supplement 1


VOC of 638 mV.We show for the firsttime DMSO-free tin-based perovskitesolarcells with a self-assembled hole selective contact (MeO-2PACz). Ourmethod provides reproducible and hysteresis-free devices with MeO-2PACz,having the best device PCE of 5.8 % with a  Hence,some obstacles need to be overcome, such as the undesirable Sn(II)oxidation, the unregulated crystallization rate, and the high hysteresismeasured after aging devices.4Perovskitesolar cells (PSC)have attracted extensive research interest for next-generation solution-processedphotovoltaic devices and have taken a big step toward commercialization.Interest in lead-free tin perovskite solar cells (Sn-PSCs) soaredonce their optoelectronic properties revealed promising alternativesto toxic lead-based PSCs like having low bandgap, large charge-carriermobility, and low exciton binding energy.pin sandwich architecturewith poly (PEDOT:PSS);2 however, the hygroscopic and acidic nature ofPEDOT:PSS significantly limits the device performance and operationalstability under ambient ultraviolet radiation and humidity.5 Otherwise, self-assembled molecules (SAMs) recentlyhave been used as hole-selective layers (HSLs) in pin structures, thanks to their low-price synthesis pathway7 and easily functionalized molecular structures,8 and demonstrated conformal coverage on large-area substrates.9 Additionally, SAM will be a promising HSLs inSn-PSCs, owing to its ability to modify the contact layers, i.e.,indium tin oxide (ITO), and enhance its charge transfer properties.The first application of SAMs as an HSL in Sn-PSCs has been reportedby Song et al. and the PCE of the best device reached 6.5 % (the efficiencydistribution is 5.2 % ± 0.6 %).10 Theymanaged to obtain a uniform dimethyl sulfoxide (DMSO)-processed FASnI3-based perovskite film via a two-step sequential depositionmethod on top of the [2-ethyl]phosphonicacid (MeO-2PACz) SAM. Despite the good performance, a preannealingstep at 400 °C for 30 min of the ITO substrates was necessary.10The high-performingSn-PSCs are generally made in the 3 perovskite can be successfully depositedon top of the MeO-2PACzSAM with a one-step method using a low-temperature and DMSO-free solventsystem of .11 Indeed, we previously showedthat DMSO acts as an oxidizing agent for Sn(II) in an acidic medium,12 and that it can oxidize Sn(II) species evenduring the synthesis of the perovskite precursor solution.14 Besides, DMSO is partially reduced to dimethyl sulfide,13 which might influence the crystallization rateof perovskite precursor owing to the low boiling point (37.3 °).We prepared DMSO-free MeO-2PACz SAM-containing Sn-PSCs and comparedtheir performance to control devices that use a water-free PEDOT complex. The hydrophilicity of MeO-2PACzfilms slightly improved with increasing spinning speed, with measuredcontact angles of 53.59 °, 50.77 °, and 47.07 ° for4000 rpm, 5000 rpm, and 6000 rpm, respectively . In contrast, PEDOT HSL has a more hydrophobicsurface (61.80 °)15 than MeO-2PACzeven after depositing the Al2O3 interlayer,which is mainly used to improve its wettability. We fabricated ITO/HSL/FASnI3/C60/BCP/Ag15pin architecture devices using MeO-2PACz SAM spin-coatedat different speeds, finding that the thinner layer (6000 rpm) showedstatistically higher efficiency (∼4.5 % average) with betteropen-circuit voltage (VOC) and fill factor(FF) . 3 than MeO-2PACz SAM, which leads to improved hole extractioncapabilities and thus increases short-circuit current density (JSC).17Initially, we spin-coated 0.1mM of MeO-2PACz SAMsolution ontocleaned ITO-covered substrates.ectively S1. In co3 perovskitethin films deposited onto both the PEDOT HSL and the MeO-2PACz SAMexhibit the typical orthorhombic structure, assigned to the crystallographicplanes (100), (120), (200), (211), (222), and (300) .18 The PEDOT-based perovskitelayer exhibits higher crystallinity compared to the MeO-2PACz-basedone, as indicated by the sharp increase in the intensity of the 100and 200 peaks. Higher film crystallinity provides much better optoelectronicproperties such as less surface trapping and higher carrier mobility.19 As we show in 20TheX-ray diffraction patterns of FASnInd (300) S3.18 Thend (300) S3.18 The21 As shown in Tables S1 and S2). The devices withPEDOT show a broader HI distribution, while SAM devices exhibit anarrower HI distribution .Determining the accurate PCE of the Sn-PSCscan be notoriouslydifficult, owing to variations in photovoltaic parameters and hysteresisover time. Although the source of hysteresis is still uncertain inSn-PSCs, ionic migration, carriers trapping–detrapping at interfaces,and choice of contact material can lead to it.ribution 2c with nJ–V) scansand photovoltaic parameters of the best PCEs with PEDOT and MeO-2PACzmeasured at a scan rate of 100 mVs–1 (from forwardto reverse bias). The efficiency distribution of PEDOT and MeO-2PACzis 7.8 ± 0.9 % and 5.0 ± 0.8 %, respectively. Notably, theperformance of both PEDOT- and MeO-2PACz-based devices increased torecord PCEs of 8.7 % and 5.8 %, respectively, after storage in a gloveboxenvironment for 2 weeks (JSC) values from incident photon current efficiencyare 18.7 and 13.7 mA/cm2 for PEDOT and MeO-2PACz, respectively,showing negligible difference with those extracted by the J–V curves . 2 weeks 2d. IntegIn summary, we demonstrated a simple one-step tin-based perovskitedeposition method with a MeO-2PACz SAM by using a DMSO-free solventsystem, which led to hysteresis-free solar cells with a champion PCEof 5.8 %. This method has great potential for improving the hysteresis-freeSAM-based Sn-PSCs performance with low-temperature processability.


Brain tumours and Gliomas, in particular, are among the primary causes of cancer mortality worldwide. Glioma diagnosis and therapy have not significantly improved despite decades of efforts. Autocrine TGF-β signalling promotes glioma proliferation, invasion, epithelial-to-mesenchymal transition (EMT), and drug resistance. Non-coding RNAs such as miRNA, lncRNA, and circRNAs have emerged as critical transcriptional and post-transcriptional regulators of TGF-β pathway components in glioma. Here, we summarize the complex regulatory network among regulatory ncRNAs and TGF-β pathway during Glioma pathogenesis and discuss their role as potential therapeutic targets for Gliomas. IDH) wild type, GBM IDH mutant, and GBM not otherwise specified (NOS) [IDH group of genes represent the most critical genetic alterations in GBM, which plays a vital role in therapeutic responses [CD133 [Gliomas are a group of brain tumours clinically divided into four types from grade I to grade IV. Grade IV gliomas, known as Glioblastoma multiforme (GBM), are the most common form of adult brain cancer ,2. The eed (NOS) ,6. Mutatesponses . GBMs liesponses . Diffuseesponses . DIPG shesponses ,10. Glios [CD133 . GSCs cas [CD133 . GSCs cos [CD133 .MGMT) can resolve some TMZ-induced alterations and thus mediate the survival of GBM cells [MGMT inhibitors are considered beneficial for improving the action of TMZ in GBM patients [MGMT increases TMZ efficiency [Current therapeutic strategies for GBM include maximum surgical resection and radio- and chemotherapy with temozolomide (TMZ) ,12. TMZ BM cells . MGMT inpatients . Localizficiency . Overallficiency , while tficiency .MAPK, ERK, and JUN kinase—in the non-canonical TGF-β pathway [TGF-β target genes [SMAD pathways include various branches of MAP kinase pathways, Rho-like GTPase signalling pathways, and phosphatidylinositol-3-kinase/AKT pathways. For example, in the ERK-mediated signal transduction, the activated TGFβRI phosphorylates and activates the ShcA protein, forming the SHCA/GRB22/SOS complex, followed by the sequential activation of c-RAF, MEK, ERK [CTGF), ZEB1, and SNAIL1 [TGF-β is a pleiotropic cytokine that regulates cell proliferation, differentiation, tissue homeostasis, motility, invasion, extracellular matrix production, angiogenesis, epithelial to mesenchyme transition (EMT), chemoresistance, and immune response in various cancers, including GBM ,18. TGF- pathway . TGF-β tet genes . Non-canMEK, ERK . In normMEK, ERK ,25,26. TMEK, ERK ,28. TGF-MEK, ERK . TGF-β pd SNAIL1 ,31,32. Td SNAIL1 . Severald SNAIL1 ,35,36. SThe human genome may be categorized into protein-coding genes (PCGs) and non-protein-coding genes (NCGs). ncRNAs constitute >90% of the human genome. The non-coding category of the genome is highly heterogeneous, consisting of small non-coding RNAs < 200 bps in length and long ncRNAs (lncRNAs) > 200 bps in length. miRNAs are small non-coding RNAs of 21–23 nucleotides in length that generally perform post-transcriptional gene silencing of their mRNA targets . They recis and trans by interacting with DNA, RNAs, and proteins [LncRNAs are transcripts longer than 200 bps with no ability to code for proteins ,51,52,53proteins . To reguproteins . They haproteins ,58. cis-acting elements and trans-acting factors [Circular RNAs are covalently closed, single-stranded RNAs produced by a non-canonical back splicing of cellular non-coding RNAs and precursor messenger RNAs ,55,56. C factors ,61,62,63 factors ,64 A sma factors ,66.The miRNAs, lncRNAs, and circRNAs are aberrantly expressed in GBM tumour tissues and regulate GBM pathogenesis  57,65,6,665,67. Several miRNAs, lncRNAs, and circRNAs have recently been identified, which modulate the TGF-β pathway to promote or repress GBM . Since TmiR-182 is over-expressed in GBM tissues and cells, whereas CYLD, a negative regulator of the NFκB pathway, is downregulated [miR-182, 7q32.1, is frequently amplified in gliomas [miR-182 is induced by TGF-β in U373MG and LN229 cells through the SMAD signalling pathway [miR-182 expression was also upregulated in Smad2/Smad4- overexpressing cells [miR-182 [miR-182 expression in glioma cells through the SMAD signalling pathway. CYLD is a target of miR-182 [miR-182 in U373MG and LN229 cells decreased the expression of CYLD. Also, miR-182 interacts with and degrades CYLD. Further, overexpression of miR-182 increased, while inhibition of miR-182 reduced the luciferase activity of NF-κB reporter and expression of NF-κB target genes. miR-182 overexpression significantly induced the phosphorylation of IKKβ, while miR-182 inhibitor reversed this effect. Importantly, in vitro kinase assay showed that endogenous IKK kinase activity was prolonged in miR-182 –transduced cells. These results indicate that miR-182 suppresses CYLD and enhances and sustains NF-κB activity in GBM [miR-182 up-regulation promotes anchorage-independent growth, colony formation, and invasion of GBM in vitro and in vivo [miR-182 inhibition reversed these effects [miR-182 overexpression was reversed with combined transfection of miR-182 mimics and IκBα dominant-negative mutant construct, indicating that miR-182 promotes GBM tumour through the NF-κB pathway [miR-182 suppression inhibits NF-κB activity and malignant properties of patient-derived glioma cells (PDGCs) [miR-182 inhibition. This indicates that TGF-β-induced miR-182 is essential for sustained NF-κB activity in GBM [miR-182, and several NF-κB target genes was observed in clinical GBM samples, which conferred poor survival of the patients [miR-182, and NF-κB target genes [miR-182 promotes GBM pathogenesis by activating and sustaining NF-κB activity by downregulating CYLD [egulated . The gen gliomas . Interes pathway . miR-182ng cells . ChIP as[miR-182 . These r miR-182 . Up-reguy in GBM . miR-182 in vivo . At the  effects . Also, t pathway . Further (PDGCs) . The stuy in GBM . Overexppatients . Also, tet genes . Upon ining CYLD . miR-15a-5p is upregulated in glioma tissues [miR-15a with SMAD7. Overexpression of miR-15a promoted migration and invasion of SHG139 cells [miR-15a displayed the opposite effect, indicating the tumour-promoting ability of miR-15a [miR-15a in SHG139 cells reduced migration and downregulated mesenchymal markers—Vimentin and N-cadherin. However, the combined knockdown of miR-15a and SMAD7 reversed these effects [miR-15a inhibition attenuated GBM tumour growth in vivo. Hence, this study indicates the oncogenic function of miR-15a in GBM by inhibiting SMAD7 activity [Guo et al., using a microarray screen, observed that  tissues . Bioinfo39 cells . Whereas miR-15a . Anti-mi effects . Furtheractivity . miR-193b levels are upregulated in glioma cell lines and GBM tumour samples [miR-193b depletion reduces the proliferation of U87 and U251 cells [miR-193b knockdown revealed G0/G1 arrest in U87 and U251 cells [miR-193b [miR-193b significantly increased the protein levels of SMAD3 in U87 and U251 cells [miR-193b regulates GBM proliferation through the TGF-β pathway, one of the primary targets of the TGF-β pathway, p21 levels were evaluated upon miR-193b inhibition. Inhibition of miR-193b and the subsequent up-regulation of SMAD3 in GBM cells displayed a significant accumulation of p21 [miR-193b is an oncogene that promotes cell growth by directly targeting SMAD3 and restricting the tumour-suppressive effects of SMAD3 through p21 down-regulation in GBM [ samples . miR-19351 cells . Cell cy51 cells . BioinfomiR-193b . Inhibit51 cells . Furthern of p21 . Also, tn of p21 . Hence, n in GBM . miR-210-3p is induced upon exposure to hypoxic conditions in GBM [HIF-1α, significantly reduced hypoxia-induced miR-210 levels, indicating the transcriptional activation of miR-210-3p by HIF-1α in GBM [HIF-1α promotes TGF-β expression, which was abrogated upon miR-210-3p inhibition. This indicates that miR-210-3p is an essential mediator in promoting HIF-1α-mediated TGF-β expression [miR-210-3p inhibition, indicating an oncogenic role of miR-210-3p in promoting hypoxia/TGF-β -mediated GBM pathogenesis [miR-210-3p promotes resistance to TMZ in U87-MG cells. However, the chemoresistance induced by miR-210-3p overexpression was abrogated upon TGF-β knockdown [miR-210-3p also promotes the transcription activity of NF-κB in U87-MG cells [miR-210-3p promotes the TGF-β expression and TGF-β-mediated migration, invasion, and TMZ resistance in GBM [miR-210-3p on NF-κB-mediated GBM pathogenesis and the involvement of TGF-β in this context are yet to be studied. s in GBM . Treatmeα in GBM . HIF-1α pression . Exposurogenesis . Overexpnockdown . HypoxiaMG cells . This ste in GBM . HoweverQKI), an essential negative regulator of the TGF-β signalling, is downregulated, and miR-148a is upregulated in GBM tissues and cell lines [QKI and overexpression of miR-148a confer poor prognosis in GBM patients [miR-148a and QKI [miR-148a reduced the protein levels of QKI, while inhibition of miR-148a reduced this effect [SKP1, a member of the E3 ubiquitin ligase complex that degrades SMAD3, is also an essential target of miR-148a [miR-148a overexpression enhanced the SMAD luciferase reporter activity and phosphorylation of SMAD2/3 [miR-148a positively correlates with the gene signatures of TGF-β signalling [miR-148a is associated with poor prognosis in GBM patients, indicating that miR-148a is an oncogenic miRNA [miR-148a are positively correlated with p-SMAD3 expression and the DNA binding activity of NF-κB [miR-148a contains multiple NF-κB binding domains. Consequently, NF-κB activation increased the expression of miR-148a in GBM cells [miR-148a promotes the invasion, migration, and aggressiveness of GBM [miR-148a oncogene, which further promotes the aggressive phenotypes of GBM by downregulating the expression of QKI and SKP1 and activating the TGF-β signalling [miR-148a establishes an essential link between NF-κB and TGF-β signalling in promoting GBM pathogenesis [Quaking . DNA methylation and histone acetylase inhibition resulted in the down-regulation of miR-127-3p in GBM tissues [miR-127-3p in LN229 and T98G cells reduced the proliferation and caused cell cycle arrest compared to control cells. However, the overexpression of miR-127-3p did not significantly affect the apoptosis of glioma cells. In vivo nude mice model with miR-127-3p overexpressing LN229 cells displayed reduced colony formation and tumour volume compared to the control group. Hence, miR-127-3p functions as a tumour suppressor in GBM [SKI, RGMA, ZWINT, SERPINB9, and SFRP1 are the primary targets of miR-127-3p. SKI is an essential negative regulator of the TGF-β pathway [miR-127-3p or knockdown of SKI promotes TGFBR1 expression, phosphorylation of SMAD3, and induces cell cycle arrest of LN229 cells [miR-127-3p is an essential tumour suppressor miRNA that attenuates GBM proliferation by inhibiting the TGF-β signalling [RNAseq analysis of GBM and normal brain tissues indicated that  tissues . miR-127 tissues . Additior in GBM . Bioinfo pathway . It bind29 cells . Hence, gnalling .miR-564 is downregulated in glioma cells and tumour tissues compared to normal human astrocytes and normal brain tissues, respectively [miR-564 mimics decreased the proliferation and invasion of U87 cells [miR-564 [miR-564 overexpression markedly decreased the mRNA and protein levels of TGF-β1 [miR-564 also reduced the expression of SMAD4 protein and phosphorylated SMAD2 protein levels [miR-564 overexpression in U87 cells [miR-564, EGFR, and MMP9 [miR-564 overexpression [miR-564 overexpression [miR-564- treated tumours than in scramble-treated tumours [miR-564 is a tumour suppressor miRNA, which restricts proliferation and invasion in GBM by targeting TGF-β1, and its downstream targets EGFR and MMP9 [ectively . miR-56487 cells . Bioinfo[miR-564 . miR-564f TGF-β1 . In addin levels . Moreove87 cells . EGFR an87 cells . Also, aand MMP9 . Cell prpression . Furtherpression . Also, t tumours . Hence, and MMP9 . LncRNA-ATB levels are higher in glioma tissues and U251, A172 cell lines than in normal brain tissues [lncRNA-ATB correlated with poor survival of GBM patients [lncRNA-ATB and miR-200a in GBM tissues. miR-200a is downregulated in GBM tissues and cell lines, and the knockdown of lncRNA-ATB significantly increased miR-200a expression in U251 and A172 cells [lncRNA-ATB and TGF-β2 are direct targets of miR-200a. Also, miR-200a inhibition results in up-regulation of TGF-β2 [LncRNA-ATB knockdown mediated a reduction in cell proliferation, colony formation, and invasion of U251 and A172 cells, which is attenuated upon miR-200a inhibition. Additionally, the knockdown of lncRNA-ATB significantly reduced the levels of TGF-β2 expression, which was rescued by miR-200a inhibition. Glioma samples show a positive correlation between lncRNA-ATB and TGF-β2 and a negative correlation between miR-200a and TGF-β2. The reduction in mRNA and protein levels of TGF-β2 upon lncRNA-ATB depletion was further downregulated upon miR-200a overexpression. In contrast, TGF-β2 expression was rescued with the combination of lncRNA-ATB knockdown and miR-200a inhibition [lncRNA-ATB depletion demonstrated a reduction in tumour volume, tumour weight, and reduced proliferation index indicated by Ki67 staining, supporting the oncogenic role of lncRNA-ATB in GBM. These results suggest that lncRNA-ATB could competitively bind miR-200a to stabilize TGF-β2 and promote TGF-β2-mediated GBM pathogenesis [ tissues . Increaspatients . Furtherpatients . The stu72 cells . Luciferf TGF-β2 . LncRNA-hibition . Studiesogenesis . lncRNA-ATB is upregulated by TGF-β1 treatment in LN18 and U251 cells [lncRNA-ATB by TGF-β treatment was abrogated upon treatment with TGF-β inhibitor, SB505124, indicating the SMAD2/3 mediated regulation of lncRNA-ATB expression [LncRNA-ATB overexpression combined with TGF-β1 treatment increases the invasion of U251 and LN18 cells. Also, lncRNA-ATB overexpression and TGF-β1 treatment promote the phosphorylation of p65, the nuclear translocation of p65, and the phosphorylation of p38. These results indicate the activation of the NF-κB and p38/MAPK pathways by TGF-β regulated lncRNA-ATB [NF-κB and p38/MAPK pathway inhibitors. These results suggest that the SMAD2/3 transcription factor induces lncRNA-ATB expression in GBM, and it promotes TGF-β-mediated GBM invasion through the NF-κB and p38/MAPK pathways. Another study by Tang et al. reported that 51 cells . The up-pression . LncRNA-cRNA-ATB . The inclncRNA-UCA1 expression in U87 and U251 cells [lncRNA-UCA1 attenuated the invasion and stemness of glioma cells induced by TGF-β [lncRNA-UCA1 knockdown downregulates SLUG, ALDH1, and NANOG, which are involved in TGF-β up-regulation. Luciferase reporter assay and Ago2 RIP suggest direct binding of lncRNA-UCA1, miR-1, and miR-203a. Moreover, miR-1 and miR-203a directly mediate SLUG down-regulation. The study also reported a positive correlation between lncRNA-UCA1 and SLUG expression in GBM tissues. LncRNA-UCA1 promotes SLUG expression by binding to and titrating miR-1 and miR-203a. Reduction in spheroid formation, SLUG expression, and ALDH1 activity upon lncRNA-UCA1 knockdown is partially rescued upon Slug overexpression. Hence, these results suggest that TGF-β induced lncRNA-UCA1 acts as a molecular sponge for miR-1 and miR-203a to promote SLUG expression and SLUG-mediated GBM cell stemness [TGF-β significantly upregulates 51 cells . Furtherby TGF-β . Particustemness . LINC00645 is upregulated in GBM patients compared to normal brain tissues [LINC00645 was high with increasing grades of glioma. Moreover, TGF-β induces LINC00645 expression in GBM [LINC00645 attenuated the malignant behaviour of GBM by decreasing proliferation, invasion, migration, and EMT in T98G and U251 cells. Notably, LINC00645 knockdown reduces ZEB1 levels, an essential target of miR-205-3p [miR-205-3p is downregulated in GBM tumour tissues and GBM cell lines compared to normal brain tissues [miR-205-3p indicates poor survival in GBM patients [miR-205-3p, and a negative correlation is observed between LINC00645 and miR-205-3p in GBM patients’ samples [miR-205-3p overexpression significantly reduced the levels of LINC00645, while depletion of LINC00645 promoted miR-205-3p expression. Luciferase reporter assay and Ago2-RIP indicate a direct interaction between LINC00645 and miR-205-3p [LINC00645 in combination with TGF-β treatment increased E-cadherin levels and reduced the expression of mesenchymal markers. Further, TGF-β treatment in combination with miR-205-3p overexpression reversed these effects [miR-205-3p is a tumour suppressor that directly targets and degrades ZEB1 [LINC00645 mediated GBM invasion, and migration is reversed upon miR-205-3p overexpression [LINC00645 sponges miR-205-3p to stabilize ZEB1 and promote GBM pathogenesis [LINC00645 also induces stemness in GBM. Western blotting analysis upon LINC00645 depletion reduced the expression of stemness factors, BMI-1, OCT-4, SOX-2, and NANOG [LINC00645 knockdown. In contrast, LINC00645 overexpression had the opposite effect [LINC00645 depletion partly decreased NESTIN expression and increased the GFAP expression in U251 cells [LINC00645 reduced tumour growth of the tumour xenograft model in vivo [LINC00645/miR-205-3p/ZEB1 axis regulates invasion, migration, and EMT in GBM, and LINC00645 also promotes stemness in GSCs [TCGA and GSEA data analysis by Li et al. indicated that  tissues . Expressn in GBM . KnockdoR-205-3p . miR-205 tissues . Low exppatients . TGF-β t samples . miR-205R-205-3p . Epithel effects . miR-205des ZEB1 . TGF-β ipression . These rogenesis . In addind NANOG . Sphere-e effect . Also, L51 cells . Depleti in vivo . LINC006 in GSCs . LINC00115 [LINC00115 expression is higher in GBM tumour samples than in normal tissues and correlates with poor patient prognosis [LINC00115 knockdown inhibits GSC proliferation and neurosphere formation in vitro and also inhibits tumour formation in the xenograft model [LINC00115 physically associates with miR-200b and miR-200c [LINC00115 depletion reduced the expression of ZEB1 and ZNF596 and reduced invasion in GSCs [miR-200b and miR-200c. Down-regulation of ZEB1 and GBM invasion upon LINC00115 knockdown was reversed upon miR-200b overexpression, indicating that LINC00115 competitively binds to miR-200b to promote ZEB1-mediated GBM invasion [LINC00115 and its target ZNG596 are co-expressed in clinical glioma samples. Concomitantly, exogenous expression of ZNF596 in LINC00115-depleted GSCs reversed the inhibition of cell proliferation caused by LINC00115 depletion, indicating that ZNF596 is the downstream effector of LINC00115-driven GBM tumourigenicity [LINC00115 binds to miR-200 to promote the expression of ZNF596 [ZNF596 indicated that EZH2 is a direct target of ZNF596. ZNF296 is a transcription factor promoting the expression of EZH2 [LINC00115 depletion results in loss of EZH2 expression, which is reversed upon ZNF596 overexpression [LINC00115 further activates STAT3 downstream of EZH2 through ZNF596, indicating that LINC00115 activates EZH2/STAT3 signalling through ZNF596, thereby promoting GSC self-renewal and tumourigenicity [LINC00115 aids GSC’s self-renewal by acting as a ceRNA for transcription factors ZEB1 and ZNF596 by sponging miR-200 [ZNF596/EZH2/STAT3 signal axis [RNA-sequencing of TGF-β treated GSCs revealed up-regulation of INC00115 . Moreoverognosis . LINC001ft model . LINC001miR-200c . Also, L in GSCs . Reporteinvasion . LINC001genicity . Also, Lf ZNF596 . CRISPR- of EZH2 . LINC001pression . LINC001genicity . LINC001 miR-200 . It alsonal axis .H19, HOXD-AS2, LINC00635, LINC00277, RP11-196G11.2, LINC00152, MALAT1, and LOC100506207) in D54, P-GBM2 cells [H19 and HOXD-AS2 through SMAD signalling [H19 and HOXD-AS2 with K-homology (KH) splicing regulatory protein (KSRP) [KSRP degrades follistatin-like 1 (FSTL1) and promotes the maturation of miR-198 in the nucleus [H19 and HOXD-AS2 competitively bind to KSRP and thus prevent its nuclear translocation [miR-198 is a tumour suppressor miRNA that promotes TMZ sensitivity in GBM by downregulating MGMT [KSRP reversed the H19 and HOXD-AS2-mediated up-regulation of MGMT expression, which could reverse TGF-β-induced TMZ resistance [H19 and HOXD-AS2 confer TMZ resistance by regulating miR-198 biogenesis by competing with KSRP [Nie et al. identified eight differentially regulated lncRNAs upon TGF-β treatment  . KSRP de nucleus . TGF-β-ilocation . miR-198ing MGMT . Overexpsistance . H19 andith KSRP .MIR4435-2HG in GBM tissue samples [MIR4435-2HG in U251 and U87 cells decreased cell proliferation, colony formation, migration, and invasion [In vivo, nude mice models also show reduced tumour volume and growth upon MIR4435-2HG depletion [MIR4435-2HG in GBM [miR-1224-5p targets MIR4435-2HG [miR-1224-5p is downregulated in LN229, U87-MG, and U251 cells compared to normal human astrocytes (NHA) [MIR4435-2HG and miR-1224-5p [MIR4435-2HG overexpression was abrogated upon miR-1224-5p mimics, indicating that the MIR4435-2HG -miR-1224-5p axis promotes GBM pathogenesis [TGFBR2, a critical oncogene, is a direct target of miR-1224-5p [MIR4435-2HG overexpression promoted the expression of TGFBR2 at mRNA and protein levels [miR-1224-5p inhibition reduced proliferation and colony formation ability in GBM, which was rescued upon TGFBR2 overexpression [TGFBR2 knockdown antagonized MIR4435-2HG overexpression-induced proliferation and colony formation in GBM, indicating that MIR4435-2HG promotes GBM proliferation by sponging miR-1224-5p and stabilizing TGFBR2 [MIR4435-2HG in GBM by targeting the miR-1224-5p/TGFBR2 axis [Xu et al. reported the high expression of  samples . Loss-ofinvasion . In vivoepletion . These rG in GBM . The sta4435-2HG . Also, ies (NHA) . Lucifer-1224-5p . Functioogenesis . Further-1224-5p . Furthern levels . Also, mpression . In addig TGFBR2 . Hence, BR2 axis .RPSAP52 is overexpressed in GBM tumour samples [RPSAP52 is associated with poor survival in GBM patients [RPSAP52 and TGF-β1expression in GBM samples [RPSAP52 increased TGF-β1 protein expression, and knockdown of RPSAP52 exhibited the opposite effect [RPSAP52 and TGF-β1 individually increased the percentage of CD133+ cells. Further, the overexpression of TGF-β1 rescued the reduction in the percentage of CD133+ cells observed upon RPSAP52 silencing [Wang et al. identified that lncRNA  samples . High expatients . Wang et samples . Furthere effect . Overexpilencing . PVT1 and down-regulation of p53 in higher grades of glioma compared to normal brain cells [PVT1 increased with increasing grades of glioma [PVT1 levels are associated with poor survival in GBM patients. Clinical GBM samples show high expression of PVT1, TGF-β, and pSMAD2/3 levels and low p53 levels [PVT1 levels in U373 cells, while overexpression of p53 showed a reverse effect [PVT1 and p53 [PVT1 promoter. Also, the dual luciferase reporter assay indicated that p53 binds to the promoter of PVT1 to attenuate the expression of PVT1 [PVT1 reduced proliferation, viability, migration, and invasion, induced cell cycle arrest at S and G2/M phases, and promoted apoptosis in GBM [lncRNA PVT1 depletion. At the same time, the knockdown of p53 had a reverse effect. Furthermore, the study demonstrated that lncRNA PVT1 overexpression increased the transcription activity of the TGF-β, as depicted in the dual luciferase reporter assay, and increased the pSMAD2/3 levels [PVT1 decreased the transcription activity of TGF-β, while p53 knockdown displayed the opposite effects [PVT1 and p53 counteracted the suppressive effects of p53 on TGF-β activity, indicating that p53 attenuated the TGF-β/SMAD pathway in GBM by targeting PVT1 [PVT1 and p53 individually suppressed the tumour growth [PVT1 and p53 counteracted the tumour-suppressive effects of p53 in GBM [lncRNA PVT1 or p53 [P53 knockdown increased PVT1 levels, TGF-β, and pSMAD2/3 levels [PVT1 knockdown displayed the opposite effects [lncRNA PVT1 depletion on TGF-β activity was counteracted by p53 knockdown [p53 potentially contributes to downregulating the oncogenic lncRNA PVT1, thereby suppressing the activation of TGF-β and TGF-β mediated GBM progression by modulating the lncRNA PVT1-TGF-β axis [Li et al. reported overexpression of lncRNA in cells . Also, tf glioma . Kaplan-3 levels . Furthere effect . RIP ass and p53 . Bioinfo of PVT1 . Loss-ofs in GBM . Howevers in GBM . Express3 levels . Knockdo effects . Howevering PVT1 . In vivor growth . In cont3 in GBM . The lev1 or p53 . P53 kno3 levels . At the  effects . Also, tnockdown . This stF-β axis .lncRNA-MUF is induced upon TGF-β treatment in T98G cells [lncRNA-MUF induction upon TGF-β treatment is observed across other GBM cell lines—LN229, U87-MG, and LN18. Moreover, levels of lncRNA-MUF are elevated in GBM tumour samples, and its expression is associated with poor survival and prognosis [LncRNA-MUF induction by TGF-β is completely abolished upon treatment with TGFBR1 inhibitor SB505124 in glioma cells. In addition, the ChIP qPCR assay demonstrates the enrichment of SMAD2/3 antibody in the promoter of lncRNA-MUF upon TGF-β stimulation [lncRNA-MUF revealed that lncRNA-MUF promotes proliferation, migration, and invasion in GBM [lncRNA-MUF sensitizes glioma cells to TMZ-induced cell death [lncRNA-MUF downregulated its cis oncogene, CAPRIN2, and various trans genes from the TGF-β ontology group  [VIMENTIN, and SNAIL1 upon lncRNA-MUF knockdown in T98G and U87-MG cells [lncRNA-MUF and miR-34a, and overexpression of miR-34a reduces lncRNA-MUF expression. miR-34a has a potential tumour-suppressor role in glioma by targeting several oncogenes, particularly SNAIL1, and it is downregulated in GBM tissues compared to normal tissues [SNAIL1 is a crucial transcription factor that promotes tumour cell invasion and EMT and is upregulated in GBM. We observed a positive correlation between MUF and SNAIL1 expression in GBM tumour samples [lncRNA-MUF knockdown was rescued upon miR-34a inhibition in T98G and U87-MG cells. These experiments indicate that TGF-β-induced lncRNA-MUF sponges miR-34a to promote SNAIL1-induced invasion in GBM [lncRNA-MUF promotes GBM invasion through the miR-34a/Snail axis [Using a genome-wide microarray screen, we identified that 8G cells . Also, lrognosis . LncRNA-mulation . Loss-ofn in GBM . In addill death . Knockdo SNAIL1) . WesternMG cells . Bioinfo tissues . SNAIL1  samples . Down-ren in GBM . Our stuail axis . LINC01711 expression in glioma cells and that the levels of LINC01711 are elevated in GBM tumour samples, and its expression is associated with poor patients’ survival and prognosis [lncRNA MUF, LINC01711 is also induced by the SMAD2/3 transcription factors downstream of TGF-β signalling. Down-regulation of LINC01711 reduces proliferation, migration, and invasion and induces apoptosis in GBM [LINC01711 knockdown results in downregulating ZEB1, a crucial transcription factor that promotes tumour cell invasion and EMT [LINC01711 also interacts with miR-34a, and ZEB1 is a target of miR-34a [LINC01711 knockdown was rescued upon miR-34a inhibition [miR-34a inhibition could reverse the reduction in invasion caused by LINC01711 knockdown in T98G and U87-MG cells [ZEB1 overexpression could partially reverse the LINC01711 knockdown-mediated reduction in invasion in GBM [LINC01711 could promote TMZ resistance in GBM. LINC01711 depletion significantly reduced proliferation and increased caspase 3/7 activity in T98G and LN229 cells, suggesting that LINC01711 promotes resistance to TMZ in GBM. Given the role of ZEB1 in TMZ resistance and that LINC01711 depletion results in ZEB1 inhibition, we evaluated if LINC01711 knockdown-mediated sensitization of GBM cells to TMZ-induced apoptosis is associated with a reduction in ZEB1 levels. ZEB1 protein levels were significantly downregulated during TMZ treatment in LINC01711-depleted cells compared to cells treated with TMZ alone [LINC01711 knockdown could induce cisplatin-mediated apoptosis in GBM. Hence, upon induction by TGF-β, LINC01711 promotes GBM proliferation, migration, invasion, and drug resistance by modulating the LINC01711/miR-34a/ZEBl signalling axis [We have shown that TGF-β induces rognosis . Like lns in GBM . LINC017 and EMT . LINC017 miR-34a . Reductihibition . FurtherMG cells . We alson in GBM . FurtherMZ alone . We founing axis .TCONS_00020456 decreased with increasing glioma grades, and the low expression of TCONS_00020456 indicated poor survival of GBM patients [TCONS_00020456 significantly promoted the invasion and migration of U251 and U87 cells [TCONS_00020456 significantly inhibited invasion and migration in GBM [TCONS_00020456 expression [TCONS_00020456 increased the expression of SMAD2, PKCα, N-cadherin, vimentin, and down-regulation of E-cadherin. Also, the phosphorylation of JNK and ERK was elevated upon TCONS_0002045 knockdown [TCONS_0002045 reversed these effects [TCONS_0002045 abrogates GBM invasion and migration by targeting SMAD2 and PKCα pathways [TCONS_0002045 in nude mice model revealed a decrease in tumour size and weight in the TCONS_0002045 overexpression group compared to the TCONS_002045 knockdown group [TCONS_002045 knockdown group compared to the TCONS_0002045 overexpression group [TCONS_0002045, SMAD2, and PKCα [miR-1279 was identified as the common miRNA target between the three. LncRNA TCONS_0002045 abrogates GBM migration and invasion by targeting SMAD2 and PKCα [SMAD2 and PKCα by TCONS_0002045 and the role of miR-1279 needs further investigation. Tang et al., using a microarray screen, identified 1759 upregulated and 1932 downregulated lncRNAs in U251 cells . Among tpatients . Further87 cells . While on in GBM . Bioinfopression . Among tpression . Furthernockdown . The ove effects . These rpathways . In vivown group . In addion group . Computaand PKCα . Among tand PKCα . HoweverRP11-838N2.4 expression is lower in TMZ-resistant cells  compared to the parental non-resistant cells  [RP11-838N2.4 down-regulation is associated with poor prognosis, a high risk of GBM relapse, and shorter postoperative survival times [lncRNA RP11-838N2.4 enhances the cytotoxic effects of TMZ in vitro and in vivo [lncRNA RP11-838N2.4 displayed low levels of miR-10a [lncRNA RP11-838N2.4 overexpression hindered the TGF-β pathway independent of miR-10a by reducing mRNA and protein levels of TGF-β1, TGFBR1, SMAD2, SMAD3, and SMAD4 levels [LncRNA RP11-838N2.4 hinders GBM proliferation and promotes TMZ sensitivity and TMZ-mediated apoptosis by sponging miR-10a and stabilizing EphA8. In addition, it downregulates the TGF-β pathway by reducing the expression of the signalling pathway’s components. However, the exact molecular mechanism of lncRNA RP11-838N2.4-mediated down-regulation of the TGF-β pathway needs further investigation.7, U251) . Moreoveal times . Overexp in vivo . Consequ miR-10a . The lnc miR-10a . Notably4 levels . LncRNA CircARID1A was highly abundant in GBM samples and GBM cell lines and was also present in the blood exosomes of GBM patients [circARID1A attenuated the migration and invasion of GBM cells [MMP 2, MMP 9, and MMP 14 were downregulated upon circARID1A knockdown, indicating that circARID1A promotes GBM cell migration and invasion [cicrARID1A interacts with miR-370-3p [circARID1A and miR-370-3p in the cytoplasm [miR-370-3p binding sites of circARID1A, co-transfected with miR-370-3p mimics, significantly reduced the luciferase reporter activity [cicrARID1A and miR-370-3p [miR-370-3p expression is downregulated in GBM tissue compared to normal brain tissues [TGFBR2 is an essential target of miR-370-3p [miR-370-3p in U87 cells showed enrichment of TGFBR2 and circARID1A, indicating the possibility of circARID1A/miR-370-3p/TGFBR2 axis in GBM [miR-370-3p increased the invasion of GBM cells [circARID1A attenuated TGFBR2 levels [miR-370-3p inhibition promoted TGFBR2 protein levels, which was reversed upon combined knockdown of miR-370-3p and circARID1A [miR-370-3p inhibition, which was reversed upon combined knockdown of miR-370-3p and circARID1A [circARID1A knockdown. Thus, circARID1A promotes GBM invasion by sponging miR-370-3p to stabilize TGFBR2 [Li et al., using microarray analysis, identified differentially expressed circular RNAs in GBM tumour tissues . CircARIpatients . shRNA-mBM cells . Also, Minvasion . BioinfoR-370-3p . FISH exytoplasm . Dual luactivity . These rR-370-3p . miR-370 tissues . BioinfoR-370-3p . RNA puls in GBM . FurtherBM cells . Also, t2 levels . miR-370rcARID1A . GBM celrcARID1A . Furthere TGFBR2 . Chen et al. performed circular RNA sequencing in LN229 and T98G GBM cells and identified several differentially expressed circular RNAs. KEGG and gene ontology analysis of the top-upregulated circular RNAs revealed that TGF-β is a vital pathway regulated by the top-candidate circular RNAs . FurtherLRRC4) is a tumour suppressor in GBM [LRCC4 promoted the generation of a circular RNA, circCD44, from the CD44 mRNA by inhibiting the interaction of CD44 pre-mRNA and SAM68 [CircCD44 expression is downregulated in GBM tissues and cell lines [circCD44 attenuated the proliferation, colony formation, and invasion of GBM cells [circCD44 [SMAD6 is an essential target of miR-326 and miR-330-5p [circCD44 sponges miR-326 and miR-330-5p to stabilize SMAD6 [LRRC4/SAM68/circCD44/miR-326/miR-330-5p/SMAD6 signalling axis is an essential regulator of GBM pathogenesis [Leucine-rich repeat-containing 4  was carried out in patients with renal-cell carcinoma, hepatocellular carcinoma, melanoma, lung cancer, and gastrointestinal stromal tumours [Many ncRNAs are in clinical trials for potential biomarkers and therapeutic targets for cancers and other diseases. LncRNA y cancer , and lncarcinoma . Resultsarcinoma . Andes-1arcinoma . A phase tumours ,109. The tumours . ASO-loa tumours . CTGF, ZEB1, and SNAIL1, are reported to promote TMZ resistance [H19, HOXD-AS2, lncRNA-MUF, LINC01711, lncRNA RP11-838N2.4, miR-210-3p contribute to TMZ resistance [TGF-β pathway promotes TMZ resistance in GBM . Severalsistance ,32. Manysistance ,96,100. PI3K/AKT/mTOR pathways, promote GBM pathogenesis. Specific lncRNAs and miRNAs establish a link between TGF-β and these non-canonical signalling pathways. For example, lncRNA-ATB, induced by TGF-β, promotes GBM invasion through the NF-κB and P38/MAPK pathways [miR-182 suppresses CYLD and promotes sustained activation of NF-κB in GBM [miR-148a oncogene. miR-148a, upon induction, promotes the GBM pathogenesis by activating the TGF-β signalling by promoting the expression of pSMAD3 and downregulating the negative regulators (QKI and SKI) of the TGF-β signalling [Furthermore, the non-canonical TGF-β downstream targets, such as NF-κB and pathways . Also, TB in GBM . SimilarmiR-129-2, lncRNAs AF086127, AF086217, AF086391, AF119852, AK021535, AK022370, AL050068, BC012548, and BC041658 occurs in DIPG [Most studies on the TGF-β modulating lncRNAs and circRNAs in GBM have focused on their ability to function as ceRNAs to sponge miRNAs. However, lncRNAs and circRNAs also function by interacting with proteins ,59. Furt in DIPG . HoweverIn summary, tumour-promoting ncRNAs involved in the TGF-β pathway have the potential to serve as attractive biomarkers and therapeutic targets for GBM.


Correction:  Harm Reduction Journal (2023) 20:3https://doi.org/10.1186/s12954-022-00716-3Following publication of the original article , the affThe original article has been corrected.


Correction: Mol Cancer 19, 25 (2020)10.1186/s12943-020-1143-7Following publication of the original article , the autBelow is the link to the electronic supplementary material.Additional file 2: Figure S2. Overexpressing SOX2 significantly reversed BCSC proliferation inhibition induced by silencing SOX2OT. a and b: Overexpressing SOX2 significantly reversed BCSC proliferation inhibition induced by silencing SOX2OT.


MET-Gen , bestehend aus 21 Exonen, ist auf Chromosom 7q21-31 lokalisiert und codiert die MET-Rezeptortyrosinkinase (180 kDa), welche zusammen mit seinem Liganden, dem „hepatocyte growth factor“ (HGF) eine wichtige Rolle in der Tumorproliferation, Angiogenese und Migration spielt [MET-Aberrationen, die zur Dysregulation des MET-Onkogens und somit zur Aktivierung verschiedener Signalwege wie MAPK, PI3K-AKT und JAK-STAT führen, wurden bereits beschrieben. Zu diesen zählen MET-Überexpression, MET-Genamplifikationen, MET-Mutationen einschließlich Exon-14-Skipping-Mutationen sowie MET-Fusionen , die sowohl als primäre Treiberaberration als auch als Resistenzmechanismus gegenüber anderen Kinaseinhibitoren auftreten, befinden sich zurzeit in zahlreichen Studien. Hierbei zeigen sich MET-Inhibitoren besonders wirksam bei hochamplifizierten („high-level“) Tumoren [MET-Amplifikation befinden sich auch Tumoren mit MET-Fusionen in klinischen Studien. Die Frequenz der MET-Fusionen ist insbesondere in NSCLC allerdings sehr gering  und die Fusionspartner sind sehr heterogen, wie z. B. TPR, CD74 und KIF5B. Ausnahme sind die Glioblastome, wo MET-Fusionen in 12 % der Fälle beschrieben sind -Verfahren angewandt. Einzelgenanalysen wie Sanger-Sequenzierung oder RT-PCR können diese Aberrationen zwar nachweisen, werden aufgrund der Vielzahl von Biomarkern, die getestet werden müssen, und der geringen Materialverfügbarkeit immer seltener angewendet. Auf DNA-Ebene kann die Mutation nach der HGVS-Nomenklatur spezifiziert werden, allerding kann der Splicingeffekt nur auf RNA-Ebene nachgewiesen werden. Zudem haben Studien gezeigt, dass die Analyse mittels amplikonbasierter NGS-Verfahren auf DNA-Ebene zu falsch negativen Ergebnissen führen kann, wenn die Mutation an der Primer-Bindungsstelle auftritt [auftritt .MET-Amplifikationen ist immer noch die Fluoreszenz-in-situ-Hybridisierung (FISH), die zurzeit DNA-basierten Verfahren immer noch überlegen ist. Zweifarbige FISH-Sonden markieren das MET-Gen und das Zentromer von Chromosom 7 (Cen7). Anhand von Auswertekriterien wie dem MET/CEN7-Verhältnis und/oder der durchschnittlichen MET-Genkopienzahl werden die Fälle in niedrig, intermediär und hochamplifiziert eingeteilt. Der Nachweis von MET-Amplifikationen bzw. Genkopienzahlveränderungen mittels DNA-basierten Verfahren befindet sich noch in der Evaluierung. Bisherige Studien haben gezeigt, dass ein geringer Tumorzellgehalt, Tumorheterogenität, fokale Amplifikationen, amplikonbasierte NGS-Verfahren und ctDNA anstelle von DNA ein Problem bei der Auswertung darstellen [Der Goldstandard für die Detektion von rstellen , 7.MET-Fusionen ist ein hybridisierungsbasiertes oder ein anchored-multiplex-PCR-basiertes NGS-Verfahren auf RNA-Ebene die erste Wahl [Für den Nachweis von ste Wahl . Hiermitste Wahl .MET-Aberrationen in NSCLC und anderen Karzinomen die wichtige Rolle dieser Kinase in der zielgerichteten Therapie und die Notwendigkeit einer flächendeckenden und qualitätsgesicherten molekularen Diagnostik.Zusammenfassend zeigt die Vielzahl an


Plant cells detect potential pathogens through plasma membrane-localized pattern recognition receptors (PRRs) that recognize microbe-associated molecular patterns (MAMPs) and activate pattern-triggered immunity (PTI). PRR-mediated MAMP perception is linked to PTI signaling by receptor-like cytoplasmic kinases (RLCKs). In tomato, Flagellin-sensing 2 (Fls2)/Fls3 interacting RLCK 1 (Fir1) is involved in PTI triggered by flagellin perception. Fir1 is necessary for regulation of jasmonic acid (JA) signaling and is involved in pre-invasion immunity. We show that Fir1 physically interacts with JASMONATE-ZIM-DOMAIN PROTEIN 3 (JAZ3), a negative regulator of JA signaling.This finding suggests that Fir1 modulates JA signaling by regulating JAZ3. Solanum lycopersicum), the PRR repertoire includes the Flagellin sensing 2 (Fls2)  and Flagellin sensing 3 (Fls3)  receptors, which recognize the bacterial flagellin-derived peptides flg22 and flgII-28, respectively. Binding of flg22 and flgII-28 by Fls2 and Fls3 triggers signaling pathways that initiate a battery of immune responses, known as pattern-triggered immunity (PTI) (Liang and Zhou 2018). The output of PTI often consists of generation of reactive oxygen species (ROS), gene transcription reprogramming, activation of mitogen-activated protein kinases (MAPKs), closure of stomata, and the accumulation of antimicrobial proteins .The plant immune system is equipped with plasma membrane-localized pattern recognition receptors (PRRs) that mediate early detection of potential pathogens by recognizing conserved microbe-associated molecular patterns (MAMPs) . In tomato plants  RESPIRATORY BURST OXIDASE HOMOLOG D (RBOHD), which is involved in ROS production, is modulated through phosphorylation by several RLCKs, including RPM1-INDUCED PROTEIN KINASE (RIPK) , BOTRYTIS-INDUCED KINASE1 (BIK1) , and avrPphB SUSCEPTIBLE 1-LIKE 13 (PBL13) . The ion channels SLOW ANION CHANNEL-ASSOCIATED 1 HOMOLOG 3 (SLAH3) and REDUCED-HYPEROSMOLALITY-INDUCED-[Ca2+] INCREASE 1.3 (OSCA1.3), are directly phosphorylated by avrPphB SUSCEPTIBLE 1-LIKE 27 (PBL27) and BIK1, respectively, to generate an ion flow that leads to stomatal closure . Immunity-associated MAPKs serve as direct substrates of several RLCKs, including PBL27 , OsRLCK185 , BRASSINOSTEROID-SIGNALING KINASE 1 (BSK1) , and members of the RLCK subfamily VII . Finally, BIK1 directly interacts in the nucleus with WRKY transcription factors to modulate gene expression associated with the jasmonic acid (JA) and salicylic acid (SA) defense hormones .In several instances, receptor-like cytoplasmic kinases (RLCKs) have been found to link PRR-mediated MAMP perception to downstream signaling pathways (Liang and Zhou 2018). RLCKs interact with and modulate the activity of a variety of proteins that participate in branches of PTI signaling. For example, activity of the Arabidopsis  DC3000. In addition, Fir1 appears to participate in pre-invasion immunity and stomatal closure. Consistent with its interaction with Fls2 and Fls3, Fir1 is required for flagellin-induced ROS production andPATHOGENESIS-RELATED 1bmRNA expression. Analysis of gene expression profiles offir1 mutant plants upon PTI activation revealed a deregulated JA response. In line with this finding,fir1 loss-of-function mutations diminished the contribution of coronatine to virulence ofPstbacteria, and enhanced resistance toBotrytis cinerea, a necrotrophic fungus, after activation of PTI. These findings suggest that Fir1 is necessary for the appropriate activation of PTI and support a model in which Fir1 regulates JA signaling to inhibit a JA response .We recently investigated the role of the tomato Fls2/3 interacting RLCK 1 (Fir1) in PTI . Fir1 was found to interact with Fls2 and Fls3, suggesting that it plays a role as an early signaling component of PTI activated by flagellin perception. Phenotypic analyses revealed that a loss-of-function mutation of thefir1-1mutant plants during activation of PTI  . In addition, we tested the interaction of Fir1 and the JA receptor JASMONIC ACID–INSENSITIVE1(JAI1) , and FER1 (homolog of Arabidopsis FERONIA), which inhibits coronatine-induced signaling to promote disease resistance . In these experiments, Fir1 was fused to the C-terminal fragment of the luciferase protein (C-LUC) and co-expressed viaAgrobacteriumtumefaciensinNicotiana benthamianaleaves with a candidate interactor fused to the N-terminal fragment of luciferase (N-LUC). As negative controls, C-LUC-Fir1 was co-expressed with either an empty N-LUC vector or with N-LUC fused to the green fluorescent protein (GFP). The luminescence emitted from leaf discs was measured 48 hours after agro-infiltration and used to quantify interactions. Leaf disks co-expressing Fir1 and JAZ3, but not any of the other protein combinations, emitted a significantly higher luminescence than the negative controls . Western blot analysis was used to validate that each fusion protein was expressed in the inoculated plant tissues .We hypothesized that Fir1 modulates the JA response by interacting with components of the JA signaling pathway. To assess this hypothesis, a split-luciferase complementation assay was employed to test the interaction between Fir1 and members of the JASMONATE-ZIM-DOMAIN (JAZ) protein family, which negatively regulate JA signaling (Pauwels and Goossens 2011), and whose transcripts were differentially expressed inin vitropull-down assay to confirm the interaction between Fir1 and JAZ3. Fir1 fused to the glutathione-S-transferase (GST) protein was expressed inE. coliand purified. Lysates of bacterial cells expressing His-tagged JAZ3 and JAZ7 (negative control) were incubated with immobilized GST-Fir1 or GST (negative control) and washed. Western blot analysis with anti-His antibodies detected His-JAZ3, but not His-JAZ7 among the proteins pulled-down by GST-Fir1 , suggesting a direct interaction between Fir1 and JAZ3.We used anPsttype III secreted effector HopBB1 interacts with the TCP14 transcription factor, a repressor of a subset of JA responses. To activate JA response genes, and consequently increase pathogen virulence, HopBB1 reduces TCP14 abundance by coupling it with JAZ3, which sends it to degradation in the SCFCOIdegradation complex . The HaRxL10 effector protein from the oomycete pathogenHyaloperonospora arabidopsidis (Hpa) targets JAZ3 to decrease its abundance. The reduction in JAZ3 abundance activates JA responses, and suppresses SA responses, which normally restrictHpa growth . Together, the results indicate that Fir1 interacts in planta andin vitro with JAZ3, which represents a candidate signaling component regulated by Fir1.JAZ3 is a negative regulator of JA signaling (Pauwels and Goossens 2011) and it has been shown to be targeted by pathogen effector proteins . For example, theBacterial strains, plant materials, and growth conditionsNicotiana benthamianaplants were cultivated at 25°C in a phytochamber under long-day conditions . The bacterial strains used were:Escherichia coliDH5a (Invitrogen),E. coliRosetta (Novagen), andAgrobacterium tumefaciensGV2260 .E. coliwas grown in Lysogeny broth (LB) medium at 37°C andA. tumefaciensin LB medium at 30°C with the required antibiotics.Split-luciferase complementation assayA. tumefaciensGV2260 bacteria, which were infiltrated intoN. benthamiana leaves for protein co-expression. Split-luciferase complementation assays were carried out as described .Genes encoding JAZ2, JAZ3, JAZ7, JAZ9, FER1, and JAI1 were PCR-amplified from tomato cDNA with primers 1-2 (JAZ2), 3-4 (JAZ3), 5-6 (JAZ7), 7-8 (JAZ9), 9-10 (FER1), or 11-12 (JAI1) (Table 1), and cloned in frame with N-LUC in the pCAMBIA1300:N-LUC plasmid . pCAMBIA1300:N-LUC carrying a GFP-N-LUC fusion and pCAMBIA1300:C-LUC carrying a C-LUC-Fir1 fusion were prepared as described by Sobol et al. (2022). The obtained vectors were transformed intoIn vitro pull-down assayE. coli strain andin vitro pull-down assays were carried out as described .Tomato cDNA was used to PCR-amplify genes encoding JAZ3 and JAZ7 using primers 15-16 (JAZ3), or 17-18 (JAZ7) (Table 1). JAZ3 and JAZ7 were cloned downstream and in frame to a 10xHis-tag in the pET-16b vector (Novagen). The pGEX-4T1 vector carrying Fir1 fused to GST (GST-Fir1) was prepared as described by Sobol et al. (2022). Plasmids carrying His-JAZ3, His-JAZ7, GST, and GST-Fir1 were transformed into the RosettaAccession numbershttps://solgenomics.net/) under the following accession numbers: Fir1 (Solyc12g099830),JAZ2(Solyc12g009220),JAZ3(Solyc03g122190),JAZ7(Solyc11g011030),JAZ9(Solyc08g036640),FER1(Solyc09g015830),JAI1(Solyc05g052620).Sequence data used in this article can be located in the Solanaceae Genomics Network database (Table 1. Commercial products used in this study.Table 2.Primers used in this study.Table 3. Plasmids used in this study