C) serves as a docking platform for export substrates and flagellar chaperones and plays an important role in hierarchical protein targeting and export. FlhAC consists of domains D1, D2, D3, and D4 and adopts open and closed conformations. Gly-368 of Salmonella FlhA is located within the highly conserved GYXLI motif and is critical for the dynamic domain motions of FlhAC. However, it remains unclear how it works. Here, we report that periodic conformational changes of the GYXLI motif induce a remodeling of hydrophobic side chain interaction networks in FlhAC and promote the cyclic open-close domain motions of FlhAC. The temperature-sensitive flhA(G368C) mutation stabilized a completely closed conformation at 42°C through strong hydrophobic interactions between Gln-498 of domain D1 and Pro-667 of domain D4 and between Phe-459 of domain D2 and Pro-646 of domain D4, thereby inhibiting flagellar protein export by the fT3SS. Its intragenic suppressor mutations reorganized the hydrophobic interaction networks in the closed FlhAC structure, restoring the protein export activity of the fT3SS to a significant degree. Furthermore, the conformational flexibility of the GYXLI motif was critical for flagellar protein export. We propose that the conserved GYXLI motif acts as a structural switch to induce the dynamic domain motions of FlhAC required for efficient and rapid protein export by the fT3SS.Flagellar structural subunits are transported via the flagellar type III secretion system (fT3SS) and assemble at the distal end of the growing flagellar structure. The C-terminal cytoplasmic domain of FlhA (FlhAIMPORTANCE Many motile bacteria employ the flagellar type III secretion system (fT3SS) to construct flagella beyond the cytoplasmic membrane. The C-terminal cytoplasmic domain of FlhA (FlhAC), a transmembrane subunit of the fT3SS, provides binding sites for export substrates and flagellar export chaperones to coordinate flagellar protein export with assembly. FlhAC undergoes cyclic open-close domain motions. The highly conserved Gly-368 residue of FlhA is postulated to be critical for dynamic domain motions of FlhAC. However, it remains unknown how it works. Here, we carried out mutational analysis of FlhAC combined with molecular dynamics simulation and provide evidence that the conformational flexibility of FlhAC by Gly-368 is important for remodeling hydrophobic side chain interaction networks in FlhAC to facilitate its cyclic open-close domain motions, allowing the fT3SS to transport flagellar structural subunits for efficient and rapid flagellar assembly. Salmonella enterica serovar Typhimurium  is divided into three structural parts: the basal body, which acts as a bi-directional rotary motor, the filament, which functions as a helical propeller to produce thrust, and the hook, which is a universal joint connecting the basal body and filament and transmits torque produced by the motor to the filament. Flagellar assembly begins with the basal body, followed by the hook and finally the filament. To construct the flagellum on the cell surface, the flagellar type III secretion system (fT3SS) transports flagellar structural subunits from the cytoplasm to the distal end of the growing flagellar structure  as the energy source to unfold and translocate export substrates across the cytoplasmic membrane . In addition, FlgN, FliS, and FliT act as export chaperones that escort their cognate substrates from the cytoplasm to the fT3SS ocation 68. The fT. The fT+properly .Salmonella FlhA is composed of an N-terminal transmembrane domain with eight transmembrane helices , a compactly folded cytoplasmic domain , and a flexible linker  connecting these two domains  and adopts open and closed conformations , the chaperone-substrate complexes can bind to the conserved hydrophobic dimple in the open form but not in the closed form , allowing the chaperone-substrate complexes to efficiently bind to the FlhAC ring  mutation inhibits the protein transport activity of the fT3SS at a restrictive temperature of 42°C but not at a permissive temperature of 30°C (–flhA(G368C) mutation. Molecular dynamics (MD) simulation has shown that the flhA(G368C) mutation restricts cyclic open-close domain motions of FlhAC at 42°C and stabilizes a completely closed conformation of FlhAC (C coupled with flagellar protein export.The  of 30°C –29. The of FlhAC . Gly-368of FlhAC . This leC combined with MD simulation. We provide evidence that the flhA(G368C) mutation stabilizes hydrophobic side chain interactions between domains D1 and D3 and those between domains D2 and D4, thereby suppressing dynamic open-close domain motions of FlhAC, and that its intragenic suppressor mutations induce the remodeling of the hydrophobic interaction networks in FlhAC, allowing FlhAC with the G368C mutation (FlhAC-G368C) to restore the dynamic open-close domain motions.To clarify this hypothesis, we carried out mutational analysis of FlhAflhA(G368C) mutation stabilizes a completely closed form of FlhAC at 42°C, we first compared the interfaces between domains D1 and D3 and those between domains D2 and D4 in the open and closed forms of FlhAC-G368C obtained in the previous MD simulation (C-G368C (right panel), we propose that the flhA(G368C) mutation may stabilize these two hydrophobic interactions at 42°C, thereby suppressing cyclic open-close motions of FlhAC.To address why the mulation   mutation in domain D3 does not affect FlhA function at all. However, the combination of the flhA(G368C) and flhA(K548C) mutations results in a loss-of-function phenotype even at 30°C (flhA(G368C/K548C) mutation inhibits flagellar protein export at 30°C, we analyzed the structure and dynamics of FlhAC-G368C/K548C by MD simulation at 27°C for 1.5 μs  between domains D2 and D4 during MD simulation .The highly conserved Lys-548 residue of FlhA is located at the interface between domains D1 and D3 and contributes to D1-D3 interactions  Fig. 1BFig. 1B.  at 30°C . To clarr 1.5 μs . DomainsC-G368C, which changes its conformation back and forth between the closed and open forms at 27°C  mutation restores motility of the flhA(G368C/K548C) mutant to a considerable degree . Gly-368 forms the highly conserved GYXLI motif along with Tyr-369, Arg-370, Leu-371, and Ile-372  mutation, which has been isolated as an intragenic suppressor mutation of the flhA(G368C) mutant grown at 42°C  and Y369G/R370G/L371G/I372G (here referred to as GGGG) mutants and analyzed their motility in 0.35% soft agar. Immunoblotting with polyclonal anti-FlhAC antibody revealed that the AAAA and GGGG mutations did not affect the steady expression level of FlhA (Hydrophobic parts of the side chain of Tyr-369 and Arg-370 make hydrophobic contacts with Pro-415 and Ala-416 in the open form of wild-type FlhA Pro-434 . To clar of FlhA . The AAA of FlhA . In agre of FlhA . The GGG of FlhA . Consist of FlhA . These oflhA(M365I), flhA(A446E), and flhA(P550S) mutations have been reported to restore motility defects in the flhA(G368C) mutant at 42°C (flhA(G368C) mutation stabilizes the completely closed form of FlhAC at 42°C through the strong hydrophobic interactions between Gln-498 and Pro-667 and those between Phe-459 and Pro-646, we next analyzed the effect of the M365I, A446E, and P550S suppressor mutations on the conformational changes of FlhAC-G368C.The intragenic  at 42°C , 28. To C-G368C but not in its open form  mutation induces a conformational change of domain D2 as judged by far-UV circular dichroism (CD) measurements of purified FlhAC with the G368C substitution  mutation may induce hydrophobic side chain interaction networks in FlhAC to stabilize a complete closed form at 42°C and that Leu-413, Pro-415, Pro-434, Phe-459, Gln-477, Leu-492, Gln-498, Lys-548, Pro-646, and Pro-667 may be involved in dynamic open-close domain motions of FlhAC (flhA(G368C) mutation affects the hydrophobic side chain interaction networks in FlhAC, we constructed flhA(G368C) mutants with either L413A, P415A, P434A, F459A, Q477A, L492A, Q498A, K548A, P646A, or P667A substitution and analyzed the motility of these double mutants at both 30°C and 42°C (C antibody (MD simulation of FlhAof FlhAC . To testand 42°C . These dantibody .flhA(G368C) mutant even at 30°C (flhA(G368C) mutant at 42°C (flhA(G368C) mutation affects the hydrophobic side chain interaction networks in FlhAC, thereby inducing conformational changes of domains D1 and D2.The P415A, P434A, Q477A, Q498A, and K548A substitutions reduced the motility of the  at 30°C . Consist at 30°C . Further at 42°C . The P41 at 42°C . TherefoflhA(G368C) mutant at both 30°C and 42°C (flhA(G368C) mutant grown at 30°C  mutant at both 30°C and 42°C (flhA(G368C) mutant grown at 30°C but not the level of FlgD secretion  mutant was much worse than that of the flhA(F459A) mutant (flhA(F459A) mutant  mutant  and 6, w) mutant , thereby) mutant . TherefoflhA(G368C) mutant at both 30°C and 42°C  mutation may limit the hinge movement between domains D1 and D3 through a remodeling of the hydrophobic side chain interaction networks of domain D1, thereby allowing Pro-646 and Pro-667 to make the strong hydrophobic contacts with Phe-459 and Gln-498, respectively  mutation limits the cyclic open-close domain motions of FlhAC at 42°C, thereby reducing the flagellar protein export by the fT3SS and therefore the cell motility  mutation induced a remodeling of hydrophobic side chain interaction networks in FlhAC at 42°C, allowing Gln-498 of domain D1 and Phe-459 of domain D2 to hydrophobically interact with Pro-667 and Pro-646 of domain D4, respectively (flhA(G368C) mutation not only suppresses the dynamic open-close domain motions of FlhAC but also stabilizes the completely closed conformation at 42°C. Intragenic M365I, R370S, A446E, and P550S suppressor mutations, which restore motility defects in the flhA(G368C) mutant to near wild-type levels  mutation  affinity chromatography have revealed that the flhA(G368C) mutation reduces the binding affinity of FlhAC for the chaperone-substrate complex   (C subunits stably when FlhAC adopts the open form in the ring structure (right panel) because FliJ can contact both FlhAC subunits. However, the FliJ binding to FlhAC in the closed form appears to be unstable (left panel) because the distance between the neighboring D4 domains is longer in the closed form (ca. 80.8 Å) than that in the open form (ca. 36.9 Å), restraining the proper contacts of FliJ with two FlhAC subunits (middle panel). Because the open and closed forms of FlhAC represent the active and inactive states of the fT3SS, respectively (C may be important for an efficient and robust energy coupling mechanism of the fT3SS.FliJ binds to FlhAig. S1B) . FliJ alaffinity . It has tructure . This suith FliD . This raD: 6WA9) . FliJ caectively , we propSalmonella strains used in this study are listed in int (flhA mutations were confirmed by DNA sequencing (Eurofins Genomics).The d in int . DNA manFresh colonies were inoculated onto soft agar plates  containing 50 μg/mL ampicillin, and then the plates were incubated at 30°C. At least six measurements were performed.Salmonella cells were grown at 30°C in 5 mL of L-broth  containing 100 μg/mL ampicillin. The cells were attached to a coverslip , and unattached cells were washed away with motility buffer . A 1-μL aliquot of polyclonal anti-FliC serum was mixed with 50 μL of motility buffer, and then 50 μL of the mixture was applied to the cells attached to the cover slip. After washing with the motility buffer, 1 μL of anti-rabbit IgG conjugated with Alexa Fluor 594 (Invitrogen) was added to 50 μL of motility medium, and then the mixture was applied. After washing with the motility buffer, the cells were observed by fluorescence microscopy  of ca. 1.2 to 1.4. Cultures were centrifuged to obtain cell pellets and culture supernatants. The cell pellets were resuspended in sodium dodecyl sulfate (SDS)-loading buffer solution  containing 1 μL of 2-mercaptoethanol. Proteins in the culture supernatants were precipitated by 10% trichloroacetic acid and suspended in a Tris/SDS loading buffer  containing 1 μL of 2-mercaptoethanol  using a transblotting apparatus (Hoefer). Then, immunoblotting with polyclonal anti-FlgD, anti-FlgK, or anti-FlhAC antibody was carried out using an iBind Flex Western device (Thermo Fisher Scientific) as described in the manufacturer’s instructions. Detection was performed with Amersham ECL Prime Western blotting detection reagent (Cytiva). Chemiluminescence signals were captured by a Luminoimage LAS-3000 analyzer (FujiFilm). At least three measurements were performed.oethanol . Both whhttp://www.ebi.ac.uk/Tools/msa/clustalo/).Multiple sequence alignment was carried out using Clustal Omega (C with either G368C/K548C or G368C/F459C/K548C substitution were conducted as described previously (C (b water molecules (MD simulations of FlhAeviously . The mutously (C . The Ambously (C  was usedolecules  and 0.17olecules  with a molecules  of AMBER


All the strains exhibited the typical DS-1-like genotype constellation, and the nucleotide sequences of the 11 genome segments showed high nucleotide similarities (>97%). Phylogenetic analyses together with representative global G2P[4] RVA showed that Zambian strains clustered into human lineages IV , V , and XXIII (for NSP4). The AA differences between the lineages where the study strains clustered and lineages of global reference strains were identified and analyzed. Selection pressure analysis revealed that AA site seven in the Viral Protein 3 (VP3) genome segment was under positive selection. This site occurs in the region of intrinsic disorder in the VP3 protein, and Zambian G2P[4] strains could potentially be utilizing this intrinsically disordered region to survive immune pressure. The Zambian G2P[4] strains from 2012 to 2016 comprised the G2P[4] strains that have been circulating globally since the early 2000s, highlighting the epidemiological fitness of these contemporary G2P[4] strains. Continuous whole-genome surveillance of G2P[4] strains remains imperative to understand their evolution during the post-vaccination period.The G2P[4] genotype is among the rotavirus strains that circulate commonly in humans. Several countries have reported its immediate upsurge after the introduction of rotavirus vaccination, raising concern about sub-optimal vaccine effectiveness against this genotype in the long term. This study aimed to gain insight into the evolution of post-vaccine Zambian G2P[4] group A rotavirus (RVA) strains and their overall genetic make-up by analysis of sequence alignments at the amino acid (AA) level. Twenty-nine Zambian G2P[4] rotavirus strains were subjected to whole-genome sequencing using the Illumina MiSeq Vaccination and, to an extent, improvement in sanitation have substantially reduced RVA disease mortality cases from ~500,000 during the pre-vaccination era to ~128,000 during the post-vaccination period , G3P[8], G9P[8], and G12P[8] typically belong to the Wa-like genogroup ,12, G3P,14, and The G2P[4] genotype has been observed to increase or maintain dominance after the introduction of the monovalent G1P[8] rotavirus vaccine in several countries such as Australia, Belgium, Brazil, Botswana, Japan, Kenya, Malawi, Saudi Arabia, South Africa, and Venezuela ,29,30,31® introduction was reported by surveillance based on conventional VP7 and VP4 genotyping  prevalence period post Rotarixnotyping . Apart fnotyping ,19, therThe Health Sciences Research Ethics Committee (HSREC) at the University of the Free State in Bloemfontein, South Africa, granted ethical permission for this study under ethics number (UFS-HSD2016/1082).n = 133) were obtained from children under the age of five years presenting with acute gastroenteritis as part of the ongoing World Health Organization Regional Office for Africa (WHO-AFRO) RVA surveillance program in Zambia. The stool samples were collected between 2011–2016 and were characterized into G and P types by conventional genotyping methods at the Diarrheal Pathogens Research Unit (DPRU), a WHO Rotavirus Reference Laboratory in South Africa (WHO RRL SA). Briefly, viral dsRNA was extracted from 10% fecal suspension using the QIAmp viral RNA extraction method . The extracted RNA was reverse transcribed and amplified using consensus primer pairs Con2/Con3 and sBeg/End9 as described previously  RVA strains undergoing negative and positive selection, we employed three evolutionary analyses method available from the DataMonkey webserver . These aAll the 29 study strains had a pure DS-1-like genotype constellation (G2-P[4]-I2-R2-C2-M2-A2-N2-T2-E2-H2). The genome segments from the 29 G2P[4] strains showed nucleotide sequence identities of >97% amongst each other .Phylogenetic analyses together with representative global G2P[4] RVAs showed that Zambian strains clustered into human lineages IV , V , and XXIII (for NSP4) .Zambian G2 sequences clustered further into sub-lineage IVa_3 . The AA We identified AA substitutions  between P[4] strains in lineages I, II, and III in respect to the P[4] strains in lineage IV, where the study P[4] sequences clustered . Within Amino acid position 159 of lineage V of R2 sequences where Zambian strains clustered featured an arginine, while other R2 lineages had a lysine . Three AIn the genome segment encoding NSP1, three AA differences were observed between the lineages I–II, V, and the lineage IV of the study strains. These differences are represented by l273F, H319R, and K429R, as shown in The AA sites in the genome segments of Zambian G2P[4] strains were undergoing purifying selection with the exception of the AA site seven in the VP3 gene, which was under diversifying/positive selection .This site was found to have two AA: isoleucine occurring in ~40% of the strains and arginine in ~60% of the strains .The study reports on whole-genome analysis of 29 G2P[4] RVA strains collected from Zambia from 2011 to 2016. All the Zambian G2P[4] gene sequences exhibited a pure DS-1-like constellation, high nucleotide similarities (>97%), and clustered alongside contemporary human G2P[4] lineages. In essence, Zambian G2P[4] strains emerged independent of interspecies-transmission events, suggesting they were of human origin. These findings are consistent with whole-genome studies of G2P[4] studies in several countries, whereby the reported G2P[4] strains evolved devoid of reassortment with animal genes ,44,52,53The Zambian G2 sequences, which were all post-vaccine, clustered into sub-lineage IVa_3, which is relatable to a South African study wherein post-vaccine G2 strains clustered in sub-lineage IVa_3, while pre-vaccine strains clustered in sub-lineage IVa_1 . In thatAccording to the literature, the isoleucine substitution for valine residue is suggested to shift the equilibrium to a more compact conformation . TherefoMost AA sites were undergoing purifying selection, probably as a strategy to purge deleterious polymorphisms that arise due to the error-prone nature of the RNA polymerase enzyme . HoweverOur analysis would have been more enriched with analysis performed in the context of pre-vaccination Zambian G2P[4] samples. However, natural fluctuations are known to impact the prevalence of circulating rotavirus strains , and samThe G2P[4] strains circulating in Zambia from 2012 to 2016 belong to the lineages of G2P[4] strains that have been circulating globally since the early 2000s, highlighting the epidemiological fitness of these contemporary strains. The analysis of AA substitutions defining the lineages of the circulating G2P[4] strains provides an insightful perspective to understanding their evolution.


EC50s were higher with E23K than with E23G and increased dramatically (138- to 446-fold) when these substitutions were combined with PA I38T, the dominant BXA resistance marker. E23G/K-substituted viruses exhibited slightly impaired replication in MDCK and Calu-3 cells, which was more pronounced with E23K. In ferret transmission experiments, all viruses transmitted to direct-contact and airborne-transmission animals, with only E23K+I38T viruses failing to infect 100% of animals by airborne transmission. E23G/K genotypes were predominantly stable during transmission events and through five passages in vitro. Thermostable PA–BXA interactions were weakened by E23G/K substitutions and further weakened when combined with I38T. In silico modeling indicated this was caused by E23G/K altering the placement of functionally important Tyr24 in the endonuclease domain, potentially decreasing BXA binding but at some cost to the virus. These data implicate E23G/K, alone or combined with I38T, as important markers of reduced BXM susceptibility, and such mutants could emerge and/or transmit among humans.Baloxavir marboxil (BXM) is approved for treating uncomplicated influenza. The active metabolite baloxavir acid (BXA) inhibits cap-dependent endonuclease activity of the influenza virus polymerase acidic protein (PA), which is necessary for viral transcription. Treatment-emergent E23G or E23K (E23G/K) PA substitutions have been implicated in reduced BXA susceptibility, but their effect on virus fitness and transmissibility, their synergism with other BXA resistance markers, and the mechanisms of resistance have been insufficiently studied. Accordingly, we generated point mutants of circulating seasonal influenza A(H1N1)pdm09 and A(H3N2) viruses carrying E23G/K substitutions. Both substitutions caused 2- to 13-fold increases in the BXA EC Baloxavir is a new and potent anti-influenza drug targeting essential functions of viral replication. Currently, the I38T polymerase acidic protein (PA) substitution is the major marker of reduced susceptibility and potential resistance to baloxavir, but the full baloxavir resistance profile remains unclear. Here, we demonstrated that PA E23G/K substitutions alone weaken baloxavir efficacy, but they also synergize with I38T to impair drug activity further. E23G/K substitutions are located close to the binding site of baloxavir and indirectly weaken key drug-binding interactions. This effect has some negative consequences for virus replication, but E23G/K viruses possess the capacity for airborne spread between naïve ferrets, the gold-standard model of human influenza transmission. Therefore, E23G/K viruses have the potential for community spread, which would adversely affect baloxavir clinical implementation. Our study supports ongoing surveillance for circulating human E23G/K viruses, and it may inform design of enhanced baloxavir-like drugs less susceptible to emergence of viral resistance. Influenza A and B viruses are highly contagious respiratory pathogens that cause annual epidemics and occasional pandemics. Vaccines are effective preventative measures, but antiviral agents play an important adjunct role in influenza treatment and control. The two clinically approved classes of influenza antivirals are the neuraminidase (NA) inhibitors (NAIs), which target the viral NA protein and inhibit virus budding, and a polymerase acidic protein (PA) endonuclease inhibitor that targets the PA protein and viral transcription. Four NAIs have been approved in various countries , whereasN). PA residue 38 is a hotspot for substitutions (I38T/M/F/L/N/S), and I38T or I38X mixtures were identified early in laboratory studies , reaching only 62% activity of the WT. While the same substitutions conferred only 87% the activity of WT in the A(H3N2) subtype, this was not statistically significant pdm09 (40–74% activity inhibited) (P ≤ 0.0001] in A(H3N2) (57% activity inhibited), and E23K activity was 14% inhibited, though the latter was not statistically significant  . E23G wanificant . E23G+I3nificant . In A by plaque reduction assays pdm09] and A/Texas/71/2017 [(A(H3N2)] viruses with E23G/K or dual E23G/K+I38T substitutions and determined 50% effective concentrations pdm09 virus 0.22 nM, while the EC50 value of the A(H3N2) virus was 0.90 nM. The dominant BXA resistance marker I38T yielded EC50 values of 18.30 nM and 69.83 nM for A(H1N1)pdm09 and A(H3N2) (a >78-fold increase relative to WT) respectively, and consistent with previous reports . From 48 hpi onward, there was no significant difference between the titers of E23G-containing viruses and those of WT, whereas the titers of E23K viruses again lagged pdm09 viruses with E23K substitution alone or in combination with I38T produced plaques 31%–46% smaller than those produced by viruses with E23G pdm09 viruses, all substitutions resulted in replication being slower than that of WT virus until 48–72 hours post infection (hpi) .Replication capacity of E23G/K and E23G/K+I38T viruses were also determined human bronchial epithelia-derived Calu-3 cells that support influenza virus replication and share characteristics of the human proximal lower airway. Similar to MDCKs, E23G-containing viruses seldom exhibited titers lower that WT past 24 hpi. In contrast, E23K-containing viruses often produced statistically lower titers than WT, as late as 96 hpi  ) for 1 h, washed, and overlaid with 0.45% immunodiffusion-grade agarose  in infection medium containing BXA (0 or 1 pM–1.5 μM). At 72 hpi, PFU/well were enumerated, and EC50s were determined with GraphPad Prism (log inhibitor vs. response logistic nonlinear regression). Separately, under no drug pressure, plaque monolayers (n = 6 wells) were prepared, and ≥87individual PFU/assay were sized by ImageJ (NIH).MDCKs (1×1050) values determined by Reed/Meunch method ×[Glide_emodel]), and Max._score. Several predicted binding modes (poses) were output; poses distant from the original BXA binding mode were excluded. Consequently, each score was calculated as the mean and median values. Docking scores were converted to + scale by ×(−1); higher scores corresponded to high activity and vice versa. Resulting scores and QSAR equations based on EC50 values are provided (Maestro software (Schrödinger) was used to perform preprocessing and energy minimization for ligand–protein complexes  in GraphPad Prism_v9. Replicates, group comparisons, and P values are listed in each figure legend.Data were analyzed using unpaired S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S1 File(XLSX)Click here for additional data file.


UBASH3A as a negative regulator of T cell receptors (TCRs) signaling is a susceptible factor in RA. The aim of this study was to determine the role of UBASH3A in RA pathogenesis, by assessing the role of super-enhancer (SE) in the control of UBASH3A expression in CD4UBASH3A loci displaying SE architecture. CD4+ T cells were transfected with UBASH3A plasmids, and cytokine levels were measured by a cytometric bead array.UBASH3A mRNA and protein levels were quantified by PCR and western blotting, respectively. The cells were treated with a locked nucleic acid to inhibit enhancer RNA (eRNA) expression. Chromatin immunoprecipitation was used to identify the factors recruited to UBASH3A was extracted as a RA susceptibility gene associated with SNPs in the SEs that are highly expressed in CD4+ T cells by in silico screening. UBASH3A mRNA and protein expression levels were lower in CD4+ T cells of RA patients than in the control. eRNA_1 and eRNA_3 knockdown reduced UBASH3A mRNA levels. RA patients exhibited accumulation of BTB and CNC homology 2 (BACH2), the silencing transcription factor, at the UBASH3A loci in CD4+ T cells, but not the SE-defining factor, mediator complex subunit 1 (MED1)/bromodomain 4 (BRD4). However, opposite changes were observed in the control. Stimulation of TCRs expressed on CD4+ T cells of RA patients resulted in interleukin (IL)-6 production, while UBASH3A over-expression significantly inhibited the production.UBASH3A is suppressed via epigenetic regulation of SE in CD4+ T cells. Low UBASH3A levels result in excessive TCR signal activation with subsequent enhancement of IL-6 production.In RA, transcription of The online version contains supplementary material available at 10.1186/s41232-022-00231-9. Rheumatoid arthritis (RA) is a systemic autoimmune disease characterized by synovitis and progressive joint destruction –3. The p+ T cells  and of 24 RA patients , with clinical disease activity index (CDAI) of 136±49, simplified disease activity index for rheumatoid arthritis (SDAI) 140±51, and disease activity score-28 (DAS28) of 6.1±1.2. The T cells were prepared using CD4+ microbeads of the CD4+ T Cell Isolation kit  (Supplementary Table + T cells were sorted into subsets by fluorescence-activated cell sorting (FACS) gating. Cells were then seeded onto six-well plates at a density of 1 × 106 cells/well and incubated in Roswell Park Memorial Institute (RPMI) 1640 medium containing 10% fetal bovine serum and 1× antibiotics at 37°C under 5% CO2 atmosphere. The positive control Jurkat cells were treated with anti-CD28 (PV1) or anti-CD3 (2C11) for 6 h to activate TCR signaling [CD4eviously . First, ignaling .+ T cells were prepared from PBMC of 3 healthy donors  and 3 patients with RA . We also used CD4+ T cell line Jurkat as a positive control. Immunoblotting was performed as described previously [CD4eviously . Briefly+ T cells of 3 RA patients  and 3 healthy donors  was collected 24 h after transfection of the cells with plasmid cloning DNA (pcDNA) 3.1-UBASH3A (courtesy of Professor Patrick Concannon) and empty vector. Next, 20 μL of the supernatant was reacted with 0.2 μL of capture beads of the Human Flex Set  and 19.8 μL of RPMI medium for 1 h at room temperature. Then, 0.2 μL of phycoerythrin detection reagent and 19.8 μL of RPMI medium were added, and the mixture was allowed to react for 1 h. The supernatant was rinsed twice with flow cytometry staining (FACS) buffer, centrifuged, and suspended in 150 μL of FACS buffer. Finally, each cytokine was detected by FACS, and its concentration was analyzed by FCAP Array software.CBA was performed as described previously . Briefly+ T cells of 3 RA patients  and 3 healthy donors  using the RNeasy Mini kit  and reverse-transcribed using SuperScript®VILO™ Master Mix (Life Technologies). On a Step One Plus system (Applied Biosystems), qPCR was performed with TaqMan®Fast Universal PCR Master Mix (Applied Biosystems) and the TaqMan® Gene Expression Assay (Applied Biosystems) primer/probe pairs to measure the expression of the following genes , IL-6, IL-17A, tumor necrosis factor-alpha (TNF-α), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH).RT-qPCR was performed as described previously . BrieflyA single patient with RA and a single patient with dermatomyositis, who met the diagnostic criteria of the two conditions, consented to provide lymph node samples. This study was conducted with the approval of the ethics committee of the University of Occupational and Environmental Health. Lymph node tissue samples were collected, fixed in 10% formaldehyde, and embedded in paraffin. Immunofluorescence staining was performed as described previously . Briefly+ T cells collected from 5 healthy donors  and 5 RA patients  were cultured in RPMI 1640 medium. Chromatin was cross-linked with 1% formaldehyde for 10 min and fragmented to 200–500 bp by sonication. DNA was extracted using the EZ ChIP Kit  using the protocol provided by the manufacturer. DNA was immunoprecipitated using a series of antibodies. PCR was performed using specific primers (Supplementary Table ChIP-PCR was performed as described previously . CD4+ T + T cells (1 × 105 cells) collected from 3 healthy donors  and 3 RA patients  using Lipofectamine 3000 and P3000 reagents . In contrast, locked nucleic acid (LNA) was designed to knock down the expression of eRNA (Supplementary Table + T cells (1 × 105 cells) with RNAi MAX (Thermo Fisher Scientific) reagents for 72 h, using the protocol provided by the manufacturer.Following the instructions provided by the manufacturer, we transfected pcDNA3.1-UBASH3A and empty vector into CD4SNP-PCR was performed according to the instructions supplied by the manufacturer to determine the genotypes of rs1893592-A/C, as described previously . On a Stt test or Dunnett’s multiple comparison test. Spearman’s test was used for the correlation analysis between two variables of interest. The statistical significance was set at P<0.05. All statistical analyses were performed using SPSS statistical software .All quantified data are expressed as mean ± standard deviation. Differences between the two groups were tested for statistical significance by the Student’s unpaired two-tailed + T cells  registered in the dbSUPER, and the SNP (rs1893592) with RA-susceptibility identified by the GWAS report [UBASH3A locus  since SE activity is dependent on the expression of eRNAs transcribed from the SE. The eRNA candidates were examined by in silico screening based on H3K27ac marks documented in lymphoblastoma in the ENCODE database, as well as the registered transcript signals obtained by RNA-seq analysis of lymphoblastoma (GM12878) and leukemia cells (K562) in dbSUPER  by qRT-PCR in CD4+ T cells -mediated RNAi approach  for BTB and CNC homology 2 (BACH2), a silencing transcription factor, by mining the binding sites for MafK, a heterodimer partner for BACH2  was observed as expected even without treatment  are present in the IL6 promoter, we performed a ChIP assay to determine whether activated NF-κB is recruited to this promoter , risk allele A of rs1893592 was significantly associated with decreased expression of UBASH3A in T cells [The presence of the RA-susceptibility SNP rs1893592) in as found . Furtheras found . The C aoduction . In cont T cells . However893592 inUBASH3A-SE based on H3K27ac and transcript peaks registered in the databases, we identified UBASH3A eRNA_1–3, as well as their expression in CD4+ T cells of healthy donors by RT-qPCR  with the RA pathogenic process. Additional subjects are clearly required, not only regarding this issue, but also with regard to other assays. Thus, the in vivo role of UBASH3A in RA pathogenesis remains to be determined. However, the presence of high IL-6 levels in RA patients is widely known in clinical practice to be involved in the progression of the pathology, such as inflammation, immune abnormalities, and joint destruction [Ubash3a-deficient mice showed significantly worse arthritis scores in the null mutants [UBASH3A gene locus displays a SE architecture, the present findings suggest that the low expression levels of UBASH3A associated with SE dysfunction promote, at least in part, IL-6-associated RA pathogenesis. Further, stimulation of CD3/CD28 on CD4+ T cells in the absence of transfection with UBASH3A induced higher rates of IL-6 expression in RA than in HD. Given that UBASH3A is a suppressor of TCR signaling, the lower endogenous UBASH3A levels in CD4+ T cells from RA patients compared to HD seemed reasonable. We further found that UBASH3A was negatively correlated with the IL-6 gene level in CD4+ T cells of RA patients. Thus, in agreement with previous studies [The present study has several limitations. First, the study included a small number of UBASH3A-stained lymph node samples collected from patients with dermatomyositis as the control group for RA patients. Second, CD4truction . Moreovetruction . Further mutants . Given t studies , we beliUBASH3A mRNA was downregulated in CD4+ T cells of RA patients. Furthermore, the recruitment of BACH2 and the lack of co-activators in CD4+ T cells reduced UBASH3A SE activity. Given that UBASH3A significantly attenuated IL-6 cytokine production in TCR signaling-activated CD4+ T cells, low levels of UBASH3A resulted in high levels of IL-6. These results suggest that UBASH3A is a potentially useful therapeutic target in RA.Additional file 1. All full-length images of Western blotting data. Uncropped full-length images of Figs. Additional file 2. The numbers shown here corresponds to ones of uncropped full-length images of Additional files Additional file 3. All full-length images of PCR data (#1). Uncropped full-length images of Fig. Additional file 4. All full-length images of PCR data (#2). Uncropped full-length images of Fig. CR data #. UncroppAdditional file 5. All full-length images of PCR data (#3). Uncropped full-length images of Fig. CR data #. UncroppAdditional file 6. All full-length images of PCR data (#4). Uncropped full-length images of Fig. Additional file 7: Supplementary Table S1. Baseline characteristics of patients with RA. Data are expressed as mean ± SD. MS, morning stiffness; TJC, Tender Joint Count; SJC, Swollen Joint Count; PGA, patient's global assessment; EGA, evaluator's global assessment; HAQ, health assessment questionnaire; CRP, C-reactive protein; ESR, erythrocyte sedimentation rate; RF, rheumatoid factor; MMP-3, matrix metalloproteinase-3; KL-6, sialylated carbohydrate antigen KL-6; ACPA, anti-citrullinated protein antibody; CDAI, Clinical Disease Activity Index; SDAI, Simplified disease activity index; DAS28, Disease Activity Score; N.D., Not determined. Supplementary Table S2. LNAs, probes and primers used in the study. * phosphorothioate backbone Oligonucleotides used for LNA transfection (5’→3’ sequence), SNP-PCR(Life Technologies assay number), ChIP-PCR (5’→3’ sequence), qPCR (Life Technologies assay number). Supplementary Figure S1. Expression level of UBASH3A in different CD4+ T cell subsets. UBASH3A mRNA expression level was quantified in Th1, Th2, Th17, and regulatory T cells by RT-qPCR. The amount of UBASH3A transcript was expressed relative to that of GAPDH. Data are mean ± standard deviation of three independent experiments. *p<0.05, vs. Th1, by Dunnett's multiple comparison test. RQ, Relative quantification. Supplementary Figure S2. Expression levels of UBASH3A protein in CD4- and CD4+ T cells of HD (n=3) and RA patients (n=3) evaluated by Western blotting. The results were quantified using ImageJ software. The amount of UBASH3A was normalized to that of β-actin used as a loading control. Three independent experiments were performed. Supplementary Figure S3. Quantification of UBASH3A protein expression in CD4+ T cells from the lymph nodes of dermatomyositis (DM) and RA patients. Lymph nodes were evaluated by immunofluorescence staining. The results were quantified using ImageJ software. The amount of UBASH3A (*, ** and *** in Fig. Supplementary Figure S4. Schematic diagram of the study findings. 1. Epigenetic dysfunction. Recruitment of BACH2 and MED1/BRD4 to UBASH3A gene is promoted and suppressed, respectively, in RA CD4+ T cells, but not the control. 2. Low levels of UBASH3A. Expression of UBASH3A gene is suppressed through the above mechanism. 3. Enhanced TCR signaling. UBASH3A confers weak ability for negative regulator of TCR signaling due to low levels of UBASH3A, resulting in enhanced TCR signaling. 4. IL-6 induction. TCR signaling activates IL-6 gene, leading to overproduction of IL-6 and sustained inflammation.


Replacement of amino acids unique to GDF11 and GDF8 into the native locus of the other ligand yields measurable, differential skeletal and muscle phenotypes, revealing distinct features between the ligands and a requirement for GDF11 in early-stage skeletal development. Gdf11-deficient mice, without apparent perturbation of skeletal or cardiac muscle development or homeostasis. These experiments uncover distinctive features between the GDF11 and GDF8 mature domains in vivo and identify a specific requirement for GDF11 in early-stage skeletal development.Growth differentiation factor 11 (GDF11) and GDF8 (MSTN) are closely related TGF-β family proteins that interact with nearly identical signaling receptors and antagonists. However, GDF11 appears to activate SMAD2/3 more potently than GDF8 in vitro and in vivo. The ligands possess divergent structural properties, whereby substituting unique GDF11 amino acids into GDF8 enhanced the activity of the resulting chimeric GDF8. We investigated potentially distinct endogenous activities of GDF11 and GDF8 in vivo by genetically modifying their mature signaling domains. Full recoding of GDF8 to that of GDF11 yielded mice lacking GDF8, with GDF11 levels ∼50-fold higher than normal, and exhibiting modestly decreased muscle mass, with no apparent negative impacts on health or survival. Substitution of two specific amino acids in the fingertip region of GDF11 with the corresponding GDF8 residues resulted in prenatal axial skeletal transformations, consistent with  The TGF-β superfamily of proteins is well known for regulating embryological development, wound healing, and adult tissue maintenance. In recent years, two highly homologous TGF-β proteins—growth differentiation factor 11 (GDF11) and GDF8 —have garnered substantial interest with evidence of their roles in aging and regenerative processes . Due to Mstn (Gdf8) results in hypermuscular, hypo-adipose phenotypes in numerous animal species, including humans  mice do not  into the analogous region of GDF8 (in place of G89 and E91) enhanced SMAD signaling activity of the hybrid GDF8 molecule by ∼50% . This reGdf11 (Gdf8 (Gdf8 double-stranded DNA (dsDNA) donor plasmid Gdf11Gdf8aa mice (with Gdf8 amino acid residues G89 and E91 replacing the corresponding residues in Gdf11) (n Gdf11) .(a)Gdf11+/8aa), bi-allelic (Gdf118aa/8aa), WT (Gdf11+/+).Mono-allelic ((2)Gdf11aaGdf8 mice (with Gdf11 amino acid residues D89 and Q91 replacing the corresponding residues in Gdf8) (in Gdf8) .(a)+/11aaGdf8), bi-allelic (11aa/11aaGdf8), WT (+/+Gdf8).Mono-allelic ((3)Gdf11MDGdf8 mice (with the Gdf11 mature domain replacing the Gdf8 mature domain in the Gdf8 locus) (8 locus) .(a)+/11MDGdf8), bi-allelic (11MD/11MDGdf8), WT (+/+Gdf8).Mono-allelic  in each chimeric line before analyses to breed out potential off-target modifications and confirmed Mendelian ratios of allele inheritance to rule out potential embryonic lethality resulting from the genetic modifications (Table S1). Targeted locus amplification (TLA) sequencing , perform11Gdf8aa , Gdf8Gdf8Gdf11aa , and Gdf8Gdf11MD  mice andGdf11Gdf8aa, Gdf8Gdf11aa, and Gdf8Gdf11MD F5 progeny in C57BL/6J background through three initial rounds of breeding.Genotype and survival distribution of Table S1 Gdf8 with Gdf11 altered circulating protein levels in vivo, we collected serum from Gdf8+/+, Gdf8+/11MD, and Gdf811MD/11MD mice at 10–14 wk of age and measured endogenous GDF11 and GDF8 levels, using a liquid chromatography–tandem mass spectrometry assay that distinguishes between GDF11 and GDF8 by detecting two differential peptide fragments between the two proteins (Gdf811MD/11MD mutants (n = 10), circulating GDF11 concentrations increased ∼50-fold above normal levels . The levels of GDF11 peptides measured in Gdf811MD/11MD mutants ..Gdf8 witl levels , whereasl levels . The sam mutants  were com+/+ mice  and to t1MD mice , suggestenotypes . Mass spfindings . They alf11 mice , with Ms ligands . Our stu WT mice . Based o WT mice , our dat WT mice , indicatGdf11 results in perinatal lethality, and both homozygous (Gdf11−/−) and heterozygous (Gdf11+/−) disruption of Gdf11 cause developmental abnormalities of the skeleton , eviscerated, and stained with Alizarin red and Alcian blue   , quadricior (TA) , and triior (TA)  muscles ior (TA)  and tibiior (TA)  in the G mutants  to asses mutants .Gdf8Gdf11aa and Gdf11Gdf8aa lines, we saw no significant differences in body weight , compared with sex-matched Gdf8+/+ mice (n = 10), with similarly significant decreases recorded in TA  as well. Although all three muscle groups in Gdf811MD/11MD mice normalized to overall body weight did not yield statistically significant differences (P < 0.01) (P < 0.05) (Gdf8+/+ mice (n = 21). Overall, we did not observe any distinct defects or malformations in muscle development or patterning at any point during development, and the chimeric mice appeared similar to WT into early adulthood. In all cases, separation by males and females resulted in shifts in the mean muscle mass between the sexes. Analysis of muscle mass in MstnGdf11/Gdf11 mice showed similar (∼10%) reductions in muscle mass in male, but not female, mice in which the mature domain of GDF8 was replaced by that of GDF11, with no differences in fiber composition   and tric < 0.05)  showed sposition . HoweverGdf11Gdf8aa, Gdf8Gdf11aa, and Gdf8Gdf11MD mutants, compared with WT mice. Interestingly, we did record a statistically significant decrease in the liver weight of Gdf811MD/11MD females (n = 9), compared with Gdf8+/+ females   , but a snt males , despite+/+ mice . This tr1aa mice .Gdf8Gdf11MD mutant mice, in line with prior reports that endogenous GDF8 negatively regulates muscle development. The observed effects were distinct from those conferred to young Gdf8Gdf11aa mutant mice by substitution of only the Gdf11-like 89/91 residues—which did not yield any significant change in skeletal muscle mass. These data indicate a clear difference in potency between the mature domains of GDF11 and GDF8, and between the full GDF11 mature domain–replaced ligand and the double amino acid–substituted ligand generated here. In addition, though we did not observe signs of kidney agenesis in the Gdf8Gdf11MD mouse line, we recorded a trending decline in liver weight, most significant in Gdf8Gdf11MD mutant females. These data suggest a possible systemic effect on the liver—which does not produce GDF8—that could reflect a direct effect on liver hepatocytes or, more likely, be connected to the local GDF8 regulation of skeletal muscle mass. In such a scenario, skeletal muscle may be more sensitive to increased potency of GDF8, and as a result, a change in muscle mass indirectly affects liver development or homeostasis.Taken together, these data indicate that the increased potency conferred to GDF8 by substitution of the GDF11 mature domain significantly impacted skeletal muscle mass in young Gdf11Gdf8aa line, we found a statistically significant increase in heart weight of Gdf118aa/8aa males (n = 13), compared with Gdf11+/+ males  and with Gdf11+/8aa males   . Howeverale mice , and thefference . Analyse+/+ mice . Further+/+ mice . We alsok of age . In all k of age  and leftk of age  were conk of age .Gdf11Gdf8aa, Gdf8Gdf11aa, and Gdf8Gdf11MD mutant hearts have function and physiology similar to WT mice. Although no heart weight phenotype has been reported for Gdf11+/− mice, a difference was found in the heart weight of bi-allelic Gdf118aa/8aa mutant males  showed no significant differences in average CSA or fiber size distribution in either 8aa mice  and S6. e points . However mutants . These d mutants  or suppl mutants .Gdf11Gdf8aa mutant mice. In addition, the heightened ligand potency achieved through modifying the full mature domain of GDF8 in Gdf8Gdf11MD mutants confers a genetically linked phenotype impacting skeletal muscle mass in adult mice. Incredibly, in bi-allelic Gdf811MD/11MD mutants, circulating GDF11 concentration increased ∼50-fold more than normal, resulting in unexpectedly high levels of circulating GDF11 in vivo. Yet these chimeric mice were viable and survived normally into adulthood. The results indicate that gene regulation differences or differences in the prodomain regions may influence changes in ligand concentrations in vivo, and that ligand activity may not reflect alterations to ligand potency alone.Altogether, these data support the notion that the modest changes in ligand potency achieved through dual amino acid mutations confer a genetically linked phenotype in early-stage skeletal development within only Gdf11 or Gdf8 mature signaling domains in vivo. Results presented here indicate that changes in GDF11 and GDF8 potency elicit differential phenotypes  deletion of Gdf11  into the analogous location in the Gdf11 mature domain (D89 and Q91) decreased the potency of mature GDF11 to that of GDF8  postnatally in male or female mutants at 10–14 wk of age. Likewise, Gdf8Gdf11aa mice did not show significant differences in either sex at 10–14 wk of age. For both chimeric lines, we saw no differences in heart or kidney weight in either the Gdf11Gdf8aa or Gdf8Gdf11aa mutants. The only statistically significant finding occurred in the heart weight of Gdf118aa/8aa mutant males, compared with Gdf11+/+ and Gdf11+/8aa mice; however, this result was not observed in mutant females of the same line. The differences in the heart weight normalized to body weight also proved insignificant in the Gdf118aa/8aa mutants. Interestingly, Gdf8Gdf11aa mutants did not exhibit physiological changes to those found in Gdf11Gdf8aa mutants during embryonic development, nor did they produce measurable anatomic differences into adulthood.Because both GDF8 and GDF11 have been shown to play critical roles in skeletal and cardiac muscle development and function, and to regulate other organ systems , furtherGdf8Gdf11aa mutants, chimeric Gdf8Gdf11MD mice also did not exhibit detectable transformations during embryonic skeletal development, either in mono-allelic Gdf8+/11MD or in bi-allelic Gdf811MD/11MD mutants. Furthermore, we detected neither cranial bone malformation nor cleft palates in the mutants. Therefore, raising the activity of GDF8 to the full potency of GDF11 did not produce measurable changes in osteogenesis. This result may reflect the different tissue-specific expression patterns of the Gdf11 and Gdf8 loci. We performed additional experiments to determine whether increased GDF8 function in Gdf8Gdf11MD mutants might result in phenotypic differences in muscle growth and function postnatally and into adulthood. In Gdf8Gdf11MD mice, we confirmed that the increase in the GDF8 potency produced by replacing the mature domain of Gdf8 with that of Gdf11 decreased skeletal muscle size/mass in several limb muscles, consistent with results from GDF8 supplementation studies .S. pyogenes Cas9 mRNA, (2) in vitro–transcribed synthetic guide RNAs (sgRNAs), which targeted the native loci near the desired integration sites, and (3) the generated Gdf11 and Gdf8 ssDNA or Gdf11 dsDNA donor template. Zygote injections were performed by the Genome Modification Facility at Harvard University. Super-ovulated C57BL/6J female mice were mated to C57BL/6J males, and the fertilized zygotes were subsequently harvested from the oviducts. Zygotic pronuclei were microinjected with (1) purified Streptococcus pyogenes Cas9 mRNA , (2) in vitro–transcribed synthetic guide RNAs, which targeted near the integration sites of the native loci, and (3) the generated Gdf11 and Gdf8 ssDNA donor construct. Microinjected zygotes were implanted into the oviducts of C57BL/6J surrogate females at 12 h post-coitum. To identify mosaic offspring that contained the desired chimeric Gdf11 and Gdf8 double amino acid–substituted sequences, progeny from the microinjected surrogate females were genotyped by Sanger sequencing and by PCR validation and subcloning. In the Gdf11 amino acid chimera, we produced 47 total pups, with 14 live pups and 33 dead pups. Of the 14 live pups, 4 exhibited a mosaic genotype, and 3 were ultimately selected as germline founders. In the Gdf8 amino acid chimera, we obtained 92 viable pups, with 0 dead pups. Of the 92 live pups, 49 showed a mosaic genotype upon screening for the targeted allele. Ultimately, four confirmed positive founders were used as breeders to establish the colony. In the Gdf8 full mature domain chimera, we obtained 80 viable pups, with two dead pups. Of the 80 live pups, 35 showed a mosaic genotype upon screening for the targeted allele. Ultimately, five confirmed positive founders were used as breeders to establish the colony. We bred these chosen mice with WT C57BL/6J mice and genotyped the resultant pups to confirm that our desired mutation was incorporated in the germline. We backcrossed the five-generation knock-in alleles in a C57BL/6J genetic background before characterization experiments. Male and female mice were selected based on gender and randomized before treatment for all proposed animal studies.The CRISPR/Cas9 system was used to generate chimeric mice carrying amino acid mutations from the GDF8 and GDF11 interchanged in the mature domain. Two rounds of microinjections into C57BL/6J zygotes were performed for each line, delivering (1) purified µl of lysis solution  with 10 µg/ml Proteinase K and incubated at 50°C for 10–12 h. Next, samples were placed in heating blocks at 98°C for 5 min to inactivate the Proteinase K. DNA was extracted using a standard phenol:chloroform/ethanol precipitation protocol, and genotyping was performed by PCR using Phusion High-Fidelity PCR Master Mix with HF Buffer. Primers used are as follows: for Gdf11Gdf8aa, FW: CCTGACCCTCAGCATCCTTTCA, RV: GGTCCTTACTTTGCCCCATCCT; and for Gdf8Gdf11aa and Gdf8Gdf11MD, FW: TGTGGTTGGTTTGTTTGTTTGT, RV: GCCTGTGGTGCTTGAATTCA. PCR products were digested with restriction enzyme AseI , with NEBuffer 3.1, at 37°C for 20–30 min, and analyzed on 1% agarose gel. Further genotyping was performed via Sanger sequencing to verify the nucleotide changes and the presence of the AseI site.Initial mouse colony breeder genotypes were verified by sequencing. Subsequent progeny tissues were collected from the tail or the ear, and mixed in 1.5-ml Eppendorf tubes containing 300 Gdf11Gdf8aa, Gdf8Gdf11aa, and Gdf8Gdf11MD F5 mutant mice was harvested, homogenized, and subjected to ACK lysis. Harvested bone marrow  was frozen and delivered to Cergentis B.V. for TLA sequencing analysis  fused to Insert: 1, head  with four inserted bases ATCCCTTTTTAGAAGTCAAGGTGACAGACACACCCAAGAGGTCCCGAAGAAACCTAGGCCTGGACTGGATGAACACTCGAGTGAGTCCCGCTGCTGCCGATATCCTCTCACAGTGGACTTTGAGGCTTTTGGCTGGACTGGATCATC; 3′ integration site: Insert: 327, tail  fused to chr1:53,066,629 (head) AACATGCTCTACTTCAATGACAAGCAGCAGATTATCTACGGCAAGATCCCTGGCATGGTGGTGGATCGATGTGGCTGCTCCTGAGCTTTGCATTAGGTTAGAAATTTTCCAAGTCATGGAAGGTCTTC. After amplification of the targeted locus, PCR amplicons of the complete Gdf11 and Gdf8 region of interest were purified and prepped for Illumina sequencing. Analysis was performed by aligning mutant sequences to the mouse mm10 reference genome sequence. In Gdf11Gdf8aa mice, two mutated nucleotides (G→C and T→C) were confirmed on chr10 in the mature domain of Gdf11, resulting in alteration of only the two targeted amino acids: Gdf11 D89 to G89 (Asp→Gly) and Q91 to E91 (Gln→Glu). In Gdf8Gdf11aa mice, two mutated nucleotides (G→A and G→C) were confirmed on chr1 in the mature domain of Gdf8, resulting in amino acid changes in Gdf8 G89 to D89 (Gly→Asp) and E91 to Q91 (Glu→Gln). In bi-allelic Gdf118aa/8aa and Gdf811aa/11aa samples, the mutated nucleotides were confirmed at 100% frequency, indicating that the mutations occurred on both alleles, whereas in mono-allelic Gdf11+/8aa and Gdf8+/11aa samples, the mutations were confirmed at ∼50% frequency, indicating occurrence of the desired mutations on only one allele. In the Gdf8Gdf11MD mice, correct integration of exon 3 of Gdf11 was confirmed on chr1 in place of the native exon 3 of Gdf8, indicating successful replacement of the GDF8 mature domain with that of GDF11. In bi-allelic Gdf811MD/11MD samples, no WT reads were present at the integration site and in the deleted region, confirming bi-allelic replacement of native GDF8, whereas in mono-allelic Gdf8+/11MD samples, WT reads were detected at the integration site and in the deleted region on one allele, confirming mono-allelic replacement of native GDF8.g for 10 min at room temperature. The upper layer of serum was then transferred to a new tube and stored at −80°C before mass spectrometry analysis. Serum from chimeric mice (minimum 100 µl) was submitted to the Brigham and Women’s Hospital Brigham Research Assay Core (BRAC) for quantitative liquid chromatography–tandem mass spectrometry detection of GDF11 and GDF8 protein concentrations. The mouse serum was denatured and alkylated, followed by pH-based fractionation, using cation ion exchange SPE. After desalting and concentrating, the peptide mix was separated via liquid chromatography, followed by mass spectrometry analysis in a positive electrospray ionization mode. GDF11 and GDF8 concentrations were determined using unique proteotypic peptides from GDF11 and GDF8 as surrogate peptides, coupled with heavy-labeled unique peptides as internal standards. Included in the analysis were GDF8 concentrations and the mean GDF11 concentrations.For serum collection, after euthanasia, blood was collected via orbital bleeding into Microtainer tubes with a serum separator (BD) and incubated for 30 min at room temperature before centrifugation at 2,000Gdf11Gdf8aa and Gdf8Gdf11aa mutants and compared with WT mice. For timed breeding, a vaginal plug observed in the female indicated embryonic day 0.5 (E0.5). Mouse embryos were harvested at E18.5, skinned, and eviscerated, and underwent washes of 100% ethanol and 100% acetone for 24 h at room temperature. The skeletons were then stained using a 0.3% Alcian blue (dyes bone) and 0.1% Alizarin red (dyes cartilage) solution at 37°C while oscillating for 72 h. Next, they were transferred to 1% KOH for 24 h on a rocker at room temperature, followed by a series of glycerol/KOH washes at (1) 20% glycerol/1% KOH, (2) 50% glycerol/1% KOH, and (3) 80% glycerol/1% KOH for 24 h at room temperature. The stained preparations were placed in 80% glycerol/1× PBS and imaged using a Nikon D750 camera attached to a Nikon SMZ1500 stereo microscope with an HR Plan APO 1× objective lens. Images were captured in NEF (Nikon Electronic Format) and processed and adjusted in the Adobe Camera Raw platform.Embryonic day 18.5 (E18.5) harvests  were per2, and overall body weights were taken postmortem. For tissue collection, the heart was dissected, washed in PBS, and dried on paper towels before weighing. The TA muscle, quadriceps muscle, and triceps muscle were harvested and weighed. The tibia bone was excised, cleaned, and measured using electronic calipers. Statistical analysis was performed using Prism 8.4.2 for macOS. The muscle weights were divided by sex and genotype within each mouse line, and the results were represented as the mean within each group by genotype and sex. The combined muscle weights were normalized to body weight and tibia length. Groups more than two were compared by one-way ANOVA with Tukey’s correction for multiple comparisons.Mice were ear-tagged and their genotype blinded at 10–14 wk of age. Adult mice were euthanized with COGdf11Gdf8aa mice  and Gdf8Gdf11aa mice , age 10–14 wk, were sedated with 0.1–0.5% inhaled isoflurane for echocardiography (iography . Mice weThe muscle cryoinjury procedure  was chosµm. Hematoxylin and eosin (H&E) staining was used to visualize cardiomyocyte cross sections and regenerating myofibers in injured muscles. Sections were stained with hematoxylin for 3 min and washed with tap water for 2 min, followed by back-to-back washes in acid alcohol. Scott’s Bluing Reagent was used for 3 min for nuclear staining, followed by another tap water wash, and finally 2 min for eosin staining. Afterward, sections were dehydrated in ethanol and xylene, and subsequently mounted in Permount Mounting Medium  and cured for 24 h before imaging. In regenerating myofibers after cryoinjury, a cross section representing the mid-belly of the TA where there was a clear representation of the injury was chosen. To quantify muscle fiber size, a series of images were taken spanning the entire regenerating area in the cross sections using a dual-head Olympus B×4 microscope with cellSens Standard software. Centrally nucleated myofibers were measured in each image using FIJI software , resulting in ∼300–1,500 fibers collectively for each animal. Comparative analyses of more than two groups were performed by one-way ANOVA with Tukey’s correction for multiple comparisons. All analyses were performed under conditions in which the analyst was blinded to sample identity.For cryosections, harvested TA muscles were flash-frozen in 2-methylbutane for 30 s followed by liquid nitrogen for 30 s. Samples were stored at −80°C before sectioning. Mouse hearts were rinsed with 1× PBS, embedded in compound at optimal cutting temperature, and frozen. TA samples and cardiomyocyte samples were sectioned at 10 n equals the number of biological replicates of animals used per experiment. The number of animals used for each group was determined based on total empirical data and anticipated completeness of datasets and was sufficient to detect differences in experimental outcomes, if present. Statistical analysis was performed using Prism 8.4.2 for macOS. Comparisons between two different experimental groups were assessed for statistical significance using a t test, and comparisons between more than two groups were performed by one-way ANOVA with Tukey’s correction for multiple comparisons. Statistical significance was accepted at P < 0.05. All experiments and analyses were performed under blinded conditions.All data are presented as mean + SEM. For all data,


Candida albicans possesses multiple virulence factors which enable colonization and infection of host tissues. Candida-related infections frequently occur in immunocompromised patients, which is related to an insufficient inflammatory response. Furthermore, immunosuppression and multidrug resistance of C. albicans clinical isolates make the treatment of candidiasis a challenge for modern medicine. The most common resistance mechanism of C. albicans to antifungals includes point mutations in the ERG11 gene, which encodes target protein for azoles. We investigated whether the mutations or deletion of the ERG11 gene influence the pathogen-host interactions. We prove that both C. albicans erg11∆/∆ and ERG11K143R/K143R exhibit increased cell surface hydrophobicity. Additionally, C. albicans KS058 has an impaired ability of biofilm and hyphae formation. Analysis of the inflammatory response of human dermal fibroblasts and vaginal epithelial cell lines revealed that altered morphology of C. albicans erg11∆/∆ results in a significantly weaker immune response. C. albicans ERG11K143R/K143R triggered stronger production of pro-inflammatory response. Analysis of genes encoding adhesins confirmed differences in the expression pattern of key adhesins for both erg11∆/∆ and ERG11K143R/K143R strains. Obtained data indicate that alterations in Erg11p consequence in resistance to azoles and affect the key virulence factors and inflammatory response of host cells.Opportunistic pathogen  Candida albicans constitutes part of the natural microbiome of healthy individuals [Candida-related infections [Candida, C. albicans remains the main cause of fungal infections. Candidemia (bloodstream infection) is responsible for 10% of nosocomial infections, and the mortality rate ranges from 30 to 60% [Candida sp. led to a high mortality rate among patients suffering from fungal infections.The polymorphous fungus ividuals . It can fections ,3. Among0 to 60% ,5. ThereC. albicans. One of them is the ability of this fungus to morphological transition from yeast (blastoconidia) to hyphae [C. albicans produces a hyphae-specific toxin , which is responsible for damage of epithelial cells and triggering the innate immune response, which results in cytokine production [C. albicans is able to form a biofilm that provides resistance to antifungals. This specialized structure is composed of C. albicans cells embedded in extracellular polysaccharide substance (EPS) attached to host tissues or abiotic surfaces [C. albicans cell wall (CW) proteins (adhesins). The best-known adhesin family is an agglutinin-like sequence (Als), e.g., Als1p or Als5p [C. albicans infection, there are several different factors that enable the successful colonization of host tissues. The quorum sensing, creation of opaque cells and changes in cell surface hydrophobicity (CSH) also affect the invasive abilities of C. albicans [Successful colonization and infection of host tissues are provided by multiple virulence factors of o hyphae . Filamenoduction ,8. Apartsurfaces . The adhor Als5p . There aor Als5p . Despitealbicans ,13.C. albicans CW is the first barrier to interfere with the host cells’ surface. According to this, CW plays a major role in initial colonization  but also stimulates the innate immune system to fight the infection [C. albicans CW is composed of a thick layer of mannans (outer CW), β-glucan and chitin (inner CW). Those components are considered pathogen-associated molecular patterns (PAMPs) [C. albicans remodels its CW to evade the host’s immune system [C. albicans PAMP β-glucan can be both exposed or masked in response to environmental changes [C. albicans infection frequently occurs in the mucosal epithelium , these cells produce inflammatory interleukins (ILs), e.g., IL-1α, IL-6, IL-10 or IL-8 [C. albicans infection [C. albicans infections [During infection nfection . C. albi (PAMPs) . Neverth (PAMPs) . Interese system . Major C changes ,19. Neve changes . Since C or IL-8 ,22. IL-8nfection . Considefections . Attractfections .C. albicans possess multiple virulence factors and strategies to evade the immune response. Additionally, considering the growing multidrug resistance of C. albicans clinical isolates, there is an uprising problem with successful treatment. The most commonly employed antifungals, like azoles , target the Erg11p (encoded by the ERG11 gene), resulting in blocking the ergosterol biosynthesis pathway and growth inhibition of the pathogen. C. albicans’ resistance strategies to azoles include point mutations in the ERG11 gene or alterations to ERG11 expression [There are many different strategies to eliminate fungal infections, but the crucial part is the recognition of PAMPs and the production of proinflammatory cytokines. On the other hand, pression ,27.K143R) in the ERG11 gene  or deletion of this gene  corresponds with changes in the plasma membrane (PM) and CW composition of C. albicans mutants [C. albicans and the immune response. Thus, we decided to investigate whether introduced mutations affect inflammatory response.We previously reported that both point mutation  and 10C1B1I1 (K143RERG11K143R/). Additionally, we analyzed the filamentation and biofilm formation of C. albicans strains in order to determine the impact of the mutations on the virulence of the pathogen. Since C. albicans lives on skin and mucosal epithelium , we investigated the inflammatory response of normal human dermal fibroblasts (NHDF) and normal vaginal epithelial (VK2 E6/E7) cell lines to C. albicans mutants’ infection. Research revealed that C. albicans KS058 and 10C1B1I1 strains lead to altered inflammatory response and different cytokines expression patterns in the NHDF and VK2 E6/E7 cell lines. Additionally, we determined the expression of adhesins for KS058 and 10C1B1I1 strains cultured in YPD medium and after co-culture with NHDF and VK2 E6/E7 cell lines. Obtained data suggest that alterations in ergosterol levels in PM lead to significant changes in the virulence of C. albicans and in triggering inflammatory responses during infection. Therefore, a continuation of the study will impact the understanding of the consequences of C. albicans’ resistance mechanisms on host-pathogen interactions.Here, we investigated the cell surface hydrophobicity (CSH) of C. albicans KS058 (erg11∆/∆) and 10C1B1I1 (K143RERG11K143R/) mutants [The aim of this study was to investigate whether changes in the CW composition of  mutants  correlatC. albicans KS058 and 10C1B1I1 strains, the CSH parameter was significantly elevated in comparison to the SC5314 (WT) strain . For this study, we used the hydrophobic CAF4-2 strain  as an internal control. The obtained data show that, for the C. albicans CAF4-2 strain, the surface hydrophobicity is 61.47%. Analysis revealed that C. albicans KS058 and 10C1B1I1 strains exhibit significantly elevated cell surface hydrophobicity . Those results indicate the crucial role of ergosterol in maintaining fungal cell physiology.For both C. albicans KS058 resulted in impaired biofilm formation compared to the SC5314 (WT) strain  strain B. After C. albicans is an essential aspect of fungal infections. The capacity for morphological transitions from yeast to hyphae enables dissemination, invasion and the damage of host tissues [C. albicans WT (SC5314), KS058 and 10C1B1I1 strains after culture in the presence of FBS are presented in Polymorphism of  tissues . Thus, wC. albicans KS058 is not able to form hyphae during 24 h of culture in the presence of FBS. This proves that lack of ergosterol affects the crucial virulence factors of C. albicans  was elevated only after 2 h of culture compared to the WT strain .According to C. albicans 10C1B1I1 compared to the WT strain. On the other hand, elevated ergosterol content (as a consequence of amino acid substitution in Erg11p) in C. albicans 10C1B1I1 mutant resulted in the formation of longer filaments than in WT strain but only in the early logarithmic phase of growth . , the percentage of hyphae in culture was lower or similar for ulture). B.C. albicans PM correlate with altered virulence potential. We employed analysis of inflammatory response to infection with C. albicans mutants. Since C. albicans usually live on skin or mucosal epithelia , we determine the cytokine release of normal human dermal fibroblasts (NHDF) and normal vaginal epithelial (VK2 E6/E7) cell lines . Interestingly, after 24 h of co-culture, we observed significantly elevated levels of MCP-1, both in control conditions (277.38 pg/mL) and after co-culture with C. albicans WT, KS058 or 10C1B1I1 strains . The level of IL-8 was comparable to control conditions for the KS058 strain . A higher concentration of IL-8 was observed in the case of NHDF co-cultured with C. albicans WT or 10C1B1I1 strains . The amount of IL-8 significantly increased after 24 h of co-culture with WT and 10C1B1I1 strains . Thus, C. albicans 10C1B1I1 strain triggers a stronger inflammatory response in the NHDF cell line after 24 h of culture than the WT strain. On the other hand, C. albicans KS058 did not stimulate NHDF to produce higher levels of the tested cytokines.Research revealed that NHDF cell lines do not produce IL-1α either in control conditions or during infection with of IL-10 . IL-6 prC. albicans WT and 10C1B1I1 strains triggered the significantly higher production of IL-1α, while for C. albicans KS058, the level of IL-1α was comparable to the control conditions after 8 h of co-culture . 24 h post-infection, the IL-1α level in co-culture supernatant increased in the case of all tested C. albicans WT, KS058 and 10C1B1I1 strains . The VK2 E6/E7 cell line, after co-culture with C. albicans mutants, also produced an increased amount of IL-6 and IL-8. We observed that the WT and 10C1B1I1 strains triggered IL-6 production more effectively than the KS058 strain after 8 h of co-culture . In 24 h of culture, we observed an increase in IL-6 levels in the presence of C. albicans KS058 (6.64 pg/mL) and the results obtained for the WT and 10C1B1I1 strains were comparable to those achieved in 8 h of culture. We observed a similar pattern in the case of IL-8; the stronger response of the VK2 E6/E7 cell line to the C. albicans WT and 10C1B1I1 strains, and the level of this chemokine was comparable among the investigated times of co-culture (8 and 24 h). Moreover, the C. albicans KS058 strain did not stimulate vaginal epithelial cells to increase production of IL-8 after 8 h of culture, but the elongation of the time of co-culture (from 8 to 24 h) with this strain resulted in elevated levels of IL-8 . Similarly to NHDF, the VK2 E6/E7 cell line after C. albicans infection produced trace amounts of IL-10 in all tested conditions. The level of MCP1 was beyond the value obtained for the control condition in the case of all C. albicans strains, while the highest level of this chemokine was noted for C. albicans KS058 strain (after 8 and 24 h of co-culture MCP1 level was as follows: 4.90 and 3.31 pg/mL).The analysis of the inflammatory response of the VK2 E6/E7 cell line revealed that co-culture with Additionally, we investigated the relative expression of genes encoding chosen cytokines  in order to confirm obtained results. The experiment was performed using the same cells harvested for analysis of cytokines production . The relC. albicans WT or 10C1B1I1 strains for 8 h results in the increased expression of IL-6 and IL-8 genes  for the purpose of determining if there are differences in adhesins’ expression profile between C. albicans mutants.Considering that adhesins are crucial for the initial steps of the C. albicans WT (SC5314), KS058 and 10C1B1I1 strains culture in the YPD medium. The results are presented in Firstly, we determined the gene expression of adhesins after C. albicans KS058 strain, we observed higher expression of all investigated genes encoding adhesins after 8 h of culture in the YPD medium . The C. albicans 10C1B1I1 strain exhibited significantly increased expression of ALS5 and HWP1 genes, while the HWP1 expression was six-fold higher than for the WT strain after 8 h of culture.In the case of the D medium A. For C.ALS5 and HWP1 genes decreased compared to 8 h of culture and is similar for all investigated strains, except the two-fold higher expression of ALS5 for C. albicans KS058 compared to the WT strain (ALS1 gene expression after 24 h was significantly higher than after 8 h of culture for KS058 (a 10-fold increase compared to the WT strain).After 24 h of culture in YPD medium, the expression of T strain B. IntereC. albicans WT (SC5314), KS058 and 10C1B1I1 strains after co-culture with NHDF and VK2 E6/E7 cell lines. The results are presented in Additionally, we determined the adhesins gene expression profile in C. albicans 10C1B1I1 strain displayed lowered ALS1 gene expression compared to the WT strain. The expression of ALS1 and ALS5 for C. albicans KS058 was elevated but not statistically significant  not only provide resistance to azoles but can also affect the morphology of fungal cells. Therefore, it is relevant to study mechanisms involved in acquiring resistance to antifungals and to determine the impact of the mutations on fungal pathogenicity.0 to 80% ,30. Bloo0 to 80% . That pre Erg11p . This prERG11 gene expression and elevated ergosterol content in C. albicans PM [C. albicans 10C1B1I1) and the lack of Erg11p  result in the exposure of chitin and β-glucan on the cell surface [ERG11 gene) result in increased azole resistance. Taking those data into account, C. albicans 10C1B1I1 and KS058 strains could be relevant models in studying the inflammatory response of human cells to infection with azole-resistant C. albicans strains.We previously have shown that amino acid substitution K143R in Erg11p results in both increased icans PM . More in surface ,32. AddiC. albicans, e.g., cell surface hydrophobicity (CSH), filamentation and biofilm formation. The surface of fungal cells is important during the initial infection of host tissues because it is the first one to interfere with human cells. The analysis revealed that, compared to C. albicans WT (SC5314), both the KS058 and 10C1B1I1 strains demonstrated significantly higher CSH values, and the CSH obtained for those mutants was even higher than for hydrophobic CAF4-2 strain  can impact the CSH of investigated strains. Our previous studies confirmed that elevated CSH results in higher activity of the Cdr1p efflux pump leading to increased fluconazole resistance [Candida CW leads to decreased CSH [Candida sp., which display high CSH values, are more virulent than hydrophilic cells in mice models, and increased CSH is highly related to disease-causing isolates [The purpose of the study was to verify whether K143R substitution in Erg11p and lack of this protein influence the key virulence factors of 2 strain A. Accord surface . The CSH surface . Therefosistance . Additioased CSH . Moreoveisolates .C. albicans is its ability to form biofilms both on biotic and abiotic surfaces, which can be critical for patients after transplantations [C. albicans KS058 has an impaired ability of biofilm formation , filamentation or CSH [One of the key virulence factors of ntations . Here, tormation B. This cns KS058 . Oppositn or CSH ,39.C. albicans WT and 10C1B1I1 strains, the formation of filaments initiated after 1 h of culture in YPD media supplemented with 20% FBS  or by the production of hyphae-specific adhesins  [The filamentation assay revealed that, for  20% FBS A. After T strain B. On the, Hwp1p) ,41. For C. albicans KS058 and 10C1B1I1 also demonstrate different patterns of expression of genes encoding adhesins. After 8 h of culture in YPD media, we observed increased expression of ALS1, ALS5, HWP1 and SAP5 for C. albicans KS058 or ALS1 and HWP1 for C. albicans 10C1B1I1  and normal vaginal epithelial VK2 E6/E7 cell lines. The chosen cell lines represent the host niches for Candida sp. We investigated the expression of adhesins, level of cytokines  and chemokines (IL-8 and MCP-1) which are produced by human cells in response to C. albicans infection . Opposite to NHDF, VK2 E6/E7 8 h co-culture led to a decrease in ALS5 and HWP1 gene expression in both KS058 and 10C1B1I1 strains compared to the WT strain  correspond with the weaker inflammatory response of the human cell lines.NHDF and VK2 E6/E7 cell lines differ in cytokines/chemokines production pattern. IL-1α is highly produced by VK2 E6/E7 cell line, especially after stimulation with  strains . This prC. albicans mutants results in altered immune reactions. In epithelial cells, IL-1α is expressed constitutively, and elevated expression of interleukin is detected in response to inflammation [C. albicans 10C1B1I1 resulted in the detection of significantly higher levels of IL-1α than the WT strain. A similar tendency was observed in the case of IL-6 and IL-8 levels, which was confirmed by the increased expression of genes encoding those cytokines , calcium chloride (CaCl2) .During this study following reagents were used: yeast extract (YE), peptone , glucose , phosphate-buffered saline (PBS) tablets, hexadecane, penicillin with streptomycin, L-Glutamine (L-Glu) ; RPMI 1640, KSFM medium with dedicated supplements (human recombinant EGF (rEGF), bovine pituitary extract (BPE)), fetal bovine serum (FBS) ; α-MEM medium ; KC. albicans strains used in this study are CAF2-1 (genotype: ura3Δ::imm434/URA3), CAF4-2 (genotype: ura3Δ::imm434/ura3Δ::imm434) and SC5314 which were a kind gift from Prof. D. Sanglard  [C. albicans KS058 was constructed by our team (the same genotype as SC5314 but erg11Δ::SAT1-FLIP/erg11Δ::FRT) [ERG11K143R::FRT/ERG11K143R::FRT) which was a kind gift from Prof. D. Rogers  [The zerland) . C. albi1Δ::FRT)  and 10C1TN, USA) . Strains2, 37 °C). For the purpose of inflammatory response studies, NHDF were seeded into 24-well culture plates  in α-MEM with 1% FBS, 1% L-Glu and without antibiotics. During C. albicans co-cultures, NHDF were cultured in α-MEM with 1% L-Glu. VK2 E6/E7 were routinely cultured in complete KSFM medium supplemented with human rEGF (0.1 ng/mL), BPE (0.05 mg/mL), CaCl2 (0.4 mM) and 1% PenStrep in a humidified incubator . For the purpose of inflammatory response studies, VK2 E6/E7 was seeded into 24-well culture plates in a complete KSFM medium without antibiotics.Cell lines NHDF  and VK2 E6/E7  were cultured according to distributor directions. NHDF was routinely cultured in complete α-MEM medium  in a humidified incubator  were washed two times  and resuspended in phosphate buffer . Cultures were adjusted to OD600 = 0.5 (±0.05) in 2 mL of phosphate buffer, and 500 μL of hexadecane was added. The probes were vigorously vortexed for 3 min and then incubated stationary for 60 min at RT. After this, OD600 of 1 mL of water phase was measured with Hach Odyssey DR/2500 spectrophotometer. The cell surface hydrophobicity (CSH) was calculated according to the formula :Cell surface hydrophobicity (CSH) was performed according to Biniarz et al. with modifications . Twenty-C. albicans was cultured for 24 h in 20 mL of YPD medium supplemented with 20% FBS (starting OD600= 0.1). At different time points , C. albicans cells were harvested, washed 2 times with PBS  and concentrated. Then, microscopic preparations were observed under Zeiss Axio Imager  A2 microscope. For the determination of hyphae length and the percentage of hyphae, at least n = 300 of C. albicans cells were analyzed in each condition.For the purpose of filamentation assay, C. albicans strains were pre-cultured in YPD for 24 h . Then cultures were washed 3-times with RPMI 1640 medium, pH 7.4  and adjusted to OD600 = 1. 200 μL of prepared inoculum were transferred into a 96-well plate  and then incubated for 90 min at 37 °C with agitation (250 rpm) using Spark microplate reader and incubator . RPMI 1640 medium alone constitutes a negative control of the experiment. After initial incubation , the medium was discarded, wells were washed with PBS, and fresh RPMI 1640 medium was added. Then cells were cultured for 24 h under the same conditions .The method was performed according to Lohse M. B. et al.’s protocol with modifications . Briefly600 measurements of cultures: before incubation (t = 0 h), after initial incubation (t = 90 min) and after final incubation  using Spark microplate reader .The biofilm formation was determined by OD6 cells per well in 500 μL of dedicated medium (2). After this, the medium was discarded, and cells were inoculated with C. albicans mutants at the multiplicity of infection (MOI) = 10 . Negative controls were human cells cultured alone in a dedicated medium.In order to determine the inflammatory response of used cell lines, the NHDF or VK2 E6/E7 were seeded into 24-well plates  at density 1 × 10d medium  and cult2 = 1.After 8 or 24 h of co-culture, the medium was harvested and centrifuged  to remove non-adherent cells from the culture supernatant. After this, supernatants were tested for the presence of cytokines  using ELISA–Milliplex MAP Kit . The experiment was performed according to manufacturer instructions in 3 independent biological replicates. For each cytokine in the experiment, a standard curve was determined and RC. albicans strains with human cell lines, adherent cells were harvested by centrifugation . After this, cells were immediately resuspended in fenosol  and kept at –20 °C until the RNA isolation. The RNA isolation was performed using a Total RNA Mini kit  according to manufacturer instructions. Then RNA was adjusted to an equal concentration of 10 ng/μL, and reversed transcription (RT) was performed using a High-Capacity cDNA Reverse Transcription kit . In order to determine the level of expression of genes encoding adhesins or cytokines, the cDNA was used as the matrix in real-time qPCR (RT-qPCR) and iTaq Universal SYBR Green Supermix . RT-qPCR reaction was performed using Step-One Plus Real-Time PCR System . The list of used primers’ sequences is presented in After co-culture of ALS1, ALS5 and HWP1; reference gene: ACT1) following thermal cycling conditions were used: the initial step at 95 °C for 10 min, followed by 40 cycles at 95 °C for 20 s, 54 °C for 20 s and 72 °C for 30 s. For the determination of cytokine genes expression , RT-qPCR was performed as follows: the initial step at 95 °C for 10 min, followed by 40 cycles at 95 °C for 20 s, 59 °C for 20 s and 72 °C for 30 s. The relative expression of target genes was calculated using the 2∆∆Ct− method.For determination of adhesins genes expression .Unless stated otherwise, data represent the means ± standard errors from at least 3 biological replicates. Statistical significance was determined using Student’s


Tumor cells exhibited phenotypic and molecular characteristics similar to their lineage progenitor cells. Liver developmental signaling pathways are showed to be associated with HCC development and oncogenesis. The similarities of expression profiling between liver progenitors (LPs) and HCC suggest that understanding the molecular mechanism during liver development could provide insights into HCC.PITX2), was characterized via in vitro and vivo functional assays. Furthermore, molecular mechanism studies were performed to unveil how PITX2C regulate the key developmental factors in LPs, thereby increasing the stemness of HCC.To profile the dynamic gene expression during liver development, cells from an in vitro liver differentiation model and two paired hepatocellular carcinoma (HCC) samples were analyzed using deep RNA sequencing. The expression levels of selected genes were analyzed by qRT-PCR. Moreover, the role of a key transcription factor, pituitary homeobox 2 (PITX2 was found to exhibit a similar expression pattern to specific markers of LPs. PITX2 consists of three isoforms (PITX2A/B/C). The expression of PITX2 is associated with tumor size and overall survival rate, whereas only PITX2C expression is associated with AFP and differentiation in clinical patients. PITX2A/B/C has distinct functions in HCC tumorigenicity. PITX2C promotes HCC metastasis, self-renewal and chemoresistance. Molecular mechanism studies showed that PITX2C could up-regulate RALYL which could enhance HCC stemness via the TGF-β pathway. Furthermore, ChIP assays confirmed the role of PITX2C in regulating key developmental factors in LP.PITX2C is a newly discovered transcription factor involved in hepatic differentiation and could increase HCC stemness by upregulating key transcriptional factors related to liver development.The online version contains supplementary material available at 10.1186/s13046-022-02424-z. Tumor cells are widely recognized to exhibit phenotypic similarity with lineage progenitor cells. Hepatocellular carcinoma (HCC) is one of the most common solid tumors worldwide with an inferior prognosis . CompareIn our previous study, an in vitro liver differentiation model from human embryonic stem cells (ES) was established . ES coulPITX2-V16 (PITX2A), PITX2-V245 (PITX2B), PITX2-V3 (PITX2C). PITX2A/B/C is produced by alternative splicing and transcription [PITX2A and PITX2B, PITX2C uses an alternative promoter [PITX2C is the dominant isoform in developing and adult left atrium [PITX2, a member of the bicoid/paired-like homeobox gene family, is a multifunctional transcription factor –14. PITXcription . Comparepromoter . All isot atrium . The rolt atrium –22. On tt atrium , 23–25. PITX2C was associated with AFP and differentiation in HCC clinical samples. PITX2C overexpression increased the stemness-related characteristics of HCC in in vitro and in vivo functional assays, while mechanistic studies revealed that PITX2C rather than PITX2A/B could activate TGF-β signaling by regulating the transcription of RALYL. Furthermore, PITX2C was found to regulate and cooperate with key factors related to hepatic differentiation and HCC tumorigenicity.In the present study, the functions of the three isoforms of PITX2 were characterized in HCC. The mRNA level of Two cohorts of patients with HCC were carefully evaluated histologically and included in this study. One cohort for checking the mRNA expression level by qRT-PCR included 93 pairs of primary HCC tissues and their matched non-tumor tissues collected after surgical resection at Sun Yat-sen University Cancer Center  between 2001 and 2005 (Cohort-1). The surgical specimens (both tumor and adjacent non-tumor tissue) were processed immediately after the operation and snap-frozen in liquid nitrogen for RNA extraction. The other cohort for pathological study of tissue microarray (TMA) is a retrospective cohort of 132 patients with HCC who also underwent hepatectomy at Sun Yat-sen University Cancer Center  between 2001 and 2008 (Cohort-2). The dissected tumor tissues were embedded in paraffin block and used for tissue microarray (TMA) construction. None of these patients were diagnosed with autoimmune diseases and received chemotherapy or radiotherapy before operation. The mean age of these enrolled HCC patients was 48 years . The median follow-up period was 34.5 months . Over 85% of these patients had hepatitis B virus infection. About 20% of patients had cirrhosis. Moreover, the clinical characteristics of these patients included age, sex, serum AFP, serum HBsAg, differentiation, tumor size, metastatic status . The stavivo functional assays was reported in our previous study [In vitro and us study . A detaiIHC staining was performed according to the standard procedure. Details could be found in The pGL3.0 luciferase expression system was purchased from Promega Corporation  and was performed according to the manufacturer’s instruction.A chromatin immunoprecipitation (ChIP) assay was performed as previously described . AntibodPITX2 expression with clinicopathological parameters as well as RALYL expression. Kaplan-Meier plots and log-rank tests were used for survival analysis. Data are presented as the mean ± SD of three independent experiments. A P value less than 0.05 was considered statistically significant.Statistical analysis was conducted using the SPSS software . Pearson’s Chi-square test was used to analyze the association of vitro hepatocyte differentiation model. The heatmap of the expression profiles together with the qRT-PCR results have demonstrated that the marker genes were highly specifically expressed in their corresponding stages [PITX2A/B/C contained varying 5′ sequences    of HCC tissues by qRT-PCR, compared with adjacent non-tumor tissues. However, PITX2C was rarely expressed, as the values ofΔCt (Ct PITX2C in tumor – Ct GAPDH in tumor) were over 25 in most of HCC cases. Only five cases showed stable expression of PITX2C  staining of HCC and their adjacent non-tumor tissues. Similarly, the transcript of PITX2C was rarely detected in non-tumor tissue  Fig. C. The prHA) Fig. D. Up-regX2C Fig. E. Specifion Fig. H. These PITX2A/B/C was examined at protein level   were increased in PITX2C overexpressing cells but reduced after PITX2 knockdown  regularly. When the subcutaneous tumor reached a similar size of approximately 5 mm in diameter, 5-Fu was administrated via intraperitoneal injection every 5 days. Tumors induced by PITX2C-transfected cells grew faster and were larger than those in the control group  to regulate hepatic differentiation  Heatmap of the expression profiles of the selected genes which were highly expressed in LP and PH. These genes included the specific genes for LP cells  and the others encoding nuclear protein which showed a similar expression pattern to LP markers. (B) Among those selected nuclear protein genes, PITX2 was located in the central of gene regulatory network (Pathway Common). (C) Screenshot from SeqMan browser (Lasergene software 7.0) showing the varing 5′ sequences of the full-length of PIT2XA/B/C . (D) Western blotting analysis confirmed the protein levels of PITX2 in immortalized liver cells and HCC cell lines. GAPDH was used as a loading control. (E) Representative images of FISH staining of PITX2C (red) in HCC cases with low, moderate and relative high expression levels of PITX2C. DAPI (blue) was used for nuclei counterstaining. (F) Kaplan-Meier overall (left) and disease-free (right) survival curve of two HCC groups in TCGA cohort: PITX2 (+), patients with higher PITX2 expression; PITX2 (−), patients with lower PITX2 expression. Supplementary Figure 2. PITX2A/B/C has distinct function in the tumorigenicity of HCC. Representative images of foci formation assay (A) and colony formation (B) in PITX2A/B/C-transfected cells and control cells. (C) Two shRNAs targeting PITX2 (shPITX2–1 and shPITX2–4) effectively decreased the mRNA level of PITX2 in PLC-8024 and SNU449 detected by qRT-PCR. Non-transfected cells were used as controls. Data are presented as the mean ± SD of 3 independent experiments.  (D) The cell proliferation between shPITX2 -transfected cells and control cells was compared by XTT assay. The results are expressed as the mean ± SD of three independent experiments. . Representative images (left) and summary bar chart (right) of foci formation assay (E) and colony formation in soft agar assay (F) in shPITX2-transfected and control cells. Values indicate the mean ± SD of 3 independent experiments . (G) Orthotopic tumor formation was performed via intrahepatic implantation experiments using PITX2A-transfected cells and control cells or shPITX2-transfected cells and control cells. The final tumor volumes are summarized in the dot chart. Average tumor volume is expressed as the mean ± SD of mice. The P value was calculated using paired Student’s t test. (H) Representative images of excised orthotopic tumor formed by intrahepatic implantation experiment using PITX2A-transfected Hep3B cells and control cells. Supplementary Figure 3. PITX2C promotes cell mobility, self-renewal and chemoresistance of HCC. (A) Representative images (top) and bar chart (bottom) of cell migration and invasion abilities in shPITX2-transfected and control cells by Transwell and Matrigel invasion assays. Migrated and invaded cells were stained with crystal violet and counted under a microscope. Values indicate the mean ± SD of three independent experiments . (B) Representative images of spheroid formation assay using shPITX2-transfected cells and control cells (left). The numbers of primary and secondary spheroids are calculated in the bar chart (right). Values indicate the mean ± SD of three independent experiments . The apoptotic indexes of PITX2C-transfected (C), shPITX2-transfected cells (D) and control cells were detected by fluorescence-activated cell sorting-based Annexin V/AAD double staining after treatment with 5-Fu or Sorafenib at the indicated concentrations for 48 h. (E) The mRNA levels of AFP and Lgr5 were compared by ΔCt in PITX2C or shPITX2- transfected cells and control cells . Supplementary Figure 4. Representatives of IHC staining images with anti-EPCAM, CD133, c-Myc and NANOG in tumors induced by 8024-Ctrl, 8024-PITX2C cells with 5-FU treatment. Red arrows indicate cancer stem cells. Supplementary Figure 5. Analysis of the ChIP sequencing data. (A) PITX2C shared similar binding motifs with several key transcription factors in LP. (B) Screenshot from the WashU epigenome browser showing PITX2C binding sites at the promoter of HNF1A, HNF4A, FOXA1, SMAD3, and ARID5B. (C) Heatmap of the expression profile for HNF4A, FOXA1, SMAD and ARID5B in the four stages  of in vitro hepatocyte differentiation model. (D) The expression of PITX2 is positively correlated with that of Wnt5α in GEPIA. Table S1. List of PCR primers for PITX2A/B/C expression.Additional file 2: Table S2. The gene ontology (GO) analysis of PITX2C ChIP-seq.