Chromatin remodelling and transcription factors play important roles in lineage commitment and development through control of gene expression. Activation of selected lineage-specific genes and repression of alternative lineage-affiliated genes result in tightly regulated cell differentiation transcriptional programmes. However, the complex functional and physical interplay between transcription factors and chromatin-modifying enzymes remains elusive. Recent evidence has implicated histone demethylases in normal haematopoietic differentiation as well as in malignant haematopoiesis. Here, we report an interaction between H3K4 demethylase JARID1A and the haematopoietic-specific master transcription proteins SCL and GATA1 in red blood cells. Specifically, we observe a direct physical contact between GATA1 and the second PHD domain of JARID1A. This interaction has potential implications for normal and malignant haematopoiesis. Histoneand a7Hoxa1, a5  [JARID1A  belongs to the KDM5 family of histone demethylases. It is an approximately 200 kDa, ubiquitously expressed nuclear protein . Given t5 and a7 . Increas5 and a7 , but theNUP98 resulted in fusion of its amino terminus with the C-terminus PHD motif (PHD3) of JARID1A [HOXA and HOXB gene clusters [Functionally, JARID1A is involved in normal and malignant haematopoiesis. Peripheral blood analysis of JARID1A knock-out mice showed neutrophilia, and analysis of their haematopoietic stem cell and myeloid compartments revealed a significant decrease in the rate of apoptosis as well as enhanced survival and cell cycling . Moreove JARID1A   cells . LMO2 isThis complex is a key driver of gene expression programmes in both embryonic and adult haematopoiesis . EmphasiIn the context of the pentameric complex, SCL serves both as a transcriptional activator and repressor in a context-dependent manner . This duGiven the documented interactions of JARID1A with LMO2, JARID1A's implication in AML and the fact that many protein partners of SCL, such as mSin3A and pRB ,26, are 2.2.1.−1 penicillin, 100 µg ml−1 streptomycin and 2 mM l-glutamine. MEL cells differentiation was induced by addition of 2% DMSO followed by incubation at 37°C for 4 days.MEL (clone 585) cells were maintained in Dulbecco's modified Eagle medium-high glucose (Gibco) supplemented with 10% FCS, 100 U ml2.2.g and washed once in 1× phosphate-buffered saline. Cell pellets were resuspended in chilled resuspension buffer , 1 mM dithiotreitol (DTT)). Cell suspension was incubated on ice for 10 min, mixed occasionally and vortexed for 10 s. The nuclei were pelleted by centrifugation for 1 min at 16 300g and resuspended in 20 mM HEPES, pH 7.9, 1.5 mM MgCl2, 420 mM NaCl, 25% glycerol, Complete EDTA free protease inhibitor (Roche), 1 mM DTT and incubated for 1 h at 4°C on a rotary shaker. Nuclear extracts were obtained by centrifugation at 16 300g for 5 min and supernatants aliquoted and stored at −80°C.For nuclear extract preparation, MEL cells were harvested by centrifugation at 10002.3.−1, 0.5 ml fractions were collected, precipitated with 72% trichloroacetic acid and subjected to western blotting.The 2.5 mg crude nuclear extracts isolated from MEL cells were subjected to fractionation on a Superose 6H/R column (Amersham Biosciences) equilibrated in 20 mM HEPES, pH 7.9, 200 mM NaCl, 1 mM DTT, 0.2 mM PMSF, 10% glycerol. At a flow rate of 0.5 ml min2.4.Western blotting analysis was performed using NuPAGE precast gels  according to the manufacturer's instructions. Primary antibodies used were: JARID1A , SCL , LDB1 , LMO2  and GATA1 . Secondary antibodies used were horseradish peroxidase conjugated anti-goat/mouse/rabbit immunoglobulin (Santa Cruz). Protein detection was carried out using ECL prime reagent kit (Amersham).2.5.−1 BSA (Pierce) and 0.4 mg ml−1 sonicated salmon sperm DNA (Life Technologies). Immunoprecipitations were performed overnight at 4°C by incubation of the pre-cleared nuclear extracts with primary antibodies  and blocked protein G Dynabeads in dilution buffer . Beads were washed five times with 4× bed volume of wash buffer  and bound material was eluted in 1× Laemmli buffer by boiling at 95°C for 10 min. Fractions were analysed by immunoblotting as described above.One milligram of nuclear extracts was diluted in dilution buffer  to attain a final concentration of 150 mM NaCl. Nuclear extracts were pre-cleared for 3 h at 4°C by incubation with normal IgG  and protein G Dynabeads (Life Technologies), blocked with 0.2 mg ml2.6.S-transferase (GST) tag or only an N-terminal hexahistidine (His) tag. The human SCL(180–253)/E47 (535–613) bHLH was cloned, expressed and purified as described [The three PHD domains of JARID1A (referred to as PHD1 (282–360), PHD2 (1156–1222) and PHD3 (1608–1659), respectively, in this paper) were amplified using JARID1A mouse cDNA (GeneArt) as template and primers 5′-aagttctgtttcagggcccgCGGAAGGGCACCCTGAGCG-3′/5′-atggtctagaaagctttaCTCGCGCACGGCCTGCTC-3′ (for PHD1), 5′-aagttctgtttcagggcccgGAACGGATCGAGGAAGTGAAGTTCTGC-3′/5′- atggtctagaaagctttaTCTGGGCCGTCTGCTCCG-3′ (for PHD2) and 5′-aagttctgtttcagggcccgGCCGTGTGTGCCGCCCAG-3′/5′-atggtctagaaagctttaGGCGCAGTTGATGCAGATGTAGTCC3′ (for PHD3) and subsequently cloned using ligation independent In-fusion system (Clontech) into pOPINJ or pOPINF vectors , encodinE47 535–6 bHLH wasEscherichia coli BL21 cells for protein expression. Cells were grown at 37°C in Luria Broth (LB) until absorbance at 600 nm reached 0.6. This was followed by induction of protein expression by addition of 1 mM isopropyl β-d-1-thiogalactopyranoside (IPTG) and growth at 20°C for 20 h. Cell lysis was carried out in lysis buffer  and the clarified lysate was mixed with glutathione beads. This was followed by overnight incubation at 4°C on a rotary shaker. Next day, beads were washed with the wash buffer . The over-expressed protein was eluted using the elution buffer . The eluted protein fractions were concentrated using a concentrator (Millipore) with a molecular weight cut-off (MWCO) appropriately chosen on the basis of the molecular weight of the protein. The concentrated proteins were further purified by size-exclusion chromatography, by loading on a HiLoad 16/60 Superdex 75 prep grade column  in 50 mM HEPES, pH 8, 200 mM NaCl, 1 mM DTT.The GST-Tag PHD1, PHD2 and PHD3 constructs were transformed into E. coli Rosetta (DE3) pLysS cells for protein expression. Cells were grown at 37°C in LB until absorbance at 600 nm reached 0.6. This was followed by induction of protein expression by addition of 1 mM IPTG and growth at 20°C for 16–20 h. Cells transformed with plasmid encoding GATA1NFCF were grown in LB autoinduction media [2). Cells were disrupted by sonication on ice and cell lysate clarified by centrifugation at 48 384g. The clarified cell lysate was incubated with Talon affinity resin (Clontech) equilibrated with lysis buffer for an hour at 4°C. The beads were subsequently washed with 10 bed volumes of 50 mM HEPES, pH 8, 400 mM NaCl, 10 mM Imidazole  and the protein was eluted in 50 mM HEPES, pH 8, 200 mM NaCl, 500 mM Imidazole . The eluant was concentrated using protein concentrator (Millipore) of appropriate MWCO (3 kDa for PHD2 and 10 kDa for GATA1NFCF). Both the proteins were further purified by size-exclusion chromatography using pre-equilibrated Superdex 75 (16/60)  in 50 mM HEPES, pH 8, 200 mM NaCl, 1 mM DTT. The purified proteins were assessed by SDS–PAGE and concentrated to be used for analytical ultracentrifugation (AUC).The PHD2 and GATA1NFCF constructs were transformed into on media  overnigh2.7.g  with 150 sample distribution scans being taken with an interval of 6 min using 280 nm absorbance optics. The 10–120th scans were analysed using SedFit software [c(s) model to calculate the sedimentation coefficients of the sedimenting species at a resolution of 100 and also using c method at a resolution of 10. The frictional coefficient is a unitless entity and is the ratio between the experimental frictional coefficient and the minimum frictional coefficient if the molecules in the sample were compact spheres (f/f0). Analysed data were then plotted using Pro Fit software  and fitted with a Gaussian function to determine peak centres, which represent the sedimentation coefficients of the species. Sedimentation coefficient (s) was plotted on the X-axis, continuous sedimentation coefficient distribution (c(s)) on the Y1-axis, molecular weight on the Y2-axis and c was plotted over a third dimension which was represented by a heat plot. All the samples were in 50 mM HEPES, pH 8, 200 mM NaCl and 1 mM DTT.Analytical ultracentrifugation sedimentation velocity experiments were performed using a Beckman Optima XL-I analytical ultracentrifuge operating in velocity mode at 20°C. All protein samples, at a molar concentration of 0.04 mM, were loaded onto 3 mm double sector-shaped centrepieces and spun at 29 024software  using co3.3.1.in vitro model of erythroid differentiation. Expression of endogenous JARID1A was detected in both undifferentiated MEL cells  and MEL cells undergoing terminal erythroid maturation (D2 and D4) (a). As SCL and protein partners are known to engage repressive activities in erythroid progenitors to prevent precocious lineage differentiation [b). Reciprocally, IP with antibodies against the components of the pentameric complex and IB with antibodies against JARID1A also revealed that SCL and GATA1, but not LDB1 or LMO2 coimmunoprecipitated with JARID1A (c). This is in contrast with previously published findings where JARID1A was found to be co-immunoprecipitated with in vitro translated radio-labelled LMO2 in MEL cells nuclear extracts [We first interrogated the possible association between JARID1A and the pentameric complex in the cell line MEL, a well-established  and D4) a. As SCLntiation ,23, we e JARID1A c. This iextracts . It is p3.2.In order to determine the molecular basis for the interaction of JARID1A with members of the pentameric complex, we first carefully selected the domains of each of these proteins likely to be involved in protein–protein interactions based on previous reports. We then analysed interactions between the selected domains by AUC.JARID1A, like other KDM5 family members, possesses an atypical insertion of a DNA-binding ARID (AT-rich) domain and a histone-binding PHD (PHD1) domain into the catalytic Jumonji domain, which separates it into two fragments (JmjN and JmjC) . It alsoScl-null background [Previous functional and structural data indicate that SCL/E47 bHLH and GATA1 zinc-finger domains mediate protein–protein interactions with ETO2/p300  and Fli-ckground , thus sta). An equimolar mixture of both proteins resulted in sedimentation coefficient peaks at 1.9 ± 0.08 and 4.3 ± 0.17 S (a). The first peak corresponds to unbound SCL/E47 bHLH, and while the second peak shows an increase in sedimentation coefficient. The model independent c and molecular weight analysis shows three species at 1.8 ± 0.27, 3.9 ± 0.2 and 4.5 ± 0.2 S representing unbound SCL/E47 bHLH, GST PHD1 and complex, respectively (d). The presence of three peaks suggests a mixture of different states and a dynamic interaction between SCL/E47 and GST PHD1. GST PHD2 (1156–1222) and SCL/E47bHLH individually showed sedimentation coefficients values of 3.8 ± 0.20 and 1.8 ± 0.10 S, respectively (b). When mixed in equimolar amounts, two well-defined peaks at 1.8 ± 0.10 and 3.8 ± 0.19 S can be observed, which overlap with the coefficients of the individual protein (b). This together with the model independent c and molecular weight analysis (e) suggests no complex formation. Apparent sedimentation coefficient analysis of GST PHD3 (1608–1659) and SCL/E47 bHLH showed peaks at 3.6 ± 0.07 and 1.9 ± 0.06 S, respectively (c). When mixed in equimolar amounts, two well-defined peaks at 1.9 ± 0.06 and 3.8 ± 0.10 S can again be observed (c). Together with the model independent c and molecular weight analysis (f), they suggest no complex formation.AUC was used to probe direct interactions between GST-fused JARID1A PHD domains and either HIS-tagged SCL(180–253)/E47(535–613) bHLH domains (SCL/E47 bHLH) or HIS-tagged GATA1 zinc-finger domains (GATA1NFCF) (201–313). We first determined the apparent sedimentation coefficients of GST PHD1 (282–360) and SCL/E47 bHLH separately, which showed sedimentation coefficients of 3.8 ± 0.09 and 1.9 ± 0.04 S (Svedbergs), respectively a. An equ± 0.17 S a. The fiectively d. The prectively b. When m protein b. This tanalysis e suggestectively c. When mobserved c. Togethanalysis f, they sa). An equimolar mixture of the two proteins yielded two peaks with values of again 1.5 ± 0.08 and 3.8 ± 0.10 S overlapping with the individual proteins' peaks, indicating no complex formation . GST PHD2 and GATA1NFCF separately displayed peaks at 3.9 ± 0.10 and 1.5 ± 0.06 S, respectively (b). When mixed in equimolar amounts, two well-defined peaks appeared at 1.5 ± 0.10 and 4.0 ± 0.30 S (b). The first peak probably originates from unbound GATA1NFCF, while the second broader peak extends further than the sedimentation coefficient peak of GST PHD2 alone. This result, together with the model independent c and molecular weight analysis (e), strongly suggests an interaction between GST PHD2 and GATA1NFCF. Finally, GST PHD3 and GATA1NFCF separately displayed peaks at 3.6 ± 0.07 and 1.5 ± 0.06 S, respectively (c). An equimolar mixture of the two proteins yielded two peaks overlapping with the individual protein peaks (c) which, together with the model independent c and molecular weight analysis (f), suggests no complex formation between GST PHD3 and GATA1NFCF.Next, we investigated interactions between the zinc-finger domains of GATA1 and the PHD domains of JARID1A. We first determined the apparent sedimentation coefficients of GST PHD1 and GATA1NFCF separately, which showed values of 3.8 ± 0.10 and 1.5 ± 0.06 S a. An equormation a,d. GST ectively b. When m± 0.30 S b. The fianalysis e, strongectively c. An equin peaks c which, analysis f, sugges15N HSQC NMR to probe the correct folding of the HIS-tag PHD2 construct. Well-dispersed, individual peaks can be distinguished in the 2D NMR spectrum  suggesting a folded protein. We then used AUC to study the apparent sedimentation coefficients of His-tag PHD2 and GATA1NFCF. HIS-tagged PHD2 and GATA1NFCF separately showed sedimentation coefficients of 1.4 ± 0.10 and 1.5 ± 0.05 S, respectively (a). An equimolar mixture of both proteins showed a sedimentation coefficient peak at 1.7 ± 0.06 S, a shift that is highly suggestive of complex formation (a). The model independent c and molecular weight analysis of absorbance data obtained from the equimolar mixture of PHD2 and GATA1NFCF resulted in two peaks (b). The first peak has a molecular weight of 15 kDa probably originating from unbound GATA1NFCF, while the second peak has a molecular weight of 20 kDa, probably deriving from the complex.To exclude any interference of the GST affinity tag, we further probed the interactions of a His-tag PHD2 domain of JARID1A. First, we used ectively a. An equormation a. The mowo peaks b. The fiIn conclusion, AUC experiments have revealed that the second PHD (PHD2) domain of JARID1A interacts directly with the zinc-finger domains of GATA1. Also, the data show that there is a possible direct interaction between the first PHD domain of JARID1A and the bHLH domains of SCL/E47, although this is likely to be highly dynamic in nature. As we have probed the interactions of the PHD (1–3) domains of JARID1A and, respectively, the bHLH domains of SCL/E47 and zinc-finger domains of GATA1, these experiments do not rule out interactions involving other parts of the proteins.4.Histone demethylase JARID1A is a recognized key factor for haematopoietic cell differentiation and proliferation and its disruption in the form of a fusion protein is prevalent in AML patients. Fusion proteins containing chromatin remodelling proteins often target histone-modifying activities to inappropriate genomic targets. An outstanding biological and mechanistic question is how JARID1A is normally targeted to the correct genomic loci.In this study, we report the interaction between JARID1A and members of the haematopoietic-specific pentameric protein complex (SCL and GATA1) in erythroid cells. When analysing putative interactions between known protein–protein interaction domains of JARID1A, SCL/E47 and GATA1, we show that this interaction could be directly mediated by GATA1 zinc-finger domains and the second PHD domain (PHD2) of JARID1A. Interestingly, the loss of both normal GATA1 and JARID1A function is linked with AMKL ,38,39, sIn conclusion, our data suggest that, in haematopoietic cells, JARID1A could be targeted to its genomic loci through interactions with a key component of the essential pentameric complex and thus participate in the control of expression of haematopoietic-specific genes and blood lineage differentiation. Further studies are required to elucidate whether JARID1A PHD2 domain is able to bind concomitantly histone tails and GATA1 and to further understand the functional relevance of JARID1A interaction with the SCL/GATA1 pentameric complex in the context of normal and aberrant haematopoiesis.


X, stimulates transcription from competence promoters, in vitro. As the only known alternative σ-factor in streptococci, σX expression is tightly controlled in each species and has a specific physiological role. Pneumococcal transformation also requires the DNA binding activity of ComW, a known σX activator and stabilizer. Mutations to the housekeeping σ factor, σA, partially alleviate the ComW requirement, suggesting that ComW is a key player in the σ factor swap during the pneumococcal competence response. However, there is no evidence of a direct ComW – RNA polymerase interaction. Furthermore, if and how ComW functions directly at combox promoters is still unknown. Here we report that a DNA-binding ComW variant, ComΔ6, can stimulate transcription from σX promoters in vitro.The alternative streptococcal σ-factor and master competence regulator, σ Streptococcus pneumoniae (pneumococcus), an inhabitant of the human nasopharynx, is a causative agent of pneumonia and meningitis in children, the elderly, and immune-compromised individuals. Natural genetic transformation (NGT) was discovered in the pneumococcus , a member of the σ70 family of proteins  basally transcribes comC leading to production of the 41 aa pro-peptide, ComC, 41 (comAB). Extracellular CSP is sensed by the histidine-kinase receptor, ComD, resulting in auto-phosphorylation and activation of its cognate response regulator, ComE, via a phospho-relay event (comCDE and other early competence genes (X (X – RNA polymerase (σX holo-enzyme) transcribes from the competence specific combox promoter, directly linking ComCDE quorum sensing to the expression of the transformation regulon  transcribe late genes and transform at levels 10- and 10,000-fold below that of wild type cells, respectively (comW cells have decreased σX levels (X weakly interact in yeast-2-hybrid assays (A that presumably decrease its affinity for RNA polymerase (RNAP) alleviate the ComW requirement, indicating that ComW functions on or with the σX holo-enzyme. Beyond this, there is currently no evidence of a physical interaction between ComW and RNAP.Although σ protein , is requ of ComW  and σX a of ComW . SigX’s ectively . FurtherX levels , and Comd assays , suggest73RGFISC78, as demonstrated with the truncated variant, ComWΔ6, although ComWΔ6 producing cells transform at levels slightly below wild type cells   . LB was X and σA were expressed from pNLI94 and pNLI114, respectively. E. coli transformed with pNLI94  or with pNLI114  were cultured in 3 L of LB medium at 37°C, 200 rpm to an OD600 0.5. To prepare a sample for SDS-PAGE, 2 mL of uninduced cells were collected and pelleted for 10 min at room temperature, and then boiled at 95°C for 2 min in a 3:1 mixture of E. coli resuspension buffer  and Laemmli’s buffer (Bio Rad), respectively, then stored at 4°C. The large culture was induced for protein expression by addition of [1 mM]f isopropyl β-D-1-thiogalactopyranoside (IPTG) (Gold Biotechnology). Induced cultures were incubated overnight at 20°C, 200 rpm. The next day, 2 mL of induced culture was prepped for analysis by SDS PAGE, and the large cultured collected at 5,000 rpm, 4°C, for 30 min. Cells were resuspended in 50 mL of E. coli resuspension buffer supplemented with 25 mM imidazole, 50 mM MgCl2, 100 μg/mL DNase, and a protease inhibitor tablet (Roche). The cell resuspension was lysed using an Emulsiflex-C3 (Avestin). The soluble fraction was separated by centrifugation at 30,000 × g for 30 min at 4°C.Pneumococcal ComWΔ6 was expressed from pNLI60 and purified according to . PneumocX purification, the soluble fraction was discarded. The pellet was washed in 15 mL of cold, filtered 20 mM L-arginine, 1 mM EDTA, 5% glycerol  supplemented with 1 mM βME and 2% w/v sodium deoxycholate (NaDOC) (Sigma Aldrich) with a homogenizer, followed by sonication for 2 min at 65% amplitude . The washed pellet was spun was 30,000 × g for 30 min at 4°C. Washing, sonication, and spinning were repeated. Then the pellet was resuspended and dissolved in 15 mL of RGE supplemented with 1 mM βME and 0.6% N-lauroyl sarcosine sodium salt (Sigma-Aldrich) at 4°C with slow stirring for up to 2 days. The dissolved inclusion body was collected by centrifugation at 30,000 × g for 30 min at 4°C. The supernatant was saved and dialyzed for 8 h at room temperature in 2 L of 25 mM Tris-HCl, pH 8.3, 500 mM NaCl, 30 mM L-arginine, 10% glycerol, and 5 mM βME (CB4), and then overnight at 4°C in 2 L of CB4. The soluble supernatant was passed through a 5 mL Ni-NTA resin (Thermo Fisher Scientific) on a column equilibrated with CB4 (no βME) via gravity flow. The column was washed with 500 mL of CB4 supplemented with 5 mM βME and 40 mM imidazole. SigX-V5H6 was eluted from the column with 10 mL of CB4 supplemented with 5 mM βME and 275 mM imidazole. Subsequently, the flow through was saved and concentrated using a Vivaspin 15R 2,000 MWCO column concentrator (Sartorius). The concentrated solution was applied to a pre-packed Superdex 200 column (GE) on an AKTA (Amersham Biosciences). SigX-V5H6 was eluted at 0.3 mL/min in CB4 supplemented with 1 mM βME and collected in 1 ml fractions for analysis on SDS-PAGE gels. The fractions were pooled and concentrated again using a VivaSpin column concentrator. Protein concentration was measured at 5.6 mg/mL using a Nanodrop. Purified SigX-V5H6 was diluted to 1.2 mg/mL in CB4 supplemented with 1 mM EDTA, 1 mM βME and 20% glycerol for storage at −20°C until use.For σA, the soluble fraction was separated by centrifugation at 30,000 × g for 30 min at 4°C. The soluble supernatant was passed through a 5 mL Ni-NTA resin (Thermo Fisher Scientific) on a column equilibrated with Column Buffer  via gravity. The column was washed with 500 mL of Column Buffer supplemented with 5 mM βME and 40 mM imidazole. σA was eluted from the column with 10 mL of column buffer supplemented with 5 mM βME and 275 mM imidazole. The eluate was concentrated using a Vivaspin 15R concentrator column into 50 mM Tris-HCl pH 8.0, 20 mM L-arginine, 500 mM NaCl, 1 mM EDTA, 10% glycerol, and 1 mM βME (DBR buffer). Protein concentration was measured as 1.85 mg/mL using a Nanodrop. SigA-V5H6 was diluted to 1 mg/mL in DBR supplemented with 20% glycerol and stored at −20°C until use.For purification of σamiA, comEA, and ssbB were taken from the genome of S. pneumoniae R6 (NC_003098.1). Oligonucleotides were synthesized by IDT . Equal amounts (1 μM) of forward and reverse primers were used to amplify amiA (NL299/NL300), comEA (NL295/NL296), and ssbB (NL228/NL229) from 50 ng of CP2137 genomic DNA with 0.2 mM dNTP (Thermo Fisher Scientific) and 1 μL of Phire Hot Start DNA Polymerase II (Thermo Fisher Scientific) in 50 μL reactions in a thermocycler. PCR products were purified with Zymo kits, eluted in diethyl pyrocarbonate (DEPC) H2O and visualized on a 1% agarose gel prior to storage at −20°C.Primers used in this study are listed in In vitro transcription from linear templates was carried out using 100 nM of E. coli RNAP obtained from NEB, pneumococcal σX and σA purified from E. coli, and ComWΔ6 purified from E. coli, when needed. To determine the minimum concentration of each σ-factor required to stimulate RNAP, initiation complexes were formed with 100 nM of E. coli RNAP, 20 nM of ssbB or amiA, and 0–200 nM of σX or σA, in 50 mM Tris-acetate, 50 mM sodium-acetate, 12.5% glycerol, 0.25 mM EDTA, 15 mM magnesium-acetate, and 35 mM βME (4X TGA buffer). Transcription from plasmid templates was carried out using 0–400 nM of E. coli RNAP and 0–320 nM of σA or σX. Solutions were incubated at 37°C for 15 min in a thermocycler, followed by addition of 1 μL of a master mix containing (120 nM) ATP, CTP, and GTP (Thermo Fisher Scientific) with αP32UTP at 0.5 μCi/μL . Runoff transcription was allowed to proceed for 6 min at 37°C in a thermocycler, followed by addition of 20 nM cold UTP (0.5 μL of a 200 nM stock) and a final elongation step at 37°C for 10 min in a final volume of 5.5 μL. Reactions were stopped by addition of one volume of formamide dye  and incubation at 65°C for 2 min in a thermocycler. The samples were placed on ice until gel loading.E. coli RNAP, 80 nM σX or σA, 20 nM of comEA, ssbB, or amiA, and 0–160 nM of ComWΔ6 were mixed in 50 mM Tris-acetate, 50 mM sodium-acetate, 12.5% glycerol, 0.25 mM EDTA, 15 mM magnesium-acetate, and 35 mM βME (4X TGA buffer). Solutions were incubated at 37°C for 15 min in a thermocycler, followed by addition of 1 μL of a master mix containing (120 nM) ATP, CTP, and GTP (Thermo Fisher Scientific) with αP32UTP at 0.5 μCi/μL . Runoff transcription was allowed to proceed for 6 min at 37°C in a thermocycler, followed by addition of 20 nM cold UTP (0.5 μL of a 200 nM stock) and a final elongation step at 37°C for 10 min in a final volume of 5.5 μL. Reactions were stopped by addition of one volume of formamide dye  and incubation at 65°C for 2 min in a thermocycler. The samples were placed on ice until gel loading.To measure transcription stimulation by ComWΔ6 on PCR templates, 100 nM of in vitro transcription using the MAXIscript T7 Transcription Kit (Invirtogen) and the RNA Century-Plus Marker Templates (Invitrogen).To visualize mRNA transcripts, 2 μL of each reaction was run on a 20 × 20, 0.375 mm thick, 6% polyacrylamide urea gel at 20 watts for 45 min. Gels were dried for 40 min at 80°C and incubated on a phosphorimager and imaged on a Typhoon 3000 (GE). The expected mRNA transcript sizes were determined using the transcriptional start sites (TSS) for each gene as determined by In vitro transcription assays confirmed that σX, in molar excess to RNAP, promotes transcription from combox promoters  assays.romoters . However in vivo . Previouectively . Althoug-factors , so exceresponse , and mutenotypes . These d partner , interac partner . We hypoX to pneumococcal RNAP (RNAPSp) in their IVT assays, making the number of molecules of σX higher than that of RNAPSp  to the pneumococcal combox and housekeeping promoters, in vitro.Luo and Morrison used a ∼1.7 molar excess of σssbB and amiA were PCR amplified for use in in vitro transcription assays  mRNA product from 20 nM of an ssbB linear template was produced, although the amount of mRNA produced  allowed for observable, yet sub-optimal transcription from 20 nM of linear ssbB DNA template containing a combox promoter, without ComW. In addition, 80 nM of σX + 100 nM RNAPEcoli did not produce an mRNA product when incubated with 20 nM of linear amiA template, a gene under control of the primary σ-factor, σA (not shown), demonstrating that σX transcription specifically initiates from the combox promoter.The promoter regions of pneumococcal n assays . At 40 nproduced . The amoA produced detectable levels of a 347-base mRNA product from the housekeeping promoter upstream of amiA when incubated with RNAPEcoli . This might result from an orphan -10 promoter region σA binding site that is present 29 bp downstream of the ssbB transcription start site, according to Ecoli to pneumococcal promoters, in vitro. In addition, as it appeared that more σX was required to stimulate RNAPEcoli activity at combox promoters than σA at housekeeping promoters, these results might indicate that more σX is needed to stimulate RNAPSp. As 80 nM of σX and σA are below saturating concentrations but still produced visible ssbB and amiA transcripts, respectively, we used 80 nM of each σ-factor in subsequent reactions.In contrast at 20 nM, pneumococcal σNAPEcoli . The amoin vitro transcription with pneumococcal σ factor, we tested transcription from plasmid templates. Supercoiling can affect bacterial gene transcription  produced 2X as much mRNA signal from the sbbB template compared to reactions with 0 nM ComWΔ6, an increase that was statistically significant  ComW might function as a component of a two-part σ factor with σX and RNAP as an active member of the holo-enzyme.The DNA – binding protein, ComW does stimulate σunctions . (A) As The datasets generated for this study are available on request to the corresponding author.NI conceived, conducted experiments and wrote the manuscript. DM edited the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Botrytis cinerea in Arabidopsis thaliana and how metabolism and reactive oxygen species (ROS) are influenced after GABA treatment and infection. We show that GABA already primes disease resistance at low concentrations (100 µM), comparable to the well-characterized priming agent β-Aminobutyric acid (BABA). Treatment with GABA reduced ROS burst in response to flg22  and oligogalacturonides (OGs). Plants treated with GABA showed reduced H2O2 accumulation after infection due to increased activity of catalase and guaiacol peroxidase. Contrary to 100 µM GABA treatments, 1 mM exogenous GABA induced endogenous GABA before and after infection. Strikingly, 1 mM GABA promoted total and active nitrate reductase activity whereas 100 µM inhibited active nitrate reductase. Sucrose accumulated after GABA treatment, whereas glucose and fructose only accumulated in treated plants after infection. We propose that extracellular GABA signalling and endogenous metabolism can be separated at low exogenous concentrations.The stress-inducible non-proteinogenic amino acid γ-aminobutyric acid (GABA) is known to alleviate several (a)biotic stresses in plants. GABA forms an important link between carbon and nitrogen metabolism and has been proposed as a signalling molecule in plants. Here, we set out to establish GABA as a priming compound against  The non-proteinogenic amino acid γ-aminobutyric acid (GABA) makes up a major part of the free amino acid pool and can be found in all living organisms. Besides its wide distribution over different kingdoms of life, the metabolic enzymes involved in GABA metabolism are also highly conserved among organisms . Plants Lemna rhizoid growth, with effects already being observed in the 1–10 µM range [In animals, it is well known that GABA is closely associated with metabolic pathways and acts as an inhibitory neurotransmitter through GABA receptors . In planµM range . GABA alµM range ,17,18,19µM range ,22,23,24µM range . GABA isµM range ,26,27. DµM range . GABA alµM range ,30,31,32Arabidopsis [Brassica napus [The complex metabolic and signalling functions of GABA are believed to be intricately linked and intertwined in plants and are thus difficult to disentangle from each other . In planbidopsis  and Brasca napus . Most ofca napus . It is tca napus ,38,39, lca napus  and 14-3ca napus .2+ [2+ in the cytosol is thus closely associated with GAD activity and GABA accumulation [B. cinerea in the leaves and fruits of plants [During stress, GABA originates from the breakdown of Glu through GAD, which is activated by an increase in cytosolic Ca2+ . Pathogemulation ,42. GABAmulation ,32. Simimulation . Such prf plants ,43. Besif plants ,43.2+-mediated activation of the GABA shunt under UV stress was responsible for ROS detoxification [Caragana intermedia plants during salt stress [A striking link exists between GABA and reactive oxygen species (ROS) homeostasis in plants. The GABA shunt effectively bypasses two enzymes of the TCA cycle that are sensitive to oxidative stress . Thus, pfication . Severalfication ,45,46. It stress . Intrigut stress ,47. Undet stress ,32. Thist stress ,48. Thust stress ,49.B. cinerea in Arabidopsis, discriminating between GABA signalling and metabolic effects. The effect of GABA pre-treatment on elicitor-induced ROS burst was also studied. We further investigated the concentration-dependent effects of exogenous GABA on sugar and nitrogen metabolism in response to B. cinerea infection, and how this correlates with endogenous GABA levels. We discuss our results in favour of low exogenous GABA acting as a stress signal and high exogenous GABA as a non-stress signal.Altogether, GABA clearly forms an integral part of the response to abiotic and biotic stresses in plants by regulating key metabolic and stress response pathways. Emerging evidence points to a dual role for GABA, both acting as a metabolite and signalling entity. The aim of this study was to further explore this duality during GABA priming against Arabidopsis thaliana plants of the Col-0 ecotype were used in all experiments. Seeds were dispensed onto square pots (9 × 9 × 8 cm) containing potting soil and vermiculite in a 3:2 ratio so that each pot contained 5 plants. Plants were grown in a Conviron®  growth chamber under cool white fluorescent lamps of 100 μmol photons m−2 s−1 with a light cycle of 12 h light (21 °C) and 12 h dark (18 °C).Arabidopsis (4–5 weeks old) soil-grown plants were used for priming experiments as explained [2O containing 0.0001% Tween-20) treatment using a spraying bottle. Each pot was gently sprayed with 5 mL of solution at a distance of +/− 15 cm until the surface of the plant was completely covered. All priming agents were prepared in sterile ddH2O containing 0.0001% Tween-20  as surfactant.xplained . PrimingB. cinerea B05.10 strain was used [B. cinerea was cultivated on 24 g/L potato dextrose agar (PDA) for 14 days in the dark at 21 °C prior to harvesting. Spores were harvested using ddH2O containing 0.0001% Tween-20. The harvested spores were then filtered through sterilized glass wool to remove any residual hyphae. Infection buffer was prepared by diluting the spores to 1 × 105 spores per mL in sterile 12 g/L potato dextrose medium. Infection control (IC) solution contained potato dextrose without spores. Spores were incubated in the infection buffer for 4 h before infection to allow synchronous germination.In all experiments, the was used ,51. To o2O, and dried by blotting with paper towel. Leaves were placed with the adaxial side upwards in a square petri dish  lined with moist paper. The leaves were infected individually by adding 5 µL of infection buffer or IC buffer to the tip of each leaf using a pipette. Plates were sealed with parafilm and placed in an infection room with a 12-h day and 12-h night cycle and maintained at 18 °C. A representative image of lesions after 72 h of infection can be found in the https://imagej.nih.gov/ij/).Infections were performed as described . Three s2O. The plate was covered in aluminium foil and incubated in the growth chamber for 12–16 h for recovery. Subsequently, water was replaced with 100 µL of incubation solution , and 1 µg/mL horseradish peroxidase  prepared in ddH2O). Baseline ROS production was determined by measuring the luminescence for 30 min using a GloMax®-Multi Detection System . Subsequently, 100 µL of assay solution (incubation solution with elicitor) were added to all the wells and luminescence measured for 60 min at 2-min intervals with an integration time of 0.5 s. Elicitors were added to final concentrations of 100 nM for flg22 [To quantify the ROS burst, a method relying on the oxidation of L-012 was used ,52. Leafor flg22 . OGs weror flg22 . Backgro2O2 was carried out by using the eFOX method [2O2. Frozen plant material was ground in 20 volumes of ice-cold 5% trichloroacetic acid (TCA) using a mortar and pestle. Extracts were centrifuged at 15,000× g at 4 °C for 10 min, and 500 µL of extract added to 500 µL of eFOX reagent , 200 mM sorbitol, 200 µM xylenol orange , and 1% EtOH prepared in 50 mM H2SO4). Reactions were incubated for 30 min at room temperature and the OD measured at 550 and 800 nm using a spectrophotometer . The concentration of H2O2 was calculated using a standard curve prepared from 30 % H2O2  in a range between 0 and 200 µM.The extraction and quantification of HX method . SamplesArabidopsis leaves were ground in liquid nitrogen using a mortar and pestle. In total, 100 mg of material were combined with 300 µL of extraction buffer (100 mM phosphate buffer (pH 7.0), 0.1% Triton X-100, 15% glycerol, 1 mM PMSF, 1 mM ascorbic acid, and 0.35 mM β-mercaptoethanol) and enzymes extracted by grinding with a plastic micro pestle inside a 1.5-mL Eppendorf tube for 30 s. Extracts were then centrifuged for 15 min at 10,000× g at 4 °C. Enzymes were kept at −80 °C until use, except for APX, which was measured immediately.Antioxidant enzymes were extracted as previously described . Arabido2O2 to start the reaction, and the decrease in OD240 measured for 5 min at 10 s intervals on a Spectronic Genesys 5 spectrophotometer . Areas of linear breakdown of H2O2 were considered to determine the activity. Blanks consisted of a general control (buffer and H2O2) and sample control (buffer and enzyme extract).Catalase (CAT) activity was measured as described previously ,57. Enzy2O2. The oxidation of ascorbic acid was measured as the decrease in OD at 290 nm for 5 min at 10 s intervals. A second blank consisted of reaction mixture without the addition of enzyme. Activity was determined in the linear range.Ascorbate peroxidase (APX) activity was assayed as described ,59. Reac2O2 to initiate the reaction. The increase in absorbance at OD480 was measured for 5 min at 10 s intervals. The second blank contained no enzyme.Guaiacol peroxidase (GPX) activity was measured using a colorimetric method with guaiacol as substrate . Previou−1, where 1 U is equal to the change in OD of 0.01 per min.For all enzyme reactions, the activity was calculated from the linear range of the reaction and enzyme activity is expressed as units (U)·mg protein2O by boiling for 15 min at 95 °C. Samples were vortexed and centrifuged for 10 min at 15,000× g and 200 µL of supernatant applied to a Dowex®  anion and cation exchange column. Columns were subsequently rinsed 6 times with 200 µL of ddH2O. The flow through was diluted 1:1 in 20 µM rhamnose H2O as the internal standard. Samples were analysed by HPAEC-IPAD Dionex 5000  with separation on a CarboPacTM PA100 column (Thermo Scientific) and a mobile phase of 90 mM NaOH as described [Soluble sugars were extracted and quantified as before . Ground 2O and boiling for 15 min. Samples were centrifuged for 10 min at 15,000× g and supernatantly diluted (1:1) with 50 µM Nor-Valine . Amino acids were analysed by reverse-phase HPLC  by derivatization using o-Phtalaldehyde before injection. Separation was carried out using a YMC Triart C18  column and mobile phases consisting of buffer A  and buffer B (Acetonitrile:MeOH:water 45:40:15) using the following gradient: 96% A and 4% B from 0 to 6 min; 92% A and 8% B from 6 to 18 min; 85% A and 15% B from 18 to 32 min; 67% A and 33% B from 32 to 50 min; 100% B from 50 to 53 min. Amino acids were detected using a fluorescence detector at λex = 230 nm and λem = 450 nm.Amino acids were extracted from 100 mg of ground leaf material by adding 1 mL of ddHv/v) glycerol, 0.1% Triton X-100, 10 mM MgCl2, 1 mM EDTA, 1 mM benzamidine, 1 mM ε-aminocapronic acid, 1 mM PMSF, 1 × cOmplete™ Protease Inhibitor Cocktail , 1 mM DTT, and 20 µM flavin adenine dinucleotide). Samples were centrifuged at 13,000× g (4 °C) for 5 min and the supernatant used in reactions. Assays were performed by adding 100 µL of extract to 450 µL of assay buffer. Assay buffers consisted of 50 mM HEPES-KOH pH 7.5, 0.04% Triton X-100, 10 µM Na2MoO4, 0.5 mM DTT, and 20 mM KNO3, with the addition of 10 mM MgCl2 for the selective NR (active) reaction and 5 mM EDTA for the total NR reaction. The reaction mixture was incubated for 2 min at 25 °C before 50 µL of 3 mM NADH was added to start the reactions. Reactions were stopped by adding 50 µL of 0.6 mM zinc acetate. Blanks for both selective and total NR reactions were included by the addition of zinc acetate before adding enzyme. Excess NADH was removed by adding 75 µL of phenyl methosulphate to the reactions and by incubating for 15 min in the dark. Finally, 300 µL of 1% sulphanilamide in 3 N HCl and 300 µL of 0.02 % N-(1-napthyl)-ethylenediamine were added and incubated for 20 min for colour development. Samples were centrifuged at 14,000× g for 5 min and the supernatant used to measure the azo-dye formed at an OD of 540 nm using a Multiskan Ascent 96/384 Plate Reader . The concentration of nitrite formed per reaction was interpolated from a standard curve prepared between 0 and 200 µM using NaNO2.Nitrate reductase (NR) activity was measured as described previously . Leaf saB. cinerea 72 h after treatment. Disease progression was analysed 72 h after infection (144 h after treatment). Samples were harvested at 3, 24, and 72 h (time of infection) after treatment and 24 h after infection (96 h after treatment). Elicitor-induced ROS burst experiments were carried out 24 h after treatment. All samples were obtained from the source leaves (rosette leaves 5–7) of plants between 4 and 5 weeks old. Examples of the lesion developed after 72 h of infection can be found in The experimental setup is depicted in https://inkscape.Org). Statistical analysis was performed using GraphPad Prism version 8.0.0 for Windows, GraphPad Software, San Diego, CA, USA, www.graphpad.com.Graphs were prepared using GraphPad Prism 8.0.0 and Inkscape 1.0  plants were sprayed with either 100 µM or 1 mM GABA and compared to a positive control β-aminobutyric acid (BABA) at the same concentration. A period extending up to 72 h between treatments and infection was considered to allow for both signalling and metabolic effects  NR activity. Our data indicate that GABA differentially regulates NR activity depending on the concentration. Contrary to 100 µM exogenous GABA, 1 mM stimulated the total NR activity 24 h after treatment A. The acB. cinerea infection, we analysed the levels of glucose (Glc), fructose (Fru), and sucrose (Suc) in the source leaves after leaf spraying plants with 100 µM and 1 mM GABA and BABA, in comparison to untreated and H2O controls.It is surprising that there are few data available on the interplay between GABA and soluble sugar metabolism and signalling. To establish whether exogenous GABA affects soluble sugar content after treatment and B. cinerea infection, plants pre-treated with GABA at both 100 µM and 1 mM showed significantly higher levels of Glc, Fru, hexoses, and total sugars  against the necrotrophic fungus B. cinerea (B. cinerea infection (Higher concentrations of exogenous GABA (1–10 mM) are able to induce resistance in tomato leaf disks and fruits against  cinerea ,72. Our  cinerea . Identicnfection A, althouCaragana intermedia [B. cinerea, the timing and distribution of plant ROS production in response to B. cinerea is crucial for plant resistance. Our data also showed a concentration-dependent regulation of NADPH-oxidases biotic stress is required to induce ROS scavenging enzymes [In contrast to BABA, treatment with GABA did not significantly affect the levels of H cinerea A,B. Thisstresses ,45,46. R cinerea C–E. Our 2+ systemic waves involved in plant defence signalling [It has been demonstrated that 10 mM exogenous D6-GABA is rapidly taken up and metabolized into D4-succinic acid in leaves, while root feeding resulted in a fast distribution of D6-GABA over the whole plant . Thus, tgnalling ,89.2+, which is known to activate several defence responses [With the notion that GABA signalling and metabolism can be separated at specific concentrations, we found that exogenous GABA at 1 mM promoted both the total and active NR activity whereas 100 µM GABA inhibited active NR activity . The incesponses ,91, but esponses . On the esponses , which aesponses . It is iConnections between GABA and sugar metabolism/signalling are poorly established. Suc signalling events are typically associated with temporal fluctuations in Suc levels, which often involves the production of secondary metabolites with antifungal activity ,94. As sB. cinerea at low concentrations. Several responses can be differentially regulated based on the concentration of GABA, suggesting GABA priming can be performed without inflicting major metabolic pathways. It will be interesting to repeat our experiments on plants lacking an array of GABA membrane transporters to better discriminate between extracellular and intracellular signalling events. In general, GABA promoted the detoxification of ROS through ROS scavenging enzymes, and increased GABA metabolism and sugar accumulation after infection with B. cinerea. This indicates that increasing GABA content signals/promotes metabolic processes and strategies that prevent toxification and cell death. Our data support a role of low GABA concentrations to be perceived extracellularly as stressors and higher concentrations to be taken up and act as a signal of favourable (nutrient availability) conditions. Knowing that mammalian GABA receptor antagonists also abolish the effect exerted by GABA in plants, it is possible that shared mechanisms are into play. As such, it is possible that GABA signalling can be translated to distal tissues and organs through constant secretion/uptake of GABA between cells, as it is the case in animals.Our data support GABA as an effective priming agent against


Bone metastases are common in prostate cancer (PCa). Fluorocholine-18 (FCH) and sodium fluoride-18 (NaF) have been used to assess PCa associated skeletal disease in thousands of patients by demonstrating different mechanism of uptake-cell membrane (lipid) synthesis and bone mineralization. Here, this difference is characterized quantitatively in detail. Our study cohort consisted of 12 patients with advanced disease (> 5 lesions) (M) and of five PCa patients with no skeletal disease (N). They had routine PET/CT with FCH and NaF on consecutive days. Skeletal regions in CT were used to co-register the two PET/CT scans. Bone 3-D volume of interest (VOI) was defined on the CT of PET with a threshold of HU > 150, and sclerotic/dense bone as HU > 600, respectively. Additional VOIs were defined on PET uptake with the threshold values on both FCH (SUV > 3.5) and NaF (SUV > 10). The pathologic skeletal volumes for each technique , NaF (SUV > 10) and FCH (SUV > 3.5) were developed and analyzed. The skeletal VOIs varied from 5.03 L to 7.31 L, whereas sclerotic bone VOIs were from 0.88 L to 2.99 L. Total choline kinase (cell membrane synthesis) activity for FCH (TCA) varied from 0.008 to 4.85 [kg] in M group and from 0.0006 to 0.085 [kg] in N group. Total accelerated osteoblastic  activity for NaF (TBA varied from 0.25 to 13.6 [kg] in M group and varied from 0.000 to 1.09 [kg] in N group. The sclerotic bone volume represented only 1.86 ± 1.71% of the pathologic FCH volume and 4.07 ± 3.21% of the pathologic NaF volume in M group, and only 0.08 ± 0.09% and 0.18 ± 0.19% in N group, respectively. Our results suggest that CT alone cannot be used for the assessment of the extent of active metastatic skeletal disease in PCa. NaF and FCH give complementary information about the activity of the skeletal disease, improving diagnosis and disease staging. Prostate cancer (PCa) is the second most common male cancer and bone is the most frequent metastatic site. Approximately 10–30% of men with prostate cancer will present with incurable advanced or metastatic disease. Patients who develop metastatic disease are commonly treated with luteinizing hormone-releasing hormone agonist/antagonist therapy, which frequently leads to tumor regression . UnfortuFluorocholine is a common radiotracer, used especially in Europe. A high progression in cancer growth demands increased lipid synthesis for the formation of new cellular membranes: this stimulates choline transporter and the cellular uptake of choline. In the cells, choline is transformed into phosphotidylcholine (lecithin) . Thus, t18F-NaF PET/CT in the US -NaF-PET and [18F]-fluorocholine-PET as a part of clinical staging on consecutive days as a part of their diagnostic program.Prostate cancers were diagnosed between 1999 and 2015. In this retrospective one institute analysis we included all those patients who had both [18F]-NaF-PET and [18F]-fluorocholine-PET in all patients; most (10 out of 12) of the patients also had pelvic MRI and diagnostic whole body CT. These patients had to have more than five identifiable active metastases on both PET studies .Twelve new consecutive patients were analyzed between August 2015–December 2015 without any further selection. Men’s ages ranged from 57 to 86 years; Gleason scores were 6–10; initial PSA values varied from 9.8 to 790 μg/L, and all patients had T3-T4 disease. In addition, all patients had skeletal metastases, and three of them had visceral metastases. At Docrates Cancer Center in Helsinki, the radiological TNM staging was performed using -NaF-PET and [18F]-fluorocholine-PET on consecutive days. The men’s ages ranged from 50 to 93 years; Gleason scores were 6–9; initial PSA values varied from 7.1 to 73 μg/L, and these patients had T1c-T4 disease.Five patients with prostate cancer who had no skeletal metastases served as controls and their characteristics are summarized in 18F-FCH-PET/CT at Docrates Cancer Center using TruePoint PET-CT . Patients were injected with 18F-FCH, using an average activity of 280 MBq (range 210–402 MBq). Imaging was done in two phases, early whole body imaging at an average time of 11 ± 4 min post injection, and late imaging of the pelvis at an average time of 64 ± 5 min post injection. Whole body imaging covered the area from the calvarium to the mid-thighs.All the patients were studied for fluorocholine 18F)-PET/CT imaging was performed at 62 min (range 58–76 min) as a whole body imaging  with an average activity of 222 MBq (range 192 to 251 MBq).For sodium fluoride . The tracers were manufactured and supplied by MAP Medical Technologies Oy, Tikkakoski, Finland.The skeleton VOI was segmented by selecting voxels with HU > 150 on the CT scans from FCH and NaF PET/CT scansA rigid registration of the two skeleton VOIs from FCH PET/CT and NaF PET/CT scans was performed. Now the two PET and CT scans are spatially co-registered in a single frame of reference. The skeleton VOI was labeled as “Skeleton” for figures.A subset VOI of sclerotic (pathologic) bone was created by selecting voxels with HU > 600 within the skeleton VOI—labeled as “Skeleton 600”.A PET based VOI labeled “FCH PET 3.5” was created that is the fusion of voxels with FCH PET SUVbw > 3.5 and the “Skeleton” VOI.A PET based VOI labeled “NaF PET 10” was created that is the fusion of voxels with NaF PET SUVbw > 10 and the “Skeleton” VOI.A PET based VOI labeled “NaF 10 FCH 3.5” was the fusion of “FCH PET 3.5” VOI and “NaF PET 10” VOI.A PET based VOI labeled “Scl FCH 3.5” was the fusion of “FCH PET 3.5” VOI and “Skeleton 600” VOI.A PET based VOI labeled “Scl NaF 10” was the fusion of “NaF PET 10” VOI and “Skeleton 600” VOI.A PET based VOI labeled “Scl NaF 10 FCH 3.5” was the fusion of “Scl FCH 3.5” VOI and “Scl NaF 10” VOI.Image segmentation for evaluation of functional volumes were performed using MIM Maestro v6.5 . The image analysis processing steps were as follows:10 criteria presented in the literature [In summary, we created eight VOIs for each patient based on spatially co-registered FCH PET/CT and NaF PET/CT scans; namely, “Skeleton”, “Skeleton 600”, “FCH PET 3.5”, “NaF PET 10”, “NaF 10 FCH 3.5”, “Scl FCH 3.5”, “Scl NaF 10”, and “Scl NaF 10 FCH 3.5”. For each VOI we computed the VOI volume (in ml). The thresholds for skeleton (HU > 150) and dense bone (sclerosis) (HU > 600) were determined from the CT component of the PET/CT study and were based on experience in more than 500 PET/CT studies. The pathologic FCH value (SUV > 3.5) was based on experience in more than 300 FCH/PET studies to indicate a pathological uptake. Tha pathological NaF value (SUV > 10) was similarly based on individual experience, but it was the same as the MDACC TFLterature .In addition to the PET based VOIs, we computed metrics analogous to total lesion glycolysis (TLG) in FDG PET scans. For the FCH PET based VOIs, the product of the VOI volume and the mean SUVbw in “FCH PET 3.5” and “Scl FCH 3.5” corresponds to the total cell membrane synthesis activity (TCA) in bone and sclerotic bone, respectively. For the NaF PET based VOIs, the product of the VOI volume and the mean SUVbw in “NaF PET 10” and “Scl NaF 10” corresponds to the total bone demineralization (TBA) in bone and sclerotic bone, respectively. An example of the patient analysis is shown in The percentage volumes of “Skeleton 600”, “FCH PET 3.5”, “NaF PET 10”, and “NaF 10 FCH 3.5” with respect to “Skeleton” were calculated. Similarly, the percentage volumes of “Scl FCH 3.5”, “Scl NaF 10”, and “Scl NaF 10 FCH 3.5” with respect to “Skeleton 600” was calculated. The mean, minimum, and maximum of the percentage volumes were calculated. The ratio of mean percentage volumes between abnormal and normal patients were calculated. A student’s t-test was performed between the percentage volumes for abnormal and normal patients.Our cohort consisted of twelve patients with advanced disease (> 5 lesions) who had had routine PET/CT both with FCH and NaF on consecutive days. An example is given in The patient characteristics are summarized in n = 12), whereas sclerotic bone volumes were from 0.88 L to 2.99 L. The sclerotic bone volume was 20.5 ± 6.8% of skeletal volume (this was defined by FCH-PET).Our results demonstrate that the skeletal VOI volumes varied from 5.03 L to 7.31 L in patients with skeletal metastases (n = 5), and sclerotic bone volumes were from 1.02 L to 1.76 L. The sclerotic bone volume was 19.9 ± 4.4% of skeletal volume.In control prostate cancer patients without skeletal metastases, the skeletal VOI volumes varied from 4.60 L to 7.90 L  in patients with multiple metastases. In the control PCa, patients pathologic FCH was only 0.08 ± 0.09% of the sclerotic bone volume (p < 0.004) and pathologic NaF volume 0.18 ± 0.19% of sclerotic bone (p < 0.001). All the differences between the metastasis group and control cancer patient group are shown in The sclerotic bone volume represented only 1.86 ± 1.71% of the pathologic FCH volume and 4.07 ± 3.21% of the TLFp < 0.020), but TBA and total sclerotic bone did not correlate with S-PSA. There was a correlation between TCA and S-PSA , and 19.9 ± 4.43% of skeletal volume in control prostate cancer patients without skeletal metastases (N). The sclerotic bone volume was defined from the CT measurements using Hounsfield Unit criteria, HU > 600. This indicates that the patient populations could be compared with each other for other skeletal phenomena because the degenerative changes were in the same level in both patient populations. The sclerotic volume ranges were from 0.88 L to 2.99 L in the metastatic group and from 1.02 L to 1.76 L in control patients without bone metastases.18FDG-PET) as total choline kinase activity  for FCH (TCA) and total accelerated osteoblastic activity  activity for NaF (TBA). The respective volumes of the whole skeletal volume are shown as Venn-Diagrams in We developed new parameters . Both NaF and FCH are useful in the evaluation of active skeletal metastatic disease, but they measure different phenomena and definitely give complementary information. Furthermore, FCH correlates positively with serum PSA concentration and is thus probably a prognostic factor.


Addition of 1.0 mM of Si-ions significantly aid cell recovery and protected from the toxic effect of 0.4 mM H2O2 on cell migration. These results suggest that ionic silicon may have a potential effect in unfavorable situations where reactive oxygen species is predominant affecting cell viability, proliferation, migration, and differentiation. Furthermore, this study provides a guide for designing Si-containing biomaterials with desirable Si-ion release for skeletal muscle regeneration.Volumetric muscle loss injuries overwhelm the endogenous regenerative capacity of skeletal muscle, and the associated oxidative damage can delay regeneration and prolong recovery. This study aimed to investigate the effect of silicon-ions on C2C12 skeletal muscle cells under normal and excessive oxidative stress conditions to gain insights into its role on myogenesis during the early stages of muscle regeneration. In vitro studies indicated that 0.1 mM Si-ions into cell culture media significantly increased cell viability, proliferation, migration, and myotube formation compared to control. Additionally, MyoG, MyoD, Neurturin, and GABA expression were significantly increased with addition of 0.1, 0.5, and 1.0 mM of Si-ion for 1 and 5 days of C2C12 myoblast differentiation. Furthermore, 0.1–2.0 mM Si-ions attenuated the toxic effects of H Althougnloading . These mnloading ,24.ROS levels at the site of muscle injury critically effects the muscle regeneration process ,28,29. AMuscle regeneration is a multistep process that starts with the activation of satellite cells, followed by myoblast proliferation and migration, and finally myoblasts fusion and differentiation to produce multinucleated myotubes to replace damaged fibers ,37. An i2O2 to determine the minimum H2O2 concentration that induces a significant oxidative damage. Then, four different concentrations of Si-ions were added to culture medium to study the effect of Si-ions on C2C12 cells under toxic oxidative stress condition (0.4 mM H2O2).Yet, the effect of Si-ions on skeletal muscle cell activity, such as proliferation, migration, differentiation, and myogenic biomarker expression, has not been explored. Thus, the effect of ionic Si released from Si-based biomaterials needs to be understood in skeletal muscle cells for the optimization of cell-based therapies and tissue engineering applications. In this study, we aim to understand the effect of Si-ions on C2C12 skeletal muscle cells under normal (no added ROS) and toxic ROS conditions to gain insight into its role on myogenesis during the early stages of muscle regeneration. First, C2C12 cell viability, proliferation, migration, and differentiation were studied following the addition of three different concentrations of Si-ions to cell culture medium to determine the optimal Si-ions dose for C2C12 cells. Second, we studied the effect of Si-ions on myogenic gene expression  and myokines  expression. Third, we studied the effects of ROS on C2C12 cell viability by culturing cells in five different concentrations of Hp < 0.05, n = 3 per group) as indicated by the MTS-assay results, expressed as optical density (OD) in p < 0.05, n = 3 per group), congruent with MTS results. Finally, following normalization of cell counts to the control it was determined that there was a 1.4-fold increase in cell counts with 0.1 mM Si.The effect of Si-ions on C2C12 myoblast cell viability was tested by using three different concentrations of Si-ions at two time points (6 and 24 h) as shown in p < 0.05) based on the OD results of the MTS assay. p > 0.5). An increased number of cells during proliferation were seen in Si ion samples compared to controls was confirmed by counting the 9 FOVs for each well. There was a significant increase in cell number in 0.1 mM Si-ion groups at day 1 (* p < 0.05) and day 2 (*** p < 0.001). Furthermore, the area covered by live C2C12 cells was calculated using the fluorescent images as shown in p < 0.001) as shown in p < 0.01 and * p < 0.05, respectively). Overall, FI was significantly increased from day 4 to day 7 for all samples including the control (*** p < 0.001). Total number of cells counted from 9 FOVs of DAPI stained nuclei indicated that all Si concentrations significantly increased the total number of cells at day 4 compared to the control with optimal concentration of 0.1 mM Si-ions, *** p < 0.001  then further increased following 5 days of differentiation to ~1.8-fold difference for 0.1 mM Si-ions and ~1.5-fold difference for 0.5 mM Si-ions  compared to control. For the high concentration 1.0 mM Si-ion group, there was a significant increase by ~2-fold in MyoG expression at day 1 which decreased to a ~1.8-fold difference compared to the control by day 5 of differentiation (*** p < 0.001). The qRT-PCR results indicated that MyoD expression was significantly increased in 0.1, 0.5 and 1.0 mM of Si-ion samples following 1 and 5 days of C2C12 myoblast differentiation as shown in p < 0.001). A 2.5-fold increase in MyoD expression for 0.1 mM Si-ion samples and a 3-fold increase in 1.0 mM Si-ion samples was observed compared to control at day 1. There was a 3-fold difference in MyoD expression in the 0.1 mM Si-ion group and only a 2.8-fold difference in the 1.0 mM Si-ion group following 5 days of differentiation. Finally, there was a 2.5-fold increase in MyoD expression in the 0.5 mM Si-ion group following 1 day of differentiation that decreased to a 2-fold difference by day 5.Expression of two key regulatory genes of myogenesis related to C2C12 myoblast differentiation were detected using qRT-PCR analysis following 1 and 5 days of differentiation. MyoG is an important myogenic regulatory factor expressed to mark cell commitment to differentiation . MyoD isp < 0.01). NRTN expression following 5 days of differentiation indicated that NRTN concentration decreased as differentiation time increased from 3 to 5 days. Although 0.5 mM and 1.0 mM of Si-ions reported the highest NRTN concentration at day 3 of differentiation, 0.5 mM and 1.0 mM Si-ions reported the lowest NRTN concentration by day 5 of differentiation. The initial increase followed by reduction in NRTN levels may indicate that NRTN secretion was increased, however, the inability to recruit motor neurites via retrograde signaling in vitro resulted in a quick reduction in NRTN concentration.Collected media were tested to determine the effect of Si-ions on C2C12 biomarker expression following 5 days of differentiation under normal conditions. NRTN expression following 3 and 5 days of differentiation was determined by ELISA assay. NRTN concentration (ng/mL) was increased in Si-ion samples at day 3 compared to the control C. NRTN ep < 0.05). At 3 and 5 days of differentiation, GABA concentration was significantly reduced for all Si-ions groups and the control compared to day 1 concentrations. GABA concentration was higher in the 0.5 mM Si-ion group compared to the other groups at day 3. Overall, the GABA concentration reduced dramatically after the first day of differentiation indicating that GABA expression decreases as differentiation time increases. Additionally, there was no significant difference between the control and Si-ions-media on GABA expression at day 5. D-BAIBA concentration was increased in the 0.5 mM Si-ion group (~0.14 ± 0.02 µM) compared to the control (~0.12 ± 0.01 µM) at day 1 of differentiation but no significant difference was observed (p < 0.05). D-BAIBA concentrations reduced dramatically at day 3 and 5 compared to day 1 of differentiation, indicating that D-BAIBA concentration decreases as differentiation time increases. The initial increase followed by reduction in both GABA and D-BAIBA levels again suggest that myokine secretion was increased, however, the lack of retrograde signaling in vitro resulted in a quick reduction in myokine concentration.The collected media was also tested to study the effect of Si-ions on aminobutyric acid expression during C2C12 differentiation. Although the analysis was performed for all isomers of aminobutyric acid , only GABA and β-aminoisobutyric acid (D-BAIBA) were detected in DM as indicated in observed B. While 2O2 on C2C12 myoblast cell viability was tested using five different concentrations  compared to the normal GM as a positive control as shown in 2O2. All H2O2 concentrations significantly decreased cell viability at 6 and 24 h compared to the control  as shown in 2O2 concentration increased from 0.2 to 0.6 mM as shown in 2O2 while the number of dead cells increased. The fluorescent images were further used to count the number of live cells per 6 FOVs for each group . The normalized cell number indicated that 0.2 mM H2O2 decreased the number of viable cells by 37% (** p < 0.01) while 0.4 mM H2O2 decreased viable cells by 70% at 24 h (*** p < 0.001). These results confirmed the results obtained by the MTS assay.The effect of Hch group B and the2O2 was the minimum concentration to induce a significant effect  on C2C12 cell viability. To study the effect of Si-ions on C2C12 under toxic oxidative stress conditions, 0.4 mM H2O2 was added to GM to induce ROS related damage, then four different concentrations of Si-ions were added to determine the optimal concentration that attenuated the effect of toxic oxidative stress. Cell viability was measured by MTS assay at 6 and 24 h as shown in 2O2 group  presented a significant decrease in cell viability at 6 and 24 h compared to the control. At 6 h, there was no significant difference in cell viability for low concentrations of Si-ions (0.1 and 0.5 mM), while higher concentrations of Si-ions (1.0 and 2.0 mM) attenuated the effect of toxic H2O2 as indicated by a significant increase in cell viability  compared to the 0.4 mM H2O2 group. At 24 h, all Si-ion concentrations attenuated the toxic effects of H2O2 resulting in increased cell viability compared to the 0.4 mM H2O2 group .As presented above, 0.4 mM H2O2 group which presented a higher number of dead cells (red). The fluorescent images were used to count the number of live cells per 6 FOVs for each group, then cell number was normalized to the 0.4 mM H2O2 group as shown in 2O2 as indicated by a significant increase in cell viability (*** p < 0.001).2O2. After determining the optimal concentration of Si-ions for attenuating the toxic effects of oxidative stress on C2C12 myoblast cells, a cell differentiation experiment was performed to study the effect of Si-ions on cell differentiation under toxic H2O2, and 0.4 mM H2O2 + 0.5 mM Si-ion groups as shown in p < 0.001). FI was increased (22 ± 5) in the 0.5 mM Si-ion group under oxidative stress compared to 0.4 mM H2O2 (18 ± 6), but no significant difference was observed. Based on the MHC-stained myotubes, the myotube area coverage (%) was calculated per 9 FOVs as shown in 2O2 group to 12.2 ± 1.6% for the 0.4 mM H2O2 + 0.5 mM Si group (** p < 0.01). 2O2 significantly decreases the MyoG (### p < 0.001) and MyoD (# p < 0.05) expression at day 1 of differentiation. Although the combined group (0.4 mM H2O2 + 0.5 mM Si) presented a significant decrease in MyoG (### p < 0.001) expression, no significant change was observed for MyoD expression at day 1. Furthermore, comparing 0.4 mM H2O2 group to the Si treated group (0.4 mM H2O2 + 0.5 mM Si) revealed that Si addition significantly increased the MyoD and MyoG expression (* p < 0.05) under ROS condition at day 1 of differentiation. On the other hand, day 5 results of MyoG and MyoD did not reveal any significant difference compared to the control.DM was used as a control to compare 0.5 mM Si-ions, 0.4 mM H2O2 only, 0.5 mM Si + 0.4 mM H2O2, and no treatment group as a control. After 1 and 5 days of differentiation, real time qRT-PCR was used to quantify the relative concentrations of NRF-2 and SOD-1 mRNA in all 4 groups (2O2 has increased the SOD-1 and NRF-2 expression with a significant increase only in NRF-2 expression at day 1 and 5 (2O2), there was a significant increase in NRF-2  and SOD-1  at day 5 compared to the control.To determine the effects of Si-ions on antioxidant activity in the presence of toxic oxidative stress, C2C12 cells were differentiated in media containing 0.5 mM Si-ions only, 0.4 mM H4 groups . At norm 1 and 5 A. For th2O2, 0.4 mM H2O2 + 1.0 mM Si-ions, and the control as shown in 2O2 concentrations . All used H2O2 concentrations significantly decreased the migration rate (*** p < 0.001) compared to the control group as shown in 2O2 conditioned media attenuated the toxic effect of H2O2 and significantly increased the cells’ migration rate compared to the non-Si-ions treatment (** p < 0.01) as shown in 2/h) and the H2O2 + Si group (rate = 0.031 ± 0.002 mm2/h), while the H2O2 alone presented a significant decrease in the migration rate (rate = 0.025 ± 0.001 mm2/h) compared to the control (*** p < 0.001). Scratch-wound healing assay was performed to compare cell migration parameters under different conditions. It is commonly used to measure cells’ migration parameters such as speed, persistence, and polarity . In this2O2 as a source of ROS. Our key results were that ionic Si enhanced the C2C12 viability, proliferation, differentiation, and myogenic gene and marker expression. Importantly, our results indicate that Si-ions at a concentration of 1.0 mM accelerate muscle wound healing by increasing the cell migration rate protecting it from the toxic oxidative damage compared to a positive control. Previous studies had indicated that antioxidants can improve muscle mass recovery [Our analyses revealed the novel effect of ionic Si-ions on C2C12 myoblasts under normal and toxic oxidative stress conditions. After confirming the positive effect of Si on myoblast functionalities, we studied its effect on C2C12 myoblasts under toxic oxidative stress conditions produced by Hrecovery , enhancerecovery , and prorecovery ,56,57. Irecovery ,47,48.Cell viability and proliferation experiments confirmed that Si-ions at concentrations of 0.1–1.0 mM supports myoblast cell growth, and 0.1 mM Si-ions is the optimal concentration to enhance C2C12 cell viability and proliferation. At early timepoints, all Si-ion concentrations resulted in higher cell numbers compared to the positive control. After 24 h, 0.1 mM of Si ions in GM significantly increased cell viability compared to the control, congruent with the results of MTS, live/dead assay, and the fluorescent image cell counting. Furthermore, all Si-ion concentrations significantly enhanced C2C12 fusion at an early stage of differentiation (day 4) compared to the control. Specifically, 0.1 mM Si-ions significantly increased myoblast fusion as indicated by FI calculations. Additionally, the size and length of the formed myotubes were more pronounced for the Si-ions groups compared to the control, determined by calculating the area of formed myotubes. It is important to note that the increase of FI and myotube area from day 4 to day 7 of differentiation occurred despite no change in the total number of cells, counted from DAPI stain, from day 4 to 7 indicating the successful differentiation of C2C12 myoblasts.This enhancement of C2C12 differentiation can be attributed to the early expression of myogenic markers such as MyoD, which was overexpressed almost 3-fold at day 1 of differentiation compared to control. The effect of Si-ions can be clearly seen from MyoD and MyoG overexpression compared to the Si-free control. MyoG was expressed at a later stage by day 5 to mark the commitment to differentiation. While MyoG expression was significantly increased by 2-fold in the high concentration (1.0 mM) Si-ion group at day 1, an almost 2-fold increase in MyoG expression was observed in both high and low concentration (0.1- and 1.0-mM Si-ions) groups compared to the control at day 5. The early expression of MyoG at day 1 can be attributed to the high dose of Si-ions. Overall, the enhancement in C2C12 cell functionalities such as proliferation and differentiation can be attributed to the antioxidant effect of Si-ions that promotes the early stage of differentiation as indicated by the myogenic marker’s expression. The results of this study indicate that certain concentrations of ionic Si promote maximal expression of myogenic biomarkers.Our results indicated that addition of Si-ions to the differentiation media can increase NRTN, BAIBA, and GABA expression at day 1 of differentiation as compared to the control without Si-ions. Although all Si-ion concentrations increased myokine expression at day 1, myokine expression was drastically reduced as differentiation time increased from day 1 to day 5 for all samples including the control. The initial increase in GABA and D-BAIBA levels followed by reduction in both suggest that myokine secretion increased, however, the lack of retrograde signaling in vitro resulted in a quick reduction in myokine concentration. On the other hand, it has been well established that ROS has a dose-dependent effect on assisting or hindering tissue regeneration. Low to moderate levels of ROS can enhance regeneration by inducing cell migration and differentiation ,30. WhilExcessive oxidative stress is a major contributor for various skeletal muscle tissue pathologies including prolonged severe traumatic injuries, sarcopenia, degenerative diseases and also other muscular atrophies . Severe 2O2 needed to induce oxidative damage on C2C12 cells, then determine the optimal Si-ion concentration to counteract or attenuate the effect of H2O2. We performed cell viability studies with five different concentrations of H2O2 to determine the minimal concentration of ROS for inducing significant cell death. All used H2O2 concentrations had a negative impact on cell viability, but 0.4 mM was the minimum concentration for inducing a strong significant difference (*** p < 0.001). Results of MTS assay and cell counting indicated that 0.2 mM H2O2 decreased the number of viable cells by 37%, while 0.4 mM H2O2 decreased the number of live cells by 70% at 24 h. Then, 0.4 mM H2O2 was used to study the effect of Si-ions in a toxic oxidative stress environment, and four different concentrations of Si-ions were tested. The results indicated that high concentrations of Si-ions (1.0–2.0 mM) can attenuate the effect of toxic ROS after only 6 h, while the low concentrations of Si-ions induced significant enhancement of cell viability after 24 h compared to control. Thus, we concluded that 0.5 mM Si-ions is the optimal concentration of Si-ions for attenuating the effect of toxic H2O2 based on a significant increase in cell viability. Furthermore, after determining the effective concentration of H2O2 (0.4 mM) and the effective concentration of Si-ions (0.5 mM), a cell differentiation study was performed to investigate if Si-ions have any effect on fusion. FI was increased in the 0.5 mM Si-ion group (22 ± 5) under oxidative stress conditions compared to 0.4 mM H2O2 (18 ± 6), but no significant difference was observed. Previous studies indicated that antioxidants reduce the deleterious effects of H2O2 on cell migration but not cell fusion [2O2 group to 12.2 ± 1.6% for the 0.4 mM H2O2 + 0.5 mM Si group. This indicates that measuring the myotubes area combined with FI calculations is a more accurate method to assess cell differentiation. This enhancement in cell differentiation under ROS condition with Si treatment was further confirmed by MyoD and MyoG gene expression analysis.Thus, our second aim was to study the effect of Si-ions on C2C12 activity under toxic oxidative stress. Our objectives were to determine the minimal concentration of Hl fusion . Althoug2O2 increased C2C12 cell migration rate while the high concentration of 0.5–1 mM hindered cell mobility [2O2 significantly decrease the cells’ migration rate that usually results in poor wound healing and prolonged healing time under severe muscle injuries. Adding 1.0 mM Si to the media with H2O2 attenuated the toxic effect and enhanced the healing rate under these conditions. It is important to mention the advantages of using the live dynamic imaging technique in such wound-healing assays , the expression was more pronounced under Si treatment with 1.7 fold change. This suggests that Si-ions may play a role in mitigating the effects of excessive reactive oxidative species by upregulating antioxidant markers. H2O2 can react with metal ions in the cells to produce hydroxyl radical which is one of the most reactive species in the biological systems [Additionally, the results of qRT-PCR assays suggest that Si may upregulate the expression of SOD-1 when in the presence of oxidative stress, however, the same effect was not observed under normal conditions or oxidative stress conditions in the absence of Si-ions. Although 0.4 mM H systems . High le systems ,62,63,642SiO3) [2O2. The cell viability, proliferation, and morphology were determined in the same manner as described above. Media was collected periodically for biomarker and gene expression analysis using qPCR and ELISA. Finally, cell migration was evaluated using a scratch test [To study the effect of Si-ions on C2C12 skeletal muscles under normal and oxidative stress conditions, a series of 2D cell culture experiments were performed. To examine the effects of Si-ions on C2C12 cells under normal conditions, cells were cultured in normal media and in media containing Si-ions at concentrations of 0.1 mM, 0.5 mM, and 1.0 mM. Si ions were obtained using sodium metasilicate powder (Na2SiO3) ,65,66,67tch test ,68 to de2SiO3, MW: 122.06 g/mol, Sigma-Aldrich Co., St. Louis, MO, USA) and hydrogen peroxide  were used as sources of Si-ions and ROS, respectively. Dulbecco’s Modified Eagle’s Medium ((DMEM-1×) with 4.5 g/L glucose, L-glutamine, and sodium pyruvate), phosphate-buffered saline (PBS-1×), penicillin-streptomycin (P/S) 10,000 U/mL each, and trypsin EDTA-1× solution were purchased from Mediatech Inc. . Fetal bovine serum (FBS) and horse serum (HS) were obtained from Thermo Fischer Scientific Inc. . C2C12 mouse myoblast skeletal muscle cell lines were obtained from The American Type Culture Collection (ATCC)  after authentication and testing for mycoplasma contamination. CellTiter 96® AQueous One Solution Cell Proliferation Assay (MTS) was obtained from Promega . Diamidino-2-phenylindole (DAPI), a blue, fluorescent nucleic acid stain, was purchased from Sigma-Aldrich Co., . Human Myosin Heavy Chain (MHC) fluorescein-conjugated antibody was purchased from R&D Systems, Inc. . Invitrogen™ LIVE/DEAD™ Viability/Cytotoxicity Kit was purchased from Thermo Fischer Scientific Inc. . Mouse Neurturin (NRTN) ELISA Kit  was purchased from Biomatik USA, LLC . (S)-α-aminobutyric acid (L-AABA) and (R)-α-aminobutyric acid (D-AABA) were purchased from Thermo Fisher Scientific Inc. . (S)-β-aminoisobutyric acid (L-BAIBA) and (R)-β-aminoisobutyric acid (D-BAIBA) were purchased from Adipogen Corp. , and GABA was purchased from Sigma-Aldrich Co., . For quantitative real-time polymerase chain reaction (qRT-PCR): the RNeasy Mini Kit was obtained from Qiagen , the reverse transcription system was obtained from Promega , and the PCR primers for the targeted genes, MyoD and MyoG, were obtained from Thermo Fischer Scientific Inc. .Sodium metasilicate powder  followed by a serial dilution, the desired concentrations of Si were obtained  in C2C12 myoblast cell growth media. Hydrogen peroxide is a form of ROS generated when the enzyme superoxide dismutase scavenges superoxide anions [2O2 was used as a source of ROS and inducing oxidative damage. 100 mM-H2O2 solution was prepared in 10 mL sterile DI water, filtered, and serially diluted in sterile DI water until the desired concentrations of H2O2  were reached in C2C12 cell growth media.Nae anions , thus H22 Corning cell culture flasks with canted neck and vented caps under normal conditions  maintaining cell density at 40–70% confluency. Cells in GM were maintained at 40–70% confluency, allowing them to proliferate but not to differentiate into myotubes. GM was changed every 48 h. For differentiation studies, after reaching 75% confluency, C2C12 myoblast cells were cultured in differentiation medium (DM), DMEM/high glucose + 2% HS + 100 U/mL P/S, which was changed every 48 h for 5–7 days until fully developed myotubes were formed.C2C12 myoblast is an immortal cell line that are easy to obtain and passage. Typically, C2C12 myoblasts differentiate rapidly under normal, Si-free, conditions, forming contractile myotubes and producing proteins with characteristics similar to endogenous muscle tissue. Using C2C12 myoblasts provides a more stable environment with fewer confounding variables than the normal physiologic environment of human primary cells . Thus, C4 cells/well were seeded in 24-well plates with 500 µL of GM and incubated as described above. Three different concentrations of Si-ions in GM were tested compared to the normal GM as control. Sample size of n = 6 was used for each group; GM, GM + 0.1 mM Si, GM + 0.5 mM Si, and GM + 1.0 mM Si-ions. These different concentrations of Si-ions were added to the media during seeding, then cells were incubated for 6 and 24 h for cell viability experiments and for 1, 2, and 3 days for cell proliferation studies. After each time point, MTS assays were performed (n = 3 for each group) using the CellTiter 96 AQueous One Solution Cell Proliferation Assay kit according to the manufacturer’s protocol. Briefly, MTS solution was prepared by adding 40 µL of CellTiter 96 reagent to 200 µL of GM for each sample to be tested. At each time point GM was removed from each sample and 240 µL of the MTS solution was added to each well of the 24-well plate. After 3 h, 100 µL samples were collected from each well twice and transferred into two separate wells of a new 96 well-plate. The colorimetric absorbance was determined using a microplate reader  at 490 nm. The remaining n = 3 for each group each time point was used for the live/dead assay using Invitrogen LIVE/DEAD Viability/Cytotoxicity Kit according to the manufacturer’s protocol. Briefly, 200 µL of live/dead solution was added to each well and incubated for 30 min. Fluorescent images were then taken using a DMi8 inverted Leica microscope , with green staining for live cells and red staining for dead cells. Three fluorescent images (20× field of view (FOV)) were taken for each well to compare the number of live and dead cells in samples. The number of live and dead cells in fluorescent images were counted using ImageJ software (NIH).To study the effect of Si-ions on C2C12 myoblast viability and proliferation, 2.5 × 104 cells/well were seeded in 6-well plates in GM for 24 h or until 75% confluency. After reaching 75% confluency, GM was removed, and each well was washed with 2 mL of PBS-1× to remove any unattached cells. Then, 2 mL of DM was added to the first group as control and Si-ions at concentrations of 0.1, 0.5, and 1.0 mM Si were added to DM for the other three groups . DM was collected for biomarker expression assays and new DM with the specified Si-ion concentration was added to each well every 48 h. After each time point, cells were fixed and immunostained following previously published protocols [To study the effect of Si-ion concentration on cell morphology and differentiation capacity, C2C12 cells were cultured for 4 and 7 days in DM containing 3 different concentrations of Si-ions compared to normal DM as control. For all differentiation experiments, 10 × 10rotocols ,75. BrieFusion index  (FI), ar2O2 as a source of ROS, the first experiment was performed to determine the minimal concentration of H2O2 that can be used to induce significant cell damage. Five concentrations of H2O2  were used to compare the effect of H2O2 to the normal GM as control. C2C12 cell viability experiment was performed by seeding 2.5 × 104 cells/well in a 24-well plate [2O2, GM + 0.4 mM H2O2, GM + 0.6 mM H2O2, GM + 0.8 mM H2O2, and GM + 1.0 mM H2O2 as the experimental H2O2 groups. Cells were incubated for 6 and 24 h. At each time point, 12 wells (n = 3 each group) were used for the MTS assay and the other 12 wells (n = 3 each group) for the live/dead assay, as previously mentioned. Fluorescent images displaying green (live cells) and red (dead cells) were used to count the total number of live cells per FOV using ImageJ.To study the C2C12 cell viability under different concentrations of Hll plate ,74. Then2O2 was used to study the effect of Si-ions on C2C12 cell viability under toxic oxidative stress. This experiment was performed to determine the optimal Si-ions concentration for preserving C2C12 cells under toxic oxidative stress. 2.5 × 104 cells/well were seeded in a 24-well plate and provided with 500 µL of specified media for each group (n = 3). GM was used as the positive control, GM + 0.4 mM H2O2 was used as the negative control, and four different Si-ion concentrations were used as following: GM + 0.4 mM H2O2 + 0.1 mM Si, GM + 0.4 mM H2O2 + 0.5 mM Si, GM + 0.4 mM H2O2 + 1.0 mM Si, and GM + 0.4 mM H2O2 + 2.0 mM Si. All plates were incubated for 6 and 24 h. After each time point MTS and live/dead assays were performed as previously mentioned. Fluorescence images (10× FOV) of stained C2C12 cells were taken to observe the cells after exposure to H2O2 and H2O2 + Si for 6 and 24 h.Based on the above cell viability experiment under ROS conditions, a concentration of 0.4 mM H2O2 and optimal Si-ion concentrations. In a 6-well plate, 10 × 104 cells/well were cultured for 24 h or until 75% confluency. After reaching 75% confluency, GM was removed, and each well was washed with 2 mL of PBS-1× to remove any unattached cells. Then, normal DM was used as a positive control compared to 0.5 mM Si, 0.4 mM H2O2, and 0.4 mM H2O2 + 0.5 mM Si-ions. Every 48 h DM was collected from each well for biomarker expression assays and new DM was added to each well as specified. After 4 days of differentiation, cells were fixed and immunostained and counterstained using MHC and DAPI and fluorescent images were captured at 10× using a Leica DMi8 inverted fluorescence microscope. FI and area covered by myotubes were determined using ImageJ as follows. First, cell count was determined using microscope software and the cell batch counter plug in to count any clustered nuclei. The cell batch counter plug in was then used to count nuclei found within myotubes to determine the FI. The image was then converted to 8-bit, made binary, and converted to mask before determining the area covered by myotubes. The image threshold was converted to black and white, with green fluorescence set to display as black. Finally, the black regions of the image were measured to determine the percent area covered by myotubes.After determining the optimal Si-ions concentration that can mitigate the effect of oxidative damage on C2C12 cells from the previous experiment, cells were cultured in media with the effective H2O2, Si supplements using 0.1, 0.5-, and 1.0-mM Si-ions, and 0.4 mM H2O2 + 1.0 mM Si, (n = 3 and triplicated tests). The experiments were performed by seeding 5 × 104 cells/well in a 12-well plate with 1 mL GM for 24 h until full confluency. Then, a 200 µL sterile pipette tip was used to scratch the cells to introduce a thin wound, the media was removed, and each well was washed twice with PBS-1×. Then, 2 mL fresh conditioned media was added according to each group as described above. The plate was placed inside the sterile chamber of a Keyence BZX-710 fluorescence live imaging microscope  and provided with 5% CO2 at 37 °C. This microscope allowed for high throughput, fully automated cell imaging at the exact same position on each well. Phase contrast images were captured every 2 min over 48 h at 10× magnification (Resolution 0.7 pixels/μm and Z plane separation = 4 µm pitch). All images, around 700 for each sample, were used to measure the wound area using the Wound Healing ImageJ software plugin [2/h) was calculated using the slope function.In this study, we have developed a scratch-wound healing assay to quantify the cells’ migration rate using dynamic live imaging microscopy (DLIM) to monitor the cells’ migration throughout scratch/wound healing. Briefly, we compared the cell migration under normal conditions using GM, oxidative stress using 0.4, 0.6, and 0.8 mM He plugin  and Image plugin . ImageJ QRT-PCR was used to detect changes in myogenic gene expression of C2C12 cells under normal conditions compared to treatment with Si-ions over 5 days. A cell differentiation experiment was performed as mentioned above . Three gDM was collected during the cell differentiation experiment over 5 days and stored at −20 °C. Later, collected media was analyzed for myokine expression. A mouse NRTN ELISA kit  was used to determine the concentration of NRTN secreted during C2C12 differentiation. NRTN is a key component in peroxisome-proliferator-activated receptor gamma coactivator-1α (PGC-1α) mediated neurite recruitment by skeletal muscle. NRTN ELISA kit was used according to manufacturer guidelines. NRTN-specific antibody was precoated onto the microplate in the kit, and standards and samples were pipetted into the wells to allow any present NRTN to bond with the immobilized antibody. Then, biotin-conjugated antibody specific for NRTN was added after removing any unbound molecules. After washing the plate with a wash buffer solution, avidin conjugated horseradish peroxidase (HRP) was added followed by another wash to remove any unbound avidin reagent. Then TMB substrate solution was added to each well and the plate was incubated for 15–30 min as previously described while protected from light. After 20 min, the stop solution was added, and optical density was determined within 5 min using a microplate reader set to 450 nm. Protein expression was determined based on a polynomial regression of the optical density and protein concentration of the provided standards using OriginPro 8.5 software.The collected media was further analyzed for any markers of aminobutyric acid expression during C2C12 differentiation. Analysis was performed for all aminobutyric acid isomers, including α-aminobutyric acid (AABA), β-aminobutyric acid (BABA), and GABA. Aminobutyric acid concentrations were determined using LC-MS/MS analysis, performed on a Shimadzu LCMS-8050 triple quadrupole mass spectrometer  at the Shimadzu Center at the University of Texas at Arlington. Analysis was performed based on a previously published LC-MS/MS method that enables baseline separation with sensitive detection of aminobutyric acid isomers in minimal amounts of biological fluid samples developed by the Brotto Research Laboratory .Immunofluorescent images were analyzed for cell number, myotube area, and FI using ImageJ. Wound Healing ImageJ software plugin ,76 and Ip < 0.05 was considered as statistical significance and * represents p < 0.05, ** for p < 0.01, and *** for p < 0.001. FI calculations were conducted blindly by the operator.OriginPro 8.5 software was used for all graphs and statistical analysis. Statistical data are presented by individual data points and a horizontal line indicating the average of each group. Bar graphs display group means and standard deviations. One-way ANOVA followed by Tukey’s post hoc was used between group comparisons. Due to the difference in variance between the groups, following a One-way ANOVA, Tukey’s post hoc analysis was conducted to compare differences between individual groups. 2O2 within 24 h resulting in increased cells’ viability and differentiation compared to the 0.4 mM H2O2 group. Also, 1.0 mM Si-ions increased the cells’ migration rate with a significant decrease in the scratch healing time to achieve faster healing under oxidative stress conditions. The observed upregulation of NRF-2 and SOD-1 in the 0.5 mM Si + 0.4 mM H2O2 group combined with the lack of a significant difference in expression for groups containing only Si-ions or only H2O2 suggest that Si-ions at optimal concentrations may enhance ROS metabolism and clearance in C2C12 cells under oxidative stress. These results indicate that ionic Si may have antioxidant and stimulatory effects on muscle tissue to promote skeletal muscle repair. Thus, this study provides novel evidence of the potential role of Si-ions that influence the cellular response during the myogenesis process and pave the way for designing Si-containing biomaterials with desirable Si-ions release for muscle tissue regeneration applications along with muscle drug delivery systems for degenerative disease conditions.In this study, we explored the effect of Si-ions on C2C12 skeletal muscle cell myogenesis, such as proliferation, migration, differentiation, and myogenic biomarker expression to gain insight into its role on myogenesis during the early stages of muscle regeneration. In vitro studies indicated the addition of 0.1 mM Si-ions to media significantly increased cells’ viability, proliferation, migration, and myotube formation compared to control. Additionally, Si-ions significantly increased MyoG and MyoD gene expression within 5 days of C2C12 myoblast differentiation. Si-ions attenuated the toxic effects of H