Ubf silencing in vivo is not viable, and these new tools allow further characterization of rRNA transcription and its coordination with cellular signaling. shUBF cells display cell cycle G1 delay and reduced 47S rRNA precursor and 28S rRNA at baseline and serum-challenged conditions. Growth-related mTOR signaling is downregulated with the fractions of active phospho-S6 Kinase and pEIF4E translation initiation factor reduced, similar to phosphorylated cell cycle regulator retinoblastoma, pRB, positive regulator of UBF availability/rRNA transcription. Additionally, we find transcription-competent pUBF (Ser484) severely restricted and its interacting initiation factor RRN3 reduced and responsive to extracellular cues. Furthermore, fractional UBF occupancy on the rDNA unit is decreased in shUBF, and expression of major factors involved in different aspects of rRNA transcription is severely downregulated by UBF depletion. Finally, we observe reduced RNA Pol1 occupancy over rDNA promoter sequences and identified unexpected regulation of RNA Pol1 expression, relative to serum availability and under UBF silencing, suggesting that regulation of rRNA transcription may not be restricted to modulation of Pol1 promoter binding/elongation rate. Overall, this work reveals that UBF depletion has a critical downstream and upstream impact on the whole network orchestrating rRNA transcription in mammalian cells.Transcription/processing of the ribosomal RNA (rRNA) precursor, as part of ribosome biosynthesis, is intensively studied and characterized in eukaryotic cells. Here, we constructed shRNA-based mouse cell lines partially silenced for the Upstream Binding Factor UBF, the master regulator of rRNA transcription and organizer of open rDNA chromatin. Full  The nucleolus is the site of transcription of the ribosomal RNA (rRNA) precursor from the 150 to 220, or more, tandem rDNA gene copies, present in megabase arrays distributed over 5 different chromosomes in the mouse genome (Such studies have highlighted the important role in rRNA transcription of UBF (Upstream Binding Factor), a conserved “architectural” transcription factor with several DNA binding domains, termed “HMG boxes” due to their similarity to High Mobility Group chromosomal proteins. UBF orchestrates the assembly of the transcription pre-initiation complex (PIC) of RNA polymerase I  on the promoter, and the establishment of active or potentially active rDNA chromatin conformation , 3; revi; revi3; via Pol1 subunits PAF53 and PAF49  and mediated by several kinases including cdk/cyclins, CKI, CKII, CDC2, GSK, and PI3K [W1]. Importantly, UBF binding to rDNA enhancer/promoter regulatory sequences or its protein interactions with the transcription machinery, including SL1 and Pol1, are dependent on those phosphorylations , 22, 23.Regarding the impact of UBF on rDNA chromatin conformation, UBF is thought to bind to ∼140 bp of DNA as a dimer, inducing a single DNA loop in a structure termed the «enhancesome» . UBF canin vitro or in vivo  and UBF2 (90 kDa), which are otherwise identical except for 37 aa in HMG box 2 of UBF2 removed by alternative splicing . Most st in vivo , 32, 33. in vivo . Both is in vivo , 36.Overall, it is clear that UBF has a multi-pronged involvement and significance in the regulation of rRNA transcription. Despite great advances with the identification of many of the key factors, their protein interactions, and mechanisms involved, important questions remain regarding the structure of the PIC, the mechanisms of its assembly, the modulation of Pol1 activity, and the co-ordination of rRNA transcriptional regulation with cell signaling and cell growth, and are thus the focus of on-going research.via shRNA-mediated lentiviral transduction. These cells maintain a haploid-like, viable level of UBF expression, and so can provide information that was inaccessible in studies with Ubf gene deletion which is embryonically lethal , using an anti-UBF antibody, to identify those with reduced UBF protein, as compared with wild-type levels (WT) in the maternal NIH 3T3 line. Two clones, named shUBF16 and shUBF72, which displayed the lowest UBF protein levels, as normalized to endogenous calnexin in this initial screen, were further analyzed in three independent experiments; these confirmed a robust reduction of total UBF to below 50% of WT in both clones (A1). In particular, UBF protein levels in shUBF16 were 0.49 ± 0.06 (relative ratio to WT control set to 1 ± SD) when normalized to same-sample calnexin levels (A2), or 0.28 ± 0.09 when normalized to total protein, as determined by whole-sample Ponceau staining of the WB membrane . The equivalent values for shUBF72 were 0.33 ± 0.06 and 0.21 ± 0.02 (A2 and A3), respectively. Both UBF1 and UBF2 were downregulated as the shRNA construct targets both alternatively spliced isoforms . Quantification of total Ubf mRNA expression in the two shUBF clones showed consistently significant reductions relative to WT, specifically 0.55 ± 0.05 for shUBF16 (B1) and 0.40 ± 0.04 for shUBF72 (B2). Thus, both shUBF16 and shUBF72 exhibited UBF levels that could be compared with a haploinsufficiency status, known to be viable in mice  while nucleolar marker fibrillarin appeared only slightly reduced (A2–B2) and the overall nucleolar organization seemed intact (A4–B4 in overlays). Consistently, quantification of P53 protein levels, phosphorylation (Ser392), and gene expression did not indicate that UBF downregulation in shUBF72 was associated with p53-dependent nucleolar stress; in particular, both at base line conditions and with experimentally induced nucleolar stress by treatment with actinomycin, P53, pP53 protein levels, and P53 mRNA were considerably reduced in shUBF72 relative to WT cells (C1–C4). Additionally, gene expression of P53 downstream target protein P21 in shUBF72 was also moderately reduced compared with WT (C4). Quantification of cell proliferation revealed significantly lower growth rates for shUBF72 cells compared with WT (D). Cell cycle analysis indicated an altered profile with a clear delay in the G1 phase for shUBF72 and a consequent increase in the fraction of the cell population in G1 by a near 40% and corresponding decreases in phases S (45%) and G2/M (59%), compared with WT (E1 and E2). Delay of cells in G1 is typically associated with impairment in ribosome biogenesis . Despite this, 47S rRNA expression in shUBF72 increased in step with serum as in WT, suggesting that the mechanisms of growth regulation still functioned normally even after UBF depletion. A similar reduction of pre-rRNA expression at all serum concentrations and dependency of UBF expression on serum concentration was also observed with qRT-PCR analysis of shUBF16 cells (data not shown).Given the significant reduction of UBF expression in the shUBF cell lines and the critical role of UBF in orchestrating the transcription of the rRNA genes, a marked reduction in the production of rRNA would be expected in these cells and we assessed this by different approaches. First, a quantitative RT-PCR analysis was conducted in 3 independent experiments, using as primers an oligonucleotide pair targeted to the extreme 5′ of the 47S rRNA precursor that can specifically detect the primary rRNA transcript . This anFig. 3B).We next employed short pulse labeling of nascent RNA with FUrd to monitor rRNA expression. Again, the experiments were conducted under the three different growth conditions , and the mean fluorescence intensity in the nucleoli of a large number of cells (n = 50 per condition in each cell line) were compared. Again, significant reductions of FUrd incorporation in shUBF72 (indicative of reduced nascent rRNA production) were observed at all serum concentrations as compared with WT, and dependency of rRNA transcription on serum concentration was registered in both WT and shUBF cells B.C1 and C2) as well as of 28S rRNA, the largest mature rRNA species resulting from the multi-step processing of the 47S rRNA precursor (D1 and D2).In complement, Northern blot analysis, which allowed the resolution of distinct rRNA species, confirmed, in four independent experiments, the reduction of 47S rRNA in shUBF72  or intracellular factors  (see introductory comments). These include different signaling cascades that target factors of the transcription machinery; for example, the major growth- and proliferation-regulating PI3K/mTOR and RAF-MEK-ERK (MAPK) pathways both modulate the phosphorylation status of UBF, among others, thus affecting rRNA transcription initiation, elongation, and chromatin configuration. We thus set up assays to quantify mTOR and MAPK signaling under UBF downregulation in a manner that would preserve native protein phosphorylation and ensure specificity (with the use of inhibitors rapamycin for mTOR C1 kinase and U0126 for MAPK), comparing WT and shUBF72 cells in independent experiments, in parallel.A1 for representative WB and A2 for quantification; compare the ratio at 0.5% and 10% serum), a near elimination in the presence of rapamycin, and substantial reduction in the presence of U0126 (A1 and A2). Serum and mTOR dependency were replicated in shUBF72 but, interestingly, at all serum concentrations and conditions, the pS6K/S6K ratio was markedly reduced . This suggested downregulation of the growth signaling axis and specifically of the mTOR kinase pathway under UBF depletion.S6 kinase (S6K) is a prominent direct downstream target of mTOR kinase; under growth stimulation, activated mTOR kinase phosphorylates S6K which, in turn, phosphorylates UBF, thus boosting rRNA transcription . ConsistFig. 4B1 and B2). In agreement with reduced growth signaling in shUBF72, the pEIF4E/EIF4E ratio was markedly lower in UBF-depleted cells at all conditions, relative to WT, and in particular at 10% serum it was, again, 50% of WT (B2). These observations would point to the downregulation of growth signaling as well as translational competence on UBF depletion.Quantification of another marker of mTOR activation, pEIF4E/EIF4E, gave similar results. Elongation initiation factor EIF4E is activated by MAPK-dependent phosphorylation and mTOR-dependent phosphorylation and release of its repressor factor 4E-BP1 , 41. Aga0% of WT B2. TheseC1 and C2). Downregulation of pRB was thus another intracellular cue linked to repressed rRNA transcription in shUBF.We next quantified a known tumor suppressor and cell cycle regulator, Retinoblastoma (RB), also known to impact rRNA transcription; non-phosphorylated RB sequesters UBF, while pRB releases it, thus boosting rRNA production , 43, 44.Together, these experiments are evidence of significantly repressed growth signaling accompanied by loss of transcriptional and translational efficiency in cells when UBF is reduced. Since this signaling is upstream of UBF, our findings suggest a bidirectional feedback between both extrinsic and intrinsic growth signaling and rRNA biosynthetic control.Ubf gene expression remained essentially unaltered under all growth conditions for both WT and shUBF cells, despite Ubf mRNA being severely downregulated to levels well below 50% in shUBF72 . The same was the case for UBF protein levels (for both UBF1 and UBF2), as observed when the average measurements at each condition were normalized to same-sample calnexin protein levels (B1 and B2), suggesting only minor fluctuations in absolute protein concentrations. We also observed stable gene expression of UBF and a constant ratio between UBF1 and UBF2 isoforms throughout the cell cycle phases in synchronized WT cells, indicating no differential pre-mRNA splicing . A trend for serum-responsive pUBF increase within WT or shUBF72 cells, also when pUBF was shown as a fraction of total UBF protein (pUBF/UBF) , was not statistically significant and it is not clear whether it is biologically relevant. These results indicated that limited absolute levels of UBF in cells were accompanied by concomitant diminution of the transcription-enhancing pUBF levels. Given that pUBF functions synergistically with factor RRN3 to assemble the PIC and initiate rRNA transcription, we also assessed RRN3 protein levels under the same experimental set up . We observed RRN3 concentration to display serum-dependent modulation and a severe decrease in shUBF72 under all serum concentrations, relative to WT cells .The finding of stable UBF expression may not be surprising given that it is well established that UBF-mediated PIC formation is, rather, dependent on UBF phosphorylation . We thusFig. 5A to monitor UBF and pUBF). UBF occupancy of rDNA chromatin, at binding sites over the whole rDNA unit as now quantified by qPCR , was clearly reduced in shUBF72 compared with WT at all positions, including regulatory and coding regions . In particular, known UBF-binding hotspots in the promoter-enhancer sequences and also binding within the 28S rRNA coding region displayed UBF occupancy that was reduced to around 40% in shUBF72 relative to WT .We next employed chromatin immunoprecipitation experiments (ChIP), which specifically allow the assessment of the chromatin-bound UBF fraction, using total input samples (“input”) from cross-linked cells, and eluted samples, following UBF immunoprecipitation for qPCR (“ChIP” samples) . The reduction of the gene fraction with permissive chromatin status combined with the reduction of UBF occupancy of rDNA chromatin detected with ChIP assays was consistent with, and explained, the decline in active 47S rRNA synthesis observed in these cells (A and B).UBF is responsible for the non-nucleosomal conformation of rDNA chromatin and this “active” or “poised” state of chromatin, which can be considered transcriptionally competent, can be directly monitored by psoralen accessibility crosslinking (PAC) , 3, 6. Tse cells , A and BEffects on promoter binding by RNA polymerases generally, including RNA Pol1, and the modulation therefore of transcription initiation rate, is a typical and well-characterized mode of transcriptional regulation. Here, we investigated what happens to the pool of available cellular Pol1 under conditions of UBF depletion. We quantified separately two distinct Pol1 subunits: PolR1E (RPA49/PAF53), a 53kDa-protein of the TFIIE/F-like heterodimer component of Pol1 that mediates its interaction with UBF, and PolR1A (RPA190) the 194 kDa largest Pol1 subunit at its catalytic core , 25.A1 and A2) and PolR1A (B1 and B2) reduced by UBF depletion, in both shUBF16 and shUBF72, to well below 50% of WT levels. This was the case when Pol1 subunit protein normalization was carried out to either calnexin (B2 and C2) or to total cellular protein as revealed by Ponceau staining  . In further support, the immunofluorescence signal for PolR1A localization in the nucleolus in shUBF72 cells was noticeably weaker and more restricted, compared with WT cells (C1 and D1).Interestingly, in three independent experiments, we found protein levels of both PolR1E (Polr1B mRNA levels in shUBF72 were reduced 5.3-fold (0.19 ± 0.07 vs. 1.0 ± 0.02 in WT), to 19% of WT levels. (A). Additionally, an upward trend in Polr1B mRNA accompanied serum elevation, in both WT and shUBF72 cells (A). These results were mirrored by equivalent experiments, quantifying the corresponding changes and serum dependency of POLR1E at the protein level (B1 and B2 and S4A). For example, at 10% serum, POLR1E protein levels were reduced to 18% of WT (0.18 ± 0.02 in shUBF72 vs. 1 ± 0.2 in WT). These combined findings, therefore, indicated that the cellular concentration of Pol1 was regulated at the transcriptional level by growth conditions/signaling as well as by the depletion of UBF.Because modulation of levels of the polymerase itself is a rather atypical manner of transcriptional regulation, we were interested in further characterizing this finding and we therefore next examined whether reduced protein levels could be attributed to enhanced protein instability (due to the downregulation of protein interactor UBF) or more upstream transcriptional control of Pol1 gene expression. Quantification of gene expression of yet another Pol1 core subunit, PolR1B (RPA135), conducted under the three serum regimes, clearly showed very strong downregulation in shUBF72 at all conditions. For instance, at 10% serum, relative  levels. A. Additi72 cells A. These C). In particular, Pol1 occupancy over the Sp/Pr and T0/Pr promoter sequences and the 28S rRNA coding sequences were, respectively, 0.18 ± 0.03 in shUBF vs. 0.50 ±0.07 in WT (or 36% of WT), 0.41 ±0.06 vs. 1.05 ± 0.07 (or 39% of WT), and 0.07 ± 0.01 vs. 0.16 ± 0.02 (or 44% of WT) (C). The reduced Pol1 occupancy in shUBF72 was consistent with the observed relative 47S rRNA synthesis in these cells.Consistent with reduced rRNA transcriptional output and reduced Pol1 protein levels, ChIP experiments using anti-pol1 antibody to POLR1A revealed reduced fractional occupancy for chromatin-associated Pol1 across the rDNA unit C. In par% of WT) C. The reD) and Neuro2 neural crest-derived cell line (E), as well as the human HeLa cell line (F). Interestingly in all these wild-type cell lines of different cell types and species, we confirmed significant changes in the protein levels of POLR1E , in step with increased serum concentration (D–F).The modulation of RNA Pol1 concentration in shUBF cells was rather unexpected and hence we wished to investigate whether this was a more generalized mechanism of rRNA transcriptional control in proliferating cells. We therefore assessed POLR1E protein levels at increasing serum concentrations in mouse or human cell lines other than NIH 3T3 fibroblasts, namely the mouse IMCD kidney cell line D and Neuell line E, as welell line F. Interentration , D–F.via direct interaction with rDNA regulatory elements , correlating its downregulation with the observed reductions in Pol1 subunits. Given the link between C-myc expression and a larger number of genes associated with Pol1 transcription by transcriptome analysis in stages of granulocyte differentiation (A–H). Specifically, we monitored the expression of Cyclin D, important for UBF S484 phosphorylation by CDK4 and re-activation of rRNA transcription after mitosis (C1 and C2), as well as transcriptional terminator/chromatin modifier TTF1 and CTCF, a non-PIC modifier of rDNA structure. In all instances, quantification showed a clear trend of increased gene expression of each of these factors, as a function of serum concentration in both WT and shUBF72 cells, and all showed a large decrease of expression in shUBF72, relative to WT, at each serum concentration (A–H).To gain some initial understanding of the molecular mechanisms involved in the regulation of Pol1 gene expression, we interrogated a potential role for the C-MYC transcription factor. C-MYC has been shown to co-ordinate gene expression of Pol1 subunit POLR1B, PIC factor RRN3, and UBF itself, through binding to their respective promoters, and has also been found to affect rRNA transcription elements , 47, 48.WT cells G, correlntiation , we furtntiation , A–H. Sp mitosis , of SL1-ntration , A–H.FigA1–A4) fully restored the protein levels of POLR1E subunit of RNA Pol1 (B2 and B3) and substantially rescued expression of C-myc, compared with shUBF72 cells transfected in parallel with an empty FLAG vector (C). Finally, in a complementary approach, we constructed a third UBF-silenced cell line, named shUBF555, now targeting a different site of the UBF1/2 transcripts than shUBF72 and shUBF16  and displaying similar levels of UBF depletion (B1 and B3). shUBF555 recapitulated the phenotypes we documented for shUBF72 and shUBF16. In particular, shUBF555 exhibited significantly reduced mRNA and protein levels of RNA pol1 (C1 and C3), markedly reduced pre-rRNA levels , as well as downregulated gene expression of Rrn3  and of upstream transcription factor C-myc .Interestingly, as we have shown here, the functional interactions affected by UBF depletion include factors that are both upstream, at the same level, and downstream of UBF. Additional confirmation of this network of functional interactions of UBF was provided by the ability of a cocktail of exogenous UBF1 and UBF2 to rescue downstream and upstream UBF depletion effects. Specifically, transient transfection of shUBF72 cells with a plasmid mix expressing shUBF72 oligo-resistant, synonymous triple mutant FLAG-tagged UBF1 and UBF2 , A1–A4 fG vector C. FinallIn the overall conclusion of our work, the coordinate downregulation of these proteins suggests that downregulation of UBF alone leads to a drastic and large-scale re-adaptation of their expression and reveals an unrecognized network coordinating the expression of the critical factors that control rRNA expression.de novo gain-of-function missense mutations in human UBF (E210K) that upregulate rRNA transcription have been identified as the cause of severe developmental neuroregression in childhood . More recently, heterozygous ildhood , and it is thought to be rapidly induced and responsive to different stimuli and signaling cascades . The eliewed by , 59). rDiewed by . Additiothe rDNA , 62. Sucthe rDNA ). In thithe rDNA . HoweverA1), both become phosphorylated at Ser486 (B1), and both are present across the active rDNA in NIH 3T3 fibroblasts . In vivo, UBF initially was purified from mammalian cells as a complex of UBF1 and UBF2  with the addition of 10% v/v fetal bovine serum (FBS) and 50 U/ml of penicillin/streptomycin. Mouse IMCD3 (IMCD) cells were cultured in DMEM/F-12 (1:1) medium with 10% v/v FBS, 2 mM glutamine, and 50 U/ml of penicillin/streptomycin. Mouse Neuro2A (N2A) cells were kept in the same conditions as NIH 3T3 but with MEMα instead of DMEM. All lines were maintained at 37 °C in 5% COUbf ORF, originally from the RNAi Consortium (TRCN0000096691), was obtained from Open Biosystems (RMM3981–201808572). The shRNA sequence targets both Ubf1 and Ubf2 mRNAs. Lentiviral shRNA production, lentivirus particle packaging and harvesting, transductions of NIH 3T3 mouse fibroblasts, and single colony selection of clones were carried out as previously described  and obtained from Open Biosystems (RMM3981–201806555)].A pLKO.1-based lentiviral shRNA vector, expressing a puromycin resistance gene and targeting the mouse escribed , 69. IniUbf1 was carried out on NIH 3T3 cells, as a validation control, using Lipofectamine 2000 (Invitrogen) and custom-made siMAX siRNA duplexes acgaaggaggugaaggacucc/ggaguccuucaccuccuucgu (MWG) at 40 nM. Cells were sampled and analyzed by WB at 48 h post-transfection.siRNA-mediated silencing of ImageJ 1.49n (NIH) and normalized using same-sample and same-membrane band intensities for the ER protein calnexin, or normalized with same-sample total protein and quantified using ImageLab 6.0.1 software (Bio-Rad) after Ponceau S staining. Means of measurements from three independent experiments for each cell line, run in parallel, were plotted, and differences were statistically evaluated using Microsoft Excel and GraphPad Prism 8.0 .Protein extraction from cells grown on 10-cm plates was carried out directly in 4X SDS-PAGE sample buffer, using a rapid extraction protocol that preserves protein phosphorylation . The lys+ RNA) was affinity-purified using the Dynabeads mRNA Direct purification kit (Invitrogen). cDNA was synthesized with a random hexamer/oligo-dT primer mix, using the iScript cDNA synthesis kit (Bio-Rad).Total RNA extraction from cell lines was carried out with the RNeasy Mini Kit (QIAGEN), including in-column DNase treatment, according to the manufacturer’s protocol. Polyadenylated RNA  was conducted on the CFX96 Real-Time PCR system (Bio-Rad) with specific oligonucleotide primers shown in B2m (β2-microglobulin) was used as a reference, unless otherwise stated. The Common Base method was used to allow direct quantitative comparisons across samples  for 10 min and permeabilized for 15 min with 0.5% v/v Triton X-100 in PHEM. For the PoLR1A staining in Zeiss Apochromat 63× NA 1.4 oil lens on a Zeiss Axiovert 200M inverted fluorescence microscope, equipped with a Zeiss AxioCam MRm camera.For immunofluorescence, cells were grown on coverslips, fixed with 3.7% w/v paraformaldehyde in PHEM buffer , nine cultures each of NIH 3T3 WT and shUBF cells were seeded, in parallel, in twelve-well plates with 5000 cells per well. For each cell type, triplicate measurements of cell counts from each of three wells were recorded with a hemocytometer and the average of nine values was calculated at 24, 48, and 72 h after seeding.To generate growth curves of cell lines D, nine c5 cells each time, fixed in 70% v/v ethanol, treated with 200 μg/ml RNAse for 45 min at 37 °C, labeled with 20 μg/ml propidium iodide, and analyzed using an S3e Cell Sorter (Bio-Rad) with the FlowJo software (Treestar). For analysis of cell cycle phases by RT-PCR or WB , they were incubated for 15 min with fresh medium containing 2 mM 5-fluorouridine (Sigma F5130). Immunofluorescence was performed as described using an anti-BrdU primary antibody. Quantitative analysis of captured images, measuring the average mean intensity of the nucleolar signal in each cell from three independent experiments , was carried out using Imaris .FUrd labeling of cells was performed as described previously . After W5’AGAGAAAAGAGCGGAGGTTCGGGACTCCAA3’ . Probes were non-radioactively labeled using DIG-oligonucleotide 3′-End Labeling kit (Roche). Chemiluminescence detection was performed using the DIG-luminescent detection kit (Roche) and acquired in ChemiDoc MP image system (Bio-Rad). Band intensities of chemiluminescence signals of rRNA bands were measured using ImageJ. Signals were expressed as band intensity per μg of total RNA loaded. Ethidium bromide-staining of rRNA bands was visualized with Syngene G:BOX (ChemiXR5).Total RNA was extracted using Trizol (Ιnvitrogen). RNA samples were analyzed by Northern blot . Single-, as per ) and 5’GSDS-PAGE and quantitative WB. At least 3 independent experiments were performed for the full set of comparisons between WT and shUBF cell lines.For assessment of components of the mTOR or RAF-MEK-ERK (MAPK) pathways, multiple cultures, seeded in parallel in an identical manner, were grown to 70% confluency in normal media with 10% FBS and then transferred for 16 h to 0.5% serum to establish the same baseline growth signaling level. Medium was then changed to either normal 10% FBS, or 10% FBS with mTOR inhibitor rapamycin at 20 mM, or 10% FBS with MAPK inhibitor U0126 at 10 mM, for 2 h and then processed for quantitative WB exactly as described in section 2, 0.5% NP-40, irradiated in the presence of 4,5,8-trimethylpsoralen  (Sigma) at 0.6 mg/ml, for 5 min on ice and repeated 4 times at 6 cm distance from the 366-nm UV light box  (based on the protocol of (Typhoon (Cytiva), using a Gaussian peak fit generated with MagicPlotPro (MagicPlot Systems LLC).Nuclei were enriched from cells lysed in 10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCltocol of ). Genomitocol of , 78, usi2, 10 mM KCl 0.1% Triton, 1 mM DTT), transferred in sonication buffer , sonicated with a Bioruptor (Diagenode) for 30 cycles of 30 s on/30 s off at high intensity and the chromatin was diluted 10-fold in immunoprecipitation (IP) buffer , followed by 1 h preclearing using Protein A sepharose beads  at RT. Each IP reaction was carried out overnight with the equivalent of 15 μg DNA in IP Buffer, at 4 °C. The antibody slurry consisted of 30 μl slurry of Protein A sepharose beads , blocked in 3 μg salmon sperm DNA and 30 μg BSA, and 5 μl antibody serum or 10 μg of Rabbit IgG per IP reaction. Beads were washed and divided into two equal parts: one was processed for ChIP (qPCR) and the other for WB analysis (“UBF ChIP” sample). For ChIP, the immunoprecipitated chromatin was eluted in elution buffer (1% SDS and 0.1 M NaHCO3) and reverse cross-linked using 200 mM NaCl containing 0.5 μg/μl RNase at 65  °C overnight. Samples were purified with the QIAquick PCR purification kit (QIAGEN) and analyzed in triplicate per reaction by quantitative real-time PCR on CFX96 Real-Time PCR system (Bio-Rad), with specific primers to the mouse rDNA (BK000964), as shown in ChIP was performed as previously described  with sevA1) in parallel with non-precipitated, input samples .For WB analysis, beads were boiled at 95 °C for 10 min in SDS-sample buffer and separated on SDS-PAGE  or 24 h (in C) after removal of the transfection medium.For the rescue experiments in  24 h in C after rt test for comparisons between two groups, not assuming equal variance; one-way ANOVA with Dunnett’s correction or two-way ANOVA with Tukey’s correction for differences of one or two variables, respectively, among more than two groups. Growth curve and cell cycle differences , <0.01 very significant (∗∗), <0.001 highly significant (∗∗∗), and <0.0001 extremely significant (∗∗∗∗). Statistical analyses were performed using the GraphPad Prism 8.0 Software .To evaluate statistical significance of differences between comparisons of values expressed as means ± SD of at least three different independent experiments run in parallel, the following statistical analysis was employed, as appropriate: Welch’s ferences  were comThe manuscript contains all data described within the text.https://www.phosphosite.org/proteinAction.action?id=1131&showAllSites=true.W1: https://www.genecards.org/cgi-bin/carddisp.pl?gene=POLR1A.W2: https://www.genecards.org/cgi-bin/carddisp.pl?gene=POLR1E.W3: https://www.proteinatlas.org/ENSG00000068654-POLR1A.W4: https://www.proteinatlas.org/ENSG00000137054-POLR1E.W5: http://www.signalingpathways.org/ominer/query.jsf?geneSearchType=gene&findMax=y&gene=POLR1A&foldChangeMin=2&foldChangeMax=30&significance=0.05&species=all&reportsBy=pathways&omicsCategory=tm&countMax=3000.W6: http://www.signalingpathways.org/ominer/query.jsf?geneSearchType=gene&findMax=y&gene=POLR1E&foldChangeMin=2&foldChangeMax=30&significance=0.05&species=all&reportsBy=pathways&omicsCategory=tm&countMax=3000.W7: This article contains The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


The doubled haploid (DH) technology is employed worldwide in various crop-breeding programs, especially maize. Still, restoring tassel fertility is measured as one of the major restrictive factors in producing DH lines. Colchicine, nitrous oxide, oryzalin, and amiprophosmethyl are common chromosome-doubling agents that aid in developing viable diploids (2n) from sterile haploids (n). Although colchicine is the most widely used polyploidy-inducing agent, it is highly toxic to mammals and plants. Therefore, there is a dire need to explore natural, non-toxic, or low-toxic cheaper and accessible substitutes with a higher survival and fertility rate. To the best of our knowledge, the advanced usage of human anticancer drugs “Paclitaxel (PTX)” and “Caffeine–Taurine (CAF–T)” for in vivo maize haploids doubling is being disclosed for the first time. These two antimitotic and antimicrotubular agents (PTX and CAF–T) were assessed under various treatment conditions compared to colchicine. As a result, the maximum actual doubling rates (ADR) for PTX versus colchicine in maize haploid seedlings were 42.1%  versus 31.9% , respectively. In addition, the ADR in maize haploid seeds were CAF–T 20.0% , PTX 19.9% , and colchicine 26.0% . Moreover, the morphological and physiological by-effects in haploid plants by PTX were significantly lower than colchicine. Hence, PTX and CAF–T are better alternatives than the widely used traditional colchicine to improve chromosome-doubling in maize crop. During the last century, the development of maize inbred lines relied on recurrent selfing and selection to reach the desired level of homozygosity . It requ2O) and antimitotic herbicides, such as benzamides (pronamide), dinitro–anilines , carbamates (chlorpropham and isopropyl N-3-chlorophenyl carbamate), and phosphor–thioimidates (aminoprophosmethyl/APM) due to its availability and well-developed protocols. These herbicides stop mitosis and cell division, leading to chromosome doubling [20, which is considered a comparatively safe and effective alternative [20 pressure chamber is relatively higher [20 exposure at 0.6 MPa  on maize at V3–V8 growth stage revealed even potential regarding the doubling of chromosomes, but treatment of adult plants requires big chambers and pots for growth [Haploid plants are generally sterile , and thedoubling , like codoubling ,19. In cdoubling  and in vdoubling , but foudoubling . A high doubling . Oryzalidoubling . Anotherdoubling  employs ernative , but they higher ,26. N20 2O, APM, and oryzalin [20 and anti-mitotic herbicides with equal efficacy of doubling chromosomes to toxic colchicine have been acknowledged. However, some basic limitations confine their usages, such as cost, availability, higher doses or concentrations, and unregistered sales, such as pronamide and APM in most African and Asian countries, limit their easy accessibility. The availability of such chemicals in high-quality pure grade is more expensive than colchicine and is limited [Taxus brevifolia. Although it was generally considered a particular metabolite of Taxus spp., it was recently found in hazel cell cultures [v/v) and cremophor EL, known as “Taxol” [Colchicine is far more toxic than pronamide, trifluralin, Noryzalin ,24,25, roryzalin ,21. “Theoryzalin ,28. Howeoryzalin . Recentlcultures . It is a “Taxol” . PTX is  “Taxol” . Microtu “Taxol” . PTX can “Taxol” . It is a “Taxol” .Taurine is a non-essential amino acid containing sulfur called 2-amino-ethane-sulfonic acid . It is aNicotiana tabacum “Bright Yellow 2”). Caffeine did not significantly affect the doubling of chromosomes in wheat anther culture. However, several caffeine treatments reported a higher frequency of chromosomes doubled plants [Caffeine can increase the early doubling of chromosomes and haploid plants, the androgenic induction, and spontaneously produce fertile plants . It can d plants . TherefoContinuous efforts proceed to optimize cheaper, innovative, non-toxic, or less toxic chemical agents to improve chromosome-doubling efficiency with higher survival rates than colchicine. To meet this challenge, we designed an advanced study to explore the ability of PTX and caffeine–taurine (CAF–T) to induce chromosome doubling in maize haploids. A systematic approach was adopted, and multiple treatments with different processing times were designed based on replicated field trials to study the chromosomal doubling efficacy of PTX and CAF–T versus colchicine. Moreover, the impact of PTX versus colchicine on the morphology and physiology of haploid plants was assessed and analyzed through seed germination, photosynthetic pigments, plant and ear height, number of silks/ears, and plant weight.Under different concentrations and processing periods, the survival rate (SR), doubling rate (DR), and actual doubling rate (ADR) of PTX-treated haploid seeds and seedlings were analyzed. In addition, anthers emergence rate (AER), partial fertility rate (PFR), complete fertility rate (CFR), and total fertility rate (TFR) were also calculated based on four different types of tassels A usuallyIn all treatments of M1, the highest DRs induced by PTX were 19.8% and 21.2% in the condition of 100 μM for 16 h and 24 h, respectively. However, both conditions for DR, ADR, PFR, CFR, and TFR were determined to be non-significantly different to each other. The M2 in PTX produced the highest DR of 54.3% and TFR of 75.2% in the condition of 400 μM for 16 h, followed by another treatment that produced a DR of 46.9% and TFR of 59.4% in the condition of 100 μM for 8 h. However, both conditions were statistically non-significant regarding ADR . While comparing both M1 versus M2, M2 is a better line not only to yield higher DR C,D but aIn Experiment 2, using different treatments and processing times, CAF–T application to maize haploid seeds was assessed through SR, DR, ADR, AER, PFR, CFR, and TFR A–G. The Two treatment methods, i.e., M1 and M2, were employed using different colchicine concentrations and processing time to calculate SR, DR, ADR, AER, PFR, CFR, and TFR A–N. In MIn M2, the highest DR induced by colchicine was calculated 43.8% with SR 72.9% in the condition of 0.5 mM for 24 h treatment B,D, reveIn addition to fertility rates, the number of seeds on DH ears subsequent from each treatment was widely variable . Mostly To establish the efficacy of PTX versus colchicine, 1478 putative haploid seedlings from two populations were used in this experiment. PTX treatment presented consistent and significantly better SR, ADR, and TFR in both genotypes as compared to colchicine treatment A,B. HoweThe plant and ear height were significantly lower for both PTX and colchicine than CK in both the genotypes. However, PTX treatment was significantly better than colchicine treatment for both plant and ear height A,B. ColcThe number of silks/ears was significantly lower for both PTX and colchicine than CK treatment in both genotypes. However, PTX treatment was significantly better than colchicine treatment for the number of silk/ear C, which Our experiment measured plant weight significantly less for PTX and colchicine than CK in both genotypes. However, PTX treatment was significantly better than colchicine treatment for plant weight D, which a (aChl), chlorophyll b (bChl), total chlorophyll (TChl), and carotenoid (xC) contents were significantly lower for both PTX and colchicine than CK in both the genotypes. However, PTX treatment was significantly better than colchicine treatment for aChl, bChl, TChl  with Stock 6 inducer for this experiment because of its higher germination of about 97%.Germination (%) Impact on Treated Seeds by PTXAcross all treatments (T1 to T12) of PTX, germination percentage decreased with increasing concentrations and processing time A. PTX-tr2.Germination (%) Impact on Treated Seeds by CAF–TCAF–T-treated seed germination percentage was found between 96.2% and 77.8% across all respective seed-soaked treatments. CAF–T exhibited a non-significant impact on seed germination percentages on T1 and T2 versus CK B.3.Germination (%) Impact on Treated Seeds by ColchicineThe colchicine-treated seed germination percentage ranged from 90.8% to 80.5% across all respective seed-soaked treatments C. In conIn conclusion, PTX revealed a lesser impact on seeds germination percentage versus colchicine.This experiment aimed to validate the chromosomes doubling induced by PTX and CAF–T in the field studies through the microscope.The M1 revealed DR ranging from 60% to 80% across all treatments of PTX based on chromosome doubling count under the microscope using root tip cells.Maximum DR in M1 achieved 80% by these treatments, i.e., PTX 800 μM 16 h, 24 h, and 48 h: PTX 200 μM 48 h A.The M3 showed DR ranging from 56.7% to 70.0% across all treatments of PTX. A maximum DR (70%) was achieved through M3 at concentrations of PTX 400 μM and 800 μM for 16 h B.In conclusion, M1 exhibited better frequency and DR than M3. In addition, we noticed 48 h treatments were not exceptionally better than 24 h treatments both in M1 and M3. Therefore, it helps deduce that 48 h treatments cannot improve DR further but decrease SR. The CK treatment showed a spontaneous doubling ratio of about 3%. Hence, PTX proved effective in doubling chromosomes, as shown in The M1 revealed DR ranging from 53.3% to 86.7% across all treatments of CAF–T based on chromosome doubling count under the microscope using root tip cells. The maximum DR in M1 was achieved at 86.7% by caffeine 2 g/L + taurine 12 g/L, 16 h C.The M3 disclosed DR ranging from 33.3% to 53.3% across all treatments of CAF–T. The maximum DR (53.3%) was achieved by M3 at a concentration of caffeine at 2 g/L and taurine at 12 g/L for 8 h; caffeine at 6 g/L and taurine at 36 g/L for 8 h; caffeine at 4 g/L and taurine at 24 g/L for 24 h; caffeine at 6 g/L and taurine at 36 g/L for 24 h; no further increases in ratios were observed D.In conclusion, CAF–T-based M1 exhibited better frequency and DR than M3. In addition, we observed that 48 h treatments cannot improve DR further but decrease SR. Hence, CAF–T verified its efficacy in doubling chromosomes, as shown in No standard set criteria are used to assess chromosomal doubling agents’ efficacy. Some studies were based on the microscopic chromosomal count , severalIn our experiments, the CAF–T-based M1 disclosed SR ranges from 80.4% to 99% across all treatments. The PTX-based M1 and M2 showed SR ranges from 73.4% to 98.5% and 56.4% to 90.1%, respectively, whereas colchicine-based M1 and M2 revealed SR varies from 69% to 90.8% and 29% to 82.6%, respectively, across all treatments. After treatment with colchicine, the established seedlings can be only 40 to 80%, in which 10–30% diploids or false positives may be observed in the field . The higThe spontaneous doubling rate was 1.4% in Experiments 1, 2, and 3, which was very low and almost equal to the reported 1.5% . HoweverOverall, M2 was more effective than M1 in achieving higher DR in our field experiments because many stem cells were exposed to chemical treatments leading to tassel fertility. M2 also proved better in tetraploids than M1 . HoweverIn M1, PTX, colchicine, and CAF–T exhibited an AER range from 4.9% to 16.6%, 4.7% to 32.8%, and 4.5% to 12.4% across all treatments. In M2-based experiments, PTX and colchicine displayed AER range from 5.9% to 28.7% and 7.8% to 35.9%, respectively, across all treatments. In diverse China and US germplasm, the reported AER with significant genetic variance varied from 9.8 to 89.8%. .In the LS field efficacy-validation experiment, PTX and colchicine-treated maize seedlings showed an ADR of 37.7% and 19.2%, respectively, in V1, 20% and 14.5%, respectively, in V2, whereas  reportedHaploid plant tassels cover a wide range of fertility, from one or two anthers shedding pollen to the complete tassel . TherefoColchicine, PTX, and CAF–T-treated M1 revealed a maximum DR of 21.2%, 29.6%, (0.08% for colchicine concentration for 8 h), and 20.9%, respectively, but colchicine induced 18% of DR at 0.06% colchicine for 12 h treatment ,58,59,60PTX and CAF–T produced comparatively higher seed counts than colchicine. According to , only 30Phlox drummondi [Prunella vulgaris and Dendrocalumus brandisii, a higher colchicine concentration with longer processing times decreased the germination of seeds [Datura stramonium L.), which decreased linearly by increasing incubation time and the concentration of colchicine. A decrease in plant height in Abelmoschus esculentusalso (okra) and Phlox drummondi was measured due to colchicine effects [Dendrocalumus brandisii. Comparatively, colchicine revealed more negative effects regarding the degradation of sucrose than starch during the germination of seeds and seedling growth [The colchicine effects on the morphology and physiology of treated maize haploid seeds or seedlings have not been reported. However, only SR of both maize haploid seeds and seedlings have been widely reported. In addition to SR, PTX unveiled less impact on seeds germination percentages as compared to colchicine. Severe effects of different colchicine concentrations were also reported in rummondi . In Prunof seeds ,65. Colcof seeds . We founof seeds  studied  effects ,66. ColcTChl directly impacts the plants’ photosynthesis capacity [Chlorophyll is the most essential pigment for photosynthesis because it largely determines the process’s capacity and plant growth. The capacity . The carcapacity . Damage capacity  because capacity . SSON prR1–nj anthocyanin marker expression on kernels. The required sample sizes were obtained from 16,400 putative haploid seeds for all experiments in this embodiment. The details of genotypes and experimental locations are demonstrated in Maize haploids seeds were produced at the College of Agriculture, Guizhou University , China. Haploid seeds were separated visually from diploids by an All treatments were replicated thrice in Experiments 1, 2, and 3. Each replication has an average of 70 putative haploids—some treatments have more than 70 seeds/seedlings to ensure equal stand with lower-dosed treatments because higher doses affect plant stands. All three replications of each treatment were blocked in a randomized complete block design (RCBD), including Experiment 4. PTX was purchased from Peptide Biotechnology Co. Ltd., Xi’an, China, CAF–T from BANNY DEER at Wuhan East–Lake High Tech Region, China, and colchicine (98%) from Shanghai Macklin Biochemical Co. Ltd., Shanghai Chemical Industry Park, Shanghai, China. MAPADA P1 UV–Visible Spectrophotometer was purchased from Shanghai Mapada Instruments Co., Ltd. Shanghai, China.2O).The putative maize haploid seeds and seedlings (including roots and stem) were treated with PTX at 28 °C for 8, 16, and 24 h. The stock solution concentrations of PTX were used for chromosomal doubling, including 100 μM (0.085 g/L), 200 μM (0.170 g/L), 400 μM (0.341 g/L), and 800 μM (0.682 g/L). PTX is highly lipophilic and has a poor water solubility of less than 0.1 μg/mL, seriously affecting its bioavailability . The met2O.The putative maize haploid seeds were soaked in CAF–T at 28 °C for 8, 16, and 24 h. The concentrations of CAF–T were used for chromosomal doubling, including caffeine at 1 g/L and taurine at 6 g/L, caffeine at 2 g/L and taurine at 12 g/L, caffeine at 4 g/L and taurine at 24 g/L, and caffeine at 6 g/L and taurine at 36 g/L, with 0.1% DMSO, which was adjusted by ddH2O.The putative maize haploid seeds and seedlings (including roots and stem) were treated with colchicine at 28 °C for 8, 16, and 24 h. The different concentrations of colchicine were used for chromosome doubling, including 0.5 mM (0.2 g/L), 1 mM (0.4 g/L), 1.5 mM (0.6 g/L), and 2 mM (0.8 g/L) [2O).LS field efficacy-based comparative study was designed to further authenticate the best treatment (concluded in Experiment 1) of the new chemical, i.e., PTX (400µM and 2% of DMSO and 1.02 mL/L of tween-80 for 16 h). The morphological and physiological measurements were also taken. The putative maize haploid seedlings from two populations, i.e., V1 and V2, were used to compare PTX with colchicine treatment (2 mM and 0.1% of DMSO for 8 h treatment) as the best treatment in Experiment 3 regarding TFR and also suggested by  and contTo verify the doubling of chromosomes under the microscope, the same concentrations of PTX and CAF–T see  were use2O for 6 h at 28 °C, and then soaked in three chemical agents, i.e., PTX, CAF–T, and colchicine for 8, 16, and 24 h at 28 °C. After chemical treatments, seeds were rinsed with ddH2O for 30 min and shifted on a wet germination paper in an incubator with a maintained temperature of 28 °C [The putative maize haploid seeds were first soaked in ddH2O at 28 °C for 6 h and shifted on a moist germination paper for 72–96 h in an incubator temperature maintained at 28 °C for germination. When the roots achieved their length up to 1 cm, a 28 °C temperature decreased to 24 °C in demand to make shoots grow. When shoots’ sizes increased up to 2 cm, the coleoptiles’ top 2–3 mm [2O for 30 min, and then shifted into trays for growing in the greenhouse.This protocol is a “standard seedling-immersion method” defined in the 1990s . The putp 2–3 mm  were cutWe followed the protocol of root immersion as suggested by . The rooAfter respective treatments, seedlings were transplanted into trays filled with suitable soil composition, sand, peat, and compost with pH 6 to 6.5. Treated seedlings were grown in a greenhouse for 15 h of photoperiod during the day and maintained at about 26–28 °C and 24–28 °C at night. Nutrients and water were provided on a regular need basis. Gradually transferring the seedlings from lower light intensity to higher light intensity and higher-to-lower humidity helped acclimate the treated seedlings .When survived seedlings achieved V2 leaf stage  more or The knob-2 probe used in our experiment was developed from tandem repeats of plasmid clones. The 180-base pair (bp) knob sequence was obtained by dividing the 180 bp knob sequence into three non-overlapping parts, each containing 59 nucleotides, and then modifying the 5’ ends with fluorescein amidites (FAM). The knob-2 probe sequence and maize chromosomes were prepared according to the protocol provided by . This isGAAGGCTAACACCTACGGATTTTTGACCAAGAAATGGTCTCCACCAGAAATCCAAAAATThe fluorescence in situ hybridization (FISH) procedure was followed as described by .Survival rate (SR) = count of survived plants at pollination/number of seedlings treated × 100 ;Doubling rate (DR) = sum of plants showed partial and/or complete fertility/count of haploid survived plants at pollination × 100;Actual doubling rate (ADR) = SR × DR/100 ;Anthers emergence rate (AER) = number of plants showed anthers only/number of haploid plants survived at pollination × 100 ,49;Partial fertility rate (PFR) = number of plants that exhibited partial fertility/number of haploid plants that survived at pollination × 100;Complete fertility rate (CFR) = number of plants presented complete fertility/number of haploid plants that survived at pollination ×100;Total fertility rate (TFR) = AER + PFR + CFR.In Experiments 1–4, the following parameters were documented: (a) Number of seeds or seedlings treated; (b) Germination percentage; (c) Number of survived Do plants at pollination; (d) Count of Do plants showed anthers only; (e) Sum of Do plants that exhibited partial fertility; (f) Number of plants presented complete fertility; (g) Number of false/wrong diploid plants; (h) Number of haploid plants. The following calculations were made in Experiments 1–4 and expressed in percentages (%).In addition, seed setting data were also collected in Experiments 1–3 and expressed as follows; (a) Percentage of DH lines with 1–5 seeds; (b) Percentage of DH lines containing 6–25 seeds; Percentage of DH lines containing > 25 seeds .0 plants/D1 ears produced 1–5 seeds/number of haploids survived plants at pollination × 100;Percentage of DH lines with 1–5 seeds = number of D0 plants/D1 ears produced 6–25 seeds/number of haploid survived plants at pollination × 100;Percentage of DH lines containing 6–25 seeds = number of D0 plants/D1 ears produced > 25 seeds/number of haploid survived plants at pollination × 100;Percentage of DH lines containing > 25 seeds = number of D0 plants produced seeds/number of D0 survived haploid plants at pollination × 100;RR = number of D0 plants produced seeds/number of putative seeds/seedlings treated with chemical agent × 100.OSR = number of DPlant and Ear Height, Plant Weight, and Number of Silks/EarFive plants per repeat were randomly selected (evading three plants at each plot beginning and end to exclude any impact) from each treatment and genotype. Plant and ear height were measured randomly from haploid and diploid  plants. Plant height was measured from the plant base or soil to the last collar leaf. Ear height measurements were obtained from the plant’s base (ground/soil) to the node-bearing upper ear. Plant and ear height data were measured from three replications and analyzed as means. Cut the plants from the ground surface and weigh them on a scale in grams (g). The mean plant weight and number of silks/ears were calculated from four replications.aChl, bChl, TChl, and xC)Measurement of Photosynthetic Pigments  Total number of maize haploid seeds/roots were treated; (b) The number of treated seeds or roots doubled with 5–10% cells. The percentage of treated seeds/roots doubled was calculated as the sum of treated seeds or roots with cells doubled up to 5–10% and divided by the number of seeds or roots treated × 100.n = 3 or n = 4) were expressed in graphs based on standard error (SE). The mean comparison was made by the “Tukey’s Honestly Significant Difference (HSD)” test at p ≤ 0.05, following the Analysis of Variance (ANOVA). All replicated data were analyzed via IBM SPSS Statistics 21.0 software. The graphics software known as “Origin” was applied to improve graphics .In Experiments 1 to 5, the results of all three or four replications  to improve chromosome-doubling success rates and survivability. The ADR for PTX and colchicine in maize haploid seedlings were 42.1%  and 31.9% , respectively. The ADR induced in maize haploid seeds by colchicine, CAF–T, and PTX were 26% , 20% , and 19.9% (100 μM for 24 h treatment), respectively. PTX and CAF–T offer occupational health, operational ease, and disposal. PTX guarantees a cost-effective and better rate of doubling haploid plants at lower concentrations. Optimizing chromosomal doubling protocols based on less toxic or bio-safe chemicals like PTX and CAF–T will further enhance the overall efficiency of DH production. The morphological and physiological effects produced in haploid plants by PTX were significantly lower than toxic colchicine. PTX will improve the quantity of D0 lines seeds, which may exterminate the requirement to increase D1 lines seeds, thereby saving time and cost of the additional cycle to multiply seeds.


Staphylococcus aureus. Using the broth microdilution method, antibacterial activities against methicillin-resistant S. aureus ATCC 43300 (MRSA) and methicillin-sensitive S. aureus ATCC 29213 (MSSA) were identified for four aurone-derived triazole compounds, AT106, AT116, AT125, and AT137, using the half-maximal inhibitory concentrations for the bacteria (IC50) and mammalian cell lines (CC50). Compounds AT125 and AT137 were identified to have pharmaceutical potential as the IC50 values against MRSA were 5.412 µM and 3.870 µM, whereas the CC50 values measured on HepG2 cells were 50.57 µM and 39.81 µM, respectively, resulting in selectivity indexes (SI) > 10. Compounds AT106 and AT116 were also selected for further study. IC50 values for these compounds were 5.439 µM and 3.178 µM, and the CC50 values were 60.33 µM and 50.87 µM, respectively; however, SI values > 10 were for MSSA only. Furthermore, none of the selected compounds showed significant hemolytic activity for human erythrocytes. We also tested the four compounds against S. aureus biofilms. Although AT116 and AT125 successfully disrupted MSSA biofilms, there was no measurable potency against MRSA biofilms. Checkerboard antibiotic assays to identify inhibitory mechanisms for these compounds indicated activity against bacterial cell membranes and cell walls, supporting the pharmaceutical potential for aurone-derived triazoles against antibiotic-resistant bacteria. Examining structure–activity relationships between the four compounds in this study and other aurone-derived triazoles in our library suggest that substitution with a halogen on either the salicyl ring or triazole aryl group along with triazoles having nitrile groups improves anti-Staphylococcal activity with the location of the functionality being very important.Infections caused by antibiotic-resistant bacteria continue to pose a significant public health threat despite their overall decreasing numbers in the last two decades. One group of compounds fundamental to the search for new agents is low-cost natural products. In this study, we explored a group of newly synthesized novel aurone-derived triazole compounds to identify those with pharmaceutical potential as inhibitors of antibiotic-resistant  Staphylococcus aureus and its antibiotic-resistant strains are well-known in medical and research communities. They are not only capable of causing illnesses with significant mortality [S. aureus (MRSA), continue to pose a notable public health threat [S. aureus is capable of forming multilayered cell masses called biofilms that increase its ability to resist antibiotics, generating a persistent source for recurring infections that are often associated with increased mortality and morbidity [ortality ,2, but wortality , these ph threat ,5,6,7. Ih threat ,8,9. Alth threat ,8,10, anh threat . Furtherorbidity ,13.S. aureus is generally a multi-stage process that begins with the attachment of planktonic (free-floating) cells to biotic or abiotic surfaces using cell wall-anchored proteins, cell wall teichoic acids, and extracellular DNA (eDNA) [S. aureus cells begin dividing, forming microcolonies and a matrix of extracellular polymeric substance (EPS), consisting primarily of polysaccharides, proteins, or eDNA [S. aureus biofilm enters a maturation and re-structuring stage [S. aureus cells [Biofilm development by A (eDNA) ,15,16,17A (eDNA) ,13,14,15ng stage ,13,16. Tus cells ,13,15,16us cells ,13,15,16us cells ,13,15,16us cells ,16,17.S. aureus poses, there are several additional factors that warrant the search for new antimicrobial agents. One key factor is that resources for research into new agents have significantly shrunk since the 1990s as large pharmaceutical companies have completely abandoned the field [S. aureus-related infections. In the US alone, HA-related infections generated an added burden of $10 billion or more annually [In addition to the threat that he field , resultiannually ,21, and annually ,23,24,25S. aureus is natural products, especially aurones (2-benzylidene-1-benzofuran-3(2H)-ones), which is a flavonoid subfamily that contributes to the yellow coloring of some flowering plants [S. aureus (MSSA) and MRSA along with toxicity and phenotypic studies that demonstrate the potential of these molecules for future pharmaceutical use.One source for potential agents with bioactivity against g plants . These sg plants ,29,30,31g plants ,33. In ag plants . An auroS. aureus, four were chosen for further study  of AT106 were less toxic compared to the other three AT compounds, higher concentrations of AT106 were required for anti-Staphylococcal activity. Conversely to their antibacterial activity where AT137 was the strongest and AT106 was the weakest performer, AT106 is the least toxic with CC50 concentrations above 60 µM whereas AT137 is the most toxic with a 2.838 µM CC50. Still, all four compounds show pharmaceutical potential as they all have selectivity towards S. aureus. Unfortunately, these initial tests also show that only AT137 and AT125 were able to meet our selectivity criteria against MRSA, but we retained AT106 and AT116 for further testing as they could still be workable in mixtures with other bioactive compounds.After screening the library of AT triazoles against er study . Table 1In addition to the toxicity for mammalian cells, we assessed the hemolytic activity of these AT compounds against human red blood cells. The results of the initial hemolysis assay in 50 values (5–25 µM), the graphs reflect our earlier distinction between strong inhibitors  and weaker inhibitors  of S. aureus growth. These results signify that AT125  shown in As an initial step to determine potential modes of action for the ATs, checkerboard assay experiments were conducted wherein mixtures of ATs and antibiotics used against S. aureus, including MRSA. The increasing resistance of microbes to existing therapies necessitates improved antimicrobials, and triazole compounds are well-established molecular scaffolds for pharmaceuticals, including antimicrobials [Our current results extend the existing set of evidence of the bioactivity of aurones by using aurone-derived triazoles with activity against crobials ,36,37,38crobials . The 1,2crobials ,43,44,45crobials . The 1,2crobials ,49,50,5150 values between 3.09–5.44 µM (1.08–1.63 µg/mL). These concentrations are comparable to MIC ranges and reported IC50 values for the reference antibiotics vancomycin and linezolid [50 values of the ATs for MRSA were between 3.87–22.38 µM (1.35–6.71 µg/mL) with only AT125 and AT137 exhibiting IC50 values less than the MICs for vancomycin and linezolid. The inhibition of MRSA by AT125 and AT137 is confirmed by the 48 h growth curves where only concentrations lower than the IC50 values allowed for the growth of MRSA.In this report, we have described the anti-Staphylococcal activity of aurone-derived 1,2,3-triazoles (ATs) formed by the reaction of aurones with sodium azide. The resulting ATs possess two sites for alkylation chemistry and the potential for modification of the ring system. To assess the pharmaceutical potential of the AT compounds, we used the standard method of calculating their selectivity index (SI), which is the ratio of their half-maximal toxicity concentration and their half-maximal inhibitory concentration. An SI higher than 10 shows that the compound has a large enough selectivity towards the bacterium, but not mammalian cells. The AT compounds chosen for this study  all had SIs of >10 with mammalian cell lines and were not hemolytic for human erythrocytes indicating efficacy for delivery in the bloodstream. All four of the ATs were effective against MSSA, exhibiting ICinezolid ,53,54,55S. aureus infections can be difficult to treat and eliminate due to multiple factors, including impermeability associated with chemical features of a drug or the physiology of the bacterial cells in the biofilm, such as reduced proliferation or metabolism, especially if dormant bacteria known as persisters are present [TM assay which contains the dyes SYTO9 and propidium iodide. SYTO9 is transported across intact cell membranes and stains the nucleic acids, resulting in the green fluorescence of living cells. Propidium iodide cannot cross membranes of live cells, thus only staining nucleic acids of dead cells, producing a red fluorescence. Comparisons of live and dead cell numbers, as well as total bacterial cell numbers, to mock-treated control biofilms, were used to assess the efficacy of AT treatment. All ATs were effective against MSSA using the 100 μM concentration. This concurs with other observations that biofilms are more resistant to antibiotics, requiring concentrations up to 1000 times greater than required for planktonic bacteria [Biofilms associated with  present . For thibacteria .S. aureus cell wall as a potential target. S. aureus cell walls are composed of peptidoglycan, which is a layered polymer of alternating N-acetylmuramic acid (MurNAc) and N-acetylglucosamine (GlcNAc) molecules connected at the MurNAc residues by a short chain of amino acids [S. aureus polysaccharide biofilms is poly-N-acetylglucosamine (PNAG), also referred to as polysaccharide intercellular adhesin (PIA) [S. aureus biofilm, and although it blocks fungal ergosterol biosynthesis, it was proposed that the itraconazole might also target bacterial polysaccharide synthesis as does another antifungal drug, caspofungin [S. aureus antibiofilm activity when combined with the antibiotic tetracycline. In this study, the authors demonstrate that the azoles act as competitive inhibitors of active efflux pumps in S. aureus biofilms, thus maintaining higher concentrations of the antibiotic in the bacterial cells by blocking the exit of the antibiotic [S. aureus biofilm activity suggests yet another potential mechanism of action for the ATs by the disruption of the quorum sensing [Staphylococcus, and we will continue to test AT anti-biofilm efficacy against other strains of S. aureus as well as biofilm-forming strains of Staphylococcus epidermidis [Although the mode of action for these AT compounds is not yet clear, the synergism of ATs observed with vancomycin in checkerboard assays suggests the no acids . The maiin (PIA) . If the pofungin . Anothertibiotic . This co sensing . The ATs sensing . Althougdermidis .® BacLightTM Bacterial Viability Kit or similar assays [Checkerboard assays with daptomycin and vancomycin were used to assess potential modes of action. For these assays, a computed FICi below 0.5 indicates synergy, whereas a score greater than 4 indicates antagonism. None of the ATs exhibited an antagonistic effect with daptomycin or vancomycin. All four ATs showed synergy with daptomycin against MRSA suggesting that these ATs target cell membranes . Althougr assays ,66,67. Sr assays ,48,49,50r assays . Again, r assays .S. aureus [The structures of these aurone-derived triazoles may also be important to their anti-Staphylococcal activity. In a study evaluating fluoro- and trifluoromethyl-substituted salicylanilide derivatives against . aureus , the aut. aureus . All fou. aureus . AT125 a. aureus . One of . aureus  also had. aureus ,73,74,75S. aureus  plates. Before use, an isolated colony was transferred from the TSA plate into Tryptic Soy Broth (TSB) and incubated 18–24 h in a humidified incubator at 37 °C. To adjust for a standard concentration of bacteria, the turbidity of the culture was measured at 600 nm, and its optical density (OD600) adjusted to be in the range 0.08–0.12 which equates to a concentration of approximately 1 × 108 CFU/mL. This suspension was diluted 1:20 in TSB. The final mixture for experiments used the bacterial suspension and AT compound mixture as a 1:10 dilution which resulted in a final 5 × 105 CFU/mL bacterial concentration [S. aureus ATCC 43300 and MSSA strain S. aureus ATCC 29213 were used for all testing in this study.MRSA strain ATCC 43300 and MSSA strain ntration . MRSA st5 CFU/mL bacterial concentration and 100, 50, 25, 12.5, 6.25, or 3.125 µM concentrations of the AT compound. Blank wells with only CAMHB, a negative control well with 10 µL of bacterium solution mixed into 90 µL CAMHB, a vehicle control well with bacteria mixed into 1% DMSO in CAMBH, and a positive cell death control with vancomycin (1 µL of stock 2 mg/mL solution in 89 µL CAMHB with 10 µL of bacteria) were included. The plate was incubated for 18–24 h at 37 °C in a humidified incubator. Before measurement, PrestoBlueTM dye  was added (10 µL per well) and incubated for 1 h with aluminum foil covering the plate. A CLARIOstar®  multi-mode plate reader was used to measure the fluorescence at wavelengths of 560 nm for excitation and 590 nm for emission. Inhibitory concentration (IC50) values were defined as half inhibitory concentration and were computed using GraphPad Prism version 9.1 software [The assay for anti-Staphylococcal activity of AT compounds was prepared in 96-well transparent, U-bottom plates. A series of 2-fold dilutions of AT concentrations was tested in triplicate, with each well containing 10 µL of prepared bacterium and 90 µL of AT mixture in CAMHB. This resulted in 100 µL of volume in each well with a 5 × 10software .2 (95% room air) in a humidified incubator. Before use, attached cell monolayers, which had reached at least 90% confluency, were detached by trypsinization with trypsin-EDTA (0.25%) and resuspension in fresh complete culture media. For experiments, the stock concentration of 1 × 105 cells/mL  was achieved by counting the trypsinized cells and adjusting to their final concentration for assays.Human HeLa epithelial (ATCC CCL-2) and HepG2 liver epithelial (ATCC HB-8065) cell lines were used to determine toxicity of AT compounds for mammalian cells. The cells were grown in a complete culture medium of RPMI 1640 with 10% heat-inactivated fetal bovine serum (FBS) at 37 °C in 5% CO5 cells/mL) in complete culture media to each well. The plate was incubated overnight at 37 °C in a 5% CO2 (95% room air) humidified incubator. After the incubation and before mixing in the AT compounds, a 100 µL volume of fresh complete culture media was added to the pre-seeded wells. The 2-fold dilutions of AT compounds in triplicates were prepared on a separate 96-well plate. Once the dilution plate was prepared, dilutions were transferred into the pre-seeded plate to achieve the same concentrations of ATs as for the inhibition assay described above. The final inoculated plate was then returned to the 5% CO2 (95% room air) humidified incubator for an additional 24 h of incubation with the diluted ATs. Controls were blank wells containing only media, wells with cells and DMSO in complete culture media, and wells with cells and complete culture media only. Measurement of toxicity was analogous to the inhibition assay except that 20 µL of PrestoBlueTM was used and the incubation was 3–6 h before reading with the plate-reader. Cytotoxicity concentration (CC50) was defined as the concentration where half of the cells remained viable and was computed using GraphPad Prism.HeLa or HepG2 cells were pre-seeded into a 96-well transparent, flat bottom plate by adding a volume of 100 µL stock cell concentration  for the assay (donor#:1WB3ALS40ML-36517). A volume of 1–2 mL of the human red blood cells (hRBCs in Alsevier’s solution) was mixed with 8–10 mL of Phosphate-Buffered Saline (PBS) in a 15 mL centrifuge tube to pellet by centrifugation at 1400× The wells of a 96-well U-bottom plate that contained AT compounds were prefilled with 50 µL of PBS. A 2-fold dilution of AT compounds was prepared. For each triplicate, an initial AT dilution was prepared using 6 µL of AT stock solution in 144 µL of PBS. This initial mixture was diluted 2-fold on a 96-well plate following the method described above for a final volume of 50 µL of diluted AT in each well. As a final step, 50 µL of the 1% hRBC solution was added to each well, except the blank wells. PBS and hRBCs were used for negative control, whereas hRBCs with 1% and 0.1% Triton-X100 were used as positive controls. After 1 h of incubation at 37 °C in a humidified incubator, the plate was centrifuged at 1000 rpm for 10 min in a Thermo Sorvall Legend XTR centrifuge with a TX-750 swinging bucket rotor  to pellet intact cells. After centrifugation, 50 µL of supernatant was transferred from each well into a new 96-well U-bottom plate and hemolysis of the hRBCs detected in a plate-reader for absorbance at 405 nm. Hemolytic activity for all concentrations of the ATs was computed following this formula:For the growth curve assay, honeycomb plates  were used with TSB as the growth medium. Using bacterial cultures as described above, growth curve measurements were carried out in quintuplicates. For positive growth controls, wells with 10 µL of bacteria mixed into 90 µL of TSB were used. For quintuplicate treatment wells, 4-fold dilutions of AT compounds were used such that AT concentrations of 100, 25, 6.25, and 1.56 µM were tested. A 10 µL inoculum of bacteria prepared as described above was added to each dilution of the ATs. The growth in each well was measured in 15 min intervals for 48 h at 37 °C in the Bioscreen C instrument . The res5 CFU/mL) concentration of bacteria in media was mixed with 2-fold dilutions of antibacterial agents to verify their inhibition potential [90—the concentration of the agent with at least 90% inhibition of the bacteria) for the antibiotics. Control wells contained 10 µL of bacteria with 90 µL of CAMHB, and the negative control was bacteria in 1% DMSO and CAMHB. Incubation, staining with PrestoBlueTM, and assessing cell viability are as described for the antibacterial assay above.This preparation for the checkerboard assay was analogous to the antibacterial assay described above in that the standard  in BHI medium to grow biofilm in 35 mm MatTEK dishes with a 20 mm glass bottom well by adding 1500 µL of overnight bacteria per dish. The dishes were incubated at 37 °C in a humidified incubator for 48 h. The old media was refreshed by adding 1500 µL of BHI for the control, or the AT mixture (15 µL of AT stock with 1485 µL BHI to achieve 100 µM AT concentration), or DMSO mixture (15 µL DMSO with 1485 µL BHI) for negative control. The dishes were then incubated in a humidified incubator for an additional 24 h at 37 °C. The media was removed, and cells were washed twice with 500 µL PBS, followed by the addition of 50 µL BacLightTM with 250 µL PBS for 15 min. Biofilms were imaged using a Zeiss AxioObserver microscope with an LSM700 confocal module . To assess biofilm disruption, Fiji [To prepare AT-treated cultures, overnight bacterial cultures were used (adjusted to 1 × 10on, Fiji  softwareon, Fiji  to clearon, Fiji , which s