In this study, we investigated the effect of preoperative prostate morphology, especially intravesical prostatic protrusion (IPP), on continence after robot-assisted radical prostatectomy (RARP).Retrospective analysis was applied to patients who underwent RARP between October 2010 and July 2014. The following parameters were assessed in all patients: age, body mass index (BMI), prostate-specific antigen, magnetic resonance imaging and pressure-flow studies findings. The impact of preoperative and intraoperative factors on postoperative urinary incontinence (UI) was assessed using multivariate logistic regression analysis. To evaluate the effects of IPP, the patients were divided into groups according to the IPP length: Group 1, < 5 mm and Group 2, ≥ 5 mm. The International Prostate Symptom Score (IPSS), Overactive Bladder Symptom Score, Quality of Life index and the number of pads used were assessed.p < 0.05) and nerve-sparing (NS)  were significant factors related to UI in the first month after RARP. Twelve months after RARP, multivariate analyses revealed that only NS is a factor related to postoperative UI . The comparison of Groups 1 and 2 indicated significant differences in age (p < 0.01), prostate volume (p < 0.01), total IPSS and voiding symptom score (p < 0.05), compliance (p < 0.01), and detrusor pressure at maximum flow (p < 0.01). Group 1 had a higher continence rate (38.0%) than Group 2 (20.8%) in the first month after RARP (p < 0.05), but the difference was no longer significant from the third month after RARP. The total IPSS and voiding symptom scores were significantly different between the two groups before RARP, however, the significant difference disappeared from the first month after RARP.A total of 119 patients were eligible for this study. Multivariate analyses showed that IPP (odds ratio (OR) 1.14, 95% confidence interval (CI) 1.02–1.28, The data suggest that IPP affects early postoperative UI. Although NS was strongly involved in UI in the early and later stages after RARP, IPP had no effect on UI in the later stages. To date there is a variety of treatments for localized prostate cancer (PCa) that can be performed, including active surveillance, radiation therapy and radical prostatectomy. In particular, the robot-assisted laparoscopic radical prostatectomy (RARP) has become a widely performed type of treatment in patients with PCa due to low blood loss and transfusion rates, shorter hospital stay, and fine surgical manipulation . AlthougSeveral studies reported that RARP provides earlier urinary continence compared to ORP and LRP . It has On the other hand, patient’s factors have also been noted regarding UI after surgery. Several studies have suggested that age, body mass index (BMI), preoperative International Prostate Symptom Score (IPSS) and prostate volume (PV) affect early UI after RARP . PatientThis study was conducted at the Division of Urology, Tottori University Hospital, Yonago, Japan. The study was approved by the Tottori University Ethics Committee (No. 2545).Patients who underwent RARP for PCa (stages cT1c–cT3b N0 M0) between October 2010 and July 2014 at our department were included in this study. All patients were signed up after being fully briefed in accordance with the institutional ethics committee. Approved informed consent was obtained and patients were informed that the data would be used anonymously for clinical research purposes. All study data were prospectively collected and retrospectively analysed. The age, BMI, prostate-specific antigen (PSA), Gleason score (GS) and clinical stage were recorded. All patients underwent PFS and magnetic resonance imaging (MRI) before RARP. IPSS, Overactive Bladder Symptom Score (OABSS), QOL index and the number of pads used per day were evaluated before RARP and 1-, 3-, 6-, 9- and 12-months after RARP.All surgical procedures were performed by six surgeons. The four grades of postero-lateral resection of the prostate were the guide for performing the nerve-sparing (NS) techniques. These included: grade 1, intrafascial dissection; grade 2, interfascial dissection; grade 3, extrafascial dissection and grade 4, wide dissection . The NS In this study, continence was defined as using no pads, and was assessed in the physician’s interview with the patient at each outpatient visit before RARP and 1-, 3-, 6-, 9- and 12-months after RARP. Urinary symptom status was analysed using the IPSS, OABSS and QOL index. The IPSS consists of seven questions to assess voiding symptoms  and storage symptoms  .PFS was performed by a single examiner using the standard method prescribed by the International Continence Society, using a Solar Gold urodynamic system  . An 8-FrThe PV, IPP, membranous urethra length (MUL), membranous urethra width (MUW) and levator thickness (LT) were measured using MRI. The PV was calculated as height × width × length × π/6 . The IPP was measured from the tip of the protruding prostate to the base of the bladder . The MUL was measured as the distance from the prostatic apex to the level of the urethra at the penile bulb on coronal T2-weighted image. The MUW was defined as the maximum diameter of the membranous urethra . LT was calculated as (the outer levator distance minus the inner levator distance)/2 on axial T2-weighted image Fig. .Fig. 1MRThe effect of IPP was investigated by stratifying the IPP into two levels. The patients were subsequently categorised into two groups based on the IPP length: Group 1 (< 5 mm) and Group 2 (≥ 5 mm). To confirm the difference between the two groups, we compared the pad-free rate, IPSS, OABSS and QOL index before RARP and at 1-, 3-, 6-, 9- and 12-months after RARP.The data were presented as median values and interquartile range (IQR) and evaluated using the Mann–Whitney U test and Yates chi-square test, where a p -value of less than 0.05 was considered significant. Logistic regression analysis was performed to determine independent predictive values of the main risk factors of UI that have been reported in previous studies  in the first month and 12 months after RARP –14. The A total of 119 patients were eligible for this study. The number of patients who did not use pads at 1-, 3-, 6-, 9- and 12-months after RARP were 37 (31.0%), 63 (52.9%), 82 (68.9%), 85 (71.4%) and 91 (76.5%), respectively.2 and 7.8 (3.2–37.1) ng/mL, respectively. The MRI evaluation revealed that the median values of PV, MUL, MUW, LT and IPP before RARP were 26.0 (9.6–66.1) mL, 12.1 (8.9–16.1) mm, 10.6 (9.8–13.5) mm, 11.1 (7.9–15.1) mm, and 3.8 (0.0–16.5) mm, respectively. In terms of the clinical stage, 108 patients had ≤ T2c, 10 patients had T3a and one patient had T3b disease. Twenty-one patients had a GS of 6, 50 patients had a score of 7 and 48 patients had scores ≥ 8. According to NCCN risk stratification, 14 patients were considered at low risk, 53 patients were at intermediate risk and 52 patients were at high risk. A NS procedure was performed bilaterally or unilaterally in 51 patients. Lymph node dissection was performed in 105 patients.The median values of patient age, BMI and PSA before RARP were 66 (48–76) years, 23.5 (18.0–30.6) kg/m2O, 144 (47–400) ml, 277 (55–470) ml and 45 (6–90) cmH2O, respectively. DO was identified in 10 patients , 4 0–18), 4 (0–12), 3 (0–10) and 3 (0–6), respectively. The median compliance, FDV, MCC and PdetQmax were 49.8 (5.0–290.2) ml/cmH8, 4 (0–1p < 0.05) and NS  remained as significant factors in the first month after RARP. At 12 months after RARP, the only factor that remained significantly associated with UI on multivariate analysis was NS   as being linked to UI in the first month and 12 months after RARP, showed that the IPP , PV (p < 0.01), total preoperative IPSS score (p < 0.05), preoperative IPSS voiding symptom score (p < 0.01), bladder compliance (p < 0.01) and PdetQmax (p < 0.01) (Table The patients were stratified into two groups according to IPP length: Group 1 (< 5 mm: n = 71) and Group 2 (≥ 5 mm: n = 48). The comparison of the two groups indicated significant differences in age (p < 0.01), but the significant difference disappeared from the first month after RARP. The IPSS storage symptom score, QOL index and OABSS were not significantly different between groups 1 and 2 both before and after RARP , 40 (56.3%), 50 (70.4%), 53 (74.6%) and 55 (77.5%), respectively. In Group 2, the number of patients who did not use pads at 1-, 3-, 6-, 9- and 12-months after RARP were 10 (20.8%), 23 (47.9%), 32 (66.7%), 32 (66.7%) and 36 (75.0%), respectively. Group 1 had a higher continence rate than Group 2 in the first month after RARP (p < 0.05); however, the significant difference disappeared 3 months onward after RARP (Fig. The post-RARP UI is influenced by the preoperative patient’s characteristics as well as the surgical technique. In a systematic review, the 12-month UI rates ranged from 4 to 31% with a mean value of 16% after RARP . It mighBesides the surgical technique, several other factors related to UI after RARP have been mentioned. Some reports show that patient age, BMI, MUL, LT, IPP, race, OABSS and severity of preoperative IPSS score are factors associated with the continence rate after RARP , 19, 20.In this study, we focused on and examined the impact of IPP on UI after RARP. In a previous study, the impact of IPP on the effects of the therapy for BPH was extensively evaluated . RARP prBesides the impact of IPP on early post-RARP UI, there were also significant differences between the two groups in bladder compliance, PdetQmax, IPSS total score, IPSS voiding symptom score and PV. A higher grade of IPP would result in a higher degree of subclinical bladder dysfunction before RARP, resulting in a lower rate of urinary continence after RARP. Yamada et al. reviewed 272 patients after RARP. Multivariable analysis showed that the presence of an unstable bladder preoperatively was an independent negative predictor of the recovery of continence within 12 months after surgery . In our In this study, the total IPSS score and voiding symptom score were preoperatively significantly different between the two groups. However, there was no significant difference after surgery. Therefore, patients with severe preoperative voiding symptoms benefit from RARP.The limitations of this study were the small number of cases, few cases of severe IPP and lack of evaluation of bladder neck preservation. Since it was difficult to perform bladder neck preservation in high-grade IPP patients, the assessment of bladder neck preservation in cases of IPP facilitates the evaluation of its effect on UI in the future.Thus, future studies assessing the effect of IPP on UI should include a larger number of severe cases of IPP to enable more detailed study.The results of this study suggest that IPP affects urinary continence in the early postoperative period. Although NS was strongly involved in urinary continence in both early and later stages after RARP, IPP had no effect in the later stages. Various preoperative factors are involved in continence after RARP, and IPP is one of those factors. More detailed evaluation of a larger number of cases in the future will help shed more light on this topic.


Embryonic stem cells (ESCs) can be maintained in the naïve state through inhibition of Mek1/2 and Gsk3 (2i). A relevant effect of 2i is the inhibition of Cdk8/19, which are negative regulators of the Mediator complex, responsible for the activity of enhancers. Inhibition of Cdk8/19 (Cdk8/19i) stimulates enhancers and, similar to 2i, stabilizes ESCs in the naïve state. Here, we use mass spectrometry to describe the molecular events  triggered by 2i and Cdk8/19i on ESCs. Our data reveal widespread commonalities between these two treatments, suggesting overlapping processes. We find that post-transcriptional de-repression by both 2i and Cdk8/19i might support the mitochondrial capacity of naive cells. However, proteome reprogramming in each treatment is achieved by different mechanisms. Cdk8/19i acts directly on the transcriptional machinery, activating key identity genes to promote the naïve program. In contrast, 2i stabilizes the naïve circuitry through, in part, de-phosphorylation of downstream transcriptional effectors. Naïve pluripotency can be stabilized through different pharmacological approaches. Here, the authors profile temporal changes of protein phosphorylation, proteome and metabolome as mESCs transition to the naïve state in response to two pharmacological treatments, revealing general and treatment-specific processes. Recently, we showed that naïve pluripotency can also be stabilized by inducing a global upregulation of key identity genes under the control of super-enhancers3. This is achieved by inhibiting Cdk8/19 (Cdk8/19i) and, thereby, relieving their negative effect on the Mediator complex at enhancers. This suggests the existence of common regulatory processes involved in the acquisition of naïve pluripotency conferred by 2i-treatment or Cdk8/19i-treatment. Understanding the pathways that regulate pluripotency is important, especially for the derivation of naïve human ESCs4.A spectrum of transitory pluripotent states occurs during early development, and these can be captured in vitro through stimulation and blockade of specific pathways. Leukemia inhibitory factor (LIF) together with inhibitors of Mek1/2 and Gsk3 (2i)5. Furthermore, while primed mEpiSCs and hESCs are mainly glycolytic, naïve mESCs use oxidative phosphorylation6. Another key feature of naïve cells is their hypomethylated DNA7, although this has deleterious implications upon long-term passages8. Importantly, a profound connection between the metabolism of these cells and their epigenetic status emerges as several metabolites have been found to determine histone and DNA methylation levels9.Compared to the more developmentally advanced states of the post-implantation embryo , known as primed state cells; or serum ESCs, which represent a metastable state that fluctuates between primed and naïve pluripotency), naïve ESCs express a set of transcription factors that reinforce the pluripotency core factors while repressing lineage specification genes12. Post-transcriptional regulation makes mRNA levels an imperfect predictor of protein abundance in mESCs13. Protein function is also rapidly altered by post-translational modifications (PTMs), and methods used to stabilize naïve cells target specific kinases, but how signaling is connected to transcriptional control remains unclear. In this work, we use mass spectrometry (MS) to dissect the establishment of naïve pluripotency by two methods: blunting extracellular signaling (2i) and stimulating enhancer transcription (Cdk8/19i). Using an integrative strategy, we analyze three interconnected regulatory layers. First, we profile the early phosphorylation cascades that are triggered in response to these inhibitors (0–6 h) to identify downstream effectors. Next, we monitor how protein levels are tuned until naïve pluripotency is stabilized (0–14 days). In addition, since the availability of metabolites can determine their epigenetic status, we also probe the metabolome of cells adapted to 2i or Cdk8/19i conditions. Our analyses reveal missing links in the molecular roadmap from primed to naïve pluripotency, and provide insights into the regulatory principles of cell identity.Despite the above-mentioned transcriptomic and epigenetic insights, our understanding of pluripotency demands direct analysis of protein-driven mechanisms, yet crucially, this has been less explored3. Using isobaric labeling, a total of 4408 proteins were quantified in all 64 samples    media. Cells were then supplemented with either 2i or Cdk8/19i, collected after 1, 2, 4, 7, 10, or 14 days of treatment, and subjected to proteomic analysis Fig. . We chosr = 0.91; p.val = 0.039) and #15 (401 proteins) , displayed remarkable similar patterns in 2i and Cdk8/19i. Nevertheless, we also found clusters exhibiting different dynamics between the treatments  as well as clusters of proteins that were only co-regulated in 2i   and the naïve circuitry Tfcp2l1) showed similar up-regulation in 2i and Cdk8/19i. Lin28a, Dppa2, Dnmt3a, and Eras, that are required in the primed state, were downregulated upon inhibition of both pathways Fig. . We foun#2) Fig. . Among t#2) Fig. . In cont#2) Fig. . In addil1 showed15, diapause16, and pluripotency17. The master transcription factor of autophagy Tfe3 rapidly translocates to the nucleus in response to 2i18, and we have shown that Cdk8/19i recapitulates this event3. Tfe3, together with Tfeb and Mitf, belong to the MiTF/Tfe family that bind the CLEAR elements present in the promoters of lysosomal biogenesis genes20. We then examined the protein dynamics of the MiTF/Tfe target genes in our data. Several lysosomal hydrolases such as Ctsb, Glb1, Asah1, Ppt1, and Scpep1 increased in both 2i and Cdk8/19i cultures compared to reference S/L  . Interestingly, cluster#3 in 2i, which mainly contained nuclear proteins , displayed an opposite kinetic to the proteostasis machinery   is the major degradation pathway for short-lived proteins. Our data revealed a co-regulation of proteasome subunits only in 2i  Fig. . Rapid dp.val = 0.039) indicating a central role for these proteins in naïve pluripotency. GO analyses revealed a massive representation of mitochondrial proteins  (Supplementary Data Another wave of proteome remodeling occurred around 4–7 days and involved the upregulation of a large fraction of proteins that remained increased afterwards (cluster#7) Fig. . Importa28. Using transmission electron microscopy, we confirmed such distinct features but did not appreciate differences in the size nor the morphology of mitochondria in 2i, Cdk8/19i cells compared to reference S/L   Fig. . We did 05) Fig. . We conf05) Fig. . These r30. Among the targets of mTOR-enhanced translation is Tfam. However, TFAM mRNA and protein levels remained unaltered in our data  and 387 (Cdk8/19i) proteins  which increased levels were not accompanied by changes in their cognate mRNAs  and the endomembrane system  . In support of this hypothesis, we found that, unlike cytosolic tRNA synthetases, their mitochondrial counterparts were predominantly upregulated . In contrast, the number of upregulated compounds was lower: 50 in 2i and 66 in Cdk8/19i, with few metabolites in common. These results suggest that cells in 2i and Cdk8/19i diverge partially in their metabolic state, which also differs from reference S/L mESCs.Our proteomic data suggested changes in mitochondrial metabolism, but also in many other routes (Supplementary Data 35. We found that glutamine synthase (Glul) was actually repressed in 2i whereas it was overexpressed in Cdk8/19i . In agreement, Atf4 and two critical effectors, Ddit3 and Eif2ak4 were only up-regulated in 2i , making SAM unavailable for other methylation reactions9. We found Nnmt mRNA expression increased 16-fold in 2i, while it diminished in Cdk8/19i .Despite the differences in the above metabolic pathways, our data also showed that 2i and Cdk8/19i caused similar alterations in other routes, such as the one carbon metabolism (OCM). While serine biosynthesis proteins  were repressed in 2i and Cdk8/19i, Tdh (involved in the catabolism of threonine into glycine) was upregulated several fold40 Fig. . Further40 Fig. . Thus, t6. Cdk8 kinase activity is required for induction of many Hif1a targets and supports glycolysis in cancer cells43. In concordance, our proteome data showed multiple Hif1a targets involved in glycolysis were downregulated by both 2i and Cdk8/19i  indicating that both drugs induced a similar lipidomic signature  and 42% (Cdk8/19i) of the downregulated metabolites belonged to lipid metabolism. Importantly, 30 of them decreased in both inhibitors  and Eef2k  . This included hypo-phosphorylated RB that, together with the increased expression of p21/p27, confirmed the elongated G1 phase typical of naïve ESCs48. Interestingly, our data also suggested that naïve cells are partially relieved from the G2/M checkpoint through inhibition of Chek1: phosphorylation of its catalytic site (S317) significantly diminished, and its motif was enriched among the downregulated p-sites   Fig. . In addi49 , and almost half of those peptides were co-phosphorylated in the priming site  including DUSPs and SPRYs but also genes from the primed state such as Otx2, Utf1, Sox3, Lin28b, and Lef159 all of which were downregulated in 2i  and overlapped with those activated by Zic3 , which promotes the transition to primed pluripotency60  as another transcriptional target that was rapidly dephosphorylated only in 2i Fig. . Similarors Fig. . Moreove62. In addition, we found Cdk8/19i-specific changes in Aebp2 and Jarid2 suggesting that Cdk8 may also regulate PRC263. The implication of Cdk8 on epigenetics in our data was further evidenced by dephosphorylation of Dnmt3l64. However, given the main role of Cdk8 in transcription, we focused on proteins involved in this process. We found dephosphorylation in Aff1 and Aff4 from the super elongation complex in Cdk8/19i but not in 2i. Moreover, Ccnt2, the cyclin of the pTEFb, and Cdk13, involved in CTD phosphorylation and elongation, were also regulated only in Cdk8/19i  in AML67. To test whether a similar mechanism plays a role in the stabilization of naïve pluripotency by our Cdk8/19 inhibitor, we checked Cdk8/19i-dependent expression of genes that are transcriptionally-regulated by SE in S/L mESCs68. Indeed, we found that Cdk8/19i, but not 2i, showed a positive co-regulation of protein and RNA levels of SE-genes  and transcriptional machineries Cdk8/19i). We propose a model in which 2i and Cdk8/19i result in a similar activation of naïve genes through different mechanisms. Although more factors might be involved, we showed that Erk inhibition results in the rapid activation of Cic and Erf . Genes regulated mainly at the translational level in Atlasi et al12. (RNA < RFP) were enriched in endoplasmic reticulum and plasma membrane (FDR = 0) but not in mitochondria. However, we found that mitochondrial proteins were indeed more upregulated at the protein than the mRNA  or RFP  levels. Moreover, in agreement with our results, divergences in mRNA-protein in Atlasi’s data were coincident with the downregulation of Lin28, underscoring the known role of this RNAbp in post-transcriptional repression in mESCs12. Given that RFP is a direct reflection of translational activity, it is intriguing that the upregulation of mitochondrial protein levels (MS) was not detected in the RFP data. Multiple explanations may account for such discrepancy including differences in bulk vs. local RFP measurements and stalled/paused polysomes (Supplementary Note 2).Once activated, the naïve circuitry entails the execution of a complex sequence of molecular events. We found that a feature of the naïve state is the upregulation of mitochondrial proteins, which seems to be partially controlled post-transcriptionally. Interestingly, these results contrast with a recent report that found that proteomic changes in the naïve state are largely driven by transcriptomic rewiring, with little contribution of other regulatory layers71, we found that either 2i or Cdk8/19i lead to de-phosphorylation of mTOR, possibly affecting catabolism/anabolism rates. It is well established that protein synthesis is repressed under stress conditions, where the translation machinery is diverted to selectively promote stress-response proteins72. A similar translational reprogramming might occur here: the substantial demand for translation of mitochondrial proteins to transition to the naïve state could be counterbalanced by tuning down global protein synthesis, as well as the recycling of cellular components through autophagy. Secondly, the over-expression of several enzymes that feed 1C units suggests that naïve cells may adapt their one carbon metabolism to cope with mitochondrial translation rates. Although this would require further examination of one-carbon fluxes by means of stable isotope tracers. Interestingly, both threonine and choline sources converge into 5,10-meTHF which is essential to produce the taurinomethyluridine base of mitochondrial tRNAs41. Therefore, it is our hypothesis that naïve ESCs might increase 5,10-meTHF levels to meet the demand of methylated mitochondrial-tRNAs needed for maintaining the high translation rates of mitochondrially-encoded proteins. Of note, these enzymes are repressed as cells progress to primed pluripotency10, and they exhibit specific expression in the morula and ICM2, further supporting their roles in pluripotency.Interestingly, the upregulation of mitochondrial proteins may be facilitated by two additional events. Firstly, in line with prior reports9. In contrast, Nnmt levels remain basal in Cdk8/19i, consistent with their lack of hypomethylation3. Another contributing factor could be the 2i-dependent degradation of Uhrf1 that impedes the recruitment of Dnmt1 leading to a passive loss of methylated bases during replication39. Although low levels of DNA methylation are considered a hallmark of naïve pluripotency given its resemblance with the demethylation wave of the ICM, it has been reported that prolonged culture in 2i can have deleterious effects on the chromosome integrity of mESCs8. In this scenario, Cdk8/19i would be an attractive alternative, as these cells exhibit similar functional capacities to 2i, while methylation is stable and not subject to progressive loss3.Despite their similarities, 2i and Cdk8/19i showed differences in several metabolic pathways. The upregulation of Nnmt in 2i diverts SAM from methylation reactions3, hence, some of the events described here might play a role in this system too. Moreover, our data might be valuable in other areas such as cancer. Inhibition of Cdk8 reduces cell proliferation in AML through activation of tumor suppressors67. In colorectal cancer cells however, Cdk8 acts as an oncogene43. Here, we show that Cdk8/19i leads to phosphorylation changes in hundreds of proteins, which may help in understanding the context-dependent functions of these kinases. While the primary function of Cdk8/19 resides in the nucleus, and potential off-targets cannot be neglected, Cdk8/19 can indeed function independent of core Mediator51 and it has been recently found in the cytoplasm73, suggesting that the target spectrum of these kinases may be larger than anticipated. On the other hand, the Erk pathway involves multistep reactions and feedback loops, that generate various behaviors. The PD0325901 used in 2i, and many other Mek inhibitors, do not disrupt the phosphorylation of the Mek1/2 activation loop, which can explain the rebound in phosphorylated Erk1/2 and pathway output that is observed with this type of inhibitors, notably in Ras-driven cancers. Indeed, one of the downstream effectors identified here, Cic, has been shown to modulate sensitivity to Mek-inhibitors74. Thus, our data might be used to identify potential vulnerabilities and resistance mechanisms to such compounds.In our previous work, we have shown that Cdk8/19i also shifts human ESCs towards the naïve stateMore detailed descriptions of the methods employed in the current study is included as Supplementary Methods in the supplementary information.The experimental design consisted of four different mouse embryonic stem cell lines: TNGA, TON, ZS and BL6. Wild-type ES cells were derived at the Transgenic Mouse Unit of CNIO from E3.5 C57BL6 blastocysts (BL6), or mixed background C57BL6/129 (V6.4) blastocysts. Nanog-GFP knock-in mouse ES cells  and were shared by the laboratory of Austin Smith; The ZS mouse ES line was a 2C-reporter shared by the laboratory of Minoru Ko. Mouse ES cells were routinely cultured on gelatin-coated plates in a base media of “Serum/LIF” (15% FBS), in DMEM (high glucose) basal media, with LIF (1000 Units/ml), non-essential amino acids, glutamax and β-mercaptoethanol plus antibiotics. Where used with mouse PSC, the “2i” two-inhibitor cocktail comprised 1 μM Mek-inhibitor  plus 3 μM GSK3β-inhibitor . The Cdk8/19i was supplemented at 1.1 μM. Cultures were routinely tested for mycoplasma. Cell pellets were collected by trypsinization at different time points after treatment with 2i or Cdk8/19i: 30 min, 1, 2, and 6 h, 1, 2, 4, 7, 10 and 14 days of treatment and washed with PBS and preserved at −80 °C for further analysis.4OH for subsequent fractionation by high pH reversed phase chromatography. Collected fractions were dried and dissolved in 50 µl of 1% FA for LC-MS/MS analysis. The Impact  was coupled online to a nanoLC Ultra system (Eksigent). 7.5 µl of each fraction were loaded onto a reversed-phase C18, 5 µm, 0.1 × 20 mm trapping column (NanoSeparations) and washed for 15 min at 2.5 µl/min with 0.1% FA. The peptides were eluted at a flow rate of 250 nl/min onto a home-made analytical column packed with ReproSil-Pur C18-AQ beads, 1.9 μm, 75 μm × 50 cm, heated to 45 °C. Solvent A was 4% ACN in 0.1% FA and Solvent B CH3CN in 0.1% FA. The MS acquisition time used for each sample was 180 min. The Q-q-TOF Impact was operated in a data dependent mode. The 20 most abundant isotope patterns exceeding a threshold of 5000 counts and with charge ≥2 and m/z > 350 from the survey scan were sequentially isolated and fragmented in the collision cell by collision induced dissociation (CID).Four mouse embryonic stem cell lines were used: TNGA, TON, ZS, and BL6. Each cell line was employed as a biological replicate, so the overall experiment consisted in four biological replicates. For time course full proteome analysis cells were lysed using 7 M urea, 2 M thiourea, 50 mM Hepes, 1:1000 (v/v) of benzonase and 1:100 (v/v) of HaltTM phosphatase and protease inhibitor cocktail 100×. Cell lysates were homogenized by sonication and cleared by centrifugation. Protein concentration was measured with Qubit® Protein Assay Kit. One hundred and ten microgram of each lysate (except for control conditions for which 220 µg were used) were digested using the filter aided sample preparation (FASP) method. A first digestion using Lys-C  for 4 h was followed by a dilution 8-fold in 50 mM TEAB and a subsequent digestion with trypsin  overnight at 37 °C. Labeling was performed using the iTRAQ® Reagent 8plex kit (AB Sciex). Labeled samples were combined and cleaned-up with C18 Sep-Pack. Eluate was dried and dissolved in 10 mM of NH3CN and 6% TFA. Titanium dioxide (TiO2) beads were prepared at 60 µg beads/µl of DHB solution (20 mg/ml DHB in 80% CH3CN 6% TFA). TiO2 beads were added to the sample in a ratio 1:2 (Sample:TiO2) and incubated for 15 minutes. Supernatant was used for a second binding with half the amount of beads. Beads from the first and second TiO2 binding were transfer to separate C8-tips and washed. Peptides were eluted with 25 µl of 5% NH4OH and 25 µl of 10% NH4OH 25% CH3CN. Eluate from the second TiO2 binding was resuspended in 22 µl 5% FA for LC-MS/MS analysis. Eluate from the first binding was fractionated with high pH reverse phase microcolumns. Sample was loaded into the tips thrice and the flow-through was collected to a vial. Next, 50 µl of phase A (20 mM NH4OH) was loaded and collected as the flow-through. Peptides were sequentially eluted increasing the percentage of Buffer B (20 mM NH3 in CH3CN). Samples were dissolved in 22 µl of 5% FA for subsequent LC-MS/MS analysis. Three replicates were run for each sample. The experiments were performed on an Ultimate 3000 RSL nano LC system (Thermo Scientific) coupled to a Q Exactive HF-X mass spectrometer (Thermo Scientific) equipped with an EASY-spray ion source (Thermo Scientific). The peptides were eluted from an Easy-Spray Column (75 µm i.d. × 50 cm) packed with PepMap RSLC C18 2 µm by application of a binary gradient consisting of 0.1% FA (buffer A) and 100% ACN in 0.1% FA (buffer B), with a flow rate of 250 nl/min during 90 min. The column was operated at a constant temperature of 45 °C. The MS survey was performed in the Orbitrap for a m/z range between 350 and 1500 m/z. The resolution was set to 60,000 FWHM at m/z 200. The 15 most abundant isotope patterns with charge ≥2 and <6 from the survey scan were selected with an isolation window of 1 m/z and fragmented in the HCD collision cell. Normalized collision energy was set to 35. The resulting fragments were detected in the Orbitrap for a m/z range between 100 and 2000 m/z with a resolution of 45,000 FWHM at m/z 200. The maximum ion injection times for the survey scan and the MS/MS scans were 45 ms and 80 ms, respectively and the ion target values were set to 3e6 and 5e4, respectively for each scan mode. Raw data was acquired using Xcalibur .For time course phosphoprotome profiling cells were lysed using 5% SDS and digested following the S-trap protocol. Samples were labeled using TMT11plex reagent following manufacturer instructions. Labeling reaction was stopped by addition of 1% hydroxylamine; then samples were combined and desalted using C18 Sep-Pack. To perform phosphopeptide enrichment, peptides were dissolved in 80% CHp-value for each condition. The p-value threshold was adjusted by Benjamini–Hochberg correction to a 5% FDR.Proteome raw files were analyzed using MaxQuant 1.5.3.30 and 1.6.0.16 for full proteome and phosphoproteome datasets, respectively, against a Mus musculus database . For total proteome sample quantification type was set to iTRAQ8plex, whilst for phospho-proteome it was set to TMT11plex. Carbamidomethylation of cysteine was included as fixed modification and oxidation of methionine and acetylation of protein N-terminal were included as variable modifications. Phosphorylation of serine, threonine, and tyrosine was also included as variable modification in the phosphoproteomics dataset. Peptides and proteins were filtered at 1% FDR. For protein assessment (FDR < 1%) in MaxQuant, at least one unique peptide was required for identification. Only unique peptides were used for protein quantification. Reverse, only identified by site and proteins or phospho-sites that did not have reporter intensity in all channels were discarded for further analysis. Reporter intensities for the full proteome time course were extracted from “proteingroups.txt” table, and the reporter intensities for the phosphorylation sites were extracted from “Phospho(STY)Sites.txt” table. Data was further processed using R and Perseus. Reporter intensities were transformed to log2 and normalized within each experiment using “normalizecyclicloess” function from limma package in R. In order to integrate the independent experiments a further interexperiment normalization was conducted using ComBat function from sva package in R.Statistical determination of differential proteins, and phosphorylation sites at each time point versus Serum/LIF initial conditions was performed using a two-sided limma approach implemented in Prostar (version 1.12.4). We set a minimum fold change of 0.1 (log2) and a maximum Four mouse embryonic stem cell lines were used: TNGA, V6.4, ZS, and BL6. Metabolomic profiling was performed in collaboration with Metabolon Inc. Samples were prepared using the automated MicroLab STAR® system from Hamilton Company. Several recovery standards were added prior to the first step in the extraction process for QC purposes. To remove protein, dissociate small molecules bound to protein or trapped in the precipitated protein matrix, and to recover chemically diverse metabolites, proteins were precipitated with methanol under vigorous shaking for 2 min (Glen Mills GenoGrinder 2000) followed by centrifugation. The resulting extract was divided into five fractions: two for analysis by two separate reverse phase (RP)/UPLC-MS/MS methods with positive ion mode electrospray ionization (ESI), one for analysis by RP/UPLC-MS/MS with negative ion mode ESI, one for analysis by HILIC/UPLC-MS/MS with negative ion mode ESI, and one sample was reserved for backup. Samples were placed briefly on a TurboVap® (Zymark) to remove the organic solvent.m/z), and chromatographic data  on all molecules present in the library. Furthermore, biochemical identifications are based on three criteria: retention index within a narrow RI window of the proposed identification, accurate mass match to the library +/− 10 ppm, and the MS/MS forward and reverse scores between the experimental data and authentic standards. The MS/MS scores are based on a comparison of the ions present in the experimental spectrum to the ions present in the library spectrum. Peaks were quantified using area-under-the-curve. To account for differences in metabolite levels due to differences in the amount of material present in each sample, we have applied an additional normalization factor based on the total protein as determined by Bradford assay. One of the samples was discarded in this step due to the high presence of missing values . Afterwards, data processing was performed within Prostar R package (version 1.12.16). Metabolites that were not quantified in at least 75% of the samples in one treatment were discarded. Data was transformed to log2 scale and missing values were imputed using Imp4p for values missing at random, and with LAPALA with upper bound 2.5 for values missing in an entire condition. Once missing values were imputed, data was normalized by quantile centering to the median. Finally, with these data we performed differential analysis using limma (v3.36.1) with a design that account for biological replicates of the same cell line, with p-value correction of 5% FDR.Raw data was extracted, peak-identified, and QC processed using Metabolon’s hardware and software. Compounds were identified by comparison to library entries of purified standards or recurrent unknown entities. Metabolon maintains a library based on authenticated standards that contains the retention time/index (RI), mass to charge ratio .16 (GSE81285), Marks et al.5 (GSE23943), Buecker et al.59 (GSE56138), Kolodziejczyk et al.75 (E-MTAB-2600), Fidalgo et al.76 (GSE81045), and Lynch et al.3 (GSE112208). Data was either downloaded directly from GEO repository or, if available, from Supplementary Material from the publication. Processed transcript instensities were log2 transformed and normalized by “Loess” (from limma package in R). Afterwards, differential genes between Naïve and Serum/LIF conditions were estimated using the workflow “GEO2R” (https://www.ncbi.nlm.nih.gov/geo/geo2r/). Transcripts were considered significant if their fold change was above or below 1  and the p-value corrected by Benjamini–Hochberg was below 0.05. Upregulated and downregulated transcripts in 2i were used as gene-sets to perform rank-based enrichment analysis using GSEA algorithm. Ranked list of protein ratios in log2 at each time point of treatment was used as input for GSEA. GSEA was performed using default values. Normalized enrichment score was employed as scoring system.Processed data from the following selected publications was used in the current publication: Bulut-Karslioglu et al.Further information on research design is available in the Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Supplementary Data 3Supplementary Data 4Supplementary Data 5Supplementary Data 6Supplementary Data 7Supplementary Data 8Supplementary Data 9Reporting Summary


In a previous study, we found that higher waist circumference (WC) and higher body mass index (BMI) both increase the risk of chronic spontaneous urticaria (CSU). The aim of this study was to determine whether WC and BMI also increase the duration of CSU. We used multivariable Cox proportional hazards models to determine the hazard ratio (HR) for longer disease duration (longer than 3 years) according to WC and BMI. A total of 52,667 subjects were enrolled and their mean age was 54.5. After adjustments for other confounding variables the high WC/high BMI group had 1.062 times higher HR  than the normal WC/normal BMI group. Interestingly, the high WC/normal BMI group showed a significantly higher HR  than the normal WC/normal BMI group, but not the normal WC/high BMI group . Taken together, our results suggest that high WC rather than high BMI is a predictive risk factor for the longer disease duration of CSU. Another population-based study from Italy found a positive correlation between CSU risk and obesity defined by BMI2. A population-based study of the natural course of CSU in Korea found that the 1-, 2-, 3-, 4-, and 5-year remission rates of CSU were 21.5%, 33.0%, 38.9%, 42.6%, and 44.6%, respectively3. A retrospective observational study of 549 CSU patients in Spain revealed that the percentage of patients who experienced disease durations of 1 year, 3 years, 5 years, and more than 5 years were 64.1%, 32.4%, 17.3% and 8.4%, respectively4. Several risk factors such as the presence of autoantibodies, age older than 45 years, and vitamin D deficiency have been reported to be associated with longer duration of CSU7. A study of 85 CSU patients in Poland revealed that higher BMI values had a trend towards longer disease duration8.In a previous study, we found that higher waist circumference (WC) and higher body mass index (BMI) both increase the risk of chronic spontaneous urticaria (CSU)9. Considering the total follow-up period of 7 years, we defined the incidence rate of longer disease duration as the percentage of patients with disease durations of longer than 3 years among the total CSU patients. We followed-up CSU patients identified by the diagnosis-based algorithm for at least 3 years after the first detection to determine whether they could be identified with the CSU diagnosis-based algorithm again, until December 2015. The clinical characteristics  of CSU patients were analysed using Student’s t-test and the chi-square test. Normal BMI was defined as < 25 kg/m2 and normal WC was defined as < 90 cm in male or < 85 cm in female, according to the Korean definition for obesity10. As WC and BMI are not always directly proportional to each other, we divided CSU patients into 4 groups as follows: normal WC/normal BMI, normal WC/high BMI (≥ 25 kg/m2), high WC /normal BMI, and high WC/high BMI. We used these categories to determine the impacts of WC and/or BMI on the duration of CSU. We used multivariable Cox proportional hazards models to determine the hazard ratio (HR) for longer duration CSU according to WC and BMI after adjustments for confounding variables. P < 0.05 was considered to be statistically significant. We performed statistical analyses using SAS version 9.4 .The aim of the current study was to determine whether WC and/or BMI also increase the duration of CSU. We conducted a nationwide, cross-sectional, population-based study using data from the Korean National Health Insurance Service (NHIS). The NHIS is a national health care program covering the entire Korean population. This study was approved by the Ethics Committee of Seoul St. Mary’s Hospital, the Catholic University of Korea (KC17ZESI0487), and was conducted according to the principles of the Declaration of Helsinki. The need of the written informed consent has waived by the ethics committee of Seoul St. Mary's Hospital, the Catholic University of Korea. The follow-up period was from January 2009 to December 2015. We used the following diagnosis-based algorithm to identify CSU patients: patients who had visited outpatient clinics twice at least 6 weeks apart and received 2 diagnoses designated by the International Classification of Disease (ICD)-10 code for idiopathic urticaria (L50.1), other specified urticaria (L50.8), or unspecified urticaria (L50.9), or patients who had visited outpatient clinics twice at least 6 weeks apart with one diagnosis of L50.1, L50.8, or L50.9 plus another diagnosis of angioneurotic edema (T78.3)P < 0.05). Patients equal or above 80 years old had the highest incidence rate of longer disease duration (47.08%), followed by patients aged 65–79 (44.70%), 40–64 (42.45%) and 20–39 (35.57%). Patients with high WC (44.59%) and high BMI (43.43%) also had significantly higher incidence rates of longer disease duration, compared with normal WC patients and normal BMI patients, respectively (P < 0.05). Other factors that were associated with significantly higher incidence rates of longer disease duration were residential area  (43.39%), diabetes mellitus (DM) (44.2%), hypertension (44.2%), and hyperlipidemia (44.2%) (P < 0.05), but not smoking, alcohol, exercise, or household income.A total of 52,667 CSU patients were enrolled in this study. Their mean age was 54.5. Number of patients in 20–30, 40–64, 65–79, and ≥ 80 years age groups were 7883 (15.0%), 30,122 (57.2%), 11,686 (22.2%), and 2976 (5.7%), respectively. Number of female patients was 28,632 (54.4%). 22,215 patients (42.18%) were found to have longer disease duration (> 3 years). Female patients had a significantly higher incidence rate of longer duration (42.62%) than male patients (41.66%)  showed significantly higher HRs  (Table 8 firstly reported that higher BMI values had a trend towards longer disease duration. In this study, we further revealed that higher WC rather than higher BMI is more related with the longer disease duration of CSU, although both WC and BMI can affect the disease duration. Nevertheless, the pathophysiology of causal relationship between obesity and CSU has not been fully elucidated so far. According to previous studies, adipokines may act on human mast cells to induce endothelial inflammation11. As endothelial inflammation progresses, atherosclerotic changes proceed12. In the atherosclerotic plaque mast cells can be activated by variable proinflammatory factors such as C3a, C5a, cytokines, monocyte chemoattractant protein-1 and oxidized low-density lipoprotein13. Activation of mast cells through this process may affect the development of CSU8. Our results that especially, high WC/normal BMI group showed a higher risk for a longer disease duration of CSU but the normal WC/high BMI group did not, may reflect that those with high WC/normal BMI may have relatively high fat mass while those with normal WC/high BMI may have relatively high muscle mass.Taken together, these results suggest that high WC rather than high BMI is a predictive risk factor for the longer disease duration of CSU.Zbiciak-Nylec et al


The effects of processing temperature on filtration performance and characteristics of retentates and permeates produced during ultrafiltration (UF) of skim milk at 5, 20, and 50 °C were investigated. The results indicate that despite higher flux at 50 °C, UF under these conditions resulted in greater fouling and rapid flux decline in comparison with 5 and 20 °C. The average casein micelle diameter was higher in retentate produced at 5 and 20 °C. The retentate analysed at 5 °C displayed higher viscosity and shear thinning behaviour as compared to retentate analysed at 20 and 50 °C. Greater permeation of calcium and phosphorus was observed at 5 and 20 °C in comparison with 50 °C, which was attributed to the inverse relationship between temperature and solubility of colloidal calcium phosphate. Permeation of α-lactalbumin was observed at all processing temperatures, with permeation of β-lactoglobulin also evident during UF at 50 °C. All UF retentates were shown to have plasmin activity, while lower activity was measured in retentate produced at 5 °C. The findings revealed that UF processing temperature influences the physicochemical, rheological, and biochemical properties of, and thereby govern the resulting quality and functionality of, retentate- and permeate-based dairy ingredients. Ultrafiltration (UF) has a wide range of applications in dairy processing, such as protein standardisation of cheese milk, production of liquid protein concentrates, high protein powders and novel milk products such as protein-enriched milk . The choA commonly used criterion to evaluate membrane filtration performance is to measure the flux, which is generally higher for high temperature UF ≈50 °C), because of lower feed and permeate viscosity. However, such high temperatures can also give rise to undesirable microbial growth in produced retentates . In addi0 °C, becFor instance, applying UF to skim milk at specific processing temperatures can impact the activity of plasmin, an indigenous milk proteinase that is technologically significant for many dairy products and high protein dairy ingredients due to its ability to hydrolyse all caseins, except κ-casein . The actMuch of the research that has been conducted has focused on studying UF performance and associated changes in skim milk, at traditionally used high temperature (50 °C), with very limited work published on applying UF at lower temperatures. Given the importance of processing temperature in filtration, this study aimed to generate new scientific information in relation to UF of skim milk across a wider range of processing temperatures, more specifically its impact on UF operation, partitioning of milk constituents, and controlling technologically significant properties of retentates and permeates. Therefore, in this study on UF of skim milk, the influence of three processing temperatures, 5, 20, and 50 °C, was evaluated on filtration performance by measuring permeate flux and membrane permeability, before and after membrane cleaning. Besides studying filtration performance, characterising retentates and permeates would provide additional scientific and technical knowledge required to realise their potential as dairy ingredients in various food applications, particularly UF permeate, which was recognised as a food ingredient by Codex Alimentarius in 2017. Therefore, the properties of generated UF retentates and permeates were investigated using extensive characterisation in terms of composition, physicochemical properties, protein profile, and enzyme activity.Low-heat skim milk powder was provided by a local Irish dairy company. Reconstituted skim milk powder was prepared by slow addition of powder to deionised water under constant magnetic stirring at 22 ± 1 °C. Once all powder was added, solutions were left stirring for 2 h and then stored overnight at 4 °C. A lactose assay kit was purchased from Abcam, Cambridge, UK. Urokinase enzyme (U4010) and N-succinyl-ala-phe-lys 7-amido-4-methyl (S0763) was purchased from Sigma-Aldrich, Arklow, Wicklow, Ireland.2/h/bar) was used as a benchmark against which subsequent water permeability measurements were compared before and after cleaning the membrane on completion of each UF run. The continuously stirring feed (2.5 L) was delivered to the filtration unit using a peristaltic feed pump . In order to understand the relationship between permeate flux and transmembrane pressure (TMP) for operation at 5, 20, and 50 °C, we carried out a flux excursion exercise before concentration experiments at each processing temperature in full recirculation mode by returning the retentate and permeate lines back to the feed vessel. The feed flow rate was kept constant during each experiment to achieve the cross flow velocity of 0.34 m/s, recommended by Crowley et al. [Ultrafiltration (UF) experiments were performed as described by Crowley et al. . A lab-sy et al. . The TMPFor concentration experiments, TMP between 0.45–0.50 bar was selected as initial pressure to operate in the sub-critical flux region. The feed temperature was maintained throughout processing at 5 ± 0.5 °C, 20 ± 0.5 °C, and 50 ± 0.5 °C by recirculating retentate back into the feed vessel. The retentate line passed through a plate heat exchanger that received water in alternate channels supplied by a water bath set at the specific processing temperature. The processing temperature, TMP, and filtration duration was monitored and measured at regular intervals of 1, 1.5, 2, 2.5, 3, and 3.5 × volume concentration factor (VCF). The Brix value was measured at each interval using a portable refractometer  . Feed vo2+) concentration was determined using a Ca ion-selective electrode at 25 °C, with a calibration curve constructed from 5 Ca2+ standard solutions  [The total protein content was determined using the Kjeldahl method with a nitrogen-to-protein conversion factor of 6.38 . Ash cond 10 mM) .−1. The data were fitted to the power law model to calculate consistency coefficient (K) and flow behaviour index (n), as described by Mun et al. [The viscosity measurement temperature was the same as the processing temperature applied during UF of skim milk. The viscosity of feed and fresh retentate samples was measured within 2 h at 5, 20, and 50 °C with a HAAKE RotoVisco 1 viscometer  using a cup and bob geometry . The visn et al. . The parwww.gelanalyzer.com) by Istvan Lazar Jr., PhD and Istvan Lazar Sr., PhD, CSc.Protein profile of feed, retentate, and permeate streams was qualitatively assessed using sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) with precast gels  under reducing conditions using an AcquaTank mini gel unit  . The finPlasmin and plasminogen-derived activity after activation of plasminogen by urokinase were determined in fresh feed, retentate, and permeate samples within 16 h from UF experiments by measuring the concentration of the fluorescent product 7-Amino-4-Methylcoumarin (AMC) released by plasmin from the fluorescent substrate, N-succinyl-l-alanyl-l-phenylalanyl-l-lysyl-7-amido-4-methyl coumarin. Plasmin and plasminogen assays were carried out as described by Richardson et al. . Samplesp < 0.05) by applying Duncan’s post-hoc test, using the software IBM SPSS Statistics 20.0 .All results shown in this study for UF of skim milk, sample composition, physicochemical properties, protein profile, and plasmin- and plasminogen-derived activity are raw mean values analysed by fixed-effects one-way ANOVA. The data represent at least duplicate analysis of samples from 3 independent runs at each processing temperature. Means were tested for statistical significance (The relationship between permeate flux and transmembrane pressure (TMP) at 5, 20, and 50 °C is shown in 2/h (LMH) for feed at 5 °C (F5), 20 °C (F20), and 50 °C (F50), respectively. The R2 values of 0.89, 0.95, and 0.99, display the linearity of flux decline at 5, 20, and 50 °C, respectively, with the rate of flux decline being much more rapid at 50 °C than 5 or 20 °C. The brix value of feed increased from 9.5 to 23.3, 18.7, and 22.0, during UF concentration, at 5, 20, and 50 °C, respectively. The processing time required to reach 3.5 × feed volume reduction was 128, 100, and 60 min, at 5, 20, and 50 °C, respectively.During UF processing, the permeate flux was measured at regular intervals, and it declined steadily with time at each processing temperature . The staThe flux decline observed in this study can be linked to a substantial role played by skim milk components  in controlling the fouling mechanisms during UF. The particle deposition on, and pore blocking of, membranes can lead to fouling, resulting in gradual flux decline during UF . The fouIt has been reported that caseins have much lower driving forces for reduction in free energy and resulting adsorption on membrane surfaces than whey proteins, and therefore whey proteins dominate the adsorption on membrane surfaces . Among mp < 0.05) lower at 50 °C than 5 and 20 °C  higher than 2.2 mM in R50. The mineral profile of retentates is shown in p < 0.05) lower than R50. This provides evidence for the  solubilisation of colloidal calcium phosphate (CCP) within micelles as the temperature is lowered [p < 0.05) higher values of total calcium and phosphorus in permeate, P5 and P20, as compared to P50. This is further supported by the significantly higher ionic calcium content in P5 and P20 than P50. The content of sodium, magnesium, potassium, and sulphur did not vary significantly in permeate samples, suggesting comparable permeation of these minerals at all three processing temperatures. The total protein content of P50 was significantly (p < 0.05) higher than P5. At high temperature, UF membranes tend to be slightly more open and permeable, which may lead to greater transmission of smaller, lower molecular weight proteins such as β-LG and α-LA, as observed in permeate P50 [The average content of total solids, protein, lactose, ash, and pH for feed, fresh retentate, and permeate produced at 5, 20, and 50 °C is shown in  lowered . During  lowered . Mineral lowered ,27. Thisp < 0.05) smaller in retentate R50 at 179 nm, than R5 at 191 nm and R20 at 190 nm. During UF, temperature-dependent changes may take place within casein micelles and CCP of skim milk, which may consequently influence size and hydration of casein micelles [After each UF run, feed and retentate samples were analysed for casein micelle size distribution and zeta potential  within 2micelles . As solumicelles . The ovemicelles . The reln) value of 1, indicating Newtonian liquid behaviour [p < 0.05) different between retentates, as shown by log–log plots of apparent viscosity and shear rate data in n <1.0; however, the extent of shear-thinning was more pronounced in R5 than R20 and R50. The K value was significantly (p < 0.05) higher for retentate R5 at 0.23 in comparison with R20 and R50, both with K values of 0.01. Concentrates containing less than 15% protein generally display Newtonian fluid behaviour [The viscosity of feed and retentate samples was analysed at the same temperature used in UF processing. The application of the Power Law model to viscosity data revealed that all feed samples had a flow behaviour index  higher in R50. The electrophoretic pattern of permeate samples revealed two protein bands in each permeate sample, generated at different processing temperatures, as shown in p < 0.05) higher in P50 as compared to P5 and P20, as also visible in corresponding intense bands shown in The protein profile of retentate and permeate samples produced at 5, 20, and 50 °C are shown in p < 0.05) among retentate samples, with R5 displaying lower activity compared to R20 and R50. As described earlier, the role of plasminogen activator in converting inactive plasminogen to plasmin is noteworthy in explaining the difference in plasmin activity between retentate samples. The duration required for temperature equilibration in starting feed batch and subsequent UF processing at 5, 20, and 50 °C could have influenced the action of plasminogen activator [The innate plasmin activity of feed and retentate samples generated at each processing temperature is shown in ctivator . The UF ctivator , which mctivator , which mThe plasminogen-derived activity was measured after incubating feed and retentate samples with added urokinase, a plasminogen activator, which can cause activation of inactive plasminogen to active plasmin, even when added at very low concentrations . The uroThe results of this study show that UF of skim milk at 50 °C resulted in higher permeate flux than at 5 and 20 °C. However, the rate of flux decline during processing was considerably greater at this higher processing temperature, which was attributed to more extensive membrane fouling at 50 °C, as indicated by low permeability of clean water immediately post UF processing. These differences in fouling impacted protein profile of the resultant filtration streams and would also be expected to impact on cleaning-in-place of such membranes. The retentate produced at 5 °C had the highest viscosity. The plasmin activity in retentates increased in the order 5 < 20 < 50 °C. UF processing temperature significantly affected permeation of soluble minerals due to temperature-induced alterations mineral equilibria. These changes directly influence the physicochemical and functional properties of dairy ingredients derived from UF retentate and permeate streams.


Elizabethkingia anophelis, an important multi-drug resistant bacterium. The tertiary structure is comprised of six α-helices and nine β-strands, and is similar with other GNATs. We identify a new and uncharacterized GNAT dimer interface, which is conserved in at least two other unpublished GNAT structures. This suggests that GNAT enzymes can form at least five different types of dimers, in addition to a range of other oligomers including trimer, tetramer, hexamer, and dodecamer assemblies. The high-resolution structure presented in this study is suitable for future in-silico docking and structure–activity relationship studies.General control non-repressible 5 (GCN5)-related N-acetyltransferases (GNATs) catalyse the acetylation of a diverse range of substrates, thereby orchestrating a variety of biological processes within prokaryotes and eukaryotes. GNAT enzymes can catalyze the transfer of an acetyl group from acetyl coenzyme A to substrates such as aminoglycoside antibiotics, amino acids, polyamines, peptides, vitamins, catecholamines, and large macromolecules including proteins. Although GNATs generally exhibit low to moderate sequence identity, they share a conserved catalytic fold and conserved structural motifs. In this current study we characterize the high-resolution X-ray crystallographic structure of a GNAT enzyme bound with acetyl-CoA from  Elizabethkingia anophelis is a newly discovered gram negative bacterium, isolated from the midgut of Anopheles gambiae mosquito2. The bacterium is emerging as an important and opportunistic human pathogen, with capacity for causing neonatal meningitis and sepsis. Multi-drug resistance profiles against several antibiotics have been reported3, although the antimicrobial susceptibility and the resistance mechanism to antibiotics remain unresolved. Recent studies have reported antibiotic resistance to ampicillin, chloramphenicol, kanamycin, streptomycin and tetracycline5, while resistance against tetracycline, trimethoprim/sulfamethoxazole, and ciprofloxacin are of high concern since these antibiotics are first-line therapies for infections caused by this pathogen6. Infections caused by E. anophelis are generally hospital-acquired and include nosocomial pneumonia, bacteremia, sepsis, meningitis, skin and soft-tissue infection, urinary tract infection, and biliary tract infection in neonates and adults with underlying other diseases including malignancies and/or immunosuppression9. Several outbreaks of E. anophelis across many countries, has led to recent interest in discovering the underlying mechanisms of antibiotic resistance and possible new drug targets to overcome resistance12.14. GNAT proteins possess a highly conserved catalytic fold, and can acetylate a wide range of substrates ranging from antibiotics to proteins16. The transfer of an acetyl group from acetyl-CoA (AcCoA) to substrates such as aminoglycoside antibiotics alter the fundamental characteristics of these molecules and can render them inactive18.General control non-repressible 5 (GCN5)-related N-acetyltransferases (GNATs) are a large superfamily of enzymes, playing prominent roles across a large number of biological processes including aminoglycoside antibiotic resistance, transcription regulation, protein acetylation and stress reaction14. Previous studies have demonstrated that GNAT enzymes of several bacteria are capable of acetylating different antibiotics20. E. anophelis strain 12012‐2PRCM has been reported to be resistant against aminoglycosides, β‐lactams, polypeptides, sulfonamides, chloramphenicols, quinolones, and tetracyclines5.Despite the relatively low number of studies on bacterial protein acetylation, evidence is gathering to suggest that this post-translational modification occurs on many bacterial virulence factors and may play a key role in bacterial virulence21. These secondary structural elements comprise four conserved sequence motifs, motif C , motif D (β2-3-α2), motif A (β4-α3), B (β5-α4))21. The N-terminus is moderately well conserved while the C-terminal region varies considerably, in part, due to this region being responsible for substrate binding22. With the emergence of multi-drug resistance (MDR) in E. anophelis, here we determine the structure of an uncharacterized GNAT family acetyltransferase from this organism, which may provide a platform for in-silico fragment screening, drug-design, and/or structure–activity relationship studies.Previous structural characterizations of GNAT proteins have led to a consensus or conserved fold common to GNAT family members. Functional GNAT proteins generally contain six (or sometimes seven) β-strands and four α-helices arranged in a (β0)-β1-α1-α2-β2-β3-β4-α3-β5-α4-β6 topology23. The expression vector provides a non-cleavable N-terminal His6-tag . The enzyme was expressed in E. coli Rosetta BL21(DE3)R3 following standard SSGCID protocols, as described previously24. Purification was performed using Ni–NTA affinity and size exclusion chromatography following standard SSCID protocols25. Briefly, the bacterial cell pellet was suspended in a buffer comprised of 25 mM HEPES, 500 mM NaCl, 5% glycerol, 30 mM imidazole, 0.025% sodium azide, 0.5% CHAPS, 10 mM MgCl2, 1 mM TCEP, 250 µg/ml AEBSF, 0.05 µg/ml lysozyme pH 7.0). Cell lysis was undertaken by sonication, and the resulting extract clarified by centrifugation and passed over a HisTrap FF 5 ml column pre-equilibrated in 25 mM HEPES, 500 mM NaCl, 5% glycerol, 30 mM imidazole, 0.025% sodium azide, 1 mM TCEP pH 7.0. The column was washed with 20 column volumes of 25 mM HEPES, 500 mM NaCl, 5% glycerol, 30 mM imidazole, 0.025% sodium azide, 1 mM TCEP pH 7.0 to remove unbound proteins. The His-tagged protein was eluted with seven column volumes of a buffer comprised of 25 mM HEPES, 500 mM NaCl, 5% glycerol, 1 mM TCEP, 250 mM imidazole and 0.025% azide pH 7.0. The protein was further purified by size exclusion chromatography using a HiLoad 26/60 Superdex 75 preparative-grade column pre-equilibrated in 25 mM HEPES, 500 mM NaCl, 5% glycerol, 2 mM DTT, 0.025% azide pH 7.0, and calibrated using the GE Healthcare Calibration Kit (28-4038-41) with conalbumin, ovalbumin and ribonuclease A. Two protein peaks, corresponding to monomer and dimer species  were pooled and analyzed by SDS–PAGE  , Microscillaceae bacterium (60% identity with 90% coverage), and Caenibacillus caldisaponilyticus (59% identity with 90% coverage). Within the top 20 Blast results, 3 GNAT proteins were aminoglycoside 6′-N-acetyltransferases: Chitinophaga eiseniae (53% identity with 98% coverage), Pedobacter nutrimenti (59% identity and 89% coverage), and Rhabdobacter roseus (50% identity and 98% coverage) , solved by a structural genomics consortium that remains to be published. The next most similar protein was 1yvk , and this was also solved by a structural genomics group and remains unpublished. The sequence identity between 1y9k and 1yvk is 59% ), revealed the most closely related proteins to be GNAT family members from 08865), r2 of surface area and mediated by 10 hydrogen bonds. Key residues involved in these hydrogen bond interactions include Ile68, Val70, Asn76, Gly78, Gly80, Gly81, Gln110, Tyr112, and Lys116 35. Approximately 62% of GNATs have been reported to contain a conserved Tyr as a general acid to initiate the catalysis, and the positioning of Tyr114 from our structural analysis is consistent with this36. Both the acetyl-CoA residues involved in H-bonding, and active site residues are shown in Fig. Our structure contained a well-ordered acetyl-CoA molecule bound in the active site Fig. AB. The i116 Fig. C, and sp44. Analysis of the interfaces present in the crystal using PISA45 suggests the protein is most likely to be a dimer  structures120 forming a salt bridge interaction with Asp146, and Gln115 hydrogen bonding with Asn105. Dimers are the most common GNAT assembly, and there are four different types of dimers that have been described in the literature21. One form involves the C-terminal β-strands interacting to form a continuous β-sheet49. A second form involves two monomers exchanging their C-terminal β-strands -Iy and ScGNA151. A third form involves two monomers forming a β-barrel (eg SmAAC(3)-Ia52, while the fourth type is formed through a four-helical bundle (e.g. pPCAF)53. The dimer interface found in our structure is distinctly different to any other dimer described previously (Fig. To test whether this interface is conserved in the most closely related GNATs, we examined whether the same dimer structures were present from proteins of different crystal space groups and distinctly different sequences. We found that the same dimer structure is present in both of the crystal structures of 1YVK and 1Y9K Fig. . Since tE. anophelis. Structural analysis suggests that the protein exhibits the hallmarks typical of a GNAT fold. Structural and sequence-based alignments suggest that this acetyltransferase may be a possible aminoglycoside 6′-N-acetyltransferase, however this needs to be determined experimentally. Residues involved in acetyl-CoA binding have been identified and are conserved in closely related GNATs. We identify a new type of dimeric interface, and this is conserved in at least two other structures that have been deposited to the PDB, but remain unpublished. The oligomerization of GNATs vary range from dimers through to dodecaomers, and oligomerization is important for function15. Thus characterization of new Gcn5-related N-acetyltransferases has the potential to expand our understanding of how these enzymes carry out acetylation of a wide range of substrates, and high-resolution structural elucidation may offer new opportunities for biotechnological applications and drug design.Here, we describe the structure of a GNAT family member from Supplementary Information.


Solanum tuberosum L.). In this study, the antiviral activity of Bacillus licheniformis strain POT1 against AMV on potato plants was evaluated. The dual foliar application of culture filtrate (CF), 24 h before and after AMV-inoculation, was the most effective treatment that showed 86.79% reduction of the viral accumulation level and improvement of different growth parameters. Moreover, HPLC analysis showed that a 20 polyphenolic compound was accumulated with a total amount of 7,218.86 and 1606.49 mg/kg in POT1-treated and non-treated plants, respectively. Additionally, the transcriptional analysis of thirteen genes controlling the phenylpropanoid, chlorogenic acid and flavonoid biosynthetic pathways revealed that most of the studied genes were induced after POT1 treatments. The stronger expression level of F3H, the key enzyme in flavonoid biosynthesis in plants, (588.133-fold) and AN2, anthocyanin 2 transcription factor, (97.005-fold) suggested that the accumulation flavonoid, especially anthocyanin, might play significant roles in plant defense against viral infection. Gas chromatography-mass spectrometry (GC-MS) analysis showed that pyrrolopyrazine-1,4-dione is the major compound in CF ethyl acetate extract, that is suggesting it acts as elicitor molecules for induction of systemic acquired resistance in potato plants. To our knowledge, this is the first study of biological control of AMV mediated by PGPR in potato plants.Alfalfa mosaic virus (AMV) is a worldwide distributed virus that has a very wide host range and causes significant crop losses of many economically important crops, including potato ( Solanum tuberosum L.) is the third-largest economic food crop in the world, including Egypt, after rice and wheat1. Under field conditions, potatoes suffer from many viral diseases infections. Among such viruses causing great economic losses and considered the major limiting factors for potato production is alfalfa mosaic virus 2. It is a worldwide-distributing virus, infecting 698 species of 167 genera in 71 families, including Solanaceae and Leguminosae4. It is transmitted by sap inoculation and by numerous aphid species in a non-persistent manner3. Most potato cultivars are susceptible to AMV infection and induce diverse symptoms, including yellow blotching and bright mottling of potato leaves resulted in calico symptoms5. AMV management is difficult and depends mainly on selecting resistant plant cultivars and/or intensive insecticide and pesticide treatments that are often used to control its vector spreading6. The application of plant growth-promoting rhizobacteria (PGPR), as bio-control agents, is a promising safe approach in crop protection against different pathogens7, including viruses6. Different Bacillus spp. have been reported to induce antiviral responses against tomato spot wilt virus (TSWV) and potato virus Y (PVY) in tomato plants8 and against cucumber mosaic virus (CMV) in tobacco9, tomato10, Arabidopsis11. Under field conditions, the application of powder and liquid formulations of three PGPRs strains  reduced the severity of sunflower necrosis virus (SNV) up to 51.4% in sunflower plants12.Potato  either by salicylic acid (SA) signaling pathway and/or jasmonic acid (JA) pathways26. Induction and expression of such genes were correlated with polyphenol content under both normal and stress conditions27. The present study evaluated the efficacy of Bacillus licheniformis strain POT1 to induce systemic resistance in potato against AMV infection and its effects on plant growth development and transcriptional levels of phenylpropanoid, chlorogenic acid and flavonoids biosynthetic pathways genes. Furthermore, identification of the metabolites in potato plant extract and the bioactive constituents of POT1 crude filtrate were performed using High-performance liquid chromatography (HPLC) and Gas chromatography-mass spectrometry (GC-MS) analysis.Potatoes are good sources of chlorogenic acid and flavonoids, which constitute the majority of polyphenolic compounds28, the most potent bacterial isolate exhibiting antiviral activity was selected and subject for morphological, physiological, biochemical characteristics  and (Acc# MH733009). The phylogenetic tree analysis indicated a high identity of B. licheniformis isolates from different parts of the world with 99% and presented as a monophyletic group of these isolates (data not shown).By using the half-leaf methodcs Table  and moleThe symptoms of AMV including yellow blotching and bright mottling ended with clear visibility of calico symptoms on non-treated infected potato plant leaves (T2) were observed at 21 dpi  in AMV-infected potato leaves. On the other hand, a significant decrease in the viral accumulation level in T3 (18.23-fold) and T4 (4.40-fold) treatment plants was observed , recording 2.33 ± 0.58 g, 22.32 ± 1.09 g, 20.12 ± 1.07 g, and 2.46 ± 0.07 g, respectively, compared to control plants , a significant up-regulation of PAL with a relative expression of 2.928- and 2.462-fold change, no significant changes, was observed in T3 and T4 treatments, respectively , foliar spraying of potato plants with CF 24 h before inoculation with AMV and 24 h after inoculation, triggered the expression of both PAL and C4H, genes.The relative expression levels of the two genes, HCT, C3H and HQT) encoding three regulatory enzymes of chlorogenic acid biosynthesis were investigated. The significant up-regulation of HCT was observed in all treatments with relative expression levels of 1.203-, 3.732- and 2.441-fold change for T2, T3 and T4, respectively, compared to control  encoding eight enzymes controlling the flavonoid biosynthesis pathway was observed  was observed in AMV-infected plants (T2) when compared with the control plants. However, POT1-treated plants before infection (T3) or before + after (T4) exhibited higher expression levels with 4.823- and 5.656-fold change, respectively  showed the best results of CHI gene expression. The expression level was induced only in T4 treat-plants with a significant relative expression level of 1.777-fold-chang, while T2 and T3 showed down-regulation with relative expression levels of 0.636-and 0.703-fold, respectively, lower than control  in T4 treated-plants reflects the higher inducing activity of both AMV and POT1 for F3H transcription. Unexpectedly, the transcript level of FLS gene was shutdown in all treatments in comparison with control  was more enhancer than T3, resulting in the highest expression levels of 18.635- and 20.440-fold increased than control for DFR and F3′H, respectively  was showed in T4 treatment, while T2 treated-plants exhibited expression level of 20.821-fold change than control  were benzoic acid , p-Hydroxy benzoic acid , chlorogenic acid , cinnamic acid  in T1, T2, T3 and T4 extracts, respectively.The HPLC chromatograms of ethanolic extracts of T1, T2, T3 and T4 potato plants were shown in Fig. p-Coumaric acid were induced only in T2, T3 and T4 with accumulation levels of , , respectively, while Ellagic acid and resvertol were induced in T4 only with 4.42 and 495.51 mg/kg, respectively, , syringic acid and The identification of the bioactive components of bacterial filtrate extract was done by the GC-MS machine. The active constituents with their retention time (RT), detected compounds, chemical formula (CF), and chemical structures were illustrated in Table 30. Due to negative impacts on public health and environmental hazards, chemical treatments including pesticide or insecticide must be managed and controlled. Biological control using PGPR (one or more strains) is being considered as an alternative or a supplemental way of handling of plant diseases better than the chemical control in agriculture34. Consequently, searching and discovering new environmental eco-friendly biocontrol agents capable to control plant viral diseases are demand8. In the current study, the antiviral activity of Bacillus licheniformis strain POT1 against AMV on potato (Solanum tuberosum L.) plant was evaluated. Moreover, the transcriptional levels of thirteen genes involved in phenylpropanoid, chlorogenic acid and flavonoid biosynthetic pathways genes, as well as bioactive constituents of POT1 crude filtrate (CF), were analyzed. Additionally, HPLC analysis was used to compare the content of polyphenol compounds with the expression levels of biosynthetic pathway genes. To our knowledge, this is the first time to deal with the effect of PGPR against AMV in potato plants.Plant viruses are among the most important plant pathogens, as near half of the emerging epidemics have a viral etiology, causing problems of food security and they are responsible for huge losses of crop productionBacillus spp., Pseudomonas spp. and Streptomyces spp., improved plant growth and increased protection against viral infection38.Under greenhouse experiments, the application of POT1 has significantly increased plant growth, yield, reduced disease severity, and virus accumulation compared to infected potato plants without any treatments. We have shown that inhibition of AMV infection can be generated by treating plants with bacterial culture filtrate either prior to or post-challenge with the virus. Our results showed that the up-regulation of almost genes triggered by bacterial filtrate resulted in increasing resistance to AMV via limiting viral accumulation, symptom severity, and growth parameters increasing. Many reports showed that the application of some In the current study, the obtained results showed that potato plants infected with AMV (T2) were significantly reduced the tuber numbers and weight as well as fresh and dry weight when compared with the control plants (T1). However, T4 treatment was significantly enhanced and improved all evaluated growth parameters recording the highest values of fresh and dry weight when compared to the other treatments and significantly reduced the negative effects resulted from the viral infection.Streptomyces spp. CF exhibited a significant reduction of PVY in potato39. Thus, POT1 activates induced systemic resistance (ISR) of potato plants against AMV infection. ISR using PGPR showed promising results against plant viruses such as tomato mottle virus, CMV, and PVY in tomato40 cucumber43 and potato45, respectively. Besides the activation of some defense genes, ISR enhanced the production peroxidase, antioxidant protective enzyme and secondary metabolites47. Among the secondary metabolites, polyphenolic compounds play vital roles in plant growth and resistance against different biotic and abiotic stresses19. Routes to the major classes of polyphenol compounds involve three pathways (i) phenylpropanoid pathway, (ii) chlorogenic acid pathway and (iii) flavonoid pathway27.The greenhouse experiment results confirmed the effective biocontrol activity of POT1 against AMV infection, which resulted in a considerable decrease in AMV concentration levels. Significant reductions in virus concentration by 45.29% and 86.79% for potato leaves of T3 and T4 treatments, respectively, were showed when compared to T2 treatment. These results suggest that CF of POT1 contains secondary metabolites that can play a notable role in SAR. In this context, the foliar application of PAL to cinnamic acid then to p-coumaric acid by cinnamic acid 4-hydroxylase (C4H) and ended by the formation of the main intermediate coumaroyl-CoA that is controlled by 4-coumarate-CoA ligase (4CL). Based on the correlation between accumulation rates and resistance degree, phenylpropanoid compounds have been proposed to play crucial roles in plant defense against different microbial pathogens50. In the current study, the expression of PAL was induced in T3 and T4 treatments, while T2 treatment showed down-regulation. On the other, C4H was induced and overexpressed with a relative expression level 6.291-fold change in treatment T4 only. Consistent with the results of the transcriptional levels, HPLC analysis revealed that p-coumaric acid was induced and accumulated with the highest amount (52.99 mg/kg) in T4 treatment. Thus, overexpression of C4H could be associated with p-coumaric acid content increasing. The down-regulation of both genes, PAL and C4H, in infected potato tissues (T2) reflects the suppressor activity of AMV. Many researchers reported that the viral infection was associated with down-regulation or decreasing in PAL and C4H activity52. Besides its role in plant defense, PAL involved in the biosynthesis of salicylic acid (SA) that plays an essential role in the induction of plant systemic resistance pathogens55. Moreover, through enhancing the accumulation of lignin in the plant cell walls, C4H plays a role in increasing plant defense57. Consequently, the induction of PAL and C4H transcripts in T3 and T4 treatment suggesting that POT1 is a good elicitor activated ISR that is associated with SA and secondary metabolites, precursors of chlorogenic acid and flavonoids, biosynthesis in potato tissues.The phenylpropanoid pathway started with the conversion of L-phenylalanine by 59. The chlorogenic acid pathway started with the conversion of p-coumaroyl CoA to shikimate through HCT catalyzing activity60. Following synthesis of p-coumaroyl shikimate by C3H, HCT catalyzes the transfer of it to the caffeoyl CoA61 ended with chlorogenic acid through HQT activity62. In the present study, although HCT showed slightly induced after AMV infection in T2 treatment (1.203-fold), the overexpression with relative expression level 3.732- and 2.441-fold change was observed in T3 and T4 treatments, respectively. Likewise, the up-regulation of C3H with the transcriptional expression level of 6.453-, 7.061- and 8.111-fold was reported in T2, T3 and T4, respectively. HCT and C3H involved in lignin biosynthesis in the plant cell wall63. Thus, the induction of transcriptional expression of these genes shows their protective role against AMV and suggests that the potato plant can use the lignifications as one of its defense to resist the viral infection and movement. On the other hand, the decreasing of chlorogenic and caffeic acids contents upon AMV-inoculation  could reflect the down-regulation of transcriptional expression levels of HQT for these treatments when compared to control. Tomato HQT overexpression was associated with increases in chlorogenic acid content and versa versa62. Based on current results, whether HQT suppression and HCT and C3H induction, AMV-infected potato plant was associated with decreasing the content of chlorogenic acid. We can assume that AMV could not make complete suppression of chlorogenic acid biosynthesis and had a suppression effect on HQT rather than HCT and C3H. Moreover, the ISR activated by POT1 may be correlated with inducing and increasing cell wall lignifications.Chlorogenic acid, which is the ester of caffeic acid and quinic acid, is one of the polyphenol compounds, phenolic acids, that improving plant disease resistance through inhibiting pathogensp-coumaroyl CoA to naringenin chalcones, through CHS catalyzing activity, which can be transformed to naringenin by the action of CHI27. These are the first two steps of the flavonoid pathway and are strictly required for chalcones and dihydrochalcones production, which considered being the primary precursors and constituting the main intermediates for a large number of flavonoids synthesis by the action of enzymes set such as F3H, FLS, DFR, F3′H, AN1 and AN265. Although AMV induced CHS (2.084-fold) in infected potato tissues (T2), increasing transcriptional expression levels in T3 and T4 by 4.823- and 5.656-fold change, respectively, were observed. On the other hand, T4 treatment showed a significant up-regulation of CHI with expression level 1.777-fold increased than control. The repression of CHI in T2 and T3 suggested that AMV infection suppressed naringenin biosynthesis, even if the potato plants treated with CF 24 h before viral infection. Interestingly the dual foliar application (T4), before and after the viral infection, showed the highest induction of both genes, CHS and CHI, that are strictly required for flavonoid production in multiple tissues of potato65.The flavonoid pathway started with the conversion of F3H, the key enzyme in flavonoid biosynthesis in plants66, was the master expressed gene among flavonoid pathway genes with the highest expressions level 116.162-, 367.092- and 588.133- fold change than the control for T2, T3 and T4, respectively. Likewise, F3′H exhibited significant up-regulation in both POT1 treatments with relative expression 7.963- and 20.440-fold increased than control. F3H converts hydroxylate naringenin to dihydroflavonol or dihydrokaempferol while F3′H, the primary enzymes responsible for the diversification of anthocyanins, transforms dihydrokaempferol into dihydroquercetin68. The stronger induction of F3H and F3′H, suggesting that they played significant roles in plant defense against viral infection and leads to induction of biosynthesis of many intermediate precursors compounds for flavonoids production. The six significant subclasses of flavonoids are the flavones, isoflavones, flavonols, flavanones, flavan-3-ols and anthocyanidins. The high expression of F3H was associated with the accumulation of both quinochalcones and flavonols in safflower plants69. Among flavonols18, myricetin and quercetin were up-regulated and accumulated in T4 plants with 438.40 and 281.80 (mg/kg), respectively. The antiphytoviral activity of quercetin against TMV70, PVX71 and Tomato ringspot virus72 were previously reported. Meanwhile, POT1 T4-treatment was associated with induction and accumulations of many flavonoids compounds.The obtained results showed that 75. DFR, a NADPH-dependent reducing enzyme, converts dihydroflavonols to leucoanthocyanidins which necessary for formation of anthocyanins in higher plants76. Quattrocchio et al.77 reported that anthocyanin pathway is regulated at the DFR step in Petunia hybrid plants. Comparing to control in this study, both T3 and T4 treatments induced DFR transcripts with relative expression values of 5.314- and 18.635-fold change, respectively. Based on this data, we are suggesting that POT1 treatments stimulated the plant immune defense system to produce more anthocyanins-related compounds. AN1 and AN2 are two transcription factors involved in the regulation of anthocyanin biosynthesis78. Thus, a significant up-regulation (1.866-fold) of AN1 in T4 treatment could be induced biosynthesis of anthocyanin-related compounds. D’Amelia et al.79 reported that AN1 expression is associated with high anthocyanin contents in leaves. On the other hand, the higher and stronger expression of AN2 with relative expression levels of 61.819- and 97.005-fold change in T3 and T4, respectively, revealed that anthocyanins played important roles in plant defense against viral infections. HPLC analysis showed that, a significant overaccumulation of the total phenolic contents in T4  rather than T2 (1606.49 mg/kg) plants. Meanwhile, D’Amelia et al.1 showed that AN2 able to regulate the production of phenolic compounds and high expression in potato tuber during drought stress was associated with increases in total phenolic levels27. The accumulation of anthocyanin in plants upon biotic stresses has been reported. Ustilago maydis triggered anthocyanin induction in maize80, and anthocyanin-enriched tomato fruits exhibited lower susceptibility to gray mold81. Moreover, antibacterial, antifungal and antiviral activities of certain anthocyanins were also reported84.Due to their powerful antioxidant properties, anthocyanins (flavonoids) protect plants against various biotic and abiotic stresses86. Moreover, it represents a new and rich source of secondary metabolites that need to be discovered. The GC-MS spectrum analysis showed that pyrrolo pyrazine-1,4-dione was the major compound in POT1 ethyl acetate extract. Pyrrole was known for a wide range of bioactivities, including antibiotics, antitumor, antifungal, anti-inflammatory, anti-angiogenesis and cholesterol reducing drugs87. Moreover, pyrrolopyrazine-1,4-dione isolated from Streptomyces spp. showed antioxidant88 and anticandidal89 activity, while that isolated from Shewanella spp. exhibited anticyanobacterial and algicidal activity90. Additionally, these compounds showed excellent protease inhibitor activity with a very good antiretroviral activity91 and have the ability to inhibit HIV-1 viruses, DNA polymerases and protein kinase activity93. The obtained results supported previous reports of pyrrolo pyrazine-1,4-dione activity in preventing viral replication. Consequently, POT1 could be useful as a preventive agent against AMV infection. However, further examinations needed for the potential field application.Bacillus species produce wide structural variability of secondary metabolites that exhibiting strong antibacterial and antifungal activitiesVirus-free potato tubers cv Spunta used in this study were kindly provided by the International Potato Center (CIP), Ministry of Agriculture and Land Reclamation, Egypt.52 and maintained continuously on Nicotiana glutinosa plants under greenhouse conditions.Alfalfa mosaic virus Kh1 isolate (Acc# MN099289) used in this study was previously isolated from infected potato plants94. The bacterial isolate showing a maximum antiviral activity was selected, and preliminary identified based on morphological and biochemical characteristics95. For the bacterial culture filtrate (CF), the selected bacterial isolate was grown in a nutrient broth medium and incubated on a shaking incubator at 30 °C for two days. The bacterial culture was centrifuged at 6,000 rpm for 10 min at 4 °C to separating bacterial cells and collecting supernatants.Soil-adhered potato roots were collected from a potato field in Alexandria governorate, Egypt. The roots were crushed in a mortar and a loopful was cultured on Nutrient Agar (NA) media and incubated at 30 °C. Different colonies were picked and assayed for antiviral activity using half-leaf method96. Briefly, PCR reaction was started with initial denaturation at 95 °C for 2 min, followed by 35 cycles at 95 °C for 30 s, 50 °C for 30 s and 72 °C for 1.5 min. An additional final extension step was carried out at 72 °C for 5 min. PCR amplified products were checked on 1.5% agarose gel electrophoresis, visualized under UV transilluminator, and purified by a PCR clean-up column kit  for sequencing. Sanger sequencing of 16 rRNA gene was performed using a BigDye Terminator v3.1 Cycle Sequencing kit and a 3130xl Genetic Analyzer system . After the sequencing process, the annotated nucleotide sequence was analyzed using NCBI-BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi), and deposited in Genbank. The phylogenetic tree was constructed based on the UPGMA statistical method with a bootstrap of 2.000 replicates using the MEGA 5 software97.Bacterial genomic DNA was isolated from bacterial culture of selected bacterial isolate using Wizard Genomic DNA Purification Kit  according to the manufacture instructions. By using 16 rRNA specific primers, forward (5`-AGAGTTTGATCCTGGCTCAG-3`) and reverse (5`-GGTTACCTTGTTACGACTT-3`), PCR reaction was performed as previously reported98. The first treatment (T1) was mock-treated plants (control), in which potato plants inoculated with free-virus inoculation buffer + foliar spraying of free bacterial broth medium. The second treatment (T2) was plants inoculated with AMV with foliar spraying of free bacterial broth medium (infected). The third treatment (T3) included plants treated by foliar spraying of CF, 24 h before inoculation of AMV. The fourth treatment (T4) was potato plants treated by twice foliar spraying of CF, 24 h before inoculation of AMV and 24 h post-inoculation of AMV. All plants were kept under insect-proof greenhouse conditions 28 °C/16 °C (day/night) and 70% relative humidity and daily observed for symptoms development recording.Potato tubers were cultivated in plastic pots (30 cm in diameter) filled with 4 kg of sterilized soil compromised of clay: sand, equal ratio (1:1). The experiment was carried out in four treatments, each comprised five replicate pots, and one potato tuber/each pot. After 35 days of growing, two true upper leaves of each potato plants were dusted with carborundum and mechanically inoculated, as previously described99. The amplified cDNA was used as a template for quantitative real-time PCR (qRT-PCR).Leaves of five biological replicates of each treatment were collected at 21 dpi and kept at -80 °C until use. Total RNA was extracted using the RNeasy plant mini kit  according to the manufacturer’s instructions. Each biological sample was a mix of five samples derived from five different plants. The extracted RNA was dissolved in DEPC-treated water, treated with RNase-free DNase to eliminate genomic DNA, quantified by NanoDrop UV spectrophotometer  and the integrity was assessed by agarose gel electrophoresis. Two micrograms of total RNA for each sample were reverse transcribed to cDNA using oligo (dT) and random hexamer primers with reverse transcriptase enzyme of Super-Script II , according to the manufacturer’s instructions. The reverse transcriptase reactions were performed in a thermal cycler , according to Behiry et al.EF1-α  with the SYBR Green PCR Master Mix  and performed according to previously reported100. The amplification program of the thermal cycler included an initial denaturation step at 95 °C for 10 min, followed by 40 cycles consisting of; denaturation at 95 °C for 15 s, annealing at 60 °C for 30 s and extension at 72 °C for 30 s. After that, the melting curves were obtained to eliminate the inclusion of non-specific products. The relative expression level of the target gene was accurately quantified and calculated according to 2-ΔΔCT algorithm101.Different primer sets specific for polyphenolic-related genes were synthesized according to previous studies Table . The hou-α Table  was usedAMV-CP (Table −1 of each primer (forward and reverse), 10 μL of 2 × SYBR Green PCR Master Mix and 7 μL of nuclease-free water. The qRT-PCR reaction was performed using a Rotor-Gene 6000 in two steps. The first step at 95 °C for 10 min as initial denaturation step and the second step cycle consisting of 40 cycles . Each biological sample was run in triplicate to guarantee data reproducibility. The relative accumulation level of AMV-CP was calculated as according to described above.By using a specific primer of CP Table , qRT-PCRIn AMV infectivity experiments, plants from each treatment were carefully uprooted, washed under running water, and assessed for the fresh weight (g), dry weight (g), tuber weight (g) and tuber number/each plant. After drying plant samples in an oven at 80 °C for 72 h, dry weights were determined and calculated in all treatments  at 70 dpi.102. Separation was achieved using a mobile phase consisting of mixture of solvent A (HPLC grade water containing 0.2% phosphoric acid), solvent B (acetonitrile) and solvent C (methanol). The injection volume was 20 μL while VWD detector was set at 284 nm. The standard polyphenolic compounds used were Quinol, Gallic acid, Catechol, p-Hydroxy benzoic acid, Chlorogenic acid, Vanillic acid, Caffeic acid, Syringic acid, p-Coumaric acid, Benzoic acid, Ferulic acid, Ellagic acid, o-Coumaric acid, Resvertol, Cinnamic acid, Rosemarinic, Catechin, Rutin, Myricetin, Quercetin, Naringenin and Kaempferol.Potato leaves of all treatments were collected, air-dried, and milled. About 2 g of the leaves were macerated and extracted with 15 mL of 96% ethanol for 5 h in a shaking water bath at 35 °C. After filtration and centrifugation, the cleared supernatant was transferred to another tube and concentrated by evaporation at a temperature below 35 °C. The ethanol extract was stored in a brown vial prior to HPLC analyses. An Agilent 1260 Infinity HPLC series, equipped with a Quaternary pump and a Zorbax Eclipse plus C18 column (100 mm × 4.6 mm i.d.) , operated at 30 °C, was used to identify the phenolic and flavonoid-type compounds according to Al-Huqail et al.103. The analyses were run on a GC-MS system  and the test carried out at the Marine Pollution Lab of National Institute of Oceanography and Fisheries, Alexandria, Egypt. The mass detector used in split mode and helium gas with a flow rate of 1 ml/min was used as a carrier. The injector was operated at 250 °C and oven temperature for initial setup was at 60 °C for 2 min, scan time 0.2 s; mass range 50–650 amu and ramp 4/min to 250 °C for 20 min. Mass spectra were taken at 70 eV, during the running time 53 min. The constituents were identified after comparing them with available data in the GC-MS library in the literature.To identify active components of bacterial culture filtrate, a 48 h bacterial culture broth was precipitated, and the supernatant was collected and mixed with ethyl acetate, as a solvent, in the ratio of 1:1 (v/v). The mixture was shaken vigorously for 20 min, and by using separating funnel, the ethyl acetate phase was separated from the aqueous phase. Ethyl acetate extract was concentrated by evaporation at 50 °C in a rotary evaporator. The residue which contained the secondary metabolites and chemical compounds was analysed using gas chromatography-mass spectroscopy (GC-MS)P ≤ 0.05 level of probability, and standard deviation (± SD) is shown as a column bar. Compared to mocked-inoculated potato tissues, the relative expression values higher than 1 was demonstrated an increase in the gene accumulation (up-regulation), while values lower than 1 means a decrease in expression levels (down-regulation).The relative expression values of three replicates for each set were analysed by one-way ANOVA using the CoStat software. The significant differences of the relative expression levels were determined according to the least significant differences (LSD)