ASCL1, NEUROD1, DLL3, NOTCH1, INSM1, MYCL1, POU2F3 and YAP1 are correlated with specific clinical and pathological characteristics. We selected a retrospective series of 46 primary pediatric neuroblastoma, composed of 30 treatment-naïve and 16 post-chemotherapy cases. Gene expression levels were explored by means of quantitative real-time PCR. An increased expression of NOTCH1 (p = 0.005), NEUROD1 (p = 0.0059), and YAP1 (p = 0.0008) was found in stage IV tumors, while the highest levels of MYCL1 and ASCL1 were seen in stages IVS and III, respectively (p = 0.0182 and p = 0.0134). A higher level of NOTCH1 (p = 0.0079) and YAP1 (p = 0.0026) was found in cases with differentiating morphology, while high mitosis-karyorrhexis index cases demonstrated significantly lower levels of POU2F3 (p = 0.0277). High expression of NOTCH1 (p = 0.008), NEUROD1 (p = 0.026), INSM1 (p = 0.010), and YAP1 (p = 0.005) together with stage IV (p = 0.043) was associated with shorter disease-free survival. In summary, our data indicate that the assessment of gene expression levels of neuroendocrine-lineage transcription factors might help to identify neuroblastoma patients with the risk of relapse.Pediatric neuroblastoma is responsible for approximately 8–10% of pediatric tumors, and it is one of the leading causes of tumor-related deaths in children. Although significant progress has been made in the characterization of neuroblastoma in recent years, the mechanisms influencing the prognosis of neuroblastoma patients remain largely unknown. Our aim was to investigate if the major neuroendocrine-associated transcriptional drivers, including The online version contains supplementary material available at 10.1007/s00428-022-03406-4. MYCN represents one of the most important driver genes in neuroblastoma, being gene amplification strongly correlated to unfavorable outcome, although in vitro and in vivo data suggest that there is no direct correlation between a high cellular MYCN protein content and aggressive tumor cell behavior or loss of differentiation [ASCL1, NEUROD1, HES1, INSM1, POU2F3, YAP1, MYCL1 [Neuroblastic tumors represent the most common extra-cranial solid malignancy in the pediatric age. Neuroblastoma is a tumor arising from developing sympathetic nervous system and is responsible for approximately 8–10% of pediatric tumors. Despite advances in molecular profiling and therapeutic options, survival of high-risk neuroblastoma patients remains poor. Neuroblastoma development mechanisms are incompletely understood, but linked to oncogene mutations and/or amplifications . In partntiation . Moreoventiation –8. In pa1, MYCL1 –12. In l1, MYCL1 –16. Some1, MYCL1 . In addi1, MYCL1 –20.. In particular MYCN gene is pivotal in neuroblastoma development and progression [NEUROD1 and ASCL1 genes [PCP4/PEP19 [ERK [NEUROD1 seems to act mainly through ALK to favor neuroblastoma cell proliferation, directly binding to the promoter region of this gene [ASCL1 is downregulated during neuroblastoma cell differentiation along with upregulation of several genes including IGF2 [ASCL1 expression with neuronal differentiation is independent from MYCN gene expression, suggesting that targeting ASCL1 might increase the efficacy of retinoic acid-based differentiating therapies in neuroblastoma [INSM1 that is activated by MYCN gene, expressed in a large fraction of neuroblastomas and associated to a shorter survival [Some genes belonging to the abovementioned families have been individually investigated in neuroblastoma and found to be overexpressed and involved in response to specific therapiesgression –4. CentrL1 genes . TranscrP4/PEP19 , and ERKP19 [ERK . NEUROD1his gene . ASCL1 iing IGF2 ; moreoveblastoma . More resurvival –30.Neuroblastic tumors encompass a spectrum of lesions with different pathological features, response to therapies and outcome. We studied the role of major drivers of neuroendocrine-associated transcriptional clustering in a retrospective series of surgically resected pediatric neuroblastomas, with the aim of exploring the correlation with specific pathological and clinical characteristics including response to treatment and survival.We selected a retrospective series of 46 cases of pediatric neuroblastoma, all primitive, operated from 2007 to 2019 at “Città della Salute e della Scienza,” Turin, and treated at the Pediatric Oncology Division of the “Regina Margherita” Children’s Hospital, with sufficient residual histological material for molecular and immunohistochemical analyses. The case series was composed 30 treatment-naïve and 16 post-chemotherapy cases. The chemotherapy consisted of combination treatment, including carboplatin and etoposide.Clinical and pathological data such as age at diagnosis, sex, tumor location, stage  stained slides. The suitability of the material was evaluated by hematoxylin and eosin staining, and care was taken to select tumor areas. Total RNA isolation was performed by commercially available RNA extraction kits designed for paraffin material according to the manufacturer’s instructions .ASCL1 (HS00269932_m1), DLL3 (HS01085096_m1), INSM1 (Hs00357871_s1), MYCL1 (Hs00420495_m1), NEUROD1 (HS01922995_s1), NOTCH1 (Hs01062014_m1), POU2F3 (Hs00205009_m1), YAP1 (Hs00902712_g1), ACTB (Hs01060665_g1) assay served as housekeeping reference gene for the analyses.RT reactions were performed using 10 ng total RNA in a volume of 15 μl with the following conditions: 16 °C for 30 min, 42 °C for 30 min, 85 °C for 5 min, and 4 °C for 5 min. Expression levels of all genes studied and internal reference were examined using a fluorescence-based real-time detection method . The following TaqMan gene expression assays (Applied Biosystems) were used according to the manufacturer’s instructions: Each measurement was performed in duplicate. The ΔΔCt values were calculated subtracting ΔCt values of sample and ΔCt value of Stratagene , and converted to ratio by the following formula: 2 − ΔΔCt.t test, Pearson’s chi-square test and Bonferroni’s correction, Wilcoxon’s rank test).Statistical analyses were carried out using the Stata 15.0 software . The differences in the distribution of the variables evaluated based on clinical-pathological parameters were analyzed using parametric and non-parametric tests  was assessed from the date of diagnosis to the date of relapse or the date of the last checkup. All dead patients were considered as events. Survival analysis was determined by the Kaplan–Meier curves and Mantel log-rank test was used to compare statistical differences. Cox regression analyses were carried out on DFI to calculate HRs and 95% CIs for the different study groups. All statistical tests were two sided. The clinical and pathological features of 46 neuroblastoma cases are summarized in Supplementary Table NOTCH1 and ASCL1 , NEUROD1 , INSM1 , and YAP1 . Moreover, ASCL1 was found significantly correlated with DLL3 , NEUROD1  and INSM1 . Furthermore, NEUROD1 was significantly correlated with MYCL1  and YAP1 , while INSM1 resulted significantly correlated with YAP1 (p = 0.0257).A strong reciprocal positive correlation was observed across the series between NOTCH1, NEUROD1, MYCL1, and YAP1 were found significantly correlated with tumor stage. In detail, NOTCH1 (p = 0.005), NEUROD1 (p = 0.0059), and YAP1 (p = 0.0008) were more expressed in stage IV tumors, while highest levels of MYCL1 and ASCL1 were seen in stages IVS and III, respectively (p = 0.0182 and p = 0.0134). Moreover, a higher level of both NOTCH1 (p = 0.0079) and YAP1 (p = 0.0026) was found in cases with differentiating morphology according to INPC classification. Finally, cases with high MKI according to Shimada demonstrated significantly lower levels of POU2F3 (p = 0.0277).As to concern clinical and pathological correlations , INSM1 (p = 0.0016), and DLL3 (p = 0.0064) in cases that relapsed during the follow-up  of up Table .Fig. 1GeINSM1 were significantly correlated with shorter disease-free interval (DFI) in the whole group , as well as when separately analyzed in the treatment-naïve  and the post-chemotherapy groups of patients . Moreover, the patients with higher levels of NOTCH1 had a shorter DFI, both in the whole cohort  and in the 30 treatment naïve patients , . In addition, considering the whole case series, patients with high expression of YAP1 and NEUROD1 had shorter DFI, compared to those with low expression . However, no significance was observed when patients were stratified according to the chemotherapy status (data not shown).As to concern univariate survival analyses, higher levels of p = 0.043) and high expression of NOTCH1 , NEUROD1 , INSM1 , and YAP1  were associated with adverse prognosis. Moreover, poorly differentiated forms  of disease were significantly associated to better DFI survival compared to undifferentiating lesions.Furthermore, Cox regression analyses to estimate HRs and 95% CIs for DFI in the different study groups shown in Table INSM1, NOTCH1, NEUROD1, and YAP1 gene expression levels according to the recurrent disease status (Supplementary Table p = 0.003).Considering the different combinations of OR = 8.75, 95% CI = 2.19–34.81, p = 0.002). However, when stratified according to the treatment status, no significance was observed the treatment naïve group  and post-chemotherapy group of patients  analyzed separately  with MYCN amplification status, and this limitation should be considered in future studies. Interestingly, in a translational view, the demonstration of NOTCH1 expression in neuroblastoma and its positive correlation with aggressiveness is paralleled by in vitro data on the efficacy of Notch1 inhibition in neuroblastoma cells. In a study on a variety of neuroblastoma cell lines, gamma-secretase inhibition (in particular GSI-I) was shown to impair cell proliferation and to promote apoptosis in vitro and in vivo through targeting Notch signaling [NOTCH1 overexpression in the adverse clinical behavior of neuroblastoma patients in our series is to be further validated in a biological perspective. However, among the possible mechanisms, Notch1 has been shown to actively maintain a stem cell phenotype in neuroblastoma cells that confer highly tumorigenic properties [The association of lastomas . At variignaling . Moreoveignaling . The impoperties .INSM1 transcription factor has emerged in vitro as a neuroblastoma biomarker that plays critical role in facilitating tumor cell growth and transformation [INSM1 gene expression levels from the present study.ormation . Its proormation . No defiormation , in lineNEUROD1 was already shown to promote cell growth in vitro [Kip1 activity in neuroblastoma cell lines SH-SY5Y and SK-N-SH [Among other markers associated with neuroblastoma aggressiveness in our series, in vitro  and tumoin vitro  in neuro SK-N-SH . Moreove SK-N-SH  and MEK- SK-N-SH  therapeuNOTCH1 and YAP1 expression was significantly higher in differentiating compared to poorly differentiated tumors according to the INPC classification. This observation cannot find a clear explanation in the current literature. A previous in vitro study is partly in contrast with our findings and shows that Notch1 inhibition prevents neurite formation in neuroblastoma cells [In terms of correlation with differentiation, ma cells . MoreoveThe potential clinical impact of our findings is twofold. First, the integration of gene expression profiles of the biomarkers investigated in the present study might assist to better predict the clinical behavior of neuroblastoma patients, and to improve a personalized approach in tailoring adjuvant chemotherapeutic regimens and/or predict response to treatment. Second, the pathways regulated by the genes herein investigated are potential targets for selective therapies. In particular, DLL3 has been tested as a target for rovalpituzumab tesirine therapy in neuroblastoma models with promising results .NOTCH1, INSM1, YAP1, and NEUROD1 might help to further characterize the risk of relapse in neuroblastoma patients.In conclusion, we identify a strong prognostic impact of neuroendocrine-lineage transcriptional profiles in neuroblastoma, and suggest that the evaluation of Supplementary file 1(DOCX 26 kb)Supplementary file 2(PNG 469 kb)High resolution image (TIF 19566 kb)Below is the link to the electronic supplementary material.


CO adsorption anddissociation on “perfect” and “defect-rich”Ir(111) surfaces were studied by a combination of surface-analyticaltechniques, including polarization-dependent (PPP and SSP) sum frequencygeneration (SFG) vibrational spectroscopy, low-energy electron diffraction(LEED), Auger electron spectroscopy, X-ray photoelectron spectroscopy(XPS), and density functional theory (DFT) calculations. CO was foundto be ordered and tilted from the surface normal at high coverageon the “perfect” surface , whereas it was less ordered and preferentially upright (θ= 4–10°) on the “defect-rich” surface forcoverages of 0.55–0.70 ML. SFG, LEED, and XPS revealed thatCO adsorption at low pressure/high temperature and high pressure/lowtemperature was reversible. In contrast, upon heating to ∼600K in near mbar CO pressure, “perfect” and even more“defect-rich” Ir(111) surfaces were irreversibly modifiedby carbon deposits, which, according to DFT, result from CO disproportionation. Whereas conventional vibrational spectroscopy woulddetect only minor frequency shifts, polarization-dependent (PD-) SFGenables us to directly monitor the effect of defects on the moleculararrangement and orientation of the CO overlayer. SFG spectroscopywas carried out at a “near ambient pressure” of 1 mbarand at temperatures up to 600 K, which induced irreversible surfacechanges, likely originating from CO dissociation. Carbon depositswere detected by AES, and the reaction pathway via CO disproportionationwas examined by DFT.22.1eff(2) ≠ 0at an anisotropic surface/interface. Polarization-dependent SFG spectraof adsorbed molecules are usually taken with PPP and SSP polarizations,which allows a quantitative analysis of molecular orientation.39 The SFG spectra can be fitted by Lorentzian lineshapes:NR(2) is the magnitudeof the non-resonant susceptibility generated by the substrate. χq, ωq, and Γq represent theresonance amplitude, frequency, and damping constant of the qth vibrational mode, respectively. When ωIR is close or equal to ωq, the SFG intensity  is enhanced and a vibrational peak appearsin the SFG spectrum. The interfacial molecular orientations can bedetermined in an SFG experiment because the measured χeff(2) is relatedto the macroscopic second-order susceptibility in the laboratory coordinates(χijk(2)) bye(ωi) refers to the unit electricfield vectorand L(ωi) is the Fresnel factor determined by the laser incidence andrefraction angles, polarizations, and refractive indices.40 Furthermore, χijk(2) is related to the microscopic hyperpolarizabilitytensor elements βi ′ j ′ k′(2) in the molecular coordinate system throughEuler transformation ⟨Rii′Rjj′Rkk′⟩by41Ns is the effective molecular surface numberdensity per unit.For CO with C∞v symmetry, the moleculeshave a random azimuthal distribution, and the surface CO orientation can be determined by measuring IPPP/ISSP for a known molecular hyperpolarizabilityratio R /βccc(2) = βbbc(2)/βccc(2)), assuminga δ-function for the orientation distribution.42IR–visible SFGis a second-order surface-specific processas the effective second-order nonlinear susceptibility χ2.243All LEED/AESand SFG experiments were carried out in a custom-designed ultrahigh-vacuum(UHV) preparation/analysis chamber coupled to a UHV-to-atmosphericpressure-compatible SFG spectroscopic cell. The experimental setuphas been described in detail previously.2.2.1–10 mbar by a turbomolecular drag pumpPfeiffer TMU, monitored by a hot-cathode gauge . The chamber is further equipped witha four-grid retractable LEED/AES optics (SPECS ErLEED DN 150 CF) anda 3000D controller with a thoria (ThO2)-coated Ir filamentthat allows for a maximum of 10–6 mbar operationpressure. LEED patterns are recorded using a CMOS-sensor camera. TheErLEED 3000D power supply provides all necessary voltages to operatea LEED optics as a retarding field analyzer (RFA) for AES. For recordingAES data, an integrated lock-in amplifier and RFC-PC software areused.The UHV chamber is a stainless-steelvessel of about 40 L, which is pumped to a routine base pressure of5 × 10+ ions , oxidation  at 800 K, and UHV annealing at 1050 K for 30min, as described in refs .For scanning tunneling microscopy (STM) images of a sputtered singlecrystal surface with three-dimensional islands exhibiting a very highdensity of steps and edges, one should refer to ref (47), with the CO overlayer structures characterized by LEED.The Ir(111) single crystal was disk-shaped, with 8 mmdiameter and 2 mm thickness. For a smooth/“perfect”surface, Ir(111) was pretreated by repeated cycles of sputtering withAr in refs , 44, 45. in refs . LEED anr to ref . CO of p2.2.2–8 mbar to 1 bar pressure and at 100–800 K. SFG measurementswere performed using a 20 ps mode-locked Nd:YAG laser system  with a fundamental radiation of 1064 nm . A tunable mid-infrared beam (with the photon energyωIR) and a visible beam with a fixed wavelength of532 nm were directed in a co-propagation geometry toward the Ir(111)surface . The polarization of IR was keptas P and that of visible and SFG signal was switched between P andS using a Glan–Taylor prism and a half-wave plate. All spectrawere normalized by the energy of visible and IR laser pulses and fittedusing Lorentzian lineshapes (The freshly pretreated Ir(111) can be directly transferred fromthe UHV chamber to the SFG cell under UHV, avoiding contaminations.The SFG cell can be operated from 2.5 × 10see refs , 42, 43)neshapes 1.2.3–10 mbar).A SPECS XR50 high-intensity nonmonochromatic Al/Mg dual-anode X-raysource and a Phoibos 100 hemispherical energy analyzer (EA) with amultichannel plate detector were used for XPS, as described in ref (α radiation (1486.61 eV) was used for the acquisition of XPS spectra.In this chamber, the sample preparation and surface order analysisby LEED followed the same procedure as described above.XPS experiments were carried out in another stainless-steel UHVchamber method as implemented in our WIEN2k code.50 The calculations of the present work used the generalized gradientapproximation by Perdew, Burke, and Ernzerhof (PBE).52 We used a plane wave cutoff parameter RMTKmax = 5 for all calculations with aC atom and a properly scaled RMTKmax for the pure Ir surface, where RMT refers to the smallest atomic sphere radius . The final resultswere checked with RMTKmax = 6. A 4 × 4 × 1 (checked with 8 ×8 × 1) k-mesh was used, and the self-consistent field calculationsand the atomic positions were fully relaxed until the forces weresmaller than 1 mRy/bohr.The disproportionationof two CO molecules on the Ir(111) surface into CO33.153) demonstrated a cleansurface. Unfortunately, the XPS C 1s analysis of C species on cleanIr(111) was somewhat hindered by overlapping satellite features ofthe Ir 4d peak due to the use of a nonmonochromatized X-ray source.15 As shown in α3 and Kα4 satellites of Ir 4d5/2 (at a binding energy of 296.3 eV) overlappedwith the C 1s region. Still, significant amounts of carbon can beexcluded.Before CO adsorption, an ordered andclean Ir(111) was confirmed by LEED, AES, and XPS. As shown in 3.219 However, in the (>1.0)mbar regime, a more complex 54 Our observations are consistent with these results.Previous LEED studies of CO on Ir(111) reportedthat S1, on the clean Ir(111) surface, a (1 ×1) LEED pattern formed, whereas upon CO chemisorption at 300 K andat 5 × 10–8 mbar pressure, –7 mbar), a 54 Further increasing the CO pressure to 1.0 mbar maintained the As shownin 3.33.3.1–7 to 1 mbar, both for PPP and SSP polarizationcombinations. A previous combined IRAS/TPD study of CO/Ir(111) deduceda relationship between the IR peak position and the CO coverage,20 which is utilized herein to convert SFG peakpositions to coverages for both surfaces as the differences in peakpositions are small. Still, the coverages on the “defect-rich”surface may be slightly underestimated. In the 10–6 mbar range, the observed peak positions are well in line with IRASstudies of CO on Ir(111) and graphene-supported Ir clusters.24–7 to 1.0mbar, the CO vibrational frequency moved from 2084 to 2094 cm–1 (coverage 0.62 to 0.77 ML) and from 2079 to 2090cm–1 (coverage 0.56 to 0.70 ML) on “perfect”, representing CO adsorbed on defects ,60 was requiredto fit the experimental spectra . Accordingly, the full width at half-maximum (FWHM = 2Γ)of the on-top CO peak was 4 cm–1 larger (∼12vs ∼8 cm–1) as the CO layer was less homogeneouson “defect-rich” surfaces.A single peak, characteristicof on-top CO, was observed on bothsurfaces. As pressure increased from 10perfect”3a,b and perfect”3c,d,resIPPP and ISSP) exhibiteddifferent trends with increasing coverage/pressure: for CO on “perfect”Ir(111), IPPP decreased, but ISSP increased, whereas for CO on “defect-rich”Ir(111), both IPPP and ISSP hardly changed but overall decreased. Above 10–3 mbar, both vibrational frequency and intensity changedmoderately because saturation was nearly reached. The fitting resultsof CO on “perfect” and “defect-rich” Ir(111)can be found in ref  indicate an increasingtilt angle (θ), with θ increasing from 25° to 36°as the coverage increased from 0.62 to 0.77 ML.25 As CO tilted closer to the surface ,it yielded a relatively strong SSP signal  but caused a drop in the PPP signal. For CO on “defect”Ir(111), SSP shows a poor signal-to-noise ratio and is mostly dueto the non-resonant background, whereas PPP was strong. Overall, thisindicates a small θ so that CO is upright on defective Ir(111). IPPP on the defective surface was stronger thanon the perfect surface, likely due to a combined effect of the COtilt angle, order, and coverage.Ourrecent work about CO on smooth Ir(111) had pointed out thata decreasing ing ISSP 3a,b,f , whichpoints to a smaller coverage change on the “defect-rich”surface that binds CO stronger. The fitting results of CO on the “perfect”and “defect-rich” Ir(111) can be found in ref  the CO coverage by increasing the surface temperaturefrom 300 to 500 K in a constant background of 1 mbar CO 4. As thed in ref  and TablIPPP first increased and then decreased,while ISSP decreased gradually. The COtilt angle decreased from 36° to 20° when the coverage decreasedfrom 0.77 to 0.44 ML,25 so the increaseof IPPP was mainly attributed to a decreasingθ, and the decreasing ISSP was dueto the decreasing coverage and decreasing θ. In contrast, forCO on “defect-rich” Ir(111), PPP obviously decreased,whereas SSP changed only slightly. Similar to IPPP was largerand ISSP was smaller, once more confirminga small tilt angle θ on the defective surface.As previously reportedin detail for CO/“perfect”Ir(111), upon temperature increase, PPP and SSP changed oppositely4a,b,f; t3.3.3IPPP slightly decreased and ISSP obviously increased. In contrast, for the defective Ir surface, IPPP was distinctively larger and ISSP was moderately larger at higher coverage 25 at 0.13 ML  with IPPP/ISSP = 520 asthe reference, R was determined to be 0.08. Accordingly,for CO/“perfect” Ir(111),25IPPP/ISSP of 150 and 65 at 0.44 and 0.70 ML coverage . Indeed, DFT calculationsof the potential energy surface as a function of tilt angle for twoCO molecules in nearest-neighbor positions in a 3 × 3 Ir(111)supercell had indicated that the energy increased dramatically whenCO molecules came closer.25 Only when theCO molecules bent to the same direction (“concerted tilting”),the curve became flat.As mentioned, the CO tilt angle can be deduced from coverage 5a,b indicoverage 6a. IndeeIPPP/ISSP was 820 at 0.70 MLcoverage .25 With R decreased to 0.07 and0.06, the maximum of IPPP/ISSP increased to 670 and 900, respectively. Therefore,using R = 0.06 for 0.70 ML CO on the defective Ir(111),θ was determined to be 4° . Table S1 summarizes all tilt angles at differentcoverages for CO on “defect-rich” Ir(111) (based onthe pressure- and temperature-dependent spectra in R =0.06) (For CO on “defect-rich” Ir(111), coverage 5c,d,andto be 4° 6b. For 0 =0.06) 6a.Summarizing the adsorption studies of CO on “defect-rich”Ir(111), the CO tilt angle was found to be small (4–10°)with only weak coverage depencence. The SFG spectral changes weremainly due to coverage changes. On the defective rough surface, theCO molecules seem to form a less-ordered overlayer and neighboringCO molecules are frequently located in different planes. This reducesthe dipole–dipole repulsion so that “concerted tilting”is not induced and θ remains small. In summary, the surfaceroughness removed the strong coverage dependence of the CO tilt angle,observed on smooth Ir(111), so that on a rough surface CO was overallquite upright.3.428 This agrees with an ultraviolet photoelectron spectroscopy (UPS)study of Ir(111) in 10–8 Torr CO, which demonstratedthat CO does not dissociate at an appreciable rate at 533 K.61 However, it was reported that heating Ir(111)to ≥650 K in ≥10–6 mbar CO19 or to ≥773 K in ≥10–8 mbar CO18 may result in significant dissociation.Therefore, in the following, a possible CO dissociation is examinedfor three different combinations of pressure and temperature by varioussurface-sensitive techniques: (i) XPS/LEED at low pressure, high temperature;(ii) SFG at high pressure, low temperature; and (iii) SFG/AES (post-reaction)at high pressure, high temperature.DFT calculations have shown thatCO cannot dissociate on Ir(111)due to a high effective barrier of 3.17 eV.3.4.1–6 mbar CO for 3600 s) at580 K  were acquired before and afterdosing 3600 L of CO  and the temperature to 890 K  the spectral intensities were at least three times smaller(Table S2), and (iii) the spectral lineshapesbecame (more) asymmetric. All features indicate a strong modificationof the Ir(111) surface, once reaching 575 K in 1.3 × 10–2 mbar CO. Strong restructuring of Ir(111) and/or CO dissociationmay be responsible for the observed effect. Similar results were alsofound in an SFG study of CO adsorption and dissociation on Pt(111).62 When Pt(111) was heated in 400 Torr CO to 823 K and then cooledto room temperature, an apparent hysteresis in CO vibrational frequencyand a decrease in intensity were observed due to carbon formationat high temperature. The AES spectra of the Pt(111) surface afterexposure to 400 Torr of CO at 673 K showed a notable carbon peak,also indicating CO dissociation on Pt(111) at this temperature.63 For Pt thin films and nanoparticles in 10 mbar CO, dissociationwas observed by SFG and near ambient pressure (NAP-)XPS upon heatingto 550 K.64 On smooth and sputtered Pd(111),no indications of CO dissociation were observed even after hours in0.1 mbar CO, likely due to the temperature limit of 400 K.67 It is well documented that carbon perturbs the electronic and geometricstructure of stepped surfaces, which leads to poisoning.6925 the expected increase in PPP intensity due toincreasing coverage seems compensated by the loss of intensity dueto an increasing tilt angle. Unfortunately, the SSP signals were tooweak for detection after heating because even before heating ISSP was >35 times smaller than IPPP(IPPP decreased three times).Upon cool-down 10b, the –1 and the lineshapes changed to asymmetric. Note that the intensitieswere reduced even ∼20 times. Apparently, although similar intendency, the changes were much stronger for the defect-rich surface,which may result from the roughness and/or the higher CO pressure(this will be further discussed below).The spectralreversibility was also investigated for the “defect-rich”Ir(111) surface. –8 mbar18 or at ≥650 K at 1.33 × 10–6 mbar.19 Accordingly, at 10–2 to 1.0mbar CO used herein, dissociation may occur at comparably lower temperature.CO may also dissociate on Ir adatoms or clusters mobilized by CO athigh pressure, that is, CO-induced surface roughening, as observedfor CO on Cu(100)70 or Pt nanoparticles71 at mbar CO pressure even around room temperature.For CO/Ir(111), previousAES studies reported significant CO dissociationat >773 K at 1028 but the barriers of CO disproportionationvia the Boudouard reaction 2CO ↔ CO2 + C on low-coordinatedsites have been shown to be much lower .CO dissociation or disproportionation may thus be responsible forthe irreversible changes that only occurred when heated higher than570 K in (near) mbar CO pressure. The activation barriers of directCO dissociation on noble metals were computed by DFT to be ratherhigh,3.4.4–1 (red) was red-shifted to 2068 cm–1 (blue), accompaniedby a 3.5-fold intensity loss. To confirm that carbon was formed onthe Ir(111) surface at 625 K, an oxidation experiment was then performed.As expected, after oxidation at 450 K in 5 × 10–3 mbar O2, not only the peak position moved to higher wavenumbers(2071 vs 2068 cm–1), but also the spectral intensityincreased (green vs blue) because carbon was  removed byoxidation. This “partly reversible” spectrum suggeststhat CO dissociation did occur at 625 K in 0.013 mbar of CO. Analogousto defect-rich Ir(111) (As seen in  Ir(111) 10c,d, a  Ir(111) 11b,c.19 while the dissociatedoxygen is removed from the surface via reaction with gaseous CO. Accordingly,a LEED pattern was measured after dosing 1.0 mbar CO at 300 K andannealing at 700 K for 60 min in the high-pressure cell. As seen in 75 or ex situ,70 before/afterhigh-pressure CO exposure is planned for the future.Carbon deposits increasethe LEED background intensity and canbe detected by AES,3.52 + C.50 The C and CO adsorption as well as the disproportionation of COwas modeled by 2 × 2 supercells of Ir(111)  with five layers ofIr (28 a preference for CO adsorption on top sitesand on hcp sites for C atoms was found (25CO dissociationon Ir(111), still being rather unexpected, was furtherinvestigated computationally. DFT was used to study the disproportionationon Ir(111): 2CO ↔ COrs ofIr 12. Ingoas found 1. The C x and y coordinates of CO are fixed, but z of all atoms can relax. In addition, the C–OCOdistance (see 2) have unfavorable energiesfor the reaction in free space and on the Ir-top position (since Con top of Ir has the lowest C adsorption energy), whereas they areslightly favored for hcp/fcc sites since C is very strongly adsorbedthere.In order to determinethe dissociation barrier for CO, we put asecond CO molecule on top but far away from the adsorbed CO /high temperature (890 K) or high pressure(1.0 mbar)/low temperature (500 K). However, upon heat-up (300 to∼600 K) and cool-down (∼600 to 300 K) in a backgroundof ∼1 mbar CO, the obtained irreversible SFG spectra impliedthat CO dissociated on smooth and especially defective Ir(111), yieldingcarbon deposits. SFG spectra upon carbon oxidation and AES spectraof Ir(111) after annealing in 1.0 mbar of CO at 700 K indirectly anddirectly confirmed the formation of surface carbon species, respectively.DFT calculations suggested that at high pressure, CO adsorbed on hcp/fccsites can react with gas-phase CO via disproportionation, formingCO2 and leaving a C atom behind on the Ir surface.We have used surface-sensitivePD-SFG, LEED/AES, XPS, and DFT calculationsto study CO adsorption and dissociation/disproportionation on bothsmooth and defect-rich Ir(111) surfaces. PD-SFG showed that, in contrastto the strong coverage dependence of the CO tilt angle on smooth Ir(111), on the defect-rich surface,CO preferred standing upright at high coverage (4° at 0.70 ML).When the coverage ranged from 0.67 to 0.55 ML on defective surfaces,the CO tilt angle remained constant at ∼10° but stillrather small. CO forms three different overlayer structures as observedby LEED:


Panax Ginseng and Ginkgo biloba extracts (CGGE) is an effective treatment for nervous system diseases, but the neuroprotective mechanism underlying CGGE remains unclear. Both network analysis and experimental research were employed to explore the potential mechanism of CGGE in treating ischemic stroke (IS). Network analysis identified a total number of 133 potential targets for 34 active ingredients and 239 IS-related targets. What’s more, several processes that might involve the regulation of CGGE against IS were identified, including long-term potentiation, cAMP signaling pathway, neurotrophin signaling pathway, and Nod-like receptor signaling pathway. Our studies in animal models suggested that CGGE could reduce inflammatory response by inhibiting the activity of Nod-like receptor, pyrin containing 3 (NLRP3) inflammasome, and maintain the balance of glutamate (Glu)/gamma-aminobutyric acid (GABA) via activating calmodulin-dependent protein kinase type Ⅳ (CAMK4)/cyclic AMP-responsive element-binding protein (CREB) pathway. These findings indicated the neuroprotective effects of CGGE, possibly improving neuroinflammation and excitotoxicity by regulating the NLRP3 inflammasome and CAMK4/CREB pathway.Stroke is a major cause of death and disability throughout the world. A combination of  Stroke is one of the leading causes of mortality and disability, especially in low-income and middle-income countries . Accordi2+ influx. The increased intracellular Ca2+ causes mitochondrial dysfunction and overproduction of reactive oxygen species (ROS), which activates inflammatory responses and results in the death of damaged neurons  and CREB (The underlying mechanism of ischemic stroke (IS) is complex. Excitotoxicity and inflammation play crucial roles in the pathophysiological process of IS. Overactivation of the glutamate receptor mediates excitotoxicity, which has been suggested as a critical event in ischemic brain injury. Glutamate (Glu) is a major excitatory neurotransmitter in the central nervous system and acts on metabotropic and ionotropic receptors  to mediate excitatory synapse transmission . While g neurons . NMDAR a neurons . Tat-NR2and CREB . Nod-likand CREB . There iand CREB . The NLRand CREB .Panax Ginseng (P.ginseng C.A.Mey.) is a kind of valuable traditional Chinese medicine (TCM) with a history of thousands of years. The neuroprotective effects of panax ginseng on neurodegeneration diseases have been widely reported in the past decades, including cerebral ischemia, Alzheimer’s disease, and Parkinson’s disease  is a natural medicine widely used to treat neurodegenerative diseases. The neuroprotective effects of ginkgo biloba on ischemic brain injury are mainly through the regulation of excitotoxicity, inflammatory pathways and oxidative damage  showed beneficially neuroprotective effects against neuronal apoptosis , which is an effective treatment for vascular dementia  and G. biloba (NO.090914) were provided by the Shineway Pharmaceutical Group Co. Ltd. . Panax ginseng extract was prepared by adding eight times water to the powder of panax ginseng roots and rhizomes, extracting with boiling water for 3 h, then drying with a vacuum rotary evaporator at 80°C (50 g extract from 1 kg P. ginseng). Ultraviolet (UV) spectroscopy or high-performance liquid chromatography (HPLC)-UV method was used to determine the extract’s total active components and main ingredients to control the quality. The quantitative analysis showed that the total ginsenoside content was 77% in ginseng extract, among which ginsenoside Rb1, Rg1, and Re are13.1% (6.55 mg/g), 5.5% (2.75 mg/g), and 3.2% (1.6 mg/g), respectively . In the extract of ginkgo biloba, total flavonoids accounted for 49%, quercetin, kaempferol, and isorhamnetin occupied 28.7% (14.35 mg/g), ginkgolide A, B, C and bilobalide accounted for 11.6% (5.8 mg/g), and ginkgolide A was 3.3% (1.65 mg/g) (Extracts of ectively . Ginkgo 65 mg/g) . Both inAdult male SD (Sprague-Dawley) rats (200–220 g) were provided by the Beijing Vital River Laboratory Animal Technology Co., Ltd. (License No. SCXK (Beijing) 2016-0011). All rats were housed at 25 ± 1°C temperature and 65% ± 5% humidity, with a 12 h light/dark cycle (lights on from 7:00 to 19:00). All rats had free access to food and water. The Ethics Review Committee for Animal Experimentation of Xiyuan Hospital, China Academy of Chinese Medical Sciences, approved the experimental protocols described in this study.n = 10): Sham group, MCE group, Tat-NR2B9C group (7.5 mg/kg), CGGE low group (15 mg/kg), and CGGE high group (30 mg/kg). The MCE rat model was established according to a previously described method  was used to identify and screen the active compounds of CGGE with a restriction of oral bioavailability (OB) ≥ 30% and drug-likeness (DL) ≥ 0.18 (https://www.uniprot.org/).The active compounds and potential targets of CGGE for treating IS were investigated based on network analysis. The Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform  ≥ 0.18 . The pot) ≥ 0.18  were selhttps://www.ncbi.nlm.nih.gov/geo/, Series: GSE22255) database. Genes with p value <0.05 and |Fold change| > 1.2 were considered to be of significant differential expression and IS-related targets.The differential expressed genes of IS patients were obtained from Gene Expression Omnibus  was used for GO enrichment analysis, and plugin ClueGO of Cytoscape 3.9.0 software was used to perform Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. The biological process, cellular component, and molecular function analysis were selected for the bar charts. Pathways with significant changes of p < 0.05 were identified for further analysis.The Database for Annotation, Visualization, and Integrated Discovery  kits. The results were expressed as the means ± standard deviation.The protein concentration was calculated by using bicinchoninic acid (BCA) assay kit (KGI Biotechnology Co. Ltd. China). Identical quantities of protein samples were denatured by protein loading buffer, separated by sodium dodecyl sulfate(SDS)-polyacrylamide gel electrophoresis, then transferred to PVDF membranes . The PVDF membranes were sealed with 5% bovine serum albumin (BSA) at room temperature for one hour. The following antibodies were incubated at 4°C overnight: anti-CAMK4 , anti-p-CREB , anti-CREB1 , anti-NLRP3 , anti-ASC , anti-CASP1 . The stained blots were detected using an ECL reagent . Each blot was quantified using Bio-Rad Image Lab™ Version 3.0 software. All the experiments reported in this study were performed three times, and the results were reproducible.® SYBR® Green PCR Master Mix  with specific primers and expression of each sample in Light Cycler®480ⅡReal-time PCR Instrument , which internally normalized against Actb. The PCR primers for Camk4, Creb1, and Actb are as follows: Camk4 forward: 5′-CACAGAAATCAGCCTGGTT-3′ and reverse: 5′- ATCTGCTTCACAGCATCGC-3′; Creb1 forward: 5′- TTCTAGTGCCCAGCAACC-3′ and reverse: 5′-GAGGACGCCATAACAACTC-3′; Actb forward: 5′- CCACCATGTACCCAGGCATT-3′ and reverse: 5′- CGGACTCATCGTACTCCTGC-3′. Relative expression fold change was performed by using the 2−△△Ct method. Each experiment was repeated three times.According to the instructions, total RNA was extracted from the brain tissue using Trizol reagent . The concentration and purity of RNA were determined by a NanoDrop 2000 spectrophotometer . The absorbance ratio (A260/280) of all samples ranged from 1.8 to 2.0, and 2 μg of total RNA was reverse transcribed into cDNA using Prime Script RT Master Mix . RT-qPCR was determined using the QuantiFastp < 0.05 was considered statistically significant.The results were expressed as the mean ± standard deviation (SD). The statistical analyses were performed using Statistical Package for the Social Sciences (SPSS) software . One-way analysis of variance (ANOVA) was used to compare all groups’ differences. The infarct volume of rats and neurological scores were determined after the acute treatment of the CGGE. As shown in p < 0.01), while the low dosage of CGGE group could only decrease the scores after 24 h (p < 0.05).Panax ginseng and 27 compounds of Ginkgo biloba were obtained. A total of 34 components were characterized after removing the compounds without targets and repetitions  with screening parameters of OB ≥ 30% and DL ≥ 0.18. Twenty-two compounds of etitions . The totThe network contained 169 nodes and 468 edges which indicated the compound-target interactions. The median degree of 34 chemical components is 9, indicating that compounds could interact with multiple targets. The information on the top 10 chemical components and targets is shown in The reliability of the test results can be significantly improved by directly selecting the human chip results and setting relevant screening conditions for analysis. The GEO database identified two hundred and thirty-nine IS-related targets (Series: GSE22255)..CGGE targets PPI network and IS-related targets PPI network were constructed. 6,275 CGGE target proteins and 5,259 IS-related target proteins were obtained . In orde.DAVID was used to perform GO analysis of the 288 identified candidate targets. GO of candidate targets was analyzed based on the domains “biological process,” “cellular component,” and “molecular function.” The top 10 terms are shown in , the mechanisms of CGGE against IS might be concerned with long-term potentiation, cAMP signaling pathway, neurotrophin signaling pathway, and NOD-like receptor signaling pathway.In order to further analyze the biological mechanisms involved with the 288 candidate targets, the ClueGO was used to perform the KEGG pathway analysis. As shown in n= 6). It was found that the Glu level was significantly higher in the MCE group than in the sham group (p < 0.01) . However, no significant differences in the GABA level were observed among all groups (p < 0.01). Treatment groups significantly reduced the Glu/GABA ratios compared to the MCE group (p < 0.01).The changes in Glu and GABA levels in the ipsilateral cortex were illustrated in  < 0.01) . The Tatl groups . The ratl groups  was founp < 0.01). CAMK4 and p-CREB expression changes were reversed following the treatments of Tat-NR2B9C and CGGE. The mRNA expression of Camk4 and Creb1 was decreased in the MCE group, whereas it increased with Tat-NR2B9C and the CGGE treatments (The effects of CGGE on the CAMK4/CREB are shown in eatments .n = 3). Results are shown in p < 0.01). Compared to the MCE group, CGGE significantly decreased the expression of NLRP3, ASC, and CASP1 proteins. In addition, CGGE exhibited concentration-dependent inhibition of the protein expression.To investigate the potential mechanism by which CGGE alleviates neuroinflammation, we examined the expression of NLRP3 inflammasome in the ipsilateral cortex  (2+/CaM activates CaM kinases (CaMKs) and CREB kinases from the surface membrane to the nucleus, which is important for synaptic potentiation, learning, and memory  . CRE count (CRE) . Ca2+/Cad memory . In varid memory . InteresCREB is a crucial transcription factor, and the inactivation of CREB leads to the inhibition of many CREs promoters, including neurotrophic factors and synaptic proteins. The activity of CREB is regulated by phosphorylation and dephosphorylation, and many protein kinases can phosphorylate CREB, such as protein kinase A (PKA) , mitogenInflammation plays a key role in the pathophysiology of ischemic stroke. NLRP3 inflammasome is the best-characterized inflammasome and one of the major contributors to neuroinflammation, which consists of NLRP3, ASC, and Caspase-1 . The KEGTaken as a whole, the present data suggested that the neuroprotective mechanism of CGGE against IS was related to excitotoxicity and neuroinflammation, which mainly through the long-term potentiation, cAMP signaling pathway, neurotrophin signaling pathway, and NOD-like receptor signaling pathway. However, there are still some limitations. Firstly, the rat model of cerebral ischemia was established by microsphere injection, which prevented an accurate assessment of the infarct sizes. Secondly, we only measured the content of amino acids 24 h after ischemic injury, while the change in amino acid level is a continuous process. More sampling points, especially during the early stage of ischemia, would help better understand the dynamic changes of the neurotransmitters and the related mechanisms. Therefore, further studies are expected to explore the deeper mechanism.in vivo experiments. The results showed that CGGE could improve neurological function via alleviating excitotoxicity and neuroinflammation. According to the network analysis, CALM1 might be the critical target of CGGE. Furthermore, the protective mechanisms of CGGE on ischemic stroke might involve long-term potentiation, cAMP signaling pathway, neurotrophin signaling pathway, and NOD-like receptor signaling pathway. The mechanisms by which CGGE exerts anti-inflammatory and anti-excitotoxicity effects involved the inhibition of NLRP3 inflammasome and upregulation of the CAMK4/CREB pathway.In conclusion, the underlying mechanisms of CGGE on IS were explored via the integration of network analysis and


Mussaenda raiateensis, MR), Vula walu , Gasau , Molikaro  and Beki ni sina  collected from mainland region of Vitilevu, Fiji Islands, were evaluated in vitro. The hydromethanolic (ME) and dichloromethane (DM) extracts of these selected MPs were investigated. The ME extracts of BO (0.102 ± 0.009 mM CE) and DC  showed a higher TPC compared with the control [vanillic acid ]. However, the TPC of MF, MR and CL were found in the range of 0.020 ± 0.009 to 0.009 ± 0.01 mM CE. The ME extracts of MF and MR inhibited α-glucosidase significantly in comparison with acarbose as evidenced from the IC50 values . Moreover, DM extracts of MR (IC50 = 1.31 ± 0.29 ng/µl) also showed significantly higher α-glucosidase inhibitory activity. In contrary, MR (IC50 = 16.18 ± 0.16 ng/µl) and CL (IC50 = 9.21 ± 0.51 ng/µl) also showed significant α-amylase inhibitory activity in ME and DM extracts, respectively. These, results suggest that Fijian MPs could be a potential source of natural inhibitors of enzymes involved in carbohydrate digestion and thus may possibly be used in managing T2DM.Medicinal plants (MPs) are natural sources of active compounds with potential therapeutic benefits in alleviating various illnesses for decades. Fijian people also are using these MPs for the management/prevention of Type 2 diabetes mellitus (T2DM) and associated complications. However, till date, none of these Fijian MP’s antidiabetic potential have been explored or evaluated. Here, we investigated the antidiabetic potential of Fijian MPs scientifically. Phytochemicals such as polyphenols were detected to inhibit the activity of α-amylase and α-glucosidase, the two key carbohydrate enzymes linked to T2DM. Therefore, in the present study, the total phenolic content (TPC), α-amylase and α-glucosidase inhibitory activity of five Fijian MPs: Vobo ( Diabetes mellitus (DM), characterized by hyperglycemia, is the most common endocrine disorder, affecting millions of people worldwide . HyperglThus, the search for natural product inhibitors that are readily available and potentially safer is the ideal choice. The earliest record of MPs to manage T2DM is more than 3,500 years  and is sFurthermore, catechin , curcumin (the major polyphenol of turmeric) and resveratrol  are also known to reduce diabetic inflammation by scavenging reactive oxygen species (ROS) . Similarin vitro of the hydro-methanolic and dichloromethane extracts prepared from Vobo , Vula walu , Gasau , Molikaro  and Beki ni sina , traditional MPs used by the herbalists in the mainland region of Vitilevu, Fiji.Therefore, it is pertinent to explore these TMPs, which can be utilized for lowering blood glucose rendering a cheaper and safer way to manage T2DM thus giving better patient management outcomes. The natural products don’t have any side effect and economically sustainable. So, people will have less economical burden. Hence to fill this gap, our study focused with an objective to investigate the antidiabetic properties of some native plants used by established local herbalists. We hypothesized that local TMPs used by herbalists for managing T2DM; (1) may have proven scientific effect and (2) may contain phenolic compounds that can significantly inhibit the activity of α-amylase and α-glucosidase and hence could help to treat T2DM. Therefore, the present study aimed to assess and compare the total phenolic content (TPC), α-amylase and α-glucosidase inhibitory properties Aspergillus oryzae , α-glucosidase from Saccharomyces cerevisiae (≥ 100 U/mg protein) and all other chemicals were bought from Merck Co . Deionized distilled water was used to prepare specific volumes and concentrations of all the reagents used in the present study.Solvents were of analytical grade and obtained from Thermofisher . Total phenolic content was determined using Bio vision Phenolic Compounds Assay Kit (Colorimetric); Catalog # K527-200, Milpitas, CA, U.S.A. α-Amylase from All the plant materials used in the present study were collected from the wild habitats from the main island of Vitilevu, Fiji, in the year 2019. Taxonomic identification was made by the botanist at the University of the South Pacific Herbarium. After harvesting a bulk sample, the plants were washed under running water to remove all contaminants. All plant samples were cut into small pieces and air-dried for 4 weeks under the shade at room temperature (RT). The dried plant material  was grinded into coarse powder texture using an electric blender  whereas the roots and stems were manually powdered using mortar and pestle. The powdered plant materials were packed in zip lock plastic bags, labeled, and kept in the dark until ready for extraction.2O mixture): methanol (1:1) to give a stock concentration of 100 µg/µl.About 20 g of each plant sample were homogenized in 70% methanol (100 ml) at RT for 24 h. Manual shaking was done after 6 and 18 h of soaking period followed by sonication for 1 h . The mixture was filtered using Whatman filter paper (Cat No. 1441-125) and the filtrate was collected in round bottom flask. The extraction was repeated twice using the marc of the previous extraction. The extracts were combined and concentrated with a rotary evaporator  to obtain a viscous liquid. Concentrated extracts were transferred to pre-weighed labeled scintillation vials and dried using SpeedVac Concentrator (Thermo Scientific - Savant SC210A) with the vials loosely capped. The extracts were further dried using freeze drier (Biobase - Type: YL 7124). About 0.05 g of the dried plant extracts were each solubilized in 500 µl of 10% dimethyl sulfoxide (DMSO/HThe methanolic extracts residues were homogenized in 100 ml of dichloromethane (DM) at RT for 24 h. Similar steps as above for preparation of ME extracts were followed thereafter to prepare DM extracts. DM plant extracts were each solubilized in 500 µl acetonitrile: ethyl acetate: methanol (1:1:1) to give a stock concentration of 100 µg/µl.From the 100 µg/µl stock solution, 0.5 µg/µl plant sample concentration was prepared for TPC analysis. TPC was measured using phenolic compounds assay kit  as per manufactures instruction . The absB = amount of diazo chromophore ; D is the sample dilution factor; V is the volume of sample added to the reaction well.where, α-Amylase inhibitory activity of ME and DM extracts were carried out according to the standard method with minor modifications ,31. In aAs = absorbance in the presence of test substance; Ac = absorbance of control.where, Acarbose was used as positive control and all measurements were performed in triplicate.2CO3 (0.1 M). The absorbance of the released p-nitrophenol was measured at 405 nm using Eliser Plate Reader (DR-200Bc: wavelength range: 400–800 nm) and compared with that of the control, which had all the components except the plant extract replaced by 20 μl of phosphate buffer. The results were expressed as percentage inhibition, which was calculated using the formula InhiAs = absorbance in the presence of test substance; Ac = absorbance of control.where, Acarbose was used as positive control and all measurements were performed in triplicate.*P value < 0.05. Correlation tests were done using Pearson correlation coefficient, −1 ≤   ≤ 1.The results were expressed as means of three independent experiments ± standard deviations . ME extract of DC (DCME) and DM extract of MF (MFDM) recorded the highest % crude yield. The difference in the % yields between ME and DM extracts could mainly be due to presence of high number of polar components compared with low polarity components in the plants chosen for the present study. Compounds such as proteins and carbohydrates that have higher solubility in water and methanol may have been extracted. The extraction yield in the present study is consistent with the literature, where for example, Limnophila aromatic gave a 75% extraction yield in aqueous methanol but low in DM [There was a higher yield of phytochemical extracted from the MPs with ME solvent (5.89–19.52%) when compared with the yield of DM (0.46–2.72%) as shown in ow in DM ,35.PPHG is the primary factor to maintain in the management of T2DM. Phenolics apart from maintaining PPHG has also shown to be powerful antioxidants (AOs) ,36. In tin vitro [BO is noted to remain among the least investigated species in literature. However, some studies have reported BO to show highest phenolic content (2095 ± 120 mg of GAE/100 g) and thus highest 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity compared with four other ferns investigated [DC, work on TPC and AO activities on other Dicranopteris species, such as on Dicranopteris linearis has been reported [O-(2″-O-β-D-glucopyranoside)-β-glucopyranoside and 4-vinyl phenol-1-O-(20-O-α-L-rhamnopyranosyl)-β-D-glucopyranoside has been isolated from this plant species [The prevention of oxidative stress (OS) from elevated production of ROS with the consumption of AOs is one of the main pathological mechanisms to lower the risk of organ damage in T2DM patients . Studiesin vitro . Our stustigated . Phenolistigated . While oreported ,55. Phen species . Our fin species ,58. Howe50  values is further shown in Supplementary Table S1a–d. IC50 value is the concentration of plant sample required for 50% enzyme inhibition. The IC50 comparative analysis between the plant extracts and acarbose for α-amylase and α-glucosidase is presented in Dietary phenols, apart from their AO activity, have also been reported to exert antihyperglycemic effects by binding to glucose transporters  and comp50 = 16.18 ± 0.16 ng/µl. On the other hand, CLDM displayed the highest α-amylase inhibitory activity of 72.98% and IC50 = 9.21 ± 0.51 ng/µl. In comparison, ME of MF  and MR  inhibited α-glucosidase remarkably and much better than acarbose . Whilst MRDM  also showed outstanding α-glucosidase inhibitory activity. At 50 ng/µl, least α-amylase inhibitory activity was observed with DCME (10.79%) indicating that higher concentration of DC is required to achieve enzyme inhibitory activity, thus low potency in the specific antidiabetic property. However, all MPs under investigation showed remarkable α-glucosidase inhibitory activity with BO showing the least inhibition (73.02%) at 50 ng/µl. Few MPs showed the potential to significantly inhibit the enzyme activity at the lowest concentration of 5 ng/µl and compared well with the standard acarbose. At 5 ng/µl, the ME of DC, MF, MR inactivated α-glucosidase at 54.60%, 75.45% and 66.67% respectively. The MRDM inactivated α-glucosidase at 69.31%. Acarbose at 5 ng/µl inhibited the two enzymes, α-amylase and α-glucosidase, at 71.53% and 60.89% accordingly. It can be suggested that these MPs may possess high amount of enzyme inhibitors even at very low concentration of the crude plant extract. Due to crude extracts being used, this interesting result may be likely because of the phytochemicals working in a synergistic way to give the entire plant extract its therapeutic efficacy. Recent studies suggest that extract from entire MP has more benefits compared with the use of isolated compounds from the same MP extract [All the MPs inactivated α-amylase and α-glucosidase enzyme in a dose dependant manner until the concentration of 25 ng/µl after which the inhibition activity neared to the saturation point. This is depicted in  extract ,66.Rubus sanctus [In our study, 95% of MPs showed better α-glucosidase inhibition activity, when compared with α-amylase inhibition. In relation to MPs for T2DM, studies elsewhere have shown that MPs that have strong inhibition on α-glucosidase enzyme and less (or no) inhibition on α-amylase have better hypoglycemic therapy potential for direct use and/or to be developed into synthetic pharmacological drugs . Thus, b sanctus . Our stuMussaenda roxburghii showed remarkable α-glucosidase inhibitory activity [Mussaenda macrophylla displayed antidiabetic and AO property [Mussaenda species whereby quercetin, rutin, hyperin, ferulic acid, sinapic acid, β-sitosterol and saponin are found in MR [Miscanthus sinensis was noted to inhibit α-glucosidase [Dicranopteris curranii showed significant α-glucosidase inhibitory activity showing the presence of flavonoids, hydroxycinnamic acid and proanthocyanidin [Dicranopteris linearis acted synergistically producing a hepatoprotective effect, which also maybe contributed to this species anti-inflammatory and AO properties [50 = 7.65 ± 0.20 ng/µl). It is reported that essential oils from CL peels display better α-glucosidase inhibitory activity (IC50 = 7.56 µg/ml) than acarbose (IC50 = 8.44 µg/ml) when compared with its α-amylase inhibitory activity (IC50 = 8.16 µg/ml) [While majority of the MPs investigated in the present study are novel, however, antidiabetic work on species of the same genus has been reported. For instance, leave extract of activity  whereas property . Iridoidnd in MR ,72. Morecosidase . The leacyanidin . Moreoveoperties ,74,75. O6 µg/ml) . Additio6 µg/ml) . Flavono6 µg/ml)  whereby 6 µg/ml) ,80. Anti6 µg/ml) . The aqu6 µg/ml) ,82. More6 µg/ml) . In one 6 µg/ml) . The fivPlant phenols are effective in preventing T2DM via several pathways, apart from their AO potential ,18,19,36r = −0.873, P=0.05, r = −0.594, P=0.29; n=5). This suggests that high percentage yield of the ME or DM plant extracts do not necessarily mean high TPC of the crude plant extract. Thus, other secondary metabolites apart from phenols may have been extracted in both solvents that has led to the increased % crude yield of the crude extracts. Also, negligible correlation between the percentage crude yield of the ME and DM extracts was seen . MPs may inhibit α-amylase and/or α-glucosidase; however, it has rarely been noted to display good inhibition for both α-amylase and α-glucosidase enzymes [r = −0.447, P=0.45, n=5) and negligible correlation between the α-amylase and α-glucosidase activity of DM plant extracts . However, there was a moderately positive association between the α-glucosidase inhibitory activity in ME and DM  and a strong positive correlation between α-amylase inhibitory activity in ME and DM  plant extracts. Our study further associates TPC moderately positive with α-amylase and α-glucosidase  in ME whereas, strongly positive with α-amylase  in DM. Thus, our findings further indicate the presence of enzyme inhibitory compounds in both ME and DM extracts. On the other hand, TPC showed negligible correlation with α-glucosidase  in DM. Other studies also reported a mixed outcome on correlation between TPC and inhibitory activity of the TMPs but does shows strong positive correlation between TPC and AO properties of TMPs [Percentage crude yield of ME and DM plant extracts exhibited strong and moderate negative correlation with TPC, respectively ( enzymes ,85. In t of TMPs .Miscanthus floridulus, Mussaenda raiateensis and Dicranopteris caudate. The present study provides the first pharmacological insight in the total phenolic content and antidiabetic potential of the selected Fijian traditional medicinal plants. Hydromethanolic extract of Blechnum orientale and Dicranopteris caudate showed highest total phenolic content compared with vanillic acid, thus displaying potential antioxidant capacity. Mussaenda raiateensis and Miscanthus floridulus inhibited α-glucosidase significantly compared to the most common drug, acarbose, indicating phytochemicals present in the extracts have potential to reduce postprandial hyperglycemia by delaying the carbohydrate digestion. The antioxidant capacity of these plants needs to be further explored using tests such as DPPH, ABTS and FRAP. Antidiabetic ability to inhibit α-amylase and α-glucosidase needs to be also further examined using in vivo experimental models for validation. Furthermore, discovery into the safety and effectiveness of the active antidiabetic compound needs to be made.This is the first enzyme inhibition study on Click here for additional data file.


Overall, these data suggest GPR23 involvement in modulating responses to GUA in tumor cell lines, although further research needs to verify whether this receptor mediates other GUA effects.Guanine-based purines (GBPs) exert numerous biological effects at the central nervous system through putative membrane receptors, the existence of which is still elusive. To shed light on this question, we screened orphan and poorly characterized G protein-coupled receptors (GPRs), selecting those that showed a high purinoreceptor similarity and were expressed in glioma cells, where GBPs exerted a powerful antiproliferative effect. Of the GPRs chosen, only the silencing of GPR23, also known as lysophosphatidic acid (LPA) 4 receptor, counteracted GBP-induced growth inhibition in U87 cells. Guanine (GUA) was the most potent compound behind the GPR23-mediated effect, acting as the endpoint effector of GBP antiproliferative effects. Accordingly, cells stably expressing GPR23 showed increased sensitivity to GUA. Furthermore, while GPR23 expression was low in a hypoxanthine-guanine phosphoribosyl-transferase (HGPRT)-mutated melanoma cell line showing poor sensitivity to GBPs, and in HGPRT-silenced glioma cells, GPR23-induced expression in both cell types rescued GUA-mediated cell growth inhibition. Finally, binding experiments using [ While adenine-based purines (ABPs), including adenosine triphosphate, diphosphate, and monophosphate , and adenosine (ADO), have been extensively studied as extracellular signalling molecules in the central nervous system (CNS) , the gua3H]-GUO on rat brain has been described  might be somehow involved in GUO-mediated neuroprotection by the stimulation of A1R/A2AR heteromers  to pinpoint those responding to GBPs. Our bioinformatic approach revealed some potential candidates within these GPCRs, which were subsequently studied in some cancer cell lines previously used for assessing GBP functionality .3H]-GUA , was purchased from Moravek Inc. .GUA, GUO, GMP, GTP and ADO were purchased from Sigma-Aldrich . In all the experiments, nucleobases and nucleosides were dissolved in 1 N NaOH and added to the culture medium at the final concentration of 0.001 N NaOH whereas nucleotides were dissolved in aqueous solution. Geneticin was from Gibco ; Deoxyribonuclease I amplification Grade  was from Invitrogen . Forodesine was purchased from D.B.A . Tritiated GUA, -GUA binding was carried out. Membranes (50 μg) were preincubated  in binding buffer  and then incubated  with [3H]GUA in a total volume of 0.5 ml binding buffer. For saturation experiments, a concentration range of 6.25–300 nM [3H]-GUA was used. Nonspecific binding was determined by adding 500 μM unlabeled GUA and specific binding was calculated by subtracting nonspecific from total binding. In competition experiments, displacing agents at different concentrations and 50 nM [3H]-GUA were added, and the reaction started by adding the membranes. After 30 min, the reaction was stopped by adding 3 ml of cold binding buffer. The samples were rapidly filtered by vacuum filtration using Whatman GF/B glass fiber filters. These filters were then washed four times (2.5 ml cold binding buffer each time), dried for 1 h at 30°C, transferred in scintillation vials filled with 5 ml of Filter count scintillation cocktail . Bound radioactivity was measured in a Beckman Coulter LS6500 Multipurpose Scintillation Counter . For saturation and displacement curves, the pooled data were fitted by a computerized nonlinear regression analysis and resolved for the presence of a single high-affinity binding site.We started with the preparation of membranes from U87 cell lines, according to the previously published procedure . Cultured method . SubsequSamples containing PNP were obtained as previously described by p < 0.05 were considered statistically significant.All experiments were usually performed from three to four times, unless differently specified. The quantitative data are expressed as mean ± SD. Time-response curves were calculated by using nonlinear regression . Statistical analyses were performed by Prism 6.0 software, using Student’s t test coupled to the Holm-Sidak method or two-way analysis of variance (ANOVA). Group differences with 1R, A2AR and A3R responsive to ADO and P2Y1R responsive mainly to ADP , were selected from the BLAST search list (http://www.guidetopharmacology.org/GRAC/FamilyDisplayForward?familyId=694), or 2) they belonged to a group of de-orphanized GPCRs  for which experimental evidence of additional unknown endogenous ligands was available  tvailable . Four re5 mRNA levels, measured 48 h after siRNA lipo-transfection into the U87 cell line, were persistently decreased by about 75%, 72%, 90%, 75% and 90%, respectively. Importantly, the downregulation of the selected receptors did not affect cell survival, except for a significant cell growth inhibition (38%) observed with GPR21 silencing (data not shown). Overall, these results validated our experimental strategy for silencing orphan GPCRs in the U87 cell line.Subsequently, the mRNA expression of the selected receptors, namely GPR3, GPR21, GPR22, GPR23 and LPAR6/P2RY5, was silenced in U87 cell line by short interference RNA (siRNA) methodology (25). To this end, a pair of specific siRNAs targeting the coding region of each receptor was designed and synthesized . Thus, ap < 0.01) in cells silenced for GPR23 . In contrast, silencing of GPR21 or GPR22 receptors did not precluded GUO or GMP antiproliferative effects, while that of GPR3 and LPAR6/P2Y5 receptors, although significant (p < 0.05), had a limited effect on GUO- and GMP-mediated cell growth inhibition (5%–15% reduction). Collectively, these results suggested that GPR23 might play a potential role in GBP-mediated antiproliferative effects of U87 cells, thus we subsequently focus on this receptor.A functional signature of GBPs, as compared to other purines, is the profound antiproliferative effect on human tumoral cell lines, including U87 cells . Accordior GPR23 . In conAs shown previously, silencing of the GPR23 had an impact in the GBP-mediated growth inhibition of U87 cells. Now, we aimed at pharmacologically characterizing this GPR23-dependent antiproliferative effect by constructing concentration-response curves for GUO, GMP and GUA. To this end, U87 cells were GPR23 silenced or mock transfected before being exposed to increasing concentrations of GUO, GMP and GUA for 72 h and the cell proliferation assayed. Interestingly, we also included GUA as this compound, which usually derives from the metabolism of GUO, has been shown to be an active GBP in other cell lines .50 values for GUA, GUO, or GMP were 40 μM, ∼ 190 and ∼170 μM, respectively. Overall, these results indicated that GPR23 silencing modified the antiproliferative efficacy of GBPs but not their potency  . Indeed, potency , thus poConversely, to support the dependence of the GUA-induced inhibition of cell growth on GPR23 activation we assessed GUA effects in U87 and U373 cells overexpressing this receptor. To this end, U87 or U373 wild type (wt) cells were transfected with lacZ control vector or with the cDNA encoding the GPR23 receptor . GPR23 protein density was monitored upon receptor transduction in U87 and U373 cells by immunofluorescence detection. This assay showed, for instance, a superior expression of V5-tagged GPR23 receptor in U87 cells clone 12 (U87cl12) . Similar50 values related to the GUA effect in any cell line assayed, a significant increment in the GUA-induced growth inhibition was observed in U87cl12, U87cl8 and U373cl18 cells , thus we assessed whether U87cells also secrete PNP. Indeed, extracellular PNP activity increases in a time dependent fashion in the culture medium of U87 cells .Importantly, when U87cells were pre-treated with forodesine, a selective PNP inhibitor, the antiproliferative effects of GMP and GUO, but not that caused by GUA, were abolished . These rSince nucleobases are recovered by cells to be reused intracellularly, we evaluated the involvement of HGPRT, a key enzyme in the purine salvage pathway, in the GUA-mediated antiproliferative effect. Interestingly, HGPRT, by converting GUA and hypoxanthine into GMP and inosine monophosphate, respectively, regulates GUA levels. To this end, C32 melanoma cells bearing an active or inactive (C32TG cells) form of this enzyme were used to assess the impact of HGPRT activity on GUA-mediated inhibition of cell growth. Interestingly, while GUA inhibited concentration-dependently the proliferation of C32 cells, it was ineffective in C32TG cells . ConversNext, to ascertain the relationship of HGPRT with GUA signaling we determined GPR23 mRNA expression in cells lacking HGPRT activity. Importantly, C32TG cells showed very low expression of GPR23 as compared to C32 cells . In addi3H]-GUA and membrane extracts from control and GPR23 silenced U87 cells. Competition binding experiments showed that GUA concentration-dependently reduced [3H]-GUA binding within 2 min (Ki = 0.028 ± 0.0048 µM) . Other GBPs  tested or adenosine (ADO), even administered at concentrations up to 10 mM, showed a low percentage of [3H]-GUA binding with a Ki ≥ 1,000 µM (data not shown).To unequivocally demonstrate that GUA interacts with GPR23 we performed radioligand binding experiments using [0048 µM) . GUO als3H]-GUA concentrations higher than 100 nM   . CollectIn the present study, we identified GPR23 as a potential GPCR mediating some of the GBP-induced biological effects reported so far . In part5 receptors, and after checking for their expression in cell lines, such as U87 and U873 glioma cell lines responsive to GBPs  is located on chromosome Xq13-q21.1, and contains an intronless open reading frame of 1113 bp encoding 370 amino acids   into GUA. Accordingly, cell exposure to the enzyme inhibitor, forodesine, curtailed the inhibitory effect of GMP or GUO on U87 cell growth, but not that of GUA see the .It has also to be mentioned that we previously reported  that the3H]-GUA binding was displaced by unlabelled GUO, but at concentrations higher than those of unlabelled GUA, suggesting that GUO could also bind GPR23 but with lower affinity. Clearly, this aspect deserves further investigation. In contrast, guanine-based nucleotides or ADO displaced tritiated GUA at so high concentrations which are usually considered as not consistent with a selective binding of these compounds to a receptor, GPR23 in our case.Finally, the last experiments showing the existence of a specific binding site for tritiated GUA in membrane of U87 cells add consistency to the other results so far discussed, demonstrating that GUA exerts its inhibitory effects on different tumoral cells lines, at least, by interacting with GPR23. Noteworthy, [1-6) have been recognized, which are distinguished into isoforms belonging to the endothelial differentiating gene (Edg) family of GPCR and non-Edg family of purinergic receptors. Among GPCRs, three receptors for LPA have been identified, termed LPA1, LPA2, and LPA3 , with LPA1/Edg2 being the first identified and most widely expressed subtype. LPA1, LPA2 and LPA3 receptors are found in many cell types, exhibit relatively close homology with each other and show a high affinity for LPA (in the nanomolar range). In contrast, LPA4 (GPR23/P2Y9), LPA5 (GPR92), and LPA6 (P2RY5) belong to a non-Edg subfamily of purinergic GPCR clusters (reviewed in Regarding specific ligands and biological functions of GPR23, previous functional studies reported that is activated by LPA . For thi+/K+-ATPase (In conclusion, findings herein reported indicate that GPR23 protein can represent a membrane receptor responsive to extracellular GUA, although the existence of intracellular mechanisms, even HGPRT-independent and potentiated by GPR23 expression could be an alternative explanation that cannot be ruled out. On the other hand, it has been reported that isolated basolateral membranes of renal proximal tubule express a Gi protein-coupled receptor for GUA, whose stimulation inhibits the activity of Na+-ATPase . To clar


Setaria italica (L.) P. Beauv.], as a rich source of folates, has been cultivated on arid infertile lands, for which N deficiency is one of the major issues. Growing environments might have a significant influence on cereal folate levels. However, little is known whether N deficiency modulates cereal folate levels. In order to obtain enriched folate foxtail millet production in nutrient-poor soil, we conducted a study investigating the content of folate derivatives of 29 diverse foxtail millet cultivars under two N regimes (0 and 150 kg N ha−1) for 2 years to explore folate potential grown under low N. The contents of total folate and most derivatives were reduced by N deficiency. The effect on total folate content caused by N was stronger than cultivar genotype did. Folate content of enriched folate cultivars was prone to be reduced by N deficiency. Structural equation models (SEMs) revealed that N fertilization had a positive indirect effect on grain folate content through influencing plant N and K accumulation. Collectively, the results indicate much more attention should be paid to N management when foxtail millet is cultivated in infertile soil, to improve foxtail millet folate contents.Foxtail millet [ Humans lack the capability of ynthesis . For thiynthesis , 3. It hynthesis . Maruvadynthesis  reportedynthesis .Genotypes and environment significantly influence the folate content in cereal. For instance, total folate content among wheat and soybean genotypes shows a remarked variation , 8. MoreArabidopsis seedlings. Furthermore, folates are assembled from pterin, ρ-aminobenzoate, and glutamate precursors. Most folates are conjugated to a c-linked polyglutamyl tail of up to eight residues. These polyglutamyl tails may help to protect folates from oxidative breakdown, and folates tend to be stabilized by polyglutamylation  is an essential, often limiting, factor in plant growth and grain development . Upon N mylation . In Arabaragines  and migharagines . These oSetaria italica (L.) P. Beauv.], as the second millet in terms of worldwide production after pearl millet  to investigate the folate content variation of the leading Chinese foxtail millet cultivars; (2) to assess the effect of N deficiency on folate content; (3) to evaluate the association of foxtail millet folate levels with nutrient accumulation, which was affected by N regimes. The findings of this study would provide essential information for folate improvement in foxtail millet and other cereals. In particular, much attention should be paid to fertilization management when cultivating elite folate cultivars, especially in infertile soil conditions.−1 total N, 6.87 mg kg−1 Olsen-P (P), 138.2 mg kg−1 available potassium (K), and 20.33 g kg−1 organic matter.Field experiments were conducted at the Dongyang Experimental Station of Shanxi Agricultural University, Shanxi, China  in 2020 and 2021. This site generally experiences a temperate continental climate with a mean annual air temperature of 9.7°C and 440.7 mm of rainfall. The precipitation during the foxtail millet growth period was 411.8 mm and 310.8 mm in 2020 and 2021, respectively . This si−1 application (N-) and 150 kg N ha−1 application (N+). Nitrogen was supplied as slow-release urea (46%-N) before sowing. For all treatments, 75 kg ha−1 P2O5 and 75 kg ha−1 K2O were applied before sowing as well. Each plot area was 30 m2 (5 × 6 m). Plants on the borders of the plots were treated for protection and not harvested. The information of sowing date, plant density, plant height with N applied, and harvest date (the mature stage of the selected cultivars) of every selected cultivar is shown in The field experiment was a twenty-nine by two [most widely used and representative twenty-nine (29) foxtail millet cultivars and two levels of N rates] factorial design with 58 combinations, in which each combination had three plots as replicates. Two N rates were included 0 kg N ha2 of each plot, then panicle weight, straw weight, and grain weight were estimated, and the thousand-grain weight was determined. The total weight of panicles and straw was used to compute aboveground biomass. Some representative plant samples  were used to collect tissue for N, P, and K analysis after drying at 75°C to maintain a constant weight. After being weighted, the samples were finely grounded to pass through a 250 μm sieve. Another portion of representative grains from each cultivar was stored in a paper bag after threshing on time. About 5 g grains were manually ground to fine powder below 4°C, and the powder was completely filtered through the screen mesh (100 μm sieve). Then powder was temporarily stored at −80°C until further analyses of the folate levels.When panicles and grains were fully matured and plant individuals were harvested from 20 mAccording to the micro-Kjeldahl method, N content in plant tissues was determined with automatic Kjeldahl Apparatus  . Further4PteGlu; 5,10–CH=THF), 5-formyl-tetrahydrofolate , 5-methyl-tetrahydrofolate , dihydrofolate , folic acid , tetrahydrofolate , and methotrexate (MTX) were purchased from Schircks Laboratories  and MeFox, an oxidation product of 5–CH3-THF, was obtained from Toronto Research Chemicals . Sodium phosphate dibasic (Na2HPO4), sodium phosphate monobasic (NaH2PO4), sodium ascorbate, and β-mercaptoethanol were obtained from the Sigma-Aldrich Chemical. The endogenous folates in rat serum were removed by incubation with one-tenth (w/w) of activated charcoal for 1 h on ice, followed by centrifuging at 13,000 rpm at 4°C for 30 min , and the supernatant was used for the following incubation experiment. Acetonitrile and formic acid (LC-MS grade) were obtained from Fisher Scientific . The HPLC analytical column  was purchased from Akzo Nobel , and an Agilent SB-C18 pre-column  was acquired from Agilent Technologies .The folate standards: 10-formyl-folic acid , 5,10-methenyl-5,6,7,8-tetrahydrofolate  was transferred to a fresh tube. Then 35 mL of rat serum was added, and the polyglutamylated tails were deconjugated by incubation at 37°C for 4 h. Consequently, the samples were boiled for 10 min, cooled on ice for 10 min, and centrifuged at 13,000 rpm at 4°C for 10 min. The supernatant was moved to 3 kDa ultra-filtration tubes (Millipore) for cleanup and centrifuged at 13,000 rpm at 4°C for 25 min. Finally, the resulting solution was collected and 100 μL was transferred into new tubes for direct folate detection. The remaining solution was stored at −80°C. Each sample was repeated three times.The chemical powder of folate standards was dissolved as 0.1 mg/mL in a solution of 20 mM ammonium acetate in methanol and water  containing 1% (w/v) L-ascorbic acid, and 0.5% (v/v) β-mercaptoethanol (pH 6.2). 5,10–CH=THF standard was prepared in a pH 4.5 buffer. All standard solutions were kept at −80°C. For spiking and calibration, working solutions were diluted with folate extraction buffer, which was made fresh and used the same day.*2.1 mm). The injection volume was 15.0 μL. The injector and column oven temperature were separately maintained at 4 and 25°C, respectively. The mobile phases were 0.1% (v/v) formic acid in water (phase A) and 0.1% (v/v) formic acid in acetonitrile (phase B). The gradient program ran for a total of 19 min. The proportion of mobile phase B increased linearly from 5 to 9% over 2 min. In the following 6 min, phase B increased to 9.6% and then sharply increased to 20% in over 0.2 min. After holding steady at 20% for 3 min, the proportion of phase B decreased to 5% in 0.2 min, followed by a subsequent equilibration.Chromatographic studies were carried out at a flow rate of 0.30 mL/min using an Agilent 1260 HPLC system  with an Akzo Nobel analytical column  interface was used for mass analyses and quantification of target analytes. The mass spectrometer was used in positive ion mode. The parameters were optimized for the target analytes with a gas temperature of 350°C, drying gas flow at 11 L/min, nebulizer pressure at 35 psi, and capillary voltage at 3500 V (+). The parameters for folate standards were m/z 456–412, 30 eV for 5,10–CH=THF, m/z 460–313, 20 eV for 5–CH3–THF, m/z 474–327, 20 eV for 5–CHO–THF, m/z 446–299, 20 eV for THF, and m/z 455–308, 30 eV for the internal control MTX. System operation and data acquisition were performed with MassHunter software. The sum of contents of different folate derivatives represents as the total folate levels. The seven folate derivatives chromatograms of representative cultivar, Jingu21, under two N treatments were exhibited in The precision, linearity, sensitivity, recovery and matrix effect were evaluated for method validation, for which a series of dilution of folate standards was prepared in the blank foxtail millet solutions. To prepare a blank foxtail millet matrix, 1 g fine sample of Jingu21 was mixed with 20 mL phosphate buffer without sodium ascorbate and β-mercaptoethanol. The mixture was boiled for 1 h, then exposed to direct sunlight to degrade endogenous folates. Ten percent activated charcoal was added to the supernatant and was incubated with shaking for 1 h, and then centrifuged at 13,000 rpm for 30 min at 4°C. The supernatant was filtered through 3KDa MWCO ultra-filtration tubes .n = 6) on the same and different days. Intra-day repeatability and inter-day repeatability was evaluated by running samples on three different days (n = 3). The linearity and correlation coefficients were calculated by plotting the peak area at different concentrations. Data analyses were performed with MassHunter software. The correlation coefficients (R2) for all these folates were approximately > 0.99 in the folate derivatives matrices (Precision was assessed based on the relative standard deviation (RSD) of peak areas of Jingu 21 . Data were exposed to the analysis of variance (ANOVA) with agricolae package and boxplots with the ggplot 2 package in R 4.0.1 . Pearson correlation was performed on the R statistical software .The data of folate derivatives in cereal samples were shown as means ± SE of three biological replicates in micrograms per 100 g of grains, with the exception of the contents of folate derivatives under different NRE levels  in foxtail millets, in which this composite variable did not alter the underlying SEM model. A direct path from N treatment to plant NPK accumulation was used to account for the effects of nutrient utilization efficiency on the plant. A direct path from plant NPK accumulation to grain NPK accumulation was used to account for the effects of crop growth and nutrient distribution on grain nutrient uptake. Folate level was assumed to be affected by all other components both directly and indirectly. We started with a priori models that included all plausible pathways between these factors. Subsequently, the significance of each path coefficient was tested by calculating its critical ratio (P < 0.05). The SEMs was performed using the software Amos Graphics v22 . The capacity of SEM to separate the direct and indirect effects of a variable on dependent variables is considered one of the most important advantages of SEM  were established to test the direct and indirect effects of N regimes, plant NPK accumulation, and grain NPK accumulation on folate levels  was supplied, the folate levels of 29 foxtail millet cultivars ranged from 42.36 ± 2.91 to 72.89 ± 0.50 μg per 100 g grains and 42.84 ± 1.60 to 69.20 ± 2.13 μg per 100 g grains for 2020 and 2021, respectively , our stuTwo-way analysis of variance showed that both two N regimes and cultivars produced highly significant variation in the total folate contents in 2 years, with N factors affecting folates contents much more strongly than that of the cultivar genotypes  . Among tThe responses of total folate content of 29 cultivars to N deficiency differed significantly. Some foxtail millet cultivars  were found to have relatively high folate contents compared with others for each N regime, but obvious differences in folate content have appeared between two N treatments for these cultivars, in which the reduction ratio of Jingu21 was 47% in 2021  when N was supplied , 5–CHO–THF (P < 0.01) or total folate (P < 0.01) under sufficient N conditions in two successive years, respectively   . This pr3-THF, THF, 10–CHO–FA and 5,10–CH=THF for 2 years. Compared with the values when N was applied, N stress resulted in diminished the contents of 5–CHO–THF, by 13.56% and 34.67% in 2020 and 2021, and the content of 5–CH3-THF by 12.71% and 22.07% in 2020 and 2021, respectively. Also, the decreased contents of THF, 10–CHO–FA and 5,10–CH=THF by 19.62%, 23.68%, and 39.05% in 2020, and 46.71%, 23.65%, and 71.54% in 2021, respectively, induced by N stress, were observed as well , which may have been caused by its high abundance in foxtail millet. The 5,10–CH=THF and total folate content had a negative correlation (r = −0.260***) under N deficiency, while had no correlation when N was supplied  was also observed between 5–CHO–THF and 5,10- CH=THF  are statistical procedures for testing measurement, functional, predictive, and causal hypotheses. We further constructed SEMs to explore the direct and indirect relationships among N Fertilization, plant nutrient uptake, grain nutrient uptake and folate levels in foxtail millets , in term−1). With the application of N, the cultivar Jingu21 recorded the highest mean folate content among the 29 foxtail millet cultivars (71.03 μg per 100 g of grains) for 2 years. Whereas folate contents of Jigu22 and Longgu25 were < 50 μg per 100 g grains. The folate content for remaining foxtail millet cultivars ranged from 50 to 70 μg per 100 g grains. The contents of total folate and derivatives in foxtail millet were significantly reduced by N deficiency. The effects of N regimes on folate contents were much more evident compared with the impacts induced by cultivars. SEMs demonstrated N fertilization affected folate content of foxtail millet positively, brought about by enhanced N and K accumulation in plant aboveground. Furthermore, foxtail millet cultivars with higher level folate might have the ability to accumulate more N at the same N application rates. Folate content of high folate enriched cultivars was prone to be reduced by N deficiency, which indicated that much attention should be paid to N management when elite folate cultivars were cultivated, especially in infertile soil conditions, to ensure foxtail millet grain quality.We used HPLC-MS/MS techniques to investigate the folate derivatives of 29 different foxtail millet cultivars for 2 years under two N treatment rates (0 and 150 kg N ha5-Formyl-tetrahydrofolate (PubChem CID: 135403648); 5-Methyl-tetrahydrofolate (PubChem CID: 135483998); Tetrahydrofolate (PubChem CID: 135444742); 10-Formyl-folic acid (PubChem CID: 135405023); 5,10-Methenyl-tetrahydrofolate (PubChem CID: 135398657); Dihydrofolate (PubChem CID: 135398604); Folic acid (PubChem CID: 135398658); Methotrexate (PubChem CID: 126941); Sodium phosphate monobasic (PubChem CID: 23672064); Sodium phosphate dibasic (PubChem CID: 24203); Sodium ascorbate (PubChem CID: 23667548); β-Mercaptoethanol (PubChem CID: 1567); Acetonitrile (PubChem CID: 6342); Formic acid (PubChem CID: 284).The data that support the findings of this study are available from the corresponding author, X-yJ, upon reasonable request.YW: data curation, formal analysis, investigation, methodology, and writing-original draft. J-sW: supervision and validation. E-wD: validation. Q-xL: visualization. L-gW: project administration. E-yC: investigation and resources. X-yJ and X-mD: conceptualization, funding acquisition, project administration, resources, and writing—review and editing. All authors contributed to the article and approved the submitted version.


Listeria monocytogenes serotype 4b strains RF01 and RF06 were isolated from raccoon feces in Japan. Here, we report the draft genome sequences of the two isolated strains; the genome sizes were 2,918,024 and 2,872,491 bp, with 535× and 510× coverage, for the RF01 and RF06 strains, respectively. Listeria monocytogenes is a causative foodborne pathogen of listeriosis and is widespread in the environment. This bacterium has also been isolated from raccoons (Procyon lotor), which are free-living animals; therefore, raccoons have been suspected to transmit L. monocytogenes by direct contact and/or indirect contamination of the environment via invasion of human residential areas  at 35°C for 24 to 48 h (de novo using CLC Genomics Workbench v.21.0 (Qiagen) and annotated using the DDBJ Fast Annotation and Submission Tool (DFAST) v.1.1.4  were obtained from the RF01 and RF06 strains, respectively. These strains were assembled into 45 and 35 contigs, with accumulated lengths of 2,918,024 and 2,872,491 bp at 535× and 510× coverage, for RF01 and RF06, respectively .L. monocytogenes ST6 has been reported to be a hypervirulent clone and is increasingly being associated with outbreaks  were identified as ST6 (RF01) and ST4 (RF06) via multilocus sequence typing (MLST) v.2.0 (utbreaks ; moreoveutbreaks . The virentified . The datPRJDB13507.Draft genomes were deposited in DDBJ/ENA/GenBank under the accession numbers provided in