The XRD results revealed that the principle crystalline phase of the samples was the cubic perovskite structure. The B-site deficiency improved the ionic and total conductivities of Sr(Ti0.6Fe0.4)x1−Oδ3−. A small polaron conduction behavior occurred in the total electrical conductivity. The XPS results showed that the oxygen vacancy value decreased with the rise in the amount of B-site deficiencies. A lower B-site deficiency amount could produce more oxygen vacancies in the lattice but resulted in the ordering of vacancies and then lower ionic conductivity. The aging behavior was caused by the ordering of oxygen vacancies and resulted in a degeneration of electrical features under a long service time. Conversely, augmentation of the B-site deficiency amount inhibited the tendency for the ordering of oxygen vacancies and then promoted the electrical performance under a long usage time. The conduction mechanism of oxygen ions through oxygen vacancies was thoroughly investigated and discussed. The current study presents a feasible approach to ameliorate the physical features of conductors through doping the B-site of the perovskite layer with Fe, which would be a fruitful approach for numerous applications, including oxygen sensors and fuel cells anodes.Perovskite materials play a significant role in oxygen sensors due to their fascinating electrical and ionic conductivities. The sol–gel technique was employed to prepare various compositions of B-site-deficient Fe-doped SrTiO 3 (iron-doped strontium titanate) or Sr(Ti0.6Fe0.4)x1−Oδ3−, where x = 0.01, 0.02, and 0.03.Sol–gel technique is employed to prepare various compositions of B-site-deficient Fe-doped SrTiO Among the cubic perovskite oxides, strontium titanate is a chemically stable perovskite material under oxidizing and reducing atmospheres. However, the pure strontium titanate material has low electronic and ionic conductivities, which prevents its practical use for sensors and as electrodes of solid oxide fuel cells (SOFCs) and other devices.iii) or Y(iii) ions on the Sr-site or Nb(v) on the Ti-site.27–31 Similarly, the ionic conductivity could be enhanced by acceptor doping on the Ti-site, such as Cr3+, Fe3+, Ni2+, and Sc3+.32–37 It has been reported that donor or acceptor doping in SrTiO3 can promote both ionic and electronic conductivity, respectively, and the two kinds of conduction behavior are generally distinct.38 Nonetheless, according to our prior study, A- or B-site loading can have a substantial impact on the ionic and electrical conductivity.39–42 The impact of A- and/or B-site loading on both the ionic and electronic conductivity, however, cannot be used to infer the dependent effect of the separate loading on the electronic and ionic conductivity of oxygen sensors.According to many researchers, the electrical conductivity could be improved or influenced by donor doping, such as La A- or B-site doping may promote the electronic and ionic conductivities simultaneously; (2) A- or B-site doping could be unfavorable to the enhancement of the electronic and ionic conductivities simultaneously; or (3) A- or B-site doping may promote the electronic (or ionic) conductivity but result in the decrease in the ionic (or electronic) conductivity. In addition, the effect of A- and/or B-site deficiencies in the perovskite structure on the electronic as well as ionic conductivity can be evident due to the fact that the deficiency can define the level and kinds of imperfections. For example, A-site deficiency can lead to a decrease in the electron content, which can cause a lower electronic conductivity. Similarly, B-site deficiency can result in a decrease of the amount of oxygen vacancies, which can cause a lower ionic conductivity.3 mixed conductor materials were synthesized by a sol–gel method. The structural characteristics, microstructure, total conductivity, and ionic conductivity of Sr(Ti0.6Fe0.4)x1−Oδ3−  were evaluated. Especially, the effect of the B-site nonstoichiometric amount on the concentration of oxygen vacancies was examined by XPS analysis. The aging behavior of materials is significantly important because it changes the electrical properties under a long usage time and certain temperature. Therefore, the aging behavior was discussed by the ordering of the oxygen vacancies in the lattice.However, we found that the influence of the deficiency on the electrical conductivity was not limited to this. Therefore, we tried to find the relation between the produced defects amount as a deficiency and the electrical conductivity. In this paper, nonstoichiometric Fe-doped SrTiO2.0.6Fe0.4)x1−Oδ3− ] were synthesized by a sol–gel route from Sr(CH3COO)2·2H2O (AR), Ti(CH3CH2CH2CH2O)4 (AR), and Fe2O3 (AR).43–47 Here, strontium acetate interplayed with butyl titanate to develop the sol and concurrently Fe2O3 dispersed homogeneously on the sol during moderate magnetic stirring. The product was obtained in the form of a powder via sol-drying at the temperature of 50 °C. The dry gelatin powder was ground using agate mortar pestle and subsequently annealed at 1100 °C for 10 h under ambient conditions. The annealed powder was compressed in a disc form (8 mm diameter and ∼2 mm thickness) in the mold by uniaxial pressing (ca. under 50 MPa). The green tablets were annealed at a temperature of 1350 °C for 5 h at room temperature to get dense specimens for further analysis, including by XRD, SEM, XPS, and electrical property measurements.Iron-doped strontium titanate with B-site deficiencies [Sr with Al Kα. The total electrical conductivity was tested by electrochemical impedance spectroscopy (EIS) between 400 °C and 950 °C at room temperature. The oxygen ionic conductivity was investigated by the electron-blocking electrode approach in the temperature range of 600 °C to 1000 °C under ambient conditions. The electrodes for the electrical property measurements were assembled by coating Pt paste on both sides of sintered tablets and further baked at 1100 °C for 1 h. The electrode setup for the ionic conductivity test has already been explained thoroughly elsewhere.39 All the analyses were carried out under the equilibrium of each temperature point. The mixed conductivity measurements  were performed for every 50 °C. The aging behavior was measured at 800 °C for 24 h and the measurement was taken in intervals of 2 h.The phase composition was achieved by XRD . Scanning electron microscopy  was used to evaluate the microstructures of the annealed specimens. The oxidation states of the existing elements within the samples were measured 3.3.1.0.6Fe0.4)x1−Oδ3−  upon being sintered at 1350 °C for 5 h in an air atmosphere was evaluated via XRD for determining the phase composition. The results are shown in 3 in PDF 79-174 and were part of the cubic perovskite structure (Pm3̄m). The peaks at 25.57°, 36.59°, 37.42°, 41.14°, 56.33°, and 60.05° corresponded to those of Fe2TiO5 in PDF 9-182 for Sr(Ti0.6Fe0.4)0.97Oδ3− and the peaks at 30.00° and 30.98° were consistent with that of SrO in PDF 1-886 for Sr(Ti0.6Fe0.4)0.99Oδ3−. Theoretically, the ionic radius of Fe3+ (0.0645 nm for six connections) is equivalent to that of Ti4+ (0.0605 nm for six connections),40 therefore Fe3+ should dissolve into the perovskite lattice to form the substitutional solid solution. However, the introduction of B-site nonstoichiometry leads to the formation of other phases for Sr(Ti0.6Fe0.4)0.97Oδ3− and Sr(Ti0.6Fe0.4)0.99Oδ3−. For further exploration of the influence of B-site deficiencies on the perovskite structure, the tolerance factor (t) should be calculated because the tolerance factor can directly indicate the reason for the structural change. The tolerance factor can be calculated by the average radii of the A-site (rA), B-site (rB) cations, and oxygen ions (rO), as shown in t < 1.04.41Sr(Ti0.6Fe0.4)x1−Oδ3− with the ‘x’ values of 0.01, 0.02, and 0.03 were 0.9967, 0.9998, and 1.0029, respectively, indicating that B-site deficiency did not affect the formation of the principal crystalline phase of the cubic perovskite structure, and showing that the crystal structure of x = 0.02 was closest to the ideal cubic perovskite structure (t = 1). Therefore, as can be seen from 0.6Fe0.4)0.98Oδ3−. Rietveld refinement of the obtained XRD spectra was used to address the influence of the B-site deficiencies on the specimens' lattice volume and parameter.The calculated tolerance factors of Sr0.97Oδ3− and Sr(Ti0.6Fe0.4)0.99Oδ3−, respectively, the lattice parameter and lattice volume of Sr(Ti0.6Fe0.4)0.97Oδ3− and Sr(Ti0.6Fe0.4)0.99Oδ3− were greater than those for Sr(Ti0.6Fe0.4)0.98Oδ3−. Therefore, according to the results of the tolerance factor and Rietveld refinement, the lattice distortion of Sr(Ti0.6Fe0.4)0.98Oδ3− was negligible.3.2.0.6Fe0.4)0.97Oδ3− specimen are exhibited in 3 structure. The SEM micrographs of the fracture surfaces of the Sr(Ti0.6Fe0.4)x1−Oδ3− samples  are shown in 3 composites.The SEM, EDS images, and elemental distribution mappings of the fractured surface of the Sr(Ti3.3.0.6Fe0.4)x1−Oδ3− samples with different nonstoichiometries and temperatures are presented in 0.6Fe0.4)x1−Oδ3−  clearly increased. In addition, the electrical conductivity of the Sr(Ti0.6Fe0.4)x1−Oδ3− samples with different nonstoichiometries increased through to a maximum at 750 °C with the temperature increasing, and then decreased slightly, which demonstrated a small polaron conduction behavior of the Sr(Ti0.6Fe0.4)x1−Oδ3− materials. A small polaron is made up of charge carriers and lattice deformation originating from the electrons or holes.42,48–50 The conductivity can be determined from the carrier concentration and carrier migration rate.The relation curves of the electrical conductivity of Sr(Ti0.6Fe0.4)x1−Oδ3−  versus temperature are delineated in Rg) and grain boundary (Rgb) diminished after increasing the applied temperature, and these phenomena were led by the decreased intercept and semicircle in the plots, according to 0.6Fe0.4)x1−Oδ3−  and the applied temperature. The ionic conductivity rose gradually as the applied temperature and B-site deficiencies increased, indicating that the B-site deficiencies possibly promoted the concentration of oxygen vacancies as well as the movement rate of oxygen ions. The imperfections in the Fe-loaded SrTiO3 lattice can be expressed by the following The migration of tiny polarons is equivalent to negative charge carriers (electrons) at low temperatures because of the weak electron–lattice distortion coupling, therefore a rise in the operating temperature (≤850 °C) resulted in an augmentation in the electrical conductivity. Although, at higher temperatures, tiny polaron hopping induced by the enhanced electron–lattice deformation recombination resulted in low mobility, so the electrical conductivity decreased with the temperature increasing (>850 °C). The Nyquist plots for the ionic conductivity of Srx1−Oδ3−. The observed binding energies of the Fe 2p1 and Fe 2p3 peaks at 723.9 and 710.1 eV corresponded to Fe3+, indicating that the valence state of the doped Fe ions was +3. Therefore, oxygen vacancies were introduced in the lattice by Fe-doping. The concentrations of oxygen vacancies in the Fe-replaced samples were calculated via the atomic content proportion obtained from the O 1s XPS spectra (42According to  spectra .423 as Sr(Ti0.6Fe0.4)0.99O2.51, Sr(Ti0.6Fe0.4)0.98O2.52, and Sr(Ti0.6Fe0.4)0.97O2.84, which agreed with 39 The partial ordering of oxygen vacancies is probably a key factor determining the ionic conductivity of perovskites.The vacancy contents were calculated are 0.49, 0.48, and 0.16 . Thus weThe formation of oxygen vacancies in the lattice and the different conduction mechanisms of oxygen ions in oxygen vacancies were thoroughly investigated, as demonstrated in x = 0.03, and the ionic conductivity was the highest, but the ordering of oxygen vacancies, as shown in The ordering of oxygen vacancies led to a decrease in the ionic conductivity. Therefore, when the oxygen vacancy concentration was within a certain range, it was accurate or even the rule that the ionic conductivity increased with the oxygen vacancy concentration increasing. Therefore, the oxygen vacancy concentration value was 0.16 for 0.6Fe0.4)x1−Oδ3−  versus time are displayed in 0.6Fe0.4)0.98Oδ3− and Sr(Ti0.6Fe0.4)0.99Oδ3− and then decreased for Sr(Ti0.6Fe0.4)0.97Oδ3− upon prolonging the time. The aging behavior resulted from the ordering of the oxygen vacancies, which resulted in a decrease in the electrical conductivity over a long time and at a certain temperature. 0.6Fe0.4)x1−Oδ3− at 800 °C as a function of the annealing time. As shown in 0.6Fe0.4)0.99Oδ3− and Sr(Ti0.6Fe0.4)0.98Oδ3− decreased gradually with the annealing time prolonging. This was different from the electrical conductivity of the two samples of Sr(Ti0.6Fe0.4)0.97Oδ3−, which were found to increase significantly under a longer service time. By contrast, the aging behavior of Sr(Ti0.6Fe0.4)0.97Oδ3− caused an increasing conductivity value after 24 h. Thus, the increase in the B-site deficiency amount inhibited the tendency for oxygen vacancies ordering and thus promoted the electrical performance under a longer usage time.The Nyquist plots for the total electrical conductivity of Sr(Ti4.3 or Sr(Ti0.6Fe0.4)x1−Oδ3−, where x = 0.01, 0.02, 0.03, perovskite layers were successfully fabricated via a sol–gel technique. The principal crystalline phase of Sr(Ti0.6Fe0.4)x1-Oδ3− with ‘x’ values of 0.01, 0.02, 0.03 calcinated at 1350 °C for 5 h at room temperature belonged to the cubic perovskite. The total conductivity was enhanced with the increase in the amount of B-site deficiencies and it exhibited a small polaron conduction behavior. The ionic conductivity was also enhanced upon increasing the B-site deficiencies. Although, the rise in B-site deficiency amount could result in a lower concentration of oxygen vacancies, the low B-site deficiency level (high oxygen vacancy concentration) could enhance the ordering of oxygen vacancies in the cubic perovskite material. Such vacancy ordering would be responsible for a lower ionic conductivity. The increase in the B-site deficiency amount inhibited the tendency for oxygen vacancy ordering, thus promoting the electrical performance under a longer usage time. In this study, a simple approach was employed to improve the physical properties of conductors by doping the B-site of the perovskite layer with Fe, which would be advantageous for several applications, such as oxygen sensors and fuel cell anodes.Mixed conductors based on Fe-doped SrTiOThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


Since NPY receptors have different receptor gene deletions in mammals and teleost fish, it is not clear whether npy2r has the similar function in fish as in mammals. In this study, we used the CRISPR/Cas9 system to establish npy2r-deficient medaka (Oryzias latipes). Unexpectedly, the deletion of npy2r resulted in the npy2r+/− medaka were all-male, therefore, npy2r homozygous mutant lines could not be established. The deletion of npy2r increased the food intake in medaka, and the expression levels of appetite stimulating genes  increased significantly, while the expression levels of anorexia factors  decreased significantly. Moreover, the absence of npy2r significantly increased the total length and body weight of medaka. The mirror test and open field test showed that npy2r+/− medaka improved sociability and reduced anxiety-like behavior, qRT-PCR analysis showed that the expression levels of anxiety related genes  in npy2r+/− medaka were significantly decreased. So far, this is the first npy2r gene knockout model established in fish and demonstrates that npy2r plays an important role in the regulation of reproduction, feeding and anxiety in fish.Neuropeptide Y receptor Y2 ( Based oent gene . In addi mammals . In partebrafish , orange ebrafish  and Chinebrafish .npy receptor knockout mice, npy1r and npy5r knockout mice exhibited decreased food intake and body weight. npy4r knockout mice showed that npy4r promotes obesity induced by high fat diet , Y2 subfamily  and Y5 subfamily (NPY5R) . Differefat diet . Deletio in mice . Injectigulation .npy2r has been cloned and characterized in mammals including human  is an important model organism, it has the advantages of transparent embryo, fast growth rate and easy cultivation. In this study, we used the CRISPR/Cas9 technology to knockout the npy2r gene in Japanese medaka, in order to further explore the physiological function of npy2r gene.In order to explore the physiological function of npy2r+/− medaka and female npy2r+/+ medaka used in this study were 5 months old.The male and female wild-type (WT) Japanese medaka HdrR (orange-red strain) were obtained from the Institute of Hydrobiology, Chinese Academy of Sciences . They were bred in the circulating water system of the Research Center of Mandarin Fish in Huazhong Agricultural University with 27°C of room temperature and 14 h of light and 10 h of darkness. Embryos were collected in the morning and incubated at 28°C incubator. The male WT, male https://www.ncbi.nlm.nih.gov/) and Ensembl (https://asia.ensembl.org/index.html) were used to search the NPY receptors genes and amino acid sequences of Japanese medaka, Human (Homo sapiens), Mouse (Mus musculus), Seabass (Dicentrarchus labrax), Swamp eel , Spotted gar (Lepisosteus oculatus), Nile tilapia (Oreochromis niloticus), Zebrafish (Danio rerio), Rainbow trout (Oncorhynchus mykiss), Atlantic cod (Gadus morhua), Chinese perch (Siniperca chuatsi), Japanese flounder , African clawed frog (Xenopus laevis), and Torafugu (Takifugu rubripes). The accession numbers are shown in npy2r among medaka and other Vertebrate were performed by using Clustal X, and the protein genealogies of NPY receptors were assessed by the Neighbor-Joining using Mega11.NCBI (https://cctop.cos.uni-heidelberg.de/) was used to design medaka single guide RNA (sgRNA). Two targets on the second exon of medaka npy2r gene were designed. The sgRNAs were then cloned into PMD-19T vector and synthesized using TranscriptAid T7 High Yield Transcription kit . Medaka embryos at the single-cell stage were microinjected with 2 nL of a mixed solution consisting of 1 μL of sgRNAs (50 ng/μL), 1 μL of Cas9 mRNA (300 ng/μL), 0.5 μL of phenol red indicator, and 2.5 μL of DEPC water. After 24 h of microinjection, genomic DNA was extracted from medaka embryos, and the effectiveness of sgRNAs were tested by PCR and sequencing. The microinjected embryos were cultured to adulthood. The caudal fin genome DNA was extracted, and the F0 mutant line was obtained by PCR amplification and agarose gel electrophoresis. After that, the F0 mutant  and WT  medaka were crossed to generate F1 heterozygous mutants. The F2 homozygous mutant medaka was obtained by crossing F1 of the same mutant type. The sgRNAs and detection primers of npy2r are shown in The CCTOP web (dmrt1bY (5′-AGAGGAGGAGCTTGGGATTTGTAG-3′) and dmrt1a (5′-CAGACGCTTCCTCGCCGTAA-3′) were then used for PCR amplification .The male WT and male npy2r+/+ and male npy2r+/− medaka were randomly selected and placed under the same experimental conditions to determine the food intake. Before the experiment, the medaka were starved for 24 h. Before feeding, the medaka were weighed and placed individually in 1-L tank. The medaka were fed an excessive amount of brine shrimp (Artemia nauplii) and allowed to eat freely for 1 h, then the weight of medaka were measured again . The movement trajectory for 10 min was recorded, EthoVision XT software was used to analyze the biting time and swimming distance.The mirror test, or mirror approaching behavior, is a good model for measuring social interaction and social anxiety . Mirror npy2r+/+ and male npy2r+/− medaka were randomly selected, one fish at a time, and slowly placed into the tank for 2 min to adapt to the environment. After that, they were allowed to explore freely without being disturbed (n = 7). The movement trajectory for 10 min was recorded, and EthoVision XT software was used to analyze the swimming distance, movement time and total freezing time.The Open-field test can be used to assess medaka anxiety levels and locomotor activity of medaka . In thisnpy2r. The total RNA was extracted by TRIzol Reagent , and then 1 μg RNA was reverse transcribed into cDNA using reverse transcription kit . SYBR  and th1 (tryptophan hydroxylase1), th2, agrp (agouti-related protein), cck (cholecystokinin), pomc (pro-opiomelanocortin), npy (neuropeptide Y), gr1 (glucocorticoid receptor 1), gr2, mr  genes specific primers were used for qRT-PCR. npy2r, th1, th2, agrp, cck, pomc, npy, gr1, gr2, and mr genes. The 20 μL RT-qPCR reaction system included 1 μL cDNA, 0.5 μL forward and reserve primers (10 mmol/μL), 10 μL SYBR, and 8 μL double distilled water (ddH2O). The conditions for PCR were as follows: 95°C for 3 min initially, followed by 40 cycles at 95°C for 10 s, 58°C for 30 s and 72°C for 30 s, and melting curve assay from 65°C gradually increasing 0.5°C s−1°C to 95°C, with acquisition data at every 6 s. The mRNA expression levels of target genes were quantified relative to the expression of β-actin using the optimized comparative Ct (2−ΔΔCt) value method  using medaka brain cDNA as template. The eye, brain, gill, heart, kidney, ovary, testis, liver and spleen from medaka were sampled for tissue expression analysis of e method .t-test. GraphPad Prism 8.0.2 used to make data analysis diagram, p < 0.05 and p < 0.01 were considered statistically significant.The SPSS 25.0 software was used for statistical analysis. All data were expressed as means ± S.E.M, and analyzed by independent-samples npy2r. The npy2r amino acid sequence of medaka showed 79.2% homology with seabass, 74.9% homology with swamp eel, 55.7% homology with human and 49.2% homology with mouse, respectively  was obtained by PCR and sequencing after hybridization of FO and WT medaka, namely 297 bp deletion, 5 bp addition and 1 bp mutation   , but the body weight was not significantly different between npy2r+/+ and npy2r+/− medaka (p > 0.05)  . By meas < 0.01) . Compare > 0.05) .npy2r+/+ and npy2r+/− medaka three-dimensional trajectory graph can clearly see npy2r+/− medaka swam back and forth in one side of the mirror, however, WT and npy2r+/+ medaka swam all over the region   .npy2r+/+ and npy2r+/− medaka are shown in npy2r+/− medaka are more inclined to explore the edges  (p < 0.05)  , while t < 0.05) .npy2r+/+ and npy2r+/− medaka, we measured the mRNA expression levels of genes associated with anxiety behavior , appetitive genes , and anorexigenic factors  by qRT-PCR. The results showed that compared with WT, the expression levels of th2, gr1, gr2, cck gene in npy2r+/− medaka were significantly decreased (p < 0.05)       , agrp, n < 0.05) , and th1 > 0.05) . Compare < 0.05) , agrp, n < 0.05) , and gr2 > 0.05) .NPY2R, NPY4R, NPY5R, NPY6R, NPY7R, and NPY8R through two whole-genome duplications in the early stage of vertebrate evolution , and significantly decreased the expression of anorexia factors , which further indicated that npy2r play an important role in feeding regulation of medaka. Previous studies have shown that npy2r knockout mice body weight and food intake were significantly increased  by qRT-PCR, the results showed that the expression levels of th1, th2, gr1, gr2, and mr gene in npy2r+/− medaka decreased significantly. Since th1 and th2 catalyze the production of dopamine, excessive exercise may lead to significantly increased expression of th1 and th2 genes in the brain (th1, th2, gr and mr gene are significantly increased when zebrafish are in a state of anxiety or stress (npy2r is involved in the regulation of anxiety behavior in medaka.The open-field test can be used to reflect the anxiety level and locomotor activity in medaka . Therefork tests , and ourrk tests . Medaka rk tests . We detehe brain . Studiesr stress . Therefonpy2r-deficient medaka. It was found that the deletion of npy2r resulted in the npy2r+/− medaka were all-male, therefore, npy2r homozygous mutant lines could not be established. The study of npy2r+/− medaka showed that the absence of npy2r can improve social interaction and reduce anxiety behavior. In addition, the deletion of npy2r gene can promote the feeding and significantly increase the total length and weight in medaka. So far, this is the first npy2r gene knockout model established in fish and demonstrates that npy2r plays an important role in the regulation of reproduction, feeding and anxiety in fish.In conclusion, in this study, we established the


Glioblastoma (GBM) is a deadly brain tumor characterized by signaling dysregulation and aberrant cell cycle control. The CDK4/6-Rb axis is dysregulated in approximately 80% of all GBM cases. In this study, the anti-GBM effect of a novel pyrimidin-2-amine, LH20 was evaluated in vitro using the primary GBM cell lines U87MG and U251. GBM cells were administered LH20 at concentrations of 0.1, 1, 4, 8, 10, 20, 100, and 200 µM for 24 and 48 h, and the proliferation rate was evaluated using a CCK8 assay. Migration, apoptosis, and cell cycle were also assessed using a wound healing assay, Annexin V-FITC/PI apoptosis assay, and cell cycle staining, respectively. The targets of LH20 were predicted using SwissTargetPrediction and molecular docking. Western blotting analysis was performed to confirm the anti-GBM mechanism of LH20. We found that at concentrations of 4, 8, and 10 µM, LH20 significantly inhibited the proliferation and migration of U87MG and U251 cells, induced late phase apoptosis, promoted tumor cell necrosis, and arrested the G2/M phase of the cell cycle. LH20 also inhibited CDK4 and CDK6 activities by decreasing the phosphorylation of Rb. Our results suggest LH20 as a potential treatment strategy against GBM. Glioblastoma multiforme (GBM) is the most common and lethal malignant brain tumor with high molecular heterogeneity, poor overall prognosis, and a meager 10-year survival rate of <1%; only 6.9% of patients survive for more than 5 years following diagnosis . HoweverCell cycle dysregulation leads to cell cycle arrest, apoptosis, and cancer . The halTo date, several CDK inhibitors have been approved by the FDA for the treatment of advanced-stage or metastatic, hormone-receptor-positive, HER2-negative breast cancers ,11, inclThis study aimed to explore the anti-GBM effects of LH20, a novel pyrimidin-2-amine compound similar to Abe, in U87MG and U251 cell lines. Our findings will provide novel insights into the treatment of GBM, which will help to improve patients’ survival and quality of life.U87MG and U251 cells treated with LH20 A exhibitThe inhibitory activity of LH20 at 4, 8, 10, 20, 100, and 200 μM was augmented when cells were treated for 48 h  is released into the culture medium. In this study, treatment with LH20 and Abe at 10 μM for 48 h significantly increased the LDH release from U87MG cells H, suggesScratched cells cultured in high-glucose Dulbecco’s Modified Eagle Medium (DMEM) for 24 and 48 h showed apparent migration A,B. U87MTo evaluate the effects of LH20 on cell apoptosis, U87MG and U251 cells were treated with LH20 at 4, 8, and 10 μM for 24 or 48 h and assessed with Annexin Ⅴ-fluorescein isothiocyanate (FITC)/propidium iodide (PI). The U87MG cells treated with 8 and 10 μM LH20 exhibited higher late apoptosis  and necrosis rates . Further analysis using molecular docking confirmed that LH20 may mimic the adenosine of ATP and binds to the hinge regions of CDK1, CKD4/6, CCNE1, and CCNA2 to form several interactions within the binding pocket   − area [24 h/48 h])/area [0 day] × 100%.The perimeter of the central cell-free zone was determined using an optical microscope (Zeiss X-Cite) . The migg for 5 min. Aliquots of 100,000 cells were suspended with 500 µL binding buffer and 5 µL staining reagent . After incubation in the dark at 37 °C for 5 min, the fluorescence intensities of FITC and PI were analyzed using a flow cytometer  [The cells treated with LH20 were harvested using trypsin  and centrifuged at 4 °C and 300× CA, USA) .g for 5 min; the supernatant was discarded. The harvested cells were resuspended with 120 µL of JC-1 staining buffer and analyzed using a flow cytometer.Cells (U87MG and U251) were treated with 0, 4, 8, or 10 µM of LH20 for 24 or 48 h, then incubated with 500 µL of JC-1 working solution at 37 °C for 20 min, and centrifuged at 300× Cell cycle analysis was performed using a Cell Cycle Staining Kit  in a 24-well plate. Cells treated with 0, 4, 8, and 10 µM of LH20 were harvested with trypsin and washed with PBS. Cells were stained with 1 mL DNA staining solution and 10 µL of permeabilization solution at 37 °C for 30 min and analyzed using flow cytometry. The cell cycle was simulated using FlowJo software.http://www.swisstargetprediction.ch, accessed on 19 September 2022). Target genes associated with glioblastoma were obtained from GeneCards . Overlapping genes between the target of LH20 and glioblastoma were retrieved using EVenn  [https://string-db.org/, accessed on 19 September 2022). Core genes were analyzed using CytoNCA  with the criteria of median DC, CC, and BC [https://david.ncifcrf.gov/tools.jsp, accessed on 19 September 2022). Statistical significance was set at p < 0.05.The canonical SMILES of LH20 was retrieved from the ChemDraw Ultra 2017 software and used for target identification in the Swiss Target Prediction database (er 2022) , while t, and BC . Gene onThe crystallographic structures of CDK1, CDK4/6, CCNE1, and CCNA2 were downloaded from the Protein Data Bank . Protein preparation was performed using the Protein Preparation Wizard and Receptor Grid Generation modules. Preparation of all LH20 was initially minimized by the LigPrep module with ionization-generated possible states at a target pH of 7.0 ± 2.0 s. For the force field option, OPLS4 and the other parameters were set to their default values. LH20 was flexibly docked into the ligand site of the co-crystal complex using the Ligand Docking module with standard settings in both standard precision (SP) modes. Only the best pose with good hydrogen bond geometries and low-energy conformations was considered for further analyses .Cells were seeded in DMEM with 10% FBS for 24 h and treated with LH20 (10 μM) and Abe (10 μM). Proteins were extracted from U87MG cells using Triton X-100 lysis buffer. Protein lysates were resolved using 10% SDS-PAGE  and transferred to PVDF  via electroblotting. Each blot was incubated for 15 min at room temperature in Quick block solution (Beyotime) and overnight at 4 °C with primary antibody , dilutedp < 0.05. Statistical analyses were performed, and figures were generated using GraphPad Prism (version 9.0.0).Data are expressed as mean ± standard deviation (SD). Normal distribution of data was tested using the Shapiro–Wilk test, and normally distributed data were analyzed using one-way analysis of variance (ANOVA) with Dunn’s test for multiple groups. Otherwise, non-normally distributed data were analyzed using the Kruskal–Wallis test. Differences were considered significant at We demonstrated that LH20 exhibited significant growth inhibitory and migratory effects against GBM cells in vitro, induced late-phase apoptosis and MMP drop, and arrested cells in the G2/M phase. Furthermore, the molecular mechanism illustrated that LH20 potentially targets CDK4/6 to inactivate Rb phosphorylation and influence mTOR expression. Overall, our study suggests LH20 as a novel treatment option for patients with GBM.


We observed a temperature-dependent oxidative degradation of the PSs with strong (40 °C), moderate (25 °C), and weak/no degradation (5 °C). Especially at elevated temperatures such as 40 °C, fast oxidative PS degradation processes were detected. In this case study, a stronger degradation and earlier onset of oxidation was observed for PS80 in comparison to PS20, detected via the fluorescence micelle assay. Additionally, degradation was found to be strongly dependent on PS concentration, with significantly less oxidative processes at higher PS concentrations. Iron impurities, oxygen in the vial headspaces, and the pH values of the formulations were identified as the main contributing factors to accelerate PS oxidation.The surfactants polysorbate 20 (PS20) and polysorbate 80 (PS80) are utilized to stabilize protein drugs. However, concerns have been raised regarding the degradation of PSs in biologics and the potential impact on product quality. Oxidation has been identified as a prevalent degradation mechanism under pharmaceutically relevant conditions. So far, a systematic stability comparison of both PSs under pharmaceutically relevant conditions has not been conducted and little is known about the dependence of oxidation on PS concentration. Here, we conducted a comparative stability study to investigate (i) the different oxidative degradation propensities between PS20 and PS80 and (ii) the impact of PS concentration on oxidative degradation. PS20 and PS80 in concentrations ranging from 0.1 mg⋅mL Polysorbate 20 (PS20) and polysorbate 80 (PS80) are non-ionic surfactants, which are used in biopharmaceuticals to ensure the stability of monoclonal antibodies/proteins and to prevent protein particle formation in drug products ,2,3,4. D•) is generated, followed by the reaction with molecular oxygen to form peroxyl radicals (ROO•), which further propagate the radical chain reaction by hydrogen abstraction or disintegration. As long as a radical reacts with a non-radical compound, the chain reaction is propagated }tetraacetic acid disodium salt; Merck KGaA, Darmstadt, Germany); H2O2 ; methanol ; sulfuric acid ; formic acid ; ammonium formate . All tested formulation compounds were analytical grade.PS20 high purity (HP) ; PS80 HP ; sodium acetate trihydrate ; acetic acid ; nitric acid ; NPN (−1 were prepared in 25 mM acetate buffer pH 5.5 and ultrapure water followed by sterile filtration using Millipak® 40 polyvinylidene fluoride (PVDF) filters . 10 mL Fiolax® clear TOPLINE glass vials  were filled with 5 mL sample volume, stoppered with D777-1 20 mm laminated butyl rubber stoppers , and sealed airtight with aluminum crimp caps . The samples were incubated at 5 °C, 25 °C/60% rh, and 40 °C/75% rh, protected from light, and retrieved at selected sampling time points. Additionally, 1 mL samples of PS20 and PS80 raw materials were filled without filtration and stored under the same conditions. Due to the high viscosities, filtration of the raw materials was not possible.PS20 HP and PS80 HP at concentrations of 0.1, 0.2, 0.4, 1, 10, 100, and 438 mg⋅mL−1 for B, 250 ng⋅mL−1 for Al, 2.5 ng⋅mL−1 for V, 100 ng⋅mL−1 for Cr, 10 ng⋅mL−1 for Mn, 100 ng⋅mL−1 for Fe, 25 ng⋅mL−1 for Ni, 10 ng⋅mL−1 for Cu, and 10 ng⋅mL−1 for Mo for the raw materials of PS20 and PS80, as well as for the 1 M acetate buffer sample. For the 100 mg⋅mL−1 samples of PS20 and PS80 the LOQ was 250 ng⋅mL−1 for B, 250 ng⋅mL−1 for Al, 2.7 ng⋅mL−1 for V, 6.57 ng⋅mL−1 for Cr, 4.94 ng⋅mL−1 for Mn, 10 ng⋅mL−1 for Fe, 25 ng⋅mL−1 for Ni, 10 ng⋅mL−1 for Cu, and 5 ng⋅mL−1 for Mo.For the raw materials of PS20 and PS80 as well as for a solution of 1 M acetate buffer pH 5.5, the metal ion concentrations were determined by ICP-MS, utilizing a 7800 ICP-MS  system equipped with an SPS 4 autosampler . PS20 and PS80 raw material samples were dissolved through acid digestion using nitric acid and measured against custom standards of the metals Al, B, Cr, Cu, Fe, Mn, Mo, Ni, and V. No-gas-mode or helium-mode was used depending on the element. The limit of quantification (LOQ) was dependent on the sample and was 250 ng⋅mLpH measurements were conducted with the samples at room temperature using a SevenCompact S220 pH-meter with an InLab Micro Pro-ISM pH electrode . The pH electrode was calibrated with pH 4.01 and pH 7.00 buffer solutions .® Automation Workstation . Here, 240 µL of FMA buffer consisting of 5 µM N-phenylnaphthalen-1-amine (NPN), 0.0015% (w/v) Brij™-35, 150 mM NaCl, 5% (v/v) acetonitrile, and 50 mM Tris at pH 8.0 were added to 10 µL sample volume and incubated at 35 °C for 1 min. The fluorescence was measured with an Infinite M200pro fluorescence plate reader  with an excitation wavelength of 350 nm and an emission wavelength of 420 nm in quadruplicates (n = 4). Measurements were conducted against PS20 (0.1–0.6 mg⋅mL−1) and PS80 (0.05–0.3 mg⋅mL−1) calibration standards. It is worth mentioning that only micelle-forming species are detected and the fluorescence intensity is dependent on the hydrophobicity of the species. Preferential degradation of certain species can result in inaccurate PS concentration determinations. The LOQ was 0.1 mg⋅mL−1 for PS20 and 0.05 mg⋅mL−1 for PS80.The FMA was adapted from Lippold et al. (2017) and Glücklich et al. (2021) ,61. FMA 2+ to Fe3+) in the formulations as described in Jaeger et al. (1994) [® Automation Workstation . In brief, the FOX reagent was prepared by mixing 30 mL of solution A (25 mM ammonium iron(II)sulfate hexahydrate and 2.5 M sulfuric acid) with 300 µL of solution B . For each sample, 30 µL sample and 300 µL FOX reagent were mixed and incubated at RT for 15 min. The absorbance at 595 nm was subsequently measured with a SpectraMax M3 plate reader . Measurements were performed in triplicates (n = 3) and conducted against H2O2 calibration standards ranging from 0.39 to 100 µM. The LOQ was 5 µM H2O2 equivalents (5 µM H2O2 ≈ 0.17 ppm H2O2).FOX measurements were performed to determine the peroxide concentration (every mol ROS oxidizes 1 mol Fe. (1994) . The ass−1. The LOQ was 10 µmol⋅mol−1 formic acid.GC measurements were performed to determine the emerging short chain formic acid content of the different formulations with advancing oxidation, as was previously described by Mahadevan et al. (1967) . A 0.5 m® UHPLC system with RS dual gradient pump, RS autosampler, and RS column compartment  coupled to an ACQUITY QDa mass detector  with an ESI source. For solid-phase extraction and protein removal a mixed-mode column  was used. A Poroshell 120 SB-C8 4.6 × 100 mm, 2.7 μm reversed-phase column  was used as the stationary phase for PS subspecies separation. Three mobile phases  were used for the analytic gradient. After PS subspecies separation by the stationary phase, the analytic gradient, delivered by the right pump (0.7 mL⋅min−1), was mixed with a 10 mM ammonium formate buffer, delivered by the left pump (0.2 mL⋅min−1), using a T-piece. The injection volume was 2 µL and the column oven was set to 50 °C (PS20) or 60 °C (PS80). The method had a run time of 45 min. The QDa detector analyzed masses between 250–1250 Da in positive mode with a sampling rate of 2 Hz. Measurements were conducted against PS20 and PS80 calibration standards ranging from 0.05 to 0.6 mg⋅mL−1 PS. The LOQ was 0.05 mg⋅mL−1 for both polysorbates.The RP-UPLC-MS method was adapted from Lippold et al. (2017) and Evers et al. (2020) ,64. MeasOxygen concentrations in the headspaces were measured with a Microx 4 trace fiber optic oxygen meter with an IMP-PSt7 microsensor  as described in Hipper et al. (2023) . The sam−1 to raw materials in acetate (AB) and ultrapure water (UW) was tracked through FMA . Together, the progress of oxidation can be evaluated for the different conditions and storage times.The degradation of PS20 and PS80 for concentrations ranging from 0.1 mg⋅mLough FMA  and FOX ough FMA  over a p−1, less degradation was observed for both PSs after 48 weeks of storage. The PS content ranges from ~52% to more than 80% for 438 mg⋅mL−1 PS formulations and raw materials after 48 weeks. For low initial PS concentrations, a strong degradation at 40 °C within the first three months was measured, resulting in a more than 80% PS degradation for concentrations ranging from 0.1 to 1.0 mg⋅mL−1. Here, a small difference between PS20 and PS80 was detected, as zero fluorescence was measured for PS80 (0.1–1.0 mg⋅mL−1), whereas up to 18% of the remaining PS content was determined for PS20. This difference between PS20 and PS80 content increased continually for 0.4, 1.0, and 10 mg⋅mL−1 PS, where PS80 revealed faster degradation kinetics  formulations exhibit a remaining PS content of 53–85% PS for the same storage conditions (48 weeks at 40 °C/75% rh). The degradation of the 438 mg⋅mL−1 PS80 formulations was more severe in comparison to PS20 and similarly a difference of ca. 40% was determined for PS80 RM in comparison to PS20 RM. However, this difference must be taken with caution, as the viscous RM had to be diluted more than 1000-fold for content analysis, leading to larger errors in comparison to the lower concentrated aqueous solutions. This is verified by the higher point-to-point variation in the RM samples, especially visible at later timepoints of the PS80 RM (−1) and formulated in water, an initial lag phase of 6–8 weeks without degradation was monitored .kinetics A. The 10onitored A. For be2O2 equivalents were measured for PS concentrations ranging from 0.1 to 100 mg⋅mL−1 after storage at 40 °C/75% rh. A representation of the H2O2 equivalent concentration per 0.1 mg⋅mL−1 PS in the formulations was chosen to allow a relative comparison between the different PS concentrations. The analyses of 438 mg⋅mL−1 formulations and of PS RM samples were omitted due to the low degradation observed in FMA measurements. In general, H2O2 equivalent concentrations in the formulations followed a roughly symmetrical pattern, with content increasing during the first weeks and decreasing between approximately 6 to 18 weeks  . The 0.1–0.4 mg⋅mL−1 PS20 UW formulations reached the highest measured values of ROS species after 12 weeks, 4–6 weeks after the equivalent PS20 AB formulations. PS20 HP ultrapure water samples (1–100 mg⋅mL−1) exhibit the highest H2O2 equivalent content after 4–8 weeks. For both PSs, a difference in the measured peroxide concentration between both solvents was detected, with the peak peroxide content of the 0.1 mg⋅mL−1 PS20 AB formulation corresponding to approx. 50% of the equivalent PS20 UW formulation. This difference is reduced with increasing PS20 concentration, resulting in a difference of 20% at 100 mg⋅mL−1. For PS80, a smaller difference between maximal H2O2 concentrations of UW and AB formulations was observed, with the largest difference of approx. 40% at 0.4 mg⋅mL−1.Complementary to the PS content, the concentrations of H18 weeks B. The ma slopes) A,B. With−1 (−1 and higher (−1) at 25 °C was delayed by 36 weeks, resulting in content ranging from 47 to 55% after 48 weeks. Meanwhile, the corresponding PS20 formulations showed only minimal PS degradation, with 81–94% PS remaining. The decreasing temperature stress at 25 °C revealed larger differences in oxidative degradation between PS20 and PS80. Independent of concentration and solvent type, a faster degradation of PS80 was determined, except for the lowest concentration of 0.1 mg⋅mL−1, where both PSs show similar degradation kinetics  was detected after approximately 8 weeks at 40 °C, revealing an approximately three-times faster oxidation than for the corresponding formulations stored at 25 °C. The delayed oxidation of the PS20 UW formulations at 0.2 and 1.0 mg⋅mL−1, which was also observed for 40 °C, is further delayed, starting between 12 to 18 weeks , which were only slightly more acidic after 48 weeks. As mentioned above, the acidic shifts in the unbuffered UW formulations were stronger than in the AB formulations, independent of PS concentration and PS type. From 0.1–10 mg⋅mL−1 PS, lower pH values were measured with increasing concentration, and the opposite trend is observed for concentrations ranging between 10–438 mg⋅mL−1 PS. The pH shifts in 0.2–1.0 mg⋅mL−1 PS20 UW formulations showed a delay of 2–4 weeks in comparison to the PS80 UW formulations. This is consistent with the content analysis via FMA and the FOX analysis, as both revealed a faster degradation of PS80  and PS80 (retention times > 22 min) are observed , capric acid (C10), lauric acid (C12), myristic acid (C14), palmitic acid (C16), stearic acid (C18), and oleic acid (C18:1) were extracted and normalized to the initial value (t = 0). Polyesters of the corresponding fatty acids were not considered, as their content is neglectable for most of the fatty acids. The POE monoester variants are, for instance, sorbitan-C12 species with 18 to 33 numbers of POE units in total. Their changes over time are illustrated in −1 PS80 AB and the results are depicted in As mentioned, MS-based detection allows for the selective quantification of individual species in the heterogeneous PS mixtures by filtering for specific mass-to-charge values and retention times. Thereby, the resilience of PS species esterified to middle-chain length fatty acids such as caprylic acid (C8) and capric acid (C10), which was observed in the peak pattern of PS20 AB 10 mg·mL−1 PS80 AB formulations were used, as the rapid oxidation processes were occurring at lower PS80 concentrations. Oxidation in the vicinity of the double bond was observed as species such as C18:1 + C18:1-OH and C18:1 + C18:1-OOH emerged due to favorable H-atom abstraction in the vicinity of the double bond, with a maximum after 4 weeks of storage at 40 °C/75% rh , sorbitan di-oleate ethoxylated with 26 POE units (2x C18:1), as well as oxidation products of sorbitan di-oleate (26 POE moieties), with one of both oleic acids oxidized to hydroxyl-C18:1 (C18:1 + C18:1-OH) or hydroperoxyl-C18:1 (C18:1 + C18:1-OOH) were extracted and plotted over time . The 10 C/75% rh . Additio−1 and 102.2 ng⋅mL−1 in RMs of PS20 and PS80, respectively) in combination with tiny amounts of H2O2 or other organic peroxides can catalyze the generation of ROS via Fenton/Fenton-like reactions [−1 boron and 12.7 ng⋅mL−1 molybdenum in the 1 M acetate buffer is not expected to initiate oxidative processes  did indeed slow down in comparison to the low-concentration PS formulations (0.1–1.0 mg·mL−1), even though significantly higher proportions of the PS material remained. Possible explanations for the surfactant concentration-dependent degradation of PS20 and PS80 could be (i) the changing environment in the concentrated solutions and (ii) the depletion of oxygen or the presence of specific ROSs. Mittag et al. (2022) showed via EPR and spin trapping experiments that the radical formation from PS80 raw material is different to aqueous dilutions of PS80 (10% (w/v)) [• and RO• species, were detected, whereas in 10% (w/v) PS80 solutions mainly HO• radicals were measured [• radicals react in a diffusion-controlled manner (k > 109 M−1·s−1) [• and superoxide radicals possess reaction constants six to nine orders of magnitude lower and might therefore decelerate the oxidative degradation in higher concentration PS formulations [• radicals react in a diffusion-controlled manner. The second way in which higher concentrations of polysorbates could affect oxidation is the availability of oxygen. Molecular oxygen is essential for oxidation reactions, as it is inserted early in the oxidative chain reaction [v/v) were detected with increasing PS concentrations (10 and 100 mg·mL−1), whereas for the lower PS concentrations (0.1–1 mg·mL−1), 8 to 12% (v/v) of remaining oxygen was detected in the headspace ) . In bulkmeasured . As HO• M−1·s−1) ,51,52, hulations ,51,52,53er k > 10 M−1·s−1 reaction . By limireaction . We analeadspace . The oxynd 40 °C . This obated see . For con compare . The FMAet al. 20 showed vThe bigger difference between PS oxidation at 25 °C in comparison to 40 °C can be explained by the temperature itself. As mentioned above, temperature has different effects on reactions, namely influencing activation energy and/or doubling the chemical reaction rates by increasing the temperature by 10 K.−1  is dependent on the initial PS concentration, with less oxidation occurring at higher PS concentrations. The formation of different radicals as well as the availability of oxygen were discussed as potential root causes.pH changes induced by oxidation are dependent on the initial concentration of poly-sorbate, as higher acidifications are observed with increasing PS concentrations, peaking at 10 mg·mL−1 A. Oxidat−1 and 10 mg·mL−1, whereas MS-based approaches revealed a stronger difference between both polysorbates, with PS80 being more susceptible to oxidation. Additionally, faster and more severe oxidation of PS20 and PS80 was observed using FMA content determinations in comparison to the MS-based method  dihydrochloride (AAPH) have normally been used to accelerate the oxidation process. For instance, AAPH is a water-soluble radical starter [−1, when, in general, less oxidation was observed, as discussed above. At lower temperatures, greater differences between PS20 and PS80 were observed. At 5 °C, no oxidative degradation was measured for PS20 formulations in UW and AB pH 5.5 (independent of the PS concentration), whereas most PS80 formulations revealed oxidation between 36 and 48 weeks  revealed a degradation delay at 40 °C, which was even increased at 25 °C. As no additional impurities such as iron or other divalent ions were found in the acetate formulations (see ICP-MS of 1 M acetate buffer), the pH difference between UW (pH > 6.0) and AB (pH 5.5) could be a potential root cause, as the Fenton reaction is more likely to occur in acidic conditions [PS20 UW formulations in the lower concentration regime  were analyzed, revealing oxidation in the vicinity of the double bond of oleic acid, as hydroxyl-C18:1 or hydroperoxyl-C18:1 species are formed from diesters of PS80 , or even purer qualities , no definitive statement can be made. For a description of the current literature, see Weber et al. (2023) , summari−1 to raw materials formulated in water and acetate buffer pH 5.5 was investigated at 5, 25, and 40 °C to achieve an in-depth comparison between PS20 and PS80, as well as to elucidate the concentration dependence of PS oxidation. Especially at 5 °C, a concentration-dependent comparison was missing in the literature, as oxidation accelerators were mainly used to compare both PS types. We showed that PS20 and PS80 oxidation is dependent on the initial concentration, with decreasing oxidative susceptibility at increasing initial PS concentrations, independent of PS type and whether they were formulated in water or acetate. Especially for concentrations above 438 mg·mL−1, only minor degradation was observed by MS detection. The availability of oxygen as well as the generation of different radicals were discussed as root causes. Mittag and colleagues (2022) reported ROO• and RO• to be the predominant species for raw materials of PS80, whereas mainly HO• radicals were measured in 10% (w/v) PS80 solutions, independent of the quality grade [-PS80 is more prone to oxidation than PS20 as it possesses an earlier onset of oxidation, independent of whether it is formulated in water or acetate buffer;-At 5 °C, PS20 showed no oxidative degradation in water and acetate buffer up to 48 weeks, whereas PS80 oxidation started after 36 weeks;-Lower temperatures (less oxidative stress) revealed larger differences between PS20 and PS80 oxidation;-Delayed oxidation was monitored for PS20 formulated in water and high pH values were discussed as a potential origin;-Oxidation is dependent on the initial PS concentration, most likely due to differences in radical generation and/or due to limiting oxygen availability;-Metal ions such as iron in the presence of oxygen are most likely the root cause of oxidation.The oxidative degradation susceptibility of different PS20 and PS80 concentrations ranging from 0.1 mg·mLty grade . Additioty grade . The absty grade ,45,46. N


Hydra actinoporin-like toxin 4 (HALT-4) differs from other actinoporins due to its N-terminal propart that contains approximately 103 additional residues. Within this region, we identified five dibasic residues and assumed that, when cleaved, they could potentially exhibit HALT-4′s cytolytic activity. We created five truncated versions of HALT-4  to investigate the role of the N-terminal region and potential cleavage sites on the cytolytic activity of HALT-4. However, our results demonstrated that the propart-containing HALT-4 (proHALT-4), as well as the truncated versions tKK1 and tKK2, exhibited similar cytolytic activity against HeLa cells. In contrast, tRK3, tKK4 and tKK5 failed to kill HeLa cells, indicating that cleavage at the KK1 or KK2 sites did not enhance cytolytic activity but may instead facilitate the sorting of tKK1 and tKK2 to the regulated secretory pathway for eventual deposition in nematocysts. Moreover, RK3, KK4 and KK5 were unlikely to serve as proteolytic cleavage sites, as the amino acids between KK2 and RK3 are also crucial for pore formation. Hydra actinoporin-like toxin (HALT) is a family of seven α-pore-forming toxins, namely HALT-1 to HALT-7 [Hydra magnipapillata, a freshwater hydrozoan [o HALT-7 . They weydrozoan . Actinopydrozoan . All HALydrozoan ,4. ThereHydra. PC3 is a calcium-dependent protease that activates precursor proteins by cleaving pairs of basic amino acids. The Hydra PC3-like protein shares 55.4% and 56.7% identity in the catalytic domain with mouse PC3 and human furin, respectively [Hydra PC3-like protein may process the precursor form of HALT-4 by targeting and cleaving one or more paired basic sites at the N-terminus of HALT-4. Although recombinant proHALT-4 was capable of killing human cells, its activity was seven-fold weaker than that of recombinant EqtII [Numerous cnidarian toxins, including equinatoxin II, possess a signal peptide followed by 9–17 residues of a propart at the N-terminus. Anderluh et al. 2000) demonstrated that several genes encoding nematocyst-associated proteins and toxins have a highly conserved signal peptide and propart that ends with KR basic residues . For insectively . This fi000 demonE. coli. The recombinant proHALT-4, tKK1, tKK2, tRK3, tKK4 and tKK5 were then expressed in the presence of 1 mM IPTG and 3% ethanol, as shown in Assuming that each of the five pairs of basic sites could potentially be cleaved to generate HALT-4 with higher cytolytic activity, we used proHALT-4 as a template to construct five truncated HALT-4, which we named tKK1, tKK2, tRK3, tKK4 and tKK5 B. The prTo evaluate the cytolytic activity of the truncated forms of HALTs-4 , cytotoxicity assays were conducted on HeLa cells. Various concentrations  of proHALT-4, tKK1, tKK2, tRK3, tKK4 and tKK5 were applied to the cells, and their cytolytic activity was assessed. Since proHALT-4 exhibited cytolytic activity, it was hypothesized that proteolytic cleavage at the paired basic site of HALT-4 is essential for enhanced activity in HeLa cells. The cytolytic activity was determined by measuring the concentration of recombinant protein that reduced cell viability by 50% (CC50). The standard deviation was taken into account for the analysis.The results showed that tKK1 displayed no significant difference in cell viability compared to proHALT-4 A. Both pHeteractis magnifica and sticholysins I and II from Stichodactyla helianthus [Actineria illosa showed a 9.2-fold increase in its hemolytic activity when expressed in E. coli BL21 (DE3) as compared to the native AvtI [It is known that the six recombinant proteins (full-length and truncated forms of HALT-4) contain an additional 40 residues originating from the pET28a vector, including a His-tag and a thrombin cleavage site. In our previous study, we observed significant cytolytic activity of recombinant HALT-4 despite the presence of additional residues from the pET28a vector [lianthus . On the ive AvtI . In bothStichodactyla helianthus, has been shown to be dependent on the thickness of lipid bilayer [The N-terminal region of actinoporins plays a critical role in initiating pore formation by inserting the N-terminal domain into the membrane . Thus, i bilayer . Accordi bilayer . As the Based on the present results, it can be concluded that RK3, KK4 and KK5 are not the proteolytic cleavage sites for HALT-4. Thus, the N-terminal region of HALT-4 is assumed to be longer than that of other HALTs and actinoporins. Regarding KK1 and KK2, cleavage at these sites did not increase the cytolytic activity of truncated HALT-4, and they may instead play a role in the regulated secretory pathway.2O, 5 µL of 10× Thermopol buffer, 1 µL of 10 mM dNTPs, 1 µL of 10 µM forward primer, 1 µL of 10 µM reverse primer, 1 µL of template (pET28a inserted with HALT-4) [Taq polymerase were incubated in the thermal cycler. The following cycles were used: 94 °C for 5 min; followed by 30 cycles of 94 °C for 30 s, 55 °C for 30 s and 72 °C for 1 min; and a final extension at 72 °C for 5 min. The primers used in this experiment are listed in Polymerase chain reaction (PCR) was performed to amplify truncated HALTs-4, KK1, KK2, RK3, KK4 and KK5. In brief, a mixture of 40 µL of ddHSalI (20 units/µL) and NotI (20 units/µL) at 37 °C for 2 h. The digested DNA fragments were purified using gel electrophoresis and the QIAquick Gel Extraction Kit . Truncated HALT-4 was ligated to pET28a using T4 DNA ligase at 16 °C overnight. The mixture was transformed into DH5α  after ligation. Plasmid was extracted from DH5α and sent for sequencing to confirm the reading frame of truncated-HALT-4 in pET28a. After the sequence was confirmed, the plasmid was transformed into Rosetta Gami 2 (DE3) .To prepare for ligation, both the PCR product and pET28a were digested with 600 0.5 to 0.6. The expression of truncated-HALT-4 was then induced with 1 mM IPTG and 3% ethanol for 3 h at 37 °C with agitation. The cell pellet was collected by centrifugation and stored at −20 °C. To purify the truncated HALT-4, the cell pellet was resuspended in denaturing lysis buffer . Soluble cell lysate was obtained by sonication and subjected to affinity chromatography of nickel-chelating resin, Ni-NTA Superflow . The purified protein was dialyzed in a series of refolding buffers  containing decreasing concentrations of urea  and finally in 1× phosphate-buffered saline (PBS) for protein refolding. The refolded proteins were examined using 12.5% SDS-PAGE.The expression and purification of truncated HALT-4 were carried out as described by Yap et al. . In brieg for 5 min at 4 °C. The supernatant was then removed, and the cells were resuspended in 1 mL MEM. The resulting mixture was transferred to a T75 flask containing 9 mL of 10% FBS–MEM and incubated at 37 °C with 5% CO2.The HeLa cell line (ATCC CCL-2) was obtained from ATCC, Manassas, VA, USA. To culture HeLa cells, the thawed cells were quickly transferred into 9 mL of minimum essential medium (MEM) and centrifuged at 200× 4 HeLa cells were seeded in 10% FBS–MEM in a 96-well plate and incubated for 16 h. After removing the FBS–MEM, the cells were treated with different concentrations of truncated HALTs-4  for 24 h. The cells were then incubated with 30 µL of 5 mg/mL 3--2,5-diphenyltetrazolium bromide (MTT) for 3 h at 37 °C. Next, the supernatant was removed and 200 µL of 100% DMSO was added into each well. The absorbance readings were measured at 570 nm with reference to 630 nm using a microplate reader . HeLa cells treated with 100% DMSO served as the positive control, while the negative control involved treating HeLa cells with 1× PBS instead of truncated HALTs-4. The assays were performed in triplicate, and the percentage of cell viability was calculated with standard deviation.To assess cell viability, 1 × 10


Staphylococcus aureus (MRSA) is a pervasive and persistent threat that requires the development of novel therapies or adjuvants for existing ones. Aptamers, small single-stranded oligonucleotides that form 3D structures and can bind to target molecules, provide one possible therapeutic route, especially when presented in combination with current antibiotic applications. BALB/c α-1, 3-galactosyltransferase (−/−) knockout (GTKO) mice were infected with MRSA via tail vein IV and subsequently treated with the αSA31 aptamer (n = 4), vancomycin (n = 12), or αSA31 plus vancomycin (n = 12), with split doses in the morning and evening. The heart, lungs, liver, spleen, and kidneys were harvested upon necropsy for histological and qPCR analysis. All mice treated with αSA31 alone died, whereas 5/12 mice treated with vancomycin alone and 7/12 mice treated with vancomycin plus αSA31 survived the course of the experiment. The treatment of MRSA-infected mice with Vancomycin and an adjuvant aptamer αSA31 reduced disease persistence and dispersion as compared to treatment with either vancomycin SA31 alone, indicating the combination of antibiotic and specifically targeted αSA31 aptamer could be a novel way to control MRSA infection. The data further indicate that aptamers may serve as a potential therapeutic option for other emerging antibiotic resistant pathogens.Methicillin-resistant  Staphylococcus aureus is a leading cause of bacteremia in the United States with 100,000 cases a year, although this pathogenic bacteria is also asymptomatically carried by 20–30% of the human population [S. aureus can manifest in a variety of ways, including skin and soft tissue infections (SSTI), bone, joint, and implant infections, and pneumonia and septicemia [S. aureus infection can also lead to chronic carriage in wounds and the onset of various toxicoses like toxic shock syndrome [S. aureus infection and carriage is not limited to humans. Many different species of animals experience comparable diseases, such as bovine mastitis, and zoonotic transmission from pigs to humans has been observed [S. aureus in both human and animal populations, it is concerning that multi-drug-resistant S. aureus is on the rise globally [observed ,5. Givenglobally .S. aureus infections, but such widespread use led to the appearance of penicillin-resistant S. aureus only a few short years after the introduction of the groundbreaking antibiotic [S. aureus (MRSA) was observed in 1961, only two years after clinics switched from using penicillin to methicillin to control S. aureus [S. aureus isolates are resistant to at least one antibiotic, with many being resistant to more than four antibiotics [In the 1950s, penicillin was successfully used to control tibiotic . Followiibiotics . More coibiotics ,9. Whileibiotics . With thThe development of antibiotic–adjuvants, compounds that increase the efficacy of antibiotics, could make existing therapies more effective at lower doses and with fewer side effects. One example is the commonly prescribed antibiotic–adjuvant mixture Augmentin, which combines the β-lactam antibiotic amoxicillin with the β-lactamase inhibitor clavulanic acid. The use of Augmentin, as opposed to amoxicillin alone, restores activity against amoxicillin-resistant bacteria and increases bacterial clearance ,12,13. N+) bacteria via activation of the complement pathway [One such alternate strategy is to leverage the patient’s own immune system and preexisting antibodies (Abs). Abs modulate the immune response and are naturally produced against foreign antigens, like the gal-α1,3-gal  epitope . α-gal i pathway .Anti-α-gal Abs readily react with α-gal on the surface of viruses, bacteria, and animal tissues consumed via diet. In a hypothetical mechanism like that of a sandwich ELISA, if the α-gal sugar were bound to a target molecule with high affinity to a pathogen (a.k.a. the primary Ab), the bound pathogen would then display α-gal from its surface. This would flag the pathogen for binding by circulating anti-α-gal Abs (a.k.a. the secondary Ab) and signal the adaptive immune system to clear the infection before mounting a new Ab response against specific pathogen-associated molecular patterns (PAMPs). Targeting molecules, the primary Ab in the ELISA metaphor, have already been suggested and studied for pathogen binding. Perdomo et al.  showed tStreptococcus bacteria has already been reported [Another class of possible targeting molecules are aptamers, single-stranded oligonucleotides (ssDNA or ssRNA) that assume three-dimensional conformations capable of binding small molecules, proteins, or whole cells. Aptamers specifically able to bind α-gal have earned the moniker alphamer, or α-mer. Aptamers are similar to Abs in that they often bind specific targets, analogous to Abs’ binding of epitopes. However, aptamers have multiple advantages over Abs including a smaller size, low immunogenic potential, higher target affinity and selectivity, and easy synthesis in vitro, and they allow for modifications that change their stability and specificity . The sucreported . Aptamerreported . The curreported ,31.S. aureus cells have already been reported by Cao et al. [S. aureus [As one possible path for combatting antibiotic resistance, the continued development of new aptamers increases the likelihood that successful target molecules could be found in tandem with the emergence of new resistant pathogens. A high-throughput selection process for novel aptamers has already been developed, known as the Systematic Evolution of Ligands by Exponential Enrichment (SELEX) , and cano et al. , establi. aureus .This present study builds on the aptamer study by Cao et al.  study anAll aptamers and α-mers were obtained from BioSearch . Aptamers were prepared from lyophilized powder in 0.9% sterile saline. Vancomycin  was obtained from the UC Davis School of Veterinary Medicine, prepared as per manufacturer’s protocol, and diluted to 20 mg/mL in 0.9% sterile saline prior to use. The GpC SA31 oligonucleotide was 5′capped with AminoC6 and produced by Integrated DNA Technologies Inc., Coralville, IA, USA.S. aureus ATCC 33591, was used in this study. S. aureus ATCC 33591 was thawed from −80 °C vials, transferred twice into BHI broth at 37 °C and grown to late log phase before use. The minimum inhibitory concentration of vancomycin for this strain was determined to be between 3 and 6 µg/mL, based on the growth inhibition of S. aureus ATCC 33591 cultured overnight in BHI with 0–15 µg/mL vancomycin.The methicillin-resistant strain, 5 cells/cm2 in a 96-well plate using DMEM/High Modified  with 16.6% fetal bovine serum (FBS) , non-essential amino acids (Thermo Scientific), 10 mM MOPS , 10 mM TES (Sigma), 15 mM HEPES (Sigma) and 2 mM NaH2PO4 (Sigma). Cells were incubated at 37 °C in 5% CO2 for 14 days post confluence to allow for differentiation to occur prior to adhesion assays [5 cells/cm2 in 48-well plates [Colonic epithelial cells  were obtained from the American Type Culture Collection  and grown as per the manufacturer’s instructions in T-25 flasks. Subsequently, for compound treatment, cells were seeded to a density of 10n assays . Human al plates ,35.2-3′ and 5′-α-gal-SA31-3C-3′) uncapped (5′-NH2-SA31-OH-3′) forms of SA31 were incubated in serum from Homo sapiens and Mus musculus, or in the presence or absence of RQ1 DNase 1 (1 unit/100 µL) , Exonuclease1 (2 units/100 µL), or T5 Exonuclease (20 units/100 µL)  at 37 °C for 0–24 h. Degradation was measured using qPCR using primers previously reported by Cao et al. [2O , and template DNA from samples at a final volume of 25uL per reaction well. All qPCR was performed using the Bio-Rad CFX96 real-time PCR instrument . Amplified products were verified using melt curve analysis from 50 °C to 95 °C with a transition rate of 0.2 °C/s and band size verification using the Agilent BioAnalyzer 2100  with the RNA chip to measure ssDNA products. Half-life was calculated using the equation t1/2 = (elapsed time × log 2)/log .A single aptamer (SA31) was used for this study based on previous binding studies as reported by Cao et al. . Differeo et al. ; amplifiS. aureus ATCC 33591 was grown as described above and re-suspended to an OD600 of 0.2 in DMEM/highly modified medium containing non-essential amino acids, 10 mM MOPS, 10 mM TES, 15 mM HEPES, and 2 mM NaH2PO4 without FBS before use in the adhesion assay. The differentiated epithelial cells were washed once with 200 µL of PBS just prior to the addition of the bacteria. The bacterial suspension (50 µL) was mixed with appropriate amounts of aptamers and mixed by inversion for 15 min prior to addition to the differentiated Caco2 cells at a final multiplicity of infection of 1:1000. The Caco2 cells treated with bacteria and aptamer treatments were incubated at 37 °C in an atmosphere containing 5% CO2 for 60 min to allow for the bacteria to associate with the epithelial cells. After incubation, each treatment was aspirated and the Caco2 monolayer washed three times with 200 µL of Tyrodes buffer (pH 7.2) [S. aureus femA gene [(pH 7.2) ,37 to re(pH 7.2)  and othe(pH 7.2) ,35, exceemA gene  with theemA gene  using unS. aureus, a growth curve was conducted with αSA31 in an automated plate reader  by measuring A600 every hour for 24 h in triplicate. Log-phase S. aureus was adjusted to an OD600 nm = 0.1 in a 96-well plate with BHI broth and 20% GTKO mouse serum. αSA31 was added to the appropriate wells at 5 ng/µL and incubated at 37 °C for 24 h. The plate was mixed for 30 s before each OD measurement.To determine if αSA31 inhibited the growth of S. aureus ATCC 33591was grown as described above to stationary phase and re-suspended to an OD600 of 2. The bacterial suspension was incubated with or without 20% serum, with or without 150 ng/µL aptamer, and finally with or without α-galactose-1,3 monoclonal IgM  diluted to 1:50, separately for 15 min each, prior to resuspension in FBS free RPMI/DMEM and addition to macrophages at a final multiplicity of infection of 1:100. S. aureus cells were washed 3X with PBS between incubations. The cells treated with pretreated S. aureus were incubated at 37 °C in an atmosphere containing 5% CO2 for 60 min to allow for the bacteria to associate with the cells. After incubation, each treatment was aspirated and the cells washed three times with 200 µL of PBS to remove non-adhered/phagocytized bacterial cells. Cells used to analyze phagocytosis were then incubated for 2 h in FBS-free media containing 100 µg/mL gentamycin. This media was removed, the cells washed once with PBS, and then mammalian and bacterial cells were lysed prior to qPCR using 50 µL of a commercial lysis buffer . CFU MRSA per cell was determined using qPCR to calculate copies S. aureus femA per copies mammalian GAPDH. The amplification parameters for the mammalian GAPDH and S. aureus femA primers were optimized for single-plate amplification to 95 °C for 5 min, followed by 40 cycles at 95 °C for 15 s, 62 °C for 30 s, and 72 °C for 30 s, then a final extension at 72 °C for 10. Phagocytized CFU/cell were subtracted from total associated CFU/cell to determine adhered cell amounts.This study was carried out in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All efforts were made to minimize animal suffering. All animal protocols received prior approval by the UC Davis Institutional Animal Care and Use Committee (Protocol Number: 16284).−/− (GTKO) mice were bred and maintained under standard conditions by the Center for Laboratory Animal Science at the University of California, Davis, which is accredited by the American Association for Accreditation of Laboratory Animal Care. Each mouse was immunized using rabbit ghost erythrocytes prepared as described by Dodge et al. [g for 20 min and washed three times with cold (4 °C) 1X PBS. The washed pellet was resuspended in 10–12 mL of cold 1X PBS. A total of 3 mL of the suspension was aliquoted into 27 mL of cold 1X PBS, incubated at 4 °C for 30 min, and centrifuged at 40,000× g for 20 min. The pellet was resuspended in 10 mL of cold sterile distilled deionized water, centrifuged at 20,000× g, 4 °C for 20 min, and washed with cold sterile distilled deionized water until the supernatant turned light pink to clear. The pellet was resuspended with cold 1X PBS and stored at 4 °C. Protein contents were quantified via the NanoDrop 2000 . Each mouse was immunized with 0.1 mL of approximately 1 mg/mL rabbit ghost erythrocyte suspension, boosted 1 week later, and monitored by α-gal Ab ELISA.BALB/c GTe et al.  before u2 overdose at the end of treatment or after loss of 20% of body weight. All blood samples were transferred into BD Microtainer Serum Separator tubes , and the serum separated as per manufacturer’s protocol. The serum was transferred into a clean 0.5 mL microcentrifuge tube and stored at −80 °C for further analysis. At necropsy, the kidneys, lungs, and spleen were collected from each mouse via dissection. Approximately one half of each organ was immediately placed in 10% buffered formalin acetate at room temperature for histology while the remaining organ tissue was placed in 1.5 mL micro-centrifuge tube and temporarily incubated on ice prior to storage at −80 °C for later analysis.Blood was collected daily from each mouse (~50 µL) via tail nick. Terminal blood draws were collected via intracardiac puncture immediately after euthanasia via COHistology was performed and scored by a single pathologist (L. Woods) blinded to the treatment groups. The scores were averaged for bacteria, inflammation, and necrosis (B/I/N) on a scale of 0–4, with 0 being healthy tissue with no bacteria and 4 being severely damaged tissue or numerous bacteria.S. aureus femA gene [Bacterial detection was carried out in each tissue using qPCR for the emA gene  with theemA gene  using unThe in vivo concentration of αSA31 was determined after injecting mice intravenously (IV) twice daily with αSA31 concentrations ranging from 150 µg/kg/day to 10,000 µg/kg/day. Serum was collected daily prior to morning dosage and qPCR, as described above, was used to determine the concentration of αSA31 in the serum of each animal.Black 96-well plates (Thermo Scientific) were coated overnight at 4 °C in a 100 mM bicarbonate/carbonate buffer (pH 9.6) with 50 µL of 2.5 mg/mL gal-α1,3-gal-human serum albumin with a 14-atom spacer . The wells were rinsed twice with phosphate-buffered saline plus 0.2% Tween 20 (pH 7.2) (PBST) and blocked with 200 µL of Superblock  overnight at 4 °C. Before use, the plates were washed twice with PBST, prior to the addition of 50 µL mouse serum was serially diluted in PBST and added to each well for 15–20 min at 37 °C while rocking; then, plates were washed twice with PBST. The secondary antibody, goat anti-mouse IgGAM conjugated to fluorescein isothiocyanate (FITC)  was diluted in PBS with goat serum, as per manufacturer’s protocol, and 50 µL was added to each well. Plates were sealed with foil and incubated for 20 min at 37 °C while rocking, washed once with PBST and washed once with PBS prior to the addition of 50 uL of PBS for fluorescence detection using a Beckman Coulter DTX 800  with an excitation wavelength of 495 nm and an emission wavelength of 535 nm. Background fluorescence from a blank well was subtracted to report ELISA response. All assays were carried out in triplicate.t-tests.Statistical analyses were performed using Prism 5 for Mac  and JMP10 for Mac . Serum αSA31 concentrations were analyzed via a repeated measures 2-way ANOVA and a Bonferroni multiple comparisons post-test. Anti-α-gal ELISA statistics were performed using 1-way ANOVA and a Bonferroni multiple comparisons post-test. Adhesion assay statistics were performed using 1-way ANOVA and a Dunnett’s multiple comparisons test to no drug control. Phagocytosis assay statistics were performed using a 1-way ANOVA and Tukey’s HSD post-test. Histology scores and aptamer half-life statistics were performed using a 2-way ANOVA and Tukey’s HSD post-test. Organ MRSA load statistics were segregated by survival and performed using nonparametric tests and Tukey’s HSD post-test or Student’s p < 0.05) association of MRSA in vitro, while a control anti-flu aptamer [αSA31 activity was initially characterized by examining in vitro the inhibition of MRSA growth and cellular adhesion. The addition of αSA31 to cell culture medium resulted in a significant decrease in the , 5′-α-gal-SA31-3C-3′ (αSA31) and 5′-NH2-SA31-OH-3′ (SA31) was tested via endonuclease and exonuclease treatment (2 and αSA31 were significantly more stable (p < 0.001) than SA31. No degradation of either αSA31NH2 or αSA31 was observed after 24 h at 37 °C. The addition of endonuclease or either exonuclease did not lead to any significant differences in degradation times between the three aptamers. T5 exonuclease was added to test if the aptamer modifications protected them against 5′ exonuclease attack. Protection against 5′ exonuclease attack was not conferred by either the 5′ α-gal or the amine linkage. In human and mouse serum, the three aptamers displayed different patterns of stability. While αSA31NH2 was significantly more stable (p < 0.01) in human serum, no significant difference was observed between the stability of αSA31NH2 and αSA31 in mouse serum.The stability of 5′-α-gal-SA31-NHreatment . In nuclp < 0.0003) the adhesion of MRSA cells to mouse macrophages when compared to MRSA pretreated with PBS. The pretreatment of MRSA with serum and SA31 showed that a similar number of MRSA cells were phagocytized, as seen with the serum-only pretreatment. Notably, over 3.5-fold more (p < 0.05) pretreated MRSA were phagocytized when also treated with αSA31, compared to all other treatments. Similar experiments were performed using human serum and human macrophages, but no significant difference was observed between the treatments  serum concentrations on days 2 and 3, compared to day 1, and showed a stabilization at approximately 2 ng/mL serum. The serum concentration of αSA31 in mice with the 10,000 µg/kg/day dose showed a marked decrease at day 4. To examine a possible mechanism of α-mer instability and to explain the observed serum concentration decrease, the level of α-gal antibody, anti-α-gal Abs, was also assessed in the serum before the first dose and after the first day. Circulating anti-α-gal was significantly lower at both dosing concentrations following α-mer treatment (p < 0.0008)  B.9 CFU/mouse dose of MRSA, as determined by preliminary experiments and prior published studies [The in vivo antimicrobial activity of αSA31 was tested via the application of αSA31 at increasing doses in combination with vancomycin to septic mice. Sepsis was induced with a 1 × 10 studies ,47,48,49MRSA and treated with up to 10,000 µg/kg/day of αSA31, with no addition of vancomycin. All mice with induced sepsis and αSA31 only treatment did not survive beyond day 2 . Septic p < 0.05). Vancomycin treatment of uninfected mice should have no effect in vivo on αSA31 stability.The in vivo stability of αSA31 was compared between infected and uninfected mice to determine the effect of an active infection on aptamer circulation. Mice infected with MRSA and treated with the vancomycin-αSA31 combination had in vivo αSA31 stability like that seen in uninfected mice on day 1 . The serp ≤ 0.05). Mice treated with vancomycin that died had a significantly higher amount of MRSA in the lungs (p < 0.05) and significantly lower amount of MRSA in the spleen (p < 0.02) compared to mice treated with vancomycin plus 10,000 µg/kg/day αSA31 (p = 0.05) and significantly lower bacterial loads in the lung, liver, and kidney  compared to mice treated with vancomycin alone. Spleens from the group treated with Vancomycin and αSA31 had three-fold more MRSA than the mice treated with vancomycin alone.The bacterial load (CFU/mg organ) in the kidney, lung, heart, liver, and spleen . Amongstay αSA31 . Mice trp < 0.02), bacterial foci than animals treated with vancomycin alone. The difference was especially pronounced in the kidney. MRSA distribution across the organs was also significantly different (p < 0.0008) between vancomycin + αSA31 and vancomycin-only animals. Though no significant difference in necrosis was detected between vancomycin + αSA31 and vancomycin treatment groups, there was a significant difference (p < 0.001) in the distribution of necrosis amongst the organs within each treatment group  blocks adhesion to epithelial cells. The capped αSA31 aptamer was also readily bound by preformed anti-α-gal Abs both in vivo and in vitro. The pretreatment of MRSA with αSA31, followed by the addition of monoclonal anti-α-gal IgM (mAb), blocked the phagocytosis of MRSA by mouse macrophages, indicating that mAb readily detected αSA31 on the surface of MRSA. Furthermore, the treatment of mice with αSA31 doses ranging from 300 µg/kg/day to 10,000 µg/kg/day resulted in the depletion of anti-α-gal Abs from serum. The reduction in free anti-α-gal Abs from serum indicates the circulating preformed Abs successfully detected and bound the αSA31 aptamer. This same trend of Abs reduction was also seen with 1200 µg/kg/day and 10,000 µg/kg/day of αSA31 plus vancomycin during MRSA infection, though the depletion in these cases was not statistically significant.Previous work from Cao et al.  showed tS. aureus, are resistant to the lysis methods of this pathway [To clear infection, ideally, host-produced serum anti-α-gal Abs would bind to the αSA31 on the surface of a MRSA cell and activate the alternative complement pathway. The alternative complement pathway activation is tightly regulated and is a primary line of defense against infection, but many Gram-positive bacteria, especially  pathway ,52,53. I pathway ,55. ThisThe in vitro characterization experiments show that αSA31 did not exhibit MRSA growth inhibition but did increase MRSA phagocytosis by macrophages, indicating a potential ability to control MRSA infection in vivo. Prior to in vivo application, however, aptamer modifications were tested for their stability and potential toxicity. Aptamer modification positively altered stability in serum, leading to the selection of a capped α-mer that was resistant to nuclease digestion for use during the rescue experiments. The in vivo application of αSA31 showed the aptamer was removed by circulating anti-α-gal Ab, but no toxicity was observed, even at 10,000 µg/kg/day. Considering the increased stability and lack of toxicity, even at high concentrations, αSA31 was applied to control MRSA infection in septic mice.9 CFU/mouse) may have been too high to show a survival effect with an indirect treatment, like αSA31, alone, as opposed to vancomycin, which acts directly on the microbe.Having demonstrated that anti-α-gal Abs can bind the aptamer in vivo and that the α-mer specifically interacts with, and increases the phagocytosis of, MRSA in vitro, we hoped to show that αSA31 alone could rescue infected mice. While complement may not result in direct lysis, opsonization via anti-α-gal Abs should result in the increased phagocytosis and killing of bacteria by immune cells ,58,59,60While survival was not affected by treatment with αSA31 alone, the overall bacterial load in the organs was significantly affected by the presence of αSA31, especially when used in combination with vancomycin. In all organs except the spleen, mice treated with vancomycin plus αSA31 had lower CFU/mg organs than mice treated with vancomycin alone. This was particularly prominent in the lung, where the bacterial load of the vancomycin alone group was significantly higher than the aptamer–vancomycin group. High loads of MRSA in the lung can result in life-threatening pneumonia and is a common complication of nosocomial MRSA infection . The redThe treatment effect seen with αSA31 application supports the hypothesis that αSA31 tags MRSA for immune detection and assists in clearing the MRSA infection, but only as an adjuvant to vancomycin. Vancomycin is a glycopeptide antibiotic that slows the growth of MRSA through the inhibition of cell-wall synthesis. The slowed growth from vancomycin treatment may increase opportunities for the interactions between αSA31-tagged MRSA and the immune system, ultimately leading to increased phagocytosis by macrophages. This may also explain the higher concentration of MRSA CFU/mg spleen observed in the combination treatment group, as macrophages with phagocytized MRSA traffic back to the spleen . The cooThere may be an even more efficient approach for signaling the immune system with α-mers than targeting the whole cell with a single aptamer. The use of multiple capped α-aptamers could lead to a higher amount of α-gal on the MRSA cell surface, as demonstrated by Cao et al., with multiple aptamers and increased fluorescence . AlternaA known evader of the immune system, MRSA employs many evasive and anti-phagocytic strategies to go undetected by the innate immune system . SeveralWhile the rescue of MRSA-infected mice was not achieved with αSA31 alone, a synergistic effect was observed when vancomycin and αSA31 were used in combination. Though further optimization of this antibiotic–adjuvant is clearly required before it can be applied clinically, the strategy of targeted aptamer and antibiotic infections could prove extremely beneficial in the treatment of sepsis for a wide range of antibiotic-resistant bacteria.


LncRNAs have been shown to be involved in and control the biological processes of multiple diseases, including preeclampsia (PE). The impairment of trophoblast cell proliferation is recognized as a significant anomaly contributing to the development of PE. LncRNA FEZF1-AS1 was found downregulated in placental tissues of PE patients. However, the precise regulatory mechanism of FEZF1-AS1 in placental trophoblast proliferation and apoptosis remains unclear.In this study, we conducted an investigation into the expression levels of FEZF1-AS1 and NOC2L in placental tissues obtained from patients diagnosed with PE. Subsequently, we employed CCK-8 and EdU assays to quantify cell proliferation, while TUNEL staining and western blot for apoptosis-related protein detection to assess apoptosis. Furthermore, the interactions between FEZF1-AS1 and ELAVL1, as well as NOC2L and ELAVL1, were confirmed through the implementation of RIP and RNA pull-down assays. We found a downregulation of lncRNA FEZF1-AS1 and NOC2L in placental tissues of PE patients. Overexpression of FEZF1-AS1 or NOC2L resulted in increased cell proliferation and inhibition of apoptosis, whereas knockdown of FEZF1-AS1 or NOC2L had the opposite effect. In addition, lncRNA FEZF1-AS1 stabilized NOC2L mRNA expression by interacting with ELAVL1. Moreover, partial reversal of the effects of FEZF1-AS1 overexpression on cell proliferation and apoptosis was observed upon suppression of ELAVL1 or NOC2L.PE related lncRNA FEZF1-AS1 could regulate apoptosis and proliferation of placental trophoblast cells through the ELAVL1/NOC2L axis. LncRNA FEZF1-AS1 and NOC2L modulates the proliferation and apoptosis of trophoblast cells.FEZF1-AS1 stabilizes the mRNA stability of NOC2L by interacting with ELAVL1.FEZF1-AS1/ELAVL1/NOC2L axis regulates trophoblast cell proliferation and apoptosis. Preeclampsia (PE), an idiopathic hypertensive syndrome, is a significant contributor to adverse outcomes such as hemorrhage, abortion, preterm birth, fetal, and maternal death, occurring after the 20th week of pregnancy . SurprisLong non-coding RNA (lncRNAs) are a class of RNA molecules whose transcript length exceeds 200nt . LncRNAsNOC2 like nucleolar associated transcriptional repressor (NOC2L) was initially discovered as a a suppressor of in vivo histone acetyltransferase. It functions as a transcriptional co-repressor of p53 and regulates the apoptosis mediated by p53 . The regTherefore, we hypothesize that FEZF1-AS1 stabilizes NOC2L by specifically binding to ELAVL1, thereby promoting trophoblast proliferation, inhibiting apoptosis, and alleviating PE disease. In other words, the lncRNA FEZF1-AS1 may alleviate PE disease by regulating trophoblast apoptosis and proliferation.We used qPCR to analyze the expression of FEZF1-AS1 in PE and control  tissues. As shown in Fig. We performed qPCR on PE and normal samples, and identified that NOC2L was significantly downregulated in PE tissues   Fig. A. Besiden/) Fig. B. To furn/) Fig. C. Furthen/) Fig. D. In sumIn order to further understand the regulatory effect of the combination of FEZF1-AS1 and ELAVL1 on NOC2L, knockdown and overexpression of FEZF1-AS1 were performed in HTR-8/SVneo cells. The knockdown of FEZF1-AS1 inhibited the mRNA and protein expression levels of NOC2L, whereas the overexpression of FEZF1-AS1 promoted the mRNA and protein expression levels of NOC2L Fig. A, B. AddTo investigate the signaling axis of FEZF1-AS1/ELAVL1/NOC2L in trophoblast cells, we transfected oe-NC, oe-FEZF1-AS1, oe-FEZF1-AS1 + si-ELAVL1, and oe-FEZF1-AS1 + si-NOC2L into HTR-8/SVneo cells. Overexpression of FEZF1-AS1 upregulated cell activity and proliferation, and inhibition of NOC2L or ELAVL1 partially reversed these changes Fig. A, B. OveThe harms of PE disease include inducing fetal death, fetal dysplasia, and damage to various maternal organs . RecentlMore and more evidence manifested that lncRNAs developed a significant role in PE . DiffereNOC2L, also known as NIR, is involved in cell proliferation and apoptosis . One posIn short, this study demonstrated that lncRNA FEZF1-AS1 is lowly expressed in PE patients, and FEZF1-AS1 acts as a protective factor by promoting trophoblast cell proliferation and inhibiting apoptosis. It promotes the upregulation of NOC2L expression by binding to ELAVL1. Thus, the FEZF1-AS1/ELAVL1/NOC2L axis may be a valuable target for therapy in patients with PE. Unfortunately, this study has not been further validated through animal experiments, which will be the focus of our future research. Additionally, the sample size would be increased, and including more patient characteristic information.LncRNA FEZF1-AS1 played a vital role in inhibiting the development of PE disease. Its possible underlying mechanism involves promoting cell proliferation and suppressing apoptosis by bolstering the stability of NOC2L mRNA through the recruitment of ELAVL1 Fig. . Our curThe placental tissue was collected from patients with PE and from normal controls (n = 30). This research was authorized by the Medical Ethics Committee of The Affiliated Taian City Central Hospital of Qingdao University (No.202118) with the written informed consent of each participant. Inclusion criteria for the PE group were as follows: (1) diastolic blood pressure ≥ 90 mmHg from two recordings or systolic blood pressure ≥ 140 mmHg; (2) a 24 h urinary protein of ≥ 0.3 g, or a random urinary protein of 1 + or more. Within 5 min of surgical removal of placental tissue, the placenta was cut into 1 cm × 1 cm × 1 cm cubes. The cubes were then washed repeatedly with phosphate buffered saline (PBS) until all blood was removed. The sterile placenta cubes were preserved at − 80 °C for subsequent experiments.2. Small interfering RNAs , overexpressed vectors , and relative negative controls  were obtained from GenePharma . According to the manufacturer’s guidelines, cells were transfected with si-RNAs or vectors for 48 h using Lipofectamine 3000 .HTR-8/SVneo cells (ATCC) were cultured in RPMI-1640 medium  supplemented with 10% FBS (Gibco), 100 U/mL penicillin, and 100 μg/mL streptomycin. All cell lines were cultured in a humidified air environment at 37 °C with 5% COIn brief, the medium was replaced with pre-warmed medium (100 µL) per well in 96-well plates. CCK-8 reagents  were added directly, and then the cells were incubated for 2 h at 37 °C. The absorbance at 450 nm was recorded using a microplate reader . The untreated cells were normalized in order to calculate the relative cell viability.−∆∆Ct method, with GAPDH serving as the internal reference.The TRIzol (Invitrogen) method was used to extract total RNA from HTR-8/SVneo cells. The PrimeScript RT Reagent Kit with gDNA Eraser  was used to reverse transcribe RNA into cDNA. The qPCR assay was performed using the SYBR Green kit (Thermo Fisher Scientific) to measure the amplification of the target genes. The primer sequences were as follows: FEZF1-AS1 , NOC2L , ELAVL1 , GAPDH . Data analysis was conducted using the 2The extracted protein samples were transferred onto polyvinylidene difluoride membranes after being loaded onto a sodium dodecyl sulfate/polyacrylamide gel for electrophoresis. The membranes were blocked with 5% bovine serum albumin for 1 h before being incubated with primary antibodies overnight at 4 °C: Bcl-2 , Bax , cleaved Caspase3 , ELAVL1 , NOC2L , and GAPDH . The secondary antibody conjugated with horseradish peroxidase was used to incubate the membranes for 1 h. The enhanced chemiluminescence substrate  was finally used to detect the protein bands.A EdU kit  was used to measure cell proliferation following standard procedures. In brief, the treated cells were uniformly seeded in 96-well plates and incubated in a constant temperature environment for 24 h to achieve 60% fusion. 200 µL of a 50 µM EdU solution was added to each well and incubated for 2 h before being discarded. After washing with PBS buffer, the cells were fixed by adding 4% paraformaldehyde for 30 min. The formaldehyde was neutralized by adding a 2 mg/mL glycine solution, and the cells were washed with a PBS buffer. Under dark conditions, 100 µL of Apollo staining reaction solution was added to each well and incubated for 30 min at room temperature. Then, the cells were permeated by adding an osmotic agent (PBS containing 0.5% Triton X-100) and subsequently incubated with DAPI (Invitrogen) to stain the nucleus. The images were captured and analyzed using a fluorescence microscope, and the percentage of EdU-positive cells was calculated.The trophoblast cells were washed twice with PBS, then fixed with 4% paraformaldehyde. After fixation, the cells were infiltrated with a balancing buffer containing 0.2% Triton X-100. Then, cell DNA fragmentation was measured using the TUNEL method , following the manufacturer's manual. Images were obtained using a fluorescence microscope, and the percentage of TUNEL-positive cells was calculated to assess apoptosis.The biotinylated FEZF1-AS1/NOC2L RNA (Bio-FEZF1-AS1-sense/antisense or NOC2L-sense/antisense) was designed and synthesized by Thermo Fisher Scientific (USA). The probes were transfected into HTR-8/SVneo cells for 48 h, then treated with streptavidin magnetic beads (Invitrogen). After washing off the unbound proteins, the RNA–protein complex is eluted from the beads using an eluent. Finally, ELAVL1 enrichment was detected by western blot.For RIP assay, the EZ-Magna RIP Kit was bought from Millipore  and utilized in line with the user guide to investigate the interaction between ELAVL1 and NOC2L. Cells were lysed in the polysome lysis buffer and incubated with ELAVL1 antibody or isotype‐matched control antibody  conjugated magnetic beads. The beads were collected and washed, followed by RNA purification from the RNA–protein complex. The co-precipitated RNA enrichment was detected by qPCR and 3 biological replicates were carried out.After transfection, cells were treated with actinomycin D (5 μg/mL) for 0, 3, and 6 h, respectively. Then, cells were collected for total RNA extraction. cDNA was synthesized through reverse transcription, and the levels of target RNA and reference RNA were detected using qPCR.All data were obtained from at least three replicate experiments and summarized as means ± standard deviation (SD). After evaluating the normal distribution with the Shapiro–Wilk test, statistical analysis was performed using GraphPad Prism 8.0 (GraphPad Software) with either one-way ANOVA for multiple groups or Student's t-test for two groups. p < 0.05 was considered statistically significant.