This cohort study assesses the memory clinic prevalence and prognosis of individuals with a β-amyloid–negative, tau-positive cerebrospinal fluid biomarker profile. What is the memory clinic prevalence and prognosis of individuals who have a β-amyloid–negative, tau-positive (A−T+) cerebrospinal fluid (CSF) profile?In a clinical cohort of 7679 individuals, prevalence of A−T+ CSF was 4.1%. Longitudinally, cognitively unimpaired individuals or individuals with mild cognitive impairment and an A−T+ CSF profile exhibited similar trajectories to individuals with an A−T− CSF profile in relation to cognitive deterioration, brain atrophy, or cerebral glucose metabolism and β-amyloid burden indexed by positron emission tomography.A CSF profile of A−T+ appears to be benign despite being classified as a pathologic change by guidelines; compared with individuals with biomarker-negative CSF, individuals with A−T+ CSF do not have higher rates of cognitive decline or faster disease progression. Knowledge is lacking on the prevalence and prognosis of individuals with a β-amyloid–negative, tau-positive (A−T+) cerebrospinal fluid (CSF) biomarker profile.To estimate the prevalence of a CSF A−T+ biomarker profile and investigate its clinical implications.This was a retrospective cohort study of the cross-sectional multicenter University of Gothenburg (UGOT) cohort (November 2019-January 2021), the longitudinal multicenter Alzheimer Disease Neuroimaging Initiative (ADNI) cohort , and 2 Wisconsin cohorts, Wisconsin Alzheimer Disease Research Center and Wisconsin Registry for Alzheimer Prevention . This was a multicenter study, with data collected from referral centers in clinical routine (UGOT) and research settings (ADNI and WISC). Eligible individuals had 1 lumbar puncture , 2 or more cognitive assessments (ADNI and WISC), and imaging (ADNI only) performed on 2 separate occasions. Data were analyzed on August 2022 to April 2023.18F]-florbetapir amyloid positron emission tomography (PET), magnetic resonance imaging (MRI), [18F]-fluorodeoxyglucose PET (FDG-PET), and cross-sectional tau-PET .Baseline CSF Aβ42/40 and phosphorylated tau (p-tau)181; cognitive tests . Exposures in the ADNI cohort included [Primary outcomes were the prevalence of CSF AT biomarker profiles and continuous longitudinal global cognitive outcome and imaging biomarker trajectories in A−T+ vs A−T− groups. Secondary outcomes included cross-sectional tau-PET.A total of 7679 individuals  were included in the UGOT cohort, 970 individuals  were included in the ADNI cohort, and 519 individuals  were included in the WISC cohort. The prevalence of an A−T+ profile in the UGOT cohort was 4.1% , being less common than the other patterns. Longitudinally, no significant differences in rates of worsening were observed between A−T+ and A−T− profiles for cognition or imaging biomarkers. Cross-sectionally, A−T+ had similar tau-PET uptake to individuals with an A−T− biomarker profile.Results suggest that the CSF A−T+ biomarker profile was found in approximately 5% of lumbar punctures and was not associated with a higher rate of cognitive decline or biomarker signs of disease progression compared with biomarker-negative individuals. In recent research criteria, low Aβ42 or Aβ42/40 ratio and increased concentration of p-tau have been proposed to support the diagnosis of AD,2 Because p-tau increases are suggested to be AD specific,5 and increases would not be expected in Aβ-negative individuals, this finding is challenging to interpret. Guidelines vary in the terminology assigned to this finding, mostly encompassing the concept of suspected non-Alzheimer pathology.6 For example, studies evaluating the prevalence of CSF biomarker abnormalities suggest that such an A−T+ CSF profile can be found in up to 20% of cognitively unimpaired (CU) older adults.10 In addition, available studies show mixed results in the progression of disease-related changes in individuals with this biomarker profile.11 Previously, various propositions have been made to explain its underlying cause, including non-AD tauopathies  and altered CSF dynamics, among others.12 Importantly, these explanations likely vary depending on the biomarker modality being used. Unlike Aβ biomarkers, which seem to provide similar information in terms of “A” status when determined by positron emission tomography (PET) or CSF, tau biomarkers present pronounced differences across imaging and fluid modalities. Although fibrillar tau aggregates targeted by tau-PET radiotracers are likely a more direct proxy of tangle pathology,14 soluble p-tau measured by immunoassays may reflect a neuronal response to Aβ pathology,18 although p-tau is also present in dystrophic neurites, suggesting that it also reflects tau pathology.20 Although evidence is accumulating that individuals with an A+T− biomarker profile  are at risk for cognitive decline,10 less is known about the clinical implications of the CSF A−T+ pattern. Because CSF biomarkers are frequently used in clinical practice, it is important to further understand the implications of patterns not expected within the currently proposed model of AD biomarker progression.Normal CSF Aβ in combination with increased p-tau is a finding sometimes seen in clinical settings.Given the lack of studies directly evaluating the clinical relevance and prevalence of an isolated increase in CSF p-tau, we aimed to assess the prevalence, as well as cognitive and biomarker progression rates, associated with the CSF A−T+ profile, using 4 large data sets.STROBE) reporting guidelines.Four cohorts were used in the present study: University of Gothenburg (UGOT), the Alzheimer Disease Neuroimaging Initiative (ADNI), Wisconsin Registry for Alzheimer Prevention (WRAP), and Wisconsin Alzheimer Disease Research Center (WI-ADRC). For each cohort, a local institutional review board for human research or ethics committee approved of the study. The UGOT cohort consisted of cross-sectional data and was retrospectively compiled using anonymized data from the clinical routine laboratory database at Sahlgrenska University Hospital, Gothenburg, Sweden, based on CSF analyses performed between November 2019 and January 2021. ADNI is a longitudinal multicenter observational study with data being collected between September 2005 and May 2022. WRAP and the Wisconsin Alzheimer Disease Research Center (WI-ADRC) are single-center, longitudinal, observational, cohort studies that collected data between February 2007 and November 2020. The present study followed the Strengthening the Reporting of Observational Studies in Epidemiology , we included only individuals who were CU or had mild cognitive impairment  at the first cognitive evaluation. In the UGOT cohort, no data on participant race and ethnicity were available, as they are not routinely collected by health care services. In the ADNI cohort, race categories included were American Indian or Alaska Native, Asian, Black, Hawaiian, multiracial, and White. In the WISC cohort, individuals self-identifying as White were reported, whereas American Indian or Alaska Native, Asian, Black or African American, or those not identifying as any of the stated races (here indicated as other), were not reported to maintain anonymity for groups with less than 3 individuals. More information on all cohorts used can be found in eMethods 1 in 23 In both the ADNI and WISC cohorts, p-tau was quantified using a fully automated Elecsys assay (Roche), also recently approved by the US Food and Drug Administration. Aβ42 and Aβ40 were measured with mass spectrometry and Elecsys assays in the ADNI and WISC cohorts, respectively. Elecsys Aβ42 is an in vitro diagnostic test approved for use in the European Union and US; Aβ40 was measured using a research use−only Elecsys assay. CSF status determined at baseline lumbar puncture was tested in the WISC analyses. In all cohorts, previously established cohort-specific cutoffs were used. In ADNI, cutoffs of 0.0138 or less and 24 pg/mL or greater were used for Aβ42/40 and p-tau, respectively. In WISC, abnormal biomarker results were determined to be 0.046 or less and 24.8 pg/mL or greater for Aβ42/40 and p-tau, respectively. More information on the CSF collection and quantification of and cutoff determination for Aβ42, Aβ40, and p-tau can be found in eMethods 2 in CSF Aβ42/40 ratio and p-tau181 (hereafter referred to as p-tau) were quantified in all participants and were used to define AT status in all 3 cohorts. In the UGOT cohort, CSF concentrations of Aβ42, Aβ40, and p-tau were quantified on the Lumipulse G1200 platform (Fujirebio), a fully automated assay recently approved by the US Food and Drug Administration.18F]-florbetapir, [18F]-fluorodeoxyglucose (FDG) PET, and hippocampal volume magnetic resonance imaging (MRI). A brief description of imaging methods can be found in eMethods 3 in 25 As a secondary analysis, we evaluated summary tau-PET measures between CSF AT groups, to investigate whether elevated p-tau in the absence of Aβ could represent an abnormal tau-PET phenotype. In the WISC sample, a subset of participants underwent [18F]-MK6240 tau-PET and were evaluated with an entorhinal cortex region of interest (ROI), as further detailed in eMethods 3 in 26 In the ADNI cohort, a subsample of the individuals herein included had available [18F]-flortaucipir tau-PET data up to 6 years after the baseline lumbar puncture, and the previously described ADNI meta-temporal ROI was used as the composite region.27In longitudinal imaging analyses in ADNI participants, we included those who had at least a baseline and a follow-up scan. We evaluated longitudinal trajectories of Aβ -MK6240 tau-PET SUVr than the A−T− group . In both the ADNI and WISC cohorts, the A+T+ group had higher tau PET SUVr  compared with the A−T+ group. Differences were less clear for the CSF A+T−group , we evaluated whether isolated p-tau positivity was associated with the deposition of fibrillar tau . In the In this cohort study, we found that the prevalence of CSF A−T+ in a clinical neurochemistry laboratory, likely to be representative of a general memory clinic population, was 4.1%, which is far lower than the prevalence of other CSF profiles. Further, compared with the A−T− biomarker profile, we found that the A−T+ profile was not associated with higher rates of cognitive deterioration, Aβ accumulation, tau PET pathology, or neurodegeneration.33 This slight discrepancy may be explained by the presence of other disorders causing cognitive symptoms in our cohort and likely reflects more similarly what may be expected in a real-world memory clinic, considering most CSF analyses are ordered by memory clinics or general neurology centers across Sweden. However, fewer studies have performed in-depth evaluations of the clinical meaning of CSF results not consistent with the current view of the chain of AD pathophysiological events. Previous studies evaluating the prevalence of CSF AT abnormalities report mixed results. Although many studies report higher figures (above approximately 15%) for the prevalence of an A−T+ profile, most of these have relied on CSF Aβ42 alone to define “A” status.35 When defining “A” with the widely recommended Aβ42/40 ratio,36 this prevalence is substantially reduced to figures like the one reported here for this large, unselected clinical chemistry cohort (approximately 4%).8 This suggests that the absence of correction for Aβ40 concentrations may lead to artificially deflated frequencies for CSF Aβ abnormalities, and, consequently, artificially inflated frequencies for a CSF A−T+ profile. This highlights the need for using CSF Aβ42/40 in clinical settings when separately assessing “A” and “T” CSF biomarkers.Several studies have attempted to determine the prevalence of AD pathophysiology in varying scenarios. We found that in the UGOT cohort, CSF Aβ abnormalities ranged from approximately 25% in the youngest individuals to roughly 60% in the oldest individuals. These estimates are lower than those found in a recently published, large, multicenter study evaluating the prevalence of Aβ positivity as determined by either PET or CSF.6 More specifically, during the early phases of AD CSF biomarker research, p-tau was considered by some studies as a biomarker of neuronal injury.10 However, it is now known that biomarker abnormalities, especially in “T” and “N” (for neurodegeneration) categories, do not provide the same information within and between modalities.35 Some authors argue that a CSF A−T+ pattern may reflect tau pathophysiology not attributable to Aβ accumulation, such as primary age-related taupathy, but this theory has become increasingly untenable due to the accumulation of fluid biomarker evidence that tangle pathology in the absence of concurrent Aβ pathology is not well reflected by p-tau,38 although data specifically addressing this question are still scarce. Importantly, we found that the A−T+ group had similar tracer retention in temporal areas as the A−T− group, suggesting that an isolated elevation of CSF p-tau is not due to fibrillar tau pathology in the absence of Aβ pathology. However, it is important to note that to date, no convincing evidence has shown that tau-PET tracers are capable of capturing PART, which remains a lingering question for the field.Furthermore, discussing the etiology underlying a CSF A−T+ profile is highly needed. For instance, individuals presenting with this profile are sometimes referred to as suspected non-Alzheimers pathology. This construct is most often biomarker defined as Aβ negativity in the presence of neurodegeneration and a clinical picture not clearly consistent with a recognizable non-AD phenotype.39 Interestingly, in the UGOT and ADNI cohorts, concentrations of Aβ40 and Aβ42 were clearly higher in the A−T+ group, compared with the A−T− group. This could potentially suggest that the CSF A−T+ profile is indeed associated altered CSF dynamics, given that within 2 Aβ-negative groups as defined by the Aβ42/Aβ40 ratio, it would not be expected that Aβ42 and Aβ40 values alone would differ between groups.In this challenging context of determining the underpinnings of a CSF A−T+ profile, a potentially better-suited explanation is that the finding of increased p-tau in the absence of brain amyloidosis reflects slower CSF turnover. As an example of how alterations in CSF dynamics can impact biomarker readings, low concentrations of all core biomarkers  are commonly seen in cases of idiopathic normal pressure hydrocephalus, not necessarily associated with AD pathophysiology.40 However, when defining A−T+ with both Aβ− and tau-PET, individuals with this profile show slightly greater risk of cognitive decline than biomarker-negative individuals.41Data from both longitudinal cohorts suggest that individuals with an A−T+ pattern had similar cognitive and biomarker trajectories as compared with participants with a CSF A−T− profile. This aligns with recent findings in individuals with subjective cognitive decline that showed that the A−T+ group was not more likely to progress to MCI or dementia compared with the biomarker negative group when defining “A” using Aβ PET or Aβ42 and “T” using p-tau.23 and concordance with amyloid PET usually reaches over 90%,42 which in turn makes cutoff determination and interpretation uncomplicated. However, p-tau typically shows no such distribution and is less clearly linked with tau-PET status.43 Here, we show that both p-tau cutoff values that optimize sensitivity and specificity, as well as higher, less sensitive cutoff values presented very similar results in our study.Lastly, the choice of cutoff values may influence the interpretation of abnormal results. For Aβ42/40, this is likely of limited relevance because it usually presents a bimodal distributionStrengths of this study include the large sample size of participants with core CSF biomarkers of Aβ and soluble tau pathology, allowing us to derive estimated prevalence of these pathologies in a real-world, unselected clinical routine sample, for whom memory clinic physicians order most CSF tests. Further, our consistent finding that CSF A−T+ individuals do not show significant cognitive decline suggests that the findings are generalizable across different populations. Limitations include limited clinical information from the UGOT cohort, as the data are derived from a database used in laboratory practice. This precludes us from defining strict inclusion/exclusion criteria and describing the clinical features of the patients included. Another potential limitation is that the longitudinal cohorts consisted primarily of self-identified White individuals. Finally, AT status in the WISC sample was based on CSF tests not necessarily coinciding with the cognitive baseline visit.In this cohort study, we found the estimated prevalence of a CSF A−T+ profile to be 4.1% in a large, real-world, clinical routine data set. Further, individuals with this profile did not present with significantly higher rates of cognitive decline or AD biomarker progression than those of individuals with an A−T− profile. We suggest that practitioners encountering this pattern in daily clinical practice should interpret such a finding similarly to CSF biomarker-negative results during the diagnostic workup of patients with cognitive complaints, and unusually high CSF Aβ42 and Aβ40 concentrations could be a recognizable feature of such a profile.


Background: Gastric cancer (GC) is a common malignant tumor with a poor prognosis. Combination treatments may prolong the survival of patients with GC. Acacetin, which is a flavonoid, exerts potent inhibitory effects on several types of cancer cells; however, the mechanisms of action remain poorly understood.Methods: Network pharmacology and RNA sequencing were used to predict the targets of acacetin, which were then verified by drug affinity responsive target stability (DARTS), cellular thermal shift assay (CETSA) and molecular docking. The biological functions of acacetin in MKN45 and MGC803 cells were investigated using TUNEL assays, crystal staining and colony formation assays. The pathways affected by acacetin were verified through reverse experiments. The in vivo antitumor efficacy of acacetin was assessed in a subcutaneous xenotransplanted tumor model.Results: In this study, we identified EGFR from more than a dozen predicted targets as a protein that directly binds to acacetin. Moreover, acacetin affected the level of phosphorylated EGFR. In vitro, acacetin promoted the apoptosis of GC cells. Importantly, EGFR agonists reversed the inhibitory effects of acacetin on the STAT3 and ERK pathways. In vivo, acacetin decreased the protein levels of pEGFR in tumors, resulting in increased GC xenograft tumor regression without obvious toxicity.Conclusion: Our findings highlight EGFR as one of the direct targets of acacetin in GC cells. Acacetin inhibited the phosphatase activity of EGFR in vitro and in vivo, which played a role in the antitumor effects of acacetin. These studies provide new evidence for the use of acacetin as a potential reagent for the treatment of GC. Gastric cancer is the fifth most common cancer and the third leading cause of cancer-related death worldwide ; nearly Acacetin is a natural flavone with therapeutic potential for many diseases, and it is widely found in plants and dietary sources. Various biological antioxidant and anti-inflammatory activities of acacetin, especially anticancer activities, have been reported, and subsequent research has already advanced the understanding of these activities . In addiAcacetin (purity 98%) was purchased from Tauto Biotech Co., Ltd.  and dissolved in DMSO. Primary antibodies against PI3K p85 (4292), EGFR (4267), Met (8198), GSK3β (9315), MMP9 , p-EGFR (4404 and 2234), Bax (2774), Bcl-2 , Bcl-xl (2764), Cleaved Caspase-3 (9661), Caspase-3 (9662), Cleaved PARP (5625), PARP (9532), p-STAT3 (9145), STAT3 (9139), p-ERK (4370), Erk1/2 (4695), PCNA , and Ki-67 (9027), as well as HRP-linked anti-rabbit IgG (7074) and HRP-linked anti-mouse IgG (7076) were purchased from Cell Signaling Technology . Antibodies against PTGS2 (D223097), HAVCR1 (D260086), TMPRSS4 (D123375), GJB4 (D260904), KRT17 (D120232), EGR1 (D120585) and AKR1B10 (D221558) were obtained from Sangon Biotech . An antibody against GAPDH  was purchased from Proteintech . Human EGF (AF-100-15) was purchased from Peprotech (United States).Two GC cell lines, namely, MKN45 cells (gastric signet-ring cell carcinoma cell line that originated from lymph node metastasis) and MGC803 cells (poorly differentiated mucinous adenocarcinoma cell line), were purchased from the Shanghai Institute of Cell Biology, Chinese Academy of Sciences . Both cell lines were grown in RPMI-1640 medium (Gibco) supplemented with 10% FBS (Gibco).http://www.swisstargetprediction.ch/) (http://lsp.nwu.edu.cn/browse.php) (https://www.genecards.org/) (https://www.uniprot.org/) or STRING database (https://string-db.org/), the target proteins were converted to gene symbols. The STRING database was used to construct a PPI (protein-protein network). The compound-target-pathway network analyses were performed using Cytoscape 3.7.2 software. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were performed with the DAVID 6.8 database (https://david.ncifcrf.gov/).The targets of acacetin were identified by Swiss target prediction (ion.ch/)  and Tradwse.php) . GC-relads.org/) . Then, bMKN45 cells were treated with solvent control (DMSO) or acacetin (40 μM) in three biological replicates for 24 h. TRIzol reagent was added to extract total cellular RNA. Total RNA was analyzed by a NanoDrop ND-2000 spectrophotometer and Agilent Bioanalyzer 4200  for quality assessment, and qualified RNA was used for subsequent library construction. The constructed libraries were analyzed to determine their concentrations and sizes. Illumina sequencing was performed on the qualified libraries to obtain the sequence information of the tested fragments. Data analysis was carried out according to established processes. The sequencing and analysis in this section were completed by Shanghai Outdo Biotech Co., Ltd.After MKN45 and MGC803 cells had reached a certain confluence, the supernatants were removed, and the cells were washed 3 times with PBS. An appropriate amount of NP-40 lysis buffer was added, and the cells were incubated on ice for 30 min. The cell lysis products were collected and centrifuged at 4°C for 10 min at 12,000 rpm. The supernatants were transferred to new precooled EP tubes, and the sample concentrations were measured by BCA assay. Different concentrations of acacetin (100 μM and 500 μM) were added to the supernatants and incubated at room temperature for 1 h. Pronase  was diluted in 1× TNC solution, and the mixture was added in different ratios according to protein concentration . The samples were incubated at room temperature for 30 min. The reactions were terminated by high-temperature denaturation with the addition of 5× loading buffer, and Western blotting was performed. The experiment was repeated ≥3 times.Cell lysates were collected and incubated with acacetin or DMSO for 1 h at room temperature. The lysate of each sample was heated for 3 min at the indicated temperature of 55°C–75°C, and then, the samples were cooled for 3 min at room temperature. The samples were centrifuged for 10 min at 12,000 rpm, and then, the supernatants were analyzed by Western blotting.https://www.rcsb.org/). The processing and optimization of virtual screening were performed using Schrödinger Maestro software. The docking results were visualized by PyMOL 2.1 software. The modes of compound and protein interactions were analyzed to identify the interaction with each target protein residue, and then, the docking of the compounds was scored to determine whether the screened compounds had a certain active effect.The structure of the small molecule compound acacetin that was used in this docking experiment was obtained from the PubChem database, and the energy of this compound was minimized and saved by the MM2 module in Chem3D software. The crystal structure of the target protein EGFR was downloaded from the protein data bank  according to the manufacturer’s instructions. The stained wells were washed with PBS three times, stained with DAPI and measured by ImageXpress Mico4 .4 cells were seeded in each well of a six-well plate and incubated until the cells adhered. The adherent cells were exposed to acacetin for 24 h and then fixed in a 6-well plate with methanol  for 1 h. Then, the cells were stained with 1% crystal violet stain solution  for another 1 h, washed with water and photographed.For crystal staining, 1 × 10Approximately 500 GC cells/well were cultured in a 6-well plate with DMEM and exposed to acacetin for 2 days. After the medium was changed to fresh medium, the cells were allowed to grow for another 10 days until colonies were formed. Then, the cells were fixed and stained with crystal violet stain solution as described above.−ΔΔCT method. The expression was normalised to the average expression of all control individuals. The housekeeping gene used was β-actin. The following primer pairs were used: EGFR, 5′-AGGCACGAGTAACAAGCTCAC-3’ (forward)and 5′-ATGAGGACATAACCAGCCACC-3’ (reverse); and β-actin, 5′-CCCATCTATGAGGGTTACGC-3’ (forward) and 5′-TTTAATGTCACGCACGATTTC-3’ (reverse).Reverse transcription of RNA to cDNA using a reverse transcription assay kit . mRNA expression was determined using the SYBR Green PCR Mix . The relative expression of each gene was calculated using the 2To extract proteins from cells, RIPA buffer (supplemented with a protease inhibitor and phosphatase inhibitor) was added to the cells. Then, the cells were incubated on ice for 30 min, sonicated for 15 s, and centrifuged at 12,000 rpm at 4°C for 10 min. The supernatants were then used for Western blotting. Tumor tissues were dissected on ice and homogenized with a tissue grinder, and then, the samples were sonicated and collected for Western blotting. In this experiment, the protein concentrations of all the samples were measured to normalize the amount of protein that was used for each sample. Different protein concentrations or cell numbers per well were tested to ensure that the signals were in the linear range. Background corrections were performed by subtracting the background signals from the blank samples. The amount of protein that was loaded in each lane was 15-30 μg. Western blotting was performed in a manner that was consistent with our previously described protocols.6 MKN45 cells. When the tumors reached 50-100 mm3, the mice were randomly divided into the control group and acacetin treatment groups (25 mg/kg and 50 mg/kg) (n = 5). Acacetin was administered every 2 days for 3 weeks via intraperitoneal injection. Tumor volume was calculated using the formula: volume (mm3) = length × width × width/2. The mice were euthanized, and the tumors, serum and major organs, such as the liver, kidney, and lung, were isolated.All the animal studies were performed according to the Guide for the Care and Use of Laboratory Animals approved by Longhua Hospital, Shanghai University of TCM. Male BALB/c-nude mice (4–6 weeks old) were obtained from GemPharmatech Co., Ltd.  and housed under specific pathogen-free (SPF) conditions. For the MKN45 xenograft experiment, xenografts were established by subcutaneous injections of 2 × 10Tissue samples were washed with PBS to remove the blood, and large tissues were cut open and fixed with 4% paraformaldehyde. Tissue samples of appropriate size were placed in an embedding box. The tissues were dehydrated and embedded in paraffin. HE and immunohistochemical staining were performed according to the manufacturer’s instructions. Images were captured under a microscope.MKN45 tumor-bearing mice were treated as described above. On Day 21, blood was collected, allowed to clot and centrifuged to obtain serum samples for the ALT and AST tests. Enzyme-linked immunosorbent assay (ELISA) was performed according to the manufacturer’s instructions.*p < 0.05, **p < 0.01 and ***p < 0.001 indicate statistically significant differences compared with the control group; similarly, #p < 0.05, ##p < 0.01 and ###p < 0.001 indicate statistically significant differences compared with the EGF-treated group.All the experiments were performed at least three times in duplicate. Student’s t-test was used to analyze differences between the two groups, and these analyses were performed with SPSS 23.0  and GraphPad Prism 8  software. For the tests, To predict potential common targets between GC and acacetin, 3070 GC-related targets were identified from the GeneCards database. In addition, the chemical structure of acacetin was obtained from PubChem, and 100 related targets were predicted by Swiss target prediction. Sixty-nine potential common gene targets were identified by filtering the overlapping targets of GC and acacetin . A protep < 0.05, log(fold change) > 2). Among these genes, 261 genes were downregulated and 154 genes were upregulated in the acacetin group . The expression of EGFR was not significantly changed in MKN45 cells after acacetin intervention, while MGC803 cells were increased in the high-dose group . Then, ip < .05)  . Howeverp < .05) . Next, ep < .05) . Therefop < .05) . The resp < .05) . In concin vivo, we established a xenograft model by subcutaneously injecting MKN45 cells into BALB/c nude mice. The results indicated that the average tumor size in the 50 mg/kg acacetin group was significantly decreased compared with that in the DMSO control group (p < .05). However, the weight of the nude mice in the acacetin group was not affected  to approximately 60% , etc. . Among tEGFR, which is associated with cell proliferation and survival, is overexpressed in many epithelial tumors . TherefoIn vitro experiments confirmed that acacetin could inhibit the growth of GC cells both in the short and long term. These results are related to the regulation of Caspase and PARP signaling by acacetin. The activation of multiple signaling pathways downstream of EGFR is closely related to apoptosis , the absolute values were still lower than 1 ng/mL, and there was no obvious pathological damage in the liver. Western blotting and immunohistochemical analyses proved that acacetin could inhibit proliferation and promote apoptosis. Combined with our previous study, these findings provide strong evidence that acacetin has the potential to exert therapeutic effects on GC.Notably, acacetin showed satisfactory therapeutic results In summary, through target prediction and experimental verification, we identified a direct target of acacetin in gastric cancer cells. Acacetin significantly promoted GC cell apoptosis by regulating the STAT3 and ERK signaling pathways. These findings highlight the important potential of acacetin to be used as a drug candidate for the treatment of GC.


Manure is a potential substitute for chemical phosphate fertilizer, especially in intensive agriculture, such as greenhouse farming, but the associations between soil phosphorus (P) availability and the soil microbial community under manure application instead of chemical phosphate fertilizers are still rarely addressed. In this study, a field experiment in greenhouse farming with manure application instead of chemical phosphate fertilizers was established, including five treatments: a control with conventional fertilization and chemical phosphate fertilizer substitution treatments using manure as the sole P resource at 25% (0.25 Po), 50% (0.50 Po), 75% (0.75 Po), and 100% (1.00 Po) of the control. Except for 1.00 Po, all the treatments applied with manure harbored similar levels of available P (AP) as the control. Most of the bacterial taxa involved in P transformation were enriched in manure treatments. Treatments of 0.25 Po and 0.50 Po significantly enhanced bacterial inorganic P (Pi) dissolution capacity, while 0.25 Po decreased bacterial organic P (Po) mineralization capacity. In contrast, the 0.75 Po and 1.00 Po treatments significantly decreased the bacterial Pi dissolution capacity and increased the Po mineralization capacity. Further analysis revealed that the changes in the bacterial community were significantly correlated with soil pH, total carbon (TC), total nitrogen (TN), and AP. These results revealed the dosage effect of the impact of manure on soil P availability and microbial P transformation capacity and emphasized that an appropriate dosage of organic manure is important in practical production. As typical intensive agriculture, greenhouse farming is a potential approach to cope with food demand in future . GreenhoP is a macroelement for plants. To increase plant production, high amounts of P fertilizers are applied to the soil. As a result, approximately 80% of global phosphate rock is consumed as fertilizer . HoweverManure is a good substitute for mineral P fertilizers, as it is rich in P. A large amount of manure produced by livestock production largely changes the P cycle in agriculture . IntegraMicrobes are the primary drivers of P transformation in soil . The mic−1·year−1), P (90 kg P2O5·ha−1·year−1), and potassium  fertilizers and cattle manure (84 t ha−1·year−1). The manure-amended treatments were fertilized with the same amount of mineral N and K fertilizers as the control but with manure instead of mineral P fertilizers. The amount of manure applied to the 0.25 Po, 0.50 Po, 0.75 Po, and 1.00 Po treatments was 33.6, 67.2, 100.8, and 134.4 t ha−1·year−1, respectively. Each treatment contained three replicate plots. The size of each plot was 20 m2 (8 m × 2.5 m). The plant system in all treatments was tomato and muskmelon rotation. Except for the fertilization strategy, all the treatments were under the same management.The design of the experiment was detailed in a previous study . The fieSoil sampling and soil bacterial community measurement were performed according to the methods described in previous studies . In totaA FastDNA Spin Kit for Soil  was used for soil total DNA extraction. DNA was extracted from 0.5 g of fresh soil following the user guide.The primer set 515F/806R was used to amplify the V4 region of the bacterial 16S rRNA gene. The polymerase chain reactions (PCRs) were performed in a 50-μL system with a thermal cycle of initial denaturation at 95°C for 10 min; 30 cycles in a series of denaturation at 94°C for 1 min, annealing at 55°C for 1 min, and extension at 72°C for 1 min; and a final extension at 72°C for 10 min. The PCR system consisted of 25 μl PCR premix (TaKaRa Ex Taq), 1 μl forward primer (10 μM), 1 μl reverse primer (10 μM), 1 μl DNA template (20 ng), and 22 μl PCR grade water. After quality checks and purification, the PCR products were sequenced using an Illumina HiSeq 2,500 System.Bioinformatic analysis of the sequencing data was primarily performed using the VSEARCH package  accordinThe functional profiles of the bacterial community were predicted using PICRUSt2  Activity Assay Kit  following the user’s manual.The inorganic P dissolution potential of the soil microbial community was measured using an incubation method according to the previous study . In shorStatistical analysis was performed using R platform (version 4.0.2) according to the previous studies , 2023a. 4+‑N) and nitrate nitrogen (NO3‑N) in 0.25 Po and 0.50 Po were significantly lower than that in the control, while 0.75 Po and 1.00 Po contained similar levels of NH4+‑N and NO3‑N as the control.As shown in All the manure treatments contained higher AP than the control, and the AP content increased with a high amount of manure application, but only the increase in AP at 1.00 Po was statistically significant.Tomato yields did not differ significantly between the control and manure treatments , and musThe bacterial community in the studied soil was dominated by Proteobacteria, Acidobacteriota, Chloroflexi, Firmicutes, Bacteroidota, Planctomycetota, Actinobacteriota, Gemmatimonadota, Myxococcota, and Verrucomicrobiota, which accounted for 92.47% of the total reads . The varChao1 richness and Heip’s evenness were calculated to assess the impact of different treatments on bacterial diversity . The ChappaX and appA genes. In addition, the dosage effect of manure on these functional genes was also observed. For example, 0.25 Po, 0.50 Po, and 0.75 Po increased, but 1.00 Po decreased, the abundance of the ppaC gene. Po and 1.00 Po increased, but 0.50 Po and 0.75 Po decreased, the abundance of the phnW gene. When grouping the genes involved in inorganic P dissolution and organic P mineralization, the total abundance of genes involved in inorganic P dissolution varied little among treatments  repository, accession number PRJEB60932.JN and RS designed the experiment. LZ, XL, ZZ, KL, and FW performed laboratory measurement. LZ and RS performed data analysis. LZ, RS, and CZ wrote the study. All authors contributed to the article and approved the submitted version.This study was supported by the Key Science and Technology Project of Anhui Province (202103a06020012) and S&T Program of Hebei (21326904D).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


PLOS Biology finds that the SCO responds to glucose by secreting signaling molecules into the cerebrospinal fluid (CSF), thereby decreasing the local ependyma-driven CSF movement.The subcommissural organ (SCO) is a secretory tissue located on the roof of the brain’s third ventricle. A new study published in  The subcommissural organ (SCO) is a secretory tissue located on the roof of brain’s third ventricle. This Primer explores a new PLOS Biology study revealing that the SCO responds to glucose by secreting signaling molecules into the cerebrospinal fluid (CSF), thereby decreasing the local ependyma-driven CSF movement. Fig 1).Glucose provides energy for the body and is the primary fuel source for neuronal metabolism. The brain senses glucose changes: for example, increased blood glucose concentration after a big meal leads to satiety. Cerebrospinal fluid (CSF) glucose fluctuates with blood glucose, with CSF glucose approximately 60% of plasma levels . The hypFig 1A), the authors investigate how the SCO could influence ependymal cells that line the ventricle to modulate their motile cilia, which actively stroke in unison to facilitate local CSF movement. To do this, they compare basal and hyperglycemic conditions in rodent models. They find that in contrast to normoglycemia, hyperglycemia facilitates release of SCO-enriched spondin protein (SCO-spondin) into CSF, which has an “immobilizing” effect on ependymal cilia. This observation is supported both by electron microscopic analyses of ependymal cilia and ex vivo ependymal flow analyses. In scanning electron micrographs, ependymal cilia under normoglycemic basal conditions appear “soft and relaxed” and are properly angled and aligned, suggesting strong ciliary beating. In contrast, ependymal cilia under hyperglycemic conditions show evidence of increased stiffness, appearing clustered and perpendicular to the ependymal wall in scanning micrographs. Furthermore, ependymal flow in the third ventricle is significantly inhibited by hyperglycemia, demonstrated by quantifying real-time speed of fluorescent microbeads moving along ependymal surfaces ex vivo. Closer examination of ex vivo ependyma through high-resolution confocal microscopy reveals that microbeads, which are only minimally detected on normal ependyma, seem to be trapped within ependymal cilia upon glucose challenge. These outcomes of altered ependymal function, structure, and signaling are at least partially mediated by the secretion of SCO-spondin and Wnt5a proteins into the third ventricle by the SCO .Nualart and colleagues focus on the SCO because of this glandular organ’s ability to take up glucose  and secrete signaling molecules (such as SCO-spondin and Wnt5a). Given the anatomical proximity of the SCO to CSF in the third ventricle  that establish connections with hypothalamic neurons? In addition, what does the SCO secretome contain beyond spondin and Wnt5a? Since the SCO is a glandular secretory tissue that releases substances directly into the CSF, a systemic understanding of its secretome may further reveal its regulatory role in brain health. Of note, GLUT2 expression in the SCO was lost in aged brains , but whe


Shape memory polymers (SMPs) are currently one of the most attractive smart materials expected to replace traditional shape memory alloys and ceramics  in some fields because of their unique properties of high deformability, low density, easy processing, and low cost. As one of the most popular SMPs, shape memory polyurethane (SMPU) has received extensive attention in the fields of biomedicine and smart textiles due to its biocompatibility and adjustable thermal transition temperature. However, its laborious synthesis, limitation to thermal response, poor conductivity, and low modulus limit its wider application. In this work, biocompatible poly(ε-caprolactone) diol (PCL-2OH) is used as the soft segment, isophorone diisocyanate (IPDI) is used as the hard segment, and glycerol (GL) is used as the crosslinking agent to prepare thermoset SMPU with a thermal transition temperature close to body temperature for convenient medical applications. The effects of different soft-chain molecular weights and crosslinking densities on the SMPU’s properties are studied. It is determined that the SMPU has the best comprehensive performance when the molar ratio of IPDI:PCL-2OH:GL is 2:1.5:0.33, which can trigger shape memory recovery at body temperature and maintain 450% recoverable strain. Such materials are excellent candidates for medical devices and can make great contributions to human health. Shape memory polymers (SMPs) have broad potential applications in aerospace , medicinε-caprolactone) (PCL) is a linear, semi-crystalline polymer formed by the ring-opening polymerization of ε-caprolactone monomer [2 groups and one polar -COO- group in the repeating unit endow it with good flexibility and processability and excellent biocompatibility and degradability, making it suitable for applications in the biomedical field. With increased molecular weight, the mechanical properties of PCL are improved, but its transition temperature (Ttrans) also increases. Excessive Ttrans makes it difficult to be used in body-temperature-responsive biomedical devices. Furthermore, linear PCL does not have a highly elastic state and melts into a liquid at high temperatures. In order to achieve a pronounced shape memory effect, peroxide chemical reaction or high-energy ray radiation is usually required to obtain crosslinking points in such materials.As one component of many PU materials, poly( monomer . The fivTm) was used as an actuator to fix the temporary shape, and the PCL-2OH with a higher Tm was used as a skeleton to maintain the permanent shape. Thus, PCL-3OH was directed to crystallize along the PCL-2OH skeleton, and a chemical, crosslinked network structure was generated between PCL-2OH and PCL-3OH.When used alone as a shape memory material, PCL usually faces the problem of poor mechanical properties. To overcome this problem, in our previous work, by selecting PCL diols (PCL-2OH) and PCL triols (PCL-3OH) as the prepolymers, a one-step method was employed to prepare a two-way shape memory polyurethane (2W-SMPU) with high reversible strain and high shape stability . The PCLTtrans of about 37 °C and a good shape memory effect. In this work, we not only accurately control the response temperature of SMPU to near body temperature by optimizing the raw materials and reaction conditions but also significantly improve its mechanical properties and shape memory effect, enabling large-scale and rapid preparation, which is conducive to the widespread application of this type of material in the biomedical field.Based on our previous work involving the use of copolymers with PCL-2OH  and cons−1. GL of analytical purity, provided by Beijing Chemical Factory, China, is a liquid at room temperature and has a molecular weight of 92 g mol−1. After removing GL’s moisture in an oven at 80 °C, some molecular sieve was added to the GL. The organic bismuth catalyst (DY-20) with a bismuth content of 20 ± 0.5 wt%, purchased from Shanghai Deyin Chemical Co., China, has the main component of bismuth new caprate and is a light yellow transparent liquid at room temperature with a density of 1.15 g·cm−3. N,N-dimethylformamide (DMF) of analytical purity was supplied by Beijing Chemical Factory, China. PCL-2OH, purchased from Shenzhen Yisheng New Materials Co. Ltd., China, is a milky white solid at room temperature and needs to be melted in a 90 °C oven before use. Detailed information on PCL-2OH with various molecular weights is shown in A predetermined amount of PCL-2OH was weighed and placed in a three-necked flask. Then, it was dried in a vacuum oven at 120 °C for 3 h. IPDI and GL in a predetermined ratio were weighed and placed in a beaker; 5 mL of DMF was then added to the flask and beaker, respectively, to form solutions for later use. The three-necked flask was taken out and electromagnetically stirred at 250 rpm for 30 min in a nitrogen environment and then naturally cooled to about 70 °C. Then, the above IPDI and GL solution was slowly added and stirred evenly for 5 min. In the flask, the solution gradually turned white from transparent. After further stirring for 2 min, the solution was poured into a beaker and then put in a vacuum oven at 25 °C for the defoaming treatment for 1 min. Afterwards, the solution was slowly poured into a preheated mold at 50 °C and placed in an oven at 50 °C for curing. The curing procedure was 50 °C for 3 h, 70 °C for 3 h, and then 100 °C for 10 h, based on the previous experimental experience . A too-h−1; the resolution was 4 cm−1; and the scanning was repeated 8 times.The Fourier transform infrared spectroscopy (FTIR) was used to conduct the infrared spectroscopy tests on the synthesized SMPU to characterize the molecular structure and functional groups of the materials. The material was tested in the air as a film without further processing. The tests were in total reflection mode; the scanning range was 4000–450 cmT-shaped sample stage with conductive adhesive, and sputtered with gold for 50 s before the observation on the SEM.Scanning electron microscopy (SEM) was used to observe the fracture morphology of the SMPU. The accelerating voltage was 5–15 kV. The sample was fractured at low temperature in liquid nitrogen, then attached to a Ttrans and melting enthalpy change (ΔHm) in SMPU, where the position of the maximum melting peak was taken as the Ttrans. The tests were in a standard mode, and the nitrogen flow rate was 50 mL·min−1. First, the thermal history of the sample was removed by increasing the temperature from room temperature to 130 °C at a heating rate of 10 °C·min−1, and then the sample was cooled down to −70 °C at a cooling rate of 10 °C·min−1. Subsequently, the sample was heated again from room temperature to 130 °C at a heating rate of 10 °C·min−1. The last heating curve was taken to determine the Ttrans and ΔHm.Differential scanning calorimetry (DSC) was used to test the −1. The nitrogen flow rate was 60 mL·min−1, and the air flow rate was 40 mL·min−1.Thermogravimetric analysis (TGA) was used to analyze the thermal stability of the samples. The tests were performed by increasing the temperature from room temperature to 800 °C at a heating rate of 10 °C·min3 rectangular splines. For the quasi-static stress–strain tests, the strain rate mode was chosen, and the temperature was raised to 55 °C at a heating rate of 10 °C min−1, and then the temperature was kept constant for 5 min to ensure the crystals melted in the materials and to make the overall temperature reach 55 °C, followed by stretching to 100% strain at a strain rate of 50% strain min−1. For the storage modulus tests, the multi-frequency-strain mode was chosen, and the frequency was set to 1 Hz. The temperature was increased from 0 °C to 85 °C at a heating rate of 5 °C min−1. For the tensile shape memory tests, the control force mode was chosen, and the temperature was first increased from room temperature to 55 °C at a heating rate of 10 °C·min−1, followed by keeping the temperature constant for 5 min. Then, stress was applied to the sample at a rate of 0.5 MPa·min−1, and the next step was performed when the deformation reached about 50–70% strain. The temperature was decreased to 0 °C at a cooling rate of 10 °C min−1, followed by keeping the temperature constant for 10 min, and then the stress was completely unloaded at a rate of 0.5 MPa min−1. Finally, the temperature was raised to 55 °C at a heating rate of 10 °C·min−1, and the temperature was kept constant for 20 min to ensure the material reached the maximum recovery degree. The shape memory performance was characterized by the shape fixation ratio Rf and the shape recovery ratio Rr, which were calculated as follows:εload was the maximum strain achieved by the material under the high-temperature load stress, εfix was the fixed strain of the material after the stress was unloaded at the low temperature, and εrec was the strain of the material after the final heating and shape recovery.Dynamic mechanical thermal analysis (DMTA) was used to test the quasi-static stress–strain, storage modulus, and shape memory properties of the samples. The samples were cut into 15.0 × 5.0 × 1.0 mm−1.X-ray diffractometry (XRD) was used to test the crystallization properties of the samples. The diffraction angle range was from 5° to 90° at a scanning speed of 10° minwi. The sample was soaked in a bottle of DMF solvent for 12 h and then taken out, dried, and weighed. Such a procedure was repeated several times until the difference from the last weighing was less than 1 wt%, and the last weight was wf. So the gel fraction (fg) was calculated as follows [The gel fraction was measured to see the degree of crosslinking of materials by their weight losses. A small part of the sample was cut and weighed to obtain the weight of  follows :(3)fg=wfTtrans, so it is mostly used as a hard segment, and polyether or polyester polyol is used as a soft segment. The thermal, mechanical, and shape memory properties of SMPU are closely related to the type and molecular weight of the soft segment. Based on the equivalent molar ratio of IPDI:PCL-2OH:GL = 2:1.5:0.33, in which the molar ratio of -NCO:-OH is exactly 1:1, we selected PCL-2OH with molecular weights of 500, 1000, 2000, and 3000 g·mol−1, respectively, to prepare the SMPU and explore the effect of soft chain length on the material properties. The prepared samples are marked as SMPU-05, SMPU-10, SMPU-20, and SMPU-30, respectively.The thermally responsive SMPU is formed by the reaction of polyisocyanates containing isocyanate groups (-N=C=O) with polyether or polyester polyols containing active hydrogen. The urethane groups generated in the reaction can interact with the urethane groups in other segments. It produces hydrogen bonding and has higher −1, and the absorption peak of the hydrogen-bonded =N-H group is around 3310 cm−1. When there are hydrogen bonds between molecules, the stretching vibration peak of the =N-H group moves to the lower wavenumber region, so the absorption peak of =N-H group is found at 3373 cm−1, which indicates that part of the =N-H group in the molecular chain is hydrogen-bonded. The =C=O group exists in the carbamate group and in the ester group of PCL-2OH, and the absorption peak of =C=O in the free state is located at 1695 cm−1. The absorption peak of hydrogen bonding in the ordered phase is at 1635–1645 cm−1, so the absorption peak at 1721 cm−1 indicates that the molecular chain =C=O is not hydrogen-bonded. The peak at 1635–1645 cm−1 is the stretching vibration absorption peak of ≡C-H in SMPU, the peak at 1523 cm−1 is the deformation vibration absorption peak of =N-H, and the peak at 1158 cm−1 is the stretching vibration absorption peak of ≡C-O-C≡ vibration absorption peak. As shown in −1, which disappears without any residue in the four synthesized SMPU products, indicating that all the –N=C=O groups have reacted with the -OH groups in the PCL-2OH and GL. Therefore, it can be preliminarily determined that the synthesized SMPU is non-toxic, and the above results show that the polyurethane (PU) materials have been successfully synthesized by the one-step method.The chemical structure of the as-synthesized SMPU was analyzed by using FTIR. As shown in Ttrans and ΔHm are listed in Ttrans, the higher the peak height, and the narrower the peak width, which means that the longer the molecular chain of PCL-2OH, the easier it is to crystallize. The enthalpy change can be used to evaluate the crystallization properties of the material. Different from the changing trend of Ttrans, the ΔHm of PCL-2OH is 70–80 J g−1, and there is no significant difference. The melting enthalpy of perfect PCL crystals is about 140 J g−1, indicating that PCL-2OH is a semi-crystalline polymer with crystallinity of 52.7–56.1%. Here, based on the ΔHm of the corresponding PCL-2OH, the crystallization melting enthalpy change value is calculated for comparison. The crystallinities of SMPU-05 to SMPU-30 are 0%, 5.8%, 20.6%, and 26.1%, respectively.Ttrans and ΔHm of the synthesized SMPU decrease significantly to different degrees, and the melting peak even completely disappears for SMPU-05. The Ttrans of SMPU-20 appears at 36.9 °C, which is very close to the temperature of the human body. The appearance of the peak is related to the melting of soft segment crystals. With the increase of the molecular weight of PCL-2OH, the Ttrans increases, and the ΔHm also increases significantly. The cold crystallization peak appears for the SMPU-20 and SMPU-30 and moves to the higher temperature region with the increase of Ttrans. The appearance of the cold crystallization peak is mainly due to the formation of a supramolecular network structure in the synthesized SMPU after the GL crosslinking, and the motion of the PCL soft segment is bound. The mobility of the chain segment is enhanced, the crystallization continues, and the exothermic phenomenon occurs during the heating, resulting in the emergence of a cold crystallization peak. The ΔHm of the SMPU is smaller than that of PCL-2OH, indicating that part of PCL-2OH in the material does not participate in the crystallization but reacts with IPDI to form the hard-segment urethane groups. The incomplete participation of PCL-2OH in the crystallization makes the prepared SMPU have lower Ttrans, but at the same time, sacrifices the crystallizability of PCL, which is manifested as lower ΔHm, and is not conducive to the expected shape memory effect.Compared with the PCL-2OH, the In order to verify that the longer soft chain makes the polymer easier to crystallize, XRD characterization was used to observe the crystallization of the materials, and the results are shown in εload under load stress, the fixed strain εfix after stress removal, and the recovered strain εrec at high temperatures. These data and the statistical calculation results of the shape fixation ratio Rf and the shape recovery ratio Rr are listed in Rf is only 3.1%. The reason for its extremely low shape-fixing function could be related to its short soft chain, which is difficult to crystallize. As the molecular weight of the soft chain increases, the Rf of the material increases rapidly, reaching 76.1% for the molecular weight of 1000 g mol−1 and obtaining almost perfect shape fixation (>95%) for the molecular weight of 2000 g mol−1. At the same time, it also has a perfect Rr (about 98.4%).Furthermore, the shape memory function of the materials was tested. Ttrans, and both the Ttrans and ΔHm of SMPU increase with the increase in the molecular weight of the soft segment.In summary, according to the above results of FTIR, DSC, XRD, and shape memory tests on the SMPU with different soft chain molecular weights, it can be seen that the shape memory performance of SMPU is closely related to the molecular weight of the soft chain that determines the crystallizability of the soft segment, thereby affecting the shape fixation ratio of the material. The soft chains with higher molecular weights are easier to crystallize and thus have a better shape fixation ratio, while those with lower molecular weights are more difficult to crystallize or do not crystallize at all, so they have a poorer shape fixation ratio. As shown in Rr > 95%) but is lacking in the Rf (<80%). The SMPU-20 and SMPU-30 have excellent shape memory performance , indicating that PCL220 and PCL230 are both suitable for the preparation of excellent SMPU. During the shape memory testing, it was also noticed that as the molecular weight of the soft segment increases, the stress required to achieve the desired strain decreases, probably due to the higher crosslinking density in the SMPU with shorter soft segments per unit volume. As a result, under the same stress conditions, the strain achieved by SMPU-30 is larger than that of SPMU-20, which to some extent, indicates its worse mechanical properties. Secondly, the Ttrans of SMPU-20 is 36.9 °C, which is closer to the human body temperature, while the Ttrans of SMPU-30 is 42.3 °C, and the human body can already feel a little burning pain at this temperature. After careful consideration, PCL220 is chosen to prepare the SMPU for further exploration.According to the shape memory test results, the SMPU-05 prepared by PCL205 exhibits pure elastic rubber behavior, which is completely unsuitable for the preparation of SMPU. In contrast, the SMPU-10 prepared by PCL210 has a good shape recovery ratio , the Ttrans reaches about 39.7 °C. The ΔHm also increases with the increase of the proportion of PCL-2OH in the material, showing the largest melting peak in SMPU(0.17), which is narrower and higher than all other peaks. DSC tests show that the lower the crosslinking density, the higher the Ttrans of the polymer, and the narrower and higher the melting peak, which should be due to the more PCL soft chains in SMPU participating in the crystallization, and the formation of crystallization is beneficial to the shape fixing in the shape memory process.The 3 °C see . As the Hm changes in the DSC tests. It can be seen from the figure that as the crosslinking density decreases, the crystallizability of the material gradually increases. The SMPU(0.67) with a higher crosslinking density does not show obvious diffraction peaks, but there is a steamed bread peak, which represents the formation of the amorphous phase. From SMPU(0.55) to SMPU(0.17), the diffraction peaks appear and gradually increase, which is caused by the increasing proportion of PCL-2OH in the crosslinking network. As schematically shown in To demonstrate the effect of crosslinking density on the crystalline properties, XRD tests are performed on the materials, and the results, as shown in Ttrans is higher, and it is more difficult to deform. Compared with SMPU(0.67) with higher crosslinking density, SMPU(0.17) is easier to break at low temperatures.Ttrans results of the DSC tests. This is due to the fact that the bulk used in the DSC test is small, with only 5–10 mg, while the spline used in the DMA tests is larger and needs to take a certain time for the heat to transfer into the material. Moreover, the use of a frequency of 1 Hz in the DMTA tests also brings a dynamic effect. So the measured Ttrans by DMTA is generally higher than the value measured by DSC, and such a difference could be as high as 15–20 °C.The storage modulus at low temperatures reflects the anti-deformation ability of the material. The higher the storage modulus, the more difficult the material is to be deformed. The storage modulus is conducive to the shape fixation of the material. The storage modulus at high temperature reflects the mechanical properties of the material during the shape recovery, such as the size of the restoring force, and a higher storage modulus at high temperature is conducive to the shape recovery of the materials. Rf of the material gradually increases, reaching a shape fixation ratio of 98.8% for the SMPU(0.33), indicating that the high content of PCL proportion is good for the shape fixation. However, for the SMPU(0.17), the Rf is decreased, which may be due to the fact that the higher content of PCL reduces the crosslinking density. This creates a viscous flow strain that causes irreversible plastic deformation. It is noticed that the SMPU(0.67) does not show obvious crystallization in the XRD and DSC tests, but it shows an Rf of 51.5% in the shape memory tests. The material will continue to creep slowly if it does not start heating at the 62nd min, so its actual Rf should be lower than 51.5%.SMPU(0.33) is exactly the SMPU-20 material discussed above, and its shape memory curve was already shown in The above results of the gel fraction, DSC, TGA, XRD, storage modulus, and shape memory performance of SMPU with different crosslinking densities show that with the decrease of crosslinking density, the proportion of PCL-2OH increases, the crystallinity of the material increases, and the shape memory performance gradually increases. However, when the crosslinking density is reduced, the shape recovery of the material is affected. After comprehensive consideration, for the SMPU(0.33) with the molar ratio of IPDI:PCL-2OH:GL of 2:1.5:0.33, the material has the best shape memory effect and comprehensive performance.3 and marked with black tape. It is stretched after heating, and then the temporary shape is fixed. The SMPU(0.33) can reach a huge strain of 450% and achieve nearly 100% shape recovery on the hot stage. Interestingly, as shown in Using a carbon dioxide laser cutting machine, a “TJU” shape is cut out on the SMPU film, as shown in Ttrans, the crystals melt, the movement restriction of the PCL-2OH segment is lifted, and the PCL-2OH segment is aligned and oriented along the stress direction under the external force. The macroscopic performance is that the material is stretched and elongated. In this process, the entropy of the polymer becomes lower, and the material is in a thermodynamically unstable state and has a tendency to return to the original shape. As the temperature decreases, part of the PCL-2OH segments recrystallizes, the segment movement is restricted, the temporary shape is fixed, and the elastic recovery energy is sequestered in the form of internal stress. Finally, when the SMPU is heated to the Ttrans again, the PCL-2OH melts again, the SMPU returns to its original shape under the traction of internal stress, and the polymer returns to a thermodynamically stable state again.Combined with the characterization results, the shape memory behavior of the prepared SMPU can be explained as schematically shown in In this study, a series of thermoset SMPU samples have been synthesized by a simple, time-saving, and industrialized one-step method using PCL-2OH, IPDI, and GL as raw materials. The effects of chain molecular weight and crosslinking density on the thermal properties, crystallinity, shape memory effect, and other properties of the materials have been investigated. The main conclusions can be drawn as follows:Ttrans of the materials increases gradually, the crystallinity increases, and the Rf of the material is also improved. The SMPU with a PCL-2OH soft chain molecular weight of 500 g mol−1 hardly has any shape memory effect, and the best shape fixation can be achieved for the molecular weights of 2000 g mol−1;(1) As the molecular weight of the soft chain increases, the Ttrans of the materials gradually decreases, the crystallinity becomes lower, the Rf decreases, and the Rr increases. When the molar ratio of GL is 0.67, the material changes from a shape memory material to a pure elastic rubber;(2) As the crosslinking density increases, the Ttrans of the materials is close to body temperature, showing the best Rf and Rr, and the best comprehensive performance, which is expected to be useful in the biological medicine and other near body temperature fields.(3) Using PCL220 and the molar ratio of IPDI:PCL-2OH:GL = 2:1.5:0.33, the


In this study, a circular conjugate of granulocyte colony-stimulatingfactor (G-CSF) was prepared by conjugating the two end-chains of poly(ethyleneglycol) (PEG) to two different sites of the protein. For the orthogonalconjugation, a heterobifunctional PEG chain was designed and synthesized,bearing the dipeptide ZGln-Gly (ZQG) at one end-chain, for transglutaminase(TGase) enzymatic selective conjugation at Lys41 of G-CSF, and analdehyde group at the opposite end-chain, for N-terminal selectivereductive alkylation of the protein. The cPEG-Nter/K41-G-CSF circularconjugate was characterized by physicochemical methods and comparedwith native G-CSF and the corresponding linear monoconjugates of G-CSF,PEG-Nter-G-CSF, and PEG-K41-G-CSF. The results demonstrated that thecircular conjugate had improved physicochemical and thermal stability,prolonged pharmacokinetic interaction, and retained the biologicalactivity of G-CSF. The PEGylation strategy employed in this studyhas potential applications in the design of novel protein-based therapeutics. It mobilizeshematopoietic and progenitor stem cells and induces and regulatesthe proliferation, differentiation, and survival of granulocyte cells,especially neutrophils, which have a fundamental role in the initialimmune response following bacterial and fungal infections.2 G-CSF interacts with its receptor, which is prevalentlyexpressed on the surface of hematopoietic precursor cells and neutrophilicgranulocytes.3 The rhG-CSF is used forthe treatment of congenital and acquired neutropenia because it improvesthe granulocyte count in neutropenic patients4 and for the mobilization of hematopoietic stem cells in case ofstem cell transplantation.5In the past few decades, protein-baseddrugs produced through recombinantDNA techniques have gained exceptional roles in several therapiesdue to their advantageous combination of selective activity, potency,and safety. Recombinant human granulocyte colony-stimulating factor(rhG-CSF) is a hematopoietic cytokine approved in 1991 by the FDA(Filgrastim) for the treatment of chemotherapy-induced neutropenia.13 thus making G-CSF one of the most studied proteins for polymer conjugation.Currently, there are two PEGylated G-CSF conjugates in clinical practice.The first has been obtained by reductive alkylation at the proteinN-terminus,14 while the second has beenobtained by glycoPEGylation, a strategy that allows polymer couplingat the protein glycan moiety through an enzymatic method.16 Consequently, this protein is a perfect model for the evaluationand comparison of any new method of polymer conjugation.The mainlimitation of G-CSF as a biological drug is its rapidkidney clearance due to its relatively low molecular weight (∼18.8kDa). For this reason, it has been an ideal candidate for polymerconjugation, especially PEGylation, aiming to increase its hydrodynamicsize for pharmacokinetic prolongation. Several chemical and enzymaticmethods of conjugation have been proposed over the years,Here,we describe a site-selective approach of PEGylation basedon the conjugation of both polymer end-chains to the same proteinunit to achieve circular polymer conjugation 1A. The iTo the best of our knowledge, this is the firsttime that a circularPEGylation based on two site-selective conjugation approaches is appliedand the results obtained warrant further investigations.2 20kDa were purchased from NOF Corporation . The H2N-PEG-COOH 20 kDa was purchased from JenKem Technology USA,Inc. . Microbial TGase (mTGase), of Streptomycesmobaraensis origin (ACTIVA M), was provided by AjinomotoCo. . Carbobenzoxy-l-glutaminyl-glycine andall of the chemicals and solvents were purchased from Merk . Trypsin and Glu-C of sequencing grade were obtained by ThermoFisher Scientific . Human G-CSF Instant ELISA Kit waspurchased from Life Technologies . Mini-PROTEAN TGX precastgels 4–15% for SDS-PAGE were obtained from Bio-Rad .The protein rhG-CSF was a kind gift of Sandoz. PEG-aldehyde 20 kDa and PEG-NH1H NMR spectra were recordedon a Bruker 400 MHz FT-NMR spectroscope equipped with a dual thermostated 1H/13C gradient probe and a Fourier 80 benchtopcryogen-f. TMS was used as internal standard.All G-CSF =0.88 and εTGase = 1.89 mL cm–1 mg–1. The values of the absorbance at 280 nm were generatedby ProtParam (http://www.expasy.org/tools/protparam.html).Protein concentrations weredetermined spectrophotometrically on a Thermo Scientific Evolution201 spectrophotometer . The concentration of proteinswas evaluated by measuring the absorbance at 280 nm and using thefollowing extinction coefficients: ε17The concentrationof protein conjugates was determined by bicinchoninic acid colorimetricassay (BCA) as described elsewhere.18 Electrophoresis was performedusing gradient 4–15% Mini-PROTEAN TGX precast gels  loaded on a Mini-PROTEAN Tetra Cell obtainedfrom Bio-Rad, and the runs were carried out using an ElectrophoresisPower Plus300 power supply  at 300 V and60 mA. The gels were first stained with barium iodine for PEG detection19 and then with Coomassie Brilliant Blue R-250for protein detection. Briefly, for the reversible iodine staining,the gels were soaked in 20 mL of perchloric acid (0.1 M), and after15 min, the gel was transferred into 10 mL of a 5% (w/v) BaCl2 solution in 1 N HCl and 4 mL of a 1.27% (w/v) I2 and 2% (w/v) KI aqueous solution, yielding the appearance of PEG-containingbands in a few minutes. The staining solution was discarded, and thegels were incubated in water for 10–15 min. The iodine stainingwas then removed by the addition of 5 mg of ascorbic acid. After 10min the gel was washed with water and, in turn, stained with a standardprocedure for Coomassie blue staining of proteins.The conjugates were analyzed by SDS-PAGE following the Laemmlimethod.MALDI-MS data were obtainedusing a REFLEX time-of-flight instrument  equipped with a SCOUT ion source, operatingin the positive linear mode. Ions generated by a pulsed UV laser beam are accelerated to 25 kV. The matrix,a saturated solution of sinapinic acid in water/ACN  with0.1% TFA (v/v), was mixed with an equal volume of sample solution,and 1 μL of the resulting mixture was spotted on the MALDI target.The preparation of ZQG-PEG-acetalwas achieved in two steps, and the derivative was then hydrolyzedto ZQG-PEG-aldehyde just before G-CSF conjugation 1.2-PEG-COOH(MW 20 kDa) was firstderivatized with ZQG at the amino group of the polymer. After purificationand recovery, the product was modified with 4-amino butyraldehydediethyl acetal at the carboxylic group on the other side chain ofthe polymer.The starting polymer NHN,N′-dicyclohexylcarbodiimide  and 143.8 mg (1.25 mmol) of N-hydroxysuccinimide were added to 168.6 mg (0.5 mmol) of ZQG (MW 337.33Da) previously dissolved in 5 mL of anhydrous DMSO. After 1 h, 500mg (0.025 mmol) of NH2-PEG-COOH (MW 20 kDa) was added and25 μL of Et3N was added. The reaction was left tostir at room temperature for 18 h, and the degree of derivatizationwas estimated by the TNBS-based test according to the Snyder and Sobocinskiassays.20 Finally, 7 μL (0.075 mmol)of acetic anhydride  was added tothe mixture to quench the reaction. The absence of free amino groupswas verified through the Snyder and Sobocinski assay. The solutionwas extensively dialyzed against a mixture of Milli-Q water and decreasingamounts of DMSO and for 18 h against Milli-Q water. The absence ofZQG was verified through RP-HPLC on an Agilent 1200 Series HPLC withonline UV detection from Agilent Technologies . RP-HPLCanalyses were performed using a Jupiter C18 column , elutedwith H2O + 0.05% TFA (eluent A) and ACN + 0.05% TFA (eluentB) at a flow rate of 1.0 mL/min applying the following gradient ofeluent B: 0′ 40%, 25′ 70%, 30′ 95%, 35′40% B. The absorbance was read at 226 nm. After lyophilization , the identity of the product ZQG-PEG20 kDa-COOH was evaluated by 1H NMR:  PEG: 4 , 3.7 , ZQG: 7.41 , 5.13 .515.6 mg (2.5 mmol) of 20 kDa-COOH  was dissolved in 4 mL of anhydrous CH2Cl2, and 20.6 mg (0.1 mmol) of DCC (MW 206.33 Da)and 5.7 mg (0.05 mmol) of NHS (MW 115.09 Da) were added. After 1 h,11.5 μL (0.06 mmol) of 4-amino butyraldehyde diethyl acetal was added and the pHof the solution was adjusted to 8. The reaction was stirred at roomtemperature for 18 h. The mixture was dropped into cold diethyl etherunder stirring, and the obtained precipitate was washed with freshdiethyl ether to remove the excess of 4-amino butyraldehyde diethylacetal. The polymer was dried under vacuum ,and the absence of 4-amino butyraldehyde diethyl acetal was confirmedon thin-layer chromatography stained with 0.2% w/v ninhydrin in EtOHsolution and using a solution of acetal as reference. The derivatizationdegree of the product ZQG-PEG20 kDa-acetal was determinedby 1H NMR by comparing the integration values of the methylenehydrogens of acetal moiety with the signal of the monomers of PEGbackbone:  PEG: 4 , 3.7 ,ZQG: 7.41 , 5.13 , 4-amino butyraldehyde diethyl acetal:1.17 ppm . The molecular weight of ZQG-PEG20 kDa-acetal was determined by MALDI-TOF.ZQG-PEG20 kDa-acetal was hydrolyzed to the aldehydegroup: 36 mg of ZQG-PEG20 kDa-acetal  was dissolved in 360 μLof 25 mM H3PO4 pH 2.1 and stirred for 2 h at60 °C. After cooling to room temperature, ZQG-PEG20 kDa-aldehyde was incubated with G-CSF at a final protein concentrationof 2 mg/mL as follows: to a stock solution of G-CSF  in 10 mM sodium acetate, 5% (w/v) sorbitol pH 4.6,ZQG-PEG20 kDa-aldehyde was added after dilutingits solution with the same buffer . After 1 h under stirring at 25 °C, 100equiv of NaCNBH3 (with respect to the protein) were added.The mixture was incubated at 25 °C and 300 rpm and, after 24h, was analyzed by RP-HPLC on an Agilent 1200 Series HPLC with onlineUV detection from Agilent Technologies . RP-HPLCanalyses were performed using a Jupiter C18 column , elutedwith H2O containing 0.05% TFA (eluent A) and ACN containing0.05% TFA (eluent B) at a flow rate of 1.0 mL/min applying the followinggradient of eluent B: 0′ 40%, 25′ 70%, 30′ 95%,35′ 40% B. The absorbance was recorded at 226 nm. Gly–Gly(3 equiv with respect to the polymer) was added to stop the reaction.After 1 h, the conjugate ZQG-PEG-Nter-G-CSF was purified by cationexchange chromatography (CEX) using a TSKgel SP-5PW column  operating at a flow rate of 1.0 mL/minand registering the absorbance at 280 nm . The separation was performed on aShimadzu HPLC with CBM-20Alite system controller, LC-20AT pump, andSPD-10A VP detector. The peak of the conjugate was collected, concentrated,and buffer-exchanged to 10 mM sodium acetate, 5% (w/v) sorbitol pH4.6 with Amicon Ultra Centrifugal filters . ZQG-PEG-Nter-G-CSF quantification was performed through theBCA colorimetric assay as reported above, and the purity of the conjugatewas verified through SDS-PAGE.The acetal moietyof ZQG-PEGN-ethylmaleimide (NEM) at a molar ratio of 1.25 equiv withrespect to mTGase. The mixture was first purified by size exclusionchromatography (SEC) on an AKTA FPLC (Amersham Biosciences) usinga Superdex 200 Increase 10/300 GL column  eluting with PBS pH 7.4 at0.5 mL/min and measuring the absorbance at 280 nm. The peak correspondingto a mixture of the conjugates ZQG-PEG-Nter-G-CSF and cPEG-Nter/K41-G-CSFwas collected, concentrated, and buffer-exchanged to 10 mM sodiumacetate pH 4.7. The conjugates were separated through cation exchangechromatography using the conditions reported above. Finally, cPEG-Nter/K41-G-CSFwas concentrated, buffer-exchanged to 10 mM sodium acetate, 5% (w/v)sorbitol pH 4.6 with Amicon Ultra Centrifugal filters (cutoff 10 kDa),and quantified by BCA. The conjugate was characterized through SDS-PAGE,MALDI-TOF, and SEC.To complete the synthesis, mTGasewas used to tether the ZQG end of the PEG conjugated to G-CSF: ZQG-PEG-Nter-G-CSF(0.16 μmol) was buffer-exchanged to 10 mM sodium phosphate pH7.2 with Amicon Ultra Centrifugal filters and mTGase was added atan enzyme/substrate (E/S) ratio of 1:25 (w/w), previously solubilizedin 0.1 M sodium phosphate buffer pH 7.2 and quantify by UV spectroscopy.The G-CSF concentration was 1 mg/mL. After 18 h at 25 °C understirring, the reaction was stopped by the addition of a solution of 4HCO3/ACN 50:50 (v/v) mixture atpH 7.8 for 10 min. After dehydration with ACN for 15 min, gel sliceswere completely dried in a speed-vac system . Native and conjugated proteins were reduced by incubationwith 200 μL of 5 mM tris(2-carboxyethyl)phosphine (TCEP) in0.1 M NH4HCO3 pH 7.8 for 10 min at 60 °Cand alkylated by adding 200 μL of 55 mM iodoacetamide (IAA)in 0.1 M NH4HCO3 pH 7.8 for 15 min at 37 °Cin the dark. Gel slices were washed twice with a 0.1 M NH4HCO3/ACN 50:50 (v/v) mixture at pH 7.8 for 10 min, dehydratedwith ACN for 15 min and again completely dried. 50 μL of trypsinprotease  were added and the digestionreaction was let to proceed overnight at 37 °C and 300 rpm ina thermomixer . Formic acid to a finalconcentration of 2.5% (v/v) was added, and the obtained peptide solutionswere desalted by PepClean C18 Spin columns  following the manufacturer’s instructions. Thedigested mixtures were analyzed using a UPLC-QTOF system. The ACQUITYUPLC H-Class  was equipped with an AdvanceBioPeptide Map Guard and AdvanceBio Peptide Mapping column , maintained at 32 °C, flow rate 0.2mL/min, detection at 280 nm, eluted with a solvent gradient of water/ACNboth containing 0.1% formic acid. Gradient 2′ – 2% ACN,38′ – 65% ACN, 40′ – 98% ACN, 43′– 98% ACN, 44′ – 2%. The Xevo G2-S QTof (Waters)was operated in the ESI positive ion, resolution mode, with a detectionwindow between 50 and 2000 m/z. Analyses were performed at a capillaryvoltage of 1.5 kV, at a cone voltage of 30.0 V, and source offsetof 80 V. MSE acquisition was performed by alternating twoMS data functions: one for the acquisition of the peptide mass spectrawith the collision cell at low energy (6 eV) and the other for thecollection of the peptide fragmentation spectra with the collisioncell at elevated energy (linear ramp 20–40 eV). Analyses wereperformed with LockSpray using a solution of 1 ng/μL LeuEnkMS in 50:50 (v/v) water/ACN containing 0.1% formic acid, sampled every45 s. MSE data were processed with MassLynx and BiopharmaLynx1.3.4 Software (Waters) setting trypsin as the digest reagent and2 missed cleavages. The MS ion intensity threshold was set at 250counts, and the MSE threshold was set to 100 counts. Boththe MS masses-match tolerance and the MSE mass-match tolerancewere set to 15 ppm.Native protein, the intermediate ZQG-PEG-Nter-G-CSF,and cPEG-Nter/K41-G-CSF (30 μg) were analyzed by SDS-PAGE. Therespective slices were isolated and washed three times with 200 μLof a 0.1 M NHcPEG-Nter/K41-G-CSF was incubated with trypsin in ammonium bicarbonatepH 8.0, at 37 °C and 300 rpm in a thermomixer. Trypsin was addedat an enzyme/substrate (E/S) ratio of 1:100 (w/w), and the final concentrationsof G-CSF and trypsin were 0.2 mg/mL and 2 μg/mL, respectively.After 18 h, the reaction mixture was analyzed by MALDI-TOF.8 to be used for a direct comparison against thecPEG-Nter/K41-G-CSF. Briefly, 3 equiv of monomethoxy PEG-aldehyde20 kDa previously solubilized in 10 mM sodium acetate, 5% (w/v) sorbitolpH 4.6 were added to a stock solution of G-CSF  in the same buffer. After stirring for 1 h at 25°C, NaCNBH3 was added (100 equiv with respect to theprotein); the final G-CSF concentration was 3 mg/mL. The mixture wasincubated at 25 °C under stirring for 24 h, and it was monitoredby RP-HPLC with a Jupiter C18 column , eluted withH2O + 0.05% TFA (eluent A) and ACN + 0.05% TFA (eluentB) at a flow rate of 1.0 mL/min applying the following gradient: from40 to 70% of ACN in 25 min. The absorbance was read at 226 nm. Gly–Gly(3 equiv with respect to the polymer) was added to stop the reaction.After 1 h, PEG-Nter-G-CSF was purified by cation exchange chromatographyusing a TSKgel SP-5PW column operating at a flow rate of 1.0 mL/min and registering the absorbanceat 280 nm . The conjugate was concentrated and buffer-exchanged to 10 mMsodium acetate, 5% (w/v) sorbitol pH 4.6 with Amicon Ultra Centrifugalfilters . G-CSF concentration wasdetermined by UV spectroscopy and BCA.PEG-Nter-G-CSF was prepared as reported elsewhere21 1-Ethyl-3-(3-(dimethylamino)propyl)carbodiimide  and 1-hydroxy benzotriazole  were added to 16.8mg (50 μmol) of ZQG (MW 337.33 Da) previously dissolved in 2.5mL of a 0.1 M borate buffer/ACN (3:2) mixture at pH 8.0. After 1 h,250 mg (12.5 μmol) of PEG-NH2 (MW 20 kDa) was added,and the pH of the solution was adjusted to 8. The reaction was letto proceed for 18 h at room temperature under stirring, and the degreeof derivatization was estimated by the Snyder and Sobocinski assays.Finally, 2.52 mg (25 μmol) of succinic anhydride (MW 100.08Da) was added to the mixture to quench the reaction and the absenceof free amino groups was verified using the TNBS assay. ACN was evaporatedunder vacuum and the remained solution was dialyzed for 24 h againstMilli-Q water. After lyophilization, PEG20 kDa-ZQGidentity was evaluated by 1H NMR:  PEG: 4 , 3.7 , ZQG: 7.41 , 5.13 .PEG-ZQG was prepared as reported elsewhere.11 and used for the comparison with cPEG-Nter/K41-G-CSF. Briefly,G-CSF  in 10 mM sodium acetatebuffer pH 4.7, 5% (w/v) sorbitol was diluted with 10 mM sodium phosphatepH 7.2, and 20 equiv of PEG-ZQG  were added, previously dissolved in 10 mM sodium phosphatepH 7.2, in order to reach a final G-CSF concentration of 2 mg/mL.mTGase, solubilized in 0.1 M sodium phosphate buffer pH 7.2, was addedat an E/S ratio of 1:50 (w/w) and the reaction was left to react understirring for 18 h at 25 °C. After stopping the reaction withNEM (1.25 equiv with respect to mTGase), the buffer was exchangedto 10 mM sodium acetate pH 4.7 and PEG-K41-G-CSF was purified as reportedfor PEG-Nter-G-CSF.PEG-K41-G-CSF was prepared as reported elsewhere2 dmol–1 by applyingthe formula Θ = Θobs(MRW)/10L[C], where Θ is the observed ellipticity indegrees, MRW is the mean residue weight of the protein, [C] is protein concentration in mg/mL, and L is theoptical path length in centimeters.Far-UV circular dichroismspectra were measured on a Jasco J-810 spectropolarimeter equippedwith a Peltier temperature control unit. G-CSF and its derivativeswere analyzed in 10 mM acetate, 5% sorbitol pH 4.6 at a protein concentrationof 0.1 mg/mL. The spectra were collected over the wavelength rangeof 200–250 nm at 25 °C with an average of 3 scans, andthe data at each wavelength were averaged for 8 s. The sample cellpath length was 1 mm. The CD data were converted to mean residue ellipticity,expressed in deg cmThe thermal denaturationwas carried out on the same samples used for CD analysis recordingthe decrease of the ellipticity signal at 222 nm as a function ofthe temperature. Thermal unfolding experiments were performed on a0.1 cm cell path length by heating the samples from 25 to 90 °Cat a rate of 2 °C/min. CD spectra in the range of 200–250nm were then collected at 25 °C after heating to 90 °C.20k-Nter/K41-G-CSF,PEG20k-Nter-G-CSF, and PEG20k-K41-G-CSF) weredetermined in female Sprague–Dawley rats weighing between 150g and 160 g . Samples were prepared in PBS pH7.4 and were intravenously injected at the dose of 100 μg/kg in the lateral tail vein. Anesthesia was performedwith 5% isoflurane gas mixed with O2 in enclosed cages.Blood samples were collected on anesthetized rats by incision fromthe tail at predetermined time points and centrifuged at 1500g for 20 min. G-CSF equivalent concentrations in serum werequantified using a Human G-CSF Instant ELISA Kit (Life Technologies)by using the corresponding conjugates as standard. Pharmacokineticdata were elaborated using 2.0 PkSolver software by applying a bi-compartmentalmodel. For each sample, a dedicated calibration curve was built usingthe testing conjugate.Pharmacokinetic profilesof G-CSF and G-CSF conjugates  purchased from CharlesRiver Laboratories.cPEG-Nter/K41-G-CSF, PEG-Nter-G-CSF, andPEG-K41-G-CSF were injected subcutaneously as a single dose of 1 mg/kgof G-CSF equivalent, while native G-CSF was administrated daily atthe dose of 0.14 mg/kg (4 mice per group). Vehicle solution (PBS)was used as control (CTRL). Myeloid cell (granulocytes and monocytes)accumulation was followed for 7 days at predetermined time points.Blood samples were collected at days 1, 3, and 5, and at day 7, afterblood collection, the mice were sacrificed, and their spleens wereharvested to be analyzed. The quantification of myeloid cell sub-populationsin the blood or the spleen was performed by FACS analysis after stainingwith specific markers and defined on live cell gate as myeloid cells(CD11b+); granulocytes (CD11b+Ly6CintLy6G+); monocytes (CD11b+Ly6ChighLy6G−).Cytofluorimetric data were acquired with a BD LSR II flow cytometerand analyzed by FlowJo software. Injected samples were prepared usingsterile water and resulted to be negative to the LAL test (endotoxinlevels < 5 EU/mL).The study protocol was approved bythe Ethics Committee of the University of Padova and the Italian Ministryof Health. The animals were handled in compliance with Italian LegislativeDecree 116/92 guidelines and the “Guide for the Care and Useof Laboratory Animals” by the National Research Council ofthe National Academies.20 kDa-acetal was synthesized starting from a heterobifunctional NH2-PEG-COOH through a two-step procedure, first involving themodification of the amino group of the starting PEG with the dipeptideZQG, following the derivatization of the carboxylic end with 4-aminobutyraldehyde diethyl acetal. The degree of derivatization with ZQGwas 91 mol %. The absence of free ZQG was assured by monitoring thedialysis through RP-HPLC. The identity of the ZQG-PEG-COOH derivativewas confirmed by 1H NMR  and compared to the unmodified NH2-PEG-COOH . In the second step, ZQG-PEG-COOHwas reacted with 4-amino butyraldehyde diethyl acetal. The absenceof 4-amino butyraldehyde diethyl acetal was verified by the ninhydrintest on TLC and the exact degree of derivatization was calculatedfrom 1H NMR spectroscopy . Acetal modification was calculated to be 88.4 mol %. The finalPEG was characterized through MALDI-TOF and resulted in a molecularweight of about 21.1 kDa .ZQG-PEGKa (6–7) of the N-terminal residueand the ε-amino group’s pKa (9–10) of the lysine residues. At pH 4.6, the ε-aminogroups are not reactive, differently, the α-amino group owingto its lower pKa is still reactive, thusallowing the modification of G-CSF at the N-terminal residue. In thesecond step, mTGase-mediated PEGylation was performed between theother end of conjugated PEG and the lone lysine residue of G-CSF,which is a substrate of mTGase. This enzyme is a widely employed toolfor obtaining site-selective monoconjugates in which the polymer isconjugated at specific glutamine or lysine residues based on the PEGused.23 In this case, the use of a PEG bearing the ZQG dipeptide,which is a Gln-containing mTGase substrate, has led to the derivatizationof G-CSF at a Lys residue. As previously demonstrated,24 mTGase has specific substrate requirements for lysines, which meansthese amino acids must be inserted into flexible regions of the proteinsequence. Only under such conditions can they reach the catalyticsites of the enzyme, following a similar pattern to that observedfor glutamines.25 G-CSF presents four lysines,with the first three  located in the firstα helix of the protein, resulting in a rigid conformation. Incontrast, the fourth lysine (Lys41) is inserted in the loop betweenthe first and second α helix, characterized by an increasedB-factor value indicating peptide flexibility.11 Furthermore, Lys35 is preceded by Glu34, whose negativecharge prevents interaction with the catalytic sites of TGase.11The circularconjugate cPEG-Nter/K41-G-CSF was synthesized by exploiting two differentsite-selective PEGylation strategies to obtain a homogeneous product.In the first step, G-CSF was selectively modified at the N-terminalresidue by reductive alkylation, exploiting the aldehyde functionalityof the polymer ZQG-PEG-aldehyde. The selectivity of this strategyrelies on the difference between the α-amino group’sp26The selective nature of TGase toward specificlysines has also been demonstrated with other proteins that containmultiple lysine residues. For instance, interferon α-2a possesses10 lysines, among which Lys164 is predominantly recognized by TGase,with minor modifications (4.7%) occurring at Lys31 as a second siteof conjugation forming the biconjugate.20 kDa-aldehyde to the N-terminusof the protein, followed by the enzymatic reaction to close the PEGcircle. This chosen protocol offers several advantages. For instance,in TGase-mediated reactions, a substantial molar excess of PEG reagentsis necessary to avoid the formation of protein concatemers resultingfrom interprotein reactions between lysines and glutamines. Specifically,in the case of G-CSF, Lys41 and Gln135 are substrates of TGase. Withouta proper excess of a PEG reagent, the formation of G-CSF concatemersbecomes a concern. However, coupling the PEG to the N-terminus ofG-CSF first overcomes this issue. The subsequent TGase-mediated reactionbenefits from having the ZQG substrate, located at the opposite endof the PEG chain, in close proximity to Lys41 of the protein, therebyensuring a high virtual concentration of the ZQG substrate near Lys41.Furthermore, the steric hindrance imposed by a 20 kDa PEG linked tothe protein prevents the formation of the undesired PEG-G-CSF-Lys41-Q135-G-CSF-PEGdimer, leading to improved reaction yields.In the presented conjugation scheme, we initiate the processbycoupling ZQG-PEG20 kDa-acetal were conjugatedfirst, the acetal group would be deprotectedby the acidic conditions during the purification step, posing thepotential risk of undesired side reactions.Lastly, it is worthnoting that if ZQG-PEGIn the current study,the so-formed cPEG-Nter/K41-G-CSF was preparedwith the focus to examine the outcomes of circular PEGylation on physicochemicalcharacteristics, thermal stability, pharmacokinetic profile, and activityof the conjugate. cPEG-Nter/K41-G-CSF was also compared with the nativeprotein and with the corresponding linear monoconjugates of G-CSFobtained by N-terminal and Lys41 PEGylation, by using mPEG-aldehyde20 kDa and mPEG-ZQG 20 kDa, respectively.20 kDa-aldehyde was analyzedby RP-HPLC , resulting in aconversion yield of 73% based on the comparisonof peak areas of G-CSF and the conjugate. At pH 4.6, the aldehydeof the polymer reacts with the N-terminal α-amino group of theprotein, yielding the intermediate monoconjugate ZQG-PEG-Nter-G-CSF.The reaction was purified from the unreacted protein and the excessof ZQG-PEG-aldehyde by cation exchange chromatography . Before proceeding to the second stepof the synthesis, the purity of ZQG-PEG-Nter-G-CSF was verified bySDS-PAGE. As shown in The conjugation reactionof G-CSF with ZQG-PEG RP-HPLC S3, resul RP-HPLC S3, resulFigure S5). The purity of the circular conjugatewas characterized by SDS-PAGE (TR 20.4 min), while cPEG-Nter/K41-G-CSFexhibited a reduced hydrodynamic volume (TR 21.6 min). This behavior could be probably attributed to the finalshape of the circular conjugate that, in solution, resulted more compactwith respect to the linear conjugates.The enzyme mTGase was used to close the PEG ringon G-CSF. mTGaseformed, as side products, crosslinked conjugates owing to the concatenationof 2–3 units of ZQG-PEG-Nter-G-CSF, which were separated fromthe monoconjugates by SEC. The monoconjugates peak corresponded toa mixture of the mono-derivative ZQG-PEG-Nter-G-CSF (unreacted startingconjugate) and the desired circular conjugate. cPEG-Nter/K41-G-CSFwas finally separated from ZQG-PEG-Nter-G-CSF through CEX S5. The pFigure S6) has revealed a molecular weight (MW)of 39.9 kDa,corresponding to the sum of the MWs of the protein (18.8 kDa) andthe polymer (21.1 kDa).The MALDI-TOF spectrum of cPEG-Nter/K41-G-CSF (E raw data were processed using BiopharmaLynx(Waters). LC–MSE peptide mapping of all of the digestedsamples resulted in a 39.4% of sequence coverage and the identityof the found peptides was confirmed by b/y fragment ions (Tables S1–S3). The obtained sequence coveragedepends on trypsin cuttings. In fact, the digestion of G-CSF withtrypsin forms the 42–147 fragment of 11.3 kDa that was undetectedby the ESI-LC-MSE method used. However, this fragment wasnot relevant to the analysis. In accordance with previous studies,the significative peptides for the identification of PEG modificationin the circular conjugate are peptides 1–17 and 36–41.As shown in the LC–MSE chromatogram of the digestednative G-CSF , the peptide 1–17 (1785.97 Da) was eluted at 22.24 min,the peptide 1–17 with the oxidation at the methionine residue(1801.97 Da) was eluted at 21.51 min and was coeluted with peptide149–167, and peptide 36–41 (754.37 Da) was eluted at13.68 min. The masses and retention times of all of the detected fragmentsof native G-CSF are reported in Table S1. The LC–MSE chromatogram of the proteolysis mixturerelative to ZQG-PEG-Nter-G-CSF conjugate . In conclusion, the mass fingerprinting analysis -PEG-(F5-6)formed by the peptide F1 (1–17), the polymer, and the peptidesF5 (36–41) and F6 (42–147) was evaluated by MALDI-TOFfollowing the incubation of cPEG-Nter/K41-G-CSF with trypsin. TheMALDI-TOF spectrum  has revealeda molecular weight of 34.9 kDa, corresponding to the sum of the MWsof peptide F1 , polymer (21.1 kDa), peptideF5 , and peptide F6 .As the PEGylated peptides cannot be detected withthe ESI-LC-MS27 that under spectroscopy analysisshow the two characteristic negative bands at 208 and 222 nm.28 The effect of the polymer on the protein’ssurface was evaluated in the far-UV, and as shown in CD analysis was conductedto investigate if the site of attachment and the conformation of thepolymer have an impact on the secondary structure of the G-CSF conjugatesand its thermal stability. The structure of G-CSF is prevalently formedby four antiparallel helices connected by two long and one short hairpin-typeloopsTm) of about 64 °C. The thermal stabilities of cPEG-Nter/K41-G-CSFand PEG-Nter-G-CSF were increased by 1.3 and 1.9 °C, respectively,compared to that of G-CSF. For PEG-K41-G-CSF, the stability was onlyslightly affected as the melting temperature decreased by ∼0.6°C . The AUC for cPEG-Nter/K41-G-CSFand PEG-Nter-G-CSF was 4.2-fold higher than that of G-CSF, while PEG-K41-G-CSFshowed a 2.6-fold increase in AUC, consistent with previous reports.Overall, the pharmacokinetic comparisonof the conjugates demonstratedthat the ring conformation in cPEG-Nter/K41-G-CSF resulted in a slightlymore extended elimination half-life and a delayed clearance, althoughthese values are not statistically significant with respect to thoseof the linear PEG-G-CSF conjugates.G-CSF specificallyinduces the proliferation and differentiation of neutrophil precursorsupon binding to its specific cell-surface receptor, and it also regulatesthe survival of mature neutrophils. In this study, we evaluated thein vivo biological activity of cPEG-Nter/K41-G-CSF, PEG-Nter-G-CSF,and PEG-K41-G-CSF conjugates by comparing their effects on the levelsof granulocytes and monocytes in mice after subcutaneous injections.Blood samples were collected at predetermined time points over 7 daysafter a single dose of the conjugates at 1 mg/kg of G-CSF equivalent.The native G-CSF was injected daily at the dose of 0.14 mg/kg andused as a positive control.Figure S8). In the blood, the peak effect of neutrophil mobilizationwas observed on day 3 and gradually decreased in the following timepoints (Our results showed that a singleadministration of all of the conjugates induced a significant increasein the levels of circulating myeloid cells, especially granulocytes,which was higher or comparable to that of the native cytokine injecteddaily 6. ThequMoreover, the circular conjugate appeared to befaster in inducingthe mobilization of myeloid cells. The higher potency of cPEG-Nter/K41-G-CSFis possibly due to the lower steric hindrance of the polymer aftercircular PEGylation. The PEG forms a closed ring with the protein,limiting its mobility and interfering less with the process of protein/receptorinteraction with respect to the methods of linear conjugation. Therefore,this approach could be advantageous in the design of G-CSF conjugatesfor therapeutic applications.PEGylation is a well-established strategyfor improving the therapeuticproperties of biologics. The cPEG-Nter/K41-G-CSF is a new G-CSF derivative,which is a mono-PEGylated conjugate with both ends of the polymerchain linked to the protein in a ring-like conformation. This conformationresults in a more compact hydrodynamic volume for the conjugate, asdemonstrated by gel filtration analysis, which might enhance its abilityto penetrate and diffuse through biological tissues and improve itstherapeutic efficacy. Additionally, the 20 kDa PEG achieved an extensionof blood circulation half-life that conferred a long-lasting effectthat is superior or comparable to the daily administration of thereference cytokine.Our results show that G-CSF circular PEGylationdid not interferewith the biological activity of the cytokine and, in fact, showeda slightly better statistically significant activity with respectto other conjugates when that was compared to G-CSF or control groups.PEGylation approaches. This improvement may be due to the lower sterichindrance of the PEG chain linked to the protein in a ring mode. Overall,this approach provides a new tool for designing optimized polymer–proteinconjugates with improved therapeutic efficacy.