Objective: To describe mean arterial blood pressure (MABP), responsiveness to dopamine, and relationship to brain injury in infants with moderate/severe hypoxic-ischemic encephalopathy (HIE) undergoing therapeutic hypothermia (TH). We hypothesized that, when utilized, dopamine would rapidly and effectively increase MABP in treated patients.Methods: Continuous arterial blood pressure measurements were prospectively recorded from infants with moderate/severe HIE undergoing TH in a multi-institutional cohort from 2010 to 2018. Treatment with dopamine was at the discretion of the medical team for hypotension/hypoperfusion. MABP values of treated infants were compared to those obtained at an equivalent time period in control infants receiving TH but not dopamine (24 h after birth). MRI was obtained per unit protocols and included T1/T2/DWI sequences. Injury was classified as no injury/mild injury or moderate/severe injury using a standardized scoring system. Seizures were confirmed with conventional EEG.Results: Eighteen infants were treated with dopamine and were similar to untreated controls (n = 36) with the exception of lower cord gas pH . Dopamine was initiated at a mean of 24 h after birth. MABP was significantly lower in the dopamine group at the start of therapy  and 1 h later . However, after 9 h of treatment, dopamine increased the MABP by an average of 9 mmHg and MABP values were similar to untreated controls for the remainder of the observation period. There were no significant differences in rates of seizures, brain injury, or death.Conclusion: Neonates with moderate/severe HIE treated with dopamine during TH had MABP significantly lower than controls. The majority of infants responded to dopamine monotherapy following adequate volume resuscitation. An association between requirement for dopamine and severity of brain injury was not detected. Hypoxic-ischemic encephalopathy (HIE) secondary to birth asphyxia is a significant cause of neonatal morbidity and mortality, affecting 1-8/1,000 live births in developed countries . AlthougTherapeutic hypothermia (TH) became the standard of care for moderate to severe HIE after multiple randomized control trials demonstrated a reduction in the combined outcome of death or moderate-severe disability –9. TH exand the treatment of HIE have the potential to negatively impact the hemodynamic status of the infant, and this is frequently manifested as hypotension  and that dopamine would rapidly and effectively increase MABP in treated patients.This retrospective, multi-center case-control study was conducted in the neonatal intensive care units (NICU) of St. Louis Children's Hospital (SLCH) and University of Virginia Children's Hospital (UVa) from 2010 to 2018. Both centers are Level IV units serving patients from urban, suburban, and rural populations. Infants were included in the study if they underwent TH for the treatment of neonatal encephalopathy as determined by a modified Sarnat exam  and had Cases were determined by exposure to dopamine and were matched to controls 1:2 by gestational age and gender. Infants in the control group were otherwise identical except for exposure to dopamine. Infants were excluded if there was a known congenital or genetic anomaly at the time of birth. To prevent confounding, infants were excluded if they were exposed to other inotropes or vasopressors . Infants were also excluded if blood pressure data were not available at the time of dopamine initiation or the comparable period in control infants.At SLCH and UVa, universal arterial and venous cord gas screening is performed at all inborn deliveries. At both institutions, standardized encephalopathy exams are performed within the first 6 h of life for infants ≥ 34 weeks who are at risk for neonatal encephalopathy (defined as a pH < 7.10 or a base deficit <12 at SLCH and pH < 7.0 or base deficit <16 at UVa). TH treatment is initiated within 6 h of birth for all infants with qualifying exams.The standard whole-body TH protocol includes 72 h of servo-controlled hypothermia at 33.5°C followed by 12–24 h of rewarming to 36.5°C. At SLCH, conventional EEG is performed for a minimum of 24 h with at least one non-sedated MRI completed within 14 days of life; at UVa, conventional EEG is performed for the entire duration of cooling and rewarming and non-sedated MRI is completed immediately after rewarming (days 3-5) or after day 10 (if unable to obtain an early MRI).To prevent shivering and maximize the benefits of TH, sedation is provided to infants at both institutions per protocol. At SLCH, morphine is utilized for sedation with an initial bolus (50 mcg/kg) followed by a continuous infusion (10 mcg/kg/h for 12 h and then decreased to 5 mcg/kg/h) for the duration of cooling. At UVa, continuous infusion of either fentanyl 0.5 mcg/kg/h or dexmedetomidine 0.2 mcg/kg/h (from 2014 to 2016) are used during cooling. At both centers, increased infusions or bolus doses of sedatives were given as needed for agitation.Dopamine is the first-line medication used to treat hypotension/hypoperfusion in an infant with HIE undergoing TH at both institutions and is often given in conjunction with or following a normal saline bolus. In this study, dopamine was initiated and escalated at the discretion of the clinical treatment team. If the infant did not respond to dopamine and a second agent was needed, blood pressure data were not analyzed during that time period.Clinical characteristics were obtained from the electronic medical record including maternal age, antenatal magnesium exposure, mode of delivery, gestational age, worst degree of encephalopathy in the first 6 h, birth weight, Apgar scores , cord gas pH, intubation and mechanical ventilation in the first 96 h, and inborn/outborn status.Both institutions utilize conventional video EEG to monitor for the presence of seizures. Electrodes are placed using the standard International 10–20 system, modified for neonates and EEG monitoring is initiated as soon as possible after cooling starts. Seizures were defined using the aCNS consensus definition, namely a rhythmic electrographic event which is of sudden onset, repetitive and evolving, with a duration of at least 10 s .A comprehensive review of the medication administration record was performed. The following information was collected for dopamine, morphine, fentanyl, dexmedetomidine, midazolam, and vecuronium: cumulative bolus dose (mg/kg or mcg/kg), infusion start date/time, infusion stop date/time, initial infusion dose , maximum infusion dose, and cumulative infusion dose. Additionally, the dose (mcg/kg/min) of dopamine at 1, 3, 6, 9, 12, 18, 24, 36, 48, and 72 h after dopamine initiation was collected. The number of normal saline boluses in addition to dates/times and dose (mL/kg) were collected. The date/time, dose (mg/kg), and frequency of hydrocortisone administration were collected. Exposure to anti-epileptic medications (dates/times and doses in mg/kg), including phenobarbital, fosphenytoin, midazolam, and levetiracetam, were also recorded. Initiation of maintenance anti-epileptic dosing was noted.The number, dates, and times of packed red blood cell (pRBC), platelet, and fresh frozen plasma (FFP) transfusions were extracted from the transfusion record.After completion of TH, all surviving neonates underwent non-contrast, non-sedated MRI examination following institutional MRI guidelines. Brain MRI imaging was performed with either a Siemens 1.5-T Avanto/Aera or 3.0-T Trio/Skyra/Prisma  and included T1/T2/DWI sequences. MRI images were reviewed by pediatric neuroradiologists blinded to blood pressure outcomes and scored using a standardized HIE scoring system . BrieflyGiven the small sample size, injury was classified as a binary variable using the categorical output of the scoring system; normal/mild injury vs. moderate/severe injury. If two scans were obtained in the same infant, the scan with the worst injury was recorded.Blood pressure data was obtained via umbilical arterial lines that were placed at the discretion of the clinical treatment team. Per standard clinical practice, umbilical arterial lines are placed in a manner so that the tip of the catheter lies between the sixth and eighth thoracic or the third and fourth lumbar vertebrae on radiograph. Continuous, invasive arterial blood pressure measurements were recorded using a pressure transducer which interfaces with the umbilical arterial catheter and patient monitor .0 was defined at the time of dopamine initiation. Empiric evaluation of the case group revealed that dopamine was consistently started around 24 h of life. For infants in the control group, T0 was defined as the point 24 h following birth.MABP data were prospectively collected with a sampling rate of 0.5 Hz and archived in a database . The files were then converted to a MATLAB  matrix for analysis. As blood pressure is known to increase in a linear fashion following birth , 26, com0, we calculated the average MABP over a 10-min interval centered at the time points of 1, 3, 6, 9, 12, 18, 24, 36, 48, and 72 h. In the instance where a MABP value was not available for an infant at a specific time point, an average MABP was calculated from the remaining infants.From TU-test for continuous variables . Statistical significance was accepted as p < 0.05.Clinical factors were compared between cases and controls by using Chi-square or Fisher's exact test for categorical variables and Mann–Whitney During the study period, 98 infants were diagnosed with moderate-severe encephalopathy and had arterial lines placed at SLCH. Of those 98 infants, 30 were excluded due to poor data quality, exposure to other inotropes, or congenital anomalies. The remaining 68 patients included seven infants treated only with dopamine. After matching controls in a 2:1 fashion, 16 controls, and seven cases remained. At UVa, 78 infants were diagnosed with moderate-severe encephalopathy and had arterial lines placed during the study period. Of those 78 infants, 28 were excluded due to the same exclusions listed above. The remaining 60 patients included 11 patients treated only with dopamine. After matching controls in a 2:1 fashion, 20 controls and 11 cases remained in the study.p < 0.01, The final cohort consisted of 54 infants with 18 cases matched to 36 controls. Many of the clinical and demographic characteristics between the cases and controls were statistically similar , but the two groups differed with respect to their cord gases with dopamine-treated infants demonstrating lower pH indicating more severe asphyxia . There were no other differences in pharmacologic interventions . AlthougThe average age at the start of the recording was 5.6 ± 3.5 h and the average length of each recording was 66.4 ± 3.5 h. The average duration of dopamine therapy was 66.1 ± 38.8 h. One dopamine-treated infant had one missing blood pressure reading at 96 h of life. The control group had 1, 3, 5, and 13 missing MABP measures at 48, 60, 72, and 96 h of life, respectively. Missing data can be attributed to discontinuation of the umbilical arterial line by the clinical treatment team prior to the end of the study period.p < 0.05, There was no statistically significant difference between dopamine-exposed and control infants in the frequency or volume of normal saline boluses given for hypotension/hypoperfusion. Similarly, there was no difference in the frequency or volume of pRBC transfusion for the treatment of anemia. In contrast, the dopamine-exposed infants had a greater frequency of exposure to platelet and FFP transfusions compared to controls  average MABP of the control infants at the 9th h of dopamine treatment.Compared to controls, the dopamine-exposed infants had a significantly lower mean MABP at TIn addition to the absolute differences in average MABP, the two groups of infants had markedly different MABP trajectories. The dopamine-treated infants had irregular changes in MABP with time, increasing more rapidly after dopamine initiation before appearing to plateau at 49–50 mmHg. This stands in contrast to the control infants who continued to have a smooth, uninterrupted increase in MABP as postnatal age increased.This study provides a comprehensive evaluation of blood pressure changes occurring in infants with neonatal encephalopathy undergoing TH with reliable data from continuous, invasive blood pressure monitoring. We found that infants with neonatal encephalopathy undergoing TH and treated with dopamine received more blood products and generally responded to dopamine treatment, although a small subset required exogenous adrenal replacement. Additionally, although there was a trend toward a greater number of seizures and increased incidence of moderate to severe brain injury for dopamine-treated infants, it did not meet statistical significance. There was no difference in mortality between the groups.Infants requiring dopamine treatment in our cohort had similar demographic and clinical characteristics compared to controls but were more severely asphyxiated as demonstrated by the lower pH in the cord blood. Despite this difference, those more severely asphyxiated received a similar frequency and volume of fluid resuscitation compared to the control group, a finding in line with previously published data . HoweverThis study is the first to report the blood pressure response to dopamine of infants diagnosed with HIE undergoing TH and is strengthened by the availability of concurrent, continuous, full EEG monitoring. Only one previous study, published by DiSessa et al., describes the cardiovascular effects of dopamine in severely asphyxiated neonates . HoweverWe noted a clinically important, although not statistically significant, difference in seizure incidence between dopamine-exposed and control infants. Seizures are a common sequela of an initial ischemic injury, portend a worse outcome –32, and By design, infants who did not respond to dopamine were excluded from this study. By using a group of control infants who received no inotropes and a case group who received a single agent, this comparison could be made with the fewest confounders and have the greatest generalizability for typical treatment of the typical infant. While dopamine non-responders deserve investigation, this line of investigation would be confounded by the small number of infants in this group and the broad diversity of second-line treatments  which have very different mechanisms of action.We did not find significant differences in frequency of brain injury on MRI between the dopamine-exposed and control infants. This result was surprising, as there is considerable evidence that hypotension/hypoperfusion states are associated with impaired cerebral autoregulation, a known risk factor for brain injury –36. DisrThere are several potential reasons for the null finding in this study. First, it is possible that need for dopamine is not linked with additional risk for brain injury and that injury is merely the result of the original hypoxic-ischemic event. More likely is that MABP is a crude measure of perfusion; MABP is captured via pressure catheter at the level of a large vessel and may not reflect the complex of factors which govern perfusion in more distal vessels. Indeed, the entire concept of normal or adequate blood pressure threshold in neonates remains in question . Of noteThe methodology of this study was practical in nature, utilizing readily available measurements of MABP. These results suggest that the interaction between systemic and cerebral blood flow requires interrogation at the cerebrovascular level. The use of transcranial Doppler ultrasound to measure cerebral blood flow velocity and evaluate the resistive index might provide one such alternative. While this approach directly measures cerebral blood flow velocity, it is not practical for continuous monitoring due to tissue heating , 39 and Additional limitations to this study include small sample size, which could have limited our power to detect clinically significant differences in seizures or brain injury, and minor practice variations between the two centers. For example, fentanyl and morphine may have different hemodynamic effects influencing the response to dopamine between the two centers. In addition, differences in timing and interpretation of MR imaging limit the external validity of these findings. It is possible that the differences in radiographic brain injury were sufficiently subtle that they could not be captured when combining infants into normal/mild or moderate/severe categories. Finally, some scans were performed during the time frame where pseudonormalization  may haveConsidering these limitations, this study highlights the limitations of current clinical practice, namely that the use of MABP and its response to hemodynamic support is inadequate to judge the status of cerebral perfusion and is not helpful for prediction of which infants will have brain injury or seizures. This underscores the urgent need for development of more specific therapeutic endpoints in this population. Future studies should include concurrent measures of cerebral autoregulation (measured using Doppler ultrasound and/or NIRS) and consistent prospective imaging to more fully establish the link between hypotension/hypoperfusion, impaired cerebral blood flow, and risk of brain injury in HIE.For infants with hypoxic-ischemic encephalopathy (HIE), both the underlying disease process and therapeutic hypothermia (TH) have the potential to negatively impact the hemodynamic status, and this is frequently manifested as hypotension/hypoperfusion. Dopamine generally increases blood pressure in patients with moderate/severe HIE undergoing TH and facilitates sustained MABP similar to untreated controls during cooling and rewarming. Future studies should include concurrent measures of cerebral autoregulation and consistent prospective imaging to more fully establish the link between hypotension/hypoperfusion, impaired cerebral blood flow, and risk of brain injury in HIE.The datasets generated for this study are available on request to the corresponding author.The study protocol was approved by the Institution Review Boards of Washington University and University of Virginia. The IRB of Washington University in St Louis waived the requirement for written informed consent from the parents/guardians of the participants.Specifically, CP participated in the following aspects of the study: initial concept, study design, data collection, data analysis, data interpretation, draft of first manuscript, and final edits after receiving feedback from other authors. CM participated in study design and data interpretation in addition to providing critical feedback of manuscript drafts. MA, SZ, and KF participated in data collection and data interpretation in addition to providing critical feedback of manuscript drafts. ZV participated in concept, study design, data interpretation, and provided critical review of manuscript drafts. All authors contributed to drafting or revising of the paper for important intellectual content and approve of the final submitted version.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.


Objectives: Glioma is the most common and aggressive type of primary central nervous system (CNS) tumor in adults and is associated with substantial mortality rates. The aim of our study was to evaluate the prognostic significance and function of the complement factor I (CFI) in glioma.Materials and Methods: The expression levels of CFI in glioma tissues and the survival of the CFIhigh and CFIlow patient groups were analyzed using The Cancer Genome Atlas (TCGA) database and Genotype-Tissue Expression (GTEx). The correlation between CFI expression and clinicopathological features of glioma was determined by univariate and multivariate Cox regression analyses in the Chinese Glioma Genome Atlas (CGGA) database. The functional role of CFI in glioma was established through routine in vitro and in vivo assays.Results: CFI is overexpressed in glioma and its high levels correlated with poor outcomes in both TCGA and CGGA datasets. Furthermore, CFI was identified as an independent prognostic factor of glioma in the CGGA database. CFI knockdown in glioma cell lines inhibited growth in vitro and in vivo, whereas its ectopic expression increased glioma cell proliferation, migration, and invasion in vitro. CFI protein levels were also significantly higher in the glioma tissues resected from patients and correlated to worse prognosis.Conclusions: CFI is a potential prognostic biomarker in glioma and drives malignant progression. Gliomas are the most common and aggressive type of primary central nervous system (CNS) tumor in adults  is a serine protease that inactivates the complement cascade by degrading C4b and C3b  and Genotype-Tissue Expression  and analyzed using R programming language. Gene Expression Profiling Interactive Analysis  was used to analyze survival differences between the CFIhigh and CFIlow glioblastoma multiform (GBM) and low-grade glioma (LGG) patients. The glioma datasets with mRNA expression and clinical data, including PRS_type, WHO grade, age, chemotherapy status, IDH_mutation_status, 1p/19q_codeletion_status, and histology grade, were acquired from the Chinese Glioma Genome Atlas  (Dataset ID: mRNAseq_693 and mRNAseq_325). The correlation between CFI expression and clinical features was evaluated by Cox regression analyses. Variables with P < 0.05 in the univariate analysis were further confirmed in the multivariate analysis.RNA-seq data of CFI in multiple tumor tissues and corresponding normal tissues were obtained from The Cancer Genome Atlas  tool  analysis using the R “survivalROC” package. The sensitivity and specificity of prognostic signatures in predicting the overall survival (OS) were evaluated in terms of the area under the curve (AUC) of ROC. A prognostic nomogram was then constructed using the rms package of R in order to predict 1-, 2-, and 3-year OS of glioma patients based on the CGGA dataset. Pearson's correlation analysis was performed to identify CFI-related genes using the CGGA dataset. The normal human astrocyte cell line NHA was purchased from Lonza, and human glioma cell lines A172, U118, LN229, U251, T98G, and U87 were obtained from the American Type Culture Collection (ATCC). All cell lines were cultured in as previously described  and western blotting were performed as described previously  were baked at 60°C for 1 h, deparaffinized in xylene, and rehydrated in graded concentrations of ethanol . The sections were placed in pH 6.0 citric buffer for 20 min, treated with 3% hydrogen peroxide in phosphate-buffered saline, and incubated with goat serum. Each section was incubated with primary and secondary antibodies separately. Chromogen was added and the specimens were counterstained with hematoxylin. Immunohistochemical images were taken using a Leica DM IRE2 microscope  and analyzed by Image-Pro Plus v6.0 software . The integrated optical density (IOD) was calculated according to the following grading system: staining extensity was categorized as 0 (≤5% positive cells), 1 (>5% and ≤25% positive cells), 2 (>25% and ≤50% positive cells), or 3 (>50% positive cells), and staining intensity was categorized as 0 (negative), 1 (weak), 2 (moderate), or 3 (strong). All the samples were examined and scored by two experienced pathologists. Cases with discrepancies in the scores were discussed to reach a consensus.The pCDNA3.1-CFI recombinant plasmid was constructed by cloning the CFI cDNA into the pCDNA3.1 vector. Two GBM cell lines stably expressing shCFI or shCtrl were established using a lentiviral packaging kit (Santa Cruz Biotechnology) and selected with 5 mg/ml puromycin 48 h after transfection. The cells were transfected using Lipofectamine 3000 (Invitrogen) according to the manufacturer's protocol.3 cells/well, and 10 μl CCK-8 reagent was added per well at 24, 48, 72, and 96 h of culture. The absorbance at 450 nm was measured to calculate the percentage of viable cells.The proliferation rate of glioma cells was examined by the CCK-8 kit (Beyotime) following the manufacturer's instructions. Briefly, the suitably transfected cells were seeded into 96-well plates at the density of 5 × 102 cells/well and cultured for 14 days. The colonies were washed thrice with PBS, fixed with 100% methanol for 20 min, and stained with 1% crystal violet in 20% methanol for 15 min. After washing thrice with PBS, the number of colonies was counted and the averages were calculated.The cells were seeded into a six-well plate at the density of 5 × 105 cells/well. The monolayer was scratched using a sterile 20-μl pipette tip, and the wound area was photographed at 0 and 24 h under a light microscope . The migration index was calculated in terms of wound coverage.The suitably treated glioma cells were seeded onto a coverslip in six-well plates at the density of 3 × 104 suitably treated glioma cells were seeded in the upper wells of the chamber in serum-free media. The lower chamber wells contained DMEM supplemented with 20% FBS. After incubation for 24 h, non-invading cells were scraped off and invading cells on the bottom cells were fixed and stained. The number of invading cells was counted in three representative fields per sample.The invasion assay was performed as described previously  and width (W) were measured with calipers, and the volume (V) was calculated as (L × W2)/2. Mice were sacrificed when they showed signs of ill health such as weight loss, rough coat, etc. Brains were harvested and further fixed with 4% paraformaldehyde at 4°C overnight. All animal studies were approved by the Animal Welfare Ethical Review Committee of Nanjing Medical University and Anhui Medical University and conducted in accordance with the National Guidelines for the Care and Use of Laboratory Animals.An orthotopic glioma model was established by intracranially injecting 5 × 10https://www.r-project.org/), and GraphPad Prism 7.0 programs were used for statistical analyses. The association between clinicopathologic features and CFI was evaluated with the Wilcoxon signed rank test and logistic regression. Pearson's correlation analysis was used to identify CFI-related genes in the CGGA database. Univariate and multivariate Cox regression analyses were used to evaluate the relationship between the clinical variables and OS. The nomogram model was created using the rms package of R software. ROC and AUC were calculated using the package of “survivalROC” in R. All experimental data was expressed in the format of mean ± standard deviation. Student's t test was used for pairwise comparison, and one-way analysis of variance and Cox regression analysis were used for multiple group comparison. Survival was analyzed using the Kaplan–Meier method with GraphPad Prism 7. P < 0.05 was considered statistically significant.SPSS 24.0 , R3.3.1 . We analyzed the CFI expression profile across diverse tumors in TCGA database and found that CFI was significantly overexpressed in COAD (colon adenocarcinoma), GBM, LGG, READ (rectum adenocarcinoma), STAD (stomach adenocarcinoma), and THCA (thyroid carcinoma) relative to the corresponding normal samples . Analysi gliomas . Consist gliomas . Taken tp-value.Furthermore, we tried to investigate the expression and prognostic value of CFI in glioma subgroups in the CGGA dataset according to WHO 2016 classification  = 1.149, 95% confidence interval (CI) = 1.079–1.222], PRS type , WHO grade , age , chemotherapy status , IDH mutation , and 1p19q co-deletion   .C-index of the risk score was 0.769. Thus, the nomogram showed moderate sensitivity and specificity for predicting 3-year OS of glioma patients.To further elucidate the predictive accuracy of the above parameters for OS in glioma patients, we next conducted an ROC analysis. The AUC of CFI was 0.734 compared to 0.681, 0.733, 0.499, 0.593, 0.457, 0.514, 0.69, and 0.615 calculated for PRS type, histology, gender, age, radiotherapy status, chemotherapy status, IDH mutation status, and 1p19q co-deletion status, respectively. Thus, CFI can predict the OS of glioma patients with moderately high sensitivity and specificity . HoweverP-value <0.05 and FDR q-value <0.05 were considered significant. As shown in high gliomas , ANXA2 , C1R , C1S , CASP4 , CP , PLAU , PROS1 , SERPINA3 , and TNFRSF12A   using the Kyoto Encyclopedia of Genes and Genomes (KEGG) sets (c2.cp.kegg.v6.2.symbols). The gene sets with normalized  gliomas  and ther< 0.001) . Further< 0.001) . These fRecent studies suggested that VEGF signaling is a complex process involving GBM growth stimulation or inhibition (Kil et al., in situ expression of CFI compared to the NBTs. Notably, grade IV gliomas had the strongest CFI expression (We validated the bioinformatics data on CFI expression level with six paired GBM and NBT specimens, as well as in glioma specimens of different grades. As shown in pression . Consistpression . Taken tin vitro. The growth rates of CFI-depleted U251 and LN229 cells were significantly lower compared to the control cells (To gain further insights into the role of CFI in gliomas, we knocked down its expression in U251 and LN229 glioma cell lines using three independent lentiviral shRNAs. As shown in ol cells . Consistol cells . Accumulol cells . Therefool cells . CollectTo further establish an oncogenic role of CFI in glioma, we overexpressed the CFI gene in A172 cells  and perfin vivo, we injected luciferase-labeled control or shCFI U251 cells intracranially into 4-−6-week-old nude mice. Tumor growth was monitored using in vivo bioluminescence imaging system. The control mice exhibited tumor growth over 21 days after inoculation, whereas the CFI-depleted glioma cells formed significantly smaller tumors (in vivo, indicating its therapeutic potential.To further explore the role of CFI in glioma progression r tumors . Represer tumors . Quantifr tumors . Consistr tumors . Finallyr tumors . Togethein vitro. Thus, CFI is a promising novel biomarker for stratifying glioma patients into prognostic groups.Gliomas are the most prevalent primary malignancy of the brain in adults and are difficult to treat, resulting in a dismal 35% 5-year OS rate (Lapointe et al., At present, glioma is diagnosed on the basis of clinicopathological features, which also serve as the indicators of disease progression (Huse and Aldape, high phenotype, indicating that CFI may drive glioma progression through the aforementioned signaling pathways. Furthermore, we provided experimental evidence that CFI may in part contribute to FAK activation in VEGF pathway. In addition, the ANXA1, ANXA2, C1R, C1S, CASP4, CP, PLAU, PROS1, SERPINA3, and TNFRSF12A genes were positively associated with CFI expression and are possibly involved in glioma progression as well. However, we did not identify the genes that are negatively correlated with CFI expression due to the limitation of gene correlation analysis. Nevertheless, silencing CFI in the glioma cells significantly decreased their proliferation, invasion, and migration abilities, whereas its ectopic expression had the opposite effect. Thus, CFI likely functions as an oncogene in glioma and promotes tumor progression. According to their clinicopathological and histological features, gliomas are classified into the IDH-mutant grade II glioma, IDH-wildtype grade II glioma, IDH-mutant and 1p/19q-codeleted grade III glioma, IDH-mutant and 1p/19q non-co-deleted grade III glioma, IDH-wildtype GBM, IDH-mutant GBM, and other subtypes (Chai et al., high and CFIlow groups in IDH-mutant grade II glioma and IDH-mutant GBM in the CGGA dataset.Functional annotation of CFI further revealed that the JAK/STAT, NOD-like receptor, pathways in cancer, T cell receptor, and VEGF pathways were significantly enriched in the CFIin vitro and in vivo assays further indicated that CFI knockdown suppressed glioma cell proliferation, invasion, and migration, whereas its overexpression had the opposite effects. Our findings provide novel molecular insights into glioma progression and recognize CFI as a promising prognostic biomarker and therapeutic target for glioma.To summarize, integrated bioinformatics analysis showed that CFI is upregulated in gliomas and its expression level is correlated to the WHO tumor grade, IDH mutation status, and other clinical variables. In addition, we identified CFI as an independent prognostic factor of OS in glioma patients. Functional The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by the ethics committee of Nanjing Medical University and Anhui Medical University. The patients/participants provided their written informed consent to participate in this study. The animal study was reviewed and approved by the Animal Welfare Ethical Review Committee of Nanjing Medical University and Anhui Medical University.XC, YiW, and YuW conceived the project and designed the study. XC, WQ, MQ, SF, and CP performed the experiments. CP and JZ collected glioma and normal brain tissue samples. XC, WQ, MQ, and SF analyzed and/or interpreted the data. XC drafted the manuscript. YiW and YuW provided study supervision. All authors have read and approved the final manuscript.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.


AGL15 and miR156 control SE induction and AGL15 has been reported to target the MIR156 genes in planta. The results showed that AGL15 contributes to the regulation of miR156 in an embryogenic culture at two levels that involve the activation of the MIR156 transcription and the containment of the abundance of mature miR156 by repressing the miRNA biogenesis genes DCL1 (DICER-LIKE1), SERRATE and HEN1 (HUA-ENHANCER1). To repress the miRNA biogenesis genes AGL15 seems to co-operate with the TOPLESS co-repressors (TPL and TPR1-4), which are components of the SIN3/HDAC silencing complex. The impact of TSA (trichostatin A), an inhibitor of the HDAC histone deacetylases, on the expression of the miRNA biogenesis genes together with the ChIP results implies that histone deacetylation is involved in the AGL15-mediated repression of miRNA processing. The results indicate that HDAC6 and HDAC19 histone deacetylases might co-operate with AGL15 in silencing the complex that controls the abundance of miR156 during embryogenic induction. This study provides new evidence about the histone acetylation-mediated control of the miRNA pathways during the embryogenic reprogramming of plant somatic cells and the essential role of AGL15 in this regulatory mechanism.The embryogenic transition of somatic cells requires an extensive reprogramming of the cell transcriptome. Relevantly, the extensive modulation of the genes that have a regulatory function, in particular the genes encoding the transcription factors (TFs) and miRNAs, have been indicated as controlling somatic embryogenesis (SE) that is induced in vitro in the somatic cells of plants. Identifying the regulatory relationships between the TFs and miRNAs during SE induction is of central importance for understanding the complex regulatory interplay that fine-tunes a cell transcriptome during the embryogenic transition. Hence, here, we analysed the regulatory relationships between AGL15 (AGAMOUS-LIKE 15) TF and miR156 in an embryogenic culture of Arabidopsis. Both  Transcription factors (TFs) and miRNAs play a central role in the response of a somatic cell transcriptome to the SE-inducing factors such as auxin treatment [The molecular mechanisms that underlie the phenomenon of the toti-/pluripotency of somatic cells remain at the centre of developmental biology. In plants, insights into somatic embryogenesis (SE) that is induced in reatment ,2,3,4. HAGL15 encodes the MADS-domain protein that selectively binds to a consensus DNA sequence, the CArG (C-A/T rich-G) motif, in order to either activate or repress the expression of the target genes [IAA30 genes [The SE-involved genes encode numerous TFs that have hormone- and stress-related functions ,6,7,8,9 et genes . The binet genes ,14. Accoet genes ,17. Accoet genes . AGL15 a30 genes ,19,20).MIR genes encoding microRNA (miRNAs) [In addition to protein-encoded genes, the (miRNAs) ,21 are a(miRNAs) . In the (miRNAs) ,24.MIRs in embryogenic cultures of Arabidopsis and other plants has indicated highly diverse expression patterns between the members of the same MIR gene family [MIR genes seems to contribute to the production of functional miRNA molecules during SE induction. Although the MIR promoters, similar to the protein-coding genes might be activated or repressed by the binding of specific regulatory proteins, only a few TFs that target specific MIR genes have been identified to date [The differential expression of numerous miRNAs, mostly hormone-related, has been attributed to SE induction in different plants including Arabidopsis ,28,29,30e family ,35,36. T to date .MIR156 genes has been reported during flower development [MIR156 genes were reported to be among the potential targets of AGL15 in an embryogenic culture [AGL15 and miR156 transcripts in the cotyledons of Arabidopsis that contribute to SE induction in this plant has been demonstrated [The contribution of AGL15 to the positive regulation of the elopment . Importa culture  and cons culture ). Moreovnstrated ,38. TakeLEC1 and LEC2 genes, which are the master regulators of both zygotic and somatic embryogenesis [The regulatory mechanism by which AGL15 might control the target genes involves interactions with the SIN3/HDAC (Swi-independent3/Histone Deacetylase) silencing complex . By recrogenesis ,43,44,45ogenesis ,42,46,47AGL15 and the abundance of miR156 in SE, we analysed the impact of AGL15 on both the transcription of the MIR156 genes and the level of mature miR156 molecules in an embryogenic culture of Arabidopsis. Insights into the gene expression profiles in different mutants and transgenic lines together with the results of the ChIP analysis indicated that AGL15 contributes to the regulation of miR156 in an embryogenic culture at two levels that involve the activation of MIR156 transcription and the containment of the abundance of mature miR156 by repressing the miRNA biogenesis genes DCL1, SERRATE, and HEN1. We also showed that to repress the miRNA biogenesis genes AGL15 seems to co-operate with other components of the SIN3/HDAC silencing complex including the TOPLESS co-repressors and HDAC6 and HDAC19 histone deacetylases. The study provides new evidence about the histone acetylation-mediated control of the miRNA pathways during the embryogenic reprogramming of plant somatic cells and the essential role of AGL15 in this regulatory mechanism.Here, in order to verify the assumption about the regulatory relationship of MIR156 genes (MIR156a and c) during flowering [AGL15 and miR156 in SE induction. To this end, the abundance of the primary miR156 transcripts (pri-miR156) of eight MIR156 (MIR156a–h) genes that are present in the Arabidopsis genome was analysed in cultures that had different AGL15 expression levels, including WT (Col-0), 35S::AGL15, and agl15 agl18. The results showed that there was no relationship between the AGL15 expression level and the transcription of seven of the MIR156 genes (MIR156a–g) and that both the overexpression (35S::AGL15) and knock-out mutation (agl15 agl18) resulted in a significant increase in pri-miR156a-g (AGL15 and a distinct downregulation (up to two-fold) in the agl15agl18 mutant culture, which suggests that AGL15 might positively regulate MIR156h during SE induction.The role of AGL15 in the activation of two lowering  motivateiR156a-g . By contAGL15 expression level. Unexpectedly, the results showed that AGL15 significantly modulated the abundance of mature miR156 and that there was a distinctly higher (from 2- to 14-fold) abundance of mature miR156 in the agl15 agl18 mutant than in the 35S::AGL15 culture , SERRATE, HUA-ENHANCER1 (HEN1) and HYPONASTIC LEAVES1 (HYL1). To this end, the expression of these genes relative to the AGL15 expression level was evaluated in embryogenic cultures of the 35S::AGL15 and agl15 agl18 double-mutant lines. The analysis indicated a significant decrease (2- to more than 3000-fold) in the DCL1, SERRATE, HEN1 and HYL1 expression in the SE culture that overexpressed AGL15 , which implies a lack of any regulatory impact of AGL15 on HYL1. In summary, the results suggest that AGL15 may negatively affect the expression of three of the miRNA biogenesis genes including DCL1, HEN1, and SERRATE.The post-transcriptional mechanism that extensively fine tunes the mature miRNA level via miRNA-processing machinery has been assumed in an embryogenic culture . Thus, wed AGL15 A and the8 mutant B. By conDCL1, HEN1, and SERRATE) in embryogenic culture via its interaction with the HDACs. To verify this hypothesis, we evaluated the effect of the HDAC inhibitor, trichostatin A (TSA), on the DCL1, SERRATE, and HEN1 gene expression in the 35S::AGL15 and agl15 agl18 mutant cultures. We found that there was a significant up-regulation (from 2- to 120-fold) of the DCL1, SERRATE and HEN1 genes in the culture that was overexpressing AGL15 and a distinctly smaller increase of the gene transcripts in the mutant culture  had a significantly impaired embryogenic response  and downstream (+300 bp) to TSS and included one to six CArG sequences  of two genes  SERRATE . The aceTPL and four TPR (TPR1-4) genes indicated that they were significantly down-regulated during embryogenic induction  in somatic embryogenesis induction.To identify any other elements of the AGL15-related complex that silence the expression of the miRNA biogenesis genes in somatic embryogenesis, we investigated the possible involvement of the TOPLESS (TPL) co-repressors, which directly interact with AGL15 . The expnduction A. Togeth culture B,C, the DCL1, HEN1, and SERRATE in the tpl, tpr1 and tpr4 mutants was analysed in order to verify this hypothesis. The analysis showed that two of the miRNA biogenesis-related genes DCL1 and SERRATE, were significantly up-regulated in the embryogenic-induced explants of all three  mutants  genes in embryogenic cultures with different levels of AGL15 expression (agl15 agl18 vs. 35::AGL15) suggested that AGL15 might positively regulate the MIR156h expression during SE induction .Complex and multi-layered regulatory interactions have been demonstrated to fine tune the components of the miRNA processing pathways ,58,62. Hagl15 agl18 mutant than in the 35S::AGL15 line (MIR167 (MIR167a) expression in an embryogenic culture [We hypothesised that the repressive function of AGL15 in miRNA biogenesis might also involve other miRNAs and in support of this, we found a substantially higher level of the mature miR167 and miR172 transcripts in the culture of the L15 line . The rol culture . Importa culture . Hence, tpl/tpr mutants acetylases and (de)methylases of histones interplay extensively in the control of gene expression and mutations in HDAC19 may affect both the acetylation and methylation of histones [HEN1 and the role of histone methylation in the control miRNA biogenesis genes needs to be investigated in the future.Although, in contrast to nificant , the inv mutants . We cannhistones ,76,77,78HDAC6 and HDAC19 deacetylases that have reported interactions with AGL15 [hdac6 and hdac6/hdac19 mutations negatively affected the embryogenic response in the explant culture  Heynh. Col-0 (WT) and two transgenic lines with opposite AGL15 (AT5G13790) expression levels were used including the 35S::AGL15 and agl15 agl18 double mutant. In addition, the insertional mutants in the TPL and HDAC genes  were studied. The 35S::AGL15 was kindly provided by Sheryn Perry , the agl15 agl18 mutant by Donna E. Fernandez  and the hdac6 and hdac6 hdac19 by Kim Boutilier . The seeds of the other genotypes that were analysed were purchased from NASC . The seeds were sown in 42-mm-diameter Jiffy-7 peat pots (Jiffy) and the plants were grown in a “walk-in” type phytotron under controlled conditions: 22 °C, 16 h/8 h (light/dark) and a light intensity of 100 µE/m2s. The cultures that were grown in vitro were maintained in a controlled growth chamber at 22 °C, 16 h/8 h (light/dark) and a light intensity of 50 µE/m2s.Plants of −1 sucrose and 8 g L−1 agar . The explant capacity for SE was evaluated in a three-week-old culture and two parameters were evaluated including SE efficiency, i.e., the percentage of the explants that had formed somatic embryos and SE productivity, i.e., the average number of somatic embryos that had been produced by embryogenic explant.Immature zygotic embryos at the late cotyledonary stage were used as the explants for the in vitro cultures according to the standard protocol . The expAt4g27090 gene encoded 60S ribosomal protein. The control gene had a constant expression pattern (CT = 18 ± 1) in all of the tissue samples that were analysed. The plant tissues for the real-time (RT) qPCR analysis were produced in three biological repetitions and two technical replicates of each repetition were carried out. The relative expression level was calculated using 2CT−∆∆, where ∆∆Cp represents ∆CTreference condition − ∆Cpcompared condition.Total RNA was isolated from the explants that had been cultured for 0, 5 and 10 d on the SE-induction E5 medium. In some experiments, the E5 medium was supplemented with 1.0 µM of trichostatin A (TSA), which is a blocker of the HDAC activity. RNA was isolated using a miRVana miRNA Isolation Kit. Depending on the age of the culture, 250 (0 d) to 50 (10 d) explant-derived cultures were used for the RNA isolation in one biological replicate. The concentration and quality of the isolated RNA was evaluated using an ND-1000 NanoDrop spectrophotometer . The miRNA-specific and oligo-dT primers and RivertAid First Strand cDNA synthesis kit  were used to synthesise the cDNA. The mature miRNAs were identified according to Speth and Laubinger . The pro® Plus, Diagenode, Denville, NJ, USA) and the complex of proteins and DNA fragments was then immunoprecipitated using the polyclonal antibodies against the acetylated forms of histone H3 . The DNA that was cross-linked to the immunoprecipitated proteins was reversed and analysed using qPCR and the gene-specific primers. A LightCycler 480  real-time detection system was used to analyse the relative acetylation level of the chromatin associated with DCL1, HEN1, and SERRATE genes. The qPCR reaction was performed as described by [DCL1, HEN1, and SE genes were localised in the TSS + 300 bp region and included the AGL15-binding CArG motif ((adjusted input—Cp (xx gene) × 100%. Three biological replicates and two technical replicates were analysed for each combination.The Yelagandula et al. ChIP method  was usedribed by . The prirG motif . The Cp t-test (p < 0.05) or a two-way ANOVA (p < 0.05) followed by Tukey’s honestly significant difference test (Tukey HSD-test) (p < 0.05) were used to calculate any significant differences between the combinations. The graphs show the averages with the standard deviation (SD); the statistical analysis was performed using the medians.The Student’s MIR156 (MIR156h) and the containment of the abundance of mature miR156 via the downregulation of the miRNA biogenesis genes DCL1, SERRATE, and HEN1  might also be expected. In support of such a scenario, a histone acetyltransferase, GCN5, besides repressing miRNA processing, was required for the expression of a subset of the MIR genes [MIR genes. The MIR-regulatory function of HDAC19 requires experimental verification.Two histone deacetylases, HDAC6 and HDAC19, were postulated to contribute to the AGL15-mediated repression of miRNA biogenesis during somatic embryogenesis. However, histone acetylation might also regulate the transcription of the g MIR156 . Given tg MIR156 ,90, the IR genes . InteresIR genes . This fiIR genes , might cHistone (de)acetylases may also regulate miRNA processing through changes in the acetylation status of the regulatory proteins of the microprocessor complex ,93. WhetIn conclusion, the present study indicates that similar to mouse and human cells ,95, hist


Three azido-terminated poly(ethylene glycol) macromonomers (ATPEGs) were synthesized from poly(ethylene glycol)s (PEGs) and characterized. The extended polytriazole (EPTA) resins were prepared from the macromonomers, azide and alkyne monomers. Toughening effect of PEGs on polytriazole resins was analyzed by means of mechanical, thermal and electronic microscope characterization. The results show that molecular weight and content of ATPEGs have great influence on the thermal and mechanical properties of cured EPTA resins. The impact strength of cured EPTA resins increases with the increase of the amount and molecular weight of ATPEGs. The flexural strength and heat resistance of cured EPTA resins decrease with the increase of addition amount and molecular weight of ATPEGs. High impact EPTA resins were obtained. In 1893, Michael  synthesiHuang group  has prepRecently, advanced polymeric composites require the resins with high toughness to meet the development of high technologies in the aeronautics and astronautics fields. At present, there are three kinds of toughening technologies for thermosetting resins: (1) adding second phase materials such as elastomers and rigid particles in a resin; (2) using interpenetrating polymer network technology; (3) introducing a flexible chain segment to molecular structures of a resin (grafting or blocking), or selecting an appropriate crosslinking agent . The metN,N-dimethylformamide (DMF) (A.R), dichloromethane(A.R), magnesium sulfate(A.R), acetone were purchased from Shanghai Titan Technology Co., Ltd. 1,1-Bisazidomethyl-4,4′-biphenyl(BAMBP) and N’,N’,N’,N’-tetrapropargyl-p,p’-diaminodiphenyl methane (TPDDM) were synthesized according to a previously published procedure ) were mixed in acetone with a solid content of 70% and stirred at 60°C till the mixture became homogeneous. The reaction continued at 60°C for 4 h. The liquid resinwas obtained after acetone was removed by evaporation. The resin prepared from ATPEG400, BAMBP and TPDDM is named as EPTA-4, from ATPEG600, BAMBP and TPDDM as EPTA-6 and from ATPEG800, BAMBP and TPDDM as EPTA-8. Taking EPTA-4-10 resin as an example, it was prepared from TPDDM and azides . PTA resin was prepared from BAMBP and TPDDM in the same way. The liquid resins obtained by evaporation were poured into a mold and put into a vacuum oven for removing the residual acetone and embedded air. The resins were cured at 80 °C for 12 h. The obtained resins were post-cured in the following procedure: 120 °C/2 h + 150 °C/2 h + 180 °C/2 h + 210 °C/2 h.TPDDM and BAMBP together with an ATPEG with the molar ratio 1.05:1.00 of alkyne group to azide group  was performed using ESI-high resolution time of flight mass spectrometer  with m/z range of 50 ~ 4000 Da. FTIR spectrum measurements were carried out on a Nicolet iS10 infrared spectrometer  in the region of 4000–400 cm−1 using KBr pellets. The elemental analysis was performed with Vario EL III elemental analyzer . Differential scanning calorimetry (DSC) analyses were performed with a Q2000  at a heating rate of 10 °C min−1 in a nitrogen atmosphere. Dynamic mechanical analysis (DMA) was carried out on a DMA 1  in the dual cantilever clamp mode under nitrogen at the frequency of 11 Hz with a programmed heating rate of 3 °C min−1 from RT to 350 °C. Thermogravimetric analysis (TGA) was conducted on a TGA/DSC 1  under nitrogen at a heating rate of 10°C min−1 from RT to 800 °C and the gas flow rate was 60 ml/min. The flexural performance test was conducted on DDL100 electronic tension machine in accordance with GB/T 2570–1997, and the loading speed was 2 mm/min. The impact performance test was carried out on the Italy CEAST9050 cantilever beam impact testing machine, pendulum energy impact range was 0.55 ~ 22.5 J. No notch impact was tested when the pendulum energy was 4 J with the sample size of 80 mm × 10 mm × 4 mm on reference to GB/T 2571–1995. Hardness was measured by GYZJ-934-1 Barcol impressor. Scanning electron microscopy (SEM) was performed using the S-4800 scanning electron microscope  with an acceleration voltage of 15 kV.All NMR spectra were obtained on a Bruker Advance 400 MHz Spectrometer  using tetramethylsilane (TMS) as an internal standard in DMSO or CDCl−1 under nitrogen atmosphere and the obtained DSC curve is shown in The curing behavior of the EPTA-4-15 was investigated by DSC at a heating rate of 10°C min∆H of the resins is relatively high (> 670 J/g), so in order to avoid explosive polymerization during the curing process, the initial curing reaction temperature should be determined to be around Ti.The DSC curves show that the initial curing temperature of EPTA resins is around 80°C, the peak temperature of exothermic peak is around 145°C, and the end temperature is around 200°C. The detailed structures of three EPTA resins are different, but their initial curing temperatures and peak temperatures are very close, indicating that the ‘Click’ reaction of alkyne and azide is less affected by the connected structures. The exothermic heat 3 and –C≡C– near 2095 cm−1 gradually decreases with the curing process, and the stretching vibration peak of carbon-hydrogen bonds on triazole rings appears near 3034 cm−1, indicating that the polymerization reaction of –N3 and –C≡C– occurs. With the increase in polymerization temperature, the characteristic absorption peak of –N3 and –C≡C– at 2095 cm−1 gradually weakens and completely disappears at 210 °C, indicating that the curing of EPTA-4-15 resin is completed. The results of FTIR characterization of EPTA-6 resins and EPTA-8 resins are similar(see SI Fiure S10-S11). FTIR characterization also shows that the resins finish the curing reactions through the curing procedure of 80 °C/12 h + 120 °C/2 h + 150 °C/2 h + 180 °C/2 h + 210 °C/2 h.The curing process of EPTA-4-15 resin is studied by FTIR analysis. The absorption of the characteristic functional groups of the EPTA-4 resin changes at different curing stages in the FTIR spectra during the process of polymerization as shown in The mechanical properties of cured EPTA resins are listed in The hardness of cured resins was measured with a Barcol impressor and shown in The molecular weight between crosslinked points affects the properties of the resin. When the molecular weight is high, the distance between crosslinking points is long, leading to lower crosslinking degree. Thereby, the resin with high molecular weight flexible chain would exhibit high impact strength and low flexural strength and hardness.The impact and flexural properties of EPTA resins toughened with various molecular weight of ATPEGs are shown in The microstructure of the resins is particularly important for their properties. The fracture morphology of resins observed by scanning electron microscopy can provide necessary information for the properties and modification of the resin. g) of the EPTA resins is significantly lower than that of PTA resin. Tg of the toughened resin system decreases by 20°C to 30°C for every 5% increase in the amount of ATPEG. For resin systems with the same amount of ATPEG addition, the Tg of EPTA resins decreases with the increase of polyether molecular weight. With the increase of the amount and molecular weight of ATPEGs, the network has more flexible chain segment and looser nets (lower crosslinking degree) [The DMA analyses of various cured EPTA resins were conducted and typical DMA curves are shown in  degree) . As a red5 among EPTA resins and Td5 is located around 340°C. A large number of rigid structures of a benzene ring and a triazole ring with high crosslinked degree by thermal crosslinking reactions in the curing process contribute excellent thermal stability of the resins. When the temperature is above 365°C, the resin decomposes rapidly. The thermal degradations of EPTA resins are mainly due to the thermal decomposition of the triazole ring and the thermal cleavage of the CH2-N bond [The thermogravimetric analysis of the cured EPTA resins was carried out under nitrogen. The TGA curve for EPTA-4-10 resin is shown in 2-N bond .Three azide-terminated poly(ethylene glycol) macromonomers  and related EPTA resins with various amount of ATPEGs were synthesized and characterized. The EPTA resins can be cured at 80 °C, and then post-cured through the procedure of 120 °C/2 h + 150 °C/2 h + 180 °C/2 h + 210 °C/2 h.Molecular weight and amount of ATPEGs have remarkable influence on the thermal and mechanical properties of EPTA resins. The impact strength of EPTA resins increases with the increase in the amount and molecular weight of ATPEGs. When the amount of ATPEG is 15% and 20%, the impact strength of EPTA resin increases first and then decreases with the increase of molecular weight of ATPEG and reaches the highest value when the molecular weight of polyether is 600. The flexural strength and heat resistance of EPTA resins decrease with the increase of addition amount and molecular weight of ATPEGs. The highly toughed EPTA resins could be obtained by control of the amount and molecular weight of polyethers.2 and 103 MPa while Tg and Td5 are 155 °C and 345 °C respectively.ATPEG600 shows better toughening effect than ATPEG800. EPTA-6-15 resin demonstrates good comprehensive properties. The impact strength and flexural strength reach 75 kJ/m


The execution phase of apoptosis is a critical process in programmed cell death in response to a multitude of cellular stresses. A crucial component of this pathway is the apoptosome, a platform for the activation of pro-caspase 9 (PC9). Recent findings have shown that autocleavage of PC9 to Caspase 9 (C9) p35/p12 not only permits XIAP-mediated C9 inhibition but also temporally shuts down apoptosome activity, forming a molecular timer. In order to delineate the combined contributions of XIAP and the apoptosome molecular timer to apoptosis execution we utilised a systems modelling approach. We demonstrate that cooperative recruitment of PC9 to the apoptosome, based on existing PC9-apoptosome interaction data, is important for efficient formation of PC9 homodimers, autocatalytic cleavage and dual regulation by XIAP and the molecular timer across biologically relevant PC9 and APAF1 concentrations. Screening physiologically relevant concentration ranges of apoptotic proteins, we discovered that the molecular timer can prevent apoptosis execution in specific scenarios after complete or partial mitochondrial outer membrane permeabilisation (MOMP). Furthermore, its ability to prevent apoptosis is intricately tied to a synergistic combination with XIAP. Finally, we demonstrate that simulations of these processes are prognostic of survival in stage III colorectal cancer and that the molecular timer may promote apoptosis resistance in a subset of patients. Based on our findings, we postulate that the physiological function of the molecular timer is to aid XIAP in the shutdown of caspase-mediated apoptosis execution. This shutdown potentially facilitates switching to pro-inflammatory caspase-independent responses subsequent to Bax/Bak pore formation. Apoptosis is the process of controlled cell death, integral for development and maintaining homoeostasis. Its dysregulation has been implicated in cancer, autoimmune and neurodegenerative diseases . Apoptos315 exposes a short peptide sequence required for the efficient inhibition of C9-35/12 by XIAP ) and active C9-apoptosome ([Active C9]) complexes using the kinetic values for their respective cleavage of PC3 (kPC9 and kC9):IETDase activity at time y0 is equal to the activity at time 0, in this case the maximum IETDase activity. The half-life plotted was obtained from the fitted coefficient k by:Screening of the concentration-dependent activity of the molecular timer was performed by screening the entire range of physiologically relevant PC9 and APAF1 concentrations as a linearly distributed 25 × 25 concentration matrix. Simulations were performed for 4 h before determination of the max activity, time to max activity, integrated activity and half-life of activity decay. Integrated activity was calculated by using the trapz function in Matlab for approximation of the area under the curve (AUC) for the IETDase activity. The half-time of decay was calculated using the Matlab nlinfit function for non-linear fitting to fit the IETDase activity to the one phase decay equation:n = 1000 for survival curves, n = 100,000 ROC curves). As CytC and ATP are generally considered non-limiting, we kept the values in line with concentrations previously determined in HeLa cells, 10 and 920 μM, respectively [Parameter screening for the effect of the molecular timer was performed by randomly sampling each protein from pre-determined physiological concentration ranges to make entirely unique combinations  [PC9–CARDAPAF1 interactions in a homodimer, thus reducing unbinding. However, extraction of the SPR curves  and unbinding (koff) kinetics demonstrated that binding was also reduced for non-dimerising PC9F404D compared to PC9-TM  . It was ves Fig.  and the -TM Fig. . Given t-TM Fig. . This co-TM Fig. . The con-TM Fig. ii whilst-TM Fig. . Moreove-TM Fig.  and XIAP-TM Fig. . Further-TM Fig. ii. This We next set out to establish if the molecular timer operated at physiological protein levels and under what circumstances is it active. Therefore, we screened previously determined protein ranges of APAF1 and PC9 and calculated the activity of the holo-apoptosome in cleaving the IETD site in PC3 (IETDase) over time , 31, 33.To characterise the concentration-dependent effects of PC9 and APAF1 on the molecular timer effect better, we calculated the maximum IETDase activity, time to maximum IETDase activity and half time of IETDase decay across 4 h for every condition Fig. . This coThe molecular timer was originally proposed as a mechanism to prevent apoptosis under circumstances of accidental low-level release of apoptotic factors . TherefoOur previous results indicate that the molecular timer is active under physiological conditions. However, it remains unknown so far if the molecular timer can prevent lethal caspase 3 activation and apoptosis execution. Moreover, determining the contribution of the molecular timer to apoptosis resistance in intact cells experimentally cannot be separated from coupled processes such as XIAP-inhibition and C3-feedback cleavage of C9 and XIAP , 34. MatThe molecular timer could prevent apoptosis only in a small subset of cases after full MOMP Fig. i. In conWe next aimed to identify the key determiners of conditions under which the molecular timer is relevant for preventing apoptosis execution. To quantify the individual relevance of each key protein involved in apoptosis execution, we employed the receiver operator curve (ROC) and the AUC for evaluating the ability to predict apoptosis competency and molecular timer dependence. As expected, low levels of pro-apoptotic proteins APAF1, PC9, PC3 and SMAC can mediate apoptosis resistance, with SMAC having a particularly strong influence AUC = 0.86) For conditions of minMOMP, XIAP was identified as the major determiner of apoptosis resistance AUC = 0.92) Fig. . The lim2 Fig. . Taken together, these results demonstrate that the molecular timer’s ability to prevent apoptosis is complementary to XIAP, with the timer gaining relevance at conditions of low XIAP activity but also actively preventing apoptosis even when XIAP is in excess.Since PC9 autocatalytic cleavage initiates the roles of both the molecular timer and XIAP in apoptosis execution, we explored the relationship of both further. We found that the molecular timer reduced C3-substrate cleavage under all conditions, after full MOMP Fig.  or minMOn = 120 patient tumours, quantified by reverse phase protein array [Previously, we demonstrated that a simplified model of apoptosis execution was prognostic for disease free and overall survival in stage III colorectal cancer patients treated with chemotherapy . We therin array , togethein array  were useWe next looked at the relative contribution of the molecular timer in this patient cohort. 18/31 (58%) of apoptosis resistant tumours were resistant due to the apoptosome molecular timer, suggesting that the molecular timer indeed contributes substantially to apoptosis resistance in cancer patients. Moreover, the reduction in C3-substrate cleavage attributable to the molecular timer demonstrated a very strong reduction of >70% in 17/18 of patients that were resistant due to the molecular timer Fig. . VaryingAnalysing the contribution of each individual protein to apoptosis decisions interestingly identified PC9 and PC3 to strongly contribute to correctly classifying apoptosis competency in colorectal cancer Fig. . Moreove315 autocatalytic cleavage, to have sufficient affinity to bind and block C9 activity [PC9-CARDAPAF1 interactions at the apoptosome inducing a conformational change removing this apparent auto-inhibition to allow PC9 dimerisation and activity [In this paper, we demonstrate that the apoptosome molecular timer acts synergistically with XIAP in the prevention of apoptosis execution and that simulating these processes can contribute to correctly prognosticate patient survival in stage III colorectal cancer. Apoptosome activity is dictated by PC9 recruitment, activation and cleavage on the apoptosome. These processes are highly complex to study experimentally and therefore are not fully understood. We employed a systems approach to demonstrate that the induced-proximity dimerisation model of PC9 homodimerisation is insufficient to produce efficient autocatalytic cleavage of PC9. Previously, it has been demonstrated using non-cleavable mutants of PC9 that this step is not necessary for its ability to cleave PC3 . Howeveractivity , 18. Furactivity , 18. Theactivity . We cannactivity . Howeveractivity –29. PC9 activity . Cooperaactivity –43.i). The improvement in prognostic ability of our model therefore indicates that more realistic modelling of complex events is important in fully understanding patient treatment responses and prognosis.Implementing cooperative binding of C9 to the apoptosome results in excellent agreement between simulation results and a multitude of previous experimental data, and allowed us to confidently screen for biological phenomena. Furthermore, we demonstrated that our model was prognostic for both disease free and overall survival in stage III colorectal cancer by separating patients into apoptotic resistant and apoptotic capable groups using C3-substrate cleavage levels. Apoptosis resistance has been implicated in both treatment resistance and tumour progression –46, ther4a330 feedback cleavage of C9, which when combined with D315 autocatalytic cleavage to form C9-35/10 fully removes the p2 fragment containing the high affinity XIAP-binding domain creating a positive feedback loop [315 cleavage and terminated by D330 feedback cleavage in the event of sufficient C3 activation. There are several plausible hypotheses for why such a mechanism would exist, which are not necessarily mutually exclusive. First, non-lethal and low-level activation of C3 has been demonstrated to have pro-tumourigenic effects, promoting DNA-instability and pro-survival responses such as NFκB [The molecular timer, whilst an interesting effect, has yet to be conclusively demonstrated to operate in real cells and to prevent apoptosis experimentally. Previous studies have used non-cleavable forms of PC9 in order to show the molecular timer effect , 18. Howack loop . Intriguack loop . It was  as NFκB , 48. Sec as NFκB –51. The  as NFκB –56. Ther as NFκB , 57, 58. as NFκB , 60.In conclusion, we have utilised a novel system biology approach to demonstrate that cooperative recruitment of PC9 to the apoptosome is a key process in regulation of apoptosis by both XIAP and the molecular timer. Moreover, employing this model in screening suggests that the molecular timer can actively prevent apoptosis after minMOMP and MOMP in physiological settings. Finally, we showed that our model has prognostic capability and indicates that the molecular timer can contribute to patient outcomes.Supplementary Material


Vibrio vulnificus (V. vulnificus) is an estuarine bacterium that is capable of causing rapidly fatal infection in humans. Proper polarization and bactericidal activity of macrophages play essential roles in defending against invading pathogens. How macrophages limit V. vulnificus infection remains not well understood. Here we report that tuberous sclerosis complex 1 (TSC1) is crucial for the regulation of V. vulnificus-induced macrophage polarization, bacterial clearance, and cell death. Mice with myeloid-specific deletion of TSC1 exhibit a significant reduction of survival time after V. vulnificus infection. V. vulnificus infection induces both M1 and M2 polarization. However, TSC1 deficient macrophages show enhanced M1 response to V. vulnificus infection. Interestedly, the absence of TSC1 in myeloid cells results in impaired bacterial clearance both in vivo and in vitro after V. vulnificus infection. Inhibition of the mammalian target of rapamycin (mTOR) activity significantly reverses V. vulnificus-induced hypersensitive M1 response and resistant bactericidal activity both in wild-type and TSC1-deficient macrophages. Moreover, V. vulnificus infection causes cell death of macrophages, possibly contributes to defective of bacterial clearance, which also exhibits in a mTORC1-dependent manner. These findings highlight an essential role for the TSC1-mTOR signaling in the regulation of innate immunity against V. vulnificus infection. Vibrio vulnificus (V. vulnificus) is an emerging estuarine bacterium of coastal waters worldwide, such as the United States, Japan, China, South Korea, and Mexico  and M2  polarization. M1 and M2 macrophages are functionally polarized in response to bacterial infection . M1 macrnfection . Additionfection . Rapamycnfection .V. vulnificus-induced the mTORC1 activation and server inflammatory responses in macrophages (V. vulnificus-induced inflammation via mTORC1 activation in macrophages remains mostly unknown. Tuberous sclerosis complex 1 (TSC1) and TSC2 are tumor suppressor genes. TSC1 and TSC2 negatively regulate mTORC1 activity. TSC1 associates with TSC2 to form a heterodimer. TSC1 is also essential for TSC2 activity by the maintenance of TSC2 protein stability  transgenic mice were purchased from The Jackson Laboratory. Tsc1 flox mice were bred with LysozymeCre mice to induce TSC1 deletion in myeloid cells.We purchased the 6- to 8-wk-old C57BL/6J mice from the Chinese Academy of Science Shanghai SLAC Laboratory Animal Center. The Tsc1 flox mice and Vibrio vulnificus CGMCC 1.1758 strain, which was grew at 37°C in brain heart infusion broth (BHI) or on the BHI blood agar plate. The procedures of V. vulnificus that were used in this study were followed from the standard biological hazards at Biosafety Level 2 and the safety procedures of Wenzhou Medical University Laboratory Safety Department.L-929 cells were purchased from the Cell Bank of the Chinese Academy of Science in Shanghai and cultured in RPMI1640 containing 10% heat-inactivated fetal bovine serum (Tianhang Bio) and penicillin-streptomycin . The China General Microbiological Culture Collection Center provided the ® Rabbit mAb, phospho-p70 S6 Kinase (Thr389) (D5U1O) Rabbit mAb, and Cleaved Caspase-3 (Asp175) (5A1E) Rabbit mAb were purchased from Cell Signaling Technology (CST). Phosphatase Inhibitor Cocktail (Millipore) was purchased from Sigma-Aldrich. Mouse TNF-α Elisa kit, Mouse IL-6 Elisa kit, Mouse IL-1β Elisa kit, LEGENDplex™ Mouse Th1/Th2 Panel (8-plex) . LIVE/DEAD Fixable Blue Dead Cell Stain Kit (for UV excitation) was purchased from Invitrogen.Anti-mF4/80-APC, anti-mCD11b-PE, PE Annexin V Apoptosis Detection Kit I andCytofix/Cytoperm™ and perm/wash solutions were purchased from BD Biosciences Pharmingen . Anti-miNOS-PE was purchased from eBioscience. Anti-mCD206-APC was purchased from BioLegend. Hamartin/TSC1 (D43E2) Rabbit mAb, phospho-S6 Ribosomal Protein (Ser235/236) (D57.2.2E) XPV. vulnificus infection or PBS treatment were used for RNA sequencing. Briefly, the cells were lysed in TRIzol and subjected to Annoroad Co. for RNA extraction, sequencing, and transcriptome profile analysis. The significantly differentially expressed genes were identified when we compared the normalized reads count between V. vulnificus and PBS groups with p < 0.05 and |Log2FoldChange| > 0.263. The significance of the gene ontology term enrichment was estimated using Fisher’s Exact Test .J774A.1 cells with 6 h 6BMMϕs from both TSC1flox/flox LysM-Cre (TSC1 KO) and C57BL/6J (WT) were equally seeded in six-well plates or 35 mm dishes and cultured at 37°C for overnight. We then added V. vulnificus at the indicated multiplicity of infection (2MOI) to the cells for 2 h and 4 h. The supernatant was collected at the indicated time for cytokine quantification. The infected cells were used for flow cytometry analysis, RT-qPCR analysis, and Western blot analysis.The bone marrow cells were isolated from femurs and tibiae and cultured them with a 10% L-929 cell culture medium, as described previously . Bone maflox/flox LysM-Cre mice with V. vulnificus. For survival experiments, we injected mice with 0.5 × 108 CFU of V. vulnificus suspended in 200 μl of PBS or PBS alone. The mice were observed every 6 h for 48 h, and the mice that survived for 48 h should have been humanely killed. We euthanized and exsanguinated the mice 4 h after infection for blood and tissue collection. The V. vulnificus engulfed by phagocytes consisting of organs could be counted by grinding the organs completely with PBS and detected with BHI blood agar.We used i.p. injection to infect male C57BL/6J and TSC1V. vulnificus for 2 h and 4 h. Total RNA was extracted with TRIzol (Omega Bio-Tek). The reverse transcription was performed with PrimeScript Reverse Transcriptase  according to the manufacturer’s instructions. Real-time qPCR was performed as previously described  with protease and phosphatase inhibitor cocktails (Sigma) for 15 min at 4°C. Protein concentration was determined by Bradford assay. Protein samples were analyzed by SDS–PAGE gel and transferred to PVDF membrane. The membrane was blocked with 5% non-fat dried milk in TBST  for 1 h, then incubated with indicated primary antibodies overnight on a shaker at 4°C. The appropriate HRP-coupled secondary antibody was then added and was detected through ECL chemiluminescence (BioRad). β-actin was used as a protein loading control.Cell lysates preparation and Western blot analysis were performed by following the previous protocol . BrieflyCytokine concentration was determined using ELISA kits for IL-1β, TNFα, and IL-6 (BioLegend). We purchased a nitric oxide Test Kit from Nanjing Jiancheng Bio to measure nitric oxide production in the supernatant. Briefly, the supernatant from cultured-cells with indicated treatment was collected and centrifuged at 4°C 2,000 g per 15 min, and then was analyzed by following the manufacturer’s instruction.The plasma inflammatory cytokines were detected by flow cytometry with LEGENDplex™ Mouse Th1/Th2 Panel  kit by following our previous protocol . BrieflyV. vulnificus for 4 h and washed with PBS. Supernatant was diluted and plated them on blood BHI plates for 12 h. Then 100 μg/ml gentamicin was added to kill extracellular bacteria. After incubation at 37°C for 30 min, cells were washed twice with PBS and treated with 0.1% Triton-X100. The cell lysis was plated on blood BHI plates for 12 h at 37°C for the bacterial count.Cells were infected with 2 MOI of V. vulnificus at 37°C for 4 h. For the M1/M2 discrimination flow cytometry experiment, cells were stained with CD206-APC and iNOS-PE after fixation and permeabilization by BD Biosciences Cytofix/Cytoperm™ and perm/wash solutions. For the cell apoptosis assay, dying cell was identified by using the BD PE Annexin V Apoptosis Detection Kit I according to the manufacturer’s protocol. Data were collected by using flow cytometry.Cells were infected with 2 MOI of After the spleen, liver, and lung were fixed in 4% paraformaldehyde, we imbedded the fixed organs in paraffin, then cut thin sections, and following standard procedures, stained them with hematoxylin and eosin.t-test. P-values less than 0.05 were considered significant.Data were presented as mean ± SEM and analyzed for statistical differences using the Prism 6.01/GraphPad software. Statistical significance was analyzed using the Student V. vulnificus. To understand how V. vulnificus causes inflammation, cell death, and activation in macrophages, we performed RNA-Seq and transcriptomic analysis in J774A.1 cells before and after V. vulnificus infection. RNA-seq analysis revealed substantial changes in the macrophages transcriptome after 6 h of infection. The KEGG pathway enrichment analysis demonstrated that the top pathways involved in TNF signaling, NOD-like receptor signaling, IL-7 signaling, and cytokine-cytokine receptor signaling pathways were overrepresented in the upregulated genes after 6 h of infection  mRNA expression, suggesting that increased NO production by V. vulnificus was due to the expression of iNOS. Additionally, as shown in V. vulnificus infection induced upregulation of the numbers of iNOS+ M1 macrophages and CD206+ M2 macrophages in WT BMMϕs. Whereas, in contrast to WT BMMϕs, TSC1 KO BMMϕs shown increased M1 macrophages but a reduction of M2 macrophages after V. vulnificus infection. Thus, these results indicated that TSC1 possibly drives V. vulnificus-induced M2 polarization and suppresses M1 polarization. Overall, these results demonstrated that TSC1 negatively regulated V. vulnificus-infected macrophages preferred polarization to M1 instead of M2.The function of macrophages depends on proper activation and polarization into distinct subtypes with individual effector functions, such as the M1 and M2 macrophage subsets . M1 macrvia inhibition of mTORC1 activity  of V. vulnificus pretreated with or without rapamycin. We observed that the upregulation of both protein and mRNA expression of TNF-α, IL-6, and IL-1β in the V. vulnificus-infected WT and TSC1 KO macrophages were significantly inhibited by rapamycin  by the production of various proinflammatory cytokines including TNF-α, IL-6, and IL-1β in the early stage of bacterial infection , Provincial Natural and Science Foundation of Zhejiang (LY13H190007 to D-LX), Wenzhou Municipal Science and Technology Bureau (Y20150113 to D-LX).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.


The methodology and performance of the novel molecular fingerprinting method is presented and the new chemically-driven persistence image representation is used to screen the GDB-9 database to suggest molecules and/or functional groups with enhanced properties.Machine learning and high-throughput computational screening have been valuable tools in accelerated first-principles screening for the discovery of the next generation of functionalized molecules and materials. The application of machine learning for chemical applications requires the conversion of molecular structures to a machine-readable format known as a molecular representation. The choice of such representations impacts the performance and outcomes of chemical machine learning methods. Herein, we present a new concise molecular representation derived from persistent homology, an applied branch of mathematics. We have demonstrated its applicability in a high-throughput computational screening of a large molecular database (GDB-9) with more than 133,000 organic molecules. Our target is to identify novel molecules that selectively interact with CO 2/N2 interaction energies at the density functional theory (DFT) level.The choice of molecular representations can severely impact the performances of machine-learning methods. Here the authors demonstrate a persistence homology based molecular representation through an active-learning approach for predicting CO Lowering CO2 emissions is therefore mandatory to meet ambitions to limit temperature increases to 1.5 ∘C2. Advancements in carbon capture and storage technology are desired for meeting these goals of lowered atmospheric greenhouse gas emissions and reduced global temperature increases year to year. At an industrial level, liquid amine-based solvents are used for separation and capture of CO2 via chemisorption, but the solvent regeneration step is an energy intensive process. Membrane-based technologies offer an alternative, cost-effective process for CO2. Unlike solvents, where chemisorption involves a reaction with binding strengths exceeding 20 kcal mol−1 through the creation of chemical bonds between CO2 and solvent, membranes utilize much weaker noncovalent interactions. Different types of materials have been suggested for the fabrication of permeable membranes including amorphous, non-porous polymeric membranes6, or crystalline materials with permanent porosity such as metal-organic frameworks (MOFs) or zeolites7.The increasing concentration of greenhouse gases has been identified as a primary factor of many facets of environmental degradation such as higher global temperature, rising sea levels, increased ocean acidity, and more extreme weather-related events. CO2 from N2, which are two atmospheric gases with similar kinetic diameters, making size-sieving a challenging task. The introduction of functional groups which selectively interact with CO2 has been a successful approach for increasing membrane performance8. Such CO2-philic functional groups  can be either introduced into the framework of porous crystalline materials  or functionalized into the repeat units of non-porous polymeric membranes. Electronic structure theory calculations between molecular units and the respective gases provide a quantification of these noncovalent interactions, as well as elucidate their nature and properties15. However, the number of potential CO2-philic groups is intractably large, which leads to an excessive study of such systems with accurate ab initio methods. In addition, the determination of gas interaction energies may require multiple calculations to evaluate competitive gas binding sites for every structure, which further increases the computational cost and expert intervention. However, high-throughput computational screening can accelerate the discovery of new, functional materials for rational synthesis through the circumvention of the expensive and time-demanding synthesis and testing process17.The understanding of how the atomistic structure of materials affects the gas selectivies is a crucial process for the development of more efficient carbon capture technologies. Most often, this involves the separation of CO19 and materials23 with enhanced properties and advanced functionality. For many applications, first-principles studies are essential to virtual screening, but the high computational cost of these methods makes the search of large parts of the chemical space cost-prohibitive. In recent years, machine learning (ML) has become a valuable tool in reducing the cost of a systematic chemical space exploration by enhancing the search for structure-property relationships26, guiding molecular design30, and predicting electronic structure properties35. ML algorithms are used for their ability to learn complicated relationships in data with high computational efficiency that can be systematically improved through additional training data, but may require extensive training set sizes before predicting out-of-sample properties accurately. The efficiency of ML depends on how these data are passed to the algorithm. For chemical applications, this occurs through molecular representations, which are the featurization of molecular compounds from their molecular structure into a vector of values. The ML algorithm then infers the relation between the structure and the property of interest. Recent developments in the formulation of molecular representations, particularly in the realm of quantum properties and structure-function relations, have increased the efficiency of ML for chemical applications42. In addition, such representations generalize to more complex instances such as reaction barriers43. Despite the inherent ability of ML to extract important features, ML-model accuracy is dependent on the molecular representation.High-throughput computational design has shown great success in identifying new molecules33. An improvement over CM is typically observed using the Bag-of-Bonds (BoB) representation, where each atomic pair is placed in specific vectors (bags) based off the elemental pairs and sorted by value34. Faber et al. have developed FCHL, a representation based on Gaussian distribution functions for the universal kernel ridge regression-based quantum machine-learning models42. In addition, the smooth overlap of atomic positions (SOAP) representation44 calculates the local density of atoms around all atoms in a given chemical environment but suffers from an increased computational cost over the pairwise CM and BoB representations.A molecular representation reduces the dimensionality of a molecular structure into a chemically meaningful format that relays important chemical information. For example, a chemical formula conveys a three-dimensional molecule as a string of characters but it is an ambiguous input for ML. Atomistic and molecular structure should be converted into a machine-readable format that can be parsed efficiently to ML algorithms as input features. One of the most prominent representations is the Coulomb matrix (CM) introduced by Rupp et al., which is a square atom-by-atom matrix containing an approximate potential energy of the free atom along the diagonal and pair Coulombic potentials on the off-diagonal terms45. The new method is used for screening a large database of organic molecules for the discovery of CO2-philic functional groups.Herein, we present a new molecular representation scheme based on persistent homology, a branch of computational topology. Application of persistent homology on molecules encodes three-dimensional structural data into two-dimensional persistence images. Since persistence images hold topological features of chemical structures, we are suggesting them as alternative molecular fingerprints which are transposed into ML input and used to identify relationships in the data. A molecular representation is introduced for encoding chemical structures, which is applied in the prediction of interaction energies of organic molecules with gas molecules. Persistence images offer a similar-size ML vectorization regardless of system size, and a numerical example is given as a proof of this concept. Whereas CM and BoB do not provide a constant-size representation by this definition, it is effectively achieved through padding empty cells. However, the input vector space takes the dimensions of the largest molecule in the data and requires significant padding for smaller molecules, whereas the introduced method has a predefined vector size regardless of number of atoms. It has been suggested that using feature vectors with sizes independent of system size may result in improved generalization between small and large systems58. A PD encodes molecular features such as bonds and rings. The PDs can then be vectorized into a persistence image59 (PI) for use as a molecular representation. However, employing persistent homology and its derivative, PI, purely focuses on detecting topological attributes but lacks explicit incorporation of key chemical information such as element identity, leading to limited applicability in molecular systems. Here, we describe the application of persistent homology with domain-specific knowledge for the generation of persistence images based on atomic properties. The basic steps are presented in the following paragraphs. Anisole was selected as a representative example because it contains two distinct functional units (phenyl- and methoxy groups).The mapping of molecular structure to a chemically driven persistence image entails several steps. First, the molecular homological features, which measure the connectedness, proximity, and the empty space among the atoms, are computed and stored. These homological features are summarized in a persistence diagram (PD)To construct a persistence diagram for a given molecule, spheres of a given radius centered at each atom are considered and, as the radius increases, the spheres intersect and lead to the evolution of homological features, called connected components and holes. The connected components encode interatomic distances, while holes describe molecular attributes such as rings and functional groups.x-axis of the PD, whereas the persistence is denoted by its location on the y-axis. Birth of connected components occurs at 0, since every atom is given an initial sphere with radius 0 at the start of the algorithm , which is a stable, computationally tractable representation2 have approximately the same bond distance (1.41 Å), and therefore generate the same PI  is of comparable size (4 Å × 4 Å). Similarly, the PD of a large structure, the main protease of the new coronavirus identified as COVID-1964 in complex with an inhibitor N3 (2500 non-hydrogen atoms) has size of 6 Å × 6 Å. A detailed analysis is given in Supplementary Note 45.Another important feature is related to the dimensionality of the PI molecular representation which remains of the same order with respect to the molecular size as we show empirically in Supplementary Figs. 2 interaction over N2. Such molecular groups can be introduced in polymeric materials for the development of the next generation of functional gas separation membranes. Since it is desirable to avoid any density functional theory (DFT)-optimized geometries as input for ML models, which introduce a significant computational bottleneck for the screening of large molecular databases, we resort to structures generated by the OpenBabel65 software package (gen3d function). Our target is to train a ML model that maps low-cost geometries with accurate quantum chemical data, so it can provide reliable interaction energies for molecular species with geometries generated on-the-fly.Here, we demonstrate the performance of the chemically driven PIs on an application relevant to green chemistry. Our aim is to screen a large molecular database in order to discover molecular groups that show a stronger affinity for CO2 and N2 were computed by means of DFT. We also wanted to compare PI with the widely utilized Coulomb matrices (CM), Bag-of-Bonds (BoB), FCHL, and Smooth Overlap of Atomic Positions (SOAP) representations, since each of these are produced with little computational burden and widely implemented in a number of programming libraries68. PI, CMs, BoBs, FCHL, and SOAP representations were generated for each structure and the performance of each representation scheme was evaluated for the prediction of gas interaction energies. For each scheme, a variety of machine-learning algorithms were tested, including random forest, Gaussian process regression, and kernel ridge regression. A detailed analysis of the optimization process is given in Supplementary Note 2 and one for N2 interaction energies. The 10-fold cross validation root-mean-squared error (RMSE) for the five trained models on the CO2 energies are shown in Fig. 2 interaction energies (see Supplementary Note −1), followed by BoB and FCHL . The most accurate models were PI with kernel ridge regression  and SOAP with kernel ridge regression , where PI showed a tighter variance, yielding a higher confidence in the predictions.We tested the performance of PI as alternative molecular representations that effectively encode chemical structures. The initial subset of 100 organic molecules was used, generated based on the procedure described in Supplementary Note 2 interactions with little computational cost. ML models trained on DFT-quality data can estimate DFT-quality results for hundreds of thousands of systems within seconds, while the explicit computation at the DFT level is a cost-prohibitive process. The GDB-9 database69 was screened, which includes 133,885 organic molecules containing no more than nine non-hydrogen atoms to determine the most promising molecules for CO2 binding.High-throughput computational screening using ML is an efficient method to survey molecules for numerous chemical applications. Here, we are applying the PI method for identifying molecules and functional groups that enhance CO2 interaction strength and further investigated by the MD/DFT scheme described in the computational details  to 43 (second iteration), and ultimately to 75 out of 120 molecules. Active learning with the PI molecular representation has systematically expanded the training set to better represent the chemical space of the dataset, yielding more reliable predictions every round. Since promising structures are rare within the dataset, this strategy allows the model to account for these rare instances within the training set in a way that would be impossible with a randomly chosen training set. In addition, the three top candidates for CO2 separations were found to demonstrate stronger interaction energies than −6.50 kcal mol−1, which are shown in Fig. 2 affinity that combine previously unknown binding motifs. In particular, we found that cooperative effects between N-containing heterocycles with amino or hydroxo groups at ortho position increases the CO2 strength. The lone electron pair of nitrogen 6induces a dipole moment on CO2 that allows stronger interactions with hydrogen atoms of the NH2− and/or OH− functional groups.Three iterations were performed with each representation scheme together with the optimized machine-learning algorithm, as it is discussed in the previous section and in Supplementary Note 2 and CO2 interaction energies are on the x- and y-axis, respectively. Only the method that utilized the PI molecular representation was able to identify 4,5-diamino-1H-imidazol-2-ol as one of the molecules with the strongest CO2 affinity as indicated with an orange dot on each plot of Fig. 2 interaction energy of −7.41 kcal mol−1. All methods agree that the majority of the molecular entries of the GDB-9 dataset have a mean CO2 interaction energy centered between −3.0 and −4.0 kcal mol−1 and a N2 interaction energy at −2.0 kcal mol−1. Most of the molecules have predicted CO2 interaction energies between −3.0 and −5.0 kcal mol−1, which emphasize the difficulty in determining new molecules with high CO2 affinity. However, CM and BoB were less effective in identifying rare instances. On the contrary, results from SOAP were significantly scattered, and predicted many cases with false CO2 interaction energies close to 0 kcal mol−1 . All screenings were performed on an Intel® i5-4278U processor.After completing three iterations of active learning, the full dataset (220 molecules) is used to create a ML model for predictions on the whole GDB-9 database. For comparison, the database was screened with the four different models, where each of them uses a different molecular representation method  and data generated from the corresponding active-learning steps. The optimum learner for each method was used, as it was discussed in the previous section, except for BoB, where the kernel ridge regression (linear) was applied . Specifically, 44 molecules were identified that exceed −6.5 kcal mol−1 CO2 interaction energy. In addition, candidates that may exhibit strong CO2 interactions while maintaining weak N2 interactions were examined, yielding a strategy for identifying species with potentially strong gas separation capabilities. Ultimately, chemically driven persistence images are promising molecular representations for larger supermolecular systems due to compact vectorization. Therefore, we believe that the chemically driven PI molecular representations can be applied in a plethora of chemical problems.A novel molecular representation utilizing persistence images with embedded chemical bonding information has been introduced for predicting DFT-quality COThe PI method described herein relies on a topological representation of a molecular compound that allows a flexible summary of the diversity of the atomic geometries. Due to this flexibility in terms of topological equivalence, the PI is robust and provides accurate predictions in contrast to other methods that need to learn rigid geometric representations. We are currently expanding the applicability of the novel molecular fingerprinting method to high-throughput screening of molecular databases for catalysis and ligand-based lanthanide/actinide separations. For this type of chemical applications, additional features are taken into consideration, such as intensity normalization when a PI is generated from the corresponding PD and predictability of properties of larger molecules from data generated from smaller ones.Supplementary InformationPeer Review File