Dietary cholesterol accelerates oxidative and pro-inflammatory processes, causing hypercholesterolemia and cardiovascular diseases. Thus, the purpose of the current study is to compare the protective effects of thymoquinone (TQ) alone or in combination with losartan (LT) against the heart damage caused by a high-cholesterol diet (HCD). HCD-fed rat groups revealed an elevated activity of indicators of cardiac enzymes in the serum. Serum and cardiac lipids were also found to be significantly higher in HCD-fed rat groups. Cardiac pro-inflammatory and oxidative markers were also increased in HCD-fed rat groups, whereas antioxidant indicators were decreased. However, all of these biochemical, inflammatory, antioxidant, and oxidative change indicators returned to levels similar to those of normal rats after treatment with TQ alone or in combination with LT administered to HCD-fed rat groups. Hypercholesterolemia considerably induced the lipid peroxidation product, thiobarbituric acid reaction substances (TBARs), and oxidative radicals in cardiac cells, which were attenuated by QT and LT treatments, particularly when combined. Finally, QT, LT, and their combination were able to reduce the histological changes changes brought on by cholesterol excess in cardiac tissues. In conclusion, administration of TQ in a combination with LT which has a better protective effect, significantly reduced the hypercholesterolemic-induced oxidative and inflammatory changes that occurred in cardiac tissue. Increased total cholesterol (TC) levels as well as elevated triglycerides (TG) and low-density lipoprotein (LDL-C) in the circulation are the marks of hyperlipidemia, also known as hypercholesterolemia, appearing as a result of an imbalance in the heart’s metabolic processes, cell signaling, and gene expression. According to past clinical investigations, hypercholesterolemia affects many persons in industrialized nations . It has The progression of cardiovascular disorders may be mostly attributed to oxidative stress, which is one of the aspects contributing to hypercholesterolemia . PreviouNigella sativa contains several beneficial chemicals, including thymoquinone (TQ), nigelin, thymohydroquinone, and terpenoids [N. sativa’s pharmacological effects are caused by TQ, which is the primary active component. Previous studies have shown that TQ may have an influence on the cardiovascular system [N. sativa plant has been used to successfully treat mild hypertension in human patients [N. sativa inhibits angiotensin-converting enzyme (ACE), it is likely that the antihypertensive effect of N. sativa is influenced in part by Ang-II, the principal product of the RAS [It has been indicated that rpenoids ,26. The r system ,28. For patients . Since t the RAS . A singl the RAS ,32,33,34Losartan is a first-generation angiotensin-II-receptor antagonist used to treat excessive blood pressure and diabetic nephropathy. Prior research has shown that losartan is better than atenolol for reducing cardiovascular disease and improving blood pressure . Losarta+ 1 °C, 50–55%, and 12 h light/dark cycles) and spent 7 days getting used to the conditions of the facility before the experiment began. Both Purina rat food and water were readily available to them. According to the Principles and Guidelines for the Care and Use of Laboratory Animals, published by the Institute for Laboratory Animal Research of the National Institutes of Health , all procedures, including euthanasia, were carried out with ethical approval (Number: 05-03-42) and guidelines from Jouf University, Saudi Arabia.King Saud University’s Experimental Animal Care Center provided Wistar albino rats weighing between 150 and 180 g. The animals were provided with controlled conditions such as temperature, humidity, and light (22 Pellets for a high-cholesterol diet (HCD) were made by mixing powdered 1% cholesterol + 0.5 percent cholic acid with powdered normal cholesterol rat chow (NCRC). Six rats were administered NCRC for six weeks, whereas thirty rats were fed an HCD. For the duration of the experiment, water and food were freely accessible.After six weeks of supplementation with NCRC or HCD, the animals were randomly distributed into six groups, with six rats in each group. The first group, the control group of rats, was NCRC-fed with rat chow and was considered the vehicle group. The second group, HCD-treated rats, was considered hypercholesteremic. The third group, HCD-treated rats, was dosed with TQ (10 mg/kg/day). The fourth group, HCD-treated rats, was dosed with LT (10 mg/kg/day). The fifth group of HCD-fed rats received TQ (5 mg/kg/day) + LT (5 mg/kg/day), and the sixth group of HCD-fed rats received atorvastatin (1.0 mg/kg/day). All treatment groups were treated for four weeks. All chemical compounds were supplied from Sigma-Aldrich, St. Louis, MO, USA and given by the College of Pharmacy, King Saud University, SA.Throughout the duration of the therapy, the HCD diet was maintained. The weekly body weight and general health of the rats were meticulously observed. Whole blood samples were obtained through cardiac puncture under mild ether anesthesia and centrifuged at 4000 rpm for ten minutes. Until analysis, serum samples were kept at −20 degrees Celsius. Finally, the rats were beheaded, and their hearts were removed, weighed, and maintained until analysis at −80 °C. A cross-sectional sample of hearts from each group was kept in 10% formaldehyde for histological analysis.1H NMR spectra, the 13C NMR spectra, and the 13C DEPT spectra were all acquired using a JEOL spectrometer with a frequency of 600 MHz .The morphological topographies of TQ and LT samples were analyzed using a field emission scanning electron microscope (FESEM) . Devices that performed scanning electron microscopy imaging in high-vacuum mode were also utilized. In addition, the structural analysis was evaluated using an ATR-FT-IR spectrometer , and the Raman spectra of the samples were measured using a Raman spectrometer . The Diagnostic kits were used to determine the concentrations of serum cardiac enzymes, such as lactate dehydrogenase (LDH), creatine kinase-B (CK-B), and creatine kinase-MB (CK-MB) . Kits manufactured by Randox, London, UK, were used to evaluate lipid profiles , triglycerides (TG), low-density lipoprotein (LDL) cholesterol (LDL-C), and high-density lipoprotein (HDL) cholesterol (HDL-C). Phospholipid and free fatty acid (FFA) levels in the blood were measured using widely available measuring kits .v/v) mixture of chloroform and methanol. Before centrifugation, tissues were homogenized with 0.74% potassium chloride (1:1 w/v) and suspended in 2 mL of a chloroform and methanol combination for 2 min. The chloroform layer was dehydrated, and the residual heart lipids were analyzed. The optimal approach of Zilversmit and Davis was used to determine the tissue phospholipid (PL) (1950). Falholt et al.’s technique was used to determine free fatty acids (FFA) (1970). Using commercially available kits , total cholesterol and triglycerides were calculated. The results were expressed in terms of mg/g of wet tissue.The lipids in the heart were extracted using the ) method Using ELISA kits , the levels of inflammatory biomarkers including IL-6, IL-1 and TNF-α, prostaglandin E-2 (PGE-2), caspase-3 and nitric oxide (NO) were determined.Using commercially obtainable ELISA rat kits , the levels of TBARS and glutathione (GSH) were determined in homogenate from heart tissue, and the post-mitochondrial supernatant of cardiac tissue was analyzed for superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and glutathione-S-transferase (GST)enzyme activity.A piece of cardiac tissue was taken from each group and preserved in 10% formalin. Using a rotary microtome, sections were cut from each sample that had been implanted in a separate paraffin block. Using hematoxylin and eosin, the sections were stained. Finally, the histology was taken by microscope and alterations in histology were analyzed.p value is less than 0.05, 0.01, and 0.001, then the corresponding letter and symbol are statistically distinct.The results were expressed as mean and standard deviation. One-way analysis of variance (ANOVA) and the Student–Newman–Keuls multiple comparisons test were used to analyze statistical differences between the groups. If the TQ and LT are characterized in p < 0.001) by more than two-fold in the HCD-fed rat groups as compared to animals fed a normal diet. TQ and LT lowered LDH blood levels in the HCD-fed rat groups , when compared to the HCD-fed rat groups. TQ and LT treatments alone decreased LDH blood levels in rats given an HCD, with p < 0.01 and p < 0.05, respectively, compared to the HCD-fed rat groups. Significant decreases in LDH levels were seen in high-cholesterol rats treated with TQ and LT (p < 0.001). Atorvastatin (AVTT), a widely prescribed statin, served as the study’s standard or reference drug. Therapeutically, atorvastatin is used to reduce bad cholesterol and increase good cholesterol. Significantly (p < 0.05) decreased serum concentrations of CK-B and CK-MB were seen in the TQ and LT groups compared to the HCD-fed rat groups. In comparison to the HCD-fed rat groups, serum CK-B and CK-MB levels were significantly lower in the TQ and LT-treated groups (p < 0.01). CK-B and CK-MB levels were equally suppressed (p < 0.01) in rats treated with atorvastatin as they were in hypercholesterolemic animals  higher than in normal rats. TQ and LT alone reduced TC serum concentrations in the HCD-fed rat groups at p < 0.01 and p < 0.05 compared to the untreated HCD-fed rat groups. Combining TQ and LT inhibited TC levels in the blood more effectively (p < 0.001) than in the untreated HCD-fed rat groups, and this impact was almost identical to that of atorvastatin, the standard cholesterol-lowering medication. Serum TG levels decreased considerably when rats with hypercholesterolemia were treated with either TQ or LT alone (p < 0.05). As a result of the combination of TQ and LT, TG levels in the serum were substantially lower than when they were given separately (p < 0.001). The serum TG levels of rats given AVTT were considerably lower than those of rats fed an HCD (p < 0.001). The FFA levels of rats given HCD were considerably (p< 0.001) higher than those of rats fed a regular diet. The TQ therapy dramatically reduced blood FFA levels compared to the HCD-fed rat groups (p < 0.01). FFA levels were also reduced (p < 0.05) after treatment with LT. Comparing the TQ and LT groups to the HCD group, blood FFA levels dropped dramatically (p < 0.001), but AVTT produced findings that were similar. Phospholipids (PL) in HCD-fed rat groups were considerably (p < 0.01) higher than in the normal group. However, individual treatments with TQ and LT significantly (p < 0.05) lowered the serum PL levels, whereas the combined (TQ + LT) treated group showed more significant change (p < 0.01) when compared to HCD-fed rat groups. Comparing TQ and LT to the HCD-fed rat groups, serum LDL-C values were considerably lower . Both TQ and LT showed greater antilipidemic activity than either agent alone, with comparable effects to AVTT. After six weeks of HCD feedings, the serum levels of HDL-C remained almost unchanged throughout the experiments. It was shown that HDL-C levels rose considerably (p < 0.05) after treatment with AVTT, in contrast to the HCD-fed rat groups.In p < 0.001) elevated, whereas those given TQ therapies were significantly (p < 0.01) lower. The combination of TQ and LT considerably (p < 0.001) reduced TC levels in cardiac tissues in hypercholesterolemic rats compared to untreated HCD-fed rat groups. Higher amounts of heart tissue TG were seen in HCD-fed rat groups, compared to normal rats (p < 0.01). TQ and LT treatment lowered cardiac TG levels in comparison to the HCD-fed rat groups (p < 0.05). Nevertheless, cardiac TG levels were higher (p < 0.01) in the TQ and LT group than in the AVTT-treated group (p < 0.01). Significantly high levels of cardiac FFA were seen in HCD-treated rats compared to normal (p < 0.001). TQ and LT therapy significantly reduced myocardial FFA levels in hypercholesterolemic rats (p < 0.05) compared to untreated HCD-fed rat groups. The combination treatment of QT and LT was shown to be more effective against hypercholesterolemia, and its results were found to be comparable to those of the AVTT therapy. Rats with hypercholesterolemia had significantly (p < 0.001) more phospholipids in their heart tissue than rats with normal cholesterol levels. Cardiomyocyte PL levels were significantly reduced after QT therapy in comparison to untreated HCD-fed rat groups (p < 0.05). The combination of TQ and LT reduced myocardial PL levels more significantly (p < 0.001) than the HCD-fed rat groups. HCD-fed rat groups given AVTT had substantially lower myocardial PL levels (p < 0.001) than untreated HCD-fed rat groups.The cardiac lipid profile is shown in p < 0.05). TQ and LT were more influential (p < 0.01) than TQ and LT alone (p < 0.05). AVTT-treated rats demonstrated a substantial reduction in TNF-α levels (p < 0.01) compared to untreated HCD-fed rat groups. TQ treatment decreased IL-6 levels in the serum considerably (p < 0.01) vs. HCD-fed rat groups. Comparing the LT groups to the HCD-fed rat groups, serum IL-6 levels in the LT group were considerably lower (p < 0.05). Nevertheless, the combination of TQ and LT significantly decreased IL-6 levels (p < 0.001), and AVTT treatment substantially decreased IL-6 levels (p < 0.001) compared to untreated HCD-fed rat groups. The serum IL-1β levels of HCD-fed rat groups were considerably (p < 0.001) higher than those of normal groups. Treatment of hypercholesterolemic rats with TQ and LT for six consecutive weeks significantly decreased (p < 0.05) serum IL-1β levels relative to the HCD-fed rat groups. Compared to the HCD-fed rat groups, the combination of TQ and LT resulted in a more significant (p < 0.01) reduction in serum IL-1β levels than TQ and LT alone; AVTT also produced comparable findings.Preliminary findings indicate that rats administered HCD showed elevated blood levels of proinflammatory cytokines such as TNF-α, IL-1, and IL-6 . TQ and p < 0.001) higher than those of normal groups. It was shown that TQ and LT significantly reduced (p < 0.05) the levels of caspase-3 in the serum of HCD-fed rat groups. Comparatively, serum caspase-3 levels were reduced (p < 0.01) by the combination of TQ and LT compared to the HCD-fed rat groups. Caspase-3 levels in the serum of AVTT-treated rats were equal to those of untreated HCD-fed rat groups (p < 0.05). When compared to normal rats, rats with hypercholesterolemia had considerably (p < 0.01) higher amounts of nitric oxide in their serum. Serum NO levels were significantly lower in the TQ treatment groups compared to the HCD-fed rat groups (p <0.05). As compared to HCD-fed rat groups, TQ and LT dramatically reduced serum NO levels (p < 0.05). Additionally, compared to the HCD-fed rat groups, serum NO levels were lower in those who received AVTT therapy. Serum nitric oxide (NO) levels did not differ substantially between the LT and HCD-fed rat groups. The HCD-fed rat groups showed considerably lower PG2 serum levels than the normal groups (p < 0.001). Hypercholesterolemic rats treated with LT for six weeks had a significant increase in serum PG2 levels (p < 0.05) compared to the HCD-fed rat groups. TQ treatment raised serum PG2 concentrations in HCD-fed rat groups (p < 0.05). On the other hand, rats treated with TQ and LT for six weeks had higher levels of serum PG2 than those in the HCD-fed rat groups. There were significant differences in the HCD groups receiving AVTT therapy, which had raised serum PG2 levels (p < 0.001) compared to HCD-fed rat groups.In p < 0.001) levels of heart TBARs, but cardiac cell GSH levels were significantly lower (p < 0.001). The TQ therapy significantly reduced the levels of TBARs in HCD-fed rat groups compared to untreated HCD-fed rat groups (p < 0.01). When compared to rats treated with HCD, those given TQ and LT had considerably (p < 0.001) reduced levels of cardiac TBAR. The AVTT therapy significantly reduced (p < 0.01) myocardial TBAR levels as compared to the HCD-fed rat groups. Compared to the HCD-fed rat groups, the TQ and LT groups had substantially higher amounts of GSH in cardiac cells . GSH levels were increased (p < 0.01) by the combination of TQ and LT. It was shown that AVTT treatment of HCD-fed rat groups significantly increased GSH levels in cardiac cells (p < 0.01) compared to untreated HCD-fed rat groups. Compared to the untreated HCD-fed rats, HCD-treated rats had substantially (p < 0.001) lower levels of SOD and CAT. SOD activity in cardiac cells was considerably increased in the TQ- and LT-treated HCD group compared to the untreated HCD-fed rat groups at p < 0.05 and p < 0.01. When compared to untreated HCD-fed rats, the SOD activity in cardiac tissue was significantly (p < 0.001) enhanced by the combination of TQ and LT. A substantial increase in myocardial SOD activity was also seen after AVTT therapy (p < 0.001). In hypercholesterolemic animals, the QT and LT treatments significantly (p < 0.05) increased CAT activity in cardiac cells. QT and LT therapy, as well as AVTT, increased CAT activity in cardiac cells in contrast to untreated HCD-fed rat groups substantially (p < 0.05 and p < 0.01). In hypercholesterolemic rats, GPx and GST cardiac enzymatic activity was considerably (p < 0.001) lower than in normal groups. The enzymatic activities of GPx and GST in cardiac cells were substantially increased (p < 0.05) in hypercholesterolemic rats treated with QT and LT compared to untreated HCD-fed rats. However, the combination of QT and LT increased GPx and GST activity considerably in cardiac cells (p < 0.001). GPx and GST activities were equally elevated in hypercholesterolemic rats treated with AVTT for six weeks compared to HCD-fed animals not receiving treatment , which leads to high concentrations of these species in tissues and cells due to the body’s inability to reduce them . The reaIn the current study, many markers of oxidative stress were examined in various kinds of cardiac tissue. Lipid peroxidation and reduced glutathione peroxidase (GSH) levels result from the oxidative stress caused by hypercholesterolemia. In addition, animals receiving HCD had reduced antioxidant enzyme activity, which protects cells from oxidative injury produced by reactive oxygen species (ROS). During the first stages of oxidative stress, the expression and activity of antioxidant enzymes increase. This is due to the fact that antioxidant enzymes safeguard cellular activities . AccordiAccording to previous research, TQ may exercise its antioxidant properties by reducing the activity of reactive oxygen species (ROS) and improving the movement of other molecules capable of scavenging radicals. Experimental rabbits showed that TQ had a curative impact on atherosclerosis caused by hypercholesterolemia and reduced oxidative stress levels . Over anIn conclusion, TQ was able to prevent the oxidative damage, inflammation, and necrosis found in hypercholesterolemic rats, either on its own or in combination with LT. However, the findings suggested that the combined therapies had a considerable synergistic protective effect. These benefits may also be the result of the activation of cellular antioxidant enzymes and/or the regulation of inflammatory cytokines, as well as the restoration of tissue-regulating histological characteristics and the abatement of cardiac oxidative stress.


Children orphaned by HIV/AIDS (“AIDS orphans”) suffer numerous early-life adverse events which have a long-lasting effect on brain function. Although previous studies found altered electroencephalography (EEG) oscillation during resting state in children orphaned by HIV/AIDS, data are limited regarding the alterations in connectivity and microstate. The current study aimed to investigate the functional connectivity (FC) and microstate in children orphaned by HIV/AIDS with resting-state EEG data. Data were recorded from 63 children orphaned by HIV/AIDS and 65 non-orphan controls during a close-eyes resting state. The differences in phase-locking value (PLV) of global average FC and temporal dynamics of microstate were compared between groups. For functional connectivity, children orphaned by HIV/AIDS showed decreased connectivity in alpha, beta, theta, and delta band compared with non-orphan controls. For microstate, EEG results demonstrated that children orphaned by HIV/AIDS show increased duration and coverage of microstate C, decreased occurrence and coverage of microstate B, and decreased occurrence of microstate D than non-orphan controls. These findings suggest that the microstate and functional connectivity has altered in children orphaned by HIV/AIDS compared with non-orphan controls and provide additional evidence that early life stress (ELS) would alter the structure and function of the brain and increase the risk of psychiatric disorders. Children orphaned by HIV/AIDS (“AIDS orphans”) were defined as children under the age of 18 years who had lost one or both parents to HIV-related illnesses . The numBrain function development after ELS has mostly been assessed using functional magnetic resonance imaging (fMRI). By assessing brain activity and connectivity, recent fMRI studies have found a reduction in the volume of the hippocampus, prefrontal cortex (PFC), and corpus callosum in children with ELS , 10. BesIn the commonly resting EEG analysis, a promising approach is a microstate. EEG microstates are defined as global patterns of scalp potential topographies which remain stable for a certain period of time (50–100 ms) before rapidly transitioning to different microstates . Most stFollowing what was previously reported, there are many techniques to estimate resting-state EEG FC , 28. AmoThis study aimed to investigate the large-scale network across the whole brain in AIDS orphans and compared it to non-orphan controls. To achieve this, the present research will use FC and the microstate approach to analyze the EEG data. The temporal parameters  will be assessed for the microstate. For FC, the PLV will be used to calculate the functional connection.Data were derived from a larger neurodevelopmental study in which a total of 91 AIDS orphans and 66 non-orphan children (controls) were recruited from the local communities and school systems in central rural China. The study was open to children at 8–18 years of age who did not have HIV/AIDS-related illnesses. Age eligibility was verified through the local community leaders, school records, or caregivers. Among these participants, 65 AIDS orphans and 66 controls completed the EEG experiment. All the subjects had a normal or adequately corrected vision, were right-handed, and reported no history of mental, medical, or neurological disorders. At the end of the experiment, they received an age-appropriate gift as a token of appreciation. Written informed consent was obtained for the study. Two AIDS orphans and one control child were excluded from further analysis due to an unfinished EEG experiment. The study protocol was approved by the Institutional Review Boards at the University of South Carolina in the United States and Henan University in China (IRB 00007212).Participants were in the eyes-closed resting state when 4-min spontaneous EEG data were collected . The EEGAfter data acquisition, offline preprocessing was performed with EEGLAB . The EEGAs a measure of synchrony, PLV is used as an indicator of FC between different brain regions. Compared with other indicators of FC, PLV does not depend on the spectral power of recorded signals and is more sensitive for measuring FC . In addiN × N synchronization matrix with N equal to 29, in which each entry Ni,j contains the value of the PLV for the channels i and j. The global mean PLV of each subject was calculated based on the N × N matrix.The current source density (CSD) method was used to transform EEG data from scalp electrode space into a reference-free montage . The EEGAs a method of studying EEG, microstate analysis regards the EEG signal as a series of quasi-stable microstates and access the global functional state of the brain by comparing the characteristic of microstate time series .http://www.thomaskoenig.ch/Download/EEGLAB_Microstates/) for EEGLAB in Matlab 2018b. The steps were as follows. First, 1,000 global field power (GFP) peaks were selected randomly and were submitted to Atomize-Agglomerate Hierarchical Clustering (AAHC) analysis. Next for each cluster number of microstate maps from 3 to 6 was determined. According to the cross-validation criterion, we found four microstates could explain the variance of 76.26 and 71.75% for two groups. Then, a similar clustering analysis was performed at the group level based on the microstate template maps of all the participants. For the statistical analysis, the three temporal features of the microstates were extracted .Microstate analysis was performed with the microstate analysis plugin . For microstate analysis, the repeated-measures ANOVA was applied with microstate class  as a within-subject factor, and group (AIDS orphans and controls) as a between-subject factor. One-way ANOVA was used to compare groups for temporal parameters of each microstate when the main effects or interactions were significant.p-values when appropriate. All analyses were calculated by SPSS 25.0.A greenhouse-Geisser correction was conducted to adjust In demographic variables, age was found to be a significant difference between the two groups and included as a covariate in subsequent analyses.F = 8.446, p = 0.004, η2 = 0.062; theta: F = 24.337, p < 0.001, η2 = 0.160; delta: F = 15.395, p < 0.001, η2 = 0.107; and beta: F = 22.989, p < 0.001, η2 = 0.152)  .The four microstate classes  of orphans and controls obtained in the whole groups had topographies comparable with those previously found in most microstate studies . These mF = 3.492, p = 0.020, F = 5.756, p = 0.001, F = 4.510, p = 0.036, We found significant class  × group (AIDS orphans and controls) interactions for coverage  = 4.234, p = 0.042, η2 = 0.033), and a significant increase of microstate C  = 6.227, p = 0.014, η2 = 0.047) than controls. The occurrences of microstate B  = 8.262, p = 0.005, η2 = 0.062) and D  = 8.613, p = 0.004, η2 = 0.064) were higher in controls than in AIDS orphans. AIDS orphans showed significantly increased microstate C duration  = 8.028, p = 0.005, η2 = 0.060) compared with controls . Second, the altered FC of large-scale brain networks in AIDS orphans was measured. The result of this study showed alterations in microstate parameters and lower FC in all frequency bands for AIDS orphans. These results provide new insight into the brain development of AIDS orphans.The results of FC suggest that the brain structure and function, as well as development, can be altered even damaged by ELS. This finding is consistent with previous studies that demonstrated ELS may have a negative effect on brain. A large body of studies has highlighted the impaired cognitive and affective functioning in children who experienced ELS –38. WangIn this study, we found an increase in duration and coverage of microstate C in AIDS orphans compared with controls. This result is consistent with previous studies which found the sensitivity to perception altered  and neurIn contrast to microstate C, we found a reduction in occurrence and coverage of microstate B and a reduction in the occurrence of microstate D in AIDS orphans. According to previous studies, microstate B and D are related to the visual network and attention network, respectively. The decrease in microstate may represent a deficit in attention of AIDS orphans –58, whicIt is possible that our results reflect impaired brain function in AIDS orphans. These findings give further support to the diatheses-stress hypotheses that the brain adapts to ELS by releasing mediators which may provoke dendritic stunting and atrophy , 63 and The present study showed decreased FC and different microstate dynamics in AIDS orphans. With two independent approaches to analyze EEG resting-state data, we found alterations in the brain function in AIDS orphans, and those alterations were likely to be caused by ELS. These results suggest that functional imaging may be used to detect latent neurodevelopmental effects of ELS exposure, facilitating a better understanding of the pathophysiology and treatment of ELS-related conditions.One limitation of this study is that we only explore the whole-brain network from two different perspectives. Other methods, such as graph theory as well as long- and short-distance FC, have been used to analyze FC in recent studies. Hence, these methods will be taken into account to investigate the large-scale brain network in further studies. In addition, the research is a cross-sectional study. According to previous studies, ELS has a sustained and life-long impact on the brain. The results of this study reveal the development trajectory of brain with individuals who are preadolescents and undergoing ELS. Thus, the developmental trajectory throughout puberty needs to be explored in a longitudinal study in future studies. Furthermore, this study only explored the characteristics of large-scale resting-state EEG and found the effect of ELS on brain function. However, the effect of ELS on specific cognitive function and its potential neural circuit have not been explored and analyzed. Therefore, in future studies, we will focus on the role of the ventral prefrontal cortex in the acquisition of threat conditions in individuals who experience ELS and exploring its subregional contributions to fear learning and extinction.jfzhao63@hotmail.com.The datasets presented in this article are not readily available due to privacy or ethical restrictions. Requests to access the datasets should be directed to JZ, The study protocol was approved by the Institutional Review Boards at University of South Carolina in the United States and Henan University in China. Written informed consent to participate in this study was provided by the participants' legal guardian.HG, XS, and HH designed the study and drafted the manuscript. XS and HH performed the study, analyzed the data, and editing of the paper. JZ and XL revised the paper. All authors contributed to the article and approved the submitted version.This work was supported by the National Social Science Foundation of China, NSSFC (Grant Number 19BSH111), the Social Science Planning Project of Henan Province (2021CJY051), the Humanities and Social Science Research Project of Henan Provincial Department of Education grant number (2020-ZDJH-026), the Science and Technology Research Project of Henan Provincial Department of Science and Technology (Grant Number 212102310985), and the Henan University Philosophy and Social Science Innovation Team (2019CXTD009).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The effect of sex and age was assessed in all groups and controlled by rigorous matching. Several IgG Fc glycan traits were found to be associated with IMN. Interestingly, among them, only IgG4-related results were specific for IMN and not for PN. Hypo-galactosylation of IgG4, already shown for IMN, was observed to occur in the absence of core fucose, in line with a probable increase of pro-inflammatory IgG. In addition, elevated levels of fucosylated IgG4, along with low levels of hybrid-type glycans, were detected. Some of these IgG4 alterations are likely to be more pronounced in high PLA2R (phospholipase A2 receptor) patients. IgG Fc glycosylation patterns associated with IMN warrant further studies of their role in disease mechanisms and may eventually enrich the diagnostic spectrum regarding patient stratification.The podocyte injury, and consequent proteinuria, that characterize the pathology of idiopathic membranous nephropathy (IMN) is mediated by an autoimmune reaction against podocyte antigens. In particular, the activation of pathways leading to abundant renal deposits of complement is likely to involve the binding of mannose-binding lectin (MBL) to aberrant glycans on immunoglobulins. To obtain a landscape of circulatory IgG Fc glycosylation characterizing this disease, we conducted a systematic N-glycan profiling study of IgG1, 2, and 4 by mass spectrometry. The cohort included 57 IMN patients, a pathological control group with nephrotic syndrome (PN) ( Membranous nephropathy (MN) is a glomerular disease with a relatively low incidence, being responsible for approximately 20% of all cases nephrotic syndrome worldwide, but with a high propensity to progress to end-stage renal disease . It is hMN can present a primary  or secondary etiology. In developed countries, approximately 75% of all MN are idiopathic, whilst the remaining 20–25% are secondary to different conditions, such as infections , malignancies , systemic autoimmune disease , and drug toxicity . DiagnosIn 2009, Beck et al. demonstrated the presence of circulating anti-PLA2R (phospholipase A2 receptor) antibodies in 26 of 37 IMN patients, performing Western blotting with extracts from normal human glomeruli as a source of antigens . TherefoCirculating anti-PLA2R antibodies of patients with IMN are predominantly, but not exclusively, of the IgG4 subclass. PLA2R and IgG4 have been shown to co-localize in the immune deposits in IMN patients at the subepithelial level and only the IgG eluted from the biopsy samples of IMN patients reacted with the PLA2R ,11. CircFc glycans of IgG are important modulators of effector functions ,17,18,19Mass spectrometric techniques have advanced considerably in recent years and this has facilitated the in-depth characterization of alterations in the glycoportion of the IgG present in human blood in a subclass-specific manner, revealing associations with a plethora of human diseases, in particular those related to inflammation and metabolic health . It has n = 57) and in subjects affected by different nephrotic syndromes (n = 20). To improve the specificity of the disease signatures, the effect of sex and age was assessed via a strict double matching for all the comparisons within a selected group of healthy controls (n = 88).Although a significant increase in serum levels of IgG4 has already been observed in IMN , no systVariations in IgG glycosylation can strongly impact effector function and can be related to the pathological status of an individual. However, as reported in the literature, changes are often also associated with other factors such as age and sex, as well as the disease . For insn = 29) or low PLA2R (n = 28) individuals. None of the PN patients showed PLA2R Ab (AntiBodies) titers (<10). The age distribution of the included healthy controls  was comparable to the disease groups (17 to 87 years for CTRLs and 19 to 81 years for IMN+PN)  . There w IMN+PN) . Due to In order to account for the changes of glycoforms related to ageing  and hormTo explore the Fc glycosylation patterns of serum IgG associated with IMN, a comparison between IMN patients and age- and sex-matched healthy controls was performed. The statistical outcomes are summarized in In particular, IgG4 hypo-galactosylation was observed independent of the PLA2R level. Interestingly, a similar decline in IgG4 galactosylation was found even in the absence of the core fucose. A decrease of galactose levels was also observed for IgG1 and IgG2. However, these effects appeared less robust and were dependent on the matching approach applied.Moreover, IMN was found to be significantly associated with an increase in hybrid type structures on IgG2 and the level of core fucosylation on IgG4. These observations remained significant with both matching approaches in all the comparisons for IgG2 hybrid-type glycans, whilst for IgG4 fucosylation in low PLA2R patients, this was only true with the clustered matching approach. Interestingly, the level of IgG4 hybrid type glycans was lower in IMN patients than in controls. This observation was made independently from the statistical approach used in the complete IMN dataset, as well as in the high PLA2R IMN subset.The clustered gender-age matching also highlighted a decrease of bisected glycoforms linked to IgG2 and of the sialylation of IgG1 in the complete IMN patient set as compared to the controls.n = 20).To explore whether the found glycosylation signatures were IMN-specific or rather a feature of general nephropathy, IgG Fc glycosylation was additionally studied in a sample set consisting of patients affected by a nephropathy not attributable to IMN, but with the same clinical presentation , which is partly mediated by Fc glycans . The diaThe specificity of the glycosylation patterns we found to be associated with IMN was validated based on their further investigation in a cohort of patients suffering from kidney disease different from IMN. The pathological control group was chosen on the basis of a clinical presentation (nephrotic syndrome) similar to that of IMN. However, the natural age and sex distribution between PN and IMN patients is large, which was also reflected in our sample set. Therefore, a direct comparison between PN and IMN was not possible. Instead, both patient groups (IMN and PN) were compared against an age- and sex- matched subset of the healthy controls, based on two different matching procedures. In both sample sets, we found the levels of IgG1 and 2 hybrid type glycans to be higher with respect to healthy controls, whilst the galactosylation levels of these subclasses was lower in the diseased individuals. This suggested that these features are connected to the general clinical phenotype of the diseases, such as renal damage or failure, or inflammation, rather than to the presence of a specific disease. Indeed, aberrant IgG N-glycan profiles have already been observed in other non-IMN nephropathies. A decrease of IgG core fucosylation was noted to be lowered in systemic lupus erythematosus (SLE) . MoreoveIgG glycosylation traits that were specific to IMN as compared to related pathologies were mainly IgG4-specific , showingInterestingly, none of the disease signatures shared between IMN and PN involved IgG4 . This daTo our knowledge, among these aberrations, IgG4 galactose deficiency was the only one to be previously associated with IMN. A galactosylation in total serum and on antigen-specific IgG is widely known to have pro-inflammatory implications and is suggested to be involved in various chronic inflammatory and autoimmune pathologies via the activation of the alternative complement pathway and antibody-mediated phagocytosis . TerminaThe only IMN-specific glycosylation trait observed for an IgG subclass other than IgG4 was the level of bisecting GlcNAc on IgG2. The presence of this GlcNAc was shown to increase FcɣRIIIa affinity independent of afucosylation and galactosylation . MoreoveSome of the IMN-specific IgG4 glycosylation features seemed to be slightly more pronounced in the individuals with high PLA2R Ab titers . HoweverThe implications of our findings appear to have a double value. On one hand, the recognition of a typical Fc glyco-identity of IMN could be attractive for biological purposes, enabling the pathological molecular mechanisms to be better clarified. On the other hand, it could provide useful clinical information, uncovering the variability of the disease. In this perspective, once validated and accompanied by an investigation on the range of IgG Fc-glycan heterogeneity in different ethnic populations, our data can be used both for enriching the diagnostic spectrum as well as to track the variability of patients, helping to drive their classification.Formic acid, ammonium bicarbonate, and TPCK -treated trypsin from bovine pancreas were obtained from Sigma-Aldrich . SupraGradient acetonitrile (ACN) was obtained from Biosolve , and ultrapure deionized water (MQ) was generated by Purelab Ultra, maintained at 18.2 M . Trifluoroacetic acid was purchased from Merck .Patients with a histological diagnosis of IMN and that were positive for PLA2R Ab in the serum, as well as patients affected by a nephropathy not attributable to IMN but with the same clinical presentation , were included. All the patients were Caucasian. The patients were enrolled at time of diagnosis. Their serum was collected at the institutions where they were diagnosed and followed. Samples were collected, processed, and tested immediately or stored at −80 °C until testing. The diagnosis of IMN and clinical evaluation of the disease activity were performed by the physician in charge of the study in each of the participating nephrology units. The study was approved by the local ethics committee .Blood samples were collected using a 5 mL serum-collection tube. After keeping the samples for 30 min at room temperature in an upright position, they were centrifuged for 15 min at 15,000 rpm. Aliquots of the serum were then stored at −80 °C until analysis.Only anti-PLA2R-positive IMN patients were included in the cohort. Anti-PLA2R antibody detection was performed using the commercial Human Embryonic Kidney (HEK)-293 transfected cell-based IIFT , as previously described ,59. The Sera from 77 different clinical samples and 88 healthy control samples were randomized in two 96-well plates, together with 12 VisuCon pooled plasma standards , 6 PBS blanks (3 per plate), and 9 sample replicates. A controlled randomization with a uniform distribution of age, sex, and case/control ratio per plate was performed, as seen in m/z peptide, 130 A, 1.7 µm, 75 µm × 100 mm, Waters) with a flow rate of 0.6 µL/min at 45 °C using a 5 min multi-step gradient from 3 to 50% of solvent B (95% Acetonitrile). The mass spectrometry (MS) device was equipped with CaptiveSpray and nanoBooster technologies , using ACN-doped nebulizing gas. Spectra were recorded in profile mode within the m/z range 550 to 1800 using a frequency of 1 Hz with the following tune parameters: collision energy of 7.0 eV, transfer time of 110 μs, and a e pre-pulse storage of 21 μs. The total runtime was approximately 11.5 min for each sample.The glycopeptide mixtures were separated and analyzed using an Ultimate 3000 high-performance liquid chromatography (HPLC) system  coupled online to an Impact HD Ultra High-Resolution flight mass spectrometer (UHR-TOF) , as already reported . Brieflym/z, respectively, using at least 7 features with a signal-to-noise ratio (S/N) greater than 100. Aligned sum spectra were then calibrated on five peaks (S/N ≥ 9). For the extraction, an analyte list was built including all glycoforms for each sub-classes. The resolution of extracted analytes was accepted if there were above 100 data points per 1 m/z for features with a charge from +2 to +4 in a 12 sec time window. Analytes were included if the following parameters were satisfied: their average S/N ratio was greater than 9, their isotopic pattern did not, on average, deviate more than 20% from the theoretical pattern, and their average mass error was within ±10 ppm. This resulted in the extraction of 56 features, divided in 21 for IgG1, 18 for IgG2, and 17 for IgG4 glycoforms  as already described . Brieflyycoforms . Low-intFor this study, we considered two matching designs with a fixed and a variable number of cases and controls: 1:1  and m:n . Each IMN case was matched to 1 or all available healthy controls, according to the following criteria: sex and age (±3 years).p-values. For each test, the level of significance was set equal to 5%. Statistical analyses were performed using the open-source R software v.3.6.0 . Descriptive statistics were reported as absolute frequencies and percentages or median and quartiles (Q1–Q3), for categorical and continuous variables, respectively. The Clustered Wilcoxon test, that accounts for the matched design, was used to compare glycoproteins between groups. The Hochberg method was considered to adjust for multiple testing and significance tests were reported in terms of adjusted Our work highlighted a typical Fc glycosylation pattern of IgG1, 2, and 4 associated with the IMN, weighting and minimizing the interfering effects of age and sex and evaluating its specificity in terms of the clinical presentation of the disease. Interestingly, the specific signatures connected to IMN were IgG4-related. In particular, the hypo-galactosylation of IgG4, already reported for IMN, was confirmed, and our data supported that this alteration occurs even in the absence of the core fucose, suggesting an enhancement of the pro-inflammatory potential of IgG4. Furthermore, a higher level of fucosylation on IgG4, along with a lower IgG4 hybrid-type glycan level, was reported to be specific for IMN.These results are potentially useful to uncover the molecular mechanisms of the disease and represent a starting point for improving the classification of patients with IMN, and thus, therefore help guide the most appropriate therapeutic treatment.


Methionine (Met) metabolism provides methyl groups for many important physiological processes and is implicated in multiple inflammatory diseases associated with the disrupted intestinal microbiota; nevertheless, whether intestinal microbiota determines Met metabolism in the host remains largely unknown. Here, we found that gut microbiota is responsible for host Met metabolism by using various animal models, including germ-free (GF) pigs and mice. Specifically, the Met levels are elevated in both GF pigs and GF mice that mainly metabolized to S-adenosine methionine (SAM) in the liver. Furthermore, antibiotic clearance experiments demonstrate that the loss of certain ampicillin- or neomycin-sensitive gut microbiota causes decreased Met in murine colon. Overall, our study suggests that gut microbiota mediates Met metabolism in the host and is a prospective target for the treatment of Met metabolism-related diseases. Indeed, process . Of note process . Moreove process . Met cyc process . TherefoFaecalibacterium prausnitzii (Akkermansia muciniphila (A. muciniphila) and Bilophila wadsworthia involved in sulfur metabolism  for decontamination. Next, the piglets were taken to rearing isolators  and their anal swabs were collected weekly for microbial testing. Another six pigs served as controls (SPF pigs) by transplanting maternal feces. Control pigs were continuously orally administered fecal bacteria suspension (1 ml/d) for 3 days at 7 days of age and kept in sterile isolators .For sample collection, on the last day of the experimental period, two groups of pigs were subjected to respiratory anesthesia with isoflurane after urine collection, and other samples were collected on a clean bench after blood collection to ensure that there was no bacterial contamination throughout the entire sampling process. Liver, bile, spleen, kidney, heart, and different intestinal segments and their contents were collected, including jejunum, ileum, cecum, colon, jejunum contents, cecal contents, colonic contents, rectal contents, and feces . SamplesGF and SPF Kunming mice were bred at the Third Military Medical University . ICR mice were purchased from Liaoning Changsheng Biotechnology Co., Ltd. . GF mice were housed in sterile isolators and SPF Kunming and ICR mice were housed in sterile individual animal colonies  with free access to drinking water and standard rodent chow.n = 10 males and 10 females) and SPF  Kunming mice were treated with the same diet for 4 weeks, then were euthanized by CO2 asphyxiation, and blood, duodenum, and colon were collected  were supplemented with ampicillin , colistin , metronidazole , neomycin , and vancomycin  in drinking water for 2 weeks. Mice were then euthanized by CO2 asphyxiation and colon samples were collected  to obtain a 100 μg/ml stock solution. The standard solution was then serially diluted to 1,000, 500, 100, 10, 1, 0.5, 0.1, 0.01, 0.001 ng/ml. All metabolites were quantified by external standard method, and all standard solutions were stored at −20°C. The processing of tissue samples and fluid samples such as serum, urine, and bile was carried out according to our previous experiments , and stoMet cycle-related metabolites were separated by Thermo Fisher Scientific UPLC system  with a C1s Hypersil Gold column . The main parameters and gradient elution procedure were based on our previous experiments .Metabolite identification refers to our previous experiments . BrieflyXcalibur converted data in an instrument-specific format (*.raw) to a common data format (.XLS) that displays information on metabolites, including calculated amounts, retention times, and peak areas. The raw data can be accessed by the Metabolights repository,t-test, Prism 6.0) if the data exhibited Gaussian distribution and equal variance, and by  if the data exhibited . If the data were not normally distributed, a nonparametric test  was performed. The Gaussian distribution of the data was analyzed by  and/or . Data were analyzed for variance by . Significant differences among 3 or more groups were passed . p < 0.05 (*), p < 0.01 (**), p < 0.001 (***), and p < 0.0001 (****) were considered statistically.All data are shown as mean ± SEM. Data between two groups were analyzed by , 0AT1B (SLC6A19), 0,+ATrBAT/b (SLC3A1/SLC7A9), +LAT14F2hc/y (SLC3A2/SLC7A7), and LAT4 (SLC43A2) primarily transports Met across the basolateral membrane , increased expression of neutral amino acid transporters Slc6a19, Slc7a8, and Slc7a15 resulted in increased serum glutamine and tryptophan levels , Met is taken up across the apical membrane of intestinal epithelial cells by various Namembrane . Previou jejunum , B0AT1 ind ileum , and rBAig ileum . LAT4 isduodenum . A recenduodenum . In addial tract . Moreoven levels . In Chd8behavior . In a piransport . Sequencransport . Thus, gntegrity , angiogentegrity , both ofntegrity , or metantegrity , or intentegrity . Whether4 ~ 107 CFU/ml), and are mainly facultative anaerobic bacteria, such as Streptococcus and Lactococcus, which are different from the large intestine  Jejunal microbial metabolism: Jejunal microbiota is mainly involved in the degradation of lipids and sugars , so aminntestine ; (3) Feantestine . And thentestine , so the In addition, we examined the Met cycle-metabolites in GF mice, further confirmed that gut microbiota depletion caused the increase of Met, which promoted its downstream metabolic responses to a decrease in hCY and SAM . A varieEscherichia coli are sensitive to ampicillin . However, when the microbes were cleared with antibiotics, ampicillin and neomycin treatment instead resulted in a reduction in intestinal Met. Since Met metabolism is closely related to various inflammatory diseases , ALD, liThe data presented in the study are deposited in the Metabolights repository, and the accession number is MTBLS6154.The animal study was reviewed and approved by the Laboratory Animal Ethical Commission of the South China Agricultural University [permit number SYXK (Guangdong) 2019–0136].WT and SL designed the research and analyzed data. DY, JS, and LG assisted in the establishment of GF pigs and GF mice. XW, ZH, and BL participated in antibiotic-animal experiments. XW and BL prepared the figures and draft the manuscript. XW, ZH, WT, and SL revised and approved the final manuscript. All authors contributed to the article and approved the submitted version.This study was supported by the National Key R&D Program of China (2021YFD1301100), and Sichuan Science and Technology Programs (2021JDYZ0001 and 2021ZDZX0009).WT is employed by Sichuan Animtech Feed Co., Ltd.The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Many proteins are anchored to the cell surface of eukaryotes using a unique family of glycolipids called glycosylphosphatidylinositol (GPI) anchors. These glycolipids also exist without a covalently bound protein, in particular on the cell surfaces of protozoan parasites where they are densely populated. GPIs and GPI-anchored proteins participate in multiple cellular processes such as signal transduction, cell adhesion, protein trafficking and pathogenesis of Malaria, Toxoplasmosis, Trypanosomiasis and prion diseases, among others. All GPIs share a common conserved glycan core modified in a cell-dependent manner with additional side glycans or phosphoethanolamine residues. Here, we use atomistic molecular dynamic simulations and perform a systematic study to evaluate the structural properties of GPIs with different side chains inserted in lipid bilayers. Our results show a flop-down orientation of GPIs with respect to the membrane surface and the presentation of the side chain residues to the solvent. This finding agrees well with experiments showing the role of the side residues as active epitopes for recognition of GPIs by macrophages and induction of GPI-glycan-specific immune responses. Protein-GPI interactions were investigated by attaching parasitic GPIs to Green Fluorescent Protein. GPIs are observed to recline on the membrane surface and pull down the attached protein close to the membrane facilitating mutual contacts between protein, GPI and the lipid bilayer. This model is efficient in evaluating the interaction of GPIs and GPI-anchored proteins with membranes and can be extended to study other parasitic GPIs and proteins and develop GPI-based immunoprophylaxis to treat infectious diseases. This linear core is modified in a cell- and tissue-dependent manner by additional sugars or phosphoethanolamine units. The complexity of the GPI core together with the modifications on the glycan and lipid moieties and the diversity of anchored proteins suggest a variety of functions for these glycolipids. Apart from their function as a stable anchor for proteins, GPIs and GPI-anchored proteins (GPI-APs) have been associated with key cellular processes such as signal transduction, cellular adhesion and communication, protein sorting and trafficking (Glycosylphosphatidylinositols (GPIs) are complex glycolipids ubiquitous in eukaryotes that are bound to the outer leaflet of the cell membrane . GPIs arfficking . This muAlthough GPI glycolipids are known for more than 30 years , yet theToxoplasma gondii GPIs using surface plasmon resonance (SPR) and showed a galectin-3 dependent recognition of parasitic GPI glycans by macrophages to induce TNF-α production  serves as a useful complementary technique to unravel structural and dynamical aspects of a system with atomic resolution. Some examples of modeling GPI-APs in membrane-like environments include analyses within the nanosecond timescale of GPIs, such as that of the Variant Surface Glycoprotein of a brucei , the NETma cruzi , and thema cruzi . Howeverma cruzi . Li et ama cruzi  conducteT. gondii (GPIs 1 and 2) and Trypanosoma congolense (GPI 3) as well as the GPIs found in mammals (GPI 4), see In our previous work, we analyzed the conformational behavior of GPI glycan (without the lipid tail) in solution using the GLYCAM06 force-field, and reported that a simulation time of at least 1 μs is required to achieve sufficient sampling . In a suzθ for each GPI with respect to the bilayer using the position vector connecting the end points of the GPI core, pointing from atom C6 of Ino to atom C4 of Man3, and the bilayer normal (z-axis), see zζ using the atom C6 of Ino and atom C4 of Glc/Gal attached to Man1 residue as end points for the position vector, see zθ for the GPIs are quite broad, reflecting their structural flexibility, however, they largely peak around 70–80° as is seen more prominently in GPIs 1 (black) and 3 (green), see zζ of GPI 2 and GPI 3 are narrower, with pronounced peaks around 70–80° and the peak of GPI 3 distribution being shifted towards higher values, see We construct the GPI model by combining the AMBER force-fields—GLYCAM06  for the → 2, 1 → 3, 1 → 4, 1 → 6). A glycosidic linkage connecting 2 sugar residues is usually represented with 2 torsion angles—φ and ψ—and an additional angle Ω (relevant for (1 → 6) linkages), see φ, ψ) and  free-energy plots were generated for all the glycosidic linkages of protein-free GPI variants studied in DMPC and POPC bilayers. The free energy associated with a probability distribution for the dihedral pair  is obtained via:Bk is the Boltzmann constant, T is temperature, and P  is the probability distribution for a dihedral angle pair . We observed that in comparison with the GPI conserved core, the distributions of all the corresponding torsions across all the GPI variants are quite similar, except for the α(1 → 6) linkage connecting the Man2–Man1 units. This (1 → 6) linkage is more flexible due to the presence of a primary hydroxyl group in the glycosidic bond and the additional rotation at the methylene group. Moreover, the Gal-β(1 → 6)-GlcNAc linkage that is the side branch component of GPI 3 also shows considerable flexibility. The variation in the Man2-α(1 → 6)-Man1 linkage across all GPI variants and the flexibility of the Gal- β(1 → 6)-GlcNAc linkage in GPI 3 are together depicted in the Ramachandran-like  plots in → 6) linkage connecting the residues Man1 and Man2, serving as a hinge between the 2 relatively rigid disaccharides on either side (Man3–Man2 and Man1–GlcN) (gg (240° < Ω < 360°) was seen to be the most populated, with only minor traces of gt (0° < Ω < 120°) and tg (120° < Ω < 240°), as a consequence of the gauche effect (→ 6) linkage, as the free energy seems to have shifted more towards the gg rotamer in the anchored GPI.The overall 3D conformation of a glycan is largely governed by the glycosidic linkages. These linkages can adopt different values depending on the type of monosaccharide, the anomer type, and also the type of linkage (1 n1–GlcN) . Out of e effect . Upon cogg being the highest, followed by gt and negligible traces of tg. As we move towards GPIs with an extra side branch residue, GPIs 2 and 3, the population of the gt rotamer relatively goes up, even to the same level as that of gg for GPI 2 in POPC and more than gg for GPI 3 in POPC. These results suggest that the lipid bilayer type certainly has some notable impact on the conformation of the GPI anchor, particularly at the flexible linkages. The β(1 → 6) Gal-GlcNAc linkage presents a profile different from the Man2-Man1 linkage in both the ψ torsion and rotamer populations. The G al-GlcNAc rotamer population distribution (gg/gt/tg) in DMPC is (56/36/9)% and in POPC is (78/20/2)%, again showing highest preference for gg. Both ψ and Ω appear more flexible in Gal-GlcNAc than in the Man2–Man1 linkage, consistent with Gal as a terminal residue. Still, rotamer populations are dominated by the gauche conformers over anti in both Man2–Man1 and Gal-GlcNAc linkages, consistent with the general trend observed in manno- and gluco-pyranosides  by recording the number of hydrogen bridges formed by each GPI residue with the lipid headgroup and with water, (ii) by counting the hydration number of each GPI residue. The presence of several hydroxyl groups in carbohydrates makes them highly prone to hydrogen bonding (H-bonding). β(1 → 6) linkage in the side branch composition of GPI 3, which allows it the conformational freedom to interact with the lipid heads, compared to its stiffer α(1 → 4) counterpart in GPI 2  linkage, for example, is forced to assume primarily its gg rotamer. Additionally, it was demonstrated that with the presence of the flexible phosphoethanolamine linker (tPEtN), the attached protein GFP retained considerable re-orientation flexibility, which is, in principle, sufficient to establish protein-glycan contacts. With the variety of GPI variants modeled in this work it is now possible to look at protein–glycan interactions in more detail. These interactions are expected to be important for the structural stability of some attached proteins  linkages, one α (main chain) and the other β (side chain), in its structure, see φ, ψ) =  for GlcN-Ino and that of cluster 2 carried  =  for Man1-GlcN, both from seemingly minor energy wells, as illustrated in Firstly, to assess the effect of the attached GFP on the GPI anchor, we performed cluster analysis on both free GPIs and GFP–GPIs. Cluster analysis filters out conformations that predominantly occur in the simulation trajectories. Each of the concatenated 3μs long MD trajectories of the free GPIs, including those from both the top and bottom leaflet of the bilayer was subjected to the analysis. Considering the high flexibility of GPIs, only the clusters with more than 40 members were analyzed, where members are the structures lying within a cluster, see Section: ‘Cluster size calculation’ in ‘Computational methods’. The population distributions of clusters for each free GPI system, shown in α(1 → 6)-Man1 linkage. For example, the rotamer population was the highest for gg in free GPI 0 both in DMPC and POPC bilayer, whereas with attached GFP, the population shifted towards gt . There was considerable deviation observed at ψ of Man2–Man1 too, going from 2 prominent energy wells in free GPIs to essentially one in GFP–GPIs. The cluster conformations in Figure S6 show that irrespective of the orientation of the attached GFP, whether erect or reclining, the GPI lies close to the membrane with the glycan head spanning across the membrane surface, although the internal arrangement of GPI residues, particularly at the flexible linkages—Man2–Man1 and Gal-GlcNAc–, may vary.The same cluster analysis was carried out on GFP–GPIs in both DMPC and POPC bilayers using concatenated 4 μs long MD trajectories with more than 30 cluster members considered for analysis, see ≈80°, except in GPI 2, where the orientation of GFP was seen to highly fluctuate. The higher degree of fluctuation of tilt angle in GPI 2 compared to GPI 1 shows that the size or molecular weight of GPI affects the orientation as GPI 2 carries 1 additional branched sugar residue. Interestingly, GPI 3, despite having the same size as 2, behaves very differently, in the sense that the tilt angle is quite stable at about 80–90° in the former as opposed to the instability of the latter. This indicates that the orientation of the attached protein could be affected not just by the size of GPI, but also the chemical composition of the side chain residues, at least within the pool of simulation data acquired here. Moreover, GPIs inserted in POPC bilayer exhibit greater fluctuation in tilt angle than those in DMPC bilayer, suggesting that the local distribution of lipids also influences the protein’s presentation. Note that GPI 2 does not naturally come with a protein, but only exists in the protein-free form on the cell surface of the parasite. Nevertheless, it was attached to the protein in this study to facilitate a systematic investigation that may guide future synthetic, in vitro experiments.The orientation of GFP with respect to the bilayer was determined by measuring the tilt angle of the axis of the GFP barrel formed with the bilayer normal, see cartoon in Understanding the interactions between the GPI anchor and its attached protein is imperative to their conformational analysis. Just as GPIs form H-bonds with the lipid headgroups, they also do so with the GFP, see Figure S7. Here, more generally we calculated the number of contacts formed between residues of GFP and GPI by counting the atoms of GFP lying within a cutoff of 0.5 nm from the reference GPI residue, according to the approach by Toxoplasma gondii (GPI 1), T. gondii low molecular weight (LMW) (GPI 2), T. congolense (GPI 3)—and Human GPI (GPI 4). We have previously demonstrated the flexibility in the solution structure (α(1 → 6)-Man1 linkage, as has also been reported by α(1 → 6)-Man1 linkage. In the smaller GPI types, i.e. GPIs 1 and 4, the relative rotamer populations of the Man2–Man1 linkage followed the same order of preference as was seen previously in GPI glycan core in solution and membrane—gg > gt > tg. However, in the GPIs with longer branches, i.e. GPIs 2 and 3, the population of gt rotamer significantly increased at the expense of gg, even to the extent where gt exceeds gg for GPI 3 in POPC, see → 6) linkage in its side branch. The case of GPI 3 also demonstrates that although the conformational preferences of the GPIs largely follow the concept of independent glycosidic linkages  by combining the force-fields GLYCAM06 and Lipid14 for MD simulations . In the tructure  and the tructure  of the Glinkages , there cβ(1 → 4) and β(1 → 3) glycosidic linkages in the core structure, resulting in their erect orientation on the membrane. However, the same does not hold true for GPIs that bear a different set of glycosidic linkages (rich of rather flexible α linkages) and furthermore, the unique presence of a positively charged glucosamine in their structure, which altogether leads to a rather different appearance of a reclining, yet flexible GPI glycan. Our results from the free GPI simulations reveal a consistent pattern across all the GPIs in the presentation of the side branches on the membrane surface. The side chain residues—GalNAc in 1, GalNAc and Glc in 2, GlcNAc in 3, and GalNAc and mPEtN in 4—are relatively less membrane-embedded, more hydrated and more solvent-interactive than the other residues, and therefore, could be the most accessible epitopes for recognition. However, the galactose  residue in 3 makes an exception by interacting profusely with the membrane heads, possibly because of the longer and more flexible (1 → 6) linkage in the side branch. The importance of the side branches has also been demonstrated in experimental studies. Using surface plasmon resonance, Debierre-Grockiego and coworkers showed that T. gondii GPIs strongly bind to galectin-3, a protein whose expression is a necessary precursor to TNF-α production by GPI-stimulated macrophages. Because galectin-3 binds specifically to β-galactosides, this finding implies that it associates with the residue GalNAc in T. gondii GPI (T. gondii LMW GPI). However, other studies report a striking difference in the immunogenicity of the 2 GPI variants of T. gondii. T. gondii isolates to reveal that the protein-free T. gondii LMW GPIs generated 4–5 times more TNF-α than the protein-bound T. gondii GPIs. This finding was further corroborated by another independent study using synthetic protein-free T. gondii GPIs whereupon difference between the 2 GPI variants was observed in the intensity of binding to IgG and IgM antibodies from the sera of infected patients. T. gondii LMW GPI bound more strongly to both antibody types than did T. gondii GPI, thereby, qualifying as a suitable biomarker to differentiate between latent and acute phases of infection that differ in the levels of IgM antibodies  catalyzes the attachment of the GPI anchor to proteins. Vainauskas and Menon showed that mPEtN is required for the recognition of GPI by human GPIT, whereas tPEtN does not play a crucial role . In factuman GPI , therebyuman GPI . As is euman GPI . Given tAs GPI anchors are often found attached to proteins, it was important to investigate the interactions between GPI and an attached protein. We chose the GFP as the substitute for natural GPI-anchored proteins. GFP by itself does not favorably interact with zwitterionic membranes (such as DMPC and POPC) as is known experimentally  and alsoThere are conflicting opinions in literature on whether GPI causes any conformational change in the attached protein. For example, it has been reported that GPI anchor brings about conformational change in Thy1 , but notUsing atomistic MD we have investigated and compared the conformational behavior of parasitic and mammalian protein-free and protein-attached GPIs inserted into pure bilayers of DMPC and POPC by extending our previously reported model of the GPI anchor core. Our results from the simulations of free GPIs indicate that the side-chain residues that branch out from the middle mannose Man1, whether sugar residue or phosphoethanolamine, are the most solvent-accessible epitopes of the whole GPI, and thus, are potential targets for recognition of parasitic GPIs by immune cells or of human GPI by the GPI transamidase. In this way, we are able to provide a new insight into the importance of the side-chain residues that has been demonstrated in experiments. The orientation and presentation of the GPI anchor with respect to lipid bilayers is qualitatively consistent across all GPI variants in that the GPIs lie parallel to the bilayer and are in contact with the lipid head-groups. Such a flop-down orientation allows for the side chain residues to project out into the solvent making them readily accessible for molecular recognition. The flop-down conformation has further consequences in that the lipid composition also makes a marked difference in the recognition process, as it was observed that GPIs are more buried in the lipid headgroup region of POPC than DMPC.We conclude from the GPI-anchored GFP simulations that the orientation of the protein on the membrane depends on several factors: (i) the molecular weight of GPI, (ii) the type of glycosidic linkage between residues, (iii) the region of the protein in contact with GPI (whether a rigid or flexible patch), (iv) the flexibility of the PEtN linker, and (v) the lipid composition of the bilayer. The use of GFP as the attached protein helped to exclude direct protein-membrane interactions, and instead reflected how the conformational preferences of the GPI glycan and its interactions with the GFP influence the latter’s lateral orientation. This enables us to demonstrate that the presentation of the protein depends even on small differences in composition between the GPI variants. We believe that this numerical atomistic model to describe GPIs and GPI-anchored proteins in a modular way is a useful tool for further investigation of these complex systems.3 for the free GPI systems and (12.5 × 12.5 × 22) nm3 for the GPI-anchored GFP systems. The smaller protein-free GPI systems consisted of 1 GPI anchor per leaflet in pure bilayers of (8 × 8) lipids, whereas the bigger systems had 1 GPI-anchored GFP embedded in 1 leaflet of pure (16 × 16) lipid bilayers. The pure bilayers considered in this work were of DMPC and POPC. Charged systems were neutralized by adding Na+ or Cl− counter ions. System construction was achieved with the LEap facility of AMBER, following which the topology and structure files were converted to GROMACS’ format using a modified version  method for charge fitting. The electrostatic potential was obtained from the optimized geometry at the HF/6-31*G level of theory. As the designed system is meant for condensed-phase simulations, a restraint weight of 0.01 was applied for the RESP charge fitting. Charges on alkyl hydrogens were fixed at 0 to maintain consistency with GLYCAM parameters. GLYCAM’s charge derivation protocol offers modular blocks of sugar residues for building long glycans with variable branching. As a result, every terminal sugar residue carries a net charge of 0.194. The total charge on the PEtN linker was also 0.194, consistent with GLYCAM’s formula. The atom types and bonded parameters  assigned to the linker were taken from GLYCAM’s database. In our previous work, partial charges were derived similarly for the PEtN linker that bonds with GPI at one end and to protein on the other. The remaining details of the methodology can be found in ref (To ensure force-field compatibility with the attached GPI glycan, we followed the approach of the original GLYCAM paper  to derivd in ref . The atogmx cluster was employed for this calculation.Cluster analysis on the conformation of GPIs was performed using the method described in the work of


In order to study the axial compression property of a GFRP (glass fiber-reinforced polymer) tube-confined coal gangue steel fiber short concrete column, a test was carried out. The whole process of deformation and failure of the specimen under axial compression load was observed, and the whole process of the stress–strain curve of the specimen was obtained. The results show that the thickness of the GFRP tube has the most significant effect on the mechanical properties. The thickness of the 7 mm tube is 4.3 times the axial ultimate stress and 21.5 times the ultimate strain of the unconstrained short column. Under a certain volume fraction, the ultimate axial strain of the wave fiber is 10.1% higher than that of the hook fiber short column, and the ductility coefficient is 9.6% higher. The fiber volume fraction significantly increases the strain of the short column, and the 3% fiber content is 50.1% higher than that of the non-fiber short column. Finally, three classical strength models of confined concrete were selected for comparative calculation, and a new stress correction model was proposed. With the vigorous development of the construction industry, the demand for concrete is gradually increasing. As an essential concrete component, the traditional aggregate resources represented by common stones are decreasing. In addition, mining coal produces a significant amount of coal gangue, which is removed from the ground and left to pile around the mine for an extended period, taking up much space and seriously harming the area’s ecology ,2,3,4. MLateral restraint can effectively improve the mechanical properties of concrete members. As a traditional concrete constraining method, steel tube confined concrete has good properties in improving both the strength and ductility of concrete. However, as a common external constraint of concrete, a steel tube has certain limitations. When the steel tube reaches yield stress, it cannot provide continuous restraint for the core concrete . In addiFiber-reinforced polymer (FRP) has the advantages of being lightweight as well as having high strength, stable properties, high mechanical strength, and corrosion resistance ,11. It iIn this paper, based on the external confinement GFRP tube to improve the compressive properties of concrete, the coal gangue concrete itself is also improved. Fiber type and fiber content can also improve the mechanical properties of coal gangue concrete. A variety of variables together have an impact on the mechanical properties of coal gangue concrete short columns, and now, there are a few relevant experimental and theoretical studies in this aspect. Through experimental study, GFRP tube thickness contributes the most to the axial compression performance of short columns. The axial compression performance of the wave fiber short column is better than that of the end hook fiber short column. Increasing the fiber volume fraction will improve the mechanical properties when the fiber volume fraction is low. The strength prediction formula of the short concrete column is explored to provide a reference for engineering applications.A total of 30 short concrete columns restrained by GFRP tubes were made in 10 groups, three in each group, with the main parameters of the wall thickness of GFRP tubes, fiber type, and volume replacement rate of steel fibers. The column height was 300 mm, and the diameter was 150 mm. The concrete specimens were produced simultaneously. The measured strength of the concrete specimens was 41.6 MPa. To prevent the failure of the end of the specimen, the upper and lower ends of the specimen were winding 1 m long, 5 cm wide CFRP cloth to strengthen the constraints. The surface treatment of the specimens and the adhesion of the fiber cloth were carried out according to the Technical Specification for Carbon Fiber Sheet Reinforced Concrete Structures  (CECS146The specific production process of the specimen was divided into eight steps. (1) GFRP tube processing: A GFRP tube was cut into 300 mm high short columns. All the tubes were fixed on the padding plate with glass glue. Tubes of the same thickness were placed in a row. (2) Concrete pouring: The test used a HJW-60 single horizontal shaft forced concrete mixer to mix concrete. The steel fiber shape is shown in Figure 2The coal gangue concrete mix proportion is shown in As shown in For confined specimens, at the initial loading stage, the lateral expansion of concrete was small, the GFRP tube did not play a restraining role, and there was no apparent change outside the specimen. With the further increase in load, the circumferential expansion of the specimen was slight, and some areas outside the GFRP tube began to whiten. When the load was close to the peak load, the sound of minor fracture of the fiber was gradually accelerated, and the GFRP tube fiber was gradually broken. Then, the GFRP tube broke instantly, and the specimen was destroyed. As shown in The stress and hoop and axial strain curves are shown in The first stage curves of different GFRP tube thickness specimens almost coincided. In the initial stage, the core concrete of each specimen ignored the lateral expansion, and the GFRP tube had almost no constraint on the core concrete. After entering the transition section, the micro-cracks inside the concrete continued to develop, and the strain growth rate of the GFRP tube accelerated. The strain change in the second stage was mainly affected by the thickness of GFRP. The slower strain growth rate was caused by the GFRP tube with greater restraint stiffness and thickness.In terms of stress–strain behavior, wave fibers had more obvious improvement than end-hook fibers. The geometry of wave fibers had the following advantages over end-hook fibers, resulting in better integrity: the contact area between wave shape and substantial cementitious material increased; the wave shape increased the bonding strength with the cementitious material; regular wave shapes evenly distributed the load transmitted by the cementitious material.Both the axial and circumferential fracture strain of GFRP increased with increasing steel fiber doping, which was attributed to the confining effect of internal steel fibers. The additional constraints provided by the internal steel fiber led to improving these stress–strain behaviors. Steel fiber inhibited the development of cracks, reduced the number of isolated prominent cracks, and reduced the influence of stress concentration on GFRP. At the same time, steel fiber also improved the lateral deformation ability of core concrete, which was conducive to the restraint effect of GFRP. The fiber volume fraction was an important parameter affecting the axial compression property of the specimen, indicating that the addition of steel fiber improved the practical constraint of GFRP. The strain of the short column with fiber was significantly higher than that without fiber, but the increase in fiber content had a limited increase in strain. Considering the engineering cost and the workability of concrete, fiber content was not recommended to exceed 2%.The unconfined gangue concrete short column for the circumferential strain was about 0.005. The GFRP-confined gangue short column with three different tube thicknesses was significantly higher than the unconfined short column. The presence of a GFRP tube improved the circumferential deformation capacity of the gangue concrete short column. Still, the change in tube thickness had less effect on the circumferential deformation capacity of the short column, which was similar to the experimental results of Deng Zongcai . For theDI is the ductility coefficient of the specimen; In the formula: In engineering applications, in addition to improving the strength of concrete, the ductility of concrete should also be considered. T3W1, T5W1 and T7W1 were selected for comparison. The specimens with a tube thickness of 5 mm were 1.03 times and 1.37 times the specimens with a wall thickness of 3 mm and 7 mm, respectively. It can be seen that the ductility of the 3 mm and 5 mm tube thickness was better than that of the 7 mm tube thickness. T5W0, T5W1, T5W2 and T5W3 were selected for comparison. The fiber content of 1%, 2% and 3% was 1.23 times, 1.31 times and 1.15 times the fiber content of 0%, respectively. When the volume fraction of the GFRP tube was 3 mm, 5 mm and 7 mm, the ductility factor of the wave fiber was 9.6%, 2.1% and 4.4% higher than that of the end hook fiber. There was no simple linear relationship between tube thickness, fiber content, fiber shape and ductility coefficient. Some matching combinations existed to maximize the ductility coefficient of the specimen. It can be seen from When calculating the stress of the GFRP tube confined coal gangue short concrete column, the following formula  was usedIn the formula: The following three models were used to calculate the axial compressive strength of confined concrete:According to William’s failure criterion, Mander  obtainedIn the formula, Deng et al.  analyzedWhen the core concrete was subjected to three-dimensional compression, it was in the spatial stress state of equal lateral pressure due to the lateral hoop force. The Mohr–Coulomb  strengthΦ is the internal friction angle of concrete, and the value range is between 30° and 35°.In the formula, The test parameters are substituted into the above three theoretical models, respectively, and the calculation results are shown in The deviation in the calculation results was that the above three formulas were based on ordinary concrete. Under the same strength, there were differences in the elastic modulus and failure mode between ordinary concrete and coal gangue concrete . Therefore, under compression, the contribution of core concrete to strength was weakened, and the contribution of external constraints to strength was enhanced. Therefore, it was not appropriate to use the above model to directly calculate the bearing capacity of GFRP confined coal gangue concrete short columns. We modified the existing model and converted the Deng model into Equation (7), and the modified model was denoted as Deng1.2 = 0.94, the fitting effect was good, and the modified model was recorded as Deng1. The results are shown in Taking To show the calculation results of different models more intuitively, the comparison of the stress test values of all specimens with the stresses calculated by the Deng1 model, the Mander model, the Deng model, and the Mohr–Coulomb model (Cou) is shown in In summary, this paper improved the bearing capacity of coal gangue concrete by external confinement GFRP, which was similar to the practice in Reference . However(1)The thickness of the GFRP tube was the key factor affecting the axial compression performance of the specimen. The stress and strain of coal gangue concrete short column increased obviously with the increase in GFRP tube thickness. Compared with the unconstrained short column, the ultimate stress of the short column with tube thickness of 3, 5 and 7 mm was increased by 2.1 times, 3.0 times and 4.3 times, respectively, and the ultimate axial strain was increased by 18.8 times, 20.5 times and 21.5 times, respectively.(2)Under a certain fiber volume fraction, the connection effect of wave fiber on concrete was more prominent. The ultimate axial strain of the wave fiber-reinforced short column of the GFRP tube thickness 3 mm specimen was 10.1% higher than that of the end hook fiber specimen. The maximum difference in ductility coefficient between the two fiber specimens was 9.6%.(3)The steel fiber volume fraction significantly affected the axial compression behavior of the specimens. With the increase in fiber volume fraction, strain increase was more evident than stress. Compared with the specimens without fiber incorporation, the ultimate circumferential strain of the specimens with fiber volume fractions of 1%, 2%, and 3% increased by 26.4%, 46.6%, and 50.1%.(4)In this paper, by comparing the typical axial stress model of the existing literature with the experimental data, it was found that they all had different degrees of error. A stress model suitable for GFRP tube-confined steel fiber-reinforced coal gangue concrete was proposed by comparing the three stress models. The modified Deng model was in good agreement with the experimental results.This paper studied the mechanical properties of GFRP tube-confined coal gangue steel fiber short concrete composite short columns. The effects of GFRP tube thickness, steel fiber shape and fiber content on the mechanical properties of GFRP columns were analyzed. The stress–strain curves obtained from the axial compression failure of GFRP columns were analyzed, and the theoretical formula calculation was compared with the experimental data. The following conclusions were obtained:Future Research: This study optimized and upgraded the existing experiments and theories, but there were still some limitations. For example, the location of the strain gauge determines that they could only detect the strain of the GFRP tube. When the GFRP tube slipped with the core concrete, the monitoring data could not truly reflect the state of the concrete. In addition, due to time constraints, only one form of GFRP fiber tube was used in the test, and no other types of fiber-reinforced polymers were tested. The type of fiber tube will be further explored in future tests.


Pakistan has high neonatal mortality rate and antibiotic utilization data in hospitalized term neonates from Pakistan is lacking. The study aimed to determine the pattern of hospital antibiotic use in term neonates using World Health Organization (WHO) Access, Watch, and Reserve classification (AWaRe).st January 2020 to 31st December 2020. Hospitalized full term newborns up to 28-days of life, of either gender were consecutively included in the study. Data on demographic characteristics, admission diagnoses and antibiotic prescribed  were extracted from clinical charts. Descriptive statistics in the form of mean ± SD and frequency and percentages were calculated.This cross-sectional chart review study was conducted at the neonatal unit of Pediatric Medicine Department of University Hospital over a period of one year from 1A total of 2276 term neonates consisting of 69% (n=1570) males were included in the study. Antibiotic prescription rate was 92.8%. Most common reason for admission was birth asphyxia (36.1%) followed by sepsis (33%). Most commonly prescribed antibiotic Ampicillin (84%) belonged to Access group of WHO – AWaRe classification. More than 97% of prescriptions were two or more antimicrobial combinations belonging to Watch group. Ampicillin and Cefoperazone was the most commonly prescribed (42.8%) two drug combination. Meropenem and vancomycin was prescribed to 20% of the neonates – mostly for sepsis and pneumonia. Mortality rate was 7.7% in current study.High antibiotic prescription rate particularly from Watch-group is demonstrated in this study. There is high need of antimicrobial stewardship program in neonatal units. Neonates and infants comprise majority of the six million annual deaths under-5 years of age around the world.6The emergence of antibiotic resistance is one of the greatest challenges to the effective management of bacterial infections globally. Antimicrobial resistance has led to the longer durations of illness, increased treatment costs and high mortality rates.9In 2017, the WHO Expert Committee on Selection and Use of Essential Medicines developed an instrument – The AWaRe classification of antibiotics, to be used at local, regional and global level for the monitoring of antibiotic utilization, setting the goals and assessing the impact of stewardship programs. Currently 258 antibiotics are included in this classification. The WHO has currently set a country level target of 60% antibiotic consumption from the Access class. Traffic light color codes have been suggested to indicate these categories – Access (green), Watch (amber), and Reserve (red).10The present study aimed to document the antibiotic utilization in term neonates using WHO AWaRe classification in Pakistan to guide prioritization of future research with regards to rational antibiotic use in neonates.st January 2020 – 31st December 2020 at Nishtar University Hospital Multan in the Neonatal unit of Pediatric Medicine Department. All consecutive charts of full term neonates  between 1 – 28 days of life, who received at least one antibiotic during their hospital stay were included. Neonates admitted for post-operative care, syndromic features  and major congenital malformations  were excluded from the study which was approved from institutional Ethics and Research Committee .This descriptive cross-sectional chart review study was conducted from 1We created Excel 2016 spreadsheet as our master database. We extracted the initial data on total admissions, age (days), gender and diagnosis of the neonates during study period from admission / discharge register. We obtained the clinical charts of included neonates from the Medical Records and Statistical Department of the hospital for information on antibiotics used i.e. generic name, class, numbers, and duration of each antibiotic. All the data entry was supervised and double checked to minimize the bias. We used Statistical Package for Social Sciences (SPSS v. 23) for data analysis after importing the data from Excel spread sheet. The descriptive statistics were run in the form of mean ± standard deviation for numerical data and frequency and percentages for categorical data.A total of 2499 term neonates were admitted during study period. Of these neonates, 179 did not get any antibiotic and were excluded. Another 44 neonates with major congenital anomalies (n=25), syndromic features (n=7) and postoperative cases (n=12) were also excluded from the study. The final study group consisted of 2276 term neonates. Majority of the neonates were admitted within 24-hours of life , 69% (n=1570) were males and 40.6 % (n= 923) were low birth weight. Most of the neonates were out born  and 78% (n=1776) of the deliveries occurred at healthcare facilities. Most common mode of delivery was normal vaginal delivery  and 54.1% (n=1231) were discharged from the hospital. Antibiotic prescription rate in term neonates over one year was 92.8%. Mean number of antibiotics prescribed / patient were 2.1 ± 0.5 and mean duration of antimicrobial therapy was 6.7± 5.1 days. Ampicillin was the most common antibiotic prescribed  followed by Cefoperazone , Cefotaxime  and Meropenem . Antimicrobial days / 100 admission days were highest for Ampicillin (71) followed by Cefotaxime (37) and Cefoperazone (36) Only 2.4% of the neonates were treated with single antimicrobial agent. Most common single agent was Ampicillin (65.5%) followed by Cefotaxime (18.2%). More than three quarter of the neonates were prescribed two antibiotics. Most common of the two antibiotic combination was Ampicillin + Cefoperazone (42.8%) followed by Ampicillin + Cefotaxime in 35.9% of the neonates. Most common of the three drug combination was Ampicillin + Cefotaxime + Vancomycin in 18% followed by Ampicillin + Cefotaxime + Meropenem in 11.5%. Of all the four antibiotic combinations Ampicillin + Cefotaxime + Meropenem + Vancomycin was most common (36.0%). Most common reason for admission was birth asphyxia  followed by sepsis  and neonatal jaundice . All the antibiotic combinations used to treat most common diagnoses comprised of Watch-category. Ampicillin + Cefoperazone was the most common antibiotic combination used in TTN (55.8%), JNN (47.3%), birth asphyxia (47.3%) and pneumonia (25%). This was second most common in meconium aspiration (38.6%) and third most common in sepsis (19.1%). Most common combination prescribed to neonates with sepsis and meconium aspiration was Ampicillin + Cefotaxime (23.9% and 41.2% respectively). Meropenem + Vancomycin was the second most commonly prescribed combination for sepsis (22.1%) and third most common for pneumonia (17.2%) Our study documents a high prescription rate of antibiotics in neonates particularly from the Watch category. Concordant to our study high antibiotic prescription has been reported from countries like Rwanda (88%),Mean antibiotic duration in our study was lower than the study from University teaching hospital of Rwanda – 8.2 ±7.7 days.Ampicillin was highest prescribed antibiotic in our study from Access class. However, contrary to standard first line therapy in the form of Ampicillin and gentamicin, only 2.4% of the neonates in our study were prescribed Ampicillin alone and none received gentamicin. In more than 90% of the cases Ampicillin was combined with third generation Cephalosporin. Polypharmacy rate in our study was comparable to study from NepalBirth asphyxia was the most common admission diagnosis. However, in a study from Nepal neonatal sepsis was common admission diagnosis - 47.3%.18Meropenem and Vancomycin were most commonly prescribed to in sepsis and pneumonia. In a study from Kenya, most common antibiotic used for neonatal sepsis was penicillin and gentamicin.Being a single center study is one of our limitations. However, a large sample size over a period of one year and being the University hospital catering for the larger population, our study is likely to be representative of antibiotic use in term neonates. Due to small number of in-born neonates we did not separately compare the antibiotic utilization with out-born neonates.High antibiotic prescription rate particularly from Watch class is demonstrated in this study. Polypharmacy is observed in 90% of the cases. The study indicates lack of standard protocols for empirical treatment of neonatal infections. There is an urgent need for justified antimicrobial utilization in neonatal units considering the antibiograms of these units. Working healthcare professionals need to be trained on judicious antimicrobial use through antimicrobial stewardship program.MK: Conceived, designed the study, did statistical analysis, editing of manuscript and final approval, responsible and accountable for the accuracy or integrity of the work.JR: Did data collection, result interpretation, manuscript writing and critical review.IN: Did data collection, literature review and wrote manuscript first draft.